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WO1991011458A1 - PEPTIDES CYCLIQUES CONTENANT UNE SEQUENCE Arg-Gly-Asp FLANQUEE DE PROLINE - Google Patents

PEPTIDES CYCLIQUES CONTENANT UNE SEQUENCE Arg-Gly-Asp FLANQUEE DE PROLINE Download PDF

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Publication number
WO1991011458A1
WO1991011458A1 PCT/US1991/000564 US9100564W WO9111458A1 WO 1991011458 A1 WO1991011458 A1 WO 1991011458A1 US 9100564 W US9100564 W US 9100564W WO 9111458 A1 WO9111458 A1 WO 9111458A1
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Prior art keywords
cys
arg
asp
pro
gly
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Inventor
Peter L. Barker
John P. Burnier
Robert A. Lazarus
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Genentech Inc
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Genentech Inc
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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to inhibitors of platelet aggregation, and more specifically to cyclic peptides capable of preventing binding of fibrinogen or other ligands to the platelet giycoprotein llbllla receptor (GP llbllla)-
  • the invention also relates to therapeutic applications of the cyclic peptide inhibitors in diseases for which blocking of platelet aggregation and intracellular adhesion is mediated by GP llbllla- .
  • Platelets are particles found in mammalian whole blood which participate in the process of thrombus formation * and blood coagulation.
  • a membrane bound giycoprotein commonly known as GP ll llla (Phillips et al., (1988) Blood, 71, 831-843), is present in platelets and can, under certain circumstances? be exposed on the ⁇ exterior surface of platelets.
  • Giycoprotein llbllla is a non-c ⁇ vale ⁇ t, calcium ion dependent heterodimer complex comprised of alpha and beta subunits (Jennings, et al., J. Biol. Chem ⁇ (1982) 257, 10458).
  • This complex is known to contribute to normal platelet function through Interactions with proteins containing the sequence Arg-Gly-Asp such as fibfth ⁇ gen.
  • the interaction of GP llbllla with fibrinogen is stimulated by certain factors released or .exposed when a blood vessel is injured.
  • Multiple factors including a variety of physiological stimuli and soluble mediators, initiate platelet activation via several pathways (Rink arid Hallam (1984) Trends in Biochemical Sciences, 715-719; Kroll and Schafer (1989) Bl ⁇ od, 74, 1181 -1195; and Colman and Walsh, (1987) Hemostasis and Thrombosis: Basic Principles and Clinical Practice, pp. 594-605, J.W. Lippincott, Philadelphia).
  • hypothrombotic state is mediated by platelet aggregation and adhesion
  • inhibition represents a good target for therapeutic intervention (Stein et al. (1989) J. Am. Ceil. Cardioi, 14, 813).
  • These disorders are a major cause of medical morbidity, leading to infarction, stroke and phlebitis and of mortality from stroke and pulmonary and cardiac emb ⁇ lir
  • Patients with atherosclerosis are predisposed to arterial thrortiboembolic phenomena for a variety of reasons.
  • Atherosclerotic plaques form niduses for platelet plugs and thrombii that lead to vascular narrowing and occlusion, resulting in myocardial and cerebral iscrremic disease. This may happen spontaneously or following procedures ⁇ ach as angioplasty or endarterectomy.
  • Thrombii that break off and are released into the circulation may cause infarction of other organs, especially the brain, extremities, heart and kidneys.
  • platelets may also play a role in venous thrombosis.
  • a large percentage of such patients have no antecedent risk factors and develop venous thrombophlebitis and subsequent pulmonary emboli without a known cause.
  • Other patients who form venous thrombi have underlying diseases that are known to predispose them to these syndromes. Some of these patients may have genetic or aquired deficiencies of factors that normally prevent hypercoagulability, such as antithrombin-3. Others have mechanical obstructions to venous flow, such as tumor masses, that may lead to low flow states and thrombosis. Patients with malignancy often have a high incidence of thrombotic phenomena for unclear reasons. Antithrombotic therapy in these situations employing currently available agents may be dangerous and is often ineffective.
  • a large category of patients including those with atherosclerosis, coronary artery disease, artificial heart valves, cancer, and a history of stroke, phlebitis, or pulmonary emboli, are candidates for limited or chronic antithrombotic therapy.
  • the number of available therapeutic agents is limited and these, for the most part, act by inhibiting or reducing levels of circulating clotting factors. These agents are frequently not effective against the patient's underlying hematologic problem, which often concerns an increased propensity for platelet aggregation and adhesion. They also cause the patient to be susceptible to abnormal bleeding.
  • Available antiplatelet agents such as aspirin, inhibit only part of the platelet activation process and are therefore often inadequate for therapy.
