CZ294292A3 - Tissue adhesive containing cryo precipitate, process of its preparation and the use of aprotinin, protease from snake poison and batroxobin for preparing such adhesive - 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

(57) The subject matter of the invention is a tissue glue which comprises component A comprising whole blood cryoprecipitate and a high amount of protease inhibitor, corresponding to 3 000 to 5 000 KlU / ml aprotinin and component A.

B containing a proteolytic enzyme that is able to specifically cleave the fibrinogen present in component A to form. fibrin polymer. In another embodiment, the present invention provides a tissue adhesive comprising component A comprising whole blood cryoprecipitate and component B comprising a proteolytic enzyme obtainable from snake venom, which enzyme is capable of specifically cleaving the fibrinogen present in component A, formation of a fibrin polymer.

Tissue adhesive and process for its manufacture

Technical field

BACKGROUND OF THE INVENTION The present invention relates to a tissue glue comprising 2 components A and Ba of a method for manufacturing the tissue glue. Further, the invention relates to the use of high amounts of aprotinin and the use of a snake venom proteolytic enzyme for the manufacture of this tissue adhesive.

BACKGROUND OF THE INVENTION

Improving local hemostasis at the site of a surgical wound with plasma proteins is a well-known idea. For example, fragments of fibrin are used for hemostasis in brain surgery. Blood plasma and thrombin were used to make a fibrin film to cover surgical wounds. In the last 20 years, there have been a number of publications describing the use of tissue glue, fibrin adhesive or fibrin sealant in most surgical disciplines. Over the last 10 years, commercially available formulations have been widely used in Europe as these adhesives. Such adhesives consist of two components which, when mixed together, form a piece of precipitate, the so-called thrombus. The first component is fibrinogen concentrate. This concentrate also contains fibronectin and factor XIII, which are important for thrombus stabilization and strength. The second component is thrombin, the active enzyme converting fibrinogen, i.e. the last component of the normal coagulation system to fibrin thromb. This procedure bypasses most stages of normal coagulation and mimics its last phase. Some manufacturers add plasminogen, an enzyme that causes lysis of thrombus after some time, while other manufacturers add aprotinin, a protease inhibitor, to prevent thrombus lysis.

in patients patients with

While these products provide satisfactory results in patients with mild bleeding bleeding, however, with severe disorders of this type such as haemophilia A or B, there is still a high risk of postoperative bleeding. Sometimes they experience delayed complications of bleeding only a few days after surgery. Also, patients who are treated with anticoagulant factors cannot receive the prior art tissue glue. Another significant disadvantage of commercially available concentrates is their high production cost.

PCT application WO 86/01814 discloses a process for producing a cryoprecipitated suspension comprising fibrinogen and factor VIII, which is a useful precursor in the manufacture of fibrin adhesive. In this process, a) fresh frozen plasma from a single donor, which may be a human or other animal that has been inspected for blood-borne diseases, is frozen for at least about 6 hours at about -80 hours ° C; b) raising the temperature of the frozen plasma, for example to a value in the range of about 0 ° C to room temperature, to form a supernatant and a cryoprecipitated suspension containing fibrinogen and factor VIII; and c) isolating the cryoprecipitated suspension. Also disclosed is a method of making a fibrin glue useful in surgical procedures, which method is performed by a) producing the cryprecipitated suspension described above; b) applying a defined volume of said suspension to the desired site; and c) applying to this site a composition containing sufficient thrombin to convert the fibrinogen in suspension into a fibrin glue that solidifies.

EP-A-0 341 007 discloses a surgical adhesive which in an aqueous preparation comprises a patient's own plasma, collagen, thrombin and optionally an antifibrinolytic agent. This adhesive is formed from the patient's own plasma, additional fibrinogen reagents. This adhesive is a two-component mixture that is mixed using.

without the use of any concentration or isolation usually processed to just immediately before

EP-A-0 253 198 discloses a one-component tissue glue comprising an aqueous solution of fibrinogen, factor VIII, a thrombin inhibitor, prothrombin factors, calcium ions and optionally a plasmin inhibitor. The tissue glue can be reconstituted from the lyophilized sample by the addition of water. The tissue glue may contain all the active substances in pasteurized form to prevent the transmission of hepatitis and HTLV III.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a tissue glue that is also suitable for patients with severe blood coagulation disorders, such as patients suffering from haemophilia A or B. It is another object of the present invention to provide a tissue glue that can also be used in patients They have already developed antibodies against bovine thrombin, which is an active factor of component B. Another object of the invention is to provide a tissue adhesive for patients who are treated with anticoagulant factors such as heparin. Given the risk of transmission of viral diseases by tissue glue components, it must be guaranteed that viruses will be inactivated in these components.

