WO1991001142A1 - Combinations and methods for treating or preventing thrombotic diseases - Google Patents
Combinations and methods for treating or preventing thrombotic diseases Download PDFInfo
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- WO1991001142A1 WO1991001142A1 PCT/US1990/004103 US9004103W WO9101142A1 WO 1991001142 A1 WO1991001142 A1 WO 1991001142A1 US 9004103 W US9004103 W US 9004103W WO 9101142 A1 WO9101142 A1 WO 9101142A1
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- leu
- amino acid
- tyrosine
- peptide
- alanine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- A61K38/166—Streptokinase
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/49—Urokinase; Tissue plasminogen activator
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2123/00—Preparations for testing in vivo
Definitions
- 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.
- 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.
- 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.
- diseases are caused by either partial or total occlusion of a blood vessel by a blood clot, which consists of fibrin and platelet aggregates.
- 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.
- heparin produces many undesirable side effects, including hemorrhaging and thrombocytopenia.
- the second category of thera ⁇ Prinic accelerates thrombolysis and dissolves the blood clot, thereby removing it from the blood vessel and unblocking the flow of blood [V. J. Marder
- thrombolytic therapies 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.,
- Thromboxane A 2 (TXA 2 ) receptor antagonists or serotonin receptor antagonists 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)].
- PGE prostaglandin E
- 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.
- 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
- 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.
- 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.
- 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.
- 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
- 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.
- 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.
- FIG. 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.
- i.v. intravenous bolus injection
- inf. constant infusion
- 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.
- 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.
- anticoagulant peptides may be employed in methods, compositions and combinations for decreasing reperfusion time, i.e., the time required
- 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.
- 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.
- 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.
- 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.
- amino acids are used throughout the present application:
- Leu - leucine lie - isoleucine
- 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.
- 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.
- any amino acid as used herein includes the L-isomers of the naturally occurring amino acids, as well as other "non-protein” ⁇ -»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.
- non-protein ⁇ -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, ⁇ -2- and 3-thieny- lal-alanine, ⁇ -2- and 3-furany1alanine, ⁇ -2-, 3- and 4-pyridylalanine,
- 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.
- the anticoagulant peptide is characterized by a sequence of amino acids consist ⁇ ing substantially of the formula: X-A ⁇ -A 2 -A 3 -A 4 -A 5 -A 6 -A ?
- 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, ⁇ -(2- and 3-thienyl)alanine, ⁇ -(2- and 3-furanyl)alanine, ⁇ -(2-, 3-and 4-pyridyl)alanine, ⁇ -(benzothienyl-2- and 3-yl) alanine, ⁇ -(l- and 2-naphthyl)alanine, Tyr or Trp;
- a 3 is Glu or Asp;
- a 4 is any amino acid; A-.
- 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
- Y is a carboxy terminal residue selected from OH, C.-Cg alkoxy, amino, mono- or di-(C.-C 4 ) alkyl substituted amino or benzylamino.
- 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 ⁇ 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,
- the anticoagulant peptide employed in the combinations, compositions and methods of this invention is characterized in that X is
- 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.
- 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.
- 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.
- 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).
- the peptide is pro ⁇ quiz played by solid phase peptide synthesis, as described in copending United States patent applications Serial
- non-protein amino acids 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.
- Sulfation of the anticoagulant peptide may be achieved either by a biological (enzymatic) or a chemical process.
- 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.
- the sulfation procedure is modified, so that gram quantities of the peptide are first dissolved in an organic sol-
- B.2412 vent preferably dimethylformamide
- 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.
- 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.
- 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.
- 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.
- 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).
- APTT activated partial thromboplastin times
- TT thrombin times
- the anticoagulant employed may be a
- hirudin peptides 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.
- fibrinolytics such as tissue plasmin ⁇ ogen activator purified from natural sources, recombinant tissue plasminogen activator, strepto ⁇ kinase, urokinase, prourokinase, anisolated strepto
- antiplatelet agent may be made from among those well known in the art.
