CA1315479C - Platelet-aggregation inhibitor peptide derivatives - Google Patents
Platelet-aggregation inhibitor peptide derivativesInfo
- Publication number
- CA1315479C CA1315479C CA000584873A CA584873A CA1315479C CA 1315479 C CA1315479 C CA 1315479C CA 000584873 A CA000584873 A CA 000584873A CA 584873 A CA584873 A CA 584873A CA 1315479 C CA1315479 C CA 1315479C
- Authority
- CA
- Canada
- Prior art keywords
- asp
- gly
- arg
- amide
- tetrapeptide derivative
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1019—Tetrapeptides with the first amino acid being basic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0815—Tripeptides with the first amino acid being basic
- C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
07-21(453)A
NOVEL PLATELET AGGREGATION
INHIBITOR PEPTIDE DERIVATIVES
Abstract of the Disclosure Novel tetrapeptide derivatives are provided which have useful activity as inhibitors of platelet aggregation. These compounds have the sequence X-Gly-Asp-Y wherein X is illustrated by arginine and Y is illustrated by O-methyltyrosine amide.
NOVEL PLATELET AGGREGATION
INHIBITOR PEPTIDE DERIVATIVES
Abstract of the Disclosure Novel tetrapeptide derivatives are provided which have useful activity as inhibitors of platelet aggregation. These compounds have the sequence X-Gly-Asp-Y wherein X is illustrated by arginine and Y is illustrated by O-methyltyrosine amide.
Description
1 31 5~7q -1- 07-21~453)A
NOVEL PLATELET~AGGREGATION
IN~IBITOR PEPTIDE DERIVATIVES
Background of the Invention This invention relakes to novel peptide derivatives and more particularly to tetrapeptide derivatives having activity as inhibitors of platelet aggregation.
Fibrinogen is a glycoprotein present as a normal component of blood plasma. It participates in platelet aggregation and fibrin formation in the blood clotting mechanism.
Platelets are cellular elements found in whole blood which also participate in blood coagulation. Fibrinogen binding to platelets is important to normal platelet function in the blood coagulation mechanism. When a blood vessel receives an injury, the platelets binding to fibrinogen will initiate aggregation and form a thrombus. Interaction of fibrinogen with platelets occurs through a membrane glycoprotein complex, known as gpIIb/IIIa; this is an important feature of the platelet function.
Inhibitors of this interaction are useful in modulating platelet thrombus formation.
It is also known that another large glycoprotein named fibronectin, which is a major extracellular matrix protein, interacts with fibrinogen and fibrin, and with other structural molecules such as actin, collagen and proteoglycans.
Various relatively large polypeptide fragments in the cell-binding domain of fibronectin have been found to have cell-attachment activity. See U.S. Patents 4,517,68Ç; 4,589, 8al; and 4,661,111. These 1 31 5~7~
NOVEL PLATELET~AGGREGATION
IN~IBITOR PEPTIDE DERIVATIVES
Background of the Invention This invention relakes to novel peptide derivatives and more particularly to tetrapeptide derivatives having activity as inhibitors of platelet aggregation.
Fibrinogen is a glycoprotein present as a normal component of blood plasma. It participates in platelet aggregation and fibrin formation in the blood clotting mechanism.
Platelets are cellular elements found in whole blood which also participate in blood coagulation. Fibrinogen binding to platelets is important to normal platelet function in the blood coagulation mechanism. When a blood vessel receives an injury, the platelets binding to fibrinogen will initiate aggregation and form a thrombus. Interaction of fibrinogen with platelets occurs through a membrane glycoprotein complex, known as gpIIb/IIIa; this is an important feature of the platelet function.
Inhibitors of this interaction are useful in modulating platelet thrombus formation.
It is also known that another large glycoprotein named fibronectin, which is a major extracellular matrix protein, interacts with fibrinogen and fibrin, and with other structural molecules such as actin, collagen and proteoglycans.
Various relatively large polypeptide fragments in the cell-binding domain of fibronectin have been found to have cell-attachment activity. See U.S. Patents 4,517,68Ç; 4,589, 8al; and 4,661,111. These 1 31 5~7~
-2~ 07-21(453)A
polypeptides include an internal amino acid sequence Arg-Gly-Asp-Ser. Certain relatively short peptide fragments from the same molecule were found to promote cell attachment to a substrate when immobilized on the substrate or to inhibit attachment when in a solubilized or suspended form. See U.S. Patents 4,578,079 and 4,614,517. These peptides were defined as X-Arg-Gly-Asp-R-Y
wherein X = H or amino acid, R = Thr or Cys;
and X-Arg-Gly-Asp-Ser-Y
wherein X = H or amino acid, Y = OH or amino acid.
In U.S. Patent 4,683,291, inhibition of platelet function is disclosed with synthetic peptides designed to be high affinity antagonists of fibrinogen binding to platelets. These synthetic peptides have up to 16 amino acid residues with Arg-Gly-Asp-Yal or Arg-Gly-Asp-Ser at the C-terminal.
Similar synthetic peptides which contain the Arg-Gly-Asp sequence and their use as inhibitors of fibrinogen binding to platelets are disclosed by Kloczewiak et al., Biochem. 23, 1767-1774 ~1984);
Plow et al., Proc. Natl. Acad. Sci. 82, 8057-8061 (1985); Ruggeri et al., Ibid. 83, 5708-5712 (1986);
Ginsberg et al., J Biol. Chem. 260 (7~, 3931-3936 (1985); and Haverstick et al., Blood 66 (4), 946-952 (1985).
rief Description of the Invention In accordance with the present invention novel tetrapeptide derivatives are provided which have useful activity as inhibitors of platelet -3- 07-21(453~A
aggregation. They are believed to act by antagonizing interactions between fibrinogen and/or extracellular matrix proteins and the platelet gpIIb/IIIa receptor. These tetrapeptide derivatives contain the sequence :
X-Gly-Asp-Y (I) NH z Il I
wherein X = H2NC-NH-(CH2)n-CH-COOH or Ac-Arg, Z ~ H, NH2 or NH-Acyl, n = 1 to 4, : ' Rl wherein Y = H2N-C-R2, Tyr-NH2 or Phe-NH2?
(CH2)m Rl ~ H, alkyl, phenyl or phenylalkyl, R2 = H, COOH, CONH2, COCH3, CH20H, CH2NH2, C(NH)CH3 or C~NH)NH2, R3 = phenyl, biphenyl or napthyl, each substituted with 1 to 3 alkyl or alkoxy groups, or an unsubstituted napthyl or pyridyl group, m = O to 2, : 30 wherein alkyl and alkoxy each have 1 to 4 carbons, provided that when Y is Tyr-NH2 or Phe-NH2, X lS Ac-Arg, and provided :: : further that when Z is NH2 or NH-Acyl, X is in the D- or L-amino acid stereo-configuration.
,:
.
polypeptides include an internal amino acid sequence Arg-Gly-Asp-Ser. Certain relatively short peptide fragments from the same molecule were found to promote cell attachment to a substrate when immobilized on the substrate or to inhibit attachment when in a solubilized or suspended form. See U.S. Patents 4,578,079 and 4,614,517. These peptides were defined as X-Arg-Gly-Asp-R-Y
wherein X = H or amino acid, R = Thr or Cys;
and X-Arg-Gly-Asp-Ser-Y
wherein X = H or amino acid, Y = OH or amino acid.
In U.S. Patent 4,683,291, inhibition of platelet function is disclosed with synthetic peptides designed to be high affinity antagonists of fibrinogen binding to platelets. These synthetic peptides have up to 16 amino acid residues with Arg-Gly-Asp-Yal or Arg-Gly-Asp-Ser at the C-terminal.
Similar synthetic peptides which contain the Arg-Gly-Asp sequence and their use as inhibitors of fibrinogen binding to platelets are disclosed by Kloczewiak et al., Biochem. 23, 1767-1774 ~1984);
Plow et al., Proc. Natl. Acad. Sci. 82, 8057-8061 (1985); Ruggeri et al., Ibid. 83, 5708-5712 (1986);
Ginsberg et al., J Biol. Chem. 260 (7~, 3931-3936 (1985); and Haverstick et al., Blood 66 (4), 946-952 (1985).
rief Description of the Invention In accordance with the present invention novel tetrapeptide derivatives are provided which have useful activity as inhibitors of platelet -3- 07-21(453~A
aggregation. They are believed to act by antagonizing interactions between fibrinogen and/or extracellular matrix proteins and the platelet gpIIb/IIIa receptor. These tetrapeptide derivatives contain the sequence :
X-Gly-Asp-Y (I) NH z Il I
wherein X = H2NC-NH-(CH2)n-CH-COOH or Ac-Arg, Z ~ H, NH2 or NH-Acyl, n = 1 to 4, : ' Rl wherein Y = H2N-C-R2, Tyr-NH2 or Phe-NH2?
(CH2)m Rl ~ H, alkyl, phenyl or phenylalkyl, R2 = H, COOH, CONH2, COCH3, CH20H, CH2NH2, C(NH)CH3 or C~NH)NH2, R3 = phenyl, biphenyl or napthyl, each substituted with 1 to 3 alkyl or alkoxy groups, or an unsubstituted napthyl or pyridyl group, m = O to 2, : 30 wherein alkyl and alkoxy each have 1 to 4 carbons, provided that when Y is Tyr-NH2 or Phe-NH2, X lS Ac-Arg, and provided :: : further that when Z is NH2 or NH-Acyl, X is in the D- or L-amino acid stereo-configuration.
,:
.
-4- 07-21(453)A
In the above general formula (I), X and Y are attached to glycine and aspartic acid, respectively, through co~ventional peptide bonds, with the N-terminal shown at the left and the C-terminal at the right.
