COMBINATIONS AND METHODS FOR TREATING OR PREVENTING THROMBOTIC DISEASES

Abstract: The present invention relates to combinations and methods which are effective in thrombolytic therapy and prophylaxis. More particularly, this invention relates to pharmaceutically effective combinations of a) a peptide which is homologous to at least a portion of the carboxyl terminal 25 amino acids of hirudin, or a derivative thereof, which displays anticoagulant activity ("anticoagulant peptide" or "hirudin peptide"); and b) a thrombolytic agent for treating or preventing thrombotic diseases. This invention also relates to methods for decreasing reperfusion time, or increasing reocclusion time, or both, in a patient treated with a thrombolytic agent by administering to the patient a combination of a thrombolytic agent and an anticoagulant peptide. And the invention relates to methods for decreasing the dosage of a thrombolytic agent required for a desired therapeutic or prophylatic effect in a patient, such as to dissolve a blood clot, by administering to the patient a combination of an anticoagulant peptide and a thrombolytic agent, the dosage of the thrombolytic agent being less thanthat required for a desired therapeutic or prophylactic effect when that agent is administered as a monotherapy.

Description: COMBINATIONS AND METHODS FOR TREATING OR PREVENTING THROMBOTIC DISEASES TECHNICAL FIELD OF THE INVENTION The present invention relates to combina¬ tions and methods which are effective in thrombolytic therapy and prophylaxis. More particularly, this invention relates to pharmaceutically effective combinations of a) a peptide which is homologous to at least a portion of the carboxyl terminal 25 amino acids of hirudin, or a derivative thereof, which displays anticoagulant activity ("anticoagulant pep¬ tide" or "hirudin peptide"); and b) a thrombolytic agent for treating or preventing thrombotic diseases. This invention also relates to methods for decreasing reperfusion time, or increasing reocclusion time, or both, in a patient treated with a thrombolytic agent by administering to the patient a combination of a thrombolytic agent and an anticoagulant peptide. And the invention relates to methods for decreasing the dosage of a thrombolytic agent reguired for a desired therapeutic or prophylactic effect in a patient, such as to dissolve a blood clot, by- administering to the patient a combination of an anticoagulant peptide and a thrombolytic agent, the dosage of the thrombolytic agent being less than that required for a desired therapeutic or pro¬ phylactic effect when that agent is administered as a monotherapy. BACKGROUND ART Acute vascular diseases, such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, and other blood system thromboses constitute major health risks. Such diseases are caused by either partial or total occlusion of a blood vessel by a blood clot, which consists of fibrin and platelet aggregates. Current methods for treatment and prophy¬ laxis of thrombotic diseases involve therapeutics which act in one of two different ways. The first type of therapeutic inhibits thrombin activity or thrombin formation, thus preventing clot formation. These drugs also inhibit platelet activation and aggregation. One such drug is heparin, a compound widely used in the treatment of conditions in which thrombin activity is responsible for the development or expansion of a thrombus, such as in venous throm- boembolism. Although effective, heparin produces many undesirable side effects, including hemorrhaging and thrombocytopenia. The second category of thera¬ peutic accelerates thrombolysis and dissolves the blood clot, thereby removing it from the blood vessel and unblocking the flow of blood [V. J. Marder B.2412 and S. Sherry, "Thrombolytic Therapy, Part 1 of 2", New England J. Med., 318, pp. 1512-20 (June 9, 1988); V. J. Marder and S. Sherry, "Thrombolytic Therapy, Part 2 of 2", New England J. Med., 318, pp. 1585-95 (June 16, 1988)]. Over the past few years, the value of thrombolysis in the treatment of acute myocardial infarction has been demonstrated [TIMI Study group, New England J. Med., 320, pp. 618-27 (March 9, 1989)], although the advantage of one thrombolytic agent over another, e.g., recombinant tissue plasminogen activator (rtPA) versus streptokinase, remains unresolved [H. D. White et al., "Effect of Intravenous Streptokinase as Compared With That of Tissue Plasminogen Activator on Left Ventricular Function After First Myocardial Infarction", New England J. Med., 320, pp. 817-21 (March 30, 1989]. Numerous limitations and complications are associated with current thrombolytic therapies. These include the narrow window of time following onset of vascular occlusion in which such agents are effective in establishing reperfusion, the occurrence of rethrombosis following reperfusion (especially evident in myocardial infarction) and bleeding asso- ciated with administration of thrombolytic agents. These drawbacks often counterbalance the advantages of thrombolysis over more conventional therapeutic regimens and make administration of high dosages of thrombolytic agents impractical. The factors which influence rethrombosis are not well understood. Despite the use of antico¬ agulants as adjuncts in thrombolytic therapy, rethrombosis occurs in 10-20% of reperfused arteries. Heparin, the anticoagulant of choice, appears to have no effect on the rate of rethrombosis. More¬ over, this agent may contribute significantly to the incidence of bleeding [G. C. T±mmis et al., B.2412 "Hemorrhage vs. Rethrombosis After Thrombolysis for Acute Myocardial Infarction", Arch. Intern. Med., 146, pp. 667-72 (1986)]. In contrast to heparin, adjunct use of antiplatelet drugs in thrombolytic therapy has proven somewhat successful in both decreasing reperfusion times and preventing rethro oosis in the treatment of experimental and clinical myocardial infarctions. In one study, a combination of streptokinase and aspirin resulted in a reduction in rethrombosis by 10% or 1%, compared with either streptokinase or aspirin therapy alone [ISIS-2 Collaborative Group, "Randomized Trial of Intravenous Streptokinase, Oral Aspirin, Both, or Neither Among 17,187 Cases of Suspected Acute Myocardial Infarction: ISIS-2", Lancet, 13, pp. 349-60 (1988)]. Other antiplatelet drugs which alter platelet eicosanoid metabolism have been tested in adjunct use with thrciibolytic agents in pre-clinical models of coronary arterial occlusion. Thromboxane A 2 (TXA 2 ) receptor antagonists or serotonin receptor antagonists, alone or together, substantially increases reocclusion time when combined with recombinant tPA (rtPA) [P. Golino et al., "Mediation of Reocclusion by Thromboxane A 2 and Serotonin After Thrombolysis With Tissue-Type Plasminogen Activator in a Canine Preparation of Coronary Thrombosis", Circulation, 77, pp. 678-84 (1988)]. In a model of rabbit jugular vein throm- bosis, prostaglandin E (PGE) was found to modestly decrease reperfusion time when used with rtPA [D. E. Vaughan et al. "PGE Accelerates Thrombolysis by Tissue Plasminogen Activator", Blood, 73, pp. 1213-17 (1989)]. Despite the efficacy of these platelet inhibitor/thrombolytic agent combinations, the adverse effect of antiplatelet agents in promoting vasodilation, hypotension and bleeding B.2412 restricts the use of such combinations in a human clinical setting. Antiplatelet agents which are antagonists of the platelet fibrinogen receptor, glycoprotein Ilb/IIIa (GPIIb/IIIa), have also been tested in com¬ bination with fibrinolytic agents. Administration of rtPA together with an anti-GPIIb/IIIa monoclonal antibody was found to attenuate thrombolysis of experimental coronary thrombi, increase reocclusion time and decrease reperfusion time in dogs [T. Yasuda et al., "Monoclonal Antibody Against the Platelet Glycoprotein (GP) Ilb/IIIa Receptor Prevents Coronary Artery Reocclusion After Reperfusion with Recombinant Tissue-Type Plasminogen Activator in Dogs", J. Clin. Invest., 81, pp. 1284-91 (1988)]. However, in a canine venous thrombosis model, the same combination failed to potentiate thrombolysis [D. Spriggs et al., "Absence of Potentiation with Murine Antiplatelet GPIIb/IIIa Antibody of Thrombolysis With Recombinant Tissue-Type Plasminogen Activator (rt-PA) in a Canine Venous Thrombosis Model", Thromb. Haemostasis, 61, pp. 93-96 (1989)]. Moreover, this combination, which resulted in demonstrable increases in bleeding times, discouraged the use of the anti-GPIIb/IIIa antibody as an adjuvant in thrombolytic therapy in humans. Therefore, the role of antiplatelet drugs as useful adjuvants in thrombolytic therapy remains in doubt. Alternate strategies focusing on those components of the thrombus which influence its own enzymatic dissolution have yet to be explored. Thrombin is known to associate with a fibrin clot, potentially influencing its growth and the dynamic reconstruction of the thrombus [C. W. Francis et al., "Thrombin Activity of Fibrin Thrombi and Soluble Plasma Derivatives", J. Lab. Clin. Med., 102, pp. 220-30 (1983); C. Y. Liu et al., "The Binding of Thrombin by Fibrin", J. Biol..Chem., 254, pp. 10421-25 B.2412 (1979)]. Upon thrombolysis, increased exposure of clot-bound thrombin may cause accelerated fibrinogen cleavage and rethrombosis. Recently, it has been demonstrated that the heparin-catalyzed inactivation of α-thrombin by antithrombin III is neutralized by fibrin II monomer, a fibrin degradation product [P. J. Hogg and C. M. Jackson, "Fibrin Monomer Protects Thrombin from Inactivation by Heparin-Antithrombin III: Implica- tions for Heparin Efficacy", Proc. Natl. Acad. Sci. USA, 86, pp. 3619-23 (1989)]. Accordingly, the efficacy of heparin in thrombolytic therapy may be counteracted by the by-products of clot dissolution. This can result in an increase in both reperfusion time and the incidence of reocclusion when heparin . is used in conjunction with a thrombolytic agent. To date, therefore, the need exists for the development of compositions, combinations and methods for the treatment or prevention of thrombotic diseases which avoid the disadvantages of conventional agents while providing effective therapy for those diseases. More particularly, the need exists for a safe and effective agent which decreases reperfusion time and increases reocclusion time when used in conjunction with a thrombolytic agent. The agent used in combination with the thrombolytic agent should not cause a significant increase in bleeding time and it should not be antigenie. DISCLOSURE OF THE INVENTION The present invention solves the problems referred to above by providing pharmaceutically effec¬ tive combinations, compositions and methods for the treatment and prevention of thrombotic diseases. According to this invention, an anticoagulant peptide which is at least partially homologous to the carboxyl terminal 25 amino acids of hirudin, or a derivative B.2412 thereof, which displays anticoagulant activity, is used in a