NO316155B1 - Enhanced tissue adhesive prepared using cryoprecipitate, method of preparation thereof, and use of a plurality of deprotease inhibitor - Google Patents

Abstract

A tissue glue is described comprising a component A which comprises a cryoprecipitate of whole blood and a component B comprising a proteolytic enzyme being capable of cleaving specifically fibrinogen present in component A, and causing the formation of a fibrine polymer, in a preferred embodiment component A of the tissue glue comprises 3,000 to 5,000 KIU/ml of aprotinin. In another preferred embodiment component B of the tissue glue is a protease derived from the venom of the South American pit viper. This protease is known under the name batroxobin.

Description

This invention relates to a tissue adhesive comprising two components A and B, a method for the production of the tissue adhesive, the use of a large amount of aprotinin and the use of a snake venom proteolytic enzyme for the production of a tissue adhesive

Improvement of local hemostasis at a surgical wound site by the use of plasma proteins is a well-known concept Thus fibnnlappers for hemostasis in brain surgeon! have been used Blood plasma and thrombin were used to produce a fibrin film over the surgical wound In the last 20 years there are many publications describing the use of "fibrin-hm" or "fibrin adhesive" or "fibrin sealant" for most surgical disciplines In the last 10 years preparations of "hm" have been widely used in Europe The "glue" is composed of two components, while a mixture of these components produces a blood clot The first component is a fibromogen concentrate This concentrate also contains fibronectin and Factor XIII which is important for clot stabilization and strength The other component is thrombin, an active enzyme that converts fibrinogen, the final component of the normal coagulation system into a fibrin clot This procedure bypasses most of the steps in normal coagulation and mimics its final phase Some manufacturers add plasminogen, which is an enzyme that will include blood clot lysis after some time, while others add aprotinin which is a protease inhibitor to prevent blood clot lysis

Nevertheless, these products provide satisfactory results in patients, albeit with mild bleeding disorders, but patients suffering from severe bleeding disorders such as hemophilia A or B still have a very high risk of postoperative bleeding Sometimes a delayed bleeding complication occurs after an average of days from surgery Patients who are treated with anticoagulant factors also cannot be treated with the tissue according to the prior art Another serious disadvantage of the commercial concentrates is the high production costs

WO 86/01814 describes a method for the preparation of a cryoprecipitated suspension containing fibrinogen and Factor VIII useful as a precursor to the preparation of a fibrin-hm which involves (a) freezing fresh frozen plasma from a single donor eg a human or other animal which has been screened for blood-borne diseases at about -80°C for at least about 6 hours, (b) raising the temperature of the frozen plasma, eg to about 0°C and room temperature, to form a supernatant and a cryoprecipitated suspension which containing fibrinogen and Factor VIII, and (c) recovery of the cryoprecipitated suspension A method for the production of a fibrin glue usable in surgical procedures is also described, comprising (a) production of a cryoprecipitated suspension as described above, (b) applying a defined volume of the suspension to a desired site, and (c) applying a mixture containing a sufficient amount of thrombin to the site to cause the fibrinogen in the su the tension is converted into the fibnn glue which then becomes solid

EP-A-0 341 007 describes a surgical adhesive, in an aqueous composition, which comprises autogenous plasma from the patient, collagen, thrombin and optionally an antifibrinolytic agent. The present adhesive is formed from the patient's plasma without the use of any added reagents for concentration or isolation of the fibrinogen Appropriately, the adhesive is designed as a two-part mixture that is mixed together immediately before use

EP-A-0 253 189 describes a one-component tissue preparation having an aqueous solution of fibrinogen, Factor VIII, a thrombin inhibitor, prothrombin factors, calcium ions and optionally a plasmin inhibitor. The tissue can be recovered from a lyophilized sample by add water Vevshmet can contain all active substances in pasteurized form to avoid hepatitis and HTLV lll transmission

WO-A-91/01762 describes a method for pasteurization of a mixture of proteins, essentially containing fibrinogen, by heating the mixture under conditions that ensure pasteurization, where said mixture is pasteurized < the presence of monosaccanth and a sugar alcohol This tissue can be prepared by start with cryoprecipitate A concentration tnnn is described after paste unsenng

