EP1030679A1 - Compositions et procedes permettant de favoriser l'internalisation et la degradation de l'activateur du plasminogene de type urokinase - Google Patents

Compositions et procedes permettant de favoriser l'internalisation et la degradation de l'activateur du plasminogene de type urokinase

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Publication number
EP1030679A1
EP1030679A1 EP98957325A EP98957325A EP1030679A1 EP 1030679 A1 EP1030679 A1 EP 1030679A1 EP 98957325 A EP98957325 A EP 98957325A EP 98957325 A EP98957325 A EP 98957325A EP 1030679 A1 EP1030679 A1 EP 1030679A1
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EP
European Patent Office
Prior art keywords
amino acid
group
acid selected
scupa
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98957325A
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German (de)
English (en)
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EP1030679A4 (fr
Inventor
Douglas Cines
Abd Al-Roof Higazi
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University of Pennsylvania Penn
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University of Pennsylvania Penn
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Publication of EP1030679A1 publication Critical patent/EP1030679A1/fr
Publication of EP1030679A4 publication Critical patent/EP1030679A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • A61K38/095—Oxytocins; Vasopressins; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • 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
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8132—Plasminogen activator inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase

Definitions

  • the field of the invention is regulation of cell adhesion and migration.
  • Plasminogen activators convert the inactive zymogen plasminogen into the broad-spectrum proteolytic enzyme, plasmin (Higgins et al., 1990, Annu. Rev. Pharmacol. Toxicol. 30:91-121; Holden, 1990, Radiology 174:993-1001; Mayer, 1990, Clin. Biochem. 23 : 197-211).
  • plasminogen activator designated urokinase- type plasminogen activator (uPA)
  • uPA urokinase- type plasminogen activator
  • uPA is the principal cell-associated plasminogen activator and has been implicated in several biological processes including angiogenesis, organogenesis, ovulation, inflammation, cancer, tumor cell invasion and metastasis, atherosclerosis, and other biological and pathological processes characterized by cell migration through physiological barriers such as fibrin and basement membranes (Gyetko et al., 1994, J. Clin. Invest. 93:1380-1387; Gyetko et al., 1996, J. Clin. Invest. 97:1818-1826; Shapiro et al., 1997, Am. J. Pathol. 150:359-369; Dado et al., 1994, Fibrinolysis 8(Su ⁇ pl. 1): 189-203).
  • uPA is synthesized as a single chain zymogen, designated single chain uPA (scuPA), which exhibits little urokinase activity (Ellis et al., 1987, J. Biol. Chem. 262:14998-15003; Petersen et al., 1988, J. Biol. Chem. 263:11189-11195; Husain, 1991, Biochemistry 30:5707-5805; Colleen et al., 1986, J. Biol. Chem. 261:1259- 1266).
  • scuPA single chain uPA
  • tcuPA Physiological formation of tcuPA from scuPA is catalyzed primarily by plasmin (Robbins et al., 1967, J. Biol. Chem. 242:2333-2342).
  • scuPA may also be activated by binding of scuPA to the cell-surface receptor, uPAR.
  • uPAR cell-surface receptor
  • PAI-1 plasminogen activator inhibitor- 1
  • SERPIN serine protease inhibitor
  • PAI-1 is thought to be the most relevant inhibitor of uPA activity in the fluid phase, due to its high second order rate constant of inhibition, 1.7 x 10 " ° M -1 -s , which is higher than any other protease inhibitor (Hekman et al., 1988, Arch. Biochem. Biophys. 262:199- 210).
  • a soluble recombinant form of uPAR designated suPAR, is known and differs from uPAR by lacking the portion of uPAR that links the receptor to the cell surface.
  • suPAR possesses the same properties as uPAR with respect to binding and activating scuPA and promoting the adhesivity of the uPA-uPAR complex (Higazi et al, 1995, J. Biol. Chem. 270:17375-17380; Higazi et al, 1996, Blood 87:3545-3549). Binding of scuPA to uPAR enhances urokinase activity of s
  • Formation of the scuPA-uPAR complex also dampens the capacity of PAI-1 to inhibit scuPA activity, relative to the capacity of PAI-1 to inhibit tcuPA activity (Higazi et al., 1996, Blood 87:3545-3549). Formation of a complex between scuPA and uPAR also alters the regulation of scuPA enzymatic activity by peptide substrates of plasmin and promotes binding of scuP A to vitronectin (Higazi et al., 1996, Thromb. Res.
  • a region of uPA comprising the protein sequence RHRGGS at amino acid positions 179-184, is required for inhibition of uPA activity by PAI-1 (Madison et al., 1990, J.
  • PAI-1 In addition to inhibiting urokinase activity of uPA, PAI-1 also promotes the internalization .and lysosomal degradation of uPA, which involves the CC2- macroglobulin receptor/low density lipoprotein-related receptor protein (o ⁇ MR/LRP; Nykjaer et al., 1994, J. Biol. Chem. 269:25668-25676).
  • the complex formed between PAI-1 and tcuPA binds to with considerably higher affinity than does either component alone.
  • scuPA bound to uPAR is active, protected from inactivation by PAI-1 , and protected from clearance from the cell surface mediated by binding of scuPA to c ⁇ MR/LRP and subsequent degradation. Furthermore, scuPA that dissociates from uPAR reverts to an inactive conformation and becomes essentially insusceptible to inactivation by PAI-1. Thus, unbound scuPA retains the capacity to rebind to uPAR and revert once again to its active conformation.
  • uPA and uPAR are abundant epidemiological data which indicate that the expression or uPA and uPAR in human tissue correlates with the conversion of cells from a benign to a neoplastic state. Furthermore, expression of uPA and uPAR is associated with a wide variety of common malignancies, and is predictive of future development of those malignancies. Interference with uPA activity by binding an antibody to uPA, by expression of an antisense oligonucleotide complementary to mRNA encoding uPA, or by overexpression of catalytically inactive forms of uPA impede tumor progression in several experimental murine models of human cancers (Ossowski, 1988, J. Cell Biol. 107:2437-2445; Ossowski et al., 1991, Cane. Res. 51:275-281; Kook et al., 1994, EMBO J. 13:3983-3991; Crowley et al., 1993, Proc.
