BG60759B2 - Thrombin inhibitors - Google Patents

Abstract

The invention relates to biologically active molecules that bind to and inhibit thrombin. These molecules have a thrombin anion-binding exosite associated moiety (aveam), a linker region of at least 18 a in length, and a thrombin catalytic site targeting moiety (csdm). The invention also relates to compositions, combinations and methods using these molecules for therapeutic, prophylactic and diagnostic purposes. 37 claims

Description

THROMBIN INHIBITORS

LITERATURE REFERENCE RELATED TO THE APPLICATION

This application is a continuation-in-part of the related U.S. patent application serial number 595,482, filed

I on 18.08.1989, currently abandoned.,'

TECHNICAL FIELD OF THE INVENTION

The present invention relates to novel biologically active molecules that bind to and inhibit thrombin. Specifically, these molecules are characterized by a thrombin anion-binding exosite-associating moiety (ABEAM); a portion of

the linker of at least 18 A in length; and a thrombin catalytic site-directed moiety (CSDM). The present invention also relates to compositions, combinations and methods using these molecules for therapeutic, prophylactic and diagnostic purposes.

STATE OF THE ART

Acute vascular diseases such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, and other thromboses of the circulatory system constitute major health risks. Such diseases are caused by partial or complete occlusion of a blood vessel by a blood clot containing fibrin and plaque.

Modern methods of treatment and prevention of thrombotic diseases require therapies that act in one of two different ways. The first type of treatment inhibits thrombin activity or thrombin formation, thereby preventing clot formation. These drugs also inhibit the activation and aggregation of plaques. The second category of therapeutics promotes thrombolysis and dissolves the blood clot, thereby removing it from the blood vessel and unblocking blood flow [ J.P.Cazenava et al., Agents Action. 15, Suppl., pp. 24-49 (1984)].

Heparin, a compound of the preceding class, has been widely used in the treatment of conditions such as venous thromboembolism, in which thrombin activity is responsible for the development and spread of the thrombus. Although active, heparin causes many undesirable side effects, including hemorrhage and thrombocytopenia. This has led to

the search for a more specific and less toxic anticoagulant.

Hirudin is a naturally occurring polypeptide produced by the blood-sucking leech Hirudo medicinalis. The substance, which is synthesized in the salivary glands of the leech, is the most potent known natural inhibitor of coagulation. Hirudin prevents blood from clotting by tightly binding to thrombin (Κ^=2χ10 M) in a 1;1 stoichiometric complex [S. R. Stone and J. Hofsteenge, Kinetics of the Inhibition of Thrombin by Hirudin, Biochemistry, 25, pp. 4622-28 (1986)]. This inhibits thrombin from catalyzing the conversion of fibrinogen to fibrin (clot), as well as inhibiting all other thrombin-mediated processes [J. W. Fenton, II, Regulation of Thrombin Generation and Functions, Semin. Thromb. Hemost., 14, pp. 254-40 (1988)].

The actual binding between hirudin and thrombin is a two-step process. Initially, hirudin binds to a low-affinity site on the thrombin molecule (IQ=1x10 M), which is separated from the catalytic site. This binding involves the interaction of a structure at the C-terminus of hirudin with an ‘anion-binding exosite (ABE)’ in thrombin [J. W. Fenton, II et al., Thrombin Anion Exosite Interactions with Heparin and Various Polyanions, Ann.New York Acad. Sci., 556, pp. 15865 (1989)]. Following the low-affinity binding, the hirudin-thrombin complex undergoes a conformational change, after which hirudin binds to the high-affinity site on thrombin [S. Kono et al.,

Analysis of Secondary Structure

of Hirudin and the Conformational Change Upon Interaction with Thrombin, Arch, iochem. Biophys., 267, pp. 158-66 (1988)]. This later site corresponds to the active site of thrombin.

The isolation, purification and chemical composition of hirudin are known in the art [P. Walsmann and F. Markwatdt, Biochemical and Pharmacological Aspects of the Thrombin Inhibitor Hirudin, Pharmazie, 56, pp. 655-60 ' I(1981)]. The complete amino acid sequence of the polypeptide has recently been revealed [J. Dodt et al., The Complete Covalent Structure of Hirudin : Localization of the Disulfide Bonds, Biol. Chem. Hoppe-Seyler. 566, pp. 575-85 (1985); S.J.T. Mao et al., Rapid Purification and Revised Amino Terminal Sequence of Hirudin : A Specific Thrombin Inhibitor of the Blood-Sucking Leech, Anal. Biochem. 161, pp. 514-18 (1987); and R.P. Harvey et al., Cloning and Expression of a c]DNA Coding for the Anti-Coagulant Hirudin from the Bloodsucking Leech, Hirudo medicinalis, Proc. Natl. Acad. Sci. USA, 85, pp. 1084-88 (1986)].

At least ten different isomorphic forms of hirudin have been sequenced and shown to differ only slightly in their amino acid sequence [D. Tripier, Hirudin: A Family of Iso-Proteins. Isolation and Sequence

Determination of New hirudins, Folia Haematol., 115, pp. 5055 (1988)]. All forms of hirudin comprise a single-chain polypeptide protein containing 65 or 66 amino acids, in which the amino terminus initially contains hydrophobic amino acids.

selenides, and the carboxy terminus typically contains polar amino acids. Specifically, all forms of hirudin are characterized by a single N-terminal domain (residues 1-59) stabilized by three disulfide bridges at the 1-2, 5-5, and 4-6 hemi-cysteinyl probes, and a highly acidic C-terminal segment (residues 40-65). In addition, the C-terminal segment of hirudin is characterized by the presence of a tyrosine residue at amino acid position 65, which is sulfated.

In animal studies, hirudin purified from leeches has shown efficacy in preventing venous thrombosis, vascular bypass occlusions, and thrombin-induced disseminated intravascular coagulation. In addition, hirudin exhibits low toxicity, low antigenicity, and a very short half-life.

Markwardt et botic Action period of removal from circulation [ F.

al., Pharmacological Studies on the Antithromof Hirudin in Experimental Animals, Thromb.

Haemost.,

47, pp.

226-29 (1982)]

In order to obtain larger quantities of hirudin,

attempts have been made to produce the polypeptide by recombinant DNA techniques

The presence of an O-sulfated tyrosine residue in native hirudin and the inability of microorganisms to effect such modification of the protein made the prospect of recombinant production of biologically active hirudin highly speculative. The observation that disulfatohirudins are almost as active as their sulfated counterparts [U.S. Pat. No. 4,654,502], however, led to the cloning and expression of hirudin in E. coli [European

-6 patent applications 158,564,168,342 and 171,024] and yeast

[European patent application 200,655]. Despite these advantages, hirudin is still relatively expensive to produce and is not widely available commercially.

Recently, efforts have been made to identify peptide fragments of native hirudin that are also effective in prolonging clotting time. An unsulfated C-terminal fragment of hirudin with 21 amino acids,

N-acetylhirudin inhibits clot formation in vitro. In addition, several other smaller, unsulfated peptides corresponding to the C-terminal 11 or 12 amino acids of hirudin (residues 55-65 and 54-65) have also demonstrated efficacy in inhibiting clot formation in vitro [J.L. Krstenansky et al., Antithrombin Properties of C-terminus of Hirudin Using Synthetic Unsulfated Nacetyl-hirudin, FEBS Lett, 211, pp. 10-16 (1987)]. Bee

Again, such peptide fragments may prove insufficiently satisfactory for dissolving blood clots in the regimen of the applied therapy, due to their low activity.

For example, N-acetyl-hirudin 45· has a specific activity four orders of magnitude lower than native hirudin.

In addition to catalyzing the formation of the fibrin clot, thrombin has several other bioregulatory roles [J.W. Fenton, II, Thrombin Bioregulatory Functions, Adv. Clin. Enzymol., 6, pp. 186-93 (1988)]. For example, thrombin directly activates plaque aggregation and initiates reactions. This

4*

means that thrombin plays a central role in acute platelet-dependent thrombosis. [S.R. Hanson and L.A. Harker, Interruption of Acute Platelet-Dependent Thrombosis by the Synthetic Antithrombin D-Phenylalanyl-L-Prolyl-L-Arginyl-chloromethylketone, Proc. Natl. Acad. Sci. USA, 85, pp. 3184-88 (1988)]. Thrombin can also directly activate the inflammatory response by stimulating the synthesis of platelet-activating factor (PAF) in endothelial cells [S. Prescott et al., Human Endothelialm Cells in Culture Procedure Platelet-Activating Factor (1-alkyl-2-acetyl-sn-glycero-3phospho-choline) When Stimulated With Thrombin, Proc. Natl. Acad. Sci. USA, 81, pp. 3534-38 (1984)]. PAF is localized on the surface of endothelial cells and serves as a ligand for neutrophil adhesion and subsequent degranulation [G.M. Vercolletti et al., Platelet-Activating Factor Primes Neutrophil Response to Agonists: Role in Preventing Neutrophil-Mediated Endothelial Damage, Blood, 71, pp. 1100-07 (1988)]. Alternatively, thrombin may promote the inflammatory process by increasing vascular permeability, which may lead to edema [P.J. Del Vecchio et al., Endothelial Monolayer Permeability to Macromolecules, Fed. Proc., 46, pp. 2511-15 (1987)]. Reagents that block the active site of thrombin, such as hirudin, interrupt the activation of platelets and endothelial cells [C.L. Knupp, Effect of Thrombin Inhibitors on Thrombin-Induced Release and' Aggregation, Thrombosis Res., 49, pp. 23-36 (1988)].

Thrombin is also involved in potentiation of hand,

-ΰ of its native digestive

based on the ability of the product, fibrin, to serve as a substrate for tumor growth [A. Falanga et al., Isolation and Characterization of Cancer Procoagulant: A Cysteine Proteinase from Malignant Tissue, Biocheistry, 24, pp. 5558-67 (1985); S.G. Gordon et al., Cysteine Proteinase Procoagulant From Amnion-Chorion, Blood, 66, pp. 1261-65 (1985); and A. Falanga et al., A New Procoagulant in Acute Leukemia, Blood, 71, pp. 870-75 (1988)]. Thrombin has also been implicated in neurodegenerative diseases based on its ability to cause neuritic reactions [D. Gurwitz et al., Thrombin Modulates and Reverses Neuvroblastoma Neurite Outgrowth, Proc Natl. Acad. Sci. USA, 85, pp. 5440-44 (1988)]. Therefore, the ability to regulate in vivo thrombin activity is of significant clinical importance.

Despite the developments to date, there is still a need for a molecule that effectively inhibits thrombin in clot formation, plaque activation, and many other thrombin-mediated processes, and that can be produced inexpensively and in commercially viable quantities.

CONTENT OF THE INVENTION

The present invention solves the above problems by providing molecules that mimic the action of hirudin by binding to both the low affinity anion-binding exosite (AVE) and the catalytic site of (/-thrombin.

These molecules are more potent than hirudin and can therefore be administered to patients at doses relatively lower than those required in a hirudin-based therapy regimen. The molecules of the invention can be used in compositions and methods for inhibiting thrombin-mediated or thrombin-related functions or processes. Pharmaceutical compositions containing these molecules, as well as methods using them for the treatment and prevention of vascular diseases, inflammatory responses, carcinomas and neurodegenerative diseases, also form part of the present invention. These molecules can also be used in compositions and methods for ex vivo modeling, for storing and treating extracorporeal blood and for coating invasive devices. Furthermore, the molecules of the present invention can be administered to patients in combination with a fibrinolytic agent, in order to increase the efficacy of the administered dose of the agent or to reduce the dose of the agent required for a given effect, so as to dissolve the blood clot.

Due to their high potency and the fact that they can be prepared by chemical synthesis techniques, the molecules of the present invention can be prepared inexpensively and in commercially viable quantities. In addition, the molecules of the present invention are significantly smaller than hirudin and are less likely to stimulate an unwanted immune response in patients treated with them. In this regard, the use of these inhibitors of

- -IQ thrombin is not limited to the treatment of acute diseases. These molecules can also be used in the treatment of chronic thromboembolic diseases, such as atherosclerosis and restenosis following angioplasty. The molecules of the present invention can also be used in a wide variety of other applications in place of natural or recombinant hirudin.

As will be emphasized by the following description, the molecules, compositions and methods of the invention are useful in the treatment and prevention of numerous diseases due to the undesirable effects of thrombin, as well as for diagnostic purposes.

BRIEF DESCRIPTION OF THE SCHEMES

Fig. 1 depicts an autoradiograph of an SDS-polyacrylamide gel demonstrating the binding of DNFB-[^Sj-sulfo-Tyr^ _? hirudin gf to human n-thrombin in the presence or absence of sulfo-Tyr^-N-acetyl-hirudin^_ _{with 4} .

Fig. 2 depicts a three-dimensional model of human -thrombin.

Fig. 3-A depicts the effects of chirulog-8 and sulfo-Tur _with chiudin ₆ * on the cleavage of Spectrozyme TH by human thrombin.

Fig. 3-B depicts a Linewaever-Burke plot of cleavage of Spectrozyme TH by human oL-thrombin in the presence or absence of Hirulog-8 or Sulfo-Tug _vz -hirudin^_ .

Fig. 4 depicts the effect of varying concentrations of Hirulog-8, hirudin or Sulfi-Tur^^-N-acetyl-hirudin on

-Η -

activated partial thromboplastin time of normal human serum.

Fig. 5-A depicts the time course of cleavage at different concentrations of Hirulog-8 by human α(-thrombin.

Fig. 5-B depicts the relationship between the concentration of Hirulog-8 and the duration of inhibition of Spectrozyme TH hydrolysis by human 12C-thrombin.

Fig-6 depicts the effect of linker length on the in-

I- the thrombin inhibitor according to the invention on the inhibition of thrombin-catalyzed hydrolysis of Spectrozyme TH.

Fig. 7 depicts the inhibitory effect of various concentrations of Hirulog-8 or sulfo-Tur _£5 -N-acetyl-hirudin^ on thrombin modification by 12C-DFP.

Fig. 8 depicts the in vivo effect of different doses of Hirulog-8 on baboon APTT.

Fig. 9 depicts the comparative inhibitory effects of Hirulog-8 or heparin on the hydrolysis of fibrinogen by soluble or clot-bound thrombin.

Fig. 10 depicts the in vivo effects of different doses of Hirulog-8 on plaque deposition on an endarterectomized segment of baboon aorta.

Fig. 11 depicts the in vivo effects of different doses of Hirulog-8 on plaque deposition on a segment of collagen-coated tubing inserted into a baboon.

Fig. 12 depicts the ratio of the in vivo effects of heparin, hirudin, or Hirulog-8 on plaque deposition on a segment of collagen-coated tubing inserted into a baboon's

-12AV deviation.

Fig. 15 depicts the in vivo effects of different doses of

Hirulog-8 on fibrin deposition on a segment of collagen-coated tubing inserted into a baboon AV shunt.

Fig.14 depicts the change in

ARTT at a time point following intravenous bolus injection of baboons with

Chirologist-8.

Fig. 15 depicts the change in APTT at a time point following subcutaneous injection of

Fig. 16 depicts a baboon with Hirulog-8. *' * The correlation of the in vivo effects of tissue plasminogen activator together with saline, heparin, hirudin or Hirulog-8 on reperfusion time in a rat model.

Fig. 17 depicts the ratio of the in vivo effects of tissue plasminogen activator together with saline, heparin, hirudin or Hirulog-8 on reocclusion time in a rat model.

Fig. 18 depicts the ratio of in vivo effects of

tissue plasminogen activator together with saline, heparin, hirudin or Hirulog-8 on APTT in a rat model.

Fig. 19 depicts the ratio of the in vivo effects of tissue plasminogen activator together with saline, heparin, hirudin or Hirulog-8 on vessel opening in a rat model.

Fig. 20 depicts the effect of different doses of Hirulog-8 on bleeding time in a baboon model.

DETAILED DESCRIPTION OF THE INVENTION

The following are the most commonly used abbreviations of amino acids:

the villages are used in the application and in the claims: Ogp - ornithine Gly - glycine Ala - alanine Vai - valine Leu - leucine lie - isoleucine Pro - proline Phe - phenylalanine Trp - tryptophan Met - methionine 1— Ser - serine Thr - threonine Cys - cysteine Tug - tyrosine Asn - asparagine Gin - glutamine Asp - aspartate Glu - glutamate Lys - lysine Arg - arginine His - histidine Nle - norleucine Hyp - hydroxyproline Pgl - phenylglycine Ac - acetyl Sue - succinyl BOC - tert-butoxycarbonyl Tos - paraToluene sulfonyl Cbz - carbobenzyloxy D-Ala - D-alanine 3,4, -dehydroRgo - 3.4,- Sar - sarcosine

dehydroproline (N-methylglycine)

Tyr(OSO^H) - tyrosine sulfate

Tyr(S0 ₃ H) - tyrosine sulfonate

3-,5-DiiodoThug - 3-,5diiodotyrosine

The term "any amino acid", as used herein, includes L-isomers of the naturally occurring in pro-44.

data amino acids, as well as other non-protein «’C amino acids, commonly used by protein chemists when preparing synthetic analogues of naturally occurring proteins. Naturally occurring amino acids are: glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartate, asparagine, glutamate, glutamine, -carboxyglutamate, arginine, ornithine, and lysine. Examples of “non-protein” ^-amino acids

are norleucine, norvaline, alloisoleucine, homoarginine, tiaproline, dehydroproline, hydroxyproline (Hyproline), homoserine, cyclohexylglycine (Chg), &C -amino-p-butyrate (Aba), cyclohexylalanine (Cha), aminophenylbutyrate (Pba), phenylalanines substituted at the ortho, meta or para position of the phenyl moiety with one or two of the following: (C1-C4) alkyl, (C1-C4) alkoxy, halogen or nitro groups or substituted with a methylenedioxide group; -2 and 3-thiophene-alanine, P-2- and 3-furanyl-alanine, -2-, 5- and

4-pyridyl-alanine, B-(benzothiophen-2- and 3-yl)alanine, B-(1- and

2-naphthyl)alanine, O-alkylated derivatives of serine, threonine or tyrosine, S-alkylated cysteine, S-alkylated homocysteine, O-sulfate, O-phosphate and O-carboxylated esters of tyrosine, 3 and 5-sulfonyl-tyrosine, 3- and 5-carbonyl-tyrosine, 3- and 5-phosphonyl-tyrosine, 4-methylsulfonyl-tyrosine, 4-methylphosphonyl-tyrosine, 4-phenylacetic acid, 3,5-diiodo-tyrosine, 3- and 5-nitrotyrosine, E-alkyllysine, delta-alkyl ornithine and the D-isomers of naturally occurring amino acids.

