WO1991011458A1

WO1991011458A1 - CYCLIC PEPTIDES CONTAINING Arg-Gly-Asp FLANKED BY PROLINE

Info

Publication number: WO1991011458A1
Application number: PCT/US1991/000564
Authority: WO
Inventors: Peter L. Barker; John P. Burnier; Robert A. Lazarus
Original assignee: Genentech, Inc.
Priority date: 1990-02-02
Filing date: 1991-01-28
Publication date: 1991-08-08
Also published as: CA2073696A1

Abstract

A small cyclic peptide is provided having high affinity for the platelet GP IIbIIIa receptor, which peptide is represented by formula α where R1 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; Xaa8 may be Met, Phe, nLeu, Ile, Asp, Lys, Arg and Gln; and Z is a linking group, either disulfide, thioether or amide, but preferably disulfide. Preferred cyclic peptides exhibiting high inhibition potency in a platelet aggregation assay are represented by formula β where Xaa2, Xaa3 and Xaa4 may be any amino acid but preferably are Arg, Ile and Pro, respectively. Xaa8 is preferably Met, Phe or nLeu; Xaa10 when present, is preferably Ala; and Xaa11, when present, is preferably Ala or Asp. These compounds may be effectively employed in a pharmaceutical composition containing a pharmaceutically acceptable excipient for reducing platelet aggregation in a mammal. The pharmaceutical composition is especially useful in treating a mammal having an increased propensity for thrombus formation, and may be used in combination with an anticoagulant or thrombolytic agent.

Description

Cyclic Peptides Containing Arg-Gly-Asp Flanked by Praline

Field of the Invention 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- . Background of the Invention

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-cόvaleήt, 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 fibfthόgen. 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) Blϋod, 74, 1181 -1195; and Colman and Walsh, (1987) Hemostasis and Thrombosis: Basic Principles and Clinical Practice, pp. 594-605, J.W. Lippincott, Philadelphia). These pathways converge in a common final step which consists of activation of the GP llbllla receptor on the platelet surface followed by binding of the receptor to fibrinogen culminating in aggregation and thrombus formation. By virtue of these interactions GP llbllla is an important component of the platelet aggregation system. Thus, inhibition of the interaction of GJ? llbllla with Arg-Gly-Asp containing ligands such as fibrinogen is a useful means of modulating thrombus -formation. Many common human disorders- are characteristically associated with a hyperthrombotic state leading to intravascular thrombi and emboli. Since the 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 embόlir 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.

In addition to being involved in arterial thrombosis, 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.

Patients whose blood flows over artificial surfaces, such as prosthetic synthetic cardiac valves or through extracorporeal perfusion devices, are also at risk for the development of platelet plugs, thrombii and emboli. It is standard practice that patients with artificial cardiac valves be treated chronically with anti-coagulation agents. However, in all instances, platelet activation and emboli formation may still occur despite adequate anticoagulation treatment.

Thus 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.

Background Prior Art Inhibition of fibrinogen binding to the GP llbllla complex has been shown to be an effective antithrombotic treatment in animals (H. K. Gold, et al., Circulation (1988) 77, 670- 677; T. Yasuda, etal., J. din. Invest. (1988) 81, 1284-1291 ; B. S. Collar, et al., Blood (1986) 68, 783-786). A number of synthetic peptides, have been disclosed as inhibitors of fibrinogen binding to platelets all of which contain the Arg-Gly-Asp sequence. See US Patent 4,683,291 ; EP 0319506 A2; Plow et al., Proc. Natl. Acad. Sci. USA (1985) 82, 8057-8061 ; Ruggeri etal., Proc. Natl Acad. Sci. USA (1986) 83, 5708-5712; Haverstick et al., Blood (1985) 66, 946-952; Plow et al., Blood (1987) 70, 110-115; and references cited in the above publications.

