FR2631237A1 - USE OF GONADOLIBERIN DERIVATIVES FOR PREPARING ANTI-TUMOR PHARMACEUTICAL COMPOSITIONS - Google Patents

Abstract

The invention relates to a process for the preparation of pharmaceutical compositions containing a gonadotropin-releasing derivative of the general formula I: Glp-His-Trp-Ser-Tyr-X1 -X2 -X3 -Pro-X4 in which X1 is a glycyl group or a D isomer of any natural or synthetic aromatic D-amino acid group; X2 represents an L-amino acid group carrying a C1-4 alkyl group or an L-phenylalanyl or L-tryptophyl group; X3 represents an L-amino acid residue bearing a C1-4 alkyl or C2-4 alkanoyl amide side chain; and X4 represents a glycylamide or (C1 - 4) alkyl amide group, with the proviso that X3 represents a group other than the leucyl group when X2 is a tryptophyl group and X1 is other than the glycyl group, its addition salts formed with pharmaceutically acceptable acids or metal complexes thereof, which comprises admixing the compound of general formula I, its acid addition salt or its metal complex prepared in a manner known per se, with carriers and / or additives commonly used in the pharmaceutical industry and their forming into a pharmaceutical composition useful for treating hormone-dependent tumors without inducing hormonal castration. In addition, the invention relates to the use of gonadotropin releasing derivatives of general formula I for preparing pharmaceutical compositions against hormone dependent tumors and clinical conditions.

Description

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  The present invention relates to the use of gonadotropin releasing derivatives for the preparation of compositions

  pharmaceutical inhibiting tumors.

  More particularly, the invention relates to the use of gonadotropin-releasing derivatives of general formula (I1, Glp-His-Trp-Ser-Tyr-X1-X2-X3-Pro-X4 (I1 in which X1 is a glycyl group or a D-isomer of any natural or synthetic aromatic D-amino acid group; X 2 represents a L-amino acid group carrying a C 1-4 alkyl group or an L-phenylalanyl or L-tryptophyl group; X 3 represents a residue of a C 1 -C 4 alkyl side-chain-borne amino acid or C2-4 alkanoyl amide and X4 represents a glycylamide or C 1-41 amide group, with the proviso that X 3 represents a group other than the leucyl group when X 2 is a tryptophyl group and X 1 is other than the glycyl group, and addition salts formed with pharmaceutically acceptable acids and (metal) complexes thereof, for the treatment of various benign and malignant tumors and clinical conditions, as well as for the preparation of com positions

  pharmaceutical products containing these compounds.

  Gonadotropin-releasing hormone [other names used in the literature are known to be: gonadotropin-releasing hormone (OnPH), luteinizing hormone-releasing hormone and follicle-stimulating hormone (LH / FSHPIH)] and its agonist derivatives stimulate the release of luteinizing hormone (LH) and follicle-stimulating hormone [FSH] from the pituitary gland and, because of this action, gonadotropin-releasing hormone regulates the model

  fundamental of the reproductive cycle.

  The regulation of reproductive functions is a very complicated system influenced by a number of diverse

  factors from one end of the hypothalamic-pituitary axis

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  gonadal. In this complex regulation, gonadotropin-releasing hormone plays a central role, since virtually every regulatory parameter has an effect on the synthesis or release of 6nPH at

  from the pituitary gland, or its action on the pituitary gland.

  Gonadotropins (LH and FSH) regulate the synthesis of steroid hormones in the gonads as well as the processes of gamete maturation and release.In females, the main function of FSH is to promote the development of follicles and ova. In animals that have been removed from the pituitary, purified FSH induces an increase in ovarian weight and increases the number of ovarian follicles, while no ovulation results.The function of LH is to regulate steroidogenesis and ovulation and subsequent luteinization, steroidogenesis is maintained by baseline LH, while ovulation and continuous meiosis of gametes are regulated by a preovulatory LH peak. LH is responsible for the induction of ovulation, it is assumed that FSH is also required for the process, so that the combined effects of both hormones must be - co The movements of the LH / FSH ratio play a regulatory role in the sexual cycle and induce alterations.

  phenotypic characteristics in the gonads.