  • fibronectin contains the Arg-Gly-Asp sequence of amino acids.
  • Large polypeptide fragments of fibronectin have been shown to have activity for cell attachment to various surfaces which has been disclosed in US Patents 4,517,686; 4,589,881 ; 3 and 4,661 ,111. These large polypeptides contain the amino acid sequence Arg-Gly-Asp-Ser in the interior portion of the polypeptide chain.
  • Short peptides derived from the large polypeptides were also found to promote cell attachment to vaHous substrates when bound on the substrate. Alternatively, the same short peptides were found to inhibit cell attachment to the same substrates when dissolved or suspended in the medium surrounding the substrate. This activity has been disclosed in US Patents 4,578,079 and 4,614,517. The short peptides were defined as
  • Q is hydrogen or an amino acid ⁇ AAI is serine, threonine, or cysteine; and B is hydroxy or an amino acid.
  • Venoms from various pit vipers have been found ' to contain proteins that contain the Arg-Gly-Asp sequence and inhibit platelet aggregation. These venoms have been described and characterized by Huang et al., J. Bibl. Che ., (19 * 87) 262, 16157-16136; Huang etal. (1989) Biochemistry, 28, 661-666; Gan et aL (1988) J. Biol. Chem., 263, 19827-19832; Chao et al. (1989) Proc. Natl. Acad. Sci. USA, 86, 8050-8054; Shebuski et al., J. Biol.
  • X is hydrogen or at least one amino acid and Y is hydroxyl or at least one amino acid.
  • HN HN CO where Pen is penicillamine. No information is provided by these authors on the effectiveness of these small cyclic peptides in inhibiting platelet aggregation.
  • Ri and R4 are from 0 to 4 amino acids; R3 is from 1 to 4 amino acids; R2 is -CH2CO- or from 1 to 4 amino acids; Xaas may be Met, Phe, nLeu, He, Asp, Lys, Arg and Gin; and Z is a linking group, either disulfide, thioether or amide, but preferably disulfide.
  • each of Xaa2, Xaa3, Xaa4, Xaaio, aan, Xaai2 are amino acids or are absent, but preferably at least Xaa2, Xaa3 and Xaa4 are present.
  • R5 is -CH2CO- or Cys;
  • Z is thioether or disulfide;
  • Ri, R , and Xaas are as defined above.
  • Xaa2, Xaa3 and Xaa4 may be any amino acid but preferably are Arg, He and Pro, respectively.
  • Xaas is preferably Met, Phe or nLeu;
  • Xaaio when present is preferably Ala;
  • Xaan when present is preferably Ala or Asp.
  • Representative preferred compounds according to this invention are conveniently selected from the following list: Cys 1 -Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys 13 ;
  • preferred compounds contain from 7 to about 16 amino acids and preferably from 10lo 13 amino acids in a ring bridged through the disulfide of cystine, and containing Pro residues flanking of the Arg-Gly-Asp sequence.
  • these compounds may be effectively employed in a pharmaceutical composition containing a pharmaceutically acceptable excipient for reducing platelet aggregation in a mammal.
  • This pharmaceutical composition is especially useful in treating a mammal having an increased propensity for thrombus formation, such as those which are post angioplasty, and may be used in combination with an anticoagulant or th ⁇ pmbolytic agent.
  • the small cyc ⁇ c peptides represente'd by Formulae I - III above are composed of L amino acids unless otherwise specified. Standard abbreviations are used for the amino acids as provided below: . * "
  • Gly glycine lie isoleucine
  • the abbreviation Xaa represents an unspecified naturally ocurring L-amino acids.
  • the small cyclic peptides of this invention are useful as inhibitors of platelet aggregation. Without intending to be limited to any particular theory or mechanism of action, it is believed these compounds exhibit this effect by acting as competitive inhibitors of the platelet GP llbllla receptor. By competing for the GP llbllla receptor, these small cyclic peptides prevent the binding, in a concentration dependent manner, of indigenous binding proteins such as fibrinogen, fibronectin, vitronectin, von Willebrands factor, as well as other Arg-Gly-Asp containing proteins. Thus, these compounds act as antagonists of the final common pathway of platelet aggregation and therefore are useful as antithrombotics. Mammals exposed to medical procedures such as angioplasty and thrombolytic therapy are particularly susceptable to thrombus formation. By preventing or modulating formation of platelet plugs, emboli, and thrombii, these compounds possess useful therapeutic properties.
  • the cyclic peptides of this invention are also useful in either inhibiting or promoting cell adhesion.
  • Members of the integrin class of adhesion receptors are capable of recognizing the Arg-Gly-Asp sequence and therefore the instant cyclic peptides can effectively block cell attachment or adhesion.
