||||||||||||III USOO5425936A United States Patent 19 11) Patent Number: 5,425,936 Maraganore et al. 45 Date of Patent: Jun. 20, 1995

54 INHIBITORS OF THROMBIN 57 ABSTRACT 75 Inventors: John M. Maraganore, Tewksbury; This invention relates to novel biologically active mole Jo-Ann M. Jablonski, cules which bind to and inhibit thrombin. These mole Middleborough; Paul R. Bourdon, cules comprise a catalytic site directed moiety (CSDM) Somerville, all of Mass. of the formula: 73) Assignee: Biogen, Inc., Cambridge, Mass. x-R-R-R-R-R-y 21 Appl. No.: 924,549 R 22 Filed: Jul. 31, 1992 wherein X is hydrogen or is characterized by a back Related U.S. Application Data bone chain consisting of from 1 to 100 atoms; R1 is 60 Division of Ser. No. 652,929, Feb. 8, 1991, Pat. No. selected from the group consisting of unsubstituted, 5,240,913, which is a continuation-in-part of Ser. No. mono-substituted, di-substituted and tri-substituted satu 549,388, Jul. 6, 1990, Pat. No. 5,196,404, which is a rated ring structures; R2 is a bond or is characterized by continuation-in-part of Ser. No. 395,482, Aug. 18, 1989. a backbone chain consisting of from 1 to 5 atoms; R3 is abandoned. a bond or is characterized by a backbone chain consist 51) Int. Cl...... C07K 5/10; A61K 38/07 ing of from 1 to 3 atoms; R4 is any amino acid; R5 is any 52 U.S. C...... 424/1.69; 530/330; L-amino acid which comprises a guanidinium- or 514/18 amino-containing side chain group; Ré is a non-amide 58 Field of Search ...... 530/330, 326; 514/13, bond; and Y is characterized by a backbone chain con 514/18; 424/1.69 sisting of from 1 to 9 atoms; or the formula: 56) References Cited U.S. PATENT DOCUMENTS x--R-R-R-R-y 4,971,953 11/1990 Krstenansky ...... 514/14 R’ FOREIGN PATENT DOCUMENTS wherein R1 is selected from the group consisting of 0019589A1 11/1980 European Pat. Off. ... C07C 103/52 unsubstituted, mono-substituted, di-substituted and tri substituted ring structures; R4 is any amino acid corn (List continued on next page.) prising a side chain group characterized by the capacity to accept a hydrogen bond at a pH of between about 5.5 OTHER PUBLICATIONS and 9.5; and X, R2, R3, R5, Ró and Y are defined as S. Bajusz et al., “Inhibition of Thrombin and Trypsin by above. Preferred thrombin inhibitors are further char Tripeptide Aldehydes”, Int. J. Peptide Protein Res., 12, acterized by a anion binding exosite associating dgmain pp. 217-221 (Oct. 1978). (ABEAM) and a linker portion of between 18A and 42A in length which connects the Y to the ABEAM. (List continued on next page.) This invention also relates to compositions, combina tions and methods which employ these molecules for therapeutic, prophylactic and diagnostic purposes. Primary Examiner-Jill Warden Assistant Examiner-Carol A. Salata Attorney, Agent, or Firm-Fish & Neave; James F. Haley, Jr.; Andrew S. Marks 8 Claims, 3 Drawing Sheets 5,425,936 Page 2

FOREIGN PATENT DOCUMENTS Coagulation Inhibitor in the Leech', DNA, 5, pp. 0188280A1 9/1984 European Pat. Off. ... C07C 103/52 511-517 (Dec. 1986). 0276014A2 7/1988 European Pat. Off...... CO7K 7/10 G. Frantzen Handeland et al., “Simplified Assay for 0291981A2 11/1988 European Pat. Off...... CO7K 7/00 Antithrombin III Activity. Using Chromogenic Peptide 0291982A2 11/1988 European Pat. Off...... CO7K 7/00 0333356A3 9/1989 European Pat. Off...... CO7K 7/00 Substrate,” Scand. J. Haematol, 31, pp. 427-436 (Nov. 0341607A2 11/1989 European Pat. Off...... CO7K 7/00 1983). WO90/O339 4/1990 WIPO ...... CO7K 7/06 B. Furie et al., "Computer-generated Models of Blood WO91/02750 3/1991 WIPO ...... CO7K 7/08 Coagulation Factor Xa, Factor IXa, and Thrombin WO91/19734 12/1991 WIPO ...... CO7K 37/64 Based upon Structural Homology with Other Serine Proteases', J. Biol. Chem., 257, pp. 3875-3882 (Apr. 10, 1982). OTHER PUBLICATIONS S. G. Gordon et al., “Cysteine Proteinase Procoagulant C. Bergmann et al., “Chemical Synthesis and Expres From Amnion-Chlorine', Blood, 66, pp. 1261-1265 sion of a Gene Coding for Hirudin, the Thrombin-S- (Dec. 1985). pecific Inhibitor from the Leech Hirudo medicinalis', D. Gurwitz et al., “Thrombin Modulates and Reverses Biol. Chem. Hoppe-Seyler, 367, pp. 731-740 (Aug. Neuroblastoma Neurite Outgrowth', Proc. Natl. Acad. 24-29, 1986). O Sci. USA, 85, pp. 3440-3444 (May 1988). W. Bode et al., “The Refined 1.9 A Crystal Structure of S. R. Hanson et al., “Interruption of Acute Platelet-de Human o-Thrombin: Interaction with D-Phe-- pendent Thrombosis by the Synthetic Antithrombin Pro-Arg Chloromethylketone and Significance of the D-phenylalanyl-L-prolyl-L-arginyl Chloromethyl Tyr-Pro-Pro-Trp Insertion Sequence', EMBO J., 8, Ketone', Proc. Natl. Acad. Sci USA, 85, pp 3184-3188 pp. 3467-3475 (Nov. 1989). (May 1988). J.-Y. Chang, "The Functional Domain of Hirudin, a R. P. Harvey et al., “Cloning and Expression of a Thrombin-Specific Inhibitor’, FEBS Lett, 164, pp. cDNA Coding for the Anticoagulant Hirudin from the 307-313 (Dec. 1983). Bloodsucking Leech, Hirudo medicinalis, Proc. Natl. J. DiMaio et al., "Bifunctional Thrombin Inhibitors Acad. Sci. USA, 83, pp. 1084-1088 (Feb. 1986). Based on the Sequence of Hirudin', J. Biol. Chem., 265, G. L. Hortin et al., “Antithrombin Activity of a Peptide pp. 21698-21703 (Dec. 15, 1990). Corresponding to Residues 54-75 of Heparin Cofactor A. Falanga et al., “Isolation and Characterization of II”, J. Biol. Chem..., 264, pp. 13979–13982 (Aug. 25, Cancer Procoagulant: A Cysteine Proteinase from Ma 1989). lignant Tissue', Biochemistry, 24, pp. 5558-5567 (Sep. J. A. Jakubowski et al., “Inhibition of coagulation and 24, 1985). Thrombin-Induced Platelet Activities by a Synthetic A. Falanga et al., “A New Procoagulant in Acute Leu Dodecapeptide Modeled on the Carboxy-Terminus of kemia', Blood, 71, pp. 870-875 (Apr. 1988). Hirudin', Blood, 75, pp. 399-406 (Jan. 1990). J. W. Fenton II, “Regulation of Thrombin Generation A. Kelly et al., “Potent Antithrombotic Effects of a and Function', Semin. Thromb. Hemost, 14, pp. Novel Hybrid Antithrombin Peptide In Vivo”, Circula 234-240 (Jul. 1988). - tion, 82, pp. III-603, Abstract 2397 (Oct. 1990). J. W. Fenton II, "Thromblin Bioregulatory Functions', C. Kettner et al., “D-Phe-Pro-ArgoH2C1-A Selective Ady. Clin. Enzymol, 6, pp. 186-193 (Sep. 16-19, 1987). Affinity Label for Thrombin”, Thrombosis Res., 14, pp. J. W. Fenton II et al., “Thrombin Anion-binding Exo 969-973 (Jun. 25, 1979). site Interactions with Heparin and Various Polyan P. Klement et al., “Effects of Heparin and Hirulog on ions, Ann. New York Acad. Sci., 556, pp. 158-165 (Jun. tPA-Induced Thrombolysis in a Rat Model, Fibrinoly 7, 1989). sis, 4, p. 9, Abstract 27 (Aug. 4-8, 1990). E. Fortkamp et al., “Cloning and Expression in Escher ichia coli of a Synthetic DNA for Hirudin, the Blood (List continued on next page.) 5,425,936 Page 3

