U.S. patent application number 10/167727 was filed with the patent office on 2002-12-12 for pharmaceutical compositions and uses for androst-5-ene-3beta, 17beta-diol.
This patent application is currently assigned to Endorecherche, Inc.. Invention is credited to Labrie, Fernand.
Application Number | 20020187968 10/167727 |
Document ID | / |
Family ID | 22256669 |
Filed Date | 2002-12-12 |
United States Patent
Application |
20020187968 |
Kind Code |
A1 |
Labrie, Fernand |
December 12, 2002 |
Pharmaceutical compositions and uses for androst-5-ene-3beta,
17beta-DIOL
Abstract
Androst-5-ene-3.beta.,17.beta. diol is used to treat or reduce
the likelihood of acquiring osteoporosis or menopausal symptoms, or
other diseases affected by estrogen receptor activity, and for
conditions which respond well to DHEA treatment, but where a higher
ratio of estrogenic to androgenic effects is desired. Combination
therapies are included, as are kits and pharmaceutical compositions
for providing the active ingredients of claimed methods and
combinations.
Inventors: |
Labrie, Fernand;
(Sainte-foy, CA) |
Correspondence
Address: |
OSTROLENK FABER GERB & SOFFEN
1180 AVENUE OF THE AMERICAS
NEW YORK
NY
100368403
|
Assignee: |
Endorecherche, Inc.
|
Family ID: |
22256669 |
Appl. No.: |
10/167727 |
Filed: |
June 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10167727 |
Jun 10, 2002 |
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09332356 |
Jun 11, 1999 |
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6432940 |
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09332356 |
Jun 11, 1999 |
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09096286 |
Jun 11, 1998 |
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Current U.S.
Class: |
514/170 ;
514/178 |
Current CPC
Class: |
A61P 15/08 20180101;
A61K 31/568 20130101; A61P 35/00 20180101; A61P 21/00 20180101;
A61K 31/565 20130101; A61P 17/00 20180101; A61P 25/00 20180101;
A61P 15/18 20180101; A61P 9/00 20180101; A61P 3/10 20180101; A61P
15/02 20180101; A61P 15/12 20180101; A61P 13/00 20180101; A61P 9/10
20180101; A61P 5/24 20180101; A61P 13/08 20180101; A61P 5/48
20180101; A61P 15/10 20180101; A61P 15/00 20180101; A61P 43/00
20180101; A61P 3/04 20180101; A61P 5/26 20180101; A61P 25/28
20180101; A61P 19/10 20180101; A61K 31/565 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
514/170 ;
514/178 |
International
Class: |
A61K 031/56 |
Claims
What is claimed is:
1. A pharmaceutical composition comprising a pharmaceutically
acceptable diluent or carrier, and a therapeutically effective
amount of androst-5-ene-3.beta.,17.beta.-diol or a prodrug
thereof.
2. The pharmaceutical composition of claim 1, further comprising a
therapeutically effective amount of an estrogen.
3. The pharmaceutical composition of claim 1, further comprising a
therapeutically effective amount of a progestin.
4. The pharmaceutical composition of claim 2, further comprising a
therapeutically effective amount of a progestin.
5. The pharmaceutical composition of claim 1, further comprising a
compound selected from the group consisting of
dehydroepiandrosterone and dehydroepiandrosterone-sulfate.
6. The pharmaceutical composition of claim 1, further comprising a
pharmaceutically acceptable diluent or carrier for percutaneous or
transmucosal administration, wherein said
androst-5-ene-3.beta.,17.beta.-- diol or prodrug thereof is present
at a concentration between approximately 2 and 30 percent by weight
relative to the total weight of the pharmaceutical composition.
7. A kit comprising: a first container having therein
androst-5-ene-3.beta.,17.beta.-diol or prodrug; and at least one
additional container having therein at least one active ingredient
selected from the group consisting of a progestin, an estrogen,
dehydroepiandrosterone and dehydroepiandrosterone-sulfate.
8. A therapeutic method for treating or reducing the likelihood of
acquiring reduced or imbalanced concentrations of sex steroids,
comprising the step of administering a therapeutically effective
amount of androst-5-ene-3.beta.,17.beta.-diol or prodrug thereof to
a patient in need thereof.
9. The method of claim 8, further comprising administering at least
one additional agent selected from the group consisting of DHEA and
DHEA-S, as part of a combination therapy.
10. A method for treating or reducing the likelihood of acquiring
symptoms of menopause, comprising the step of administering a
therapeutically effective amount of
androst-5-ene-3.beta.,17.beta.-diol or prodrug thereof to a patient
in need thereof.
11. A method for treating or reducing the likelihood of acquiring
vaginal atrophy, comprising the step of administering a
therapeutically effective amount of
androst-5-ene-3.beta.,17.beta.-diol or a prodrug thereof to a
patient in need thereof.
12. A method for treating or reducing the likelihood of acquiring
hypogonadism, comprising the step of administering a
therapeutically effective amount of
androst-5-ene-3.beta.,17.beta.-diol or prodrug to a patient in need
thereof.
13. A method for treating or reducing the likelihood of acquiring
osteoporosis, comprising the step of administering a
therapeutically effective amount of
androst-5-ene-3.beta.,17.beta.-diol or prodrug to a patient in need
thereof.
14. A method for treating or reducing the likelihood of acquiring
diminished libido, comprising the step of administering a
therapeutically effective amount of
androst-5-ene-3.beta.,17.beta.-diol or prodrug to a patient in need
thereof.
15. A method for treating or reducing the likelihood of acquiring
skin atrophy, comprising the step of administering a
therapeutically effective amount of
androst-5-ene-3.beta.,17.beta.-diol or prodrug to a patient in need
thereof.
16. A method for treating or reducing the likelihood of acquiring
urinary incontinence, comprising the step of administering a
therapeutically effective amount of
androst-5-ene-3.beta.,17.beta.-diol or prodrug to a patient in need
thereof.
17. A method for reducing the likelihood of acquiring ovarian
cancer, comprising the step of administering a therapeutically
effective amount of androst-5-ene-3.beta.,17.beta.-diol or prodrug
to a patient in need thereof.
18. A method for reducing the likelihood of acquiring uterine
cancer, comprising the step of administering a therapeutically
effective amount of androst-5-ene-3.beta.,17.beta.-diol or prodrug
to a patient in need thereof.
19. A method for treating or reducing the likelihood of acquiring
skin dryness, comprising the step of administering a
therapeutically effective amount of
androst-5-ene-3.beta.,17.beta.-diol or prodrug to a patient in need
thereof.
20. A method of contraception, comprising the step of administering
a therapeutically effective amount of
androst-5-ene-3.beta.,17.beta.-diol or prodrug to a patient in need
thereof.
21. The method of claim 20, further comprising administering a
therapeutically effective amount of an estrogen.
22. The method of claim 20, further comprising administering a
therapeutically effective amount of a progestin.
23. A method for treating breast cancer, uterine cancer, ovarian
cancer or endometriosis comprising the step of administering a
therapeutically effective amount of
androst-5-ene-3.beta.,17.beta.-diol or prodrug thereof to a patient
in need thereof in association with at least one of the following
compounds: antiestrogen, androgen, DHEA, inhibitor of aromatase,
inhibitor of 17.beta.-hydroxysteroid dehydrogenase, LHRH agonist or
antagonist.
24. A method for treating breast cancer, uterine cancer, ovarian
cancer or endometriosis comprising the step of administering a
therapeutically effective amount of dehydroepiandrosterone (DHEA)
to a patient in need thereof in association with at least one of
the following compounds: antiestrogen, androgen, inhibitor of
aromatase, inhibitor of 17.beta.-hydroxysteroid dehydrogenase, LHRH
agonist or antagonist.
25. A method for treating or reducing the likelihood of acquiring
an indication selected from the group consisting of obesity,
cardiovascular disease, atherosclerosis, insulin resistance, loss
of muscle mass, loss of energy or fatigue, loss of memory,
Alzheimer disease, comprising administering a therapeutically
effective amount of the pharmaceutical composition of claim 1.
26. A transdermal patch which continuously delivers
androst-5-ene-3.beta.,17.beta.-diol or prodrug, said patch having a
means for attaching to skin, a reservoir for said compound and a
means for conducting said compound into contact with skin.
27. The pharmaceutical composition of claim 1, further comprising a
pharmaceutically acceptable diluent or carrier for oral
administration, wherein said androst-5-ene-3.beta.,17.beta.-diol or
prodrug of androst-5-ene-3.beta.,17.beta.-diol is present at a
concentration between approximately 5 and 99 percent by weight
relative to the total weight of the pharmaceutical composition.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to pharmaceutical
compositions, kits and methods for preventing and treating reduced
or imbalanced concentrations of sex steroids and conditions which
can respond favorably to increased activity of androgens and/or
estrogens. The invention utilizes
androst-5-ene-3.beta.,17.beta.-diol (hereinafter 5-DIOL) or
compounds converted in vivo to 5-DIOL
[0003] 2. Description of the Related Art
[0004] 5-DIOL is a compound biosynthesized from DHEA through the
action of reductive 17.beta.-hydroxysteroid dehydrogenase
(17.beta.-HSD) and is a weak estrogen. It has an 85-fold lower
affinity than 17.beta.-estradiol (E.sub.2) for the estrogen
receptor in rat anterior pituitary gland cytosol (Simard and
Labrie, J. Steroid Biochem., 26: 539-546, 1987), further confirming
size data obtained on the same parameter in human myometrial and
breast cancer tissue (Kreitmann and Bayard, J. Steroid Biochem. 11:
1589-1595, 1979; Adams et al., Cancer Res., 41: 4720-4926, 1981;
Poulin and Labrie, Cancer Res., 46: 4933-4937, 1986).