  The release of gonadotropins takes place in two phases. In the basal stage, the release is continuous, has a pulsating character and regulates the basal functions of the sexual organs. Ovulation, however, requires high levels of short-lived gonadotropins and is known as a peak

preovulatory gonadotropins.

  GnPlH has long been considered a non-specific hormone of a species until the early eighties, when it was learned that the structure of gonadotropin-releasing hormone of certain species of fish and birds is structurally

  different from that of mammals IJ.A King and P.P. Millar J. Biol.

  Chem. 257, 10722 (1982); N. Sherwood et al .: Proc. Natl. Acad. Sci. USA 80, 2794 (1983)]. These differences are found in the

  amino acids positions 7 and / or 8 of the 6nRH :.

  Novel GnPlH analogs derived from fish- or bird-specific GnPH analogues have been developed and used to induce ovulation and folliculogenesis in various species of fish and mammals (Hungarian Patent No. 190.207;

U.S. Patent No. 4,647,555.

  It has been found that two of these analogs [(D-Phe8, GIn8) -

  3nPH and (D-Phe6, GlnSdesGly 10-GnPH-ethylamide)] were particularly potent in stimulating reproductive functions in fish. Using these two peptides, it was possible to obtain induced artificial reproduction of fish that previously could not be artificially reproduced. The same analogs were also capable of inducing folliculogenesis and ovulation in fish outside the breeding season. fry

  (Hungarian Patent No. 189,394 and US Patent No. 4,647,552).

  Because of their potent folliculogenic activity, the effect of these analogs has been tested in various mammals. They were able to induce folliculogenesis and ovulation even in sexually immature animals, overcome physiological anoestrus, treat spermatogenesis disorders, and treat other sexual disorders in various animals. The use for the treatment of mammals is described in detail in Hungarian Patent No. 194,913 and US Patent No.

4753928).

  The possibility of a particular mechanism of action and direct gonadal action has been raised by the fact that these novel BnRH analogues induce follicular maturation and ovulation in cases where nnPFH

  mammals were ineffective.

  The presence of GnIH-like factors and BnPH receptors in the gonads and ovary has been well studied in recent years and it has been suggested that these may be

  form a paracrine system [see A.J. W. Hsueh and J.M.

  Schaeffer: J. Steroid Biochim. 23, 757 1985)].

  Recently, the use of analogues - superactive

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  GnPH for the treatment of various hormone-sensitive tumors has been widely accepted and the results are very promising. The application of superactive GnPH analogues extends to both benign and malignant neoplasms, and includes breast and prostate cancer, chondrosarcomas and osteosarcomas, pancreatic cancer, pituitary tumors, ovarian cancer and neoplasms of the female genital tract as well as other hormone-dependent tumors. About the mechanism of action of these superactive analogues of SnRH, it is known that a single acute administration of these substances induces a marked and prolonged release of LH and FSH, whereas a chronic treatment produces significant inhibitory effects by a method of "down-regulation" of pituitary membrane receptors for BnRH, desensitization of pituitary gonadotropes and reduction of gonadal receptors for LH and FSH. Thus, repeated administrations of GnRH agonists lead to a marked decrease in the level of LH and FSH in the circulation, while the concentration of estrogen, progesterone and testosterone falls to castration levels. This deprivation of castration-like sexual steroids and the elimination of the stimulatory effects of estrogen and testosterone are fundamental for the use of super-active 13nPH analogs in the treatment of hormone-dependent tumors. Currently, no GnPH agonist is known which exerts antitumor activity via a different mechanism of action. The object of the present invention is to use the GnPH analogues of general formula (I) for treating hormone-dependent tumors. without inducing a complete hormonal castration. The invention is based on the finding that the foregoing purpose can be achieved, i.e., the growth of hormone-dependent tumors can be inhibited by the use of the compounds of general formula I1). This finding is surprising, since all known BnPlH analogues

  now in the literature and possessing anti-

  tumor, are superactive, that is to say 50 to 200 times more active than the natural hormone and exert their effect by desensitization [M. Eisenberger et al .: J. Clin. Oncology 4, 414 (1986) J. Waxman: British Medical Journal 295, 1084 (198711). The desensitization phenomenon induced by superactive analogs of mammalian nRH has been extensively studied in both in vitro and in vitro experiments. It has been established that gonadropic cells of the pituitary gland are desensitized even by a single higher dose of superactive mammalian-BnRH analogs against the releasing effects of LH and FSH from subsequent SnRH flares. The effect is distinguished on the one hand by the internalization of the hormone-receptor complex, that is to say by penetration into the inner part of the cells and, on the other hand, by an effect inhibiting the biosynthesis or the secretion, respectively ,

  gonadotropins [Keri et al .: Mol. Cell. Endocrinol. 30, 109 (19831).