OTHER PUBLICATIONS J. L. Krstenansky et al., "Hirudin and Hirullin C-Ter C. L. Knupp, "Effect of Thrombin Inhibitors on minal Domains: Structural Comparisons and Anti Thrombin-Induced Platelet Release and Aggregation', thrombin Properties”, Circulation, 82, p. III-659, Ab Thrombosis Res., 49, pp. 23-36 (Jan. 1, 1988). stract 2619 (Oct. 1990). S. Konno et al., "Analysis of the Secondary Structure of J. M. Maraganore et al., “Anticoagulant Activity of Hirudin and the Mechanism of Its Interaction with Synthetic Hirudin Peptides', J. Biol. Chem., 264, pp. Thrombin”, Arch. Biochem. Biophys, 267, pp. 158-166 8692-8698 (May 25, 1989). (Nov. 15, 1988). J. M. Maraganore et al., “Design and Characterization J. L. Krstenansky et al., “Antithrombin Properties of of Hirulags: A Novel Class of Bivalent Peptide Inhibi C-Terminus of Hirudin Using Synthetic Unsulfated tors of Thrombin', Biochemistry, 29, 7095–7101 (Jul. 31, N-acetylhirudina3-45", FEBS Lett. 211, pp. 10-16 1990). (Jan. 1987). T. J. Owen et al., "N-Terminal Requirements of Small J. L. Krstenansky et al., "Anticoagulant Peptides: Na Peptide Anticoagulants Based on Hirudins4-65', J. Med. ture of the Interaction of the C-Terminal Region of Hirudin with a Noncatalytic Binding Site on Throm Chem., 31, pp. 1009-1011 (May 9, 1988). bin', J. Med. Chem., 30, pp. 1688-1691 (Sep. 1987). T. J. Rydel et al., “The Structure of a Complex of Re J. L. Krstenansky et al., "Comparison of Hirudin and combinant Hirudin and Human a-Thrombin', Science, Hirudin PAC-Terminal Fragments and Related Ana 249, pp. 277-280 (Jul. 20, 1990). logs as Antithrombin Agents”, Thrombosis Res., 52, pp. M. Scharfet al., “Primary Structures of New Iso-Hiru 137-141 (Oct. 15, 1988). dins', FEBS Lett., 255, pp. 105-110 (Sep. 11, 1989). J. L. Krstenansky et al., “C-Terminal Peptide Alcohol, S. R. Stone et al., “Kinetics of the Inhibition of Throm Acid and Amide Analogs of Desulfato Hirudin3445 as bin by Hirudin', Biochemistry, 25 pp. 4622-4628 (Aug. . Antithrombin Agents”, Thrombosis Res., 54, pp. 12, 1986). 319-325 (May 15, 1989). M. Tsiang, “Equilibrium Binding of Thrombin to Re J. L. Krstenansky et al., “Development of MDL 28, combinant Human Thrombomodulin: Effect of Hiru 050, a Small Stable Antithrombin Agent Based on a din, Fibrinogen, Factor Va, and Peptide Analogues', Functional Domain of the Leech Protein, Hirudin', Biochemistry, 29, pp. 10602-10612 (Nov. 27, 1990). Thrombosis & Hemostasis, 63, pp. 208-214 (Apr. 12, Managamore et al. Biochemistry, Jul. 31, 1990 pp. 1990). 7095-101. U.S. Patent June 20, 1995 Sheet 1 of 3 5,425,936

