EA041509B1 - USE OF SEX STEROID HORMONES PRECURSORS IN COMBINATION WITH A SELECTIVE ESTROGEN RECEPTOR MODULATOR FOR THE TREATMENT OF DISEASES AND CONDITIONS IN POST-MENOPAUSE WOMEN - Google Patents

Description

The field of technology to which the invention belongs

The present invention provides novel methods for administering and dosing dehydroepiandrosterone (DHEA) to exploit positive androgenic effects (e.g., in the vaginal lamina propia and/or muscularis layer) without causing unwanted systemic estrogenic effects. In addition to DHEA, other sex steroid precursors (eg, dehydroepiandrosten sulfate, androst-5-en-3b,17b-diol and 4-androsten-3,17-dione) can be used.

State of the art

Many hormone-related therapies are known. For example, many provide the sex steroid hormone estrogen or androgen systemically and/or to the target tissue. In addition to the direct administration of androgens and/or estrogens, sex steroid precursors that can be converted to estrogen and/or androgen in a particular tissue have also been used for many conditions. Both androgens and estrogens can be beneficial in some contexts and harmful in others. This depends, inter alia, on the target tissue, the specific needs presented by the patient, and the extent to which non-target tissue may be affected. Some therapies, although targeted, may still have undesirable activity elsewhere in the body (for example, local administration of this pharmaceutical agent results, however, in an increased systemic presence of either the pharmaceutical agent or one of its metabolites. Mechanism of Action is also not always fully understood, especially the relative contributions of androgens and estrogens.

The essence of the invention

Thus, the purpose of this invention is the use of specific doses of compositions and methods of administration to better achieve the beneficial effects of sex steroid hormones and better eliminate their undesirable side effects.

In one aspect, this invention provides a method for treating and/or reducing the likelihood of acquiring vaginal diseases or conditions associated with hormonal imbalances in postmenopausal (postmenopausal) women, said method comprising administering a sex steroid precursor selected from the group consisting of dehydroepiandrosterone , dehydroepiandrosterone sulfate, androsten-5-en-3b,17b-diol and 4-androsten-3,17dione, to a patient in need of said treatment, wherein said said sex steroid hormone precursor is administered in a therapeutic amount that increases the level of circulating androgen metabolites without increases in estradiol levels above values found in healthy postmenopausal women.

In another aspect, this invention provides a method for treating and/or reducing the likelihood of acquiring symptoms or diseases associated with menopause (menopause) in postmenopausal women, said method comprising administering a sex steroid hormone precursor selected from the group consisting of dehydroepiandrosterone, sulfate dehydroepiandrosterone, androsten-5-en-3b,17b-diol and 4-androsten-3,17-dione, to a patient in need of said treatment, wherein said said sex steroid precursor is administered in a therapeutic amount that increases the level of circulating androgen metabolites without increasing estradiol levels above values found in healthy postmenopausal women to avoid the risk of breast cancer and uterine cancer.

In another aspect, this invention provides a method for treating and/or reducing the likelihood of acquiring symptoms or diseases associated with menopause (menopause) in postmenopausal women, said method comprising administering a sex steroid hormone precursor selected from the group consisting of dehydroepiandrosterone, sulfate dehydroepiandrosterone, androsten-5-en-3b,17b-diol and 4-androsten-3,17-dione, to a patient in need of said treatment, wherein said said sex steroid hormone precursor is administered in a therapeutic amount that increases the level of circulating androgen metabolites, and further comprises administering, as part of a combination therapy, a therapeutically effective amount of a Selective Estrogen Receptor Modulator to avoid the risk of breast and uterine cancer commonly present in a postmenopausal woman and to prevent bone thinning (osteoporosis), fat accumulation, and diabetes type a 2.

In another aspect, this invention provides a method for treating vaginal conditions of the Iamina propia layer or muscularis layer, comprising vaginal administration at a daily dose of 3-13 mg.

In another aspect, this invention provides a pharmaceutical composition comprising a sex steroid precursor selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androsten-5-en-3b,17b-diol and 4-androsten-3,17-dione, and additionally containing a pharmaceutically acceptable excipient, diluent or carrier selected from the group consisting of triglycerides of saturated acids C ₁₂ -C ₁₈ with varying parts of the corresponding partial glycerides (solid fat, Witepsol), butter, mixed triglycerides of oleic, palmitic and stearic acids (cocoa oil), partially hydrogenated cotton

- 1 041509 saturated fatty acids (C12-C16), triglycerides from palm, palm kernel oil and coconut oils with self-emulsifying glyceryl monostearate and polyoxyl stearate (Fattibase), Hexaride Base 95, high-melting fractions of coconut and palm kernel oils (Hydrokote), rearranged hydrogenated vegetable oils (S-70-XX95 and S-070-XXA), eutectic mixtures of mono-, di-, triglycerides obtained from natural vegetable oils (Suppocire), Tegester-triglycerides, Tween 61, triglycerides derived from coconut oil (Wecobee), cocoa butter, semi-synthetic glycerides (Japocire, Ovucire), mixtures of tri-, di- and monoglycerides of saturated fatty acids (Massa Estarinum ) and combinations of the above (see Allen et al., 2008) Any vehicle, including liquid, in which DHEA and other precursors are soluble, including yuchen in this invention.

In another aspect, this invention provides a vaginal suppository containing 0.25-2.00 wt.%, more specifically, 0.5 wt.% DHEA, based on the total weight of the suppository, and further containing a lipophilic excipient. A particularly suitable excipient is witepsol H-15.

By providing the desired androgenic effects without estrogenic systemic effects, the systemic side effects of estrogen, such as increased risk of breast and endometrial cancer, found with existing estrogen-based topical and systemic estrogen replacement therapies can be avoided (Labrie, Cusan et al., Menopause , in press).

Along with other forms of administration of precursors, this invention provides vaginal suppositories and vaginal creams formulated with preferred excipients and preferred precursor concentrations.

Vaginal administration is preferred because topical action can provide the desired androgenic effects on the desired vaginal layers at much lower doses than other routes of administration. Administration of doses by other routes of administration may also be used with changes to previous doses and concentrations for known variation between these routes of administration. The attending physician should adjust the dosage as appropriate according to the response of the individual patient.

In preferred embodiments, the sex steroid precursor is DHEA.

Brief description of the figures

Fig. 1 shows serum levels of DHEA and 5-diol on day 1 or day 7 in 40-75 year old postmenopausal women after daily administration of vaginal suppositories containing 0, 0.5, 1.0, or 1.8% DHEA. Data are expressed as means±SEM (n=9 or 10).

Fig. 2 shows serum levels of Dough and DHT on day 1 or day 7 in 40-75 year old postmenopausal women after daily administration of vaginal suppositories containing 0, 0.5, 1.0, or 1.8% DHEA (n=8). Data are expressed as means±SEM (n=8-9). Testo (testosterone) levels from one patient in the placebo group were excluded due to unexplained high Testo levels not reflected in the case of any other steroid.

Fig. 3 shows serum levels of E1 and E2 on day 1 or day 7 in 40-75 year old postmenopausal women after daily administration of vaginal suppositories containing 0, 0.5, 1.0, or 1.8% DHEA. Data are expressed as means±SEM (n=9 or 10).

Fig. 4 shows serum levels of E ₁ -S and DHEA-S on day 1 or day 7 in 40-75 year old postmenopausal women after daily administration of vaginal suppositories containing 0, 0.5, 1.0, or 1.8% DHEA. Data are expressed as means±SEM (n=9 or 10).

Fig. 5 shows serum levels of 4-dione and ADT-G on day 1 or day 7 in 40-75 year old postmenopausal women following daily administration of vaginal suppositories containing 0, 0.5, 1.0, or 1.8% DHEA. Data are expressed as means±SEM (n=9 or 10).

Fig. 6 shows serum levels of 3α-diol-3G and 3α-diol-17G on day 1 or day 7 in 40-75 year old postmenopausal women after daily administration of vaginal suppositories containing 0, 0.5, 1.0, or 1.8% DHEA. (n=8). Data are expressed as means±SEM (n=8-10).

Fig. 7 shows the mean 24-hour serum concentration (AUC _o _ _244/24 ) of DHEA, 5-diol, DHEA-S, 4-dione, Dough and DHT measured on day 1 or day 7 in 40-75 year old postmenopausal women after daily administration of vaginal suppositories containing 0, 0.5, 1.0 or 1.8% DHEA. Data were expressed as means±SEM (n=8-10). Testo levels from one patient in the placebo group were excluded (n=8 in this group). Serum steroid hormone concentrations measured in 30-35 year old postmenopausal women are added as a reference. Data were expressed as mean (n=47) with 5th and 95th percentiles (dotted lines). *, p<0.05, **, p<0.01, experimental data (day 7) vs. placebo (day 7).

Fig. 8 shows the mean 24-hour serum concentration (AUC _o _ _244/24 ) of ADT-G, 3a-diol 3G, 3α-diol-17G, E1, E2 and E1-S measured on day 1 or day 7 at 40-75 -year postmenopausal

- 2 041509 women after daily administration of vaginal suppositories containing 0, 0.5, 1.0 or 1.8% DHEA. Data were expressed as means±SEM (n=9 or 10). Serum steroid hormone concentrations measured in 30-35 year old postmenopausal women are added as a reference. Data were expressed as mean (n=47) with 5th and 95th percentiles (dotted lines). *, p<0.05, **, p<0.01, experimental data (day 7) vs. placebo (day 7).

Fig. 9 shows changes in serum levels of the sum of androgenic metabolites ADT-G, 3αdiol-17G in postmenopausal women with vaginal atrophy following intravaginal administration of increasing doses of DHEA. These data are expressed as the percentage of serum levels of the same steroid metabolites observed in young adults (30-35 year old) menstruating premenopausal (premenopausal) women. The conversion rate is obtained by dividing the sum of the serum levels of ADT-G, 3α-diol-3G and 3α-diol-17G in women who received doses of 0.5, 1.0 and 1.8% DHEA by the values found in premenopausal ( premenopausal) women (data from Labrie et al., 2006). Changes in serum DHEA compared to healthy premenopausal women are also shown as a comparison to show conversion efficiency (0-0) and () background levels of androgen metabolites and DHEA, respectively.

Fig. 10 shows maturity index (A) and vaginal pH (B) measured on day 1 and day 2 in 40-75 year old postmenopausal women after daily administration of vaginal suppositories containing 0, 0.5, 1.0, or 1.8% DHEA. Data were expressed as means±SEM (n=9 or 10). *, p<0.05, **, p<0.01, data on day 7 vs data on day 1.

Fig. 11 shows the time course in serum of dehydroepiandrosterone (DHEA) (A) and androst-5ene-3b,17b-diol (5-diol) (B) after a single oral administration of two 50 mg DHEA capsules or application of 4 g of a 10% cream or DHEA gel for postmenopausal women.

