MXPA99007545A - Immobilized activity stabilized lhrh antagonist complexes, method for the production thereof - Google Patents

Abstract

The invention relates to a retard system for LHRH antagonists, specially cetrorelix, based on complexation with suitable biophile carriers enabling targeted release of the active substance over a period of several weeks. Acidic polyamino acids, polyasparaginic acids and polyglutaminic acids are selected for complexation with cetrorelix. The cetrorelix polyamino acid complexes are produced from aqueous solutions by combining the solutions and precipitating the complexes which are subsequently centrifuged off and vacuum dried by P2O5, lyophilization proving to be a suitable method. The acidic polyamino, polyglutaminic and polyasparaginic acids display good retarding properties in a static liberation system depending on the hydrophobicity and the molar mass of the polyamino acids. Animal testing demonstrates the principal efficacy of the cetrorelix polyamino acid complexes as a depot system. By complexation of cetrorelix with polyamino acids, testosterone suppression can be achieved in male rats over a period of 600 hours. Active substance release can thus be controlled according to polymer type and molar mass.

Description

COMPLEXES IMMOBILIZED AND STABILIZED LHRH ANTAGONISTS, AND PROCESSES FOR PREPARATION Description of the invention The invention relates to complexes of LHRH antagonists of stabilized activity and retarders, such as antide, antarelix, azaline, A-75998, ganirelix, Nal-Glu antagonist with polyamino acids, in particular polyglutamic acid and polyaspartic acid, as well as the preparation thereof and medicaments containing them. Complexes of peptide hormones-elaborated polyamino acids can be applied in medicine, for example, for the therapy of tumors sensitive to hormones, such as, for example, breast and prostate carcinomas, benign hypertrophy of the prostate, and in gynecology for the treatment of endometriosis. hysteroscopy and for the treatment of fertility disorders. In the patent briefs DD 257197, DD 269785 and DD 299265 describe processes for the preparation of immobilized peptide preparations, stabilized in their biological activities and modified in their pharmacological properties, whose most essential characteristic is the formation of complexes of the respective peptide with polyamino acids, for insulin and other biologically active proteohormones. In the patents cited, preparation processes are described in which the complexes are formed under the action of formic acid and organic solvents, such as chloroform, and under drastic production conditions. However, there is a risk that the peptide hormone is partially inactivated or its stability is reduced. Literature was described for the first time in 1981 in patent literature EP 0042753 and EP 0049628 hardly soluble or complex salts for LHRH antagonists. The preparation of these complexes was carried out with a view to the development of pharmaceutical products for various medicinal applications. In 1989, ORSOLINI described in patent DE 38 22 459 the preparation of water-insoluble polypeptides by forming complexes of LHRH analogs and embonic acid, tannin and stearic acid. The hard-to-soluble complexes obtained are additionally included in a polymer matrix of lactic acid-glycolic acid copolymer. Other processes for the elaboration of the complex-cetrorelix included in a copolymer of (lactic acid-glycolic acid) were described in 1993 by ORSOLINI and HEIMGARTNER in patents DE 42 23 282 and 42 23 284.