EP-A-0 305 243 describes a tissue serum comprising a component A containing fibrinogen, fibrnectin, Factor VIII and 10,000 KIU/m! of aprotinin and component B consisting of thrombin

An aim of the present invention is to provide a tissue HM that is also suitable for patients with severe blood coagulation disorders, e.g. hemophilia A or B A further aim of the present invention is to provide a tissue HM that can also be used for patients who have already developed antibodies against bull -thrombin which is the active factor in component B Yet another aim of the present invention is to provide a tissue HM to patients who are treated with anticoagulant factors similar to hepan. Due to the risk of transmission of viral diseases together with the components of the tissue glue, it must be ensured that fractions of the tissue is virus-inactivated

The two-component tissue adhesive with separate components A and B according to the invention comprises a component A where component A comprises a cryoprecipitate from whole blood and an amount of protease inhibitor corresponding to aprotinin in amounts of 3000 to 5000 KIU/ml units, said component A can be obtained by thawing of cryopaste, dissolving in a buffer at pH 7.0 to 7.2, preheating to 30 to 35°C, adjusting pH to 7.0 to 7.2, adding aluminum hydroxide under tube, centrifugation and removal of precipitate, addition of CaCl2, vtrus inactivation, concentration by ultrafiltration to a protein concentration of 60 to 100 mg/ml, addition of an amount of protease inhibitor corresponding to 3000 to 5000 KIU/ml units of aprotinin, and a component B comprising a proteolytic enzyme capable of to cleave fibrinogen specifically in the presence of component A and to cause the formation of a fibrin polymer

Commercially available cryoprecipitate can be used to produce the tissue according to the invention. However, it may be advantageous to concentrate the cryoprecipitate between a factor of 2 and 5, preferably a factor of 3

The addition of a protease inhibitor in sufficient concentration corresponding to an amount of 3,000 to 5,000 KIU/ml units of aprotinin to the cryoprecipitate renders the tissue according to the invention suitable for use in patients with severe bleeding disorders. The preferred protease inhibitor is aprotinin, which is commercially available under the trade name Trasylol® or Antagosan®

The cryoprecipitate can be obtained from the patient himself by donating an autologous unit of blood before surgery. This approach prevents the risk of transmission of viral infections by blood transfusions. However, to have a suitable commercial product, the cryoprecipitate must be virus-inactivated. A method for viral inactivation is described in the PCT /EP 91/00503 The basic principle is treatment of the cryoprecipitate with special detergents and removal of the detergent later from the cryoprecipitate

The second component, component B, in the tissue adhesive according to the present invention is produced by a solution of a proteolytic enzyme capable of specifically cleaving fibrinogen. Generally, thrombin has been used which was isolated from plasma from humans or mammals, e.g. from ox This thrombin can be supplied in a lyophilized form The recovery of thrombin occurs with a 40 mmol solution of calcium chloride The preferred concentration of thrombin is 50 to 200 u/ml

For the production of a fast tissue glue, the thrombin solution of roughly 100 u/ml calcium chloride will be prepared. For the production of a slow hm, e.g. when filling a cavity, i.e. tooth extraction or closing the cavity after transphenoidal hypophysectomy, the thrombin will be dissolved further to a concentration of 25 u/ ml with the appropriate calcium chloride solution

Another embodiment of the improved tissue adhesive according to the invention comprises as component B a proteolytic enzyme that is isolated from snake venom. This embodiment is advantageous because patients who have developed antibodies against thrombin can also be treated. Furthermore, patients who have been pre-treated with hepan can be treated with the tissue adhesive in according to the invention, because hepan has no influence on the reaction with the snake venom enzyme In a particularly preferred embodiment of the present invention, the snake venom enzyme used is batroxobin which can be isolated from the South Amencan cave snake Bothrops moujeni Preferably component B contains 0.5 to 10 u/ml of the respective proteolytic enzyme of snake venom

Chemically, batroxobin is a single-chain glycopeptide with a molecular weight of about 36,000. Defibrase® causes the cleavage of is 16 Arg/17 Gly bound in fibrinogen which causes the release of fibrnopeptide A and the formation of monomeric fibrin I

