SK294292A3 - Tissue adhesive and method of its production - 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

Tissue adhesive and process for its production

Technical field

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

BACKGROUND OF THE INVENTION

The improvement of local hemostasis to local surgical wounds by plasma proteins is a well known idea. For example, a piece of fibrin is used for hemostasis in brain surgery. Blood plasma and thrombin were used for the production of 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 such adhesives. Such adhesives consist of two components which, when mixed together, form a precipitate piece, the so-called " Thromb. 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, an effective fibrinogen converting enzyme, i. the last component of a normal coagulation system for fibrin thromb. This procedure bypasses most stages of normal coagulation and mimics its last phase. Some manufacturers have added plasminogen, an enzyme that causes lysis of thrombus for some time, while others have added aprotinin, a protease inhibitor, to prevent glacial throbbing.

in patients patients with

Although these products provide satisfactory results in patients with mild bleeding bleeding but 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 until a few days after surgery. Also, patients who are treated with anticoagulant factors cannot be given prior art tissue glue. Another significant drawback of commercially available concentrates is their high production cost.

PCT Application WO 86/01814 describes a process for the production of a cryoprecipitated suspension containing fibrinogen and factor VIII, which is a useful precursor in the manufacture of fibrin adhesive. This is done by: (a) fresh frozen plasma from a single donor, which may be a human or other animal that has been inspected for at least about 6 hours of blood-borne diseases, is frozen at approximately - 80 ° 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 process is performed by: a) producing the cryoprecipitated suspension described above; b) applying a defined volume of said suspension to the desired site; (c) a preparation containing a sufficient amount of thrombin is applied to this site to convert the fibrinogen in suspension into a fibrin glue which solidifies.

EP-A-0 341 007 discloses a surgical adhesive which comprises in an aqueous formulation 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 which is mixed using.

without the use of any concentration or isolation usually processed to only 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. Tissue glue may contain all active substances in pasteurized form to prevent 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 It is a further object of the invention to provide a tissue glue 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 whole blood cryoprecipitate and a high amount of protease inhibitor corresponding to 3000-5000 units KUU / ml aprotinin, preferably aprotinin and a component B comprising a proteolytic enzyme capable of specifically cleaving the fibrinogen present in component A and ensure the formation of a fibrin polymer.

A commercially available cryoprecipitate can be used to make the tissue glue of the 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 KUU / ml aprotinin to the cryoprecipitate makes the tissue glue of the invention suitable for use in a patient suffering from severe bleeding. A preferred protease inhibitor is aprotinin, a product available under the tradename Trasylol ( ^R ) or Antagosan ( ^R ).

The cryoprecipitate may be from a patient who donates his / her own blood before surgery. This approach avoids the risk of transmitting viral diseases with blood derivatives. However, viruses must be inactivated in the cryoprecipitate to produce a suitable commercial product. A method of virus inactivation 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 laterone from the cryoprecipitate.

The second component, component B, of the tissue adhesive of the present invention is prepared by dissolving a proteolytic enzyme that is capable of specifically cleaving fibrinogen. Usually, thrombin is used which has been isolated from the plasma of a human or mammal, 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 having a concentration of approximately 100 u / ml in a calcium chloride solution. For the production of slow glue that can

For example, to fill cavities in 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 antibodies against thrombin are to be treated. In addition, patients who have been pretreated with heparin can be treated with such tissue glue of 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 is formed by 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 formation of monomeric fibrin I. When large quantities of aprotinin are used in 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. The use of large amounts of aprotinin is 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 protolytic enzyme isolated from snake venom.

The present invention also provides a process for the manufacture of a fibrin adhesive according to the invention. In this process, component A is produced by converting the cryoprecipitate into a cryocrystalline in several stages, which include

- virus inactivation,

- removal of the virucidal agent,

the 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. Then, the solution is cooled to room temperature, 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 filter stage is included. Calcium chloride is then added in an amount corresponding to its desired final concentration.

The virus is inactivated 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 at elevated temperature for several hours.

The virucidal agents are removed by adding castor oil and gently stirring 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 in the range of 7.0 to 7.2. The protein solution is 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. Particular preference is given to a lyophilized thrombin or a lyophilized snake venom fraction originating 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.

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

The tissue glue of the invention has the advantage that it can be used in a patient with severe blood coagulation disorders more cheap 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 success rates above 80%. This means that only about one fifth of the patients require infusion, due to bleeding after extraction. In addition, patients previously treated with heparin can be treated with the tissue glue of the invention. Another advantage is that, with the tissue glue share of the invention wherein thrombin is replaced 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 a patient in a 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 do not limit the scope of the invention in any way.

DETAILED DESCRIPTION OF THE INVENTION

Human fibrinogen (L-grade) comes from Kabi (Stockholm, Sweden), and 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 are diluted with 0.015M Tris buffer, 0.15M NaCl, pH 7.4. The fibrinogen is dialyzed in Tris buffer, the concentration of which is determined from Abs _2θ , using a conversion factor E of ^1% 280 = 15.

