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WO1992011866A1 - SEQUENCE DE LAMININE UTILES COMME ACTIVATEUR DE tPA - Google Patents

SEQUENCE DE LAMININE UTILES COMME ACTIVATEUR DE tPA Download PDF

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Publication number
WO1992011866A1
WO1992011866A1 PCT/US1992/000353 US9200353W WO9211866A1 WO 1992011866 A1 WO1992011866 A1 WO 1992011866A1 US 9200353 W US9200353 W US 9200353W WO 9211866 A1 WO9211866 A1 WO 9211866A1
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WIPO (PCT)
Prior art keywords
sequence
peptide
laminin
tpa
plasminogen activator
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PCT/US1992/000353
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English (en)
Inventor
Salvatore V. Pizzo
Sharon Stack
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Duke University
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Duke University
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Publication date
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Publication of WO1992011866A1 publication Critical patent/WO1992011866A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/06—Use of macromolecular materials
    • A61L33/12—Polypeptides, proteins or derivatives thereof, e.g. degradation products thereof
    • A61L33/128—Other specific proteins or polypeptides not covered by A61L33/122 - A61L33/126

Definitions

  • compositions 10 for plasminogen activation The field of this invention concerns compositions 10 for plasminogen activation.
  • the naturally occurring products are provided by natural intravascular processes, different from administration as a drug of the same product.
  • the physiological process localizes admin ⁇ istration at the site requiring the particular product.
  • a high localized concentration is provided and the clergy protein is rapidly degraded or deactivated as it moves from the site where it is needed.
  • Nature by providing t for specific ligand signals, provides that a particular activity be localized.
  • the drugs may have numerous effects, desirable as well as undesirable, so that there is an interest in providing the lowest amount of the drug to achieve the desired purpose. Also, there is interest in being able to enhance or diminish the lifetime of the activity provided by the drug, depending upon the nature of the drug.
  • a pentapeptide sequence IKVAV from the A chain of laminin has been identified which stimulates neurite outgrowth, cell adhesion and cell migration (Tashiro (1989) J. Biol. Chem. 264:16167-182). This pentapeptide is contained within an 18-amino acid fragment comprising residues 2091-2108 of the laminin A chain (Sasaki et al., (1988) J. Biol. Chem. 263:16536-544).
  • compositions are provided combining plasminogen activator and a laminin fragment or analog thereof, whereby the properties of the plasminogen activator are modified.
  • the composition may be administered to a mammalian host for preventing the formation of or rapidly dissolving any clots or embolisms.
  • compositions comprising tissue plasminogen activator or analogs thereof with a peptide fragment of laminin or functional analogs thereof, where the resulting composition has enhanced plasminogen activation activity as compared to the same amount of plasminogen activator in the absence of the laminin associated peptide.
  • These compositions may be formulated in accordance with conventional techniques employed for formulation of plasminogen activator and administered analogously to a host susceptible to the formation of an embolism or having an embolism.
  • tissue plasminogen activator which is employed may be from any mammalian source, particularly primate, more particularly human, and may be the naturally occurring tissue plasminogen activator, alleles thereof, mutants thereof, including deletions, insertions and substitutions, or active fragments thereof, where such active fragments are capable of activating plasminogen.
  • tissue plasminogen activator tPA
  • EPA 0 357 296 EPA 0 178 105
  • EPA 0 373 896 EPA 117 981
  • WO 90/03 436 tissue plasminogen activator
  • the laminin associated sequence will, for the most part, include the sequence R-K-Q-A-A-S-I-K-V-A-V, more preferably include the sequence (C-)S-R-A-R-K-Q-A-A-S- I-K-V-A-V-S-A-D-R, where the ( ) indicates optional presence.
  • the indicated sequences there will be not more than 2 single mutations, including deletions, insertions or substitutions.
  • substitutions will be conservative, where amino acids having substantially the same conformation and polarity as the naturally present amino acid will be used for the substitution.
  • Illustrative conservative substitutions include groups such as G,A; V,I,L; S,G,M,C; D,E; K,R; N,Q; and F,W,Y,H. Conservative substitutions may be extended, where N,Q may be used for substitution of D,E and vice versa or S,T,M and vice versa.
  • sequences are sequences from the human laminin A chain and may be modified in a number of ways.
  • one or more different groups may be attached at an end for convenience of isolation and purification, to stabilize the peptide, or the like.
  • one or more of the amino acids may be substituted by the unnatural D-stereoisomer.
  • one or more alanines may be substituted.
