WO1989001512A1 - Enzyme d'eponge ayant une activite analogue a la transglutaminase - Google Patents

Enzyme d'eponge ayant une activite analogue a la transglutaminase Download PDF

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Publication number
WO1989001512A1
WO1989001512A1 PCT/US1988/002857 US8802857W WO8901512A1 WO 1989001512 A1 WO1989001512 A1 WO 1989001512A1 US 8802857 W US8802857 W US 8802857W WO 8901512 A1 WO8901512 A1 WO 8901512A1
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WIPO (PCT)
Prior art keywords
transglutaminase
enzyme
sponge
marine sponge
fibronectin
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Application number
PCT/US1988/002857
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English (en)
Inventor
Laszlo Lorand
Gerald Weissmann
Original Assignee
The Liposome Company, Inc.
New York University
Northwestern University
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Publication date
Application filed by The Liposome Company, Inc., New York University, Northwestern University filed Critical The Liposome Company, Inc.
Publication of WO1989001512A1 publication Critical patent/WO1989001512A1/fr

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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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/38—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing enzymes
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII

Definitions

  • the present invention is directed to the isolation and use of an enzyme having transglutaminase-like activity, isolated from marine sponges, and the use of transglutaminases from other sources, for the treatment of bleeding conditions.
  • Such conditions may result from surgical or dental procedures, or bleeding disorders such as hemophilia or hemophilia-like disorders.
  • Marine sponge enzyme (MSE) isolated by the methods of the present invention may be used in the treatment of such disorders.
  • transglutaminase shall be taken to include transglutaminase-like substances, such as MSE.
  • the process of clot formation is initiated when either intrinsic or extrinsic pathways are activated.
  • the intrinsic pathway is initiated by the blood itself, such as when blood comes into contact with a substance such as glass, or in vivo, when antigen-antibody reactions or other trauma to the blood occur.
  • the extrinsic pathway is initiated by the contact of blood with traumatized vascular wall or extravascular tissues, e.g., in injury.
  • the enzyme prothrombin is converted to thro bin by a complex of substances known as prothrombin activator.
  • Thrombin, a proteolytic enzyme then catalyzes the reaction of fibrinogen, a soluble circulating protein, into insoluble fibrin.
  • the mechanism by which this occurs is the removal of four low molecular weight peptides from each fibrinogen molecule, forming a fibrin monomer, which then polymerizes with other fibrin monomers to form long fibrin threads.
  • Such threads form the substance (the reticulum) of the clot.
  • weak non-covalent bonds hold the threads together and they thus may be easily broken apart.
  • thrombin activates fibrin-stabilizing factor (Factor XIII) to Factor XIII (activated form), which causes formation of covalent bonds as well as multiple cross-linkings between the fibrin threads, increasing the clot strength.
  • transglutaminase activity is present in species without blood, hemolymph, or closed coelom.
  • the lack of blood products employed by this invention makes it an improved starting material for a transglutaminase-like substance, as such blood products have of late been the source of serious contaminants such as virus particles and byproducts (e.g., hepatitis and HTLV-III virus) that are found in the final products.
  • virus particles and byproducts e.g., hepatitis and HTLV-III virus
  • pro-clotting enzymes to the patient has been via intravenous injection (in the case of hemophiliacs), or topical administration in the case of surgical or dental patients.
  • the substance is administered to the bleeding site via application on gauze or sponges.
  • Such administration promotes the formation of a clot at the bleeding site.
  • Transglutaminase is such a pro-clotting enzyme but has not been previously so employed.
  • This application is continued as needed to the site until bleeding is arrested.
  • any supporting matrix that supports enzymatic activity for example, a balloon, a heteropolymer, a polymer matrix, a gauze applicator, a sponge, or a bead, to name a few, may be used.
  • the MSE may be administered topically.
  • the use of a delivery system that sustains the release of the substance at the site is a possible enhanced mechanism for its application.
  • Such a delivery system for example, is liposomes.
  • Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unila ellar vesicles (possessing a single membrane bilayer) or multilameller vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer) .
  • the bilayer is composed of two lipid monolayers having a hydrophobia "tail” region and a hydrophilic "head” region.
  • the structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient towards the center of the bilayer while the hydrophilic "heads" orient towards the aqueous phase.
  • the original liposome preparation of Bangham et al. involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell,” and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means.
