CA2321498A1 - Pharmaceutical preparations for the selectively supplementing oestrogen deficiency in the central nervous system - Google Patents
Pharmaceutical preparations for the selectively supplementing oestrogen deficiency in the central nervous system Download PDFInfo
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- CA2321498A1 CA2321498A1 CA002321498A CA2321498A CA2321498A1 CA 2321498 A1 CA2321498 A1 CA 2321498A1 CA 002321498 A CA002321498 A CA 002321498A CA 2321498 A CA2321498 A CA 2321498A CA 2321498 A1 CA2321498 A1 CA 2321498A1
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- cycloprop
- estradiol
- beta
- tetraene
- estra
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/12—Drugs for genital or sexual disorders; Contraceptives for climacteric disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/24—Antidepressants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
- A61P5/30—Oestrogens
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
i , 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 et~ective 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, a wherein R, is a hydrogen atom, a hydroxyl group, or an alkyloxy group of 1-S carbon atoms, R, is a hydrogen atom, an alkyl group of 1-5 carbon atoms, an acyl group of l-5 carbon atoms, a group of the general formula SO,NR,~R", 4vith R", and R" being in each instance a hydrogen WO 991:12108 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, R3 is a hydrogen atom or a hydroxyl group, Ra is a hydrogen atom, a hydroxyl group, or an alkyl group of up to 5 carbon atoms, RS and R6 are in each instance and independently of each other a hydrogen atom or a halogen atom, R, is a hydrogen atom or a methyl group, R8 is a hydrogen atom and a hydroxyl group, an oxo group or a grouping of general formula CR,,R,3, in which the R,, and the R,3 are in each instance and independently of each other a hydrogen atom or a halogen atom, R9 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 ~3 position, R, being in the (3 position, if >CR;R6 is in the a position, and nice 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).
t The best-known clinical symptoms of estrogen deticiency 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, C., Levasseur, M.C., 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," l ~~e A~~~i~y~r Male l (Suppl. l ), 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 deticiency s}mdrome" in aging males.
The brain is an extremely important target organ for the etl'ects of estrogen.
Estrogens have a decisive physiological influence on many neurobiolo~ical processes. Generally speaking, their effects can be classified in two major groups: organizing and activating (NIcEwen 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 pi~ysiological secretion of the gonads -, -____ _1_..___. ...............
s s 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, G., "Gonadotropic secretion and its control," in Knobil, E. Neil, J.D., (eds), T7re Physiology oJ'IZc.~prod~rctiorz, Raven Press, New York, pp. 1349-1376, 1988);
- control of sexual behaviour, Baum, M.J., et al., "Hormonal basis of proceptivity and receptivity in female primates," Arch. Sex. Behav. ~ : pp. I 7 ~- I 92, I
977);
- regulation of the neuroendocrine reactivity to stress, (Viau, V., Meaney, M.J., "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, M.F., et al., "Estrogen at~ects performance of ovariectomized rats in a two-choice water-escape working memory task," P.SyCflOlle?rl'OG'i)dOCI'liIOJO~~'~' ? l : pp. 51-65, I 996);
- maintenance of reaction readiness of neurochemical mechanisms that are indispensable for ensuring vigilance and adequate information processing, Fink, G., 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 of interneural contacts in the brain structures with a decisive role for cognitive performance and the emotional status, (Wooley, C.S., 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).
_:1_ 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, R.C., Sohrabi, F., Toran-Allerand, C.D., "Presumptive estrogen target neurons express mRNA 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 si~,;nal transmission in the central nervous system (Luine, V.N., "Estradiol increases choline acetyltransferase activity in specific basal forebrain nuclei and projection areas of female rats," Exp. Neurol. 89; pp. 489-490, 1985); (Weiland, N., "Glutamic acid decarboxylase messenger ribonucleic acid is regulated by estradiol and progesterone in the hippocampus," Er~docr~inulcyry 1 3 l : pp. 2697-2702, 1992); Bosse, R., Di Paolo, T., "The Modulation of brain dopamine and GABA;, receptors by estradiol: a clue for CNS changes occurring at menopause, Cell. Nlol. Neurobiol 16: pp. 199-212, 1996) - and increasing the resistance of nerve cells relative to pathological etJ:ects (Goodman, Y., 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, V. W., et al., "Estrogen replacement therapy _;_ ' 4 s in older women: comparisons between Alzheimer's disease cases and controls,"
Arch. Neurol. 51:
pp. 896-900, 1994); (Paganini-Hill, A., Henderson, V. W., "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 (e.g., oxytocin and arginine vasopressin) play an important role in the control of emotional behaviour components (Adan, R.A., Burbach, J.P., "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, E., Nakano, M., "Estrogens as antioxidants," ivlethods Enzymol. 186:
pp. 330-333, 1990);
(Lacort, M., et al., "Protective effects of estro~ens and catecholestro~ens against peroxidative 1S membrane damage in vitro, Lipids 30: pp. 141-146, 1990. 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, C.D., 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, T.G., Zigmond, M.J., "Neurodegenerative disease and oxidative stress: insights from an animal model of Parkinsonism" in: Fiskum, G. (ed) Neuro-degenerative Di.oeases, Plenum Press, New York, pp 37-46, 1996). For this reason, estrogen _6_ a WO 99/42108 PCTlDE99/00353 supplementation is also believed to play a role in the sense of maintaining and increasing endogenic antioxidative capacity (Betel, C., et al., "17b-estradiol protects neurons from oxidative stress-induced cell death in vitro," Biochem. Biophys. Res. Commun. 216; pp.
473-482, 1995).
S At present, estrogen supplementation is effected with natural and synthetic estrogen, the effects of which occur in all organs that contain estrogen receptors, i.e., 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, B.A., Ross, R.K., Henderson, B.E., "Relationship of hormone use to cancer risk," J. Natl. Cancer Inst., Monograph 12: pp. 137-147, 1992).
1S The proliferative effects of estrogen can be considered to be immediate risk factors for the occurrence of a benign prostatic hyperplasia and/or ~ynacomasty in the male (Knabbe, C., "Endokrine Therapie von Prostataerkrankungen," [Endocrine therapy of prostate diseases], in Allolio, B., Schulte, H.M. (eds), Prakti~~che I;IICI(l(.'l'IIIIIIIJ~~T~' [Practical Endocrinology], Urban &
Schwarzenberg, Munich, pp. 64S-651, 1996). For this reason, estrogen supplementation for males has never been given serious consideration, despite the indications that have been proven.
_7_ a 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 97/036b1, DE 43 38 314 C1.
- 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, cerebra( 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 C 1 describes steroids with phenolic :A-ring structure whose radical-capture and antioxidative properties are independent from the extent of its estrogen-like effectiveness.
_g_ These compounds can be used for prophylaxis and therapy ot'radical mediated cellular damage.
In all of these patent specifications, the therapeutic and neuroprotective etI-iciency 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 tactors;
- 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 tree radicals and reactive oxygen species liberated as a consequence of a traumatic or neurotoxic et~ect.
However, none of the cited patent specifications describes steroids with selective estrogen-like neurotropic transcription effects, i.e., 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 (ulS
s,s~4,601 and WO
95/12402, the etlect of l7a-estradiol--a substance that exhibits a reduced estrogeneity in the genital tract (Clark, J.H. et al., "Effects of estradiol I 71 on nuclear occupancy of the estrogen WO 99/2108 PCTlDE99/00353 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 I 7a-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 et~ects. WO 97/03361 and DE
43 38 314 Cl interpret the cytoprotective effects ol'estrogen as being a consequence ofthe 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 pharmaceinical 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 atlecting other organs and systems.
WO 99/x2108 PCT/DE99100333 The steroids are characterized in that they have a selective neurotropic, estrogen-like transcription effect, unlike systemically active natural and synthetic estogens, including 17x-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 physiolo~,ical parameters;
- exhibit central nervous system specific transcription et~ects at doses that have no biological effects in the tissues of the reproductive system;
- exhibit central nervous system specific transcription et~ects at doses in which neither 17b-estradiol or 17x-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 fi~rmuia I
R
R,L
in which in which Rr is a hydrogen atom, a hydroxyl group, or an alkyloxy group of 1-5 carbon atoms, RZ is a hydrogen atom, an alkyl group of l-5 carbon atoms, an acyl group of 1-5 carbon atoms, a group of the general formula SO,NR,~R", with R", and R" 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, R~ is a hydrogen atom or a hydroxyl group, R; is a hydrogen atom, a hydroxyl group, or an alkyl group of up to 5 carbon atoms, RS and R~ are in each instance and independently of each other a hydrogen atom or a halogen atom, R, is a hydrogen atom or a methyl group, R~ is a hydrogen atom and a hydroxyl group, an oxo group or a grouping of general tormula C: R,,R, ~, in which the R,, and the R,3 are in each instance and independently of each other a I~vdro~~en 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 >CRSR6 is either in the a or [3 position, R, being in the (3 position, if >CR;Ri, is in the a position, and nice versa.