- 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.
- 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) .
- 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.
- 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.
- 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.
- the anticoagulant,- peptide is administered
- 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.
- 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.
- compositions are most preferably administered intravenously either in a bolus form or as a constant infusion.
- 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.
- 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.
- 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.
- 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.
- 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
- 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.
- 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.
- an anticoagulant peptide when 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.
- 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.
- an anticoagulant dosage of aspirin is normally 50-300 mg/patient.
- 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.
- peptide samples (20-100 ⁇ g) 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 ⁇ .O ml/min.
- TFA trifluoroacetic acid
- 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 ⁇ client 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.
- 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.
- N-acetylation of the hirudin peptides of this invention was achieved directly during pep ⁇ tide synthesis.
- N-acetyl hirudin 53 _ g4 was synthesized by the basic procedure used to syn ⁇ thesize hirudin 53 _ 64 described in Example 1.
- N-acetyl-asparagine for 2 mmoles of asparagine in the final cycle of peptide synthesis.
- 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
- 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).
- 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.
- 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).
- Sulfonyl-Tyr 63 -N-acetyl- hirudin 53 _ 64 was a side reaction product obtained during the large-scale sulfation reaction described in that example.
- 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).
- 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:
- Semi-peptidic peptidomimetic analogs of the hirudin peptides may be prepared to stabilize a loop, tur: or helical conformation of the parent peptide.
- 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
- 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
- Fully non-peptidic peptidomimetic analogs may also be produced, taking into consideration the above-described strategies relating to constrained peptide compounds.
- 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.
- DNDFB dinitrodifluorobenzene
- B.2412 was cleaved from the resin and purified by HPLC on a Vydac C4 column, as described in Fi-gure 1.
- 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 ⁇ ,_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.
- 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).
- FPA fibrinopeptide A
- heparin 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.
- N-acetyl Sulfo-Tyr g3 hirudin 53 _g 4 The synthesis of N-acetyl Sulfo-Tyr 63 hirudin 53 _ 64 is described in Example 2.
- N- acetyl Sulfo-Tyr 63 hirudin 53 _ 64 was equally effective in inhibiting both clot-free thrombin and clot-bound thrombin.
- 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.
- 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.
- antithrombin Ill- independent thrombin inhibitors are capable of neutralizing clot-bound thrombin to the same extent as free thrombin.
- Example 7 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%.
- PPACK D-Phe-Pro-Arg-CH 2 Cl
- 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 ⁇ g/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).
- rtPA recombinant tPA
- Coronary thrombi were established in the left anterior descending (LAD ⁇ artery as follows.
- reperfusion time may be decreased " when that agent is administered in combina ⁇ tion with a hirudin peptide rather than a conven ⁇ tional anticoagulant.
- 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.
- 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.
- 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.
