+

WO1996033218A1 - Anti-haemorrhagic peptides - Google Patents

Anti-haemorrhagic peptides Download PDF

Info

Publication number
WO1996033218A1
WO1996033218A1 PCT/IB1996/000358 IB9600358W WO9633218A1 WO 1996033218 A1 WO1996033218 A1 WO 1996033218A1 IB 9600358 W IB9600358 W IB 9600358W WO 9633218 A1 WO9633218 A1 WO 9633218A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
peptide
amino acid
haemorrhage
blocked
Prior art date
Application number
PCT/IB1996/000358
Other languages
French (fr)
Inventor
Beatrice Beaubien
Clement E. Burrowes
Zhi-Jie Yang
Original Assignee
Allelix Biopharmaceuticals Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Allelix Biopharmaceuticals Inc. filed Critical Allelix Biopharmaceuticals Inc.
Priority to AU51612/96A priority Critical patent/AU5161296A/en
Publication of WO1996033218A1 publication Critical patent/WO1996033218A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu

Definitions

  • the present invention relates to peptides having anti-haemorrhagic properties.
  • the present invention relates to an anti-haemorrhagic peptide derived from thrombospondin, and a method for reducing haemorrhage in a mammal.
  • Haemorrhage is associated with many disease conditions.
  • Thrombocytopenia for example, a disorder in which platelet number is reduced to below normal. Platelets, the cell components which control blood clotting and play a role in the repair of blood vessels, are important in preventing haemorrhage from occurring. Thrombocytopenia may stem from decreased platelet production, sequestration of platelets by the spleen, increased platelet destruction or dilution of platelets.
  • Thrombocytopenic Purpura a thrombocytopenic condition having both thrombotic and immunologic idiopathic forms, is a severe, potentially chronic condition in which sudden relapses can result in life- threatening bleeding.
  • Thrombocytopenia can also be induced by certain drugs, including heparin, quinidine, sulfa preparations, oral anti-diabetic agents and rifampin. Whatever the cause, the thrombocytopenic individual is much less tolerant of bleeding since they lack the ability to prevent excessive bleeding through clotting.
  • plasma therapy can be used to treat thrombocytopenic individuals that must undergo surgery, this type of treatment is not feasible for widespread use due to the limits imposed by plasma supply.
  • Vasculitis is a disorder in which inflammation within the vasculature occurs resulting in varying degrees of vessel destruction, necrosis or scarring in one or more layers of the vessels. Subsequent loss of vessel wall integrity results from formation of fibrin plugs and can cause red blood cell leakage into surrounding tissue, i.e. haemorrhage.
  • Diseases characterized by vasculitis include von Willebrand's Disease, Wegener's Granulomatosis and Systemic Lupus Erythematosis. Individuals inflicted with von Willebrand's Disease, for example, present with bleeding over several hours from small skin cuts and abnormal bleeding following small surgical procedures such as tooth extraction and tonsillectomy.
  • An agent capable of reducing the occurrence of haemorrhage in conditions such as those mentioned above is desirable, particularly in those instances in which haemorrhage can be reasonably predicted or expected, for example, in conditions where relapse is possible (Thrombocytopenic Purpura), in individuals having vasculitis where infection, cuts or surgery can result in excessive bleeding, and in individuals having drug-induced thrombocytopenia where alternative drug therapy is not available.
  • the present invention provides a method for reducing haemorrhage in a mammal inflicted with a haemorrhagic-causing condition, said method comprising the step of administering to said mammal an anti-haemorrhagic amount of a peptide of formula (I), or a functional equivalent thereof, wherein formula
  • X j and X 5 are selected from the group consisting of cysteine and alanine;
  • X 2 , X 3 , and X 4 are the same or different amino acid residues selected from the group consisting of valine, threonine, serine, and arginine;
  • X 6 is an amino acid selected from the group consisting of lysine, glycine and arginine;
  • R ] is selected from the group consisting of H, a blocking group, a blocked or an unblocked amino acid residue and the desamino form of said amino acid;
  • R 2 is selected from the group consisting of NH 2 , a blocking group, a blocked or an unblocked amino acid residue and the carboxyamide or alkylamide form of said amino acid.
  • Figure 1 graphically illustrates inhibition in the Local Shwartzman Reaction of haemorrhage on administration of a hexapeptide in accordance with the present invention
  • Figure 2 is a bar graph showing dose-dependent increase in airway haemorrhage in the rat lung injury animal model
  • Figure 3 graphically illustrates inhibition of airway haemorrhage in the animal model of Fig. 2 on administration of increasing concentrations of the hexapeptide referred to in Fig. 1;
  • Figure 4 is a bar graph comparison of the effects of the hexapeptide of Fig. 1 in the rat lung injury model to the effects of a scrambled hexapeptide.
  • a composition comprising an anti-haemorrhagic peptide capable of reducing haemorrhage in a mammal inflicted with a haemorrhage-causing condition.
  • haemorrhage is used herein to refer to the escape of blood, or bleeding, from vessels.
  • the term refers to both external bleeding, i.e. escape of blood from the body, and internal bleeding, i.e. escape of blood from vessels into the body.
  • haemorrhage also refers to instances of bleeding that vary in the amount of blood involved and which can be characterized as petechiae (very small), purpura (up to one centimetre) or ecchymoses (larger).
  • anti-haemorrhagic refers to a peptide which is capable of treating haemorrhage in a mammal, either by interrupting or obstructing haemorrhage that has already presented, by preventing or reducing haemorrhage that may occur or is anticipated, or by reducing the incidence of haemorrhage particularly in mammals inflicted with a condition in which haemorrhage occurs at frequent intervals.
  • the anti-haemorrhagic effect of a peptide can be determined using animal models such as those described herein, e.g.
  • the Schwartzman model and the rat lung injury model in which a haemorrhagic event is caused and reduction in haemorrhage is determined subsequent to treatment with a given peptide by calculating blood volume at the affected site in comparison to the blood volume at the affected site of an untreated control.
  • Haemorrhage-causing condition is meant to encompass those conditions which result in haemorrhage, either internally or externally.
  • conditions that are haemorrhage-causing include, but are not limited to, conditions of thrombocytopenia such as drug-induced thrombocytopenia, immunologic idiopathic thrombocytopenic purpura and thrombotic thrombocytopenic purpura; conditions of vasculitis such as autoimmune vasculitis, Wegener's Granulomatosis, Systemic Lupus Erythematosis and von Willebrand's Disease; and conditions of intravascular coagulation such as desseminated intravascular coagulation.
  • Anti-haemorrhagic peptides in accordance with the present invention are encompassed by the following general formula (I):
  • X j and X 5 are selected from the group consisting of cysteine and alanine;
  • X 2 , X 3 , and X 4 are the same or different amino acid residues selected from the group consisting of valine, threonine, serine, and arginine;
  • X 6 is an amino acid selected from the group consisting of lysine, glycine and arginine;
  • R j is selected from the group consisting of H, a blocking group, a blocked or an unblocked amino acid residue and the desamino form of said amino acid; and R 2 is selected from the group consisting of NH 2 , a blocking group, a blocked or an unblocked amino acid residue and the carboxyamide or alkylamide form of said amino acid.
  • the most preferred peptide in either blocked or unblocked form, is the hexapeptide, CSVTCG (SEQ ID NO: l), derived from the Type I repeat of thrombospondin; however, peptides which are functionally equivalent to the hexapeptide and to peptides encompassed by formula (1) above, can also be prepared which are useful as anti-haemorrhagic agents.
  • the term "functionally equivalent”, as it is used herein, is meant to encompass peptides which differ from the hexapeptide, and other peptides encompassed by formula (I), by addition, deletion, replacement or modification of one or more of its amino acid residues but which retain the anti- haemorrhagic property thereof, i.e.
  • hexapeptide may include additional amino acid residues at either end which do not affect its anti-haemorrhagic activity.
  • one or more of the amino acids may be deleted from the hexapeptide, such as a terminal amino acid, without compromising activity.
  • the hexapeptide can include amino acid replacements of native amino acids such as conservative amino acid replacements, e.g. an amino acid of the hexapeptide may be replaced by an amino acid of similar charge and size such as replacement of threonine with serine, without loss of activity.
  • Non-conservative amino acid replacements are also tolerated, for example, replacement of a cysteine residue by alanine, or replacement of a glycine residue by lysine or arginine.
  • amino acids of the hexapeptide can be modified or derivatized, as described in more detail herein, to yield a peptide which retains anti- haemorrhagic activity.
