CA2162997A1 - Topical fibrinogen complex - Google Patents

Abstract

A composition which, upon reacting with thrombin, functions as a fibrin sealant and is characterized as being free of detectable levels of lipid enveloped virus activity, free of prothrombin complex and active thrombin, and contains noprotease inhibitors or other non-human proteins. Also, described is a method forproducing the composition

Description

, TOPICAL FIBRINOGEN COMPLEX

This ar~plic~lion is a continuation-in-part of Inl~r"atio"al Application PCT/US92/07493, which was filed as a continuation-in-part of U.S. Serial No. 755,156, filed Se~le,),ber 5, 1991, now abandoned.

BACK~;ROUND OF THE INVENTION

Field of The Invention The present invention relates to a fibrinogen composition and its method of preparation, wherein the composition can be used for wound closure in conjunction with thrombin and calcium.

Related Art The attempt to use fibrinogen to achieve topical i ,e" ,ostasis was investigated as far back as the early 20th Century when fibrinogen patches for hemostasis were used in cer~br~l surgery. Later, mixtures of plasma and ll ,n)l "l,in were used for skin grafting and intracavity injections in the therapy of tuberculosis. However, these early allelll,,~s had two major drawbacks: since the source of fibrinogen was plasma, the concentration of fibrinogen was low which resulted in a fibrin film of insufficient strength; and it was not possible to inhibit the normal physiologic process of fibrinolysis such that the fibrin film was degraded relatively quickly.

Prior attempts to develop an effective fibrin sealant have also been hampered by the fact that most of these preparations contain high levels - of plasminogen which required these compositions to additionally contain an anti-fibrinolytic agent in order to prevent premature degradation of the fibrin seal. Because anti-fibrinolytic agents are typically derived from a non-human source the possibility of a patient having an adverse reaction to such foreign proteins is significant, especially upon multiple exposure to these agents. Although Rose, et al., (U.S. 4,627,879) report the production of a fibrin adhesive which does not necessarily require the W O 95/25748 2 1 6 2 9 9 7 PCTrUS95/03451 presence of an anti-fibrinolytic additive, the composition disclosed in this reference does not deal with another major drawback of these prior fibrin sealing compositions, namely, the possible presence of i"rectious agents, such as He~ ilis B or Hl\~ in the plas",a. As a conseq.lence, the Rose reference requires that the co",positions described therein be derived from a single donor, in order to avoid the l,d"s",ission of infectious agents which might be associated with pooled plasma.

Thus, there is considerable need for a fibrin sealant which can be derived from pooled plasma and which is free of anti-fibrinolytic compounds, animal prole;ns, and infectious agents such as viruses. The present invention addlt:sses these needs by providing such cor~,lJosilions.

WO 95/25748 - . ` t, , PCTtUS95tO3451 .

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that pooled plasma, even when subst~ntially depleted of Factor Vlll, can be processed to prpduce a fibrinogen p,~pa,dlion which, when reacted with lhr~mL,in and calcium, will produce a fibrin sealant that can be used to promote he" losl~is.

In detail, the invention provides a fibrinogen composition which, in addition to being essentially free of Factor Vlll and plasminogen, does not require the use of an anti-fibrinolytic agent and has been treated to eliminate the presence of infectious agents, such as lipid enveloped viruses. A further advantage of the composition is that essentially all of the prolc"1s present in the composition are of human origin.

The composition of the invention, through its transient in vivo presence, provides a matrix which persists for a period of time sufficient to achieve a medical effect, essentially lacks host toxicity upon degradation, and provides mechanical strength to promote hemostasis.

WO 95/25748 ~ 2 ~ 6 2 9 9 7 PCT/US95/03451 BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 Schematic representation for prepa,~lion of Topical Fibrinogen Complex.

FIGURE 2 Effect of calcium ion concer,l,dlion on fibrin polymer formation.
Fibrinogen (90 mg total protein/ml) and Ihr~",L,i" (500 U/ml) were mixed and allowed to incubate for 10 minutes. Lane A: MW markers; Lane B:
Control fibrinogen; Lane C: OmM Ca++; Lane D: 1mM Ca++; Lane E:
3mM Ca++; Lane F: 6mM Ca++; Lane G: 10mM Ca++; Lane H: 20mM
Ca++; Lane l: 30mM Ca++; Lane J: MW markers.

FIGURE 3 Effect of calcium ion concel~ lion on fibrin polymer formation.
Fibrinogen (130 mg total protein/ml) and ll ,ro" Ibil~ (500 U/ml) were mixed and allowed to incllbate for 10 minutes. Lane A: MW markers; Lane B:
Control fibrinogen; Lane C: 0 mM Ca++; Lane D: 1mM Ca++; Lane E:
3mM Ca++; Lane F: 6mM Ca++; Lane G: 10mM Ca++; Lane H: 20mM
Ca++; Lane l: 30mM Ca++; Lane J: MW markers.

FIGURE 4 Rate of fibrin polymeri~alion. Fibrinogen (130 mg total protein/ml) and thrombin (500 U/ml) were mixed in the presence of calcium ion (40mM CaCI2). Lane A: MW markers; Lane B: 0 min; Lane C:
1 min; Lane D: 3 min; Lane E: 5 min; Lane F: 10 min; Lane G: 30 min;
Lane H: 60 min; Lane l: 2 hr; Lane J: 4 hr; Lane K: 8 hr; Lane L: 24 hr;
Lane M: fibrinogen control; Lane N: MW markers.

woss/2s74s ; 2 1 62 9 9 7 PCT/US95/03451 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The inventors have devised compositions which, when reacted with thrombin, will produce a fibrin sealant that represents a significant improvement over the prior art CGlllpOSitiGllS inle"de~ to accGnl,~lisll this effect. These ColllposiliGIls (collectively, "Topical FiL,ri,-Ggen Complex (TFC)"), are advdnl~geolJs because, like prior art cG""~osili~ns, they will effectively induce hemostasis in human tissue but, unlike prior art compositions, can do so without risk of causing an immunologically adverse response in the treated person to non-human proteins and without substantial risk of viral infection.

r,efer,~d blood fractions for producing the compositions of the invention are plasma, cryopreci,citdle, and/or Factor Vlll-depleted cold-precipitate.
Because the pre~er,~d blood fraction for use as a sldllil19 material is human plasma, the starting ",alerial will her~a~ler be referred to as plasma, although it will be understood by those of skill in the art that the compositions of the invention can be produced by sla,li"g with any human blood-derived fraction which has not been significantly depleted of fibrinogen. Generally, the process involves the formation of a cryoprecipi-tate from plasma which is high in Factor Xlll (F Xlll) and fibrinogen. This step may (and pr~feral,ly will) be followed by cold-precipitation of pr~tei"s from the cryoprec;pi1ale. The product of the cold-precipitation process, if any, (i.e, the cold-p,~c;,.~ilale), will typically CGI ,lain a high concentration of fibrinogen and very low levels of F Vlll.

More specifically, the ,urt:~r,~d method for producing the TFC
compositions of the present invention uses frozen human plasma from one or more donors as a starting material. Brefeldbly, the plasma to be used will have been screened using con\,~nliol ~al assay techniques for the presence of infectious viral cGnldl~;.1ants, such as hepalilis B and human immunodeficiency virus (HIV) to eliminate plasma for use as a starting material which CGnlai"S detectable levels of such cGI ~Idminants.

