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NZ725538B2 - Compositions and methods for modulating apolipoprotein (a) expression - Google Patents
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NZ725538B2 - Compositions and methods for modulating apolipoprotein (a) expression - Google Patents

Compositions and methods for modulating apolipoprotein (a) expression Download PDF

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Publication number
NZ725538B2
NZ725538B2 NZ725538A NZ72553814A NZ725538B2 NZ 725538 B2 NZ725538 B2 NZ 725538B2 NZ 725538 A NZ725538 A NZ 725538A NZ 72553814 A NZ72553814 A NZ 72553814A NZ 725538 B2 NZ725538 B2 NZ 725538B2
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New Zealand
Prior art keywords
certain embodiments
compound
modified
group
oligonucleotide
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NZ725538A
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NZ725538A (en
Inventor
J Graham Mark
P Seth Punit
Eric E Swayze
P Prakash Thazha
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Ionis Pharmaceuticals Inc
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Publication date
Application filed by Ionis Pharmaceuticals Inc filed Critical Ionis Pharmaceuticals Inc
Priority to NZ740338A priority Critical patent/NZ740338A/en
Priority claimed from NZ631512A external-priority patent/NZ631512A/en
Publication of NZ725538A publication Critical patent/NZ725538A/en
Publication of NZ725538B2 publication Critical patent/NZ725538B2/en

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    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
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Abstract

Discloses a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12-30 contiguous nucleobases comprising a portion of at least 8 contiguous nucleosides complementary to an equal length portion of nucleobases of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide complementary to SEQ ID NO: 1; and wherein the conjugate group and sequences are as defined in the complete specification. he nucleobase sequence of the modified oligonucleotide complementary to SEQ ID NO: 1; and wherein the conjugate group and sequences are as defined in the complete specification.

Description

COMPOSITIONS AND METHODS FOR MODULATING APOLIPOPROTEIN (a) EXPRESSION SEQUENCE LISTING The present ation is being filed along with a Sequence Listing in electronic format. The ce Listing is provided as a file entitled BIOLOZSOWOSEQ_ST25.txt, created on May 1, 2014, which is 432 Kb in size. The ation in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION The principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates the amount, activity, and/or function of the target nucleic acid. For example in certain instances, antisense compounds result in d transcription or translation of a . Such modulation of expression can be achieved by, for example, target mRNA degradation or occupancy-based inhibition. An e of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense nd. Another e of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi refers to antisense-mediated gene silencing through a mechanism that utilizes the RNA-induced siliencing complex . An additional e of tion of RNA target function is by an occupancy-based mechanism such as is employed naturally by microRNA. MicroRNAs are small non-coding RNAs that regulate the expression of protein- coding RNAs. The binding of an antisense compound to a microRNA prevents that NA from binding to its messenger RNA targets, and thus interferes with the function of the microRNA. MicroRNA mimics can e native microRNA function. Certain antisense compounds alter splicing of pre-mRNA.
Regardless of the specific mechanism, ce-specificity makes antisense compounds attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively te the expression of genes involved in the pathogenesis of diseases. nse technology is an effective means for modulating the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. Chemically modified nucleosides may be incorporated into nse compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target nucleic acid. In 1998, the antisense compound, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, CA) was the first antisense drug to achieve marketing clearance from the US. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in AIDS patients.
New chemical modifications have improved the potency and efficacy of antisense nds, uncovering the potential for oral delivery as well as enhancing subcutaneous administration, decreasing potential for side s, and g to improvements in patient convenience. Chemical modifications increasing potency of antisense compounds allow administration of lower doses, which reduces the potential for toxicity, as well as decreasing overall cost of therapy. Modifications increasing the resistance to ation result in slower clearance from the body, allowing for less nt dosing. Different types of chemical modifications can be combined in one compound to further optimize the compounds y. oteins are globular, e-like particles that consist of a non-polar core of acylglycerols and cholesteryl esters surrounded by an amphiphilic coating of n, phospholipid and cholesterol.
Lipoproteins have been classified into five broad categories on the basis of their fimctional and physical properties: chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Chylomicrons transport dietary lipids from intestine to tissues. VLDLs, IDLs and LDLs all transport lglycerols and terol from the liver to tissues. HDLs transport endogenous cholesterol from tissues to the liver Lipoprotein particles undergo continuous metabolic processing and have variable properties and compositions. Lipoprotein ies increase without increasing le diameter because the density of their outer coatings is less than that of the inner core. The protein components of lipoproteins are known as apolipoproteins. At least nine apolipoproteins are distributed in significant amounts among the various human lipoproteins.
The lipoprotein(a) [Lp(a)] particle was identified nearly 50 years ago and is comprised of a highly unique LDL particle in which one apolipoprotein B (apoB) protein is linked via a disulfide bond to a single apolipoprotein(a) [apo(a)] protein. The apo(a) protein shares a high degree of homology with plasminogen particularly within the kringle IV type 2 repetitive domain. Levels of ating Lp(a) are inversely proportional to the number of kringle IV type 2 variable repeats present in the molecule and, as both s are co-expressed within individuals, can display heterozygous plasma isoform profiles (Kraft et al., Eur J Hum Genet, 1996; 4(2): 74-87). It is t that this kringle repeat domain in apo(a) may be responsible for its pro-thrombotic and anti-fibrinolytic properties, potentially enhancing atherosclerotic progression.
Apo(a) is transcriptionally regulated by IL-6 and in studies in rheumatoid arthritis patients d with an IL-6 inhibitor (tocilizumab), plasma levels were reduced by 30% after 3 month treatment (Schultz et al., PLoS One 2010; 5:el4328).
Apo(a) has been shown to preferentially bind oxidized phospholipids and potentiate ar inflammation (Bergmark et al., J Lipid Res 2008; 49:2230—2239; Tsimikas et al., Circulation. 2009; ll9(l3):l7l 1—1719).
Further, studies suggest that the Lp(a) particle may also stimulate endothelial permeability, induce plasminogen activator inhibitor type-l expression and activate macrophage interleukin-8 secretion insky and Marcovina, Curr Opin Lipidol 2004; 15:167—174). Importantly, recent genetic association studies revealed that Lp(a) was an independent risk factor for myocardial infarction, , eral vascular e and abdominal aortic aneurysm (Rifai et al., Clin Chem 2004; 50:1364—71; Erqou et al., JAMA 2009;302:412–23; Kamstrup et al., Circulation 2008;117:176–84). Further, in the recent Precocious Coronary Artery Disease (PROCARDIS) study, Clarke et al. (Clarke et al., NEJM (2009)361; 2518-2528) described robust and independent associations between coronary heart disease and plasma Lp(a) trations. Additionally, Solfrizzi et al., suggested that increased serum Lp(a) may be linked to an increased risk for mer’s Disease (AD) (Solfrizzi et al., J Neurol urg Psychiatry 2002, 72:732- 736. Currently, in the clinic setting, examples of indirect apo(a) inhibitors for treating cardiovascular disease include aspirin, Niaspan, Mipomersen, Anacetrapib, Epirotirome and Lomitapide which reduce plasma Lp(a) levels by 18%, 39%, 32%, 36%, 43% and 17%, tively. Additionally, Lp(a) apheresis has been used in the clinic to reduce apo(a) containing Lp(a) particles.
To date, therapeutic strategies to treat cardiovascular disease by directly ing apo(a) levels have been limited. Ribozyme oligonucleotides (U.S. Patent 5,877,022) and nse ucleotides (WO 00201; ; WO2013/177468; US20040242516; U.S. Patent Nos. 8,138,328, 8,673,632 and 7,259,150; Merki et al., J Am Coll Cardiol 2011; 57:1611–1621; each publication incorporated by reference in its entiretly) have been developed but none have been approved for commercial use.
Thus, there remains a clear unmet l need for novel agents which can potently and selectively reduce apo(a) levels in patients at enhanced risk for cardiovascular events due to chronically elevated plasma Lp(a) levels.
SUMMARY OF THE INVENTION The present application is a divisional application out of NZ . NZ 740338 is in turn a divisional of the present application. The complete description of the inventions of NZ 631512, the present application and NZ 740338 are retained herein for clarity and completeness.
Provided herein are compositions and methods for modulating sion of apo(a) mRNA and protein. In n embodiments, the apo(a) specific inhibitor decreases expression of apo(a) mRNA and protein. Provided herein are compositions and methods for ting expression of Lp(a) levels. In a particular aspect, the present invention provides a compound comprising a modified oligonucleotide and a conjugate group, wherein the ed ucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 1; wherein the conjugate group comprises: [FOLLOWED BY PAGE 3a] HOOH O O O N HO 4 H AcHN O HOOH O O O O O N O N N HO 4 O 4 H H H AcHN O HOOH O O N HO O AcHN , and wherein the compound is not: a compound sing a ed oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 20 contiguous nucleobases complementary to an equal length portion of nucleobases 3901 to 3920 of SEQ ID NO: 1, wherein the base sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 1; and wherein the conjugate group comprises: HOOH O O O N HO 4 H AcHN O HOOH O O O O O N O N N HO H H 4 O 4 H AcHN O HOOH O O N HO O AcHN .
In n embodiments, the composition is an apo(a) specific inhibitior. In certain ments, the apo(a) specific inhibitor is a nucleic acid, protein, or small molecule. In certain ments, the apo(a) specific inhibitor is an antisense oligonucleotide targeting apo(a) with a conjugate. In certain embodiments, the apo(a) specific inhibitor is a modified oligonucleotide and a conjugate, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising a n of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 3901 to 3920 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% [FOLLOWED BY PAGE 3b] complementary to SEQ ID NO: 1. In certain embodiments, the apo(a) specific inhibitor is a modified oligonucleotide and a conjugate, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a base sequence sing at least 8, least 9, least 10, least 11, at least 12, least 13, at least 14, at least 15, at least 16, least 17, least 18, least 19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 1-130, 133, 134. In certain embodiments, the apo(a) specific inhibitor is a modified oligonucleotide and a ate, wherein the modified oligonucleotide consists of 20 linked nucleosides and [FOLLOWED BY PAGE 4] has a base sequence comprising at least 8 contiguous nucleobases of any of SEQ ID NO: 5 8, wherein the modified oligonucleotide comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5’ wing segment consisting of five linked nucleosides; (c) a 3’ wing segment consisting of five linked nucleosides; and wherein the gap segment is positioned between the 5’ wing segment and the 3’ wing segment, wherein each nucleoside of each wing segment comprises a 2’-O-methoxyethyl sugar, wherein at least one intemucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5- methylcytosine.
Certain embodiments provide a composition comprising a ated antisense compound bed herein, or a salt thereof, and a pharmaceutically acceptable r or diluent.
In certain embodiments, the modulation of apo(a) expression occurs in a cell or tissue. In certain embodiments, the tions occur in a cell or tissue in an animal. In certain embodiments, the animal is a human. In certain embodiments, the modulation is a reduction in apo(a) mRNA level. In certain embodiments, the modulation is a reduction in apo(a) protein level. In certain embodiments, both apo(a) mRNA and protein levels are reduced. In certain embodiments, the modulation is a reduction in Lp(a) level.
Such reduction may occur in a time-dependent or in a dose-dependent manner.
Certain embodiments provide conjugated antisense compositions and methods for use in therapy.
Certain embodiments provide compositions and methods for preventing, ng, delaying, slowing the progression and/or ameliorating apo(a) related diseases, disorders, and conditions. Certain ments provide compositions and s for preventing, treating, delaying, slowing the progression and/or rating Lp(a) related diseases, disorders, and conditions. In certain embodiments, such diseases, disorders, and conditions are inflammatory, cardiovascular and/or lic diseases, disorders, and conditions. In certain embodiments, the compositions and methods for therapy include administering an apo(a) specific inhibitor to an individual in need thereof. In certain embodiments, the apo(a) specific inhibitor is a nucleic acid. In n embodiments, the nucleic acid is an antisense nd. In certain embodiments, the antisense nd is a modified oligonucleotide. In certain embodiments, the nse compound is a modified oligonucleotide with a conjugate.
In certain embodiments, the present disclosure provides conjugated antisense compounds. In certain embodiments, the t disclosure provides ated antisense compounds comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide complementary to a nucleic acid ript. In certain embodiments, the present disclosure provides methods comprising ting a cell with a conjugated antisense compound comprising an antisense oligonucleotide and reducing the amount or activity of a nucleic acid transcript in a cell.
The asialoglycoprotein receptor (ASGP-R) has been described previously. See e. g., Park et al., PNAS vol. 102, No. 47, pp 17129 (2005). Such receptors are sed on liver cells, particularly hepatocytes. Further, it has been shown that compounds comprising clusters of three N- acetylgalactosamine (GalNAc) ligands are capable of binding to the ASGP-R, resulting in uptake of the nd into the cell. See e.g., Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, pp 5216-5231 (May 2008). ingly, conjugates comprising such GalNAc clusters have been used to facilitate uptake of certain compounds into liver cells, specifically hepatocytes. For example it has been shown that certain GalNAc-containing conjugates increase activity of duplex siRNA nds in liver cells in vivo. In such ces, the GalNAc-containing conjugate is typically attached to the sense strand of the siRNA duplex.
Since the sense strand is ded before the antisense strand ultimately hybridizes with the target nucleic acid, there is little concern that the conjugate will interfere with activity. Typically, the conjugate is attached to the 3’ end of the sense strand of the siRNA. See e.g., U.S. Patent 8,106,022. Certain conjugate groups described herein are more active and/or easier to synthesize than conjugate groups previously described.
In certain embodiments of the present invention, conjugates are attached to single-stranded nse compounds, including, but not limited to RNase H based antisense compounds and antisense compounds that alter splicing of a pre-mRNA target nucleic acid. In such embodiments, the conjugate should remain attached to the antisense compound long enough to provide benefit (improved uptake into cells) but then should either be cleaved, or otherwise not interfere with the subsequent steps necessary for activity, such as hybridization to a target nucleic acid and ction with RNase H or enzymes associated with splicing or splice tion. This balance of ties is more important in the g of single-stranded antisense compounds than in siRNA nds, where the conjugate may simply be attached to the sense strand.
Disclosed herein are ated single-stranded antisense compounds having improved potency in liver cells in vivo compared with the same antisense compound lacking the conjugate. Given the ed balance of properties for these compounds such improved potency is surprising.
In certain ments, conjugate groups herein comprise a cleavable moiety. As noted, without wishing to be bound by ism, it is logical that the conjugate should remain on the compound long enough to provide enhancement in uptake, but after that, it is desirable for some portion or, ideally, all of the conjugate to be cleaved, releasing the parent compound (e. g., antisense compound) in its most active form. In certain ments, the cleavable moiety is a cleavable nucleoside. Such embodiments take age of endogenous ses in the cell by attaching the rest of the conjugate (the cluster) to the antisense oligonucleotide through a nucleoside via one or more cleavable bonds, such as those of a phosphodiester linkage. In certain embodiments, the cluster is bound to the cleavable nucleoside through a phosphodiester linkage. In certain embodiments, the cleavable nucleoside is attached to the antisense oligonucleotide (antisense compound) by a phosphodiester linkage. In certain embodiments, the conjugate group may comprise two or three cleavable nucleosides. In such ments, such cleavable sides are linked to one another, to the antisense compound and/or to the cluster via cleavable bonds (such as those of a phosphodiester linkage). Certain conjugates herein do not comprise a cleavable nucleoside and instead comprise a cleavable bond. It is shown that that sufficient cleavage of the conjugate from the oligonucleotide is provided by at least one bond that is vulnerable to cleavage in the cell (a cleavable bond).
In certain embodiments, conjugated antisense compounds are prodrugs. Such prodrugs are administered to an animal and are ultimately metabolized to a more active form. For example, conjugated nse compounds are cleaved to remove all or part of the conjugate resulting in the active (or more active) form of the antisense compound lacking all or some of the conjugate.
In certain embodiments, conjugates are attached at the 5’ end of an oligonucleotide. Certain such 5’- conjugates are cleaved more efficiently than counterparts having a similar conjugate group attached at the 3’ end. In certain embodiments, improved activity may correlate with ed cleavage. In certain embodiments, oligonucleotides comprising a conjugate at the 5’ end have greater efficacy than oligonucleotides sing a conjugate at the 3’ end (see, for example, Examples 56, 81, 83, and 84). r, 5’-attachment allows r oligonucleotide synthesis. Typically, oligonucleotides are synthesized on a solid support in the 3’ to 5’ direction. To make a 3’-conjugated oligonucleotide, typically one attaches a pre—conjugated 3’ nucleoside to the solid t and then builds the oligonucleotide as usual. However, attaching that conjugated nucleoside to the solid t adds complication to the synthesis. Further, using that approach, the conjugate is then present throughout the synthesis of the oligonucleotide and can become degraded during subsequent steps or may limit the sorts of reactions and reagents that can be used. Using the structures and techniques described herein for jugated oligonucleotides, one can synthesize the oligonucleotide using standard automated techniques and uce the conjugate with the final (5 ’-most) nucleoside or after the oligonucleotide has been cleaved from the solid support.
In view of the art and the present disclosure, one of ordinary skill can easily make any of the conjugates and ated oligonucleotides herein. er, synthesis of certain such ates and conjugated oligonucleotides disclosed herein is easier and/or requires few steps, and is therefore less expensive than that of conjugates previously disclosed, providing advantages in manufacturing. For example, the synthesis of certain conjugate groups ts of fewer synthetic steps, resulting in increased yield, relative to conjugate groups previously described. Conjugate groups such as GalNAc3-10 in e 46 and GalNAc3-7 in Example 48 are much simpler than previously bed conjugates such as those described in US. 8,106,022 or US. 7,262,177 that require assembly of more chemical intermediates . Accordingly, these and other conjugates described herein have ages over previously described compounds for use with any oligonucleotide, ing single-stranded oligonucleotides and either strand of -stranded oligonucleotides (e. g., siRNA).
Similarly, sed herein are conjugate groups having only one or two GalNAc ligands. As shown, such conjugates groups improve activity of nse compounds. Such compounds are much easier to prepare than conjugates comprising three GalNAc ligands. Conjugate groups comprising one or two GalNAc ligands may be attached to any antisense compounds, including single-stranded oligonucleotides and either strand of double-stranded oligonucleotides (e. g., siRNA).
In certain embodiments, the conjugates herein do not substantially alter certain measures of bility. For example, it is shown herein that conjugated antisense compounds are not more immunogenic than unconjugated parent compounds. Since potency is improved, embodiments in which tolerability remains the same (or indeed even if tolerability worsens only slightly compared to the gains in potency) have improved properties for y.
In certain embodiments, conjugation allows one to alter nse compounds in ways that have less attractive consequences in the absence of conjugation. For example, in certain embodiments, replacing one or more phosphorothioate linkages of a fully phosphorothioate antisense compound with phosphodiester es results in improvement in some measures of tolerability. For example, in certain instances, such antisense compounds having one or more odiester are less immunogenic than the same compound in which each linkage is a phosphorothioate. However, in certain instances, as shown in Example 26, that same replacement of one or more phosphorothioate es with phosphodiester es also results in reduced cellular uptake and/or loss in potency. In certain embodiments, conjugated antisense compounds described herein tolerate such change in linkages with little or no loss in uptake and potency when compared to the conjugated hosphorothioate rpart. In fact, in certain embodiments, for example, in Examples 44, 57, 59, and 86, oligonucleotides sing a ate and at least one phosphodiester internucleoside linkage actually exhibit increased potency in vivo even relative to a full phosphorothioate counterpart also comprising the same conjugate. Moreover, since conjugation results in ntial increases in uptake/potency a small loss in that substantial gain may be acceptable to achieve improved tolerability.
Accordingly, in certain ments, conjugated antisense compounds comprise at least one phosphodiester linkage.
In certain embodiments, conjugation of antisense compounds herein results in increased delivery, uptake and activity in hepatocytes. Thus, more compound is delivered to liver tissue. However, in certain embodiments, that sed delivery alone does not explain the entire increase in activity. In certain such embodiments, more compound enters cytes. In certain ments, even that increased hepatocyte uptake does not explain the entire increase in activity. In such embodiments, productive uptake of the conjugated compound is increased. For example, as shown in Example 102, certain ments of GalNAc-containing conjugates increase enrichment of antisense oligonucleotides in cytes versus non- parenchymal cells. This ment is beneficial for oligonucleotides that target genes that are expressed in hepatocytes.
In certain embodiments, conjugated antisense compounds herein result in reduced kidney exposure.
For example, as shown in Example 20, the concentrations of antisense oligonucleotides comprising n embodiments of GalNAc-containing conjugates are lower in the kidney than that of antisense oligonucleotides lacking a GalNAc-containing conjugate. This has several beneficial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding benefit. Moreover, high concentration in kidney typically results in loss of compound to the urine resulting in faster clearance. Accordingly for non-kidney targets, kidney accumulation is undesired.
In certain embodiments, the present disclosure provides conjugated antisense compounds ented by the formula: A—B—C—D—eE—F) Wherein A is the antisense oligonucleotide; B is the cleavable moiety C is the conjugate linker D is the ing group each E is a tether; each F is a ligand; and q is an integer between 1 and 5.
In the above diagram and in similar diagrams herein, the branching group “D” branches as many times as is necessary to accommodate the number of (E-F) groups as indicated by q” Thus, Where q = l, the formula is: A—B—C—D—E—F Where q = 2, the formula is: Where q = 3, the a is: Where q = 4, the formula is: Where q = 5, the formula is: A—B—C—D In certain ments, conjugated antisense compounds are provided having the structure: Targeting moiety /—/% HO OH wwflwfi O:P70H NH2 0 N VJ”0 «fr/" NHAc HO0H 0 o Mfl£0%,“ C? NHAc ice—1 Cleavable moiety Ligand Tether :eeww Branching group NHAc In certain embodiments, conjugated antisense compounds are provided having the structure: Cell targeting moiety HO /\ " /P\ Cleavable m01ety' ACHN | 0 >| NH N 2 HOOH O o O H (H) 0 (1:117% OH “ ACHN 0 Q Tether Ligand 0—on o \ HO OH ii 0 O\/\/\/\0/ A80 NHAC Branching group PCT/USZOl4/036460 In certain embodiments, conjugated antisense compounds are provided having the structure: ASO Cleavable moiety Cell targeting moiety I—(gl—I—l HO OW\/\ (H) 04.30 OH ACHN 0 j 0 HO OH 0 (<2; Conjugate o 9 | linker HO /P\ 0—1320 0 do 0 I ACHN 0 OH Tether I—l HO 0“ (ii 0 O\/\/\/\O (5-0 NHAC Branching group In certain ments, ated antisense compounds are provided having the structure: Ligand Cl bl _ (I) Tether eava e m01ety HO—P=O HOOH HO%/O H l O N 0 4 2 O HOOH o Hog Vlir/O H ( ) O N 3 2 O ACHN O Conjugate HOOH HO¥/ WV linker O H O N 2 O ACHN O Branching group %/—J Cell targeting moiety The present disclosure provides the following non-limiting numbered embodiments: In embodiments having more than one of a particular variable (e.g., more than one “m” or “n”), unless otherwise indicated, each such particular variable is selected independently. Thus, for a structure having more than one n, each n is selected independently, so they may or may not be the same as one another.
In certain embodiments, the present disclosure provides conjugated antisense compounds ented by the following structure. In certain embodiments, the nse compound comprises modified oligonucleotide ISIS 494372 with a 5’-X, wherein X is a conjugate group comprising GalNAc. In certain embodiments, the antisense compound consists of d oligonucleotide ISIS 494372 with a 5’-X, wherein X is a conjugate group comprising GalNAc.
WO 79625 In n embodiments, the present disclosure provides conjugated antisense compounds ented by the following structure. In certain embodiments, the antisense compound comprises the conjugated modified ucleotide ISIS 681251. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 681251.
HO&W WHO HN O ‘5 N O O N WNH / N O In certain embodiments, the present disclosure provides conjugated nse compounds ented by the following structure. In certain embodiments, the antisense compound ses the conjugated modified oligonucleotide ISIS 681257. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 681257. 2014/036460 In n embodiments, the present disclosure provides conjugated antisense compounds represented by the ing structure. In certain embodiments, the antisense compound comprises a modified oligonucleotide With SEQ ID NO: 58 With a NAc With variability in the sugar mods of the Wings. In certain embodiments, the antisense compound consists of a modified oligonucleotide With SEQ ID NO: 58 with a 5’-GalNAc with variability in the sugar mods of the Wings.
Wherein either R1 is —OCH2CHZOCH3 (MOE)and R2 is H; or R1 and R2 together form a bridge, wherein R1 is —O- and R2 is —CH2-, -CH(CH3)-, or -CH2CH2-, and R1 and R2 are directly connected such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and —O-CH2CH2-; And for each pair of R3 and R4 on the same ring, independently for each ring: either R3 is selected from H and -OCH2CH20CH3 and R4 is H; or R3 and R4 er form a , wherein R3 is —O-, and R4 is — CH2-, -CH(CH3)-, or -CH2CH2-and R3 and R4 are directly connected such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and —O-CH2CH2-; And R5 is selected from H and —CH3; And Z is selected from S' and O'.
The present sure provides the following non-limiting ed embodiments: DETAILED DESCRIPTION It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means r” unless stated otherwise. rmore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and ents that comprise more than one subunit, unless specifically stated otherwise.
The section headings used herein are for zational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.
A. Definitions Unless specific definitions are provided, the lature used in connection with, and the procedures and ques of, analytical chemistry, synthetic c chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such ques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society Pharmaceutical , Washington DC, 1994; ”Remington's Sciences,” Mack hing Co., Easton, Pa., 21St edition, 2005; and “Antisense Drug Technology, Principles, Strategies, and ations” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2“1 Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published ations and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.
Unless otherwise indicated, the following terms have the following meanings: As used herein, “nucleoside” means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, lly occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.
As used herein, “chemical modification” means a chemical difference in a compound when compared to a naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and intemucleoside linkage modifications. In reference to an ucleotide, chemical modification does not include differences only in nucleobase sequence.
As used herein, “furanosyl” means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.
As used herein, “naturally occurring sugar moiety” means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in lly occurring DNA.
As used herein, “sugar moiety” means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
As used herein, “modif1ed sugar moiety” means a substituted sugar moiety or a sugar ate.
As used herein, “substituted sugar moiety” means a furanosyl that is not a naturally occurring sugar moiety. tuted sugar moieties include, but are not d to furanosyls sing substituents at the 2’-position, the 3’-position, the 5’-position and/or the 4’-position. Certain substituted sugar es are bicyclic sugar moieties.
As used herein, “2’-substituted sugar moiety” means a furanosyl comprising a substituent at the 2’- position other than H or OH. Unless otherwise indicated, a stituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2’-substituent of a 2’-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.
As used , “MOE” means -OCH2CH20CH3.
As used herein, “2’-F nucleoside” refers to a nucleoside comprising a sugar comprising fluorine at the 2’ position. Unless otherwise indicated, the fluorine in a 2’-F nucleoside is in the ribo position (replacing the OH of a natural ribose).
As used herein the term ”sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the ing nucleoside sub-units are capable of linking together and/or linking to other nucleosides to form an oligomeric nd which is capable of hybridizing to a complementary oligomeric compound. Such structures e rings sing a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e. g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a ement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e. g., 6-membered carbocyclic ic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring s of peptide nucleic acid). Sugar surrogates include Without limitation morpholinos, cyclohexenyls and eXitols.
As used , “bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2’-carbon and the 4’-carbon of the furanosyl.
As used herein, “nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid es ribonucleic acids (RNA), deoxyribonucleic acids (DNA), -stranded nucleic acids (ssDNA), double-stranded nucleic acids (dsDNA), small interfering ribonucleic acids (siRNA), and microRNAs (miRNA). A nucleic acid may also comprise any combination of these elements in a single molecule.
As used herein, “nucleotide” means a side further comprising a phosphate linking group. As used herein, “linked sides” may or may not be linked by phosphate linkages and thus includes, but is not limited to “linked nucleotides.” As used herein, “linked sides” are nucleosides that are connected in a continuous sequence (i.e. no additional sides are t between those that are linked).
As used herein, ”nucleobase” means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and Wherein the group of atoms is e of bonding With a complementary naturally occurring nucleobase of r oligonucleotide or nucleic acid.
Nucleobases may be naturally occurring or may be modified. As used herein, ”nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, or nucleobase ation.
As used herein the terms, ”unmodified nucleobase” or ”naturally ing nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) ding 5-methyl C), and uracil (U).
As used herein, “modified base” means any nucleobase that is not a naturally occurring nucleobase.
As used herein, “modified nucleoside” means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.
As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety.
As used herein, rained ethyl side” or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4’-CH(CH3)-O-2’bridge.
As used herein, “locked nucleic acid nucleoside” or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4’-CH2-O-2’bridge.
As used herein, “2’-substituted nucleoside” means a nucleoside sing a substituent at the 2’- position other than H or OH. Unless otherwise indicated, a 2’-substituted nucleoside is not a bicyclic nucleoside.
As used , “deoxynucleoside” means a nucleoside sing 2’-H furanosyl sugar moiety, as found in naturally ing deoxyribonucleosides (DNA). In certain embodiments, a 2’-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e. g., uracil).
As used herein, ”oligonucleotide” means a compound comprising a plurality of linked nucleosides.
In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more d nucleosides.
As used herein nucleoside” means an oligonucleotide in which none of the intemucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides.
As used herein, “modified ucleotide” means an ucleotide sing at least one modif1ed nucleoside and/or at least one modif1ed intemucleoside linkage.
As used herein, “linkage” or “linking group” means a group of atoms that link together two or more other groups of atoms.
As used herein “intemucleoside linkage” means a covalent linkage n adjacent nucleosides in an oligonucleotide.
As used herein “naturally ing internucleoside linkage” means a 3' to 5' phosphodiester linkage.
As used , “modified cleoside linkage” means any intemucleoside linkage other than a naturally occurring intemucleoside linkage.
As used herein, “terminal internucleoside linkage” means the linkage between the last two nucleosides of an oligonucleotide or defined region thereof.
As used herein, “phosphorus linking group” means a linking group comprising a phosphorus atom.
Phosphorus linking groups include without limitation groups haVing the formula: Rb:1:)_Rc wherein: R2, and Rd are each, independently, O, S, CH2, NH, or NJ 1 wherein J1 is C1-C6 alkyl or tuted C1- C6 alkyl; Rb is O or S; RC is OH, SH, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, amino or substituted amino; and J1 is Rb is O or S.
Phosphorus linking groups include without limitation, phosphodiester, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, orothioamidate, thionoalkylphosphonate, phosphotriesters, thionoalkylphosphotriester and boranophosphate.
As used herein, “internucleoside phosphorus linking group” means a phosphorus linking group that directly links two nucleosides.
As used herein, “non-internucleoside phosphorus g group” means a phosphorus linking group that does not directly link two nucleosides. In n embodiments, a non-internucleoside phosphorus linking group links a nucleoside to a group other than a nucleoside. In n embodiments, a non- intemucleoside phosphorus linking group links two groups, neither of which is a nucleoside.
As used herein, ”neutral linking group” means a linking group that is not charged. Neutral linking groups include without limitation phosphotriesters, phosphonates, MMI (-CHg-N(CH3)-O-), amide-3 (- CH2-C(=O)-N(H)-), amide-4 (-CHz-N(H)-C(=O)-), formacetal (-O-CH2), and thioformacetal (-S-CH2).
Further neutral g groups include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: ydrate Modifications in Antisense Research; Y.S. SanghVi and PD. Cook Eds. ACS ium Series 580; Chapters 3 and 4, (pp. 40-65)). Further neutral linking groups include nonionic es comprising mixed N, O, S and CH2 component parts.
As used herein, “internucleoside neutral g group” means a neutral linking group that directly links two nucleosides.
As used herein, nternucleoside neutral linking group” means a neutral linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside neutral linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-internucleoside neutral linking group links two groups, neither of which is a nucleoside.
As used herein, ”oligomeric compound” means a polymeric structure comprising two or more sub- structures. In n embodiments, an eric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric nd consists of an oligonucleotide. Oligomeric compounds also include naturally occurring nucleic acids. In certain embodiments, an oligomeric compound comprises a backbone of one or more linked ric subunits where each linked monomeric subunit is directly or indirectly attached to a heterocyclic base moiety. In certain embodiments, oligomeric compounds may also include monomeric subunits that are not linked to a heterocyclic base moiety, y providing abasic sites.
In certain embodiments, the linkages joining the monomeric subunits, the sugar moieties or surrogates and the heterocyclic base moieties can be independently modified. In certain ments, the linkage-sugar unit, which may or may not include a heterocyclic base, may be substituted with a mimetic such as the rs in peptide nucleic acids.
As used herein, “terminal group” means one or more atom attached to , or both, the 3’ end or the 5’ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain WO 79625 ments, a terminal group comprises one or more terminal group nucleosides.
As used herein, “conjugate” or “conjugate group” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, tion, cellular distribution, cellular uptake, charge and/or clearance properties.
As used herein, “conjugate linker” or “linker” in the context of a conjugate group means a portion of a conjugate group comprising any atom or group of atoms and which covalently link (1) an oligonucleotide to another portion of the conjugate group or (2) two or more portions of the ate group.
Conjugate groups are shown herein as radicals, providing a bond for forming covalent attachment to an oligomeric compound such as an antisense oligonucleotide. In certain embodiments, the point of attachment on the oligomeric compound is the 3'-oxygen atom of the 3'-hydroxyl group of the 3’ terminal nucleoside of the oligomeric compound. In certain embodiments the point of attachment on the oligomeric compound is the 5'-oxygen atom of the 5'-hydroxyl group of the 5’ terminal nucleoside of the oligomeric compound. In n embodiments, the bond for forming attachment to the oligomeric compound is a ble bond. In certain such embodiments, such cleavable bond constitutes all or part of a cleavable moiety.
In n embodiments, conjugate groups comprise a cleavable moiety (e.g., a cleavable bond or cleavable nucleoside) and a carbohydrate cluster portion, such as a GalNAc cluster portion. Such carbohydrate cluster portion comprises: a ing moiety and, optionally, a conjugate linker. In certain embodiments, the carbohydrate cluster portion is identified by the number and identity of the ligand. For e, in certain embodiments, the carbohydrate cluster portion ses 3 GalNAc groups and is ated “GalNAc3”. In certain embodiments, the carbohydrate cluster portion comprises 4 GalNAc groups and is designated “GalNAc4”. Specific carbohydrate cluster portions (having specific tether, branching and conjugate linker groups) are described herein and designated by Roman numeral followed by subscript “a”. Accordingly “GalNac3-la” refers to a specific carbohydrate cluster portion of a conjugate group having 3 GalNac groups and specifically identified tether, branching and linking groups. Such carbohydrate cluster fragment is attached to an oligomeric compound via a cleavable moiety, such as a cleavable bond or cleavable side.
As used herein, able moiety” means a bond or group that is capable of being split under physiological conditions. In certain embodiments, a ble moiety is d inside a cell or sub-cellular compartments, such as a lysosome. In n embodiments, a cleavable moiety is cleaved by endogenous enzymes, such as nucleases. In certain embodiments, a cleavable moiety ses a group of atoms having one, two, three, four, or more than four ble bonds.
As used herein, “cleavable bond” means any chemical bond capable of being split. In certain embodiments, a cleavable bond is ed from among: an amide, a polyamide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a ate, a di-sulfide, or a peptide.
As used herein, ”carbohydrate cluster” means a compound having one or more carbohydrate residues attached to a scaffold or linker group. (see, e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugated to a alent Carbohydrate Cluster for Cellular Targeting,” Bioconjugate try, 2003, (14): 18-29, which is incorporated herein by reference in its entirety, or Rensen et al., “Design and Synthesis of Novel N—Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein or,” J. Med. Chem. 2004, (47): 5798-5808, for examples of carbohydrate conjugate clusters).
As used herein, “modif1ed carbohydrate” means any carbohydrate having one or more chemical modifications relative to naturally occurring carbohydrates.
As used herein, “carbohydrate derivative” means any compound which may be synthesized using a ydrate as a starting material or intermediate.
As used herein, hydrate” means a naturally ing carbohydrate, a d carbohydrate, or a carbohydrate derivative.
As used herein cting group” means any compound or protecting group known to those having skill in the art. Non-limiting examples of protecting groups may be found in ”Protective Groups in Organic Chemistry”, T. W. Greene, P. G. M. Wuts, ISBN 062301-6, John Wiley & Sons, Inc, New York, which is incorporated herein by reference in its entirety.
As used , “single-stranded” means an oligomeric compound that is not hybridized to its complement and which lacks sufficient self-complementarity to form a stable self-duplex.
As used , “double stranded” means a pair of oligomeric compounds that are hybridized to one another or a single self-complementary oligomeric compound that forms a hairpin structure. In certain embodiments, a double-stranded oligomeric compound ses a first and a second oligomeric compound.
As used herein, “antisense compound” means a compound comprising or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.
As used , “antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity includes modulation of the amount or activity of a target nucleic acid transcript (e. g. mRNA). In certain ments, antisense activity includes modulation of the splicing of pre-mRNA.
As used herein, “RNase H based antisense compound” means an antisense compound wherein at least some of the antisense activity of the nse compound is utable to hybridization of the nse compound to a target nucleic acid and subsequent cleavage of the target nucleic acid by RNase H.
As used herein, “RISC based nse compound” means an antisense compound wherein at least some of the antisense activity of the antisense compound is attributable to the RNA Induced Silencing Complex (RISC).
As used herein, “detecting” or “measuring” means that a test or assay for detecting or measuring is med. Such detection and/or ing may result in a value of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of ing or measuring the activity has nevertheless been med.
As used herein, “detectable and/or measureable activity” means a statistically significant activity that is not zero.
As used herein, “essentially unchanged” means little or no change in a particular parameter, particularly relative to another parameter which changes much more. In n embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target c acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.
As used herein, “expression” means the process by which a gene ultimately results in a n.
Expression includes, but is not limited to, transcription, ranscriptional modification (e. g., splicing, polyadenlyation, addition of 5’-cap), and translation.
As used herein, ”target nucleic acid” means a nucleic acid molecule to which an antisense compound is intended to hybridize to result in a desired antisense activity. Antisense oligonucleotides have sufficient complementarity to their target nucleic acids to allow ization under physiological conditions.
As used herein, “nucleobase mentarity” or ementarity” when in reference to nucleobases means a nucleobase that is e of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, mentary nucleobase means a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that base pair.
Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still e of nucleobase complementarity.
As used herein, “non-complementary” in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one r.
As used herein, “complementary” in reference to oligomeric compounds (e.g., linked nucleosides, ucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity.
Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside.
Rather, some ches are tolerated. In n embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% mentary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary. In certain embodiments, complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% mentary.
As used herein, “mismatch” means a nucleobase of a first oligomeric compound that is not capable of pairing with a nucleobase at a corresponding position of a second oligomeric compound, When the first and second oligomeric compound are aligned. Either or both of the first and second eric compounds may be oligonucleotides.
As used herein, ”hybridization” means the pairing of complementary oligomeric compounds (e. g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves en bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen en bonding, between complementary nucleobases.
As used herein, “specifically izes” means the ability of an oligomeric compound to hybridize to one nucleic acid site With greater affinity than it hybridizes to r nucleic acid site.
As used , “fully complementary” in nce to an oligonucleotide or portion thereof means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing With a nucleobase of a complementary nucleic acid or contiguous portion thereof. Thus, a fully mentary region comprises no mismatches or unhybridized nucleobases in either strand.
As used herein, “percent complementarity” means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. t mentarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.
As used , “percent identity” means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.
As used herein, ”modulation” means a change of amount or quality of a molecule, function, or activity When ed to the amount or quality of a molecule, function, or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or ion) in gene sion. As a r example, modulation of expression can include a change in splice site ion of pre-mRNA processing, resulting in a change in the te or relative amount of a particular splice-variant compared to the amount in the absence of modulation.
As used herein, “chemical motif” means a pattern of chemical modifications in an oligonucleotide or a region f. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligonucleotide.
As used herein, “nucleoside motif” means a pattern of nucleoside modifications in an oligonucleotide or a region thereof. The linkages of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.
As used herein, “sugar motif” means a pattern of sugar modifications in an oligonucleotide or a region thereof.
As used , ge motif” means a pattern of linkage modifications in an oligonucleotide or region thereof. The nucleosides of such an oligonucleotide may be d or fied. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.
As used herein, “nucleobase modification motif” means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase ce.
As used herein, nce motif” means a pattern of bases ed along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical ations and thus may have any combination of al modifications, including no al modifications.
As used herein, “type of modification” in reference to a nucleoside or a nucleoside of a “type” means the chemical ation of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a “nucleoside having a modification of a first type” may be an unmodified nucleoside.
As used herein, “differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified.
Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2’-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2’-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.
As used herein, “the same type of modifications” refers to modifications that are the same as one r, including absence of modifications. Thus, for e, two unmodified DNA nucleosides have “the same type of modification,” even though the DNA side is unmodified. Such nucleosides having the same type modification may comprise different nucleobases.
As used herein, ate regions” means portions of an ucleotide wherein the chemical modifications or the motif of al modifications of any neighboring portions e at least one difference to allow the separate regions to be distinguished from one another.
As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.
As used herein the term “metabolic disorder” means a disease or condition principally characterized by ulation of metabolism — the complex set of al reactions associated with breakdown of food to produce energy.
As used herein, the term “cardiovascular er” means a e or condition principally characterized by impaired function of the heart or blood vessels.
As used herein the term ”mono or polycyclic ring system” is meant to include all ring systems selected from single or polycyclic radical ring systems wherein the rings are fused or linked and is meant to be inclusive of single and mixed ring systems individually ed from aliphatic, alicyclic, aryl, aryl, aralkyl, kyl, cyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such mono and poly cyclic structures can contain rings that each have the same level of saturation or each, independently, have varying degrees of saturation ing fully saturated, partially saturated or fully unsaturated. Each ring can comprise ring atoms selected from C, N, O and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the fused ring has two nitrogen atoms. The mono or polycyclic ring system can be further tuted with substituent groups such as for example imide which has two =0 groups attached to one of the rings. Mono or polycyclic ring s can be attached to parent molecules using various strategies such as directly through a ring atom, fused through multiple ring atoms, through a substituent group or through a bifunctional linking moiety.
As used herein, “prodrug” means an ve or less active form of a compound which, when administered to a subject, is metabolized to form the active, or more active, compound (e. g., drug).
As used herein, ”substituen ” and ”substituent group,” means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e. g., a modified 2’- substuent is any atom or group at the 2’-position of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, compounds of the t disclosure have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.
Likewise, as used herein, “substituent” in reference to a al functional group means an atom or group of atoms that differs from the atom or a group of atoms ly present in the named functional group. In certain embodiments, a substituent es a hydrogen atom of the functional group (e. g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups le for use as substituents include without limitation, halogen, yl, alkyl, alkenyl, alkynyl, 2014/036460 acyl (-C(O)Raa), carboxyl (-C(O)O-Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (-O-Raa), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (-N(Rbb)(RCC)), imino(=NRbb), amido (-C(O)N(Rbb)(RCC) or -N(Rbb)C(O)Raa), azido (-N3), nitro (-N02), cyano (-CN), carbamido (-OC(O)N(Rbb)(RCC) or -N(Rbb)C(O)ORaa), ureido (-N(Rbb)C(O)N(Rbb)(Rcc)), thioureido (-N(Rbb)C(S)N(Rbb)- (Rcc)), guanidinyl (-N(Rbb)C(=NRbb)N(Rbb)(RCC)), amidinyl (-C(=NRbb)N(Rbb)(RCC) or -N(Rbb)C(=NRbb)(Raa)), thiol (-SRbb), yl (-S(O)Rbb), sulfonyl (-S(O)2Rbb) and amidyl (-S(O)2N(Rbb)(RCC) or -N(Rbb)S- (0)2Rbb). n each Raa, Rbb and RCC is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including t limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, l, heteroaryl, alicyclic, cyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive .
As used , ”alkyl,” as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, l, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (Cl-Cu alkyl) with from 1 to about 6 carbon atoms being more preferred.
As used , ”alkenyl,” means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, l-methylbuten—l-yl, dienes such as l,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.
As used herein, "alkynyl," means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and haVing at least one carbon-carbon triple bond. Examples of alkynyl groups e, without limitation, ethynyl, l-propynyl, l-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.
As used herein, ”acyl," means a radical formed by removal of a hydroxyl group from an organic acid and has the general a X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, tic sulfonyls, aromatic sulf1nyls, tic sulf1nyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further tuent groups.
As used herein, ”alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is tic. Preferred alicyclics include rings haVing from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups. 2014/036460 As used herein, ”aliphatic” means a straight or branched arbon radical containing up to twenty four carbon atoms wherein the tion between any two carbon atoms is a single, double or triple bond.
An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms e without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.
As used herein, ”alkoxy” means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent le. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert—butoxy, n- pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further tuent groups.
As used herein, ”aminoalkyl” means an amino tuted C1-C12 alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.
As used herein, ”aralkyl” and ”arylalkyl” mean an aromatic group that is covalently linked to a C1-C12 alkyl radical. The alkyl radical portion of the ing aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.
As used herein, ”aryl” and ”aromatic” mean a mono- or polycyclic carbocyclic ring system ls haVing one or more aromatic rings. es of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. red aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.
As used herein, ”halo” and ”halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.
As used herein, ”heteroaryl,” and ”heteroaromatic,” mean a radical comprising a mono- or poly- cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is ic and es one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, en or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, olyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, WO 79625 quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.
As used herein, “conjugate compound” means any atoms, group of atoms, or group of linked atoms le for use as a conjugate group. In certain embodiments, conjugate compounds may possess or impart one or more properties, including, but not limited to codynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.
As used herein, unless otherwise indicated or modified, the term “double-stranded” refers to two separate oligomeric compounds that are hybridized to one another. Such double stranded compounds may have one or more or bridizing nucleosides at one or both ends of one or both s (overhangs) and/or one or more internal non-hybridizing nucleosides (mismatches) provided there is sufficient complementarity to in hybridization under physiologically relevant conditions.
As used herein, “5’ target site” refers to the tide of a target nucleic acid which is complementary to the 5’-most nucleotide of a particular antisense compound.
As used herein, “About” means within 210% of a value. For example, if it is stated, “a marker may be increased by about 50%”, it is implied that the marker may be increased between 45%-55%.
As used herein, “administered itantly” refers to the co-administration of two agents in any manner in which the pharmacological s of both are manifest in the patient at the same time.
Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be nsive.
As used herein, “administering” or “administration” means providing a pharmaceutical agent to an individual, and includes, but is not limited to, stering by a medical professional and self-administering.
Administration of a pharmaceutical agent to an individual can be continuous, chronic, short or intermittent.
Administration can parenteral or non-parenteral.
As used herein, “agent” means an active substance that can provide a eutic benefit when administered to an animal. “First agent” means a therapeutic compound of the ion. For example, a first agent can be an antisense oligonucleotide targeting apo(a). “Second agent” means a second therapeutic compound of the invention (e.g. a second nse oligonucleotide targeting apo(a)) and/or a non-apo(a) therapeutic compound.
As used herein, “amelioration” or “ameliorate” or “ameliorating” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. The severity of tors can be ined by subjective or objective measures, which are known to those skilled in the art.
WO 79625 As used herein, “animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and nzees.
As used herein, )” means any nucleic acid or protein sequence encoding apo(a). For example, in n embodiments, apo(a) includes a DNA sequence encoding apo(a), a RNA sequence transcribed from DNA encoding apo(a) (including genomic DNA sing introns and exons), a mRNA sequence encoding apo(a), or a peptide sequence encoding apo(a).
As used herein, “apo(a) nucleic acid” means any nucleic acid encoding apo(a). For example, in certain embodiments, an apo(a) nucleic acid includes a DNA sequence encoding apo(a), a RNA sequence transcribed from DNA encoding apo(a) (including genomic DNA comprising introns and exons), and a mRNA sequence encoding apo(a).
As used , “apo(a) mRN ” means a mRNA encoding an apo(a) protein.
As used herein, “apo(a) protein” means any protein sequence encoding Apo(a).
As used herein, “apo(a) specific inhibitor” refers to any agent capable of specifically inhibiting the expression of an apo(a) nucleic acid and/or apo(a) protein. For example, apo(a) specific inhibitors include nucleic acids ding antisense nds), es, antibodies, small molecules, and other agents capable of inhibiting the expression of apo(a) nucleic acid and/or apo(a) protein. In certain ments, by specifically ting apo(a) nucleic acid expression and/or apo(a) protein expression, apo(a) specific inhibitors can affect other components of the lipid transport system including downstream components. rly, in certain embodiments, apo(a) specific inhibitors can affect other molecular processes in an animal.
As used herein, “atherosclerosis” means a hardening of the arteries affecting large and medium-sized arteries and is characterized by the presence of fatty deposits. The fatty deposits are called ”atheromas” or “plaques,” which consist mainly of cholesterol and other fats, calcium and scar tissue, and damage the lining of arteries.
As used herein, ary heart disease (CHD)” means a narrowing of the small blood s that supply blood and oxygen to the heart, which is often a result of atherosclerosis.
As used , “diabetes mellitus” or “diabetes” is a syndrome terized by disordered lism and abnormally high blood sugar (hyperglycemia) resulting from insufficient levels of insulin or reduced insulin sensitivity. The characteristic ms are excessive urine production (polyuria) due to high blood glucose levels, excessive thirst and increased fluid intake (polydipsia) attempting to compensate for increased urination, blurred vision due to high blood glucose effects on the eye's optics, unexplained weight loss, and lethargy.
As used herein, “diabetic dyslipidemia” or “type 2 diabetes with idemia” means a condition characterized by Type 2 diabetes, reduced HDL-C, ed triglycerides (TG), and elevated small, dense LDL particles.
As used herein, “diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For e, the t in an injected composition can be a liquid, e. g. saline solution.
As used herein, “dyslipidemia” refers to a disorder of lipid and/or lipoprotein metabolism, ing lipid and/or lipoprotein overproduction or deficiency. Dyslipidemias can be manifested by elevation of lipids such as chylomicron, terol and triglycerides as well as lipoproteins such as low-density lipoprotein (LDL) cholesterol.
As used herein, “dosage unit” means a form in which a pharmaceutical agent is provided, e.g. pill, tablet, or other dosage unit known in the art. In certain embodiments, a dosage unit is a Vial containing lyophilized nse oligonucleotide. In certain embodiments, a dosage unit is a Vial containing reconstituted nse ucleotide.
As used herein, “dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose can be administered in one, two, or more boluses, tablets, or injections. For example, in n ments where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections can be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses can be stated as the amount of pharmaceutical agent per hour, day, week, or month. Doses can also be stated as mg/kg or g/kg.
As used herein, “effective amount” or “therapeutically effective amount” means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an indiVidual in need of the agent. The effective amount can vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual’s medical condition, and other nt factors.
As used herein, “fully complementary” or “100% complementary” means each nucleobase of a nucleobase ce of a first nucleic acid has a complementary nucleobase in a second nucleobase sequence of a second nucleic acid. In certain ments, a first nucleic acid is an antisense compound and a second nucleic acid is a target nucleic acid.
As used herein, “glucose” is a monosaccharide used by cells as a source of energy and inflammatory intermediate. “Plasma glucose” refers to e present in the plasma.
As used , “high density lipoprotein-C” or “HDL-C” means cholesterol associated with high y lipoprotein particles. Concentration of HDL-C in serum (or plasma) is typically fied in mg/dL or nmol/L. “Serum HDL-C” and “plasma HDL-C” mean HDL-C in serum and , respectively.
As used , “HMG-CoA reductase inhibitor” means an agent that acts through the inhibition of the enzyme HMG-CoA reductase, such as atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin. 2014/036460 As used herein, “hypercholesterolemia” means a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL-cholesterol and VLDL-cholesterol, as per the guidelines of the Expert Panel Report of the al Cholesterol Educational Program (NCEP) of Detection, Evaluation of Treatment of high cholesterol in adults (see, Arch. Int. Med. (1988) 148, .
As used herein, “hyperlipidemia” or “hyperlipemia” is a ion characterized by ed serum lipids or circulating (plasma) lipids. This condition manifests an abnormally high concentration of fats. The lipid fractions in the circulating blood are cholesterol, low density oteins, very low density lipoproteins, chylomicrons and triglycerides. The Fredrickson fication of hyperlipidemias is based on the pattern of TG and cholesterol-rich lipoprotein les, as measured by electrophoresis or ultracentrifugation and is commonly used to characterize primary causes of hyperlipidemias such as hypertriglyceridemia (Fredrickson and Lee, Circulation, 1965, 31 :321-327; ckson et al., New Eng J Med, 1967, 276 (1): 34—42).
As used herein, triglyceridemia” means a condition characterized by elevated triglyceride levels. Its etiology includes primary (i.e. genetic causes) and secondary (other underlying causes such as diabetes, metabolic syndrome/insulin resistance, obesity, physical inactivity, cigarette smoking, excess alcohol and a diet very high in carbohydrates) s or, most often, a combination of both (Yuan et al.
CMAJ, 2007,176:1113-1120).
As used herein, “identifying” or “selecting an animal with metabolic or cardiovascular disease” means identifying or ing a subject prone to or having been diagnosed with a metabolic disease, a cardiovascular disease, or a lic syndrome; or, identifying or selecting a subject having any symptom of a metabolic disease, cardiovascular disease, or metabolic syndrome including, but not limited to, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertension sed insulin resistance, decreased insulin sensitivity, above normal body weight, and/or above normal body fat content or any combination thereof Such identification can be accomplished by any method, including but not limited to, standard clinical tests or assessments, such as measuring serum or circulating (plasma) cholesterol, ing serum or circulating (plasma) glucose, measuring serum or circulating (plasma) triglycerides, measuring blood-pressure, measuring body fat content, measuring body weight, and the like.
As used herein, “improved cardiovascular outcome” means a reduction in the ence of adverse cardiovascular events, or the risk thereof. Examples of adverse cardiovascular events include, without limitation, death, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiac arrest, and atrial dysrhythmia.
As used herein, iately adjacent” means there are no intervening elements between the immediately nt elements, for e, between regions, segments, nucleotides and/or nucleosides.
As used herein, “increasing HDL” or “raising HDL” means increasing the level of HDL in an animal after administration of at least one compound of the invention, compared to the HDL level in an animal not administered any compound.
As used herein, idual” or “subject” or “animal” means a human or non-human animal selected for treatment or therapy.
As used herein, “individual in need thereof” refers to a human or non-human animal selected for treatment or y that is in need of such treatment or therapy.
As used herein, “induce”, “inhibit”, “potentiate”, “elevate”, “increase”, “decrease”, “reduce” or the like denote quantitative differences between two states. For example, “an amount ive to inhibit the ty or expression of apo(a)” means that the level of activity or sion of apo(a) in a treated sample will differ from the level of apo(a) activity or expression in an untreated sample. Such terms are d to, for example, levels of expression, and levels of activity.
As used herein, “inflammatory condition” refers to a e, disease state, syndrome, or other condition resulting in inflammation. For example, rheumatoid arthritis and liver fibrosis are inflammatory conditions. Other examples of inflammatory conditions include sepsis, myocardial ischemia/reperfusion injury, adult respiratory distress syndrome, nephritis, graft rejection, inflammatory bowel disease, multiple sclerosis, arteriosclerosis, atherosclerosis and vasculitis.
As used herein, “inhibiting the sion or activity” refers to a reduction or blockade of the expression or activity of a RNA or protein and does not necessarily te a total elimination of expression or activity.
As used herein, “insulin resistance” is defined as the condition in which normal amounts of insulin are inadequate to produce a normal insulin response from fat, muscle and liver cells. Insulin resistance in fat cells results in hydrolysis of stored triglycerides, which elevates free fatty acids in the blood plasma. Insulin resistance in muscle reduces glucose uptake whereas insulin resistance in liver reduces glucose e, with both effects serving to elevate blood glucose. High plasma levels of insulin and glucose due to insulin resistance often leads to metabolic syndrome and type 2 es.
As used herein, “insulin sensitivity” is a measure of how effectively an individual ses glucose.
An individual having high insulin sensitivity effectively processes glucose whereas an individual with low insulin sensitivity does not effectively process glucose.
As used herein, “lipid-lowering” means a reduction in one or more lipids (e. g., LDL, VLDL) in a subject. “Lipid-raising” means an increase in a lipid (e. g., HDL) in a subject. Lipid-lowering or lipid-raising can occur with one or more doses over time.
As used , “lipid-lowering therapy” or “lipid lowering agent” means a therapeutic regimen provided to a subject to reduce one or more lipids in a t. In certain embodiments, a lipid-lowering therapy is provided to reduce one or more of apo(a), CETP, apoB, total cholesterol, LDL-C, VLDL-C, IDL- C, non-HDL-C, triglycerides, small dense LDL particles, and Lp(a) in a subject. Examples of lipid-lowering therapy include, but are not limited to, apoB inhibitors, statins, fibrates and MTP inhibitors.
As used , rotein”, such as VLDL, LDL and HDL, refers to a group of proteins found in the serum, plasma and lymph and are ant for lipid ort. The chemical composition of each lipoprotein differs, for example, in that the HDL has a higher proportion of n versus lipid, whereas the VLDL has a lower proportion of protein versus lipid.
As used herein, “Lp(a)” comprises apo(a) and a LDL like particle containing apoB. The apo(a) is linked to the apoB by a disulfide bond.
As used herein, “low density lipoprotein-cholesterol (LDL-C)” means cholesterol carried in low density otein particles. Concentration of LDL-C in serum (or plasma) is typically quantified in mg/dL or . “Serum LDL-C” and a LDL-C” mean LDL-C in the serum and plasma, respectively.
As used herein, “major risk factors” refers to factors that bute to a high risk for a particular disease or condition. In certain embodiments, major risk factors for coronary heart disease e, without limitation, cigarette smoking, hypertension, high LDL, low HDL-C, family history of coronary heart disease, age, and other s disclosed .
As used herein, “metabolic disorder” or “metabolic disease” refers to a condition characterized by an alteration or disturbance in metabolic function. “Metabolic” and “metabolism” are terms well known in the art and generally include the whole range of biochemical processes that occur within a living sm.
Metabolic disorders include, but are not limited to, hyperglycemia, prediabetes, diabetes (type 1 and type 2), y, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes.
As used herein, “metabolic syndrome” means a condition characterized by a clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin. In certain embodiments, metabolic syndrome is identified by the presence of any 3 of the following factors: waist circumference of greater than 102 cm in men or greater than 88 cm in women; serum triglyceride of at least 150 mg/dL; HDL-C less than 40 mg/dL in men or less than 50 mg/dL in women; blood pressure of at least 130/85 mmHg; and fasting e of at least 110 mg/dL. These determinants can be readily ed in clinical practice (JAMA, 2001, 285: 2486-2497). teral administration” means administration through injection or infusion. Parenteral administration includes subcutaneous stration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e. g. intrathecal or intracerebroventricular administration. Administration can be continuous, chronic, short or intermittent.
As used herein, “peptide” means a molecule formed by g at least two amino acids by amide bonds. Peptide refers to ptides and proteins.
As used herein, “pharmaceutical agent” means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeted to apo(a) is a pharmaceutical agent.
As used herein, “pharmaceutical composition” or “composition” means a e of substances suitable for administering to an dual. For example, a pharmaceutical composition can comprise one or more active agents and a pharmaceutical carrier e. g., a sterile aqueous solution.
As used herein, “pharmaceutically acceptable derivative” encompasses derivatives of the compounds described herein such as solvates, hydrates, esters, prodrugs, polymorphs, isomers, isotopically labelled variants, pharmaceutically acceptable salts and other derivatives known in the art.
As used herein, “pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of nse nds, i.e., salts that retain the desired ical activity of the parent compound and do not impart undesired toxicological effects thereto. The term aceutically acceptable salt” or “salt” es a salt prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic or organic acids and bases. “Pharmaceutically acceptable salts” of the compounds described herein may be prepared by methods well-known in the art. For a review of pharmaceutically acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, ion and Use -VCH, Weinheim, Germany, 2002). Sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic administration to . Accordingly, in one embodiment the compounds described herein are in the form of a sodium salt.
As used herein, “portion” means a defined number of contiguous (i.e. linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.
As used herein, “prevent”or ”preventing” refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing risk of developing a disease, disorder, or ion.
As used herein, “raise” means to increase in amount. For example, to raise plasma HDL levels means to increase the amount of HDL in the .
As used , “reduce” means to bring down to a smaller extent, size, amount, or number. For example, to reduce plasma triglyceride levels means to bring down the amount of triglyceride in the plasma.
As used herein, “region” or “target region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. For example, a target region may encompass a 3’ UTR, a 5’ UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation ation region, or other defined nucleic acid . The structurally defined regions for apo(a) can be obtained by accession number from ce databases such as NCBI and such information is incorporated herein by reference. In certain ments, a target region may encompass the sequence from a 5’ target site of one target segment within the target region to a 3’ target site of another target segment within the target .
As used herein, “second agent” or “second eutic agent” means an agent that can be used in combination with a “first agent”. A second therapeutic agent can include, but is not limited to, antisense oligonucleotides targeting apo(a) or apoB. A second agent can also include anti- apo(a) antibodies, apo(a) peptide inhibitors, cholesterol lowering agents, lipid lowering agents, glucose lowering agents and anti- inflammatory agents.
As used , “segments” are d as smaller, sub-portions of regions within a nucleic acid. For example, a “target segment” means the sequence of nucleotides of a target nucleic acid to which one or more antisense compounds is targeted. “5’ target site” refers to the t nucleotide of a target segment. “3’ target site” refers to the 3’-most tide of a target segment. Alternatively, a “start site” can refer to the 5’ most nucleotide of a target segment and a “stop site” refers to the 3’-most nucleotide of a target segment. A target t can also begin at the “start site” of one sequence and end at the “stop site” of another sequence.
As used herein, “statin” means an agent that inhibits the activity of HMG-CoA reductase.
As used herein, taneous administration” means administration just below the skin.
As used herein, “subject” means a human or non-human animal selected for treatment or therapy.
As used herein, “symptom of cardiovascular disease or disorder” means a phenomenon that arises from and accompanies the cardiovascular disease or disorder and serves as an indication of it. For example, angina; chest pain; shortness of breath; palpitations; weakness; dizziness; nausea; sweating; tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling in the lower extremities; cyanosis; fatigue; fainting; numbness of the face; numbness of the limbs; claudication or cramping of muscles; bloating of the abdomen; or fever are ms of cardiovascular e or disorder.
As used herein, “targeting” or “targeted” means the process of design and selection of an antisense compound that will specifically ize to a target nucleic acid and induce a desired effect.
As used herein, peutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
As used herein, peutic lifestyle change” means y and lifestyle changes intended to lower fat/adipose tissue mass and/or cholesterol. Such change can reduce the risk of developing heart disease, and may includes recommendations for dietary intake of total daily calories, total fat, saturated fat, polyunsaturated fat, saturated fat, carbohydrate, protein, cholesterol, insoluble fiber, as well as recommendations for physical actiVity.
As used herein, “treat” or “treating” refers to administering a compound described herein to effect an alteration or improvement of a disease, disorder, or condition.
As used , “triglyceride” or “TG” means a lipid or neutral fat consisting of ol combined with three fatty acid molecules.
As used herein, “type 2 diabetes,” (also known as “type 2 diabetes mellitus”, “diabetes mellitus, type 2”, “non-insulin-dependent diabetes”, “NIDDM”, “obesity related diabetes”, or “adult-onset diabetes”) is a lic disorder that is ily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia.
Certain Embodiments In certain embodiments, a compound comprises a siRNA or antisense oligonucleotide targeted to apolipoprotein(a) (apo(a)) known in the art and a conjugate group described herein. Examples of antisense oligonucleotides targeted to apo(a) suitable for conjugation include but are not d to those disclosed in ; US 8,673,632; US 7,259,150; and US Patent Application Publication No. US 2004/0242516; which are incorporated by reference in their entireties herein. In certain embodiments, a compound comprises an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 12- 130, 133, 134 disclosed in WC 2013/177468 and a conjugate group described . In certain embodiments, a compound comprises an nse oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 11-45 and 85-96 disclosed in US 8,673,632 and a conjugate group described . In certain embodiments, a compound comprises an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 11-45 disclosed in US 7,259,150 and a conjugate group described herein. In certain embodiments, a compound comprises an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 7-41 disclosed in US Patent Application Publication No. US 2004/0242516 and a ate group described herein. The nucleobase sequences of all of the aforementioned referenced SEQ ID NOs are incorporated by reference herein.
Certain embodiments provide a compounds and methods for decreasing apo(a) mRNA and protein expression. In certain embodiments, the compound is an apo(a) specific inhibitor for treating, preventing, or ameliorating an apo(a) associated disease. In certain embodiments, the compound is an antisense oligonucleotide targeting apo(a). In certain embodiments, the compound is an nse oligonucleotide targeting apo(a) and a conjugate group.
Certain embodiments provide a compounds and methods for sing Lp(a) levels. In n embodiments, the compound is an apo(a) specific inhibitor for treating, preventing, or ameliorating an Lp(a) associated e. In certain embodiments, the compound is an antisense ucleotide ing apo(a). In certain ments, the compound is an antisense ucleotide targeting apo(a) and a conjugate group.
Certain embodiments provide a nd comprising a modified oligonucleotide ing apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides. In certain embodiments, the modified ucleotide with the conjugate group ts of 15 to 30, 18 to 24, 19 to 22, 13 to 25, 14 to 25, 15 to 25 linked nucleosides. In n embodiments, the modified oligonucleotide with the ate group comprises at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or 30 linked nucleosides. In certain embodiments, the modified oligonucleotide with the conjugate group consists of 20 linked nucleosides.
Certain ments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases complementary to an equal length portion of any of SEQ ID NOs: 1-4.
Certain embodiments provide a nd comprising a modified oligonucleotide targeting an apo(a) segment and a conjugate group, wherein the modified ucleotide comprises at least 8, at least 9, at least , at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or contiguous bases complementary to an equal length portion of any of the target segments shown in, for example, Examples 114 and 117. In the tables, the “Start Site” refers to the 5’-most nucleotide of a target segment and “Stop Site” refers to the 3’-most nucleotide of a target segment. A target segment can range from the start site to the stop site of each sequence listed in the tables. Alternatively, the target segment can range from the start site of one sequence and end at the stop site of another sequence. For example, as shown in Table 125, a target segment can range from 3901-3920, the start site to the stop site of SEQ ID NO: 58. In r example, as shown in Table 125, a target segment can range from 3900-3923, the start site of SEQ ID NO: 57 to the stop site of SEQ ID NO: 61.
Certain embodiments provide a nd comprising a d oligonucleotide targeting apo(a) and a conjugate group, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to any of SEQ ID NOs: 1-4. Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, n the nucleobase sequence of the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to any of the target segments shown in, for example, Examples 1 14 and 1 17.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising a portion of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases mentary to an equal length portion of nucleobases 3901 to 3920 of SEQ ID NO: 1, wherein the base sequence of the d oligonucleotide is at least 80% complementary to SEQ ID NO: 1.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence sing at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or 30 contiguous nucleobases complementary to an equal length portion of bases 3900 to 3923 of SEQ ID NO: 1, wherein the nucleobase sequence of the d oligonucleotide is at least 80% complementary to SEQ ID NO: 1.
Certain embodiments provide a compound comprising a modified ucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has 2014/036460 a base sequence sing at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 12-130, 133, 134. In certain embodiments, the modified oligonucleotide has a nucleobase sequence sing at least 8 contiguous nucleobases of any one of the nucleobase sequences of SEQ ID NOs: 12-130, 133, 134. In certain embodiments, the nd consists of any one of SEQ ID NOs: , 133, 134 and a conjugate group.
Certain embodiments provide a compound sing a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a base sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 12-20, 22-33, 35-44, 47-50, 51, 53, 57-62, 65-66, 68, 70-79, 81, 85- 86, 89-90, 92-94, 97, 105-110, 103-104, 133-134. In certain embodiments, the nd consists of any of the nucleobase sequences of SEQ ID NOs: 12-20, 22-33, 35-44, 47-50, 51, 53, 57-62, 65-66, 68, 70-79, 81, 85-86, 89-90, 92-94, 97, 105-110, 103-104, 133-134 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 12-19, 26-30, 32, 35, 38-44, 46-47, 50, 57-58, 61, 64-66, 68, 72-74, 76-77, 92-94, 103-110. In certain embodiments, the compound consists of any of the base sequences of SEQ ID NOs: 12-19, 26-30, 32, 35, 38-44, 46-47, 50, 57-58, 61, 64-66, 68, 72-74, 76-77, 92-94, 0 and a conjugate group.
Certain embodiments provide a compound comprising a modified ucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the base ces of SEQ ID NOs: 111, 114-121, 123-129. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 111, 114-121, 123-129 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: l4, l7, 18, 26-28, 39, 71, 106-107. In certain embodiments, the compound ts of any ofthe nucleobase sequences of SEQ ID NOs: l4, l7, 18, 26-28, 39, 71, 106-107 and a conjugate group. 2014/036460 Certain embodiments e a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, n the modified oligonucleotide ts of 12 to 30 linked sides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 14, 26-29, 39-40, 82. In certain ments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 14, 26-29, 39-40, 82 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 14, 16-18. In certain ments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 14, 16-18 and a ate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase ce comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the base sequences of SEQ ID NOs: 26-27, 107. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 26-27, 107 and a conjugate group.
Certain embodiments e a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the base sequences of SEQ ID NOs: 28-29, 39-40, 47. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: : 28-29, 39-40, 47 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide ing apo(a) and a conjugate group, wherein the modified ucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 28, 93, 104, 134. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 28, 93, 104, 134 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 58. In certain embodiments, the modified oligonucleotide with the conjugate group has a base sequence comprising at least 8 uous nucleobases of the nucleobase sequence of SEQ ID NO: 5 8. In certain embodiments, the compound consists of SEQ ID NO: 5 8 and a conjugate group.
In certain embodiments, the present disclosure es conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises the modified oligonucleotide ISIS 494372 with a 5’-X, wherein X is a conjugate group comprising . In certain embodiments, the antisense compound consists of the modified oligonucleotide ISIS 494372 with a 5’-X, wherein X is a conjugate group comprising GalNAc.
In certain embodiments, the present disclosure es conjugated antisense compounds represented by the following structure. In certain embodiments, the nse nd comprises the conjugated modified oligonucleotide ISIS 681251. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 681251.
HO&W WHO HN O ‘5 N O O N WNH / N O In certain embodiments, the present disclosure provides conjugated nse nds represented by the following structure. In certain embodiments, the antisense compound comprises the conjugated d oligonucleotide ISIS 681257. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 681257.
In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises a d oligonucleotide with the nucleobase sequence of SEQ ID NO: 58 with a 5’-GalNAc with variability in the sugar mods of the wings. In certain ments, the antisense compound consists of a d oligonucleotide with the nucleobase seuquence of SEQ ID NO: 58 with a 5’-GalNAc with variability in the sugar mods of the wings.
HOOH o Hofiowflflll O wNH O HOOH O HomOWHk/o IoNH 0 HOOH Infiwofibmo fi IoNH Wherein either R1 is —OCH2CHZOCH3 (MOE) and R2 is H; or R1 and R2 er form a bridge, wherein R1 is —O- and R2 is —CH2-, -CH(CH3)-, or -CH2CH2-, and R1 and R2 are directly ted such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and —O-CH2CH2-; And for each pair of R3 and R4 on the same ring, independently for each ring: either R3 is ed from H and -OCH2CH20CH3 and R4 is H; or R3 and R4 together form a bridge, wherein R3 is —O-, and R4 is — CH2-, -CH(CH3)-, or -CH2CH2-and R3 and R4 are ly connected such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and —O-CH2CH2-; And R5 is ed from H and —CH3; And Z is selected from S' and 0'.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a ate group, wherein the d oligonucleotide is single-stranded.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein at least one intemucleoside linkage is a modified intemucleoside linkage. In certain embodiments, the modified intemucleoside linkage is a orothioate intemucleoside linkage. In certain embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 internucleoside linkages of said modified oligonucleotide are phosphorothioate intemucleoside linkages. In certain ments, each intemucleoside linkage is a phosphorothioate intemucleoside linkage. In certain embodiments, the modified oligonucleotide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 phosphodiester intemucleoside linkages. In certain embodiments, each intemucleoside linkage of the modified oligonucleotide is selected from a phosphodiester internucleoside linkage and a orothioate intemucleoside linkage.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein at least one nucleoside ses a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine.
Certain embodiments provide a nd comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide comprises at least one modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the d sugar comprises a 2’-O-methoxyethyl, a ained ethyl, a ro-HNA or a 4’- (CH2)n-O-2’ bridge, wherein n is l or 2.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified ucleotide consists of 12 to 30 linked nucleosides and comprises: (a) a gap segment consisting of linked deoxynucleosides; (b) a 5’ wing segment ting of linked nucleosides; (c) a 3’ wing segment consisting of linked nucleosides; and wherein the gap segment is positioned between the 5’ wing segment and the 3’ wing t and wherein each side of each wing segment ses a modified sugar.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, n the modified oligonucleotide consists of 20 linked nucleosides and comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5’ wing segment consisting of five linked nucleosides; (c) a 3’ wing segment consisting of five linked nucleosides; and wherein the gap segment is oned between the 5’ wing segment and the 3’ wing segment, wherein each nucleoside of each wing segment comprises a ethoxyethyl sugar, wherein at least one intemucleoside linkage is a phosphorothioate linkage and wherein each ne residue is a 5-methylcytosine.
Certain embodiments e a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and has a nucleobase sequence comprising at least 8 contiguous bases of any of SEQ ID NOs: 12-130, 133, 134, wherein the modified oligonucleotide ses: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5’ wing segment consisting of five linked nucleosides; (c) a 3’ wing segment consisting of five linked nucleosides; and wherein the gap segment is positioned between the 5’ wing segment and the 3’ wing segment, wherein each nucleoside of each wing segment comprises a 2’-O- methoxyethyl sugar, wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine.
