JPH05194263A - Improved tissue glue manufactured by using cold sediment - Google Patents

Abstract

PURPOSE: To obtain a tissue glue suitable even to patients suffered from serious blood coagulation disorder. CONSTITUTION: This tissue glue is composed of A-component comprising whole blood's cold sediment and a large quantity of protease inhibitory factor corresponding to 3,000-5,000KIU/mL unit aprotinin, and B-component consisting of a protease capable of forming fibrin polymer through specifically cutting off fibrinogen existing in the A-component. In another version, this improved tissue glue is composed of A-component consisting of whole blood's cold sediment and B-component consisting of a protease obtained from snake venom, an enzyme capable of forming fibrin polymer through specifically cutting off fibrinogen existing in the A-component.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention comprises two components, A and B.
And a method for producing a tissue glue, the use of a large amount of aprotinin for producing a tissue glue, and the use of a snake venom proteolytic enzyme.

[0002]

BACKGROUND OF THE INVENTION Improving local hemostasis at the surgical wound site by the application of plasma proteins is a well known concept. Therefore, the heparin patch is used for hemostasis in cerebral surgery. Plasma and thrombin have been used to make hebrin films on surgical wounds. In the last two decades, there are numerous publications discussing the application of "Fibrin glue" or "Fibrin glue" or "Fibrin sealant" for most surgical training. Past 10
Over the years, "Glue" commercial products have been widely used in Europe. A "glue" consists of two components, but a mixture of these components produces an agglomerate. The first component is the fibrinogen concentrate. This concentrate also contains fibronectin and factor XIII, which are important for clot stability and strength. The second component is thrombin, the last component of the normal coagulation system, the active enzyme that converts fibrinogen into fibrin clots. This method bypasses most of the usual coagulation processes and mimics its final stage. Some manufacturers add plasminogen, an enzyme that induces clot lysis after a while, while others add aprotinin, an inhibitor of proteases, to prevent clot lysis.

However, these products, although accompanied by mild bleeding disorders, give satisfactory results to the patient,
Patients suffering from severe bleeding disorders such as hemophilia A or B still have a very high risk of post-operative bleeding. Occasionally, late bleeding complications occur after an average of days after surgery. Patients treated with anticoagulants also cannot be treated with prior art tissue glue. Another significant drawback of commercial concentrates is their high manufacturing costs.

WO 86/01814 discloses fibrinogen and VIII which are useful as precursors for the formation of fibrin glue.
Disclosed is a method of preparing a cryoprecipitated suspension containing a factor, comprising: (a) fresh freezing from a single donor, such as a human or other animal selected for a blood-borne disease. Freeze the plasma at about -80 ° C for at least about 6 hours;
(B) raising the temperature of the frozen plasma, for example between about 0 ° C. and room temperature, whereby the supernatant and fibrinogen and
Forming a cryoprecipitation suspension containing Factor VIII; and (c) recovering the cryoprecipitation suspension. Also disclosed is a method of producing a surgically beneficial fibrin glue, which comprises: (a) preparing a cryoprecipitation suspension as described above; (b) applying a defined amount of suspension at the desired site; And (c) applying a composition containing a sufficient amount of thrombin to the site, converting fibrinogen in the suspension into fibrin glue, and then fibrin glue coagulates.

EP-A-0341007 discloses a surgical adhesive consisting of a patient's autologous plasma, collagen, thrombin, and optionally an antifibrinolytic drug in an aqueous composition. This adhesive does not require any additional reagents for concentration or isolation of fibrinogen,
Made from patient plasma. For convenience, the adhesive is formulated as a two-part composition, which is mixed together shortly before use.

EP-A-0253198 discloses a one-component tissue glue with an aqueous solution of fibrinogen, factor VIII, thrombin inhibitor, prothrombin factor, calcium ions and optionally plasmin inhibitor. Tissue glue can be reconstituted from lyophilized samples by adding water. Tissue glue
In order to avoid hepatitis and HTLVIII transfer, all active substances may be contained in pasteurized form.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a tissue glue suitable for patients with severe blood coagulation diseases such as hemophilia A and B. Another object of the present invention is to provide a tissue glue that can be used in patients who have already developed an antibody to the bovine thrombin, which is the B component activator. Another object of the invention is to provide a tissue glue for patients treated with anticoagulant factors such as heparin. Due to the risk of infectious viral diseases associated with the components of the tissue glue, the fraction of the tissue glue must be guaranteed to be virus inactivated.

