HK1037329B - PHARMACEUTICAL COMPOSITIONS AND USES FOR ANDROST-5-ENE-3β,17β-DIOL - Google Patents

Description

Pharmaceutical composition of androst-5-ene-3 beta, 17 beta-diol and use thereof

Background

Technical Field

The present invention relates to pharmaceutical compositions, kits and methods for preventing and treating conditions of reduced or unbalanced steroid concentration and a good response to increased androgen and/or estrogen activity. The present invention uses androst-5-ene-3 β, 17 β -DIOL (hereinafter referred to as 5-DIOL) or a compound that can be converted to 5-DIOL in vivo.

Description of the Related Art

5-DIOL is a compound biosynthesized from DHEA by the action of a reducing 17 β -hydroxysteroid dehydrogenase (17 β -HSD) and is a weak estrogen. It has a higher affinity for the estrogen receptor in the cytoplasm of the anterior pituitary gland in rats than 17 beta-estradiol (E)₂) 95-fold lower (Simard and Labrie, j. steroidbiochem, 26: 539-546, 1987), further confirming data on the same parameters measured from human myometrium and breast cancer tissues (Kreitmann and Bayard, j. step biochem., 11: 1589 1595, 1979; adams et al, Cancer res, 41: 4720 4926, 1981; poulin and Labrie, Cancer res, 46: 4933-4937, 1986).

Concentrations in the range of plasma levels found in adult females, 5-DIOL, increased cell proliferation and progesterone receptor levels in human breast tumor ZR-75-1 cells (which lack 3 β -hydroxysteroid dehydrogenase/D5-D4 isomerase activity) (Poulin and Labrie, Cancer Res., 46: 4933-4937, 1986) and increased estrogen-dependent synthesis of the 52kDa glycoprotein in MCF-7 cells (Adams et al, Cancer Res., 41: 4720-4926, 1981).

In general, it is believed that DHEA and DHEA-S serum levels decrease with age and correspondingly significantly reduce androgen and estrogen formation in the surrounding target tissues and significantly reduce the biochemical and cellular functions induced by sex steroids. Accordingly, DHEA and DHEA-S are useful in the treatment of a variety of conditions associated with reduced and/or unbalanced sex steroid levels. Recently, we found that serum levels of 5-DIOL decreased significantly with increasing age.

Osteoporosis, a condition affecting both men and women, is associated with a decrease in androgens and estrogens. Estrogens are known to reduce the rate of bone degradation, while androgens increase bone mass.

Menopausal symptoms are also associated with a lack of estrogen, and low doses of estrogen, commonly used in premenopausal and menopausal women, can relieve vasomotor symptoms, urogenital atrophy, irritability, insomnia, absence of mental, osteoporosis, and other symptoms associated with menopause.

In addition, breast cancer, cardiovascular disease and insulin resistance are also associated with decreased serum levels of DHEA and DHEA-S, which have been proposed for the prevention or treatment of these conditions. DHEA is also suggested to be beneficial in the treatment and/or prevention of obesity, diabetes, arteriosclerosis, chemically induced breast, skin and colon cancer, autoimmune diseases, alzheimer's disease, memory loss, aging, and to maintain vitality, muscle mass and longevity. The use of DHEA and the benefits of androgen and estrogen therapy are disclosed in International patent publication WO 94/16709.

DHEA and DHEA-S have been proposed to be more beneficial in the treatment of these conditions than standard androgen and estrogen therapy because the action of DHEA and DHEA-S targets tissues that are capable of converting DHEA and DHEA-S into specific sex steroids. However, high doses of DHEA are required to achieve the desired estrogenic and androgenic effects. Most importantly, the androgenic effects of DHEA predominate, and thus the present invention provides a better ratio of estrogen to androgen action, particularly in women, for conditions where a higher ratio of estrogen is required or where the problem of side effects of androgens is present.

The closest current technology is as follows:

WO 9416709A (ENDORECHERCHE INC)1994, 8, 4 (1994-08-04)

WO 9856386A (CAMPONOVO TIZIANO; SUNNIMEX LTD (GB)) 12 months and 17 days 1998-12-17

US-A-5387583(LORIA ROGER M)1995 2, 7 (1998-02-07)

US-A-5-206008(LORLA ROGER)1993 at 27/4 (1993-04-27)

FR 672M (VISMARA) 7, 17 days 1961 (1961-7-17)

US-A-5461042(LORIA ROGER M) 10 months and 24 days 1995 (1995-10-24)

C E BIRD et al: "δ 5-androstenediol: kinetics of postmenopausal metabolism and binding to plasma proteins in normal women "ACTA ENDOCRINOLOGICA, NO, SCANDINAVIAN UNIVERSITYPASS, OSLO, vol.99, No.2, p. 309-313, ISSN: 0001-5598

DAUVOIS et al: "androstenedione and androst-5-ene-3 β, 17 β -diol stimulation of DMBA-induced mammary tumor-aromatase in rats", mammary cancer research and treatment, US, NIGHOFF, BOSTON, vol.13, No.1, pages 61-69, ISSN: 01647-6806

Taurinaire: MED, vol 15, No.9, 1974, pages 583-

ARANEO et al: dehydroepiandrosterone to reduce progressive cutaneous ischemia due to thermal injury J.SURG.RES., vol.59, No.2, 1995, page 250-262

KUIPER et al: "estrogen receptor beta phenotype: a novel estrogen-like mediator IN neuroendocrine systems, FRONTERS IN NEUROENDOCRINOLOGY, vol.19, No.4, 1998, pp.253-286

EP-A-0424954(NIPPON ZOKI PHARMACEUTICAL CO)1991, 5 months and 2 days (1991-05-02)

DORGAN J F et al: "relationship between serum Dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate, AND androstene-3 β, 17 β -diol AND risk of breast cancer in post-menopausal women" canceramidsiology, BIOMARKERS AND PREVENTION (3/1997) 6(3)177-81

YOSHIMOTO S et al: "clinical efficacy of methanon F on women with climacteric symptoms after menopause: its relationship TO the serum level of hormones "HORUMON TO RINSHO. CLINICALENDCORINOLOGY" (8 months of 1983) 31(8) 815-22.

Summary of The Invention

It is an object of the present invention to provide methods of pharmaceutical compositions and kits comprising 5-DIOL or a prodrug capable of being converted to 5-DIOL in vivo. In some embodiments, the pharmaceutical composition consists essentially of 5-DIOL.

It is another object of the present invention to increase 5-DIOL in a patient in need of such treatment or prevention and to provide a method for treating or preventing an imbalance or reduction in sex steroid hormone (androgen and/or estrogen) levels.

It is another object of the present invention to provide methods of treating or reducing the risk of developing conditions responsive to estrogen activity, including vaginal atrophy, hypogonadism, decreased libido, skin atrophy, urinary incontinence, imbalances in fat and lipoprotein, atherosclerosis, cardiovascular disease, alzheimer's disease, parkinson's disease, psychiatric disorders, and climacteric symptoms (hot flashes, insomnia, depression, memory loss) by administering 5-DIOL. It is another object of the invention to provide methods for preventing and treating conditions responsive to androgen administration, including breast cancer, ovarian cancer, endometrial cancer, decreased libido, skin atrophy, dry skin, osteoporosis, and climacteric symptoms, by administering 5-DIOL. Many diseases affected by sex steroids (e.g. osteoporosis) respond well to androgens and estrogens.

Patients in need of treatment or reduced risk of developing the above-mentioned diseases are those diagnosed with or predisposed to the diseases.

Except where specifically noted or otherwise evident from the context, dosage as used herein refers to the weight of active ingredient unaffected by the pharmaceutical excipient, diluent, carrier, or other component, although such additional components are also intended to be included (as shown in the examples herein). Any dosage form commonly used in the pharmaceutical industry (capsules, tablets, injections, etc.) is suitable for the present invention, and the terms "excipient", "diluent" or "carrier" include those inactive ingredients, typically included with the active ingredient in the dosage form of the industry. For example, capsules, tablets, enteric coatings, solid or liquid diluents or excipients, flavoring agents, preservatives and the like are typically included

The invention also includes the use of an active agent in the manufacture of a medicament for the treatment of a disease described herein (e.g. susceptible to such a drug), or the use of a component of a composition for the manufacture of a medicament for the treatment of a disease, wherein the treatment further comprises another component of the claimed combination therapy.

