JP7849826B2 - Composition for posterior ocular delivery - Google Patents

Description

This invention relates to a composition for efficiently delivering drugs such as nucleic acids to the posterior segment of the eye without the intervention of medical technology, a pharmaceutical composition (particularly an eye drop) comprising the composition and a drug, and the treatment and prevention of posterior segment diseases using the pharmaceutical composition.

Intractable posterior segment eye diseases include age-related macular degeneration, retinal vein occlusion, retinitis pigmentosa, proliferative vitreoretinopathy, and diabetic retinopathy. Conventional treatments for these diseases have included laser photocoagulation (PDT) and vitrectomy, which are forms of medical intervention.

Regarding drug treatment, anti-inflammatory drugs and steroids have been attempted. Furthermore, in recent years, anti-vascular endothelial growth factor (VEGF) drugs such as pegaptanib (Macugen®), ranibizumab (Lucentis®), bevacizumab (Avastin®), and aflibercept (Eylea®) have been developed and their effectiveness has been demonstrated. However, these high molecular weight bioactive substances, even when administered as eye drops, are not delivered to the posterior segment where the retina is located due to barrier mechanisms such as the tear film and aqueous humor drainage. Therefore, currently, the administration of these drugs is limited to intravitreal injection, which is highly invasive. Implantable steroid-containing preparations (implants, etc.) have also been developed, but these still require medical technology intervention, and the invasiveness problem remains unresolved.

Liposome formulations are widely used as carriers for delivering macromolecules such as antibodies, proteins, and nucleic acids to target cells, and are also being investigated as drug delivery systems (DDS) for the posterior segment of the eye (Non-Patent Documents 1 and 2). However, their effectiveness is not yet sufficient, and no formulation capable of delivering drugs to the posterior segment of the eye via eye drops has yet been commercialized.

Int. J. Mol. Sci., 18: 2076 (2017) YAKUGAKU ZASSHI, 132(12): 1365-1370 (2012)

As mentioned above, all treatments for posterior segment diseases are,
1) It is highly invasive, raising concerns about decreased compliance;
2) Regarding drug therapy administered intravitreously, there is a significant burden on the patient due to the fear associated with direct injection into the eye, the need for frequent administration, and the potential for side effects such as endophthalmitis and retinal detachment.
3) Current treatments do not restore the condition of the retina or vision, and cannot restore lost nerve cells;
4) Conventional sustained-release injectable and implantable drugs require medical technology intervention, and their high cost raises concerns from a healthcare economics perspective;
These are some of the problems, and there is still an unmet medical need for treatments for posterior segment diseases.
Therefore, the object of the present invention is to provide an effective drug delivery system (DDS) for the posterior segment of the eye that enables the treatment of posterior segment diseases by eye drops.

To achieve the above objective, the inventors diligently investigated liposome formulations that can deliver the drug to the posterior segment of the eye by eye instillation, using the model polymer FITC-dextran (FD-10) with a molecular weight of approximately 10,000. As a result, they revealed that by optimizing the lipid composition and particle size, the drug can be efficiently delivered to the posterior segment of the eye by eye instillation. Furthermore, they succeeded in improving the delivery to the retinal region by surface-modifying the liposomes with cationic polymers, cell membrane-permeable peptides, folic acid-binding lipid derivatives, etc. In particular, they confirmed that surface modification improves the persistence of nucleic acid delivery. Furthermore, they revealed that matching the properties of the liposome particles and the nucleic acid is necessary when applying actual nucleic acids. In addition, they demonstrated that the liposome formulation actually suppresses retinal edema using a posterior segment disease (RVO) model mouse.
Based on these findings, the inventors conducted further research and, as a result, completed the present invention.

In other words, the present invention is as follows:
[1] (A) to (D) below:
(A) One or more phospholipids selected from the group consisting of distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, egg yolk lecithin, and hydrogenated versions thereof;
(B) Compounds having one or more steroid skeletons selected from the group consisting of cholesterol, cholesterol esters, cholestanol, and dehydrocholesterol;
(C) One charged substance selected from the group consisting of stearylamine, didodecyldimethylammonium bromide, and dicetyl phosphate; and (D) A surface modifier selected from the group consisting of folic acid derivatives, octaarginine, polylysine, and intracellular penetration peptides;
A liposome formulation comprising the following components for delivering a drug to the posterior segment of the eye by eye instillation, wherein the average particle diameter is 600 nm or less.
[2] The liposome formulation according to [1], wherein the average particle size is 200 nm or less.
[3] The liposome formulation according to [1] or [2], wherein component (D) is a folic acid derivative, octaarginine, or an intracellular penetration peptide.
[4] A liposome formulation according to any one of [1] to [3], wherein the surface potential is +20 to 60 mV.
[5] A liposome formulation according to any one of [1] to [4], wherein the drug is a nucleic acid.
A prophylactic or therapeutic agent for posterior segment diseases, comprising a liposome formulation according to any one of [6], [1] to [4], in which a polymer effective for the prevention or treatment of posterior segment diseases is encapsulated.
[7] The agent according to [6], wherein the polymer is a nucleic acid.
[8] The agent according to [7], wherein the nucleic acid is siRNA, single-stranded nucleic acid, or modified nucleic acid.
[9] An eye drop preparation, as described in any of [6] to [8].

The liposome formulation of the present invention allows for the efficient delivery of high molecular weight physiologically active substances, such as nucleic acids, to the posterior segment of the eye via eye drops, thereby providing a non-invasive and effective therapeutic agent for posterior segment diseases.