When aprotinin is used in the quantities according to the invention, the tissues that comprise purified fibrinogen, fibronectin and Factor XIII can also be used as component A. The risk of post-bleeding is thereby dramatically reduced

When proteolytic proteases from snake venom are used to produce component B, "conventional" components A that have fibrinogen, fibronectin and Factor XIII can also be used. The use of large amounts of aprotinin according to the invention is preferred. A particularly preferred embodiment is the combination of component A in according to the invention formed from cryoprecipitate with or without large amounts of aprotinin and component B according to the invention which has the proteolytic enzyme isolated from snake venom

The method for producing the fibnn glue according to the invention comprises tnnn with the production of component A which comprises steps of producing a cryosolution from cryoprecipitate,

vimsin activation,

removal of virucidal agent,

addition of the protease inhibitor and

preparation of a suitable protease solution of a protease as component B

A cryopaste is thawed overnight at 4 to 10°C The cryopaste is dissolved in a buffer containing sodium chloride-sodium citrate and glycine and has a pH of 7.0 to 7.2 and then warmed to 30-35°C The cryopaste should dissolve light, otherwise it is not suitable for the preparation The dissolution can be made faster by cutting the cryopaste into small pieces after thawing After cooling the solution to almost room temperature and adjusting the pH to a value of 7.0 to 7.2, aluminum hydroxide is added under examination The precipitate centrifuged and discarded If necessary, a filter cleaning step is carried out The calcium clot is added up to the desired final concentration of calcium chloride

For the virus inactivation, the solution is heated to 30°C Then the detergents are added After sterilizing for some time, the solution is transferred to a virus-free container and left at slightly elevated temperatures for several hours without sterilizing

The virucidal agents are removed by adding a quantity of ncinus oil and gentle steaming for several minutes. Once the oil/water phases have separated, the solution is cooled to room temperature. The aqueous layer is then collected in a virus-proof container, and the oil layer is discarded. The aqueous layer is clarified by the filter pH must be checked to be 7.0 to 7.2 Then the protein solution is pumped through a reverse phase column at room temperature After measuring the protein content (in the range 10 to 60 mg/ml) the eluate is concentrated by ultrafiltration to a protein content of 60 to 100 mg/ml and dialyzed against a buffer which is identical to the buffer mentioned above but which also has a relatively high concentration of calcium chloride Then the protease inhibitor is added A standard filtration is carried out, and the sample is filled and deep frozen in suitable containers

Component B is preferably a freeze-dried protease Particularly preferred is lyophilized thrombin or lyophilized fraction of the South Amencan cave snake Bothrops moujeni The proteolytic enzyme is known under the trade name Reptilase and is the enzyme batroxobin

The proteolytic enzymes are dissolved in a calcium chloride buffer

The application of the two components A and B is carried out by using a double-syringe technique, e.g. through a plastic coupling. When the two components are mixed, a blood clot will form. The application can occur via a needle or can be sprayed into a three-lumen -catheter Each of the two components is injected into a separate lumen, and an air pressure source in the range of a few atmospheres is connected to the third lumen to spray the mixture

The tissue adhesive according to the invention is advantageous because it can be used in patients with severe blood coagulation disorders and is still cheaper than the known tissue adhesives. Patients with severe hemophilia can also, for example, have teeth extracted without preventive infusions of Factor VIll concentrates with a success rate of over 80% This means that only about 1/5 of the patients need infusions due to post-extraction bleeding Furthermore, such patients who have been pre-treated with hepan can be treated with the tissue according to the invention Another advantage is that people who have formed antibodies against thrombin, the second component of the tissue can be treated with a tissue glue according to the invention, where thrombin is substituted with a protease from snake venom, especially Defibrase®, which is late protease batroxobin isolated from the venom of the South Amencan cave snake Bothrops moujeni

The invention is further described in the following examples

Human fibnogen (brand L) was from Kabl (Stockholm), bovine thrombin from Merz-Dade Chromogenic substrate (N.-α-benzoyl-DL-arginine-p-nitroanihd (BAPNA) and analytical brand reagents were from Sigma (St Louis, MO) Reagents and salts were diluted with 0.15M tris, 0.15M NaCl, at pH 7.4 Fibrinogen was dialyzed in tns buffer with concentration determined from Abs2go using a conversion factor of E<1%>280 = 15