It will be seen that X in the above formula is arginine when Z = NH2 and n = 3; homoarginine ~hen Z - NH2 and n = 4; and guanidinobutyric acid when Z = H and n = 2. Alternatively, this ~-NH2 of arginine can be replaced with H or N-acyl, preferably N-acetyl. The methylene chain can range from one to four units, as shown, but preferably is three CH2 units in length.
It will also be seen that Y in the above formula ~I) generally is an unnatural aromatic amino acid derivative, preerably a tyrosine derivative in which the C-terminal is H, COOH, CONH2, COCH3, CH20H, CH2NH2, C(NH)CH3 or C~NH)NH2, and the aromatic or benzenoid ring is substituted with one to three lower alkyl or alkoxy groups. The lower alkyl substituents in the general formula ~I) can be, for example, methyl, ethyl, iso-propyl or~butyl.
A most preferred compound is Arg-Gly-Asp-~
(O-methyltyrosine)-amide.
O O
11 : 1~
Arg-Gly-Asp-c-~-cH-c-NH2 This compound, in which the p-hydroxyphenyl ; group of tyrosine lS methylated and the C-terminal i6 carboxyamide, unexpectedly is substantially more active as an inhibitor of platelet aggregation than 1 31 5~79 -5- 07-21~453)A
ei~her Arg-Gly-Asp-Ser or Arg-Gly-Asp-Tyr. This advantage has been demonstrated in platelet-rich plasma assays and with in vivo assays where, by comparison, Arg-Gly-Asp~Ser is relatively ineffective.
Moreover, Arg-Gly-Asp-(O-methyltyrosine~-amide is effective in preventing thrombosis in a rat carotid artery assay model, thereby demonstrating its use as an antithrombotic agent.
In those instances where the C-terminal amino acid Y in the above formula (I) is a natural amino acid, it is an amide derivative of tyrosine or phenylalanine and the NH2-terminal amino acid X is acetyl-arginine. Thus, it has been surprisingly found that acetyl-RGDY-amide is very effective in the in VlVO thrombocytopenia assay (85% inhibition) whereas the non-acetylated RGDY-amide is relatively ineffective (only 19~ inhibition).
Other preferred compounds of the invention are Arg-Gly-Asp-(O-ethyltyrosine)-amide, des-amino-Arg~Gly-Asp-(O-methyltyrosine)-amide, des-amino-(homoarginine)-Gly-Asp-(O-methyltyrosine)-amide, acetyl-Arg-Gly-Asp-(O-methyltyrosine)-amide, acetyl-(D-Arg)-Gly-Asp-(0-methyltyrosine)-amide Arg-Gly-Asp-(4-metho~y-1-napthylalanine)-amide, Arg-Gly-Asp-~2,6-dimethyl-O-methyltyrosine)amide, and Arg-Gly-Asp-(p-phenyl-phenylalanine)-amide.
Detailed Description of the Invention The noveI peptides of this invention can be made by conventional methods of peptide synthesis. A
preferred method is the solid phase synthesis of Merrifield, J. Amer. ChemO Soc. 85, 2149-2154 (1963);
Science 150, 178-185 (1965); Ibid , 232, 341-347 (1986).
1 3 1 547q -6~ 07-21(453~A
Solid phase synthesis is generally commenced from the C-terminus of the peptide by coupling a protected alpha amino acid to a suitable resin, e.g., chloromethylated polystyrene resin or p-methylbenzhydrylamine resin when synthesizing a peptide amide derivative. In the present invention, the tyrosine derivative as described above can be used as the C-terminal peptide for initiating the solid phase synthesis. The three remaining alpha amino acids are then coupled stepwise in the desired order to obtain an intermediate peptide coupled to the resin. During this synthesis, suitable protecting groups are used as needed. Thus, aspartic acid is protected on the ~-carboxyl group as the ben~yl ester and arginine is protected on the guanidino group by tosyl. Each a-amino group is protectd with the t-butyloxycarbonyl group (BOC).
After the desired tetrapeptide sequence has been completed, the intermediate peptide is cleaved from the resin and protecting groups are removed by treatment with a reag~nt such as HF. The peptide can then be purified by high performance liquid chromatography (HPLC) or other such methods of protein purification.
Background information on the established solid phase synthesis procedure which can be used for the prepartion of the tetrapeptide derivatives herein can be had by reference to the treatise by Stewart and Young, I'Solid Phase Peptide Synthesis," W. H. Freeman & Co., San Francisco, 1969, and the review chapter by Merrifield in Advances in Enzymology 32, pp. 221-296, F. F. Nold, Ed., Interscience Publishers, New York, 1969; and Erickson and Merrifield, The Proteins, Vol.
2, p. 255 et seq. (ed. Neurath and Hill~, ~cademic Press, New York, 1976.
131~479 -7- 07-21(453)A
As used herein, the peptide sequences are shown by conventional single or three letter abbreviations for the constituent amino acids as follows:
Abbreviated Designation ~ Amino Acid A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan ~Y Tyr Tyrosine *Not to be confused with the C-terminal Y in the general formula ~I) for the tetrapeptide derivatives as defined herein.
In the above general formula (I), X and Y are attached to glycine and aspartic acid, respectively, through co~ventional peptide bonds, with the N-terminal shown at the left and the C-terminal at the right.
It will be seen that X in the above formula is arginine when Z = NH2 and n = 3; homoarginine ~hen Z - NH2 and n = 4; and guanidinobutyric acid when Z = H and n = 2. Alternatively, this ~-NH2 of arginine can be replaced with H or N-acyl, preferably N-acetyl. The methylene chain can range from one to four units, as shown, but preferably is three CH2 units in length.
It will also be seen that Y in the above formula ~I) generally is an unnatural aromatic amino acid derivative, preerably a tyrosine derivative in which the C-terminal is H, COOH, CONH2, COCH3, CH20H, CH2NH2, C(NH)CH3 or C~NH)NH2, and the aromatic or benzenoid ring is substituted with one to three lower alkyl or alkoxy groups. The lower alkyl substituents in the general formula ~I) can be, for example, methyl, ethyl, iso-propyl or~butyl.
A most preferred compound is Arg-Gly-Asp-~
(O-methyltyrosine)-amide.
O O
11 : 1~
Arg-Gly-Asp-c-~-cH-c-NH2 This compound, in which the p-hydroxyphenyl ; group of tyrosine lS methylated and the C-terminal i6 carboxyamide, unexpectedly is substantially more active as an inhibitor of platelet aggregation than 1 31 5~79 -5- 07-21~453)A
ei~her Arg-Gly-Asp-Ser or Arg-Gly-Asp-Tyr. This advantage has been demonstrated in platelet-rich plasma assays and with in vivo assays where, by comparison, Arg-Gly-Asp~Ser is relatively ineffective.
Moreover, Arg-Gly-Asp-(O-methyltyrosine~-amide is effective in preventing thrombosis in a rat carotid artery assay model, thereby demonstrating its use as an antithrombotic agent.
In those instances where the C-terminal amino acid Y in the above formula (I) is a natural amino acid, it is an amide derivative of tyrosine or phenylalanine and the NH2-terminal amino acid X is acetyl-arginine. Thus, it has been surprisingly found that acetyl-RGDY-amide is very effective in the in VlVO thrombocytopenia assay (85% inhibition) whereas the non-acetylated RGDY-amide is relatively ineffective (only 19~ inhibition).
Other preferred compounds of the invention are Arg-Gly-Asp-(O-ethyltyrosine)-amide, des-amino-Arg~Gly-Asp-(O-methyltyrosine)-amide, des-amino-(homoarginine)-Gly-Asp-(O-methyltyrosine)-amide, acetyl-Arg-Gly-Asp-(O-methyltyrosine)-amide, acetyl-(D-Arg)-Gly-Asp-(0-methyltyrosine)-amide Arg-Gly-Asp-(4-metho~y-1-napthylalanine)-amide, Arg-Gly-Asp-~2,6-dimethyl-O-methyltyrosine)amide, and Arg-Gly-Asp-(p-phenyl-phenylalanine)-amide.
Detailed Description of the Invention The noveI peptides of this invention can be made by conventional methods of peptide synthesis. A
preferred method is the solid phase synthesis of Merrifield, J. Amer. ChemO Soc. 85, 2149-2154 (1963);
Science 150, 178-185 (1965); Ibid , 232, 341-347 (1986).
1 3 1 547q -6~ 07-21(453~A
Solid phase synthesis is generally commenced from the C-terminus of the peptide by coupling a protected alpha amino acid to a suitable resin, e.g., chloromethylated polystyrene resin or p-methylbenzhydrylamine resin when synthesizing a peptide amide derivative. In the present invention, the tyrosine derivative as described above can be used as the C-terminal peptide for initiating the solid phase synthesis. The three remaining alpha amino acids are then coupled stepwise in the desired order to obtain an intermediate peptide coupled to the resin. During this synthesis, suitable protecting groups are used as needed. Thus, aspartic acid is protected on the ~-carboxyl group as the ben~yl ester and arginine is protected on the guanidino group by tosyl. Each a-amino group is protectd with the t-butyloxycarbonyl group (BOC).
After the desired tetrapeptide sequence has been completed, the intermediate peptide is cleaved from the resin and protecting groups are removed by treatment with a reag~nt such as HF. The peptide can then be purified by high performance liquid chromatography (HPLC) or other such methods of protein purification.
Background information on the established solid phase synthesis procedure which can be used for the prepartion of the tetrapeptide derivatives herein can be had by reference to the treatise by Stewart and Young, I'Solid Phase Peptide Synthesis," W. H. Freeman & Co., San Francisco, 1969, and the review chapter by Merrifield in Advances in Enzymology 32, pp. 221-296, F. F. Nold, Ed., Interscience Publishers, New York, 1969; and Erickson and Merrifield, The Proteins, Vol.