  • the compounds of the present invention are preferably used to inhibit thrombus formation following angioplasty, or may be used in combination with thrombolytic agents such as tissue plasminogen activator and its derivatives (US patents 4,752,603; 4,766,075; 4,777,043; EP 199,574; EP 0238,304; EP 228,862; EP 297,860; PCT WO89/04368; PCT WO89/00197), streptokinase and its derivatives, or urokinase and its derivatives to prevent arterial reocclusion following thrombolytic therapy.
  • tissue plasminogen activator and its derivatives US patents 4,752,603; 4,766,075; 4,777,043; EP 199,574; EP 0238,304; EP 228,862; EP 297,860; PCT WO89/04368; PCT WO89/00197
  • streptokinase and its derivatives or urokinase and its derivative
  • Mammals exposed to renal dialysis, blood oxygenation, cardiac catheterization and similar medical procedures as well as mammals fitted with certain prosthetic devices are also susceptible to thromboembolic disorders. Physiologic conditions, with or without known cause may also lead to thromboembolic disorders.
  • the compounds described herein are useful in treating thromboembolic disorders in mammals.
  • the compounds described herein may also be used as adjuncts to anticoagulant therapy, for example in combination with aspirin, heparin or warfarin and other anticoagulant agents.
  • the application of the compounds described herein for these and related disorders will be apparent to those skilled in the art. Preparation of the Peptides
  • the small cyclic peptides represented by Formulae I - III may be prepared by known methods, either recombinant or chemical.
  • an oligonucle ⁇ tide encoding the peptide is synthesized using standard phosphoramidate, phosphotriester or phosphonate methods (see e.g., Adams et al. (1983) J. Amer. Chem. Soc, 105, 661 ; Froehler et al. (1983) Tetrahedron Lett., 24, 3171 ; German Offenlegungsshrift 2644432; Froehler *t al., European Pub. No.219342, published 22 April 1987).
  • a second oligonucleotide complementary to the first oligonucleotide sequence is also synthesized either chemically or enzymajtcally and the two oligonucleotides are hybridized under standard conditions to generate a double stranded DNA molecule.
  • hybridization procedures for oligonucleotides may be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Coldspring Harbor Laboratory Press, New York, 1989.
  • the oligonucleotide may be directly cloned into a plasmid expression vector such as pNH ⁇ a (Stratagene, La Jolla, CA).
  • the vector js first linearized using a restriction endonuclease such as Hind III, and the ends are*f ⁇ lled in blunted with DNA polymerase I large fragment (Klenow).
  • the oligonucleotide is then blU it-ligated into the vector using the enzyme T4 ligase under appropriate conditions (Sagarameilar and Khorana (1972) J. Mol. Biol., 72, 427; Ferretti and Sagarameilar (1981) Nuc. Acids. Re ⁇ ., 9, 3695).
  • The'vector containing the oligonucleotide insert is transformed as described in Sambrook, supra, into a particular strain of .
  • -_2U host cells engineered to 'accept' the foreign plasmid, and to produce the polypeptide encoded by the oligonucleotide.
  • Exemplary host cell strains appropriate for plasmid pNH ⁇ a are E. coli D1210HP and D1210 (Stratagene, La Jolla, CA). Transfected host cells producing the polypeptide in significant quantity are lysed and the polypeptide purified employing appropriate purification procedures.
  • the preferred synthetic method " for the small cyclic peptides of the instant invention is by chemical synthesis.
  • the small linear peptides are first synthesized with a commercially available automated peptide synthesizer (e.g., Milligen- Biosearch 9500 automated peptide synthesizer) or by manual solid phase synthesis (see e.g., Merrifield, J. Am. Chem. Soc. (1964) 85, 2149 or Houghten, Proc. Natl. Acad. Sc/.(1985) 82, 5132).
  • a commercially available automated peptide synthesizer e.g., Milligen- Biosearch 9500 automated peptide synthesizer
  • solid phase synthesis see e.g., Merrifield, J. Am. Chem. Soc. (1964) 85, 2149 or Houghten, Proc. Natl. Acad. Sc/.(1985) 82, 5132.
  • solid phase synthesis peptide synthesis is initiated at the C-terminus of the
  • linear peptide is .synthesized, it is isolated and may be further purified using, for example, chromatographic methods such as high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the linear peptide then is cycled to compounds of Formulae I - III using standard chemical methods well known in the art. Which cyclization procedure is employed will depend on the type of bond to be formed. .Exemplary cyclization procedures are set forth below.