The term patient as used in the present specification refers to any mammal, and in particular a human.

The term anionic amino acid, as used herein, means a meta, para or ortho, mono- or di-substituted phenylalanine, cyclohexylalanine or tyrosine containing a carboxyl, phosphoryl or sulfonyl moiety, as well as S-alkylated cysteine, S-alkylated homocysteine, β-carboxyglutamate, β-alkyl lysine, delta-alkyl ornithine, glutamate and aspartate. Examples of anionic amino acids are phosphothreonine, phosphoserine, phosphotyrosine, 3-, 4- or 5-sulfotyrosine, 3-methylphosphonyl tyrosine and 3-methylsulfonyl tyrosine.

The terms catalytic site, active site, and active site pocket, as used herein, each refer to any or all of the following sites in thrombin:

the substrate-binding or S^ site; the hydrophobic binding or oily site; and the site at which the cleavage of the substrate actually occurs (the actual carrier site).

The term, as used here, refers to

to the nitrogen of the side chain of ornithine. The term refers to any nitrogen of the side chain of arginine.

The term N*^ refers to the pZ-amino group of an amino acid. The term psi, as used in the application and claims, refers to the replacement of an amide bond with the atoms indicated in parentheses, according to the nomenclature described in J. Rudinger, In Drug Design, Vol. II, E.J. Ariens, ed., Academic Press, New York, p. 319 (1971).

The term backbone, as used herein, refers to the portion of a chemical structure that defines the smallest number of consecutive bonds that can be written from one end of the chemical structure to the other. The atomic components included in the backbone may include any atom capable of forming bonds with at least two other atoms.

For example, each of the following chemical structures is characterized by a main chain of 7 atoms (the atoms that are included in the main chain are indicated in bold):

The term calculated length, as used in the

The present description refers to assumed measurements resulting from the summation of the bond lengths between the atoms that make up the main chain. The bond lengths between any two given atoms are well known in the art [see, for example, CRC Handbook of Chemistry and Physics, 65th Edition, R.C. Weist, ed., CRC Press, Inc., Boca Raton, Fla., pp. F-166-70 (1984)].

The present invention relates to molecules that bind to and inhibit thrombin. These molecules are characterized by

They consist of three domains: a catalytic site-directed moiety (CSDM), a linker region, and an anion-binding exosite associated moiety (ABEAM).

According to the present invention, the first domain, CSDM, binds to the catalytic site of thrombin, located at or near Ser-195, and inhibits or slows the amidolytic or esterolytic activity of thrombin. Preferably, CSDMs, according to the present invention, are selected from one of three main groups: those that reversibly bind to thrombin and are slowly cleaved; those that reversibly bind to thrombin and cannot be cleaved; and those that

which bind irreversibly to thrombin. Reversible inhibitors bind to the active site of thrombin through noncovalent bonds, such as ionic bonds, hydrophobic bonds, or hydrogen bonds. Irreversible CSDMs form covalent bonds with thrombin.

According to the preferred embodiment, CSDMs that bind reversibly to thrombin and are cleaved slowly have the formula:

X-A ₄ -A _Λ -A ₃ -Y where X is hydrogen or is characterized by a main chain consisting of 1 to 35 atoms; A^ is Arg, Lys or Orn; Ao is a non-amide bond; A^ is characterized by a main chain consisting of 1 to 9 atoms; and Y is a bond.

The non-amide linkage component of the present invention can be formed by chemically modifying an amide linkage. This can be achieved by methods well known in the art [M. Szelke et al., Potent New Inhibitors of Human Renin, Nature, 299, pp 55557 (1982); D.H.Coy et al. Facile Solid Phase Preparation of Proteins Containing the CH^-NH Peptide ond Isostere and Application to the Synthesis of Somatoststin (SRIF) Octapeptide Analogue, Peptides 1986, D. Theodoropoulos, Ed., Walter DeGruyter & Co., Berlin, pp. 143-46 1987)]. When a non-amide linkage is formed in this way, it is preferable that the chemical modification be carried out before the addition of the dipeptide containing this linkage to the other components of the CSDM or to the remainder of the thrombin-inhibiting molecule. On this one

-4H-

In this way, _the -A^-Aj - is added en bloc to the remainder of the molecule, in a single step of the synthesis.

In a most preferred embodiment, A is Arg and _A is Pro, D-Pro or Sar. In this arrangement, it is a naturally occurring amide bond that is slowly cleaved by thrombin. This avoids the need for conversion of the non-amide bond and allows A to be added sequentially to the remainder of the molecule rather than en bloc.

As previously stated, CSDMs of the present invention can bind irreversibly to thrombin. Examples of irreversible CSDMs include, but are not limited to, general serine proteinase inhibitors such as phenylmethylsulfonylfluoride (PMSF), diisopropylfluorophosphate (DFP), tosylprolylchloromethylketone (TPCK) and tosylsilylchloromethylketone (TLCK); heterocyclic protease inhibitors such as isocoumarins; thrombin-specific inhibitors such as D-Phe-Pro-ArgCHCl (PPAK); and transition state analogs such as difluoroketomethylene.

According to another preferred embodiment of the present invention, non-cleavable, reversible CSDMs consist of the formula:

^X - ^C ^- ^C _a - ^A 3- ^Y where C^ is a derivative of Arg, Lys or Arg, characterized by a reduced carboxylate moiety or a carboxylate moiety that is displaced relative to the -carbon of a chemical structure characterized by a main chain of 1 to 10 atoms; C^ is a non-cleavable bond; and X, Y and A^ are as previously defined. Examples of C components are ^2 -homoarginine; arginine containing a reduced carboxylate moiety, such as Asg[psiCH NH] ; β -homolysine and β-homoornithin.

Other non-cleavable, reversible CSDMs that can be used in the thrombin inhibitors of the invention are benzamidine, DAPA, NAPAP and argatroban (argipidine).

For those thrombin inhibitors of the invention which possess CSDM regions characterized by either a C-1 or C-2 linkage, the term P-2 sequence as used herein refers to the two chemical structures associated with said linkage.

The X component of the CSDM, which is not involved in the actual binding to the catalytic site, may be of unlimited length and of varying structure. However, for convenience and to reduce the cost of synthesis, X is preferably characterized by a backbone consisting of 1 to 55 atoms and does not exceed a calculated length of 56 Å. It is preferred that X is a peptide, particularly preferably D-Phe-Pro. This most preferably

A preferred variant allows the X component to fit into a groove on thrombin that is adjacent to the active site.

[S. Bajust et al. ··Inhibition of Thrombin and Trypsin by

Tripeptide

Aldehydes, Int

Peptide Protein Res., 12, pp. 217-21 (1978); C. Kettner et al. , D-Phe-Pro-Arg-CH Cl-A selective

Affinity Label for

Thrombin, Thromb. Res.,

14, pp. 969-75 (1979)]. This allows the CSDM component and therefore ^the molecules according to the present invention to bind thrombin with an advantageously high degree of affinity.

-21affinity and optimal specificity.

According to the present invention, the second component of the thrombin inhibitors of the present invention is a linker region. Since the role of this region of the molecule is to provide a bridge between CSDM and ABEAM, the length of the linker is of primary importance rather than its structure. The calculated length of the main chain characterizing the linker should be at least about 18 Å - the distance between the catalytic site and the anion-containing exosite of thrombin - and less than 42 Å.

The backbone of the linker may contain any atoms capable of bonding to at least two other atoms. Preferably, the backbone is comprised of any chemically feasible combination of atoms selected from oxygen, carbon, nitrogen, and sulfur. Those skilled in the art will appreciate which combination of the atoms in the backbone above falls within the desired length based on known bond distances [see, for example, R.T. Morisson and R.N. Boyd, Organic Chemistry, 5th Edition, Allyn and Bacon, Inc. Boston, Mass. (1977)]. In a preferred embodiment, the linker is a peptide containing the amino acid sequence Gly-Gly-Gly-Asn-Gly Asp-Phe. Preferably, the amino acid bond to the ABEAM component is Phe.

The third domain of the thrombin inhibitors of the present invention is ABEAM, which binds to the anion-binding exosite of thrombin. Preferably, ABEAM has the following formula:

where W is a bond; B^ is an anionic amino acid; B^ is any amino acid; B^ is Ile, Al, Leu, Nle or Phe; B^ is Pro, Hyp, 3,4- dehydroPro, thiazolidine-4-carboxylate, amino acid; B θ is an anionic amino acid; B? is a lipophilic amino acid selected from the group consisting of Trp, Trp, Phe, Leu, Nle, He, Val, Cha, Pro or a dipeptide consisting of one of these lipophilic amino acids and any amino acid; B& is a bond or peptide containing from one to five residues of any amino acid; Z is a carboxy terminal residue selected from OH, C^-C^. alkoxy, amino, mono- or di-(C^-C^) alkyl substituted amino or benzylamino.

Peptides homologous to the carboxy-terminal region of hirudin have been shown to bind to the anion-binding exosite of thrombin [ copending U.S. patent application Ser. No. 314,756 and J.M. Maraganore et al., Anticoagulant Activity of Synthetic Hirudin Peptides, J. Biol. Chem., 264^ pp. 8692-98 (1989); both of which are incorporated herein by reference].

According to a preferred embodiment of the invention, ABEAM is homologous to amino acids 56-64 of hirudin, i.e. B^ is Glu; B^ is Glu; Bj is He; B^_ is Pro; B^ Glu; B^. is Glu; is Tyr-Leu, Tyr(SO^ H)-Leu or Tyr(OSOjH)-Leu or (3-,5-DiiodoTyr)-Leu; B _β is a bond; and Z is OH. It should be noted that native hirudin contains Tyr(OSOjH) at position 63. However, carboxy-terminal peptides of hirudin that contain Tyr(SOjH) have anticoagulant activity identical to those containing native Tyr(OSOjH) [see ifpending US application Ser. No. 314,756].

Other ABEAM components within the scope of the present invention may include those regions of any molecule known to bind to the anion-binding site of thrombin. These include amino acids 1675-1686 of Factor V, amino acids 272-285 of plaque glycoprotein 1b, amino acids 426-444 of thrombomodulin, amino acids 245-259 of prothrombin fragment 2, and amino acids 30 to 44 of the fibrinogen A ?-chain. In addition, the ABEAM component may be selected from any of the hirudin peptide analogs described by J.L. Krstenansky et al., Development of MDL28,050, A small Stable Antithrombin Agent Based on a Functional Domain of the Leech Protein, Hirudin, Thromb. Haemostash, 63, pp. 208-14 (1990).

The preferred thrombin inhibitors of the present invention are referred to as Hirulogs and are described in the following examples. The most preferred Hirulogs are Hirulog-8, Hirulog-12, Hirulog-18a, Hirulog-18b and Hirulog-33. Hirulog-8, -12 and -33 are reversible thrombin inhibitors that are slowly cleaved. Hirulog-18a and -18b are reversible inhibitors that are not cleaved.

The thrombin inhibitors of the present invention can be synthesized by various techniques well known in the art. These include enzymatic cleavage of natural or recombinant hirudin, recombinant DNA techniques, solid-phase peptide synthesis,

liquid-phase peptide synthesis, organic chemical synthesis techniques or a combination of these techniques. The choice of synthesis technique will, of course, depend on the composition of the individual inhibitor. In a preferred embodiment of the invention, the thrombin inhibitor is entirely peptide and is synthesized by solid-phase peptide synthesis techniques, liquid-phase peptide synthesis or a combination thereof, which constitute the most cost-effective ways to produce industrial quantities of these molecules.

When non-protein amino acids are contained in the thrombin inhibitor molecule, they can either be directly added to the growing chain during peptide synthesis or prepared by chemical modification of the entire synthesized peptide, depending on the nature of the desired non-peptide amino acid. Those skilled in chemical synthesis are well aware of which non-peptide amino acids can be directly added and which must be synthesized by chemical modification of the entire peptide chain after protein synthesis.

The synthesis of those thrombin inhibitors of the invention which contain both non-amino acid and peptide moieties is preferably carried out by a mixed heterologous/solid phase technique. This technique involves the solid phase technique of all or most of the peptide moieties of the molecule, followed by the addition of non-amino acid components which are synthesized by liquid phase techniques. The non-amino acids may be

- 2F coupled to the peptide region by solid-phase or liquid-phase methods. Similarly, any remaining peptide region can also be added by solid-phase or liquid-phase methods.

The molecules of the present invention exhibit potent anticoagulant activity. This activity can be assayed in vitro using any conventional technique. Preferably, an assay for anticoagulant activity involves directly determining the thrombin inhibitory activity of the molecule. Such techniques measure the inhibition of thrombin-catalyzed cleavage of colorimetric substrates, or more preferably, the increase in thrombin time or the increase in activated partial thromboplastin time of blood plasma. A further assay identifies factors in the intrinsic coagulation pathways. Alternatively, the assay used may employ purified thrombin and fibrinogen to measure the inhibition of fibrinopeptide A or B residue by radioimmunoassay or ELISA.

The anti-plaque activity of the molecules of the present invention can also be measured by any conventional plaque assay. Preferably, the assay should measure the change in the degree of plaque aggregation or the change in the residual secretory component of the plaque in the presence of thrombin. The above can be measured by an aggregometer. The latter can be measured using RIA or ELISA techniques specific for the secretory component;

S.

the removed components.

The molecules of the present invention are useful in compositions, combinations or methods for the treatment and prevention of various diseases due to thrombin-mediated or thrombin-related functions or processes. These include myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, restenosis following arterial injury or invasive cardiac procedures, acute or chronic atherosclerosis, edema and inflammation, with various cellular regulatory processes (e.g., secretion, shape change, proliferation), cancer and metastasis, and neurodegenerative diseases.

The thrombin inhibitors of the present invention can be formed using conventional methods for preparing pharmaceutically useful compositions, such as adding a pharmaceutically acceptable carrier. These compositions and methods using them can be used to treat or prevent thrombotic diseases.

the patient's condition.

According to a modified embodiment of the present invention, thrombin inhibitors can be used in combinations, compositions and methods for the treatment of thrombotic diseases and to reduce the dose of thrombolytic agent required to effect reperfusion or to prevent reocclusion of the patient. In addition, thrombin inhibitors according to the invention can be used: in combinations, compositions and methods for reducing the time to reperfusion or to

-27increasing the time to reocclusion in a patient treated with a thrombolytic agent. These combinations and compositions comprise a pharmaceutically effective amount of a thrombin inhibitor according to the present invention and a pharmaceutically effective amount of the thrombolytic agent.

In these combinations and compositions, the thrombin inhibitor and the thrombolytic agent work in a complementary manner to dissolve blood clots, resulting in reduced reperfusion time and increased reocclusion time in patients treated therewith. Specifically, the thrombolytic agent dissolves the clot, while the thrombin inhibitor prevents newly formed, clot-bound or clot-bound thrombin from regenerating the clot. The use of the thrombin inhibitor in the combinations and compositions of the present invention advantageously allows the administration of the thrombolytic agent to be administered.

in doses discussed above, as too low to lead to thrombolytic effects if used alone. This avoids the undesirable side effects associated with the use of thrombolytic agents, such as bleeding complications.

The thrombolytic agents that can be used in the combinations and compositions according to the present invention are those known in the art.

Such agents include, but are not limited to, tissue plasminogen activator purified from natural sources, recombinant tissue plasminogen activator, streptokinase, urokinase, prurokinase, and isolated streptokinase plasminogen.

gene activator complex (ASPAC), animal salivary gland plasminogen activators and known, biologically active derivatives of any of the above.

The term combination, as used herein, includes a single dosage form containing at least one thrombin inhibitor according to the present invention and at least one thrombolytic agent; a multiple dosage form in which the thrombin inhibitor and the thrombolytic agent are administered separately but concurrently; or a multiple administration form in which the two components are administered separately but sequentially. In sequential administration, the thrombin inhibitor may be administered to the patient within a period of 5 hours before to 5 hours after administration of the thrombolytic agent. Preferably, the thrombin inhibitor is administered to the patient within a period of 2 hours to 2 hours after administration of the thrombolytic agent.

Alternatively, the thrombin inhibitor and the thrombolytic agent may be in the form of separate, conjugated molecules. The conjugation of the two components may be accomplished by standard cross-linking techniques well known in the art. The separate molecule may also take the form of a recombinant fusion protein if both the thrombin inhibitor and the thrombolytic agent are proteins.

Various dosage forms can be used to administer the compositions and combinations of the invention. These include, but are not limited to, parenterally in

-29 administration, oral administration and topical application. The compositions and combinations according to the present invention can be administered to the patient in any pharmaceutically acceptable dosage form, including those which can be administered to the patient intravenously as a bolus or by continuous infusion, intramuscularly - including paravertebral and periarticularly - subcutaneously, intracutaneously, intraarticularly, intrasynovially, intrathecally, intralesionally, periosteally or by oral, nasal or topical routes. Such combinations and compositions are preferably adapted for nasal oral or parenteral administration, but most preferably for parenteral administration.

