JP2668762B2 - Improved tissue adhesive produced 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

DETAILED DESCRIPTION OF THE INVENTION

[0001]

This invention relates to two components A and B
(Herein referred to as glue) tissue adhesive composed of, the use of large amounts of aprotinin for producing the preparation and tissue glue of the tissue glue.

[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, a hebrin 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, commercial products of Glue have been widely used in Europe. "Glue" consists of two components, but a mixture of these components produces a coagulum. The first component is a fibrinogen concentrate. This concentrate also contains fibronectin and factor XIII, which are important for clot stability and strength. The second component is the last component of the normal coagulation system, thrombin, an active enzyme that converts fibrinogen into fibrin clot. This method skips most of the usual solidification steps and mimics its final stage. Some manufacturers add plasminogen, an enzyme that induces coagulation after a while, while others add aprotinin, a protease inhibitor, to prevent coagulation.

[0003] However, these products, with minor bleeding disorders, give satisfactory results to patients,
Patients suffering from severe bleeding disorders such as hemophilia A or B still have a very high risk of postoperative bleeding. Occasionally, late bleeding complications occur after an average of days after surgery. Patients who have been treated with anticoagulants also cannot be treated with prior art tissue glues. Another significant disadvantage of commercial concentrates is their high production costs.

WO86 / 01814 discloses fibrinogen and VI which are useful as precursors for the formation of fibrin glue.
Disclosed is a method for preparing a cryoprecipitated suspension containing factor II, comprising: (a) fresh from a single donor, such as a human or other animal, screened for a blood-borne disease; Frozen plasma is frozen at about −80 ° C. for at least about 6 hours; (b) the temperature of the frozen plasma is raised, for example to between about 0 ° C. and room temperature, whereby the supernatant and cryoprecipitation containing fibrinogen and Factor VIII. Forming a suspension; and (c) collecting the cryoprecipitation suspension. Also disclosed are methods of producing a surgically useful fibrin glue that (a) prepare a cryoprecipitated suspension as described above; (b) apply a defined amount of suspension to the desired site; And (c) applying a composition containing a sufficient amount of thrombin to the site to convert fibrinogen in the suspension into fibrin glue, which then coagulates.

[0005] EP-A-0341007 discloses a surgical adhesive consisting of a patient's autologous plasma, collagen, thrombin, and any antifibrinolytic drug in an aqueous composition. This adhesive can be used without any additional reagents for the concentration or isolation of fibrinogen.
Made from the patient's plasma. For convenience, the adhesive is formulated as a two-part composition, which is mixed together immediately before use.

EP-A-0253198 discloses a one-component tissue glue having an aqueous solution of fibrinogen, factor VIII, thrombin inhibitor, prothrombin factor, calcium ions and optionally plasmin inhibitor. Tissue glue can be reconstituted from a lyophilized sample by adding water. Tissue glue may contain all actives in pasteurized form to avoid hepatitis and HTLV VIII metastasis.

[0007]

It is an object of the present invention to provide a tissue glue which is also suitable for patients with severe blood clotting diseases such as hemophilia A and B. It is another object of the present invention to provide a tissue glue that can be used in patients who have already developed antibodies to bovine thrombin, an activator of the B component. It is another object of the present invention to provide a tissue glue for a patient who has been treated with an anticoagulant factor such as heparin. Due to the risk of infectious viral disease associated with the components of the tissue glue, it must be ensured that the fraction of the tissue glue is virus inactivated.

[0008]

The tissue glue according to the present invention comprises a concentrated cryoprecipitate of whole blood, 3000-500
Proteolysis capable of specifically cleaving the A component consisting of a large amount of a protease inhibitor corresponding to 0 KIU / ml of aprotinin, preferably aprotinin, and fibrinogen present in the A component to cause the formation of a fibrin polymer. It is composed of an enzyme B component.