pharmaceutically effective combination with a thrombolytic agent, for treating or prevent¬ ing thrombotic diseases. The thrombolytic agent dissolves the clot, while the anticoagulant peptide neutralizes the newly exposed thrombin, thus pre¬ venting rethrombosis. According to one embodiment of this invention, the dosage of the thrombolytic agent is less than that conventionally required for a desired therapeutic or prophylactic effect when that agent is administered as a monotherapy. This, in turn, decreases the risk of undesirable side effects associated with the use of thrombolytic agents. Moreover, the anticoagulant peptide com- ponent of the combination exhibits a- saturable effect on clotting time, resulting in a drastically reduced risk of bleeding. This inventicn also provides methods, compositions and combinations for decreasing the dose of a thrombolytic agent required for a desired therapeutic or prophylactic effect in a patient, such as dissolving a blood clot. And this invention provides methods, compositions and combinations for both increasing reocclusion time and decreasing reperfusion time in a patient treated with a throm¬ bolytic agent. As will be appreciated from the disclosure to follow, the combinations, compositions and methods of the present invention are safer and more effective in the treatment and prevention of thrombotic diseases than conventional therapies. And the combinations, compositions and methods of the present invention provide more efficient and more effective throm¬ bolytic therapy than conventional regimens. The use of the combinations, compositions and methods of this invention advantageously reduces the dosage of thrombolytic agent which wou-l^ " be required to achieve B.2412 a desired therapeutic or prophylactic effect in therapy regimens based upon the use of that agent alone. And the combinations, compositions and methods of this invention decrease reperfusion time and increase reocculsion time for a given dose of thrombolytic agent. Accordingly, the combinations, compositions and methods of this invention reduce or eliminate the potential side effects often associ¬ ated with conventional single thrombolytic agent therapies, while not interfering with the throm¬ bolytic activity of those agents. And by employing hirudin peptides as anticoagulant agents used in combination with the thrombolytic agent, the com¬ binations, compositions and methods of this inven- tion avoid the side effects of conventional anti¬ coagulants, such as heparin. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the purification of Sulfo- Tyr 63 hirudin 53 _ 64 by reverse-phase HPLC. Figure 2 depicts HPLC chromatograms illus¬ trating the relative efficiency of the sulfation process described herein, (Figure 2c) as compared with conventional sulfation processes (Figures 2a, 2b) for the treatment of large quantities of peptide. Figure 3 depicts the HPLC chromatographic elution profile of a mixture of Sulfonyl-Tyr 63 hirudin 53 _ 64 and Sulfo-Tyr 63 hirudin 53 _ 64 . Figures 4A-4C depict the synthesis of hirulog-1, hirulog-2 and hirulog-3, peptidomimetic analogs of hirudin peptides. Figure 5A depicts the synthesis of hirulog-4, a peptidomimetic analog of a hirudin peptide. Figures 6A and 6B depict the synthesis of hirulog-5 and hirulog-6, peptidomimetic analogs of hirudin peptides. B.2412 Figure 7 depicts the synthesis of hirulog-7, a peptidomimetic analog of a hirudin peptide. Figure 8 depicts the comparative effect of heparin and Sulfo-Tyr 63 -hirudin 53 _ 64 administered as either an intravenous bolus injection ("i.v.") or a constant infusion ("inf.") on in vivo fibrin accretion in rabbits. DETAILED DESCRIPTION OF THE INVENTION This invention relates to therapeutic or prophylactic combinations, compositions and methods for treating or preventing thrombotic diseases. More particularly, this invention relates to phar¬ maceutically effective combinations and compositions comprising a peptide which is homologous to at least a portion of the carboxy terminal 25 amino acids of hirudin, or a derivative thereof, which displays anticoagulant activity ("anticoagulant peptide" or "hirudin peptide") and a thrombolytic agent. Accord- ing to one embodiment of this invention, the dosage of the thrombolytic agent in the combination or composition is less than that required for a desired therapeutic or prophylactic effect when that agent is administered as a monotherapy. According to an alternate embodiment, the combinations, compositions and methods of this invention effectively decrease reperfusion time, or prevent reocclusion by increasing occlusion time, or both, for a given dose of thrombolytic agent (as compared with the corresponding times established when the thrombolytic agent is used with a conven¬ tional anticoagulant), thus minimizing the extent of tissue damage due to lack of blood flow. More specifically, anticoagulant peptides may be employed in methods, compositions and combinations for decreasing reperfusion time, i.e., the time required B.2412 to reestablish blood flow following initiation of thrombolytic therapy in a patient treated with a thrombolytic agent. Alternatively, anticoagulant peptides in combination with a thrombolytic agent may also be used in methods, compositions and combi¬ nations for increasing reocclusion time, i.e., the time in which rethrombosis of a reperfused clot or embolus occurs in a patient, or for preventing thrombin mediated rethrombosis of reperfused arterial emboli. And anticoagulant peptides may be used in methods, compositions and combinations for decreasing the dosage of a thrombolytic agent required to achieve reperfusion, avoid reocclusion or both, in a patient. The use of an anticoagulant peptide in the ' combinations, compositions and methods of this inven¬ tion advantageously permits the administration of thrombolytic agents in dosages formerly considered too low to result in thrombolytic effects if given alone. And such combinations advantageously avoid the side effects of high level dosages of throm¬ bolytic agents. The combinations, compositions and methods of this invention are useful for treating or pre- venting vascular diseases attributed to blood system thromboses that may arise from any disease state. Examples of such thrombotic diseases include, but are not limited to, myocardial infarction, deep venous thrombosis, pulmonary embolism, and other peripheral vascular thromboembolic occlusions. The methods, combinations and compositions of this invention may be used for the treatment or pre¬ vention of thrombotic diseases in patients including mammals and, in particular, humans. The methods of this invention comprise the step of treating a patient in a pharmaceutically acceptable manner with B.2412 a pharmaceutically effective combination of an anticoagulant peptide and a thrombolytic agent for a period of time sufficient to prevent or lessen the effects of thrombotic disease. The following common abbreviations of amino acids are used throughout the present application: Cha - cyclohexylalanine Orn - ornithine Git - glutaryl Mai - maleyl NpA - beta-(2-naphthyl)alanine Gly - glycine Ala - alanine Val - valine Leu - leucine lie - isoleucine Pro - proline Phe - phenylalanine Trp - tryptophan Met - methionine Ser - serine Thr - threonine' Cys - cysteine Tyr - tyrosine Asn - asparagine Gin - glutamine Asp - aspartic acid Glu - glutamic acid Lys - lysine Arg - arginine His - histidine Nle - norleucine Hyp - hydroxyprolme Pgl - phenylglycine Tyr(S0 3 H) - tyrosine sulfonate D-Ala - D-alanine Ac - acetyl Sue - succinyl 3,4,-dehydroPro — 3,4-dehydroproline Tyr(OS0 3 H) — O-sulfate ester of tyrosine NMePgl — N-methyl-phenylglycine Sar — sarcosine (N-methylglycine) SubPhe — ortho, meta, para, mono- or di-substi- tuted phenylalanine pSubPhe — para substituted phenylalanine pClPhe — para-chloro-phenylalanine pN0 2 Phe — para-nitro-phenylalanine. As used in this application, an "alkyl group" and the "alkyl portion of an alkoxy group" includes straight, branched, or cyclic alkyl groups; for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl , pentyl, isopentyl, sec-pentyl, B.2412 cyclopentyl, hexyl, isohexyl, cyclohexyl and cyclo- pentyl ethyl. An "acyl group" of from 2 to 10 carbon atoms includes straight, branched, cyclic, saturated and unsaturated acyl groups having 1 or 2 carbonyl moieties per group — for example acetyl, benzoyl, maleyl, glutaryl and succinyl. A "halogen group" is a fluoro, chloro, bromo or iodo group. The term "any amino acid" as used herein includes the L-isomers of the naturally occurring amino acids, as well as other "non-protein" α-»amino acids commonly utilized by those in the peptide chemistry arts when preparing synthetic analogues of naturally occurring amino peptides. The "naturally occurring amino acids" are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine; phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glu¬ tamic acid, glutamine, arginine, ornithine and lysine. Examples of "non-protein" α-amino acids are norleucine, norvaline, alloisoleucine, homoarginine, thiaproline, dehydroproline, hydroxyproline (Hyp), homoserine, cyclohexylglycine (Chg), a-amino-n-buty- ric acid (Aba), cyclohexylalanine (Cha), aminophenyl- butyric acid (Pba), phenylalanine substituted at the ortho, meta, or para position of the phenyl moiety with one or two of the following: a (C 1 -C 4 ) alkyl, a (C,-C 4 ) alkoxy, halogen or nitro groups or substi¬ tuted with a methylenedioxy group, β-2- and 3-thieny- lal-alanine, β-2- and 3-furany1alanine, β-2-, 3- and 4-pyridylalanine, β-(benzothienyl-2- and 3-yl)alanine, β-(l- and 2-naphthyl)alanine, O-alkylated derivatives of serine, threonine or tyrosine, S-alkylated cys¬ teine, S-alkylated homocysteine, O-sulfate, O-phos¬ phate and O-carboxylate 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, B.2412 3,5-diiodotyrosι..e, and the D-isomers of the naturally occurring amino acids. The anticoagulant peptide, i.e., hirudin peptide, used in the combinations, compositions and methods of this invention is at least partially homologous to the carboxy terminal 25 amino acids of hirudin. Specifically, the anticoagulant peptide is characterized by a sequence of amino acids consist¬ ing substantially of the formula: X-A χ -A 2 -A 3 -A 4 -A 5 -A 6 -A ? - A Q -A g -A χQ - wherein X is a hydrogen, one or two alkyl groups of from 1 to 6 carbon atoms, one or two acyl groups of from 2 to 10 carbon atoms, carbobenzyloxy or t-butyloxy carbonyl; A, is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A 2 is Phe, SubPhe, β-(2- and 3-thienyl)alanine, β-(2- and 3-furanyl)alanine, β-(2-, 3-and 4-pyridyl)alanine, β-(benzothienyl-2- and 3-yl) alanine, β-(l- and 2-naphthyl)alanine, Tyr or Trp; A 3 is Glu or Asp; A 4 is any amino acid; A-. is lie, Val, Leu, Nle or Phe; A, is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carb- oxylate, Sar, NMePgl or D-Ala; A.