Figure 1 is a graph showing the delivery of FD-10 to the inner plexiform layer of mice after ophthalmic administration of FD-10-encapsulated PLA-modified liposomes (mean ± S.E.M., n = 7-9). * P < 0.05 versus untreated, †† P < 0.01 versus FD-10 solution. Figure 2 shows the retinal delivery of siRNA by ophthalmic administration of siRNA-encapsulated unmodified liposomes and folic acid (FA)-modified liposomes. Figure 2A shows the epifluorescence microscope image of the retina 30 minutes after ophthalmic administration. Figure 2B shows the relative fluorescence intensity in the inner plexiform layer 30 minutes after ophthalmic administration. Figure 3 shows the retinal delivery of bonac nucleic acid to mice via ophthalmic administration of unmodified (SA-containing) liposomes containing bonac nucleic acid. Figure 3A shows the epifluorescence microscope image of the retina 30 minutes after ophthalmic administration (scale bar: 50 μm). Figure 3B shows the relative fluorescence intensity in the inner plexiform layer 30 minutes after ophthalmic administration (mean ± S.E.M., n = 7-8). ** P < 0.01 versus siRNA solution. Figure 4 shows the retinal delivery of bonac nucleic acid to mice via ophthalmic administration of bonac nucleic acid-encapsulated R8-modified liposomes. Figure 4A shows a fluorescence microscopy image of the retina 30 minutes after ophthalmic administration (scale bar: 50 μm). Figure 4B shows the relative fluorescence intensity in the inner plexiform layer 30 minutes after ophthalmic administration (mean ± S.E.M., n = 7-8). Figure 5 shows fluorescence microscopy images of the retina 30 minutes after administration of Bonac nucleic acid-encapsulated unmodified (SA-containing) liposomes and R8-modified liposomes to a retinal vein occlusion (RVO) model mouse, demonstrating the retinal delivery of Bonac nucleic acid (scale bar: 50 μm). Figure 6 shows the therapeutic effect of administering modified liposomes containing nucleic acids with anti-VEGF activity to RVO mice via ophthalmic instillation. The group administered with VEGF siRNA-encapsulated folic acid (FA)-modified liposomes showed suppressed thickening of the inner granular layer compared to the group administered with the media solution (n=6-9) (Figure 6A). Furthermore, the group administered with Bonac nucleic acid-encapsulated R8-modified liposomes also showed suppressed thickening of the inner granular layer compared to the group administered with the media solution (Figure 6B). Lipid composition: Egg yolk lecithin (EPC)/Chol. = 7/3, Lipid concentration: 20.4 mM, R8 concentration: 2.5 mol%

The present invention will be described in detail below. Unless otherwise specified, terms used herein may be used in the sense commonly used in the art.

1. The present invention provides a liposome formulation for delivering a drug to the posterior segment of the eye by eye drop (hereinafter sometimes referred to as "the liposome formulation of the present invention"). The liposome formulation of the present invention contains the following components (A) to (D).

(A) Phospholipids The phospholipids used in the liposome formulation of the present invention are not particularly limited as long as they are pharmaceutically acceptable, for example, phosphatidylcholine (e.g., dioleoyl phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, etc.), phosphatidylglycerol (e.g., dioleoyl phosphatidylglycerol, dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol) Examples include phosphatidylethanolamine (e.g., dioleoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylglycerophosphoethanolamine, etc.), phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, natural lipids derived from egg yolk, soybeans, and other plants and animals (e.g., egg yolk lecithin, soybean lecithin, etc.), or hydrogenated versions thereof. Preferably, examples include phosphatidylcholine (e.g., distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, etc.), egg yolk lecithin, or hydrogenated versions thereof. These phospholipids may be used individually or in combination of two or more.

(B) Sterols The compounds having a steroid skeleton (sterols) used in the liposome formulation of the present invention are not particularly limited as long as they are pharmaceutically acceptable. Examples include animal-derived sterols such as cholesterol, its fatty acid esters (cholesterol esters), cholestanol (dihydrocholesterol), dehydrocholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, and desmosterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol, and brassicasterol; and microbial-derived sterols such as thymosterol and ergosterol. Preferably, cholesterol, cholesterol esters, cholestanol, and dehydrocholesterol can be used. These sterols may be used individually or in combination of two or more.

The mixing ratio (molar ratio) of component (A) and component (B) in the liposome formulation of the present invention is not particularly limited as long as it does not impair the drug delivery performance to the posterior segment of the eye of the liposome formulation of the present invention. For example, if component (A) is a phospholipid composed of saturated fatty acids, then (A)/(B) is less than 8, preferably less than 5, more preferably 2 to 4, even more preferably 2 to 3, and particularly preferably 2 to 2.5.

(C) Charged substance The liposome formulation of the present invention is further characterized by containing a charged substance. The charged substance has a charge opposite to that of the drug to be encapsulated, thereby enhancing the interaction between the drug and the liposome through electrostatic interaction and improving the drug encapsulation rate. For example, if the drug is a nucleic acid, since nucleic acids are negatively charged, a positively charged substance can be incorporated. Examples of positively charged substances include fatty acid amides such as stearylamine and oleylamine, cationic lipids such as didodecyldimethylammonium bromide, dioctadecyldimethylammonium chloride, N-(2,3-oleyloxy)propyl-N,N,N-trimethylammonium, didodecylammonium bromide, 1,2-dioleyloxy-3-trimethylammonium propane, 3β-N-(N',N'-dimethylaminoethane)carbamol cholesterol, 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium, and cationic lipids such as 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneammonium trifluoroacetate. Preferably, these are stearylamine, didodecyldimethylammonium bromide, etc. On the other hand, if the drug has a positive charge, a negatively charged substance can be added. Examples of negatively charged substances include dicetyl phosphate.

