WO1990003391A1 - Peptides d'hirudine - Google Patents

Peptides d'hirudine Download PDF

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Publication number
WO1990003391A1
WO1990003391A1 PCT/US1989/000848 US8900848W WO9003391A1 WO 1990003391 A1 WO1990003391 A1 WO 1990003391A1 US 8900848 W US8900848 W US 8900848W WO 9003391 A1 WO9003391 A1 WO 9003391A1
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Prior art keywords
peptide
hirudin
glu
thrombin
group
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Application number
PCT/US1989/000848
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English (en)
Inventor
John M. Maraganore
Original Assignee
Biogen, Inc.
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Filing date
Publication date
Application filed by Biogen, Inc. filed Critical Biogen, Inc.
Publication of WO1990003391A1 publication Critical patent/WO1990003391A1/fr
Priority to FI904330A priority Critical patent/FI904330A0/fi
Priority to DK210590A priority patent/DK210590A/da
Priority to NO90903833A priority patent/NO903833L/no

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to novel, biologic- ally 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 peptidomimetic and covalent analogs of these peptides which display anticoagulant activity. This invention also relates to compositions, combinations and methods using such peptides or analogs for
  • this invention relates to novel methods for sulfating tyrosine residues contained within a
  • 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.
  • 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.
  • the second category of therapeutic accelerates thrombolysis and dissolves the blood clot, thereby removing it from the blood vessel and unblocking the flow of blood [J. P. Cazenave et al., Agents Action, 15, Suppl., pp. 24-49 (1984)].
  • Heparin a compound of the former class, has been widely used to treat conditions, such as venous thromboembolism, in which thrombin activity is responsible for the development or expansion of a thrombus. Although effective, heparin produces many undesirable side effects, including hemorrhaging and thrombocytopenia. This has led to a search for a more specific and less toxic anticoagulant.
  • Hirudin is a naturally occurring polypeptide which is produced by the blood sucking leech
  • Hirudo medicinalis This compound, which is produced in the salivary gland of the leech, is the most potent natural inhibitor of coagulation known. Hirudin prevents blood from coagulating by binding tightly to thrombin (K d ⁇ 2 x 10 -11 M) in a 1:1 stoichiometric complex [S. R. Stone and J. Hofsteenge, "Kinetics of the Inhibition of Thrombin by Hirudin", Biochemistry, 25, pp. 4622-28 (1986)]. This, in turn, inhibits thrombin from catalyzing the conversion of fibrinogen to fibrin (clot).
  • Hirudin is believed to be a selective and specific inhibitor of thrombin which exhibits no inhibitory activities toward other coagulation factors. It has been suggested, however, that hirudin inhibits both Factor IXa activation of Factor X and Factor Xa activation of prothrombin [E. W. Davie et al., "Blood Coagulation", Adv. Enzymol., 48, pp. 277-318 (1979); J. S. Rosenberg et al., "Activation of Human Prothrombin by Highly Purified Human Factors V and Xa in the Presence of Human Antithrombin", J. Biol.
  • hirudin binds to Factor Xa; and that purified
  • hirudin preparations may be the equivalent of antistasin, a protein which has been purified from the Mexican leech, Haementeria officinalis, and which selectively inhibits Factor Xa [G. P. Tuszynski et al., "Isolation and Characteristics of Antistasin", J. Biol. Chem., 262, p. 9718 (1987)].
  • antistasin unlike hirudin, inhibits the cleavage of S-2222 by Factor Xa. This implies that the inhibition of Factor Xa by hirudin occurs via a different mechanism than the Factor Xa inhibition by antistasin.
  • hirudin binds to a "low" affinity site on the thrombin molecule (K d ⁇ 1 x 10 -8 M) which is separate from the catalytic site.
  • hirudin undergoes a conformational change and then binds to the "high" affinity site on thrombin. This latter site corresponds to the active site of
  • hirudin At least two different isospecific forms of hirudin, HV-1 and HV-2, have been sequenced and have been shown to differ slightly in amino acid sequence [R. P. Harvey et al., supra]. Both forms of hirudin comprise a single polypeptide chain protein containing 65 amino acids in which the amino terminus primarily comprises hydrophobic amino acids and the carboxy terminus typically comprises polar amino acids. More specifically, all forms of hirudin are characterized by an N-terminal domain (residues 1-39) stabilized by three disulfide bridges in a 1-2, 3-5, and 4-6 half-cysteinyl pattern and a highly acidic C-terminal segment (residues 40-65). In addition, the C-terminal segment of hirudin is characterized by the presence of a tyrosine residue at amino acid position 63 which is sulfated.
  • hirudin In animal studies, hirudin, purified from leeches, has demonstrated efficacy in preventing venous thrombosis, vascular shunt occlusion and thrombin-induced disseminated intravascular coagulation. In addition, hirudin exhibits low toxicity, little or no antigenicity and a very short clearance time from circulation [F. Markwardt et al., "Pharmacological Studies on the Antithrombotic Action of Hirudin in Experimental Animals", Thromb. Haemostasis, 47, pp. 226-29 (1982)].
  • hirudin Despite hirudin's effectiveness, however, studies have shown that hirudin prolongs bleeding time in a dose-dependent manner, thus making the determination and administration of proper dosages critically important. Furthermore, the high cost and low supply of the naturally occurring product has prevented its widespread use.
  • N ⁇ -acetylhirudin 45-65 has a specific activity four orders of magnitude lower than native hirudin.
  • thrombin In addition to catalyzing the formation of a fibrin clot, thrombin has several other bioregulatory roles [J. W. Fenton, II, "Thrombin Bioregulatory
  • thrombin directly activates platelet aggregation and release reactions. This means that thrombin plays a central role in acute platelet-dependent
  • thrombosis [S. R. Hanson and L. A. Harker, "Interruption of Acute Platelet-Dependent Thrombosis by the Synthetic Antithrombin D-Phenylalanyl-L-Prolyl-L-Arginylchloro- methylketone", Proc. Natl. Acad. Sci. USA, 85, pp. 3184- 88 (1988)].
  • Thrombin can also directly activate an
  • PAF platelet activating factor
  • thrombin such as hirudin
  • the present invention solves the problems referred to above by providing peptides characterized by the biological activity of native hirudin.
  • the peptides and analogs of this invention are effective as anticoagulants which cause an increase in blood clotting time.
