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EP0483261A1 - Derives de peptides d'hirudine - Google Patents

Derives de peptides d'hirudine

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Publication number
EP0483261A1
EP0483261A1 EP19900911670 EP90911670A EP0483261A1 EP 0483261 A1 EP0483261 A1 EP 0483261A1 EP 19900911670 EP19900911670 EP 19900911670 EP 90911670 A EP90911670 A EP 90911670A EP 0483261 A1 EP0483261 A1 EP 0483261A1
Authority
EP
European Patent Office
Prior art keywords
patient
tyrosine
amino acid
hirudin
glu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19900911670
Other languages
German (de)
English (en)
Inventor
John M. Maraganore
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biogen Inc
Original Assignee
Biogen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biogen Inc filed Critical Biogen Inc
Publication of EP0483261A1 publication Critical patent/EP0483261A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/0005—Use of materials characterised by their function or physical properties
    • A61L33/0047—Enzymes, e.g. urokinase, streptokinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/06—Use of macromolecular materials
    • A61L33/12—Polypeptides, proteins or derivatives thereof, e.g. degradation products thereof
    • A61L33/128—Other specific proteins or polypeptides not covered by A61L33/122 - A61L33/126
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo
    • 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, biologically active peptide derivatives which inhibit the fibrinolytic, but not the amidolytic activity of thrombin.
  • the peptide derivatives which display partial homology to the carboxy terminal portion of hirudin, are characterized by a modified tyrosine or cysteine residue at a predetermined site in the molecule.
  • This invention also relates to compositions and methods employing such peptide derivatives for therapeutic, prophylactic or diagnostic purposes.
  • This invention also relates to platelet and endothelial cell inhibitory compositions and methods characterized by the above-described peptide derivatives or similar peptide derivatives which do not contain a modified cysteine or tyrosine residue at a corresponding locus in the molecule.
  • 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 contains fibrin and aggregated platelets.
  • 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, Sup l.. 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 he orrhaging 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 gl-nd of the leech, is the most potent natural inhibitor of coagulation known.
  • hirudin undergoes a conformational change and then binds to the "high” affinity site on thrombin. This latter site corresponds to the active site of thrombin.
  • hirudin At least ten different isomorphic forms of hirudin have been sequenced anfi have been shown to differ slightly in amino acid sequence [D. Tripier, "Hirudin: A Family of Iso-Proteins. Isolation and Sequence Determination of New Hirudins", Folia Haematol.. 115, pp. 30-35 (1988)]. All forms of hirudin comprise a single polypeptide chain protein containing 65 or 66 amino acids in which the amino terminus primarily comprises hydrophobic amino acids and the carboxy terminus typically comprises polar amino acids.
  • hirudin 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) .
  • 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 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. Hae ostasis. 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 determi- nation and administration of proper dosages critically important. Furthermore, the high cost and low supply of the naturally occurring product has prevented its widespread use.
  • hirudin is still moderately expensive to produce and it is not wideiy available commercially. Recently, efforts have been made to identify peptide fragments of native hirudin which are also effective in lowering clotting time.
  • An unsulfated 21 amino acid C-terminal fragment of hirudin, N ⁇ acetyl- hirudin 45 . 65 inhibits clot formation in vitro.
  • several other smaller, unsulfated peptides corresponding to the C-terminal 11 or 12 amino acids of hirudin have also demonstrated efficacy in inhibiting clot formation in vitro [J. L.
  • thrombin In addition to catalyzing the formation of a fibrin clot, thrombin has several other bioregulatory roles [J. . Fenton, II, "Throabin Bioregulatory Functions", Adv. Clin. Enzvmoi.. 6, pp. 186-93 (1988)]. For example, 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.
  • Thrombin can also directly activate an inflammatory response by stimulating the synthesis of platelet activating factor (PAF) by endothelial cells [S. M. Prescott et al. , "Human Endothelial Cells in Culture Produce Platelet-Activating " Factor (l-alkyl-2- acetyl-sn-glycero-3-phosphocholine) When Stimulated with Thrombin, Proc. Natl. Acad. Sci. USA. 81, pp. 3534-38 (1984)].
  • PAF is exposed on the surface of endothelial cells and serves as a ligand for neutrophil adhesion and subsequent degranulation [G. M. Vercolletti et al. , "Platelet-Activating Factor Primes Neutrophil Responses to Agonists: Role in Promoting Neutrophil-Mediated Endothelial Damage", Blood. 71, pp. 1100-07 (1988)].
  • thrombin activates platelets and endothelial cells
  • a receptor The mechanism by which thrombin activates platelets and endothelial cells involves a receptor and is effected at a lower concentration than that required for fibrinogen cleavage [J. T. Harmon and G. A. Ja ieson, "The Glycocalcin Portion of Glycoprotein lb Expresses Both High and Moderate Affinity Receptor Sites for Thrombin", J. Biol. Chem.. 28, pp. 13224-29 (1986)].
  • Reagents which block the active site of thrombin such as hirudin, interrupt the activation of platelets and endothelial cells [C. L.
  • the present invention solves the problems . referred to above by providing peptide derivatives ' which inhibit the fibrinolytic activity, but not the amidolytic activity of thrombin.