  • terminal amino acids may be employed having the unnatural chirality.
  • one may provide for a terminal amide or a terminal acylated amino, particularly acetylated, or alkylated, particularly methylated, amino acids.
  • the cysteine may be alkylated or unsubstituted on the mercaptan group.
  • Substituents may be aliphatic, alicyclic, aromatic, heterocyclic or substituted derivatives thereof, generally from 1 to 12 carbon atoms, usually 1 to 6, and 0 to 5, usually 0 to 3 heteroatoms, such as 0, N, S, P or the like.
  • the peptides of this invention will be fewer than 60 amino acids, preferably fewer than 30 amino acids, generally ranging from about 10-30, more usually from about 15-25 amino acids. Usually, not more than 10% of the amino acids present in the group will be modified by mutation, particularly substitutions.
  • the subject peptides may be prepared in a variety of conventional ways, including synthesis on a support, recombinant technology, or any other convenient means. For the most part, synthesis will be used, particularly where the amino acid sequence is fewer than about 30 amino acids. By employing synthesis, one may introduce various unnatural amino acids along the chain, more readily modify the N- or C-terminus or the like. In this way, great flexibility is introduced to allow for modifications which will enhance the lifetime of the peptide, modulate its activating effect, improve stability and formulation, or the like.
  • the subject peptide will usually be combined with tPA at a ratio of at least about 5 ⁇ g, more usually at least about 10 ⁇ g and up to about 500 ⁇ g, more usually not more than about 200 ⁇ g per 0.1 pmole of tPA or its analog.
  • the formulations may be prepared in various ways.
  • the tPA or analog thereof and laminin associated peptide may be combined in an appropriate aqueous medium comprising buffer, stabilizers, saline, or the like, such as phosphate buffered saline, where the various components are physiologically acceptable, and the mixture lyophilized to provide for an intimate mixture of the tPA and laminin associated peptide.
  • each of the components may be separately formulated in physiologically acceptable solutions and mixed prior to use.
  • various physiologically acceptable excipients may be employed, where the tPA may be formulated in conventional manners. The level of administration of the tPA can be substantially diminished, depending upon the amount of laminin associated peptide included.
  • tPA increases in activity of the tPA of 10-fold or more may be achieved, so that the amount of tPA which is administered will be at least about 2-fold less than presently prescribed, preferably at least about 5-fold less, and usually not more than about 30-fold less, more usually not more than about 20-fold less.
  • the amount of tPA which is administered will also vary depending upon the manner of administration, the length of time administered, the desired rapidity of response, and the like. Thus, substantial reductions in the amounts as to each of these methods of administration can be achieved.
  • the amount of tPA administered will generally be in the range of about 20,000 to 200,000 IU/kg hr.
  • the peptide can also be administered in the absence of exogenous tPA to stimulate the activity of endogenous tPA.
  • the peptide may be coated or bound covalently to the surface of devices, such as stents, catheters and other such devices, which are inserted in human blood vessels or tissue to prevent fibrin deposition on these objects.
  • the peptide may also be administered intraperitoneally at the time of abdominal surgery to prevent adhesions which form as a consequence of fibrin formation on serosal surfaces.
  • Synthetic peptides IKVAV-NH2 and SRARK-NH2 were purchased from the University of Louisville Peptide Synthesis Facility. All other peptides were from Multiple Peptide Systems, San Diego, CA. The composition and sequence of all peptides were verified using an Applied Biosysterns 420A derivatizer/analyzer amino acid analysis system and an Applied Biosystems 477A pulse liquid phase sequencer with "on-line" 120A PTH amino acid analysis. Peptides with biological activity were further puri ⁇ fied by reverse-phase HPLC.
  • the synthetic substrates D-Val-Leu-Lys-p-nitroanilide (VLK-pNA) and D-Ile-Pro- Arg-p-nitroanilide (IPR-pNA) and human fibrinogen were purchased from Helena Laboratories, Beaumont, TX. Fibrinogen was rendered plasminogen-free by chroma- tography on L-Lysine-Sepharose (Deutsch and Mertz, (1970) Science 170:1095-1096) and CNBr fragments of fibrinogen were generated according to the method of Blomback et al., (1968) Nature, 218:130-134.
  • Urokinase was purchased from Calbiochem, San Diego, CA and coupled to CNBr activated Sepharose according to the method of Cuatrecasas et al. , (1968) Proc. Natl. Acad. Sci. USA, 6J.:636-643.
  • Two chain recombinant tissue plasminogen activator (t-PA) was supplied by Dr. Henry Berger at Wellcome Research Laboratories, Research Triangle Park, NC.
  • Recombinant human fibroblast collagenase type I was kindly supplied by Dr. Jerry McGeehan at Glaxo Research Laboratories, Research Triangle Park, NC.