Whether or not the plasma has been previously screened, the cryop-~c;pilale or cold-prec;,~ilale product of the plasma to be used in the compositions of the invention will be treated as further described below to reduce the viral activity therein to undetectable levels. For purposes of this disclosure, the phrase "undetectable levels" will refer to levels of viral activity which can be detected by viral assay protocols which are well known to those of ordinary skill in the art, such as delectiG" of plaque forming units in infected tissue or cells (~e, e.g., Example 5). A redlJction in the known viral activity level in a given composition will be conventionally desc, il~ed herein as the "log,0 redlJction factor". Generally, using the available techniques for reducing viral activity in a protein containing composition which are known in the art and/or described herein (~e, e.g., Example 5), it can be expected that the compositions of the inventions will have a log,0 reduction factor for lipid enveloped viruses of at least 4 logs (pr~ferably at least 6 logs) and a lesser reduction factor for other viral pathogens.

AS described in Exal l l~!e 1, to process the frozen plasma to a cryoprec;~ e, the plasma is thawed in a controlled environment. The resulting cryoprecipitate may be further processed to TFC without first being converted to a cold-precipitate. However, to limit the total protein con~enl of the compositions of the invention to fibrinogen, residu~l amounts of fibronectin and any added protein components (such as albumin), the cryoprecipitate will ~.referdbly be further processed to a cold-precipit~te.

To that end, a cryoprecipitate can be produced by freezing the blood fraction (for example, plasma) which is thered~ler warmed to a temperature not exceeding about +6C. The cryoprecipitate is dissolved in distilled water at about 20C-35C. Calcium chloride is added at a conce"L,dle from about 1,uM to about 1000 IlM. rreferdbly, calcium chloride is added at this step at a concen~rdtion of about 40 ~lM and the pH adjusted to 6.8 + 0.3 to enhance the precipitation of fibrinogen.

The dissolved cryoprecipitate is then cooled to about 10C with mixing, whereupon the cold-precipitate forms. The precipitate is removed from solution by centrifugation at, for example, 5600 9 to 7200 9. The precipitate may be stored at -60C or lower if desired. Where cold-WO 95/25748 . . ' ~ 2 1 6 2 9 9 7 PCTtUS95/03451 pr~c;,~ dlion is used, the addition of a calcium ion source during the process will enhance the preci,c ildlion of fibrinogen, as well as fibronectin, thereby inc,easin~ the co,1cel,lralion of these subsL~nces in the cold-,ur~-,;pi1~le. The fibrinogen can also be further concenl,~dle-~ by the acldilion of polyethylene glycol (PEG) to the cold-prec;pita~e.

The cryoprec;pilale or cold-prec;pilale will usually be dissol~d in buffer with mixing and the pH of the cold-prec;pitdle adjusted to about 7.2, preferably to 7.2 + 0.1. To prevent interaction between the fibrinogen of the cryoprecipitate or cold-precipitate and any lhlol~lbil) which may be present therein, the cryo- or cold-precipitate will ,urererdbly be resuspen-ded in the presence of a protease inhibitor, such as PPACK (D-Phe-L-Pro-L-Arg-chloromethylketone), heparin cofactor ll, hirudin or anti-thrombin lll (ATIII) to inhibit lhr~n,bin which may be pr~sen~. Most pre~n~d as thrombin inhibitor is PPACK at a concenl,dlion from about 0.75 ~M to about 1.75 ~M. The product of a composition of the invention which has been treated with an effective amount of a thrombin inhibitor will be considered to be essentially free of active lhlo~ . If used, the thrombin inhibitor will be removed from the compositions of the invention before Iyoph~ lio,) by, ~r~ferdbly, PEG precipitation and, optionally, also the DEAE column step of the procedure ~e, FIGURE 1 and Example 1).
Those of skill in the art will know of other protease inlliLJitol~ of thrombin suitable for use in the compositions of the invention and their effective concenl~lions.

To remove any ,c,roll,r~mbin complex which may be present in the cryo-or cold-precipitate, the precipitate suspension is ~ns~r~d to a buffer solution containing a salt such as tri-calcium phosphate. Exposure to the salt-con~ lil lg buffer will minimize the likelihood of ,uroll ,rombin conversion to thrombin which, if such a reaction were to occur, could lead to the conversion of fibrinogen to fibrin. In this state, the resulting composition can be considered to be essentially free of prolhrumbin complex. The calcium phosphate, in turn, is removed from the process by centri~ugation and/or filtration. Additional techniques for removal of plulhr~ bin are described by Murano (Prothrombin and Other Vitamin K
Proteins, Vol. Il, Seegers and Walz, eds., CRC Press, Inc. Boca Raton, Fl., WO 95125748 ' 2 1 6 2 9 9 7 PCT/US95103451 1986); Heystek, et a/., (V~ Sang., 25:113, 1973); Banowcliffe, et al., (V~
Sang., 25:426, 1973); Chandra, et a/., (V~K Sang., 41:257, 1981); and Chanas, et a/., (US. 4,465,623), incor~.ordled herein by reference. Thus, by removing prull,rur,,bi,, and inhibiting Ihru,,lL,in in the compositions of the invention, the final compositions will be essentially free of fibrin molecules; i.e., all of the fibrinogen therein will be unreacted until se,ua,~dlely ex~ose.l to thrombin (~Jrt:rerdbly in vivo) to produce a fibrin sealanl.

In a pre~er,~d embodiment, the dissolved cryo- or cold-prec;pil~te is warmed to about 23-27C and contacted with a Iysine affinity column, such as the matrix column product sold commercially under the tradename Iysine-Sepharose 4B. Residu~l plasminogen present in the cryo- or cold-precipitate will be adsorbed by the matrix while fibrinogen will not, thus rendering the resulting solution essentially plasminogen free.
For example, using the Iysine-Sepharose ",alerial referred to above, the resulting final TFC composition will contain no more than 10~9 plasminogen/milliliter of TFC.

The removal of plas"linogen is important for two reasons. First, plasi" ,i, logen, when converted to plasmin, will break down ~ibri,1ogen and fibrin molecules. The latter are formed from interaction between fibrinogen and thrombin in the fibrin sealant to be produced from the compositions of the invention. Second, although fairly effective plasminogen inhibitors are known in the art, their use requires additional manufacturing steps (to deter" ,ine the concer,l,clio,1 of plasminogen ,uresenl and concenlralion of inhibitor needed). More importantly, presently commercially available plasminogen inhibitors are of non-human origin and therefore pose the risk of immunologically adverse reactions to composilions which contain them, particularly on repeated ~I,plic~lions thereof. This risk is avoided by removal of plasminogen from the compositions of the invention.

The solution collected from the Iysine matrix will preferably be concel,l,~led by, for example, the addition of polyethylene glycol (PEG).
For use in humans, the PEG used to concenl~le the eluate will have a molecular weight in a range which is not toxic to humans (e.g., 3000-WO 9512S748 2 ~ 6 2 9 q 7 PCT/US95/03451 .
g 6000) and will be added to a final conce~ ,l,dlion from about 3% to about 7%, pr~ferdl)ly about 4% (w/w). Most ,c,ererdLly the resulting PEG
pl~c;pil~e will be dissol~d in a buffer solution, filtered and the pH thereof adjusted to an alkaline level; e.g., about 8.6, ,~r~terdbly 8.6 + 0.1. In an alternate embodiment of the invention, it is also possible to perform the PEG prec;pil~lion step prior to adsor~lion with the Iysine matrix.

In all embodiments, the corrlpositisns (pr~r~rdbly the concentrate solution) will be Irt:aled with an effective amount of a viral activity reducingagent, such as a detergent, which, typically, acts by disrupting the lipid envelope of such viruses as Hepatitis B, Hl\! and HTL\C The term "effective amount of viral activity reducing" agent means that the conce~lr~lio,1 of viral activity reducing agent added to the composition is sufficient to reduce the viral activity in the compositions of the invention to undetectable levels. Of course, the conce"l,dlion of viral activity reducing agent should not significantly inhibit the ability of the composition to form a fibrin sealant in the presence of thrombin; i.e., the viral activity reducing agent used will not denature fibrinogen.

