U.S. patent application number 11/518516 was filed with the patent office on 2007-06-28 for substances and agents for positively influencing collagen.
Invention is credited to Alfred Schmidt, Heinrich Wieland.
Application Number | 20070148123 11/518516 |
Document ID | / |
Family ID | 7918298 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070148123 |
Kind Code |
A1 |
Wieland; Heinrich ; et
al. |
June 28, 2007 |
Substances and agents for positively influencing collagen
Abstract
The invention relates to the use of a substance and/or
substances or a composition containing said substance and/or
substances, whereby said substance and/or substances inhibit(s) the
formation and/or the effect of estrogen to stabilize, increase
and/or restore collagen. The appropriate substances are aromatase
inhibitors and/or anti-estrogens. Optionally, 5-alpha-reductase
inhibitors are also used.
Inventors: |
Wieland; Heinrich; (St.
Peter, DE) ; Schmidt; Alfred; (Hamburg, DE) |
Correspondence
Address: |
CHRISTENSEN, O'CONNOR, JOHNSON, KINDNESS, PLLC
1420 FIFTH AVENUE
SUITE 2800
SEATTLE
WA
98101-2347
US
|
Family ID: |
7918298 |
Appl. No.: |
11/518516 |
Filed: |
September 7, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10049968 |
Feb 13, 2002 |
|
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PCT/EP00/07315 |
Jul 28, 2000 |
|
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11518516 |
Sep 7, 2006 |
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Current U.S.
Class: |
424/74 ;
424/757 |
Current CPC
Class: |
A61K 36/48 20130101;
A61K 31/5685 20130101; A61P 13/12 20180101; A61Q 19/08 20130101;
A61K 8/9789 20170801; A61K 31/00 20130101; A61K 2800/782 20130101;
A61P 35/00 20180101; A61Q 19/00 20130101; A61P 17/00 20180101; A61Q
7/02 20130101; A61P 9/10 20180101; A61P 19/10 20180101 |
Class at
Publication: |
424/074 ;
424/757 |
International
Class: |
A61K 36/48 20060101
A61K036/48; A61K 8/97 20060101 A61K008/97 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 13, 1999 |
DE |
199 38 421.5 |
Claims
1. Use of a substance which inhibits the production and/or the
effect of estrogens, or a composition containing said substance,
for the stabilization, the increase and/or the restoration of
collagen.
2. Use according to claim 1, characterized in that the substance is
selected from the group consisting of aromatase inhibitors and
anti-estrogens.
3. Use according to claim 1, characterized in that the substance is
an aromatase inhibitor.
4. Use according to claim 1, characterized in that the substance is
derived from soya glycine.
5. Use according to claim 4, characterized in that the substance
from soya glycine had been treated by oxidation.
6. Use according to any of the preceding claims, characterized in
that the substance is contained in the composition in an amount of
0.001 to 5 wt.-%, relative to the total amount of the
composition.
7. Use according to any one of the preceding claims, characterized
in that the composition comprises both an aromatase inhibitor and
an anti-estrogen.
8. Use according to any one of the preceding claims, characterized
in that an inhibition of the production and/or the effect of
dihydrotestosterone is performed at the same time or in
addition.
9. Use according to claim 8, characterized in that an aromatase
inhibitor is combined with a 5-alpha-reductase inhibitor.
10. Use according to claim 7, characterized in that a substance is
used which has both an inhibitory effect on aromatase and on
5-alpha-reductase.
11. Use according to any one of the preceding claims, characterized
in that a local application is performed.
12. Use according to any one of the preceding claims, characterized
in that it serves for cosmetic purposes.
13. Use according to any one of the preceding claims, characterized
in that it serves for the stabilization, the increase and/or the
restoration of collagen in one or more of the following body
regions: skin, ligaments, fasciae, tendons, cartilages, bones,
dentine and the vessel walls of arteries, veins and urinary
passageways.
14. Use according to any one of the preceding claims, characterized
in that the substance is contained in a pharmaceutical formulation
which is suitable for the topical application, for injection, for
inhalation or for transdermal therapy.
15. Use of a substance which inhibits the production and/or the
effect of estrogens, or a composition containing said substance,
for the prophylaxis of myocardial infarction and brain
infarction.
16. Use of a substance which inhibits the production and/or the
effect of estrogens, or a composition containing said substance,
for the prophylaxis or therapy of osteoporosis.
17. Use of a substance which inhibits the production and/or the
effect of estrogens, or a composition containing said substance,
for the prophylaxis or therapy of arteriosclerosis.
18. Use of a substance which inhibits the production and/or the
effect of estrogens, or a composition containing said substance,
for the prophylaxis or therapy of urinary incontinence.
19. Use of a substance which inhibits the production and/or the
effect of estrogens, or a composition containing said substance,
for the prophylaxis or therapy in the case of excessive production
of glucocorticoids in the body, or as a therapy accompanying the
therapeutic use of glucocorticoids for the decrease of side effects
which are associated with glucocorticoids.
20. Use of a substance which inhibits the production and/or the
effect of estrogens, or a composition containing said substance,
for the decrease of hair growth in women.
21. Use of a substance which inhibits the production and/or the
effect of estrogens, or a composition containing said substance,
for the cosmetic treatment of wrinkles and/or strias of the skin as
well as atony of the upper skin.
22. Use of a substance which inhibits the production and/or the
effect of estrogens, or a composition containing said substance,
for the amelioration of sun exposure to the skin.
23. Use according to any one of claims 15 to 22, characterized in
that a substance or a composition as defined in any one of claims 2
to 10 is used.
24. Cosmetic composition for topical application, characterized in
that the composition comprises one or more substance(s) such that
the production and/or the effect of estrogens and, at the same
time, the production and/or the effect of dihydrotestosterone is
inhibited.
25. Cosmetic composition according to claim 24, characterized in
that an aromatase inhibitor is combined with a 5-alpha-reductase
inhibitor.
26. Cosmetic composition according to claim 24, characterized in
that the composition comprises a substance which has both an
inhibitory effect on aromatase and on 5-alpha-reductase.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0001] FIG. 1 shows a micrograph of a skin biopsy taken from a 43
year old women prior to treatment with the cream lotion described
in Example 3;
[0002] FIG. 2 shows a micrograph of a skin biopsy taken from the 43
year old women after 16 weeks of treatment with the cream lotion
described in Example 3;
[0003] FIG. 3 is a photograph showing the stretched strias in the
region of thighs and buttock of a 25 year old women before
treatment with the gel described in Example 4; and
[0004] FIG. 4 is a photograph showing the region of the thighs and
buttock of the 25 year old women after 10 weeks of treatment with
the gel described in Example 4.
DESCRIPTION
[0005] The present invention relates to the use of substances and
agents, which positively influence the peripheric-local, tissue- or
organ-cell specific generation of sexual hormones, and to the
therapeutic or prophylactic applications related thereto. The
present invention particularly relates to the use of such
substances and agents for positively influencing collagen. For this
purpose, applications for collagen-containing parts of the body are
contemplated, such as skin, tendons, fasciae, ligaments,
cartilages, bones, dentine, arteries and veins, urinary vessels and
other vessel walls. Extremely useful possibilities follow therefrom
for the prophylaxis and therapy of various diseases. "Positive
influencing" in the terms of the invention means essentially a
stabilization, an increase and/or a restoration of collagens or
collagen fibres.
[0006] As to the background of the invention, the context of the
peripheric local, tissue- or organ-cell specific production of
sexual hormones will be described in the following.
[0007] Testosterone is the essence of the male sexual hormones. Its
effects are exhibited via the so-called androgen receptor. Like all
steroid hormones, testosterone acts together with its
transcriptional factor (Roy 1995), which regulates the
transcription. Androgen receptors change their structure upon
binding of androgen and move to the cell nucleus towards the
corresponding genes, the expressions of which are influenced by
androgens. Testosterone itself does not bind particularly strong to
the androgen receptor, but first has to be slightly chemically
modified within the cell. The enzyme 5-alpha-reductase removes the
single double-bond in the steroid hormone molecule, and
dihydrotestosterone (DH) is made, which has a tenfold higher
affinity to the androgen receptor than testosterone (Grino, 1990).
Dihydrotestosterone may also be made from
5-alpha-dihydroandrostendione, which itself was made from
androstendione by means of 5-alpha-reductase. This route strongly
predominates e.g. in the genital skin of men and women (Stanczyk
1990).
[0008] Thus, this conversion, which occurs not before the target
organ, i.e. in the periphery, strongly enhances the
testosterone-derived effect. For example, in the skin, the effect
comprises the promotion of hair growth (except for the scalp) and
the increase of the activity of the sebaceous glands. Only cells
which have both 5-alpha-reductase-activity and androgen receptors,
are allowed to be correspondingly stimulated by testosterone in
physiological amounts. The testosterone merely originates from the
testicle and, at a minor proportion, also from the adrenal gland.
The testosterone level of men is 280 to 1100 nano grams/milli liter
and of women 15 to 70 nano grams/milli liters (estradiol: up to
0.45 nano grams/milli liter), resp.
[0009] Not only for dihydrotestosterone, but also for testosterone
itself, there is an intracellular synthesis route in the periphery
(Labrie 1995). Testosterone produced there, however, is not
delivered to the blood stream, but acts within the cell where it
was made after its conversion into DHT within the same cell. The
precursor of the testosterone produced in the periphery is released
from the adrenal gland into the blood. The precursor is
dehydroepiandrosterone (DHEA), which is present in the blood in
micro molar concentrations. Dehydroepiandrosterone is converted
into another molecule within the cell, where it has been invaded
easily through the cell membrane by simple diffusion. To this end,
the 3-beta-hydroxyl group is converted into a keto group, and the
double-bond of ring B is transferred into ring A
(3-betahydroxysteroid-dehydrogenase/isomerase, 3-beta HSD).
