EP0333356A2
Expired / Lapsed
EP
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Hirudin peptides
Assignee: Biogen Inc
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Filing: 1989-03-03
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Granted: —
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Expires: 2009-03-03
Abstract
Abstract This invention relates to novel, biologically active peptides which display the anticoagulant activity of hirudin. More specifically, it relates to peptides which are homologous to at least a portion of the carboxy terminal 26 amino acids of hirudin. Such peptides may also be characterized by a modified tyrosine residue. This invention further relates to covalent and peptidomimetic analogs of these peptides which display anticoagulant activity. This invention also relates to compositions, combinations and methods using such peptides or analogs for therapeutic, prophylactic or diagnostic purposes. And this invention relates to novel methods for sulfating tyrosine residues contained within a peptide or polypeptide.
Inventors
- 👤 John M. Maraganore
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-24 00:29
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/81; C07K14/815; A; A61; A61K; A61K38/00
Claims Analysis
Total Claims: 50
Claim 1
Independent Claim
52 words
A peptide displaying anticoagulant activity, wherein said peptide is selected from the group consisting of: (a) peptides characterized by a sequence of amino acids consisting substantially of the formula: Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-X; and (b) D-retro forms of the peptides of (a); wherein X is selected from the group consisting of COOH, Leu and Leu-Gln.
Claim 2
Dependent Claim
52 words
A peptide displaying anticoagulant activity, wherein said peptide is selected from the group consisting of: (a) peptides characterized by a sequence of amino acids consisting substantially of the formula: Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-X; and (b) D-retro forms of the peptides of (a); wherein X is selected from the group consisting of COOH, Leu and Leu-Gln.
Claim 3
Dependent Claim
99 words
A peptide displaying anticoagulant activity, wherein said peptide is selected from the group consisting of: (a) a peptide characterized by a sequence of amino acids consisting substantially of the formula: Y-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Z; and b) D-retro forms of the peptides of (a); wherein Y is selected from the group consisting of NH₂, an amino protecting group, at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp, and at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Asn-Pro-Glu-Ser-His-Asn-Asn-Gly-Asp; Z is selected from the group consisting of COOH, Leu and Leu-Gln; and the tyrosine residue contains a negatively charged side group.
Claim 4
Dependent Claim
99 words
A peptide displaying anticoagulant activity, wherein said peptide is selected from the group consisting of: (a) a peptide characterized by a sequence of amino acids consisting substantially of the formula: Y-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Z; and b) D-retro forms of the peptides of (a); wherein Y is selected from the group consisting of NH₂, an amino protecting group, at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp, and at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Asn-Pro-Glu-Ser-His-Asn-Asn-Gly-Asp; Z is selected from the group consisting of COOH, Leu and Leu-Gln; and the tyrosine residue contains a negatively charged side group.
Claim 5
Dependent Claim
28 words
The peptide according to claim 2, wherein the negatively charged side group is selected from the group consisting of sulfate, phosphate, carboxylate, sulfonate, phosphonate, carbonate, methylsulfonate and methylphosphonate.
Claim 6
Dependent Claim
22 words
The peptide according to claim 2, wherein the negatively charged side group is selected from the group consisting of sulfate and sulfonate.
Claim 7
Dependent Claim
25 words
The peptide according to claim 4, wherein Y is Asn-Gly-Asp, Z is Leu and said negatively charged side group is sulfate, said peptide being Sulfo-Tyr₆₃hirudin₅₃₋₆₄.
Claim 8
Dependent Claim
25 words
The peptide according to claim 4, wherein Y is Asn-Gly-Asp, Z is Leu and said negatively charged side group is sulfonate, said peptide being Sulfonyl-Tyr₆₃hirudin₅₃₋₆₄.
Claim 9
Dependent Claim
16 words
The peptide according to claim 1 or 2, wherein the amino terminal amino acid is N-acetylated.
Claim 10
Dependent Claim
19 words
The peptide according to claim 7, wherein said peptide is selected from the group consisting of Sulfo-Tyr₆₃-N-acetyl-hirudin₅₃₋₆₄ and N-acetyl-Sulfonyl-Tyr₆₃hirudin₅₃₋₆₄.
Claim 11
Dependent Claim
16 words
The peptide according to claim 1 or 2, wherein the amino terminal amino acid is dinitrofluorobenzylated.
Claim 12
Dependent Claim
93 words
A peptide displaying anticoagulant activity, said peptide being capable of forming a covalent bond with thrombin, wherein said peptide is selected from the group consisting of: (a) a peptide characterized by a sequence of amino acids consisting essentially of the formula : Y-Phe-Glu-Glu-Ile-Pro-Glu-Glu-W; and (b) D-retro forms of the peptides of (a); wherein Y is selected from the group consisting of NH₂, an amino protecting group, at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp, and at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Asn-Pro-Gln-Ser-His-Asn-Asn-Gly-Asp: and W is nitroanisole.