Preferred compounds are the following:
15[3H,3'H-cycloprop[14,15]-extra-1,3,5( 10),8-tetraene-_~,17a-diol, IS~iH,3'H-cycloprop[14,15]-18x-homo-extra-1,3,x( 10),8-tetraene-3,17x-ol, 17x-hydroxy-15(3H,3'H-cycloprop[14, 15]-extra-l, ~,5( 10),8-tetraene -3-yl-pentanoate, 17-methylene-15(3H,3'H-cycloprop(14, 15]-extra-I,s,s( 10),8-tetraene-s-ol, 15[3H,3'H-3',3'-difluoro-cycloprop[14,15)-extra- I ,s,~( I 0),8-tetraenes, I
7a-diol, 17-methylene-15[iH,3'H-cyclcprop[14,15]-extra-I,.>. ~( 10),S-tetraene-~-yl-sultamate WO 99/:12108 PCTIDE99/00353 17-difluoromethylene-15(3H.3'H-cycloprop[ 14 I s]-estra- l , ~,~( 10),8-tetraene-3-ol, 3-methoxy-IS[3-methyl-3'H-cycloprop[14,15]-estra-l, ~ 5( 10) 8-tetraene->-ol, 15a-methyl-3'H-cycloprop[14,15)-estra-1,3,x( 10),8-tetraene-s, l7a-diol, 17-difluoromethylene-l5(3H 3'H-cycloprop[ 14,1 ~)-estra-I, ~,~( 10),8-tetraenee-3-yl-(tetramethyleneimino) sulfonate, 17-methylene-3'H-cycloprop[8,9]-15[3H,3'H-cycloprop[ 14, I ~]-estra- I , ~,~( 10)-triene-3-ol.
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 reduction ufco';nitive performance, age-mediated and perimenopausal dysphoria, premenstrual syndrome, neuroses and neurasthenia, anxiety states and neuroses, hot Clashes after estru~en deprivation (menopause, gonodectomy, treatment with GnRH analogues) and the psycho~enic inhibition of sexual activity.
It was established that when this is done, the risk of dama~,.:in~; hormone-sensitive tissues of the reproductive system (endometrium, myumetrium, prostate, and mammary glands in the sense of uncontrolled proliferation and carcino~,enesis is lar~,ely precluded.
- I .s -r WO 99I=12108 PCT/DE99/00353 The object of the present invention is also pharmaceutical preparations for 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 t'ormulations:
- 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 ? m'.: daily;
- subcutaneous implants with a daily release capacity ti~om 0 05 to ? mg daily;
- gels and cremes with transdermal release li-om 0 05 to ? m~ 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.
-l4-Based on the example of 15(3H,3'H-cycloprop[I=4, I >]-estra-l, >,~( 10),8-tetraene-3,17a-diol (compound of general formula I: R,=R~=R3=R,=K; =R~,=R,=RH=a-OH, [3-H; ER,=CH3;
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 17b-estradiol and 17a-estradiol.
Example I
The effect on uterus weight aher chronic subcutaneous application in vivo Sexually mature (3 months old, weight 2~Ot30 ~) female Wistar rats (Tierzucht Schonwalde GmbH, Germany) were ovariectomized when under I:etamine narcosis. At'ter l4 days, the animals were implanted subcutaneously with osmotic minipumps (Alzett, USA) that released a daily dose of0.01, 0.1, 0.3, 1, 3, 30, and l00 ltg of the substance to be investigated (lS~iH,3'H-cycloprop[14, I S]-estra-1,3,5(10),8-tetraene-3,17a-diol, 17b-estradiol, 17a-estradiol) for seven days; the controls received an corresponding volume of vehicle (pr«pyleneglycol). On the seventh day ofthe treatment, the animals were killed and the uterus wet wei~lo was determined (relative to 100 g body weight).
_15-WO 99/x2108 PCT/DE99/00353 Figure 1 shows the uterotropic effect of the various doses of 17b-estradiol (square symbol), 17a-estradiol (circle symbol), and 15[3H,3'H-cycloprop[ l4,15]-estra-I, 3,5( 10),8-tetraene-3,17x-diol (triangle symbol) on the ovariectomized rats. Each point represents the average value t standard error (xtSEM) 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 enlar';ement otthe uterus was achieved at daily does rates of 0.03-0.1 pg. In order to achieve a comparable uterotropic effect, one would require daily doses of 100 ttg 17x-estradiol or _~0 Ey, of 1 s[3H,s'f-f-cvcloprop[ 14,15]-estra-1,3,5( 10),8-tetraene-3,17x-diol.
The results show that the effectiveness of I s[31-l, ~'1-i-cycloprop[ 14,1 ~]-~atra-1,3,x(10),8-tetraene-3,17x-diol in the female genital tract is some 1000-told less than the etlectiveness of 17b-estradiol and is comparable to the effectiveness of 17x-estradiol.
Example Activation of the transcription of an a-estro<,en receptor-dependent reporter gene in vitro -lo-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) of vitellogenin, a thymidinekinase promotor, and the luciferase coding gene of Photinrr.s pyralis. Prior to the start of the experiment, the cell culture was cultivated in steroid-free medium for seven days and then inc~rbated for 48 Fours with 17b-estradiol, 17a-estradiol, or I S[3H,3'H-cyclo-prop[ 14,15]-estra- I ,_s,s( 10),S-tetraene-s,17a-diol in four different concentrations ( 10-"0, 10-"', 10'x, and 10-x Nf). 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 transtired estrogen-dependent reporter gene (luciferase) in estrogen-receptor eapressin~; VICE-7 breast cancer cells after 48 hours of treatment with different doses of 17b-estradiol (square symbol), 17a-estradiol (circle symbol), and I 5(3H,3'H-cycloprop[ 14, I S]-estra- I ,s, ~( 10),S-tetraene-~,17a-diol (triangle symbol).
The figure represents the average values from tvvo 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 1 ~(3H, ~'H-cycloprop[ 14, I 5]-estra-1,3,5(10),8-_l7_ tetraene-3,17x-diol and 17a-estradiol is one order of magnitude ( 10 times) less than the ef~'ectiveness of 17b-estradiol.
The results show that the 15(3H,3'H-cycloprop[ 14,15]-estra-I,s,~( 10),8-tetraene-3,17a-diol has an estrogen effect that is many times weaker in breast cancer tissue.
Example.
Stimulation of the transcription of the oxytocin gene in the brain after chronic treatment in vivo, at doses that have no et~ect on the uterus Sexually mature (3 months old, weight 25030 <,j female ~~Vistar rats (Tierzucht Schonwalde GmbH, Germany) were ovariectomized when under ketamine narcosis. After 14 days, the animals were implanted subcutaneously with osmotic rninipumps (Alzett, USA) that released a daily dose of 0.01, 0.1, and 1 pg of the substance to be investigated ( I ~(3t-1,3'H-cycloprop[14,15J-estra-1,3,5( 10),8-tetraene-3,17x-diol, l 7a-estradiol, I 7b-estradiol ) t'or seven days; the controls received an corresponding volume of vehicle (propylene~;lycol). Immediately after the animals _Ig_ had been killed, the uterus wet weight was determined (relative to l 00g of body weight). The messenger ribonucleic acid mRNA, which codes the biosynthesis of oxytocin, was shown by in-situ hybridization with a specific radioactive-marked oligodeoxynucleotide probe, using an established method (Fisher, D., 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. 1208-121 ~, 199s j, I X95) in the hypothalamic nucleus paraventricularis (PVN). Changes in the transcription ot~the oaytocin gene that were governed by the treatment were quantified by densitometric nteasuremerrts of the specific hybridizing signals within the defined anatomic structure.
Figure 3 shows the induction of oxytocin-codin'; transcripts (OT mRNA; upper drawing) in the hypothalamic paraventricular nucleus of ovariecton~ized rats at'ter chronic subcutaneous treatment with 17b-estradiol (circle symbol), 17a-estradiol (square symbol), and I5(3H,3'H-cycloprop[14, I SJ-estra-1,3,5(10),8-tetraene-3,17x-diol ~trian~le 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 xtSENI of ~-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).
The results show that 15[3H,3'H-cycloprop[14, l5]-estra-1,3,5( 10),8-tetraene-3,17a-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 17b-estradiol. At all events, the neurotropic transcription effects of 15~3H,3'H-cycloprop[ 1=(,1 s]-estra-1, 3,~( 10),S-tetraene-3,17a-diol, unlike that of 17b-estradiol, are not associated W1L11 e11I11~!~~illellt of the uterus. .fit the doses that were used, the 17a-estradiol had no affect on the concentrations of oxytocin mRNA
in the hypothalamic PVN.
These results document a selective, estrogen-like etlect of l ~(3H, 3'1-1-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-3,17a-diol in the brains of female rats.