[0005] At concentrations well within the range of the plasma levels
found in adult women, 5-DIOL enhances cell proliferation and
progesterone receptor levels in human mammary tumor ZR-75-1 cells
which lack 3.beta.-hydroxysteroid dehydrogenase/D5-D4 isomerase
activity (Poulin and Labrie, Cancer Rest., 46: 4933-4937, 1986) and
increases the estrogen-dependent synthesis of the 52 kDa
glycoprotein in MCF-7 cells (Adams et al., Cancer Res., 41:
4720-4926, 1981).
[0006] In general, it is known that the serum levels of DHEA and
DHEA-S decrease with age and correspondingly, that there is a
dramatic reduction in the formation of androgens and estrogens in
peripheral target tissues and a marked decrease in the biochemical
and cellular functions induced by sex steroids. As a result, DHEA
and DHEA-S have been used in the treatment of a variety of
conditions which are associated with decrease and/or imbalances in
the levels of sex steroids Recently, we have found that the serum
levels of 5-diol decrease markedly with age.
[0007] Osteoporosis, a condition which affects both men and women,
is associated with a decrease in androgens and estrogens. Estrogens
have been shown to decrease the rate of bone degradation while
androgens have been shown to build bone mass.
[0008] Menopausal symptoms have also been associated with a loss of
estrogens, and low dose estrogen therapy is commonly used in
perimenopausal and menopausal women to relieve vasomotor symptoms,
urogenital atrophy, irritability, sleep problems, loss of energy,
osteoporosis, and other symptoms associated with menopause.
[0009] In addition, breast cancer, cardiovascular disease, and
insulin resistance have been associated with decreased serum levels
of DHEA and DHEA-S and both DHEA and DHEA-S have been suggested to
prevent or treat these conditions. DHEA has also been suggested to
have beneficial effects in the treatment and/or prevention of
obesity, diabetes, atherosclerosis, chemically-induced breast,
skin, and colon cancer, autoimmune diseases, Alzheimer's disease,
loss of memory, aging and to support energy, muscle mass, and
longevity. Uses of DHEA as well as the benefits of androgen and
estrogen therapy are discussed to International Patent Publication
WO 94/16709.
[0010] DHEA and DHEA-S have been suggested to be better for the
treatment of these conditions than standard estrogen and androgen
therapy since the action of DHEA and DHEA-S is targeted to the
tissues which can covert DHEA and/or DHEA-S to specific sex
steroids, However, high doses of DHEA are required to get the
necessary estrogenic and androgenic effects. Most importantly, the
androgenic effects of DHEA are predominant and therefore, for
conditions i which a higher proportion of estrogens is desired or
where androgenic side effects are a problem, especially in women,
the present invention permits a better proportion of estrogenic
versus androgenic effects.
SUMMARY OF THE INVENTION
[0011] It is an object of the present invention to provide
pharmaceutical compositions and kits which include 5-DIOL or
prodrugs converted thereto in vivo. In some embodiments, the
pharmaceutical compositions consist essential of 5-DIOL.
[0012] It is also an object of the present invention to provide
methods of treating and preventing imbalances of reductions in the
levels of sex steroid hormones (androgens and/or estrogens) raising
5-DIOL levels in a patient in need of such treatment or
prevention.
[0013] It is a further object of this invention to provide methods
of or treating or reducing the risk of onset of conditions which
respond favorably to estrogenic activity, including vaginal
atrophy, hypogonadism, diminished libido, skin atrophy, urinary
incontinence, lipid, and lipoprotein imbalance, atherosclerosis,
cardiovascular disease and symptoms of menopause (hot flushes,
sleep disorders, Alzheimer's disease, Parkinson's disease, mental
disorders, depression, loss of memory) by administering 5-DIOL. It
is a further object of this invention to provide methods of
preventing or treating conditions which respond favorably to
androgenic activity, including breast cancer, ovarian cancer,
endometrial cancer, diminished libido, skin atrophy, skin dryness,
osteoporosis and symptoms of menopause by administering 5-DIOL. A
number of diseases that are affected by sex steroids (e.g.
osteoporosis) respond favorably to both androgens and
estrogens.
[0014] A patient in need of treatment or reducing the risk of onset
of a given disease is one who has either been diagnosed with such
disease or one who is susceptible to acquiring such disease.
[0015] Except where otherwise noted or where apparent from context,
dosages herein refer to weight of active compounds unaffected by
pharmaceutical excipients, diluents, carries or other ingredients,
although such additional ingredients are desirable included, as
shown in the examples herein. Any dosage form (capsule, tablet,
injection or the like) commonly used in the pharmaceutical industry
is appropriate or use herein, and the terms "excipient," "diluent"
or "carrier" include such non-active ingredients as are typically
included, together with active ingredients in such dosage forms in
the industry. For example, typical capsules, pills, enteric
coatings, solid or liquid diluents or excipients, flavorants,
preservatives, or the like are included.
[0016] The invention also includes use of an active agent in the
manufacture of a medicament for treatment of a disease specified
herein as susceptible to that agent, or one component of a
combination in the manufacture of a medicament for treatment of a
disease, where the treatment further includes another component of
the claimed combination therapy.
[0017] It is an additional object of this invention to provide
novel contraceptive methods which include administering 5-DIOL.
[0018] 5-DIOL is a metabolite of dehydroepiandrosterone (DHEA). It
has now been discovered that 5-DIOL has an unexpected variation
(relative to DHEA) in its androgenic and estrogenic effects. In
particular, 5-DIOL is five-fold more estrogenic than DHEA while its
androgenic activity is only one- to two-fold higher than that of
DHEA, thus giving an estrogenic to androgenic ratio of
approximately 3.0 for 5-DIOL compared with DHEA. On the other hand,
at higher doses, 5-DIOL produces maximal effects less androgenic
than DHEA. Thus, the relative estrogenic versus androgenic effects
of administering a 5-DIOL lie more toward estrogenic effect than
does a corresponding dosage of DHEA. Therefore, 5-DIOL is
particularly useful in treating and preventing conditions involving
low levels of sex steroids where the estrogen level, in particular,
has fallen (i.e. more so than the androgen level). Indeed the
invention is useful wherever an estrogenic effect is desired to a
greater extent than is an androgenic effect. As explained below,
5-DIOL may be administered alone or in combination with other
therapeutic agents such as antiestrogens, androgens, progestins,
estrogens, DHEA, DHEA-sulfate, LHRH agonists or antagonists,
inhibitors of 17.beta.-hydroxysteroid dehydrogenase, aromatase
inhibitors, inhibitors of gonadal sex steroid secretion, as part of
a combination therapy.
[0019] In the context of the invention, any prodrugs of 5-DIOL may
be used in place of 5-DIOL, including 5-DIOL-fatty acids, as these
will also increase the serum levels of 5-DIOL.
[0020] Other features and advantages of the present invention will
become apparent from the following description of the invention
which refers to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered percutaneously twice daily for 7 days, on
the uterine weight in ovariectomized rats, an estrogen-sensitive
parameter. Intact animals are used as additional controls. The
compounds were dissolved in 50% ethanol-50% propylene glycol and
were administered in 0.1 ml on the dorsal skin area (2 cm.times.2
cm).
[0022] FIG. 2 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered percutaneously twice daily for 7 days, on
the serum luteinizing hormone (LH) concentration in ovariectomized
rats, a measure of androgenic and/or estrogenic effect. Intact
animals are used as additional controls. The compounds were
dissolved in 50% ethanol-50% propylene glycol and were administered
in 0.1 ml on the dorsal skin area (2 cm.times.2 cm).
[0023] FIG. 3 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered percutaneously twice daily for 7 days, on
the serum DHEA concentration in ovariectomized rats. Intact animals
are used as additional controls. The compounds were dissolved in
50% ethanol-50% propylene glycol and were administered in 0.1 ml on
the dorsal skin area (2 cm.times.2 cm).
[0024] FIG. 4 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered percutaneously twice daily for 7 days, on
the serum 5-DIOL concentration in ovariectomized rats. Intact
animals are used as additional controls. The compounds were
dissolved in 50% ethanol-50% propylene glycol and were administered
in 0.1 ml on the dorsal skin area (2 cm.times.2 cm).
[0025] FIG. 5 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered percutaneously twice daily for 7 days, on
the serum androstenedione (4-dione) concentration in ovariectomized
rats, a measure of androgenic and/or estrogenic effect. Intact
animals are used as additional controls. The compounds were
dissolved in 50% ethanol-50% propylene glycol and were administered
in 0.1 ml on the dorsal skin area (2 cm.times.2 cm).
[0026] FIG. 6 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered percutaneously twice daily for 7 days, on
the serum testosterone concentration in ovariectomized rats. Intact
animals are used as additional controls. The compounds were
dissolved in 50% ethanol-50% propylene glycol and were administered
in 0.1 ml on the dorsal skin area (2 cm.times.2 cm).
[0027] FIG. 7 is a graph of the effect of increasing doses so DHEA
or 5-DIOL, administered percutaneously twice daily for 7 days, on
the serum dihydrotestosterone (DHT) concentration in ovariectomized
rats. Intact animals are used as additional controls. The compounds
were dissolved in 50% ethanol-50% propylene glycol and were
administered in 0.1 ml on the dorsal skin area (2 cm.times.2
cm).
[0028] FIG. 8 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered percutaneously twice daily for 7 days, on
the ventral prostate weight in orchiectomized rats, a measure of
androgenic effect. Intact animals are used as additional
controls.
[0029] FIG. 9 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered percutaneously twice daily for 7 days, on
the seminal vesicle weight in orchiectomized rats, a measure of
androgenic effect. Intact animals are used as additional
controls.
[0030] FIG. 10 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered percutaneously twice daily for 7 days, on
the concentration of the mRNA encoding the C1 component of
prostatic binding protein (PBP-C1) in the ventral prostate of
orchiectomized rats, a measure of androgenic effect. Intact animals
are used as additional controls.
[0031] FIG. 11 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered percutaneously twice daily for 7 days, on
the mRNA encoding the C3 component of prostatic binding protein
(PBP-C3) in the ventral prostate of orchiectomized rats, a measure
of androgenic effect. Intact animals are used as additional
controls.