2, p. 255 et seq. (ed. Neurath and Hill~, ~cademic Press, New York, 1976.
131~479 -7- 07-21(453)A
As used herein, the peptide sequences are shown by conventional single or three letter abbreviations for the constituent amino acids as follows:
Abbreviated Designation ~ Amino Acid A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan ~Y Tyr Tyrosine *Not to be confused with the C-terminal Y in the general formula ~I) for the tetrapeptide derivatives as defined herein.
-8- 07-21(453)A
The platelet-binding inhibitor activity of the peptide derivatives of this invention is demonstrated by various assays. In one assay, the peptides are tested for their inhibition of thrombin-induced platelet aggregation in washed human platelets. The % inhibition is determined for the test peptide by comparing the e~tent of platelet aggregation in the presence and absence of the peptide.
In another assay, platelet aggregation is examined in platelet-rich plasma which also is rich in fibrinogen and other plasma proteins.
In yet another test, the effect of the peptide on collagen induced thrombocytopenia (platelet aggregation) is measured in vivo in the rat. Again, the % inhibition is determined for the test peptlde and compared against a saline or ethanol vehicle in the absence of peptide.
- In these assays, the test compound results were then compared with the activity of the known active inhibitor tetrapeptide Arg-Gly-Asp-Ser.
:
Finally, a most preferred compound of this invention was tested in a rat carotid artery thrombosis bioassay. In this test, a thrombus is induced to form in a rat carotid artery by applying an electrical current to the artery for 5 minutes.
In the presence of infused saline, a clot forms and occludes the artery in about 8-9 minutes. Infusion of the preferred inhibitor compound of this invention, Arg-Gly-Asp-(O-methyltyrosine)-amide, significantly delayed or prevented occlusion whereas, by comparison, infusion o~ the known inhibitor Axg-Gly-Asp-Ser lengthened the time to occlusion only slightly.
1 31 5~79 -9- 07-21(453)A
The following examples will further illustrate the invention in greater detail although it will be appreciated that the invention is not limited to these specific examples.
S Example 1 The novel peptide derivatives of -this invention were made by conventional solid phase synthesis. This synthesis is illustrated by ; preparation of Arg-Gly-Asp-(O-methyltyrosinej-N~I2 as follows:
20 grams of p-methylbenzhydryl amine resin (containing 14 mmoles of amino groups) was shaken with 2 e~uivalents (eg.) of BOC-tyrosine methyl ether and 2 eq. of dicyclohexyl carbodiimide (DCC) in methylene chloride for 4 hours. The resin was filtered and washed repeatedly, with dimethyl-formamide (DMF3, followed by methanol and then methylene chloride. The BOC group was removed by treatment with 50% trifluoroacetic acid (TFA) in methylene chloride for 30 minutes and the rssin was washed with methylene chloxida, neutralized with 10%
diisopropylamine and washed again. The resin was then ready for reaction with 2 eq. o~ the next amino acid BOC-~-benzyl-aspartic~acid. The cycle as above described was repeated for each amino acid except that ~; ~ 2 eq. of hydroxybenzotriazole was added to the BOC-tosylarginine and~DCC.
The resulting tetrapeptide was removed from 10.0 grams of resin and deprotected with 88 ml. of 10% anisole in liquid HF at 0C, and ollowing evapor-ation of the HF, the peptide was taken up in 30%
aqueous acetic acid and lyophilized. The peptide product was purified by HPLC on a 700 ml. column of 15-20 ~m (300 A) *Vydac C18 reverse phase packing (The Separations Group, Hesperia, California) using a 0-50%
gradient in acetonitrile (0.1% TFA). Fractions *~rade Mark 1315~79 -10- 07-21(453)A
containing product, as ascertained by analytical HPLC, were pooled and lyophilized to afford about 1.0 gram of pure tetrapeptide from 10 grams of resin.
Substantially similar synthesis procedures were used for the solid phase synthesis of other peptide derivatives of this invention by substituting equivalent amounts of other BOC-tyrosine derivatives for the BOC tyrosine methyl ether, and/or des-amino arginine, homoarginine or acetyl arginine for arginine in the above example.
Example 2 The peptide derivatives prepared in E~ample 1 were tested for their platelet-binding inhibitor activity by the following standard protocol:
Inhibition of Thrombin-Induced Platelet Aggregation in Washed Human Platelets Platelet Preparation: 60 ml of whole blood is freshley drawn and anticoagulated with l/lOth volume of CCD(100 mM Na Citrate and 136 mM glucose, pH 6.5 with HCl) at room temperature (RT). The blood is divided into 2 disposable 60 ml plastic centrifuge tubes and centrifuged for 3 minutes at 1000 x g, allowing the centrifuge to coast to a stop (no brake). The platelet rich plasma (PRP) is withdrawn, being careful that no white cells are taken~and is placed in a 60 ml centrifuge tube. The tube is immediately placed on ice for 15 minutes. After the 15 minutes at 0C.,~ 1/2 volume of ice cold CCD is added (i.e. 15 ml CCD/30 ml of PRP). The tube is mixed and the contents are divided equaIly into two centrifuge tubes. These are then centrifuged at 0C
13154~
~11- 07-21(453)A
for 10 minutes at 900 x g (no brake~. The supernatant is carefully poured off. The platelet pellet is gently resuspended in 1/2 the original volume of PRP in a 0C modified Tangen-Hepes-BSA
buffer, pH 7.4, consisting of 145 mM NaCl, 5 mM KCl, O.05 mM CaCl2, 0.1 mM MgCl2, 11 mM glucose, 15 mM
Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid~, 1 mg/ml bovine serum albumin, (p~ adjusted with NaOH). The resuspended pellets are combined into 1 cen-trifuge tube and incubated undisturbed at 37~C for 30 minutes. After the incubation, the platelet suspension is removed and an aliquot is quickly counted in a hemocytometer or a Coulter~
Counter (Coulter Electronics, Hialeah, Fl.). The platelet count is adjusted to 3 x 108 cells/ml with the Tangen-~epes-BSA buffer.
Com~ound Testing: Aggregation studies are accomplished using a Payton aggregometer (Payton Scientific, Inc., Buffalo, N.Y~). The control compound used was RGDS (Arg-Gly-Asp-Ser) purchased from Peninsula Laboratories, Cal.; the thrombin was purchased from Parke-Davis, N.J. and prepared at a working concentration of 0.5 units/ml with the Tangen-Hepes-BSA buffer supplemented with 16 mM CaCl2, 16 mM MgCl2. All compounds are diluted to a working concentration Gf 10 3 M with double distilled water.
The reaction mixture consisted of 400 ~1 of 3 x 108/ml washed platelets, 50 ~1 of buffer(control) or test compound at 10 3 M with a final concentration of 10 4 M. Platelets, test compound or buffer are placed in cuvettes in the aggregometer for a 1 minute pre-incubation before adding thrombin. 50 ~1 of 0.5 U/ml thrombin is added to cuvettes and the aggregation is monitored for 1 minute, which is the time for maximal 1 31 5~7q -12- 0'7-21(453)A
aggregation of the platelets. All compounds are run in duplicate. The entire test is run within 3 hours, since this is the maximal viability of the platelets.
Results are calculated as follows: % of control =
[(maximal OD minus initial OD of compound) divided by (maximal OD minus initial OD of control)] x 100. %
inhibition = 100 minus (% of control).
The compounds tested and their activity results in % inhibition at 10 4 M and IC50's were as recorded in Table I. IC50's (if a compound showed 50%
inhibition) were calculated by linear regression of the dose response curve.
Table I
No. Peptide Sequence I % Inhi~ition ¦ IC50(M) I at lO M
RGDS 71 4 x 10 5 2 GRGDSP-NH2 55 8 x 10 4 Ac-RGDY-NH2 68 5 x 10 RGDF-NH2 77 1x 10 5 6 RGD~o-methyl-Tyr)-NH2 79 2x 10 5 7 RGD(o-ethyl-Tyr)-NH2 54 -- -5 8 Ac-RGD(0-methyl-TYr)~NH2 78 2x 10 5 9 RGD(2,6-dimethyl 78 2x 10 O-methyl-Tyr)-N~2 -5 10 des-NH2-RGD(O-methyl-Tyr)-NH2 90 1 x 10 5 11 des-NH2-(homo Arg)-GD- 82 1 x 10 (0-methyl-TYr)-NH2 Ac ~ Acetyl -13- 07-21(453)A
Several peptide derivatives prepared in Example 1 were further tested for their platelet-binding in platelet-rich plasma (PRP) by the following standard protocol:
In Vitro ~uman Platelet Aggrec~ation in PRP
Healthy male or female donors who have not taken any antiplatelet drugs for at least 2 weeks were fasted for 8 hours prior to drawing blood; then 30 ml whole blood was collected using a butterfly needle and 30 cc plastic syringe with 3 ml of 0.129 M
buffered sodium citrate ~3.8%). The syringe is rotated carefully as blood is being dra~l to mix the citrate. Platelet~rich plasma (PRP) is prepared by centrifugation at 100 x g for 10 minutes at room temperature, allowing the centrifuge to coast to a stop without braking. The PRP is removed fxom the blood with a plastic pipette and placed in a plastic, capped, 50 ml Corning conical sterile centrifuge tube. The tube is capped and placed at room temperature. Platelet poor plasma (PPP) is prepared by centrifuging the remaining blood at 2000 x g for 15 minutes at room temperature allowing the centrifuge to coast to a stop without bra~ing. The PRP is adjusted with PPP to a count of 2-3 x 108 pla-telets per ml. 400 ~l of the PRP preparation and 50 ~l of the compound to be tested or saline were preincubated for 1 minute at 37C. in a Payton aggregom~ter (Payton Scientific, Inc., Buffalo, NY~. 50 ~l of adenosine 5'diphosphate (ADP) (50 ~M) is added to the cuvettes and the aggregation is monitored for 1 minute. All compounds are tested in duplicate. The antire procedure is run within 3 hours, since this is the maximal viability of the platelets. The saline instead of compound is used to determine the maximal 1 3 1 5~7q -14- 07-21(453)A
aggregation. Results are calculated as follows:
Percent of control = [(maximal OD minus initial OD of compound~ divided by (maximal OD minus initial OD of control)] x 10Q. % inhibition = 100 - tpercent of control).