  • these peptides inhibit thrombin-induced platelet aggregation and platelet release (hereinafter “platelet activation”) and the release of inflammatory substances from endothelial cells (hereinafter “platelet activation”) and the release of inflammatory substances from endothelial cells (hereinafter “platelet activation”) and the release of inflammatory substances from endothelial cells (hereinafter “platelet activation”) and the release of inflammatory substances from endothelial cells (hereinafter “platelet activation”) and the release of inflammatory substances from endothelial cells (hereinafter “platelet activation”) and the release of inflammatory substances from endothelial cells (hereinafter
  • endothelial activation without significantly increasing clotting time.
  • the relatively small size of these peptides advantageously allows them to be produced synthetically. Thus, they may be produced in extremely high yields and are easily purified, as compared to either native hirudin or its full length recombinant DNA counterpart.
  • the peptides of the present invention unlike hirudin, exhibit a saturable effect on clotting time.
  • the therapeutic and prophylatic uses of these peptides avoid the harmful and potentially fatal consequences of an overdose associated with conventional anticoagulants, such as heparin.
  • the small size of the peptides of this invention decreases the possibility of an adverse antigenic response in patients treated with them.
  • the peptides, compositions, combinations and methods of this invention are useful in the treatment, prevention or diagnosis of vascular diseases attributed to the undesirable effects of
  • FIG. 1 depicts the purification of
  • Figure 2 depicts HPLC chromatograms illustrating the relative efficiency of the sulfation process of this invention (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 .
  • Figure 4 depicts the anticoagulant activity of hirudin 53-64 and Suifo-Tyr 63 hirudin 53-64 over a range of peptide concentrations.
  • Figure 5 is a table which displays the covalent structures of hirudin peptides according to this invention, as well as their anticoagulant activity.
  • the amino acids are represented by single letter codes as follows
  • Figure 6 depicts the inhibition of thrombin times by varying amounts of native hirudin and Sulfo- Tyr 63 hirudin 53-54 .
  • Figure 7 depicts the effects of varying amounts of Sulfo-Tyr 53 hirudin 53-64 on bovine
  • Figure 8 depicts the effects of varying amounts of heparin alone, Sulfo-Tyr 63 hirudin 53- 64 alone, and a 1:1 combination of heparin and Sulfo- Tyr 63 hirudin 53-64 on activated partial thrombo- plastin times.
  • Figure 9 depicts the in vivo anticoagulant activity of Sulfo-Tyr 63 hirudin 53-64 in a dose response study in Balb/c mice.
  • Figure 10 depicts the inhibition of Factor IXa activation of Factor X by Sulfo-Tyr 63
  • Figure 11 depicts an SDS-polyacrylamide gel demonstrating the inhibition of Factor Xa activation of prothrombin by Sulfo-Tyr 63 hirudin 53-64 .
  • Figures 12A-12C depict the synthesis of hirulog-1, hirulog-2 and hirulog-3, peptidomimetic analogs of the peptides according to this invention.
  • Figure 13A depicts the synthesis of hirulog-4, a peptidomimetic analog of the peptides according to this invention.
  • Figures 14A and 14B depict the synthesis of hirulog-5 and hirulog-6, peptidomimetic analogs of the peptides according to this invention.
  • hirulog-7 a peptidomimetic analog of the peptides according to this invention.
  • Figure 16 depicts the effects of low doses of Sulfo-Tyr 63 hirudin 53-64 on thrombin-induced platelet aggregation and clotting time.
  • Figure 17 depicts the effects of Arg 53 - hirudin 53-64 on ADP- and collagen-induced platelet aggregation.
  • the present invention relates to peptides which correspond to the amino acid sequence of the carboxyl terminal portion of hirudin and which display the biological avtivity of native hirudin.
  • the peptides of this invention are homologous to at least a portion of the carboxy terminal 26 amino acids of native hirudin. According to one embodiment of this invention, such peptides are derivatized at the single tyrosine residue by the addition of a negatively charged side group.
  • Hirudin peptides of this invention include, but are not limited to: 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 D-retro forms thereof; wherein X is selected from the group consisting of COOH, Leu and Leu-Gin.
  • hirudin peptides include those which are characterized by a sequence of amino acids consisting substantially of the formula: Y-Phe-Glu-Glu-Ile-Pro- Glu-Glu-Tyr-Z, and D-retro forms thereof; wherein Y is selected from the group consisting of NH 2 , 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-Gin; and the tyrosine residue is characterized by the presence of a negatively charged side group.
  • the negatively charged side group may be selected from the group consisting of sulfate, phosphate, carboxylate, sulfonate, phosphonate, carbonate, methyl sulfonate, methyl phosphonate and variants thereof.
  • such peptides include those in which Y is Asn-Gly-Asp and Z is Leu and the tyrosine residue is sulfated.
  • the peptides of the present invention may be characterized by the presence of an N-acetyl group on the amino terminal amino acid.
  • the production of the peptides of this invention may be achieved by a variety of methods known in the art.
  • the peptides may be derived from the intact hirudin molecule by proteolysis using specific endopeptidases in combination with exopeptidases, Edman degradation, or both.
  • the intact hirudin molecule in turn, may be purified from its natural source, H. medicinalis, using conventional methods.
  • hirudin may be produced by known recombinant DNA techniques using cDNAs [R. P. Harvey et al., Proc. Natl. Acad. Sci. USA, 83, pp. 1084-88 (1986); J. Dodt et al.,
  • the peptides of the present invention are produced directly, thus eliminating the need for an entire hirudin molecule as a starting material. This may be achieved by well known recombinant DNA techniques wherein only those DNA sequences which encode the desired peptides are expressed in transformed hosts.
  • the peptides of this invention may be produced by conventional chemical synthesis techniques.
  • hirudin peptides are synthesized by solution phase or solid phase peptide synthesis and, optionally, digested with carboxypeptidase (to remove C-terminal amino acids) or degraded by manual Edman degradation (to remove N-terminal amino acids). Peptides produced in this way may then be purified by separation techniques widely known in the art, preferably utilizing reverse phase HPLC.
  • the use of solution phase synthesis advantageously allows for the direct addition of derivatized amino acids to the growing peptide chain. This obviates the need for a subsequent derivatization step to modify the tyrosine residue of the peptides of this invention.
  • Derivatization of the hirudin peptides of the invention may involve the addition of a negatively charged side group onto either the free phenolic hydroxyl or the benzoyl meta carbon of the single tyrosine residue.
  • the derivatization may involve the addition of a variety of negatively charged side groups which are known in the art.
  • Derivatization methods include, but are not limited to, sulfation, methyl sulfonation, phosphorylation, methyl phosphonation and carboxylation of the tyrosine hydroxyl group and sulfonation, phosphonation and carbonation of the tyrosine benzoyl meta carbon. Techniques for performing these reactions are also well known in the art.