To some extent, viral contaminants may be removed from the compositions of the invention by virtue of process steps which do not involve the addition of a viral activity reducing agent to the composition.
Because viral activity reducing agents typically affect only lipid enveloped viruses (by disrupting the inl~y, ily of the lipid envelope), the process steps will likely be the principal means given the current state of the art by which any viruses present which lack a lipid envelope will be removed from the compositions of the invention. Such process steps will be known to, or can readily be ascertained by, one of ordinary skill in the art and include viral partitioning and adsorption/filtration with calcium phosphate.

Nondenaturing detergents which are useful as such agents can be selected by one of ordinary skill in the art from such recognized groups as anionic, cationic, and non-ionic detergents. Examples include sulfated WO 9S125748 2 ~ 6 2 9 9 7 PCT/US95/03451 alcohols and sodium acid salts such as sulfated oxyethylated alkylphenol (sold co",r"e,~ially under the tradenames "Triton W-30" and "Triton X-100") sodium dodecylbe"~e"sulrul,dle (sold col"",er~ially under the tradename "Nacconol NR") sodium 2-sulfoethyl oleate (sold co""~,er~;ally underthetradename "Igepon A') sodium cholale sodium deoxycholate sodium dodecylsulfo"ale dodecyldimethylbenzylan "~ ,onium chloride (sold col"",ercially under the tradename "Triton K-60"), oxyethylated amines (sold commercially under the tradename "Ethomeen") N-dodecylamino-ethanesulfonic acid ethylene oxide-propylene oxide conde"saLes ("Pluronic" copolymers) polyoxyethylated derivatives of esters (sold commercially under the tradenames "Tween 80" and "Polysorbate 80") polyoxyethylene fatty alcohol ethers (sold commercially under the tradenames "Br3 35") tetramethylsutylphenyl ethers of polyethylene glycols (sold commercially as "octoxynols") as well as detergents sold commercially under the tradenames "nonidet P-40" and "Lubrox PX".
Those of skill in the art will know of other suitable nonionic detergents for use as all or part of the viral activity reducing agent and may wish to refer for examples to U.S. Patent Nos. 4481189 4 540 573 4 591 505, 4314997 and 4315919.

In the preferred embodiment the viral activity reducing agent will consist of an organic solvent ,urt:fer~bly tri-n-butyl phosphate (TNBP) mixed with nonionic delerye"ls ,c,r~f~rdL,ly polysorbate 80 and octoxynol 9. As desc, iL,ed in Examples 1 and 5 undenatured compositio"s of the invention in which viral activity is at undeteclable levels can be produced using a ~ fer,~d viral activity reducing agent co",~rised of TNBP
(concentration 0.03-0.3%), polysorbate 80 (conce,lt,~lion 0.03-0.3%) and octoxynol 9 (concent,alion 0.1-1.0%). In addition chaotropic agents may also be utilized to inactivate viruses providing the agent does not denature fibrinogen.

Alternatively the concenlrclion of organic solvent and detergent used in the practice of the prefel,t:d embodiments of the invention can vary depending upon the composition to be treated and upon the solvent or delergenl selected. The alkyl phosphates can be used in concenl-alions from about 0.10 mg/ml of mixture treated to 1.0 mg/ml pr~ferdbly WO95125748 ; ` ` 2 1 62 9 97 PCT/US95/034~1 between about 0.1 mg/ml to about 10 mg/ml. The amount of detergent or wetting agent utilized is not crucial since its function is to improve the conlact between the organic solvent and the virus. For most of the nonionic " ,ale, ials which are useful, the wetting agent can vary from about 0.001% to 10%"ur~erdbly from about 0.01% to about 2% of the aqueous mixture, depending upon the amount of fatty malerial in the treated aqueous mixture.

Whatever viral activity reducing agent is used, it will be removed after it is conlacLed with the compositions of the invention for a period of time sufficient to reduce the viral activity in the composition to undetectable levels (at least one minute). In the preferred embodiment, DEAE
diethylaminoethyl cellulose (sold commercially under the tradename "DE
52") is the matrix utilized for the removal of the solvent/detergent from the fibrinogen composition. The fibrinogen binds to the diethylaminoethyl cellulose and, after thorough washing to remove unbound material and deleryenl~ is eluted with, for example, 0.3M NaCI. Other ion exchange male,ials which can be utilized for removal of the solvent/detergent include virtually any of the commercially avn ;~ 'Q anion e;cchange mal, ices including, but not limited to, cellulose and agarose ~al~ices. The specific parameters for binding and eluting from these various ion exchange r"alerials are known to those of skill in the art, or can be readily asce, lai, led without undue experimentation.

The stability of the TFC co"".osilions of the invention may be enhanced through the use of such excipienls as human serum albumin (HSA), hydroxyethyl starch, deAl,an, or combinations thereof. The solubility of the compositions may also be enhanced by the addition of a nondenaturing nonionic detergent, such as polysorbate 80. Suitable concer,L,dLions of these compounds for use in the compositions of the invention will be known to those of skill in the art, or can be readily ascertained without undue experimentation. The compositions of the invention are, however, sufficiently stable to be stored and used without the use of a st~hi~i~er. Thus, to minimize the non-fibrinogen protein content of the compositions, the most ,ur~er,ed embodiment of the compositions will either contain no added stabilizer or will contain a nGnprc"ei. ,aceous stabilizer.

Typically, after formulation the bulk is concel,l,dted from about 20% to about 50% of its original eluate volume, then diluted to the pre-conce,lt,dlion eluate volume. The bulk may then be conc6"l,aled to a final total protein cGncenl~dlion of about 4g + 1g/dL CGIl~positio~, (w/v) before sterile ~.,ucessi,lg and Iyophilization. As noted above, a pre:rer,~d composition of the invention is one which, when r~consli1, ~ed, will CGn5i51 essentially of fibrinogen; i.e., the ,cr~teins in the composition will be fibrinogen, no more than a residual amount of fibronectin (i.e., 20 mg/ml or less, ~r~fe,dbly 10 ~lg/ml or less), no more than a residual amount of plasminogen (no more than 10 ~g/ml"ur~rerdbly no more than 5 ~g/ml) and from about 1 to 40 Units/ml of Factor Xlll (,cr~fer~L,ly more than 10 Units/ml). However, in aller"alive embodiments of the invention, the composition may also contain components such as a protein stabilizer;
e.g., human serum albumin. Thus, the fibrinogen component of the composition may comprise from about 50% to 100% of the total protein in a TFC composition of the invention (w/v), and ,ult:rerdbly will comprise at least 75% of the composition (w/v).

In the prt:rerlt:d embodiment, concenl,dliGn of the protein(s) in the composition is accomplished by ullldrillldlion using a mell,l)rdl1e with a molecular weight exclusion large enough to allow NaCI to be removed, but small enough to retain protein molecules. This filtration is most pr~ferdbly performed using a mel"brd,1e with a 30,000 MW exclusion. When the TFC c~m~osition of the invention is Iyopl~ ed, the pre-lyophili~aliG"
voiume is usually y,~a~er than the volume to which the Iyophilizate is resuspended at time of use.

If desired, the compositions of the invention can be modified to include non-proteinaceous as well as proteinaceous drugs. The term "non-proteinaceous drugs" encompasses compounds which are cl~-ssically referred to as drugs, such as mitomycin C, daunorubicin, and vinblastine, as well as antibiotics.

