AU739071B2

AU739071B2 - Pharmaceutical preparations for the selectively supplementing estrogen deficiency in the central nervous system

Info

Publication number: AU739071B2
Application number: AU29209/99A
Authority: AU
Inventors: Michael Oettel; Vladimir Patchev; Wolfgang Romer; Sigfrid Schwarz; Ina Thieme
Original assignee: Jenapharm GmbH and Co KG
Current assignee: Jenapharm GmbH and Co KG
Priority date: 1998-02-20
Filing date: 1999-02-10
Publication date: 2001-10-04
Anticipated expiration: 2019-02-10
Also published as: PL342313A1; EP1056460B1; CN1291102A; EE200000477A; KR20010041107A; AU2920999A; NZ506049A; CZ291764B6; JP2002503694A; DE59907446D1; BG104689A; EA002707B1; CZ20002938A3; WO1999042108A1; TR200002400T2; EP1056460A1; CA2321498A1; ATE252388T1; NO20004153L; HUP0100742A3

Abstract

Selected steroids are used to produce pharmaceutical preparations for selectively supplementing oestrogen deficiency in the central nervous system (CNS) without influencing other organs or systems. These steroids are characterised in that they have a selective, neurotropic, oestrogen-like transcription effect, unlike the systemically active natural and synthetic oestrogens, including 17a-estradiol. It has been surprisingly discovered that the selected steroids, when used according to the invention, selectively influence the transcription of oestrogen-dependent genes in the central nervous system and cause alterations of the corresponding physiological parameters; have transcription effects specific to the central nervous systems in doses which have no biological effects on the tissues of the reproductive system; have transcription effects specific to the central nervous system at doses at which neither 17b-estradiol nor 17a-estradiol have any effect; and do not influence the transcription of oestrogen-dependent genes in the central nervous system to a greater extent than the secondary 17b-estradiol.

Description

WO 99/42108 PCT/DE99/00353 Pharmaceutical Preparations for the Selectively Supplementing Estrogen Deficiency in the Central Nervous System The present invention relates to the use of selected steroids to produce pharmaceutical preparations for selectively supplementing estrogen deficiencies in the central nervous system without affecting other organs or systems.

These steroids are characterized in that, unlike systemically effective natural and synthetic estrogens, which include 17a-estradiol, they possess a selectively neurotropic, estrogen-like transcription effect.

These steroids are compounds of general formula I, Re

R

RO R wherein R, is a hydrogen atom, a hydroxyl group, or an alkyloxy group of 1-5 carbon atoms, R, is a hydrogen atom, an alkyl group of 1-5 carbon atoms, an acyl group of 1-5 carbon atoms, a group of the general formula SONR,,R,,, with and being in each instance a hydrogen I C

-I

kftZT O« WO 99/42108 PCT/DE99/00353 atom, an alkyl group of 1-5 carbon atoms, independently of each other, or together with the nitrogen standing for a pyrrolidino, piperidino or morpholino group,

R

3 is a hydrogen atom or a hydroxyl group,

R

4 is a hydrogen atom, a hydroxyl group, or an alkyl group of up to 5 carbon atoms, R, and R 6 are in each instance and independently of each other a hydrogen atom or a halogen atom,

R

7 is a hydrogen atom or a methyl group,

R

8 is a hydrogen atom and a hydroxyl group, an oxo group or a grouping of general formula CR,2R 13 in which the R 12 and the R, 3 are in each instance and independently of each other a hydrogen atom or a halogen atom, R, is a methyl or ethyl group, Z is a C,C double bond or a substituted or unsubstituted cyclopropane ring and the >CR,R, is either in the a or p position, R 7 being in the P position, if>CR 5

R

6 is in the a position, and vice versa in order to produce pharmaceutical preparations for selectively supplementing estrogen deficiency in the central nervous system without affecting other organs or systems.

An abrupt or gradual reduction of the estrogen concentrations in the bodies of both men and women can occur under conditions that can be physiological (increasing age, menopause) or pathological conditions (gonadectomy, the use of GnRH analogues as supplemental cancer therapy).

WO 99/42108 PCT/DE99/00353 The best-known clinical symptoms of estrogen deficiency include disruption of temperature regulation, in the form of hot flashes, osteoporosis, and an increased disposition to cardiac and vascular diseases (Netter, A. "The Menopause," in Thibault, Levasseur, Hunter, R.H.F.

(eds), Reproduction in Mammals andMan, Ellipses, Paris, pp. 627-642, 1993).

The most recent clinical studies (van den Beld, A.W. et al., "The role of estrogen in physical and psychosocial well-being in elderly men," The Aging Male (Suppl. p. 54, 1998) contain clear evidence of a reduction of the serum estrogen level as men age. This emphasizes the existence and the pathophysiological relevance of an "estrogen deficiency syndrome" in aging males.

The brain is an extremely important target organ for the effects of estrogen. Estrogens have a decisive physiological influence on many neurobiological processes. Generally speaking, their effects can be classified in two major groups: organizing and activating (McEwen B.S. et al., "Steroid hormones as mediators of neural plasticity," J. Steroid. Biochem. Mol. Biol 39: pp. 223- 232, 1991).

The former apply mainly to the gender-specific organization of neural substrates during early ontogenesis.