Abstract
Description
Claims
Applications Claiming Priority (2)
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US38336089A | 1989-07-20 | 1989-07-20 | |
US383,360 | 1989-07-20 |
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WO1991001142A1 true WO1991001142A1 (en) | 1991-02-07 |
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PCT/US1990/004103 WO1991001142A1 (en) | 1989-07-20 | 1990-07-20 | Combinations and methods for treating or preventing thrombotic diseases |
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EP (1) | EP0482069A1 (en) |
AU (1) | AU6054090A (en) |
CA (1) | CA2064231A1 (en) |
WO (1) | WO1991001142A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0669394A1 (en) * | 1993-07-15 | 1995-08-30 | Grünenthal GmbH | Bifunctional derivatives of Urokinase with improved fibrinolytic activity and Thrombin inhibiting activity |
US5656600A (en) * | 1993-03-25 | 1997-08-12 | Corvas International, Inc. | α-ketoamide derivatives as inhibitors of thrombosis |
KR970061266A (en) * | 1996-02-27 | 1997-09-12 | 슈탸인, 라우페 | Pharmaceutical compositions comprising hirudin and methods for preparing the same |
EP0850648A2 (en) * | 1994-03-25 | 1998-07-01 | Roche Diagnostics GmbH | Combination of thrombolytically active proteins and anticoagulants and uses thereof |
US5976841A (en) * | 1994-11-17 | 1999-11-02 | Gruenenthal Gmbh | Proteins having fibrinolytic and coagulation--inhibiting properties |
US6060451A (en) * | 1990-06-15 | 2000-05-09 | The National Research Council Of Canada | Thrombin inhibitors based on the amino acid sequence of hirudin |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE4005591A1 (en) * | 1990-02-22 | 1991-09-05 | Behringwerke Ag | THE HERBAL INHIBITING PEPTIDES, METHOD FOR THEIR PRODUCTION AND THEIR USE |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0265129A1 (en) * | 1986-10-09 | 1988-04-27 | Smithkline Beecham Corporation | Thrombolytic therapy |
EP0328957A2 (en) * | 1988-02-13 | 1989-08-23 | BASF Aktiengesellschaft | Mixture of a thrombolytically acting and an antithrombolytically acting substance |
-
1990
- 1990-07-20 EP EP19900911071 patent/EP0482069A1/en not_active Withdrawn
- 1990-07-20 AU AU60540/90A patent/AU6054090A/en not_active Abandoned
- 1990-07-20 WO PCT/US1990/004103 patent/WO1991001142A1/en not_active Application Discontinuation
- 1990-07-20 CA CA 2064231 patent/CA2064231A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0265129A1 (en) * | 1986-10-09 | 1988-04-27 | Smithkline Beecham Corporation | Thrombolytic therapy |
EP0328957A2 (en) * | 1988-02-13 | 1989-08-23 | BASF Aktiengesellschaft | Mixture of a thrombolytically acting and an antithrombolytically acting substance |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6060451A (en) * | 1990-06-15 | 2000-05-09 | The National Research Council Of Canada | Thrombin inhibitors based on the amino acid sequence of hirudin |
US5656600A (en) * | 1993-03-25 | 1997-08-12 | Corvas International, Inc. | α-ketoamide derivatives as inhibitors of thrombosis |
US5670479A (en) * | 1993-03-25 | 1997-09-23 | Corvas International, Inc. | α-ketoamide derivatives as inhibitors of thrombosis |
EP0669394A1 (en) * | 1993-07-15 | 1995-08-30 | Grünenthal GmbH | Bifunctional derivatives of Urokinase with improved fibrinolytic activity and Thrombin inhibiting activity |
US5681721A (en) * | 1993-07-15 | 1997-10-28 | Gruenenthal Gmbh | Bifunctional urokinase variants with improved fibrinolytic characteristics and thrombin inhibiting effect |
US5747291A (en) * | 1993-07-15 | 1998-05-05 | Gruenenthal Gmbh | Bifunctional urokinase variants with improved fibrinolytic characteristics and thrombin inhibiting effect |
EP0850648A2 (en) * | 1994-03-25 | 1998-07-01 | Roche Diagnostics GmbH | Combination of thrombolytically active proteins and anticoagulants and uses thereof |
EP0850648A3 (en) * | 1994-03-25 | 2004-03-31 | Roche Diagnostics GmbH | Combination of thrombolytically active proteins and anticoagulants and uses thereof |
US5976841A (en) * | 1994-11-17 | 1999-11-02 | Gruenenthal Gmbh | Proteins having fibrinolytic and coagulation--inhibiting properties |
KR970061266A (en) * | 1996-02-27 | 1997-09-12 | 슈탸인, 라우페 | Pharmaceutical compositions comprising hirudin and methods for preparing the same |
Also Published As
Publication number | Publication date |
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AU6054090A (en) | 1991-02-22 |
CA2064231A1 (en) | 1991-01-21 |
EP0482069A1 (en) | 1992-04-29 |
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