-, is any amino acid; Ag is any amino acid; A g is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, lie, Val, Cha, Pro, or a dipeptide con¬ sisting of one of these lipophilic amino acids and any amino acid; A, Q is a bond or a peptide containing from one to five residues of any amino acid; and Y is a carboxy terminal residue selected from OH, C.-Cg alkoxy, amino, mono- or di-(C.-C 4 ) alkyl substituted amino or benzylamino. Preferably, the anticoagulant peptide employed in the combinations, compositions and methods of the present invention is characterized in that X is hydrogen or N-acetyl; A χ is Asn-Gly-Asp; A 2 is Phe; A 3 is Glu; A 4 is Glu; A-. is lie; Ag is Pro; A ? is Glu; A g is Glu; A gf -is a dipeptide selected B.2412 from the group consisting of S-alkylated cysteine-Leu, S-alkylated homocysteine-Leu, tyrosine-O-sulfate-Leu, tyrosine-O-phosphate-Leu, tyrosine-O-carboxylate-Leu, 3-sulfonyl tyrosine-Leu, 5-sulfonyl tyrosine-Leu, 3-carbonyl tyrosine-Leu, 5-carbonyl tyrosine-Leu, 3-phosphonyl tyrosine-Leu, 5-phosphonyl tyrosine-Leu, 4-methylsulfonyl tyrosine-Leu, 4-methylphosphonyl tyrosine-Leu, 4-phenylacetic acid-Leu, and 3- or 5- nitrotyrosine-Leu and 3,5-diiodotyrosine-Leu; A, Q is a bond; and Y is OH. These preferred peptides exhibit a higher anticoagulant activity than the other peptides. Most preferably, the anticoagulant peptide employed in the combinations, compositions and methods of this invention is characterized in that X is N-acetyl; A-, is Asn-Gly-Asp; A 2 is Phe; A 3 is Glu; A 4 is Glu; Ag is lie; Ag is Pro; is Glu; A g is Glu; Ag is the dipeptide tyrosine-O-sulfate-Leu; A, 0 is a bond; and Y is OH. This most preferred peptide advantageously displays a ten-fold greater anticoagulant activity over the other peptides. The anticoagulant peptide employed in the methods, compositions and combinations of the present invention may be prepared by a variety of techniques known to those of skill in the art. These include enyzmatic cleavage of natural hirudin. Alternatively, anticoagulant peptides may be produced directly, via recombinant DNA techniques, or by conventional chem¬ ical synthesis techniques, such as solid-phase peptide synthesis, solution-phase peptide synthesis or a combination of these techniques. Optionally, the synthesized peptides may be digested with carboxypep- tidase (to remove C-terminal amino acids) or degraded by manual Edman degradation (to remove N-terminal amino acids). Most preferably, the peptide is pro¬ duced by solid phase peptide synthesis, as described in copending United States patent applications Serial B.2412 Nos. 164,178, 251,150 and 314,756, in J. M. Maraganore et al., "Anticoagulant Activity of Synthetic Hirudin Peptides", J. Biol. Chem., 264, pp. 8692-98 (1989) and in European patent application 276,014, the disclosures of which are herein incorporated by reference. When "non-protein" amino acids are con¬ tained in the anticoagulant peptide, they may either be added directly to the growing chain during peptide synthesis or prepared by chemical modification of the complete synthesized peptide, depending on the nature of the desired "non-protein" amino acid. For example, derivatization of a tyrosine residue at position A g must be performed after peptide synthesis. Derivatization methods include, but are not limited to, sulfation, methyl sulfonation, phosphorylation, methyl phosphonation and carboxylation of the tyrosine hydroxyl group and sulfonation, phosphoration and carbonation of the tyrosine benzoyl meta carbon. Detailed techniques for sulfating, sulfonating, carboxylating, carbonylating, phosphorylating, and phosphonating a tyrosine are described in copending United States patent applications Serial Nos. 164,178, 251,150, and 314,756. Those of skill in the chemical synthesis art are well aware of which "non-protein" amino acids may be added directly and which must be synthesized following peptide synthesis. Sulfation of the anticoagulant peptide may be achieved either by a biological (enzymatic) or a chemical process. Preferably, a purified anti¬ coagulant peptide is reacted concurrently with dicyclohexylcarbodiimi.de and sulfuric acid in an organic solvent. Sulfonation of the meta carbon results as a side reaction of this sulfation process. For large-scale sulfations, the sulfation procedure is modified, so that gram quantities of the peptide are first dissolved in an organic sol- B.2412 vent, preferably dimethylformamide, and then reacted with a dehydrating agent, preferably dicyclohexyl- carbodiimide. The dehydrated tyrosine residue of the peptide is then sulfated by reaction with sul- furic acid. The reaction is complete upon formation of an insoluble dicyclohexyl urea salt. This modi¬ fication results in high yields of sulfated peptide on a large scale. This sulfation technique may be used to sulfate the tyrosine residues of any peptide or polypeptide, whether isolated and purified or present in a crude preparation. Following either sulfation reaction, the sulfated peptide may be separated from any sulfonated peptide, as well as from unreacted peptide, by HPLC, DEAE chromatography, or any of several other conventional separation techniques. Sulfation may also be achieved by reacting an anticoagulant peptide with sulfur trioxide-tri- ethylamine salt in pyridine. In addition, a tyrosyl- sulfotransferase activity, either as a crude preparation or as a purified enzyme, may be used to sulfate the tyrosine residue [R. W. H. Lee and W. B. Huttner, "Tyrosine 0 Sulfated Proteins of PC-12 Pheo Chromo Cytoma Cells and Their Sulfation By a Tyrosyl Protein Sulfo ransferase", J. Biol. Chem. , 258, pp. 11326-34 (1983)]. Phosphorylation or carboxyla¬ tion of the anticoagulant peptides may be achieved by reactions similar to those described above for sulfation, with the substitution of phosphoric acid or formic acid, respectively, for sulfuric acid. In those reactions, phosphonation or carbonation will occur, respectively, as a side reaction. Alterna¬ tively, enzymatic methods may be employed for carboxylation or phosphorylation of the anticoagulant peptides. Methyl sulfonation and methyl phosphona¬ tion of the anticoagulant peptides may be achieved B.2412 by methods well-known in the art including, but not limited to, alkylation with chlorosulfonic or chloro- phosphonic acid, respectively. The extent of the sulfation reaction may be followed spectrophotometrically. The absorbance spectra of sulfated peptides reveal a shift in maxi¬ mal absorbance from approximately 275 nm to approxi¬ mately 250-265 nm. Confirmation of derivatization may be obtained by desulfating the peptide with 30% trifluoroacetic acid at 60°C for 30 minutes. This will result in an increase of maximal absorbance back to 275 nm. Anticoagulant peptides may also be deriva¬ tized at their amino terminus by the addition of an N-acetyl group. N-acetylation may be achieved by any of a number of techniques that are known to those of skill in the art. Preferably, acetylation is achieved by using an N-acetyl amino acid deriva¬ tive in the synthesis of the peptides. Alterna- tively, N-acetylation may be achieved by reacting the peptide with acetic anhydride. The activity of the anticoagulant peptides may be assayed using any conventional technique. For example, the assay employed may use purified thrombin and fibrinogen and measures the inhibition of release of fibrinopeptides A or B by radioimmuno- assay or ELISA. Alternatively, the assay may involve direct determination of the thrombin-inhibitory activity of the peptide. Such assays measure the inhibition of thrombin-catalyzed cleavage of colori- metric substrates or, more preferably, the increase in activated partial thromboplastin times (APTT) and increase in thrombin times (TT). The latter assays measure factors in the "intrinsic" pathway of coagulation. According to an alternate embodiment of this invention, the anticoagulant employed may be a B.2412 peptidomimetic analog of any of the hirudin peptides described above. Analogs of the hirudin peptides may be either semi-peptidic or non-peptidic in nature. These analogs may be characterized by the presence of a dinitrofluorobenzyl group attached to the amino terminus of the peptides. Alternatively, these analogs are characterized by the replacement of tyro¬ sine or derivatized tyrosine, as well as any other more caboxy terminal residues, with nitroanisole. All of these analogs may be employed as the anticoag¬ ulant in the compositions, combinations and methods of this invention, in the same way as their peptide counterparts. The thrombolytic agent utilized the methods, compositions and combinations of the present invent tion may be selected from those thrombolytic agents which are known in the art. These include, but are not limited to, fibrinolytics, such as tissue plasmin¬ ogen activator purified from natural sources, recombinant tissue plasminogen activator, strepto¬ kinase, urokinase, prourokinase, anisolated strepto¬ kinase plasminogen activator complex (ASPAC), animal salivary gland plasminogen activators and known, biologically active derivatives of any of the above. According to another embodiment of this invention, the combinations for treating or preventing thrombotic disease may also comprise an antiplatelet agent. The choice of antiplatelet agent may be made from among those well known in the art. Examples of antiplatelet agents which may be employed in the combinations of this invention include prostaglandins, such as prostaglandin E and stable prostacyclin derivatives; theophylline; small platelet inhibitory peptides, such as Arg-Gly-Asp-containing peptides; cyclooxygenase inhibitors, such as aspirin; naturally occurring antiplatelet agents, such as those isolated from snake venom; small non-p.eptide platelet inhib- B.2412 itors, such as ticlopidine, dipyridamole and sulphin- pyrazone; inhibitors of platelet surface components, such as inhibitors of glycoprotein Ilb/IIIa, inhibi¬ tors of glycoprotein lb, antibodies against glycopro- tein Ilb/IIIa, and antibodies against glycoprotein lb; inhibitory eicosanoids, such as iloprost; hemato- poietic factors, such as erythropoetin; analogues of any of the above compounds and combinations of any of the above compounds. The most preferred anti- platelet agent is aspirin. The compositions and combinations used in the methods of this invention may be formulated using conventional methods to prepare pharmaceuti¬ cally useful compositions and combinations. Such compositions preferably include at least one phar-- maceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences (E. W. Martin) . In addition, the compositions and combinations preferably include a pharmaceutically acceptable buffer, preferably phosphate buffered saline, together with a pharmaceutically acceptable compound for adjusting isotonic pressure, such as sodium chloride, mannitol or sorbitol. As defined herein, the term "combination" includes a single dosage form containing at least one anticoagulant peptide and at least one throm¬ bolytic agent, a multiple dosage form wherein the