Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/745—Blood coagulation or fibrinolysis factors
  • C07K14/75—Fibrinogen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
  • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
  • A61L24/043—Mixtures of macromolecular materials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
  • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
  • A61L24/10—Polypeptides; Proteins
  • A61L24/106—Fibrin; Fibrinogen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• 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.
• the present invention is based upon the discovery that pooled plasma, even when substantially depleted of Factor VIII, can be processed to produce a fibrinogen preparation which, when reacted with thrombin and calcium, will produce a fibrin sealant that can be used to promote hemostasis.
• the invention provides a fibrinogen composition which, in addition to being essentially free of Factor VIII 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 proteins present in the composition are of human origin.
• 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.
• FIGURE 1 Schematic representation for preparation of Topical Fibrinogen Complex.
• FIGURE 2 Effect of calcium ion concentration on fibrin polymer formation.
• Fibrinogen 90 mg total protein/ml
• thrombin 500 U/ml
• 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 I 30mM Ca + +
• Lane J MW markers.
• FIGURE 3 Effect of calcium ion concentration on fibrin polymer formation.
• Fibrinogen 130 mg total protein/ml
• thrombin 500 U/ml
• 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 I 30mM Ca + +
• Lane J MW markers.
• FIGURE 4 Rate of fibrin polymerization. Fibrinogen (130 mg total protein/ml) and thrombin (500 U/ml) were mixed in the presence of calcium ion (40mM CaCI 2 ). 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 I: 2 hr; Lane J: 4 hr; Lane K: 8 hr; Lane L: 24 hr;
• Lane M fibrinogen control
• Lane N MW markers.
• compositions which, when reacted with thrombin, will produce a fibrin sealant that represents a significant improvement over the prior art compositions intended to accomplish this effect.
• compositions (collectively, 'Topical Fibrinogen Complex
• TFC tissue-derived fibroblasts
• Preferred blood fractions for producing the compositions of the invention are plasma, cryoprecipitate, and/or Factor Vlll-depleted cold-precipitate. Because the preferred blood fraction for use as a starting material is human plasma, the starting material will hereafter 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 starting 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 XIII (F XIII) and fibrinogen. This step may (and preferably will) be followed by cold-precipitation of proteins from the cryoprecipitate. The product of the cold-precipitation process, if any, (i.e, the cold-precipitate), will typically contain a high concentration of fibrinogen and very low levels of F VIII.
• F XIII Factor XIII
• the preferred method for producing the TFC compositions of the present invention uses frozen human plasma from one or more donors as a starting material.
• the plasma to be used will have been screened using conventional assay techniques for the presence of infectious viral contaminants, such as hepatitis B and human immunodeficiency virus (HIV) to eliminate plasma for use as a starting material which contains detectable levels of such contaminants.
• infectious viral contaminants such as hepatitis B and human immunodeficiency virus (HIV)
• cryoprecipitate or cold-precipitate 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.
• 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 detection of plaque forming units in infected tissue or cells (see, e.g., Example 5).
• a reduction in the known viral activity level in a given composition will be conventionally described herein as the "log 10 reduction factor".
• compositions of the inventions will have a log 10 reduction factor for lipid enveloped viruses of at least 4 logs (preferably at least 6 logs) and a lesser reduction factor for other viral pathogens.
• cryoprecipitate As described in Example 1, to process the frozen plasma to a cryoprecipitate, 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 content of the compositions of the invention to fibrinogen, residual amounts of fibronectin and any added protein components (such as albumin), the cryoprecipitate will preferably be further processed to a cold- precipitate.
• a cryoprecipitate can be produced by freezing the blood fraction (for example, plasma) which is thereafter warmed to a temperature not exceeding about +6°C.
• the cryoprecipitate is dissolved in distilled water at about 20°C-35°C.
• Calcium chloride is added at a concentrate from about 1 ⁇ M to about 1000 ⁇ M.
• calcium chloride is added at this step at a concentration of about 40 ⁇ M and the pH adjusted to 6.8 ⁇ 0.3 to enhance the precipitation of fibrinogen.
• the dissolved cryoprecipitate is then cooled to about 10°C with mixing, whereupon the cold-precipitate forms.
• the precipitate is removed from solution by centrifugation at, for example, 5600 g to 7200 g.
