Classifications

• • 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
  • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
  • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
  • A61L24/043—Mixtures of macromolecular materials

Definitions

• This invention relates to fibrin based tissue sealant compositions and more specifically, to thromboplastin-containing tissue sealant compositions which are capable of effectuating formation of a fibrin based clot in situ..
• Blood coagulation is the end result of a complex cascade of multiple proteins and other cofactors, culminating in the formation of fibrin strands.
• Fibrin formed from the precursor fibrinogen, is the protein which holds blood clots together.
• the coagulation cascade is described as consisting of separate pathways: the extrinsic pathway and the intrinsic pathway. (Rapaport and Rao Thromb. Haemostasis 74.7-17 (1995 ⁇ 1.
• the "extrinsic” pathway is dependent upon thromboplastin, and the "intrinsic” pathway is independent of thromboplastin.
• the pathways converge upon the generation of thrombin, which in combination with calcium ions, converts fibrinogen to fibrin, and Factor XIII to
• a fibrin sealant exploits this final stage ofthe coagulation cascade and historically has been designed as a two-component system, analogous to a two-component epoxy adhesive.
• the first component consists of fibrinogen and Factor XIII; which can be equated to the "resin" component of an adhesive composition.
• Thrombin and calcium ions make up the second component, which acts ast the catalyst ofthe resin.
• the two components may be applied sequentially or simultaneously by a syringe or by spraying. When they come in contact with one another, fibrin is formed from the fibrinogen. Fibrin sealants are used for hemostasis, as well as tissue sealing in patients on heparin or with coagulation deficiencies.
• Fibrin sealants can partially or totally preclude the use of sutures and thereby avoid inflammatory reactions (DePalma et al., Transfusion 33:717-720 (1993)).
• fibrin sealant adhesive systems see Sierra, J. Biomaterials Appl. 7:309-352 (1993).
• U.S. Patent No. 5,290,552 describes a dual-component system comprising fibrinogen, Factor XIII and collagen as one component; and thrombin and calcium ions as the other component. These two components are then mixed together just prior to use.
• U.S. Patent No. 4,600,574 describes a surgical adhesive comprising a flat web-like sheet of collagen, gelatin or polysaccharide which is coated with a solution of fibrinogen and Factor XIII, followed by lyophilization to form a matrix.
• 4,453,939 describes a composition for the healing of wounds which comprises a web-like carrier comprised of collagen which is coated on one side with a mixture of: (1) a fibrinogen-containing component which contains fibrinogen and Factor XIII; and (2) a thrombin-containing component. Coagulation is initiated upon insertion ofthe web into the patient which results in hydration and activation.
• Thromboplastin also referred to as tissue factor protein (TF)
• TF tissue factor protein
• U.S. Patent No. 5,091,363 describes a composition and method for the treatment of hemophilia A.
• thromboplastin is used to determine the thromboplastin time (PT) of a patient as an indicator of clotting efficiency.
• U.S. Patent No. 5,110,730 and PCT International Publication No. WO 94/11029 describe DNA segments defining a structural gene coding for a human tissue factor heavy chain protein and a precursor form ofthe protein. Methods of producing tissue factor protein recombinantly and the recombinant protein also are disclosed. The protein is disclosed to be useful for modulating the binding of Factor VH VIIa by tissue factor in vivo. Diagnostic uses for detecting the presence of a thrombus or the amount of tissue factor in a body sample are disclosed.
• European Patent Application Publication No. 0 278 776 discloses a tissue factor protein capable of correcting various bleeding disorders by inducing coagulation which is distinct from tissue thromboplastin because it lacks the naturally occurring lipid portion of the molecule.
• DNA isolates coding for tissue factor protein and derivatives thereof, recombinant expression systems for recombinant expression ofthe DNA are disclosed. Methods of treating coagulation disorders using the compositions also are disclosed.
• European Patent Application No. 0 347 262 discloses the sequence of a cDNA coding for human tissue factor and its use for the construction of recombinant expression vectors which in transformed hosts, produce human tissue factor apoprotein, soluble human tissue factor and truncated human tissue factor for clinical and diagnostic use.
• the present invention relates to tissue sealant composition that are useful to promote tissue adhesion and/or hemostasis via fibrin formation at the site of administration.