  • the peptides of this invention are derivatized by sulfation.
  • a preferred peptide of this invention is Sulfo-Tyr 63 hirudin 53-64 (subscript numbers represent the corresponding amino acid position in the native hirudin molecule), a 12 amino acid peptide, having a sulfated tyrosine residue, that is homologous to residues 53-64 of native hirudin.
  • Another preferred peptide is Sulfonyl- Tyr 63 hirudin 53-64 which has a sulfonated tyrosine residue. This sulfonated peptide may possess a longer biological half-life than its sulfated counterpart.
  • Sulfation of the hirudin peptides of this invention may be achieved either by a biological (enzymatic) or a chemical process.
  • a purified peptide of this invention is reacted concurrently with dicyclo- hexylcarbodiimide 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 solvent, preferably dimethylformamide, and then reacted with a dehydrating agent, preferably dicyclohexyl- carbodiimide.
  • a dehydrating agent preferably dicyclohexyl- carbodiimide.
  • the dehydrated tyrosine residue of the peptide is then sulfated by reaction with sulfuric acid.
  • the reaction is complete upon formation of an insoluble dicyclohexyl urea salt. This modification results in high yields of sulfated peptide on a large scale.
  • This novel 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 a peptide of this invention with sulfur trioxide- triethylamine salt in pyridine.
  • a tyrosylsulfotransferase 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 O Sulfated Proteins of PC-12 Pheo Chromo Cytoma Cells and Their Sulfation By a Tyrosyl Protein Suifotransferase", J. Biol. Chem., 258, pp. 11326-34 (1983)].
  • Phosphorylation or carboxylation of the hirudin peptides of this invention 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. Alternatively, enzymatic methods may be employed for carboxylation or phosphorylation of the hirudin peptides of this invention.
  • Methyl sulfonation and methyl phosphonation of the hirudin peptides of this invention may be achieved by methods well-known in the art including, but not limited to, alkylation with chlorosulfonic or chlorophosphonic acid, respectively.
  • the extent of the sulfation reaction may be followed spectrophotometrically.
  • the absorbance spectra of sulfated peptides reveal a shift in maximal absorbance from approximately 275 nm to approximately 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.
  • hirudin peptides may also be derivatized 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.
  • acetylation is achieved by using an N-acetyl amino acid derivative in the synthesis of the peptides of this
  • N-acetylation may be achieved by reacting the peptide with acetic
  • N-acetylated hirudin peptides of this invention advantageously demonstrate increased biostability as compared to their corresponding unacetylated peptide counterparts.
  • the anticoagulant activity of the hirudin peptides of this invention may be assayed using any conventional technique.
  • the assay is preferably, the assay
  • thrombin- inhibitory activity of the peptide involves direct determination of the thrombin- inhibitory activity of the peptide.
  • Such assays measure the inhibition of thrombin-catalyzed cleavage of colorimetric 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 latter assays measure factors in the "intrinsic" pathway of coagulation.
  • the assay employed may use purified thrombin and fibrinogen and measures the inhibition of release of fibrinopeptides A or B by radioimmunoassay or ELISA.
  • the anticoagulant potency of the peptides of the present invention depends, in part, on their in vivo half-life. Accordingly, this invention also relates to pharmaceutical compositions, either covalent or non-covalent, comprising hirudm peptides coupled to pharmaceutically acceptable polymers which increase the biological half-life of those peptides.
  • a hirudin peptide of this invention may be coupled to an activated derivative of polyethyl- eneglycol (PEG) using conventional techniques.
  • a PEG N-succinimidyl succinate is attached to the ⁇ -amino moiety of the peptide.
  • Such attachment is effected by reacting the peptide with the PEG N-succinimidyl succinate reagent (SS-PEG) in an organic solvent or a buffered solution having a pH greater than about 7.0.
  • SS-PEG PEG N-succinimidyl succinate reagent
  • the hirudin peptides of this invention are useful alone, or in compositions, combinations and methods for the treatment and prophylaxis of vascular diseases attributed to blood system thromboses.
  • the peptides of this invention, and compositions and combinations containing them may be used for heparin replacement for prophylactic purposes, heparin replacement in the treatment of thrombocytopenia, treatment of disseminated intravascular coagulation and treatment of vascular thrombi that may arise from any disease state.
  • the hirudin peptides of this invention, as well as compositions and combinations containing them may be used in the treatment or prophylaxis of vascular diseases in patients including mammals and, in particular, humans.
  • the hirudin peptides of this invention may be administered in combination with heparin or low molecular weight heparin.
  • Such combinations advantageously lower the dosage of heparin or low molecular weight heparin required to produce a desired anticoagulant effect when either compound is used alone.
  • these combinations advantageously reduce the potential for hemorrhagic complications often associated with heparin use.
  • Such combinations surprisingly demonstrate greater anticoagulant activity than that exhibited in monotherapies based upon either of the individual components.
  • the term "combination" includes a single dosage form containing at least one peptide of the present invention and heparin or low molecular weight heparin, a multiple dosage form wherein the two agents are administered separately, but concurrently, or a multiple dosage form wherein the two agents are administered separately, but sequentially.
  • the hirudin peptides of this invention may be formulated using conventional methods to prepare pharmaceutically useful compositions and combinations. Such compositions preferably include at least one pharmaceutically acceptable carrier. See, e.g.,
  • compositions of the present invention typically contain, in addition to the hirudin peptides, a pharmaceutically acceptable buffer,
  • compositions of the present invention may also contain, in addition to the above components, a low dosage of heparin or low molecular weight heparin, preferably less than about 2,500 to 5,000 units.
  • hirudin peptide-containing compositions and combinations of this invention include, but are not limited to, parenteral administration, oral administration and topical application.
  • the dosage and dose rate will depend on a variety of factors such as the specific composition, the object of the treatment, i.e., therapy or prophylaxis, and the judgment of the treating physician.
  • compositions for injection may be in the form of lyophilizates or solutions.
  • Compositions formulated for topical application may, for example, be in aqueous jelly, oily suspension or emulsified ointment form. The resulting formulations will contain an amount of hirudin peptide effective to prevent or reduce blood clots.
  • a therapeutic amount of the peptides of this invention will normally be in the daily dosage range of about 0.2 to 75 mg/kg body weight, preferably in the range of about 0.5 to 10 mg/kg body weight.