The tissue glue of the present invention comprises a component A comprising a whole blood cryoprecipitate and a high amount of protease inhibitor corresponding to 3000 to 5,000 units of KIU / ml aprotinin, preferably aprotinin and a component B comprising a proteolytic enzyme capable of specifically cleaving fibrinogen present in component A and ensure the formation of a fibrin polymer.

A commercially available cryoprecipitate may be used to make the tissue glue of the present invention. However, it may be advantageous to concentrate the cryoprecipitate 2 to 5 times, preferably 3 times.

The addition of a protease inhibitor at a concentration of 3,000 to 5,000 units KIU / ml aprotinin to the cryoprecipitate makes the tissue glue of the invention suitable for use in patients suffering from severe bleeding. The preferred protease inhibitor is aprotinin, which is a product which is commercially available under the trademark Trasylol @) b _{no of} AntagosanR ^^.

The cryoprecipitate may be from a patient who donates blood before surgery. This approach avoids the risk of transmission of viral diseases by blood derivatives. However, viruses must be inactivated in the cryoprecipitate to produce a suitable commercial product. A method for inactivating viruses is described in PCT / EP 91/00503. The essence of this process is to treat the cryoprecipitate with special detergents and to remove the detergent lateron from the cryoprecipitate.

The second component, component B, of the tissue glue of the present invention is prepared by dissolving a proteolytic enzyme that is capable of specifically cleaving fibrinogen. Typically, thrombin is used which has been isolated from human or mammalian plasma, such as cattle. The thrombin may be supplied in lyophilized form. Thrombin is reconstituted with a 40 mM calcium chloride solution. The preferred thrombin concentration is 50 to 200 u / ml.

For the production of quick tissue adhesives, it is suitable to produce a thrombin solution of about 100 µ / ml in a calcium chloride solution. For the production of a slow adhesive, which may for example be used to fill cavities during tooth extraction or to seal the cavity after removal of the pituitary gland, the thrombin may be further diluted with a suitable calcium chloride solution to a concentration of 25 µ / ml.

In another embodiment, the improved tissue adhesive of the invention comprises as component B a proteolytic enzyme that is isolated from snake venom. This embodiment is advantageous if patients who have developed anti-thrombin antibodies are to be treated. In addition, patients pretreated with heparin can be treated with such tissue glue according to the invention since heparin does not affect the snake venom enzyme response. In a particularly preferred embodiment of the present invention, batroxobin is used as a snake venom enzyme that can be isolated from the South American snake Bothrpos moujeni. Component B preferably contains 0.5 to 10 µ / ml of such a snake venom proteolytic enzyme.

Chemically, batroxobin consists of a single chain glycopeptide with a molecular weight of approximately 36,000. Defibrase ( ^R ) causes cleavage of alpha 16 Arg / 17 Gly, resulting in the release of fibrinopeptide A and the formation of monomeric fibrin I.

When large amounts of aprotinin are used according to the invention, purified fibrinogen, fibronectin and factor XIII can also be used as component A of the tissue adhesive of the invention. The risk of additional bleeding is dramatically reduced in this case.

Using proteolytic proteases from snake venom in the production of component B, it is also possible to use conventional components A containing fibrinogen, fibronectin and factor XIII. Use large. amounts of aprotinin are preferred according to the invention. Particularly preferred is an embodiment of the invention which combines component A of the invention derived from cryoprecipitate with or without the use of a high amount of aprotinin with component B of the invention comprising a proteolytic enzyme isolated from snake venom.

