CA2064231A1 - Combinations and methods for treating or preventing thrombotic diseases - Google Patents

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

2 0 6 ~ 2 3 ~. ; PcTlus~olo4~o3 COMBINATIONS AND METHODS FOR
TREATING OR PREVE2JTING l~ROMBOTIC DISEASES

.

TECHNICAL FIELD OF T9E INVENTION :
The pxesent 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 ~or decreasing reperfusion time, or increasing reocclusion time, or both, in a patient treated wi~h a thrombolytic agent , . " ... .. , ~ . .

W091/01142 2 0 ~ '' ~2- PCT/US90/04103 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 prophylactic effect in a patient, such as to dissolve a blood clot, by administering to the patient a combination of an anticoag~lant 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 myocardi~l 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 a~d prophy-laxis of thrombotic diseases involve therapeutics which act in one of two different ways. The first ty~s of therapeutic inhibits thrombin activity or thrombin formation, thus preventing clot formation.
These drugs also inhibit platelet actlvation 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 o~ 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 thromboiysis and dissolves the blood clot, thereby removing it from the blood vessel and unblocking the flQw of blood [V. J. Marder B.2412 ;, ~

WO91/01142 2 0 6 ~ 2 3 ~ PCT/USgo/04103 - -3~
~ ~.! ,, ,..` ;, . :
and S. Sherry, "Thrombolytic Therapy, Part 1 of 2", New Enqland J. M~d., 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 ~he past few years, the value of thrombolysis in the treatment of acute myocardial infarction has been demonstrated [TIMI Study group, New En~land J. Med., 320, pp. 618-27 (March 9, l9B9)], although the advantage of one thrombolytic agent over another, e.g., recombinant tissue pl~sminogen 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 En~and J.
Med., 320, pp. 817-21 (March 30, 1989].
Numerous limitations and complications are associated with current thrombolyti 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 bleedin~ 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 agen~s 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 o~ bleeding [G. C. T~mmis et al., B.2412 WO91/01142 ~ . PCT/US90/04103 t ~ _ 2~ 4 "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 ~ecreasing reperfusion times and preventing rethromDosis in the treatment of experimental and clinical myocardial infarctions.
In one study, a combination of streptokinase and asplrln resulted in a reduction in rethrombosis ~y 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 thrr~bolytic agents in pre-clinical models of coronary arterial occlusion. Thromboxane A2 (TXA2) receptor antagonists or serotonin receptor antagonists, alone or together, substantially increases reocclusion time when combined with recombinant tPA
(rt~A) [P. Golino et al., 'IMediation of Reocclusion by Thromboxane A2 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 modestlydecrease reperfusion time when used wi~h 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 agent-- in promoting vasodilation, hypot~nsion and Dleeding B.2412 _5_ 2~0~
restricts the use of such combinations in a human clinical setting.
Antiplatelet agents which are antagom sts of the platelet fibrinogen receptor, glycoprotein IIb/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 thro~bi, increase reocclusion time and decrease reperfusion time in dogs [T. Yasuda et al., "Monoclonal ~ntibody Against the Platelet Glycoprotein (GP) IIb/IIIa Receptor Prevents Coronary Artery Reocclusion After Reperfusion with Recombinant Tissue-Type Plasminogen Activator in Dogs", J. Clln.
Invest., 81, pp. 1284-9l (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 G~IIb/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 dynami~
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 (l483); C. Y. Liu et al., "The Binding of Thrombin by Fibrin", J. Biol.~hem., 254, pp. 10421-25 B.2412 WOgl/01]42 ~ ; PCT/US90/04103 206423i 6-(1979)]. Upon thrombolysis, increased exposure of clot~bound thrombin may cause accelerated fibrinogen cleavage and rethrombosis.
Recently, it has been demonstrated that S the heparin-catalyzed inactivation of ~ thrombin by antithrombin III is neutralized by fibrin II monomer, a fibrin degradation product [P. J. Hogg and C. M.
Jackson, "Fibr-n 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 antigenic.
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.
Accordin~ to this inve~tion, an anticoagulant peptide which is at least partially homologous to the carboxyl terminal 25 amino acids of hir~din, or a derivative B.2412 ..

'~'' '' ' ' ~ ., ' ' , ' : , .. . .

WO91/01142 2 0 6 ~ ~ 3 ~ PCT/US9o/o4lo3 -7- ~
thereof, which displays anticoagulant activity, i5 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 reguired for a desired therapeutic or prophylactic effect when that agent is administered as a monotherapy. This, in turn, decreases 'he risk of undesirable side effects associated with the use of thrombolytic agents. Moreover, the anticoagulant peptide com-ponent of the combination exhibits a saturableefect on clotting time, resulting in a drastically reduced risk of bleeding.
This invention also provides methods, compositions and combinations for decreasing the dose of a thrombolytic a~ent required for a desired therapeutic or prophylactic effect in a patient, such as dissolving a blood clot. And this invention pro~ides 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 e~fective in the treatment and prevention of thrombotic diseases than conventional therapie~. 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 ~ be required to achieve B.2412 WO91/01142 2 0 6 ~ 2 ~ 1: PCT/VS90/04103 ~ 8-a desired therapeutic or prophylactic effect in therapy regimens based upon the use of that agent alone. And the combinations, compositions and me~hods 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 age-.t, the com-binations, compositions and methods of this inven-tion avoid the side effects of conventional anti-coagulants, such as hepaxin.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the purification:of Sulfo-Tyr63hirudins3_64 by reverse-phase ~PLC.
Figure 2 depicts ~PLC chromatograms illus-trating the relative efficiency of the sulfation process described herein, (Fisure 2c) as compared with conventional sulfation processes (Figures 2a, 2b) for the treatment of large ~uantities of peptide.
Figure 3 depicts the HPLC chromatographic elution profile of a mi~ture of Sulfonyl-Tyr63 hirudin53_64 and Sulfo-Tyr63hirUdin53_64 Figures 4A-4C depict the synthesis of hirulog-l, 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 WO91/01]42 , ~ PCT/US90/04103 - 9 20~ 12~1 -Figure 7 depicts the synthesls of hirulog-7, a peptidomimetic analog of a hirudin peptide.
Figure 8 depicts the comparative effect of heparin and sulfo-Tyr63~hirUdin53_64 admini as either an intravenous bolus injection ("i.v.") or a constant infusion ("inf.") on ln 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 embod,ment of ~his 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 establishedwhen 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 ~06~231 -lO-to reestablish blood flow followln~ 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 m~thods, 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.
lS 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 i 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 systemthromboses 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 paxticular, humans. The methods of this invention comprise the step of treating a patient in a pharmaceutically acceptable manner with B.2412 WO 91/01142 PCl'/US~0/0~103 ~0&~231:

a pharmaceutically effectlve comblnation 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 Glt - glutaryl Mal - maleyl NpA - b ta-(2-naphthyl)alanine Gly - glycine Ala - alanine Val - valine Leu - leucine Ile - isoleucine Pro - proline Phe - phenylalanine Trp - tryptophan Met - methionine Ser - serine ~hr - threonine - Cys - cysteine Tyr - tyrosine Asn - asparagine Gln - glutamine Asp - aspartic acid Glu - glutamic acid ~ys - lysine Arg - a~ginine His - histidine Nle - norleucine Hyp - hydroxyprol ne Pgl - phenylglycine Tyr(SO3H~ - tyxosine sulfonate D-Ala - D-alanine Ac - acetyl Suc - succinyl

3,4,-dehydroPro -- 3,4-dehydroproline Tyr(OSO3H) -- 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 pNO2Phe -- 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, ~.2412 .. ; . ,. ~ ..... .. .

WOgl/01142 PCT/US90/04103 '45~ 1 - _ cyclopentyl, hexyl, isohexyl, cyclohexyl and cyclo pentylmethyl. 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" a-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" a-amino acids are norleucine, norvaline, alloisoleucine, homoarginine, thiaproline, dehydroproline, hydroxyproline (Hyp), homoserine, cyclohexylglycine (Chg), ~-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 (C1-C4) alkyl, a (Cl-C4) alkoxy, halogen or nitro groups or substi-tuted with a methylenedioxy group, ~-2- and 3-thieny-lal-alanine, ~-2- and 3-furanylalanine, ~-2-, 3- and

4-pyridylalanine, ~-(benzothienyl-2- and 3-yl)alanine, ~-(1- 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 ~yrosine, 4-methylphosphonyl tyrosine, ~-phenylacetic acid, B.2412 ', ~ ! . . i . ' -13- 206~3~';
3,5-diiodotyrosl..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 seguence of amino acids consist-ing substantially of the formula:
Al A2 A3-A4-A5-A6~A7-As-As-Alo_y wherein X is a hydrogen, one or two alkyl groups of from l to 6 carbon atoms, one or two acyl groups of from 2 to l0 carbon atoms, carbobenzyloxy or t-butyloxy carbonyl; Al is a bond or is a peptide containing from l to ll residues of any amino acid:
A2 is Phe, SubPhe, ~-(2- and 3-thienyl)alanine, ~-(2-and 3-furanyl)alanine, ~-(2-, 3-and 4-pyridyl)alanine, ~-~benzothienyl-2- and 3-yl) alanine, ~ and 2-naphthyl)alanine, Tyr or Trp; A3 is Glu or Asp; A4 is any amino acid; A5 is Ile, Val, Leu, Nle or Phe;
A6 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carb-oxylate, Sar, NMePgl or D-Ala; A7 is any amino acid;
A8 is any amino acid; Ag is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, Pro, or a dipeptide con-sisting o~ one of these lipophilic amino acids and any amino acid; Alo 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, Cl-C6 alkoxy, amino, monor or di-(Cl-C4) 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; Al is Asn-Gly-Asp;
A2 is Phe; A3 is Glu; A4 is Glu; A5 is Ile; A6 is Pro; A7 is Glu; A8 is Glu; Agjls a dipeptide selected B.2412 . . , ,:;: . :, . . . .

::::: : -: :: .~ .. : : . .:. . ,: ; :, WO91/01142 ; PCT/US90/04103 2 ~ 2$ 1 `: -14-from the group consisting of S-alkylated cysteine-Leu, S-alkylated homocysteine- Leu, tyrosine-O-sul~ate-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; Alo 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 o~ this inven~ion is characterized in that X is N-acetyl; Al is Asn-Gly Asp; A2 is Phe; A3 is Glu;
A4 is Glu; A5 is Ile; A~ is Pro; A7 is Glu; A8 is Glu; Ag is the dipeptide tyrosine-O-sulfate-Leu;
Alo 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 -onventional 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 de~radation (to remove N-terminal amino acids). Most preferably, the peptide is pro-duced by solid phase peptide synthesis, as described in copending United States pa~ent applications Serial B.2412 .. ,; . ~ .~ ,. . .
. .. . .: . .
: , . . . .. ., . - ~.
. ~ . . - .- ,, .. .. ~ . . -- .. . . .~ .