Androstendione is produced thereby, from which it is only a little
step to testosterone (conversion of the keto group at C-17 into a
hydroxyl group by means of 17-betahydroxysteroid-dehydrogenase).
This testosterone is then, within the same or other cells,
converted into the potent androgen DHT. Using the precursor which
is present in the blood at a relatively high concentration, the
cells which have the above-mentioned two enzymes therefore can
themselves produce, further modify or release testosterone to the
environment, where it is contacted with cells which have
5-alpha-reductase and therefore can make DHT.
[0010] The extent of conversion of DHEA into androstendione in the
peripheral tissue depends from the DHEA level on the one hand, but
also from the activity of 3-beta-HSD on the other hand. The latter
is probably stimulated by the luteotropic hormone (LH) not only in
the testicle, but also in the peripheral tissue (Venencie
1999).
[0011] Particular structures in the brain can sensitize
testosterone concentrations. When these sensitize them as being too
low, a demand is directed to the pituitary gland to the effect that
LH is more released. Because of this, the production of
androstendione or testosterone in the corresponding peripheral
tissue, such as e.g. skin, is also stimulated. If 5-alpha-reductase
is present, the androgen effects are then more exerted.
[0012] In women, the brain determines the concentration of
estrogens instead of that of androgens. A decrease of them also
leads, via LH release, to an enhancement of the peripheric
conversion of DHEA into androstendione in the correspondingly
established tissue. Besides testosterone, androstendione may also
be converted into estrone. The enzyme which is responsible therefor
is aromatase, which is not as much ubiquitous as the testosterone
producing enzyme 17-beta-HSD. Aromatase is also present in the skin
(Thiboutot, 1998, Theintz, 1989, Milevich 1988, Svenstrup 1990,
Milevich 1990, Dijkstra 1987), but also in other tissue- or
organ-specific cells. Estrone is only a weakly effective estrogen
and therefore, like testosterone, must be first converted to an
active hormone in the target cell in order to fully display its
effects. This is done by 17-beta-HSD, which converts estrone in the
much more active estradiol. The mechanism of action of estradiol on
the cellular level corresponds to that of DHT. It acts via
intracellular hormone receptors, which selectively activate as
transcription factors the corresponding genes. However, estradiol
may also be made intracellularily from androstendione not only via
estrone, but also via testosterone, which can also be converted
into an estrogen by means of aromatase. This time, it is converted
directly into an active estrogen, estradiol. Within a particular
target cell, testosterone may also be converted into a highly
active androgen or a highly active estrogen by means of a single
enzymatic catalytic step, resp., depending on which enzyme is more
active. Therefore, in order to provide hormone effects from
estrogens or androgens in the peripheral tissue, the organism needs
neither ovaries nor testicles, but only an adrenal gland (Labrie
1995, Labrie 1997). Therefore, both men and women can produce in
corresponding tissues both estradiol and testosterone. Since the
production of highly active hormones and their effects may occur
within the same cell, we speak of "intracrinology" (Labrie
1991).
[0013] Since aromatase is distributed less ubiquitary than
17-beta-HSD, it is possible that steroids which have to be
aromatized have been produced in other cells than estrone or
estradiol. Accordingly, the theca cells in the ovary are
specialized for the production of androgenic estrogen precursors,
and estradiol is produced from them in the adjacent granulosa cells
(Tamaoka 1987, Roberts 1990). This is reasonable, because in this
case only those cells, the growth of which is connected to the
growth of the follicle, release estradiol into the blood, the
concentration of which indicates the size of the follicle to the
brain. At a specific concentration of estradiol in the blood,
ovulation occurs. This is caused by a sudden increase of the
concentration of FSH in the blood which is effected by the excess
of a specific threshold concentration of estradiol in the blood.
The direct production of estrogens from DHEA takes place strongly
during pregnancy in the placenta. The essential precursor is the
sulfate form, DHEA-S. DHEA-S does not derive from the adrenal gland
of the pregnant, but from that of the fetus (Gips 1980). If the
delivery or the conversion is ineffective, a diagnostically
distinctive decrease of particular degeneration products of
estrogens (17-keto-steroids) occur in the urine. If the sulfatase
which releases DHEA is missing (in-born ichthyose, which occurs
only in boys) , there is no increase of the estrogen concentration
within the blood of the mother. The extragonadale sexual hormone
production in the skin apparently plays a role already within the
fetus. A further condition, under which a normal spontaneous birth
occurs in spite of hardly present estrogens in the mother blood, is
the inborn deficiency of aromatase. The placenta does not convert
any more androgens into estrogens, and a moustache even grows in
the mother. This may be based on a stimulation of the extragonadale
sexual hormone production within the skin of the mother, which
results from human choriongonadotropin (hCG). This increased
production is apparently responsible for the effect that at those
locations, where estradiol is necessary for a normal birth, this
production also results from the precursors. The estrogens which
are mainly produced by the placenta are biologically quite
ineffective. Their increased production is apparently based both on
a high concentration of fetal precursors in the funiculus blood and
on the stimulus of aromatase by means of the pregnancy-hormone hCG,
the beta-chain of which is almost identical with that of FSH and
therefore also displays identical biological effects. In the
pregnant woman, the peripheral production of sexual steroids is
also enhanced in the organs which have LH/hCG-receptors (or
possibly receptors for another messenger agent). Certainly, the
skin belongs to such organs (Venencie 1999, You 2000).
[0014] The object of the invention is to favourably influence the
extragonadal sexual hormone production in order that therapeutic
effects are enabled through the peripheric-local, tissue- or
organ-cell specific presence or absence of sexual hormones.
[0015] According to the present invention, it was surprisingly
found that collagen can be positively influenced in the
collagen-containing body parts concerned by using a substance which
is capable of inhibiting the production and/or the effect of
estrogens. After the application of the substance or a composition
containing this substance, the collagen is stabilized, increased
and/or restorated in the collagen-containing body parts.
[0016] With this concept, the present invention follows a
fundamentally new approach. The central issue of this concept is a
targeted intervention in the peripheric-local, tissue- or
organ-cell specific production of sexual hormones by means of
sub-stances which are particularly suitable therefor, that is
essentially those substances which inhibit the production and/or
the effect of estrogens. Since aromatase was found to be a key
enzyme in this context, aromatase inhibitors serve as particularly
suitable substances of the invention for being used according to
the present invention. The concurrent or additional inhibition of
the production of dihydrotestosterone may also be preferably
effective, particularly when applied to the skin. To this end,
preferably 5-alpha-reductase inhibitors, but also alpha-receptor
blockers are contemplated.
[0017] It was surprisingly found that said substance(s) or the
composition containing this (these) substance(s) exhibit as a
consequence of their action a positive influence on the collagen,
particularly on the content of the collagen fibres within the
collagen containing body region such as the skin, thereby rendering
these body region more tight or firm. By means of biopsies it was
found that the proportion of collagen fibres increased. It is
considered that--in fundamental contrast to the natural influence
via the estrogen level in the blood as well as in contrast to the
known estrogen replacement therapy (HRT) --a positive influence on
the collagen can be achieved at the specific target location when
the local extragonadal estrogen production and/or the local effect
of estrogens is reduced or inhibited by using the specific
substance or the composition containing this substance according to
the use of the present invention.
[0018] In a further aspect of the invention it was found that
negative influencing factors, which may disadvantageously affect
the content and stability of collagens, can be compensated at least
partially by using the substance or the composition containing this
substance, and harmful effects on the human body can be ameliorated
thereby. As such negative influencing factors, there were
identified particularly the increased release of LH, the production
of vitamin D as a result of an exposure to sun illumination, and
the excessive presence or the administration of
glucocorticoids.
[0019] As a result of the concept of the invention as mentioned
above, significant implications and useful cosmetic and therapeutic
uses are provided which are exemplified in further detail below.
For the medical applications, the suitable substances can be used,
together with the pharmaceutically acceptable additives which are
typical for the respective mode of application, for the production
of an agent or a pharmaceutical formulation and can be applied in
the therapeutic uses.
[0020] The term "estrogens" shall be construed to mean all natural,
female sexual hormones which have estrogen-like effects, such as
estradiol, estrone and estrol.
[0021] As substances which are inhibitory in terms of production
and/or effect of estrogens, particularly two classes of substances
which will be described in further detail in the following are
contemplated.
[0022] On the one hand, these are anti-estrogens, i.e. substances
which block estrogen receptors and therefore inhibit the effect of
estrogen as antagonists.
[0023] Furthermore; these are substances which may inhibit the
extragonadal production of estrogens locally. To this end,
steroidal and non-steroidal inhibitors of the
(cytochrome-p450)-aromatase are contemplated. Aromatase is the
central enzyme which catalyses the chemical conversion of the
precursor molecules (such as dehydroepiandrosterone (DHEA) and
androstendione), which derive from the adrenal gland and are
transported via the blood, into estrogens. As a consequence, the
inhibition of this enzyme leads to a local in situ inhibition of
the estrogen production. Because of their particularly beneficial
course of action, the aromatase inhibitors are preferred for the
application of the use according to the present invention.