The present invention also provides a method of making the fibrin adhesive of the present invention. In this process, component A is produced by converting the cryoprecipitate into a cryoprotectant in several steps, including

- virus inactivation,

- removal of the virucidal agent,

addition of a protease inhibitor, and

- producing a suitable protease solution.

Preferably, the cryopaste is thawed overnight at 4-10 ° C and then dissolved in a buffer containing sodium chloride, trisodium citrate and glycine at pH 7.0-7.2, then the solution is heated to 30 ° C. to 35 ° C. The cryopaste should dissolve readily, otherwise it is not suitable for this preparation. Dissolution can be accelerated by cutting the cryopaste into small pieces before melting. The solution is then cooled to near room temperature and the pH is adjusted to 7.0 to 7.0

7.2 and aluminum hydroxide was added with stirring. The precipitate is centrifuged and discarded. If necessary, a filtration step is included. Calcium chloride is then added in an amount corresponding to its desired final concentration.

Virus inactivation is carried out as follows: The solution is heated to 30 ° C, detergents are added, the mixture is stirred for some time, and then the solution is transferred to a virus-free container where it is left without stirring for several hours at elevated temperature.

The virucidal agents are removed by the addition of castor oil and gentle mixing for several minutes, after which the oil phase is allowed to separate from the aqueous phase. The separated aqueous solution is cooled to room temperature and transferred to a virologically safe container. The oil phase is discarded. The aqueous phase is clarified by filtration, maintaining the pH between 7.0 and 7.2. The protein solution was then pumped through the reverse phase column at room temperature. The protein content is measured (ranging from 10 to 60 mg / ml) and the eluate is concentrated by diafiltration to a protein content of 60 to 100 mg / ml and then dialyzed against a buffer which is the same as the buffer mentioned above but contains a relatively high concentration calcium chloride. The protease inhibitor is then added and the mixture is filtered under sterile conditions. The samples are placed in suitable containers and deep-frozen.

Component B is preferably a lyophilized protease. Particularly preferred is a lyophilized thrombin or a lyophilized snake venom fraction which originates from the South American species of the snake Bothrpos moujeni. This proteolytic enzyme is known as Reptilase and is a batroxobin enzyme.

Proteolytic enzymes are dissolved in a calcium chloride buffer.

Application of components A and B is carried out using a technique with two syringes, which are, for example, connected in a plastic connector. When both components are mixed, a thrombus clot is formed. Administration can be by cannula or by injection with a three-vein catheter. Two separate adhesive components are injected into the two cores and a compressed air source (pressure in the range of several tenths of MPa) is used to spray the mixture.

The tissue glue of the invention has the advantage that it can be used in patients with severe blood coagulation disorders cheaper than known tissue adhesives. For this reason, patients with severe haemophilia may be subjected to surgical procedures such as tooth extraction without preventive infusion of factor VIII concentrate, with a success rate above 80%. This means that only about one fifth of patients require infusion, due to bleeding after extraction. In addition, patients previously treated with heparin can be treated with the tissue glue of the present invention. Another advantage is that by using tissue glue according to the invention wherein thrombin is substituted by a protease from snake venom especially Defibrase ^ ^R which is the serine protease batroxobin isolated from the venom of the South American pit viper Bothrpos moujeni, it is possible to treat patients who have developed antibodies against thrombin as a second component of tissue glue.

The invention is illustrated by the following examples. These examples are illustrative only and are not to be construed as limiting the scope of the invention in any way.

DETAILED DESCRIPTION OF THE INVENTION

Human fibrinogen (L-grade) comes from Kabi (Stockholm, Sweden), bovine thrombin from Merz-Dade. The chromogenic substrate, Na-benzoyl-DL-arginine-p-nitroanilide (ΒΑΡΝΑ) and analytical grade reagents are from Sigma (St. Louis, Missouri, USA). Reagents and salts were diluted with 0.015M Tris buffer, 0.15M NaCl, pH 7.4. Fibrinogen is dialyzed in Tris buffer, its concentration being determined from Abs ₂ g ₀ , using a conversion factor E of ^1% 280 = 15.