It is another object of the present invention to provide novel methods of contraception which comprise administering 5-DIOL.

5-DIOL is a metabolite of Dehydroepiandrosterone (DHEA). It has been found that 5-DIOL shows unexpected changes in its androgenic and estrogenic effects (compared to DHEA). Specifically, 5-DIOL is 5 times more estrogenic than DHEA, while its androgenic activity is only 1-2 times higher than DEHA, so that the ratio of estrogenic to androgenic effects obtainable from 5-DIOL is about 3.0 compared to DHEA. On the other hand, at higher doses, 5-DIOL produced less than the androgenic effect of DHEA. Thus, administration of 5-DIOL produced estrogen in a preference for androgenic effects over the corresponding dose of DHEA. Thus, 5-DIOL is useful in the treatment and prevention of conditions involving low sex steroid levels, especially in conditions in which estrogen levels are reduced (i.e., greater than androgen levels). Indeed, the present invention may be used in conditions where a greater degree of estrogenic effects than androgenic effects are desired. As described below, 5-DIOL may be administered alone or as part of a combination therapy in combination with other therapeutic agents, such as antiestrogens, androgens, progestins, estrogens, DHEA sulfate, LHRH agonists or antagonists, inhibitors of 17 β -hydroxysteroid dehydrogenase, inhibitors of aromatase, inhibitors of gonadal steroid secretion.

In the context of the present invention, any prodrug of 5-DIOL can be substituted for 5-DIOL, including 5-DIOL fatty acids, since they also increase the level of 5-DIOL in serum.

Other features and advantages of the present invention will be apparent from the following detailed description of the invention, taken in conjunction with the accompanying drawings.

Brief Description of Drawings

FIG. 1 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered transdermally (twice daily for 7 days) on uterine weight in ovariectomized rats, a parameter sensitive to estrogen. Intact animals served as a secondary control. The compound was dissolved in 50% ethanol-50% propylene glycol and administered to the back skin (2 cm. times.2 cm) at 0.1 ml.

FIG. 2 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered transdermally (twice daily for 7 days) on serum Luteinizing Hormone (LH) concentration in ovariectomized rats, a measure of estrogen and/or estrogenic effects. Intact animals served as a secondary control. The compound was dissolved in 50% ethanol-50% propylene glycol and administered to the back skin (2 cm. times.2 cm) at 0.1 ml.

FIG. 3 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered transdermally (twice daily for 7 days) on serum DHEA concentration in ovariectomized rats. Intact animals served as a secondary control. The compound was dissolved in 50% ethanol-50% propylene glycol and administered to the back skin (2 cm. times.2 cm) at 0.1 ml.

FIG. 4 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered transdermally (twice daily for 7 days) on serum 5-DIOL concentrations in ovariectomized rats. Intact animals served as a secondary control. The compound was dissolved in 50% ethanol-50% propylene glycol and administered to the back skin (2 cm. times.2 cm) at 0.1 ml.

FIG. 5 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered transdermally (twice daily for 7 days) on serum androstenedione (4-dione) concentration in ovariectomized rats, a measure of estrogen and/or estrogenic effects. Intact animals served as a secondary control. The compound was dissolved in 50% ethanol-50% propylene glycol and administered to the back skin (2 cm. times.2 cm) at 0.1 ml.

FIG. 6 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered transdermally (twice daily for 7 days) on serum testosterone levels in ovariectomized rats. Intact animals served as a secondary control. The compound was dissolved in 50% ethanol-50% propylene glycol and administered to the back skin (2 cm. times.2 cm) at 0.1 ml.

FIG. 7 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered transdermally (twice daily for 7 days) on serum Dihydrotestosterone (DHT) concentration in ovariectomized rats. Intact animals served as a secondary control. The compound was dissolved in 50% ethanol-50% propylene glycol and administered to the back skin (2 cm. times.2 cm) at 0.1 ml.

FIG. 8 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered transdermally (twice daily for 7 days) on the weight of the ventral prostate of testicular-removed rats, a measure of androgenic effect. Intact animals served as a secondary control.

FIG. 9 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered transdermally (twice daily for 7 days) on the weight of testis from rats removed, a measure of androgenic effect. Intact animals served as a secondary control.

FIG. 10 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered transdermally (twice daily for 7 days) on the concentration of mRNA encoding the C1 component of prostate-binding protein (PBP-C1) in the ventral prostate of testicular-depleted rats, a measure of androgenic effect. Intact animals served as a secondary control.

FIG. 11 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered transdermally (twice daily for 7 days) on the concentration of mRNA encoding the C3 component of prostate-binding protein (PBP-C3) in the ventral prostate of testicular-depleted rats, a measure of androgenic effect. Intact animals served as a secondary control.

FIG. 12 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered subcutaneously (twice daily for 7 days) on uterine weight in ovariectomized rats, a measure of estrogenic effect. Intact animals served as a secondary control.

FIG. 13 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered subcutaneously (twice daily for 7 days) on serum LH concentrations in ovariectomized rats, a measure of androgenic and/or estrogenic effects. Intact animals served as a secondary control.

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 testicular-removed rats, a measure of androgenic effect. Intact animals served as a secondary control.

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 testicular-removed rats, a measure of androgenic effect. Intact animals served as a secondary control.

FIG. 16 is a graph of the effect of increasing doses of DHEA or 5-DIOL administered subcutaneously (twice daily for 7 days) on serum LH concentrations in testicular-deprived rats, a measure of androgenic and/or estrogenic effects. Intact animals served as a secondary control.

FIG. 17 is the concentration of DHEA (ng/mL) in plasma of male rats after single oral ingestion of androst-5-ene-3 β, 17 β -diol prodrug (150 μmol/rat) (Y-axis as a function of time (X-axis)). In the panels, the 24 hour AUC of DHEA induced by the following compounds is shown.

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 bisbenzoate

EM-1410 androst-5-ene-3 beta, 17 beta-diol dipropionate

EM-1474-D androst-5-ene-3 beta, 17 beta-diol bis-hemisuccinate

FIG. 18 is a graph of the plasma concentration (ng/mL) of androst-5-ene-3 β, 17 β -diol (Y-axis as a function of time (X-axis)) following single dose oral absorption of androst-5-ene-3 β, 17 β -diol prodrug (150 μmol/rat) in male rats. In the frame, the 24 hour AUC is for androst-5-ene-3 β, 17 β -diol induced by the following compounds.

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 bisbenzoate

EM-1410 androst-5-ene-3 beta, 17 beta-diol dipropionate

EM-1474-D androst-5-ene-3 beta, 17 beta-diol bis-hemisuccinate

The bioavailability of androst-5-ene-3 beta, 17 beta-diol prodrug was measured in vivo.

1) Principle of

Bioavailability of androst-5-ene-3 β, 17 β -diol prodrug was determined by single dose oral dosing to determine the concentration of androst-5-ene-3 β, 17 β -diol prodrug in plasma from male Sprague Dawley rats.

2) Animals and treatments

Male Sprague-Dawley rats (Crl: CD (SB) Br) weighing 275-350g were obtained from Charles-River Canada Inc. and housed two per cage during the acclimation phase, which was housed separately during the study phase. The animals were maintained in 12 hour bright, 12 hour dark conditions (08:00 bright). Animals were fed certified rodent chow (Lab Diet #5002, pellets) and were given tap water ad libitum. Rats were fasted (water only) starting the night before dosing.

Each compound tested (suspension in 0.4% methylcellulose) was administered to three animals by oral gavage (150 μmol/rat). Blood samples of-0.7 ml were collected from jugular vein of rats anesthetized with isoflurane induction at 1, 2, 3, 4, and 7 hours after gavage. Blood samples were quickly transferred to a frozen 0.75ml mini-reservoir (Microtainer) containing EDTA and kept in an ice-water bath until centrifugation at 3000rpm for 10 minutes. Plasma separation was performed rapidly (within 50 minutes after blood sample collection). 0.25ml portions of plasma were then transferred to a borosilicate glass tube (3X 100) and rapidly frozen on dry ice. Plasma samples were stored at-80 ℃ until the concentration of plasma sex steroid or sex steroid precursor was determined by GC-MS.

Detailed Description

The present invention believes that 5-DIOL can be used to treat any known disease that responds well to DHEA treatment, with other benefits due to its more favorable estrogen to androgen activity ratio and lower maximum androgen activity (compared to DHEA). According to the present invention, the direct administration of 5-DIOL has a number of advantages over the administration of DHEA (as described herein).