Fig. 12 shows the time course in serum of androstenedione (4-dione) (A) and testosterone (B) after a single oral administration of two 50 mg DHEA capsules or 4 g of a 10% DHEA cream or gel for postmenopausal women.

Fig. 13 shows the time course in serum of estrone (E1) (A) and 17β-estradiol (E ₂ ) (B) after a single oral administration of two 50 mg DHEA capsules or 4 g of a 10% DHEA cream or gel for postmenopausal women.

Fig. 14 shows the time course in serum of dehydroepiandrosterone sulfate (DHEA-S) (A) and estrone sulfate (E1-S) (B) after a single oral administration of two 50 mg DHEA capsules or application of 4 g of 10% DHEA cream or gel for postmenopausal women.

Fig. 15 shows the time course in serum of androsteronglucuronide (ADT-G) (A) and androstone 3a,17b-diolglucuronide (3α-diol-G) (B) after daily oral administration of two 50 mg DHEA capsules or application of 4 g of 10% cream or DHEA gel for postmenopausal women.

Fig. 16 shows the time course in serum of dehydroepiandrosterone (DHEA) (A) and androst-5ene-3b,17b-diol (5-diol) (B) after daily oral administration of two 50 mg DHEA capsules or application of 4 g of 10% cream or DHEA gel for postmenopausal women. Measurements were taken on day 14 of dosing.

Fig. 17 shows the time course in serum of androstenedione (4-dione) (A) and testosterone (B) after daily oral administration of two 50 mg DHEA capsules or 4 g of 10% DHEA cream or gel for postmenopausal women. Measurements were taken on day 14 of dosing.

Fig. 18 shows the time course in serum of estrone (E1) (A) and estradiol (E2) after daily oral administration of two 50 mg DHEA capsules or 4 g of 10% DHEA cream or gel for postmenopausal women. Measurements were taken on day 14 of dosing.

Fig. 19 shows the time course in serum dehydroepiandrosterone sulfate (DHEA-S) (A) and estrone sulfate (E1-S) (B) after daily oral administration of two 50 mg DHEA capsules or application of 4 g of 10% DHEA cream or gel for postmenopausal women. Measurements were taken on day 14 of dosing.

Fig. 20 shows the time course in serum of androsteronglucuronide (ADT-G) (A) and androst-5ene-3β,17β-diol-G (3α-diol-G) (B) after daily oral administration of two 50 mg DHEA capsules or application of 4 g 10% DHEA cream or gel for postmenopausal women. Measurements were taken on day 14 of dosing.

Fig. 21 shows the ratios of the AUC _o _ ₂₄₄ values of DHEA and its metabolites on day 14 of dosing compared to pre-treatment baseline values. The corresponding numerical values can be found in Table. 5.

Fig. 22 shows the effect of daily intravaginal application of 0.0, 0.25, 0.5 and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on the percentage of vaginal parabasal cells in postmenopausal women. Data are expressed as means±SEM.

Fig. 23 shows the effect of daily intravaginal application of 0.0, 0.25, 0.5 and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on the percentage of vaginal surface cells in postmenopausal women. Data are expressed as means±SEM.

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Fig. 24 shows the effect of daily intravaginal application of 0.0, 0.25, 0.5 and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on vaginal pH in postmenopausal women. Data are expressed as means±SEM.

Fig. 25 shows the effect of daily intravaginal application of 0.0, 0.25, 0.5 and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on changing the severity of the symptom of vaginal atrophy, rated by women as the most disturbing symptom . Values are compared relative to day 1 and are expressed as mean ±SEM.

Fig. 26 shows the effect of daily intravaginal application of 0.0, 0.25, 0.5 and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on the change in vaginal secretions as assessed by vaginal examination. Data are expressed as means±SEM.

Fig. 27 shows the effect of daily intravaginal application of 0.0, 0.25, 0.5 and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on vaginal discoloration as assessed by vaginal examination. Data are expressed as means±SEM.

Fig. 28 shows the effect of daily intravaginal application of 0.0, 0.25, 0.5 and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on change in the integrity of the vaginal epithelium, as assessed by vaginal examination. Data are expressed as means±SEM.

Fig. 29 shows the effect of daily intravaginal application of 0.0, 0.25, 0.5 and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on the change in the thickness of the vaginal epithelium, assessed by vaginal examination. Data are expressed as means ±SEM,

Fig. 30 shows mean 24-hour serum concentrations (AUC _o-244 /24) of DHEA, 5-diol, DHEA-S, Ei, E2 and E ₁ -S measured on days 1 and 7 after once-daily administration of vaginal ovulation (dosage form) containing 0.5% DHEA. Data are expressed as means±SEM (n=10). Serum concentrations of steroid hormones measured in 30-35 year old premenopausal women (n=47) as well as in 55-65 year old postmenopausal women (n=369) were added as reference data, which were expressed as mean values and 5th and 95th percentiles (dashed lines). *, p<0.05, **, p<0.01, experimental data against background. (Data from Labrie, Cusan et al. 2008).

Fig. 31 shows mean 24-hour serum concentrations (AUC _0-2 4 _H /24) of 4-dione, testosterone, DHT ADT-G, 3α-diol-3G, and 3α-diol-17G measured on days 1 and 7 post-dose. once daily vaginal ovulation (dosage form) containing 0.5% DHEA. Data are expressed as means±SEM (n=10). Serum concentrations of steroid hormones measured in 30-35 year old premenopausal women (n=47) and in 55-65 year old postmenopausal women (n=369) were added as reference data, which were expressed as mean values and 5- th and 95th percentiles (dashed lines). *, p<0.05, **, p<0.01, experimental data against background. (Data from Labrie, Cusan et al., 2008).

Detailed description of the invention

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Vaginal dryness is found in 75% of postmenopausal women (Wines and Willsteed 2001; N.A.M.S. 2007). Due to various reasons, in particular the risk of estrogen-induced complications, only 20-25% of symptomatic women with vaginal atrophy seek medical treatment (Pandit and Ouslander 1997; N.A.M.S. 2007). Thus, there is a clear medical need and a major opportunity to improve the quality of life of the large population of women who suffer from vaginal atrophy for much of their lifespan. It may be mentioned that although hot flashes (hot flashes) decrease spontaneously with time, the symptoms of vaginal atrophy, namely vaginal dryness, vulvovaginal irritation/itching, and dyspareunia, increase in severity over time in the absence of treatment.

Based on the well-known fact that estrogen secretion by the ovaries stops at menopause, systemic and local estrogens have been the only approach to treat vaginal atrophy so far. However, systemic estrogen plus progestin (HRT) and estrogen alone (ERT) have been shown to increase the risk of breast cancer (Steinberg, Thacker et al., 1991; Sillero-Arenas, Delgado-Rodriguez et al., 1992; Colditz, Egn et al., 1993; Colditz, Hankinson et al., 1995; Collaborative Group on Hormonal Factors in Breast Cancer 1997; Hulley 2002; Beral 2003; Chlebowski, Hendrix et al., 2003; Holmberg and Anderson 2004; Lyytinen, Pukkala et al. ., 2006; Corrao, Zambon et al., 2008; Holmberg, Iversen et al., 2008; Li, Plummer et al., 2008), ovarian cancer (Garg, Kerlikowske et al., 1998; Coughlin, Giustozzi et al. , 2000; Lacey, Mink et al., 2002; Riman, Dickman et al., 2002; Rodriguez, Patel et al., 2002; Rossouw, Anderson et al., 2002; Lyytinen, Pukkala et al., 2006), a also endometrial cancer (estrogen only) (Gambrell, Massey

- 14 041509 et al., 1980; Persson, Adami et al., 1989; Voigt, Weiss et al., 1991; Jick, Walker et al., 1993; Grady, Gebretsadik et al., 1995; Beral, BuU et al., 2005). The publicity followed by the women's health initiative (Rossouw, Anderson et al., 2002) had the strongest effect in calling into question the safety of available treatments for menopausal symptoms (Archer 2007).

Although intravaginal preparations have been developed to eliminate systemic estrogen exposure, a long series of studies have unanimously demonstrated that all intravaginal estrogen preparations result in relatively high estrogen levels, measured directly or through their systemic effects (Englund and Johansson 1978; Rigg/Hermann et al., 1978; Martin, Yen et al., 1979; Furuhjelm, Karlgren et al., 1980; Deutsch, Ossowski et al., 1981; Mandel, Geola et al., 1983; Nilsson and Heimer 1992; Nachtigall 1995; Ayton, Darling et al., 1996; Dugal, Hesla et al., 2000; Rioux, Devlin et al., 2000; Manonai, Theppisai et al., 2001; Notelovitz, Funk et al., 2002; Ponzone, Biglia et al., 2005; Weisberg, Ayton et al., 2005; Galhardo, Soares et al., 2006; Kendall, Dowsett et al., 2006; Long, Liu et al., 2006; Bachmann, Lobo et al., 2008). These data showing a significant increase in estrogen levels clearly indicate that the use of intravaginal estrogen preparations is also potentially associated with an increased risk of breast and uterine cancer (Kvorning and Jensen 1986; Mattson, Culberg et al. 1989; Rosenberg, Magnusson et al. ., 2006; N.A.M.S. 2007). In fact, there has been official concern about the stimulatory effects of vaginal estrogen preparations on the endometrium ((N.A.M.S. 2007).

The earliest measurements of serum estradiol (E2) levels following intravaginal estrogens used radioimmunoassays, a technology lacking specificity, accuracy, validity, and sensitivity (Rinaldi, Dechaud et al., 2001). The present inventors measured serum estrogens using GLP (Good Laboratory Practice) validated mass spectrometric assays following intravaginal administration of two of the most common estrogen preparations (Labrie, Cusan et al., 2008). This study was able to definitively show that both E2 pill (25 μg E ₂ /day) and conjugated estrogens cream (1 g 0.625 mg conjugated estrogens/day), after one week of once daily treatment, caused approximately 5-fold increase in serum E2 in postmenopausal women. Such data indicate that the actions of estrogens applied locally to the vagina are unlikely to be limited to that vagina and that a systemic effect is expected, as previously suggested (Englund and Johansson 1978; Rigg, Hermann et al., 1978; Martin, Yen et al. , 1979; Furuhjelm, Karlgren et al., 1980; Deutsch, Ossowski et al., 1981; Mandel, Geola et al., 1983; Nilsson and Heimer 1992; Nachtigall 1995; Ayton, Darling et al., 1996; Dugal, Hesla et al., 2000; Rioux, Devlin et al., 2000; Manonai, Theppisai et al., 2001; Notelovitz, Funk et al., 2002; Ponzone, Biglia et al., 2005; Weisberg, Ayton et al., 2005 ; Galhardo, Soares et al., 2006; Kendall, Dowsett et al., 2006; Long, Liu et al., 2006; Bachmann, Lobo et al., 2008).