Beef thrombin comes from commercial sources (Merz-Dade or Parke Davis) and has a degree of activity given by the manufacturer. Reptilase, a snake venom that releases only FPA, is from Pentapharm (Basel, Switzerland). The proteolytic activity of Reptilase ^{® R} is normalized to thrombin activity by comparing proteolysis rates of non-specific chromogenic substrates ΒΑΡΝΑ (0.25 mM) at 37 ° C in a mixture of Tris buffer and sodium chloride solution at pH 8.0, followed by 15 minutes at 405 nm.

Based on esterolytic activity, the unit activity of Reptilase is normalized to thrombin activity.

The tissue glue is essentially made using a two syringe method. One syringe contains pure or cryoprecipitate fibrinogen substrate and the other contains Reptilase (20 U / ml) or thrombin with calcium chloride (20mM).

The Clotting Time (CT) is determined using a Research Model 300-R ACL Coagulation Analyzer (IL, Milan, Italy). Viscoelasticity (TEG) was determined using a Heiliger Thromboelastograph 3-channel apparatus at 37 ° C. The tensile strength (BS) of the adhesive, expressed in grams, is determined by placing the adhesive components between two pieces of roughly interwoven synthetic fiber mesh (0.5 χ 1 cm), leaving a gel that completely penetrates the two pieces of mesh . After two hours at 24 ° C, the tear force of the sieve-glue-sieve is measured using an Accuforce Cadet Tensionometer (AMATEK, Mansfield & Greene, USA).

Sterile cryoprecipitate (cryo) is prepared from frozen (-30 ° C) human plasma, which is thawed at 4 ° C, after which the plasma forming supernatant is separated. Four unit amounts were pooled to determine protein and fibrinogen concentrations using the Buiret method before and after precipitation of the cryoprecipitate (diluted 1: 5) with 2 U / ml thrombin. Factor XIII is determined by measuring the incorporation of ( ³ H) -putrescine into dimethylated casein, after activation of the sample with 4 U / ml bovine thrombin at 22 ° C for 10 minutes.

A noteworthy feature of the coagulation rate vs. fibrinogen (CT-fibrinogen) curve is that it is biphasic at a predetermined level of thrombin or Reptilase (i.e., 1 U / ml), see Figure 1A) and reaches a minimum at 1-8 mM fibrinogen. This value differs somewhat from the maximum turbidity value (after 10 minutes), an experiment of 20 to show the mM fibrinogen dependence. Conversion CT at the level of thrombin or

Reptilase. This curve shows that there is an almost linear inverse of the gelation rate at low enzyme levels (less than 2 U / ml), while at higher levels the gelation rate stabilizes (plateau formation).

The development of viscoelasticity of pure fibrin is somewhat slower than that of its turbidity. Divalent calcium is a major factor in gel reinforcement by covalent protein chains by Factor XIIa.

Such gel cross-linking is a major source of its mechanical strength and stabilizes after 20 minutes.

kofaktorem intertwined

The finding that Reptilase has the ability to induce factor XIIIa seems reasonable.

The pooled cryoprecipitate, prepared from five unit parts, provides the following mean protein concentration values:

protein fibrinogen factor XIII mg / ml 36 mg / ml

4.10 U / ml

Coagulation rates

The rate of thrombus formation, i.e. the coagulation rate (CT) of the cryoprecipitate, is directly proportional to the concentration of thrombin or Reptilase. However, at values above 3 U / ml, increasing enzyme concentration has little effect on the coagulation rate. At the determined enzyme concentration, serial dilution of cryoprecipitate gives a two-phase CT curve? thus, the dependence is the same as in the case of fibrinogen in a pure fibrin system.

Viscoelasticity (TEG) and tensile strength (BS) of cryoprecipitate adhesives

The development of viscoelasticity in cryoprecipitate adhesives was investigated using either thrombin or Reptilase. A longer time is required for the development of this parameter than for the development of turbidity. However, cryoprecipitate adhesives manufactured using excess calcium chloride and either thrombin or Reptilase achieve the same TEG values in about the same time. After the start of gelation, the crosslinking induced by factor XIIIa appears to reinforce the gel fiber structure, so that the TEG values of both adhesives converge within 1 hour. This is similar to the final BS value of both cryoprecipitate adhesives formed in the presence of excess calcium chloride. The tear of both cryoprecipitate adhesives occurs at 50 to 60 g. These experiments show that the gel fibers in the adhesive are reinforced by covalent cross-linking, which is induced by factor XIIa.