The mixing ratio (molar ratio) of component (C) in the liposome formulation of the present invention is not particularly limited as long as it does not impair the drug delivery performance to the posterior segment of the eye of the liposome formulation of the present invention. For example, the ratio of the sum of components (A) and (B) to component (C) is 10:1 to 30:1, preferably 15:1 to 25:1, more preferably 18:1 to 25:1, and particularly preferably 18:1 to 22:1.

(D) Surface Modifier The liposome formulation of the present invention is characterized by further containing a surface modifier. Examples of surface modifiers include substances that have affinity for molecules expressed on the surface of target cells, such as cells of the posterior segment of the eye, such as the retina, choroid, sclera, and optic nerve (e.g., folate receptors, heparan sulfate proteoglycans, etc.). Specific examples of surface modifiers include folate derivatives, polymers of basic amino acids such as polyarginine and polylysine, and intracellular penetration peptides. Examples of folate derivatives include, but are not limited to, FA-PEGylation reagents in which the end of a linker such as polyethylene glycol (e.g., molecular weight 1,000 to 20,000) is modified with folate. There are no particular restrictions on polylysine or polyarginine as long as they have a molecular weight of 500 or more. In the case of polylysine, for example, polylysine having molecular weight distributions of 500 to 2000, 5000 to 15000, 15000 to 30000, and 30000 to 70000 can be used, preferably polylysine having distributions of 5000 to 15000, 15000 to 30000, and 30000 to 70000. Polyarginine with similar molecular weights can also be used, but polyarginine with a molecular weight of 500 to 2000, such as octaarginine, can also be preferably used. Examples of intracellular penetration peptides include AntP from Drosophila, TAT from HIV (Frankel, A. et al, Cell 55, 1189–93 (1988); Green, M. & Loewenstein, PM Cell 55, 1179–88 (1988)), Penetratin (Derossi, D. et al, J. Biol. Chem. 269, 10444–50 (1994)), Buforin II (Park, CB et al. Proc. Natl Acad. Sci. USA 97, 8245–50 (2000)), Transportan (Pooga, M. et al. FASEB J. 12, 67–77 (1998)), and MAP (model amphipathic peptide) (Oehlke, J. et al. Biochim. Biophys. Acta. 1414). 127-39 (1998)), K-FGF (Lin, YZ et al. J. Biol. Chem. 270, 14255-14258 (1995)), Ku70 (Sawada, M. et al. Nature CellBiol. 5, 352-7 (2003)), Prion (Lundberg, P. et al. Biochem. Biophys. Res. Commun. 299, 85-90 (2002)), pVEC (Elmquist, A. et al. Exp. Cell Res. 269, 237-44 (2001)), Pep-1 (Morris, MC et al. Nature Biotechnol. 19, 1173-6 (2001)), Pep-7 (Gao, C. et al. Bioorg. Med. Chem. 10, Examples include, but are not limited to, those using the cell-penetrating domains of proteins such as 4057-65 (2002), SynBl (Rousselle, C. et al. MoI. Pharmacol. 57, 679-86 (2000)), HN-I (Hong, FD & Clayman, G L. Cancer Res. 60, 6551-6 (2000)), and VP22 derived from HSV. These surface modifiers may be used individually or in combination of two or more.
The modification of liposomes with a surface modifier can be carried out by chemically bonding the surface modifier to the surface of the prepared liposomes using a method known to the present day, or by mixing the surface modifier, which has been pre-bonded to lipids, with the liposomes.

The mixing ratio (molar ratio) of component (D) in the liposome formulation of the present invention is not particularly limited as long as it does not impair the drug delivery performance to the posterior segment of the eye of the liposome formulation of the present invention. For example, as a mole fraction of the total formulation, it is 0.01 to 10 mol%, preferably 0.1 to 1 mol%. Alternatively, the amount of component (D) in the liposome formulation of the present invention can be adjusted so that the surface potential of the liposomes after surface modification is +20 to 60 mV.

The liposome formulation of the present invention, in addition to components (A) to (D), also contains, for example, glycolipids (e.g., glyceroglycolipids such as sphingomyelin, sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, glycosyldiglyceride, sphingoglycolipids such as galactosylcerebroside, lactosylcerebroside, ganglioside, etc.), long-chain fatty acids or long-chain aliphatic alcohols (e.g., fatty acids having 10 to 20 carbon atoms or their alcohols (e.g., palmitic acid, stearic acid, lauric acid)). It may also contain saturated fatty acids such as myristic acid, pentadecyl acid, arachidic acid, margaric acid, and tuberculinostearic acid; unsaturated fatty acids such as palmitoleic acid, oleic acid, arachidonic acid, vaccenic acid, linoleic acid, linolenic acid, arachidonic acid, and eleostearic acid; oleyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, linolyl alcohol, etc.; glycerin fatty acid esters (e.g., monoacylglycerides, diacylglycerides, triacylglycerides, etc.).

The liposomes of the present invention may be monolayer or multilayer, but are preferably monolayer. Furthermore, the average particle size of the liposome formulation of the present invention is not particularly limited as long as it does not impair the drug delivery to the posterior segment of the eye, but is, for example, less than 1 μm, preferably 600 nm or less, more preferably 400 nm or less, and even more preferably 200 nm or less. The lower limit of the average particle size is also not particularly limited, but is, for example, 10 nm or more, preferably 50 nm or more, and even more preferably 100 nm or more. Therefore, the preferred range of average particle size for the liposome formulation of the present invention is, for example, 10 to 600 nm, more preferably 50 to 400 nm, even more preferably 50 to 200 nm, and particularly preferably 100 to 200 nm.

