Pharmaceutical composition which comprises a low soluble salt of a lhrh antagonist

The invention relates to a pharmaceutical composition, containing a poorly soluble salt of an LHRH analogue, useful in the treatment of tumors dependent on sex hormone and endometriosis.

It is known that, in therapy, peptides can be used without risk and with high biological efficacy only through parenteral injections, while, if administered orally, they are destroyed by enzymatic degradation; In the case of nasal application, a small percentage of the dose used is absorbed, and in the case of dermal application, there is no absorption.

Peptides have a very short lifespan in the body, so parenteral administration of drugs containing peptides, for example, LHRH analogues, such as certain LHRH superagonists and antagonists, should be performed daily to inhibit LH and FSH in both groups. substances.

The result, in men, is the reduction of testosterone secretion, and in women, it is the reduction of oestradiol secretion below a predefined, specifically defined value. For this method, the term chemical castration is used.

By LHRH analogues are meant both superagonists, such as cetrorelix (INN), antide (INN) or ganirelix (INN). Goserelin and the synthesis of goserelin are described in Drugs of the Future 5 (4), (1980), p. 191. Buserelin and the synthesis of busereline are described in Drugs of the Future, 4 (3), (1979), p. 173 and Drugs ofToday, 21, (305), (1985).

The decapeptide and the synthesis of the decapeptide are described in Drugs of the Future 3, (9), (1978), p. 645. Leuprolide and the synthesis of leuprolide is presented in Drugs of the Future 7 (12), (1982), p. 883.

Azaline B is described on pages 13-26 - Thie State of the Art (1993), Parthenon Publishing Groups Ed., B. Lunenfeld, V. Insler.

Attempts have been made to develop processes that use LHRH analogues with prolonged action in formulations having an effect of intensifying release. For example, GB 2052258 discloses a formulation containing LHRH analogues in the form of prolonged-release injections, containing peptide in the form of zinc salts, sesame oil and aluminum stearate.

The difference between super-agonists and antagonists is that, in the case of super-agonists, a mechanism of reverse reaction leads to the effect that in the first weeks of therapy is associated with a high secretion of hormones, the so-called explosion, which must be compensated with additional medication. In contrast, in the case of antagonists of which one is centrorelix (INN), the pharmacological effect occurs immediately and there is no explosion. Long-term reduction of the level of the sex hormone is the standard therapy in the palliative treatment of prostate and breast carcinoma, in order to reduce the growth of the tumor in the case of tumors dependent on sex hormones and also represents a curative treatment in endometriosis. From a chemical point of view, LHRH superagonists and antagonists are nona- or decapeptides.

An LHRH antagonist that is effective in the above method is cetrorelix, a decapeptide having the following amino acid sequence:

Ac-DNal-DPCI-Phe-DPal-Ser-Tyr-DCit-Leu-Arg-Pro-D-Ala-NH₂.

Its synthesis and pharmacological properties are described in EP 299402. Cetrorelix acetate has been identified as being physiologically acceptable salt. In preclinical and clinical studies, it was found that aqueous cetrorelix acetate solution should be administered daily to reduce the level of testosterone or oestradiol to a desired concentration, until the next injection. The duration of the action cannot be extended after reaching a threshold although weekly application instead of daily application would already be a step forward for chronic and unpatientized patients.

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The use of LHRH analogues consisting of administration of water-soluble salts 50 as a solution for injections or nasal administration in the form of nasal drops of Buserelin Acetate (Nasal Suprefact) or Nafarelin Acetate is currently sold only in US. As described above, it is difficult for these dosage forms to be used frequently. The injections can only be done by the doctor, and the drops for the nose should be used several times a day. Both dosage forms are not suitable for the treatment of chronic diseases.

DE-OS 4223282.1 describes the preparation of a prolonged-release formulation from the cetrorelic embryo by microencapsulation. Implants are forms of administration that have longer intervals of use. For example, if the implant of a cylinder under the skin of a poly (lactic-glycolic acid) copolymer containing goserelin acetate is made, it can effectively lower the level of testosterone (Zoladex deposit). Monthly injection of a suspension of biologically degradable particles containing leuprorelin acetate as active substance also decreases the level of sex hormones in the blood during this period (Enantone Monatsdepot^R).

Both forms of extended release are described in the following patents and patent applications 65. Below are the disadvantages, besides the advantage of the intervals between administrations.