  • peptides in accordance with formula (I) have the following amino acid sequences: CSVTCG (SEQ ID NO: 1) CSVTCR (SEQ ID NO:2) CSTSCR (SEQ ID NO:3) CSTSCG (SEQ ID NO:4) CRVTCG (SEQ ID NO:5) RCRVTCG (SEQ ID NO:6)
  • ASVTAR SEQ ID NO:7
  • amino acid residues represented by X, and X 2 are both deleted and R j and R 2 are other than an amino acid, i.e. is H or NH 2 , respectively, or a blocking group, to provide a blocked or unblocked peptide comprising four amino acid residues.
  • a preferred peptide in this regard is VTCG (SEQ ID NO: 13).
  • the peptides may incorporate amino acid residues which are modified without affecting activity.
  • the termini may be derivatized to include blocking groups, i.e. chemical substituents suitable to protect and/or stabilize the N- and C-termini from "undesirable degradation", a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound as an anti-haemorrhagic agent, i.e. sequential degradation of the compound initiated at a terminal end thereof.
  • Blocking groups include protecting groups conventionally used in the art of peptide chemistry which will not adversely affect the in vivo activities of the peptide.
  • suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus.
  • suitable N-terminal blocking groups include C ⁇ -C 5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof, such as the acetamidomethyl (Acm) group.
  • Desamino analogs of amino acids are also useful N-terminal blocking groups, and can either be coupled to the N-terminus of the peptide or used in place of the N-terminal residue.
  • Suitable C-terminal blocking groups in which the carboxyl group of the C- terminus is either incorporated or not, include esters, ketones or amides.
  • Ester or ketone-forming alkyl groups particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (-NH 2 ), and mono- and di-alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups.
  • Descarboxylated amino acid analogues such as agmatine are also useful C- terminal blocking groups and can be either coupled to the peptide's C-terminal residue or used in place of it.
  • N- and C-terminal blocking groups of even greater structural complexity may alternatively be incorporated to protect the N- and C-terminal ends of the present peptides from attack provided that the anti-haemorrhagic activity of the compound is not adversely affected by the incorporation thereof.
  • Internal amino acids of the peptide can also be modified by derivatization without affecting anti-haemorrhagic activity.
  • derivatizations can be made to the side chains of the amino acids.
  • the side chains can derivatized by incorporation of blocking groups as described above.
  • and X 5 are cysteine residues derivatized by addition of the acetamidomethyl (Acm) blocking group.
  • Cyclization can be effected between both terminal and internal amino acid residues in the peptide.
  • Cyclization can be via a disulfide linkage, for example, between two cysteine residues.
  • cyclization can be via a peptide linkage between the amino and carboxyl groups of terminal amino acid residues of the peptide, or between amino and carboxyl groups of the side chains of terminal or internal amino acid residues.
  • Other modifications can also be incorporated without adversely affecting anti- haemorrhagic activity and these include, but are not limited to, substitution of one or more of the amino acids in the natural L-isomeric form with amino acids in the D- isomeric form.
  • the peptide may include one or more D-amino acid residues, or may comprise amino acids which are all in the D-form.
  • Retro-inverso forms of peptides in accordance with the present invention are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms.
  • retro-inverso peptides in accordance with the present invention are the retro-inverso form of the CSVTCG hexapeptide, specifically the D-substituted peptide, GCTVSC (SEQ ID NO:14), and the retro-inverso form of its Ala derivative, namely the peptide,
  • peptides in the form of acid addition salts are also contemplated as functional equivalents.
  • peptides of the present invention may be readily prepared by standard, well- established solid-phase peptide synthesis (SPPS) as described by Stewart et al. in Solid
  • a suitably protected amino acid residue is attached through its carboxyl group to a derivatized, insoluble polymeric support, such as cross-linked polystyrene or polyamide resin.
  • "Suitably protected” refers to the presence of protecting groups on both the ⁇ -amino group of the amino acid, and on any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used throughout the synthesis, and are removable under conditions which will not affect the final peptide product.
  • Stepwise synthesis of the oligopeptide is carried out by the removal of the N- protecting group from the initial amino acid, and coupling thereto of the carboxyl end of the next amino acid in the sequence of the desired peptide.
  • This amino acid is also suitably protected.
  • the carboxyl of the incoming amino acid can be activated to react with the N-terminus of the support-bound amino acid by formation into a reactive group such as formation into a carbodiimide, a symmetric acid anhydride or an "active ester" group such as hydroxybenzotriazole or pentafluorophenyl esters.
  • solid phase peptide synthesis methods include the BOC method which utilizes tert-butyloxycarbonyl as the ⁇ -amino protecting group, and the FMOC method which utilizes 9-fluorenylmethyloxycarbonyl to protect the ⁇ -amino of the amino acid residues, both methods of which are well-known by those of skill in the art.
  • N- and/or C- blocking groups can also be achieved using protocols conventional to solid phase peptide synthesis methods.
  • synthesis of the desired peptide is typically performed using, as solid phase, a supporting resin that has been chemically modified so that cleavage from the resin results in a peptide having the desired C-terminal blocking group.
  • a supporting resin that has been chemically modified so that cleavage from the resin results in a peptide having the desired C-terminal blocking group.
  • synthesis is performed using a p-methylbenzhydrylamine (MBHA) resin so that, when peptide synthesis is completed, treatment with hydrofluoric acid releases the desired C-terminally amidated peptide.
  • MBHA p-methylbenzhydrylamine
  • N-methylaminoethyl-derivatized DVB resin which upon HF treatment releases a peptide bearing an N-methylamidated C-terminus.
  • Blockage of the C-terminus by esterification can also be achieved using conventional procedures. This entails use of resin/blocking group combination that permits release of side-chain protected peptide from the resin, to allow for subsequent reaction with the desired alcohol, to form the ester function.
  • FMOC protecting groups in combination with DVB resin derivatized with methoxyalkoxybenzyl alcohol or equivalent linker, can be used for this pu ⁇ ose, with cleavage from the support being effected by TFA in dicholoromethane. Esterification of the suitably activated carboxyl function e.g. with DCC, can then proceed by addition of the desired alcohol, followed by deprotection and isolation of the esterified peptide product.
  • Inco ⁇ oration of N-terminal blocking groups can be achieved while the synthesized peptide is still attached to the resin, for instance by treatment with a suitable anhydride and nitrile.
  • a suitable anhydride and nitrile for instance, the resin-coupled peptide can be treated with 20% acetic anhydride in acetonitrile.
  • the N-blocked peptide product can then be cleaved from the resin, deprotected and subsequently isolated.
  • amino acid composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the peptide.
  • amino acid content of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequenators, which sequentially degrade the peptide and identify the amino acids in order, may also be used to determine definitely the sequence of the peptide.
  • the peptide Prior to its use as an anti-haemorrhagic agent, the peptide is purified to remove contaminants. In this regard, it will be appreciated that the peptide will be purified so as to meet the standards set out by the appropriate regulatory agencies. Any one of a number of conventional purification procedures may be used to attain the required level of purity including, for example, reversed-phase high-pressure liquid chromatography (HPLC) using an alkylated silica column such as C -, C 8 - or C 18 - silica.
  • HPLC reversed-phase high-pressure liquid chromatography
  • a gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid.
  • Ion-exchange chromatography can also be used to separate peptides based on their charge.
  • compositions comprising the anti-haemorrhagic peptide of the present invention are prepared for use in treating mammals inflicted with such haemorrhage-causing conditions as thrombocytopenia and vasculitis.
  • mammal as it is used herein is meant to encompass humans, domestic animals such as cats, dogs and horses, livestock such as cattle, pigs, goats, and sheep, and non- domesticated mammals that may be in need of anti-haemorrhagic treatment.
  • the anti-haemorrhagic compositions comprise an anti-haemorrhagic amount of peptide together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means acceptable for use in the pharmaceutical and veterinary arts, i.e. a carrier which is non-toxic and which does not adversely affect the activity of the peptide as an anti-haemorrhagic agent.