[0008]

The tissue glue according to the present invention comprises a cryoprecipitate of whole blood and 3000-5000 KIU /
From a proteolytic enzyme capable of specifically cleaving an A component consisting of a large amount of a protease inhibitor corresponding to a unit of aprotinin, preferably aprotinin, and fibrinogen present in the A component to cause the formation of a fibrin polymer. The B component is

Commercially available cryoprecipitates can be used to produce the tissue glue of the present invention. However, it may be advantageous to concentrate the cryoprecipitate at a magnification of between 2 and 5, preferably at a magnification of 3. 3000-5000K
Adding a sufficient concentration of protease inhibitor to the cryoprecipitate, corresponding to an amount of aprotinin in IU / ml,
The tissue glue of the present invention is suitable for use in patients suffering from severe bleeding disorders. Preferred protease inhibitors are aprotinin, which is commercially available under the trademark Trasylol (Trasylol ^R) or Antagozan (Antagosan ^R).

The cryoprecipitate can be obtained from the patient himself by donating autologous blood units prior to surgery.
This attempt prevents the risk of transmission of viral infections by blood derivatives. However, to obtain a suitable commercial product,
The cryoprecipitate must be virus inactive. The procedure for virus inactivation is PCT / EP91 / 00503.
It is described in. The basic principle is the treatment of the cryoprecipitate with a special detergent and the subsequent removal of the detergent from the cryoprecipitate.

The B component, which is the second component of the tissue glue of the present invention, is prepared by a solution of a proteolytic enzyme capable of specifically cleaving fibrinogen. Usually, thrombin isolated from plasma of mammals such as human or bovine is used. The thrombin can be supplied in pasteurized form. Reconstitution of thrombin occurs with a 40 mMol solution of calcium chloride. The preferred concentration of thrombin is 50-200 u / ml.

In order to produce fast tissue glues, approximately 100 u / ml thrombin solution of calcium chloride is prepared. To produce slow glues, for example by filling voids, ie extraction teeth or sealing the cavity of transsphenoidal hypophysectomy, thrombin is further dissolved in a suitable calcium chloride solution to a concentration of 25 u / ml.

Another aspect of the improved tissue glue of this invention consists of a proteolytic enzyme isolated from snake venom as the B component. This aspect is advantageous because it can treat patients who are developing antibodies to thrombin. Furthermore, since heparin does not affect the reaction of the snake venom enzyme, heparin-pretreated patients can be treated with the tissue glue according to the invention. In a highly preferred embodiment of the present invention, the snake venom enzyme batroxobin, which can be isolated from the South American viper, Bothrpos Moujeni, is used. Preferably, the B component contains 0.5-10 u / ml of each snake venom proteolytic enzyme.

Chemically, batroxobin has a molecular weight of about 3
It is 6,000 single chain glycopeptides. Defibrase ^R causes cleavage of α16Arg / 17Gly bound in fibrinogen, causing the release of fibrinopeptide A and the formation of monomeric fibrin I.

If aprotinin is used in the amounts according to the invention, a tissue glue consisting of purified fibrinogen, fibronectin and factor XIII can also be used as component A. The risk of post-bleeding is thereby significantly reduced. If a proteolytic protease from snake venom is used to prepare the B component, a "conventional" A component with fibrinogen, fibronectin and factor XIII can also be used. The use of large amounts of aprotinin according to the invention is preferred. A highly preferred embodiment comprises the A component of the invention obtained from a cryoprecipitate, with or without high amounts of aprotinin, and a B component of the invention having a proteolytic enzyme isolated from snake venom. It is a combination.

The method of producing fibrin glue of the present invention comprises the step of preparing a cold solution from a cold precipitate, production of component A, inactivation of viruses, removal of virucidal agents, addition of protease inhibitors. , And-preparation of a suitable protease solution.