U.S. Patent June 20, 1995 Sheet 2 of 3 5,425,936

U.S. Patent June 20, 1995 Sheet 3 of 3 5,425,936

5OO

45O

4OO

35O

3OO

25O

2 O O

OO HRUGEN REC-HRUDN H RULOG-8 D-CHA-HRULOG

FIG 3 5,425,936 1. 2 dependent (arterial) thrombosis S. R. Hanson and L. A. NHBTORS OF THROMBIN Harker, "Interruption of Acute Platelet-Dependent Thrombosis by the Synthetic Antithrombin D CROSS-REFERENCE TO PRIOR Phenylalanyl-L-Prolyl-L-Arginylchloromethylke APPLICATIONS 5 tone', Proc. Natl. Acad. Sci USA, 85, pp. 3184-88 This application is a divisional of application Ser. No. (1988) and fibrin-dependent (venous) thrombosis. 652,929, filed Feb. 8, 1991, now U.S. Pat. No. 5,240,913, Thrombin has several other bioregulatory roles J. issued Aug. 31, 1993, which is a continuation-in-part of W. Fenton, II, “Thrombin Bioregulatory Functions”, application Ser. No. 549,388, filed Jul. 6, 1990, now U.S. Adv. Clin. Enzymol, 6, pp. 186-93 (1988). For example, Pat. No. 5,196,404, issued Mar. 23, 1993, which is a 10 thrombin can directly activate an inflammatory re continuation-in-part of application Ser. No. 395,482, sponse by stimulating the synthesis of platelet activating filed Aug. 18, 1989, now abandoned. factor (PAF) in endothelial cells S. Prescott et al., "Human Endothelial Cells in Culture Produce Platelet TECHNICAL FIELD OF INVENTION Activating Factor (1-alkyl-2-acetyl-sn-glycero-3-phos This invention relates to novel biologically active 15 phocholine). When Stimulated With Thrombin, Proc. molecules which bind to and inhibit thrombin. These Natl. Acad. Sci USA, 81, pp. 3534-38 (1984)). PAF is molecules comprise a catalytic site directed moiety exposed on the surface of endothelial cells and serves as (CSDM) of the formula: a ligand for neutrophil adhesion and subsequent degran ulation G. M. Vercolletti et al., “Platelet-Activating 20 Factor Primes Neutrophil Responses to Agonists: Role x--R-R-R-R-y in Promoting Neutrophil-Mediated Endothelial Dam Ril age', Blood, 71, pp. 1100-07 (1988). Alternatively, thrombin may promote inflammation by increasing wherein X is hydrogen or is characterized by a back vascular permeability which can lead to edema P. J. bone chain consisting of from 1 to 100 atoms; R1 is 25 selected from the group consisting of unsubstituted, Del Vecchio et al., “Endothelial Monolayer Permeabil mono-substituted, di-substituted and tri-substituted satu ity To Macromolecules', Fed. Proc., 46, pp. 2511-15 rated ring structures; R2 is a bond or is characterized by (1987). Reagents which block the active site of throm a backbone chain consisting of from 1 to 5 atoms; R3 is bin, such as hirudin, interrupt the activation of platelets a bond or is characterized by a backbone chain consist 30 and endothelial cells C. L. Knupp, “Effect of Throm ing of from 1 to 3 atoms; R4 is any amino acid; R5 is any bin Inhibitors on Thrombin-Induced Release and Ag L-amino acid which comprises a guanidinium- or gregation', Thrombosis Res., 49, pp. 23-36 (1988). amino-containing side chain group; R6 is a non-amide Thrombin has also been implicated in promoting bond; and Y is a bond or is characterized by a backbone cancer, based on the ability of its native digestion prod 35 uct, fibrin, to serve as a substrate for tumor growth A. chain consisting of from 1 to 9 atoms; or the formula: Falanga et al., “Isolation and Characterization of Can cer Procoagulant: A Cysteine Proteinase from Malig X--R-R-R-R-y nant Tissue, Biochemistry, 24, pp. 5558-67 (1985); S. G. R1 Gordon et al., “Cysteine Proteinase Procoagulant From 40 Amnion-Chorion, Blood, 66, pp. 1261-65 (1985); and wherein R1 is selected from the group consisting of A. Falanga et al., “A New Procoagulant In Acute Leu unsubstituted, mono-substituted, di-substituted and tri kemia', Blood, 71, pp. 870-75 (1988). And thrombin has substituted ring structures; R4 is any amino acid com been implicated in neurodegenerative diseases based on prising a side chain group characterized by the capacity its ability to cause neurite retraction D. Gurwitz et al., to acceptahydrogen bond at a pH of between about 5.5 45 "Thrombin Modulates and Reverses Neuroblastoma and 9.5; and X, R2, R3, R5, R6 and Y are defined as Neurite Outgrowth', Proc. Natl. Acad. Sci. USA, 85, pp above. Preferred thrombin inhibitors are further char 3440-44 (1988). Therefore, the ability to regulate the acterized by a anion binding exosite associating domain in vivo activity of thrombin has many important clinical (ABEAM) and a linker portion of between 18A and implications. 42A in length which connects Y to the ABEAM. This SO One route to the successful treatment or prevention invention also relates to compositions, combinations of acute vascular disease is the inhibition of thrombin. and methods which employ these molecules for thera Many types of thrombin inhibitors are already known in peutic, prophylactic and diagnostic purposes. the art. Heparin, an indirect inhibitor of thrombin, is widely used to treat venous thrombosis. Although ef. BACKGROUND ART 55 fective against fibrin-dependent clot formation, heparin Acute vascular diseases, such as myocardial infarc has little efficacy in inhibiting thrombin-induced activa tion, stroke, pulmonary embolism, deep vein thrombo tion of platelets. Therefore, this drug is not utilized in sis, peripheral arterial occlusion, and other blood sys the treatment of arterial thromboses. Moreover, heparin ten thromboses constitute major health risks. Such produces many undesirable side effects, including hem diseases are caused by either partial or total occlusion of 60 orrhaging and thrombocytopenia. a blood vessel by a blood clot, which contains fibrin and Hirudin is a naturally occurring polypeptide which is platelets. produced by the blood sucking leech Hirudo medicina Thrombin is the naturally occurring protein which lis. This compound, which is synthesized in the salivary catalyzes the conversion offibrinogen to fibrin, the final gland of the leech, is the most potent natural inhibitor of step in blood clot formation. In addition to catalyzing 65 coagulation known. Hirudin prevents blood from coag the formation of a fibrin clot, thrombin also activates ulating by binding tightly to thrombin platelet aggregation and release reactions. This means (Kd=2x 10-11M) in a 1:1 stoichiometric complex S. that thrombin plays a central role in both acute platelet R. Stone and J. Hofsteenge, “Kinetics of the Inhibition 5,425,936 3. 4. of Thrombin by Hirudin', Biochemistry, 25, pp. 4622-28 are also smaller than hirudin and therefore less anti (1986). This, in turn, inhibits thrombin from catalyzing genic. These inhibitors are also produced synthetically, the conversion of fibrinogen to fibrin (clot), as well as allowing for the production of commercially feasible inhibiting all other thrombin-mediated processes J. W. quantities at reasonable costs. Fenton, II, “Regulation of Thrombin Generation and Despite the developments to date, there is an ongoing Functions', Semin. Thromb. Hemost, 14, pp. 23440 need for even more potent thrombin inhibitors which (1988). can be produced inexpensively and in commercially Hirudin inhibits thrombin by binding to the latter at feasible quantities. Such inhibitors would not only be two separate sites. Initially, the C-terminus of hirudin effective in treating and preventing vascular disease, but interacts with an "anion-binding exosite' (ABE) in O may also be therapeutically useful in treating cancer, thrombin J. W. Fenton, II et al., “Thrombin Anion neurodegenerative disease and inflammation. Binding Exosite Interactions with Heparin and Various Polyanions', Ann. New York Acad. Sci, 556, pp. 158-65 SUMMARY OF THE INVENTION (1989). Following this low affinity binding, the hirudin The present invention provides molecules which are thrombin complex undergoes a conformational change 15 potent inhibitors of thrombin. These molecules have and amino terminal portion of hirudin is able to bind to been designed based on the three-dimensional X-ray the catalytic site of thrombin S. Kono et al., “Analysis crystallographic structure of a thrombin-inhibitor com of Secondary Structure of Hirudin and the Conforma plex. Because of this, the inhibitors of the present inven tional Change Upon Interaction with Thrombin', Arch. tion are spatially configured to provide the best fit into Biochem. Biophys, 267, pp. 158-66 (1988). 20 the three-dimensional spaces in and around the catalytic The isolation, purification and amino acid sequence site of thrombin. This results in molecules which have of hirudin are known in the art P. Walsmann and F. optimal thrombin inhibitory activity. Markwardt, "Biochemical and Pharmacological As The invention further provides thrombin inhibitors pects of the Thrombin Inhibitor Hirudin', Pharmazie, which additionally comprise a moiety that binds to the 36, pp. 653-60 (1981); J. Dodt et al., "The Complete 25 Covalent Structure of Hirudin: Localization of the Di anion binding exosite of thrombin. These inhibitors sulfide Bonds', Biol. Chem. Hoppe-Seyler, 366, pp. qualitatively mimic the action of hirudin. Because these 379-85 (1985); S. J. T. Mao et al., “Rapid Purification molecules are designed for optimal spatial configura and Revised Amino Terminal Sequence of Hirudin: A tion, they are more potent than hirudin. The high po Specific Thrombin Inhibitor of the Blood-Sucking 30 tency of the inhibitors of this invention allows them to Leech', Anal. Biochem, 161 pp 514-18 (1987); and R. P. be administered to patients in dosages which are com Harvey et al., “Cloning and Expression of a cDNA paratively lower than those required in hirudin-based Coding for the Anti-Coagulant Hirudin from the Blood therapy regimens. sucking Leech, Hirudo medicinalis', Proc. Natl. Acad. The molecules of this invention may be utilized in Sci. USA, 83, pp. 1084-88 (1986). 35 compositions and methods for inhibiting any thrombin In animal studies, hirudin, purified from leeches, has mediated or thrombin-associated function or process. demonstrated efficacy in preventing venous thrombosis, Pharmaceutical compositions containing these mole vascular shunt occlusion and thrombin-induced dissemi cules, as well as methods of treatment or prophylaxis of nated intravascular coagulation. In addition, hirudin vascular diseases, inflammatory responses, carcinomas, exhibits low toxicity and a very short clearance time 40 and neurodegenerative diseases using these inhibitors from circulation F. Markwardt et al., “Pharmacologi are also part of the present invention. These molecules cal Studies on the Antithrombotic Action of Hirudin in may also be employed in compositions and methods for Experimental Animals', Thromb. Haemost, 47, pp. ex vivo imaging, for storing and treating extracorporeal 226-29 (1982). blood and for coating invasive devices. And the mole Hirudin has more recently been cloned and expressed 45 cules of this invention may be administered to a patient in E. coli European patent applications 158,564,168,342 in combination with a fibrinolytic agent to increase the and 171,024 and yeast European patent application efficacy of a given dose of that agent or to lower the 200,655. Despite these advances, hirudin is still moder dose of that agent required for a given effect, such as ately expensive to produce and it is not widely available dissolving a blood clot. commercially. 