GB2218335A

GB2218335A - Use of gonadoliberin derivatives for preparing tumour-inhibiting pharmaceutical compositions

Info

Publication number: GB2218335A
Application number: GB8910724A
Authority: GB
Inventors: Gyoergy Keri; Robert I Nicholson; Istvan Teplan; Tamas Gulyas; Aniko Horvath; Gyoergyi Boekoenyi; Balazs Szoeke; Agnes Balogh; Orsolya Csuka
Original assignee: Innofinance Altalanos Innovacios Penzintezet
Current assignee: Innofinance Altalanos Innovacios Penzintezet
Priority date: 1988-05-10
Filing date: 1989-05-10
Publication date: 1989-11-15
Anticipated expiration: 2009-05-10
Also published as: FI892275A; DE3915317A1; IL89998A0; CH678013A5; GR890100315A; BE1004932A3; HU199694B; IT1229251B; FI892275A0; LU87516A1; IT8920435A0; SE8901660D0; GB8910724D0; KR900017610A; DK228289D0; NL8901159A; GB2218335B; DK228289A; FR2631237A1; JPH0215097A

Description

1 1 1 9 22 18%_135 USE OF GONADOLIBERIN DERIVATIVES FOR PREPARING

TUMOUR-INHIBITING PHARMACEUTICAL COMPOSITIONS This invention relates to the use of gonadoliberin derivatives for the preparation of tumour-inhibiting pharmaceutical compositions.

More particularly, the invention relates to the use of gonadoliberin derivatives of general formula (I), G1p-His-Trp-Ser-Tyr-Xl-X 2_ X 3_ Pro-X4 wherein X 2 X1 (I) 5tnds for a glycyl group or a D-isomer of any native or synthetic aromatic D-2mino acid group; stands for an L-amino acid group bearing a C 1-4 alkyl or L-phenylalanyl or L-tryptophyl group; stands for an L-amino acid residue bearino a C 1-4 alkyl or C 2-4alkanoyl amide side chain; and X 4 means a glycyl amide or C 1-4 alkyl amide group, with the proviso that X 3 means a group different Irom leucyl when X 2 stands for a tryptophyl group and X 1 is other than glycyl group, and addition salts formed with pharmaceutically acceptable acids and (metal) complexes thereof for the treatment of various benign and malignant tumours and clinical conditions as well as for the preparation of pharmaceutical compositions containing these compounds.

It is known that gonadoliberin L-other names us'ed in the literature are: gonadotropin releasing hormone (GnRH), luteinizing and folliclestimulating hormone-releasing hormone M/FSH-RH)7 and its agonist derivatives stimulate the release of the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland and, due to this action, gonadoliberin regulates the basic pattern of the reproductive cycle.

The regulation of the reproductive functions is a very complicated system influenced by a number of various factors throughout the hypothalamopituitary-gonadal axis. In this complicate regulation, gonadoliberin plays a central role since nearly each of the regulatory parameters effect either the synthesis or the release of GnRH from the hypothalamus, or its action on the pituitary gland.

Gonadotropins M and FSH) regulate the synthesis of steroid51 hormones in the gonads as well as the maturation processes of gametes and their release. In females, the primary function of FSH consists in the promotion of development of follicles and ova. In hypophysectomized animals, purified FSH induces an increase in the weight of the ovaries and enhances the number of antrum follicles while 1 no ovulation ensues. The function of LH is to regulate the steroidogenesis and to induce ovulation and subsequent luteinization. Steroidogenesis is maintained by basic LH i level whereas ovulation and continued meiosis of gametes are regulated by a preovulatory LH-peak. Although LH is. commonly thought to be responsible for induction of ovulation, it is assumed that FSH is also required for the process, so the conjugated effects of both hormones have to be considered. The shifts in the LH/FSH ratio plays a regulatory role in the sexual cycle and induce characteristic phenotypical alterations in the gonads.

The release of gonadotropins proceeds in two phases. In the baS21 stage the release is continuous, has a pulsating character and regulates the basal functions of the sexual organs. Ovulation, however, requires high levels of gonadotropins with a short duration and is known as the preovulatory peak of gonadotropins.