  • compositions useful for modulating the activity of uPA in a mammal particularly in a human, and for methods of using those compositions to treat pathological conditions attributable to undesirable uPA activity in the mammal.
  • compositions and methods for promoting internalization and degradation of scuPA which act independently of activation of scuPA by plasmin, independently of binding of scuPA to uPAR, and independently of inactivation of soluble or uPAR-bound scuPA by PAI-1.
  • the invention includes a composition comprising a peptide having the amino acid sequence X 1X2X3X4X5X5X7X3, wherein: X j is hydrogen, .an amino-terminal blocking group, or one to twenty amino acid residues;
  • X2 is an amino acid selected from the group consisting of D, E, H, K, and R;
  • X3 is an amino acid selected from the group consisting of E and D;
  • X4 is an amino acid selected from the group consisting of I, L, and V;
  • X5 is an amino acid selected from the group consisting of I, L, .and V;
  • Xg is .an amino acid selected from the group consisting of M;
  • X7 is an amino acid selected from the group consisting of D, E, H, K, and R;
  • Xg is hydrogen, a carboxyl-terminal blocking group, or one to twenty amino acid residues.
  • X j is hydrogen or .an .amino-terminal blocking group;
  • X2 is an amino acid selected from the group consisting of D, E, and R;
  • X is an amino acid selected from the group consisting of D and E;
  • X 4 is I;
  • X j is hydrogen
  • composition of the invention further comprising a pharmaceutically acceptable carrier.
  • Also included in the invention is a method of affecting a biological process characterized by abnormal cell migration through a physiological barrier.
  • the method comprises administering the composition of the invention to a mammal experiencing the biological process in an amount to affect the biological process.
  • the biological process is selected from the group consisting of angiogenesis, organogenesis, ovulation, inflammation, cancer, tumor cell invasion and metastasis, and atherosclerosis.
  • the mammal is a human.
  • the invention further includes a method of inhibiting PAI-1 -dependent adhesion of a cell to a tissue of a mammal, the method comprising administering to the tissue the composition of the invention in an amount to inhibit adhesion of the cell to the tissue.
  • the tissue is in vivo in the m.ammal.
  • the mammal is a human.
  • a method of promoting clearance of scuPA from the surface of a mammalian cell comprising administering the composition of claim 1 to the cell in an amount to promote clearance of the scuPA from the cell.
  • the cell is a human cell.
  • the composition is administered in vivo in the human.
  • the invention includes a method of impeding pathological migration of a cell in a mammal.
  • the method comprises administering to the mammal the composition of the invention in an amount effective to impede pathological migration of the cell.
  • the composition is administered to the mammal at the site of a tumor in the mammal.
  • the mammal is a human.
  • the invention yet further includes a method of inhibiting PAI-1 activity in a tissue of a mammal.
  • the method comprises administering to the tissue the composition of the invention in an amount effective to inhibit PAI-1 activity in the tissue.
  • the mammal is a human.
  • the composition is administered in vivo in the human.
  • the invention also includes a kit comprising a peptide having the amino acid sequence X j X2X3X4X5X X7X , wherein:
  • X j is hydrogen, .an amino-terminal blocking group, or one to twenty .amino acid residues
  • X2 is an amino acid selected from the group consisting of D, E, H, K, and R;
  • X3 is an amino acid selected from the group consisting of E and D;
  • X4 is an amino acid selected from the group consisting of I, L, and V
  • X5 is an amino acid selected from the group consisting of I, L, and V
  • Xg is an amino acid selected from the group consisting of M
  • X7 is an amino acid selected from the group consisting of D, E, H, K, and R;
  • Xg is hydrogen, a carboxyl-terminal blocking group, or one to twenty amino acid residues, and an instructional material for using the kit. Also included is a composition comprising a combination of a peptide having the amino acid sequence X j X2X3X4X5XgX7X , wherein:
  • X j is hydrogen, an amino-terminal blocking group, or one to twenty amino acid residues
  • X2 is an amino acid selected from the group consisting of D, E, H, K, and R;
  • X3 is an amino acid selected from the group consisting of E and D;
  • X4 is an amino acid selected from the group consisting of I, L, and V
  • X5 is an amino acid selected from the group consisting of I, L, .and V
  • Xg is an amino acid selected from the group consisting of M
  • X7 is an amino acid selected from the group consisting of D, E, H, K, and R;
  • Xg is hydrogen, a carboxyl-terminal blocking group, or one to twenty .amino acid residues, and a thrombolytic agent.
  • the thrombolytic agent is selected from the group consisting of tissue plasminogen activator, streptokinase, urokinase, the streptokinase derivative and staphylokinase.
  • composition comprising a combination of a peptide having the amino acid sequence X 1X2X3 X4X5XgX Xg, wherein: X j is hydrogen, .an .amino-terminal blocking group, or one to twenty .amino acid residues;
  • X2 is an amino acid selected from the group consisting of D, E, H, K, and R;
  • X3 is an amino acid selected from the group consisting of E and D;
  • X4 is an amino acid selected from the group consisting of I, L, and V;
  • X5 is an amino acid selected from the group consisting of I, L, and V;
  • Xg is an amino acid selected from the group consisting of M;
  • X7 is an amino acid selected from the group consisting of D, E, H, K, and R; and
  • Xg is hydrogen, a carboxyl-terminal blocking group, or one to twenty amino acid residues, and an anti-coagulating agent.
  • the anti-coagulating agent is selected from the group consisting of .an agent which inhibits platelet function, .and agent which inhibits the activity of thrombin, and agent which promotes the activity of activated protein kinase
  • tissue factor pathway inhibitor an anti-thrombin III agent, and a tissue factor pathway inhibitor.