  • MLVs multilamellar vesicles
  • Unilamellar vesicles may be produced using an extrusion apparatus by a method described in Cullis et al., PCT Publication No. 87/00238, January 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles” incorporated herein by reference. Vesicles made by this technique, called LUVETS, are extruded under pressure through a membrane filter. Vesicles may also be made by an extrusion technique through a 200 n filter; such vesicles are known as VET...S.
  • liposomes that may be used are those characterized as having substantially equal lamellar solute distribution.
  • This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Patent No. 4,522,803 to Lenk, et al., monophasic vesicles as described in U.S. Patent No. 4558,579 to Fountain, et al., and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle; this procedure is described in Bally et al., PCT Publication No.
  • vesicles include those that form reverse-phase evaporation vesicles (REVs), Papahadjopoulos et al., U.S. Patent No. 4,235,871, issued November 25, 1980.
  • REVs reverse-phase evaporation vesicles
  • Papahadjopoulos et al. U.S. Patent No. 4,235,871, issued November 25, 1980.
  • a bioactive agent such as a drug is entrapped in or associated with the liposome and then administered to the patient to be treated.
  • a bioactive agent such as a drug
  • U.S. Patent No. 3,993,754 Sears, U.S. Patent No. 4,145,410; Papahadjopoulos et al., U.S. Patent No. 4,235,871; Schnieder, U.S. Patent No. 4,114,179; Lenk et al., U.S. Patent No. 4,522,803; and Fountain et al., U.S. Patent No. 4,588,578.
  • the transglutaminase-like substance once purified from the sponge, may be entrapped in the aqueous space of the liposomes by any of the known procedures for forming the liposomes, and administered to the subject.
  • transglutaminases in another aspect of the invention, we disclose the topical application of transglutaminases and transglutaminase-like substances to promote clotting of the blood at epithelial sites.
  • transglutaminases are shown in the present invention to form complexes with fibronectin in blood plasma. Such complex formation was detected by a shift in transglutaminase electrophoretic (anodic) mobility, when mixtures of plasma (containing fibronectin) and transglutaminase were applied to electrophoretic gels.
  • This binary system of transglutaminase and fibronectin can enhance the activity of topically-acting clot formation of the transglutaminase.
  • (1) tertiary and (2) quarternary complexes can be formed with (1) transglutaminase, fibronectin, and liposomes, and (2) transglutaminase, fibronectin, liposomes and a gelatin, or any combination of these components.
  • the present invention relates to the isolation of a marine sponge enzyme having properties similar to transglutaminase, from the tissues of the marine sponge, specifically Microciona prolifera.
  • the transglutaminase-like substance can be used in the treatment of bleeding disorders, such as hemophilia, or in the treatment of excess bleeding as may arise in a surgical or dental procedure.
  • the transglutaminase can be administered alone or in the form of a sustained delivery vehicle, such as in liposomes.
  • a binary complex of transglutaminase and fibronectin can be prepared, or a tertiary complex of transglutaminase, fibronectin, and a liposome, or a tertiary complex of transglutaminase, fibronectin, and gelatin. Further, a quarternary complex of transglutaminase, fibronectin, a liposome, and gelatin can be administered. Such compositions can be administered topically to a subject in need of such treatment.
  • FIGURE 1 depicts two reactions that are catalyzed by transglutaminase.
  • A Protein crosslinking via gamma-glutamyl:epsilon-lysine bonds.
  • B A ine incorporation into proteins (e.g. dimethylcasein)
  • FIGURE 2 is a graph demonstrating the incorporation of putrescine into dimethylcasein as it varies with time and transglutaminase enzyme concentration.
  • FIGURE 3 is a bar graph demonstrating the effect of primary amines on the inhibition of transglutaminase.
  • a - Control B&C - dansylcadaverine, D&E - histamine, F&G - [Di-allyl] [amino-propionyl] [benzothiophene] , H&I - dimethyldansylcadaverine.
  • FIGURE 4 is a graph demonstrating that the effect of transglutaminase on clotting of (A) lobster plasma and (B)
  • FIGURE 5 is a graph demonstrating that uptake of putrescine is possible only when both transglutaminase and substrate are present together.
  • A 1:1 Mix 8-7 dialysate, 8-5 Fraction 16,
  • B 8-7 dialysate,
  • C 8-5 Fraction 16.
  • the present invention is concerned with the isolation and purification of MSE from the marine sponge, and the method of use of this substance for the medical use of clotting of blood.
  • the invention also involves the method of treating abnormal clotting disorders such as hemophilia or hemophilia-like disorders, and treatment of bleeding during trauma or surgical procedures using this substance. It also involves the administration of the substance in a sustained delivery system, for example, in liposomes.
  • a further embodiment is the use of a tra sglutaminase-fibronectin (binary) complex at a topical site to promote and enhance clot formation.
  • tertiary and quarternary complexes including transglutaminase, fibronectin and liposomes, and transglutaminase, fibronectin, liposomes, and gelatin, respectively can be formed, and delivered to a subject requiring treatment with clot-promoting agents.