WO91/01]42 2 0 ~ 4 2 3 1 PCT/US90/04103 -15~ ?,~
Nos. 16~,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 t.yrosine residue at position A9 must be performed after peptide synthesis.
Derivatization methods include, but are not limitei 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 technigues for sulfating, sulfonating, carboxylating, carbonylating, phosphorylating, and phosphonating a tyrosine are described in copending United States patent applications Serial Nos. 16g,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 ~iological (enzymatic) or a chemical process. Preferably, a puriXied anti-coagulant peptide is reacted concurrently with dicyclohexylcarbodiimide and sulfuric acid in an organic solvent. Sulfonation o~ 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 dissol~d in an organic sol-B.2412 g~S~3~ 16- Pcr/US90/04103 vent, preferably dimethylformamide, and then reacted with a dehydratin~ agent, preferably dicyclohexyl-carbodiimide. The dehydrated tyrosine residue of the peptide is then sulfated by reaction with sul-furic acid. The reactlon is complete upon formationof an insoluble dicyclohexyl urea salt. This modi-fication results in high yields of sulfated peptide on a large scale. This sulfation technigue 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-et~ylamine salt in pyridine. In addition, a tyrosyl-sulfotransferase activity, either as a crudepreparation or as a purified enzyme, may be used to sulfate the tyrosine residue [R. W. H. Lee and W. B.
~uttner, "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 phosphorlc acid or formic acid, respectively, for sulfuric acid. In those reactions, phosphonatio~ or carbonation will occux, 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 pep~ides may be achieved B.2412 O91/01142 ~ 0S~ 2 ~ 1 PCT/US9o/041~3 -17- ! :
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 60C for 30 minutes. This will result in an increase of maximal absorbance back to 2~5 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 mea~ures 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 anticoagSrant employed may be a B.2412 . .. .

WO91/01142 ~ 18- PCr/US90/04103 peptidomimetic analog of any of the hirudin peptides described above. Analogs of the hirudin peptides may be either semi-peptldic or non-peptidic in nature.
These analogs may be characterïzed 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 counL~rparts.
The thrombolytic agent utilized the methods, compositions and combinations of the present inven-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 ~SPAC), animal salivary ~land 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~ptide platelet inhib-B.2412 , . ., ,-. , . : ., , .: .

,, : - :;.. . , .:

WO91/01142 2 0 5 g 2 ~ PCT/US90/04l03 itors, such as ticlopidine, dipyridamole and sulphin-pyrazone; inhibitors of platelet surface components, such as inhibitors of glycoprotein IIb/IIIa, inhlbi-tors of glycoprotein Ib, antibodies against glycopro-tein IIb/IIIa, and antibodies against glycoproteinIb; 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 inthe 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., Reminqton~s Pharmaceutical Sciences ~E. W. Martin).
In addition, the compositions and combinatlons 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 anticoagulan~ peptide is administered B.2412 ... ~.: . " . .

. - :i:. -:. .. . . .

WO91tO1142 , ~ ~ ~ PCT/US90/04~03 to a patient during the time period ranging from about 2 hours prior to about 2 hours following administration of the thrombolytic agent. Othex administration schedules may also be employed.
Alternatively, the anticoagulant peptide and the thrombolytic agent may be in the form of a 'f 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 ln 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 compositionsand 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 andparenteral administration, but, most preferably, are formulated for parenteral adm~nistration.

B.2412 WO91/01142 2 ~6 ~2 Parenteral compositions are most preferably administered intravenously either in a bolus form or as a constant infusion. For parenteral administra~
tion, fluid uni~ dose forms are prepared which contain a composition of the present invention and a s~erile vehicle. The anticoagulant peptide and thrombolytic agent components of the pharm~ceutically 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 local anesthetic, preservatives and buffering agen~s 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, ~illers, diluents, tableting agents, lubri~
cants, disintegrants, and wetting agents. ~he 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 disintegxants include, but are not limited to, starch, polyvinylpyrroli~one and starch deriva-B.2412 ,. ,.~ , .. , . .... - . -, wo 91/01]42 2 b~¢ ~ 22- Pcr/lJS90/0~103 tives, such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate.
Suitable wetting agents include sodium lauryl sulfate.
Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, 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 seguentially 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 seguentially to the patient, with the anticoagulant peptide being administered before, after, or both before and after treatment with ~he 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 i~vention include those - B.2412 .

, ,, . ... .. : . : :.: . . . . . ... .. .. .
: ; .. . .. .. . - - ~; . ;. .

W~91/01142 -23- 2 Q 6 ~ 2 3 1 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 un}ts/patient;
streptokinase - 140,000 to 2,500,000 units/patient;
tPA - 0.5 to S.0 mg/kg body weight; ASPAC - O.l to lO units/kg body weight.
Most preferably, the compositions of the present invention comprise between about O.l 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 -~5 range. For example, an anticoagulant dosage of aspirin is normally 50-300 mg/patient.

B.2412 .; - . .,"
, WO91/01142 i ,t PCT/US90/04~03 '~0~231 -24- ~
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 administxation, or both, may be reducea, 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, reguire inter-mittent treatment upon any recurrence of diseasesymptoms.
In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these lS 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 110C 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-l00 ~g) were applied to a Vydac C4 column (0.46 x 25 cm) or an Aquapore RP-300 C8 column (0.46 x 3.0 cm) using a Beckman Liguid Chromatographic system or an Applied Biosystems 150A Chromatographic System, respectively.
The Vydac C4 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 ~.0 ml/min. The B.2412 ,,, ,, ~ . . ;, ., ~ .,!,.. .
" , '. ,' . . ` ', , ' .' ' ' ';, ' ' '; ' " ' ' `;' ' ~, ' ' ' ' . ':' . ' ' ' ' . ' ' , , / '. .' ' , 1 `, .' . '.. ,', . .' ~, ' , ' ' ' ,; . ~' ' 1 ., . '; ' ' ' ' ' '~ ~ ' ' ' " ' ' ; ' ' ' ' I . ' .,.. . , ,, .. ' WO91/01142 2 0 ~ ~ 2 3 1 PcT/US~o/04103 effluent stream was monitored at 215 nm for absorb-ance. The Aquapore C8 column was equilibrated in water containing 0.1% TFA and developed wlth 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.