[0032] FIG. 12 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered subcutaneously twice daily for 7 days, on
the uterine weight in ovariectomized rates, a measure of estrogenic
effect. Intact animals are used as additional controls.
[0033] FIG. 13 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered subcutaneously twice daily for 7 days, on
the serum LH concentration in ovariectomized rats, a measure of
androgenic and/or estrogenic effect. Intact animals are used as
additional controls.
[0034] FIG. 14 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered subcutaneously twice daily for 7 days, on
prostate weight in orchiectomized rats, a measured of androgenic
effect. Intact animals are used as additional controls.
[0035] FIG. 15 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered subcutaneously twice daily for 7 days, on
seminal vesicle weight in orchiectomized rats, a measure of
androgenic effect. Intact animals are used as additional
controls.
[0036] FIG. 16 is a graph of the effect of increasing doses of DHEA
or 5-DIOL, administered subcutaneously twice daily for 7 days, on
the serum LH concentration in orchiectomized rats, a measure of
androgenic and/or estrogenic effect. Intact animals are used as
additional controls.
[0037] FIG. 17 shows the plasma concentration of DHEA (ng/mL) (Y
axis in function of time (X-axis) after a single oral absorption of
prodrugs of andros-5-ene-3.beta.,17.beta.-diol (150 .mu.mol/rat) in
male rats. In the box, AUC 24 h of DHEA induced by these compounds
is reported.
1 EM-760 dehydroepiandrosterone EM-900 androst-5-ene-3.beta.,
17.beta.-diol EM-1304 androst-5-ene-3.beta., 17.beta.-diol 3-
acetate EM-1305-CS androst-5-ene-3.beta., 17.beta.-diol diacetace
EM-1397 androst-5-ene-3.beta., 17.beta.-diol acetate 17 benzoate
EM-1400 androst-5-ene-3.beta., 17.beta.-diol dibenzoate EM-1410
androst-5-ene-3.beta., 17.beta.-diol dipropionate EM-1474-D
androst-5-ene-3.beta., 17.beta.-diol dihemisuccinate
[0038] FIG. 18 shows the plasma concentration of
androst-5-ene-3.beta.,17.- beta.-diol (ng/mL) (Y axis) in function
of time (X-axis) after a single oral absorption of prodrugs of
androst-5-ene-3.beta.,17.beta.-diol (150 .mu.mol/rat) in male rats.
In the box, AUC 24 h of androst-5-ene-3.beta.,17.beta.-diol induced
by these compounds is reported.
2 EM-760 dehydroepiandrosterone EM-900 androst-5-ene-3.beta.,
17.beta.-diol EM-1304 androst-5-ene-3.beta., 17.beta.-diol 3-
acetate EM-1305-CS androst-5-ene-3.beta.,17.beta.-diol diacetate
EM-1397 androst-5-ene-3.beta., 17.beta.-diol acetate 17 benzoate
EM-1400 androst-5-ene-3.beta., 17.beta.-diol dibenzoate EM-1410
androst-5-ene-3.beta., 17 .beta.-diol dipropionate EM-1474-D
androst-5-ere-3.alpha.,17.alpha.-diol dihemisuccinate
[0039] In Vivo Assays of Bioavailability of the Prodrugs of
Androst-5-ene-3.beta.,17.beta.-diol
[0040] 1) Principle
[0041] The assays of the bioavailability of prodrugs of
androst-5-ene-3.beta.,17.beta.-diol were performed in male Sprague
Dawley rats by measuring the plasma concentrations of the compounds
after single oral administration of the compounds.
[0042] 2) Animals and Treatment
[0043] Male Sprague-Dawley rats [Crl:CD(SB)Br] weighing 275-350 g
were obtained from Charles-River Canada Inc. and housed 2 per cage
during the acclimation period and individually during the study
period. The animals were maintained under a regimen of 12 hours
light; 12 hours dark (lights on at 08:00). Animals received
certified Rodent feed (Lab Diet # 5002, pellets) and tap water ad
libitum. Rats were fasted (access to water only) starting on the
evening prior to dosing.
[0044] Each compound to be tested was administered to three animals
as a suspension in 0.4% methylcellulose by oral gavage at a dose of
150 .mu.mol/rat. One blood sample of .about.0.7 ml was collected
from the jugular vein of rats under Isoflurane-induced anesthesia
at 1, 2, 3, 4 and 7 hours post-gavage. Blood samples were
immediately transferred into a refrigerated 0.75 ml Microtainer
containing EDTA and kept in an ice-water bath until centrifugation
at 3000 rpm for 10 minutes. Plasma separation was performed rapidly
(less than 50 minutes) after blood collection). One aliquot of 0.25
ml of plasma was then transferred into a borosilicate tube
(3.times.100) and was rapidly frozen on dry-ice. Plasma samples
were kept at .about.80.degree. C. until measurement of plasma
concentration of the sex steroid or sex steroid precursors by
GC-MS.
DETAILED DESCRIPTION OF THE INVENTION
[0045] It is believed that 5-DIOL may be used, in accordance with
the invention, for the treatment of any disease known to respond
favorably to treatment with DHEA with the added benefits related to
its more favorable ratio of estrogenic versus androgenic activities
and its lower maximal androgenic activity relative to DHEA.
Administering 5-DIOL directly in accordance with the invention, has
a number of advantages over administering DHEA, as discussed
herein.
[0046] Applicant has discovered that 5-DIOL produces significantly
different androgenic and estrogenic effects than does DHEA. In
particular, 5-DIOL is shown to produce less potential androgenic or
masculinizing effects for a given production of estrogenic effects
than does DHEA. Therefore, 5-DIOL is particularly beneficial in
treating conditions which require estrogenic activity with minimal
androgenic activity. In fact, after menopause, women have a deficit
of both androgens and estrogens although the ratio of
estrogen/androgen is lower than before menopause. Women thus
require a more favorable estrogenic-androgenic ratio to compensate
the loss of estradiol secretion by the ovaries. DHEA cannot
compensate for this ovarian estrogenic deficit but will only
replace the lowered secretion of DHEA-S and DHEA by the
adrenals.
[0047] In particular, the production of the active androgen DHT and
its precursor 4-dione by administering DHEA ranged, for increasing
dosages, from 30 to 125% greater than obtained with 5-DIOL
administration. On the other hand, a 53% greater production of
testosterone was obtained with DHEA than by administering 5-DIOL.
4-dione is a steroid which is itself a weak androgen but is
particularly efficiently transformed intracellularly into the more
potent androgens testosterone, and DHT. The data shows that the
maximum level of serum DHT reached after 5-DIOL was less than half
of the maximum level reached with DHEA, and that at a relatively
low dosage levels, the androgenic effect of 5-DIOL reaches a
plateau of maximum activity much lower than that achieved by
DHEA.
[0048] FIGS. 10 and 11 confirm the relatively, higher level of
androgenic activity of DHEA as compared to the activity of 5-DIOL.
The data shows, depending on dosage, that DHEA is two to five times
more potent than 5-DIOL in stimulating prostate binding protein-C1
(PBP-C1) and prostate binding protein-C3 (PBP-C3) mRNA levels. The
levels of PBP-C1 and PBP-C3 mRNA are particularly sensitive
parameters of androgenic action and the concentration of the mRNAs
encoding these proteins is regulated by androgens. Measurements of
PBP-C1 and PBP-C3 mRNA levels are indicative of the androgenic
activity since androgens act at the transcriptional level to
increase steady state levels of the mRNAs encoding the subunit
components of PBP.
[0049] Because both DHEA and 5-DIOL administered percutaneously
have almost identical effects of estrogenic parameters at a given
dosage, DHEA produces two to three times more androgenic activity
than 5-DIOL where equivalent estrogenic effects are provided.
[0050] It has also been found that a major difference between DHEA
and 5-DIOL is that oxidative 17.beta.-HSD(s) which transform(s) 5
-DIOL into DHEA has an extremely low level of activity after
systemic administration of 5-DIOL (see FIGS. 3 and 4). On the other
hand, the similar levels of serum 5-DIOL following administration
of DHEA and 5-DIOL indicate that the reductive 17.beta.-HSD has a
relatively high level of activity. In addition, since no
significant DHEA appears available to be transformed directly into
4-dione by 3.beta.-HSD, following 5-DIOL administration,
androstenedione must derive from the oxidation of testosterone.
This is in agreement with the fact that levels of serum 4-dione, a
highly efficient precursor of androgens, are much lower after
5-DIOL treatment than after DHEA treatment.
[0051] Without intending to be bound by theory, it is believed that
one of the reasons DHEA produces a different androgenic response
than does 5-DIOL (at equal dosage) is that DHEA and 5-DIOL are
metabolized differently. 5-DIOL's metabolism (more so than DHEA's
metabolism) leads to less formation and/or more inactivation of
certain steroids (e.g. testosterone and DHT) before they can exert
androgenic activity. Thus, 5-DIOL leads to lower exposure to these
potent androgens than does an equal dose of DHEA where the oxidized
steroids predominates as a reservoir for formation of the active
androgens.
[0052] Further, as shown in FIGS. 14 and 15, where both compounds
were administered by the subcutaneous route for an assessment of
maximal bioavailability, the stimulation of androgen-sensitive
parameters by 5-DIOL reached a plateau at a lower value than
achieved with DHEA. It is thus clear that the difference of maximal
androgenic activity of DHEA and 5-DIOL observed after percutaneous
administration of the two compounds (FIGS. 8, 9, 10, and 11) is not
due to difference rates of absorption through the skin.
[0053] In general, the estrogenic activities of 5-DIOL under
conditions of maximal bioavailability were shown to be greater than
those of the same doses of DHEA. In fact, after subcutaneous
administration, 5-DIOL is about 5 times more potent than DHEA to
stimulate uterine growth. Therefore, as discussed above, as a
dosage where the estrogenic effects are the same, the androgenic
effects produced by 5-DIOL will be significantly lower than the
effects produced by DHEA. Moreover, the androgenic effects of
5-DIOL reach a plateau of maximal effect which is significantly
lower than the maximal stimulation achieved with DHEA.