Example =1 Stimulation of the transcription of the antiapoptotic 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 -? 0-and acts counter to the programmed cell death (apoptosis) (Merry, D E., Korsmeyer, S.J., "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, Nl. 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 gyros is a component of the hippocampal formation in which the neurogenesis persists in the rat, even at a mature age (could, h , et al., "Proliferation of granule cell precursors in the dentate gyros of adult monkeys is diminished by stress," I'rc» ;'~atl Acad. Sci. USA 96: pp.
3168-317, 1998) and bcl-2 is expressed. Bcl-? transcripts were shown in brain sections by in-.sitcr hybridization with a specific oligonucleotide probe (Clark R.S.B., 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 Figure 4 shows the effect of three different doses ot~ 17b-estradiol (circle symbol), 17a-estradiol (square symbol), and 15~3H,3'H-cycloprop[ I ~, I 5]-estra- I ,3, 5( I 0),8-tetraene-3,17a-diol (triangle symbol) on the expression of bcl-2 in the dentate ~;vrus ot~ the hippocamp~rs of ovariectomized rats; the symbols and abbreviations used are the sane as those in Fi~~ure s _2 I _ Treatment with S~H,3'H-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-3,17~G-diol resulted in a 25 dose-dependent stimulation bcl-2 expression in the dentate gyros. The effect was the same as that mediated by the same dose of 17b-estradiol. At the dose rates that were used, 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, 15~H,3'H-cycloprop[14,15]-estra-1,3,5(10),8-3o tetraene-3,17~G-diol affects the transcription of the antiapoptotic gen bcl-2 in the central nervous system according to an estrogen-like mode, without any effect occurring at the uterus.
Example 5 Dissociated induction of oxytocin receptors in the brain and myometrium Binding sites with identical biochemical characteristics for tle peptide hormone oxytocin are present in the myometrium and in the central nervous system. 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 amygdaloid nuclear complex. Estrogen-mediated induction of oxytocin receptors in these structures is causally - ~z -S
n, ~ v related to the formation of prosocial behaviour patterns, includin~~ sexual behaviour (Inset, T.R., "Oxytocin - a neuropeptide for affiliation: evidence from behavioral, receptor autoradiographic, and comparative studies," Psychone~~roet~c%crvrrolcyl~.~ 17: pp. 3-35, 1992) In order to determine the density of oxytocin receptors in defined anatomical structures, the method of choice is autoradiographic representation by binding of the radioactively marked oxytocin receptor antagonists d(CH,);-Tyr(Me)-', Thr~, Orn~-['wl]Tyn'-vasotocin (r''l-OVTA) (Kremarik, P., et al., "Histoautoradiographic detection of oxytocin and vasopressin binding sites in the telencephalon of the rat," J.Comp.Neurol. _>;s pp 34 3-3s9, 199x).
Frozen sections of the brain and uterus of ovariectomized rats that Itad received a daily subcutaneous dose of 1 ~tg of 15~3H,3'H-cycloprop[ I-l, I s]-~stra- I,s,~( 10),8-tetraene-3,17a-diol, 17b-estradiol, or 17a-estradiol for a period of seven daw (:oml~arc Example 3) were incubated with r''51-OVTA (N EN DuPont, Germany) at at i;Oll~eltti'~IIOn Uf ~0 pNl Subsequently, filmautoradiograms were made, and these were then used for densitometric determination of the oxytocin binding sites, using an established method ( fatchev, V. K , et al., "Oxytocin binding sites in rat limbic and hypothalamic structures: site specilic modulation by adrenal and gonadal steroids," Nc.~rrro.scie~~ce 57: pp. 537-X43, 199;) _; _ , S
Figure 5 shows the specific binding of a'''1-marked li~and of the oxytocin receptor (''-Sl-OVT) in the myometrium and in two estrogen-sensitive brain structures, the hypothalamic ventromedial nucleus (VMN) and the nucleus interstitialis striae terminalis (BLAST), after a seven-day treatment with 17b-estradiol (black columns), 1?a-estradiol (hatched columns), and 15~3H,3'H-cycloprop[14,15]-estra-1,3,~(10),S-tetraene-3,17x-dial (grey columns), at a daily dose rate of 1 pg. The star symbols indicate significant <lifterences (p~=0 05) as compared to placebo-treated ovariectomized rats (OVX). The right-I~ami ~Irawirn; ~huw~ tlw etlects of the substances that were tested on the proliferation of the endometrium Each column represents xtSEM of 4-5 individual determinations.
The results of this test are also shown in Figure 5. ~freatmer~t with 17b-estradiol and 15(3H,3'H-cycloprop[14,1 S]-estra-1,3,5(10),8-tetraene-3,17x-diol resulted in a significant increase in the density of oxytocin receptors in all the brain structures that were examined, with 15~3H,3'H-cycloprop[14,15]-estra-1,3,x(10),8-tetraene- 3, I ?a-dial eYhibitin'; a weaker et~ect than 17b-estradiol in the VMN . At the doses that were administered. floe 17a-estradiol was not effective in any brain structures. In the myometrium, 17b-~stradiol caused a struper induction of oxytocin binding sites, whereas 1?a-estradiol and I5(3H,3'H-cycloprop[14,15)-extra-1,3,500),8-tetraene-3,17x-diol exhibited a significantly smaller et~tect ~('l~e computer-aided measurement of the WO 99/:2108 PCT/DE99/00353 strength of the endometrium in the uterus pl~~p'dl~iltlOlls ~110VVt;d that the daily dose of l~tg 17b-estradiol caused a significant endometrial proliferation, whereas at an equivalent dose 15~3H,3'H-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-3,17x-diol and l 7a-estradiol did not influence the thickness of the endometrium.
In summary, the results presented in Example C~ shuvv that I ~(3l-l, 3'I-1-cycloprop[ 14,15]-estra-1,3,5(10),8-tetraene-3,17a-diol influences a biochemical parameter--the oxytocin receptor--that is characteristic for the reproductive system (myometrium) atld for the central nervous system, mainly in the central nervous system, and that because ot'this selectise neurotropic effect it differs qualitatively from the natural estro~ens 17b-estradical amt ! 7a-estractiul Example b The effects on cognitive funi:tions utter cl~r~niv treatment It is known that a reduction of estrogen concentrations is associated with diminished learning and memory functions (Kopera, H., "Estrogen and psychic functions. Aging and estrogens," Front Hormone Res. ?: pp. I 18-133, 1973).
;-A correlation between serum-estrogen level and cognitive performance has also been demonstrated in the animal-test model (Kondo, 1'., Suzuki, I<., Sakun~a, Y., "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 15(3H,3'H-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-~,17a-diol, 17b-estradiol, and 17a-estra-diol on cognitive performance:
Sexually mature female Wistar rats (weight ?40t?0 ~) were ovariectomized when under Nembutal narcosis. One week after the operation the follov~ ip; substance were administered subcutaneouslv .at the dose rates shown: 17b-mtracliol, I u~" 17a-eatradiol, 100 ltg; 15~3H,3'H-cycloprop .[14, I 5]-estra-1,3,5(10),8-tetraene-3,17x-diol, s0 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 G., et al., "Influence of the estrous cycle, ovariectomy anct estradiol replacement upon the acquisition of conditioned avoidance responses in rats," Physiol. E3ehav. 4C~: pp. ;97--t0 I
, 1989). Each animal was exposed SO times in each session to a combination of an unconditioned response (electric t,_ t 4 shock) and two conditioned stimuli (light and sound si',nal). Retention of the learned behaviour pattern was tested on the seventh day of the treatment .~lt'ter n 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 lh~: COndIClolltn~
signals) out of 50 sequential exposures was used as the criterion for evaluating the retention or extinction ofthe learned behaviour.
Figure 6 shows the effect of 17b-estradiol (black columns), 17a-estradiol (shaded columns), and 15(3H,3'H-cycloprop[14,15]-estra-1,3,x(10),8-tetraene-s,17a-ciiol (rev columns) on the acquisition and retentionof a new behaviour })attel~n I11 ()1%a i'1(:CTOI111Zf;d rats (open columns; OVX).
The star symbols indicate significant differences to the placebo-treated animals (OVX) on the corresponding test day. The following uterus ~vei'~hts (xtSEM; n = 8-10 per treatment group;
data in m<r 100 g body weight were determined utter fourteen days of treatment; OVX, 5312;
17b-estradiol 18719; 17a-estradiol, IOOy~; s(31-i,:~'11-c~cloprol~( I-l, l ~J-estra-1,3,500),8-tetraene-3,17a-diol, 10814.
