HUT67051A - Improved tissue glue prepared by using cryoprecipitate - 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

BACKGROUND OF THE INVENTION The present invention relates to a tissue binding agent consisting of two components, A and B, to a process for preparing a tissue fixator and to the use of a high amount of aprotinin and a snake venom proteolytic enzyme.

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, general 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 factor XIII, which is required for the stabilization and growth of the clots. The second component is thrombin, an active enzyme that is converted to fibrinogen, the last component of fibrin clots 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 to the product, which induces coagulation lysis over time.

-3 enzymes, while others add aprotinin, which is 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 / 01814 discloses a process for the preparation of a cryoprecipitated suspension containing fibrinogen and factor VIII, which is a precursor to the production of a fibrin fixative comprising the steps of: (a) fresh blood-borne disease-controlled human or animal donors; freezing plasma at -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. A process for preparing a fibrin fixator for surgical interventions has also been described, comprising the steps of: (a) preparing a cryoprecipitated suspension

-4a according to the above procedure; (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 reconstituted from the lyophilized solution by the addition of water. The tissue fixator contains pasteurized active ingredients to prevent hepatitis and HTLV III transmission.

It is an object of the present invention to provide a tissue fixator for use in patients suffering from severe blood coagulation disorders such as haemophilia A or B. The present invention further provides a tissue fixator that can be used in patients who have already developed 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 by the tissue fixation component, it must be ensured that the virus is in an inactivated state in the tissue fixation components.

The tissue fixator of the present invention consists of a component A comprising a cryoprecipitate of whole blood and a large amount of a protease inhibitor corresponding to an aprotinin unit of 3000-5000 KIV, preferably aprotinin - and a component B - specific for cleavage of fibrinogen present in component A capable and fibrin-forming proteolytic enzyme.

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 invention suitable for the treatment of patients with severe bleeding disorders. Preferred protease inhibitor

-6R R is aprotinin available commercially under the trademarks Trasylol and Antagosan.

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. Generally, thrombin of human or animal origin, such as bovine, is used. Thrombin is provided in lyophilized form. The 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. In order to obtain a slow tissue fixator, for example to remove body cavities, i.e., tooth extraction, or in the case of transphenoidal hypophisectomy (cavity closure), thrombin is further diluted to 25 U / ml with the appropriate calcium chloride solution.

The improved tissue fixator of the present 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 comprises 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 in fibrinogen results in the cleavage of the 16 Arg / 17 Gly bond which causes release of fibrinopeptide and formation of fibrin I monomer.

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.

If a snake venom proteolytic protease is used to produce component B,

-8 Traditional traditional components A, fibrinogen, fibronectin, and factor XIII are also used. It is preferred to use a large amount of aprotinin according to the invention. 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 is pre-melted overnight at 4-10 ° C. The cryopaste (cryopaste) was dissolved in a buffer containing sodium chloride, trisodium citrate, and glycine, pH 7.0-7.2, and then heated 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 the mixture to at least room temperature and pH

It was adjusted to -97.0-7.2, and 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 mixture was heated to 30 ° C for virus inactivation. Then the detergent is added. Alternatively, the material mixed for a while is transferred to a virus-free container and kept on a slightly rising thermometer without mixing.

The viroid material is removed by addition of castor oil and gently stirring 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 discarded. The aqueous layer was purified by filtration. The pH should be controlled between 7.0 and 7.2. The protein solution is then passed through a reversed phase column at ambient temperature. After determination of the protein content (10-60 mg / ml concentration), the eluate is concentrated by diafiltration to 60-100 mg / ml protein content and dialyzed against the aforementioned buffer, except that the buffer has a relatively high CaCl ₂ concentration. A protease inhibitor is then added to the system. Sterile filtration is performed and then »» f »· * ·

-10a 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 using a cannula or a 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 less expensive than known 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 boars of these patients require infusion after tooth extraction. A ♦ * * · · * • 4 4 4 ♦ ♦ · «» 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 * * *

Patients treated with -11heparin can also be treated with the tissue fasteners of the present invention. A further advantage of the tissue fixers of the invention is that individuals producing antibodies to the component of the tissue fixator can be treated with a tissue fixator of the invention in which thrombin is replaced by a snake venom, Defibrase, which is isolated from the poison of batroot vipera.

The invention is illustrated without limitation by the following examples.

Human fibrinogen (quality L) was from Kabi (Stockholm), bovine thrombin from Mertz-Dade. Na-benzoyl-DL-arginine-p-nitroanilide (ΒΑΡΝΑ) chromogenic substrate and analytical reagents were purchased from Sigma (St. 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 ₂ g ₀ using the E 280 conversion factor.

Bovine thrombin was obtained from general sources (Merz-Dade or Parke-Davis), the manufacturer's

activity values. Reptylase, a snake venom that only releases FPA, was derived from Pentapharm (Basel) p. The proteolytic activity of Reptylase with thrombin was normalized to a nonspecific * * 4 9 * 99 * f # ».

-12 chromogenic substrate, BAPNA (0.25 mM) at 37 ° C,

Proteolysis ratios were compared at 405 nm in Tris / saline, pH 8.0, and monitored over 15 minutes.