[0024] Examples of aromatase inhibitors include the following
substances:
[0025] steroidal aromatase inhibitors: [0026]
4-hydroxyandrost-4-ene-3,17-dione (Formestan und Lentaron), [0027]
6-methylene-androstra-1,4-diene-3,17-dione (Exemestan), [0028]
10-(2-propynyl)estr-4-ene-3,17-dione (MDL 18962) [0029] 7-alpha
substituted androstendionee-derivatives [0030]
1,4,6-androstatriene-3,17-dione (ATD) [0031] 10-oxirane- and
10-thiirane substituted androgens [0032]
10-propargylestr-4-ene-3,17-dione [0033]
10-propargylestr-4-ene-3,17-propionate 10-(2-propynyl)-derivative
[0034] 13-retro-antiprogestine [0035]
14-alpha-hydroxy-4-androstene-3,6,17-trione (14 alpha-OHAT) [0036]
16- or 19-substituted androst-4-ene [0037]
19-(cyclopropylamino)-androst-4-ene-3,17-dione [0038]
19-(ethyldithio)-androst-4-ene-3,17-dione (ORG 30958) [0039]
19-oxiranyl- and 19-thiiranyl-steroids [0040] 19-thiomethyl- and
19-azido-androstenedione [0041]
1-methyl-androsta-1,4-diene-3,17-dione (Atamestan) [0042]
2,2-dimethyl-4-hydroxy-4-androstene-3,17-dione [0043]
3-alpha-methoxyandrost-4-ene-6,17-dione [0044]
3-beta-hydroxyandrost-4-ene-6-one-derivatives [0045]
3-deoxyandrogen-19-oxygenated derivatives of
3-oxo-17-beta-carboxamido steroids [0046]
4-(phenylthio)-4-androstene-3,17-dione [0047]
4-(thio-substituted)-4-androstene-3,17-dione [0048]
4-acetoxy-4-androstene-3,17-dione [0049] 4-amino-androstenedione
[0050] 4-androstene-3,6,17-trione [0051] 4-hydroxyandrostenedione
(4-OHA, CGP 32349) [0052] 4-methoxy-4-androstene-3,17-dione [0053]
4-oxygenated androst-5-en-17-one and their 7-oxo-derivatives [0054]
4-thiosubstituted derivatives of 4-androstene-3,17-dione [0055]
4-thiosubstituted-4-androstene-3,17-dion-derivatives [0056]
5-alpha-dihydro-norethindrone (a metabolite of norethindrone)
[0057] 5-alpha-reduced C19-steroids [0058]
5-alpha-androstane-17-ones with or without a carbonyl functionality
at C-3 and/or C-6 [0059] 6-alpha,7-alpha-cyclopropane derivatives
of androst-4-ene [0060] 6-alpha-fluorotestosterone [0061]
6-beta-propynyl substituted steroids [0062] 6,7-aziridinyl steroid
und related compounds [0063] 6-alkyl analogs of
delta-1,4,6-androgens [0064] 6-alkyl analogs of delta 4,6-androgens
[0065] 6-alkyl- and 6-arylandrost-4-ene-3,17-dione [0066]
6-alkylandrost-4-ene-3,17-dione of 7-alpha- and
7-beta-arylalkyl-substituted androst-4-ene-3,17-diones [0067]
6-alkylandrosta-4,6-dien-3,17-dione und their 1,4,6-triene-analogs
[0068] 6-alkyl-substituted androgens [0069]
6-phenylalkyl-substituted C19-Steroids with 1,4-diene-, 4,6-diene-
or 1,4,6-triene-structure [0070] 6-bromoandrostenedione [0071]
6-hydroxy-iminoandrostenedione [0072]
6-methylenandrosta-1,4-diene-3,17-dione (FC 24304) [0073]
6-phenylalkyl-substituted androst-4-ene-3,17-dione [0074]
6-substituted androst-4-ene-analogs [0075]
7-alpha-(4'-amino)phenylthio-4-androstene-3,17-dione [0076]
7-alpha-substituted androsta-1,4-diene-3,17-dione [0077]
7-alpha-substituted androstenedione [0078]
7-alpha-(4'-amino)phenylthio-4-androstene-3,17-dione [0079]
7-alpha-arylalkylated androsta-1,4-diene-3,17-dione [0080]
7-alpha-substituted androstenediones [0081] 7-substituted
4,6-androstadiene-3,17-dione [0082] 7-substituted steroids [0083]
androst-4-ene-3,6-dione derivatives [0084]
androst-5-ene-7,17-dione-19-nor- and
5-beta,6-beta-epoxy-derivatives [0085] A- or B-ring-substituted
derivatives of androst-4-ene-3,6,17-trione [0086] A-ring linked
steroids [0087] bromoacetoxy-4-androstene-3-one [0088]
delta-1,4,6-androgens [0089] delta-4,6-androgens [0090] epimeric
6-hydroperoxyandrostendionees [0091] estr-4-ene-3,17-dione (MDL 18
962), [0092] estr-4-ene-3,6,17-trione [0093] flavonoids [0094]
RU486
[0095] Non-steroidal aromatase inhibitors: [0096]
6-[(4-chlorophenyl)(1H-1,2,4-triazole-1-yl)-methyl]-1-methyl-1H-benzotria-
zole (Vorazol), [0097]
2,2'-[5-(1H-1,2,4-triazole-1-yl-methyl)-1,3-phenylene]bis(2-methylproprio-
nitrile) (Arimidex), [0098]
4-[1-(cyanophenyl)-1-(1,2,4-triazolyl)methyl]benzonitrile
(Letrozol), [0099]
{4-(5,6,7,8-tetrahydro-imidazo-[1,5a]-pyridine-5-yl)-benzonitrile
monohydrochloride (Fadrozol) [0100] pyridoglutethimide
(Rogletimid). [0101] aminogluthetimide [0102]
1,2-imidazolyl-methyl-cyclopentanol-derivatives [0103]
1-[(benzofurane-2-yl)phenylmethyl]-triazole and -tetrazole [0104]
1-[benzofuran-2-yl)-phenylmethyl]-imidazole (substituted) [0105]
1-(benzofurane-2-ylmethyl)imidazole of
N,N-disubstituted-5-aminopyrimidine-derivatives [0106]
1-imidazolyl(alkyl)-substituted di- and tetrahydroquinoline [0107]
1-pentyl-3-(4-aminophenyl)pyrrolidine-2,5-dione [0108]
1-phenyl-3-azabicyclo[3.1.0]hexane-2,4-dione [0109]
1-phenyl-3-azabicyclo[3.1.0]hexane-2,4-dione and analogs [0110]
3-alkylated 3-(4-aminophenyl)piperidine-2,6-dione [0111]
3-cycloalkyl-substituted 3-(4-aAminophenyl)piperidine-2,6-dione
[0112] 3-ethyl-3-(4-pyridyl)piperidine-2,6- and 5-alkylderivatives
[0113] 3-ethyl-3-(4-pyridyl)piperidine-2,6-dione-analogs [0114]
4-amino-4H-1,2,4-triazole-derivatives [0115] 4-cyclohexylaniline
[0116] aminoglutethimid [0117] benzimidazole-und
imidazole-compounds [0118] delta-1,4-bis-norcholadienic acid [0119]
delta-1-testolactone [0120] imidazole derivatives of pyrrolidonic
and piperidonic [0121] imidazolyl-1,3,5-triazines [0122] MR 20492
and MR 20494 (two indolizinone derivatives) [0123]
pyridyl-substituted indanone, indane and tetralines [0124]
s-triazine derivate SEF19 [0125] substituted pyridines [0126]
testololactone
[0127] Other aromatase inhibitors: [0128] 8-bromo-cyclic adenosine
mono-phosphate [0129] FR901537 [0130] hexamethyl melamine
derivative (SAE9) [0131] insulin sensitizers troglitazone and
ketoconazole [0132] Letrozole (CGS 20267) [0133] Mefloquin [0134]
MPV-2213ad [0135] N-n-octanoyl-nornicotine and other nor-nicotine
derivatives [0136] Org 33201 [0137] R 76713 and R 76713 [0138]
sesqui-terpene lactones [0139] SH 489 [0140] TAN-931 [0141] thyroid
hormones [0142] tobak alkaloid derivatives [0143] YM511
[0144] As to the specifications of these substances as well as
their availability, it is referred, for example, to "Rote Liste",
Editio Cantor, Aulendorf (DE) (1999).
[0145] Such aromatase inhibitors are known as such, mainly as
systemically applied therapeutic agents for the medical therapeutic
treatment of breast cancer. In this connection, it is referred to
the review article of A. M. H. Brodi in: "J. Steorid Biochem.
Molec. Biol.", Vol. 49, No. 4-6, pp. 281-287 (1994), P. E. Goss and
K. M. E. H. Gwyn in. "Journal of Clinical Oncology", Vol. 12, No.
11, pp. 2460-2470 (1994). For the determination of the aromatase
inhibition and the subsequent decrease of estrogens, it is referred
to the further literature citations which are indicated in the
mentioned review articles, see for example A. M. H. Brodi et al.
in: "J. Steroid Biochem. Molec. Biol.", Vol. 7, pp. 787-793 (1976),
and D. A. Marsh et al. in: "J. Med. Chem.", Vol. 28, pp. 788-795
(1985). [0146] Specific azole derivatives and their aromatase
inhibiting and anti-mycotic effect are further described in EP-A-0
575 210.