[0054] As will be discussed in detail below, 5-DIOL can be
administered to treat or prevent conditions in patients who have
insufficient levels or imbalanced concentrations of sex steroids,
namely androgens and/or estrogens. In particular, 5-DIOL is
believed useful in treating and reducing risk of acquiring
conditions which respond favorably to estrogenic activity and in
which lower androgenic activity than provided by DHEA is desired.
Because 5-DIOL is metabolized qualitatively to the same sex
steroids as is DHEA, 5-DIOL may be used for any purpose that
responds favorable to DHEA, with the benefit of different
androgenic versus estrogenic effects than would result from using
DHEA. Where the desired estrogen/androgen ratio lies between what
is achievable with DHEA versus 5-DIOL, then a mixture of DHEA and
5-DIOL may be used to provide the desired ratio.
[0055] Individuals who will benefit from treatment with 5-DIOL
include all those suffering from conditions treatable with DHEA,
including those with abnormally low levels of 5-DIOL, estrogen, or
androgen. Reducing the risk of acquiring such conditions is also
possible and the recommended 5-DIOL dosage and target serum levels
is the same as for the therapeutic uses of 5-DIOL herein.
Individuals who could benefit from the invention can be identified
by measuring serum levels of 5-DIOL, DHEA, sex steroids and their
metabolites (especially androsterone glucuronide and
androsterone-3a,17.beta.-diol glucuronide for androgen and
estrone-sulfate and estradiol-sulfate for estrogens) as described
by Blanger et al., in Steroid Formation, Degradation and Action in
Peripheral, Normal and Neoplastic tissues (H. Bradlow, L.
Castagnetta, S. d'Aquino, L. Gogliotti, eds) Ann. N.Y. Acad. Sci.
586: 93-100, 1990. Serum IGF-1 levels can be measured as described
(Furlanetto et al., J. Clin. Invest. 60: 648, 1977).
[0056] In accordance with one aspect of the invention, once the
deficiency or imbalance is determined, 5-DIOL is preferably
administered at a dosage sufficient to raise and maintain serum
5-DIOL, concentrations up to 3 times above the normal range of
young adults. Serum Concentrations of 5-DIOL between 4.0 nM and 10
nM for women and 10 to 20 nM for men are preferred, e.g. 7.0 nM for
women and 15 nM for men. Naturally, the attending clinician may
raise or lower dosage based on patient response which may vary
significantly. Intermittent or continuous administration of a
progestin (e.g. medroxyprogesterone acetate, 5-10 mg/day orally)
may alleviate possible unwanted side effects on the endometrium of
the 5-DIOL treatment in premenopausal women
[0057] In some preferred embodiments, serum concentration is
between 4.0 and 7.0 or between 7.0 and 15 nM for women and men,
respectively. However, for purposes of contraception or for
prevention of ovarian or uterine cancer, concentrations up to 15 nM
(e.g. between 10 to 13) may be preferred for women. For
contraception, an estrogen may be added (e.g. estradiol giving
serum estradiol levels between 50 and 200 nanograms per liter), and
an added progestin may be particularly appropriate. Preferred
dosages discussed herein may be increased as appropriate to achieve
desired serum concentrations, e.g. with variations for individuals
patient response as monitored by the attending clinician.
[0058] When 5-DIOL is administered by the percutaneous or
transmucosal technique, the delivered dosage may be raised or
lowered in known ways, i.e. by altering the location to which the
lotion, ointment, cream, gel or patch is applied by altering the
size of the surface area to which it is applied, by altering the
concentration of the active ingredient, or by altering the vehicle
or carrier. For example, increasing the surface area will normally
increase the dosage of active ingredient delivered if the
concentration of active ingredient remains constant. In the same
manner, dosage delivered increases with increased concentration of
active ingredient in the delivery base, and decreases with
decreased concentration. Dosage delivered into the bloodstream also
varies in a known manner with respect to the body region at which
the transdermal penetration system is applied to the skin. Changing
the vehicle or carrier can also alter he delivered dosage in known
ways.
[0059] Preferably, serum 5-DIOL concentration is measured before
treatment begins, and a dosage is selected to quickly raise serum
5-DIOL concentration to he preferred target range between 4.0 and
10 nM for women and 10 to 20 nM for men. Subsequently, the patient
is monitored both symptomatologically and by circulating 5-DIOL or
sex steroid metabolite concentrations to verify that the desired
serum concentration and symptomatic relief have been obtained.
5-DIOL is then maintained at a constant concentration in the
circulation. For a typical postmenopausal patient, for example,
this dosage is the equivalent of application of 400 mg of the
5-DIOL, as part of a 10 percent composition in 50% ethanol-50%
propylene glycol, to a 200 square centimeter area of the abdomen or
thighs two times daily per 50 kg of body weight. If oral
administration is chosen, 500 mg should be administered twice daily
per 50 kg of body weight.
[0060] As used in the invention, 5-DIOL may be administered with or
without additional carrier or diluent by the oral route but
requires an additional carrier or diluent when administered by the
percutaneous or transmucosal route. In a pharmaceutical composition
for oral administration, 5-DIOL is preferably present in a
concentration between 5 and 99% by weight relative to total weight
of the composition, more preferably between 50 and 99 percent,
especially between 80 and 99 percent.
[0061] When prepared for percutaneous administration, 5-DIOL is
preferably present in a concentration between 2 and 20% by weight
relative to the total weight of the composition, more preferably
between 5 and 15%, especially between 5 and 10%.
[0062] The 5-DIOL active ingredient may be obtained from Steraloids
Inc. (P.O. Box 310, Wilton, N.H., 03086, USA). Preferred 5-diol
prodrugs are the two compounds set forth below, commercially
available from Steraloids Inc. 1
[0063] 5-DIOL may be administered alone or may be administered in
combination with other active compounds, such as antiestrogens,
progestins, androgens, estrogens, DHEA or DHEA-S, inhibitors of
17.beta.-hydroxysteroid dehydrogenase, aromatase inhibitors, LHRH
agonists or antagonists and other inhibitors of gonadal steroid
secretion. Both DHEA and 5-DIOL are metabolized to androgens and
estrogens, but in different proportions. In addition, 5-DIOL
possesses weak intrinsic estrogenic activity. That, in conjunction
with the different inherent action DHEA or 5-DIOL have on the
androgen or estrogen receptor, is believed to impart the different
ratios of androgenic versus estrogenic activity provided by 5-DIOL
versus DHEA. It is not necessary to utilize only DHEA's
androgen/estrogen ratio for only 5-DIOL's androgen/estrogen ratio.
DHEA and 5-DIOL may be used together to provide an optimally
effective androgenic/estrogenic response ratio that is between the
ratio for DHEA and the ratio or 5 -DIOL. The relative amounts of
DHEA versus 5-DIOL may be varied depending on whether the desired
androgen/estrogen response ratio lies closest to that of DHEA or to
that of 5-DIOL.
[0064] For the treatment of breast cancer, endometrial cancer,
ovarian cancer, endometriosis or other estrogen-sensitive disease
requiring blockade of estrogen formation and/or action, at least
one of the following, namely an antiestrogen, an aromatase
inhibitor, an androgenic compound, a progestin, an LHRH agonist or
antagonist or another inhibitor of gonadal sex steroid secretion,
an inhibitor of 17.beta.-hydroxysteroid dehydrogenase activity can
be used in combination with 5-DIOL. 5-DIOL alone or with DHEA would
provide the androgenic component required to stimulate
androgen-sensitive functions, particularly bone formation and
inhibition of androgen-sensitive cancer growth (e.g. breast and
endometrial cancer). 5-DIOL could then be used alone or in
combination with any of the compounds mentioned above useful in the
combination. In some cases, DHEA in the absence of 5-DIOL could be
used in combination with any of the compounds mentioned above.
[0065] In another embodiment, 5-DIOL is combined with an
antiestrogen, which is preferably EM-800
((-)-7-pivaloyloxy-3-(4'-pivaloyloyloxyphenyl)- -4-methyl-2
(4'"-2(2-piperininoethoxy)phenyl)-2H-benzopyran), ICI 182, 780
(7a-[9-4(4,4,5,5,5-pentafluoro-pentylsulphinyl)nonyl]oestra-1,3,5(10)-tri-
ene-3,17.beta.-diol) or any other antiestrogen for the treatment of
breast cancer, endometrial cancer, ovarian cancer, cardiovascular
diseases, atherosclerosis, and other estrogen-sensitive diseases.
Non-steroidal antiestrogens such as EM-800 tend to be selective
estrogen receptor modulators which act as estrogen receptor
antagonists in breast tissue, yet provide estrogen-like beneficial
effects on cholesterol, lipids and atherosclerosis.
[0066] The dosage of 5-DIOL can vary. The blood level of 5-DIOL and
of other sex steroids and their metabolites is indicative of the
adequate dosage taking into account individual variation in
absorption, metabolism, and sensitivity of response. Preferably,
the attending clinician will, especially at the beginning of
treatment, monitor an individual patient's overall response and
serum levels of 5-DIOL (in comparison to the preferred serum
concentration discussed above), and monitor the patient's overall
response to treatment, adjusting dosages as necessary where a given
patient's metabolism or reaction to treatment is atypical. For
combination therapies, one approach would be to start treating with
5-DIOL alone and to add the other compounds only if necessary. For
treatment of breast cancer, endometrial cancer, ovarian cancer, and
endometriosis, treatment with antiestrogen+5-DIOL or
antiestrogen+5-DIOL +DHEA or antiestrogen+DHEA are started
simultaneously. If DHEA androgen, progestin or estrogen is added,
similar monitoring of overall serum levels, both of the active
ingredients and androgenic or estrogenic metabolites is preferred
during early stages of treatment and as judged useful by the
physician at later time intervals.