The compounds tested and 1heir activity results in % inhibition at 10 4 M and IC50's were as recorded in Table II. IC~o's (if a compound showed 50%
inhibition) were calculated by linear regression of the dose response curve.
Table II
No.Peptide Sequence 1 % Inhi~ition I IC50~M) 1 RGDS 25 1 x 10 2GRGDSP-~I2 23 2 x 10_5 3RGDF-NH2 100 2 x 10 4Ac-RGDY-NH2 48 __ 5RGD(o-methyl-Tyr)-NH2 lQ0 3 x 10 S
: 6Ac-RGD(O-methYl-TY~)-NH2 100 2 x 10 5 7RGD(2,6-dimethyl- 53 9 x 10 O-methyl-Tyr)-NH2 -5 8des-NH~-RGD(O-methyl-Tyr)-NH2 100 2 x 10 5 9des-N}I2-(homo Arg)GD- 100 2 x 10 (O-methyl-Tyr)-NH2 10RGD(4-methoxy-1- 100 1 x 10 `
napthyl-Ala)-NH2 -5 11des-NH2-RGD(O-methyltyramine)-NH2 100 2 x lO
12Ac-(D-Arg)GD(O-methyl-Tyr)-NH2 71 __ 13RGD(p-phenyl-phe)-NH2 100 From the above results in Table II it will be seen that compounds 4 to 13 were from 2 to 4 times - 35 as effective in % inhibition of platelet aggregation in plat01et-rich plasma n vitro compared to the control compound Nos. 1 and 2. While compound 3 also was effective in this in vitro test, it was less effecti~e in the in vivo test of Example 4, below.
1 31 5~7q 15- 07-21(453)~
Several peptide derivatives prepared in Example 1 were further tested for their effect on collagen induced thrombocytopenia ln vivo in the rat as follows:
In Vivo Rat Thrombocytopenia Male rats (Charles River, CRL:CD(SD), 400-450 g~ were used. The rats were anesthetized with Na pentabarbital ~65 mg/kg, Vet Labs, Limited, Inc., Lenexa, KA). Two incisions were made to expose both jugular veins. Using an infusion pump (Harvard Apparatus, South Natick, Mass.) and a 5 cc syringe with a 19 g. butterfly, the test compound or vehicle was infused into the left jugular vein at a rate of 0.39 ml/min for 3 min. After 2 min of compound/vehicle infusion, collagen (60 ~g/kg) (Helena Laboratories, Beaumont, TX) was injected with a 1 ml syringe into the right jugular vein. The body cavity was opened and the vena cava was exposed for blood sampling. One min after the collagen injection, compound in~usion was stopped and blood was sampled from the vena cava (withi~ 30 sec) with a 3 cc syringe containing 0.3 mg of 4.5% EDTA/Tris (0.1 M) (pH 7.35~ plus 15Q ~M indomethacin. Platelet rich plasma (PRP~ was prepared by centrifuging the blood at 126 x g for lQ min. Five ~l of PRP was counted in 20 ml of Isoton~ III in a Coulter Countex.
-16- 07-21(453)A
Percent inhibition of collagen induced aggregation was calculated by comparison of the number of platelets counted in treat~d animals with numbers from animal~
receiving no collagen and with counts from animals receiving vehicle and collagen. Estimation of potency was based on inhibition of collagen-induced thrombocytopenia.
The % inhibition of platelet aggregation in vivo and the maximum inhibition % of the test compounds is set forth in the following Table III.
: Four animals were tested with each compound to give the data shown.
Table III
-No. Peptide Sequence . % Inhibit~on Maximum at l mg~kg Inhibition %
1 RGDS 29 ~ 6 30 2 GRGDSP-NH2 18 ~ 3 20 3 RGDY-NH2 l9 + 12 20 4 RGDF-NH2 40 __ 5 Ac-RGDY-NH2 85 __ 6RGD(0-methyl Tyr)-NH2 75 ~ 4 80 7Ac-RGD(O-methyl-Tyr)-NH2 86 __ 8RGD(2,6-dimethyl- 70 __ O-methyl-Tyr)-NH2 9 des-NH2-RGD(o-methyl-Tyr)-NH2 64 __ 10 des-NH2-(homo Arg~GD-~ 60 __ (0-methyl-Tyr)-NH2 From the above resuIts in Table III it will be seen that preferred compounds of the invention (Nos. 5 to 10) were from about 2 to 4 times as effective in % inhibition of platelet aggregation 3 5 in vivo compared to the control compound Nos. 1 to 3. Compound 4 had intermediate effectiveness.
1 31 51~7q -17- 07-21~453)A
E~ample 5 The tetrapeptide derivative Arg-51y-Asp-(O-methyltyrosine)~amide was still further tested for its activity against rat carotld artery thrombosis as follows:
Rat Carotid Artery Thrombosis Male Sprague-Dawley rats (300-450 gms) are anesthetized with sodium pentabarbital i.p. 30 mg/kg.
A mid-line incision is made in the neck through which the trachea, jugular vein and carotid artery are exposed and isolated. The trachea is cannulated and the animal is allowed to breathe 2 enriched room air.
The jugular vein is cannulated for i.v. infusion. The carotid artery is stripped of its sheath and all vagal fibers for a distance of 1.5-2.0 cm and fitted on the proximal end with an appropriately sized Carolina Medical Electronics electro magnetic flow probe.
Recording of blood flow is done on a Gould recorder via the Carolina Medical Electronics flow meter. A
mechanical zero flow is determined by momentarily clamping the artery distal to the flow probe. A few millimeters distal to th flow probe, a bipolar electrode is placed on the artery and positioned so ` that it touches only the artery.
After I5 minutes for stabilization after the surgical preparation, an i.v. infusion of the desired dose of peptide is begun into the jugular vein (using a Harvard infusion pump) and allowed to run for 5 minutes at which time th flowmeter lS
turned off and an electrical current of 2.5 mA is applied to the external arterial wall (Grass stimulator and constant current unit~ for 5 minutes.
The infusion of peptide is allowed to run throughout the test time period of 30 minutes.
1 31 547~
-18- 07-21(453)A
Immediately after discontinuing the electrical current the flow meter is turned on and measurements o flow amplitude are taken. At the point of a 20% decrease in systolic flow, thrombus formation has begun and the time from end of current to 20% flow decrease is noted. This is the "time in minutes to onset of thrombus formation." When flow declines to the predetermined 0 flow, time in minutes from onset of thrombus is noted and this time is called "Time in minutes to 0 flow from onset of thrombus." The sum of these times is "time from injury to 0 flow." The latter time (or time to occlusion) for the test compound compared to that of the known inhibitor Arg-Gly-Asp-Ser is set forth in the following Table IV.
Table IV
Peptide Number I Time to (Dose infused) of Animals Occlusion (minutes) Saline 6 8.9 ~ 0.8 RGDS
(0.05 mg/kg/min.) 6 13 ~ 1 (1.0 mg/kg/min.) 6 12 ~ 2 RGD (O-methyl-Tyr)~NH2 (1.0 mg/kg/min.) 4 15 (with 2 ~ ~ (with 2 animals) Occlusion did not occur even 30 minutes after infusion was stopped.
-19- 07-21~453)A
The novel tetrapeptide derivatives of this invention can be used for administration to humans by conventional means, preferably in formulations with pharmaceutically acceptable diluents or carriers.
The preferable route of administration as a platele-t aggregation inhibitor is parenteral, especially intravenous. Intravenous administration of the tetrapeptide derivatives in solutioIl with normal physiological saline, human albumin and other such diluents and carriers is illustrative. Other suitable formulations of the active tetrapeptide derivatives in pharmaceutically acceptable diluents and carriers in therapeutic dosage form can be prepared by reference to general texts in the pharmaceutical field such as, for example, Reminyton's Pharmaceutical Sciences, Ed. Arthur Osol, 16th ed., 1980, Mack Publishing Co., Easton, Pennsylvania.
Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such examples be included with th scope of the appended claims.
The platelet-binding inhibitor activity of the peptide derivatives of this invention is demonstrated by various assays. In one assay, the peptides are tested for their inhibition of thrombin-induced platelet aggregation in washed human platelets. The % inhibition is determined for the test peptide by comparing the e~tent of platelet aggregation in the presence and absence of the peptide.
In another assay, platelet aggregation is examined in platelet-rich plasma which also is rich in fibrinogen and other plasma proteins.
In yet another test, the effect of the peptide on collagen induced thrombocytopenia (platelet aggregation) is measured in vivo in the rat. Again, the % inhibition is determined for the test peptlde and compared against a saline or ethanol vehicle in the absence of peptide.
- In these assays, the test compound results were then compared with the activity of the known active inhibitor tetrapeptide Arg-Gly-Asp-Ser.
:
Finally, a most preferred compound of this invention was tested in a rat carotid artery thrombosis bioassay. In this test, a thrombus is induced to form in a rat carotid artery by applying an electrical current to the artery for 5 minutes.
In the presence of infused saline, a clot forms and occludes the artery in about 8-9 minutes. Infusion of the preferred inhibitor compound of this invention, Arg-Gly-Asp-(O-methyltyrosine)-amide, significantly delayed or prevented occlusion whereas, by comparison, infusion o~ the known inhibitor Axg-Gly-Asp-Ser lengthened the time to occlusion only slightly.