anticoagulant peptide and the thrombolytic agent are administered separately but concurrently, or a multiple dosage form wherein the anticoagulant peptide and the thrombolytic agent are administered separately, but sequentially. For example, the anticoagulant peptide may be administered to a patient during the time period ranging from about 5 hours prior to about 5 hours following administration of the thrombolytic agent. Preferably, the anticoagulant,- peptide is administered B.2412 to a patient during the time period ranging from about 2 hours prior to about 2 hours following administration of the thrombolytic agent. Other administration schedules may also be employed. Alternatively, the anticoagulant peptide and the thrombolytic agent may be in the form of a single conjugated molecule. Conjugation of an anti¬ coagulant peptide to a thrombolytic agent may be achieved by standard cross-linking methods which are well known in the art. The anticoagulant peptide and the thrombolytic agent may also be present as a single molecule, i.e., in the form of a fusion pro¬ tein, produced by recombinant DNA techniques or by in vitro synthesis. The dosage and dose rate of both the anti-< coagulant peptide and the thrombolytic agent will depend on a variety of factors, such as the specific composition, the object of the treatment, i.e., therapy or prophylaxis, the nature of the thrombotic disease to be treated, and the judgment of the treating physician. Various dosage forms may be employed to administer the compositions and combina¬ tions of this invention. These include, but are not limited to, parenteral administration, oral adminis- tration and topical application. The compositions and combinations may be administered to the patient in any pharmaceutically acceptable dosage form, including those which may be administered to a patient intravenously as bolus or by continued infusion, intramuscularly — including para- vertebrally and periarticularly — subcutaneously, intracutaneously, intra-articularly, intrasynovially, intrathecally, intra-lesionally, periostally or by oral or topical routes. Such compositions and com- binations are preferably adapted for oral and parenteral administration, but, most preferably, are formulated for parenteral administration. B.2412 Parenteral compositions are most preferably administered intravenously either in a bolus form or as a constant infusion. For parenteral administra¬ tion, fluid unit dose forms are prepared which contain a composition of the present invention and a sterile vehicle. The anticoagulant peptide and thrombolytic agent components of the pharmaceutically acceptable composition may be either suspended or dissolved, depending on the nature of the vehicle and the nature of the component. Parenteral compositions are normally prepared by dissolving both the anticoagulant peptide and the thrombolytic agent in a vehicle, optionally together with other components, and filter sterilizing before filling into a suitable vial or ampule and sealing. Preferably, adjuvants such as- a local anesthetic, preservatives and buffering agents are also dissolved in the vehicle. The composition may then be frozen and lyophilized to enhance stability. Parenteral suspensions are prepared in substantially the same manner, except that one or both of the active components are suspended rather than dissolved in the vehicle. Sterilization of the compositions is preferably achieved by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the components. Tablets and capsules for oral administra- tion contain conventional excipients, such as binding agents, fillers, diluents, tableting agents, lubri¬ cants, disintegrants, and wetting agents. The tablet may be coated according to methods well known in the art. Suitable fillers which may be employed include cellulose, mannitol, lactose and other similar agents. Suitable disintegrants include, but are not limited to, starch, polyvinylpyrrolidone and starch deriva- B.2412 tives, such as sodium starch glycolate. Suit»able 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, emul¬ sions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid prepara- tions may contain conventional additives. These include suspending agents; such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats; emulsifying agents which include lecithin, sorbitan monooleate or acacia; non-aqueous vehicles, such as almond oil, fraction¬ ated coconut oil, and oily esters; and preservatives, such as methyl or propyl p-hydroxybenzoate or sorbic acid. In accordance with this invention, the anticoagulant peptide and the thrombolytic agent are administered sequentially or concurrently to the patient. The anticoagulant peptide and the thrombo¬ lytic agent may be administered to the patient at one time or over a series of treatments. More particularly, the anticoagulant peptide and the thrombolytic agent may be administered sequentially to the patient, with the anticoagulant peptide being administered before, after, or both before and after treatment with the thrombolytic agent. Concurrent administration involves treatment with the anticoagu¬ lant peptide at least on the same day (within 24 hours) of treatment with the thrombolytic agent. Other dosage regimens are also useful. According to one embodiment of this inven¬ tion, typical daily dosages of the compos _ions and combinations of the present invention include those B.2412 in which the concentration of the anticoagulant peptide is between about 0.005 mg/kg body weight of the patient to be treated ("body weight") and about 15 mg/kg body weight and in which the concentration of the thrombolytic agent is between about 10% to about 80% of the conventional dosage range. The "conventional dosage range" of a thrombolytic agent is the daily dosage of the thrombolytic agent used when that agent is administered in thrombolytic therapy as a monotherapy (Physician's Desk Reference 1989, 43rd Edition, Edward R. Barnhart, publisher). That conventional dosage range will, of course, vary depending on the thrombolytic agent employed. Examples of normal dosages ranges 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/kg body weight. Most preferably, the compositions of the present invention comprise between about 0.1 mg/kg and about 2.5 mg/kg body weight of the anticoagulant peptide and between about 10% to about 70% of the conventional dosage range of a thrombolytic agent. Alternatively, when an anticoagulant peptide is administered with a thrombolytic agent in order to decrease reperfusion time or to increase reocculsion time, or both, in a patient treated with a thrombo¬ lytic agent, conventional dosages of the thrombolytic agent may be employed in conjunction with the above- described dosages of anticoagulant peptides. In compositions according to this invention which additionally comprise an antiplatelet agent, that antiplatelet agent is preferably present in about 10% to about 70% of the conventional dosage range. For example, an anticoagulant dosage of aspirin is normally 50-300 mg/patient. B.2412 Once improvement of the patient's condition has occurred, a maintenance dose of a combination or composition of this invention is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require inter- mittent treatment upon any recurrence of disease symptoms. In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner. In all of the examples of peptide synthe¬ ses described below, we carried out amino acid analysis of the synthesized peptides. Amino acid hydrosylates were prepared by treatment of samples in 6 N hydrochloric acid, in vacuo, at 110°C for 24 hours, followed by ion-exchange chromatography employing a Beckman System 6300 analyzer. We routinely analyzed purity of the syn¬ thetic peptides by reverse-phase HPLC. Unless otherwise specified, peptide samples (20-100 μg) were applied to a Vydac C 4 column (0.46 x 25 cm) or an Aquapore RP-300 C Q column (0.46 x 3.0 cm) using a Beckman Liquid Chromatographic System or an Applied Biosystems 150A Chromatographic System, respectively. The Vydac C 4 column was equilibrated in water con¬ taining 0.1% trifluoroacetic acid (TFA) and deve¬ loped with a gradient of increasing acetonitrile concentration from 0 to 80% in the same TFA- containing solvent. The gradient was developed over 30 minutes at a flow rate of ϊ.O ml/min. The B.2412 effluent stream was monitored at 215 nm for absorb¬ ance. The Aquapore C g column was equilibrated in water containing 0.1% TFA and developed with an increasing gradient of acetonitrile concentration from 0 to 70% in a 0.085 % TFA solvent. The gra¬ dient was developed for 45 minutes at a flow rate of 0.5 ml/min. The effluent stream was then monitored at 214 nm for absorbance. EXAMPLE 1 Synthesis Of Hirudin 53 _ 64 And Hirudin 4g _ 64 Hirudin 53 _ 64 (subscript numbers represent the corresponding amino acid position in the native hirudin molecule) has the amino acid formula: H 2 N-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu- COOH. Hirudin 4 g_ 64 has the amino acid formula: H 2 N-Glu-Ser-His-Asn-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro- Glu-Glu-Tyr-Leu-COOH. We prepared these peptides as part of a single synthesis by solid-phase peptide synthesis employing an Applied Biosystems 430 A Peptide Synthesizer (Applied Biosystems, Foster City, California) . Specifically, we reacted 0.259 meq of Boc-Leu-O-resin (1% divinylbenzene resin (DVB)) sequentially with 2 mmoles of protected amino acids. Following 11 cycles of synthesis, 0.42 g of wet resin were removed from the reaction vessel. The remain¬ ing 0.43 g aliquot of wet resin was reacted with two times 2 mmoles of protected amino acids for four cycles. Hirudin 53 _ 64 and hirudin 4g _ 64 thus syn¬ thesized were fully deprotected and cleaved from the resin by treatment with anhydrous HF: p-cresol: ethyl methyl sulfate (10:1:1, v/v/v). Yield of the peptides was 56% and 53% for hirudin 4g _ 64 and hirudin 53 _ 64 , respectively. B.2412 Individual HPLC analysis of the peptides revealed a high degree of purity and single predomi¬ nant peaks of 214 nm-absorbing material eluting at 16.1 min and 16.3 min, respectively, for hirudin 4g _ 64 and hirudin 53 _ 64 . EXAMPLE 2 Synthesis Of N-Acetyl Hirudin 53 _ 64 N-acetylation of the hirudin peptides of this invention was achieved directly during pep¬ tide synthesis. For example, N-acetyl hirudin 53 _ g4 was synthesized by the basic procedure used to syn¬ thesize hirudin 53 _ 64 described in Example 1. In order to carry out N-acetylation, however, we modi- fied the procedure by substituting 2 mmoles of N-acetyl-asparagine for 2 mmoles of asparagine in the final cycle of peptide synthesis. EXAMPLE 3 Sulfation Of Hirudin Peptides Hirudin 53 _ 64 was O-sulfated at the tyrosine residue to prepare Sulfo-Tyr 63 hirudin 53 _g 4 , using the chemical modification procedure of T. Nakahara et al., "Preparation of Tyrosme-0-[ 35S] Sulfated Cholecystokinin Octapeptide from a Non-Sulfated Precursor Peptide", Anal. Biochem. , 