  • anti-haemorrhagic amount means an amount of the peptide sufficient to reduce haemorrhage in a mammal inflicted with a haemorrhage-causing condition as determined using suitable models such as the animal models described in the specific examples herein.
  • compositions for in vivo administration include conventional carriers used in formulating peptide-based drugs, such as diluents, excipients and the like. Reference may be made to "Remington's
  • compositions in accordance with the present invention will depend on the administrable form to be used to treat the inflicted individual.
  • the compounds are formulated for administration by intravenous injection and are accordingly provided as aqueous solutions in sterile and pyrogen-free form and optionally buffered or made isotonic.
  • the compounds may be administered in distilled water or, more desirably, in saline or 5% dextrose solution.
  • Water solubility of these and other compounds of the invention may be enhanced, if desired, by inco ⁇ orating into the composition a solubility enhancer, such as cetyltrimethylammonium bromide or chloride, or by preparing the acid addition salt thereof.
  • Lyoprotectants such as mannitol, sucrose or lactose and buffer systems, such as acetate, citrate and phosphate may also be included in the formulation, as may bulking agents such as serum albumin.
  • the present invention provides in another of its aspects a package, in the form of a sterile-filled vial or ampoule, that contains an anti-haemorrhagic amount of a peptide in accordance with the present invention, in either unit dose or multi-dose amounts, wherein the package inco ⁇ orates a label instructing use of its contents for treating haemorrhage.
  • the package contains the peptide and the desired carrier, as an administration-ready formulation.
  • the package provides the anti-haemorrhagic peptide in a form, such as a lyophilized form, suitable for reconstitution in a suitable carrier, such as phosphate-buffered saline.
  • a suitable carrier such as phosphate-buffered saline.
  • the package is a sterile-filled vial or ampoule containing an injectable solution which comprises an effectve amount of an anti- haemorrhagic peptide of the formula, R1-CSVTCG-R2, wherein Rl and R2 are as defined in formula (I), dissolved in neutral phosphate buffer (pH 6.5-7.5) to a peptide concentration ranging from microgram to milligram quantities per millilitre buffer.
  • an injectable solution which comprises an effectve amount of an anti- haemorrhagic peptide of the formula, R1-CSVTCG-R2, wherein Rl and R2 are as defined in formula (I), dissolved in neutral phosphate buffer (pH 6.5-7.5) to a peptide concentration ranging from microgram to milligram quantities per millilitre buffer.
  • the compounds of the present invention may be formulated for administration by other routes.
  • Compositions for topical application such as eye drops, creams, lotions, or ointments may be useful, as may aerosol inhalable formulations.
  • Oral dosage forms such as tablets, capsules and the like, formulated in accordance with standard pharmaceutical practise, may also be employed.
  • the peptide, H-C(Acm)-SVTC(Acm)-G (hereinafter referred to as the Acm- hexapeptide), was prepared as a single peptide chain by solid phase peptide synthesis using 1.00 mmol scale FMOC chemistry on an FMOC-Gly preloaded 2-methoxy-4- alkoxybenzyl alcohol resin (Sasrin Resin, Bachem Biosciences In., Philadelphia) with an Applied Biosystems 433 A peptide synthesizer (Foster City, CA).
  • the peptide-resin was dried under vacuum overnight and cleavage of the peptide from the resin was achieved by mixing a cooled solution of 9.5mL trifluoroacetic acid (TFA), 0.5mL water, 0.5mL thioanisole and 0.25mL 2-ethanedithiol (lmL per lOOmg of peptide-resin) with the peptide-resin for 2 to 2.5 hours at room temperature.
  • TFA trifluoroacetic acid
  • the resin was removed by filtration and washed with 1-3 mL of TFA to obtain 8-10 mL of a clear yellow liquid.
  • This liquid was slowly dropped into 45 mL of cold tert-butyl ether in a 50 mL conical polypropylene centrifuge tube and formed a white precipitate.
  • the precipitate was centrifuged at 7000 rpm, O°C for 5 minutes (Sorvall RT6000, Dupont), decanted and washed two more times with tert-butyl ether.
  • the unblocked hexapeptide, CSVTCG was also synthesized as described above.
  • the cysteine residue starting material was FMOC-Cys(trityl)-OH instead of FMOC-Cys(Acm)-OH.
  • the LSR is a model of vasculitis that is not mediated by immune complex deposition.
  • a thrombo-haemorrhagic event is induced by a first intradermal injection of endotoxin from gram negative bacteria, followed by a second intravenous injection of the endotoxin (the provocative dose) 18-24 hours later.
  • Thrombo-haemorrhagic lesions develop rapidly following the intravenous challenge.
  • Small veins and venules show microthrombi composed of platelets, fibrin and polymo ⁇ honuclear leucocytes (PMNs).
  • PMNs polymo ⁇ honuclear leucocytes
  • mice Female New Zealand white rabbits, weighing 2.5 to 3.0 kg, were used in these studies. They were acclimatized for three days during which time they had access to commercial rabbit chow and tap water.
  • Endotoxin lipopolysaccharide Escherichia coli serotype 055:B5 obtained from Sigma
  • sterile saline Baxter
  • CSVTCG hexapeptide (1 mg/kg dissolved in 3.0 ml saline) was administered i.v. to the rabbits.
  • Control rabbits were given 3.0 ml of saline. All animals received 7.5 ml of In-erythrocytes (prepared as described below) 5 min before the i.v. endotoxin challenge (provocative dose).
  • the provocative dose (10 ⁇ g/kg endotoxin in 2 ml of saline) was administered i.v. 18-20 h after the intradermal injection.
  • the skin lesions were allowed to develop for 4 hours. Five minutes prior to sacrifice of the animals (by sodium pentabarbital), a blood sample was taken from each animal via cardiac puncture. Following sacrifice of the animals, the skin of the affected area was removed, cleaned and the lesions were punched out with a 17 mm punch. Radioactivity was assayed in a gamma counter (Canberra Packard, Cobra II) and the volume of blood ( ⁇ lJlesion) was calculated using the radioactivity in the blood as a reference.
  • Fig. 1 The results are illustrated in Fig. 1 and show that haemorrhage at the affected site was significantly reduced in animals to which CSVTCG hexapeptide was administered.
  • cysteine Acm-labelled peptide (Acm)CSVTC(Acm)-G, was tested using the LSR model in the manner described above. This peptide was also found to reduce haemorrhage.
  • An IgG-immune complex-induced rat lung injury model was used to further exemplify the anti-haemorrhagic effects of the present peptides.
  • the antigen bovine serum albumin (BSA, obtained from Sigma)
  • BSA bovine serum albumin
  • rabbit anti-BSA IgG obtained from Organontechnika
  • haemorrhage in the lung was evaluated by determining the haemoglobin content of bronchoalveolar airway lavage. Haemorrhage response was reproducible in this model and the extent of haemorrhage was dependent on the dose of antibody delivered.
  • Fig. 2 The dose-dependent increase in red blood cells recovered from bronchoalveolar lavage (BAL) is illustrated in Fig. 2.
  • Rats were anaesthetized with pentobarbital (50 - 65 mg/kg, i.p.)(MTC Pharmaceuticals). Then the trachea of each anaesthetized rat was intubated with an AngiocathTM catheter (G18) through the mouth. Anti-BSA IgG (2 mg in 300 ⁇ l PBS (phosphate buffered saline, pH: 7.4)) was instilled into the airway through the catheter. An equal volume of vehicle solution (300 ⁇ l PBS) was instilled into the airway of control rats.
  • pentobarbital 50 - 65 mg/kg, i.p.
  • AngiocathTM catheter G18
  • Anti-BSA IgG 2 mg in 300 ⁇ l PBS (phosphate buffered saline, pH: 7.4)
  • An equal volume of vehicle solution 300 ⁇ l PBS was instilled into the airway of control rats.
  • BSA (10 mg in 200 ⁇ l saline containing 0.1 ⁇ Ci I-HSA, human serum albumin obtained from Amersham) was injected intravenously via a penovein.
  • the anti-haemorrhagic peptide (CSVTCG) dissolved in a small amount of sterile dH 2 O and then diluted with PBS to the required concentration, was co-injected intravenously with BSA.
  • Concentrations of peptide tested ranged from 2.5 ⁇ g/kg to 2.5 mg/kg.
  • a vehicle-treated immune complex-injured rat was always included in each experiment as a positive control to compensate for variation from batch to batch and day to day experiments. Results were evaluated by comparing the percent changes of the tested animals to their vehicle controls. At least four rats were used in each treatment.
  • the rat was re-anaesthetized as set out above and a blood sample was withdrawn by cardiac puncture. The rat was then sacrificed by overdose of pentobarbital (50 mg/kg, i.v ). Airways were lavaged by infusion of 4 ml PBS through an intratracheal cannula (PE-
  • Airway haemorrhage was monitored by BAL haemoglobulin (Hb) index.
  • Hb index BAL samples were sonicated, centrifuged and three dilutions were prepared from each sample (serial dilutions of lOx). Optical densities (O.D.) of the resulting supernatants were determined at 414 nm. Hb index was calculated as the average value of the absorbances.
  • Anti-haemorrhagic CSVTCG peptide was also co-instilled with anti-BSA IgG intratracheally. A dose-dependent inhibition of airway haemorrhage also occurred in this case as illustrated in Fig. 3.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Genetics & Genomics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