[0009] Commercially available cryoprecipitates can be used to produce the tissue glue of the present invention. However, this cryoprecipitate is used concentrated. It can be advantageous to concentrate at a magnification of between 2 and 5, preferably at a magnification of 3.
The addition of a sufficient concentration of protease inhibitors corresponding to an aprotinin amount of 3000-5000 KIU / ml units to the cryoprecipitate makes the tissue glue of the invention suitable for use in patients suffering from severe bleeding disorders. . Preferred protease inhibitors are aprotinin, Trasylol (Trasylol ^R) or Antagozan (A
commercially available under the trademark ntagosan ^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 infection by blood derivatives. However, in order to obtain a suitable commercial product,
The cryoprecipitate must be virus inactive. The procedure for virus inactivation is described in PCT / EP91 / 00503.
It is described in. The basic principle is the treatment of the cryoprecipitate with a special detergent and the removal of the detergent from the cryoprecipitate later.

[0011] The second component of the tissue glue of the present invention, component B, is prepared with a solution of a proteolytic enzyme capable of specifically cleaving fibrinogen. Usually, thrombin isolated from plasma of mammals such as humans or cattle 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.

To produce a fast tissue glue, an approximately 100 u / ml thrombin solution of calcium chloride is prepared. For example, to produce a slow glue by filling the cavity, ie, filling the extraction or sealing the cavity of the transsphenoidal pituitary, the thrombin is further dissolved in a suitable calcium chloride solution to a concentration of 25 u / ml.

If aprotinin is used in the amount of the present invention, a tissue glue composed of purified fibrinogen, fibronectin and factor XIII can also be used as the A component. The risk of post-bleeding is thereby significantly reduced. The use of large amounts of aprotinin according to the invention is preferred.

The method for producing fibrin glue of the present invention comprises the step of preparing a concentrated cold solution from a cold precipitate, production of component A, inactivation of viruses, removal of virucidal agents, protease inhibition. The addition of factors and the preparation of a suitable protease solution.

Preferably, the cold paste is pre-thawed overnight at 4-10 ° C. The cold paste contains sodium chloride, trisodium citrate and glycine and contains p
After dissolving in H 7.0-7.2 buffer, 30-3
Heat to 5 ° C. Cold pasta should be melted quickly. Otherwise it is not suitable for preparation. Dissolution can be accelerated by dividing the cold pasta into small pieces after thawing.
After cooling the solution to about room temperature and adjusting the pH to a value between 7.0 and 7.2, aluminum hydroxide is added with stirring. The precipitate is centrifuged and discarded. Optionally performing a filtration step. Calcium chloride is then added to bring the calcium chloride to the desired final concentration.

For virus inactivation, the solution is
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, cool the solution 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 has to be checked that the pH is between 7.0 and 7.2. Next, the protein solution is pumped through a reversed phase column at room temperature. After measuring the protein content (ranging from 10 to 60 mg / ml), the eluate was filtered by diafiltration at 60-100 mg / ml.
It is concentrated to a protein content of ml and dialyzed against the same buffer as above but with a considerably higher concentration of calcium chloride. The protease inhibitor is then added. Perform sterile filtration, and place the sample in a suitable container and freeze-freeze.

The B component is preferably a lyophilized protease. Particularly preferred is lyophilized thrombin. The proteolytic enzyme is dissolved in the 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, a coagulum is formed.
Application can occur via cannula or can be sprayed onto a three lumen catheter. Each of the two components is injected into a separate lumen, and a source of compressed air in the range of several atmospheres is connected to the third lumen to spray the mixture.

The tissue glue of the present invention is advantageous because it can be used in patients with severe blood clotting disorders and is less expensive than known tissue glues.
Patients with severe haemophilia can subsequently extract teeth 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 the patients need infusion for bleeding after tooth extraction. In addition, such patients pre-treated with heparin can be treated with the tissue glue of the present invention. Another advantage is that people who have developed antibodies against thrombin, the second component of the tissue glue, can be treated with the tissue glue according to the invention.

The present invention is further illustrated by the following non-limiting examples. Human fibrinogen (grade L)
Was from Kabi (Stockholm) and bovine thrombin was from Merz-Dade. Chromogenic substrate Na-benzoyl-DL-
Arginine-p-nitroanilide (BAPNA) and analytical reagents are Sigma (St. Louis, St. Louis,
MO). Reagents and salts were diluted to pH 7.4 with 0.015 M Tris, 0.15 M NaCl. Fibrinogen in Tris buffer, E ^1% 28
Dialysis was performed at a concentration determined from Abs ₂₈₀ using a conversion factor of 0 = 15.

Bovine thrombin is commercially available (Mel-Tude or Parke Davis).
And had an activity evaluated by the manufacturer.