154, pp. 194-99 (1986). We dissolved 1.5 mg of hirudin 53 _ 64 , as prepared in Example 1, in 50 μl of dimethylformamide and dried the solution under N 2 - The peptide was then redissolved in 40 μl of dimethylformamide (DMF) containing 2 x 10 moles of sulfuric acid. To this we added 10 μl of a solution containing 50 μg N,N'-dicyclohexylcarbodiimide in 40 μl DMF (7.0 x 10 moles). The reaction was allowed to proceed for about 5-10 min at 25°C before the addition of 750 μl of deionized water. Aiy insoluble reaction B.2412 products were removed by centrifugation in a micro- fuge apparatus prior to further purification. Sulfo-Tyr 63 hirudin 53 _ 64 was purified away from other peptide and reaction components by reverse- phase HPLC employing a Vydac C, g column (4.6 x 25 cm) and an Applied Biosystems, Inc., liquid chromatographic system. The column was equilibrated in a 0.1% TFA- water solvent and developed with a linear gradient of increasing acetonitrile concentration from 0 to 35% over 90 min at a flow rate of 0.8 ml/min with a 0.085% TFA-containing solvent. Fractions were collected, dried in a speed-vac apparatus and redis¬ solved in deionized water. On HPLC analysis, large number of peaks of 214 nm absorbing material were resolved (Figure 1). By assaying the peak fractions for anti¬ coagulant activity, we identified two potential Sulfo-Tyr 63 hirudin 53 _g 4 -containing fractions (peaks A and B; Figure 1). Ultraviolet spectral analysis of peak A at neutral pH revealed a maximal absorbance at 258-264 nm, indicating the presence of a modified tyrosine residue. .Amino acid analysis of the peptide in peak A confirmed the hirudin 53< _ 64 structure. These data demonstrated that peak A contained Sulfo- Tyr 63 hirudin 53 _ 64 . We confirmed the presence of Sulfo-Tyr 63 hirudin 53-64 by treating the peptide of peak A with 30% TFA at 60°C for 1 hour to remove the sulfate group. We then dried the peptide, redissolved it in water and subjected it to reverse-phase HPLC. We carried out HPLC analysis of the desulfated Sulfo- Tyr 63 hirudin 53< _ 64 using an Aquapore RP-300 C g column (0.46 x 3.0 cm) and an Applied Biosystems 150A HPLC system. The column was equilibrated in water con- taining 0.1% TFA and developed with a gradient of increasing acetonitrile concentration from 0 to 70% over 45 minutes at a flow rat≥ of 0.5 ml/min in a B.2412 0.085% TFA-containing solvent. The peptide showed HPLC chromatographic behavior identical to that of unsulfated hirudin 53 _ 64 . In addition, peak absorb¬ ance of the treated peptide returned to 275-280 nm, typical for a peptide containing an unmodified tyro¬ sine residue. We then applied the above-described sulfa¬ tion procedure to large quantities of the correspond¬ ing N-acetylated peptide. As shown in the HPLC chromatogram of Figure 2a, treatment of 25 mg of N-acetyl-hirudin 53 _ 64 (as prepared in Example 2) by the Nakahara procedure produced an 80.1% yield of the desired Tyr-sulfated product. However, efforts to scale the reaction proportionally to 50 mg of N-acetyl-hirudin 53-64 only resulted in a 48.5% yield' of the Tyr-sulfated derivative (Fig. 2b). Accordingly, we significantly modified the chemistry of the Nakahara procedure to achieve high yield of the Tyr-sulfated derivative in a large scale sulfation reaction. More specifically, we dissolved 1 g of N-acetyl-hirudin 53 _ 64 in 40 ml of dimethyl¬ formamide in the presence of 5.0 ml of N,N'-dicycl- ohexylcarbodiimide (0.2 g / J.16 ml dimethyl formamide). The mixture was stirred at 0°C and 0.5 ml of con- centrated sulfuric acid was added dropwise to the reaction mixture until a precipitate formed. Fol¬ lowing 5 minutes, the reaction was stopped by the addition of 40 ml water. Reverse-phase HPLC separa¬ tion of the reaction mixture (Fig. 2c) indicated a 81.7% yield of the sulfated peptide, Sulfo-Tyr 63 -N- acetyl-hirudin 53 _ 64 . Large-scale purification of the sulfated hirudin peptide was then achieved by a one-step anion exchange chromatography. Specifically, crude Sulfo-Tyr 63 -N-acetyl-hirudin 53 _g 4 was purified on a column of DEAE-Sepharose (250 ml wet resin/5 g crude peptide). The column was pre-equilibrated and B.2412 the sample was loaded in 20 mM sodium acetate, pH 5.0. The column was developed with a linear NaCl gradient (0-0.4 M). The Sulfo-Tyr 63 -N-acetyl- hirudin 53 _ 64 eluted at approximately 0.2-0.3 M NaCl, after the unsulfated peptide, but prior to the sul- fonated side product Sulfo-Tyr 63 -N-acetyl- hirudin 53 _ 64 . EXAMPLE 4 Sulfonation Of Hirudin Peptides N-acetyl-hirudin 53 _ 64 was modified to its Tyr-sulfohated derivative, Sulfonyl-Tyr 63 -N-acetyl- hirudin 53 _ 64 , during the preparation of Sulfo- Tyr 63 -N-acetyl-hirudin 53 _ 64 , as described in Exam¬ ple 3. Sulfonyl-Tyrg 3 -N-acetyl-hirudin 53 _ 64 was a side reaction product obtained during the large-scale sulfation reaction described in that example. Accord¬ ingly, Sulfonyl-Tyr 63 -N-acetyl-hirudin 53 _ 64 was obtained at between 30 to 40% yield and was found to elute prior to Sulfo-Tyr 63 -hirudin 53 _g 3 in reverse- phase HPLC separations (see Figure 3). EXAMPLE 5 Peptidomimetic Analogs Of Hirudin Peptides Hirudin peptides, preferably Sulfo-Tyr 63 hirudin 53 _ 64 , may be used to produce semi-peptidic or non-peptidic peptidomimetic analogs, synthetic molecules which exhibit antithrombin and anticoagu¬ lant activities. Such peptidomimetic analogs, here¬ inafter referred to as "hirulogs", are represented by the following chemical structures: Hirulog-1: Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu \ / Gly Tyr(SO,) \ - / d Asn-Cys-S-S-Cys-Leu B.2412 Hirulog-2: Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu \ \ Gly Tyr(SO-) / / Asn-Cys-S-S-(CH 2 ) n -S-S-Cys-Leu Hirulog-3: Hirulog-4: Hirulog-5: Asn-Gly-Asp-Phe-Glu-Glu-Ala / \ 0 Pro I I C=0 Glu \ / Leu-(S0 3 )Tyr-Glu Hirulog-6: Asn-Gly-Asp-Phe-Glu-Glu-Ala-Pro I I O Glu \ / Leu-(S0 3 )Tyr-Ser B.2412 Hirulog-7 : Cys-Gly-Asp-Cys-Glu-Glu-Ile-Pro-Glu-Glu-Tyr ( SO, ) -Leu I I 3 S S \ / S- ( CH 2 ) 2 -S Semi-peptidic peptidomimetic analogs of the hirudin peptides may be prepared to stabilize a loop, tur: or helical conformation of the parent peptide. For example, a loop structure is constructed by the addition of cysteiny_ or lysyl residues at both the N- and C-terminal ends of Sulfo-Tyr 63 hirudin 53 _ 64 . Terminal cysteinic residues are crosslinked by oxida- tion to produce Hirulog-1 (Figure 4a), oxidation with an aliphatic dithiol to produce. Hirulog-2 (Figure 4b), or alkylation with aliphatic dihaloace- tate or propionate to produce Hirulog-3 (Figure 4c). Terminal lysyl residues are crosslinked with any of a number of imidate agents which vary in spacer length or with dihydroxysuccinimidyl aliphatic reagents, resulting in production of Hirulog-4 (Figure 5). A turn structure around Pro-8 of Sulfo- Tyr 63 hirudin 53 _ 64 is constrained by replacement of lle-7 with chloroalanine, with or without concomitant replacement of Glu-9 or Glu-10 with (L) or (D)-serine. Peptidomimetic analogs containing chloroalanine alone would yield cross-linking of Glu-9 or Glu-10 to Ala-7 with a ketone linkage to produce Hirulog-5 (Figure 6a). Derivatives with serine at positions 9 or 10 would yield crosslinking via an ether linkage, producing Hirulog-6 (Figure 6b). A helical structure in the peptidomimetic analogs of this invention can be constrained by substituting cysteinyl residues at position (n) and (n+3) of the hirudin peptide and crosslinking by either direct oxidation, oxidation with an aliphatic dithiol, or alkylation with afi aliphatic dihalo B.2412 acetate. For example, replacement of Asn-1 and Phe-4 of Sulfo-Tyr 63 hirudin 53 _g 4 with cysteines and oxidation via ethanedithiol (Figure 7) would con¬ strain a helical turn in the NH 2 -terminal side of the derivative and, thus, seed a stable helical structure in the peptide derivative. This is exemplified by Hirulog-7. Fully non-peptidic peptidomimetic analogs may also be produced, taking into consideration the above-described strategies relating to constrained peptide compounds. EXAMPLE 6 Synthesis Of Hirudin Peptide Analogs The hirudin analog dinitrofluorobenzyl- Sulfo-Tyr 63 hirudin 54 _ 64 (DNFB-Sulfo-Tyr 63 hirudin 54 _ 64 ) was synthesized by reacting stoichiometric quantities of Sulfo-Tyr 63 hirudin 54 _g 4 with dinitrodifluorobenzene (DNDFB) in dimethylformamide. We incubated the reac¬ tion mix for 24 hours at 22°C and then dried it under vacuum and redissolved in 0.1% TFA in water. The resulting products were separated by HPLC chromatog¬ raphy employing an Aquapore RP-300 C8 octasilyl column (0.46 x 3.0 cm). The column was first equilibrated in 0.1% TFA in water (solvent A). After loading the sample, we developed the column with a 0 - 50% linear gradient of solvent B (0.085% TFA/70% acetonitrile) over 45 minutes. The effluent stream was monitored for adsorbance at 214 nm. The DNFB-Sulfo-Tyr 63 hirudin 54 _ 64 eluted at 48% solvent B. We synthesized the hirudin analog nitro- anisoleg 3 hirudin 53 _ 63 in a two-step method. First, we synthesized methoxytyrosylg 3 hirudin 53 _ 63 by the solid phase synthesis techniques, as described in Example 1, substituting Boc-O-methoxytyrosine resin for Boc-O-Leu resin in the synthesis. The peptide B.2412 was cleaved from the resin and purified by HPLC on a Vydac C4 column, as described in Fi-gure 1. We next nitrated the purified methoxytyrosylg 3 hirudin 5 , 63 by adding to it an excess of tetranitromethane in 20 mM Tris-HCl, pH 8.0. The reaction was incubated at 27°C for 4 hours. The resulting products are lyophilized, redissolved in 20 mM ammonium bicar¬ bonate and desalted on a Biogel P-4 column (1 x 30 cm) which was equilibrated and eluted in 20 mM ammonium bicarbonate. The separation of nitroanisole c Ό,_hiru- din 53 _ 63 was achieved by HPLC on an Aquapore RP-300 C8 octasilyl column (0.46 x 3.0 cm) as described above. EXAMPLE 7 Failure Of Heparin To Neutralize Clot-Bound Thrombin We analyzed the heparin-catalyzed, anti¬ thrombin Ill-dependent inactivation of non clot- associated thrombin. Thrombin activity was deter- mined via radioimmunoassay for fibrinopeptide A (FPA), a cleavage product of the thrombin-catalyzed digestion of fibrinogen (Diagnostica Stago, Asnieres, France). Specifically, citrated, normal human plasma (George King Biomedical, Inc., Kansas), maintained at 37°C, was incubated with varying concentrations of heparin (Upjohn, Kalamazoo, Michig»an; 0.01 - 1.0 U/ml) for 1 minute. We then added varying concentra¬ tions of human -thrombin (a gift from Dr. John Fenton, Albany, New York) (0.005 - 0.05 U/ml) and continued incubation for another 5 minutes. The plasma samples were then assayed for fibrinopeptide A content, as previously discussed [H. L. Nossel et al. , "Measurement of Fibrinopeptide A in Human Blood", J. Clin. Invest., 54, pp. 43-53 (1974)]. The results are shown in the table below: B.2412 As demonstrated above, a heparin concen¬ tration of 0.1 U/ml caused an approximately 50% inhibition of