Preferably, cold pasta is pre-thawed overnight at 4-10 ° C. The cold pasta contains sodium chloride, trisodium citrate and glycine and has a pH of 7.
After being dissolved in a buffer solution of 0 to 7.2, it is heated to 30 to 35 ° C. Cold pasta should dissolve quickly. Otherwise it will not be suitable for preparation. Dissolving the cold pasta into small pieces after thawing can accelerate the dissolution. After cooling the solution to about room temperature and adjusting the pH to a value of 7.0-7.2, aluminum hydroxide is added with stirring. Centrifuge and discard the precipitate. Optionally perform a filtration step. Calcium chloride is then added to bring the calcium chloride to the desired final concentration.

To inactivate the virus, the solution was
Heat to 0 ° C. Next, a cleaning agent is added. After stirring for a while, the solution is transferred to a virus-free container and left at a slightly elevated temperature for several hours without stirring.

The virucidal agent is removed by adding a certain amount of lysine oil and gently stirring for a few minutes. Once the oil / water phase has separated, the solution is cooled to room temperature. Remove the water layer into a virus-free container and discard the oil layer. The aqueous layer is clarified by filtration. It must be checked for a pH of 7.0-7.2. The protein solution is then pumped through the reverse phase column at room temperature. After measuring the protein content (ranging from 10 to 60 mg / ml), the eluent was diafiltered to 60 to 100 mg / ml.
Concentrate to a protein content of ml and dialyze against the same buffer as above but with a much higher concentration of calcium chloride. The protease inhibitor is then added. Sterile filtration is performed, and the sample is placed in an appropriate container and deep-frozen.

Component B is preferably a lyophilized protease. Particularly preferred is lyophilized thrombin, or the lyophilized fraction of the South American viper Bosphorus mougeni. The proteolytic enzyme is known under the trade name of Reptilase and is the enzyme batroxobin. The proteolytic enzyme is dissolved in calcium chloride buffer.

The application of the two components A and B is carried out using the double-syringe method, for example via a plastic connector. When the two components are mixed together, a coagulum is formed.
Application can occur by cannula or can be sprayed onto a three lumen catheter. Each of the two components is infused into a separate lumen, and a source of compressed air in the range of several atmospheres is connected to the third lumen to atomize the mixture.

The tissue glue of the present invention is advantageous because it can be used in patients suffering from severe blood coagulation disorders and is cheaper than known tissue glues.
Patients with severe hemophilia can subsequently be extracted with a success rate of 80% or more without prophylactic infusion of factor VIII concentrate, for example. This means that only about one-fifth of patients require infusion due to bleeding after tooth extraction. In addition, such patients pretreated with heparin can be treated with the tissue glue of the present invention. Another advantage is that those who develop antibodies to the second component of the tissue glue, thrombin, can be treated with the tissue glue according to the invention, wherein instead of thrombin a protease from snake venom, in particular a South American viper, Defibrase, a serine protease batroxobin isolated from the venom of Bosphorus mougeni, is used.

The invention will be further clarified by the following non-limiting examples. Human fibrinogen (grade L)
Was from Kabi (Stockholm) and bovine thrombin was from Merz-Dade. Chromogenic substrate N-a-benzoyl-DL-arginine-p-
Nitroanilide (BAPNA) and analytical reagents were from Sigma (St. Louis, MO).
Reagents and salts 0.015M Tris, 0.15M N
Diluted with aCl to pH 7.4. Fibrinogen was dialyzed against Tris buffer at a concentration determined from Abs ₂₈₀ using a conversion factor of E ^1% 280 = 15.

Bovine thrombin was from a commercial source (Merz-Dade or Parke Davis) and had activity assessed by the manufacturer. A snake venom which liberate FPA only reptilase (Reptilase ^R) were those Pentapharm of (Pentapharm) (Basel). By comparing the proteolytic activity of leptylase at 37 ° C. in Tris / Sarin at pH 8.0 at 405 nm for 15 minutes, the proteolytic activity of the non-specific chromogenic substrate BAPNA (0.25 mM) was compared, Normalized to that of thrombin. The unit activity of reptilase was normalized to that of thrombin based on the esterification degradation activity.