  • Cyclic Disulfide For example, the linear peptide may be cyclized by dissolution in water at pH 7.5 to 8 followed by exposure of the solution to air until disulfide formation is complete. Alternatively, chemical oxidants may be used such as I2 or potassium ferricyanide. Cyclic Thioethers
  • Cyclic thioethers are prepared by coupling bromoacetic acid to the amino terminus of the peptide on the resin and following removal of the peptide from the resin cyclization under dilute conditions is afforded by adjusting the pH to between 5 and 9.5. Cyclic Amides
  • Cyclic amides can be prepared by using side chain protecting groups which can be removed independently of the other side chain protecting groups as well as the link to the resin.
  • side chain protecting groups such groups as ally! esters or allyl carbamates would suffice and in the case of Boc chemistry the Fmoc or fluorenylmethyl esters would allow such differential protection.
  • Upon removal of these groups amide bond formation is achieved with standard coupling procedures. Then the cyclized peptide can be deprotected and removed from the resin.
  • equivalent peptides may be prepared by substituting amino acid analogues or other bifunctional ligands at the bridging positions (i.e., R2 and R3 of Formula I).
  • Exemplary equivalent bridging pairs would include Cys-pen, or pen-pen, where pen is penicillamine.
  • Alternative exemplary bridging pairs would include amino acids having free ⁇ amino and carboxyl groups or pairs containing free side chain amino and carboxyl groups.
  • Ri and R4 of Formula I can be blocking groups. Specifically, Ri may be acyl, either aryl or alkyl and R4 may be an amine or substituted amine.
  • the compounds described in this invention may be isolated as such or converted to salts of various inorganic and organic acids and bases.
  • Such salts are within the scope of this invention.
  • Examples of such salts include ammonium, metal salts like sodium, potassium, calcium and magnesium; salts with organic bases like dicyclohexylamine, N-methyi-D- glucamine and the like; and salts with amino acids like arginine or lysine.
  • Salts with inorganic and organic acids may be likewise prepared, for example, using hydrochloric- hydrobromic, sulfuric, phosphoric, trifluoroacetic, methanesulfonic, malic, maleic, fumaric and the like.
  • Non- toxic and physiologically compatible salts are particularly useful although other less desireable salts may have use in the processes of isolation and purification.
  • a number of methods are useful for the preparation of the salts described above and are known to those skilled in the art. For example, reaction of the free acid or free base form of a compound selected from Formulae I - III with one or more molar equivalents of the desired acid or base in a solvent or solvent mixture in which the salt is insoluble; or in a solvent like water after which the solvent is removed by evaporation, distillation or freeze drying.
  • the free acid or base form of the product may be passed over an ion exchange resin to form the desired salt or one salt form of the product may be converted to another using the same general process.
  • Microtiter plates were coated with fibrinogen (10 ⁇ g/ml) and then blocked with TACTS buffer containing 0.5% bovine serum albumin (BSA).
  • TACTS buffer contains 20mM Tris.HCI, pH 7.5, 0.02% sodium azide, 2 mM calcium chloride, 0.05% Tween 20, 150 mM sodium chloride.
  • PBS phosphate buffered saline
  • solubilized GP llbllla receptor 40 ⁇ g/ml
  • platelet aggregation assays may be performed in human platelet rich plasma r (PRP).
  • PRP human platelet rich plasma r
  • Fifty milliliters of whole human blood (9 parts) is drawn on 3.8% sodium citrate (1 part) from a donor who has not taken aspirin or related medicatio rns for at least two weeks. Thet ⁇ lood is centrifuged at 160 x g for 10 min at 22° C and then allowed to stand for 5 min after which the PRP is decahted.
  • Platelet poor plasma (PPP) is isolated from the remaining' blood fter centrifugation at 2000 x g for 25 min.
  • the platelet count Baker 9000 hematology analyzer of the PRP was diluted to ca.
  • Results of the receptor binding and platelet inhibition assays for many of the cyclic peptides synthesized as described above are set forth in Tables 1 and 2 of Example 25.
  • the first compound in Table 1 represents a linear peptide containing Arg-Gly-Asp which binds to platelet GP llbllla- This compound was selected as the reference or standard compound to which the other cyclic peptides provided in Tables 1 and 2 were compared.
  • the ratio value provided in the first column adjacent each peptide entry is the ratio of the binding affinity for that compound verses the standard.
  • a ratio value greater than 1 represents a compound which binds GP llbllla with a lower affinity than the reference compound, while a ratio value lower than 1 represents a compound that has a higher affinity than the reference compound.
  • compounds having a ratio greater than 1 were not considered sufficiently potent to be considered candidates for a platelet aggregation inhibition assay. Therefore, generally, preferred compounds of the instant invention have a ratio value less than 1.
