HK1025255B - Immobilized activity stabilized lhrh antagonist complexes method for the production thereof - Google Patents

Description

Immobilized, activity-stable LHRH-antagonist complexes and methods for their preparation

The invention relates to LHRH-antagonists, such as, for example, antiendocridin, aprezrin, Azaline, A-75998, Ganrelix, Nal-glutamate antagonists, to slow-release, active-stable complexes of polyaminoacids, in particular poly-glutamate and poly-aspartate, to a process for their preparation and to medicaments containing these complexes.

The peptide hormone-polyamino acid complexes described can be used in medicine, for example, for the treatment of hormone-sensitive tumors, such as breast and prostate cancer, benign prostatic hyperplasia, hypertrophy, and in gynecology for the treatment of endometriosis, hysteroscopy and for the treatment of fertility disorders.

In patent specifications DD 257197, DD 269785 and DD 299265, methods for the preparation of fixed, biologically active stable and pharmacologically modified peptide preparations are described for insulin and for other biologically active protein hormones, the most essential feature of which is that the corresponding peptides form complexes with polyamino acids.

The preparation of the formulations is described in the cited patents, i.e. the generation of the complex under the action of formic acid and organic solvents such as chloroform and under severe preparation conditions. These methods have the risk of partial inactivation and reduced stability of the peptide hormones.

The literature describes, for the first time in 1981, sparingly soluble salts or complexes of LHRH-analogues in EP 0042753 and EP 0049628. The preparation of these complexes refers to the preparation of pharmaceutical products adapted to various medical uses.

ORSOLINI in 1989 described in DE patent 3822459 the preparation of water-insoluble polypeptides by forming complexes of LHRH-analogues with methylenepamoic acid (Embonsure), tannins and stearic acid. The poorly soluble complex obtained is also embedded in a polymer matrix made of (lactic-glycolic acid) -copolymers.

Further methods for the preparation of cetrorelix complexes embedded in (lactic-glycolic acid) -copolymers were described in 1993 by ORSOLINI and HEIMGARTNER in DE patents 4223282 and 4223284. The patent lists poorly soluble cetrorelix-complexes with methylenepamoic acid, tannins, stearic acid and palmitic acid.

The object of the present invention is to produce a sustained-release preparation which has improved, controllable, sustained-release properties and increased stability against premature proteolytic action of LHRH antagonists, for use in the treatment of a range of diseases known here, such as hormone-sensitive tumors, for example breast and prostate cancer, benign prostatic hyperplasia, endometriosis, hysteroscopy and for the treatment of fertility disorders, and to provide a simple, easy and environmentally friendly process for the preparation of these preparations.

The object of the invention is to provide a novel sustained-release preparation of LHRH-antagonists, such as, for example, anti-oviridin, aprezilex, Azaline, A-75998, Ganrelix, Nal-glutamate antagonists, but preferably cetrorelix, with biodegradable polymers having improved controllable sustained-release properties, and to provide a process for the preparation thereof.

The object of the invention is achieved by the preparation of a fixed, active, stable peptide hormone preparation for parenteral administration from a complex of an LHRH-antagonist with a polyamino acid, in particular with polyglutamic acid and polyaspartic acid, wherein the polyamino acid-peptide hormone complex is precipitated from an aqueous solution in the absence of organic solvents, preferably by freeze-drying the aqueous solution, to prepare a polyamino acid-peptide hormone complex with a controlled hormone content. The release of the active substance can be regulated by the type and molar mass of the polyamino acid, by introducing hydrophobic amino acids into the polymer backbone, or by partial esterification (fig. 2 and 3).

The compounds of the invention are used in medicine for the treatment of hormone-sensitive tumors, in particular for the treatment of breast and prostate cancer, benign prostatic hyperplasia, hypertrophy, and in gynaecology for the induction of ovulation, in vitro fertilization and endometriosis, and in combination with hysteroscopy.

The concept of "complex" in the context of the present invention means: two or more components are combined into a poorly soluble system that has no proven stoichiometric basis. Here, overlapping of interactions is involved, mainly due to hypovalent binding.

The poorly soluble peptide complexes are also sometimes referred to in the literature as "salts". This designation is likewise inaccurate in many cases, since, as already mentioned, substances of defined composition are not involved.

Ionic interactions also occur in peptides and proteins, but are not the only cause of structural changes or changes in aggregation state.

For peptides and proteins, the concept of "complexes" and "salts" can be understood broadly due to the wide variety of functional groups, as a result of the superposition of various interactions that produce the peptide and protein structures.

The polyamino acids used are suitable as a peptide-type, biocompatible carrier substance. It is important according to the invention that the active substance is not chemically bound to the polymer, but is merely immobilized on the polymer by divalent binding and hydrophobic interactions.

We have surprisingly found that: the LHRH-antagonist cetrorelix has a very high binding affinity especially for polyamino acids, especially for polyglutamic acid and polyaspartic acid. Such a high affinity for cetrorelix is surprising, since it cannot be foreseen on the basis of the literature to date and because of the structure of peptides.

The spontaneously precipitated complexes contained a defined, reproducible amount of hormone.

If, conversely, the hormone content of the complex is to be varied and precisely determined, a suitable method is freeze-drying.

These preparation conditions are much milder than those described in the previous patents and avoid possible hormonal inactivation.

The interactions between the molecules which occur during the mixing of the solutions lead to the production of stable complexes which have a regulatable release pattern of the active substance and an increased proteolytic stability.