• the precipitate may be stored at -60°C or lower if desired.
• the addition of a calcium ion source during the process will enhance the precipitation of fibrinogen, as well as fibronectin, thereby increasing the concentration of these substances in the cold- precipitate.
• the fibrinogen can also be further concentrated by the addition of polyethylene glycol (PEG) to the cold-precipitate.
• PEG polyethylene glycol
• the cryoprecipitate or cold-precipitate will usually be dissolved in buffer with mixing and the pH of the cold-precipitate adjusted to about 7.2, preferably to 7.2 ⁇ 0.1.
• the cryo- or cold-precipitate will preferably be resuspen- ded in the presence of a protease inhibitor, such as PPACK (D-Phe-L-Pro- L-Arg-chloromethylketone), heparin cofactor II, hirudin or anti-thrombin III (ATIII) to inhibit thrombin which may be present.
• PPACK D-Phe-L-Pro- L-Arg-chloromethylketone
• ATIII anti-thrombin III
• thrombin inhibitor is PPACK at a concentration 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 thrombin. If used, the thrombin inhibitor will be removed from the compositions of the invention before lyophilization by, preferably, PEG precipitation and, optionally, also the DEAE column step of the procedure (see. FIGURE 1 and Example 1).
• the precipitate suspension is transferred to a buffer solution containing a salt such as tri-calcium phosphate. Exposure to the salt-containing buffer will minimize the likelihood of prothrombin 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 prothrombin complex.
• the calcium phosphate is removed from the process by centrifugation and/or filtration. Additional techniques for removal of prothrombin are described by Murano (Prothrombin and Other Vitamin K Proteins, Vol.
• the final compositions will be essentially free of fibrin molecules; i.e., all of the fibrinogen therein will be unreacted until separately exposed to thrombin (preferably in vivo) to produce a fibrin sealant.
• the dissolved cryo- or cold-precipitate is warmed to about 23-27°C and contacted with a lysine affinity column, such as the matrix column product sold commercially under the tradename lysine-Sepharose 4B. Residual 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.
• a lysine affinity column such as the matrix column product sold commercially under the tradename lysine-Sepharose 4B.
• Residual 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.
• the resulting final TFC composition will contain no more than 10 ⁇ g plasminogen/milliliter of TFC.
• plasiminogen when converted to plasmin, will break down fibrinogen 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.
• the solution collected from the lysine matrix will preferably be concentrated by, for example, the addition of polyethylene glycol (PEG).
• PEG polyethylene glycol
• the PEG used to concentrate the eluate will have a molecular weight in a range which is not toxic to humans (e.g., 3000- 6000) and will be added to a final concentration from about 3% to about 7%, preferably about 4% (w/w).
• Most preferably the resulting PEG precipitate will be dissolved in a buffer solution, filtered and the pH thereof adjusted to an alkaline level; e.g., about 8.6, preferably 8.6 ⁇ 0.1.
• an alkaline level e.g., about 8.6, preferably 8.6 ⁇ 0.1.
• compositions preferably the concentrated solution
• a viral activity reducing agent such as a detergent, which, typically, acts by disrupting the lipid envelope of such viruses as Hepatitis B, HIV and HTLV
• a viral activity reducing agent such as a detergent
• an "effective amount of viral activity reducing” agent means that the concentration 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.
• the concentration 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.
• compositions of the invention 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.
• 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 alcohols and sodium acid salts, such as sulfated oxyethylated alkylphenol (sold commercially under the tradenames "Triton W-30” and “Triton X- 100"), sodium dodecylbenzensulfonate (sold commercially under the tradename "Nacconol NR”), sodium 2-sulfoethyl oleate (sold commercially under the tradename “Igepon A'), sodium cholate, sodium deoxycholate, sodium dodecylsulfonate, dodecyldimethylbenzylammonium chloride (sold commercially under the tradename "Triton K-60”), oxyethylated amines (sold commercially under the tradename "Ethomeen”), N-do
• the viral activity reducing agent will consist of an organic solvent, preferably tri-n-butyl phosphate (TNBP) mixed with nonionic detergents, preferably polysorbate 80 and octoxynol 9.
• TNBP tri-n-butyl phosphate
• nonionic detergents preferably polysorbate 80 and octoxynol 9.