In addition to the above concerns regarding the safety of both systemically and locally administered estrogens, recent data clearly demonstrated that women not only were estrogen deficient during menopause, but that they were also progressively deficient of androgens produced in the body from the age of thirty onwards. specific peripheral target tissues via intracrine conversion of dehydroepiandrosterone (DHEA) to androgens and/or estrogens (Labrie, Belanger et al., 1988; Labrie 1991; Labrie, Luu-The et al., 2003; Labrie, Luu-The et al. , 2005). Indeed, serum DHEA and DHEA sulfate decrease progressively from a maximum observed at age 30 (Orentreich, Brind et al., 1984; Labrie, Belanger et al., 1997; Labrie, Luu-The et al., 2003), to 60% lower during menopause (Labrie, Belanger et al., 2006).

Regarding the role of androgens in women, it is important to mention that women secrete 50% of androgens compared to the observed levels in men (Labrie, Belanger et al., 1997; Labrie, Luu-The et al., 2005). Since serum DHEA is the predominant source of androgens that perform a number of physiological functions in women (Labrie, Luu-The et al., 2003; Labrie 2007), a 60% decrease in circulating DHEA found already at menopause results in a similar 60% increase in total androgen pool (Labrie, Belanger et al., 2006) with potential signs and symptoms of hypoandrogenicity in bone, muscle, breast, vagina, brain, and glucose, insulin, and lipid metabolism (Labrie, Luu-The et al. , 2003; Labrie 2007). Recent evidence has shown that among these androgenic target tissues, the vagina is androgen-responsive after administration of DHEA in the rat, with beneficial effects not only on the superficial epithelial layer of the vagina, but also on collagen fibers in the Iamina propria and on the muscularis. (muscular lamina of the mucosa) (Berger, El-Alfy et al., 2005).

Based on authors' preclinical studies (Sourla, Flamand et al., 1998; Berger, El-Alfy et al., 2005) and authors' clinical studies (Labrie, Diamond et al., 1997; Labrie, Cusan et al., 2008) showing beneficial effects on the vagina of DHEA administered subcutaneously or locally, this clinical trial is an exploratory (prospective) randomized and placebo-controlled study of the effect of three doses of intravaginal DHEA administered once a day for 12 weeks on changes in superficial and parabasal cells, pH and most disturbing

- 15 041509 symptom of vaginal atrophy as primary targets. These results clearly show that locally administered DHEA is very effective and rapid in correcting all signs and symptoms of vaginal atrophy, with almost maximum effect achieved as early as 2 weeks at a dose of DHEA that does not cause significant changes in estrogens or androgens, while all other steroidal hormones remain unchanged or well within the range found in healthy postmenopausal women.

By administering DHEA locally to the vagina, the beneficial effects of estrogens and androgens produced locally in the vagina are achieved without any significant release of estradiol or testosterone into the blood (Labrie, Cusan et al., J. Ster. Biochem. Mol. Biol. In press ). In the formation of androgens and/or estrogens from DHEA through the process of intracrinology, any tissue is unpredictable, since this reaction depends on the activity of the enzymatic apparatus specifically present in each cell of each tissue. Thus, it is not possible to predict, from androgens and estrogens that are produced from DHEA in one tissue, the extent to which similar androgens and estrogens may be produced in another tissue.

The results of the ERC-210 clinical trial (Example 3) clearly demonstrate for the first time that topical administration of DHEA as precursor hormone replacement therapy (HPRT) is highly effective and rapid in correcting all symptoms and signs of vaginal atrophy in postmenopausal women. Most importantly, this is achieved at a dose (0.5%) of DHEA that does not increase serum levels of active estrogens and androgens, and with no or minimal changes in serum DHEA and any of its metabolites, all of which remain well within the range. the range of values found in healthy postmenopausal women (Labrie, Cusan et al., 2008).

Although 75% of postmenopausal women suffer from vaginal atrophy (Wines and Willsteed 2001; N.A.M.S. 2007), thus affecting their quality of life during most of their lifespan, only 20% seek treatment. (Pandit and Ouslander 1997). Fear of breast cancer associated with increased levels of estrogen in the blood is the main suspected cause. Since the secretion of estrogen into the systemic circulation is exclusively from the ovaries and ceases at menopause, administration of estrogens to postmenopausal women does not appear to be physiological. After the WHI, the scientific challenge is to explore alternative types and drugs of hormone therapy that could provide all of the postmenopausal benefits of estrogens while improving women's quality of life, minimizing risks, and maximizing benefits (Archer 2007). Because non-estrogen-based therapies have not been shown to be convincingly effective (Nelson, Vesco et al., 2006; Suckling, Lethaby et al., 2006), women and their clinicians have been left without a safe way to treat vaginal atrophy.

Various forms of estrogens are effective in the treatment of vulvovaginal atrophy (Pandit and Ouslander 1997; Utian, Shoupe et al., 2001). Indeed, E ₂ vaginal tablet has shown similar efficacy to E ₂ vaginal ring (Weisberg, Ayton et al., 2005) as well as conjugated estrogen cream (Rioux, Devlin et al. 2000; Manonai, Theppisai et al., 2001).

This new HPRT is in marked contrast to the 5-fold increase in serum E2 measured by mass spectrometry after treatment with intravaginal _E2 or conjugated estrogens (Labrie, Cusan et al., 2008). These recent results on serum estrogens support a long series of studies showing that all intravaginal estrogen preparations lead to elevated serum estrogen concentrations, measured directly by radioimmunoassays or through their systemic effects (Englund and Johansson 1978; Rigg, Hermann et al., 1978; Martin , Yen et al., 1979; Furuhjelm, Karlgren et al., 1980; Deutsch, Ossowski et al., 1981; Mandel, Geola et al., 1983; Nilsson and Heimer 1992; Nachtigall 1995; Ayton, Darling et al., 1996; Dugal, Hesla et al., 2000; Rioux, Devlin et al., 2000; Manonai, Theppisai et al., 2001; Notelovitz, Funk et al., 2002; Ponzone, Biglia et al., 2005; Weisberg, Ayton et al., 2005; Galhardo, Soares et al., 2006; Kendall, Dowsett et al., 2006; Long, Liu et al., 2006; Bachmann, Lobo et al., 2008).

The most common adverse events reported with the use of vaginal estrogens are vaginal bleeding and breast pain, both of which are secondary to elevated serum estrogens (Suckling, Lethaby et al., 2006). These side effects have been reported for the E ₂ ring, conjugated estrogen cream, and E2 tablets (Ayton, Darling et al. 1996; Weisberg, Ayton et al., 2005). As mentioned above, there is also concern about the stimulatory effects of vaginal estrogens on the endometrium (NAMS 2007). Uterine bleeding, breast pain, and perineal pain were reported in 9% of women who took the vaginal tablet for 24 weeks, while 34% complained of the same symptoms in the conjugated estrogen vaginal cream group (Rioux, Devlin et al., 2000 ). Suckling, Lethaby et al., 2006 reported no difference between different vaginal estrogen formulations.

It is well known that atrophic vaginitis in postmenopausal women could be aggravated or induced by the use of aromatase inhibitors for the treatment of breast cancer. Indeed, these drugs show their benefits in the case of breast cancer.

- 16 041509 decrease in E2 biosynthesis in all tissues, thereby increasing the frequency and severity of menopausal symptoms (Fallowfield, Cella et al., 2004; Morales, Neven et al., 2004). In a recent study where seven patients with breast cancer who were treated with aromatase inhibitors received Vagifem at a daily dose of 25 mcg for 2 weeks and then twice a week thereafter, serum E2 increased from a median of 3 pmol/l to 72 pmol/L at 2 weeks (range 3 - 232) (Kendall, Dowsett et al., 2006). Serum E2 levels usually decreased thereafter to values of 40 pmol/l or less, although at weeks 7-10 values of 137 and 219 pmol/l were found. The patient who received Premarin cream had serum E ₂ levels of 83 pmol/l at 2 weeks. It should be mentioned that blood sampling for E2 measurement was performed at the time of the patient visit, so the timing probably did not correspond to the highest serum E2 levels after administration of Vagifem. Therefore, it is more likely that the values reported in Kendall, Dowsett et al., 2006 underestimate, to an unknown extent, the true increase in serum E2 following administration of Vagifem intravaginal pill or Premarin cream. These authors concluded that the use of Vagifem with aromatase inhibitors is contraindicated. These findings in women treated with aromatase inhibitors raise a serious concern regarding the use of any vaginal, as well as any oral or transdermal estrogen preparation, in postmenopausal women.

The relatively high increase in serum E ₂ after treatment with various vaginal estrogen preparations, leading to an increased risk of breast cancer, is a widely recognized problem (Rosenberg, Magnusson et al., 2006). Although a low-event, short-monitoring study (4.7% subgroup of 1472 women) found no statistically significant difference in remission in the subgroup of women who used vaginal estrogen (Dew, Wren et al., 2003), it does not seem it is reasonable or acceptable to increase serum levels of E2 during breast cancer therapy, when the goal of treatment with aromatase inhibitors is precisely to achieve maximum inhibition of E2 biosynthesis.

In one early study with Vagifem, the E ₂ tablet, when administered at a dose of 25 μg, resulted in serum E ₂ levels of 80 pmol/l with values less than 50 pmol/l at 14 h and later (Kvorning and Jensen 1986). In a more recent study with Vagifem, peak and mean serum _E2 concentrations at 24 h measured were 180±99 pmol/L and 84 pmol/L for the 25 µg dose, while values were 81±62 pmol/L and 40 pmol/L. l, respectively, were found for a dose of 10 μg (Notelovitz, Funk et al., 2002). Other vaginal estrogen tablets and creams even resulted in higher serum estrogen levels (Schiff, Tulchinsky et al., 1977; Rioux, Devlin et al., 2000).

With 10 μg and 25 μg E ₂ vaginal tablets, serum E ₂ was found to increase to peak values of approximately 90 and 160 pmol/L, respectively, from a background of approximately 35 pmol/L (Nilsson and Heimer 1992). Serum E ₂ has been reported with Vagifem at _{a Cmax} of 51±34 pg/ml on day 1, with this value being virtually unchanged on days 14 (47±21 pg/ml) and 84 (49±27 pg/ml) (Vagifem, Physician PackageInsert 1999).

In another study, it was reported that after 52 weeks of treatment with 25 μg Vagifem, serum E ₂ levels remained unchanged from 10.3±21.5-9.9 pg/ml (Bachmann, Lobo et al., 2008). These results may be explained by the fact that blood sampling was most likely performed 3 or 4 days after the administration of Vagifem. It is also important to mention that the elevated serum E2 levels in this study are most likely due to the lack of specificity of the immunoassays used, since normal serum E2 levels measured by mass spectrometry in postmenopausal women are two to three times lower (Labrie, Belanger et al., 2006).