  • IC50 values in the platelet aggregation assay are considered a more accurate measure of a compounds utility as an antithrombotic.
  • a compound should be at least about 5 times more potent in the platelet aggregation assay than the reference or standard compound.
  • preferred compounds of the instant invention have an IC50 in platelet aggregation inhibition assays of less than about 15 ⁇ M, while most preferred compounds have an IC50 of less than 5 ⁇ M, and very most preferred compounds have an IC50 of less than about 1 ⁇ M.
  • preferred compounds of the instant invention may be represented by Formulae I - III or are cyclic peptides containing from 7 to about 16 amino acids in a ring containing the sequence Arg-Gly-Asp flanked on both sides by at least one proline.
  • flanked means immediately adjacent or within about 3 residues of the Arg-Gly-Asp sequence.
  • Other apparently similar compounds described in the prior art as effective cell adhesion inhibitors have so far proved to be relatively impotent as platelet aggregation inhibitors.
  • an alternative preferred method for reducing platelet aggregation in a mammal is to administer to the mammal a pharmaceutically effective amount of a cyclic peptide having from about 7 to about 16 amino acids in the cycle, the cycle containing the sequence Arg-Gly-Asp and at least one proline, where the cyclic peptide exhibits an IC50 in a platelet aggregation inhibition assay of less than about 15 ⁇ M.
  • the compounds of this invention may be utilized in compositions such as tablets, capsules or elixers for oral administration, suppositories for rectal administration.'sterile-solutions or suspensions for injectable administration, and the like.
  • Animals in need of treatment using compounds of this invention can be administered dosages that will provide optimal efficacy.
  • the dose and method of adminstration will vary from animal to animal and be dependent upon such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
  • Dosage formulations of the cyclic polypeptides of the present invention are prepared for storage or administration by mixing the the cyclic polypeptide having the desired degree of purity with physiologically acceptable carriers, excipients, or stabilizers.
  • physiologically acceptable carriers excipients, or stabilizers.
  • Such materials are non-toxic to the recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino a'clds such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccha ⁇ des, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents
  • Dosage formulations of the cyclic polypeptides of the present invention to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes such as 0.2 micron membranes. Cyclic polypeptide formulations ordinarily will be stored in lyophilized form or as an aqueous solution.
  • the pH of the cyclic polypeptide preparations typically will be between 3 and 11 , more preferably from 5 to 9 and most preferably from 7 and 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of cyclic polypeptide salts. While the preferred route of administration is by hypodermic injection needle, other methods of administration are also anticipated such as suppositories, aerosols, oral dosage formulations and topical formulations such as ointments, drops and dermal patches.
  • Therapeutic cyclic polypeptide formulations generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by hypodermic injection needle.
  • Therapeutically effective dosages may be determined by either in vitro or in vivo methods.
  • One method of evaluating therapeutical ly effective dosages is illustrated in Table I where the cyclic polypeptide Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys, Cys 1 - Cys 13 disulfide was determined to have a 50% inhibitory concentration (IC50) of 18 nM using the method of Example 21 (a ratio of 0.5 to Gly-Arg-Gly-Asp-Val) when inhibiting fibrinogen binding to the GP llbllla platelet receptor.
  • IC50 50% inhibitory concentration
  • a therapeutically effective dosage range may be determined. For each particular cyclic polypeptide of the present invention, individual determinations may be made to determine the optimal dosage required. The range of therapeutically effective dosages will naturally be influenced by the route of administration. For injection by hypodermic needle it may be assumed the dosage is delivered into the body's fluids. For other routes of administration, the absorption efficiency must be individually determined for each cyclic polypeptide by methods well known in pharmacology.
  • the range of therapeutic dosages may range from about 0.001 nM to about 1.0 mM, more preferably from about 0.1 nM to about 100 ⁇ M, and most preferably from about 1.0 nM to about 50 ⁇ M.
  • a typical formulation of compounds of Formulae I - III as pharmaceutical compositions contain from about 0.5 to 500 mg of a compound or mixture of compounds as either the free acid or base form or as a pharmaceutically acceptable salt. These compounds or mixtures are then compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, or flavor, etc., as called for by accepted pharmaceutical practice. The amount of active ingredient in these compositions is such that a suitable dosage in the range indicated is obtained.
  • Typical adjuvants which may be incorporated into tablets, capsules and the like are a binder such as acacia, corn starch or gelatin; an excipient such as microcrystalline cellulose; a disintegrating agent like corn starch or alginic acid; a lubricant such as magnesium stearate; a sweetening agent such as sucrose or lactose; a flavoring agent such as peppermint, wintergreen or cherry.