It is clear that at the dose rate that was used, the 5(3f-l,s'H-cycloprop[ 14, I S]-estra-1,3,5(10),8-tetraene-3,17x-diol has an estrogen-like stimulating et~ect on the retention of the behaviour pattern that was learned, the uterotropic effect being ~i~nificantly less than the effect of 17b-estradiol at a daily dose rate of 1 ug. This result indicates that 5(3H, ~'H-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-3,17x-diol affects cognitive performance like an estrogen, whereas it exhibits a smaller proliferative effect in the reproductive or<,ans Example 7 Biotransformation of 17a-hydroxy-14, I sa-methylene-extra-a,5 ( IOj,S-tetraene-3-of and 17a-estradiol to 17b-estradiol A daily dose of 100 ug 17a-estradiol, 30 ~y ~(3(-I,s'H-cycloprop[ 14,1 S]-estra-1,3,5(l0),8-tetraene-3,17x-diol, or 1 pg 17b-estradiol was administered to ovariectomized rats for seven days (compare Example 6). On the last day of treatment, the serum concentrations of 17b-estradiol in the three groups was determined and compared to the concentrations in controls that had been treated with the vehicle.
S_ Figure 7 shows the serum values for 17b-estradiol after seven days of subcutaneous treatment of ovariectomized rats with 17b-estradiol (black columns), 17a-estradiul (shaded columns) and 5(3H,3'H-cycloprop[ 14,15]-estra-t,3,5( l0),S-tetraene-s, l 7cc-cliol (;rev columns) at the cited dose rates. The star symbols indicate significant ditierences compared to the values measures in placebo-treated animals; the latter were below the minimum detection limit for the methods; each treatment group comprises seven animals.
It is clear that after the application of l7b-estradiul and 17a-estradiol at the dose rates discussed, measurable concentrations of 17b-estradiol are registered in the serum.
Chronic subcutaneous treatment with 5(3H,3'H-cycloprop[14,15]-estra-I, ~,5( 10),x-tetraene-s,17a-diol causes no increase of the endogenous 17b-estradiol Thi, rewlt imlicttes that thmbserved pharmacological effects after administration of 5~3H,3'H-cycluhr:yj i-J. I ~)-mtru-I,.~.~( It>).5-tetraene-3,17a-diol cannot be attributed to a biotransformation ol'the substance to I 7b-estraciiol.
-
These steroids are characterized in that, unlike systemically et~ective 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, a wherein R, is a hydrogen atom, a hydroxyl group, or an alkyloxy group of 1-S carbon atoms, R, is a hydrogen atom, an alkyl group of 1-5 carbon atoms, an acyl group of l-5 carbon atoms, a group of the general formula SO,NR,~R", 4vith R", and R" being in each instance a hydrogen WO 991:12108 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, R3 is a hydrogen atom or a hydroxyl group, Ra is a hydrogen atom, a hydroxyl group, or an alkyl group of up to 5 carbon atoms, RS and R6 are in each instance and independently of each other a hydrogen atom or a halogen atom, R, is a hydrogen atom or a methyl group, R8 is a hydrogen atom and a hydroxyl group, an oxo group or a grouping of general formula CR,,R,3, in which the R,, and the R,3 are in each instance and independently of each other a hydrogen atom or a halogen atom, R9 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 ~3 position, R, being in the (3 position, if >CR;R6 is in the a position, and nice 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).
t The best-known clinical symptoms of estrogen deticiency 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, C., Levasseur, M.C., 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," l ~~e A~~~i~y~r Male l (Suppl. l ), 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 deticiency s}mdrome" in aging males.
The brain is an extremely important target organ for the etl'ects of estrogen.
Estrogens have a decisive physiological influence on many neurobiolo~ical processes. Generally speaking, their effects can be classified in two major groups: organizing and activating (NIcEwen 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 pi~ysiological secretion of the gonads -, -____ _1_..___. ...............
s s 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, G., "Gonadotropic secretion and its control," in Knobil, E. Neil, J.D., (eds), T7re Physiology oJ'IZc.~prod~rctiorz, Raven Press, New York, pp. 1349-1376, 1988);
- control of sexual behaviour, Baum, M.J., et al., "Hormonal basis of proceptivity and receptivity in female primates," Arch. Sex. Behav. ~ : pp. I 7 ~- I 92, I
977);
- regulation of the neuroendocrine reactivity to stress, (Viau, V., Meaney, M.J., "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, M.F., et al., "Estrogen at~ects performance of ovariectomized rats in a two-choice water-escape working memory task," P.SyCflOlle?rl'OG'i)dOCI'liIOJO~~'~' ? l : pp. 51-65, I 996);
- maintenance of reaction readiness of neurochemical mechanisms that are indispensable for ensuring vigilance and adequate information processing, Fink, G., 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 of interneural contacts in the brain structures with a decisive role for cognitive performance and the emotional status, (Wooley, C.S., 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).
_:1_ 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, R.C., Sohrabi, F., Toran-Allerand, C.D., "Presumptive estrogen target neurons express mRNA 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 si~,;nal transmission in the central nervous system (Luine, V.N., "Estradiol increases choline acetyltransferase activity in specific basal forebrain nuclei and projection areas of female rats," Exp. Neurol. 89; pp. 489-490, 1985); (Weiland, N., "Glutamic acid decarboxylase messenger ribonucleic acid is regulated by estradiol and progesterone in the hippocampus," Er~docr~inulcyry 1 3 l : pp. 2697-2702, 1992); Bosse, R., Di Paolo, T., "The Modulation of brain dopamine and GABA;, receptors by estradiol: a clue for CNS changes occurring at menopause, Cell. Nlol. Neurobiol 16: pp. 199-212, 1996) - and increasing the resistance of nerve cells relative to pathological etJ:ects (Goodman, Y., 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, V. W., et al., "Estrogen replacement therapy _;_ ' 4 s in older women: comparisons between Alzheimer's disease cases and controls,"
Arch. Neurol. 51:
pp. 896-900, 1994); (Paganini-Hill, A., Henderson, V. W., "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 (e.g., oxytocin and arginine vasopressin) play an important role in the control of emotional behaviour components (Adan, R.A., Burbach, J.P., "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, E., Nakano, M., "Estrogens as antioxidants," ivlethods Enzymol. 186:
pp. 330-333, 1990);
(Lacort, M., et al., "Protective effects of estro~ens and catecholestro~ens against peroxidative 1S membrane damage in vitro, Lipids 30: pp. 141-146, 1990. 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, C.D., 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, T.G., Zigmond, M.J., "Neurodegenerative disease and oxidative stress: insights from an animal model of Parkinsonism" in: Fiskum, G. (ed) Neuro-degenerative Di.oeases, Plenum Press, New York, pp 37-46, 1996). For this reason, estrogen _6_ a WO 99/42108 PCTlDE99/00353 supplementation is also believed to play a role in the sense of maintaining and increasing endogenic antioxidative capacity (Betel, C., et al., "17b-estradiol protects neurons from oxidative stress-induced cell death in vitro," Biochem. Biophys. Res. Commun. 216; pp.
473-482, 1995).
S At present, estrogen supplementation is effected with natural and synthetic estrogen, the effects of which occur in all organs that contain estrogen receptors, i.e., 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, B.A., Ross, R.K., Henderson, B.E., "Relationship of hormone use to cancer risk," J. Natl. Cancer Inst., Monograph 12: pp. 137-147, 1992).
1S The proliferative effects of estrogen can be considered to be immediate risk factors for the occurrence of a benign prostatic hyperplasia and/or ~ynacomasty in the male (Knabbe, C., "Endokrine Therapie von Prostataerkrankungen," [Endocrine therapy of prostate diseases], in Allolio, B., Schulte, H.M. (eds), Prakti~~che I;IICI(l(.'l'IIIIIIIJ~~T~' [Practical Endocrinology], Urban &
Schwarzenberg, Munich, pp. 64S-651, 1996). For this reason, estrogen supplementation for males has never been given serious consideration, despite the indications that have been proven.
_7_ a 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 97/036b1, DE 43 38 314 C1.
- 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, cerebra( 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 C 1 describes steroids with phenolic :A-ring structure whose radical-capture and antioxidative properties are independent from the extent of its estrogen-like effectiveness.
_g_ These compounds can be used for prophylaxis and therapy ot'radical mediated cellular damage.
In all of these patent specifications, the therapeutic and neuroprotective etI-iciency 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 tactors;
- 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 tree radicals and reactive oxygen species liberated as a consequence of a traumatic or neurotoxic et~ect.
However, none of the cited patent specifications describes steroids with selective estrogen-like neurotropic transcription effects, i.e., 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 (ulS
s,s~4,601 and WO
95/12402, the etlect of l7a-estradiol--a substance that exhibits a reduced estrogeneity in the genital tract (Clark, J.H. et al., "Effects of estradiol I 71 on nuclear occupancy of the estrogen WO 99/2108 PCTlDE99/00353 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 I 7a-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 et~ects. WO 97/03361 and DE
43 38 314 Cl interpret the cytoprotective effects ol'estrogen as being a consequence ofthe 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 pharmaceinical 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 atlecting other organs and systems.