  At the same time, the strong release of LH and FSH following mammalian BnRH super-analogue administration induces gonad desensitization against subsequent releases of LH and FSH which are also developed as a result of intracellular and at the level of the receptor [J. Waxman: The Releaser 1, 7 (1986)]. This double desensitizing effect leads to a significant decrease in the secretion or biosynthesis, respectively, of gonadal steroids, so that tumors dependent on steroid hormones are inhibited or reduced. As the presence of steroids is absolutely necessary for the survival of tumor cells of hormone-dependent tumors in the competitive environment, surgical or hormonal desensitizing castrations, respectively, are of decisive therapeutic importance in the treatment of these tumors despite the painful side effects of castration symptoms. Treatments with the known superactive analogues of mammalian nnPH led to a decrease in the amount of estradiol, progesterone, and other sex steroids up to the level of castration, so that inhibition of ovarian function and atresia

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  follicles, which could be established unambiguously from the image

histological of the ovary.

  In the case of the pharmaceutical compositions used according to the invention, the active components are analogs of chicken 6npH or salmon 3nPH analogues, respectively, or their derivatives which are not superactive for mammals and whose mechanism of action therefore does not rely on desensitization. In in vitro pituitary culture studies, it has been established that chicken nnPRH analogs or salmon GnPH analogues do not behave as superactive analogues in a rat pituitary culture and are not therefore have no desensitizing effect in the corresponding dose range. On the other hand, a novel mechanism of action exhibited in mammals by Salmon GnPlH or salmon -3nRH analogs, respectively, used by the Applicant, was indicated by the fact that very young animals could be stimulated by these analogues until sexual maturation, or that oligospermic animals could be stimulated at a physiological level under zoo conditions. Due to the desensitization phenomenon, this could not be achieved with superactive 6nPH analogues of

  mammals (see Hungarian Patent No. 194.913).

  Considering all these facts and the known mechanism of action of the superactive analogues of mammalian 3nPH

  in the case of hormone-dependent tumors, the non-

  Desensitizing the anti-tumor activity exerted on mammals by chicken OnPH analogs or Salmon IFP, respectively, used as active components in the method according to the invention, is unexpected and novel. Although these compositions decrease the estradiol level (from 85 pmol / l to 34

  pmoles / I at an effective dose from the point of view of anti-

  not only is the progesterone level not decreased, but it is even decisively stimulated (from 19 pmol / l to 38 pmol / l), while the testosterone level is lowered to the level of castration (10 pmoles / 1)] by Zoladex [(DSer / But /,

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  AzGly 1 0] -nlnPHil. Even more importantly, after long-term treatment with mammalian GnPH super-analogs, the histological image of the ovary shows inhibition of ovarian function and follicular atresia, while an effective dose 1GnRh analog of chicken or salmon nRHPi, respectively, does not result in any inhibition of the histological image of the ovary but that yellow bodies and follicles under development can be observed. This effect can not be explained by current knowledge, but it rests in

  any case on a new mechanism of action.

  The preparation of the compounds of the invention of the general formula (1) is known [see US Patent No. 4,410,514; Horvath and

coll .: BBPC 138, 419 (1986) 1].

  The invention therefore relates to a process for the preparation of pharmaceutical compositions useful for the treatment of hormone-dependent tumors. According to the process of the invention, a GnPH analogue of general formula I [1 or a salt or metal complex thereof prepared in a known manner is mixed with carriers and / or additives commonly used in the pharmaceutical industry and transformed into a composition

  pharmaceutical agent does not induce hormonal castration.

  In addition, the invention relates to the use of compounds of general formula (I) or of their salts or metal complexes

  for the treatment of tumors and clinical conditions.