Synthesis of Hirudin53_64 And Hirudin49_64 Hirudin53_64 (subscript numbers represent the corresponding amino acid position in the native hirudin molecule) has the amino acid formula:
H2N-Asn-Gly-Asp-Phe-~,lu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH. Hirudingg 64 has the amino acid formula:
H2N-Glu-Ser-His-Asn-Asn-Gly~Asp-Phe-~,lu-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)) 25 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. Hirudin53_64 and hirudin49_64 thus syn-thesized were fully deprotected and cleaved from ~he 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 hirudin49_64 and hirudin53-64' respectively-B.2412 , . ;, . . ,; . . - . . - ., . ~ , . . .

WO91/01142 ~t'~ PCT/US~0/04103 20~ ~2~ 1 -26-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.l min and 16.3 min, respectively, for hirudin49 64 and hirUdins3-64 Synthesis Of N-Acetyl Hirudin53_64 N-acetylation of the hirudin peptides of this invention was achieved directly during pep-tide synthesis. For example, N-acety~ hirudin53 $4 was synthesized by the basic procedure used to s~n-thesize hirudin53 64 described in Example lo 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.

Sulfation Of Hirudin Peptides Hirudin53_64 was O-sulfated at the tyrosine residue to prepare Sulfo-Tyr63hirudin53 64' usiny the chemical modification procedure of T. Nakahara et al., "Preparation of Tyrosine-0-~35S] Sulfated Cholecystokinin Octapeptide from a Non-Sulfated Precursor Peptide", Anal. Biochem., 154, pp. 194~99 (1986). We dissolved 1.5 mg of hirudin53 64~ as prepared in Example l, in 50 ~l of dimethylformamide and dried the solution under N2. The peptide wa~
then redissolved in 40 ~l of dimethylformamide (DMF) containing 2 x lO 5 moles of sulfuric acid. To this we added lO ~l of a solution containin~ 50 ~g N,N'-dicyclohexylcarbodiimide in 40 ~l DMF (7.0 x l0 5 moles). The reaction was allowed to proceed for about 5-lO min at 25C be~ore the addition of 750 ~l of deionized water. ~ny insoluble reaction B.2412 -27- ';
products were xemoved by centrifugation in a micro-fuge apparatus prior to further purification.
SUlf-TYr63hirUdins3~64 was purified away from other peptide and reaction components by reverse-phase HPLC employing a Vydac C18 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 gradlent 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-Tyr63hirudin53-64-containins 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 hirudin53_64 structure-These data demonstrated that peak A contained Sulfo TYr63hirUdins3-64-We confirmed the presence of Sulfo-Tyr63 hirudin53_64 by treating the peptide of peak A with 30% TFA at 60C for l hour to remove the sulfate group. We then dried the peptide, redissolved it in water and subjected it to reverse-phase ~PLC. We carried out HPLC analysis of the desulfated Sulfo-TYr63hirUdin53_64 using an Aquapore RP-300 C column ~O.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 ofincreasing acetonitrile concentration from 0 to 70%
over 45 minutes at a flow ra~ of 0.5 ml/min in a B.2412 . - : .

~ ., . :

,;, i~ ,`'.i `~ .~

2 0 ~ ~23 ~ -28-0.085% TFA-contalning solvent. The peptide showed HPLC chromatographlc behavior ldentical to that of unsulfated hirudin53_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, treatme~t of 25 mg of N-acetyl-hirudin53_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-hirudin53_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-hirudin53_6~ in 40 ml of dimethyl-formamide in the presence of 5.0 ml of N,N'odicycl-ohexylcarbodiimide (0.2 g~.16 ml dimethyl formamide).
The mixture was stirred at 0C and 0.5 ml of con-- 25 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 ~PLC separa-tion of the reaction mixture (Fig. 2c) indicated a ~0 81.7% yield of the sulfated peptide, Sulfo-Tyr63-N-acetyl-hirudins3-64 Large-scale purification of the sulfated hirudin peptide was then achieved by a one-step anion exchange chromatography. Specifically, crude Sulfo-Tyr63-N-acetyl-hirudin53_64 was purified on a column of DEAE-Sepharose (250 ml wet resin/5 g crude peptide). The column w~ pre-equilibrated and B.2412 . . . . ....... . ..
;
"~:, ' '', ,,, " ,,"~-",,s., ,,, ,,, ~" ~,,,,,,,~ : ,, , : . ~ . . , .... : : .; .

2~6~231 -29- ; ! .;~
the sample was loaded in 20 mM sodium acetate, pH
5.o. The column was developed with a linear NaCl gradient (0-0.4 M). The Sulfo-Tyr63-N-acetyl-hirudin53 64 eluted at approximately 0.2-0.3 M NaCl, after the unsulfated peptide, but prior to the sul-fonated side product Sulfo-Tyr63-N-acetyl-hirudin53-64 -Sulfonation Of ~irudin Peptides N-acetyl-hirudin53_64 was modified to its Tyr-sulfo~ated derivative, Sulfonyl-Tyr63-N-acetyl-hirUdin53-64~ during the preparation of Sulfo-Tyr63-N-acetyl-hirudin53_64, as described in Exam-ple 3. Sulfonyl-Tyr63-N-acetyl-hirudin53 64 was a side reaction product obtained during the large-scale sulfation reaction described in that example. Accord-ingly, Sulfonyl-Tyr63-N-acetyl-hirudin53 64 was obtained at between 30 to 40% yield and was:~ou~d to elute prior to sulfo-Tyr63-hirudin53-63 in reverse-phase HPLC separations (see Figure 3).
EX~M2LE 5 Peptidomimetic Analogs Of Hirudin Peptides Hirudin peptides, preferably Sulfo-Tyr63 hirudin53_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-l:
Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu Gly Tyr(SO3) \ _ /
Asn-Cys-S-S-Cys-Leu B.2412 .
: .
, ~ . . .