Ox thrombin was from commercial sources (Merz-Dade or Parke Davis) with activity graded by the manufacturer Reptilase®, a snake venom releasing only FPA was from Pentapharm (Basel) The proteolytic activity of Reptilase® was normalized to the activity of thrombin by comparing the rates of proteolysis t a non-specific chromogenic substrate BAPNA (0.25 mM) at 37°C in tns/saline, pH 8.0, measured at 405 nm 115 min

On the basis of their esterolytic activity, the unit activity of the reptilase was normalized to the unit activity of thrombin

Fibnn glue was essentially formed by a double syringe method with pure or cryoprecipitate fibnnogen substrate in one syringe, and reptilase (20 U/ml) or thrombin with CaCl2 (20 mM) in the other

Coagulation time (CT) was determined with a Research Model 300-R ACL Coagulation Analyzer (IL, Milan) Viscoelasticity (TEG) was determined on a 3-channel Heihger Thromboelastograph at 37°C Breakdown strength (BS) of the adhesives (in grams) was determined by mixing the adhesive components between two pieces of coarse woven synthetic fiber (0.5 x 1 cm), allowing the formation of tissue to be totally woven between the two pieces of coarse mesh and after 2 hours at 24°C the assembly of mesh- glue-mesh pulled apart using an Accuforce Cadet Tensionometer

(AMATEK, Mansfteld & Greene, USA)

Sterile cryoprecipitate (cryoprecipitate) was prepared from frozen (-30°C) human plasma that was thawed at 4°C, and supernatant plasma removed. Five such units were collected to determine protein, and fibronogen concentrations were determined by the "Buiref method before and after clumping of the cryoprecipitate (diluted 1 5)

with 2 U/ml thrombin Factor XIII was determined by measuring [^H]-putrescine incorporation into dimethylated casein after activation of the samples with 4 U/ml ox thrombin, 10 mm , 22°C

A noticeable feature of the CT fibrinogen curve is that it is biphasic for a fixed level of thrombin or reptilase (ie 1 U/ml, Fig 1 A) and reaches a minimum in the 11 - 8 mM fibrinogen range. This is somewhat different from the maximum turbidity (after 10 mm ) which peaks in the range of 20 to 40 mM fibrinogen. forming a plateau above at higher levels

The development of viscoelasticity in pure fiber is somewhat slower than its turbidity Ca(ll) is a major cofactor in gel re-strengthening through factor Xlla-induced covalent blocking of protein chains Such gel cross-linking is a major source of mechanical strength of the gel, forming a plateau after 20 mm

A comment on the ability of reptilase to induce Factor XIIla activity seems appropriate

Protein levels for collected cryoprecipitate

Pooled cryo prepared from 5 units gave the following average values

Coagulation rates

The clotting time (CT) of cryo is linearly dependent on thrombin or reptilase levels, whereas, above 3 U/ml, increasing enzyme levels exert little effect on CT For a fixed level of enzyme, serial dilution of cryo produces a biphasic CT curve equivalent to the fibnogen dependence seen in the pure fibnn system

Viscoelasticity (TEG) and breakdown strength (BS) of cryo-adhesives

The development of viscoelasticity of cryoglue was investigated with either thrombin or reptilase This parameter takes much longer to develop than turbidity However, cryoglue prepared with excess CaCl2 and either thrombin or reptilase achieves equivalent TEG values in roughly the same time frame It appears that after the initial application of gel formation, Factor Xlla-induced cross-linking supports up under the gel fiber structure, so that the TEG values for both adhesives converge within 1 hour Similarly, the final BSs in both cryo-adhesives were formed with an excess of CaCl2 Both cryo-glues break at 50 to 60 g These experiments indicate that the gel fibers in the glue are re-reinforced by Factor Xlla-induced covalent cross-linking

Preparation of a cryo-solution

Commercially, cryopaste is dried overnight at 4-10°C. 1 kg of cryo is dissolved in 2 liters of buffer A (120 mM/l NaCl, 10 mM/l sodium citrate, 120 mM/l glycine and pH 7.0 to 7.2 ) and preheated to 30-35°C The cryopaste should dissolve easily, otherwise it is not suitable for the preparation To speed up the dissolution, cut the body paste into small pieces after thawing Then the solution is cooled to 20°C to 22°C, and the pH is checked If necessary, it must be adjusted to pH 7.0 to 7.2 by adding diluted sodium hydroxide or acetic acid. Add 100 ml of aluminum hydroxide and stir in