  • the peptide derivatives of this invention are effective as anticoagulants which cause an increase in blood clotting time.
  • These peptide derivatives are characterized by partial homology to the C-terminus of native hirudin and by the presence of a derivatized tyrosine or cysteine residue at a functionally critical locus in the moleucle.
  • the present invention also provides compositions and methods which inhibit thrombin-induced platelet aggregation and platelet release (hereinafter “platelet activation”) and the release of inflammatory substances from endothelial cells (hereinafter “endothelial activation”) .
  • platelet activation thrombin-induced platelet aggregation and platelet release
  • endothelial activation inflammatory substances from endothelial cells
  • the relatively small size of the peptides derivatives of this invention and the other peptide derivatives which characterize the compositions of this invention advantageously permits production using conventional peptide synthesis techniques. 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.
  • these peptide derivatives unlike hirudin, exhibit a saturable effect on clotting time.
  • the therapeutic and prophylatic uses of the peptides avoid the harmful and potentially fatal consequences of an overdose associated with conven ⁇ tional anticoagulants, such as heparin.
  • the small size of the peptide derivatives employed in this invention decreases the possibility of an adverse antigenic response in patients treated with them.
  • compositions and methods of ths invention are useful in the treatment, prevention or diagnosis of vascular and other diseases attributed to the undesirable effects of thrombin, as well as in the treatment of extracorporeal blood and in in vivo diagnosis techniques.
  • compositions characterized the peptide derivatives of this invention are also useful for coating invasive devices to be inserted into a patient.
  • Figure 1 depicts the effects of various concentrations of N-acetyl-TNBCys 63 hirudin 53 .
  • M N-acetyl- (3,5-diiodoTyr) H hirudin 53 _ 6 _, N-acetyl-SCMCys 63 hirudin H _ M , N-acetyl-SCECys 63 hirudin H _ 6 _, N-acetyl-hirudin
  • M on the activated partial thromboplastin times of normal human plasma.
  • Leu - leucine lie - isoleucine
  • 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, cyclopentyl, hexyl, isohexyl, cyclohexyl and cyclo pentylmethvl.
  • 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, aleyl, glutaryl and succinyl.
  • a "halogen group” is a fluoro, chloro, bro o 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, glutamic acid, glutamine, arginine, ornithine and lysine.
  • non-protein ⁇ -amino acids are norleucine, norvaline, alloisoleucine, homoarginine, thiaproline, dehydroproline, hydroxyproline (Hyp) , homoserine, cyclohexylglycine (Chg) , ⁇ -amino-n-butyric acid (Aba) , cyclohexylalanine (Cha) , aminophenylbutyric acid (Pba) , phenylalanine substituted at the ortho, meta, or para position of the phenyl moiety with one or two of the following: a (C,-C t ) alkyl, a (C,-C 4 ) alkoxy, halogen or nitro groups or substituted with a methylenedioxy group, ⁇ -2- and 3-thienylal-alanine, ⁇ -2- and 3- furanylalanine, ⁇ -2-, 3- and 4-pyridylalanine
  • derivatized amino acid refers to O-sulfate esters of tyrosine, O-
  • antiplatelet as used herein means an inhibitor of both platelet aggregation and platelet release reactions.
  • the present invention relates to peptide derivatives of the formula:
  • 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 0 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 is Glu or Asp;
  • A is Glu or Asp; A.
  • a 5 is 5 any amino acid;
  • a 5 is lie, Val, Leu, Nle or Phe;
  • _ is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4- carboxylate, Sar, NMePgl or D-Ala;
  • a 7 is any amino acid;
  • a s is any amino acid;
  • A is a derivatized amino acid selected from the group consisting of O-sulfate esters of tyrosine, O-phosphate esters of tyrosine, O-carboxylate esters of tyrosine, 3-sulfonyl tyrosine, 5-sulfonyl tyrosine, 3- phosphonyl tyrosine, 5-phosphonyl tyrosine, 3-carbonyl tyrosine, 5-carbonyl tyrosine, 4-methylsulfonyl tyrosine, 4-methylphosphonyl tyrosine, 4-phenylacetic acid, 3,5-
  • a I0 is a bond.
  • Leu or Leu- Gln; and Y is OH.
  • Preferred peptide derivatives according to this invention are characterizexl by an A, component selected from the group consisting of 3,5-diiodo- diiodotyrosine, 3-nitrotyrosine, 5-nitrotyrosine, S- carboxymethylcysteine, S-carboxyethylcysteine and thionitrobenzoic acid cysteine.
  • A component selected from the group consisting of 3,5-diiodo- diiodotyrosine, 3-nitrotyrosine, 5-nitrotyrosine, S- carboxymethylcysteine, S-carboxyethylcysteine and thionitrobenzoic acid cysteine.