Parenteral compositions are preferably administered intravenously, as a bolus or as a continuous infusion. If the thrombin inhibitor is used as an antiplaque compound, a continuous infusion is preferred. If the thrombin inhibitor is used as an anticoagulant, a subcutaneous or intravenous bolus injection is preferred. For parenteral administration, liquid unit dosage forms are prepared which contain a thrombin inhibitor according to the present invention and a sterile carrier (vehicle). The thrombin inhibitor may be either suspended or dissolved, depending on the nature of the carrier and the nature of the thrombin inhibitor being administered. Parenteral compositions are generally prepared by dissolving the thrombin inhibitor in the carrier, optionally together with other components, and filter-sterilizing prior to filling into suitable vials or

-30 ampoules and sealed. Preferably, adjuvants such as local anesthetics, preservatives and buffering agents are also dissolved in the vehicle. The composition may then be frozen.

zen and lyophilized, to enhance stability. Parenteral suspensions are prepared in exactly the same way way, except of cases where the active component

it is suspended rather than dissolved in the carrier.

Sterilization of the compositions is preferably carried out

by exposure to ethylene oxide before suspension in a sterile vehicle. Preferably, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of its components.

Tablets and capsules for oral administration may contain conventional carriers such as binders, fillers, diluents, tabletting agents, lubricants, disintegrants and wetting agents. The tablet may be coated according to methods well known in the art. Suitable fillers which may be used include cellulose, mannitol, lactose and the like. Suitable disintegrants include, but are not limited to, starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable wetting agents include sodium lauryl sulfate.

Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for

-U1~

reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives. These include suspending agents such as sorbitol, glucose, methyl cellulose, gelatin, hydroxyethyl cellulose, carboxyethyl cellulose, aluminum stearate gel or hydrogenated edible fats; emulsifying agents which include lecithin, sorbitan monooleate, polyethylene glycols or acacia; non-aqueous vehicles such as almond oil, fractionated coconut oil and fatty esters; and preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid.

Compositions formulated for topical administration may, for example, be aqueous jellies, oily suspensions, or emulsified ointment forms.

The dosage and dose rate of the thrombin inhibitor will depend on various factors, such as the size of the patient, the specific pharmaceutical compositions being used, the entity being treated, e.g., therapy or prophylaxis, the nature of the thrombotic disorders being treated, and the judgment of the attending physician.

According to the present invention, the preferred pharmaceutically effective daily dose of the thrombin inhibitor according to the invention is between 1 g/kg of the weight of the patient being treated (body weight) and about 5 mg/kg of body weight. In compositions containing a thrombolytic agent, the pharmaceutically effective daily dose of the thrombolytic is between 10¾ and 80¾ of the conventional dosage level. The conventional dosage level of the thrombolytic agent is the daily dose

3A used when this agent is administered as monotherapy. [Physician’s Desk Reference 1989, 43rd Edition, Edward R. Barnhart, publisher]. This conventional dosage level will depend, of course, on the thrombolytic agent used. Examples of conventional dosage levels are as follows: urokinase - 500,000 to 6,250,000 units/patient; streptokinase - 140,000 to 2,500,000 units/patient; tPA - 0.5 to 5.0 mg/kg body weight; ASPAC - 0.1 to 10 units/body weight.

Preferably, the therapeutic and prophylactic compositions of the present invention comprise a thrombin inhibitor in doses between 10 μg/kg body weight and 500 μg/kg body weight. Most preferred combinations comprise the same amount of thrombin inhibitor and between 10% and 70% of the conventional dosage level of the thrombolytic agent. It should also be understood that the daily pharmaceutically effective dose of the thrombin inhibitors of the invention or of the thrombolytic agent present in the compositions of the invention may be less than or greater than the specific levels recited above.

After improvement in the patient's condition is established, a maintenance dose of the combination or composition according to the invention is administered as needed. Therefore, the dose or frequency of administration, or both, may be limited as a function of the symptoms, to the extent that the improved condition is maintained. Once the symptoms have been alleviated to the desired level, treatment should be discontinued. Patients

The patient may, however, require periodic treatment if any symptoms of the disease appear.

According to an alternative embodiment of the invention, thrombin inhibitors can be used in compositions and methods for coating the surfaces of invasive devices, thereby reducing the risk of clot formation or plaque activation in patients receiving such devices. The surfaces that can be coated with the compositions

According to the invention, include, for example, prostheses, artificial valves, vascular grafts, stents and catheters. Methods and compositions for coating these devices are known to those skilled in the art. This includes chemical crosslinking or physical absorption of the thrombin inhibitor-containing composition onto the surface of the devices.

According to another embodiment of the present invention, thrombin inhibitors can be used for ex vivo imaging of a thrombus in a patient _. In this embodiment, the thrombin inhibitor is labeled with a radioisotope. The choice of radioisotope is based on well-known _factors such as toxicity, biological half _- life, and detectability. Preferred radioisotopes include, but are not limited to, I, I, and In. Techniques for labeling the thrombin inhibitor are well known in the art. The radioisotope I is preferred, and labeling is accomplished using I-Bolton-Hunter reagent. The labeled thrombin inhibitor is administered to a patient and allowed to bind to thrombin contained in the clot. After

‘34- this is observes the clot, when used well famous techniques h4. discovery like a camera capable of re- hides radioactivity, combined with computer graphics

system. This technique also allows images of plaque-bound thrombin and meisothrombin.

The invention also relates to compositions comprising the thrombin inhibitors of the present invention and methods of using such compositions in the treatment of tumor metastases. The efficacy of the thrombin inhibitors of the present invention in the treatment of tumor metastases is demonstrated by the inhibition of the growth of the metastases. This is based on the presence of a procoagulant enzyme in some cancer cells. This enzyme activates the conversion of Factor Xa into a coagulation cascade, culminating in the deposition of fibrin, which in turn serves as a substrate for tumor growth. By inhibiting the deposition of fibrin by inhibiting thrombin, the molecules of the present invention serve as effective agents against tumor metastases. Examples of metastatic tumors that can be treated with the thrombin inhibitors of the present invention include, but are not limited to, brain carcinoma, liver carcinoma, lung carcinoma, osteocarcinoma, and neoplastic plasma cell carcinoma.

The invention also relates to methods and compositions using the above-described thrombin inhibitors to inhibit thrombin-induced activation of endothelial cells. This inhibition includes repression of the synthesis of factor for

- Plaque activation factor (PAF) by endothelial cells. These compositions and methods have important applications in the treatment of diseases characterized by thrombin-induced inflammation and edema, which are believed to be mediated by PAF. Such diseases include, but are not limited to, senile respiratory distress syndrome, septic shock, septicemia, and reperfusion injury.

The early stages of septicemia

Shock includes interrupted, acute inflammatory and coagulopathic responses. It has previously been shown that injection of baboons with a lethal dose of live

E. coli causes a shift in neutrophil count, blood pressure, and hematocrit. Changes in blood pressure and hematocrit are due in part to the generation of disseminated intravascular coagulopathy (DIC) and are indicated for concurrent fibrinogen consumption.

[F.B. Taylor et al., “Protein C

Prevents the

Coagulation and Lethal Effects of Escherichia coll infusion in,, the Baboon, J. Clin. Invest., 79, pp. 91825 (1987)]. Neutropenia is due to an acute inflammatory response caused by septic shock, which causes a marked increase in the level of tumor necrosis factor. The thrombin inhibitors, according to the invention, can be used in compositions and methods for the treatment or prevention of DIC in septicemia and other diseases.

The present invention also relates to the use of the above-described thrombin inhibitors or compositions comprising them as anticoagulants for extracorporeal blood.

As used herein, the term extracorporeal blood includes

-Blood separated from the patient, treated outside the body, and then returned to the patient, in such processes as dialysis, blood filtration, or blood bypass surgery. The term also includes blood products that are stored outside the body for possible use.

applying to a patient and taking blood from the patient for use in various tests. Such products include whole blood, plasma, or any blood fraction in which inhibition of coagulation is desired.

The amount or concentration of thrombin inhibitor in this type of composition is based on the volume of blood to be treated or, preferably, its thrombin content.

/

Preferably, an effective amount of the thrombin inhibitor of the invention for preventing coagulation in blood is from about 1 g/60 ml of extracorporeal blood to about 5 mg/60 ml of extracorporeal blood.

Thrombinovite© inhibitors of the invention can be used to inhibit clot-associated thrombin, which is believed to contribute to clot growth. This is particularly important since commonly used antithrombin agents such as heparin and low molecular weight heparin are ineffective against clot-associated thrombin.

Finally, the thrombin inhibitors of the invention can be used in compositions and methods for treating neurodegenerative diseases. Thrombin is known to cause neurite retraction, a process involving rounding-37 in the shape changes of brain cells and implicated in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

In order that the invention described herein may be more fully understood, the following examples are provided. It is to be understood that these examples are for illustrative purposes only and are not to be construed as limiting the present invention in any way.

EXAMPLE 1

We have prepared this peptide by solid-phase peptide synthesis using

Applied Biosystems 430 A Peptide Synthesizer (Applied Biosystems, Foster City, Calif.).

Specifically, 0.259 mol of BOC-O-Leu resin (1% DVB resin) was reacted sequentially with 2 mmol of the protected amino acids. After 10 cycles of synthesis, we deprotected the peptide and cleaved it from the DVB resin by treatment with anhydrous HF: p-cresol: ethyl methyl sulfate (10:1:1, v/v/v). The peptide was then purified on a Vydac C^g reversed-phase HPLC column (22 mm x 25 cm), which was pre-calibrated with 0.1% TFA in water. Before loading the peptide onto the column, we dissolved it in 2.0 ml of 0.1% TFA in water. If necessary, an additional 1 ml of 6M guanidine chloride was added to the sample to increase solubility. After loading the sample, the column was washed with an increasing linear gradient of acetonitrile (0-80%) in 0.1% TFA over 45 minutes at a flow rate of 4.0 mL/min. The effluent was monitored at 229 nm and fractions were collected manually.

We sulfate the resulting purified peptide at the single tyrosine residue using standard procedures [T. Nakahara et al., Preparation of Tyrosine-0-[ ^3S ~ SjSulfated Cholecystokinin Octapeptide From A Non-Sulfated Precursor Peptide, Anal. Biochem., 154, pp. 194-99 (1986)]. Sulfo- _Tur

Hirudin^^ is then purified from other peptides and reaction components by reverse phase HPLC using

Vydac Cd column (4.6 x 25 cm) and Applied Biosystems liquid chromatography system. The column was calibrated with 0.1% TFA/water solvent and washed with acetonitrile at a flow rate of 0.8 ml/min with a 0.085% TFA-containing solution. Fractions were examined for absorbance at 214 nm with an increasing linear gradient of up to 35% over 90 min.

EXAMPLE 2

I Cross-linking of human thrombin with sulfo-Tug-ez dinitrofluorobenzyl-hirudin ₆

Sulfo-Tur-dinitrofluorobenzyl-hirudin (2.0 mg; prepared as in Example 1) was prepared with a stoichiometric amount of difluorodinitrobenzene (Pierce Chemical Co., Rockford, Ill.) in dimethyl formamide (DMF) for 18 hours at room temperature. The sample was then subjected to analytical HPLC separation using an Applied Biosystems 150 A Liquid Chromatographic System and a Brownlee RP-500 C& ko- |

-32* lon (0.46 x 10 cm) to determine the degree of randomness. The column was calibrated with 0.1% TFA in water (solvent A) and washed with a 0-50% linear gradient of 0.085% TFA/70% acetonitrile (solvent B) over 45 minutes and then a 50-100% linear gradient of solvent B over 15 minutes.

We use a constant flow rate of 1.0 ml/min.

The effluent is examined for absorbance at 214 nm and 310

nm. The peptide derivatized with difluorodinitrobenzene reagent absorbs at 310 nm. We found that the above reaction gives sulfo-Tug

-DNFB-hirudin at 15 to 30% yield.

After synthesis, sulfo-Tur-DNFB-hirudin was stored in the same dimethylformamide solvent at 20 ^° C for up to 1 month.

A 10-fold molar excess of sulfo-Tur-DNFB-hirudin 53^4 was reacted with human thrombin (12.5 mg) for 18 hours at room temperature in phosphate-buffered saline. The extent of cross-linking was determined by analyzing the reaction mixture on an SDS-polyacrylamide gel. SDS-PAGE showed a decrease in the relative mobility of the thrombin band, reflecting an increase in molecular weight of 1000-2000 daltons (Da). This change was due to the cross-linking of thrombin with sulfo-Tur-DNFB-hirudin^.^4. at a single site.

We confirm that this conformation between the covalent complex of sulfo-Tyr$ ₃ -DNFB-hirudin and human thrombin is specific by using [^*S]-sulfo-Tyr^ -DNFB-hirudin. [ ³⁵ δ]-sulfo-Tyr^j -DNFB-hirudin was prepared essentially as described above, using

-^0-

instead of Η·£ SO^ in the Nakahara sulfation process [see related U.S. patent application ser. Nos. 164,178, 251,150, 280,618 and 514,756 and J.M. Maraganore et al., Anticoagulant Activity of Synthetic Hirudin Peptides, J. Biol. Cjem., 264, pp. 8692-98 (1989), all of which are incorporated herein by reference].

[ S]-sulfo-Tyr-DNFB-hirudin^^ was reacted with human s^-thrombin in the presence and absence of a 5 or 20-fold molar (above the thrombin concentration) excess of sulfo-Tyr^-N-acetyl-hirudin (prepared as in Example 1, with the addition of N-acetyl asparagine as the final step of the peptide synthesis). Incubation at room temperature for 18 hours followed by SDS-PAGE and autoradiography. The results (Fig. 1) show that the [^S]-labeled peptide is incorporated into the band representing thrombin and that the presence of cold, unlabeled hirudin peptide reduces the size of the formed

The covalent complex is reduced to < 10^. Thus, the reaction of sulfo-Tur-DNFB-hirudin thrombin results in a 1:1 stoichiometric binding of the hirudin peptide to the specific binding site.

In order to identify the site on thrombin where sulfo-Tyr-DNFB-hirudin binds, the thrombin/sulfo-Tyr-dinitrobenzyl (DNB)-hirudin complex (1.0 mg) was applied to a Sephadex G-50 column (1.5 x 45 cm), calibrated and washed with 7M urea, 20 mM Tris, pH 7.5. This chromatography separated any unreacted sulfo-Tyr-DNFB-hirudin. It was isolated

-Cpk containing thrombin/sulfo-Tyr-DNB-hirudin in the free volume fractions, pooled and reduced by addition of

10^dl ^-mercapto ethanol.

After reduction, we subjected the complex to S-carboxy methylation using s iodoacetic acid as described above [J. M. Maraganore et al., A New Class of Phospholipases Aj£ > with Lysine in Place of Aspartate-49, J. Biol. Chem. 259, pp. 13839-43 (1984C. The reduced S-alkylated protein was then dialyzed extensively against 5% acetic acid at room temperature. After dialysis, the complex was digested with pepsin (2% w/v) for 4 hours at 37°C. The peptide fragments of the reduced S-carboxy methylated thrombin (sulfo-Tyr-DNB-hirudin) were purified by reverse-phase HPLC using an Aquapore Rp-300 C& column (0.46 x 10 cm). The column was calibrated with 0.1% TFA in water and washed with an increasing gradient of 0.08536 TFA/70% acetonitrile (0-606) over 80 minutes at a flow rate of 1.0 ml/min. The effluent was monitored for absorbance as at 214 nm.

and at 310 nm. Fractions of 10 ml are automatically collected.

HPLC separation of the peptide fragments allows the separation of a single highest peak at 214 as well as at 310 nm absorbing material. Due to its far UV absorption, this fragment contains the linked sulfo-Tur _-ez -DNFB-hirudin^_

The fragment was then subjected to automated Edman degradation using an Applied Biosystems 470 A gas-phase sequencer equipped with a 900A data system. Phenylthiohydantoin (PTH) amino acids were analyzed on-line using an Applied Biosystems 120A PTH analyzer and a PTH-C^g column (2.1 x 220 mm).

The following is a table showing repeatable yields of sequencing analysis:

cycle amino acid pmola 1 Lys i- 858.5 2 Glu 629.2 5 Thr 557.6 4 Trp 276.5 5 Thr 289.0 6 Ala 474.4 7 Asn 569.0 8 Yes 490.7 9 Gly 296.1 10 (x) (-) 1 1 Gly 267.2 12 Gin 208.8 15 Pro 105.5 14 Ser 21 .6 15 Yes 25.5

Thrombin Active Site Regions

Semin. Thromb. Hemostasis

12, pp. 200-08 (1986)]. Peptide cleavage occurs at the Leu-Lys and Val-Leu bonds, according to the specificity of the enzyme.

-4 '3 ~

During sequence analysis, the amino acid _? corresponding to Lys-149 (cycle 10) could not be identified or quantified. This is most likely due to the derivatization (randomness) of the £ -NH^ group of this amino acid with the dinitrofluorobenzyl moiety of sulfo-Tur^ DNFB-hirudin.

Thus, Lys-149 is the largest site where sulfo-Tug ₽3

-DNFB-hirudin^ reacts with d, -thrombin.

EXAMPLE 3

Model of a thrombin inhibitor capable of blocking the catalytic site and binding to the anion-binding exosite

Carboxy-terminal hirudin peptides effectively block thrombin-catalyzed fibrinogen hydrolysis, but not chromogenic substrate hydrolysis [J.M. Maraganore et al., J. Biol. Chem., 264, pp. 8692-98 (1989)]. Furthermore, hirudin peptides do not neutralize thrombin-catalyzed activation of Factors V and VIII [J.W. Fenton, II, et al., Hirudin Inhibition by Thrombin, Angio. Archiv. Biol., 18, p. 27 (1989)].

