CA2247133A1 - Tissue sealant compositions and methods of use thereof - Google Patents

Abstract

Novel tissue sealant compositions are disclosed herein which can be formulated as a single-component. The compositions contain as essential elements thromboplastin and fibrinogen. Additional blood clotting factors such as Factors II, V, VII, X, and XIII can also be added. Also provided are methods for promoting tissue adhesion and/or hemostatus by administering the tissue adhesive compositions described herein.

Description

..

TISSUE SEALANT COMPOSITIONS
AND METHODS OF USE THEREOF

This application claims priority from provisional application No. 60/011,973, filed S February 20, 1997, incorporated herein in its entirety.

TECE~ICAL FTFr 1 ) OF THE INVFNTION
This invention relates to fibrin based tissue sealant compositions and more specifically, to thromboplastin-contzlining tissue sealant compositions which are capable of effectll~ting formation of a fibrin based clot in si~u R~ACKGROUND Al~T
Blood coagulation is the end result of a complex cascade of multiple proteins and other cofactors, clllmin~tinp 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 con.C~i~tin~ of separate p~lhw~y~; the extrinsic pathway and the intrinsic pathway. (Rapaport and Rao Thromb. EIaemostasis 74:7-17 (1995)). The "extrinsic" pathway is dependent upon thromboplastin, and the "intrinsic" pathway is independent of thromboplastin. The p~lhw~y~ converge upon the generation of thrombin, which in combination with calcium ions, converts fibrinogen to fibrin, and Factor XIII to Factor XIIIa.
A fibrin sealant exploits this final stage of the coagulation e~C~c~e and historically has been ~lPc~igne~l as a two-component system, analogous to a two-component epoxy adhesive. The first component consists of fibrinogen and Factor XIII; which can be e~uated to the "resin" component of an adhesive composition. Thrombin and calcium ions make up the second component, which acts ast the catalyst of the resin. The two components may be applied sequentially or c imlllt~neously 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 hemost~ , as well as tissue sealing in patients on heparin or with coagulation deficiencies. They promote wound healing by decreasing oozing and control air leaks by producing a fluid-tight seal at wound sites. Fibrin sealants can partially or SUBSTITUTE SHEET (RULE 26) WO 97129792 PCT/US97/0261i totally preclude the use of sutures and thereby avoid infl~mm~tory reactions (DePalma et al., Tr~n~fusion 33:717-720 (1993)). For a detailed review of the history and use of fibrin sealant adhesive systems, see Sierra, J. Biorn~t~r;~ls Appl. 7:309-352 (1993).
Early surgical adhesives contained a high content of fibrinogen (about 8-10%) which could only be prepared with difficulty from fibrinogen lyophili7~tes. They were generally unstable and therefore required storage at -20~C to 5~C until use. Examples of these early adhesives include compositions marketed under the tradenarnes "Tisseel"(~) or "Tissucol"(g) (Immuno AG of Vienna, Austria), Beriplast(~) (Behringwerke AG, Marburg, Germany) and Biocoll~) (LFB, Lille, France). These were prepared from large quantities of screened, pooled donor sourced human plasma.
Patient autologous and single-donor sourced fibrin sealants were developed in the United States in response to concerns over v*al disease tr~n~mi~ion of the commercial, pooled donor sourced fibrin scal~nt~. These efforts were focused on producing concentr~tetl fibrinogen, which could then be used in conJurlction with readily available 1 ~ bovine thrombin.
In 1983, Gestring and Lerner, described a cryoprecipitation production method which utilized small amounts of patient autologous blood. (Gestring and Lerner, Vas~
17:294-304 (1983).) This method was modified for large-scale production. (See U.S.
Patent 4,627,879.) A&esive compositions incorporating other blood factor components have been described. U.S . Patent No. 4,061,731 describes a composition comprising patientautologous plasma and microcrystalline collagen and/or gelatin in combination with endogenous thrombin. These compositions and methods of use are limited by the lack of large commerically available amounts of patient blood, pre~lion time, which can range from an hour to ovemipht, and the equipment and expertise of trained hospital personnel.
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, 3û gelatin or polys~cch~ritle which is coated with a solution of fibrinogen and Factor XIII, followed by lyo~hili7~tion to form a matrix. U.S. Patent No. 4,453,939 describes a composition for the healing of wounds which comprises a web-like carrier comprised of SUBSTITUTE SHEET (RULE 26) collagen which is coated on one side with a mixture of: (1) a fibrinogen-co..~ gcomponent which contains fibrinogen and Factor XIII; and (2) a thrombin-co~t~ g component. Coagulation is initiated upon insertion of the web into the patient which results in hydration and activation.
Thromboplastin (also referred to as tissue factor protein ~rF)) as a therapeutic or diagnostic agent for coagulation disorders has been described. U.S. Patent No. ~,091,363 describes a composition and method for the trç~tment of hemophilia A. In addition, 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 Tnt~ tional Publication No. WO 94/11029 describe DNA se~ment~ clçfining a structural gene coding for a human tissue factor heavy chain protein and a precursor form of the protein. Methods of producing tissue factor protein recombinantly and the recombinant protein also are disclosed. The protein is disclosed to be useful for mod~ ting the binding of Factor VII/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 inc1llring 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 of the 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 apopl~leill, soluble human tissue factor and truncated human tissue factor for clinical and diagnostic use.
The above prior art, however, does not describe or disclose a fibrin based tissue sealant composition which utilizes thromboplastin as an initiator of fibrin formation.
Indeed, the er~;~iv~;ness of thromboplastin as a topical hemostatic agent has been reported to be limited when compared to conventional agents (such as thrombin). (See Figure 3 in PC~ WO 941Q2I72).