WO 99/x2108 PCT/DE99100333 The steroids are characterized in that they have a selective neurotropic, estrogen-like transcription effect, unlike systemically active natural and synthetic estogens, including 17x-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 physiolo~,ical parameters;
- exhibit central nervous system specific transcription et~ects at doses that have no biological effects in the tissues of the reproductive system;
- exhibit central nervous system specific transcription et~ects at doses in which neither 17b-estradiol or 17x-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 fi~rmuia I
R
R,L
in which in which Rr is a hydrogen atom, a hydroxyl group, or an alkyloxy group of 1-5 carbon atoms, RZ is a hydrogen atom, an alkyl group of l-5 carbon atoms, an acyl group of 1-5 carbon atoms, a group of the general formula SO,NR,~R", with R", and R" 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, R~ is a hydrogen atom or a hydroxyl group, R; is a hydrogen atom, a hydroxyl group, or an alkyl group of up to 5 carbon atoms, RS and R~ are in each instance and independently of each other a hydrogen atom or a halogen atom, R, is a hydrogen atom or a methyl group, R~ is a hydrogen atom and a hydroxyl group, an oxo group or a grouping of general tormula C: R,,R, ~, in which the R,, and the R,3 are in each instance and independently of each other a I~vdro~~en 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 >CRSR6 is either in the a or [3 position, R, being in the (3 position, if >CR;Ri, is in the a position, and nice versa.
Preferred compounds are the following:
15[3H,3'H-cycloprop[14,15]-extra-1,3,5( 10),8-tetraene-_~,17a-diol, IS~iH,3'H-cycloprop[14,15]-18x-homo-extra-1,3,x( 10),8-tetraene-3,17x-ol, 17x-hydroxy-15(3H,3'H-cycloprop[14, 15]-extra-l, ~,5( 10),8-tetraene -3-yl-pentanoate, 17-methylene-15(3H,3'H-cycloprop(14, 15]-extra-I,s,s( 10),8-tetraene-s-ol, 15[3H,3'H-3',3'-difluoro-cycloprop[14,15)-extra- I ,s,~( I 0),8-tetraenes, I
7a-diol, 17-methylene-15[iH,3'H-cyclcprop[14,15]-extra-I,.>. ~( 10),S-tetraene-~-yl-sultamate WO 99/:12108 PCTIDE99/00353 17-difluoromethylene-15(3H.3'H-cycloprop[ 14 I s]-estra- l , ~,~( 10),8-tetraene-3-ol, 3-methoxy-IS[3-methyl-3'H-cycloprop[14,15]-estra-l, ~ 5( 10) 8-tetraene->-ol, 15a-methyl-3'H-cycloprop[14,15)-estra-1,3,x( 10),8-tetraene-s, l7a-diol, 17-difluoromethylene-l5(3H 3'H-cycloprop[ 14,1 ~)-estra-I, ~,~( 10),8-tetraenee-3-yl-(tetramethyleneimino) sulfonate, 17-methylene-3'H-cycloprop[8,9]-15[3H,3'H-cycloprop[ 14, I ~]-estra- I , ~,~( 10)-triene-3-ol.
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 reduction ufco';nitive performance, age-mediated and perimenopausal dysphoria, premenstrual syndrome, neuroses and neurasthenia, anxiety states and neuroses, hot Clashes after estru~en deprivation (menopause, gonodectomy, treatment with GnRH analogues) and the psycho~enic inhibition of sexual activity.
It was established that when this is done, the risk of dama~,.:in~; hormone-sensitive tissues of the reproductive system (endometrium, myumetrium, prostate, and mammary glands in the sense of uncontrolled proliferation and carcino~,enesis is lar~,ely precluded.
- I .s -r WO 99I=12108 PCT/DE99/00353 The object of the present invention is also pharmaceutical preparations for 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 t'ormulations:
- 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 ? m'.: daily;
- subcutaneous implants with a daily release capacity ti~om 0 05 to ? mg daily;
- gels and cremes with transdermal release li-om 0 05 to ? m~ 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.
-l4-Based on the example of 15(3H,3'H-cycloprop[I=4, I >]-estra-l, >,~( 10),8-tetraene-3,17a-diol (compound of general formula I: R,=R~=R3=R,=K; =R~,=R,=RH=a-OH, [3-H; ER,=CH3;
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 17b-estradiol and 17a-estradiol.
Example I
The effect on uterus weight aher chronic subcutaneous application in vivo Sexually mature (3 months old, weight 2~Ot30 ~) female Wistar rats (Tierzucht Schonwalde GmbH, Germany) were ovariectomized when under I:etamine narcosis. At'ter l4 days, the animals were implanted subcutaneously with osmotic minipumps (Alzett, USA) that released a daily dose of0.01, 0.1, 0.3, 1, 3, 30, and l00 ltg of the substance to be investigated (lS~iH,3'H-cycloprop[14, I S]-estra-1,3,5(10),8-tetraene-3,17a-diol, 17b-estradiol, 17a-estradiol) for seven days; the controls received an corresponding volume of vehicle (pr«pyleneglycol). On the seventh day ofthe treatment, the animals were killed and the uterus wet wei~lo was determined (relative to 100 g body weight).
_15-WO 99/x2108 PCT/DE99/00353 Figure 1 shows the uterotropic effect of the various doses of 17b-estradiol (square symbol), 17a-estradiol (circle symbol), and 15[3H,3'H-cycloprop[ l4,15]-estra-I, 3,5( 10),8-tetraene-3,17x-diol (triangle symbol) on the ovariectomized rats. Each point represents the average value t standard error (xtSEM) 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 enlar';ement otthe uterus was achieved at daily does rates of 0.03-0.1 pg. In order to achieve a comparable uterotropic effect, one would require daily doses of 100 ttg 17x-estradiol or _~0 Ey, of 1 s[3H,s'f-f-cvcloprop[ 14,15]-estra-1,3,5( 10),8-tetraene-3,17x-diol.
The results show that the effectiveness of I s[31-l, ~'1-i-cycloprop[ 14,1 ~]-~atra-1,3,x(10),8-tetraene-3,17x-diol in the female genital tract is some 1000-told less than the etlectiveness of 17b-estradiol and is comparable to the effectiveness of 17x-estradiol.
Example Activation of the transcription of an a-estro<,en receptor-dependent reporter gene in vitro -lo-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) of vitellogenin, a thymidinekinase promotor, and the luciferase coding gene of Photinrr.s pyralis. Prior to the start of the experiment, the cell culture was cultivated in steroid-free medium for seven days and then inc~rbated for 48 Fours with 17b-estradiol, 17a-estradiol, or I S[3H,3'H-cyclo-prop[ 14,15]-estra- I ,_s,s( 10),S-tetraene-s,17a-diol in four different concentrations ( 10-"0, 10-"', 10'x, and 10-x Nf). 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 transtired estrogen-dependent reporter gene (luciferase) in estrogen-receptor eapressin~; VICE-7 breast cancer cells after 48 hours of treatment with different doses of 17b-estradiol (square symbol), 17a-estradiol (circle symbol), and I 5(3H,3'H-cycloprop[ 14, I S]-estra- I ,s, ~( 10),S-tetraene-~,17a-diol (triangle symbol).
The figure represents the average values from tvvo 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 1 ~(3H, ~'H-cycloprop[ 14, I 5]-estra-1,3,5(10),8-_l7_ tetraene-3,17x-diol and 17a-estradiol is one order of magnitude ( 10 times) less than the ef~'ectiveness of 17b-estradiol.
The results show that the 15(3H,3'H-cycloprop[ 14,15]-estra-I,s,~( 10),8-tetraene-3,17a-diol has an estrogen effect that is many times weaker in breast cancer tissue.
Example.
Stimulation of the transcription of the oxytocin gene in the brain after chronic treatment in vivo, at doses that have no et~ect on the uterus Sexually mature (3 months old, weight 25030 <,j female ~~Vistar rats (Tierzucht Schonwalde GmbH, Germany) were ovariectomized when under ketamine narcosis. After 14 days, the animals were implanted subcutaneously with osmotic rninipumps (Alzett, USA) that released a daily dose of 0.01, 0.1, and 1 pg of the substance to be investigated ( I ~(3t-1,3'H-cycloprop[14,15J-estra-1,3,5( 10),8-tetraene-3,17x-diol, l 7a-estradiol, I 7b-estradiol ) t'or seven days; the controls received an corresponding volume of vehicle (propylene~;lycol). Immediately after the animals _Ig_ had been killed, the uterus wet weight was determined (relative to l 00g of body weight). The messenger ribonucleic acid mRNA, which codes the biosynthesis of oxytocin, was shown by in-situ hybridization with a specific radioactive-marked oligodeoxynucleotide probe, using an established method (Fisher, D., 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. 1208-121 ~, 199s j, I X95) in the hypothalamic nucleus paraventricularis (PVN). Changes in the transcription ot~the oaytocin gene that were governed by the treatment were quantified by densitometric nteasuremerrts of the specific hybridizing signals within the defined anatomic structure.