The second group includes specific changes in the function of neural control loops under the influence of estrogen concentrations, which result from the physiological secretion of the gonads R -3- WO 99/42108 PCT/DE99/00353 after sexual maturity. The activating effects of estrogen in the central nervous system are expressed by the following physiological processes, amongst others: gender-specific regulation of gonadotropic secretion (Fink, "Gonadotropic secretion and its control," in Knobil, E. Neil, (eds), The Physiology of Reproldclion, Raven Press, New York, pp. 1349-1376, 1988); control of sexual behaviour, Baum, et al., "Hormonal basis of proceptivity and receptivity in female primates," Arch. Sex. Behav. 6 pp. 173- 192, 1977); regulation of the neuroendocrine reactivity to stress, (Viau, Meaney, "Variations in the hypothalamic-pituitary-adrenal response to stress during the estrous cycle in the rat," Endocrinology, 129: pp. 2503-2511, 1991); learning and retention of behaviour patterns with adaptive relevance, (O'Neal, et al., "Estrogen affects performance of ovariectomized rats in a two-choice water-escape working memory task," Psychoneuiroendocrinology 21: pp. 51-65, 1996); -maintenance of reaction readiness of neurochemical mechanisms that are indispensable for ensuring vigilance and adequate information processing, Fink, et al., "Estrogen control of central neurotransmission: effects on mood, mental state, and memory," Cell. Mol. Neurobiol.

16; pp. 325-344, 1996), dynamic changes to the density ofinterneural contacts in the brain structures with a decisive role for cognitive performance and the emotional status, (Wooley, McEwen, B.S., "Estradiol mediates fluctuations in hippocampal synapse density during the estrous cycle in the adult rat," J. Neurosci. 12: pp. 2549-2554, 1992).

S-4- WO 99/42108 PCT/DE99/00353 The enormous neurotropic potential of estrogen finds expression in their ability to induce the expression of a series of genes specific to the central nervous system, the products of which are critical to the survival of nerve cells (Miranda, Sohrabi, Toran- Allerand, "Presumptive estrogen target neurons express nmRNA for both the neurotrophins and neurotriphin receptors: a basis for potential development interactions of estrogen with the neurotrophins," Mol. Cell. Neurosci. 4: pp. 510-525, 1993), for ensuring the diversity and quality of signal transmission in the central nervous system (Luine, "Estradiol increases choline acetyltransferase activity in specific basal forebrain nuclei and projection areas of female rats," Exp. Neurol. 89; pp. 489-490, 1985); (Weiland, "Glutamic acid decarboxylase messenger ribonucleic acid is regulated by estradiol and progesterone in the hippocampus," Endocrinology 13 1: pp. 2697-2702, 1992); Boss6, Di Paolo, "The Modulation of brain dopamine and GABA, receptors by estradiol: a clue for CNS changes occurring at menopause, Cell. Mlol. Neurobiol 16: pp. 199-212, 1996) and increasing the resistance of nerve cells relative to pathological effects (Goodman, et al., "Estrogens attenuate and corticosterone exacerbates excitotoxicity, oxidative injury, and amyloid b-peptide toxicity in hippocampal neurons," J. Neurochem. 66: pp. 1836-1844, 1996).

Clinical results suggest estrogen deficiency as a causal factor in the pathogenesis of Alzheimer's disease and point to the possibility of an estrogen substitution being able to halt the clinical manifestation or progress of this illness (Henderson, et al., "Estrogen replacement therapy WO 99/42108 PCT/DE99/00353 in older women: comparisons between Alzheimer's disease cases and controls," Arch. Neurol. 51: pp. 896-900, 1994); (Paganini-Hill, Henderson, "Estrogen deficiency and risk of Alzheimer's disease," Am. J. Epidemiol. 140: pp. 256-261, 1994).

A series of neuropeptides, whose gene transcription is affected by physiological estrogen quantities oxytocin and arginine vasopressin) play an important role in the control of emotional behaviour components (Adan, Burbach, "Regulation of vasopressin and oxytocin gene expression by estrogen and thyroid hormones," Progr. Brain Res. 92: pp. 127-136, 1992).

Reports in the professional literature point to the tact that estrogen deficiency is accompanied by a clear decline in the organism's ability to eliminate reactive species of oxygen and free radicals (Niki, Nakano, "Estrogens as antioxidants," Methods Enzymol. 186: pp. 330-333, 1990); (Lacort, et al., "Protective effects of estrogens and catecholestrogens against peroxidative membrane damage in vitro, Lipids 30: pp. 141-146, 1995). The excess of free radicals is implied in the mechanisms of cellular damage in several organs and systems, and is related to the pathogenesis of neurodegenerative diseases (Smith, et al., "Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer's disease," Proc. Natl. Acad. Sci. USA 88: pp. 10540-10543, 1991); (Hastings, Zigmond, "Neurodegenerative disease and oxidative stress: insights from an animal model of Parkinsonism" in: Fiskum, G. (ed) Neurodegenerative Diseases, Plenum Press, New York, pp. 37-46, 1996). For this reason, estrogen 6- WO 99/42108 PCT/DE99/00353 supplementation is also believed to play a role in the sense of maintaining and increasing endogenic antioxidative capacity (Behl, et al., "17b-estradiol protects neurons from oxidative stress-induced cell death in vitro," Biochem. Biophys. Res. Commun. 216, pp. 473-482, 1995).

At present, estrogen supplementation is effected with natural and synthetic estrogen, the effects of which occur in all organs that contain estrogen receptors, for all practical purposes, within the whole of the body. However, since even small pharmacological doses of these estrogens cause marked cellular proliferation in the tissues of the female genital tract (endometrium) and in the mammary lymph epithelium, which ultimately degenerates into carcinogenic non-differentiation, their use for treating the symptoms of an estrogen deficiency in the central nervous system is limited by several counter-indications (Bernstein, Ross, Henderson, B.E., "Relationship of hormone use to cancer risk," J. Natl. Cancer Inst., Monograph 12: pp. 137-147, 1992).

The proliferative effects of estrogen can be considered to be immediate risk factors for the occurrence of a benign prostatic hyperplasia and/or gynacomasty in the male (Knabbe, C., "Endokrine Therapie von Prostataerkrankungen," [Endocrine therapy of prostate diseases], in Allolio, Schulte, H.M. (eds), Praklische L-'ndocrinology [Practical Endocrinology], Urban Schwarzenberg, Munich, pp. 645-651, 1996). For this reason, estrogen supplementation for males has never been given serious consideration, despite the indications that have been proven.