WO 95/25748 ` .: ` 2 1 6 2 9 9 7 PCT/US95/03451 The proteinaceous drugs which can be added to the fibrinogen comrositions of the invention include immunomo~ tors and other bicl-g;c-' r~spOnSe modifiers. The term "biological ,~spo"se modifiers"
is meant to enco",,uass suL~slances which are involved in modifying a - 5 biological response, such as the immune ,t:s,uo"se or tissue growth and repair, in a manner which enhances a particular lesi, ~.1 therapeutic effect, for example, the cytolysis of bacterial cells or the growth of epidermal cells. Examples of response modifiers include such compounds as Iymphokines. Examples of Iymphokines include tumor necrosis factor, the interleukins, Iymphotoxin, macrophage activating factors, mig,dlion inhibition factor, colony stimulating factors, and the inlel rer~ns. In addition, peptide or polysaccharide fragments derived from these proteinaceous drugs, or independently produced, can also be incorporated into the fibrinogen compositions of the invention. Those of skill in the art will know, or can readily ascertain, other substances which can act as proteinaceous or non-prulei, ,aceous drugs.

The compositions of the invention can also be modified to incorporate a diagnostic agent, such as a radiop~-llJe agent. The presence of such agents allow the physician to monitor the progression of wound healing occurring internally, such as at the liver, gall bladder, urinary tract, bronchi, lungs, heart, blood vessels, and spinal canal. Such compounds include barium sulfate as well as various organic compounds containing iodine. Examples of these latter compounds include iocetamic acid, iodipamide, iodoxamate meglumine, iopanoic acid, as well as dial,i~oate derivatives, such as dial,i~oate sodium. Other contrast agents which can be utilized in the compositions of the invention can be readily asce, lail1ed by those of skill in the art.

The concer,L,dlion of drug or diagnostic agent in the composition will vary with the nature of the compound, its physiological role, and desired therapeutic or diagnostic effect. The term "therapeutically effective amount" means that the therapeutic agent is present in a sufficient concenlfdlion to minimize toxicity, but display the desired effect. Thus, for example, the co"ce"l,~dlion of an antibiotic used in providing a cytolytic therapeutic effect will likely be clif~t:r~nl from the concenl,dlion of an immune r~spo"se modulator where the therapeutic effect is to stimulate the ,ur~lif~,dli~n of immune cells at the site of application of the fibrinogen complex. The term "diagnostically effective amount" denotes that co"cenl,dlion of diagnostic agent which is effective in allowing the fibrin glue to be n,o"ito,ed, while minimizing pote,ltial toxicity. In any event, the c~esi,~d conce~lt~dlioll in a particular instance for a particular compound is readily ascel lainable by one of skill in the art.

The above ~iscloslJre generally describes the pr~:senl invention. A further unde,~lan.ling can be obtained by reference to the following specific exar"ples which are provided herein for purposes of illusLIdliGn only, and are not intended to be limiting unless otherwise specified.

WO 9S/25748 ~` 2 ~ 6 2 9 9 7 PCT/US9S/034Sl PREPARATION OF TOPICAL FIBRINOGEN COMPLEX

Topical fibrinogen complex (TFC) was pror~uce~ by the initial ~ r~lJa~lio"
of a cr~ Jr~c;~;t~e of plasma. The cryoprec;pilate for such use was prepared by two difrer~"l techniques depending upon the physical form of the plasma.

In one technique, sealed plastic bottles of frozen plasma were thawed in a controlled env;,~"ment by contact with a heat exc~ange medium, such as air or water. The thaw was col ,I,."ed by programming the temperature and flow of the heat-exchange medium so that the maximum te",,uerdlure of the plas",a did not exceed +6C. The containers were then opened and the contents pooled into a jacketed stainless steel thawing tank. In the thawing tank, the plasma was gently warmed (while being mixed) to melt the remaining ice. The thawed plasma was then pumped directly to a centrifuge or into a jacketed stainless steel holding tank where it was maintained at 2.5C + 3.5C. The plasma was centrifuged to remove the cryoprecipitate. The cryoprecipit~te, so pr~par~d, may be stored at or below -25C or immediately processed to antihemophilic factor. The cryo-poor plasma was collected in a jacketed -~lain'~ss steel reaction tank.

Alternatively, cryoprec;~,ilale was prepared by placing sealed plastic bags of frozen ~.lasma in a liquid nitrogen bath for several seconds. The bags were removed from the bath and the crisp, cracked bags were stripped from the plasma. The ,ulasma was then placed into a jacketed stainless steel thawing tank. Alteratively, sealed plastic bags of frozen plasma were arranged so as to warm the bags so that the frozen plasma would break away from the plastic. The containers were then opened and the contents pooled into a jacketed stainless steel thawing tank. In the thawing tank the plasma was gently warmed, while being mixed, to melt the remaining ice. The thawed plasma was pumped directly to a centrifuge or into a stainless steel holding tank where it was maintained at 2.5C + 3.5C.
The plasma was centrifuged to remove the cryoprecipitate. The cryoprecipitate, so prepared, may be stored at or below -25C, or immediately be processed to anlihe" lophilic factor. The cryo-poor plasl "a was collected in a jacketed stainless steel reactioi1 tank.

After the cryoprec;pil~e was prepared, it was dissol~d in distilled water at 20C to 35C. This part of the prolocol is illustrated schematically in FIGURE 1. Sumcient calcium chloride was added to obtain a minimum calcium conce, Itldliol ~ of about 40 ~M and the pH adjusted to 6.8 + 0.3.
This solution was cooled to 10C + 2C while mixing. The prec~ le which forms was removed by centrifugation (56009-72009). The prt:c;~.ilaLe may be stored at or below -60C or processed directly to TFC.
The precipitate was then suspended in Process Solution I at a ratio of four liters of Process Solution I per kg of prec;pil~le. Process Solution I
comprises: (a) 0.5M glycine, 0.5M sodium chloride, and 0.1M sodium citrate; pH ~djusted to 7.2 + 0.1 with NaOH, (b) protease inhibitor: 0.75-1.75 ~M PPACK (D-phe-L-pro-L-arg-chloromethyl ketone) or equivalent, and (c) 0.6 + 0.1 U/ml heparin. The temperature was adjusted to 24-32C and the suspension stirred for approximately one hour.

After the precipitate was suspended in Process Solution 1, the suspension was l,~ns~er,ed into a tank containing 200 1 of process Solution ll a 10-15C and stirred for at least 30 minutes. Process Solution ll co~,prises: 7 + 1 mM sodium phosphate monobasic monohydrate, 18 + 2 mM sodium phosphate dibasic heptahydrate, calcium phosphate Il ibasic 0.25% (w/v). The suspension was then allowed to settle undisturbed for at least 30 minutes. The suspension may be centrifuged (56009-72009) at this step to remove some precipitate. The suspension was then clarified by filtration first through a 0.45~ filter, then through a filter of at least 0.2~1 in pore size.

The filtrate was warmed to 23-27C and applied to a Lysine Sepharose 4B
column or equivalent. The gel was packed in a chromatography column and equilibrated with 5 column volumes of Process Solution lll (25 mM
phosphate buffer: 7 + 1 mM sodium phosphate monobasic monohydrate, 18 + 2 mM sodium phosphate dibasic heptahydrate).