  • a binder such as acacia, corn starch or gelatin
  • an excipient such as microcrystalline cellulose
  • a disintegrating agent like corn starch or alginic acid
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose or lactose
  • a flavoring agent such as peppermint, wintergreen or cherry.
  • a liquid carrier such as a fatty oil.
  • Other materials of various types may be used as coatings or as modifiers of the physical form of the dosage unit.
  • a syrup or elixer may contain the active compound, a sweetener such as sucrose, preservatives like propyl paraben, a coloring agent and a flavoring agent such as cherry.
  • a sweetener such as sucrose
  • preservatives like propyl paraben a coloring agent
  • a flavoring agent such as cherry.
  • Sterile compositions for injection can be formulated according to conventional pharmaceutical practice. For example, dissolution or suspension of the active compound in a vehicle such as water or naturally occuring vegatable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.
  • amino acids are described by the standard three letter amino acid code when refering to inte.mediates and final products.
  • the linear peptides and intermediates described herein are prepared by the solid phase method of peptide synthesis (R. Merrifield, J. Am. Chem. Soc. 1964, 85, 2149 and M. Bodansky, "Principles of Peptide Synthesis.” Springer-Verlag, 1984).
  • Peptides were assembled using a Milligen-Biosearch 9500 automated peptide synthesizer.
  • Protected amino acids were obtained from from Milligen-Biosearch as the pentafluorophenyl esters or dihydrooxobenzotriazine esters (threonine and serine) except for Fmoc-Arg(Pmc) which was obtained from Calbiochem.
  • Side chain protection was Asp (t-butyl ester), Cys (Trt), and Arg (Pmc).
  • the peptide was then deprotected and removed from the resin by stirring the peptide-resin in 20 ml of a mixture of TFA (95) : triethylsilane (2.5) : anisole (2.0) : water (0.5) for 1 hr at room temperature. The mixture was then filtered and the resin washed with TFA. The filtrate and washings were concentrated under vacuum. The isolated residue was triturated with ether then filtered. The recovered peptide was taken up in 10% acetic acid in water and lyopjiilized. The crude linear peptide (230 mg) was purified by HPLC.
  • the peptide was dissolved in 0.1% TFA in water, loaded onto a 2.5 cm x 50 cm C-18 reversed phase column (15 micron, 300A) and eluted with a shallow gradient of increasing acetonitrile.
  • the elution conditions consisted of a 0% to 40% acetonitrile (containing 0.1% TFA) gradient at 0.5% min -1 .
  • the aqueous phase was 0.1% TFA in water.
  • Product containing fractions were then lyophilized to afford the pure title peptide.
  • Example 21 BrCf ⁇ CO-Arg-lle-Pro-Ajg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys.
  • the title compound is-prepared using the procedure in .Example 1 except that bromoacetic acid is used in place of S-trityl N-Fmoc cysteine pentafluorophenyl ester.
  • Example 21 The compound prepared in Example 21 is dissolved in deionized water (1 mg/ml) and the pH of the solution is adjusted to 8.0 - 8.5 with ammonium hydroxide. After stirring for 4 hr at ambient temperature the reaction solution is acidified to pH 3.0 - 3.5 with trifluoroacetic acid and then lyophilized. The resulting p ⁇ rde product is purified by HPLC using the conditions described in -Example 1. The desired titletcompound elutes after 32 minutes. FAB mass spectrum: calc. 1469.7; obs. 1470.7 (M+ ). In the Gp llbllla binding assay of Example 23 the title compound exhibits a ratio of 0.27 against the control compound (see note 1 of Table 1).
  • Example 23 inhibition of Immobilized fibrinogen binding to GP llbllla Microtiter plates are coated with fibrinogen (10 ⁇ g/ml) and then blocked with TACTS buffer containing 0.5% BSA.
  • TACTS buffer contains 20m M Tris.HCI, pH 7.5, 0.02% sodium azide, 2 mM calcium chloride, 0.05% Tween 20, 150 mM sodium chloride.
  • the plate is washed with phosphate buffered saline containing 0.01% Tween 20 and a dilution of the sample to be determined was added, followed by addition of solubilized llbllla receptor (40 ⁇ g/ml) in TACTS, 0.5% BSA.
  • the procedure is repeated at several dilutions of the test sample and the concentration of test sample which inhibits 50% of the binding of GPIIbllla receptor to fibrinogen IC50 is determined, using a four parameter fit (Marquardt, J. Soc. Indust. Appl. Math. (1963) 11 : 431-441) which is the IC50.