[0147] It was found that substances having aromatase inhibiting
properties are contained in soya glycines (INCI name according to
the Linne-system), and that these soya glycine derived aromatase
inhibitors can be used according to the present invention. These
soya glycine derived aromatase inhibitors can be readily obtained
by providing "glycine soya" (soy bean oil or soy bean extract, or
soya sterole), and subsequently isolating the component having
aromatase inhibiting effects by means of typical separation
methods, such as liquid chromatography, particularly by means of
HPLC.
[0148] It was further found that the aromatase inhibiting effect of
soya glycine may be enhanced, when the soya glycine is treated by
oxidation. The synthesis of this oxidized form derived from soya
glycines is readily carried out by means of oxidation of soya
glycine (soy bean oil or soy bean extract, or soya sterol), and the
subsequent isolation of the component with aromatase inhibiting
effect by means of typical separation methods, such as liquid
chromatography, particularly by means of HPLC. The oxidation can be
carried out by an enzymatic approach, for example according to the
method described by Y. Fujimoto et al. in: "J. Am. Chem. Soc.",
Vol. 104, pp. 4718-4720 (1982), or by a chemical approach, for
example according to the method described by P. Welzel in:
"Tetrahedron", Vol. 41, No. 20, pp. 4509-4517 (1985).
[0149] As examples for substances of the class of anti estrogens,
there are mentioned particularly the non-steroidal estrogen
antagonists Tamoxifen
(Z-2-[4-(1,2-diphenyl-1-butenyl)-phenoxy]-N,N-dimethylamine) and
aminoglutethimide (3-(4-aminophenyl)-3-ehtyl-2,6-piperidin-dione)
as well as their analogs and derivatives, for example
3-hydroxytamoxifen, 4-hydroxytamoxifen and
7-.alpha.-alkyl-sulfinyl-tamoxifen-analogs (ICI 182,780).
[0150] Concerning the specifications of these substances, their
availability as well as further suitable anti estrogens, see, for
example, "Rote Liste", Edition Cantor, Aulendorf (DE) (1999).
[0151] Likewise, these anti estrogens have been hitherto described
mainly in connection with the systemic therapeutic treatment of
breast cancer.
[0152] In order that the extragonadal, cellular production or
effect of sexual hormones can be enabled more specific and be
better adjusted, one or more of the substances described above for
the inhibition of the production and/or the effect of estrogens may
be combined with a further effect principle such that the
production and/or the effect of dihydrotestosterone is inhibited
additionally or concurrently. This is effected by the use of a
5-alpha-reductase inhibitor or an alpha-receptor blocker, wherein
the use of a 5-alpha-reductase inhibitor is particularly
preferred.
[0153] Examples of 5-alpha-reductase inhibitors, distinguished
according to the respective type, include:
[0154] Type 1 Inhibitors: [0155] LY191704 (benzochinolinone) [0156]
4,7-beta-dimethyl-4-azacholestane-3-one (MK-386) and related
4-azasteroids [0157] benzo[c]chinolizine-3-one
[0158] Type 2 Inhibitors: [0159] benzophenone- and indole
carboxylic acids
N-tert-butyl-3-oxo-4-aza-5.alpha.-androst-1-ene-17-.beta.-carboxami-
de (Finasterid)
[0160] Dual Inhibitors (Type 1 and Type 2): [0161]
3-carboxy-20-keto-steroids [0162] 6-azasteroid [0163]
4-aza-3-oxo-5-alpha-androst-1-ene-17-beta-N-aryl-6-azasteroids
[0164] FK143
[0165] Non-Steroidal Inhibitors: [0166] 4-(1-benzoyl
indole-3-yl)butyric acid [0167]
4-[3-[3-[bis(4-isobutylphenyl)methylamino]benzoyl]-1H-indole-1-yl]-butyri-
c acid [0168] benzanilide-derivates [0169]
carbamoylalkenyl-phenyloxy carboxylic acid derivates [0170]
ethyl-4-(1-methyl-2-oxopiperid-5-yl)benzoate [0171] FK143 [0172]
N,N-bis(1-methylethyl)-4-[3-(1,2-dihydro-1-methyl-2-oxopyrid-5-yl)propyl]-
benzamide [0173] phenoxybenzoic acid derivatives [0174]
carboxamide- und phenylalkyl-substituted pyridones and piperidones
[0175]
natrium-4-[2-(2,3-dimethyl-4-[1-(4-isobutylphenylethoxy]benzolamino)pheno-
xy]butyrate (ONO-3805) [0176]
(Z)-4-2-[[3-[1-(4,4'-difluorobenzhydryl)indole-5-yl]-2-pentenoyl]-amino]p-
henoxy]butyric acid (KF20405)
[0177] Steroidal Inhibitors: [0178]
17-beta-(N,N-diisopropylcarbamoyl)estra-1,3,5(10)-triene-3-sulfonic
acid [0179] 17-beta-carbamoyl-1,3,5(10)-estratriene-3)-carboxylic
acid [0180]
17-beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5-alpha-androstan-3-o-
ne) (4-MA [0181]
17-beta-N-(2-methyl-2-propyl)-carbamoyl-androst-3,5-diene-3-carboxylic
acid [0182] 3-androstene-3-carboxylic acid (steroidal acrylates),
[0183] 3-carboxy-17-beta-substituted steroid [0184]
4-aza-3-oxo-steroid family [0185] 4-hydroxy-androstenedione [0186]
4-methyl-4-aza-5-alpha-pregnan-3-one-20(S)-carboxylate [0187]
6-methylene-progesterone-, -androstene- and -androstane derivatives
[0188] Finasterid [0189] progesteron [0190]
natrium-4-methyl-3-oxo-4-aza-5-alpha-pregnane-20(S)-carboxylate
[0191] steroidal A-ring arylcarboxylic acids [0192] TZP-4238
(steroidal antiandrogen)
[0193] As an example for an alpha-receptor blocker,
R-(-)-5-{2-[2-(2-ethoxyphenoxy)ethylamino]peropyl]-2-methoxy-benzolsulfon-
amid (Tamsulosin) may be mentioned.
[0194] These substances for the inhibition of the production and/or
effect of dihydrotestosterone are also known as such, but only for
the treatment of benign prostate hyperplasy (see "Rote Liste",
Editio Cantor, Aulendorf (DE), (1999)).
[0195] As particularly suitable substances, those are contemplated
which exhibit both an inhibitory effect on aromatase and on
5-alpha-reductase. As examples for substances having this
bifunctional property, there can be mentioned the sterol
4-hydroxy-androstendionee, which is similar to androstenedione, and
its derivatives, e.g. Formestan mentioned above, or the soya
sterols.
[0196] Since, according to the present invention, the concurrent
inhibition of the production or effect of both estrogens and
dihydrotestosterone was found to be novel and significant
predominantly for topical applications to the skin and for
controlling hair growth, as will be described in further detail
below, the present invention further provides a topic composition,
i.e. a composition which is determined to be applied to the skin,
which composition comprises one or more substance(s) such that the
production and/or the effect on estrogens and, at the same time,
the production and/or the effect of dihydrotestosterone are
inhibited. Such a composition is particularly cosmetically
applicable in a topical formulation to be applied locally. This
combined effect principle may be obtained by a combination of
substances which have the property of inhibiting the production or
effect of estrogens, on the one hand, and the property of
inhibiting the production or effect of dihydrotestosterone, on the
other hand, respectively, wherein the substances having the
respective functions mentioned above are suitable. Because of an
improved controllability, a combination of an aromatase inhibitor
with a 5-alpha-reductase inhibitor is preferred in this context. To
this end, an embodiment is particularly preferred, wherein an
applied substance is bifunctional as explained above and has
properties of aromatase inhibition and 5-alpha-reductase
inhibition.
[0197] The substance or the substances described above can be
administered in typical pharmaceutical formulations. Accordingly,
as desired and as selected, the pharmaceutical formulations contain
the typical and known additives for an oral or a topical
application, for injections, for inhalations or for transdermal
therapy, respectively. A pharmaceutical formulation is preferably
designed to be suitable for a topical application or for a
transdermal therapy, for injection or for inhalation.
[0198] The content of the therapeutically effective substance for
the inhibition of the production or effect of estrogens in such
formulations is not problematic and may be adjusted according to
the respective treatment case. For example, a content of
therapeutic substance in the total composition is suitable from
0.0001 to 10 percent by weight (wt. %), preferably 0.001 to 5 wt. %
and particularly 0.3 to 2 wt. %. The further, optionally present
additives may be used in amounts which are typical for the
respective formulations. Corresponding considerations are valid for
the optional additional use of the therapeutic substance for the
inhibition of the production or effect of dihydrotestosterone.
[0199] If the different effect mechanisms for the control of
production or effect of estrogens and, optionally, the production
or effect of dihydrotestosterone shall mutually supplement and
favourably influence each other, the substances suitable therefor,
which have been described above, are used in a balanced proportion
in order to achieve a desired effect. The content proportion which
is to be applied in this combination may be adjusted to the
respective demands. Accordingly, either the substance of one type
or the substance of the other type may for example predominate,
depending on which mode of action is primarily desired. The weight
proportion of the amounts of the one to the other type of
substance, for example, lies in a range of 90/10 to 10/90,
particularly in a range of 60/40 to 40/60.
[0200] Various aspects of the present invention are based on
particular conditions in the human body, which exhibit one or more
of the following criteria: [0201] increased activity of aromatase,
[0202] increased expression or production of LH and/or hCG in the
human body, [0203] increased expression or production of
glucocorticoids in the human body, or the therapeutic
administration of glucocorticiods, and [0204] increased production
of vitamin D, for example as a consequence of exposure to the
sun.