  • Figure 1 is a pair of graphs which depict the effects which individual peptides had on binding of scuPA to LM-TK " cells ( ⁇ 2MR + /LRP, uPAR " ), wherein each individual peptide had a sequence selected from the group consisting of EEIIMD, REIIMD, and EEIIMR.
  • Figure 1 A the effect of the presence of the indicated peptide is shown, wherein "No Addition" means that none of the three peptides was added to the reaction mixture.
  • Figure IB the effect of the presence of the indicated concentration of peptide EEIIMD is shown.
  • Figure 2 is a bar graph which depicts the effect of peptide EEIIMD on binding of the scuPA in the presence or absence of PAI-1 to LM-TK " cells.
  • Figure 3 is a bar graph which depicts the effect of PAI-1 and the effect of peptide EEIIMD on the binding of the scuPA-suPAR and the tcuPA-suPAR complexes to LM-TK " cells.
  • Figure 4 is a graph which depicts the effect of the presence of peptide
  • Figure 5 is a pair of bar graphs which depict the effects of the presence of peptide EEIIMD on binding of scuPA to o ⁇ MR/LRP and to LM-TK " cells.
  • the results depicted in Figure 5 A were obtained by assessing binding of labeled scuPA to purified o ⁇ MR/LRP in the absence or presence of rRAP and in the absence or presence of the peptide. Open boxes represent data collected in the absence of rRAP, and striped boxes represent data collected in the presence of rRAP.
  • the data depicted in Figure 5B were obtained by assessing binding of labeled scuPA to LM-TK " cells in the absence or presence of the peptide.
  • Data representing binding in the presence of 4 milhmolar Ca ⁇ are represented by open boxes.
  • Data representing binding in the presence of 400 nanomolar rRAP are indicated by grey boxes.
  • Figure 6 is a bar graph which depicts the effect of the presence of peptide EEIIMD on the internalization and degradation of scuPA by LM-TK " cells.
  • Control refers to data collected in experiments wherein cells were incubated in the absence of rRAP and anti-o ⁇ MR/LRP.
  • rRAP refers to data collected in experiments wherein cells were incubated in the presence of rRAP.
  • Ab refers to data collected in experiments wherein cells were incubated in the presence of an anti-o ⁇ MR/LRP antibody. Data collected in experiments performed in the absence of the peptide are indicated by open boxes. Data collected in experiments performed in the presence of the peptide are indicated by striped boxes.
  • Figure 7 is a graph depicting a comparison of the distribution of labeled fibrin micro-emboli and radiolabeled fibrinogen.
  • Figure 8 is a graph depicting clearance of micro-emboli from the lungs of wild type animals and various animals having deletion mutations in tissue-type plasminogen activator (tPA “ ' “ ), urokinase-type plasminogen activator (uPA “ ' “ ), .and the urokinase receptor (uPAR “/_ ).
  • Figure 9 is a graph depicting data obtained when the phenotype of the urokinase-type plasminogen activator (uPA " ' " ) mice was rescued by infusion of two chain uPA. Clot lysis in the uPA _/" mice was complete.
  • the invention is based on the discovery of a peptide which promotes binding of scuPA to LM-TK " cells and thereby promotes internalization and degradation of scuPA by those cells.
  • the peptide of the invention also promotes binding of scuPA to purified CC2MR/LRP.
  • the peptide of the invention is useful for affecting the physiological availability of scuPA, and is thus useful for affecting biological processes characterized by abnormal cell migration through a physiological barrier, such biological processes including, but not being limited to, angiogenesis, organogenesis, ovulation, inflammation, cancer, tumor cell invasion and metastasis, and atherosclerosis.
  • Peptide EEIIMD promoted binding of scuPA to purified o ⁇ MR/LRP. Peptide EEIIMD also accelerated the rate of internalization and degradation of scuPA by LM-TK " cells. Binding of scuPA to LM-TK " cells and internalization of scuPA by those cells in the presence of peptide EEIIMD were inhibited by the presence of either rRAP or an anti- ⁇ 2MR/LRP antibody. Peptide EEIIMD had no effect on binding of tcuPA to either LM-TK " cells or purified o ⁇ MR/LRP, and did not affect scuPA-uPAR complex formation.
  • peptide EEIIMD regulates binding of scuPA to o ⁇ MR/LRP, .and EEIIMD-promoted binding of scuPA to c ⁇ MR/LRP represents a method of promoting internalization and degradation of scuPA by cells comprising c ⁇ MR/LRP independently of both activation of scuPA by plasmin and binding of scuPA to uPAR.
  • EEIIMD was observed to be a competitive inhibitor of PAI-1 binding to tcuPA. This peptide also bound to scuPA under experimental conditions under which PAI-1 did not bind to scuPA. It was furthermore observed that peptide EEIIMD promoted internalization and degradation of scuPA by means of binding of scuPA to cellular ⁇ 2 MR/LRP.
  • the peptide of the invention as described herein including, but not limited to, peptide EEIIMD and peptides and peptidomimetics derived therefrom, may be used to promote clearance of scuPA from cell surfaces, thereby impeding pathologic cell migration.
  • the peptide of the invention may be used synergistically with inhibitors of uPA-uPAR complex formation.
  • Inhibitors of uPA-uPAR complex formation are known in the art and include those that are peptide- or antibody-based, those which are directed to the binding sequences in either uPA or uPAR, those based on sequences from other proteins believed to interact with the uPA-uPAR complex, organic inhibitors, antisense-based inhibitors, .and the like.
  • Suramin may be used to inhibit complex formation.
  • inhibitors of uPA-uPAR complex formation are used alone in a subject experiencing a biological process characterized by abnormal cell migration through a physiological barrier, a biologically relevant amount of scuPA remains bound to uPAR or in the cellular environment near uPAR.