  • Methods for determining effective dosages are known to those skilled in the art. However, those dosages are approximately equal to or less than those currently used with other thrombolytic agents, e.g., about 3.5 mg/kg body weight, followed by a maintenance dose of
  • the PEG derivatized hirudin peptides of this invention display a longer half-life as compared to native hirudin, as well as the underivatized peptides, their dosage amounts are advantageously well below those specified above for the underivatized peptides.
  • compositions and combinations of this invention may additionally contain other components which are effective in fibrinolytic therapy. These include, but are not limited to, tissue plasminogen activator, urokinase and streptokinase. These other compounds may be present as separate components in the hirudin peptide-containing compositions of this invention. Alternatively, the hirudin peptides may be conjugated to such fibrinolytic agents. Conjugation may be achieved by any of the methods known in the art. The hirudin peptides and fibrinolytic agents may also be present as a single molecule, i.e., in the form of a fusion protein, produced by recombinant DNA techniques or by in vitro synthesis.
  • This invention also relates to compositions containing the hirudin peptides of this invention and methods of using such compositions for the treatment of tumor metastases.
  • the efficacy of the hirudin peptides of this invention for the treatment of tumor metastases via inhibition of metastatic growth is based upon the presence of a procoagulant enzyme present in certain cancer cells [A. Falanga and S. G. Gordon, "Isolation and Characterization of Cancer Procoagulant: A Cysteine Proteinase from
  • This enzyme activates the conversion of Factor X to Factor Xa in the coagulation cascade, resulting in fibrin deposition which, in turn, serves as a substrate for tumor growth.
  • the hirudin peptides of the present invention serve as effective anti-metastatic tumor agents.
  • metastatic tumors which may be treated by the peptides of this invention include, but are not limited to, carcinoma of the brain, carcinoma of the liver, carcinoma of the lung, osteocarcinoma and neoplastic plasma cell carcinoma.
  • This invention also relates to methods and compositions employing low dosages of the peptides and analogs of this invention to inhibit thrombininduced platelet activation and thrombin-induced endothelial cell activation.
  • the inhibition of endothelial cell activation effected by the methods and compositions of this invention includes the repression of platelet activating factor (PAF) synthesis by these cells.
  • PAF platelet activating factor
  • This mechanism of inhibition has important implications in the treatment of diseases characterized by thrombin-induced inflammation, which is thought to be mediated by PAF.
  • the present invention also relates to compositions comprising low dosages of the peptides and analogs of this invention and methods which employ them for the treatment of patients suffering from diseases characterized by thrombin-induced inflammation.
  • Such diseases include, but are not limited to, adult respiratory distress syndrome, septic shock, septicemia and reperfusion damage.
  • the use of low dosages of hirudin peptides surprisingly and unexpectedly permits the above- described inhibitory activities to be achieved in a treated patient, with minimal anticoagulant effects. Accordingly, the use of low dosages of hirudin peptides is advantageous in circumstances where inhibition of activation of platelets and endothelial cells or treatment of diseases characterized by thrombin-induced inflammation are desirable without concomitant anticoagulant effects.
  • compositions for inhibiting platelet and endothelial cell activation comprise between about 0.0001 mg/kg body weight and about 0.099 mg/kg body weight. More preferably, these compositions comprise between about 0.001 mg/kg body weight and about 0.05 mg/kg body weight.
  • This invention also relates to the use of hirudin peptides, or compositions containing them, as anticoagulants for extracorporeal blood.
  • extracorporeal blood includes blood removed in line from a patient, subjected to extracorporeal treatment, and then
  • compositions are similar to the above-described injectable preparations.
  • the amount or concentration of active peptide in these types of compositions will be based on the volume of blood to be treated or, more preferably, its thrombin content.
  • This invention also relates to the bioanalytic use of hirudin peptides , or compositions containing them, for determining the concentration of Factor IXa, Factor Xa, thrombin, or mixtures
  • peptides and compositions may be used in a manner similar to that of reagents employed in conventional assays.
  • the peptides of this invention may be
  • the peptides of the present invention may be used for ex vivo thrombus imaging in humans and other mammals.
  • the hirudin peptides are radiolabeled with a radioisotope.
  • the choice of radioisotope is based upon a number of well-known factors, for example, toxicity, biological half-life and detectability.
  • radioisotopes include, but are not limited to, 125 I, 123 l and 111 In.
  • Techniques for labeling peptides are well known in the art. Most preferably, the radioisotope is 123 I and labeling is achieved using 123 I-Bolton Hunter Reagent.
  • the labeled peptide is administered to a patient, preferably by an intravenous route, and allowed to bind to the thrombin contained in a fibrin clot. The clot is then observed by utilizing well-known detecting means, such as a camera capable of detecting radioactivity coupled to a computer imaging system. This technique also
  • This invention also relates to peptidomimetic analogs of the hirudin peptides described
  • the analogs of the hirudin peptides may be either semi-peptidic or non-peptidic in nature. These peptidomimetic analogs advantageously exhibit increased shelf-life and biostability when compared to the parent compound. Moreover, the bioavailability of the peptidomimetic analogs of the present invention may be greater than the corresponding peptides when administered by oral or topical routes. Furthermore, these analogs may exhibit increased anticoagulant activity.
  • This invention also relates to analogs of the hirudin peptides of this invention which are capable of forming covalent bonds to thrombin.
  • these analogs are characterized by the presence of a dinitrofluorobenzyl group attached to the amino terminus of the
  • these analogs are characterized by the replacement of tyrosine or derivatized tyrosine, as well as any other more caboxy terminal residues, with
  • hirudin peptide analogs possess the ability to form a covalent bond with thrombin and therefore have greater affinity for that molecule. As a result of this greater affinity, these hirudin peptide analogs may be substantially more potent than the other hirudin peptides of this invention which bind to thrombin via ionic interactions.
  • peptidomimetic and covalent analogs of this invention are characterized by biological activities that are similar to those of the hirudin peptides described herein. Accordingly, these analogs may be employed in compositions, combinations, and methods for diagnosis, therapy and prophylaxis in the same manner as the peptides of this invention.
  • This invention also relates to hirudin peptides which are identical to the above-described peptides, except they are characterized by the replacement of Asp 53 or Asn 53 with an arginine residue.
  • These peptides contain an Arg 53 -Gly 54 -Asp 55 sequence which binds to and inhibits the platelet surface glycoprotein Hb/IIIa.
  • These peptides surprisingly and unexpectedly display both inhibitory acitivity against platelet activation induced by any agonist, as well as anticoagulant activity.
  • the presence of the Arg-Gly-Asp sequence serves to target these peptides to the site of a platelet-rich clot.