[0205] These conditions may be present spontaneously or
permanently, or in addition inherently by means of natural or
pathologic circumstances, or may occur by means of external
effects. The potential stimulation of the enzymes which are
involved in the extragonadal sexual hormone production, including
aromatase and possibly also 5-alpha-reductase, may be seen as the
common basis for these influencing criteria. However, the use of
the substances mentioned above according to the present invention
counteracts a decrease or weakening of collagen as a cause of this
stimulation. Among the organs or tissues which contain aromatase,
there are skin, connective tissue, bones, vessel walls (even the
vena cava contains in its wall aromatase [Sasano 1999]), blood
cells (particularly macrophages), muscles, uterus, brain and
others.
[0206] Therefore, taking account of the mentioned criteria,
profiles of therapeutic uses having a high priority are brought
about which have great importance due to the high prevalence of the
underlying diseases, such as osteoporosis, varicose veins and ulcus
cruris, arteriosclerosis, myocardial infarction and urinary
incontinence. In this connection, the skin, tendons, bones and the
walls of vessels and of downstream urinary passages are of
particular importance due to their collagen contents.
[0207] Likewise, particular groups of persons may be especially
affected, e.g. postmenopausal women.
[0208] The cause for this is eventually not ascertained, but the
following may be assumed: the activity of the enzymes, which are
involved in the extragonadal production of sexual hormones, is
allowed to be stimulated by LH or hCG. Naturally, aromatase belongs
to this group of enzymes. The extragonadal production of sexual
hormones is allowed to be stimulated not only by an enhancement of
the involved enzymes via LH, but also through the provision of
higher amounts of precursors. LH-receptors are present in the
adrenal gland. These are located on steroidogenic cells, which
produce DHEA (Pabon 1996). The activity of aromatase is further
allowed to be stimulated specifically by means of glucocorticoids
(Harada 1992), which enhance the transcription of the m-RNA of
aromatase. In the cells of the skin, the stroma cells of the fatty
tissues and in osteoblasts, the promoter of exon 1, which is
expressed in a tissue specific manner, has a
glucocorticoid-responsive element, to which the glucocorticoid
receptor binds and enhances the transcription of the aromatase gene
(Zhao 1995). The postmenopausal phase is a condition with a
permanently increased concentration of LH and its releasing hormone
LHRH or GnRH, which controls the release of LH from the hypophysis.
Permanently increased LH concentration should lead to a permanently
increased local production of estradiol in tissues which may react
hereto and have aromatase. This would result in a decrease of their
contents of collagen fibers.
[0209] In the following, individual examples will be described for
particularly suitable application fields and methods of
treatment.
[0210] Skin
[0211] An essential change of the skin during pregnancy are
pregnancy strias which are interpreted as stretched strias or
streaks. Similar strias are likewise found in Morbus Cushing, which
is a condition associated with permanently increased blood levels
of glucocorticoids caused by the replacement of cyclic variations
of blood ACTH levels by an even level of the ACTH concentration. A
characteristic of the skin of pregnancy strias is a decrease of
collagen content. The gene for type I and type II collagen are
expressed only at 10% compared to normal skin (Lee 1994). Aromatase
is present in the skin both in fibroblasts and in keratinozytes
(Berkowitz 1984, Fujimoto 1986, Bisat 1989, Harada 1992, Hughes
1997, Lachgar 1999). In both cells, their expression can be
enhanced by glucocorticoids (Harada 1992, Berkowitz 1992, Ida 1991,
Svenstrup 1990, Berkowitz 1981, Hughes 1997). Accordingly, a review
article of the year 1984 concludes that glucocorticoids, but also
estradiol have collagen decreasing effects (Borel 1984). Recently,
the collagen decreasing effect of estradiols on the molecular level
could be shown directly in the mesangium cells of renal glomerula
(Neugarten 1999, Silbiger 1999, Kwan 1996).
[0212] The established scholarship opinion, however, is that the
skin is an estrogen dependent organ, wherein estrogen should not
lead to a decrease, but to an increase of collagen fibres within
the skin.
[0213] Collagen of the skin essentially is of type I. Accordingly,
M. L. Barklink et al. ("J. Appl. Physiol." 1993, 74(2), p. 727-732)
have found that the bone mass and the collagen content of the skin
decrease with increasing age. It was found that a correlation
exists between a decreased content of collagen within the skin and
the decreased estrogen level in the blood which is associated in
the menopausal phase with increasing age. D. Gruber et al ("Klin.
Wochenschrift" (Wien) 1995, 107, p. 622-625) report that the
estrogen dependent, postmenopausal decrease of the collagen tissue
can be assessed by means of sonography, and they assume that a
successful therapy is possible by the optimization of a dose for an
estrogen replacement therapy (hormone replacement therapy, HRT).
Other experts (see e.g. "Therapie" 1996, 51, p. 67-70, and
"Dermatology" 1996, 193, p. 289-294) also try to respond to the
skin aging which occurs particularly in the postmenopausal
phase.
[0214] However, it was not confirmed that estrogens may increase
the collagen content of the skin. Accordingly, implants of
estradiol lead to a significant reduction of the immature
"crosslinks" hydroxylsinorleucine (Holland 1994). The percentage of
collagen content and the proportion of major "crosslinks"
(hitidino-hydroxylsinorleucine) of the skin do not change.
[0215] The HRT (hormone replacement therapy) neither changes the
amount nor the synthesis ratio of collagens in the skin (Haapasaari
1997). It is found occasionally that the relative proportion of
collagen type III shall increase after administration of estrogen
(Savvas 1993, Schmidt 1996).
[0216] When treating collagen deficiency conditions of the outer
skin, such as e.g. anatony or slackness of outer skin, the
formation of wrinkles and stretched strias, it was found according
to the present invention that the positive influence on the
collagen is effected locally in the cutis, that is in epidermis and
corium. Thus, contrary to the official scholarship opinion, but
being founded by experimental studies, the finding is established
that estradiol has a direct local collagen decreasing effect, while
it is realized according to the present invention that the content
of collagen fibres of the skin is increased by means of a local
inhibition of aromatase activity within the skin by using the
substances described above or by corresponding measures for locally
inhibiting the production or effect of estrogens.
[0217] The topic inhibition of aromatase within the skin may be
utilized to increase the percentage of collagen fibres of the skin
locally. This is of importance when cosmetically treating wrinkles
in the face and at open-necked regions or when cosmetically
influencing pregnancy strias or stretched strias at the lower
abdomen, the thighs and the buttock (which is well visible
especially in the case of dark skin color).
[0218] A part of the substances and their ability for treating
disordered connective fatty tissue of the inner skin, such as
cellulite, or their use for smoothening and/or reducing fat cell
containing body regions are already known from WO-A-97/36570 and
WO-A-99/17712. However, the phenomena described there occur in the
inner skin fatty connective tissue of the subcutis, whereas this
skin aspect of the present invention is directed to the part of the
skin which particularly contains collagen, that is the cutis
(epidermis and corium) as well as other parts of the body which
strongly contains collagen. In particular, no hints are to be found
in these references on the surprisingly found correlation between
the inhibition of the production, on the one-hand, and the effect,
on the other hand, of local estrogens and a positive, direct
influence on the collagen locally in the cutis, which is both
anatomically and functionally different from the subcutis.
[0219] Consequently, a complete functional penetration of the skin
down to the subcutis is not necessary according to the present
invention in order to display the described effects in the cutis
itself. The positive influence of the collagen according to the
present invention is effected via the relative deficiency of
estrogens directly in the epidermis and in the corium of the cutis.
This is because the cutis of the skin is capable of forming
estrogens from androgens (Bulun 1998), because both fibroblasts
(Macdiarmid 1994, Toda 1994, Staib 1994, Jakob 1995, Isurugi 1996)
and kerationzytes (Hughes 1997) have the aromatase enzyme.
Furthermore, epidermis and corium are estrogen-dependent skin
layers and, therefore, must also have estrogen receptors (Hughes
1997 on keratinozytes, Dieudonne 1998 on fibroblasts). Since the
aromatase activity of the skin (particularly of the female skin) is
expressed constitutionally and estrogens decrease the content of
collagen fibres, the female skin constitutionally does not contain
as much collagen fibres as the male skin, which is also much
thicker. Upon a decrease of the local estrogen concentration in the
female skin, the collagen fibres are increased. Since the estrogen
concentration of the female skin is predominantly effected by means
of the local activity of aromatase, a decrease of the local
estrogen concentration is achieved by means of an inhibition of the
aromatase activity in the keratinozytes and the skin fibroblasts.
Therefore, the inhibition of the skin aromatase leads to an
increase of collagen fibres, especially those of type I, and
accordingly to an increase of the thickness and the tightness of
the skin. These phenomena are noticed by the female probands tested
within the framework of the invention and were experimentally
demonstrated after about 4 weeks treatment duration.
[0220] The topical inhibition of aromatase in the skin results in
that only the route to testosterone is open for androstendione.
Testosterone itself also cannot be aromatized to estradiol, but is
converted into the strong androgen DHT when 5-alpha-reductase is
active. The skin is rich in 4-alpha-reductase (Mestayer 1996,
Courchay, Luu-The 1994) and thereby can exhibit the typical male
appearance (Bart, sebaceous glands, thickness). Estradiol is
capable of inhibiting 5-alpha-reductase (Cassidenti 1991). Now, if
aromatase is inhibited, there is a depletion of estradiol of the
skin. The inhibition and the estradiol deficiency are two factors
which provide a remarkable predominance to 5-alpha-reductase.