Certain embodiments provide a compound comprising a d oligonucleotide targeting apo(a) and a conjugate group, wherein the d oligonucleotide consists of 20 linked nucleosides and has a nucleobase ce comprising at least 8 contiguous nucleobases of SEQ ID NO: 58, wherein the modified ucleotide comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5’ wing segment consisting of five linked nucleosides; (c) a 3’ wing segment consisting of five linked nucleosides; and wherein the gap segment is positioned between the 5’ wing segment and the 3’ wing segment, wherein each nucleoside of each wing segment comprises a 2’-O-methoxyethyl sugar, wherein at least one intemucleoside linkage is a phosphorothioate linkage and wherein each ne residue is a 5 - methylcytosine.
Certain embodiments provide a d oligonucleotide ing apo(a) and a ate group, wherein the modified oligonucleotide consists of 20 linked nucleosides with the nucleobase ce of SEQ ID NO: 58, wherein the modified ucleotide comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5’ wing segment consisting of five linked nucleosides; (c) a 3’ wing segment consisting of five linked nucleosides; and wherein the gap segment is positioned between the 5’ wing segment and the 3’ wing segment, wherein each nucleoside of each wing segment comprises a 2’-O- methoxyethyl sugar, wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine.
In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 5’ end of the modified oligonucleotide. In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 3’ end of the modified oligonucleotide.
In certain embodiments, the conjugate group comprises one or more s. In certain embodiments, the conjugate group comprises two or more ligands. In certain ments, the conjugate group comprises three or more ligands. In certain ments, the conjugate group comprises three ligands. In certain embodiments, each ligand is selected from among: a polysaccharide, modified polysaccharide, mannose, galactose, a mannose derivative, a ose derivative, D-mannopyranose, L-Mannopyranose, D-Arabinose, L-Galactose, D-Xylofuranose, L-XlefUI‘aIIOSC, D-glucose, L-glucose, D-Galactose, L-Galactose, nnofuranose , B-D-Mannofuranose, (x-D-Mannopyranose, B-D-Mannopyranose, (x-D-Glucopyranose, B-D- yranose, (x-D-GlucofiJranose, B-D-GlucofiJranose, (x-D-fructofiJranose, (x-D-fructopyranose, lactopyranose , B -D-Galactopyranose, (x-D-Galactofuranose, B -D-Galactofuranose, glucosamine, sialic acid, (x-D-galactosamine, N—Acetylgalactosamine, 2-Amino0-[(R)-l-carboxyethyl]—2-deoxy-B-D- glucopyranose, 2-Deoxymethylamino-L-glucopyranose, deoxyformamido-2,3-di- O-methyl-D- mannopyranose, 2-Deoxysulfoamino-D-glucopyranose, N—Glycoloyl-(x-neuraminic acid, 5-thio-B-D- glucopyranose, methyl 2,3,4-tri- O-acetyl-l-thioO-trityl-(x-D-glucopyranoside, 4-Thio-B-D- galactopyranose, ethyl 3,4,6,7-tetraacetyldeoxy-l ,5-dithio-0t-D-gluc0-heptopyranoside, 2,5-Anhydro- D-allononitrile, ribose, D-ribose, Dthioribose, L-ribose, Lthioribose. In certain embodiments, each ligand is N—acetyl galactosamine.
In certain embodiments, each ligand is N—acetyl galactosamine.
In certain embodiments, the conjugate group comprises: HO OH In certain embodiments, the conjugate group comprises: 2014/036460 In certain embodiments, the conjugate group comprises: WO 79625 HoOH ' O (3ng HO 3 O O AcHN | HOOH o N OW o AcHN l o=P-OH HoOH " o N HO 3 o [20 E AcHN In certain embodiments, the conjugate group comprises at least one phosphorus linking group or neutral linking group.
In certain embodiments, the conjugate group comprises a structure selected from among: OH OH E—o—E—o eke o—E—o—g é—o—E—o 0&0 o 5H V3 “rt \ (5H 5H m “”2 e” o o t‘ mgr-HII 5 E s ”M H OH 0 o and wherein n is from 1 to 12; and wherein m is from 1 to 12.
In certain embodiments, the ate group has a tether having a structure selected from among: 0 Z1 6’5 L 5'3. {95% )W/L E n L is either a phosphorus linking group or a neutral linking group; Z1 is C(=O)O-R2; Z2 is H, C1-C6 alkyl or substituted C1-C6 alky; R2 is H, C1-C6 alkyl or tuted C1-C6 alky; and each ml is, independently, from O to 20 wherein at least one ml is greater than 0 for each tether.
In certain embodiments, conjugate group has a tether having a structure selected from among: (3 3L 0 COOH 9H 5,1 o—P—o and “‘21‘ m1 (5H m1 fiwN/K(o—fi—o—Qm1m1 H O wherein Z2 is H or CH3; and each ml is, independently, from O to 20 wherein at least one ml is greater than 0 for each tether.
In certain embodiments, the conjugate group has tether having a structure selected from among: Walden/$71 ; EMHJLN o wherein n is from 1 to 12; and wherein m is from 1 to 12.
In n embodiments, the conjugate group is covalently attached to the modified oligonucleotide.
In certain embodiments, the compound has a structure represented by the formula: A—B—C—D+E—F) wherein A is the modified oligonucleotide; B is the cleavable moiety C is the conjugate linker D is the branching group each E is a tether; each F is a ligand; and q is an integer n 1 and 5.
In certain embodiments, the compound has a structure represented by the formula: A—éBfiCfiDfiE_F>Cl wherein: A is the modified oligonucleotide; B is the cleavable moiety C is the conjugate linker D is the branching group each E is a tether; each F is a ligand; each n is independently O or 1; and q is an integer n 1 and 5.
In n embodiments, the compound has a ure represented by the formula: A—B—c+E—F> wherein A is the modified oligonucleotide; B is the cleavable moiety; C is the conjugate linker; each E is a tether; each F is a ligand; and q is an integer between 1 and 5.
In certain ments, the compound has a structure represented by the formula: A—C_D_6E—F) A is the modified oligonucleotide; C is the conjugate linker; D is the branching group; each E is a tether; each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, the compound has a structure represented by the formula: A—c+E—F> wherein A is the ed ucleotide; C is the conjugate linker; each E is a tether; each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, the compound has a structure represented by the formula: A—B—D+E—F> wherein A is the modified ucleotide; B is the cleavable moiety; D is the branching group; each E is a tether; each F is a ; and q is an integer between 1 and 5.
In certain embodiments, the compound has a structure represented by the formula: Awe—a wherein A is the modified oligonucleotide; B is the ble moiety; each E is a ; each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, the compound has a structure represented by the formula: A nee—F) wherein A is the modified oligonucleotide; D is the branching group; each E is a tether; each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, the conjugate linker has a structure ed from among: 0 O A .fiifrpfiks Wherein each L is, independently, a phosphorus linking group or a neutral linking group; and each n is, independently, from 1 to 20.
In certain embodiments, the conjugate linker has a ure selected from among: 0 O O O EkNMN/YH a H /‘14,. JV“ . EkNW” ;"77_ W;; O HO’ 0 O O N\/\></\/\f‘H H r‘;W H 0 0 ’ H s’ N\/\ NY, 0 o W o o O O \n/\/ \915’ W \/\O O 5’5 2014/036460 In certain embodiments, the conjugate linker has the followingstructure: Ova/”‘1 In certain ments, the conjugate linker has a structure selected from among: {a\o/\/\e_‘s; éf\O/\/\O/\/\éé ;and £\O/\/\O/\/\O/\/\€‘s _ In certain embodiments, the conjugate linker has a structure selected from among: OH OH $030 0&0_ _ _ 030;_ _ _ and $030_ _ _ 04/0 0 5H W3 \ 5H V3 “Pg 5H V3 9" In certain embodiments, the ate linker has a structure ed from among: 0 o 3’ o—P—o—§ e3 _ 3 NH/\(\/)/631, 3 ”M6 (IDH ’ and E 2 ”M In certain embodiments, the conjugate linker comprises a pyrrolidine. In certain ments, the conjugate linker does not comprise a pyrrolidine. In certain embodiments, the conjugate linker comprises PEG. In certain embodiments, the ate linker comprises an amide. In certain embodiments, the ate linker comprises at least two amides. In certain embodiments, the conjugate linker does not comprise an amide. In certain embodiments, the conjugate linker comprises a ide. In certain embodiments, the conjugate linker comprises an amine. In certain embodiments, the conjugate linker comprises one or more disulfide bonds. In certain embodiments, the conjugate linker comprises a protein binding moiety. In certain embodiments, the protein binding moiety comprises a lipid.
In certain embodiments, the n binding moiety is selected from among: cholesterol, cholic acid, adamantane acetic acid, l-pyrene butyric acid, dihydrotestosterone, l,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, l, menthol, 1,3-propanediol, heptadecyl group, ic acid, myristic acid, O3-(oleoyl)lithocholic acid, eoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a Vitamin (e.g., folate, Vitamin A, Vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic component, a d (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g., sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid), or a cationic lipid.
In n embodiments, the protein binding moiety is selected from among: a C16 to C22 long chain saturated or unsaturated fatty acid, cholesterol, cholic acid, Vitamin E, adamantane or l-pentafluoropropyl.
In certain embodiments, the conjugate linker has a structure selected from among: H H w E—NH N N | '31/ W 0”; 9 0 R023 [1V0 O—P—OH N | N 0 \‘f ”luv ' H \ i N N ” O 3r flfigo ; ( " m ( )n é;\o ”Tu Ill Cl) ii 0,, OH , ’ O p "'1 1H. n ”r“ “I” 0 'NW 04 04.. if ‘H’ ‘M’ Mn MAN *0}; H n n n W , ’ .LLL/N\/\S/S n O "”4.
J\/|VV JWV O o o WO‘CK /,0 \..... o\ ,p 5 N 0’? OH OH o N _ ,P O I n W \l.... w o\ d n 0 Q E—s H n o N ,and. :1 N HNfl/g ”W n Ili/ n 0 wherein each n is, independently, is from 1 to 20; and p is from 1 to 6.
In certain embodiments, the conjugate linker has a structure selected from among: ”CO 0,, )5. 0V0)" O o N O N H H SN NNo ; I777- S O I111 n n n i n O ITIH o O H N W gulf/WN n HMN O ’ ”\J “ $3 n O ; and wherein each n is, independently, from 1 to 20.
In certain embodiments, the conjugate linker has a structure ed from among: “i“ Q ”T” ('3 O!» W I o \..... o\ ,o O. 5 ,P OH O\ O I H is 0V0 S_S/\/\( O\HI X! ”LLL/ N\/\ ,s o N \g‘. N s d 3 o , M ,an E H E 10 0 \NW 5 o In certain embodiments, the conjugate linker has a structure selected from among: 14:“ ”S, 9w 0 like}: 3W0 and EWO wherein n is from 1 to 20.
In certain embodiments, the ate linker has a structure selected from among: 5W“,AM?EHO——E 3L and EWfi/W In certain ments, the conjugate linker has a structure selected from among: szAM:—EHo——E and EWMW‘ wherein each n is independently, O, l, 2, 3, 4, 5, 6, or 7.
In certain embodiments, the ate linker has the following structure: 0 O In certain embodiments, the branching group has one of the following structures: wherein each Al is independently, O, S, C=O or NH; and each n is, independently, from 1 to 20.
In certain embodiments, the branching group has one of the following structures: wherein each Al is independently, O, S, C=O or NH; and each n is, independently, from 1 to 20.
In certain embodiments, the branching group has the following structure: /O\a—/"Ha, In certain embodiments, the branching group has the following ure: O N / #O/E In n embodiments, the branching group has the following structure: M/;LN}5‘ “In,” ' In certain embodiments, the ing group has the following structure: ,1”, 55‘“ In certain embodiments, the branching group comprises an ether.
In certain embodiments, the branching group has the following structure: each n is, independently, from 1 to 20; and m is from 2 to 6.
In certain ments, the branching group has the following structure: “at 0 EL o 0 KW“! “a .
’ HJ~rfit/\NjV\/\AN)‘ H O Kfo O ’ NH;rr "’w" 0 o O o o E—NH )2 O . EANNNflf o , o ; AN 21 e“ ;and 'gZ/NH m o In certain embodiments, the branching group has the following structure: O O In certain ments, the branching group comprises: O o EWNH EWNH )n )n O AM O H H 1’1 on (n NH EMNH ‘5 0r o , ; wherein each j is an integer from 1 to 3; and wherein each n is an integer from 1 to 20.
In n embodiments, the branching group comprises: HN}L 0 EWLNH In certain embodiments, each tether is selected from among: 0 Z1 6’5 L .771. £19k Jfi/L 1%.
Wherein L is selected from a phosphorus linking group and a neutral linking group; Z1 is C(=O)O-R2; Z2 is H, C1-C6 alkyl or tuted Cl-C6 alky; R2 is H, C1-C6 alkyl or substituted Cl-C6 alky; and each ml is, independently, from O to 20 wherein at least one ml is greater than 0 for each tether.
In certain embodiments, each tether is selected from among: 0 3L 0 COOH OH 0—3—0 I “L “”2 6H “”2 ng/Kfl—fi—O‘Mfi:m2H O Wherein Z2 is H or CH3; and each m2 is, independently, from O to 20 Wherein at least one m2 is greater than 0 for each tether.
In certain embodiments, each tether is selected from among: /$71 ; EMHJLN o “(WK/NJ}? n n is from 1 to 12; and Wherein m is from 1 to 12.
In certain embodiments, at least one tether comprises ethylene glycol. In certain embodiments, at least one tether comprises an amide. In n embodiments, at least one tether comprises a polyamide. In certain embodiments, at least one tether comprises an amine. In certain embodiments, at least two tethers are different from one another. In certain embodiments, all of the s are the same as one another. In certain embodiments, each tether is selected from among: O H 1%.
EMNWOVhOAH/Ei ; Lat/NW2 ; aw J EWOW'T; ; gWONM;o a ,4;”WW5; mew;H g H H E _N\<H:imwii ;£MOfiOk/)nn\g §_H n DWI/“WE ; ; 2 p E—H o o O O EWMWK)‘; w; WW wherein each n is, independently, from 1 to 20; and each p is from 1 to about 6.
In certain embodiments, each tether is selected from among: “ta/\AN/VOwO/VLL‘I ; gl/HWE ; HHV\/\[('15- ; ELMO/ff ; ”JR/\ONOVRL ; zl/Nmegmnd Emoaéy In certain embodiments, each tether has the following ure: ,6 NH 3, wherein each n is, ndently, from 1 to 20.
In n embodiments, each tether has the following structure: 0 O 2014/036460 In certain embodiments, the tether has a structure selected from among: 0 O Wmfifi or ”mi: , ; wherein each n is independently, O, l, 2, 3, 4, 5, 6, or 7.
In certain embodiments, the tether has a structure selected from among: «r.
In certain embodiments, the ligand is galactose. In certain embodiments, the ligand is mannose phosphate.
In certain embodiments, each ligand is selected from among: Hofio OH HO O 0 Ho OH HO 0—; R R1 and 1 wherein each R1 is ed from OH and NHCOOH.
In certain embodiments, each ligand is selected from among: HOOH OH HO HO HO&/ \ HOFE;§::;§x/O OH OH O O -O O '0 HO O HO ”s ; #5 . \ HO , NHAC OH HO : HE HOOH ”kw \t’ Wm“HO N HO OH HOOH mom 0“ OH HO \ - /*2: , HO HO OH OH .0 In certain embodiments, each ligand has the following structure: HOOH In certain ments, each ligand has the following structure: HOOH Ho&cofi NHAC _ In certain embodiments, the conjugate group comprises a cell-targeting moiety.
In n embodiments, the conjugate group comprises a cell-targeting moiety having the following structure: HOOH HO O A? ACHN O5}? HOOH \<\\ ) n “0% WHITE/Hm)o 0 O H o ACHN OH HOOH Hofiw Wo O (‘1)5 Al) | \O n NHAC wherein each n is, independently, from 1 to 20.
In certain embodiments, the argeting moiety has the following structure: HOOH HO O\/\/\/\ 9 ACHN O/EEIOK HOOH HOmOWO/fiO/moo o ACHN OH O NHAC In certain embodiments, the cell-targeting moiety has the following structure: 2014/036460 HO OH NHAc 0 Ho HN H o o N NHAc 0 wherein each n is, independently, from 1 to 20.
In certain embodiments, the cell-targeting moiety has the following structure: HO OH H o O HN\/\/N NHAc HO OH o O H H O N_|H HO N\/\/N\H/\/o NHAc o HO HN H o o N HO WY NHAc In certain embodiments, the cell-targeting moiety comprises: HO OH 0 o AcHN WY HO OH H o HN 5 HO M o AcHN 0 HO OH Hog/OM0 NH AcHN o In certain embodiments, the cell-targeting moiety ses: HoOH ' O N O o AcHN | OZT-OH HoOH O N HO 0% O o AcHN | O=Fl>—OH HoOH HOWO N AcHN _§ In certain embodiments, the cell-targeting moiety comprises: HOOH O o N o HO W AcHN HOOH O HOW WHO o N /‘7‘ AcHN HoOH o N o HO /Wfi:_H AcHN In certain embodiments, the cell-targeting moiety comprises: HO OH O NH AcHN HO OH sew N o ACHN I2 2 SgtwHOOH ZI NH AcHN 0 HO OH @WNACHN In certain embodiments, the cell-targeting moiety comprises: OH OH H0 00 O AcHN WNH 0th in mfiw O AcHN W E N/\/\N N o FWH “06090OHH0 NHAC In certain embodiments, the cell-targeting moiety comprises: H o OH ”NH 0 0 NHAc In certain embodiments, the cell-targeting moiety comprises: HoOH O O o N H0 10 H AcHN o HoOH O 0 ) o N N:3 H0 10 H H AcHN o HoOH O Ii O N HO O H NHAc In certain embodiments, the cell-targeting moiety comprises: HOOH HOWOVWNQ/ “PK0,15; AcHN o o HoOH O’BRMOaVOYo mow,”o 0/ O o AcHN 0 0 638 HO OH ,O/ HO 0 NHAc In certain embodiments, the cell-targeting moiety comprises: 14/179625 HOEE:%A/o o/W7kN N 4 HA$E\H AcHN HoOH o N H0%Q/o Ngk o N H 4 HWZ/ O AcHN HoOH O o/\(V>JLN 0 Ho 4 HAM?” AcHN HoOH o Hog/OWNMN3 o H H AcHN HoOH o N A o N»\// M 3 H O AcHN HoOH O O N/\/\ O Ho 3 H N AcHN HoOH O o o N HO 4 H N AcHN H HoOH o N .a N”\// N O o H HO 4 H o AcHN HoOH O O o NMN O HO 4 H H AcHN In certain embodiments, the cell-targeting moiety comprises: HOOH ' HO 0mN 0 o ACHN O—FI’ OH HOOH O DAWN HO 3 o o ACHN O—FI’ OH HOOH O OWN HO 3 0 _§ AcHN HOOH H O O OWNWNQ HO O O ACHN O—|=|’-OH HOOH o 0%“MN HO O O AcHN O‘I-OH HOOH O o”$§\fHMN3 HO O 0—; AcHN In n embodiments, the cell-targeting moiety comprises: HOOH //\OH WWW 1O0 N AcHN I AcHN I HOOH O OWN HO 3 O 0—; AcHN In certain embodiments, the cell-targeting moiety comprises: HoOH H O /_/ Hog/W0 o 3 lo In certain embodiments, the cell-targeting moiety comprises: OH OH :50» O Ho OWOL ACHN NH OH OH HOflOWNAcHN HY In n embodiments, the cell-targeting moiety comprises: OH OH HO O AcHN In certain ments, the cell-targeting moiety comprises: \/\/\/O\P\/OHO AcHN 0' Y 0‘ 2 ’0 O Y O~p\ I OH fl0’ Y I"QAcHN wherein each Y is selected from O, S, a substituted or unsubstituted Cl-CIO alkyl, amino, substituted amino, azido, alkenyl or alkynyl.
In certain embodiments, the conjugate group comprises: HO$&/O\/\/\/O\P/OHO AcHN O" \ O“ I Y O\P’O\/\O’P\‘fs\ o O” Y O~p\ I OH fl0’ Y H9AcHN wherein each Y is selected from O, S, a substituted or unsubstituted Cl-CIO alkyl, amino, substituted amino, azido, alkenyl or alkynyl.
In certain embodiments, the ate group comprises: HO$g/O\/\/\/O\p/EHO ACHN o" ‘Y wherein each Y is selected from O, S, a substituted or unsubstituted Cl-CIO alkyl, amino, substituted amino, azido, alkenyl or alkynyl.
In certain embodiments, the conjugate group comprises: o 71.
O O\/\/\/u\ .mO ACHN In n embodiments, the conjugate group comprises: OVV\/U\ .mOH ACHN In certain embodiments, the conjugate group comprises: 0 _\\o—§ ACHN NWH In certain embodiments, the conjugate group comprises: O ,,\OH Ho OWNH/\/\/\n/N ACHN O 0—? In certain embodiments, the ate group comprises a cleavable moiety selected from among: a phosphodiester, an amide, or an ester.
In certain embodiments, the conjugate group comprises a phosphodiester cleavable moiety.
In certain ments,the conjugate group does not comprise a cleavable moiety, and wherein the conjugate group comprises a phosphorothioate linkage between the conjugate group and the oligonucleotide.
In certain embodiments, the conjugate group comprises an amide cleavable moiety. In certain ments, the conjugate group comprises an ester cleavable .
In certain embodiments, the compound has the following structure: HOOH HO 0 4(3) ACHN OéHo ) HOOH \<\\“ o 0 O " o O || 0 BX ACHN OH OH O 0'“ Q13 HOOH 9. JJ) HO-I"=O 0 OW/P\o n I 11 OH A NHAC wherein each n is, independently, from 1 to 20; Q13 is H or O(CH2)2-OCH3; A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain ments, the compound has the following structure: HOOH Ho OW\/\ 1‘5? ACHN O/|\O OH K HO OH O O 9 i)? o O BX OW\/\ x \ o_ — HO O 1|) 0M0 (5H «(J ACHN OH O o Q13 wherein each n is, independently, from 1 to 20; Q13 is H or O(CH2)2-OCH3; A is the modified oligonucleotide; and BX is a heterocyclic base .
In certain embodiments, the compound has the following structure: HO-l|3=O ('3‘ Q13 HO OH (61 HO O (I? WO’RM A HN E o 0 OH ) 0 \Z HO O“ n o 0 o 0 || o ACHN OH O 5H “0 O” 9 0 O 0’1|)\Oii)n HO H OH NHAC wherein each n is, independently, from 1 to 20; Q13 is H or O(CH2)2-OCH3; A is the modified oligonucleotide; Z is H or a linked solid support; and BX is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure: HO-l|3=O OWBX 65 QB HO—I|’=O HO OH 0 HO O\/A\/“»/”\ / \% 0 (5H0 §—\b ACHN 0 \z ( )3 HO OH 0 O 9 0—3-0 HO /\o’1|)‘o/\/\O/}V I— ACHN 0H 0 NHAC wherein each n is, independently, from 1 to 20; Q13 is H or O(CH2)2-OCH3; A is the modified oligonucleotide; Z is H or a linked solid support; and BX is a heterocyclic base moiety. 2014/036460 In certain embodiments, the compound has the ing structure: OH OH ACHN Ol'bH in “0%0 O K AcHN WNMM N M H “fig H‘H/O\ 6 HO—llazo 0 Fr flNH O Q13 “0%on Ho—1||>=o HO A NHAC wherein Q13 is H or O(CH2)2—OCH3; A is the modified oligonucleotidegand Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the ing structure: wherein Q13 is H or O(CH2)2—OCH3; A is the modified oligonucleotidegand Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure: HOOH O Hog/ WHJIO o N AcHN o HOOH O O O AcHN o HO"?=O wherein Q13 is H or O(CH2)2-OCH3; A is the modified oligonucleotide; and BX is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure: HOOH mkmfi043‘“I HoOH o &::f/ o o HOdfO HO r o 0 AcHN 0 §\ _‘\i_lr1k HO OH (:1/063 9f HO _ O O\/\/\“/N _I'D—O HO O A NHAc wherein Q13 is H or 2-OCH3; A is the modified oligonucleotide; and BX is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure: wherein Q13 is H or O(CH2)2—OCH3; A is the modified oligonucleotide; and BX is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure: HOOH O O O OAHJLNMN HO 3 H H ACHN H O O HOOH O N JJ\/\/u\ N N O OAHJLNV H HW? HO 3 H O =0 ACHN HOOH O O M /\/\ 0 Owa HO¥Q/O $.- 3 M M ('3‘ ACHN HO—1ID20 wherein Q13 is H or O(CH2)2—OCH3; A is the modified oligonucleotide; and BX is a heterocyclic base moiety.
In certain embodiments, the compound has the ing structure: HoOH O o O OWNMN 4 H H ACHN H O O HOOH O W N N O OWN/V H HWO\ 4 H O HO—1":-O ACHN O HOOH O O Owa O N/\/\ O \5' HO Q13 4 H M 9 ACHN HO—TZO wherein Q13 is H or O(CH2)2—OCH3; A is the modified oligonucleotide; and BX is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure: HoOH 0 CW”? HO 3 O O ACHN O—FI’ OH HoOHO HOWOW”QC A HNC O:—F:’—OH O HOA::O HOOH HOfiwoAH/WN Q13\QBX ACHN wherein Q13 is H or O(CH2)2—OCH3; A is the modified oligonucleotide; and BX is a cyclic base moiety.
In certain embodiments, the compound has the following structure: 2014/036460 HoOH O HOEEZiA/Iflfijgo O NMNQH 3 AcHN CIT-OH HoOH H O H0§Q/m 3 O AcHN O=T—OH p A HoOH HNV”§%_£;:O HOfiTO O o/Tii§ o O HO 3 0 _\(;ZBX AcHN \\\\\\\\c§ Q13 wherein Q13 is H or O(CH2)2—OCH3; A is the modified oligonucleotide; and BX is a heterocyclic base moiety.
In certain embodiments, the nd has the following structure: AcHN I O=P-OH HoOH F‘/3 Ho—rqa o () HQ¥Q/ BX o o”IfirwrN ‘q 3 0 L0 0‘“ 13 AcHN \\\\\\i-o wherein Q13 is H or O(CH2)2—OCH3; A is the modified oligonucleotide; and BX is a heterocyclic base moiety.
In n embodiments, the compound has the following structure: HoOH H o /—/ HO mvaLN3 10 AcHN I o=FI>—OH HoOH H O HO mNx/WLN3 10 AcHN OH1|% o Ho—1|>=o HoOH H O 7/ O O BX HO mNVWLN \q.. 3 LO 08 Q13 AcHN l wherein Q13 is H or O(CH2)2—OCH3; A is the modified oligonucleotide; and BX is a heterocyclic base .
In certain embodiments, the conjugate group comprises: HO OH NHAC wherein Q13 is H or O(CH2)2—OCH3; A is the modified oligonucleotide; and BX is a heterocyclic base moiety.
In certain embodiments, the conjugate group comprises: wherein Q13 is H or O(CH2)2—OCH3; A is the modified oligonucleotide; and BX is a heterocyclic base moiety.
In certain embodiments, the conjugate group comprises: wherein Q13 is H or 2—OCH3; A is the modified oligonucleotide; and BX is a heterocyclic base moiety.
In certain embodiments, BX is selected from among from adenine, guanine, thymine, uracil, or cytosine, or 5-methyl ne. In certain embodiments, BX is e. In certain embodiments, BX is thymine. In certain ments, Q13 is O(CH2)2-OCH3. In certain embodiments, Q13 is H.
In certain embodiments, the compound is in a salt form. In further embodiments, the nd r comprises of a pharmaceutically acceptable carrier or diluent. In certain embodiments, the compound comprises a modified oligonucleotide targeting apo(a) and a conjugate group, or a salt thereof, and a pharmaceutically acceptable carrier or diluent.
Certain embodiments provide a composition comprising a conjugated antisense compound as described herein, wherein the viscosity level of the compound is less than 40 centipoise (cP). In certain embodiments, the conjugated antisense compounds as described herein are efficacious by virtue of having a viscosity of less than 40 CF, less than 35 CF, less than 30 CF, less than 25 CF, less than 20 cP or less than 15 cP when measured by the parameters as described in Example 125.
Certain embodiments provide compositions and methods comprising administering to an animal a conjugated antisense compound or composition disclosed herein. In certain embodiments, administering the conjugated antisense compound prevents, treats, rates, or slows ssion of a cardiovascular, metabolic and/or inflammatory disease Certain embodiments provide compositions and s for use in therapy to treat an apo(a) related disease, disorder or condition. Certain embodiments provide compositions and methods for use in therapy to treat an Lp(a) related disease, disorder or condition. In certain embodiments, apo(a) and/or Lp(a) levels are elevated in an animal. In certain embodiments, the composition is a compound sing an apo(a) specific inhibitor. In certain ments, the apo(a) specific tor is a nucleic acid. In certain embodiments, the nucleic acid is an antisense compound. In certain embodiments, the antisense nd is a modified oligonucleotide targeting apo(a). In certain embodiments, the antisense compound is a modified oligonucleotide targeting apo(a) and a conjugate group. In certain embodiments, the modified oligonucleotide targeting apo(a) with the conjugate group, is used in treating, preventing, slowing progression, ameliorating a cardiovascular and/or metabolic disease, disorder or condition. In certain embodiments, the compositions and methods for therapy include administering an apo(a) specific tor to an individual in need thereof.
Certain embodiments provide compositions and methods for ng apo(a) levels. Certain ments provide itions and methods for reducing Lp(a) levels. In certain embodiments, reducing apo(a) levels in a tissue, organ or subject es the ratio of LDL to HDL or the ratio of TG to HDL.
Certain embodiments provide compositions and methods to reduce apo(a) mRNA or protein expression in an animal sing administering to the animal a conjugated nse compound or composition disclosed herein to reduce apo(a) mRNA or protein expression in the animal. Certain embodiments provide compositions and methods to reduce Lp(a) levels in an animal comprising administering to the animal a conjugated antisense compound or composition disclosed herein to reduce apo(a) mRNA or protein expression in the animal.
Certain embodiments provide compositions and methods for preventing, treating, delaying, slowing the progression and/or rating apo(a) related diseases, disorders, and conditions in a subject in need thereof. Certain embodiments e itions and methods for ting, treating, delaying, slowing the progression and/or ameliorating Lp(a) related diseases, ers, and conditions in a subject in need thereof. In certain ments, such diseases, ers, and ions include inflammatory, cardiovascular and/or metabolic diseases, disorders, and conditions. n such cardiovascular diseases, disorders or conditions include, but are not limited to, aortic stenosis, aneurysm (e.g., abdominal aortic aneurysm), , arrhythmia, atherosclerosis, cerebrovascular disease, coronary artery disease, coronary heart disease, dyslipidemia, hypercholesterolemia, hyperlipidemia, hypertension, hypertriglyceridemia, myocardial infarction, peripheral vascular disease (e.g., peripheral artery disease, eral artery occlusive disease), retinal vascular occlusion, or stroke. Certain such metabolic diseases, disorders or conditions include, but are not limited to, hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome and diabetic dyslipidemia. Certain such inflammatory diseases, ers or conditions include, but are not limited to, aortic stenosis, coronary artey disease (CAD), Alzheimer’s Disease and thromboembolic diseases, disorder or conditions. n thromboembolic diseases, disorders or conditions include, but are not limited to, stroke, thrombosis (e.g., venous thromboembolism), myocardial infarction and peripheral vascular disease. n embodiments provide itions and methods for ting, treating, delaying, slowing the ssion and/or ameliorating aortic stenosis.
Certain embodiments e a method of ng at least one symptom of a cardiovascular disease, disorder or condition. In n embodiments, the ms e, but are not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever.
Certain embodiments provide a method of reducing at least one symptom of aortic stenosis.
In certain embodiments, the modulation of apo(a) or Lp(a) expression occurs in a cell, tissue or organ. In certain embodiments, the modulations occur in a cell, tissue or organ in an animal. In n embodiments, the modulation is a reduction in apo(a) mRNA level. In certain embodiments, the modulation is a reduction in apo(a) protein level. In certain embodiments, both apo(a) mRNA and n levels are reduced. In certain ments, the modulation is a reduction in Lp(a) level. Such reduction may occur in a ependent or in a dose-dependent manner.
In certain embodiments, the subject or animal is human.
In certain embodiments, the conjugated nse compound is parenterally administered. In further embodiments, the parenteral administration is subcutaneous.
In certain embodiments, the conjugated antisense compound or composition is co-administered with a second agent or therapy. In certain embodiments, the conjugated antisense compound or composition and the second agent are administered concomitantly.
In certain ments, the second agent is a e-lowering agent. In certain embodiments, the second agent is a LDL, TG or cholesterol lowering agent. In certain embodiments, the second agent is an anti-inflammatory agent. In certain embodiments, the second agent is an Alzheimer e drug. In certain embodiments, the second agent can be, but is not limited to, a non-steroidal anti-inflammatory drug (NSAID e. g., aspirin), niacin (e. g., Niaspan), nicotinic acid, an apoB inhibitor (e. g., Mipomersen), a CETP inhibitor (e. g., Anacetrapib), an apo(a) tor, a thyroid hormone analog (e. g., Eprotirome), a HMG-CoA reductase inhibitor (e. g., a statin), a fibrate (e.g., Gemflbrozil) and an microsomal triglyceride transfer protein inhibitor (e. g., Lomitapide). The therapy can be, but is not limited to, Lp(a) apheresis. Agents or therapies can be co- administered or administered concomitantly. Agents or therapies can be sequentially or subsequently stered.
Certain embodiments provide use of a conjugated antisense compound targeted to apo(a) for decreasing apo(a) levels in an animal. Certain embodiments provide use of a conjugated nse compound targeted to apo(a) for decreasing Lp(a) levels in an animal. Certain embodiments provide use of a conjugated nse compounds targeted to apo(a) for the treatment, prevention, or amelioration of a disease, er, or condition associated with apo(a). Certain embodiments provide use of a conjugated antisense nds ed to apo(a) for the treatment, prevention, or amelioration of a disease, disorder, or condition ated With Lp(a).
Certain embodiments provide use of a conjugated antisense compound targeted to apo(a) in the ation of a medicament for decreasing apo(a) levels in an . Certain embodiments provide use of a conjugated antisense compound targeted to apo(a) in the preparation of a medicament for decreasing Lp(a) levels in an . n embodiments provide use of a conjugated antisense compound for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, er, or condition ated With apo(a). Certain ments provide use of a conjugated antisense compound for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated With Lp(a).
Certain embodiments provide the use of a conjugated antisense compound as described herein in the manufacture of a medicament for ng, ameliorating, delaying or preventing one or more of a disease related to apo(a) and/or Lp(a).
Certain embodiments provide a kit for treating, preventing, or ameliorating a disease, disorder or condition as described herein Wherein the kit comprises: (i) an apo(a) specific inhibitor as described herein; and optionally (ii) a second agent or therapy as described herein.
A kit of the t invention can further include instructions for using the kit to treat, prevent, or ameliorate a disease, disorder or condition as described herein by combination therapy as bed herein.
B. Certain Compounds In certain embodiments, the ion provides conjugated antisense compounds comprising antisense oligonucleoitdes and a conjugate. a. n Antisense Oligonucleotides In certain embodiments, the invention provides antisense oligonucleotides. Such antisense oligonucleotides comprise linked nucleosides, each nucleoside comprising a sugar moiety and a nucleobase.
The structure of such antisense oligonucleotides may be ered in terms of chemical features (e.g., modifications and patterns of modifications) and nucleobase sequence (e.g., sequence of antisense oligonucleotide, idenity and sequence of target nucleic acid). i. Certain Chemistry Features In n embodiments, antisense oligonucleotide comprise one or more ation. In certain such embodiments, antisense oligonucleotides comprise one or more modified nucleosides and/or modified internucleoside linkages. In certain embodiments, modified nucleosides comprise a d sugar moirty and/or modifed base. 1. Certain Sugar Moieties In n embodiments, compounds of the disclosure comprise one or more modifed nucleosides comprising a modifed sugar moiety. Such compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced se stability or increased binding affinity with a target nucleic acid relative to an ucleotide comprising only nucleosides comprising naturally occurring sugar moieties. In certain embodiments, modified sugar moieties are substitued sugar moieties. In certain embodiments, modified sugar es are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.
In certain ments, modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not d to substituents at the 2’ and/or 5’ positions.
Examples of sugar substituents suitable for the 2’-position, include, but are not limited to: 2’-F, 2'-OCH3 (“OMe” or “O-methyl”), and 2'-O(CH2)20CH3 ). In certain ments, sugar substituents at the 2’ position is selected from allyl, amino, azido, thio, O-allyl, O-Cl-Clo alkyl, O-Cl-Clo substituted alkyl, OCF3, O(CH2)ZSCH3, O(CH2)2-O-N(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), Where each Rm and Rn is, ndently, H or substituted or unsubstituted C1-C10 alkyl. Examples of sugar tuents at the 5’- position, include, but are not limited to:, 5’-methyl (R or S); 5'-Vinyl, and 5’-methoxy. In certain embodiments, substituted sugars comprise more than one non-bridging sugar substituent, for example, 2'-F- 5'-methyl sugar moieties (see,e.g., PCT International Application WO 01 157, for additional 5', 2'-bis substituted sugar moieties and nucleosides).
Nucleosides comprising 2’-substituted sugar moieties are referred to as 2’-substituted nucleosides. In certain embodiments, a 2’- substituted nucleoside comprises a 2'-substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)- alkynyl, lenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, 2SCH3, O-(CH2)2-O- N(Rm)(Rn) or O-CH2-C(=O)-N(Rm)(Rn), Where each RIn and RI1 is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl. These stituent groups can be further substituted With one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (N02), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
In certain embodiments, a 2’- substituted nucleoside comprises a 2’-substituent group selected from F, NHg, N3, OCF3, O-CHg, 0(CH2)3NH2, CHg-CHZCHz, O'CHQ'CHZCHZ, QOCHg, O(CH2)2$CH3, )2-O-N(Rm)(Rn), O(CH2)20(CH2)2N(CH3)2, and tituted acetamide (O-CHg-C(=O)-N(Rm)(Rn) Where each RIn and R11 is, ndently, H, an amino protecting group or substituted or tituted C1-C10 alkyl.
In certain embodiments, a 2’- substituted nucleoside comprises a sugar moiety comprising a 2’- substituent group ed from F, OCFg, O-CH3, OCHQCHQOCH3, O(CH2)2SCH3, O-(CH2)2-ON (CH3)2, -O(CH2)20(CH2)2N(CH3)2, and O-CHg-C(=O)-N(H)CH3.
In certain embodiments, a 2’- substituted side ses a sugar moiety comprising a 2’- substituent group selected from F, O-CH3, and OCHQCHQOCH3.
Certain modifed sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms. Examples of such 4’ to 2’ sugar substituents, include, but are not limited to: '[C(Ra)(Rb)]n-, '[C(Ra)(Rb)]n'O', -C(RaRb)-N(R)-O- or, —C(RaRb)-O-N(R)-; 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2',. 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt) and 4'-CH(CH20CH3)-O-2',and analogs thereof (see, e.g., US. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH3)(CH3)-O-2'and analogs thereof, (see, e.g., WO2009/006478, published January 8, 2009); 4'- CHZ-N(OCH3)-2' and analogs thereof (see, e.g., /150729, published December 11, 2008); 4'-CH2-O- -2' (see, e.g., US2004/0171570, hed September 2, 2004 ); 4'-CH2-O-N(R)-2', and 4'-CH2-N(R)- O-2'-, Wherein each Ris, independently, H, a protecting group, or C1-C12 alkyl; 4'-CH2-N(R)-O-2', Wherein R is H, C1-C12 alkyl, or a protecting group (see, US. Patent 7,427,672, issued on September 23, 2008); 4'-CH2- C(H)(CH3)-2' (see, e.g., Chattopadhyaya, et al., J. Org. Chem.,2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' and analogs thereof (see, published PCT International Application WC 2008/154401, published on December 8, 2008).
In certain embodiments, such 4’ to 2’ bridges independently comprise from 1 to 4 linked groups independently ed from -[C(Ra)(Rb)]n-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=O)-, -C(=S)-, -O-, - Si(Ra)2-, -S(=0)x-, and 'N(Ra)'; Wherein: X is 0, l, or 2; nis l, 2, 3, or4; each R2, and Rb is, independently, H, a protecting group, yl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ 1, NJ1J2, SJ 1, N3, COOJ1, acyl - H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or yl (S(=O)-J1); and each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 l, C2-C12 alkynyl, substituted C2-C12 l, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)- H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted 2014/036460 C1-C12 aminoalkyl, or a protecting group.
Nucleosides comprising ic sugar moieties are referred to as bicyclic nucleosides or BNAs.
Bicyclic nucleosides include, but are not limited to, (A) (x-L-Methyleneoxy (4’-CH2-O-2’) BNA , (B) B-D- Methyleneoxy (4’-CH2-O-2’) BNA (also referred to as locked nucleic acid or LNA) , (C) Ethyleneoxy (4’- (CH2)2-O-2’) BNA , (D) Aminooxy (4’-CH2-O-N(R)-2’) BNA, (E) Oxyamino (4’-CH2-N(R)-O-2’) BNA, (F) Methyl(methyleneoxy) (4’-CH(CH3)-O-2’) BNA (also referred to as ained ethyl or cEt), (G) methylene-thio (4’-CH2-S-2’) BNA, (H) methylene-amino (4’-CH2-N(R)-2’) BNA, (1) methyl carbocyclic (4’-CH2-CH(CH3)-2’) BNA, and (J) ene yclic (4’-(CH2)3-2’) BNA as depicted below. wherein BX is a nucleobase moiety and R is, independently, H, a protecting group, or C1-C12 alkyl.
Additional ic sugar es are known in the art, for example: Singh et al., Chem. Commun, 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci.
U. S. A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J.
Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol, 2001, 8, 1-7; Orum et al., Curr. Opinion Mo]. Ther., 2001, 3, 239-243; US. Patent Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 06356, WO 1994/14226, WO 2005/021570, and ; US. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; US. Patent Serial Nos. 12/129,154, ,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International Applications Nos. , , and .
In certain embodiments, ic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by ic configuration. For example, a nucleoside comprising a 4’-2’ ene-oxy bridge, may be in the (x-L configuration or in the B-D configuration. usly, (x-L- methyleneoxy (4’-CH2-O-2’) bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., c Acids Research, 2003, 21, 6365-6372).
In certain embodiments, substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar tuent (e. g., 5’-substituted and 4’-2’ bridged sugars). (see, PCT International ation , published on 11/22/07, wherein LNA is substituted with, for example, a 5'-methyl or a 5'-Vinyl group).
In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the naturally occuring sugar is substituted, e. g., with a , carbon or nitrogen atom. In certain such embodiments, such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above. For example, certain sugar surrogates se a 4’-sulfer atom and a substitution at the 2'-position (see,e.g., hed US. Patent Application /0130923, published on June 16, 2005) and/or the 5’ position. By way of onal example, yclic bicyclic nucleosides having a 4'-2' bridge have been described (see, e. g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).
In certain embodiments, sugar surrogates comprise rings haVing other than 5-atoms. For e, in certain embodiments, a sugar surrogate ses a morphlino. Morpholino compounds and their use in oligomeric compounds has been reported in numerous patents and published articles (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and US. Patents 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following structure: ijiBx In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are d to herein as “modifed morpholinos.” For another example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, CJ. . & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula VI: q1 q2 T3_0 q3 q7 C14 C16 BX / R1 q5 wherein independently for each of said at least one tetrahydropyran nucleoside analog of a VI: BX is a nucleobase moiety; T3 and T4 are each, independently, an ucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense nd and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'—terminal group; q, (12, q3, q4, q5, q6 and q7 are each, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl, and each of R1 and R2 is independently ed from among: en, halogen, substituted or tituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, wherein X is O, S or NJ1, and each J1, J2, and J3 is, independently, H or C1-C6 alkyl.
In certain embodiments, the modified THP nucleosides of Formula VI are provided wherein ql, C12, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of ql, C12, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of ql, C12, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides of Formula VI are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is fluoro and R2 is H, R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.
Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, e. g., review article: Leumann, J.
C, Bioorganic & Medicinal Chemistry, 2002, 10, 4).
Combinations of modifications are also provided without limitation, such as 2'-F-5'-methyl substituted sides (see PCT International Application 157 Published on 8/21/08 for other disclosed 5', 2'-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see published US. Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid (see PCT International Application , published on 11/22/07 n a 4'—CH2-O-2' bicyclic nucleoside is fiirther substituted at the 5' position with a hyl or a 5'-vinyl . The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e. g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
In certain embodiments, the present disclosure provides oligonucleotides comprising modified nucleosides.
Those modified nucleotides may include modified sugars, modified nucleobases, and/or modified es.
The specific modifications are selected such that the resulting oligonucleotides possess desireable teristics. In n embodmiments, oligonucleotides comprise one or more RNA-like nucleosides. In certain embodiments, ucleotides comprise one or more DNA-like nucleotides. 2. Certain Nucleobase Modifications In certain embodiments, nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present sure comprise one or more modifed nucleobases.
In certain ments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, xpanded bases, and fiuorinated bases as defined . 5-substituted pyrimidines, 6-azapyrimidines and N—2, N—6 and 0-6 substituted purines, including opropyladenine, 5- propynyluracil; 5-propynylcytosine; oxymethyl cytosine, xanthine, nthine, 2-aminoadenine, 6- methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-CEC- CH3) uracil and cytosine and other alkynyl tives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioa1kyl, 8-hydroxyl and other 8- tuted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, aguanine and 7-deazaadenine, aguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, xpanded bases, and fiuorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine( [5,4-b][1,4]benzoxazin— 2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e. g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin—2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indolone), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3- d]pyrimidin—2-one). Modified nucleobases may also include those in Which the purine or dine base is replaced With other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2- pyridone. Further nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, WitZ, J.I., Ed., John Wiley & Sons, 1990, 85 8-859; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.
Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include Without limitation, US. 3,687,808; 4,845,205; ,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; ,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; ,763,588; 5,830,653 and 6,005,096, certain of which are ly owned with the instant application, and each of which is herein incorporated by reference in its entirety. 3. Certain Internucleoside Linkages In certain embodiments, the t disclosure provides oligonucleotides comprising linked sides. In such embodiments, nucleosides may be linked together using any intemucleoside linkage.
The two main s of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. entative phosphorus containing intemucleoside linkages include, but are not limited to, phosphodiesters (PO), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (PS). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-OC (O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine N(CH3)-N(CH3)-). ed linkages, compared to l phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the ucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be ed as a racemic mixture, or as separate enantiomers. Representative chiral linkages e, but are not limited to, alkylphosphonates and phosphorothioates. s of preparation of phosphorous-containing and non-phosphorous-containing internucleoside es are well known to those skilled in the art.
The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, reomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), a or b such as for sugar anomers, or as (D) or (L) such as for amino acids etc.
Included in the antisense compounds provided herein are all such le isomers, as well as their racemic and optically pure forms.
Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI 2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), and thioformacetal (3'-S-CH2-O-5'). Further neutral ucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and PD. Cook, Eds., ACS ium Series 580; Chapters 3 and 4, . Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts. 4. Certain Motifs In certain embodiments, nse oligonucleotides comprise one or more modified side (e.g., nucleoside comprising a modified sugar and/or modified nucleobase) and/or one or more modified internucleoside linkage. The pattern of such modifications on an oligonucleotide is referred to herein as a motif. In certain embodiments, sugar, nucleobase, and linkage motifs are independent of one another. a. Certain sugar motifs In certain embodiments, oligonucleotides comprise one or more type of ed sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modif1cation motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.
In certain embodiments, the ucleotides comprise or consist of a region haVing a gapmer sugar motif, which comprises two external s or “wings” and a central or internal region or “gap.” The three regions of a gapmer sugar motif (the 5’-wing, the gap, and the 3’-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the sides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3’-most nucleoside of the 5’-wing and the 5’-most nucleoside of the 3’-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the ry n the wings and the gap. In n embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In n embodiments, the sugar motifs of the two wings are the same as one another (symmetric sugar gapmer). In certain embodiments, the sugar motifs of the 5'-wing differs from the sugar motif of the g (asymmetric sugar gapmer). i. Certain 5’-wings In certain embodiments, the 5’- wing of a gapmer consists of l to 8 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer consists of l to 7 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer consists of l to 6 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer ts of l to 5 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain ments, the 5’- wing of a gapmer consists of 3 to 5 linked nucleosides.
In n embodiments, the 5’- wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer consists of l to 3 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer consists of l or 2 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer consists of 2 or 3 linked nucleosides.
In certain embodiments, the 5’- wing of a gapmer consists of 3 or 4 linked nucleosides. In certain ments, the 5’- wing of a gapmer consists of l nucleoside. In certain ments, the 5’- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the ’- wing of a gapmer consists of 6 linked nucleosides.
In certain embodiments, the 5’- wing of a gapmer comprises at least one bicyclic nucleoside. In certain ments, the 5’- wing of a gapmer comprises at least two bicyclic nucleosides. In certain embodiments, the 5’- Wing of a gapmer ses at least three bicyclic nucleosides. In certain embodiments, the 5’- Wing of a gapmer comprises at least four bicyclic nucleosides. In certain embodiments, the 5’- Wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain ments, the 5’- Wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 5’- Wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 5’- Wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 5’- Wing of a gapmer is a LNA nucleoside.
In certain ments, the 5’- Wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In n embodiments, the 5’- Wing of a gapmer comprises at least one 2’-substituted nucleoside. In certain embodiments, the 5’- Wing of a gapmer comprises at least one 2’-MOE side. In certain embodiments, the 5’- Wing of a gapmer comprises at least one 2’-OMe nucleoside. In certain embodiments, each nucleoside of the 5’- Wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 5’- Wing of a gapmer is a 2’-substituted side. In certain embodiments, each nucleoside of the 5’- Wing of a gapmer is a 2’-MOE nucleoside. In certain embodiments, each nucleoside of the 5’- Wing of a gapmer is a 2’-OMe nucleoside.
In certain embodiments, the 5’- Wing of a gapmer comprises at least one 2’-deoxynucleoside. In certain embodiments, each nucleoside of the 5’- Wing of a gapmer is a 2’-deoxynucleoside. In a certain embodiments, the 5’- Wing of a gapmer comprises at least one ribonucleoside. In n embodiments, each nucleoside of the 5’- Wing of a gapmer is a ribonucleoside. In certain embodiments, one, more than one, or each of the nucleosides of the 5’- Wing is an RNA-like nucleoside.
In certain embodiments, the 5’-Wing of a gapmer comprises at least one ic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5’-Wing of a gapmer ses at least one bicyclic side and at least one 2’-substituted nucleoside. In certain embodiments, the 5’-Wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2’-MOE nucleoside. In certain embodiments, the 5’-Wing of a gapmer ses at least one bicyclic nucleoside and at least one 2’-OMe nucleoside. In certain embodiments, the 5’-Wing of a gapmer comprises at least one bicyclic nucleoside and at least one xynucleoside.
In certain embodiments, the 5’-Wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic d nucleoside. In certain embodiments, the 5’-Wing of a gapmer ses at least one constrained ethyl nucleoside and at least one 2’-substituted nucleoside. In certain embodiments, the 5’-Wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2’-MOE nucleoside. In certain embodiments, the 5’-Wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2’-OMe nucleoside. In certain embodiments, the 5’-Wing of a gapmer comprises at least one ained ethyl side and at least one 2’-deoxynucleoside. ii. Certain 3’-wings In certain embodiments, the 3’- wing of a gapmer ts of l to 8 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer consists of l to 7 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer consists of l to 6 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer consists of l to 5 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer consists of 3 to 5 linked nucleosides.
In n embodiments, the 3’- wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer ts of 1 to 4 linked nucleosides. In certain ments, the 3’- wing of a gapmer consists of l to 3 linked nucleosides. In n embodiments, the 3’- wing of a gapmer consists of l or 2 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer consists of 2 or 3 linked nucleosides.
In certain ments, the 3’- wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer consists of l side. In certain embodiments, the 3’- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer ts of 31inked nucleosides. In certain embodiments, the 3’- wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer ts of 5 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer consists of 6 linked nucleosides.
In n ments, the 3’- wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 3’- wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 3’- wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 3’- wing of a gapmer is a ic nucleoside. In certain ments, each nucleoside of the 3’- wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 3’- wing of a gapmer is a LNA nucleoside.
In n embodiments, the 3’- wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 3’- wing of a gapmer comprises at least two non-bicyclic modified nucleosides. In certain embodiments, the 3’- wing of a gapmer comprises at least three non-bicyclic modified nucleosides. In certain embodiments, the 3’- wing of a gapmer comprises at least four non-bicyclic modified nucleosides. In certain embodiments, the 3’- wing of a gapmer comprises at least one 2’-substituted nucleoside. In certain embodiments, the 3’- wing of a gapmer comprises at least one 2’-MOE nucleoside. In certain embodiments, the 3’- wing of a gapmer comprises at least one 2’-OMe nucleoside. In certain embodiments, each nucleoside of the 3’- wing of a gapmer is a non-bicyclic modified nucleoside. In n embodiments, each nucleoside of the 3’- wing of a gapmer is a 2’-substituted nucleoside. In certain embodiments, each nucleoside of the 3’- wing of a gapmer is a 2’-MOE nucleoside. In certain embodiments, each nucleoside of the 3’- wing of a gapmer is a 2’-OMe nucleoside.
In certain embodiments, the 3’- wing of a gapmer ses at least one 2’-deoxynucleoside. In n embodiments, each nucleoside of the 3’- wing of a gapmer is a 2’-deoxynucleoside. In a certain embodiments, the 3’- wing of a gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 3’- Wing of a gapmer is a ribonucleoside. In certain ments, one, more than one, or each of the nucleosides of the 5’- Wing is an RNA-like nucleoside.
In certain embodiments, the 3’-Wing of a gapmer ses at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2’-substituted nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2’-MOE nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2’-OMe side. In n embodiments, the 3’-Wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2’-deoxynucleoside.
In certain embodiments, the 3’-Wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2’-substituted nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2’-MOE nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one ained ethyl nucleoside and at least one 2’-OMe nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2’-deoxynucleoside.
In n embodiments, the 3’-Wing of a gapmer ses at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3’-wing of a gapmer comprises at least one LNA nucleoside and at least one 2’-substituted nucleoside. In certain ments, the 3’-Wing of a gapmer comprises at least one LNA nucleoside and at least one 2’-MOE nucleoside. In certain ments, the 3’-Wing of a gapmer ses at least one LNA nucleoside and at least one 2’-OMe nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one LNA nucleoside and at least one 2’- deoxynucleoside.
In certain embodiments, the 3’-Wing of a gapmer comprises at least one bicyclic nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2’-deoxynucleoside. In certain ments, the 3’- Wing of a gapmer ses at least one constrained ethyl nucleoside, at least one cyclic modified nucleoside, and at least one 2’-deoxynucleoside. In certain embodiments, the 3’-wing of a gapmer comprises at least one LNA side, at least one cyclic modified nucleoside, and at least one 2’- deoxynucleoside.
In certain embodiments, the 3’-Wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2’-substituted nucleoside, and at least one 2’-deoxynucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2’-substituted nucleoside, and at least one 2’-deoxynucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one LNA nucleoside, at least one 2’-substituted nucleoside, and at least one 2’-deoxynucleoside.
In certain embodiments, the 3’-Wing of a gapmer ses at least one bicyclic nucleoside, at least one 2’-MOE nucleoside, and at least one 2’-deoxynucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2’-MOE nucleoside, and at least one 2’-deoxynucleoside. In certain embodiments, the 3’-Wing of a gapmer ses at least one LNA nucleoside, at least one 2’-MOE nucleoside, and at least one 2’-deoxynucleoside.
In certain embodiments, the g of a gapmer comprises at least one bicyclic nucleoside, at least one 2’-OMe nucleoside, and at least one 2’-deoxynucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2’-OMe nucleoside, and at least one 2’-deoxynucleoside. In n embodiments, the g of a gapmer comprises at least one LNA nucleoside, at least one 2’-OMe nucleoside, and at least one 2’-deoxynucleoside. iii. Certain Central Regions (gaps) In certain embodiments, the gap of a gapmer consists of 6 to 20 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 15 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 12 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to linked nucleosides. In n embodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides. In certain ments, the gap of a gapmer consists of 6 to 8 linked sides. In certain embodiments, the gap of a gapmer consists of 6 or 7 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 9 linked nucleosides. In certain ments, the gap of a gapmer consists of 7 or 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 to 10 linked nucleosides. In n embodiments, the gap of a gapmer consists of 8 or 9 linked nucleosides. In certain embodiments, the gap of a gapmer ts of 6 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 10 linked nucleosides. In certain ments, the gap of a gapmer ts of 11 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 12 linked nucleosides.
In certain embodiments, each nucleoside of the gap of a gapmer is a 2’-deoxynucleoside. In certain ments, the gap ses one or more d nucleosides. In certain embodiments, each nucleoside of the gap of a gapmer is a 2’-deoxynucleoside or is a modified nucleoside that is “DNA-like.” In such embodiments, “DNA-like” means that the nucleoside has similar characteristics to DNA, such that a duplex comprising the gapmer and an RNA molecule is capable of ting RNase H. For example, under certain conditions, 2’-(ara)-F have been shown to support RNase H activation, and thus is DNA-like. In certain embodiments, one or more nucleosides of the gap of a gapmer is not a 2’-deoxynucleoside and is not DNA- like. In certain such embodiments, the gapmer nonetheless supports RNase H activation (e.g., by Virtue of the number or placement of the non-DNA nucleosides).
In certain embodiments, gaps comprise a stretch of unmodified 2’-deoxynucleoside interrupted by one or more modified nucleosides, thus resulting in three sub-regions (two stretches of one or more 2’- deoxynucleosides and a stretch of one or more interrupting modified nucleosides). In certain embodiments, no stretch of unmodified 2’-deoxynucleosides is longer than 5, 6, or 7 nucleosides. In certain embodiments, such short stretches is achieved by using short gap regions. In certain embodiments, short stretches are achieved by interrupting a longer gap region.
In certain embodiments, the gap comprises one or more modified nucleosides. In certain embodiments, the gap ses one or more modified nucleosides selected from among cEt, FHNA, LNA, and 2-thio-thymidine. In certain embodiments, the gap comprises one modified nucleoside. In certain embodiments, the gap comprises a 5’-substituted sugar moiety selected from among 5’-Me, and 5’-(R)-Me.
In n embodiments, the gap comprises two modified nucleosides. In certain embodiments, the gap comprises three modified nucleosides. In certain embodiments, the gap comprises four modified nucleosides.
In certain embodiments, the gap comprises two or more modified nucleosides and each modified nucleoside is the same. In certain embodiments, the gap comprises two or more modified nucleosides and each modified nucleoside is different.
In certain embodiments, the gap comprises one or more d linkages. In certain embodiments, the gap comprises one or more methyl phosphonate linkages. In certain ments the gap comprises two or more modified linkages. In certain embodiments, the gap comprises one or more modified linkages and one or more modified nucleosides. In certain embodiments, the gap comprises one modified linkage and one modified nucleoside. In certain embodiments, the gap comprises two modified linkages and two or more modified nucleosides. b. Certain Internucleoside Linkage Motifs In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif In n embodiments, ucleotides of the present sure comprise a region of uniformly d internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In n embodiments, the oligonucleotide is uniformly linked by phosphorothioate internucleoside es. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and orothioate. In certain ments, each internucleoside linkage of the oligonucleotide is selected from odiester and phosphorothioate and at least one ucleoside linkage is phosphorothioate.
In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 7 phosphorothioate internucleoside es. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the ucleotide comprises at least 9 orothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 11 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 12 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 13 phosphorothioate internucleoside es. In certain embodiments, the oligonucleotide comprises at least 14 phosphorothioate internucleoside linkages.
In certain embodiments, the ucleotide comprises at least one block of at least 6 utive orothioate ucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 7 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 9 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive orothioate internucleoside linkages. In certain ments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3’ end of the oligonucleotide.
In certain such embodiments, at least one such block is d Within 3 nucleosides of the 3’ end of the oligonucleotide.In certain embodiments, the oligonucleotide ses less than 15 phosphorothioate internucleoside linkages. In n ments, the ucleotide comprises less than 14 phosphoro- thioate internucleoside linkages. In certain embodiments, the ucleotide comprises less than 13 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 12 phosphorothioate internucleoside es. In n embodiments, the oligonucleotide comprises less than 11 phosphorothioate internucleoside linkages. In certain ments, the oligonucleotide comprises less than 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 9 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 8 phosphorothioate internucleoside linkages. In certain ments, the oligonucleotide comprises less than 7 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 5 phosphorothioate internucleoside es. c. Certain Nucleobase Modification Motifs In certain embodiments, oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif. In certain such embodiments, base modifications are arranged in a gapped motif. In certain embodiments, nucleobase modifications are arranged in an alternating motif. In certain ments, each nucleobase is modified. In n embodiments, none of the nucleobases is chemically modified.
In certain embodiments, oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3’-end of the oligonucleotide. In certain embodiments the block is Within 3 nucleotides of the 3’-end of the oligonucleotide. In certain such embodiments, the block is at the 5’-end of the oligonucleotide. In certain embodiments the block is Within 3 nucleotides of the 5’-end of the oligonucleotide.
In certain embodiments, nucleobase modifications are a on of the natural base at a particular position of an oligonucleotide. For example, in certain embodiments each purine or each pyrimidine in an oligonucleotide is modified. In certain embodiments, each e is modified. In certain ments, each guanine is modified. In n embodiments, each thymine is modified. In certain embodiments, each cytosine is modified. In certain embodiments, each uracil is modified.
In certain embodiments, some, all, or none of the ne moieties in an oligonucleotide are 5- methyl ne moieties. Herein, 5-methyl cytosine is not a “modified nucleobase. :9 Accordingly, unless otherwise indicated, unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl. In certain ments, the ation state of all or some cytosine nucleobases is specified.
In certain embodiments, chemical ations to nucleobases comprise attachment of certain conjugate groups to nucleobases. In certain embodiments, each purine or each pyrimidine in an oligonucleotide may be optionally modified to comprise a conjugate group. d. Certain Overall Lengths In certain embodiments, the present disclosure provides oligonucleotides of any of a variety of ranges of lengths. In certain ments, oligonucleotides t ofX to Y linked nucleosides, Where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14,15,16,17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that XSY. For example, in certain embodiments, the oligonucleotide may consist of8 to 9, 8 to 10, 8 toll, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9to 11, 9to 12, 9to 13, 9to 14, 9t015, 9to 16, 9to 17, 9to 18, 9to 19, 9to20, 9to21, 9to22, 9to23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30,10 toll, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16,10 to 17, 10 to 18, 10 to 19, 10 to 20,10 to 21,10 to 22,10 to 23,10 to 24,10 to 25,10 to 26,10 to 27, to 28, 10to 29, 10to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to ,11to21,11to22,11to23,11to24,11to25,11to26,11to27,11to28,11to29,11to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20,12 to 21,12 to 22,12 to 23,12 to 24,12 to ,12 to 26,12 to 27,12 to 28,12 to 29,12 to 30,13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20,13 to 21,13 to 22,13 to 23,13 to 24,13 to 25,13 to 26,13 to 27,13 to 28,13 to 29,13 to 30,14 to ,14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20,14 to 21,14 to 22,14 to 23,14 to 24,14 to 25,14 to 26, 14 to 27,14 to 28,14 to 29,14 to 30,15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20,15 to 21,15 to 22,15 to 23,15 to 24,15 to 25,15 to 26,15 to 27,15 to 28,15 to 29,15 to 30,16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21,16 to 22,16 to 23,16 to 24,16 to 25,16 to 26,16 to 27,16 to 28,16 to 29,16 to 30,17 to 18, 17 to 19,17 to 20,17 to 21,17 to 22,17 to 23,17 to 24,17 to 25,17 to 26,17 to 27,17 to 28,17 to 29,17 to 30, 18 to 19, 18 to 20,18 to 21,18 to 22,18 to 23,18 to 24,18 to 25,18 to 26,18 to 27,18 to 28,18 to 29,18 to ,19 to 20,19 to 21,19 to 22,19 to 23,19 to 24,19 to 25,19 to 26,19 to 29,19 to 28,19 to 29,19 to 30, to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to , 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments Where the number of sides of an oligonucleotide of a compound is limited, Whether to a range or to a specific number, the compound may, nonetheless further comprise additional other substituents.
For example, an ucleotide sing 8-30 sides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an ucleotide may further comprise, for example one or more ate groups, terminal groups, or other substituents. r, Where an oligonucleotide is described by an overall length range and by regions having specified lengths, and Where the sum of specified s of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range.
. Certain Antisense Oligonucleotide Chemistry Motifs In certain embodiments, the chemical ural features of antisense oligonucleotides are characterized by their sugar motif, internucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, each internucleoside linkage of an ucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. Thus, the internucleoside linkages Within the Wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region. Likewise, such sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. One of skill in the art Will appreciate that such motifs may be combined to create a variety of oligonucleotides.
In certain embodiments, the selection of internucleoside e and nucleoside ation are not independent of one another. i. Certain Sequences and Targets In certain embodiments, the invention provides antisense oligonucleotides having a sequence mentary to a target nucleic acid. Such antisense compounds are capable of izing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds 2014/036460 specifically ize to one or more target nucleic acid. In certain embodiments, a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid or reduce non-specific hybridization to non-target nucleic acid sequences under conditions in which specific ization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro ). In n embodiments, oligonucleotides are selective between a target and non-target, even though both target and non-target comprise the target sequence. In such embodiments, selectivity may result from relative accessibility of the target region of one nucleic acid molecule compared to the other.
In certain embodiments, the present sure provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the ucleotide. In certain embodiments, ucleotides are 99% complementary to the target nucleic acid.
In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such ucleotides are 90% complementary to the target nucleic acid.
In certain ments, such ucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully complementary to a target nucleic acid and is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is from 6 to 14 nucleobases in length.
In certain embodiments, ucleotides comprise a hybridizing region and a terminal region. In certain such ments, the hybridizing region consists of 12-30 linked nucleosides and is fully complementary to the target nucleic acid. In certain embodiments, the hybridizing region includes one mismatch relative to the target nucleic acid. In certain embodiments, the hybridizing region includes two mismatches relative to the target nucleic acid. In certain embodiments, the hybridizing region includes three mismatches ve to the target nucleic acid. In certain embodiments, the terminal region ts of 1-4 terminal nucleosides. In certain embodiments, the terminal nucleosides are at the 3’ end. In certain embodiments, one or more of the terminal nucleosides are not complementary to the target nucleic acid.
Antisense mechanisms e any mechanism involving the hybridization of an oligonucleotide with target nucleic acid, wherein the hybridization results in a ical effect. In certain embodiments, such hybridization results in either target nucleic acid degradation or occupancy with concomitant inhibition or stimulation of the cellular machinery involving, for example, translation, transcription, or ng of the target nucleic acid.
One type of antisense mechanism involving degradation of target RNA is RNase H mediated antisense. RNase H is a ar endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNase H activity in mammalian cells. Activation of RNase H, therefore, results in cleavage of the RNA , thereby greatly enhancing the ency of DNA-like oligonucleotide-mediated inhibition of gene expression.
In certain embodiments, a conjugate group comprises a cleavable moiety. In certain embodiments, a conjugate group comprises one or more ble bond. In certain embodiments, a conjugate group comprises a linker. In n embodiments, a linker comprises a protein binding moiety. In certain embodiments, a conjugate group comprises a cell-targeting moiety (also ed to as a cell-targeting group).
In certain embodiments a cell-targeting moiety comprises a branching group. In certain ments, a cell- targeting moiety comprises one or more tethers. In certain embodiments, a cell-targeting moiety comprises a carbohydrate or carbohydrate cluster. ii. Certain ble Moieties In certain ments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety comprises a cleavable bond. In certain embodiments, the ate group comprises a cleavable moiety. In certain such embodiments, the cleavable moiety attaches to the antisense oligonucleotide. In certain such embodiments, the ble moiety attaches ly to the cell-targeting moiety. In certain such embodiments, the cleavable moiety attaches to the conjugate linker. In certain ments, the ble moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a ble nucleoside or side analog. In n embodiments, the nucleoside or nucleoside analog comprises an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, the cleavable moiety is a side comprising an ally protected heterocyclic base selected from uracil, thymine, cytosine, 4-N- benzoylcytosine, 5-methylcytosine, 4-N-benzoylmethylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N—isobutyrylguanine. In certain embodiments, the cleavable moiety is 2'-deoxy nucleoside that is attached to the 3' position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is 2'- deoxy adenosine that is attached to the 3' position of the nse oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is 2'-deoxy adenosine that is attached to the 3' position of the antisense oligonucleotide by a odiester linkage and is attached to the linker by a phosphodiester linkage.
In certain embodiments, the cleavable moiety is attached to the 3' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the 5' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is ed to a 2' position of the antisense oligonucleotide. In n embodiments, the cleavable moiety is attached to the antisense oligonucleotide by a phosphodiester linkage. In certain embodiments, the cleavable moiety is attached to the linker by either a phosphodiester or a phosphorothioate linkage. In certain embodiments, the cleavable moiety is attached to the linker by a phosphodiester linkage. In certain embodiments, the conjugate group does not include a cleavable moiety.
In certain embodiments, the cleavable moiety is cleaved after the complex has been administered to an animal only after being internalized by a targeted cell. Inside the cell the cleavable moiety is cleaved thereby releasing the active antisense ucleotide. While not g to be bound by theory it is believed that the cleavable moiety is d by one or more ses within the cell. In certain embodiments, the one or more nucleases cleave the odiester linkage between the ble moiety and the . In certain embodiments, the cleavable moiety has a structure selected from among the following: O=Fl’-OH l s5 O=Fl’-OH O=F|’-OH O O O BX1 O BX2 ‘ d o O 2 OH: — O=F:’-OH | O=F|’-OH O O O Bx O BX2 O BX3 ; and C? C? C? O: -OH O=P-OH O: -OH wherein each of BX, BX1, BXQ, and BX3 is independently a heterocyclic base moiety. In certain embodiments, the cleavable moiety has a structure selected from among the following: O N <’ 1‘“ O N NJ (.5: 0: -OH iii. Certain Linkers In certain embodiments, the conjugate groups comprise a linker. In certain such embodiments, the linker is covalently bound to the cleavable moiety. In certain such embodiments, the linker is covalently bound to the antisense oligonucleotide. In certain embodiments, the linker is covalently bound to a cell- ing moiety. In n embodiments, the linker further comprises a covalent attachment to a solid support. In certain embodiments, the linker further comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker r ses a covalent ment to a solid t and further ses a covalent attachment to a protein binding moiety. In certain embodiments, the linker es multiple positions for ment of tethered ligands. In certain embodiments, the linker includes multiple positions for attachment of tethered ligands and is not attached to a branching group. In certain embodiments, the linker further comprises one or more cleavable bond. In certain embodiments, the conjugate group does not e a linker.
In certain embodiments, the linker includes at least a linear group comprising groups selected from alkyl, amide, 1de, hylene , ether, thioether (-S-) and hydroxylamino (-O-N(H)-) . In certain embodiments, the linear group comprises groups selected from alkyl, amide and ether groups. In certain ments, the linear group ses groups selected from alkyl and ether groups. In certain embodiments, the linear group comprises at least one phosphorus linking group. In certain embodiments, the linear group comprises at least one phosphodiester group. In certain embodiments, the linear group includes at least one neutral linking group. In certain embodiments, the linear group is covalently attached to the cell- targeting moiety and the cleavable moiety. In certain ments, the linear group is covalently ed to the cell-targeting moiety and the antisense oligonucleotide. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety, the cleavable moiety and a solid support. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety, the cleavable moiety, a solid support and a protein binding moiety. In certain embodiments, the linear group includes one or more cleavable bond.
In certain embodiments, the linker includes the linear group covalently attached to a scaffold group.
In certain embodiments, the scaffold includes a branched aliphatic group comprising groups selected from alkyl, amide, disulf1de, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the scaffold includes a branched aliphatic group comprising groups selected from alkyl, amide and ether groups. In certain ments, the scaffold includes at least one mono or polycyclic ring .
In certain embodiments, the ld includes at least two mono or clic ring systems. In certain embodiments, the linear group is covalently attached to the scaffold group and the ld group is covalently attached to the cleavable moiety and the linker. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker and a solid support. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker and a protein binding moiety. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker, a protein binding moiety and a solid support. In certain embodiments, the scaffold group includes one or more cleavable bond.
In certain embodiments, the linker includes a protein binding . In certain embodiments, the protein binding moiety is a lipid such as for example including but not limited to cholesterol, cholic acid, adamantane acetic acid, l-pyrene butyric acid, dihydrotestosterone, l,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, l, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a Vitamin (e.g., folate, Vitamin A, Vitamin E, biotin, pyridoxal), a e, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic component, a steroid (e.g., uvaol, hecigenin, diosgenin), a e (e.g., triterpene, e.g., sarsasapogenin, friedelin, epifriedelanol tized lithocholic acid), or a cationic lipid. In certain ments, the protein binding moiety is a C16 to C22 long chain saturated or unsaturated fatty acid, cholesterol, cholic acid, n E, adamantane or l-pentafluoropropyl.
In certain embodiments, a linker has a structure selected from among: WO 79625 H H «vw E—NH N N | 21/ W 0/, (I? 0 R02 o—P—OH N [k0 N | N O \3 "'W - H \ 1 N ; ( )n W o le <5\ I 0 N | ii O,,_ mO—Ifi-OH ; OH - ; ’ aMo p T ”l“ ”T” o w 0, H H H H H [ko\ E/WnflnwnN N N N NMJL \o N 5“ n :3 H kHz/$S/S ’ 1 no O "'4, JVI'W wherein each n is, independently, from 1 to 20; and p is from 1 to 6.