The dispersion of the liposome formulation of the present invention can be prepared, for example, by mixing components (A) to (C) and dispersing them in an aqueous solution by a conventional method. Appropriate equipment such as an ultrasonic dispersion device or an emulsification dispersion device can be used for dispersion. The average particle size of the liposomes can be adjusted to a desired value by passing the obtained dispersion through a filter with an appropriate pore size, for example, using an extruder.

2. Pharmaceutical Composition The present invention also provides a posterior segment disease prevention or treatment agent (hereinafter also referred to as "the pharmaceutical composition of the present invention") comprising a liposome formulation of the present invention encapsulated with a polymer effective for the prevention or treatment of posterior segment diseases. In this specification, when "encapsulated," the nucleic acid molecule of the present invention may be held in the inner phase of the liposome or in the lipid bilayer. In this specification, "prevention" includes not only suppression of onset but also delay of onset, and "treatment" is used as a concept that includes not only complete cure but also symptom reduction and suppression of progression.

The "posterior segment diseases" targeted by the pharmaceutical composition of the present invention generally refer to diseases of the vitreous humor, retina, choroid, sclera, or optic nerve. Examples include, but are not limited to, age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinal vein occlusion, and uveitis.

The active ingredient contained in the pharmaceutical composition of the present invention is not particularly limited as long as it is a high-molecular-weight physiologically active substance effective in preventing or treating posterior segment diseases. However, since many posterior segment diseases are deeply related to neovascularization, examples of such high-molecular-weight physiologically active substances include polymers having anti-VEGF activity. Examples of polymers having anti-VEGF activity include nucleic acids such as siRNA, shRNA, and miRNA that suppress the expression of VFGF against VDGF, proteins or nucleic acids that suppress the function of VEGF, such as anti-VEGF antibodies or fragments thereof, VEGF antagonists containing the VFGF-binding domain of the VEGF receptor, and aptamers that bind to VEGF or its receptor. In a preferred embodiment, the drug encapsulated in the liposome formulation of the present invention is a nucleic acid, more specifically, a nucleic acid molecule or derivative thereof having 10 to 100 nucleotides in a single molecule, which exerts physiological activity in cells of the posterior segment of the eye, such as retinal cells (e.g., retinal pigment epithelial cells), and inhibits the function of target DNA, RNA, or proteins expressed therein. Preferably, the structure consists of a single-stranded nucleic acid molecule, a nucleic acid molecule containing a double or multi-strand within a single molecule, or a combination thereof (e.g., a double-stranded nucleic acid molecule formed from two single-stranded nucleic acid molecules), with oligonucleotides being a preferred example.

The nucleotides constituting the oligonucleotides used in this invention may be DNA, RNA, DNA/RNA chimeras, or derivatives and analogs thereof. Specific examples of oligonucleotides include, but are not limited to, siRNA, shRNA, microRNA, antisense nucleic acids, ribozymes, and peptides.

In another preferred embodiment, the oligonucleotide may include, but is not limited to, "single-stranded nucleic acid molecules containing repression sequences that suppress the expression of target genes," as described in International Publication Nos. 2012/017919, 2013/103146, 2012/005368, 2012/077446, and 2013/133393, etc.

The oligonucleotides used in this invention are not limited to their natural form, and at least a portion of the sugar or phosphate backbone constituting the nucleotide may be modified to enhance in vivo stability, such as nuclease resistance. When modified, desirable modifications include modification of the 2' position of the sugar, modification of other parts of the sugar, and modification of the phosphate backbone of the oligonucleotide. Examples of 2' position modifications of the sugar include substituents such as OR, R, R'OR, SH, SR, _NH2 , NHR, _{NR2, N3 ,} CN, F, Cl, Br, and I. Here, R represents an alkyl or aryl alkyl group, preferably a C1-C6 alkyl group, and R' represents an alkylene, preferably a C1-C6 alkylene. Examples of modifications of other parts of the sugar include the 4'-thio form. Examples of modifications of the phosphate backbone of the oligonucleotide include phosphorothioates, phosphorodithioates, alkylphosphonates, and phosphoramidates.

Oligonucleotides can be synthesized in solid phase or liquid phase by the known phosphoamidite method or triester method. The most common embodiment is the solid-phase synthesis method using the phosphoamidite method, which can be performed using an automated nucleic acid synthesizer or manually. After solid-phase synthesis is complete, the product is removed from the solid phase, protected groups are removed, and the target product is purified. Purification yields nucleic acids with a purity of 90% or higher, preferably 95% or higher.

The pharmaceutical composition of the present invention can be prepared by forming a complex between the above-mentioned nucleic acid, preferably oligonucleotide, and the liposome of the present invention as described above.

Since the liposomes of the present invention contain a charged substance (component (C)), a complex can be easily formed with an active ingredient having the opposite charge by appropriately selecting the charged substance. For example, if the active ingredient is a negatively charged nucleic acid, a positively charged substance can be incorporated as component (C). Accordingly, the pharmaceutical composition of the present invention can be produced by first dispersing components (A) to (C) in an aqueous solvent to form liposomes, then adding nucleic acid and dispersing again, or by dispersing components (A) to (C) and nucleic acid in an aqueous solvent, passing the resulting dispersion through a filter with an appropriate pore size as described above to obtain liposomes with a desired average particle size, and then surface modifying with component (D) as described above.
Alternatively, the active ingredient (e.g., nucleic acid) can be encapsulated in liposomes using known methods such as the lipid film method (vortex method), reverse-phase evaporation method, surfactant removal method, freeze-thaw method, and remote loading method.