Fibrin glue is essentially a double-syringe method in which one syringe is provided with pure or cryoprecipitated fibrinogen substrate and the other with reptilase (20 U).
/ Ml) or thrombin with CaCl ₂ (20 mM).

Clot time (CT) is calculated by research model 30.
Determined using a 0-RACL coagulation analyzer (IL, Milan). Viscoelasticity (TEG) was determined on a 3-channel Heliger Thromboelastograph at 37 ° C. The breaking strength (BS) of the glue (several grams) is obtained by mixing the glue component between two pieces of the woven synthetic fiber (0.5 × 1 cm),
A fully woven gel was formed between the two pieces of rough mesh, and after 2 hours at 24 ° C. a mesh-glue mesh ensemble was placed on the Acuforce Cadet tensiometer (AMATEK, Mansfield and Green, USA). ) Was used for peeling.

Sterile cryoprecipitate (cryo) was prepared by thawing human frozen plasma (-30 ° C) at 4 ° C to remove the supernatant plasma. Five such units were pooled for protein determination, and the fibrinogen concentration before and after agglomeration of the cryoprecipitate (diluted 1: 5) by billet method with 2 U / mL thrombin. Were determined. Factor XIII was determined by measuring [ ³ H] -putrescine incorporation into dimethylated casein after activating the sample with 4 U / ml bovine thrombin for 10 minutes at 22 ° C.

A notable feature of the CT-fibrinogen curve is that for fixed levels of thrombin or reptilase (ie 1 U / ml), the curve is two-step, reaching a minimum in the 1-8 mM fibrinogen range. This differs somewhat from the maximum turbidity (after 10 minutes) which peaks in the 20-40 mM fibrinogen range. Contrary experiments show that CT depends on both thrombin and reptilase levels. This curve shows a near linear inverse dependence of gelation rate at low enzyme levels (less than 2 U / mL) and plateaus at the top at higher levels.

The viscoelastic development of pure fibrin is slightly slower than its turbidity. Ca (II) is the major cofactor in gel strengthening by covalent ligation of protein chains induced by Factor XIIIa. Such gel cross-linking is the major cause of the mechanical strength of the gel, which plateaus after 20 minutes. A note about the ability of reptilases to induce Factor XIIIa activity appears to be relevant.

Protein level of stored cold sediment: 5
Pooled cryoprecipitates prepared from individual units showed the following mean values: Protein: 75 mg / mL Fibrinogen: 36 mg / mL Factor XIIIa: 4.10 U / mL

Coagulation rate: Clotting time (CT) of cold sediment
Is linearly dependent on the level of thrombin or reptilase. However, above 3 U / mL, increasing enzyme levels had little effect on CT. At fixed levels of enzyme, serial dilutions of cryoprecipitate show a two-step CT curve comparable to the fibrinogen dependence noted for pure fibrin system.

Viscoelasticity (TEG) and breaking strength (BS) of cryoprecipitate glue. The viscoelastic development of cold sediment glue was studied with both thrombin and reptilase. This parameter takes much longer than turbidity to develop. However,
Cryoprecipitate glues produced with excess CaCl ₂ and either thrombin or reptilase achieve comparable TEG values at approximately the same time frame. After the early onset of gelation, the TEG values of both glues appear to converge within 1 hour because Factor XIIIa-induced cross-linking reinforces the gel fiber structure. The same is true for the final BS of both cryoprecipitate glues formed with excess CaCl ₂ . Both cryoprecipitate glues break at 50-60 g. These experiments show that gel fibers within the glue become enhanced by factor XIIIa-induced covalent cross-linking.