Production of cryosolution

A commercially available cryopaste is pre-melted overnight at 4-10 ° C. 1 kg cryoprecipitate is dissolved in 2 L of buffer A (120 mmol / l NaCl, 10 mmol / l trisodium citrate, 120 mmol / l glycine, pH 7.0-7.2) and preheated to 30-35 ° C. The cryopaste should dissolve easily, otherwise it is not suitable for preparation. To accelerate dissolution, the cryopaste can be broken into small pieces before thawing. The solution was then cooled to 20-22 ° C and the pH was measured. The pH may optionally be adjusted to lie in the range of 7.0 to 7.2 by addition of dilute sodium hydroxide or acetic acid. 100 ml of aluminum hydroxide are added to the mixture and stirring is continued for 30 minutes. The precipitate is centrifuged and discarded. The supernatant is filtered using a 1 µm filter. Calcium chloride is added to maintain a final calcium ion concentration of 1 mmol / L. The pH value should be checked again.

Virus inactivation

The solution was warmed to 30 ° C and TNBP at 10g / L and Triton X 100 at 10g / L were added. The mixture was gently stirred for 0.5 hour and the resulting solution was transferred to a virus-free container where it was left without stirring at 30 ° C for 3.5 hours.

Removal of virucidal agents

To the mixture prepared as above was added 150 ml of castor oil, and the resulting mixture was gently stirred for 30 minutes. The solution is cooled to 20 ° C and the oil phase is separated from the aqueous phase (30 to 45 minutes). The aqueous layer is transferred to a virologically safe container and the oil layer discarded. The aqueous layer was clarified by filtration on a 1 and 0.45 µm filter cascade. The protein solution is then pumped through a reverse phase column (column C-18) at a flow rate

1 / h, at room temperature. The eluate is monitored by UV irradiation and collected until absorbance returns to

%. The measured protein concentration is about 40 mg / ml.

The eluate was concentrated by diafiltration to a protein content of 70-80 mg / ml and dialyzed against a sufficient amount of Buffer B (the same ingredients as for Buffer A, but additionally 1 mmol / L calcium chloride). 4 Mio is then added. The KIU of aprotinin per L of solution and the solution is filtered under sterile conditions using a filter cascade with filters having a pore size of 0.45 and 0.2 µm. The solution is then filled with plastic bags, which are deep-frozen (and optionally lyophilized).

Preparation of thrombin solution

The lyophilized thrombin was dissolved in a 40 mM calcium chloride solution. The amount of thrombin in the adhesive is 100 U / ml. For a fast-acting adhesive, which is applied, for example, by spraying on the wound area, a thrombin concentration of 100 U / ml in calcium chloride solution will suffice. For a slow adhesive to be used, for example, to fill the cavity after tooth extraction or to seal the cavity after the pituitary gland is removed, the thrombin solution is further diluted to a final concentration of 25 U / ml by adding more calcium chloride solution.

The preparation of the Reptilase solution is similar to the preparation of the thrombin solution. However, the amount of Reptilase is approximately 2 U / ml.

Clinical report planned techniques, after cleaning on both PT and APTT's

The patient, age 21, has a strong tendency to bleed from the obtained thrombin inhibitor. This problem cannot be explained by any associated disease (i.e., cancer or autoimmune disease). A laboratory test, confirmed by two external laboratories, shows that the patient has a high level of anti-thrombin IgG antibodies. Over the past year he has had repeated attacks of kidney colic caused by a large stone in the left kidney pelvis. Electro lithotripsy was an ultrasound. Based on the fact that IgG binds to the protein affinity column