1 31 5~79 -9- 07-21(453)A
The following examples will further illustrate the invention in greater detail although it will be appreciated that the invention is not limited to these specific examples.
S Example 1 The novel peptide derivatives of -this invention were made by conventional solid phase synthesis. This synthesis is illustrated by ; preparation of Arg-Gly-Asp-(O-methyltyrosinej-N~I2 as follows:
20 grams of p-methylbenzhydryl amine resin (containing 14 mmoles of amino groups) was shaken with 2 e~uivalents (eg.) of BOC-tyrosine methyl ether and 2 eq. of dicyclohexyl carbodiimide (DCC) in methylene chloride for 4 hours. The resin was filtered and washed repeatedly, with dimethyl-formamide (DMF3, followed by methanol and then methylene chloride. The BOC group was removed by treatment with 50% trifluoroacetic acid (TFA) in methylene chloride for 30 minutes and the rssin was washed with methylene chloxida, neutralized with 10%
diisopropylamine and washed again. The resin was then ready for reaction with 2 eq. o~ the next amino acid BOC-~-benzyl-aspartic~acid. The cycle as above described was repeated for each amino acid except that ~; ~ 2 eq. of hydroxybenzotriazole was added to the BOC-tosylarginine and~DCC.
The resulting tetrapeptide was removed from 10.0 grams of resin and deprotected with 88 ml. of 10% anisole in liquid HF at 0C, and ollowing evapor-ation of the HF, the peptide was taken up in 30%
aqueous acetic acid and lyophilized. The peptide product was purified by HPLC on a 700 ml. column of 15-20 ~m (300 A) *Vydac C18 reverse phase packing (The Separations Group, Hesperia, California) using a 0-50%
gradient in acetonitrile (0.1% TFA). Fractions *~rade Mark 1315~79 -10- 07-21(453)A
containing product, as ascertained by analytical HPLC, were pooled and lyophilized to afford about 1.0 gram of pure tetrapeptide from 10 grams of resin.
Substantially similar synthesis procedures were used for the solid phase synthesis of other peptide derivatives of this invention by substituting equivalent amounts of other BOC-tyrosine derivatives for the BOC tyrosine methyl ether, and/or des-amino arginine, homoarginine or acetyl arginine for arginine in the above example.
Example 2 The peptide derivatives prepared in E~ample 1 were tested for their platelet-binding inhibitor activity by the following standard protocol:
Inhibition of Thrombin-Induced Platelet Aggregation in Washed Human Platelets Platelet Preparation: 60 ml of whole blood is freshley drawn and anticoagulated with l/lOth volume of CCD(100 mM Na Citrate and 136 mM glucose, pH 6.5 with HCl) at room temperature (RT). The blood is divided into 2 disposable 60 ml plastic centrifuge tubes and centrifuged for 3 minutes at 1000 x g, allowing the centrifuge to coast to a stop (no brake). The platelet rich plasma (PRP) is withdrawn, being careful that no white cells are taken~and is placed in a 60 ml centrifuge tube. The tube is immediately placed on ice for 15 minutes. After the 15 minutes at 0C.,~ 1/2 volume of ice cold CCD is added (i.e. 15 ml CCD/30 ml of PRP). The tube is mixed and the contents are divided equaIly into two centrifuge tubes. These are then centrifuged at 0C
13154~
~11- 07-21(453)A
for 10 minutes at 900 x g (no brake~. The supernatant is carefully poured off. The platelet pellet is gently resuspended in 1/2 the original volume of PRP in a 0C modified Tangen-Hepes-BSA
buffer, pH 7.4, consisting of 145 mM NaCl, 5 mM KCl, O.05 mM CaCl2, 0.1 mM MgCl2, 11 mM glucose, 15 mM
Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid~, 1 mg/ml bovine serum albumin, (p~ adjusted with NaOH). The resuspended pellets are combined into 1 cen-trifuge tube and incubated undisturbed at 37~C for 30 minutes. After the incubation, the platelet suspension is removed and an aliquot is quickly counted in a hemocytometer or a Coulter~
Counter (Coulter Electronics, Hialeah, Fl.). The platelet count is adjusted to 3 x 108 cells/ml with the Tangen-~epes-BSA buffer.
Com~ound Testing: Aggregation studies are accomplished using a Payton aggregometer (Payton Scientific, Inc., Buffalo, N.Y~). The control compound used was RGDS (Arg-Gly-Asp-Ser) purchased from Peninsula Laboratories, Cal.; the thrombin was purchased from Parke-Davis, N.J. and prepared at a working concentration of 0.5 units/ml with the Tangen-Hepes-BSA buffer supplemented with 16 mM CaCl2, 16 mM MgCl2. All compounds are diluted to a working concentration Gf 10 3 M with double distilled water.
The reaction mixture consisted of 400 ~1 of 3 x 108/ml washed platelets, 50 ~1 of buffer(control) or test compound at 10 3 M with a final concentration of 10 4 M. Platelets, test compound or buffer are placed in cuvettes in the aggregometer for a 1 minute pre-incubation before adding thrombin. 50 ~1 of 0.5 U/ml thrombin is added to cuvettes and the aggregation is monitored for 1 minute, which is the time for maximal 1 31 5~7q -12- 0'7-21(453)A
aggregation of the platelets. All compounds are run in duplicate. The entire test is run within 3 hours, since this is the maximal viability of the platelets.
Results are calculated as follows: % of control =
[(maximal OD minus initial OD of compound) divided by (maximal OD minus initial OD of control)] x 100. %
inhibition = 100 minus (% of control).
The compounds tested and their activity results in % inhibition at 10 4 M and IC50's were as recorded in Table I. IC50's (if a compound showed 50%
inhibition) were calculated by linear regression of the dose response curve.
Table I
No. Peptide Sequence I % Inhi~ition ¦ IC50(M) I at lO M
RGDS 71 4 x 10 5 2 GRGDSP-NH2 55 8 x 10 4 Ac-RGDY-NH2 68 5 x 10 RGDF-NH2 77 1x 10 5 6 RGD~o-methyl-Tyr)-NH2 79 2x 10 5 7 RGD(o-ethyl-Tyr)-NH2 54 -- -5 8 Ac-RGD(0-methyl-TYr)~NH2 78 2x 10 5 9 RGD(2,6-dimethyl 78 2x 10 O-methyl-Tyr)-N~2 -5 10 des-NH2-RGD(O-methyl-Tyr)-NH2 90 1 x 10 5 11 des-NH2-(homo Arg)-GD- 82 1 x 10 (0-methyl-TYr)-NH2 Ac ~ Acetyl -13- 07-21(453)A
Several peptide derivatives prepared in Example 1 were further tested for their platelet-binding in platelet-rich plasma (PRP) by the following standard protocol:
In Vitro ~uman Platelet Aggrec~ation in PRP
Healthy male or female donors who have not taken any antiplatelet drugs for at least 2 weeks were fasted for 8 hours prior to drawing blood; then 30 ml whole blood was collected using a butterfly needle and 30 cc plastic syringe with 3 ml of 0.129 M
buffered sodium citrate ~3.8%). The syringe is rotated carefully as blood is being dra~l to mix the citrate. Platelet~rich plasma (PRP) is prepared by centrifugation at 100 x g for 10 minutes at room temperature, allowing the centrifuge to coast to a stop without braking. The PRP is removed fxom the blood with a plastic pipette and placed in a plastic, capped, 50 ml Corning conical sterile centrifuge tube. The tube is capped and placed at room temperature. Platelet poor plasma (PPP) is prepared by centrifuging the remaining blood at 2000 x g for 15 minutes at room temperature allowing the centrifuge to coast to a stop without bra~ing. The PRP is adjusted with PPP to a count of 2-3 x 108 pla-telets per ml. 400 ~l of the PRP preparation and 50 ~l of the compound to be tested or saline were preincubated for 1 minute at 37C. in a Payton aggregom~ter (Payton Scientific, Inc., Buffalo, NY~. 50 ~l of adenosine 5'diphosphate (ADP) (50 ~M) is added to the cuvettes and the aggregation is monitored for 1 minute. All compounds are tested in duplicate. The antire procedure is run within 3 hours, since this is the maximal viability of the platelets. The saline instead of compound is used to determine the maximal 1 3 1 5~7q -14- 07-21(453)A
aggregation. Results are calculated as follows:
Percent of control = [(maximal OD minus initial OD of compound~ divided by (maximal OD minus initial OD of control)] x 10Q. % inhibition = 100 - tpercent of control).
The compounds tested and 1heir activity results in % inhibition at 10 4 M and IC50's were as recorded in Table II. IC~o's (if a compound showed 50%
inhibition) were calculated by linear regression of the dose response curve.
Table II
No.Peptide Sequence 1 % Inhi~ition I IC50~M) 1 RGDS 25 1 x 10 2GRGDSP-~I2 23 2 x 10_5 3RGDF-NH2 100 2 x 10 4Ac-RGDY-NH2 48 __ 5RGD(o-methyl-Tyr)-NH2 lQ0 3 x 10 S
: 6Ac-RGD(O-methYl-TY~)-NH2 100 2 x 10 5 7RGD(2,6-dimethyl- 53 9 x 10 O-methyl-Tyr)-NH2 -5 8des-NH~-RGD(O-methyl-Tyr)-NH2 100 2 x 10 5 9des-N}I2-(homo Arg)GD- 100 2 x 10 (O-methyl-Tyr)-NH2 10RGD(4-methoxy-1- 100 1 x 10 `
napthyl-Ala)-NH2 -5 11des-NH2-RGD(O-methyltyramine)-NH2 100 2 x lO
12Ac-(D-Arg)GD(O-methyl-Tyr)-NH2 71 __ 13RGD(p-phenyl-phe)-NH2 100 From the above results in Table II it will be seen that compounds 4 to 13 were from 2 to 4 times - 35 as effective in % inhibition of platelet aggregation in plat01et-rich plasma n vitro compared to the control compound Nos. 1 and 2. While compound 3 also was effective in this in vitro test, it was less effecti~e in the in vivo test of Example 4, below.