WO 95125748 . - : ` 2 1 6 2 9 9 7 PCT/US95/03451 The unbound ",alerial was collected in a tank. The column was washed with at least two gel volumes of Process Solution lll. The unbound f~d~ions were pooled. Next, the temperature of the bulk containing the pooled unbound ~,actions was adjusted to 14 + 4C. Polyethylene glycol 3350 was added to a final concer,L,dlion of 4% (w/w). The suspension was mixed for at least 30 minutes and the pr~zc;f il~le removed by centrifugation (87009) at 10-18C. The resulting PEG ~r~c;r.ilale was dissolved in Process Solution IV (39 mM tris~ hos~l ~dle with pH adjusted to 8.6 + 0.1 with phosphoric acid) approximately 15 I/kg precipitate. The protein conce"l,~lion of the suspension was adjusted to 0.6 + 0.2 9%
(w/v), then the suspension was clarified by filtration. A mixture of Triton X-100, tri (N-butyl) phosphate (TNBP) and Polysorbate-80 was added to the solution to a final concentration of 1.0% (v/v), 0.3% (v/v), and 0.3%
(v/v), respectively. The protein-detergent solution was mixed for 1 hour.

The bulk solution was then applied to a c~"on,alography column conlaining DE 52 ion exchange cellulose resin or equivalent. The resin was packed in a chromatography column and regenerated with 3 column volumes of 1.0M NaCI, then 3 column volumes of 0.5M HCI, 3 column volumes of 0.9% saline, and then 3 column volumes of 0.5M NaOH, and equilibrated with 3 column volumes of 0.5M tris-phosphate buffer (pH
adjusted to 8.6), and 3 column volumes of Process Solution IV.

The ion exchange column was then washed with a minimum of 20 column volumes of Process Solution V (0.02M histidine, 0.01 M NaCI, pH adjusted to 7.0 + 0.1 with 6N HCI). The fibrinogen was eluted with Process Solution Vl (0.02M histidine, 0.3M NaCI, pH adjusted to 7.0 + 0.1 with 6N
HCI).

The fibrinogen was then supplemented with human serum albumin (5%
or 25% human albumin released for therapeutic use) to a concentration of about 80 mg of albumin or less per gram of protein. Polysorbate-80 was added to a final concentration of 15 mg per gram of protein.

W095/25748 2 1 6 2 9 9 7 PCT/US95tO3451 Finally, the fibrinogen was co"cet,lrdled to about 25% of the original volume by ult~ ldtion using a 30,000 MW cut-off ,,e,,~Lrcl, ,e, then diluted to its preconcenl,~lion volume with Process Solution Vll (0.02M histidine, pH ~d3usterl to 7.0 + 0.1). The bulk was again concer,l,dled by ultl~fill,alion to achieve a final protein conce"l,dlio" of 4 + 1 9% (w/v).
The bulk was sterile filtered (0.2~), aseptically filled into sterile final containers, Iyophilized under aseptic cor,clilions, and closed with sterile closures.

TFC in vftro STUDIES

A. Clotting Evaluation Studies were done to determine the TFC composition which will produce fast clotting in the presence of thrombin (i.e., on formation of a fibrin sealant (FS)). Fast cl~lil ,y was arbil,drily defined as 1-2 seconds.
Shorter time periods generally result in clotting within the delivery device and longer times lead to a loose mixture of the components that flows with ill-defined direction.

In performing these experiments, a clean pyrex glass plate was positioned at ~30 from the l,ori~onlal axis. A 2" line was drawn on the underside to define the area of FS application.

Human fibrinogen was tested at 50-130 mg/ml total protein with bovine ll"on,bin (Armour Pharmaceutical Co.) at 100-1000 NIH U/ml without Ca+ +. For the purpose of this study the concentration of fibrinogen in the TFC composition was considered to equal the value of total proteins, although it is likely that a residual amount of fibronectin was also present.
The effect of adding [Ca++] to a fibrin sealant (FS, TFC, lhrur,,bin and calcium salt) was studied at 10, 20, 40, and 60 mM.

The FS was delivered using an experimental dual syringe device (Fenwal) following the 2" line described above, starting at the upper end and 3û moving downward.

W O 95/25748 : . ` ' ;- 2 1 6 2 9 9 7 p~lr/lJSg5l0345l Table 1 summarizes the data obtained by testing fibrinogen at 50-130 mg/ml with II,ru,nL,in at 100-1000 NIH U/ml in the absence of calcium.
- Each data point is the average of four determinations.

AV E R A G E C L~JI IIN G TIM E S IN S t~Ol~D S ~ S D) Tl,,u,,,b n in NIH U / m l Total PrDtein ( m g/ ml) 100 250 500 1000 5.3+ 1.3 2.5+ 0.4 2.3+ 0.41.2(N = 1) 3. a 0.4 2.4_ 0.4 1.9~ 0.41.4(N = 1) 110 5.3+ 1.6 2.2+ 0.5 1.6~ 0.1 *
130 3.6_ 0.6 1.8+ 0.2 0.8(N = 1 ) *

* Not Determined - clotting occurred too fast for time measurement.
~N~ refers to the mean of the 4 m easu,e",erlt~ made) As Table 1 shows clulling times at the lower thrombin concel ,l,dlion (e.g.
100 NIH U/ml) and low protein content (e.g. 50 mg/ml) were long. The mixture was also observed to be runny. Higher conce"lr~lions of ll,ru,,,bin (e.g. 1000 NIH U/ml) generally clotted within the delivery device and therefore were considered unsuitable. Addition of CaCI2 improved the appearance of the clot and generally shortened the clolli"g time.

Table 2 shows the effect of [CA++] in the range of 0-60 mM. CaCI2 solution was used to reco~slilute the thrombin so the final [Ca++] in the 1:1 mixture of FS is half that reported in the Table.

WO 95/25748 . - 2 1 6 2 9 9 7 PCT/US95/03451 EFFECT OF lCa++] ON TIME TO CLOT
Thrombin in NIH U/ml Total Prot~i., mg/ml 100 250 500 1. [Protein]=50 mg/ml [calcium ion]
0 mM 4.3+0.7 2.6+0.2 2.0+0.6 10 mM 2.4+0.3 1.8+0.2 *
20 mM 2.3+0.2 1.2+0.5 *
40 mM 2.6+0.2 * *
60 mM 3.0_0.5 * *
2. [Protein]=90 mg/ml [calcium ion]
0 mM 4.1 +1.1 2.2+0.2 1.8_0.3 10 mM 2.9+0.3 2 8+0 3 *
20 mM 2.0+0.4 2 6+01 *
40 mM 2.5+0.2 1.9_0.1 *
60 mM 3.2+0.1 * *
3. [Protein] = 110 mg/ml [calcium ion]
0 mM 4.8_1.8 2.4+0.5 1.7+0.3 10 mM 2.9_0.4 2.4+1.2 *
20 mM 2.6+0.2 * *
40 mM 2.6+0.7 * *

4. [Protein]=130 mg/ml [calcium ion]
0 mM 3.1 +0.8 1.5+0.1 *

* Not Determined - clotting occurred too fast for time measurement [Protein] refers to the conce"L,~lion of total protein in mg/ml TFC.
Based on the above results it was concluded that a protein range of 90-130 mg/ml and a Ll"~o",bin concentration of 250-500 NIH U/ml are appropriate for further studies. It was aiso apparent that additional WO 95125748 : ; 2 1 6 2 9 9 7 PCT/US95103451 calcium ion was needed to enhance the clot. Consequently, Ca++
co"ce"l,dlion was incl~Jded as a variable in later c\,~lu~tions.

B. Rate of Cross-Linking These studies focused on deler,~ ing the role of Ca ions and the effect of [Ca+ +] on the extent of fibrin polymeri~aLion as well as the rate of cross linking with time due to fibrin polymerization.

The cross-linking reaction of fibrin was tested in a system under reduced conditions which utilized SDS/PAGE. The resolving gels at 7.5% and the stacking gels at 3.75% were cast as described by Schwartz, et al., (Journal of Clinical Investigation, 50: 1506, 1971).