50 Due to the fact that the molecules of the present Recently, efforts have been made to identify peptide invention may be prepared by chemical synthesis tech fragments of native hirudin or derivatives thereof niques, commercially feasible amounts may be pro which are also effective in prolonging clotting times. duced inexpensively. Moreover, because the molecules Such compounds are described in European patent of the present invention are significantly smaller than application Nos. 276,014,291,982, 333,356, 341,607 and 55 the thrombin inhibitors presently employed in medical 372,670. The molecules described in these patent appli treatment, they are less likely to stimulate an undesir cations demonstrated varying efficacy in inhibiting clot able immune response in patients treated with them. formation, but were all 2 to 4 orders of magnitude less Accordingly, the use of these thrombin inhibitors is not potent than hirudin. Such peptide fragments, therefore, limited to the treatment of acute disease. These mole may not be fully satisfactory to dissolve blood clots in cules may also be utilized in therapy for chronic throm on-going therapy regimens. boembolic diseases, such as atherosclerosis and resteno More recently, compounds which mimic the action sis following angioplasty. The molecules of the present of hirudin by binding to both the anion binding exosite invention may also be utilized in a variety of other appli and the catalytic site of thrombin have been described cations in place of known thrombin inhibitors, espe U.S. patent application Ser. Nos. 395,482, now aban 65 cially heparin or hirudin. doned, and 549,388, now U.S. Pat. No. 5,196,404. As will be appreciated from the disclosure to follow, These compounds demonstrate thrombin inhibitory the molecules, compositions and methods of this inven activity equal to or greater than native hirudin. They tion are useful in the treatment and prevention of vari 5,425,936 5 6 ous diseases attributed to the undesirable effects of phosphonic acid ester of tyrosine, 4-phenylacetic acid, thrombin, as well as for diagnostic purposes. 3,5-diiodotyrosine, 3- and 5-nitrotyrosine, e-alkyl lysine, BRIEF OESCRIPTION OF THE DRAWINGS delta-alkyl ornithine, and the D-isomers of any of the above amino acids. Unless specifically indicated, all FIG. 1 depicts a space filling model of the Hirulog-8- 5 amino acids referred to in this application are in the thrombin complex. L-form. FIG. 2 is a schematic depiction of the interaction between the 1-position D-Phe and 2-position proline of The compounds referred to herein as tyrosine-O-sul Hirulog-8 and the hydrophobic pocket adjacent the fate, Tyr(OSO3H) and O-sulfate ester of tyrosine are catalytic site of thrombin. 10 identical and have the structural formula: FIG. 3 depicts the comparative anticoagulant activity of hirugen, recombinant hirudin, hirulog-8 and D-Cha hirulog. COOH (H-CH: O-SO-Hi DETAILED DESCRIPTION OF THE 15 INVENTION NH The following common abbreviations of the amino The compounds referred to herein as Tyr(SO3H), acids are used throughout the specification and in the 3-sulfo-tyrosine and 5-sulfo-tyrosine are identical and claims: have the structural formula: 20 Orin - ornithine Gly-glycine Ala - alanine Wall - vaine Leu - leucine e - isoleucine Pro - proline Phe- phenylalanine CH-CH2 OH Trp - tryptophan Met - Inethionine 25 ser - serine hir - threonine cys - cysteine Tyr - tyrosine ASn - asparagine Glin -glutamine Asp - aspartic acid Glu - glutamic acid Lys - lysine Arg - arginine The term "patient' as used in this application refers His - histidiae Nile - norleucine 30 to any mammal, especially humans. Nipa - napthylalanine Cha - cyclohexylalanine The term "anionic amino acid' as used herein means Hyp - hydroxyproline TPro - thioproline Ac-acetyl Suc - succinyl a meta, para or ortho, mono- or di-substituted phenylal BOC - tertibutoxycarbonyl Tos - ratoluenesulfonyl anine, cyclohexylalanine or tyrosine containing a nega Cbz - Carbobenzyloxy Inp - isonipecotic acid tively charged moiety, as well as S-alkylated cysteine, 3,4-dehydroPro - 3,4- Sar - sarcosine dehydroproline (N-methylglycine) 35 S-alkylated homocysteine, y-carboxyglutamic acid, TyrCSO3H) - tyrosine-O- TyrcSO3H) - 3-sulfo e-alkyl lysine, delta-alkyl ornithine, glutamic acid, and sulfate tyrosine aspartic acid. Examples of anionic amino acids are O 3-,5-diiodoTyT - 3,5-diiodotyrosine sulfate, O-phosphate and O-carboxylate esters of tyro sine, 3- and 5-sulfo-tyrosine, 3- and 5-carbo-tyrosine, 3 The term 'any amino acid' as used herein includes 40 and 5-phospho-tyrosine, 4-methane sulfonic acid ester the L-isomers of the naturally occurring amino acids, as of tyrosine, 4-methane phosphonic acid ester of tyro well as other "non-protein’ a-amino acids commonly sine, 4-phenylacetic acid, 3,5-diiodotyrosine and 3- and utilized by those in the peptide chemistry arts when 5-nitrotyrosine. preparing synthetic analogs of naturally occurring amino peptides. The naturally occurring amino acids 45 The terms “catalytic site”, “active site' and “active are glycine, alanine, valine, leucine, isoleucine, serine, site pocket' as used herein, each refer to any or all of methionine, threonine, phenylalanine, tyrosine, trypto the following sites in thrombin: the substrate binding or phan, cysteine, proline, histidine, aspartic acid, aspara "S1’ site; the hydrophobic binding or 'oily' site; and gine, glutamic acid, glutamine, y-carboxyglutamic acid, the site where cleavage of a substrate is actually carried arginine, ornithine and lysine. Examples of “non- 50 out ("charge relay site'). protein' d-amino acids include norleucine, norvaline, The term “backbone chain' as used herein, refers to alloisoleucine, homoarginine, thioproline, dehydropro the portion of a chemical structure that defines the line, hydroxyproline (Hyp), isonipecotic acid (Inp), smallest number of consecutive bonds that can be traced homoserine, cyclohexylglycine (Chg), a-amino-n- from one end of that chemical structure to the other. butyric acid (Aba), cyclohexylalanine (Cha), amino- 55 The atomic components that make up a backbone chain phenylbutyric acid (Pba), phenylalanines substituted at may comprise any atoms that are capable of forming the ortho, meta, or para position of the phenyl moiety bonds with at least two other atoms. with one or two of the following: a (C1-C4) alkyl, a For example, each of the following chemical struc (C1-C4) alkoxy, halogen or nitro groups or substituted tures is characterized by a backbone chain of 7 atoms with a methylenedioxy group; S-2- and 3-thienylal-ala- 60 (the atoms which comprise the backbone chain are nine, 3-2- and 3-furanylalanine, 3-2-, 3- and 4 pyridylalanine, R-(benzothienyl-2- and 3-yl)alanine, indicated in boldface): A-(1- and 2-naphthyl)alanine, O-alkylated derivatives of serine, threonine or tyrosine, S-alkylated cysteine, S H H H H alkylated homocysteine, O-sulfate, O-phosphate and 65 O-carboxylate esters of tyrosine, 3- and 5-sulfo tyrosine, H H H H 3- and 5-carboxy-tyrosine, 3- and 5-phosphotyrosine, H H H 4-methane sulfonic acid ester of tyrosine, 4-methane 5,425,936 7 8 -continued um- or amino-containing side chains are arginine, lysine H O R and ornithine. H it Preferably, the saturated homocyclic or heterocyclic N C C O H 1N1C N1N1N H ring structure is supplied by a D-cyclohexyl-alanine (D-Cha), a mono-substituted D-Cha, a di-substituted R D-Cha or a tri-substituted D-Cha residue (i.e., X is H2N; R1 is selected from the group consisting of unsubsti tuted, mono-substituted, di-substituted and tri-sub stituted cyclohexane; R2 is CH2-CH; and R3 is C=O). 10 Most preferably, X is H2N; R is cyclohexane; R2 is CH2-CH; R3 is C=O; R4 is proline; R5 is arginine and Y is proline. The presence of a non-amide bond at R6 retards or prevents cleavage of the inhibitor by thrombin. The 5 non-amide bond component may be formed by chemi cally modifying an amide bond. This may be achieved by methods well known in the art M. Szelke et al., "Potent New Inhibitors of Human Renin', Nature, 299, pp. 555-57 (1982); D. H. Coy et al., “Facile Solid Phase Preparation of Proteins containing the CH2-NH Pep The term "calculated length' as used in this applica tide Bond Isostere and Application to the Synthesis of tion, refers to a predicted measurement derived by sum Somatostatin (SRIF) OctapeptideAnalogues', Peptides ming up the bond lengths between the atoms that com 1986, D. Theodropoulos, Ed., Walter Gruyter & Co., prise the backbone chain. Bond lengths between any Berlin, pp. 143-46 (1987). When a non-amide bond is two given atoms are well known in the art see, for 25 formed in this manner, it is preferable that the chemical example, CRC Handbook of Chemistry and Physics, 65th modification be performed prior to the addition of the Edition, R. C. Weist, ed., CRC Press, Inc., Boca Raton, portion of the molecule containing this bond to the rest Fla., pp. F-166-70 (1984)). of the thrombin inhibitor. In this manner, the portion Applicants have analyzed the structure of a throm containing this non-amide bond can be added en bloc, in bin-Hirulog-8 complex by three-dimensional X-ray 30 a single synthesis step, to the rest of the inhibitor. crystallography. Hirulog-8 is an inhibitor that binds to In the most preferred embodiment R5 is Arg and Y is both the anionic binding exosite and the catalytic site of Pro. In this embodiment Ró is a naturally occurring thrombin. It has the formula: (D-Phe)-Pro-Arg-Pro imide bond, which is slowly cleaved by thrombin. This (Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu avoids the necessity of pre-forming the non-amide bond Tyr-Leu. The synthesis of this compound is described in 35 and allows Y and R5 to be added to the rest of the inhibi U.S. patent application Ser. No. 395,482, now aban tor sequentially rather than en bloc. doned, and in Example 2 of this application. This crys Further analysis of the Hirulog-8-thrombin crystallo tallographic data revealed several structural features in graphic structure revealed that the proline bound to the and around the active site of thrombin that were crucial D-Phe of Hirulog-8 was within 3.46A of the hydroxyl in designing the improved thrombin inhibitors of this group of Tyr76 of thrombin. Because this distance was invention. close enough to form hydrogen bonds, the substitution One of these structural features is a hydrophobic of an amino acid comprising a side chain group charac pocket in thrombin adjacent to its catalytic center. In terized by the capacity to accept a hydrogen bond at a Hirulog-8, the N-terminal D-Phe residue, specifically pH of between about 5.5 and 9.5 for Pro at this position the phenyl ring of that amino acid, occupies this space. 