HK1241741A1 - Treatment of hot flushes, vasomotor symptoms, and night sweats with sex steroid precursors in combination with selective estrogen receptor modulators - Google Patents

Abstract

Novel methods for reduction or elimination of the incidence of hot flushes, vasomotor symptoms, and night sweats while decreasing the risk of acquiring breast, uterine or endometrial cancer and furthermore having beneficial effect by inhibiting the development of osteoporosis, hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, insulin resistance, diabetes type 2, loss of muscle mass, adiposity, Alzheimer's disease, loss of cognition, loss of memory, or vaginal dryness in susceptible warm-blooded animals including humans involving administration of an amount of a sex steroid precursor, particularly dehydroepiandrosterone (DHEA) and an antiestrogen or a selective estrogen receptor modulator, particularly benzopyran compounds. Pharmaceutical compositions for delivery of active ingredient(s) and kit(s) useful to the invention are also disclosed.

Description

Treatment of hot flashes, vasomotor symptoms and night sweats with sex steroid precursors in combination with selective estrogen receptor modulators

The application is a divisional application of Chinese patent application 201080027160.5 with the application date of 2010, 6 months and 16 days.

Cross reference to related applications

The present application claims priority from united states provisional application No. 61/187,549 filed on 6/16 2009 and united states official application No. 12/791,174 filed on 6/1 2010, the contents of which are hereby incorporated by reference.

Technical Field

The present invention relates to a new treatment for female hot flushes, vasomotor symptoms and night sweats. In particular, the therapy comprises a sex steroid precursor in combination with the administration of a Selective Estrogen Receptor Modulator (SERM) for reducing the risk of acquiring breast or endometrial cancer. The invention also provides kits and pharmaceutical compositions for practicing the foregoing compositions. Administration of the foregoing composition in a patient reduces or eliminates the occurrence of hot flashes, vasomotor symptoms, night sweats, and sleep disorders. Furthermore, it is believed that the risk of breast and/or endometrial cancer in patients receiving the combination therapy is reduced. Additional benefits such as reducing the likelihood or risk of developing osteoporosis, hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, alzheimer's disease, cognitive loss, memory loss, insomnia, cardiovascular disease, insulin resistance, diabetes, and obesity (particularly abdominal obesity) are also provided.

Background

The following is a complete citation of the following discussion and use of the references in a more abbreviated citation format.

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A variety of diseases, conditions and adverse symptoms are known to respond well to the administration of exogenous steroids or their precursors. For example, estrogens are believed to decrease the rate of bone loss, while androgens have been shown to increase bone mass by stimulating bone formation. Hormone replacement therapy (e.g., administration of estrogen) can be used to treat menopausal symptoms. Lutein is commonly used to counteract endometrial cancer proliferation and estrogen-induced endometrial cancer risk. The use of estrogens, androgenic compounds and/or lutein for the therapeutic or prophylactic purpose of a wide variety of symptoms and disorders is associated with various disadvantages. Treatment of women with androgen compounds may have adverse side effects that cause some masculinizing side effects. Also, administration of sex steroids to patients may increase the risk of the patient for some diseases. For example, estrogenic activity causes the worsening of breast cancer in women.

Furthermore, androgen compounds have been found to be beneficial in the treatment of breast pain often caused by HRT (Pye et al, 1985, Japanese). In fact, estrogen replacement therapy may cause severe breast pain, which may lead to treatment discontinuation.

There is a need for more effective hormonal therapies and reduced side effects and risks. The combination therapies of the present invention and the pharmaceutical compositions and kits useful in such therapies are believed to be responsive to such needs.

Disclosure of Invention

It is an object of the present invention to provide methods of treating or reducing the occurrence or risk of hot flashes, vasomotor symptoms, night sweats and sleep disorders.

It is another object to provide a method of treating or reducing the risk of developing the above diseases while minimizing the risk of developing breast cancer and/or endometrial cancer, osteoporosis, cardiovascular disease, hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, insulin resistance, diabetes, obesity (particularly abdominal obesity), and vaginal dryness.

It is another object to provide kits and pharmaceutical compositions suitable for use in the above methods. The products are preferably packaged with instructions for using their contents to reduce or eliminate the effects of symptoms selected from the group consisting of hot flashes, vasomotor symptoms and night sweats.

In one embodiment, the present invention provides methods of reducing or eliminating the occurrence of hot flashes, vasomotor symptoms, night sweats, and sleep disorders comprising administering to a patient in need of such elimination or reduction a therapeutically effective amount of a sex steroid precursor or prodrug thereof in combination with administering to the patient a therapeutically effective amount of a selective estrogen receptor modulator or an antiestrogen or prodrug thereof.

The sex steroid precursor is preferably selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-ene-3 β,17 β -diol, 4-androstene-3, 17-dione and prodrugs of any of the foregoing additional agents.

In another embodiment, the invention provides additional beneficial effects or reducing the risk of a condition selected from the group consisting of osteoporosis, hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, alzheimer's disease, insulin resistance, diabetes, muscle wasting, obesity, the beneficial effects being achieved by administering to a patient in need thereof a therapeutically effective amount of a sex steroid precursor or prodrug thereof in combination with administering to the patient a therapeutically effective amount of a selective estrogen receptor modulator or prodrug thereof.

In another embodiment, the present invention provides a pharmaceutical composition comprising:

a) a pharmaceutically acceptable excipient, diluent or carrier;

b) a therapeutically effective amount of at least one sex steroid precursor or prodrug thereof; and

c) a therapeutically effective amount of at least one selective estrogen receptor modulator or one antiestrogen or prodrug.

In another embodiment, the present invention provides a pill, a lozenge, a capsule, a gel, a cream, an ovoid suppository, or a suppository comprising:

a) a pharmaceutically acceptable excipient, diluent or carrier;

b) a therapeutically effective amount of at least one sex steroid precursor or prodrug thereof; and

c) a therapeutically effective amount of at least one selective estrogen receptor modulator or one antiestrogen or prodrug.

In another embodiment, the present invention provides a kit comprising a first container containing a pharmaceutical formulation comprising a therapeutically effective amount of at least one sex steroid precursor or prodrug thereof; and the kit further comprises a second container containing a pharmaceutical formulation comprising a therapeutically effective amount of at least one selective estrogen receptor modulator or an antiestrogen or prodrug thereof.

In another embodiment, the invention relates to a method of treating or reducing the incidence of hot flashes, vasomotor symptoms, night sweats, and sleep disorders by increasing the level of sex steroid precursors selected from the group consisting of Dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), androst-5-ene-3 β,17 β -diol (5-diol), and 4-androstene-3, 17-dione in a patient in need of such treatment or such reduction, and further comprising administering to the patient a therapeutically effective amount of a Selective Estrogen Receptor Modulator (SERM) as part of a combination therapy.

By "pure SERM" as used herein, it is meant that the SERM, at physiological or pharmaceutical concentrations, does not have any estrogenic activity in the breast and endometrial tissues.

In another embodiment, the invention provides a kit comprising a first container containing a therapeutically effective amount of at least one sex steroid precursor, and further comprising a second container containing a therapeutically effective amount of at least one selective estrogen receptor modulator.

In another embodiment, the present invention provides, in a container, a pharmaceutical composition comprising:

a) a pharmaceutically acceptable excipient, diluent or carrier;

b) a therapeutically effective amount of at least one sex steroid precursor; and

c) a therapeutically effective amount of at least one selective estrogen receptor modulator.

In another embodiment, the invention provides a method of reducing or eliminating the occurrence of a symptom selected from the group consisting of hot flashes, vasomotor symptoms, and night sweats, the method comprising administering to a patient in need of the elimination or reduction (i) a therapeutically effective amount of a sex steroid precursor or prodrug thereof in combination with (ii) a therapeutically effective amount of a selective estrogen receptor modulator or an antiestrogen or prodrug of either.

In another embodiment, the present invention provides a pharmaceutical composition for reducing or eliminating a symptom selected from the group consisting of hot flashes, vasomotor symptoms, and night sweats, comprising:

a) a pharmaceutically acceptable excipient, diluent or carrier;

b) at least one sex steroid precursor or prodrug thereof; and

c) at least one selective estrogen receptor modulator or one antiestrogen or a prodrug of either;

wherein the pharmaceutical composition is provided in a package that directs use of the composition to reduce or eliminate at least one symptom selected from the group consisting of hot flashes, vasomotor symptoms, and night sweats.

In another embodiment, the present invention provides a kit for reducing or eliminating a symptom selected from the group consisting of hot flashes, vasomotor symptoms, and night sweats comprising (i) a first container having therein at least one sex steroid precursor or prodrug thereof; (ii) a second container having therein at least one selective estrogen receptor modulator or an antiestrogen or prodrug of any of the foregoing; and (iii) instructions for using the kit to reduce or eliminate at least one symptom selected from the group consisting of hot flashes, vasomotor symptoms, and night sweats.

As used herein, a compound that is administered to a patient "in combination with" another compound is administered sufficiently close to the administration of the other compound that the patient simultaneously acquires the physiological effects of both compounds, even if the compounds are not administered at the same time. When the compounds are administered as part of a combination therapy, they are administered in combination with each other. Preferred selective estrogen receptor modulators as discussed herein are preferably used in combination with preferred sex steroid precursors, namely dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-ene-3 β,17 β -diol or 4-androstene-3, 17-dione and in particular dehydroepiandrosterone.

Estrogen supplementation is commonly used in postmenopausal women to prevent and treat disorders resulting from menopause, i.e. osteoporosis, hot flashes, vaginal dryness, coronary heart disease (Cummings, in 1991, b.), but this therapy presents some adverse effects associated with long-term estrogen administration. In particular, the increased perceptual risk of uterine and/or breast cancer due to estrogen production (Judd, Meldrum et al, 1983, second, Colditz, Hankinson et al, 1995) is a major drawback of this therapy. The authors of the present invention have found that the adverse effects are suppressed by the addition of a Selective Estrogen Receptor Modulator (SERM) in the administration of sex steroid precursors.

On the other hand, SERMs themselves have little or no beneficial effect on some menopausal symptoms, such as hot flashes and night sweats. Applicants believe that the addition of a sex steroid precursor to SERM therapy for menopausal symptoms reduces or even eliminates hot flashes and night sweats. It is important to indicate hot flashes and night sweats as the initial manifestations of menopause, and patient acceptance of menopause therapy is generally dependent on whether a successful reduction in hot flashes and night sweats is achieved.

A Selective Estrogen Receptor Modulator (SERM) as used herein is a compound that acts as an estrogen receptor antagonist ("antiestrogen") in breast tissue directly or via its active metabolites, while providing estrogenic or estrogenic-like effects in bone tissue and at serum cholesterol levels (i.e., by lowering serum cholesterol). A non-steroidal compound that acts as an estrogen receptor antagonist in vitro or in human or rat breast tissue (especially if the compound acts as an antiestrogen on human breast cancer cells), may act as a SERM. In contrast, steroidal antiestrogens tend not to act as SERMs, as they have a tendency not to exhibit any beneficial effects on serum cholesterol. Get through meThe nonsteroidal antiestrogens found to act as SERMs include EM-800, EM-652.HCl, Raloxifene (Raloxifene), Tamoxifen (Tamoxifen), 4-hydroxy-Tamoxifen (Tamoxifen), Toremifene (Tormeifene), 4-hydroxy-Toremifene (Tormeifene), Droloxifene (Drolooxifene), LY353381, LY 335563, GW-5638, Lasofoxifene (Lasofoxifene), Bazedoxifene (TSE 424; WAY-TSE 424; WAY 140424; 1- [ [4- [2- (hexahydro-1H-azafeifene)-1-yl) ethoxy]Phenyl radical]Methyl radical]-2- (4-hydroxyphenyl) -3-methyl-1H-indol-5-ol), penoxifene (pivoxifene) (ERA 923; 2- (4-hydroxyphenyl) -3-methyl-1- [ [4- [2- (1-piperidinyl) ethoxy]Phenyl radical]Methyl radical]-1H-indol-5-ol) and Idoxifene (Idoxifene), but are not limited to these compounds.

However, we have also found that not all SERMs react in the same way, and that they can be divided into two sub-classes: "pure SERM" and "mixed SERM". Thus, some SERMs, such as EM-800 and EM-652.HCl, at physiological or pharmaceutical concentrations do not have any estrogenic activity in breast and endometrial tissues, and have cholesterol-and blood lipid-lowering effects in rats. Such SERMS may be referred to as "pure SERMS. The ideal SERM is a pure SERM of the type EM-652.HCl, due to its potent and pure antiestrogenic activity in the mammary gland. Others such as Raloxifene (Raloxifene), Tamoxifen (Tamoxifen), Droloxifene (Droloxifene), 4-hydroxy-Tamoxifen (Tamoxifen) (1- (4-dimethylaminoethoxyphenyl) -1- (4-hydroxyphenyl) -2-phenyl-but-1-ene), Toremifene (Toremifene), 4-hydroxy-Toremifene (Toremifene) [ (Z) - (2) -2- [4- (4-chloro-1- (4-hydroxyphenyl) -2-phenyl-1-butenyl) phenoxy ] -N, n-dimethylethylamine), LY353381, LY 335563, GW-5638, Lasofoxifene (Lasofoxifene), Idoxifene (Idoxifene), and Bazedoxifene (Bazedoxifene) have some estrogenic activity in the breast and endometrium. This second series of SERMs may be referred to as a "hybrid SERM". As shown in the in vitro tests of FIGS. 5 and 6 and an in vivo test of breast cancer of FIG. 7, the unwanted estrogenic activity of the "mixed SERMs" can be suppressed by the addition of pure "SERMs". Since human breast cancer xenografts in nude mice are the closest available model to human breast cancer, we therefore compared the effect of EM-800 and Tamoxifen (Tamoxifen), administered alone and in combination, on ZR-75-1 breast cancer xenograft growth in nude mice.

The applicants believe that it is important that the SERMs of the present invention act as pure antiestrogens in breast, uterine and endometrial tissues, as the SERMs must counteract the possible side effects of estrogen, particularly those that arise from exogenous steroid precursors that may increase the risk of cancer in these tissues. In particular, applicants believe that the benzopyran derivatives of the present invention, having the absolute configuration 2S at position 2, are more suitable than their racemic mixtures. Thus, in U.S. patent No. 6,060,503, optically active benzopyran antiestrogens with the 2S configuration are disclosed for the treatment of estrogen-deteriorated breast and endometrial cancers and these compounds are shown to be significantly more effective than racemic mixtures (see fig. 1-5 of U.S. patent No. 060,503).

It is difficult to obtain the 2S enantiomer in pure form industrially, and the applicants believe that it is preferable to dope the 2R enantiomer to a degree of less than 10 wt%, preferably less than 5 wt% and more preferably less than 2 wt%.

Brief description of the drawings

Figure 1 shows the effect of treatment with DHEA (administered transdermally once daily) or EM-800 (administered orally 75 micrograms once daily) alone or in combination for 9 months on serum triglyceride (a) and cholesterol (B) levels in rats. Data are presented as mean ± SEM. **: p of the experimental group relative to the individual control group was < 0.01.

FIG. 2 shows the effect on total serum cholesterol levels of 37 weeks of treatment in ovariectomized rats with increasing doses (0.01, 0.03, 0.1, 0.3, and 1 mg/kg) of administered EM-800 or Raloxifene (Raloxifene). comparison of ovariectomized rats with 17 β -estradiol (E)₂) Cutting of implantsA de-ovariectomized animal; p < 0.01 in experimental versus Ovariectomized (OVX) control rats.

FIG. 3 shows: A) effect of increasing doses (0.3 mg, 1.0 mg, or 3.0 mg) of DHEA on mean ZR-75-1 tumor size administered transdermally twice daily in Ovariectomized (OVX) nude mice supplemented with estrone. The control group OVX mice receiving vehicle only were used as an additional control group. Based on the initial tumor size of 100%. DHEA is administered transdermally (p.c.) to the dorsal skin as a 0.02 ml solution of 50% ethanol-50% propylene glycol. B) Effect on ZR-75-1 tumor weight in OVX nude mice supplemented with estrone treatment with increasing doses of DHEA or EM-800(a SERM of the invention) administered alone or in combination for 9.5 months. **: p < 0.01 in the treated group compared to control OVX mice supplemented with estrone.

Figure 4 shows the effect of increasing oral dose (B) of the antiestrogen EM-800(15 microgram, 50 microgram or 100 microgram) or increasing dose of DHEA administered transdermally in combination EM-800(15 microgram) or administered alone of EM-800(A) on the mean ZR-75-1 tumor size in Ovariectomized (OVX) nude mice supplemented with estrone at 9.5 months. Based on the initial tumor size of 100%. The control group OVX mice receiving vehicle only were used as an additional control group. Estrone is administered once daily at a dose of 0.5 microgram subcutaneously, while DHEA is dissolved in 50% ethanol-50% propylene glycol and administered twice daily in a volume of 0.02 ml to the dorsal skin area. Also compared to OVX animals receiving vector only.

FIG. 5 shows the effect of increasing concentrations of EM-800, (Z) -4-hydroxy-Tamoxifen (Tamoxifen), (Z) -4-hydroxy-Toremifene (Tormeifene), and Raloxifene (Raloxifene) on alkaline phosphatase activity in human Shichuan (Ishikawa) cells. At 1.0nM E₂The activity of alkaline phosphatase was measured after 5 days of exposure to increasing concentrations of the indicated compounds in the presence or absence. Data are mean ± SEM of four wells. When the SEM overlaps with the symbol used, only the symbol is shown (Simard, Sanchez et al, 1997, Uighur).

FIG. 6 shows the stimulatory effect of blocking (Z) -4-hydroxy-Tamoxifen (Tamoxifen), (Z) -4-hydroxy-Toremifene (Tormemifene), Droloxifene (Droloxifene) and Raloxifene (Raloxifene) on alkaline phosphatase activity in human Stokes' (Ishikawa) malignant cells by the antiestrogens EM-800. Alkaline phosphatase activity was measured after 5 days of exposure to 3 or 10nM of the indicated compound in the presence or absence of 30 or 100 nMEM-800. Data are presented as mean ± SD of 8 wells, except that data for the control group was taken from 16 wells (Simard, Sanchez et al, 1997 en).

Figure 7 shows the stimulatory effect of Tamoxifen (Tamoxifen) on human breast cancer ZR-75-1 xenograft growth, completely blocked by concurrent administration of EM-652. In the absence of Tamoxifen (Tamoxifen), EM-652.HCl, which is consistent with its pure antiestrogenic activity, is not itself effective for tumor growth.

Figure 8 shows a comparison of the effect of standard ERT (estrogen) or HRT (estrogen + lutein) and combinations of dehydroepiandrosterone with SERM Acolbifene (Acolbifene) on the menopause parameters. The addition of acobifene (Acolbifene) to dehydroepiandrosterone will counteract the possible negative effects of estrogen formation from dehydroepiandrosterone.

Figure 9 shows rat mammary sections:

A) untreated animals. The leaflet (L) comprises several alveoli. Insert figure. High magnification reveals the alveoli.

B) Animals were treated with EM-800(0.5 mg/kg body weight daily) for 12 weeks. The size of the leaflets (L) is reduced. Insert figure. High magnification shows atrophic acinar cells.

Figure 10 shows rat endometrial sections:

A) untreated animals. Luminal epithelial cells (LE) are characterized by columnar epithelial cells, while glandular epithelial cells (GE) are fairly cuboidal. The matrix contains several cellular molecules and collagen fibers.

B) Animals treated with EM-800(0.5 mg/kg body weight daily) over a 12 week period. The height of luminal epithelial cells was significantly reduced. Glandular epithelial cells have unstained cytoplasm with no evidence of viability. The matrix is highly porous due to the reduction of intercellular molecules of the matrix.

Figure 11 shows the effect on uterine weight of increasing concentrations of EM-652.HCl, Lasofoxifene (the free base; the active and inactive enantiomers) and Raloxifene (Raloxifene) administered orally for 9 days in ovariectomized mice concurrently treated with estrone. Is relative to warp E₁The control group treated had p < 0.05, and p < 0.01.

Figure 12 shows the effect of increasing concentrations of EM-652.HCl, Lasofoxifene (free base; active and inactive enantiomers) and Raloxifene (Raloxifene) administered orally for 9 days on vaginal weight in ovariectomized mice concurrently treated with estrone. Is relative to warp E₁The p of the treated control group was < 0.01.

Figure 13 shows the effect on uterine weight of 1 microgram and 10 microgram of EM-652.HCl, Lasofoxifene (free base; active and inactive enantiomers), and Raloxifene (Raloxifene) administered orally for 9 days in ovariectomized mice. P < 0.01 relative to OVX control.

Figure 14 shows the effect on vaginal weight of 1 microgram and 10 microgram of EM-652.HCl, Lasofoxifene (free base; active and inactive enantiomers), and Raloxifene (Raloxifene) administered orally for 9 days in ovariectomized mice. P < 0.01 relative to OVX control.

Figure 15 shows the effect on trabecular bone volume in ovariectomized rats for 12 months of treatment with either Dehydroepiandrosterone (DHEA) alone or in combination with Flutamide (Flutamide) or EM-800. Animals without ovariectomy were added as an additional control group. Data are presented as mean ± SEM, p < 0.01 relative to OVX control.

Figure 16 shows the effect on trabecular number in ovariectomized rats for 12 months of treatment with either Dehydroepiandrosterone (DHEA) alone or in combination with Flutamide (Flutamide) or EM-800. Animals without ovariectomy were added as an additional control group. Data are presented as mean ± SEM, p < 0.01 relative to OVX control.

Figure 17 shows the proximal tibial metaphysis from the non-ovariectomized control group (a), the ovariectomized control group (B) and ovariectomized rats treated with DHEA alone (C) or in combination with Flutamide (D) or EM-800 (E). The decrease in trabecular bone (T) volume in the ovariectomized control animals (B) and the marked increase in trabecular bone volume (T) following DHEA administration (C) were observed. The addition of Flutamide to DHEA partially blocks the effect of DHEA on trabecular volume (D), while the combination of DHEA with EM-800 provides complete protection against bone loss associated with ovariectomy. Mansen-goddner (Masson-Goldner) modified trichrome bone dye, magn.x 80. T: trabecular, GP: and (5) growing the plate.

FIG. 18 shows the effect of antiestrogens on ZR-75-1 tumor growth. Effect of treatment with 7 antiestrogens for 161 days on estrone-induced breast tumor growth in human ZR-75-1 in ovariectomized nude mice. Tumor size is expressed as a percentage of the initial tumor area (100% on day 1). Data are presented as mean ± SEM (n-18 to 30 tumors/group); # is p < 0, 01 relative to EM-652. HCl; p < 0, 01 relative to OVX. Antiestrogens were administered once daily at an oral dose of 50 μ g/mouse under estrone stimulation from a 0.5 cm subcutaneous silica gel implant containing estrone and cholesterol in a ratio of 1: 25.

FIG. 19 shows the effect of antiestrogens on ZR-75-1 tumor growth. Effect of treatment with 7 antiestrogens for 161 days on growth of human ZR-75-1 breast tumors in ovariectomized nude mice. Tumor size is expressed as a percentage of the initial tumor area (100% on day 1). Data are presented as mean ± SEM (n-18 to 30 tumors/group); # is p < 0, 01 relative to EM-652. HCl; p < 0, 01 relative to OVX. The antiestrogen is administered once daily at an oral dose of 100 micrograms/mouse in the absence of estrogen stimulation.