1 31 5~7q 15- 07-21(453)~
Several peptide derivatives prepared in Example 1 were further tested for their effect on collagen induced thrombocytopenia ln vivo in the rat as follows:
In Vivo Rat Thrombocytopenia Male rats (Charles River, CRL:CD(SD), 400-450 g~ were used. The rats were anesthetized with Na pentabarbital ~65 mg/kg, Vet Labs, Limited, Inc., Lenexa, KA). Two incisions were made to expose both jugular veins. Using an infusion pump (Harvard Apparatus, South Natick, Mass.) and a 5 cc syringe with a 19 g. butterfly, the test compound or vehicle was infused into the left jugular vein at a rate of 0.39 ml/min for 3 min. After 2 min of compound/vehicle infusion, collagen (60 ~g/kg) (Helena Laboratories, Beaumont, TX) was injected with a 1 ml syringe into the right jugular vein. The body cavity was opened and the vena cava was exposed for blood sampling. One min after the collagen injection, compound in~usion was stopped and blood was sampled from the vena cava (withi~ 30 sec) with a 3 cc syringe containing 0.3 mg of 4.5% EDTA/Tris (0.1 M) (pH 7.35~ plus 15Q ~M indomethacin. Platelet rich plasma (PRP~ was prepared by centrifuging the blood at 126 x g for lQ min. Five ~l of PRP was counted in 20 ml of Isoton~ III in a Coulter Countex.
-16- 07-21(453)A
Percent inhibition of collagen induced aggregation was calculated by comparison of the number of platelets counted in treat~d animals with numbers from animal~
receiving no collagen and with counts from animals receiving vehicle and collagen. Estimation of potency was based on inhibition of collagen-induced thrombocytopenia.
The % inhibition of platelet aggregation in vivo and the maximum inhibition % of the test compounds is set forth in the following Table III.
: Four animals were tested with each compound to give the data shown.
Table III
-No. Peptide Sequence . % Inhibit~on Maximum at l mg~kg Inhibition %
1 RGDS 29 ~ 6 30 2 GRGDSP-NH2 18 ~ 3 20 3 RGDY-NH2 l9 + 12 20 4 RGDF-NH2 40 __ 5 Ac-RGDY-NH2 85 __ 6RGD(0-methyl Tyr)-NH2 75 ~ 4 80 7Ac-RGD(O-methyl-Tyr)-NH2 86 __ 8RGD(2,6-dimethyl- 70 __ O-methyl-Tyr)-NH2 9 des-NH2-RGD(o-methyl-Tyr)-NH2 64 __ 10 des-NH2-(homo Arg~GD-~ 60 __ (0-methyl-Tyr)-NH2 From the above resuIts in Table III it will be seen that preferred compounds of the invention (Nos. 5 to 10) were from about 2 to 4 times as effective in % inhibition of platelet aggregation 3 5 in vivo compared to the control compound Nos. 1 to 3. Compound 4 had intermediate effectiveness.
1 31 51~7q -17- 07-21~453)A
E~ample 5 The tetrapeptide derivative Arg-51y-Asp-(O-methyltyrosine)~amide was still further tested for its activity against rat carotld artery thrombosis as follows:
Rat Carotid Artery Thrombosis Male Sprague-Dawley rats (300-450 gms) are anesthetized with sodium pentabarbital i.p. 30 mg/kg.
A mid-line incision is made in the neck through which the trachea, jugular vein and carotid artery are exposed and isolated. The trachea is cannulated and the animal is allowed to breathe 2 enriched room air.
The jugular vein is cannulated for i.v. infusion. The carotid artery is stripped of its sheath and all vagal fibers for a distance of 1.5-2.0 cm and fitted on the proximal end with an appropriately sized Carolina Medical Electronics electro magnetic flow probe.
Recording of blood flow is done on a Gould recorder via the Carolina Medical Electronics flow meter. A
mechanical zero flow is determined by momentarily clamping the artery distal to the flow probe. A few millimeters distal to th flow probe, a bipolar electrode is placed on the artery and positioned so ` that it touches only the artery.
After I5 minutes for stabilization after the surgical preparation, an i.v. infusion of the desired dose of peptide is begun into the jugular vein (using a Harvard infusion pump) and allowed to run for 5 minutes at which time th flowmeter lS
turned off and an electrical current of 2.5 mA is applied to the external arterial wall (Grass stimulator and constant current unit~ for 5 minutes.
The infusion of peptide is allowed to run throughout the test time period of 30 minutes.
1 31 547~
-18- 07-21(453)A
Immediately after discontinuing the electrical current the flow meter is turned on and measurements o flow amplitude are taken. At the point of a 20% decrease in systolic flow, thrombus formation has begun and the time from end of current to 20% flow decrease is noted. This is the "time in minutes to onset of thrombus formation." When flow declines to the predetermined 0 flow, time in minutes from onset of thrombus is noted and this time is called "Time in minutes to 0 flow from onset of thrombus." The sum of these times is "time from injury to 0 flow." The latter time (or time to occlusion) for the test compound compared to that of the known inhibitor Arg-Gly-Asp-Ser is set forth in the following Table IV.
Table IV
Peptide Number I Time to (Dose infused) of Animals Occlusion (minutes) Saline 6 8.9 ~ 0.8 RGDS
(0.05 mg/kg/min.) 6 13 ~ 1 (1.0 mg/kg/min.) 6 12 ~ 2 RGD (O-methyl-Tyr)~NH2 (1.0 mg/kg/min.) 4 15 (with 2 ~ ~ (with 2 animals) Occlusion did not occur even 30 minutes after infusion was stopped.
-19- 07-21~453)A
The novel tetrapeptide derivatives of this invention can be used for administration to humans by conventional means, preferably in formulations with pharmaceutically acceptable diluents or carriers.
The preferable route of administration as a platele-t aggregation inhibitor is parenteral, especially intravenous. Intravenous administration of the tetrapeptide derivatives in solutioIl with normal physiological saline, human albumin and other such diluents and carriers is illustrative. Other suitable formulations of the active tetrapeptide derivatives in pharmaceutically acceptable diluents and carriers in therapeutic dosage form can be prepared by reference to general texts in the pharmaceutical field such as, for example, Reminyton's Pharmaceutical Sciences, Ed. Arthur Osol, 16th ed., 1980, Mack Publishing Co., Easton, Pennsylvania.
Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such examples be included with th scope of the appended claims.
Claims (15)
1. A tetrapeptide derivative having inhibitory activity toward platelet aggregation selected from the group consisting of X-Gly-Asp-Y
wherein X = or Ac-Arg, Z = H, NH2 or NH-Acyl, n = 1 to 4, wherein Y = , Tyr-NH2 or Phe-NH2, R1 = H, alkyl, phenyl or phenylalkyl, R2 = H, COOH, CONH2, COCH3, CH2OH, CH2NH2, C(H)CH3 or C(NH)NH2, R3 = phenyl, biphenyl or napthyl, each substituted with 1 to 3 alkyl or alkoxy groups, or an unsubstituted napthyl or pyridyl group, m = 0 to 2, wherein alkyl and alkoxy each have 1 to 4 carbons, provided that when Y is Tyr-NH2 or Phe-NH2, X is Ac-Arg, and provided further that when Z is NH2 or NH-Acyl, X is in the D- or L-amino acid stereo-configuration.
-21- 07-21(453)A
wherein X = or Ac-Arg, Z = H, NH2 or NH-Acyl, n = 1 to 4, wherein Y = , Tyr-NH2 or Phe-NH2, R1 = H, alkyl, phenyl or phenylalkyl, R2 = H, COOH, CONH2, COCH3, CH2OH, CH2NH2, C(H)CH3 or C(NH)NH2, R3 = phenyl, biphenyl or napthyl, each substituted with 1 to 3 alkyl or alkoxy groups, or an unsubstituted napthyl or pyridyl group, m = 0 to 2, wherein alkyl and alkoxy each have 1 to 4 carbons, provided that when Y is Tyr-NH2 or Phe-NH2, X is Ac-Arg, and provided further that when Z is NH2 or NH-Acyl, X is in the D- or L-amino acid stereo-configuration.