Other proteins such as fibronectin contain 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-Arg-Gly-Asp-AA1-B

wherein 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. Chem., (1989) 264: 21550-21556; and Friedman et al., published European Application 338 634/A2. The final authors disclose relatively large (ca. 5,0J0 to ca. 7,000 dalton) platelet aggregation inhibitors isolated from venoms represented by the^general formula

X-Cys-R-R-R-Arg-Gly-Asp-R-R-R-R-R-Cys-Y

where X is hydrogen or at least one amino acid and Y is hydroxyl or at least one amino acid.

These authors report that a platelet aggregation inhibitor encompassed by the above structural formula isolated from Echis carinatυs loses its inhibitory activity in a platelet aggregation assay upon treatment with 80 mM dithiothreitol. Thus, the authors conclude one or more intrachain disulfide bonds are necessary for activity.

Small (1 ,000 - 2,000 dalton) synthetic cyclic peptides containing the Arg-Gly-Asp sequence capable of inhibiting cell attachment to fibronectin and vitronectin have also been described by Pierschbacher et al. in WO 89/05150. These authors describe two cyclic peptides capable of inhibiting cell adhesion represented by the formula

Gly-

and

Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly

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.

From the forgoing, it will be appreciated that a need exists for an inhibitor that would prevent binding of Arg-Gly-Asp containing proteins such as fibrinogen (and/or related ligands such as fibronectin, vitronectin and von Willebrands factor) with the platelet GP llbllla receptor. Such an inhibitor would antagonize the final common pathway of platelet aggregation and act as a potent antithrombotic. Ideally, the inhibitor should be small in size to minimize antigenicity, simple to construct and exhibit high inhibition potency in a platelet aggregation assay. Summary of the Invention

These and other needs are achieved by providing a small cyclic peptide having a high affinity and specificity for the platelet GP llbllla receptor represented by Formula I

R₁ -R₂-Arg-Gly-Asp-Xaa₈-Pro-R₃-R₄

where 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.

A preferred embodiment of the instant invention having surprisingly high affinity for the GP llbllla receptor may be represented by Formula II

where each of Xaa2, Xaa3, Xaa4, Xaaio, aan, Xaai2 are amino acids or are absent, but preferably at least Xaa2, Xaa3 and Xaa4 are present. In this embodiment R5 is -CH2CO- or Cys; Z is thioether or disulfide; and Ri, R , and Xaas are as defined above.

The most preferred cyclic peptides of this invention, exhibiting especially high inhibition potency in a platelet aggregation assay are represented by Formula III

Cys-Xaa₂-Xaa₃-Xaa₄-Arg-Gly- Asp-Xaa₈-Pro-Xaa₁₀-Xaa-, ₁ -Xaa! ₂-Cys i I S S

where 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; and Xaan when present is preferably Ala or Asp. Representative preferred compounds according to this invention are conveniently selected from the following list: Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys¹³;

Cys¹-Ala-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys¹³;

Cys¹-Arg-Ala-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys¹³;

Cys¹-Arg-lle-Ala-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys¹³; Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp-Arg-Cys¹³;

Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Ala-Arg-Cys¹³;

Cys¹-Arg-lle-Pro-Arg-Gly-Asp-M'et-Pro-Asp-Asp-Ala-Cys¹³;

Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Ala-Arg-Cys¹³;

Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Phe-Pro^Ala-Asp-Arg-Cys¹³; Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Leu-Pro-Ala-Asp-Arg-Cys¹³;

Cys¹-Arg-lle.-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp-Tyr-Cys¹³;

Cys¹-Arg-lle-Pro-Arg-Gly-Asp-nLeu-Pro-Ala-Asp-Arg-Cys¹³;

Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp-Cys¹²;

Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Cys1¹ ; and Cys¹ -Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Cys¹ ° where each Cys¹-Cys¹³, Cys¹-Cys¹², Cys¹-Cys¹¹, and Cys¹-Cys¹⁰ is linked through a disulfide.

In an alternative embodiment ofrthe invention 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.

