HU211631A9 - Improved tissue glue prepared by using cryoprecipitate - Google Patents

Description

The present invention relates to a tissue fixative consisting of two components, A and B. a method of producing a tissue fixer and using a large amount of aprotinin and a proteolytic enzyme derived from snake venom to produce a tissue fixator.

The use of plasma proteins to improve local hemostasis at the site of surgical wounds is a well-known process. For example, fibrin patches have been used in brain surgery for hemostasis for a long time. Blood plasma and thrombin are used at the site of the surgical wound to create a layer of fibrin. In the last twenty years, numerous publications have described the use of "fibrin fixative", "fibrin adhesive" or "fibrin sealant" in surgery. Over the last ten years, generic "fixation" formulations have been used in Europe. The "fastener" consists of two components, the mixture of the two components forming a curd. The first component is a fibrinogen concentrate. This concentrate contains fibronectin and contains factor XIII, which is required for stabilization and growth of the clot. The second component is thrombin. is an active enzyme that is fibrinogenic. is converted to the last component of the fibrin clot in the normal coagulation system. This procedure avoids most of the steps of normal coagulation, but resembles only its last phase. Some manufacturers add plasminogen, an enzyme that induces clot lysis over time, while others add aprotinin, a protease inhibitor that prevents clot lysis.

Although these products provide satisfactory results in patients with mild bleeding disorders, patients with severe bleeding disorders such as haemophilia A or B are at increased risk of postoperative bleeding. Delayed bleeding complications often occur within a few days of surgery. Thus, patients treated with anticoagulation factors cannot be treated with tissue fixators to date. Another disadvantage of conventional concentrates is the high production cost.

WO / Ol 814 discloses a fibrinogen. and Vili factor, which is a precursor to the production of a fibrin fixative comprising the steps of: (a) freezing fresh plasma from human or animal donors for blood-borne diseases to -80 ° C. for at least six hours; (b) raising the temperature of the frozen plasma to between 0 ° C and room temperature so as to form a supernatant and form a cryoprecipitated suspension containing fibrinogen and factor VIII; (c) recovering the cryoprecipitated suspension. Also disclosed is a process for the preparation of a fibrin fixator for surgical interventions comprising the steps of: (a) preparing a cryoprecipitated suspension by the above process; (b) applying a volume suspension at the desired site; (c) applying to the site a composition comprising a sufficient amount of thrombin such that the fibrinogen is converted into a subsequent solidifying fibrin fixative in the suspension.

EP-A-0 341 007 discloses a surgical adhesive in the form of an aqueous composition comprising the patient's autogenous plasma, thrombin, and optionally an antifibrinolytic agent. Said adhesive is formed from the patient's plasma without the addition of an additional reagent to concentrate or isolate fibrinogen. Thus, the adhesive consists of two ingredients which are mixed together before use.

EP-A-0 253 198 discloses a one-component tissue fixator which is an aqueous solution of fibrinogen, factor VIII, a thrombin inhibitor, prothrombin factors, calcium ions, and an optional plasma inhibitor. The tissue fixator may be reconstructed from the lyophilized solution by the addition of water. The tissue fixator contains pasteurized active ingredients to prevent hepatitis and HTLV III transmission.

The present invention relates to such a tissue fixator. which can be used in patients with severe blood coagulation disorders such as haemophilia A or B. The present invention also provides a tissue fixator that can be used in patients who have already produced antibodies to bovine thrombin, the active component of component B. It is a further object of the present invention to provide a tissue fixator for patients treated with anticoagulation factors such as heparin. Due to the risk of transmission of viral diseases to the tissue attachment component by the animal, it must be ensured that the virus is in an inactivated state in the tissue attachment components.

The tissue fixator of the present invention comprises a component A, which is a cryoprecipitate of whole blood and contains a large amount of a protease inhibitor corresponding to 3000 units of KIV aprotinin units, preferably aprotinin, and a component B capable of specifically cleaving the fibrinogen present in component A, and a proteolytic enzyme that causes fibrin formation.

Generally available cryoprecipitates can be used to make the tissue fixator of the present invention. However, it is advantageous to concentrate the cryoprecipitates in a factor of 2 to 5, preferably in a factor of 3.