WO 99/42108S PCT/DE99/00353 The use of natural and synthetic estrogens that have a systemic effect--i.e., in all the body's organs and systems--in order to treat neurodegenerative diseases is claimed by the following patents: US 4,897,389, US 5,554,601, and WO 95/12402, WO 97/03661, DE 43 38 314 Cl.

US 4,897,389 protects the use of estradiol, estron, and estriol, either singly or in combination with gonadotropin, androgens, anabolic androgens, or human growth hormone, for the treatment of senile dementia, Parkinson's disease, cerebral atrophy, Alzheimer's disease, cerebellar atrophy, senile or essential tremor.

US 5,554,601, and WO 95/12402 protect the use of estrogen substances, including those that display slight "sexual activity," for protecting nerve cells against progressive damage and cellular death, and for the treatment of neurodegenerative diseases. 17a- estradiol is cited as an example of a substance with slight "sexual activity" and a neuroprotective effect.

WO 97/03661 protects the use of non-estrogen substances that have at least two ring structures in their overall structure, at least one thereof being a terminal phenolic ring, and whose molecular weight is less thatn 1000 Dalton, for ensuring neuroprotection.

-DE 43 38 314 Cl describes steroids with phenolic A-ring structure whose radical-capture and antioxidative properties are independent from the extent of its estrogen-like effectiveness.

WO 99/42108 PCT/DE99/00353 These compounds can be used for prophylaxis and therapy of radical mediated cellular damage.

In all of these patent specifications, the therapeutic and neuroprotective efficiency of the substances that were obtained was said to be based on one or more of the following end effects: stimulation of the biosynthesis of natural neuronal growth factors; stimulation of the activity of acetylcholine-synthesizing enzymes or the uptake of substrates of the acetylcholine synthesis; direct cytoprotection by increasing the resistance of nerve cells relative to the removal of nutrient substrates or growth factors; reduction of the sensitivity of nerve cells to fi-ee radicals and reactive oxygen species liberated as a consequence of a traumatic or neurotoxic effect.

However, none of the cited patent specifications describes steroids with selective estrogen-like neurotropic transcription effects, such as those that at an in vivo dosage display no significant biological effect that influences transcription of estrogen-like genes in the central nervous system in an estrogen-like mode.

It is, in particular, appropriate to emphasize that. as described in US 5,554.601 and WO 95/12402, the effect of 17a-estradiol--a substance that exhibits a reduced estrogeneity in the genital tract (Clark, J.H. et al., "Effects ofestradiol 17a on nuclear occupancy of the estrogen WO 99/42108 PCT/DE99/00353 receptor, stimulation of nuclear type II sites, and uterine growth," J.Steroid. Biochem 16: pp.

323-328, 1982), relates only to the protection of cultivated nerve cells prior to cell death induced by the removal of nutrient, the relative strength of 17a-estradiol not being evaluated with that of 17b-estradiol. From this, one can conclude that there are no indications for a selective neurotropic effect of 17a-estradiol or its derivatives in the investigations and patent specifications published up to now, whereas it is known that 17b-estradiol exhibits no central nervous system selectivity, and is thus to be classified as an estrogen with systemic effects. WO 97/03361 and DE 43 38 314 C1 interpret the cytoprotective effects of estrogen as being a consequence of the radical-capture properties of its terminal phenolic A-rings. A dissociated neurotropic effect of 17a-estradiol based on influencing the transcription of estrogen-sensitive genes has not been investigated in patent specifications, nor been the subject of reports in the literature.

It is the objective of the present invention to describe pharmaceutical preparations for selectively supplementing estrogen deficiencies in the central nervous system without affecting other organs and systems.

According to the present invention, this objective has been achieved in that selected steroids are used to produce pharmaceutical preparations that ensure the supplementation of the estrogen deficiency in the central nervous system without atfecting other organs and systems.

WO 99/42108 PCT/DE99/00353 The steroids are characterized in that they have a selective neurotropic, estrogen-like transcription effect, unlike systemically active natural and synthetic estogens, including 17a-estradiol.

Surprisingly, it has been discovered that the selected steroids, when used according to the present invention, selectively influence the transcription of estrogen dependent genes in the central nervous system and modify the corresponding physiological parameters: -exhibit central nervous system specific transcription effects at doses that have no biological effects in the tissues of the reproductive system; exhibit central nervous system specific transcription effects at doses in which neither 17bestradiol or 17a-estradiol have any effect; and do not affect the transcription of estrogen-dependent genes in the central nervous system to a greater extent that the secondary 17b-estradiol.

These steroids are compounds of the general Ibrinula 1.

R

RR

R_ O- (f -1 1- WO 99/42108 PCT/DE99/00353 in which in which R, 1 is a hydrogen atom, a hydroxyl -roup, or an alkyloxy group of 1-5 carbon atoms, R 2 is a hydrogen atom, an alkyl group of 1-5 carbon atomns, an acyl group of 1-5 carbon atoms, a group of the general formula SONR, 0

R

1 with .Rj, and R, 1 being in each instance a hydrogen atom, an alkyl group of 1-5 carbon atoms, independently of each other, or together with the nitrogen standing for a pyrrolidino, piperidion or m-orphiolino grIoLIP. R 3 isa hydrogen atom or a hydroxyl group, R, is a hydrogen atom, a hydroxyl group, Or- anl alkyl group of up to 5 carbon atoms, R, and R 6 are in each instance and independently of each other a hydrogen atom or a halogen atom, Rt 7 is a hydrogen atom or a methyl ,roulp, R, is a hydrogen atom and a hydroxyl group, an oxo group or a grouping of general formu1Lla CR 1 in which the Rt 12 and the R, 3 are in each instance and independently of each other a hvdrogen atoin or a halogen atom, is a methyl or ethyl group, Z is a C,C double bond or a SLibstitLited or- un1substituted cyclopropane ring and the >CR 5

R

6 is either in the u or P3 position, R, being in the f3position, if >CR 5

R,

6 is in the (x position, and v'ice versa.