45 should increase the binding affinity of the inhibitor. Substituting a saturated ring for the unsaturated ring Accordingly, another embodiment of this invention is a increases the lipophilic interactions with thrombin, thus thrombin inhibitor of the formula: increasing inhibitory potency. Therefore, according to one embodiment of this invention, the thronbin inhibi tor has the formula: 50 x--R-R-R-R-y R1 x-R-R-R-R-R-y wherein R1 is selected from the group consisting of R unsubstituted, mono-substituted, di-substituted and tri 55 substituted ring structures; R2 is a bond or is character wherein X is hydrogen or is characterized by a back ized by a backbone chain consisting of from 1 to 5 bone chain consisting of from 1 to 100 atoms; R1 is atoms; R3 is a bond or is characterized by a backbone selected from the group consisting of unsubstituted, chain consisting of from 1 to 3 atoms; R4 is any amino mono-substituted, di-substituted and tri-subsituted satu acid comprising a side chain group characterized by the rated homocyclic or heterocyclic ring structures; R2 is a capacity to accept a hydrogen bond at a pH of between bond or is characterized by a backbone chain consisting about 5.5 and 9.5; R5 is any L-amino acid which com of from 1 to 5 atoms; R3 is a bond or is characterized by prises a guanidinium- or amino-containing side chain a backbone chain consisting of from 1 to 3 atoms; R4 is group; R5 is a non-amide bond; and Y is characterized any amino acid; R5 is any L-amino acid which con by a backbone chain consisting of from 1 to 9 atoms. prises a guanidinium- or amino-containing side chain 65 Amino acids comprising a side chain group charac group; Risis a non-amide bond; and Y is characterized terized by the capacity to accept a hydrogen bond at a by a backbone chain consisting of from 1 to 9 atoms. pH of between about 5.5 and 9.5 are well known in the Examples of L-amino acids which comprise a guanidini art. For example, histidine (which contains an 5,425,936 9 10 imidazolium nitrogen), thioproline (which contains a 8692-98 (1989); both of which are herein incorporated thiol group) and isonipecotic acid (which contains a by reference. carboxylate side group) are each known to be hydrogen According to a preferred embodiment of this inven bond acceptors at pH 5.5 to 9.5. tion, ABEAM is homologous to amino acids 56-64 of More preferred thrombin inhibitors according to this hirudin, i.e., B1 is Glu; B2 is Glu; B3 is Ile; B4 is Pro; B5 embodiment comprise an unsubstituted, mono-sub is Glu; B6 is Glu; B7 is Tyr-Leu, TyrCSO3H)-Leu or stituted, di-substituted and tri-substituted cyclohexane Tyr(OSO3H)-Leu, or (3-,5-diiodoTyr)-Leu; B8 is a at the R1 position. Most preferably X is H2N, R1 is bond; and Z is OH. It should be noted that native hiru cyclohexane, R2 is CH2-CH and R3 is C-O and the din contains Tyr(OSO3H) at position 63. However, resulting amino acid formed by X, R1,R2 and R3 is in O carboxy terminal hirudin peptides which contain Tyr(- the D-configuration (i.e., D-Cha) SO3H) have identical anticoagulant activity as those In another preferred embodiment the thrombin inhib which contain the native TyrcCSO3H) see U.S. patent itor of this invention additionally consists of an anion application Ser. No. 314,756, now abandoned. binding exosite associating moiety (ABEAM), and a Other ABEAM components within the scope of this linker bound to Y at one end and the ABEAM at the 15 other. Similar thrombin inhibitors have been described invention may comprise those portions of any molecule in U.S. application Ser. Nos. 549,388, filed Jul. 6, 1990, known to bind to the anion binding site of thrombin. now U.S. Pat. No. 5,196,404, and 395,482, filed Aug. 18, These include amino acids 1675-1686 of Factor V, 1989, now abandoned, both of which are herein incor amino acids 272-285 of platelet glycoprotein Ib, amino porated by reference, but the present preferred inhibi acids 415-428 of thrombomodulin, amino acids 245-259 tors are surprisingly and unexpectedly more potent. 20 of prothrombin Fragment 2 and amino acids 30 to 44 of In this embodiment, the linker region of the inhibitor fibrinogen Aa chain. In addition, the ABEAM compo provides a bridge between the CSDM and the nent may be selected from any of the hirudin peptide ABEAM. Accordingly, it is the length of the linker, analogues described by J. L. Krstenansky et al., “Devel rather than its structure, that is of prime importance. opment of MDL-28,050, A Small Stable Antithrombin The calculated length of the backbone chain which 25 Agent Based On A Functional Domain of the Leech characterizes the linker must be at least about 18A-the Protein, Hirudin', Thromb. Haemostas, 63, pp. 208-14 distance between the catalytic site and the anign binding (1990), particularly those comprising the sequence Asp exosite of thrombin-and less than about 42A. Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-Cha-(D-Glu). The backbone chain of the linker may comprise any The thrombin inhibitors of the present invention may atoms which are capable of bonding to at least two 30 be synthesized by various techniques which are well other atoms. Preferably, the backbone chain consists of known in the art. These include organic chemical syn any chemically feasible combination of atoms selected thesis techniques, solid-phase peptide synthesis, solu from oxygen, carbon, nitrogen and sulfur. Those of skill tion-phase peptide synthesis or a combination of these in the art are aware of what combination of the above 35 techniques. Portions of some of the inhibitors of this backbone chain atoms falls within the required length invention may also be produced by other methods, such based on known distances between various bonds see, as enzymatic cleavage of natural or recombinant hiru for example, R. T. Morrison and R. N. Boyd, Organic din or recombinant DNA techniques. These portions Chemistry, 3rd Edition, Allyn and Bacon, Inc., Boston, may then be bound to the synthetically produced por Mass. (1977). According to a preferred embodiment, tions of the inhibitor to produce the final product ac the linker is a peptide which comprises the amino acid cording to this invention. The choice of synthesis tech sequence Gly-Gly-Gly-Asn-Gly-Asp-Phe. Preferably, nique will, of course, depend upon the composition of the amino acid bound to the ABEAM component is the particular inhibitor. Phe. In a preferred embodiment of this invention, the The third domain of these preferred thrombin inhibi 45 thrombin inhibitor is synthesized via a mixed heterolo tors is the ABEAM which binds to the anion binding gous/solid phase technique. This technique involves the exosite of thrombin. Preferably the ABEAM has the solid-phase synthesis of all or most of the peptide por formula: tion of the molecule, followed by the addition of the non-amino acid components which are synthesized by 50 solution phase techniques. The non-amino acid may be wherein W is a bond; B1 is an anionic amino acid; B2 is coupled to the peptidic portion via solid-phase or solu any amino acid; B3 is Ile, Val, Leu, Nile or Phe: B4 is tion-phase methods. Similarly, any remaining peptidic Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, portions may also be added via solid-phase or solution Sar, any N-methylamino acid or D-Ala; B5 is an anionic phase methods. This constitutes the most cost-efficient amino acid; B6 is an anionic amino acid; B7 is a lipophilic 55 procedures for producing commercial quantities of amino acid selected from the group consisting of Tyr, these molecules. Trp, Phe, Leu, Nile, Ile, Val, Cha, Pro, or a dipeptide When "non-protein’ amino acids are contained in the consisting of one of these lipophilic amino acids and any thrombin inhibitor of this invention, they may be either amino acid; B8 is a bond or a peptide containing from added directly to the growing chain during peptide one to five residues of any amino acid; and Z is OH or 60 synthesis or prepared by chemical modification of the is characterized by a backbone chain consisting of be complete synthesized peptide, depending on the nature tween 1 and 6 atoms. of the desired "non-protein' amino acid. Those of skill Peptides which are homologous to the carboxy termi in the chemical synthesis art are well aware of which nal portion of hirudin have been shown to bind to the "non-protein’ amino acids may be added directly and anion binding exosite on thrombin U.S. patent applica 65 which must be synthesized by chemically modifying the tion Ser. No. 314,756, now abandoned, and J. M. complete peptide chain following peptide synthesis. Maraganore et al., “Anticoagulant Activity of Syn The molecules of the present invention display potent thetic Hirudin Peptides”, J. Biol. Chem., 264, pp. anticoagulant activity. This activity may be assayed in 5,425,936 11 12 vitro using any conventional technique. Preferably, an able side effects associated with the use of thrombolytic assay for anticoagulant activity involves direct determi agents, such as bleeding complications. nation of the thrombin-inhibitory activity of the mole Thrombolytic agents which may be employed in the cule. Such techniques measure the inhibition of thron combinations and compositions of the present invention bin-catalyzed cleavage of colorimetric substrates or, are those known in the art. Such agents include, but are more preferably, the increase in thrombin times or in not limited to, tissue plasminogen activator purified crease in activated partial thromboplastin times of from natural sources, recombinant tissue plasminogen human plasma. The latter assay measures factors in the activator, streptokinase, urokinase, prourokinase, aniso "intrinsic' pathway of coagulation. Alternatively, the lated streptokinase plasminogen activator complex (AS assay employed may use purified thrombin and fibrino O PAC), animal salivary gland plasminogen activators gen to measure the inhibition of release of fibrinopep and known, biologically active derivatives of any of the tides A or B by radioimmunoassay or ELISA. above. The antiplatelet activity of the molecules of this in The term “combination' as used herein, includes a vention may also be measured by any of a number of single dosage form containing at least one thrombin conventional platelet assays. Preferably, the assay will 15 inhibitor of this invention and at least one thrombolytic agent; a multiple dosage form, wherein the thrombin measure a change in the degree of aggregation of plate inhibitor and the thrombolytic agent are administered lets or a change in the release of a platelet secretory separately, but