This patent specifies difficultly soluble complexes of cetrorelix with embonic acid, tannin, stearic acid and palmitic acid. The object of the invention is to provide depot preparations with improved and controllable retarding properties, and greater stability against premature proteolytic disintegration of LHRH antagonists, for therapy in the fields known for this, such as hormone-sensitive tumors, such as for example breast and prostate carcinomas, benign hypertrophy of the prostate, endometriosis, hysteroscopy, and for the treatment of fertility disorders, and indicate a process of easy execution and non-polluting for the preparation of these preparations. The task of the invention is to provide new depot preparations of LHRH antagonists with improved and controllable retarding properties, such as antide, antarelix, azaline, A-75998, ganirelix, Nal-Glu antagonist, however preferably cetrorelix with biologically disintegrable polymers , as well as indicate a process for its elaboration. In accordance with the invention, the task is solved by elaborating immobilized and stabilized, parenterally applicable preparations of peptide hormones from LHRH antagonist complexes. with polyamino acids, in particular polyglutamic acid and polyaspartic acid, proceeding in such a way that the polyamino acid-peptide hormone complex is precipitated from aqueous solutions avoiding organic solvents. Advantageously, the polyamino acid-peptide hormone complexes can also be produced with a controllable content of hormones by lyophilization of the aqueous solutions. The rate of release of the active substance can be controlled by the type and molecular mass of the polyamino acids, by incorporating hydrophobic amino acids into the polymer structure, or by partial esterification (Figures 2 and 3). The application of the complexes according to the invention in medicine has its field in the therapy of hormone-sensitive tumors, in particular for the treatment of mammary and prostate carcinomas, benign hypertrophy of the prostate, as well as in the gynecology for the induction of ovulation, in-vitro fertilization and endometriosis, and in combination with hysteroscopy. The concept of "complex" within the frame of this invention comprises the joining of two or more components to form a hardly soluble system that is not subject to any proven stoichiometry. This The result is a superposition of interactions, with secondary valence bonds having an important role. In the literature, difficultly soluble peptide complexes are sometimes also referred to as "salt". This denomination also in many cases is inaccurate, because it is not about substances with a defined composition, as previously mentioned. It is true that in the case of peptides and proteins, ionic interactions occur, but nevertheless they are not only responsible for a change in structure or state of aggregation. For peptides and proteins, the concept of "complex" and "salt" must be broadened by virtue of the large number of functional groups, given that several interactions that lead to the formation and structure of peptides and proteins overlap. Polyamino acids that are suitable as biophilic carrier materials for the peptides were employed. Furthermore, it is a fundamental part of the invention that the active substances are not chemically bound to the polymer, but are only fixed to the polymer by means of secondary valence bonds and hydrophobic interactions. Surprisingly it was found that especially the LHRH antagonist cetrorelix shows a very large binding affinity to polyamino acids, in particular to polyglutamic acid and polyaspartic acid. Such a large affinity of cetrorelix was not predictable based on the current state of the literature, and was surprising by virtue of the structure of the peptide. Complexes that precipitate spontaneously have a reproducible defined hormonal content. If, on the other hand, it is necessary to vary and precisely fix the hormonal content in the complexes, it was found that lyophilization is the appropriate method. The processing conditions are substantially more benign than those described in the above patents, and therefore prevent a possible deactivation of the hormone. The reciprocal actions that occur between the molecules when mixing the solutions lead to stable complexes that present a controllable profile of release of active substance and a greater proteolytic stability. Accordingly, the polyamino acids do not only influence the delay behavior, but at the same time offer protection against undesirable premature proteolytic disintegration. This aspect is above all advantageous with a view to the application to of delayed effect of said preparations. In the delay behavior of the complexes, the type and molecular mass of the polyamino acids can be influenced substantially, the incorporation into the polymer structure of amino acids with hydrophobic side chains, and by the partial esterification of the existing carboxylic groups. The invention is explained in more detail on the basis of the following exemplary embodiments, without however limiting its scope. Preparation of polyamino acid-peptide complexes by precipitation: Example 1 50 mg of polyamino acid is dissolved in 5 ml of H0, in poly-glu with addition of IN NH4OH, slight heating at 40 ° C and ultrasonic treatment. 50 mg of cetrorelix (as acetate) are dissolved in 4 ml of H20. The polyamino acid solution is stirred and the cetrorelix solution is added in one step, and then it is stored for 4 hours at 4 ° C. After the precipitate is separated by centrifugation at 4000 rpm for 5 minutes, the supernatant is removed and the precipitate is dried for 24 hours under vacuum over P2O5. In view of the fact that there are no stoichiometric complexes, the yield was related to the sum of the starting substances.

Figure 1 shows various release curves in the static release system as a function of molecular mass (releasing agent: 0.01 m of ammonium acetate, pH 7.0). Performance: 59-65% of the theory. For the content of cetrorelix in the complex, we refer to Table 1. Table 1: Composition of the cetrorelix-poly-amino acid complexes.

