Note: Descriptions are shown in the official language in which they were submitted.
CA 02321498 2000-08-18
i
,
WO 99/42108 PCT/DE99/00353
Pharmaceutical Preparations for the Selectively Supplementing Estrogen
Deficiency in the Central Nervous System
The present invention relates to the use of selected steroids to produce
pharmaceutical
preparations for selectively supplementing estrogen deficiencies in the
central nervous system
without affecting other organs or systems.
These steroids are characterized in that, unlike systemically 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
CA 02321498 2000-08-18
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).
CA 02321498 2000-08-18
t
WO 99142108 PCT/DE99/00353
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
-, -
____ _1_..___. ...............
CA 02321498 2000-08-18
s
s
WO 99142108 PCT/DE99100353
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_
CA 02321498 2000-08-18
WO 99/42108 PCT/DE99/00333
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
_;_
CA 02321498 2000-08-18
' 4
s
WO 99142108 FCT/DE99/00353
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_
CA 02321498 2000-08-18
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_
CA 02321498 2000-08-18
a
WO 99/42108 PCT/DE99/00353
The use of natural and synthetic estrogens that have a systemic effect--i.e.,
in all the body's organs
and systems--in order to treat neurodegenerative diseases is claimed by the
following patents:
US 4,897,389, US 5,554,601, and WO 95/12402, WO 97/036b1, 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_
CA 02321498 2000-08-18
WO 99/42108 PCT/DE99/00353
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
-9-
CA 02321498 2000-08-18
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.
-10-
CA 02321498 2000-08-18
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
CA 02321498 2000-08-18
WO 99/42108 PCT/DE99/00353
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
CA 02321498 2000-08-18
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 -
CA 02321498 2000-08-18
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-
CA 02321498 2000-08-18
WO 99/42108 PCT/DE99/00353
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-
CA 02321498 2000-08-18
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-
CA 02321498 2000-08-18
WO 99/42108 PCT/DE99/00353
MCF-7/2A breast cancer cells that express the alpha-isoform of the estrogen
receptor (Era) were
stably transfixed with the EREwtcLUC reporter plasmid. The reporter contains
the estrogen
response element (ERE) 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_
CA 02321498 2000-08-18
WO 99/42108 PCTIDE99100353
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_
CA 02321498 2000-08-18
WO 99/42108 PCT/DE99/00353
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).
-19-
CA 02321498 2000-08-18
WO 99142108 PCT/DE99100353
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-
CA 02321498 2000-08-18
WO 99/12108 PCT/DE99/00353
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 _
CA 02321498 2000-08-18
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 -
CA 02321498 2000-08-18
S
n, ~ v
W0 99/42108 PCT/DE99100353
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;)
_; _
CA 02321498 2000-08-18
, S
WO 99/42108 PCT/DE99/00353
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
CA 02321498 2000-08-18
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).
;-
CA 02321498 2000-08-18
WO 99/42108 PCT/DE99/00353
A correlation between serum-estrogen level and cognitive performance has also
been
demonstrated in the animal-test model (Kondo, 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,_
CA 02321498 2000-08-18
t 4
WO 99142108 PCT/DE99/00353
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.
CA 02321498 2000-08-18
WO 99142108 PCT/DE91/00353
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_
CA 02321498 2000-08-18
WO 99/42108 PCTIDE99100353
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.
-