Preparation of cold solution. Commercial cold pasta 4 ~
Pre-thaw at 10 ° C overnight. 2 kg of 1 kg of cold sediment
1 liter of buffer A (120 mM / l NaCl, 10
Dissolved in mM / l trisodium citrate, 120 mM / l glycine and pH 7.0-7.2), 30-
Preheat to 35 ° C. Cold pasta should dissolve immediately. Otherwise it will not be suitable for preparation. After thawing, cold pasta is cut into small pieces for faster dissolution. The solution is then cooled to 20 ° C-22 ° C and the pH is checked. Optionally add dilute sodium hydroxide or acid to pH
It must be adjusted to 7.0-7.2. 100 ml
Add aluminum hydroxide and stir for another 30 minutes.
Centrifuge and discard the precipitate. The supernatant is filtered using a 1 μm filter. 0.1M / l CaCl ₂ is added to ^bring the final concentration of Ca ²⁺ to 1 mM / l. The pH has to be checked again.

Virus inactivation. The solution is heated to 30 ° C. 1% w / v TNBP
And 1% w / v Triton X100. The mixture is gently stirred for 0.5 hours. The solution is then transferred to a virus-free container and left at 30 ° C. for 3.5 hours without stirring.

Removal of virucidal agents. To the mixture prepared as above, 150 ml of lysine oil is added and gently stirred for 30 minutes. While waiting for the oil / water separation (30-45 minutes), the solution is cooled to 20 ° C. Remove the water layer into a virus-free container, while discarding the oil layer.
The aqueous layer is clarified by filtration through a 1 μm / 0.45 μm filter cascade. The protein solution is then pumped through a reverse phase column (C-18-column) at a rate of 3 l / h at room temperature. The flow through is monitored by UV and collected until the absorbance is restored to 50%. The fraction contains approximately 40 mg / ml as determined by protein analysis.

The eluate was concentrated by diafiltration to a protein content of 70-80 mg / ml and a sufficient amount of buffer B (same component as buffer A but with additional 1 mM / l calcium chloride). ) And dialyse. Then 4 × 10 ⁶ KIU of aprotinin is added per liter of solution. Then, sterile filtration is performed using a 0.45 μm + 0.2 μm cascade. The solution is then placed in a plastic bag, deep-frozen and optionally lyophilized.

Preparation of thrombin solution. Lyophilized thrombin is dissolved in a 40 mM / l calcium chloride solution. The amount of thrombin is 100U in glue
/ Ml. For a fast acting glue, for example for spraying the glue to the wound area, 100 U of calcium chloride /
ml thrombin solution will be sufficient. For slow glues, eg for filling voids by tooth extraction or sealing the cavity of transsphenoidal hypophysectomy, the addition of large amounts of CaCl ₂ will further dissolve thrombin to a final concentration of 25 U / ml. .. Preparation of reptilase is similar to that of thrombin. However, the amount of leptylase is approximately 2 U / ml.

Report of Clinical Case A 21 year old patient, MY (21 year old male), suffered from severe hemorrhagic predisposition due to an acquired inhibitor of thrombin. No underlying disease (ie tumor or autoimmune disease) could explain this problem. Laboratory tests confirmed by two external laboratories have shown that MY has high levels of antithrombin IgG antibodies. Last year, he suffered from repeated attacks of renal pain due to a large stone in his left pelvis. Selective lithotripsy with ultrasound was planned. Patients were subjected to immunosuppressive therapy in combination with in vitro immunoadsorption, based on the method that IgG binds to protein A affinity columns. After 8 treatments, 60 L of patient plasma was processed through the protein A column with an inhibitor titer of 9
It decreased to 8%. This was determined by measuring the thrombin time (TT) of normal pooled plasma with purified MJ plasma before and after affinity. Nonetheless, TT extended as well as PT and APTT.
At this point, the kidney stones migrated to the urethra, causing the complete blockage of the kidneys made by hydronephrosis. The patient received another 10 times of immunoadsorption therapy (approx. 80 liters of plasma) followed by a thorough plasmapheresis (approx. 50 liters) and a heavy immunoglobulin infusion (2 g / k).
g). At this point, the level of thrombin inhibitor was reduced to 0.5%. PTT is 47 "(8 before treatment
5 to 90 "), TT is 35" (90 "before treatment)
And against a normal control 27 "). Using a biological adhesive made from cold sediment and high levels of thrombin (200 U / mL) (cold sediment glue), the stones were surgically removed. In this mixture, the cryoprecipitate gelled immediately upon spraying in this mixture, however gelation did not occur in this patient and local hemostasis was achieved by suturing. 6 hours later, the patient was bleeding from the surgical drain, immunoadsorbing 10 liters of plasma, but effective in actually increasing bleeding. There was no