EP 0058481 describes the composition and preparation of implants sold under the trade name Zoladex *. The disadvantage of the dosage form consists of the expensive process of making the cylinders, using special extrusion machines and packing machines, which 70 introduce the cylinders into syringes specially designed with extremely thick cannulae. The shape of the cylinder - the diameter of 1 mm and the length of several mm - causes great pain and hematomas, when implanted in the patient's body. A less painful dosage form is desirable.

EP 0052510 describes the composition and preparation of microparticles, for example, with 75% naphthalene. In the presence of chlorine hydrocarbons, the active substance is incorporated into poly (lactic acid, glycolic acid) copolymers. The use of chlorinated hydrocarbons cannot be avoided in this composition, because the polymers used, which have the biodegradation performance in vivo, are dissolved only in them.

The high residual solvent content of the dosage form prepared in this way 80 is approximately = 1000 ppm and is a disadvantage. According to the latest knowledge, chlorinated hydrocarbons are carcinogenic, the residual solvent content in raw materials and in medicines is limited to below 50 ppm according to the current proposal for European pharmacopeia (Pharm Europe, vol. 4, no. 1, March 1992). The process also has the disadvantage: the amount of active substance that is actually incorporated in microcapsules 85 is small, due to the loss of peptide in the aqueous phase.

Reducing the residual solvent content below this limit value, to use the risk-free drug, is only possible with laborious post-treatment. For example, Syntex has filed a patent application for a process for reducing the residual solvent with CO.₂ subcritical. 90

DE 4023134 applies a process for incorporating the peptide, as described above, into biodegradable polymers such as poly- (lactic acid, glycolic acid) copolymers. According to this patent application, a water-insoluble peptide salt is used, instead of incorporating the peptide acetate salt into the polyester, to reduce the large losses of active substance in this process. The peptide salts cited as being insoluble in water are pamogates, 95 tansit-Siearai-and palmitate. As mentioned above, the process has the disadvantage that carcinogenic hydrocarbons must be used and contain chloromethane or chloroform as physiologically unacceptable residual solvents.

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EP 145240 contains other ways of incorporating water-soluble peptide salts. In a complicated process, the active substance is incorporated with significant losses in the yield of the active substance, starting from solutions of aqueous active substances to biologically degradable poly (lactic acid, glycolic acids) copolymers through multiple emulsions, again having the disadvantage the use of chlorinated hydrocarbons.

According to EP 505966, buserelin acetate is embedded in poly (lactic acid, glycolic acid) copolymers by spray drying a solution of active substance-polymer containing chlorinated hydrocarbons. This process also has the disadvantage of using carcinogenic chlorinated hydrocarbons as solvents.

When using chlorinated hydrocarbons, the disadvantage lies not only in the high amounts of residual solvent in the medicines, but, from an environmental point of view, the use of these solvents, in itself, creates problems for the organization and deposition of the chlorinated hydrocarbons at the manufacturing site and the risks for employees.

According to WO 9214449, slightly soluble peptides, such as growth hormones, are incorporated, for example, into lauric acid by mixing the active substance with lauric acid, melting and grinding after cooling the mixture into 100 µm particles. US 5137669 discloses extended release forms for LHRH agonists to inject these particles as suspensions for delayed release of the active substance. A common factor for both formulations is that they require support materials for a longer period of release of the active substance and the injections are only obtained through a laborious process. Until now, it has been impossible to prove the tolerance of fat deposits, their safety and the reproducible release of the active substance. In case of fat incorporation, side effects may occur, instead of application, in the sense that encapsulation cannot be tolerated in a treatment lasting several years.

For bromocriptine mesylate, an active substance that is very difficult to prepare in long-term release forms, DE 3430852 synthesizes a new polyester, for example, a glucose polyester with lactic acid and glycolic acid to achieve the desired release form. of the active substance.

In this case, toxicological tests to prepare a time-release form were costly, and this objective could not be achieved through a cheaper and simpler dosage form.

Commonly used in pharmaceutical formulations is embonic acid (4,4'methylenbis acid (3-hydroxy-2-naphthenic) to form salts with low drug dissolution ability. These salts with low dissolution potential remain in body for a longer time and give the drug a prolonged release effect (Rompps Chemie-Lexicon, Stuttgart, 1976, p. 1007).