In certain embodiments, a linker has a structure ed from among: “<0 0 O o N O N H H 1'7- "LLL NMO ’ n W8SNOn i n O Q» ”a O/ is O Q, n H o o 00 0A NkflAN/31 O NJ: V n H NWN/E o, I“? n ’4 9 q Uvo-so . UV ‘I’ ,NM 0 OH E n O IILLWn O ,and o N H s‘ H \N o SHN‘En wherein each n is, independently, from 1 to 20.
In n embodiments, a linker has a structure selected from among: wherein n is from 1 to 20.
In certain embodiments, a linker has a structure selected from among: Wherein each L is, independently, a phosphorus linking group or a neutral linking group; and each n is, ndently, from 1 to 20.
In certain embodiments, a linker has a structure selected from among: WO 79625 EL/NfiLHWNvflg/KOH H WO 79625 JVIW In n embodiments, a linker has a structure selected from among: 0 O O O H H EkNMM/EKE; EkNWM/E ;“51)J\/N\gWKFJH ; O O 2014/036460 In certain embodiments, a linker has a structure selected from among: 0 O O O O H EkNWM/f;H a EkNll/WHEH / )va [771. M; ; OH m, o o H A N HN o o W f “QB, ; 3;” , , WE 2014/036460 In certain embodiments, a linker has a structure selected from among: w; “i” UV0A moat N 0 N 1W0 and 3W0 n n is from 1 to 20.
In certain embodiments, a linker has a structure selected from among: \/\‘g; 5\O/\/\O/\/\‘g ;and €9\O/\/\O/\/\O/\/\€é .
In certain embodiments, a linker has a structure selected from among: OH OH OH 3 OH 3 In certain embodiments, a linker has a structure selected from among: O 0 E‘WMNS EH0_II_ _E e; 1% and WNW In certain embodiments, the conjugate linker has the structure: In certain embodiments, the conjugate linker has the structure: 0 O In certain embodiments, a linker has a structure selected from among: EHO——E “a and fiWfi/M In certain embodiments, a linker has a structure selected from among: szAMS‘'P—o——: WNWOH g‘ “a and 0 wherein each n is ndently, O, l, 2, 3, 4, 5, 6, or 7. iv. Certain Cell-Targeting Moieties In certain embodiments, conjugate groups comprise cell-targeting moieties. Certain such cell-targeting es increase cellular uptake of antisense compounds. In certain embodiments, cell- targeting moieties comprise a branching group, one or more tether, and one or more ligand. In certain embodiments, cell-targeting moieties comprise a branching group, one or more tether, one or more ligand and one or more cleavable bond. 1. Certain Branching Groups In certain embodiments, the conjugate groups comprise a targeting moiety comprising a branching group and at least two tethered ligands. In certain embodiments, the branching group attaches the conjugate linker. In certain embodiments, the branching group attaches the ble moiety. In n embodiments, the ing group attaches the antisense oligonucleotide. In certain embodiments, the branching group is covalently attached to the linker and each of the tethered ligands. In certain embodiments, the branching group comprises a branched tic group comprising groups ed from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the branching group comprises groups selected from alkyl, amide and ether groups. In certain embodiments, the branching group comprises groups selected from alkyl and ether groups. In certain ments, the branching group comprises a mono or polycyclic ring . In certain ments, the branching group comprises one or more cleavable bond. In certain embodiments, the conjugate group does not include a branching group.
In certain embodiments, a ing group has a structure selected from among: WO 79625 o ( E/NH 3L NH O n H 11W,“ N )9. ' and H H N ’ ( L( n E/NH 0 wherein each n is, independently, from 1 to 20; j is from 1 t0 3; and m is from 2 t0 6.
In certain embodiments, a branching group has a structure selected from among: bLLI— ml 0 o 0% | NH ('3' O o O “51W” 11 . HO O—P—O E _ EANWNMJK; ; wherein each n is, independently, from 1 to 20; and m is from 2 to 6.
In certain ments, a branching group has a structure selected from among: 3L 0 “a EWMi/Y‘iO E O O #5 I/m E ; N O . o ITJH Tl?) o ’ NH ra‘ W W ’ijjo ’;\N 377. E/NH Hf HN/ii O E £51“ J: HM}; WE/MNH itWkNH H J\ EWLN H N593 ; and at M H o o if 9" NH 2”“ WW In certain embodiments, a branching group has a structure selected from among: \ | A1 A1 ‘7‘," ( /A1 A1 .111“ ) nE TAN—E §_A1 ( n n A A 1 1 / and | WO 79625 wherein each A1 is independently, O, S, C=O or NH; and each n is, ndently, from 1 to 20.
In certain embodiments, a branching group has a structure selected from among: WIW ”Iw ”IV" A A1 A1 AF; )n AF; )n AF; ’ n n —A1 §_A1 A1 nA1 and §_A1 n( )n wherein each A1 is independently, O, S, C=O or NH; and each n is, independently, from 1 to 20.
In certain embodiments, a branching group has a structure selected from among: wherein A1 is O, S, C=O or NH; and each n is, independently, from 1 to 20.
In certain embodiments, a branching group has a structure selected from among: 0 "m o Nl-I In n embodiments, a branching group has a structure selected from among: /O\g—/“m In certain embodiments, a branching group has a structure ed from among: 2. Certain Tethers In certain embodiments, conjugate groups comprise one or more tethers covalently attached to the branching group. In certain embodiments, ate groups comprise one or more tethers covalently attached to the linking group. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amide and polyethylene glycol groups in any combination. In certain embodiments, each tether is a linear tic group comprising one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amide, phosphodiester and polyethylene glycol groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether and amide groups in any combination. In certain ments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, phosphodiester, ether and amide groups in any combination. In n ments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester in any combination.
In certain embodiments, each tether comprises at least one phosphorus linking group or neutral g group.
In certain embodiments, the tether includes one or more cleavable bond. In certain embodiments, the tether is attached to the branching group through either an amide or an ether group. In certain embodiments, the tether is attached to the branching group h a phosphodiester group. In certain embodiments, the tether is attached to the branching group through a phosphorus linking group or neutral linking group. In certain embodiments, the tether is attached to the branching group through an ether group.
In n ments, the tether is attached to the ligand h either an amide or an ether group. In n embodiments, the tether is attached to the ligand through an ether group. In certain embodiments, the tether is attached to the ligand through either an amide or an ether group. In certain embodiments, the tether is attached to the ligand through an ether group.
In certain embodiments, each tether comprises from about 8 to about 20 atoms in chain length between the ligand and the branching group. In certain embodiments, each tether group comprises from about 10 to about 18 atoms in chain length between the ligand and the branching group. In certain embodiments, each tether group comprises about 13 atoms in chain length.
In certain embodiments, a tether has a structure selected from among: 0 H 3L EMNWOVhOAH/i ; “3/wa ; 1W ; “H‘Mfoflfi ; fimo/Yoflh re: WNW; E H H 1% n n ’ p N “O n J W W1W , WWmWE “WWW?O H ; ”OWHWH‘ ; 2 p H O O ;M ”Wok - mgM and E “M3 n n ’ ’ n v65 n Y wherein each n is, independently, from 1 to 20; and each p is from 1 to about 6.
In certain embodiments, a tether has a ure selected from among: “gt/\AMNOWOA/R— - - NW w: - , HL/ 9:" , WYE , EMOAEF ; ‘JJJV\O/\/OVBLLL ; E/Nwmfsfilnd _ H‘:\/\O/\5ré In certain ments, a tether has a structure ed from among: ,6 NH 1 wherein each n is, independently, from 1 to 20.
In certain ments, a tether has a structure ed from among: 0 21 .a‘ L 1, 2. d 39L JYLN wherein L is either a phosphorus linking group or a neutral linking group; 21 is C(=O)O-R2; Zg is H, C1-C6 alkyl or substituted C1-C6 alky; R2 is H, C1-C6 alkyl or substituted C1-C6 alky; and each m1 is, ndently, from O to 20 wherein at least one m1 is greater than 0 for each tether.
In certain embodiments, a tether has a structure selected from among: In certain embodiments, a tether has a structure selected from among: 0 ~91 fWkNJfi,o—fi—o—(—9fi::‘0 COOH OH 0—5—0 I “a m1<5H m1 m1H O wherein Zg is H or CH3; and each m1 is, independently, from O to 20 wherein at least one m1 is greater than 0 for each tether.
In certain embodiments, a tether has a structure selected from among: 0 0 ”mior “mi: , ; wherein each n is independently, O, l, 2, 3, 4, 5, 6, or 7.
In certain embodiments, a tether comprises a phosphorus linking group. In certain ments, a tether does not comprise any amide bonds. In n embodiments, a tether comprises a phosphorus linking group and does not comprise any amide bonds. 3. Certain Ligands In n embodiments, the present disclosure provides ligands wherein each ligand is covalently attached to a tether. In certain embodiments, each ligand is selected to have an affinity for at least one type of receptor on a target cell. In certain embodiments, ligands are selected that have an affinity for at least one type of receptor on the surface of a mammalian liver cell. In certain embodiments, ligands are selected that have an affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is, ndently ed from galactose, N—acetyl galactoseamine, mannose, glucose, glucosamone and fucose. In certain embodiments, each ligand is N—acetyl galactoseamine (GalNAc). In certain embodiments, the ing moiety comprises 2 to 6 ligands. In certain embodiments, the targeting moiety comprises 3 ligands. In n embodiments, the ing moiety comprises 3 N—acetyl oseamine ligands.
In certain embodiments, the ligand is a carbohydrate, carbohydrate derivative, d carbohydrate, multivalent carbohydrate cluster, polysaccharide, d polysaccharide, or polysaccharide derivative. In certain embodiments, the ligand is an amino sugar or a thio sugar. For e, amino sugars may be selected from any number of compounds known in the art, for example glucosamine, sialic acid, (1-D- galactosamine, N—Acetylgalactosamine, 2-acetamidodeoxy—D-galactopyranose (GalNAc), 2-Amino0- [(R)-l-carboxyethyl]deoxy—B-D-glucopyranose (B-muramic acid), 2-Deoxy—2-methylamino-L- glucopyranose, 4,6-Dideoxy—4-formamido-2,3-dimethyl-D-mannopyranose, ysulfoamino-D- glucopyranose and N—sulfo-D-glucosamine, and N—Glycoloyl-(x-neuraminic acid. For example, thio sugars may be ed from the group consisting of 5-Thio-B-D-glucopyranose, Methyl 2,3,4-triacetyl-l-thio O-trityl-(x-D-glucopyranoside, 4-Thio-B-D-galactopyranose, and ethyl 3,4,6,7-tetraacetyldeoxy—l,5- dithio-(x-D-gluco-heptopyranoside.
In certain embodiments, “GalNac” or “Gal-NAc” refers to 2-(Acetylamino)deoxy—D- galactopyranose, commonly referred to in the literature as N—acetyl galactosamine. In certain ments, “N—acetyl galactosamine” refers to tylamino)deoxy-D-galactopyranose. In certain embodiments, “GalNac” or “Gal-NAc” refers to 2-(Acetylamino)deoxy-D-galactopyranose. In certain embodiments, “GalNac” or Ac” refers to 2-(Acetylamino)deoxy—D-galactopyranose, which includes both the B- form: 2-(Acetylamino)deoxy-B-D-galactopyranose and (x-form: 2-(Acetylamino)deoxy—D- galactopyranose. In certain embodiments, both the B-form: 2-(Acetylamino)deoxy-B-D-galactopyranose and (x-form: 2-(Acetylamino)deoxy—D-galactopyranose may be used interchangeably. Accordingly, in structures in which one form is depicted, these structures are intended to include the other form as well. For example, where the structure for an (x-form: 2-(Acetylamino)deoxy—D-galactopyranose is shown, this structure is intended to include the other form as well. In certain embodiments, In certain preferred embodiments, the B-form 2-(Acetylamino)-2—deoxy—D-galactopyranose is the preferred embodiment. o OH HO J‘M o ””1 k HO N 2-(Acetylamino)deoxy-D-galactopyranose HO 0—; NHAC 2-(Acetylamino)deoxy-B-D-galactopyranose NHAC 2-(Acetylamino)deoxy-(x-D-galactopyranose In certain ments one or more ligand has a structure selected from among: HO O H$4/ 0—; HO R1 ‘% R1 and R1 O O wherein each R1 is selected from OH and NHCOOH.
In certain embodiments one or more ligand has a structure selected from among: HOOH HO HO&S/ \ OH o 0H0 O HO,/1§;:::/O '0 ' HO HO o HO NHAcrrrr ; “f . \ HO , OH HO : 5; HOOH “0%HO N HO OH HOOH \H“ ’ Wom“ \ Mm 0“ OH HO - HO \%?:é73 /a HO *5 , 0 ,and OH OH HO OH OH -O In certain ments one or more ligand has a structure selected from among: HOOH HO&£w\HH NHAC In certain embodiments one or more ligand has a structure selected from among: HOOH Home}: NHAC _ i. Certain Conjugates In n embodiments, conjugate groups comprise the structural features above. In certain such embodiments, conjugate groups have the following structure: HO OH wherein each n is, independently, from 1 to 20.
In certain such embodiments, conjugate groups have the following structure: HO OH O H O O HN\/\/ NHAc O HO OH o O H H H 0 NWN 0 N_| NHAc WY o HO HN H\/\/ o o N NHAc In certain such embodiments, conjugate groups have the following structure: HO OH _ O H H o OH N N o 9H 6 NHAc W\9: ” = 0 HO 0H 0 H H ‘ N N o O qN 0I HO \9/ n o—P=X “ n ” NHAc (IDH o o o HW/HN o HO n NHAc wherein each n is, independently, from 1 to 20; Z is H or a linked solid support; Q is an antisense compound; X is O or S; and BX is a heterocyclic base moiety.
In certain such embodiments, conjugate groups have the following structure: HO OH _ O H H O o OH O=Fl’—OH HO \/ : HO OV\H/ \/ O—FI’ZX NHAc OH o 0 0 H HN O N\/ NHAC O 2014/036460 In certain such embodiments, conjugate groups have the following structure: HO OH _ O H O 0 2 OH: — N N oWM 9H N Ho < NHAc = O N NHAc o H0 W 3 NHAc HOOH HO O i? “ 0 (5H0 ACHN ) HOOH n mm Wakomoo 0 0 ll o ACHN OH HOOH Q O 0%’3‘04") 11 H0 11 OH NHAC In certain such embodiments, conjugate groups have the following structure: NHAC In certain such ments, conjugate groups have the following structure: HOOH HO O i? ACHN O/|\O OH ) HOOH 11 NHZ HOW Wolomo \ O 0 O 0 0 " newH O N /P\ JAN _ _ be ACHN OH OH O 0‘ HOOH o 0 SP). fl) Ho—r}=o We}? “ HO 9 NHAC In certain such embodiments, conjugate groups have the following structure: HOOH HO OV\/\/\ i? O’ |\O ACHN OH k HO OH O 0 0 (II) o {If HO O\/\/\/\ /l3l\ /\/\ O—P—OW N’J O | O 0 (5H \ ACHN 0H 0 Ho—13=o HO 0“ 15L ('3 O DMD/('31? — NHAC In certain such embodiments, conjugate groups have the following structure: o (3 o HO WO/flKO/egh ('3 O O ACHN OH HOOH O Hofig/ W0 0 / fl) O | O n NHAC In certain such embodiments, conjugate groups have the following structure: HO—I|’=O HOOH 0 HO OW\/\ (H) /P\ OH AcHN I O OH 0 HO OH O O 9 0 (13—0 _ HO O\/\/\/\O/II)\O/\/\O/a\/ I AcHN 0H OH In n embodiments, conjugates do not comprise a pyrrolidine.
In certain such embodiments, conjugate groups have the following structure: I N O- W HOOH .- o H H o ('3‘ HO :VYNWNf O=P-O' ACHN o HOOH \/\/\n/N\/\/N\H/\/OO N AcHN H O HO O\/\/\n/N\/\/ ACHN In certain such embodiments, conjugate groups have the ing structure: ACHN O Q o /JKJ O:I_O HO OH it W O O\/\/\/\O (5.0 NHAC In certain such embodiments, conjugate groups have the following ure: HO OH NHAC In certain such embodiments, conjugate groups have the following structure: HoOH o o N Ho 4 Hkk AcHN Hog/o o : O N ”W0 s H M ‘ AcHN o HoOH o /£’J o N Ho O 4 H AcHN In certain such embodiments, conjugate groups have the following structure: HO OH H O o N O HO W ACHN HOOH O o o O o N HO WH QWQWO 5 ACHN HOOH HO 4 ACHN In certain such embodiments, conjugate groups have the following structure: HO OH H O o N O HO W ACHN HOOH O o o O OAmi}N O HO ”WMWO_P_§ AcHN HOOH O O HO Ami—H ACHN In certain such embodiments, conjugate groups have the ing structure: ACHN OHOH HO O O H O H O OWN N NWN‘9; § AcHN H O H O OH “NH O O NHAC OHOH O O H O H O? ACHN H O H O O O NHAC In certain such embodiments, conjugate groups have the following structure: HOOH ' o N HO 0% O O ACHN O—FI’ OH HOOH o N OW O ACHN O‘FI’ OH HOOH o N HO 0%20 o E AcHN In certain such ments, conjugate groups have the following structure: HoOH ' Home;Q0o N ACHN | OZT-OH HOOH o N HO O/*%:?; o ACHN | O=T—OH HOOH o N ACHN ('3 In certain embodiments, the cell-targeting moiety of the ate group has the following structure: HoOH HO O\\X AcHN \\ HoOH Hog/OO i —X 0 /3‘ ACHN HOOH X/ o o// ACHN wherein X is a substituted or unsubstituted tether of six to eleven utively bonded atoms.
In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure: HoOH HO O\\X AcHN \\ HoOH Hog/Oo _#_X___0 ”’3‘ AcHN HOOH X/ o o// AcHN wherein X is a substituted or unsubstituted tether of ten consecutively bonded atoms.
In n embodiments, the cell-targeting moiety of the conjugate group has the ing structure: HoOH HO O\\X AcHN \\ HoOH Hog/Oo _#_X___0 ”’3‘ AcHN HOOH X/ o o// AcHN wherein X is a substituted or unsubstituted tether of four to eleven consecutively bonded atoms and wherein the tether comprises exactly one amide bond.
In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure: HoOH HO O\Y AcHN ‘jz \N 2/0 HoOH O Hog/Oo __Y\N/u\Z,O ,% AcHN H z\ HOOH O Y/ \W O O/ o AcHN wherein Y and Z are independently selected from a C1-C12 substituted or unsubstituted alkyl, alkenyl, or l group, or a group comprising an ether, a ketone, an amide, an ester, a carbamate, an amine, a piperidine, a phosphate, a odiester, a phosphorothioate, a triazole, a pyrrolidine, a disulfide, or a thioether.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure: HOOH Hoj§:%~L/O\YO AcHN ‘jz \N 2/0 HoOH O Hoj§:%~X/Oo _YkNJLZ,O )5 AcHN H z\ HOOH Y/‘WV O O O/ o ACHN wherein Y and Z are independently selected from a C1-C12 substituted or unsubstituted alkyl group, or a group comprising exactly one ether or exactly two ethers, an amide, an amine, a piperidine, a ate, a phosphodiester, or a phosphorothioate.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure: HOOH Hoj§:%~L/O\YO AcHN ‘jz \N 2/0 HoOH O Hoj§:%~X/Oo Z,O )5 AcHN H z\ HOOH Y/‘WV O O O/ o ACHN wherein Y and Z are independently selected from a C1-C12 substituted or unsubstituted alkyl group.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the ing structure: HoOH O HomoflmN/UffoH AcHN O “ wherein m and n are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.
In certain such embodiments, the cell-targeting moiety of the ate group has the following structure: HOOH O Homo/amN/UT’IOo H n HOOH W0 )1 mo N m " IZ AcHN wherein m is 4, 5, 6, 7, or 8, and n is l, 2, 3, or 4.
In certain ments, the cell-targeting moiety of the conjugate group has the following structure: H00H HO O\ HOOH X AcHN o X HO 3% AcHN I2 Howe/X AcHN wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms, and wherein X does not comprise an ether group.
In certain embodiments, the argeting moiety of the conjugate group has the following structure: wherein X is a substituted or unsubstituted tether of eight utively bonded atoms, and wherein X does not comprise an ether group.
In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure: HOOH HQOH HO \X AcHN O X HO NE AcHN H OHOH HOJE%:i/O//x AcHN wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms, and wherein the tether ses y one amide bond, and wherein X does not comprise an ether group.
In certain ments, the cell-targeting moiety of the conjugate group has the following structure: AcHN wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms and wherein the tether consists of an amide bond and a substituted or unsubstituted C2-C11 alkyl group.
In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure: HOOH H O O—Y’N O AcHN HoOH O O /Y\ o N 2 HO H ” AcHN HoOH O O—Y/M O AcHN wherein Y is selected from a C1-C12 substituted or unsubstituted alkyl, alkenyl, or alkynyl group, or a group comprising an ether, a ketone, an amide, an ester, a ate, an amine, a piperidine, a phosphate, a phosphodiester, a phosphorothioate, a triazole, a pyrrolidine, a ide, or a thioether.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure: HOOH H O 0—y—N O AcHN HoOH O O /Y\ o N 2 HO H ” AcHN HoOH C) o——Y/’N O AcHN wherein Y is selected from a C1-C12 substituted or unsubstituted alkyl group, or a group comprising an ether, an amine, a piperidine, a ate, a phosphodiester, or a phosphorothioate.
In certain such embodiments, the cell-targeting moiety of the ate group has the following structure: HOOH H O O—Y’N O AcHN HoOH O O /Y\ o N 2 HO H ” AcHN HoOH O O-—Y’/H O AcHN wherein Y is selected from a C1-C12 substituted or unsubstituted alkyl group.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the ing structure: HOOH H O o N O HO fin AcHN HoOH O Hofifi/ flEHo o N A AcHN HoOH O O‘49\n N O HO H AcHN WMmmnmLL&&i@Z&%lQHmHZ In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure: HOOH H OoNfan O AcHN HoOH o O oIfw Ni HO H AcHN HoOH HO H AcHN wherein n is 4, 5, 6, 7, or 8. b.Certain c0n°u ated antisense com ounds In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2’, 3’, of 5’ on of the nucleoside. In certain ments, a conjugated antisense compound has the following structure: A———B———C———D——6E-—-a wherein A is the antisense oligonucleotide; B is the cleavable moiety C is the conjugate linker D is the branching group each E is a tether; each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, a conjugated antisense nd has the following structure: §E_F) wherein A is the antisense oligonucleotide; C is the conjugate linker D is the branching group each E is a tether; each F is a ligand; and q is an integer between 1 and 5.
In certain such embodiments, the ate linker comprises at least one cleavable bond.
In certain such embodiments, the branching group comprises at least one cleavable bond.
In n embodiments each tether comprises at least one cleavable bond.
In n embodiments, the conjugates are bound to a side of the nse oligonucleotide at the 2’, 3’, of 5’ position of the nucleoside.
In certain embodiments, a conjugated antisense compound has the following structure: A—B—c+E—F> wherein A is the antisense oligonucleotide; B is the cleavable moiety C is the conjugate linker each E is a ; each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2’, 3’, of 5’ position of the nucleoside. In certain embodiments, a conjugated antisense compound has the following structure: A—C+E—F> wherein A is the antisense oligonucleotide; C is the conjugate linker each E is a tether; each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, a conjugated antisense nd has the ing structure: A—B—D+E—F> wherein A is the nse oligonucleotide; B is the cleavable moiety D is the branching group each E is a tether; each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, a conjugated antisense nd has the following structure: A owe—F) wherein A is the antisense oligonucleotide; D is the branching group each E is a tether; each F is a ligand; and q is an integer between 1 and 5.
In certain such embodiments, the conjugate linker comprises at least one cleavable bond.
In certain embodiments each tether comprises at least one ble bond.
In certain embodiments, a conjugated antisense compound has a structure selected from among the following: Targeting moiety HO OH WWW"; HOOHM iJfll PO Ligand Tether WU Cleavable moiety $wa Branching group In certain embodiments, a conjugated nse compound has a structure selected from among the following: Cell targeting moiety Branching group In certain embodiments, a conjugated antisense compound has a structure ed from among the following: 2014/036460 Cleavable m01ety Cell targeting moiety | HO OH 0’ : OH ACHN ((2:01 0 HOOH O 233 Conjugate O—lID—O linker ACHN O OH Tether I—l Ligand ZOE—lO O O NHAC Branching group In certain embodiments, the conjugated antisense compound has the following structure: Representative United States patents, United States patent ation ations, and international patent application publications that teach the ation of certain of the above noted conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties as well as other modifications include Without limitation, US 5,994,517, US 319, US 6,660,720, US 6,906,182, US 7,262,177, US 7,491,805, US 8,106,022, US 7,723,509, US 2006/0148740, US 2011/0123520, WO 2013/033230 and , each of Which is incorporated by reference herein in its entirety.
Representative ations that teach the preparation of certain of the above noted conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties as well as other modifications include without limitation, BIESSEN et al., ”The Cholesterol Derivative of a Triantennary Galactoside with High Affinity for the Hepatic Asialoglycoprotein Receptor: a Potent Cholesterol ng Agent” J. Med. Chem. (1995) 38:1846-1852, BIESSEN et al., ”Synthesis of Cluster Galactosides with High Affinity for the c Asialoglycoprotein Receptor” J. Med. Chem. (1995) 38:1538-1546, LEE et al., ”New and more efficient multivalent ligands for asialoglycoprotein receptor of mammalian cytes” Bioorganic & Medicinal Chemistry (2011) 19:2494-2500, RENSEN et al., ”Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein or on Hepatocytes in Vitro and in Vivo” J. Biol. Chem. (2001) 276(40):37577-37584, RENSEN et al., ”Design and sis of Novel N—Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem. (2004) 47:5798-5 808, SLIEDREGT et al., ”Design and Synthesis of Novel Amphiphilic Dendritic osides for ive Targeting of Liposomes to the Hepatic Asialoglycoprotein or” J. Med. Chem. (1999) 42:609-618, and Valentijn et al., “Solid-phase synthesis of lysine-based cluster galactosides with high affinity for the Asialoglycoprotein Receptor” Tetrahedron, 1997, 53(2), 759-770, each of which is incorporated by nce herein in its entirety.
In certain embodiments, conjugated antisense compounds comprise an RNase H based oligonucleotide (such as a gapmer) or a splice modulating oligonucleotide (such as a fully modified oligonucleotide) and any conjugate group comprising at least one, two, or three GalNAc groups. In certain ments a conjugated antisense compound comprises any conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., IntJPep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 261; Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol, 2001, 11, 821-829; Rensen et al., JBiol Chem, 2001, 276, 37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38- 43; lind et al., Glycoconj J, 2004, 21, 227-241; Lee et al., Bioorg Med Chem Lett, 2006, 16(19), 5132- 5135; Maierhofer et al., Bioorg Med Chem, 2007, 15, 7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Lee et al., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., Analyt Biochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012, 51, 448; Biessen et al., JMed Chem, 1995, 38, 1846-1852; egt et al., JMed Chem, 1999, 42, 609-618; Rensen et al., JMed Chem, 2004, 47, 5798- 808; Rensen et al., Arterioscler Thromb Vasc Biol, 2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464; Sato et al., JAm Chem Soc, 2004, 126, 14013-14022; Lee et al., JOrg Chem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792; Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., Methods Enzymol, 2000, 313, 1; Maier et al., Bioconjug Chem, 2003, 14, 18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan, Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; MerWin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013, 21, 5275-5281; International applications WOl998/013381; WO2011/038356; /046098; WO2008/098788; WO2004/101619; WO2012/037254; WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947; WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046; WO2009/082607; WO2009/134487; WO2010/144740; /148013; WOl997/020563; WO2010/088537; WO2002/043771; WO2010/129709; /068187; WO2009/126933; WO2004/024757; WO2010/054406; WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; US. Patents 4,751,219; 8,552,163; 6,908,903; 177; 5,994,517; 6,300,319; 022; 7,491,805; 805; 7,582,744; 8,137,695; 6,383,812; 6,525,031; 720; 7,723,509; 8,541,548; 125; 772; 8,349,308; 467; 8,501,930; 601; 7,262,177; 6,906,182; 916; 8,435,491; 8,404,862; 7,851,615; Published US. Patent Application Publications US2011/0097264; US2011/0097265; US2013/0004427; /0164235; US2006/0148740; US2008/0281044; US2010/0240730; US2003/0119724; US2006/0183886; /0206869; US2011/0269814; US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393; US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075; US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938; US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968; US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; each of Which is incorporated by reference in its entirety.
C. Certain Uses and Features In certain embodiments, conjugated antisense compounds exhibit potent target RNA reduction in vivo. In certain embodiments, unconjugated antisense compounds accumulate in the kidney. In certain embodiments, conjugated antisense compounds accumulate in the liver. In certain embodiments, ated antisense compounds are well tolerated. Such properties render conjugated nse compounds particularly useful for inhibition of many target RNAs, including, but not limited to those involved in metabolic, cardiovascular and other diseases, disorders or conditions. Thus, provided herein are methods of treating such diseases, disorders or conditions by contacting liver tissues With the conjugated antisense compounds targeted to RNAs associated With such diseases, disorders or conditions. Thus, also provided are methods for ameliorating any of a variety of metabolic, cardiovascular and other es, disorders or conditions With the conjugated antisense compounds of the present invention.
In certain embodiments, conjugated antisense compounds are more potent than unconjugated counterpart at a particular tissue concentration. Without g to be bound by any theory or mechanism, in certain embodiemtns, the conjugate may allow the conjugated antisense compound to enter the cell more efficiently or to enter the cell more productively. For example, in certain embodiments conjugated antisense compounds may exhibit greater target reduction as compared to its unconjugated counterpart Wherein both the conjugated antisense compound and its unconjugated counterpart are present in the tissue at the same concentrations. For example, in certain embodiments conjugated antisense compounds may exhibit greater target reduction as compared to its ugated counterpart wherein both the conjugated antisense compound and its unconjugated rpart are present in the liver at the same concentrations.
Productive and oductive uptake of oligonucleotides has beed discussed previously (See e. g.
Geary, R. S., E. Wancewicz, et al. (2009). t of Dose and Plasma Concentration on Liver Uptake and Pharmacologic ty of a 2'-MethoxyethylModif1ed Chimeric Antisense Oligonucleotide Targeting PTEN.” Biochem. Pharmacol. 78(3): ; & Koller, E., T. M. Vincent, et al. (2011). ”Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes.” Nucleic Acids Res. 39(11): 4795-807). Conjugate groups bed herein may improve productive uptake.
In n embodiments, the conjugate groups described herein may further improve potency by increasing the affinity of the conjugated antisense compound for a particular type of cell or tissue. In certain embodiments, the conjugate groups described herein may further improve potency by increasing recognition of the conjugated antisense compound by one or more cell-surface receptors. . In certain ments, the conjugate groups described herein may further improve potency by facilitating endocytosis of the ated antisense compound.
In certain embodiments, the cleavable moiety may further improve potency by allowing the conjugate to be cleaved from the nse oligonucleotide after the conjugated nse compound has entered the cell. Accordingly, in certain embodiments, ated antisense compounds can be administed at doses lower than would be necessary for unconjugated antisense oligonucleotides.
Phosphorothioate es have been incorporated into antisense oligonucleotides previously. Such phosphorothioate linkages are resistant to nucleases and so improve ity of the oligonucleotide. Further, phosphorothioate linkages also bind certain proteins, which results in accumulation of antisense oligonucleotide in the liver. Oligonucleotides with fewer phosphorothioate linkages accumulate less in the liver and more in the kidney (see, for example, Geary, R., “Pharmacokinetic Properties of 2’-O-(2- Methoxyethyl)-Modif1ed Oligonucleotide Analogs in Rats,” Journal ofPharmacology and Experimental Therapeutics, Vol. 296, No. 3, 890-897; & cological Properties of2 ’Methoxyethyl Modified Oligonucleotides in Antisense a Drug logy, Chapter 10, Crooke, S.T., ed., 2008) In certain ments, oligonucleotides with fewer phosphorothioate intemculeoside linkages and more phosphodiester internucleoside es accumulate less in the liver and more in the kidney. When treating diseases in the liver, this is undesibable for several reasons (1) less drug is getting to the site of desired action (liver); (2) drug is escaping into the urine; and (3) the kidney is exposed to relatively high concentration of drug which can result in toxicities in the . Thus, for liver diseases, phosphorothioate linkages provide important benefits.
In certain embodiments, however, administration of oligonucleotides uniformly linked by oro- thioate intemucleoside linkages induces one or more proinflammatory reactions. (see for example: J Lab Clin Med. 1996 Sep; l28(3):329-3 8. “Amplification of antibody production by phosphorothioate oligodeoxynucleotides”. Branda et al.; and see also for e: logic Properties in Antisense a Drug Technology, Chapter 12, pages 342-351, Crooke, S.T., ed., 2008). In certain embodiments, administration of oligonucleotides n most of the internucleoside linkages comprise phosphorothioate intemucleoside linkages induces one or more proinflammatory reactions.
In certain embodiments, the degree of proinflammatory effect may depend on several variables (e. g. backbone modification, off-target effects, nucleobase modifications, and/or nucleoside modifications) see for example: Toxicologic Properties in nse a Drug Technology, Chapter 12, pages 342-351, , S.T., ed., 2008). In certain embodiments, the degree of proinflammatory effect may be mitigated by adjusting one or more variables. For example the degree of proinflammatory effect of a given oligonucleotide may be mitigated by replacing any number of phosphorothioate intemucleoside linkages with phosphodiester intemucleoside linkages and thereby reducing the total number of phosphorothioate internucleoside es.
In certain embodiments, it would be desirable to reduce the number of phosphorothioate linkages, if doing so could be done without losing stability and without shifting the distribution from liver to kidney. For example, in certain embodiments, the number of phosphorothioate linkages may be reduced by replacing phosphorothioate linkages with odiester linkages. In such an embodiment, the antisense compound having fewer phosphorothioate linkages and more phosphodiester linkages may induce less proinflammatory reactions or no proinflammatory reaction. Although the the nse compound having fewer phosphoro- thioate linkages and more phosphodiester linkages may induce fewer proinflammatory reactions, the antisense compound having fewer phosphorothioate linkages and more phosphodiester linkages may not accumulate in the liver and may be less efficacious at the same or similar dose as ed to an antisense compound having more phosphorothioate linkages. In certain embodiments, it is ore desirable to design an antisense compound that has a ity of phosphodiester bonds and a plurality of orothioate bonds but which also possesses stability and good distribution to the liver.
In certain embodiments, conjugated antisense compounds accumulate more in the liver and less in the kidney than ugated counterparts, even when some of the phosporothioate linkages are replaced with less ammatory odiester intemucleoside linkages. In certain embodiments, conjugated nse compounds accumulate more in the liver and are not excreted as much in the urine compared to its unonjugated counterparts, even when some of the phosporothioate linkages are replaced with less proinflammatory phosphodiester intemucleoside linkages. In certain embodiments, the use of a conjugate allows one to design more potent and better tolerated antisense drugs. Indeed, in certain emobidments, conjugated antisense compounds have larger therapeutic indexes than unconjugated rparts. This allows the conjugated antisense nd to be administered at a higher te dose, because there is less risk of proinflammatory response and less risk of kidney toxicity. This higher dose, allows one to dose less frequently, since the clearance (metabolism) is expected to be similar. Further, because the compound is more potent, as described above, one can allow the concentration to go lower before the next dose without losing therapeutic activity, allowing for even longer periods between dosing.
In n embodiments, the inclusion of some orothioate linkages remains desirable. For example, the terminal linkages are vulnerable to exonucleoases and so in certain embodiments, those linkages are phosphorothioate or other modified linkage. Intemucleoside linkages linking two deoxynucleosides are vulnerable to endonucleases and so in certain ments those those linkages are phosphorothioate or other modif1ed linkage. Intemucleoside linkages between a modified nucleoside and a deoxynucleoside where the deoxynucleoside is on the 5’ side of the linkage deoxynucleosides are vulnerable to endonucleases and so in certain embodiments those those linkages are phosphorothioate or other modif1ed e.
Internucleoside es between two modified nucleosides of certain types and n a deoxynucleoside and a modified nucleoside of certain typ where the modified nucleoside is at the 5’ side of the linkage are sufficiently resistant to nuclease digestion, that the linkage can be phosphodiester.
In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 16 phosphorthioate linkages. In certain ments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 15 phosphorthioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 14 phosphorthioate linkages. In certain embodiments, the nse oligonucleotide of a conjugated antisense compound comprises fewer than 13 phosphorthioate linkages. In certain embodiments, the antisense ucleotide of a conjugated antisense compound comprises fewer than 12 phosphorthioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense nd comprises fewer than 11 phosphorthioate linkages. In certain embodiments, the antisense ucleotide of a ated antisense nd comprises fewer than 10 phosphorthioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound ses fewer than 9 phosphorthioate linkages. In certain embodiments, the nse oligonucleotide of a conjugated antisense compound comprises fewer than 8 phosphorthioate linkages.
In certain ments, antisense nds comprsing one or more conjugae group bed herein has increased ty and/or potency and/or tolerability compared to a parent antisense nd lacking such one or more conjugate group. Accordingly, in certain embodiments, attachment of such conjugate groups to an oligonucleotide is desirable. Such conjugate groups may be attached at the 5’-, and/or 3’- end of an oligonucleotide. In certain instances, attachment at the 5’-end is synthetically desireable.
Typically, oligonucleietides are synthesized by attachment of the 3’ terminal nucleoside to a solid support and sequential coupling of nucleosides from 3’ to 5’ using techniques that are well known in the art. ingly if a conjugate group is desred at the 3’-terminus, one may (I) attach the conjugate group to the 3’-terminal nucleoside and attach that conjugated nucleoside to the solid support for subsequent preparation of the ucleotide or (2) attach the ate group to the 3’-terminal nucleoside of a completed oligonucleotide after synthesis. Niether of these approaches is very nt and thus both are costly. In particular, attachment of the conjugated nucleoside to the solid support, while trated in the Examples herein, is an inefficient process. In certain embodiments, attaching a conjugate group to the 5’-terminal nucleoside is tically easier than attachment at the 3’-end. One may attach a non-conjugated 3’ terminal nucleoside to the solid support and prepare the ucleotide using standard and well characterized reastions. One then needs only to attach a 5’nucleoside having a conjugate group at the final coupling step.
In certain embodiments, this is more efficient than attaching a conjugated nucleoside ly to the solid support as is typically done to prepare a 3’-conjugated oligonucleotide. The Examples herein demonstrate attachment at the 5’-end. In on, certain conjugate groups have synthetic advantages. For Example, certain conjugate groups comprising phosphorus linkage groups are synthetically r and more efficiently ed than other conjugate , including conjugate groups reported previously (e.g., WO/2012/037254).
In certain embodiments, conjugated antisense compounds are administered to a subject. In such embodiments, antisense compounds comprsing one or more ae group described herein has increased activity and/or potency and/or tolerability compared to a parent antisense compound lacking such one or more conjugate group. Without being bound by mechanism, it is believed that the conjugate group helps with distribution, ry, and/or uptake into a target cell or tissue. In n embodiments, once inside the target cell or tissue, it is desirable that all or part of the conjugate group to be d to releas the active oligonucleitde. In n embodiments, it is not necessary that the entire conjugate group be cleaved from the oligonucleotide. For example, in Example 20 a conjugated oligonucleotide was administered to mice and a number of different chemical s, each comprising a different portion of the conjugate group remaining on the oligonucleotide, were detected (Table 23a). Thisconjugated antisense compound demonstrated good potency (Table 23). Thus, in certain embodiments, such metabolite profile of multiple partial cleavage of the conjugate group does not interfere with activity/potency. Nevertheless, in certain embodiments it is desirable that a prodrug (conjugated oligonucleotide) yield a single active compound. In certain instances, if multiple forms of the active nd are found, it may be necessary to determine relative amounts and activities for each one. In certain embodiments where regulatory review is required (e.g., USFDA or counterpart) it is desirable to have a single (or predominantly single) active species. In certain such embodiments, it is desirable that such single active species be the antisense oligonucleotide lacking any portion of the conjugate group. In certain embodiments, conjugate groups at the 5’-end are more likely to result in complete metabolism of the conjugate group. Without being bound by mechanism it may be that nous enzymes responsible for metabolism at the 5’ end (e.g., 5’ nucleases) are more active/efficient than the 3’ counterparts.
In certain embodiments, the specific conjugate groups are more le to metabolism to a single active species. In certain embodiments, certain conjugate groups are more amenable to lism to the oligonucleotide.
D. Antisense In certain embodiments, oligomeric compounds of the t invention are antisense compounds.
In such embodiments, the oligomeric compound is complementary to a target nucleic acid. In certain embodiments, a target nucleic acid is an RNA. In n embodiments, a target nucleic acid is a non-coding RNA. In certain ments, a target nucleic acid encodes a protein. In certain embodiments, a target nucleic acid is selected from a mRNA, a pre-mRNA, a microRNA, a ding RNA, including small non- coding RNA, and a promoter-directed RNA. In certain embodiments, oligomeric compounds are at least partially complementary to more than one target nucleic acid. For example, oligomeric compounds of the present invention may be microRNA mimics, Which typically bind to multiple targets.
In n embodiments, antisense compounds comprise a portion having a nucleobase sequence at least 70% complementary to the nucleobase ce of a target c acid. In certain embodiments, antisense compounds comprise a portion having a nucleobase sequence at least 80% complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, antisense compounds comprise a portion having a base sequence at least 90% complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, nse nds comprise a portion having a nucleobase sequence at least 95% complementary to the nucleobase sequence of a target nucleic acid. In n embodiments, nse compounds comprise a portion having a nucleobase sequence at least 98% mentary to the nucleobase ce of a target nucleic acid. In certain embodiments, antisense compounds comprise a portion having a nucleobase sequence that is 100% complementary to the nucleobase ce of a target nucleic acid. In certain embodiments, antisense compounds are at least 70%, 80%, 90%, 95%, 98%, or 100% complementary to the nucleobase sequence of a target nucleic acid over the entire length of the antisense compound.
Antisense mechanisms include any mechanism involving the hybridization of an oligomeric compound With target nucleic acid, Wherein the hybridization results in a biological effect. In certain embodiments, such hybridization results in either target nucleic acid degradation or occupancy With concomitant inhibition or stimulation of the cellular machinery involving, for example, ation, transcription, or polyadenylation of the target nucleic acid or of a nucleic acid With Which the target nucleic acid may otherwise interact.
One type of nse mechanism ing degradation of target RNA is RNase H mediated antisense. RNase H is a cellular endonuclease Which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds Which are “DNA-like” elicit RNase H activity in mammalian cells. Activation of RNase H, ore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of ke oligonucleotide-mediated inhibition of gene expression.
Antisense mechanisms also include, without limitation RNAi mechanisms, which utilize the RISC pathway. Such RNAi isms include, without limitation siRNA, ssRNA and NA mechanisms.
Such isms include creation of a microRNA mimic and/or an anti-microRNA.
Antisense mechanisms also include, without limitation, mechanisms that hybridize or mimic non- coding RNA other than NA or mRNA. Such non-coding RNA includes, but is not limited to promoter-directed RNA and short and long RNA that effects transcription or translation of one or more nucleic acids.
In certain embodiments, ucleotides comprising ates described herein are RNAi compounds. In certain embodiments, oligomeric ucleotides comprising conjugates described herein are ssRNA compounds. In certain embodiments, oligonucleotides comprising conjugates described herein are paired with a second oligomeric compound to form an siRNA. In certain such embodiments, the second oligomeric compound also comprises a conjugate. In certain embodiments, the second oligomeric compound is any modified or f1ed nucleic acid. In certain embodiments, the oligonucleotides comprising conjugates described herein is the antisense strand in an siRNA compound. In certain embodiments, the oligonucleotides comprising conjugates described herein is the sense strand in an siRNA compound. In embodiments in which the conjugated oligomeric compound is double-stranded siRnA, the conjugate may be on the sense strand, the antisense strand or both the sense strand and the antisense strand.
C. Apolipoprotein 1a) gapog an In certain embodiments, conjugated antisense compounds target any apo(a) nucleic acid. In certain embodiments, the target nucleic acid encodes an apo(a) target protein that is ally nt. In such embodiments, tion of the target nucleic acid results in clinical benefit.
The targeting process usually includes determination of at least one target region, segment, or site within the target nucleic acid for the nse interaction to occur such that the desired effect will result.
In certain ments, a target region is a structurally def1ned region of the nucleic acid. For example, in certain such embodiments, a target region may encompass a 3’ UTR, a 5’ UTR, an exon, an intron, a coding region, a translation initiation region, translation termination region, or other def1ned nucleic acid region or target segment.
In certain embodiments, a target segment is at least about an 8-nucleobase portion of a target region to which a conjugated antisense compound is targeted. Target segments can e DNA or RNA sequences that comprise at least 8 consecutive nucleobases from the 5'-terminus of one of the target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5'-terminus of the target segment and continuing until the DNA or RNA comprises about 8 to about 30 nucleobases). Target segments are also ented by DNA or RNA sequences that comprise at least 8 consecutive nucleobases from the 3'-terminus of one of the target segments (the remaining nucleobases being a consecutive h of the same DNA or RNA beginning immediately downstream of the 3'-terminus of the target segment and continuing until the DNA or RNA comprises about 8 to about 30 nucleobases). Target ts can also be represented by DNA or RNA sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of a target t, and may extend in either or both directions until the conjugated nse compound comprises about 8 to about 30 nucleobases.
In certain embodiments, antisense compounds targeted to an apo(a) nucleic acid can be modified as described herein. In certain embodiments, the antisense compounds can have a modified sugar moiety, an unmodified sugar moiety or a mixture of modified and unmodified sugar moieties as described herein. In certain embodiments, the antisense compounds can have a modified intemucleoside linkage, an unmodified cleoside linkage or a mixture of modified and unmodified intemucleoside linkages as described herein. In certain embodiments, the antisense compounds can have a modified nucleobase, an unmodified nucleobase or a mixture of modified and unmodified nucleobases as described herein. In certain embodiments, the antisense compounds can have a motif as described herein.
In certain ments, antisense compounds targeted to apo(a) nucleic acids can be conjugated as described herein.
One apo(a) protein is linked via a de bond to a single apolipoprotein B (apoB) protein to form a lipoprotein(a) (Lp(a)) le. The apo(a) n shares a high degree of homology with plasminogen particularly within the kringle IV type 2 repetitive domain. It is thought that the kringle repeat domain in apo(a) may be responsible for its rombotic and brinolytic properties, potentially enhancing atherosclerotic ssion. Apo(a) is transcriptionally regulated by IL-6 and in studies in rheumatoid arthritis ts treated with an IL-6 inhibitor izumab), plasma levels were reduced by 30% after 3 month treatment. Apo(a) has been shown to preferentially bind ed phospholipids and potentiate vascular inflammation. Further, studies suggest that the Lp(a) particle may also stimulate endothelial permeability, induce plasminogen activator inhibitor type-l sion and activate hage interleukin-8 secretion. Importantly, recent genetic association s ed that Lp(a) was an independent risk factor for myocardial infarction, stroke, peripheral vascular disease and abdominal aortic aneurysm. Further, in the Precocious Coronary Artery Disease (PROCARDIS) study, Clarke et al. described robust and independent associations n coronary heart disease and plasma Lp(a) concentrations. Additionally, Solfrizzi et al., suggested that increased serum Lp(a) may be linked to an increased risk for Alzheimer’s Disease (AD).
Antisense compounds targeting apo(a) have been previously disclosed in WOZOOS/OOOZOl and US2010- WO 79625 0331390, herein incorporated by reference in its entirety. An antisense oligonucleobase targeting Apo(a), ISIS-APOARX, was assessed in a Phase I clinical trial to study it’s safety profile.
Certain Conjugated Antisense Compounds Targeted to an Apo(a) Nucleic Acid In certain ments, conjugated antisense compounds are targeted to an Apo(a) nucleic acid having the sequence of GENBANK® Accession No. NM_005577.2, incorporated herein as SEQ ID NO: 1; GENBANK Accession No. NT_007422. 12 truncated from nucleotides 3230000 to 3380000, incorporated herein as SEQ ID NO: 2; GENBANK ion No. NT_025741.15 truncated from nucleotides 65120000 to 65258000, designated herein as SEQ ID NO: 3; and GENBANK Accession No. NM_005577.1, incorporated herein as SEQ ID NO: 4. In certain such ments, a conjugated antisense compound is at least 90%, at least 95%, or 100% complementary to any of the nucleobase ces of SEQ ID NOs: 1-4.
In certain embodiments, a conjugated antisense compound targeted to any of the nucleobase ces of SEQ ID NOs: 1-4 comprises an at least 8 consecutive nucleobase sequence selected from the nucleobase sequence of any of SEQ ID NOs: 12-130, 133, 134. In certain ments, a conjugated antisense compound targeted to any of SEQ ID NOs: 1-4 ses a nucleobase sequence selected from the nucleobase sequence of any of SEQ ID NOs: 12-130, 133, 134.
Table A: Antisense Compounds targeted to Ap0(a) SEQ ID NO: 1 Target Start ,_ , . SEQ ID ISIS N0 Sequence (5 3 ) Motlf Site NO 494372 3901 TGCTCCGTTGGTGCTTGTTC eeeeeddddddddddeeeee 58 494283 1323 TCTTCCTGTGACAGTGGTGG eeeeeddddddddddeeeee 26 2294 3320 85 494284 —13;; TTCTTCCTGTGACAGTGGTG eeeeeddddddddddeeeee 27 3321 87 494286 1613 GGTTCTTCCTGTGACAGTGG dddddddddeeeee 29 1955 2297 494301 T CGACTATGCGAGTGTGGTGT eeeeeddddddddddeeeee 3 8 2014/036460 1312 1654 1996 2338 2680 3022 1313 494302 —133: CCGACTATGCGAGTGTGGTG eeeeeddddddddddeeeee 39 2339 2681 3023 Ap0(a) Therapeutic Indications In certain embodiments, the invention provides methods for using a conjugated antisense compound targeted to an apo(a) nucleic acid for modulating the expression of apo(a) in a subject. In n embodiments, the expression of apo(a) is reduced.
In certain ments, provided herein are methods of treating a subject comprising administering one or more pharmaceutical itions as described herein. In certain embodiments, the invention provides s for using a conjugated antisense compound ed to an apo(a) nucleic acid in a pharmaceutical ition for treating a subject. In certain embodiments, the individual has an apo(a) related disease. In certain embodiments, the individual has an Lp(a) related disease. In certain embodiments, the individual has an inflammatory, cardiovascular and/or a metabolic disease, disorder or condition.
In n embodiments, the subject has an inflammatory, cardiovascular and/or metabolic disease, disorder or condition.
In certain ments, the cardiovascular diseases, disorders or conditions include, but are not limited to, aortic stenosis, aneurysm (e. g., abdominal aortic aneurysm), , arrhythmia, atherosclerosis, cerebrovascular disease, coronary artery disease, coronary heart e, dyslipidemia, hypercholesterolemia, hyperlipidemia, hypertension, hypertriglyceridemia, myocardial infarction, peripheral vascular disease (e. g., peripheral artery disease), stroke and the like.
In certain embodiments, the compounds targeted to apo(a) bed herein modulate physiological markers or phenotypes of the cardiovascular e, disorder or condition. For example, administration of the compounds to animals can decrease LDL and cholesterol levels in those animals compared to untreated animals. In certain embodiments, the modulation of the physiological markers or phenotypes can be associated with tion of apo(a) by the compounds.
In certain embodiments, the physiological markers of the cardiovascular disease, disorder or condition can be quantifiable. For example, LDL or cholesterol levels can be measured and quantified by, for 2014/036460 example, standard lipid tests. For such markers, in n embodiments, the marker can be decreased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or arange defined by any two of these values.
Also, ed herein are methods for preventing, treating or ameliorating a symptom ated with the cardiovascular disease, disorder or condition in a subject in need thereof. In certain embodiments, provided is a method for reducing the rate of onset of a symptom associated with the cardiovascular disease, disorder or condition. In certain embodiments, provided is a method for reducing the severity of a symptom associated with the cardiovascular disease, disorder or condition. In such embodiments, the s comprise stering a therapeutically effective amount of a compound targeted to an apo(a) nucleic acid to an individual in need thereof The cardiovascular disease, er or condition can be characterized by numerous physical symptoms. Any symptom known to one of skill in the art to be associated with the cardiovascular disease, disorder or condition can be prevented, treated, ameliorated or otherwise modulated with the compounds and methods described herein. In certain embodiments, the symptom can be any of, but not limited to, angina, chest pain, ess of breath, palpitations, weakness, dizziness, , sweating, ardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the n or fever.
In certain embodiments, the metabolic es, disorders or conditions include, but are not d to, hyperglycemia, betes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome and diabetic dyslipidemia.
In certain embodiments, compounds targeted to apo(a) as described herein modulate physiological markers or phenotypes of the metabolic e, disorder or condition. For example, administrion of the compounds to animals can decrease glucose and insulin resistance levels in those animals compared to ted animals. In certain embodiments, the modulation of the physiological s or phenotypes can be associated with inhibition of apo(a) by the compounds.
In certain embodiments, physiological markers of the metabolic disease, disorder or condition can be quantifiable. For example, glucose levels or insulin resistance can be measured and quantified by standard tests known in the art. For such markers, in certain embodiments, the marker can be decreased by about 5, 10, , 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two ofthese values. In another example, n sensitivity can be measured and quantified by rd tests known in the art. For such markers, in certain embodiments, the marker can be increase by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two ofthese values.
Also, provided herein are methods for preventing, treating or ameliorating a symptom associated with the metabolic disease, disorder or condition in a subject in need f In certain embodiments, provided is a method for reducing the rate of onset of a symptom associated with the lic disease, disorder or condition. In certain embodiments, provided is a method for reducing the severity of a symptom associated with the metabolic disease, disorder or condition. In such embodiments, the methods comprise administering a therapeutically ive amount of a nd targeted to an apo(a) nucleic acid to an individual in need thereof.
The metabolic disease, disorder or condition can be characterized by numerous physical symptoms.
Any m known to one of skill in the art to be associated with the metabolic disease, disorder or condition can be prevented, treated, ameliorated or otherwise modulated with the compounds and methods described . In certain embodiments, the symptom can be any of, but not limited to, excessive urine production (polyuria), excessive thirst and increased fluid intake (polydipsia), blurred vision, unexplained weight loss and lethargy.
In certain ments, the inflammatory diseases, disorders or conditions include, but are not limited to, aortic stenosis, ry artey disease (CAD), Alzheimer’s Disease and thromboembolic diseases, disorder or conditions. n thromboembolic es, disorders or conditions include, but are not limited to, , thrombosis, myocardial infarction and peripheral vascular disease.
In n embodiments, the compounds targeted to apo(a) described herein modulate physiological markers or phenotypes of the inflammatory disease, disorder or condition. For example, administration of the compounds to animals can decrease inflammatory cytokine or other atory markers levels in those animals ed to untreated animals. In certain ments, the modulation of the physiological markers or phenotypes can be associated with inhibition of apo(a) by the compounds.
In certain embodiments, the physiological s of the inflammatory disease, disorder or condition can be quantifiable. For example, cytokine levels can be measured and quantified by standard tests known in the art. For such markers, in certain embodiments, the marker can be decreased by at least about 5%, 10%, %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or a range defined by any two of these values.
Also, provided herein are methods for preventing, treating or ameliorating a symptom associated with the inflammatory disease, disorder or condition in a subject in need thereof In certain embodiments, ed is a method for reducing the rate of onset of a symptom associated with the inflammatory disease, disorder or condition. In certain ments, provided is a method for reducing the severity of a m ated with the inflammatory disease, disorder or condition. In such embodiments, the methods comprise administering a therapeutically effective amount of a compound targeted to an apo(a) nucleic acid to an dual in need thereof.
In certain embodiments, provided are methods of treating an individual with an apo(a) related disease, disorder or condition comprising administering a therapeutically effective amount of one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has elevated apo(a) levels. In certain embodiments, provided are methods of treating an individual with an Lp(a) related disease, disorder or condition comprising administering a therapeutically effective amount of one or more pharmaceutical itions as described herein. In certain embodiments, the individual has elevated Lp(a) 2014/036460 levels. In certain ments, the individual has an inflammatory, cardiovascular and/or metabolic disease, disorder or condition. In n ments, administration of a therapeutically effective amount of an antisense compound targeted to an apo(a) c acid is accompanied by monitoring of apo(a) or Lp(a) levels. In certain embodiments, administration of a eutically effective amount of an antisense compound targeted to an apo(a) nucleic acid is accompanied by monitoring of markers of inflammatory, cardiovascular and/or metabolic disease, or other disease process ated With the expression of apo(a), to determine an dual’s response to the antisense compound. An individual’s response to administration of the antisense compound targeting apo(a) can be used by a physician to determine the amount and duration of eutic intervention with the compound.
In certain embodiments, administration of an antisense compound targeted to an apo(a) nucleic acid results in reduction of apo(a) expression by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or a range defined by any two of these values. In certain embodiments, apo(a) sion is reduced to at least S 100 mg/dL, S 90 mg/dL, S 80 mg/dL, S 70 mg/dL, S 60 mg/dL, S 50 mg/dL, S 40 mg/dL, S 30 mg/dL, S20 mg/dL or S 10 mg/dL.
In certain ments, administration of an antisense compound targeted to an apo(a) c acid results in reduction of Lp(a) expression by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or a range defined by any two of these values. In certain embodiments, Lp(a) expression is reduced to at least S 200 mg/dL, S 190 mg/dL, S 180 mg/dL, S 175 mg/dL, S 170 mg/dL, S 160 mg/dL, S 150 mg/dL, S 140 mg/dL, S 130 mg/dL, S 120 mg/dL, S 110 mg/dL, S 100 mg/dL, S 90 mg/dL, S 80 mg/dL, S 70 mg/dL, S 60 mg/dL, S 55 mg/dL, S 50 mg/dL, S 45 mg/dL, S 40 mg/dL, S 35 mg/dL, S 30 mg/dL, S 25 mg/dL, S 20 mg/dL, S 15 mg/dL, or S 10 mg/dL.
In certain embodiments, the ion provides methods for using a conjugated nse compound targeted to an apo(a) nucleic acid in the preparation of a medicament. In certain embodiments, pharmaceutical compositions comprising a conjugated antisense compound targeted to apo(a) are used for the preparation of a medicament for treating a patient suffering or susceptible to an inflammatory, cardiovascular and/or a metabolic disease, disorder or condition.
Ap0(a) Treatment tions Certain subjects with high Lp(a) levels are at a significant risk of various diseases (Lippi et al., Clinica Chimica Acta, 2011, 412:797-801; Solfrizz et al.). In many subjects with high Lp(a) levels, current ents cannot reduce their Lp(a) levels to safe levels. Apo(a) plays an important role in the formation of Lp(a), hence reducing apo(a) can reduce Lp(a) and prevent, treat or ameliorate a disease associated with Lp(a).
In certain embodiments, treatment with the compounds and methods disclosed herein is indicated for a human animal with elevated apo(a) levels and/or Lp(a) levels. In certain embodiments, the human has apo(a) levels 2 10 mg/dL, Z 20 mg/dL, Z 30 mg/dL, Z 40 mg/dL, Z 50 mg/dL, Z 60 mg/dL, Z 70 mg/dL, Z 80 mg/dL, Z 90 mg/dL or Z 100 mg/dL. In certain embodiments, the human has Lp(a) levels 2 10 mg/dL, Z mg/dL, Z 20 mg/dL, Z 25 mg/dL, Z 30 mg/dL, Z 35 mg/dL, Z 40 mg/dL, Z 50 mg/dL, Z 60 mg/dL, Z 70 mg/dL, Z 80 mg/dL, Z 90 mg/dL, Z 100 mg/dL, Z 110 mg/dL, Z 120 mg/dL, Z 130 mg/dL, Z 140 mg/dL, Z 150 mg/dL, Z 160 mg/dL, Z 170 mg/dL, Z 175 mg/dL, Z 180 mg/dL, Z 190 mg/dL, Z 200 mg/dL.
D. Certain Pharmaceutical itions In certain embodiments, the present disclosure provides pharmaceutical compositions comprising one or more nse compound. In certain embodiments, such pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain ments, a pharmaceutical ition comprises a sterile saline on and one or more antisense compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and sterile water. In certain embodiments, a pharmaceutical composition ts of one or more antisense compound and sterile water.
In certain embodiments, the sterile saline is ceutical grade water. In n embodiments, a ceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition ts of one or more antisense compound and sterile phosphate-buffered saline (PBS). In n embodiments, the sterile saline is pharmaceutical grade PBS.
In certain embodiments, antisense compounds may be admixed With pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations.
Compositions and methods for the formulation of pharmaceutical compositions depend on a number of ia, including, but not limited to, route of administration, extent of disease, or dose to be administered.
Pharmaceutical compositions comprising antisense compounds ass any pharmaceutically acceptable salts, , or salts of such esters. In certain embodiments, pharmaceutical compositions comprising antisense compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other ivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
A g can include the incorporation of additional nucleosides at one or both ends of an oligonucleotide Which are cleaved by endogenous nucleases Within the body, to form the active antisense oligonucleotide.
Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the c acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA xes With mono- or poly-cationic lipids are formed Without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase bution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
In certain embodiments, pharmaceutical compositions ed herein se one or more modified oligonucleotides and one or more ents. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, n, lactose, amylase, magnesium stearate, talc, silicic acid, s paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system.
Examples of delivery s include, but are not limited to, liposomes and ons. Certain delivery systems are useful for preparing certain pharmaceutical itions including those comprising hydrophobic compounds. In certain embodiments, certain organic ts such as dimethylsulfoxide are used.
In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue- specific delivery les designed to deliver the one or more pharmaceutical agents of the present sure to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated With a tissue-specific antibody.
In certain embodiments, a pharmaceutical composition provided herein comprises a co-solvent system. Certain of such co-solvent s comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, Which is a solution of absolute ethanol comprising 3% W/V benzyl alcohol, 8% W/V of the nonpolar surfactant Polysorbate 80““ and 65% W/V polyethylene glycol 300. The proportions of such vent systems may be varied considerably Without significantly altering their solubility and toxicity characteristics.
Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80““; the fraction size of hylene glycol may be varied; other biocompatible polymers may replace polyethylene , e. g., polyvinyl pyrrolidone; and other sugars or ccharides may substitute for dextrose.
In certain embodiments, a pharmaceutical composition provided herein is ed for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration.
In certain embodiments, a pharmaceutical composition is prepared for administration by ion (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's on, Ringer's solution, or physiological saline buffer. In certain ments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives).
In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain ceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as ding, stabilizing and/or sing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the sion, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such sions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated ons.
In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration.
In certain of such embodiments penetrants appropriate to the barrier to be ted are used in the formulation. Such ants are generally known in the art.
In certain embodiments, a pharmaceutical composition ed herein comprises an oligonucleotide in a therapeutically effective amount. In certain embodiments, the eutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to g the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
In certain embodiments, one or more ed oligonucleotide provided herein is formulated as a prodrug. In certain embodiments, upon in viva administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an oligonucleotide. In certain embodiments, gs are useful because they are easier to administer than the corresponding active form.
For example, in certain instances, a g may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In n instances, a prodrug may have improved lity compared to the corresponding active form. In n embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a g is an ester.
In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain ces the carboxylic acid containing compound is the corresponding active form. In n embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form.
In certain embodiments, the present disclosure provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a rodent. In certain embodiments, the animal is a primate. In certain embodiments, the animal is a non-human primate. In certain embodiments, the animal is a human.
In certain embodiments, the present disclosure provides methods of administering a pharmaceutical ition comprising an oligonucleotide of the present disclosure to an animal. Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e. g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical intrathecals are stered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect (e.g., into the liver).
Nonlimiting disclosure and incorporation by reference While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain ments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the nces, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety. gh the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of al modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in n instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2’-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2’-OH for the natural 2’-H of DNA) or as an RNA having a modified base (thymine (methylated ) for l uracil of RNA).
Accordingly, nucleic acid ces provided herein, including, but not limited to those in the ce listing, are intended to ass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligonucleotide having the nucleobase sequence TCG” encompasses any oligonucleotides having such nucleobase ce, whether modified or unmodified, including, but not limited to, such nds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligonucleotides having other modified bases, such as GAUCG,” wherein meC indicates a cytosine base comprising a methyl group at the 5-position.
EXAMPLES The ing examples illustrate certain embodiments of the present disclosure and are not limiting.
Moreover, where specific embodiments are provided, the ors have contemplated generic application of those c ments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular ffinity modification appears at a ular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.
Example 1: General Method for the Preparation of Phosphoramidites, Compounds 1, 1a and 2 O O BX ’BX DMTOWBx Q’ \c S "1 /\/OMG H3C S; \: o O | o o o x P\ Px 1 l a 2 Bx is a heterocyclic base; Compounds 1, 1a and 2 were prepared as per the procedures well known in the art as described in the specification herein (see Seth et al., Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J. Org. Chem., 2010, 75(5), 1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554); and also see published PCT International Applications (, , WO2010/036698, /143369, WO 2009/006478, and ), and US patent 7,569,686).
Example 2: Preparation of Compound 7 AcOOAc AcOOAc O 0 AC0 WK 0 HO TMSOTf, 50 °C O/\© 5 AC0 OAc —» o CICHzCHzCI N\ AcHN TMSOTf, DCE 3 (93%) 4 (66%) AcOOAc AcOOAc ACO¥/ \/\/\”/ VG O O H2/Pd O OH 0 o —» ACO¥/ V\/\”/ MeOH A HNC 0 AcHN o (95%) 6 7 Compounds 3 (2-acetamido-l,3,4,6-tetraacetyldeoxy-B-Dgalactopyranose or galactosamine pentaacetate) is commercially available. Compound 5 was prepared according to published procedures (Weber et al., J. Med. Chem, 1991, 34, 2692). 2014/036460 Example 3: Preparation of Compound 11 EtO\n/\\ NC/\\ HOHO O EtO /\/CN 9 HCI, EtOH NH —>2 NC/\/O NH —>2 aq. KOH Reflux, rt, WEI/DQ7W20 EtO HO 1 4-dioxane, O (56%) 8 (40%) NC\) 10 ON 11 Compounds 8 and 9 are commercially available.
Example 4: Preparation of Compound 18 EtO EtO W n O O O benzylchloroformate, EtO NAG LIOH, H20_ o NH Dioxane, Na2C03 O 2 —> BOW/V Dioxane O (86") EtO O 030 ( 910/0) ON N 12 11 H H N N HO >(FE“ WO F V}Ho OAQ HBTU DIEA DMF HO o (69%) N + iN/\/\0'quo O 13 AcOOAc H2N H O \/\/N o OH \n/\\ A00¥/W 17 H o o AcHN o H 2 \/\/N\n/\/O\%7HJOK GAE: HBTU,D|EA, HOBt 95% O O DMF 16 (64%) HQNMH O AcOOAc O H H ACHN AcOOAc OMNWNfiO 0 OVVYHWHYVO A00 NAG AcHN do O O O AcOOAc H HN’CO 0 Nfl ACO O\/\/\n/ AcHN 18 2014/036460 Compound 11 was prepared as per the procedures illustrated in Example 3. Compound 14 is commercially available. Compound 17 was prepared using similar procedures reported by Rensen et al., J.
Med. Chem, 2004, 47, 5798-5808.
Example 5: Preparation of Compound 23 O O o H3co)L(‘/):LOH 21 oj/ b 1TBDMScI TBDMSO |EA N DMF, ImIdazode, rt (95 %) DMF, rt (65%) HO —> 2. Pd/C, H2, MeOH, rt 2. TEA.3HF TEA THF -,,OH 87% OTBDMS (7é%) DMTO HO 0 o 1 DMTCI pyr rt (7)5% N OCH3 —> 2. LiOH, Dioxane (97%) a 22 Compounds 19 and 21 are commercially available. 2014/036460 Example 6: Preparation of Compound 24 ACOOAC 1. H2, Pd/C, MeOH (93%) AcowovaHWHWI/Vog’hlko 2. HBTU, DIEA, DMF (76%) /«T:j O O /ODMT H HOJ%afi\ f W4 ”9 23 O 18 AnI—IN Compounds 18 and 23 were prepared as per the procedures illustrated in Examples 4 and 5. 2014/036460 Example 7: Preparation of Compound 25 AcOOAc O H H ACO \/\/\n/N\/\/N\EO ACHN O AcOOAc O /ODMT H O O =_ 1. Succinic anhydride,DMAP, DCE ACO $:ro OWNWHTVogimwNQ ACHN O O O 2. DMF, HBTU, EtN(iPr)2, PS-SS AcOOAc HN”: AcOmOMNflH O AcHN AcOOAc O H o H 0 A00 \/\/\n/N\/\/ K ACHN o ODMT AcOOAc o O O ’ o H H AcO W \/\/N\n/\/ %H0 NWa N NH AcHN o o o ( o AcOOAc HN AcHN Compound 24 was prepared as per the procedures illustrated in Example 6.
Example 8: Preparation of Compound 26 AcOOAc O H o H 0 A00 \/\/\n/N\/\/ K ACHN O AcOOAc o /ODMT H O O OWYNWNYVQgiuwNQH O :__ AcO Phosphitylation ACHN O O O AcOOAc H HN’CO o N\/\/ A00 O\/\/\n/ AcHN AcOOAc O H o H 0 A00 \/\/\n/N\/\/ K ACHN O AcOOAc O /ODMT Agog/O H o H o O 0%NWNQ=__ WYN\/\/N\g/V H s ACHN O O < O HN “Owe/P‘NaPr» HN\/\/ O 0 OR Compound 24 is prepared as per the ures illustrated in Example 6.
Example 9: General preparation of conjugated ASOs comprising 3-1 at the 3’ terminus, Compound 29 ACOOAC O H o H o ACO \/\/\n/N\/\/ K ACHN O ACOOA 0 .5 ACO VVT \/\/NY» %H0 NWa N NH ACHN o o o 1. DCA, DCM ACOOAC HN’: H\/\/ O 2. DCI,N1\/H, ACN O O\/\/\n/N Phosphoramidite DNA/RNA ACO building block 1 automated synthesizer ACHN 3. Capping 4. t-BuOOH DMTOAfi, ACOOAC O H o H ('3 CN ACO /VENo 0=P—o/\’ ACHN O ACOOA H\/\/ ACHN O 0QOHN 1. DCA, DCM 2. DCI, N1VH, ACN ACOOAC H O Phosphoramidite DNA/RNA O Ole/Nx/V building bIOCk 1a automated synthesizer AcO 3- Capping 27 4. t-BuOOH ACHN DMTOWBX ('3‘. beMe \/CN ACOOAC o H H o (I) ACHN O ACOOACO o P O O : ACO OW\H/N\/\/N M’lLNQ NH H a ACHN \CIDI/V O 1. DCA, DCM AcOOAc H\/\/ o 2. DCI, NMI, ACN O Phosphoramidite DNA/RNA ACO O\/\/\n/N automated synthesize O ng bIOCkS 28 3. Capping 4. xanthane hydride or t-BuOOH . Et3N/CH3CN (1:1) 6. Aqueous NH? (cleavage) (IDH OLIGO X=P\-O o o BX BX — Heterocycllc base._ VfOMe0 X=OorS | O=|={—o HOOH o H H o ('3 HO WY \/\/ K OzF|>-O ACHN O HOOH o ,0 H O O ’ O H HO WY WNYvogiMMNQ ACHN O O O HOOH H HN’CO HO OWY ACHN Wherein the protected GalNAc3-1 has the structure: E—Ff—o 0 «JANN Na HOOH o H H o 9 HO W\n/ \/\/ K O=F|"O' ACHN O HOOH o ,0 O O o H H 2' HO W WNYVOgiuwNQ ACHN o o o HOOH H HN’CO HO O\/\/\n/ ACHN The GalNAc3 cluster n of the conjugate group 3-1 (GalNAc3-1a) can be combined With any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc3-1a has the formula: HOOH O H o H o HO WYNW T; ”in, ACHN O HOOH HNW“ o o _,O HO WY 0 NW \n/V H 8 Na AcHN o o )3 OH The solid support bound protected GalNAc3-1, Compound 25, was prepared as per the procedures illustrated in Example 7. Oligomeric Compound 29 comprising GalNAc3-1 at the 3’ terminus was prepared using standard procedures in ted DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks, Compounds 1 and la were prepared as per the procedures illustrated in Example 1. The phosphoramidites illustrated are meant to be entative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare oligomeric compounds haVing a ermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described herein.
Such gapped oligomeric compounds can have ermined ition and base sequence as dictated by any given target. e 10: General preparation conjugated ASOs comprising GalNAc3-1 at the 5’ terminus, Compound 34 ODMT 1. Capping (Ac20,NMprr) 1. DCA,DCM OLIGO g géislgrcfiuOOHi := UNL—ODMT , 2. DCI, NMI, ACN O Phosphoramidite | 4. DCI, NMI, ACN Q\E UNL O-P~ /\/CN Phosphoramidite~ ~ _ O 1 building blocks DNARNA DNA/RNA 31 automated synthes1zer. automated synthesizer 1. g (A020, NMI, pyr). DMTO/\<:7’BX 2. t-BuOOH NC Q 3. DCA, DCM \/\o—1l> 4. DCI, NMI, ACN Phosphoramidite 26 OLIGO DNA/RNA X = O, or S automated synthesizer (I) BX = Heterocylic base Q UNL—O—R‘O/VCN AcOOAc O H o H o ACO \/\/\n/N\/\/ K AcHN 0 A00 OAC O /ODMT $0 H O o H 1" w\/\/N 0 NW wah N AcHN O O O NC\/\O/ROWE); AcOOAc Hfl” 0 0 CM 0‘“ NC\/\ I AcO O — O I'D—O AcHN OLIGO 1. Capping (A020, NMI, pyr) (I) 2. H _ _| /\/CN 3. Et3N:CH3CN (1:1 v/v) Q UNL O FPO 4. DCA, DCM . NH4, rt (cleavage) HOOH O H o H o HO \/\/\n/N\/\/ K AcHN O HoOH o xOH O O o H H HO WT WN 0 NW \H/V 8 N AcHN o o o - | O‘R O BX O Nfl 0 Q H0 [31/ 'O-ll3=O AcHN 34 The UnylinkerTM 30 is commercially available. Oligomeric Compound 34 comprising a GalNAc3-1 cluster at the 5’ us is prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks, Compounds l and la were prepared as per the procedures illustrated in e 1. The phosphoramidites rated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid t can be adjusted to prepare gapped oligomeric compounds as described herein. Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target.