An anti-haemorrhagic peptide is provided having the amino acid sequence of the general formula (I): R1-X1-X2-X3-X4-X5-X6-R2 wherein: X1 and X5 are selected from the group consisting of cysteine and alanine; X2, X3, and X4 are the same or different amino acid residues selected from the group consisting of valine, threonine, serine, and arginine; X6 is an amino acid selected from the group consisting of lysine, glycine and arginine; R1 is selected from the group consisting of H, a blocking group, a blocked or an unblocked amino acid residue and the desamino form of said amino acid; and R2 is selected from the group consisting of NH2, a blocking group, a blocked or an unblocked amino acid residue and the carboxyamide or alkylamide form of said amino acid.

Description

ANTI-HAEMORRHAGIC PEP TIDES
FIELD OF THE INVENTION
The present invention relates to peptides having anti-haemorrhagic properties. In particular, the present invention relates to an anti-haemorrhagic peptide derived from thrombospondin, and a method for reducing haemorrhage in a mammal.
BACKGROUND OF THE INVENTION
Haemorrhage is associated with many disease conditions. Thrombocytopenia, for example, a disorder in which platelet number is reduced to below normal. Platelets, the cell components which control blood clotting and play a role in the repair of blood vessels, are important in preventing haemorrhage from occurring. Thrombocytopenia may stem from decreased platelet production, sequestration of platelets by the spleen, increased platelet destruction or dilution of platelets. Thrombocytopenic Purpura, a thrombocytopenic condition having both thrombotic and immunologic idiopathic forms, is a severe, potentially chronic condition in which sudden relapses can result in life- threatening bleeding. Thrombocytopenia can also be induced by certain drugs, including heparin, quinidine, sulfa preparations, oral anti-diabetic agents and rifampin. Whatever the cause, the thrombocytopenic individual is much less tolerant of bleeding since they lack the ability to prevent excessive bleeding through clotting. Although plasma therapy can be used to treat thrombocytopenic individuals that must undergo surgery, this type of treatment is not feasible for widespread use due to the limits imposed by plasma supply.
Vasculitis is a disorder in which inflammation within the vasculature occurs resulting in varying degrees of vessel destruction, necrosis or scarring in one or more layers of the vessels. Subsequent loss of vessel wall integrity results from formation of fibrin plugs and can cause red blood cell leakage into surrounding tissue, i.e. haemorrhage. Diseases characterized by vasculitis include von Willebrand's Disease, Wegener's Granulomatosis and Systemic Lupus Erythematosis. Individuals inflicted with von Willebrand's Disease, for example, present with bleeding over several hours from small skin cuts and abnormal bleeding following small surgical procedures such as tooth extraction and tonsillectomy.
An agent capable of reducing the occurrence of haemorrhage in conditions such as those mentioned above is desirable, particularly in those instances in which haemorrhage can be reasonably predicted or expected, for example, in conditions where relapse is possible (Thrombocytopenic Purpura), in individuals having vasculitis where infection, cuts or surgery can result in excessive bleeding, and in individuals having drug-induced thrombocytopenia where alternative drug therapy is not available.
SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for treating haemorrhage-causing conditions in mammals.
Accordingly, in one aspect, the present invention provides a method for reducing haemorrhage in a mammal inflicted with a haemorrhagic-causing condition, said method comprising the step of administering to said mammal an anti-haemorrhagic amount of a peptide of formula (I), or a functional equivalent thereof, wherein formula
(I) is as follows:
R1-X X2-X3-X4-X5-X6-R2 (I) wherein:
Xj and X5 are selected from the group consisting of cysteine and alanine; X2, X3, and X4 are the same or different amino acid residues selected from the group consisting of valine, threonine, serine, and arginine; X6 is an amino acid selected from the group consisting of lysine, glycine and arginine;
R] is selected from the group consisting of H, a blocking group, a blocked or an unblocked amino acid residue and the desamino form of said amino acid; and
R2 is selected from the group consisting of NH2, a blocking group, a blocked or an unblocked amino acid residue and the carboxyamide or alkylamide form of said amino acid. In another aspect of the present invention, there is provided a package containing an anti-haemorrhagic amount of a peptide as defined above, which bears a label instructing use of the peptide to reduce haemorrhage.
These and other aspects of the invention will be described in detail by reference to the following figures in which:
BRIEF REFERENCE TO THE DRAWINGS
Figure 1 graphically illustrates inhibition in the Local Shwartzman Reaction of haemorrhage on administration of a hexapeptide in accordance with the present invention;
Figure 2 is a bar graph showing dose-dependent increase in airway haemorrhage in the rat lung injury animal model;
Figure 3 graphically illustrates inhibition of airway haemorrhage in the animal model of Fig. 2 on administration of increasing concentrations of the hexapeptide referred to in Fig. 1; and
Figure 4 is a bar graph comparison of the effects of the hexapeptide of Fig. 1 in the rat lung injury model to the effects of a scrambled hexapeptide.
DETAILED DESCRIPTION OF THE INVENTION
A composition is provided comprising an anti-haemorrhagic peptide capable of reducing haemorrhage in a mammal inflicted with a haemorrhage-causing condition.
The term "haemorrhage" is used herein to refer to the escape of blood, or bleeding, from vessels. The term refers to both external bleeding, i.e. escape of blood from the body, and internal bleeding, i.e. escape of blood from vessels into the body. The term haemorrhage also refers to instances of bleeding that vary in the amount of blood involved and which can be characterized as petechiae (very small), purpura (up to one centimetre) or ecchymoses (larger).
As used herein, the term "anti-haemorrhagic" refers to a peptide which is capable of treating haemorrhage in a mammal, either by interrupting or obstructing haemorrhage that has already presented, by preventing or reducing haemorrhage that may occur or is anticipated, or by reducing the incidence of haemorrhage particularly in mammals inflicted with a condition in which haemorrhage occurs at frequent intervals. The anti-haemorrhagic effect of a peptide can be determined using animal models such as those described herein, e.g. the Schwartzman model and the rat lung injury model, in which a haemorrhagic event is caused and reduction in haemorrhage is determined subsequent to treatment with a given peptide by calculating blood volume at the affected site in comparison to the blood volume at the affected site of an untreated control.
"Haemorrhage-causing condition" is meant to encompass those conditions which result in haemorrhage, either internally or externally. Examples of conditions that are haemorrhage-causing include, but are not limited to, conditions of thrombocytopenia such as drug-induced thrombocytopenia, immunologic idiopathic thrombocytopenic purpura and thrombotic thrombocytopenic purpura; conditions of vasculitis such as autoimmune vasculitis, Wegener's Granulomatosis, Systemic Lupus Erythematosis and von Willebrand's Disease; and conditions of intravascular coagulation such as desseminated intravascular coagulation.
Anti-haemorrhagic peptides in accordance with the present invention are encompassed by the following general formula (I):
RJ-XJ-X2-X3-X4-X5-X -R2 (I) wherein:
Xj and X5 are selected from the group consisting of cysteine and alanine; X2, X3, and X4 are the same or different amino acid residues selected from the group consisting of valine, threonine, serine, and arginine; X6 is an amino acid selected from the group consisting of lysine, glycine and arginine;
Rj is selected from the group consisting of H, a blocking group, a blocked or an unblocked amino acid residue and the desamino form of said amino acid; and R2 is selected from the group consisting of NH2, a blocking group, a blocked or an unblocked amino acid residue and the carboxyamide or alkylamide form of said amino acid.
The most preferred peptide, in either blocked or unblocked form, is the hexapeptide, CSVTCG (SEQ ID NO: l), derived from the Type I repeat of thrombospondin; however, peptides which are functionally equivalent to the hexapeptide and to peptides encompassed by formula (1) above, can also be prepared which are useful as anti-haemorrhagic agents. The term "functionally equivalent", as it is used herein, is meant to encompass peptides which differ from the hexapeptide, and other peptides encompassed by formula (I), by addition, deletion, replacement or modification of one or more of its amino acid residues but which retain the anti- haemorrhagic property thereof, i.e. the ability to prevent or reduce haemorrhage in a mammal. Functional equivalents of the hexapeptide, thus, may include additional amino acid residues at either end which do not affect its anti-haemorrhagic activity. Likewise, one or more of the amino acids may be deleted from the hexapeptide, such as a terminal amino acid, without compromising activity. Alternatively, the hexapeptide can include amino acid replacements of native amino acids such as conservative amino acid replacements, e.g. an amino acid of the hexapeptide may be replaced by an amino acid of similar charge and size such as replacement of threonine with serine, without loss of activity. Non-conservative amino acid replacements are also tolerated, for example, replacement of a cysteine residue by alanine, or replacement of a glycine residue by lysine or arginine. Further, amino acids of the hexapeptide can be modified or derivatized, as described in more detail herein, to yield a peptide which retains anti- haemorrhagic activity.
In specific embodiments of the present invention, peptides in accordance with formula (I) have the following amino acid sequences: CSVTCG (SEQ ID NO: 1) CSVTCR (SEQ ID NO:2) CSTSCR (SEQ ID NO:3) CSTSCG (SEQ ID NO:4) CRVTCG (SEQ ID NO:5) RCRVTCG (SEQ ID NO:6)
ASVTAR (SEQ ID NO:7)
CSVTCK (SEQ ID NO:8)
CSTSCK (SEQ ID NO:9)
CSRTCG (SEQ ID NO:10) CRTSCG (SEQ ID NO:l l)
PCSVTCR (SEQ ID NO: 12)
In another emobodiment, the amino acid residues represented by X, and X2 are both deleted and Rj and R2 are other than an amino acid, i.e. is H or NH2, respectively, or a blocking group, to provide a blocked or unblocked peptide comprising four amino acid residues. A preferred peptide in this regard is VTCG (SEQ ID NO: 13).
It will be appreciated, of course, that the peptides may incorporate amino acid residues which are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e. chemical substituents suitable to protect and/or stabilize the N- and C-termini from "undesirable degradation", a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound as an anti-haemorrhagic agent, i.e. sequential degradation of the compound initiated at a terminal end thereof.
Blocking groups include protecting groups conventionally used in the art of peptide chemistry which will not adversely affect the in vivo activities of the peptide.
For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus. Examples of suitable N-terminal blocking groups include Cι-C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof, such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful N-terminal blocking groups, and can either be coupled to the N-terminus of the peptide or used in place of the N-terminal residue. Suitable C-terminal blocking groups, in which the carboxyl group of the C- terminus is either incorporated or not, include esters, ketones or amides. Ester or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (-NH2), and mono- and di-alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups. Descarboxylated amino acid analogues such as agmatine are also useful C- terminal blocking groups and can be either coupled to the peptide's C-terminal residue or used in place of it. Further, it will be appreciated that the free amino and carboxyl groups at the termini can be removed altogether from the peptide to yield desamino and descarboxylated forms thereof without affect on peptide activity. Of course, N- and C-terminal blocking groups of even greater structural complexity may alternatively be incorporated to protect the N- and C-terminal ends of the present peptides from attack provided that the anti-haemorrhagic activity of the compound is not adversely affected by the incorporation thereof.