Bovine 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 the 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 the 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) is determined using a Heiliger Thromboelastograph triple channel device 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 x 1 cm) leaving a gel that completely permeates the two pieces of mesh . After two hours at 24 ° C, the force required to rupture the web-adhesive-web 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, then 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 was determined by measuring the incorporation of ^H-putrescine into dimethylated casein, after activation of the sample with 4 U / ml bovine thrombin at 22 ° C for 10 minutes.

A notable feature of the curve depending on the rate of coagulation on fibrinogen concentration (CT-fibrinogen) is that it is biphasic at a determined level of thrombin or Reptilase (i.e. 1 U / ml), see FIG. IA) and reaches a minimum at concentrations of 1 to 8 mM fibrinogen. This value differs somewhat from the maximum turbidity value (after 10 minutes) of 20 and the experiment shows the mM fibrinogen dependence. Conversion CT at thrombin or

ReptilaseR. This curve shows that there is an almost linear indirect proportion of the gelation rate at low levels of the enzyme (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 through covalent protein chains by factor XIIa.

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

cofactor intertwining

The finding that Reptilase has the ability to induce factor XIIa appears to be justified.

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

protein fibrinogen factor XIII ng / ml 36 mg / ml

4.10 U / ml

Coagulation rates

Thrombus formation rate, t. j. 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 a determined enzyme concentration, serial dilution of cryoprecipitate provides a two-phase CT curve; thus, the dependence here is the same as that of fibrinogen in a pure fibrin system.

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

The development of viscoelasticity in cryoprecipitate adhesives is 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 produced using an excess of calcium chloride and either thrombin or Reptilase achieve the same TEG values in about the same time. After the initiation of the cross-linking gelation induced by factor XIIIa, it appears that it reinforces the gel fiber structure so that the TEG values of both adhesives converge within 1 hour. This is similar to the final BS of both cryoprecipitate adhesives formed in the presence of excess calcium chloride. Both cryoprecipitate adhesives break at 50 to 60 g. These experiments show that the gel fibers in the adhesive are reinforced by covalent crosslinking, which is caused by

XIIIa.

Production of cryosolution

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 speed up the dissolution, the cryopaste can be divided into small pieces before thawing. The solution was then cooled to 20-22 ° C and the pH was measured. The pH may, if necessary, be adjusted to be 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 was added to maintain a final calcium ion concentration of 1 mmol / L. The pH should be checked again.

Virus inactivation

The solution is warmed to 30 ° C and TNBP at 10 g / L and Triton X 100 at 10 g / L are 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 stirred gently 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 to 0.4 µm filter cascade. The protein solution is then pumped through a reverse phase column (column C-18) at a flow rate of 3 L / h, at room temperature. The eluate is monitored by UV irradiation and collected until the absorbance returns to 50%. The measured protein concentration is about 40 mg / ml.

The eluate was concentrated by diafiltering to a protein content of 70-80 mg / ml and dialyzed against a sufficient amount of buffer B (the same ingredients as in buffer A, but additionally 1 mM calcium chloride). 4 Mio is then added. The KIU of aprotinin per liter of solution is filtered under sterile conditions using a filter cascade with filters having pore sizes 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 a calcium chloride solution will suffice. For a slow adhesive, for example to fill the cavity after tooth extraction or to seal the cavity after the cerebral gland has been 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

The patient, aged 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. In the last year he had repeated attacks of kidney colic caused by a large stone in the left renal pelvis. Elective lithotripsy by ultrasound was planned. - Based on the technique that IgG binds to the protein A affinity column, the patient has undergone immunosuppressive treatment associated with extracorporeal immunoadsorption. After eight treatment cycles in which 60 L of patient 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 normal pooled plasma before and affinity columns of the patient. 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 (approximately 80 L of plasma), followed by intensive plasmaferesis (approximately 50 L) and a high dose immunoglobulin (2 g / kg) infusion. At this point, the level of thrombin inhibitors 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 cryoprecipitate and a large amount (200 U / ml). However, the patient did not experience hemostasis by stitching. The wound looked dry after the surgery. However, after purification to both PT and APTT with thrombin. for immediate and local hours, the patient started bleeding from surgical drainage. Immunoadsorption of 10 L of plasma was performed, but without any effect on bleeding, which has increased.

The patient was reoperated to determine the source of bleeding. No surgical bleeding was detected, 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 wound surface 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 glue of the invention.

Claims (14)

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 a proteolytic enzyme which is fibrinogen present in the polymer component . 2. Tissue adhesive characterized by being concentrated. 3. The tissue glue characterized in that it is aprotinin, which is present in KUU / ml units. capable of specifically cleaving A to form fibrin share claim 1 m that component A is a share claim 1 wherein the protease inhibitor is an amount of from 3,000 to 5,000 4. The tissue glue of any one of claims 1 to 3, wherein the proteolytic 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 the South American snake 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 units KU / 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 - converting the cryoprecipitate to a cryoprotectant to form component A, - virus inactivation is carried out, - virucidal agents are removed, - a protease inhibitor is added and an appropriate solution of a suitable protease, as component B, is prepared. 10. A tissue glue comprising, as component A, fibrinogen, fibronectin and factor XIII and a protease inhibitor in an amount corresponding to 3,000 to • 5000 KUU / ml aprotinin. C F The tissue glue of 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 3,000 to 5,000 KU / ml units 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.