[0067] Treatment in accordance with the invention is suitable for
indefinite continuation. It is expected that 5-DIOL treatment will
usually simply maintain this natural steroid within a range of 4 to
10 nM and 10 to 20 nM serum concentration for women and men,
respectively. Undesirable side effects from sustained 5-DIOL
treatment are expected to be either minimal or nonexistent.
Avoiding unlikely side effects from sustained estrogen use may be
achieved in ways already known to the art, for example, by
intermittent (or in some embodiments continuous) administration of
a progestin (e.g. medroxyprogesterone acetate) at a daily oral dose
of 2 to 10 mg. Any androgenic side effects should be minimal due to
the relatively low androgenic effects of 5-DIOL and the already low
levels of DHEA in most patients undergoing the method of the
invention (FIG. 18).
[0068] In order to facilitate the combination therapy aspect of the
invention, for any indication discussed herein, the invention
contemplates pharmaceutical compositions which include both 5-DIOL
and a second or subsequent active compound(s) in a single
composition for simultaneous administration. The composition may be
suitable for administration in any traditional manner including but
not limited to oral administration, percutaneous administration or
transmucosal administration. In other embodiments, a kit is
provided wherein the kin includes 5-DIOL and a second compound in
separate containers. Additional active compounds discussed herein
may also be included. In addition to other modes of administration,
the second compound as well as 5-DIOL may also be administered
transdermally in accordance with the invention as discussed in more
detail below. Thus, the kit may include appropriate materials for
transdermal administration, e.g., ointments, lotions, gels, creams,
sustained release patches and the like. The same strategy applies
to the progestin, antiestrogen, androgen, estrogen, DHEA, DHEA-S,
inhibitor of 17.beta.-hydroxysteroid dehydrogenase, aromatase
inhibitor or inhibitor of gonadal sex steroid secretion which can
be administered orally (or by injection for the LHRH agonist or
antagonist).
[0069] Although, it is anticipated that in some circumstances
5-DIOL may be administered by injection, this method is not
favored. Since treatment with 5-DIOL will often be of prolonged and
indefinite duration, repeated delivery by injection is
inconvenient.
[0070] It is believed that the preferred routes for therapeutic
administration of 5-DIOL are percutaneous, transmucosal or oral,
since the discomfort and inconvenience of administering 5-DIOL by
injection are avoided.
[0071] Any of a number of art-recognized transdermal penetration
systems may be utilized for the delivery of 5-DIOL. For example,
5-DIOL may be prepared as part of an ointment, lotion, gel or cream
for rubbing onto a patient's skin or mucosa. The active ingredient
is preferably present from approximately 5% to 20% by weight
relative to the total weight of the pharmaceutical composition and
more preferably is between approximately 5 to 12% by weight.
Alternatively, the active ingredient may be placed into a
transdermal patch having structures known in the art, for example,
structures such as those set forth in E.P. Patent No. 0279982.
[0072] When formulated as an ointment, lotion, gel, cream or the
like, the active compound is admixed with a suitable carrier which
enhances transdermal or transmucosal penetration of the compound
through the skin or mucosa. Suitable carriers are known in the art
and include but are not limited to Klucel HF and Glaxal base which
is available from Glaxal Canada Limited Company. Other suitable
vehicles can be found in Koller and Buri, S.T.P. Pharma 3(2),
115-124, 1987. The carrier is preferably one in which the active
ingredient(s) is(are) soluble at ambient temperature at the
concentration of active ingredient that is used. The carrier should
have sufficient viscosity to maintain the precursor on a localized
area of skin or mucosa to which the composition has been applied,
without running or evaporating for a time period sufficient to
permit substantial penetration of the precursor through the
localized area of skin or mucosa and into the bloodstream where it
will cause measurable and desired increase in serum 5-DIOL
concentration. The carrier is typically a mixture of several
components, e.g. pharmaceutically acceptable solvents and a
thickening agent. A mixture of organic and inorganic solvents can
aid hydrophilic and lipophilic solubility, e.g. water and an
alcohol such as ethanol.
[0073] Desirably, the carrier is one which, if formulated as 10%
5-DIOL and 90% carrier (by weight) and applied twice daily in an
amount providing 100 mg of 5-DIOL to the abdominal area, will
elevate serum concentration of 5-DIOL in a typical patient by at
least 1.0 nM per 50 kg of body weight above serum levels prior to
treatment, and thereafter maintain relatively constant serum levels
of 5-DIOL.
[0074] The carrier may include various additives commonly used in
ointments, lotions, gels, and creams and well known in the cosmetic
and medical arts. For example, fragrances, antioxidants, perfumes,
gelling agents, thickening agents such as carboxymethylcellulose,
surfactants, stabilizers, emollients, coloring agents and other
similar agents may be present. When used to treat systemic
diseases, the site of application on the skin is preferably changed
periodically to avoid potential excess local concentration of
steroids and possible overstimulation of he skin and sebaceous
glands by androgenic metabolites of 5-DIOL.
[0075] 5-DIOL or derivatives can also be administered, by the oral
route, and may be formulated with conventional pharmaceutical
excipients, e.g. spray dried lactose and magnesium stearate into
tablets or capsules or oral administration at concentrations
providing easy dosage in a range from 0.050 to 2.5 grams per day
per 50 kg of body weight.
[0076] The active substance can be worked into tablets or dragee
cores by being mixed with solid, pulverulent carrier substances,
such as sodium citrate, calcium carbonate or dicalcium phosphate,
and binders such as polyvinyl pyrrolidone, gelatin or cellulose
derivatives, possibly by adding also lubricants such as magnesium
stearate, sodium lauryl sulfate, "Carbowax" or polyethylene glycol.
Of course, taste-improving substances can be added in the case of
oral administration forms. The active substance can be also
administered in solid dispersion state in appropriate carriers.
Such carriers may be chosen from the group consisting of
polyethylene glycols of molecular weight varying from 1000 to 20000
and polyvinylpyrrolidone (Providone purchased from American
Chemicals Ltd., Montral, Canada).
[0077] As further forms, one can use plug capsules, e.g. of hard
gelatin, as well as closed soft-gelatin capsules comprising a
softener or plasticized, e.g. glycerine. The plug capsules contain
the active substance preferably in the form of granulate, e.g. in
mixture with fillers, such as lactose, saccharose, mannitol,
starches, such as potato starch or amylopectin, cellulose
derivatives or highly dispersed silicic acids. In solf-gelatin
capsules, the active substance is preferably dissolved or suspended
in suitable liquids, such as vegetable oils or liquid polyethylene
glycols.
[0078] The concentration of active ingredients in the ointment,
cream, gel or lotion is typically from about 2 to 20 percent
preferably between 5 and 15 percent and preferably between 5 and 10
percent (by weight relative to the total weight of the lotion,
cream, gel or ointment). Within the preferred ranges, higher
concentrations allow a suitable dosage to be achieved while
applying the lotion, ointment, gel or cream to a lesser surface
area of the skin than would be possible at lower concentrations and
allow more freedom in choosing the body parts to which the ointment
or lotion will be applied. For example, it is well known in the art
that a compound which is capable of transdermal penetration
normally penetrates more efficiently at some points in the body
than in others. For example, penetration is very efficient on the
forearm and considerable less efficient on the palms.
[0079] The lotion, ointment, gel or cream should be thoroughly
rubbed into the skin so that no excess is plainly visible, and the
skin would not be washed in that region until most of the
transdermal penetration has occurred, preferably, at least 15
minutes and, more preferable, at least 30 minutes after
application.
[0080] A transdermal patch may be used to deliver 5-DIOL in
accordance with known techniques. It is typically applied for a
much longer period, e.g. 0.5 to 4 days, but typically contacts
active ingredients to a smaller surface area, allowing a slow and
constant delivery of active ingredient.
[0081] A number of transdermal drug delivery systems that have been
developed, and are in use, are suitable or delivering the active
ingredient of the present invention. The rate of release is
typically controlled by a matrix diffusion, or by passage of the
active ingredient through a controlling membrane.
[0082] Mechanical aspects of transdermal devices are well known in
the art, and are explained, for example, in U.S. Pat. Nos.
4,162,037, 5,154,922, 5,135,480, 4,666,441, 4,624,665, 3,742,951,
3,797,444, 4,568,343, 4,064,654, 5,071,644, 5,071,657, the
disclosures of which are incorporated herein by reference.
Additional background is provided by European Patent 0279982 and
British Patent Application 2185187.
[0083] The device may be any of the general types known in the art
including adhesive matrix and reservoir-type transdermal delivery
devices. The device may include drug-containing matrixes
incorporating fibers which absorb the active ingredient and/or
carrier. In a reservoir-type device, the reservoir may be defined
by a polymer membrane impermeable to the carrier and to the active
ingredient.
[0084] In a transdermal device, the devise itself maintains active
ingredient in contact with the desired localized skin surface. In
such a device, the viscosity of the carrier for active ingredient
is of less concern than with a cream or gel. A solvent system for a
transdermal device may include, for example, oleic acid, linear
alcohol lactate and dipropylene glycol, or other solvent systems
known in the art. The active ingredient may be dissolved or
suspended in the carrier.
[0085] For attachment to the skin, a transdermal patch may be
mounted on a surgical adhesive tape having a hole punched in the
middle. The adhesive is preferably covered by a release liner to
protect it prior to use. Typical material suitable for release
includes polyethylene and polyethylene-coated paper, and preferably
silicone-coated for ease of removal. For applying the device, the
release liner is simply peeled away and the adhesive attached to
the patient's skin. In U.S. Pat. No. 4,135,480, the disclosure of
which is incorporated by reference, Bannon et al. described an
alternative device having a non-adhesive means for securing the
device to the skin.