-21- 07-21(453)A
2. The tetrapeptide derivative of Claim 1 having the sequence Arg-Gly-Asp-(O-methyltyrosine)-amide.
3. The tetrapeptide derivative of Claim 1 having the sequence Arg-Gly-Asp-(O-ethyltyrosine)-amide.
4. The tetrapeptide derivative of Claim 1 having the sequence acetyl-Arg-Gly-Asp-(O-methyl tyrosine)-amide.
5. The tetrapeptide derivative of Claim 1 having the seguence acetyl-(D-Arg)-Gly-Asp-O-methyltyrosine)-amide.
6. The tetrapeptide derivative of Claim 1 having the sequence Arg-Gly-Asp-(4-methoxy-1-napthylalanine)-amide.
7. The tetrapeptide derivative of Claim 1 having the sequence Arg-Gly-Asp (2,6-dimethyl-O-methyltyrosine)-amide.
8. The tetrapeptide derivative of Claim 1 having the seguence desamino-Arg-Gly-Asp-(O-methyltyrosine)-amide.
9. The tetrapeptide derivative of Claim l having the sequence desamino-(homoarginine)-Gly-Asp-(O-methyl-tyrosine)-amide.
10. The tetrapeptide derivative of Claim 1 having the sequence Arg-Gly-Asp-(p-phenyl-phenylalanine)-amide.
11. The tetrapeptide derivative of claim 1 having the sequence acetyl-Arg-Gly-Asp-Tyr-amide.
12. The tetrapeptide derivative of claim 1, wherein Y is a tyrosine derivative.
13. The tetrapeptide derivative of claim 1, wherein n = 3.
14. Use of the tetrapeptide derivative of any one of claims 1 to 11 for inhibiting platelet aggregation.
15. Use of the tetrapeptide derivative of any one of claims 1 to 11 for inhibiting thrombus formation.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/128,379 US4857508A (en) | 1987-12-03 | 1987-12-03 | Novel platelet-aggregation inhibitor peptide derivatives |
| US128,379 | 1987-12-03 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1315479C true CA1315479C (en) | 1993-03-30 |
Family
ID=22435083
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000584873A Expired - Fee Related CA1315479C (en) | 1987-12-03 | 1988-12-02 | Platelet-aggregation inhibitor peptide derivatives |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US4857508A (en) |
| EP (1) | EP0319506B1 (en) |
| JP (1) | JPH01190699A (en) |
| AT (1) | ATE102629T1 (en) |
| CA (1) | CA1315479C (en) |
| DE (1) | DE3888310T2 (en) |
| ES (1) | ES2061733T3 (en) |
Families Citing this family (79)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5041380A (en) * | 1982-08-04 | 1991-08-20 | La Jolla Cancer Research Foundation | Tetrapeptide |
| US20030143733A1 (en) * | 1985-06-17 | 2003-07-31 | La Jolla Cancer Research Foundation | Tetrapeptide |
| CA1311583C (en) * | 1986-12-15 | 1992-12-15 | Gerard Armand Marguerie De Rotrou | Peptide derivatives and their application, in particular in therapy |
| US5827821A (en) * | 1987-12-10 | 1998-10-27 | The Burnham Institute | Conformationally stabilized cell adhesion peptides |
| EP0394326B1 (en) * | 1987-12-10 | 1996-07-31 | La Jolla Cancer Research Foundation | Methods for the production of conformationally stabilized cell adhesion peptides |
| ZW6189A1 (en) * | 1988-05-09 | 1990-05-09 | Smithkline Beckman Corp | Anti-aggregatory peptides |
| US4879313A (en) * | 1988-07-20 | 1989-11-07 | Mosanto Company | Novel platelet-aggregation inhibitors |
| US5037808A (en) * | 1988-07-20 | 1991-08-06 | Monsanto Co. | Indolyl platelet-aggregation inhibitors |
| US5053393A (en) * | 1988-07-20 | 1991-10-01 | Monsanto Company | Novel platelet-aggregation inhibitor |
| US5654267A (en) * | 1988-12-20 | 1997-08-05 | La Jolla Cancer Research Center | Cooperative combinations of ligands contained within a matrix |
| KR910700063A (en) * | 1988-12-20 | 1991-03-13 | 알. 더글라스 암스트롱 | Polypeptide-polymer conjugates having wound healing activity |
| CA2008116C (en) * | 1989-02-23 | 2001-11-20 | Thomas Weller | Glycine derivatives |
| US5318899A (en) * | 1989-06-16 | 1994-06-07 | Cor Therapeutics, Inc. | Platelet aggregation inhibitors |
| US5344783A (en) * | 1989-06-16 | 1994-09-06 | Cor Therapeutics, Inc. | Platelet aggregation inhibitors |
| US5686568A (en) * | 1989-06-16 | 1997-11-11 | Cor Therapeutics, Inc. | Platelet aggregration inhibitors |
| US5851839A (en) * | 1989-06-16 | 1998-12-22 | Cor Therapeutics, Inc. | Platelet aggregation inhibitors |
| US5384309A (en) | 1989-07-17 | 1995-01-24 | Genentech, Inc. | Cyclized peptides and their use as platelet aggregation inhibitors |
| US5061693A (en) * | 1989-07-28 | 1991-10-29 | Merck & Co., Inc. | Fibrinogen receptor antagonists |
| US5328840A (en) * | 1989-08-15 | 1994-07-12 | The Research Foundation Of The State University Of New York | Method for preparing targeted carrier erythrocytes |
| US5120859A (en) * | 1989-09-22 | 1992-06-09 | Genentech, Inc. | Chimeric amino acid analogues |
| US4952562A (en) * | 1989-09-29 | 1990-08-28 | Rorer Pharmaceutical Corporation | Anti-thrombotic peptides and pseudopeptides |
| EP0494248A4 (en) * | 1989-09-29 | 1992-08-26 | Rhone-Poulenc Rorer International (Holdings) Inc. | Anti-thrombotic peptides and pseudopeptides |
| US5332726A (en) * | 1989-09-29 | 1994-07-26 | Rhone-Poulenc Rorer Pharmaceuticals Inc. | Antithrombotic peptides and pseudopeptides |
| US5051405A (en) * | 1989-10-10 | 1991-09-24 | Rhone-Poulenc Rorer Pharmaceuticals Inc. | Anti-thrombotic peptides and pseudopeptides |
| NZ235564A (en) * | 1989-10-13 | 1993-10-26 | Merck & Co Inc | Fibrinogen receptor antagonist and pharmaceutical compositions |
| US5338723A (en) * | 1989-10-13 | 1994-08-16 | Merck & Co., Inc. | Fibrinogen receptor antagonists |
| US5643872A (en) * | 1989-10-23 | 1997-07-01 | Smithkline Beecham Corporation | Cyclic anti-aggregatory peptides |
| US5053392A (en) * | 1989-12-01 | 1991-10-01 | Rhone-Poulenc Rorer Pharmaceuticals Inc. | Novel arginine, glycine, aspartic acid derivatives as platelet-aggregation inhibitors |
| US5780303A (en) * | 1990-04-06 | 1998-07-14 | La Jolla Cancer Research Foundation | Method and composition for treating thrombosis |
| CA2079606A1 (en) * | 1990-04-06 | 1991-10-07 | Michael D. Pierschbacher | 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 |
| US5612311A (en) * | 1990-04-06 | 1997-03-18 | 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 |
| US5648330A (en) * | 1990-04-06 | 1997-07-15 | La Jolla Cancer Research Foundation | Method and composition for treating vascular graft occlusion |
| WO1992007872A1 (en) * | 1990-10-29 | 1992-05-14 | California Institute Of Biological Research | Tenascin related peptides |
| JPH06502643A (en) * | 1990-11-01 | 1994-03-24 | スミスクライン・ビーチャム・コーポレイション | γ-turn peptidomimetic compounds as fibrinogen antagonists |
| US5179082A (en) * | 1990-11-13 | 1993-01-12 | Merck & Co., Inc. | Method for blocking platelet adhesion to collagen |
| US5561220A (en) * | 1991-02-08 | 1996-10-01 | Diatech, Inc. | Technetium-99m labeled peptides for imaging inflammation |
| DK0570493T3 (en) * | 1991-02-08 | 2000-06-26 | Diatide Inc | Technetium-99m-labeled polypeptides for imaging. |
| US6107459A (en) * | 1991-02-08 | 2000-08-22 | Diatide, Inc. | Technetium-99m labeled peptides for diagnostic imaging |
| US6019958A (en) * | 1991-02-08 | 2000-02-01 | Diatide, Inc. | Technetium-99m labeled peptides for imaging inflammation |
| US5645815A (en) * | 1991-02-08 | 1997-07-08 | Diatide, Inc. | Radiolabled compounds for thrombus imaging |
| KR100210206B1 (en) * | 1991-03-06 | 1999-07-15 | 윌리암스 로저 에이 | Phenylamidines derivatives useful as platelet aggregation inhibitors |
| CA2106314A1 (en) * | 1991-04-05 | 1992-10-06 | John P. Burnier | Platelet aggregation inhibitors having high specification for gpiibiiia |
| WO1992018117A1 (en) * | 1991-04-11 | 1992-10-29 | Rhone-Poulenc Rorer International (Holdings), Inc. | Anti-thrombotic peptide and pseudopeptide derivatives |
| EP0513675B1 (en) * | 1991-05-13 | 1996-09-11 | Fujisawa Pharmaceutical Co., Ltd. | New peptide compound and a process for the preparation thereof |
| US5220050A (en) * | 1991-05-17 | 1993-06-15 | G. D. Searle & Co. | Peptide mimetic compounds useful as platelet aggregation inhibitors |
| AU2296792A (en) * | 1991-06-28 | 1993-01-25 | Corvas International, Inc. | Novel inhibitors of platelet aggregation |
| DE4126277A1 (en) * | 1991-08-08 | 1993-02-11 | Cassella Ag | HYDANTO DERIVATIVES |
| IL103252A (en) | 1991-09-30 | 1997-03-18 | Du Pont Merck Pharma | CYCLIC COMPOUNDS USEFUL AS INHIBITORS OF PLATELET GLYCOPROTEIN IIb/IIIa AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