FIG. 20 shows the effect of antiestrogens on ZR-75-1 tumor growth. Effect on growth of ZR-75-1 breast tumor in humans in ovariectomized nude mice on 161 days of treatment with anti-estrogens Tamoxifen (Tamoxifen), EM-652.HCl (acobifene), and a combination of Tamoxifen (Tamoxifen) and EM-652. HCI. Tumor size is expressed as a percentage of the initial tumor area (100% on day 1). Data are presented as mean ± SEM (n-18 to 30 tumors/group); # is p < 0, 01 relative to EM-652. HCl; p < 0, 01 relative to OVX. The antiestrogen is administered once daily at an oral dose of 200 micrograms/mouse in the absence of estrogen stimulation.

Figure 21 shows the effect of antiestrogens on the class of responses. Effects of 7 anti-estrogens administered for 161 days on the response class of human ZR-75-1 breast tumors in ovariectomized nude mice. Complete regression refers to those tumors that were undetectable at the end of treatment; partial regression corresponds to regression of tumors greater than or equal to 50% of their original size; a stable response refers to a tumor that regresses by less than 50% or worsens by less than 50%; and worsen means that they worsen by more than 50% compared to their original size. Antiestrogens were administered once daily at an oral dose of 50 μ g/mouse under estrone stimulation from a 0.5 cm subcutaneous silica gel implant containing estrone and cholesterol in a ratio of 1: 25.

Figure 22 shows the effect of antiestrogens on the class of responses. Effects of 7 anti-estrogens administered for 161 days on the response class of human ZR-75-1 breast tumors in ovariectomized nude mice. Complete regression refers to those tumors that were undetectable at the end of treatment; partial regression corresponds to regression of tumors greater than or equal to 50% of their original size; a stable response refers to a tumor that regresses by less than 50% or worsens by less than 50%; and worsen means that they worsen by more than 50% compared to their original size. The antiestrogen is administered once daily at an oral dose of 200 micrograms/mouse in the absence of estrogen stimulation.

Figure 23 shows the effect of antiestrogens on the class of responses. Effects of anti-estrogens Tamoxifen (Tamoxifen), EM-652.HCl (acobifene), and a combination of Tamoxifen (Tamoxifen) and EM-652.HCl on the response class of human ZR-75-1 breast tumors in ovariectomized nude mice for 161 days. Complete regression refers to those tumors that were undetectable at the end of treatment; partial regression corresponds to regression of tumors greater than or equal to 50% of their original size; the stable response refers to tumor regression of < 50% or worsening of < 50%; and worsen means that they worsen by more than 50% compared to their original size. The antiestrogen is administered once daily at an oral dose of 200 micrograms/mouse in the absence of estrogen stimulation.

Figure 24 shows a study design of a phase II-III placebo-controlled study evaluating the effect of DHEA on vasomotor symptoms (hot flashes) in menopausal women.

Figure 25 shows the effect of daily doses of DHEA or placebo on the average number of moderate to severe hot flashes over a 16 week treatment period (, DHEA vs. placebo p < 0.05).

Figure 26 shows the effect of treatment with DHEA or placebo at a daily dose of 50 mg on the average number of all hot flashes (mild, moderate and severe) (p < 0.05 for DHEA versus placebo) over a 16 week treatment period.

Figure 27 shows the maturation index measured on days 1 and 7 for women after 40 to 75 years of age of menopause after daily administration of pessaries containing 0%, 0.5%, 1.0%, or 1.8% DHEA. Data are presented as mean ± SEM (n ═ 9 or 10). Data at day 7 was p < 0.05, p < 0.01 relative to data at day 1.

Figure 28 shows the measured vaginal pH on days 1 and 7 after daily administration of pessaries containing 0%, 0.5%, 1.0%, or 1.8% DHEA for women after 40 to 75 years of age amenorrhea. Data are presented as mean ± SEM (n ═ 9 or 10). Data at day 7 was p < 0.05, p < 0.01 relative to data at day 1.

Detailed Description

Advantageous Effect of DHEA

The most widely recognized fact about menopause is that the estrogen secretion from the ovaries is progressively reduced and eventually stopped, the cessation of estrogen secretion from the ovaries is caused by circulating 17 β -estradiol (E)₂) A significant decrease in the level is exemplified. The cycle E can be easily measured₂The changes in levels coupled with the beneficial effects of estrogen on menopausal symptoms and bone resorption have led most hormone replacement therapy studies to focus on various forms of estrogen and combinations of estrogen and lutein to avoid the potentially harmful stimulatory effects of estrogen alone on the endometrium that may cause endometrial hyperplasia and cancer.

At the time of menopause, 17 β -estradiol (E) is circulated₂) The rapid decrease, coupled with the beneficial effects of exogenous estrogen on menopause symptoms and bone resorption, has led most hormone replacement therapy studies to focus on various forms of estrogen and combinations of estrogen and lutein to avoid the risk of endometrial cancer induced by estrogen administration alone.

Hormone Replacement Therapy (HRT) of estrogen and lutein in postmenopausal women is used for acute symptoms, especially hot flashes and night sweats, due to estrogen deficiency and for the long-term prevention of osteoporosis and possibly cardiovascular diseases. While lutein effectively protects the uterus from the stimulatory effects of chronic estrogen exposure, it also has its own side effects, particularly dysfunctional uterine bleeding (Archer et al, 1999, b.). It is a common side effect and a common reason for women to stop hormone supplementation early in the first 6 to 12 months. Following 5.2 years of data that show that a combination of pramelin (Premarin) and prevela (Provera) (timekeeper (Prempro)) results in a 26% increase in the incidence of breast cancer and has a potential negative impact on cardiovascular events (Women's health care study in 2002), traditional HRT has recently been seriously questioned, and many Women even abandon this therapy.

We believe that what we call cytoendocrinology has increased understanding of The formation and role of androgens and hormones in tissues of peripheral targets (Labrie in 1991, Egypt a, Labrie et al in 1992, Egypt b, Labrie et al in 1994, Egypt, Labrie et al in 1995, Luu-The et al in 1995, Egypt a, Labrie et al in 1996, Egypt, Labrie et al in 1997, Egypt a, Labrie et al in 1997, Egypt b, Labrie et al in 1997, Egypt c, Labrie et al in 1997, 1997d), and our recent observations show that the dominant role played by androgens in preventing bone loss after ovariectomy in rats is to outweigh the advantages of estrogens (Martel et al, B1998) and the similar situation observed in postmenopausal women (Labrie et al, B c 1997), has paved the way for timely and possibly highly significant advances in the fields of sex steroid supplementation and aging. Our observations support this possibility very well.

In Berger et al (2005) b, DHEA was shown to have a particularly interesting effect on the three-layered vaginal wall of the rat vagina, a highly viscous liquefied epithelial cell, resulting in increased muscle layer thickness and increased collagen fiber tightness in the lamina propria. Thus DHEA exhibits both androgenic and estrogenic effects on the vaginal mucosa, providing a more physiological complementary therapy.

The present invention is therefore based on our recent progress towards understanding sex steroid physiology in both men and women (Labrie in Japanese 1991; Labrie et al in Japanese a 1992; Labrie et al in Japanese b 1992; Labrie et al in Japanese 1994; Labrie et al in Japanese a 1995; Luu-The et al in Japanese a 1995; Labrie et al in Japanese a 1997; Labrie et al in Japanese b 1997; Labrie et al in Japanese c 1997; Labrie et al in 1997 b), and to recognize that women lack estrogen activity during menopause due not only to reduced ovarian activity, but also have been in reduced androgen exposure for years. In fact, normal women produce amounts of androgen equivalent to two-thirds that secreted by men (Labrie et al, 1997, Eveno a).

Androgen stores in women progressively decrease from age 30, and a parallel decrease in serum concentrations of DHEA and DHEA-S occurs (Labrie et al, 1997, b). Thus, it seems logical to use a concurrent androgen and estrogen replacement therapy during and after peri-menopausal periods, thereby maintaining the physiological balance of both classes of sex steroids in each cell and tissue, whereas this goal is only achieved in peripheral tissues by local formation of androgens and estrogens from the steroid precursor DHEA.

Principal source of DHEA-androgens

Role played by DHEA in the formation of peripheral steroids

Humans and some other primates are unique among animal species in that their adrenal glands secrete large amounts of the inactive precursor steroids DHEA and especially DHEA-S, which are converted in peripheral tissues into potent androgens and/or estrogens. DHEA-S levels in adult and female plasma are 500-fold higher than testosterone and 10,000-fold higher than estradiol, thus supplying large amounts of substrates for androgenic and/or estrogenic effects. As mentioned above, the local synthesis and effect of sex steroids in peripheral target tissues has been termed cytoendocrinology (Labrie et al, Japanese 1988; Labrie, Japanese 1991). Recent and rapid progress in this field has been made possible by structural elucidation of most of The tissue-specific genes encoding The steroidogenic enzymes responsible for The local conversion of DHEA-S and DHEA to androgens and/or estrogens in peripheral tissues (Labrie et al, Eveno a 1992; Labrie et al, 1992 c; Labrie et al, Eveno 1995; Luu-The et al, Eveno 1995, b; Labrie et al, Eveno 1996; Labrie et al, 1997 d).

The major importance of DHEA and DHEA-S in human sex steroid physiology can be explained by the estimation that about 50% of the total androgens in adult males are derived from these adrenal steroid precursors (Labrie et al, Japanese 1985; Beerlanger et al, Japanese 1986; Labrie et al, Japanese 1993); in women, however, our best estimate is that the intracellular effects of estrogen in peripheral tissues reach a level of 75% before menopause and nearly 100% after menopause (Labrie in 1991, b.).

The focus of almost exclusively focusing on the role played by ovarian estrogens has shifted attention away from a dramatic 70% reduction in circulating DHEA that has occurred between the ages of 20 and 30 and between the ages of 40 and 50 (Migeon et al, Japanese 1957; Vermeulen and Verdonck, Japanese 1976; Vermeulen et al, Japanese 1982; Orentreich et al, Japanese 1984; Beereger et al, Japanese 1994; Labrie et al, Japanese 1997B). This reduction in serum DHEA and DHEA-S, due to the conversion of DHEA to both androgens and estrogens in peripheral tissues, explains why women are deficient in not only estrogens but also androgens during menopause as described above.

As noted above, recent data have shown that lutein has a negative impact on breast cancer (Clarke and Sutherland, 1990, B; Musgrove et al, 1991, 1992, B), and reports have shown an increased risk of the disease (Colditz et al, 1995, B). In this context, it is important to point out that if the stimulatory effect of DHEA on the endometrium in humans is absent (Labrie et al, 1997, b.c.), the need to administer a lutein to neutralize the potent effect of estrogen on the endometrium is eliminated.

In terms of the breast, DHEA is known to prevent the development of and inhibit the growth of dimethylbenzanthracene breast tumors in rats (Luo et al, 1997, B). In addition, DHEA inhibited the growth of human breast cancer xenografts in nude mice (see example 1 and Couillard et al, 1998 b.). Thus, in contrast to estrogen and lutein, which exhibit stimulatory effects, DHEA is expected to inhibit both the development and growth of female breast cancer.

As clearly demonstrated in our previous studies, supplementation with physiological amounts of exogenous DHEA allows the biosynthesis of androgens and estrogens only in appropriately targeted tissues containing specific steroidogenic enzymes. The active androgens and estrogens synthesized in this way remain in the native cells and leak very little into the circulation. In fact, the most significant effect of DHEA administration is the circulating levels of glucuronic acid derivatives of DHT metabolites, ADT-G and 3 α -diol-G, which are produced in peripheral endocrine tissues that possess the appropriate steroidogenic enzymes to synthesize DHT from the adrenal precursors DHEA and DHEA-S and then to further metabolize DHT into inactive conjugates (Labrie in 1991, Ev.; Labrie et al in 1996, Ev.). The local biosynthetic effects and effects of androgens in target tissues eliminate exposure of other tissues to androgens and thus minimize the risk of undesirable androgenic or other androgen-related side effects. This same discussion also applies to estrogens, although we believe that reliable parameters for total estrogen secretion (compared to glucuronides for androgens) are not currently available.

Role played by androgens and estrogens in bone physiology

Relevant literature on the major role played by androgens in bone physiology is complete (Labrie et al 1997 Evc; Martel et al 1998 Evh.) indeed, both testosterone and DHT increase transcription of α (I) procollagen mRNA in osteoblastic osteosarcoma cells (Benz et al 1991 Evh.) in castrated rats treatment with DHT has also been shown to stimulate internalized bone development (Kapur and Reddi in 1989 Evh.) furthermore, bone density measured in lumbar, femoral tuberosity and systemically during 24 months of treatment in postmenopausal women is increased by estrogen + testosterone implants over that measured by E alone₂Increased (Davis et al, second 1995).

In addition, anabolic steroids have been reported to help prevent bone loss in severe osteoporosis (Hennernan and Wallach, 1957). Similarly, subcutaneous E has been found₂Compared to the use of a sterone implant, it is more effective than oral estrogen in preventing osteoporosis in postmenopausal women (Savvas et al, Japanese 1988). Although the differences observed in this study have been attributed to different estrogen administration routes, the cause of the differences may also be the effect of testosterone. An increase in osteocalcin, a marker of bone formation in serum, was found in postmenopausal women receiving methyltestosterone plus estrogen, compared to estrogen-alone, as an indicator of increased bone formation (Raisz et al, 1996, b.). Similar stimulatory effects on serum osteocalcin were observed in postmenopausal women after 12 months of treatment with transdermal DHEA (Labrie et al, 1997, egen c). In addition, androgen therapy, as observed in nandrolone decanoate (nandrolone), has been found to increase spinal mass density in postmenopausal women (Need et al, 1989, b.). Androgens are increasingly gaining acceptance due to their unique role in postmenopausal women, however a masculinizing effect has been observed in the use of testosterone (Burger et al, Japanese 1984; Studd et al, Japanese 1987).

DHEA and abdominal obesity

Abdominal obesity is associated with insulin resistance, type 2 diabetes mellitus and increased risk of atherosclerosis (Shimokata et al, 1989, 1995, Ferranini et al, 1997, 2000). Hormonal changes and in particular a reduction of the secretion of DHEA and DHEA-S by the adrenal glands are considered to be among the factors involved, among others (Tcherof et al 1996, B.). In both rat and mouse models, DHEA administration reduced visceral fat accumulation in diet-induced obesity (Yen et al, 1977; Cleary and Zisk, 1986; Mohan et al, 1990; Hansen et al, 1997). A beneficial effect of DHEA in reducing insulin resistance with aging has also been observed (Han et al, 1998B).

In a study conducted on postmenopausal women who received a DHEA cream for 12 months, we have found that insulin resistance is reduced and subcutaneous fat in the thigh area is also reduced (Diamond et al, 1996, Ill.). In addition, 50 mg daily administration of DHEA for 6 months in 65 to 78 year old men and women reduced abdominal visceral fat by 10.2% in women and 7.4% in men (Villareal and Holloszy in 2004 b). In the same study, abdominal subcutaneous fat was reduced by 6% in both women and men. Furthermore, the reactivity of serum insulin to the glucose tolerance test was reduced by 13% without change in the glucose response, thus resulting in a 34% improvement in the insulin sensitivity index after DHEA administration. Improvement in DHEA effect has also been found in middle-aged men with hypercholesterolemia (Kawano et al, second 2003).

In a study previously conducted by this same team, 6 months of DHEA administration reduced the overall fat mass by 1.4 kg, while the non-fat mass increased by 0.9 kg (Villareal et al, 2000 b). The effects of androgens on libido, hot flashes, and quality of life.

Cell-based studies have shown that female self-reported sexual dysfunction is from 8% to 50%. Indeed, hypoactive sexual desire and sexual dysfunction increase with age in women starting at age 30 (Laumann et al, 1999, Egypt) and after ovariectomy (Nathorst-Boos and von Schoultz, 1992, Egypt). Although low levels of excitation and libido are implicated in psychosocial and health factors (Dennerstein et al 1997B), it is believed that androgens play an independent role (Bachmann et al 2002B; Miller et al 2004B).

Androgens are known to play a role in the female sexual excitement, pleasure, and the intensity and ease of orgasm. Androgens are also involved in neurovascular smooth muscle responses with increased swelling and lubrication (Basson, second 2004). Estrogens affect the engorgement response of the vulva and vagina. Because estrogens also affect mood, they have an effect on sexual arousal (Basson in second 2004). It should be remembered that DHEA is converted in the vagina to both androgens and estrogens (Sourla et al, 1998 b) (Berger et al, 2005 b).

In addition, the detailed benefits of androgen addition to ERT or HRT for overall well being, physical strength, mood and overall quality of life have been described (Sherwin and Gelfand, Japanese 1985; Sherwin, Japanese 1988). Improvements in the major psychological and psychosomatic symptoms, namely irritability, nervousness, memory and insomnia, have been observed following the addition of androgens to Estrogen Replacement Therapy (ERT) (Notelovitz et al, 1991, article b).

Loss of libido and/or sexual satisfaction is common early in the post-menopausal phase. The addition of androgens in Hormone Replacement Therapy (HRT) is known to have a beneficial effect on the problems. Shifren et al (ethin 2000) have found that testosterone improves sexual frequency, pleasure and mood in women who have been surgically aborted by the transdermal administration of a patch. This effect was observed at a testosterone dose of 300 μ g daily, which resulted in serum testosterone levels at the upper limit of normal values. Testosterone therapy has also been studied in non-androgen deficient women complaining of decreased libido (Goldstat et al, japanese 2003). This treatment with testosterone improved sexual desire, sexual function and quality of life compared to placebo. Similarly, the addition of methyl testosterone to estrogen increases sexual desire and frequency in postmenopausal women with normal androgen levels compared to estrogen administered alone (Lobo et al, japanese 2003). Among women with sexual arousal, sexual desire dysfunction, androgen therapy has been suggested for those women with free serum testosterone levels within the low quantile of the reference range (Bachmann et al, 2002 b.). In fact, the treatment of sexual desire deficiency (HSDD) with testosterone has increased (Sherwin and Gelfand, 1987, Edis 1995, Shifren et al 2000, and Goldstat et al 2003). These randomized clinical trials demonstrated that testosterone has utility in women with HSDD.

The androgenic effect of DHEA should also be suitable for reducing hot flashes. In fact, androgen therapy successfully reduced hot flashes in hypogonadal men (DeFazio et al, Japanese 1984) and in women in the transition phase of menopause (Overlie et al, Japanese 2002). Furthermore, in women who fail to achieve satisfactory results by administering estrogen alone, the addition of androgens has been found to effectively relieve hot flashes (Sherwin and Gelfand, japanese 1984). Hot flashes are one of the primary reasons women initially sought HRT therapy, and estrogens are very effective in alleviating this condition.

By the case of adrenal insufficiency, a clear example is provided regarding the nature of androgen deficiency originating from the adrenal gland. (Arlt et al, 1999, second) have studied the effect of administering 50 mg daily DHEA with placebo for 4 months in a population of women suffering from adrenal insufficiency. Treatment with DHEA increased serum testosterone, which was in the low normal range. The treatment increases the frequency, sexuality and satisfaction of sexual thoughts. Also improves well-being, melancholia and anxiety. In a study in which DHEA was administered daily at a high dose of 300 mg, the subjective mental (p < 0.016) and limb (p < 0.030) responses to pornographic video tapes were observed to be greater (cockbert and Heiman, 2002 b.). A study in women who received 50 mg daily DHEA, improved libido was observed in women over the age of 70, but not in women between the ages of 60 and 70 (baulie in 1999, article b). DHEA has also been shown to have a beneficial effect on hot flashes (baulie in 1999 et al, japan 2000). According to a recent canadian survey, 70.8% of the practitioners add androgens to estrogens to improve quality of life (Gelfand, 2004, b).

Other potential benefits of DHEA

During aging, the formation of DHEA and DHEA-S by the adrenal glands is reduced by 70 to 95%, resulting in a significant reduction in the androgenic and estrogenic effects in peripheral target tissues, which is most likely involved in the pathogenesis of diseases associated with aging, such as insulin resistance (Coleman et al, 1982, B; Schriock et al, 1988) and obesity (Nestler et al, 1988, B; MacEwen and Kurzman, 1991; Tchern et al, 1995). In fact, circulating levels of DHEA-S and DHEA have been found to be low in patients with breast cancer (Zumoff et al, 1981, B), and DHEA has been found to exhibit antitumor gene activity in a range of animal models (Schwartz et al, 1986, B; Gordon et al, 1987, B; Li et al, 1993, B). DHEA has also been shown to have immunomodulatory effects in vitro (Suzuki et al, 1991, b.) and in vivo in fungal and viral diseases (Rasmussen et al, 1992, b.) including HIV (Henderson et al, 1992 b). On the other hand, the stimulatory effect of DHEA on the immune system in postmenopausal women was described (Casson et al, 1993, B).

Previous data obtained with DHEA in women

The use of estrogen replacement therapy requires the addition of lutein to counteract the estrogen-induced endometrial hyperplasia, while both estrogen and lutein increase the risk of breast cancer (Bardon et al, Japanese 1985; Colditz et al, 1995). To avoid the limitations of standard Estrogen (ERT) or Hormone Replacement Therapy (HRT), we have studied the effects of DHEA administration for 12 months in 60 to 70 year old women on bone density, parameters of bone formation and metabolism, serum lipids, glucose and insulin, adipose tissue mass, muscle mass, physical strength, well-being, and on vaginal and endometrial tissue structure (Diamond et al, 1996, EvenP; Labrie et al, 1997, EvenP C). DHEA is administered transdermally to avoid the first effect of steroid precursors through the liver.

We therefore evaluated the effect of long-term supplementation by daily administration of 10% DHEA cream for up to 12 months in women 60 to 70 years old (N ═ 15). Human measurements showed no change in body weight at 12 months, but a 9.8% reduction in subcutaneous fat thickness (p < 0.05) (Diamond et al, 1996, second). Bone density in the hip increased by 2.3%, Fahrenheit (Ward) triangle increased by 3.75%, and lumbar increased by 2.2% (both p < 0.05). These changes in bone density were accompanied by a significant decrease in urinary hydroxyproline and plasma bone alkaline phosphatase by 38% and 22%, respectively, at 12 months (both p < 0.05). An increase of 135% (p < 0.05) in plasma osteocalcin over the control group was concomitantly observed, thus showing a stimulatory effect of DHEA on bone formation.

Fat and muscle area in the mid-thigh were measured by computer tomography and showed a 3.8% reduction in thigh fat (p < 0.05) and a 3.5% increase in thigh muscle area (p < 0.05) at 12 months (Diamond et al, 1996, Ev.). There was no significant change in abdominal fat measurements. These changes in body fat and muscle surface area were accompanied by a 12% (p < 0.05) decrease in fasting plasma glucose and a 17% (p < 0.05) decrease in fasting plasma insulin levels. Treatment with DHEA had no adverse effect on the lipid or lipoprotein profile. In fact, the overall trend in total cholesterol and its lipoprotein fraction is a 3% to 10% reduction. Plasma triglycerides are not affected.

The index of sebum secretion increased 79% after 12 months of DHEA treatment and returned to pre-treatment values after 3 months of discontinuation of treatment. For 8 out of 10 women with a maturation value of zero at the beginning of the treatment, DHEA administration had the effect of stimulating the maturation of vaginal epithelial cells, while the stimulation was also observed in 3 women with intermediate vaginal maturation prior to treatment. Most importantly, the estrogen-stimulated effect observed in the vagina was not found in the endometrium, and the endometrium of all women remained completely atrophic after 12 months of DHEA treatment (Labrie et al, 1997, egen c).

