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WO1992008476A1 - Peptides inhibant la liaison plaquettaire de molecules d'ashesion - Google Patents

Peptides inhibant la liaison plaquettaire de molecules d'ashesion Download PDF

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Publication number
WO1992008476A1
WO1992008476A1 PCT/US1991/008328 US9108328W WO9208476A1 WO 1992008476 A1 WO1992008476 A1 WO 1992008476A1 US 9108328 W US9108328 W US 9108328W WO 9208476 A1 WO9208476 A1 WO 9208476A1
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WO
WIPO (PCT)
Prior art keywords
arg
peptide
asp
gly
bond
Prior art date
Application number
PCT/US1991/008328
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English (en)
Inventor
Zaverio M. Ruggeri
Richard A. Houghten
Original Assignee
The Scripps Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
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Publication of WO1992008476A1 publication Critical patent/WO1992008476A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to thrombotic disorders, and, more particularly to peptides that
  • the extracellular matrix is the extracellular space in tissue that is filled by a network of macromolecules.
  • the macromolecules comprise a variety
  • adhesion molecules such as fibronectin, vitronectin, thrombospondin, fibrinogen, and von Willebrand factor (vWF) , all contain the tripeptide seguence Arg-Gly-Asp (RGD) .
  • This seguence is present in other naturally occurring peptides 0 including but not limited to collagen ⁇ 1(1) and ⁇ 2 (I), thrombin, ⁇ lytic protease, and lambda receptor protein.
  • the RGD sequence is recognized by one or more integrins on the cell surface and thus constitutes the integrin recognition sequence.
  • Integrins are a superfamily of 5 adhesion molecule receptors [Hynes, Cell 48:549-554 (1987) ]. These receptors play a major role in binding of cells to other cells and binding of cells to the extracellular matrix. Integrins include platelet membrane glycoprotein lib/Ilia, fibronectin receptor, vitronectin receptor, MAC-1, and LFA-1. All of the integrins form ⁇ / ⁇ heterodimers on the cell surface and some share a common ⁇ subunit. Many of the adhesion molecules either bind to or constitute part of the extracellular matrix in addition to other structural molecules such as collagen, glycosaminoglycans, and proteoglycans.
  • the extracellular matrix includes the subendothelial matrix.
  • This invention relates generally to regulation of cell-to-cell aggregation or regulation of cells binding to the extracellular matrix, and relates specifically to the inhibition of binding of fibrinogen and other adhesion molecules such as, von Willebrand Factor (vWF) , fibronectin, and vitronectin, to platelets and other cells expressing the membrane glycoprotein Ilb/IIIa (GPIIb/IIIa) integrin or i munologically or structurally related integrins, which results inter alia in inhibition or prevention of cell-to-cell or platelet-to-platelet aggregation and thrombosis.
  • fibrinogen and other adhesion molecules such as, von Willebrand Factor (vWF) , fibronectin, and vitronectin
  • Platelets are specialized circulating cell fragments that perform the biological functions of adhering to areas of blood vessel wall injury and aggregating with one another to form a hemostatic plug. Both adhesion and aggregation are mediated by the interaction of specific molecules with membrane glycoprotein (GP) receptors on the platelet surface.
  • GP membrane glycoprotein
  • the adhesive properties of platelets are of central importance in normal hemostasis as well as in the development of pathological vascular occlusion. There is significant evidence that rupture of an atherosclerotic plaque with superimposed thrombosis occurs in the majority of cases of unstable angina, myocardial infarction and sudden death of ischemic origin [Davies et al.. Br. Heart J. 63:363 (1985); Fuster et al. , Circulation 77:1213 (1988); Badimon et al.. Arteriosclerosis 6:312 (1986); Ver ylen et al.. Science 238:491 (1987)].
  • Platelet adhesion to surfaces may be mediated by several different binding sites on the platelet membrane which may interact with distinct adhesion molecules.
  • platelet aggregation the interaction of platelets with one another which results in the mass of the thrombus
  • fibrinogen is considered the physiological GP lib/Ilia ligand
  • evidence is accumulating from ex vivo experiments ⁇ 5 that vWF is involved in mediating platelet aggregation.