  • novel peptides may be employed in compositions and methods for achieving the dual effects of increasing clotting time and inhibiting platelet activation.
  • one or a combination of any of the hirudin peptides or analogs may be used in compositions and methods for coating invasive devices to be inserted into a patient. These compostions and methods result in lower risk of thrombotic complications in patients receiving such devices.
  • Surfaces that may be coated according to the methods and compositons of this invention are exemplified by those of prostheses, artificial valves, vascular grafts, stents and catheters. Methods for achieving the coating of these surfaces are known in the art. These include chemical cross-linking or physical adsorption of the hirudin peptide- or analog- containing compositions to the surfaces of the devices.
  • peptide samples (20-100 ⁇ g) were applied to a Vydac C 4 column (0.46 x 25 cm) or an Aquapore RP-300 C 8 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 containing 0.1% trifluoroacetic acid (TFA) and developed 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 1.0 ml/min.
  • TFA trifluoroacetic acid
  • Hirudin 53-64 has the amino acid formula: H 2 N-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu- COOH.
  • Hirudin 49-64 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.
  • Des(Tyr-Leu) hirudin 53-62 has the amino acid formula: H 2 N-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro- Glu-Glu-COOH.
  • carboxypeptidase A essentially as described by R. P. Ambler, "Enzymatic Hydrolysis with Carboxypeptidases", Methods Enzymol., 25, part B, pp. 143-54.
  • the enzyme 1.0 mg was suspended in 1.0 ml of deionized water at 4°C and centrifuged in a microfuge apparatus. We discarded the supernatant and added 100 ⁇ l of 1% sodium
  • Peptide fragments were purified by reverse phase HPLC employing an Aquapore RP-300 C 8 column (0.46 x 3.0 cm) and an Applied Biosystems 150 A liquid chromatographic system. The column was equilibrated in water containing 0.1% TFA and
  • Hirudin 57-64 has the amino acid formula: H 2 N-Glu-Glu-Ile-Phe-Glu-Glu-Tyr-Leu-COOH.
  • To synthe- size hirudin 57-64 we followed the procedure outlined in Example 1, except that we used 0.55 mmoles of Boc-Leu-OCH 2 -PAM resin (1% DVB) (Applied Biosystems) in place of the Boc-Leu-O-resin (1% DVB). We then added 2 mmoles of protected amino acids at each cycle of coupling. The yield of crude peptide was 17.9%.
  • HPLC analysis revealed a single predominant peak eluting at 14.2 min in the gradient.
  • HF p-cresol: ethyl methyl sulfate (10:1:1, v/v/v). Approximately 100 mg of peptide was recovered by extraction of the resin with 30% acetic acid. The yield of hirudin 45-64 was 17%.
  • HPLC analysis revealed a high degree of purity (>90%) in the product and a single predominant peak of 214 nm absorbing material at 14.4 min in the acetonitrile gradient.
  • Hirudin 55-64 has the amino acid formula:
  • Hirudin 55-64 was prepared by the procedure outlined in Example 1, using 0.0259 meq of Boc-Leu-O-resin (1% DVB). We then added 2 mmoles of protected amino acids to the growing peptide at each cycle of coupling. Deprotection and cleavage from the resin was achieved as in the previous examples. The recovery of this peptide was 30%.
  • HPLC analysis revealed a high degree of purity (>95%) in the sample and a single predominant peak at 16.1 min in the acetonitrile gradient.
  • Hirudin 64-45 has the amino acid formula: H 2 N-Leu-Tyr-Glu-Glu-Pro-Ile-Glu-Glu-Phe-Asp-Gly- Asn-Asn-His-Ser-Glu-Pro-Asn-Pro-Thr-COOH.
  • the peptide was fully deprotected and uncoupled from the DVB resin by treatment with anhydrous HF. Following extraction with 30% acetic acid, 120 mg of peptide was recovered and the yield of hirudin 64-45 was 19.
  • HPLC analysis of the peptide revealed a high degree of purity in the preparation (>90%) and a single predominant peak eluting at 13.7 min in the acetonitrile gradient.
  • Hirudin 54-64 has the amino acid formula:
  • hirudin 45-64 hirudin 49-64 and hirudin 53-64 , as prepared above, were purified to homogeneity by preparative reverse-phase HPLC employing a Waters Associates (Milford, MA) liquid chromatography
  • hirudin 53-64 were dissolved in 2.0 ml of 0.1% TFA in water. An additional 1.0 ml of 6 M guanidinium chloride was added to crude samples of hirudin 49-64 and hirudin 53-64 to increase solubility. The samples were separately injected on a Vydac C 18 column
  • hirudin peptides may be similarly prepared and purified.
  • N-acetylation of the hirudin peptides of this invention was achieved directly during peptide synthesis.
  • N-acetyl hirudin 53-64 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.
  • Other peptides of the present invention may be similarly N-acetylated by substituting the N-acetyl form of the amino terminal amino acid for the unacetylated form in the last cycle of peptide synthesis.
  • Hirudin 53-64 was O-sulfated at the tyrosine residue to prepare Sulfo-Tyr 63 hirudin 53-64 , using the chemical modification procedure of T. Nakahara et al . , "Preparation of Tyrosme-O- [ 35 S ] Sulfated Cholecystokinin Octapeptide from a Non-Sulfated
  • N,N'-dicyclohexylcarbodiimide in 40 ⁇ l DMF (7.0 x 10 -5 moles). The reaction was allowed to proceed for about 5-10 min at 25°C before the addition of 750 ⁇ l of deionized water. Any insoluble reaction products were removed by centrifugation in a microfuge 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 18 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
  • peak A contained Sulfo- Tyr 63 hirudin 53-64 .
  • the column was equilibrated in water containing 0.1% TFA and developed with a gradient of increasing acetonitrile concentration from 0 to 70% over 45 minutes at a flow rate of 0.5 ml/min in a 0.085% TFA-containing solvent.
  • the peptide showed HPLC chromatographic behavior identical to that of unsulfated hirudin 53-64 .
  • peak absorbance of the treated peptide returned to 275-280 nm, typical for a peptide containing an unmodified tyrosine residue.
  • N-acetyl-hirudin 53-64 (as prepared in Example 9) by the Nakahara procedure produced an 80.1% yield of the desired Tyr-sulfated product.
  • N-acetyl-hirudin 53-64 only resulted in a 48.5% yield of the Tyr-sulfated derivative (Fig. 2b).