The current data clearly show that DHEA therapy, via its conversion to androgens and/or estrogens in specific tissues of the intracellular endocrine target, exhibits beneficial effects in postmenopausal women without significant side effects. The lack of stimulation of the endometrium by DHEA eliminates the need for lutein supplementation therapy and is therefore free from lutein-induced fear of breast cancer. The observed stimulating effect of DHEA on bone density and a marker for increased bone formation in serum, osteocalcin, are particularly beneficial for the prevention and treatment of osteoporosis and show the unique activity of DHEA in bone physiology, i.e., bone formation, whereas ERT and HRT only reduce the rate of bone loss.

Androgens have been proposed to play a role in depression, memory loss, cognitive loss, and brain cell activity (Almeida et al, 2008 et al, Azad et al, 2003, et al, 2008 et al). Estrogens, also synthesized in the brain from DHEA, have been shown to play a beneficial role in alzheimer's disease, memory loss and cognitive loss (Rocca et al, 2007 b). Three combinatorial analyses have shown that the risk of alzheimer's disease is reduced by 20 to 40% in women who use estrogen after menopause (Yaffe et al, 1998, Leblanc et al, 2001, Hogovorst et al, 2000). Estrogens reduce the deposition of beta-amyloid in the brain, while luteinizing hormones have the opposite effect (Xu et al, 1998, B, Huang et al, 2004).

Laboratory data confirm the association between estrogen deficiency and cognitive impairment or dementia. In ovariectomized rats, estrogen potentiates synaptogenesis on the dendritic ridges in the hippocampus (Mc Ewen and Alves in 1999, B., Monk and Brodatz in 2000). In addition, estrogen enhances cerebral blood flow and glucose metabolism, and it may act as an antioxidant (Mc Ewen and Allves, 1999B., Monk and Brodatz, 2000B., Gibbs and Aggamal, 1998B.). Estrogens have also been found to prevent B-amyloid 1-42 from triggering an increase in intracellular calcium and from causing mitochondrial damage (Chen et al, B2006, Morrison et al, B2006).

There is currently substantial evidence from clinical studies showing that there is a critical age range for the beneficial effects of estrogens in neuroprotection (Rocca et al, 2007), cardiovascular disease (Manson et al, 2006), and total mortality (Rocca et al, 2006). When with E₂Treatment ofTreatment was initiated early after menopause with the best benefits observed, and sometimes ineffective or negative effects when treatment was initiated late after menopause (women health care (WHI) study). Estrogen reduction β -amyloid deposition in the brain, whereas luteinizing hormone has the opposite effect (Xu et al, second 1998, Huang et al, second 2004).

Benefits of DHEA: combination of estrogen-like and androgenic effects

It has been observed that androgens exhibit a direct antiproliferative activity for the growth of ZR-75-1. Androgen has also been shown to inhibit growth of breast cancer induced by DMBA in rats, while concomitant administration of the pure antiandrogen Flutamide (Flutamide) would reverse the inhibition (Dauvois et al, 1989, article b). Taken together, the data show that the androgen receptor is involved in the inhibitory effect of DHEA on human breast cancer cells in vitro in breast cancer, and that this inhibitory effect of androgens is an inhibitory effect of secondary antiestrogens (Poulin and Labrie in 1986, b.; Poulin et al in 1988, b.). Similar inhibitory effects in vivo have been observed on ZR-75-1 xenografts in nude mice (Dauvois et al, 1991, second).

We have shown that DHEA exhibits beneficial effects on bone in both female rats (Luo et al, 1997, B) and postmenopausal women (Labrie et al, 1997, B, c). Thus, treatment with DHEA increased bone density (BMD) in full skeletal, lumbar and femoral bone in ovariectomized female rats (Luo et al, 1997 en b).

The present invention is based on our recent advances in the understanding of female sex steroid physiology and recognizes that women are not only deficient in estrogen at menopause due to the cessation of estrogen secretion from the ovaries, but have been under reduced androgen exposure for years. In fact, normal women produce amounts of androgen equivalent to two-thirds that secreted by men (Labrie et al, 1997, Eveno a). Androgen stores in women progressively decrease from age 30, and a parallel decrease in serum concentrations of DHEA and DHEA-S occurs (Labrie et al, 1997, b). Thus, it seems logical to use a concurrent androgen and estrogen replacement therapy during and after peri-menopausal periods, thereby maintaining the physiological balance of both classes of sex steroids in each cell and tissue, whereas this goal is only achieved in peripheral tissues by local formation of androgens and estrogens from the steroid precursor DHEA. The addition of a SERM, such as Acolbifene (Acolbifene), is a positive effect that increases the protection against breast cancer and other benefits of the administration of the SERM. In fig. 8, the positive and negative effects are compared to the conventional ERT.

Previous data show that DHEA therapy, via its conversion to androgens and/or estrogens in specific tissues of the intracellular endocrine target, exhibits beneficial effects in postmenopausal women without significant side effects. In fact, our data obtained in rats clearly demonstrate that DHEA can provide beneficial effects that are absent when a Selective Estrogen Receptor Modulator (SERM) is used alone.

Advantageous effects of acobifene (Acolbifene):

it can be seen in figure 7 that the stimulatory effect of Tamoxifen (Tamoxifen) on tumor growth was completely blocked by concurrent treatment with EM-652 HCl. The EM-652.HCl does not exhibit any stimulatory effects on the growth of human breast cancer ZR-75-1 xenografts in nude mice, in terms of its pure antiestrogenic activity.

We have tested the steroid antiestrogens fulvestrant (falodex), ICI182, 780, and found to act as an antiestrogen rather than a SERM, fulvestrant (fluvistran) also being useful in combination with DHEA for the prevention of breast cancer in the present invention. In accordance with the present invention, the SERMs may be administered in the same dosages as known in the art, even though the art uses them as antiestrogens rather than SERMs.

We have also observed a correlation between the beneficial effects of SERMs on serum cholesterol and the beneficial estrogenic or estrogenic-like effects on bone. SERMs also have beneficial effects on hypertension, insulin resistance, diabetes and obesity, especially abdominal obesity. Without wishing to be bound by theory, it is believed that many of these are preferably SERMs having two aromatic rings linked by one or two carbon atoms, which are expected to interact with the estrogen receptor by virtue of the aforementioned portion of the molecule that is most recognized by the receptor. Preferred SERMs have side chains that selectively elicit antagonistic properties in breast and typically uterine tissue, while not having significant antagonistic properties in other tissues. Thus, SERMs may suitably act as antiestrogens in the breast, while acting as estrogens (or providing estrogenic-like activity) in bone and blood (where the concentrations of lipids and cholesterol are favorably affected), unexpectedly and suitably. The beneficial effects on cholesterol and lipids translate into a beneficial effect against atherosclerosis, and atherosclerosis is known to be adversely affected by inappropriate cholesterol and lipid levels.

As illustrated in figure 9, significant atrophy of the mammary gland was observed, although circulating levels of 17 β -estradiol in the non-ovariectomized animals rose from 95.9 ± 32.4 pg/ml to 143.5 ± 7.8 pg/ml (50% rise in animals treated for 12 weeks with daily oral administration of 0.5 mg/kg EM-800). Similarly, in figure 10, significant atrophy of the endometrium was observed in animals receiving EM-800(0.5 mg/kg). In these ovariectomized animals, which received the pure antiestrogen EM-800, the inhibitory effect of estrogen at the hypothalamic-pituitary level was removed, thus causing an increase in LH, which in turn causes an increase in the secretion of 17 β -estradiol by the ovaries.

Hot flashes, cardiovascular symptoms, alzheimer's disease, loss of cognitive function and insomnia apparently involve estrogen receptors located in the central nervous system. Perhaps, low levels of estrogen in the brain may at least partially explain the conditions. Exogenous estrogens and particularly those formed by administration of sex steroid precursors (i.e., estradiol) may interact with normal estrogens through the brain barrier, and by binding to estrogen receptors. On the other hand, the SERMs of the present invention, and in more detail those from the Acolbifene family, fail to pass through the brain barrier, as shown in example 8. Thus, they do not antagonize the positive effects of estrogen in the brain, but they antagonize the negative effects of estrogen in breast, uterus and endometrial tissues, making this composition (SERM + sex steroid precursor) particularly attractive for use in treating or reducing the risk of acquiring the above conditions.

As mentioned above, androgens have also been proposed to play a role in all of these symptoms. In fact, DHEA can provide both estrogen and androgen in the brain, depending on physiological needs.

Overall added benefit of combining a sex steroid precursor with a SERM or an antiestrogen

The main reason women consult their physicians during menopause is the occurrence of hot flashes, a problem that is well known to be solved by estrogen replacement therapy. Since the address responsible for the hot flashes is the Central Nervous System (CNS) and EM-652 has a very poor ability to enter the CNS (as in the appended data), it is expected that the administration of sex steroid precursors will increase the estrogen concentration in the CNS, and thus will control the hot flashes without interference from SERMs. On the other hand, SERMs will eliminate all negative effects of estrogen at other sites, especially the risk of breast and uterine cancer. In fact, the addition of EM-652 to sex steroid precursors will block the stimulating effect of the formed estrogen on the mammary and uterine glands, while EM-652 will exhibit its own beneficial effects in other tissues, such as its effect on bone density which is partially reversed by ovariectomy in bone.

Since our data show that DHEA reduces hot flashes, vasomotor symptoms and night sweats, we reduced the risk of breast cancer by removing E2. However, DHEA can be slightly converted to estrogen, so a SERM is needed.

While EM-652 should exhibit significant beneficial effects in the prevention and treatment of breast and uterine cancers, no adverse effect on either parameter was observed.

Preferred SERMs or antiestrogens mentioned herein are related to: (1) all diseases susceptible to the present invention; (2) therapeutic and prophylactic applications; and (3) preferred pharmaceutical compositions and kits.

A patient in need of treatment for, or at reduced risk of developing, a particular disease is one who has been diagnosed with, or is predisposed to, the disease.

Unless otherwise indicated, the preferred dosages (concentrations and modes of administration) of the active compounds of the invention for therapeutic and prophylactic purposes are the same. The dosages of the active ingredients in question here are the same regardless of the disease to be treated (or of the disease which reduces its probability of occurrence).

Unless otherwise indicated or evident from the context, dosage herein refers to the weight of the active compound unaffected by the pharmaceutical excipient, diluent, carrier or other ingredient, although such additional ingredients are suitably included as is appropriate and desired as shown in the examples herein. Any dosage form commonly used in the pharmaceutical industry (capsules, pills, tablets, injections, etc.) is suitable for use herein, and the terms "excipient", "diluent" or "carrier" are intended to include the inactive ingredients of such dosage forms as are typically included in the industry with active ingredients. For example, typical capsules, pills, enteric film coatings, solid or liquid diluents or excipients, flavoring agents, preservatives, and the like may be included.

All active ingredients used in any of the therapies discussed herein can be formulated in a pharmaceutical composition that also includes one or more other active ingredients. Optionally, their administration may be separate but sufficiently simultaneous in time whereby a patient ultimately has elevated blood levels or otherwise enjoys the benefit of both active ingredients (or strategies). For example, in some preferred embodiments of the invention, one or more active ingredients are formulated as a single pharmaceutical composition. In other embodiments of the invention, a kit is provided comprising at least two separate containers, wherein the contents of at least one container are completely or partially different from the contents of at least one other container with respect to the active ingredient contained therein.

The combination therapy as referred to herein also includes the use of an active ingredient (of the composition) in the manufacture of a medicament for the treatment (or reduction of risk) of the disease in question, wherein the therapeutic or prophylactic effect further includes another active ingredient of the composition according to the invention. For example, in one embodiment, the invention provides the use of a SERM in the preparation of a medicament for use in combination with a sex steroid precursor in the treatment of any of the diseases for which the combination therapy of the invention is believed to have utility (i.e., hot flashes, night sweats, irregular menstruation and any symptoms associated with menopause) in vivo.

It is well known that estrogen stimulates proliferation of mammary epithelial cells, and that cell proliferation itself is thought to increase cancer risk by accumulating random genetic errors that may cause neoplasms (Preston Martin et al, 1990B.). Based on this concept, antiestrogens have been introduced for the prevention of breast cancer, with the goal of reducing the rate of estrogen-stimulated cell division.

We have also investigated the potential interaction of the novel co-administration of antiestrogens (EM-800) and sex steroid precursors (DHEA) for the inhibitory effects of both drugs on the growth of human ZR-75-1 breast cancer xenografts in nude mice. Figures 3 and 4 show that DHEA itself induces 50 to 80% inhibition of tumor growth at the doses used, whereas the nearly complete inhibition of tumor growth achieved with low doses of antiestrogens is not affected by DHEA.

The limitations of Bone Mass Density (BMD) measurement are well known. For example, BMD measurements showed no change in rats treated with steroid antiestrogen ICI182780 (Wakeling, 1993 b), whereas inhibitory changes were observed by tissue morphometry (Gallagher et al, 1993 b). Similar differences have been reported for Tamoxifen (Tamoxifen) (Jordan et al, Japanese 1987; Sibonga et al, Japanese 1996).

It should be noted that the decrease in bone density is not the only abnormality associated with a decrease in bone strength. It is therefore important to analyze the biochemical parameter changes of bone metabolism induced by various compounds and therapies to gain a better understanding of their effects.

Of particular importance is the indication that the combination of DHEA with EM-800 exhibits unexpectedly beneficial effects on important biochemical parameters of bone metabolism. In fact, DHEA itself does not affect the urinary hydroxyproline/creatinine ratio, which is a marker of bone resorption. Furthermore, no effect of DHEA on daily urinary calcium or phosphorus excretion could be detected (Luo et al, 1997, Ev.). EM-800 reduced the urinary hydroxyproline/creatinine ratio by 48%, and similar to DHEA, no effect of EM-800 on urinary calcium or phosphorus excretion was observed. In addition, EM-800 had no effect on a marker of bone formation, serum alkaline phosphatase activity, while DHEA increased the value of this parameter by about 75% (Luo et al, 1997, B).

One of the unexpected effects of the combination of DHEA and EM-800, which was related to the marker of bone resorption, i.e., the urinary hydroxyproline/creatinine ratio, was a 69% decrease when both DHEA and EM-800 were combined, which was statistically different (p < 0.01) from the 48% inhibition achieved by EM-800 alone, while DHEA itself did not show any effect. Thus, the addition of DHEA to EM-800 increased the inhibitory effect of EM-800 on bone resorption by 50%. Most importantly, another unexpected effect of adding DHEA to EM-800 was a decrease in urine calcium of about 84% (from 23.17 + -1.55 to 3.71 + -0.75 micromoles/24 hours/100 grams (p < 0.01) and a decrease in urine phosphorus of 55% (from 132.72 + -6.08 to 59.06 + -4.76 micromoles/24 hours/100 grams (p < 0.01), respectively (Luo et al, 1997, Uighur).

Importantly, treatment with a combination of EM-800 and DHEA for 12 months had a beneficial effect on bone morphometric measurements in ovariectomized rats. Trabecular bone volume is particularly important for bone strength and for preventing fractures. Thus, in the study described above, the trabecular bone volume of the tibia in ovariectomized rats administered DHEA alone increased from 4.1 ± 0.7% to 11.9 ± 0.6% (p < 0.01), while the addition of EM-800 in DHEA further increased the trabecular bone volume to 14.7 ± 1.4%, similar to that seen in the non-ovariectomized control group (fig. 15).

Treatment with DHEA resulted in a 137% increase in trabecular bone number from 0.57 ± 0.08 per mm in ovariectomized rats compared to ovariectomized controls. The stimulating effect of DHEA thus amounts to 1.27. + -. 0.1 per mm; while treatment with EM800 and DHEA resulted in an additional 28% increase in trabecular bone number (p < 0.01) compared to that achieved with DHEA alone (fig. 16). Similarly, the addition of EM-800 in DHEA therapy resulted in an additional 15% reduction in trabecular bone spacing (p < 0.05) compared to that achieved with DHEA alone, so the resulting values were not different from those seen in the unresectable ovarian control group.

As illustrated by the numerical data presented in supplementary figures 15, 16 and 17, the increase in proximal tibial metaphyseal trabecular volume elicited by DHEA in the treated ovariectomized animals (C) and the partial inhibition of the stimulatory effect of DHEA following the addition of Flutamide (D) in DHEA therapy, compared to the ovariectomized control group (B). On the other hand, administration of DHEA in combination with EM-800 completely prevented bone mass reduction due to ovariectomy (E), whereas trabecular bone volume was comparable to that seen in the ovariectomized control group (a).

Table 1

Table 2

Effect on bone markers and serum lipids in 12 months of treatment with Dehydroepiandrosterone (DHEA) alone or in combination with Flutamide (FLU) or EM-800

P < 0.05 relative to OVX control; p < 0.01.

The importance of the androgen component in the stimulatory effect of DHEA on bone tissue morphology measurements was also confirmed by the effect of DHEA on markers of bone formation and resorption. Serum alkaline phosphatase concentration, a marker of bone formation (Meuner et al, Japanese 1987, and Lauffenburger et al, Japanese 1977), increased from 51. + -.4 International units/liter in the OVX control group to 201. + -.25 International units/liter in the DHEA-treated animals, indicating a stimulating effect of DHEA on bone formation (Table 2). FLU reversed the stimulatory effect of DHEA for this parameter by 65%, while EM-800 had no significant effect. On the other hand, since hydroxyproline released during collagen degradation is not reused in collagen synthesis, it is a useful marker for collagen metabolism or bone resorption. In this study, the urinary hydroxyproline/creatinine ratio was reduced from 11.7. + -. 1.2 micromoles/mmol in the OVX control group to 7.3. + -. 1.0 micromoles/mmol (p < 0.05) in DHEA-treated rats (Table 2). The inhibitory effect of DHEA on this parameter was completely prevented by the administration of FLU, while the effect of EM-800 on DHEA was not statistically significant.

Furthermore, treatment with DHEA reduced serum cholesterol by 22% from 2.29 ± 0.16 to 1.78 ± 0.16 mmol/l (p < 0.05), while concomitant treatment with pure anti-androgen FLU neutralized this effect. On the other hand, the addition of pure antiestrogen EM-800 further reduced the total serum cholesterol to 0.63. + -. 0.09 mmol/l (p < 0.01), thus achieving a 65% inhibitory effect. No statistically significant changes in serum triglyceride levels were observed with either treatment used (table 2).

It is also advantageously observed that concurrent treatment with DHEA does not prevent the potent inhibitory effect of EM-800 on serum cholesterol (Luo et al, 1997 en).

The bone loss observed in women during menopause is believed to be associated with an increase in the rate of bone resorption that cannot be completely compensated for by a secondary increase in bone formation. In fact, in osteoporosis, parameters of both bone formation and bone resorption are increased, and both bone resorption and formation are inhibited by estrogen replacement therapy. It is therefore believed that the inhibitory effect of estrogen supplementation on bone formation results from a coupled mechanism between bone resorption and bone formation, whereby estrogen-induced initial bone resorption is reduced, which leads to a reduction in bone formation (Parfitt, b.1984).

The strength of the cancellous bone and subsequent resistance to fracture is not dependent solely on the total amount of cancellous bone, but rather on the trabecular microstructure as determined by the number, size and distribution of trabeculae. Ovarian loss in postmenopausal women is accompanied by a significant decrease in total trabecular volume (Melsen et al, 1978; Vakamatsou et al, 1985, Ev.), which is primarily associated with a decrease in number, and less frequently, with a decrease in trabecular width (Weinstein and Hutson, 1987, Ev.).

To facilitate the combination therapy aspect of the invention, the invention contemplates pharmaceutical compositions comprising a SERM and a sex steroid precursor in a single composition for simultaneous administration for any of the indications discussed herein. The composition may be adapted for any conventional mode of administration, including, but not limited to, oral, subcutaneous, intramuscular, or transdermal administration. In other embodiments, a kit is provided, wherein the kit comprises one or more SERMs and sex steroid precursors in separate containers or in one container. The kit may include suitable materials for oral administration such as tablets, capsules, syrups and the like, and suitable materials for transdermal administration such as ointments, lotions, gels, creams, sustained release patches and the like.

Applicants believe that the administration of a SERM or antiestrogen and sex steroid precursor has utility in treating and/or reducing the incidence of hot flashes and night sweats. The active ingredients (SERM, antiestrogen or precursor or others) of the present invention can be formulated and administered in a variety of ways. When administered together as in the present invention, the active ingredients may be administered simultaneously or separately.

The active ingredient for transdermal or transmucosal administration is preferably from 0.01% to 1% DHEA or 5-diol. Optionally, the active ingredient may be placed in a pessary or a transdermal patch of construction known in the art, such as those described in european patent No. 0279982, or in an intravaginal cream, gel, pessary or suppository.

When formulated as an ointment, lotion, gel, cream, pessary, suppository, or the like, the active compound is admixed with a suitable carrier that is compatible with and enhances the transdermal or transmucosal penetration of the compound through the skin or mucosa of a human. Suitable carriers are known in the art and include, but are not limited to, krusel (Klucel) HF and Glaxal (Glaxal) matrices. Some are commercially available, such as the Gerassa (Glaxal) matrix available from GlaxalCanada Limited Company, Canada. Other suitable carriers can be found in Koller and Buri, journal of 1987, "S.T.P.Pharma", 3(2), p.115-124, B. The carrier is preferably one in which the active ingredient is soluble at ambient temperature and at the use concentration of the active ingredient. The carrier has a viscosity sufficient to maintain the inhibitor at a localized area of skin or mucosa to which the composition is applied, without running or evaporating for a period of time sufficient to allow the precursor to substantially permeate through the localized area of skin or mucosa and into the blood stream and produce a desired clinical effect thereat. The carrier is typically a mixture of several components such as a pharmaceutically acceptable solvent and a thickening agent. Mixtures of organic and inorganic solvents, such as water, with an alcohol, such as ethanol, can contribute to hydrophilic and lipophilic solubility.

When formulated as an ovoid suppository, a pessary, or the like, the active compound is admixed with a suitable carrier compatible with the human vaginal mucosa. Preferred carriers are stearines (a mixture of glycerides of saturated fatty acids), particularly witness (Witepsol), and especially Witepsol (Witepsol) H-15 matrix (available from medica, inc. of montelukast, canada). Any other lipophilic base may be used, such as other compositions of Fiblebee (Fattibase), Wicobee (Wecobee), cocoa butter or Witepsol (Witepsol) bases.

A preferred sex steroid precursor is Dehydroepiandrosterone (DHEA) (available, for example, from Proquina, Inc. of orixaba (Orizaba), Veracruz, Mexico).

The carrier may also include various additives commonly used in ointments, lotions, and suppositories, and well known in the cosmetic and pharmaceutical arts. For example, perfumes, antioxidants, perfumes, gelling agents, thickening agents such as carboxymethylcellulose, surfactants, stabilizers, softeners, colorants and other like agents may be present.

The treatment according to the invention is adapted to last indefinitely. The SERM or antiestrogen compound and sex steroid precursor can also be administered by oral route, and can be formulated with conventional pharmaceutical excipients such as spray-dried lactose, microcrystalline cellulose and magnesium stearate into tablets or capsules for oral administration.