  • ⁇ Hawiger et al. report the use of synthetic peptides as a method of inhibiting the binding of vWF to platelets (U.S. Patent No. 4,666,884, issued May 19, 1987) and Ruoslahti et al. report a tetrapeptide containing Arg- Gly-Asp that has cell attachment-promoting-activity when
  • bonds preferably covalent bonds such as disulfide bonds or amide bonds, of the present invention are superior to the prior art small synthetic peptides in their ability to inhibit fibrinogen-platelet binding, vWF-platelet binding, and platelet aggregation due to greater
  • the peptides of the present invention can be effective at lower concentrations in the blood, resulting in less likelihood of undesired side effects and lower cost of administration.
  • the background information for these studies is predicated upon the knowledge obtained previously from Ruggeri et al. Proc. Nat'l Acad. Sci. USA 83:5708-5712 (1986) and from Zimmerman et al. U.S. Patent No. 4,683,291 issued July 28, 1987, Reexam certificate issued July 3, 1990, the contents of which are hereby incorporated by reference.
  • the peptides of the present invention include 0 peptides that inhibit binding of adhesion molecules to cells expressing integrins comprising multimers having at least a first and second subunit, the sequence of each subunit containing at least the Arg-Gly-Asp sequence, and the subunits are joined by a interchain stable bond 5 preferably a covalent bond, more preferably a disulfide and amide bond.
  • the bond may be located on the amino terminal, carboxyl terminal side or alternating sides of the Arg-Gly-Asp sequence of each subunit.
  • the sequence of each subunit may be identical or different.
  • Peptides of the present invention are formed from subunits to make di ers, trimers, tetramers, pentamers, or the like.
  • the present invention has the utility of preventing or retarding the formation of a clot or thrombus, both venous and arterial, in the blood.
  • the peptides of the present invention have higher inhibitory activity than 0 previously known synthetic peptides and therefore may be superior anti-thrombotic agents.
  • the another aspect of this invention relates to pharmaceutical compositions using peptides of the present - invention as the active ingredient in a pharmaceutically acceptable medium for intravenous, subcutaneous, intraperitoneal, oral or nasal administration into mammals.
  • Another aspect of this invention relates to methods o f° r inhibiting the aggregation of cells to each other, or inhibiting the binding of adhesion molecules to cells or inhibiting the binding of cells to extracellular matrices, by contacting the cells with a peptide of the present invention in an amount of said peptide effective 5 to inhibit such aggregation or binding.
  • the cells of the present methods are preferably cells expressing integrins, most perferably platelets or tumor cells and the adhesion molecules of the present methods are preferably vWF or fibrinogen.
  • Another aspect of this invention relates to methods for preventing and /or treating thrombosis in mammals using peptides of the present invention, alone or in combination with thrombolytic agents.
  • Another aspect of this invention relates to methods 5 for detecting thrombi or platelet aggregates using the peptides of the present invention.
  • Figure 1 shows the correlation between number of arginine residues in a synthetic peptide and inhibitory activity.
  • Figure 2 shows the effect of the residue preceding the Arg-Gly-Asp sequence on the inhibitory activity of synthetic peptides.
  • Figure 3 shows the effect of the number of residues intervening between the polyarginine chain and the Arg- Gly-Asp sequence on the inhibitory activity of synthetic peptides.
  • Figure 4 shows the dose-response curves of various nc synthetic peptides. Dimeric peptides have the lowest IC 50 values.
  • each amino acid residue can be in the (L) or (D) configuration, preferably the (L) configuration.
  • the peptides of the present invention have the utility of inhibiting the binding of adhesion molecules to cells, and specifically inhibiting fibrinogen-platelet or vWF-platelet binding, which results inter alia in inhibition or prevention of cell-to-cell or platelet-to- platelet binding.
  • the present invention has the utility of preventing or retarding the formation of a clot or thrombus, both venous and arterial, in the blood.
  • the peptides of the present invention have higher inhibitory activity than previously known synthetic peptides and therefore may be superior anti-thrombotic agents.
  • the peptides of the present invention comprise peptides that inhibit binding of adhesion molecules to cells expressing integrins comprising multimers having at least a first and second subunit, the sequence of each subunit contains at least the Arg-Gly-Asp sequence, and the subunits are joined by an interchain stable bond preferably a covalent bond, more preferably a disulfide and amide bond.