| US5635477A (en) * | 1991-09-30 | 1997-06-03 | The Dupont Merck Pharmaceutical Company | Cyclic compounds useful as inhibitors of platelet glycoprotein IIB/IIIA |
| US5254573A (en) * | 1991-10-15 | 1993-10-19 | Monsanto Company | Substituted heterocyclic derivatives useful as platelet aggregation inhibitors |
| US5625093A (en) * | 1991-10-15 | 1997-04-29 | G. D. Searle & Co. | Substituted β-amino acid derivatives useful as platelet aggregation inhibitors |
| US5239113A (en) * | 1991-10-15 | 1993-08-24 | Monsanto Company | Substituted β-amino acid derivatives useful as platelet aggregation inhibitors and intermediates thereof |
| WO1993008818A1 (en) * | 1991-11-07 | 1993-05-13 | The University Of Southern California | Compositions and methods for preventing adhesion formation |
| US5441974A (en) * | 1991-12-13 | 1995-08-15 | G. D. Searle & Co. | Phenyl amidines lactones useful as platelet aggregation inhibitors |
| US5424334A (en) * | 1991-12-19 | 1995-06-13 | G. D. Searle & Co. | Peptide mimetic compounds useful as platelet aggregation inhibitors |
| US5264457A (en) * | 1992-02-14 | 1993-11-23 | G. D. Searle & Co. | Phenyl amidines sulfonamides useful as platelet aggregation inhibitors |
| LT3284B (en) | 1992-04-23 | 1995-06-26 | Merck & Co Inc | Fibrinogen receptor antagonists |
| US5504106A (en) * | 1992-06-25 | 1996-04-02 | G. D. Searle & Co. | Phenyl amidine alkanoic acids and lactones useful as platelet aggregation inhibitors |
| CA2101599A1 (en) * | 1992-08-31 | 1994-03-01 | Wilhelm Bannwarth | Tri- and tetracyclic compounds |
| EP0672059A1 (en) * | 1992-11-18 | 1995-09-20 | The Du Pont Merck Pharmaceutical Company | CYCLIC COMPOUNDS LINKED BY A HETEROCYCLIC RING USEFUL AS INHIBITORS OF PLATELET GLYCOPROTEIN IIb/IIIa |
| US5409939A (en) * | 1993-02-12 | 1995-04-25 | G. D. Searle & Co. | Phenyl amidine thio derivatives useful as platelet aggregation inhibitors |
| US5614106A (en) * | 1993-03-12 | 1997-03-25 | Baxter International Inc. | Method and apparatus for collection of platelets |
| AU6395094A (en) * | 1993-03-15 | 1994-10-11 | G.D. Searle & Co. | Urea derivatives useful as platelet aggregation inhibitors |
| US5612355A (en) * | 1993-06-23 | 1997-03-18 | G. D. Searle & Co. | Phenyl amidine lactones useful as platelet aggregation inhibitors |
| US5780590A (en) * | 1993-10-15 | 1998-07-14 | Rhone-Poulenc Rorer Pharmaceuticals Inc. | Antithrombotic azacycloalkylalkanoyl peptides and pseudopeptides |
| RU2134695C1 (en) * | 1993-10-15 | 1999-08-20 | Рон-Пуленк Рорер Фармасьютикалз Инк. | Antithrombotic azacycloalkylakanoyl peptides |
| US5770575A (en) * | 1994-03-16 | 1998-06-23 | Ortho Pharmaceutical Corporation | Nipecotic acid derivatives as antithrombotic compounds |
| US6387880B1 (en) | 1994-10-24 | 2002-05-14 | G.D. Searle & Co. | Transdermal N-[N-[5-[4-(aminoiminomethly)phenyl]-1-oxopentyl]-L-α-aspartyl]-L-phenylalainine or its esters and their pharmaceutically acceptable salts |
| US5602155A (en) * | 1995-01-17 | 1997-02-11 | G. D. Searle & Co. | Platelet aggregation inhibitors |
| US5639765A (en) * | 1995-01-17 | 1997-06-17 | G. D. Searle & Co. | Guanidinoalkyl glycine β-amino acids useful for inhibiting bone loss |
| US5674894A (en) * | 1995-05-15 | 1997-10-07 | G.D. Searle & Co. | Amidine derivatives useful as platelet aggregation inhibitors and vasodilators |
| US5681820A (en) * | 1995-05-16 | 1997-10-28 | G. D. Searle & Co. | Guanidinoalkyl glycine β-amino acids useful for inhibiting tumor metastasis |
| PL331772A1 (en) * | 1996-08-21 | 1999-08-02 | Rhone Poulenc Rorer Pharma | Stable non-hygroscopic crystalline form of n-[n-n-(4-piperidin-4-yl)-n-ethylglycin] compounds |
| SI0946185T2 (en) * | 1996-11-27 | 2010-08-31 | Aventis Pharma Inc | PHARMACEUTICAL COMPOSITION COMPRISING A COMPOUND HAVING ANTI-Xa ACTIVITY AND A PLATELET AGGREGATION ANTAGONIST COMPOUND |
| US5932693A (en) * | 1996-12-10 | 1999-08-03 | Washington University | Antithrombotic peptides |
| ID26082A (en) | 1998-03-19 | 2000-11-23 | Briston Myers Squib Company | DWIFASA CONTROLLED REMOVAL DELIVERY SYSTEM FOR HIGH SOLUTION PHARMACY SUBSTANCES AND ITS METHODS |
| RU2016125229A (en) | 2010-07-09 | 2018-12-04 | БиЭйчВи ФАРМА, ИНК. | COMBINED DELIVERY SYSTEM WITH IMMEDIATE / SLOW-DELIVERY FOR MEDICINES WITH A SHORT HALF-HOUR, INCLUDING REMOGLYPHLOSIN |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3795667A (en) * | 1970-09-30 | 1974-03-05 | Squibb & Sons Inc | Novel peptide intermediates in the preparation of secretin |
| US4517686A (en) * | 1982-08-04 | 1985-05-21 | La Jolla Cancer Research Foundation | Polypeptide |
| US4661111A (en) * | 1982-08-04 | 1987-04-28 | La Jolla Cancer Research Foundation | Polypeptide |
| US4614517A (en) * | 1982-08-04 | 1986-09-30 | La Jolla Cancer Research Foundation | Tetrapeptide |
| US4578079A (en) * | 1982-08-04 | 1986-03-25 | La Jolla Cancer Research Foundation | Tetrapeptide |
| US4589881A (en) * | 1982-08-04 | 1986-05-20 | La Jolla Cancer Research Foundation | Polypeptide |
| GB8327966D0 (en) * | 1983-10-19 | 1983-11-23 | Nyegaard & Co As | Chemical compounds |
| US4590003A (en) * | 1984-03-23 | 1986-05-20 | Oncogen | Platelet related growth regulator |
| US4683291A (en) * | 1985-10-28 | 1987-07-28 | Scripps Clinic And Research Foundation | Platelet binding inhibitors |
-
1987
- 1987-12-03 US US07/128,379 patent/US4857508A/en not_active Expired - Fee Related
-
1988
- 1988-12-02 JP JP63305879A patent/JPH01190699A/en active Pending
- 1988-12-02 DE DE3888310T patent/DE3888310T2/en not_active Expired - Fee Related
- 1988-12-02 CA CA000584873A patent/CA1315479C/en not_active Expired - Fee Related
- 1988-12-02 ES ES88870179T patent/ES2061733T3/en not_active Expired - Lifetime
- 1988-12-02 EP EP88870179A patent/EP0319506B1/en not_active Expired - Lifetime
- 1988-12-02 AT AT88870179T patent/ATE102629T1/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| DE3888310T2 (en) | 1994-08-11 |
| EP0319506A2 (en) | 1989-06-07 |
| EP0319506A3 (en) | 1990-08-16 |
| ATE102629T1 (en) | 1994-03-15 |
| ES2061733T3 (en) | 1994-12-16 |
| JPH01190699A (en) | 1989-07-31 |
| EP0319506B1 (en) | 1994-03-09 |
| DE3888310D1 (en) | 1994-04-14 |
| US4857508A (en) | 1989-08-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1315479C (en) | Platelet-aggregation inhibitor peptide derivatives | |
| US4683291A (en) | Platelet binding inhibitors | |
| EP0578728B1 (en) | PLATELET AGGREGATION INHIBITORS HAVING HIGH SPECIFICITY FOR GP IIbIIIa | |
| EP0352249B1 (en) | Novel platelet-aggregation inhibitors | |
| CA1311583C (en) | Peptide derivatives and their application, in particular in therapy | |
| CA2021951A1 (en) | Fibrinogen receptor antagonists | |
| CA2027315A1 (en) | Fibrinogen receptor antagonists | |
| EP0462960A1 (en) | Novel platelet-aggregation inhibitor | |
| US5061693A (en) | Fibrinogen receptor antagonists | |
| EP0410537A1 (en) | Fibrinogen receptor antagonists | |
| EP0410541A1 (en) | Fibrinogen receptor antagonists | |
| EP0411833A1 (en) | Fibrinogen receptor antagonists | |
| US5066592A (en) | Trigramin - a platelet aggregation inhibiting polypeptide | |
| US5498601A (en) | Platelet aggregation-inhibiting peptides | |
| JPH03118331A (en) | Cyclic fibrinogen receptor antagonist | |
| EP0382451A3 (en) | Viper venom polypeptides and variants | |
| CA2168964C (en) | Novel peptide, and platelet aggregation-inhibiting agents, blood coagul ation-inhibiting agents for extracorporeal circulation, cell adhesion-inhibiting agents, tumor metastasis-inhibiting agents, agents for protecting platelet preparations for blood transfusion, platelet preparations and platelet preparation packs for transfusion using said novel peptides | |
| JPH09501910A (en) | Methods and compositions for the treatment of thrombosis | |
| US6521594B1 (en) | Method and composition for treating thrombosis | |
| US6017877A (en) | Method and composition for treating thrombosis | |
| JP2000503649A (en) | Antithrombotic agents and usage | |
| AU661778B2 (en) | New peptide compounds active in cell adhesion processes, a process for the preparation thereof, and pharmaceutical compositions containing them | |
| JPH06179696A (en) | Anticoagulant peptide and medicine for improving thrombosis | |
| CA2021953A1 (en) | Fibrinogen receptor antagonists |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed | ||
| MKLA | Lapsed |
Effective date: 19970401 |