[0086] Except for the higher dosage indications noted above (e.g.
contraception), the target serum concentration of 5-DIOL is
comparable regardless of whether 5-DIOL is being used as part of a
combination therapy for treatment of menopause or is being used (by
itself or in combination with antiestrogens, androgens, progestins,
estrogens and inhibitors of 17.beta.-hydroxysteroid dehydrogenase,
aromatase inhibitors, inhibitors of gonadal sex steroid formation,
LHRH agonists or antagonists, DHEA and/or DHEA-S) for the treatment
of cardiovascular diseases, osteoporosis, skin deterioration,
menopause, vaginal atrophy, urinary incontinence, uterine cancer,
ovarian cancer, osteoporosis, endometriosis, hypogonadism or
diminished libido in accordance with the invention or for the
treatment of any conditions related to decreases or imbalances in
the levels of sex steroids, in particular 5-DIOL and its
metabolites.
[0087] 5-DIOL is particularly useful in treating conditions in
which a minimal androgenic effect is desired since the androgenic
effects of 5-DIOL are lower than produced by DHEA for an equal
estrogenic effect and the maximal androgenic effect achieved with
5-DIOL is lower than that achieved with DHEA. 5-DIOL is especially
preferred for the treatment of conditions in women that respond to
estrogen therapy (or therapy with an estrogen precursor, such as
DHEA) since the androgenic action of 5-DIOL is lower than that of
DHEA and therefore, the potential androgenic or masculinizing
effects are reduced, while the desired estrogenic activity is
provided. Moreover, in postmenopausal women, in general, a greater
estrogenic/androgenic ratio than that provided by DHEA is
required.
[0088] Since 5-DIOL is a natural source of estrogens and androgens
and the secretion of this compound markedly decreases during aging
(FIG. 17), its replacement should have minimal unwanted side
effects. Its intrinsic estrogenic activity should compensate for
the loss of estrogen secretion by the ovaries after menopause, an
effect not achievable by DHEA. The invention is useful for many
diseases wherein activation of the estrogen receptor will have
beneficial effects, especially osteoporosis and menopausal
symptoms, including vaginal atrophy, insomnia, irritability,
cardiovascular disease, urinary incontinence, and loss of libido.
In addition, the invention is useful for treating and preventing
diseases which are responsive to the activation of the androgen
receptor, e.g. bone loss, obesity, breast cancer, endometrial
cancer, ovarian cancer, urinary incontinence, hypogonadism, loss of
libido, loss of muscle mass, loss of energy, insulin resistance and
other aging processes. Further, 5-DIOL can be used to treat or
prevent any condition which responds favorably to an improvement in
the overall balance of circulating sex steroids, namely estrogens
and androgens.
[0089] Conditions expected to respond to the treatments herein may
be diagnosed in conventional ways. For example, the appearance of
breast cancer is usually detected by self breast examination,
clinical breast examination by the physician and/or mammography.
Endometrial cancer, on the other hand, is usually diagnosed by
endometrial biopsy. Both cancers can be diagnosed and evaluated by
standard physical methods well known to those skilled in the art,
e.g. bone scan, chest X-Ray, skeletal survey, ultrasonography of
the liver and liver scan (if needed), CAT scan, MRI and physical
examination.
[0090] The onset of menopause is generally first recognized by the
occurrence of hot flashes. Further characterization of the
menopause can be determined in accordance with known techniques.
See for Example, The Menopause (Herbert J. Buchsbaurm, ed),
Springer Verlag, New York (1983), pp. 222. Vaginal atrophy is often
indicated by dyspareunia and vaginal infections. Vaginal atrophy,
hypogonadism, diminished libido, insomnia, irritability,
depression, and urinary incontinence are all characterized in
well-known ways. For the above-indicated disease, see, or example,
Korenman, Stanley G, "Sexual Dysfunctions" in Williams Textbook of
Endocrinology, Jean D. Wilson and Daniel W. Foster, eds), WB
Saunders Co., Philadelphia, pp. 1033-1048, 1992.
[0091] Bone density, on the other hand, can be measured by standard
methods well known to those skilled in the art, e.g. QDR
(Quantitative Digital Radiography), dual photon absorptiometry and
computerized tomography. Plasma and urinary calcium and phosphate
levels, plasma alkaline phosphatase, osteocalcin, calcitonin and
parathormone concentrations, as well as urinary hydroxyproline,
deoxyprrolidine, and calcium/creatinin ratios are useful parameters
of bone formation and resorption.
[0092] Loss of collagen or connective tissues in the skin often
accompanies aging, especially in persons over 50 years of age. It
may be evidenced by wrinkling of the skin and/or low elasticity.
Skin status can be assessed by visual inspection, palpation and,
with more precision, by punch biopsy and standard histological
examination.
[0093] The normal range of body weight is well known to those
skilled in the art, while cholesterol and lipoproteins are
routinely measured by standard techniques (Nestler et al. J. Clin.
Endocrinol. Metab. 66: 57-61, 1988 for references).
[0094] In addition, 5-DIOL is useful as a female contraceptive. In
the prior art, female contraception usually involves administering
an estrogen, which at increased circulating levels, reduces LHRH
secretion from the hypothalamus which, in turn, decreases LH
secretion from the pituitary. The resultant reduction in LH
secretion decreased ovarian function, and in particular ovulation.
Addition of a progestin controlled the growth of the endometrium
and transformed the vaginal and cervical secretions into an
unfavorable environment for sperm capacitation and fertility.
[0095] In the present invention, 5-DIOL provides estrogen for
contraception while simultaneously and desirably providing
minimally increased levels of androgens which will contribute to
contraception since androgens also inhibit LHRH and LH secretion.
These androgens can, especially in women at perimenopause (as well
as in postmenopausal women when contraception is no longer
required), provide much needed stimulation of bone formation and
resistance to bone loss. In addition to being a weak estrogen by
itself, the estrogens produced from the administered 5-DIOL also
contribute to reducing bone loss. As with other uses discussed
herein, use of 5-DIOL instead of a sex steroid (here estrogen)
avoids externally administering relatively high doses of estrogens
and this avoids giving such estrogens extensive access to all
tissues, many of which do not require estrogens. By substituting
5-DIOL, estrogens and/or androgens are instead produced by natural
processes in the same tissues where estrogens and/or androgens are
needed and that normally convert 5-DIOL to estrogens and/or
androgens. The relative proportions of estrogen and androgen also
remain substantially at natural levels in each specific tissue.
[0096] Because ovarian function is diminished by the contraception
technique described herein, the ovarian production of estrogen an
progesterone will be decreased. Thus, a progestin (e.g.
medroxyprogesterone acetate, megestrol acetate, norethynodrel,
L-norgestrel) may be administered as part of the contraceptive
method to prevent endometrial hypertrophy when high doses of 5-DIOL
are needed. The progestin may be administered in a pharmaceutical
composition that includes the 5-DIOL or separately. In certain
embodiments, the progestin may be administered intermittently every
month for 12-14 days, or 12-14 days every few months (e.g. every
2-5 months) or continuously, depending upon the dose of 5-DIOL used
which may well have no stimulatory effect on the endometrium at
physiological dose. Progestin dosage may be in the range utilized
in the prior art but is preferably lower for reasons explained
below.
[0097] Since 5-DIOL is converted to estrogen in many tissues, it is
unlikely that estrogen will need to be added to the contraceptive
therapy to compensate for the decreased estrogen production in the
ovaries. However, minimum doses can be given, if necessary.
Preferred dosage of added estrogen, when used in the contraceptive
method is an amount effective to achieve between 50 and 300
nanograms estradiol per liter or equivalent. Preferably the ratio
of added estradiol to 5-DIOL (w/w) will range from 100:1 to
10,000:1, preferably, 200:1 to 5,000:1 and especially 300:1 to
3000:1. As with added progestin, added estrogen may be administered
as part of a pharmaceutical composition that includes the 5-DIOL
(or, where used, a prodrug of 5-DIOL) or separately.
[0098] In some embodiments, 5-DIOL, progestin and estrogen are all
administered, together or separately, as part of a combination
therapy. A combination therapy results whenever a regimen of
treatment elevates blood levels of each active agent
simultaneously. This simply requires that the active agents be
administered sufficiently close in time that elevated blood levels
of these agents are concurrent.
[0099] The use of combination contraceptives containing estrogens
and progestins has not been shown to reduce the risk of breast
cancer (Romiev et al., 1990, Cancer 66: 2253-2263). These data are
consistent with a known mitogenic effect of both estrogen and
progesterone on breast cell epithelial proliferation, thus
explaining a peak of cell proliferation at mid-luteal phase
(Masters et al., J. Natl. Cancer Inst. 1977, 58: 1263-1265;
Anderson et al., 1982, Brit. J. Cancer 46 376-382). In fact, total
breast cell proliferation rate in premenopausal women using
contraceptives is not different from that of untreated cycling
women (Potter et al., 1988; Brit. J. Cancer 58: 163-170; Going et
al., 1988; Am. J. Pathol. 130: 193-204). The androgenic component
of 5-DIOL should reduce this potential harmful effect of estrogens
and progestins.
[0100] Osteoblasts (bone Forming cells) contain the enzymes which
convert 5-DIOL to estrogens and androgens. Therefore, 5-DIOL can be
used instead of (or in addition to) androgen, estrogen, or DHEA in
the treatment or prevention of osteoporosis. Sufficient quantities
of androgens are produced in the bone (by conversion of the
administered 5-DIOL) to stimulate bone formation and reduce bone
loss. Furthermore, the low estrogenic activity of 5-DIOL and the
estrogens produced from the administered 5-DIOL contribute to
reducing bone loss.
[0101] Since bone mass density has been shown to be stimulated at
particularly low doses of androgens, 5-DIOL, which has a lower
ratio of androgenic/estrogenic activity than DHEA should be able to
have maximal beneficial effects on the bone at a dosage which
produces minimal risks of hyperandrogenism.