  • the bond may be located on the amino terminal or carboxyl terminal side of the Arg-Gly-Asp sequence.
  • the sequence of each subunit may be identical or different.
  • the present invention also includes peptides that inhibit binding of adhesion molecules to cells expressing integrins comprising multimers of at least a first and second subunit held together by interchain stable bonds having the following general formula:
  • the stable bonds that link subunits include covalent bonds, preferably amide bonds formed between Glu and Lys, Asp and Lys, Glu and Orn, or Asp and Orn and disulfide bonds formed between Cys residues on each subunit.
  • the bonds may be formed on the
  • Peptides of the present invention are formed from j e subunits to make dimers, trimers, tetramers, pentamers, and the like.
  • the peptides of the present invention also includes multimers of at least a first and second subunit held together by interchain stable bonds further defined by o the formula:
  • (Cx) is a chain of 1 to 18 amino acid residues 5 selected from the group consisting of Ala, Arg, Asn, Asp,
  • w is an integer from 0 to 1
  • y is an integer from 2 to 5
  • the sequence of each subunit is either identical or different.
  • the stable bonds that link 0 subunits include covalent bonds, preferably amide bonds formed between Glu and Lys, Asp and Lys, Glu and Orn, or
  • bonds formed between Cys residues on each subunit.
  • the bonds may be formed on the amino terminal side, the carboxyl terminal side , or 5 alternating sides of the Arg-Gly-Asp sequence of each subunit.
  • These peptides are formed from subunits to make dimers, trimers, tetramers, pentamers, and the like.
  • the peptides of the present invention further comprises peptides that inhibit binding of adhesion molecules to cells expressing integrins comprising multimers of at least a first and second subunit held together by interchain stable bonds having the following formula:
  • n is an integer from 1 to 5
  • z is an integer from 1 to 4
  • X is any amino acid or combination of amino acids
  • (Cx) is a chain of 1 to 18 amino acid residues selected from the group consisting of Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Met, Phe, Pro, Trp, Tyr, Val, or Orn
  • w is an integer from 0 to 1
  • y is an integer from 2 to 5; and the sequence of each subunit is identical or different.
  • the stable bond that link subunits include covalent bonds, preferably amide bonds formed between Glu and Lys, Asp and Lys, Glu and Orn, or Asp and Orn and disulfide bonds formed between Cys residues on each subunit.
  • the bonds may be formed on the amino terminal side, the carboxyl terminal side , or alternating sides of the Arg-Gly-Asp sequence of each subunit.
  • the peptides are formed from subunits to make dimers, trimers, tetramers, pentamers, and the like.
  • the peptides of the present invention comprises peptides that inhibit binding of fibrinogen and other adhesion molecules to cells expressing integrins such as platelets comprising a multimer of at least a first and second subunit held together by an interchain stable bond having the following formula:
  • n is an integer from l to 5
  • z is an integer from 1 to 4
  • x is selected from the amino acids Gin, Phe, Pro, Thr, Leu, Ala, Gly, or Ser
  • Cx is Val
  • w is an integer from 0 to 1
  • y is an integer from 2 to 5.
  • the peptides of the present invention includes peptides that inhibit binding of adhesion molecules to cells expressing integrins comprising multimers of at least a first and second subunit held together by an interchain stable bond having the following formula:
  • the stable bonds that link subunits include covalent bonds, preferably amide bonds formed between Glu and Lys, Asp and Lys, Glu and Orn, or Asp and Orn and disulfide bonds ,c formed between Cys residues on each subunit.
  • the bonds may be formed on the amino terminal side, the carboxyl terminal side, or alternating sides of the Arg-Gly-Asp sequence of each subunit.
  • the peptides are formed from subunits to make dimers, trimers, tetramers, pentamers, o and the like.
  • the peptides of the present invention comprises a peptide that inhibits binding of adhesion molecules to cells expressing integrins comprising multimers of at least a first and second 5 subunit held together by interchain stable bond having the following formula:
  • the invention further comprises a method for 5 inhibiting the binding of adhesion molecules to cells with integrins comprising contacting the cells with a interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.
  • the invention further comprises a method for inhibiting the binding of adhesion molecules to platelets comprising contacting the platelets with a interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.
  • the invention further comprises a method for inhibiting the binding of fibrinogen to platelets comprising contacting platelets with an interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.