Surprisingly, it has been found that the formulation of the invention according to claim 1, results in an unexpected prolongation of the action and improved efficiency, without the use of biologically degradable polymers or fats. Not only was the duration of the action prolonged, as measured by the duration of the hormone formation prevented, but the growth of the tumor was also stopped, to a greater extent than the proportion.

The formulations of the invention can also be used as LHRH agonists, for example, in leuprolide, buserelin, goserelin and tryptorelin. The formulations of the invention may also be used in bombesin antagonists, somatostatin antagonists and GHRH analogues.

The problem solved by the invention is to make a pharmaceutical composition, containing a poorly soluble salt of an LHRH analog, in which the particle diameter is between 5 and 200 μm.

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The pharmaceutical composition according to the invention removes the above disadvantages, in that it comprises an LHRH analog chosen from cetrorelix, antarelix, ganirelix, antide or A-75998, isotonic agents and does not contain biodegradable polymers or fats.

By applying the invention, the following advantages are obtained:

- the duration of action of the drug has increased; 150

- the loads are more uniform;

- variations in the quality of the drug prepared with the composition of the invention are reduced.

The invention relates to a composition with prolonged action, for injections, which contains therapeutically active peptides, in the form of amorphous and crystalline salts, with reduced solubility.

The experimental working method was performed according to the following procedure. 155

Inhibitory effect on DMBA-induced breast cancer (7,12-dimethylbenz / a / -antracen) in Spraque-Dawley rats.

Method:

Sprague-Dawley 50-day-old female rats (animal diet: Altromin R, water at discretion) were treated with 20 mg of 7,12-dimethylbenzene / a / -anthracene orally dissolved in 160 ml of 1 ml of Measles using a stomach tube. Tumor occurrence is monitored by weekly palpation of animals. About 90% of animals develop tumors between day 35 and day 70 after adequate induction for experimental testing.

Tumor weight was determined using the method of Druckrey, H., Steinhoff, D., Nakayama, M., preussmann, R., Anger, K. (1983). 165 "Experimentelle Beitrăge zum Dose-Problem in der Krebs-Chemotherapie und zur Wirkungsweise von Endoxan (Experimental contributions to the dosage problem in cancer chemotherapy and to the action of endoxane), Dtsch. Med. Wschr. 88; 651.

The method was validated by comparing the tumor weights determined by palpation and the tumor weights determined by weighing (after tumor excision). 170

The correlation coefficient was 0.98. After the total weights of the tumors reached about 1 g, the animals were sorted and 7 animals were assigned for control and for the treatment group. Treatment was started immediately thereafter by subcutaneous injection of test substances.

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The hormonal status of the animals was determined using 175 methylene blue staining of the vaginal cell and evaluated according to Jones, TC, Mohr, U, Hunt, RD (1972): The genital system, in: Monographs on pathology of laboratory animals, sponsored from International Life Science Institute (Springer, NY, London).

The composition of the invention is an X-ray amorphous precipitate of cetrorelix decapeptide as an embonic acid salt. The aqueous suspension of this precipitate, which may possibly contain isotonic additives, exhibits a clear prolonged action action in an animal model, compared to the aqueous solution of the peptide. It was surprisingly found that the duration of action was approximately the same as that of an injection suspension, which contained the peptide embonate precipitate, in a biologically degradable polymer such as poly (lactic acid, glycolic acid) copolymers. This result was particularly unexpected, as, as shown above, 185 only very laboriously prepared extended release dosage forms, which generally contain the active substance in biodegradable polymers, have so far shown sufficient duration of action. long.

This finding was also surprising because according to J.

Pharm. Pharmavol. 47, 878-883 (1985), pyrimethamine, a 2,4-diaminopyrimidine derivative, 190 did not show any difference compared with the embonic acid salt in terms of pharmacokinetic behavior, course of plasma level and AUC after subcutaneous injection at mice.

Similarly, imipramine HCI showed no difference from imipramine embonate after oral administration (Indian Journal of Physiology and Pharmacology 25, (4), 331-338 (1989). Crystal suspensions for injections of certain substances medicines, without peptides, such as prednisolone or triamcinolone, are known to have prolonged-release forms, such as crystalline zinc insulin suspensions used to treat diabetes. Insulin has 51 amino acids. The latter are all in crystalline state, while X-ray diffractometric analysis shows that the dosage form of the invention is amorphous. The particle size of the formulation of the invention is between 5 pm and 200 pm. A cetrorelix pad with a particle size of less than 5 µm has a prolonged release effect lower than that of the formulation of the invention. Similarly, a cetrorelix pad with a particle size above 200 µm has a prolonged release effect, smaller than that of the formulation according to the invention.