.. : . . . -.... . .

... . .

WO91/01142 ,~ , PcT/US9o/o41o3 23~ 30 Hiruloq-2:
Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu Gly Tyr~SO3) Asn-Cys-S-S-(CH2)n-S-S-Cys-Leu Hiruloq-3:
Asp-Phe-Glu-Glu-Ile~Pro-Glu Glu Gly Tyr(S03) Asn-Cys-S fH~ ( CH2 )n-~H-S-Cys-Leu COOH COOH

Hiruloq-4:
Asp-Phe-Glu-Glu-Ile-Pro-Glu Glu Gly H ~ Tyr(SO3) Asn-Lys N-C-(C~2)n-C-N-LYs-LeU
O

Hiruloq-5:
Asn-Gly-Asp-Phe-Glu~Glu-Ala / \
O Pro C=O Glu Leu-(SO3)Tyr-Glu Hirulo~-6:
Asn-Gly-Asp-Phe-Glu-Glu-Ala-Pro O Glu Leu-(SO3)Tyr-Ser B.24~2 ,., . : .: . . . . : .~ , .~ . ,,. .. . , . ~.

O91/01]42 2 0 6 ~ 2 3 1 PCT/US90/04103 Hirulog-7:
Cys-Gly-Asp-Cys-Glu-Glu-I le-Pro-Glu-Glu-Tyr(sO3)-Leu S (CH2)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-Tyr63hirudin53 64.
Terminal cysteinic residues are crosslinked by oxida-tion to produce Hirulog-l (Figure 4a), oxidation with an aliphatic di-:~iol to produc~ Hirulog-2 (Figure 4b), or alkylation with aliphatic dihaloace-tate or propionate to produce Hirulog-3 (Figure 4c).
Terminal ly yl 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-TYr63hirUdin53_64 is Constrained by replacement of Ile-7 with chloroalanine, with or wi~hout concomitant replacement of Glu-9 or Glu-lO with (L) or ~D)-serine.
Peptidomimetic analogs containing chloroalanine alone would yield cross-linking of Glu-9 or Glu-lO to Ala-7 with a ketone linkage to produce ~irulog-5 (Figure 6a). Derivatives with serine at positions 9 or lO 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 ~p aliphatic dihalo B.2412 WO91/01142 . : PCT/US90/04103 2~423i~ s -32-acetate. For example, replacement of Asn-1 a~d Phe-4 of Sulfo-Tyr63hirudin53_64 with cysteines and oxidation via ethanedithiol (Figure 7) would con-strain a helical turn in the NH2-terminal side of the derivative and, thus, seed a stable helical structure in the peptide derivative. This is exemplified by ~irulog-7.
Fully non-peptidic peptidomimetlc analogs may also be produced, taking into consideration the above-described strategies relating to constrained peptide compounds.
r Synthesis Of ~irudin Peptide Analoqs The hirudin analog dinitrofluorobenzyl-lS SUlfo-Tyr63hirudin54_64 (DNFB-sulfo-T~r63hirudin54 64) was synthesized by reacting stoichiometric quantities of Sulfo-Tyr63hirudin54_64 with dinitrodifluorobenzene (DNDFB) in dimethylformamide. We incubated the reac-tion mix for 24 hours at 22C 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-Tyr63 hirudin54_64 eluted at 48% solvent B.
We synthesiæed the hirudin analog nitro-anisole63hirudin53_63 in a ~wo-step method- First~
we synthesized methoxytyrosyl63hirudin53 63 by the solid phase synthesis techniques, as described in Example 1, substituting Boc-0-methoxytyrosine resin for Boc-0-Leu resin in the synthesis. The peptide B.2412 -. , , ;, .. , . . . ~

WO91/01142 20 6~2 3l was cleaved from the resin and purlfied by HPLC on a Vydac C4 column, as described in Figure l. We next nitrated the purified methoxytyrosyl63hirudin53 63 by adding to it an excess of tetranitromethane in 20 mM Tris-HCl, pH 8Ø The reaction was incubated at 27C for 4 hours. The xesulting products are lyophilized, redissolved in 20 mM ammonium bicar- ~`
bonate and desalted on a Biogel P-4 column (l x 30 cm) which was equilibrated and eluted in 20 mM ammonium bicarbonate. The separation of nitroanisole63hiru-din53 63 was achieved by HPLC on an Aquapore RP-300 C8 octasilyl column (0.46 x 3.0 cm) as described above.
ExAr~LE ?
Failure Of Heparin To Neutralize Clot Bound Thrombin We analyzed the heparin-catalyzed, ~nti-thrombin III-dependent inactivation of non ~lot-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 37C, was incubated with varying concentrations of heparin (Upjohn, Kalamazoo, Michigan; O.Ol - l~0 U/ml) for l 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 ~0 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.24l2 2~23;~ n~ ~

[Thrombin] [Heparin]FPA release (U/ml) (U/ml) (nM) 0.005 0 48.5 0.0l 38.9 0.l0 25.2 l.0 3.3 0.0l0 0 94.3 0.~l 70.7 ' 0.l0 48.9 l.0 8.9 : 0.050 0 653.0 0.0l 517.2 0.l0 326.9 l.0 54.4 lS As demonstrated above, a heparin concen-tration of 0.l U/ml caused an approximately 50%
inhibi~ion of thrombin-cataly~ed 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 CaCl2 to citrated, normal human plasma in the presence of both l25I-fibrinogen (25,000 cpm/ml), which was labelled by the IODOGEN method follwing manufacturer's 25 directions (Pierce, Rockford, Illinois) and a copper ~:
coil. We removed the resulting radiolabelled fibrin clot which formed on the copper coil after l0 minutes.
The clot was washed exte~sively with phosphate buffered saline and then dialyzed against 4 liters ~f the same buffer for 24 hours to remo~e any non-adsorbed thrombin and soluble FPA.
We assayed clot-bound thrombin by adding the washed clot to human plasma maintained at 37C, incubating for 5 minutes and then measuring FPA
~5 release as described above. All assays were normal-ized for fibrin content by measurement of l25I-fibrinogen incorporation, assuming that the amount of ~hrombin adsorbed to the clot was proportional to ~.2~12 .. .. . . -. .. ..