Hirudin peptides such as sulfo-Tyr^ -N-acetyl-hirudin^^/ express a strong inhibitory effect against thrombin-induced platelet activation in vitro [J.A. Jakubowsky and J.M. Maraganore, Inhibition of Thrombin-Induced Platelet Activi

ties by a Synthetic 12 Amino Acid Residue Sulfated Peptide (Hirugen), Blood, p. 1213 (1989)]. However, a thrombin inhibitor capable of blocking the active site may be needed to inhibit planned thrombosis in vivo if the activation of Factors V and VIII are rate-limiting steps. This conclusion is based on the results obtained with the irreversible thrombin inhibitor (D-Phe)-Pro-Arg-CHCl [S. R. Hanson and L>A Harker, Interruption of Acute PlateletDependent Thrombosis by the Synthetic Antithrombin D-Phenylalanyl-L-Arginyl Chloromethyl Ketone, Proc. Natl. Acad. Sci. USA, 85, pp. 3184-88 (1988)] and other reversible thrombin inhibitors [J.F. Eidt et al., Thrombin is an Important Mediator of Platelet Aggregation in Stenosed Canine Coronary Arteries with Endothelial Injury, J. Clin. Invest., 84, pp. 18-27 (1989)].

Using the above information that the KH-terminus of the chirodin peptides is proximal to Lys-149, we used a three-dimensional model of thrombin (Fig. 2), [B. Furie,, et al., Computer-Generated Models of Blood Coagulation Factor Xa, Factor IXa, and Thrombin Based Upon Structural Homology with Other Serine Protease, J. Biol. Chem. 257, pp. 3875-82 (1982)] to design an agent ₅ that: 1 \ binds to the anion-binding exosite of thrombin; 2\ is capable of blocking the active site pocket of thrombin and inhibiting the function of the catalytic residues contained therein.

We determined that the minimum distance of £ -NH^ on l ·°

Lys-149 from the 7? -hydroxyl of Ser-195 is 18-20 A. Based-4.5on a length of

A/ amino acid residue, we calculated that at least about 4-7 amino acids would be needed to bind the hirudin peptide, as well as sulfo-Tug.,

-hirudin^ ₃ _ , to a domain containing the active site structure of the inhibitor. The linker composition is indicated as glycine. Glycine was chosen to design the highest mobility of the linker for these preliminary studies. It should be understood, however, that other* stronger polymeric linkers may also be used.

We chose the sequence (D-Phe)-Pro-Arg-Pro as the active site inhibitor because thrombin exhibits specificity towards

Arg as the P^ amino acid in substrate cleavage. Pro following Arg results in a bond that is cleaved very slowly by thrombin. We denote alternating peptides by replacing Pro (following P^ or N-methyl-alanine amino acid or by

Arg) with sarkosyl chemical reduction of Arg-Gly cleaved (sissified) bond.

EXAMPLE 4

Synthesis of Hirulog-8

The formula of Chirulog-8 is: H-(D-Phe)-Pro-Arg-Pro-(Gly )^_Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH.

We synthesized Hirulog-8 by conventional solid-phase peptide synthesis using an Applied Biosystems 43OA Peptide Synthesizer. This peptide was synthesized using BOC-Lleucine-O-divinyl-benzene resin. Additionally, t-BOC-amino acids were used; (Peninsula Laboratories, Belmont, CA-46-

lif.), including BOC-O-2,6-dichlorobenzyl tyrosine, BOC-L-glutamate (7-benzyl ester), BOC-L-proline, BOC-L-isoleucine, BOC-L-phenylalanine, BOC-L-aspartate (B-benzyl ester), BOC-glycine, BOC-L-asparagine, BOC-D-phenylalanine, and BOC-D-arginine. In order to obtain higher yields in the synthesis, the (Gly)^. linker segment was attached in two cycles by manual addition of BOC-glycylglycine (Beckman Biosciences, Inc., Philadelphia, Pa.). After completion of the synthesis, the peptide was completely deprotected and separated from the divenylbenzene resin by treatment with anhydrous HF: p-cresol: ethylmethyl sulfate (10:1:1, v/v/v). After separation from the resin, the pep

The solution is lyophilized to dryness.

Crude Hirulog-8 was purified by reverse-phase HPLC using an Applied Biosystems 151A liquid chromatography system and a Vydac column (2.2 x 25 cm). The column was calibrated with 0.1% TFA/water and washed with a linear gradient of increasing acetonitrile concentration from 0 to 80% over 45 min in 0.1% TFA at a flow rate of 4.0 mL/min. The effluent was monitored for absorbance at 229 nm and fractions were collected manually. 25-30 mg of crude Hirulog-8 was purified by HPLC to yield 15-20 mg of pure peptide.

We confirmed the structure of purified Hirulog-8 by amino acid and sequence analysis. Amino acid hydrolysates were prepared by treating the peptide with 6N HCl, in vacuo, at 110°C for 24 hours. We then analyzed the hydrolysates by ion exchange chromatography and subsequent ninhydrin derivatization/determination using a Beckman 6500 automated analyzer. We performed sequence analysis using automated Edman degradation with an Applied Biosystems 470A gas-phase sequencer equipped with a Model 900A data system. Phenylthiohydantoin (PTH) amino acids were analyzed on-line using an _Applied Biosystems 120A pTH analyzer and a PTH-C column (2.1 x 220 mm).

EXAMPLE 5

Synthesis of Hirulog-9

The formula of Hirulog-9 is: H-(D-Phe)-Pro-Arg-D-Pro-(Gly)^

-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. We synthesized this peptide in the same manner as described in Example 4, using 1 BOC-D-proline (Peninsula Laboratories) at cycle 15 instead of BOC-L-proline. Purification and characterization were performed as described in Example 4.

EXAMPLE 6

Synthesis of Hirulog-10

The formula of Hirulog-10 is: H-(D-Phe)-Pro-Arg-Sar-(Gly)-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. The peptide was synthesized as in Example 4 using

BOC-sarcosine (Sigma Chemical Co., St. Louis, Mo.) at cycle 16. Purification and characterization were performed as described in Example 4.

EXAMPLE 7

Synthesis of Hirulog-11

The formula of Hirulog-11 is: H-(D-Phe)-Pro-Arg-Pro-(Gly)4.

-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-(5,5-diiodoTyr)Leu-OH. This peptide was synthesized as in Example 4 using BOC-3,5-diiodo-L-tyrosine (Sigma) in run 2. Purification and characterization were performed as described in Example 4.

EXAMPLE 8

Synthesis of Hirulog-12

The formula of Hirulog-12 is: H-(D-Phe)-Pro-Arg-Pro-(Gly)-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(OSO j )-Leu-OH. This peptide was synthesized by reacting 1.0 mg of Hirulog-8 in dimethylformamide (80 wt. L) with dicyclohexylcarbodiimide solution (1.25 g/mL, 0.007 mL) and concentrated sulfuric acid (0.5 L) at 0°C for 10 min. The reaction was quenched by the addition of water (1.0 mL).

The reaction mixture can be subjected to reverse-phase HPLC using an Applied Biosystems 150A liquid chromatography system and an Aquapore RP-300 Cg (0.46 x 10 cm). The column is calibrated with solution A (0.1% TFA/water) and washed with increasing concentrations of solution B (0.085% TFA/70% acetonitrile) from 0 to 50% over 45 minutes at a flow rate of 1.0 mL/min.

The effluent was examined for absorbance at 214 nm.

EXAMPLE 9

Inhibition of thrombin-catalyzed hydrolysis of a synthetic p-nitroanilide substrate by

Chirologist 8

We then analyzed the effects of Hirulog-8 on

-4$human χ. thrombin-catalyzed hydrolysis of Spectrozyme TH (tosyl-C1y-Pr0-Ar-p-nitroanilide; American Diagnostica, New York, NY). Specifically, we measured the initial rates in the presence or absence of Hirulog-8 over a range of substrate concentrations from 2.2 to 22 μM. The thrombin-catalyzed rate was measured on a Cary 19 spectrophotometer at 405 nm and recorded continuously as a function of

The kinetics were performed at room temperature (25 ± 1°C) in 0.05 M Tris, pH 7.5, 0.1 M NaCl buffer.

For a typical enzymatic reaction, 1.0 ml of buffer is added to the sample and control cuvettes. Thrombin (3.2 x 10 M, final concentration) and Hirulog-8 (0-4 x 10 M) are added to the sample cuvette before the addition of Spectrozyme TH (2.2-22 µM). Immediately after the addition of the substrate, the contents of the sample cuvette are mixed using a plastic pipette. The reaction is monitored spectrophotometrically for 5-15 minutes.

The initial values of the speeds at each con-

The substrate concentration is expressed as moles of Spectrozyme TH hydrolyzed/sec/mole of thrombin. This was determined during the initial linear phase of the reaction (3=15% total substrate hydrolyzate) by measuring the slope of the hydrolysis reaction. Accordingly, Lineweaver-Burke plots are constructed by plotting the reciprocal of the initial rate against the reciprocal of the substrate concentration. The results show that the -thrombin-catalyzed hydrolysis of Spectrozyme TH has Υ^,^χ· = 17 moles of hydrolyzate

-50s/sec/mol thrombin and K at 1.19 x 10 M. Fig. 3 A and B show that increasing concentrations of Hirulog-8 lead to a significant dose-dependent increase in K _m , with a negligible increase in for Spectrozyme TH hydrolysis. Therefore, inhibition of the thrombin-catalyzed reaction by Hirulog-8 is mediated by mixed competitive/non-competitive components with respect to Spectrozyme TH hydrolysis.

The Ki of Hirulog-8 for -thrombin was determined using the equation:

where

is the slope of the thrombin-catalyzed reaction in the presence of Hirulog-8: [Hirulog-8] is the molar concentration of the peptide;

and IHI yIRDY

is the thrombin-catalyzed reaction, in the presence of inhibitor; Ki is the molar inhibition constant for Hirulog-8 with

-5ί-

human thrombin. The Ki for Hirulog-8 was calculated to be 1.95 ± 0.11 x 10 M.

EXAMPLE 10

Specificity of Hirulog-8 to the hirudin-peptide binding site and the active site of human thrombin

Hirulog-8 is reported to be an analog that binds human thrombin through its hirudin-peptide binding site, thereby blocking the thrombin catalytic site. We tested the catalytic ability of Hirulog-8 to perform these functions through various assays described below.

The kinetics of Hirulog-8 inhibition of human thrombin were investigated primarily as described above, using human β-thrombin as an example. The β-thrombin catalyzed reaction against Spectrozyme TH showed a ζ Ί of 14 mol hydrolyzed/sec/mol thrombin and a K = 1.1 x 10 M. These results confirm that β-thrombin, the proteolytic form of thrombin, exhibits almost complete catalytic activity, although this form lacks significant masking (enveloping) activity.

[S.D. Lewis et al., Catalytic Competence of Human - and y^-Thrombins in the Activation of Factor Xiii, Biochemistry,

26, pp. 7597-7603 (1987)]. The inhibition of -thrombin by Hi was investigated above the level of peptide concentrations from rulog-8.

2.7 x 1O'^&

-8 exhibits an increasing Ki by 5 orders of magnitude relative to <£— g>

to 6.8 x 10 M. As shown below, Hirulog thrombin. This absence of -thrombin is due to an intact anion-binding exosite (AVE) in the high Ki

-52thrombin [J.W. Fenton, II, et

-Thrombin et al., Anion-Binding Exosite of

Fibtin(ogen) Recognition, (1988)]. ^^-thrombin is formed by proteolysis of the B-chain of ω-thrombin at LysHuman

Biochemistry, 27, pp. 7106-12

149 and Arg-78.

The inhibition of human c>L-thrombin by Hirulog-8 is known

significantly decreased in the presence of sulfo-Tyr^-N-acetyl-hyru-έ. -s' din^ at concentrations from 2.6 x 10 M to 129 x 10 M.

This is because sulfo-Tug ₆ ^ N-acetyl hirudin^ ₃ _ competes with Hirulog-8 for binding to the AVE of thrombin.

This was also demonstrated by adding phenylmethylsulfonyl-β-thrombin (PMS-β-thrombin; 18 nM, final) to the reactions of Hirulog-8 with human β-thrombin. The addition of this modified thrombin resulted in a significant reduction in the ability of Hirulog-8 to inhibit β-thrombin.

PMS-α-thrombin possesses intact AVE but is covalently branched at its active site. This modified thrombin releases Hirulog-8 into the reaction mixture and therefore reduces the amount of peptide capable of inhibiting intact, catalytically active human α-thrombin.

We also investigated the effect of salt concentrations on the Ki of Hirulog-8 for thrombin, as described above in Example 9. We measured the Ki, in the presence or absence of Hirulog-8 (11.5 x 10 M), in buffers containing

0.1, 0.25 and 0.5 M NaCl. As shown in the table below, the inhibition of α-thrombin by Hirulog-8 increases at lower salt concentrations. This result confirms that the interaction of the highly anionic hirudin peptide portion of Hirulog-8 with the positively charged site surrounding Lys149 of thrombin is essential for Hirulog-8 inhibition of thrombin-catalyzed hydrolysis by Specrozyme HT.

Enzyme Terms and Conditions Hirulog-8, Ki, nM human^/ - 0.05M Tris, pH 7.5 1 .95 thrombin 0.1M NaCl (buffer) human 7^- buffer 1 .080 thrombin human/ ” buffer + 2.bu< M 25.5 thrombin sulfo-Thug^-N-acetyl human ok - hirudin^-s^ buffer + 1 2.9^ M > 2.000 thrombin sulfo-T3-N-acetyl hirudin 5-3- 6 f human*/ - buffer + PMS- 9.90 thrombin -thrombin human - 0.05M Tris, pH 7.5 2.09 thrombin 0.025 M NaCl human/ - 0.05 M Tris, pH 7.5 3.72 thrombin 0.5 M NaCl

-EXAMPLE 1 1

Anticoagulant activity of Hirulog-8: Comparison with hirudin and sulfo-Tyr^j -N-acetyl-hirudin^

We studied the anticoagulant activity of Hirulog-8, pooled normal blood plasma (George King Biomedical, Overland Park, Kan.), and Coag-A-Mate XC instruments (General Diagnostics, Organon Technica, Oklahoma City, Okla.). Activity was assayed using an activated partial thromboplastin time (APTT) assay with CaCl2 and phospholipid solutions obtained from the manufacturer. Hirulog-8, hirudin, or sulfo-Tyr-3-N-acetyl-hirudin were then added to the APTT assay wells at final concentrations of 10 to 32,300 ng/ml in a total volume of 25 µl before addition of 100 µl of plasma.

The control APTT (no inhibitor) was 29.6 sec (mean, n=8, SEM<0.5%). Fig. 4 shows the results of these dose-response studies. Hirulog-8 is 2 to 3 times more potent than hirudin and 100 to 150 times more potent than sulfo-Tyr^j -N-acetyl-hirudin^. Hirulog-8 and hirudin increase plasma APTT to values that are too high to be measured. This is in contrast to sulfo-Tyr^j -N-acetyl-hirudin, which shows a saturable dose-response in APTT to 200-25056 of the control valves [J.M. Maraganore et al., «J. Bio. Chem. 264, pp. 8692-98, (1989)]. This result indicates that Hirulog-8 can block the active site of thrombin in plasma, as well as in vitro in chromogenic assays, in a manner similar to hirudin.

-5S’

EXAMPLE 12

Inhibition of thrombin-induced plaque activation by Hirulog-8

Thrombin-induced platelet activation assays were performed at 37°C using a Biodata PAP planar aggregometer. Platelet-rich plasma (PRP) was obtained from a normal, healthy volunteer who had not taken any drugs known to alter platelet function for at least one week prior to the assay. PRP was prepared as described by J.A. Jakubovski et al., Modification of Human Platelet by a Diet Enriched in Saturated or Polyunsaturated Fat, Atherosclerosis, 31, pp. 335-44 (1978). Various concentrations of Hirulog-8 (0-500 ng/ml in 50 µl water) were added to 0.4 ml of prewarmed (37°C) PRP. One minute later, human oZ-thrombin was added to the platelet suspension to a final concentration of 0.2, 0.25 or 0.5 units/ml of total assay volume.

Aggregation was assayed as an increase in light transmission 5 minutes after the addition of thrombin. We then calculated % inhibition as (% aggregation πρρβΎν ) / aggregation control ) x 100. This assay demonstrates that Hirulog-8 blocks thrombin-induced plaque activation in vitro.

EXAMPLE 13

Use of Hirulog-8 in thrombus imaging

Hirulog-8 is modified by covalent attachment of an I-containing chemical group. Specifically, Hirulog-8 (as prepared in Example 4) is reacted with ⁴ ^I-Bolton Hunter's reagent (New England Nuclear, Boston, Mass.) in 0.1 M sodium borate, pH 9.0. The labeled c I molecule (with a specific activity >5 p. Ci/yU g) is desalted on a Biogel P2 column calibrated with phosphate buffer in saline.

Ex vivo modeling of experimental thrombi was performed primarily as described by T.M. Palabrica et al., Thrombus Imaging in a Primate Model with Antibodies Specific for an External Membtane .JProtein of Activated Plate lets, Proc. Natl. Acad. Sci. USA. 86, pp. 1036-40 (1989). Specifically, modeling was performed in baboons using an external Ticoflex shunt between the femoral artery and femoral vein. An experimental thrombus was formed by placing a segment of precoated Dacron graft into the shunt. I-labeled thrombin inhibitor was injected into the venous portion of the Ticoflex shunt. A series of 0.5 to 1 hour images were taken using an Ohio

Nuclear Series 100 Gamma Camera with PDP-11/34 computer. Kinetics of I-thrombin inhibitor, increased by graft-

The donation and pooled blood results from the radionuclide images obtained in this way.