  • the Sulfo-Tyr 63 -N-acetyl- hirudin 53-64 eluted at approximately 0.2-0.3 M NaCl, after the unsulfated peptide, but prior to the sulfonated side product Suifo-Tyr 63 -N-acetyl- hirudin 53-64 .
  • hirudin peptides of this invention may be sulfated, purified and analyzed by procedures identical to those set forth above.
  • N-acetyl-hirudin 53-64 was modified to its Tyr-sulfonated derivative, Suifonyl-Tyr 63 -N-acetyl- hirudin 53-64 , during the preparation of Sulfo- Tyr 63 -N-acetyl-hirudin 53-64 , as described in Example 10.
  • Sulfonyl-Tyr 63 -N-acetyl-hirudin 53-64 was a side reaction product obtained during the large-scale sulfation reaction described in that example.
  • This example illustrates the inhibitory activity of the hirudin peptides of this invention towards thrombin, as measured by the inhibition of activated partial thromboplatin times (APTT).
  • Anticoagulant activity was determined by assaying APTT of pooled, normal human plasma (George King
  • hirudin peptides were mixed with stock solutions of hirudin peptides at concentrations ranging from 750 ⁇ g/ml in water, for non- sulfated hirudin peptides, to 35 ⁇ g/ml in water for sulfated hirudin peptides.
  • the plasma and hirudin peptides were mixed to a final volume of 125 ⁇ l prior to each APTT determination.
  • Figure 5 shows a comparison of the MCT 50
  • Hirudin 53-64 demonstrated an MCT 50 of 0.77.
  • Sulfo-Tyr 63 hirudin 53-64 advantageously caused an almost 10-fold decrease in MCT 50 .
  • thrombin times were measured using the tilt-tube method. Specifically, we dissolved human ⁇ -thrombin (Diagnostica Stago, Asniere, France) in 0.01 M Tris-HCl, pH 7.4, containing 0.15 M NaCl and 10 mM CaCl 2 at a concentration of 2 .5 U/ml . We mixed 100 ⁇ l of the thrombin solution with 200 ⁇ l of normal human plasma (George King Biomedical, Inc., KA) that had been premixed with 0.- 10 ⁇ g/ml of
  • Sulfo-Tyr 63 hirudin 53-64 (0.8 U/ml final concentration of thrombin).
  • the thrombin time of normal plasma was typically 15 seconds.
  • Figure 6 demonstrates a comparison of the increase in thrombin times by hirudin (Sigma
  • Sulfo-Tyr 63 hirudin 53-64 A concentration of 3.3 x 10 -8 M Sulfo-Tyr 63 hirudin 53-64 increased TT from 15 to 55 sec. By comparison to hirudin, Sulfo-Tyr 63 hirudin 53-64 exhibited an approx- imately 50-fold greater molar specific inhibitory activity towards thrombin. These are the same relative inhibitory activities observed in the APTT assay.
  • hirudin 53-62 demonstrated a 10-fold increase in clotting time as compared to its unsulfated counterpart.
  • Thrombomodulin is an endothelial cell- surface glycoprotein which forms a stoichiometric complex with thrombin [C. T. Esman, "The Regulation of Natural Anticoagulant Pathways", Science, 235, pp. 1348-52 (1987)]. Identification of homology between the C-terminal segment in hirudin and residues 150-155 in the thrombomodulin precursor suggests that this segment in thrombomodulin participates in complex formation with thrombin. It has been
  • hirudin also impairs thrombin's catalytic activity it is unlikely to share in this property. Accordingly, we believe that the anticoagulant activity of the hirudin peptides of this invention may derive from binding of the peptide to a non-catalytic site of thrombin, thus yielding an increased K m for thrombin-catalyzed hydrolysis of fibrinogen.
  • hirudin peptides for human a-thrombin was determined by comparing their relative inhibitory activities towards both human a-thrombin and bovine of-thrombin in a TT assay.
  • Figure 7 demonstrates that Suifo-Tyr 63 -N-acetyl- hirudin 53-64 is 4 to 5 times more active as an inhibitor of human or-thrombin than bovine a-thrombin.
  • Human a-thrombin and bovine a-thrombin are highly similar in structure, except for the presence of a unique lysine residue at position 149 of the ⁇ -chain in the human molecule. This residue defines the site of ⁇ -autocatalytic degradation.
  • Lys-149 of human ⁇ -thrombin is an important structural determinant for the binding of the hirudin peptides of this invention to thrombin.
  • Figure 8 shows the results of a dose-dependent APTT assay using the hirudin peptide
  • thrombin solution bovine plasma thrombin, Calbiochem, Behring Diagnostics, La Jolla, CA
  • hirudin 45-64 demonstrated no measurable inhibitory effect on thrombin- catalyzed hydrolysis of the artificial substrate.
  • the mouse plasma was assayed for activated partial thromboplastin times (APTT) by the methods described in Example 12.
  • Figure 9 demonstrates that Sulfo-Tyr 63 hirudin 53-64 produced a dose-dependent increase in vivo in the APTT of mouse plasma.
  • Example 10 prepared in Example 10, in 200 ⁇ l of 20 mM sodium borate, pH 9.0 and reacted it with a 50-fold molar excess of SS-PEG. The reaction was let stand at room temperature overnight and then applied to reverse phase HPLC for purification and characterization.
  • Reverse-phase HPLC was performed using an Aquapore RP-300 C 8 column (0.46 x 3.0 cm) using an Applied Biosystems 150A chromatographic system.
  • the column was equilibrated in water containing 0.1% TFA and developed with an increasing gradient of acteonitrile from 0 to 50% in a 0.085% TFA solvent over 45 minutes at a flow rate of 0.5 ml/min.
  • the effluent stream was monitored at
  • the SS-PEG-Sulfo-Tyr 63 hirudin 53-64 is an active derivative of the peptides of present invention that advantageously exhibits an expected increase in circulating half-life when compared to its underivatized counterpart.
  • Figure 10 demonstrates that Sulfo- Tyr 63 hirudin 53-64 exhibits a dose-dependent inhibition of S-2222 cleavage in this assay. Because this peptide does not inhibit Factor Xa cleavage of S-2222, this result shows that increasing amounts of peptide cause decreasing production of Factor Xa. Therefore, Sulfo-Tyr 63 hirudin 53-64 must inhibit Factor IXa activation of Factor X. Inhibition of Factor IXa was complete at 6 x 10 -6 M concentration of the peptide.
  • samples prepared as described above were analyzed for resulting thrombin activity using Chromozym TH (Boehringer-Mannheim, Indianapolis, IN) as a substrate.