Internal amino acids of the peptide can also be modified by derivatization without affecting anti-haemorrhagic activity. Such derivatizations can be made to the side chains of the amino acids. For example, the side chains can derivatized by incorporation of blocking groups as described above. In one embodiment, X| and X5 are cysteine residues derivatized by addition of the acetamidomethyl (Acm) blocking group.
Another modification that may be incorporated in peptides of the present invention to yield a functional equivalent is cyclization of the peptide. Cyclization can be effected between both terminal and internal amino acid residues in the peptide.
Cyclization can be via a disulfide linkage, for example, between two cysteine residues.
Alternatively, cyclization can be via a peptide linkage between the amino and carboxyl groups of terminal amino acid residues of the peptide, or between amino and carboxyl groups of the side chains of terminal or internal amino acid residues. Other modifications can also be incorporated without adversely affecting anti- haemorrhagic activity and these include, but are not limited to, substitution of one or more of the amino acids in the natural L-isomeric form with amino acids in the D- isomeric form. Thus, the peptide may include one or more D-amino acid residues, or may comprise amino acids which are all in the D-form. Retro-inverso forms of peptides in accordance with the present invention are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms. Examples of retro-inverso peptides in accordance with the present invention are the retro-inverso form of the CSVTCG hexapeptide, specifically the D-substituted peptide, GCTVSC (SEQ ID NO:14), and the retro-inverso form of its Ala derivative, namely the peptide,
GATVSA (SEQ ID NO: 15).
The present peptides in the form of acid addition salts are also contemplated as functional equivalents. Thus, a peptide in accordance with the present invention treated with an inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organic acid such as acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic and the like, to provide a water soluble salt of the peptide is suitable for use as an anti-haemorrhagic agent.
The peptides of the present invention may be readily prepared by standard, well- established solid-phase peptide synthesis (SPPS) as described by Stewart et al. in Solid
Phase Peptide Synthesis. 2nd Edition, 1984, Pierce Chemical Company, Rockford,
Illinois; and as described by Bodanszky and Bodanszky in The Practice of Peptide
Synthesis. 1984, Springer- Verlag, New York. At the outset, a suitably protected amino acid residue is attached through its carboxyl group to a derivatized, insoluble polymeric support, such as cross-linked polystyrene or polyamide resin. "Suitably protected" refers to the presence of protecting groups on both the α -amino group of the amino acid, and on any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used throughout the synthesis, and are removable under conditions which will not affect the final peptide product. Stepwise synthesis of the oligopeptide is carried out by the removal of the N- protecting group from the initial amino acid, and coupling thereto of the carboxyl end of the next amino acid in the sequence of the desired peptide. This amino acid is also suitably protected. The carboxyl of the incoming amino acid can be activated to react with the N-terminus of the support-bound amino acid by formation into a reactive group such as formation into a carbodiimide, a symmetric acid anhydride or an "active ester" group such as hydroxybenzotriazole or pentafluorophenyl esters.
Examples of solid phase peptide synthesis methods include the BOC method which utilizes tert-butyloxycarbonyl as the α -amino protecting group, and the FMOC method which utilizes 9-fluorenylmethyloxycarbonyl to protect the α -amino of the amino acid residues, both methods of which are well-known by those of skill in the art.
Incorporation of N- and/or C- blocking groups can also be achieved using protocols conventional to solid phase peptide synthesis methods. For incoφoration of C-terminal blocking groups, for example, synthesis of the desired peptide is typically performed using, as solid phase, a supporting resin that has been chemically modified so that cleavage from the resin results in a peptide having the desired C-terminal blocking group. To provide peptides in which the C-terminus bears a primary amino blocking group, for instance, synthesis is performed using a p-methylbenzhydrylamine (MBHA) resin so that, when peptide synthesis is completed, treatment with hydrofluoric acid releases the desired C-terminally amidated peptide. Similarly, incoφoration of an N-methylamine blocking group at the C-terminus is achieved using
N-methylaminoethyl-derivatized DVB resin, which upon HF treatment releases a peptide bearing an N-methylamidated C-terminus. Blockage of the C-terminus by esterification can also be achieved using conventional procedures. This entails use of resin/blocking group combination that permits release of side-chain protected peptide from the resin, to allow for subsequent reaction with the desired alcohol, to form the ester function. FMOC protecting groups, in combination with DVB resin derivatized with methoxyalkoxybenzyl alcohol or equivalent linker, can be used for this puφose, with cleavage from the support being effected by TFA in dicholoromethane. Esterification of the suitably activated carboxyl function e.g. with DCC, can then proceed by addition of the desired alcohol, followed by deprotection and isolation of the esterified peptide product.
Incoφoration of N-terminal blocking groups can be achieved while the synthesized peptide is still attached to the resin, for instance by treatment with a suitable anhydride and nitrile. To incoφorate an acetyl blocking group at the N- terminus, for instance, the resin-coupled peptide can be treated with 20% acetic anhydride in acetonitrile. The N-blocked peptide product can then be cleaved from the resin, deprotected and subsequently isolated.
To ensure that the peptide obtained from either chemical or biological synthetic techniques is the desired peptide, analysis of the peptide composition should be conducted. Such amino acid composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the peptide. Alternatively, or additionally, the amino acid content of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequenators, which sequentially degrade the peptide and identify the amino acids in order, may also be used to determine definitely the sequence of the peptide.
Prior to its use as an anti-haemorrhagic agent, the peptide is purified to remove contaminants. In this regard, it will be appreciated that the peptide will be purified so as to meet the standards set out by the appropriate regulatory agencies. Any one of a number of conventional purification procedures may be used to attain the required level of purity including, for example, reversed-phase high-pressure liquid chromatography (HPLC) using an alkylated silica column such as C -, C8- or C18- silica. A gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid. Ion-exchange chromatography can also be used to separate peptides based on their charge.
In one aspect, compositions comprising the anti-haemorrhagic peptide of the present invention are prepared for use in treating mammals inflicted with such haemorrhage-causing conditions as thrombocytopenia and vasculitis. The term "mammal" as it is used herein is meant to encompass humans, domestic animals such as cats, dogs and horses, livestock such as cattle, pigs, goats, and sheep, and non- domesticated mammals that may be in need of anti-haemorrhagic treatment.
The anti-haemorrhagic compositions comprise an anti-haemorrhagic amount of peptide together with a pharmaceutically acceptable carrier. In this context, the term "pharmaceutically acceptable" means acceptable for use in the pharmaceutical and veterinary arts, i.e. a carrier which is non-toxic and which does not adversely affect the activity of the peptide as an anti-haemorrhagic agent. The term "anti-haemorrhagic amount" means an amount of the peptide sufficient to reduce haemorrhage in a mammal inflicted with a haemorrhage-causing condition as determined using suitable models such as the animal models described in the specific examples herein.
Pharmaceutically acceptable carriers useful to prepare compositions for in vivo administration include conventional carriers used in formulating peptide-based drugs, such as diluents, excipients and the like. Reference may be made to "Remington's
Pharmaceutical Sciences", 17th Ed., Mack Publishing Company, Easton, Penn., 1985, for guidance on drug formulations generally. As will be appreciated, the pharmaceutical carriers used to prepare compositions in accordance with the present invention will depend on the administrable form to be used to treat the inflicted individual.
According to one embodiment of the invention, the compounds are formulated for administration by intravenous injection and are accordingly provided as aqueous solutions in sterile and pyrogen-free form and optionally buffered or made isotonic. Thus, the compounds may be administered in distilled water or, more desirably, in saline or 5% dextrose solution. Water solubility of these and other compounds of the invention may be enhanced, if desired, by incoφorating into the composition a solubility enhancer, such as cetyltrimethylammonium bromide or chloride, or by preparing the acid addition salt thereof. Lyoprotectants, such as mannitol, sucrose or lactose and buffer systems, such as acetate, citrate and phosphate may also be included in the formulation, as may bulking agents such as serum albumin.
For use in treating individuals with a haemorrhage-causing condition, precise dosage sizes of the anti-haemorrhagic composition appropriate for treatment are established in appropriately controlled trials, and will correspond to an amount of peptide that reduces haemorrhage without causing intolerable side effects. It is anticipated that an effective treatment regimen for patients will involve the intravenous administration of dosages in the range of 0.1 μg - 10 mg/kg, and more specifically, dosages in the range of 1 μg - 10 mg/kg. It will be appreciated, however, that the exact dosage sizes required to attain the desired anti-haemorrhagic effect will vary according to the route and frequency of administration. It will also vary with the specific individual being treated and the haemorrhage-causing condition with which the individual is inflicted.
For use in treating haemorrhage in a mammal including a human, the present invention provides in another of its aspects a package, in the form of a sterile-filled vial or ampoule, that contains an anti-haemorrhagic amount of a peptide in accordance with the present invention, in either unit dose or multi-dose amounts, wherein the package incoφorates a label instructing use of its contents for treating haemorrhage. In one embodiment of the invention, the package contains the peptide and the desired carrier, as an administration-ready formulation. Alternatively, and according to another embodiment of the invention, the package provides the anti-haemorrhagic peptide in a form, such as a lyophilized form, suitable for reconstitution in a suitable carrier, such as phosphate-buffered saline.
In a preferred embodiment, the package is a sterile-filled vial or ampoule containing an injectable solution which comprises an effectve amount of an anti- haemorrhagic peptide of the formula, R1-CSVTCG-R2, wherein Rl and R2 are as defined in formula (I), dissolved in neutral phosphate buffer (pH 6.5-7.5) to a peptide concentration ranging from microgram to milligram quantities per millilitre buffer.