Thrombin (Armour Pharmaceutical) was reconstituted with or without CaCI2 solution at the desired molarity, i.e., 0, 2, 6, 12, 20, 40, or 60 mM.
Fibrinogen was reconstituted with water, quickly mixed with the thrombin in a 12 x 75 mm test tube and sampled at the appropriate time periods for the studies. The clots were rinsed with 0.15M NaCI then dissolved in three times the clot volume of 9M urea co,llaini.1g 3% SDS and 3%
13-mercaptoethanol by boiling in a water bath at 95 ~ 5C. The dissolved clot solutions were then stored at 5C until the gel electrophoresis was performed.

The effect of CaCI2 conce,1l,dlio,- was tested with thrombin at 500 NIH
U/ml and fibrinogen at 90 mg/ml and 130 mg/ml after 10 minutes clotting time. The effect of Ca++ conceut,dlion on the rate of disalJpearance of the y-band to form the y-y dimer is shown in FIGURES 2 and 3.

As illustrated in FIGURES 2 and 3, the presence of Ca+ + is necessary for complete fibrin polymerization. As calcium ion in the range of 20-60 mM
gives comparable results, a midpoint concentration of 40 mM was chosen to ensure optimal polymerization. Also, there was no si5y~ icanl difference in fibrin polymerization between the two fibrinogen concentrations (90 mg/ml and 130 mg/ml) when measured at the time point studied (10 minutes~.

WO 95/25748 ` 2 1 6 2 9 9 7 PCT/US95/0345l Four co",l)inaLions of Ll"o~"bin at 250 NIH U/ml or 500 NIH U/ml in 40 mM CaCI2 and ~ibri"oye" at 90 mg/ml or 130 mg/ml were tested to study the effect of varying the concenL,aLions of the co",ponenls. Samples were taken for gel electrophoresis after 10 minutes of clotting time. Gel electrophGr~sis of the four combi~aLiol~s of Ll,r~mL)in (in 40 mM CaCI2) and fibrinogen did not reflect any significant dif~r~"ces at the time point studied (10 minutes).

The time study was conducted and sampled over 24 hours. FIGURE 4 shows the fibrin polymerization reaction as it progresses through a 24 hour period. Formation of the y-y dimer occurs very rapidly in the presence of Ca++ (within one minute) as is shown in FIGURE 4. The polymer is not cleLecLal,le by this system until 10 minutes incubation time.
As the a polymer increases with increasing incubation time (up to 24 hours) the a monomer band shows a cor,~sponding decrease in inLel IsiLy.
To summarize the time study demonstrates that initial polyme, i~aLion (y ~y dimer) occurs almost insLanLa,1eously as the reactants are mixed with the a polymers forming more slowly. From this study it can be concluded that the presence of Ca ion is necessary for polymeri~aLio,1 and that the results are similar to those previously I~JOl Led in the literature (T. Seelich J.Head&NeckPathol.,3:65-69 1982;M.Schwartz etal. J.ClinicalInu, 50:1506-1513 1971).

C. Tensile St~

The tensile strength of the fibrin sealant was cv~luated by applying strain to the clot until rupture of the bulk material was observed and measuring the force needed in a tensile stress-strain system. In addition the change in rupture stress as a function of varying the components in the polymerization mixture used to produce the sealant was studied.

WO 95/25748 . : :` 2 ~ 6 2 9 9 7 PCT/US95/03451 To study the tensile sl,~nylh of the FS, a mold was designed based on that descriL,ed by Nowotny, et al., (Biomaterials, 2:55, 1981) with some ~odi~icalions. The newly designed mold was ~aL,ricated of transparent plastic to facilitate visual inspection of clot rO m~aLio~ ,. Clotting was allowed to proceed in disposable clot holders for ease of cleaning.

The clot holders were obtained by cutting plastic disposable l,~"srer pipettes (SAMCO, San Fernando Mfg. Co.). Two small pieces of moistened sponge were used to anchor the clotting mixture at both ends.
Dispos~hle clot holders with the sponges in place were inserted through the end holders and into the mold, (end holders were included in the mold). A te"sior"eter instrument (T10, Monsanto) was used to measure and record the peak rupture stress of the clots. A~apte,~ for the T10 grippers were fabricated to hold the end holders.

The clots were formed by injecting equal volumes of fibrinogen and thrombin (with or without CaCI2) using a dual syringe acJn,inisl,dlion device (Fenwal) and a 3 inch 22 gauge needle. All bubbles were removed prior to placing the syringes in their holder. The needle was inserted through one sponge "top", through the mold and into the other sponge "bottom". Parafilm (American Can Co.) placed under the entire mold prevented leakage of excess mixture. As the clotting mixture filled the mold, the needle was withdrawn.

Approximately 2 to 5 minutes before testing, the clot was removed from the mold and placed in the T10 grippers. At testing time, the clot was stretched at a rate of 100 mm/min. The gauge length was set (somewhat arbitldrily) at 6.0 cm and the cross sectional area of the clot was 0.049 cm2. The T10 reported the stress values in Kgf/cm2.

W095/25748 2 1 6 2 9 9 7 PCT/USg5/03451 EFFECT OF CaC~ ON TENSILE STRENGTH OF FIBRIN SEALANT

Tensile St~e~ tl~ KgF/cm2 Fgn.* Conc. CaC~ ~ 250 NIH U/ml ~? 500 NIH U/ml mg~ml mMol Tl--u~ . - (N) Tl.. u,.. ~ln (N) go 0 0.93+ 0.136 (9) 1.23_ 0.165 (8) 110 0 1.07+ 0.150 (10) 1.29~0.237 (9) 130 0 0.94+0.157 (14) 1.36+0.177 (16) 1.6~:0.380 (12) 1.92~0.350 (8) 110 10 2.6t~0.540 (8) 2.8~0.610 (8) 130 10 2.3~0.760 (11) 3.24_1.170 (8) 1.65+ 0.406 (8) 2.1 ~ 0.540 (8) 110 20 2.26~0.530 (8) 4.11+1.080 (8) 130 20 2.23+ 0.489 (8) 3.54i 1.030 (8) go 40 1.74+0.410 (8) 2.36+0.390 (11) 110 40 2.36+ 0.660 (8) 3.79~ 0.626 (8) 130 40 2.63+0.670 (12) 4.0 +0.940 (9) 2.1 Q~ 0.38 (8) 2.29~ 0.450 (8) 110 60 3.0Q~0.690 (8) 4.13+0.896 (8) 130 60 3.69~0.787 (10) 3.57+1.160 (9) Fibrinogen Lot #2830R129 Measurements of tensile strength (peak stress) were taken 10 minutes after injection of the clotting mixture. The results in Table 3 show the effect of varying the CaCI2 concentration when II,r~i"bin is 250 NIH U/ml or 500 NIH U/ml. Three concenl~dlions of fibrinogen (90 110 and 130 mg total protein/ml) were tested. Each measurement of peak stress was the average of N determinations. A minimum of 8 readings were taken per point.

As shown in Table 3 in the absence of Ca++ the clots had the lowest peak stress values at both thrombin concentrations and at all fibrinogen levels. Addition of calcium ions at 10-60 mM increased the tensile sl,~nylh for all thrombin/fibrinogen concentrations. Generally higher .

values were observed at the higher thrombin concer,l,~lion, i.e., 500 NIH
U/ml of thrombin, which also gave somewhat similar values for [Ca+ +] in the range of 20-60 mM of calcium ions. The lowest standard deviation (SD) values were observed at 40 mM CaCI2.