  • the most preferred peptides according to the present invention have the amino acid formulae:
  • N-Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu- » (3,5-diiodoTyr)-Leu-OH herein referred to as "N- Ac-(diiodoTyr) 63 hirudin 53 _ 64 ") ;
  • N-Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu- (3-nitroTyr)-Leu-OH herein referred to as "N-Ac- (3-nitroTyr) 63 hirudin 53 . 6 ” ;
  • N-Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu- (5-nitroTyr)-Leu-OH herein referred to as "N-Ac- (5-nitroTyr) ⁇ 3 hirudin familiar. 64 ") ; N-Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-
  • SCMCys (SCMCys) -Leu-OH (herein referred to as "N-Ac- SCMCys 63 hirudin manufacture. 6 " ) ;
  • N-Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu- (SCECys) -Leu-OH herein referred to as "N-Ac- SCECys 63 hirudin S3 . 64 " ) ;
  • N-Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu TNBCys
  • this increase is 3 to 10-fold in anticoagulant potency and up to 100-fold in antiplatelet activity.
  • the peptide derivatives of the present invention may be synthesized by various techniques which are well known in the art. These include recombinant DNA techniques, solid-phase peptide synthesis, solution-phase peptide synthesis, organic chemical synthesis techniques, or a combination o f these techniques. The choice of synthesis techniques will, of course, depend upon the composition of the particular peptide derivative. In the preferred embodiments of this invention, the peptide derivative is synthesized entirely by solid-phase synthesis techniques, which constitute the most cost-efficient procedure for producing commercial quantities of these molecules [see, for example, European patent application 276,104, copending United States patent application 314,756 and J. M.
  • non-protein amino acids When “non-protein” amino acids are contained in the peptide derivatives, 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. Those of skill in the chemical synthesis art are well aware of which "non-protein” amino acids may be added directly and which must be synthesized by chemically modifying the complete molecule following synthesis [see, copending United States patent application 314,756].
  • non-protein amino acids which may be added directly during synthesis are diiodoTyr, norleucine, and norvaline. Those amino acids which must be synthesized by reacting the fully synthesized molecule are exemplified by SCMCys, SCECys, 3- or 5-nitroTyr and TNBCys.
  • the anticoagulant activity of the peptide derivatives of the invention 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 radioimmunoassay 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 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 peptide derivatives of this invention are useful in compositions, combinations and methods for the treatment and prophylaxis of vascular diseases attributed to blood system thromboses.
  • the peptide derivatives of this invention, and composi ⁇ tions containing them may be used for heparin replace ⁇ ment 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 peptide derivatives of this invention, as well as compositions containing them may be used in the treat ⁇ ment or prophylaxis of vascular diseases in all mammals and, in particular, humans.
  • the peptide derivatives of this invention may be administered in combination with heparin or low molecular weight heparin.
  • 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.
  • combi- nations 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 derivative of the present invention or at least one other peptide derivative which lacks the derivatized amino acid at A, and heparin or low molecular weight heparin, a multiple dosage form wherein the peptide derivative and the heparin are administered separately, but concurrently, or a multiple dosage form wherein the two agents are administered separately, but sequentially.
  • the above-described peptide derivatives, as well as other similar peptide derivatives which lack the derivatized amino acid at position A may be formulated into compositions for inhibiting platelet activation ("antiplatelet”) .
  • the antiplatelet activity of either of the above groups of peptide derivatives may be measured by any of a number of conventional platelet assays.
  • the assay measures a change in the degree of aggregation of platelets or a change in the release of a platelet secretory component in the presence of thrombin.
  • the former may be measured in a commercially available aggregometer.
  • the latter may be measured using RIA or ELISA techniques specific for the secreted component.
  • compositions of the present invention are also useful in inhibiting coagulation or platelet activation in extracorporeal blood.
  • extracorporeal blood includes blood removed in line from a patient, subjected to extracorporeal treatment and then returned to the patient in such processes as dialysis procedures, blood filtration, or blood bypass during surgery.
  • the term also includes blood products which are stored extracorporeally for eventual administration to a patient, as well as blood collected from a patient to be used for various assays. Such products include whole blood, plasma, platelets, or any blood fraction in which inhibition of either coagulation or platelet activation is desired.
  • the pharmaceutically acceptable compositions and methods for inhibiting platelet activation in a treated patient or in extracorporeal blood may further comprise standard dosage amounts of other platelet inhibitors such as metal chelaters, prostaglandins, other small platelet inhibitory peptides, cyclooxy- genase inhibitors, small non-peptide platelet inhibitors, inhibitors of platelet surface components, antibodies to platelet surface components, hemato- poietic factors, analogs of any of the above compounds or combinations thereof.
  • platelet inhibitors such as metal chelaters, prostaglandins, other small platelet inhibitory peptides, cyclooxy- genase inhibitors, small non-peptide platelet inhibitors, inhibitors of platelet surface components, antibodies to platelet surface components, hemato- poietic factors, analogs of any of the above compounds or combinations thereof.
  • the most preferred additional platelet inhibitors are aspirin, ticlopidine, dipyrida- mole, sulphinpyrazone, prostaglandin E, or known analogs thereof, stable prostacyclin derivatives, monoclonal antibodies against glycoprotein lib/Ilia or natural inhibitors of glycoprotein lib/Ilia, monoclonal antibodies against glycoprotein lb, natural inhibitors of glycoprotein lb, erythropoetin, arg-gly-asp- containing peptides and derivatives of arg-gly-asp- containing peptides.
  • This invention also relates to methods and compositions for inhibiting thrombin-induced endothelial cell activation.
  • Such inhibiton 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.