• undenatured compositions of the invention in which viral activity is at undetectable levels can be produced using a preferred viral activity reducing agent comprised of TNBP
• chaotropic agents may also be utilized to inactivate viruses, providing the agent does not denature fibrinogen.
• the concentration of organic solvent and detergent used in the practice of the preferred embodiments of the invention can vary, depending upon the composition to be treated, and upon the solvent or detergent selected.
• the alkyl phosphates can be used in concentrations from about 0.10 mg/ml of mixture treated to 1.0 mg/ml, preferably 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 contact between the organic solvent and the virus.
• the wetting agent can vary from about 0.001 % to 10%, preferably from about 0.01 % to about 2% of the aqueous mixture, depending upon the amount of fatty material in the treated aqueous mixture.
• 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 detergent, is eluted with, for example, 0.3M NaCI.
• ion exchange materials which can be utilized for removal of the solvent/detergent include virtually any of the commercially available anion exchange matrices including, but not limited to, cellulose and agarose matrices. The specific parameters for binding and eluting from these various ion exchange materials are known to those of skill in the art, or can be readily ascertained without undue experimentation.
• the stability of the TFC compositions of the invention may be enhanced through the use of such excipients as human serum albumin (HSA), hydroxyethyl starch, dextran, or combinations thereof.
• HSA human serum albumin
• the solubility of the compositions may also be enhanced by the addition of a nondenaturing nonionic detergent, such as polysorbate 80.
• Suitable concentrations 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 stabilizer.
• the most preferred embodiment of the compositions will either contain no added stabilizer or will contain a nonproteinaceous stabilizer.
• the bulk is concentrated from about 20% to about 50% of its original eluate volume, then diluted to the pre- concentration eluate volume.
• the bulk may then be concentrated to a final total protein concentration of about 4g ⁇ 1g/dL composition (w/v) before sterile processing and lyophilization.
• a preferred composition of the invention is one which, when reconstituted, will consist essentially of fibrinogen; i.e., the proteins in the composition will be fibrinogen, no more than a residual amount of fibronectin (i.e., 20 mg/ml or less, preferably 10 ⁇ g/ml or less), no more than a residual amount of plasminogen (no more than 10 ⁇ g/ml, preferably no more than 5 ⁇ g/ml) and from about 1 to 40 Units/ml of Factor XIII (preferably more than 10 Units/ml).
• the composition may also contain components such as a protein stabilizer; e.g., human serum albumin.
• 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 preferably will comprise at least 75% of the composition (w/v).
• concentration of the protein (s) in the composition is accomplished by ultrafiltration using a membrane with a molecular weight exclusion large enough to allow NaCI to be removed, but small enough to retain protein molecules. This filtration is most preferably performed using a membrane with a 30,000 MW exclusion.
• the pre-lyophilization volume is usually greater than the volume to which the lyophilizate is resuspended at time of use.
• compositions of the invention can be modified to include non-proteinaceous as well as proteinaceous drugs.
• non- proteinaceous drugs encompasses compounds which are classically referred to as drugs, such as mitomycin C, daunorubicin, and vinblastine, as well as antibiotics.
• the proteinaceous drugs which can be added to the fibrinogen compositions of the invention include immunomodulators and other biological response modifiers.
• biological response modifiers is meant to encompass substances which are involved in modifying a biological response, such as the immune response or tissue growth and repair, in a manner which enhances a particular desired therapeutic effect, for example, the cytolysis of bacterial cells or the growth of epidermal cells. Examples of response modifiers include such compounds as lymphokines.
• lymphokines include tumor necrosis factor, the interleukins, lymphotoxin, macrophage activating factors, migration inhibition factor, colony stimulating factors, and the interferons.
• 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-proteinaceous drugs.
• compositions of the invention can also be modified to incorporate a diagnostic agent, such as a radiopaque agent.
• a diagnostic agent such as a radiopaque agent.
• 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 diatrizoate derivatives, such as diatrizoate sodium.
• Other contrast agents which can be utilized in the compositions of the invention can be readily ascertained by those of skill in the art.
• the concentration 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 concentration to minimize toxicity, but display the desired effect.
• the concentration of an antibiotic used in providing a cytolytic therapeutic effect will likely be different from the concentration of an immune response modulator where the therapeutic effect is to stimulate the proliferation of immune cells at the site of application of the fibrinogen complex.