The same technique can be used to obtain ex vivo images of deep vein thrombi caused by stasis in the femoral vein of baboons. Because Hirulog-8 binds to thrombin with high specificity, the use of this molecule allows for precise ex vivo imaging of thrombi. Also, the small size of Hirulog-8, in contrast to native hirudin or thrombin antibodies,

-bl

provide the ability for the radiolabeled thrombin inhibitor to image plaque-bound thrombin and meisothrombin, as well as thrombin contained in the fibrin clot.

EXAMPLE 14

Antimetastatic activity of thrombin inhibitors

The antimetastatic activity of the thrombin inhibitors of the present invention, preferably Hirulog-8, was tested using T241 sarcoma cells [L.A. Liotta et al., Nature, 284, pp. 67-68 (1980)] and syngeneic C57BL/6 mice (Jackson Laboratory, Bar Harbor Me.). Mice were injected either intravenously or subcutaneously with 0-250 g/kg Hirulog-8, prepared as in Example 4, followed by injection with 10 -10® T241 tumor cells. After 15 days, the animal was sacrificed and lung tumor colonies were measured. The antimetastatic activity of Hirulog-8 was measured as the percentage reduction in tumor colonies compared to a placebo-treated control mouse. Hirulog-8 demonstrated antimetastatic activity in this assay.

EXAMPLE 15

I

Inhibition of Endothelial Cells by Thrombin Inhibitor

The ability of the thrombin inhibitors of the present invention to prevent thrombin-induced synthesis of plaque activating factor (PAF) was investigated by

cultured human umbilical vein endothelial cells (HUVECs) were used. HUVECs were isolated from human umbilical cord by collagenase digestion according to the established procedure [M.A. Gimborn, Jr., Culture of Vascular Endothelium, Prog. Hemost. Thromb., 3, pp. 1-28 (1976)]. HUVECs were cultured to confluence in a 96-well microtiter plate in the presence of [ 3 H]acetate. Cells cultured in this manner produced [ ³ H]acetyl-PAF, which could be quantified by extraction from HUVEC membrane phospholipids.

Hirulog-8 (0-1 μg/ml) was added to [3H]-acetate-loaded HUVECs 1 min before the addition of thrombin (final concentration 1000 IU/ml). The cells were incubated for 5 min and the supernatant was removed. Medium containing 0.1% gelatin, 50 mM acetic acid in methanol (2:1 v/v) was then added to the KUVECs. PAF was then extracted and quantified using conventional techniques [T.M. McIntyre et al., Cultured Endothelial Cells

Synthesize Both Platelet-Activating Factor and Prostacyclin in Response to Histamine, Bradykinin and Adenosine Triphosphate, J. Clin. Invest., 76, pp. 271-80 (1985)]. IC values were then calculated. Hirulog-8 inhibited the synthesis of PAF by HUVECs in this assay.

The effect of Hirulog-8 on thrombin-induced polymorphonuclear leukocyte (PMN) adhesion to HUVECs can be demonstrated as follows. HUVECs were cultured to confluence in MEM containing 1% fetal calf serum in 24-well group plates. The medium was then removed, the cells were washed twice with fresh serum-free medium and incubated at <?

The same medium was incubated for 10-50 minutes at 57°C until serum products were separated. PMNs (2.5 x 10 in 1 ml), which had been pre-warmed to 37°C, were then added to each well. The PMNs were allowed to settle onto the HUVEC monolayer for 2 minutes. Hirulog-8 (5 μg/ml) or saline was added to each well immediately after the addition of -thrombin (0.1 or 1 U/ml).

Io

Cells were incubated for 5 minutes at 37°C, washed twice, and examined by phase-contrast microscopy. Adherent PMNs were counted directly. Samples incubated with Hirulog-8 had significantly fewer adherent PMNs than those treated with saline.

EXAMPLE 16

Synthesis of Hirulog-13

The formula of Hirulog-13 is as follows: H-(D-Phe)-Pro-ArgPro-(Gly)-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-LeuOH. We synthesized, purified, and characterized this peptide essentially as described in Example 4, except that only one BOC-glycylglycine cycle was used to produce the diglycine segment.

EXAMPLE 17

Synthesis of Hirulog-14

The formula of Hirulog-14 is as follows: H-(D-Phe)-Arg-Pro(Gly )^. -Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH.

- έ/ύ Hirulog-14 was synthesized, purified and characterized using the methods described in Example 4, except that one cycle of BOC-glycine addition was used after the two cycles of BOC-glycineglycine addition to produce the pentaglycine segment.

EXAMPLE 18

Synthesis of Hirulog—15

The formula of Hirulog-15 is as follows: H-(D-Phe)-Pro-ArgPro-(Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-LeuOH. Hirulog-15 was synthesized, purified, and characterized using the methods described in Example 4, except that three cycles of BOC-glycylglycine addition were used to prepare the hexaglycine segment.

EXAMPLE 19

Synthesis of Hirulog-16

The formula of Hirulog-16 is as follows: H-(D-Phe)-Pro-ArgPro-(Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-LeuOH. Hirulog-16 was prepared, purified and characterized as described in Example 4, except that four cycles of BOC-glycylglycine addition were used to prepare the octaglycine segment.

EXAMPLE 20

Synthesis of Hirulog-17

The formula of Hirulog-17 is as follows: H-(D-Phe)-Pro-Arg-Ci'

Pro-Gly-Gly-Glu-Gly-His-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-ProGlu-Glu-Tyr-Leu-OH. Hirulog-17 was synthesized essentially as described in Example 4, except that Gly-Gly-GluGly-His-Gly replaced the Gly^ segment present in Hirulog-8. This sequence was added to the growing peptide chain by the sequential addition of BOC-glycine, BOC-L-histidine, BOC-glycine, BOC-L-glutamate, and BOC-glycylglycine during cycles 13-17 of the synthesis. Purification and characterization were performed as described in Example 4.

EXAMPLE 21

Synthesis of Chirulog-18a, -18b and -18c

The formula of Hirulog-18a is as follows: H-(D-Phe)-Pro-(homoarginine)-(Gly)^--Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-GluTyr-Leu-OH. The formula of Hirulog-18b is as follows: H-(D-Phe)Pro-(-homoarginine)-(Gly)-Asn-Gly-Asp-Phe-Glu-Glu-Ile-ProGlu-Glu-Tyr-Leu-OH. The formula of Hirulog-18c is as follows: H(D-Phe )-Pro-(-homoarginine)-Val-(Gly)y-Asn-Gly-Asp-Phe-GluGlu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. Hirulog-18a was synthesized using a mixed homogeneous/solid-phase procedure. Residues 5-20 were prepared by solid-phase synthesis as described in Examples 4 and 17. The resulting resin-bound intermediate was reacted with a BOC-β-homoarginine-Gly protected intermediate, which was synthesized by a multi-step reaction scheme depicted below and described immediately thereafter.

CA/ / )= N

Η- N \

'vos

H -N

With 0CH ₃

(2l>z

) \CO2 Η wax

chem, Torrance, CA) and 2.1 mL (19.1 mmol) N-methylmorpholine (Aldrich, Milwaukee, Wis.) in 100 mL anhydrous tetrahydrofuran (THF) under argon for 5 min at room temperature.

The solution was then cooled to -15°C and 2.8 ml (21.6 mmol) of isobutyl chloroformate (Aldrich) was added.

We continue to stir the reaction mixture at -15°C for 5 minutes, then filter it through a pad of Celite/MgSO^. Then we add the filtrate

to an ice-cold ethereal solution of diazomethane (150 mM, obtained from

32.4 g Diazald; Aldrich). The solution was stirred and allowed to gradually reach ambient temperature overnight. The solvent was then removed in vacuo and the residue was dissolved in 200 mL chloroform. The organic solution was then washed successively with 200 mL saturated NaHCO 3 , followed by 200 mL saturated NaCl, dried over anhydrous MgSO 4 , and concentrated again to an oily residue.

The residue was then purified by flash chromatography on a 4 x 17 cm silica gel column using a step gradient of acetone in chloroform (10% acetone in 2 L chloroform followed by 20% acetone in 3 L chloroform). The fractions containing the desired product were combined and dried to dryness. The diazomethyl ketone product was purified as a pale yellow foam (6.54 g).

N -BOC-N^-Tos-t-homoarginine methyl ester

We dissolve the previously prepared diazomethyl ketone in 100

-64ml of anhydrous methanol and pass the resulting solution under argon while a solution of silver benzoate catalyst (165 mg in 400 ml of triethylamine) is added dropwise. After 50 minutes,

The refluxing solution was cooled to room temperature, slurried with Norit and filtered through Celite. The solvent was then removed in vacuo and the oily residue was purified by flash chromatography on silica gel. Elution was carried out with 4 L of 10% acetone in chloroform. The desired product, γ-homoarginine methyl ester, was purified in this manner as a light yellowish-brown foam (6.45 g).

Everything

[ -BOC-N^-Tos- F-homoarginine | from the above methyl ester is dissolved in 100 ml of methanol, then it is allowed to react with a solution of LiOH (1.48 g in 50 ml of water) overnight at room temperature under argon and with constant stirring. The methanol is removed under vacuum, the precipitate is dissolved in water and washed in ethyl acetate. Then a saturated solution of citric acid is added until the solution reaches pH 4.

Then we extract the resulting carboxylic acid with ethyl acetate.

The extraction was repeated at pH 5 and the combined organic phases were dried over MgSO4 and concentrated in vacuo. The resulting crude acid was collected as a white foam (4.9 g). The acid was then purified on Vydac C4.

reverse-phase HPLC column as described in Example 4, except that the effluent was monitored at 214 nm.

—&5After lyophilization

Tos-^-homoarginine, of the desired fractions, the product, is obtained as a white amorphous

N-BOC-N solid phase.

Sample of N -BOC-N<

I^Tos-y^-homoarginine is hydrolyzed in HF assay.

used as a standard for

The retention time of β-homoarginine is identical to that of the amino acid arginine, but the peak intensity is significantly lower than expected.

N -BOC-N^-Tos-p ⁵ -homoargininylglycine benzyl ester

Next, 4.06 g (9.2 mmol) of the above carboxylic acid was combined with 2.04 mL of N-methylmorpholine in 25 mL of anhydrous THF. The mixture was stirred under argon at -5°C. A cooled solution of isobutyl chloroformate (2.4 mL in 25 mL of THF) was then added dropwise to the solution over 10 minutes. After addition, the reaction mixture was stirred for 1 h.

minutes at -5 C. For Hirulog-18a, a solution of glycine benzyl ester (4.9 g in 40 mL THF; 27.6 mmol) was then added and the reaction mixture was allowed to reach room temperature. The solvent was then removed in vacuo and the resulting precipitate was dissolved in 100 mL ethyl acetate. The solution was extracted successively with 100 mL saturated NaHCO3 and saturated NaCl, dried over MgSO4, and concentrated in vacuo. The resulting crude dipeptide ester was purified on a 4 x 20 cm silica gel column with a methanol step gradient in chloroform containing 10 drops of NH4OH per 100 mL (2 L 1% methanol in chloroform, followed by 3 L 2% methanol in chloroform). Fractions (25 mL) were collected,

The TLC and product extracts were combined and the solvent removed in vacuo. The resulting product N-BOC-N-TosP-homoarginineglycine benzyl ester was isolated as a white foam (3.9 g).

For Hirulog-18b and -18c the above reaction is identical, except for the following modifications: for Hirulog-18b the glycine benzyl ester is replaced with proline benzyl ester and the reaction is carried out at 1.8 mmol scale; for Hirulog-18c the glycine benzyl ester is replaced with valine benzyl ester and the reaction is carried out at 3.0 mmol scale.

-homoargininylglycine

The above benzyl ester was dissolved in 50 mL of methanol and hydrogenated at atmospheric pressure over 1.0 g of 10% palladium/carbon for 17 hours. The resulting solution was filtered through Calite and the solvent was removed in vacuo. The reaction yield was 2.9 g of crude N-BOC-N-Tos--xomoargininylglycine, which was purified on a Vydac C/ρ HPLC column as described above.

The above N-BOC-N-Tos--homoargininylglycine (1.02 g) was dissolved in 1 ml of anhydrous DMF and cooled in an ice bath. Then, 5 ml of 0.5M hydroxybenztriazole in DMF (Applied Biosystems Inc, Foster City. Calif.) and 5.5 ml of 0.5M dicyclohexylcarbodiimide in CH2Cl2 (Applied Biosystems) were added sequentially to this solution. After 1 hour, the cold suspension of the symmetric anhydride of the dipeptide unit rapidly

- 67 ~ filtered through a plug of glass wool to remove dicyclohexyl urea.

Meanwhile, a suspension of N-BOC-(Gly-Asn-Gly-AspPhe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-O-PAM (0.2 mmol in CH2Cl2) was activated by standard peptide synthesis methods. A Kaiser test on the resulting product indicated a free terminal amino group.

The activated -homoarginylglycine dipeptide is then added to the resin-bound hexadecapeptide.

The resulting octadecapeptide was then coupled sequentially to N-BOC-Pro and N-BOC-(D-Phe) using a standard coupling procedure. The resulting peptide Hirulog-18a was purified and characterized as described in Example 4.

A similar scheme is used in the synthesis of Hirulog18b and Hirulog-18c.

EXAMPLE 22 •s

Synthesis of Hirulog-19

The formula of Hirulog-19 is as follows: H-(D-Phe)-Pro-Arg[psiCH-NH]-(Gly)^-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-GluTyr-Leu-OH. Residues 4-20 of this peptide were assembled by solid-state peptide synthetic procedures as described in Example 4. The next added residue -BOC-N^-tosyl-argininal was prepared as depicted and described below.

-

Supreme Court of the Russian Federation

N -BOC-N- ⁷ -Tos-argininal

N-BOC-N7-Tos-arginine (Bachem Inc.; 10 g) was added ι o

to 80 ml of anhydrous THF and the suspension is cooled to 0 °

C. Then add 1,1'-carbonyldiimidazole (Aldrich; 3.61 g) all at once and continue stirring for 20 minutes. The resulting clear solution is partially immersed in a dry ice/acetone bath to maintain a temperature of -20 o

to -30 C during the dropwise addition of a suspension of lithium aluminum hydride (Aldrich; 1.8 g in 80 ml THF) over 45 min with constant stirring. The reaction mixture was

Stir for an additional 30 minutes at -20° C. and then quench by dropwise addition of 63 ml of 2N HCl at -10° C. Filter the resulting solution through a medium glass funnel and concentrate the resulting filtrate in vacuo.

The resulting crude aldehyde, collected as a white foam, (11.5

d), was suspended in 100 ml chloroform, washed with water (2 x 50 ml) and the organic layer was then dried over sodium sulfate and concentrated in vacuo. The crude aldehyde (7.7 g) was dissolved in 100 ml chloroform and purified by flash chromatography over a 5 x 20 cm flash column containing 350 ml silica gel (Merck Grade 60, 230-400 holes, 60 A). Elution was carried out using a step gradient of 0.5% methanol in 500 ml chloroform, 1% methanol in 1 L chloroform and 1.5% methanol in 1 L chloroform. This procedure gave a yield of

8.9 g N -BOC-N^-Tos-argininal.

N^-BOC-N^-Tos-aprHHHHanbT (258 mg) was then added to the resin-bound (Gly )^- -Asn-Gly-Asp-Phe-Glu-GluIle-Pro-Glu-Glu-Tyr-Leu-O-PAM under solid-phase reductive alkylation conditions (40 mg sodium cyanoborohydride for 24 hours) using the method of D.H. Coy et al., Solid-Phase Synthesis of Peptides In Peptides, Vol. 8 pp. 119-121 (1978). Following the reaction of the resin-bound

The peptide resin with the protected argeninal, peptide synthesis is completed with a BOC-proline incorporation cycle and a BOC-(D-phenylalanine) incorporation cycle. After completion of synthesis, Hirulog19 is released from the protector and removed from the resin, as described in Example 4.

Hirulog-19 was purified by reverse-phase HPLC using an Applied Biosystems 151A liquid chromatography system and an Aquapore C& column (10 x 22 cm). The column was calibrated with one part 70% acetonitrile/50% water containing 0.85% TFA (buffer B) and 4 parts water containing 1% TFA (buffer A). The column was washed with a linear gradient of increasing concentration of buffer B (20-50%) over 120 minutes at a flow rate of 4.0 ml/minute. The effluent was monitored for absorbance at 214 nm and fractions were collected manually. Subsequent purification was performed using CINT£5 HA

Mr. Et

M

-7/ ~ performed under isocratic conditions using 20% buffer B/80% buffer A.

EXAMPLE 23

Synthesis of Hirulog-21

The formula of Hirulog-21 is as follows: H-(D-Phe)-Pro-ArgPro-(Gly)4. -Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu(Gly)^ -Lys-OH. Hirulog-21 was synthesized using the methods described in Example

4, using the appropriate BOC amino acids.

The purification and characterization of HiruLog-21 was carried out according to the methods described in Example 4.

EXAMPLE 24

Synthesis of Hirulog-25

The formula of Hirulog-25 is as follows: H-(D-Phe)-Pr-[4-argininyl-2,2-difluoro]malonylglycyl-(Gly)-[2,3-Asn-Gly-AspPhe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. The hexadecapeptide (Gly)-[2,3-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, was synthesized as described above and allowed to bind to the resin. The next residue (3-argininyl-2,2-difluoro)malonylglycine was synthesized according to the scheme described and illustrated below (p. 71).