SUBS~ITUTE SHEET (RULE 26) SUMMA~Y OF THF INVl~NTION
The present invention relates to tissue sealant composition that are useful to pron1ote tissue adhesion and/or hemostasis via fibrin formation at the site of ~Aministration. In a preferred embodiment of the present invention, the tissue sealant composition is prepared as a single component cont~inin~ thromboplastin and fibrinogen, which can be supplied in purified form or by adding plasma. In addition to these two essential components, 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.
In another embodiment of the present invention, the tissue sealant composition is prepared in two parts (i.e. it is a "dual component system) which are mixed together before 7~1mini~tration: one co~ fibrinogen, and the other co~ thromboplastin. In addition to these essential components, any or all of the aforementioned optional components can also be included in either of the two components. Preferably, mixing of the two components does not initiate fibrin formation, in which case the composition would necess~rily have to be ~ inist.qred before complete gelling had occurred.
Using the simplest embodiment cont~ining only thromboplastin and fibrinogen in asuitable vehiGle, the tissue sealant composition reacts at the site of ~r1ministration 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. When any of these optional components are not present in sufficient ~uantities at the site of ~rimini~tration to effect fibrin formation, they are added to the tissue sealant composition prior to ~ ni~tration.
The present invention additionally relates to a method for promoting tissue adhesion andJor hemostasis using any of the aforementioned compositions as fi~rther 2~ described below.

P~RTFF DFS~TPTION OF THF D~AVVINGS
Figure 1 is a flow chart showing the blood coagulation cascade. ~From Enzyme Research Labolat~,lics, Inc., South Bend, IN.) The abbreviations for the various blood coagulation factors are included in Table I.