The active substances can be formulated into tablets or dragee cores by mixing with solid, pulverulent carrier substances such as sodium citrate, calcium carbonate or dicalcium phosphate and binders such as polyvinylpyrrolidone, gelatin or cellulose derivatives, or by adding lubricants such as magnesium stearate, sodium lauryl sulfate, Carbowax or polyethylene glycol. Of course, in the case of oral administration forms, taste-improving substances can be added.

In other forms, embolic capsules, such as hard gelatin, and closed soft gelatin capsules containing a softening or plasticizing agent, such as glycerin, may be used. The embolic capsules contain the active substance, preferably in granular form, for example in admixture with a filler such as lactose, sucrose, mannitol, a starch such as potato starch or amylopectin, a cellulose derivative or highly dispersed silicic acid. In soft gelatin capsules, the active substance is preferably dissolved or suspended in a suitable liquid such as vegetable oil or liquid polyethylene glycol.

The lotion, ointment, gel or cream should be thoroughly rubbed into the skin, whereby the residual amount is not clearly visible, and the skin in this area should not be washed until most of the transdermal penetration has occurred, preferably for at least 4 hours and more preferably for at least 6 hours.

Transdermal patches may be used to deliver the precursors according to known techniques. They are typically applied for an extended period of time, e.g., 1 to 4 days, but typically have a small surface area with which the active ingredient is contacted to allow for slow and constant delivery of the active ingredient.

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

The mechanical aspects of transcutaneous devices are well known in the art and are described, for example, in U.S. Pat. Nos. 5,162,037, 5,154,922, 5,135,480, 4,666,441, 4,624,665, 3,742,951, 3,797,444, 4,568,343, 5,064,654, 5,071,644, 5,071,657, the disclosures of which are incorporated herein by reference. European patent No. 0279982 and uk patent application No. 2185187 provide additional background.

The device may be of any general type known in the art, including transdermal delivery devices of the adhesive matrix and reservoir type. The device may include a drug-containing matrix having incorporated therein fibers and/or carriers that absorb the active ingredient. In reservoir type devices, the reservoir may be defined by a polymer membrane impermeable to the carrier and active ingredient.

In transdermal devices, the device itself maintains the active ingredient in contact with the intended topical skin surface. In such devices, the viscosity of the carrier for the active ingredient is not of concern to the extent that it is in the case of a cream or gel. Solvent systems for transdermal devices 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, transdermal patches may be affixed to a surgical tape with a hole punched in the middle. The tape is preferably covered by a release film to provide protection prior to use. Typical materials suitable for release include polyethylene and polyethylene coated paper, and preferably silicone coated for ease of removal. In applying the device, the release film is simply peeled off and the tape is adhered to the patient's skin. In U.S. patent No. 5,135,480, the disclosure of which is incorporated herein by reference, Bannon et al describe an alternative device having a non-adhesive means for securing the device to the skin.

The SERM, antiestrogen, and sex steroid precursor need only be administered in a manner and in a dose sufficient to allow the serum concentration of each to reach the desired level. In accordance with the combination therapy of the present invention, while the concentration of the sex steroid precursor is maintained within the desired parameters, the concentration of the SERM is also maintained within the desired parameters.

A preferred sex steroid precursor is DHEA, although DHEA-S and the analogs described below are particularly effective for the reasons described below.

The molecular formula of the selective estrogen receptor modulator has the following characteristics: a) two aromatic rings separated by 1 to 2 intervening carbon atoms, the two aromatic rings being unsubstituted or substituted with a hydroxyl group or a group that is converted to a hydroxyl group in vivo; and b) a side chain having an aromatic ring and a tertiary amine functionality or salts thereof.

A preferred SERM of the invention is acobifene (Acolbifene):

acobifene (Acolbifene) (also known as EM-652. HCl; EM-1538) is the hydrochloride salt of the potent antiestrogen EM-652. Which is disclosed in U.S. patent No. 6,710,059B 1. Another preferred SERM is lasofoxifene (Lasoxifene) (Oporia); CP-336, 156; (-) -cis- (5R, 6S) -6-phenyl-5- [4- (2-pyrrolidin-1-ylethoxy) phenyl ] -5, 6,7, 8-tetrahydronaphthalene-2-ol, D- (-) -tartrate) (available from Pfizer, Inc., U.S.A.).

Another preferred SERM is developed by Wyeth eyes (USA) and disclosed in Japanese patent No. 10036347 (American household products corporation) and Bazedoxifene approved in the United states for the prevention of postmenopausal osteoporosis (TSE 424; WAY-TSE 424; WAY 140424; 1- [ [4- [2- (hexahydro-1H-aza-1H) -as-1-yl) ethoxy]Phenyl radical]Methyl radical]-2- (4-hydroxyphenyl) -3-methyl-1H-indol-5-ol, acetate), and the non-steroidal estrogen derivatives described in WO 97/32837. Other preferred SERMs of the invention include Tamoxifen ((Z) -2- [4- (1, 2-diphenyl-1-butenyl) phenoxy)]-N, N-dimethylethylamine) (available from Zeneca, Jiekang, England), Toremifene (Tormeifene) ((Z) -2- [4- (4-chloro-1, 2-diphenyl-1-butenyl) phenoxy]N, N-dimethylethylamine) available under the trademark of Fries (Fareston) or Schering-Plough from Orion, Finland, Droloxifene (Droloxifene) ((E) -3- [1- [4- [2- (dimethylamino) ethoxy ] ethoxy)]Phenyl radical]-2-phenyl-1-butenyl]Phenol) and Raloxifene (Raloxifene) ([2- (4-hydroxyphenyl) -6-hydroxybenzo [ b ]) from elily (EliLilly) usa]Thien-3-yl][4- [2- (1-piperidinyl) ethoxy group]Phenyl radical]-methanone hydrochloride), LY335124, LY 326315, LY 335563 (6-hydroxy-3- [4- [2- (1-piperidinyl) ethoxy)]Phenoxy radical]-2- (4-hydroxy)Phenyl) benzo [ b]Thiophene hydrochloride) and Arzoxifene (Arzoxifene) (LY353381, 6-hydroxy-3- [4- [2- (1-piperidinyl) ethoxy)]Phenoxy radical]-2- (4-methoxyphenyl) benzo [ b]Thiophene hydrochloride). Another preferred SERM is Idoxifene (Idoxifene) ((E) -1- [2- [4- [1- (4-iodophenyl) -2-phenyl-1-butenyl)]Phenoxy radical]Ethyl radical]Pyrrolidine) (Smith Kline Beecham, usa); levomeoxifene (Levorgeloxifene) (3, 4-trans-2, 2-dimethyl-3-phenyl-4- [4- (2- (2- (pyrrolidin-1-yl) ethoxy) phenyl]-7-methoxy) (3, 4-trans-2, 2-dimethyl-3-phenyl-4- [4-2- (2- (pyrolidin-1-y 1) ethoxy) phenyl]7-methoxychroman (Novo Nordisk, A/S) Inc.) and disclosed in WO 97/25034, WO 97/25035, WO 97/25037, WO 97/25038 to Shalmi et al and WO 97/25036 to Korsgaard et al; GW5638 (described in Willson et al, 1997, B) and indole derivatives (disclosed by Miller et al in European patent No. 0802183A 1). Also included are the epothilones (Iproxifene) (TAT 59; (E) -4- [1- [4- [2- (dimethylamino) ethoxy ] forms from Taiho (Taiho) Inc. (Japan)]Phenyl radical]-2- [4- (1-methylethyl) phenyl]-1-butenyl radical]Dihydric phenol phosphate), Ospemifene (Ospemifene) available from orlian-valmus pharmaceutical (Orion-Farmos pharmaceutical) finland (FC 1271; ((Z) -2- [4- (4-chloro-1, 2-diphenyl-1-butenyl) phenoxy]Ethanol), SERM 3471, HMR3339 and HMR3656 from Sanofi-Aventis (france), SH646 from Schering AG, pefoxifene (ERA923) developed by wyeth eyers, the non-steroidal estrogen derivatives described in WO 97/3283, nonperfifene (Fispemifene) developed by QuatRx (usa) and CC 8490 developed by Celgene (Celgene) usa.

Any SERM may be used as required for efficacy, as suggested by the manufacturer. Appropriate dosages are known in the art. Any other non-steroidal antiestrogen available commercially may be used in accordance with the present invention. Any compound having activity similar to a SERM (e.g., Raloxifene (Raloxifene)) can be used.

The SERM administered according to the present invention, when administered orally, is preferably administered in a dosage range of between 0.01 and 10 mg/kg body weight per day (preferably 0.05 to 1.0 mg/kg), preferably 5 mg per day in two divided doses for subjects with normal body weight, in particular 10 mg per day; or, when administered parenterally (i.e., intramuscularly, subcutaneously or transdermally), preferably in a dosage range of between 0.003 to 3.0 mg/kg body weight per day (preferably 0.015 to 0.3 mg/ml), and for subjects of normal body weight preferably in two divided doses of 1.5 mg, especially 3.0 mg per day. The SERM is preferably administered with a pharmaceutically acceptable diluent or carrier as described below.

Preferred antiestrogens of the present invention are fulvestrant (Faslodex); ICI 1827807; alpha- [9- (4, 4,5, 5, 5-pentafluoro-pentylsulfinyl) nonyl ] estra-1, 3, 5(10) -triene-3, 17 beta-diol administered intramuscularly at a monthly dose of 250 mg, and are available from AstraZeneca Canada Inc., of Mississauga (Ontario), Canada.

For all doses suggested herein, the attending clinician should supervise the response of the individual patient and adjust the dose accordingly.

Examples of the invention

Example 1

In the mammary gland, androgens are formed from the steroid precursor Dehydroepiandrosterone (DHEA). Clinical evidence shows that androgens have inhibitory effects on breast cancer. On the other hand, estrogen stimulates the development and growth of breast cancer. We investigated the effect of DHEA, alone or in combination with the aforementioned pure antiestrogen EM-800, on the growth of tumor xenografts formed by human breast cancer cells ZR-75-1 in ovariectomized nude mice.

Mice received 0.5 micrograms daily of estrone (an estrogen hormone) by subcutaneous injection immediately after ovariectomy. EM-800(15, 50 or 100 micrograms) was administered orally once daily. DHEA was administered twice daily to the dorsal skin alone (total dose 0.3, 1.0 or 3.0 mg) or in combination with an oral dose of EM-800 of 15 micrograms per day. Tumor size changes for treatment response were assessed periodically relative to measurements taken on day 1. At the end of the experiment, tumors were removed and weighed.

A 9.4-fold increase in tumor size was observed in 9.5 months in ovariectomized mice receiving estrone alone compared to those not receiving estrone. Administration of 15, 50 or 100 micrograms of EM-800 in the ovariectomized group supplemented with estrone resulted in 88%, 93% and 94% tumor size inhibition, respectively. In another aspect, DHEA at doses of 0.3, 1.0 or 3.0 mg inhibits the final tumor weight by 67%, 82% and 85%, respectively. Comparable inhibition in tumor size was obtained with an oral dose of EM-800 of 15 micrograms per day with or without different doses of DHEA administered transdermally. DHEA, independently of EM-800, inhibited the growth of estrone-stimulated ZR-75-1 mouse xenograft tumors in nude mice. The inhibitory effect of EM-800 was not altered by administration of a defined dose of DHEA.

Materials and methods

ZR-75-1 cells

ZR-75-1 human breast cancer cells were obtained from the American Type Culture Collection (Rockville, Md.) and routinely cultured as described (Poulin and Labrie, 1986, et. B.; Poulin et. al, 1988) in RPMI 1640 medium supplemented with 2 mML-amic acid, 1mM sodium pyruvate, 100 International units of penicillin/ml, 100 micrograms of streptomycin/ml, and 10% fetal bovine serum in 37 ℃ humidified gas environment at 95% air/5% carbon dioxide. Weekly in a batch of 0.05% trypsin: after treatment with 0.02% EDTA (weight/volume), cell subcultures were performed. The cell culture used in this report in the experiments was a ZR-75-1 cell line derived from passage 93.

Animal(s) production

Female homozygously-vaccinated Harland Schorge-Doley (Sprague-Dawley) (nu/nu) athymic mice (28 to 42 days old) were obtained from HSD (Indianapolis, Ind.). Mice were housed in vinyl cages with an air filter top placed in a laminar flow hood and maintained under pathogen limiting conditions. The cage, liner and food are autoclaved before use. The water was autoclaved, acidified to pH 2.8, and available ad libitum.

Cell seeding

One week prior to inoculation of tumor cells under anesthesia achieved by intraperitoneal injection of 0.25 ml/animal of Avertin (Avertin) (pentanol: 0.8 g/100 ml of 0.9% sodium chloride; and tribromoethanol: 2 g/100 ml of 0.9% sodium chloride), mice were ovariectomized on both sides (OVX). After treatment of the monolayer with 0.05% trypsin/0.02% EDTA (weight/volume), 1.5x10 was harvested⁶ZR-75-1 cells in logarithmic growth phase were suspended in 0.1 ml of medium containing 25% Matrigel (Matrigel) and subcutaneously inoculated into the flank of the animal using a 1-inch long 20-gauge needle as described previously (Dauvois et al, 1991, et al). To promote tumor growth, each animal was injected daily subcutaneously with 10 micrograms estradiol (E) in a vehicle consisting of 0.9% sodium chloride, 5% ethanol, 1% gelatin₂) For a total of 5 weeks. After the palpable ZR-75-1 tumor appeared, tumor diameters were measured with calipers, and mice with tumor diameters between 0.2 and 0.7 cm were selected for use in this study.

Hormone therapy

All animals except the OVX control group were injected daily subcutaneously with 0.5 microgram of estrone (E) in 0.2 ml of 0.9% sodium chloride, 5% ethanol, 1% gelatin₁). In the group shown, a dose of DHEA of 0.3, 1.0 or 3.0 mg/animal was administered transdermally twice daily in a volume of 0.02 ml to the dorsal skin area outside the tumor growth zone. DHEA was dissolved in 50% ethanol-50% propylene glycol. As previously described (Gauthier et al inJournal of 1997 "J.Med.chem." No. 40, p. 2117-2122), in the department of medical chemistry of the molecular Endocrinology laboratory at the CHUL research center, EM-800((+) -7-trimethylacetoxy-3- (4 '-trimethylacetoxyphenyl) -4-methyl-2- (4 "- (2"' -N-piperidinylethoxy) phenyl) -2H-benzopyran) was synthesized. EM-800 was dissolved in 4% (v/v) ethanol, 4% (v/v) polyethylene glycol (PEG)600, 1% (w/v) gelatin, 0.9% (w/v) sodium chloride. Animals in the indicated group received either a single daily dose of 15 micrograms, 50 micrograms or 100 micrograms of oral EM-800 or the combination DHEA, whereas animals in the OVX group received only the vehicle (0.2 ml of 4% ethanol, 4% PEG 600, 1% gelatin, 0.9% sodium chloride). Tumors were measured weekly with a vernier caliper. Two perpendicular diameters (L and W) were recorded in centimeters and the tumor area (square centimeter) was calculated using the formula L/2xW/2x π (Dauvois et al, in Eden 1991). The area measured on day 1 of treatment was 100%, while the tumor size change was expressed as a percentage of the initial tumor area. In the case of a typical subcutaneous tumor, it is not possible to correctly acquire the three-dimensional volume of the tumor, and therefore only the tumor area is measured. The animals were sacrificed after 291 days (or 9.5 months) of treatment.

The reaction categories were evaluated as described (Japanese a 1989 by Dauvois et al; Japanese b 1995 by Labrie et al). In short, partial regression corresponds to tumor regression to a degree equal to or greater than 50% of their original size; a stable response means that tumors regress to less than 50% of their original size or worsen to less than 50% of their original size, while complete regression means that the tumors are undetectable at the end of treatment. Worsening refers to tumor progression of more than 50% compared to their original size. At the end of the experiment, all animals were killed by decapitation. Tumors, uterus and vagina were immediately removed, connective and adipose tissue was removed, and weighed.

Statistical analysis

Using an analysis of variance (ANOVA) to assess the effects due to DHEA, EM-800 and time, statistical significance of the effect of treatment on tumor size was assessed, and repeated measures (individual factors within the group) were taken in the same animals at the beginning of treatment and at the end of treatment. Repeated measurements at 0 and after 9.5 months of treatment constituted a random set of regions for the animals. Thus time is analyzed as intra-regional effects and the two treatments as inter-regional effects. All interactions between major effects are included in this pattern. The significance of the interaction of the therapeutic factors with them was analyzed using the individuals within the group as error terms. A logarithmic conversion of the data is performed. The assumption underlying ANOVA is that the normality of the residuals and the homogeneity of the variance are assumed.

A post hoc pairwise comparison was performed using the Fisher (Fisher) assay for the least significant difference. The main effects and interactions of treatment on body weight and organ weight were analyzed using standard two-way ANOVA with interaction. All ANOVA (SAS Institute) was performed using the SAS program (saishi computer software of kary, north carolina, usa). The significance of the difference was demonstrated using a two-tailed assay with an overall level of 5%. The class data were analyzed by a Kruskall-Wallis assay for graded class response variables (complete response, partial response, stable response, and tumor progression). After global evaluation of a therapeutic effect, critical p-values for multiple comparisons were adjusted and a subset of the results shown in table 4 was analyzed. The exact p-value was calculated using the StatXact program (Western Picture (Cytel) Inc. of Cambridge, USA). Data are presented as mean ± standard deviation of mean (SEM) of 12 to 15 mice in each group.

Results

As illustrated in panel 3A, human ZR-75-1 tumors increased 9.4-fold over 291 days (9.5 months) in ovariectomized nude mice treated with a daily dose of 0.5 micrograms estrone administered subcutaneously; whereas in the control OVX mice receiving vehicle only, the tumor size decreased to 36.9% of the initial value during the study.

Treatment with DHEA at increasing doses administered transdermally, for menses E₁Stimulated ZR-75-1 tumor growth results in progressive inhibition. 50.4%, 76.8% and 80.0% inhibition was achieved with DHEA doses of 0.3 mg, 1.0 mg and 3.0 mg daily in each animal over a 9.5 month treatment period (FIG. 3A). Consistent with the reduction in total tumor burden, treatment with DHEA resulted in a significant reduction in the average weight of tumor left at the end of the experiment. In fact, the mean tumor weight is self-complementing E₁The dose of the ovariectomized control group of nude mice was reduced to 0.37 ± 0.12 g (P ═ 005), 0.20 ± 0.06 g (P ═ 001), and 0.17 ± 0.06 g (P ═ 0009), respectively, in the groups of animals receiving daily doses of 0.3, 1.0, and 3.0 mg of DHEA (fig. 3B).

The inhibition of estrogen stimulated tumor size by doses of antiestrogen EM-800 at 15 micrograms, 50 micrograms and 100 micrograms daily, when compared to the tumor size at 9.5 months in control animals, was 87.5% (P <. 0001), 93.5% (P <. 0001) and 94.0% (P ═ 0003), respectively (fig. 4A). The tumor sizes reduced at the three EM-800 doses did not differ significantly from each other. As illustrated in panel 3B, tumor weight at the end of the 9.5 month study was a supplement from control E₁Is reduced to 0.08 + -0.03 g, 0.03 + -0.01 g and 0.04 + -0.03 g in animals treated with doses of EM-800 of 15 microgram, 50 microgram and 100 microgram daily, respectively, (at all doses of EM-800 relative to the supplemental E)₁P <.0001) of OVX.

As mentioned above, the daily oral dose of 15 microgram of antiestrogen EM-800, measured at 9.5 months, showed an inhibition of estrone stimulated tumor growth of 87.5%. The addition of DHEA to the three doses used had no significant effect on the already significant tumor size inhibition achieved with the daily dose of 15 μ g of antiestrogen EM-800 (FIG. 4B). Thus, the mean tumor weight was surprisingly reduced from 1.12 ± 0.26 g in control group estrone supplemented mice to 0.08 ± 0.03 g (P <. 0001), 0.11 ± 0.04 g (P ═ 0002), 0.13 ± 0.07 g (P ═ 0004) and 0.08 ± 0.05 g (P <. 0001) in animals receiving 15 micrograms daily antiestrogen doses alone or DHEA doses in combination of 0.3, 1.0 and 3.0 milligrams, respectively (no significant difference was observed between the 4 groups) (fig. 3B).

It is also of interest to examine the type of response achieved with the above treatments. Thus, although not reaching statistically significant levels (P ═ 088), increasing doses of DHEA treatment reduced the number of worsening tumors from 87.5% in OVX control animals supplemented with estrone to values of 50.0%, 53.3% and 66.7% in animals treated with daily doses of 0.3, 1.0 or 3.0 milligrams of DHEA (table 3). On the other hand, the full-blown response was 0% in mice self-supplemented with estrone, increasing to 28.6%, 26.7% and 20.0% in animals receiving daily transdermal DHEA doses of 0.3, 1.0 and 3.0 mg. On the other hand, in supplement E₁The control mice showed a steady response of 12.5%, 21.4%, 20.0% and 13.3% in the three groups receiving the above DHEA dose. In the ovariectomized control mice, the rates of complete, partial and stable responses were measured as 68.8%, 6.2% and 18.8%, respectively, while only a worsening phenomenon was observed in 6.2% of the tumors (table 3).

In animals receiving either the antiestrogen EM-800(P ═ 0006) (15 micrograms) alone or in combination with 0.3 mg, 1.0 mg or 3.0 mg DHEA, the resulting full response or tumor disappearance was 29.4%, 33.3%, 26.7% and 35.3%, respectively. On the other hand, in the same animal group, a worsening phenomenon was observed in 35.3%, 44.4%, 53.3% and 17.6% of tumors, respectively. There were no significant differences between groups treated with EM-800 alone or DHEA in combination.

No significant effect of DHEA or EM-800 treatment was observed on body weights adjusted for tumor weight. OVX mice were treated with estrone such that uterine weight increased from 28 ± 5 mg to 132 ± 8 mg (P <.01) in OVX control mice, whereas the stimulatory effect of ascending doses of DHEA on estrone caused a progressive but relatively small inhibitory effect, which reached 26% at the highest dose of DHEA used (P ═ 0008). In the same figure, it can be seen that estrone stimulated uterine weight is 132 + -8 mg in control mice self-supplemented with estrone, reduced to 49 + -3 mg, 36 + -2 mg and 32 + -1 mg (all doses relative to p <.0001 in control) of 15, 50 or 100 microgram daily oral dose of EM-800 (overall p <.0001), respectively. Uterine weights measured at 15 μ g EM-800 in combination with a daily dose of 0.3 mg, 1.0 mg, or 3.0 mg DHEA were 46 + -3 mg, 59 + -5 mg, and 69 + -3 mg DHEA, respectively.

On the other hand, estrone treatment increased vaginal weight from 14 ± 2 mg to 31 ± 2 mg (P <. 01) in OVX animals, whereas addition of DHEA had no significant effect. After treatment with doses of EM-800 at 15, 50 or 100 micrograms daily, vaginal weight decreased to 23 + -1 mg, 15 + -1 mg and 11 + -1 mg, respectively (all doses were <. 0001 relative to the overall P and paired P of the control). Vaginal weights determined for the combined DHEA and EM-800 doses of 0.3 mg, 1.0 mg or 3.0 mg were 22 ± 1 mg, 25 ± 2 mg and 23 ± 1 mg, respectively (none of the groups were significant relative to 15 μ g of EM-800). It should be mentioned that EM-800 reduces uterine weight in OVX animals supplemented with estrone to a value that is not different from that of the OVX control group at the highest dose used, i.e. 100. mu.g daily, whereas vaginal weight is reduced to a value that is lower than that measured for the OVX control group (P <.05). DHEA may partially counteract the effect of EM-800 on uterine and vaginal weight, possibly due to its androgenic effect.