Applicants have found that the androgenic and estrogenic effects of 5-DIOL production are significantly different from those of DHEA production. Specifically, 5-DIOL produces less of the potential androgenic or masculinizing effects (compared to DHEA) for the same estrogenic effects. Thus, 5-DIOL is particularly advantageous for treating conditions requiring estrogenic activity but requiring minimal androgenic activity. In fact, postmenopausal women are deficient in both androgen and estrogen, although the estrogen/androgen ratio is lower than premenopausal. Women need a more favorable ratio of estrogen to androgen to compensate for the lack of ovarian estrogen secretion. DHEA cannot compensate for this ovarian estrogen deficiency and only replaces the reduced DHEA-S and DHEA secretion by the adrenal glands.

In particular, the active androgen DHT and its precursor 4-dione are produced by administering DHEA (in the range of 30-125% dose increase over the amount obtained with 5-DIOL administration). On the other hand, testosterone production with DHEA was 53% more than with 5-DIOL. 4-dione is a steroid which is itself a weak androgen but which is particularly efficiently converted intracellularly to the stronger androgens testosterone and DHT. The data show that the highest level of serum DHT achieved with 5-DIOL is less than half of the highest level achieved with DHEA, and that at relatively low dosage levels, the plateau (plateau) level of maximum androgenic activity of 5-DIOL is lower than the level produced by DHEA.

FIGS. 10 and 11 demonstrate that the level of DHEA androgenic activity is relatively high compared to the activity of 5-DIOL. The data show that DHEA stimulates mRNA levels of prostate binding protein C1(PBP-C1) and prostate binding protein C3(PBP-C3) 2-5 fold stronger (depending on dose) than 5-DIOL. The levels of PBP-C1 and PBP-C3mRNA are particularly sensitive parameters of androgen action, and the concentration of mRNA encoding these proteins is regulated by androgen. Measurement of the levels of PBP-C1 and PBP-C3mRNA is indicative of androgen activity, since androgen acts at the transcriptional level, increasing the steady state levels of mRNA encoding the PBP subunit components.

Because transdermal DHEA and 5-DIOL (given doses) have nearly the same effect on estrogenic parameters, DHEA produces 2-3 times more androgenic activity than 5-DIOL (the estrogenic effects are the same).

The greatest difference between DHEA and 5-DIOL has been found to be the oxidative 17 β -hsd(s) which, following systemic administration of 5-DIOL, converts 5-DIOL to particularly low active levels of DHEA (fig. 3, 4). On the other hand, similar serum 5-DIOL levels after DHEA and 5-DIOL administration indicate that reducing 17 β -HSD has a relatively high level of activity. In addition, since no significant amount of DHEA is available for direct conversion to 4-dione by 3 β -HSD, androstenedione must be oxidatively derived from testosterone after administration of 5-DIOL. This is consistent with the fact that serum 4-dione, a highly potent androgen precursor, levels after treatment with 5-DIOL are much lower than after treatment with DHEA.

Without wishing to be bound by theory, it is believed that one reason DHEA produces a different androgen response than 5-DIOL (same dose) is that DHEA and 5-DIOL are metabolized differently. 5-DIOL metabolism (and more so than DHEA metabolism) results in less formation and/or more inactivation of some steroids (such as testosterone and DHT) before they produce androgenic activity. Thus, 5-DIOL results in lower exposure to these potent androgens compared to the same dose of DHEA, where the oxidized steroid primarily serves as a depot for the formation of active androgens.

As further shown in figures 14 and 15, the two compounds were administered by the subcutaneous route to assess maximum bioavailability, with a dose below DHEA that reached a plateau from the 5-DIOL stimulated androgen sensitive parameter. It can thus be seen that the difference in the maximum androgen activity of the two compounds observed following transdermal administration of DHEA and 5-DIOL (figures 8, 9, 10 and 11) is not due to differences in the rate of absorption through the skin.

Generally, 5-DIOL shows greater estrogenic activity than the same dose of DHEA under conditions of maximum bioavailability. In fact, 5-DIOL stimulated uterine growth 5-fold more strongly than DHEA after subcutaneous administration. Thus, as noted above, at doses with the same estrogenic effect, the androgenic effect produced by 5-DIOL is significantly lower than that produced by DHEA. In addition, the maximum plateau level achieved by androgen action of 5-DIOL is significantly lower than the maximum stimulatory effect produced by DHEA.

5-DIOL can be administered to treat or prevent conditions in patients with inadequate or unbalanced sex steroid (androgen and/or estrogen) concentration levels, as described below. In particular, it is believed that 5-DIOL is useful in treating and reducing the risk of developing conditions which respond well to estrogenic activity and for which lower androgenic activity than that produced by DHEA is desirable. Since 5-DIOL is metabolized to the same sex steroid as DHEA, 5-DIOL can be used for any purpose that responds well to DHEA and results in a more favorable androgenic effect on estrogens than does DHEA. Mixtures of DHEA and 5-DIOL may be used to provide the desired ratio when the desired estrogen/androgen ratio is between that produced by DHEA and 5-DIOL.

Subjects who would benefit from treatment with 5-DIOL are all those with conditions treatable with DHEA, including those with abnormally low levels of 5-DIOL, estrogen or androgen. The risk of developing these conditions may also be reduced and the recommended 5-DIOL dose and target serum levels are the same as those described herein for treatment with 5-DIOL. The level of 5-DIOL, DHEA, sex steroids and their metabolites in serum (androsterone glucuronide and androstane-3 alpha, 17 beta-DIOL glucuronide for androgens; estrone sulfate and estradiol sulfate for estrogens) can be determined by measuring the levels of 5-DIOL, DHEA, sex steroids and their metabolites in serum (H.Bradlow, L.Castagnetta, S.d' Aquino, L.Gogliotti, eds) Ann.N.Y.Acad.Sci.586: 93-100, 1990, to identify individuals who would benefit from the present invention. Serum IGF-1 levels were determined as described (Furlanetto et al, J.Clin.invest.60: 648, 1977).

In accordance with the present invention, once a deficiency or imbalance is found, the 5-DIOL is preferably administered in a dose sufficient to raise and maintain the serum 5-DIOL concentration within 3-fold of the normal range for young adults. Preferably, the serum 5-DIOL concentration is between 4.0nM and 10nM for females and between 10nM and 20nM for males, e.g., 7.0nM for females and 15nM for males. Of course, the attending physician may also decrease or increase the dosage in accordance with the patient's response, and this variation may be quite large. Intermittent or continuous administration of a progestogen (e.g., medroxyprogesterone acetate, 5-10 mg/day, orally) can alleviate adverse side effects on the endometrium when 5-DIOL is used in the treatment of premenopausal women.

In some embodiments, the serum concentration is 4.0-7.0, 7.0-15nM for women and men, respectively. However, for contraceptive or prophylactic purposes against ovarian or uterine cancer, it is preferred that the concentration for women be within 15nM (e.g., between 10-13). For contraception, either an estrogen (e.g., estradiol, with serum estradiol levels between 50-200 nanograms per liter) or a progestin may be added. The preferred dosages described herein may be increased as appropriate to achieve the desired serum concentration, as monitored by the attending physician for changes in response in each patient.

When the 5-DIOL is administered by transdermal or transmucosal techniques, the delivered dose can be increased or decreased by known methods, i.e., by changing the site of the lotion, ointment, cream, gel or patch (patch) for application, by changing the size of the surface area for application, by changing the concentration of the active ingredient, or by changing the carrier or vehicle. For example, if the active ingredient concentration is kept constant, increasing the surface area will generally increase the dose of active ingredient delivered. In the same way, in a delivery matrix, increasing the concentration of the active ingredient will increase the delivered dose and decreasing the concentration will decrease the delivered dose. Varying the amount of the transdermal osmotic system delivered to the bloodstream may also be accomplished by varying the area of the body surface to which the formulation is applied by known means. Varying the carrier or vehicle by known methods will also vary the dosage delivered.