concurrently; or a multiple dosage form component in the presence of thrombin. The former wherein the two components are administered sepa may be measured in an aggregometer. The latter may be rately, but sequentially. In sequential administration, the measured using RIA or ELISA techniques specific for thrombin inhibitor may be given to the patient during the secreted component. the time period ranging from about 5 hours prior to The molecules of the present invention are useful in about 5 hours after administration of the thrombolytic compositions, combinations and methods for the treat agent. Preferably, the thrombin inhibitor is adminis ment and prophylaxis of various diseases attributed to 25 tered to the patient during the period ranging from 2 thrombin-mediated and thrombin-associated functions hours prior to 2 hours following administration of the and processes. These include myocardial infarction, thrombolytic agent. stroke, pulmonary embolism, deep vein thrombosis, Alternatively, the thrombin inhibitor and the throm peripheral arterial occlusion, restenosis following arte bolytic agent may be in the form of a single, conjugated rial injury or invasive cardiological procedures, acute 30 molecule. Conjugation of the two components may be or chronic atherosclerosis, edema and inflammation, achieved by standard cross-linking techniques well various cell regulatory processes (e.g. secretion, shape known in the art. The single molecule may also take the changes, proliferation), cancer and metastasis, and neu form of a recombinant fusion protein, if both the throm rodegenerative diseases. bin inhibitor and the thrombolytic agent are peptidic. The thrombin inhibitors of the present invention may 35 Various dosage forms may be employed to administer be formulated using conventional methods to prepare the compositions and combinations of this invention. pharmaceutically useful compositions, such as the addi These include, but are not limited to, parenteral admin tion of a pharmaceutically acceptable carrier. These istration, oral administration and topical application. compositions and the methods employing them may be The compositions and combinations of this invention used for treating or preventing thrombotic diseases in a 40 may be administered to the patient in any pharmaceuti patient. cally acceptable dosage form, including those which According to an alternate embodiment of the present may be administered to a patient intravenously as bolus invention, the thrombin inhibitors may be employed in or by continued infusion, intramuscularly-including combinations, compositions, and methods for treating paravertebrally and periarticularly-subcutaneously, thrombotic disease, and for decreasing the dosage of a 45 intracutaneously, intra-articularly, intrasynovially, in thrombolytic agent required to establish reperfusion or trathecally, intra-lesionally, periostally or by oral, nasal, prevent reocclusion in a patient. Additionally, the or topical routes. Such compositions and combinations thrombin inhibitors of this invention may be used in are preferably adapted for topical, nasal, oral and paren combinations, compositions, and methods for decreas teral administration, but, most preferably, are formu ing reperfusion time or increasing reocclusion time in a 50 lated for parenteral administration. patient treated with a thrombolytic agent. These combi Parenteral compositions are most preferably adminis nations and compositions comprise a pharmaceutically tered intravenously either in a bolus form or as a con effective amount of a thrombin inhibitor of the present stant infusion. If the thrombin inhibitor is being used as invention and a pharmaceutically effective amount of a an antiplatelet compound, constant infusion is pre thrombolytic agent. 55 ferred. If the thrombin inhibitor is being used as an In these combinations and compositions, the throm anticoagulant, a subcutaneous or intravenous bolus in bin inhibitor and the thrombolytic agent work in a com jection is preferred. For parenteral administration, fluid plementary fashion to dissolve blood clots, resulting in unit dose forms are prepared which contain a thrombin decreased reperfusion times and increased reocclusion inhibitor of the present invention and a sterile vehicle. times in patients treated with them. Specifically, the The thrombin inhibitor may be either suspended or thrombolytic agent dissolves the clot, while the throm dissolved, depending on the nature of the vehicle and bin inhibitor prevents newly exposed, clot-entrapped or the nature of the particular thrombin inhibitor. Paren clot-bound thrombin from regenerating the clot. The teral compositions are normally prepared by dissolving use of the thrombin inhibitor in the combinations and the thrombin inhibitor in a vehicle, optionally together compositions of this invention advantageously allows 65 with other components, and filter sterilizing before the administration of a thrombolytic reagent in dosages filling into a suitable vial or ampule and sealing. Prefera previously considered too low to result in thrombolytic bly, adjuvants such as a local anesthetic, preservatives effects if given alone. This avoids some of the undesir and buffering agents are also dissolved in the vehicle. 5,425,936 13 14 The composition may then be frozen and lyophilized to hibitor. Most preferred combinations comprise the same enhance stability. amount of the thrombin inhibitor and between about Parenteral suspensions are prepared in substantially 10% and about 70% of the conventional dosage range the same manner, except that the active component is of a thrombolytic agent. It should also be understood suspended rather than dissolved in the vehicle. Steriliza- 5 that a daily pharmaceutically effective dose of either the tion of the compositions is preferably achieved by expo thrombin inhibitors of this invention or the thrombo sure to ethylene oxide before suspension in the sterile lytic agent present in combinations of the invention, vehicle. Advantageously, a surfactant or wetting agent may be less than or greater than the specific ranges cited is included in the composition to facilitate uniform dis above. tribution of its components. O Once improvement in the patient's condition has Tablets and capsules for oral administration may occurred, a maintenance dose of a combination or corn contain conventional excipients, such as binding agents, position of this invention is administered, if necessary. fillers, diluents, tableting agents, lubricants, disinte Subsequently, the dosage or the frequency of adminis grants, and wetting agents. The tablet may be coated tration, or both, may be reduced, as a function of the according to methods well known in the art. Suitable 15 symptoms, to a level at which the improved condition is fillers which may be employed include cellulose, manni retained. When the symptoms have been alleviated to tol, lactose and other similar agents. Suitable disinte grants include, but are not limited to, starch, polyvinyl the desired level, treatment should cease. Patients may, pyrrolidone and starch derivatives, such as sodium however, require intermittent treatment upon any re starch glycolate. Suitable lubricants include, for exam. 20 currence of disease symptoms. pie, magnesium stearate. Suitable wetting agents in According to an alternate embodiment of this inven clude sodium lauryl sulfate. tion, thrombin inhibitors may be used in compositions Oral liquid preparations may be in the form of aque and methods for coating the surfaces of invasive de ous or oily suspensions, solutions, emulsions, syrups or vices, resulting in a lower risk of clot formation or elixirs, or may be presented as a dry product for recon- 25 platelet activation in patients receiving such devices. stitution with water or another suitable vehicle before Surfaces that may be coated with the compositions of use. Such liquid preparations may contain conventional this invention include, for example, prostheses, artificial additives. These include suspending agents; such as valves, vascular grafts, stents and catheters. Methods sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl and compositions for coating these devices are known cellulose, carboxymethylcellulose, aluminum stearate 30 to those of skill in the art. These include chemical cross gel or hydrogenated edible fats; emulsifying agents linking or physical adsorption of the thrombininhibitor which include lecithin, sorbitan monooleate, polyethyl containing compositions to the surfaces of the devices. ene glycois, or acacia; non-aqueous vehicles, such as According to a further embodiment of the present almond oil, fractionated coconut oil, and oily esters; and invention, thrombin inhibitors may be used for ex vivo preservatives, such as methyl or propyl p-hydroxyben- 35 thrombus imaging in a patient. In this embodiment, the Zoate or sorbic acid. thrombin inhibitor is labelled with a radioisotope. The Compositions formulated for topical administration choice of radioisotope is based upon a number of well may, for example, be in aqueous jelly, oily suspension or known factors, for example, toxicity, biological half-life emulsified ointment form. and detectability. Preferred radioisotopes include, but The dosage and dose rate of the thrombin inhibitor 40 are not limited to, 125I, 123I and 111 In. Techniques for will depend on a variety of factors, such as the size of labelling the thrombin inhibitor are well known in the the patient, the specific pharmaceutical composition art. Most preferably, the radioisotope is 123 and the used, the object of the treatment, i.e., therapy or pro labelling is achieved using 123I-Bolton-Hunter Reagent. phylaxis, the nature of the thrombotic disease to be The labelled thrombin inhibitor is administered to a treated, and the judgment of the treating physician. 