SUBSTITUTE Sl IEET (RULE 26) MODE(S~ FOR CARRY~ OUT THF INVFNTION
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.
TABLE I
MolecularPlasma Plasma Component WeightConc~ r~lion Concelltration (daltons)(~lg/ml) ~M) Fibrinogen (~) 330,000 3000 9.09 Prothrombin ~II) 72,000 100 1.388 Factor V 330,000 10 0.03 Factor V~I 50,000 0.5 0.01 Factor VIII 330,000 0.1 0.0003 Factor IX 56,000 5 0.08928 Factor X 58,800 8 0.13605 Fac~or XI 160,000 5 0.031 Factor XII 80,000 30 Q.375 Factor X~II 320,000 10 0.03124 Prote~nC 62,000 4 0.0645 Protein ~ 69,00010(free) 0.1449 Protein Z 62,000 2.2 0.0355 Prekallikrein 86,000 50 0.5814 HK 1 10,000 70 0.6363 Fibronectin 450,000 300 0.6667 Allti~ vLllbin III58,000 290 5 Pl~nninogen 90,000 216 2.4 Urokinase 53,000 0.1 0.001887 Heparin Cofactor II66,000 90 1.3636 Alpha2-Antiplasmin63,000 60 0.9524 Protein C Inhibitor57,000 4 0.0702 Alpha2-Macroglobulin725,0002100 2.8966 An exogenous thrombin-free, fibrin based tissue sealant is described herein, which can be prepared as a single-component system. As used herein, the term "single-component" is inten~le-l to mean that the tissue sealant composition can be used alone to cause the conversion of fibrinogen to fibrin at the site of ~-lmini~tration. In contrast, SUBSTITUT~ SHEET (RULE 26~

conventional "dual-component" systems provided as two separate compositions require contacting the two compositions with one another prior to or simultaneous with 57~1min;~stration to activate the coagulation cascade which results in in situ fibrin formation.
The term "tissue sealant" as used herein refers to a composition which is effective to act as a tissue adhesive and/or hemostatic agent.
The use of thromboplastin as the initiator of fibrin clot formation may improve the hemostatic yualities of the adhesive by virtue of the extrinsic pathway's efficiency in forming thrombi. In this pathway, as depicted in Figure 1, thromboplastin complexes with and converts Factor VII to VIIa, and then, in the presence of calcium ions, converts Factor X into Factor Xa, and Factor V into Va creating a ''~ -binase complex." In turn,Factor Xa (which uses Factor Va as a cofactor) converts Factor II ~rolhrolllbin~ into thrombin. Thrombin then converts fibrinogen into fibrin which forms a clot, and also converts Factor XIII into Factor X~IIa in the presence of calcium ions. Factor XIIIa in turn causes covalent cros~linking of the fibrin clot which makes it more stable both mechanically and proteolytically.
Accordingly, 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 cont~ining only thromboplastin, fibrinogen and calcium ions in a suitable delivery vehicle, 2û all other factors ~and other requirements~ which are necessary to effect fibrin formation are ~ound 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 of the necessary factors required for fibrin ~ormation, i.e. thromboplastin, fibrinogen, calcium ions and Factors II, V, VII, X and ~III.
In a p.~f~led embodiment, the tissue sealant compositions are prepared as a single-component system. There are advantages of the single-component system over conventional two-component fibrin sealant systems now in place. No exogenous thrombin SUBSTITUTE SHEET (RULE 26) CA 02247l33 l998-08-l8 wo 97/29792 PCT/US97/02614 of either human or other source is required, especially in large quantities. This elimin~tes concerns over irnmunologically in~ se~l coagulopathies from bovine sourced thrombin aldlions. The need for large quantities of exogenous thrombin are elimin~ted in that very small amounts of thromboplastin enable the conversion of pLO~ vlllbin to thrombin.
Another important advantage of the composition and its use is its that it elimin~t~s the need for catalysis of the "resin", which facilitates uniform mi~in~, curing and subsequent strength of the material. The convenience is improved in that only one delivery device or dosage unit is required to prepare and apply the material.