  • a biologically relevant amount of scuPA may furthermore be capable of binding to uPAR owing to persistent local synthesis of scuPA.
  • a biologically relevant amount of scuPA means an amount of scuPA which is capable of influencing the migration of a cell through a physiological barrier.
  • a peptide of the invention when used alone or in combination with an inhibitor of uPA-uPAR complex formation, may be used to efficiently promote clearance of scuPA from cell surfaces.
  • a peptide of the invention and an inhibitor of uPA-uPAR complex formation are used in combination, a synergistic effect results, the peptide of the invention causing internalization of scuPA by means of scuPA binding to ( ⁇ MR/LRP and subsequent internalization and degradation of scuPA, and the uPA-uPAR binding inhibitor preventing sequestration of a biologically relevant amount of scuPA bound to uPAR.
  • the peptide of the invention including, but not limited to, peptide
  • EEIIMD is also useful as an inhibitor of PAI-1 activity.
  • PAI-1 activity There is abundant human epidemiologic and experimental data which link PAI-1 activity with thrombosis.
  • arterial clots are relatively resistant to thrombolytic agents, in part because thrombin binding to fibrin promotes the release of PAI-1 by activated platelets which become trapped in the fibrin meshwork.
  • PAI-1 regulates cell adhesion mediated by the uPA-uPAR complex.
  • the peptide of the invention including, but not limited to peptide EEIIMD and peptides and peptidomimetics derived therefrom, is a competitive inhibitor of PAI-1 -binding to scuPA and to tcuPA.
  • the presence of the peptide of the invention in the extracellular milieu inhibits cleavage of cell-surface scuPA by PAI-1 by removing scuPA so that it cannot be converted to tcuPA.
  • the peptide of the invention promotes endogenous thrombolysis, reducing the risk of thrombosis in a mammal.
  • Promotion of endogenous thrombolysis is a useful therapeutic and prophylactic treatment of individual subjects afflicted with a wide variety of diseases and disorders involving thrombogenesis.
  • diseases and disorders include, but are not limited to, heart attack, stroke, deep venous thrombosis, pulmonary embolus, and the presence in an individual of an abnormally large amount of PAI-1.
  • the subject may be any mammal, and is preferably a human subject.
  • the peptide of the invention may be used alone or in combination with known thrombolytic agents, thereby permitting the use of far lower and safer concentrations of known thrombolytic agents.
  • known thrombolytic agents include, by way of example, tissue plasminogen activator, streptokinase, urokinase, the streptokinase derivative APSAC, and staphylokinase.
  • the peptide of the invention may also be used synergistically with known anticoagulants.
  • the anticoagulants with which the peptide of the invention may be used include, but are not limited to, those which inhibit platelet function, those which inhibit the activity of thrombin, those which promote the activity of, for example, activated protein kinase C, anti-thrombin III, a tissue factor pathway inhibitor, .and the like.
  • Combined use of the peptide of the invention with a known anticoagulant is effective to prevent thrombus formation and to promote thrombolysis of existing clots.
  • the peptide of the invention may be used to inhibit PAI-1 -dependent cell adhesion, a critical control point during cell migration through tissues.
  • the peptide of the invention may be used to inhibit angiogenesis, metastasis, the ingrowth of smooth muscle cells into atherosclerotic plaques, infiltration of leukocytes into inflamed or damaged tissue, ovulation, binding of spermatozoa to ova, placental development, and other types of cell migration, particularly undesirable cell migration.
  • amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:
  • the present invention also provides for analogs of the peptides described herein. Analogs can differ from the peptides described herein by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both.
  • conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • Modifications which do not alter the primary sequence of the peptide of the invention may be used in the compositions and methods described herein. Modifications which do not normally alter primary sequence include in vivo, or in vitro chemical derivativization of polypeptides, e.g., acetylation, or carboxylation.
  • glycosylation e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes.
  • sequences which have phosphorylated .amino acid residues e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
  • polypeptides which have been modified using ordinary chemical or molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a pharmaceutical agent.
  • Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids.
  • the peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.
  • the peptide of the invention has the general formula
  • X j is hydrogen, .an amino-terminal blocking group, or one to twenty amino acid residues
  • X2 is an amino acid selected from the group consisting of D, E, H, K, and R;
  • X is an amino acid selected from the group consisting of E and D;
  • X4 is an amino acid selected from the group consisting of I, L, .and V;
  • X5 is an amino acid selected from the group consisting of I, L, .and V;
  • Xg is .an amino acid selected from the group consisting of M;
  • X7 is an amino acid selected from the group consisting of D, E, H, K, and R;
  • Xg is hydrogen, a carboxyl-terminal blocking group, or one to twenty .amino acid residues.
  • X j is hydrogen or .an amino-terminal blocking group
  • X2 is .an .amino acid selected from the group consisting of D, E, and R
  • X3 is an amino acid selected from the group consisting of D and E
  • X4 is I
  • X5 is I
  • Xg is M
  • X7 is .an amino acid selected from the group consisting of D and E
  • Xg is hydrogen or a carboxyl-terminal blocking group.
  • X j is hydrogen
  • X2 is E
  • X3 is E
  • X4 is I
  • X5 is I
  • Xg is M
  • X7 is D
  • Xg is hydrogen.
  • the peptide of the invention is EEIIMD.
  • the peptide of the invention is also referred to herein as the "PAI-1" peptide.
  • the invention encompasses the preparation and use of pharmaceutical compositions comprising the peptide of the invention as an active ingredient.
  • a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. Administration of one of these pharmaceutical compositions to a subject is useful for treating a variety of diseases or disorders as described elsewhere herein.
  • the active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
  • the term "pharmaceutically acceptable carrier” means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.
  • physiologically acceptable ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.
  • compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • compositions suitable for administration to humans are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, .and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs, birds including commercially relevant birds such as chickens, ducks, geese, and turkeys, fish including farm-raised fish and aquarium fish, and crustaceans such as farm-raised shellfish.
  • compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration.
  • Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% .and
  • a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.
  • additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers.
  • Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
  • a formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient.
  • Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.
  • an "oily" liquid is one which comprises a carbon- containing liquid molecule and which exhibits a less polar character than water.
  • a tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent.
  • Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture.
  • compositions used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents.
  • Known dispersing agents include, but are not limited to, potato starch and sodium starch glycollate.
  • Known surface active agents include, but are not limited to, sodium lauryl sulphate.
  • Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate.
  • Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid.
  • binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.
  • Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.
  • Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient.
  • a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets.
  • tablets may be coated using methods described in U.S. Patents numbers 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets.
  • Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.
  • Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
  • an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
  • Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin.
  • Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.
  • Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.
  • Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle.
  • Aqueous vehicles include, for example, water and isotonic saline.
  • Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents.
  • Oily suspensions may further comprise a thickening agent.
  • suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose.
  • Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively).
  • Known emulsifying agents include, but are not limited to, lecithin and acacia.
  • Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid.
  • Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.
  • Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.
  • Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent.
  • Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent.
  • Aqueous solvents include, for example, water and isotonic saline.
  • Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
  • a pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in- water emulsion or a water-in-oil emulsion.
  • the oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these.
  • Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.
  • These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration.
  • a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.
  • Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e. about 20 °C) and which is liquid at the rectal temperature of the subject (i.e. about 37 °C in a healthy human).
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, .and various glycerides.
  • Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
  • Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier.
  • enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for vaginal administration. Such a composition may be in the form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or a solution for vaginal irrigation.
  • Methods for impregnating or coating a material with a chemical composition include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e. such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.
  • Douche preparations or solutions for vaginal irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier.
  • douche preparations may be administered using, and may be packaged within, a delivery device adapted to the vaginal anatomy of the subject.
  • Douche preparations may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subj ect and administration of the pharmaceutical composition through the breach in the tissue.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, .and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g. sterile pyrogen-free water
  • compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable . formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions.
  • Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent.
  • Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers.
  • Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low- boiling propellant in a sealed container.
  • such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers.
  • Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, .and the active ingredient may constitute 0.1 to 20% (w/w) of the composition.
  • the propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).
  • compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension.
  • Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate.
  • the droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.
  • formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention.
  • Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
  • Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100%) (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration.
  • Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient.
  • Such powdered, aerosolized, or aerosolized formulations, when dispersed preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration.
  • Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier.
  • Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein.
  • Other ophthalmalmically- administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA, which is incorporated herein by reference.
  • dosages of the peptide of the invention which may be administered to an animal, preferably a human, range in amount from 1 ⁇ g to about 100 g per killogram of body weight of the animal. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. Preferably, the dosage of the compound will vary from about 1 mg to about 10 g per killogram of body weight of the animal. More preferably, the dosage will vary from about 10 mg to about 1 g per killogram of body weight of the animal.
  • the compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even lees frequently, such as once every several months or even once a year or less.
  • the frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type .and severity of the disease being treated, the type and age of the animal, etc.
  • the invention also includes a kit comprising the peptide of the invention and an instructional material which describes adventitially administering the peptide to a cell or a tissue of a mammal.
  • this kit comprises a (preferably sterile) solvent suitable for dissolving or suspending the peptide of the invention prior to administering the compound to the mammal.
  • an "instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the peptide of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein.
  • the instructional material may describe one or more methods of alleviation the diseases or disorders in a cell or a tissue of a mammal.
  • the instructional material of the kit of the invention may, for example, be affixed to a container which contains peptide of the invention or be shipped together with a container which contains the peptide. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
  • Recombinant scuPA and recombinant suPAR were purified as reported and were obtained from Abbott Laboratories (Abbott Park, IL; Higazi et al., 1996, Blood 87:3545-3549; Higazi et al., 1997, J. Biol. Chem. 272:5348-5353).
  • Peptides EEIIMD, REIIMD, and EEIIMR were synthesized using methods well known in the art of peptide synthesis.
  • Glu-plasminogen, tcuPA, PAI-1, .and the plasmin chromogenic substrate Spectrazyme PLTM were obtained from American Diagnostica, Inc. (Greenwich, CT).
  • LM-TK cell line
  • scuPA, suPAR, and tcuPA were radiolabeled with 125 I as described (Barnathan et al., 1990, J. Biol. Chem. 265:2865-2872) using iodobeads obtained from the Pierce Chemical Company (Rockford, IL).
  • suPAR and scuPA were incubated together at a molar ratio of 1.25 to 1, respectively, for one hour at 37 °C at ten times the desired final concentration in binding buffer (comprising PBS supplemented with 1.5% (w/v) BSA).
  • PAI-1 was added to a solution comprising binding buffer and scuPA, tcuPA, the scuPA/suP AR complex, or the tcuPA/suPAR complex at a 1 : 1 molar ratio, and the solution was incubated for thirty minutes at 37 °C. Protein complexes were diluted to a desired working concentration immediately before use.
  • a solution comprising 5 nanomolar tcuPA, 0 or 200 micromolar peptide EEIIMD, and 0 or 200 micromolar peptide REIIMD was incubated for thirty minutes. The solution was then added to a reaction mixture comprising 25 nanomolar PAI-1 , 50 nanomolar Glu-plasminogen, and 50 micromolar chromogenic substrate, and the optical density of the mixture at 405 nanometers was measured continuously, as described (Higazi et al., 1996, Blood 87:3545-3549).
  • Ligand Binding of radiolabeled ligands, including scuPA and tcuPA, to cells was measured as described (Higazi et al., 1996, Blood 88:542-551). Briefly, LM-TK " cells were suspended in DMEM (GIBCO, Grand Island, NY) containing 10% (v/v) fetal calf serum and were grown to confluence at 37 °C overnight in 96-well FalconTM multiwell tissue culture dishes (Becton Dickinson, Lincoln P.ark, NJ). The cells were chilled for one hour on ice .and were washed twice with chilled binding buffer.