And, the patient was subjected to immunosuppressive treatment associated with extracorporeal immunoadsorption. After eight treatment cycles in which 60 L of the patient's plasma was processed by passing through the protein A column, the inhibitor titer decreased by 98% as determined by measuring the thrombin time (TT) of the patient's normal pooled plasma prior to and the affinity column. However, TT values as well have prolonged. At this time, the kidney stone moved to the urethra and caused complete blockage of the kidney, accompanied by hydronephrosis. The patient was subjected to 10 additional cycles of immunoadsorption (about 80 L of plasma), followed by intensive plasmaferesis (about 50 L) and a high dose immunoglobulin (2 g / kg) infusion. At this point, the level of thrombin inhibitor decreased to 0.5%. PTT decreased to 47 seconds (versus 85 to 90 seconds before treatment) and TT was 35 seconds (compared to 90 seconds before treatment and 27 seconds for normal control). It was decided to remove the stone surgically using a biological adhesive (cryoprecipitate adhesive) made from cryoprecitrite and a large amount (200 U / ml). However, the patient did not gelate hemostasis was achieved by stitching. The wound looked dry after the surgery. However, after thrombin. for immediate and local hours, the patient began bleeding from surgical drainage. Immunoadsorption of 10 L of plasma was performed, but without any effect on the bleeding, which increased.

The patient was reoperated to determine the source of bleeding. No surgical bleeding was found, but diffuse bleeding from the entire wound surface. A mixture of cryoprecipitate and Reptilase (2 U / ml; Defibrase) was now injected onto the wound. The spray mixture immediately formed a clot, the surface of the wound became cloudy and the bleeding stopped. The patient was subjected to immunoadsorption treatment for a further 5 days with no bleeding. This assay clearly demonstrates the advantages of using a snake venom protease as component B of the tissue adhesive of the invention.

Claims (12)

PATENT CLAIMS CLAIMS 1. Tissue adhesive comprising component A comprising whole blood cryoprecipitate and a high amount of protease inhibitor corresponding to 3000 to 5000 KUU / ml aprotinin and component B comprising proteolytic enzyme, fibrinogen present polymer. which is able to cleave specifically in component A to form fibrin 2. Tissue adhesive according to Claim 1 characterized is three m that component A is concentrated. 3. Tissue adhesive according to Claim 1 characterized is three wherein said protease inhibitor is aprotinin, which is present in an amount from 3 000 to 5 000 KUU / ml. 4. Tissue glue according to any from claims 1 to 3, characterized is three m that proteolytic the enzyme is thrombin of mammalian or human origin. 5. A tissue adhesive comprising component A comprising whole blood cryoprecipitate and component B containing a proteolytic enzyme obtainable from snake venom, which enzyme is capable of specifically cleaving the fibrinogen present in component A to form a fibrin polymer. 6. The tissue glue of claim 5, wherein the snake venom enzyme is batroxobin isolated from the venom of a South American snake of the species Bothrpos moujeni. Tissue adhesive according to claim 5 or 6, characterized in that it contains a high amount of protease inhibitor, preferably aprotinin, in an amount corresponding to 3,000 to 5,000 KUU / ml aprotinin. ‘ Tissue adhesive according to any one of claims 1 to 7, characterized in that the viruses in the cryoprecipitate are inactivated. A process for the manufacture of a fibrin adhesive according to any one of claims 1 to 8, characterized in that it is: - converting the cryoprecipitate into a cryoprotectant to form component A, - virus inactivation is carried out, - remove virucidal agents, - protease inhibitor is added and an appropriate solution of the appropriate protease is prepared as component B. 10. Tissue adhesive comprising as component A fibrinogen, fibronectin and factor i XIII and a protease inhibitor in an amount corresponding to 3000 to 5000 KUU / ml aprotinin. Tissue adhesive according to claim 10, characterized in that component B is a proteolytic enzyme according to claim 5 and / or 6 or thrombin. Tissue adhesive comprising as component A fibrinogen, fibronectin and factor XIII and as component B a proteolytic enzyme according to claim 5 and / or 6. Use of a high amount of aprotinin in the range of 3000 to 5000 KUU / ml in combination with a cryoprecipitate or a combination of fibrinogen, fibronectin and factor XIII for the manufacture of tissue glue. Use of a snake venom protease for the manufacture of tissue glue. Use of batroxobin isolated from the snake Bothrpos moujeni for the manufacture of tissue glue.