MXPA99006754A - A stabilized mixture comprising fibrinogen - Google Patents

Abstract

A stabilized solution of fibrinogen containing samples, in particular a stabilized solution of the biological active component (BAC) which is a solution of proteins derived from blood plasma comprising fibrinogen, tranexamic acid and arginine or lysine or mixtures of arginine and lysine, their pharmaceutically acceptable salts, as well as, optionally, substances forming a buffered solution in aqueous medium.

Description

A STABILIZED MIXTURE THAT COMPRISES FIBRINOGEN DESCRIPTION OF THE INVENTION The invention pertains to a mixture comprising fibrinogen, particularly to a stabilized solution of the Biological Active Component (BAC), a two-component tissue glue comprising separately components A and B as well as a fibrin clot obtainable by mixing component A and component B. The fibrinogen contained in the samples can be used for various applications, for example in two-component tissue adhesives or fibrin glue. The Biologically Active Component (BAC) is a viscous solution of proteins that normally contains fibrinogen in amounts of approximately 50 mg of fibrinogen per ml, which has been derived from the cryoprecipitation of plasma. Such BAC was described in WO 94/22503. Since the procedures employed for virus inactivation and concentration result in an increase in proteolytic activity in cryoprecipitation, stabilization of the final product with antiproteolytic agents is required. Some of these enzymes Ref .: 30741 proteolytics are in a zymogen form (proenzyme) and their activation is promoted by the minute amount of activated enzymes present in the cryoprecipitation. The temperature interval employed during the separation of the cryoprecipitation has been found to improve the activation of the precursors of the proteolytic enzymes. This activation presumably takes place by means of a "cascade" sequence, by means of which the proteases activate the precursor forms of other proteolytic enzymes, including the generation of the fibrinolytic plasmid of the plasminogenated precursor. Inhibition of fibrinolysis and other proteolytic activity has been found to reduce the degradation of factor VIII and other coagulated and adhesive proteins. It may happen that the BAC coagulants spontaneously already after storage for a day of 2 6 ° C. At lower temperatures this process is slower; Following storage for several months at -18 ° C, BACs without further treatment form a solid clot after melting and can not be reconstituted. The same phenomenon has been observed with cryoprecipitate without purification. During the production process, plasminogen and other proteases dependent on vitamin K are removed by absorption of aluminum hydroxide. However, some proteases are left in the final product. It is desirable to stabilize the fibrinogen comprised in a mixture, in particular a BAC solution in order to ensure a careful use of the product when applied in clinical operations. Surprisingly, this object is achieved by a mixture comprising fibrinogen, 4- (aminomethyl) cyclohexane-carboxylic acid as well as arginine, lysine or combinations thereof. Preferably, the mixture is present as a solution of fibrinogen, tranexamic acid (4- (aminomethyl) -cyclohexane-carboxylic acid) as well as arginine, lysine or combinations thereof. In a preferred embodiment of the invention comprising a solution of the biological active component (BAC) which has been stabilized with a combination of tranexamic acid its physiologically acceptable salts and arginine or lysine or combinations of lysine and arginine. Optionally, the solution is buffered to a physiologically compatible pH value. Tranexamic acid is a protease inhibitor which the scientific name is 4- (aminomethyl) cyclohexane-carboxylic acid. According to the invention, a buffer containing glycine is preferred. Arginine as well as lysine can be used according to the invention as a common salt, for example, as hydrochloride. The BAC is preferably obtained from the concentrated cryoprecipitate after being worked up as described in EP-A-534 178. Arginine has been described in the art as a stabilizer in therapeutic protein concentrates. Nevertheless, the synergistic effect provided by the combination of tranexamic acid and arginine as well as lysine is surprising. Preferably, the amount of tranexamic acid in the BAC solution is from about 1-20% by weight. The amount of arginine hydrochloride or lysine is preferably from about 0.1-4% by weight. More preferred are amounts of tranexamic acid of about 5-15% by weight, particularly preferred from about 8-12% by weight. Normally about 10% by weight can be used. The amount of arginine