[0102] In addition, since 5-DIOL (or prodrugs thereof, if desired)
is transformed to androgens and estrogens only by natural
mechanisms exclusively in tissues that normally perform such
transformation according to their local needs, side effects are
greatly reduced or eliminated relative to externally administered
active sex steroids of the prior art which have access to many
tissues that neither produce nor require a given androgen or
estrogen. The physiological balance of sex steroids in those
tissues are thus not disturbed in accordance with the present
invention, contrary to all hormone replacement therapies of prior
art. The relative ratio of androgens and estrogens produced from
the 5-DIOL is also a substantially normal ratio instead of being an
abnormally elevated ratio of one type of sex steroid as occurs when
that active sex steroid is directly administered exogenously, thus
causing exposure of all tissues, including those having no need for
such therapy.
[0103] In one preferred treatment for menopausal symptoms, the
invention seeks to simultaneously maintain blood levels of 5-DIOL,
androgens, and estrogens within normal premenopausal parameters.
Without intending to be bound by theory, it is believed that
maintenance of appropriate precursor levels will better enable
natural enzymes, such as 17.beta.-hydroxysteroid dehydrogenase,
3.beta.-hydroxysteroid hydrogenase, aromatase and
5.alpha.-reductase to regulate production of androgens and
estrogens and to maintain them in a matter more closely resembling
their absolute and relative levels prevailing prior to menopause.
Hence, the invention contemplates that not only estrogens but also
androgens will be kept in better balance. In fact, the target
tissues possess the enzymatic machinery necessary to synthesize and
inactivate androgens and/or estrogens according to local needs
(Labrie, Mol. Cell. Endocrinol. 78, C113-C118, 1991).
[0104] As discussed above, 5-DIOL can be administered with
estrogens. However, since compared to DHEA, a relatively higher
proportion of estrogens than androgens are produced and 5-DIOL is
itself a weak estrogen, it should be possible to attain the desired
level of estrogens without the addition of estrogens and without
producing unwanted androgenic side effects accompanying high levels
of DHEA, a more androgenic compound. Similarly, since 5-DIOL is a
weak estrogen, progestin therapy may not be required.
[0105] However, if it is determined that additional estrogens are
needed, the estrogen and 5-DIOL may be administered simultaneously
or separately. In addition, it is necessary only that both the
5-DIOL and estrogen be administered in a manner and at a dosage
sufficient to allow blood serum concentration of each to obtain
desired levels. In accordance with the combination therapy of the
invention, concentration of the 5-DIOL is maintained within desired
parameters at the same time that estrogen concentration is
maintained within desired parameters. Where estradiol is used,
serum estradiol concentration should typically be maintained
between 50 and 200 nanograms per liter, preferably between 100 and
175 nanograms per liter and most preferably between 125 and 175
nanograms per liter. Where another estrogen is used, serum
concentration may be varied in a known manner to account for the
known difference in estrogenic activity relative to estradiol and
in order to achieve normal premenopausal estrogen levels. A lesser
concentration is needed, for example, if Mestranol is used.
Adequate serum estrogen levels can also be assessed by
disappearance of the symptoms of menopause. Serum concentration of
the 5-DIOL is typically maintained between 4 and 10 nM for women
and between 10 and 20 nM for men or in some embodiments between 4.0
and 7.0 nM or women or between 7.0 and 15 nM for men.
[0106] If estrogen is combined with 5-DIOL, it is preferably
estradiol, but may be estrone sulfate or another compound which
acts as an estrogen receptor agonist directly or following proper
conversion. When administered separately, commercially available
estrogen supplements may be used, e.g., PREMARIN, available from
Ayerst (St. Laurent, Qubec, Canada). For typical patients, the
appropriate dosage of estrogen to achieve desired serum
concentrations is between 0.3 and 2.5 milligrams of PREMARIN per
day per 50 kg of body weight when administered orally. In certain
embodiments of the invention, the estrogen may be
17.beta.-estradiol administered percutaneously in a patch which is
available from CIBA under the name ESTRADERM wherein the daily
doses is between 0.05 and 0.2 milligrams per day per 50 kg of body
weight. For typical patients, the appropriate dosage of the sex
steroid precursor 5-DIOL to achieve desired serum concentration of
the precursor is between 0.10 and 2.5 grams per day per 50 kg of
body weight when administered orally. Other prodrugs will be
administered at a dosage that depends on their in vivo conversion
rate to 5-DIOL. 5-DIOL may also be administered transdermally or
transmucosally, as described in detail above, in a sufficient
amount to achieve target serum concentration.
[0107] In another embodiment, menopause is treated with 5-DIOL as
set forth above, in combination with periodic administration of a
progestin such as medroxyprogesterone acetate (e.g., Provera) which
is preferably administered intermittently, e.g. at a dosage of 2-10
mg per day for 12 consecutive days, said 12-day periods being
spaced 20 days to 5 months apart. A combination therapy using
5-DIOL, an estrogen and a progestin may also be used, preferably at
the dosages discussed herein for each component.
[0108] The same doses of 5-DIOL will be used for all indications,
except contraception and prevention of ovarian and endometrial
cancer where serum levels of about to 15 nM 5-DIOL will be
preferred.
[0109] For all other indications when androgens and/or estrogens
are needed, the usual dosage mentioned above will be used.
Similarly, when used in combination with an antiestrogen for the
treatment or prevention of breast cancer, endometriosis, as other
estrogen-sensitive disease, the same dose of 5-DIOL will be used.
In some cases, where an aromatase inhibitor, an inhibitor of
17.beta.-hydroxysteroid dehydrogenase, an androgen, a progestin, an
inhibitor of gonadal steroid formation, an LHRH agonist or
antagonist is used, the same dose of 5-DIOL is recommended.
[0110] The following examples demonstrate the androgenic and
estrogenic effects of 5-DIOL and provide a comparison of the
relative activities of 5-DIOL and DHEA. As discussed above, the
preferential estrogenic activity relative to androgenic activity of
5-DIOL compared to DHEA is shown.
[0111] Materials and Methods
[0112] Animals
[0113] Male and female Sprague-Dawley rats [Crl:CD(SD)Br] weighing
225-250 g and 175-200 g, respectively, were obtained from Charles
River Canada Inc. (St-Constant, Qubec) and housed 2 per cage under
a regimen of 14 h of light/day (lights on at 07:15 h). Animals
received Purina rat chow and water ad libitum. The animal studies
were conducted to the "Guideline for Care and Use of Experimental
Animals".
[0114] Treatment
[0115] The animals were randomly divided into the indicated groups
(8 and 10 rats per group for subcutaneous and topical
administration, respectively). The animals of the appropriate
groups were bilaterally ovariectomized (OVX) or orchiectomized
(ORCH) under ether anesthesia while other rats were used as intact
controls. DHEA and 5-DIOL obtained from Steraloids (Wilton, N.H.,
USA) were dissolved in 50% ethanol-50% propylene glycol and applied
twice daily (0.5 ml) on the dorsal skin area (2 cm.times.2 cm) for
7 days starting on the day of OVX or ORCH. For subcutaneous
administration, DHEA and 5-DIOL were dissolved in 0.5 ml 10%
ethanol-1% gelatin-0.9% NaCl and injected twice daily in the dorsal
area for 7 days starting also on the day of OVX or ORCH.
[0116] Seven days after starting treatment or approximately 12 h
after last administration of the steroid, the animals were killed
by decapitation. Blood samples were collected individually and
serum was frozen at -20.degree.C. until assayed. Uteri, ovaries,
ventral prostates, dorsal prostates and seminal vesicles were
immediately removed, freed from connective and adipose tissue,
weighed, frozen in liquid nitrogen, and stored at -80.degree.C.
until assayed. Three rats from the indicated treatment groups were
perfused with paraformaldehyde for in situ hybridization.
[0117] Steroid Analysis
[0118] Steroid extraction, Ethanol (5 ml was added to 1 ml serum
and centrifugation at 2000.times.g was performed for 15 min. The
resulting pellet was further extracted with 2 ml ethanol and, after
a second centrifugation at 2000.times.g for 15 min, the two
supernatants were combined. The suspension was recentrifuged as
described above and the supernatant was decanted and combined with
the previously obtained ethanol extracts. The organic solvent was
then evaporated under nitrogen and the residue was dissolved in 1
ml water: methanol (95:5, v/v). The C-18 columns (Bound-Elut,
Amersham, Bucks, U.K.) were conditioned by passing consecutively 10
ml methanol, 10 ml water and 10 ml methanol/water (5:95, v/v). The
extracts solubilized in water: methanol (95:5, v/v) were t hen
deposited on the C-18 columns. After washing the columns with 2 ml
water: methanol (95:5, v/v) and 5 ml methanol: water (50:50, v/v),
5 ml methanol: water (85:15, v/v) were added to eluate the
non-conjugated steroids
[0119] Chromatography on LH-20 Columns and Radioimmunoassay.
[0120] Chromatography on Sephadex LH-20 columns (Pharmacia,
Uppsala, Sweden) was performed as previously described (Blanger et
al., 1988). In brief, steroids were solubilized in 1 ml
isooctane/toluene/methanol (90:5:5, v:v:v) and deposited on the
LH-20 columns. Fractions were collected and, after evaporation of
the organic solvent, the concentration of the various steroids was
determined by radioimmmunoassay as previously described (Blanger et
al., 1980; Blanger et al., 1988; Blanger et al., 1990).
[0121] Preparation of the cDNA Probes
[0122] The plasmid containing the DNA fragment complementary to the
mRNA encoding PBP-C1 was kindly provided by Dr. Malcolm G. Parker
(Imperial Cancer Research Fund, London, United Kingdom). The
434-basepair Pst-I restriction fragment of the PBP-C1 cDNA was
purified by electroelution (Bio-Rad electro-eluter, model 422,
Bio-Rad, Richmond, Calif.; after electrophoresis on a 5% (wt/vol)
polyacrylamide gel. The purified fragment was radiolabeled with
[a-.sup.35S]dCTPaS (Amersham, Oakville, Ontario, Canada) to high
specific activity (10.sup.9 dpm/.mu.g) by the random primer method
(Feinberg and Vogelstein, 1983).