  • liposomes in combination with gelatin matrices for delivery of drug and other substances contained therein is disclosed in U.S. Patent No. 4,708,861, toffy et al., issued Nov. 24, 1987, relevant portions of which are hereby incorporated by reference.
  • transgluta inase-fibronectin binary complex, and MSE-fibronectin complexes, disclosed in this invention enable enhancement of the clotting capabilities of transglutaminase by maintaining a clotting-effective amount of the transglutaminase at the bleeding site by use of fibronectin, a substance normally present at clotting sites, or by the use of novel drug delivery systems such as liposomes and gelatins, alone or in combination.
  • fibronectin a substance normally present at clotting sites
  • another binary complex such as transglutaminase and liposomes can also be used.
  • the transglutaminase can be entrapped in or associated with the liposomes.
  • a tertiary complex such as transglutaminase, fibronectin and liposomes can be used to both enhance delivery, both topically and parenterally, to the desired site, and to enhance the clotting capabilities by the presence of fibronectin.
  • a quarternary complex such as for example, transglutaminase, fibronectin, liposomes, and a gelatin, within which the other components can be contained, is also embodied by the present invention.
  • any compatible transglutaminase or transglutaminase-like substance can be employed, the MSE of the present invention being among those transglutaminases.
  • These complexes may be formed by simple admixing of the components, for example, combination of the transglutaminase and the fibronectin.
  • the transglutaminase and alternatively or additionally, the fibronectin can be entrapped in or associated with the liposomes.
  • gelatin any or all of the components, such as the transglutaminase alone or in combination with the fibronectin or the liposomes can be admixed with the gelatin and administered to the site.
  • the formation of these binary, tertiary, or quarternary complexes requires the use of compatible solvents and ingredients to achieve the complex.
  • clumped marine sponge cells resemble a blood clot, evidenced by a clump of cells surrounded by a fibrous matrix.
  • a stabilizing factor as is present in blood clots, such substance being the transglutaminases.
  • transglutaminases There are two reactions that are known to be catalyzed by transglutaminase; the first is protein crosslinking via gamma glutamyl-epsilon lysine bridges, and the second amine (primary amine) incorporation into proteins (Figure 1).
  • the marine sponge cells contain transglutaminase-like activity (according to procedures in Lorand et al., 1984, Molec. Cell.
  • Unbroken cells show no such activity, but supematants of centrifuged, sonicated cells show transglutaminase activity as demonstrated by standard qualitative test criteria.
  • the enzyme is substantially free of sponge cells.
  • the enzyme-like activity is also present in preparations containing the enzyme on the surface of the cells. In such case, the enzyme is exogenous to the sponge cells.
  • Several tests were carried out on the supematants of sonicated sponge cells; such tests are standard assays for the presence of transglutaminase in cells.
  • Another test is that of inhibition by low molecular weight inhibitors, primary amines (e.g., histamine, dansylcadaverine) but not tertiary amines (dimethyldansylcadaverine).
  • primary amines e.g., histamine, dansylcadaverine
  • tertiary amines dimethyldansylcadaverine
  • the marine sponge enzyme was first isolated by the following technique as outlined in the purification table (Table 1): The sponge cell extract containing the transglutaminase was isolate*! by a process involving several steps, one of which is the disruption of the sponge cells. About 100 ml of sponge are dissociated in 50 ml calcium/magnesium free sea water (CMFSW) (460mM NaCl, 7mM a 2 S0 4 , lOmM KC1, lO M Hepes, 2.5mM EDTA) to make 150 ml total of thick cell suspension.
  • CMFSW calcium/magnesium free sea water
  • the cells are then separated into 12 centrifuge tubes containing 12.0 ml each of cell suspension, and are then centrifuged at approximately 2000 rpm for 5 minutes in a clinical centrifuge, and the supernatant decanted. About 10.0 ml of the CMFSW is added to the pellet, and the pellet resuspended, centrifuged as before, and the supernatant again decanted. A solution of 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5 was added to each tube, and the tubes resuspended. The tubes were spun as above and the supernatant decanted.
  • the cells are then centrifuged a third time in 10 mM EDTA/10 mM Benzamidine/50mM Tris, and sonicated at moderate power for 15 one-second bursts separated by four-second periods of rest.
  • the samples were then ultracentrifuged at 150,000g for 60 minutes at 4°C and the supernatant dialyzed for 18 hours against four separate washes of 2.0 liters of 0.8 M sucrose, 1 mM EDTA, 0.5mM Benza idine, 15mM Tris-HCl, at pH 7.5.
  • the dialysate was then further purified on a Sephadex G100 column to obtain an enzyme product that had peak activity when concentrated purified greater than 25 times over the starting (cell homogenate) material.
  • the resulting material is MSE, substantially free of marine sponge cells.
  • Figure 4B were identical.