hydrochloride or lysine is more preferred in the range of about 1-3% by weight, particularly about 2% by weight is preferred. The sample comprises fibrinogen, in particular a solution of BAC is preferably derived from a cryoprecipitate which was concentrated by ultra-distillation as described in Patent OA-94/22503. The BAC preferably comprises a fibrinogen content of from about 15-150 mg / ml in particular from 20-80 mg / ml. The amount of fibrinogen can be measured according to the Clauss Method (Claues, A., "Gerinnungsphysiologische Schnell ethode zur Bestimmung des Fibrinogens", Acta. Haematol., 17, 237-247, 1957). The use of a BAC derived from the concentrated cryoprecipitate is advantageous from such a fraction which also contains fibrinogen also valuable blood components which play an important role for blood coagulation when a proteolytic enzyme such as a human thrombin is bound with a BAC solution. . The valuable components are factor VIII, factor XIII, fibronectin, vitronectin, von Willebrand factor (vWF), etc. Preferably, the components are derived from the cryoprecipitate in particular concentrated cryoprecipitates. However, it is also possible that the fibrinogen components, factor VIII, factor XIII, fibronectin, - the von factor. Willebrand (vWF), the vitronección have been prepared by recombinant methods. Such preparations, for example, for factor XIII are commercially available. It was found that the combination of tranexamic acid and arginine or lysine stabilizes a mixture containing fibrinogen in particular a solution of the biological active component BAC, (2-d ° C for at least 14 days by keeping at least 50% of activated fibrinogen ). When only one of the components was used, the sample is significantly more unstable. Furthermore, it was surprising that the use of tranexamic acid and arginine hydrochloride or lysine did not adversely affect the properties of a blood clot obtained from stabilized BAC according to the invention. For example, the maximum elongation of the clot was also maintained after 14 days of storage of the respective samples as well as the tensile strength remained almost at the same level. Also the activity of factor VIII in BAC was not negatively affected when tranexamic acid and arginine or lysine were added. In particular, the stabilized solution of the biological active component BAC according to the invention is well suited for the preparation of a two-component tissue adhesive. A tissue glue according to the invention is understood as a system which can be applied with or in a patient in need thereof, for example, to avoid severe bleeding during surgical operations. The tissue glue was also handled as a fibrin glue and was basically analogous to the natural blood coagulation cascade. Tissue glue is derived from two components prior to application in surgical operations. A component that contains fibrinogen which upon exposure to a proteolytic enzyme such as human thrombin forms fibrin which is the polymer that forms the basic material of the natural blood clot. During the surgical operations the two components are applied, for example, by two syringes which are emptied simultaneously to mix the two components as fast as possible and avoiding the blocking of the supply lines. The solution according to the invention is advantageous for preparing a two-component fibrin glue since the fibrinogen solution has not been freshly prepared but can be stored in a refrigerator at -18 ° C, neither decreasing its coagulant capacity nor its remarkable mechanical properties. The degrees of fibrinogen activity can be balanced by providing a higher amount of fibrinogen in the solution so that the proper use of the fibrinogen-containing solution (BAC) is not impeded. t Therefore, the object of the present invention is also a two-component tissue glue comprising separately components A and B wherein the component A comprises a solution according to the embodiments described in claims 1 to 10 and a component B which comprises a solution of a proteolytic enzyme which is capable of reacting with fibrinogen (or BAC respectively) to the fibrin form. Preferably, the proteolytic enzyme is human thrombin in particular has an activity from about 2 to 4,000 IU / ml. The activity of thrombin is measured according to the coagulant assay (European Pharmacopoeia). It is understood by a skilled person that a fibrin glue can be defined by its coagulable protein content instead of the definition based on coagulable fibrinogen. In order to provide a balanced solution of the mixed components A and B it may be advantageous to add to the component B approximately the same concentration of tranexamic acid and arginine.