  • Chromozym TH Boehringer-Mannheim, Indianapolis, IN
  • a 5 ⁇ l aliquot of each sample was added to a cuvette containing 1.0 ml of 0.01 M Tris-HCl, pH 7.5, 0.15 M NaCl and 4 ⁇ g/ml Chromozym TH.
  • Thrombin activity was monitored continuously at 420 nm. Analysis of thrombin activity in this manner demonstrated a four-fold reduction in esterolytic activity generated via Factor Xa activation of prothrombin at a Sulfo-Tyr 63 hirudin 53-64 concentration of 70 ⁇ g/ml.
  • the anti-metastatic activity of hirudin peptides may be assayed using sarcoma T241 cells [L. A. Liotta et al., " Nature, 284, pp. 67-68 (1980)] and syngeneic C57BL/6 Mice (Jackson Laboratory, Bar Harbor, ME).
  • the mice are injected either intravenously or subcutaneously with 0 - 250 g/kg of Sulfo-Tyr 63 hirudin 53-64 , as prepared according to Example 10, followed by intravenous injection of 10 4 -10 6 T241 tumor cells. After 15 days, the animal is terminated and lung tumor colonies are quantitated.
  • Anti-metastic activity of the hirudin peptide is measured as percent reduction in tumor colonies compared to a placebo-treated control.
  • the peptides of this invention may be used to produce semipeptidic or non-peptidic peptidomimetic analogs, synthetic molecules which exhibit antithrombin and anticoagulant activities. These peptidominetic analogs display the inhibitory activity toward thrombin hydrolysis of fibrinogen, Factor IXa
  • peptidomimetic analogs of this invention hereinafter referred to as “hirulogs”, are represented by the following chemical structures:
  • Semi-peptidic peptidomimetic analogs of the hirudin peptides of this invention may be prepared to stabilize a loop, turn, or helical conformation of the parent peptide.
  • a loop structure is constructed by the addition of cysteinyl or lysyl residues at both the N- and C-terminal ends of Sulfo- Tyr 63 hirudin 53-64 . Terminal cysteinic residues are crosslinked by oxidation to produce Hirulog-1
  • Hirulog-3 ( Figure 12c). 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 13).
  • a turn structure around Pro-8 of Sulfo- Tyr 63 hirudin 53-64 is constrained by replacement of
  • 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 an aliphatic dihalo
  • Hirulog-7 exemplified by Hirulog-7.
  • Hirudin peptides such as Sulfo-Tyr 63 hirudin 53-6 4 , may be modified by covalent attachment of 123 I, 125 I- or 111 In-containing chemical groups.
  • hirudin 53-64 (as prepared in Example 10) may be
  • the 125 I-labelled peptide (with a specific radioactivity of >5 ⁇ Ci/ ⁇ g) is then desalted on a column of Biogel P2 which is equilibrated in a phosphate- buffered saline.
  • the 125 I-labelled peptide may
  • imaging is performed in baboons with an external Ticoflex shunt between the femoral artery and femoral vein.
  • An experimental thrombus is "formed by placement of a segment of preclotted Dacron graft in the shunt. 125 I-labelled Sulfo-
  • Tyr 63 hirudin 53-64 is injected in the venous part of the Ticoflex shunt.
  • Serial anterior images are then obtained for 0.5 - 1 hr using an Ohio Nuclear Series
  • the same technique may be used to obtain ex vivo images of a deep venous thrombus obtained by stasis in the femoral vein of baboons.
  • Sulfo-Tyr 63 hirudin 53-64 binds to thrombin with high specificity, the use of radiolabelled hirudin peptides allows precise ex vivo images of experimental thrombi. Also, the small size of hirudin peptides, in contrast to native hirudin or antibodies to thrombin, provides the potential that the radiolabelled-peptide will yield images of platelet-bound thrombin and meizothrombin, as well as thrombin contained in the fibrin clot.
  • HUVECs were extracted from human umbilical cords by collagenase digestion according to established procedures [M. A. Gimbrone, Jr., "Culture of Vascular Endothelium” , Prog. Hemost. Thromb . , 3 ,
  • [ 3 H]-acetate Cells cultured in this manner produced [ 3 H]-acetyl-PAF, which can be quantitated by extraction of HUVEC membrane phospholipids.
  • hirudin 53-64 (0 - 200 ⁇ g/ml) or Sulfo-Tyr 63 hirudin 53-64 (0 - 8 ⁇ g/ml) to the [ 3 H]-acetate loaded HUVECs 1 minute prior to the addition of thrombin (final concentration of 1 U/ml).
  • thrombin final concentration of 1 U/ml.
  • medium containing 0.1% gelatin, 50 mM acetic acid in methanol (2:1, v/v) was extracted and quantitated PAF using standard techniques [T. M. Mclntyre et al., "Cultured Endothelial Cells Synthesize Both Platelet- Activating Factor and Prostacyclin in Response to
  • PMN polymorphonuclear leukocyte
  • HUVECs HUVECs. We grew HUVECs in MEM containing 1% fetal calf serum to confluency in 24-well cluster plates.
  • N-acetyl-Arg 53 hirudin 53-64 was prepared by solid phase peptide synthesis and isolated to >95% purity by the procedures described in Example 1.
  • This peptide contains an Arg-Gly-Asp sequence which is also prevalent in a wide number of adhesive proteins which interact with platelet glycoprotein Hb/IIIa [M. D. Pierschbacher and E. Ruoslahti, "Cell Attachment
  • Fibronectin can be Duplicated by Small Synthetic Fragments of the Molecule", Nature, 309, pp. 30-33 (1984)].
  • N-acetyl-Arg 53 hirudin 53-64 was assessed using an APTT assay as described in Example 12.
  • N-acetyl-Arg 53 hirudin 53-64 demonstrated 78.9% of the anticoagulant activity of N-acetyl-hirudin 53-64 .
  • Example 22 except that platelet aggregation was stimulated by the addition of either collagen
  • Figure 17 demonstrates that N-acetyl-Arg 53 hirudin 53-64 inhibited platelet aggregation in a dose-dependent manner, with an
  • IC 50 of approximately 190 ⁇ g/ml for collagen-induced aggregation and 490 ⁇ g/ml for ADP-induced aggregation.
  • N-acetyl- Arg 53 hirudin 53-64 is capable of blocking thrombin- mediated thrombosis, whether via cleavage of fibrinogen or activation of platelets, as well as plate- let-dependent thrombosis caused by collagen, ADP, epinepherine, thromboxane A 2 , or any other compound that activates platelets.