• the tissue sealant composition is prepared as a single component containing thromboplastin and fibrinogen, which can be supplied in purified form or by adding plasma.
• the composition may further comprise calcium ions, and addition factors (also supplied in purified form or by adding plasma) such as Factors II, V, VII, X and XIII.
• the tissue sealant composition is prepared in two parts (i.e. it is a "dual component system) which are mixed together before administration: one containing fibrinogen, and the other containing thromboplastin.
• any or all ofthe aforementioned optional components can also be included in either ofthe two components.
• mixing of the two components does not initiate fibrin formation, in which case the composition would necessarily have to be administered before complete gelling had occurred.
• the tissue sealant composition reacts at the site of administration with tissues and/or blood, which supply an effective amount of calcium ions, Factors II, V, VII, X and XIII to cause the fibrinogen to be converted to fibrin.
• tissue and/or blood which supply an effective amount of calcium ions, Factors II, V, VII, X and XIII to cause the fibrinogen to be converted to fibrin.
• any of these optional components are not present in sufficient quantities at the site of administration to effect fibrin formation, they are added to the tissue sealant composition prior to administration.
• the present invention additionally relates to a method for promoting tissue adhesion and/or hemostasis using any ofthe aforementioned compositions as further described below.
• Figure 1 is a flow chart showing the blood coagulation cascade. (From Enzyme Research Laboratories, Inc., South Bend, IN.) The abbreviations for the various blood coagulation factors are included in Table I. MODE S-1 FOR CARRYING OUT THE INVENTION Blood coagulation is a complex cascade of events that results in formation of fibrin strands. Figure 1 illustrates the blood coagulation cascade. The various factors, their molecular weights and typical plasma concentrations are given in Table I.
• tissue sealant refers to a composition which is effective to act as a tissue adhesive and/or hemostatic agent.
• thromboplastin as the initiator of fibrin clot formation may improve the hemostatic qualities ofthe adhesive by virtue ofthe extrinsic pathway's efficiency in forming thrombi.
• this pathway as depicted in Figure 1, thromboplastin complexes with and converts Factor VII to Vila, and then, in the presence of calcium ions, converts Factor X into Factor Xa, and Factor V into Va creating a "prothrombinase complex.”
• Factor Xa (which uses Factor Va as a cofactor) converts Factor II (prothrombin) into thrombin. Thrombin then converts fibrinogen into fibrin which forms a clot, and also converts Factor XIII into Factor Xllla in the presence of calcium ions. Factor Xllla in turn causes covalent crosslinking ofthe fibrin clot which makes it more stable both mechanically and proteolytically.
• the tissue sealant composition contains, as its primary elements, an effective amount of thromboplastin and fibrinogen. Additionally, the tissue sealant composition preferably contains calcium ions. In the case of a tissue sealant composition containing only thromboplastin, fibrinogen and calcium ions in a suitable delivery vehicle, all other factors (and other requirements) which are necessary to effect fibrin formation are found at the application site. However, not all application sites, especially those which are not actively bleeding, provide a sufficient source of these additional components. In this situation, the tissue sealant preferably contains all ofthe necessary factors required for fibrin formation, i.e. thromboplastin, fibrinogen, calcium ions and Factors II, V, VII, X and XIII.
• the tissue sealant compositions are prepared as a single- component system.
• the single-component system over conventional two-component fibrin sealant systems now in place.
• No exogenous thrombin of either human or other source is required, especially in large quantities.
• the need for large quantities of exogenous thrombin are eliminated in that very small amounts of thromboplastin enable the conversion of prothrombin to thrombin.
• Another important advantage ofthe composition and its use is its that it eliminates the need for catalysis ofthe "resin", which facilitates uniform mixing, curing and subsequent strength ofthe material.
• the convenience is improved in that only one delivery device or dosage unit is required to prepare and apply the material.
• Thromboplastin can be obtained from brain or tissue extracts, or it can be prepared using recombinant techniques. Thromboplastin which is obtained from natural sources contains an amount of lipid associated therewith which is necessary for thromboplastin activity. Thromboplastin which is produced from recombinant techniques must be "lipidated” to restore its native activity. As used herein, the term "thromboplastin” refers to thromboplastin from natural sources, as well as lipidated thromboplastin produced using recombinant techniques. See, for example, U.S. Patent No. 5,314,695 which describes the lipidation procedure.