Preferred compounds are the following: I 5fH,3''H-cycloprop[14, 15]-estra-l1,3),S( 0),S-tetiraenie-3,17a.-dliol, 15PH,3)'H-cycloprop[14,15]-l8a-homo-estra- 0),8-telraene-'3, I 7aX-ol, 1 7a-hydroxy-1I SH,3''H-cycloprop[ 14, 1 S]-esti-a- 1,3,5(1 0),S-tetraeie -3-yl-penitamoate, 1 7-methylene-1I5f31-I,3H-cycloprop( 14, 1 SI-esti-a- 1,3,5(1 0),8-tetr-aenie-3-oI, 1 SPH,3 H-3',3'-difluoro-cycloprop[ 14,1 0),8-tetr-aenle3. I7a-diol, 1 7-methylene-1I5f3H,3''H-cyclcprop[ll4, 15]-est-a- 1.3. 5(1 0),8-tetraenie-3)-yI-Suilfamate 1 5 T-12- WO 99/42108 WO 9942108PCT/D E99/00353 17-difluoro met hyl ene- 1 5PH.3 H-cycloprop[ I 5]-estra- 1,3, 5( 1 0),8-tetraene-3-oI, P-methyI-3 'H-cycloprop[ 1 4, 15]-estra 351)Sttan--l 15a-methyl-3'H-cycloprop[14,1I5]-estra- 10),8-tetraerie-3, I 7a-diolI, I 7-difluoromethylene-1 P fH YH-cycloprop[ 14,1I5)-estra-1I,3,5( I 0),S-tetraenee-3-yl- (tetramethyleneimino) sulfonate, 17-methylene-3'H-cycloprop[8,9]- 15j3H,3''H-cyclopr-op[ 14. I S-estr-a- I 10)-triene-3)- O1.

One advantageous embodiment of the present Invention is the use according to the present invention of the compounds to produce pharmaceutical preparations For the prophylaxis and therapy of the age-mediated reduJCtI1 inofcowi i1ti ye performiance, agemediated and perimenopausal dysphoria, pi-emenstruLal syndrome. nleitroses and neurasthenia, anxiety states and neuroses, hot flashies after estrogen deprivation (menopause, gonodectomy, treatment with GnRH1 analop~es) and the psychogenic inhibition Of Sexual activity.

It was established that when this is done, the risk of daniaging, hormione-sensitive tissues of the reproductive system (endometriurn, m~yometium,11, prostate, and mammi-ary glands; in the sense of uncontrolled proliferation and carcinog-enesis Is largely p)reclulded.

WO 99/42108 PCT/DE99/00353 The object of the present invention is also pharmaceutical preparations tor oral and parenteral, including topical, rectal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, intravaginal, intrabuccal or sublingual application that in addition to the usual carriers and dilutants contain a compound as set out in Claim 1 as the active agent.

The following can be used as pharmaceutical formulations: tablets or dragees, from 0.1 to 2 mg daily, orally; ampules from 0. 1 to 2 mg daily, as subcutaneous injection plasters with transdermal release, from 0.05 to 2 mg daily; subcutaneous implants with a daily release capacity from 0.05 to 2 mg daily; gels and cremes with transdermal release from 0.05 to 2 mg daily; buccal release systems with a daily release from 0. I to I mg daily.

The medications according to the present invention are produced with the usual solid or liquid carriers or dilutants as well as with the pharmaceutical-technical accessory agents that correspond to the type of application that is desired at a suitable dose rate, in the usual manner.

WO 99/42108 WO 9942108PCT/D E99/00353 Based on the example of 15 PH, 3'H-cyclo prop[ 14, 1 5]-estra- 1,3),5(1 0),8-tetraene-3, I 7a-diol (compound of general formula 1: R,=RR 3 =RRR(,=R=Ra-O H, f3-l- R,=CH_3; Z=C,C double bond)--documented below as the prototype Substance-- the selective estrogen-like effect according to the present invention will be demonstrated experimentally in comparison to 17bestradiol and I 7a-estradiol.

Example I The effect on uterus weight after chronic subcu~tan~eous application in vivo Sexually mature (3 months old, weight 250±30 g) Ifemale Wistar rats (TierZu~cht Schonwalde GmbH, Germany) were ovariectomized Whenl under ketaiffe narcosis. After 14 days, the animals were Implanted subcutaneouIsly With osmo1tic mninipumips (Alzett, USA) that released a daily dose of 0.01, 0.1, 0.3, 1, 3, 30, and 100 jag of the substance to be investigated (l5I3H,3)'Hcycloprop[14,1I5]-estra-1,3',5(lO),8-tetraene-3',1I7a-diol, I 7b-estradiol, I 7a-estradiol) for seven days; the controls received an correspondingI volume1 of'vCehiCle (pro0pylCeegycol). On the seventh day of the treatment, the animals were killed and thle uteruLs wet wvehiht was determined (relative to 100 g body weigzht).

WO 99/2108 PCT/DE99/00353 Figure 1 shows the uterotropic effect of the various doses of 17b-estradiol (square symbol), 17aestradiol (circle symbol), and 15pH,3'H-cycloprop[ 14,15]-estra- 1,3,5( 10),8-tetraene-3,17a-diol (triangle symbol) on the ovariectomized rats. Each point represents the average value standard error (x±SEM) of 7-10 test animals; the shaded field shows the dispersion range of this parameter in placebo-treated animals (OVX).