Therefore, it is possible that if only aromatase is topically
inhibiting, some virilization of the skin may occur. Additionally,
testosterone already provides a half maximum inhibition of
aromatase in small amounts (6 nano mole/L) (Berkovitz 1990). By
inhibiting 5-alpha-reductase, an enrichment of the skin with
testosterone and therefore also an inhibition of the aromatase are
achieved.
[0221] It is desired that a virilization is avoided particularly in
the face. Therefore, it is very advantageous that 5-alpha-reductase
is inhibited together with the aromatase, if the collagen content
of the skin is desired to be increased locally. A lack of
inhibiting 5-alpha-reductase apparently would enable an even
further increase of the collagen content of the skin, but with the
expense of virilization. Since a local collagen increase is
produced in the skin when inhibiting both enzymes, the estrogen
depletion alone appears to be sufficient therefor. An accompanying
inhibition of both the aromatase and the 5-alpha-reductase may
occur either with corresponding soya steroles or with known drug
substances (Hsiang 1987, Brodie 1989, Brodie 1989). Both the
aromatase and the 5-alpha-reductase act at the 3=4-double bond in
ring A of the sterole core structure. It is described in the
literature that 4-hydroxyandrostendione in higher concentrations (3
micro mole/L) can also inhibit 5-alpha-reductase. The critical
concentration for aromatase lies at about 3 nano mole/L. Therefore,
e.g. soya steroles which inhibit aromatase also exhibit an
inhibiting effect on 5-alpha-reductase.
[0222] For positively influencing collagen in the cutis of the
skin, a formulation suitable for the usable substance to be applied
for the topical administration of the described therapeutic
substance(s) or the composition may be selected, e.g. an ointment,
a cream, a gel, an emulsion (lotio), a powder, an oil, etc. For
this purpose, the composition comprises additives which are typical
for the corresponding formulations as ointment, cream, gel,
emulsion, powder or oil, etc. Known as well as commercially
available, conventional skin care agents are well suitable in the
respective formulations for use in the present invention. As
typical additives for such formulations, there are, for example,
plant oils such as almond oil, olive oil, peachstone oil, peanut
oil, castor oil and the like, plant extracts, etheric oils, vitamin
oils, fatty and fat-like substances, lipoids, phosphatides,
hydrocarbons, such as paraffins, vaseline, lanoline, waxes and the
like, detergents, further skin agents such as lecithine, lanoline,
alcohols, carotin and the like, skin nutritions, perfumes, cosmetic
agents, alcohols, glycerol, glycols, urea, talcum, conservation
agents, sun protection agents, dyes such as titan white and zinc
white, antioxidants, etc. Generally, water serves as the basic
substance, thereby usually producing, with the addition of
emulsifiers such as fatty alcohol sulfate, alkali soaps, lecithine,
triethanolamine and the like, an O/W- or W/O emulsion.
[0223] Other Collagen Containing Body Regions
[0224] Based on the surprisingly found possibility of stabilizing,
increasing and/or restorating collagen in the cutis of the skin by
means of the specific substances, the invention is applicable not
only there, but also to other, particularly collagen-containing
parts of the body in terms of local target areas of application,
such as cartilages, tendons, ligaments, fasciae, vessel walls of
arteries and veins, such as varicose veins, or of urinary
passageways, collagen-containing ulcii such as ulcus cruris,
dentine, bones, the collagen capsules of arteriosclerotic plaques,
and the like.
[0225] The rupture of the anterior sacrum of the knee is a typical
injure of sportive women which affect the binding tissue. This
occurs much more often in women then in men (Powell 2000, de Loes
2000). This is particularly true for the time of ovulation (Wojtys
2000). Then, the highest LH level in the blood is also found and
therefore the highest extragonadal formation of sexual steroids in
women in the premenopausal phase. Apparently, a somewhat higher
local estrogen concentration within the ligament makes it more
susceptible to injuries. The ligaments of the joint of the knee
have estrogen receptors (Sciore 1998).
[0226] Accordingly; the substance or the composition containing
this substance may optionally be used in support of surgical
operations of such body parts having high collagen contents. The
use for cosmetic purposes is preferred, also within the framework
of cosmetic surgery. However, the substance or composition to be
used according to the present invention is also suitable as an
alternative to surgical measures, for example to support or produce
cartilage mass, or to strengthen tendons and ligaments. A further
beneficial field of application therefore is sports medicine, where
aberrations or even injuries of tendons, ligaments, muscles and
cartilages caused by sport stresses are the subject of
counteractions.
[0227] For such applications, in particular topical formulations as
those which have been described above in connection with the skin
applications, or injections of physiologically acceptable liquids
or suspensions, which contain one or more of the described
substances, in or at the desired target location are particularly
suitable. Concerning the injections, the kind of carrier liquid and
the other components are determined according to the respective
place of application and are well-known to the person skilled in
the art.
[0228] Osteoporosis
[0229] During the menopausal phase, there is a continuous chronic
lost of bone substance. Since the supportive proteins of the bone
decrease continuously, the bone suffers from binding less calcium
phosphate. This results in a decrease of bone density which can be
determined by X-ray methods. This development could be retarded
conventionally by the administration of estrogens or of estrogens
and gestagens (hormone replacement therapy; HRT). The
administration of androgens also has a beneficial effect. The
mechanism of protecting bones by means of very low concentrations
of estrogens or androgens in the blood, which are, besides, present
there by being bound to carrier proteins, is not known.
[0230] Since osteoblasts contain aromatase (Shozu 1998, Tanaka
1996), the considerations explained above allowed to expect that
the decrease of bone mass is based on the collagen decreasing
effect of the increased local production of estrogens. The stimulus
of this, i.e. the postmenopausally increased concentration of LH,
would be remarkably lowered by the administration of hormones, and
the resorption by the bones would therefore be diminished--which
explains the effect of the conventional HRT. In fact, the aromatase
activity in the bones correlates with the extent of osteoporosis
(Nawata 195).
[0231] Following the new concept of the present invention, it is
now possible to stop the continuation of osteoporosis by means of
inhibiting aromatase. Optionally the aromatase inhibition can be
carried out topically, by means of diffusing the steroidal
hydrophobic drug through the skin, by a transdermal system or by an
inhibitor introduced into the blood systemically, by means of
injecting a depot of a drug in the muscular system, or by
inhalating the drug. For topical applications, the
4-hydroxy-androstendione, which is market-approved (trade name:
Lentaron), is particularly suitable. As a sterol being almost
identical to androstendione, it easily permeates the skin and
occupies upon contact with aromatase irreversibly in the active
center thereof (suicide inhibition). An oral administration is
therefore obsolete.
[0232] The effect of estrogens may also be realized by means of
blocking the estrogen receptors via the anti-estrogens mentioned
above. Tamoxifen, which is applied in the hormone therapy of breast
cancer, is offered here as the most wide-spread substance.
[0233] Excessive Production of Glucocorticoid in the Body and the
Avoidance of Side Effects in Glucocorticoid Therapy
[0234] Like in skin fibroblasts, glucocorticoids also stimulate
aromatase expression in osteoblasts (Shozu 1998, Tanaka 1996).
According to established scholarship opinion, this should properly
be favourable for bones. However, clinical experience clearly shows
that the systemic treatment with glucocorticoids leads to an all
too soon osteoporosis (Jardinet 2000, Lespessailles 2000). This is
well explained by the considerations made above, but not by the
established scholarship opinion.
[0235] An abnormal, excessive production of glucocorticoids in the
body, or a therapy with glucocorticoids affects in an undermining
manner other components of the connective tissue. The collagen
fibres in the skin have already been mentioned in connection with
the stretched strias in M. Cushing. The correlation between the
stability of tendons and ligaments, which essentially consist of
collagen fibres, and glucocorticoids becomes also clear. During
glucocorticoid therapy, tendons disrupt more often (Leppilahti
1998). Since glucocorticoids stimulate the expression of aromatase,
it is plausible that the vulnerability of tendons such as the
Achilles tendon is exerted via this mechanism. Glucocorticoids
result, via a decrease of collagen synthesis in the skin, in an
atrophy thereof (Oikarinen 1998). It is plausible that this is at
least partially based on the stimulus of aromatase expression in
skin fibroblasts, so that the accompanying, optionally topical or
systemic inhibition of aromatase according to the present invention
is capable of avoiding the disadvantageous effects of a therapy
with glucocorticoids, in any case those side effects which affect
the skin and the connective tissue.
[0236] Myocardial Infarction, Brain Infarction and Arteriosclerotic
Plaques
[0237] Already in the childhood, it occurs that lipids are
deposited at the inner wall of arteries. These whitish lipids form
together with macrophages, which are partially contained therein, a
so-called "fatty streak". It predominantly consists of cholesterol
esters. Arteriosclerotic plaques may develop from this "fatty
streak" in the course of time (Schwartz 1993). This is a relatively
large protrusion within the intimae which is surrounded by a
capsule of collagen fibres and consists in the interior of
amorphous cholesterol esters and living or dead macrophages (Libby
2000). As long as the collagen fibre capsules are intact, the
arteriosclerotic plaque is clinically not noticeable. However, if
it disrupts, the blood comes in contact with its content which may
quickly cause an intravasal hemostasis with a subsequent
fibrinogenic thrombus. This thrombus may then block the artery
vessel and thereby stop oxygen delivery for downstream tissue
regions and therefore may result in myocardial infarction or brain
infarction. The capsule disrupts the easier the thinner it is, the
less viscous the content of the plaques is, and the more
macrophages the plaques contain which may secerne the collagen
digesting enzymes (Libby 1996, Davies 1994). An important
therapeutic aim according to the present invention is the
stabilization of the plaques. It is less important to decrease the
size of the plaques, but to lower their cell and lipid content as
far as possible and thus to stabilize them.