• Recombinant thromboplastin is commercially available from Ortho Diagnostics (Raritan, N.J.) sold under the tradename RecombinPlastin® and Baxter Healthcare
• thromboplastin extract sold under the tradename Innovin ® (Miami, FI.)
• Methods of preparing purified thromboplastin are well known in the art and are described, for example in U.S. Patent Nos.: 5,254,350; 4,755,461; 5,270,451; 3,522,148; 3,522,148 and European Patent Publication No. 524 803 A2.
• Purified constituents are commercially available or readily obtainable from human and animal blood fractions or can be recombinantly produced using methods well known to those of skill in the art. It should be appreciated that the constituents as noted herein can be obtained from any suitable animal source, e.g., human, bovine or porcine.
• bovine fibrinogen is commercially available from Sigma Chemical Co. (Saint Louis, MO); Factors V, VII and XIII are commercially available from American Diagnostics Inc. (Greenwich, CT); Factor IX (human and bovine) is commercially available from Accurate Chemical & Scientific Corp. (Westbury, N.Y.) and American Diagnostic Inc. (Greenwich, CT); human and rabbit Factor VIII is commercially available from Accurate Chemical &
• human Factor X can be purchased from American Diagnostics Inc (Greenwich, CT) or bovine Factor X can be purchased from Sigma Chemical Co. (St. Louis, MO).
• Plasma can be used "as is" as a source for the various constituents after removal ofthe cellular components of blood by centrifugation.
• plasma can be processed to prepare a plasma cryoprecipitate by freezing, thawing and further centrifugation, which can be used as a source of fibrinogen and Factor XIII.
• Various factors can also be isolated from plasma which is in crystalline or amorphous form, or as a lyophilizate.
• Factors II, V, VII and X can be obtained from a cryosupernatant of plasma.
• Fibrinogen and Factor XIII can also be obtained from allogeneic or autologous plasma preparations.
• Fibrinogen and Factor XIII may be obtained from the "resin" component of commercially available dual-component fibrin sealant compositions.
• bovine fibrinogen can be obtained from a fibrin sealant preparation such as Tisseel ® (Immuno AG,
• Factors II, V, VII and X can be obtained from anti-hemophilia B therapeutic agents (Octapharma, Immuno AG, Alpha Therapeutics, Baxter-Hyland and Armour Bayer).
• Factor II may be produced by recombinant expression techniques as described in
• the desired constituents are initially produced in soluble form and, where appropriate, are virally deactivated.
• the components are purified from a native or natural source, they are provided in purified or substantially purified form.
• Purified shall mean that the protein or factor of interest is substantially free of cellular and other biological components normally associated with the protein or factor in its native or natural environment in the cell or body fluid.
• purified can be used to describe proteins and factors isolated from their native environment or isolated from a biological, non-naturally occurring environment such as when they are recombinantly produced in a host cell such as a Chinese Hamster Ovary cell which is commercially available from the American Type Culture Collection ("ATCC”; Rockville, MD).
• ATCC American Type Culture Collection
• compositions of this invention can include, in addition to the factors in forms as they appear in nature, i.e., in a "purified” state, analogs, muteins, conjugates, and homologues ofthe proteins or factors, provided that the biological activity of the factor is not substantially impaired.
• “Substantially impaired” would include a greater than 50% reduction in the biological activity ofthe analog, homologue or mutein, as compared to native or natural protein or factor.
• use of a term such as "thromboplastin”, in addition to thromboplastin from natural sources is intended to encompass all alternative forms of thromboplastin having a biological activity which is not substantially impaired, i.e. "thromboplastin equivalents.”
• the biological activity of a protein or factor includes any feature of the polypeptide determined by suitable experimental investigation, including, but not limited to the experiments set forth herein relating to coagulation time and the ability to promote the formation of fibrin in situ.