A second lot of human fibrinogen was tested to confi"" these findings and the data were compared at 40 and 60 mM CaCI2 and 500 NIH U/ml ll"oi,lbin. The results of testing a second lot of fibrinogen showed generally simiiar values of peak stress particularly at the higher fibrinogen concentrations of 110 and 130 mg/ml and also showed higher values at 90 mg/ml.

Time studies of the clot tensile strength were performed over a 24 hour time period using fibrinogen at 90 mg/ml and 130 mg/ml with thrombin concentration at 500 NIH U/ml and CaCI2 at 40 mM. Over a 24 hour period, the tensile strength showed no signi~icanl decrease in value. A
gradual increase in peak stress was expected to occur as cross linking continued with time.

D. Clot Lysis Studies The length of time that a fibrin clot will remain solid when incubated at 37C under sterile, moist conditions, with and without a plasminogen activator, was determined. Also tested was the effect on the clot longevity of adding protease inhibitor (Apr~tini,)) to the reaction mixture.

Sterile human fibrinogen solution was prepared using one of the following diluents:

a. sterile water (i.e., zero KlU/ml Aprotinin) b. Aprotinin solution at 1000 KlU/ml c. Aprotinin solution at 3000 KlU/ml to yield one of three concentrations; 90, 110, or 130 mg/ml of total protein.

Thrombin was prepared by reconstituting with a 40 mM CaCI2 solution to produce either a 250 or 500 NIH U/ml. Thus, six combinations of ll,,u,,,bin and fibrinogen were tested. Urokinase (Abbott) was prepared at 5 U/ml in normal saline.

Fibrin clots were tOI " ,ed by mixing equal volumes of fibrinogen (in H20 or Aprotinin) and thrombin (in CaCI2 solution) in cylindrical silicone tubing (5 mm inner diameter). The mixture was delivered using a dual syringe administration device (Fenwal). All the delivery devices and the silicone tubing were slerili~ed by autoclaving.

A 10 cm length of silicone tubing was sealed at one end using parafilm.
Holding the tubing about 10 from vertical, the Fenwal device was used to inject the fibrinogen and Ihror"bin rapidly into the tubing with the (22 9) needle tip barely penetrating the parafilm. After the clot had solidified for 20 minutes, the 10 cm silicone tubing was cut into 3 cm lengths to yield a clot volume of 590 ~I. Each 3 cm segment was cut in half and the two halves were placed in one well of a sterile 24-well plate (Corning). Any segment that was found to contain air bubbles was discarded. The clot was extruded from the tubing by gently squeezing the tube at one end.
It was rinsed with 1 ml of sterile saline then 1 ml of either urokinase or saline was added to the well and the plate was placed in a sterile, moist, 37C incubator. Every 24 hours, the super~ala,1ls from each well were removed for testing using the Fibrin(ogen) Degradation Products agglutination kit (Baxter Dade).

The urokinase and saline solutions were replaced daily with fresh reagents before returning the plate to the 37C incubator. All preparations and sampling of supernatants were performed under sterile conditions. The clots were visually inspected and their appearance noted. After 14 days the experiment was terminated. Total number of conditions tested was 36 and all conditions were performed in duplicate.

W O 95/25748 2 1 6 2 9 q 7 PCT~US95/03451 CLQT IYSIS TIMES
~+ UROKINASE. NO APROTININI

CONDITION TIME CLOT LOST CYLINDRICAL SHAPE
130 mg/ml riLri"ogen + 500 U/ml Illlu~ I Day 10 130 mg/ml ri~ril,ogen + 250 U/ml Illlu~ , Day 8 110 mg/ml fibrinogen + 500 U/ml Illlulll~.l Day 7 110 mg/ml ~iL.,i"ogen + 250 U/ml Ill~nl ~, Day 11 90 mg/ml tiL.ri"ogen + 500 U/ml thrombin Day 8 1~ 90 mg/ml ~il,ri"ogel~
+ 250 U/ml Ll llul I l~, Day 8 Table 4 sl""",a,i~es the clot Iysis time (defined as the time clots lost their cylindrical shape) in the presence of urokinase when no Aprotinin was included. The observed range was 7-11 days with a mean of 8.66 +
1.5d. Measurements of Fibrin(ogen) Degradation Products (FDP) showed a peak in activity that generally con~sponded to or soon followed the time when the clots lost their well-defined shape.

When urokinase was deleted and the clots were incuh~ted in normal saline only, in presence or absence of A,ur~lin;n, the clots maintained their integrity during the entire observation period (i.e., 14 days). FDP
meas~"e,ne"ls confirmed the absence of significant clot Iysis.

This study shows that when the clot is not influenced by any plasminogen activators in situ, it should be expected to last for at least 14 days. When urokinase is present, the clots last a minimum of 7 days. These time periods may be sufficient for the healing mechanism to play its natural role. Thus, based on these results, it can be concluded that the trace WO 95125748 PCTtUS95/03451 2~ 62997 levels of plasminogen in the fibrinogen ~ Jardlio"s do not adversely affect the clot longevity and that the addition of a protease inhibitor, such as Aprotinin, is not necessary.

TFC in vivo TESTING

Studies were done to cva'~ e the optimal concenl,~liG" of TFC using an in vivo model. Swiss Webster mice (20-25 9) were ar,~nged in 10 groups of five for testing. In the protocol which was utilized, each animal was anesll,eLi~ed, weighed, and a small piece of skin was removed from the back of the animal. The skin specimen was dipped in a saline solution and attached to a Gottlob device. Equal volumes of TFC and thrombin at various concel IlldliOl ls (Table 2) were then added simultaneously to the wound, the skin replaced onto the animal, and held in place for approximately two minutes.

The anesthetized animal was placed face down on a ,cldl~orm which was then positioned on a tensiometer (Monsanto Company) and the Gottlob device attached to the grippers. The tensiometer parameters were set to:
(1) area: 1.76 cm2; (2) speed: 10.0 mm/min.; (3) gauge: 1.0 cm; (4) stress range: 500.0%. The force required to separate the piston (with the skin specimen) from the back of the animal was recorded in g/cm2. The data from these experiments were ~Idlislically evaluated using RS1/Discover software (BBN Software Corp., Cal),bridge, MA). Analysis of the results of this study indicated that TFC at 120-130 mg/mL and thrombin at 250 U/mL gave maximal adhesion responses.

The ability of the clot to adhere to tissue in vivo is important in maintaining hemostasis. In this experiment, a maximum adhesion response occurred within the range of reagents tested, collri"~ling the in vitro findings of Example 2.

wo gsl25748 2 1 6 2 9 9 7 PCTIUS95/03451 ._ ' !

CHARACTERISTICS OF TFC
A TFC composition was prepared yener~lly according to the process steps described in Example 1. The chara.:teri~lics of TFC identified below were analyzed with the following results:

TEST RESULTS
SOLUBILITY 1050 seconds pH 7.2 STERILITY STERILE
PYROGEN NON-PYROGENIC
HEPATITIS B tHBsAg) NON-REACTIVE
TOTAL PROTEIN 10.4 g/dL
CLOTTABLE PROTEIN 86 %
F-XIII 32.4 U/ml PLASMINOGEN <0.1 mg/dL
PEG 0.0 g/dL
TRITON X-100 0.4 PPM
TNBP 1.1 PPM
TWEEN-80 0.1 %
ALBUMIN 0.64 g/dL
HISTIDINE 55.9 mM
SODIUM 213 mg/L

W095125748 ~` 2 1 PCT/US95/03451 E)(AMPLE 5 REDUCTION OF VIRAL ACTIVITY IN
TFC PROCESS INTERMEDIATES TO UNDETECTABLE LEVELS

TFC process intermedidles ~e, Example 1) were assayed for activity of 5 lipid enveloped viruses. The lipid enveloped viruses assayed were:
Pseudorabies ("PRV", as a model for herpes viruses), HIV Types 1 and 2, Sindbis ("SIN", as a model for he,ualiLis C virus), and Vesicular Stomatis Virus ("VSV", as a model for RNA viruses). The process intermediates were obtained after the resuspension of PEG pr~c;,~,~ale in tris-phosphate buffer, pH 8.6 + 0.3 (without buffer, the viricidal activity of the viral activity reducing agent would be reduced and an agent of greater concentration would be used).