  • the ratio of the IC50 of the test sample to that of the control compound (Gly-Arg-Gly-Asp-Val) is then determined and reported in Table 1. The smaller the ratio in Table I, the more potently the test compound inhibits immobilized fibrinogen binding to GP llbllla-
  • Example 24 Inhibition of platelet aggregation. Fifty milliliters of whole human blood (9 parts) is drawn on 3.8% sodium citrate (1 part) from a donor who has not taken aspirin or related medications for at least two weeks. The blood is centrifuged at 160 x g for 10 min at 22° C and then allowed to stand for 5 min after which the PRP is decanted. Platelet poor plasma (PPP) is isolated from the remaining blood after centrifugation at 2000 x g for 25 min. The platelet count of the PRP was diluted to ca. 300000 per microliter with PPP.
  • PPP Platelet poor plasma
  • a 225 uL aliquot of PRP plus 25 uL of either a dilution of the test sample or a control (PBS) is incubated for 5 min in a Chrono-log Whole Blood Aggregometer at 25° C.
  • Adenosine diphosphate (ADP, 8 ⁇ M) is added and the platelet aggregation recorded.
  • the IC50 value represents the concentration of test compound needed to inhibit the maximum aggregation response to 50% of its normal value. The results of this test are recorded in Table 1.
  • the present invention may have application in the treatment of a large group of disorders associated with, or characterized by, a hyperthrombotic state.
  • disorders are genetic or aquired deficiencies of factors which normally prevent a hyperthrombotic state; medical procedures such as angioplasty and thrombolytic therapy; 17 mechanical obstructions to blood flow, such as tumor masses, prosthetic synthetic cardiac valves, and extracorporeal perfusion devices; atherosclerosis; and coronary artery disease.
  • Ratio of IC50 of test compound to IC50 of Gly-Arg-Gly-Asp-Val is typically about 40nM. Errors can range to about ⁇ 50%. Table 2

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Abstract

L'invention se rapporte à un petit peptide cyclique, qui développe une affinité élevée pour le récepteur GP IIbIIIa de plaquettes et qui est représentée par la formule α;où R1 et R4 représentent 0 à 4 acides aminés; R3 représente 1 à 4 acides aminés; R2 représente -CH2CO- ou 1 à 4 acides aminés; Xaa8 peut être Met, Phe, nLeu, Ile, Asp, Lys, Arg et Gln; et Z représente un groupe de liaison, soit disulfure soit thioéther soit amide, mais de préférence disulfure. Les peptides cycliques préférés, qui présentent un pouvoir inhibiteur élevé dans des tests d'aggrégation de plaquettes, sont représentés par la formule β; où Xaa2, Xaa3 et Xaa4 peuvent représenter n'importe quel acide aminé mais sont de préférence Arg, Ile et Pro respectivement. Xaa8 est de préférence Met, Phe ou nLeu; Xaa10, lorsqu'il est présent, est de préférence Ala; et Xaa11, lorsqu'il est présent, est de préférence Ala ou Asp. Ces composés peuvent être utilisés efficacement dans une composition pharmaceutique contenant un excipient pharmaceutiquement acceptable en vue de réduire l'aggrégatiotion des plaquettes chez un mammifère. Une telle composition pharmaceutique est particulièrement utile pour traiter un mammifère ayant une propension accrue à la formation de thrombus et peut être utilisée en combinaison avec un agent anticoagulant ou thrombolitique.
PCT/US1991/000564 1990-02-02 1991-01-28 PEPTIDES CYCLIQUES CONTENANT UNE SEQUENCE Arg-Gly-Asp FLANQUEE DE PROLINE Ceased WO1991011458A1 (fr)

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WO1993007169A1 (fr) * 1991-10-04 1993-04-15 Chiron Corporation Inhibiteurs peptidiques de l'adhesion plaquettaire
US5227490A (en) * 1992-02-21 1993-07-13 Merck & Co., Inc. Fibrinogen receptor antagonists
EP0538399A4 (fr) * 1990-07-09 1994-05-04 Tanabe Seiyaku Co., Ltd.