  The compounds of the general formula (II) are preferably used in the form of pharmaceutical compositions, for example in the form of a solution, a powder, an injection or in a continuous release form.

  administered by intramuscular, subcutaneous, intra-

peritoneal or intravenous.

  The pharmaceutical compositions according to the invention are suitably used at a daily dose of 0.5 to 5000 μg / kg, preferably 1 to 500 μg / g, more preferably 5 to kg / kg of body weight. An interval of at least 8 hours should be allowed between repeated treatments. The administration is properly continued until the cessation of the

  tumor growth and improvement.

  The pharmaceutical composition prepared according to the method of the present invention inhibits tumor growth by inhibiting cell division [mitosel in hormone-dependent tumors. This effect is probably partially achieved by the stimulation of specific differentiation functions of these

  hormone sensitive tumor cells.

  The main advantages of the process according to the invention can be summarized as follows: a) The growth of benign and malignant tumors can

  to be inhibited and a remission can be obtained.

  b) The method of the invention is suitable for inhibiting the growth of tumors without bringing complete deprivation of sex steroids in the bloodstream, that is to say without inducing a "hormonal castration". This means that the treatment is not accompanied by the physiological effects and

  psychological disadvantage of hormonal castration.

  c) The GnRH analogs used in the method exert their anti-tumor activity by a mechanism of action which is different from that of the superactive SnPh analogues used until now. This means that the process can be used either alone,

  either in an alternating treatment.

  dl In the case of breast cancer and gonadal tumors, inhibition of tumor growth becomes possible without reduction of ovarian weight and inhibition of follicular maturation. e) In contrast to a treatment with superactive mammalian GnPH analogues, the method of the invention does not

  not decrease libido and spermatogenesis.

  The tumor growth is lowered even in cases where the tumor cells are steroid receptor negative, which means that by using the pharmaceutical compositions prepared in the invention, breast cancer and gonadal tumors can be treated in a controlled manner. g range much wider than with compositions known until now; thus, steroid-negative tumors, which are more

  aggressive in most cases, can also be treated ..

  g) The tumor growth is inhibited and the remission is stabilized. Thus, the method of the invention can be used as a complementary treatment with other drugs, such as cis-platinum [PttNH3) 2CI2] which inhibits mitosis, but is accompanied by

toxic side effects.

  The present invention is further illustrated using

  non-limiting examples.

Example I

  Use against breast cancer induced by

dimethyl benzanthracene (OMBA).

  Sprague-Dawley rats of 15-20 weeks (weighing about 200 g) with DMBA-induced breast tumor were treated intramuscularly (im) twice daily for 3 weeks with 10 μg / day GnRH analogue. (D-Phe6, BlnS, desily 10) -1nRHH-ethylamide. The characteristic tumor weights and dimensions were measured directly before treatment and then 1, 2 and 3 weeks after the start of treatment. 16 hours after the final injection, the animals' ovaries and blood samples were taken for hormone estimates. The ovaries were weighed and treated for

determine their histology.

  Treatment of rats with D-Phe6, Gln8, Silyl O) -BnRH-ethylamide produced a decrease over time in the size of DMBA-induced ovarian-dependent breast tumors. At the end of the 3-week treatment period, the average tumor volume had decreased by 10%. compared to their original volume. Decreases in tumor size produced by D-Phe6,6ln8, des6ly 10) -BnPiHethylamide were equivalent in the rate and extent of remission, to those observed three weeks after surgical castration, or to improvement. noted in animals receiving the

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  same dose of [O-Ser (But) 6, Azgly 10) -OnPH (ICI 118630), a mammalian GnPH agonist used for the treatment of advanced breast cancer. The evaluation of ovarian histology and sex steroid hormone levels in animals treated with D-Phe6, Iln8, Gly10) -Sn-methylamide. indicated follicular maturation and development of normal corpora lutea and continued maintenance of ovarian function. Although the concentration of circulating estradiol decreased relative to intact untreated controls, the significance of this decrease was not as clear as in the case of surgically castrated animals. Progesterone levels and ovarian weight were

  not reduced by treatment with D-Phe6, Gln8, desely 10) -

  NDOs-ethylamide. In contrast, ICI 1186630 significantly delayed follicular maturation, reduced ovarian weights, and decreased plasma concentrations of both estradiol and

  progesterone at castrate levels.