  • diseases include, but are not limited to, adult respiratory distress syndrome, septic shock, septicemia and reperfusion damage.
  • compositions and combinations used in the methods of this invention may be formulated using conventional methods to prepare pharmaceutically useful compositions.
  • Such composi ⁇ tions and combinations preferably include at least one pharmaceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences (E. W. Martin) .
  • the compositions and combinations preferably include a pharmaceutically acceptable buffer, prefer ⁇ ably phosphate buffered saline, together with a pharma ⁇ ceutically acceptable compound for adjusting isotonic pressure, such as sodium chloride, mannitol or sor- bitol.
  • compositions and combinations of the present invention are dependent on a variety of factors, such as the specific composition employed, the object of the treatment, i.e., therapy or prophylaxis, the nature of the thrombotic disease to be treated, antiplatelet or anticoagulant activity and the judgment of the treating physician.
  • Various dosage forms may be employed to administer the compositions and combinations of this invention. These include, but are not limited to, parenteral administration, oral administration 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 paravertebrally and periarticularly — subcutaneously, intracutan- eously, intra-articularly, intrasynovially, intra- thecally, intra-lesionally, periostally or by oral or topical routes.
  • Such compositions and combinations are preferably adapted for oral and parenteral administration, but, most preferably, are formulated for parenteral administration.
  • compositions and combinations 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 peptide derivative compo ⁇ nent of the pharmaceutically acceptable composition or combination may be either -uspended or dissolved in the vehicle, depending on the nature of the vehicle and the nature of the component.
  • Parenteral compositions and combinations may be prepared by dissolving the peptide derivative 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 may be prepared in substantially the same manner, except that the active component is 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 may advantageously be included in the composition to facilitate uniform distribution of the components.
  • Tablets and capsules for oral administration contain conventional excipients such as binding agents, fillers, diluents, tab eting agents, lubri- - 20 -
  • 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 derivatives, such as sodium starch glycolate.
  • Suitable lubricants include, for example, magnesium stearate.
  • Suitable wetting agents include sodium lauryl sulfate.
  • Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or another suitable vehicle before use.
  • Such liquid preparations may contain conventional additives. These include suspending agents; such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxy- methylcellulose, aluminum stearate gel or hydrogenated edible fats; emulsifying agents which include lecithin, sorbitan monooleate or acacia; non-aqueous vehicles, such as almond oil, fractionated coconut oil, and oily esters; and preservatives, such as methyl or propyl p- hydroxybenzoate or sorbic acid.
  • suspending agents such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxy- methylcellulose, aluminum stearate gel or hydrogenated edible fats
  • emulsifying agents which include lecithin, sorbitan monooleate or acacia
  • non-aqueous vehicles such as almond oil, fractionated coconut oil, and oily esters
  • preservatives such as methyl or propy
  • a pharmaceutically effective daily dose of the peptide derivatives of this inveniton is between about 0.1 mg/kg body weight of the patient to be treated ("body weight") and about 100 mg/kg body weight. More preferably, the daily dose of the peptide derivative is between about 0.5 mg/kg body weight to about 10 mg/kg body weight.
  • a pharmaceutically effective dosage of either of those compounds is less than that required to achieve a desired anticoagulant result when either is administered as a monotherapy.
  • the dosage of heparin or low molecular weight heparin is less than about 5,000 units/patient/day.
  • an pharmaceutically effective amount of the peptide derivatives of this invention is between about 0.1 mg/600 ml extracorporeal blood and about 100 mg/600 ml extracorporeal blood. More preferably, the dose is between about 0.5 mg/600 ml extracorporeal blood and about 10 mg/600 ml extracorporeal blood.
  • a pharmaceutically effective dose of the peptide derivatives of this inveniton is between about 0.0001 mg/kg body weight of the patient to be treated ("body weight") and about 100 mg/kg body weight. More preferably, the dose of the peptide derivative is between about 0.001 mg/kg body weight to about 0.05 mg/kg body weight. These lower dosages allow inhibition of platelet or endothelial cell activation without concurrent anticoagulant effects.
  • an pharmaceutically effective amount of the peptide derivatives of this invention is between about 0.0001 mg/600 ml extracorporeal blood and about 100 mg/600 ml extracorporeal blood. More preferably, the dose is between about 0.001 mg/600 ml extracorporeal blood and about 0.05 mg/600 ml extracorporeal blood.
  • compositions and combinations of this invention may additionally contain standard dosages of other components which are effective in fibrinolytic therapy.
  • tissue plasminogen activator purified from natural sources, recombinant tissue plasminogen activator, anisolated streptokinase plasminogen activator complex (ASPAC) , animal salivary gland plasminogen activator, urokinase, streptokinase and known, biologically active derivatives of any of the above.
  • ASPAC anisolated streptokinase plasminogen activator complex
  • animal salivary gland plasminogen activator animal salivary gland plasminogen activator
  • urokinase streptokinase
  • streptokinase and known, biologically active derivatives of any of the above.
  • tissue plasminogen activator purified from natural sources
  • ASPAC anisolated streptokinase plasminogen activator complex
  • animal salivary gland plasminogen activator animal salivary gland plasminogen activator
  • urokinase streptokinase
  • streptokinase and
  • compositions either co- valent or non-covalent, comprising the above-described peptide derivatives coupled to pharmaceutically acceptable polymers which increase their biological half-life.