Figure 3 shows the induction of oxytocin-codin'; transcripts (OT mRNA; upper drawing) in the hypothalamic paraventricular nucleus of ovariecton~ized rats at'ter chronic subcutaneous treatment with 17b-estradiol (circle symbol), 17a-estradiol (square symbol), and I5(3H,3'H-cycloprop[14, I SJ-estra-1,3,5(10),8-tetraene-3,17x-diol ~trian~le 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 xtSENI of ~-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).
The results show that 15[3H,3'H-cycloprop[14, l5]-estra-1,3,5( 10),8-tetraene-3,17a-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 17b-estradiol. At all events, the neurotropic transcription effects of 15~3H,3'H-cycloprop[ 1=(,1 s]-estra-1, 3,~( 10),S-tetraene-3,17a-diol, unlike that of 17b-estradiol, are not associated W1L11 e11I11~!~~illellt of the uterus. .fit the doses that were used, the 17a-estradiol had no affect on the concentrations of oxytocin mRNA
in the hypothalamic PVN.
These results document a selective, estrogen-like etlect of l ~(3H, 3'1-1-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-3,17a-diol in the brains of female rats.
Example =1 Stimulation of the transcription of the antiapoptotic 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 -? 0-and acts counter to the programmed cell death (apoptosis) (Merry, D E., Korsmeyer, S.J., "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, Nl. 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 gyros is a component of the hippocampal formation in which the neurogenesis persists in the rat, even at a mature age (could, h , et al., "Proliferation of granule cell precursors in the dentate gyros of adult monkeys is diminished by stress," I'rc» ;'~atl Acad. Sci. USA 96: pp.
3168-317, 1998) and bcl-2 is expressed. Bcl-? transcripts were shown in brain sections by in-.sitcr hybridization with a specific oligonucleotide probe (Clark R.S.B., 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 Figure 4 shows the effect of three different doses ot~ 17b-estradiol (circle symbol), 17a-estradiol (square symbol), and 15~3H,3'H-cycloprop[ I ~, I 5]-estra- I ,3, 5( I 0),8-tetraene-3,17a-diol (triangle symbol) on the expression of bcl-2 in the dentate ~;vrus ot~ the hippocamp~rs of ovariectomized rats; the symbols and abbreviations used are the sane as those in Fi~~ure s _2 I _ Treatment with S~H,3'H-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-3,17~G-diol resulted in a 25 dose-dependent stimulation bcl-2 expression in the dentate gyros. The effect was the same as that mediated by the same dose of 17b-estradiol. At the dose rates that were used, 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, 15~H,3'H-cycloprop[14,15]-estra-1,3,5(10),8-3o tetraene-3,17~G-diol affects the transcription of the antiapoptotic gen bcl-2 in the central nervous system according to an estrogen-like mode, without any effect occurring at the uterus.
Example 5 Dissociated induction of oxytocin receptors in the brain and myometrium Binding sites with identical biochemical characteristics for tle peptide hormone oxytocin are present in the myometrium and in the central nervous system. 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 amygdaloid nuclear complex. Estrogen-mediated induction of oxytocin receptors in these structures is causally - ~z -S
n, ~ v related to the formation of prosocial behaviour patterns, includin~~ sexual behaviour (Inset, T.R., "Oxytocin - a neuropeptide for affiliation: evidence from behavioral, receptor autoradiographic, and comparative studies," Psychone~~roet~c%crvrrolcyl~.~ 17: pp. 3-35, 1992) In order to determine the density of oxytocin receptors in defined anatomical structures, the method of choice is autoradiographic representation by binding of the radioactively marked oxytocin receptor antagonists d(CH,);-Tyr(Me)-', Thr~, Orn~-['wl]Tyn'-vasotocin (r''l-OVTA) (Kremarik, P., et al., "Histoautoradiographic detection of oxytocin and vasopressin binding sites in the telencephalon of the rat," J.Comp.Neurol. _>;s pp 34 3-3s9, 199x).
Frozen sections of the brain and uterus of ovariectomized rats that Itad received a daily subcutaneous dose of 1 ~tg of 15~3H,3'H-cycloprop[ I-l, I s]-~stra- I,s,~( 10),8-tetraene-3,17a-diol, 17b-estradiol, or 17a-estradiol for a period of seven daw (:oml~arc Example 3) were incubated with r''51-OVTA (N EN DuPont, Germany) at at i;Oll~eltti'~IIOn Uf ~0 pNl Subsequently, filmautoradiograms were made, and these were then used for densitometric determination of the oxytocin binding sites, using an established method ( fatchev, V. K , et al., "Oxytocin binding sites in rat limbic and hypothalamic structures: site specilic modulation by adrenal and gonadal steroids," Nc.~rrro.scie~~ce 57: pp. 537-X43, 199;) _; _ , S
Figure 5 shows the specific binding of a'''1-marked li~and of the oxytocin receptor (''-Sl-OVT) in the myometrium and in two estrogen-sensitive brain structures, the hypothalamic ventromedial nucleus (VMN) and the nucleus interstitialis striae terminalis (BLAST), after a seven-day treatment with 17b-estradiol (black columns), 1?a-estradiol (hatched columns), and 15~3H,3'H-cycloprop[14,15]-estra-1,3,~(10),S-tetraene-3,17x-dial (grey columns), at a daily dose rate of 1 pg. The star symbols indicate significant <lifterences (p~=0 05) as compared to placebo-treated ovariectomized rats (OVX). The right-I~ami ~Irawirn; ~huw~ tlw etlects of the substances that were tested on the proliferation of the endometrium Each column represents xtSEM of 4-5 individual determinations.
The results of this test are also shown in Figure 5. ~freatmer~t with 17b-estradiol and 15(3H,3'H-cycloprop[14,1 S]-estra-1,3,5(10),8-tetraene-3,17x-diol resulted in a significant increase in the density of oxytocin receptors in all the brain structures that were examined, with 15~3H,3'H-cycloprop[14,15]-estra-1,3,x(10),8-tetraene- 3, I ?a-dial eYhibitin'; a weaker et~ect than 17b-estradiol in the VMN . At the doses that were administered. floe 17a-estradiol was not effective in any brain structures. In the myometrium, 17b-~stradiol caused a struper induction of oxytocin binding sites, whereas 1?a-estradiol and I5(3H,3'H-cycloprop[14,15)-extra-1,3,500),8-tetraene-3,17x-diol exhibited a significantly smaller et~tect ~('l~e computer-aided measurement of the WO 99/:2108 PCT/DE99/00353 strength of the endometrium in the uterus pl~~p'dl~iltlOlls ~110VVt;d that the daily dose of l~tg 17b-estradiol caused a significant endometrial proliferation, whereas at an equivalent dose 15~3H,3'H-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-3,17x-diol and l 7a-estradiol did not influence the thickness of the endometrium.
In summary, the results presented in Example C~ shuvv that I ~(3l-l, 3'I-1-cycloprop[ 14,15]-estra-1,3,5(10),8-tetraene-3,17a-diol influences a biochemical parameter--the oxytocin receptor--that is characteristic for the reproductive system (myometrium) atld for the central nervous system, mainly in the central nervous system, and that because ot'this selectise neurotropic effect it differs qualitatively from the natural estro~ens 17b-estradical amt ! 7a-estractiul Example b The effects on cognitive funi:tions utter cl~r~niv treatment It is known that a reduction of estrogen concentrations is associated with diminished learning and memory functions (Kopera, H., "Estrogen and psychic functions. Aging and estrogens," Front Hormone Res. ?: pp. I 18-133, 1973).
;-A correlation between serum-estrogen level and cognitive performance has also been demonstrated in the animal-test model (Kondo, 1'., Suzuki, I<., Sakun~a, Y., "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 15(3H,3'H-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-~,17a-diol, 17b-estradiol, and 17a-estra-diol on cognitive performance:
Sexually mature female Wistar rats (weight ?40t?0 ~) were ovariectomized when under Nembutal narcosis. One week after the operation the follov~ ip; substance were administered subcutaneouslv .at the dose rates shown: 17b-mtracliol, I u~" 17a-eatradiol, 100 ltg; 15~3H,3'H-cycloprop .[14, I 5]-estra-1,3,5(10),8-tetraene-3,17x-diol, s0 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 G., et al., "Influence of the estrous cycle, ovariectomy anct estradiol replacement upon the acquisition of conditioned avoidance responses in rats," Physiol. E3ehav. 4C~: pp. ;97--t0 I
, 1989). Each animal was exposed SO times in each session to a combination of an unconditioned response (electric t,_ t 4 shock) and two conditioned stimuli (light and sound si',nal). Retention of the learned behaviour pattern was tested on the seventh day of the treatment .~lt'ter n 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 lh~: COndIClolltn~
signals) out of 50 sequential exposures was used as the criterion for evaluating the retention or extinction ofthe learned behaviour.
Figure 6 shows the effect of 17b-estradiol (black columns), 17a-estradiol (shaded columns), and 15(3H,3'H-cycloprop[14,15]-estra-1,3,x(10),8-tetraene-s,17a-ciiol (rev columns) on the acquisition and retentionof a new behaviour })attel~n I11 ()1%a i'1(:CTOI111Zf;d rats (open columns; OVX).