It can be seen that using 17b-estradiol, a significant enlargement of the uterus was achieved at daily does rates of 0.03-0.1 ig. In order to achieve a comparable uterotropic effect, one would require daily doses of 100 ug 17a-estradiol or 30 .g of 15pH,3'H-cycloprop[14,15]-estra- 1,3,5(10),8-tetraene-3,17a-diol.

The results show that the effectiveness of I 5p -1,3l-l-cyclopropf 14,15 -estra-1,3,5(10),8-tetraene- 3,17a-diol in the female genital tract is some 1000-fold less than the effectiveness of 17b-estradiol and is comparable to the effectiveness of 17a-estradiol.

Example 2 Activation of the transcription of an a-estrogen receptor-dependent reporter gene in vitro WO 99/42108 PCT/DE99/00353 MCF-7/2A breast cancer cells that express the alpha-isoform of the estrogen receptor (Era) were stably transfixed with the EREwtcLUC reporter plasmid. The reporter contains the estrogen response element (ERE) ofvitellogenin, a thymidinekinase promotor, and the luciferase coding gene ofPhotinus pyralis. Prior to the start of the experiment, the cell culture was cultivated in steroid-free medium for seven days and then incubated for 48 hours with 17b-estradiol, 17aestradiol, or 15pH,3'H-cyclo-prop[ 14,1 5]-estra- 10),8-tetraene-3, 17c-diol in four different concentrations (10 10 10" 9 and 10' MN). The cells were lysed and the transcription of the luciferase reporter gene was established by determining the luciferase activity with a specific test batch (Serva/Promega, Germany).

Figure 2 shows the induction of the transcription of a stably transfixed estrogen-dependent reporter gene (luciferase) in estrogen-receptor expressing MCF-7 breast cancer cells after 48 hours of treatment with different doses of 17b-estradiol (square symbol), 17a-estradiol (circle symbol), and 15p[H,3'H-cycloprop[ 14, 15]-estra- 1.3.5(1 0).8-tetraene-3.17a-diol (triangle symbol).

The figure represents the average values from two independent trials It is clear that the substances that were tested stimulate the transcription of the reporter as a function of dose rate. The effectiveness of 15[3H,3'H-cycloprop[ 14, 15]-estra- ,3,5(10),8- -17- WO 99/42108 PCT/DE99/00353 tetraene-3,17a-diol and 17a-estradiol is one order of magnitude (10 times) less than the effectiveness of 17b-estradiol.

The results show that the 1 5PH,3'H-cycloprop[14, I 5]-estra- 1 1 0),8-tetraene-3, 17a-diol has an estrogen effect that is many times weaker in breast cancer tissue.

Example 3 Stimulation of the transcription of the oxytocin gene in the brain after chronic treatment in vivo, at doses that have no efYect on the uterus Sexually mature (3 months old, weight 250±30 g) female Wistar rats (Tierzucht Schonwalde GmbH, Germany) were ovariectomized when Under ketamine narcosis. After 14 days, the animals were implanted subcutaneously with osmotic minipumps (Alzett, USA) that released a daily dose of 0.01, 0.1, and I pg of the substance to be investigated I 53H,3'H-cycloprop[14,15]estra-1,3,5(10),S-tetraene-3,17a-diol, 17a-estradiol. 1 7b-estradiol) for seven days; the controls received an corresponding volume of vehicle (propyleneglycol). Inimmediately after the animals

-IS-

WO 99/42108 PCT/DE99/00353 had been killed, the uterus wet weight was determined (relative to 00g of body weight). The messenger ribonucleic acid mRNA, which codes the biosynthesis of oxytocin, was shown by insitu hybridization with a specific radioactive-marked oligodeoxynucleotide probe, using an established method (Fisher, et al., "Lactation as a modell of naturally reversible hypercorticalism: plasticity in the mechanism governing hyphthalamo-pituitary-adrenal activity in the rat," J. Clin. Invest. 96: pp. 1208-1215, 1995), 1995) in the hypothalamic nucleus paraventricularis (PVN). Changes in the transcription of the oxytocin gene that were governed by the treatment were quantified by densitometric measurements of the specific hybridizing signals within the defined anatomic structure.

Figure 3 shows the induction ofoxytocin-coding transcripts (OT mRNA, upper drawing) in the hypothalamic paraventricular nucleus of ovariectomized rats after chronic subcutaneous treatment with 17b-estradiol (circle symbol), 17a-estradiol (square symbol), and I 5pH,3'Hcycloprop[14,15]-estra-1,3,5(10),8-tetraene-3, 17a-diol (triangle symbol) at three different dose rates. The lower drawing shows the effects of the substances that were tested on the weight of the uterus. Each point represents x+SEM of 5-7 individual determinations. The shaded field shows the dispersion range of the corresponding parameters in rats treated with the vehicle. The stars indicate significant differences (p<0.05) as compared to the control group (OVX).

WO 99/42108 PCT/DE99/00353 The results show that 1 5PH,3'H-cycloprop[ 14, I5]-estra- 1,3,5(1 O),8-tetraene-3, 1 7ac-diol stimulates the transcription of the oxytocin gene in the PVN as a function of dose, the stimulation effect being very similar to that achieved with 1 7b-estradiol. At all events, the neurotropic transcription effects of 1 5PH,3'H-cycloprop( 14, 1 5]-estra- 1,3,5(1 0),8-tetraene-3, 17a-diol, unlike that of 17b-estradiol, are not associated withli enlargement of the uterus. At the doses that were used, the 17a-estradiol had no affect on the concentrations ofoxytocin niRNA in the hypothalamic

PVN.