, ~, , , , . , .,, .. ~ . , ~. ' . ! , . .. .. . . ..

Wo91/01142 PCT/US90/04103 2~231~ `

the size of the clot and, hence, the radioisotope r concentration.
We determined the degree of inhibition of clot-bound thrombin by heparin by adding various 5 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:
[Heparin] FPA release Inhibition (U~ml) ~nM) o 175.5 0 0.1 124.5 0.8 2.5 57.0 54.6 5.0 24.2 80.7 7.~ 13.1 89.6 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-20 thrombin III complex was more than lO-fold less efficient in neutralizing clot-bound rather than clot-free thrombin. It is much more difficult for the heparin-an~ithrombin III complex to access thrombin when adsorbed to fibxin. Thus, heparin 25 demonstrates a limited antithrombotic effect in the course of thrombosis and fibrin accumulation.

Inhibition Of Free And Clot-Bound Thrombin By Hirudin Pe~tides We next analyzed the ability of a synthetic Tyr-sulfated hirudin peptide, N~Ac-Asn-~ly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(OS03H)-Leu-OH ("N-acetyl Sulfo-Tyr63 hirudin53~64"), to n~utralize free thrombin and clot-bound thrombin. The synthesis of -~5 N-acetyl Sulfo-Tyr63 hirudin53_64 is described in Example 2.

B.2412 , - , : ,, ., .. ,,., - , .
.. . : ,: ; . . : ,: . ., :
.. ,., .... . ,- .. " . . ~ .. .. ..... . .. .. ..
. - ~ .. ~ .,: : ::: . .: .,: , . , , :

.

. ;' ~ ' , ' ~ ~ , , . . A . . ~

~06~L231 WO 91/01142 PCr/US90/04103 --3 7 ~
thrombolytic therapy, or clot extension, such as that observed in deep vein thrombosis.
E ~ LE 9 Inhibition of Clot-Bound Thrombin By 5Other Antithrombin III-Independent Inhibitors The failure of heparin to efficiently inhibit clot-bound thrombin may be due to steric hindrance relat?d to the size of the hepaxln-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 III-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-CH2Cl (PPACK) to neutralize thrombin. We found that a concentration of 3.0 nM PPACK inhibits both free and clot-bound thrombin by over 70%.

25Inhibition By N-Acetyl Sulfo-Tyr Hirudin Of Clot-Bound Thro~ ~n In A Rabb~ del Of Thrombus Accretion .. . .
Having observed the ln vitro efficacy of N-acetyl Sulfo-Tyr63hirudin53_64 in blocking thrombin adsorbed to fibrin clots, we next examined the activity of that peptide in ln VlVO studies of fibrin accumulation on pre-existing thrombi.
We anesthetized New Zealand White rabbits (provided by Drs. Michael Buchanan and Jack ~irsh, McMaster University, Hamilton Ontario) with 30 mg/kg of sodium pentobarbitol inje~ted into the marginal ~2412 WO~1/01142 PCT/US90/04103 2 0 6 ~ %lU~ 38-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 l.O ml of blood from a carotid cannula into a 1.O ml tuberculin syringe containing l unit of human a-thrombin. We immediately injected 150 ~l 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 injected l.0 mg of l25I-rabbit fibrinogen 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 regim~n 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-Tyr63hirudin53_64 at a rate of 0.5 ~g/kg/hr (Group III). The final group received a single i.v.
bolus injection of 2.0 mg/kg N-acetyl Sulfo-Tyr63 hirudin53_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 l25I-fibrin. In addition, we monitored the anti-coagulant effects of the ~arious 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-Tyr63hirudi~53_64 (Group III) inhibited thrombus B.24l2 WO~]/01142 2 ~ 6 4 231 ', ,,, ' ' growth by 43.7%, while i.v. bolus injection of N-acetyl Sulfo-Tyr63hirudin53 ~ caused a 62.4%
inhibition. The difference between the control group (Group I) and Group IV was statistically significant (Stud~nt'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 retnrombosis of reperfused arterial emboli when - hirudin peptides are used in combination with throm-bolytic agents.
EXAMPLE ll Effects Of Adjuvant Use Of ~irudin Peptides With Recombinant tPA In Reperfusion Of Experimental Coronary Thrombi In Doqs The preparation of coronary thrombosis and its lysis by recombi~ant tPA (rtPA) was performed essentially as described previously [P. Golino et al., 25 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 anestheti~ed the dogs by injecting th~m 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 wer~ established in the left anterior descending (LAD5 artery as follows.

B.2412 .. . . . . .
. . . : . ., . ~ . . ;. . ;. ~

~ o ~ 4 ~ 3 1 40 Pcr/usso/04103 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 5 the segment A No. 7F Amplatz Ll left coronary cath-eter (Cordls 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., Bloomington, Indiana3 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 min~les or until reperfusion occurred. A second group of eight dogs (Group II) was infused with 2.0 mg/kg/hr of N-acetyl Sulfo-Tyr63hirudin53_64. p was monitored by hemodynamic measurements and reper fusion time was defined as the time re~uired to reestablish blood flow through the LAD following initiation of thrombolytic therapy. The results of these treatments are shown be~w:

B.2412 .. ... . .

:,....... , ., " :: , .. ,.. , , .. : .