Claim 13
Dependent Claim
93 words
A peptide displaying anticoagulant activity, said peptide being capable of forming a covalent bond with thrombin, wherein said peptide is selected from the group consisting of: (a) a peptide characterized by a sequence of amino acids consisting essentially of the formula : Y-Phe-Glu-Glu-Ile-Pro-Glu-Glu-W; and (b) D-retro forms of the peptides of (a); wherein Y is selected from the group consisting of NH₂, an amino protecting group, at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp, and at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Asn-Pro-Gln-Ser-His-Asn-Asn-Gly-Asp: and W is nitroanisole.
Claim 14
Dependent Claim
102 words
A peptide displaying anticoagulant and antiplatelet activity, wherein said peptide is selected from the group consisting of: (a) a peptide characterized by a sequence of amino acids consisting essentially of the formula: V-Arg-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Z; and (b) D-retro forms of the peptides of (a); wherein V is selected from the group consisting of NH₂, an amino protecting group, at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn, and and at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Asn-Pro-Gln-Ser-His-Asn; Z is selected from the group consisiting of COOH, Leu and Leu-Gln; and the tyrosine residue contains a negatively charged side group.
Claim 15
Dependent Claim
102 words
A peptide displaying anticoagulant and antiplatelet activity, wherein said peptide is selected from the group consisting of: (a) a peptide characterized by a sequence of amino acids consisting essentially of the formula: V-Arg-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Z; and (b) D-retro forms of the peptides of (a); wherein V is selected from the group consisting of NH₂, an amino protecting group, at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn, and and at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Asn-Pro-Gln-Ser-His-Asn; Z is selected from the group consisiting of COOH, Leu and Leu-Gln; and the tyrosine residue contains a negatively charged side group.
Claim 16
Dependent Claim
23 words
The peptide according to any one of claims 1, 2, 10 or 11, wherein said peptide is conjugated with a pharmacologically acceptable polymer.
Claim 17
Dependent Claim
13 words
The peptide according to claim 12, wherein said polymer is polyethyleneglycol N-succinimidyl succinate.
Claim 18
Dependent Claim
22 words
The peptide according to any one of claims 1, 2, 10 or 11, wherein said peptide is conjugated with a fibrinolytic agent.
Claim 19
Dependent Claim
23 words
A peptidomimetic analog of the peptide of claim 1 or 2, wherein said analog inhibits thrombin hydrolysis of fibrinogen and displays anticoagulant activity.
Claim 20
Dependent Claim
25 words
The peptidomimetic analog according to claim 15, wherein said analog is selected from the group consisting of hirulog-1, hirulog-2, hirulog-3, hirulog-4, hirulog-5, hirulog-6 and hirulog-7.
Claim 21
Dependent Claim
19 words
The peptidomimetic analog according to claim 15 or 16, wherein said peptidomimetic analog is conjugated with a fibrinolytic agent.
Claim 22
Dependent Claim
57 words
A pharmaceutically acceptable composition for increasing blood clotting time in a patient or in extracorporeal blood which comprises a pharmaceutically effective amount of at least one component selected from the group consisting of a peptide according to any one of claims 1 to 14, and a peptidomimetic analog according to any one of claims 15 to 17.
Claim 23
Dependent Claim
55 words
The composition according to claim 18, further comprising a compound selected from the group consisting heparin and low molecular weight heparin, wherein the dosage of said heparin or low molecular weight heparin is less than that required to achieve a desired result when said heparin or low molecular weight heparin is administered as a monotherapy.
Claim 24
Dependent Claim
25 words
The composition according to claim 19, wherein the dose of said heparin or low molecular weight heparin is less than about 2,500 to 5,000 units.
Claim 25
Dependent Claim
24 words
The composition according to any one of claims 18 to 20, wherein said composition further comprises a pharmaceutically effective amount of a fibrinolytic agent.
Claim 26
Independent Claim
42 words
A method for increasing blood clotting time in a patient or in extracorporeal blood comprising the step of treating said patient or said extracorporeal blood in a pharmaceutically acceptable manner with a composition according to any one of claims 18 to 21.
Claim 27
Independent Claim
34 words
A method for preventing or treating vascular disease in a patient, comprising the step of treating said patient in a pharmaceutically acceptable manner with a composition according any one of claims 18 to 21.
Claim 28
Dependent Claim
22 words
The use of a composition according to any one of claims 18 to 21 for the prevention or treatment of vascular disease.
Claim 29
Dependent Claim
35 words
A composition for determining the concentration of Factor IXa, Factor Xa, thrombin or mixtures thereof, in a biological sample, said composition comprising at least one peptide according to any one of claims 1 to 6.
Claim 30
Dependent Claim
35 words
A method for determining the concentration of Factor IXa, Factor Xa, thrombin or mixtures thereof, in a biological sample, said method comprising the step of contacting said sample with a composition according to claim 25.
Claim 31
Dependent Claim
36 words
A diagnostic kit for assaying the concentration of Factor IXa, Factor Xa, thrombin or mixtures thereof, in a biological sample, said kit comprising at least one peptide according to any one of claims 1 to 6.