  • 125 _ labeled ligands with or without 50-fold molar excess of non-labeled ligands, were added to the cells in the presence or absence of 0-300 micromolar peptide, and the cell cultures were incubated for two hours at 4°C. The cells were washed four times with binding buffer, solubilized in 0.1 N NaOH, and the cell extract was assessed for radioactivity. In other experiments, binding of labeled ligands was performed in the absence or presence of 400 nanomolar rRAP diluted in TBS containing 4 millimolar Ca , or in the presence of 100 micrograms per milliliter affinity purified IgG anti- o ⁇ MR/LRP .antibody. Assays were repeated at least three times, and data presented herein represent the mean and standard deviation.
  • a 96-well microtiter plate was incubated with 3 micrograms per milliliter purified t ⁇ MR/LRP or with 3 micrograms per milliliter BSA in TBS containing 4 millimolar Ca 2+ overnight at 4°C.
  • the buffer was removed, and the non-reacted sites on the plate were blocked using a blocking solution comprising TBS, 4 millimolar Ca , 0.05% (v/v) Tween-20, and 3% (w/v) BSA.
  • the wells of the plate were filled with the blocking solution and incubated for one hour at 4°C.
  • LM-TK " cells were grown overnight at 37 °C to confluence in 48-well FalconTM Multiwell tissue culture dishes (Becton Dickinson, Lincoln Park, NJ). Cells were prechilled on ice for one hour, washed twice with a buffer comprising TBS, 4 millimolar Ca 9+ , -and 3% (w/v) BSA, and incubated for one hour at room temperature with the same buffer or with buffer supplemented with either 400 nanomolar rRAP or 100 micrograms per milliliter IgG anti-c ⁇ MR/LRP.
  • the buffer was removed, 125 I- scuPA was added to the cells, and the cells were incubated for two hours at 4 °C in the presence of 0-300 micromolar peptide, 0 or 400 nanomolar rRAP, and 0 or 100 micrograms per milliliter anti-o ⁇ MR/LRP. Unbound ligand was removed and cells were washed five times with the buffer.
  • DMEM comprising 4 millimolar Ca 2+ , 0 or 400 nanomolar rRAP, and 0 or 100 micrograms per milliliter anti-o ⁇ MR/LRP was added to the cells, and the cells were incubated for eighteen hours at 37 °C.
  • the medium was removed from each cell culture after the eighteen hour incubation period, trichloroacetic acid was added to a final concentration of 10% (v/v), precipitated protein was separated by centrifugation, and radioactivity in the supernatant was assessed.
  • EEIIMD concentration equal to approximately 50 micromolar, the near-maximal effect being observed using approximately 300 micromolar peptide, as depicted in Figure IB.
  • peptides having the general formula XEIIMD may be used as effective promoters of scuPA binding to LM-TK " cells, wherein X is an amino acid residue selected from the group consisting of D, E, H, K, and R.
  • X is an amino acid residue selected from the group consisting of D, E, H, K, and R.
  • X is an amino acid residue selected from the group consisting of D, E, H, K, and R.
  • X is an amino acid residue selected from the group consisting of D, E, H, K, and R.
  • X is an amino acid residue selected from the group consisting of D, E, H, K, and R.
  • X is an amino acid residue selected from the group consisting of D, E, H, K, and R.
  • X is D or E. Most preferably, X is E.
  • the charge of the amino acid residue at the carboxyl terminus of peptide EEIIMD is important to the magnitude, but not to the existence of the capacity of the peptide to promote scuPA binding to LM-TK " cells. It has been demonstrated herein that if the carboxyl terminal amino acid residue has a positive charge, the peptide will have a reduced, but non-negligible, capacity to promote scuPA binding to LM-TK " cells, relative to peptide EEIIMD.
  • peptides having the general formula EEIIMZ are included in the invention and may be used as effective promoters of scuP A binding to LM-TK " cells, wherein Z is .an .amino acid residue selected from the group consisting of D, E, H, K, and R.
  • Z is D, E, or R.
  • Z is D or E.
  • Z is E.
  • PAI-1 and peptide EEIIMD The effect of PAI-1 and peptide EEIIMD on the binding of the scuPA- suPAR complex and of tcuPA to LM-TK " cells was examined. As depicted in Figure 3, the presences of PAI-1 and peptide EEIIMD each caused a minimal increase in binding of the scuPA-suPAR complex to LM-TK " cells. PAI-1 clearly stimulated binding of tcuPA, as reported by others (Nykjaer et al., 1994, J. Biol. Chem. 269:25668-25676; Nykjaer et al., 1992, J. Biol. Chem. 267:14543-14546).
  • peptide EEIIMD had no effect on binding of the tcuPA-suPAR complex to LM-TK " cells.
  • the presence of peptide EEIIMD also failed to stimulate binding of tcuPA to LM-TK " cells.
  • the failure of peptide EEIIMD to stimulate binding of tcuPA or the tcuPA-suPAR complex to LM-TK " or to more than minimally stimulate binding of the scuPA-suPAR complex to LM-TK " cells likely results from either the inability of the peptide to bind to the site of the scuPA protein to which PAI-
  • peptide EEIIMD binds or the conformational state of scuPA.
  • the capacity of the peptide to competitively inhibit PAI-1 activity was assessed using tcuPA as the substrate of PAI-1.
  • the data depicted in Figure 4 indicate that peptide EEIIMD inhibits PAI-1 activity.
  • the control peptide EEIIMR did not inhibit PAI-1 activity.
  • peptide EEIIMD and PAI-1 appear to interact with at least one common portion of scuPA.
  • Peptide EEIIMD promoted binding of scuPA to purified 0C2MR/LRP, and the promoting effect of peptide EEIIMD was inhibited by the presence rRAP, as depicted in Figure 5 A.