Example 11: Preparation of Compound 39 A OOAc° 1. HoWfiko/D AcOOAc AC0 35 TMSOTf,DCE o NH2o 8 Ni‘ 2. H2/Pd, MeOH AcHN 36 A60 OAc AcO 1. H2,Pd/C,MeOH HBTU, DMF, EtN(iPr)2 o Compound 13 AcHN W” 2. HBTU DIEA DMF :ZVMWHOAc mo Compound 23 w9%“? NHAc AcHN 0 ODMT E Phosphitylation NHAc o O AGO p o 38 A00 OWNH ACHN ACO OAc A00 /ODMT WHWN AcHN 8 OMNqo Acogx/ACO OWN\H/\O/figiNH NC\/\O’Fl’NUP02 NHAc AcO p o NH 39 A00 w AcHN Compounds 4, 13 and 23 were prepared as per the procedures rated in Examples 2, 4, and 5.
Compound 35 is prepared using similar procedures published in Rouchaud et al., Eur. J. Org. Chem, 2011, 12, 2346-2353.
Example 12: Preparation of Compound 40 A00 OAc o /ODMT AcHN WHW 0A M“QOH Ac$53M“wW?“ NHAC 1. Succinic anhydride, DMAP, DCE A00 p - 0 NH 2. DMF,HBTU, EtN(iPr)2, PS-SS ACO WV8 AcHN ACO OAc 0 ODMT NHAc o 0 A00 W 40 o NH ACO w AcHN Compound 38 is ed as per the procedures illustrated in Example 11.
Example 13: Preparation of Compound 44 ACOOAC HBTU, DMF, EtN(iPr)2 o NH2 AcHN 36 V }NLo HOWfO 41 ACO OAc AcHN WWHN8 O O }N>=0H 1. H2, Pd/C,MeOH 2. HBTU,D|EA,DMF Op Compound 23 OAC 0 Acow WV8ACO O O NH ACHN ACO OAc AcHN 8 jZI\n/\\::NNMNam-I Phosphitylation O 43 o p A00 OVHWNH ACHN ACO OAc MHVo /ODMT AcHN 8 5 AcomowpNHOAcACO 44 ACHN Compounds 23 and 36 are prepared as per the procedures illustrated in es 5 and 11.
Compound 41 is prepared using similar procedures published in WO 2009082607.
Example 14: Preparation of Compound 45 A00 OAc Acog/OMHo {ODMT AcHN 8 o : O 43 A00 W 1. Succinic anhydride, DMAP, DCE AcO i?) OMNH AcHN 2. DMF, HBTU, EtN(iPr)2, Ps-ss ACO OAc AcOk/OMHo {ODMT AcHN 8 o : 0A0 45 A00 OWN“ ACHN Compound 43 is ed as per the ures illustrated in Example 13.
Example 15: Preparation of Compound 47 O DMTO HO >_ / o < > N 1. DMTCI, pyr 2. Pd/C, H2, MeOH HO: 46 Compound 46 is commercially available. 2014/036460 Example 16: Preparation of Compound 53 HBTU, EtN(iPr)2’ DMF H3COWNHZ O / H3COWNO 49 BZ/NH ,CBz H3COW\NO mN/CBZ . 1. TFA H 1.LIOH,MeOH 2- HBTU EtN(I'Pr)2 DMF 2. HBTU, EtN(iPr)2’DMF /CzB Compound 47 O \CBz ,CBz DMTO HN 1. H2,Pd/C [082 2_ tN(iPr)2,DMF Compound 17 How. N—mN NH N O H HN‘CBz OACOAc AcO 0/\/\)'|\NH NHAc $§%%oMN “N OHMNPIH‘OH p:(§:&,OMN“ ODMT NHAc Compounds 48 and 49 are commercially available. Compounds 17 and 47 are prepared as per the procedures illustrated in Examples 4 and 15.
Example 17: Preparation of Compound 54 NHAC A:E§::SL__O/A\v/“\v/fi\N 0 HM ...\OH N HN N NHAC O Ego“NH ODMT NHAC Phosphitylation OACOAc A00 /\/\)j\ 0 NH NHAC OAC 0 \/\ 0 HM ....0' CN 0 W N HN N A00 7 NHAC O OACOAC o ODMT A00 0 NHAC Compound 53 is prepared as per the procedures illustrated in Example 16.
Example 18: Preparation of Compound 55 0 Wk 0 NH NHAC OWJ\HN N HN N NHACO O A::C§ACO:83 O/\/\/uo—NH ODMT NHACO 1. ic anhydride, DMAP, DCE 2. DMF, HBTU, EtN(iPr)2, PS-SS 0 Wk 0 NH NHAC SSQLMW‘LH NHAC O AfigoI O/\/\/UO—NH ODMT NHACO Compound 53 is prepared as per the procedures illustrated in Example 16.
Example 19: General method for the preparation of conjugated ASOs comprising GalNAc3-1 at the 3’ position via solid phase techniques (preparation of ISIS 647535, 647536 and ) Unless otherwise stated, all reagents and solutions used for the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid support are used for incorporation nucleoside residues which include for example T, A, G, and InC residues. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for B-D-Z’-de0xyrib0nucle0side and 2’- MOE.
The ASO syntheses were performed on ABI 394 synthesizer (1-2 umol scale) or on GE Healthcare Bioscience AKTA oligopilot sizer (40-200 umol scale) by the phosphoramidite coupling method on an GalNAc3-1 loaded VIMAD solid t (110 umol/g, GuzaeV et al., 2003) packed in the column. For the 2014/036460 coupling step, the phosphoramidites were red 4 fold excess over the g on the solid support and phosphoramidite condensation was carried out for 10 min. All other steps followed standard protocols supplied by the manufacturer. A solution of 6% dichloroacetic acid in toluene was used for removing oxytrityl (DMT) group from 5 ’-hydroxyl group of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) in anhydrous CH3CN was used as activator during coupling step. Phosphorothioate linkages were introduced by sulfurization with 0.1 M solution of xanthane hydride in 1:1 pyridine/CH3CN for a contact time of 3 minutes.
A solution of 20% tert—butylhydroperoxide in CH3CN containing 6% water was used as an oxidizing agent to provide phosphodiester intemucleoside linkages with a contact time of 12 s.
After the desired sequence was assembled, the cyanoethyl phosphate protecting groups were deprotected using a 1:1 (v/v) e of ylamine and itrile with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %) and heated at 55 0C for 6 h.
The unbound ASOs were then filtered and the ammonia was boiled off The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 um, 2.54 x 8 cm, A = 100 mM ammonium acetate in 30% aqueous CH3CN, B = 1.5 M NaBr in A, 0- 40% of B in 60 min, flow 14 mL min-1, k = 260 nm). The residue was desalted by HPLC on a reverse phase column to yield the desired ASOs in an isolated yield of 15-30% based on the initial loading on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.
Antisense ucleotides not comprising a conjugate were synthesized using standard oligonucleotide synthesis procedures well known in the art.
Using these methods, three separate antisense compounds targeting ApoC III were prepared. As summarized in Table 17, below, each of the three antisense compounds targeting ApoC III had the same nucleobase sequence; ISIS 304801 is a 55 MOE gapmer having all phosphorothioate linkages; ISIS 647535 is the same as ISIS 304801, except that it had a GalNAc3-1 conjugated at its 3’end; and ISIS 647536 is the same as ISIS 647535 except that certain intemucleoside linkages of that compound are phosphodiester linkages. As further summarized in Table 17, two separate antisense compounds targeting SRB-l were synthesized. ISIS 440762 was a 22 cEt gapmer with all orothioate ucleoside es; ISIS 651900 is the same as ISIS 440762, except that it included a GalNAc3-1 at its 3’-end.
Table 17 Modified ASO targeting ApoC III and SRB-l CalCd Observed , , AesGesmCesTesTesmCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmCds TesTesTesAesTe 3 (£31880 1 Afi‘l’c 7165.4 7164.4 ISIS mCesTesTesmCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmCdsTesTesTesAesTeoAdo" APOC 647535 3_la -9239.5 9237.8 136 ISIS AesGeomCeoTeoTeomCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmCdsTeoTeoTesAesTeoAdo" APOC 647536 GalNAC3_la -9142.9 9140.8 136 44017862 TkskasAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTkska 46470 46464 - ISIS SR1} TkskasAdsGdsTdsmCdsAd5TdsGdsAdsmCdsTdsTkskaoAdoa-GalNAc3-la 672 1. 1 67 19.4 1 38 651900 Subscripts: “e” indicates 2’-MOE modified side; “d” indicates B-D-2’-deoxyribonucleoside; “k” indicates 6’-(S)-CH3 bicyclic nucleoside (e. g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “0” indicates odiester intemucleoside linkages (PO); and “o’” indicates -O-P(=O)(OH)-.
Superscriptm1ndicates66m” ' 5-methylcytosines. “GalNAc3-1” indicates a conjugate group having the structure shown previously in Example 9. Note that GalNAc3-1 comprises a cleavable adenosine which links the ASO to der of the conjugate, which is designated c3-1a.” This nomenclature is used in the above table to show the full nucleobase sequence, including the ine, which is part of the conjugate. Thus, in the above table, the sequences could also be listed as ending with “GalNAc3-1” with the “Ado” omitted. This convention of using the subscript 66a” to indicate the portion of a conjugate group lacking a cleavable nucleoside or cleavable moiety is used hout these Examples. This portion of a conjugate group lacking the cleavable moiety is referred to herein as a “cluster” or “conjugate cluster” or “GalNAc3 cluster.” In certain instances it is convenient to describe a conjugate group by separately providing its r and its cleavable moiety. e 20: Dose-dependent antisense inhibition of human ApoC III in huApoC III transgenic mice ISIS 304801 and ISIS 647535, each targeting human ApoC III and described above, were separately tested and evaluated in a dose-dependent study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.
Treatment Human ApoCIII transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow. Animals were acclimated for at least 7 days in the ch facility before initiation of the experiment. ASOs were prepared in PBS and sterilized by filtering through a 0.2 micron filter. ASOs were dissolved in 0.9% PBS for ion.
Human ApoC III transgenic mice were ed intraperitoneally once a week for two weeks with ISIS 304801 or 647535 at 0.08, 0.25. 0.75, 2.25 or 6.75 umol/kg or with PBS as a l. Each ent group consisted of 4 animals. Forty-eight hours after the administration of the last dose, blood was drawn from each mouse and the mice were sacrificed and tissues were collected.
ApoC [[1 mRNA is ApoC III mRNA levels in the mice’s livers were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. ApoC III mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of ApoC III mRNA levels for each treatment group, normalized to PBS-treated control and are denoted as “% PBS”. The half maximal effective dosage (ED 50) of each ASO is also presented in Table 18, below.
As illustrated, both antisense nds reduced ApoC III RNA relative to the PBS control.
Further, the antisense nd conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 ate (ISIS 304801).
Table 18 Effect ofASO treatment on ApoC III mRI\A levels in human ApoC III transgenic mice ($331.) (£353., 3’ 13:21:35; SE18.
PBS 0 100 -- - -- 0.08 95 :31% 0.77 None PS/20 135 0.08 50 0.75 15 ISIS 0.074 GalNAc3-1 PS/20 136 647535W 6.75 8 ApoC [[1 Protein Analysis (Turbidometric Assay) Plasma ApoC III protein analysis was determined using procedures reported by Graham et al, ation ch, published online before print March 29, 2013.
Approximately 100 pl of plasma isolated from mice was ed without dilution using an Olympus al Analyzer and a commercially available turbidometric ApoC III assay (Kamiya, Cat# KAI-006, Kamiya Biomedical, Seattle, WA). The assay protocol was performed as described by the vendor.
As shown in the Table 19 below, both antisense nds reduced ApoC III protein relative to the PBS control. Further, the antisense compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the 3-1 conjugate (ISIS 304801).
Table 19 Effect ofASO treatment on ApoC III plasma protein levels in human ApoC III transgenic mice Dose % EDSO Internucleoside AS0 3, C , SEQ ID on uJ gate (umol/kg) PBS (umol/kg) Linkage/Length N0- ---———_ ISIS 0.08 ISIS —— 019 GalNAc 1 PS/20 136 Plasma triglycerides and cholesterol were extracted by the method of Bligh and Dyer (Bligh, E.G. and Dyer, W.J. Can. J. Biochem. Physiol. 37: 7, 1959)(Bligh, E and Dyer, W, Can JBiochem Physiol, 37, 911-917, 1959)(Bligh, E and Dyer, W, Can JBiochem Physiol, 37, 911-917, 1959) and measured by using a Beckmann Coulter clinical er and commercially ble reagents.
The triglyceride levels were measured relative to PBS ed mice and are denoted as “% PBS”. Results are presented in Table 20. As illustrated, both antisense compounds lowered triglyceride levels. Further, the nse compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801).
Table 20 Effect ofASO treatment on triglyceride levels in transgenic mice Dose % EDSO 3’ ucleoside SEQ ID (”“30ng) PBS kg) Conjugate Linkage/Length No.
PBS 0 100 -- -- __ 0.08 87 ISIS 0.75 46 0.63 None PS/20 135 304801 2.25 21 6.75 12 0.08 65 ISIS 0.75 9 0.13 GalNAc3-1 PS/20 136 647535 2.25 8 6.75 9 Plasma samples were analyzed by HPLC to determine the amount of total cholesterol and of different ons of cholesterol (HDL and LDL). Results are presented in Tables 21 and 22. As rated, both antisense compounds lowered total cholesterol levels; both lowered LDL; and both raised HDL. Further, the antisense compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801). An increase in HDL and a decrease in LDL levels is a cardiovascular beneficial effect of antisense inhibition of ApoC III.
Table 21 Effect ofASO ent on total cholesterol levels in transgenic mice Dose Total Cholesterol Internucleoside AS0 3’ SEQ (umol/kg) (mg/dL) Conjugate Linkage/Length ID No.
PBS 0 257 __ __ 0.08 226 35213801H None PS/20 135 6.75 82 0.08 230 64131525 2:: :: GalNAc3-1 PS/20 136 6.75 99 Table 22 Effect ofASO treatment on HDL and LDL cholesterol levels in transgenic mice Dose HDL LDL 3 ’ Intemucleoside SEQ (umol/kg) (mg/dL) (mg/dL) Conjugate Llnkage/Length ID No.
PBS 0 17 28 -- -- 0.08 17 23 35213801# None PS/20 135 6.75 45 2 0.08 21 21 1515W GalNAc3-1 PS/20 136 647535W 6.75 58 2 Pharmacokinetics Analysis (PK) The PK of the ASOs was also evaluated. Liver and kidney samples were minced and extracted using standard protocols. Samples were ed on MSD1 utilizing C-MS. The tissue level (pg/g) of full-length ISIS 304801 and 647535 was measured and the results are provided in Table 23. As illustrated, liver concentrations of total full-length antisense nds were similar for the two antisense compounds.
Thus, even though the GalNAc3-1 -conjugated nse compound is more active in the liver (as demonstrated by the RNA and protein data above), it is not present at substantially higher concentration in the liver. Indeed, the calculated ECSO (provided in Table 23) confirms that the observed increase in potency of the ated compound cannot be entirely attributed to increased accumulation. This result suggests that the conjugate improved y by a mechanism other than liver accumulation alone, possibly by improving the productive uptake of the antisense compound into cells.
The results also show that the concentration of GalNAc3-1 conjugated antisense compound in the kidney is lower than that of antisense compound lacking the GalNAc conjugate. This has several beneficial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding benefit. Moreover, high tration in kidney typically results in loss of compound to the urine resulting in faster clearance. Accordingly, for non-kidney targets, kidney accumulation is red. These data suggest that 3-1 conjugation s kidney accumulation.
Table 23 PK is of ASO treatment in transgenic mice Internucleoside Dose L'1VeI‘ K'd1 1’1ey L'1Ve ECI‘ .3 ’ 5° SEQ ASO Linkage/Length (umol/kg) (jig/g) (jig/g) (jig/g) ate ID NO. 0.1 5.2 2.1 ISIS 0.8 62.8 119.6 —53 None PS/20 135 304801 2.3 142.3 191.5 6.8 202.3 337.7 0.1 3.8 0.7 ISIS 0'8 72'7 34'3 —3 8. G lNAa c3-1 PS/20 136 647535 2.3 106.8 111.4 6.8 237.2 179.3 Metabolites of ISIS 647535 were also identified and their masses were confirmed by high resolution mass spectrometry analysis. The cleavage sites and structures of the observed metabolites are shown below.
The relative % of full length ASO was calculated using standard procedures and the results are presented in Table 23a. The major lite of ISIS 647535 was full-length ASO lacking the entire conjugate (i.e. ISIS ), which results from cleavage at cleavage site A, shown below. Further, additional metabolites resulting from other cleavage sites were also observed. These results suggest that introducing other cleabable bonds such as esters, peptides, disulfides, phosphoramidates or acyl-hydrazones between the 3-1 sugar and the ASO, which can be cleaved by enzymes inside the cell, or which may cleave in the reductive environment of the cytosol, or which are labile to the acidic pH inside endosomes and lyzosomes, can also be Table 23a Observed full length metabolites of ISIS 647535 Metabolite ASO Cleavage site Relative % 2 ISIS 304801 + dA B 10.5 3 ISIS 647535 minus [3 GalNAc] C 16.1 ISIS 647535 minus 4 D 17 6 [3 GalNAc -- 1 5-hydroxy-pentan0ic acid tether] ' ISIS 647535 minus D 9 9 [2 GalNAc -- 2 oxy-pentan0ic acid tether] ' ISIS 647535 minus D 6 [3 GalNAc -- 3 5-hydroxy-pentan0ic acid tether] 9-8 ASO 304801 Cleavage Sites Cleavage site A HO OH Cleavage siteC O:F"OH ge site D o OH (N NHAC CleavagesiteCOQ01—Cleavage site B \0 \“WWo if0 NHAC WCleavage site D O HO HN/:OON site C NHAc Cleavage ASO 304801 O:P*OH NH2 ASO 304801 ‘ _ . /N Metabohte 1 ] Metabohte 2 OH OWN/I?” 2014/036460 I‘ASO 304801 O:P*OH NH2 H 0 / OH 1 o O C n H L HOWWW0 H o 3:0 Metabohte 3 HN A50 304801 HOWNWH 0 O o:F‘fOH NH2 H o HZNWN (5H (NfN K0,,“ ’N/J o 0 d n n ‘7 HOW \/\/ W0 H 0 3:0 Metabohte 4 HN ASO 304801 HOWH/N c‘) o:FfOH NH2 H 0 OH HZNWN E KOgN(“‘ng’ g g N O o o5 H \ H2N\/\/NWO m O F":0 Metabohte 5.
H“ Aso304801 HOWH/NWH o O:P*OH NH2 H o / N HZNWN (gm 0 <N 1NJ o o o‘: H \ ”NW\H/V0 u o 1:0 Metabolite 6 Example 21: Antisense inhibition of human ApoC III in human ApoC III enic mice in single administration study ISIS 304801, 647535 and 647536 each targeting human ApoC III and described in Table 17, were further evaluated in a single administration study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.
Treatment Human ApoCIII transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow. Animals were ated for at least 7 days in the research facility before initiation of the experiment. ASOs were prepared in PBS and sterilized by ng through a 0.2 micron filter. ASOs were dissolved in 0.9% PBS for ion.
Human ApoC III transgenic mice were injected intraperitoneally once at the dosage shown below with ISIS , 647535 or 647536 (described above) or with PBS treated control. The treatment group consisted of 3 animals and the control group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were ced 72 hours following the last administration .
Samples were collected and analyzed to determine the ApoC III mRNA and n levels in the liver; plasma triglycerides; and cholesterol, including HDL and LDL fractions were assessed as bed above (Example 20). Data from those analyses are presented in Tables 24-28, below. Liver transaminase levels, alanine ransferase (ALT) and aspartate aminotransferase (AST), in serum were ed relative to saline injected mice using standard protocols. The ALT and AST levels showed that the antisense compounds were well tolerated at all administered doses.
These results show improvement in potency for antisense compounds comprising a GalNAc3-1 conjugate at the 3’ terminus (ISIS 647535 and 647536) compared to the antisense compound lacking a GalNAc3-1 conjugate (ISIS 304801). Further, ISIS 647536, which comprises a GalNAc3-1 conjugate and some odiester linkages was as potent as ISIS 647535, which comprises the same conjugate and all internucleoside linkages within the ASO are phosphorothioate.
Table 24 Effect ofASO treatment on ApoC III mRNA levels in human ApoC III transgenic mice Dose 0 ED50 3’ Internucleoside SEQ ID ASO A) PBS (mg/kg) (mg/kg) ate linkage/Length No.
PBS 0 99 -- - -- 1 104 ISIS 3 92 13.2 None PS/20 135 304801W 40 ISIS Oi3 :3 1 9' GalNAc —1 647535 3 PS/20 136 WO 79625 647536 Table 25 Effect ofASO treatment on ApoC III plasma protein levels in human ApoC III transgenic mice Dose 0 EDso 3’ Intemucleoside SEQ ID ASO APBS (mg/kg) (mg/kg) Conjugate Linkage/Length N0, PBS 0 99 -- -- __ A23.2 ISIS 3 92 N‘me 135/20 135 40 0.3 98 2.1 ISIS 1 70 GalNAc3-1 PS/2O 136 647535fl 20 M1.8 ISIS 1 60 GalNAc3-1 PS/PO/2O 136 647536E 21 Table 26 Effect ofASO treatment on triglyceride levels in transgenic mice Dose 0 EDso Intemucleoside SEQ ID ASO APBS 3 , Conjugate- (mg/kg) (mg/kg) Linkage/Length No.
PBS 0 98 -- -- __ ISIS 3 92 29-1 None PS/20 135 304801W 47 ISIS 1 70 2-2 GalNAc3-1 PS/20 136 23 ISIS 1 66 1-9 Gama-1 PS/PO/ZO 136 647536E 23 Table 27 Effect ofASO treatment on total cholesterol levels in transgenic mice PBS 0 96 —— __ ISIS 3 96 None PS/20 135 304801W 72 ISIS 1 85 GalNAc3-1 PS/20 136 647535 3 61 53 0.3 115 ISIS 1 79 3-1 20 136 647536fi 54 Table 28 Effect ofASO treatment on HDL and LDL cholesterol levels in transgenic mice Dose HDL LDL 3’ Internucleoside SEQ ID (mg/kg) % PBS % PBS ate Linkage/Length No.
PBS 0 131 90 —- __ 1 130 72 ISIS 3 186 79 None PS/20 135 304801 10 226 63 240 46 ISIS 1 214 67 GalNAc3-1 PS/20 136 218 35 1818 1 187 56 GalNAc3-1 PS/PO/20 136 647536W 221 34 These results confirm that the GalNAc3-1 conjugate improves potency of an antisense compound.
The results also show equal potency of a GalNAc3-1 conjugated nse compounds where the antisense oligonucleotides have mixed linkages (ISIS 647536 which has six phosphodiester linkages) and a full phosphorothioate version of the same antisense compound (ISIS 647535).
Phosphorothioate linkages provide several properties to antisense nds. For example, they resist nuclease digestion and they bind proteins resulting in accumulation of compound in the liver, rather than in the kidney/urine. These are desirable properties, particularly when treating an indication in the liver.
However, phosphorothioate linkages have also been associated with an inflammatory response. Accordingly, ng the number of phosphorothioate linkages in a compound is expected to reduce the risk of inflammation, but also lower tration of the compound in liver, increase concentration in the kidney and urine, decrease stability in the presence of nucleases, and lower l potency. The present results show that a 3-1 conjugated antisense compound where certain phosphorothioate linkages have been replaced with phosphodiester linkages is as potent against a target in the liver as a counterpart having full phosphorothioate linkages. Such compounds are expected to be less proinflammatory (See Example 24 describing an ment showing reduction of PS results in reduced atory effect).
Example 22: Effect of GalNAc3-1 conjugated modified ASO targeting SRB-l in vivo ISIS 440762 and 651900, each targeting SRB-l and described in Table 17, were evaluated in a dose- dependent study for their ability to t SRB-l in Balb/c mice.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900 or with PBS treated l. Each treatment group consisted of 4 s. The mice were sacrificed 48 hours following the final administration to determine the SRB-l mRNA levels in liver using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard ols. SRB-l mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are ted as the average t of SRB-l mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”.
As illustrated in Table 29, both antisense compounds lowered SRB-l mRNA levels. Further, the antisense nd comprising the GalNAc3-1 conjugate (ISIS 651900) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 440762). These results demonstrate that the potency benefit of GalNAc3-1 conjugates are observed using antisense oligonucleotides complementary to a different target and having different chemically modified nucleosides, in this ce modified nucleosides comprise constrained ethyl sugar moieties (a bicyclic sugar moiety).
Table 29 Effect ofASO ent on SRB-l mRNA levels in Balb/c mice Internucleosid Dose Liver ED50 , . e ASO 3 conlugate (mg/kg) %PBS (mg/kg) linkage/Lengt _-___———_ IsIs —— W PS“ 440762E: WO 79625 1s1s 0.3 GalNAc3-1 PS/14 138 651900 Example 23: Human Peripheral Blood Mononuclear Cells (hPBMC) Assay Protocol The hPBMC assay was performed using BD Vautainer CPT tube method. A sample of whole blood from volunteered donors with informed consent at US HealthWorks clinic (Faraday & El Camino Real, Carlsbad) was obtained and collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat.# BD362753). The imate starting total whole blood volume in the CPT tubes for each donor was recorded using the PBMC assay data sheet.
The blood sample was remixed immediately prior to centrifugation by gently inverting tubes 8-10 times. CPT tubes were centrifuged at rt (18-25 0C) in a horizontal (swing-out) rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman Allegra 6R). The cells were ved from the buffy coat interface (between Ficoll and polymer gel layers); transferred to a sterile 50 m1 conical tube and pooled up to 5 CPT tubes/50 m1 l onor. The cells were then washed twice with PBS (Ca++, Mg++ free; GIBCO). The tubes were topped up to 50 ml and mixed by inverting several times. The sample was then centrifuged at 330 x g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) and aspirated as much supernatant as possible t disturbing pellet. The cell pellet was dislodged by gently swirling tube and resuspended cells in RPMI+10% FBS+pen/strep (~1 ml / 10 ml starting whole blood volume). A 60 ul sample was pipette into a sample vial (Beckman Coulter) with 600 pl VersaLyse reagent (Beckman Coulter Cat# A09777) and was gently vortexed for 10-15 sec. The sample was allowed to incubate for 10 min. at rt and being mixed again before counting. The cell sion was d on Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cell type (dilution factor of 1:11 was stored with other parameters). The live cell/ml and viability were ed. The cell suspension was d to 1 x 107 live PBMC/ml in RPMI+ 10% FBS+pen/strep.
The cells were plated at 5 x 105 in 50 ul/well of 96-well tissue e plate (Falcon Microtest). 50 ul/well of 2x concentration oligos/controls diluted in RPMI+10% FBS+pen/strep. was added ing to experiment template (100 ul/well total). Plates were placed on the shaker and allowed to mix for approx. 1 min. After being incubated for 24 hrs at 37 0C; 5% C02, the plates were centrifuged at 400 x g for 10 minutes before removing the supernatant for MSD cytokine assay (i.e. human lL-6, lL-10, lL-8 and MCP-l).
Example 24: Evaluation of Proinflammatory Effects in hPBMC Assay for GalNAc3-1 conjugated ASOs The antisense oligonucleotides (ASOs) listed in Table 30 were evaluated for proinflammatory effect in hPBMC assay using the protocol described in Example 23. ISIS 353512 is an internal standard known to be a high responder for lL-6 release in the assay. The hPBMCs were isolated from fresh, volunteered donors and were treated with ASOs at 0, , 0.064, 0.32, 1.6, 8, 40 and 200 uM concentrations. After a 24 hr ent, the cytokine levels were measured.
The levels of IL-6 were used as the primary t. The ECSO and Emax was calculated using standard procedures. Results are expressed as the average ratio of Emax/ECSO from two donors and is denoted as “EmaX/ECSO.” The lower ratio indicates a relative se in the proinflammatory response and the higher ratio tes a relative increase in the proinflammatory response.
With regard to the test compounds, the least proinflammatory compound was the PS/PO linked ASO (ISIS 616468). The GalNAc3-1 conjugated ASO, ISIS 647535 was slightly less proinflammatory than its non-conjugated counterpart ISIS 304801. These results indicate that incorporation of some PO linkages reduces proinflammatory reaction and addition of a GalNAc3-1 conjugate does not make a compound more proinflammatory and may reduce proinflammatory response. Accordingly, one would expect that an antisense compound comprising both mixed PS/PO linkages and a GalNAc3-1 conjugate would produce lower proinflammatory responses relative to full PS linked antisense nd with or without a GalNAc3-1 conjugate. These results show that GalNAc3_1 conjugated antisense compounds, particularly those having reduced PS content are less proinflammatory.
Together, these results suggest that a GalNAc3-1 conjugated compound, particularly one with reduced PS content, can be administered at a higher dose than a rpart full PS antisense compound lacking a GalNAc3-1 conjugate. Since half-life is not expected to be ntially different for these compounds, such higher administration would result in less frequent dosing. Indeed such administration could be even less frequent, because the GalNAc3-1 conjugated compounds are more potent (See Examples -22) and ing is necessary once the concentration of a compound has dropped below a d level, where such desired level is based on potency.
Table 30 Modified ASOS ASO Sequence (5’ to 3’) Target SEIEJD ISIS GesmCesTesGesAesTdsTdsAdsGdSAdsGds TNFoc 139 104838 AdsGdsAdsGdsGesTesmCeSmCeSmCe ISIS Tes Ces Ces TdSTdSTdS CdsAdsGdS CRP 140 3 5 3 5 12 GdsAdsGdsAdsmCdsmCdsTesGesGe ISIS AeSGeSmCeSTesTeSmCdSTdSTdSGdSTdS ApoC III 135 304801 mCdSmCdsAdSGdsmCds TeSAeSTe ISIS AeSGeSmCeSTesTeSmCdSTdSTdSGdSTdS ApoC III 136 64753 5 mCdsmCdSAdsGdsmCdSTesTesTesAesTeoAdoa-GalNAC3-1 a ISIS AesGeomCeoTeoTeomCdsTdsTdsGdsTds ApoC III 135 616468 dsAdsGdsmCdSTaoTaoTesAesTe Subscripts: cc :9 e indicates 2’-MOE modified nucleoside; “d” indicates B-D-2’- deoxyribonucleoside; “k” indicates 6’-(S)-CH3 bicyclic nucleoside (e. g. cEt); “s” indicates phosphorothioate 2014/036460 intemucleoside linkages (PS); “0” indicates phosphodiester internucleoside linkages (PO); and “0’” indicates O)(OH)-. Superscript “m” indicates 5-methylcytosines. “AdOs-GalNAc3-1a” indicates a conjugate having the structure GalNAc3-l shown in Example 9 attached to the 3’-end of the antisense oligonucleotide, as indicated.
Table 31 Proinflammatory Effect of ASOs targeting ApoC III in hPBMC assay EC50 Emax 3’ cleoside SEQ ID ASO EmaX/ECSO (uM) (uM) Conjugate Linkage/Length No.
ISIS 353512 0.01 265.9 None PS/20 140 , 26,590 (high responder) ISIS 304801 0.07 106.55 1,522 None PS/20 135 ISIS 647535 0.12 138 1,150 GalNAc3-1 PS/20 136 ISIS 616468 0.32 71.52 224 None PS/PO/20 135 Example 25: Effect of 3-1 conjugated modified ASO targeting human ApoC III in vitro ISIS 304801 and 647535 described above were tested in vitro. Primary hepatocyte cells from transgenic mice at a density of 25,000 cells per well were treated with 003,008, 0.24, 0.74, 2.22, 6.67 and 20 uM concentrations of modified oligonucleotides. After a ent period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR and the hApoC III mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
The IC50 was calculated using the standard methods and the results are presented in Table 32. As illustrated, comparable potency was observed in cells treated with ISIS 647535 as compared to the control, ISIS 304801.
Table 32 Modified ASO ing human ApoC III in primary hepatocytes , . Intemucleoside SEQ ASO IO” (”M) 3 conlugate linkage/Length ID No. rsrs 0.44 None PS/20 135 304801 rsrs 0.31 GalNAc3-1 PS/20 136 647535 In this experiment, the large potency benefits of GalNAc3-1 conjugation that are ed in vivo were not observed in vitro. uent free uptake experiments in primary hepatocytes in vitro did show increased potency of oligonucleotides comprising various GalNAc conjugates relative to oligonucleotides that lacking the GalNAc conjugate.(see Examples 60, 82, and 92) Example 26: Effect of PO/PS linkages 0n ApoC 111 A80 ty Human ApoC III transgenic mice were injected intraperitoneally once at 25 mg/kg of ISIS 304801, or ISIS 616468 (both described above) or with PBS treated control once per week for two weeks. The treatment group consisted of 3 animals and the control group consisted of 4 s. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours ing the last stration.
Samples were collected and analyzed to determine the ApoC III protein levels in the liver as described above (Example 20). Data from those es are presented in Table 33, below.
These results show reduction in potency for antisense compounds with PO/PS (ISIS 616468) in the wings relative to full PS (ISIS 304801).
Table 33 Effect ofASO treatment on ApoC III protein levels in human ApoC III transgenic mice Dose 3’ Internucleoside 0 SEQ ID ASO A) PBS (mg/kg) Conjugate linkage/Length No.
PBS 0 99 - -- 1s1s mg/kg/wk 24 None Full PS 135 304801 for 2 wks 1s1s mg/kg/wk 40 None 14 PS/6 PO 135 616468 for 2 wks Example 27: Compound 56 Compound 56 is cially available from Glen Research or may be prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.
Example 28: Preparation of Compound 60 AcO OAc AcO OAc WOBH 57 O ACO H0 AGO OvW\ —>H2/Pd —’ OBH MeOH o TMSOTf, DCE 58 N\ AcHN (quant) (71%) AcO OAc N(iPr)2)PC1, AcO OAc N(£02 EDIP O | CN 0 A00 OWO/P\O/\/ AcO \/\/\/\OH CHZCIZ AcHN 59 (80%) ACHN 60 Compound 4 was prepared as per the procedures illustrated in Example 2. nd 57 is commercially available. Compound 60 was confirmed by structural is.
Compound 57 is meant to be representative and not intended to be limiting as other monoprotected substituted or unsubstituted alkyl diols including but not limited to those ted in the specification herein can be used to prepare phosphoramidites having a predetermined ition.
Example 29: Preparation of Compound 63 ODMT 1. BnCl OH 1. DMTC1,pyr 0 % HO >—CH3 2. KOH DMSO 2. Pd/C H ’ BnO —2» OH 033/0 ODMT OO 3. HCl, MeOH 3. Phosphitylatlon I ODMT 4. Ncho3 NOPUZ 62 63 Compounds 61 and 62 are prepared using procedures similar to those reported by Tober et al., Eur. J.
Org. Chem, 2013, 3, 7; and Jiang et al., edron, 2007, 63(19), 3982-3988.
Alternatively, Compound 63 is prepared using procedures similar to those reported in scientific and patent literature by Kim et al., Synlett, 2003, [2, 1838-1840; and Kim et al., published PCT International Application, WO 2004063208.Example 30: Preparation of nd 63b OH ODMT /_/ CN 0 S O TPDBSOon/VOH 1. DMTCl, pyr 2. TBAF 3. Phosphitylatlon. . O\P/O\/E\O/\/ODMT O I N(iPr)2 63a OH ODMT Compound 63a is prepared using procedures similar to those reported by Hanessian et al., Canadian Journal ofChemistry, 1996, 74(9), 1731-1737.
Example 31: Preparation of Compound 63d O N(iPr)2 l. DMTCl, pyr DMTO O P HOWO O/\/\OBn 2. Pd/C,H2 \/\/ o/\/\o/ \o/\/CN O Phosphitylation O —/_/ 63c f 63d DMTO nd 63c is prepared using procedures similar to those reported by Chen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.
Example 32: Preparation of nd 67 COZBn AcO OAc ACOSL €~§O/OMOH AcO 0A0 H N/WOTBDMS CO Bn 2 2 OWLN/K(OTBDMS —>ACO%O ACHN HBTU, DIEA AcHN R : H or CH3 A 00AC C 1. TEA.3HF, THF O COZBn A O OWL O\ /O\/\ 2. Phosphitylation C E P CN ACHN R N(iPr)2 Compound 64 was prepared as per the procedures illustrated in Example 2. Compound 65 is prepared using procedures similar to those reported by Or et al., published PCT International Application, WO 2009003009. The protecting groups used for nd 65 are meant to be representative and not intended to be limiting as other protecting groups including but not limited to those presented in the specification herein can be used. e 33: Preparation of Compound 70 H N/\(0Bn2 AcO OAc 68 MOW 0 AcO OAc O M CH3 OH HBTU DIEA AcO 35/130,OWLN ACHN C4 N/YOBH AcHN CH3 A 00AC C 1. 2 OM O\/\ 2. Phosphitylation m%o N/YOP CN AcHN CH3 N(iPr)2 Compound 64 was prepared as per the ures illustrated in Example 2. Compound 68 is commercially available. The protecting group used for nd 68 is meant to be representative and not intended to be limiting as other protecting groups including but not limited to those presented in the specification herein can be used.
Example 34: Preparation of Compound 75a O CF3 1. TBDMSCI, pyr Y 2. Pd/C,H2 N('Pr)1 2 /\/O HNWO I NC 3. CF3C02Et,MeOH H /P\ /\/CN NC/\/O OH FgchWO 0 0 NC\/\O 4. TEA.3HF, THF O . Phosphitylatlon 0 75 A 75a 0 CF3 Compound 75 is prepared according to published procedures reported by Shchepinov et al., c Acids Research, 1997, 25(22), 4447-4454.
Example 35: Preparation of Compound 79 DMTOWO HOWO 1- BnCl NaH DCL NMI, ACN \/O ’ OH , HO\/\/O OBn Phosphoramidite 60 DMTOMO 2. DCA, CH2C12 HOMO 76 77 AcO OAC NC 0 I AGO OW\/\ (I) o/P\O AcHN ch K 1. HZ/Pd, MeOH ACO OAC O \/\/\/\O/P\O/\/\O/a\/OBH(I) 2. Phosphitylation AcHN o ACO 1‘3\ 0 O 0/ 0 NHAC ACO OAC NC 0 1 Aco OW/ \1? O 0 AcHN ACO OAC \LO ACO%/ P\O/V\O/3\/O\P/ \/\CN|0O I ACHN O N(iPr)2 NHAC Compound 76 was prepared according to published procedures ed by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.
Example 36: Preparation of Compound 79a HOWO\3/\OBIIHOWO 1. FmocCl, pyr FmocOWo 171(1'Pr)2 2. Pd/C, H2 FmOCO\/\/O\g/\O/P\O/\/CN HOMO 3. Phosphitylation FmocO/\/\O 77 79a Compound 77 is prepared as per the ures illustrated in Example 35.
Example 37: General method for the preparation of ated oligomeric compound 82 comprising a phosphodiester linked GalNAc3-2 conjugate at 5’ terminus via solid support (Method 1) ODMT O\/\/ o O/\/\ODMT DMT0’\<_7’BX O/\/\ODMT e o 0- NC 1|)\ 0 BX NC\/\ _._ \/\o’ o 0 13-0 1.DCA,DCM ’\(_7’ 0 2. DCI, NMI, ACN o‘ NC\/\0_15=0 Phosphoramidite 56 OLIGO | DNA/RNA 0 O automated synthesizer OLIGO . I :2 o—rlko/VCN 79b (I) Q VIMAD—O-l’l~O/\/CN X = S' or O' X BX = Heterocylic base 80 1. Capping (Ac20, NMI, pyr) 2. t-BuOOH 3. DCA, DCM 4. DCI, NMI, ACN AC0 OAC NC\\\ Phosphoramidite 60 OW\/\/ \I" AcHN 0 NC 7} AcOOAC 1 o of O 0I | O BX OW\/\O/P\O/\/\O O_ —O ACO (:1; W AcHN O O NC~\\O _ll)_0 NHAC (I) Q: VIMAD—O-l’l\0/\/CN 1. Capping (AczO, NMI, pyr) 81 2. t-BuOOH 3. 20% Et2NH inToluene (V/V) 4. NH4, 55 0C, NHAC 32 wherein GalNAc3-2 has the structure: HOOH HO O\/\/\/\ (I; AcHN ('3- OK HoOH O O O BX oW ‘ — ,('1)?\ o (I; o AcHN O Q o f O:IILO HO OH it NW NHAc The GalNAc3 r portion of the conjugate group GalNAc3-2 (GalNAcg-Za) can be combined With any cleavable moiety to provide a variety of conjugate groups. n GalNAc3-2a has the formula: AcHN o HO OH (1)5 f NHAC The VlMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). The phosphoramidite Compounds 56 and 60 were prepared as per the procedures illustrated in Examples 27 and 28, respectively. The phosphoramidites illustrated are meant to be representative and not intended to be ng as other phosphoramidite building blocks ing but not limited those presented in the specification herein can be used to prepare an oligomeric compound haVing a phosphodiester linked conjugate group at the 5’ terminus. The order and quantity of phosphoramidites added to the solid t can be adjusted to e the oligomeric compounds as described herein haVing any predetermined sequence and composition.
Example 38: Alternative method for the preparation of oligomeric compound 82 sing a phosphodiester linked 3-2 conjugate at 5’ terminus (Method 11) o‘ 1. DCA, DCM .' 2. DCI, NMI, ACN Phosphoramidite 79 OLIGO DNA/RNA ted synthes1zer- VIMAD—O-llko/VCN X = S' or O' BX = Heterocyclic base NHAC l. Capping 2. t-BuOOH 3. Et3NzCH3CN(1:1V/V) 83 4. NH4, 55 °C Oligomeric Compound 82 The VlMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). The GalNA03-2 cluster oramidite, Compound 79 was prepared as per the procedures illustrated in Example . This alternative method allows a one-step installation of the phosphodiester linked GalNAc3-2 conjugate to the oligomeric compound at the final step of the synthesis. The oramidites illustrated are meant to be representative and not intended to be limiting, as other phosphoramidite building blocks including but not limited to those presented in the specification herein can be used to prepare oligomeric compounds having a phosphodiester conjugate at the 5’ us. The order and ty of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition. 2014/036460 Example 39: General method for the preparation of oligomeric compound 83h comprising a GalNAc3- 3 Conjugate at the 5’ Terminus (GalNAc3-1 modified for 5' end attachment) via Solid t AcO o H 1. H2, Pd/C, MeOH (93%) I? o BnO OH H o OgiNJLOAQ 2_ M 0 O O O O HBTU, DIEA, DMF, 76% NHAc HNMN 3. H2,Pd/C,MeOH H 0 o OAc o AcO AcO AcO o H NHAc N H AcHN WNW o o F I? O O F o N H \/\/ O OH \COCF3 OAC M AGO T» 5L“ 83b 0 o O o F ACO ‘F— NHAc HNMN 830 Pyridine, DMF NHAc AcO 836 o H 3' 5, N H fi AcHN W WNW F F OLIGO o o O_F|, O_(CH2)6_NH2 H O O N N F —>H OAc M W If»0 NH O Borate buffer, DMSO, pH 8.5, rt A oc o 0 0 O F F NHAc HNMN H O Aco\gof/AcO o NHAc ACO OAC AcO o Aqueous ammonia HO OH HO o H H “W“ 0 NH )6—o—fi-o—- H0 O mgry0H0 OW o HN/\/\N Compound 18 was prepared as per the procedures illustrated in Example 4. Compounds 83a and 83b are commercially available. eric Compound 83e comprising a odiester linked mine was prepared using standard oligonucleotide synthesis procedures. Treatment of the protected oligomeric compound With aqueous ammonia provided the 5'-GalNAc3-3 conjugated oligomeric compound (83h).
Wherein GalNAc3-3 has the structure: HO OH NHAC The GalNAc3 cluster portion of the conjugate group GalNAc3-3 (GalNAc3-3a) can be combined With any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc3-3a has the formula: WO 79625 HO OH 2014/036460 Example 40: General method for the preparation of oligomeric compound 89 comprising a phosphodiester linked GalNAc3-4 conjugate at the 3’ terminus via solid support ODMT 0\/\/ l. DCA A\ $2 UNL—ODMT 0 O/\/\0Fmoc 2. DCI, NMI, ACN , | CN F N(z'Pr)2 .E UNL—O—lfi‘o/V mocOWO | O /P\ /\/CN 85 DMTOWO O O 3. Capping ODMT CN OFmoc 4. t-BuOOH O\/\/ I /_/_ O O OFmoc 1. 2%P1per1d1ne,. . . ’1') /—/— 2% DBU, 96% DMF 0 0M0 0 JO_/0Fmoc 3. DCI,NMI,ACN O Phosphoramidite 79a DNA/RNA 1. Capping automated s thesizer g. til/31119012.0 1per1 me, AcO OAC 2% DBU, 96% DMF A00 4. DCI, NMl, ACN O Phosphoramidite 60 AcHN O DNA/RNA automated s thes1zer.
Z 5. Capping.
AcO OAc O O’P\ O\/\/ P20 O | 87 .”? t-BuOOHDCAOligo sis (DNA/RNA automated synthesizer)CappingOxidationEt3NzCH3CN (1:1, V/V) A00 OAc AcHN 0 0O/\O\/\/OO7P' 13:0 9 f0 AGO \/\/\/\Or \ _O o o 0 oPOMO AGO DMT /\/CN NHAC 5OL—Go3 Q UNL—0—P—ON NH 55°C HO 4’ Ho\‘\\7L\LOAcHN HO OH OOLLH wk*0 0P7\,,0 O\/\/\/\ 0- o A HN0 159 ' 89 00/-\0\/\/ O 1|3=Q HO \ o o\/\/\/\O’ HO O/\/\0 NHAc / OLIGO 5V 3‘ Wherein GalNAc3-4 has the structure: HO OH ACHN O HO OH IO 0—1) HO o / \ O O- O\/\/ ACHN \/\/\/\ p o- O O\ / HO \O \ $0 0- HO APO NHAC g -/0 Wherein CM is a cleavable moiety. In certain embodiments, cleavable moiety is: The GalNAc3 cluster portion of the conjugate group GalNAc3-4 (GalNAc3-4a) can be combined With any cleavable moiety to provide a y of conjugate groups. Wherein GalNAc3-4a has the formula: HO OH AcHN O HO OH IO HO 0 0—5/ 0 o o \/\/\/\ W0 AcHN o 0- 0 O\/ 9 f 0W0 W0 /O/\/\o OH HO "”“b The protected Unylinker functionalized solid support Compound 30 is cially available.
Compound 84 is prepared using procedures similar to those reported in the literature (see Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454; Shchepinov et al., Nucleic Acids Research, 1999, 27, 3035-3041; and Hornet et al., Nucleic Acids Research, 1997, 25, 4842-4849).
The oramidite ng , Compounds 60 and 79a are prepared as per the procedures illustrated in Examples 28 and 36. The phosphoramidites illustrated are meant to be representative and not intended to be ng as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a phosphodiester linked conjugate at the 3’ terminus With a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.
Example 41: General method for the preparation of ASOs comprising a phosphodiester linked GalNAc3-2 (see Example 37, Bx is adenine) conjugate at the 5’ position via solid phase ques (preparation of ISIS 661134) Unless ise stated, all reagents and solutions used for the synthesis of oligomeric nds are purchased from commercial s. Standard phosphoramidite building blocks and solid t are used for incorporation nucleoside residues Which include for example T, A, G, and InC residues.
Phosphoramidite compounds 56 and 60 were used to synthesize the phosphodiester linked GalNAc3-2 2014/036460 conjugate at the 5’ terminus. A 0.1 M solution of phosphoramidite in anhydrous itrile was used for B- D-2’-deoxyribonucleoside and .
The ASO syntheses were performed on ABI 394 synthesizer (1-2 umol scale) or on GE Healthcare Bioscience AKTA oligopilot synthesizer 0 umol scale) by the phosphoramidite coupling method on VIMAD solid support (110 umol/g, Guzaev et al., 2003) packed in the column. For the coupling step, the phosphoramidites were delivered at a 4 fold excess over the l loading of the solid support and phosphoramidite coupling was carried out for 10 min. All other steps followed rd protocols supplied by the manufacturer. A solution of 6% dichloroacetic acid in toluene was used for removing the dimethoxytrityl (DMT) groups from roxyl groups of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) in anhydrous CH3CN was used as tor during the coupling step. Phosphorothioate linkages were introduced by sulfurization with 0.1 M solution of xanthane hydride in 1:1 pyridine/CH3CN for a contact time of 3 minutes. A solution of 20% tert—butylhydroperoxide in CH3CN containing 6% water was used as an oxidizing agent to e phosphodiester internucleoside linkages with a contact time of 12 minutes.
After the desired sequence was assembled, the cyanoethyl phosphate protecting groups were deprotected using a 20% diethylamine in toluene (v/v) with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %) and heated at 55 0C for 6 h.
The unbound ASOs were then filtered and the ammonia was boiled off The residue was purified by high pressure liquid chromatography on a strong anion ge column (GE Healthcare Bioscience, Source 30Q, um, 2.54 x 8 cm, A = 100 mM ammonium acetate in 30% aqueous CH3CN, B = 1.5 M NaBr in A, 0-40% of B in 60 min, flow 14 mL min-1, k = 260 nm). The residue was desalted by HPLC on a reverse phase column to yield the desired ASOs in an isolated yield of 15-30% based on the initial g on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.
Table 34 A80 comprising a phosphodiester linked 3-2 conjugate at the 5’ position ing SRB-l Observed SEQ ID ISIS No. Sequence (5 to 3 ) CalCd Mass Mass No.
GalNAc3'2a'o'AdoTkskasAdsGdsTdsmCdsAdsTds 661134 6482.2 6481.6 141 GdsAdsmCdsTdsTkska Subscripts: cc :9 e indicates 2’-MOE modified nucleoside; “(1” indicates B-D-2’- deoxyribonucleoside; “k” indicates 6’-(S)-CH3 bicyclic nucleoside (e. g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “0” indicates phosphodiester internucleoside linkages (PO); and “0’” indicates -O-P(=O)(OH)-. Superscript “m” indicates 5-methylcytosines. The ure of GalNAc3-2a is shown in Example 37.
WO 79625 e 42: General method for the preparation of ASOs comprising a GalNAc3-3 conjugate at the 5’ position via solid phase techniques (preparation of ISIS 661166) The synthesis for ISIS 661166 was performed using similar procedures as illustrated in Examples 39 and 41.
ISIS 661166 is a 55 MOE gapmer, wherein the 5’ position ses a GalNA03-3 conjugate.
The ASO was characterized by ion-pair-HPLC d MS analysis with Agilent 1100 MSD system.
Table 34a ASO comprising a GalNAc3-3 conjugate at the 5’ position via a hexylamino phosphodiester linkage targeting Malat—l INSIS Conjugate Calcd Observed --3GalNAC3a-o’mCesGesGesTesGes 661 166 mCdsAdsAdsGdSGdSmCdsTdSTdsAdsGds 5’-GalNAc3-3 8992.16 8990. 51 GesAesAes TesTe Subscripts: “e” indicates 2’-MOE modified nucleoside; “(1” indicates B-D-Z’-deoxyribonucleoside; 66 S” indicates phosphorothioate internucleoside linkages (PS); 66 039'1ndicates phosphodiester ucleoside linkages (PO); and “o”’ indicates -O-P(=O)(OH)-. Superscript “m” indicates 5-methylcytosines. The structure of “5’-Ga1NA03-3a” is shown in Example 39.
Example 43: Dose-dependent study of phosphodiester linked GalNAc3-2 (see examples 37 and 41, Bx is adenine) at the 5’ terminus targeting SRB-l in vivo ISIS 661134 (see Example 41) comprising a phosphodiester linked GalNAc3-2 conjugate at the 5’ terminus was tested in a dose-dependent study for nse tion of SRB-l in mice. Unconjugated ISIS 440762 and 651900 (GalNA03-1 ate at 3’ us, see Example 9) were included in the study for comparison and are described previously in Table 17.
Treatment Six week old male Balb/c mice (Jackson tory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS , 651900, 661134 or with PBS treated control. Each ent group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. SRB-l mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”. The EDsos were measured using similar s as described usly and are presented below.
As illustrated in Table 35, treatment with antisense ucleotides lowered SRB-l mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides comprising the phosphodiester linked GalNAc3-2 conjugate at the 5’ terminus (ISIS 661134) or the GalNAc3-1 conjugate linked at the 3’ us (ISIS ) showed substantial improvement in potency compared to the unconjugated antisense ucleotide (ISIS 440762). Further, ISIS 661134, which comprises the phosphodiester linked GalNAc3-2 conjugate at the 5’ terminus was equipotent compared to ISIS 651900, which ses the GalNAc3-1 conjugate at the 3’ terminus.
Table 35 ASOs containing GalNAc3-1 0r GalNAc3-2 targeting SRB-l ISIS Dosage SRB-l mRNA EDSO Conjugate SEQ ID NO' No. (mg/kg) levels (% PBS) (mg/kg) PBS 0 100 -- -- 0.2 116 0.7 91 440762 2 69 2.58 No conjugate 137 7 22 5 0.07 95 0.2 77 651900 0.7 28 0.26 3’ GalNAc3-1 138 2 11 7 8 0.07 107 0.2 86 661134 0.7 28 0.25 5’ GalNAc3-2 141 2 10 7 6 ures for 3’ GalNAc3-1 and 5’ GalNAc3-2 were described previously in Examples 9 and 37.
Pharmacokinetics Analysis (PK) The PK of the ASOs from the high dose group (7 mg/kg) was examined and evaluated in the same manner as illustrated in Example 20. Liver sample was minced and extracted using rd protocols. The full length metabolites of 661134 (5’ GalNAc3-2) and ISIS 651900 (3’ GalNAc3-1) were identified and their masses were confirmed by high resolution mass spectrometry analysis. The results showed that the major metabolite detected for the ASO comprising a odiester linked GalNAc3-2 conjugate at the 5’ terminus (ISIS 661134) was ISIS 440762 (data not shown). No additional metabolites, at a detectable level, were ed. Unlike its counterpart, additional metabolites similar to those reported usly in Table 23a were observed for the ASO having the GalNAc3-1 conjugate at the 3’ terminus (ISIS 651900). These results t that having the phosphodiester linked GalNAc3-1 or GalNAc3-2 conjugate may improve the PK profile of ASOs without compromising their potency.
Example 44: Effect of PO/PS linkages on antisense inhibition of ASOs sing GalNAc3-1 conjugate (see Example 9) at the 3’ terminus targeting SRB-l ISIS 655861 and 655862 comprising a GalNA03-l conjugate at the 3’ terminus each targeting SRB-l were tested in a single administration study for their ability to inhibit SRB-l in mice. The parent unconjugated compound, ISIS 353382 was included in the study for comparison.
The ASOs are 55 MOE gapmers, wherein the gap region comprises ten 2’-deoxyribonucleosides and each wing region comprises five 2’-MOE modified nucleosides. The ASOs were prepared using similar methods as illustrated usly in Example 19 and are described Table 36, below.
Table 36 Modified ASOs comprising GalNAc3-1 conjugate at the 3’ terminus targeting SRB-l Chemistry SEQ ISIS No. Sequence (5’ to 3’) ID 353382 GesmCesTCSTesmCesAdsGdsTdsmCdSAdSTdSGdSAdS Full PS no conjugate 143 (parent) InCdSTdSTeSmCeSmCeSTeSTe sTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds Full PS Wlth 144 655 861 mCdsTdsTesmCesmCesTesTeoAd0"GalNAC3-1 a GalNAC3-1 conjugate GesmCeoTeoTeomCeoAdsGdsTdsmCdsAdsTdsGdsAds Mixed PS/PO With 144 655 862 InC:dsTdsTeomC:eomC:esTesTeoAdowc;alNAc3'1 a GalNAc3-1 conjugate Subscripts: “e” indicates 2’-MOE modified nucleoside; “d” indicates -deoxyribonucleoside; “s” indicates phosphorothioate internucleoside linkages (PS); “0” indicates phosphodiester internucleoside linkages (PO); and “0’” indicates -O-P(=O)(OH)-. Superscript “m” indicates ylcytosines. The structure of “GalNAc3-l” is shown in e 9.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 353382, , 655862 or with PBS treated control. Each treatment group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were ed. The mice were sacrificed 72 hours following the final administration to ine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. SRB-l mRNA levels were determined relative to total RNA (using Ribogreen), prior to ization to PBS-treated control. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, ized to PBS-treated control and is denoted as “% PBS”. The EDsos were measured using similar methods as bed previously and are reported below.
As illustrated in Table 37, treatment with nse oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner compared to PBS treated control. Indeed, the antisense oligonucleotides comprising the GalNAc3-1 conjugate at the 3’ terminus (ISIS 655861 and 655862) showed substantial improvement in y comparing to the ugated antisense oligonucleotide (ISIS 353382). Further, ISIS 655862 with mixed PS/PO linkages showed an improvement in potency relative to full PS (ISIS 655861).
Table 37 Effect of PO/PS linkages 0n antisense inhibition of ASOs comprising GalNAc3-1 conjugate at 3’ terminus targeting SRB-l ISIS Dosage SRB-l mRNA EDSO Chemlsu'y. SEQ ID NO' No. (mg/kg) levels (% PBS) ) PBS 0 100 -- -- 3 76.65 :?) 10 52.40 10.4 Full PS without conjugate 143 24.95 0'5 8122 Fu11 PS 'th G lNA 1 1.5 63.51 W. a 03' —5 2.2 conjugate 144 24.61 14.80 0.5 69.57 1.5 45.78 Mixed PS/PO with 655862 1'3 144 19.70 GalNA03-1 conjugate 12.90 Liver transaminase , alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Organ weights were also evaluated. The results demonstrated that no elevation in transaminase levels (Table 38) or organ weights (data not shown) were observed in mice treated with ASOs compared to PBS control. Further, the ASO with mixed PS/PO linkages (ISIS 655 862) showed similar transaminase levels compared to full PS (ISIS 655 861).
Table 38 Effect of PO/PS linkages 0n transaminase levels of ASOs comprising GalNAc3-1 conjugate at 3’ terminus targeting SRB-l ISIS Dosage ALT AST Chemlstry_ SEQ ID No, No. (mg/kg) (U/L) (U/L) PBS 0 28.5 65 __ 3 50.25 89 .31233)W “flag?“. 143 p J g 27.3 97 0528—55-7 1-5 30 78 Full PS with 655861 144 #GalNAc3—1 28.8 67.8 0.5 50 75.5 , . 29.3 69 ____—— Example 45: Preparation of PFP Ester, Compound 110a n OAC OAc , 2 OAc oOAC EtOAC’ MeOH 103a; ":1 O o/VHWN 3 —> AcO 103k); n= 7 A00 —>AcHN N 104a; n=1 7/0 104b; n= 7 4 0A0 AcHN o 0 OAc OAc 0Ac AcOfi/O OAc Wm 0 WNH PFPTFA o n —>AcO AcHN DMF, pyr AcHN OWNHn N02 105a; n=1 Compound 90 0 A00 0 O AcHN 106a; n=1 106b; n= 7 0AACO%O0A0CAcHN OVWN o Ra-Ni H2 O0Ac H HBTU, DIEA, DMF —’> ACO WNH —> MeOH, EtOAc ACHNACONgQ/On 0A0 0A0 0 Wm ’8n AcHN 99 107a;n 1 107b; n 7 AcHN 0 AcO ‘ngOAc OVWNH NH AcHN NH ACONgA/On 0 0A0 0A0 o WHN AcHN 108a; n=1 0 108b; n= 7 I /\H/V\0A0 Pd/C, H2, WACHSA 108a; n=1 EtOAc, MeOH 108b; n= 7 AcO ACHNfi/CWnWNH Acofi/OWOACW ACHN 109a; n= 1 109b; n= 7 AcHN PFPTFA,DMF, o 0A0 0A0 —> O A00 0/\/\/\/HN O 109a AcHN 110a o F F F F F Compound 4 (9.5g, 28.8 mmoles) was d with compound 103a or 103b (38 moles), individually, and TMSOTf (0.5 eq.) and molecular sieves in dichloromethane (200 mL), and d for 16 hours at room temperature. At that time, the c layer was filtered thru celite, then washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced under reduced pressure. The resultant oil was purified by silica gel chromatography (2%-->10% methanol/dichloromethane) to give compounds 104a and 104b in >80% yield. LCMS and proton NMR was consistent with the structure.
Compounds 104a and 104b were treated to the same conditions as for compounds 100a-d (Example 47), to give compounds 105a and 105b in >90% yield. LCMS and proton NMR was consistent with the structure.
Compounds 105a and 105b were treated, dually, with compound 90 under the same conditions as for compounds 901a-d, to give compounds 106a (80%) and 106b (20%). LCMS and proton NMR was consistent with the structure.
Compounds 106a and 106b were treated to the same conditions as for compounds 96a-d (Example 47), to give 107a (60%) and 107b (20%). LCMS and proton NMR was consistent with the structure.
Compounds 107a and 107b were treated to the same conditions as for compounds 97a-d (Example 47), to give compounds 108a and 108b in 40-60% yield. LCMS and proton NMR was consistent with the structure.
Compounds 108a (60%) and 108b (40%) were treated to the same ions as for compounds 100a- d le 47), to give compounds 109a and 10% in >80% yields. LCMS and proton NMR was consistent with the structure.
Compound 109a was treated to the same conditions as for compounds 101a-d (Example 47), to give Compound 110a in 30-60% yield. LCMS and proton NMR was consistent with the structure. Alternatively, Compound 110b can be prepared in a similar manner starting with Compound 10%.
Example 46: General Procedure for Conjugation with PFP Esters nucleotide 111); Preparation of ISIS 666881 (GalNAc3-10) A 5’-hexylamino modified oligonucleotide was synthesized and purified using standard solid-phase oligonucleotide procedures. The 5’-hexylamino modified oligonucleotide was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 uL) and 3 equivalents of a selected PFP fied GalNAc3 r dissolved in DMSO (50 uL) was added. If the PFP ester precipitated upon addition to the A80 solution DMSO was added until all PFP ester was in solution. The reaction was complete after about 16 h of mixing at room temperature. The ing solution was diluted with water to 12 mL and then spun down at 3000 rpm in a spin filter with a mass cut off of 3000 Da. This process was ed twice to remove small molecule impurities. The solution was then lyophilized to s and redissolved in concentrated aqueous a and mixed at room temperature for 2.5 h followed by concentration in vacuo to remove most of the ammonia.
The conjugated oligonucleotide was purified and desalted by RP-HPLC and lyophilized to e the GalNAc3 conjugated oligonucleotide.
HO1%OH o 83e 3 . 5. | AcHN o o OLIGO O-F|’-O-(CH2)6-NH2 OH OH \/\/\/\H 110a +> NH 1. Borate buffer, DMSO, pH 8.5, rt AcHN 2. NH3 (aq) rt , O OH OH HOfi/OO /\/\/\/HN O OLIGO o 4 Oligonucleotide 111 is conjugated with GalNAc3-10. The GalNAc3 cluster portion of the conjugate group 3-10 c3-10a) can be combined with any cleavable moiety to provide a y of conjugate groups. In n embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)- as shown in the oligonucleotide (ISIS 666881) synthesized with GalNAc3-10 below. The structure of GalNAc3-10 (GalNAc3-10a-CM-) is shown below: Homwflffi NWNAMAO A.” Following this l procedure ISIS 666881 was ed. 5’-hexylamino modified oligonucleotide, ISIS 660254, was synthesized and purified using standard solid-phase oligonucleotide procedures. ISIS 660254 (40 mg, 5.2 umol) was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 uL) and 3 equivalents PFP ester (Compound 110a) dissolved in DMSO (50 uL) was added. The PFP ester precipitated upon addition to the ASO solution requiring additional DMSO (600 uL) to fully dissolve the PFP ester. The reaction was complete after 16 h of mixing at room temperature. The solution was diluted with water to 12 mL total volume and spun down at 3000 rpm in a spin filter with a mass cut off of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was lyophilized to dryness and redissolved in concentrated aqueous ammonia with mixing at room temperature for 2.5 h followed by concentration in vacuo to remove most of the ammonia. The ated oligonucleotide was purified and desalted by C and lyophilized to give ISIS 666881 in 90% yield by weight (42 mg, 4.7 umol).
GalNAc3-10 conjugated oligonucleotide NH2(CH2)6'oAdoGesmCesTesTesmCesAdsGdsTds ISIS 660254 Hexylamlne- 145 InC:dslAdsTdsC}d31AdsmC:dsTdsTesmC:esmC:esTesTe GalNAC3-10a'o’Ad0GesmCesTesTesmCesAdsGdsTds ISIS 6668 81 GalNAC3-10 InC:dslAdsTdsC}d31AdsmC:dsTdsTesmC:esmC:esTesTe Capital letters indicate the nucleobase for each nucleoside and InC indicates a 5-methyl cytosine.
Subscripts: “e” indicates a 2’-MOE modified nucleoside; “d” indicates a B-D-Z’-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “0” tes a phosphodiester internucleoside linkage (PO); and “0’” indicates -O-P(=O)(OH)-. Conjugate groups are in bold.
Example 47: Preparation of Oligonucleotide 102 Comprising GalNAc3-8 HZNMNHBOC HANn O 91a; n= 1 91b n—2 BocHNMNH N02 M, PFPTFA DIPEA DMF BocHNVWHN o 92a; n=1 92b, n=2 HZNAHANH OAC OAc N02 ; O TMSOTf, DCM AcO OAc —> AcHN HZNV®VHN o 93a; n=1 93b, n=2 94a; m=1 94b, m=2 O OAC OAC O HOW0,Bn O0Ac m A OC A oC % —> AcHN OMOHm Pd/C. H2 64, m=2 AmwgmOWN ”W 0 OAc —938(93b) ACOfi/OAWNMNH Ra-Ni, H2 HBTU, DIPEA, DMF ACACHN N02 AcOfi/OOAcOMNWHN o AcHN n 96a; n=1, m=1 96b; n=1, m=2 960; n=2, m=1 96d: n=2. m=2 2014/036460 mow/*0 0 AcHN o N O m H N n HBTU,D|EA,DMF 0A0 0A0 O H Acofi/OWH0 a9“NHn NH2 AcHN O ODMTr 0Ac 0 OAc HO AGO OWNWHN 0 ’7 AcHN n N O -, 97a; n=1, m=1 97b; n=1, m=2 97c; n=2, m=1 97d; n=2, m=2 ACO\%DAWAcHN O N 0 OAc H OOAC 0 /\H/\ H O ODMTr AcO OWN NH n N AcHN ) 0A0 O 0A0 7 H N O KMWGVHN Aco o "0H AcHN n 98a; n=1, m=1 98b; n=1, m=2 980; n=2, m=1 98d' n=2 m=2 WO 79625 AchK/WVLV o HBTU, DIEA, DMF 97a; n=1,m=1OOAc O 97b; n=1, m=2 —> 97c; n=2, m=1 AcO N/WNH MN O 0 97d, n—2, m=2 HOzC/flifio’ ” AcHNO ‘Bn B 760%»)OWNWHN0A0 o 99 m AcHN '1 100a; n=1, m=1 100b; n=1, m=2 100c; n=2, m=1 100d; n=2, m=2 Pd(OH) 2/c, o 0 H2, EtOAc, OOAC , DMF, _MeQH_. AC0 d?NI-In MS OH pyr AcHN Acofi/o0A0 OAc 0 101a; n=1, m=1 AcHN OMNWHNn 101b; n=1, m=2 O 101c; n=2, m=1 101d; n=2, m=2 (QACJK:WW,“ (DAG/W OAc OAc AcO 0M 102a; n=1, m=1 AcHN 102b; n=1, m=2 102c; n=2, m=1 102d; n=2, m=2 The triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120 mL) and N,N—Diisopropylethylamine (12.35 mL, 72 moles). Pentafluorophenyl trifluoroacetate (8.9 mL, 52 moles) was added dropwise, under argon, and the reaction was allowed to stir at room temperature for 30 minutes. Boc-diamine 91a or 91b (68.87 mmol) was added, along with N,N—Diisopropylethylamine (12.35 mL, 72 moles), and the reaction was allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was d by silica gel chromatography (2%-->10% methanol/dichloromethane) to give nds 92a and 92b in an approximate 80% yield. LCMS and proton NMR were consistent with the structure.
Compound 92a or 92b (6.7 ) was treated with 20 mL of dichloromethane and 20 mL of trifluoroacetic acid at room temperature for 16 hours. The resultant solution was evaporated and then ved in ol and treated with DOWEX-OH resin for 30 minutes. The resultant on was filtered and reduced to an oil under reduced pressure to give 85-90% yield of compounds 93a and 93b.
Compounds 7 or 64 (9.6 mmoles) were treated with HBTU (3.7g, 9.6 mmoles) and N,N— Diisopropylethylamine (5 mL) in DMF (20 mL) for 15 minutes. To this was added either compounds 93a or 93b (3 moles), and d to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under d pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and d to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (5%-->20% methanol/dichloromethane) to give compounds 96a-d in 20-40% yield.
LCMS and proton NMR was consistent with the structure.
Compounds 96a-d (0.75 mmoles), individually, were enated over Raney Nickel for 3 hours in Ethanol (75 mL). At that time, the catalyst was removed by filtration thru celite, and the ethanol removed under reduced pressure to give nds 97a-d in 80-90% yield. LCMS and proton NMR were consistent with the ure.
Compound 23 (0.32g, 0.53 mmoles) was treated with HBTU (0.2g, 0.53 ) and N,N— Diisopropylethylamine (0.19 mL, 1.14 mmoles) in DMF (30mL) for 15 minutes. To this was added compounds 97a-d (0.38 mmoles), individually, and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced re, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure.
The resultant oil was purified by silica gel chromatography 20% methanol/dichloromethane) to give compounds 98a-d in 30-40% yield. LCMS and proton NMR was consistent with the structure.
Compound 99 (0.17g, 0.76 mmoles) was treated with HBTU (0.29 g, 0.76 mmoles) and N,N— Diisopropylethylamine (0.35 mL, 2.0 mmoles) in DMF (50mL) for 15 minutes. To this was added compounds 97a-d (0.51 mmoles), individually, and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure.
The resultant oil was purified by silica gel chromatography (5%-->20% methanol/ dichloromethane) to give compounds 100a-d in 40-60% yield. LCMS and proton NMR was tent with the structure.
Compounds 100a-d (0.16 mmoles), dually, were enated over 10% Pd(OH)2/C for 3 hours in methanol/ethyl acetate (1 :1, 50 mL). At that time, the catalyst was removed by filtration thru celite, and the organics removed under reduced pressure to give compounds 101a-d in 80-90% yield. LCMS and proton NMR was consistent with the structure.
Compounds 101a-d (0.15 mmoles), individually, were dissolved in DMF (15 mL) and pyridine (0.016 mL, 0.2 mmoles). uorophenyl trifluoroacetate (0.034 mL, 0.2 mmoles) was added dropwise, under argon, and the reaction was allowed to stir at room temperature for 30 minutes. At that time, the DMF was reduced by >75% under reduced re, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography 5% methanol/dichloromethane) to give compounds lOZa-d in an approximate 80% yield. LCMS and proton NMR were consistent with the ure. 3' 5' ('3' -o-F|>-o-(CH2)6NH2 Borate buffer, DMSO, pH 8.5, rt 102d —> 2. aq. ammonia, rt HoOH o o Ho%O o’fka 4 HM?” AcHN o o HOOH O O HO¥Q/ W o O”Tka N Wm N H 4 4 HwH AcHN HoOH o Hog/O O/TTKN”r¥\N O 4 H 2 H 102 AcHN eric Compound 102, comprising a GalNAc3-8 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc3 cluster portion of the ate group GalNAc3- 8 (GalNAc3-8a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a preferred embodiment, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.
The structure of GalNAc3-8 (GalNAc3-8a-CM-) is shown below: Example 48: Preparation of ucleotide 119 Comprising GalNAc3-7 ACOOAC ACO OAC 0 0 A00 TMSOT“! DOE A00 OWNHCBZ Pd(OH)2/C —> 4 —> Ni? HO/VHg/NHCBZ AcHN H2, MeOH, EtOAc 4 35b 112 HO\H/\\ HBTU, DIEA $0: 0 NHCBZ —, OWNH HO A00 2 + % AcHN o 105a p A00 OAC ACO%Q/o\/H\/NHo o AcHN A00 OAc o H 0 A00 o AcHN \/H4\/N\n/\/o\%NHCBZ o 0 A00 OAc p ACO%Q/O\/H4\/NHO AcHN ACO OAC O oWH O A HNC ACO OAc Pd/C, H2, 0 114 CHsOH AGO%Q/O\/H4\/NH\n/\/O%NH2ACHN O 0 A00 OAC p ACO%O\/H4\/NHO ACHN A00 OAC O\/H\/NHO O HBTU, DIEA, DMF A HNC O O ACO OAC —>ACO%O\/H4\/NH\”/VO\%NHOO WOBn ACHN O O HO 0Q \n/V\n/ O ACO OAC O O 0 H A00 O\/H4\/NH ACHN Compound 112 was sized following the procedure described in the literature (J. Med. Chem. 2004, 47, 5798-5808).