In one early study, oral and vaginal administration of 1.25 mg Premarin resulted in serum levels of E ₂ and estrone of at least 100 and 1000 pg/mL, respectively, during 24 hours post-administration, with these levels being somewhat higher after vaginal administration. Serum gonadotropin levels decreased in most subjects (Englund and Johansson 1978). Similar results have been reported (Rigg, Hermann et al., 1978). In one recent study, after 3 months of daily oral or intravaginal administration of 0.625 mg Premarin, serum _E2 levels increased to 83.1 and 58.6 pg/mL, respectively (Long, Liu et al., 2006), illustrating the very an important systemic effect after both intravaginal and oral estrogen administration, as serum E ₂ was only 36% lower after intravaginal administration compared to oral administration of conjugated estrogens. In a 12-week study with Premarin vaginal cream at a daily dose of 2 g, three times a week, 21% of women experienced bleeding after a progestogen test (Nachtigall 1995). In addition, of these women, 12% showed an increase in endometrial thickness on sonography.

No increase in serum levels of E1, _E2 , or _E3 has been reported with vaginal ring use (Nachtigall 1995; Gupta, Ozel et al., 2008), although significant increases in E1-S and _E2 have been observed in women over 60 years of age (Naessen, Rodriguez-Macias et al., 2001). In the EST ring group of a recent study, serum _E2 increased from 16±22 pmol/L to 49±64 pmol/L at week 24 (Weisberg, Ay

- 17 041509ton et al., 2005). In the Vagifem group, on the other hand, serum E ₂ increased from 15±33 pmol/l to 36±51 pmol/l. However, these authors reported that serum E ₂ remained within or near the values found in healthy postmenopausal women. At 48 weeks of treatment with the EST ring or Vagifem, 30-32% complained of urinary frequency and 18-33% complained of dyspareunia (Weisberg, Ayton et al., 2005).

Three studies have documented that the vaginal E ₂ ring allows for low serum E ₂ during the 90-day period, with the exception of the serum estrogen flare, which reaches the lower flare region seen in women with normal menstrual cycles, or 100-200 pmol/L during the first 0.5-8 hours after ring insertion (Holmgren, Lindskog et al., 1989; Schmidt, Andersson et al., 1994) (Baker and Jaffe 1996). That a daily intravaginal delivery of 7.5 µg E2 has systemic effects is shown by the observation of a significant increase in bone mineral density in the entire thigh and lumbar spine after 2 years of treatment with this intravaginal dose of _E2 (Salminen, Saaf et al., 2007 ).

As mentioned above, there is concern about the stimulatory effects of vaginal estrogens on the endometrium (N.A.M.S. 2007). After 12 weeks of treatment of 32 women with 25 mcg intravaginal E2 (Vagifema), one patient had simple hyperplasia without atypia (Bachmann, Lobo et al., 2008). In a 24-week study of 80 women, one case of proliferative endometrium was found (Rioux, Devlin et al., 2000), and in another 52-week study of 31 women, two had proliferative endometrium (Mettler and Olsen 1991).

In a 12-week study with Premarin vaginal cream at a dose of 2 g, three times a week, 21% of women experienced bleeding after a progestogen test (Nachtigall 1995). Of these, 12% showed an increase in the thickness of the endometrium according to sonography. The use of a dose of 0.3 mg of conjugated estrogens administered intravaginally three times a week can induce endometrial proliferation, although rarely, since endometrial proliferation was observed in only one in twenty cases (Nachtigall 1995).

An extended release estradiol ring (EST ring) induced endometrial proliferation similar to that induced by 0.625 mg Premarin cream (Ayton, Darling et al., 1996) but less than 1.25 mg Premarin cream (Nachtigall 1995). In fact, both the vaginal ring (EST ring) and conjugated estrogens cream (Premarin cream) have been shown to induce endometrial proliferation (Nachtigall 1995; Ayton, Darling et al., 1996). Two cases of moderate endometrial proliferation or hyperplasia in an endometrial polyp were found with the E ₂ ring (Nachtigall 1995), while two cases of hyperplasia (one simple and one complex, without atypia) were found with conjugated estrogens cream in the conjugated estrogens cream trial versus tablet E ₂ (Rioux, Devlin et al., 2000). E2 vaginal tablet was associated with endometrial hyperplasia similar to estradiol vaginal tablet (Dugal, Hesla et al., 2000; Manonai, Theppisai et al., 2001), but to a lesser extent than conjugated estradiol cream (Manonai, Theppisai et al., 2001).

Although serum estrogen levels increase to a lower extent after topical intravaginal application compared with oral or subcutaneous HRT or ERT, the risk of breast cancer remains a concern and the safety of intravaginal estrogens is questionable (Suckling, Lethaby et al., 2006; N.A.M.S. 2007) . Indeed, although the increase in serum estrogens is lower after the intravaginal route of administration compared with the oral or transdermal route, it is significantly elevated above normal postmenopausal levels for all intravaginal estrogen preparations (Ponzone, Biglia et al., 2005).

In addition to the increased risk of breast cancer associated with estrogen administration, it is important to remember that the true hormonal difference between postmenopausal women who do not suffer from vaginal atrophy (estimated at 25% of the postmenopausal population) and the remaining 75% of postmenopausal women who do suffer from vaginal atrophy (Wines and Willsteed 2001; N.A.M.S. 2007) is not related to the secretion of estrogens into the systemic circulation, as estrogen secretion by the ovaries ceases in all women during menopause (menopause). Despite this, a deficiency in estrogen secretion is not a valid explanation for the presence of symptoms of vaginal atrophy in most postmenopausal women.

However, the formation of sex steroid hormones does not stop with the cessation of ovarian function at menopause. Recent progress in understanding endocrine physiology in women indicates that after menopause, DHEA secreted by the adrenal glands is the only source of sex steroid hormones produced exclusively in target tissues (Labrie 1991). Unlike ovarian-derived estrogens, which are secreted into the general circulation where they can be measured, DHEA is an inactive precursor that is converted in peripheral tissues at different rates according to the level of expression of steroidogenic enzymes in each tissue. This process of intracrinology allows local interstitial production of active sex steroid hormones without significant release of these active steroids into the bloodstream (Labrie, Dupont et al., 1985; Labrie, Belanger et al., 1988; Labrie 1991; Labrie, Luu-The et al. , 2005).

- 18 041509

However, DHEA secretion appears to decrease with age, with a 60% decrease already observed by menopause (Labrie, Luu-The et al., 2003; Labrie, Belanger et al., 2005; Labrie, Luu-The et al., 2005; Labrie , Belanger et al., 2006; Labrie, Luu-The et al., 2006; Labrie 2007). The only difference between symptomatic and asymptomatic women is the amount of DHEA secreted by the adrenal glands or the sensitivity of the vaginal tissue to DHEA. This difference in the sensitivity of different women seems to be related, to an unknown extent, to the level of activity of the tissue type-specific enzymatic apparatus in each tissue (Labrie 1991; Labrie, Belanger et al., 2005). With this understanding, it is clear that DHEA, and not estrogens, is the physiological hormone replacement therapy for postmenopausal women.

As well demonstrated in previous studies by the authors of this invention (Labrie 1991; Labrie, Luu-The et al., 2003; Labrie, Luu-The et al., 2005; Labrie, Belanger et al., 2007), the addition of physiological amounts of exogenous DHEA allows the biosynthesis of androgens and/or estrogens only in suitable target tissues that contain the required steroidogenic enzymes of intracrinology (Labrie, Luu-The et al., 2005). Active androgens and estrogens synthesized locally from DHEA in peripheral target tissues exhibit their effects in the same cells in which they are formed. Most importantly, there is very little leakage of these active sex steroid hormones into the bloodstream, which explains the marked beneficial effects seen in the vagina without a significant change in circulating estrogens or androgens (Labrie, Cusan et al., 2008). This local biosynthesis, action, and inactivation of estrogens and androgens in target tissues eliminates exposure of other tissues to excess sex steroids and therefore eliminates the increased risks of unwanted side effects from increased estrogen exposure, including breast cancer, ovarian cancer, and uterine cancer (Gambrell, Massey et al., 1980; Persson, Adami et al., 1989; Steinberg, Thacker et al., 1991; Voigt, Weiss et al., 1991; Sillero-Arenas, DelgadoRodriguez et al., 1992; Colditz, Egn et al. ., 1993; Jick, Walker et al., 1993; Colditz, Hankinson et al., 1995; Grady, Gebretsadik et al., 1995; Collaborative Group on Hormonal Factors in Breast Cancer 1997; Garg, Kerlikowske et al., 1998; Coughlin, Giustozzi et al., 2000; Hulley 2002; Lacey, Mink et al., 2002; Riman, Dickman et al., 2002; Rodriguez, Patel et al., 2002; Rossouw, Anderson et al., 2002; Beral 2003 ; Chlebowski, Hendrix et al., 2003; Holmberg and Anderson 2004; Beral, Bull et al., 2005; Lyytinen, Pukkala et al., 2006; Corrao, Zambon et al., 2008; Holmberg, Iversen et al., 2008; Li, Plummer et al., 2008).

Change in pH is now recognized as a valid (significant) parameter that reflects the beneficial effects of therapy for vaginal atrophy. After 12 weeks of intravaginal treatment with 25 μg E2, the percentage of patients with a pH less than 5.0 was 51% compared to 21% in the placebo group (Bachmann, Lobo et al., 2008). However, against background, 11.2% and 13% of the women had a pH below 5.0 in these two respective groups. In the ERC-210 clinical study (Example 3), no patient had a pH below 5.0 at the start of therapy and 12%, 36%, 46% and 48% had pH values below 5.0 at 12 weeks in groups 0, 0.25, 0.5 and 1.0% DHEA, respectively.

In the ERC-210 clinical trial (Example 3), this effect of DHEA on the maturation of vaginal epithelial cells was particularly rapid: with vaginal ovulation 0.5% DHEA, 79% of the maximum effect on parabasal cells was already observed at 2 weeks, while 48% of the maximum stimulatory effects appeared on surface cells at the same time interval. On the other hand, 85% of the maximum stimulatory effect of 0.5% DHEA on the percentage of surface cells was achieved at 4 weeks. Similarly, 63% of the maximum effect of 0.5% DHEA on the most disturbing symptom was observed at 2 weeks and 87% was achieved at 4 weeks. In addition, only 17.8% of women reported no change in their most distressing symptom at 12 weeks in the 0.5% DHEA group compared to 48.8% in the placebo group.

The action of DHEA on parabasal cells is rapid as the % of parabasal cells decreased to less than 20% at one month with the three doses of DHEA used. The effect on % of surface cells is also very fast with 100% effect seen at 2 weeks with a high (1%) dose of DHEA. In a study with a vaginal cream or estrogen tablet, approximately 50% of the effect measured at 12 weeks was observed at 2 weeks (Rioux, Devlin et al., 2000). These results indicate that the rate of effect of DHEA is not the worst, but perhaps the best relative to the effect of E ₂ vaginal preparations and conjugated estrogens.