  • a peptide derivative may be coupled to an activated derivative of polyethylene- glycol (PEG) using conventional techniques.
  • PEG polyethylene- glycol
  • a PEG N-succinimidyl succinate is attached to an ⁇ -amino moiety of the peptide derivative.
  • SS-PEG PEG N-succinimidyl succinate reagent
  • the peptide derivatives may be used in compositions and methods for in vivo thrombus imaging in a patient.
  • the peptide derivatives used in these compositions and methods are labeled with a radioisotope.
  • the choice of radioisotope is based upon a number of well-known factors, for example, toxicity, biological half-life and detectability.
  • Preferred radioisotopes include, but are not limited to, '"I, '"I and "'In. Techniques for labeling peptides and peptide derviatives are well known in the art. Most preferably, the radioisotope is '"I and labeling is achieved using '"I-Bolton Hunter Reagent.
  • the labeled peptide derivative 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 yields images of platelet- bound thrombin and eizothrombin.
  • one or a combination of any of the peptide derivatives 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 chem cal cross-linking or physical adsorption of the peptide derivative-containing compositions to the surfaces of the devices.
  • This invention also relates to compositions containing the peptide derivatives of this invention and methods of using such compositions for the treatment of tumor metastases.
  • the efficacy of the peptide derivatives 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 Malignant Tissue", Biochemistry. 24, pp. 5558-67 (1985); S. G. Gordon et al., "Cysteine Proteinase Procoagulant from Amnion-Chorion", Blood, 66, pp. 1261-65 (1985); and A.
  • peptide derivatives of the present invention serve as effective anti-metastatic tumor agents.
  • metastatic tumors which may be treated by the peptide derivatives 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.
  • N-Acetyl-(3.5-DiiodoTyrAhirudin., .. N-Acetyl-3,5-DiiodoTyr 63 hirudin 53 . 64 (herein referred to as "N-Ac-(diiodoTyr) ⁇ 3 hirudin S3 _ 64 "; in which the subscript numbers represent the corresponding amino acid position in the native hirudin molecule) has the amino acid formula: N-Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile- Pro-Glu-Glu-(3,5-diiodoTyr)-Leu-OH.
  • Boc-L- diiodotyrosine Boc-L-glutamic acid (r-benzyl ester)
  • Boc-L-proline Boc-L-isoleucine
  • Boc-L-phenylalanine Boc- ⁇ -aspartic acid ( ⁇ -benzyl ester)
  • Boc-glycine Boc-L-N-acetyl-asparagine.
  • the column was equilibrated in 0.1% TFA in water and developed with a linear gradient of increasing acetonitrile concentration from 0 to 80 in 0.1% TFA over 45 minutes at a flow-rate of 4.0 ml/mm.
  • the effluent stream was monitored for absorbance at 229 nm and fractions were collected manually.
  • 64 (herein referred to as "N-Ac-SCMCys 63 hirudin 53 _ 6 ”) has the amino acid formula: N-Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile- Pro-Glu-Glu-SCMCys-Leu-OH.
  • N-Ac-SCMCys 63 hirudin 53 _ 6 has the amino acid formula: N-Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile- Pro-Glu-Glu-SCMCys-Leu-OH.
  • Boc-L-cysteine for Boc-L- diiodotyrosine at the second step of synthesis.
  • the peptide was then deprotected and purified by reverse phase HPLC in an identical manner as for the peptide in Example 1. Fractions containing the peptide were evaporated to dryness. We then dissoved 5 ⁇ g of HPLC-purified N- acetyl-Cys 63 hirudin 53 _ M in 1.0 ⁇ l of 0.1 M Tris-HCl, pH 7.5 and immediately reacted the peptide with a 10- fold molar excess of iodoacetic acid, which had previously been dissolved in 1.4 N NaOH. The reaction proceeded for 5 hours at 25°C and was then stopped by the addition of acetic acid to a final concentration of 5%.
  • N-acetyl-S-carboxymethylcysteinyl ⁇ 3 - hirudin 53 . 6 was purified by reverse phase HPLC on an Aquap ⁇ re C. RP-300 column (0.46 x 10 cm) which had been equilibrated in solvent A (0.1% TFA in water). The column was developed with an increasing concentration of solvent B (0.085% TFA/70% acetonitrile) from 0 to 50% over 45 minutes at a flow rate of 1.0 ml/min. The effluent stream was monitored at 214 nm for absorbance. N-acetyl-S-carboxymethyl-cysteinyl 63 hirudin 53 . 64 eluted after the unmodified N-acetyl-Cys 63 hirudin 53 . 6 .
  • N-Ac-SCECys 63 hirudin 53 . 64 has the amino acid formula: N-Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile- Pro-Glu-Glu-SCECys-Leu-OH.
  • N-acetyl-Cys 63 hirudin 53 . 6 prepared as in Example 2, in 100 ⁇ l of dimethylformamide (DMF) and then reacted the peptide with a 50-fold molar excess of 3-iodopropionic acid at 37°C for 24 hours.