~ollrces of Composition Components 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 neces~ y for thromboplastin activity. Thromboplastin which is produced from recombinant techniques must be lS "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 ~Rarit~n, N.J.) sold under the tr~enzlme RecombinPlastin(~ and Baxter He~lth~re (: orpor~tion, Dade Division provides a thromboplastin extract sold under the tr~clen~me ~nnovin'lD (Miami, Fl.) Methods of preparing purified thromboplastin are well known in the art and are described, for exarnple in U.S. Patent Nos.: 5,254,35~); 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 a~imal blood fractions or can be recombinantly produced using methods well known to tlhose 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. For example, SUBSTIT-UTE SHEET (RULE 26) WO 97l29792 PCT/US97/02614 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 con"l,elcially available from Accurate Chemical & Scientific Corp. (Westbury, N.Y.) and Arnerican Diagnostic Inc. (Greenwich, CT); human and rabbit Factor VIII is commercially available from Accurate Chemical &
Scientific Corp. (Westbury, N.Y); and human Factor X can be purchased from Arnerican Diagnostics Inc (Greenwich, CT) or bovine Factor X can be purchased from Sigrna Chemical Co. (St. Louis, MO).
Human or animal plasma can be used "as is" as a source for the various con~titllent~ after removal of the cellular components of blood by centrifugation. For example, 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 f~om plasma which is in crystalline or amorphous fo}m, or as a lyophi~i7~te Also, Factors II, V, VII and X can be obtained from acryo~u~L. ~ t of plasma.
Fibrinogen and Factor XIII can also be obtained from allogeneic or autologous plasma lJle~udLions.
Fibrinogen and Factor XIII may be obtained from the L'resin" component of cornrnercia}ly available dual-component fibrin sealant compositions. For exarnple, bovine fibrinogen can be obtained from a fibrin sealant ~ dtion such as Tisseel~ (Irnmuno AG, Vienna, Austria.) In addition, Factors II, V, VII and X can be obtained from anti-hemophilia B
therapelltic agents (Oct~ph~ , Irnmuno AG, Alpha Therapeutics, Baxter-Hyland andArmour Bayer).
Factor II may be produced by recombinant expression techniques as described in U.S. Paten~ No. ~,476,777. In addition, purification methods for Factor II are described in Miletich et al. Meth Fn7;yrnol. 80:221-228 (1981) and U.S. Patent No. 5,378,365.

SUBSTITUTE SHEET (RULE 26) Factor VIII can also be produced by the method disclosed in Eulopec~ Patent No.
085,923, incorporated herein by reference. Additional methods of ~lc~uing and isolating recombinant Factor VIII as well as purifying Factor VIII are well known to those of skill in the art as evidenced by Wood et al. Nature 312:330-336 (1984); U.S. Patent No.
5,422,260; U.S. Patent No. 5,422,250, U.S. Patent No. 5,410,022; and U.S. Patent No.
5,738,612.
Methods of producing, isolating and purifying Factor IX are well known to those of skill in the art as evidenced by U.S. Patent No. 5,286,849; U.S. Patent No. 5,171,569; and n J. Biol. Chem. 261:9622-9628 (1986).
Factor X can be produced as described in Miletich et al. ~E_-P~ a,dlion ofthe Tissue S~ nt Compositions To produce the tissue sealant, the desired consti~lent~ are initially produced in soluble form and, where a~,o~liate, are virally deactivated. As is known to those of skill in the art, when 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. Thus, the term "purified" can be used to describe proteins and factors isolated from their native environment or isolated from a biological, non-n~tllriqlly occurring environment such as when they are recoml~ lly produced in a host cell such as a Chinese Harnster Ovary cell which is commercially available from the American Type Culture Collection ("ATCC"; Rockville, MD).
~ It should be understood, although not always explicitly stated, that the 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 of the proteins or factors, provided that the biological activity of the factor is not substantially impaired. "Substantially impaired" would include a greater than 50%

SUBSTITUTE SHEEt (RULE 2~

CA 02247l33 l998-08-l8 reduction in the biological activity of the analog, homologue or mutein, as compared to native or natural protein or factor. Accordingly, use of a term such as "thromboplastin", in addition to thromboplastin from natural sources, is int~ncled to encompass all alternative forms of thromboplastin having a biological activity which is not substantially impaired, S i.e. "thromboplastin equivalents." The biological activity of a protein or factor includes any feature of the polypeptide ~Içt~rm;ned 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 ~r~;r~ d single-component composition which contains thromboplastin, fibrinogen and all of the necessary extrinsic pathway factors (Factors II, V, VII, X and XIII) is ~l~a~ed by mixing the con~tiiuPnt~ together either without calcium ions or an amount of calcium ions which is insufficient to effect gelation (<3mM3 within an hour.
Such compositions remain flowable for at least one hour, or until they come in contact with tissue and/or blood after ~-imini~tration. The additional Ca2+ from blood or tissue permits the extrinsic pathway reactions to occur, which results in fibrin clot formation.
Alternative embodiments of the tissue sealant compositions of the present invention are described below in Table II:

SUBST!TUTE SHEET (RULE 26 TABLE II

Single/Dual First Second Missing Reaction Component Component* Optional Component(s) Started by:
Component **
Single Fibrinogen None Additional Administration <3mM Ca2 Ca2 (sufficient to sitewhich Factor II to bring total provides Factor V concentration additional Ca2+
Factor VII to > SrnM) Factor X
Factor XIII
Single Fibrinogen None Factor II A-lmini~tration <3mM Ca Factor V to site which Factor VII provides all Factor X mi.~sing Factor XIII components Additional Ca2 Dual ~3 mM Ca T Fibrinogen FactorII AddingPlasma Factor IX Factor V co Factor XIII Factor VII mi~in~
Factor X components***
Additional ca2+
Single Factor XIII None Factor II Administration Fibrinogen Factor V to site which >5mM Ca2+ FactorVII provides all Factor X mi~.~ing components Dual > 5 mM Ca ~ Fibrinogen Factor V Atlmini~tration Factor XIII Factor II to site which Factor II Factor X provides all mi~in~
components * In add tion to thromboplastin.
** The mi~in~ component(s) is what is found at the application site that triggers the extrinsic pathway reactions to occur, resulting in conversion of fibrinogen to fibrin.
*** Must be ~(lmini.~tered immediately after adding plasma.

SlJBS~U~E SHEET (RUI_E 26) "An effective arnount" of the 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 forrnation.
Suitable concentrations for most of the 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" of the components are used and incorporated into the compositions of this invention.

TABLE III

Component Effective Concentration P~f~ d Range (mglmL) Concentration Ran~e (m~/mL) Fibrinogen 1-200 2-120 Factor II 0.001-1.0 0.10-0.50 bin) Factor V 0.0001-0.03 0.001-0.02 Factor X 0.001-0.08 0.03-0.07 Factor XIII 0.0001-0.04 0.001-0.05 Factor VII 0.00001-0.004 0.0001-0.003 thromboplastin 0.00005-5 0.0001-.001 Calcium ion (C~a(II)) 0.5-30 mM 2-20 mM