As an alternative to injectable formulations, the compounds of the present invention may be formulated for administration by other routes. Compositions for topical application, such as eye drops, creams, lotions, or ointments may be useful, as may aerosol inhalable formulations. Oral dosage forms, such as tablets, capsules and the like, formulated in accordance with standard pharmaceutical practise, may also be employed.
Specific embodiments of the present invention will be described in more detail in the following specific examples which are not to be construed as limiting.
Example 1 - Synthesis of the hexapeptide. H-Cf Acm.-SVTC(AcmVG
The peptide, H-C(Acm)-SVTC(Acm)-G (hereinafter referred to as the Acm- hexapeptide), was prepared as a single peptide chain by solid phase peptide synthesis using 1.00 mmol scale FMOC chemistry on an FMOC-Gly preloaded 2-methoxy-4- alkoxybenzyl alcohol resin (Sasrin Resin, Bachem Biosciences In., Philadelphia) with an Applied Biosystems 433 A peptide synthesizer (Foster City, CA). Derivatized amino acid residues FMOC-Cys(Acm)-OH, FMOC-Ser(tBu)-OH, FMOC-Val-OH and FMOC-Thr(tBu)-OH (Bachem) were incoφorated at the appropriate step of chain elongation.
The peptide-resin was dried under vacuum overnight and cleavage of the peptide from the resin was achieved by mixing a cooled solution of 9.5mL trifluoroacetic acid (TFA), 0.5mL water, 0.5mL thioanisole and 0.25mL 2-ethanedithiol (lmL per lOOmg of peptide-resin) with the peptide-resin for 2 to 2.5 hours at room temperature. The resin was removed by filtration and washed with 1-3 mL of TFA to obtain 8-10 mL of a clear yellow liquid. This liquid was slowly dropped into 45 mL of cold tert-butyl ether in a 50 mL conical polypropylene centrifuge tube and formed a white precipitate. The precipitate was centrifuged at 7000 rpm, O°C for 5 minutes (Sorvall RT6000, Dupont), decanted and washed two more times with tert-butyl ether.
The precipitate was dried under vacuum and then dissolved in water. The solution was frozen in acetone-dry ice and lyophilized overnight to yield 659 mg of crude peptide (10 mL). The resulting white powder was dissolved in water, filtered through a 0.45 μm syringe filter (Gelman Acrodisc 3 CR PTFE), and purified by reversed-phase HPLC (Beckman System Gold) with a C18 column (Waters RCM 25 x 10) using 1% TFA in water as buffer A and 1% TFA in acetonitrile as buffer B. The column was equilibrated with 100:0 buffer A:buffer B and eluted with a linear gradient in 30 minutes at 1 mlJmin to 100% buffer B. Fractions were re-analysed on the HPLC and pooled according to matching profiles. The pure fractions were frozen in acetone-dry ice and lyophilized 12 hours to give a white powder.
The unblocked hexapeptide, CSVTCG, was also synthesized as described above. In this case, the cysteine residue starting material was FMOC-Cys(trityl)-OH instead of FMOC-Cys(Acm)-OH.
Example 2 - The Local Shwartzman Reaction LSK)
The LSR is a model of vasculitis that is not mediated by immune complex deposition. A thrombo-haemorrhagic event is induced by a first intradermal injection of endotoxin from gram negative bacteria, followed by a second intravenous injection of the endotoxin (the provocative dose) 18-24 hours later. Thrombo-haemorrhagic lesions develop rapidly following the intravenous challenge. Small veins and venules show microthrombi composed of platelets, fibrin and polymoφhonuclear leucocytes (PMNs). Necrotizing vasculitis develops as the reaction progresses resulting in swollen endothelial cells and extravasation of red blood cells in the surrounding tissue.
Female New Zealand white rabbits, weighing 2.5 to 3.0 kg, were used in these studies. They were acclimatized for three days during which time they had access to commercial rabbit chow and tap water.
The backs of the rabbits were shaved. Endotoxin (lipopolysaccharide Escherichia coli serotype 055:B5 obtained from Sigma), dissolved in sterile saline (Baxter), was injected intradermally into the rabbits at concentrations of 0, 12.5, 25 and 50 μg in 0.2 ml volumes. Fifteen minutes prior to injection of the provocative dose,
CSVTCG hexapeptide (1 mg/kg dissolved in 3.0 ml saline) was administered i.v. to the rabbits. Control rabbits were given 3.0 ml of saline. All animals received 7.5 ml of In-erythrocytes (prepared as described below) 5 min before the i.v. endotoxin challenge (provocative dose). The provocative dose (10 μg/kg endotoxin in 2 ml of saline) was administered i.v. 18-20 h after the intradermal injection.
The skin lesions were allowed to develop for 4 hours. Five minutes prior to sacrifice of the animals (by sodium pentabarbital), a blood sample was taken from each animal via cardiac puncture. Following sacrifice of the animals, the skin of the affected area was removed, cleaned and the lesions were punched out with a 17 mm punch. Radioactivity was assayed in a gamma counter (Canberra Packard, Cobra II) and the volume of blood (μlJlesion) was calculated using the radioactivity in the blood as a reference.
The results are illustrated in Fig. 1 and show that haemorrhage at the affected site was significantly reduced in animals to which CSVTCG hexapeptide was administered.
The cysteine Acm-labelled peptide, (Acm)CSVTC(Acm)-G, was tested using the LSR model in the manner described above. This peptide was also found to reduce haemorrhage.
Preparation of ] ** •In-erythrocytes:
Blood was drawn from the central ear artery of a donor rabbit into 3.8% citrate (1:10). The citrated blood was centrifuged at 1800 φm (Sorvall RT, 6000B) at room temperature for 10 min. The plasma was removed together with the bufϊy coat. The RBCs were washed twice with tyrode buffer (80 g NaCl, 2 g KC1, 0.5 g NaH2PO4, 10 g NaHCO3, 10 g D(+)glucose, per litre H2O, pH 7.4) and resuspended in 15.0 ml of tyrode buffer. 20 μCi of 1 ** 'in-Cl was incubated with 100 μl of 2-mercaptopyridine-N- oxide (a chelating agent) for 5 min at room temperature. The RBCs were added to the chelated indium and incubated at room temperature for 15 min with intermittent shaking. Following, incubation, the cells were washed with 35 ml of platelet poor plasma (PPP) and centrifuged at 2000 φm. The supernatant was discarded, the cells were washed for a second time with 35 ml of tyrode buffer, and then resuspended in 15 ml of the buffer. Example 3 - The Rat Lung Injury Model
An IgG-immune complex-induced rat lung injury model was used to further exemplify the anti-haemorrhagic effects of the present peptides. In this model, the antigen, bovine serum albumin (BSA, obtained from Sigma), was injected intravenously (i v.), and antibody, rabbit anti-BSA IgG (obtained from Organon Technika), was instilled intratracheally (i.t.). Four hours later, haemorrhage in the lung was evaluated by determining the haemoglobin content of bronchoalveolar airway lavage. Haemorrhage response was reproducible in this model and the extent of haemorrhage was dependent on the dose of antibody delivered.
To establish the rat model of IgG immune complex-mediated lung injury, six groups of animals with 3 rats in each group were used. They were, respectively, instilled intratracheally with:
1) vehicle control (PBS/saline),
2) anti-BSA IgG at 0.5 mg, 3) anti-BSA IgG at 1.Omg,
4) anti-BSA IgG at 2.0 mg,
5) anti-BSA IgG at 3.0 mg, and
6) irrelevant control immunoglobin (rabbit IgG) at 2.4 mg respectively.
The dose-dependent increase in red blood cells recovered from bronchoalveolar lavage (BAL) is illustrated in Fig. 2.
EXPERIMENTAL:
Male Long Evans rats with body weights of 350 - 400 g were obtained. They were housed in plastic cages in a laminar-flow BioClean Duo-Flo unit (Lab Products, Maywood, NJ) and fed ad libitum with rat pellets and water. They were allowed a 5 day acclimation period prior to treatment. a) Administration by i.v. instillation
Rats were anaesthetized with pentobarbital (50 - 65 mg/kg, i.p.)(MTC Pharmaceuticals). Then the trachea of each anaesthetized rat was intubated with an Angiocath™ catheter (G18) through the mouth. Anti-BSA IgG (2 mg in 300 μl PBS (phosphate buffered saline, pH: 7.4)) was instilled into the airway through the catheter. An equal volume of vehicle solution (300 μl PBS) was instilled into the airway of control rats. Immediately following this instillation, BSA (10 mg in 200 μl saline containing 0.1 μCi I-HSA, human serum albumin obtained from Amersham) was injected intravenously via a penovein. The anti-haemorrhagic peptide (CSVTCG), dissolved in a small amount of sterile dH2O and then diluted with PBS to the required concentration, was co-injected intravenously with BSA. Concentrations of peptide tested ranged from 2.5 μg/kg to 2.5 mg/kg. A vehicle-treated immune complex-injured rat was always included in each experiment as a positive control to compensate for variation from batch to batch and day to day experiments. Results were evaluated by comparing the percent changes of the tested animals to their vehicle controls. At least four rats were used in each treatment.
Four hours after introducing the BSA and anti-BSA IgG/peptide solutions, the rat was re-anaesthetized as set out above and a blood sample was withdrawn by cardiac puncture. The rat was then sacrificed by overdose of pentobarbital (50 mg/kg, i.v ). Airways were lavaged by infusion of 4 ml PBS through an intratracheal cannula (PE-
240). This wash was repeated 3 times and the lavages were collected and combined for analysis.
Airway haemorrhage was monitored by BAL haemoglobulin (Hb) index. To obtain the Hb index, BAL samples were sonicated, centrifuged and three dilutions were prepared from each sample (serial dilutions of lOx). Optical densities (O.D.) of the resulting supernatants were determined at 414 nm. Hb index was calculated as the average value of the absorbances.
When the CSVTCG peptide was co-injected intravenously with BSA at time 0, a dose-dependent inhibition of airway haemorrhage occurred as illustrated in Fig. 3. b) Administration by i t. instillation
Anti-haemorrhagic CSVTCG peptide was also co-instilled with anti-BSA IgG intratracheally. A dose-dependent inhibition of airway haemorrhage also occurred in this case as illustrated in Fig. 3.
A comparison of the anti-haemorrhagic effects of the CSVTCG peptide and a scrambled peptide, VCTGSC (SEQ ID NO: 16), was also conducted in the rat lung injury model. The peptides were co-instilled i.t. at a concentration of 2.5 mg/kg with anti-BSA IgG as described above. Figure 4 illustrates the results of this comparison. The CSVTCG peptide significantly inhibits haemorrhage while the scrambled peptide does not.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Beaubien, Beatrice Yang, Jean Burroweβ, Clement
(ii) TITLE OF INVENTION: AN ANTI-HAEMORRHAGIC AGENT
(iii) NUMBER OF SEQUENCES: 14
(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Nikaido, Marmelstein, Murray & Oram
(B) STREET: 655 Fifteenth Street N.W. Suite 330
(C) CITY: Washington
(D) STATE: D.C.
(E) COUNTRY: U.S.A. (F) ZIP: 20005-5701
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION: (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 638-5000
(B) TELEFAX: (202) 638-4810
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Cys Ser Val Thr Cys Gly 1 5
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Cys Ser Val Thr Cys Arg 1 5
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Cys Ser Thr Ser Cys Arg
1 5
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Cys Ser Thr Ser Cys Gly 1 5
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Cys Arg Val Thr Cys Gly 1 5 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Arg Cys Arg Val Thr Cys Gly 1 5
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ala Ser Val Thr Ala Arg 1 5
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Cys Ser Val Thr Cys Lys 1 5
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Cys Ser Thr Ser Cys Lys
1 5
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Cys Ser Arg Thr Cys Gly
1 5
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Cys Arg Thr Ser Cys Gly 1 5
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12;
Pro Cys Ser Val Thr Cys Arg
1 5 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION? SHQ ID NO:13:
Val Thr Cys Gly 1
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Gly Cys Thr Val Ser Cys 1 5
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Gly Ala Thr Val Ser Ala 1 5
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Val Cys Thr Gly Ser Cys 1 5