Preparation of polyamino acid-peptide complexes by lyophilization: Example 2 Cetrorelix complex with 50% peptide content 50 mg of polyamino acid are dissolved in 5 ml of H0, in poly-glu with addition of NH40H IN, slight heating at 40 ° C and ultrasonic treatment. 50 mg of cetrorelix (as acetate) are dissolved in 4 ml of H20. The polyamino acid solution is stirred and the cetrorelix solution is added all at once and stirred for a further 2 minutes. The complex formed is frozen at -20 ° C and then lyophilized. In view of the fact that there are no stoichiometric complexes, the yield was related to the sum of the starting substances. Yield: 90-95% of the theory Amount of cetrorelix in the complex: 45-50% Example 3 By means of the corresponding modification of the amount of polyamino acid and cetrorelix a 70% cetrorelix complex was analogously made. Example 4 It is possible to obtain an increase in hydrophobicity combined with an increase in the delay behavior, inter alia, by partial esterification of the carboxylic groups. In figure 2 represent the curves of release of complexes of cetrorelix with methyl esters of polyglutamic acid.

Figure 3 shows the release curves of the cetrorelix complexes of polyglutamic acid with leucine and fenvialanin. EXAMPLE 5 To verify the in-vitro release tests, the complexes of cetrorelix-polyamino acid in aniomals were tested in this case of the complexes of cetrorelix with the following polyamino acids polyglutamic acid, M 5000 g / mol polyglutamic acid, M 16000 g / mol polyaspartic acid, M 7300 g / mol Figure 4 shows the suppression of testosterone in male rats after a single subcutaneous injection of 1.5 mg / kg. For each test group, 5 animals were examined. With these results it was possible to demonstrate that the complexes investigated produce a long-term effect of more than 600 hours in the suppression of testosterone (figure 4) . The effectiveness and suitability of the principle of cetrorelix-polyamino acid complexes as deposit preparation was checked

Claims (1)

1. CLAIMS New complexes of LHRH antagonists with polyamino acids, in particular polyglutamic acid and polyaspartic acid. Complex according to claim 1, characterized by an average molecular mass of polyamino acids of 2000-20000 g / mol. Complex according to claim 1 or 2, characterized in that antide, antarelix, azaline, A-75998, ganirelix, Nal-Glu antagonist, but in particular cetrorelix are used as LHRH antagonists. Process for the preparation of immobilized preparations and stabilized, parenterally applicable activity of peptide hormones of complexes of LHRH antagonists with polyamino acids, in particular polyglutamic acid and polyaspartic acid of average molecular masses dse 2000-20000 g / mol, characterized by the fact that that the polyamino acid-peptide hormone complex is precipitated from aqueous solutions. Process according to claim 4, characterized in that the polyamino acid-peptide hormone complexes are elaborated with a desired hormonal content controllable by lyophilization of aqueous solutions. Process according to claim 4, characterized in that the release of the active substance is controlled by the incorporation of hydrophobic amino acids into the polymer structure or by partial esterification. Medicament characterized by a content of one or several compounds according to claim 1-6, as well as optionally auxiliary and structural substances and stabilizers. Medicine to be used in the therapy of hormone-sensitive tumors, in particular for the treatment of mammary, ovarian and prostate carcinomas, benign prostatic hypertrophy, fertility disorders, endometriosis and in combination with hysteroscopy , as well as for in-vitro fertilization, characterized by a content of compounds according to claim 1-6. SUMMARY Within the framework of the present invention, it was desired to develop a delay system for LHRH antagonists, in particular for cetrorelix, by complexing with suitable biofilic carriers, allowing the active substance to be released in a specific manner over several weeks. The acid polyamino acids that are polyglutamic acid and polyaspartic acid were chosen to form the complexes with cetrorelix. The cetrorelix-polyamino acid complexes are prepared from aqueous solutions by pooling the solutions and precipitation of the complexes, which are then separated by centrifugation and dried under vacuum over P2O5. If you want to obtain complexes with a defined composition, it was found that lyophilization is the appropriate method. The cetrorelix-carboxylic acid complexes were also made from the aqueous solutions. The poly-glu and poly-asp acid polyamino acids exhibited good retardation properties in the static release system as a function of the hydrophobicity and the molecular mass of the polyamino acid. In the animal tests, the principle effectiveness of the cetrorelix-polyamino acid complexes as dit systems could be verified. For example, it was possible to achieve a suppression of testosterone during 600 hours in male rats through the formation of cetrorelix complexes with polyamino acids. In addition, the release of the active substance can be controlled by the type and molecular mass of the polymers.