Given their high affinity for the pjatelet GP llbllla receptor, 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 thβpmbolytic agent.

Detailed Description of the Invention

The small cycϋc 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: . * "

Abbreviation Amino acid

Ala alanine

Arg - arginine

Asp _r aspartic acid

Cys - cysteine

Gly , glycine lie isoleucine

Leu leucine nLeu norleucine Lys lysine

Met methionine

Phe phenylalanine

Pro praline

Tyr tyrosine

Val valine

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. When used in combination with the above thrombolytic agents, the compounds of the present invention may be administered prior to, simultaneously with, or subsequent to the antithrombolytic agent. 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. Thus, 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.

To produce a linear peptide 'capable of cyclization using recombinant DNA methodology, an oligonucleøtide 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. A detailed discussion of 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 pNHδa (Stratagene, La Jolla, CA). The vector js first linearized using a restriction endonuclease such as Hind III, and the ends are*fιlled 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 pNHδa 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. According to this preferred method, 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). When solid phase synthesis is employed peptide synthesis is initiated at the C-terminus of the putative peptide by coupling an appropriately protected amino acid to any suitable resin (see e.g., U.S. Patent Nos.4,244,946; 4,305,872; and 4,316,891). Solid phase synthesis is then carried out as described in the above references and as provided in Vale et al., Science (1981) 213, 1394-1397 and Marke et al., J. Am. Chem. Soc. (1981)_,103, 3178.

However 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). 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. In the case of Fmoc chemistry 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.

It is believed that for the preferred compounds containing from about 10 to about 13 amino acids in the cyclic portion of the peptide, there are no significant conformational differences among peptides cyclized through a disulfide, thioether, amide or other analogous bridging groups. Thus, it is contemplated that 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 α amino and carboxyl groups or pairs containing free side chain amino and carboxyl groups. Examples of the latter would include basic residues such as D/L ornithine or D/L lysine and acidic residues such as D/L Glu or D/L Asp. It is also contemplated that 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. 9

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. Alternatively, 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.

GP ll llla • Fibrinogen ELISA and Platelet Aggregation Assays The evaluation of inhibitors, of the fibrinogen - platelet interaction is guided by in vitro receptor binding assays and in vitro platelet aggregation inhibition assays. in vitro biological activity of the compounds of Formulae I - III were monitored using a modified fibrinogen - GP llbllla ELISA based on the method of Nachman and Leung {J. din.

Invest. (1982) 69, 263-269) which measures the inhibition of fibrinogen binding to purified human platelet GP llbllla receptor. Human fibrinogen was prepared by the method of Lipinska, et al. J. Lab. Clin. Med. (1974) 84, 509-516. Platelet GP llbllla was prepared by the method of Fitzgerald, etal. (Anal. Biochem. (1-985) 1 1, 169-177).

Microtiter plates were coated with fibrinogen (10 μg/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.) The plate was washed with phosphate buffered saline (PBS) containing 0.01% Tween 20 and the sample to be determined as added, followed by addition of solubilized GP llbllla receptor (40 μg/ml) in TACTS, 0.5% BSA. After a one-hour incubation, the plate was washed and 1 μg/ml of murine anti GP Mia monoclonal antibody AP3 (P. J. Newman et al. Blood (1985) 65, 227-232) was added. After a one-hour incubation and another wash, a goat anti-mouse IgG conjugated to horseradish peroxidla^e was added. A final wash was performed and developing reagent buffer (0.67 mg/ml o-phenylenediamine dihydrochloride, 0.02% hydrogen peroxide, 0.22 mM citrate, 50 mM phosphate, pH 5.0) were added and the mixture was incubated until color developed. The reaction was stopped with 1N sulfuric acid and the absorbance at 492 nm was recorded.