Preferably, the concentration of serum 5-DIOL is measured prior to the start of treatment and a dose is selected to rapidly raise the serum 5-DIOL concentration to the preferred target range (female, between 4.0-10nM, male, between 10-20 nM). Subsequently, the ideal serum concentration and symptom relief was confirmed by monitoring the symptoms and circulating 5-DIOL or sex steroid metabolite concentrations. The 5-DIOL in the circulation was then maintained at a constant concentration. For example, for a typical post-menopausal patient, the dose is 400mg of 5-DIOL applied as a 10% composition formulated with 50% ethanol-50% propylene glycol, 200cm per 50kg body weight²Abdomen or legs, twice a day. If oral administration is selected, 500mg should be administered per 50kg body weight (twice daily).

As used herein, 5-DIOL for oral administration may or may not be accompanied by an auxiliary carrier or diluent, but must be accompanied by an auxiliary carrier or diluent when administered by the transdermal or transmucosal routes. In pharmaceutical compositions for oral administration, the 5-DIOL is preferably present in a concentration of 5-99% (by weight of the total composition), preferably 50-99%, more preferably 80-99%.

When preparing a composition for transdermal administration, the 5-DIOL is preferably present in a concentration of 2-20% (by weight of the total composition), preferably between 5-15%, more preferably between 5-10%.

The 5-DIOL active ingredient is available from Steraloids Inc (p.o. box 310, Wilton, n.h., 03086, USA). Preferred 5-DIOL prodrugs are the two compounds listed below, which are commercially available from Steraloids Inc.

5-DIOL can be administered alone or in combination with other active ingredients such as antiestrogens, progestins, androgens, estrogens, DHEA or DHEA-S, inhibitors of 17 beta-hydroxysteroid dehydrogenase, inhibitors of aromatase, 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 has weak intrinsic estrogenic activity. Due to the different intrinsic effects of DHEA or 5-DIOL on androgen or estrogen receptors, it is believed that 5-DIOL is produced which gives different ratios of androgen/estrogen activity to DHEA. It is not necessary to use only the androgen/estrogen ratio of DHEA or only the androgen/estrogen ratio of 5-DIOL. DHEA and 5-DIOL can be used together to obtain the optimum effective androgen/estrogen response ratio between that obtained for DHEA and that obtained for 5-DIOL. The relative amount of DHEA to 5-DIOL may vary depending on whether the expected androgen/estrogen response ratio is close to DHEA or 5-DIOL.

For the treatment of breast cancer, endometrial cancer, ovarian cancer, endometriosis, and other estrogen-sensitive diseases where it is desirable to block estrogen formation and/or action, 5-DIOL may be used in combination with at least one of the following: antiestrogens, aromatase inhibitors, androgenic compounds, progestins, LHRH agonists or antagonists, or other inhibitors of gonadal steroid secretion, inhibitors of 17 beta-hydroxysteroid dehydrogenase activity. The desired androgen function can be obtained by using 5-DIOL alone or in combination with DHEA to stimulate androgen-sensitive functions, particularly bone formation and inhibit growth of androgen-sensitive cancers such as breast and endometrial cancers. The 5-DIOL may be used alone or in combination with any of the ingredients described above in the composition. In some cases, DHEA without 5-DIOL can be used in combination with any of the components described above.

In another embodiment, 5-DIOL is combined with an antiestrogen, preferably EM-800((+) -7-pivaloyloxyphenyl-3- (4 '-pivaloyloxyphenyl) -4-methyl-2 (4' "-2 (2-piperidinoethoxy) phenyl) -2H-benzopyran), ICI 182, 780(7 α - [9-4(4, 4,5, 5, 5-pentafluoro-pentylsulfinyl) nonyl ] estra-1, 3, 5(10) -trien-3, 17 β -DIOL) or any other antiestrogen to treat breast cancer, endometrial cancer, ovarian cancer, cardiovascular disease, atherosclerosis and other diseases sensitive to estrogens. Non-steroidal antiestrogens, such as EM-800, tend to act as selective estrogen receptor modulators of estrogen receptor antagonists in breast tissue and also provide estrogen-like beneficial effects on cholesterol, fat and atherosclerosis.

The dosage of 5-DIOL may be varied. Plasma levels of 5-DIOL and other sex steroids and their metabolites indicate doses that account for individual differences in absorption, metabolism and response sensitivity. Preferably, the attending physician, especially at the beginning of treatment, should monitor the overall response and serum 5-DIOL levels (as compared to the preferred serum concentrations described above) for each patient and monitor the overall response of the patient to treatment, and adjust the dosage as necessary when the patient's metabolism or response to treatment is irregular. For combination therapy, treatment was initiated with 5-DIOL treatment alone, supplemented with other compounds only when needed. For the treatment of breast cancer, endometrial cancer, ovarian cancer and endometriosis, treatment with anti-estrogen +5-DIOL or anti-estrogen +5-DIOL + DHEA or anti-estrogen + DHEA may be initiated simultaneously. If DHEA, androgen, progestin or estrogen is added, total serum levels are similarly monitored, and it is desirable to monitor the active ingredient and androgen or estrogen metabolites at an early stage of treatment, which is also considered useful for later evaluation by the physician.

The treatment according to the invention is suitable for an indefinite period of time. It is generally expected that 5-DIOL treatment will simply maintain the serum concentration of the native steroid in the range of 4-10nM (female) and 10-20nM (male), respectively. Sustained treatment of 5-DIOL is expected to produce minimal or no adverse side effects. The adverse side effects of continuous estrogen use can be avoided by methods known in the art, such as by intermittent (or in some embodiments continuous) administration of a progestin (e.g., medroxyprogesterone acetate) at 2-10mg per day. The side effects of androgens are minimized with the method of the present invention because of the relatively low androgenic effects of 5-DIOL and already low DHEA levels in most patients (FIG. 18).

To facilitate the combination therapy of the present invention, the present invention contemplates pharmaceutical compositions comprising 5-DIOL and another or subsequent active compound in a single composition for simultaneous administration for any of the indications described herein. The compositions are suitable for any conventional form of administration including, but not limited to, oral, transdermal or transmucosal administration. In other embodiments, a kit is provided wherein the kit comprises 5-DIOL and a second compound in a separate container. Other active compounds described herein may also be included. This second compound and 5-DIOL can be administered transdermally (as described herein below), among other forms of administration. Thus, the kit may include suitable materials for transdermal administration, such as ointments, lotions, gels, creams, sustained release patches, and the like. The same strategy can also be used for progestogens, antiestrogens, androgens, estrogen DHEA, DHEA-S, inhibitors of 17 β -hydroxysteroid dehydrogenase, aromatase inhibitors or gonadal steroid secretion inhibitors (orally administrable) (or injected LHRH agonists or antagonists).

While it is expected that in some cases 5-DIOL will be administered by injection, this approach is not optimal. Because treatment with 5-DIOL is usually prolonged and indefinite, repeated administration by injection is inconvenient.

It is believed that the preferred method of therapeutically administering 5-DIOL is transdermal, transmucosal or oral administration, because of the discomfort and inconvenience of injecting 5-DIOL.

5-DIOL can be delivered using a number of art-recognized transdermal osmotic systems. For example, the 5-DIOL can be made as part of an ointment, lotion, gel or cream for rubbing on the skin or mucosa of a patient. The active ingredient is preferably present in an amount of about 5% to about 20% (by weight of the total pharmaceutical composition), preferably between about 5% to about 12% (by weight). Alternatively, the active ingredient may be placed in a transdermal patch having a structure known in the art, for example, as disclosed in European patent No. 0279982.

When formulated as ointments, lotions, gels, creams and the like, the active ingredient is mixed with a suitable carrier which is compatible with human skin or mucous membranes and which enhances the transdermal or transmucosal permeability of the compound through the skin or mucous membranes. Suitable carriers are known in the art and include, but are not Limited to, Klucel HF and Glaxal matrix (available from Glaxal Canada Limited Company). Other suitable carriers can be found in Koller and Buri, S.T.P.Pharma 3(2), 115-124, 1987. Suitable carriers are those which dissolve the active ingredient at ambient temperature at the concentration of active ingredient used. Suitable carriers should have sufficient viscosity to maintain the precursors on the skin or mucosal area of the coating composition without running off or evaporating over a period of time sufficient for the precursors to sufficiently penetrate the area of application on the skin or mucosa and enter the bloodstream to be produced and increase the serum 5-DIOL concentration as desired. The carrier is typically a mixture of several compounds, such as pharmaceutically acceptable solvents and thickeners. Mixtures of organic and inorganic solvents can aid in the solubility of both hydrophilic and lipophilic properties, for example, water and alcohols (e.g., ethanol).