Claim 32
Dependent Claim
54 words
A pharmaceutically acceptable composition for inhibiting the growth of metastatic tumors, wherein said composition comprises a pharmaceutically effective amount of at least one component selected from the group consisting of a peptide according to any one of claims 1 to 14 and a peptidomimetic analog according to any one of claims 15 to 17.
Claim 33
Dependent Claim
34 words
A method for inhibiting the growth of metastatic tumors in a patient, wherein said method comprises the step of treating said patient in a pharmaceutically acceptable manner with a composition according to claim 28.
Claim 34
Dependent Claim
34 words
The method according to claim 29, wherein said metastatic tumor is selected from the group consisting of carcinoma of the brain, carcinoma of the lung, carcinoma of the liver, osteocarcinoma and neoplastic cell carcinoma.
Claim 35
Dependent Claim
33 words
The peptide according to any one of claims 1, 2, 7, 9, 10 or 11, wherein said peptide is labeled with a radioisotope selected from the group consisting of ¹²³I, ¹²⁵I and ¹¹¹In.
Claim 36
Dependent Claim
22 words
A composition for ex vivo imaging of a fibrin or platelet thrombus in a patient comprising a peptide according to claim 31.
Claim 37
Dependent Claim
39 words
A method for ex vivo imaging of a fibrin or platelet thrombus in a patient comprising the steps of: (a) administering to said patient a composition according to claim 32; and (b) using detecting means to observe said composition.
Claim 38
Dependent Claim
54 words
A composition for coating the surface of an invasive device to be inserted into a patient, wherein said composition comprises at least one component selected from the group consisting of a peptide according to any one of claims 1 to 14 and a peptidomimetic analog according to any one of claims 15 to 17.
Claim 39
Dependent Claim
32 words
A method for coating the surface of an invasive device to be inserted into a patient, said method comprising the step of contacting said surface with a composition according to claim 34.
Claim 40
Dependent Claim
29 words
A pharmaceutical composition for increasing blood clotting time and inhibiting platelet activation in a patient, said composition comprising a pharmaceutically effective amount of a peptide according to claim 11.
Claim 41
Dependent Claim
39 words
A method for increasing blood clotting time and inhibiting platelet activation in a patient, wherein said platelet activation is induced by any agonist, said method comprising the step of treating said patient with a composition according to claim 36.
Claim 42
Dependent Claim
53 words
A pharmaceutical composition for inhibiting thrombin-induced platelet activation and thrombin-induced endothelial cell activation in a patient, said composition comprising a pharmaceutically effective low dosage amount of a peptide according to any one of claims 1 to 8, said low dosage being between about 0.0001 mg/kg body weight and about 0.099 mg/kg body weight.
Claim 43
Dependent Claim
31 words
A method for inhibiting thrombin-induced platelet activation and thrombin-induced endothelial cell activation in a patient, said method comprising the step of treating said patient with a composition according to claim 38.
Claim 44
Dependent Claim
48 words
A pharmaceutically acceptable composition for treating thrombin-induced inflammation in a patient, said composition comprising a pharmaceutically effective low dosage amount of a peptide according to any one of claims 1 to 8, said low dosage being between about 0.0001 mg/kg body weight and about 0.099 mg/kg body weight.
Claim 45
Dependent Claim
25 words
A method for treating thrombin-induced inflammation in a patient, said method comprising the step of administering to said patient a composition according to claim 40.
Claim 46
Dependent Claim
31 words
The method according to claim 41, wherein said thrombin-induced inflammation is caused by a disease selected from the group consisting of adult respiratory distress syndrome, septic shock, septicemia and reperfusion damage.
Claim 47
Independent Claim
72 words
A method for sulfating a tyrosine residue of a peptide or a polypeptide, said method comprising the steps of: (a) dissolving said peptide or polypeptide in an organic solvent; (b) reacting said dissolved peptide or polypeptide with a dehydrating reagent to dehydrate the tyrosine residue of said peptide or polypeptide; and (c) sulfating said dehydrated tyrosine residue by adding sulfuric acid dropwise to said dissolved peptide or polypeptide until a precipitate forms.
Claim 48
Independent Claim
72 words
A method for sulfating a tyrosine residue of a peptide or a polypeptide, said method comprising the steps of: (a) dissolving said peptide or polypeptide in an organic solvent; (b) reacting said dissolved peptide or polypeptide with a dehydrating reagent to dehydrate the tyrosine residue of said peptide or polypeptide; and (c) sulfating said dehydrated tyrosine residue by adding sulfuric acid dropwise to said dissolved peptide or polypeptide until a precipitate forms.
Claim 49
Dependent Claim
20 words
The method according to claim 43, wherein said peptide is selected from the group consisting of N-acetyl-hirudin₅₃₋₆₄ and hirudin ₅₃₋₆₄.
Claim 50
Dependent Claim
18 words
The method according to claim 43, wherein said organic solvent is dimethylformamide and said dehydrating reagent is dicyclohexylcarodiimide.
References Cited (Backward Citations)
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