  • N-Acetyl-Thionitrobenzoic Acid Cysteinyl confronthirudin S3 . 6 (herein referred to as "N-Ac- TNBCys H hirudin 53 _ #4 ") has the amino acid formula: N-Ac- Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-TNBCys-Leu-OH.
  • N-acetyl-3-nitroTyr 63 hirudin 53 _ M has the amino acid formula: N-Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro- Glu-Glu-(3-nitroTyr)-Leu-OH.
  • 64 has the amino acid formula: N-Ac- Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-(5-nitroTyr)- Leu-OH.
  • This example illustrates the enhanced inhibitory activity of the peptides of this invention on activated partial thromboplastin times (APTT) , as compared to control peptides N-acetyl-hirudin 53 . 64 and N- acetyl-Cys 63 hirudin makeup. ⁇ 4 .
  • Anticoagulant activity was determined by assaying APTT of pooled, normal human plasma (George King Biomedical, Inc., Overland Park, Kansas) (diluted 4:1, v/v, plasma:water) with a Coag-A- Mate 2001 instrument (General Diagnostics, Inc. , Morris Plains, New Jersey) .
  • diluted plasma was mixed with varying amounts of either N-acetyl-TNB- Cys 63 hirudin administrat_ 6 , N-acetyl-(diiodoTyr) 63 hirudin 53 . 64 , N- acetyl-SCMCys 63 hirudin 53 . ⁇ , N-acetyl-SCECys 63 hirudin household. M , N- acetyl-hirudin 53 . M or N-acetyl-Cys 63 hirudin 53 . 6 (0.2 -293.3 ⁇ g/ml final peptide concentration).
  • M are weak anticoagulants in comparison to the peptides of this invention.
  • the s-carboxymethylation or S- carboxyethylation of the Cys ⁇ 3 residue of N-acetyl- Cys 63 hirudin 53 . 64 resulted in an approximately ten-fold increase in anticoagulant potency.
  • Thionitrobenzoation of the cysteine residue of N-acetyl-Cys 63 hirudin 53 . 64 resulted in a 2- to 3-fold increase in anticoagulant activity.
  • diiodination of the tyrosine residue of N-acetyl-hirudin 53 are weak anticoagulants in comparison to the peptides of this invention.
  • the s-carboxymethylation or S- carboxyethylation of the Cys ⁇ 3 residue of N-acetyl- Cys 63 hirudin 53 . 64 resulted in an approximately
  • N-acetyl- (diiodoTyr) 63 hirudin 53 . ⁇ 4 On the inhibition of platelet aggregation caused by thrombin.
  • varying amounts of N-acetyl-(diiodoTyr) 63 hirudin 53 . 64 (0 - 3.3 ⁇ g/ml final concentration), contained in 0.05 ml water, were added to 0.4 ml of prewarmed (37°C) platelet-rich plasma. The peptide/platelet mixture was incubated for 1 minute at 37°C.
  • We then added 0.05 ml of human ⁇ -thrombin (a gift of * Dr. J.
  • N-acetyl-(diiodoTyr) 63 - hirudin 53 ⁇ blocked thrombin-induced platelet aggregation in vitro at low concentrations in a dose-dependent manner with an IC 50 of approximately 2.4 ⁇ g/ml (the concentration of peptide required to reduce platelet aggregation to 50% of maximum) .
  • IC 50 approximately 2.4 ⁇ g/ml (the concentration of peptide required to reduce platelet aggregation to 50% of maximum) .
  • N-acetyl- (diiodoTyr) 63 hirudin 53 _ M was approximately 15 times as potent as hirudin 53 . 64 in inhibiting platelet aggregation (data not shown) .
  • compositions according to this invention which comprise other peptides described herein which similarly inhibit platelet aggregation. And, those compositions which comprise peptides containing a derivatized cysteine or tyrosine at the A, position exhibit significantly greater inhibitory activity than corresponding peptides which lack a derivatized amino acid at that position.
  • N-acetyl-(diiodoTyr) 63 hirudin 53 . 64 on thrombin-induced platelet secretion may be analyzed by assaying inhibition of serotonin release and throm- boxane A 2 generation.
  • the serotonin release assay is performed as follows. Approximately 20 ml of platelet- rich plasma, prepared as described in Example 7 , is loaded with u C-serotonin by incubating the plasma with 27 nCi/ml of 5-[2- u C]serotonin binoxalate (60 mCi/mmole; DuPont-NewEngland Nuclear, Boston, Massachusetts) at 37°C for 30 minutes. Under these conditions, platelets incorporate >90% of the added serotonin, resulting in a specific activity of approx ⁇ imately 10,000 counts/minute/ml of platelet-rich plasma.
  • Platelet-rich plasma containing u C-serotonin loaded platelets (0.4 ml) is mixed in an aggregometer with varying concentrations (0 - 11 ⁇ g/ml total assay volume) of N-acetyl-(diiodoTyr) complicathirudin 53 .
  • M contained in 0.05 ml saline for 1 minute at 37°C.
  • Human ⁇ -thrombin (0.05 ml) is then added to a final concentration of either 0.25 or 0.5 U/ml of the total assay volume and the mixture is incubated at 37°C for 4 more minutes.