GnRH had long been considered to be a non-species-specific hormone until it became known in the early eighties that the structure of the gonadoliberin of certain fish and bird species is structurally different from that of the mammals /-J. A. King and R. P. Millar: J. Biol. Chem. 257, 10722 (1962); N. Sherwood et al.: Proc. Natl. Aced. Sci. USA 80, 2794 (1983)7. These differences are found in the amino acids of positions 7 and/or 6 of GnRH.

Novel GnRH analogues derived from fish- or bird-specific GnRH analogues have been developed and used for inducing ovulation and folliculogenesis in various species of fish and mammals / - Hungarian patent specification No. 190,207; United States patent specification No. 4,647,553).

Twro of these analogues /-(O-Phe 6 Gln 8)-GnRH and (D-Phe 6 Gln 8 desGly 10)-GnRH-ethylamide)7 were found to be particularly potent in stimulating reproductive functions of fish. By using these two peptides, the induced artificial propagation of fish could be achieved which could not artificially be propagated earlier. The same analogues were also capable to induce folliculogenesis and ovulation in fish out of the season of spawning (Hungarian patent specification No. 189,394 and United States patent specification No. 4,647,552).

Due to their potent folliculogenic activity, the effect of these analogues was tested in various mammals. They were able to induce folliculogenesis and ovulation even in sexually immature animals, to overcome physiological anestrus, to cure disturbances in spermatogenesis and to treat other sexual disorders in various mammals. The use for treating mammals is described in detail in the Hungarian patent specification No. 194,913 and U.S. patent specification No. 4,753,928.

The possibility of a particular mechanism of action and a direct gonadal action was raised by the fact that these novel GnRH-analogues induced follicular maturation and ovulation in cases where the GnRH-analogues of mammals proved to be ineffective.

The presence of GnRH-like factors and GnRH receptors in the gonads and the ovary has been well documented in the recent years and it has been suggested that they may form a paracrine system /-for review see: A. J. W. Hsueh and J. M. Schaeffer: J. Steroid Biochem. 23, 757 (19B5)7.

1 1 1 Recently, the use of 5uperactive GnRH-analogue5 for treating various hormone-sensitive tumours has widely been accepted and the results are very promising. The application of superactive GnRH-analoques extends to the field of both the benign and malignant neoplasms and includes the prostate and breast cancer, chondrosarcomas and osteosarcomas, pancreatic cancef, pituitary tumours, ovarian cancer and neoplasms of the female genital tract as well as other hormone-dependent tumours. Regarding the mechanism of action of these superactive GnRH-analogues, 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 dramatic inhibitory effects through a process of "down-regulation" of pituitary membrane receptors for GnRH, desensitization of the pituary gonadotrops and reduction in gonadal receptors for LH and FSH. Thus, repeated administrations of GnRH agonists lead to a marked decrease in Lli and FSH level in the circulation whereas the concentration of estrogen, progesterone and testosterone falls to castration levels. This castration-like sexual steroid deprivation and the elimination of the stimulatory effects of estrogen and testosterone form the basis for the use of superactive GnRH-analoques in the treatment of hormone-dependent tumours. At present, no GnRH agonist is known which exerts antitumour activity via a different mechanism of action.

The aim of the present invention is to use Ithe GnRH- analogues of the general formula (I) for curing hormone-dependent tumours without inducing complete hormonal castration.

The invention is based on the recognition that the above aim can be achieved, i.e. the growth of hormone-dependent tumours can be inhibited by using the compounds of the general formula (I). This recognition is surprising since all the GnRH-analogues known from the literature up to now and possessing antitumour activity are superactive, i.e. 50 to 200 times -more active than the native hormone and exert their effect through desensitization /-M. A. Eisenberger et al.: J. Clin. Oncology 4, 414 (1986); J. Waxman: British Medical Journal 295, 1084 (1987)7. The desensitization phenomenon induced by superactive mammalian GnRHanalogues was widely investigated both in vitro and in vivo experiments. It has been stated that the gonadotropic cells of the pituitary-gland were desensitized even by a single higher dose of superactive mammalian GnRH-analoques against the LH- or FSH-releasing effects of the subsequent GnRH pulses; and this effect stands out, on the one hand, by the internalisation of the hormone-receptor complex, i.e. by the penetration into the inner part of the cells,and, on the other hand, through an effect inhibiting the biosynthesis or secretion, respectively, of the gonadotropins /-Kdri et al.: Mol. Cell. Endocrinol. 30, 109 (19B327. Simultaneously, the high LH and FSH pulses following the administration of the superactive mammalian GnRH-analoque 11 1 .7 C induces a desensitization also on the level oil gonads against the subsequent LH and FSH pulses which are also developed as a consequence of a receptor-level and intracellular action /-J. Waxman: The Releaser 1, 7 (1986)7. This double desensitizing effect leads to a significant decrease in the secretion or biosynthesis, respectively, of the gonadal steroids whereby the steroidal hormone-dependent tumours are inhibited or remitted. Since the presence of steroids is indispensably needed for the survival of the tumour cells of hormone-dependent tumours in the competitive environment, the surgical or hormonal-desensitizing castration, respectively, have a decisive therapeutical importance in the treatment of such tumours despite of the inconvenient side effects of the castration symptoms. Treatments carried out with the known superactive mammalian GnRH-analogues led to the diminution of the amount of estradiol, progesterone and other sexual steroids to the castration level whereby inhibition of the ovarian function and follicle atresia occurred which could unambiguously be stated on the histological picture of the ovary.