• diagnostically effective amount denotes that concentration of diagnostic agent which is effective in allowing the fibrin glue to be monitored, while minimizing potential toxicity.
• the desired concentration in a particular instance for a particular compound is readily ascertainable by one of skill in the art.
• Topical fibrinogen complex was produced by the initial preparation of a cryoprecipitate of plasma.
• the cryoprecipitate for such use was prepared by two different techniques depending upon the physical form of the plasma.
• sealed plastic bottles of frozen plasma were thawed in a controlled environment by contact with a heat exchange medium, such as air or water.
• a heat exchange medium such as air or water.
• the thaw was controlled by programming the temperature and flow of the heat-exchange medium so that the maximum temperature of the plasma did not exceed +6°C.
• 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 2.5°C ⁇ 3.5°C.
• the plasma was centrifuged to remove the cryoprecipitate.
• the cryoprecipitate, so prepared, may be stored at or below -25°C or immediately processed to antihemophilic factor.
• the cryo-poor plasma was collected in a jacketed stainless steel reaction tank
• cryoprecipitate was prepared by placing sealed plastic bags of frozen plasma 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 plasma 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 2.5°C ⁇ 3.5°C.
• the plasma was centrifuged to remove the cryoprecipitate.
• the cryoprecipitate, so prepared may be stored at or below -25°C, or immediately be processed to antihemophilic factor.
• the cryo-poor plasma was collected in a jacketed stainless steel reaction tank.
• cryoprecipitate After the cryoprecipitate was prepared, it was dissolved in distilled water at 20°C to 35°C. This part of the protocol is illustrated schematically in FIGURE 1. Sufficient calcium chloride was added to obtain a minimum calcium concentration of about 40 ⁇ M and the pH adjusted to 6.8 ⁇ 0.3. This solution was cooled to 10°C ⁇ 2°C while mixing. The precipitate which forms was removed by centrifugation (5600g-7200g). The precipitate may be stored at or below -60°C 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 precipitate.
• Process Solution I comprises: (a) 0.5M glycine, 0.5M sodium chloride, and 0.1 M sodium citrate; pH adjusted 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
• Process Solution II comprises: 7 ⁇ 1 mM sodium phosphate monobasic monohydrate, 18
• the suspension was then allowed to settle undisturbed for at least 30 minutes.
• the suspension may be centrifuged (5600g-7200g) 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 ⁇ in pore size.
• the filtrate was warmed to 23-27°C 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 III (25 mM phosphate buffer: 7 ⁇ 1 mM sodium phosphate monobasic monohydrate,
• Process Solution V (0.02M histidine, 0.01 M NaCI, pH adjusted to 7.0 ⁇ 0.1 with 6N HCI).
• Process Solution VI (0.02M histidine, 0.3M NaCI, pH adjusted to 7.0 ⁇ 0.1 with 6N
• 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. Finally, the fibrinogen was concentrated to about 25% of the original volume by ultrafiltration using a 30,000 MW cut-off membrane, then diluted to its preconcentration volume with Process Solution VII (0.02M histidine, pH adjusted to 7.0 ⁇ 0.1). The bulk was again concentrated by ultrafiltration to achieve a final protein concentration of 4 ⁇ 1 g% (w/v).
• the bulk was sterile filtered (0.2 ⁇ ), aseptically filled into sterile final containers, lyophilized under aseptic conditions, and closed with sterile closures.
• the FS was delivered using an experimental dual syringe device (Fenwal) following the 2" line described above, starting at the upper end and moving downward.
• Table 1 summarizes the data obtained by testing fibrinogen at 50-130 mg/ml with thrombin at 100-1000 NIH U/ml in the absence of calcium. Each data point is the average of four determinations.
• clotting times at the lower thrombin concentration e.g., 100 NIH U/ml
• low protein content e.g., 50 mg/ml
• the mixture was also observed to be "runny”.
• Higher concentrations of thrombin e.g., 1000 NIH U/ml
• Addition of CaCI 2 improved the appearance of the clot and generally shortened the clotting time.
• Table 2 shows the effect of [CA + + ] in the range of 0-60 mM. CaCI 2 solution was used to reconstitute the thrombin so the final [Ca + + ], in the 1:1 mixture of FS is half that reported in the Table. TABLE 2 EFFECT OF [Ca + + ] ON TIME TO CLOT
• Protein refers to the concentration of total protein in mg/ml TFC.