It is expected that if one vehicle, made up of 10% 5-DIOL and 90% vehicle (by weight), is applied twice daily to the abdominal region at 100mg 5-DIOL, the patient's serum 5-DIOL concentration will be elevated by at least 1.0nM per 50kg body weight (compared to pre-treatment) and relatively stable serum 5-DIOL levels will be maintained thereafter.

The carrier may include various carriers commonly added to ointments, lotions, gels and creams and well known in the cosmetic and pharmaceutical arts. For example, perfumes, antioxidants, fragrances, gelling agents, thickeners (such as carboxymethylcellulose), surfactants, stabilizers, emollients, colorants, and the like may be present. When used to treat systemic diseases, the site of application on the skin should preferably be varied periodically to avoid potential local steroid overdose and possible overstimulation of the skin and sebaceous glands by 5-DIOL androgen metabolites.

The 5-DIOL or derivative may also be administered orally and may be formulated with conventional pharmaceutical excipients, such as spray dried lactose and magnesium stearate incorporated into tablets or capsules, or orally administered to provide a dose in the range of 0.050-2.5 grams per day per 50kg body weight.

The active substances can be formulated into tablets or dragee cores by means of solid, pulverulent carrier substances, such as sodium citrate, calcium carbonate or dicalcium phosphate, binders, such as polyvinylpyrrolidone, gelatin or cellulose derivatives, and also lubricants, such as magnesium stearate, sodium lauryl sulfate, "Carbowax" or polyethylene glycol. Of course, flavoring agents may also be added if they are administered orally. The active substance may also be administered as a solid dispersion in a suitable carrier. These carriers may be selected from polyethylene glycol (molecular weight 1000-.

Other dosage forms, such as an insertable capsule, e.g., hard gelatin, and a closed soft gelatin capsule containing a softening or plasticizing agent, e.g., glycerin. The active substance contained in the push-fit capsules is preferably in the form of granules, for example in admixture with fillers such as lactose, sucrose, mannitol, starches (e.g. potato starch or amylopectin), cellulose derivatives or highly disperse silicic acids. In soft gelatin capsules, the active substance is preferably dissolved or dispersed in a suitable liquid, such as vegetable oil or liquid polyethylene glycol.

The concentration of the active ingredient in the ointment, cream, gel or lotion is generally about 2-20%, preferably 5-15%, more preferably 5-10% (by total weight of the lotion, cream, gel or ointment). In preferred ranges, higher concentrations are more likely to result in suitable dosages when applying lotions, ointments, gels or creams to smaller skin surfaces than lower concentrations, and allow more freedom in the choice of body area to which the ointment or lotion is applied. For example, as is well known in the art, compounds are generally more effectively permeated (transdermally permeable) in certain parts of the body than in others. For example, penetration of the forearm is highly efficient, while penetration of the palm is relatively poor.

The lotion, ointment, gel or cream should be rubbed thoroughly into the skin so that there is no clearly visible excess and the skin in the area cannot be rinsed until most of the transdermal penetration is complete, preferably at least 15 minutes, more preferably 30 minutes (after application).

The 5-DIOL can be delivered by transdermal patches according to known techniques. Which is typically for a longer period of time, such as 0.5-4 days, the active ingredient is typically contacted with a smaller surface area, resulting in a slow, sustained delivery of the active ingredient.

A number of transdermal drug delivery systems have been developed and are in use and are suitable for delivering the active ingredients of the present invention. The release rate is typically controlled by matrix diffusion or by passage of the active ingredient through a controlling membrane.

The mechanical aspects of transdermal devices are well known in the art and can be explained, for example, by 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, all of which are incorporated herein by reference. Other background information is provided in european patent 0279982 and british patent application 2185187.

The device may be any of the usual types known in the art, including adhesive matrix and reservoir type transdermal devices. The device may include a matrix containing the drug mixed with fibers capable of absorbing the active ingredient and/or the carrier. In reservoir type devices, the reservoir is a polymeric membrane that is impermeable to the carrier and active ingredient.

Transdermal devices, which themselves maintain the active ingredient in contact with the desired skin surface. In such devices, the carrier viscosity of the active ingredient is less important than a cream or gel. The solvent system of the 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.

For adhesion to the skin, a transdermal patch may be placed on a surgical adhesive plaster with a hole punched in the middle. Preferably, a release liner is used to protect the adhesive prior to use. Materials generally suitable for release include polyethylene and polyethylene coated paper, preferably silicone coated paper to facilitate release. In use of the device, the peel ply can be easily peeled off to bond the adhesive to the skin of the patient. U.S. patent No.4,135,480 to Bannon et al discloses a device that secures the device to the skin without adhesive.

In addition to the higher dose indications described above (e.g., contraception), the target serum 5-DIOL concentration is similar whether 5-DIOL is used as part of a combination therapy for the treatment of menopause or (alone or in combination with antiestrogens, androgens, progestins, inhibitors of estrogen and 17 β -hydroxysteroid dehydrogenase, inhibitors of aromatase, inhibitors of gonadal steroid formation, agonists or antagonists of LHRH, DHEA and/or DHEA-S) for the treatment of cardiovascular disease, osteoporosis, skin atrophy, menopause, vaginal atrophy, urinary incontinence, uterine cancer, ovarian cancer, osteoporosis, endometriosis, hypogonadism or hypoactive sexual desire, or for the treatment of any condition associated with reduced or unbalanced levels of sex steroids (particularly 5-DIOL and its metabolites).

5-DIOL is particularly useful in the treatment of conditions where minimal androgenic effects are desired, since the androgenic effects of 5-DIOL are lower than those produced by DHEA (which is the same estrogenic effect) and the maximum androgenic effects produced by 5-DIOL are lower than those produced by DHEA. 5-DIOL is particularly preferred for treating conditions in women responsive to estrogen therapy (or treatment with an estrogen precursor such as DHEA) because 5-DIOL has less androgenic effects than DHEA, thereby reducing potential androgenic or androgenic effects while providing the desired estrogenic activity. In addition, women prior to menopause typically require a greater estrogen/androgen ratio than DHEA can provide.

Since 5-DIOL is a natural source of estrogen and androgen, secretion of this compound decreases significantly with increasing age (FIG. 17), and its replacement should have minimal adverse side effects. Its intrinsic estrogenic activity should compensate for the lack of secretion of postmenopausal ovarian estrogen, an effect which DHEA does not achieve. The invention is useful for a number of diseases where estrogen receptor activation has beneficial effects, particularly osteoporosis and climacteric disorders, including vaginal atrophy, insomnia, irritability, cardiovascular disease, urinary incontinence and decreased libido. In addition, the invention is useful for the treatment and prevention of disorders responsive to estrogen receptor activation, such as bone loss, obesity, breast cancer, endometrial cancer, ovarian cancer, urinary incontinence, hypogonadism, decreased libido, decreased muscle mass, decreased vitality, insulin resistance, and other aging conditions. In addition, 5-DIOL can be used to treat or prevent any condition that responds well to an improvement in the overall balance of circulating sex steroids (i.e., estrogen and androgen).

Diseases that are expected to respond to such treatment as described herein can be diagnosed using routine methods. For example, breast cancer typically manifests itself as a self-examination of the breast, a clinical mammogram examination by a physician, and/or a mammography examination. Endometrial cancer, on the other hand, is typically diagnosed by endometrial biopsy. Cancer can be diagnosed and evaluated by standard physical examination methods well known to those skilled in the art, such as bone scans, chest X-ray examinations, skeletal examinations, liver ultrasonography, and liver scans (if necessary), CAT scans, MRI, and physical examinations.

The onset of menopause is usually diagnosed first from hot flashes. Other characteristics of the menopause can be determined by known methods. See, for example, menopause (Herbert J. Buchsbaurm, ed), Springer Verlag New York (1983), page 222. Vaginal atrophy is often manifested by sexual impotence and vaginal infections. Vaginal atrophy, hypogonadism, hypoactive sexual desire, insomnia, irritability, depression and urinary incontinence can be identified by well-known methods. For such diseases see, for example, Korenman, Stanley G, Williams Textbook of Endocrinology, "sexual dysfunction", Jean D.Wilson and Daniel W.Foster, eds, WB Saunders Co., Philadelphia, pp.1033-1048, 1992.