In one study of oral estrogens in 71 postmenopausal women, daily administration of 0.3 mg of oral synthetic conjugated estrogens reduced parabasal cells by 23 to 2.3%, while superficial cells increased by 2.1 to 15.9%. . (Marx, Schade et al., 2004). In one study comparing 0.3 and 0.625 mg doses of conjugated equine estrogens (Utian, Shoupe et al., 2001), the 0.625 mg dose showed a greater effect on % of surface cells.

In one recent study, vaginal maturity value (VMV) increased from background 27.45 to 56.85 (p<0.0001) in the estrogen-treated group (Simon, Reape et al., 2007). The percentage of superficial cells increased by 17.15% from background, while the percentage of parabasal cells decreased by 41.66% in the estrogen-treated group. In the same study, vaginal pH decreased

- 19 041509 from 6.64 at baseline to 5.05 (1.69 or 24% decrease in the estrogen group). The severity of the most distressing symptoms decreased from 2.58 to 1.04 (-1.54) in the estrogen group compared to a decrease from 2.59 to 1.84 (-0.75) in the placebo group. These results observed with estrogens are comparable to a 1.56 reduction in the severity of most bothersome symptoms at 12 weeks in the 0.5% DHEA group and a 0.67 reduction in the placebo group observed in the ERC-210 clinical trial (Example 3).

At week 12, 11% of subjects in the EST-ring group and 24% of subjects in the Vagifem group had persistent atrophic epithelium. At week 48, the corresponding values were 8% and 14% (Weisberg, Ayton et al., 2005). At 48 weeks of treatment with Vagifem or the EST ring, vaginal dryness was still present in 33% of women (Weisberg, Ayton et al., 2005). On the other hand, vulvar itching was present in 15% and 20% of women after treatment with the EST-ring and Vagifem, respectively, while 33% and 28% of women still had dyspareunia after treatment with the EST-ring and Vagifem, respectively. Bleeding after the progestogen test was 7% in the Vagifem group and 0% in the EST ring group.

After 3 months of daily administration of 0.625 mg of Premarin orally or intravaginally (as a cream), a 70.6 and 75% improvement in dyspareunia was observed (Long, Liu et al., 2006). This study concluded that 1 g 0.625 mg Premarin was the minimum dose for the treatment of sexual dysfunction.

In women who received 25 μg E ₂ intravaginally, dyspareunia persisted in 12.4% of cases after 12 months of treatment (Simunic, Banovic et al., 2003). The success rate of therapy with topical E2 tablets was 84.5% as assessed by patients and 86.1% as assessed by physicians (Simunic, Banovic et al., 2003). Bachman et al., 1992 (Bachmann, Notelovitz et al., 1992) reported that 40-50% of women receiving oral estrogen replacement therapy had constant complaints of vaginal dryness.

As previously reported, after 12 months of DHEA treatment (Labrie, Diamond et al., 1997), a clinical trial of ERC-210 (Example 3) showed no effect on endometrial histology after 3 months of intravaginal administration of the hormone precursor DHEA, as shown by histopathological examination of endometrial biopsies. obtained before and after 12 weeks of treatment. These findings are consistent with the absence of aromatase activity in human endometrium (Baxendale and Reed et al., 1981; Bulun, Lin et al., 2005). These findings are strongly supported by the widely accepted clinical observation that endometrial atrophy is a characteristic of postmenopause despite lifelong continuous DHEA secretion (Labrie, Luu-The et al., 2005; Labrie, Belanger et al., 2006). The absence in the human endometrium of the steroidogenic enzymes required to convert DHEA to estrogens is consistent with the physiological role of the endometrium, which is active exclusively during the reproductive years, when its function is essentially controlled by hormones derived from the ovaries and placenta. After the onset of menopause, there is no physiological role for the endometrium to correct for any continued action of estrogen after ovarian cessation of estrogen secretion. Thus, the enzymes necessary for the synthesis of estrogens from DHEA are not expressed in the endometrium, which is a tissue completely dependent on estrogens derived from the ovaries.

It has long been known that estrogens given alone stimulate endometrial proliferation (Smith, Prentice et al., 1975), while progestins given in combination with estrogens inhibit the stimulatory effect of estrogens (Feeley and Wells 2001). Since androgen receptors are expressed in the human endometrium and stroma (Mertens, Heineman et al., 1996), it is interesting to mention that a clinical study that investigated the effects of androgens showed no effect on the endometrium of a relatively high dose of testosterone in postmenopausal women (testosterone undecanoate, 40 mg every other day) (Zang, Sahlin et al., 2007). In women who received estradiol valerate (2 mg/day), Ki-labeling increased by 50% at 3 months of treatment, while the simultaneous administration of testosterone reduced proliferation by up to 28%. Ki67-labeling increased only in the two estrogen-treated groups, but decreased with the addition of testosterone to the stroma. Although there was no stimulatory effect on endometrial proliferation in women, testosterone appears to have some antiestrogenic effect in the endometrium.

While FDA guidelines encourage sponsors to develop the lowest doses and effects for both estrogens and progestins, it is the opinion of the inventors of this invention that while estrogens are effective in correcting symptoms of vaginal atrophy and vasomotor symptoms, systemic estrogens are not physiological hormones that allow 25% of postmenopausal women to avoid moderate to severe symptoms of vaginal atrophy. These women remain relatively asymptomatic throughout their menopausal years because the only source of sex steroid hormones in postmenopausal women, both symptomatic and asymptomatic, is the local biosynthesis of estrogen and androgen from adrenal DHEA, via the mechanisms of intracrinology. DHEA replacement therapy is the only physiological approach that can provide women suffering from postmenopausal symptoms with the missing amount of DHEA they need.

- 20 041509 veins for their symptoms. Using this approach, called precursor hormone replacement therapy (HPRT), vaginal atrophy and vasomotor symptoms should be managed at a risk no greater than that of similar postmenopausal women who do not have symptoms of vaginal atrophy due to higher exposure to DHEA and sex steroids. hormones produced intracellularly by the process of intracrinology.

The sex steroid precursors administered in accordance with this invention are preferably administered in the dose range of (1) 0.5-100 mg per day (preferably 3-50 mg per day and most preferably 3-13 mg per day), when administered intravaginally ; (2) in the dosage range of 15-200 mg per day (preferably 30-100 mg per day) when administered to the skin; (3) in the dosage range of 1-200 mg per day (preferably 25-100 mg per day), eg 75 mg per day, by oral administration; or (4) in the dose range of 1.0-25 mg per day (preferably 3.25-20 mg per day) when administered parenterally (ie, intramuscular or subcutaneous).

In a pharmaceutical composition for vaginal administration, DHEA or another precursor is preferably present in a concentration of 0.1-10 wt.% relative to the total weight of this composition, more preferably 0.2-3.0%, in particular 0.25-2.0% . For example, 1.3 milliliters (ml) of a vaginal suppository having 0.5 wt.% DHEA (relative to the weight of the entire composition), administered once a day, provides desirably 6.5 mg/day of DHEA. Larger or smaller suppositories may be used, as well as different concentrations, while maintaining the dose within this desired range.

In a pharmaceutical composition for administration to the skin, DHEA or other precursor is preferably present in a concentration of 0.1-10 wt.% relative to the total weight of this composition, more preferably 0.2-2.0%, in particular 0.3-1, 5%.

In a pharmaceutical composition for oral administration, DHEA or another precursor is preferably present in a concentration of 5-98 wt.% relative to the total weight of this composition, more preferably 10-50%, in particular 15-40%.

In a pharmaceutical composition for parenteral administration (i.e. intramuscular or subcutaneous), DHEA or other precursor is preferably present at a concentration of 0.2-25 mg/ml, more preferably 0.65-15 mg/ml, in particular 2-10 mg /ml

An example of the effectiveness of the invention

Example 1

Clinical study of ERC-213.

Bioavailability of DHEA after administration of vaginal suppositories in postmenopausal women with vaginal atrophy in a randomized, placebo-controlled phase I trial.

Pharmacokinetics and local effects of daily administration of FHEA vaginal suppositories for one week.

The primary purpose of this invention was to evaluate the systemic bioavailability of DHEA and its metabolites following daily intravaginal administration of suppositories at four different concentrations of DHEA. This study was a randomized, placebo-controlled, double-blind study with 10 subjects per study arm. Thus, forty postmenopausal women were randomized to receive a daily dose of one suppository of the following DHEA concentrations: 0.0, 0.5% (6.5 mg DHEA/suppository), 1.0% (13 mg DHEA/suppository), or 1.8 % (23.4 mg DHEA/suppository).

Maturity index as well as vaginal pH was measured during pre-treatment as well as after 7 days of treatment to provide an indication of local DHEA action over a short period of time.

As illustrated in FIG. 1B, in table. 1 and 2, daily intravaginal administration of 1.3 ml of a suppository containing 0.5, 1.0 and 1.8% DHEA resulted in a progressive increase in serum DHEA with AUC ₀ values. _24m 24.8±4.8 ng-h/ml, 56.2±8.9 ng-h/ml (p<0.05), 76.2±10.3 ng-h/ml (p<0 .01) and 114.3±9.97 ng-h/ml (p<0.01), respectively. Thus, there were increases of 127, 207 and 361% over control at doses of 0.5, 1.0 and 1.8% DHEA, respectively. As observed for all other steroids, similar AUC ₀ values. _24m on days 1 and 7.

Indeed, the mean value of 4.76 ± 0.42 ng/mL DHEA after treatment with the highest dose (Table 2) is similar to the value of 4.47 ± 2.19 ng/mL found in forty-seven (47) 30-35 -year-old premenopausal healthy women (Labrie, Belanger et al., 2006). This serum DHEA after any of the doses of DHEA used remains within the boundaries of healthy premenopausal women, as well illustrated in FIG. 7A.

As previously observed after oral or subcutaneous administration of DHEA (Labrie, Belanger et al., 2007), serum 5-diol exhibits a pattern almost consistent with that of DHEA, although much lower concentrations are observed. Indeed, the value of AUC _o _ ₂₄₄ increases from 5.60±0.60 ng-h/ml in the placebo group on day 7 to 9.83±1.14 (p<0.05), 13.8±1.87 (p<0.01) and 21.0±1.66 (p<0.01) at doses of 0.5, 1.0 and 1.8% DHEA, respectively (1D, Table 1). These changes correspond to 75%, 147% and 276% increases over control. Only a dose of 1.8% DHEA causes an increase in serum 5

- 21 041509 diol, exceeding the values found in healthy premenopausal women (Fig. 7B) during 24 hours after daily intravaginal administration of DHEA on day 7.