45 patient and allowed to bind to the thrombin contained According to the present invention, a preferred phar in a clot. The clot is then observed by utilizing well maceutically effective daily dose of the thrombininhibi known detecting means, such as a camera capable of tor of this invention is between about 0.5 nmoles/kg detecting radioactivity coupled to a computer imaging body weight of the patient to be treated (“body system. This technique also yields images of platelet weight”) and about 2.5 umoles/kg body weight. In 50 bound thrombin and neizothrombin. combinations containing a thrombolytic agent, a phar This invention also relates to compositions containing maceutically effective daily dose of the thrombolytic is the thrombin inhibitors of this invention and methods between about 10% and 80% of the conventional dos for using such compositions in the treatment of tumor age range. The “conventional dosage range' of a metastases. The efficacy of the thrombin inhibitors of thrombolytic agent is the daily dosage used when that 55 this invention for the treatment of tumor metastases is agent is employed in a monotherapy. Physician's Desk manifested by the inhibition of metastatic growth. This Reference 1989, 43rd Edition, Edward R. Barnhart, is based upon the presence of a procoagulant enzyme in publisher. That conventional dosage range will, of certain cancer cells. This enzyme activates the conver course, vary depending on the thrombolytic agent em sion of Factor X to Factor Xa in the coagulation cas ployed. Examples of conventional dosage ranges are as 60 cade, resulting in fibrin deposition which, in turn, serves follows: urokinase-500,000 to 6,250,000 units/patient; as a substrate for tumor growth. By inhibiting fibrin streptokinase-140,000 to 2,500,000 units/patient; deposition through the inhibition of thrombin, the mole tPA-0.5 to 5.0 mg/kg body weight; ASPAC-0.1 to cules of the present invention serve as effective anti 10 units/kg body weight. metastatic tumor agents. Examples of metastatic tumors Most preferably, the therapeutic and prophylactic 65 which may be treated by the thrombin inhibitors of this compositions of the present invention comprise a dos invention include, but are not limited to, carcinoma of age of between about 5 nmoles/kg body weight and the brain, carcinoma of the liver, carcinoma of the lung, about 250 nmoles/kg body weight of the thrombin in Osteocarcinoma and neoplastic plasma cell carcinoma. 5,425,936 15 16 The invention also relates to methods and composi for illustrative purposes only and are not to be con tions employing the above-described thrombin inhibi strued as limiting this invention in any manner. tors to inhibit thrombin-induced endothelial cell activa tion. This inhibition includes the repression of platelet EXAMPLE 1. activation factor (PAF) synthesis by endothelial cells. 5 Design Of A Thrombin Inhibitor Capable Of Blocking These compositions and methods have important appli The Catalytic Site And Binding To The Anion Binding cations in the treatment of diseases characterized by Exosite thrombin-induced inflammation and edema, which is Carboxy terminal hirudin peptides effectively block thought to be mediated be PAF. Such diseases include, thrombin-catalyzed fibrinogen hydrolysis, but not chro but are not limited to, adult respiratory distress syn O drome, septic shock, septicemia and reperfusion dam mogenic substrate hydrolysis J. M. Maraganore et al., age. J. Biol. Chem, 264, pp. 8692-98 (1989). In addition, Early stages of septic shock include discrete, acute hirudin peptides do not neutralize thrombin-catalyzed inflammatory and coagulopathic responses. It has previ activation of Factors V and VIII J. W. Fenton, II, et ously been shown that injection of baboons with a lethal 15 al., “Hirudin Inhibition by Thrombin', Angio. Archiv. dose of live E. coli leads to marked declines in neutro Biol, 18, p. 27 (1989). phil count, blood pressure and hematocrit. Changes in Hirudin peptides, such as Tyré3-O-sulfate-N-acetyl blood pressure and hematocrit are due in part to the -hirudins3-64 (“hirugen”), exhibit potent inhibitory ef generation of a disseminated intravascular coagulopa fects toward thrombin-induced platelet activation in thy (DIC) and have been shown to parallel consump vitro J. A. Jakubowski and J. M. Maraganore, "Inhibi tion of fibrinogen F. B. Taylor et al., “Protein C Pre tion of Thrombin-Induced Platelet Activities By A vents the Coagulopathic and Lethal Effects of Escher Synthetic 12 Amino Acid Residue Sulfated Peptide ichia coli Infusion in the Baboon', J. Clin. Invest, 79, pp. (Hirugen)', Blood, p. 1213 (1989). Nevertheless, a 918–25 (1987). Neutropenia is due to the severe inflam thrombin inhibitor capable of blocking the active site matory response caused by septic shock which results in 25 may be required for inhibition of platelet thrombosis in marked increases in tumor necrosis factor levels. The vivo, if activation of Factors V and VIII are critical and rate-limiting steps. This conclusion is warranted from thrombin inhibitors of this invention may be utilized in results obtained with the irreversible thrombin inhibitor compositions and methods for treating or preventing (D-Phe)-Pro-Arg-CH2Cl S. R. Hanson and L. A. DIC in septicemia and other diseases. 30 This invention also relates to the use of the above Harker, “Interruption of Acute Platelet-Dependent described thrombin inhibitors, or compositions com Thrombosis by the Synthetic Antithrombin D prising them, as anticoagulants for extracorporeal Phenylalanyl-L-Prolyl-L-Arginyl Chloromethyl Ke blood. As used herein, the term "extracorporeal blood” tone', Proc. Natl. Acad. Sci. USA, 85, pp. 3184-88 includes blood removed in line from a patient, subjected 35 (1988) and other reversible thrombin inhibitors J. F. to extracorporeal treatment, and then returned to the Eidt et al., “Thrombin is an Important Mediator of patient in such processes as dialysis procedures, blood Platelet Aggregation in Stenosed Canine Coronary filtration, or blood bypass during surgery. The term also Arteries with Endothelial Injury”, J. Clin. Invest, 84, includes blood products which are stored extracorpore pp. 18-27 (1989)). ally for eventual administration to a patient and blood Using the above knowledge that the NH2-terminus of collected from a patient to be used for various assays. hirudin peptides is proximal to Lys-149, we employed a Such products include whole blood, plasma, or any three-dimensional model of thrombin B. Furie, et al., blood fraction in which inhibition of coagulation is “Computer-Generated Models of Blood Coagulation desired. Factor Xa, Factor IXa, and Thrombin Based Upon The amount or concentration of thrombin inhibitor in 45 Structural Homology with Other Serine Proteases', J. these types of compositions is based on the volume of Biol. Chem, 257, pp. 3875-82 (1982) to design an agent blood to be treated or, more preferably, its thrombin which: 1) binds to the anion binding exosite of throm content. Preferably, an effective amount of a thrombin bin; and, 2) is capable of blocking the active site pocket inhibitor of this invention for preventing coagulation in of thrombin and inhibiting the function of catalytic extracorporeal blood is from about 0.5 nmoles/60 ml of 50 residues contained therein. extracorporeal blood to about 2.5 moles/60 ml of We determined that the minimal distance from the extracorporeal blood. e-NH2 of Lys-149 to the g-hydroxylate of Ser-195 is The thrombin inhibitors of this invention may also be 18-20A. Based on a 3A/amino acid residue length, we used to inhibit clot-bound thrombin, which is believed calculated that at least about 4-7 amino acids would be to contribute to clot accretion. This is particularly im 55 required to linkahirudin peptide, such as Tyrs3-O-sul portant because commonly used anti-thrombin agents, fate-hirudins3-64, to a domain comprising an active-site such as heparin and low molecular weight heparin, are inhibitor structure. The composition of the linker was ineffective against clot-bound thrombin. designed to be glycine. Glycine was chosen in order to Finally, the thrombin inhibitors of this invention may engineer the greatest flexibility of a linker for these be employed in compositions and methods for treating 60 preliminary investigations. It should be understood, neurodegenerative diseases. Thrombin is known to however, that other, more rigid biopolymer linkers may cause neurite retraction, a process suggestive of the also be employed. rounding in shape changes of brain cells and implicated We chose the sequence (D-Phe)-Pro-Arg-Pro as the in neurodegenerative diseases, such as Alzheimer's dis active site inhibitor because thrombin exhibits specific ease and Parkinson's disease. 65 ity for Arg as the P1 amino acid in the cleavage of sub In order that the invention described herein may be strates. A Pro following the Arg (the P1 amino acid) more fully understood, the following examples are set yields a bond that is cleaved very slowly by thrombin. forth. It should be understood that these examples are We designed alternate peptides by replacing that Pro 5,425,936 17 18 with a sarcosyl- or N-methyl-alanine amino acid or by Rydel et al., “The Structure of a Complex of Recombi chemical reduction of an Arg-Gly scissile bond. nant Hirudin and Human o-Thrombin', Science, 249, EXAMPLE 2 pp. 277-80, (1990) complexes. As shown in FIG. 1, the structure of Hirulog-8 bound to thrombin was resolved Synthesis Of Hirulog-8 5 allowing resolution of the D-Phe-Pro-Arg sequence of Hirulog-8 has the formula: H-(D-Phe)-Pro-Arg-Pro the CSDM and the Asp-Phe-Glu-Glu-Ile segment of (Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu the ABEAM. Tyr-Leu-OH. We synthesized Hirulog-8 by conven In FIG. 1, thrombin is displayed in white, except for tional solid-phase peptide synthesis employing an Ap its active site, which is shown in dense dots. Hirulog-8 plied Biosystems 430 A Peptide Synthesizer. This pep-10 is depicted in sparse dots. The left-hand portion of Hiru tide was synthesized using BOC-L-Leucine-O-divinyl log-8 nearest the thrombin active site is the CSDM. The benzene resin. Additional t-BOC-amino acids (Penin right-hand portion is the ABEAM. Other amino acids sula Laboratories, Belmont, Calif.) used included BOC O-2,6-dichlorobenzyl tyrosine, BOC-L-glutamic acid of Hirulog-8 are not shown in FIG. 1 because electron (y-benzyl ester), BOC-L-proline, BOC-L-isoleucine, 15 densities corresponding to then could not be assigned. BOC-L-phenylalanine, BOC-L-aspartic acid (3-benzyl Examination of the CSDM portion of the Hirulog ester), BOC-glycine, BOC-L-asparagine, BOC-D- 8:thrombin structure showed the positioning of the phenylalanine, and BOC-L-arginine. In order to 1-position amino acid (D-Phe) in a hydrophobic pocket achieve higher yields in synthesis, the (Gly)4 linker formed by His57, Tyró0A, Trp60D, Leu99, Ile174, and segment was attached in two cycles of manual addition 20 Trp215 of thrombin. The D-Phe residue formed close of BOC-glycylglycine (Beckman Biosciences, Inc., van der Waals contacts with Leu99, Ile174, and Trp215 Philadelphia, Pa.). After completion of synthesis, the (FIG. 2). In FIG. 2, thrombin is displayed in solid lines peptide was fully deprotected and uncoupled from the and Hirulog-8 is in dashed lines. The positioning of the divinylbenzene resin by treatment with anhydrous HF: D-Phe residue within the pocket suggested that substi p-cresol: ethylmethylsulfate (10:1:1, v/v/v). Following 25 tutions at the 1-position which enhance lipophilic removal from the resin, the peptide was lyophilized to contacts would lead to an increased binding affinity of dryness. the CSDM moiety in the thrombin inhibitors of this Crude Hirulog-8 was purified by reverse-phase invention. Accordingly, we replaced the D-Phe residue HPLC employing an Applied Biosystems 151A liquid