In addition to the GP llbllla ELISA assay, platelet aggregation assays may be performed in human platelet rich plasma^r(PRP). 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. Thetølood 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. 300000 per icroliter with PPP. A 225 μL aliquot of PRP plus 25 μL 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. An aggregating agent (collagen, 1 μg/ml; U46619, 100 ng/ml; or ADP, 8 μM) is added and the . platelet aggregation was measured by recording the change in light transmittance. Inhibition of platelet aggregation was measured at the maximum aggregation response.

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. Generally, 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.

The IC50 values in the platelet aggregation assay (column 2, Table 1) are considered a more accurate measure of a compounds utility as an antithrombotic. Generally, to be considered suitable 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. Thus, preferred compounds of the instant invention have an IC50 in platelet aggregation inhibition assays of less than about 15 μM, while most preferred compounds have an IC50 of less than 5 μM, and very most preferred compounds have an IC50 of less than about 1μM. Accordingly, 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. As used herein, 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. For example, the cyclic peptide Gly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala, Pen- ^' Cys disulfide described by Piershbacher et al., J. Biol. Chem. (1987) 262, 17294-17298 exhibited a binding ratio of 7 (i.e., about 7 times lower affinity for platelet GP llbllla than the reference compound) with an IC50 in the platelet aggregation inhibition assay of >300 μM. In view of the foregoing discoveries, 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 μM.

,

- 11

The most preferred compounds for use ϊrrlhe above described method comprises the compounds represented by Formulae I - III. Dosage Formulations

In the management of thromboembolic disorders 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. 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, disacchaήdes, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sobitol; counterions such as sodium and/or nonionic surfactants such as Tween, Pluronics or polyethyleneglycol.

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, Cys¹ - Cys¹³ 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. Similarly, in a platelet aggregation assay in Example 22 using the same cyclic peptide, the IC50 was found to be 8 μM as shown in Table I. Based upon such in vitro assay techniques, 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 μM, and most preferably from about 1.0 nM to about 50 μM. 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. When the dosage form is a capsule, in addition to the above materials it may also contain 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. 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.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and illustrative examples, make and utilize the present invention to ^• the fullest extent. The following working examples therefore specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way of the remainder of the disclosure.

Examples In the following EExamples, 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). Abbreviations used are as follows: Floureny Im et y loxycarbony I (Fmoc); 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc); trityl (Trt); benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP); trifluoroacetic acid (TFA); dimethylacetamide (DMA); and dimethylformamide (DMF).

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).

Example 1 Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys. The peptide was assembled on 1.5 g (0.145 mmol) of Fmoc-Cyc(Trt) PepSyn KA resin (Milligen-Biosearch). The coupling protocols were those recommended by Milligen- Biosearch with the exception of Arg which was coupled using BOP and that DMA was used instead of DMF as solvent. After chain assembly the N-terminal Fmoc group was removed using 20% piperidine in DMA. The peptide resin was then washed with dichloromethane followed by methanol and then dried under vacuum. 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. FAB mass spectrum: Calc. M + H =1533.7; found 1533.9.

Example 2 Cys-Arg-lle-Pro-Arg-Gly-Asp- et-Pro-Asp-Asp-Arg-Cys, Cys¹ • Cys¹³ disulfide. The purified linear peptide (85 mg) was dissolved in 200 ml of water and the pH adjusted to 8.5 with ammonium hydroxide. The resulting solution was stirred for 2 days at room temperature. When the linear peptide was completely oxidized, as determined by HPLC, the solution was acidified to pH 3 with TFA, loaded directly onto a reverse phase chromatography column and purified as described in Example 1. The product containing fractions were lyophilized to afford the pure title compound. FAB mass spectrum: Calc. M + H = 1531.7; found 1531.8. HPLC _R = 30 min. Following the procedures in Examples 1 and 2, the compounds of Examples 3 - 21 were prepared.

Example 3 Cys-Arg-Arg-AIa-Arg-Gly-Asp-Asp-Leu-Asp-Asp-Tyr-Cys, Cys¹ - Cys¹³ disulfide. FAB mass spectrum: Calc. M + H = 1555.6; found 1555.7. HPLC .R = 30 min. Eixample 4

Cys-Ala-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide. FAB mass spectrum: Calc. M + H = 1446.6; found 1446.6. HPLC tR = 32 min.