The patient was re-operated to find the cause of the bleeding. However, no surgical bleeding was found. Diffuse bleeding was observed throughout the wound surface area. At this time, a mixture of cryoprecipitate and leptylase (2 U / mL; defibrase) was sprayed onto the wound. The spray immediately coagulated, the wound surface appeared cloudy, and bleeding stopped.
The patient continued to receive immunoadsorption therapy daily for another 5 days. There was no bleeding. This demonstrates the advantage of using snake protease as the B component of the tissue glue of the present invention.

[0040]

The tissue glue of the present invention is advantageous because it can be used in patients suffering from severe blood coagulation disorders and is cheaper than known tissue glues. Patients with severe hemophilia can subsequently be extracted with a success rate of greater than 80% without prophylactic infusion of factor VIII concentrate, for example. This means that only about one-fifth of patients require infusion due to bleeding after tooth extraction. In addition, such patients pretreated with heparin can be treated with the tissue glue of the present invention. Another advantage is that people who develop antibodies to the second component of the tissue glue, thrombin, can be treated with the tissue glue according to the invention, where instead of thrombin, a protease from snake venom, in particular a South American viper. , Defibrase, a serine protease batroxobin isolated from the venom of Bosphorus mougeni.

Claims (15)

[Claims] 1. A cryoprecipitate of whole blood and 3000-500
A component consisting of a large amount of a protease inhibitor corresponding to 0 KIU / ml of aprotinin, and a B component consisting of a proteolytic enzyme capable of specifically cleaving fibrinogen present in the A component to cause the formation of a fibrin polymer, An organizational glue composed of. 2. The tissue glue according to claim 1, wherein the cryoprecipitate of the component A is concentrated. 3. A protease inhibitor of 3000 to 50
The tissue glue according to claim 1, which is aprotinin in an amount of 00 KIU / ml unit. 4. The tissue glue according to any one of claims 1 to 3, wherein the proteolytic enzyme is thrombin obtained from mammals or humans. 5. A proteolytic enzyme obtained from snake venom, which is an enzyme capable of specifically cleaving A component consisting of a cryoprecipitate of whole blood and fibrinogen present in the A component to cause the formation of a fibrin polymer. B component consisting of, and a tissue glue composed of. 6. The tissue glue according to claim 5, wherein the snake venom enzyme is batroxobin isolated from the venom of the South American viper, Bosulpos mougeni. 7. A large amount of a protease inhibitor corresponding to aprotinin of 3000 to 5000 KIU / ml.
The tissue glue according to claim 5 or 6, which preferably comprises aprotinin. 8. The tissue glue according to any one of claims 1 to 7, wherein the cryoprecipitate is virus inactivated. 9. Preparation of component A, which comprises the step of preparing a cold solution from a cold precipitate, inactivation of the virus, removal of virucidal agents, addition of protease inhibitors, and suitable as component B. A method for producing fibrin glue according to any one of claims 1 to 8, which comprises the step of preparing a suitable solution of protease. 10. A component of fibrinogen, fibronectin and factor XIII, and 3000 to 5
A tissue glue composed of a protease inhibitor corresponding to an aprotinin amount of 000 KIU / ml. 11. The tissue glue according to claim 10, wherein the B component is a protease or thrombin according to claim 5 and / or 6. 12. A tissue glue which comprises fibrinogen, fibronectin and factor XIII as component A and component B is a proteolytic enzyme according to claim 5 and / or 6. 13. 3000 to 300 in combination with a cryoprecipitate or in combination with fibrinogen, fibronectin and factor XIII to produce a tissue glue.
How to use a large amount of aprotinin of 5000 KIU / ml. 14. A method of using a snake venom protease for the production of tissue glue. 15. Bosulpos for manufacturing tissue glue
Use of batroxobin isolated from Mougeni.