1-[(2'-carboethoxy 1', 1'-difluoro)θηπ]Ν -BOC-N benzyl-N ° ^GI -Cbgornitinol

A solution of 3.1 g (7.1 mmol) N -BOC- N -benzyl- N , _g C6gornitinol [F. Salituro et al. , ·'Inhibition of Aspartic Proteinases by Peptides Containing Lysine and Ornithine Side 'BiChain Analogues of Statins, J. Med. Chem., 30, pp. 286-95 (1987)], and 1.56 mL (9.23 mmol) ethyl bromodifluoroacetate in anhydrous 15 mL THF was added over 90 min under reflux

to the suspension of 786 mg Zn powder (Fluka) in 15 ml THF under argon. After 4 hours in reflux and 2 hours at room temperature- perature, mix cooled and partitioned between ethyl acetate

and saturated NaCl/KHSO4 (200 mL each). The organic phase was isolated, dried over MgSO4, and concentrated in vacuo. The resulting oily residue was purified on silica gel using CHCl4 : methanol (90 : 10) plus 100 drops/L NH4.

OH as eluent.

-[(2’-carboethoxy 1’,

1'-difluoro)ethyl]I

-BOC ornithinol tetrabutyldimethylsilyl ether

The resulting compound 1-[(2'-carboethoxy 1', 1'-difluoro )ethyl]N-[(2'-carboethoxy 1', ^1' -difluoro )ethyl]N-[(2'-carboethoxy 1', 1'-difluoro )ethyl]N-[(2'-carboethoxy 1', 1'-difluoro ) ^ethyl]N-[ (2'-carboethoxy 1', 1'-difluoro )ethyl]N-[(2', 1

the cedure of E.J. Corey et al., Protection of Hydroxyl Groups as tertButyldimethylsilyl Derivatives, J. Amer. Chem. Soc.,

94, pp. 6190-91, (1972). The orthogonally protected amine was then dissolved in methanol and hydrogenated over Pd(OH) at 30 psi for 18 hours. The catalyst was then removed by filtration and the filtrate was concentrated in vacuo to give 1-[(2'-carboethoxy 1', 1'-difluoro)ethyl]-BOC-ornithinol tert-Butyldimethylsilyl ether.

- [(2'-carboethoxy 1', 1'-difluoro)ethyl]-BOC-N^Tos argininol-tert-Butyldimethylsilyl ether

The compound prepared above was then reacted with 6.8 equivalents each of 1-guanyl-3,5-dimethylpyrazole and triethylamine in <2% water at 105°C for 24 hours. The mixture was then lyophilized and the residue was subjected to preparative HPLC as described in Example 4. The fractions containing the desired guanidine compound (tested by TLC) were combined and dried in vacuo. The residue was dissolved in H2O:acetone (1:4), cooled in an ice bath and adjusted to pH 12 with 50% w/v NaOH. To this solution was added a solution of 5 equivalents of para toluene sulfonyl chloride in acetone over 60 minutes until the pH was maintained at 11-12 with NaOH. The solution was allowed to reach room temperature and stirred overnight. The acetone was then removed in vacuo and the remaining aqueous solution was washed with ether. The ether layer was separated and re-extracted with saturated NaHCO3. The aqueous phases were combined and acidified to pH 5 with 2N HCl. The resulting acidic solution was then extracted twice with ethyl acetate, dried and concentrated in vacuo to give the desired product.

-[(2'-carboethoxy 1', 1'-difluoro)ethyl]N -BOC-N^TTos-Argininol

The resulting compound 1-[(2'-carboethoxy 1', 1'-difluoro)ethyl]N-BOC-H$-T3-argininol tert-Butyldimethylsilyl ether was desilylated by treatment with 3 equivalents of tetra-n-butylammonium fluoride in THF at room temperature, as described by E.J. Corey et al., supra. The compound obtained by this process was then saponified by treatment with 2.5 equivalents of LiOH in methanol/water at room temperature under argon overnight. The reaction mixture was then washed with ethyl acetate and acidified with citric acid to pH 4. The resulting acid was extracted with ethyl acetate, the organic phase was dried, and concentrated in vacuo.

The crude acid is then purified at Vydac

C^g reversed phase HPLC column under the conditions described in Example 4.

< a.

1-[(2'-carboethoxy 1', 1'-difluoro)ethyl]N -BOC-N Tos-argininone

The alcohol function of the previous compound was converted to a ketone by adding 1 equivalent of pyridine dichromate in CH2Cl2 containing 0.5% glacial acetic acid in the presence of molecular sieves [N. Peet et al., Synthesis of Peptidyl and Fluoromethyl Ketones and Peptidyl©/-Keto Esters as Inhibitors of Porcine Pancreatic Elastase, Human

Neutrophil Elastase, and Rat and Human Neutrophil Cathepsin G, J. Med. Chem., 55, pp. 594-407 (1990)]. After stirring under argon for 15 hours, the reaction mixture was filtered and the solvent was removed in vacuo. The resulting 1-[(2'-carboethoxy 1', 1'-difluoro)ethyl]-BOC-N-Tosargininone was collected as an oily residue and then purified by HPLC according to the conditions given in Example 4.

The free carboxylic acid is converted to the symmetrical anhydride and reacted with the resin-bound hexadecapeptide, ~Ι5 as described in Example 21. The two N-terminal residues of Hirulog-25, BOC-Pgo and BOC-(D-Phe) are added under standard peptide synthesis conditions and the resulting peptide is then cleaved with HF.

EXAMPLE 25

Synthesis of Hirulog-26

The formula of Hirulog-26 is as follows: H-(D-Phe)-Pro-Arg oxopropionylglycyl-(Gly)^ -Asn-Gly-Asp-Phe-Glu-Glu-Ile-ProGlu-Glu-Tyr-Leu-OH. The hexadecapeptide (Gly)^ -Asn-Gly-AspPhe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH was synthesized as previously described and allowed to bind to the resin. The next residue, N -BOC-argoxopropionylglycine, was synthesized according to the reaction scheme depicted and described below.

down.

-

3-(C-amino)2-oxo-3-{3-[(N-Tos)guanidyl]-propyl)-di-tert-butylmalonate

Prepare a batch of N ^4, -Cbz-N y -Tos-argininediazomethyl <4b ketone in the same manner as N -BOC-N ^y -Tos-argininediazomethyl ketone, described in Example 21, except for the substitution of N -Cbz-N -Tos-arginine for N -BOC-N^-Tos-arginine. Dissolve 4.5 mg of N -Cbz-N^-Tos-argininediazomethyl ketone in

Q

200 mL of CH2Cl2 was added to the flask and the solution was cooled to -70°C in a dry ice/acetone bath with stirring. Anhydrous HBr gas was then bubbled through the solution at a medium air flow rate for 15 minutes. The solution was stirred for an additional 15 minutes at -70°C and then concentrated in vacuo. The resulting product, N-Cbz-N-Tos-ArgCOCH2Br, was collected as 5.0 g of yellow crystals.

Meanwhile, a solution of sodium hydride (36 mg; 80% oil dispersion) in 1 ml DMF and 1.2 ml hexamethylphosphoramide (HMPA) was added to a solution of 259 mg ditertbutoxymalonate in 4 ml DMF. The mixture was stirred at room temperature for 40 min and then added dropwise over 20 min to a solution of 1 mmol N^-Cbz-N^-Tos-Arg-COCH^Br in 1 ml DMF/0.13 ml HMPA. The reaction was allowed to proceed for 3 h, then the solution was poured into 50 ml water and extracted with 2 x 50 ml ethyl acetate. The organic phase was isolated, dried and concentrated in vacuo to an oily residue. The residue was purified by

This was applied to a 3x10 cm silica gel column, which was eluted sequentially with 400 ml of 5% acetone in chloroform, 400 ml of 10% acetone in chloroform, and 200 ml of 20% acetone in chloroform. Fractions (25 ml) were collected and analyzed by TLC. Fractions containing the desired product were combined and concentrated to give 3-(C12amino)2-oxo-3-[3-[(N^-Tos)guanidyl]-propyl]-di-tert-butyl malonate.

fl·

5-(N-Cbamino)4-oxo-5-{3-[(N-Tos)guanidyl]-propyl)

pentanoylglycine benzyl ester

The above di-t-butyl ester was stirred in 1.2 equivalents of 1N HCl for 2 hours at room temperature.

with

It is then decarboxylated in excess pyridine at 100 C for 15 minutes. The solvent is then removed in vacuo and the residue is purified by silica gel chromatography as described above. The resulting carboxylic acid is acylated with glycine benzyl ester according to the method described in Example 21.

-?&

pentnoylglycine

The resulting ester was dissolved in 500 mL of methanol and hydrogenated overnight at 1 atm of hydrogen gas over 600 mg of 10% palladium-carbon catalyst. The reaction mixture was then filtered through Celite and concentrated in vacuo to a solid residue (155 mg). The resulting amino acid was re-

in example 4.

the conditions described

5-( N -BOCamino)-4-oxo-5-{3-[ ( N -Tos)guanidyl]-propyl) pentnoyl lysine

The above amino acid was converted to its corresponding BOC derivative by dissolving in dioxane/water (2:1, v/v) and cooling to RT with stirring. The pH was adjusted to 10 with 0.1N NaOH, and then 1.1 equivalents of di-tert-butyl dicarbonate (in dioxane) were added. The reaction was stirred at 0 C to 20 C for 4 hours and then evaporated in vacuo. The residue was then partitioned between ethyl acetate/1% citric acid (2:1). The organic phase was isolated by extracting once with 1% citric acid and then three times with saturated NaCl. The organic phase was dried over MgSO ₄ , filtered and concentrated in vacuo to give the BOC-protected product.

The resulting deprotected pseudopeptide carboxylate is then coupled to the resin-bound hexadecapeptide using standard peptide coupling techniques.

synthesis. This is followed by sequential addition of BOC-D-Phe and BOCPR0 to the resin-bound peptide. The completed (full) Hirulog-26 is then cleaved from the resin, deprotected and purified as described in Example 4.

EXAMPLE 26

Synthesis of Hirulog-27

The formula of Hirulog-27 is as follows: H-(D-Phe)-Pro-Arg(CO-CH^ )-Pro-(Gly)|-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-GluTyr-Leu-OH. The (Gly)^ -Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-GluTyr-Leu hexadecapeptide was synthesized as previously described and allowed to bind to the resin. The remainder of the molecule was synthesized according to the reaction scheme depicted

and described below.

benzyl ester ^ = 1-1 = Η

Dissolve 720 mg of proline benzyl ester (HCl salt) in 25 ml of THF. The solution is then cooled to -78°C in an acetone/dry ice bath with stirring under argon. Lithium diisopropylamide (8.0 ml of a 0.75 M hexane suspension) is then added and stirred for an additional 5 minutes. To this is added dropwise 1.08 g of N-Cl2-L-T3-arginine bromomethyl ketone in 10 ml of THF, prepared as described in Example 25, over 20 minutes. The reaction proceeds with stirring for an additional 2 minutes and the solution is then allowed to warm to room temperature with stirring. The reaction is quenched by adding 10 ml of saturated NaCl, allowing the phases to separate, and isolating the organic phase. This phase was then dried over MgSO4, filtered and evaporated under vacuum.

N^-BOC-N^-Tos-aprHHHH( COCH,, )proline

The above benzyl ester (1.5 g) was hydrogenated using the palladium-carbon procedure described in Example 25.

The resulting pseudodipeptide was BOC-protected following the procedure described in Example 25 to produce the desired product.

The purified, protected pseudopeptide is then coupled to the resin-bound hexadecapeptide by standard peptide synthesis techniques. Hirulog-27 is released from the protector, cleaved from the resin, and purified by the techniques described in Example 4.

EXAMPLE 27

Synthesis of Hirulog-28

The formula of Hirulog-28 is as follows: H-(D-Phe)-Pro-Arg ( _CHN )-Pro-(Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-GluTyr-Leu-OH. (Gly)^-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-GluTyr-Leu-O-PAM The hexadecapeptide was synthesized as previously described and allowed to bind to the resin. The remainder of the molecule was synthesized according to the reaction scheme depicted and described below.

V —

N -BOC-N^-Tos-arginine [psiCH^N]proline benzyl ester o

One gram of crushed 3 A molecular sieve (Aldrich) was added to a stirred solution of 5.25 g of proline benzyl ester free base (Schweizerhall, Inc.) in 10 mL of anhydrous THF and 2 mL of anhydrous ethanol under argon at room temperature. 1.45 mL of 5N methanolic HCl and 1.5 g of N-BOC-N-Tos-argininal (prepared as described in Example 22) were added to this mixture and stirred for 1 hour. One 85 mg portion of sodium cyanoborohydride was added to the mixture, followed 1 hour later by a second 85 mg portion of sodium cyanoborohydride. The reaction was stirred for 20 hours and filtered. 1 mL of water was added and

0.9 ml of 1 N HCl was added to the filtrate with stirring, after which the solution was concentrated in vacuo to yield 6.2 g of N BOC-N^-Arg[psiCH^H]-Pro benzyl ester as a light oil.

The oil was then purified by flash chromatography through a 5 cm flash column containing 350 ml of silica gel (Merck

Grade 60, 230-400 holes, 60 A). The product was obtained by sequential elution with 0.25%, 0.75% and 1.5% methanol in chloroform.

N -BOC-N -Tos-arginine [psiCH^NjnponHH

The resulting benzyl ester was hydrogenated over palladium-carbon and purified as described in Example 25. This process gave 160 mg of N-BOC-N-Tos-Arg[p2CH2N]proline free acid, which was then purified using HPLC.

chromatographic system described in Example 4, except that elution was performed with an isocratic 26% buffer B/74% buffer A system previously described in Example 22. The final yield of dipeptide was 86 mg.

The dipeptide is then coupled to the resin-bound hexadecapeptide, followed by a BOC-Pho incorporation cycle and a BOC-(D-Phe) incorporation cycle. Deprotection, cleavage, and purification of the fully synthesized Hirulog-28 are accomplished by the methods described in Example 4.

EXAMPLE 28

Synthesis of Hirulog-29

The formula of Hirulog-29 is as follows: 4-chloro-isocoumarin-3-carboxyethoxy-(Cl)^- -Asn-Gly-Asp-Phe-Glu-Glu-Ile-ProGlu-Glu-Tyr-Leu-OH. (Gly)^. -Asn-Gly-Asp-Phe-Glu-Glu-Ile-ProGlu-Glu-Tyr-Leu The heptadecapeptide was synthesized as previously described and allowed to bind to the resin. The 4-chloroisocoumarin-3-carboxyalkoxy moiety was synthesized according to the reaction scheme described and depicted below.

Ethyl 2-bromo-homophthalate

Homophthalic acid (10.0 g), 2-bromoethanol (21.0 g) and benzene (200 ml) were mixed. 12-15 drops of sulfuric acid were then added and heated to reflux for 2.5 hours. The solution was then filtered and concentrated in vacuo. The precipitate was washed with 250 ml of ether/hexane (1:1) and filtered on a glass funnel. The resulting light brown solid was dried to give approximately 15.0 g of product.

4-Chloro-3-[2-bromoethyloxy]-isocoumarin

-QF Ethyl 2-bromo homophthalate, prepared as previously described (4 g) was mixed with phosphorus pentachloride (8.2 g) and benzene (100 ml). The mixture was refluxed for 4.5 hours, filtered hot and evaporated in vacuo. The reddish-brown oily residue was immediately chromatographed on a 24 mm x 175 mm silica gel column using dichloromethane as eluent. 20 ml fractions were collected and examined by TLC. 4-chloro-3-[2-bromoethyloxy]isocoumarin eluted in fractions 2-6. The fractions were combined, evaporated in vacuo and the resulting residue was collected as a clear, light yellow oil (2.2 g).

4-Chloro-3-[3-oxypropionate]-isocoumarin

4-Chloro-3-[2-bromoethyl]-isocoumarin (1.4 g), prepared above, was dissolved in anhydrous THF and directly added to the refluxing solution of magnesium turnings (170 mg)

and a few crystals of iodine in 15 ml of anhydrous THF, stirring under argon. The mixture was allowed to reflux for 1.5 h. It was then poured over very dry ice into a 400 ml beaker. The mixture was left at 20 C until all the excess CO2 had sublimed, and then approximately 100 ml each of diethyl ether and THF were added to the mixture, resulting in a yellow solution containing a large amount of white, coarse precipitate.

oh

Anhydrous HCl is bubbled through this mixture at 20 C, which dissolves most of the precipitate. The solution is then filtered and evaporated in vacuo to give the crude product. It is then recrystallized overnight from

8 in DCM.

The resulting 4-chloro-3-[3-oxypropionate]-isocoumarin was coupled to the glycine benzene ester and the resulting product was catalytically hydrogenated over palladium-carbon as described in Example 25. This pseudodipeptide was then coupled to the resin-bound hexadecapeptide, (Gly).-Asn-GlyAsp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, by standard peptide synthesis techniques.

EXAMPLE 29

Synthesis of Hirulog-ZO

The formula of Hirulog-30 is as follows: -4-chloro-3-[2-aminoethanol]-isocoumarin-(Gly)-Asn-Gly-Asp-Phe-Glu-Glu-Ile-ProGlu-Glu-Tyr-Leu. The hexadecapeptide (Gly)^-Asn-Gly-Asp-PheGlu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu was synthesized as previously described and allowed to bind to the resin.

The 4-chloro-3-[2-aminoethanol]-isocoumarin moiety was prepared in a manner analogous to that described in Example 28 for the synthesis of 4-chloro-3-[2-bromoethanol]-isocoumarin, except that 2-aminoethanol was used instead of 2-bromoethanol in the first esterification step of homophthalic acid.

The urea linkage is formed by reacting the amino group of 4-chloro-3-[2-aminoethanol]-isocoumarin with the activating agent, carbonyldiimidazole (CDI). The resulting imidazolide intermediate is not isolated but is reacted with the resin-bound hexadecapeptide to produce Hirulog-30. Hirulog

- £7-50 is then released from the protector, cleaved from the resin and purified by the techniques described in Example 4.