  • the invention further comprises a method for inhibiting the binding of von Willebrand factor to platelets comprising contacting platelets with an interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.
  • Tne invention further comprises a method for inhibiting the binding of cells with integrins to extracellular matrices comprising contacting cells with a interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.
  • the invention further comprises a method for inhibiting the binding of platelets to extracellular matrices comprising contacting platelets with an interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.
  • the invention further comprises a method for inhibiting aggregation of cells to each other comprising contacting the cells with an interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.
  • the invention further comprises a method for inhibiting aggregation of platelets to each other comprising contacting the platelets with an interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.
  • Peptides of formula Arg m -Arg-Gly-Asp-Val were found to give progressively lower 1C 50 values (the concentration of the peptide which would inhibit binding by 50%, as determined by the procedure described below) ._ when the value of m increased between 1 and 8 (Fig. 1). This was in agreement with results published previously by Ruggeri et al.. Proc Natl. Acad. Sci USA 83:5708 (1986) .
  • Peptides within the scope of the present invention are peptides covalently linked by interchain amide bonds.
  • Peptides are synthesized in a manner analogous to that used to prepare the peptides in Table 2. In place of 0 Cys. , a Glu, Lys, Asp, or Orn is substituted and the amide bond is made using an amide condensation agent. Examples of such interchain amide bond linked peptides are as follows (Table 3) . 5 It Table 3
  • Interchain covalent bonds may be located on the amino terminal or carboxyl terminal side of the Arg-Gly- Asp peptide sequence to form dimers, trimers, tetramers, pentamers and the like.
  • dimers that are formed by placement of the bond on the carboxyl side of the Arg-Gly-Asp sequence are as follows (Table 4) .
  • the location of the interchain covalent bond may be on the amino terminal side of of the first subunit and on the carboxyl terminal side of the second subunit, or vise versa, as generally illustrated in the following formula:
  • X is an amino acid capable of forming a stable bond, preferably a covalent bond.
  • Interchain covalent bonds for trimers can be similarly arranged as generally illustrated in the following formula: -Arg-Gly-Asp-X
  • X is an amino acid capable of forming a stable bond, preferably a covalent bond.
  • the second subunit is bonded at two locations, one bond connects the first subunit with the second and the other bond connects the third subunit with the second subunit.
  • Interchain covalent bonds between subunits forming tetramers, pentamers and the like can be similarly arranged.
  • sequences identified above are specific examples of peptides falling within the sequences defined in formulas and should not be interpreted as limiting the scope of the present invention.
  • Interchain covalently-linked peptides of the present invention also have utility in inhibiting binding of other proteins including other adhesion molecules to platelets or other integrin containing cells.
  • the peptides of the present invention were quite effective in inhibiting binding of von Willebrand factor to platelets (Table 5) . o Table 5
  • Peptides are synthesized by the method of simultaneous multiple peptide synthesis (SMPS) as described in detail by Houghten, Proc. Natl. Acad. Sci. USA 82:5131 (1985) and as generally set forth in US Patent No. 4,683,291, issued July 28, 1987, Reexam. issued July 3, 1990, the disclosure of which is incorporated herein by reference, or by other suitable methods. All peptides were purified by reversed phase high performance liquid chromatography (HPLC) using methodology reported by Ruggeri et al.. Proc. Natl. Acad. Sci. USA 83:5708 (1986) although other equivalent purification techniques may also be used.
  • HPLC reversed phase high performance liquid chromatography
  • a purity of at least 95 to 99% (based on all peptides present) is reasonably obtainable and preferable.
  • Formation of intermolecular disulfide bonds to obtain homodimeric peptides was achieved by air oxidation of peptide solutions prepared directly into 25 mM ammonium acetate, pH 8.5, at a concentration of 1 x 10 ⁇ 3 moles/liter, although other methods of forming disulfide bonds may be used including oxidation using potassium ferricyanide or iodine. After overnight exposure to air, isolation of dimers from monomeric species was obtained by reversed- phase HPLC chromatography. A purity of 95 to 99% dimers is expected and preferred. Trimers, tetramers, pentamers and the like may be similarly obtained.
  • amide condensation agents such as diisopropylcarbodiimide and the like.
