HU199694B - Process for producing citostatic pharmaceutical compositions containing gonadoliberin derivatives - Google Patents

Description

The present invention relates to a process for the preparation of antineoplastic pharmaceutical compositions containing gonadoliberin derivatives.

More particularly, the present invention relates to a process of formula (I)

Glp-His-Trp-Ser-Tyr-X ₁ -X ₂ -X ₃ -Pro-X ₄ (I)

- where

X 1 is a glycyl group or a D-phenylalanyl group,

X ₂ is L-leucyl or L-tryptophyl, X ₃ is L-glutaminyl or L-leucyl, and

X ₄ is a glycyl amide or ethyl amide group, provided that when X _{2 is a} tryptophyl group and X is a non-glycyl group, then X _{3 is a} non-leucyl group only - gonadoliberin derivatives or their pharmaceutically acceptable acids and its (metal) complexes for the treatment of a variety of benign and malignant tumors and clinical conditions.

Gonadoliberin (also known in the literature as gonadotropin releasing hormone, GnRH, luteinizing and follicle stimulating hormone-releasing hormone, LH / FSH-RH) and its agonist derivatives are luteinizing (LH) and follicle-stimulating hormone (FSH). release of the pituitary gland, and through this action, gonadoliberin regulates the essential reproductive processes.

The regulation of the reproductive process is a very complex system that is influenced by many different factors through the hypothalamic-pituitary-gonadal system. Gonadoliberin plays a central role in this complex regulation, since almost all of the regulatory parameters affect the synthesis or release of gonadoliberin in the hypothalamus, or affect the pituitary.

Gonadotropins (LH and FSH) regulate the steroid synthesis of the gonads and the maturation and release of gametes. In females, the primary role of FSH is in promoting the development of follicles and ova. Purified FSH in pituitary anesthetized animals causes an increase in ovarian weight and an increase in the number of secondary follicles as soon as ovulation does not follow. The role of LH is to regulate steroidogenesis and to induce ovulation and subsequent luteinisation. The synthesis of steroids is maintained by basal LH levels, while ovulation and germ cell division are preceded by so-called ovulation. preovulatory LH peak. Although LH is generally considered to be responsible for inducing ovulation, it is likely that FSH is also required for the process, ie the combined effect of both hormones must be considered. Changes in the LH / FSH ratio play a regulatory role in the sexual cycle and induce typical phenotypic changes in the sex glands.

The release of gonadotropins is biphasic. At baseline, release is continuous, pulsating, and regulates the basic functions of the genitals. However, ovulation requires short term high concentrations (pre-ovulatory peak) of gonadotropins.

GnRH was considered a non-species-specific hormone for a long time, until it became known in the early eighties that certain fish and / or fish were the structure of the gonadoliberin of avian species is different from that of mammals (JA King and RP Millar, J. Biol. Chem. 257: 10722-28 (1982); N. Sherwood et al., Proc. Natl. Acad. Sci. USA 80, 2794-2798 (1983)). )). These differences can be found at the 7 and / or 8 amino acid positions of GnRH.

Fish and fish new GnRH analogs related to avian-specific GnRH have been developed and used to induce ovulation and folliculogenesis in various fish and mammals (U.S. Patent Nos. 190,207 and 4,747,553).

Two of these analogs ((D-Phe ⁶ , Gin ⁸ ) -GnRH and (D-Phe ⁶ , Gin ⁸ , desGly ¹⁰ ) -GnRH-ethylamide) have been shown to be particularly effective in enhancing the reproductive function of fish. With these two peptides we were able to induce artificial reproduction of fish that could not be artificially propagated before. The same analogues have also been shown to be able to induce folliculogenesis and ovulation in fish outside the drinking period (Hungarian Patent Nos. 189,394 and 4,647,552).

Due to their potent follicular stimulating activity, the effect of these analogs in mammals was also investigated. Even in sexually immature animals, the analogs were capable of inducing folliculogenesis and ovulation, halting physiological anesthesia, curing sperm production disorders, and other sexual disorders. Use in the treatment of mammals is described in Hungarian Patent No. 193,607.

The fact that these new GnRH analogues induced follicular maturation and ovulation in cases where the mammalian GnRH analogs were ineffective raised the possibility of a specific mechanism of action and a direct effect on the sex glands.

Research in recent years has shown the presence of GnRH-like factors and GnRH receptors in the gonads, and it is believed that they form a paracrine system (summary:

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A. J. W. Hsueh and J. M. Schaeffer, J. Steroid Biochem. 23: 757-764 (1985).