-Example 5 Cys-Arg-Ala-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys, Cys¹ • Cys¹³ disulfide. FAB mass spectrum: Calc. M + H = 1489.6; found 1489.6. HPLC tR = 25 min.

Example 6 Cys-Arg-lle-Ala-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide. FAB mass spectrum: Calc. M + H = 1505.6; found 1505.6. HPLC tR = 29 min.

Example 7 Cys-Arg-lle-Pro-Arg-Gly-Asp-Ala-Pro-Asp-Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide.

FAB mass spectrum: Calc. M + H = 1471.7; found 1471.7. HPLC tR = 27 min.

[Example 8 Cys-Arg-lle-Pro-Arg-Gly-Asp- et-Ala-Asp-Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide. FAB mass spectrum: Calc. M + H = 1505.7; found 1505.7. HPLC tR = 29 min. Example 9

Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide. FAB mass spectrum: Calc. M + H = 1487.7; found 1487.7. HPLC tR = 30 min.

Example 10 Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Ala-Arg-Cys, Cys¹ - Cys¹³ disulfide. FAB mass spectrum: Calc. M + H = 1487.7; found 1487.7. HPLC tR = 31 min.

Example 11 Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Ala-Cys, Cys^{1 ■} Cys¹³ disulfide. FAB mass spectrum: Calc. M + H = 1446.6; found 1446.6. HPLC tR = 32 min.

Example 12 Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Cys, Cys¹ - Cys¹⁰ disulfide.

FAB mass spectrum: Calc. M + H = 1145.5; found 1145.5. HPLC t = 33 min.

{Example 13 Cys-Aig-lle-Pro-Arg-Gly-Asp- et-Pro-Ala-Ala-Arg-Cys, Cys¹ - Cys¹³ disulfide. FAB mass spectrum: Calc. M + H = 1443.7; found 1444.0. HPLC tR = 31 min. ^Example 14 Cys-Arg-lle-Pro-Arg-Gly-Asp-Pha-Pro-Ala-Asp'Arg-Cys, Cys¹ - Cys¹³ disulfide.

FAB mass spectrum: Calc. M + H = |503.7; found 1504.0. HPLC tR = 34 min.

^Example 15 Cys-Arg-lle-Pro-Arg-Gly-Asp-Leu-Pro-Ala-Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide.

FAB mass spectrum: Calc. M + H = 1469.7; found 1470.0. HPLC tR = 32 min.

.Example 16 Cys-Arg-lle-Pro-Arg-Gly-Asp-Mfeϊ-Pro-Ala-Asp-Tyr-Cys, Cys¹ - Cys¹³ disulfide.

FAB mass spectrum: Calc. M _- H = 1494^6; founts 495.0. HPLC tR = 31 min. •^{> ♦}* Example_17

Cys-Arg-lie-Pro-Arg-Gly-Asp-nLeu-Pro-Ala-Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide. FAB mass spectrum: Calc. M .+ H = 1469^7; found 146918. HPLC tR = 34 min.

Example 18 Cys-Arg-lle-Pro-Lys-Gly-Asp- et ro-Asp-Asp-Arg-Cys, Cys^{1 ■} Cys¹³ disulfide. FAB mass spectrum: Calc. M + H = 1503,7; f υnd- 1503.8. HPLC tR = 30 min.

^■ -Example 19 Cys-Arg-lle-Pro-Lys-Gly-Asp-MetrPro-Ala-Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide.

FAB mass spectrum: Calc. M +.H = 1459.7;,. ound"1460.4. HPLC tR = 30 min.

^*" Exajnple 20 Cys-Arg-lle-Pro-Arg-Gly-Asp-tøet'-Pro-Ala-Asp-Cys, Cys¹ - Cys¹² disulfide.