Claims

CLAIMSWe Claim:
1. A method for treating a mammal inflicted with a haemorrhage-causing condition, said method comprising the step of administering to said mammal an anti- haemorrhagic amount of a peptide of formula (I), or a functional equivalent thereof, wherein formula (I) is as follows:
1- J-X2- 3-X4-X5-X5- 2 (I) wherein:
Xj and X5 are selected from the group consisting of cysteine and alanine; X2, X3, and X4 are the same or different amino acid residues selected from the group consisting of valine, threonine, serine, and arginine; X6 is an amino acid selected from the group consisting of lysine, glycine and arginine;
Rj is selected from the group consisting of H, a blocking group, a blocked or an unblocked amino acid residue and the desamino form of said amino acid; and
R2 is selected from the group consisting of NH2, a blocking group, a blocked or an unblocked amino acid residue and the carboxyamide or alkylamide form of said amino acid.
2. A method as defined in claim 1, wherein said blocked or unblocked peptide is selected from the group consisting of: CSVTCG, CSVTCR, CSTSCR, CSTSCG, CRVTCG, RCRVTCG, ASVTAR, CSVTCK, CSTSCK, CSRTCG, CRTSCG and PCSVTCR.
3. A method as defined in claim 2, wherein said peptide is CSVTCG.
4. A method as defined in claim 2, wherein said peptide is Acm-CS VTC(Acm)-G.
5. A method as defined in claim 1, wherein X] and X2 are deleted, Ri is selected from the group consisting of H and a blocking group, and R2 is selected from the group consisting of NH2 and a blocking group.
6. A method as defined in claim 5, wherein said peptide is VTCG.
7. A method as defined in claim 1, wherein said peptide is cyclized.
8. A method as defined in claim 7, wherein said peptide is cyclized via a disulfide linkage.
9. A method as defined in claim 1, wherein said peptide is administered at a dose of 0.1 μg - 10 milligrams per kilogram body weight.
10. A method as defined in claim 3, wherein said peptide is administered at a dose of 0.1 μg - 10 milligrams per kilogram body weight.
11. A method as defined in claim 1, wherein said peptide is administered intravenously.
12. A method as defined in claim 1, wherein said peptide is administered intratracheally.
13. A method as defined in claim 1, wherein said haemorrhage-causing condition is selected from the group consisting of a condition of thrombocytopenia and a condition of vasculitis.
14. A method as defined in claim 1, wherein said haemorrhage-causing condition is a thrombocytopenic puφura.
15. A method as defined in claim 1, wherein said haemorrhage-causing condition is Wegener's Granulomatosis.
16. A package containing an anti-haemorrhagic amount of a peptide as defined in claim 1 , wherein the package bears a label instructing use of its contents to treat haemorrhage.
17. A package according to claim 16, which is a vial or ampoule.
18. A package according to claim 16, further comprising a pharmaceutically acceptable carrier.
19. A package according to claim 18, wherein said carrier is an aqueous buffer.
20. A package according to claim 16, comprising the peptide R1-CSVTCG-R2, or a pharmaceutically acceptable acid addition salt thereof.
21. Use of a peptide of formula (I),
RJ-XJ-X2- 3- 4-X5-X6- 2 (I)
wherein.
X] and X5 are selected from the group consisting of cysteine and alanine; X2, X3 and X4 are the same or different amino acid residues selected from the group consisting of valine, threonine, serine and arginine;
X6 is an amino acid selected from the group consisting of lysine, glycine and arginine;
Rj is selected from the group consisting of H, a blocking group, a blocked or an unblocked amino acid residue and the desamino form of said amino acid; and
R2 is selected from the group consisting of NH2, a blocking group, a blocked or an unblocked amino acid residue and the carboxyamide or alkylamide form of said amino acid, or a functional equivalent thereof, in the preparation of a medicament.
22. Use of a peptide of formula (I),
1" 1 2" 3" 4 5"^^~^*2 */ wherein:
Xj and X5 are selected from the group consisting of cysteine and alanine; X2, X3 and X4 are the same or different amino acid residues selected from the group consisting of valine, threonine, serine and arginine;
X6 is an amino acid selected from the group consisting of lysine, glycine and arginine;
Ri is selected from the group consisting of H, a blocking group, a blocked or an unblocked amino acid residue and the desamino form of said amino acid; and
R2 is selected from the group consisting of NH2, a blocking group, a blocked or an unblocked amino acid residue and the carboxyamide or alkylamide form of said amino acid, or a functional equivalent thereof, in the preparation of a pharmaceutical for the treatment of a haemorrage-causing condition.
23. Use of claim 22, wherein the haemorrage-causing condition is selected from the group consisting of thrombocytopenia and vasculitis.
24. Use of claim 22, wherein the haemorrage-causing condition is thrombocytopenic puφura.
25. Use of claim 22, wherein the haemorrage-causing condition is Wegener's Granulomatosis.
PCT/IB1996/000358 1995-04-21 1996-04-19 Anti-haemorrhagic peptides WO1996033218A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU51612/96A AU5161296A (en) 1995-04-21 1996-04-19 Anti-haemorrhagic peptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US42607895A 1995-04-21 1995-04-21
US08/426,078 1995-04-21