thrombin-catalyzed FPA release over the complete range of thrombin concentrations added to plasma. We then prepared clot-bound thrombin by adding a final concentration of 20 mM CaCl 2 to citrated, normal hum»an plasma in the presence of both 125I-fibπnogen (25,000 cpm/ml), which was labelled by the IODOGEN method follwing manufacturer's directions (Pierce, Rockford, Illinois) and a copper coil. We removed the resulting radiolabelled fibrin clot which formed on the copper coil after 10 minutes. The clot was washed extensively with phosphate buffered saline and then dialyzed against 4 liters of the same buffer for 24 hours to remove any non- adsorbed thrombin and soluble FPA. We assayed clot-bound thrombin by adding the washed clot to human plasma maintained at 37°C, incubating for 5 minutes and then measuring FPA release as described above. All assays were normal- ized for fibrin content by measurement of 125I- fibrinogen incorporation, assuming that the amount of thrombin adsorbed to the clot was proportional to B.2412 the size of the clot and, hence, the radioisotope concentratio . We determined the degree of inhibition of clot-bound thrombin by heparin by adding various amounts of heparin (0.1 - 7.5 U/ml) to the plasma 1 minute prior to the addition of the clot. The results are shown in the table below: As demonstrated above, concentrations of heparin in excess of 1.0 U/ml were required to neutralize clot-bound thrombin by greater than 50%. Comparison of the results obtained for free thrombin and bound thrombin suggested that the heparin-anti- thrombin III complex was more than 10-fold less efficient in neutralizing clot-bound rather than clot-free thrombin. It is much more difficult for the heparin-antithrombin III complex to access thrombin when adsorbed to fibrin. Thus, heparin demonstrates a limited antithrombotic effect in the course of thrombosis and fibrin accumulation. EXAMPLE 8 Inhibition Of Free And Clot-Bound Thrombin By Hirudin Peptides We next analyzed the ability of a synthetic Tyr-sulfated hirudin peptide, N-Ac-Asn-Gly-Asp-Phe- Glu-Glu-Ile-Pro-Glu-Glu-Tyr(OS0 3 H)-Leu-OH ("N-acetyl Sulfo-Tyr g3 hirudin 53 _g 4 "), to neutralize free thrombin and clot-bound thrombin. The synthesis of N-acetyl Sulfo-Tyr 63 hirudin 53 _ 64 is described in Example 2. B.2412 When we added 2.8 μg/ml of N-acetyl Sulfo- Tyrg 3 hir din 53 _ 64 to plasma one minute before adding "free" thrombin, thrombin-catalyzed FPA release was inhibited by over 70%. As demonstated in the table below, this inhibition was observed over a range of thrombin concentrations, from 0.0025 to 0.04 U/ml. [Thrombin] 0 0.0025 0.0025 0.005 0.005 0.01 0.01 0.02 0.02 0.04 0.04 This data demonstrates that a 2.8 μg/ml concentration of N-acetyl Sulfo-Tyr 63 hirudin 5 ,_ 64 was equivalent to a heparin concentration of 0.1 U/ml in inhibiting FPA release. We next examined the inhibition of clot- bound thrombin by N-acetyl Sulfo-Tyr 63 hirudin-., β4 . We again used a fixed concentration of 2.8 μg/ml of hirudin peptide added to plasma 1 minute prior to the addition of a radiolabeled, washed fibrin clot prepared as in Example 7. FPA release was substan¬ tially reduced from 125.5 nM (control) to 36.0 nM. In contrast to the results obtained with heparin, N- acetyl Sulfo-Tyr 63 hirudin 53 _ 64 was equally effective in inhibiting both clot-free thrombin and clot-bound thrombin. These results demonstrate that antithrom- botic dosages of hirudin peptides are more effective than heparin in inhibiting clot-bound thrombin. Accordingly, hirudin peptides are more effective than heparin in blocking either rethrombosis following B.2412 thrombolytic therapy, or clot extension, such as that observed in deep vein thrombosis. EXAMPLE 9 Inhibition Of Clot-Bound Thrombin By Other Antithrombin Ill-Independent Inhibitors The failure of heparin to efficiently inhibit clot-bound thrombin may be due to steric hindrance related to the size of the heparin-anti- thrombin III complex. Another possibility is that the charge of the complex may cause electrostatic repulsion, A third possibility is that thrombin may bind to the fibrin clot in such a manner as to bury a critical structure involved in formation of the thrombin-antithrombin III-heparin tertiary complex. We next examined whether other, antithrombin Ill- independent thrombin inhibitors are capable of neutralizing clot-bound thrombin to the same extent as free thrombin. Using the methods described in Example 7, we examined the ability of D-Phe-Pro-Arg-CH 2 Cl (PPACK) to neutralize thrombin. We found that a concentration of 3.0 nM PPACK inhibits both free and clot-bound thrombin by over 70%. EXAMPLE 10 Inhibition By N-Acetyl Sulfo-Tyr β _ Hirudin 53 _ 4 Of Clot-Bound Thrombin In A Rabbit Model Of Thrombus Accretion Having observed the in vitro efficacy of N-acetyl Sulfo-Tyr 63 hirudin 53 _ 64 in blocking thrombin adsorbed to fibrin clots, we next examined the activity of that peptide in in vivo studies of fibrin accumulation on pre-existing thrombi. We anesthetized New Zealand White rabbits (provided by Drs. Michael Buchanan and Jack Hirsh, McMaster University, Hamilton Ontario) with 30 mg/kg of sodium pentobarbitol i jedted into the marginal B.2412 ear vein. We next surgically exposed both jugular veins and clamped off a 2 cm segment from each vein. The isolated segments were then emptied of blood and blood flow was temporarily occluded by proximal and distal clamps. We collected 1.0 ml of blood from a carotid cannula into a 1.0 ml tuberculin syringe containing 1 unit of human -thrombin. We immediately injected 150 μl of the clotting blood into the iso¬ lated venous segments via a 25 gauge needle. A 3-0 suture was then passed through the forming thrombus and the vessel wall to deepen the thrombus in place and to prevent embolization. We removed the clamps 15 minutes after the injection of clotting blood and iinnjjeecctt<ed 1.0 mg of 125I-rabbit fibπnogen into the animal- Rabbits treated as described above were then separated into four groups. The first group received an infusion of saline (Group I). The second group was treated with a high dose of heparin, repre- sentative of a high-dose therapeutic regimen of heparin in humans (70 U/kg loading dose, followed by infusion at a rate of 30 U/kg/hr) (Group II). The third group received an infusion of N-acetyl Sulfo- Tyr 63 hirudin 53 _ 64 at a rate of 0.5 μg/kg/hr (Group III). The final group received a single i.v. bolus injection of 2.0 mg/kg N-acetyl Sulfo-Tyr g3 hirudin 53 _ 64 (Group IV). After four hours, we removed the thrombi from all four groups of animals and determined the extent of fibrin accretion by radioisotope counting of 125I-fibπn. In addi.ti.on, we monitored the anti¬ coagulant effects of the various treatments by ex vivo determinations of thrombin time (TT) and acti¬ vated partial thromboplastin time (APTT). Figure 8 demonstrates that high-dose heparin blocked thrombus accretion by 38.5%. Infused N-acetyl Sulfo-Tyr 63 hirudin 5 _ g 4 (Group III) inhibited thrombus B.2412 growth by 43.7%, while i.v. bolus injection of N-acetyl Sulfo-Tyr, 3 hirudin 53< _g 4 caused a 62.4% inhibition. The difference between the control group (Group I) and Group IV was statistically significant (Student's t-test; p = 0.081). These results demonstrate that rapid neutralization of clot-bound thrombin by i.v. bolus injection of a hirudin peptide markedly alleviated the growth of a thrombus for up to 4 hours. This property indicates the utility of such peptides in the treatment of deep venous thrombosis and pulmonary embolization. We believe that such neutralization of clot-bound thrombin would lead to a decrease in reperfusion time and prevent thrombin-mediated rethrombosis of reperfused arterial emboli when hirudin peptides are used in combination with throm¬ bolytic agents. EXAMPLE 11 Effects Of Adjuvant Use Of Hirudin Peptides With Recombinant tPA In Reperfusion Of Experimental Coronary Thrombi In Dogs The preparation of coronary thrombosis and its lysis by recombinant tPA (rtPA) was performed essentially as described previously [P. Golino et al., Circulation, 77, pp. 678-84 (1988)]. We performed the study with 16 open-chest mongrel dogs (provided by Dr. John Willerson, South West Medical Foundation, Dallas, Texas) weighing between 25 and 35 kg. We anesthetized the dogs by injecting them with 30 mg/kg sodium pentobarbitol. The dogs were then intubated and ventilated through a Harvard respirator. We monitored arterial pressure with a polyethylene cath¬ eter inserted into the right carotid artery. Fluids and drugs were administered through a catheter in the right jugular vein. Coronary thrombi were established in the left anterior descending (LAD} artery as follows. B.2412 We performed a left thoractomy on the dogs at the fifth intercostal space and suspended the heart in a pericardial cradle. We next isolated a segment of the LAD and placed a pulsed Doppler flow probe around the segmen÷- A No. 7F Amplatz LI left coronary cath¬ eter (Cordis Corp., Miami, Florida) was then posi¬ tioned into the left coronary ostium via a left carotid arterial cutdown. Fluoroscopy was used to assist in the insertion of the catheter. We then positioned a 0.025 inch Teflon-coated guidewire (Cook Co., Bloo ington, Indiana) in the LAD and removed the catheter. We placed a copper coil of 24-gauge into the LAD, immediately distal to the flow probe. The guidewire was then removed and lidocaine (2 mg/kg loading dose; 1 mg/min infusion) was administered. After positioning the copper coil in the LAD, a persistent coronary thrombus developed within approx¬ imately 2 minutes. The thrombus was associated with cyanosis and systemic bulging of the portion of the myocardial tissue supplied by the LAD. Fifteen minutes after occlusion, we initiated thrombolytic reperfusion with an i.v. bolus injection of 0.05 mg/kg rtPA (Activase™, Genentech, San Francisco, California), followed by infusion of 0.005 mg/kg/min rtPA for up to 90 minutes or until reperfusion was established. Once reperfusion was established, all dogs received an hourly bolus of 150 U/kg heparin. Eight dogs (Group I) received an additional infusion of saline for up to 90 minutes or until reperfusion occurred. A second group of eight dogs (Group II) was infused with 2.0 mg/kg/hr of N-acetyl Sulfo-Tyr 63 hirudin 53 __g 4 . Reperfusion was monitored by hemodynamic measurements and reper¬ fusion time was defined as the time required to reestablish blood flow through the LAD following initiation of thrombolytic therapy. The results of these treatments are shown belfcw: B.2412 Reperfusion Time Group Treatment (min) I control (saline 32 ± 4 infusion) II hirudin peptide 19 ± 6 infusion This data demonstrates that adjuvant use of a hiruαin peptide with rtPA in thrombolysis of coronary thrombi significantly reduced the time required to establish reperfusion. Thus, for a given dose of thrombolytic agent, reperfusion time may be decreased " when that agent is administered in combina¬ tion with a hirudin peptide rather than a conven¬ tional anticoagulant. Alternatively, the reper- fusion time established for a thrombolytic agent administered with a conventional anticoagulant may be realized at a lower dose of the thrombolytic agent when that agent is administered in combination with a hirudin peptide. This demonstration supports the use of hirudin peptides in combination with thrombolytu agents as a means to rapidly achieve reperfusion in a patient, thus decreasing the extent of damage to the myocardial tissue resulting from infarction. Moreover, the adjuvant use of hirudin peptides with thrombolytic agents permits the use of lower doses of the thrombolytic agent than those employed when that agent is administered as a monotherapy. This, in turn, decreases both the risk of bleeding and the ultimate cost of treatment. While we have hereinbefore presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the methods and compositions of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claimέ appended hereto rather B.2412 than the specific embodiments which have been pre ¬ sented hereinbefore by way of example. B.2412