  • promotion by peptide EEIIMD of binding of scuPA to LM-TK " cells was inhibited approximately 70% by the presence of 400 nanomolar rRAP and approximately 85% by the presence of affinity purified .anti-c ⁇ MR/LRP IgG, as depicted in Figure 5B.
  • EEIIMD promoted both internalization and degradation of scuPA by LM-TK " cells, as depicted in Figure 6.
  • peptide EEIIMD fails to stimulate cellular binding of tcuPA, even though this peptide binds to tcuPA, because the binding of peptide EEIIMD to tcuPA does not induce tcuPA to assume a secondary structure including the site recognized by o ⁇ MR/LRP.
  • a similar mechanism may also account for the failure of peptide EEIIMD to induce cellular binding of the scuPA-suPAR complex to c ⁇ MR/LRP.
  • the binding site for o ⁇ MR/LRP on scuPA may be induced and shielded by uPAR, consistent with the capacity of uPAR to block interaction of scuPA and the tcuPA-PAI-1 complex with ⁇ 2 MR/LRP (Nykjaer et al, 1994, J. Biol. Chem. 269:25668-25676; Higazi et al., 1996, Blood 88:542-551).
  • conversion of scuPA to tcuPA is associated with the loss of the ability of peptide EEIIMD to induce the structure recognized by o ⁇ MR/LRP, presumably as a result of the loss of coordinate interaction between different portions of the molecule.
  • scuPA can be rendered susceptible to binding by c ⁇ MR/LRP without the need to first convert scuPA to tcuPA. No physiological analogue of the activity exhibited by peptide EEIIMD, as described herein, has been reported.
  • Example 2 Models for Testing the Potency of PAI-1 peptide in vivo
  • Plasma Plasma (2.5 mL) was mixed with 0.1 mL trace labeled ⁇ l -human fibrinogen (40 mg; 10 mg/mL final concentration in a glass tube.
  • CaCl2 (20 mM final concentration) and human thrombin (Sigma; 0.2 U/mL, final concentration) were added at room temperature for 1 hour .and maintained overnight at 4°C. All subsequent steps were performed at 4°C as well.
  • the clots were decanted on plastic lids, cut into small pieces and resuspended in 2 mL Kreb's Ringer's buffer (KRB).
  • the clots were homogenized for 30 seconds using a PT-10/35 Polytron homogenizer (Brinkmann Instr., Westbury, NY) at mid-speed. After homogenization, the samples were centrifuged at 2000x g for 15 min. The supernatant was removed and the pellet was resuspended in KRB. A second homogenization was performed at higher speed for 30 seconds and the pellet was washed as described. After the final homogenization, the micro-emboli were suspended in 13 mL KRB-bovine serum albumin (BSA) (3 mg/mL). 12 ⁇ I-micro-emboli aliquots were stored at 4°C and were used within 24 hour.
  • BSA KRB-bovine serum albumin
  • the preparation was mixed by repetitive pipetting and allowed to sediment for 5 minutes just prior to use to eliminate any larger particles which remained.
  • the supernatant was split into 200 ⁇ l aliquots immediately prior to injection. Random aliquots were selected to characterize the size distribution of the micro-emboli using a ZM Coulter counter (Coulter Electronics, LTD. Hialeah, FL). Mice.
  • mice having genetic deletions in tissue-type plasminogen activator (tPA “ ' “ ), urokinase-type plasminogen activator (uPA “ ' “ ), and the urokinase receptor (uPAR “ ' “ ), each on a 25% Swiss/75%C57/black background and their respective littermate controls were used (Carmeliet et al., 1994, Nature 369:419_424; Dwerchin et al., 1996, J. Clin. Invest. 97:870-878).
  • tPA _ " on a C57/black background wild type C57/black mice and wild type Balb/c mice were obtained from Jackson Laboratories (Bar Harbor, ME).
  • mice All of the mice weighed 20-30 g at the time of study. There was no influence of the various genetic backgrounds of the wild type mice with respect to endogenous fibrinolysis. Plasma clearance 125 I-micro-emboli (200 ⁇ l aliquots containing 15-
  • mice were resuspended by pipetting several times immediately before loading a 21 gauge syringe. Mice were injected via the tail vein and returned to their cages until sacrifice. At various times after injection (10 minutes, 1, 3 and 5 hours), the mice were anesthetized using metofane, and 100 ⁇ l of blood was withdrawn by retroorbital puncture into a heparinized capillary pipette. The mice were sacrificed by cervical dislocation. The major organs were harvested immediately, rinsed in saline, dried on Whatman paper and the radioactivity in each tissue was measured. The exact dose (cpm) injected into each mouse was calculated by subtracting the residual radioactivity remaining in the tube .and syringe after injection.
  • Radioactivity in the tail of each mouse was counted to verify that the injection was complete.
  • the lungs were obtained at 10 minutes after injection and were fixed in formalin for immunohistochemical staining.
  • the lungs were exposed to X-ray film to determine the distribution of radioactivity.
  • the individual lobes from each lung were isolated and the radioactivity therein was assessed.
  • tcuPA Two chain urokinase
  • uPA ' mice were anesthetized by intraperitoneal injection of Nembutal (50 mg/kg).
  • the 1 1 9 ,J 1 -micro-emboli were injected into the tail vein as described above 5 minutes after the infusion was started.
  • FIG 8 there is shown the clearance of the micro-emboli from the lungs of wild type animals and animals having various deletion mutations in tissue-type plasminogen activator (tPA “ ' “ ), urokinase-type plasminogen activator (uPA “ ' “ ), .and the urokin.ase receptor (uPAR “ ' “ ).