No. 3 table

Effect of transdermal administration of DHEA or oral administration of EM-800 alone or in combination for 9.5 months on the response (full, partial, stable and worsening) of human ZR-75-1 breast tumor xenografts in nude mice

E₁Estrone; DHEA ═ dehydroepiandrosterone; ovariectomy

Example 2

Examples of the Synthesis of preferred Compounds of the invention

Synthesis of (S) - (+) -7-hydroxy-3- (4 ' -hydroxyphenyl) -4-methyl-2- (4 ' - (2 ' -N-piperidinylethoxy) phenyl) -2H-1-benzopyran hydrochloride EM-01538(EM-652, hydrochloride)

Scheme 1

Step A: BF (BF) generator₃·Et₂O, toluene; 100 ℃; for 1 hour.

And C: 3, 4-dihydropyran, p-toluenesulfonic acid monohydrate, ethyl acetate; after 16 hours at 25 ℃ under nitrogen, it is crystallized from isopropanol.

Steps D, E and F:

(1) piperidine, toluene, Dean Stark apparatus, refluxed under nitrogen;

(2)1, 8-diazabicyclo [5, 4, 0] undec-7-ene, DMF, at reflux for 3 hours;

(3)CH₃MgCl, THF, -20 to 0 ℃, then 24 hours at room temperature;

step G, H: (1S) - (+) -10-camphorsulfonic acid, acetone, water, toluene at room temperature for 48 hours.

Step HH: 95% ethanol, 70 ℃, then at room temperature for 3 days.

Step HHR: recovering the mother liquor and the cleaning solution of the step HH; (S) -10-camphorsulfonic acid, refluxing; 36 hours, then 16 hours at room temperature.

Step I:

(1) aqueous DMF, sodium carbonate, ethyl acetate;

(2) ethanol, dilute hydrochloric acid;

(3) and (3) water.

2-tetrahydro-pyranoxy-4-hydroxy-2 '- (4' -tetrahydro-pyranoxyphenyl) acetophenone (4). 2, 4-dihydroxy-2' - (4 "-hydroxyphenyl) acetophenone 3(97.6 g, 0.4 mol) (available from Kaiser scientific Laboratories, Inc. of Lenexa, Inc.) was treated at about 25 ℃ with p-toluenesulfonic acid monohydrate (0.03 g, 0.158 mmol) in a suspension of 3, 4-dihydropyrane (218 ml, 3.39 mol) and ethyl acetate (520 ml). The reaction mixture was stirred under nitrogen for about 16 hours without external heating. The mixture was then washed with a solution of sodium bicarbonate (1 g) and sodium chloride (5 g) in water (100 ml). The phases were separated and the organic phase was washed with brine (20 ml). Each rinse was back-extracted with 50 ml of ethyl acetate. All organic phases were combined and filtered through sodium sulfate. The solvent was removed by distillation at atmospheric pressure (about 600 ml) and isopropanol (250 ml) was added. Additional solvent (about 300 ml) was distilled off at atmospheric pressure, and isopropanol (250 ml) was added. Additional solvent (about 275 ml) was distilled off at atmospheric pressure and isopropanol (250 ml) was added. The solution was cooled at about 25 ℃ under stirring, and after about 12 hours, the crystalline solid was filtered, washed with isopropanol and dried (116.5 g, 70%).

Synthesis of 4-hydroxy-4-methyl-2- (4 ' - [2 ' -N-piperidinyl ] -ethoxy) phenyl-3- (4 ' -tetrahydropiper inoxyl) phenyl-7-tetrahydropiper inoxyl-alkane (10). Under nitrogen, a solution of 2-tetrahydropyranyloxy-4-hydroxy-2' - (4 "-tetrahydropyranyloxyphenyl) acetophenone 4(1 kg, 2.42 mol), 4- [2- (1-N-piperidinyl) ethoxy ] benzaldehyde 5(594 g, 2.55 mol) (available from Chemsyn science laboratories, leneca, usa) and piperidine (82.4 g, 0.97 mol) (available from Aldrich chemical limited of milwaki, wisconsin., usa) in toluene (8 liters) was refluxed with a Dean Stark apparatus until one equivalent of water was collected (44 ml). Toluene (6.5 l) was removed from the solution by distillation at atmospheric pressure. Dimethylformamide (6.5 l) and 1, 8-diazabicyclo [5, 4, 0] undec-7-ene (110.5 g, 0.726 mol) were added. The solution was stirred at room temperature for about 8 hours to isomerize chalcone (chalcone)8 to alkanone 9, then added to a mixture of water and ice (8 liters) and toluene (4 liters). The phases were separated and the toluene layer was washed with water (5 liters). The combined aqueous washes were extracted with toluene (3 × 4 liters). The combined toluene extracts were finally washed with brine (3 × 4 l), concentrated to 5.5 l at atmospheric pressure and then cooled to-10 ℃. Under nitrogen, a 3M solution of methylmagnesium chloride in THF (2.5 liters, 7.5 moles) (available from Aldrich chemical ltd, milwaki, wi) was added with constant external cooling and stirring to maintain the temperature below 0 ℃. After all the Grignard reagent was added, the external cooling was removed and the mixture was allowed to warm to room temperature. The mixture was stirred at this temperature for about 24 hours. The mixture was cooled again to about-20 ℃ and saturated ammonium chloride solution (200 ml) was added slowly with continued external cooling and stirring, maintaining the temperature below 20 ℃. The mixture was stirred for 2 hours, then saturated ammonium chloride solution (2 l) and toluene (4 l) were added and stirred for 5 minutes. The phases were separated and the aqueous layer was extracted with toluene (2 × 4 liters). The combined toluene extracts were washed with dilute hydrochloric acid until the solution was homogeneous and then washed with brine (3 × 4 liters). Finally the toluene solution was concentrated to 2 l at atmospheric pressure. This solution was used directly in the next step.

Synthesis of (2R, S) -7-hydroxy-3- (4 ' -hydroxyphenyl) -4-methyl-2- (4 ' - [2 ' -N-piperidinyl ] ethoxy) phenyl) -2H-1-benzopyran (1S) -10-camphorsulfonate (+ -12). To a toluene solution of 4-hydroxy-4-methyl-2- (4 '- [2 "-N-piperidinyl ] -ethoxy) -phenyl-3- (4' -tetrahydropiperanyloxy) phenyl-7-tetrahydropiperanyloxy (10) were added acetone (6 liters), water (0.3 liters) and (S) -10-camphorsulfonic acid (561 g, 2.42 moles) (available from Aldrich chemical Co., Milwaukee, Wis., U.S.A.). The mixture was stirred under nitrogen for 48 hours, after which the solid (2R, S) -7-hydroxy-3- (4 '-hydroxyphenyl) -4-methyl-2- (4 "- [ 2"' -N-piperidinyl ] ethoxy) phenyl) -2H-1-benzopyran (1S) -10-camphorsulfonate (12) was filtered, washed with acetone and dried (883 g). This material was used in the next (HH) step without further purification.

Synthesis of (2S) -7-hydroxy-3- (4 ' -hydroxyphenyl) -4-methyl-2- (4 ' - [2 ' -N-piperidinyl ] ethoxy) phenyl) -2H-1-benzopyran (1S) -10-camphorsulfonate (13, (+) -EM-652(1S) -CSA salt). A suspension of (2R, S) -7-hydroxy-3- (4 ' -hydroxyphenyl) -4-methyl-2- (4 ' - [2 ' -N-piperidinyl ] ethoxy) phenyl) -2H-benzopyran (1S) -10-camphorsulfonate + -12 (759 g) in 95% ethanol was heated to about 70 ℃ with stirring until the solids dissolved. The solution was allowed to cool to room temperature with stirring and then some crystals of (2S) -7-hydroxy-3- (4 ' -hydroxyphenyl) -4-methyl-2- (4 ' - [2 ' -N-piperidinyl ] ethoxy) phenyl) -2H-1-benzopyran (1S) -10-camphorsulfonate 13 were implanted. The solution was stirred at room temperature for a total of about three days. The crystals were filtered, washed with 95% ethanol and dried (291 g, 76%). The de of the product was 94.2% and the purity 98.8%.

Synthesis of (S) - (+) -7-hydroxy-3- (4 ' -hydroxyphenyl) -4-methyl-2- (4 ' - (2 ' -N-piperidinylethoxy) phenyl) -2H-1-benzopyran hydrochloride EM-01538(EM-652, hydrochloride). A suspension of compound 13(EM-652- (+) -CSA salt, 500 mg, 0.726 mmol) in dimethylformamide (11 μ l, 0.15 mmol) was treated with 0.5M aqueous sodium carbonate (7.0 ml, 3.6 mmol) and stirred for 15 min. The suspension was treated with ethyl acetate (7.0 ml) and stirred for 4 hours. The organic phase was then washed with saturated aqueous sodium carbonate (2 × 5 ml) and water (1 × 5 ml), dried over magnesium sulfate and concentrated. A solution of the resulting pink foam (EM-652) in ethanol (2 mL) was treated with 2N hydrochloric acid (400. mu.L, 0.80 mmol) and stirred for 1 hour, with distilled water (5 mL) and stirred for 30 minutes. The resulting suspension was filtered, washed with distilled water (5 ml), dried in air and under high vacuum (65 ℃) to give a solidOpalescent powder (276 mg, 77%) (fine yellow-white powder) scanning calorimetry (melting peak at 219 ℃ C.,. DELTA.H 83J/g; in 10 mg/ml methanol [ α%]²⁴D＝154°；¹H NMR(300MHz，CD₃OD) (ppm)1.6 (broad, 2H, H-4 '), 1.85 (broad, 4H, H-3 ' and 5 '), 2.03(s, 3H, CH)₃) 3.0 and 3.45 (broad, 4H, H-2 "", and 6 ""), 3.47(t, J ═ 4.9Hz, 2H, H-3 "'), 4.26(t, J ═ 4.9Hz, 2H, H-2"'), 5.82(s, 1H, H-2), 6.10(d, J ═ 2.3Hz, 1H, H-8), 6.35(dd, J ═ 8.4, 2.43Hz, 1H, H-6), 6.70(d, J ═ 8.6Hz, 2H, H-3 ', and H-5'), 6.83(d, J ═ 8.7Hz, 2H, H-3 ", and H-5"), 7.01(d, J ═ 8.5Hz, 2H, H-2 ', and H-6'), 7.12(d, J ═ 8.5, 8, 6H, 6 ═ 8, 6H ″, and H-6 ″;¹³C RMN(CD₃OD, 75MHz) ppm14.84, 22.50, 23.99, 54.78, 57.03, 62.97, 81.22, 104.38, 109.11, 115.35, 116.01, 118.68, 125.78, 126.33, 130.26, 130.72, 131.29, 131.59, 134.26, 154.42, 157.56, 158.96, 159.33. elemental composition: the theoretical values of carbon, hydrogen, nitrogen and chlorine are 70.51, 6.53, 2.84 and 7.18%, and the actual values are 70.31, 6.75, 2.65 and 6.89%.

Example 3

Materials and methods

Animal(s) production

Female BALB/c mice (BALB/cAnNCrlBR) weighing 18 to 20 grams were obtained from Charles River (Charles-River, Inc) Inc. (St-Constant), Quebec, Canada, and were kept in an environment of controlled temperature (23. + -. 1 ℃) and light (12 hours/day light period, starting from 7: 15) only for 5 cages. The mice were fed with the ad libitum feeding of the rodent chow and tap water. Animals were Ovariectomized (OVX) under Isoflurane (Isoflurane) anesthesia through bilateral flank incisions and randomly assigned to groups of 10 animals each. 10 mice without ovariectomy were kept as a control group.

Treatment of

In the first experiment (figures 11 to 14), starting 2 days after ovariectomy, doses of 1, 3 or 10 micrograms/animal of test compound, i.e., EM-652.HCl, Lasofoxifene (Lasofoxifene) (free base form; active and inactive enantiomers) and Raloxifene (Raloxifene) were administered orally by gastric tube feeding daily for up to 9 days. In the second experiment (table 6), TSE424 doses of 1, 3, 10 or 30 micrograms/animal were administered orally by gastric tube feeding once daily for 9 days starting 2 days after ovariectomy. In both experiments, to assess antiestrogenic activity, estrone (0.06 microgram of E per day by subcutaneous injection) was initiated 5 days after ovariectomy₁Two) and a total of 6 days of administration. The compound was dissolved in ethanol (final concentration of 4%) and placed in 0.4% methyl cellulose for administration. Mice in the non-ovariectomized and OVX control groups received vehicle only (4% ethanol-0.4% methylcellulose) during the 9 day period. On the 11 th morning after ovariectomy, animals were killed by exsanguination of the abdominal aorta. The uterus and vagina were removed rapidly, weighed, and preserved in 10% buffered formalin for further histological examination.

Item I. results

Experiment 1:

as illustrated in figure 11, EM-652.HCl administered daily at oral doses of 1 microgram, 3 microgram and 10 microgram elicited 24%, 48% and 72% inhibition, respectively, for estrone-stimulated uterine weight (all doses relative to p < 0.01 for the control group), while Raloxifene (Raloxifene), administered at the same dose, elicited 6% (not significant), 14% (p < 0.01) and 43% (p < 0.01) inhibition, respectively, for this parameter. On the other hand, Lasofoxifene (free base form) does not have an inhibitory effect at the lowest dose used, but it causes 25% (p < 0.01) and 44% (p < 0.01) inhibition of estrone-stimulated uterine weight at doses of 3 and 10 micrograms daily, respectively. The inactive enantiomer of Lasofoxifene (Lasofoxifene) did not exhibit inhibitory effects on this parameter at any of the doses used.

The above compounds exhibit similar effects on vaginal weight. Oral administration of EM-652.HCl at 1, 3, and 10 microgram daily doses (figure 12) resulted in 10% (no significant), 25%, and 53% inhibition of vaginal weight (p < 0.01 for the two highest doses), respectively, whereas Raloxifene (Raloxifene) exhibited a significant 24% (p < 0.01) inhibitory effect for this parameter only at the highest dose (10 microgram). Like Raloxifene (Raloxifene), Lasofoxifene (free base form) caused a significant 37% (p < 0.01) inhibitory effect only at the highest dose used, while the inactive enantiomer did not have an inhibitory effect on vaginal weight at any of the doses used.

When the compounds were administered alone (in the absence of estrone) at oral doses of 1 microgram and 10 microgram per day in ovariectomized mice, EM-652.HCl had no significant stimulatory effect on uterine weight at both doses used, while treatment with 10 microgram of Lasofoxifene (Lasofoxifene) and Raloxifene (Raloxifene) resulted in 93% (p < 0.01) and 85% (p < 0.01) stimulation of uterine weight, respectively (fig. 13), thus showing the estrogenic effect of these latter compounds on this parameter. Similarly, EM-652.HCl did not exhibit significant stimulatory effects on vaginal weight (fig. 14), while administration of 10 micrograms of Lasofoxifene (Lasofoxifene) and Raloxifene (Raloxifene) resulted in 73% (p < 0.01) and 56% (p < 0.01) stimulatory effects, respectively, on vaginal weight. On the other hand, the inactive enantiomer of Lasofoxifene (Lasofoxifene) does not have a stimulatory effect on uterine and vaginal weight.

Experiment 2:

as shown in table 4, TSE424 administered at an oral dose of 1 microgram, 3 microgram, 10 microgram or 30 microgram daily resulted in 12% (not significant), 47%, 74% and 94% inhibition, respectively, of estrone stimulated uterine weight (the three highest doses relative to E)₁P < 0.01 for the control group). On the other hand, 3, 10 and 30 microgram of T are administered orally dailySE424 dose, resulted in 16% (not significant), 56% (p < 0.01) and 93% (p < 0.01) inhibition of vaginal weight, respectively.

When the compound was administered alone (in the absence of estrone) at oral doses of 3 micrograms and 30 micrograms per day in ovariectomized mice, TSE424 did not have a significant stimulatory effect on uterine and vaginal weight at the two doses used (table 4).

4 th table

In ovariectomized mice, treated with or without estrone, increasing concentrations of TSE424 were administered orally for 9 days of effect on uterine and vaginal weight. P < 0.01 relative to control group treated with E1.

Example 4

Preventive effect on bone loss, serum lipids and total fat

Animals and treatments

Female smigra-dowley (sprague dawley) rats 10 to 12 weeks old weighing about 220 to 270 grams at the start of treatment (Crl: cd (sd) Br ((Charles River Laboratory) of St constantan (St-Constant) quebeck, canada.) prior to the start of the experiment, the animals were acclimated to ambient conditions (temperature 22 ± 3 ℃; humidity is 50 +/-20%; 12 hours light-12 hours dark cycle, light period starting at 07: 15) for at least 1 week, animals were individually housed and free access to tap water was allowed to be provided with a granular certified rodent chow (Lab Diet 5002 from Ralston Purina, St. Louis, Mo.) the experiments were conducted according to CCAC's guidelines for laboratory animal management and use, in an animal agency under the codes of the Canadian animal Care Committee (CCAC) and the International Association for the administration and accreditation of laboratory animals (AAALAC).

In the first experiment, 154 rats were randomly allocated to 11 groups of 14 animals each, which groups were as follows: 1) a control group without ovariectomy; 2) OVX control group; 3) OVX + E₂(1 mg/kg); 4) OVX + EM-652.HCl (2.5 mg/kg); 5) OVX + E₂+ EM-652. HCl; 6) OVX + dehydroepiandrosterone (DHEA; 80 mg/kg); 7) OVX + DHEA + EM-652. HCl; 8) OVX + DHEA + E₂；9)OVX+DHEA+E₂+EM-652.HCl；10)OVX+GW 5638；11)OVX+E₂+ GW 5638. On study day 1, animals in the appropriate group were resected on both Ovaries (OVX) under Isoflurane (Isoflurane) anesthesia. DHEA was topically applied to the dorsal skin as a solution in 50% ethanol-50% propylene glycol, while the other test compounds were administered as a suspension in 0.4% methylcellulose by oral-gastric tube feeding. Treatment was initiated on study day 2 and was performed once daily for a period of 3 months.

In a second experiment, 132 rats were randomly allocated to 9 groups of 14 or 15 animals each, the groups being as follows: 1) a control group without ovariectomy; 2) OVX control group; 3) OVX + pramelin (Premarin) (0.25 mg/kg); 4) OVX + EM-652.HCl (2.5 mg/kg); 5) OVX + pramelin (Premarin) + EM-652. HCl; 6) OVX + TSE424 (2.5 mg/kg); 7) OVX + pramelin (Premarin) + TSE 424; 8) OVX + Lasofoxifene (Lasofoxifene) (tartrate; a racemate; 2.5 mg/kg); 9) OVX + pramelin (Premarin) + Lasofoxifene (Lasofoxifene). On study day 1, animals in the appropriate group were resected on both Ovaries (OVX) under Isoflurane (Isoflurane) anesthesia. The test compounds were administered as a suspension in 0.4% methylcellulose by oral gastric tube feeding. Treatment was initiated on study day 2 and was performed once daily for a period of 26 weeks. In both experiments, animals not receiving the test substance were treated with the appropriate vehicle only during the same period.

Bone density measurement

After 3 months (experiment 1) or 26 weeks (experiment 2) of treatment, rats were individually scanned for their whole body bones and lumbar spine under Isoflurane (Isoflurane) anesthesia using a dual energy X-ray absorptiometry (DEXA; QDR4500A of hololojie (holgic) corporation of Waltham, ma, usa) and a regional high resolution scanning software. Bone density (BMD) and total body composition (percent fat) were determined for the lumbar vertebrae (vertebrae L2-L4).

Serum analysis

After 3 months (experiment 1) or 26 weeks (experiment 2) of treatment, blood samples were collected from the jugular vein of overnight fasted animals (under Isoflurane anesthesia). Samples were processed for serum preparation and frozen at-80 ℃ prior to analysis. Serum cholesterol levels and alkaline phosphatase Activity (ALP) were determined using a heiliman 911 analyzer (Hitachi) analyzer (Boehringer Mannheim Diagnostic) corporation (Boehringer Mannheim laboratory system).

Statistical analysis

Data are presented as mean ± SEM. Statistical significance was determined according to the Duncan-Kramer multiple domain assay (Kramer CY in journal "Biometrics" 1956, 12 th page 307-310 Enoted text).

Results

As shown in table 5, lumbar BMD in OVX control animals was 10% lower (p < 0.01) than in the non-ovariectomized control group 3 months after ovariectomy. Administration of estradiol alone and EM-652.HCl at the doses used prevented lumbar BMD loss by 98% (p < 0.01) and 65% (p < 0.05), respectively, while E was administered₂Combination treatment with EM-652.HCl prevented OVX-induced reduction in lumbar BMD by 61% (p < 0.05). On the other hand, when the administration effect of DHEA alone protects lumbar BMD to 43% (p < 0.05), DHEA + E is added₂Concurrent treatment with + EM-652.HCl prevented OVX-induced reduction in lumbar BMD by 91% and resulted in BMD values that were not different from those of the non-ovariectomized control group.

In table 6, after 26 weeks of ovariectomy,lumbar BMD was 18% lower than that of the control group without ovariectomy (p < 0.01). Administration of pramlinum (Premarin), EM-652.HCl, TSE424, and Lasofoxifene (Lasofoxifene) alone protected lumbar BMD by 54%, 62%, 49%, and 61%, respectively (relative to all p < 0.01 for the OVX control). Addition of pramiline (Premarin) to EM-652.HCl, TSE424, or Lasofoxifene (Lasofoxifene) resulted in lumbar BMD values that were not significantly different from those resulting from administration of each SERM alone (table 6). Similarly, at E₂Or addition of DHEA to EM-652.HCl completely prevented OVX-induced reduction of lumbar BMD (Table 5). The positive effect of DHEA on BMD was also demonstrated by its effect on serum alkaline phosphatase Activity (ALP), a marker of bone formation and turnover. ALP activity was increased from 73 + -6 International units/liter in OVX control animals to DHEA, DHEA + EM-652.HCl, DHEA + E₂And DHEA + E₂+ EM-652.HCl treatment of animals 224 ± 18 international units/liter, 290 ± 27 international units/liter, 123 ± 8 international units/liter and 261 ± 20 international units/liter (all p < 0.01), thus showing a stimulating effect of DHEA on bone formation (table 7).

In addition to the prophylactic effects on bone loss, EM-652.HCl, TSE424, Lasofoxifene (Lasofoxifene), GW5638, DHEA and E₂Exhibit some beneficial effects on overall fat percentage and serum lipids. After 3 months of ovariectomy, overall fat increases by 22% (p < 0.05; Table XXX 6). Administration of EM-652.HCl completely prevented OVX-induced fat percentage increase, while DHEA and/or E were added to the SERM₂Resulting in a lower fat percentage value than that observed in the non-ovariectomized control animals. After 26 weeks of ovariectomy, the increase in fat due to estrogen deficiency was 40% reversed after administration of pramlins (Premarin), EM-652.HCl, TSE424 or Lasofoxifene (Lasofoxifene) by 74%, 78%, 75% and 114%, respectively, while addition of pramlins (Premarin) to each SERM completely prevented the increase in fat percentage due to OVX (table 8).