• Thrombin (Armour Pharmaceutical) was reconstituted with or without CaCI 2 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 containing 3% SDS and 3% ⁇ -mercaptoethanol by boiling in a water bath at 95 ⁇ 5°C. The dissolved clot solutions were then stored at 5°C until the gel electrophoresis was performed.
• FIGURE 4 shows the fibrin polymerization reaction as it progresses through a 24 hour period. Formation of the y- dimer occurs very rapidly in the presence of Ca + + (within one minute) as is shown in FIGURE 4. The ⁇ polymer is not detectable by this system until 10 minutes incubation time. As the ⁇ polymer increases with increasing incubation time (up to 24 hours) the ⁇ monomer band shows a corresponding decrease in intensity. To summarize, the time study demonstrates that initial polymerization ( ⁇ -y dimer) occurs almost instantaneously as the reactants are mixed, with the ⁇ polymers forming more slowly.
• the tensile strength of the fibrin sealant was evaluated 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.
• a mold was designed based on that described by Nowotny, et al., (Biomaterials, 2:55, 1981) with some modifications. The newly designed mold was fabricated of transparent plastic to facilitate visual inspection of clot formation. Clotting was allowed to proceed in disposable clot holders for ease of cleaning.
• the clot holders were obtained by cutting plastic disposable transfer pipettes (SAMCO, San Fernando Mfg. Co.). Two small pieces of moistened sponge were used to anchor the clotting mixture at both ends. Disposable 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 tensiometer instrument (T10, Monsanto) was used to measure and record the peak rupture stress of the clots. Adapters 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 CaCI 2 ) using a dual syringe administration 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.
• 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 arbitrarily) at 6.0 cm and the cross sectional area of the clot was 0.049 cm 2 . The T10 reported the stress values in Kgf/cm 2 . TABLE 3 EFFECT OF CaCU ON TENSILE STRENGTH OF FIBRIN SEALANT
• a second lot of human fibrinogen was tested to confirm these findings and the data were compared at 40 and 60 mM CaCI 2 and 500 NIH U/ml thrombin.
• the results of testing a second lot of fibrinogen showed generally similar 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.
• Sterile human fibrinogen solution was prepared using one of the following diluents:
• Thrombin was prepared by reconstituting with a 40 mM CaCI 2 solution to produce either a 250 or 500 NIH U/ml. Thus, six combinations of thrombin and fibrinogen were tested.
• Urokinase (Abbott) was prepared at 5 U/ml in normal saline.
• Fibrin clots were formed by mixing equal volumes of fibrinogen (in H 2 0 or
• a 10 cm length of silicone tubing was sealed at one end using parafilm.
• the Fenwal device was used to inject the fibrinogen and thrombin rapidly into the tubing with the (22 g) needle tip barely penetrating the parafilm.
• the 10 cm silicone tubing was cut into 3 cm lengths to yield a clot volume of 590 ⁇ l. 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,
• Table 4 summarizes the clot lysis 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 corresponded to or soon followed the time when the clots lost their well-defined shape.
• FDP Fibrin(ogen) Degradation Products
• mice (20-25 g) were arranged in 10 groups of five for testing. In the protocol which was utilized, each animal was anesthetized, 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 concentrations (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 platform 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 cm 2 ; (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/cm 2 .
• the data from these experiments were statistically evaluated using RS1 /Discover software (BBN Software Corp., Cambridge, 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.
• TFC composition was prepared generally according to the process steps described in Example I.
• the characteristics of TFC identified below were analyzed with the following results:
• TFC process intermediates (see. Example 1) were assayed for activity of 5 lipid enveloped viruses.
• the lipid enveloped viruses assayed were:
• PRV Pseudorabies
• HIV Types 1 and 2 Sindbis
• Sindbis Sindbis
• VSV Vesicular Stomatis Virus
• 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).
• 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 appropriate cell lines (porcine kidney 13 ("PK-13”), buffalo green monkey kidney (“BGMK”) and Vero, respectively) to determine 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 (TCID- of the virus before and after treatment with the viral activity reducing agent.
• TID- tissue culture infectious dose end point
• the pyrogenicity of the TFC composition of Example 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 monitored 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.

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