30 min more The precipitate is centrifuged and discarded The supernatant is filtered using a 1 fjm filter 0.1 M/l CaCI2 is added to give a final concentration of Ca<2+> of 1 mM/l Again the pH must be checked

Virus inactivation

The solution is heated to 30°C 1% w/v TNBP and 1% w/v Tnton® X 100 are added The mixture is gently stirred for 11/2 hours The solution is then transferred to a virus-fn container and left at 30°C for 13.5 hours without x-ray

Removal of antiviral agents

150 ml of ncinus oil is added to the mixture prepared as described above and stirred gently for 30 min. While waiting for the oil/water separation (30 to 45 min ), the solution is cooled to 20°C. The aqueous layer is extracted into a virus-fn container, while the oil layer is discarded The aqueous layer is clarified by filtration on a 1 pm/0.45 fjm filter cascade The protein solution is then pumped through a reverse phase column (C-18 column) at a rate of 3 I/hr at room temperature The throughput is measured by UV and collected until the absorbance has returned to 50% The fraction contains roughly 40 mg/ml as measured in a protein measurement

The eluate is concentrated by ultrafiltration to a protein content of 70-80 mg/ml and dialysis against a sufficient amount of a buffer B (same ingredients as buffer A but in addition 1 mM/l calcium chloride) Then 4 million KIU of aprotinin per liter of solution is added Afterwards a Stenl filtration by using a 0.45 j/m + 0.2 fjm cascade The solution is then filled and deep-frozen in plastic bags, possibly lyophilized

Preparation of a thrombin solution

Lyophilized thrombin is dissolved in a solution of 40 mM/l calcium chloride The amount of thrombin is 100 U/ml in the glue For a fast-acting glue, e.g. for spraying the glue on the wound area, a thrombin solution of 100 U/ml in calcium chloride will be sufficient For a slow glue, e.g. filling a hole during a tooth extraction or closing the hole after a transphenoidal hypophysectomy, thrombin will be further dissolved to a final concentration of 25 U/ml by adding large amounts of CaCU

The production of reptilase is hk the production of thrombin However, the amount of reptilase is roughly 2 U/ml

Report from a clinical case

The patient (a 21-year-old man), suffered from the age of 21 years of severe bleeding diathesis due to acquired inhibitor against thrombin No underlying disease (i.e. tumor or autoimmune disease) could explain this problem Laboratory test, confirmed by two external laboratories, indicated that MY had high levels of anti-thrombin IgG antibody In the past year he had suffered from repeated attacks of renal colic caused by a large stone in the left renal pelvis Elective htotnpsi by ultrasound was planned Based on the technique that IgG binds to protein- A-affinity columns, the patient was placed on immunosuppressive therapy combined with extracorporeal immunoadsorption After 8 treatments, where 6 liters of the patient's plasma were passed through passage on the protein-A column, the inhibitor titer was reduced by 98% This was determined by measuring the thrombin time (TT ) for normally collected plasma, with pre- and post-affinity-purified MJ plasma Nevertheless, TT as well as PT and APTT values were prolonged On the at that time the kidney stone moved clockwise and caused complete blockage of the kidney followed by hydronephrosis. The patient received 10 more immunoadsorption treatments (about 80 liters of plasma) followed by intensive plasmapheresis (about 50 liters) and high dose immunoglobulin infusion (2 g/ kg) At this point thrombin inhibitor level decreased to 0.5% PTT was decreased to 47 sec (vs 85-90 sec pretreatment) and TT was 35 sec (vs 90 sec pretreatment and 27 sec normal control) It was decided to remove the stone in surgery, using biological adhesive (cryo-glue) made from cryoprecipitate and high levels (200 U/ml) of thrombin With this mixture, the cryogen gelled immediately when sprayed However, gelling did not occur in the patient and local hemostasis was achieved by to suture At the end of the surgery the wound looked "dry" Nevertheless, 6 hours later the patient was bleeding from the surgical drains Immunoabsorption of 10 liters of plasma was performed but without effect on the bleeding which actually increased