of Hirulog-8 with either D-napthylalanine (D-NPA) or chromatographic system and a Vydac C18 column 30 D-cyclohexylalanine (D-Cha) to form D-NPA (2.2X25 cm). The column was equilibrated in 0.1% Hirulog-8 and D-Cha-Hirulog-8, respectively. TFA/water and developed with a linear gradient of The binding of the CSDM of Hirulog-8 to the cata increasing acetonitrile concentration from 0 to 80% over 45 minutes in the 0.1% TFA at a flow-rate of 4.0 lytic site of thrombin was also found to include apolar ml/min. The effluent stream was monitored for absor- 35 interactions between the first proline of the inhibitor bance at 229 nm and fractions were collected manually. (adjacent to D-Phe) and a pocket defined by His57, We purified 25-30 mg of crude Hirulog-8 by HPLC and Tyró0A and Trp60D of thrombin (FIG. 2). Further recovered 15-20 mg of pure peptide. more, that proline was found to be within 3.46A of the We confirmed the structure of purified Hirulog-8 by phenolic hydroxyl group of thrombin Tyró0A (FIG. 2). amino acid and sequence analyses. Amino acid hydroly- 40 In FIG. 2, thrombin is depicted in solid lines and Hiru sates were prepared by treating the peptide with 6.N log-8 in dashed lines. HCl, in vacuo, at 110° C. for 24 hrs. We then analyzed The proximity of this proline to Tyró0A of thrombin the hydrolysates by ion-exchange chromatography and suggested the potential for hydrogen bond formation subsequent ninhydrin derivatization/detection using a between the two. By substituting proline with an amino Beckman 6300 automated analyzer. We performed se- 45 acid capable of forming hydrogen bonds, the stability of quence analysis using automated Edman degradation on the CSDM binding to the thrombin active site can be an Applied Biosystems 470A gas-phase sequencer increased. This, in turn, would increase the inhibitory equipped with a Model 900A data system. Phenylthi activity of such a molecule. Therefore, we replaced the ohydantoin (PTH) amino acids were analyzed on-line proline of Hirulog-8 with either L-histidine (His2 using an Applied Biosystems 120A PTH-analyzer and a 50 Hirulog-8), L-thioproline (TPro2-Hirulog-8) or isonipe PTH-C8 column (2.1X220 mm). cotic acid (Inp2-Hirulog-8). Each of these substitutions EXAMPLE 3 created a hydrogen bond acceptor at the 2-position (R4 component) of the thrombin inhibitors of this invention Design Of 1- And 2-Position Substituted Hirulogs (i.e., an imidazolium nitrogen, a thiol and a carboxylate, We obtained the X-ray crystallographic structure of 55 respectively). the Hirulog-8:thrombin complex by the following steps. First, we generated Hirulog-8:thrombin complex crys EXAMPLE 4 tals of suitable quality to obtain a high resolution dif Synthesis of 1-Position Substituted Hirulogs fraction pattern. We next collected diffractometer data employing these crystals. Finally, we determined the 60 D-Npa-Hirulog-8 was synthesized in the same man 3-dimensional structure of the Hirulog-8:thrombin com ner as Hirulog-8 (Example 2), except that Boc-D-map plex using molecular replacement rotation/translation thylalanine (Bachem Inc., Torrance, Calif.) was en methods employing the coordinates of the PPACK:th ployed in place of D-Phe at the last cycle of synthesis. rombin W. Bode et al., “The Refined 1.9A Crystal D-Cha-Hirulog-8 was similarly prepared using Boc-D- Structure of Human cy-Thrombin: Interaction. With 65 cyclohexylalanine (Bachem Biosciences, Philadelphia, D-Phe-Pro-Arg-Chloromethylketone and Significance Pa.) at the last cycle of synthesis. of the Tyr-Pro-Pro-Trp Insertion Segment”, EMBO.J., Both 1-position substituted peptides were purified as 8, pp. 3467-75 (1989) and hirudin:thrombin T. J. described for Hirulog-8 in Example 2. The purified 5,425,936 19 20 - - - - peptides were characterized by amino acid analysis and D-Cha increases the binding affinity of the CSDM in by FAB-MS. the inhibitors of this invention. The failure of D-Npa Hirulog-8 to decrease Kindicates that the presence of a EXAMPLE 5 saturated ring structure at this position causes the in Synthesis of 2-Position Substituted Hirulog Derivatives creased binding affinity. D-Cha contains such a satu Substitutions at the 2-position were designed having rated ring, while D-Npa contains an unsaturated ring. the formula: (D-Cha)-X-Arg-HPro-(Gly)4-Asn-Gly Molecules containing the 2-position substitutions de Asp -Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu or (D- scribed in Example 5 will display similar surprising and Phe)-X-Arg-HPro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu unexpected decreases in Ki. Ile-Pro-Glu-Glu-Tyr-Leu, wherein X is either histidine, O thioproline or isonipecotic acid. These peptides are EXAMPLE 7 f synthesized essentially as described in Example 2 and Anticoagulant Activity of A1-Substituted Hirulogs Example 4 except for incorporation of Boc-L-hydroxy proline (Bachem, Inc.) instead of Boc-L-proline at cycle We compared the anticoagulant activity of Tyré3-O- 16 of the synthesis and either Boc-N-im-CBZ-L-histi 15 sulfate-N-acetyl-hirudins3-64 ("hirugen'), recombinant dine, Boc-L-thioproline, or Boc-isonipecotic acid (all hirudin (American Diagnostica), Hirulog-8, and the obtained from Bachem, Inc.) instead of Boc-L-proline 1-position substituted hirulogs of the present invention at cycle 18. HPro is used at the 4-position to slow down using pooled, normal human plasma (George King Bio the rate of cleavage of the inhibitor by thrombin. The medical, Overland Park, Kans.) and a Coag-A-Mate XC peptides are purified and characterized as described in instrument (General Diagnostics, Organon Technica, Example 2. Oklahoma City, Okla.). Activity was monitored using the activated partial thromboplastin time (APTT) assay EXAMPLE 6 with CaCl2 and phospholipid solutions obtained from Characterization Of Antithrombin Activities Of the manufacturer. Recombinant hirudin (American 1-Position Substituted Hirulogs 25 Diagnostica), Hirulog-8, D-Cha-Hirulog-8 or hirugen We compared the inhibition of thrombin-catalyzed was then added to the APTT determination wells at a hydrolysis of Spectrozyme TH (tosyl-Gly-Pro-Arg-p- final concentrations of 10 to 32,300 ng/ml in a total nitroanilide; American Diagnostica, New York, N.Y.) volume of 25 pull prior to addition of 100 pil of plasma. of Hirulog-8, D-Cha-Hirulog-8 and D-Npa-Hirulog-8 in As shown in FIG. 3, D-Cha-Hirulog-8 prolonged an assay. Specifically, we measured the initial rate ve APTT to 470% of control values at a concentration of locities in the presence or absence of each inhibitor over 1 g/ul. This increase was significantly greater than the a range of substrate concentrations from 2.2 to 22 u.M. increases in APTT caused by hirugen, recombinant The thrombin-catalyzed rate was monitored in a Cary hirudin or Hirulog-8 at the same concentration. Thus, in 19 spectrophotometer at 405 nm and recorded continu addition to showing enhanced antithrombin activities in ously as a function of time. Kinetics were performed at 35 vitro over Hirulog-8, D-Cha-Hirulog-8 also showed a room temperature (25-1 C.) in a 0.05M sodium bo significantly increased anticoagulant effect in plasma rate buffer, pH 8.4, containing 0.1M NaCl. assays over Hirulog-8. For a typical enzyme reaction, 1.0 ml of buffer was Molecules containing the 2-position substitutions de added to both the sample and reference cuvettes. scribed in Example 5 will display increases in APTT Thrombin (3.2X 109M, final concentration) and the that are greater than Hirulog-8. hirulog (0-4x108M) were added to the sample cu While we have hereinbefore presented a number of vette prior to addition of Spectrozyme TH (2.2-22 embodiments of this invention, it is apparent that our uM). Immediately following addition of substrate, the basic construction can be altered to provide other em contents of the sample cuvette were mixed by use of a 45 bodiments which utilize the molecules, compositions, plastic pipette. The reaction was monitored spectropho combinations and methods of this invention. Therefore, tometrically for 5-15 minutes. it will be appreciated that the scope of this invention is Initial rate velocities at each substrate concentration were expressed as moles Spectrozyme TH hy to be defined by the claims appended hereto rather than drolyzed/sec/mole thrombin. This was determined the specific embodiments which have been presented during the initial linear phase of the reaction (s.15% 50 hereinbefore by way of example. total hydrolysis of substrate) by measuring the slope of We claim: the hydrolytic reaction. Lineweaver-Burke plots were 1. A catalytic site-directed thrombin inhibitor charac constructed accordingly, by plotting the inverse of the terized by the formula: initial velocity against the inverse of the substrate con centration. Shown below are the inhibitory constants 55 obtained for Hirulog-8 and derivatives of the present x--R-R-R-R-y invention. R1 wherein X is HN; R1 is cyclohexyl; R2 is CH2-CH; R3 Derivative Ki, nM is C=O; R4 is Pro or any amino acid comprising a side Hirulog-3 1.4 chain group characterized by the capacity to accept a D-Cha-Hirulog-8 0.12 hydrogen bond at a pH of between 5.5 and 9.5; R5 is any D-Npa-Hirulog-8 4.3 L-amino acid which comprises a guanidinium- or amino-containing side chain group; R6 is a non-amide AS can be seen from these results, substitution of D-Phe 65 bond that retards or prevents the cleavage of said inhibi in Hirulog-8 with D-Cha results in a surprising and tor by thrombin; and Y is selected from the group con unexpected decrease in Ki by one order of magnitude. sisting of sarcosine, N-methyl alanine, hydroxyproline This finding indicates that substitution of D-Phe by and any naturally occurring L-a-amino acid; and the 5,425,936 21 22 amino acid defined by components X, R1, R2 and R3 is pharmaceutically acceptable buffer and a thrombin in the D configuration. inhibitor according to claim 4. 2. The thrombin inhibitor according to claim 1, hav- 6. A method for imaging of a fibrin or a platelet ing the amino acid sequence: thrombus in a patient comprising the steps of: 5 (a) administering to said patient a composition ac D-Cha-Pro-Arg-Pro. cording to claim 5; and (b) using ex vivo detecting means to observe the 3. The thrombin inhibitor according to claim 1, thrombin inhibitor present in said composition. wherein R is selected from the group consisting of 7. The method according to claim 6, wherein said histidine, thioproline and isonipecotic acid. 10 patient is a human. 4. The thrombin inhibitor according to claim 1, 8. The thrombin inhibitor according to claim 4, wherein said inhibitor is labelled with a radioisotope. wherein said radioisotope is selected from the group 5. A composition for imaging of a fibrin or a platelet consisting of 123, 125I and 111 In. thrombus in a patient, said composition comprising a k : :: *k 5

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