Recently, the use of superactive GnRH analogs for the treatment of various hormone-dependent tumors has become widely accepted and the results are very encouraging. Applications include benign and malignant neoplasms, including prostate and breast cancers, chondrosarcoma and osteosarcoma, pancreatic cancer, pituitary tumors, ovarian cancer and neoplasm of female genital organs. Regarding the mechanism of action of these superactive GnRH analogues, single, acute administration of such agents results in a definite and prolonged release of LH and FSH, whereas chronic treatment results in dramatic inhibition both by sensitization of gonadotropic cells in the pituitary gland, both by reducing the number of LH and FSH receptors in the gonads. Accordingly, repeated repeated administration of GnRH agonists results in a significant reduction in circulating LH and FSH levels, while estrogen, progesterone and testosterone levels fall to castration levels. Such a reduction in the level of sex steroids and thus the elimination of the stimulatory effects of estrogen and testosterone form the basis for the use of superactive GnRH analogs in the treatment of hormone-dependent tumors. There is currently no known GnRH agonist that exerts its anticancer activity through other types of mechanism of action.

It is an object of the present invention to use novel GnRH analogs of formula I for the treatment of hormone-dependent tumors without inducing total hormonal castration.

The present invention is based on the discovery that using the compounds of the formula I, the above object can be achieved, i.e., the growth of hormone-dependent tumors can be stopped. This finding is surprising because GnRH analogues with hitherto known antitumor activity have been shown to be superactive, i.e. 50 to 200 times more active than the native hormone, and have been exerted by desensitization (MA Eisenberger et al., J. Clin. Oncology 4). / 3; 414-424 (1986); J. Waxman: British Medical Journal 295, 1084-85 (1987). The phenomenon of desensitization induced by superactive mammalian GnRH analogs has been extensively studied in vitro and in vivo. It was found that the administration of superactive mammalian GnRH analogues at a single higher dose desensitizes gonadotropic cells of the pituitary gland to the LH and / or GHR pulses of subsequent GnRH pulses. FSH-releasing effects, and that this effect is partly due to complex internalization of the hormone receptor, ie intracellular uptake, and partly intracellularly, via the biosynthesis of gonadotro4 pins. (Kéri et al., 1983, Mol. Cell. Endocrinol. 30: 109-120). However, the high LH and FSH pulse following administration of the mammalian superactive GnRH analogue also causes desensitization at the gonadal level to the subsequent LH and FSH pulses, which is also due to receptor-level and intracellular activity (J. Waxman, The Releaser 1 , 7-11 (1986). This dual desensitization effect leads to a significant reduction in the secretion and biosynthesis of gonadal steroids, resulting in inhibition and inhibition of steroid hormone-dependent tumors. results in regression. Because the presence of steroids in hormone-dependent tumors is essential for the survival of tumor cells in a competitive environment, surgical and surgical treatment of these tumors is essential. despite the unpleasant side effects of castration symptoms, hormone-desensitization castration is a dominant medical phenomenon. Treatments with known mammalian superactive GnRH analogues resulted in a reduction in the levels of estradiol, progesterone and other sex steroids to castration, resulting in inhibition of ovarian function, follicular atresia, which was also evident in the histology of the ovary. . For the active ingredient of the pharmaceutical compositions of the present invention, chicken GnRH or g. salmon-GnRH analogue, respectively. they are derivatives thereof which are not super-active in a mammalian animal and thus do not act by the mechanism of desensitization. In vitro experiments on pituitary cell culture revealed that chicken and GnRH analogs of salmon do not behave as superactive analogs in rat hopophyseal cell culture and thus do not desensitize in the appropriate dosage range. The chicken GnRH produced by us and the gnRH, respectively. however, the novel mechanism of action of salmon-GnRH analogues in mammals was suggested by the fact that these analogues were able to stimulate infantile animals to sexual maturity and, under zoo conditions, oligospermic animals could be stimulated to physiological levels, which was not possible due to see Hungarian Patent Specification No. 194,913).