  • Peptide solutions for testing were prepared by carefully weighing a lyophilized dry peptide and dissolving it into a buffer composed of 20 mM HEPES, 150 mM NaCl, pH 7.35, to give the desired final concentration.
  • the composition and concentration of single amino acids was verified with an automated amino acid analyzer (LKB) following complete hydrolysis after 24 hours in 6 M HCl at 110°C , as described by Ruggeri et al.. Proc. Natl. Acad. Sci. USA 83:5708 (1986).
  • LLB automated amino acid analyzer
  • a purity of 95 to 99% is expected and preferred.
  • Most of the peptides of the present invention may also be obtained using recombinant DNA techniques in prokaryotic or eucaryotic expression systems.
  • Fibrinogen binding to platelets and inhibition of binding was performed as previously described by Ruggeri et al.. Proc Natl. Acd. Sci. USA 83:5708 (1986).
  • Fibrinogen was purified from freshly collected plasma using the glycine precipitation method of Kazal et al.. Proc. Soc. Exp. Biol. Med. 113: 989 (1963) .
  • Traces of contaminating vWF were removed by gel filtration through an 80 x 5 cm Sepharose 4B-C1 column (Pharmacia) .
  • Purified fibrinogen was labeled with 125 I using Iodogen (Pierce) following the procedure described by Fraker and Speck, Biochem. Biophys. Res. Commun.
  • Platelet suspensions devoid of plasma proteins were prepared by the albumin density gradient centrifugation technique of Walsh et al.. Br. J. Haematol. 36:281 (1977) with modifications described by Trapani-Lombardo et al.. J. Clin. Invest.76:1950 (1985). Binding of radiolabeled fibrinogen to platelets was measured after platelet stimulation with alpha-thrombin (a generous gift of Dr. John W. Fenton, II) at 22-25°C for 10 minutes (3.125 x 10 - 1 platelets per liter and thrombin at 0.5 NIH units/ml).
  • alpha-thrombin a generous gift of Dr. John W. Fenton, II
  • Hirudin (Sigma) was then added at a 25-fold excess (unit/unit) for 5 minutes before addition of the radiolabeled ligand and any competing ligand. After these additions, the final platelet count in the mixture was 1 x 10 11 /liter. After incubation for an additional 30 minutes at 22-25°C, bound and free ligand were separated by centrifuging 50 ⁇ l of the mixture through 300 ⁇ l of 20% sucrose at 12,000 x g for 4 minutes as described by Ruggeri et al.. J. Clin. Invest. 72:1-12 (1983). The platelet pellet was separated from the rest of the mixture to determine platelet-bound radioactivity.
  • Nonspecific binding was arbitrarily defined as that measured in the presence of a saturating concentration of the anti-GP Ilb-IIIa monoclonal antibody LJ-CP3 (U.S. Patent application, Serial No. 294,471 filed Jan. 6, 1989, ATCC Accession No. HB 10046) .
  • the nonspecific binding corresponded typically to less than 10% of the total binding, or 0.5% of added counts.
  • Von Willebrand factor was purified from human cryoprecipitate, iodinated and the von Willebrand factor binding studies performed as previously described by Ruggeri et al. J. Clin. Invest. 72:1-12 (1983).
  • the peptides of the present invention can be formulated into pharmaceutical preparations for therapeutic, diagnostic or other uses in mammals.
  • the peptides of the present invention are especially useful u in prevention and/or treatment of vascular disorders including venous or arterial thromboembolisms, thrombotic occlusion or stenosis in coronary, cerebral or perepheral arteries, and vascular grafts and in disease states predisposed to thrombosis such as disseminated intravascular coagulation, major trauma, major surgery, cancer, acute myocardial infarction, and thrombotic stroke, and hereditary diseases such as anti-thrombin III deficiencies, congenital protein C deficiencies or other hereditary diseases associated with platelet aggregation and thrombus formation.
  • Other candidates for treatment 0 with peptides of the present invention include mammals with coronary heart disease or those in high risk categories for heart attack or stroke such as those with high blood pressure, diabetes, high cholesterol, or 5 smokers.
  • the peptides may be used alone or in combination with a thrombolytic agent or more than one thrombolytic agent for prevention or treatment of thrombosis.