EXAMPLE 30

Synthesis of chirulog-31

The formula of Hirulog-31 is as follows: agripidyl-( Gly )^_Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. The hexadecapeptide -Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-GluTyr-Leu was synthesized by standard peptide synthesis techniques previously described and allowed to bind to the resin. The agripidylglycine moiety of this Hirulog was synthesized by the reaction scheme described and depicted below.

V

- 89 3

Dehydro-1P-nitro-argipidine is synthesized essentially by the method for synthesizing argipidine, which is described in U.S. Patent No. 4,258,192, incorporated herein by reference. The only differences are that the guanidine group is protected from the nitro function and the heterocyclic ring of the quinoline remains unsaturated. The intermediate is used . to acylate t-butyl glycine by the method described in Example 21. The t-butyl ester is removed by standard acid hydrolysis techniques. The resulting free acid is reacted with the hexadecapeptide using standard coupling methods. The resulting peptide is deprotected, cleaved from the resin, and purified by the techniques described in Example 4.

The peptide was then subjected to the hydrogenation procedure described in patent 4,258,192 and purified by HPLC technique described in example 4.

EXAMPLE 51

Synthesis of Hirulog-52

The formula of Hirulog-52 is as follows: H-(D-Phe)-Pro-Arg(Gly)^- -Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. Hirulog-52 was synthesized, purified and characterized using the methods described in Example 4, except that BOC-glycine was used instead of BOC-proline in the cycle following the two cycles of BOC-glycylglycine addition.

EXAMPLE 52

Synthesis of Hirulog-55 to The formula of Hirulog-33 is as follows: N-aixeTHn-Gly-AspPhe-Leu-Ala-Glu-(Gly-Vai-Arg-Pro-(Gly)-Asn-Gly-Asp-PheGlu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. Hirulog-33 was synthesized, purified and characterized by standard peptide techniques.

day synthesis used in Example 4, with appropriate BOC-amino acid substitutions. The CSDM region of Hirulog-33 has an amino acid sequence identical to a segment of the fibrinopeptide A sequence from the Aβ-chain of human fibrinogen.

EXAMPLE 33

Cleavage of various Hyruloses by thrombin

The inhibition of thrombin by Hirulog-8 was found to be transient, due to the slow cleavage of the Arg-Pro bond by thrombin. After this cleavage, thrombin was observed to

regains full hydrolytic activity towards the chromogenic substrate. Therefore, Hirulog-8 is characterized as a slow-substrate thrombin inhibitor.

The cleavage of Hirulog-8, as well as the other Hirulogs according to the present invention, by human β-thrombin was demonstrated in in vitro studies. Reaction mixtures containing β-thrombin (1.6 nM) and various concentrations of Hirulog-8, Hirulog-10, Hirulog-18a, Hirulog-18b, Hirulog-18c, Hirulog-19, Hirulog-32 or Hirulog-33 (80 to 160 nM) were prepared in 20 mM Tris-HCl, pH 7.4, containing 0.1 M NaCl. Aliquots (0.975 ml) of the reaction mixtures were removed at various times and mixed in a cuvette with 0.025 ml Spectrozyme

-31 TH (11 JA. M final concentration), chromogenic substrate. The initial level of the reaction is determined and based on the control mixtures containing thrombin in the absence of Hirulog, 10% inhibition is calculated.

An alternative method uses _reverse -phase HPLC separation of this aliquot of the Hirulog/thrombin reaction mixture. When human α-thrombin (0.25 μM final concentration) is added to the above Hirulogs

Aliquots (50/L) were separated and added to the reaction vessel containing one (12.5 µM final concentration).

both before and at various times after binning. Aliquots were frozen in flasks or injected directly onto an HPLC column. The HPLC system used an Applied Biosystems liquid chromatography system equipped with an Aquapore C column (0.46 x 10 cm). The column was calibrated with 70% solvent A (0.1% TFA in water) and 30% solvent B (0.085% TFA/70% acetonitrile) and developed with a linear gradient from 30 to 50% solvent B over 30 minutes at a flow rate of 1 ml/minute. The effluent was monitored at 214 nm. Peptide concentration was determined by measuring peak heights.

Both of the above studies allow the determination of the degree of hydrolysis of Hirulog by thrombin (expressed in M/min) and the reverse rate (k^.^; expressed in min). Both methods give comparable k _cci t values, which are shown in the following table.

INHIBITOR р _ pi А А SEQUENCE -/ < ^min ) Chirulog-8 Arg-Pro 0.31-0.5 Chirulog-10 Arg-Sar 10 Chirulog-18a β-homo-Arg-Gly <0.01 Surgeon-18b p—homo—Arg—Pro <0.01 Cyclist-18s p-homo-Arg-Val <0.01 Cycologist-19 ArgfpsiCHNH]-Gly <0.01 Chirulog-32 Arg-Gly 535 Chirulog-33 Arg-Pro 0.056

As shown above, Hirulog-8, -10, -32, and -33 are cleaved by thrombin with values ranging from 0.056 min ^- ^ (slow cleavage) to 535 min ^- ' (fast cleavage). In contrast,

ca, Hirulog-18a, -18b, -18c and -19 appear to be resistant to cleavage by thrombin.

Fig. 5, A and B, show more detailed analyses of the cleavage of Hirulog-8 by thrombin. As depicted in Fig.

5A, Hirulog-8 concentrations in slight excess over thrombin showed transient inhibitory activity (greater than or equal to minutes, dependent on the concentration of

Chirologist).

Progressively increasing concentrations of Hirulog-8 showed prolonged inhibitory effects. A linear relationship between the duration of inhibition and the concentration of

-y 3

Hirulog-8 is shown in Fig. 5B. From these data it is calculated

I -4 the reciprocal time or k at 0.37 min

Purification and sequence analysis of the products obtained from the digestion with Hirulog-8 obtained in the above reactions determined that Hirulog-8 is cleaved slowly by thrombin at the Arg-Pro bond. This is a highly unusual cleavage site for serine proteases, and it is suggested that cleavage by Hirulog-8 is possible due to the high affinity of the t peptide for thrombin.

Effect

Hirulog-8, visible between

EXAMPLE 34 of the linker length on the activity of Hirulog

Hirulog-13, Hirulog-15 and Hirulog-16, are distributed only along the length of the polyglycine region of their respective linker segments. In order to determine the effect of linker length on antithrombin activity, the inhibition of human OC-thrombin by each of these Hirulogs was compared. The following table gives the linker length of each of these Hirulogs:

peptide o

linker length (A)

Chirulog-824

Chirulog-1318

Chirologist-1530

Hirulog-1636

The antithrombin activities of these Hyrulogs were measured primarily against the thrombin-catalyzed hydrolysis of Spectrozyme TH, described in Example 9. Fig. 6 depicts the relationship between linker length and K ₄ for the inhibition of Hyrulog by this thrombin-catalyzed reaction. This figure shows that Hyrulog-8, -15 and -16 have comparable inhibitory activities, while Hyrulog-13, with an 18X linker length, has activity reduced by more than 10-fold. This confirms that linker lengths >18 Å and <42 Å do not affect the activity of Hyrulog. While not hoping to find a theoretical explanation, applicants believe that this is due to the fact that the Hyrulog linker is as disordered when free in solution as when bound to thrombin. Applicants also believe that there is little cooperativity in the binding of the CSDM and ABEAM regions of the thrombin inhibitors of the invention to thrombin.

EXAMPLE 55 e Inhibition of Thrombin-Catalyzed Hydrolysis by Various Hyruloses

We compare the inhibitory activity of various thrombin inhibitors according to the present invention against thrombin-catalyzed hydrolysis of a tripeptidyl-p-nitroanilide substrate. The antithrombin activities of Hirulog-10, Hirulog-18a, Hirulog-18b, Hirulog-18c, Hirulog-19, Hirulog52 and Hirulog-35 were tested according to the method described in Example 9 using Spectrozyme TH as the substrate. The following table

-95‘ lists the calculated Ki values .as well as the P. -P.* ’ sequence of each of these thrombin inhibitors.

Inhibitor r. -r/ * Sequence K (mM) Chirulog-8 Arg-Prq, 1.9 1.4 Chirulog-10 Arg-Sar > 2,000 Chirulog-18a P-HomoArg-Gly 7.4 Surgeon-18b β-HomoArg-Pro 4.6 Cyclist-18s y3 -HomoArg-Val 205.0 Cycologist-19 Arg[psiCH NH]-Gly 20.0 Chirulog-32 Arg-Gly > 2,000 Chirulog-33 Arg-Pro 3.6

As stated above, Hirulog-10 and Hirulog-52 are

weak inhibitors of thrombin-catalyzed hydrolysis of Spectrozyme TH. This is consistent with the fact that each of these inhibitors is rapidly cleaved by thrombin at the ₽ ₁ -P^ ' linkage. In Hirulog-19, where this linkage is reduced to a ρβίΗ,-NH linkage and rendered non-cleavable by thrombin, effective inhibition of thrombin hydrolysis is observed.

Studies of cjB-homoarginine-containing inhibitors (Hirulog-18a, -18b and -18c) show that this amino acid derivative can replace arginine in the inhibitors, according to y·

-9G efficacy. Furthermore, the invention, without prejudice to this, shows that displacement of the amide bond by one methylene does not significantly reduce thrombin inhibitory activity. The 30- to 50-fold increase in Ki for Hirulog-18c compared to Hirulog-18a and -18b, respectively, suggests that the structure of the P' amino acid is important for inhibitory activity. While not wishing to be bound by theory, applicants believe that the presence of phipsi angles in the ' amino acid (Gly in Hirulog-18a; Pro in Hirulog-18b), as well as conformational reasons (such as those caused by proline in Hirulog-18b), contribute to the potency of the inhibitors according to the present invention. Alternatively, the 3 -branched side chain

I ^J chain of the P^ * amino acid Vai in Hirulog-18c may oddly link the CSDM region of this molecule to the thrombin reaction center due to ethereal reasons.

EXAMPLE 36

Binding of Hirulog-8 to the active site of thrombin

Diisopropylfluorophosphate (DFP) is a well-known inhibitor of serine proteases, including thrombin, which acts by covalently modifying the hydroxyl group of Ser-195. We added a 270-fold excess of C-DFP to thrombin in 0.1 M sodium borate, pH 8.0. After a ten-minute reaction, formation of a thrombin complex was demonstrated by SDS-PAGE and fluorographic analyses (Fig. 7, lane 1). When

37the reaction is carried out in tylo-hirudin£ ₃ (at 500 and presence of sulfo-Tur-N-acebj

3000-fold molar excess over thrombin), the modification of thrombin by [C]-DFP was not significantly altered (Fig. 7, lanes 4 and 5). However, when the reaction was carried out in the presence of Hirulog-8 (at 3 or 30-fold molar excess over thrombin) the incorporation of [C]-DFP into the catalytic site of thrombin was completely blocked (Fig. 7, lanes 2 and 5). These data demonstrate that the CSDMs of the thrombin inhibitors of the invention are capable of binding to the catalytic site of thrombin and inhibiting the catalytic activity.

EXAMPLE 37

Comparison between the antithrombin activity of

Hirulog-8 and the synthetic catalytic site-directed pentapeptide (D-Phe-Pro-Arg-Gly)

As indicated in Fig. 1, Hirulog-8, unlike its constituent anion-binding exosite associated moiety, sul®o-Tyr _f3 -N-acetyl-hirudin £5_, is capable of inhibiting thrombin-catalyzed hydrolysis of small p-nitroanilide substrates. Similarly, the ability of (DPhe)-Pro-Arg-Gly pentapeptide to inhibit thrombin catalytic reactivity was tested.

The pentapeptide was synthesized as described in Example 4 using BOC-glycine-divinylbenzene resin. The peptide was purified and characterized as described in Example 4.

The effects of both Hirulog-8 and this pentapeptide on the thrombin-catalyzed hydrolysis of Spectrozyme TH were studied as described in Example 9, using fixed peptide concentrations of 50 nM or

Yu^M, respectively.

The results indicate that

Hirulog-8 can inhibit the thrombin-catalyzed reaction at nanomolar concentrations, while concentrations of the pentapeptide as high as 10 M have negligible effect on thrombin catalysis. These data indicate that the CSDM component of the thrombin inhibitors of the invention is itself a weak inhibitor of the catalytic functions of thrombin.

EXAMPLE 38

Anticoagulant activity of Hirulog-8 in vivo

The in vivo anticoagulant activity of Hirulog-8 was determined after intravenous administration of this peptide in baboons. Different dosages of Hirulog-8 _were used, from 0.002 to 0.2 mg/kg/min. Baboons (male, 10-15 kg) were sedated with ketamine hydrochloride before administration of Hirulog-8. Whole blood from treated and control animals was collected via a catheter placed in the femoral vein and collected in 3.8% sodium citrate (9:1; blood:sodium citrate). Plasma was isolated by standard methods and the APTT was recorded by the methods described in Example 10. As shown in Fig. 8, Hirulog-8 resulted in a dose-dependent increase in APTT. A 200% increase in APTT (considered a therapeutic level) was achieved with the lowest dose of Hirulog (0.002 mg/kg/min infusion).

EXAMPLE 39

Inhibition of clot-associated thrombin by Hirulog-8

It is known that thrombin can bind to the fibrin clot and, once absorbed, continue to cleave additional fibrinogen produced by clot growth. It has been demonstrated that clot-bound thrombin is resistant to neutralization by the heparin-antithrombin III complex [P.J. Hogg et al., Fibrin Monomer Protects Thrombin from Inactivation By Heparin-Antithromb-in III : Implications for Heparin Efficacy, Proc. Natl. Acad.

Sci. USA. 86, pp. 3619-23 (1989)], but can be inhibited by antithrombin III-independent inhibitors, such as PPARγ, hirudin, or sulfo-Tyr-N-acetyl-hirudin. Clot-associated thrombin has been suggested to play a role in thrombus growth and in rethrombosis following thrombolytic therapy.

We compared the abilities of Hirulog-8 and heparin to inhibit clot-bound thrombin using the method described by J.I. Weitz et al., Clot-Bound Thrombin Is Protected from Heparin Inhibition-A Potential Mechanism for Rethrombosis After Lytic Therapy, Blood, 74, p. 136a, (1989).

A clinically relevant dose of heparin (0.1 U/ml) inhibits fibrinopeptide A (FPA) cleavage catalyzed by soluble thrombin by about 70%. While a similar dose has no effect on FPA cleavage catalyzed by clot-bound thrombin. Conversely, Hirulog-8 has approximately identical inhibitory effects on FPA cleavage catalyzed by either soluble or clot-bound thrombin (Fig. 9).

This study shows that Hirulog-8, as well as other thrombin inhibitors according to the invention, are more effective than conventional drugs in blocking thrombus growth, increasing the rate of thrombolytic reperfusion and protecting against rethrombosis following thrombolytic treatment.

EXAMPLE 40

The effect of Hirulog-8 in in vivo plaque-dependent thrombosis

Because baboons are known to have similar coagulation and hemostatic responses to humans, a baboon model was used to determine the ability of Hirulog-8 to interrupt plaque-dependent thrombosis. Specifically, various thrombogenic surfaces were placed in chronically displaced AV shunts in animals. These surfaces included segments of endarterectomized baboon aorta, collagen-coated silastic tubing, collagen-coated Gortex, and Dacron blood grafts. After placement in the shunt, the surfaces were exposed to native blood flow to induce thrombus formation. Thrombus formation was measured for 60 minutes by examining the position of plaques on the thrombogenic surface. Measurements were recorded by external gamma camera imaging following preinfusion.

- ΊΟ4~ of the tested animal with autologous In-labeled plaques.

Placement of a 5 cm segment of endarterectomized baboon aorta into the external AV shunt in the absence of Hirulog-8 resulted in time-dependent plaque deposition. This accumulation reached a plateau after 60 minutes, at which time a total of 14.0 ± 5.1 x 10 plaques/cm were detected deposited on the endarterectomized segment. Doses of 0.002 and 0.01 mg/kg/min of Hirulog-8 inhibited plaque deposition by 53.6% and 75.5%, respectively. These results are depicted in Fig. 10. for

Hirulog-08 (the dose required to reduce plaque deposition by 50%) in this model system is 0.002 mg/kg/min.

When 5 cm segments of collagen-coated silastic tubing were placed in the AV shunt, 12-6 + 5.0 x 10® plaques/cm were deposited after 60 minutes in the absence of Hirulog-8. Administration of Hirulog-8 resulted in a dose-dependent inhibition of plaque deposition. A dose of 0.04 mg/kg/min of Hirulog-8 completely inhibited plaque deposition. The results of this part of the experiment are depicted in Fig. 11. The ED^ of Hirulog-8 in this system was calculated to be 0.004 mg/kg/min.

Both collagen-coated Gortex and Dacron blood grafts are known to be more thrombogenic than sIa elastic tubing. A total of 35.0+6.0 x 10 plaques/cm were deposited on Gortex after 60 minutes of exposure to native blood in the absence of Hirulog-8. Hirulog-8 was again found to demonstrate a dose-dependent antithrombotic effect on plaque thrombus formation. A dose of 0.2

702 ut/ylvIiawa Hirulog-8 caused 62.9% inhibition of plaque deposition. The ED-p for Hirulog-8 in the Gortex system was 0.155 mg/kg/min. A similar result was obtained for the Dacron grafts. The highest dose of Hirulog-8 required to inhibit plaque deposition on these two surfaces was expected due to their high thrombogenic activity.