Options/l Component This invention also provides compositions comprising a single tissue a&esive in combination with other constituents, such as stabilizers, ples~l v~lives, therapeutics collagen, collagen analogs and collagen conjugates. Any stabilizer that fimctions to m~int~;n the activity of the tissue adhesive upon ~fimini~tration to the patient can be used in practicing the invention. Examples of such stabilizers include, but are not limited to SUBSTITUTE SHEET (RULE 26) WO 97/29792 PCT/USg7/~2614 Tris (trishydroxymethylaminomethane), PIPES (Piperazine-N,N-bis(2-ethane-sulfonic acid, 1.5 sodium sali), imic~7c-1e, and MOPS (3-(N-Morpholine) prop~ne~lllfonic acid).
Suitable preservatives include sodium azide, thimerosal, BHA, BHT. Other prescl v~Lives that function to prevent the growth of microorg~ni~m~ that would darnage the component system is suitably added to the adhesive components.
Collagen, a collagen analog or a collagen-co~ i"g conjugate can be added to increase the rate of gelation, and also to thicken the adhesive composition and ~ m~nt 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 1~ 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 dlions also are available cornmercially from a supplier such as Collagen Corp. (Palo Alto, CA) under the tr~ on~me Zyderm II(~. Other biom~t~ri~l~ may be used to ~lgment either the physical performance of the sealant or its application in a specific repair site.
For example, hydroxylapatite or tri-calcium phosphate can be incorporated for repairs in bony tissue. Attachment factors such as RGD peptide sequences can be added as well.
Additional biom~t~ori~l~ include, but are not limited to bone or hard tissue materials, plastics, particulates and metals. As used herein, an analog is int~n~led to include the m~t~ri~l~ as described above having similar and dir~elll chemical or physical entities of the same m~tPri~l as naturally occurring in nature or purified from a native source. An ~nalog can consist of hybridized or conjugated proteins~ as described in published PCT
~nt~ ional Publication No. WO 94/16085.
Therapeutic agents can also be added, in which case the tissue sealant additionally 2~ serves as a vehicle for delivery of these components. For example, components such as antibiotics, metabolic substances, cells and growth factors can be added. Growth factors such as EGF, TGF-a, TGF-13, FGF, PDGP can be added. Cytokines such as interleukin or stem cell factor also can be suitably added. Antibiotics can be added and are particularly SUBSTITUTE SHEET (RULE 26) WO 97/2g792 PCT/US97/02614 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 t~he art, the amount of an added component will vary with the use of the adhesive and the recipient but is easily S determined by the keating physician.
Factors VIII and IX are optionally added to overcome tissue factor pathway inhibitor (TFPI) which shuts down Factor X activation by the ~IIa/thromboplastincomplex that may occur in a physiologically complex mixture. However, this interaction is preferably minimi~d in the tissue sealant compositions of the present invention by using sufficiently purified materials.
Factor VIII and IX can be obtained from plasma as described above for the other factors using known methods. In addition, Factor VIII can be obtained from anti-hemophilia A therapeutic agents (Octapharma, Tmmlln~ AG, Alpha Therapeutics, Baxter-Hyland and Arrnour Bayer).
A calcium ion chelator can also be incorporated in an amount effective to prevent formation of a fibrin clot prior to ~1mini~kation while still allowing the formation of clot upon z~rnini.ctration. The amount of chelator will vary based on the source of the reagents ~ and lllt;m~te use of the composition. Any calcium ion chelator that functions similarly to ethylene~ min~tetraacetic acid (EDTA) in binding/chelating ions or other metal ions can be used in the practice of this invention. Exarnples of suitable chelators include, but are not limited to cikrate, salts of citrate, ethylene-bis (oxyethylenenitolo~ tekaacetic acid ~EGTA) and salts of EGTA.
At1rnin;~tr~tion and Use ~he tissue sealants of the present invention are applied to human (or animal) tissue .
which provides physiological calcium from the surrounding tissue (and optionallyadditional necessary components), resulting in fibrin formation.
The tissue sealants of this invention can be used in a wide variety of procedures and su~gical indications, adjunctively as a replacement for sutures, hemostatic agents, p~cking SUBSTITUTE SHEET (RULE 26) CA 02247l33 l998-08-l8 WO 97/2g792 PCT/US97/02614 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.
Historically, fibrin sealants have been used in tissue remodeling and wound repair.
They also have been shown to act as osteogenic or osteostim~ tory agents.
Cardiovascular applications are numerous. ~or exarnple, fibrin se~ nt~ have been used as a hemostatic sealant for vascular graft ~ rhment cardiovascular patches, heart valve attachment and to preclot vascular grafts.
Fibrin sealants are also used to deliver drugs and antibiotics. ~rafting of skin with fibrin sealants has been succe~ful for burn patients, face lifts and in rhinophyma repair.
Accordingly, the compositions described herein can be used to initiate the formation of fibrin or a fibrin clot in situ. l~urther provided are methods of treating bleeding disorders and treating wounds in a patient. These methods require ~lmini~tering to the patient a therapeutically effective amount of the single-component adhesive composition as described herein to form fibrin.
As used herein, "~imini~tering" shall mean providing the recipient or patient with the tissue sealant topically for local therapy or ~lmini~tered by injection intravascularly (if the sealant is to be used as an embolic agent) by combining the adhesive with a suitable ph~ ceu~ically acceptable carrier such as phosphate buffered saline. Other suitable ph~rm~ceutical carriers, stabilizers and preservatives are well known to those of skill in the art and are described for example in Remir~ton's Ph~rrn. Sci. 15th Ed. (Mack Publ. Co., Easton (1~75)).
Local ~qtlmini~tration of the adhesive can be in a single dose or multiple doses as ~let~rmined by the treating physician. In any case, the dosage must contain an effective amount of thromboplastin and fibrinogen, as well as any other optional components, such that 7(~1mini~tration causes fibrin formation.
As is a~ ellt to those of skill in the art to which this invention pertains, thecompositions of this invention can be combined with standard carriers and preservatives to SUBSTITUTE SHEET (RULE 26) form pharmaceutical compositions, which are also within the scope of this invention.
Accordingly, the use of the 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 of the above composition for theS pl~dlalion of medicaments for inducing the formation of fibrin in situ in an animal such as a rat, guinea pig, rabbit or a human patient.
As used herein, the term "comprising" is intended to mean that the 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 ess~nti~l significance to the combination. Thus, a composition con~i~tin~
essentially of the elements as defined herein would not exclude trace cont~min~nt~ from the isolation and purification method and ph~rm~çeutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. It could, however, exclude other, non-specified blood coagulation factors that can impair or substantially alter the ability of the formulation to remain flowable prior to arlministration and/or can substantially alter or impair the ability to promote the formation of fibrin in sit., e.g., TFPI, thrombin, arld fibrin catalysts. "Consisting of" shall mean excluding more than trace elements of other ingredients and substantial method steps for ~rlministering the compositions of th;s invention. Embodiments defined by each of these transition terms are within the scope of this invention.