Compound 112 (5 g, 8.6 mmol) was dissolved in 1:1 methanol/ethyl acetate (22 mL/22 mL).
Palladium hydroxide on carbon (0.5 g) was added. The reaction mixture was stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite and washed the pad with 1:1 ol/ethyl acetate. The filtrate and the washings were combined and concentrated to dryness to yield nd 105a (quantitative). The structure was confirmed by LCMS.
Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol) and DIEA (2.8 mL, 16.2 mmol) were dissolved in anhydrous DMF (17 mL) and the reaction mixture was d at room temperature for 5 min. To this a solution of Compound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 mL) was added. The reaction was stirred at room temperature for 6 h. Solvent was removed under reduced pressure to get an oil. The residue was dissolved in CHgClg (100 mL) and washed with aqueous saturated NaHC03 on (100 mL) and brine (100 mL). The organic phase was separated, dried (Na2S04), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 10 to 20 % MeOH in romethane to yield Compound 114 (1.45 g, 30%). The structure was confirmed by LCMS and 1H NMR analysis.
Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1 ol/ethyl acetate (4 mL/4 mL).
Palladium on carbon (wet, 0.14 g) was added. The reaction mixture was flushed with hydrogen and stirred at room ature under en for 12 h. The reaction mixture was filtered through a pad of celite. The celite pad was washed with methanol/ethyl acetate (1:1). The filtrate and the washings were combined together and evaporated under reduced pressure to yield Compound 115 (quantitative). The structure was confirmed by LCMS and 1H NMR analysis.
Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol) and DIEA (0.26 mL, 1.5 mmol) were ved in anhydrous DMF (5 mL) and the reaction mixture was stirred at room temperature for 5 min. To this a solution of Compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF was added and the reaction was stirred at room temperature for 6 h. The t was removed under reduced pressure and the e was dissolved in CHgClg. The organic layer was washed aqueous ted NaHC03 solution and brine and dried over anhydrous Na2S04 and filtered. The organic layer was concentrated to dryness and the residue obtained was purified by silica gel column chromatography and eluted with 3 to 15 % MeOH in dichloromethane to yield Compound 116 (0.84 g, 61%). The structure was confirmed by LC MS and 1H NMR analysis.
ACO OAC ACHN Pd/C, H2, ACO OAC 116 EtOAC, MeOH 0 OH —,Aco§wovswknmo()0ng: ACHN ACO OAC H ACO%Q/O\/H4\/NHO 117 ACHN ACO OAC ACOE “ago /O\/H\/NH O F 4 F ACHN PFPTFA DMF , v Pyr ACO OAC KO MOI: AGO0%OVHYNH 0%NH F ACHN \n/V F ACO OAC (p Inflow/OWNH 118 ACHN Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1 methanol/ethyl acetate (5 mL/S mL).
Palladium on carbon (wet, 0.074 g) was added. The on mixture was flushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The on e was filtered through a pad of celite. The celite pad was washed with methanol/ethyl acetate (1:1). The filtrate and the washings were combined together and evaporated under reduced pressure to yield compound 117 (0.73 g, 98%). The structure was confirmed by LCMS and 1H NMR analysis.
Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous DMF (3 mL). To this solution N,N— Diisopropylethylamine (70 uL, 0.4 mmol) and uorophenyl trifluoroacetate (72 uL, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 12 h and poured into a aqueous saturated NaHC03 solution. The mixture was extracted with dichloromethane, washed with brine and dried over anhydrous Na2SO4. The romethane solution was concentrated to dryness and purified with silica gel column chromatography and eluted with 5 to 10 % MeOH in dichloromethane to yield nd 118 (0.51 g, 79%). The structure was confirmed by LCMS and 1H and 1H and 19F NMR. 3- 5' | OLIGO O-T-O-(Csz'NHz 1. Borate buffer, DMSO, pH 8.5, rt 118 —> 2. aq. a, rt HO%HOOH ACHN HW/flAJk/‘gwfiocm oueo oWHN O 119 Oligomeric Compound 119, comprising a GalNAc3-7 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc3 cluster portion of the conjugate group GalNAc3- 7 (GalNAc3-7a) can be combined with any ble moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.
The structure of GalNAc3-7 (GalNAc3-7a-CM-) is shown below: HoOH o o N Ho 4 Hkk AcHN Hog/o o : O N s 4 H M ”W0 ‘ AcHN o HoOH o t o N Ho O 4 H AcHN Example 49: Preparation of Oligonucleotide 132 Comprising GalNAc3-5 Er HN’BOC Boc\Eh 80:15YN OH Boc\N H HBTU TEA L'OH H20 DMF MeOH, THF HN\ 120 122 78% 123 Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) were dissolved in ous DMF (80 mL). Triethylamine (11.2 mL, 80.35 mmol) was added and stirred for 5 min. The reaction mixture was cooled in an ice bath and a solution of compound 121 (10 g, mol) in anhydrous DMF (20 mL) was added. Additional triethylamine (4.5 mL, 32.28 mmol) was added and the reaction mixture was stirred for 18 h under an argon atmosphere. The reaction was red by TLC (ethyl acetate:hexane; 1:1; Rf = 0.47).
The t was removed under reduced pressure. The residue was taken up in EtOAc (300 mL) and washed with 1M NaHSO4 ( 3 X 150 mL), aqueous saturated NaHC03 solution (3 X 150 mL) and brine (2 X 100 mL).
Organic layer was dried with . Drying agent was removed by filtration and organic layer was concentrated by rotary evaporation. Crude mixture was purified by silica gel column chromatography and eluted by using 35 — 50% EtOAc in hexane to yield a compound 122 (15.50 g, ). The structure was confirmed by LCMS and 1H NMR analysis. Mass m/z 589.3 [M + H]+.
A solution of LiOH (92.15 mmol) in water (20 mL) and THF (10 mL) was added to a cooled solution of Compound 122 (7.75 g,13.16 mmol) dissolved in methanol (15 mL). The reaction mixture was stirred at room temperature for 45 min. and monitored by TLC :hexane; 1:1). The reaction mixture was concentrated to half the volume under reduced pressure. The remaining solution was cooled an ice bath and neutralized by adding concentrated HCl. The reaction mixture was diluted, extracted with EtOAc (120 mL) and washed with brine (100 mL). An emulsion formed and cleared upon ng overnight. The c layer was separated dried (NaZSO4), filtered and evaporated to yield Compound 123 (8.42 g). Residual salt is the likely cause of excess mass. LCMS is consistent with structure. Product was used without any r purification. M.W.cal:574.36; M.W.fd:575.3 [M + H]+.
S? e O o @S-OH - H20 '4stij HZNMOH || 0 + HO O ' —> (I? /\© Toluene, Reflux ('5 124 125 126 99.6% Compound 126 was synthesized following the procedure described in the literature (J. Am. Chem.
Soc. 2011, 133, 958-963). 2014/036460 HOBt DIEA CI"20'2 PyBop, Bop, DMF CF3COO AcO OAC O OH 3W0 AcHN 7 o CF3COO'@H3N —> HATU, HOAt, DIEA, DMF CF3COO' @NH3 128 A00 OAc AcHN W0 Acog/ O AcHN A00 OAC AcHN o AcO OAc o o AcHN WY Pd/C H M OH e 129 ’ 2’ o A 0 OAcC H or HN N AcHN A00 OAC 0 NH AcHN 130 AcO OAc o A00 0 AcHN W0 PFPTFA, DMF, Pyr AcO OAC Acog/0O:k5\(NO MW: AcHN AcO OAC AcO CW AcHN o Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82 mmol) and compound 126 (6.33 g, 16.14 mmol) were dissolved in and DMF (40 mL) and the resulting reaction mixture was cooled in an ice bath. To this N,N—Diisopropylethylamine (4.42 mL, 25.82 mmol), PyBop (8.7 g, 16.7 mmol) followed by Bop coupling reagent (1.17 g, 2.66 mmol) were added under an argon atmosphere. The ice bath was removed and the solution was allowed to warm to room temperature. The reaction was completed after 1 h as determined by TLC (DCM:MeOH:AA; 89:10:1). The reaction mixture was concentrated under d pressure. The residue was dissolved in EtOAc (200 mL) and washed with 1 M NaHSO4 (3X100 mL), s saturated NaHC03 (3X100 mL) and brine (2X100 mL). The c phase separated dried (Na2S04), filtered and concentrated. The residue was purified by silica gel column chromatography with a gradient of 50% hexanes/EtOAC to 100% EtOAc to yield Compound 127 (9.4 g) as a white foam. LCMS and 1H NMR were consistent with ure. Mass m/z 778.4 [M + H] +.
Trifluoroacetic acid (12 mL) was added to a solution of compound 127 (1.57 g, 2.02 mmol) in dichloromethane (12 mL) and stirred at room temperature for 1 h. The reaction mixture was co-evaporated with e (30 mL) under reduced pressure to dryness. The residue obtained was porated twice with acetonitrile (30 mL) and toluene (40 mL) to yield Compound 128 (1.67 g) as trifluoro acetate salt and used for next step t further purification. LCMS and 1H NMR were consistent with structure. Mass m/z 478.2 [M + H] i Compound 7 (0.43 g, 0.963 mmol), HATU (0.35 g, 0.91 mmol), and HOAt (0.035 g, 0.26 mmol) were combined er and dried for 4 h over P205 under reduced pressure in a round bottom flask and then dissolved in anhydrous DMF (1 mL) and stirred for 5 min. To this a solution of compound 128 (0.20 g, 0.26 mmol) in anhydrous DMF (0.2 mL) and N,N—Diisopropylethylamine (0.2 mL) was added. The on mixture was stirred at room temperature under an argon atmosphere. The reaction was complete after 30 min as determined by LCMS and TLC (7% MeOH/DCM). The on mixture was concentrated under reduced pressure. The residue was dissolved in DCM (30 mL) and washed with 1 M NaHSO4 (3x20 mL), aqueous saturated NaHC03 (3 x 20 mL) and brine (3x20 mL). The organic phase was separated, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography using 5-15% MeOH in dichloromethane to yield Compound 129 (96.6 mg). LC MS and 1H NMR are consistent with structure. Mass m/z 883.4 [M + 2H]+.
Compound 129 (0.09 g, 0.051 mmol) was dissolved in ol (5 mL) in 20 mL scintillation vial.
To this was added a small amount of 10% Pd/C (0.015 mg) and the reaction vessel was flushed with H2 gas.
The reaction mixture was stirred at room temperature under H2 atmosphere for 18 h. The reaction mixture was filtered through a pad of Celite and the Celite pad was washed with methanol. The filtrate washings were pooled together and concentrated under reduced pressure to yield Compound 130 (0.08 g). LCMS and 1H NMR were consistent with structure. The product was used without r purification. Mass m/z 838.3 [M + 2H]+.
To a 10 mL d round bottom flask were added compound 130 (75.8 mg, 0.046 mmol), 0.37 M ne/DMF (200 uL) and a stir bar. To this solution was added 0.7 M pentafluorophenyl trifluoroacetate/DMF (100 uL) drop wise with stirring. The reaction was completed after 1 h as determined by LC MS. The solvent was removed under reduced pressure and the residue was dissolved in CHC13 (N 10 mL). The organic layer was partitioned against NaHSO4 (1 M, 10 mL) saturated NaHC03 (10 mL) , aqueous and brine (10 mL) three times each. The organic phase separated and dried over Na2SO4, filtered and trated to yield Compound 131 (77.7 mg). LCMS is consistent with structure. Used without further purification. Mass m/z 921.3 [M + 2H]+.
HO OH 0 HO 3' 5' I AcHN W0 -O-F|’-o-(CH2>6-NH2 1. Borate buffer, DMSO, pH 8.5, rt 131—> 2. aq. ammonia, rt HO OHE HO%/O ACHN HO OH HO Memo—r- AcHN Oligomeric Compound 132, comprising a GalNAc3-5 conjugate group, was prepared using the l procedures rated in Example 46. The GalNAc3 cluster portion of the conjugate group 3- (GalNAc3-5a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.
The structure of GalNAc3-5 (GalNAc3-5a-CM-) is shown below: HO OH AcHN WYO HO OH N “N NH HO OM O AcHN HO OH o NH HO VVY O NAM/\o—I—E H 4 AcHN o Example 50: Preparation of Oligonucleotide 144 Comprising GalNAc4-11 DMTO Fmoc 1. TBTU, DIEA DMTO Fmoc KC)? .
ACN, VIMAD Resin K617 pipiDBU1DMF —> —> O O 2. A020 Capping (222296) 2,OGOH .3 Kaiser: Negetive O HN’FmOC DMTO KG Fmoc\N/W\n/OH“ o O DMTr\ 136 O b . N HBTU, DIEA, DMF 135 ’O NH-Fmoc DMTr| 1. pip-:DBUzDMi:_ 1. 2% hydrazine/DMF : Posmve —> kHCH Kaiser. Positive 2. Dde-Lys(Fmoc)—OH (138) . 2. Fmoc—Lys(Fmoc)—OH (140) HATU, DIEA, DMF 0: ”0101—, HATU DIEA DMF Kaiser: Negative Kaiser. ve O /Fmoc N \Fmoc l_|N\Fmoc AcO OAC AcHN OWNH AcO OAC AcO OW}N N ACHN 1. pip:DBU:DMF O H Kalser: Posmve 2. 7, HATU, DIEA, AcO OAc K ' alser: N ega Ivet' AcO OWN AcHN O o AcO OAC AcHN OWNH Synthesis of Compound 134. To a Merrifield flask was added aminomethyl VIMAD resin (2.5 g, 450 umol/g) that was washed with acetonitrile, dimethylformamide, dichloromethane and acetonitrile. The resin was swelled in acetonitrile (4 mL). Compound 133 was pre-activated in a 100 mL round bottom flask by adding 20 (1.0 mmol, 0.747 g), TBTU (1.0 mmol, 0.321 g), itrile (5 mL) and DIEA (3.0 mmol, 0.5 mL). This solution was allowed to stir for 5 min and was then added to the Merrifield flask with shaking.
The suspension was allowed to shake for 3 h. The reaction mixture was drained and the resin was washed with acetonitrile, DMF and DCM. New resin loading was quantitated by measuring the ance of the DMT cation at 500 nm (extinction coefficient = 76000) in DCM and determined to be 238 umol/g. The resin was capped by suspending in an acetic anhydride solution for ten s three times.
The solid support bound nd 141 was synthesized using iterative Fmoc-based solid phase peptide synthesis methods. A small amount of solid support was withdrawn and ded in aqueous ammonia (28-30 wt%) for 6 h. The cleaved compound was analyzed by LC-MS and the observed mass was consistent with structure. Mass m/z 1063.8 [M + 2H]+.
The solid support bound compound 142 was synthesized using solid phase peptide synthesis methods.
AcO OAC ACHN OW>\NH A00 OAC AcO OVV>\ O AcHN o H DNA syntesizer O H 142—> 3 A00 OAC AcO OWN AcHN o 0 A00 OAC AGO$¢OWNHO 143 ACHN HO OH ACHN O\/\/>\NH HO OH Hog/OW}O H o N N AcHN o H pH aqueous NH3 0 H —> 3 N HO OH o Hog/O O H NH | AcHN o o HO OH ACHN WNH The solid support bound nd 143 was synthesized using standard solid phase synthesis on a DNA synthesizer.
The solid support bound compound 143 was ded in aqueous ammonia (28-30 wt%) and heated at 55 0C for 16 h. The solution was cooled and the solid support was filtered. The e was concentrated and the residue dissolved in water and purified by HPLC on a strong anion exchange column. The fractions containing full length compound 144 were pooled together and desalted. The resulting GalNAc4-11 WO 79625 conjugated eric compound was analyzed by LC-MS and the observed mass was tent with structure.
The GalNAc4 cluster portion of the conjugate group GalNAc4-ll c4-lla) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.
The structure of GalNAc4-ll (GalNAc4-l la-CM) is shown below: HO OH EEWWMACHN HO OH EtwACHN ET“: AcHN o HO OH HO$Q/ WNHO O ACHN Example 51: Preparation of Oligonucleotide 155 Comprising GalNAc3-6 Q 0 H BrQL Q o o O N NH2 OTNWIVEOHH ‘5 OH O OH 0 2M NaOH o OH Compound 146 was synthesized as described in the literature (Analytical Biochemistry 1995, 229, 54- 60).
O AcO OAC H O 35b O 4 p A00 J\O TMS—OTf, 4 A molecular sieves, CHZCIZ, rt H AcHN Q 0 H A00 OAC O\n/N\)J\OH H2, Pd(OH)2 /C O O 147 —>ACO W\/\NH2 EtOAc/MeOH AcHN 105a HBTU, DIEA, DMF, rt AcO OAC Agog/OWNJK/ \n/O\/© —>HO H 0 H2, Pd(OH)2/C, EtOAc/MeOH AcHN 148 0 A00 OAC O O\/\/\/\N)l\/ AcHN Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol) were dissolved in CHgClg (200 ml). Activated molecular sieves (4 A. 2 g, powdered) were added, and the reaction was allowed to stir for 30 minutes under en atmosphere. TMS-OTf was added (4.1 ml, 22.77 mmol) and the reaction was allowed to stir at room temp overnight. Upon completion, the reaction was quenched by pouring into solution of saturated aqueous NaHC03 (500 ml) and crushed ice (N 150 g). The c layer was separated, washed with brine, dried over MgSO4, filtered, and was concentrated to an orange oil under reduced pressure. The crude material was purified by silica gel column chromatography and eluted with 2-10 % MeOH in CHgClg to yield Compound 112 (16.53 g, 63 %). LCMS and 1H NMR were consistent with the expected compound.
Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1 MeOH/EtOAc (40 ml). The reaction mixture was purged by ng a stream of argon through the on for 15 minutes. Pearlman’s catalyst (palladium ide on carbon, 400 mg) was added, and hydrogen gas was bubbled through the solution for s. Upon completion (TLC 10% MeOH in CHgClg, and LCMS), the catalyst was removed by filtration h a pad of celite. The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 105a (3.28 g). LCMS and 1H NMR were consistent with desired product.
Compound 147 (2.31 g, 11 mmol) was dissolved in anhydrous DMF (100 mL). N,N— Diisopropylethylamine (DIEA, 3.9 mL, 22 mmol) was added, followed by HBTU (4 g, 10.5 mmol). The reaction mixture was allowed to stir for N 15 minutes under nitrogen. To this a solution of compound 105a (3.3 g, 7.4 mmol) in dry DMF was added and stirred for 2 h under nitrogen atmosphere. The reaction was diluted with EtOAc and washed with saturated aqueous NaHC03 and brine. The organics phase was separated, dried ), filtered, and concentrated to an orange syrup. The crude material was purified by column chromatography 2-5 % MeOH in CH2C12 to yield Compound 148 (3.44 g, 73 %). LCMS and 1H NMR were consistent with the expected product.
Compound 148 (3.3 g, 5.2 mmol) was dissolved in 1:1 MeOH/EtOAc (75 ml). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman’s st (palladium hydroxide on ) was added (350 mg). Hydrogen gas was bubbled through the solution for 30 s. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration through a pad of celite. The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 149 (2.6 g). LCMS was consistent with d product. The residue was dissolved in dry DMF (10 ml) was used immediately in the next step.
ACO \/\(\/)/\NAJ\/ N ACHN 3 H O 146 —> AcO OAc o HBTU, DIEA,DMF o OWN/[V ACO 3 H NHAC ACO OAC o o AGO OWNJK/H O AC0 OAC Pd(OH)2/C,H2 ACHN 3 ’ Acog/ WNX/ \ll/\No £1 O N ji/mNH2 MeOH,EtOAc ACHN 3 H O ACO OAC o O OWN/kw 3 H NHAC Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF (20 ml). To this DIEA (450 uL, 2.6 mmol, 1.5 eq.) and HBTU (1.96 g, 0.5.2 mmol) were added. The on mixture was d to stir for 15 minutes at room temperature under nitrogen. A solution of compound 149 (2.6 g) in anhydrous DMF (10 mL) was added. The pH of the reaction was adjusted to pH = 9-10 by addition of DIEA (if ary). The reaction was allowed to stir at room temperature under nitrogen for 2 h. Upon completion the reaction was diluted with EtOAc (100 mL), and washed with aqueous ted aqueous NaHC03, followed by brine. The organic phase was ted dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel column chromatography and eluted with 2-10 % MeOH in CHgClg to yield Compound 150 (0.62 g, %). LCMS and 1H NMR were consistent with the desired product.
Compound 150 (0.62 g) was dissolved in 1:1 MeOH/ EtOAc (5 L). The reaction mixture was purged by bubbling a stream of argon h the solution for 15 minutes. Pearlman’s catalyst (palladium hydroxide on carbon) was added (60 mg). Hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration ge-tip Teflon filter, 0.45 um). The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 151 (0.57 g). The LCMS was consistent with the desired product. The product was dissolved in 4 mL dry DMF and was used immediately in the next step.
WO 79625 )J\/\/U\ o H O N BnO 0H Acog/ WNJK/ \ll/\N M 3 H OBn 83a O 151 —, H AcHN 3 o PFP-TFA DIEA DMF ’ ’ AcO 0A0 0 O OWN/[V A00 3 H NHAc A00 OAC o o o H AcO \/\M/\ N A00 AcHN 3 H O O o H Pd(OH)2/C, H2 0 N M —> Acog/OVWNJK/ \“/\N 3 H OH MeOH, EtOAc AcHN 3 H 0 AcO 0A0 0 L70 A00 0A0 AcHN 3 H m o o F PFP-TFA, DIEA N —>AC 3 3 H O DMF AcHN Lfo F AcO 0A0 0 O OWN/[V 3 H NHAc Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 mL) and N,N— Diisopropylethylamine (75 uL, 1 mmol) and PFP-TFA (90 uL, 0.76 mmol) were added. The reaction mixture turned a upon t, and gradually turned orange over the next 30 minutes. Progress of reaction was monitored by TLC and LCMS. Upon completion tion of the PFP ester), a solution of compound 151 (0.57 g, 0.33 mmol) in DMF was added. The pH of the reaction was adjusted to pH = 9-10 by addition of isopropylethylamine (if necessary). The reaction mixture was stirred under nitrogen for N min. Upon completion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CHgClg and washed with aqueous saturated NaHC03, followed by brine. The organic phase separated, dried over MgSO4, filtered, and concentrated to an orange syrup. The residue was purified by silica gel column chromatography (2-10 % MeOH in CHgClg) to yield nd 152 (0.35 g, 55 %). LCMS and 1H NMR were consistent with the desired product.
Compound 152 (0.35 g, 0.182 mmol) was dissolved in 1:1 MeOH/EtOAc (10 mL). The reaction mixture was purged by bubbling a stream of argon thru the solution for 15 minutes. Pearlman’s catalyst (palladium hydroxide on carbon) was added (35 mg). Hydrogen gas was bubbled thru the solution for 30 minutes. Upon tion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration (syringe-tip Teflon filter, 0.45 um). The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 153 (0.33 g, quantitative). The LCMS was consistent with desired product. nd 153 (0.33 g, 0.18 mmol) was dissolved in anhydrous DMF (5 mL) with stirring under nitrogen. To this N,N—Diisopropylethylamine (65 uL, 0.37 mmol) and A (35 uL, 0.28 mmol) were added. The reaction mixture was stirred under nitrogen for N 30 min. The reaction mixture turned magenta upon t, and gradually turned orange. The pH of the reaction mixture was maintained at pH = 9-10 by adding more N,-Diisopropylethylamine. The progress of the reaction was monitored by TLC and LCMS.
Upon tion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CH2C12 (50 mL), and washed with saturated aqueous NaHC03, followed by brine. The organic layer was dried over MgSO4, filtered, and concentrated to an orange syrup. The e was purified by column chromatography and eluted with 2-10 % MeOH in CHgClg to yield Compound 154 (0.29 g, 79 %). LCMS and 1H NMR were consistent with the desired product. 3' 5' || HOOH O -O-P-O-(CH2)6NH2 O OH 0%” AcHN HN HOOH o 1. Borate buffer, DMSO, o 154 pHssn—’ 0 H ' JV“ H Ho mm rm MNWO 2. aq. ammonia, 5 rt 0 4 AcHN O O HoOH £0] 0 HO oAHfHN O AcHN eric nd 155, comprising a 3-6 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc3 r portion of the conjugate group GalNAcg- 6 (GalNAc3-6a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.
The structure of GalNAc3-6 (GalNAc3-6a-CM-) is shown below: HoOH o WNJHo AcHN HN AcHN Example 52: Preparation of Oligonucleotide 160 Comprising GalNAc3-9 AcOOAC AcOOAcO Meow TMSOTf 50 °C /\M%OA© AcHN CICHZCHZCI rt 93% 4\i TMSOTf DCE 66% AcO OAC AcO OAc “0&0WW H2, Pd/C MeOH, 95A:0 “0&0W0“ AcHN AcHN 156 157 HBTU, DMF, EtN(iPr)2 Phosphitylation DMTO AcOOAc 81% AcHN ODMT H0: 47 NC Ooj_P/ \N(iPr)2 ACOWWNAcOOAC AcHN ODMT Compound 156 was synthesized ing the procedure described in the literature (J. Med. Chem. 2004, 47, 5798-5808).
Compound 156, (18.60 g, 29.28 mmol) was dissolved in methanol (200 mL). Palladium on carbon (6.15 g, 10 wt%, loading (dry basis), matrix carbon powder, wet) was added. The reaction mixture was stirred at room temperature under hydrogen for 18 h. The reaction e was filtered through a pad of celite and the celite pad was washed thoroughly with methanol. The combined e was washed and concentrated to dryness. The residue was purified by silica gel column chromatography and eluted with 5-10 % methanol in dichloromethane to yield Compound 157 (14.26 g, 89%). Mass m/z 544.1 [M-H]'.
Compound 157 (5 g, 9.17 mmol) was dissolved in anhydrous DMF (30 mL). HBTU (3.65 g, 9.61 mmol) and N,N—Diisopropylethylamine (13.73 mL, 78.81 mmol) were added and the reaction e was stirred at room ature for 5 s. To this a solution of compound 47 (2.96 g, 7.04 mmol) was added.
The reaction was stirred at room temperature for 8 h. The reaction mixture was poured into a saturated NaHC03 aqueous solution. The mixture was extracted with ethyl acetate and the organic layer was washed with brine and dried (Na2S04), filtered and evaporated. The residue obtained was purified by silica gel column chromatography and eluted with 50% ethyl acetate in hexane to yield compound 158 (8.25g, 73.3%).
The structure was confirmed by MS and 1H NMR analysis.
Compound 158 (7.2 g, 7.61 mmol) was dried over P205 under reduced pressure. The dried compound was dissolved in anhydrous DMF (50 mL). To this razole (0.43 g, 6.09 mmol) and N- methylimidazole (0.3 mL, 3.81 mmol) and 2-cyanoethyl-N,N,N’,N’-tetraisopropyl phosphorodiamidite (3.65 mL, 11.50 mmol) were added. The on mixture was stirred t under an argon atmosphere for 4 h. The on mixture was diluted with ethyl acetate (200 mL). The reaction mixture was washed with saturated NaHC03 and brine. The organic phase was separated, dried (Na2S04), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 50-90 % ethyl acetate in hexane to yield Compound 159 (7.82 g, 80.5%). The structure was confirmed by LCMS and “P NMR analysis.
HOOH ' O 0%“ HO 9 o o ACHN O—Fl> OH HOoH ' 1.DNAsynthesizer 159 0 MN 2. aq. NH4OH HO o o ACHN o-F'> OH HoOH O OWNED Oligomeric Compound 160, comprising a GalNAc3-9 conjugate group, was prepared using standard oligonucleotide synthesis procedures. Three units of compound 159 were coupled to the solid support, followed by nucleotide phosphoramidites. Treatment of the protected oligomeric compound with aqueous a yielded compound 160. The 3 cluster portion of the ate group GalNAc3-9 (GalNAcg- 9a) can be combined With any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The ure of GalNAc3-9 (GalNAc3- 9a-CM) is shown below: HoOH ' Hog/OmOo N ACHN 0—; OH HoOH o N HO o”$§j§ o ACHN 0-; OH HoOH ififiLg/O”*£\Wo N 0 s AcHN Example 53: Alternate procedure for preparation of Compound 18 (GalNAc3-1a and GalNAc3-3a) /\/\ H2N NHR H TMSOTf R = H or Cbz HO\/\/\n/N\/\/NHR OAC o OAC 161 = 0 CszI, Et3N E E: g’bgefiaezb AGO 4 Ny/O PFPO OAc h OAc O O 0 —> A00 o\/\/\n/N\/\/NHR + NHAc PFPOWOQ‘NHCBZ o o O O R = Cbz, 163a Pol/C, H2 l— PFPOM A00 (3%ng H NHAC OAc WNW/j ACO%/O‘§’)%l—NWNYVO%OAC O O O H H NHCBZ NHAC o O O Lactone 161 was reacted with diamino propane (3-5 eq) 0r Mono-B00 protected diamino propane (1 eq) to provide alcohol 162a 0r 162b. When unprotected propanediamine was used for the above reaction, the excess diamine was removed by evaporation under high vacuum and the free amino group in 162a was protected using CszI to provide 162b as a white solid after purification by column chromatography.
Alcohol 162b was further d with compound 4 in the presence of TMSOTf to e 163a which was converted to 163b by removal of the Cbz group using catalytic hydrogenation. The uorophenyl (PFP) ester 164 was prepared by reacting d 113 (see Example 48) with PFPTFA (3.5 eq) and ne (3.5 eq) in DMF (0.1 to 0.5 M). The triester 164 was directly reacted with the amine 163b (3—4 eq) and DIPEA (3—4 eq) to provide Compound 18. The above method greatly facilitates purification of intermediates and minimizes the formation of byproducts which are formed using the procedure bed in Example 4.
Example 54: ate procedure for preparation of nd 18 c3-1a and GalNAc3-3a) HOZC/\\ PFPTFA PFPO O DMF, pyr O /\/O NHCBZ O NHCBZ PFPO H020 W o o O HOZCJ 113 H 164 BocHN\/\/N7]/\\O 1. HCI or TFA —.BocHN H NHCBZ —» W Wl/V0% DIPEA /\/\ M BOCHN H ACO0%OWk OPFF 165 NHAC OACOOACQ: 166 O 1. 1 6-hexanediol AcO 0% H ’ 4 HN N or 1,5-pentane-dlol NHAc W 77/\\ TMSOTf + compound 4 2. TEMPO O O 3. PFPTFA, pyr H H o NHCBZ \/\/ (V NHAc o o 0 OACOAc HNWNW O H A00 0% NHAc The triPFP ester 164 was prepared from acid 113 using the ure outlined in example 53 above and reacted with mono-Boc protected diamine to provide 165 in essentially quantitative yield. The Boc groups were removed with hydrochloric acid or trifluoroacetic acid to provide the triamine which was reacted with the PFP activated acid 166 in the presence of a suitable base such as DIPEA to provide Compound 18.
The PFP protected Gal-NAG acid 166 was prepared from the corresponding acid by treatment with PFPTFA (l-l.2 eq) and pyridine (l-l.2 eq) in DMF. The precursor acid in turn was prepared from the corresponding alcohol by oxidation using TEMPO (0.2 eq) and BAIB in acetonitrile and water. The precursor alcohol was prepared from sugar intermediate 4 by on with 1,6-hexanediol (or 1,5-pentanediol or other diol for other n values) (2-4 eq) and TMSOTf using conditions described previously in example 47.
Example 55: Dose-dependent study of oligonucleotides comprising either a 3' or 5'-conjugate group rison of GalNAc3-1, 3, 8 and 9) targeting SRB-l in vivo The ucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice. Unconjugated ISIS 353382 was included as a rd. Each of the various GalNA03 conjugate groups was attached at either the 3' or 5' us of the respective ucleotide by a phosphodiester linked 2'-deoxyadenosine nucleoside (cleavable moiety).
Table 39 d ASO targeting SRB-l ASO Sequence (5’ to 3’) Motif Conjugate ID NO.
ISIS 3533 82 GCSmCCSTCSTCSmCCSACSGCSTCSmCCSACSTCSGCSACS / 1 0/5 none 143 (parent) mCdsTdsTesmCesmCesTesTe GCSmCCSTCSTCSmCCSACSGCSTCSmCCSACSTCSGCSACS ISIS 655 861 5/ 1 0/5 GalNAc3-1 144 sTesmCesmCesTesTeoAdo"GalNAc3' a GCS CCSTCSTCS CCSACSGCSTCS CCSACSTCSGCSACS ISIS 664078 5/10/5 GalNAC3-9 144 mCdsTdSTesmCesmCesTesTeoAdoa-GalNAC3- a GalNAc3-3a-0sAd0 ISIS 661 161 GesmCesTesTesmCesAcchsTcschsAcsTcchsAcs 5/ l 0/5 GalNAc3-3 145 InC:dsTdsTesmC:esmC:esTesTe GalNAc3-8a-0sAd0 ISIS 665001 GesmCesTesTesmCesAcchsTcschsAcsTcchsAcs 5/ l 0/5 GalNAc3-8 145 InCdSTdSTesmCesmCesTesTe Capital letters indicate the nucleobase for each nucleoside and InC indicates a 5-methyl cytosine.
Subscripts: “e” indicates a 2’-MOE modified nucleoside; “(1” indicates a B-D-2’-deoxyribonucleoside; “s” indicates a phosphorothioate ucleoside linkage (PS); “0” indicates a odiester internucleoside linkage (PO); and “0’” indicates -O-P(=O)(OH)-. Conjugate groups are in bold.
The structure of GalNAC3-la was shown previously in Example 9. The structure of GalNA03-9 was shown previously in Example 52. The structure of GalNA03-3 was shown previously in Example 39. The structure of 3-8 was shown previously in Example 47.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 353382, , 664078, 661161, 665001 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.
As illustrated in Table 40, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides comprising the odiester linked GalNAc3-1 and GalNAc3-9 conjugates at the 3’ terminus (ISIS 655861 and ISIS 664078) and the 3-3 and GalNAc3-8 conjugates linked at the 5’ terminus (ISIS 661161 and ISIS 665001) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 353382). rmore, ISIS 664078, comprising a GalNAc3-9 conjugate at the 3' us was ially equipotent ed to ISIS 655861, which comprises a GalNAc3-1 conjugate at the 3’ terminus. The 5' conjugated antisense oligonucleotides, ISIS 661161 and ISIS , comprising a GalNAc3-3 or GalNAc3-9, respectively, had increased potency compared to the 3' conjugated antisense oligonucleotides (ISIS 655861 and ISIS 664078).
Table 40 ASOs containing GalNAc3-1, 3, 8 0r 9 targeting SRB-l Dosage SRB-l mRNA ISIS N0. Conjugate. (m /k ) (% Saline) Saline n/a 100 3 88 353382 10 68 none 36 0.5 98 1'5 76 655861 GalNac3 -1 (3') 31 20 0.5 88 1.5 85 664078 GalNac3-9 (3 ), 46 20 0.5 92 1.5 59 661161 GalNac3-3 (5), 19 11 0.5 100 1.5 73 665001 GalNac3-8 (5 ), 29 13 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group.
ALTs, ASTs, total bilirubin and BUN values are shown in the table below.
Table 41 Dosage Total ISIS N0. ALT AST BUN Conjugate. mg/kg Bilirubin Saline 24 59 0.1 37.52 3 21 66 0.2 34.65 353382 10 22 54 0.2 34.2 none 22 49 0.2 33.72 0.5 25 62 0.2 30.65 1.5 23 48 0.2 30.97 655861 —5GalNac3-1 (3), 28 49 0.1 32.92 40 97 0.1 31.62 0.5 40 74 0.1 35.3 1.5 47 104 0.1 32.75 664078 —5GalNac3-9 (3 ), 43 0.1 30.62 38 92 0.1 26.2 0.5 101 162 0.1 34.17 1.5 g 42 100 0.1 33.37 661161 GalNac3-3 (5), g 23 99 0.1 34.97 53 83 0.1 34.8 0.5 28 54 0.1 31.32 1.5 42 75 0.1 32.32 665001 —5GalNac3-8 (5 ), 24 42 0.1 31.85 32 67 0.1 31.
Example 56: Dose-dependent study of oligonucleotides comprising either a 3' 0r 5'-c0njugate group rison of GalNAc3-1, 2, 3, 5, 6, 7 and 10) targeting SRB-l in vivo The ucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various GalNA03 conjugate groups was attached at the 5 ' terminus of the respective oligonucleotide by a phosphodiester linked 2'-deoxyadenosine nucleoside (cleavable moiety) except for ISIS 655861 which had the GalNA03 conjugate group attached at the 3’ terminus.
Table 42 Modified ASO targeting SRB-l ASO ce (5 ’ to 3 ’) Motif Conjugate ID NO.
ISIS 3 53 3 82 GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds / 1 0/5 ‘ no conjugate 143 (parent) mCdsTdsTesmCesmCesTesTe GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds ISIS 655 861 mCdsTdsTesmCesmCesTesTeoAdo"GalNAc3-1a 5/ 1 O/5 3-1 144 GalNAc3'2a'o’AdoGesmCesTesTesmCesAdsGdsTds ISIS 664507 5/ 1 O/5 GalNAc3-2 145 mCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe GalNAc3-3a-0sAd0 ISIS 661 161 GesmCesTesTesmCesAdSGdSTdSmCdSAdSTdSGdSAdS 5/ 1 O/5 GalNAc3-3 145 mCdsTdsTesmCesmCesTesTe ISIS 666224 GalNAc3-5a-oaAdoGesmCesTesTesmCesAdsGdsTdS 5/ 1 O/5 GalNAc3-5 145 2014/036460 InCdslAdsTdsCIdsIAdsmCdsTdsTesmCesmCesTesTe GalNAc3'6a'0’Ad0GesmCesTesTesmCesAdsGdsTds ISIS 666961 5/10/5 GalNAc3-6 145 InCdslAdsTdsCIdsIAdsmCdsTdsTesmCesmCesTesTe GalNAc3'7a'0’Ad0GesmCesTesTesmCesAdsGdsTds ISIS 666981 5/10/5 GalNAc3-7 145 InCdslAdsTdsCIdsIAdsmCdsTdsTesmCesmCesTesTe 3-10a'0’AdoGesmCesTesTesmCesAdsGdsTds ISIS 666881 5/10/5 GalNAc3-10 145 InCdslAdsTdsCIdsIAdsmCdsTdsTesmCesmCesTesTe Capital letters indicate the nucleobase for each nucleoside and InC indicates a 5-methyl cytosine.
Subscripts: “e” tes a 2’-MOE modified nucleoside; “d” indicates a -deoxyribonucleoside; “s” indicates a phosphorothioate intemucleoside e (PS); “0” indicates a phosphodiester intemucleoside linkage (PO); and “0’” indicates -O-P(=O)(OH)-. Conjugate groups are in bold.
The structure of GalNAc3-1a was shown usly in Example 9. The ure of GalNAc3-2a was shown usly in Example 37. The structure of GalNAc3-3a was shown previously in Example 39. The ure of GalNAc3-5a was shown previously in Example 49. The structure of GalNAc3-6a was shown previously in Example 51. The structure of GalNAc3-7a was shown previously in Example 48. The structure of GalNAc3-10a was shown previously in Example 46.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected aneously once at the dosage shown below with ISIS , 655861, 664507, 661161, 666224, 666961, 666981, 666881 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the e percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.
As illustrated in Table 43, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner. Indeed, the conjugated nse oligonucleotides showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 353382). The 5' conjugated antisense oligonucleotides showed a slight increase in potency compared to the 3' conjugated antisense oligonucleotide.
Table 43 Dosage SRB-l mRNA ISIS N0. Conjugate. (mg/kg) (% Saline) Saline n/a 100.0 —-E_ 353382 none 655861 GalNaC3-1 (3') 1.5 81.2 33.9 15.2 0.5 102.0 1.5 73.2 664507 —5 GalNac3-2 (5 ), 31-3 10.8 0.5 90.7 1.5 67.6 661161 —5 GalNac3-3 (5), 24.3 11.5 0.5 96.1 1.5 61.6 666224 GalNac3-5 (5), 25.6 11.7 0.5 85.5 1.5 56.3 666961 —534-2 GalNAc3-6 (5), 13.1 0.5 84.7 1.5 59.9 666981 —524.9 GalNAc3-7 (5), 8.5 0.5 100.0 1.5 65.8 666881 —5 GalNAc3-10 (5 ), 26.0 13.0 Liver transaminase levels, alanine aminotransferase (ALT) and ate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group.
ALTs, ASTs, total bilirubin and BUN values are shown in Table 44 below.
Table 44 Dosage Total ISIS N0. ALT AST BUN Conjugate. mg/kg Bilirubin Saline 26 57 0-2 27 3 25 92 0.2 27 353382 10 23 40 0.2 25 none 29 54 0.1 28 0.5 25 71 0.2 34 1.5 28 60 0.2 26 655861 , —526 GalNac3-1 (3) 63 0‘2 28 25 61 0.2 28 0.5 25 62 0.2 25 1.5 24 49 0.2 26 664507 —5GalNac3-2 (5 ), 21 50 0.2 26 59 84 0.1 22 0.5 20 42 0.2 29 1.5 g 37 74 0.2 25 661161 GalNac3-3 (5), g 28 61 0.2 29 21 41 0.2 25 0.5 34 48 0.2 21 1.5 23 46 0.2 26 666224 —5GalNac3-5 (5), 24 47 0.2 23 32 49 0.1 26 0.5 17 63 0.2 26 1.5 23 68 0.2 26 666961 —5GalNAc3-6 (5 ), 66 0.2 26 29 107 0.2 28 0.5 24 48 0.2 26 1.5 30 55 0.2 24 666981 —5GalNAc3-7 (5), 46 74 0.1 24 29 58 0.1 26 0.5 20 65 0.2 27 1.5 23 59 0.2 24 666881 GalNAc3-10 (5 ), 45 70 0‘2 26 21 57 0.2 24 Example 57: Duration of action study of oligonucleotides comprising a 3'-conjugate group targeting ApoC III in vivo Mice were injected once with the doses indicated below and monitored over the course of 42 days for ApoC-III and plasma triglycerides (Plasma TG) . The study was med using 3 transgenic mice that express human APOC-III in each group.
Table 45 Modified ASO targeting ApoC III Sequence (5’ to 3’) SEIEJD ISIS AesGesmCesTesTesmCdsTdsTdsGdsTds PS 13 5 304801 mC:dslAdstdsmChsTesTesTeslAesTe 64753 5 AdsGdSmCdSTesTesTesAesTeoAdos-GalNAc3-1 a AesGeomCeoTeoTeomCdsTdsTdsGdsTdsmCdsmCds 64753 6 AdSGdSmCdSTeoTeoTesAesTeoAdoa-GalNAc3-1 a Capital letters indicate the nucleobase for each nucleoside and InC indicates a 5-methyl cytosine.
Subscripts: “e” indicates a 2’-MOE modified nucleoside; “(1” indicates a B-D-2’-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “0” indicates a phosphodiester internucleoside linkage (PO); and “0’” indicates O)(OH)-. ate groups are in bold.
The structure of 3-la was shown previously in Example 9.
Table 46 ApoC III mRNA (% Saline on Day 1) and Plasma TG Levels (% Saline on Day 1) ASO Dose Target Day 3 Day 7 Day 14 Day 35 Day 42 Saline 0 mg/kg II 98 100 100 95 116 ISIS 304801 30 mg/kg ApoC-III 28 30 41 65 74 ISIS 647535 10 mg/kg ApoC-III 16 19 25 74 94 ISIS 647536 10 mg/kg ApoC-III 18 16 17 35 51 Saline 0 mg/kg Plasma TG 121 130 123 105 109 ISIS 304801 30 mg/kg Plasma TG 34 37 50 69 69 ISIS 647535 10 mg/kg Plasma TG 18 14 24 18 71 ISIS 647536 10 mg/kg Plasma TG 21 19 15 32 35 As can be seen in the table above the duration of action increased with addition of the 3'-conjugate group compared to the unconjugated oligonucleotide. There was a further increase in the duration of action for the conjugated mixed PO/PS oligonucleotide 647536 as compared to the conjugated full PS oligonucleotide 647535.
Example 58: Dose-dependent study of oligonucleotides comprising a 3'-conjugate group (comparison of GalNAc3-1 and 4-11) targeting SRB-l in vivo The oligonucleotides listed below were tested in a dose-dependent study for antisense tion of SRB-l in mice. Unconjugated ISIS 440762 was included as an unconjugated standard. Each of the conjugate groups were attached at the 3' terminus of the respective oligonucleotide by a phosphodiester linked 2'-deoxyadenosine nucleoside cleavable moiety.
The structure of GalNAc3-1a was shown previously in e 9. The structure of GalNAc3-11a was shown previously in e 50.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900, 663748 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-l mRNA levels using real-time PCR and EEN® RNA quantification t (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the e percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.
As illustrated in Table 47, treatment with antisense oligonucleotides d SRB-l mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising the phosphodiester linked GalNAc3-1 and GalNAc4-11 conjugates at the 3’ terminus (ISIS 651900 and ISIS 663748) showed substantial improvement in potency compared to the unconjugated nse ucleotide (ISIS 440762). The two conjugated oligonucleotides, GalNAc3-1 and GalNAc4-11, were equipotent.
Table 47 Modified ASO targeting SRB-l 9 9 % Saline SEQ ID ASO Sequence (5 to 3 ) Dose mg/kg control No.
Saline 100 0.6 73.45 m m rsrs 440762 Egg ghfidfii‘g“ CdSAdSTdSGdSAdS 2 59.66 137 ds ds ks k 6 2350 0.2 62.75 TkskasAdsGdsTdSmCdSAdSTdSGdSAdS 0.6 29.14 ISIS 651900 13 8 mCdsTdSTkskaoAdol-GalNAC3-1a 2 8.61 6 5.62 0.2 63.99 TkskasAdsGdsTdsmCdsAdsTdsGdsAds 0-6 33-53 ISIS 663748 138 InCdSTdsTksmclmAdo.-GalNAc4-11a 2 7.58 6 5.52 Capital letters indicate the nucleobase for each side and InC indicates a 5-methyl cytosine.
Subscripts: “e” indicates a 2’-MOE modified nucleoside; “k” indicates 6’-(S)-CH3 bicyclic nucleoside; “d” indicates a B-D-2’-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “0” indicates a phosphodiester internucleoside linkage (PO); and “0’” indicates -O-P(=O)(OH)-. Conjugate groups are in bold.
Liver transaminase levels, e aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was ted with no significant change from the saline group.
ALTs, ASTs, total bin and BUN values are shown in Table 48 below.
Table 48 Dosage Total ISIS N0. ALT AST BUN Conjugate. mg/kg Bilirubin Saline 30 76 0-2 40 0.60 32 70 0.1 35 440762 2 26 57 0.1 35 none 6 31 48 0.1 39 0.2 32 115 0.2 39 0.6 33 61 0.1 35 651900 GalNac3-1 (3), 2 30 50 0‘1 37 6 34 52 0.1 36 0.2 28 56 0.2 36 663748 0.6 34 60 0.1 35 GalNac4-11 (3') 2 44 62 0.1 36 6 38 71 0.1 33 Example 59: Effects of 3-1 conjugated ASOs targeting FXI in vivo The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of FXI in mice. ISIS 404071 was included as an unconjugated standard. Each of the conjugate groups was attached at the 3' terminus of the respective oligonucleotide by a phosphodiester linked 2'-de0xyaden0sine nucleoside cleavable .
Table 49 Modified ASOs targeting FXI SEQ ID AS0 Sequence (5’ t0 3’) Linkages 404071 TdsTdsTdsmCdsAesGesAesGesGe 656172 TdSTdSTdSmCdSAesGesAesGesGeoAdo’-GalNAc3-1 a ISIS TesCleocjeoTeolAeolAdsTdsmC:dsmC:dslAdsmChs PO/PS 656173 TdsTdsTdsmcdsAeoGeersGesGeoAdos-Ga1NAc3-1a Capital letters indicate the nucleobase for each side and InC indicates a 5-methyl cytosine.
Subscripts: “e” indicates a 2’-MOE modified nucleoside; “(1” indicates a B-D-Z’-de0xyrib0nucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “0” indicates a phosphodiester ucleoside linkage (PO); and “0’” indicates -O-P(=O)(OH)-. Conjugate groups are in bold.
The structure of GalNA03-1a was shown previously in Example 9.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously twice a week for 3 weeks at the dosage shown below with ISIS 404071, 656172, 656173 or with PBS treated control. Each treatment group consisted of 4 s. The mice were sacrificed 72 hours following the final administration to determine the liver FXI mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent ular Probes, Inc. Eugene, OR) according to standard protocols. Plasma FXI n levels were also ed using ELISA. FXI mRNA levels were ined relative to total RNA (using RIBOGREEN®), prior to normalization to PBS-treated control. The results below are presented as the average percent of FXI mRNA levels for each treatment group. The data was ized to PBS-treated control and is denoted as “% PBS”. The EDsos were measured using similar methods as described previously and are presented below.
Table 50 Factor XI mRNA (% Saline) Dose ASO % Control Conjugate Linkages mg/kg WO 79625 Saline 100 none 3 92 $148071 10 40 none PS 15 0.7 74 ISIS 2 33 GalNAc3-1 PS 656172 6 9 0.7 49 ISIS 656173 2 i2 GalNAc3-1 PO/PS As illustrated in Table 50, treatment with nse oligonucleotides lowered FXI le\A levels in a dose-dependent manner. The oligonucleotides comprising a 3'-GalNAc3-1 conjugate group showed substantial improvement in y compared to the ugated antisense oligonucleotide (ISIS 404071).
Between the two conjugated oligonucleotides an improvement in potency was further provided by substituting some of the PS linkages with P0 (ISIS 656173).
As illustrated in Table 50a, treatment with antisense oligonucleotides lowered FXI protein levels in a ependent manner. The oligonucleotides comprising a 3'-GalNAc3-1 conjugate group showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071).
Between the two conjugated oligonucleotides an improvement in potency was r provided by substituting some of the PS linkages with P0 (ISIS 656173).
Table 50a Factor XI n (% Saline) A80 1131;; 1232:3321) (% Conjugate Linkages Saline 100 none $1.807. To PS 3 1s1s GalNAc3-1 PS 656172 2 23 6 1 1s1s GalNAc3-1 PO/PS 656173 2 6 6 0 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate ransferase (AST), in serum were measured relative to saline injected mice using standard ols. Total bilirubin, total albumin, CRE and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in the table below.
Table 51 Dosage Total Total ISIS No. ALT AST CRE BUN Conjugate. m /k Albumin Bilirubin Saline 71.8 84.0 3.1 0.2 0.2 22.9 3 152.8 176.0 3.1 0.3 0.2 23.0 404071 10 73.3 121.5 3.0 0.2 0.2 21.4 none 82.5 92.3 3.0 0.2 0.2 23.0 0.7 62.5 111.5 3.1 0.2 0.2 23.8 656172 2 33.0 51.8 2.9 0.2 0.2 22.0 GalNac3-1 (3') 6 65.0 71.5 3.2 0.2 0.2 23.9 0.7 54.8 90.5 3.0 0.2 0.2 24.9 656173 2 85.8 71.5 3.2 0.2 0.2 21.0 GalNac3-1 (3') 6 114.0 101.8 3.3 0.2 0.2 22.7 e 60: Effects of conjugated ASOs targeting SRB-l in vitro The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of SRB-l in primary mouse hepatocytes. ISIS 353382 was ed as an unconjugated standard. Each of the conjugate groups were attached at the 3' 0r 5' terminus of the respective ucleotide by a odiester linked 2'-de0xyaden0sine nucleoside cleavable moiety.
Table 52 Modified ASO targeting SRB-l ASO Sequence (5’ to 3’) Motif Conjugate fggo.
ISIS 353382 S§:¥::$:Eg:gg:fif¥fdSmCdSAdsTdsGdsAds 5/10/5 none 143 ISIS 655861 SC;EECmECAgfingcag‘kigdlAd 5/10/5 GalNAc3-1 144 ISIS 655862 SC;Egcmgc‘ififidfidmcégghf‘i‘id 5/10/5 GalNAc3-1 144 ISIS 661161 gaileXdidadchmInchTZTTm?méd(T}dT 5/10/5 3-3 145 ISIS 665001 $32233::ajigmccdfii:;E::%:$:Te 5/10/5 GalNAc3-8 145 ISIS 664078 SC;EECmECAgfingcég‘kiggAd 5/10/5 GalNAc3-9 144 ISIS 666961 $33?!ngGfdAfmZSTETTmZCm‘édng 5/10/5 GalNAc3-6 145 ISIS 664507 Egflfiaédlgdmgflfgmgmgf‘fdgfld 5/10/5 GalNAc3-2 145 ISIS 666881 SaifiifidlcgwfigcfrjfiEchZCTA‘iGde 5/10/5 GalNAc3-10 145 ISIS 666224 Eé‘jififiédfigflffém2???“ 5/10/5 GalNAc3-5 145 ISIS 666981 Egjifidédsdmgdifgmg:23)??de 5/10/5 GalNAc3-7 145 Capital s indicate the nucleobase for each nucleoside and InC indicates a 5-methyl cytosine.
Subscripts: “e” indicates a 2’-MOE modified nucleoside; “d” indicates a B-D-2’-deoxyribonucleoside; “s” tes a phosphorothioate intemucleoside linkage (PS); “0” indicates a phosphodiester intemucleoside linkage (PO); and “0’” indicates -O-P(=O)(OH)-. Conjugate groups are in bold.
The structure of GalNAc3-la was shown usly in Example 9. The structure of GalNAc3-3a was shown previously in Example 39. The structure of GalNAc3-8a was shown previously in e 47. The ure of GalNAc3-9a was shown previously in e 52. The structure of GalNAc3-6a was shown usly in Example 51. The structure of GalNAc3-2a was shown previously in Example 37. The structure of GalNAc3-10a was shown usly in Example 46. The structure of GalNAc3-5a was shown previously in Example 49. The ure of GalNAc3-7a was shown previously in Example 48.
Treatment The oligonucleotides listed above were tested in vitro in primary mouse hepatocyte cells plated at a density of 25,000 cells per well and treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 or 20 nM modified oligonucleotide. After a treatment period of imately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR and the SRB-l mRNA levels were adjusted according to total RNA content, as ed by RIBOGREEN®.
The IC50 was calculated using standard methods and the results are presented in Table 53. The results show that, under free uptake conditions in which no reagents or electroporation techniques are used to artificially e entry of the oligonucleotides into cells, the oligonucleotides comprising a GalNAc conjugate were significantly more potent in hepatocytes than the parent oligonucleotide (ISIS 353382) that does not comprise a GalNAc conjugate.
Table 53 Internucleoside SEQ ID ASO ICso (nM) Conjugate linkages N0.
ISIS 353382 19021 PS none 143 ISIS 655861 11a PS GalNAc3-1 144 ISIS 655862 3 PO/PS GalNAc3-1 144 ISIS 661161 15a PS GalNAc3-3 145 ISIS 665001 20 PS GalNAc3-8 145 ISIS 664078 55 PS GalNAc3-9 144 ISIS 666961 22a PS GalNAc3-6 145 ISIS 664507 30 PS GalNAc3-2 145 ISIS 666881 30 PS GalNAc3-10 145 ISIS 666224 3021 PS GalNAc3-5 145 ISIS 666981 40 PS GalNAc3-7 145 21Average of multiple runs.
Example 61: Preparation of oligomeric compound 175 comprising GalNAc3-12 2014/036460 AcO B \ 00 ”MNHZ O 0A0 pprJKAA/o 0 o A Oc OAC 91a 0A0 —> \ /\/\ O HN N N O Ac H H OAc 166 HN 167 \AC HOOC /N N A00 CBZ 0 ¥COOH TFA 169 COOH _. HZNMN — H OAC DCM HN\AC HBTU DIEA DMF A00 OAc ©\/O\n/NH (LNW O A O0 N\ O OAc /\/\ W0 0 O N N OAc o HN H H HN AcO o o 170 HN A0 A00 OAc M0O OAC Pd(0H)2/C,H2 HN o H ”MAC MeOH/EtOAc —> RLNJV O AcO H2N N 0 W0:[ OAc A”MHWO0 HN AcO o 0 171 ”N 2014/036460 benzyl (perfluorophenyl) glutarate AcO OAc MO0 OAc HN HN\ o o N N o HN H H HN AcO O 0 1 72 AcO OAc JOK/VVO 0 OAc HN HN\ Pd(OH)2/C,H2 “\J/\J Ac 172 ——————————> H |/»/ OAC O A“) HOWT/\v/\WVN N o OAc O 0 WI ANMNWOQQOA‘ o HN H H HN AcO o 0 173 HN\ AcO OAc PFPIFA —> o o DEA DMF )LV/~\/“\/O OAc “N HN o H ‘Ac O A oc 174 HN\ 3' 5' || OLIGO O-(CH2)e—NH2 1. Borate 174 buffer, DMSO, pH 8.5, rt 2. aq. ammonia, rt HO O ACHN M O O NHAC nd 169 is commercially available. Compound 172 was prepared by addition of benzyl (perfluorophenyl) glutarate to compound 171. The benzyl (perfluorophenyl) ate was prepared by adding PFP-TFA and DIEA to 5-(benzyloxy)oxopentanoic acid in DMF. Oligomeric compound 175, comprising a GalNAc3-12 conjugate group, was prepared from compound 174 using the general procedures illustrated in Example 46. The GalNAc3 cluster portion of the conjugate group GalNAc3-12 (GalNAc3-12a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a certain embodiments, the ble moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-12 (GalNAc3-12a-CM-) is shown below: WO 79625 OH OH HofiowvokACHN Ol'bH in HO%O O O j:O/\/\ o H H H ‘H/ a H 5 Fr 0 OH JJJ’NH HO o 0 H056 NHAC Example 62: Preparation of oligomeric compound 180 comprising GalNAc3-13 2014/036460 OOAc AcHN OMOH 0M0 HATU, HOAt DIEA, DMF 0A0 0A0 A00 0M AcHN OOAc H2, Pd/C AcO OWN —> AcHN (DH/MO 0A0 0A0 O 177 A00 OW AcHN 0 0A0 0A0 A00 o\/\/\/U\ AcHN “0&0OAco\/\/\/ICJ:N PFPTFA TEA AcHN OH/VWOH OAC OAC 178 ACHN O 0A0 0A0 A00 0M AcHN NH OAC OAC ACOfiOWOk O F Ojku/m/EI/ D?FH O F AcHN H 0A0 0A0 ACHN O 3' 5' || -O-F|’—O-<CH2>6‘NH2 1. Borate , DMSO, pH 8.5, rt 2. aq. ammonia, rt OH OH Hofi/OMOO AcHN NH OH OH E45: 0 O AcHN N HM W0 OLIGO o o ACHN Compound 176 was prepared using the general procedure shown in Example 2. Oligomeric compound 180, comprising a GalNAc3-l3 conjugate group, was prepared from compound 177 using the general procedures illustrated in Example 49. The GalNAc3 cluster portion of the conjugate group GalNAc3-l3 c3-l3a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a certainembodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-13 (GalNAc3-13a-CM-) is shown below: OHOH HOW \/\/\)OJ\0 NH AcHN OHOH HO 0 O H O H o O\/\/\)LN N NWN‘6; a AcHN H O H O OH “NH o o Example 63: Preparation of oligomeric compound 188 sing GalNAc3-14 1816 N N NHCB O HOW/V0%NHCBZ Ho(/\);5 3’ Z 4 7/ HBTU DIEA Op DMF HO (3);) HO VZH 13 182 OAc OAc A00 A00 ONGNWO Acofl/ONGWHN AcO OAc NHAC AOOAC NHAC O 0 A00 CNN C NHCBZ Pd/C, H2 AcO \fl/VO A00 OWle/VO\%NH26 o o NHAC :N’<—/O NchAC AGO W OAc OWN AGO 0H6 0 A00 6H AcO NHAC Op “3%?m“AcO ON 1. Pd/C, H2 HO\n/\/\n/ OAC NHAC 03%NZ’LO—> 2 PFP.TFA AcO IDYr, —> ACO HBTU, NHACAAC DIEA, AcO DMF 0 A00 GHW NHAC ACOAcofl/ONGH \H/\\O F A:O&/Of\%/ 2:9ng F NHAc NHAC 83e HO 3' 5' II OLIGO O-F|’-O-(CH2)6-NH2 %oNHN HOHSH 6 EDI/\b m NHAc H CH O N O 1. Borate buffer, DMSO, pH 8.5, rt HO\é 0% TI/VN H H“ OLIGO —, NHAc O o 0 2. aq. ammonia, rt HO H o N HO 6'" 188 NHAc Compounds 181 and 185 are commercially available. Oligomeric compound 188, comprising a 3-14 conjugate group, was prepared from compound 187 using the general procedures illustrated in Example 46.
The GalNAc3 cluster portion of the conjugate group GalNAc3-14 c3-14a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of 3-14 (GalNAc3-14a-CM-) is shown below: HOOH O O o N AcHN o HOOH O O O H0%O O NJK/‘O JJ\/\/u\ é H M ”W0 < AcHN o HoOH O t o N O H0 10 H AcHN Example 64: Preparation of oligomeric nd 197 comprising GalNAc3-15 AcO 0A0 OTBS OTBS “0%” K] A00 OAC O A HNC N H ”gem” HBTU, DIEA ACHN 7 N NH3/MeOH 8220, DMAP HO OH HO O ACHN OTBS ACHN AcHN Phosphitylation B::OOB%/o Q? AcHN DMTO / O O—P \/\/O DMTO 5' DMTO 3.
/\/\O DMTO 88, DNA sizer 196 1. 194, DNA synthesizer AcHN —> O /O N \P/ 2. Aq NH355 O0, 18h o |\OH O O // \/\/O HO OH O\ O U . W HO aw“ / OH /\/\O NHAC O OH 0 Compound 189 is commercially available. Compound 195 was prepared using the general procedure shown in Example 31. Oligomeric compound 197, comprising a GalNAc3-15 conjugate group, was prepared from compounds 194 and 195 using standard oligonucleotide synthesis procedures. The GalNAc3 r portion of the conjugate group GalNAc3-15 (GalNAc3-15a) can be combined with any cleavable moiety to e a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.
The structure of GalNAc3-15 (GalNAc3-15a-CM-) is shown below: HOOH kWU0,15:“PkAcHN O’P‘OIQMO/aVO-\m—E NHAC Example 65: Dose-dependent study of oligonucleotides comprising a 5’-conjugate group (comparison of GalNAc3-3, 12, 13, 14, and 15) targeting SRB-l in vivo The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice. Unconjugated ISIS 353382 was included as a standard. Each of the GalNA03 conjugate groups was attached at the 5' terminus of the respective oligonucleotide by a phosphodiester linked 2'- denosine nucleoside (cleavable moiety).
Table 54 Modified ASOs targeting SRB-l ISIS Sequences (5’ to 3’) Conjugate SEQ N0. ID —N°- 3 5 3 3 82 GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAdsmIEdsTdsTesmCesmCesTesTe none 143 661161 GalNAc30aAd0GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAdsmCdSTdS Gal\ACa-3 145 Te:nCe:nCesTesTe 671144 GalNAc3-12a-OaAd0GesmCesTeSTesmCesAdsGulsTdSmC,1,AdsT,1,GulsAdsmCdsTdS Gamma-12 145 smCCSTCST 670061 GalNAc30aAd0GesmCesTesTesmCesAdsGdsTdSmCdsAdsTdsGdsAdsmCdSTdS Gal\ACa-13 145 TesmcesmCCSTCST 671261 GalNAc3-14a-OaAd0GesmCesTeSTesmCesAdsGulsTdSmC,1,AdsT,1,GulsAdsmCdsTdS Gal\ACa-14 145 Te:nCe:nCesTesTe 671262 GalNAc3-15a-oaAdoGesmCCSTesTesmCesAdsGdsTdSmCdsAdsTdsGdsAdsmCdsTds Gal\ACa-15 145 Tes Ces Te Capital letters indicate the nucleobase for each nucleoside and InC indicates a 5-methyl cytosine. Subscripts: ‘6 633 indicates a 2’-MOE d nucleoside; “(1” indicates a B-D-2’-deoxyribonucleoside; 66 S33'1ndicates phosphorothioate ucleoside linkage (PS); 66 033'1ndicates a phosphodiester ucleoside e (PO); and “0 indicates -O-P(=O)(OH)-. Conjugate groups are in bold- The structure of GalNAc3-3a was shown previously in Example 39. The structure of GalNAc3-12a was shown previously in Example 61. The structure of GalNAc3-13a was shown previously in Example 62.
The structure of GalNAc3-14a was shown previously in Example 63. The structure of GalNAc3-15a was shown previously in Example 64.
Treatment Six to eight week old C57bl6 mice (Jackson Laboratory, Bar , ME) were injected subcutaneously once or twice at the dosage shown below with ISIS 353382, 661161, 671144, , 671261, 671262, or with saline. Mice that were dosed twice received the second dose three days after the first dose. Each ent group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA fication reagent (Molecular Probes, Inc. Eugene, OR) ing to standard protocols. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.
As rated in Table 55, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner. No significant differences in target knockdown were observed between s that ed a single dose and animals that received two doses (see ISIS 353382 s 30 and 2 x 15 mg/kg; and ISIS 661161 dosages 5 and 2 x 2.5 . The antisense oligonucleotides comprising the phosphodiester linked GalNAc3-3, 12, 13, 14, and 15 conjugates showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 335382).
Table 55 SRB-l mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-l mRNA (% ED50 (mg/kg) Conjugate Saline) Saline n/a 100.0 n/a n/a 3 85.0 69.2 353382 22.4 —3034-2 none 2 x 15 36.0 0.5 87.4 1.5 59.0 661161 5 25.6 2.2 GalNAc3-3 2 x 2.5 27.5 17.4 0.5 101.2 671144 —;'533(1) 3.4 GalNAc3-12 17.6 0.5 94.8 670061 1.5 57.8 2.1 GalNAc3-13 20.7 13.3 0.5 110.7 1.5 81.9 671261 4.1 GalNAcg-l4 39.8 14.1 671262 GalNAc3- 1 5 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The changes in body weights were ted with no significant differences from the saline group (data not shown). ALTs, ASTs, total bilirubin and BU\I values are shown in Table 56 below.
Table 56 Total Dosage ALT . . . BUN ISIS No. AST (U/L) B111rub1n Conjugate. (mg/kg) (U/L) ) (mg/(1L) Saline n/a 28 60 0.1 39 n/a 3 30 77 0.2 36 25 78 0.2 36 353382 “one 28 62 0.2 35 2 x 15 22 59 0.2 33 0.5 39 72 0.2 34 1.5 26 50 0.2 33 661161 5 41 80 0.2 32 GalNAc3-3 2 x 2.5 24 72 0.2 28 32 69 0.2 36 0.5 25 39 0.2 34 1.5 26 55 0.2 28 671144 GalNAc3-12 48 82 0.2 34 23 46 0.2 32 0.5 27 53 0.2 33 1.5 24 45 0.2 35 670061 3-13 23 58 0.1 34 24 72 0.1 31 0.5 69 99 0.1 33 1.5 34 62 0.1 33 671261 GalNAc3-14 43 73 0.1 32 32 53 0.2 30 0.5 24 51 0.2 29 1.5 32 62 0.1 31 671262 GalNAc3-15 30 76 0'2 32 31 64 0.1 32 Example 66: Effect of various cleavable moieties on antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising a NAc3 cluster WO 79625 The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice. Each of the GalNAc3 conjugate groups was attached at the 5' terminus of the respective oligonucleotide by a phosphodiester linked nucleoside (cleavable moiety (CM)).
Table 57 Modified ASOs ing SRB-l Mew—(WW No. Cluster ID No. 661161 GalNAc3-3a-0:AdoGeSmCCSTCSTCSmCCSAdSGdSTdSmCdSAdSTu1S Gal\ACa-3a Ad 145 GCSACSmccsrcsresmcesmcesresre 670699 GalNAc303TdoGeSmCeOTCOTCOmCeoAdSGdSTdSmCdSAdSTdS Gal\ACa-3a Ta 148 GCSACSmccsrcsreomceomcesresre 670700 GalNAc30:AeoGeSmCCOTeoTeomCeoAdSGdSTdSmCdSAdSTdS Gal\ACa-3a Ac 145 GCSACSmccsrcsreomceomcesresre 670701 GalNAc30aTeoGesmCeOTCOTeomCeoAdSGdSTdsmCdsAdSTds a-3a Te 148 GCSACSmccsrcsreomceomcesresre 671165 GalNAc30sAdOGesmCeoTeoTeomCeoAdsGdsTdsmCdSAdSTdS Gal\AC3-13a Ad 145 GCSACSmccsrcsreomceomcesresre Capital s indicate the nucleobase for each nucleoside and InC indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2’-MOE modified nucleoside; “d” indicates a B-D-Z’-deoxyribonucleoside; “s” tes a phosphorothioate intemucleoside linkage (PS); “0” indicates a phosphodiester intemucleoside linkage (PO); and “0’” indicates -O-P(=O)(OH)-. Conjugate groups are in bold.
The structure of GalNAc3-3a was shown usly in Example 39. The structure of GalNAc3-13a was shown previously in Example 62.
Treatment Six to eight week old C57bl6 mice (Jackson Laboratory, Bar , ME) were injected aneously once at the dosage shown below with ISIS 661161, 670699, 670700, 670701, 671165, or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The s below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.
As illustrated in Table 5 8, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent . The antisense oligonucleotides comprising various cleavable moieties all showed r potencies.
Table 58 SRB-l n1RNA(% Saline) ISIS No. Dosage (mg/kg) SRB-l mRNA GalNAc3 CM (% Saline) Cluster Saline n/a 100.0 n/a n/a 0.5 87.8 1.5 61.3 661161 GalNAc3-3a Ad 33.8 14.0 0.5 89.4 1.5 59.4 670699 GalNAc3-3a Td 313 17.1 0.5 79.0 1.5 63.3 670700 GalNAc3-3a AC 32.8 17.9 0.5 79.1 1.5 59.2 670701 3-3a Te 35.8 17.7 0.5 76.4 1.5 43.2 671165 GalNAc3-13a Ad 22.6 10.0 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline ed mice using standard protocols. Total bilirubin and BUN were also evaluated. The s in body weights were evaluated with no significant differences from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 56 below.
Table 59 Dosage ALT AST BUN GalNAc3 ISIS No ' B{gain (mg/kg) (U/L) (U/L) ) Cluster (mg/(1L) Saline n/a 24 64 0.2 31 n/a n/a 0.5 25 64 0.2 31 1.5 24 50 0.2 32 27 52 0.2 31 0.5 42 83 0.2 31 1.5 33 58 0.2 32 670699 GalNAc3-3a Td 26 70 0.2 29 25 67 0.2 29 0.5 40 74 0.2 27 1.5 23 62 0.2 27 670700 —5GalNAc3-3a AC 24 49 0‘2 29 25 87 0.1 25 0.5 30 77 0.2 27 670701 —15 GalNAc3-3a Te 22 55 0.2 30 WO 79625 81 101 0.2 25 3 1 82 O 2. 24 - 671165 GalNAC _13a3 Ad e 67: Preparation of oligomeric compound 199 comprising GalNAc3-16 0::ASFTK:WNN/NAN OOAC /\M/2\NH /ODMTr Aco OWN AcHN o AcOOCfi/OOWNWW0 OH4<:;>)LN{O1. Succinic anhydride,DMAP DCE 0A0 2. DMF, HBTU, DIEA, PS-SS AcHN AcOOAc O H H AGO OMNMVN o 2 2 AHN o C OD'V'T AcOOAc o H "- H ' o N N 1.DNA Synthesizer AcO WW NM N —> H 8 Z 2.aq.NH3 AcHN o O AcOOAc H o 0 OMWN A0o 2 AcHN 198 HOOH O H H HO OMNMVN O HOOH AcHN O m o /O o —. o H H 0 N N Ho WW N H 8 Q AcHN o O HOOH H o 0 OMWN Ho 2 AcHN eric compound 199, comprising a GalNA03-l6 conjugate group, is prepared using the general procedures illustrated in Examples 7 and 9. The GalNA03 cluster portion of the conjugate group GalNA03-l6 03-l6a) can be combined with any cleavable moiety to provide a variety of conjugate . In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.The structure of GalNA03-l6 (GalNA03-l6a-CM-) is shown below: OHOH O o HogomOWN/fl“/\H/\N4 H 2 H /-—E HAcHN H o o ’0 O N “WNW OAcHN 4 H 2 o NWNQ HO%HoOH o o/\(\/))LN O 4 H/WM AcHN Example 68: Preparation of oligomeric nd 200 comprising GalNAc3-17 A00 OOAC O 3' 5' (Ijl AcHN o N/\/\N o F OLIGO H O-F|>-O-(CH2)6-NH2 OAC F OAc o H O O F OH 0 OWNMNH 1.Borate buffer, DMSO, pH 8.5,rt A00 NMO F A HN H H —> 0A0 F 0A0 O 2. aq. ammonia, rt 0 N A00 0 WHN o AcHN W 102a HoOH O o o N HO /\(V)JL /\/\3 H H AcHN o 0 HoOH o o O NWNWO OLIGO o N/\/\N H H HO 3 H H AcHN HoOH o HOWO o/\(V)JL /\/\N 0 3 H H AcHN Oligomeric compound 200, comprising a GalNA03-l7 conjugate group, was prepared using the l procedures illustrated in Example 46. The GalNA03 cluster portion of the conjugate group GalNA03-l7 03-l7a) can be combined with any cleavable moiety to e a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNA03-l7 (GalNA03-l7a-CM-) is shown below: WO 79625 HoOH o o o N/\/\N 3 H H ACHN H HO 0 N m N/\// N N”$¢A\ 3 o H H O_III__ 3 H o AcHN HoOH o O N/\/\N O 3 H H AcHN ACO GAO O 0 83e AcHN o’Aij§\/kN’\\/\N O F 3 5' 3 GAO oOAC o F F OLKBO o—Twowcsz—NHZ H o AcHN Oo/$¢\VE~N/\/“NH N/J\/\/Kb F 1. Borate buffer, DMSO, pH 8.5, rt OAC OOAC 0 2. aq. ammonia, rt A00 0 H\/\/HN O AcHN MN 102b O N/\/\ 4 H H AcHN O O HOOH O O Home JJ\/\/u\ NMN H ”W0 OLIGo 4 H H AcHN O N/\/\ O 4 H H AcHN Oligomeric compound 201, comprising a 3-l8 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc3 cluster portion of the conjugate group GalNAc3-l8 (GalNAc3-18a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In n embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-l8 (GalNAc3-18a-CM-) is shown below: HoOH o o m::$\\/O )LN/\/\N N 4 HH H Oo/liiNV N N H I-1/\iv)‘7\‘3—III—_E ACHN HOoOH AcHN e 70: ation of oligomeric compound 204 comprising GalNAc3-19 AcO OAc AcOOAc O O 0 ON 0 0M HBTU DMF DIEA AcO '—>A00 OH N .....OH AcHN DMTO AcHN 64 NH 202 DMTO ' 47 AcOOAc PhosphityIation \/\)J\ , N NC 1. DNA synthesizer A HN .....0\ C O —’ p/ \J l 2. aq. NH3 DMTO (iPr)2N HOOH How 0 AcHN OOZT—OH| HOOH HowAWN?0 AcHN | OZT—OH HOOH HowAMEN/Q0 .cm -OL|GO AcHN Oligomeric compound 204, comprising a GalNA03-l9 conjugate group, was prepared from compound 64 using the general ures illustrated in Example 52. The GalNA03 cluster portion of the conjugate group GalNA03-l9 (GalNA03-l9a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNA03-l9 (GalNA03-l9a-CM-) is shown below: 2014/036460 AcHN 9 OZT—OH HoOH HO o/*%§:§N AcHN 9 O=T—OH HoOH o N HO 0% Example 71: Preparation of oligomeric nd 210 comprising GalNAc3-20 F F H EtN(iPr)2, CH3CN N FfiNNM: F? MN ""'OH DMTO 0 206 DMTO AcOOAc 0 A00 E: \/\/\)J\ K2co3/Methanol HZNMN AcHN 166 ””OH DMTO ACOOAC O Phosphitylation O OM 3 N ""'OH —> AcO NH AcHN DMTO 1. DNA syntheSIzer. AcO OAC 0 0M 3 N ""‘O NO —’ AcO NH \P/OV 2. aq. NH3 AcHN l DMTO (iPr)2N OH .