  • the thrombin-induced serotonin release reaction is stopped and reuptake by the platelets is halted by the addition of a 1/lOth volume of an ice-cold cocktail containing 3.3% EDTA, 10 mM theophylline, 1 ⁇ g/ml prostaglandin E,, and 500 ⁇ M imipramine (hereinafter "ETPI") .
  • the first three components are commonly used to prevent the platelet release reaction [J. A. Jakubowski and N. G. Ardlie, "Further Observations on the Effects of Dietary Fatty Acid Composition on Platelet Reactivity and Blood
  • Imipra ine is a serotonin receptor agonist which prevents reuptake during sample handling.
  • EPTI inhibits serotonin uptake and release by >95%.
  • the platelets are removed by centrifugation at 12,000 x g for 2 minutes in a Sorval rotor. Serotonin release is assayed by measuring U C- radioactivity in the supernatant using liquid scintillation counting (Tri-Carb 1500; Packard Instruments). N-acetyl-(diiodoTyr) 63 hirudin 53 . 64 inhibits serotonin release in a dose-dependent manner, similar to that observed for inhibition of platelet aggregation.
  • Platelets contained in platelet-rich plasma are stimulated with thrombin in the presence of increasing concentrations of N-acetyl-(diiodoTyr) 63 hirudin 53 .
  • Peptide dissolved in 0.5 ml saline, is added to 0.4 ml of platelet-rich plasma and the mixture is incubated for 1 minute at 37°C.
  • Thrombin contained in 0.05 ml saline, is then added to a final concentration of either 0.5 or 0.25 U/ml. The mixture is then incubated for 4 minutes at 37°C. Platelet thromboxane A 2 generation is quenched by the addition of ice-cold indomethacin to a final concentration of 10 ⁇ M, followed by centrifugation at 12,000 x g for 2 minutes.
  • Thromboxane A 2 released into the supernatant is assayed by a radioimmunoassay that detects thromboxane B 2 , a stable hydrolytic product and indicator of thromboxane A 2 [J. A. Jakubowski et al., "Cumulative Antiplatelet Effect of Low-Dose Enteric Coated Aspirin", Br. J. Haematol.. 60, pp. 635-42 (1985)].
  • compositions -ccording to this invention which comprise other peptides similarly inhibit platelet release reactions.
  • Those compositions which comprise peptides containing a derivatized cysteine or tyrosine at the A, position exhibit a significantly greater inhibitory activity than corresponding peptides which lack a derivatized amino acid at that position.
  • N-acetyl-(diiodoTyr) 63 - hirudin 53 S4 in the treatment of patients suffering from heparin-induced thrombocytopenia is illustrated by various in vitro assays.
  • the platelet-rich plasma used in these assays is obtained from patients after cessation of heparin therapy and recovery from heparin-induced thrombocytopenia.
  • Platelet-rich plasma obtained from " various patients and prepared as in Example 7, is incubated with varying concentrations of porcine lung sodium heparin (0.05 - 0.5 U/ml; Elkins-Sinn, Cherry Hill, New Jersey) or N-acetyl-(diiodoTyr * ) complicathirudin 53 .
  • M 0.8 - 55 ⁇ g/ml).
  • Platelet aggregation is monitored turbido- metrically, as in Example 7. Heparin induces aggregation of platelets in the plasma of patients who have fully recovered from heparin-induced thrombocytopenia. N-acetyl-(diiodoTyr) 63 hirudin 53 . 6 does not induce platelet aggregation in similar plasma. The extent of thromboxane A 2 generation from similar platelets incubated with either heparin or N- acetyl-(diiodoTyr) 63 hirudin !3-64 may also be examined. Platelet-rich plasma from patients who have recovered from heparin-induced thrombocytopenia is incubated with either heparin or N-acetyl-(diiodoTyr) disregardhirudin 53 . M
  • Thromboxane A 2 secretion is stopped by the addition of indomethacin, followed by centrifugation and determined by assaying for throm ⁇ boxane B 2 , as in Example 8. Heparin causes release of thromboxane A 2 from the platelets of patients with heparin-induced thrombocytopenia, while N-acetyl- (diiodoiyr) 63 hirudin 53 . ⁇ does not.
  • compositions of the present invention which comprise N-acetyl-(diiodoTyr) 63 hirudin understand_ 64 anticoagulate plasma without causing platelet activation in patients afflicted with this autoimmune disorder.
  • compositions according to this invention which comprise other peptides described herein are similarly effective in increasing APTT, without causing platelet activation in patients who are suffering or who have suffered form heparin-induced thrombocyto ⁇ penia. Therefore, in these patients, the compositions of the present invention constitute a safe and effective alternative to heparin as anticoagulant agents and agents which inhibit platelet aggregation.
  • the anti-metastatic activity of the peptides of this invention preferably, N-acetyl-(diiodoTyr) 63 - hirudin 53 .
  • 64 is 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 N-acetyl-(diiodoTyr) ⁇ 3 hirudin 53 .
  • Example 2 as prepared according to Example 1, 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 peptide is measured as percent reduction in tumor colonies compared to a placebo-treated control.