[0123] Measurement of PBP-C1 mRNA Levels by in Situ
Hybridization
[0124] In situ hybridization of prostatic sections with the PBP-C1
probe was performed as described previously (Pelletier et al.,
1988). In brief, rats were perfused with fixation buffer consisting
of 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). The
ventral prostates freed from fat and connective tissue, were
postfixed in fixation buffer for 2 h at 4 C and subsequently soaked
in 0.05 M PBS containing 15% (wt/vol) sucrose. Thereafter, the
ventral prostates were rapidly frozen in isopentane chilled in
liquid nitrogen. Multiple (six to eight) 10 .mu.M tissue sections
from each ventral prostate were mounted on gelatin-coated glass
slides. Prehybridization buffer contained 50% formamide,
5.times.SSPE (1.times.SSPE=0.18 M NaCl, 10 mM NaH.sub.2PO.sub.1,
and 1 mM EDTA, pH 7.4), 0.1% sodium dodecyl sulfate, 0.1% BSA, 0.1%
Ficoll, 0.1% polyvinyl pyrrolidone, 0.2 mg/ml yeast tRNA, 0.2 mg/ml
denatured salmon testis DNA, and 2 .mu.g/ml poly(A). The slides
were hybridized in prehybridization buffer containing, in addition,
4% dextran sulfate and saturating concentrations
(1.0-1.5.times.10.sup.7 cpm/ml) of the PBP-C1 cDNA probe for 48 h
at 37 C. The sections were then washed twice for 30 min in
2.times.SSC (1.times.SSC=0.15 M NaCl and 0.015 M sodium citrate, pH
7.0), dehydrated, and exposed for autoradiography. To determine the
amount of nonspecific background hybridization, prostatic sections
for each treatment group were treated with pancreatin
ribonuclease-A (20 .mu.g/ml) for 1 h at room temperature before
prehybridizing sections from rat brain, pituitary, kidney, and
liver with the PBP-C1 probe. No specific hybridization could be
observed (data not shown).
[0125] LH Radioimmunoassay
[0126] Serum LH was measured by double-antibody radioimmunoassay
using rat hormones (LH-I-6 for iodination; LH-RP-2 as standard),
and the rabbit antisera anti-r-LH-S-8, generously supplied by the
National Pituitary Program, Baltimore, USA.
[0127] Statistical Analyses
[0128] Radioimmunoassay data were analyzed using a program based on
model II of Rodbard and Lewald (Rodbard, 1974). Plasma steroid
levels are shown as the means.+-.SEM of duplicate determinations of
individual samples. In situ hybridization data were obtained as
follows: for each prostatic tissue section, 20 randomly selected
areas measuring 0.25 mm.sup.2 (excluding acinar lumen) were
analyzed using an Image Research Analysis System (Amersham,
Arlington Heights, Ill.), and the mean optical density value for
each section was calculated. Data are shown as the means.+-.SEM of
20 readings from 6-8 prostatic sections originating from ventral 3
prostates. Statistical significance was measured according to the
multiple range test of Duncan-Kramer (Kramer, 1956).
[0129] As shown in FIG. 1, DHEA and 5-DIOL produced similar
stimulatory effects on uterine weight, an indicator of estrogenic
activity. In particular, the administration of the 10 mg and 30 mg
doses of DHEA and 5-DIOL produced a comparable stimulation of
uterine weight in the ovariectomized rat, while 1 and 3 mg doses
had no significant effect. However, at 100 mg, the highest dose
used, 5-DIOL produced a greater stimulatory effect with a maximal
52% reversal of the inhibitory effect of the ovariectomy as
compared to the maximal 42% reversal produced by DHEA.
[0130] The effects of 5-DIOL and DHEA on serum LH are shown in FIG.
2. Serum LH is known to be a sensitive indicator of both androgenic
and estrogenic activity based on the findings that serum LH
increases rapidly upon the removal of the predominant inhibitory
feedback action of sex steroids after gonadectomy in male as well
as female animals (Ferland et al., In: Labrie, F., Meites, J., and
Pelletier, G. (eds), Hypothalamus and Endocrine Functions, pp.
191-209. New York: Plenum Press, 1976). At the 10 mg dose, 5-DIOL
produced greater effects and completely reversed the potent
stimulatory effect of ovariectomy to 0.86.+-.0.17 ng/ml while the
corresponding dose of DHEA only caused a 61% (p<0.01) inhibitory
effect. However, at higher doses, DHEA and 5-DIOL were found to
have similar effects, with the 30 mg and 100 mg doses,
respectively, inhibiting serum LH levels by approximately 35% and
70% below the value found in intact control animals.
[0131] The effects on the circulating levels of the primary
steroids and precursors produced by the administration of 5-DIOL
and DHEA are shown in FIGS. 3 through 7. In particular, as shown in
FIG. 3, treatment with DHEA caused serum DHEA to increase from
undetectable levels in control ovariectomized animals to
1.74.+-.0.30 nM (p<0.01), 3.67.+-.0.59 nM (p<0.01),
12.9.+-.3.69 nM (p<0.01) and 39.2.+-.6.5 nM (p<0.01) after
administration of the 3, 10, 30 and 100 mg dosages, respectively.
On the other hand, a relatively constant but low level of serum
DHEA was produced with the same dosages of 5-DIOL.
[0132] FIG. 4 shows the serum levels of 5-DIOL which are produced
after administration with 5-DIOL and DHEA. While at the lower
doses, 1 and 3 mg, 5-DIOL produced higher levels of 5-DIOL, at the
higher doses, there was no significant difference in the levels of
5-DIOL produced by 5-DIOL and DHEA.
[0133] The serum concentrations of 4-dione, testosterone and
dihydrotestosterone (DHT) produced after administration of DHEA and
5-DIOL are shown in FIGS. 5 through 7. The levels of serum 4-dione,
an androgenic indicator, produced by DHEA, ranged, depending on the
dosage, from 30% to 125% higher than produced by 5-DIOL.
[0134] Consistent results were obtained with serum DHT and
testosterone, both indicators of androgenic activity. Specifically,
DHEA produced 30% to 125% higher serum DHT levels than 5-DIOL and
at the highest dose, DHEA produced a 53% greater stimulatory effect
on testosterone than the same dose of 5-DIOL. Furthermore, the
levels of serum DHT produced by 5-DIOL remained substantially
constant and relatively low across the dosage range.
[0135] FIGS. 8 through 11 are directed to the effects of DHEA and
5-DIOL on a variety of well-recognized androgen-sensitive
parameters in the orchiectomized rat. FIG. 8 shows the effects on
ventral prostate weight, wherein at the 10 mg dose, DHEA was able
to reverse by about 75% the inhibitory effect of orchiectomy while
a 150% higher dose (15 mg) of 5-DIOL was only able to produce a 50%
reversal of the effect of castration. In addition, DHEA was 1-fold
more potent in increasing seminal vesicle weight, as shown in FIG.
9.
[0136] Furthermore, as shown in FIGS. 10 and 11, DHEA produced two
to five times the effect produced by 5-DIOL on the concentration of
the mRNAs encoding the C1 and C3 components of prostate binding
protein (PBP) which, as discussed above, are precise indicators of
androgenic activity. Moreover, the maximal levels of C1 and C3 PBP
mRNAs achieved with the highest doses of 5-DIOL were only 17% to
37% of the levels obtained by DHEA.
[0137] FIGS. 12 through 15 show the effects of subcutaneous
administration of DHEA and 5-DIOL on selected parameters described
above. Since the two steroids were injected subcutaneously, these
results provide a more direct measure of the relative estrogenic
and androgenic activities of DHEA and 5-DIOL under conditions of
optimal bioavailability. In particular, as shown in FIG. 12,
5-DIOL, at the lowest dose used, namely 0.3 mg, reversed by 90% the
effect of 1-week ovariectomy on uterine weight (an
estrogen-sensitive parameter) while DHEA, at the same dose, had no
significant effect. However, at the higher dosages, the maximal
stimulatory effect achieved by the two steroids was similar with a
2.8 to 2.9-fold stimulation. Furthermore, as calculated from the
doses giving half-maximal stimulation of uterine weight (.about.2.5
mg for DHEA and 0.5 mg for 5-DIOL), 5-DIOL is approximately 5.0
times more uterotrophic than DHEA following subcutaneous
administration. In addition, as shown in FIG. 13, 5-DIOL was found
to be 5- to 7-fold more potent than DHEA in inhibiting the elevated
serum LH levels indicated by castration.
[0138] As shown in FIG. 14, the maximal stimulatory effects on
prostate and seminal vesicle weights (androgen-sensitive
parameters) obtained with 5-DIOL were about 70% of the values
achieved with DHEA. However, as calculated from the doses giving
half-maximal reversal of the effect of orchiectomy (ED.sub.50),
DHEA and 5-DIOL had approximately an equal potency (i.e. an
ED.sub.50 value of 1 mg). On the other hand, the ED.sub.50 values
of maximal DHEA and 5-DIOL effects were calculated at 2.5 mg and
1.2 mg, respectively, for an estimated 2-fold higher potency of
5-DIOL compared to DHEA. Similarly, as shown in FIG. 15, for
seminal vesicle weight, the maximal stimulatory effect of 5-DIOL
was approximately 70% that of DHEA with half-maximal reversals of
the effect of orchiectomy estimated at 2.5 and 1.2 mg,
respectively, for DHEA and 5-DIOL for an estimated 2-fold high
potency of 5-DIOL. On the prostatic concentrations of C1 and C3 PBP
mRNAs, 5-DIOL was about twice as potent as DHEA. FIG. 16 shows the
effects on serum LH concentration, with 5-DIOL being approximately
10 times more potent than DHEA.
[0139] From the foregoing, it may be seen that, at every
concentration, the ratio of estrogenic to androgenic effects
provided by DHEA is more heavily weighted toward androgenic effects
than is that ratio for 5-DIOL.
[0140] Although the present invention has been described in
relation to particular embodiments thereof, many other variations
and modifications and other uses will be apparent to those skilled
in the art.
* * * * *