The addition of a protease inhibitor at a concentration of 3000-5000 KIV aprotinin units to the cryoprecipitate makes the tissue fixator of the present invention suitable for the treatment of patients with severe bleeding disorders. A preferred protease inhibitor is aprotinin, which is commonly available under the trademarks Trasylol ^R and Antagosan ^R , respectively.

The cryoprecipitate can also be obtained from the patient himself by administering autologous blood prior to surgery. This solution reduces the risk of blood-borne viral infection. However, for the actual product, the cryoprecipitate must be inactivated for viruses. A method for inactivating viruses is described in PCT / EP 91/00503. The process is based on treating the cryoprecipitate with a special detergent and removing the detergent from the cryoprecipitate.

The second component B of the tissue fixator of the present invention may be prepared from a solution of a proteolytic enzyme capable of specifically cleaving fibrinogen.

HU 211 631 A9

Generally, thrombin of human or animal origin, such as bovine, is used. Thrombin is provided in lyophilized form. Thrombin is reconstituted with 40 mM calcium chloride solution. Preferably, the concentration of thrombin is 50-2000 u / ml.

For rapid tissue fixation, a solution of calcium chloride is prepared from approximately 100 U / ml thrombin. Thrombin is further diluted to 25 U / ml with the appropriate calcium chloride solution to produce a slow tissue fixation, for example, to remove body cavities, i.e., tooth extraction or transphenoidal hypophisectomy.

The improved tissue fixator of the invention may further comprise a proteolytic enzyme, component B, isolated from a snake venom. This is advantageous in that it will also treat patients producing antibodies to thrombin. In addition, heparin-pretreated patients will also be treated with the tissue fixators of the invention, since heparin does not interfere with the snake venom enzyme response. A great advantage of the present invention is that the snake venom enzyme used is batroxobin, which can be isolated from the South African Bothrpos moujeni viper. Preferably, component B contains from 0.5 to 10 U / ml of the snake venom proteolytic enzyme.

Batroxobin is a single chain glycopeptide of approximately 36,000 molecular weights. Defibrase ^R in fibrinogen results in the cleavage of the 16 Arg / 17 Gly bond. causing Fibrinopeptide release and fibrin I monomer formation.

When a high amount of aprotinin according to the invention is used, a tissue fixative containing purified fibrinogen, fibronectin, and factor XIII is used as component A. This significantly reduces the risk of subsequent bleeding.

When a snake venom proteolytic protease is used to produce component B, "conventional" components A, fibrinogen, fibronectin, and factor XIII are also used. The use of a large amount of aprotinin according to the invention is preferred. In a very preferred embodiment, component A from the cryoprecipitate is mixed with a large amount of aprotinin with component B of the invention, a poteolytic enzyme isolated from snake venom.

In the process of making the tissue recorder of the present invention, the steps of manufacturing component A comprise the steps of preparing a cryoprecipitate solution from the cryoprecipitate,

- a virus inactivation,

- removal of viroid material,

- addition of a protease inhibitor,

- preparation of the appropriate protease solution.

Preferably, a cryopaste (cryopaste) was pre-melted overnight at 4-10 ° C. The cryopaste (cryopaste) is dissolved in a buffer containing sodium chloride, trisodium citrate, and glycine, heated to pH 7.0-7.2 and then warmed to 30-35 ° C. The cryopaste must be easily soluble, otherwise it is not suitable for the preparation of the composition. The dissolution may also be accelerated by cutting the cryop pulp into small pieces after freezing. After cooling to at least room temperature and adjusting the pH to 7.0-7.2, aluminum hydroxide was added with stirring. The precipitate was centrifuged and set aside. Optionally, a filtration step may be performed. The calcium chloride is then added in an amount corresponding to the desired final concentration of calcium chloride.

The virus was warmed to 30 ° C for virus inoculation. Then the detergent is added. Alternatively, the material mixed for a time is transferred to a virus-free container and kept at a slightly elevated temperature without stirring.

The viroid is removed by adding castor oil and stirring gently for a few minutes. The mixture was cooled to room temperature to separate the oil / water phase. The aqueous layer is placed in a virus-safe (virussafe) container and the oily layer is killed. The aqueous layer was purified by filtration. The pH should be maintained under the control range of 7.0-7.2. The protein solution is then passed through a reversed phase column at ambient temperature. After determining the protein content (10-60 mg / ml concentration), the eluate is diafiltered to a protein content of 60-100 mg / ml and dialyzed against the aforementioned buffer, with the exception that the concentration of CaCl _{2 in} the buffer is relatively high. A protease inhibitor is then added to the system. Sterile filtration is performed and the sample is frozen in appropriate containers.