The AUC ₀ _ _24m serum testosterone (Testo) did not show a significant change at a dose of 0.5% (2.79±0.30 ng-h/ml vs. 2.58±0.33 ng-h/ml in the placebo group) (Fig. 2B). At doses of 1.0% and 1.8%, AUC values _{of o-244} were found to be 4.54 ± 0.91 ng-h / ml (p < 0.05) and 5.97 ± 0.69 ng-h / ml (p<0.01) (Table 1). These values correspond to the mean serum levels of Testo 0.11±0.01 (NS), 0.12±0.01 (NS), 0.19±0.04 (p<0.05) and 0.25±0, 03 (p<0.01) ng/ml, respectively. Even at the highest dose of 1.8% DHEA used, serum Dough levels remained within the normal range of premenopausal women measured at 0.18±0.07 ng/mL (0.06-0.31, 5th - 95th percentile) (Labrie, Belanger et al., 2006) (Fig. 7E). On the other hand, the 1.0% dose (0.18±0.07 ng/mL) corresponds exactly to the values found in healthy premenopausal women, namely 0.19±0.4 (FIG. 7E).

In FIG. 2C and D, serum DHT increased from an AUC _o _ ₂₄₄ of 0.58±0.07 ng-h/ml in the placebo group on day 7 to 0.93±0.11 (NS), 1.31±0.26 (p<0.05) and 1.93±0.23 (p<0.01) ng-h/mL in the 0.5, 1.0 and 1.8% DHEA groups, respectively (Table 2). These values correspond to mean serum DHT levels of 0.02±0.01, 0.04±0.01, 0.05±0.01, and 0.08±0.01 ng/mL (Table 2), thus reaching , at the highest dose of DHEA, normal serum DHT levels of 0.07±0.03 ng/ml observed in premenopausal women (Labrie, Belanger et al., 2006) (Fig. 7F).

Mean serum E1 levels were measured at 12.6±1.41 ng/ml in the placebo group on day 7 (Table 2), while there was no significant change at the 0.5% DHEA dose (15.4±2, O4 ng/ml). An increase to 24.1±3.54 ng/ml (p<0.01) and 25.0±2.85 ng/ml (p<0.01) was observed at doses of 1.0 and 1.5% DHEA, respectively. The corresponding AUC _o _ ₂₄₄ values are shown in FIG. 3B and are listed in Table. 1.

Mean serum E2 levels were measured at 2.77±0.29 pg/ml and 4.04±0.69 pg/ml (N.S.) in the placebo and 0.5% DHEA groups, respectively (Table 2). Mean serum E2 concentrations of 6.01±1.31 pg/ml (p<0.05) and 5.68±0.84 pg/ml (p<0.05) were found on day 7 in women who received doses 1 .0 and 1.8% DHEA, with absolute increases of 3.18 and 2.85 pg/mL over placebo, respectively. Comparable findings were observed for mean serum levels of E1-S with mean serum levels of O.12±O.O2 and 0.13 ng/mL (N.S.) in the placebo group and the 0.5% DHEA group, respectively (Table 2). Values of 0.18±0.03 and 0.25±0.25 ng/mL were measured in the 1.0% and 1.8% DHEA groups, respectively. Only the 1.8% DHEA group showed a statistical difference (p<0.01) with the placebo group.

As can be seen in 4B and D, a comparable pattern was observed for both E1-S and DHEA-S. Serum DHEA-S AUC ₀ _ _24m was measured at 8.35±2.22 ng-h/ml in the placebo group and 13.3±3.16 ng-h/ml in the 0.5% DHEA group (NS) . With the two higher doses of DHEA, AUC ₀ _ _24m values were measured at 16.5±2.71 ng-h/mL (NS) and 19.3±3.59 ng-h/mL (p<0.05), respectively (Fig. 4D, Table 1). These DHEA-S values at all DHEA doses remain below the serum DHEA-S levels observed in premenopausal women, which show a mean of 1.27 ± 0.62 ng-h/mL (7C).

As illustrated in FIG. 5V, AUC values ₀ . _{The 24m} serum 4-dione after DHEA administration on day 7 was measured at 6.34±0.80 and 8.71±0.84 ng-h/mL (NS) in the placebo group and the 0.5% DHEA group, respectively. With these two higher doses of DHEA, the AUC ₀ values. _24m 4-dione slightly increased to 11.1±1.51 (p<0.01) and 11.9±0.81 (p<0.01) ng-h/mL, respectively. As can be seen in 7D and Table. 2, all of these serum 4-dione values remained much lower than the mean serum 4-dione concentrations observed in healthy premenopausal women. Indeed, the highest dose of DHEA resulted in mean serum 4-dione concentrations of 0.50 ± 0.03 ng/ml, while the mean in 30-35 year old menstruating women is 0.96 ± 0.35 ng/ml ( Labrie, Belanger et al., 2006) (Appendix T), thus achieving only 50% of the serum 4-dione levels seen in premenopausal women.

Given the critical role of measurements of serum levels of ADT-G, 3α-diol-3G and 3α-diol-17G (Labrie, Belanger et al., 2006), it is interesting to note that in 5D and Table. 2 serum levels of ADT-G increased from a mean of 6.97±1.20 ng/ml in the placebo group to 19.2±3.99 ng-h/ml in the 0.5% DHEA group (p<0.01) . Values of 19.7±2.48 and 25.7±2.88 ng-h/mL were measured in the 1.0% and 1.8% DHEA groups, respectively (p<0.01 vs placebo groups for both DHEA-treated groups). Similar changes can be seen for the minor androgen metabolites 3α-diol-3G and 3α-diol-17G (6B, 6D, 8B and 8C, Table 1 and Table 2). It is important to point out that, as shown in FIG. 8, even at the highest dose of DHEA used, mean serum levels of ADT-G, 3α-diol-3G, and 3α-diol-17G remained 36, 11, and 6% below the mean serum levels found in premenopausal women.

As shown in Table. 2, the sum of androgen metabolite glucuronides measured over a 24 hour period on day 7 of administration of a 1.3 ml suppository containing 1.8% DHEA (23.4 mg DHEA) is only 28.2 ng/ml, while the mean serum concentration of the same metabolite in 30-35 year old premenopausal women is 42.8 ng/ml (Labrie, Belanger et al., 2006) (Appendix 2). Thus, the highest dose of DHEA used results in only 65.7% of the value corresponding to total androgen metabolites found in healthy menstruating subjects.

- 22 041509 women. On the other hand, doses of 0.5 and 1.0% DHEA result in androgen metabolite sums of 21.02 and 21.53 ng/mL, respectively, corresponding therefore to only 49.0 and 50.2% of the values observed in premenopausal women (Fig. 9). The authors of this invention have previously found that daily oral administration of 100 mg of DHEA leads to 74% of the levels found in premenopausal women (Labrie, Belanger et al., 2007).

The inventors have observed that after oral or subcutaneous administration of DHEA, changes in serum DHEA are approximately 100% overestimated for changes in steroid production reflected by changes in serum ADT-G, 3a-diol-3C, and 3a-diol-17C (Labrie , Belanger et al., 2007). As can be seen in FIG. 9, mean serum DHEA levels varied from 23% of the value observed in premenopausal women in the placebo group to 52%, 71% and 106% in women who received doses of 0.5%, 1.0% and 1.8% DHEA, respectively. The data of Fig. 9 show that changes in serum DHEA after intravaginal DHEA administration are also an overestimate of changes in androgen production, and perhaps even more so in estrogen production, as illustrated by even lower serum E _r S levels (Table 1). Indeed, at the 1.0% dose, serum androgen metabolites increased by 31.6% of the value found in premenopausal women, while serum DHEA increased by 49.1% (55% excess). At the highest dose of DHEA, serum androgen metabolites increased by 47.1%, while serum DHEA increased by 83.5 (over 77%).

Table 1. Areas under the curve (AUC0.244) of DHEA and its eleven metabolites on days 1 and 7 of daily administration of intravaginal suppositories in 40-75 year old postmenopausal women with vaginal atrophy

Group Value DHEA 5-DIOL TESTO DHT E1 E2 Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day I ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml placebo Average SEN 24.47 4.80 24.82 4.77 5.55 0.59 5.60 0.60 2.71" 0.34 2.58 0.33 0.61 0.08 0.58 0.07 305.58 34.56 301.92 33.77 69.51 7.63 66.49 6.90 DHEA 0.5% Average SEM 65.49 7.80 56.17 8.94 10.91 1.03 9.83 1.14 2.79 0.29 2.79 0.30 0.91 0.10 0.93 0.11 336.52 37.96 369.69 48.86 87.79 11.34 96.93 16.46 DHEA 1.0% Average SEM 74.82 6.71 76.22 10.28 12.09 1.66 13.84 1.87 3.79 0.70 4.54 0.91 1.11 0.23 1.31 0.26 418.08 70.91 578.59 84.90 101.57 22.97 144.34 31.47 DHEA 1.8% Average SEM 123.52 9.43 114.30 9.96 18.98 1.05 21.04 1.66 5.13 0.72 5.97 0.69 1.62 0.19 1.93 0.23 433.74 37.68 600.93 68.35 89.76 11.65 136.28 20.27 Group El-S DHEA-S 4-D1ONE ADT-G 3a-DIOL-3G 3a-DIOL-17G Day 1 ng.h/ml Day 7 pg.h/ml Day 1 mcg.h/ml Day 7 mcg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml placebo 3.15 0.62 2.93 0.47 8.71 2.41 8.35 2.22 6.23 0.71 6.34 0.80 176.53 30.86 167.39 28.87 12.00 0.00 12.00 0.00 12.53 0.53 12.20 0.20 DHEA 0.5% 3.19 0.60 3.24 0.63 13.59 3.42 13.29 3.16 9.03 0.98 8.71 0.84 474.10 126.99 461.15 95.77 16.01 2.02 16.73 1.97 24.68 4.70 26.74 5.02 DHEA 1.0% 3.14 0.44 4.37 0.60 14.42 3.07 16.49 2.71 10.28 1.35 11.06 1.51 417.73 66.09 471.54 59.54 17.12 2.28 20.14 3.26 20.88 4.42 24.94 4.75 DHEA 1.8% 4.23 0.76 5.93 1,I 14.99 2.62 19.33 3.59 10.61 0.63 11.94 0.81 510.77 52.78 617.73 69.01 20.36 2.31 26.02 3.38 22.00 2.68 32.23 4.35

a: Single patient data excluded.