The polyamino acids thus not only influence the sustained release state, but at the same time also have a protective effect against undesired premature proteolysis. This is advantageous in particular in the case of long-term use of these preparations.

The sustained-release state of the complex is mainly influenced by the type and molar mass of polyamino acids, the introduction of amino acids with hydrophobic side chains onto the polymer backbone, and by the partial esterification of the existing carboxyl groups.

The invention is further illustrated by means of the following examples, without being limited thereto.

The polyamino acid-peptide-complex is prepared by precipitation.

Example 1

50mg of polyamino acid are heated to 40 ℃ and sonicated (1N NH in the case of polyglutamic acid)₄OH) was dissolved in 5ml of water. 50mg of cetrorelix (as acetate) was dissolved in 4ml of water. The polyamino acid solution was stirred, and cetrorelix solution was added at once, followed by storage at 4 ℃ for 4 hours. The precipitate was then centrifuged at 4000rpm for 5 minutes, the supernatant removed, and the precipitate was passed over P in vacuo₂O₅And drying for 24 hours. Since no stoichiometric complex is present, the yield is calculated as the total amount of starting material. Figure 1 shows various release profiles with respect to molar mass in a statistical release system. (Release Medium 0.01m ammonium acetate, pH7.0)

Yield: 50-65% of theory

The content of the complex in Western and Western Quyleigh grams is shown in Table 1.

Table 1: composition of precipitated cetrorelix-polyamino acid complexes

Polyamino acid	Average molar mass [ g/mol ]]	Hormone content [% ] in the complex]Relative error is 5%	PAS in molar ratio	Molar ratio hormone: free carboxyl
					Polyglutamic acid	5000	86	1∶0.05	1∶1.9
16000	85	1∶0.016	1∶2.1
				50000		60	1∶0.02	1∶8.2
The methylation degree of the polyglutamic acid methyl ester is 2.2 percent and 24.4 percent	16000	81	1∶0.02						1∶2.8
				16000	58	1∶0.06	1∶6.7
	Polyaspartic acid	7300	86					1∶0.03	1∶2.2
14000				78	1∶0.03	1∶3.8
		28000	69				1∶0.023	1∶6.1
Poly [ (glutamic acid, phenylalanine)/4: 1]				45000	65	1∶0.017			1∶4.5
	Poly [ (glutamic acid, leucine)/4: 1]	70000	79				1∶0.005	1∶2.4

Preparation of polyamino acid-peptide-complexes with defined peptide content by lyophilization

Example 2

Cetrorelix complexes with a peptide content of 50%

Heating to 40 deg.C and ultrasonic treating (adding 1NNH in the case of polyglutamic acid)₄OH) 50mg of polyamino acid are dissolved in 5ml of water. 50mg of cetrorelix (as acetate) was dissolved in 4ml of water. The polyamino acid solution was stirred, the cetrorelix solution was added at once, and stirring was continued for 2 minutes. The resulting complex was frozen at-20 ℃ and then freeze-dried. Since no stoichiometric complex is present, the yield is calculated as the total amount of starting material.

Yield: 90-95% of theory

The content of the compound of Chinese and western trityleigh: 45-50 percent.

Example 3

A similar procedure produces 70% cetrorelix complexes by varying the amounts of polyamino acid and cetrorelix accordingly.

Example 4

It is also possible to increase hydrophobicity and increase sustained release properties by partially esterifying the carboxyl group. FIG. 2 shows the release profile of cetrorelix complex with poly (methyl glutamate). FIG. 3 shows the release profile of the cetrorelix complex of polyglutamic acid with leucine and phenylalanine.

Example 5

For release detection in vitro experiments, cetrorelix-polyamino acid-complexes were tested in animal experiments.

The invention relates to cetrorelix complexes 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 testosterone inhibition after one subcutaneous injection to male rats at a dose of 1.5mg/kg, 5 animals were tested per experimental group.

These results indicate that the long-lasting effect of the tested complexes in testosterone suppression is over 600 hours (figure 4).

The basic role and applicability of cetrorelix polyamino acid complexes as sustained release formulations was demonstrated.

Claims (6)

1. A complex of a polyamino acid consisting of polyglutamic acid and polyaspartic acid having an average molecular weight of 2000-20000g/mol and an LHRH-antagonist cetrorelix.

2. Method for the preparation of fixed and activity stable parenterally administered peptide hormone formulations using a complex according to claim 1, namely a complex of polyglutamic acid and polyaspartic acid with an average molecular weight of 2000-20000g/mol and the LHRH-antagonist cetrorelix, characterized in that the complex of polyamino acid and LHRH-antagonist cetrorelix is precipitated from an aqueous solution.

3. Method according to claim 2, characterized in that the complex of polyamino acids and the LHRH-antagonist cetrorelix, containing the desired controllable hormone content, is prepared from an aqueous solution by lyophilization.

4. A method according to claim 2, characterized in that the release rate of the active substance is controlled by the hydrophobic amino acids in conjunction with the polymer structure or by partial esterification.

5. Use of one or more complexes according to claim 1 or 2 for the preparation of a medicament for the treatment of hormone sensitive tumours.

6. Use of one or more complexes according to claim 1 or 2 for the preparation of a medicament for the treatment of breast, ovarian and prostate cancer, benign prostatic hyperplasia hypertrophy, fertility disorders or endometriosis, for use in combination with hysteroscopy and for in vitro fertilization.