WO1994025482A1 (fr) * 1993-04-23 1994-11-10 Evans Herbert J Polypeptides comprenant des groupes contraignant la conformation qui sont adjacents a un site d'interaction proteine-proteine
WO1994029349A1 (fr) * 1993-06-11 1994-12-22 Merrell Pharmaceuticals Inc. Peptides trifonctionnels antithrombiniques et antiplaquettaires
US5643872A (en) * 1989-10-23 1997-07-01 Smithkline Beecham Corporation Cyclic anti-aggregatory peptides
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5686569A (en) * 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5780595A (en) * 1989-06-16 1998-07-14 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5928896A (en) * 1993-04-23 1999-07-27 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein--protein interaction site
US5952465A (en) * 1993-04-23 1999-09-14 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
US5965698A (en) * 1993-04-23 1999-10-12 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein--protein interaction site
US6017877A (en) * 1990-04-06 2000-01-25 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6084066A (en) * 1993-10-29 2000-07-04 Virginia Commonwealth University Polypetides that include conformation-constraining groups which flank a protein-protein interaction site
RU2163242C2 (ru) * 1994-12-13 2001-02-20 Хехст АГ Циклогексапептиды, их смеси, способ их получения
US6258550B1 (en) 1993-04-23 2001-07-10 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
EP1205754A3 (fr) * 2000-11-07 2002-07-17 Advanced Gene Technology Méthode pour criblage les extraits végétaux pour agents actifs
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
JP2013542952A (ja) * 2010-11-01 2013-11-28 インダストリー−アカデミック コーポレーション ファウンデーション，ヨンセイ ユニバーシティ 血栓溶解用組成物及びこれを含む血管狭窄又は閉塞性疾患の治療用医薬組成物
WO2020037729A1 (fr) * 2018-08-24 2020-02-27 深圳翰宇药业股份有限公司 Procédé de synthèse d'un thioéther d'integrilin

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US5807825A (en) * 1989-06-16 1998-09-15 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5795867A (en) * 1989-06-16 1998-08-18 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5968902A (en) * 1989-06-16 1999-10-19 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5843897A (en) * 1989-06-16 1998-12-01 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5851839A (en) * 1989-06-16 1998-12-22 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5795868A (en) * 1989-06-16 1998-08-18 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5807828A (en) * 1989-06-16 1998-09-15 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5686569A (en) * 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5686567A (en) * 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5686571A (en) * 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5686566A (en) * 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5756451A (en) * 1989-06-16 1998-05-26 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5759999A (en) * 1989-06-16 1998-06-02 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5780595A (en) * 1989-06-16 1998-07-14 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5786333A (en) * 1989-06-16 1998-07-28 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5643872A (en) * 1989-10-23 1997-07-01 Smithkline Beecham Corporation Cyclic anti-aggregatory peptides
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6017877A (en) * 1990-04-06 2000-01-25 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
EP0538399A4 (fr) * 1990-07-09 1994-05-04 Tanabe Seiyaku Co., Ltd.
WO1993007169A1 (fr) * 1991-10-04 1993-04-15 Chiron Corporation Inhibiteurs peptidiques de l'adhesion plaquettaire
US5227490A (en) * 1992-02-21 1993-07-13 Merck & Co., Inc. Fibrinogen receptor antagonists
US6258550B1 (en) 1993-04-23 2001-07-10 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
US6111069A (en) * 1993-04-23 2000-08-29 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
US5965698A (en) * 1993-04-23 1999-10-12 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein--protein interaction site
WO1994025482A1 (fr) * 1993-04-23 1994-11-10 Evans Herbert J Polypeptides comprenant des groupes contraignant la conformation qui sont adjacents a un site d'interaction proteine-proteine
US5928896A (en) * 1993-04-23 1999-07-27 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein--protein interaction site
US5948887A (en) * 1993-04-23 1999-09-07 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein--protein interaction site
US6100044A (en) * 1993-04-23 2000-08-08 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
US5952465A (en) * 1993-04-23 1999-09-14 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
US6147189A (en) * 1993-04-23 2000-11-14 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
WO1994029349A1 (fr) * 1993-06-11 1994-12-22 Merrell Pharmaceuticals Inc. Peptides trifonctionnels antithrombiniques et antiplaquettaires
US6084066A (en) * 1993-10-29 2000-07-04 Virginia Commonwealth University Polypetides that include conformation-constraining groups which flank a protein-protein interaction site
RU2163242C2 (ru) * 1994-12-13 2001-02-20 Хехст АГ Циклогексапептиды, их смеси, способ их получения
EP1205754A3 (fr) * 2000-11-07 2002-07-17 Advanced Gene Technology Méthode pour criblage les extraits végétaux pour agents actifs
JP2013542952A (ja) * 2010-11-01 2013-11-28 インダストリー−アカデミック コーポレーション ファウンデーション，ヨンセイ ユニバーシティ 血栓溶解用組成物及びこれを含む血管狭窄又は閉塞性疾患の治療用医薬組成物
EP2636412A4 (fr) * 2010-11-01 2014-06-25 Univ Yonsei Iacf Composition pour la dissolution de thrombi, et composition pharmaceutique pour le traitement de maladies vasculaires sténotiques ou occlusives qui la comprend
WO2020037729A1 (fr) * 2018-08-24 2020-02-27 深圳翰宇药业股份有限公司 Procédé de synthèse d'un thioéther d'integrilin

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