Component B is preferably a freeze-dried protease. Particularly preferred is the lyophilized thrombin or the lyophilized fraction isolated from the South African Bothrpos moujeni viper. The proteolytic enzyme is known as Reptilase, the enzyme itself is batroxobin.

The proteolytic enzyme is dissolved in calcium chloride buffer.

The use of the two components, A and B, can be accomplished by the double syringe method. When the two components are mixed together, a clot is formed. The material may be applied with a cannula or three-lumen catheter. In the latter process, the two components are injected into a separated lumen and some sources of atmospheric pressure are connected to the third lumen so that the mixture becomes a spray.

The tissue fastener of the present invention is advantageous because it is useful in patients with severe bleeding disorders and is cheaper than prior art tissue fasteners. Patients with severe haemophilia can then, for example, undergo tooth extraction without receiving an infusion of inhibitory factor VIII concentrate in excess of 80%. Thus, only 5% of these patients require infusion after tooth extraction. Patients treated with heparin can also be treated with the tissue fasteners of the present invention. A further advantage of the tissue fixators of the present invention is that they are antibodies to component 2 of the tissue fixator

Individuals producing A9 can be treated with a tissue fixator of the invention in which thrombin is replaced by a snake venom, Defibrobar ^R , which is a batroxobin isolated from the poison of Bothrpos rnoujeni vipera in South Africa.

The invention is illustrated without limitation by the following examples.

Human fibrinogen (quality L) came from "Kabi" (Stockholm) and bovine thrombin from "Mertz-Dade". Na-benzoyl-DL-arginine-p-nitroanilide (ΒΑΡΝΑ) chromogenic substrate and analytical reagents were purchased from Sigma (Sl. Louis, MO). Reagents and salts were diluted with 0.015 M Tris and 0.15 M NaCl, pH 7.4. Fibrinogen was dialyzed against Tris buffer and its concentration was determined from Abs ₂₃₀ using the E ^1% 280 conversion factor.

Bovine thrombin was obtained from general sources (Merz-Dade or Parke-Davis) with activity values as reported by the manufacturer. Reptilase ^R , a snake venom that emits only FPA, came from Pentapharm (Basel). The proteolytic activity of Reptylase ^R with thrombin was normalized by the addition of a nonspecific chromogenic substrate, BAPNA (0.25 mM), at 37 ° C in Tris / saline. pH 8.0. At 405 nm, the proteolysis ratios were compared and checked within 15 minutes.

Based on the esterolytic activity, the unit of reptilase activity was normalized similarly to atrombin.

The fibrin fixative was prepared by the double syringe method, using either pure or cryoprecipitated fibrinogen in one syringe and reptilase (20 U / ml) or CaCl _{2 in} thrombin (20 mM).

The clotting time was determined using a Research Model 300-R ACL Coagulation Analyzer (IL, Milan). Viscoelasticity (TEG) was determined using a three-channel Heiliger Thromboelastograph. 37 “C-on. The break length of the fastener (BS) (in grams) was determined by mixing the fastener components between two coarse woven synthetic filters (0.5 x 1 cm) to allow gel formation between the two coarse woven meshes and two hours. after 24 ° C, the mesh fastener mesh assembly was separated on an Accuforce Cadet Tensiometer (AMATEK, Mansfield & Greene, USA).

Sterile cryoprecipitate (cryo) was prepared from frozen (-30 ° C) human plasma which was thawed at 4 ° C and plasma supernatant removed. Five such units were pooled to determine protein and fibrinogen concentrations, as determined by the Biuret method, before and after the cryoprecipitate arrest (1: 5 dilution) using 2 U / ml thrombin. Factor XIII was determined by the accumulation of [ ³ H]-putrescine in dimethyl casein after the activation of the samples with 4 U / ml bovine thrombin at 22 ° C for 10 minutes.