These results document a selective, estrogen-like effect of 15PH-.,3'H-cycloprop[14,15]-estra- 1,3,5(10),8-tetraene-3,17a-diol in the brains of female rats.

Example 4 Stimulation of the transcription of the antiapoptoic gene bcl-2 in the hippocampus after chronic treatment in vivo with doses that display no uterotropic effect The material tested originated from animals treated in the experiment described in Example 3.

The bcl-2 gene codes the synthesis of a protein involved in the cascade of cellular proliferation WO 99/42108 PCT/DE99/00353 and acts counter to the programmed cell death (apoptosis) (Merry, Korsmeyer, "Bcl-2 genes family in the nervous system," Ann. Rev. Neurosci. 20: pp. 245-267, The transcription of these genes is stimulated by estrogen (Kandouz, M. et. al., "Antagonism between estradiol and pregetine on Bcl-2 expression in breast cancer cells." Int. J. Cancer 68: pp. 120-125, 1996).

The dentate gyrus is a component of the hippocampal formatiol in which the neurogenesis persists in the rat, even at a mature age (Gould. et al., "Prolileration of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress," Proc Nail. Acad. Sci. USA 96: pp.

3168-317, 1998) and bcl-2 is expressed. Bcl-2 transcripts were shown in brain sections by in-siti hybridization with a specific oligonucleotide probe (Clark et al.. "Apoptosis suppressor.

gene bcl-2 expression after traumatic brain injury in rats," J. Neurosci. 17: pp. 9172-9182, 1997), and quantified densitometrically by using the method described in Example 3.

Figure 4 shows the effect of three different doses of 17b-estradiol (circle symbol), 17a-estradiol (square symbol), and 15pH,3'H-cycloprop[ 14, 15]-estra- 1,3,5(1 0),8-tetraene-3, I 7a-diol (triangle symbol) on the expression of bcl-2 in the dentate gyrus of the hippocampus of ovariectomized rats; the symbols and abbreviations used are the same as those in Figure 3 -21- WO 99/42108 PCT/DE99/00353 Treatment with 5 PH,3'H-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-3, 17a-diol resulted in a dose-dependent stimulation bcl-2 expression in the dentate gyrus. The etffect was the same as that mediated by the same dose of 17b-estradiol. At the dose rates that were Lused, the 17a-estradiol had no effect on the transcription of bcl-2, as can be seen from Figure 4.

These results show that at the dosage used, I 5PH,3'H-cycloprop[14,1 5]-estra-1,3,5(10),8tetraene-3,17a-diol affects the transcription of the antiapoptotic gen bcl-2 in the central nervous system according to an estrogen-like mode, wVithoult any efftect occu1rrin at the uterus.

Examl)e Dissociated induction of oxytocin receptors in the brain and myometrium Binding sites with identical biochemical characteristics for the peptide hormone oxytocin are present in the myometrium and in the central nervous systemn. Acute or chronic estrogen treatment causes an increase in the number (density) of oxytocin receptors in both organs. The brain structures in which this parameter reacts to estrogen with particular sensitivity are the nucleus interstitialis striae terminalis, nucleus ventromedialis. and the amvgdaloid nuclear complex. Estrogen-mediated induction of oxYvtoci receptors in these structures is causally WO 99/42108 PCT/DE99/00353 related to the formation of prosocial behaviour patterns, including sexual behaviour (Insel, T.R., "Oxytocin a neuropeptide for affiliation: evidence from behavioral, receptor autoradiographic, and comparative studies," Psychone-uroedic/ocrinolfogy 17: pp. 3-35, 1992) In order to determine the density of oxytocin receptors in detined aniiatomical structures, the method of choice is autoradiographic representationii by binding of the radioactively marked oxytocin receptor antagonists d(CH,),-Tyr(Me) 2 Thr 4 Orn-[' 2 "l]Tyr"-vasotocin 2 1-OVTA) (Kremarik, et al., "Histoautoradiographic detection of oxytocin and vasopressin binding sites in the telencephalon of the rat," J.Comp.Neurol. 333: pp. 343-359, 1993).

Frozen sections of the brain and uterus of ovariectomized rats that had received a daily subcutaneous dose of 1 lg of 15PH,3'H-cyclopop[ 14, 15]-estra- 1,3,5( 10),8S-tetraene-3, I 7a(x-diol, 17b-estradiol, or 17a-estradiol for a period of seven days (compare Example 3) were incubated with 2 1-OVTA (NEN DuPont, Germany) at at coniicentration of 50 pN. Subsequently, filmautoradiograms were made, and these were then used for deiisitometric determination of the oxytocin binding sites, using an established method (Patchev, et al., "Oxytocin binding sites in rat limbic and hypothalamic structures: site specific modulation by adrenal and gonadal steroids," Neuroscience 57: pp. 537-543, I1993) WO 99/42108 PCT/DE99/00353 Figure 5 shows the specific binding of a 2 'l-mnarked ligand of the oxytocin receptor (12 5 1-OVT) in the myometrium and in two estrogen-sensitive brain structures, the hypothalamic ventromedial nucleus (VMN) and the nucleus interstitialis striae terminals (BNST), after a seven-day treatment with 17b-estradiol (black columns), '17a-estradiol (hatched columns), and 15 H,r'H-cycloprop[14,15]-estra-l,c,5(10),8-tetraene-3,y17a-dio (grey columns), at a daily dose rate of 1 [ig. The star symbols indicate significant differences (p<0.05) as compared to placebotreated ovariectomized rats (OVX). The right-hand drawin shliows theil effects of the substances that were tested on the proliferation of the endometrium. Each column represents x±SEM of individual determinations.