For purposes of the assay, the samples were separately spiked with four lipid enveloped viruses in the presence of the following viral activity reducing agent: a mixture of TNBP, octoxynol 9 and polysorbate 80, in respective ratios of 0.3%:1%:0.3% (v/v).

PRV, VSV and SIN were assayed by incubation with a~u,ur~priale cell lines (porcine kidney 13 ("PK-13"), buffalo green monkey kidney ("BGMK") and Vero, respectively) to deler",i,1e plaque forming units ("pfu") before and after treatment with the viral activity reducing agent. HIV-1 was assayed by incubation with susceptible T cells (H9) to determine 50% of the tissue culture infectious dose end point (TCID50) of the virus before and after treatment with the viral activity reducing agent. The results of these assays are tabulated below, which demonstrate that the use of the viral activity reducing agent during the manufacture of TFC is very effective in reducing the activity of lipid envelope viruses therein.

WO 95/25748 2 1 6 2 9 ~ 7 PCT/US95/03451 VIRUS RECOVERY

HIV-1 ID5~sv Lopg~vpfu/mL
Virus + TFC Solution 12.5 9.0 8.9 8.9 Virus + TFC Solution Negative <1.9 ~1.9 <1.9 After Incub2tion LOg,n Reduction Factor 12.5 7.1 7.1 6.9 No plaques were detected. The theoretical detection point of the assay was used to calculate the virus recovery. Reagent cytotoxicity due to solvent/deterge"l mixture (i.e. the viral activity reducing agent) has been taken into account for these calculations.

PYROGENICITY OF TFC

The pyrogenicity of the TFC composition of Exa~r"~le 4 was tested using the well known rabbit pyrogen test. Three rabbits were injected with a dose of 0.5 ml/kg and their body temperatures ~,o"itur~d over a course of three hours following the injection. The results of this test are tabulated below which strongly indicate that TFC is non-pyrogenic.

WO 95/25748 `:. 2 1 6 2 9 ~ 7 PCT/US95/03451 Rabbit TEST TEMPERATURE (C) Weight Dose Pre-injectionMaximum # (kg) (ml) Temperature (C) 1 hr 2 hr 3 hr Rise (C) 3.23 1.7 39.2 39.1 39.1 39.1 0.0 2 2.70 1.4 39.1 39.0 39.0 39.1 0.0 103 3.11 1.6 39.6 39.5 39.5 39.5 0.0 Sum of Maximum Rise: 0.0 The preceding examples and description are provided to assist in understanding the present invention and, as such, are intended to be exemplary only, not limiting. Those of skill in the art will recognize that other materials or methods may be used, depending on the circumstances, and still remain within the spirit and scope of the present invention.

Claims (24)

1. A method for preparing a hemostasis promoting composition derived from human plasma or plasma fractions wherein the human plasma or plasma fractions comprise fibrinogen, Factor XIII, and plasminogen, comprising:
(a) providing a cryoprecipitated plasma preparation from the human plasma or plasma fractions;
(b) separating the cryoprecipitate from the cryoprecipitated plasma preparation;
(c) suspending the product of step (b) in a salt-cotaining buffer to eliminate prothrombin complex from the product of step (b);
(d) treating the supernatant obtained from the suspension in step (c) by affinity-chromatography to allow plasminogen to adsorb thereon;
(e) collecting the fraction essentially free of plasminogen;
(f) contacting the fraction of step (e) with a viral activity reducing effective amount of an viral activity reducing agent;
(g) removing the viral activity reducing agent from the material obtained in step (f); and (h) recovering a fibrinogen-containing composition.

2. The method of claim 1, further including a step (b') wherein the cryoprecipitate in step (b) is further processed to form a cold-precipitate.

3. The method of claims 1 or 2, wherein step (b) or (b'), respectively, further comprises treating the cold-precipitate with a protease inhibitor in a concentration sufficient to inhibit thrombin activity.

4. The method of claim 3, wherein the protease inhibitor is selected from the group consisting of PPACK, heparin cofactor II, hirudin, and anti-thrombin III (AT III).

5. The method of claim 1, wherein in step (d) the affinity-chromatography consists of a lysine-bound solid matrix as the adsorbent.

6. The method of claim 5 wherein the solid matrix is agarose.

7. The method of claim 1 further including the step of (e ) comprising treating the fractions by addition of polyethylene glycol (PEG).

8. The method of claim 1 or 2 wherein the viral activity reducing agent comprises at least one nondenaturing detergent.

9. The method of claim 8 wherein at least one of the detergents is non-ionic.

10. The method of claim 8 wherein a mixture of at least one detergent and at least one organic solvent is utilized.

11. The method of claim 10 wherein the mixture further includes an alkyl phosphate.

12. The method of claim 10 wherein the mixture further includes a polyoxyethylated ester derivative.

13. The method of claim 12 wherein the alkyl phosphate is tri (n-butyl) phosphate.

14. The method of claim 1 wherein the viral activity reducing agent is removed by adsorbing fibrinogen on an anionic exchange resin and by washing any agent not bound to the resin.

15. The method of claim 14 wherein the anionic exchange moiety is diethylaminoethyl (DEAE).

16. The method of claim 1 which further comprises the steps of:
(i) adding a stabilizer and/or a solubilizer to the recovered composition of step (h);
(l) concentrating the composition of step (i) to about 20% to about 50% of its original volume;
(k) diluting the concentrate of step a) to its preconcentration volume;

(l) concentrating the composition of step (k) to about 3 9% to about 5 9% (w/v), and (m) sterile processing the composition.

17. The method of claims 1 or 16, wherein in step (h) or step (m), respectively, the composition is additionally lyophilized.

18. The method of claim 17 wherein the volume of the composition when reconstituted is less than the pre-lyophilization volume of the composition.

19. A hemostasis promoting composition derived from human plasma or plasma fractions characterized as:
(a) having fibrinogen in an amount of about 50% to about 100% of the total protein concentration;
(b) from about 10 to about 40 units/ml F XIII;
(c) being free of detectable levels of lipid enveloped virus activity;
(d) being substantially non-pyrogenic, (e) having a maximum of about 10 µg/ml plasminogen, (f) being essentially free of prothrombin complex and active thrombin; and (g) being free of fibrinolysis inhibitors.

20. The composition of claim 19, wherein the composition is lyophilized.

21. The composition of claim 19, which further comprises a stabilizer and/or a solubilizer.

22. The composition of claim 19, which further comprises a therapeutically effective amount of a drug.

23. The composition of claim 19, which further comprises a diagnostically effective amount of a diagnostic agent.

24. A fibrin sealant comprising a mixture of:
i. a hemostasis promoting composition derived from human plasma or plasma fractions characterized as:
(a) having fibrinogen in an amount of about 50% to about 100% of the total protein concentration;
(b) from about 10 to about 40 units/ml F XIII;
(c) being free of detectable levels of lipid enveloped virus activity;
(d) being substantially non-pyrogenic, (e) having a maximum of about 10 µg/ml plasminogen, being essentially free of prothrombin complex and active thrombin; and (g) being free of fibrinolysis inhibitors, ii. a catalytically effective amount of thrombin; and, iii. calcium ion.