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

The fibrin fixative was prepared by a double syringe procedure, using either one syringe pure or cryoprecipitated with fibrinogen and the other using reptilase (20 U / ml) or a mixture of thrombin with CaCl ₂ (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 at 37 ° C. The breakage length (BS) of the fastener (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 after two hours 24 At ° C, the mesh fastener mesh assembly was separated on an Accuforce Cadet Tensiometer (AMATEK, Mansfield & Greene, USA).

Sterile cryoprecipitate (cryo; cryo) was prepared from frozen (-30 ° C) human plasma which was 4θΟ-οη * · · · · * • * ·· ·· · _ν «« «·« 4 ··

-13 were thawed and plasma supernatant was removed. Five such units were pooled to determine protein and fibrinogen concentration, as determined by the Biuret method, before and after coagulation of the cryoprecipitate (1: 5 dilution) using 2 U / ml thrombin.

Factor XIII was determined by the accumulation of [H]-putrescine in dimethyl casein after 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 around 20-40 mM of 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. Ca (II) is one of the major factors resulting from covalent crosslinking of protein chains induced by factor XIII during gel formation. These cross-links are the main source of mechanical reinforcement of the gel, which reaches the plateau after 20 minutes.

I

-14A reptilase seemed to have a good inducing effect on factor XIII activity.

* ·· · * · «·« a • · 94 | • · · «V« ♦ * · <α ** ·· »« · ^ ··

Protein levels of the pooled cryoprecipitate:

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

Protein:

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 form a specific enzyme color, a cryo-dilution series was prepared which gives a two-phase CT curve equivalent to the fibrinogen dependence mentioned for the pure fibrin system.

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

The viscoelasticity of the -15A cryoprotectants was determined by thrombin or reptilase. This parameter develops much slower than the turbidity. However, cryoprotectants prepared with excess CaCl ₂ and thrombin or reptilase achieved an equivalent TEG over approximately the same time period. Prior to the start of gelation, factor XIII-induced crosslinking appeared to occur between the gel fibers so that the TEG values of all attachments converged within one hour. Similarly, the final BS of all cryoprotectant with excess CaCl ₂ was the same. All cryo fixers are broken at 50-60 g. These experiments showed that the gel fibers became stiffer with the anchor due to the covalent crosslinking induced by factor XIII.

Preparation of the cryo solution

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 / l trisodium citrate, and 120 mM / Ι glycine, pH 7.0-7.2) 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. The pH is adjusted to 7.0-7.2

-16 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 χ / m filter. To the + mixture was added 0.1 M / L CaCl ₂ to give a final Ca concentration of 1 mM / Ι. The pH is 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 mixing.

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 cascade of 1 Z / m / 0.45 χ / m. The protein solution was then passed through a reverse phase column (C-18 column) at neutral temperature with a flow rate of 3 L / hr. The passage was followed by a UV monitor and

After reaching an absorbance of -1750%, 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 substances as buffer A and an additional 1 mM / Ι CaCl ₂ ). Then 4 mio per liter. KIU of aprotinin was added. Sterile filtration was performed using a 0.45 μη + 0.2 μπ \ 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 ₂ 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 of thrombin solution is appropriate. Thrombin is further diluted to a concentration of 25 u / ml in a slow-acting tissue fixator such as tooth extraction for occlusion or in transphenoidal hypophysectomy for cavity closure.

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

-18U / ml.

Clinical case description

A 21-year-old patient, MY (a 21-year-old male patient), received severe diathesis (bleeding) 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, recurrent kidney seizures suffered from seizures of 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 immuno-19adsorption treatments (about 80 liters of plasma) followed by intensive plasmapheresis (about 50 liters) and a high-dose infusion of immunoglobulin (2g / kg). The thrombin inhibitor was then reduced to 0.5%. PTT was reduced to 47 (pretreatment 85-90, see above), and TTT was reduced to 35 (pretreatment 90, and normal control 27, above).

It was decided that the stone would be surgically removed using a biological adhesive consisting of cryoprecipitate and high 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 (2U / ml; Defibrase) was 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)

A tissue fixator, characterized in that a component A comprising a concentrated cryoprecipitate of whole blood and a large amount of a protease inhibitor corresponding to 3,000-5,000 IU / ml of aprotinin and a component B specific for cleavage of fibrinogen present in component A is a capable proteolytic enzyme and allows the formation of a fibrin polymer. Tissue fixator according to claim 1, characterized in that the protease inhibitor is aprotinin in an amount of 3000 to 5000 KlU / ml. Tissue fixator according to claim 1 or 2, characterized in that the proteolytic enzyme is mammalian or human thrombin. Tissue fixator according to any one of claims 1 to 3, characterized in that the cryoprecipitate is an inactivated virus. A process for producing tissue fixers according to any one of claims 1 to 4, characterized in that: - preparing component A by converting the cryoprecipitate to a concentrated cryo solution, inactivating 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 comprises fibrinogen, fibronectin, and factor XIII, and a protease inhibitor corresponding to 3,000-5,000 IU / ml aprotinin. 7. Tissue fixator according to claim 1, characterized in that component B is a proteolytic enzyme or thrombin according to claims 4 and / or 5. Tissue fixator, characterized in that component A contains fibrinogen, fibronectin and factor XIII and component B contains the proteolytic enzyme according to claims 4 and / or 5. 9. Use of a concentrated cryoprecipitate according to claims 1-4.