Against this background, the non-desensitizing nature of the antitumor activity of chicken and salmon GnRH analogues used as active ingredients in the compositions of the present invention and the known mechanism of action of mammalian superactive GnRH analogs in hormone dependent tumors are unexpected. These formulations reduce estradiol levels (from 85 pmol / l to 34 pmol / l) at an effective dose for antitumor activity, but reduce progesterone levels by 3

Not only does it not reduce, but it is also strongly stimulated (from 19 pmol / l to 38 pmol / l), ICI's formulation Zoladex ([DSer / Bu '), AzGly' °] -GnRH) reduces progesterone to a castration value (10 pmol / l). More importantly, however, after prolonged treatment with mammalian superactive GnRH analogues, the histology of the ovary shows inhibition of ovarian function and follicular atresia, as well as in chicken and chicken. however, the active dose of salmon-GnRH analog does not inhibit ovarian histology, developing follicles and corpus luteum. At present, this effect cannot be explained, but it is based on a novel mechanism of action.

The preparation of compounds of formula (I) is known from Hungarian Patent No. 190,207 and the corresponding U.S. Pat. No. 4,410,514 and from an article by Horváth et al., Biochem, Biophys. )). These sites describe the beneficial effects of the compounds on the reproductive process of animals.

Accordingly, the present invention provides a process for the preparation of a pharmaceutical composition comprising the GnRH analogue of formula (I), an acid addition salt or a metal complex thereof as an active ingredient. According to the present invention, the active ingredient of the present invention is admixed with carriers and / or excipients conventionally used in the preparation of pharmaceuticals and converted into a pharmaceutical composition suitable for the treatment of hormone-dependent tumors but without causing hormonal castration.

The term "known in the art" encompasses methods disclosed prior to the priority date of the invention.

The compounds of formula I are preferably used in the form of solutions, powders, injections or depot formulations. The route of administration may be intramuscular, subcutaneous, intraperitoneal, or intravenous.

The pharmaceutical compositions of the present invention are preferably administered in a daily dose of 0.5 to 5000, preferably 1 to 500, more preferably 5 to 150 pg / kg body weight. It is advisable to allow at least 8 hours between repetitive treatments. It is advisable to continue dosing until tumor growth has stopped and no improvement has occurred.

The pharmaceutical composition of the present invention suppresses tumor growth by inhibiting the proliferation of hormone-dependent tumor cells. This effect is likely to be achieved, in part, by stimulating the specific differentiation functions of these hormone-sensitive tumor cells.

The main advantages of the process according to the invention are as follows:

a) It allows stopping the growth of benign and malignant tumors and thus improving the condition of the cancer patient.

b) The method is capable of inhibiting tumor growth by completely reducing the circulating levels of steroid sex hormones, i.e., "without causing hormonal castration. This means that the treatment does not have the adverse physiological and psychological effects of hormonal castration.

c) The GnRH analogs used in the method exert their antitumor activity by a mechanism of action other than the superactive GnRH analogs used hitherto. This means that the procedure can be used as a standalone or alternative therapy.

(d) In the case of breast cancer and gonadal cancer, it shall permit the elimination of tumor growth without reducing ovarian weight and inhibiting follicular maturation.

e) The composition of the present invention does not reduce libido and spermatogenesis as opposed to treatment with superactive GnRH analogs.

f) It reduces tumor growth even when tumor cells do not contain steroid receptor. This means that the pharmaceutical compositions of the present invention can be used to treat a broader range of breast and gonadal cancers than previously known hormonal formulations, i.e., the more aggressive steroid-negative tumors.

g) Inhibits tumor growth and stabilizes healing. Thus, it can be used as adjunctive therapy in combination with other drugs, e.g. cis-platinium [Pt (NH ₃ ) ₂ Cl ₂ ], which inhibits mitosis and has toxic side effects.

The invention is illustrated by the following non-limiting examples.

Example 1

Antitumor activity against dimeric 1-benzanthracene (DMBA) induced breast cancer

15-20 weeks, approx. Female Sprague-Dawley rats (200 g) which induced breast cancer with dimethylbenzanthracene (DMBA) were treated twice daily for 3 weeks with (D-Phe ⁶ , Gin ⁸ , desGly ¹⁰ ) GnRH-ethylamide analog (10 pg / day, intramuscular). injection). Tumor weights and typical sizes were measured immediately prior to treatment, and 1, 2, and 3 weeks after treatment. 16 hours after the last injection, the ovaries were removed and blood samples were taken to determine the levels of various hormones. The weight of the ovaries removed was measured and the tissue prepared for histological examination.

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Treatment of female rats (DPhe ⁶ , Gin ⁸ , desGly 10) with GnRH-ethylamide reduced the size of the ovarian-dependent breast cancer induced by DMBA in proportion to the treatment time. By the end of the three-week treatment period, the average tumor volume had fallen to 10% of its original volume. GnRH-ethylamide-mediated tumor size reduction (D-Phe, Gin ⁸ , desGly 10) was equivalent in both rate and extent to the regression observed at 3 weeks post-surgical castration or in animals dosed at the same dose. , a mammalian GnRH agonist used for the treatment of advanced breast cancer: (D-Ser / But / ⁶ , Azgly ¹⁰ ) GnRH (ICI 118630).