As shown in table 7, inAfter 3 months of ovariectomy, a 22% increase in serum cholesterol levels (p < 0.01) was observed in OVX control rats compared to the non-ovariectomized control. In fact, serum cholesterol was increased from 2.01 ± 0.11 mmol/l in the non-ovariectomized animals to 2.46 ± 0.08 mmol/l in the OVX control group. Administration of E alone₂Or DHEA administration reduces serum cholesterol levels to 1.37 + -0.18 mM and 1.59 + -0.10 mM, respectively, EM-652.HCl alone or combination E₂And/or DHEA administration results in significantly lower cholesterol levels (between 0.65 and 0.96 mmol/l) than those observed in ovariectomized animals (2.01 ± 0.11 mmol/l). Similarly, GW5638, TSE424 and Lasofoxifene (Lasofoxifene) alone or in combination with E₂Or administration of pramlins (Premarin), completely prevented OVX-induced increases in serum cholesterol levels and resulted in values lower than those observed in ovariectomized animals (tables 7 and 8).

5 th table

Effect of estradiol, EM-652.HCl, GW5638, or DHEA treatment on prevention of bone loss 3 months after ovariectomized female rats administered alone or in combination

P < 0.05 for rats in experimental versus OVX control; p < 0.01.

6 th table

Effect on prevention of bone loss after 26 weeks of treatment of ovariectomized female rats with Premartin (Premarin), EM-652.HCl, TSE424 or Lasofoxifene (Lasofoxifene) administered alone or in combination with Premartin (PREMARIN)

P < 0.01 for the experimental group versus OVX control rats.

7 th table

Effect of estradiol, EM-652.HCl, GW5638, or DHEA treatment on the percentage of total fat, serum cholesterol levels, and alkaline phosphatase activity after 3 months in ovariectomized female rats

P < 0.05 for rats in experimental versus OVX control; p < 0.01.

8 th table

Effect on Total fat percentage, serum cholesterol levels and alkaline phosphatase Activity after 26 weeks of ovarian female rats treated with Premarlin (Premarin), EM-652.HCl, TSE424 or Lasofoxifene (Lasofoxifene) administered alone or in combination

P < 0.05 for rats in experimental versus OVX control; p < 0.01.

Example 5

Prophylactic effect on bone loss following treatment of ovariectomized female rats with Selective Estrogen Receptor Modulators (SERM) EM-652.HCl, TSE-424 and ERA-923 administered alone and in combination with DHEA

Animals and treatments

Female smigra-dowley (Sprague Dawley) rats 10 to 12 weeks old weighing about 220 to 270 grams at the start of treatment (Crl: cd (sd) Br ((Charles River Laboratory) of St constantan (St-Constant) quebeck, canada.) prior to the start of the experiment, the animals were acclimated to ambient conditions (temperature 22 ± 3 ℃; humidity is 50 +/-20%; 12 hours light-12 hours dark cycle, light period starting at 07: 15) for at least 1 week, animals were individually housed and free access to tap water was allowed to be provided with a granular certified rodent chow (Lab Diet 5002 from Ralston Purina, St. Louis, Mo.) the experiments were conducted according to CCAC's guidelines for laboratory animal management and use, in an animal agency under the codes of the Canadian animal Care Committee (CCAC) and the International Association for the administration and accreditation of laboratory animals (AAALAC).

126 rats were randomly assigned to 9 groups of 14 animals each, which were as follows: 1) a control group without ovariectomy; 2) OVX control group; 3) OVX + EM-652.HCl (2.5 mg/kg); 4) OVX + TSE-424(EM-4803, 2.5 mg/kg); 5) OVX + ERA-923(EM-3527, 2.5 mg/kg); 6) OVX + dehydroepiandrosterone (DHEA; 80 mg/kg); 7) OVX + DHEA + EM-652. HCl; 8) OVX + DHEA + TSE-424; 9) OVX + DHEA + ERA-923. On study day 1, animals in the appropriate group were resected on both Ovaries (OVX) under Isoflurane (Isoflurane) anesthesia. DHEA was topically applied to the dorsal skin as a solution in 50% ethanol-50% propylene glycol, and the test compound was administered as a suspension in 0.4% methylcellulose by oral gastric tube feeding. Treatment was initiated on study day 2 and was performed once daily over a 5 week period.

Bone density measurement

After 5 weeks of treatment, rats were individually scanned for lumbar, femoral and tibial using a dual energy X-ray absorptiometry (DEXA; QDR4500A from hololojie (Hologic) of Waltham, ma) and a regional high resolution scanning software under Isoflurane (Isoflurane) anesthesia. Bone density (BMD) was determined for lumbar vertebrae (vertebrae L2 to L4), remote femoral metaphysis (DFM) and Proximal Tibial Metaphysis (PTM).

Statistical analysis

Data are presented as mean ± SEM. Statistical significance was determined according to the Duncan-Kramer multiple domain assay (KramerCY in 1956).

Results

As shown in table 9, lumbar BMD in OVX control animals was 9% lower than non-ovariectomized controls 5 weeks after ovariectomy. At the doses used, SERM was administered alone: administration of EM-652, HCl, TSE-424, or ERA-923 prevented lumbar BMD loss by 86%, 53%, and 78%, respectively. On the other hand, administration of DHEA alone prevented lumbar BMD loss by 44%, while combined treatment with DHEA + EM-652 HCl, DHEA + TSE-424, or DHEA + ERA-923 prevented OVX-induced lumbar BMD reduction by 94%, 105%, and 105%, respectively.

Bone density at the distant metaphysis of femur (DFM) decreased by 10% after 5 weeks of ovariectomy (table 9). The administration of SERM EM-652.HCl, TSE-424, or ERA-923 alone prevented DFM BMD loss by 95%, 70%, and 83%, respectively. On the other hand, the effect of DHEA alone prevented DFM BMD loss by 71%, whereas concurrent treatment with DHEA + EM-652.HCl, DHEA + TSE-424, or DHEA + ERA-923 completely prevented OVX-induced reduction in DFM BMD and resulted in higher values of DFM BMD than observed in the non-ovariectomized control animals. Similar results were obtained at proximal tibial metaphyseal BMD (table 9).

Example 6

Effect of Compounds of the invention on alkaline phosphatase Activity in human endometrial carcinoma Stone Chuan (Ishikawa) cells

Material

Maintenance of seed cell culture

From the West Neissan medical center of New York, N.Y. (mountain Sina medical)Center) provided a human shikawa (Ishikawa) cell line derived from a well-differentiated endometrial cancer. Shichuan (Ishikawa) cells are routinely maintained in Eagle's (Eagle) Minimal Essential Medium (MEM) containing 5% (v/v) FBS (fetal bovine serum) supplemented with 100 units/ml penicillin, 100. mu.g/ml streptomycin, 0.1mM solution of non-essential amino acids. Cells were plated at 37 ℃ at 1.5X10⁶The density of individual cells was plated on a Firek (Falcon) T75 flask.

Cell culture experiments

24 hours before the start of the experiment, the medium of near-densely grown Shichuan (Ishikawa) cells was replaced with fresh estrogen-free basal medium (EFBM) consisting of a 1: 1 (vol: vol) mixture of phenol red-free Han's (Ham) F-12 medium and Dulbecco's (Dulbecco) modified Eagle's (Eagle) medium (DMEM) and supplemented with 100 units/ml penicillin, 100. mu.g/ml streptomycin, 2mM glutamic acidamide and 5% FBS treated twice with polydextrose-coated charcoal to remove endogenous steroids. Cells were then harvested by 0.1% pancreatin (Sigma) and 0.25 mhepes, resuspended in EFBM, plated in a volume of 100 μ l and a density of 2.2x104 cells/well in a felon (Falcon) 96-well flat bottom microtiter dish and allowed to adhere to the dish surface for 24 hours. Thereafter, the medium was replaced with fresh EFBM containing the indicated compound concentration in a final volume of 200. mu.l. Cells were cultured for 5 days with medium changes every 48 hours.

Alkaline phosphatase assay

At the end of the incubation period, the microtiter plates were inverted and the growth medium was poured. The dishes were rinsed in 200. mu.l PBS (0.15M sodium chloride, 10mM sodium phosphate, pH 7.4) per well. The PBS was then removed from the culture dish while carefully leaving some residual PBS, and the washing procedure was repeated once. The buffered saline solution was then poured and the inverted petri dish was blotted gently on a paper towel. After replacing the cover, the dish was left at-80 ℃ for 15 minutes, followed by thawing at room temperature for 10 minutes. The petri dish was then placed on ice and 50 microliters of an ice-cold solution containing 5mM p-nitrophenyl phosphate, 0.24mM magnesium chloride and 1M diethanolamine (pH 9.8) was added. The dish was then warmed to room temperature and the yellow color due to p-nitrophenyl production was allowed to develop (8 minutes). The plate was observed at 405nm in a plate reader for enzyme-linked immunosorbent assay (2550 EIA reader from Burry (BIO-RAD) Co.).

Computing

Dose-response curves and IC were calculated using a weighted iterative nonlinear quadratic regression₅₀Numerical values.

Example 7

Effect of EM-652.HCl, TSE424 and Lasofoxifene (Lasofoxifene) on the proliferation of human breast cancer MCF-7 cells

The method comprises the following steps:

maintenance of seed cell culture

MCF-7 human breast cancer cells from the American Type Culture Collection, passage 147 of # HTB 22 were obtained and routinely grown in phenol red free Dulbecco's modified Eagle (Eagle) -Han (Ham) F12, the supplement described above and 5% FBS medium. The MCF-7 human breast adenocarcinoma cell line is a pleural effusion derived from a white female patient of 69 years old. MCF-7 cells were used between passage 148 and 165, and subcultured weekly.

Study of cell proliferation

Cells in late logarithmic growth phase were harvested with 0.1% pancreatin (Sigma) and resuspended in appropriate medium containing 50 ng of bovine insulin/ml and 5% (v/v) FBS treated twice with polydextrose-coated charcoal to remove endogenous steroids. Cells were plated at the indicated densities on 24-well Verkang (Falcon) plastic petri dishes (2 cm/well) and allowed to adhere to the surface of the petri dishes for 72 hours. Thereafter, the medium was replaced with fresh medium containing the indicated concentration of compound diluted from a 1000-fold stock solution in 99% redistilled ethanol in the presence or absence of E2. Control cells received ethanol vehicle only (0.1% ethanol, v/v). Cells were cultured for a specified period and medium was changed every 2 or 3 days. The number of cells was determined by measuring the DNA content.

Calculation and statistical analysis

Dose-response curves and C were calculated using a weighted iterative non-linear least squares regression₅₀Numerical values. All results are expressed as mean ± SEM.

No. 11 watch

Experiment 1

Experiment 2

Example 8

Comparison of the Effect of EM-652.HCL, TAMOXIFEN (TAMOXIFEN), TOREMIFENE (TOREMIFENE), DROLOXIFENE (DROLOXIFENE), IDOXIFENE (IDOXIFENE), GW-5638 and RALOXIFENE (RALOXIFENE) on the growth of human RZ-75-1 mammary tumors in nude mice

The goal of this example was to compare the effector and antagonist effects of EM-652.HCl with six other oral antiestrogens (SERMs) on the growth of well characterized estrogen sensitive ZR-75-1 breast cancer xenografts in ovariectomized nude mice.

Materials and methods

Human ZR-75-1 breast cancer cells

ZR-75-1 human breast cancer cells were obtained from the American Type culture Collection (Rockville, Md.) and cultured in phenol red free RPMI-1640 medium. Cells were supplemented with 2mM L-glutaminic acid, 1mM sodium pyruvate, 100 International units penicillin/ml, 100. mu.g streptomycin/ml and 10% (v/v) fetal bovine serum, and cultured at 37 ℃ in a humidified gas atmosphere of 95% air/5% carbon dioxide. Cells were subcultured weekly and harvested at 85 to 90% compactness using 0.083% pancreatin/0.3 mM EDTA.

Animal and tumor vaccination

Homozygous female nu/nu Br athymic mice (28 to 42 days old) were obtained from Charles River, Inc (Saint-Constant), Inc. Mice (5 per cage) were housed in vinyl cages fitted with air filter covers, placed in laminar flow hood and maintained under pathogen-limiting conditions. The photoperiod was 12 hours light and 12 hours dark (light period started from 07: 15). The cages, liners and food (pro-Lab R-M-H feed #4018 from Agway) were autoclaved prior to use. The water is autoclaved and is available at will. Bilateral ovariectomy was performed under anesthesia induced by Isoflurane (Isoflurane). In ovariectomy, estradiol (E) is subcutaneously inserted₂) Implant to stimulate initial tumor growth. In a1 cm long silicone tube (0.062 inch inner diameter and 0.095 inch outer diameter), E was prepared containing a 1: 10 (weight/weight) mixture of estradiol and cholesterol at 0.5 cm₂An implant. After 1 week of ovariectomy, the Ovariectomized (OVX) mice were treated with a 2.5 cm-long 22-gauge injection needleFlank, were inoculated subcutaneously with 2X10 in 0.1 ml of RPMI-1640 medium + 30% Matrigel (Matrigel)⁶Individual ZR-75-1 (passage 93) cells. After 4 weeks, the E in all animals was replaced with the same size of an estrone-containing implant (weight: 1: 25 for E1: chol)₂An implant. Randomized cohort and treatment began after 1 week.

Treatment of

255 will carry an average area of 24, 4 + -0, 4 square millimeters (ranging from 5, 7 to 50, 7 square millimeters) the day before treatment is initiatedTumor mice were randomly assigned to 17 groups (in terms of tumor size), each group including 15 mice (29 or 30 tumors in total). Group 17 included 2 controls (OVX and OVX + estrone), 7 groups were supplemented with one estrone implant and treated with one antiestrogen, and the other 8 groups received only one antiestrogen. The estrone implants were then removed from animals in the ovariectomized control group (OVX) and the group that will receive anti-estrogen only. The estrone-containing implants in the other 9 groups were then replaced every 6 weeks. EM-652.HCl, Raloxifene (Raloxifene), Droloxifene (Droloxifene), Idoxifene (Idoxifene) and GW5638 were synthesized in the medical chemistry department of the research center for oncology and molecular endocrinology. Tamoxifen (Tamoxifen) is commercially available from plystein (plantax) corporation (Netanya), while Toremifene (Toremifene) citrate is commercially available from orlian (Orion) corporation (Espoo, finland). Under estrone stimulation, an oral dose of 50 μ g (2 mg/kg on average) of an antiestrogen suspended in 0.2 ml of 0.4% (w/v) methylcellulose was administered daily. Animals were treated once daily by the oral route with 200 μ g (8 mg/kg average) of each antiestrogen in the absence of estrone stimulation. Animals in both control groups received only 0.2 ml of vector. Suspensions of appropriate concentrations of antiestrogens were prepared each month, stored at 4 ℃ and used with constant stirring. The powdery material preparation is carried out in an airtight mannerStored at 4 deg.C (Idoxifene), Raloxifene (Raloxifene), Toremifene (Tormeifene), GW5638, Droloxifene (Droloxifene)) or at room temperature (Tamoxifen, EM-652. HCl).

Tumor measurement and autopsy

Two perpendicular diameters were recorded, and the formula used: the tumor area (mm square) was calculated at L/2xW/2x π. The area measured on day 1 of treatment was 100%.

After 161 days of treatment, the remaining animals were anesthetized with isaflurane (Isoflurane) and killed by exsanguination. To further characterize the effects of estrogen and antiestrogen, estrogen-responsive tissues such as uterus and vagina were immediately removed, connective and adipose tissues were removed and weighed. The uterus was prepared to evaluate endometrial thickness by Image analysis using Image Pro-Plus (Media Cybernetics, inc.) of Media nebrode, illinois. Briefly, the uterus was fixed in 10% formalin and embedded in paraffin. Sections of mouse uterus stained with hematoxylin and eosin were analyzed. 4 images per uterus (2 per uterine horn) were analyzed. The average epithelial cell height was measured for all animals in each group.

Reaction standard

Tumor responses were assessed at the end of the study, or at the death of each animal if dead during the experiment. In this case, only data from mice that survived at least half of the study period (84 days) were used in the tumor response analysis. In short, complete regression refers to those tumors that were undetectable at the end of the experiment; partial regression is that corresponding to tumors that regress by more than or equal to 50% of their original size; a stable response refers to tumors with a regression degree of < 50% or a worsening degree of < 50%; and worsening refers to tumors with a degree of deterioration of greater than or equal to 50% compared to their original size.

Statistical analysis

Changes in total tumor surface area between day 1 and day 161 were analyzed according to ANOVA for repeated measurements. The pattern includes treatment, time and time-treatment interaction effects plus terms that account for random stratification. The significance of the different therapeutic effects at 161 days was therefore examined by time-treatment interaction. Residual analysis shows that the original scale measurements are not suitable for analysis by ANOVA, nor for any of the transformations attempted. The grade for this analysis is selected accordingly. The effect of these treatments on epithelial thickness was evaluated by one-way ANOVA, which also included random stratification. A post-hoc pairwise comparison is made using the least squares means statistic. The overall-error rate (α) was controlled at 5% to indicate the significance of the difference. All calculations were performed on SAS software (sainstitute computer software, kary saishi, north carolina, usa) using Proc MIXED.

Results

Antagonistic Effect on ZR-75-1 tumor growth

Estrone itself (OVX + E1) caused a 707% increase in ZR-75-1 tumor size over a 23-week treatment period (FIG. 18). In the estrone-stimulated mice, the administration of the pure antiestrogen EM-652.HCl was administered daily at an oral dose of 50 μ g, completely preventing tumor growth. In fact, not only was tumor growth prevented, the tumor size was 26% smaller than the initial value at the beginning of the treatment (p < 0.04) after 23 weeks of treatment. This value obtained after EM-652.HCl treatment was not statistically different compared to the tumor size observed after Ovariectomy (OVX) alone, which decreased to 61% less than the initial tumor size. The other 6 antiestrogens did not reduce the initial mean tumor size at the same dose (50 μ g) and treatment period. Tumors in these groups were significantly higher than in OVX control and EM-652 HCl treated groups (p < 0, 01). Indeed, 23 weeks of treatment with Droloxifene (Droloxifene), Toremifene (Toremifene), GW5638, Raloxifene (Raloxifene), Tamoxifen (Tamoxifen) and Idoxifene (Idoxifene) resulted in mean tumor sizes that were 478%, 230%, 227%, 191%, 87% and 86%, respectively, higher than the pre-treatment values (fig. 18).

Effect on ZR-75-1 tumor growth

After 161 days of treatment, a dose of 200 μ g per day of Tamoxifen (Tamoxifen) in the absence of estrone supplementation resulted in an increase in mean tumor size of 196% from baseline (p < 0, 01 relative to OVX) (fig. 19). On the other hand, the mean tumor size of mice treated with Idoxifene (Idoxifene) increased (125%) (p < 0, 01), while the tumor size of mice treated with Toremifene (Toremifene) increased 86% (p < 0, 01) (fig. 19). The addition of 200. mu.g of EM-652.HCl to 200. mu.g of Tamoxifen (Tamoxifen) completely inhibited the proliferative effects observed when Tamoxifen (Tamoxifen) was administered alone (FIG. 20). On the other hand, treatment with EM-652 · HCl (p ═ 0, 44), Raloxifene (Raloxifene) (p ═ 0, 11), Droloxifene (Droloxifene) (p ═ 0, 36), or GW5638 (p ═ 0, 17) alone did not significantly change ZR-75-1 tumor size compared to OVX controls at the end of the experiment (fig. 19).

Effect on class response

The effect of 50 μ g of antiestrogen on estrone stimulation. In addition to the effect on tumor size, the class of response achieved by each individual tumor at the end of the experiment is an important parameter for the efficacy of the treatment. In ovariectomized mice, complete, partial and stable responses were achieved in 21%, 43% and 38% of tumors, respectively, and there was no tumor progression. On the other hand, 100% of tumors worsened in OVX animals supplemented with estrone (figure 21). In the EM-652.HCl treatment group of OVX animals supplemented with estrone, complete, partial and stable responses were observed in 17%, 17% and 60% of tumors, respectively, and only 7% (2 out of 30 tumors) worsened. Treatment with any of the other antiestrogens at a daily dose of 50 μ g failed to reduce the percentage of tumors in the exacerbations to below 60% under the same conditions of estrone stimulation. In fact, 65% of tumors (17 out of 26) were worsened in the Tamoxifen (Tamoxifen) treated group; while 89% (25 out of 28) worsened with Toremifene (Tormeifene), 81% (21 out of 26) worsened with Raloxifene (Raloxifene), 100% (23 out of 23) worsened with Droloxifene (Droloxifene), 71% (20 out of 28) worsened with Idoxifene (Idoxifene) and 77% (20 out of 26) worsened with GW5638 (fig. 21).

Effect of 200 microgram of antiestrogens on the Category response in the absence of estrone stimulation

As illustrated in FIG. 22, the proportion of tumors in the exacerbations by Tamoxifen (Tamoxifen), Idoxifene (Idoxifene) and Toremifene (Tormemifene) in the absence of estrone stimulation is higher than that of other antiestrogens. In fact, tumors in the malignant category were 62% (16 out of 26), 33% (8 out of 24) and 21% (6 out of 28), respectively, after treatment with Tamoxifen (Tamoxifen), Idoxifene (Idoxifene) and Toremifene (Toremifene) at a dose of 200 micrograms per day. As can be seen in figure 23, with 200 micrograms of EM-652.HCl added to Tamoxifen (Tamoxifen), the percentage of tumors in exacerbations was from 62% (16 out of 26) when Tamoxifen (Tamoxifen) was administered alone, to 7% (2 out of 28) when EM-652.HCl was added to Tamoxifen (Tamoxifen).

Effect of antiestrogens on uterine epithelial cell thickness

The height of endometrial epithelial cells was measured as the most direct parameter for the effector and antagonist effects of each compound in the endometrium.

Effect of 50 microgram daily of antiestrogen on uterine epithelial cell thickness in the Presence of estrone stimulation

At an oral dose of 50 μ g per day, EM-652.HCl inhibited the stimulatory effect of estrone on epithelial height by up to 70%. The efficacy of the other 6 antiestrogens tested was significantly lower (p < 0, 01). Indeed, Droloxifene (Droloxifene), GW5638, Raloxifene (Raloxifene), Tamoxifen (Tamoxifen), Toremifene (Toremifene) and Idoxifene (Idoxifene) inhibited estrone stimulation by 17%, 24%, 26%, 32%, 41% and 50%, respectively (table 12).

Effect of 200 microgram daily of antiestrogen on uterine epithelial cell thickness in absence of estrone stimulation

In the absence of estrone stimulation, only EM-652.HCl and Droloxifene (Droloxifene) did not significantly increase the height of epithelial cells in the tested compounds (114% and 101% of the value of the OVX control group, respectively). Tamoxifen (155%), Toremifene (Toremifene) (135%) and Idoxifene (Idoxifene) (176%) exhibited significant stimulatory effects on uterine epithelium height (p < 0, 01 relative to OVX control). Raloxifene (Raloxifene) (122%) and GW5638 (121%) also exhibited statistically significant stimulatory effects on uterine epithelium height (p < 0,05 (table 12) relative to OVX control group-the agonistic and antagonistic effects of antiestrogens measured on uterine and vaginal weight were consistent with the pattern observed on uterine epithelial cell thickness (data not shown).