Ho HVHjL o N HO 3 o o AcHN | CIT—OH OH 0 ~‘ o “MN HO 3 o o AcHN | O=F|>—0H OH 0 Compound 205 was prepared by adding PFP-TFA and DIEA to 6-(2,2,2-trifluoroacetamido)hexanoic acid in acetonitrile ,which was ed by adding triflic anhydride to 6-aminohexanoic acid. The reaction e was heated to 80 0C, then lowered to rt. Oligomeric compound 210, comprising a GalNAc3-20 conjugate group, was prepared from compound 208 using the general procedures rated in Example 52. The GalNAc3 cluster portion of the conjugate group GalNAc3-20 (GalNAc3-20a) can be combined with any cleavable moiety to provide a variety of conjugate . In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-20 (GalNAc3-20a-CM-) is shown below: OH _.
HO o Hog/OWHO NMN AcHN (I) o:F|>—OH 0 50 HO ‘ HO OW 3 AcHN (I) O=F|>—OH 0 50 e 72: Preparation of oligomeric compound 215 comprising GalNAc3-21 1 AcOOAc O OH N H O R Acog/OM AcOOAcO AcHN 176 OWJKNH BOP, EtN(iPr)2, 1 2,-dichloroethane ACHN \\\OH ODMT AcOOAc DMTCI, Pyridine, rt 0 OWJ\Nr/ Phosphitylation —>A00 —> AcHN 10” r/ P\ 1. DNA synthesizer ACOOAC ACO¥/0 o N(IPr)2 —> 0 M 2. aq . NH3 AcHN \\\ ODMT AcHN | o:F|>—0H 0 N Ho 0W L AcHN Cl) O—T—OH 0 N HO 0W LO AcHN OLIGO Compound 21] is commercially available. Oligomeric compound 215, comprising a GalNAc3-2l conjugate group, was prepared from compound 213 using the general procedures illustrated in Example 52. The GalNAc3 cluster portion of the conjugate group GalNAc3-2l (GalNAc3-2la) can be combined with any cleavable moiety to provide a variety of ate . In n embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-21 (GalNAc3-2la-CM-) is shown below: HO H O N AcHN ('3 OZT—OH HO H O N Ho ow L AcHN (I) O=F|’—OH “kO N Ho WL o I E Example 73: Preparation of oligomeric compound 221 sing GalNAc3-22 O O H H\ /\/OH H o F F R 211 o OH R 205 F F 216 OH DIEA ACN O K2003 DMT'C' —FacTNMNNODMTr —» R MEOH / H20 pyridine O 217 OH HZNM /\/ODMTr 0A0 F N o F 3 O 0W 218 NHAc F F OH 166 OAc H A00goe/O/VVYNWLN/\/ODMTr Phosphitylation A00 0 —> NHAc OAc H O O/W\n/NMN/VODMTI‘ A00 0 NHAc NC ,P\ . 220 \/\0 NW» OH H OHQ‘MWWNMWc HO 0 NHAc 1. DNA Synthesizer OH “\NJK I ,O OH O’P\OH 2. Aq. NH3 N/\/ HOgoe/OWW0 NHAc % goe/OWWHWJ OH O/P<OH HO O NHAc % 221 m Compound 220 was prepared from nd 219 using diisopropylammonium tetrazolide. Oligomeric compound 221, comprising a GalNAc3-2l conjugate group, is prepared from compound 220 using the general procedure illustrated in Example 52. The GalNAc3 cluster portion of the conjugate group GalNAc3-22 (GalNAc3-22a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-22 c3-22a-CM-) is shown below: NMH OH990W NWOH HO o NHAc OH “Mi |,’o OHEzgjic/O/N\/”\V/\Tr O’P\OH N/\\/ HO O NHAc Lj Mi OIP<OH N/\/ HO 0 NHAc Lj O E Example 74: Effect of various cleavable es on antisense inhibition in vivo by ucleotides targeting SRB-l comprising a 5’-GalNAc3 conjugate The oligonucleotides listed below were tested in a ependent study for antisense inhibition of SRB-l in mice. Each of the GalNAc3 conjugate groups was attached at the 5' terminus of the respective oligonucleotide.
Table 60 Modified ASOs targeting SRB-l ISIS Sequences (5 , GalNAc3 SEQ to 3 , ) CM No. Cluster ID No.
CAGTmCA TGAmCTT 353382 es es es es es dsIn ds rriis ds ds ds ds ds ds ds es n/a n/a 143 Ces CesTesTe GalNAcaA GmC T TmCA G T mc AT3 30 661161 domes es esmes Ines ds ds ds ds ds ds GalNA03—3a Ad 145 GdsAds CdsTdsTes Ces CesTesTe GalNAc3G mc T T me A G T me A T3 a” 666904 3: es es 3: 2: ds ds ds ds ds ds 3-3a PO 143 GdsAds CdsTdsTes Ces Te GalNAcsA G mc T T mc A G T mc A T3 30 67544] din es es eIsn esIn es ds ds ds ds ds ds GalNA03—l7a Ad 145 GdsAds CdsTdsTes Ces CesTesTe GalNAc3A G mc T T mc A G T mc A T3 30 675442 din es es eIsn esIn es ds ds ds ds ds ds GalNA03—l8a Ad 145 GdsAds CdsTdsTes Ces CesTesTe In all , capital letters indicate the nucleobase for each nucleoside and InC indicates a 5-methyl cytosine.
Subscripts: “e” indicates a 2’-MOE modified nucleoside; “(1” indicates a B-D-Z’-deoxyribonucleoside; “s” indicates a phosphorothioate cleoside linkage (PS); “0” indicates a phosphodiester intemucleoside linkage (PO); and “0’” indicates -O-P(=O)(OH)-. Conjugate groups are in bold.
The structure of GalNAc3-3a was shown previously in Example 39. The structure of GalNAc3-l7a was shown previously in Example 68, and the ure of GalNAc3-l 8a was shown in Example 69.
Treatment Six to eight week old C57BL/6 mice on Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with an oligonucleotide listed in Table 60 or with saline.
Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final stration to determine the SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-l mRNA levels for each ent group, normalized to the saline control.
As illustrated in Table 61, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising a GalNAc conjugate showed similar ies and were significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.
Table 61 SRB-l mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-l mRNA GalNAc3 CM (% Saline) Cluster 100-0 3 79.38 353382 10 68.67 n/a n/a 40.70 0.5 79.18 1.5 75.96 661161 —5 GalNAc3-3a Ad .53 12.52 0.5 91.30 1.5 57.88 666904 —5 GalNAc3-3a P0 21.22 16.49 0.5 76.71 1.5 63.63 675441 GalNAc3-17a Ad 29.57 13.49 0.5 95.03 1.5 60.06 675442 —531.04 GalNAc3-18a Ad 19.40 Liver transarninase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also ted. The change in body weights was evaluated with no significant change from the saline group (data not shown). ALTs, ASTs, total bin and BUN values are shown in Table 62 below.
Table62 Dosage ALT AST 311133151111 BUN GalNAc3 ISIS N0 ' (mg/kg) (U/L) (U/L) ) Cluster (mg/dL) Saline n/a 26 59 0.16 42 n/a n/a 3 23 58 0.18 39 353382 10 28 58 0.16 43 n/a n/a 20 48 0.12 34 0.5 30 47 0.13 35 1.5 23 53 0.14 37 661161 —5GalNAc3-3a Ad 26 48 0.15 39 32 57 0.15 42 0.5 24 73 0.13 36 1.5 21 48 0.12 32 666904 —5GalNAc3-3a P0 19 49 0.14 33 20 52 0.15 26 0.5 42 148 0.21 36 1.5 60 95 0.16 34 675441 —5GalNAc3-17a Ad 27 75 0.14 37 24 61 0.14 36 0.5 26 65 0.15 37 1.5 25 64 0.15 43 675442 —5GalNAc3-18a Ad 27 69 0.15 37 30 84 0.14 37 Example 75: Pharmacokinetic analysis of oligonucleotides comprising a 5’-conjugate group The PK of the ASOs in Tables 54, 57 and 60 above was evaluated using liver samples that were obtained following the ent procedures described in Examples 65, 66, and 74. The liver samples were minced and extracted using standard ols and analyzed by IP-HPLC-MS ide an internal standard.
The combined tissue level (ug/g) of all metabolites was measured by integrating the appropriate UV peaks, and the tissue level of the full-length ASO missing the conjugate (“parent,” which is Isis No. 353382 in this case) was ed using the appropriate extracted ion tograms (EIC).
Table 63 PK Analysis in Liver ISIS N0. Dosage Total Tissue Level Parent ASO Tissue GalNA03 CM (mg/kg) by UV (Hg/g) Level by EIC (ug/g) Cluster 353382 3 8.9 8.6 22.4 21.0 n/a n/a 54.2 44.2 661161 5 32.4 20.7 Gal\AC3-3a Ad 632 441 671144 5 20.5 19.2 Gal\A03-12a Ad 486 41.5 670061 5 31.6 28.0 Gal\AC3-l3a Ad 67.6 555 Gal\A03-l4a Ad 64.7 49.1 Gal\AC3-15a Ad 52.3 24.2 670699 5 16.4 10.4 Gal\Acg-3a T01 31.5 225 Gal\Acg-3a Ae 38.1 200 670701 5 21.8 8.8 Gal\AC3-3a Te 352 161 671165 5 27.1 26.5 Gal\ACg-13a Ad 48.3 44.3 666904 5 30.8 24.0 Gal\Ac3-3a PO 52.6 37.6 675441 5 25.4 19.0 Gal\A03-17a Ad 542 42-1 675442 5 22.2 20.7 Gal\AC3-183 Ad 39.6 290 The results in Table 63 above show that there were greater liver tissue levels of the oligonucleotides comprising a GalNA03 conjugate group than of the parent ucleotide that does not comprise a 3 conjugate group (ISIS 353382) 72 hours following oligonucleotide administration, particularly when taking into consideration the differences in dosing between the ucleotides with and without a GalNA03 conjugate group. Furthermore, by 72 hours, 40-98% of each oligonucleotide comprising a GalNA03 conjugate group was metabolized to the parent compound, indicating that the GalNAc3 conjugate groups were cleaved from the oligonucleotides.
Example 76: Preparation of eric compound 230 comprising GalNAc3-23 ToSCI NaN3 HO/\/O\/\O/\/OH —> HO/\/ /OTSO 222 223 4 TMSOTf HO/\/ \/\O/\/O N OAC O O/\/O\/\O/\/N3 NHAC Pd(OH)2 OAcOAC ACN —’ o NH —, O 2 H2, EtOAc,MeOH o/\/ \/\o/\/ F F NHAC F F F O C—No2 OAC H OAc N 0 OAc NHAc H N02 1) Reduce O\/\ /\/ 2) Couple Diacid 0 ON 0 OAc 3) Pd/C o o OAC 4) PFPTFA NHAC OAC O O/\/O\/\O/\/NH NHAc OAc H o OACOAC NHAc H NH 0 F O O/\/O\/\O/\/ M OAC O O O o F F NHAc OAC F 0 O/\/O\/\O/\/ 3' 5' H -‘O_F|"O‘(CH2)6'NH2 1. Borate buffer, DMSO, pH 8.5, rt 2. aq. ammonia, rt N O O O/\/O\/\O/\/ OHOH NHAC H NH N MHvHx/O 4 .-CM o OMAN 0. O O O O NHAC OH O o/\/O\/\O/\/NH NHAC 230 Compound 222 is commercially available. 44.48 ml (0.33 mol) of compound 222 was treated with tosyl chloride (25.39 g, 0.13 mol) in pyridine (500mL) for 16 hours. The reaction was then evaporated to an oil, dissolved in EtOAc and washed with water, sat. NaHC03, brine, and dried over Na2S04. The ethyl acetate was concentrated to s and purified by column chromatography, eluted with EtOAc/hexanes (1 :1) followed by 10% methanol in CHZCIZ to give compound 223 as a colorless oil. LCMS and NMR were tent with the structure. 10 g (32.86 mmol) of 1-Tosyltriethylene glycol (compound 223) was treated with sodium azide (10.68 g, 164.28 mmol) in DMSO ) at room temperature for 17 hours. The reaction mixture was then poured onto water, and extracted with EtOAc. The c layer was washed with water three times and dried over Na2S04. The organic layer was concentrated to dryness to give 5.3g of compound 224 (92%). LCMS and NMR were consistent with the structure. 1-Azidotriethylene glycol (compound 224, 5.53 g, 23.69 mmol) and compound 4 (6 g, 18.22 mmol) were d with 4A molecular sieves (5g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100mL) under an inert atmosphere.
After 14 hours, the reaction was filtered to remove the sieves, and the organic layer was washed with sat.
NaHC03, water, brine, and dried over . The organic layer was concentrated to dryness and purified by column chromatography, eluted with a gradient of 2 to 4% ol in dichloromethane to give compound 225. LCMS and NMR were tent with the structure. Compound 225 (11.9 g, 23.59 mmol) was hydrogenated in EtOAc/Methanol (4:1, 250mL) over Pearlman's catalyst. After 8 hours, the catalyst was removed by filtration and the solvents removed to dryness to give nd 226. LCMS and NMR were consistent with the structure.
In order to generate compound 227, a solution of nitromethanetrispropionic acid (4.17 g, 15.04 mmol) and Hunig’s base (10.3 ml, 60.17 mmol) in DMF (100mL) were treated dropwise with pentaflourotrifiuoro acetate (9.05 ml, 52.65 mmol). After 30 minutes, the on was poured onto ice water and extracted with EtOAc. The organic layer was washed with water, brine, and dried over Na2S04. The organic layer was trated to dryness and then recrystallized from heptane to give compound 227 as a white solid. LCMS and NMR were consistent with the structure. Compound 227 (1.5 g, 1.93 mmol) and compound 226 (3.7 g, 7.74 mmol) were stirred at room temperature in acetonitrile (15 mL) for 2 hours. The on was then evaporated to dryness and d by column chromatography, eluting with a gradient of 2 t010% ol in dichloromethane to give compound 228. LCMS and NMR were consistent with the structure. Compound 228 (1.7 g, 1.02 mmol) was d with Raney Nickel (about 2g wet) in ethanol (100mL) in an atmosphere of hydrogen. After 12 hours, the catalyst was removed by filtration and the organic layer was evaporated to a solid that was used directly in the next step. LCMS and NMR were consistent with the structure. This solid (0.87 g, 0.53 mmol) was treated with benzylglutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and DIEA (273.7 ul, 1.6 mmol) in DMF (5mL). After 16 hours, the DMF was removed under reduced pressure at 65°C to an oil, and the oil was dissolved in dichloromethane. The organic layer was washed with sat. NaHC03, brine, and dried over Na2SO4. After evaporation of the organic layer, the compound was purified by column tography and eluted with a gradient of 2 to 20% methanol in dichloromethane to give the coupled product. LCMS and NMR were consistent with the ure. The benzyl ester was deprotected with Pearlman’s catalyst under a hydrogen atmosphere for 1 hour. The catalyst was them d by filtration and the solvents removed to dryness to give the acid.
LCMS and NMR were consistent with the structure. The acid (486 mg, 0.27 mmol) was dissolved in dry DMF (3 mL). Pyridine (53.61 ul, 0.66 mmol) was added and the reaction was purged with argon.
Pentaflourotrifiouro acetate (46.39 ul, 0.4 mmol) was slowly added to the reaction mixture. The color of the on changed from pale yellow to burgundy, and gave off a light smoke which was blown away with a stream of argon. The reaction was allowed to stir at room temperature for one hour (completion of reaction was confirmed by LCMS). The solvent was removed under reduced re (rotovap) at 70 OC. The residue was diluted with DCM and washed with 1N NaHSO4, brine, saturated sodium bicarbonate and brine again. The organics were dried over Na2S04, filtered, and were concentrated to dryness to give 225 mg of nd 229 as a brittle yellow foam. LCMS and NMR were consistent with the ure.
Oligomeric compound 230, comprising a GalNAc3-23 conjugate group, was prepared from compound 229 using the l procedure illustrated in Example 46. The GalNAc3 cluster portion of the 3-23 conjugate group (GalNAc3-23a) can be combined with any cleavable moiety to provide a variety of conjugate groups. The structure of GalNAc3-23 (GalNAc3-23a-CM) is shown below: N O O O/\/O\/\O/\/ 0H0“ NHAC H NH “We 0 o/\/O\/\o/\/ M 4.; OH O O O O NHAC OH O O/\/O\/\O/\/NH NHAC Example 77: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising a GalNAc3 conjugate The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice.
Table 64 d ASOs targeting SRB-l ISIS Sequences (5 , GalNA03 SEQ t0 3 , ) CM N0. Cluster ID No.
GalNAcaA GmC T TmCA G T mc AT3 30 661161 domes es es es es ds ds ds ds ds ds GalNA03—3a Ad 145 GcsAcs CcsTcsTesmCesmCesTesTe GalNAc3G mc T T mc A G T mc A T3 30 666904 613: es es es es ds ds ds ds ds ds 3—3a PO 143 GcsAcs CcsTcSTesmCesmCesTesTe GalNAcsA G mc T T mc A G T mc A T3 30 673502 dom es e0 epn e0In e0 ds ds ds ds ds ds GalNA03-10a Ac 145 GcsAcs CcsTcsTeo Ceo CesTesTe GalNAcaA GmC T T mc A G T mc A T3 30 677844 domes es esmes Ines ds ds ds ds ds ds GalNA03—9a Ac 145 GcsAcs CcsTcsTes Ces Te GalNAc3-23a-03Ad0G C T T mc A G T mc A T 677843 es es eIsn esIn es ds ds ds ds ds ds In GalNA03—23a Ac 145 CS CS CTSTCSCC CTTCS C S CS CS 6 GemCTTmCAGTmCATGAmCTTmC 655861 3 es es es Hes cs ds ds ds ds ds ds ds ds ds es es GalNA03—la Ac 144 CesTesTe do"GalNAc3'1a GmCTTmCAGTmCATGAmCTTmC 677841 es es es es Ines cs ds ds ds ds ds ds ds ds ds es es GalNA03—l9a Ac 144 CesTesTe do"GalNAc3'19a GmCTTmCAGTmCAT GAmCTTmC 677842 es es es es es ds ds ds ds ds ds ds ds ds ds es es GalNA03—20a Ac 144 CesTesTe lNAc3-20a The ure of 3-la was shown previously in Example 9, GalNA03-3a was shown in Example 39, GalNA03-9a was shown in Example 52, GalNA03-10a was shown in Example 46, GalNA03-19a was shown in Example 70, GalNA03-20a was shown in Example 71, and GalNA03-23a was shown in Example Treatment Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were each injected subcutaneously once at a dosage shown below with an oligonucleotide listed in Table 64 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent ular Probes, Inc. Eugene, OR) according to rd protocols. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, ized to the saline control.
As illustrated in Table 65, ent with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner.
Table 65 SRB-l mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-l mRNA GalNAc3 CM (% Saline) Cluster Saline n/a 100.0 n/a n/a 0.5 89.18 1.5 77.02 661161 GalNAc3-3a Ad 29-10 12.64 0.5 93.11 1.5 55.85 666904 —5 GalNAc3-3a P0 2129 13.43 0.5 77.75 1.5 41.05 673502 —5 GalNAc3-10a Ad 19-27 14.41 0.5 87.65 1.5 93.04 677844 GalNAc3-9a Ad 40-77 16.95 0.5 102.28 1.5 70.51 677843 —530.68 GalNAc3-23a Ad 13.26 0.5 79.72 1.5 55.48 655861 —526.99 3-1a Ad 17.58 0.5 67.43 1.5 45.13 677841 —5 GalNAc3-19a Ad 27-02 12.41 0.5 64.13 1.5 53.56 677842 —5 GalNAc3-20a Ad -47 10.23 Liver transaminase , alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were also measured using standard protocols. Total bilirubin and BUN were also evaluated. Changes in body weights were evaluated, with no significant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 66 below.
Table66 Dosage ALT AST Bgfiflm BUN GalNAc3 ISIS N0 ' (mg/kg) (U/L) (U/L) (mg/dL) Cluster (mg/(1L) Saline n/a 21 45 0.13 34 n/a n/a 0.5 28 51 0.14 39 1.5 23 42 0.13 39 661161 GalNAc3-3a Ad 22 59 0.13 37 21 56 0.15 35 0.5 24 56 0.14 37 1.5 26 68 0.15 35 666904 —523 GalNAc3-3a P0 77 0.14 34 24 60 0.13 35 0.5 24 59 0.16 34 1.5 20 46 0.17 32 673502 —524 GalNAc3-10a Ad 45 0.12 31 24 47 0.13 34 0.5 25 61 0.14 37 1.5 23 64 0.17 33 677844 —5GalNAc3-9a Ad 58 0.13 35 22 65 0.14 34 0.5 53 53 0.13 35 1.5 25 54 0.13 34 677843 —5GalNAc3-23a Ad 21 60 0.15 34 22 43 0.12 38 0.5 21 48 0.15 33 1.5 28 54 0.12 35 655861 —5GalNAc3-1a Ad 22 60 0.13 36 21 55 0.17 30 0.5 32 54 0.13 34 1.5 24 56 0.14 34 677841 Ac3-19a Ad 23 92 0.18 31 24 58 0.15 31 0.5 23 61 0.15 35 1.5 24 57 0.14 34 677842 GalNAc3-20a Ad 41 62 0.15 35 24 37 0.14 32 Example 78: nse tion in vivo by oligonucleotides targeting Angiotensinogen comprising a GalNAc3 conjugate The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of Angiotensinogen (AGT) in normotensive Sprague Dawley rats.
Table 67 Modified ASOs targeting AGT 1s1s , , GalNAc3 SEQ InC:eslkesmC:esTesC}eSIAdsTdsTdsTds'TdsTdsChsmC:dsmCdsmCdsAesGes 552668 esTe InC:es14esmC:esTesC}eSIAdsTdsTdsTds'TdsTdsChsmC:dsmCdsmCdsAesGes 669509 GesAesTeoAdoa-GalNAc3-1a The structure of GalNAc3-la was shown previously in Example 9.
Treatment Six week old, male Sprague Dawley rats were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 67 or with PBS. Each treatment group consisted of 4 animals. The rats were sacrificed 72 hours following the final dose. AGT liver mRNA levels were measured using ime PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. AGT plasma n levels were measured using the Total Angiotensinogen ELISA (Catalog # 2, IBL International, Toronto, ON) with plasma diluted 120,000. The s below are presented as the average percent of AGT mRNA levels in liver or AGT protein levels in plasma for each treatment group, normalized to the PBS control.
As illustrated in Table 68, treatment with antisense oligonucleotides d AGT liver mRNA and plasma protein levels in a dose-dependent manner, and the oligonucleotide comprising a GalNAc conjugate was significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.
Table 68 AGT liver mRNA and plasma protein levels ISIS Dosage (mg/kg) AGT liver AGT plasma GalNAc3 Cluster CM No. mRNA (% PBS) n (% PBS) PBS n/a 100 100 n/a n/a 3 95 122 85 97 552668 n/a n/a 46 79 90 8 11 0.3 95 70 l 95 129 669509 GalNAc3-la Ad 3 62 97 9 23 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in plasma and body s were also measured at time of sacrifice using standard protocols. The results are shown in Table 69 below.
Table 69 Liver transaminase levels and rat body weights Body CM ISIS No. (figs/1g: ALT (U/L) AST (U/L) Weight (% g (Egg? of baseline) PBS n/a 51 81 186 n/a n/a 3 54 93 183 51 93 194 552668 n/a n/a 59 99 182 90 56 78 170 0.3 53 90 190 1 51 93 192 669509 GalNAc3-1a Ad 3 48 85 189 56 95 189 Example 79: Duration of action in vivo of oligonucleotides targeting APOC-III comprising a GalNAc3 The oligonucleotides listed in Table 70 below were tested in a single dose study for duration of action in mice.
Table 70 Modified ASOs targeting APOC-III ISIS , , GalNAc3 SEQ sequences (5 t0 3 ) CM No. Cluster ID No.
AesGes CesTesTes CdsTdsTdsGdsTds Cds CdsAdsGds Tes 304801 n/a n/a 135 TesAesTe AesGesmCesTesTesmCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmCdsTesTes 647535 G lNAa C3 -1 a Ad 136 TesAesTeoAdoa-GalNAa-la GalNAc3'3a'o’Ad0AesGesmCesTesTesmCdsTdsTdsGdsTdsmCds 663083 G lNAa C3 -3 a Ad 151 InC:dslAdsCldsmC:dsTesTes TesAesTe TdsGdsTds Cds 674449 93-7211);AdolAesC-le:n CesTesTes GalNA03—7a Ad 151 CdsAdsGds CdsTesTes TesAesTe GalNAC3-1oago’AdersG: CesTesTes CdsTdsTdsGdsTds Cds 674450 GalNA03—1Oa Ad 151 CdsAdsGds Tes TesAesTe GalNAc3'13a'o’AdoAesGesmCesTesTesmCdsTdsTdsGdsTdsmCds 674451 G lNAa C3-13 a Ad 151 lAdsCldsmC:dsTesTes TesAesTe The structure of GalNAc3-1a was shown previously in Example 9, GalNAc3-3a was shown in Example 39, GalNAc3-7a was shown in Example 48, GalNAc3-10a was shown in Example 46, and GalNAc3-13a was shown in Example 62.
WO 79625 Treatment Six to eight week old transgenic mice that express human APOC-III were each injected subcutaneously once with an oligonucleotide listed in Table 70 or with PBS. Each treatment group consisted of 3 animals. Blood was drawn before dosing to determine baseline and at 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results below are presented as the average percent of plasma triglyceride and APOC-III levels for each ent group, normalized to baseline levels, showing that the oligonucleotides comprising a GalNAc conjugate group exhibited a longer duration of action than the parent oligonucleotide without a conjugate group (ISIS 304801) even though the dosage of the parent was three times the dosage of the oligonucleotides comprising a GalNAc conjugate group.
Table 71 Plasma ceride and II protein levels in enic mice T1me pomt ISIS Dosage Triglycerides AFDC-IE GalNAc3 CM (days p05“ prom (A No' (mg/kg) (% baseline) Cluster dose) baseline) 3 97 102 7 101 98 14 108 98 PBS n/a 21 107 107 n/a n/a 28 94 91 88 90 42 91 105 3 40 34 7 41 37 14 50 57 304801 30 21 50 50 n/a n/a 28 57 73 68 70 42 75 93 3 36 37 7 39 47 14 40 45 647535 10 21 41 41 GalNAc3-1a Ad 28 42 62 69 69 42 85 102 3 24 18 7 28 23 14 25 27 663083 10 21 28 28 GalNAc3-3a Ad 28 37 44 55 57 42 60 78 674449 10 g g: g? GalNAc3-7a Ad 14 38 41 21 44 44 28 53 63 69 77 42 78 99 3 33 3O 7 35 34 14 31 34 674450 10 21 44 44 GaDiAcyloa Ad 28 56 61 68 7O 42 83 95 3 35 33 7 24 32 14 4O 34 674451 10 21 48 48 GaDiAcy13a Ad 28 54 67 65 75 42 74 97 Example 80: Antisense inhibition in vivo by oligonucleotides targeting Alpha-1 Antitrypsin (AlAT) comprising a GalNAc3 Conjugate The oligonucleotides listed in Table 72 below were tested in a study for ependent inhibition of AlAT in mice.
Table 72 Modified ASOs targeting AlAT ISIS ces (5 , , GalNAc3 SEQ ID t0 3 ) CM No. Cluster No.
Aes Ces Ces CesAesAdsTdsTds CdsAdsGdsAdsAdsGdsGdsAesAes 476366 n/a n/a 152 GesGesAe AesmCesmCesmCesAesAdsTdsTdsmCdsAdsGdsAdsAdsGdsGdsAesAes 656326 G lNAc -1a 3 a Ac 153 GesGesAeoAdos-GalNAa-la GalNAc3'3a'o’AdoAesmCesmCesmCesAesAdsTdsTdsmCdsAdsGdsAds 678381 G lNAa C3 -3 a Ac 154 AdsGdsGdsAesAes GesGesAe 3'7a'o’AdoAesmCesmCesmCesAesAdsTdsTdsmCdsAdsGdsAds 678382 G DQAa C3 -7a A{ 154 AdsGdsGdsAesAes GesGesAe GalNAC3-10a'o’AdersmCesmCesmCesAesAdsTdsTdsmCdsAdsGds 678383 G lNAa C3-10a Ac 154 GdsGdsAesAes GesGesAe GalNAc3'13a'o’Aders Ces Ces CesAesAdsTdsTds CdsAdsGds 678384 GalNA03—13a Ac 154 AdsAdsGdsGdsAesAes GesGesAe The structure of GalNAc3-1a was shown previously in Example 9, Gall\Ac3-3a was shown in Example 39, GalNAc3-7a was shown in Example 48, GalNAc3-10a was shown in Example 46, and Gal\IAc3-13a was shown in Example 62.
Treatment Six week old, male C57BL/6 mice (Jackson tory, Bar Harbor, ME) were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 72 or with PBS. Each treatment group ted of 4 animals. The mice were sacrificed 72 hours following the final administration. A1AT liver mRNA levels were ined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. A1AT plasma protein levels were determined using the Mouse Alpha 1-Antitrypsin ELISA (catalog # 41-A1AMS-E01, Alpco, Salem, NH). The results below are presented as the e percent of A1AT liver mRNA and plasma protein levels for each treatment group, normalized to the PBS control.
As illustrated in Table 73, treatment with antisense oligonucleotides lowered A1AT liver mRNA and A1AT plasma protein levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent (ISIS 476366).
Table 73 AlAT liver mRNA and plasma protein levels ISIS Dosage (mg/kg) A1AT liver A1AT plasma GalNAc3 Cluster CM No. mRNA (% PBS) protein (% PBS) PBS n/a 100 100 n/a n/a 86 78 476366 73 61 n/a n/a 45 30 38 0.6 99 90 2 61 70 656326 GalNAc3-1a Ad 6 15 30 18 6 10 0.6 105 90 678381 2 53 60 GalNAc3-3a Ad 6 16 20 18 7 13 0.6 90 79 2 49 57 678382 GalNAc3-7a Ad 6 21 27 18 8 11 0.6 94 84 2 44 53 678383 GalNAc3-10a Ad 6 13 24 18 6 10 0.6 106 91 2 65 59 678384 GalNAc3-13a Ad 6 26 31 18 11 15 Liver minase and BUN levels in plasma were ed at time of sacrifice using standard protocols. Body weights and organ weights were also ed. The results are shown in Table 74 below.
Body weight is shown as % relative to baseline. Organ weights are shown as % of body weight relative to the PBS control group.
Table 74 ISIS Dosage ALT AST BUN BOdy .Lwer Kldney Spleen we1ght (% we1ght (Rel we1ght (Rel we1ght (Rel No‘ (mg/kg) (U/L) (U/L) (mg/dL) ne) % BW) % BW) % BW) PBS n/a 25 51 37 119 100 100 100 34 68 35 116 91 98 106 476366 15 37 74 30 122 92 101 128 45 30 47 31 118 99 108 123 0.6 29 57 40 123 100 103 119 2 36 75 39 114 98 111 106 656326 6 32 67 39 125 99 97 122 18 46 77 36 116 102 109 101 0.6 26 57 32 117 93 109 110 2 26 52 33 121 96 106 125 678381 6 40 78 32 124 92 106 126 18 31 54 28 118 94 103 120 0.6 26 42 35 114 100 103 103 2 25 50 31 117 91 104 117 678382 6 30 79 29 117 89 102 107 18 65 112 31 120 89 104 113 0.6 30 67 38 121 91 100 123 2 33 53 33 118 98 102 121 678383 6 32 63 32 117 97 105 105 18 36 68 31 118 99 103 108 0.6 36 63 31 118 98 103 98 2 32 61 32 119 93 102 114 678384 6 34 69 34 122 100 100 96 18 28 54 30 117 98 101 104 Example 81: Duration of action in vivo of oligonucleotides targeting AlAT comprising a 3 cluster The oligonucleotides listed in Table 72 were tested in a single dose study for on of action in mice.
Six week old, male C57BL/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 72 or with PBS. Each treatment group consisted of 4 s. Blood was drawn the day before dosing to determine baseline and at 5, 12, 19, and 25 days following the dose. Plasma AlAT protein levels were measured via ELISA (see Example 80). The results below are presented as the average percent of plasma AlAT protein levels for each treatment group, normalized to baseline levels. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent and had longer duration of action than the parent lacking a GalNAc conjugate (ISIS 476366). Furthermore, the oligonucleotides comprising a 5 ’- GalNAc conjugate (ISIS , 678382, 678383, and 678384) were lly even more potent with even longer duration of action than the oligonucleotide comprising a 3’-GalNAc conjugate (ISIS 656326).
Table 75 Plasma AlAT n levels in mice ISIS Dosage Time point AlAT (% GalNAc3 CM No. (mg/kg) (days post- baseline) Cluster dose) 93 12 93 PBS n/a n/a n/a —19 90 97 38 12 46 476366 100 n/a n/a —1962 77 33 12 36 656326 18 —19GalNAcg-la Ad 72 21 12 21 678381 18 —19GalNAc3-3a Ad 48 21 12 21 678382 18 GalNAc3-7a Ad 19 39 60 24 12 21 678383 18 —19GalNAc3-10a Ad 73 29 12 34 678384 18 —19GalNAc3-13a Ad 76 Example 82: Antisense inhibition in vitro by oligonucleotides targeting SRB-l comprising a GalNAc3 conjugate Primary mouse liver hepatocytes were seeded in 96 well plates at 15,000 cells/well 2 hours prior to treatment. The oligonucleotides listed in Table 76 were added at 2, 10, 50, or 250 nM in Williams E medium and cells were incubated overnight at 37 0C in 5% C02. Cells were lysed 16 hours following oligonucleotide addition, and total RNA was purified using RNease 3000 BioRobot (Qiagen). SRB-l mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.
Eugene, OR) ing to standard protocols. IC50 values were ined using Prism 4 software (GraphPad). The results show that oligonucleotides comprising a variety of different GalNAc conjugate groups and a variety of different cleavable moieties are significantly more potent in an in vitro free uptake experiment than the parent ucleotides lacking a GalNAc conjugate group (ISIS 353382 and 666841).
Table 76 Inhibition of SRB-l expression in vitro ISIS , , . GalNAc 1C50 SEQ Sequence (5 to 3 ) Llnkages CM No. cluster (nM) ID No. 3 53 3 82 CesTesges CesAdfdsTnds CdsAdsTdsGdsAds PS n/a n/a 250 143 CdsTdsTes Ces CesTesTe GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds Ga1\AC3 655861 PS AC 40 144 InC:dsTdsTesmC:esmC:esTesTeoAdo’'(;alNAc3'1a ' 1 Gal\AC3 661 161 GEINAC3'3a'0’Adognes CesTesTIfis CgsAdsGdsTds PS AC 40 145 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe '33. 661 162 3-3a-03Adocifs CCOTCOTIEO CIEOAdSGdSTdS Ga1\AC3 PO/PS Ac 8 145 CdsAdsTdsGdsAds CdsTds Teo Ceo CesTesTe '33.
GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds Ga1\AC3 664078 PS AC 20 144 InC:dsTdsTesmC:esmC:esTesTeoAdo’'GalNAc3'9a '93.
GalNAc3'8a'o’AdoGesmCesTesTesmCesAdsGdsTds Ga1\AC3 665001 PS AC 70 145 lAdsTdsC}d31AdsmC:dsTdsTesmC:esmC:esTesTe ' 83.
GalNAc3'5a'o’AdoGesmCesTesTesmCesAdsGdsTds Ga1\AC3 666224 PS AC 8O 145 InC:dslAdsTdsC}d31AdsmC:dsTdsTesmC:esmC:esTesTe '53. 666841 C6°T6°HTC6° 3?:ngan fiSédsTdsGdsAds PO/PS n/a n/a >250 143 ds ds e0 e0 es es e GalNAc3-10a'o’Ad0GesmCesTesTesmCesAdsGdsTds Gal\AC3 6668 81 PS Ad 3 O 145 InC:dslAdsTdsC}d31AdsmC:dsTdsTesmC:esmC:esTesTe "103. 666904 GalNAC3-3a-03Gein TesmCesfideGdsTds Cds Gal\Ac3 PS PO 9 143 GdsAds CdsTds Tes Ces CesTesTe '33. 666924 GSINAC3-3a-0’Tdogrfs CCSTCSTIES CIisAdsGdsTds Gal\Ac3 PS Tc 15 148 CdsAdsTdchsAcs CcsTds Tes Ces CesTesTe '33.
GalNAc3'6a'0’AdoGesmCesTesTesmCesAdsGdsTds Ga1\AC3 666961 PS AC 150 145 InC:dSIAdsTdsC}c SACschsTdsTesmCesmCesTesTe '63.
GalNAc3'7a'0’AdoGesmCesTesTesmCesAdsGdsTds Ga1\AC3 666981 PS AC 20 145 InC:dSIAdsTdsC}c SACschsTdsTesmCesmCesTesTe '73. 670061 GilNAc3'13a'0’Adoges CesTesEles gesAdsGdsTds Gal\AC3 PS Ac 3 O 145 CdsAdsTdchsAcs CcsTds Tes Ces CesTesTe "133.
GalNAc -3a-0,T 0G mc T T me A G T3 ‘3 6° 6° 66 66 ‘6 G31\AC3 670699 m if H66 51" PO/PS Tc 15 148 CdsAdsTds GcsAcs Cc sTdsTeo Ceo CesTesTe '33.
GalNAc -3a-0sAe0G mc T T me A G T3 ‘6 Gal\AC° 670700 6° 6° 66 66 m 6; 6: 6;: PO/PS AC 30 145 Tds GdsAds CdsTdsTeo Ceo sT '33.
GalNAc -3a-0aTe0G mc T T me A G T3 670701 65 6° 6° 51" if 66 66 ‘6 Gal\AC° m PO/PS Te 25 148 CdsAdsTds GdsAds CdsTdsTeo Ceo CesTesTe '33.
GilNAcfa'lZa'o’Adog-les CesTesEles gesAdsGdsTds Gal\AC3 671 144 PS Ad 40 145 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe _123' GalNAc -13,-0,A 0G mc T T me A G T3 d Gal\AC3 671165 6° 6° H?" ‘15 d5 ‘15 m H? n?" PO/PS A, 8 145 CdsAdsTds GdsAds CdsTdsTeo Ceo CesTesT '13a 671261 GigNAcfa'l‘I'a'o’Adoges CeSTeSTneS geSAdSGdSTdS Ga1\AC3 PS Ac >250 145 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe '14a 671262 GilgNAc3-1 Sa'o’Adoges CeSTeSTneS geSAdSGdSTdS 3 PS Ac >250 145 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe '15a 673501 GilNAc3'7a'0’Ad0Cigs CCOTCO'TIifO sGdsTds 3 PO/PS Ac 3 O 145 CdsAdsTdsGdsAds CdsTdsTeo Ceo Te '7a GiinlNAcfa-loa-O’Adoges CeoTeoTneO geoAdsGdsTds Ga1\AC3 673502 PO/PS Ac 8 145 TdsGdsAds CdsTds Teo Ceo CesTesTe _10a 675441 GigNAch7a'0’Ad0ges CesTesTnes gesAdsGdsTds Gal\AC3 PS Ac 3 O 145 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe '17a 675442 GigNAcfa'lsa'o’Adog-les CesTesTnes GdsTds Gal\AC3 PS Ac 20 145 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe _1 8a GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds 3 677841 PS A“ 40 144 mcdsTdsTesmCesmCesTesTeoAdoa-Ga1NAc3-19a -19a GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds Gal\AC3 677842 PS A“ 30 144 mcdsTdsTesmCesmCesTesTeoAdoa-Ga1NAc3-20a -2oa 677843 GigNAc3'23a'o’Adog-les CesTesTnes gesAdsGdsTds Gal\AC3 PS AC 40 145 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe '23a The ure of GalNAcg-la was shown previously in Example 9, GalNAc3-3a was shown in Example 39, GalNAc3-5a was shown in Example 49, GalNAc3-6a was shown in Example 51, GalNAc3-7a was shown in Example 48, GalNAc3-8a was shown in Example 47, GalNAc3-9a was shown in e 52, GalNAcg-lOa was shown in Example 46, GalNAc3-12a was shown in Example 61, GalNAc3-13a was shown in Example 62, GalNAc3-14a was shown in Example 63, GalNAc3-15a was shown in Example 64, GalNAc3-17a was shown in Example 68, GalNAc3-18a was shown in Example 69, GalNAc3-19a was shown in Example 70, GalNAcg-ZOa was shown in Example 71, and GalNAc3-23a was shown in Example 76.
Example 83: Antisense inhibition in vivo by oligonucleotides ing Factor XI sing a GalNAc3 cluster The oligonucleotides listed in Table 77 below were tested in a study for dose-dependent inhibition of Factor X1 in mice.
Table 77 Modified oligonucleotides targeting Factor XI 1s1s GalNAc , , SEQ WW6 to” TesGesGesTesAesAdsTds C: (giséds CdsTdsTdsTds CdsAesGes 404071 TesCleoCleoTeolAeolAdsTdsmcdsmcdsAdsmCdsTdsTdsTdsmCdsAeoGeo 663086 GalNAc3-3a-oaAdoTesGeoGeoTeeroAdsTdsmCdsmCdsAdsmCdsTdS GalNAc3-3a TdsTdsmCdslAeoC}eolAesC-lesC}e 3'7a'0’Ad0TesGeoGeoTeeroAdsTdsmCdsmCdsAdsmCdsTds 678347 GalNAC3'7a Ad 155 TdsTdsmCdsAeoGeersGesGe GalNAc3-10a'0’AdoTesGeoGeoTeeroAdsTdsmCdsmCdsAdsmCds 678348 GalNAC3_10a TdsTdsTdsmCdslAeoCleOIAesC-lescje GalNAc3-13a'o’Ad0TesCleoCleoTeolAeOIAdsTdsmCdsmCdslAdsmCds 678349 3_13a TdsmCdslAeoCleOIAesC-lescje The structure of GalNAc3-1a was shown previously in Example 9, GalNAc3-3a was shown in Example 39, GalNAc3-7a was shown in Example 48, GalNAc3-10a was shown in Example 46, and GalNAc3-13a was shown in Example 62.
Six to eight week old mice were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed below or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final dose. Factor XI liver mRNA levels were measured using real-time PCR and normalized to cyclophilin according to standard protocols.
Liver transaminases, BUN, and bilirubin were also measured. The results below are presented as the average percent for each treatment group, normalized to the PBS control.
As illustrated in Table 78, treatment with antisense oligonucleotides lowered Factor XI liver mRNA in a dose-dependent manner. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 404071). Furthermore, the oligonucleotides comprising a 5 ’-GalNAc conjugate (ISIS 663086, 678347, 678348, and 678349) were even more potent than the ucleotide comprising a 3’-GalNAc conjugate (ISIS ).
Table 78 Factor XI liver mRNA, liver minase, BUN, and bilirubin levels ISIS Dosage Factor XI ALT AST BUN Bilirubin GalNAc3 SEQ No. (mg/kg) mRNA (% PBS) (U/L) (U/L) (mg/dL) ) Cluster ID No.
PBS n/a 100 63 70 21 0.18 n/a n/a 3 65 41 58 21 0.15 404071 33 49 53 23 0.15 n/a 146 17 43 57 22 0.14 0.7 43 90 89 21 0.16 656173 2 9 36 58 26 0.17 GalNAcg-la 147 6 3 50 63 25 0.15 0.7 33 91 169 25 0.16 663086 2 7 38 55 21 0.16 GalNAc3-3a 155 6 1 34 40 23 0.14 0.7 35 28 49 20 0.14 678347 2 10 180 149 21 0.18 GalNAc3-7a 155 6 1 44 76 19 0.15 0.7 39 43 54 21 0.16 678348 —25 GalNAc3-10a 155 38 55 22 0.17 6 2 25 38 20 0.14 0.7 34 39 46 20 0.16 umGalNA03-13a _—-__ Example 84: Duration of action in vivo of oligonucleotides targeting Factor XI comprising a GalNAc3 Conjugate The oligonucleotides listed in Table 77 were tested in a single dose study for duration of action in mice.
Treatment Six to eight week old mice were each injected subcutaneously once with an oligonucleotide listed in Table 77 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn by tail bleeds the day before dosing to determine baseline and at 3, 10, and 17 days following the dose. Plasma Factor XI protein levels were measured by ELISA using Factor XI capture and biotinylated detection antibodies from R & D Systems, Minneapolis, MN (catalog # AF2460 and # BAF2460, respectively) and the OptEIA Reagent Set B (Catalog # 550534, BD ences, San Jose, CA). The results below are ted as the e percent of plasma Factor XI protein levels for each treatment group, normalized to baseline levels. The s show that the oligonucleotides comprising a GalNAc conjugate were more potent with longer duration of action than the parent lacking a GalNAc conjugate (ISIS 404071). rmore, the oligonucleotides comprising a ’-GalNAc conjugate (ISIS 663086, , 678348, and 678349) were even more potent with an even longer duration of action than the oligonucleotide comprising a 3’-GalNAc ate (ISIS 656173).
Table 79 Plasma Factor XI protein levels in mice ISIS (mg/kgg)Dosa e TimepolsDt-dos(e) yoint da s Factor XI %baseline) CM SE ID GalNA03 Cluster No. 130. 3 123 PBS n/a 10 56 n/a n/a n/a 17 100 3 11 404071 30 10 47 n/a n/a 146 17 52 3 1 656173 6 10 3 GalNA03-1a Ad 147 17 21 3 1 663086 6 10 2 GalNA03-3a Ad 155 17 9 3 1 678347 6 10 1 GalNA03-7a Ad 155 17 8 WO 79625 678348 10 1 GalNAcg-10a Ad 155 678349-== GalNAC3-13a Example 85: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising a GalNAc3 Conjugate Oligonucleotides listed in Table 76 were tested in a dose-dependent study for antisense inhibition of SRB-l in mice.
Treatment Six to eight week old C57BL/6 mice were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 76 or with saline. Each ent group consisted of 4 animals. The mice were sacrificed 48 hours following the final administration to determine the SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of liver SRB—l mRNA levels for each treatment group, normalized to the saline control.
As rated in Tables 80 and 81, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a ependent manner.
Table 80 SRB-l mRNA in liver ISIS No. Dosage (mg/kg) SRB-l mRNA (% GalNA03 r CM Saline) Saline n/a 100 n/a n/a 0.1 94 655861 %GalNAcg-la Ad 3 32 0.1 120 661161 (1)3 £7 GalNAcg-3a Ad 3 26 0.1 107 666881 cg-10a Ad 3 27 0.1 120 666981 %GalNAcg-7a Ad 3 21 0.1 118 670061 WGalNAcg-13a Ad 3 18 - 677842 GalNAC3'ZOa Ad Table 81 SRB-l mRNA in liver ISIS No. Dosage (mg/kg) SRB-l mRNA (% GalNA03 r CM Saline) 0.1 107 661161 (1)3 2: GalNAcg-3a Ad 3 18 0.1 110 677841 (1)3 :3 g-19a Ad 3 25 Liver transarninase levels, total bilirubin, BUN, and body weights were also ed using standard protocols. Average values for each treatment group are shown in Table 82 below.
Table 82 ISIS Dosage ALT AST Bilirubin BUN Body Weight GalNA03 CM No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) (% baseline) Cluster Saline n/a 19 39 0.17 26 118 n/a n/a 0.1 25 47 0.17 27 114 0.3 29 56 0.15 27 118 655861 GalNAcg-la Ad 1 20 32 0.14 24 112 3 27 54 0.14 24 115 0.1 35 83 0.13 24 113 0.3 42 61 0.15 23 117 661161 GalNAcg-3a Ad 1 34 60 0.18 22 116 3 29 52 0.13 25 117 0.1 30 51 0.15 23 118 0.3 49 82 0.16 25 119 666881 GalNAcg-10a Ad 1 23 45 0‘14 24 117 3 20 38 0.15 21 112 0.1 21 41 0.14 22 113 0.3 29 49 0.16 24 112 666981 GalNAcg-7a Ad 1 19 34 0.15 22 111 3 77 78 0.18 25 115 0.1 20 63 0.18 24 111 0.3 20 57 0.15 21 115 670061 GalNAcg-13a Ad 1 20 35 0.14 20 115 3 27 42 0.12 20 116 0.1 20 38 0.17 24 114 677842 0.3 31 46 0.17 21 117 GalNAcg-20a Ad 1 22 34 0.15 21 119 3 41 57 0.14 23 118 Example 86: Antisense inhibition in vivo by oligonucleotides targeting TTR comprising a GalNAc3 cluster Oligonucleotides listed in Table 83 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.
Treatment Eight week old TTR transgenic mice were each ed subcutaneously once per week for three weeks, for a total of three doses, with an ucleotide and dosage listed in the tables below or with PBS.
Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Tail bleeds were performed at s time points throughout the experiment, and plasma TTR protein, ALT, and AST levels were measured and reported in Tables 85-87. After the animals were sacrificed, plasma ALT, AST, and human TTR levels were measured, as were body weights, organ weights, and liver human TTR mRNA levels. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, CA). ime PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. , OR) were used according to standard protocols to determine liver human TTR mRNA levels.
The results ted in Tables 84-87 are the average values for each treatment group. The mRNA levels are the average values relative to the average for the PBS group. Plasma protein levels are the e values relative to the average value for the PBS group at baseline. Body weights are the e percent weight change from baseline until sacrifice for each individual treatment group. Organ weights shown are normalized to the animal’s body , and the average normalized organ weight for each treatment group is then presented relative to the average normalized organ weight for the PBS group.
In Tables 84-87, “BL” indicates baseline, measurements that were taken just prior to the first dose.
As illustrated in Tables 84 and 85, treatment with antisense oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent g a GalNAc conjugate (ISIS 420915). Furthermore, the ucleotides comprising a GalNAc conjugate and mixed PS/PO internucleoside linkages were even more potent than the oligonucleotide comprising a GalNAc conjugate and full PS linkages.
Table 83 ucleotides targeting human TTR GalNAc SEQ 151s No.. Sequence 5 , , . to 3 L1nkages cluster ID No.
TesmCesTesTesGesGdsTdsTdsAdsmCdsAdsTdsGdsAdsAds 420915 AesTesmCesmCesmCe TesmCesTesTesGesGdsTdsTdsAdsmCdsAdsTdsGdsAdsAds 682883 GalNAc3-3HaTesmCeoTeoTeoGeoGdSTdsTdsAdsmCdSAdS PS/PO GalNAc3-3a m- TdsGdsAdsAdsAeoTeomCesmCesmCe GalNAc3'7a-0’TesmCeoTeoTeoGeoGdsTdsTdsAdsmCdsAds 682884 PS/PO GalNAcg-7a PO TdsGdsAdsAdsAeoTeomCesmCesmCe GalNAc3-10a-o’TesmCeoTeoTeoGeoGdsTdsTdsAdsmCds 682885 PS/PO GalNAcg- 10a “- AdsTdsGdsAdsAdsAeoTeomCesmCesmCe GalNAc3-13a—o’TesmCeoTeoTeoCleoCldsTdsTdSIAdsmCds 682886 PS/PO 3-13a fl- AdsTdsGdsAdsAdsAeoTeomCesmCesmCe TesmCeoTeoTeoCleoCldsTdsTdsIAdsmCdslAdsTdsChslAdslAds 684057 PS/PO GalNAc3-19a AeoTeomCesmCesmCeoAdo"GalNAc3'19a The legend for Table 85 can be found in Example 74. The structure of GalNAc3-1 was shown in Example 9.
The structure of 3-3a was shown in Example 39. The structure of GalNAc3-7a was shown in Example 48. The structure of GalNAc3-10a was shown in Example 46. The structure of GalNAc3-13a was shown in Example 62. The structure of GalNAc3-19a was shown in Example 70.
Table 84 Antisense tion of human TTR in vivo . Dosage TTR mRNA (% Plasma TTR protein SEQ 1s1s No. GalNAc cluster CM (mg/kg) PBS) (% PBS) ID No.
PBS n/a 100 100 n/a n/a 6 99 95 420915 20 48 65 n/a n/a 156 60 18 28 0.6 113 87 2 4O 56 660261 GalNAcg-la Ad 157 6 20 27 9 11 Table 85 Antisense inhibition of human TTR in vivo Plasma TTR prote1n (A) PBS at BL)' TTR 0 SEQ . Dosage GalNAc 1s1s No. mRNA Day 17 CM 1D (mg/kg) BL Day 3 Da 10 (% PBS) y cluster (After sac) No.
PBS n/a 100 100 96 90 114 n/a n/a 6 74 106 86 76 83 420915 20 43 102 66 61 58 n/a n/a 156 60 24 92 43 29 32 0.6 60 88 73 63 68 682883 2 18 75 38 23 23 Gallglfcg- 156 6 10 80 35 11 9 0.6 56 88 78 63 67 682884 2 19 76 44 25 23 Gal??? PO 6 15 82 35 21 24 0.6 60 92 77 68 76 682885 2 22 93 58 32 32 @3130? 156 6 17 85 37 25 20 682886 0.6 57 91 70 64 69 GalNAc3- PO 156 2 21 89 50 31 mfimm—Zi102 GalNAC3_ 684057_____— “____— Table 86 Transaminase levels, body weight changes, and ve organ weights 2;): ALT (U/L) AST (U/L) Body Liver Spleen Kidne SEQ 1515 NO- (mg Day Day Day Day Day Day (% (% (% y (% ID BL BL 3 10 /kg) 17 3 10 17 BL) PBS) PBS) PBS) N0.
PBS n/a 33 34 33 24 58 62 67 52 105 100 100 100 n/a 6 34 33 27 21 64 59 73 47 115 99 89 91 420915 20 34 30 28 19 64 54 56 42 111 97 83 89 156 60 34 35 31 24 61 58 71 58 113 102 98 95 0.6 33 38 28 26 70 71 63 59 111 96 99 92 2 29 32 31 34 61 60 68 61 118 100 92 90 660261 157 6 29 29 28 34 58 59 70 90 114 99 97 95 33 32 28 33 64 54 68 95 114 101 106 92 Table 87 Transaminase levels, body weight changes, and relative organ weights 2;): ALT (U/L) AST (U/L) Body Liver Spleen Kidne SEQ 1515 NO- (% (% (% y (% ID (mg Day Day Day Day Day Day BL BL 3 10 17 3 10 17 BL) PBS) PBS) PBS) N0. /kg) PBS n/a 32 34 37 41 62 78 76 77 104 100 100 100 n/a 6 32 30 34 34 61 71 72 66 102 103 102 105 420915 20 41 34 37 33 80 76 63 54 106 107 135 101 156 60 36 30 32 34 58 81 57 60 106 105 104 99 0.6 32 35 38 40 53 81 74 76 104 101 112 95 682883 2 38 39 42 43 71 84 70 77 107 98 116 99 156 6 35 35 41 38 62 79 103 65 105 103 143 97 0.6 33 32 35 34 70 74 75 67 101 100 130 99 682884 2 31 32 38 38 63 77 66 55 104 103 122 100 156 6 38 32 36 34 65 85 80 62 99 105 129 95 0.6 39 26 37 35 63 63 77 59 100 109 109 112 682885 2 30 26 38 40 54 56 71 72 102 98 111 102 156 6 27 27 34 35 46 52 56 64 102 98 113 96 0.6 30 40 34 36 58 87 54 61 104 99 120 101 682886 2 27 26 34 36 51 55 55 69 103 91 105 92 156 6 40 28 34 37 107 54 61 69 109 100 102 99 0.6 35 26 33 39 56 51 51 69 104 99 110 102 684057 2 33 32 31 40 54 57 56 87 103 100 112 97 157 6 39 33 35 40 67 52 55 92 98 104 121 108 Example 87: on of action in vivo by single doses of oligonucleotides targeting TTR comprising a GalNAc3 cluster ISIS numbers 420915 and 660261 (see Table 83) were tested in a single dose study for duration of action in mice. ISIS numbers 420915, , and 682885 (see Table 83) were also tested in a single dose study for duration of action in mice.
Treatment Eight week old, male transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. 420915 or 13.5 mg/kg ISIS No. 660261. Each treatment group consisted of 4 s. Tail bleeds were med before dosing to determine baseline and at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels were measured as described in Example 86. The results below are ted as the average percent of plasma TTR levels for each treatment group, normalized to baseline levels.
Table 88 Plasma TTR protein levels ISIS Dosage Time point TTR (A) baselme)0 . GalNAc3 CM SEQ ID NO' No. (mg/kg) (days ose) Cluster 3 30 7 23 35 420915 100 —17 n/a n/a 156 24 75 39 100 3 27 7 21 22 660261 13.5 GalNAc3-1a Ad 157 17 36 24 48 39 69 Treatment Female transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. 420915, 10.0 mg/kg ISIS No. 682883, or 10.0 mg/kg 682885. Each treatment group consisted of 4 animals. Tail bleeds were performed before dosing to ine baseline and at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels were measured as described in Example 86.
The results below are presented as the average percent of plasma TTR levels for each treatment group, normalized to baseline levels.
Table 89 Plasma TTR protein levels ISIS g)Dosa e Time oint . 3 CM TTR (% baselme) SEQ ID NO' N0. (days polsDt-dose) Cluster 3 48 7 48 420915 100 10 48 n/a n/a 156 17 66 31 80 3 45 7 37 682883 10.0 10 38 GalNAc3-3a PO 156 17 42 31 65 3 40 7 33 682885 10.0 10 34 GalNAc3-10a PO 156 17 40 31 64 The results in Tables 88 and 89 show that the oligonucleotides comprising a GalNAc conjugate are more potent with a longer duration of action than the parent ucleotide lacking a ate (ISIS 420915).
Example 88: Splicing modulation in vivo by oligonucleotides targeting SMN comprising a GalNAc3 conjugate The ucleotides listed in Table 90 were tested for splicing modulation of human survival of motor neuron (SMN) in mice.
Table 90 Modified ASOs targeting SMN ISIS , , GalNAc3 SEQ Sequences (5 t0 3 ) CM N0. Cluster ID No.
Tes CesAes CesTesTesTes gesAesTesAesAesTesGes CesTesGes 3 87954 n/a n/a 158 GalNAc3'7a'0’AesTesTesmCesAesmCesTesTesTesmCesAesTesAesAes 6998 1 9 G INAa C3 -7a PO 158 TesC}esmcesTesC}esC}e GalNAc3'73—0’AesTeoTeomCeeromCeoTeoTeoTeomCeeroTeero 699821 G lNAa C3 -7a PO 158 AeoTeoGeomCeoTesGesGe AesTesTes CesAes CesTesTesTes CesAesTesAesAesTesGes CesTesGes 700000 GalNAc3-1a Ad 157 GeOAdoa-GalNAc3-1 a 703421 X-ATTmCAmCTTTmCATAATGmCTGG n/a n/a 15 8 703422 GalNAc3-7b-X-ATTmCAmCTTTmCATAATGmCTGG GalNAC3-7b n/a 15 8 The structure of GalNAc3-7a was shown previously in Example 48. “X” indicates a 5’ primary amine generated by Gene Tools (Philomath, OR), and GalNAc3-7b indicates the structure of GalNAc3-7a lacking the —NH-C6-O portion of the linker as shown below: HoOH o O N Ho 4 Hfl AcHN HoOH O O O O NJK/‘O NW“ HO I“ 4 H H AcHN o HOOH O i O N HO O 4 H AcHN ISIS numbers 703421 and 703422 are morphlino oligonucleotides, wherein each nucleotide of the two oligonucleotides is a morpholino nucleotide.
Treatment Six week old transgenic mice that express human SMN were injected subcutaneously once with an oligonucleotide listed in Table 91 or with saline. Each treatment group consisted of 2 males and 2 s.
The mice were sacrificed 3 days following the dose to determine the liver human SMN mRNA levels both with and without exon 7 using real-time PCR according to standard protocols. Total RNA was measured using Ribogreen t. The SMN mRNA levels were normalized to total mRNA, and further normalized to the averages for the saline treatment group. The ing average ratios of SMN mRNA including exon 7 to SMN mRNA missing exon 7 are shown in Table 91. The s show that fully modified oligonucleotides that modulate splicing and comprise a GalNAc conjugate are significantly more potent in ng splicing in the liver than the parent oligonucleotides lacking a GlaNAc ate. Furthermore, this trend is maintained for multiple modification chemistries, including 2’-MOE and morpholino modified oligonucleotides.
Table 91 Effect of oligonucleotides targeting human SMN in vivo 113:8 Dose (mg/kg) +Exon 7 / -Exon 7 (31111:? CM 118330.
Saline n/a 1.00 n/a n/a n/a 387954 32 1.65 n/a n/a 158 387954 288 5.00 n/a n/a 158 699819 32 7.84 3-7a PO 158 699821 32 7.22 GalNAc3-7a PO 158 700000 32 6.91 GalNAc3-1a Ad 159 703421 32 1.27 n/a n/a 158 703422 32 4.12 GalNAc3-7b n/a 158 Example 89: Antisense inhibition in vivo by oligonucleotides targeting Apolipoprotein A (Apo(a)) comprising a GalNAc3 conjugate The oligonucleotides listed in Table 92 below were tested in a study for dose-dependent inhibition of Apo(a) in transgenic mice.
Table 92 Modified ASOs targeting Apo(a) -ISIS GalNA03 SEQ ID Sequences <5 ”3) TesC}esmC:esTesmC:esmC:dsCidSTdsTdscjdscjdsTds(}dsmC:ds 494372 HI, c}es'-[‘esTesmC:e GalNAc3'7a'0’TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds 68 1257 GalNAc -7a3 P0 58 Tdsc}dsmC:ds TdsTeoC}eoTesTesmC:e The structure of GalNA03-7a was shown in Example 48.
Treatment Eight week old, female 6 mice (Jackson Laboratory, Bar Harbor, ME) were each injected subcutaneously once per week at a dosage shown below, for a total of six doses, with an oligonucleotide listed in Table 92 or with PBS. Each treatment group consisted of 3-4 s. Tail bleeds were performed the day before the first dose and weekly following each dose to determine plasma Apo(a) protein levels. The mice were sacrificed two days following the final administration. Apo(a) liver mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to rd protocols. Apo(a) plasma protein levels were determined using ELISA, and liver transaminase levels were determined. The mRNA and plasma protein results in Table 93 are presented as the treatment group average percent relative to the PBS treated group. Plasma n levels were further normalized to the baseline (BL) value for the PBS group. Average absolute transaminase levels and body weights (% ve to baseline averages) are ed in Table 94.
As illustrated in Table 93, treatment with the oligonucleotides lowered Apo(a) liver mRNA and plasma n levels in a dose-dependent manner. Furthermore, the oligonucleotide comprising the GalNAc conjugate was significantly more potent with a longer duration of action than the parent oligonucleotide lacking a GalNAc conjugate. As illustrated in Table 94, minase levels and body weights were unaffected by the oligonucleotides, indicating that the oligonucleotides were well tolerated.
Table 93 Apo(a) liver mRNA and plasma protein levels ISIS Dosage Apo(a) mRNA Apo(a) plasma protein (% PBS) N... (mg/kg) <% PBs> —‘1_-—m— 494372 681257 Table 94 Dosage (mg/kg) ALT (U/L) AST (U/L) Body weight (% baseline) 3 28 68 494372 10 22 55 ——__ 681257 Example 90: Antisense inhibition in vivo by oligonucleotides targeting TTR comprising a GalNAc3 cluster Oligonucleotides listed in Table 95 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.
TTR transgenic mice were each injected subcutaneously once per week for three weeks, for a total of three doses, with an oligonucleotide and dosage listed in Table 96 or with PBS. Each treatment group consisted of 4 animals. Prior to the first dose, a tail bleed was performed to determine plasma TTR protein levels at baseline (BL). The mice were sacrificed 72 hours following the final administration. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN® RNA quantification reagent ular Probes, Inc. Eugene, OR) were used according to standard protocols to determine liver human TTR mRNA levels. The results ted in Table 96 are the average values for each treatment group. The mRNA levels are the average values relative to the average for the PBS group. Plasma protein levels are the average values ve to the average value for the PBS group at ne. “BL” tes baseline, measurements that were taken just prior to the first dose. As illustrated in Table 96, treatment with nse oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 420915), and oligonucleotides comprising a phosphodiester or denosine cleavable moiety showed significant ements in potency compared to the parent lacking a conjugate (see ISIS numbers 682883 and 666943 vs 420915 and see Examples 86 and 87). 2014/036460 Table 95 Oligonucleotides targeting human TTR Isis No. Sequence 5’ to 3’ Linkages (:figgc CM 118330. 420915 T“Inc“TCSTesGei:i:.:E::fig:ggfdsTdsGdSAdSAdS PS n/a n/a 156 682883 GalNAc3gZ;gifgkfifggjgagfifdsmc“Ads PS/PO GalNAc3-3a PO 156 666943 Galfiéfihéfldzg$.EEE‘ETflnEdAd PS/PO GalNAc3-3a Ad 160 682887 Gallfiacgzné‘fldtigmcégEngdAd PS/PO 3-7a Ad 160 682888 Galflégl‘iongjgdeAfTngEEECTdAd PS/PO GalNAc3-10a Ad 160 682889 Galflégl‘iidgjgdxigmgEEECTdAd PS/PO 3-13a Ad 160 The legend for Table 95 can be found in Example 74. The structure of GalNAc3-3a was shown in Example 39. The structure of 3-7a was shown in Example 48. The structure of GalNAc3-10a was shown in Example 46. The structure of GalNAc3-13a was shown in Example 62.
Table 96 Antisense inhibition of human TTR in vivo Isis No. Dosage (mg/kg) TTR mRNA (% PBS) TTR protein (% BL) GalNAc cluster CM PBS n/a 100 124 n/a n/a 6 69 114 420915 20 71 86 n/a n/a 60 21 36 0.6 61 73 682883 2 23 36 GalNAc3-3a PO 6 18 23 0.6 74 93 666943 2 33 57 GalNAc3-3a Ad 6 17 22 0.6 60 97 682887 2 36 49 GalNAc3-7a Ad 6 12 19 0.6 65 92 682888 2 32 46 GalNAc3-10a Ad 6 17 22 0.6 72 74 682889 2 38 45 GalNAc3-13a Ad 6 16 18 Example 91: nse inhibition in vivo by oligonucleotides targeting Factor VII comprising a GalNAc3 conjugate in non-human primates Oligonucleotides listed in Table 97 below were tested in a non-terminal, dose escalation study for antisense tion of Factor VII in monkeys.
Treatment Non-naive monkeys were each injected subcutaneously on days 0, 15, and 29 with escalating doses of an oligonucleotide listed in Table 97 or with PBS. Each treatment group consisted of 4 males and 1 female. Prior to the first dose and at various time points thereafter, blood draws were performed to determine plasma Factor VII n levels. Factor VII protein levels were ed by ELISA. The results presented in Table 98 are the average values for each treatment group relative to the e value for the PBS group at baseline (BL), the measurements taken just prior to the first dose. As illustrated in Table 98, treatment with antisense oligonucleotides d Factor VII expression levels in a dose-dependent manner, and the oligonucleotide sing the GalNAc conjugate was significantly more potent in monkeys compared to the oligonucleotide lacking a GalNAc conjugate.
Table 97 Oligonucleotides targeting Factor VII . , , . GalNAc SEQ rAesT‘esCIesmC:eslAesT‘ds(his(LETds(EdsIAdST‘dSCLiSInCLiS'Tds 407935 TesmCesTesGesAe —-- GalNAC3-10a-o’AesTesGesmCesAesTdsGdsGdsTdsGds 686892 :-:GalNAC3 10a P0 AdsTdsGdsmCdsTds TesmCesTesGesAe The legend for Table 97 can be found in Example 74. The structure of GalNA03-10a was shown in Example Table 98 Factor VII plasma protein levels ISIS N0. Day Dose (mg/kg) Factor VII (% BL) 0 n/a 100 10 87 22 n/a 92 407935 29 3O 77 36 n/a 46 43 n/a 43 O 3 100 10 56 22 n/a 29 686892 29 3O 19 36 n/a 15 43 n/a 11 Example 92: Antisense inhibition in primary hepatocytes by antisense oligonucleotides ing Apo- CIII sing a GalNAc3 conjugate Primary mouse hepatocytes were seeded in 96-well plates at 15,000 cells per well, and the oligonucleotides listed in Table 99, targeting mouse ApoC-III, were added at 0.46, 1.37, 4.12, or 12.35, 37.04, 111.11, or 333.33 nM or 1.00 uM. After incubation with the oligonucleotides for 24 hours, the cells were lysed and total RNA was d using RNeasy (Qiagen). I mRNA levels were ined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.) according to standard protocols. ICso values were determined using Prism 4 software (GraphPad). The results show that regardless of whether the cleavable moiety was a phosphodiester or a phosphodiester-linked deoxyadensoine, the oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent oligonucleotide lacking a conjugate.
Table 99 Inhibition of mouse APOC-III expression in mouse primary cytes 113:8 Sequence (5’ t0 3’) CM (lg/[50) 118330. 440670 mCesAesGesmCeSTeSTdsTdsAdsTdsTdsAdsGdsGdSGdsAdsmCesAesGeSmCesAe n/a 1 3 .20 1 62 661180 mCesA65GeSEfiZEHTCEiiifflffigjfiggfid:GdSAdSmCeS Ad 1.40 163 GalNAc3-3Ha CeSAeSGes 680771 CXCTEZ$E::::STdsTdsAdsGdsGdsGdsAds Ces PO 0.70 162 680772 GalNAc3-7a_0amCesAssGesmCXCTEZ$E::::STdSTdSAdSGdSGdSGdSAdSmCes PO 1.70 162 GalNAc3-10a_0s CeSAeSGes ’ijfedSTdsTdsAdsGdsGdSGdSAdS Ces 680773 PO 2.00 162 680774 3-13HamCesAesGesmfifi:?sc1::dfldSTdSAdSGdSGdsGdsAdsmCes PO 1‘ 50 162 GalNAc3-3Ha CeSAeOGeo CXCngisgiidSTdSTdSAdSGdSGdSGdSAdS Ceo 681272 PO < 0.46 162 681 273 GalNAc3-3a-oaAdOmCesA,31(25,::IAC;g“e6:13“é::::AdSTdSTdSAdSGdSGdSGdSAdS Ad 1 . 1 0 1 64 683733 CCSACSGCSAEGTmEdX‘Xngglfi‘fi:(1};GdAd C65 Ad 2.50 163 The ure of GalNA03-1a was shown previously in Example 9, GalNA03-3a was shown in Example 39, GalNA03-7a was shown in Example 48, GalNA03-10a was shown in Example 46, GalNAc3-13a was shown in Example 62, and GalNA03-19a was shown in Example 70.
Example 93: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising mixed wings and a 5’-GalNAc3 conjugate The oligonucleotides listed in Table 100 were tested in a dose-dependent study for antisense inhibition of SRB-l in mice.