[0238] An important cause for the drastical increase of myocardial
infarctions after the menopause may be that the aromatase in the
vessel wall, in particular the macrophages of the plaques, is
stimulated by the permanently increased LH concentrations, and that
the produced estrogens influence the collagen metabolism of the
plaques such that it becomes instable. In fact, the smooth muscle
cells of the artery walls contain quite an amount of aromatase
(Harada 1999. The coronary arteria can synthesize estradiol and
have estrogen receptors (Diano 1999). An inhibition of aromatase,
or a blocking of estrogen receptors therefore does not only have a
protective effect on the bones, but also on the arteriosclerotic
plaques and therefore may help to lower the risk of myocardial
infarctions, particularly in postmenopausal women.
[0239] Urinary Incontinence
[0240] Within four weeks after sterilization, big female dogs often
get urinary incontinence (Arnold 1997). This is also a frequent
problem of elder women (Distler 2000). This effect is explained for
dogs that the mucous membrane of the urinary passageways underlies
similar cyclic variations of their constructions by sexual hormones
like the mucous membrane of the uterus. It is to be assumed that
the dog is in the status of anoestrus for half a year, that is the
estrogen production within the ovaries is not stimulated most of
the time. Therefore, it is questionable as to how the estrogens may
contribute to urinary incontinence. Estrogen therapy does not have
a clear improving effect (Jackson 1999). Possibly, there are
further hormones from the ovary and the hypothalamus or the
pituitary gland which mutually influence each other and which may
partially stimulate the local estrogen synthesis.
[0241] Since the thickness of the wall of the urethral passageways
and therefore possibly also the closure mechanism should depend on
a sufficient proportion of collagen fibres, the treatment of
urinary incontinence in older women by means of the use of
substances inhibiting the production or effects of estrogens
according to the present invention, such as the systemic therapy
with aramotase inhibitors, is indicated.
[0242] The systemic therapy with an aromatase inhibitor having low
side effects would have the additional advantage, contrary to the
hormone replacement therapy, that the coagulation system is not
negatively influenced and that a hemorrhage of the mucous membrane
of the uterus is prevented.
[0243] Excessive Production of Vitamin D and "After
Sun"-Application
[0244] Besides glucocorticoids, the expression of aromatase may
also be stimulated by fibroblasts and keratinozytes of the skin
(Hughes 1997). Vitamin D is formed in the skin by means of exposure
to sun irradiation. Therefore, it is plausible that sunbathing
decreases the collagen content of the skin.
[0245] Therefore, a further useful use of the present invention is
to treat at least the exposed parts of the body with a topical
application of the substance inhibiting the production or effect of
estrogens in order to retard the accelerated formation of wrinkles.
This is suitably carried out, in an "after sun" application, after
the light or sun exposure which occurred during the day, such as in
a topical formulation for the night. The topical formulations which
have already been described for the skin applications are suitable,
wherein the aromatase inhibitors, and in this connection especially
the soya steroles and their analogs, are again preferred due to the
proper capability of being absorbed by the skin. Again, from
counteracting virilizing effects, the concurrent inhibition of both
the aromatase and the 5-alpha-reductase is advantageous.
[0246] Influencing Hair Growth in Men and Women
[0247] Since the 5-alpha-reductase, and, by the way, both iso
enzymes, can be completely inhibited in the skin, it is possible to
exhibit by this measure, and by means of concurrently inhibiting
aromatase, an influence on the rate of hair growth of the body hair
in men and women. The 5-alpha-reductase is also responsible for the
boldness formation in men. This is ascertained, because it is
possible to stimulate the hair growth on the scalp or to slow down
hair loss by means of a systemic administration of Finasterid
(Whiting 1999, Brenner 1999). Minoxidil increases the activity of
17-beta-HSD within the scalp, whereby the reconversion of
testosterone into androstendione is enhanced. At the same time,
however, 5-alpha-reductase is stimulated (Sato 1999). Finasterid
and the anti-androgen flutamid lend themselves for a topical
administration on the hair-loosing head. This was successfully
performed until now with a scalp model which was transplanted onto
nude mice (Sintov 2000) and at the flank organ of gold hamster in
order to stop sebaceous gland production there (Chen 1998). Also
other 5-alpha-reductase inhibitors were topically well effective
there (Chen 1998). Topical administrations of Finasterid and an
inhibitor of the type I enzyme were also tested in another
sebaceous gland model, namely the male fuzzy rat. Finasterid was
little more effective than MK 386. Both substances had much lower
effects than an anti-estrogen receptor blocker (RU58841) (Ye 1997).
A substance which inhibits the 5-alpha-reductase and, at the same
time, acts as an anti-estrogen (4-MA) was applied to the head of
monkeys over a period of 27 weeks. The treated monkeys did not have
any signs of a boldness, contrary to the untreated individuals
(Rittmaster 1987).
[0248] That a topical application of a 5-alpha-reductase inhibitor
in cases of an excessive hair formation was hitherto not
contemplated is clear from the situation that Finasterid, which is
an agent for treating prostate hypertrophy, must be administered
systemically, in order to achieve the desired effects on the scalp.
Finasterid is also administered systemically for the treatment of
hirsutism (Muderis 2000).
[0249] According to the present invention it was found that the
hair growth in women in the face and on the legs after epilation
can be significantly slowed down by the concurrent inhibition of
aromatase and 5-alpha-reductase. A combination is also advantageous
in view of the situation that the production of estrogens in the
skin would increase in the skin of the extremities, if
5-alpha-reductase would be inhibited alone. In addition,
testosterone, which inhibits aromatase, would be diminished faster
(it is converted to dihydrotestosterone). The thus increased
production of estrogen in the skin would make it more thin and, at
the same time, would increase the subcutaneous fatty tissue.
Therefore, it is reasonable to inhibit aromatase at the same
time.
[0250] Depending on the type of 5-alpha-reductase which is
inhibited in addition to the aromatase, the desired hair growth
effect can be realized. For example, with an additional inhibition
of both types of 5-alpha-reductase, such as with a sterol
(4-hydroxy-androstendione) which is very similar to androstendione,
possibly not only an inhibition of the growth of the beard, but
also of the cutic hairs and of the axillary hairs can be
achieved.
EXAMPLES
[0251] The invention will be explained by some examples in the
following.
Example 1) Ointment for Eye Wrinkles (25 ml)
[0252] TABLE-US-00001 cetylsteryl alcohol 3.5 ml natriumlauryl
sulfate 0.75 ml paraffin having low viscosity 5.0 ml white vaseline
15.5 ml oxydized soja glycine having 0.15 ml aromatase inhibitory
action
[0253] 1.1A 60-year-old man with a strongly expressed wrinkle
formation in the eye region, especially at the upper and the lower
eyelid: [0254] After a ten week treatment once per day, a
smoothening in the area of the eyelids, and an almost disappearance
of the wrinkles occurs. [0255] 1.2 A 50-year-old woman with strong
wrinkle formation around the eyes, a status 5 years after a lifting
treatment. [0256] Application of the composition of example (1) two
times per day: [0257] Already after eight weeks of treatment, there
is a strong smoothening of the upper skin in the region of the
wrinkle formation around the eyes; after sixteen weeks, the
wrinkles have disappeared such that the woman indicates that she
has a better appearance than after the lifting procedure.
Example 2) Cream for the Face (50 ml)
[0258] TABLE-US-00002 propylene glykole 12.5 ml isopropyl myristate
3.0 ml sorbitane monostorate 0.5 ml Polysorbate 80 1.0 ml stearyl
alkohol 1.0 ml cetylsteoryl alcohol 3.0 ml glycerol monostearate
0.5 ml oxydized soja glycine having 0.25 ml aromatase inhibitory
action aqua dest. ad 50.0 ml
[0259] A 47-year-old woman, strong formation of wrinkles in the
lower region of the face, especially in the region of the cheek and
the chin:
[0260] After six weeks of treatment twice a day with the
composition of example 2, a smoothening of the wrinkles was
noticeable and visible; after 12 weeks, only slight wrinkles were
visible.
Example 3) Cream Lotion of the Upper Arm (100 ml)
[0261] TABLE-US-00003 Span 80 1.0 ml Span 60 5.0 ml Tween 60 9.0 ml
propylene glykol 15.0 ml palmitinic acid 9.0 ml oxydized soja
glycine having 0.4 ml aromatase inhibitory action aqua dest. ad
100.0 ml
[0262] A 43-year-old woman with wrinkles in the outer skin layer of
the upper arms; treatment two times a day with the cream lotion:
after 4 weeks, a remarkable smoothening is noticeable; after 8
weeks, a clear smoothening is noticeable and visible; after 12
weeks, the wrinkles are strongly reduced; after 16 weeks, wrinkles
are practically invisible. (Biopsies before and after the treatment
show in micrographs the increase of collagen fibres within the
skin: FIG. 1 before the treatment, FIG. 2 after a treatment of 16
weeks.)