The results of this test are also shown in Figure 5. Treatment witlih 1 7b-estradiol and 15PH,3'Hcycloprop[1 4 I5]-estra-1,3,5(10),8-tetraene-3,17a-diol resulted in a significant increase in the density of oxytocin receptors in all the brain structures that were examined, with 15PH,3'Hcycloprop[14, I15]-estra-,3,5(10 O),S-tetraene-3,17 -dil exhibiting a weaker effect than 17bestradiol in the VMN At the doses that were administered. the 1 7a-estradiol was not effective in any brain structures. In the myomrnetrium, I 7b-cstradiol caused a stronger induction of oxytocin binding sites, whereas 17a-estradiol and I 5PH,3'H-cycloprop[ 14, I5]-estra- 1,3,5(1 0),8-tetraene- 3,17a-diol exhibited a significantly smaller effect. Thile computer-aided measurement of the WO 99/42108 PCT/D E99/00353 strength of the endometrium in the uterus preparations showed that the daily dose of 1pg 17bestradiol caused a significant endometrial proliferation, whereas at an equivalent dose 15PH,3'Hcycloprop[14,15]-estra-1,3,5(10),8-tetraene-3, I7c7a-diol and 1 7a-estradiol did not influence the thickness of the endometrium.

In summary, the results presented in Example 6 show that I 15[3HI-,3'-l-cycloprop[ 14,15]-estra- 1,3,5(10),8-tetraene-3,17a-diol influences a biocihemical parameter--the oxytocin receptor--that is characteristic for the reproductive system (myometrium) and for the central nervous system, mainly in the central nervous system, and that because of this selective neurotropic effect it differs qualitatively from the natural estrogens 1 7b-estradil and 17a-estradiol.

Example 6 The effects on cognitive liunctions after chronic treatment It is known that a reduction of estrogen concentrations is associated with diminished learning and memory functions (Kopera, "Estrogen and psychic functions. Aging and estrogens," Front Hormone Res. 2: pp. 1 18-133, 1973).

WO 99/42108 PCT/DE99/00353 A correlation between serum-estrogen level and cognitive performance has also been demonstrated in the animal-test model (Kondo, Suzuki, Sakuma, "Estrogen alleviates cognitive dysfunction following transient brain ischemia in ovariectomized gerbils," Neurosci.

Lett. 238: pp.45-48, 1997).

The following experiment was conducted for purposes of a comparative trial of the effects of 15pH,3'H-cycloprop[14,15]-estra-l,3,5(10),8-tetraene-3,17a-diol, 17b-estradiol, and 17a-estradiol on cognitive performance: Sexually mature female Wistar rats (weight 240±20 g) were ovariectomized when under Nembutal narcosis. One week after the operation the following substance were administered subcutaneously at the dose rates shown: 17b-cstradiol, I Ig, 17a-estradiol, 100 pg; 15pH,3'Hcycloprop[14,15]-estra-l,3,5(10),8-tetraene-3, 17ca-diol, 30 ug. The overall duration of the treatment was 14 days. On the fifth and sixth day of the treatment, training sessions were started for learning a conditioned escape behaviour, using and established method (Diaz-Veliz et al., "Influence of the estrous cycle, ovariectomy and estradiol replacement upon the acquisition of conditioned avoidance responses in rats," Physiol. Behav. 46: pp. 397-401, 1989). Each animal was exposed 50 times in each session to a combination of an unconditioned response (electric WO 99/42108 PCT/DE99/00353 shock) and two conditioned stimuli (light and sound signal). Retention of the learned behaviour pattern was tested on the seventh day of the treatment. After a break of six days in the learning sessions, on the fourteenth day of the treatment, extinction of the learned conditioned response was established. The number of correct behavioral reactions (Escape into the "safe" section of the apparatus within three seconds after presentation of the conditioning signals) out of 50 sequential exposures was used as the criterion for evaluating the retention or extinction of the learned behaviour.

Figure 6 shows the effect of 17b-estradiol (black columns), 17a-estradiol (shaded columns), and 15pH,3'H-cycloprop[14, 15]-estra-1,3,5(1 0),8-tetraene-3, 7a-diol (grey columns) on the acquisition and retentionof a new behaviour pattern in ovariectomized rats (open columns; OVX).

The star symbols indicate significant differences to the placebo-treated animals (OVX) on the corresponding test day. The following uterus weights (x±SEM; n 8-10 per treatment group; data in mg/100 g body weight were determined after fourteen days of treatment; OVX, 53±2; 17b-estradiol 187±9; 17a-estradiol, 100±5; 5(3 1. 'H--cycloprop[ 14,15 -estra-1,3,5(10),8-tetraene- 3,17a-diol, 108±4.

WO 99/42108 WO 9942108PCTID E99/00353 It is clear that at the dose rate that was used, the 5f3-3FU -cycloprop[ 14,1 5]-estra-l,3,5(10),8tetraene-3,17a-diol has an estrogen-like stimulating effect on the retention of the behaviour pattern that was learned, the uterotropic effect being significantly less thani the effect of 17bestradiol at a daily dose rate of I ltg. This result indicates that 513H,'H-cycloprop[ 14,1 1 1O),8-tetraene-3), 17a-diol affects cognitive performnance like ani estrogen, whereas it exhibits a smaller proliferative effect in the reproductive 01r11tans.

Example 7 Biotransformation of I17a-hydroxy- 14,1I Sa-iiethylenie-estra-3,5 (l0),8-tetraene-3)-ol and 17aestradiol to I 7b-estr-adiol A daily dose of 100 .ig I 7a-estradiol, 30 Liu 5Pl-l,3F-cycloprop[ 14,1I S-estr-a- I tetraene-3 ,I 7a-diol, or I gig 17b-estradiol was administered to ovariectomnized rats for seven days (compare Example On the last day of treatmlent, the ser-Um concentrations of I 7b-estradiol in the three groups was determined and compared to the concentratins In controls that had been treated with the vehicle.