Examples of aqueous solvents used for complex formation include electrolyte solutions such as sterile water for injection, distilled water for injection, and physiological saline, as well as sugar solutions such as glucose solution and maltose solution.

Dispersion processing methods include, for example, homomixers, homogenizers, ultrasonic dispersers, ultrasonic homogenizers, high-pressure emulsification dispersers, microfluidizers (product names), nanomizers (product names), ultimateizers (product names), DeBEE2000 (product name), and Manton-Gaurin type high-pressure homogenizers. Processing conditions, processing time, and processing temperature can be selected as appropriate. Furthermore, this dispersion processing can be carried out in several stages, such as through coarse dispersion.

The pharmaceutical composition of the present invention can also be formulated with any carrier, such as a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include, but are not limited to, excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, lubricants such as magnesium stearate and aerosil, fragrances such as citric acid and menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinylpyrrolidone, dispersants such as surfactants, diluents such as water and physiological saline, and base waxes.

The pharmaceutical composition of the present invention can be administered topically to mammals via ocular administration, but ophthalmic administration is particularly preferable.

Suitable formulations for local ocular administration include eye drops (aqueous eye drops, non-aqueous eye drops, suspension eye drops, emulsion eye drops, etc.), ointments, lotions, creams, etc. When the formulation of the present invention is an eye drop, a suitable base material can be used. Examples of base materials used in eye drops include phosphate buffer, Hanks buffer, physiological saline, irrigation solution, and artificial tears.

The pharmaceutical composition of the present invention may contain, for example, a buffering agent, an isotonic agent, a solubilizer, a preservative, a viscous base, a chelating agent, a cooling agent, a pH adjuster, an antioxidant, and the like, as appropriate.
Examples of buffering agents include phosphate buffers, borate buffers, citrate buffers, tartaric acid buffers, acetate buffers, and amino acids.
Examples of isotonic agents include sugars such as sorbitol, glucose, and mannitol; polyhydric alcohols such as glycerin and propylene glycol; salts such as sodium chloride; and boric acid.
Examples of solubilizers include polyoxyethylene sorbitan monooleate (e.g., polysorbate 80), polyoxyethylene hydrogenated castor oil, nonionic surfactants such as tyroxapole and pluronic acid, and polyhydric alcohols such as glycerin and macrogol.
Examples of preservatives include quaternary ammonium salts such as benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride; parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate; benzyl alcohol; sorbic acid and its salts (sodium salt, potassium salt, etc.); thimerosal (trade name); chlorobutanol; and sodium dehydroacetate.
Examples of viscous bases include water-soluble polymers such as polyvinylpyrrolidone, polyethylene glycol, and polyvinyl alcohol, as well as celluloses such as hydroxyethylcellulose, methylcellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose.
Examples of chelating agents include sodium edetate and citric acid.
Examples of cooling agents include l-menthol, borneol, camphor, and eucalyptus oil.
Examples of pH adjusting agents include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, boric acid or its salts (borax), hydrochloric acid, citric acid or its salts (sodium citrate, sodium dihydrogen citrate, etc.), phosphoric acid or its salts (disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.), acetic acid or its salts (sodium acetate, ammonium acetate, etc.), tartaric acid or its salts (sodium tartrate, etc.).
Examples of antioxidants include sodium bisulfite, anhydrous sodium sulfite, sodium pyrosulfite, and concentrated mixed tocopherols.

The molar ratio of the active ingredient (e.g., nucleic acid) to the liposome components in the pharmaceutical composition of the present invention is typically 1/100,000 to 1/1,000. Furthermore, the amount of liposomes containing the active ingredient within the liposome formulation is not particularly limited, as long as it does not cause aggregation of the liposome particles and is sufficient to exert a sufficient therapeutic effect; it is typically 10 to 100 mM.

The dosage of the pharmaceutical composition of the present invention varies depending on the administration method, the type of posterior segment disease, the severity, and the circumstances of the patient (sex, age, weight, etc.). For example, when administering nucleic acids with anti-VEGF activity as eye drops to adults, it is generally desirable to administer 0.01 to 1000 μg, preferably 0.05 to 100 μg, and more preferably 0.1 to 50 μg of nucleic acid as a single dose, once to 10 times a day, preferably 5 to 10 times.

The present invention will be described in detail below with reference to examples, etc., but the present invention is not limited to these examples.

Reference Example 1: Preparation of liposomes encapsulated with a polymer model substance (FITC-labeled dextrin, molecular weight approximately 10,000; FD-10), surface modification, and evaluation of retinal delivery capability (1) Preparation of FD-10 (FD) encapsulated liposomes
FD-encapsulated multilayer liposomes (MLVs) were prepared by thin-film hydration. Distearoylphosphatidylcholine (DSPC, Nippon Oil & Fats Co., Ltd.) and cholesterol (Chol., Sigma) were weighed into a round-bottom flask, dissolved in an appropriate amount of chloroform, and the solvent was removed under reduced pressure on a 40°C water bath using a rotary evaporator to prepare a thin film. After drying under reduced pressure overnight, this film was hydrated with an FD solution dissolved in Milli-Q in a 70°C water bath, incubated at 10°C for 30 minutes, and FD-encapsulated MLVs were prepared.
Submicron-sized liposomes (ssLip) were prepared by passing the prepared FD-encapsulated MLV through a 100 nm pore size filter (Nuclepore® Track-Etch Membrane, Whatman) 41 times at a pressure of 150–200 kPa using an extruder (LiposoFast™-Pneumatic, AVESTIN). Subsequently, the prepared ssLip was frozen in a pre-freezing chamber at -60°C, thawed at 40°C, and subjected to sonication, a process that was repeated for 4 cycles.
FD-encapsulated ssLips were prepared by mixing an equal volume of FD-encapsulated ssLips with a PLA Poly-L-arginine (PLA, Sigma) solution under stirring to prepare FD-encapsulated PLA-modified liposomes. The prepared FD-encapsulated ssLips were then mixed in an equal volume with HBSS-MES buffer (pH 6.0) that did not contain the polymer to obtain unmodified liposomes.