Table 100 Modified ASOs targeting SRB-l ISIS Sequences (5 ’ to 3 ’) Gal\IAc3 CM SEQ No. Cluster ID No. 449093 TksTkskasAdsGdsTdsmCds AdsTds Gds STdsTkskaska n/a n/a 165 699806 GalNAC3'3a'o’TksTkskasAdsGdsTdsmCds AdsTds GdsAdsmCds Gal\AC3'3a PO TCSTkSkaSka 6998O7 GalNAC3'7a'0’TksTkskasAdsGdsTdsmCds AdsTds GdsAdsmCds Gal\AC3'73 PO TCSTkSkaSka 699809 GalNAC3'7a'0’ TksTkskasAdsGdsTdsmCds AdsTds Gds AdsmCds Gal\AC3'7a PO TcsTeSmCesmCe 69981 1 3'7a'0’TesTesmCesAdsGdsTdsmCds AdsTds GdsAdsmCds Gal\AC3'7a PO TCSTkSkaSka 699813 GalNAC3'7a'0’TksTdskasAdsGdsTdsmCds AdsTds GdsAdsmCds Gal\AC3'7a PO TCSTkSmCdSka 699815 GalNAC3'7a-0’TesTkskasAdsGdsTdsmCds AdsTds GdsAdsmCds Gal\AC3'7a PO TcsFlemC1§sInCe The ure of 3-3a was shown previously in Example 39, and the structure of GalNAc3-7a was shown previously in Example 48. Subscripts: “e” indicates 2’-MOE modified nucleoside; “(1” indicates B-D- 2’-deoxyribonucleoside; “k” indicates 6’-(S)-CH3 bicyclic nucleoside (cEt); “s” indicates phosphorothioate intemucleoside linkages (PS); “0” indicates phosphodiester intemucleoside linkages (PO). Supersript “m” indicates 5-methylcytosines.
Treatment Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with an oligonucleotide listed in Table 100 or with saline.
Each treatment group ted of 4 animals. The mice were sacrificed 72 hours following the final administration. Liver SRB-l mRNA levels were measured using real-time PCR. SRB-l mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented as the average percent of SRB-l mRNA levels for each ent group relative to the saline control group. As illustrated in Table 10], treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose- ent manner, and the gapmer oligonucleotides sing a GalNAc conjugate and having wings that were either full cEt or mixed sugar modifications were cantly more potent than the parent oligonucleotide lacking a conjugate and comprising full cEt modified wings.
Body weights, liver transaminases, total bilirubin, and BUN were also ed, and the average values for each treatment group are shown in Table 101. Body weight is shown as the average t body weight relative to the baseline body weight (% BL) ed just prior to the oligonucleotide dose.
Table 101 SRB-l mRNA, ALT, AST, BUN, and total bilirubin levels and body weights ISIS Dosage SRB-l mRNA ALT AST Body weight Bil BUN No. (mg/kg) (% PBS) (U/L) (U/L) (% BL) PBS n/a 100 31 84 0.15 28 102 1 111 18 48 0.17 31 104 449093 3 94 20 43 0.15 26 103 36 19 50 0.12 29 104 0.1 114 23 58 0.13 26 107 699806 0.3 59 21 45 0.12 27 108 1 25 30 61 0.12 30 104 0.1 121 19 41 0.14 25 100 699807 0.3 73 23 56 0.13 26 105 1 24 22 69 0.14 25 102 0.1 125 23 57 0.14 26 104 699809 0.3 70 20 49 0.10 25 105 1 33 34 62 0.17 25 107 0.1 123 48 77 0.14 24 106 699811 0.3 94 20 45 0.13 25 101 1 66 57 104 0.14 24 107 0.1 95 20 58 0.13 28 104 699813 0.3 98 22 61 0.17 28 105 1 49 19 47 0.11 27 106 0.1 93 30 79 0.17 25 105 699815 0.3 64 30 61 0.12 26 105 1 24 18 41 0.14 25 106 Example 94: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising 2’-sugar modifications and a 5’-GalNAc3 conjugate The oligonucleotides listed in Table 102 were tested in a dose-dependent study for antisense inhibition of SRB-l in mice.
Table 102 Modified ASOs targeting SRB-l ISIS Sequences (5’ to 3 ’) GalNA03 CM SEQ No. Cluster ID No. 3 5 3 3 82 GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTesmCesmCes n/a n/a TesTe 700989 GmsCmsUmsUmsCmsAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsUmsCmsCms n/a n/a 1 66 UmsUm 666904 GalNAC3-33'0’GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds GalNAC3_3 a PO InC:dsTdsTesmC:esmC:esTesTe 700991 GalNAc3'7a'0’GmsCmsUmsUmsCmsAdsGdsTdsmCdsAdsTdsGds GalNAC3_7a PO 1 66 AdsmCdsTdsUmsCmsCmsUmsUm Subscript “m” indicates a 2’-O-methyl modified side. See Example 74 for te table . The structure of GalNAC3-3a was shown previously in Example 39, and the structure of GalNA03-7a was shown usly in Example 48.
Treatment The study was completed using the protocol described in Example 93. Results are shown in Table 103 below and show that both the 2’-MOE and 2’-OMe modified oligonucleotides comprising a GalNAc conjugate were significantly more potent than the respective parent oligonucleotides lacking a ate. The results of the body weights, liver transaminases, total bilirubin, and BUN ements indicated that the compounds were all well tolerated.
Table 103 SRB-l mRNA ISIS No. Dosage ) SRB-l mRNA (% PBS) PBS n/a 100 116 353382 15 58 45 27 120 700989 15 92 45 46 1 98 666904 3 45 17 1 118 700991 3 63 14 Example 95: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising bicyclic nucleosides and a 5’-GalNAc3 conjugate The oligonucleotides listed in Table 104 were tested in a dose-dependent study for antisense inhibition of SRB-l in mice.
Table 104 Modified ASOs targeting SRB-l 11338 Sequences (5’ to 3 ’) (Egg? CM $330 440762 TkskasAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTkska n/a n/a 1 3 7 666905 GalNAc3-3a'o’TkskasAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTkska GalNA03'3 a PO 1 3 7 699782 GalNAC3-7a-0’TkskaSAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTkska GalNA03'7a PO 1 3 7 699783 GalNAC3-3a-oaTlsmclsAdsGdsTdsmcdsAdsTdsGdsAdsmCdSTdsTlsmcl 3'3a PO 137 653 621 TlsmclsAdsGdsTdsmcdsAdsTdsGdsAdsmCdSTdsTlsmcloAdO’-GalNAc3-1 a GalNA03' 1 a Ad 1 3 8 439879 TgmchdsGdSTdsmCdsAdsTd GdsAdsmCdsTdSTngg n/a n/a 137 699789 GalNAc3-3a-0aTgsmCgsAdsGdsTdsmCdsAdSTd mCdSTdsTngg 3'3a PO 137 Subscript “g” indicates a fluoro-HNA nucleoside, subscript “1” indicates a locked nucleoside comprising a 2’- O-CH2-4’ bridge. See the e 74 table legend for other abbreviations. The structure of GalNAc3-1a was shown previously in Example 9, the structure of GalNAc3-3a was shown usly in Example 39, and the structure of GalNAc3-7a was shown previously in Example 48.
Treatment The study was completed using the protocol bed in Example 93. Results are shown in Table 105 below and show that oligonucleotides sing a GalNAc conjugate and various bicyclic nucleoside modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and sing bicyclic nucleoside modifications. Furthermore, the oligonucleotide comprising a GalNAc ate and fluoro-HNA modifications was significantly more potent than the parent lacking a conjugate and comprising fluoro-HNA ations. The s of the body weights, liver transaminases, total bilirubin, and BUN measurements indicated that the compounds were all well tolerated.
Table 105 SRB-l mRNA, ALT, AST, BUN, and total bilirubin levels and body weights ISIS No. Dosage (mg/kg) SRB-l mRNA (% PBS) PBS n/a 100 1 104 440762 3 65 35 0.1 105 666905 0.3 56 1 18 0.1 93 699782 0.3 63 1 15 0.1 105 699783 0.3 53 1 12 0.1 109 653621 0.3 82 1 27 1 96 439879 3 77 37 0.1 82 699789 0.3 69 1 26 Example 96: Plasma protein binding of antisense oligonucleotides comprising a GalNAc3 conjugate group Oligonucleotides listed in Table 70 targeting ApoC-IH and oligonucleotides in Table 106 targeting Apo(a) were tested in an ultra-filtration assay in order to assess plasma protein binding.
Table 106 d oligonucleotides targeting Apo(a) ISIS sequences (5 , to 3 , GalNAc3 SEQ ) CM No. Cluster ID No Tescjes CesTes Ces 494372 CdsGdsTdf‘TLIiggdsGdsTdsGds CdsTdsTesGesTes n/a n/a 58 Tescjeo CeoTeo Ceo CdsTdsTeoGeoTes 693401 Tdfs‘TfilsgdsGdsTdsGds n/a n/a 58 GalNAc3'7a'o’TesGes CesTes Ces 681251 CdsidsTdsTdsGdsGdsTdsGds Cds 3—7a P0 58 TdsTescjesTesTes Ce GalNAc3'7a'o’TesGeo CeoTeo Ceo 681257 CdsgdsTdsTdsGdsGdsTdsGds Cds GalNAc3—7a P0 58 cjeoTesTes Ce See the Example 74 for table legend. The structure of GalNAc3-7a was shown previously in Example 48.
Ultrafree-MC ltration units (30,000 NMWL, low-binding regenerated cellulose membrane, Millipore, Bedford, MA) were pre-conditioned with 300 uL of 0.5% Tween 80 and centrifuged at 2000 g for minutes, then with 300uL of a 300 ug/mL solution of a control oligonucleotide in H20 and centrifuged at 2000 g for 16 s. In order to assess non-specific binding to the filters of each test oligonucleotide from Tables 70 and 106 to be used in the studies, 300 uL of a 250 ng/mL solution of oligonucleotide in H20 at pH 7.4 was placed in the pre-conditioned filters and centrifuged at 2000 g for 16 minutes. The unfiltered and filtered samples were analyzed by an ELISA assay to ine the oligonucleotide concentrations. Three replicates were used to obtain an average concentration for each sample. The average concentration of the filtered sample relative to the unfiltered sample is used to determine the percent of oligonucleotide that is recovered through the filter in the absence of plasma (% recovery).
Frozen whole plasma samples collected in K3-EDTA from , drug-free human volunteers, cynomolgus monkeys, and CD-1 mice, were purchased from Bioreclamation LLC (Westbury, NY). The test oligonucleotides were added to 1.2 mL aliquots of plasma at two concentrations (5 and 150 ug/mL). An aliquot (300 uL) of each spiked plasma sample was placed in a pre-conditioned filter unit and incubated at 37°C for 30 minutes, immediately followed by centrifugation at 2000 g for 16 minutes. Aliquots of filtered and unfiltered spiked plasma samples were ed by an ELISA to ine the oligonucleotide concentration in each sample. Three replicates per concentration were used to determine the average percentage of bound and unbound oligonucleotide in each sample. The average concentration of the filtered sample relative to the tration of the unfiltered sample is used to determine the percent of oligonucleotide in the plasma that is not bound to plasma proteins (% unbound). The final unbound oligonucleotide values are corrected for non-specific binding by dividing the % unbound by the % recovery for each oligonucleotide. The final % bound oligonucleotide values are determined by cting the final % unbound values from 100. The results are shown in Table 107 for the two concentrations of oligonucleotide tested (5 and 150 ug/mL) in each species of plasma. The results show that GalNAc ate groups do not have a significant impact on plasma n binding. Furthermore, oligonucleotides with filll PS intemucleoside linkages and mixed PO/PS es both bind plasma ns, and those with full PS linkages bind plasma ns to a somewhat greater extent than those with mixed PO/PS linkages.
Table 107 Percent of modified oligonucleotide bound to plasma proteins ISIS Human plasma Monkey plasma Mouse plasma No. 5 ug/mL 150 ug/mL 5 ug/mL 150 ug/mL 5 ug/mL 150 ug/mL 304801 99.2 98.0 99.8 99.5 98.1 97.2 663083 97.8 90.9 99.3 99.3 96.5 93.0 674450 96.2 97.0 98.6 94.4 94.6 89.3 494372 94.1 89.3 98.9 97.5 97.2 93.6 693401 93.6 89.9 96.7 92.0 94.6 90.2 681251 95.4 93.9 99.1 98.2 97.8 96.1 681257 93.4 90.5 97.6 93.7 95.6 92.7 Example 97: Modified oligonucleotides targeting TTR comprising a GalNAc3 conjugate group The oligonucleotides shown in Table 108 comprising a GalNAc conjugate were ed to target TTR.
Table 108 Modified oligonucleotides targeting TTR ces (5 , GalNAc3 SEQ ID ISIS No. to 3 , ) CM Cluster No GalNAc3'3a-0’Ado Tes InC:es Tes Tes Ges Gds Tds Tds Ads InC:ds 666941 GalNAC3_3 Ad 160 Ads Tds Gds Ads Ads Aes Tes InC:es InC:es InC:e Tes mCeo Te0 Te0 Geo Gds Tds Tds Ads InC:ds Ads Tds Gds Ads Ads 666942 GalNAcg-l Ad 157 A60 T60 mCes mCes mCeo Ado"GalNAc3-3a GalNAc3'3a-0’Tes InC:es Tes Tes Ges Gds Tds Tds Ads InC:ds Ads Tds 682876 GalNA03—3 PO 156 Gds Ads Ads Aes Tes mCes mCes mCe GalNAc3'7a-0’Tes InC:es Tes Tes Ges Gds Tds Tds Ads InC:ds Ads Tds 682877 GalNA03-7 PO 156 Gds Ads Ads Aes Tes mCes mCes mCe GalNAc3'10a-0’Tes InC:es Tes Tes Ges Gds Tds Tds Ads InC:ds Ads 682878 GalNA03—10 PO 156 Tds Gds Ads Ads Aes Tes mCes mCes mCe GalNAc3'13a-0’Tes InC:es Tes Tes Ges Gds Tds Tds Ads InC:ds Ads 682879 GalNAcg-13 PO 156 Tds Gds Ads Ads Aes Tes mCes mCes mCe GalNAc3'7a-0’Ado Tes InC:es Tes Tes Ges Gds Tds Tds Ads InC:ds 682880 GalNAC3_7 Ad 160 Ads Tds Gds Ads Ads Aes Tes InC:es InC:es InC:e 3'10a-0’Ado Tes InC:es Tes Tes Ges Gds Tds Tds Ads InC:ds 682881 GalNAC3_10 Ad 160 Ads Tds Gds Ads Ads Aes Tes InC:es InC:es InC:e GalNAc3'13a-0’Ado Tes InC:es Tes Tes Ges Gds Tds Tds Ads InC:ds 682882 GalNAC3_13 Ad 160 Ads Tds Gds Ads Ads Aes Tes InC:es InC:es InC:e Tes InC:es Tes Tes Ges Gds Tds Tds Ads InC:ds Ads Tds Gds Ads Ads 684056 GalNA03-19 Ad 157 Aes Tes mCes mCes mCeo Adoa-GalNAc3-19a The legend for Table 108 can be found in Example 74. The structure of GalNAcg-l was shown in Example 9.
The structure of GalNAC3-3a was shown in Example 39. The structure of GalNAcg-7a was shown in Example 48. The structure of GalNAcg-IOa was shown in Example 46. The structure of g-13a was shown in Example 62. The structure of GalNAcg-19a was shown in Example 70.
Example 98: Evaluation of pro-inflammatory effects of ucleotides comprising a GalNAc conjugate in hPMBC assay The oligonucleotides listed in Table 109 and were tested for pro-inflammatory effects in an hPMBC assay as described in Examples 23 and 24. (See Tables 30, 83, 95, and 108 for descriptions of the ucleotides.) ISIS 353512 is a high responder used as a positive control, and the other oligonucleotides are described in Tables 83, 95, and 108. The results shown in Table 109 were obtained using blood from one volunteer donor. The results show that the oligonucleotides comprising mixed PO/PS internucleoside linkages produced significantly lower pro-inflammatory responses compared to the same oligonucleotides having full PS linkages. Furthermore, the GalNAc conjugate group did not have a significant effect in this assay.
Table 109 ISIS No. Emax/ECSO GalNA03 cluster Linkages CM 353512 3630 n/a PS n/a 420915 802 n/a PS n/a 682881 1311 GalNA03-10 PS Ad 682888 0.26 GalNA03-10 PO/PS Ad 684057 1.03 GalNA03-19 PO/PS Ad Example 99: Binding affinities of oligonucleotides comprising a GalNAc ate for the asialoglycoprotein receptor The binding affinities of the oligonucleotides listed in Table 110 (see Table 76 for ptions of the oligonucleotides) for the asialoglycoprotein receptor were tested in a competitive receptor binding assay. The competitor ligand, (XI-acid glycoprotein (AGP), was incubated in 50 mM sodium acetate buffer (pH 5) with 1 U neuraminidase-agarose for 16 hours at 37°C, and > 90% desialylation was med by either sialic acid assay or size exclusion chromatography (SEC). Iodine monochloride was used to iodinate the AGP according to the procedure by Atsma et a1. (see J Lipid Res. 1991 Jan; 32(1):]73-81.) In this method, desialylated or]- acid glycoprotein (de-AGP) was added to 10 mM iodine de, Na125I, and 1 M glycine in 0.25 M NaOH.
After incubation for 10 minutes at room temperature, 125I ed de-AGP was separated from free 125I by concentrating the e twice utilizing a 3 KDMWCO spin column. The protein was tested for labeling efficiency and purity on a HPLC system equipped with an Agilent SEC-3 column (7.8x300mm) and a 13- RAM r. Competition experiments utilizing 125I -labeled de-AGP and various GalNAc-cluster containing ASOs were performed as follows. Human HepG2 cells (106 cells/ml) were plated on 6-well plates in 2 ml of riate grth media. MEM media supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine and 10mM HEPES was used. Cells were incubated 16-20 hours @ 37°C with 5% and 10% C02 respectively. Cells were washed with media t FBS prior to the experiment. Cells were incubated for 30 min @37°C with 1ml competition mix containing appropriate growth media with 2% FBS, 10'8 M 125 l d de-AGP and GalNAc-cluster containing ASOs at concentrations ranging from 10'11 to 10'5 M. Non- specific binding was ined in the presence of 10'2 M GalNAc sugar. Cells were washed twice with media without FBS to remove unbound 1251 -labeled de-AGP and competitor GalNAc ASO. Cells were lysed using Qiagen’s RLT buffer containing 1% B-mercaptoethanol. Lysates were transferred to round bottom assay tubes after a brief 10 min freeze/thaw cycle and assayed on a y-counter. Non-specific binding was subtracted before dividing 1251 n counts by the value of the lowest GalNAc-ASO concentration counts.
The tion curves were fitted according to a single site competition binding equation using a nonlinear regression algorithm to ate the binding affinities (KD’s).
The results in Table 110 were obtained from experiments med on five different days. Results for oligonucleotides marked with superscript “a” are the average of experiments run on two different days.
The results show that the oligonucleotides comprising a GalNAc conjugate group on the 5’-end bound the asialoglycoprotein receptor on human HepG2 cells with 1.5 to d greater affinity than the oligonucleotides comprising a GalNAc ate group on the 3’-end.
Table 110 Asialoglycoprotein receptor binding assay results Oligonucleotide end to ISIS No. GalNAc conjugate which GalNAc conjugate KD (nM) is attached 661161a GalNAc3-3 5’ 3.7 666881a GalNAc3-10 5’ 7.6 666981 3-7 5’ 6.0 670061 GalNAc3-13 5’ 7.4 655861a GalNAc3-1 3’ 11.6 677841a GalNAc3-19 3’ 60.8 Example 100: Antisense inhibition in vivo by oligonucleotides comprising a GalNAc conjugate group targeting Apo(a) in vivo The oligonucleotides listed in Table 111a below were tested in a single dose study for duration of action in mice.
Table 111a Modified ASOs targeting APO(a) 113:8 Sequences (5’ to 3’) (31111:? CM 118330. 681251 GalNA“JEEZSEEZi::G:ejr:Tdfnd§dSTdSGdSGds GalNAc3-7a P0 58 681257 GalNA“3'73;E}:§a§:§:§c}:e°TE§aTdSTdSGdSGdS 3-7a P0 58 The structure of GalNAc3-7a was shown in Example 48.
Treatment Female transgenic mice that s human Apo(a) were each injected subcutaneously once per week, for a total of 6 doses, with an oligonucleotide and dosage listed in Table 111b or with PBS. Each treatment group consisted of 3 animals. Blood was drawn the day before dosing to determine baseline levels of Apo(a) protein in plasma and at 72 hours, 1 week, and 2 weeks following the first dose. Additional blood draws will occur at 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the first dose. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 111b are presented as the e percent of plasma Apo(a) protein levels for each treatment group, normalized to baseline levels (% BL), The s show that the oligonucleotides comprising a GalNAc conjugate group exhibited potent reduction in Apo(a) expression. This potent effect was ed for the oligonucleotide that comprises full PS internucleoside linkages and the oligonucleotide that comprises mixed PO and PS linkages.
Table 111b Ap0(a) plasma protein levels Apo(a) at 72 hours Apo(a) at 1 week Apo(a) at 3 weeks ISIS No. Dosage ) (% BL) (% BL) (% BL) PBS n/a 116 104 107 0.3 97 108 93 1.0 85 77 57 681251 3.0 54 49 11 .0 23 15 4 0.3 114 138 104 1.0 91 98 54 681257 3.0 69 40 6 .0 30 21 4 Example 101: Antisense inhibition by oligonucleotides comprising a GalNAc cluster linked via a stable moiety The oligonucleotides listed in Table 112 were tested for tion of mouse APOC-III expression in vivo. C57Bl/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 112 or with PBS. Each treatment group consisted of 4 animals. Each mouse treated with ISIS 440670 received a dose of 2, 6, 20, or 60 mg/kg. Each mouse d with ISIS 680772 or 696847 received 0.6, 2, 6, or 20 mg/kg. The GalNAc conjugate group of ISIS 696847 is linked via a stable moiety, a phosphorothioate linkage instead of a readily cleavable phosphodiester containing linkage. The animals were sacrificed 72 hours after the dose. Liver APOC-III mRNA levels were measured using real-time PCR. APOC-III mRNA levels were normalized to cyclophilin mRNA levels according to standard ols. The results are presented in Table 112 as the average percent of APOC-III mRNA levels for each treatment group ve to the saline control group. The results show that the ucleotides comprising a GalNAc conjugate group were significantly more potent than the oligonucleotide lacking a conjugate group. Furthermore, the ucleotide comprising a GalNAc conjugate group linked to the oligonucleotide via a cleavable moiety (ISIS 680772) was even more potent than the oligonucleotide comprising a GalNAc conjugate group linked to the oligonucleotide via a stable moiety (ISIS ).
Table 1 12 Modified oligonucleotides ing mouse APOC-III Dosage APOC-III 11:18 SEQ Sequences (5 ’ to 3 ’) CM (mg/kg) mRNA (% 0. ID No.
PBS) 2 92 InC:es146:3ClesmC:esTesTdsTdslAdsTdsTdslAds 6 86 440670 n/a 162 GdsGdsGdsAdsmCes AesGes InC:eslAe 20 59 60 3 7 0.6 79 GalNAc3'7a-0’mCesAesGesmCesTesTdsTdsAds 2 5 8 680772 PO 162 TdSTdSAdSGdS dsladsmcks AesGesmCesAe 6 3 1 1 3 0 . 6 83 GalNAc3'7a-s’mCesAesGesmCesTesTdsTdsAdsTds 2 73 696847 n/a (PS—) 162 TdsAdsGdsGdsGdsAdsmCes AesGesmCesAe 6 4O 28 The structure of GalNAc3-7a was shown in Example 48.
Example 102: Distribution in liver of antisense oligonucleotides comprising a GalNAc conjugate The liver distribution of ISIS 353382 (see Table 36) that does not se a GalNAc conjugate and ISIS 655861 (see Table 36) that does se a GalNAc conjugate was evaluated. Male balb/c mice were subcutaneously injected once with ISIS 353382 or 655861 at a dosage listed in Table 113. Each treatment group consisted of 3 s except for the 18 mg/kg group for ISIS 655861, which consisted of 2 animals.
The animals were sacrificed 48 hours following the dose to determine the liver distribution of the oligonucleotides. In order to measure the number of antisense oligonucleotide molecules per cell, a Ruthenium (II) tris-bipyridine tag (MSD TAG, Meso Scale Discovery) was conjugated to an oligonucleotide probe used to detect the antisense oligonucleotides. The results presented in Table 113 are the average concentrations of oligonucleotide for each ent group in units of millions of oligonucleotide molecules per cell. The results show that at equivalent doses, the ucleotide comprising a GalNAc conjugate was present at higher concentrations in the total liver and in hepatocytes than the ucleotide that does not comprise a GalNAc conjugate. Furthermore, the oligonucleotide comprising a GalNAc conjugate was present at lower concentrations in non-parenchymal liver cells than the oligonucleotide that does not se a GalNAc conjugate. And while the concentrations of ISIS 655 861 in hepatocytes and non-parenchymal liver cells were similar per cell, the liver is approximately 80% hepatocytes by volume. Thus, the majority of the ISIS 655 861 oligonucleotide that was present in the liver was found in hepatocytes, whereas the majority of the ISIS 353382 oligonucleotide that was present in the liver was found in non-parenchymal liver cells.
Table 113 ISIS Dosage Concentration in whole Concentration in Concentration in non- llver (molecules*10A6 hepatocytes parenchymal 11ver cells No' (mg/kg) per cell) (molecules*10A6 per cell) (molecules*10A6 per cell) 3 9.7 1.2 37.2 17.3 4.5 34.0 23.6 6.6 65.6 353382 29.1 11.7 80.0 60 73.4 14.8 98.0 90 89.6 18.5 119.9 0.5 2.6 2.9 3.2 1 6.2 7.0 8.8 655861 3 19.1 25.1 28.5 6 44.1 48.7 55.0 18 76.6 82.3 77.1 Example 103: Duration of action in vivo of oligonucleotides targeting APOC-III comprising a GalNAc3 conjugate The oligonucleotides listed in Table 114 below were tested in a single dose study for duration of action in mice.
Table 1 14 Modified ASOs targeting APOC-III ISIS Sequences (5 ’ to 3 ’) GalNAc3 CM SEQ No. Cluster ID No. 3 04 8 01 AesGesmcesTCSTesmCdSTdSTdsGdSTdSmCdsmCdsAdSGdsmCdsTesTes n/a n/a 13 5 663 O84 GalNAc3-3a-O’AdersGeomCCOTCOTCOmCdSTdSTdSGdSTdSmCdS GalNAC3'3 3 Ad 151 mCdsAdsGdsmCdsTeoTeo TesAesTe 679241 AesGeomCeoTeoTeomCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmCdsTeoTeo GalNAc3-19a Ad 13 6 TesAesTeoAdoa-GalNAc3-19a The structure of 3-3a was shown in Example 39, and 3-19a was shown in Example 70.
Treatment Female transgenic mice that express human APOC-HI were each injected subcutaneously once with an oligonucleotide listed in Table 114 or with PBS. Each treatment group consisted of 3 animals. Blood was drawn before dosing to determine baseline and at 3, 7, 14, 21, 28, 35, and 42 days following the dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results in Table 115 are ted as the average t of plasma triglyceride and APOC-III levels for each treatment group, normalized to baseline levels. A comparison of the results in Table 71 of e 79 with the results in Table 115 below show that oligonucleotides comprising a mixture of phosphodiester and phosphorothioate internucleoside linkages exhibited increased duration of action than equivalent oligonucleotides comprising only phosphorothioate internucleoside linkages.
Table 115 Plasma triglyceride and APOC-III protein levels in transgenic mice T1me pomt ISIS Dosage Triglycerides AFDC-HI 3 CM (days p05“ prom (% No' (mg/kg) (% baseline) Cluster dose) ne) 3 96 101 7 88 98 14 91 103 PBS n/a 21 69 92 n/a n/a 28 83 81 65 86 42 72 88 3 42 46 7 42 51 14 59 69 304801 30 21 67 81 n/a n/a 28 79 76 72 95 42 82 92 3 35 28 7 23 24 14 23 26 663084 10 21 23 29 GalNAc3-3a Ad 28 30 22 32 36 42 37 47 3 38 30 7 31 28 14 30 22 679241 10 21 36 34 Gag“? Ad 28 48 34 50 45 42 72 64 2014/036460 Example 104: Synthesis of ucleotides comprising a 5’-GalNAc2 conjugate HN'BOC HN’BOC HBTU, HOBt O + H TFA BomN OH H2N\/\/\)LO —> BomN NMO 4’ H DIEA, DMF H o o mDOM 120 126 85% 231 0A0 0A0 F NH\/\/\)OJ\ 0F D'EA o + A00 ONgt o ACHN 0 166 F 0A0 0A0 moi/[4’0 0A0 0A0 55* O mo 0W ACHN ACHN NH 1. H2 Pd/C MeOH 0A0 2 PFPTFA DMF 0A0 0A0 F F o 0 A00IfA20 OWN ONO/w A00 OW NWI5go ACHN ACHN N O F o F O 83e OH OH 3' 5' H O O OLIGO 0_ _Fl’ 0_(CH2)6 NH2— Hofiow ACHN NH 1. Borate buffer, DMSO, pH 8.5, rt OH OH HOE go o N\/\/\)L 2. aq. ammonia, rt HAOCHN OWN HMO -m-OLIGO Compound 120 is commercially available, and the synthesis of compound 126 is described in Example 49. Compound 120 (1 g, 2.89 mmol), HBTU (0.39 g, 2.89 mmol), and HOBt (1.64 g, 4.33 mmol) were dissolved in DMF (10 mL. and sopropylethylamine (1.75 mL, 10.1 mmol) were added. After about 5 min, aminohexanoic acid benzyl ester (1.36 g, 3.46 mmol) was added to the reaction. After 3h, the reaction mixture was poured into 100 mL of 1 M NaHSO4 and extracted with 2 x 50 mL ethyl acetate.
Organic layers were combined and washed with 3 x 40 mL sat NaHC03 and 2 x brine, dried with Na2S04, filtered and concentrated. The product was purified by silica gel column chromatography (DCM:EA:Hex 1:1 :1) to yield compound 231. LCMS and NMR were consistent with the structure. Compounds 231 (1.34 g, 2.438 mmol) was dissolved in dichloromethane (10 mL) and racetic acid (10 mL) was added. After stirring at room ature for 2h, the reaction mixture was concentrated under reduced pressure and c0- evaporated with toluene ( 3 x 10 mL). The residue was dried under reduced pressure to yield compound 232 as the oracetate salt. The synthesis of compound 166 is described in e 54. Compound 166 (3.39 g, 5.40 mmol) was dissolved in DMF (3 mL). A solution of compound 232 (1.3 g, 2.25 mmol) was dissolved in DMF (3 mL) and N,N—diisopropylethylamine (1.55 mL) was added. The reaction was stirred at room temperature for 30 minutes, then poured into water (80 mL) and the aqueous layer was extracted with EtOAc (2x100 mL). The organic phase was separated and washed with sat. aqueous NaHC03 (3 x 80 mL), 1 WO 79625 M NaHSO4 (3 x 80 mL) and brine (2 x 80 mL), then dried (NaZSO4), filtered, and concentrated. The e was purified by silica gel column chromatography to yield compound 233. LCMS and NMR were consistent with the structure. Compound 233 (0.59 g, 0.48 mmol) was dissolved in methanol (2.2 mL) and ethyl acetate (2.2 mL). Palladium on carbon (10 wt% Pd/C, wet 0.07 g) was added, and the reaction mixture was stirred under hydrogen atmosphere for 3 h. The reaction mixture was d through a pad of Celite and concentrated to yield the carboxylic acid. The carboxylic acid (1.32 g, 1.15 mmol, cluster free acid) was dissolved in DMF (3.2 mL). To this N,N—diisopropylehtylamine (0.3 mL, 1.73 mmol) and PFPTFA (0.30 mL, 1.73 mmol) were added. After 30 min stirring at room ature the reaction mixture was poured into water (40 mL) and extracted with EtOAc (2 x 50 mL). A standard work-up was completed as described above to yield compound 234. LCMS and NMR were consistent with the structure. Oligonucleotide 235 was prepared using the general ure described in Example 46. The GalNAcZ cluster portion (GalNAc2-24a) of the conjugate group 2-24 can be ed with any cleavable moiety present on the oligonucleotide to e a variety of conjugate groups. The structure of GalNAc2-24 (GalNAc2-24a-CM) is shown below: OH OH ACHNHAOC%’Ox/\/\)J\; OHOH ON\/\/\j\NAM/\OWE Example 105: Synthesis of oligonucleotides sing a GalNAc1-25 conjugate O_|O| 83e —5.OL—GO —o—(CH2)6-NH2 OACOAc | Rog/owJfl;1. Borate buffer DMSO pH 8.5OH rt AcHN 2. aq. ammonia, rt OH OH 0MN14-om AcHN The synthesis of compound 166 is described in Example 54. Oligonucleotide 236 was prepared using the general procedure described in Example 46.
Alternatively, oligonucleotide 236 was synthesized using the scheme shown below, and compound 238 was used to form the oligonucleotide 236 using procedures described in Example 10.
OAC /OH OAc ACOQVLOWOA H2N 0A AGO OW + PFPTFA NHAc N/\/\/\/OH NHAc OH TEA, Acetonltrlle_ _ H tetrazole, 1-Methylimidazole, DMF O O AGO OW Y N/\/\/\/o N 2-cyanoethyltetraisopropyl phosphorodiamidite NHAc \p’ Y H | 238 01 OH OH Oligonucleotide synthesis HO O ’ O\/\/\)J\ fi OLIGO N o ACHN H 6 The GalNAc1 cluster portion (GalNAc1-25a) of the conjugate group GalNAc1-25 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc1-25 c1-25a-CM) is shown below: OH OH HO 0 OMNAO,- 3 ACHN H 6 Example 106: Antisense inhibition in vivo by oligonucleotides ing SRB-l comprising a 5’- GalNAcz or a 5’-GalNAc3 conjugate Oligonucleotides listed in Tables 116 and 117 were tested in dose-dependent studies for nse inhibition of SRB-l in mice.
Treatment Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once with 2, 7, or 20 mg/kg of ISIS No. 440762; or with 0.2, 0.6, 2, 6, or 20 mg/kg of ISIS No. 686221, 686222, or 708561; or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Liver SRB-l mRNA levels were measured using real- time PCR. SRB-l mRNA levels were normalized to hilin mRNA levels according to standard protocols. The antisense ucleotides lowered SRB-l mRNA levels in a dose-dependent manner, and the EDSO results are presented in Tables 116 and 117. Although previous studies showed that trivalent GalNAc- ated ucleotides were significantly more potent than nt GalNAc-conjugated oligonucleotides, which were in turn significantly more potent than monovalent GalNAc conjugated oligonucleotides (see, e.g., Khorev et al., Bioorg. & Med. Chem, Vol. 16, 5216-5231 (2008)), treatment with antisense oligonucleotides comprising monovalent, divalent, and trivalent GalNAc clusters lowered SRB-l mRNA levels with similar potencies as shown in Tables 116 and 117.
Table 1 1 6 Modified oligonucleotides targeting SRB-l ISIS , , ED50 SEQ Sequences (5 to 3 ) GalNAc Cluster No. (mg/kg) ID No 440762 TkskasAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTkska n/a 4. 7 l 3 7 686221 GalNAc2-24a-0’Adogks gdsTds CdSAdSTdSGdSAdS Z'24a O39 141 CdsTdsTks Ck 686222 GalNAc3-13a'0’AdOEks CkSAdsgdsTds CdSAdSTdSGdSAdS GalNAC3-13a 041 141 CdsTdsTks Ck See Example 93 for table legend. The structure of GalNAc3-13a was shown in Example 62, and the structure of GalNAc2-24a was shown in Example 104.
Table 117 d oligonucleotides targeting SRB-l ISIS Sequences (5 , to 3 , ED50 SEQ ) GalNAc Cluster No. (mg/kg) ID No 440762 AdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTkska 1 3 7 GalNAcl'Zsa'o’TkI: tnsTds CdsAdsTdsGdsAds 708561 GalNAc125a CdsTdsTks Ck See Example 93 for table legend. The structure of GalNAc1-25a was shownIn Example 105.
The concentrations of the ucleotides in Tables 116 and 117 in liver were also assessed, using procedures described in Example 75. The results shown in Tables 117a and 117b below are the average total antisense oligonucleotide tissues levels for each treatment group, as measured by UV in units of ug oligonucleotide per gram of liver . The results show that the ucleotides comprising a GalNAc conjugate group accumulated in the liver at significantly higher levels than the same dose of the oligonucleotide lacking a GalNAc conjugate group. Furthermore, the antisense oligonucleotides comprising one, two, or three GalNAc ligands in their respective conjugate groups all accumulated in the liver at similar levels. This result is surprising in view of the Khorev et al. literature reference cited above and is consistent with the activity data shown in Tables 116 and 117 above.
Table 1 17a Liver concentrations of oligonucleotides comprising a GalNAcz or GalNAc3 ate group [Antisense oligonucleotide] (ug/g) GalNAc cluster 440762 7 13.1 n/a n/a 31.1 0.2 0.9 0.6 2. 7 686221 GalNAc2-24a Ad 2 120 6 26.5 686222 ' ' GalNAcg-13a Table 117b Liver concentrations of ucleotides comprising a 1 conjugate group ISIS N0. fits/1:6) [Antisense oligonucleotide] (ug/g) GalNAc cluster CM 2 2.3 440762 7 8.9 n/a n/a 23.7 0.2 0.4 0.6 1.1 708561 2 5.9 GalNAcl-25a PO 6 23.7 53.9 Example 107: Synthesis of oligonucleotides comprising a GalNAc1-26 or GalNAc1-27 conjugate Ho? §OH o W O O\/\/\)J\ ..nO ACHN Oligonucleotide 239 is synthesized via coupling of compound 47 (see Example 15) to acid 64 (see Example 32) using HBTU and DIEA in DMF. The ing amide containing compound is phosphitylated, then added to the 5’-end of an oligonucleotide using procedures described in Example 10. The GalNA01 cluster portion cl-26a) 0f the conjugate group GalNAcl-26 can be combined With any cleavable moiety t on the oligonucleotide to provide a variety of conjugate . The structure of GalNAcl-26 (GalNAcl-26a-CM) is shown below: o -E Hog/O MO ,IIIO ACHN In order to add the GalNAq conjugate group to the 3’-end of an oligonucleotide, the amide formed from the reaction of compounds 47 and 64 is added to a solid support using procedures described in Example 7. The oligonucleotide sis is then completed using procedures described in Example 9 in order to form oligonucleotide 240.
O .mOH AcHN 240 3' 5' The GalNAc1 cluster portion (GalNAc1-27a) of the conjugate group GalNAc1-27 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of ate . The ure of GalNAc1-27 (GalNAc1-27a-CM) is shown below: 0\/\/\/U\ .-I\OH ACHN o 2 Example 108: Antisense inhibition in vivo by oligonucleotides comprising a GalNAc conjugate group targeting Apo(a) in vivo The oligonucleotides listed in Table 118 below were tested in a single dose study in mice.
Table 118 Modified ASOs targeting APO(a) 1s1s , , GalNAc3 SEQ sequences (5 t0 3 ) CM No. Cluster ID No.
TesCles CesTes Ces 494372 CdsGdsTdsTLIidesGdsTdsGds Cds n/a n/a 58 TdsTesClesTesTes Ce CesTes Ces 681251 GalNAc3'7a'o’Tesges CdsglndsTdsTdsGdsGds GalNAc3—7a P0 58 TdsClds CdsTdsTesGes TesTes Ce 681255 GalNAc3'3a'o’Tesgeo CeoTeo Ceo CdsgdsTdsTdsGdsGds GalNAc3—3a P0 58 TdsClds CdsTdsTeoGeo TesTes Ce 681256 GalNAc3'10a'o’TesIE-leo CeoTeo Ceo CdsgdsTdsTdsGdsGds GalNAc3-10a P0 58 TdsClds CdsTdsTeoGeo TesTes Ce GalNAc3'7a'o’Tesgeo CeoTeo Ceo 681257 CdsgdsTdsTdsGdsGds GalNAc3—7a P0 58 s CdsTdsTeoGeo TesTes Ce 681258 3'13a'o’TesIE-leo CeoTeo Ceo CdsgdsTdsTdsGdsGds GalNAc3— 1 3a P0 58 TdsClds CdsTdsTeoGeo TesTes Ce TesCleomC:eoTeomC:eomC:dsCldsTdsTdsC}dsC}ds smChSTdsTeoC}eo 681260 G lNAa C3-193 Ad 167 TesTesmCeoAdoa-GalNAQ-l9 The structure of GalNAc3-7a was shown in Example 48.
Treatment Male transgenic mice that express human Apo(a) were each injected subcutaneously once with an oligonucleotide and dosage listed in Table 119 or with PBS. Each treatment group consisted of 4 animals.
Blood was drawn the day before dosing to determine baseline levels of Apo(a) n in plasma and at 1 week following the first dose. Additional blood draws will occur weekly for approximately 8 weeks. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 119 are presented as the average percent of plasma Apo(a) protein levels for each treatment group, normalized to baseline levels (% BL), The results show that the antisense oligonucleotides d Apo(a) protein expression. Furthermore, the oligonucleotides comprising a GalNAc conjugate group exhibited even more potent reduction in Apo(a) sion than the oligonucleotide that does not comprise a conjugate group.
Table 119 Ap0(a) plasma protein levels Apo(a) at 1 week ISIS No. Dosage (mg/kg) (% BL) PBS n/a 143 494372 50 58 681251 10 15 681255 10 14 681256 10 17 681257 10 24 681258 10 22 681260 10 26 Example 109: Synthesis of oligonucleotides sing a GalNAc1-28 0r GalNAc1-29 conjugate OH 5' 3' HO 0\- HO MN N AcHN HWY 241 OH Oligonucleotide 241 is synthesized using procedures r to those described in e 71 to form the phosphoramidite intermediate, followed by procedures described in e 10 to synthesize the oligonucleotide. The GalNAc1 cluster portion (GalNAc1-28a) of the conjugate group GalNAc1-28 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups.
The structure of GalNAc1-28 (GalNAc1-28a-CM) is shown below: HO “o H0 W\)‘\ N ACHN NW In order to add the 1 conjugate group to the 3’-end of an oligonucleotide, procedures similar to those described in Example 71 are used to form the hydroxyl intermediate, which is then added to the solid support using procedures described in Example 7. The oligonucleotide synthesis is then completed using ures described in Example 9 in order to form ucleotide 242.
HO OH O .\\ Ho Mo N ACHN ”WY 3' 5' 242 O_- W The GalNAc1 cluster portion (GalNAc1-29a) of the conjugate group GalNAc1-29 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc1-29 (GalNAc1-29a-CM) is shown below: O _‘\OH HO M0 N ACHN ”WY —&3 Example 110: Synthesis of oligonucleotides comprising a GalNAc1-30 conjugate OAc OAc A00 A00 o HO OTBDPS 0 A00 AcO O\/\/\/OTBDPS TMSOTf N ACHN 7/0 243 1. NH /MeOH ODMTr 2_ DM§FrC| AcO 1. TBAF 3_ ACZO, pyr O 2. Phosphltllatlon ACO O\/\/\/OTBDPS —’ ODMTr 1. Couple to 5'-end of A80 A00 O\/\/\/O\P’OCE —> ACHN 245 '{mpoz 2. ect and purify ASO using DMT-on ation methods WO 79625 Ho§fi/O 5' 3' HO O\/\/\/O\p/o\ ACHN 0/; \Y Oligonucleotide 246 comprising a GalNAc1-3O conjugate group, wherein Y is selected from O, S, a tuted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The 1 cluster portion c1-3Oa) of the conjugate group GalNAc1-3O can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, Y is part of the cleavable . In certain embodiments, Y is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc1-3Oa is shown below: HO O\/\/\/O\’; AcHN Example 111: Synthesis of oligonucleotides comprising a GalNAc2-31 0r GalNAc2-32 conjugate DMTrO 1. DMTI'CI OCE Couple to 5'-end of A80 2. Phosphitilation / O-P\ —> DMTrO H0 247 248 1. Remove DMTr groups DMTrO “(k/é; 2. Couple amidite 245 Ol/P\ 3. Deprotect and purify ASO using O‘Oligo \ DMT-on DMTrO purification methods Ooh/J10?)6V Oligonucleotide 250 comprising a GalNAc2-3l conjugate group, n Y is selected from O, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, l or alkynyl, is synthesized as shown above. The GalNAcz cluster portion (GalNAc2-3la) of the conjugate group GalNAc2-3l can be combined with any cleavable moiety to provide a variety of conjugate . In certain embodiments, the Y- containing group directly adjacent to the 5’-end of the oligonucleotide is part of the cleavable moiety. In certain embodiments, the Y-containing group directly adjacent to the 5’-end of the oligonucleotide is part of a stable moiety, and the cleavable moiety is present on the ucleotide. The structure of GalNAcz-31a is shown below: HO$g/O\/\/\/O\p/OHO AcHN o” \Y O~P\ OH \/\/\/ 6’ Y HO§OA/oHO ACHN The synthesis of an oligonucleotide comprising a GalNAc2-32 conjugate is shown below. 1. DMTrCI 2. Allyl Br 3- 0304, NaIO4 1. Couple to 5'-end of A80 DMTrO HO 4. NaBH4 2. Remove DMTr groups . itilatlon__ _ 3. Couple amidite 245 OH O—\_ 0\ 4. Deprotect and purify ASO using DMTrO _ HO ,P_N(|Pr)2 DMT-on purification s 247 CEO OH fl(5’ Y NHAc Oligonucleotide 252 sing a GalNAc2-32 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, l or alkynyl, is synthesized as shown above. The GalNAcz cluster portion (GalNAc2-32a) of the conjugate group GalNAc2-32 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain ments, the Y- containing group directly adjacent to the 5’-end of the oligonucleotide is part of the cleavable moiety. In certain embodiments, the Y-containing group directly adjacent to the 5’-end of the oligonucleotide is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc2-32a is shownbelow: HO$\\/O\/\/\/O\PIOHO O AcHN OMY Os ,0 /,P\ W0)“ 0 O Y O‘P: 0'I Y OH fl NHAc Example 112: Modified oligonucleotides comprising a GalNAc1 conjugate The oligonucleotides in Table 120 targeting SRB-l were synthesized with a GalNAc1 conjugate group in order to further test the potency of oligonucleotides sing conjugate groups that contain one GalNAc ligand.
Table 120 GalNAc , , SEQ ISIS N0. Sequence (5 to 3 ) CM cluster ID NO. 711461 GalNAcl'ZSa-O’Ado Ges InC:es Tes Tes InC:es Ads Gds Tds InC:ds Ads Tds G31\AC '253, Ad 145 Gcs Ads InC:ds Tds Tes InC:es InC:es Tes Te 711462 GalNAcl'ZSa-O’Ges InC:es Tes Tes InC:es Ads Gds Tds InC:ds Ads Tds Gds G31\AC "253, PO 143 Acs InC:ds Tds Tes InC:es InC:es Tes Te 711463 Gal.\IAc1-25a_0aGes InCeo T60 T60 InCeo Ads Gds Tds mCds Ads Tds Gal\Ac -25a PO 143 Gcs Ads InC:ds Tds Te0 InC:eo InC:es Tes Te 711465 GalNAcl'26a-0’Ado Ges InC:es Tes Tes InC:es Ads Gds Tds InC:ds Ads Tds G31\AC '263, Ad 145 Gcs Ads InC:ds Tds Tes InC:es InC:es Tes Te 711466 GalNAcl'26a-0’Ges InC:es Tes Tes InC:es Ads Gds Tds InC:ds Ads Tds Gds G31\AC '26a PO 143 Acs InC:ds Tds Tes InC:es InC:es Tes Te 711467 GalNAc1-26HaGes InCeo T60 T60 InCeo Ads Gds Tds mCdS Ads Tds Gal\Ac -26a PO 143 Gcs Ads InC:ds Tds Te0 InC:eo InC:es Tes Te 711468 l'ZSa-O’Ado Ges InC:es Tes Tes InC:es Ads Gds Tds InC:ds Ads Tds G31\AC '283, Ad 145 Gcs Ads InC:ds Tds Tes InC:es InC:es Tes Te 711469 l'ZSa-O’Ges InC:es Tes Tes InC:es Ads Gds Tds InC:ds Ads Tds Gds G31\AC '283, PO 143 Acs InC:ds Tds Tes InC:es InC:es Tes Te 71 1470 GalNAcl'ZSa-O’Ges InC:eo Te0 Te0 InC:eo Ads Gds Tds InC:ds Ads Tds G31\AC '28a PO 143 Gcs Ads InC:ds Tds Te0 InC:eo InC:es Tes Te 713844 Ges InC:es Tes Tes InC:es Ads Gds Tds InC:ds Ads Tds Gds Ads InC:ds Tds G31\AC -27a PO 143 Tes InCes InCes Tes T60s_GalNAc1-27a 713845 Ges InC:eo Te0 Te0 InC:eo Ads Gds Tds InC:ds Ads Tds Gds Ads InC:ds Tds G31\AC -27a PO 143 Teo InCeo InCes Tes T60s_GalNAc1-27a 713846 Ges InC:eo Te0 Te0 InC:eo Ads Gds Tds InC:ds Ads Tds Gds Ads InC:ds Tds G31\AC "273. Ad 144 Teo InCeo InCes Tes T60 Ados_GalNAc1-27a 713847 Ges InC:es Tes Tes InC:es Ads Gds Tds InC:ds Ads Tds Gds Ads InC:ds Tds G31\AC -29a PO 143 Tes InCes InCes Tes Teoa_GalNAc1-29a 713848 Ges InC:eo Te0 Te0 InC:eo Ads Gds Tds InC:ds Ads Tds Gds Ads InC:ds Tds G31\AC -29a PO 143 Teo InCeo InCes Tes alNAc1-29a 713849 Ges InC:es Tes Tes InC:es Ads Gds Tds InC:ds Ads Tds Gds Ads InC:ds Tds G31\AC '293, Ad 144 Tes InCes InCes TeS Teo Adoa_GalNAc1-29a 713850 Ges InC:eo Te0 Te0 InC:eo Ads Gds Tds InC:ds Ads Tds Gds Ads InC:ds Tds G31\AC "293. Ad 144 Te0 InC:eo InC:es Tes Te0 Ado’-(;alNAcl'2'9a Example 113: Dose-dependent antisense inhibition of human apolipoprotein (a) (apo(a)) in human primary hepatocytes Selected gapmer antisense oligonucleotides from a previous ation (W02005/000201, the content of which is incorporated by reference in its entirety herein) were tested in a single dose assay in human primary hepatocytes. Cells were obtained from Tissue ormation Technologies (BD Biosciences, Franklin Lakes, NJ) and treated with 150 nM of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR. Human apo(a) primer probe set )3’ (forward sequence ACAGCAATCAAACGAAGACACTG, designated herein as SEQ ID NO: 5; reverse sequence AGCTTATACACAAAAATACCAAAAATGC, designated herein as SEQ ID NO: 6; probe sequence TCCCAGCTACCAGCTATGCCAAACCTT, designated herein as SEQ ID NO: 7) was used to measure mRNA levels. Additionally, mRNA levels were also ed using human apo(a) primer probe set hAPO(a)12kB (forward sequence CCACAGTGGCCCCGGT, designated herein as SEQ ID NO: 8; reverse sequence CTTTTCTCAGGTGGT, designated herein as SEQ ID NO: 9; probe sequence CCAAGCACAGAGGCTCCTTCTGAACAAG, ated herein as SEQ ID NO: 10). Apo(a) mRNA levels were normalized to GAPDH mRNA expression. Results are presented in the table below as percent inhibition of apo(a), relative to untreated control cells.
Table 121 nse inhibition of human apo(a) in human primary hepatocytes % inhibition % inhibition ISIS No (hAPO(a)3’ (hAPO(a)12kB PPset) PPset) 144367 68 77 144368 42 59 144369 43 69 144370 80 75 144371 42 57 144372 87 54 144373 63 49 144374 45 80 144375 33 11 144376 62 82 144377 42 72 144378 0 72 144379 73 46 144380 75 78 144381 63 64 144382 0 58 144383 63 79 144384 38 O 144385 40 94 144386 47 61 144387 38 60 144388 0 57 144389 52 39 144390 12 0 144391 73 57 144392 43 50 144393 83 82 144394 40 76 144395 80 84 144396 53 72 144397 23 64 144398 7 33 144399 43 44 144400 70 75 144401 87 72 Several antisense oligonucleotides were ed for r testing in a dose response assay.
The selected nse oligonucleotides were tested in human primary hepatocytes with 25 nM, 50 nM, 150 nM, or 300 nM trations of antisense oligonucleotide, as specified in the table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR. Human apo(a) primer probe set hAPO(a)3’ was used to measure mRNA levels. Apo(a) mRNA levels were normalized to GAPDH mRNA expression. Results are presented as percent inhibition of apo(a), relative to untreated control cells.
Table 122 Dose-dependent antisense inhibition of human apo(a) in human primary hepatocytes, as measured with hAPO(a)3 ’ ISIS No 25nM 50nM 150nM 300nM ————n 144395 17 9 8 32 ISIS 1443 67 demonstrated better efficacy and dose-dependency than the other nse oligonucleotides. Hence, ISIS 144367 was considered the benchmark antisense oligonucleotide to compare the potency of newly designed antisense oligonucleotides disclosed herein.
Example 114: Antisense inhibition of human apo(a) in transgenic mouse primary hepatocytes Antisense oligonucleotides were newly designed targeting an apo(a) nucleic acid and were tested for their effects on apo(a) mRNA in vitro. The antisense oligonucleotides were tested for potency in a series of parallel ments that had similar culture conditions. Primary hepatocytes from human apo(a) transgenic mice (Frazer, K.A. et al., Nat. Genet. 1995. 9: 424-431) were used in this study. Hepatocytes at a density of 35,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a ent period of approximately 24 hours, RNA was isolated from the cells and apo(a) mRNA levels were ed by quantitative real-time PCR. Human primer probe set hAPO(a)12kB was used to measure mRNA levels. Apo(a) mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results for each experiment are presented in separate tables shown below. ISIS 144367 from was used as a benchmark for the new antisense oligonucleotides and also included in the studies.
Results are presented as t inhibition of apo(a), relative to untreated control cells. A total of 1,511 gapmers were tested under these culture ions. Only those antisense oligonucleotides that were selected for further study are presented in the table below with each table representing a separate experiment.
The newly designed chimeric antisense oligonucleotides were designed as 55 MOE gapmers.
The gapmers are 20 nucleosides in length, wherein the l gap segment comprises of ten 2’- deoxynucleosides and is flanked by wing segments on the 5’ direction and the 3’ ion comprising five nucleosides each. Each nucleoside in the 5’ wing segment and each nucleoside in the 3’ wing segment has a 2’-MOE modification. The internucleoside linkages hout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
The apo(a) target sequence contains le Kringle repeat ces, therefore, an antisense oligonucleotide may target one or more regions of apo(a) depending whether on the oligonucleotide targets a Kringle sequence or not. “Start site” indicates the 5’-most nucleoside to which the gapmer is ed in the human ce. “Stop site” indicates the 3’-most nucleoside to which the gapmer is targeted human sequence. An apo(a) antisense oligonucleotide may have more than one “Start site” or “Stop site” ing on whether or not it targets a Kringle repeat.
Most gapmers listed in the tables are targeted with 100% complementarity to one or more regions of either the human apo(a) mRNA, ated herein as SEQ ID NO: 1 (GENBANK Accession No.
NM_005577.2) or the human apo(a) genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_OO7422.12 truncated from nucleotides 3230000 to 3380000), or both. ‘n/a’ indicates that the antisense oligonucleotide does not target that ular sequence With 100% complementarity.
Table 123 SEQID SEQ SEQ SEQID SEQ ISIS DH):1 96 HDNO: HDNO: DH):1 Sequence ID 1J0 Sunfi inhibition 2 Start 2 Stop Stop Sfie NO S1te Site Site 144367 249 268 GGCAGGTCCTTCCTGTGACA 90 21210 21229 11 238 257 21199 21218 580 599 26690 26709 922 941 32237 32256 494157 ACAGTGGTGGAGTA 95 12 1606 1625 43330 43349 1948 1967 48874 48893 2290 2309 54420 54439 3316 3335 72037 72056 239 258 21200 21219 581 600 26691 26710 923 942 32238 32257 494158 1607 1626 TCCTGTGACAGTGGTGGAGT 95 43331 43350 13 1949 1968 48875 48894 2291 2310 54421 54440 3317 3336 72038 72057 241 260 21202 21221 583 602 26693 26712 925 944 32240 32259 1609 1628 43333 43352 494159 1951 1970 CTTCCTGTGACAGTGGTGGA 97 48877 48896 14 2293 2312 54423 54442 3319 3338 72040 72059 4663 4682 94404 94423 5005 5024 115515 115534 242 261 21203 21222 494160 4664 4683 CCTTCCTGTGACAGTGGTGG 97 94405 94424 15 5006 5025 115516 115535 243 262 21204 21223 494161 4665 4684 TCCTTCCTGTGACAGTGGTG 96 94406 94425 16 5007 5026 115517 115536 244 263 21205 21224 3664 3683 77585 77604 494162 GTCCTTCCTGTGACAGTGGT 95 17 4666 4685 94407 94426 5008 5027 115518 115537 494163 245 264 GGTCCTTCCTGTGACAGTGG 96 21206 21225 18 4667 4686 94408 94427 246 265 21207 21226 494164 TTCCTGTGACAGTG 93 19 —46684687 —9440994428 247 266 21208 21227 494165 —4669 CAGGTCCTTCCTGTGACAGT 91 4688 —9441094429 20 494166 248 267 GCAGGTCCTTCCTGTGACAG 89 21209 21228 21 494167 250 269 TGGCAGGTCCTTCCTGTGAC 92 21211 21230 22 494168 251 270 TTGGCAGGTCCTTCCTGTGA 89 21212 21231 23 494169 252 271 CTTGGCAGGTCCTTCCTGTG 92 21213 21232 24 494170 253 272 GCTTGGCAGGTCCTTCCTGT 88 21214 21233 25 Table 124 SEQ ID SEQ SEQ ID SEQ ID NO: 1 ID NO: 0A) SEQ ISIS NO Sequence . . NO: 2 NO: 2 Start 1 Stop 1nh1b1t10n ID NO . . Start S1te. Stop S1te.
S1te S1te 144367 249 268 TCCTTCCTGTGACA :1 21210 21229 11 584 603 26694 26713 926 945 32241 32260 1610 1629 43334 43353 494283 TCTTCCTGTGACAGTGGTGG 93 26 —19521971 —4887848897 2294 2313 54424 54443 3320 3339 72041 72060 585 604 26695 26714 927 946 32242 32261 1611 1630 43335 43354 494284 —1953 TTCTTCCTGTGACAGTGGTG 95 1972 —4887948898 27 2295 2314 54425 54444 3321 3340 72042 72061 586 605 26696 26715 928 947 32243 32262 1612 1631 43336 43355 494285 —1954 GTTCTTCCTGTGACAGTGGT 95 28 1973 —4888048899 2296 2315 54426 54445 3322 3341 72043 72062 587 606 26697 26716 929 948 32244 32263 494286 1613 1632 GGTTCTTCCTGTGACAGTGG 95 43337 43356 29 1955 1974 48881 48900 2297 2316 54427 54446 588 607 26698 26717 930 949 32245 32264 494287 1614 1633 AGGTTCTTCCTGTGACAGTG 95 43338 43357 30 1956 1975 48882 48901 2298 2317 54428 54447 589 608 26699 26718 494288 931 950 CAGGTTCTTCCTGTGACAGT 91 32246 32265 31 1615 1634 43339 43358 WO 79625 1957 1976 48883 48902 2299 2318 54429 54448 2983 3002 66500 66519 592 611 26702 26721 934 953 32249 32268 1618 1637 43342 43361 494290 TGGCAGGTTCTTCCTGTGAC 90 32 1960 1979 48886 48905 2302 2321 54432 54451 2986 3005 66503 66522 593 612 26703 26722 935 954 32250 32269 1619 1638 43343 43362 494291 TTGGCAGGTTCTTCCTGTGA 89 33 1961 1980 48887 48906 2303 2322 54433 54452 2987 3006 66504 66523 594 613 26704 26723 936 955 32251 32270 1620 1639 43344 43363 494292 CTTGGCAGGTTCTTCCTGTG 94 35 1962 1981 48888 48907 2304 2323 54434 54453 2988 3007 66505 66524 596 615 26706 26725 938 957 32253 32272 1622 1641 43346 43365 494294 AGCTTGGCAGGTTCTTCCTG 90 36 1964 1983 48890 48909 2306 2325 54436 54455 2990 3009 66507 66526 626 645 26736 26755 968 987 32283 32302 1310 1329 37830 37849 1652 1671 43376 43395 494299 ACTATGCGAGTGTGGTGTCA 91 37 1994 2013 48920 48939 2336 2355 54466 54485 2678 2697 60021 60040 3020 3039 66537 66556 627 646 26737 26756 969 988 32284 32303 1311 1330 37831 37850 1653 1672 43377 43396 49800 GACTATGCGAGTGTGGTGTC 93 38 “”5 mn4 4&fl1 4&MO 2337 2356 54467 54486 2679 2698 60022 60041 3021 3040 66538 66557 49501 628 647 CGACTATGCGAGTGTGGTGT 93 26B8 fifl57 39 970 989 32285 32304 1312 1331 37832 37851 1654 1673 43378 43397 1996 2015 48922 48941 2338 2357 54468 54487 2680 2699 60023 60042 3022 3041 66539 66558 629 648 26739 26758 971 990 32286 32305 1313 1332 37833 37852 1655 1674 43379 43398 494302 CCGACTATGCGAGTGTGGTG 94 40 1997 2016 48923 48942 2339 2358 54469 54488 2681 2700 60024 60043 3023 3042 66540 66559 630 649 26740 26759 972 991 32287 32306 1314 1333 37834 37853 1656 1675 43380 43399 494303 TCCGACTATGCGAGTGTGGT 93 41 1998 2017 48924 48943 2340 2359 54470 54489 2682 2701 60025 60044 3024 3043 66541 66560 631 650 26741 26760 973 992 32288 32307 1315 1334 37835 37854 1657 1676 43381 43400 494304 CTATGCGAGTGTGG 94 42 1999 2018 48925 48944 2341 2360 54471 54490 2683 2702 60026 60045 3025 3044 66542 66561 632 651 26742 26761 974 993 32289 32308 1316 1335 37836 37855 1658 1677 43382 43401 494305 GGTCCGACTATGCGAGTGTG 93 43 2000 2019 48926 48945 2342 2361 54472 54491 2684 2703 60027 60046 3026 3045 66543 66562 633 652 26743 26762 975 994 32290 32309 494306 GGGTCCGACTATGCGAGTGT 92 44 1317 1336 37837 37856 1659 1678 43383 43402 2001 2020 48927 48946 2343 2362 54473 54492 2685 2704 60028 60047 3027 3046 66544 66563 494307M CTGCTCAGTCGGTGCTTGTT 91 n/a n/a 45 2558 2577 1212 494310 —1193 CCTCTGCTCAGTCGGTGCTT 90 n/a n/a 46 2561 2580 1213 494311 —1194 GCCTCTGCTCAGTCGGTGCT 88 37733 47 2562 2581 59905 59924 1286 494334 —1267 CTTCCAGTGACAGTGGTGGA 90 —3778737806 48 2635 2654 59978 59997 1269 1288 37789 37808 494336 TTCTTCCAGTGACAGTGGTG 90 49 2637 2656 59980 59999 1270 1289 37790 37809 494337 —2638 GTTCTTCCAGTGACAGTGGT 95 50 2657 —5998160000 1271 1290 37791 37810 494338 —2639 GGTTCTTCCAGTGACAGTGG 91 133 2658 —5998260001 494521 6393 6412 GACCTTAAAAGCTTATACAC 82 140049 140068 51 494525 6397 6416 GTCAGACCTTAAAAGCTTAT 84 140053 140072 52 494530 6402 6421 TGTCAGTCAGACCTTAAAAG 82 140058 140077 53 494535 6407 6426 GAATTTGTCAGTCAGACCTT 85 140063 140082 54 494536 6408 6427 AGAATTTGTCAGTCAGACCT 83 140064 140083 55 494544 6417 6436 CCTTAATACAGAATTTGTCA 82 140073 140092 56 Table 125 SE 1D SE ISIS SNE8:11D SNE8:11D % N8: 2 ID N?) SEQ Sequence NO 1nh1b1t10n. . Start 2 Stop ID NO Start S1te. Stop S1te.
Site Site 144367 249 268 GGCAGGTCCTTCCTGTGACA 84 21210 21229 11 494371 3900 3919 GCTCCGTTGGTGCTTGTTCA 93 n/a n/a 57 494372 3901 3920 TGCTCCGTTGGTGCTTGTTC 93 n/a n/a 5 8 494373 3902 3921 TTGCTCCGTTGGTGCTTGTT 83 n/a n/a 59 494374 3903 3922 TTTGCTCCGTTGGTGCTTGT 89 n/a n/a 60 494375 3904 3923 CTTTGCTCCGTTGGTGCTTG 85 n/a n/a 61 494386 3977 3996 TCCTGTAACAGTGGTGGAGA 86 81985 82004 62 494387 3978 3997 TAACAGTGGTGGAG 82 81986 82005 63 494388 3979 3998 CTTCCTGTAACAGTGGTGGA 86 81987 82006 64 494389 3980 3999 CCTTCCTGTAACAGTGGTGG 92 81988 82007 65 494390 3981 4000 TCCTTCCTGTAACAGTGGTG 92 81989 82008 66 494391 3982 4001 GTCCTTCCTGTAACAGTGGT 84 81990 82009 67 494392 3983 4002 TGTCCTTCCTGTAACAGTGG 81 81991 82010 68 Table 126 WO 79625 SEQ ID SEQ ID SEQ ID SEQ ID ISIS NO NO: 1 NO: 1 Sequence inhibgfi n SEQ NO: 2 NO: 2 0 ID NO Start Site Stop Site Start Site Stop Site 144367 249 268 GGCAGGTCCTTCCTGTGACA 86 21210 21229 11 498369 3203 3222 TGGAGCCAGAATAACATTCG 91 70667 70686 69 498379 3213 3232 CCTCTAGGCTTGGAGCCAGA 85 70677 70696 70 498408 3323 3342 AGTTCTTCCTGTGACAGTGG 86 72044 72063 71 498433 3367 3386 GTCCGACTATGCTGGTGTGG 87 72088 72107 72 498434 3368 3387 GGTCCGACTATGCTGGTGTG 86 72089 72108 73 498435 3369 33 88 GGGTCCGACTATGCTGGTGT 83 72090 72109 74 Table 127 SEQ ID SEQ ID 0 SEQ ID SEQ ID 113%; NO: 1 NO: 1 Sequence ition NO: 2 NO: 2 151313) Start Site Stop Site Start Site Stop Site 144367 249 268 GGCAGGTCCTTCCTGTGACA 90 21210 21229 11 498229 2871 2890 CCTCTAGGCTTGGAATCGGG 90 65117 65136 75 498238 2883 2902 GTTCAGAAGGAGCCTCTAGG 93 65129 65148 76 498239 2884 2903 TGTTCAGAAGGAGCCTCTAG 94 65130 65149 77 2887 2906 498240 GCTTGTTCAGAAGGAGCCTC 98 n/a n/a 78 4573 4592 2888 2907 498241 TGCTTGTTCAGAAGGAGCCT 94 n/a n/a 79 4574 4593 2889 2908 498242 GTGCTTGTTCAGAAGGAGCC 96 n/a n/a 80 4575 4594 2890 2909 498243 GGTGCTTGTTCAGAAGGAGC 97 n/a n/a 81 4576 4595 2891 2910 498244 TGGTGCTTGTTCAGAAGGAG 92 n/a n/a 82 4577 4596 498251 2898 2917 GCTCAGTTGGTGCTTGTTCA 90 n/a n/a 83 498252 2899 2918 TGCTCAGTTGGTGCTTGTTC 90 n/a n/a 84 Table 128 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ISIS NO NO: 1 NO: 1 Sequence inhibition NO: 2 NO: 2 ID Start Site Stop Site Start Site Stop Site NO 1443 67 249 268 GGCAGGTCCTTCCTGTGACA 91 21210 21229 11 498517 3548 3567 GCTTGGATCTGGGACCACCG 89 76233 76252 85 Table 129 SEQ ID SEQ ID SEQ ID SEQ ID Sequence NO: 2 NO: 2 inhibition Start Site Stop Site Table 130 SEQ ID SEQ ID ISIS % Sequence NO: 2 NO: 2 NO tion Start Site Stop Site -——————— -——————m 499041 6318 6337 CGTTTGATTGCTGTCTATTA 139974 139993 91 Table 131 SEQID SEQID SEQID SEQID 113138 NO: 1 NO: 1 Sequence inhilfition0 NO: 2 NO: 2 112E130 Start Site Stop Site Start Site Stop Site 144367 249 268 GGCAGGTCCTTCCTGTGACA 91 21210 21229 11 498523 3554 3573 CTCTGTGCTTGGATCTGGGA 94 76239 76258 92 498524 3555 3574 CCTCTGTGCTTGGATCTGGG 96 76240 76259 93 498525 3556 3575 GCCTCTGTGCTTGGATCTGG 94 76241 76260 94 498529 3560 3579 AGAAGCCTCTGTGCTTGGAT 89 76245 76264 95 498535 3566 3585 TTCAGAAGAAGCCTCTGTGC 89 76251 76270 96 498550 3582 3601 GCTCCGTTGGTGCTTCTTCA 90 n/a n/a 97 498553 3585 3604 TTTGCTCCGTTGGTGCTTCT 87 n/a n/a 98 3587 3606 498555 GCTTTGCTCCGTTGGTGCTT 90 n/a n/a 99 3905 3924 3588 3607 77509 77528 498556 GGCTTTGCTCCGTTGGTGCT 89 100 3906 3925 81914 81933 3589 3608 77510 77529 498557 GGGCTTTGC CCGT TTGG GCT 89 1 0 1 3907 3926 81915 81934 498579 3662 3681 CCTTCCTGTGACAGTGGTAG 87 77583 77602 102 498580 3663 3682 TCCTTCCTGTGACAGTGGTA 92 77584 77603 103 3665 3684 77586 77605 498581 TCCTGTGACAGTGG 94 104 5009 5028 115519 115538 Table 132 SEQID SEQID SEQID SEQID NO: 1 NO: 1 Sequence NO: 2 NO: 2 inhibition Start Site Stop Site Start Site Stop Site 494230 CCTCTAGGCTTGGAACCGGG 95 105 1503 1522 42020 42039 1845 1864 47564 47583 2187 2206 53110 53129 2529 2548 58662 58681 494 513 836 855 1178 1197 494243 1520 1539 TGCTTGTTCGGAAGGAGCCT 93 n/a n/a 106 1862 1881 2204 2223 2546 2565 495 514 837 856 1179 1198 494244 1521 1540 GTGCTTGTTCGGAAGGAGCC 95 n/a n/a 107 1863 1882 2205 2224 2547 2566 Table 133 SEQ ID SEQ ID SEQ ID SEQ ID ISIS NO NO: 1 NO: 1 Sequence NO: 2 NO: 2 inhibition Start Site Stop Site Start Site Stop S1te ————mq494466 4208 4227 AACTGGGACCACCG 95 8513 8 85157 ———————&494470 4212 4231 CTGTGCTTGGAACTGGGACC 85142 85161 Example 115: Dose-dependent antisense inhibition of apo(a) in transgenic mouse primary hepatocytes Gapmers from the studies described above exhibiting significant in vitro inhibition of apo(a) mRNA were ed and tested at various doses in transgenic mouse primary cytes in a series of parallel studies with similar culture conditions. Cells were plated at a density of 35,000 per well and transfected using electroporation with 0.0625 uM, 0.125 uM, 0.25 “M, 0.500 uM, or 1.000 uM concentrations of antisense ucleotide. After a ent period of approximately 16 hours, RNA was isolated from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR. Apo(a) primer probe set hAPO(a)12kB was used to measured mRNA levels. Apo(a) mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as t inhibition of apo(a), relative to untreated control cells.
The results of each of the studies are depicted in the tables presented below with each table representing a separate experiment. The half maximal inhibitory concentration (leo) of each oligonucleotide is also presented in the tables. Apo(a) mRNA levels were significantly d in a dose-dependent manner in antisense oligonucleotide d cells. The potency of the newly designed oligos was compared with the benchmark oligonucleotide ISIS 144367.