Table 2. Mean levels of serum DHEA steroids and eleven of its metabolites on days 1 and 7 of daily administration of intravaginal suppositories in 40-75 year old postmenopausal women with vaginal atrophy

Group Value DHEA 5-DIOL TESTO DHT E1 E2 Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml placebo Average 1.02 1.03 0.23 0.23 0.1 g 0.11 0.026 0.024 12.73 12.58 2.90 2.77 SEM 0.20 0.20 0.02 0.02 0.01 0.01 0.003 0.003 1.44 1.41 0.32 0.29 DHEA Average 2.73 2.34 0.45 0.41 0.12 0.12 0.038 0.039 14.02 15.40 3.66 4.04 0.5% SEM 0.33 0.37 0.04 0.05 0.01 0.01 0.004 0.004 1.58 2.04 0.47 0.69 DHEA Average 3.12 3.18 0.50 0.58 0.16 0.19 0.046 0.055 17.42 24.11 4.23 6.01 1.0% SEM 0.28 0.43 0.07 0.08 0.03 0.04 0.010 0.011 2.95 3.54 0.96 1.31 DHEA Average 5.15 4.76 0.79 0.88 0.21 0.25 0.068 0.081 18.07 25.04 3.74 5.68 1.8% SEM 0.39 0.42 0.04 0.07 0.03 0.03 0.008 0.010 1.57 2.85 0.49 0.84

-23 041509

30-35 year old premenopausal women (n=7) Mean SD Median (min,max) 4.47 2.19 4.14 1.53-9.14 (1.41-10.37) 0.49 0.20 0.44 0.25-0.84 (0.25-0.96) 0.18 0.07 0.17 0.06-0.31 (0.09-0.32) 0.07 0.03 0.07 0.03-0.14 (0.03-0.17) 53.96 23.28 49.47 23.74-87.46 (79.23-123.50) 82.05 42.19 71.38 22.0-159.97 (17.71-181.14) Group Value El-S DHEA-S 4-DIONE ADT-G 3a-DIOL-3G 3a-DIOL-17G Day 1 ng.h/ml Day 7 pg.h/ml Day 1 mcg.h/ml Day 7 mcg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml Day 1 ng.h/ml Day 7 pg.h/ml placebo Average SEM 0.13 0.03 0.12 0.02 0.36 0.10 0.35 0.09 0.26 0.03 0.26 0.03 7.66 1.29 6.97 1.20 0.50 0.00 0.50 0.00 0.52 0.02 0.51 0.01 DHEA 0.5% Average SEM 0.13 0.02 0.13 0.03 0.57 0.14 0.55 0.13 0.38 0.04 0.36 0.03 19.75 5.29 19.21 3.99 0.67 0.08 0.70 0.08 1.03 0.20 1.11 0.21 DHEA 1.0% Average SEM 0.13 0.02 0.18 0.03 0.60 0.13 0.69 0.11 0.43 0.06 0.46 0.06 17.41 2.76 19.65 2.48 0.71 0.10 0.84 0.14 0.87 0.18 1.04 0.20 DHEA 1.8% Average SEM 0.18 0.03 0.25 0.05 0.62 0.11 0.81 0.15 0.44 0.03 0.50 0.03 21.28 2.20 25.74 2.80 0.85 0.10 1.08 0.14 0.92 0.11 1.34 0.18 30-35 year old premenopausal women (n-7) Mean SD Median (min.- Max) 1.19 0.93 0.87 0.31-3.50 (0.21-4.40) 1.27 0.62 1.04 0.56-2.65 (0.45-2.71) 0.96 0.35 0.92 0.45-1.64 (0.31-1.77) 40.21 29.31 31.62 12.17-118.2 (6.86-132.6) 1.21 0.83 1.06 0.25-7.78 (0.25-4.33) 1.43 0.93 1.35 0.25-2.56 (0.25-5.71)

a: Single patient data excluded.

These values were obtained by dividing the AUC0.244 values measured on days 1 and 7 by 24, thereby obtaining the mean serum concentration of each steroid over a 24 hour period. Serum steroid concentrations measured in 30-35 year old premenopausal women are added as a reference. (Labrie, Belanger et al., 2006).

However, it should be mentioned that, as shown in Table. 3 observed a strong trend towards lower pre-treatment values for many steroids in the placebo group. This applies in particular to the low values in the placebo group for DHEA, DHEA-S, 4-dione, Testo (testosterone), DHT, E ₂ , ADT-G and Za-diol-17C. remain within or well below the values found in healthy premenopausal women after administration of doses of 0.5 and 1.0% DHEA, no attempt has been made to correct this apparent deviation. It is interesting to mention that the average 24-hour serum levels of all steroids measured on day 7 of daily administration of the 0.5% DHEA suppository correspond almost exactly to the values measured in healthy 5 5-65-year-old women, while the suppository with 1.0 % DHEA results in values within the range observed in 55-65 year old healthy women (Labrie, Belanger et al., 2006).

Since androgen metabolites are the most reliable criterion for the conversion of exogenous DHEA to active androgens, the results presented show that even the highest dose of DHEA used in this invention satisfies FDA requirements for serum steroid levels that remain within the normal range found in healthy premenopausal women. women.

-24041509

Table 3. Baseline serum steroid levels on days 1 and 7 of daily intravaginal escalating doses of DHEA expressed in ng/mL, except for E ₁ and E2 (pg/mL) and DHEA-S (µg/mL)

Steroid placebo DHEA 0.5% DHEA 1.0% DHEA 1.8% DHEA Day 1 0.72 + 0.14 1.09 + 0.24 0.94 + 0.19 0.99 + 0.15 Day 7 0.69 + 0.14 1.29±0.26 1.43±0.19 1.83±0.13 5-diol Day 1 0.22 + 0.02 0.26±0.03 0.26±0.05 0.25±0.02 Day 7 0.22 + 0.02 0.31+0.05 0.36±0.05 0.46 ± 0.04 DHEA-S Day 1 0.372 + 0.102 0.543 + 0.157 0.572±0.144 0.447 ± 0.094 Day 7 0.368 + 0.100 0.592 + 0.160 0.717±0.125 0.805±0.143 4-dion Day 1 0.18 + 0.02 0.21+0.03 0.23±0.04 0.22 ± 0.03 Day 7 0.16 + 0.02 0.25 + 0.03 0.34±0.06 0.38±0.03 Testo Day 1 0.10 + 0.01 0.09 + 0.01 0.12±0.03 0.15±0.03 Day 7 0.09 + 0.01 0.10+0.01 0.18±0.03 0.23 ± 0.03 DHT Day 1 0.024 ± 0.003 0.026 ± 0.003 0.037±0.010 0.029 + 0.002 Day 7 0.023 + 0.002 0.035 ± 0.004 0.047±0.010 0.062 ± 0.006 Day 1 11.98+1.65 11.83 + 1.28 14.72±2.79 13.59± 1.88 Day 7 11.71+1.19 13.53+1.66 22.15±3.21 23.77 + 3.35 E ₂ Day 1 3.00 + 0.44 3.13 + 0.37 4.30±1.38 3.42±0.62 Day 7 2.75 + 0.28 3.94 + 0.65 5.98±1.26 6.00 + 1.10 EIS Day 1 0.137 + 0.024 0.133 + 0.029 0.117 ± 0.016 0.164 + 0.033 Day 7 0.143 + 0.025 0.151 + 0.034 0.203±0.016 0.259 ± 0.049 ADT-G Day 1 7.42+1.48 13.65 + 3.71 11.49 + 2.10 9.44+1.23 Day 7 6.72 + 1.17 17.02 + 4.39 16.34 + 2.30 19.26±1.96 Za-diol- Day 1 0.50 ^a 0.61+0.07 0.71 +0.14 0.58±0.05 3G Day 7 0.50 ^a 0.75 + 0.11 0.96±0.25 0.96 + 0.15 Za-diol- Day 1 0.50 ^a 0.84 + 0.16 0.85±0.22 0.65±0.06 17G Day 7 0.50 ^a 0.95 + 0.16 1.18±0.30 1.27±0.19

a: Steroid levels are below the limit of quantitation for all subjects (limit of quantitation=0.50 ng/mL).

After just one week of daily administration of DHEA suppositories, the maturity index increased by 107% (p<0.01), 75% (p<0.05) and 150% (p<0.1) in groups 0.5, 1 .0 and 1.8% DHEA, respectively (FIG. 10A). 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), respectively, in groups 0.5, 1.0 and 1.8% DHEA (FIG. 10B). In the placebo group, no change in vaginal pH was observed.

Example 2

Bioavailability and metabolism of oral and subcutaneous dehydroepiandrosterone in postmenopausal women.

1. Introduction.

Humans, along with other primates, are unique among animal species in that they have adrenal glands that secrete large amounts of inactive steroid precursors DHEA, and in particular DHEA-S, which are converted to active androgens and/or estrogens in peripheral tissues. (Labrie, 1991; Labrie, Belanger et al., 1995; Labrie, Luu-The et al., 1997; Labrie, Simard et al., 1996; Labrie, Luu-The et al., 2005; Labrie, Poulin et al. ., 2006 and Simpson 2000). Indeed, plasma levels of DHEA-S in adult males and females are 100-500 times higher than testosterone levels and 1000-10000 times higher than estradiol levels, providing a large supply of substrate for conversion to androgens and/or or estrogens in peripheral intracrine tissues that have the enzymatic machinery necessary to convert DHEA to active sex steroid hormones (Labrie 1991 and Labrie, Luu-The et al., 2005). Indeed, the term intracrinology was first coined in 1988 (Labrie, Belanger et al., 1988) to describe the synthesis of active steroids produced in the same cells in which they exert their action, with no or minimal release into the extracellular space. and general blood flow before their inactivation (Labrie, 1991).

The marked decrease in adrenal DHEA-S production during aging (Belanger et al., 1994; Vermeulen and Verdonck, 1976; and Migeon et al., 1957) leads to a dramatic decrease in androgen and estrogen production in peripheral target tissues, a situation potentially associations

- 25 041509 associated with age-related diseases such as insulin resistance (Schriock et al., 1988 and Coleman et al., 1982) and obesity (Nestler et al., 1988; MacEwen and Kurman, 1991, and Tchernof et al., 1995). In addition, much attention has been paid to the benefits of DHEA administered to postmenopausal women, especially on bone, skin, vagina, glucose and insulin metabolism, fat mass, and post-oral well-being (Villareal and Holloszy, 2004; Baulieu et al., 2000; Morales , et al., 1994; and Kawano et al., 2003) and subcutaneous (Diamond et al., 1996 and Labrie Diamond et al., 1997) administration. Thus, it has become particularly important to gain more accurate knowledge of the bioavailability, pharmacokinetics and metabolism of DHEA after these two routes of administration.

Since the present inventors have already shown, using a pharmacological dose of transdermal DHEA for 2 weeks, that measurements of serum testosterone (dough) and estradiol (E2) levels do not provide a reliable estimate of the true intracellular pool of androgens and estrogens (Labrie, Belanger et al. , 1997; Labrie, Belanger et al., 2006 and Labrie, Belanger, et al, 2007b), the authors of this invention compared the serum levels of DHEA and nine steroids known to be most closely associated with active androgens and estrogens and their metabolites . A detailed analysis of 24-hour changes in serum steroid levels was performed on the first day and after 2 weeks of daily administration of DHEA by the oral route, as well as transdermally using DHEA cream or gel.

2. Subjects and methods.

Thirty-six healthy 60-70 year old postmenopausal women participated in this study after IRB approval and the provision of their written informed consent. Body weight was within ±20% of normal body weight according to the Metropolitan Life Tables.