  • the thrombin- mediated thrombosis whether via cleavage of fibrinogen or activation of platelets, as well as plate- let-dependent thrombosis caused by collagen, ADP, epinepherine, thromboxane A 2 , or any other compound that activates platelets.
  • Arg-Gly-Asp sequence may serve to target this peptide to a thrombus and thus increase the local concentration of a thrombin inhibitor at that site.
  • hirudin analog dinitro- fluorobenzyl-Sulfo-Tyr 63 hirudin 54-64 (DNFB-Sulfo- Tyr 63 hirudin 54-64 ) by reacting stoichiometric quantities of Sulfo-Tyr 63 hirudin 54-64 with dinitrodi- fluorobenzene (DNDFB) in dimethylformamide.
  • DNDFB dinitrodi- fluorobenzene
  • the column was first equilibrated in 0.1% TFA in water (solvent A). After loading the sample, we developed the column with a 0 - 50% linear gradient of solvent B (0.085% TFA/70% acetonitrile) over 45 minutes. The effluent stream was monitored for adsorbance at 214 nm. The DNFB-Sulfo-Tyr 63 hirudin 54-64 eluted at 48% solvent B. We synthesized the hirudin analog nitro- anisole 63 hirudin 53-63 in a two-step method.
  • methoxytyrosyl 63 hirudin 53-63 by the solid phase synthesis techniques, as described in Example 1, substituting Boc-O-methoxytyrosine resin for Boc-O-Leu resin in the synthesis.
  • the peptide was cleaved from the resin and purified by HPLC on a Vydac C4 column, as described in Figure 1.

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Abstract

Sont décrits de nouveaux peptides biologiquement actifs qui présentent l'activité anticoagulante de l'hirudine. Plus précisément, l'invention concerne des peptides qui sont homologues d'au moins une partie des aminoacides de l'hirudine à terminaison carboxy 26. Ces peptides peuvent également être caractérisés par un résidu tyrosine modifié. La présente invention décrit également des analogues covalents et peptidomimétiques de ces peptides qui présentent une activité anticoagulante. L'invention se rapporte également à des compositions, combinaisons et procédés mettant en ÷uvre ces peptides ou analogues à des fins thérapeutiques, prophylactiques ou diagnostiques. Sont également décrits de nouveaux procédés pour sulfater les résidus tyrosine contenus à l'intérieur d'un peptide ou d'un polypeptide.
PCT/US1989/000848 1988-09-29 1989-04-24 Peptides d'hirudine WO1990003391A1 (fr)

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FI904330A FI904330A0 (fi) 1988-09-29 1990-09-03 Hirudinpeptider.
DK210590A DK210590A (da) 1988-09-29 1990-09-03 Hirudinpeptider
NO90903833A NO903833L (no) 1988-09-29 1990-09-03 Hirudinpeptider.

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WO1992013879A1 (fr) * 1991-02-07 1992-08-20 Basf Aktiengesellschaft Nouveaux peptides a activite anticoagulante
US5240913A (en) * 1989-08-18 1993-08-31 Biogen, Inc. Inhibitors of thrombin
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WO2010054503A1 (fr) * 2008-11-17 2010-05-20 中国人民解放军军事医学科学院生物工程研究所 Polypeptides anticoagulants et leurs applications
WO2011027175A1 (fr) * 2009-09-04 2011-03-10 King's College London Antithrombotiques
US20230151079A1 (en) * 2017-03-21 2023-05-18 Fundação Butantan Recombinant protein and fragments thereof, method for producing said recombinant protein, synthetic gene and use of sculptin or recombinant protein for preparing a medicament or pharmaceutical composition for the prophylaxis and/or treatment of thromboembolic diseases or as a direct and specific inhibitor of thrombin
CN117088966A (zh) * 2022-12-29 2023-11-21 江苏诺泰澳赛诺生物制药股份有限公司 一种比伐芦定杂质的合成方法

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JP2002112782A (ja) * 2000-10-04 2002-04-16 Ajinomoto Co Inc 抗血栓活性を有する蛋白質及びその製造法
WO2010075983A1 (fr) * 2008-12-29 2010-07-08 Lonza Braine Sa Procédé pour la production de bivalirudine
JP6174367B2 (ja) * 2013-04-26 2017-08-02 株式会社Ｌｓｉメディエンス トロンビン含有試薬及び測定方法

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FR2667071A1 (fr) * 1990-09-26 1992-03-27 Adir Nouveaux derives de peptides et de pseudopeptides therapeutiquement actifs dans la cascade de coagulation sanguine, leur procede de preparation et les compositions pharmaceutiques qui les contiennent.
EP0482966A2 (fr) * 1990-09-26 1992-04-29 Adir Et Compagnie Dérivés stabilisés de fragments d'hirudine, leur procédé de préparation et leurs compositions pharmaceutiques
EP0482966A3 (en) * 1990-09-26 1992-06-24 Adir Et Compagnie Stabilised derivatives of fragments of hirudin, their preparation and pharmaceutical preparations
US5242810A (en) * 1990-12-07 1993-09-07 Biogen, Inc. Bifunctional inhibitors of thrombin and platelet activation
WO1992013879A1 (fr) * 1991-02-07 1992-08-20 Basf Aktiengesellschaft Nouveaux peptides a activite anticoagulante
US5393873A (en) * 1991-02-07 1995-02-28 Basf Aktiengesellschaft Peptides with anticoagulant activity
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US20090269422A1 (en) * 2004-04-12 2009-10-29 Cheng-Der Tony Yu Methods for controlling angiogenesis and cell proliferation
WO2010054503A1 (fr) * 2008-11-17 2010-05-20 中国人民解放军军事医学科学院生物工程研究所 Polypeptides anticoagulants et leurs applications
WO2011027175A1 (fr) * 2009-09-04 2011-03-10 King's College London Antithrombotiques
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US20230151079A1 (en) * 2017-03-21 2023-05-18 Fundação Butantan Recombinant protein and fragments thereof, method for producing said recombinant protein, synthetic gene and use of sculptin or recombinant protein for preparing a medicament or pharmaceutical composition for the prophylaxis and/or treatment of thromboembolic diseases or as a direct and specific inhibitor of thrombin
CN117088966A (zh) * 2022-12-29 2023-11-21 江苏诺泰澳赛诺生物制药股份有限公司 一种比伐芦定杂质的合成方法

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HUT55799A (en) 1991-06-28
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HU894117D0 (en) 1991-05-28
DK210590D0 (da) 1990-09-03
FI904330A0 (fi) 1990-09-03

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