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In the following, there are presented 5 embodiments of the invention in connection with FIG. 1 ... 5, which represents:

- Fig. 1, the experimental results. Tumor weight curve for untreated control animals, showing uninhibited growth. Curves 1 (*) and 2 (0) show the treatment with cetrorelix acetate in two different carrier substances. Prolonged curve 3 shows a strong reduction of tumor weight after embonate treatment.

Since the treatment, in this case, was only a single dose, the tumor continues to grow because the single dose treatment is not sufficient to destroy all tumor cells.

-fig.2 shows the testosterone level during 300 hours in four animals (no. 11-14) after applying the same dose of cetrorelix, in the form of suspension of cetrorelix embonate (D-20762). Testosterone suppression is also performed at 6 h after application, levels rise above 1 ng / ml in one animal after 192 h (eight days), in the other three animals it continues until the ninth day.

-fig.4 shows the testosterone level during 350 hours in four animals (No. 3134), after applying the same dose of cetrorelix, in the form of suspension of cetrorelix embonate (D-20762) as microparticles, prepared according to DE 4023134 A1 , quoted above. Testosterone suppression was achieved at 6 h after application, levels increased to over 1 ng / ml in two animals after approximately 150h (six days), in the other two animals the suppression lasted until the end of the test.

-fig.5 shows the evolution of testosterone level in rats treated with embonate according to the invention (particle size: 80 pm-125 pm).

Comparison of Figs. 2-4 with Fig. 5 highlights the advantage of the formulation according to the invention.

Example 1. In an equimolar ratio of peptide (calculated as free base) and embonic acid, an aqueous solution of embonic acid, containing excess alkali, is combined with the cetrorelix acetate solution, the embonic acid precipitating as yellow crystals. Upon addition of dilute sodium hydroxide solution to pH 7-7.5, the embonic acid is dissolved and precipitated with decapeptide as an aqueous salt of cetrorelix embonate, which has the peptide molar composition: 2: 1 embonic acid (Mol / mol). The precipitate is separated by filtration, washing with H₂And dry.

Example 2. Cetrorelix acid and embonic acid are dissolved in equimolar proportions in dimethylacetamide and the solution is dripped into water. The white precipitate of cetrorelix embonate peptide: 2: 1 embonic acid (Mol / Mol) is filtered and dried.

Example 3. Ketrorelic and embonic acid are dissolved in a molar ratio of 1: 1.6 in a mixture of dimethyl acetamide and, eventually, water and the solution is dripped into water. The yellow precipitate is separated by filtration and dried. The precipitate obtained is mixed with 70% ethanol, dried at 35 ° C and sieved through a mesh size of 80 to 125 µm.

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Example 4. The alkaline embonate solution is added to the ethanolic peptide acetate solution, in a peptide molar ratio: 2: 1 embonic acid. The white precipitate is separated by filtration and dried. The dry precipitate is moistened with 50% ethanol, dried in a 245 vacuum drying chamber and sieved. The white product contains 2: 1 peptide embonate salt (Mol / Mol).

Example 5. The alkaline embonate solution is added to the ethanol aqueous solution of peptide acetate in a molar ratio peptide: embonic acid 1: 1,6. The yellow precipitate is filtered and dried. The dry precipitate is moistened with 50% ethanol, dried in a vacuum drying room at 35 ° C and seated. The yellow product contains 2: 1 250 peptide embonate salt (Mol / Mol) in addition to the excess embonic acid.

Test on the duration of action in the animal.

Subcutaneously, precipitate suspensions are applied to male rats at the dose of 0.5 mg cetrorelix / kg body and the effect of the peptide on the testosterone level in plasma is determined. The effect of cetrorelix is ​​to reduce testosterone levels. For reference, 255 was also tested for a suspension injection, prepared according to DE 4023134 A1 and containing the peptide coating in poly (lactic acid, glycolic acid) copolymers. The duration of the prolonged release dosage form action of cetrorelix was determined by examining the aqueous solution of cetrorelix acetate.