Alternatively, bone density can be determined by standard methods well known to those skilled in the art, such as QDR (quantitative digital radiography), two-photon absorption measurements, and computed tomography. Plasma and urine calcium and phosphate levels, plasma alkaline phosphatase, osteocalcin, calcitonin and parathyroid hormone concentrations, and urine hydroxyproline, deoxypyrrolidine and calcium/creatinine ratios are useful bone formation and absorption parameters.

Loss of collagen and connective tissue in the skin is often accompanied by aging, especially in people over the age of 50. This may be indicated by wrinkles and/or a deterioration in the elasticity of the skin. The condition of the skin can be estimated by visual inspection, palpation, and more carefully, by needle biopsy and standard histological examination.

The normal range of body weight is well known to those skilled in the art, and cholesterol and lipoproteins can be routinely checked by standard methods (Nestler et al, J.Clin.Endocrinol.Metab.66: 57-61, 1988 for reference).

In addition, 5-DIOL can be used as a contraceptive for women. Previous female contraceptives in the art generally included estrogen administration which reduced LHRH secretion by the hypothalamus and thereby decreased LH secretion by the pituitary as circulating water levels increased. A decrease in LH secretion will reduce ovarian function, especially ovulation. The addition of progestagen controls endometrial growth, alters vaginal and cervical secretions to create an environment that is not conducive to sperm capacitation and fertilization.

In the present invention, 5-DIOL provides an estrogen for contraception which simultaneously and ideally provides a minimal increase in androgen levels, which is also beneficial for contraception, as androgens also inhibit secretion of LHRH and LH. These androgens are capable of providing many of the required stimulation of bone formation and resistance to bone loss, especially in pre-menopausal women (and post-menopausal women where contraception is no longer required). In addition to being a weak estrogen in itself, 5-DIOL administration produces estrogen which also reduces bone loss. As with other uses described herein, 5-DIOL replacement steroids (referred to herein as estrogens) can be used to avoid externally administering relatively high doses of estrogen and to avoid having estrogen widely contact all tissues, since many tissues do not require estrogen. By replacing with 5-DIOL, estrogen and/or androgen are no longer produced by the natural pathway in tissues in which they are needed, while 5-DIOL is converted to estrogen and/or androgen. The relative proportions of estrogen and androgen in each tissue are also substantially maintained at natural levels.

Because the methods of contraception described herein reduce ovarian function, the ovaries will reduce estrogen and progesterone production. Thus, where a high dose of 5-DIOL is required, a progestin (e.g., medroxyprogesterone acetate, megestrol acetate, norethindrone, norgestrel) can be administered as part of a contraceptive regimen to prevent endometrial overgrowth. The progestogen can be administered in a pharmaceutical composition comprising 5-DIOL or separately. In some embodiments, the progestogen can be administered intermittently, 12-14 days per month or 12-14 days per several months (e.g., every 2-5 months), or continuously, depending on the 5-DIOL dose used, with no irritation (at physiological doses) to the endometrium. The range of progestogen dosage can be in the ranges used in the prior art, but lower dosages are preferred for reasons described below.

Because 5-DIOL is converted to estrogen in many tissues, it is not necessarily necessary to add estrogen to compensate for the reduced ovarian estrogen secretion in contraceptive therapy. However, extremely small doses may be administered if necessary. When used in a contraceptive method, the estrogen is preferably added in a dose of 50-300 nanograms per liter of estradiol or in an equivalent effective amount. Preferably, the ratio (w/w) of estrogen to 5-DIOL is added in the range of 100: 1 to 10,000: 1, preferably 200: 1 to 5,000: 1, more preferably 300: 1 to 3000: 1. When a progestin is added, the added estrogen can be administered as part of a pharmaceutical composition that includes 5-DIOL (or a prodrug of 5-DIOL), or separately.

In some embodiments, the 5-DIOL, progestin, and estrogen are administered together or separately as part of a combination therapy. The result of the combination therapy is that at any time the therapy simultaneously increases the plasma level of each active substance. This only requires that the active drug be administered sufficiently timely to simultaneously raise the plasma levels of these substances.

The risk of breast Cancer has not been shown to be reduced with contraceptive compositions containing estrogen and progestin (Romiev et al, 1990, Cancer 66: 2253-2263). These data are consistent with the known mitogenic effects of estrogens and progestagens on mammary epithelial proliferation, and thus account for the high peak of mid-luteal cell proliferation (Masters et al, j.natl.cancer inst.1977, 58: 1263-1265; Anderson et al, 1982, brit.j.cancer 46: 376-382). In fact, the rate of total breast cell proliferation in premenstrual women with contraceptives is not different from that of untreated cycling (cycling) women (Potter et al, 1988; Brit. J. cancer 58: 163-170; Going et al, 1988; am. J. Pathol. 130: 193-204). The androgenic effects in 5-DIOL will diminish this potentially harmful effect of estrogens and progestogens.

Osteoblasts (bone forming cells) contain enzymes that convert 5-DIOL into estrogen and androgen. Thus, 5-DIOL can be used in place of (or in addition to) androgen, estrogen or DHEA for the treatment or prevention of osteoporosis. Sufficient amounts of androgen are produced in the bone (by conversion of the administered 5-DIOL) to stimulate bone formation and reduce bone loss. In addition, the low estrogenic activity of 5-DIOL and the administration of 5-DIOL-derived estrogens also contribute to the reduction of bone loss.

Since particularly low androgen doses have been shown to stimulate bone density, 5-DIOL, which is less active than DHEA androgen/estrogen, has the greatest beneficial effect on bone at doses where the risk of hyperandrogenism is minimal.

In addition, since 5-DIOL (or a prodrug thereof, if desired) is converted to androgens and estrogens in tissues solely by natural mechanisms, such conversion of these tissues in accordance with their local requirement greatly reduces or eliminates side effects, whereas previously externally administered active steroids in this field would enter many tissues where neither the given androgens nor estrogens are produced. Thus, the physiological balance of sex steroids in these tissues is not disturbed in the present invention, unlike all previous hormone replacement therapies in this field. The relative proportions of androgen and estrogen produced by 5-DIOL are also substantially normal, rather than abnormally elevated proportions of a certain type of sex steroid that occur when an active sex steroid is administered exogenously directly, and thus expose all tissues, including those that do not require such treatment.

In the treatment of preferred climacteric symptoms, the present invention seeks to simultaneously maintain plasma levels of 5-DIOL, androgen and estrogen at normal pre-menopausal parameters. Without wishing to be bound by theory, it is believed that maintaining suitable precursor levels better allows natural enzymes (such as 17 β -hydroxysteroid dehydrogenase, 3 β -hydroxysteroid hydrogenase, aromatase, and α -5 reductase) to regulate androgen and estrogen production and in some way maintain them at absolute and relative levels more similar to those that are ubiquitous prior to menopause. Thus, the present invention is expected to maintain a better balance of not only estrogen but also androgen. Indeed, the target tissue possesses the enzymatic mechanisms required to synthesize androgens and/or estrogens as locally needed and inactivate them (Labrie, mol.cell. endocrinol.78, C113-C118, 1991).

As described above, 5-DIOL can be administered with estrogen. However, since 5-DIOL produces a higher proportion of estrogen than androgen and is itself a weak estrogen compared to DHEA, it is possible to achieve the desired estrogen levels without the addition of estrogen and without the undesirable androgenic side effects associated with high levels of DHEA, a compound with greater androgenic activity. Similarly, since 5-DIOL is a weak estrogen, progestin therapy may not be required.

However, if it is determined that estrogen is required, the estrogen and 5-DIOL can be administered simultaneously or separately. In addition, only 5-DIOL and estrogen need be administered in a manner and in amounts sufficient to achieve the desired levels of both in serum. The combination therapy according to the present invention maintains the concentration of estrogen at the desired parameter while maintaining the concentration of 5-DIOL at the desired parameter. When estradiol is used, serum estradiol levels are typically maintained between 50 and 200 nanograms/L, preferably between 100 and 175 nanograms/L, and more preferably between 125 and 175 nanograms/L. When another estrogen is used, the serum concentration can be varied in a known manner to account for known differences in estrogenic activity relative to estradiol and to achieve normal pre-menopausal estrogen levels. For example, if Mestranol (Mestranol) is used, a smaller concentration is required. Appropriate serum estrogen levels can also be assessed by the disappearance of climacteric symptoms. Serum 5-DIOL concentrations are typically maintained between 4-10nM (female) and 10-20nM (male), or in some embodiments between 4.0-7.0nM (female) or between 7.0-15nM (male).