Figure 7 shows the serum values for 17b-estradiol after seven days of subcutaneous treatment of ovariectomized rats with 17b-estradiol (black column11s), 17ii-estradiol (shaded columns) and 5PH,3)'H-cycloprop[ 14,1 51-estra- I,3,5( I O).S-tetricne-3). I 7a-diol coIlumns) at the cited dose rates. The star symbols indicate significant ditfierences compared to the values measures in placebo-treated animals; the latter were below the minimium detection limit for the methods, each i: treatment group comprises seven animals.

Iti la htatr h.plcto..I betalladI7aetaila h os ae icsed mesral cocnrtoso.7-srdo r estdi h eu.Crncsbuaeu a. ctnistcle tatiuer te appictasfation of ethe subtance to I7a -estradiol. oertsdsusd Th m erble onntrios oft1in estradiociarearegisteredtin the serum. Chronictsubcutaneou icnraofedend oanyoofs -sugredion tharet incts that theatfompr oferve pharmaoloia geea knwegei.usrla

Claims

1. Use of steroids of the general formula 1 is a hydrogen atom, a hydroxyl group, or an alkyloxy group of 1-5 carbon atoms, is a hydrogen atom, an alkyl group of 1-5 carbon atoms, an acyl group of 1-5 carbon atoms, a group of the general formula SONRI,,IR, with and being in each instance a hydrogen atom, an alkyl group of 1-5 carbon atoms, independently of each other, or together with the nitrogen standing for a pyrrolidino, piperidion or morpholino group, is a hydrogen atom or a hydroxyl group. is a hydrogen atom, a hydroxyl group, or an alkyl group of up to 5 carbon atoms, WO 99/42108 PCT/DE99/00353 R, and R 6 are in each instance and independently of each other a hydrogen atom or a halogen atomn, R 7 is a hydrogen atom or a methyl grIouIp. R 8 is a hydrogen atom and a hydroxyl grIoup, an oxo grIoLup Or aI grouping of general formula C.R 12 R 13 in which thle R, 2 and the are in each instance and independently of each other a hydrogen atom or a halogen atom, R.9 is a mnethyl or ethyl group, Z is a C,C double bond or a suibstIIut[Cd 01- iinSUbStitUted cyclopropane ring and thle >CR)R, is either in the a or 13 position. beilu-. In til [3 position, if >CR5R6 is in the a position, and ice Vel'S6 in order to produce pharmaceutical p~reparations For selectively supplemnenting estrogen deficiency in thle central nervous system ithu N'110. La11fecting other or-~ans or systems.

2. Use of steroids as defined in Claim 1, these steroids being": 1 5PH,3)H-cycloprop[ 14,1 5]-estra- 1,3,5( 1 O),8-tetraerie-3, I 7a-diol, 1 5PH,3l--cycloprop[ 14,1l5]-I1 Sa-homlo-estra- I ,3t5( I O),S-teiraieie-3, I 7aX-ol, I 7ct-hydroxy- 1 5H,3'H-cycloprop[ 14, I Si-estr-a- 1,3,5(1 O),8-teti-aene -3'-yl-pentanoate, WO 99/42108 PCT/D E99100353 1 7-methylene-1 5PH,3''H-cycloprop( 14, I 5]-estra-1I,3,5(1I0),8-tetr-aene-3-oI, 15~H3H-3,3'-ifluro-cc~opop[14,1 5)-estra- 1,3,5(1 0),8-tetraene', I 7ax-diol, I 7-methylene- 1 5PH,3)'H-cyclcprop[ 14.1I 5]-estra- 1 5( 10),8-tetraene-3-yl-sulfamate

17-difluoromethylene- 1 5P3H.3H-cycloprop[ 14. 1 Si-estra- 1, 1.5( I 0),8-tetraene-3)-oI, .3-methioxy-15f3-methyl-'3'H-cycloprop[ 14,1I5]-estra- 5(10) 8-tetraene-3)-oI, 15a-mnethyl-3'H-cycloprop[ 14,1I5]-estra- 1,3,5(1 0),8-tetraene-3', I 7a-diol, I 7-difluoromethylene- I 5f3H '1--cycloprolp[ 14.. 1 5)-estra- 1 0),S-tetraenee-3)-yl- (tetrarnethyleneimino) sulfoniate, 17-m-ethylene-3)'H-cycloprop[S,9]-1I5f31-,'-cyclopriop[ 14, I51-estra- triene-3 -ol. 3. Use of steroids as defined in Claim I and Claim 2, fo1 proingC' pharmaceutical preparations for prophylaxis and therapy of' age-m-ediated reduction of cognitive performance, age-mediated and perimenopausal dysphoria, pre-menstrual syndrome, neuroses and neurasthenia, anxiety states and neuroses, P\WPDOCS\Hjw\Specs\7512530pgs.doc-08/08/01 -33- hot flashes after estrogen deprivation (menopause, gondectomy, treatment with GnRH analogues), psychogenic inhibition of sexual behaviour. 4. A method for selectively supplementing estrogen deficiency in the central nervous system of a mammal without affecting other organs or systems, which method includes the administration of one or more steroids according to claim 1 or 2. 5. A method for the treatment or prophylaxis of age-mediated reduction of cognitive performance, age-mediated and perimenopausal dysphoria, pre-menstrual syndrome, neuroses and neurasthenia, anxiety states and neuroses, hot flashes after estrogen deprivation (menopause, gonadectomy, treatment with GnRH analogues), and/or psychogenic inhibition of sexual behaviour, 20 in a mammal which method includes the administration of one or more steroids according to claim 1 or claim 2. DATED this 8th day of August 2001 JENAPHARM GMBH CO. KG By its Patent Attorneys DAVIES COLLISON CAVE