[0023] Fibrin glue is basically prepared by the double syringe method in one syringe with pure or cryoprecipitated fibrinogen substrate and in another with leptylase (20 U
/ Ml) or thrombin with CaCl ₂ (20 mM).

The coagulation time (CT) is calculated using the research model 30
Determined using a 0-RACL coagulation analyzer (IL, Milan). Viscoelasticity (TEG) was determined on a 3 channel Heriger thromboelastograph at 37 ° C. The breaking strength (BS) of the glue (several grams) is determined by mixing the glue component between two pieces of synthetic woven fibers (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 an Acuforce Cadet tensiometer (AMATEK, Mansfield and Green, USA). ).

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

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. Opposite experiments indicate that CT is dependent on both thrombin and leptylase levels. The curve shows a near linear inverse dependence of the gelation rate at low enzyme levels (less than 2 U / mL), with a plateau 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 enhancement by factor XIIIa-induced covalent ligation of protein chains. Such gel crosslinks are a major cause of the mechanical strength of the gel and plateau after 20 minutes.

Protein levels of pooled cryoprecipitate: Pooled cryoprecipitates prepared from 5 units showed the following mean values: Protein: 75 mg / mL Fibrinogen: 36 mg / mL Factor XIIIa: 4.10U / mL

Coagulation rate: The coagulation time (CT) of cryoprecipitates is linearly dependent on the level of thrombin. However, above 3 U / mL increasing enzyme levels had little effect on CT. At a fixed level of enzyme, serial dilutions of the cryoprecipitate show a two-step CT curve comparable to the fibrinogen dependence noted in pure fibrin systems.

The viscoelasticity (TEG) and breaking strength (BS) of the cryo-glue. The viscoelastic development of cryoprecipitate glue was studied with thrombin. This parameter takes much longer to develop than turbidity. However, cryo-glue generated with excess CaCl ₂ and thrombin achieves comparable TEG values in approximately the same time frame. After the initial onset of gelation, the TEG values of both glues appear to converge within one hour as factor XIIIa-induced cross-linking strengthens the gel fiber structure. The same is true for the final BS of both cryo-glue glues formed with excess CaCl ₂ . Both cryo-glues break at 50-60 g. These experiments show that the gel fibers within the glue become enhanced by the covalent crosslinking induced by factor XIIIa.

Preparation of a cold solution. 4 to cold pasta on the market
Pre-thaw overnight at 10 ° C. One kilogram of cold sediment
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 be dissolved promptly. Otherwise it is not suitable for preparation. Cut the cold pasta into small pieces after thawing to speed dissolution. The solution is then cooled to 20-22C and the pH is checked. Optionally, add dilute sodium hydroxide or acid to pH
It must be adjusted to 7.0-7.2. 100ml
Of aluminum hydroxide and stirred for another 30 minutes.
The precipitate is centrifuged and discarded. The supernatant is filtered using a 1 μm filter. 0.1 M / l CaCl ₂ is added to ^bring the final concentration of Ca ²⁺ to 1 mM / l. PH again
Must be checked.

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

Removal of the viricidal agent. 150 ml of lysine oil is added to the mixture prepared above and gently stirred for 30 minutes. While waiting for the oil / water separation (30-45 minutes), cool the solution to 20 ° C. Remove the aqueous 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 permeate is monitored by UV and collected until the absorbance returns to 50%. The fraction contains approximately 40 mg / ml as determined by protein analysis.

The eluate is concentrated by diafiltration to a protein content of 70-80 mg / ml and a sufficient amount of buffer B (of the same composition as buffer A, but with an additional 1 mM / l calcium chloride) Dialysis with). Next, add 4 × 10 ⁶ KIU aprotinin per liter of solution. Thereafter, sterile filtration is performed using a 0.45 μm + 0.2 μm cascade. The solution is then deep-frozen in plastic bags and optionally lyophilized.

Preparation of a thrombin solution. Lyophilized thrombin is dissolved in a 40 mM / l calcium chloride solution. 100U thrombin in glue
/ Ml. For example, in a fast acting glue for spraying glue onto a wound site, 100 U /
A ml of thrombin solution will be sufficient. For slow glues, for example filling voids by tooth extraction or sealing the cavity of transsphenoidal pituitary resection, the addition of large amounts of CaCl ₂ will further dissolve thrombin to a final concentration of 25 U / ml. .