SUBSTITUTE SHEET (RIJL-E 26) CA 02247l33 l998-08-l8 THF FOT T OWING FXAMPLES ARE OFFFRFn BY WA~ OF TT r USTRATION AN~
NOT BY WAY OF LIMITATION.

q FXAl~PLJ~ I
J S ~ec~s of Varying Composiffon Components and Conc~ .lions This example shows the effect of adding calcium ions and/or collagen to the tissue sealant composition. It also shows that the formulations are stable at room t~ d~UlC;
and are effective, in vivo, as well as the impact of increasing the fibrinogen concentration on gelation time. In general, 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 of the method of (Gesking and Lerner Vasc. Surg. 17:294-304 (1983)) and was mixed 1:1 (v/v) with reconstituted human reference plasma which served as a source of Factor II, V, VII, X, and as an additional source of fibrinogen and Factor XIII (Sigma Chemical Co., Product #A7432). The plasma Lul~ thus formed was then mixed with the following additional componenent.~ to achieve the concentrations given below in Table III: thromboplastin (TP) (RecombiPlastin (lipidated) from Ortho Diagnostics) having a nominal thromboplastin concentration of 200 ng/ml; and Zyderm II collagen (65 mg/ml)(Collagen Corp., Palo Alto, CA). The components were mixed at room temperature in a culture tube by vortexing. The time for the mixture to form a gel was observed and recorded after the addition of 0.2 mL of a 40 mM CaCl2 solution and the results are shown in Table IV.

SUBST~lUTE StlEET (RULE 26) TABLE IV
Plasma Clyoppt. Collagen TP inTBS lCa2+] GelTimes (mL) (mL) (mL) (mL) (mM) (seconds) 0.5 0 0 1.0 0 no gel 0.5 0 0 1.0 5 30 0.25 0.25 0 1.0 5 20 0.25 0.25 0.5 1.0 5 15 These results show that the reference plasma did not gel upon the addition of TPalone. When the calcium ion concentration was raised to S mM, gelation occurred at 30 seconds. The addition of more fibrinogen and Factor XIII as well as collagen increased the gelation rate of the sealant.

li'XAl\IPT.~, II
In Vitro Stability of Differen Compositions To assess the in vitro stability of ~ lions cont~ining thromboplastin, the protocol as outlined above was followed, except forrnulations were allowed to incubate for 1 hour with Ca2+ at 2.0 mM, then 0.2 mL of 40 mM CaCl2 solution were added (net ~Ca2+]= 4.6 mM), and the results are shown in Table V.