. . . ;. . .. . ,: , . " . .
.. ,:: .. :: .. ..... ,., ..... :.

WO91/01142 PCT/US90/Oq~03 -41- 2~ 6~23l Reperfusion Time Treatment (min) I control (sallne 32 ~ 4 infusion) II hirudin peptide l9 ~ 6 lnfuslon This data demonstrates that adjuvant use of a hiruàin peptide with rtPA in thrombolysis of coronary thrombi significantly reduced the tlme required to establish reperfusion. Thus, for a given dose of thrombolytic agent, reperfusion time may be decreased when that agent i5 admlnistered 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 o~
hirudin peptides in combination with thrombolyt;_ agents as a means to rapidly achieve reperfusion in a patient, thus decreasing the extent of damage to the myocardial tissue ~esulting from infarction.
Moreover, the adjuvant use of hirudin peptides withthrombolytic agents permits the use of lower doses o 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 3~ ultima~e 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 o this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claim~ appended hereto rather B.24l2 WO91/01142 ; ,~ PCT/US90/04103 ~06~%3~ 42- ~: ~
than the specific embodiments which have been pre-sented hereinbefore by way of example.

B . 2412 : : . , ' .: . `. .;: . ~; .., . ' `, ", ' ' ' ,'., , . . ,. . .,.,:' , , , , ,,: :. . ~ ... .. ..

WO91/01142 PCr/US90/04103 2 3 1 I claim:
1. A pharmaceutically effective combi-nation for treating or preventing thrombotic disease n a patient comprlsing: : .
a) a peptide characterized by a sequence of amino acids consisting substantially of the formula:
1 A2 A3 A4-A5-A6-A7-A8-Ag~A1o~y wherein X is a hydrogen, one or two alkyl gr~ups of from l to 6 carbon atoms, o~e or two acyl groups of from 2 to 10 carbon atoms, carbobenzyloxy or t-butyl-oxy carbonyl; A1 is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A2 is Phe, :~
SubPhe, ~-(2- and 3-thienyl)alanine, ~-(2- and 3-furanyl)alanine, ~-(2-, 3- and 4-pyridyl)alanine, ~-(benzothienyl~2- and 3-yl) alanine, ~-(1- and 2-naphthyl)alanine, Tyr or Trp; A3 is Glu or Asp;
A4 is any amino acid; A5 is Ile, Val, Leu, ~le or Phe; A6 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, Sar, NMePgl or D-Ala; A7 is any amino acid; A8 is any amino acid; Ag is a lipophilic amino .
acid selected from ~he group consisting of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; ~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, Cl-C6 alkoxy, amino, mono- or di-(Cl-C4) alkyl substituted amino or benzylamino; said peptide displaying anticoagula~t 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 ~equired for a desired B.2412

Claims (42)

2. WO 91/01142 PCT/US90/04103 therapeutic or prophylactic effect when that agent is administered as a monotherapy.
3. 3. The combination according to claim 1 or 2, wherein the peptide is characterized in that X
   is hydrogen or N-acetyl ; A1 is Asn-Gly-Asp; A2 is Phe; A3 is Glu; A4 is Glu; A5 is Ile; A6 is Pro; A7 is Glu; A8 is Glu; A9 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; A10 is a bond; and Y is OH.
4. 4. The combination according to claim 3, wherein the peptide is characterized in that X is N-acetyl and A9 is tyrosine-O-sulfate-Leu.
5. 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. 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. 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 80% of the dosage required for a desired therapeutic or prophylactic effect when that agent is administered as a monotherapy.
8. 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. 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. 10. The combination according to claim 1 or 2, further comprising a pharmaceutically effective amount of an antiplatelet agent.
11. 11. The combination according to claim 10, wherein said antiplatelet agent is aspirin.
12. 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. 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:
    
    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; A1 is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A2 is Phe, SubPhe, .beta.-(2- and 3-thienyl)alanine, .beta.-(2- and 3-furanyl)alanine, .beta.-(2-, 3- and 4-pyridyl)alanine, .beta.-(benzothienyl-2- and 3-yl) alanine, .beta.-(1- and 2-naphthyl)alanine, Tyr or Trp; A3 is Glu or Asp;
    A4 is any amino acid; A5 is Ile, Val, Leu, Nle or Phe; A6 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, Sar, NMePgl or D-Ala; A7 is any amino acid; A8 is any amino acid; A9 is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A10 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, C1-C6 alkoxy, amino, mono- or di-(C1-C4) alkyl substituted amino or benzylamino; said peptide displaying anticoagulant activity; and b) a thrombolytic agent.
14. 14. The method according to claim 13, wherein the peptide is characterized in that X is hydrogen or N-acetyl; A1 is Asn-Gly-Asp; A2 is Phe; A3 is Glu; A4 is Glu; A5 is Ile; A6 is Pro; A7 is Glu; A8 is Glu; A9 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, and 3,5-diio-dotyrosine-Leu; A10 is a bond; and Y is OH.
15. 15. The method according to claim 14, wherein the peptide is characterized in that X is N-acetyl and A9 is tyrosine-O-sulfate-Leu.
16. 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:
    