FAB mass spectrum: Calc. M + H =^ 3^*34.6; found 1331.5. HPLC tR = 32 min.

Example 21; BrCføCO-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 22

¹

CO-Arg-llθ-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys i ; i

CH₂ : : ; S

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 pαrde 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 μg/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 μg/ml) in TACTS, 0.5% BSA. After one-hour incubation, the plate is washed and murine monoclonal anti-platelet antibody AP3 (1 μg/ml) added. After one-hour incubation and another wash, goat anti-mouse IgG conjugated to horseradish peroxidase is added. A final wash is performed and developing reagent buffer (0.67 mg/ml o-phenylenediamine dihydrochloride, 0.02% hydrogen peroxide, 0.22 mM citrate, 50 mM phosphate, pH 5.0) is added and then incubated until color develops. The reaction is stopped with 1N sulfuric acid and the absorbance at 492 nm is recorded. 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.

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 μM) 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.

In view of the efficacy of these cyclic polypeptides as inhibitors of fibrinogen binding to GP llbllla. and the feasibility as demonstrated herein of producing these cyclic polypeptides, the present invention may have application in the treatment of a large group of disorders associated with, or characterized by, a hyperthrombotic state. Representative of such 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.

EXAMPLE 25 Results of GP llallib - Fibrinogen ELISA and Platelet Aggregation Assays

TABLE 1

Inhibition of Immobilized Fibrinogen Binding to GP llhll and

Inhibition of Platelet Aggregation

GP llbllla - Fibrinogen ELISA

Peptide IC50 Ratio¹ . ICδo(μ

Platelet Aggreg

Gly-Arg-Gly-Asp-Val 1 75

Cys-Arg-lle-Pro-Arg-Gly-Asp- et-Pro-Asp-Asp- 0.5 8

Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-Arg-Ala-Arg-Gly-Asp-Asp-Leu-Asp- 5.2 300

Asp-Tyr-Cys, Cys¹ - Cys¹³ disulfide

Cys-Ala-lle-Pro-Arg-Gly-Asp- et-Pro-Asp-Asp- 0.43 9

Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-Ala-Pro-Arg-Gly-Asp- et-Pro-Asp- 0.54 13

Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Ala-Arg-Gly-Asp-Met-Pro-Asp-Asp- 0.28 3

Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Ala-Pro-Asp-Asp- 1.0 43

Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp_' et-Ala-Asp-Asp- 1.5 39

Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp- 0.11 0.5

Arg-Cys, Cys¹ - Cys¹³ disulfide Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Ala- 0.16 3

Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lie-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp- 0.23 9

Ala-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Cys, Cys¹ 0.21 4.3

- Cys¹⁰ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Ala- 0.04 0.5

Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Phe-Pro-Ala-Asp- 0.3 1.0

Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Leu-Pro^'-Ala-Asp- 0.2 1.3

Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp- ^' 0.35 3.7

Tyr-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-nLeu-Pro-Ala-Asp- 0.1 1.9

Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-ile-Pro-Lys-Gly-Asp-Met-Pro-Asp-Asp- .46. <

Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-IIe-Pro-Lys-Gly-Asp-Met-Pro-Ala-Asp- 0.5 Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp- 0.13 0.8

Cys, Cys¹ - Cys¹² disulfide

1. Ratio of IC50 of test compound to IC50 of Gly-Arg-Gly-Asp-Val. The IC50 of Gly-Arg-Gly-Asp-Val is typically about 40nM. Errors can range to about ± 50%. Table 2

Inhibition of Fibrinogen Binding to GP llhllla bv Cyclic Peptides.¹ GP llbllla - Fibrinogen ELISA

Peptide IC50 Ratio²

Cys-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp- 0.8 Arg-Cys, Cys¹ - Cys¹² disulfide

Cys-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg- 1.1 Cys, Cys¹ - Cys¹¹ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala- 0.13 Cys, Cys¹ - Cys¹¹ disulfide