No. 12 watch

Example 9

Female rats are in¹⁴Radioactivity in brain following a single oral dose (20 mg/kg) of C-EM-800

Example 8 shows rats in¹⁴C-EM-800 is the radioactivity in the brain following a single oral dose (20 mg/kg) of a SERM of the invention. For comparison purposes, values from blood, plasma, liver (table 13) and uterus of each of the animals were included. Single administration to male and female Lang-Evans rats¹⁴Tissue distribution and excretion of radioactivity following oral dose of C-EM-800 (20 mg/2 ml/kg). These values show the total amount of drug-derived radioactivity in the brain of female Lang-Evans ratsIs often low (in nanogram equivalents/gram of tissue) and is undetectable after 12 hours of administration. At 2 hours, the radioactivity in the brain was 412 times lower than that in the liver, 21 times lower than that in the uterus, 8.4 times lower than that in the blood, and 13 times lower than that in the plasma. Since an unknown proportion of total brain radioactivity is due to contamination by blood radioactivity, the values for brain radioactivity shown in the X1 table are for the brain tissue itself¹⁴Overestimation of C (EM-800) associated radioactivity levels. This data shows that the anti-estrogen levels in brain tissue are too low to counteract the effects of exogenous estrogen. It is important to observe a portion of the detected radioactivity in brain tissue, possibly due to residual blood in the tissue (table 14). In addition, for this study¹⁴The radiochemical purity of C-EM-800 is a minimum of 96.25%.

No. 13 watch

Example 10

Clinical trial ERC-205

DE PHASE II-IIIAVECPLACEBOPOURLES EFFETS DE LA DHEA SUR LESVASOMOTEURS(DECHALEUR) -phase II-III placebo-controlled study to assess the effects of DHEA on vasomotor symptoms (hot flashes) in postmenopausal women

Summary of research design

As shown in figure 24, the study was a randomized placebo-controlled study to evaluate the effect of DHEA on reducing vasomotor symptoms (hot flashes) compared to placebo administration. Postmenopausal women who experienced ≧ 50 moderate or severe hot flashes per week (as measured by two-week diary) were randomly assigned to receive a daily dose of placebo or 50 mg of DHEA. 50 evaluable participants (25 patients per group) were treated for 4 months and daily hot flash assessments were recorded by each participant in a diary.

Post-menopausal women aged 40 to 70 who had moderate or severe hot flashes ≧ 50 per week, confirmed by two-week hot flash screening logs, were included in the study after signing post-notice consent. The procedure was approved by the human testing committee (IRB) at the university hospital center of Laval (Laval) and the health department of canada.

The woman must meet the conditions of a or b or c:

a. menses has stopped for at least one year; or

b. FSH levels of ≥ 40 milli-international units (within 60 days prior to day 1) for women who have amenorrhea for more than 6 months but less than 12 months, or women who have had their uterus resected prior to menopause at the point of hysterectomy; or

c. Previous bilateral ovariectomy.

Within 12 months of random assignment, normal cervical smears (which include inflammatory changes) and normal bilateral mammograms must be acquired.

The thickness of the endometrium detected by vaginal ultrasonography is required to be less than 4 mm.

After 4 months of treatment, the primary indicator is the change from baseline in the frequency of moderate to severe hot flashes per week at week 16. Goals also include changes from baseline in the frequency of all hot flashes per week and weekly weighted severity scores.

Secondary indicators are safety assessments of DHEA and quality of life.

The response index is a daily written paper log specifying the number and type of hot flashes:

0 is none.

1 slightly hot but no sweating was felt.

Moderate 2-heat and sweating were felt without stopping activity.

3 Severe, feeling hot and sweating requires discontinuation of activity. Which includes night sweats.

The hot flashes journal begins as a screening journal two weeks before random assignment, whereby the patient must daily fill the journal to record the number and severity of hot flashes. Patients who recorded moderate or severe hot flashes an average of more than 50 times per week over a two week period could be eligible for enrollment (i.e., at least 100 hot flashes were recorded on a two week screening log).

Once randomly assigned, the patient completed eight two week hot flash logs at the start of study dosing. The log must be filled out daily. The first journal is completed in the first two weeks and is handed back in two weeks of return visits. The second journal was completed during the second two weeks of the first four week treatment period and was handed back at the four week return visit. Two-week logs of hot flashes were collected at 4, 8, 12, and 16 weeks of return visits.

The diary and blinded medication began on the same day (i.e., day 1. on the same day that the patient was scheduled to begin taking study medication, she began recording hot flashes as soon as she woke up).

As illustrated in fig. 25 and table 15, the number of moderate to severe hot flashes decreased from 70.7 ± 4.5 per week at screening to 50.1 ± 5.7 at week 4 (not significant relative to placebo), 40.2 ± 6.1 at week 8, 34.7 ± 5.8 at week 12 (p < 0.05 relative to placebo) and 32.2 ± 5.8 at week 16 (p < 0.05 relative to placebo). Placebo resulted in a 32.9% reduction compared to 54.5% for DHEA.

A similar effect was observed in the frequency of all hot flashes (fig. 26, table 16), from a reduction in the value of 75.5 ± 4.4 hot flashes per week before screening to 55.3 ± 5.8 at week 4 (not significant relative to placebo), 44.7 ± 6.3 at week 8, 39.5 ± 5.9 at week 12 (p < 0.05 relative to placebo) and 36.0 ± 5.7 at week 16 (p < 0.05 relative to placebo). Placebo resulted in a 34.9% reduction compared to 52.4% for DHEA.

When mild as 1, moderate as 2 and severe as 3 gave a hot flash score, a decrease from 187.1 + -13.9 to 87.2 + -15.8 was observed in the DHEA group at week 16 compared to a decrease from 196.3 + -13.6 to 130 + -14.1 (p < 0.05) in the placebo group. Placebo resulted in an 18.0% reduction compared to 53.4% for DHEA, thus showing a 3.0-fold higher potency for DHEA.

As illustrated in table 18, the effect of DHEA is exhibited to a greater extent on moderate to severe hot flashes, preferably illustrated as a weighted severity score, where DHEA reduced the value by 99.1 ± 15.6 and placebo by 68.6 ± 15.6. This effect is better illustrated in Table 19, where the mean number of hot flashes per week at screening was reduced from 94.7 + -7.9 to 57.8 + -8.3 in the placebo group and from 88.5 + -7.1 to 31.6 + -11.6 in the group of women receiving DHEA, in women over 71. This data shows that DHEA is inhibitory up to 65% in women most affected by vasomotor symptoms (64.3% DHEA versus 39.0% placebo). In fact, for women with moderate to severe hot flashes per week between 50 and 70, the number of women receiving DHEA decreased from 56.7 ± 1.3 per week before screening to 32.7 ± 6.2 (43.3% decrease) at week 6, compared to 24.7% decrease of placebo, thus showing a 43% inhibition of DHEA over placebo effect.

Similar conclusions can be observed in table 20, due to the lower number of mild hot flashes (compare tables 18 and 19). The number of hot flashes in women with mild, moderate plus severe hot flashes, which exceeded 70, decreased from 91.0 + -7.0 weekly to 36.3 + -11.5 weekly (60% decrease) before screening in women receiving DHEA. In women receiving placebo, the number of all hot flashes decreased from 100.4 ± 7.9 at screening to 61.3 ± 8.9 at week 16, a 39.9% decrease. The data show that DHEA is 50% more potent in women with the highest total number of hot flashes of various severity.

Similar conclusions can be drawn when a weighted severity score is used computationally (table 21). When considering women with hot flashes more than 70 times per week, the score in women receiving DHEA decreased from 241.6 ± 21.1 at screening to 95.2 ± 34.3 at week 16 (a 61.6% decrease), while the value decreased from 242.0 ± 21.6 for placebo to 141.3 ± 20.1 at week 16 (a 41.6% decrease). For women between 50 and 70 hot flashes per week at screening, this value was a decrease of 81.6 + -13.6 (43.5%) from 144.3 + -6.7 at screening to week 16 compared to 154.2 + -3.7 and 118.8 + -20.2 (33.0%) for placebo.

Conclusion

Current data, as assessed by a significant reduction in the total number of moderate to severe hot flashes or all hot flashes and by a weekly hot flash severity-weighted score, demonstrate the efficacy of 50 mg DHEA treatment for relief of vasomotor symptoms.

Example 11

Clinical trial ERC-213

DHEA bioavailability following vaginal suppository administration in postmenopausal women with vaginal atrophy

Summary of research design

The main goal of this study was to measure the maturation value of vaginal epithelial cells after daily intravaginal administration of DHEA. 40 post-menopausal women were randomly assigned daily doses of an ovoid suppository having the following DHEA concentration: 0.0%, 0.5% (6.5 mg DHEA/pessary), 1.0% (13 mg DHEA/pessary), or 1.8% (23.4 mg DHEA/pessary) for 7 days. The systemic bioavailability of DHEA and its metabolites was also measured.

Results

After only one week of daily administration of the DHEA suppository, the maturation index increased 107% (p < 0.01), 75% (p < 0.05) and 150% (p < 0.01) in the groups of 0.5%, 1.0% and 1.8% DHEA, respectively (FIG. 27). No change was observed in the placebo group between day 1 and day 7. On the other hand, vaginal pH decreased from 6.29. + -. 0.21 to 5.75. + -. 0.27(p < 0.05), from 6.47. + -. 0.23 to 5.76. + -. 0.22(p < 0.01) and from 6.53. + -. 0.25 to 5.86. + -. 0.28(p < 0.05) in the 0.5%, 1.0% and 1.8% DHEA groups, respectively (FIG. 28). No change in vaginal pH was observed in the placebo group.

Conclusion

Current data show that intravaginal administration of DHEA allows the beneficial effects against vaginal atrophy to be rapidly achieved without significant changes in serum estrogen, thus avoiding the increased risk of breast cancer associated with current intravaginal or systemic estrogen formulations, and adding to all layers in the vagina the local benefits of the newly identified androgenic moiety in the DHEA effects of this tissue.

Examples of pharmaceutical compositions

Several pharmaceutical compositions using the preferred active SERM acobipene (Acolbifene) (EM-652. HCl; EM-1538) and the preferred active steroid precursor dehydroepiandrosterone (DHEA, dehydroisoandrosterone) are illustrated by way of example and without limitation as follows. Other compounds of the invention or combinations thereof may be used in place of (or in addition to) acobifene (Acolbifene) or dehydroepiandrosterone. The concentration of the active ingredient may vary within the broad ranges set forth herein. The amounts and types of other ingredients that may be included are well known in the art.

Example A

Pharmaceutical composition for oral administration (capsule)

Example B

Pharmaceutical compositions (lozenges) for oral administration

Example C

Local administration effect (cream)

Example D

Vaginal suppository or ovoid suppository for vaginal administration

DHEA suppositories are prepared using Witepsol H-15 base (Medisca, monteluro, canada) as the base. Any other lipophilic base such as stearin, fischer-tropsch (Fattibase), wecobie (Wecobee), cocoa butter or a Witepsol (Witepsol) base may be used. Preferred SERMs are EM-800 and Acolbifene (Acolbifene).

Example of the kit

Several kits using the preferred active SREM acobipene (Acolbifene), the preferred antiestrogenic farnesoid (Faslodex) and the preferred active steroid precursor DHEA are described below by way of example and without limitation. The concentration of the active ingredient may vary within the broad ranges set forth herein. The amounts and types of other ingredients that may be included are well known in the art.

Example D

Reagent kit

The SERM and sex steroid precursor are non-steroidal antiestrogen compositions (capsules) for oral administration

+

DHEA compositions (gelatin capsules) for oral administration

In the above formulations, Acolbifene (Acolbifene) can be substituted with other SERMs, and DHEA can be substituted with other sex steroid precursors. More than one SERM or more than one sex steroid precursor may be included, in which case the combined weight percentages are preferably the weight percentages of a single sex steroid precursor or a single SERM as shown in the above examples.

Example E

Reagent kit

The SERM is orally administered and the sex steroid precursor is a SERM composition (capsule) for intravaginal administration for oral administration

+

Vaginal suppository

DHEA suppositories are prepared using Witepsol H-15 base (Medisca, monteluro, canada) as the base. Any other lipophilic base such as stearin, fischer-tropsch (Fattibase), wecobie (Wecobee), cocoa butter or a Witepsol (Witepsol) base may be used.

Example F

Reagent kit

The SERM and sex steroid precursor are intravaginally administered pessaries

+

Vaginal suppository

Acobifene (Acolbifene) suppositories are prepared using stearin (Witepsol). Other compositions of any other base such as Fiberbs (Fattibase), Wicobee (Wecobee), cocoa butter or stearin may be used.

Example G

The SERM is orally administered and the sex steroid precursor is transdermally administered for oral administration

+

Sex steroid precursor composition for oral administration (gels)

Or

Sex steroid precursor composition for oral administration (cream)

Example H

Reagent kit

The antiestrogen is administered intramuscularly and the sex steroid precursor is administered orally as a commercially available steroid antiestrogen falodex (Faslodex)

+

DHEA compositions (gelatin capsules) for oral administration

In the above formulations, acobipene (Acolbifene) may be replaced by other SERMs (Torremifene (Tormeifene), Ospemifene (Ospemifene), Raloxifene (Raloxifene), Arzoxifene (Arzoxifene), Lasofoxifene (Lasofoxifene), TSE-424, ERA-923, EM-800, SERM3339, GW-5638), and DHEA may be replaced by other sex steroid inhibitors. More than one SERM or more than one precursor may be included, in which case the combined weight percentages are preferably the weight percentages of a single precursor or a single SERM as shown in the above examples.

The present invention has been described in terms of preferred embodiments and examples, but is not limited thereto. Those skilled in the art will recognize the broader applicability and scope of the invention, which is limited only by the claims that follow.

Claims (89)

1. Use of (i) a sex steroid precursor or a prodrug thereof and (ii) a selective estrogen receptor modulator or an antiestrogen or a prodrug of either, in the manufacture of a medicament for reducing or eliminating the occurrence of at least one symptom selected from the group consisting of hot flashes, vasomotor symptoms and night sweats.
2. 2. The use of claim 1, wherein the sex steroid precursor is selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-ene-3 β,17 β -diol, 4-androstene-3, 17-dione, and prodrugs of any of the foregoing precursors.
3. 3. A pharmaceutical composition for reducing or eliminating a symptom selected from the group consisting of hot flashes, vasomotor symptoms, and night sweats, comprising: a) a pharmaceutically acceptable excipient, diluent or carrier; b) at least one sex steroid precursor or prodrug thereof; and c) at least one selective estrogen receptor modulator or one antiestrogen or a prodrug of either; wherein the pharmaceutical composition is provided in a package that directs use of the composition to reduce or eliminate at least one symptom selected from the group consisting of hot flashes, vasomotor symptoms, and night sweats.
4. 4. The pharmaceutical composition of claim 3, wherein the sex steroid precursor and the selective estrogen receptor modulator or the antiestrogen are both formulated together in a pharmaceutically delivered form selected from the group consisting of a pill, a lozenge, a cream, a gel, an intravaginal suppository, and an intravaginal suppository.
5. 5. A kit for reducing or eliminating a symptom selected from the group consisting of hot flashes, vasomotor symptoms, and night sweats comprising (i) a first container having therein at least one sex steroid precursor or prodrug thereof; (ii) a second container having therein at least one selective estrogen receptor modulator or an antiestrogen or a prodrug of any of the foregoing; and (iii) instructions for using the kit to reduce or eliminate at least one symptom selected from the group consisting of hot flashes, vasomotor symptoms, and night sweats.
6. 6. The use as claimed in claim 1, wherein the selective estrogen receptor modulator has a formula with the following characteristics:

   a) two aromatic rings separated by 1 to 2 intervening carbon atoms, the aromatic rings being unsubstituted or substituted with a hydroxyl group or a group that is converted to a hydroxyl group in vivo;

   b) a side chain having an aromatic ring and a tertiary amine functionality or salt thereof.
7. 7. The use of claim 6, wherein the side chain is selected from the group consisting of:
8. 8. the use of claim 1, wherein the selective estrogen receptor modulator is selected from the group consisting of triphenylethylene derivatives, indole derivatives, benzopyran derivatives, tamoxifen, toremifene, CC 8490, SERM 3471, HMR3339, HMR3656, raloxifene, LY335124, LY 326315, arzoxifene (LY 353381), penoxifene (ERA923), bazedoxifene (TSE424, WAY140424), oropoline (lasofoxifene), and Senocolomen derivatives.
9. 9. The use as claimed in claim 1, wherein the selective estrogen receptor modulator is a benzothiophene derivative compound having the formula:

   a) wherein R is₁And R₂Is independently selected from the group consisting of hydrogen, hydroxyl, and a moiety that is converted to hydroxyl in vivo;

   b) wherein R is₃And R₄Is (a) independently C₁-C₄Alkyl, or (b) a moiety in combination with the nitrogen to which they are attached, selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, and morpholinyl;

   c) wherein A is selected from the group consisting of-CO-, -CHOH and-CH₂-the group consisting of;

   d) wherein B is selected from the group consisting ofPhenylene, dihydropyridinylidene and-ring C₄H₂N₂-the group consisting of.
10. 10. The use of claim 1, wherein the selective estrogen receptor modulator is selected from the group consisting of raloxifene, azoxifene (LY 353381), LY353381, and LY 335563.
11. 11. The use as claimed in claim 1, wherein the selective estrogen receptor modulator is a triphenylethylene or diphenylhydronaphthalene derivative compound having the formula:

    a) wherein D is-OCH₂CH₂N(R₃)R₄、-OCH₂CH₂OH or-CH-COOH (R)₃And R₄Is independently selected from the group consisting of C₁-C₄Alkyl, or R₃、R₄Together with the nitrogen atom to which they are attached is a ring structure selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, and morpholinyl);

    b) wherein E and K are independently hydrogen or hydroxy, phosphate or lower alkyl, and wherein J is hydrogen or halogen.
12. 12. The use according to claim 1, wherein the selective estrogen receptor modulator is tamoxifen, hydroxy-tamoxifen, droloxifene, toremifene, idoxifene, lasofoxifene, breloxifene, FC1271, and GW 5638.
13. 13. The use as claimed in claim 1, wherein the selective estrogen receptor modulator is an indole derivative compound having the formula:

    a) wherein D is selected from the group consisting of-OCH₂CH₂N(R₇)R₈、-CH＝CH-CO N(R₇)R₈、-CC-(CH₂)n-N(R₇)R₈Group (R) of₇And R₈Is independently selected from the group consisting of C₁-C₆Alkyl, or R₇、R₈Together with the nitrogen atom to which they are attached is a ring structure selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholinyl;

    b) wherein X is selected from the group consisting of hydrogen and C₁-C₆Alkyl groups;

    c) wherein R is₁、R₂R₃、R₄、R₅And R₆Is independently selected from hydrogen, hydroxy, C₁-C₆Alkyl groups and a moiety that is converted to a hydroxyl group in vivo.
14. 14. The use as claimed in claim 1, wherein the selective estrogen receptor modulator is selected from the group consisting of bazedoxifene (TSE 424; WAY-TSE 424; WAY 140424; 1- [ [4- [2- (hexahydro-1H-aza-1H) -azan-1-yl) ethoxy]Phenyl radical]Methyl radical]-2- (4-hydroxyphenyl) -3-methyl-1H-indol-5-ol) with penciclovir (ERA 923; 2- (4-hydroxyphenyl) -3-methyl-1- [ [4- [2- (1-piperidinyl) ethoxy]Phenyl radical]Methyl radical]-1H-indol-5-ol).
15. 15. The use as claimed in claim 1, wherein the selective estrogen receptor modulator is a senkelomon derivative compound having the formula:

    a) wherein R is₁And R₂Is independently selected from the group consisting of hydrogen, hydroxyl, and a moiety that is converted to hydroxyl in vivo;

    b) wherein R is₅And R₆Is independently hydrogen or C₁-C₆An alkyl group;

    c) wherein D is-OCH₂CH₂N(R₃)R₄(R₃And R₄Is independently selected from the group consisting of C₁-C₄Alkyl, or R₃、R₄Together with the nitrogen atom to which they are attached is a ring structure selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholinyl).
16. 16. The use as claimed in claim 1, wherein the senkoloman derivative is (3, 4-trans-2, 2-dimethyl-3-phenyl-4- [4- (2- (2- (pyrrolidin-1-yl) ethoxy) phenyl ] -7-methoxychroman.
17. 17. The use according to claim 1, wherein the selective estrogen receptor modulator has the formula:

    a) wherein R is₁And R₂Is independently hydrogen, hydroxy, or a moiety that is converted to hydroxy in vivo;

    b) wherein Z is absent or selected from the group consisting of-CH₂-, -O-, -S-and-NR₃-(R₃Hydrogen or lower alkyl);

    c) wherein R is₁₀₀Is a divalent moiety that separates L from the B ring by 4 to 10 intervening atoms;

    d) wherein L is a divalent or trivalent moiety selected from the group consisting of-SO-, -CON <, -N < and-SON <;

    e) wherein G is₁Is selected from hydrogen, C₁To C₅Hydrocarbons with G₂A divalent moiety in combination, and L is a 5-to 7-membered heterocycle and a halo or unsaturated derivative of the foregoing;

    f) wherein G is₂Absent or selected from hydrogen, C₁To C₅Hydrocarbons with G₁(ii) the divalent moieties in combination, and L is a 5-to 7-membered heterocyclic ring and halogenated or unsaturated derivatives of the foregoing;

    g) wherein G is₃Is selected from the group consisting of hydrogen, methyl and ethyl.
18. 18. The use according to claim 1, wherein the selective estrogen receptor modulator is a benzopyran compound having the following general structure:

    or a pharmaceutically acceptable salt thereof,

    a) wherein D is-OCH₂CH₂N(R₃)R₄(R₃And R₄Is independently selected from the group consisting of C₁-C₄Alkyl, or R₃、R₄Together with the nitrogen atom to which they are bonded, is a ring structure selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholinyl);

    b) wherein R is₁And R₂Is independently selected from the group consisting of hydrogen, hydroxyl, and a moiety that is converted to hydroxyl in vivo; and

    c) wherein G is₃Is selected from the group consisting of hydrogen, methyl and ethyl.
19. 19. The use as claimed in claim 1, wherein the selective estrogen receptor modulator is a benzopyran compound having optical activity due to the fact that most of its stereoisomers have the absolute configuration S at carbon 2, having the following molecular structure:

    or a pharmaceutically acceptable salt thereof,

    a) wherein R is₁And R₂Is independently selected from the group consisting of a hydroxyl group and a moiety convertible in vivo to a hydroxyl group;

    b) wherein R is₃Is selected from the group consisting of saturated, unsaturated or substituted pyrrolidinyl; saturated, unsaturated or substituted piperidino; saturated, unsaturated or substituted piperidinyl; saturated, unsaturated or substituted morpholinyl; a nitrogen-containing cyclic moiety; a nitrogen-containing polycyclic moiety; and NRaRb (Ra and Rb are independently hydrogen, straight or branched C₁-C₆Alkyl, straight or branched C₂-C₆Alkenyl and straight-chain or branched C₂-C₆Alkynyl) groups.
20. 20. The use of claim 19, wherein the compound or salt substantially lacks the (2R) -enantiomer.
21. 21. The use as claimed in claim 1, wherein the selective estrogen receptor modulator is:

    and is optically active because most of its stereoisomers have 2S configuration.
22. 22. The use of claim 19, wherein the selective estrogen receptor modulator is a benzopyran salt of an acid selected from the group consisting of acetic acid, adipic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, fumaric acid, hydroiodic acid, hydrobromic acid, hydrochloric acid, hydrochlorobenzothia-c acid, hydroxy-naphthoic acid, lactic acid, maleic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, nitric acid, palmitic acid, trimethylacetic acid, phosphoric acid, propionic acid, succinic acid, sulfuric acid, tartaric acid, terephthalic acid, p-toluenesulfonic acid, and valeric acid.
23. 23. The use as claimed in claim 1, wherein the selective estrogen receptor modulator is:

    and is optically active due to its majority of enantiomers having the 2S configuration; and wherein the sex steroid precursor is dehydroepiandrosterone.
24. 24. The use as claimed in claim 1, wherein the selective estrogen receptor modulator has no estrogenic activity in breast, uterine or endometrial tissues.
25. 25. The use as claimed in claim 1, wherein the medicament also reduces the risk of the patient suffering from breast, uterine or endometrial cancer.
26. 26. The use as claimed in claim 1, wherein the medicament also inhibits the onset of osteoporosis, hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, insulin resistance, type 2 diabetes, muscle loss, obesity, alzheimer's disease, cognitive loss, memory loss or vaginal dryness.
27. 27. The use as claimed in claim 1, wherein the antiestrogen is farad (ICI 182780, fulvestrant, 7 α - [9- (4, 4,5, 5, 5-pentafluoro-pentylsulfinyl) nonyl ] estra-1, 3, 5(10) -triene-3, 17 β -diol) or SH 646.
28. 28. The use as claimed in claim 1, wherein the selective estrogen receptor modulator is for intravaginal administration.
29. 29. The use as claimed in claim 21, wherein the selective estrogen receptor modulator is for intravaginal administration.
30. 30. The use as claimed in claim 1, wherein the selective estrogen receptor modulator is for oral administration.
31. 31. The use as claimed in claim 1, wherein the selective estrogen receptor modulator is for transdermal administration.
32. 32. The use as claimed in claim 1, wherein the selective estrogen receptor modulator is selected from the group consisting of: tamoxifen, toremifene, CC 8490, SERM 3471, HMR3339, HMR3656, raloxifene, LY335124, LY 326315, azoxifene (LY 353381), pentoxifene (ERA923), bazedoxifene (TSE424, WAY140424), Europeria (lasofoxifene), EM-652, EM-800, EM-652-HCl (acobiprofen, EM-1538), 4-hydroxy-tamoxifen, 4-hydroxy-toremifene, droloxifene, LY 335563, GW-5638, idoxifene, levomeloxifene, Aprofen (TAT-59), oximiphene (FC 1271), fispemifene, and clokoloman.
33. 33. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator has a molecular formula with the following characteristics:

    a) two aromatic rings separated by 1 to 2 intervening carbon atoms, which are unsubstituted or substituted with a hydroxyl group or a group that is converted to a hydroxyl group in vivo;

    b) a side chain having an aromatic ring and a tertiary amine functionality or salt thereof.
34. 34. The pharmaceutical composition of claim 33, wherein the side chain is selected from the group consisting of:
35. 35. the pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is selected from the group consisting of triphenylethylene derivatives, indole derivatives, benzopyran derivatives, tamoxifen, toremifene, CC 8490, SERM 3471, HMR3339, HMR3656, raloxifene, LY335124, LY 326315, azoxifene (LY 353381), penoxifene (ERA923), bazedoxifene (TSE424, WAY140424), oropoli (lasofoxifene), and Senocolomen derivatives.
36. 36. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is a benzothiophene derivative compound having the formula:

    a) wherein R is₁And R₂Is independently selected from the group consisting of hydrogen, hydroxyl, and a moiety that is converted to hydroxyl in vivo;

    b) wherein R is₃And R₄Is (a) independently C₁-C₄Alkyl, or (b) a moiety in combination with the nitrogen to which they are attached, selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, and morpholinyl;

    c) wherein A is selected from the group consisting of-CO-, -CHOH and-CH₂-the group consisting of;

    d) wherein B is selected from the group consisting of phenylene, dihydropyridinylidene, and-Ring C₄H₂N₂-the group consisting of.
37. 37. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is selected from the group consisting of raloxifene, azoxifene (LY 353381), LY353381, and LY 335563.
38. 38. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is a triphenylethylene or diphenylhydronaphthalene derivative compound having the formula:

    a) wherein D is-OCH₂CH₂N(R₃)R₄、-OCH₂CH₂OH or-CH-COOH (R)₃And R₄Is independently selected from the group consisting of C₁-C₄Alkyl, or R₃、R₄Together with the nitrogen atom to which they are attached is a ring structure selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, and morpholinyl);

    b) wherein E and K are independently hydrogen or hydroxy, phosphate or lower alkyl, and wherein J is hydrogen or halogen.
39. 39. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is tamoxifen, hydroxy-tamoxifen, droloxifene, toremifene, idoxifene, lasofoxifene, breloxifene, FC1271, and GW 5638.
40. 40. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is an indole derivative compound having the formula:

    a) wherein D is selected from the group consisting of-OCH₂CH₂N(R₇)R₈、-CH＝CH-CO N(R₇)R₈、-CC-(CH₂)n-N(R₇)R₈Group (A) ofGroup (R)₇And R₈Is independently selected from the group consisting of C₁-C₆Alkyl, or R₇、R₈Together with the nitrogen atom to which they are bonded, is a ring structure selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholinyl;

    b) wherein X is selected from the group consisting of hydrogen and C₁-C₆Alkyl groups;

    c) wherein R is₁、R₂R₃、R₄、R₅And R₆Is independently selected from hydrogen, hydroxy, C₁-C₆Alkyl groups and a moiety that is converted to a hydroxyl group in vivo.
41. 41. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is selected from the group consisting of bazedoxifene (TSE 424; WAY-TSE 424; WAY 140424; 1- [ [4- [2- (hexahydro-1H-aza-1H) -azaphen [ ]-1-yl) ethoxy]Phenyl radical]Methyl radical]-2- (4-hydroxyphenyl) -3-methyl-1H-indol-5-ol) with penciclovir (ERA 923; 2- (4-hydroxyphenyl) -3-methyl-1- [ [4- [2- (1-piperidinyl) ethoxy]Phenyl radical]Methyl radical]-1H-indol-5-ol).
42. 42. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is a Senolomen derivative compound having the formula:

    a) wherein R is₁And R₂Is independently selected from the group consisting of hydrogen, hydroxyl, and a moiety that is converted to hydroxyl in vivo;

    b) wherein R is₅And R₆Is independently hydrogen or C₁-C₆Alkyl radical；

    c) Wherein D is-OCH₂CH₂N(R₃)R₄(R₃And R₄Is independently selected from the group consisting of C₁-C₄Alkyl, or R₃、R₄Together with the nitrogen atom to which they are attached is a ring structure selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholinyl).
43. 43. The pharmaceutical composition of claim 3, wherein the senkoloman derivative is (3, 4-trans-2, 2-dimethyl-3-phenyl-4- [4- (2- (2- (pyrrolidin-1-yl) ethoxy) phenyl ] -7-methoxychroman).
44. 44. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator has the formula:

    a) wherein R is₁And R₂Is independently hydrogen, hydroxy, or a moiety that is converted to hydroxy in vivo;

    b) wherein Z is absent or selected from the group consisting of-CH₂-, -O-, -S-and-NR₃-(R₃Hydrogen or lower alkyl);

    c) wherein R is₁₀₀Is a divalent moiety that separates L from the B ring by 4 to 10 intervening atoms;

    d) wherein L is a divalent or trivalent moiety selected from the group consisting of-SO-, -CON <, -N < and-SON <;

    e) wherein G is₁Is selected from hydrogen, C₁To C₅Hydrocarbons with G₂A divalent moiety in combination, and L is a 5-to 7-membered heterocycle and a halo or unsaturated derivative of the foregoing;

    f) wherein G is₂Absent or selected from hydrogen, C₁To C₅Hydrocarbons with G₁Divalent moieties in combinationAnd L is a 5-to 7-membered heterocycle and the aforementioned halo or unsaturated derivatives;

    g) wherein G is₃Is selected from the group consisting of hydrogen, methyl and ethyl.
45. 45. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is a benzopyran compound having the following general structure:

    or a pharmaceutically acceptable salt thereof,

    a) wherein D is-OCH₂CH₂N(R₃)R₄(R₃And R₄Is independently selected from the group consisting of C₁-C₄Alkyl, or R₃、R₄Together with the nitrogen atom to which they are bonded, is a ring structure selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholinyl);

    b) wherein R is₁And R₂Is independently selected from the group consisting of hydrogen, hydroxyl, and a moiety that is converted to hydroxyl in vivo; and

    c) wherein G is₃Is selected from the group consisting of hydrogen, methyl and ethyl.
46. 46. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is a benzopyran compound having optical activity due to a majority of its stereoisomers having the absolute configuration S at carbon 2, having the following molecular structure:

    or a pharmaceutically acceptable salt thereof,

    a) wherein R is₁And R₂Is independently selected from the group consisting of a hydroxyl group and a moiety convertible in vivo to a hydroxyl group;

    b) wherein R is₃Is selected from the group consisting of saturated, unsaturated or substituted pyrrolidinyl; saturated, unsaturated or substituted piperidino; saturated, unsaturated or substituted piperidinyl; saturated, unsaturated or substituted morpholinyl; a nitrogen-containing cyclic moiety; a nitrogen-containing polycyclic moiety; and NRaRb (Ra and Rb are independently hydrogen, straight or branched C₁-C₆Alkyl, straight or branched C₂-C₆Alkenyl and straight-chain or branched C₂-C₆Alkynyl) groups.
47. 47. The pharmaceutical composition of claim 48, wherein the compound or salt substantially lacks the (2R) -enantiomer.
48. 48. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is:

    and is optically active because most of its stereoisomers have 2S configuration.
49. 49. The pharmaceutical composition of claim 48, wherein the selective estrogen receptor modulator is a benzopyran salt of an acid selected from the group consisting of acetic acid, adipic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, fumaric acid, hydroiodic acid, hydrobromic acid, hydrochloric acid, hydrochlorothiazide acid, hydroxy-naphthoic acid, lactic acid, maleic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, nitric acid, palmitic acid, trimethylacetic acid, phosphoric acid, propionic acid, succinic acid, sulfuric acid, tartaric acid, terephthalic acid, p-toluenesulfonic acid, and valeric acid.
50. 50. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is:

    and is optically active due to its majority of enantiomers having the 2S configuration; and wherein the sex steroid precursor is dehydroepiandrosterone.
51. 51. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator has no estrogenic activity in breast, uterine or endometrial tissues.
52. 52. The pharmaceutical composition of claim 3, wherein the treatment further reduces the risk of the patient suffering from breast cancer, uterine cancer, or endometrial cancer.
53. 53. The pharmaceutical composition of claim 3, wherein the treatment also inhibits the onset of osteoporosis, hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, insulin resistance, type 2 diabetes, muscle wasting, obesity, Alzheimer's disease, cognitive loss, memory loss, or vaginal dryness.
54. 54. The pharmaceutical composition of claim 3, wherein the antiestrogen is farad (ICI 182780, fulvestrant, 7 α - [9- (4, 4,5, 5, 5-pentafluoro-pentylsulfinyl) nonyl ] estra-1, 3, 5(10) -triene-3, 17 β -diol) or SH 646.
55. 55. The pharmaceutical composition of claim 3, wherein the composition is in a dosage form for intravaginal administration.
56. 56. The pharmaceutical composition of claim 48, wherein the composition is in a dosage form for intravaginal administration.
57. 57. The pharmaceutical composition of claim 3, wherein the composition is in a dosage form for oral administration.
58. 58. The pharmaceutical composition of claim 3, wherein the composition is in a dosage form for transdermal administration.
59. 59. The pharmaceutical composition of claim 3, wherein the selective estrogen receptor modulator is selected from the group consisting of: tamoxifen, toremifene, CC 8490, SERM 3471, HMR3339, HMR3656, raloxifene, LY335124, LY 326315, azoxifene (LY 353381), pentoxifene (ERA923), bazedoxifene (TSE424, WAY140424), Europeria (lasofoxifene), EM-652, EM-800, EM-652-HCl (acobiprofen, EM-1538), 4-hydroxy-tamoxifen, 4-hydroxy-toremifene, droloxifene, LY 335563, GW-5638, idoxifene, levomeloxifene, Aprofen (TAT-59), oximiphene (FC 1271), fispemifene, and clokoloman.
60. 60. The kit of claim 5, wherein the selective estrogen receptor modulator has a molecular formula with the following characteristics:

    a) two aromatic rings separated by 1 to 2 intervening carbon atoms, which aromatic rings are unsubstituted or substituted with a hydroxyl group or a group that is converted to a hydroxyl group in vivo;

    b) a side chain having an aromatic ring and a tertiary amine functionality or salt thereof.
61. 61. The kit of claim 60, wherein the side chain is selected from the group consisting of:
62. 62. the kit of claim 5, wherein the selective estrogen receptor modulator is selected from the group consisting of triphenylethylene derivatives, indole derivatives, benzopyran derivatives, tamoxifen, toremifene, CC 8490, SERM 3471, HMR3339, HMR3656, raloxifene, LY335124, LY 326315, arzoxifene (LY 353381), penoxifene (ERA923), bazedoxifene (TSE424, WAY140424), oropoline (lasofoxifene), and Senocolomen derivatives.
63. 63. The kit of claim 5, wherein the selective estrogen receptor modulator is a benzothiophene derivative compound having the formula:

    a) wherein R is₁And R₂Is independently selected from the group consisting of hydrogen, hydroxyl, and a moiety that is converted to hydroxyl in vivo;

    b) wherein R is₃And R₄Is (a) independently C₁-C₄Alkyl, or (b) a moiety in combination with the nitrogen to which they are attached, selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, and morpholinyl;

    c) wherein A is selected from the group consisting of-CO-, -CHOH and-CH₂-the group consisting of;

    d) wherein B is selected from the group consisting of phenylene, dihydropyridinylidene, and-Ring C₄H₂N₂-the group consisting of.
64. 64. The kit of claim 5, wherein the selective estrogen receptor modulator is selected from the group consisting of raloxifene, azoxifene (LY 353381), LY353381, and LY 335563.
65. 65. The kit of claim 5, wherein the selective estrogen receptor modulator is a triphenylethylene or diphenylhydronaphthalene derivative compound having the formula:

    a) wherein D is-OCH₂CH₂N(R₃)R₄、-OCH₂CH₂OH or-CH-COOH (R)₃And R₄Is independently selected from the group consisting of C₁-C₄Alkyl, or R₃、R₄Together with the nitrogen atom to which they are attached is a ring structure selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, and morpholinyl);

    b) wherein E and K are independently hydrogen or hydroxy, phosphate or lower alkyl, and wherein J is hydrogen or halogen.
66. 66. The kit of claim 5, wherein the selective estrogen receptor modulator is tamoxifen, hydroxy-tamoxifen, droloxifene, toremifene, idoxifene, lasofoxifene, breloxifene, FC1271, and GW 5638.
67. 67. The kit of claim 5, wherein the selective estrogen receptor modulator is an indole derivative compound having the formula:

    a) wherein D is selected from the group consisting of-OCH₂CH₂N(R₇)R₈、-CH＝CH-CON(R₇)R₈、-CC-(CH₂)n-N(R₇)R₈Group (R) of₇And R₈Is independently selected from the group consisting of C₁-C₆Alkyl, or R₇、R₈Together with the nitrogen atom to which they are bonded are selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl,A ring structure selected from the group consisting of methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, and morpholinyl;

    b) wherein X is selected from the group consisting of hydrogen and C₁-C₆Alkyl groups;

    c) wherein R is₁、R₂、R₃、R₄、R₅And R₆Is independently selected from hydrogen, hydroxy, C₁-C₆Alkyl groups and a moiety that is converted to a hydroxyl group in vivo.
68. 68. The kit of claim 5, wherein the selective estrogen receptor modulator is selected from the group consisting of bazedoxifene (TSE 424; WAY-TSE 424; WAY 140424; 1- [ [4- [2- (hexahydro-1H-aza-1-) ]-1-yl) ethoxy]Phenyl radical]Methyl radical]-2- (4-hydroxyphenyl) -3-methyl-1H-indol-5-ol) with penciclovir (ERA 923; 2- (4-hydroxyphenyl) -3-methyl-1- [ [4- [2- (1-piperidinyl) ethoxy]Phenyl radical]Methyl radical]-1H-indol-5-ol).
69. 69. The kit of claim 5, wherein the selective estrogen receptor modulator is a senkelomon derivative compound having the formula:

    a) wherein R is₁And R₂Is independently selected from the group consisting of hydrogen, hydroxyl, and a moiety that is converted to hydroxyl in vivo;

    b) wherein R is₅And R₆Is independently hydrogen or C₁-C₆An alkyl group;

    c) wherein D is-OCH₂CH₂N(R₃)R₄(R₃And R₄Is independently selected from the group consisting of C₁-C₄Alkyl, or R₃、R₄With themThe nitrogen atoms to which they are bonded together are a ring structure selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholinyl).
70. 70. The kit of claim 5, wherein the senkoloman derivative is (3, 4-trans-2, 2-dimethyl-3-phenyl-4- [4- (2- (2- (pyrrolidin-1-yl) ethoxy) phenyl ] -7-methoxychroman.
71. 71. The kit of claim 5, wherein the selective estrogen receptor modulator has the formula:

    a) wherein R is₁And R₂Is independently hydrogen, hydroxy, or a moiety that is converted to hydroxy in vivo;

    b) wherein Z is absent or selected from the group consisting of-CH₂-, -O-, -S-and-NR₃-(R₃Hydrogen or lower alkyl);

    c) wherein R is₁₀₀Is a divalent moiety that separates L from the B ring by 4 to 10 intervening atoms;

    d) wherein L is a divalent or trivalent moiety selected from the group consisting of-SO-, -CON <, -N < and-SON <;

    e) wherein G is₁Is selected from hydrogen, C₁To C₅Hydrocarbons with G₂A divalent moiety in combination, and L is a 5-to 7-membered heterocycle and a halo or unsaturated derivative of the foregoing;

    f) wherein G is₂Absent or selected from hydrogen, C₁To C₅Hydrocarbons with G₁(ii) the divalent moieties in combination, and L is a 5-to 7-membered heterocyclic ring and halogenated or unsaturated derivatives of the foregoing;

    g) wherein G is₃Is selected from the group consisting of hydrogen, methyl and ethyl.
72. 72. The kit of claim 5, wherein the selective estrogen receptor modulator is a benzopyran compound having the following general structure:

    or a pharmaceutically acceptable salt thereof,

    a) wherein D is-OCH₂CH₂N(R₃)R₄(R₃And R₄Is independently selected from the group consisting of C₁-C₄Alkyl, or R₃、R₄Together with the nitrogen atom to which they are bonded, is a ring structure selected from the group consisting of pyrrolidinyl, dimethyl-1-pyrrolidinyl, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholinyl);

    b) wherein R is₁And R₂Is independently selected from the group consisting of hydrogen, hydroxyl, and a moiety that is converted to hydroxyl in vivo; and

    c) wherein G is₃Is selected from the group consisting of hydrogen, methyl and ethyl.
73. 73. A kit according to claim 5 wherein the selective estrogen receptor modulator is a benzopyran compound having optical activity due to its majority of its stereoisomers having the absolute configuration S at carbon 2, having the following molecular structure:

    or a pharmaceutically acceptable salt thereof,

    a) wherein R is₁And R₂Is independently selected from the group consisting of a hydroxyl group and a moiety convertible in vivo to a hydroxyl group;

    b) wherein R is₃Is selected from the group consisting of saturated, unsaturated or substituted pyrrolidinyl; saturated, unsaturated or substituted piperidinesA group; saturated, unsaturated or substituted piperidinyl; saturated, unsaturated or substituted morpholinyl; a nitrogen-containing cyclic moiety; a nitrogen-containing polycyclic moiety; and NRaRb (Ra and Rb are independently hydrogen, straight or branched C₁-C₆Alkyl, straight or branched C₂-C₆Alkenyl and straight-chain or branched C₂-C₆Alkynyl) groups.
74. 74. The kit of claim 73, wherein the compound or salt substantially lacks the (2R) -enantiomer.
75. 75. The kit of claim 5, wherein the selective estrogen receptor modulator is:

    and is optically active because most of its stereoisomers have 2S configuration.
76. 76. The kit of claim 73, wherein the selective estrogen receptor modulator is a benzopyran salt of an acid selected from the group consisting of acetic acid, adipic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, fumaric acid, hydroiodic acid, hydrobromic acid, hydrochloric acid, hydrochlorothiazide acid, hydroxy-naphthoic acid, lactic acid, maleic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, nitric acid, palmitic acid, trimethylacetic acid, phosphoric acid, propionic acid, succinic acid, sulfuric acid, tartaric acid, terephthalic acid, p-toluenesulfonic acid, and valeric acid.
77. 77. The kit of claim 5, wherein the selective estrogen receptor modulator is:

    and is optically active due to its majority of enantiomers having the 2S configuration; and wherein the sex steroid precursor is dehydroepiandrosterone.
78. 78. The kit of claim 5, wherein the selective estrogen receptor modulator has no estrogenic activity in breast, uterine or endometrial tissues.
79. 79. The kit of claim 5, wherein the treatment further reduces the risk of the patient suffering from breast cancer, uterine cancer, or endometrial cancer.
80. 80. The kit of claim 5, wherein the treatment further inhibits the onset of osteoporosis, hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, insulin resistance, type 2 diabetes, muscle wasting, obesity, Alzheimer's disease, cognitive loss, memory loss, or vaginal dryness.
81. 81. The kit of claim 5, wherein the antiestrogen is farad (ICI 182780, fulvestrant, 7 α - [9- (4, 4,5, 5, 5-pentafluoro-pentylsulfinyl) nonyl ] estra-1, 3, 5(10) -triene-3, 17 β -diol) or SH 646.
82. 82. The kit of claim 5, wherein at least one component of the kit is in a dosage form for intravaginal administration.
83. 83. The kit of claim 75, wherein at least one component of the kit is in a dosage form for intravaginal administration.
84. 84. The kit of claim 5, wherein at least one component of the kit is in a dosage form for oral administration.
85. 85. The kit of claim 5, wherein at least one component of the kit is in a dosage form for transdermal administration.
86. 86. The kit of claim 5, wherein the selective estrogen receptor modulator is selected from the group consisting of: tamoxifen, toremifene, CC 8490, SERM 3471, HMR3339, HMR3656, raloxifene, LY335124, LY 326315, azoxifene (LY 353381), pentoxifene (ERA923), bazedoxifene (TSE424, WAY140424), Europeria (lasofoxifene), EM-652, EM-800, EM-652-HCl (acobiprofen, EM-1538), 4-hydroxy-tamoxifen, 4-hydroxy-toremifene, droloxifene, LY 335563, GW-5638, idoxifene, levomeloxifene, Aprofen (TAT-59), oximiphene (FC 1271), fispemifene, and clokoloman.
87. 87. The pharmaceutical composition of claim 3, wherein the sex steroid precursor is selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-ene-3 β,17 β -diol, and 4-androstene-3, 17-dione.
88. 88. The kit of claim 5, wherein said sex steroid precursor is selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-ene-3 β,17 β -diol, and 4-androstene-3, 17-dione.
89. 89. The kit of claim 75, wherein the selective estrogen receptor modulator is in a dosage form for intravaginal administration.