Histological examination of the ovaries of animals treated with (D-Phe ⁶ , Gin ⁸ , desGly 10 °) GnRH-ethylamide and analysis of steroid sex hormone blood levels showed normal follicular development and corpus luteum development and continued ovarian function. Although circulating levels of estradiol were reduced compared to intact, untreated controls, the degree of reduction was not as pronounced as in surgically castrated animals. Progesterone levels and ovarian weight were not reduced (treatment with D-Phe ⁶ , Gin ⁸ , desGly'jGnRH-ethylamide. In contrast, ICI 118630 significantly slowed follicular maturation, decreased ovarian weight, and decreased estradiol and progesterone plasma levels. concentration to the castration level.

These data indicate that (D-Phe ⁶ , Gln ⁸ , desGly ¹⁰ ) GnRH-ethylamide is an effective anticancer agent in DMBA-induced breast cancer, and this anticancer effect is not accompanied by complete inhibition of ovarian function.

Example 2

Inhibition of cell division in a human breast cancer cell line

Detailed studies of the MDA MB 231 tumor cell line from human breast carcinoma show that it is an estrogen receptor negative cell line. Cells from this cell line were cultured in RPMI medium supplemented with 10% FCS (Fetal calf serum) (manufactured by GIBCO (Hoofddorp, The Netherlands). 24 hours after transplantation, cells were treated with 5, 10 and 25 pg / ml (D- Phe ⁶ , Gin ⁸ , desGly ¹⁰ ) with GnRH-ethylamide in the presence of 1 pCi ³ H-thymidine Cells were incubated for 24 hours and media was removed, washed twice with phosphate buffered saline and precipitated with 10% trichloroacetic acid. The substance insoluble in trichloroacetic acid was dissolved in concentrated formic acid and its radioactivity was measured by a scintillation counter.

Pg peptide reduced the incorporation of ³ H-thymidine by 32%, 10pg by 65%, and 25 pg by 71% in the insoluble fraction of 10® db / ml cells.

These results clearly show that (D-Phe, Gin ⁸ , desGly 10) GnRH-ethylamide inhibits the growth of these estrogen-independent human breast cancer cells.

Example 3

Inhibition of cell division of the human breast cancer cell line

MDA-MB 231 tumor cell line from human breast carcinoma was tested for cell proliferation activity of (Gin ⁸ ) -GnRH, (DPhe ⁶ , Gin ⁸ ) -GnRH according to the experimental conditions described in Example 2, but for 72 hours. (DPhe ⁶ , Gln ⁸ , desGly 10) GnRH-ethylamide, (Trp'Leu ⁸ ) -GnRH.

All peptides were used at a dose of 10 pg. The incorporation of ³ H-thymidine was inhibited by the above peptides as follows: * (Gin ⁸ ) -GnRH 36% (DPhe ⁶ , Gin ⁸ ) -GnRH 60% (DPhe ⁶ , Gin ⁸ , desGly ^{, 0} ) -GnRH-EA 70% (Trp'.Leu ⁸ ) -GnRH 38%

Claims (2)

PATENT CLAIMS A process as an active ingredient of formula (I) 35 Glp-His-Trp-Ser-Tyr-X, -X ₂ -X ₃ -Pro-X ₄ (i) - wherein X 1 is a glycyl group or D-phenyl 1-a la η i I group, X ₂ is L-leucyl or L-tryptophyl. 4q X ₃ is L-glutaminyl or L-leucyl and X ₄ is a glycyl amide or ethyl amide group, provided that when X _{2 is a} tryptophyl group and X is a non-glycyl group, then X _{3 is a} non-leucyl group only - gonadoliberin derivatives or their pharmaceutically acceptable acids. the pharmaceutical compositions containing addition salts or metal complexes acceptable thereof, wherein the active ingredient prepared in a known manner are mixed and for the treatment of hormone-dependent tumors the established pharmaceutical vivőés / or excipients, converted to free ⁵⁵ but hormonal castration inducing any pharmaceutical composition. The method of claim 1, wherein the pharmaceutical composition comprises 0.0035 to 350 µg per unit dose of θθ, wherein X 1, X ₂ , X ₃ and X ₄ are as defined in claim 1. - in free form or as a salt or metal complex.