Example 4) Gel for Stretched Strias or Pregnancy Strias (100
ml)
[0263] TABLE-US-00004 microcristalline cellulose 4.0 ml
polyethylene glykol 400 5.0 ml cetyl alcohol 10.0 ml oxydized soja
glycine having 0.4 ml aromatase inhibitory action aqua dest. ad
100.0 ml
[0264] 4.1 A 27-year-old woman, mother of two children and having
strong pregnancy strias in the region of the abdomen; application
of the gel-cream 2 times a day. Findings: after 6 weeks, a slight
smoothening is noticeable; after 12 weeks, the pregnancy strias are
strongly reduced; after 18 weeks, no pregnancy strias are visible
any more. [0265] 4.2 A 25-year-old woman after multiple diets;
stretched strias in the region of thighs and buttock. Findings:
after 5 weeks, reduction of stretched strias especially at the
thighs; already after 10 weeks, reduction, the stretched strias on
the thighs and the buttock being (almost) diminished; after 15
weeks, all stretched strias are practically not visible any more:
FIG. 3 before the treatment and FIG. 4 after 10 weeks of
treatment.
Example 5) Injection Formulation for the Intrafocal Administration
(1 ml)
[0265] [0266] 15 mg 4-hydroxy-androstendionee (Formestan) [0267]
Auxiliary agents: benzyl alcohol, carmellose-Nd, polysorbate,
natrium chloride, water [0268] A 30-year-old female tennis player
(federal league) having the appearance of strong overstretchings in
the joints of both knees (ligaments and tendons); strong pain;
playing tennis being only possible with bandages on both sides and
undergoing medical treatment with non-steroidal anti-inflammatory
substances (NSAID). Injection of the composition of example 5) into
the joints of the knee with 2 weeks intervals over a period of 10
weeks: after the 3.sup.rd. injection (4 weeks), there is already a
remarkably reduced NSAID-consumption; after 10 weeks, playing
practically without pain (without NSAID!) being possible, there is
a feeling of stabilization of the joints of the knees.
LITERATURE
[0268] [0269] Arnold S, Schweiz Arch Tierheilkd 1997: 139(6):271-6
[0270] Benson J R et al., Br J Cancer 1996 August; 74(3):352-8
[0271] Berkovitz G D et al., J Clin Endocrinol Metab 1984 October:
59(4):665-71 [0272] Berkovitz G D et al., J Clin Endocrinol Metab
1988 May: 66(5):1029-36 [0273] Berkovitz G D et al., Mol Cell
Endocrinol 1990 March 5; 69(2-3):187-97 [0274] Berkovitz G D et
al., J Clin Endocrinol Metab 1992 March 74(3):629-34 [0275] Bisat T
et al., In Vitro Cell Dev Biol 1989 September: 25(9):806-12 [0276]
Borel JP et al., Pathol Biol (Paris) 1984 September: 32(7):795-812
[0277] Brenner S and Matz H, Int J Dermatol 1999 December:
38(12):928-30 [0278] Brodie A M et al., Cancer Res 1989 Dec. 1;
49(23):6551-5; Published erratum appears in Cancer Res 1990 Jan.
15: 50(2):449 [0279] Cassidenti D L et al., Obstet Gynecol 1991
July; 78(1):103-7 [0280] Chen C et al., J Invest Dermatol 1998
August; 111(2):273-8 [0281] Chen W et al., Dermatology 1996;
193(3):177-84 [0282] Chen C et al., J Invest Dermatol 1995
November: 105(5):678-82 [0283] Courchay G et al., Skin Pharmacol
1996 : 9(3):169-76 [0284] Cummings S R et al., JAMA 1999 Jun. 16:
281(23):2189-97 [0285] Davies M J et al., Basic Res Cardiol 1994:
89 Suppl 1:33-9 [0286] de Loes M et al., Scand J Med Sci Sports
2000 April: 10(2):90-7 [0287] Diano S et al., Menopause 1999
Spring: 6(1):21-8 [0288] Dijkstra A C et al., J Invest Dermatol
1987 July: 89(1):87-92 [0289] Distler W, Z Arztl Fortbild
Qualitatssich 2000 April: 94(3):211-5 [0290] Fujimoto M et al., J
Clin Endocrinol Metab 1986 August: 63(2):468-74 [0291] Gottlieb B
et al., Am J Med Genet 1999 Dec. 29: 89(4):210-7 [0292] Gips H et
al., J Endocrinol Invest 1980 January-March: 3(1):51 [0293] Grino
PB et al., Endocrinology 1990 February: 126(2):1165-72 [0294]
Hamada K et al., J Invest Dermatol 1996 May: 106(5):1017-22 [0295]
Harada N, Biochem Biophys Res Commun 1992 Dec. 15: 189(2):1001-7
[0296] Harada N et al., Circ Res 1999 Jun. 11: 84(11):1285-91
[0297] Hsiang Y H et al., J Steroid Biochem 1987 January:
26;(1):131-5 [0298] Hughes S V et al., Endocrinology 1997
September: 138(9):3711-8 [0299] Iida S et al., J Clin Endocrinol
Metab 1991 July: 73(1):192-6 [0300] Itami S et al., J Dermatol Sci
1994 July; 7 Suppl:S98-103 [0301] Jackson S et al., Br J Obstet
Gynaecol 1999 July: 106(7):711-8 [0302] Jardinet D et al.,
Rheumatology (Oxford) 2000 April: 39(4):389-92 [0303] Kwan G et
al., Kidney Int 1996 October: 50(4):1173-9 [0304] Labrie F et al.,
Ann Endocrinol (Paris) 1995: 56(1):23-9 [0305] Labrie F, Mol Cell
Endocrinol 1991 July: 78(3):C113-8 [0306] Labrie F et al., J Clin
Endocrinol Metab 1997 August: 82(8):2403-9 [0307] Lachgar S et al.,
J Investig Dermatol Symp Proc 1999 December: 4(3):290-5 [0308] Lee
KS et al., Clin Exp Dermatol 1994 July: 19(4):285-8 [0309]
Leppilahti J and Orava S, Sports Med 1998 February: 25(2):79-100
[0310] Lespessailles E et al., Joint Bone Spine 2000: 67(2):119-26
[0311] Libby P et al., Curr Opin Lipidol 1996 October: 7(5):330-5
[0312] Libby P, J Intern Med 2000 March: 247(3):349-58 [0313]
Luu-The V et al., Invest Dermatol 1994 February: 102(2):221-6
[0314] Mestayer C et al., J Clin Endocrinol Metab 1996 May:
81(5):1989-93 [0315] Marttunen M B et al., Cajcif Tissue Int 1999
November: 65(5) :365-8 [0316] Milewich L et al., Placenta 1990
March-April: 11(2):95-108 [0317] Milewich L et al., Ann N Y Acad
Sci 1988: 548:66-89 [0318] Muderris II et al., Fertil Steril 2000
May: 73(5):984-7 [0319] Nawata H et al., J Steroid Biochem Mol Biol
1995 June: 53(1-6):165-74 [0320] Neugarten J et al., Am J Physiol
1999 December: 277(6 Pt 2):F875-81 [0321] Oikarinen A et al., Br J
Dermatol 1998 December: 139(6):1106-10 [0322] Powell J W and
Barber-Foss K D, Am J Sports Med 2000 May-June: 28(3):385-91 [0323]
Rittmaster R S et al., J Clin Endocrinol Metab 1987 July;
65(1):188-93 [0324] Roberts A J et al., Crit Rev Eukaryot Gene Expr
1995: 5(2):157-76 [0325] Sasano H et al., Endocr J 1999 April;
46(2):233-42 [0326] Sato T et al., J Dermatol Sci 1999 February;
19(2):123-5 [0327] Schwartz C J, Am J Cardiol 1993 Feb. 25:
71(6):9B-14B [0328] Sciore P et al., J Orthop Res 1998 September:
16(5):604-10 [0329] Setnikar I et al., Arzneimittelforschung 1999
August: 49(8):708-15 [0330] Shozu M and Simpson E R, Mol Cell
Endocrinol 1998 Apr. 30: 139(1-2):117-29 [0331] Silbiger S et al.,
Kidney Int 1999 April: 55(4):1268-76 [0332] Sintov A et al., Int J
Pharm 2000 Jan. 20: 194(1):125-34 [0333] Stanczyk F Z et al., J
Steroid Biochem Mol Biol 1990 September; 37(1):129-32 [0334]
Svenstrup B et al., J Steroid Biochem 1990 May: 35(6):679-87 [0335]
Tamaoka Y, Nippon Sanka Fujinka Gakkai Zasshi July 1987:
39(7):1129-36 [0336] Tanaka S et al., Endocrinology May 1996:
137(5):1860-9 [0337] Thiboutot D et al., J Invest Dermatol
September 1998: 111(3):390-5 [0338] Theintz G E et al. Horm Res
1989: 32(4):124-9 [0339] Whiting D A et al., J Investig Dermatol
Symp Proc 1999 December: 4(3):282-4 [0340] Wojtys E M et al., Am J
Sports Med 1998 September-October: 26(5):614-9 [0341] Venencie P Y
et al. Br J Dermatol 1999 September: 141(3):438-46 [0342] Winkler E
M et al., Am J Phys Anthropol 1993 October: 92(2):155-64 [0343] Ye
F et al., Skin Pharmacol 1997; 10(5-6):288-97 [0344] You S et al.,
Biol Reprod 2000 January: 62(1):108-16; [0345] Zhao Y et al., Mol
Endocrinol 1995 March: 9(3):340-9;
* * * * *