In case of the pharmaceutical compositions as used in compliance with the invention, the active ingredients are chicken-GnRH analogues or salmon-GnRH analogues, respectively, or their derivatives which are not superactive for mammals and thus, their mechanism of action is not based on desensitization. In the course of investigations carried out on a pituitary cell culture in vitro, it has been stated that the chicken-GnRH or salmon-GnRH ana- i logues do not behave as superactive analogues in a rat pituitary cell culture and thus have no desensitizing effect in the corresponding dose range. On the other hand, a novel mechanism of action shown on mammals by the chicken-GnRH or salmon-GnRH analogues, respectively, used by us has been indicated by the fact that infantile animals could be stimulated by these analogues up to sexual maturation, or oligospermic animals could be stimulated to the physiological level under zoological garden circumstances. Owing to the desensitization phenomena, this could not e achieved by using superactive mammalian GnRH-analogues (see the Hungarian patent specification No. 194,913).

By considering all the above facts and on the basis of the known mechanism of action of the mammalian superactive GnRH-analoques in the case of hormone-dependent tumours, the non-desensitizing character of the antitumour activity exerted on mammals by the chicken-GnRbl or salmon-GnRH analogues, respectively, used as active ingredients in the process according to the invention is unexpected and novel.

Though these compositions decrease the estradiol level (from 85 pmol/1 to 34 pmol/1) in a dose being effective from the viewpoint of antitumour activity, the progesterone level is not only not decreased but even decisively stimulated (from 19 pmol/1 to 38 pmol/1), whereas the progesterone level is lowered to the castration level (10 pmol/1) by Zoladex /_(DSer/Bu t /, AzGly 10)-GnRH7. It is even more important that, after a long-lasting treatment with superactive mammalian GnRHanaloques, the histological picture of the ovary shows -v.

1 inhibition of the ovarian function and follicular atresia, whereas an efficient dose of the chicken or salmon GnRH---analogue, respectively, does not result in any inhibition in the histological picture of the ovary but developing follicles and yellow bodies can be observed. This effect cannot be explained by our present knowledge but it is based in any case on a novel mechanism of action.

The preparation of the compounds of the general formula (I) is known /see United States patent specification No. 4,410,514; Horv6th et al.: BBRC 138, 419 (198C7.

Thus, the present invention relates to a process for the preparation of pharmaceutical compositions being useful for the treatment of hormonedependent tumours. According to the process of the invention, a GnRHanalogue of the general formula (I) or its salt or metal complex prepared in a known way is mixed with carriers and/or additives commonly used in the pharmaceutical industry and transformed to a pharmaceutical composition not inducing hormonal castration.

Furthermore, the invention relates to the use of the compounds of general formula (I) or their salts or metal complexes for the treatment of tumours and clinical conditions.

The compounds of the general formula (I) are preferably used in the form of pharmaceutical compositions, e.g. in the form of a solution, powder, injection or in a sustained-release form. These compositions may be administered in an intramuscular, subcutaneous, intraperitoneal or intravenous route.

1 The pharmaceutical compositions according to the invention are suitably used in a daily dose of 0.5 to 5000 /uglkg, preferably 1 to 500 /ug/kg, more preferably 5 to 150 /ug/kg,of body-weight. It is suitable to allow at least 8-hour intervals between the repeated treatments. The administration is suitably continued until the growth of the tumour stops and an improvement is achieved.