  The above data show that D-Phe6, GIn8, desGly1 0] -OnPethylamide is an effective anti-tumor agent

  against mammary tumors induced by DMBA; this anti-effect

  tumor is not accompanied by complete inhibition of ovarian function.

Example 2

  Use to inhibit cell proliferation in a human breast cancer cell line An MDA MB 231 human tumor cell line from human breast carcinoma is a well-known estrogen receptor negative cell line. Cells of this cell line were cultured in PPMI supplemented with 10% fetal calf serum (FCSI (manufactured by 6IBCO, Hoofdorp, The Netherlands) 24 hours after application to a plate, the cells in exponential growth phase were treated

  with 5, 10 and 25 μg / ml, respectively, of D-Phe6,8ln6, desGlyl0) -

  nPH-ethylamide in the presence of 1 μCi of 3H-thymidine. The II cells were incubated for 24 hours, then the medium was removed and the cells were washed twice with phosphate buffered saline and then precipitated by the addition of 10% trichloroacetic acid. The TCA insoluble material was dissolved in concentrated formic acid and the radioactivity was determined in a scintillation counter. The incorporation of 3H-thymidine into the acid-insoluble fraction of 10 6 cells / ml was reduced by 32% with 5 peptide pigs, 65% with 10 μg peptide and 71X. with 25 ipg

of peptide.

  The results show unequivocally that D-Phe6, 6in8, desly 10 (6) -PNHH-ethylamide inhibits the proliferation of these human mammary tumor cells that do not depend on

I'oestrogène.

Example 3

  Use to inhibit cell proliferation in a human breast cancer cell line The activity of inhibiting cell proliferation of the

  (1In8) -6nR1H, (D-Phe6, Gln8} -QnPH, (D-Phe6,6ln8, desily 101) -

  GnRH-ethylamide and (Trp7, Leua) -nnPH respectively, was studied on a tumor cell line derived from human breast carcinoma MDAMB 231 according to the experimental conditions described in Example 2, except that the incubation was 72 hours. ; All peptides were administered at a dose of 10 μg. The significance of the inhibition of 3H-thymidine incorporation by the above peptides was as follows: (ln8] -OBnPH 36% (D- Phe6, Bln8) -nnPH 60% (D-Phe6.6ln8, desily 10) -61nRH-EA 70% (Trp7, Leu8) -SnRH 38%

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Claims (4)

PEVENDICATIONS   A process for the preparation of pharmaceutical compositions containing a gonadotropin-releasing derivative of general formula (1), Blp-His-Trp-Ser-TyrX1-X2-X3-Pro-X4 (II) wherein X1 is a glycyl group or a D-isomer of any natural or synthetic aromatic O-amino acid group; X 2 represents an L-amino acid group bearing a C 1-4 alkyl group or an L-phenylalanyl or L-tryptophyl group; X3 represents an L-amino acid residue carry a C 1-4 alkyl side chain or C 2-4 alkanoyl amide; and X4 represents a glycylamide or (1-4C) alkyl amide group, with the proviso that X3 represents a group other than the leucyl group when X2 is a tryptophyl group and X1 is other than the glycyl group, its addition salts formed with pharmaceutically acceptable acids or its metal complexes, characterized in that it comprises mixing the compound of the general formula (1), its acid addition salt or its metal complex prepared in a known manner in itself, with carriers and / or additives commonly used in the pharmaceutical industry and their forming a pharmaceutical composition useful for treating hormone-dependent tumors without inducing hormonal castration. 2. Process according to claim 1, characterized in that it comprises the preparation of a unit dose containing from 0.0035 to 350 μg of a compound of general formula (1), in which X1, X2, X3 and X4. are as defined in claim 1, in   as a pharmaceutical composition.   3. Use of gonadotropin-releasing derivatives of general formula II), wherein X 1, X 2, X 3 and X 4 are as defined in claim 1, for preparing pharmaceutical compositions suitable for treating tumors and hormonally dependent clinical conditions.   4.- Use of gonadotropin-releasing derivatives   of formula (I), wherein X1, X2, X3 and X4 are as defined in claim 1, for the manufacture of medicaments useful for treating tumors and   hormono-dependent clinical conditions.