The effect of Hirulog-8 on both the lowest and highest offset, plaque-dependent thrombus formation was also determined using a dual-chamber device that allows simultaneous measurement of both offset conditions. The device consists of two 2 cm segments of collagen-coated Gortex, followed by two 2 cm segments of wider diameter. Using this device, thrombus formation is initiated by exposing a segment of collagen-coated Gortex with a large offset to native blood flow. This part of the experiment simulates arterial-like conditions. When blood enters the segments with wider diameter,

diameter, low deviation is maintained, vortex conditions, ca-

This simulates venous thrombosis. In control animals, a total number of 9.3-^ 2.3 x 10 and 6.1 -#» 0.5 x 10 ^plaques /cm2 accumulated after 40 minutes in the high and low displacement segments, respectively. Hirulog-8 inhibited plaque deposition in both the high and low displacement segments in a dose-dependent manner. A dose of 0.05 mg/kg/min inhibited plaque accumulation by 42.6% in the low displacement and 29.0% in the high displacement.

-Ί03EXAMPLE 41

Comparison of Hirulog-8 with other antithrombotic agents in inhibiting acute plaque-dependent thrombosis

The effects of heparin, low molecular weight heparin and recombinant hirudin on plaque deposition in collagen-coated silastic tubes/baboon model/of displaced AV shunt described in Example 40 were examined.

It has been previously shown that heparin administered as a 160 U/kg bolus injection followed by a 160 U/kg/h infusion inhibits plaque deposition to an extent approximately 80% of that observed in saline-treated control animals. Low molecular weight heparin administered as a 53 anti-Xa U/kg bolus injection followed by an infusion of 53 anti-Xa U/kg/h produces similar results.

[Y. Cadroy,

In vivo Mechanism of

Thrombus Formation.

Studies Using a Primate

L’Universite Paul Sabatier de

Model, Doctoral Thesis,

Toulouse (Sciences) (1989)].

At equivalent molar doses (5 nmol/kg/min) recombinant hirudin [A.B. Kelly et al., Recombinant Hirudin Inter ruption of Platelet-Dependent Thrombus Formation, Circulation, 78, p. 11-511 (1988)] and Hirulog-8 both inhibited platelet-dependent thrombus formation by 60-70% compared to control. These results are depicted in Fig.

12. Other thrombin inhibitors have been previously tested in the baboon model [A.B. Kelly et al., Comparison of Antithrombotic and Antihemostatic Effects Produced by Antithrombins in Primate Models of Artrial Thrombosis, Thromb. and Hemostas., 62, p. 42 (1989)]. The reported ED^ doses on collagen-coated surfaces for these agents, as well as our ED definitions, are summarized in the following table:

Agent ED _y(7 RRASK 75 nmol/kg/min Gyki 14,451 500 Benzamidine 3000 Argypidine (MD805) 550 ges-Hirudin <5 Chirulog-8 <5

EXAMPLE 42

Effect of Hirulog-8 on fibrin deposition

The effect of Hirulog-8 on fibrin(ogen) deposition in thrombi formed in the endarterectomized aorta and collagen-coated segmental silastic tube model systems described in Example 40 was measured. Fibrin deposition was determined by measuring ¹¹ & 1-fibrin(ogen) 30 days after completion of the In-plaque study described above. This allowed the In labeling to decay to a non-interfering level.

Fig. 13 demonstrates that in the absence of Hirulog-8, 0.17 mg/cm fibrin minute

Doses from 0.01

- 46F is deposited on the collagen-coated tubes after exposure to the blood stream described in Example 40.

and 0.04 mg/kg/min completely inhibited fibrin(ogen) deposition. Similar results were obtained with the endarterectomized aortic segment model.

These results indicate that the thrombin inhibitors of the invention are effective in reducing thrombus-associated fibrin(ogen) deposition as well as blocking acute, plaque-dependent thrombus formation.

EXAMPLE 43

Measurement of clearance time for Hirulog-8

A baboon model was used to determine the clearance time of Hirulog-8, following intravenous infusion, single bolus intravenous infusion, and single bolus subcutaneous injection.

ARTT assays, performed as described in Example III, were used to examine clearance time.

Various dosages of Hirulog-8 (0.002-0.2 mg/kg/min) were administered to baboons by systemic intravenous infusion over 60 minutes. APTT was measured after the 60 minute infusion and at various time intervals thereafter.

The average elimination half-life for Hirulog-8 is determined to be 9.2 -»

3.3 minutes.

To determine the clearance time after a single bolus injection, baboons were injected intravenously or subcutaneously with doses of Hirulog-8 at various mg/kg. APTT measurements were taken at time intervals after injection.

‘100Fig. 14 demonstrates that APTT increases to a peak of 57% of the control value, 2 minutes after intravenous injection. The half-life of Hirulog-8 after intravenous injection is 14 minutes.

Fig. 15 demonstrates that at early time points following subcutaneous injection of Hirulog-8 (15 minutes), APTT increased by approximately 200% relative to control. Clearance via the subcutaneous route was prolonged to half the t time of 340 minutes. Hirulog-8 administered subcutaneously was found to be completely absorbed.

EXAMPLE 44

Effect of Hirulog-8 in a baboon model of disseminated intravascular coagulation

Septicemia was induced in baboons by injecting a lethal dose of live E.

Taylor et al., J. Clin.

s coll, according to the method described by F.B.

invest., 79 pp. 918-25 (1987). Hirulog-8 was infused at a dose of 0.08 mg/kg/hour starting 15 minutes before the injection of

E. coli for over 6 hours after injection.

In the absence of

Hirulog-8, E. coli induced septic shock resulted in significant decreases in neutrophil count, blood pressure and hematocrit. Control animals showed a decrease in hematocrit of 70% of baseline and in blood pressure to 20% of baseline after 3 hours. Administration of Hirulog-8 completely attenuated the decrease in hematocrit and limited the peak drop in blood pressure to 60% of baseline.

- 4&t'

Despite the attenuation of DIC by Hirulog-8, lethal infusion of E. coli still resulted in death. At necropsy of both control and Hirulog-8-treated animals, massive tissue edema was found in both groups. However, only the control group showed intravascular thrombosis. The autopsy results suggest that simply interrupting the coagulopathic stage of septicemia is not sufficient to prevent death due to septic shock.

* —

EXAMPLE 45

Effect of a combination of tPA and Hirulog-8 on thrombolysis

To determine the effect of Hirulog-8 on the enhancement of tPA-induced thrombolysis, a rat model of arterial thrombolysis was used. In this model, an experimental thrombus was formed in the abdominal aorta after balloon catheter stripping and high-grade stenosis (95¾). Blood flow and blood pressure were recorded distal to the site of injury and stenosis. Rats were recruited to receive tPA (1.0 mg/kg bolus followed by

1.0 mg/kg/hour infusion) together with one of the following saline, heparin (10 U/kg bolus followed by i.5 U/kg/min infusion), recombinant hirudin (1.0 mg/kg bolus followed by 0.02 mg/kg/hour infusion) or Hirulog-8 (0.6 mg/kg bolus followed by 0.02 mg/kg/hour infusion). The antithrombotic agent or saline is administered simultaneously with tPA and for an additional 50 minutes following the end of the tPA infusion.

~ 408-

Fig. 16 depicts the results of these experiments.

Animals treated with tPA plus saline showed a reperfusion time of 16.2 minutes. Heparin reduced the reperfusion time to 12.2 minutes, while recombinant hirudin reduced it to 15.0 minutes. Neither of these reductions was statistically significant (p<0.005). The combination of Hirulog 8 with tPA resulted in a reduction in reperfusion time to 4.4 minutes (p<0.01), thus accelerating the fibrinolytic effect of tPA by a factor of four.

Heparin, hirudin, and Hirulog 8 all significantly prevented reocclusions compared with saline-treated controls (Fig. 17). Each of these agents also prolonged APTT to values of 600%, 500%, and 400%, respectively, above control values (Fig. 18). Finally, heparin, hirudin, and Hirulog 8 each increased vessel patency time to values of 80.2%, 82%, and 95.1%, respectively (controls showing that the inhibitors were superior to

45.6%) (Fig. 19). These results demonstrate that thrombin, according to the present invention, outperforms other known antithrombotic agents in increasing the efficacy of tPA.

EXAMPLE 46

Effect of Hirulog-8 and other antithrombotic agents on bleeding time in baboons

A template measurement of bleeding time was used to investigate the effects of Hirulog-8 on hemostasis.

Different dosages of Hirulog-8 (0.002 to 0.2 mg/kg/

min) were analyzed for their effect on bleeding time. Doses of 0.002 to 0.04 mg/kg/min did not cause a significant increase in bleeding time. The results of this study are depicted in Fig. 20. At a dose of 0.1 mg/kg/min, Hirulog-8 caused a two-fold increase in bleeding time over control values. At 0.2 mg/kg/min of Hirulog-8, bleeding time increased three-fold over control values. The results clearly show that the doses required to inhibit clot-dependent thromboses (0.002 mg/kg/min; see Example 40) did not have a significant effect on hemostatic plug formation.

The effect of various other agents on the patterned bleeding time in baboons, as well as on systemic anticoagulant effects (as measured by APTT), was also tested. The results are summarized below:

Agent (% of ARTT control) bleeding time (min) Chirulog-8 500.6 5.5 ges-hirudin 595.9 12.1 RRASK 287.9 1 2 Gyki 14,451 459.4 14 benzamidine 757.6 10 argipidine (MD805) >900 >50 heparin 706.1 10

As we have presented above several preferred embodiments of the present invention, it is clear that our basic structure can be changed so as to provide other variants that use the molecules, compositions, combinations and methods of the present invention. It should therefore be appreciated that the scope of the present invention will be defined by the claims appended hereto rather than the specific variants that have been presented as examples.

Claims (33)

PATENT CLAIMS 1. Thrombin inhibitor, consisting of: a) a catalytic site-targeting moiety that binds to and inhibits the active site of thrombin; wherein said catalytic site-targeting moiety is selected from a series of proteinase inhibitors, heterocyclic proteinase inhibitors, thrombin-specific inhibitors, transition state analogs, benzamidine, DAPA, NAPAP, argipidine or moieties of the formulae: X-A^-A^-Aj-Y or XC ₄ -C ₂ -A ₃ -Y where X is hydrogen or is characterized by a backbone consisting of 1 to 35 atoms; is Arg, Lys or Orn; is a non-amide bond; A^ is characterized by a backbone consisting of 1 to 9 atoms, Y is a bond; is a derivative of Arg, Lys or Orn containing a carboxylated moiety that is reduced or replaced by the «{-carbon of a moiety characterized by a backbone consisting of 1 is a non-cleavable bond; b) a linker portion characterized by having a calculated length of <9 to 42 Å; and up to 10 atoms; and % backbone, is about 18 Å and an anion-binding exosite associated moiety; -HI said catalytic site-targeting moiety is linked to said linker moiety, and said linker moiety is linked to said anion-binding exosite associated moiety; wherein said inhibitor is capable of simultaneously binding to the catalytic site and the anion-binding exosite of thrombin. 2. The thrombin inhibitor of claim 1, wherein said anion-binding exosite moiety consists of the formula WB ₄ -Β^-Β,-Β^-Β,-Β^-Ζ where W is a bond; B. is an anionic amino acid B^ is any amino acid Bj is lie, Vai, Nle or Phe; Rgo, Nur, 3,4-dehydro Pro thiazolidine-4-carboxylate, Sar is any N-methyl amino acid or D-Ala; B is an anionic amino acid; _B6 is an anionic amino acid; B7 is a lipophilic amino acid selected from the group consisting of Tu, Tgr, Phe, Leu, No, No, No, Cha, Pro or a dipeptide consisting of one of these lipophilic amino acids and any amino acid; B& is a bond or peptide containing from one to five residues of any amino acid; Z is a carboxy terminal residue selected from OH, -C _is alkoxy, amino, mono- or di-(Cy ^-C4 )alkyl substituted amino or benzylamino. 3. A thrombin inhibitor according to claim 1 2, where Leu Leu is Glu; Tug In^ is No; In ₇ it is Tug(SOjH)-Leu, B^ is Pro; In^. is Glu; Bg, is Glu; Tyr-(OSO ₃ H)-Leu or (3-,5-iodoTyr) bond; Z is OH 4. The thrombin inhibitor of claim 1, wherein 443said main chain of said linker moiety consists of any combination of atoms selected from the group consisting of carbon, nitrogen, sulfur and oxygen. 5. A thrombin inhibitor according to claim 4, wherein said linker comprises the following amino acid sequence: Gly-Gly-Gly-Asn-Gly-Asp-Phe. 6. The thrombin inhibitor of claim 1, wherein said catalytic site-targeting moiety binds reversibly to and is slowly cleaved by thrombin. 7. The thrombin inhibitor of claim 1, wherein said catalytic site-targeting moiety binds reversibly and cannot be cleaved by thrombin. 8. The thrombin inhibitor of claim 1, wherein said catalytic site-targeting moiety binds irreversibly to thrombin. 9. The thrombin inhibitor of claim 1, wherein X is D-Phe-Pro; A. is Arg; and Ag is D-Pro, Pro or Sar. 10. The thrombin inhibitor of claim 9, wherein said thrombin inhibitor is selected from the group consisting of Hirulog-8 and Hirulog-12. A thrombin inhibitor according to claim 1, the group wherein X is N-aneTHn-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val; Aj is Arg; and A2 is Pro. 12. A thrombin inhibitor according to claim 1, selected from the group consisting of Hirulog-18a and Hirulog-18b. 13. A pharmaceutically acceptable composition comprising an amount of a thrombin inhibitor according to claim 1 _effective for inhibiting thrombin-mediated function in a patient or in extracorporeal blood and a pharmaceutically acceptable carrier. 14. A pharmaceutically acceptable composition according to claim 13, wherein said pharmaceutically effective amount is between about 1 µg/kg body weight/day to about 5 mg/kg body weight/day. between about 10uC 15. A pharmaceutically acceptable composition according to claim 114, wherein said pharmaceutically effective amount is g/kg body weight/day to about 500 µg/kg body weight/day. 16. A composition for coating the surface of invasive instruments to be inserted into a patient, wherein said composition comprises a suitable buffer and at least one thrombin inhibitor, according to claim 1. 17. A pharmaceutically acceptable combination for the treatment or prevention of thrombotic diseases in patients, including: a) a thrombin inhibitor according to claim 1; b) a thrombolytic agent; and c) a pharmaceutically acceptable carrier. 18. A pharmaceutically acceptable combination according to claim 17, wherein said thrombin inhibitor is Hirulog8 and said thrombolytic agent is tPA. 19. The combination according to claim 17, wherein the daily dose of said thrombin inhibitor is between 1000 IU/kg body weight and 5 mg/kg body weight and wherein the daily dose -HS- of said thrombolytic agent is between about 10% and about 80% of the conventional dosage level of said thrombolytic agent. 20. The combination according to claim 19, wherein the daily dose of said thrombin inhibitor is between 100 µg/kg body weight and 500 µg/kg body weight and wherein the daily dose of said thrombolytic agent is between about 10% and about 70% of the conventional dosage level of said thrombolytic agent. 21. The thrombin inhibitor of claim 2, wherein said linker moiety is characterized by a backbone having a calculated length of between about 18 Å and 36 Å and is selected from the group consisting of an acyl group of about 17 to 35 carbon atoms, an alkyl group of about 17 to 35 backbone bonds, a peptide having about 2 to 12 residues of any amino acid, and combinations thereof. 22. A thrombin inhibitor according to claim 5, wherein: B is Tug(SO.H)-Leu or Tug(OSO.H)-Leu; the linker is a peptide of about 8 to 10 amino acids, the amino acid of said linker closest to the anion-binding exosite moiety being Phe; and the catalytic site-targeting moiety consists of the following formula: X-Arg-R, where X is selected from the group consisting of DPhe-Pro and tosyl-Gly; and R is selected from the group consisting of -4i6 from Pro, Sar, and N-methyl Ala. 25. The thrombin inhibitor of claim 11, wherein said thrombin inhibitor is Hirulog-55. 24. A composition according to any one of claims 13-15 or 16, wherein said thrombin inhibitor is Hirulog-8. 25. A combination according to any one of claims 17, 1.9 or 20, wherein said thrombin inhibitor is Hirulog- 8. 26. A method for reducing the dose of a thrombolytic effective for administering reperfusion or daily reocclusion to a patient, said method comprising the step of administering the thrombolytic agent to said patient as part of a combination according to claim 18. 27. A method for reducing reperfusion time and increasing reocclusion time in a patient treated with a thrombolytic agent, said method comprising the step of administering to said patient a composition according to claim 13, wherein said composition is administered to said patient for about 5 hours before, up to 5 hours after, treatment of the patient with the thrombolytic agent. 28. The method of claim 27, wherein said composition is administered to said patient ^for 2 hours before to 2 hours after treatment of the specified patient with the specified thrombolytic agent. 29. A method for inhibiting thrombin catalytic and receptor-mediated functions in a patient or in extracorporeal blood, comprising the step of treating said patient or said extracorporeal blood with the composition of claim 13. 30. The method of claim 29, wherein said method is used to treat or prevent thrombotic disease in patients. 31 . The method of claim 29, wherein said method is used _to treat or prevent thrombin-induced inflammation in a patient. 32. Method according to prescription 31, wherein said inflammation is caused by a disease selected from the group consisting of adult respiratory distress syndrome, septic shock, septicemia, and reperfusion injury. 33. The method of claim 29, wherein said method is used to inhibit thrombus growth in patients caused by clot-associated thrombin. 34. Method according to claim 29, wherein said method is used to inhibit plaque-dependent thrombosis in a patient 55. Method according to claim 29, wherein said method is used _to treat or prevent disseminated intravascular coagulation in patients. 36A method according to any claim of 26-28 or 29-35, wherein said patient is a human. A method according to any one of claims 1 to 5. 26-28 or 29-35, wherein said thrombin inhibitor used in said composition or combination is Hirulog-8.