The star symbols indicate significant differences to the placebo-treated animals (OVX) on the corresponding test day. The following uterus ~vei'~hts (xtSEM; n = 8-10 per treatment group;
data in m<r 100 g body weight were determined utter fourteen days of treatment; OVX, 5312;
17b-estradiol 18719; 17a-estradiol, IOOy~; s(31-i,:~'11-c~cloprol~( I-l, l ~J-estra-1,3,500),8-tetraene-3,17a-diol, 10814.
It is clear that at the dose rate that was used, the 5(3f-l,s'H-cycloprop[ 14, I S]-estra-1,3,5(10),8-tetraene-3,17x-diol has an estrogen-like stimulating et~ect on the retention of the behaviour pattern that was learned, the uterotropic effect being ~i~nificantly less than the effect of 17b-estradiol at a daily dose rate of 1 ug. This result indicates that 5(3H, ~'H-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-3,17x-diol affects cognitive performance like an estrogen, whereas it exhibits a smaller proliferative effect in the reproductive or<,ans Example 7 Biotransformation of 17a-hydroxy-14, I sa-methylene-extra-a,5 ( IOj,S-tetraene-3-of and 17a-estradiol to 17b-estradiol A daily dose of 100 ug 17a-estradiol, 30 ~y ~(3(-I,s'H-cycloprop[ 14,1 S]-estra-1,3,5(l0),8-tetraene-3,17x-diol, or 1 pg 17b-estradiol was administered to ovariectomized rats for seven days (compare Example 6). On the last day of treatment, the serum concentrations of 17b-estradiol in the three groups was determined and compared to the concentrations in controls that had been treated with the vehicle.
S_ Figure 7 shows the serum values for 17b-estradiol after seven days of subcutaneous treatment of ovariectomized rats with 17b-estradiol (black columns), 17a-estradiul (shaded columns) and 5(3H,3'H-cycloprop[ 14,15]-estra-t,3,5( l0),S-tetraene-s, l 7cc-cliol (;rev columns) at the cited dose rates. The star symbols indicate significant ditierences compared to the values measures in placebo-treated animals; the latter were below the minimum detection limit for the methods; each treatment group comprises seven animals.
It is clear that after the application of l7b-estradiul and 17a-estradiol at the dose rates discussed, measurable concentrations of 17b-estradiol are registered in the serum.
Chronic subcutaneous treatment with 5(3H,3'H-cycloprop[14,15]-estra-I, ~,5( 10),x-tetraene-s,17a-diol causes no increase of the endogenous 17b-estradiol Thi, rewlt imlicttes that thmbserved pharmacological effects after administration of 5~3H,3'H-cycluhr:yj i-J. I ~)-mtru-I,.~.~( It>).5-tetraene-3,17a-diol cannot be attributed to a biotransformation ol'the substance to I 7b-estraciiol.
-
Claims (4)
1. Use of steroids of the general formula I
R1 is a hydrogen atom, a hydroxyl group, or an alkyloxy group of 1-5 carbon atoms, R2 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 SO2NR10R11, with R10, and R11 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, R3 is a hydrogen atom or a hydroxyl group.
R4 is a hydrogen atom, a hydroxyl group, or an alkyl group of up to 5 carbon atoms, R5 and R6 are in each instance and independently of each other a hydrogen atom or a halogen atom, R7 is a hydrogen atom or a methyl group, R8 is a hydrogen atom and a hydroxyl group, an oxo group or a grouping of general formula CR12R13, in which the R12 and the R13 are in each instance and independently of each other a hydrogen atom or a halogen atom, R9 is a methyl or ethyl group, Z is a C,C double bond or a substituted or unsubstituted cyclolpropane ring and the >CR5R6 is either in the .alpha. or .beta. position, R7 being in the .beta. position, if >CR5R6 is in the .alpha. 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.
R1 is a hydrogen atom, a hydroxyl group, or an alkyloxy group of 1-5 carbon atoms, R2 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 SO2NR10R11, with R10, and R11 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, R3 is a hydrogen atom or a hydroxyl group.
R4 is a hydrogen atom, a hydroxyl group, or an alkyl group of up to 5 carbon atoms, R5 and R6 are in each instance and independently of each other a hydrogen atom or a halogen atom, R7 is a hydrogen atom or a methyl group, R8 is a hydrogen atom and a hydroxyl group, an oxo group or a grouping of general formula CR12R13, in which the R12 and the R13 are in each instance and independently of each other a hydrogen atom or a halogen atom, R9 is a methyl or ethyl group, Z is a C,C double bond or a substituted or unsubstituted cyclolpropane ring and the >CR5R6 is either in the .alpha. or .beta. position, R7 being in the .beta. position, if >CR5R6 is in the .alpha. 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.
2. Use of steroids as defined in Claim 1, these steroids being:
15.beta.H,3'H-cycloprop[14,15]-estra-1,3,5(10), S-tetraene-3,17.alpha.-diol, 15.beta.H,s'H-cycloprop[14,15]-18a-homo-estra-1,3,5(10),8-tetraene-3,17.alpha.-ol, 17a-hydroxy-15.beta.H,3'H-cycloprop[14,15]-extra-1,3,5(10),8-tetraene-3-yl-pentanoate, 17-methylene-15.beta.H,3'H-cycloprop[14, 15]-estra-1,3,5(10),8-tetraene-3-ol, 15.beta.H,3'H-3',3'-difluoro-cycloprop[14, 15]-estra-1,3,5(10),8-tetraene3, 17.alpha.-diol, 17-methylene-15.beta.H,3'H-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-3-yl-sulfamate 17-difluoromethylene-15.beta.H.3'H-cycloprop[14, 15]-extra-1,3,5(10),8-tetraene-3-ol,
15.beta.H,3'H-cycloprop[14,15]-estra-1,3,5(10), S-tetraene-3,17.alpha.-diol, 15.beta.H,s'H-cycloprop[14,15]-18a-homo-estra-1,3,5(10),8-tetraene-3,17.alpha.-ol, 17a-hydroxy-15.beta.H,3'H-cycloprop[14,15]-extra-1,3,5(10),8-tetraene-3-yl-pentanoate, 17-methylene-15.beta.H,3'H-cycloprop[14, 15]-estra-1,3,5(10),8-tetraene-3-ol, 15.beta.H,3'H-3',3'-difluoro-cycloprop[14, 15]-estra-1,3,5(10),8-tetraene3, 17.alpha.-diol, 17-methylene-15.beta.H,3'H-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-3-yl-sulfamate 17-difluoromethylene-15.beta.H.3'H-cycloprop[14, 15]-extra-1,3,5(10),8-tetraene-3-ol,
3-methoxy-15.beta.-methyl-3'H-cycloprop[14, 15]-estra-1,3,5(10) 8-tetraene-3-ol, 15.alpha.-methyl-3'H-cycloprop[14,15]-estra-1,3,5(10),8-tetraene-3,17.alpha.-diol, 17-difluoromethylene-15.beta.H 3'H-cycloprop[14, 15]-extra-1,3,5(10),8-tetraenee-3-yl-(tetramethyleneimino) sulfonate, 17-methylene-3'H-cycloprop[8,9]-15.beta.H, 3'H-cycloprop[14,15]-extra-1,3,5(10)-triene-3-ol.
Use of steroids as defined in Claim 1 and Claim 2, for producing pharmaceutical preparations for prophylaxis and therapy 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), psychogenic inhibition of sexual behaviour.
Keys to diagrams:
Figure 1:
1 prototype substance 2 Uterus weight (mg/100g) 3 Dose (µg/day s.c.) Figure 2:
1 prototype substance 2 Luciferase induction (% of basal) 3 Dose (log M) Figure 3:
Top:
1 Hybridizing signal (µCi/g) Bottom:
1 Prototype substance 2 Uterus weight 3 Dose (µg/day s.c.) Figure 4:
Top:
1 Hybridizing signal (µCi/g) 2 bcl-2 mRNA
Bottom:
1 Prototype substance 2 Uterus weight 3 Dose (µg/day s.c.) Figure 5:
Left:
1 125l-OVT binding (µCi/g) 2 Prototype substance Right:
1 Signal-positive particle Figure 6:
1 Correct reactions (of 50) 2 17.beta.-estradiol (1 µg/day) 3 17.alpha.-estradiol (100µg/day)
Use of steroids as defined in Claim 1 and Claim 2, for producing pharmaceutical preparations for prophylaxis and therapy 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), psychogenic inhibition of sexual behaviour.