Claims: CLAIMS I claim: 1. A pharmaceutically effective combi¬ nation for treating or preventing thrombotic disease ,n a patient comprising: a) a peptide characterized by a sequence of amino acids consisting substantially of the formula: X-A χ -A 2 -A 3 -A 4 -A 5 -Ag-A 7 -Ag-A g -A 10 -Y wherein X is a hydrogen, one or two alkyl groups of from 1 to 6 carbon atoms, one or two acyl groups of from 2 to 10 carbon atoms, carbobenzyloxy or t-butyl- oxy carbonyl; A. is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A 2 is Phe, SubPhe, β-(2- and 3-thienyl)alanine, ~ β-(2- and 3-furanyl)alanine, β-(2-, 3- and 4-pyridyl)alanine, β-(benzothienyl-2- and 3-yl) alanine, β-(l- and 2-naphthyl)alanine, Tyr or Trp; A 3 is Glu or Asp; A 4 is any amino acid; A ς is lie, Val, Leu, Nle or Phe; Ag is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4- carboxylate, Sar, NMePgl or D-Ala; A- is any amino acid; A g is any amino acid; A g is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, lie, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A 1Q is a bond or a peptide containing from one to five residues of any amino acid; and Y is a car¬ boxyl terminal residue selected from OH, C- ι -C 6 alkoxy, amino, mono- or di-(C,-C 4 ) alkyl substituted .amino or benzylamino; said peptide displaying anticoagulant activity; and b) a thrombolytic agent. 2. The combination according to claim 1, wherein the amount of the thrombolytic agent in the combination is less than that required for a desired B.2412 therapeutic or prophylactic effect when that agent is administered as a monotherapy. 3. The combination according to claim 1 or 2, wherein the peptide is characterized in that X is hydrogen or N-acetyl; A, is Asn-Gly-Asp; A 2 is Phe; A 3 is Glu; A 4 is Glu; A ς is lie; Ag is Pro; A- is Glu; A is Glu; A g is a dipeptide selected from the group consisting of S-alkylated cysteine-Leu, S-alkylated homocysteine-Leu, tyrosine-O-sulfate-Leu, tyrosine-O-phosphate-Le , tyrosine-O-carboxylate-Leu, 3-sulfonyl tyrosine-Leu, 5-sulfonyl tyrosine-Leu, 3-carbonyl tyrosine-Leu, 5-carbonyl tyrosine-Leu, 3-phosphonyl tyrosine-Leu, 5-phosphonyl tyrosine-Leu, 4-methylsulfonyl tyrosine-Leu, 4-methylphosphonyl tyrosine-Leu, 4-phenylacetic acid-Leu, 3-nitrotyro- sine-Leu, 5-nitrotyrosine-Leu and 3,5-diiodotyrosine- Leu; A 1Q is a bond; and Y is OH. 4. The combination according to claim 3, wherein the peptide is characterized in that X is N-acetyl and A g is tyrosine-O-sulfate-Leu. 5. The combination according to claim 1 or 2, wherein the thrombolytic agent is selected from the group consisting of tPA, rtPA, streptokinase, urokinase, prourokinase, APSAC, animal salivary gland plasminogen activators and derivatives thereof. 6. The combination according to claim 1 or 2, wherein the amount of the peptide in the combi¬ nation is between about 0.005 mg/kg body weight and about 15 mg/kg body weight of said patient. 7. The combination according to claim 1 or 2, wherein the amount of the thrombolytic agent in the combination is between about 10% and about B.2412 80% of the dosage required for a desired therapeutic or prophylactic effect when that agent is administered as a monotherapy. 8. The combination according to claim 6, wherein the amount of the peptide in the combination is between about 0.1 mg/kg body weight and 2.5 mg/kg body weight of said patient. 9. The combination according to claim 7, wherein the amount of the thrombolytic agent in the combination is between about 10% and about 70% of the dosage required for a desired therapeutic or prophylactic effect when that agent is administered as a monotherapy. 10. The combination according to claim 1 or 2, further comprising a pharmaceutically effective amount of an antiplatelet agent. 11. The combination according to claim 10, wherein said antiplatelet agent is aspirin. 12. A method for treating or preventing thrombotic disease in a patient comprising the step of treating said patient in a pharmaceutically acceptable manner with a combination according to any one of claims 1 to 11. 13. A method for decreasing reperfusion time or increasing reocclusion time in a patient treated with a thrombolytic agent, said method com¬ prising the step of treating said patient in a pharmaceutically acceptable manner with a pharma¬ ceutically effective combination comprising: B.2412 a) a peptide characterized by a sequence of amino acids consisting substantially of the formula: wherein X is a hydrogen, one or two alkyl groups of from 1 to 6 carbon atoms, one or two acyl groups of from 2 to 10 carbon atoms, carbobenzyloxy or t-butyl- oxy carbonyl; A, is a be:A or is a peptide containing from 1 to 11 residues of any amino acid; A 2 is Phe, SubPhe, β-(2- and 3-thienyl)alanine, β-(2- and 3-furanyl)alanine, β-(2-, 3- and 4-pyridyl)alanine, β-(benzothienyl-2- and 3-yl) alanine, β-(l- and 2-naphthyl)alanine, Tyr or Trp; A 3 is Glu or Asp; A 4 is any amino acid; A,- is lie, Val, Leu, Nle or Phe; Ag is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4r carboxylate, Sar, NMePgl or D-Ala; A- is any amino acid; A g is any amino acid; A g is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, lie, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A, Q is a bond or a peptide containing from one to five residues of any amino acid; and Y is a car¬ boxyl terminal residue selected from OH, C-,-Cg alkoxy, amino, mono- or di-(C,-C 4 ) alkyl substituted amino or benzylamino; said peptide displaying anticoagulant activity; and b) a thrombolytic agent. 14. The method according to claim 13, wherein the peptide is characterized in that X is hydrogen or N-acetyl; A, is Asn-Gly-Asp; A 2 is Phe; A 3 is Glu; A 4 is Glu; A- is lie; A g is Pro; A 7 is Glu; A g is Glu; A g is a dipeptide selected from the group consisting of S-alkylated cysteine-Leu, S-alkylated homocysteine-Leu, tyrosine-O-sulfate-Leu, tyrosine-O-phosphate-Leu, tyrosine-O-carboxylate-Leu, 3-sulfonyl tyrosine-Leu, 5-sulfonyl tyrosine-Leu, B.2412 3-carbonyl tyrosine-Leu, 5-carbonyl tyrosine-Leu, 3-phosphonyl tyrosine-Leu, 5-phosphonyl tyrosine-Leu, 4-methylsulfonyl tyrosine-Leu, 4-methylphosphonyl tyrosine-Leu, 4-phenylacetic acid-Leu, and 3,5-diio- dotyrosine-Leu; A, Q is a bond; and Y is OH. 15. The method according to claim 14, wherein the peptide is characterized in that X is N-acetyl and A g is tyrosine-O-sulfate-Leu. 16. A method for decreasing the dose of a thrombolytic agent required to establish reperfusion or to prevent reocclusion in a patient, said method comprising the step of treating said patient in a pharmaceutically acceptable manner with a pharma¬ ceutically effective combination comprising: a) a peptide characterized by a sequence of amino acids consisting substantially of the formula: X"A l "A 2 "A 3 'A 4~ A 5 "A 6 "A 7 "A 8 "A 9 "A l0 "Y wherein X is a hydrogen, one or two alkyl groups of from 1 to 6 carbon atoms, one or two acyl groups of from 2 to 10 carbon atoms, carbobenzyloxy or t-butyl- oxy carbonyl; A, is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A 2 is Phe, SubPhe, β-(2- and 3-thienyl)alanine, β-(2- and 3-furanyl)alanine, β-(2-, 3- and 4-pyridyl)alanine, β-(benzothienyl-2- and 3-yl) alanine, β-(l- and 2-naphthyl)alanine, Tyr or Trp; A 3 is Glu or Asp; A 4 is any amino acid; A 5 is lie, Val, Leu, Nle or Phe; Ag is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4- carboxylate, Sar, NMePgl or D-Ala; A- is any amino acid; A g is any amino acid; A g is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, He, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A 1Q is a bond or _ \ peptide containing B.2412 from one to five residues of any amino acid; and Y is a carboxyl terminal residue selected from OH, C 1~ C 6 a lko y, amino, mono- or di-(C 1 -C 4 ) alkyl sub¬ stituted amino or benzylamino; said peptide display¬ ing anticoagulant activity; and b) a thrombolytic agent, the dosage of the thrombolytic agent being less than that required for a desired therapeutic or prophylactic effect when that agent is administered as a mono¬ therapy. 17. The method according to claim 16, wherein the peptide is characterized in that X is hydrogen or N-acetyl; A, is Asn-Gly-Asp; A 2 is Phe; A 3 is Glu; A 4 is Glu; A ς is He; A g is Pro; A 7 is Glu; A g is Glu; A g is a dipeptide selected from the group consisting of S-alkylated cysteine-Leu, S-alkylated homocysteine-Leu, tyrosine-O-sulfate-Leu, tyrosine-O-phosphate-Leu, tyrosine-O-carboxylate-Leu, 3-sulfonyl tyrosine-Leu, 5-sulfonyl tyrosine-Leu, 3-carbonyl tyrosine-Leu, 5-carbonyl tyrosine-Leu, 3-phosphonyl tyrosine-Leu, 5-phosphonyl tyrosine-Leu, 4-methylsulfonyl tyrosine-Leu, 4-methylphosphonyl tyrosine-Leu, 4-phenylacetic acid-Leu, 3-nitrotyro¬ sine-Leu, 5-nitrotyrosine-Leu and 3,5-diiodotyrosine- Leu; A 1Q is a bond; and Y is OH. 18. The method according to claim 17, wherein the peptide is characterized in that X is N-acetyl and A g is tyrosine-O-sulfate-Leu. 19. The method according to any one of claims 12, 13 and 16, wherein the patient is a human. 20. The method according to any one of claims 12, 13 and 16, wherein the thrombolytic agent is selected from the group consisting of tPA, rtPA, B.2412 streptokinase, urokinase, prourokinase, APSAC, animal salivary gland plasminogen activators and derivatives thereof. 