Preferably, components A and B are applied in such a way that the equal volumes of the two components are mixed and applied in the patient to the side of the respective wound. Of course, it should be understood that the two tissue adhesives of the component can be used not only during surgical operations but also in other situations where bleeding must stop. The two components are preferably applied in a ratio of 1: 1. The following examples illustrate the advantages of the stabilizing combination of tranexamic acid and arginine. These examples in no way limit but explain the invention in greater detail.

Example 1 Preparation of the test substance 800 ml of a 0.8 g sample of sodium azide (0.1%) will be added as a powder to the volume to control bacteriological growth, immediately after receiving the sample from the production line. The addition of a bacteriostat is necessary to prevent contamination of the high concentration of fibrinogen in the sample that is difficult to filter. Each test concentration will be prepared extemporaneously by adding a mixture of two solid components to 100 ml of an aliquot of the sample. The addition of tranexamic acid and arginine-HCl will be carried out in a bucket with moderate agitation (multipoint magnetic stirring plate) for 10 minutes. All test formulations will be prepared in parallel and their fibrinogen concentration will be adjusted to 50 mg / ml by the addition of buffer B.

After further stirring for 5 minutes, the 10 ml glass jars with silicone will be filled with 5 ml of aliquots. All flasks will be frozen simultaneously and stored at -80 ° C until used. Before experimentation, they will, at a temperature of 2 6 ° C (day "0", reference time). Two bottles of each group will be stored at -80 ° C as positive controls. All the experiments will be carried out in duplicates and the bottles will be labeled according to the stabilizing concentrations as indicated below: GROUP A: without stabilizer-bottles Ai - A20 GROUP B: 0% tranexamic acid and 2% of arginine monohydrochloride-fragments Bi-B20 GROUP C: 5% of tranexamic acid and 2% of arginine monohydrochloride - Ci - Cao GROUP D: 10% tranexamic acid and 2% of arginine monohydrochloride-phraseos Di-D20 GROUP E: 10% tranexamic acid and 0% of arginine monohydrochloride-phraseos Ei-E20 GROUP F: 10% tranexamic acid and 1 Arginine monohydrochloride-phraseos Fi-F20% GROUP G: 10% tranexamic acid and 4% arginine monohydrochloride-Gi "G-20 The extra amounts of the sample will take place as the retention samples and used if necessary. Before testing all frozen bottles at -80 ° C, they will melt at 37 ° C for 15 minutes and set at 2 - 6 ° C (day "O") .Two bottles from each group will be turned at room temperature for 15 minutes at day 0 and its mechanical properties and biochemical tests will be carried out and evaluated. The procedure will be repeated on days 2, 4, 7, 9, 11, 14 and 30.

Mechanical properties The elongation test of the fibrin glue will be done in a CHATILLON MODEL TCD-200 tension machine with a CHATILLON Force Gauge of 1,000 g. The data will be handled by a computer program. This instrument is an engine driven tension and compression test designed to test the elasticity, pints of performance and breakthrough strength of various products and materials. A special molding was designed in order to produce standardized clots of the solidified glue in a form which could easily be attached to the stress testing machine. This consists of two conical aluminum molds, each 2.5 cm in height, which are placed one above the other (see figure 1). The liquid components of the glue are injected into the mold where they solidify in a standard 12.7 mm x 5 mm clot. Figure 1 illustrates the special mold designed to retain the liquid glue until solidification. The molds are then anchored to the stress testing machine, and the tensile strength and the elongation of the glue cylinder within the size of the mold by extracting the two molds separately. A computer program checks the elongation and tension at every 0.2 s. The points are plotted on two axes: the elongation against the force in grams in a given time.