• the preferred single-component composition which contains thromboplastin, fibrinogen and all ofthe necessary extrinsic pathway factors (Factors II, V, VII, X and
• XIII is prepared by mixing the constituents together either without calcium ions or an amount of calcium ions which is insufficient to effect gelation ( ⁇ 3mM) within an hour. Such compositions remain flowable for at least one hour, or until they come in contact with tissue and/or blood after administration. The additional Ca from blood or tissue permits the extrinsic pathway reactions to occur, which results in fibrin clot formation.
• an effective amount ofthe individual components is an amount that, when combined as described herein and brought into contact with body tissues in situ, will induce the conversion of fibrinogen to fibrin which results in fibrin clot formation. Suitable concentrations for most ofthe factors correspond to a range present in normal human plasma and as provided in Table III, below. It should be assumed, although not always explicitely stated, that "effective amounts" ofthe components are used and inco ⁇ orated into the compositions of this invention.
• compositions comprising a single tissue adhesive in combination with other constituents, such as stabilizers, preservatives, therapeutics collagen, collagen analogs and collagen conjugates.
• stabilizers Any stabilizer that functions to maintain the activity ofthe tissue adhesive upon administration to the patient can be used in practicing the invention.
• stabilizers include, but are not limited to Tris (trishydroxymethylaminomethane), PIPES (Piperazine-N,N-bis(2-ethane-sulfonic acid, 1.5 sodium salt), imidazole, and MOPS (3-(N-Mo ⁇ holine) propanesulfonic acid).
• Suitable preservatives include sodium azide, thimerosal, BHA, BHT. Other preservatives that function to prevent the growth of microorganisms that would damage the component system is suitably added to the adhesive components.
• Collagen a collagen analog or a collagen-containing conjugate can be added to increase the rate of gelation, and also to thicken the adhesive composition and augment cohesiveness.
• the amount of collagen to be added can be easily determined by varying the amount of collagen and chosing that concentration of collagen which gives the desired result.
• the collagen may be atelopeptide collagen or telopeptide collagen.
• Animal or human-based collagen is suitably used and can be purified using methods well known to those of skill in the art and described in U.S. Patent No. 4,233,360. These collagen preparations also are available commercially from a supplier such as Collagen Co ⁇ . (Palo Alto, CA) under the tradename Zyderm II®.
• Other biomaterials may be used to augment either the physical performance ofthe sealant or its application in a specific repair site.
• hydroxylapatite or tri-calcium phosphate can be inco ⁇ orated for repairs in bony tissue.
• Attachment factors such as RGD peptide sequences can be added as well.
• Additional biomaterials include, but are not limited to bone or hard tissue materials, plastics, particulates and metals.
• an analog is intended to include the materials as described above having similar and different chemical or physical entities of the same material as naturally occurring in nature or purified from a native source.
• An analog can consist of hybridized or conjugated proteins, as described in published PCT International Publication No. WO 94/16085.
• tissue sealant additionally serves as a vehicle for delivery of these components.
• components such as antibiotics, metabolic substances, cells and growth factors can be added.
• Growth factors such as EGF, TGF- ⁇ , TGF- ⁇ , FGF, PDGF can be added.
• Cytokines such as interleukin or stem cell factor also can be suitably added.
• Antibiotics can be added and are particularly useful when the adhesive is applied to exposed wound sites such as mouth sores and burns.
• the tissue sealant compositions can also be mixed with cells, autologous, cultured or modified, allogeneic or xenogeneic. As is apparent to those skilled in the art, the amount of an added component will vary with the use ofthe adhesive and the recipient but is easily determined by the treating physician.
• TFPI tissue factor pathway inhibitor
• Factor VIII and IX can be obtained from plasma as described above for the other factors using known methods.
• Factor VIII can be obtained from anti- hemophilia A therapeutic agents (Octapharma, Immuno AG, Alpha Therapeutics, Baxter- Hyland and Armour Bayer).
• a calcium ion chelator can also be inco ⁇ orated in an amount effective to prevent formation of a fibrin clot prior to administration while still allowing the formation of clot upon administration. The amount of chelator will vary based on the source ofthe reagents and ultimate use ofthe composition. Any calcium ion chelator that functions similarly to ethylenediaminetetraacetic acid (EDTA) in binding/chelating ions or other metal ions can be used in the practice of this invention. Examples of suitable chelators include, but are not limited to citrate, salts of citrate, ethylene-bis (oxyethylenenitolo) tetraacetic acid (EGTA) and salts of EGTA.