  • N- acetyl-(diiodoTyr) practichirudin 53 _ ⁇ 4 may be modified by covalent attachment of ,W I, ,25 I- or "'In-containing chemical groups. Specifically, N-acetyl-(diiodoTyr) 63 - hirudin 53 . 6 (as prepared in Example 1) is reacted with ,23 I-Bolton-Hunter Reagent (New England Nuclear, Boston, MA.) in 0.1 M sodium borate, pH 9.0.
  • the 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 ,2 I- labelled peptide is then tested in APTT assays to monitor any loss in antithrombin activity.
  • Ex vivo imaging of experimental thrombi is performed essentially as described by T. M. Palabrica et al., "Thrombus Imaging in a Primate Model with Antibodies Specific For an External Membrane Protein of Activated Platelets", Proc. Natl. Acad. Sci. USA. 86, pp. 1036-40 (1989) .
  • 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.
  • '"i-labelled N- acetyl-(diiodoTyr) 63 hirudin S3 _ 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 100 Gamma Camera with a PDP-11/34 computer.
  • the kinetics of 123 l-peptide uptake by the graft and blood pool are derived from the radionuclide images thus obtained.
  • N- acetyl-(diiodoTyr) 63 hirudin 53 _ M as well as the c-her peptides of this invention, binds to thrombin with high specificity, the use of radiolabelled peptides of this invention allows precise ex vivo images of experimental thrombi.
  • the small size of the peptides of this invention in contrast to native hirudin or antibodies to thrombin, provides the potential that the radio- labelled-peptide will yield images of platelet-bound thrombin and meizothrombin, as well as thrombin con ⁇ tained in the fibrin clot.
  • N-acetyl-(diiodoTyr) 63 hirudin 53-6 to inhibit thrombin-induced synthesis of platelet activating factor (PAF) is assayed using cultured human umbilical vein endothelial cells (HUVEC) .
  • HUVECs are extracted from human umbilical cords by collagenase digestion according to established procedures [M. A. Gimbrone, Jr. , "Culture of Vascular
  • HUVECs are grown to confluence in a 96-well microtiter plate in the presence of [ 3 H]-acetate, ells cultured in this manner produce [ 3 H]-acetyl-PAF, which can be quantitated by extraction of HUVEC membrane phospholipids.
  • N-acetyl-(diiodoTyr) 63 hirudin 53 . 64 on thrombin-induced polymorp.ionuclear leukocyte (PMN) adhesion to HUVECs may be observed as follows. HUVECs are grown in MEM containing 1% fetal calf serum to confluency in 24-well cluster plates. The medium is removed and the cells are washed two times with fresh, serum--ree medium and incubated in the same medium for 10 - 30 minutes at 37°C to remove serum products. One ml of PMNs (2.5 x lO ' /ial) , pre-equilibrated at 37°C, is then added to each well.
  • PMN polymorp.ionuclear leukocyte
  • the PMNs are allowed to settle onto the HUVEC monolayer for 2 minutes.
  • N- acetyl-(diiodoTyr) 63 hirudin 53 _ M (5 ⁇ g/ml) or saline is then added to each well, immediately followed by the addition of thrombin (0.1 or 1.0 U/ml).
  • the cells are incubated for 5 minutes at 37°C, washed twice and then examined by phase- contrast microscopy.
  • Adherent PMNs are counted directly.
  • PMN adherence is significantly decreased in samples containing N-acetyl- (diiodoTyr) 63 hirudin 53 . ⁇ 4 .
  • the other peptides of this invention exhibit a similar effect on PMN adherence.

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Abstract

L'invention concerne de nouveaux dérivés de peptides présentant une activité biologique, inhibant l'activité fibrinolytique mais pas l'activité amidolytique de la thrombine. Les dérivés peptidiques, présentant une homologie partielle avec la partie terminale carboxy de l'hirudine, se caractérisent par un résidu de tyrosine ou de cystéine modifié au niveau d'un site prédéterminé situé dans la molécule. L'invention concerne également des compositions et des procédés employant lesdits dérivés peptidiques à des fins thérapeutiques, profilactiques ou diagnostiques. L'invention concerne également des compositions inhibant les cellules endothéliales, ainsi que des procédés caractérisés par les dérivés peptidiques ci-dessus décrits ou des dérivés peptidiques similaires ne contenant pas de résidu de cystéine ou de tyrosine modifié à un endroit correspondant dans la molécule. Ces procédés et compositions sont avantageusement utiles pour diminuer ou prévenir l'aggrégation plaquettaire et l'activation plaquettaire chez un patient ou dans du sang extracorporel, notamment chez des patients ayant une thrombocytopénie induite par l'héparine dans leurs antécédents.
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US6060451A (en) * 1990-06-15 2000-05-09 The National Research Council Of Canada Thrombin inhibitors based on the amino acid sequence of hirudin
DE4103649A1 (de) * 1991-02-07 1992-08-13 Basf Ag Neue antikoagulatorisch wirksame peptide
ATE142642T1 (de) * 1991-03-05 1996-09-15 Japan Energy Corp Hirudinanalog oder dessen salz, dessen herstellung und das als aktiven bestandteil enthaltende antikoagulanz
AU659432B2 (en) * 1991-03-08 1995-05-18 Novartis Ag A method for the inhibition or prevention of tumor cell metastasis with hirudin

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