A significant advantage of the CT-fibrinogen curve is that it achieves a minimum between two phases (i.e., 1 U / ml in Figure 1) and 1-8 mM reptilase for a given amount of thrombin or reptilase. This is slightly different from the maximum turbidity (after 10 minutes), which peaks at about 20-40 mM fibrinogen. Another experiment shows the dependence of CT on thrombin or reptilase levels. This curve, which has a plateau at higher enzyme levels, shows a nearly linear inverse relationship in the rate of gelling at low enzyme levels.

The formation of viscoelasticity of pure fibrin is slightly slower than that of turbidity. ACa (II) is one of the major factors resulting from covalent crosslinking of protein chains induced by factor XIIa during gel formation. These cross-links are the main source of mechanical reinforcement of the gel, which reaches the plateau after 20 minutes.

The reptilase seemed to have a satisfactory inducing effect on factor XIIa activity.

Protein levels of apooled cryoprecipitate:

The cryo produced from the five units gave the following main values:

Protein: 75 mg / ml

Fibrinogen: 36 mg / ml

Factor XIII: 4.10 U / ml.

Coagulation rates:

The cryo clotting time (CT) is linearly dependent on thrombin and reptilase levels. However, above 3 U / ml, elevated enzyme levels had little effect on CT. To obtain a specific enzyme level, a cryobar dilution series was prepared which gives a biphasic CT curve equivalent to the fibrinogen dependence mentioned for the pure fibrin system.

Viscoelasticity (TEG) and fracture length (BS) of the cryo-anchor

The formation of the viscoelasticity of the cryoprotectants was determined by thrombin or reptilase. This parameter develops much slower than the turbidity. At the same time, cryoprotectants made with excess CaCl ₂ were used. and thrombin or reptilase achieved an equivalent TEG over approximately the same time period. Prior to the start of gelation, the factor XIIIIa-induced cross-linking between the gel fibers appeared to occur, with the TEG values of all anchors converging within one hour. Similarly, the final BS of all cryoprotectant with excess CaCl ₂ was the same. All cryo fixers are 50-60 gauge broken. These experiments showed that the gel fibers became stiffer with the anchor due to covalent crosslinking induced by factor XIIa.

Preparation of the cryo solution

The general cryopaste (cryopaste) is pre-thawed overnight at 4-10 ° C. One kilogram of cryopaste is dissolved in two liters of buffer (120 mM / Ι NaCl, 10 mM / Ι trisodium citrate, and 120 mM / Ι glycine, pH 7.07.2) and preheated to 30-35 ° C. The cryopaste must be easily soluble, otherwise it is not suitable for the preparation of the composition. In order to accelerate dissolution, the cryop pulp is cut into small pieces before thawing. It is then cooled to 20-22 ° C and its pH is measured. ApH 7.04

It is adjusted to 7.2 by the addition of dilute sodium hydroxide or acetic acid. To the mixture was added 100 ml of aluminum hydroxide and the mixture was stirred for an additional 30 minutes. The precipitate is centrifuged and set aside. The supernatant was filtered through a 1 µm filter. 0.1 M / L CaCl3 was added to give a final Ca ²⁺ concentration of 1 mM / Ι. The pH was again measured.

Virus inactivation

The mixture was heated to 30 ° C. 1% w / v TNBP and 1% w / v Triton X 100 were added. Stir the mixture gently for half an hour. It was then placed in a virus-free container and kept at 30 ° C for an additional 3.5 hours without stirring.

Removal of viroid material

To the mixture described above was added 150 ml of castor oil and stirred gently for half an hour. While waiting for the oil / water to separate (30-45 minutes), the mixture was cooled to 20 ° C. The aqueous layer was placed in a virus-free container and the oil layer discarded. The aqueous layer was filtered through a filter casing with a diameter of 1 µm / 0.45 µm. The protein solution was then passed through a reversed phase column (C-18 column) at neutral temperature with a flow rate of 3 L / h. The passage was monitored with a UV monitor and after reaching 50% absorbance, the fractions were collected. The fractions contained approximately 40 mg / ml protein as determined by protein measurement.