  • thrombolytic agents include but are not limited to o tissue-type plasminogen activator or derivatives thereof, streptokinase or derivatives thereof, urokinase or derivatives thereof, prourokinase or derivatives thereof, an acylated form of plasminogen or plasmin and derivatives thereof, and activated protein C and 5 derivatives thereof.
  • Derivatives include natural products, fragments, synthetic peptides or recombinant products that have the desired anti-thrombotic activity of the natural product.
  • the biologically active peptides produced by peptide 0 synthesis or by the prokaryotic or eukaryotic expression of cloned peptide genes purified in accordance with the present invention can be used for the in vivo treatment of mammalian species by physicians and/or veterinarians.
  • the amount of active peptide will, of course, depend upon 5 the severity of the condition being treated, the route of administration chosen, and the specific activity of the active peptides, and ultimately will be decided by the attending physician or veterinarian.
  • a concentration of peptide which is 3 to 4 times the concentration of the IC 100 value, using the platelet inhibition assay, would be an approximate minimum dose.
  • the active peptide may be administered by any route appropriate to the condition being treated including intravenous, intraperitoneal, intramuscular, subcutaneous, oral, nasal and the like.
  • the . peptide is injected into the blood stream of the mammal being treated. It will be readily appreciated by those skilled in the art that the preferred route will vary with the condition being treated.
  • the active peptide While it is possible for the active peptide to be i c administered as the pure or substantially pure compound, it is preferable to present it as a pharmaceutical formulation or preparation.
  • formulations of the present invention both for veterinary and for human use, comprise an active peptide, o as above described, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious 5 to the recipient thereof.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the pharmaceutical art.
  • All methods include the step of bringing into 0 association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid 5 carriers or both, and then, if necessary, shaping the product into the desired formulation.
  • Formulations suitable for intravenous, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions which are preferably isotonic with the blood of the recipient.
  • Such formulations may be conveniently prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride (e.g. 0.1-2.0 M) , glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering
  • formulations of the present invention may be any suitable formulations of the present invention.
  • stabilizers are polyethylene gly ⁇ ol, proteins, saccharides, amino acids, inorganic acids, and organic acids which may be used either on their own or as admixtures. These stabilizers are preferably incorporated in an amount of 0.11-10,000
  • 25 pressure of such aqueous solutions is generally in the range of 0.1-3.0 osmoles, preferably in the range of 0.8- 1.2.
  • the pH of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8.
  • anti-adsorption agent may be used.
  • compositions may be combined with typical carriers, such as lactose, sucrose, starch, talc magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose,
  • surface active agents used in accordance with the present invention may be those which promote the absorption of peptides through the nasal mucous membrane, that is to say, those which work as absorption promotors.
  • Such surface active agents include bile salts, cationic, anionic, nonionic, and amphoteric agents and phospholipids and the like that are well known in the art.
  • the amount of such surface active agent may usually be in the range from 0.001 to 10% w/v and preferably 0.01 to 1% w/v, the amount depending on ._ the specific surfactant used.
  • Aqueous or nonaqueous media may be used either alone or in admixture in the form of solution, suspension, emulsion or ointment, if applicable to nasal mucosa.
  • the preparations of the present invention may also contain other additives, such j e as stabilizers, adsorption preventing agents and preservatives.
  • the preparations of the present invention may be formulated in the form of a gel by adding gel bases such as cellulose derivatives, natural gums, vinyl polymers, acrylic acid polymers, and 0 tne like.
  • the peptides of the present invention also have utility for the in vivo or in vitro detection of platelet aggregates or thrombi, and other aggregates of cells containing integrins, and for measuring cell surface 5 expression of integrins, and the like.
  • the peptides of the invention may be used in diagnostic tests, such as immunoassays, for detecting platelet aggregates.
  • diagnostic tests include for example, enzyme-linked immunosorbent assays, radioimmunoassays, fluorescence 0 immunoassays and other techniques in which peptides are labelled with a detectable tag.
  • Such tags include radioactive, enzymes, biotin, fluorochromes, electron dense reagents and the like.
  • the method for detecting platelet aggregates or thrombi includes exposing the 5 suspected platelet aggregate or thrombus to an effective amount of the tagged peptide of the present invention and then measuring the presence of bound, tagged peptides.