The pharmaceutical composition prepared by using the process according to the invention inhibits the tumour growth by inhibiting the cell division (mitosis) in hormone-dependent tumours. Likely, this effect is partly achieved by stimulating the specific differentiating functions of these hormone-sensitive tumour cells.

The main advantages of the process according to the invention may be summarized as follows:

a) The growth of both benign and malignant tumours can be inhibited and remission can be achieved. - b) The process according to the invention is suitabl to inhibit tumour growth without the complete deprivation of sex steroids from the bl-ood circulation, i.e. without inducing "hormonal castration". This means that the treatment is not accompanied by the disadvantageous physiological and psychological effects of hormonal castration.

c) The GnRH-analogues used in the process exert their antitumour activity by a mechanism of action being different from that of the superactive GnRH-analoques used till now. This means that the process can be used either alone or as an alternative therapy.

11 1 e tP d) In the cases of breast cancer and gonadal tumours the inhibition of tumour growth becomes possib-1e without any decrease in the ovarian weight and inhibition of the follicular maturation.

e) In contrast to a treatment with superactive mammalian GnRH-analoques, the claimed process does not decrease the libido and spermatogenesis.

f) The tumour growth is lowered even in cases where the tumour cells are steroid receptor negative which means that, by using the pharmaceutical compositions prepared by the process of the invention, breast cancer and gonadal tumours can be treated in a much wider range than by using hormonal compositions known till now; thus, steroid-negative tumours, which are in most cases more aggressive, can also be treated.

9) The tumour growth is inhibited and the remission is stabilized. Thus, the claimed process can be used as a supplementing therapy together with other drugs such as cis-platinum /_Pt(NH 3)2 cl 2-7 which inhibits the mitosis but is accompanied by toxic side effects.

The process of the invention is illustrated in detail by the aid of the following non limiting Examples.

Example 1

Use against dimethylbenzanthracene- (DMBA-) induced breast cancer 15-20 weeks old female Sprague-Dawley rats (weighing approximately 200 9) suffering from DMBA-induced mammary 1 - 12 tumour were intramuscularly (i.m.) treated twice daily for 3 weeks with 10 /ug/day of the (D-Phe 6 Glne,desGly 10)GnRh-ethy.lamide analogue. The weight and characteristic dimensions of the tumours were measured directly before the treatment and then 1, 2 and 3 weeks after beginning the treatment. 16 hours after the final injection the animals were ovaryectomized and blood samples were withdrawn for hormone estimations. The ovaries were weighed and processed for histology.

The treatment of female rats with (D-Phe 6 Gln 8 desGly 10)GnRHethylamide produced a time-dependent decrease in the size of the ovarydependent mammary tumours induced by DMBA. At the end of the 3-week treatment period, the average volume of the tumours decreased to 10% of their original volumes. The decreases in tumour size produced by (D-Phe 6 Gln 8 desGly 10)GnRH-ethylamide were equivalent in rate and extent of remission to those observed at 3 weeks after surgical castration, or to the improvement observed in animals receiving the same dose of /-DSer(But) 6 Azgly 10 7GnRH (ICI 118630), a mammalian GnRH agonist used for the treatment of advanced breast cancer.

Evaluation of the ovarian histology and sex steroid hormone levels in animals treated with (D-Phe 6 Gln 8 desGly 10 SnRH-EA indicated a normal follicular maturation and development of corpora lutea as well as the continuous maintenance of the ovarian function. Although the concentration of the circulating estradiol decreased in comparison to the intact untreated controls, the extent of this decrease was not as ,I 1 C 7 pronounced as in case of surgically castrated animals. The progesterone level and ovarian weight were not diminished by a treatment with W-Phe 6 Gln 8 desGly 10)GnRH-ethylamide. In contrast, ICI 118630 significantly retarded follicular maturation, reduced ovarian weights and decreased plasma concentrations of both estradiol and progesterone to the castrate levels. The above data indicate (D-Phe6,Glns,desGlylo)GnRH-ethylamide to be an effective antitumour agent against mammary tumours induced by DMBA; this antitumour effect is not accompanied by the complete inhibition of the ovarian function.