The eluate was concentrated by diafiltration to a protein content of 70-80 mg / ml against a sufficient amount of Buffer B (the same active ingredients as Buffer A and an additional 1 mM CaCl ₂ ). Then 4 mio per liter. KIU of aprotinin was added. Sterile filtration was performed using a 0.45 pm + 0.2 pm diameter filter cascade. The solution was then collected, frozen in plastic bags and lyophilized as desired,

Preparation of the thrombin solution

Lyophilized thrombin was dissolved in 40 mM CaCl 3 solution. The amount of thrombin in the tissue fixator is 100 U / ml. For fast acting tissue fixator, for example when applied to the wound area by spray application, 100 U / ml thrombin solution is appropriate. For slow-acting tissue fixators, such as tooth extractions during tooth extraction or transphenoidal hypophisectomy, the thrombin is further diluted to 25 u / ml with the appropriate calcium chloride solution.

Reptilase is produced in a manner similar to thrombin. However, the amount of reptilase is approximately 2 U / ml.

Clinical case description

A 21-year-old patient, MY (a 21-year-old male patient), received severe diathesis due to thrombin inhibitors. No other background problem (i.e., tumor or autoimmune disease) justified the disease. Laboratory tests, confirmed by two external laboratories, showed that MY had high levels of anti-thrombin IgG antibody. Last year, he suffered from repeated kidney seizures due to a large kidney stone in his left pelvis. The patient was to be treated by elective lithotripsy. Based on the binding of IgG to protein A on the affinity column, the patient was treated with immunosuppressive therapy in combination with extra-corporeal immunoadsorption. After 8 treatments of 60 liters of patient plasma on the protein A column, the inhibitor titer was reduced to 98%. This was determined by measuring the thrombin time (TT) of normal pooled plasma using pre- and post-affinity purified MJ plasma. Both TT, PT, and APTT values increased. However, the kidney stone was moved to the urethra, which completely blocked the hydronephrosis kidney. The patient received a further 10 immunoadsorption treatments (about 80 liters of plasma) followed by intensive plasma apheresis (about 50 liters) and a high dose infusion of immunoglobulin (2 g / kg). The thrombin inhibitor level was then reduced to 0.5%. PTT was reduced to 47 "(pre-treatment 85-90, see above) and TTT was reduced to 35" (pre-treatment 90 "and normal control 27", see above). It was decided to remove the stone surgically using a biological adhesive consisting of a cryoprecipitate and a large amount of thrombin (200 U / ml). With this mixture, the cryo gelated immediately after spraying. However, the patient did not undergo gelation and hemostasis occurred during suturing. After the surgery, the wound looked "dry." Six hours later, the patient started bleeding through the drain tube. Ten liters of plasma were immuno-adsorbed, but this did not affect the increase in bleeding.

The patient was again operated on to determine the cause of the bleeding. No surgical bleeding was found. Diffuse bleeding was observed on the entire surface of the wound surface. A cryo-reptilase mixture (2 U / ml; Defibrase) was then sprayed onto the wound. The spray coagulated immediately, the wound surface became cloudy and the bleeding stopped. The patient received immunoadsorption treatment for a further five days without bleeding. This example illustrates the advantage of the snake protease used as component B in the tissue fixator of the present invention.

Claims (9)

PATENT CLAIMS A tissue fixator comprising a component A comprising a concentrated whole blood criaprecipitate and a large amount of a protease inhibitor corresponding to 30005000 KlU / ml of aprotinin, and a component B which is a proteolytic enzyme capable of specifically cleaving fibrinogen in component A, and allows the formation of an ftbrin polymer. The tissue fixator of claim 1, wherein the protease inhibitor is aprotinin in an amount of 3000-5000 IU / ml. Tissue fastener according to claim 1 or 2, A9 wherein the proteolytic enzyme is a mammalian or human subject thrombin. 4. Tissue fixator according to any one of claims 1 to 3, wherein the cryoprecipitate is an inactivated virus. Procedure 1-3. A fabric fastener according to any one of claims 1 to 6, wherein - preparing component A by converting the cryoprecipitate to a concentrated cryo solution, - we activate a virus, removing the viroid material, and adding a protease inhibitor and preparing the appropriate solution using the appropriate protease as component B. The tissue fixator of claim 1, wherein component A is fibrinogen, fibronectin, and factor XIII, and a protease inhibitor corresponding to 3000-5000 IU / ml of aprotinin. The tissue fixator of claim 6, wherein component B is thrombin. 8. A method for promoting blood coagulation comprising the steps of 1-4. A fabric fastener according to any one of claims 1 to 5 is used. The tissue fixator or method of making it according to claim 1, essentially with reference to the examples.