  • tPA tissue-type plasminogen activator
  • uPA urokinase-type plasminogen activator
  • uPAR urokin.ase receptor
  • PAI-1 plays an important role in regulating in tumor cell adhesion and thereby regulating tumor metastasis. There is evidence to suggest that this effect is mediated by competition between PAI-I .and vironectin for the urokinase receptor, an important mediator of certain ⁇ -integrins (Stefansson et al., 1996, Nature 383:441-443; Deng et al., 1996, J. Cell Biol. 134:1563-1571). Recent data indicates that mice with a targeted deletion in PAI-1 exhibit fewer metastases as a result of impaired tumor adhesion (Bajou et al., 1998, Nature Med. 4:923-928).
  • PAI- 1 peptide will compete with native PAI-I thereby having an anti-metastatic effect on cells. Further, as the data presented herein establish, the PAI-I peptide accelerates the internalization of uPA by a MR/LRP which represents a second locus of anti-metastatic activity.
  • the wells are washed, the cells are lysed and the released radioactivity is determined.
  • the potency of the anti-adhesive effect is based on a comparison of the IC50 versus 1 nM scuPA.
  • the effect of the PAI-1 peptide on the migration of endothelial cells through collagen coated membranes as a model of pathologic angiogenesis may be examined.
  • Receptor-associated scuPA may be responsible for urokinase activity on endothelial cells and on other cell types (Barnathan et al., 1990, J. Biol. Chem. 265:2865-2872; Manchanda et al., 1991, J. Biol. Chem.
  • a third step the anti-metastatic capacity of the PAI-1 peptide in vivo may be demonstrated.
  • stable tumor cell lines that express uPA which is PAI-1 resistant have been generated.
  • a syngeneic cell line has been developed that coexpresses human uPAR and a plasmin-insensitive scuPA (scuPA-glu 158 ).
  • This mutation prevents conversion of uPA to two chain urokinase and thereby assures that any biological activity of this molecule is due to receptor bound and activated single chain uPA.
  • Data have been obtained which establish that scuPA- glu 158 bound to uPAR is enzymatically active and insensitive to PAI-I in vitro.
  • the tumor cells are injected into syngeneic rats in the presence/absence of PAI-1 peptide or a scrambled peptide control.
  • the growth of the primary tumor and formation of pulmonary metastases is monitored over the subsequent fourteen days. It is predicted that these tumor cells will metastasize readily in the absence of PAI-I peptide but that the peptide will inhibit both their local growth and metastatic capacity through an effect on uPA-mediated cell adhesion and proteolysis.

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Abstract

L'invention se rapporte à des compositions et à des procédés permettant de favoriser l'internalisation et la dégradation de l'activateur du plasminogène de type urokinase.
EP98957325A 1997-10-17 1998-10-15 Compositions et procedes permettant de favoriser l'internalisation et la degradation de l'activateur du plasminogene de type urokinase Withdrawn EP1030679A4 (fr)

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WO2002034776A2 (fr) * 2000-10-26 2002-05-02 K.U.Leuven Research And Development Epitopes de pai-1
US20030211095A1 (en) * 2002-05-08 2003-11-13 Abd. Al-Roof Higazi Peptide for regulation of urokinase plasminogen activator and method of optimizing therapeutic efficacy
US7271143B1 (en) * 2001-07-10 2007-09-18 Thrombotech Ltd. Peptides for regulation of urokinase (uPA) and tissue type (tPA) plasminogen activator and method of optimizing therapeutic efficacy
EP2173371B1 (fr) * 2007-07-24 2011-11-02 Thrombotech Ltd. Peptides issus de l'inhibiteur 1 de l'activateur du plasminogène et leurs utilisations

Citations (3)

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EP0320840A2 (fr) * 1987-12-18 1989-06-21 BEHRINGWERKE Aktiengesellschaft Peptides, leur procédé de préparation, anticorps contre ceux-ci, et utilisation de ces anticorps pour bloquer l'activité PAI-1 du sang humain
WO1994028145A2 (fr) * 1993-06-01 1994-12-08 Chiron Corporation Expression d'inhibiteurs de l'activateur de l'urokinase-plasminogene
WO1995002413A1 (fr) * 1993-07-16 1995-01-26 Cancerforskningsfondet Af 1989 Suppression d'inhibiteurs

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NL8902454A (nl) * 1989-10-03 1991-05-01 Stichting Centraal Lab Mutanten van de humane plasminogeen activator inhibitor 1 (pai-1), hun bereiding en toepassing, en recombinante polynucleotiden die voor deze mutanten coderende genetische informatie omvatten.
US5674978A (en) * 1990-09-21 1997-10-07 The Regents Of The University Of California Peptides derived from glutamic acid decarboxylase
ZA928954B (en) * 1991-12-24 1993-05-19 Akzo Nv Peptides immunochemically reactive with antibodies directed against hepatitis non-A, non-B virus.
US5674078A (en) * 1996-01-23 1997-10-07 The Whitaker Corporation Multi-directional interface header assembly

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0320840A2 (fr) * 1987-12-18 1989-06-21 BEHRINGWERKE Aktiengesellschaft Peptides, leur procédé de préparation, anticorps contre ceux-ci, et utilisation de ces anticorps pour bloquer l'activité PAI-1 du sang humain
WO1994028145A2 (fr) * 1993-06-01 1994-12-08 Chiron Corporation Expression d'inhibiteurs de l'activateur de l'urokinase-plasminogene
WO1995002413A1 (fr) * 1993-07-16 1995-01-26 Cancerforskningsfondet Af 1989 Suppression d'inhibiteurs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AL-ROOF HIGAZI A ET AL: "SINGLE-CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR BOUND TO ITS RECEPTOR IS RELATIVELY RESISTANT TO PLASMINOGEN ACTIVATOR INHIBITORTYPE 1" BLOOD, W.B. SAUNDERS, PHILADELPHIA, VA, US, vol. 87, no. 9, 1 May 1996 (1996-05-01), pages 3545-3549, XP001037493 ISSN: 0006-4971 *
See also references of WO9920295A1 *

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