Publications (1)

Publication Number Publication Date
WO1996033218A1 true WO1996033218A1 (en) 1996-10-24

Family

ID=23689198

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB1996/000358 WO1996033218A1 (en) 1995-04-21 1996-04-19 Anti-haemorrhagic peptides

Country Status (2)

Country Link
AU (1) AU5161296A (en)
WO (1) WO1996033218A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008100140A1 (en) * 2007-02-12 2008-08-21 Biotempt B.V. Treatment of trauma hemorrhage with short oligopeptides
US7560433B2 (en) 2001-12-21 2009-07-14 Biotempt B.V. Treatment of multiple sclerosis (MS)
USRE43140E1 (en) 2000-03-29 2012-01-24 Biotempt B.V. Immunoregulator
USRE43279E1 (en) 2000-03-29 2012-03-27 Biotemp B.V. Compositions capable of reducing elevated blood urea concentration
USRE43309E1 (en) 2000-03-29 2012-04-10 Biotempt B.V. Immunoregulatory compositions

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61277614A (en) * 1985-06-04 1986-12-08 Showa Denko Kk Antiplasmin agent
EP0443404A1 (en) * 1990-02-22 1991-08-28 W.R. Grace & Co.-Conn. Peptide fragments and analogs of thrombospondin
EP0478101A2 (en) * 1990-09-24 1992-04-01 W.R. Grace & Co.-Conn. Therapeutic use of peptides having thrombospondin-like activity
WO1992017499A1 (en) * 1991-04-08 1992-10-15 Cornell Research Foundation, Inc. A unique hexapeptide derived from thrombospondin and its uses
EP0514721A1 (en) * 1991-05-22 1992-11-25 W.R. Grace & Co.-Conn. Peptides having thrombospondin-like activity and their therapeutic use
WO1993011156A1 (en) * 1991-12-06 1993-06-10 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Heparin- and sulfatide-binding peptides from the type i repeats of human thrombospondin
WO1993022340A1 (en) * 1992-04-30 1993-11-11 Schering Corporation Modulation of thrombospondin-cd36 interactions

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61277614A (en) * 1985-06-04 1986-12-08 Showa Denko Kk Antiplasmin agent
EP0443404A1 (en) * 1990-02-22 1991-08-28 W.R. Grace & Co.-Conn. Peptide fragments and analogs of thrombospondin
EP0478101A2 (en) * 1990-09-24 1992-04-01 W.R. Grace & Co.-Conn. Therapeutic use of peptides having thrombospondin-like activity
WO1992017499A1 (en) * 1991-04-08 1992-10-15 Cornell Research Foundation, Inc. A unique hexapeptide derived from thrombospondin and its uses
EP0514721A1 (en) * 1991-05-22 1992-11-25 W.R. Grace & Co.-Conn. Peptides having thrombospondin-like activity and their therapeutic use
WO1993011156A1 (en) * 1991-12-06 1993-06-10 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Heparin- and sulfatide-binding peptides from the type i repeats of human thrombospondin
WO1993022340A1 (en) * 1992-04-30 1993-11-11 Schering Corporation Modulation of thrombospondin-cd36 interactions

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
C.H.SCHNEIDER E.A.: "PEPTIDES 1992;PROC.EUR.PEPT.SYMP.,22ND", 1993, ESCOM, LEIDEN, XP002011515 *
DATABASE WPI Section Ch Week 8703, Derwent World Patents Index; Class B05, AN 87-018966, XP002011516 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE43140E1 (en) 2000-03-29 2012-01-24 Biotempt B.V. Immunoregulator
USRE43279E1 (en) 2000-03-29 2012-03-27 Biotemp B.V. Compositions capable of reducing elevated blood urea concentration
USRE43309E1 (en) 2000-03-29 2012-04-10 Biotempt B.V. Immunoregulatory compositions
US7560433B2 (en) 2001-12-21 2009-07-14 Biotempt B.V. Treatment of multiple sclerosis (MS)
WO2008100140A1 (en) * 2007-02-12 2008-08-21 Biotempt B.V. Treatment of trauma hemorrhage with short oligopeptides
AU2008215193B2 (en) * 2007-02-12 2014-01-16 Biotempt B.V. Treatment of trauma hemorrhage with short oligopeptides

Also Published As

Publication number Publication date
AU5161296A (en) 1996-11-07

Similar Documents

Publication Publication Date Title
CA1311583C (en) Peptide derivatives and their application, in particular in therapy
US6319897B1 (en) Peptides which inhibit complement activation
EP0319506B1 (en) Novel platelet-aggregation inhibitor peptide derivatives
DE68928321T2 (en) Antiaggregative peptides
US6852697B1 (en) Peptides for treatment of inflammation and shock
WO1996040781A1 (en) CYCLIC PEPTIDE INHIBITORS OF β1 AND β2 INTEGRIN-MEDIATED ADHESION
RU2115659C1 (en) Derivatives of nonapeptides or their salts with pharmaceutically acceptable acids, method of their synthesis and pharmaceutical composition
WO1987006835A1 (en) Combinations having synergistic growth hormone releasing activity and methods for use thereof
US4659694A (en) Prothymosin alpha
EP0564588B1 (en) Hexapeptide anaphylatoxin-receptor ligands
Schwyzer et al. The chemistry and pharmacology of angiotensin
EP0647656B1 (en) Motilin-like polypeptides that inhibit gastrointestinal motor activity
RU2235099C2 (en) Antagonistic analogues of growth hormone releasing-hormone (gh-rh) inhibiting insulin-like growth factors (igf-i and -ii)
CA1187869A (en) Deca- undeca- dodeca and tridecapeptides with thymic activity
PT92710A (en) PROCESS FOR THE INSULATION OF POLIPEPTIDOS FROM THE POISON OF THE SHORT HOLOLENA SPIDER
JPH08504820A (en) Insulin analogue
US7049282B2 (en) Inhibition of complement action
EP0639202B1 (en) Stable polypeptide composition
WO1996033218A1 (en) Anti-haemorrhagic peptides
US5338725A (en) Anti-aggregatory agents for platelets
US6682740B1 (en) Peptides derived fram complement peptide C3a sequence and antiallergic compositions comprising them
WO1996038480A1 (en) Anti-inflammatory thrombospondin-derived peptides
US5837686A (en) Peptides and antibodies for treatment of rheumatoid arthritis
WO1993000108A1 (en) Novel inhibitors of platelet aggregation
JPH08511518A (en) Trifunctional antithrombotic and antiplatelet peptides

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载