If 5-DIOL is used in combination with an estrogen, estradiol is preferred, but estrone sulfate or other compounds which can act as estrogen receptor agonists either directly or after appropriate transformation are also possible. When administered separately, estrogen supplements are available commercially, such as conjugated estrogens ("PREMARIN") available from Ayerst (st. laurent, Quebec, Canada). For the average patient, the appropriate dosage of estrogen to achieve the desired serum concentration is 0.3-2.5 mg conjugated estrogen per day per 50kg body weight (oral administration). In some embodiments of the invention, the estrogen may be 17 β -estradiol (available from CIBA under the trade name "ESTRADERM") in a patch that is administered transdermally at a daily dose of 0.05-0.2 mg/day/50 kg of body weight. For the average patient, a suitable dose of sex steroid precursor 5-DIOL to achieve the desired concentration of serum precursor is between 0.10 and 2.5 g/day/50 kg body weight (oral administration). The dose of the other prodrugs administered depends on the rate at which they are converted to 5-DIOL in vivo. 5-DIOL can also be administered transdermally or transmucosally, as described herein above, in an amount sufficient to achieve the target serum concentration.

In other embodiments, the menopause is treated with 5-DIOL as described above in combination with the periodic administration of a progestogen, such as medroxyprogesterone acetate (e.g., Provera), the latter preferably being administered intermittently, e.g., at a dose of 2-10 mg/day for 12 consecutive days, with 20 days to 5 months between 12 days. Combination therapy with 5-DIOL is also possible with an estrogen and a progestin, with preferred dosages as described for each compound herein.

The dose of 5-DIOL is the same for all indications except for contraception and prevention of ovarian and endometrial cancer (the preferred serum level of 5-DIOL is about 15 nM).

For all other indications where androgens and/or estrogens are required, the usual dosages mentioned above may be used. Similarly, when used in combination with an antiestrogen to treat or prevent breast cancer, endometriosis, and other estrogen-sensitive diseases, the same dose of 5-DIOL can be used. In some cases, the same dose of 5-DIOL is recommended when using aromatase inhibitors, 17 β -hydroxysteroid dehydrogenase inhibitors, androgens, progestins, inhibitors of gonadal steroid formation, LHRH agonists or antagonists.

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 described above, 5-DIOL was shown to have greater estrogenic activity relative to androgenic activity compared to DHEA.

Materials and methods

Animal(s) production

Male and female Sprague-Dawley rats (Crl: CD (SD) Br) obtained from Charles River Canada Inc. (St-Constant, Quebec) weighing 225-. Animals were fed Purina rat chow ad libitum. Animals were studied as "Guideline for Care and Use of Experimental Animals".

Treatment of

Animals were randomized into groups (8 and 10 rats per group for subcutaneous and topical administration, respectively). Under ether anesthesia, appropriate groups of animals were ovariectomized bilaterally (OVX) or testicles (ORCH), while other rats served as intact controls. DHEA and 5-DIOL obtained from Steraloid (Wilton, NH, USA) were dissolved in 50% ethanol-50% propylene glycol and applied twice daily (0.5ml) to back skin (2cm × 2cm) for 7 days (from OVX or ORCH day). For subcutaneous administration, DHEA and 5-DIOL were dissolved in 0.5ml 10% ethanol-1% gelatin-0.9% NaCl and injected twice daily into the dorsal area for 7 days (also from the day of OVX or ORCH).

Animals were sacrificed by decapitation seven days after the start of treatment or about 12 hours after the last steroid administration. Blood samples were collected separately and sera were frozen at-20 ℃ until analysis. The uterus, ovaries, ventral prostate, dorsal prostate and seminal vesicles were removed rapidly, cleaned of connective and adipose tissue, weighed, frozen in liquid nitrogen, and stored at-80 ℃ until analysis. Three rats in each treatment group were perfused with paraformaldehyde for in situ hybridization.

Steroid analysis

The steroid is extracted. Ethanol (5ml) was added to 1ml of serum and centrifuged at 2000 Xg for 15 minutes. The resulting precipitate was further extracted with 2ml of ethanol, centrifuged again at 2000 Xg for 15 minutes, and the supernatants from the two times were combined. The suspension was centrifuged as above and the supernatant decanted and mixed with the previously obtained ethanol extract. The organic solvent was then evaporated under nitrogen and the residue was dissolved in 1ml of water: methanol (95: 5, v/v). The C-18 column (Bound-Elut, Amersham, Bucks, U.K.) was conditioned by passing 10ml of methanol, 10ml of water and 10ml of methanol/water (95: 5, v/v) in succession. The extract dissolved in methanol/water (5: 95, v/v) was then applied to a C-18 column. After washing the column with 2ml water: methanol (95: 5, v/v) and 5ml methanol: water (50: 50, v/v), 5ml methanol: water (85: 15, v/v) was added to elute unbound steroids.

Chromatography and radioimmunoassay on LH-20 column

Chromatography on a "SEPHADEX" LH-20 column (Pharmacia, Uppsala, Sweden) was performed as described by Belanger et al, 1988. Briefly, the steroid was dissolved in 1ml of isooctane/toluene/methanol (90: 5, v/v/v) and applied to a LH-20 column. Fractions were collected and, after evaporation of the organic solvent, the concentrations of the various steroids were determined by radioimmunoassay (as described by Belanger et al, 1980; Belanger et al, 1988; Belanger et al, 1990).

Preparation of cDNA Probe

A plasmid containing a DNA fragment complementary to the mRNA encoding PBP-C1 was provided by Dr.Malcolm G.Parker (Imperial Cancer Research Fund, London, United kingdom). After electrophoresis on a 5% (wt/vol) polyacrylamide gel, the 434 base pair Pst-I restriction fragment of PBP-C1cDNA was purified by electroelution (Bio-Rad electroelution apparatus, model 422, Bio-Rad, Richmond, Calif.). The purified fragments were purified by random priming method (Feinberg and Vogelstein, 1983) to [. alpha. -³⁵S]dCTPaS (Amersham, Oakville, Ontario, Canada) was radiolabeled to obtain high specific activity (10)⁹dpm/μg)。

In situ hybridization assay for PBP-C1 mRNA levels

In situ hybridization of prostate sections was performed using the PBP-C1 probe as described by Pelletier et al, 1988. Briefly, rats were perfused with 4% paraformaldehyde in a fixation buffer in 0.1M phosphate buffer (pH 7.4). The ventral prostate was cleared of adipose and connective tissue, pre-fixed (post-fixed) in fixation buffer for 2 hours (4 ℃), and then soaked in 0.05M PBS containing 15% (wt/vol) sucrose. Thereafter, the ventral prostate was rapidly frozen in chilled isopentane in liquid nitrogen. Multiple (6-8) 10 μ M tissue sections (taken from each ventral prostate) were placed on gelatin-coated slides. Prehybridization buffer contained 50% formamide, 5 xsspp (1 xsspp ═ 0.18M NaCl，10mM NaH₂PO₄And 1mM EDTA, pH7.4), 0.1% sodium lauryl sulfate, 0.1% BSA, 0.1% Ficoll (water-soluble Ficoll), 0.1% polyvinylpyrrolidone, 0.2mg/ml yeast tRNA, 0.2mg/ml denatured salmon testis DNA, 0.2g/ml poly (A). The slide contained 4% dextran sulfate and a saturation concentration (1.0-1.5X 10)⁷cpm/ml) of PBP-C1cDNA probe for 48 hours (37 ℃). The sections were then washed in 2 × SSC (1 × SSC ═ 0.15M NaCl and 0.015M sodium citrate, pH 7.0) for a total of 2 minutes, dehydrated, and then exposed to autoradiography. To determine the amount of non-specific background hybridization, sections of prostate from each treatment group were treated with pancreatin ribonuclease-A (20. mu.g/ml) for 1 hour at room temperature before pre-hybridizing sections of rat brain, pituitary, kidney and liver with PBP-C1 probe. No specific hybridization was observed (data not shown).

LH radioimmunoassay

Serum LH was measured using a double antibody radioimmunoassay of rat hormone (LH-I-6 for iodination; LH-RP-2 as standard) and rabbit antiserum anti-r-LH-S-8 (generously provided by National Pituitary Program, Baltimore, USA).