• EDTA ethylenedi
• tissue sealants ofthe present invention are applied to human (or animal) tissue which provides physiological calcium from the surrounding tissue (and optionally additional necessary components), resulting in fibrin formation.
• tissue sealants of this invention can be used in a wide variety of procedures and surgical indications, adjunctively as a replacement for sutures, hemostatic agents, packing materials and to deliver various therapeutic agents.
• the tissue sealants can be used in any application where the formation of a fibrin clot is desired, hemostasis is required or where prior art surgical and fibrin sealants were previously used.
• fibrin sealants have been used in tissue remodeling and wound repair. They also have been shown to act as osteogenic or osteostimulatory agents.
• Cardiovascular applications are numerous.
• fibrin sealants have been used as a hemostatic sealant for vascular graft attachment, cardiovascular patches, heart valve attachment and to preclot vascular grafts.
• Fibrin sealants are also used to deliver drugs and antibiotics. Grafting of skin with fibrin sealants has been successful for burn patients, face lifts and in rhinophyma repair.
• compositions described herein can be used to initiate the formation of fibrin or a fibrin clot in situ. Further provided are methods of treating bleeding disorders and treating wounds in a patient. These methods require administering to the patient a therapeutically effective amount ofthe single-component adhesive composition as described herein to form fibrin.
• administering shall mean providing the recipient or patient with the tissue sealant topically for local therapy or administered by injection intravascularly (if the sealant is to be used as an embolic agent) by combining the adhesive with a suitable pharmaceutically acceptable carrier such as phosphate buffered saline.
• suitable pharmaceutical carriers, stabilizers and preservatives are well known to those of skill in the art and are described for example in Remington's Pharm. Sci. 15th Ed. (Mack Publ. Co., Easton (1975)).
• Local administration ofthe adhesive can be in a single dose or multiple doses as determined by the treating physician.
• the dosage must contain an effective amount of thromboplastin and fibrinogen, as well as any other optional components, such that administration causes fibrin formation.
• compositions of this invention can be combined with standard carriers and preservatives to form pharmaceutical compositions, which are also within the scope of this invention. Accordingly, the use ofthe compositions described herein to prepare medicaments for promoting and/or inducing the formation of fibrin in situ, is further within the scope of this invention.
• This invention furhter provides uses ofthe above composition for the preparation of medicaments for inducing the formation of fibrin in situ in an animal such as a rat, guinea pig, rabbit or a human patient.
• compositions and methods include the recited elements, but not excluding others.
• Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination.
• a composition consisting essentially ofthe elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
• compositions show the effect of adding calcium ions and/or collagen to the tissue sealant composition. It also shows that the formulations are stable at room temperature and are effective, in vivo, as well as the impact of increasing the fibrinogen concentration on gelation time.
• the compositions were prepared as follows: bovine source citrated plasma which served as a source of fibrinogen and Factor XIII, was recycled three times (cryoprecipitate) as prepared by a modification ofthe method of (Gestring and Lerner Vase. Surg.
• thromboplastin (RecombiPlastin (lipidated) from Ortho Diagnostics) having a nominal thromboplastin concentration of 200 ng/ml; and Zyderm II collagen (65 mg/ml)(Collagen Co ⁇ ., Palo Alto, CA).
• TP thromboplastin
• Zyderm II collagen 65 mg/ml
• the suitability ofthe single-component tissue sealant as a hemostatic agent was evaluated in a standardized rodent liver incision model and the results are shown in Table VI.
• a midline incision transecting the abdomen was made in an anesthetized rat.
• the liver was elevated and exposed.
• a lobe was completely bisected anterior to posterior.
• the sealant was applied along the incision and the time for visible bleeding to stop was recorded.
• Fibrin sealant and a fibrin-collagen composite tissue adhesive containing thrombin was also tested.
• Prior Art Fibrin Sealant (fibrinogen, 60 mg/mL; thrombin, 15 200 U/mL; and CaCl 2 , 40 mM)
• the single component sealant is nearly as effective as the two component
• tissue adhesive is more effective than the prior art fibrin sealant.

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  • 1997-02-19 EP EP97907707A patent/EP0885020A1/de not_active Withdrawn
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