Cys-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp- 0.8 Arg-Cys, Cys¹ - Cys¹² disulfide

Cys-Pro-Arg-Gly-Asp-Met-Pro-Aia-Asp-Arg- 0.4 Cys, Cys¹ - Cys^{1 1} disulfide

Cys-Arg-Gly-Asp-Met-Pro-Ala-Asp-Arg-Cys, 4 Cys¹ - Cys¹⁰ disulfide

Cys-Arg-Gly-Asp-Met-Pro-Cys, Cys¹ - Cys⁷ 1 disulfide

Cys-Lys-Arg-Ala-Arg-Gly-Asp-Asp-Met-Asp- 4.6 Asp-Tyr-Cys, Cys¹ - Cys¹³ disulfide

Cys-lle-Pro-Arg-Gly-Asp-Met-Pro-Cys, Cys¹ - 0.8 Cys⁹ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Pro-Met-Ala- 7 Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-lle-Pro-Ala-Asp- 16 Arg-Cys, Cys¹ - Cys¹³ disulfide Cys-Arg-lle-Pro-Arg-Gly-Asp-Gln-Pro-Ala- 0.8 Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Ser-Pro-Ala- 12 Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Met-(D-Pro)- 25 Ala-Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Ala-Gly-Asp-Met-Pro-Ala- 149 Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Ala-Asp-Met-Pro-Ala- 0.9 Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Ala-Met-Pro-Ala- >10000 Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Glu-Met-Pro-Ala- >10000 Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Arg-Pro-Ala- 05 Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-Arg-lle-Pro-Arg-Gly-Asp-Lys-Pro-Ala- 1.3 Asp-Arg-Cys, Cys¹ - Cys¹³ disulfide

Cys-lle-Pro-Arg-Gly-Asp-Met-Pro-(D-Ala)- 0.6 Asp-Arg-Cys, Cys¹ - Cys¹² disulfide

Cys-lle-Pro-Arg-Gly-Asp-Met-Pro-Gly-Asp- 0.4 Arg-Cys, Cys¹ - Cys¹² disulfide

Cys-lle-Pro-Arg-Gly-Asp-Met-Pro-Ser-Asp- 0.4 Arg-Cys, Cys¹ - Cys¹² disulfide

Cys-Ile-Pro-Arg-Gly-Asp-Met-Pro-Val-Asp- 0.5 Arg-Cys, Cys¹ - Cys¹² disulfide 21

1. The compounds in this Table were prepared following the procedures of .Examples 1 and 2.

2. See Note 1 of Table 1

* * * * *

While the invention has necessarily been described in conjunction with preferred embodiments, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and alterations to the subject matter set forth herein, without departing from the spirit and scope thereof. Hence, the invention can be practiced in ways other than those specifically described herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the appended claims and equivalents thereof.

Claims

What is claimed is:

1. A compound represented by the formula

R ₁ -R₂-Arg-Gly-Asp-Xaa₈-Pro-R₃-R₄

wherein

Ri and R are independently selected from 0 to 4 amino acids;

R3 is independently selected from 1 to 4 amino acids;

R2 is -CH2CO- or from 1 to 4 amino acids;

Xaaβ is selected from Met, Phe, nLeu, Leu, lie, Asp, Lys, Arg and Gin;

Z is a linking group selected from disulfide, thioether and amide bond; and pharmaceutically acceptable salts thereof. The compound of Claim 1 wherein

R2 is -Rs-Xaa2-Xaa3-Xaa4 provided that R5 is bonded to Ri and Z; R3 is -Xaaιo-Xaaιι-Xaai2-Cys- provided that Cys is bonded to R4 and Z; where each Xaa2, Xaa3, Xaa4, Xaaio, aan and Xaai2 is independently selected from amino acids and null; and R5 is Cys or -CH2CO-.