(2) The liposome suspension (ssLip) containing the FD obtained in (1) above was administered to mice as eye drops, and the mouse eyeballs were excised 30 minutes later. The excised eyeballs were washed with a large excess of physiological saline, fixed by immersion in 4% paraformaldehyde overnight, and then immersed in 20% sucrose solution (in phosphate buffer) for 1 to 2 days. Subsequently, tissue freezing solution (Tissue-Tek(R) OCT Compound, Sakura Finetech Japan Co., Ltd.) was added in Cryomold No. 1 (Tissue-Tek(registered trademark) Cryomold, Sakura Finetech Japan Co., Ltd.) to fix the eyeballs, and the eyeballs were rapidly frozen in liquid nitrogen and embedded. The embedded eyeballs were horizontally cut using a Cryostat (Leica CM1850), and thin sections with a thickness of 10 μm were cut at the position where the optic nerve was exposed to prepare sections for observation. The retina, located 500 μm from the optic nerve of the prepared section, was observed using an epifluorescence microscope (BX51, Olympus). The median density of the internal plexiform layer (IPL) in a specific area (50 × 50 pixels) within the obtained retinal image was quantified using ImageJ (National Institute of Mental Health). The average fluorescence intensity was expressed as a value from 0 to 255, with the fluorescence intensity of an untreated eyeball set to 1.
The results are shown in Figure 1. In the FD-encapsulated ssLip administration group, fluorescence derived from FD-10 was observed in the retina. Furthermore, stronger fluorescence was observed in the PLA-modified ssLip administration group compared to the other groups.

Example 1 Preparation of nucleic acid (siRNA or Bonac nucleic acid)-encapsulated liposomes and evaluation of their physical properties (1) Preparation of nucleic acid-encapsulated liposomes Folic acid-modified DY547-labeled siRNA-encapsulated liposomes were prepared by the thin-film hydration method. DSPC, Chol., and stearylamine (SA) were weighed into a round-bottom flask, dissolved in an appropriate amount of chloroform, and the solvent was removed under reduced pressure on a 40°C water bath using a rotary evaporator to prepare a thin film. After drying this under reduced pressure overnight, it was hydrated in a 60°C water bath with a solution of DY547-labeled nucleic acid (vascular endothelial growth factor-small interfering RNA (VEGF-siRNA, Nippon Gene) or single-stranded nucleic acid in which the guide strand and passenger strand of VEGF siRNA are linked by a non-nucleotide linker technology owned by Bonac Co., Ltd. (hereinafter referred to as "Bonac nucleic acid")) dissolved in Milli-Q, and incubated at 10°C for 30 minutes to prepare nucleic acid-encapsulated MLVs.
Submicron-sized liposomes (ssLip) were prepared by passing the prepared nucleic acid-encapsulated MLV through a 100 nm pore size filter (Nuclepore® Track-Etch Membrane, Whatman) 41 times at a pressure of 150–200 kPa using an extruder (LiposoFast ^™ -Pneumatic, AVESTIN).
Equal volumes of siRNA-encapsulated ssLips were mixed with an aqueous dispersion of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (FA-PEG-DSPE, Avanti), and equal volumes of Bonac nucleic acid-encapsulated ssLips were mixed with an aqueous dispersion of Stearoyl-Octa-Arginine (St-R8, BEX Co., Ltd.). These mixtures were incubated at 40°C for 1 hour to prepare siRNA-encapsulated folate-modified liposomes and Bonac nucleic acid-encapsulated R8-modified ssLips. The prepared nucleic acid (siRNA or Bonac nucleic acid)-encapsulated ssLips were then mixed with equal volumes of HBSS-MES buffer (pH 6.0) or HBSS-MEPES buffer (pH 7.4) to prepare unmodified liposomes.
(2) Evaluation of Particle Properties of siRNA-Embedded Liposomes Table 1 shows the particle properties of folate-modified DY547-siRNA-embedded liposomes. Liposomes were prepared using two different mixing ratios (molar ratios) with DSPC/Chol./SA as the lipid composition. siRNA was encapsulated within the liposomes by electrostatic interaction between the positive charge of SA and the negative charge of siRNA. In all liposomes, the drug encapsulation rate was high at approximately 100%, and the drug encapsulation rate was improved compared to when FD was used as the model drug. This is thought to be because the interaction between the liposome and siRNA was high due to electrostatic interaction. Therefore, it is suggested that the drug encapsulation rate when using siRNA includes siRNA that is not encapsulated within the liposome but interacts with and adheres to the liposome surface. Liposomes with a DSPC/Chol. molar ratio of 7/3 had a smaller average particle size and better particle quality than those with a DSPC/Chol. molar ratio of 8/1. The increase in particle size is thought to be due to aggregation caused by electrostatic interactions between unencapsulated DY547-siRNA and liposomes. It has been reported that phosphatidylcholine containing saturated fatty acids, such as DSPC, eliminates phase transitions and increases liposome membrane fluidity when Chol. is added. In the case of DSPC/Chol. molar ratio of 7/3, it is thought that the increased Chol. content made it easier for DY547-siRNA to enter the inner aqueous phase of the liposome membrane, resulting in a decrease in the amount of unencapsulated DY547-siRNA.
In folate-modified liposomes, the zeta potential decreased with FA-PEG-DSPE modification compared to unmodified liposomes, confirming that folate was added to the liposome surface. Furthermore, the drug encapsulation rate decreased with folate modification. This is likely because folate is negatively charged and caused electrostatic repulsion with siRNA that was not encapsulated within the liposome but attached to the liposome surface. Based on these results, a DSPC/Chol molar ratio of 7/3 is considered preferable for this purpose.