Example 2 Use for inhibitinq cell proliferation in a cell line of human breast cancer MDA MB 231 human tumour cell line arising from a human breast carcinoma is a well documented etrogen receptor negative cell line. The cells of this cell line were cultivated in an RPMI medium supplemented with 10% of foetal calf serum (FCS) (manufactured by GIBCO, Hoofddorp, Netherlands). 24 hours after plating in exponential growth phase the cells were treated with 5, 10 and 25 /ug/ml, respectively, of (D-Phe 6 Glne,desGlylO)GnRH-ethylamide in the presence of I /uCi 3H-thyridine. The cells were incubated for 24 hours, then the medium was removed and the cells were washed twice with physiological saline solution buffered by phosphate and then precipitated by adding 10% trichloroacetic acid (TCA). The TCA-insoluble material was dissolved in concentrated formic acid and the radioactivity was determined in a scintillation counter.

The incorporation of 3 H-thymidine into the acid-insoluble fraction of 106 cell/ml was decreased by 32% on using 5 /ug of peptide, by 65% on using 10 /ug of peptide and by 71% on using 25 /ug of peptide.

These results unequivocally show (D-Phe 6 G1n8,desGlylo)GnRH-ethylamide to inhibit the proliferation of these estrogen-independent human mammary tumour cells.

Example 3 Use for inhibiting the cell-proliferation in a cell line of human breast cancer The cell-proliferation inhibiting activity of (G1n8)-GnRH, (D-Phe 6 G1n8)- GnRH, (D-Phe6 GlnB,desGly 10 -GnRH-ethylamide and (Trp 7 LeuB)-GnRH, respectively, was investigated on a tumour cell line arising from MDA-MB 231 human breast carcinoma according to the experimental conditions described in Example 2, except that incubation for 72 hours was used.

All peptides were administered in a 10 / ug dose. The extent of inhibiting the incorporation of 3H-thymidine by the above peptides was as follows:

(G1n 8)-GnRH 36% (D-Phe6,G1n8)-GnRH 60% 1 1 (D-Phe 6,GlnB,desGlylo)-GnRH-EA (Trp 7 Leue)-GnRH 70% 38%

Claims

C 1 a i m S 1. A process for the preparation of pharmaceutical compositions containing a gonadoliberin derivative of general formula (I), G1p-His-Trp-Ser-Tyr-Xl-X 2_ X 3-Pro-X4 wherein stands for a glycyl group or a D-isomer of any native or synthetic aromatic 0-amino acid group; (I) X 2 stands for an L-amino acid group bearing a Cl-4 alkyl or Lphenylalanyl or L-tryptophyl group; X 3 stands for an L-amino acid residue bearing a Cl-4 alkyl or C2-4 alkanoyl amide side chain; and X 4 means a glycyl amide or Cl-4 alkyl amide group, with the proviso that X3 means a group different Ir-om leucyl when X2 stands for a tryptophyl group and X 1 is other than glycyl group, its addition salts formed with pharmaceutically acceptable acids or metal complexes, which c o m p r i s e s mixing the compound of the general formula (I), its acid addition salt or metal complex prepared in a manner known per se, with carriers and/or additives commonly used in the pharmaceutical industry and transforming them to a pharmaceutical composition useful for treating hormone-dependent tumours without inducing any hormonal castration.

1 1

2. A process as claimed in claim 1, which c o m p r i s e s preparing as pharmaceutical composition a dosage unit containing 0.0035 to 350 /ug of a compound of the general formula (I), wherein X,, X 21 X 3 and X 4 are as defined in claim 1.

3. Use of gonadoliberin derivatives of the general formula (I), wherein X, , X 21 X 3 and X 4 are as defined in claim 1, for preparing pharmaceutical compositions for treating tumours and clinical conditions.

4. Use of gonadoliberin derivatives of the general formula (I), wherein X, , X 21 X 3 and X 4 are as defined in claim 1, for treating tumours and clinical conditions.

5. A process as claimed in claim 1 or claim 2,or use as claimed in claim 3 or claim 4, wherein the compound of the general Formula (I) is as set out in any one of Examples 1 to 3.

PubhBhed 1989 atThe Patent Office, State House, 66/71 High Holborn, London WClR 4TP. Further copiesmasybe obtainedfrom The Patent Office. Was Branch, St Mary Cray, Orpington, Kent BR5 3RD. Printed by Multiplex techniques ltd, St Mary Cray, Kent, Con. 1/87