The patient was reoperated to find the source of bleeding However, no surgical bleeding was found Diffuse bleeding was observed from the entire wound surface area At this point a mixture of cryo and reptilase (2 U/ml, Defibrase) was sprayed onto the wound The spray immediately clumped, the wound surface appeared turbid and bleeding stopped The patient continued to receive daily immuno-adsorption therapy for 5 more days, without bleeding This shows the advantage of using the snake protease as component B of the tissue glue according to the invention

Claims (13)

1 Two-component tissue adhesive with two separate components A and B, characterized in that it comprises component A where component A comprises a cryoprecipitate from whole blood and an amount of protease inhibitor corresponding to aprotinin in amounts of 3000 to 5000 KIU/ml units, said component A can is achieved by thawing of cryopasta, solution in a buffer at pH 7.0 to 7.2, preheating to 30 to 35°C, adjusting the pH to 7.0 to 7.2, addition of aluminum hydroxide under the X-ray, centrifugation and removal of precipitate, addition of CaCb, virus inactivation, concentration by ultrafiltration to a protein concentration of 60 to 100 mg/ml, addition of an amount of protease inhibitor corresponding to 3000 to 5000 KIU/ml units of aprotinin, and a component B comprising a proteolytic enzyme capable of cleaving fibrinogen specifically in the presence of component A and causing the formation of a fibrin polymer 2 Tissue mucus according to claim 1, characterized in that the protease inhibitor is aprotinin in amounts of 3000 to 5000 KIU/ml units 3 Tissue mucus according to any of claims 1 and 2, characterized in that the proteolytic enzyme is thrombin derived from mammals or humans 4 Tissue slime according to claim 1, characterized in that it comprises a component A which comprises a concentrated cryoprecipitate of whole blood, and a component B which comprises a proteolytic enzyme which can be obtained from snake venom, which enzyme is capable of cleaving the fibnogen present in component A , specifically and cause the formation of a fibrin polymer 5 Tissue slime according to claim 4, characterized in that the snake venom enzyme is batroxobin isolated from the venom of the South Amencan cave snake Bothrops moujeni 6 Tissue mucus according to claims 4 or 5, characterized in that it has a high amount of protease inhibitor, preferably aprotinin, corresponding to 3000 to 5000 KIU/ml units of aprotinin 7 Tissue mucus according to any one of claims 1 to 6, characterized in that the cryoprecipitate is virus inactivated 8 Process for the production of fibrin in accordance with claims 1 to 7, characterized by thawing of cryopaste, solution in a buffer at pH 7.0 to 7.2, preheating to 30 to 35°C, adjusting the pH to 7.0 to 7.2, addition of aluminum hydroxide under the X-ray, centrifugation and removal of precipitate, addition of CaCb, vimsin activation, concentration by ultrafiltration to a protein concentration of 60 to 100 mg/ml, addition of an amount of protease inhibitor corresponding to 3000 to 5000 KIU/ml units of aprotinin, and preparing solution of a suitable protease for component B as defined in claims 1 to 7 9 Tissue mucus according to claim 1, characterized in that it comprises as component A fibronogen, fibronectin and factor XIII and a protease inhibitor corresponding to an aprotinin amount of from 3000 to 5000 KIU/ml 10 Tissue composition according to claim 9, characterized in that component B is a proteolytic enzyme according to claims 4 and/or 5 or thrombin 11 Application of an amount of protease inhibitor corresponding to 3000 to 5000 KIU/ml units of aprotinin in combination with concentrated cryoprecipitate which can be obtained by thawing of cryopasta, solution in a buffer at pH 7.0 to 7.2, preheating to 30 to 35°C, adjusting the pH to 7.0 to 7.2, addition of aluminum hydroxide under the X-ray, centrifugation and removal of precipitate, addition of CaCI2, virus inactivation, concentration by ultrafiltration to a protein concentration of 60 to 100 mg/ml, for the production of a tissue adhesive according to one of claims 1 to 7 12 Use according to claim 11, in which the protease inhibitor is aprotinin 13 Use of concentrated cryoprecipitate for the production of tissue glue according to any one of claims 1 to 6