  • Plasminogen (Pg) was purified from human plasma by affinity chromatography on L-Lys- Sepharose (Deutsch and Mertz, (1970) Science 170:1095- 1096) and separated into isoforms 1 and 2 by affinity chromatography on concanavalin A-Sepharose (Gonzalez- Gronow and Robbins, (1984) Biochemistry 23:190-194) . Affinity chromatography purified form 2 was utilized for all experiments. Plasmin (Pm) was generated by incubating 100 ⁇ g of Pg with 100 ⁇ l of urokinase- Sepharose in 20 mM Hepes, pH 7.4 for 1 h at 25°C followed by centrifugation to remove the resin.
  • Lys77-Pg was prepared by limited proteolysis of Glu-Pg with Pm in a molar ratio of 10:1 for 30 min at 25°C followed by chromatography on pancreatic trypsin inhibitor-Sepharose to remove Pm (Castellino and Power, (1981) Meth. Enzymology 80:365-378). Protein concentrations were determined spectrophotometrically at 280 nm using an A ⁇ %/icm value of 16.8 and molecular weights of 92,000, 83,000 and 81,000 for Glu-Pg, Lys77-Pg and Pm, respectively (Castellino, (1981) Chem. Rev. 81:431-436).
  • Type IV collagenase/gelatinase was purified from the serum free conditioned medium of porcine synovial membranes stimulated with phorbol 12-myristate 13- acetate by inhibitor-affinity chromatography (Stack and Gray, (1988) FASEB J. A1006). Briefly, pooled conditioned medium was concentrated by ultrafiltration and the metalloproteinase zy ogens activated by incubation with 0.7 /*M p-aminophenylmercuric acetate for 4 h at 35°C.
  • Type I collagenase and type IV collagenase were separated from other proteins using an affinity matrix consisting of N-[l(R,S)-carboxy-n- butyl]-Leu-Phe-Ala coupled through the C-terminus to EAH-Sepharose 4B (Pharmacia) . Chromatography on DEAE- Sephacel was used to separate type I collagenase from type IV collagenase.
  • Coupled assays were used to evaluate the initial rate of Pg activation by t-PA or u-PA by monitoring the amidolytic activity of generated Pm (Wohl et al. , (1980) J. Biol. Chem. 255:2005-2013).
  • Lys77-Pg was incubated in 96-well microtitre plates at 37°C in 20 mM Hepes, pH 7.4 in a total volume of 175 ⁇ l with the Pm substrate VLK-pNA (0.3 mM final concentration) .
  • Pg activation was initiated by the addition of 0.11 nM (4 IU/ l) t-PA or 0.8 nM (2 IU/ml) u-PA and the Pm hydrolysis of VLK-pNA was monitored by measuring the change in absorbance at a wavelength of 405 nm at timed intervals using an Anthos Labtech Instruments model 2001 plate reader.
  • Initial veloci- ties (v.) were calculated from the slope (b) of plots of A4.
  • K m is the apparent Michaelis constant for VLK-pNA hydrolysis by Pm (0.3 mK)
  • k e is the empirically determined catalytic rate constant for Pm hydrolysis (3.2 x 10 M min-1 mol Pm-1)
  • E is the molar extinction coefficient of pNA at 405 nm (8800 M-l cm-1, Erlanger et al. , (1965) Arch. Biochem. Biophys.
  • Fluorometric measurements were performed with an SLM-Aminco SPF-500C spectrofluorometer.
  • the initial rate of substrate hydrolysis was determined by monitoring the increase in fluorescence emission at a wavelength of 346 nm using an excitation wavelength of
  • LamA 2091"2108 The effect of LamA 2091"2108 on two structurally and mechanistically related collagenases was determined by incubating human fibroblast type I collagenase (30 nM) or porcine synovial type IV collagenase/gelatinase (2.7 pM) with the synthetic substrate followed by addition of increasing amounts of LamA 2091"2108 (0.3-100/tM) .
  • Enzymes were incubated with peptide (500 ig/ml) for 10 min at 37° followed by addition of the indicated substrate as described in Methods.
  • LamA had a stimulatory effect on Pg activation.
  • An unrelated laminin-derived peptide YIGSR also had no effect on Pg activation (Table II H) .
  • the k . /K of t-PA catalyzed Pg activation calculated cat m ⁇ . from the data increases from 3.2 ⁇ K ⁇ sec ⁇ in the absence of peptide to 69.2 ⁇ K -1sec-1 in the presence of the peptide, resulting in an overall 22-fold increase in activation efficiency.
  • Fibrinogen fragments potentiate t-PA dependent Pg activation, with maximum stimulation at a fragment concentration of approximately 20 g/ml (Nieuwenhuizen et al., (1983) Biochem. Biophys. Acta. 755:531-533).
  • the effect of LamA 2091—2108 on fibrinogen stimulation of Pg activation was determined. Although fibrinogen fragments result in a greater overall stimulation of Pg activation, simultaneous addition of LamA 2091—2108 and fibrinogen fragments abolishes the additional stimulatory effect achieved with fibrinogen alone, indicating that LamA " and fibrinogen may compete for similar binding sites on the Pg molecule.
  • LamA 2091—2108 Effect of LamA 2091—2108 on collagenase activity.
  • the effect of LamA 2091"2108 on the activity of purified interstitial and type IV collagenase was determined.
  • LamA 2091"2108 had an inhibitory effect on collagenase activity with an IC go of 3 ⁇ K for type I collagenase and 43 ⁇ K for type IV collagenase. Since free Cys is a weak inhibitor of collagenase activity (IC 50 of 3 mM, Darlak et al. , (1990) J. Biol. Chem. 2 ⁇ 5:5199-5205), the amino-terminal Cys of LamA 2091"2108 was alkylated with N-ethylmaleimide (Glazer et al. , 1975). The alkylated peptide retained slight inhibitory activity with an IC 5Q of approximately 100 ⁇ K for both collagenases.