Statistical analysis

Radioimmunoassay data were analyzed using a program based on model II of Rodbard and Lewald (Rodbard, 1974). Mean ± SEM of duplicate determinations of each sample showed plasma steroid levels. Data for in situ hybridization were obtained as follows: 20 randomly selected 0.25mm sections of each prostate tissue section were analyzed using Image Research Analysis System (Amersham, Arlington Heights, IL)²Except for the acinar cavity, and the mean value of the optical density values for each section was calculated. Data shown are mean ± SEM of 20 readings from 6-8 prostate sections (obtained from the dorsal prostate). Statistical significance was determined using a multi-range test with Duncan-Kramer (Kramer, 1956).

As shown in FIG. 1, DHEA and 5-DIOL produced similar stimulation of uterine weight, an indicator of estrogenic activity. Specifically, similar stimulation was produced in the uterine weights of ovariectomized rats when DHEA and 5-DIOL were administered at doses of 10mg and 30mg, whereas the doses of 1 and 3mg had no significant effect. However, at a dose of 100mg (highest dose used), 5-DIOL produced greater stimulation (52% of the ovariectomy inhibition was maximally reversed), whereas DHEA was only 42% reversible at the maximum.

FIG. 2 shows the effect of DHEA and 5-DIOL on serum LH. Serum LH is known to be a sensitive indicator of androgenic and estrogenic activity, since it is found that serum LH increases rapidly after gonadectomectomy In male as well as female animals due to elimination of the significant feedback inhibition of sex steroids (Ferland et al, In: Labrie, F., Meites, J., and Pelletier, G (eds.), hypothalamic and endocrine functions, p.199-209, New York; Plenum Press, 1976). At the 10mg dose, 5-DIOL produced a greater effect and completely reversed the strong stimulatory effect of ovariectomy to 0.86. + -. 0.17ng/ml, whereas the corresponding dose of DHEA produced only 61% (p < 0.01) inhibition. However, at higher doses, DHEA and 5-DIOL were found to produce similar effects, inhibiting serum LH levels by about 35% and 70% at 30mg and 100mg, respectively, less than that found in intact control animals.

Figures 3-7 show the effect on circulating levels of major steroids and precursors resulting from administration of 5-DIOL and DHEA. Specifically, as shown in FIG. 3, serum DHEA increased 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 DHEA at doses of 3, 10, 30, and 100mg, respectively. On the other hand, 5-DIOL produced relatively stable but lower serum DHEA levels with the same dose.

FIG. 4 shows the serum 5-DIOL levels produced following administration of 5-DIOL and DHEA. At lower doses, 1 and 3mg of 5-DIOL produced higher levels of 5-DIOL, whereas at higher doses, 5-DIOL and DHEA produced no significant difference in 5-DIOL levels.

FIGS. 5-7 show the concentration of serum 4-dione, testosterone, and Dihydrotestosterone (DHT) produced following administration of DHEA and 5-DIOL. Serum 4-dione (an androgen indicator) levels (range dependent on the dose used) produced by DHEA were 30-125% higher than those produced by 5-DIOL.

Consistent results were obtained from serum DHT and testosterone (both indicators of androgen activity). Specifically, DHEA produced 30% -125% higher serum DHT levels than 5-DIOL (same dose), while DHEA produced 53% higher testosterone stimulation than 5-DIOL (same dose). In addition, the levels of serum DHT produced by 5-DIOL were essentially stable and relatively low throughout the dose range.

FIGS. 8-11 show the effect of DHEA and 5-DIOL on various easily recognized androgen sensitive parameters in testicular rats. FIG. 8 shows the effect on ventral prostate weight, DHEA reverses the inhibitory effect of orchiectomy by about 75% at a 10mg dose, whereas 5-DIOL at a 150% dose (15mg) produces only a 50% reversal of castration. In addition, DHEA was 1-fold stronger in increasing seminal vesicle weight as shown in fig. 9.

In addition, as shown in FIGS. 10 and 11, DHEA produced 2-5 times more effect on the concentration of mRNA encoding Prostate Binding Protein (PBP) C1 and C3 components (as described above, an accurate indicator of androgen activity) than 5-DIOL produced. In addition, the highest dose of 5-DIOL produced maximum levels of C1 and C3 PBP mRNA that were only 17% -37% of the levels obtained with DHEA.

FIGS. 12-16 show the effect of subcutaneous administration of DHEA and 5-DIOL on the above parameters. Since these two steroids are injected subcutaneously, these results provide a more direct measure of relative estrogenic and androgenic activity (under optimal bioavailability conditions) on DHEA and 5-DIOL. Specifically, as shown in figure 12, at the lowest dose used (0.3mg), 5-DIOL reversed 90% of the effect of ovariectomy (1 week) on uterine weight (an estrogen-sensitive parameter), whereas the same dose of DHEA had no significant effect. However, at higher doses, the maximal stimulatory effects produced by these two steroids were similar (2.8-2.9 fold stimulation). In addition, based on the calculated dose for half-maximal stimulation of uterine weight (-2.5 mg DHEA, 0.5mg 5-DIOL), uterine trophism (chronotropic) of 5-DIOL was approximately 5.0 times higher than DHEA (after subcutaneous administration). In addition, as shown in FIG. 13, 5-DIOL was found to be 5-7 times stronger than DHEA in suppression of serum LH levels increased by castration.

As shown in FIG. 14, the maximal stimulation of prostate and seminal vesicle weight (androgen-sensitive parameters) with 5-DIOL was about 70% of that obtained with DHEA. However, the calculated half-maximal reversal dose (ED) for orchiectomy effects₅₀) The intensity of DHEA and 5-DIOL are substantially the same (i.e., ED)₅₀The value was 1 mg). On the other hand, maximum effective ED of DHEA and 5-DIOL₅₀The values were 2.5mg and 1.2mg, respectively, with 5-DIOL estimated to be 2 times stronger than DHEA. Similarly, as shown in FIG. 15, the maximal stimulatory effect of 5-DIOL was approximately half the maximal reversal dose of 70% orchiectomy effect of DHEA for seminal vesicle weight, which was estimated to be 2.5mg DHEA, 1.2mg 5-DIOL, and 2-fold the activity of DHEA as 5-DIOL. For C1 and C3 PBP mRNA concentrations in the prostate, 5-DIOL was approximately DHEA 2-fold intense. FIG. 16 shows that 5-DIOL is about 10-fold more potent on serum LH concentrations than DHEA.

Taken together, it can be seen that at each concentration, DHEA produces a greater proportion of estrogen to androgen action than 5-DIOL produces.

While the invention has been described in terms of specific embodiments herein, many other variations, modifications and uses will become apparent to those skilled in the art.

Claims (6)

1. A pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier, and a therapeutically effective amount of androst-5-ene-3 β, 17 β -diol in combination with at least one active ingredient selected from the group consisting of: dehydroepiandrosterone, dehydroepiandrosterone sulfate, and estrogen, wherein the pharmaceutical composition is used to treat or reduce the likelihood of developing any condition selected from the group consisting of a reduced or unbalanced concentration of a sex steroid, menopausal symptoms, vaginal atrophy, hypogonadism, osteoporosis, decreased libido, skin atrophy, dry skin, urinary incontinence, obesity, cardiovascular disease, atherosclerosis, insulin resistance, memory loss, and alzheimer's disease.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises androst-5-ene-3 β, 17 β -diol in combination with a therapeutically effective amount of estrogen.

3. The pharmaceutical composition of claim 1, wherein said pharmaceutical composition comprises androst-5-ene-3 β, 17 β -diol in combination with a therapeutically effective amount of a compound selected from the group consisting of dehydroepiandrosterone and dehydroepiandrosterone-sulfate.

4. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable diluent or carrier is for transdermal or transmucosal administration and the concentration of androst-5-ene-3 β, 17 β -diol is from 2 to 20 weight percent of the total weight of the pharmaceutical composition.

5. A kit, comprising:

a first container containing androst-5-ene-3 β, 17 β -diol and at least one further container containing at least one active ingredient selected from the group consisting of estrogen, dehydroepiandrosterone and dehydroepiandrosterone sulfate.

6. Use of a pharmaceutical composition according to claim 1 for the preparation of a medicament for the treatment or prevention of obesity, cardiovascular disease, atherosclerosis, insulin resistance, memory loss, or alzheimer's disease.