TABLE V

Plasma Cryoppt Collagen TP in TBS Gel Time (mL) (mL) (mL) (mL) (seconds) 0.5 0 0 1.0 25 0.25 0.25 0 1.0 25 0.25 0.25 0.5 1 .0 1 5 These results show that gelation times decreased from 25 to 15 seconds in the presence of collagen, and that the mz~terizil did not gel prematurely at a low Ca concentration in less than one hour.
.

SllBSTlTUTE SHEET (RULE 26) WO 97/29792 PCT/USg7/02614 ~X~lVlP~,h' II
Efficacy of the Tissue Sealant as a Hemostatic Agent The suitability of the single-component tissue sealant as a hemostatic agent wasevaluated in a standardized rodent liver incis;on 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.
Once hemorrh~gin~ was established, 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 cont~ining thrombin was also tested.

TABLE VI

Composition Time to Hemostasis (seconds) Prior Art Fibrin Sealant (fibrinogen, 60 mg/rnL; thrombin, 15 200 U/mL; and CaCl2, 40 mM) Prior Art Two Component Tissue Adhesive (thrombin, 20010 U/mL, fibrinogen, 30 mg/mL, collagen 20 mg/mL, and CaCl2, 40 mM. See U.S. Pat. No. 5,290,552) Single Component Sealant (as describd in the first example 12 given in Table V) As shown, the single component sealant is nearly as effective as the two component tissue adhesive, and is more effective than the prior art fibrin sealant.

Throughout this application, various disclosures, including published patent applications, issued patents, journal articles and textbooks, are referenced. The disclosures of these materials are incorporated by reference into this application, as if each reference SUBSTITUTE SHEET (RULE 26) were individually indicated to be incorporated by reference, to more fully describe the state of the art to which this invention pertains.

As is ~ Lell~ to one of on;li~ skill in the art, many changes and modifications can be made to the above embodiments without departing from the spirit and scope of the following claims.

SUBSTITUTE SHEET (RULE 26)

Claims (13)

What is claimed is:

1. A single-component tissue sealant composition comprising thromboplastin and fibrinogen.

2. The composition according to claim 1 further comprising Ca2+ ions at a concentration less than or equal to 3.0 mM.

3. The composition according to claim 2 further comprising collagen.

4. The composition according to claim 1 further comprising Factors II, V, VII, X and XIII.

5. The composition according to claim 1 further comprising plasma, wherein said plasma comprises Factors II, V, VII, X and XIII.

6. A dual-component tissue sealant composition comprising an admixture of:
(a) a first component comprising an effective amount of fibrinogen; and (b) a second component comprising an effective amount of thromboplastin.

7. The dual-component tissue sealant composition according to claim 6 wherein:
(a) said first component further comprises Ca2+ in an amount greater than or equal to 5 mM; and (b) said second component further comprises Factors II, V, VII, X and XIII.

8. A method for promoting tissue adhesion and/or hemostasis at a local tissue site in an animal comprising administering a single-component tissue sealant composition to said site, wherein said tissue sealant composition comprises thromboplastin and fibrinogen.

9. The method according to claim 8, wherein said tissue sealant composition further comprises Ca2+ ions in a concentration less than or equal to 3 mM.

10. The method according to claim 9, wherein said tissue sealant composition further comprises collagen.

11. The method according to claim 8, wherein said tissue sealant composition further comprises Factors II, V, VII, X and XIII.

12. The method according to claim 8, wherein said tissue sealant composition further comprises plasma, wherein said plasma comprises Factors II, V, VII, X and XIII.

13. A method for promoting tissue adhesion and/or hemostasis at a local tissue site in an animal comprising administering a dual-component tissue sealant composition to said site, wherein said dual-component tissue sealant composition comprises an admixture of:
(a) a first component comprising fibrinogen; and (b) a second component comprising thromboplastin.