Table 134 0.0625 0.125 0.250 0.500 1.000 1C50 ISIS N0 HM HM HM HM HM (HM) 144367 11 27 46 62 80 0.31 494157 11 47 53 76 87 0.23 494158 19 57 75 84 88 0.13 494159 41 65 77 84 92 0.07 494160 44 69 76 85 91 0.06 494161 40 64 74 85 91 0.08 494162 36 63 76 87 88 0.09 494163 20 59 75 85 92 0.13 494164 3 45 62 74 90 0.21 494165 25 39 57 71 75 0.19 494166 17 30 47 59 76 0.31 494167 30 43 55 72 80 0.18 494168 25 36 44 59 75 0.28 494169 19 39 51 61 81 0.25 Table 135 0.0625 0.125 0.250 0.500 1.000 1C50 ISIS N0 HM HM HM HM HM (HM) 144367 23 40 58 76 88 0.19 494170 38 34 60 76 84 0.13 494230 55 71 89 95 97 0.03 494243 47 73 87 92 97 0.05 494244 58 73 86 92 96 0.03 494283 54 70 84 93 94 0.05 494284 45 62 83 92 95 0.07 494285 56 70 84 92 95 0.04 494286 51 70 87 93 95 0.05 494287 32 60 67 87 91 0.11 494288 26 41 61 79 88 0.17 494290 30 43 64 81 87 0.15 494291 29 40 56 75 85 0.18 WO 79625 PCT/USZOl4/036460 Table 136 0.0625 0.125 0.250 0.500 1.000 1C50 ISIS N0 HM HM HM HM HM (HM) 144367 10 38 62 68 84 0.23 494292 17 36 74 85 90 0.17 494294 10 34 53 80 91 0.22 494299 32 29 56 77 88 0.16 494300 34 46 76 86 90 0.12 494301 44 56 72 86 89 0.09 494302 42 59 78 88 89 0.08 494303 37 58 70 86 89 0.10 494304 46 71 78 89 90 0.05 494305 39 58 62 85 87 0.10 494306 31 52 65 79 88 0.13 494307 23 23 39 65 78 0.34 494310 14 29 62 70 88 0.25 Table 137 0.0625 0.125 0.250 0.500 1.000 1C50 ISIS N0 HM HM HM HM HM (HM) 144367 0 29 45 73 92 0.27 494311 28 53 65 85 95 0.13 494334 20 44 66 86 96 0.16 494336 15 38 54 84 97 0.20 494337 28 50 77 90 98 0.12 494338 21 40 68 91 98 0.15 494371 19 0 71 89 97 0.15 494372 33 44 77 91 97 0.12 494373 15 36 65 83 95 0.19 494374 3 17 51 83 90 0.24 494375 1 34 56 80 93 0.23 494386 13 26 46 73 91 0.25 494387 17 27 45 67 88 0.28 Table 138 0.0625 0.125 0.250 0.500 1.000 1C50 ISIS N0 HM HM HM HM HM (HM) 144367 35 42 62 70 91 0.15 494537 19 34 54 79 90 0.21 494544 10 38 73 86 94 0.17 WO 79625 498229 36 58 80 92 97 0.10 498238 41 57 75 91 97 0.09 498239 56 71 79 90 94 0.03 498240 91 94 98 99 100 <0.06 498241 75 84 91 96 98 <0.06 498242 11 27 42 47 63 0.49 498243 91 93 96 98 99 <0.06 498244 4 0 0 13 43 >1.00 498251 30 30 42 73 89 0.26 498252 37 33 58 80 92 0.20 498369 22 22 10 22 34 >100 Table 139 0.0625 0.125 0.250 0.500 1.000 1C50 ISIS N0 HM HM HM HM HM (HM) 144367 15 32 54 75 90 0.22 498379 29 48 71 80 95 0.13 498408 38 57 77 88 96 0.09 498433 29 36 70 88 96 0.15 498434 49 43 50 78 90 0.19 498435 27 39 57 78 93 0.18 498517 64 72 82 93 98 <0.06 498721 77 84 88 96 97 <0.06 498833 73 78 91 95 99 <0.06 498859 7 24 37 62 75 0.36 498868 7 14 39 63 81 0.36 498875 16 21 33 55 81 0.39 499020 7 24 23 55 78 0.36 499041 6 16 33 64 83 0.35 Table 140 0.0625 0.125 0.250 0.500 1.000 1C50 ISIS N0 HM HM HM HM HM (HM) 144367 14 47 64 79 91 0.14 498523 36 50 80 87 95 0.11 498524 43 79 87 93 97 0.01 498525 32 49 75 86 96 0.12 498529 21 49 57 78 90 0.17 498535 20 34 55 76 86 0.21 498550 12 50 69 84 96 0.11 2014/036460 ——————n Table 141 0.0625 0.125 0.250 0.500 1.000 ICso ISIS N0 HM HM HM HM HM (HM) 144367 0 9 26 49 77 0.47 494388 0 0 21 33 55 0.89 494389 0 15 22 50 79 0.46 494390 5 20 37 68 81 0.33 494391 7 20 32 54 68 0.46 494392 18 24 40 57 76 0.35 494466 33 45 58 69 82 0.16 494470 45 58 68 79 87 0.08 494472 37 50 60 69 83 0.13 494521 0 0 0 15 54 0.17 494525 0 0 2 28 65 0.85 494530 0 6 27 51 80 0.46 494535 0 7 24 53 74 0.49 494536 0 2 15 42 67 0.63 Table 142 0.0625 0.125 0.250 0.500 1.000 ICso ISIS N0 HM HM HM HM HM (HM) 144367 0 4 16 26 77 0.65 498379 12 18 27 32 63 0.81 498408 0 11 46 50 77 0.41 498433 22 30 46 60 83 0.27 498434 39 29 25 47 78 0.40 498435 21 28 26 43 73 0.50 498517 44 48 63 70 84 0.11 498721 54 54 66 75 89 <0.06 498833 44 51 58 67 83 0.11 498859 0 29 14 35 66 0.69 498868 0 12 9 26 60 1.07 498875 0 30 31 53 78 0.40 ——————n ——————-I As presented in the tables above, ISIS 494157 (SEQ ID NO: 12), ISIS 494158 (SEQ ID NO:13), ISIS 494159 (SEQ ID NO:14), ISIS 494160 (SEQ ID NO: 15), ISIS 494161 (SEQ ID NO:16), ISIS 494162 (SEQ ID NO: 17), ISIS 494163 (SEQ ID NO: 18), ISIS 494164 (SEQ ID NO: 19), ISIS 494165 (SEQ ID NO: 20), ISIS 494167 (SEQ ID NO: 22), ISIS 494168 (SEQ ID NO: 23), ISIS 494169 (SEQ ID NO: 24), ISIS 494170 (SEQ ID NO: 25), ISIS 494230 (SEQ ID NO: 105), ISIS 494243 (SEQ ID NO: 106), ISIS 494244 (SEQ ID NO: 107), ISIS 494283 (SEQ ID NO: 26), ISIS 494284 (SEQ ID NO: 27), ISIS 494285 (SEQ ID NO: 28), ISIS 494286 (SEQ ID NO: 29), ISIS 494287 (SEQ ID NO: 30), ISIS 494288 (SEQ ID NO: 31), ISIS 494290 (SEQ ID NO: 32), ISIS 494291 (SEQ ID NO: 33), ISIS 494292 (SEQ ID NO: 35), ISIS 494294 (SEQ ID NO: 36), ISIS 494299 (SEQ ID NO: 37), ISIS 494300 (SEQ ID NO: 38), ISIS 494301 (SEQ ID NO: 39), ISIS 494302 (SEQ ID NO: 40), ISIS 494303 (SEQ ID NO: 41), ISIS 494304 (SEQ ID NO: 42), ISIS 494305 (SEQ ID NO:43), ISIS 494306 (SEQ ID NO: 44), ISIS 494311 (SEQ ID NO: 47), ISIS 494334 (SEQ ID NO: 48), ISIS 494336 (SEQ ID NO: 49), ISIS 494337 (SEQ ID NO: 50), ISIS 494338 (SEQ ID NO: 133), ISIS 494371 (SEQ ID NO: 57), ISIS 494372 (SEQ ID NO: 58), ISIS 494373 (SEQ ID NO: 59), ISIS 494374 (SEQ ID NO: 60), ISIS 494375 (SEQ ID NO: 61), ISIS 494386 (SEQ ID NO: 62), ISIS 494389 (SEQ ID NO: 65), ISIS 494390 (SEQ ID NO: 66), ISIS 494392 (SEQ ID NO: 68), ISIS 494466 (SEQ ID NO: 108), ISIS 494470 (SEQ ID NO: 109), ISIS 494472 (SEQ ID NO: 110), ISIS 494521 (SEQ ID NO: 51), ISIS 494530 (SEQ ID NO: 53), ISIS 498229 (SEQ ID NO: 75), ISIS 498238 (SEQ ID NO: 76), ISIS 498239 (SEQ ID NO: 77), ISIS 498240 (SEQ ID NO: 78), ISIS 498241 (SEQ ID NO: 79), ISIS 498243 (SEQ ID NO: 81), ISIS 498379 (SEQ ID NO: 70), ISIS 498408 (SEQ ID NO: 71), ISIS 498433 (SEQ ID NO: 72), ISIS 498434 (SEQ ID NO: 73), ISIS 498435 (SEQ ID NO: 74), ISIS 498517 (SEQ ID NO: 85), ISIS 498523 (SEQ ID NO: 92), ISIS 498524 (SEQ ID NO: 93), ISIS 498525 (SEQ ID NO: 94), ISIS 498550 (SEQ ID NO: 97), ISIS 498580 (SEQ ID NO: 103), ISIS 498581 (SEQ ID NO: 104), ISIS 498721 (ATGCCTCGATAACTCCGTCC; SEQ ID NO: 134), ISIS 498833 (SEQ ID NO: 86), ISIS 498875 (SEQ ID NO: 89), and ISIS 499020 (SEQ ID NO: 90) were more potent than ISIS 144367 (SEQ ID NO: 11).
Example 116: Dose-dependent nse inhibition of apo(a) in transgenic mouse primary cytes Potent gapmers from the studies described above were further selected and tested at various doses in enic mouse primary hepatocytes in a series of studies with similar culture conditions. Cells were plated at a density of 35,000 per well and transfected using electroporation with 0.049 uM, 0.148 uM, 0.444 uM, 1.333 uM, or 4.000 uM concentrations of antisense oligonucleotide, as specified in tables below. After a ent period of approximately 16 hours, RNA was isolated from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR. Apo(a) primer probe set hAPO(a)12kB was used to measured mRNA . Apo(a) mRNA levels were adjusted according to total RNA content, as measured by EEN®. Results are presented as percent inhibition of apo(a), relative to untreated control cells.
The results of each of the studies are depicted in the tables presented below with each table representing a separate experiment. The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in the . Apo(a) mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The potency of the newly designed oligos was compared with the benchmark oligonucleotide, ISIS . As presented in the tables below, ISIS 494157 (SEQ ID NO: 12), ISIS 494158 (SEQ ID NO:13), ISIS 494159 (SEQ ID NO:14), ISIS 494160 (SEQ ID NO: 15), ISIS 494161 (SEQ ID NO:16), ISIS 494162 (SEQ ID NO: 17), ISIS 494163 (SEQ ID NO: 18), ISIS 494164 (SEQ ID NO: 19), ISIS 494230 (SEQ ID NO: 105), ISIS 494243 (SEQ ID NO: 106), ISIS 494244 (SEQ ID NO: 107), ISIS 494283 (SEQ ID NO: 26), ISIS 494284 (SEQ ID NO: 27), ISIS 494285 (SEQ ID NO: 28), ISIS 494286 (SEQ ID NO: 29), ISIS 494287 (SEQ ID NO: 30), ISIS 494290 (SEQ ID NO: 32), ISIS 494292 (SEQ ID NO: 35), ISIS 494300 (SEQ ID NO: 38), ISIS 494301 (SEQ ID NO: 39), ISIS 494302 (SEQ ID NO: 40), ISIS 494303 (SEQ ID NO: 41), ISIS 494304 (SEQ ID NO: 42), ISIS 494305 (SEQ ID NO: 43), ISIS 494306 (SEQ ID NO: 44), ISIS 494310 (SEQ ID NO: 46), ISIS 494311 (SEQ ID NO: 47), ISIS 494337 (SEQ ID NO: 50), ISIS 494371 (SEQ ID NO: 57), ISIS 494372 (SEQ ID NO: 58), ISIS 494375 (SEQ ID NO: 61), ISIS 494388 (SEQ ID NO: 64), ISIS 494389 (SEQ ID NO: 65), ISIS 494390 (SEQ ID NO: 66), ISIS 494392 (SEQ ID NO: 68), ISIS 494466 (SEQ ID NO: 108), ISIS 494470 (SEQ ID NO: 109), ISIS 494472 (SEQ ID NO: 110), ISIS 498238 (SEQ ID NO: 76), ISIS 498239 (SEQ ID NO: 77), ISIS 498433 (SEQ ID NO: 72), ISIS 498434 (SEQ ID NO: 73), ISIS 498435 (SEQ ID NO: 74), ISIS 498523 (SEQ ID NO: 92), ISIS 498524 (SEQ ID NO: 93), ISIS 498525 (SEQ ID NO: 94), ISIS 498580 (SEQ ID NO: 103), and ISIS 498581 (SEQ ID NO: 104) were more potent than ISIS 144367 (SEQ ID NO: 11).
Table 143 0.049 0.148 0.444 1.333 4.000 1C50 ISIS N0 HM HM HM HM HM (HM) 144367 0 26 67 89 92 0.32 494157 23 50 83 96 96 0.15 494158 26 62 85 96 96 0.11 494159 42 65 87 95 94 0.07 494160 51 70 88 94 94 <0.05 494161 36 67 87 95 96 0.08 494162 40 69 89 94 95 0.07 494163 41 57 87 95 94 0.08 494164 15 43 75 93 96 0.20 494230 39 77 94 99 99 0.05 494243 39 76 92 98 99 0.06 494244 58 79 91 97 99 0.02 WO 79625 Table 144 0.049 0.148 0.444 1.333 4.000 leo ISIS N0 HM HM HM HM HM (HM) 144367 21 40 79 94 93 0.18 494285 53 68 90 97 97 <0.05 494286 46 69 89 96 97 0.05 494287 31 38 79 94 95 0.15 494290 22 53 74 93 94 0.16 494292 37 51 81 93 95 0.11 494294 22 40 72 91 94 0.19 494299 15 43 75 93 95 0.20 494300 25 38 79 95 95 0.17 494301 23 48 82 92 95 0.15 494302 26 59 86 93 94 0.12 494303 10 58 84 92 91 0.16 494304 25 62 83 93 93 0.12 Table 145 0.049 0.148 0.444 1.333 4.000 leo ISIS N0 HM HM HM HM HM (HM) 144367 23 40 70 90 94 0.19 494305 20 48 82 93 95 0.16 494306 26 53 78 91 92 0.14 494310 36 50 79 88 92 0.12 494311 38 50 74 93 95 0.12 494334 20 42 73 90 94 0.19 494336 5 39 74 92 95 0.23 494337 23 51 87 96 96 0.14 494338 12 42 82 93 95 0.19 494371 28 49 82 94 94 0.14 494372 28 54 81 93 88 0.13 494373 21 28 67 86 92 0.25 494375 26 40 77 85 92 0.18 Table 146 WO 79625 0.049 0.148 0.444 1.333 4.000 1C50 1s1s No HM HM HM HM HM (HM) 144367 5 33 65 78 81 0.32 494388 30 32 60 82 86 0.25 494389 30 45 69 84 84 0.17 494390 32 47 67 83 87 0.16 494392 23 38 54 79 82 0.31 494466 48 67 86 91 95 0.04 494470 74 87 92 96 98 <0.05 494472 69 84 92 96 97 <0.05 494544 5 18 49 74 79 0.48 498238 25 51 76 92 96 0.15 498239 25 62 83 93 97 0.12 498379 5 21 53 71 81 0.55 498408 1 38 63 79 80 0.32 498433 23 43 70 77 79 0.21 Table 147 0.049 0.148 0.444 1.333 4.000 1C50 ISIS N0 HM HM HM HM HM (HM) 498434 32 44 64 78 84 0.20 498435 24 42 64 77 79 0.23 498517 28 23 53 81 85 0.45 498523 50 64 81 90 93 <0.05 498524 53 70 84 93 96 <0.05 498525 38 55 80 92 96 0.09 498550 12 18 62 81 83 0.33 498557 13 33 67 79 83 0.33 498579 6 42 69 80 85 0.31 498580 6 46 76 82 83 0.23 498581 5 40 78 81 84 0.25 498721 40 31 58 78 83 0.35 498833 21 20 58 80 90 0.44 e 117: Antisense inhibition of human apo(a) in transgenic mouse primary hepatocytes Additional antisense oligonucleotides were newly designed targeting an apo(a) nucleic acid and were tested for their effects on apo(a) mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. Primary hepatocytes from human apo(a) transgenic mice were used in this study. Hepatocytes at a density of 35,000 cells per well were transfected using electroporation with 1,000 nM nse oligonucleotide. After a treatment period of imately 24 hours, RNA was ed from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR. Human primer probe set hAPO(a)12kB was used to measure mRNA levels. Apo(a) mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN® The results for each experiment are presented in separate tables shown below. ISIS 1443 67 was also included in the studies for comparison.
Results are presented as percent inhibition of apo(a), relative to untreated control cells. A total of 231 antisense oligonucleotides were tested under these e ions. Only those antisense oligonucleotides that were selected for further studies are ted below.
The newly designed chimeric antisense ucleotides were designed as 34 MOE gapmers.
The gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2’- deoxynucleosides and is flanked by wing segments on the 5’ direction and the 3’ direction comprising three sides and four sides respectively. Each nucleoside in the 5’ wing segment and each nucleoside in the 3’ wing segment has a 2’-MOE modification. The ucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
The apo(a) target sequence contains multiple Kringle repeat sequences, therefore, an antisense oligonucleotide may target one or more regions of apo(a) depending r on the oligonucleotide targets a Kringle sequence or not. “Start site” indicates the 5’-most nucleoside to which the gapmer is targeted in the human sequence. “Stop site” indicates the 3 ’-most nucleoside to which the gapmer is targeted human sequence. An apo(a) antisense oligonucleotide may have more than one “Start site” or “Stop site” depending on r or not it targets a Kringle repeat.
Most gapmers listed in the tables are targeted with 100% complementarity to multiple regions of either the human apo(a) mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No.
NM_005577.2) or the human apo(a) genomic sequence, designated herein as SEQ ID NO: 2 NK Accession No. NT_007422. 12 truncated from nucleotides 3230000 to 3380000), or both. ‘n/a’ indicates that the antisense oligonucleotide does not target that particular sequence with 100% complementarity.
Table 148 SEQ ID SEQ ID SEQ ID SEQ ID ISIS % SEQ ID NO: 1 NO: 1 Sequence NO: 2 NO: 2 NO inhibition NO Start Site Stop Site Start Site Stop Site 144367 249 268 GGCAGGTCCTTCCTGTGACA 64 21210 21229 11 241 257 CCTGTGACAGTGGTGGA 21202 21218 583 599 CCTGTGACAGTGGTGGA 26693 26709 925 941 CCTGTGACAGTGGTGGA 32240 32256 510542 79 111 1609 1625 CCTGTGACAGTGGTGGA 43333 43349 1951 1967 CCTGTGACAGTGGTGGA 48877 48893 2293 2309 CCTGTGACAGTGGTGGA 54423 54439 3319 3 335 CCTGTGACAGTGGTGGA 72040 72056 4&8 M89 CCTGTGACAGTGGTGGA 94404 94420 5005 5021 CCTGTGACAGTGGTGGA 115515 115531 242 258 GACAGTGGTGG 21203 21219 84 600 TCCTGTGACAGTGGTGG 26694 26710 926 942 TCCTGTGACAGTGGTGG 32241 32257 1610 1626 TCCTGTGACAGTGGTGG 43334 43350 510543 1952 1968 TCCTGTGACAGTGGTGG 75 48878 48894 112 2294 2310 TCCTGTGACAGTGGTGG 54424 54440 3320 3336 TCCTGTGACAGTGGTGG 72041 72057 4664 4680 TCCTGTGACAGTGGTGG 94405 94421 5006 5022 TCCTGTGACAGTGGTGG 115516 115532 243 259 TGACAGTGGTG 21204 21220 85 601 TTCCTGTGACAGTGGTG 26695 26711 927 943 TTCCTGTGACAGTGGTG 32242 32258 161 1 1627 TTCCTGTGACAGTGGTG 43335 43351 510544 1953 1969 TTCCTGTGACAGTGGTG 73 48879 48895 113 2295 231 1 TTCCTGTGACAGTGGTG 54425 54441 3321 3337 TTCCTGTGACAGTGGTG 72042 72058 4665 4681 TTCCTGTGACAGTGGTG 94406 94422 5007 5023 TTCCTGTGACAGTGGTG 115517 115533 244 260 CTTCCTGTGACAGTGGT 21205 21221 586 an CTTCCTGTGACAGTGGT 26696 26712 928 944 CTTCCTGTGACAGTGGT 32243 32259 1612 1628 CTTCCTGTGACAGTGGT 43336 43352 1954 1970 CTTCCTGTGACAGTGGT 48880 48896 510545 65 114 2296 2312 CTTCCTGTGACAGTGGT 54426 54442 3322 3338 CTTCCTGTGACAGTGGT 72043 72059 3664 3680 CTTCCTGTGACAGTGGT 77585 77601 M86 M82 CTTCCTGTGACAGTGGT 94407 94423 5008 5024 CTTCCTGTGACAGTGGT 115518 115534 245 261 TGTGACAGTGG 21206 21222 3665 3681 CCTTCCTGTGACAGTGG 77586 77602 510546 74 115 M87 M83 CCTTCCTGTGACAGTGG 94408 94424 5009 5025 CCTTCCTGTGACAGTGG 115519 115535 246 262 TCCTTCCTGTGACAGTG 21207 21223 3666 3682 TCCTTCCTGTGACAGTG 77587 77603 510547 77 116 4&8 46$! CTGTGACAGTG 94409 94425 5010 5026 TCCTTCCTGTGACAGTG 115520 115536 247 263 GTCCTTCCTGTGACAGT 21208 21224 3667 3683 GTCCTTCCTGTGACAGT 77588 77604 510548 73 117 M89 M85 GTCCTTCCTGTGACAGT 94410 94426 501 1 5027 GTCCTTCCTGTGACAGT 115521 115537 W0 2014/179625 248 264 GGTCCTTCCTGTGACAG 21209 21225 510549 67 118 4670 4686 GGTCCTTCCTGTGACAG 9441 1 94427 632 648 CCGACTATGCGAGTGTG 26742 26758 974 990 CCGACTATGCGAGTGTG 32289 32305 1316 1332 ATGCGAGTGTG 37836 37852 1658 1674 CCGACTATGCGAGTGTG 43382 43398 510595 76 119 2000 2016 CCGACTATGCGAGTGTG 48926 48942 2342 2358 CCGACTATGCGAGTGTG 54472 54488 2684 2700 CCGACTATGCGAGTGTG 60027 60043 3026 3042 CCGACTATGCGAGTGTG 66543 66559 634 650 GTCCGACTATGCGAGTG 26744 26760 976 992 GTCCGACTATGCGAGTG 32291 32307 1318 1334 GTCCGACTATGCGAGTG 37838 37854 1660 1676 GTCCGACTATGCGAGTG 43384 43400 510597 70 120 2002 2018 GTCCGACTATGCGAGTG 48928 48944 2344 2360 CTATGCGAGTG 54474 54490 2686 2702 GTCCGACTATGCGAGTG 60029 60045 3028 3044 GTCCGACTATGCGAGTG 66545 66561 635 651 GGTCCGACTATGCGAGT 26745 26761 977 993 GGTCCGACTATGCGAGT 32292 32308 1319 1335 GGTCCGACTATGCGAGT 37839 37855 1661 1677 GGTCCGACTATGCGAGT 43385 43401 510598 70 121 2003 2019 GGTCCGACTATGCGAGT 48929 48945 2345 2361 GGTCCGACTATGCGAGT 54475 54491 2687 2703 GGTCCGACTATGCGAGT 60030 60046 3029 3045 ACTATGCGAGT 66546 66562 Table 149 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ISIS % NO: 1 NO: 1 Sequence NO: 2 NO: 2 ID NO tion Start S1te Stop Slte Start Site Stop Slte NO 144367 249 268 GGCAGGTCCTTCCTGTGACA 83 21210 21229 1 1 510783 6400 6416 GTCAGACCTTAAAAGCT 75 140056 140072 122 512944 3561 3577 AAGCCTCTGTGCTTGGA 81 76246 76262 123 512947 3560 3576 AGCCTCTGTGCTTGGAT 85 76245 76261 124 512958 3559 3575 GCCTCTGTGCTTGGATC 82 76244 76260 125 512959 3585 3601 GCTCCGTTGGTGCTTCT 77 n/a n/a 126 Table 150 ISIS NO 8158: I1D SE18: 11) Sequence inhiffition 811318212) 811318: I2D 81:??0113 Start Site Stop Site Start Site Stop Site 144367 249 268 GGCAGGTCCTTCCTGTGACA 76 21210 21229 11 510701 4217 4233 CTCTGTGCTTGGAACTG 78 85147 85163 127 219 235 21180 21196 561 577 26671 26687 903 919 32218 32234 1245 1261 37765 37781 510702 1587 1603 TGCCTCGATAACTCTGT 79 43311 43327 128 1929 1945 48855 48871 2271 2287 54401 54417 2613 2629 59956 59972 4299 4315 86472 86488 563 579 26673 26689 905 921 32220 32236 1247 1263 37767 37783 1589 1605 43313 43329 510704 1931 1947 TGTGCCTCGATAACTCT 80 48857 48873 129 2273 2289 54403 54419 2615 2631 59958 59974 4301 4317 86474 86490 4985 5001 115495 115511 510757 4929 4945 GCTCAGTTGGTGCTGCT 74 Ifla tfla 130 Example 118: Dose-dependent antisense inhibition of apo(a) in transgenic mouse primary hepatocytes Potent s from the s described above were further selected and tested at various doses in transgenic mouse primary hepatocytes in a series of studies with r culture conditions. Cells were plated at a density of 35 ,000 per well and transfected using electroporation with 0.156 1.1M, 0.313 MM, 0.625 uM, 1.250 uM, 2.500 HM, or 5.000 uM concentrations of antisense oligonucleotide, as ed in the tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR. Apo(a) primer probe set hAPO(a)12kB was used to measured mRNA levels. Apo(a) mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN® Results are presented as percent inhibition of apo(a), relative to untreated control cells.
The results of each of the s are depicted in the tables presented below with each study represented in a separate table. The half maximal inhibitory concentration (leo) of each oligonucleotide is also presented in the tables.
Table 151 0.156 0.312 0.625 1.250 2.500 5.000 1C50 1s1s NO HM NM NM MM HM MM (MM) 144367 28 55 70 83 90 92 0.31 510542 33 58 75 87 89 90 0.27 WO 79625 510543 33 45 68 78 89 89 0.34 510544 33 50 65 78 88 90 0.33 510545 33 58 76 87 91 90 0.26 510546 39 62 76 87 89 91 0.22 510547 36 66 82 84 86 91 0.22 510548 50 70 82 91 88 90 0.13 510549 32 59 73 85 86 90 0.27 510595 26 57 78 88 90 90 0.29 510597 30 53 76 85 89 89 0.30 Table 152 0.156 0.312 0.625 1.250 2.500 5.000 1C50 ISIS No HM HM HM HM HM HM (HM) 144367 36 52 78 87 93 94 0.26 510598 48 58 81 88 93 92 0.18 510701 45 59 78 87 95 95 0.18 510702 49 63 75 90 94 95 0.15 510704 55 67 80 93 94 95 <0.16 510757 34 48 68 79 90 93 0.33 510783 21 32 51 58 78 84 0.69 512944 57 72 81 91 96 97 <0.16 512947 64 74 86 92 96 97 <0.16 512958 48 69 83 91 96 97 0.13 512959 39 59 76 84 93 93 0.22 Table 153 0.156 0.312 0.625 1.250 2.500 5.000 1C50 ISIS No HM HM HM HM HM HM (HM) 144367 41 58 75 81 88 87 0.22 510542 38 54 69 74 85 83 0.27 510545 21 43 73 77 80 78 0.39 510546 37 58 73 81 83 81 0.24 510547 38 58 72 79 84 86 0.24 510548 40 63 77 79 81 84 0.21 510549 37 47 67 77 81 83 0.31 510595 34 66 73 81 80 75 0.23 510597 39 59 74 83 76 77 0.23 Table 154 0.156 0.312 0.625 1.250 2.500 5.000 1C50 ISIS No HM HM HM HM HM HM (HM) 144367 33 60 72 83 81 81 0.26 510598 47 62 75 75 76 76 0.18 510701 41 67 80 87 92 91 0.19 510702 51 64 77 80 80 83 0.13 510704 54 61 77 84 89 80 0.12 512944 71 74 81 88 92 94 0.02 512947 65 77 86 90 93 95 0.03 512958 63 73 84 92 93 96 0.06 512959 39 62 80 82 86 82 0.22 Apo(a) mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide-treated cells. The potency of the newly designed oligonucleotides was compared with the benchmark oligonucleotide, ISIS 144367. As ted in the tables above, ISIS 510542 (SEQ ID NO: 111), ISIS 510545 (SEQ ID NO: 114), ISIS 510546 (SEQ ID NO: 115), ISIS 510547 (SEQ ID NO: 116), ISIS 510548 (SEQ ID NO: 117), ISIS 510549 (SEQ ID NO: 118), ISIS 510595 (SEQ ID NO: 119), ISIS 510597 (SEQ ID NO: 120), ISIS 510598 (SEQ ID NO: 121), ISIS 510701 (SEQ ID NO: 127), ISIS 510702 (SEQ ID NO: 128), ISIS 510704 (SEQ ID NO: 129), ISIS 512944 (SEQ ID NO: 123), ISIS 512947 (SEQ ID NO: 124), ISIS 512958 (SEQ ID NO: 125), and ISIS 512959 (SEQ ID NO: 126) were more potent than ISIS 144367 (SEQ ID NO: 11). e 119: Effect of in vivo antisense inhibition of human apo(a) in human apo(a) transgenic mice Transgenic mice with the human apo(a) gene (Frazer, K.A. et al., Nat. Genet. 1995. 9: 424-431) were utilized in the studies described below. ISIS antisense ucleotides that demonstrated statistically significant inhibition of apo(a) mRNA in vitro as described above were ted further in this model.
Study 1 Female human apo(a) transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum normal lab chow. The mice were divided into treatment groups ting of 4 mice each. The groups received intraperitoneal injections of ISIS 494159, ISIS 494160, ISIS 494161, ISIS 494162, ISIS 494163, ISIS 494230, ISIS 494243, ISIS 494244, ISIS , ISIS 494284, ISIS 494285, ISIS 494286, ISIS 494301, ISIS , ISIS 494304, ISIS 494466, ISIS 494470, ISIS 494472, ISIS 498239, ISIS 498408, ISIS 498517, ISIS 494158, ISIS 494311, ISIS 494337, ISIS 494372, ISIS 498238, ISIS 498523, ISIS 498525, ISIS 510548, ISIS 512944, ISIS 512947, or ISIS 512958 at a dose of 25 mg/kg twice a week for 2 weeks. One group of mice received intraperitoneal injections of PBS twice a week for 2 weeks. The PBS group served as the control group. Two days following the final dose, the mice were euthanized, organs harvested and analyses done.
Inhibition n apo(a) mRNA Total RNA was extracted from the livers of some of the treatment groups, and human apo(a) mRNA was quantitated by RT-PCR. The results are presented in the table below, expressed as t inhibition of apo(a) mRNA compared to the PBS control.
Table 155 Percent inhibition of human apo(a) mRNA in transgenic mice ISIS N0 inhilfition 144367 98 494159 100 494160 95 494161 98 494162 100 494163 100 494230 96 494243 99 494244 99 494283 100 494284 100 494285 100 494286 98 494301 99 494302 96 494304 94 494466 97 494470 93 494472 98 498239 72 498408 100 498517 98 The data demonstrates significant inhibition of apo(a) mR\IA by several ISIS oligonucleotides. ISIS 494159 (SEQ ID NO: 14), ISIS 494162 (SEQ ID NO: 17), ISIS 494163 (SEQ ID NO: 18), ISIS 494243 (SEQ ID NO: 106), ISIS 494244 (SEQ ID NO: 107), ISIS 494283 (SEQ ID NO: 26), ISIS 494284 (SEQ ID NO: 27), ISIS 494285 (SEQ ID NO: 28), ISIS 494301 (SEQ ID NO: 39), and ISIS 498408 (SEQ ID NO: 71) were more potent than the benchmark ISIS 144367 (SEQ ID NO: 11).
Inhibition ofhuman apo(a) protein Plasma human apo(a) protein was measured from all ent groups using an Apo(a) ELISA kit (Mercodia 1001, a, Sweden). The results are presented in the table below, expressed as percent inhibition of apo(a) mRNA compared to the PBS control.
Table 156 Percent inhibition of human apo(a) protein in transgenic mice ISIS 96 No inhibition 144367 86 494159 86 494160 0 494161 82 494162 84 494163 82 494230 60 494243 84 494244 87 494283 98 494284 98 494285 89 494286 89 494301 93 494302 88 494304 83 494466 76 494470 73 494472 72 498239 54 498408 84 498517 56 494158 71 494311 83 494337 80 494372 78 498238 58 498523 47 498525 58 510548 74 512944 18 512947 65 512958 72 The data demonstrates significant inhibition of apo(a) mRNA by several ISIS ucleotides.
ISIS 494159 (SEQ ID NO: 14), ISIS 494244 (SEQ ID NO: 82), ISIS 494283 (SEQ ID NO: 26), ISIS 494284 (SEQ ID NO: 27), ISIS 494285 (SEQ ID NO: 28), ISIS 494286 (SEQ ID NO: 29), ISIS 494301 (SEQ ID NO: 39), and ISIS 494302 (SEQ ID NO: 40) were as potent as or more potent than the benchmark ISIS 144367 (SEQ ID NO: 11)..
Study 2 ISIS 494159, ISIS 494161, ISIS 494162, ISIS 494163, and ISIS 494243 were further evaluated in this transgenic model. ISIS 144367 was included for comparison. ent Female human apo(a) transgenic mice were divided into treatment groups consisting of 4 mice each. The groups received intraperitoneal injections of ISIS 144367, ISIS 494159, ISIS 494161, ISIS 494162, ISIS 494163, or ISIS 494243 at doses of 1.5 mg/kg, 5 mg/kg, 15 mg/kg, or 50 mg/kg twice a week for 2 weeks. One group of mice received intraperitoneal injections of PBS twice a week for 2 weeks. The PBS group served as the control group. Two days following the final dose, the mice were euthanized, organs harvested and analyses done.
Inhibition n apo(a) mRNA Total RNA was extracted from the livers of the treatment groups, and human apo(a) mRNA was quantitated by RT-PCR. The results are presented in the table below, expressed as t inhibition of apo(a) mRNA compared to the PBS l.
Table 157 Dose-dependent inhibition of human apo(a) mRNA in transgenic mice Dose % ISIS N0 ED” (mg/kg/wk) inhibition 100 71 42 144367— 31 0 3 5 100 91 67 494159— 5 48 3 39 494161 100 82 6 49 61 100 9O 67 494162 5 8 100 83 66 494163 58 100 8O 26 494243 32 The data demonstrates significant inhibition of apo(a) mRNA by several ISIS oligonucleotides. ISIS 494159 (SEQ ID NO: 14), ISIS 494161 (SEQ ID NO: 16), 494162 (SEQ ID NO:17), and ISIS 94163 (SEQ ID NO: 18) were more ious than the benchmark ISIS 144367 (SEQ ID NO: 11).Reduction ofhuman apo(a) protein levels Blood was collected from the ent groups, and human apo(a) protein levels were quantitated by an Apo(a) ELISA kit (Mercodia 1001, Uppsala, Sweden). The results are presented in the table below, expressed as percent reduction of apo(a) protein levels compared to the PBS control.
Table 158 Dose-dependent inhibition of human apo(a) protein in enic mice Dose % ISIS No ED50 (mg/kg/wk) inhibition 100 73 O 144367 71 6 3 69 100 88 88 494159 85 3 36 100 90 85 494161— 2 73 3 44 100 89 78 494162— 3 76 3 24 100 90 494163M 3 6O 3 37 100 61 494243 —30 174 0 3 0 The data demonstrates significant reduction of apo(a) plasma protein levels by several ISIS oligonucleotides. ISIS 494159 (SEQ ID NO: 14), ISIS 494161 (SEQ ID NO: 16), ISIS 494162 (SEQ ID NO: 17), and ISIS 494163 (SEQ ID NO: 18) were more efficacious than the benchmark ISIS 144367 (SEQ ID NO: 11).
Study 3 ISIS 494244, ISIS 494283, and ISIS 494284 were further evaluated in this model. ISIS 144367 was included for comparison.
Treatment Female human apo(a) transgenic mice were divided into treatment groups consisting of 4 mice each. The groups received intraperitoneal injections of ISIS 144367, ISIS 494244, ISIS 494283, or ISIS 494284 at doses of 0.75 mg/kg, 2.5 mg/kg, 7.5 mg/kg, or 25 mg/kg twice a week for 2 weeks. One group of mice received eritoneal injections of PBS twice a week for 2 weeks. The PBS group served as the control group. Two days following the final dose, the mice were euthanized, organs harvested and analyses done.
Inhibition ofhuman apo(a) mRNA Total RNA was extracted from the livers of the treatment , and human apo(a) mRNA was tated by RT-PCR. The results are presented in the table below, sed as percent tion of apo(a) mRNA compared to the PBS control.
Table 159 Dose-dependent inhibition of human apo(a) mRNA in transgenic mice Dose % ISIS N0 ED” (mg/kg/wk) inhibition 50 75 60 144367 22 0 1.5 0 50 73 41 494244W 18 1.5 0 50 74 52 494283 16 24 1.5 0 50 73 494284¥ 16 17 1.5 2 The data demonstrates significant tion of apo(a) le\A by several ISIS oligonucleotides. ISIS 494244 (SEQ ID NO: 107), ISIS 494283 (SEQ ID NO: 26), and ISIS 494284 (SEQ ID NO: 27) were more efficacious than the benchmark, ISIS 144367 (SEQ ID NO: 11).
Reduction ofhuman apo(a) protein levels Blood was ted from the treatment groups, and human apo(a) protein levels were quantitated by an Apo(a) ELISA kit (Mercodia 1001, Uppsala, Sweden). The results are presented in the table below, expressed as percent reduction of apo(a) protein levels compared to the PBS l.
Table 160 Dose-dependent inhibition of human apo(a) plasma protein in transgenic mice Dose % ISIS N0 ED” /wk) inhibition 50 64 14 144367 16 0 1.5 0 50 67 60 494244 2 —558 1.5 0 494283 50 64 4 =—“64 494284_= 4 The data demonstrates significant reduction of apo(a) plasma protein levels by several ISIS oligonucleotides. ISIS 494244 (SEQ ID NO: 107), ISIS 494283 (SEQ ID NO: 26), and ISIS 494284 (SEQ ID NO: 27) were more efficacious than the benchmark, ISIS 144367 (SEQ ID NO: 11).
Study 4 ISIS 494285, ISIS 494286, ISIS 494301, ISIS 494302, and ISIS 494311 were further evaluated in this model.
Treatment Male human apo(a) transgenic mice were divided into treatment groups consisting of 4 mice each.
Each such group received intraperitoneal injections of ISIS 494285, ISIS 494286, ISIS 494301, ISIS 494302, or ISIS 494311 at doses of 5 mg/kg, 15 mg/kg, or 50 mg/kg once a week for 2 weeks. One group of 3 mice ed intraperitoneal injections of PBS once a week for 2 weeks. The PBS group served as the control group. Two days following the final dose, the mice were euthanized, organs harvested and analyses done. tion ofhuman apo(a) mRNA Total RNA was extracted from the livers of the treatment groups, and human apo(a) mRNA was tated by RT-PCR. The results are presented in the table below, expressed as percent inhibition of apo(a) mRNA compared to the PBS control. The data demonstrates significant inhibition of apo(a) mRNA by ISIS 494285 (SEQ ID NO: 28), ISIS 494286 (SEQ ID NO: 29), ISIS 494301 (SEQ ID NO: 39), ISIS 494302 (SEQ ID NO: 40) and ISIS 494311 (SEQ ID NO: 47).
Table 161 Dose-dependent tion of human Apo(a) mRNA in enic mice Dose % (mtg/Wk) 494285 1 4,4286 1 80 50 98 494301 15 96 3 59 50 98 494302 15 88 2 72 50 99 494311 15 96 1 87 Reduction ofhuman apo(a) protein levels Blood was collected from the ent groups, and human apo(a) protein levels were tated by an Apo(a) ELISA kit (Mercodia 1001, Uppsala, Sweden). The results are presented in the table below, expressed as percent reduction of apo(a) protein levels compared to the PBS control. The data demonstrates significant reduction of apo(a) plasma protein levels by ISIS 494285, ISIS 494286, ISIS 494301, ISIS 494302 and ISIS 494311.
Table 162 Dose-dependent inhibition of human apo(a) protein in transgenic mice Dose % ISIS N0 ED” (mg/kg/wk) inhibition 50 88 494285 15 88 2 72 50 90 494286 15 85 2 75 50 89 494301 15 86 5 38 50 90 494302 15 82 3 61 50 90 494311 15 82 3 69 Study 5 ISIS 494372, ISIS 498524, ISIS , ISIS 498721, and ISIS 498833 were r evaluated in this model.
Treatment Female human apo(a) transgenic mice were divided into ent groups consisting of 4 mice each. The groups received intraperitoneal injections of ISIS 494372, ISIS 498524, ISIS 498581, ISIS 498721, or ISIS 498833 at doses of 5 mg/kg, 15 mg/kg, or 50 mg/kg once a week for 2 weeks. One group of 3 mice ed intraperitoneal injections of PBS once a week for 2 weeks. The PBS group served as the control group. Two days following the final dose, the mice were euthanized, organs harvested and es done.
Inhibition ofhuman apo(a) mRNA Total RNA was extracted from the livers of the treatment groups, and human apo(a) mRNA was quantitated by RT-PCR. The s are presented in the table below, expressed as percent inhibition of apo(a) mRNA compared to the PBS control. The data demonstrates significant inhibition of apo(a) mRNA by ISIS 494372 (SEQ ID NO: 28), ISIS 498524 (SEQ ID NO: 93), ISIS 498581 (SEQ ID NO: 104), and ISIS 498721 (ATGCCTCGATAACTCCGTCC; SEQ ID NO: 134).
Table 163 Dose-dependent inhibition of human Apo(a) mRNA in transgenic mice ISIS No (111535731) inhilfition ED” 50 88 494372W 18 0 50 83 498524 15 74 8 34 50 98 498581 15 58 7 48 50 97 498721 15 68 14 0 50 61 498833 15 0 155 17 Reduction ofhuman apo(a) protein levels Blood was ted from the treatment groups, and human apo(a) protein levels were quantitated by an Apo(a) ELISA kit (Mercodia 1001, Uppsala, ). The results are presented in the table below, expressed as percent reduction of apo(a) protein levels compared to the PBS control. The data demonstrates significant reduction of apo(a) plasma protein levels by ISIS 494372 (SEQ ID NO: 28), ISIS 4985 81 (SEQ ID NO: 104), and ISIS 498721 (ATGCCTCGATAACTCCGTCC; SEQ ID NO: 134).
Table 164 Dose-dependent inhibition of human apo(a) protein in transgenic mice Dose % ISIS N0 ED” (mg/kg/wk) inhibition 50 68 494372 15 25 32 12 50 38 498524 15 0 118 0 50 79 498581 15 52 9 49 50 81 498721W 10 29 50 15 498833 15 0 738 67 Example 120: Tolerability of antisense oligonucleotides targeting human apo(a) in rodent models Gapmer antisense oligonucleotides targeting human apo(a) were selected from the studies bed above for tolerability studies in CD1 mice and in Sprague Dawley rats. Rodents do not express endogenous apo(a), hence these studies tested the tolerability of each human antisense oligonucleotide in an animal rather than any phenotypic changes that may be caused by inhibiting apo(a) in the animal.
Tolerability in CD1 mice: Study 1 CD1® mice (Charles River, MA) are a urpose mice model, frequently ed for safety and efficacy g. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and ted for changes in the levels of various plasma chemistry markers.
Treatment Groups of male CD1 mice were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 494159, ISIS 494161, ISIS 494162, ISIS 494244, ISIS 494283, ISIS 494284, ISIS 494285, ISIS 494286, ISIS 494301, ISIS 494302, ISIS 494311, ISIS 494337, ISIS 494372, and ISIS . One group of six- week old male CD1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
Plasma chemistry markers To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bin, albumin, creatinine, and BUN were measured using an automated clinical try analyzer (Hitachi Olympus AU400e, Melville, NY). The results are presented in the table below. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the ed range for antisense oligonucleotides were excluded in r s.
Table 165 Plasma chemistry markers of CD1 mice ALT AST Albumin BUN Creatinine Bilirubin (IU/L (IU/L) (g/dL) (mg/dL) ) (mg/dL) PBS 38 71 2.9 25.2 0.16 0.15 ISIS 494159 615 525 2.7 23.9 0.11 0.20 ISIS 494161 961 670 2.6 23.7 0.15 0.14 ISIS 494285 582 436 2.3 25.4 0.16 0.11 ISIS 494286 191 227 2.5 21.1 0.12 0.15 ISIS 494301 119 130 2.7 26.4 0.15 0.12 ISIS 494302 74 96 2.8 24.8 0.14 0.15 ISIS 494311 817 799 2.7 28.7 0.12 0.17 ISIS 494337 722 397 2.5 20.0 0.13 0.11 ISIS 494372 73 164 2.6 28.5 0.16 0.11 ISIS 510548 2819 2245 3.1 26.0 0.15 0.15 Organ weights Liver, spleen and kidney weights were measured at the end of the study, and are presented in the table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.
Table 166 Organ weights of CD1 mice (g) Kidney Liver Spleen PBS 0.68 2.0 0.13 ISIS 494159 0.68 3.0 0.21 ISIS 494161 0.62 3.5 0.20 ISIS 494162 0.60 3.3 0.20 ISIS 494283 0.65 2.8 0.24 ISIS 494284 0.69 2.7 0.29 ISIS 494285 0.59 3.2 0.21 ISIS 494286 0.64 2.8 0.25 ISIS 494301 0.72 3.0 0.43 ISIS 494302 0.63 2.3 0.23 ISIS 494311 0.61 3.2 0.19 ISIS 494337 0.56 2.3 0.17 ISIS 494372 0.60 2.5 0.27 ISIS 510548 0.55 3.7 0.20 bility in Sprague Dawley rats Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. The rats were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma try s.
Treatment Groups of male Sprague Dawley rats were injected aneously twice a week for 8 weeks with 30 mg/kg of ISIS 494159, ISIS 494161, ISIS 494162, ISIS 494244, ISIS 494283, ISIS 494284, ISIS 494285, ISIS 494286, ISIS 494301, ISIS 494302, ISIS 494311, ISIS 494337, ISIS 494372, and ISIS 510548. One group of six male e Dawley rats was injected subcutaneously twice a week for 8 weeks with PBS. Rats were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
Plasma chemistry markers To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The s are presented in the table below. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.
Table 167 Plasma chemistry markers of Sprague Dawley rats ALT AST Bilirubin n BUN Creatinine (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) (mg/dL) PBS 30 82 0.09 3.2 19 0.28 ISIS 494159 182 208 0.14 3.4 22 0.35 ISIS 494161 36 86 0.13 3.4 23 0.35 ISIS 494162 102 158 0.17 2.6 28 0.32 ISIS 494283 53 156 0.13 2.9 24 0.32 ISIS 494284 34 113 0.08 2.0 28 0.32 ISIS 494285 110 294 0.10 1.4 110 0.52 ISIS 494286 40 83 0.07 1.6 48 0.44 ISIS 494301 38 132 0.08 3.0 18 0.33 ISIS 494302 47 105 0.09 3.2 19 0.34 ISIS 494311 93 185 0.51 2.7 23 0.30 ISIS 494372 54 119 0.12 3.0 19 0.33 ISIS 510548 116 181 0.11 1.7 65 0.66 Kidneyfunction To evaluate the effect of ISIS oligonucleotides on kidney function, urine levels of total protein and creatinine were measured using an automated clinical chemistry analyzer (Hitachi s AU400e, le, NY). Results are presented in the table below, sed in mg/dL.
Table 168 Kidney function markers (mg/dL) in Sprague-Dawley rats Total Creatinine protein 1818 494162 58 925 1818 494284 97 2519 1818 494285 38 2170 1818 494286 51 625 1818 494301 62 280 1818 494302 101 428 1818 494311 48 1160 1818 494372 46 154 1818 510548 55 2119 Organ weights Liver, spleen and kidney weights were measured at the end of the study, and are presented in the table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for nse oligonucleotides were excluded from further studies.
Table 169 Organ weights of Sprague Dawley rats (g) Kidney liver Spleen PBS 3.5 13.1 0.9 ISIS 494159 3.1 11.7 1.6 ISIS 494161 2.8 12.5 2 ISIS 494162 3.1 14.2 1.6 ISIS 494283 3.3 12.9 2.3 ISIS 494284 4.1 15.8 2.7 ISIS 494285 3.8 13.4 0.8 ISIS 494286 4.2 16.7 2.5 ISIS 494301 3.2 12.1 2.3 ISIS 494302 3.4 13.3 2.4 ISIS 494311 3.5 17.4 3.2 ISIS 494372 3.6 12.9 3.2 ISIS 510548 6.4 21.2 1.5 The finding from the rodent tolerability s showed that in general, taking into consideration all the tolerability markers screened, ISIS 4943 72 was the best tolerated antisense compound in both the CD1 mouse model and the Sprague Dawley rat model.
Example 121: Pharmacokinetics of antisense oligonucleotide in CD1 mice CD1 mice were treated with ISIS oligonucleotides and the oligonucleotide concentrations in the liver and kidney were evaluated.
Treatment Groups of four CD1 mice each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 494283, ISIS 494284, ISIS 494286, ISIS , ISIS 494302, or ISIS 494372. The mice were sacrificed 2 days following the final dose. Livers were harvested for analysis.
Measurement ofoligonucleotide concentration The concentration of the total oligonucleotide tration was measured. The method used is a modification of usly published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform d-liquid) extraction followed by a solid phase extraction. An al standard (ISIS 355868, a 27-mer 2’-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 131) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of tation (LLOQ) of approximately 1.14 ug/g. Half-lives were then calculated using WinNonlin software (PHARSIGHT).
The s are presented in the table below, expressed as ug/g liver or kidney tissue. The data indicates that ISIS 494372 was at an acceptable concentration in the liver and kidneys.
Table 170 Oligonucleotide concentration (ug/g tissue) of ISIS oligonucleotides in CD1 mice ISIS No Liver Kidney 494288 494284 494288 494894 494892 494872 Example 122: Pharmacokinetics of antisense Oligonucleotide in Sprague Dawley rats Male Sprague Dawley rats were treated with ISIS oligonucleotides and the oligonucleotide concentrations in the liver and kidney were evaluated.
Treatment Groups of four rats each were injected aneously twice per week for 3 weeks with 10 mg/kg of ISIS 494283, ISIS 494284, ISIS 494286, ISIS 494301, ISIS 494302, or ISIS . The rats were sacrificed 2 days ing the final dose. Livers were harvested for analysis.
Measurement ofOligonucleotide concentration The concentration of the total oligonucleotide concentration was measured. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) tion followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2’-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 131) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 ug/g. Half-lives were then calculated using WinNonlin software (PHARSIGHT).
The results are presented in the table below, expressed as ug/g liver or kidney tissue. The data indicates that ISIS 494372 was at an able concentration in the liver and s.
Table 171 Oligonucleotide concentration (ug/g tissue) of ISIS oligonucleotides in Sprague Dawley rats IsIsNo 494301 279 540 494302 205 387 494372 288 663 e 123: Effect of ISIS antisense ucleotides targeting human ap0(a) in cynomolgus monkeys Cynomolgus monkeys were treated with ISIS antisense oligonucleotides selected from studies described above. At the time this study was undertaken, the cynomolgus monkey genomic sequence was not available in the National Center for Biotechnology Information (NCBI) se; therefore, cross-reactivity with the cynomolgus monkey gene ce could not be confirmed. Instead, the sequences of the ISIS antisense oligonucleotides used in the cynomolgus s was compared to a rhesus monkey sequence for homology. It is expected that ISIS ucleotides with homology to the rhesus monkey sequence are fully cross-reactive with the cynomolgus monkey sequence as well.
The human antisense oligonucleotides tested are also cross-reactive with the rhesus mRNA sequence (XM_001098061.1; designated herein as SEQ ID NO: 132). The greater the complementarity between the human oligonucleotide and the rhesus monkey sequence, the more likely the human oligonucleotide can cross-react with the rhesus monkey sequence. The start and stop sites of each oligonucleotide to SEQ ID NO: 132 is presented in the table below. Each antisense ucleotide targets more than one region in SEQ ID NO:132 and has multiple start sites. “Start site” indicates the t nucleotide to which the gapmer is targeted in the rhesus monkey sequence. ‘Mismatches’ indicates the number of nucleotides mismatched between the human oligonucleotide sequence and the rhesus sequence.
Antisense oligonucleotide bility, as well as their pharmacokinetic profile in the liver and kidney, was evaluated.
Table 172 Antisense oligonucleotides complementary to SEQ ID NO: 132 ISIS No Start Site Mismatches 278 2 620 2 923 2 1265 2 1607 1 494283 1949 1 2267 1 2609 1 2951 1 3293 1 279 1 621 1 924 1 1266 1 1608 1 494284 1950 1 2268 1 2610 1 2952 1 3294 1 1268 1610 494286 1952 2270 2612 2954 3296 494301 967 1309 1651 494302 1310 1652 1186 494372 1870 2188 t—‘t—‘NNt—‘NNNt—‘NNNNNNNNNt—‘t—‘t—‘H Treatment Prior to the study, the monkeys were kept in quarantine for at least a 30-day period, during which the s were ed daily for general health. The monkeys were 2-4 years old and weighed between 2 and 4 kg. Seven groups of four randomly assigned male cynomolgus monkeys each were injected subcutaneously with ISIS oligonucleotide or PBS using a stainless steel dosing needle and syringe of appropriate size into the one of four sites on the back of the monkeys. The ions were given in clock-wise rotation; one site per dosing. The monkeys were dosed four times a week for the first week (days 1, 3, 5, and 7) as loading doses, and subsequently once a week for weeks 2-12, with 40 mg/kg of ISIS 494283, ISIS 494284, ISIS 494286, ISIS 494301, ISIS 494302, or ISIS 494372. A control group of 8 cynomolgus monkeys was injected with PBS subcutaneously thrice four times a week for the first week (days 1, 3, 5, and 7), and uently once a week for weeks 2-12.
During the study period, the monkeys were observed at least once daily for signs of s or distress. Any animal experiencing more than momentary or slight pain or distress due to the treatment, injury or s was treated by the veterinary staff with approved analgesics or agents to relieve the pain after consultation with the Study Director. Any animal in poor health or in a possible moribund condition was identified for further monitoring and possible euthanasia. For ce, one animal in the treatment group of ISIS 494302 was found moribund on day 56 and was ized. Scheduled euthanasia of the animals was conducted on days 86 and 87 by uination under deep anesthesia. The protocols described in the Example were approved by the utional Animal Care and Use Committee (IACUC).
Target Reduction RNA analysis On day 86, RNA was extracted from liver tissue for real-time PCR analysis of apo(a) using human primer probe set ABI Hs009l669l_ml (Applied Biosystems, Carlsbad CA). Results are presented as percent inhibition of apo(a) mRNA, relative to PBS control. As shown in the table below, treatment with ISIS antisense oligonucleotides ed in significant reduction of apo(a) mRNA in comparison to the PBS control.
The mRNA levels of plasminogen, another kringle-containing protein, were also measured.
Treatment with ISIS 4943 72 did not alter the mRNA levels of plasminogen.
Table 173 Percent Inhibition of apo(a) mRNA in the cynomolgus monkey liver relative to the PBS control ISIS No 494283 494284 “ 494286 E- 494301 494302 “ 494372 93 Protein analysis On different days, one mL of blood was collected from the cephalic, saphenous, or femoral vein of all study monkeys. The blood samples were put into tubes containing A for plasma separation. The tubes were centrifuged at 3,000 rpm for 10 min at room temperature to obtain plasma. Apo(a) n levels were analyzed by an Apo(a) ELISA kit (Mercodia 1001, a, Sweden). s are ted as percentage change of levels from the ne. As shown in the table below, treatment with several ISIS antisense oligonucleotides resulted in significant reduction of apo(a) protein levels in comparison to the PBS control. Specif1cally, treatment with ISIS 494372 reduced cynomolgous plasma protein levels of apo(a).
The protein levels of apoB were also measured in the study groups. Antisense inhibition of apo(a) had no effect on apoB levels.
Table 174 Apo(a) plasma protein levels (% inhibition over baseline values) in the cynomolgus monkey Day 16 Day 30 Day 44 Day 56 Day 72 Day 86 PBS 0 0 10 0 0 0 ISIS 494283 78 79 81 66 66 70 ISIS 494284 92 95 95 93 93 94 ISIS 494286 92 95 96 94 94 94 ISIS 494301 41 45 52 20 17 29 ISIS 494302 17 0 2 0 0 20 ISIS 494372 67 80 83 79 78 81 Tolerability studies Body and organ weight ements To evaluate the effect of ISIS ucleotides on the overall health of the animals, body and organ weights were measured at day 86. Body weights were measured and are presented in the table below. Organ weights were measured and the data is presented in the table below. The results indicate that treatment with ISIS 494372 was well tolerated in terms of the body and organ s of the monkeys.
Table 175 Body weights (g) in the cynomolgus monkey ————-- ISIS 494372 2719 2877 2985 2997 3037 3036 Table 176 Organ weights (% body weight) in the cynomolgus monkey Spleen Kidneys Liver Heart Lungs PBS 0.14 0.38 2.2 0.33 0.51 ISIS 494283 0.24 0.95 2.8 0.33 0.49 ISIS 494284 0.19 0.60 2.6 0.36 0.55 ISIS 494286 0.22 0.63 2.7 0.38 0.55 ISIS 494301 0.38 0.81 3.0 0.36 0.61 ISIS 494302 0.17 0.95 2.5 0.39 0.57 ISIS 494372 0.18 1.16 2.6 0.36 0.56 Liverfunction 2014/036460 To evaluate the effect of ISIS oligonucleotides on c on, monkeys were fasted overnight prior to blood collection. Approximately 1.5 mL of blood was collected from each animal and put into tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 min and then centrifuged at 3,000 rpm for 10 min at room temperature to obtain serum. Levels of various liver function markers were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan).
Plasma levels of ALT and AST were measured and the results are ted in the table below, expressed in IU/L. Bilirubin, a liver on marker, was similarly measured and is presented in the table below, expressed in mg/dL. The results indicate that treatment with ISIS 4943 72 was well tolerated in terms of the liver function in monkeys.
Table 177 Liver function markers in cynomolgus monkey plasma ALT AST Bilirubin (IU/L) (IU/L) (mg/dL) PBS 33 43 0.20 ISIS 494283 75 73 0.12 ISIS 494284 115 79 0.17 ISIS 494286 67 73 0.13 ISIS 494301 129 90 0.15 ISIS 494302 141 75 0.15 ISIS 494372 46 75 0.17 C—reactive protein level analysis To evaluate any inflammatory effect of ISIS oligonucleotides in cynomolgus monkeys, blood samples were taken for analysis. The monkeys were fasted overnight prior to blood collection.
Approximately 1.5 mL of blood was collected from each animal and put into tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a m of 90 min and then centrifuged at 3,000 rpm for 10 min at room temperature to obtain serum. C-reactive protein (CRP), which is synthesized in the liver and which serves as a marker of inflammation, was measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). The results te that treatment with ISIS 4943 72 did not cause any inflammation in monkeys.
Table 178 tive protein levels (mg/L) in cynomolgus monkey plasma 2014/036460 PBS 1.4 ISIS 494283 14.7 rsrs 494302 1s1s494372 Complement C3 analysis To evaluate any effect of ISIS oligonucleotides on the complement pathway in cynomolgus monkeys, blood samples were taken for analysis on day 84 (pre-dose) and day 85 (24 hours post-dose). Approximately 0.5 mL of blood was collected from each animal and put into tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 min and then centrifuged at 3,000 rpm for 10 min at room temperature to obtain serum. C3 was measured using a Toshiba 200FR NEO try analyzer (Toshiba Co., Japan). The results indicate that ent with ISIS 4943 72 did not cause any effect on the complement pathway in monkeys.
Table 179 Complement C3 levels (mg/dL) in lgus monkey plasma Hematology To evaluate any effect of ISIS ucleotides in cynomolgus monkeys on hematologic parameters, blood samples of approximately 0.5 mL of blood was collected on day 87 from each of the available study animals in tubes ning Kg-EDTA. Samples were analyzed for red blood cell (RBC) count, white blood cells (WBC) count, as well as for platelet count, using an ADVIA120 hematology analyzer (Bayer, USA).
The data is presented in the table below.
The data indicate that treatment with ISIS 494372 was well tolerated in terms of the hematologic parameters of the monkeys.
Table 180 Blood cell counts in cynomolgus monkeys WBC RBC et (x 103/uL) (x 10mm) (x 103/uL) PBS 15 6.3 329 ISIS 494283 16 5.3 456 ISIS 494284 13 6.3 330 ISIS 494286 14 5.5 304 ISIS 494301 15 6.0 392 ISIS 494302 12 6.3 305 ISIS 494372 11 6.1 447 e 124: Characterization of the pharmacological activity of ISIS 494372 in cynomolgus monkeys The pharmacological activity of ISIS 4943 72 was characterized by measuring liver apo(a) mRNA and plasma apo(a) levels in monkeys administered the compound over 13 weeks and allowed to recover for another 13 weeks.
Treatment Five groups of 14 randomly assigned male and female cynomolgus monkeys each were injected subcutaneously with ISIS oligonucleotide or PBS using a ess steel dosing needle and syringe of appropriate size into the one of four sites on the back (scapular region) of the monkeys. The monkeys were dosed four times a week for the first week (days 1, 3, 5, and 7) as loading doses, and subsequently once a week for weeks 2-13 as maintenance doses, as shown in the table below. The loading dose during the first week is expressed as mg/kg/dose, while the maintenance doses on weeks 2-13 are sed as mg/kg/week.
Table 181 Dosing groups in cynomolgus monkeys Number of animals for necropsy Group TeSt M1016 Dose m Terminal Recovery 1 PBS - 4 6 4 2 4 3 ISIS 8 4 494372 12 4 6 4 4O 4 6 4 Liver samples from animals were taken at the interim, terminal and recovery phases of the study for the es of apo(a) mRNA. In addition, plasma s were collected on different days to measure apo(a) protein levels. This non-clinical study was conducted in accordance with the United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations, 21 CFR Part 58.
RNA analysis Liver samples were collected from monkeys on days 30, 93, and 182, and frozen. Briefly, a piece (0.2 g) of frozen liver was homogenized in 2 mL of RLT solution (Qiagen). The resulting lysate was applied to Qiagen RNeasy mini columns. After purification and quantification, the tissues were subjected to RT-PCR analysis. The Perkin-Elmer ABI Prism 7700 Sequence Detection System, which uses real-time fluorescent RT-PCR detection, was used to quantify apo(a) mRNA. The assay is based on a target-specific probe labeled with fluorescent reporter and quencher dyes at opposite ends. The probe was yzed through the 5’- exonuclease activity of Taq DNA polymerase, leading to an increasing fluorescence emission of the er dye that can be detected during the reaction. A probe set (ABI Rhesus LPA probe set ID Rh02789275_m1, Applied Biosystems, Carlsbad CA) targeting position 1512 of the rhesus monkey apo(a) mRNA ript GENBANK Accession No XM_001098061.2 (SEQ ID NO: 132) sequence was used to measure cynomolgus monkey liver apo(a) mRNA expression levels. Apo(a) expression was normalized using RIBOGREEN®.
Results are presented as percent inhibition of apo(a) mRNA, relative to PBS control.
As shown in the table below, treatment with ISIS 494372 ed in a dose-dependent reduction of apo(a) mRNA in comparison to the PBS l. At day 30, hepatic apo(a) mRNA expression was reduced in a ependent manner by 74% and 99% in the 12 week and 40 mg/kg/week dosing cohorts, respectively. These reductions are tically significant by one-way ANOVA (Dunnett’s multiple comparison test, P<0.05).
Apo(a) mRNA levels were also measured during the recovery phase. Liver expression levels at day 88 after the last dose were still reduced 49% and 69% in the 12 mg/kg/week and 40 mg/kg/week dosing cohorts, respectively.
Table 182 Percent inhibition levels of liver apo(a) mRNA in the dosing phase in cynomolgus monkeys treated with ISIS 494372 Dose Protein analysis Approximately 20 ul of plasma was analyzed using a commercially ble apo(a) ELISA kit (Mercodia 1001, Uppsala, Sweden). The assay protocol was med as described by the manufacturer. The results are presented in the tables below as percentage change from Day 1 pre—dose apo(a) plasma protein concentrations. Statistically significant differences from Day 1 ne plasma apo(a) using the Dunnett’s multicomparison test are marked with an sk.
Maximal reduction in plasma apo(a) protein was observed in all dosing cohorts by Day 93. In the recovery phase, apo(a) plasma protein levels in the 40 mg/kg/week dosing cohort were at 22% and 93% of the baseline after 4 and 13 weeks (Days 121 and 182) of recovery, respectively. The rate of ry in the 12 mg/kg/week cohort was similar to that seen in the 40 mg/kg/week cohort.
Table 183 Apo(a) plasma protein levels as a percent of Day 1 levels in the dosing phase in cynomolgus monkeys treated with ISIS 494372 Dose Day 04’ (mg/kg/wk) 4 93 8 70 12 49 40 15* 4 73 8 56 12 32* 40 11* WO 79625 Table 184 Apo(a) plasma n levels as a percent of Day 1 levels in the recovery phase in cynomolgus monkeys treated with ISIS 494372 Dose Day 04’ (mg/kg/wk) 12 3 8 * 40 22* 12 84 40 93 Example 125: ement of viscosity of ISIS antisense oligonucleotides targeting human Ap0(a) The viscosity of select antisense oligonucleotides from the studies described above was measured with the aim of screening out nse oligonucleotides which have a viscosity more than 40 centipoise (cP).
Oligonucleotides having a viscosity greater than 40 cP would have less than optimal viscosity.
ISIS oligonucleotides (32-35 mg) were weighed into a glass vial, 120 uL of water was added and the antisense oligonucleotide was dissolved into solution by heating the vial at 500C. Part (75 uL) of the pre- heated sample was pipetted to a micro-viscometer (Cambridge). The temperature of the micro-viscometter was set to 250C and the viscosity of the sample was measured. Another part (20 uL) of the pre-heated sample was pipetted into 10 mL of water for UV reading at 260 nM at 850C (Cary UV instrument). The results are ted in the table below and indicate that most of the antisense oligonucleotides solutions are optimal in their viscosity under the criterion stated above. Those that were not optimal are marked as ‘viscous’.
Specifically, ISIS 494372 was optimal in its viscosity under the criterion stated above.
Table 185 Viscosity and concentration of ISIS antisense oligonucleotides targeting human Apo(a) ISIS No Motif Vlizgilty Concentration 494158 55 MOE 9.0 350 494159 55 MOE 11.7 325 494161 55 MOE 12.0 350 494162 55 MOE 25.8 350 494163 55 MOE Viscous 275 494243 55 MOE 28.4 325 494244 55 MOE 19.2 300 2014/036460 494283 34 VIOE 13.4 300 494284 55 VIOE 13.4 350 494285 55 VIOE 23.1 350 494286 55 VIOE 16.5 275 494301 55 VIOE 17.1 325 494302 55 VIOE 24.3 350 494304 55 VIOE 49.3 275 494311 55 VIOE 10.8 325 494337 55 VIOE 29.5 325 494372 55 VIOE 12.5 350 494466 55 VIOE Viscous 275 494470 55 VIOE 16.7 350 494472 55 VIOE 23.6 350 498408 55 VIOE 31.5 300 510548 55 VIOE 9.0 350 512947 34 VIOE 6.8 350 512958 55 VIOE 26.0 350

Claims (40)

CLAIMS :
1. A compound comprising a modified oligonucleotide and a ate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 1; wherein the conjugate group comprises: and wherein the compound is not: a compound comprising a modified ucleotide and a conjugate group, wherein the modified ucleotide consists of 20 contiguous nucleobases complementary to an equal length portion of nucleobases 3901 to 3920 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 1; and wherein the ate group comprises:
2. The compound of claim 1, wherein the modified oligonucleotide has a nucleobase sequence comprising at least an 8 nucleobase portion of one of SEQ ID NOs: 11-57, 59-133 and 135.
3. The compound of claim 2, wherein the modified oligonucleotide has a nucleobase sequence comprising at least a 12 nucleobase portion of one of SEQ ID NOs: 11-57, 59-133 and 135.
4. The nd of any of claims 1-3, n the modified ucleotide consists of 15 to 30 linked nucleosides.
5. The compound of claim 4, wherein the modified oligonucleotide consists of 15 to 25 linked nucleosides.
6. The nd of claim 5, wherein the ed oligonucleotide consists of 19 to 22 linked nucleosides.
7. The compound of claim 6, wherein the modified oligonucleotide consists of 20 linked nucleosides.
8. The compound of any of claims 1-7, wherein the nucleobase sequence of said modified oligonucleotide is at least 80% complementary to SEQ ID NO: 1 to an equal portion of SEQ ID NO: 1.
9. The compound of claim 8, wherein the nucleobase sequence of said modified oligonucleotide is at least 85% complementary to SEQ ID NO: 1.
10. The compound of claim 9, wherein the nucleobase sequence of said modified ucleotide is at least 90% complementary to SEQ ID NO: 1.
11. The compound of claim 10, wherein the base sequence of said modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1.
12. The compound of claim 11, wherein the nucleobase sequence of said modified oligonucleotide is 100% mentary to SEQ ID NO: 1.
13. The compound of any of claims 1-12, wherein the modified oligonucleotide comprises at least one modified sugar.
14. The nd of claim 13, wherein at least one modified sugar is a bicyclic sugar.
15. The nd of claim 13, wherein at least one modified sugar comprises a ethoxyethyl, a constrained ethyl, a 3’-fluoro-HNA or a 4’- (CH2)n-O-2’ bridge, wherein n is 1 or 2.
16. The compound of claim 13, wherein at least one modified sugar is 2’-O-methoxyethyl.
17. The compound of any of claims 1-16, wherein at least one nucleoside comprises a modified nucleobase.
18. The compound of claim 17, wherein the modified nucleobase is a 5-methylcytosine.
19. The compound of any of claims 1-18, wherein the conjugate group is linked to the ed oligonucleotide at the 5’ end of the modified ucleotide.
20. The compound of any of claims 1-18, wherein the conjugate group is linked to the modified oligonucleotide at the 3’ end of the modified ucleotide.
21. The compound of any of claims 1-20, wherein each internucleoside linkage of the modified oligonucleotide is selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.
22. The compound of claim 21, wherein the modified oligonucleotide comprises at least 5 phosphodiester internucleoside linkages.
23. The compound of claim 21, wherein the modified oligonucleotide ses at least 2 phosphorothioate internucleoside linkages.
24. The compound of any of claims 1-23, wherein the modified oligonucleotide is single-stranded.
25. The compound of any of claims 1-23, wherein the modified oligonucleotide is double stranded.
26. The compound of any of claims 1-25, wherein the modified oligonucleotide ses: a gap segment consisting of linked deoxynucleosides; a 5’ wing segment consisting of linked nucleosides; a 3’ wing segment consisting of linked nucleosides; wherein the gap segment is positioned between the 5’ wing segment and the 3’ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
27. The compound of claim 26, wherein each internucleoside linkage in the gap segment of the modified ucleotide is a phosphorothioate linkage.
28. The nd of claim 27, wherein the modified ucleotide further comprises at least one phosphorothioate internucleoside linkage in each wing segment.
29. The compound of claim 26, wherein the modified oligonucleotide comprises: a gap segment consisting of ten linked deoxynucleosides; a 5’ wing segment ting of five linked nucleosides; a 3’ wing t consisting of five linked nucleosides; wherein the gap segment is oned between the 5’ wing segment and the 3’ wing segment, n each nucleoside of each wing segment comprises a 2’-O-methoxyethyl sugar, and wherein each cytosine residue is a 5-methylcytosine.
30. The compound of claim 29, wherein each internucleoside linkage in the gap segment of the modified oligonucleotide is a phosphorothioate linkage.
31. The compound of claim 30, wherein the modified oligonucleotide further comprises at least one orothioate internucleoside linkage in each wing segment.
32. The compound of claim 30, wherein the internucleoside linkages are phosphorothioate es between nucleosides 1-2, nucleosides 6-16 and sides 17-20 of the modified oligonucleotide, wherein nucleosides 1-20 are positioned 5’ to 3’.
33. The compound of claim 30, wherein the 2nd, 3rd, 4th, and 5th internucleoside linkage from the 5’- end is a phosphodiester internucleoside linkage, n the 3rd and 4th internucleoside linkage from the 3’-end is a phosphodiester internucleoside linkage, and wherein each ing internucleoside linkage is a phosphorothioate internucleoside linkage.
34. The compound of any ing claim, wherein the compound is in a salt form.
35. A pharmaceutical composition comprising a compound of any one of claims 1-34, and a ceutically acceptable diluent or carrier.
36. The pharmaceutical composition of claim 35, n the compound is in a salt form.
37. A method comprising administering to a non-human animal a compound according to any one of claims 1-34, or a composition according to claim 35 or claim 36, wherein administering the compound, or the composition, treats, prevents or slows progression of a disease related to elevated apo(a) and/or elevated Lp(a).
38. Use of a compound according to any one of claims 1-34, or a composition according to claim 35 or claim 36, in the manufacture of a ment for treating, preventing, or g progression of a disease related to elevated apo(a) and/or elevated Lp(a).
39. The method of claim 37, or the use of claim 38, wherein the disease is an inflammatory, cardiovascular or metabolic disease, disorder or condition.
40. The method or the use of claim 39, wherein the cardiovascular e, disorder or condition is aortic stenosis.
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US201361818442P 2013-05-01 2013-05-01
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US201361843887P 2013-07-08 2013-07-08
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US201361871673P 2013-08-29 2013-08-29
US61/871,673 2013-08-29
US201361880790P 2013-09-20 2013-09-20
US61/880,790 2013-09-20
US201461976991P 2014-04-08 2014-04-08
US61/976,991 2014-04-08
US201461986867P 2014-04-30 2014-04-30
US61/986,867 2014-04-30
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