3. The compound of Claim 2 wherein each Xaa2, Xaa3 and Xaa4 is an amino acid. 4. The compound of Claim 3 wherein R5 is Cys and Z is disulfide. 5. The compound of Claim 4 wherein Ri and R4 are null. a The compound of Claim 5 wherein Xaag is selected from Met, Phe and nLeu.

7. The compound of Claim 6 wherein Xaa4 is Pro. a The compound of Claim 7 wherein Xaaio is Ala.

9. The compound of Claim 8 wherein Xaan is selected from Ala and Asp.

10. The compound of Claim 9 wherein Xaa2 is Arg and Xaa3 is lie.

11. The compound of Claim 1 selected from the group: Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys¹³; Cys¹-Ala-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys¹³; Cys¹-Arg-Ala-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys¹³; Cys¹-Arg-lle-Ala-Arg-Gly-Asp-Met-Pro-Asp-Asp-Arg-Cys¹³; Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp-Arg-Cys¹³; Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Ala-Arg-Cys¹³; Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Asp-Asp-Ala-Cys¹³; Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Ala-Arg-Cys¹³; Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Phe-Pro-Ala-Asp-Arg-Cys¹³; Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Leu-Pro-Ala-Asp-Arg-Cys¹³; Cys¹ -Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp-Tyr -Cys¹³; Cys¹ -Arg-lle-Pro-Arg-Gly-Asp-nLeu-Pro-Ala-Asp-Arg-Cys¹³; _ 23

Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp-Cys¹²; Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Cys¹¹ ; and Cys¹ -Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Cys¹ ° where each Cys¹-Cys¹³, Cys¹-Cys¹², Cys^τ-Cys¹¹, and Cys¹-Cys¹⁰ is linked through a disulfide.

12. The compound of Claim 11 selected from the group Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp-Arg-Cys¹³; Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Ala-Arg-Cys¹ ; and Cys¹-Arg-lle-Pro-Arg-Gly-Asp-Met-Pro-Ala-Asp-Cys¹², where each Cys¹-Cys¹³, and Cys¹-Cys¹² is linked through a disulfide.

13. A peptide comprising from 7 to about 16 amino acids in a ring bridged through the disulfide of cystine, the ring containing the sequence Arg-Gly-Asp flanked by Proline.

14. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the compound of Claim 1. 15. A method of inhibiting platelet aggregation which method comprises administering a platelet aggregation inhibiting amount of a compound of Claim 1.

16. A method for reducing platelet aggregation in a mammal, comprising administering 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 further containing the sequence Arg-Gly-Asp and at least one proline, the cyclic peptide exhibiting an IC50 in a platelet aggregation inhibition assay of less than about 15μM.

17. A method for reducing platelet aggregation in a mammal, comprising administering a pharmaceutically effective amount of the composition of matter as defined by Claim 1 to said mammal. 18. The method of Claim 17, further comprising administering said composition of matter to said mammal in admixture with a pharmaceutically acceptable carrier. 19. A method for treating a mammal who has an increased propensity for thrombus formation, comprising administering a pharmaceutiacally effective amount of the composition of matter as defined by Claim 1 to said mammal. 20. A composition of matter for reducing platelet aggregation in a mammal, comprising the composition of matter as defined by Claim 1.

21. A composition of matter for treating a mammal who has an increased propensity for thrombus formation, comprising ihe composition of matter as defined by Claim 1.

22. A_^composition of matter for inhibiting fibrinogen binding to platelets in a mammal, comprising the composition of matter as defined by Claim 1.

23. A method for treating a mammal who has an increased propensity for thrombus formation, comprising administering a pharmaceutically effective amount of the composition of matter as defined by Claim 1 in combination with a thrombolytic agent. 24. A method for treating a mammal who has an increased propensity for thrombus formation, comprising administering a pharmaceutically effective amount of the composition of matter as defined by Claim 1 in combination with an anticoagulant.

25. A method for treating a mammal who has an increased propensity for thrombus formation, comprising administering a pharmaceutically effective amount of the composition of matter as defined by Claim 1 following angioplasty.