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Abstract

Une séquence de laminine de C-S-R-A-R-K-Q-A-A-S-I-K-V-A-V-S-A-D-R-NH2 ou ses analogues sont utilisés en association avec le tPA pour augmenter considérablement l'activation de plasminogène par le tPA. Des formulations combinent le fragment de laminine ou son analogue avec le tPA ou son analogue fonctionnel pour être administré à des niveaux sensiblement inférieurs à ceux requis par le tPA lui-même tout en assurant une protection contre les embolies.
PCT/US1992/000353 1991-01-14 1992-01-14 SEQUENCE DE LAMININE UTILES COMME ACTIVATEUR DE tPA Ceased WO1992011866A1 (fr)

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US64061691A 1991-01-14 1991-01-14
US640,616 1991-01-14

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009033731A3 (fr) * 2007-09-11 2009-09-03 Mondobiotech Laboratories Ag Utilisation d'un peptide comme agent thérapeutique

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4839169A (en) * 1986-08-05 1989-06-13 Burroughs Wellcome Co. Synergistic combination of t-PA and prostacyclin
US4968617A (en) * 1985-05-28 1990-11-06 Burroughs Wellcome Co. Solid hydrochloride salt of t-PA

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4968617A (en) * 1985-05-28 1990-11-06 Burroughs Wellcome Co. Solid hydrochloride salt of t-PA
US4839169A (en) * 1986-08-05 1989-06-13 Burroughs Wellcome Co. Synergistic combination of t-PA and prostacyclin

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Vol. 29, issued 02 May 1990, STACK et al., "Regulation of Plasminogen Activation by Components of the Extracellular Matrix", pages 4966-4970. *
FEBS, Vol. 172, issued June 1984, SALONEN et al., "Laminin Interacts with Plasminogen and its Tissue-type Activators", pages 29-32. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 260, No. 18, issued 25 August 1985, SILVERSTEIN el al., "Activation of Immobilized Plasminogen by Tissue Activator", pages 10346-10352. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009033731A3 (fr) * 2007-09-11 2009-09-03 Mondobiotech Laboratories Ag Utilisation d'un peptide comme agent thérapeutique

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