  • Figure 4a also shows that the purified enzyme reacted with dimethylcasein, but not DMDC, as before.
  • the MSE as disclosed in the present invention may be used in any situation where a clotting factor is indicated; for example, it may be used topically to arrest bleeding during surgical or dental procedures. It may also be used in vivo in the intravenous treatment of bleeding disorders such as hemoplllia or hemophilia-like disorders. Since persons with hemophilia or hemophilia-like disorders often experience arthritic symptoms due to bleeding into the joints, the MSE of the invention may be injected intrasynovially (injected directly into the joint), thereby arresting the bleeding into the joint. The MSE likewise may be injected into any other similarly closed space in the body. The MSE may be used in its purified form alone, or in combination with other pharmaceutical carriers or solutions. It may also be incorporated into a delivery system, such as liposomes, and delivered by any of the methods known for administering liposomes.
  • a delivery system such as liposomes, and delivered by any of the methods known for administering liposomes.
  • the MSE resulting from the processes of the present invention whether used in their free-, liposome-encapsulated, fibronectin-complexed, gelatin-associated or other binary, tertiary, or quarternary forms can be used therapeutically in mammals, including man, in the treatment of conditions which favor or require use of a clotting agent.
  • a clotting agent such as thrombin.
  • the transglutaminase may be used in combination with fibronectin, as a transglutaminase-fibronectin complex has been shown to form in mixtures of purified and transglutaminase-containing lysate of membrane-depleted red blood cells with fibronectin-containing plasma.
  • These complexes were detected by nondenaturing electrophoresis, by observation of a shift in the mobility of transglutaminase in three electrophoretic systems as a function of transglutaminase admixture with fibronectin-containing human blood plasma, according to the methods of Lorand et al., 1988, Proc. Natl. Acad.
  • transglutaminase the erythrocyte protein
  • the relative ratio of plasma to erythrocyte lysate was increased, the free transglutaminase species was gradually replaced by a slower migrating transglutaminase species (Lorand et al., supra.).
  • transglutaminase in the electrophoretic gel area between the two discrete electrophoretic zones (fast and slow moving transglutaminase) can be taken as a sign of tight complex formation.
  • the mode of administration of the preparation may determine the sites and cells in the organism to which the compound will be delivered.
  • the liposomes of the present invention can be administered alone but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the preparations may be injected parenterally, for example, intra-arterially or intravenously.
  • the preparations may also be administered via oral, subcutaneous, or intrasynovial, or intramuscular routes.
  • parenteral administration they can be used, for example, in the form of a sterile aqueous solution which may contain other solutes, for example, enough salts or glucose to make the solution isotonic.
  • a sterile aqueous solution which may contain other solutes, for example, enough salts or glucose to make the solution isotonic.
  • Other uses, depending * upon the particular properties of the preparation, may be envisioned by those skilled in the art.
  • the liposomes of the present invention may be incorporated into dosage forms such as gels, oils, emulsions, and the like. Such preparations may be administered by direct application as a cream, paste, ointment, gel, lotion or the like.
  • a number of sustained drug delivery systems can be employed, for example, liposomes, transdermal systems, nasal delivery, carrier molecules, monoclonal antibodies, osmotic pumps, and the like.
  • liposomes as the drug delivery system is preferred.
  • the prescribing physician will ultimately determine the appropriate dosage for a given human subject, and this can be expected to vary according to the age, weight, and response of the individual as well as the nature and severity of the patient's disease.
  • the dosage of the transglutaminase or transglutaminase-like substance of the present invention will be that necessary to cause clotting of the blood.
  • the dosage of the transglutaminase or transglutaminase-like substance in liposomal form will be about that employed for the free drug. In some cases, however, it may be necessary to administer dosages outside these limits.
  • CMFSW calcium/magnesium free sea water
  • the cells were then separated into 12 centrifuge tubes containing 12.0 ml each of cell suspension, and then centrifuged at 2000 rp for 5 minutes in a clinical centrifuge, and the supernatant was decanted.
  • About 10.0 ml of the CMFSW was added to the pellet, and the pellet resuspended, centrifuged as before, and the supernatant again decanted.
  • the samples were then ultracentrifuged at 150,000g for 60 minutes at 4 C C and the supernatant dialyzed for 18 hours against four separate washes of 2.0 liters of 0.8 M sucrose, 1 mM EDTA, 0.5mM Benzamidine, 15mM Tris-HCl, at pH 7.5.
  • the dialysate was then further purified on a Sephadex G100 column to obtain an enzyme product that had peak activity when concentrated purified greater than 25 times over the starting (cell homogenate) material.