(3) Evaluation of retinal delivery of nucleic acids by nucleic acid-encapsulated liposomes The nucleic acid-encapsulated liposome suspension (ssLip) obtained in (1) above was administered as eye drops to mice, and 30 minutes later the mouse eyeballs were extracted. Fluorescence microscopy observation and fluorescence intensity quantification were performed in the same manner as in Reference Example 1 (2).
Figure 2(A) shows images of the retina 30 minutes after the first administration of DY547-siRNA solution and DY547-siRNA-encapsulated liposomes. No fluorescence was observed in the DY547-siRNA solution group, while fluorescence derived from DY547-siRNA was observed in the retina of the DY547-siRNA-encapsulated liposome group. Furthermore, stronger fluorescence was observed in the folate-modified liposome group compared to the other groups.
Figure 2(B) shows the results of quantifying the fluorescence intensity of retinal IPL. Compared to the DY547-siRNA solution administration group, the fluorescence intensity was significantly increased in the folate-modified DY547-siRNA-encapsulated liposomes, indicating good retinal penetration. One possible reason for this is that surface modification of the liposomes with folate conferred targeting properties to folate receptors present in retinal pigment epithelial cells.

Figure 3 shows the retinal migration of Bonac nucleic acid-encapsulated liposomes. In the SA-containing liposome administration group, the fluorescence intensity increased significantly compared to the medium solution, demonstrating retinal migration.
Figure 4 shows the retinal migration of Bonac nucleic acid-encapsulated R8-modified liposomes. R8-modified liposomes also showed excellent retinal migration, with a significant increase in fluorescence intensity compared to the medium solution and unmodified liposomes (SA-containing liposomes).
These results demonstrate that by encapsulating nucleic acid drugs in liposomes modified with folic acid or R8, nucleic acids can be delivered to the retina with high efficiency even through eye drops.

Example 2 Evaluation of retinal delivery of nucleic acids by ophthalmic administration of Bonac nucleic acid-encapsulated liposomes in a retinal vein occlusion (RVO) model mouse (1) Preparation of retinal vein occlusion model mouse An 8-week-old male ddY mouse (Nippon SLC Co., Ltd.) was used as the animal. The mouse was anesthetized by intramuscular administration of a combination of ketamine (120 mg/kg, Daiichi Sankyo Propharma) and xylazine (6 mg/kg, Bayer Healthcare), and then administered rose bengal (20 mg/kg, Wako Pure Chemical Industries, Ltd.), a photosensitizer, into the tail vein. To dilate the pupil, 5 μL of Midrin® P (0.5% tropicamide and 0.5% phenylephrine hydrochloride, Santen Pharmaceutical Co., Ltd.) was administered by micropipette. The laser was applied using a Micron4 (Phoenix Research lab.) fundus imaging device, equipped with a laser irradiation device (MERIDIAN AG, Bierigutstrasse). The retinal vein was occluded by irradiating it with the laser at a location three optic disc systems away from the optic disc in the right eye of the mouse (wavelength: 532 nm, spot size: 50 μm, irradiation time: 5 s, laser power: 50 mW). Three veins were occluded in each eye, and each vein was irradiated with the laser 20 to 30 times. Complete occlusion of the irradiated vein was confirmed in the fundus image. In addition, to prevent dryness of the mouse eyes, an appropriate amount of Hyalein (registered trademark) (purified sodium hyaluronate: hyalein, Santen Pharmaceutical Co., Ltd.) was administered as eye drops as needed.

(2) Evaluation of retinal delivery of nucleic acids by ophthalmic administration of Bonac nucleic acid-encapsulated liposomes Bonac nucleic acid-encapsulated liposome suspension was administered as eye drops to the RVO model mice obtained in (1) above. Thirty minutes later, the eyes of the mice were extracted, and retinal transfer was examined by fluorescence microscopy in the same manner as in Reference Example 1 (2).
The results are shown in Figure 5. Compared to untreated mice, the groups administered with liposomes containing Bonac nucleic acid (SA-containing liposomes) and R8-modified liposomes showed a significant increase in fluorescence intensity and demonstrated good retinal penetration. R8 modification further improved the retinal penetration of Bonac nucleic acid.

Example 3 Evaluation of the efficacy of nucleic acid (siRNA or bonac nucleic acid) encapsulated liposomes administered by eye instillation in RVO model mice (1) Administration conditions Nucleic acid encapsulated liposomes were administered by eye instillation in RVO model mice prepared in the same manner as in Example 2 (1) as described below. Two different administration times were investigated.
1) To examine both the preventive and therapeutic effects, the drug was administered to the right eye of mice three times at 5-minute intervals by eye drops 1, 3, 6, 12, 24, 36, and 48 hours before laser irradiation. Subsequently, the veins in the retina of the mice were occluded by laser irradiation, and the drug was administered to the right eye of the mice three times at 5-minute intervals 1, 3, and 6 hours later by eye drops.
2) The drugs were administered repeatedly by eye drops into the right eye of mice 2, 3, 6, 12, 18, 24, 30, and 36 hours after laser irradiation. For eye drop administration, 3 μL of liposome suspension containing various VEGF-siRNAs was administered into the right eye using a micropipette. The group of RVO model mice that did not receive the drug was given the same amount of HBSS-HEPES buffer and designated as the vehicle group.