Clinical Case Report A 21-year-old patient, MY (a 21-year-old man), suffered from severe hemorrhagic diathesis 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 repeated attacks of renal pain due to a large stone in the left kidney pelvis. Selective lithotripsy by ultrasound was planned. Based on the method by which IgG binds to the protein A affinity column, patients were subjected to immunosuppressive therapy combined with in vitro immunoadsorption. After eight treatments, 60 L of the patient's plasma had been processed through the protein A column, but the inhibitor titer was 9
8%. This was determined by measuring the thrombin time (TT) of normal pooled plasma with purified MJ plasma before and after affinity. Nevertheless, TT has grown as well as PT and APTT.
At this point, the kidney stones had migrated to the urethra, causing complete renal blockage caused by hydronephrosis. The patient received 10 more immunoadsorption therapies (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 the thrombin inhibitor had decreased to 0.5%. PTT is 47 "(8 before treatment
TT is 35 "(for 5" to 90 ") (90" before treatment)
And a normal control 27 "). Using a biological adhesive made from cold sediment and a high level (200 U / mL) thrombin (cryoglue glue), the stones were surgically removed. In this mixture, the cryoprecipitate gelled immediately upon spraying with this mixture, however, gelation did not occur in this patient and local hemostasis was achieved by suturing. Nonetheless, after 6 hours, the patient was bleeding from the surgical drain.Immunosorbed 10 liters of plasma and was effective against the actually increasing bleeding. There was no.

The patient was re-operated to find the cause of the bleeding. However, no surgical bleeding was found. Extensive bleeding was observed throughout the wound surface area. At this time, a mixture of the cryoprecipitate and reptilase (2 U / mL; defibrase) was sprayed onto the wound. The spray immediately clotted, the wound surface appeared cloudy and bleeding stopped.
The patient continued to receive immunosorbent therapy daily for another 5 days. There was no bleeding.

[0038]

The tissue glue of the present invention is advantageous because it can be used in patients with severe blood clotting disorders and is less expensive than known tissue glues. Patients with severe haemophilia will subsequently receive, for example,
Teeth can be extracted with a success rate of 80% or more without preventive infusion of factor concentrates. This means that only about one-fifth of patients require infusion due to bleeding after tooth extraction. In addition, such patients pre-treated with heparin can be treated with the tissue glue of the present invention.
Another advantage is that people who have developed antibodies against thrombin, the second component of the tissue glue, can be treated with the tissue glue according to the invention.

Continuation of front page (56) Reference JP-A-55-110556 (JP, A) JP-A-2-71747 (JP, A)

Claims (7)

(57) [Claims] 1. A concentrated cryoprecipitate of whole blood, comprising:
A component consisting of a large amount of a protease inhibitor corresponding to 0 to 5000 KIU / ml of aprotinin, and a proteolytic enzyme capable of specifically cleaving fibrinogen present in the A component to cause formation of a fibrin polymer B An ingredient, consisting of a tissue adhesive . 2. The method according to claim 1, wherein the protease inhibitor is 3,000 to 50.
The tissue adhesive according to claim 1, which is aprotinin in an amount of 00 KIU / ml unit. 3. The tissue adhesive according to claim 1, wherein the proteolytic enzyme is thrombin obtained from mammals or humans. 4. The tissue adhesive according to claim 1, wherein the cryoprecipitate is virus-inactivated. 5. Production of component A, which comprises the step of preparing a concentrated cold solution from a cold precipitate, inactivation of viruses, removal of virucidal agents, addition of protease inhibitors, and protease suitable as component B. The method for producing a fibrin adhesive according to any one of claims 1 to 4, comprising the step of preparing a suitable solution of: 6. The component A comprises fibrinogen, fibronectin and factor XIII, and 3000 to 50.
The tissue adhesion according to claim 1, comprising a protease inhibitor corresponding to an amount of aprotinin of 00 KIU / ml.
Agent . 7. The component A comprises fibrinogen, fibronectin and factor XIII, and 3000 to 500.
The tissue adhesive according to claim 1, which is composed of a protease inhibitor corresponding to an aprotinin amount of 0 KIU / ml, and wherein the B component is thrombin obtained from a mammal or a human.