  • Detection means for the labelled peptides include radioimagery, spectroscopic, photochemical, immunochemical, biochemical or chemical means.
  • Methods for detecting the presence of platelet aggregates or thrombi bound by tagged peptides of the present invention, in a mammal include, contrast angiography, arteriography, computerized axial tomography scanning and the like. Such methods of detecting platelet aggregates or thrombi are useful in diagnosing thrombotic diseases such as stroke, myocardial infarction or the like.
  • Such in vitro and in vivo detection methods may be quantitative or qualitative for the presence of platelet aggregates, thrombi, or other aggregates of cells containing integrins.

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Abstract

L'invention concerne des peptides comprenant des multimères à liaisons interchaînes contenant la séquence Arg-Gly-Asp maintenue solidaire par des liaisons interchaînes stables, lesdits peptides inhibent la liaison du fibrinogène ou d'autres molécules d'adhésion à des plaquettes ou à des cellules exprimant des intégrines, ils empêchent l'aggrégation plaquette à plaquette ou cellule à cellule et ils sont utiles dans la prévention, le retardement ou la détection de la formation de thrombus.
PCT/US1991/008328 1990-11-07 1991-11-07 Peptides inhibant la liaison plaquettaire de molecules d'ashesion WO1992008476A1 (fr)

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US61036390A 1990-11-07 1990-11-07

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994015958A3 (fr) * 1993-01-08 1994-09-29 Tanabe Seiyaku Co Inhibiteurs peptidiques de l'adhesion cellulaire
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6017877A (en) * 1990-04-06 2000-01-25 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
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US8969299B2 (en) 2011-06-08 2015-03-03 Kai Pharmaceuticals, Inc. Therapeutic agents for regulating serum phosphorus
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US8999932B2 (en) 2009-07-29 2015-04-07 Kai Pharmaceuticals, Inc. Therapeutic agents for reducing parathyroid hormone levels
CN110590919A (zh) * 2017-05-24 2019-12-20 中国海洋大学 含鸟氨酸的短肽及其应用
CN118620088A (zh) * 2024-06-20 2024-09-10 首都医科大学 Asn-Gly-Pro的三聚体及其制备和应用

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US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6017877A (en) * 1990-04-06 2000-01-25 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
WO1994015958A3 (fr) * 1993-01-08 1994-09-29 Tanabe Seiyaku Co Inhibiteurs peptidiques de l'adhesion cellulaire
US8987200B2 (en) 2006-11-16 2015-03-24 Kai Pharmaceuticals, Inc. Polycationic calcium modulator peptides for the treatment of hyperparathyroidism and hypercalcemic disorders
US9278995B2 (en) 2009-07-29 2016-03-08 Kai Pharmaceuticals, Inc. Therapeutic agents for reducing parathyroid hormone levels
US8999932B2 (en) 2009-07-29 2015-04-07 Kai Pharmaceuticals, Inc. Therapeutic agents for reducing parathyroid hormone levels
US9567370B2 (en) 2009-07-29 2017-02-14 Kai Pharmaceuticals, Inc. Therapeutic agents for reducing parathyroid hormone levels
US9701712B2 (en) 2009-07-29 2017-07-11 Kai Pharmaceuticals, Inc. Therapeutic agents for reducing parathyroid hormone levels
KR101781841B1 (ko) * 2009-07-29 2017-09-26 카이 파마슈티컬즈 부갑상선 호르몬 수준을 감소시키기 위한 치료제
US10280198B2 (en) 2009-07-29 2019-05-07 Kai Pharmaceuticals, Inc. Therapeutic agents for reducing parathyroid hormone levels
US8969299B2 (en) 2011-06-08 2015-03-03 Kai Pharmaceuticals, Inc. Therapeutic agents for regulating serum phosphorus
CN110590919A (zh) * 2017-05-24 2019-12-20 中国海洋大学 含鸟氨酸的短肽及其应用
CN110590919B (zh) * 2017-05-24 2022-05-24 中国海洋大学 含鸟氨酸的短肽及其应用
CN118620088A (zh) * 2024-06-20 2024-09-10 首都医科大学 Asn-Gly-Pro的三聚体及其制备和应用
CN118620088B (zh) * 2024-06-20 2025-07-29 首都医科大学 Asn-Gly-Pro的三聚体及其制备和应用

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