Test procedure Bottles containing BAC were incubated 37 ° C for 15 minutes and then turned at room temperature for 15 minutes. The particular design of the aluminum molds (as described above) were filled with a combined solution of 0.5 ml of BAC and 0.5 ml of thrombin (8 IU / ml).

The molds were left at an ambient temperature for 45 minutes to allow the solidification of the glue, and then they were mounted on the indicator and the resulting clots were tested for the maximum elongation capacity and tensile strength (the tensile force required for break the clot).

Performing biochemical tests The following proteins were tested for stability by the methods described: The activity of the coagulant Fibrinogen: The fibrinogen was measured quantitatively by the coagulation method according to Clauss.

Example 2 - The Effect of Various Concentrations of Tranexamic Acid and Arginine-HCl on Force / mm (Declination) of the Clot or.

Conclusion: In freezing either the Arg. Or TEA improves the decline of the coagulum (the clot is stronger than without the stabilizers but TEA has a stabilizing effect on the coagulum strength over time when incubated at 4 - 8 ° C Example 3 The Effect of Various Concentrations of Tranexamic Acid and Arginine-HCl on the Content of Coagulable Fibrinogen s.

Conclusion: Stabilized coagulable fibrinogen of TEA Example 4, The Effect of Various Concentrations of Tranexamic Acid and Arginine-HCl on Factor VIII Activity Conclusion: The stabilized Factor VIII of Arg. In freezing and the stabilized Factor VIII of TEA during the incubation of 4 - 8 ° C.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers. Having described the invention as above, it is -claimed as property contained in the following:

Claims (2)

1. A mixture characterized in that it comprises fibrinogen, (4-aminomethyl) -cyclohexane-carboxylic acid (tranexamic acid) as well as arginine, lysine or combinations thereof, wherein the amount of tranexamic acid is about 1% -20% by weight and the amount of arginine or lysine is about 0.1% - 4% by weight of arginine hydrochloride or lysine 2. The mixture according to claim 1, characterized in that it comprises a solution of fibrinogen, tranexamic acid as well as arginine, lysine and / or combinations thereof. 3. The mixture according to claim 2, characterized in that the solution comprises a biological active component (BAC) which is a protein solution derived from the blood plasma comprising fibrinogen, tranexamic acid and arginine or lysine or mixtures or arginine and lysine, its pharmaceutically acceptable salts, as well as, optionally, substances that form a buffer solution in an aqueous medium. 4. The mixture according to any of claims 2 - 3, characterized in that the amount of tranexamic acid is approximately 5% - 15% by weight and the amount of lysine or arginine hydrochloride is about 1% - 3% by weight. 5. The mixture according to claim 3, characterized in that the BAC was derived from a concentrated cryoprecipitate. 6. The compliance solution csn any of claims 2 to 5, characterized in that the BAC comprises a fibrinogen content from about 15 to 150 mg / ml 7. The mixture according to any of claims 2 to 6, characterized in that the BAC further comprises factor VIII, factor XIII, fibronectin, von Willebrand factor (vWF), vitronectin. 8. The mixture according to claim 7, characterized in that factors VIII, XIII, fibronectin, von Willebrand factor (vWF), vitronectin have been prepared by recombinant methods. 9. The solution according to any of claims 2 to 8, characterized in that the buffer solution is a glycine buffer. 10. A two-component tissue glue comprising separately components A and B, characterized in that component A comprises a mixture of one of claims 2 to 9, and component B comprises a solution of a proteolytic enzyme which is capable of forming fibrin when reacted with fibrinogen. 11. The two-component tissue adhesive according to claim 10, characterized in that component B comprises human thrombin in an activity of approximately 2 4000 international units per ml measured according to the Clauss method. 12. The tissue glue of two components according to any of claim 11 or 12, characterized in that the component B is stabilized with tranexamic acid and arginine.

2 . The fibrin clot characterized in that it is obtained by mixing component A and component B in an appropriate ratio, preferably 1: 1.