21. The use of an anticoagulant peptide and a thrombolytic agent for the production of a combination which is pharmaceutically effective for the treatment or prevention of thrombotic disease, said anticoagulant peptide being charac¬ terized by a sequence of amino acids consisting substantially of the formula: X- 1 -A 2 -A 3 -A 4 -A 5 -Ag-A 7 -Ag-A g - A 1 Q -Y wherein X is a hydrogen, one or two alkyl groups of from 1 to 6 carbon atoms, one or two acyl groups of from 2 to 10 carbon atoms, carbobenzyloxy or t-butyl,- oxy carbonyl; A, is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A 2 is Phe, SubPhe, β-(2- and 3-thienyl)alanine, β-(2- and 3-furanyl)alanine, β-(2-, 3- and 4-pyridyl)alanine, β-(benzothienyl-2- and 3-yl) alanine, β-(l- and 2-naphthyl)alanine, Tyr or Trp; A 3 is Glu or Asp; A 4 is any amino acid; A 5 is He, Val, Leu, Nle or Phe; Ag is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4- carboxylate, Sar, NMePgl or D-Ala; A 7 is any amino acid; Ag is any amino acid; A g is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, He, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A, Q is a bond or a peptide containing from one to five residues of any amino acid; and Y is a car¬ boxyl terminal residue selected from OH, C 1 -C 6 alkoxy, amino, mono- or di-(C,-C 4 ) alkyl substituted amino or benzylamino. 22. The use according to claim 21, wherein the amount of the thrombolytic agent in the combination is less than that required for a desired B.2412 therapeutic or prophylactic effect when that agent is administered as a monotherapy. 23. The use according to claim 21 or 22, wherein the peptide is characterized in that X is hydrogen or N-acetyl; A, is Asn-Gly-Asp; A 2 is Phe; A 3 is Glu; A 4 is Glu; A 5 is He; Ag is Pro; A 7 is Glu; Ag is Glu; A g is a dipeptide selected from the group consisting of S-alkylated cysteine-Leu, S-alkylated homocysteine-Leu, tyrosine-O-sulfate-Leu, tyrosine-O-phosphate-Leu, tyrosine-O-carboxylate-Leu, 3-sulfonyl tyrosine-Leu, 5-sulfonyl tyrosine-Leu, 3-carbonyl tyrosine-Leu, 5-carbonyl tyrosine-Leu, 3-phosphonyl tyrosine-Leu, 5-phosphonyl tyrosine-Leu, 4-methylsulfonyl tyrosine-Leu, 4-methylphosphonyl tyrosine-Leu, 4-phenylacetic acid-Leu, 3-nitrotyro- sine-Leu, 5-nitrotyrosine-Leu and 3,5-diiodotyrosine- Leu; A 1Q is a bond; and Y is OH. 24. The use according to claim 23, wherein the peptide is characterized in that X is N-acetyl and A g is tyrosine-O-sulfate-Leu. 25. The use according to claim 21 or 22, wherein the thrombolytic agent is selected from the group consisting of tPA, rtPA, streptokinase, uro- kinase, prourokinase, APSAC, animal salivary gland plasminogen activators and derivatives thereof. 26. The use according to claim 21 or 22, wherein the amount of the peptide in the combination is between about 0.005 mg/kg body weight and about 15 mg/kg body weight of said patient. 27. The use according to claim 26, wherein the amount of the peptide in the combination B.2412 is between about 0.1 mg/kg body weight and 2.5 mg/kg body weight of said patient. 28. The use according to claim 21 or 22, wherein the amount of the thrombolytic agent in the combination is between about 10% and about 80% of the dosage required for a desired therapeutic or prophylactic effect when that agent is administered as a monotherapy. 29. The use according to claim 28, wherein the amount of the thrombolytic agent in the combina¬ tion is between about 10% and about 80% of the dosage required for a desired therapeutic prophylactic effect when that agent is administered as a monotherapy. 30. A pharmaceutically effective combi¬ nation for treating or preventing thrombotic disease in a patient comprising: a) a peptidomimetic analog of a peptide characterized by a sequence of amino acids consisting substantially of the formula: X ~ A i "A 2 "A 3 "A 4 "A 5~ A 6 "A 7 "A 8 "A 9~ A l0~ Y wherein X is a hydrogen, one or two alkyl groups of from 1 to 6 carbon atoms, one or two acyl groups of from 2 to 10 carbon atoms, carbobenzyloxy or t-butyl- oxy carbonyl; A, is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A 2 is Phe, SubPhe, β-(2- and 3-thienyl)alanine, β-(2- and 3-furanyl)alanine, β-(2-, 3- and 4-pyridyl)alanine, β-(benzothienyl-2- and 3-yl) alanine, β-(l- and 2-naphthyl)alanine, Tyr or Trp; A 3 is Glu or Asp; A 4 is any amino acid; A ς is He, Val, Leu, Nle or Phe; Ag is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4- carboxylate, Sar, NMePgl or D-Ala; A 7 is any amino acid; Ag is any amino acid; A^ is a lipophilic amino B.2412 acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, He, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A, Q is a bond or a peptide containing from one to five residues of any amino acid; and _ is a car¬ boxyl terminal residue selected from OH, C,-C 6 alkoxy, amino, mono- or di-(C,-C 4 ) alkyl substituted amino or benzylamino; said analog displaying anticoagulant activity; and b) a thrombolytic agent. 31. The combination according to claim 30, wherein the amount of the thrombolytic agent in the combination is less than that required for a desired therapeutic or prophylactic effect when that agent- is administered as a monotherapy. 32. The combination according to claim 30 or 31, wherein the analog is selected from the group consisting of hirulog-1, hirulog-2, hirulog-3, hirulog-4, hirulog-5, hirulog-6 and hirulog-7. 33. The combination according to claim 30 or 31, wherein the thrombolytic agent is selected from the group consisting of tPA, rtPA, streptokinase, urokinase, prourokinase, APSAC, animal salivary gland plasminogen activators and derivatives thereof. 34. The combination according to claim 30 or 31, further comprising a pharmaceutically effective amount of an antiplatelet agent. 35. A method for treating or preventing thrombotic disease in a patient comprising the step of treating said patient in a pharmaceutically acceptable manner with a combination according to any one of claims 30 to 34. B.2412 36. A method for decreasing reperfusion time or increasing reocclusion time in a patient treated with a thrombolytic agent, said method com¬ prising the step of treating said patient in a pharmaceutically acceptable manner with a pharma¬ ceutically effective combination comprising: a) a peptidomimetic analog of a peptide characterized by a sequence of amino acids consisting substantially of the formula: X ~ A l "A 2 "A 3 "A 4 'A 5 "A 6 "A 7 "A 8~ A 9 -A lθ "Y wherein X is a hydrogen, one or two alkyl groups of from 1 to -6 carbon atoms, one or two acyl groups of from 2 to 10 carbon atoms, carbobenzyloxy or t-butyl- oxy carbonyl; A, is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A 2 is Phe, SubPhe, β-(2- and 3-thienyl)alanine, β-(2- and 3-furanyl)alanine, β-(2-, 3- and 4-pyridyl)alanine, β-(benzothienyl-2- and 3-yl) alanine, β-(l- and 2-naphthyl)alanine, Tyr or Trp; A 3 is Glu or Asp; A 4 is any amino acid; A 5 is He, Val, Leu, Nle or Phe; Ag is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4- carboxylate, Sar, NMePgl or D-Ala; A 7 is any amino acid; A g is any amino acid; A g is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, He, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A 10 is a bond Dr a peptide containing from one to five residues of any amino acid; and Y is a car¬ boxyl terminal residue selected from OH, C-^-Cg alkoxy, amino, mono- or di-(C,-C 4 ) alkyl substituted amino or benzylamino; said analog displaying anticoagulant activity; and b) a thrombolytic agent. 37. A method for decreasing the dose of a thrombolytic agent required to establish reperfusion or to prevent reocclusion in a patient, said method B.2412 comprising the step of treating said patient in a pharmaceutically acceptable manner with a pharma¬ ceutically effective combination comprising: a) a peptidomimetic analog of a peptide characterized by a sequence of amino acids consisting substantially of the formula: X ~ A 1 "A 2 "A 3 "A 4 "A 5 "A 6 "A 7 "A 8 "A 9 "A l0 "Y wherein X is a hydrogen, one or two alkyl groups of from 1 to 6 carbon atoms, one or two acyl groups of from 2 to 10 carbon atoms, carbobenzyloxy or t-butyl- oxy carbonyl; A., is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A 2 is Phe, SubPhe, β-(2- and 3-thienyl)alanine, β-(2- and 3-furanyl)alanine, β-(2-, 3- and 4-pyridyl)alanine, β-(benzothienyl-2- and 3-yl) alanine, β-(l- and 2-naphthyl)alanine, Tyr or Trp; A 3 is Glu or Asp; A 4 is any amino acid; Ag is He, Val, Leu, Nle or Phe; Ag is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4- carboxylate, Sar, NMePgl or D-Ala; A 7 is any amino acid; A g is any amino acid; A g is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, He, Val, Cha, Pro. or a dipeptide consisting of said lipophilic amino acid and any amino acid; A, 0 is a bond or a peptide containing from one to five residues of any amino acid; and Y is a carboxyl terminal residue selected from OH, c l " C 6 a lk° χ Y' amino, mono- or di-(C,-C 4 ) alkyl sub¬ stituted amino or benzylamino; said analog display¬ ing anticoagulant activity; and b) a thrombolytic agent, the dosage of the thrombolytic agent being less than that required for a desired therapeutic or prophylactic effect when that agent is administered as a mono¬ therapy. 38. The method according to any one of claims 35 to 37, wherein the βatient is a human. B.2412 39. The method according to any one of claims 35 to 37, wherein the thrombolytic agent is selected from the group consisting of tPA, rtPA, streptokinase, urokinase, prourokinase, APSAC, animal salivary gland plasminogen activators and derivatives thereof. 40. The use of a peptidomimetic analog of a peptide and a thrombolytic agent for the production of a combination which is pharmaceutically effective for the treatment or prevention of thrombotic disease, said analog displaying anticoagulant activity and being an analog of a peptide characterized by a sequence of amino acids consisting substantially of the formula: X-A 1 -A 2 -A 3 -A 4 -A 5 -Ag-A 7 -A g -A g - A λ ~-Y wherein X is a hydrogen, one or two alkyl groups of from 1 to 6 carbon atoms, one or two acyl groups of from 2 to 10 carbon atoms, carbobenzyloxy or t-butyl- oxy carbonyl; A 1 is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A ? is Phe, SubPhe, β-(2- and 3-thienyl)alanine, β-(2- and 3-furanyl)alanine, β-(2-, 3- and 4-pyridyl)alanine, β-(benzothienyl-2- and 3-yl) alanine, β-(l- and 2-naphthyl)alanine, Tyr or Trp; A 3 is Glu or Asp; A 4 is any amino acid; Ac is He, Val, Leu, Nle or Phe; Ag is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4- carboxylate, Sar, NMePgl or D-Ala; A 7 is any amino acid; A g is any amino acid; A g is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, He, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A 10 is a bond or a peptide containing from one to five residues of any amino acid; and Y is a car¬ boxyl terminal residue selected from OH, C,-Cg alkoxy, amino, mono- or di-(C 1 -C 4 ) alkyl substituted amino or benzylamino. B.2412 41. The use according to claim 40, wherein the amount of the thrombolytic agent in the combina¬ tion is less than that required for a desired thera¬ peutic or prophylactic effect when that agent is administered as a monotherapy. 42. The use according to claim 40 or 41, wherein the thrombolytic agent is selected from the group consisting of tPA, rtPA, streptokinase, uro- kinase, prourokinase, APSAC, animal salivary gland plasminogen activators and derivatives thereof. B.2412

Inventors: MARAGANORE JOHN M, MARAGANORE, JOHN, M.
Applicants: BIOGEN INC, BIOGEN, INC.
Application year: 1990
Main classification: A61K37
IPC codes: A61K38