Keys to diagrams:
Figure 1:
1 prototype substance 2 Uterus weight (mg/100g) 3 Dose (µg/day s.c.) Figure 2:
1 prototype substance 2 Luciferase induction (% of basal) 3 Dose (log M) Figure 3:
Top:
1 Hybridizing signal (µCi/g) Bottom:
1 Prototype substance 2 Uterus weight 3 Dose (µg/day s.c.) Figure 4:
Top:
1 Hybridizing signal (µCi/g) 2 bcl-2 mRNA
Bottom:
1 Prototype substance 2 Uterus weight 3 Dose (µg/day s.c.) Figure 5:
Left:
1 125l-OVT binding (µCi/g) 2 Prototype substance Right:
1 Signal-positive particle Figure 6:
1 Correct reactions (of 50) 2 17.beta.-estradiol (1 µg/day) 3 17.alpha.-estradiol (100µg/day)
4 Extinction Figure 7:
1 Estradiol (pg/ml) 2 17.beta.-estradiol (1 µg/day) 3 17.alpha.-estradiol (100µg/day) 4 Prototype substance (30 µg/day) Test detection limit
1 Estradiol (pg/ml) 2 17.beta.-estradiol (1 µg/day) 3 17.alpha.-estradiol (100µg/day) 4 Prototype substance (30 µg/day) Test detection limit
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19807264.3 | 1998-02-20 | ||
| DE19807264 | 1998-02-20 | ||
| DE1998121831 DE19821831A1 (en) | 1998-05-15 | 1998-05-15 | Selectively supplementing estrogen deficiency in the central nervous system, using 14,15-cyclopropano estrane steroid compounds |
| DE19821831.1 | 1998-05-15 | ||
| PCT/DE1999/000353 WO1999042108A1 (en) | 1998-02-20 | 1999-02-10 | Pharmaceutical preparations for selectively supplementing oestrogen deficiency in the central nervous system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2321498A1 true CA2321498A1 (en) | 1999-08-26 |
Family
ID=26044035
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002321498A Abandoned CA2321498A1 (en) | 1998-02-20 | 1999-02-10 | Pharmaceutical preparations for the selectively supplementing oestrogen deficiency in the central nervous system |
Country Status (24)
| Country | Link |
|---|---|
| EP (1) | EP1056460B1 (en) |
| JP (1) | JP2002503694A (en) |
| KR (1) | KR20010041107A (en) |
| CN (1) | CN1291102A (en) |
| AT (1) | ATE252388T1 (en) |
| AU (1) | AU739071C (en) |
| BG (1) | BG104689A (en) |
| BR (1) | BR9909205A (en) |
| CA (1) | CA2321498A1 (en) |
| CZ (1) | CZ291764B6 (en) |
| DE (1) | DE59907446D1 (en) |
| EA (1) | EA002707B1 (en) |
| EE (1) | EE03906B1 (en) |
| HU (1) | HUP0100742A3 (en) |
| ID (1) | ID27069A (en) |
| IL (1) | IL137727A0 (en) |
| IS (1) | IS5571A (en) |
| NO (1) | NO20004153L (en) |
| NZ (1) | NZ506049A (en) |
| PL (1) | PL342313A1 (en) |
| SK (1) | SK12152000A3 (en) |
| TR (1) | TR200002400T2 (en) |
| UA (1) | UA57830C2 (en) |
| WO (1) | WO1999042108A1 (en) |
Families Citing this family (2)
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|---|---|---|---|---|
| ES2253318T3 (en) | 2001-11-07 | 2006-06-01 | Schering Ag | IN VITRO LIGAND CROSSING OF THE STROGEN RECEIVER. |
| CN112189624A (en) * | 2020-09-08 | 2021-01-08 | 山东第一医科大学(山东省医学科学院) | Method for constructing and identifying female AD model by using receptor gene silencing technology |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4791099A (en) * | 1984-10-29 | 1988-12-13 | Chaovanee Aroonsakul | Method of treatment for central nervous system diseases such as Alzheimer's's disease |
| US4897389A (en) * | 1984-10-29 | 1990-01-30 | Chaovanee Aroonsakul | Treating central nervous system diseases |
| DE4239946C2 (en) * | 1992-11-27 | 2001-09-13 | Jenapharm Gmbh | Estrane derivatives with a 14alpha, 15alpha-methylene group and process for their preparation |
| US5554601A (en) * | 1993-11-05 | 1996-09-10 | University Of Florida | Methods for neuroprotection |
| DE4338314C1 (en) * | 1993-11-10 | 1995-03-30 | Jenapharm Gmbh | Pharmaceutical preparations for the prophylaxis and therapy of radical-mediated cell damage |
| DE4429397C2 (en) * | 1994-08-09 | 2003-11-20 | Jenapharm Gmbh | Estra-1,3,5 (10) -triene derivatives, processes for their preparation and pharmaceutical compositions containing these compounds |
| DE19524937A1 (en) * | 1995-07-08 | 1997-01-09 | Jenapharm Gmbh | Pharmaceutical preparations for the prophylaxis and therapy of radical-mediated cell damage and for drug substitution in men |
| JPH11510144A (en) * | 1995-07-24 | 1999-09-07 | ユニバーシティー・オブ・フロリダ・リサーチ・ファンデーション・インコーポレーテッド | Use of non-estrogenic polycyclic phenolic compounds for the manufacture of a medicament for imparting neuroprotection to cells |
-
1999
- 1999-02-10 PL PL99342313A patent/PL342313A1/en unknown
- 1999-02-10 CZ CZ20002938A patent/CZ291764B6/en not_active IP Right Cessation
- 1999-02-10 SK SK1215-2000A patent/SK12152000A3/en unknown
- 1999-02-10 EP EP99910118A patent/EP1056460B1/en not_active Expired - Lifetime
- 1999-02-10 ID IDW20001592A patent/ID27069A/en unknown
- 1999-02-10 EE EEP200000477A patent/EE03906B1/en not_active IP Right Cessation
- 1999-02-10 TR TR2000/02400T patent/TR200002400T2/en unknown
- 1999-02-10 NZ NZ506049A patent/NZ506049A/en unknown
- 1999-02-10 KR KR1020007009148A patent/KR20010041107A/en not_active Application Discontinuation
- 1999-02-10 DE DE59907446T patent/DE59907446D1/en not_active Expired - Fee Related
- 1999-02-10 HU HU0100742A patent/HUP0100742A3/en unknown
- 1999-02-10 CA CA002321498A patent/CA2321498A1/en not_active Abandoned
- 1999-02-10 IL IL13772799A patent/IL137727A0/en unknown
- 1999-02-10 JP JP2000532123A patent/JP2002503694A/en not_active Abandoned
- 1999-02-10 WO PCT/DE1999/000353 patent/WO1999042108A1/en not_active Application Discontinuation
- 1999-02-10 AU AU29209/99A patent/AU739071C/en not_active Ceased
- 1999-02-10 EA EA200000858A patent/EA002707B1/en not_active IP Right Cessation
- 1999-02-10 CN CN99803170A patent/CN1291102A/en active Pending
- 1999-02-10 BR BR9909205-0A patent/BR9909205A/en not_active IP Right Cessation
- 1999-02-10 AT AT99910118T patent/ATE252388T1/en not_active IP Right Cessation
- 1999-10-02 UA UA2000095399A patent/UA57830C2/en unknown
-
2000
- 2000-07-24 IS IS5571A patent/IS5571A/en unknown
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- 2000-08-18 NO NO20004153A patent/NO20004153L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| SK12152000A3 (en) | 2001-02-12 |
| CN1291102A (en) | 2001-04-11 |
| AU739071C (en) | 2002-04-11 |
| EP1056460A1 (en) | 2000-12-06 |
| JP2002503694A (en) | 2002-02-05 |
| IS5571A (en) | 2000-07-24 |
| BG104689A (en) | 2001-04-30 |
| UA57830C2 (en) | 2003-07-15 |
| NO20004153D0 (en) | 2000-08-18 |
| HUP0100742A2 (en) | 2001-09-28 |
| TR200002400T2 (en) | 2000-11-21 |
| PL342313A1 (en) | 2001-06-04 |
| ATE252388T1 (en) | 2003-11-15 |
| KR20010041107A (en) | 2001-05-15 |
| NZ506049A (en) | 2002-09-27 |
| BR9909205A (en) | 2000-11-14 |
| EP1056460B1 (en) | 2003-10-22 |
| NO20004153L (en) | 2000-10-09 |
| HUP0100742A3 (en) | 2002-07-29 |
| EE03906B1 (en) | 2002-12-16 |
| DE59907446D1 (en) | 2003-11-27 |
| EA002707B1 (en) | 2002-08-29 |
| IL137727A0 (en) | 2001-10-31 |
| EA200000858A1 (en) | 2001-04-23 |
| CZ291764B6 (en) | 2003-05-14 |
| AU739071B2 (en) | 2001-10-04 |
| EE200000477A (en) | 2001-12-17 |
| AU2920999A (en) | 1999-09-06 |
| CZ20002938A3 (en) | 2001-01-17 |
| ID27069A (en) | 2001-02-22 |
| WO1999042108A1 (en) | 1999-08-26 |
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