    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; A1 is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A2 is Phe, SubPhe, .beta.-(2- and 3-thienyl)alanine, .beta.-(2- and 3-furanyl)alanine, .beta.-(2-, 3- and 4-pyridyl)alanine, .beta.-(benzothienyl-2- and 3-yl) alanine, .beta.-(1- and 2-naphthyl)alanine, Tyr or Trp; A3 is Glu or Asp;
    A4 is any amino acid; A5 is Ile, Val, Leu, Nle or Phe; A6 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, Sar, NMePgl or D-Ala; A7 is any amino acid; A8 is any amino acid; A9 is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A10 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, C1-C6 alkoxy, amino, mono- or di-(C1-C4) 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. 17. The method according to claim 16, wherein the peptide is characterized in that X is hydrogen or N-acetyl; A1 is Asn-Gly-Asp; A2 is Phe;
    A3 is Glu; A4 is Glu; A5 is Ile; A6 is Pro; A7 is Glu; A8 is Glu; A9 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; A10 is a bond; and Y is OH.
18. 18. The method according to claim 17, wherein the peptide is characterized in that X is N-acetyl and A9 is tyrosine-O-sulfate-Leu.
19. 19. The method according to any one of claims 12, 13 and 16, wherein the patient is a human.
20. 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, streptokinase, urokinase, prourokinase, APSAC, animal salivary gland plasminogen activators and derivatives thereof.
21. 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:
    
    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; A1 is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A2 is Phe, SubPhe, .beta.-(2- and 3-thienyl)alanine, .beta.-(2- and 3-furanyl)alanine, .beta.-(2-, 3- and 4-pyridyl)alanine, .beta.-(benzothienyl-2- and 3-yl) alanine, .beta.-(1- and 2-naphthyl)alanine, Tyr or Trp; A3 is Glu or Asp;
    A4 is any amino acid; A5 is Ile, Val, Leu, Nle or Phe; A6 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, Sar, NMePgl or D-Ala; A7 is any amino acid; A8 is any amino acid; A9 is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A10 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, C1-C6 alkoxy, amino, mono- or di-(C1-C4) alkyl substituted amino or benzylamino.
22. 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 therapeutic or prophylactic effect when that agent is administered as a monotherapy.
23. 23. The use according to claim 21 or 22, wherein the peptide is characterized in that X is hydrogen or N-acetyl; A1 is Asn-Gly-Asp; A2 is Phe;
    A3 is Glu; A4 is Glu; A5 is Ile; A6 is Pro; A7 is Glu; A8 is Glu; A9 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; A10 is a bond; and Y is OH.
24. 24. The use according to claim 23, wherein the peptide is characterized in that X is N-acetyl and A9 is tyrosine-O-sulfate-Leu.
25. 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. 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. 27. The use according to claim 26, 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.
28. 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. 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. 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:
    
    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; A1 is a bond or is a peptide containing from 1 to 11 residues of any amino acid: A2 is Phe, SubPhe, .beta.-(2- and 3-thienyl)alanine, .beta.-(2- and 3-furanyl)alanine, .beta.-(2-, 3- and 4-pyridyl)alanine, .beta.-(benzothienyl-2- and 3-yl) alanine, .beta.-(1- and 2-naphthyl)alanine, Tyr or Trp; A3 is Glu or Asp;
    A4 is any amino acid; A5 is Ile, Val, Leu, Nle or Phe; A6 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, Sar, NMePgl or D-Ala; A7 is any amino acid; A8 is any amino acid; A9 is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A10 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, C1-C6 alkoxy, amino, mono- or di-(C1-C4) alkyl substituted amino or benzylamino; said analog displaying anticoagulant activity; and b) a thrombolytic agent.
31. 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. 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. 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. 34. The combination according to claim 30 or 31, further comprising a pharmaceutically effective amount of an antiplatelet agent.
35. 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.
36. 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:
    
    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; A1 is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A2 is Phe, SubPhe, .beta.-(2- and 3-thienyl)alanine, .beta.-(2- and 3-furanyl)alanine, .beta.-(2-, 3- and 4-pyridyl)alanine, .beta.-(benzothienyl-2 and 3-yl) alanine, .beta.-(1- and 2-naphthyl)alanine, Tyr or Trp; A3 is Glu or Asp;
    A4 is any amino acid; A5 is Ile, Val, Leu, Nle or Phe; A6 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, Sar, NMePgl or D-Ala; A7 is any amino acid; A8 is any amino acid; A9 is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A10 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, C1-C6 alkoxy, amino, mono- or di-(C1-C4) alkyl substituted amino or benzylamino; said analog displaying anticoagulant activity; and b) a thrombolytic agent.
37. 37. 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 peptidomimetic analog of 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; A1 is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A2 is Phe, SubPhe, .beta.-(2- and 3-thienyl)alanine, .beta.-(2- and 3-furanyl)alanine, .beta.-(2-, 3- and 4-pyridyl)alanine, .beta.-(benzothienyl-2- and 3-yl) alanine, .beta.-(1- and 2-naphthyl)alanine, Tyr or Trp; A3 is Glu or Asp;
    A4 is any amino acid; A5 is Ile, Val, Leu, Nle or Phe; A6 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, Sar, NMePgl or D-Ala; A7 is any amino acid; A8 is any amino acid; A9 is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, Pro or a dipeptide consisting of said lipophilic amino acid and any amino acid; A10 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, C1-C6 alkoxy, amino, mono- or di-(C1-C4) 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. 38. The method according to any one of claims 35 to 37, wherein the patient is a human.
39. 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. 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:
    
    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; A1 is a bond or is a peptide containing from 1 to 11 residues of any amino acid; A2 is Phe, SubPhe, .beta.-(2- and 3-thienyl)alanine, .beta.-(2- and 3-furanyl)alanine, .beta.-(2-, 3- and 4-pyridyl)alanine, .beta.-(benzothienyl-2- and 3-yl) alanine, .beta.-(1- and 2-naphthyl)alanine, Tyr or Trp; A3 is Glu or Asp;
    A4 is any amino acid; A5 is Ile, Val, Leu, Nle or Phe; A6 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, Sar, NMePgl or D-Ala; A7 is any amino acid; A8 is any amino acid; A9 is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, Pro, or a dipeptide consisting of said lipophilic amino acid and any amino acid; A10 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, C1-C6 alkoxy, amino, mono- or di-(C1-C4) alkyl substituted amino or benzylamino.
41. 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. 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.
    
    FIG.1