(2) Observation by Optical Coherence Tomography (OCT)
In eye drop schedule 1), mice were anesthetized by intramuscular administration of a combination of ketamine (120 mg/kg, Daiichi Sankyo Propharma) and xylazine (6 mg/kg, Bayer Healthcare) 24 hours after laser irradiation, and in eye drop schedule 2), mice were anesthetized by intramuscular administration of a combination of ketamine (120 mg/kg, Daiichi Sankyo Propharma) and xylazine (6 mg/kg, Bayer Healthcare) 48 hours after laser irradiation. 5 μL of Midrin® P (0.5% tropicamide and 0.5% phenylephrine hydrochloride, Santen Pharmaceutical Co., Ltd.) was administered as eye drops, and OCT images of the right eye were observed using Micron4 (Phoenix Research lab.) (830 nm). In addition, to prevent dryness of the mice's eyes, an appropriate amount of Hyalein® (purified sodium hyaluronate, Santen Pharmaceutical Co., Ltd.) was administered as eye drops as needed.

(3) Preparation and evaluation of frozen ocular tissue sections
After observation by OCT, euthanasia was performed by cervical dislocation, and the eyeballs were removed. The removed eyeballs were washed with a large excess of physiological saline, immersed in 4% paraformaldehyde for 2 days to fix the tissue, then immersed in 5%, 10%, 15%, and 20% sucrose solutions (in phosphate buffer) every 2 hours, followed by immersion overnight in 25% sucrose solution (in phosphate buffer). Subsequently, the tissue was fixed by adding tissue freezing solution (Tissue-Tek(R) OCT Compound, Sakura Finetech Japan Co., Ltd.) in Cryomold No. 1 (Tissue-Tek(R) Cryomold, Sakura Finetech Japan Co., Ltd.), and the eyeballs were rapidly frozen in liquid nitrogen and embedded. The embedded eyeballs were horizontally cut using a Cryostat (Leica CM1850), and thin sections with a thickness of 20 μm were cut at the position where the optic nerve was exposed to prepare observation sections. The prepared sections were stained with hematoxylin and eosin, and the entire retina was observed using an all-in-one microscope (BZ-9000, KEYENCE). The thickness of the inner granular layer (INL) of the retina at 240 μm intervals from the optic nerve in the obtained images was quantified using ImageJ (National Institute of Mental Health).
The results are shown in Figure 6. In the group administered with VEGF-siRNA-encapsulated folate-modified liposomes, thickening of the inner granular layer (INL) of the retina was significantly suppressed compared to the group administered with the media solution (Figure 6A). Similarly, in the group administered with Bonac nucleic acid-encapsulated R8-modified liposomes, INL thickening was significantly suppressed compared to the media solution (Figure 6B).
These results demonstrate that when nucleic acids with anti-VEGF activity are encapsulated in folic acid or octaarginine (R8) modified liposomes and administered as eye drops, the nucleic acids are efficiently delivered to the retina, exhibiting a therapeutic effect against retinal diseases.

The liposomal formulation of the present invention allows for efficient delivery of the encapsulated drug (preferably nucleic acid) to the posterior segment of the eye via ophthalmic administration. Therefore, it is expected to facilitate the development of eye drops for treating posterior segment diseases, which are not yet commercially available. In other words, it enables non-invasive treatment of posterior segment diseases and addresses an unmet medical need, making it extremely useful.

This application is based on Japanese Patent Application No. 2019-053908, filed in Japan on March 20, 2019, and by reference thereto, all contents of that application are incorporated herein.

Claims (7)

A liposomal formulation for suppressing thickening of the inner granular layer (INL) of the retina, wherein the formulation comprises (A) one or more phospholipids selected from the group consisting of distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, egg yolk lecithin, and hydrogenated versions thereof;
(B) Compounds having one or more steroid skeletons selected from the group consisting of cholesterol, cholesterol esters, cholestanol, and dehydrocholesterol; and (C) One charged substance selected from the group consisting of stearylamine, didodecyldimethylammonium bromide, and dicetyl phosphate;
A liposomal formulation containing the following components, with a molar ratio (A)/(B) of component (A) to component (B) of 2 to 3, which delivers the drug to the posterior segment of the eye by instillation and is used to treat retinal diseases.
Here,
(a) A formulation having an average particle size of 600 nm or less, wherein the surface of the liposomes is modified with folic acid, octaarginine, polylysine, or intracellular penetration peptide;
(b) The drug is a nucleic acid;
Liposome formulation. A liposome formulation according to claim 1, wherein the average particle diameter is 200 nm or less. The liposome formulation according to claim 1 or 2, wherein the surface of the liposome is modified with folic acid, octaarginine, or an intracellular penetration peptide. A liposome formulation according to any one of claims 1 to 3, wherein the surface potential is +20 to 60 mV. A liposome formulation according to any one of claims 1 to 4, wherein the nucleic acid is siRNA, single-stranded nucleic acid, or modified nucleic acid. A liposome formulation according to any one of claims 1 to 5, wherein the nucleic acid is VEGF-siRNA. A liposome formulation according to any one of claims 1 to 6, which is an eye drop.