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Abstract

La présente invention concerne l'isolation d'une enzyme d'éponge marine ayant des propriétés similaires à la transglutaminase, à partir des tissus de l'éponge marine, spécifiquement de la Microciona prolifera. On peut utiliser les transglutaminases et les substances analogues à la transglutaminase telles que celle-ci, dans le traitement de troubles relatifs aux saignements, tels que l'hémophilie, ou dans le traitement de saignements excessifs comme cela peut se produire dans une opération chirurgicale ou dentaire. On peut administrer la transglutaminase seule ou sous la forme d'un véhicule de diffusion étalée, tel que dans les liposomes, et dans une variante, avec ou sans fibronectine.
PCT/US1988/002857 1987-08-17 1988-08-17 Enzyme d'eponge ayant une activite analogue a la transglutaminase WO1989001512A1 (fr)

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USNOTFURNISHED 1990-08-30

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EP0598133A1 (fr) * 1992-04-21 1994-05-25 Ajinomoto Co., Inc. Remede contre les plaies
WO1994011022A1 (fr) * 1992-11-12 1994-05-26 Zymogenetics, Inc. Procede et utilisation du facteur xiii en application locale pour empecher une hemorragie
WO1994028949A1 (fr) * 1993-06-03 1994-12-22 Orthogene, Inc. Composition d'adhesif biologique et procede pour promouvoir l'adherence entre des surfaces de tissus
WO1995016000A1 (fr) * 1993-12-09 1995-06-15 The Research Foundation Of State University Of New York Procedes et peintures anti fouling marines
EP0686401A2 (fr) * 1994-06-10 1995-12-13 Ajinomoto Co., Inc. Colle pour tissu vivant et coagulant sanguin
US5989323A (en) * 1993-12-09 1999-11-23 The Research Foundation Of State University Of New York Aquatic antifouling compositions and methods
WO2002036155A1 (fr) * 2000-11-03 2002-05-10 Zymogenetics, Inc. Utilisation du facteur xiii de coagulation sanguine dans le traitement de l'hemophilie a
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WO2004070386A2 (fr) * 2003-02-05 2004-08-19 The Nottingham Trent University Nouvelle methode de criblage
US20090264347A1 (en) * 2000-11-03 2009-10-22 Zymogenetics Inc. Use of Blood Coagulation Factor XIII for Treating Hemophilia A

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US4265233A (en) * 1978-04-12 1981-05-05 Unitika Ltd. Material for wound healing
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Cited By (18)

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Publication number Priority date Publication date Assignee Title
EP0598133A1 (fr) * 1992-04-21 1994-05-25 Ajinomoto Co., Inc. Remede contre les plaies
EP0598133A4 (fr) * 1992-04-21 1996-03-06 Ajinomoto Kk Remede contre les plaies.
US5525335A (en) * 1992-04-21 1996-06-11 Ajinomoto Co., Inc. Wound healing agent
WO1994011022A1 (fr) * 1992-11-12 1994-05-26 Zymogenetics, Inc. Procede et utilisation du facteur xiii en application locale pour empecher une hemorragie
US5736132A (en) * 1993-06-03 1998-04-07 Orthogene, Inc. Method of promoting adhesion between tissue surfaces
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US5549904A (en) * 1993-06-03 1996-08-27 Orthogene, Inc. Biological adhesive composition and method of promoting adhesion between tissue surfaces
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