None of the subjects suffered from a significant metabolic or endocrine disorder, coronary disease, or hypertension. No woman was treated with androgens or anabolic steroids within 6 months prior to the screening visit. All participants had a medical history, a complete physical examination, and a serum biochemical profile including lipids, CBC, urinalysis, and detailed serum hormone determinations during the screening phase of this protocol.

3. Research program, treatment and measurements.

This study was a randomized, open-label study with 12 subjects per study arm. After obtaining written informed consent and determining that the women were eligible for the study, each subject was randomized to receive DHEA via cream, gel, or oral. Daily, before breakfast, for 14 days, subjects received, at this research clinic, either 4 g of 10% DHEA cream applied to a total area of 30 cm x 30 cm of the thigh or two 50 mg DHEA capsules orally before breakfast.

Blood sampling was performed at 08:00-09:00 h at screening and before DHEA administration, on the first day of dosing, and on days 2, 4, 7, 10, and 14. On days 1 and 14, blood samples received at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 12 and 24 hours after DHEA administration.

4. Analysis of serum steroids.

DHEA, DHEA-S, androst-5-en-3b,17b-diol (5-diol), testosterone, androstenedione (4-dione), 17βestradiol (E ₂ ), estrone (E1), estrone sulfate (E1-S) , androsterone glucuronide (ADT-G) and androstan-3b,17b-diol glucuronide (3α-diol-G) were measured by gas chromatography/mass spectrometry (DHEA, 5diol, 4-dione, testosterone, E1 and E2) using electron ionization (electron impact) or chemical ionization and liquid chromatography/tandem mass spectrometry using turboionspray (DHEA-S, E1-S, ADT-G and 3α-diol-G) as described (Labrie, Belanger et al., 2006; Labrie, Belanger, et al., 2007b and Swanson et al., 2007).

5. Calculations and statistical analysis.

On days 1 and 14, the area under the curve of serum concentration of each steroid was measured between 0 h and 24 h (AUC _o _ ₂₄₄ ). These areas under the curves were calculated in a linear trapezoidal fashion (model independent). The relative bioavailability of DHEA gel, DHEA cream and DHEA capsules was calculated based on the mean difference in log-transformed AUC values. All calculations were performed using the SAS program (SAS Institute, Cary, NQ USA).

6. Results.

Oral administration of two 50 mg DHEA capsules resulted in an increase in serum DHEA from 2.3±0.3 ng/mL to a maximum of 15.6±2.5 ng/mL at 1 hour, with a progressive decrease thereafter to 5.7±0. .5 ng/ml at 6 h followed by a plateau at 24 h (FIG. 11A). When applying 4 g of 10% DHEA gel or cream to a 30 x 30 cm area of the skin of the thighs, serum DHEA levels began to increase only at 12 h, reaching values of 8.2 ± 2.0 and 8.0 ± 1.2 nmol/l, respectively, at 24 h (FIG. 11A). There was no significant difference between cream or gel in serum DHEA levels at any of the time intervals examined up to 24 hours after the first application of this precursor steroid to the skin.

When measuring serum 5-diol after oral administration of DHEA, the concentration of 5-diol

- 26 041509 increased from a pre-treatment concentration of 0.31±0.03 ng/ml to a maximum of 1.19±0.13 ng/ml at 1 hour, followed by a progressive decrease thereafter reaching 0.79±0.05 ng /ml at 24 h (Fig. 11B). In the same figure, it can be seen that serum levels of 5-diol increased much more slowly after administration of transdermal DHEA via cream or gel, achieving first statistically different values of 1.00±0.14 ng/mL for cream and 0.72±0 .14 ng/mL for gel at 24 h.

Following oral DHEA, serum 4-dione increased from 0.6 ± 0.1 ng/mL to a maximum of 9.5 ± 2.2 ng/mL at 1 h followed by a rapid decrease to values that remained at a plateau of approximately 1 .2 ng/ml between 8 and 24 h (Fig. 12A). On the other hand, after administration of DHEA via cream or gel, the first significant increase in serum 4-dione was observed only at 24 h at values of 0.9±0.1 and 0.8±0.1 ng/mL for cream and gel, respectively. .

A comparable picture was observed for serum testosterone. Indeed, after oral administration of two 50 mg DHEA capsules, serum testosterone increased from 0.38 ± 0.03 ng/mL to a maximum value of 0.79 ± 0.14 ng/mL at 1 hour. This increase was followed by a rapid decrease to 0.30±0.08 ng/ml at 6 h followed by a plateau thereafter until 24 h (Fig. 12B). When applying DHEA in the form of a cream or gel, the first increase was observed at 24 h at a value of approximately 0.45 ng/ml. As can be seen in FIG. 13A and B, the first oral or transdermal administration of DHEA had no statistically significant effect on serum E1 or _E2 levels during the first 24 hours.

On the other hand, serum DHEA-S had a pattern, although slightly delayed, comparable to DHEA and 5-diol after oral administration of two 50 mg capsules (FIG. 14A). Thus, serum DHEA-S increased from 0.4±0.1 µg/mL to 7.7±1.0 µg/mL at 1 h and to a maximum of 8.4±0.6 µg/mL at 2 h with a progressive decrease to 2.7±0.3 μg/ml at 24 hours. No significant change in serum DHEA-S was observed during the first 24 hours after administration of DHEA cream or gel. On the other hand, serum E1-S did not change significantly during the first 24 hours after the first injection.

Serum ADT-G, the major androgen metabolite, increased from 14±3 ng/mL to 760±150 ng/mL at 1 h and 790±140 ng/mL at 2 h followed by a progressive decrease to 92±5 ng/mL at 12 h. h and up to 70±5 ng/ml at 24 h (Fig. 15A). On the other hand, serum 3α-diol-G increased from 2.2±0.5 ng/ml to 14.5±2.0 ng/ml at 2 h (FIG. 15B). However, the decrease observed thereafter for 3α-diol-G was much slower than the decrease in ADT-G, with a decrease of only about 40% observed between 2 and 24 hours after oral administration of DHEA. After applying 4 g of 10% DHEA to the skin, there was no significant change in serum ADT-G or 3α-diol-G up to 24 h (Fig. 15B).

By repeating measurements of the same kinetic parameters on day 14 of daily dosing, it could be seen that administration of two 50 mg DHEA capsules resulted, from a pre-dose value of 4.2 ± 0.4 ng/mL, to a maximum concentration of 14. 8±4.4 ng/ml DHEA at 1 h followed by a progressive decrease thereafter to 4.5±0.4 ng/ml at 24 h (FIG. 16A). On the other hand, when DHEA was administered via cream or gel, no significant change was observed during the 24 hour period, and serum DHEA remained between 10 and 15 ng/mL after cream application and between 7 and 11 ng/mL after gel application.

Similarly, when measuring serum 5-diol on the 14th day of treatment, the serum concentration of this steroid increased from 0.46 ± 0.04 to 1.37 ± 0.21 ng/ml at 1 h with a slow decrease thereafter until reaching 0.64±0.06 ng/ml at 24 h (Fig. 16B). As observed for DHEA, serum 5-diol remained nearly constant over a 24 hour period at approximately 1.5-1.9 ng/mL after cream application and 1.0-1.3 ng/mL after gel application.

When measuring serum 4-dione on the 14th day of dosing, the serum concentration of this steroid increased from 1.3 ± 0.2 ng/ml to a maximum value of 9.8 ± 1.7 ng / ml at 1 hour, followed by a rapid decrease to 1.5±0.1 ng/ml at 6 hours with a value of 1.2±0.1 ng/ml measured at 24 hours (FIG. 17A). Following application of DHEA to the skin as a cream or gel, there was a non-significant increase in serum 4-dione to approximately 2.5 ng/mL at 2 h, with values thereafter remaining flat at 1.0-1.6 ng/mL to 24 hours (FIG. 17A).

Serum testosterone increased on day 14 of dosing after oral administration of 100 mg DHEA from 0.31±0.04 ng/mL to a maximum value of 0.83±0.11 ng/mL at 1 hour followed by a progressive decrease to a value of 0 .37±0.04 ng/mL at 24 h (Fig. 17B). Following application of DHEA cream or gel, serum testosterone levels remained unchanged over a 24-hour period at approximately 0.3 ng/mL and did not differ significantly from pre-treatment values. As observed on the first day, there was no significant change in serum levels of E1 (FIG. 18A) or E ₂ (FIG. 18B) during 24 hours after 14 days of oral or transdermal DHEA administration.

- 27 041509

Serum DHEA-S increased from pre-dose levels of 1.95±0.15 µg/mL to 8.3±0.4 µg/mL at 1 hour followed by a progressive decrease to 2.6±0.3 µg/mL at 24 hours (FIG. 19A). No significant change in serum DHEA-S was observed after application of DHEA to the skin. On the other hand, serum E ₁ -S did not change during the 24 hours after 14 days of oral or transdermal administration of DHEA.

At an initial higher level on day 14 than day 1, serum ADT-G increased rapidly from 66 ± 1 to 996 ± 105 ng/mL at 1 h, with a rapid decrease thereafter to 116 ng/mL at 12 h and 91 ± 15 ng/ml at 24 h (FIG. 20A). After application of DHEA to the skin, no significant change in serum levels of ADT-G was observed. On the other hand, serum 3α-diol-G increased from 12±2.5 ng/mL to 29.4±5.5 ng/mL at 2 h with a slow decrease thereafter until reaching 13±3.0 ng/mL at 24 hours after 14 days of oral administration of 100 mg DHEA. After transdermal administration of DHEA, no significant change in serum 3α-diol-G was observed (FIG. 20B).

Then, in order to obtain a more accurate criterion for the accumulation of DHEA and its metabolites, the authors of this invention compared the areas under the curves of serum steroid concentrations (AUCo- ₂₄₄ values) measured on the 1st and 14th days of dosing. As can be predicted from FIG. 11, 12, 13, 14, 15, 16, 17, 18, 19 and Figs. 20, these AUC _o _ ₂₄₄ values of all steroids, with the exception of estrogen metabolites (E ₁ -S) and androgens (ADT-G and 3α-diol-G), due to some accumulation of these steroids, are similar in the 1st and 14- days of oral administration of DHEA (Table 4). On the other hand, after transdermal administration, due to the slower absorption of DHEA after administration as a cream or gel, 155 and 86% higher AUC _o _ ₂₄₄ DHEA values are observed on the 14th day compared to the first day of dosing, respectively. Higher values are also observed for all other steroids, except for the values for E ₁ , E2 and testosterone, which were not detected.

Table 4. AUC _o _ ₂₄₄ values measured on days 1 and 14 of dosing and their ratio

Steroid DHEA (ng.h/ml) 5-diol (ng.h/ml) 4-dione (ng.h/ml) Testosterone (ng.h/ml) E1 (pg.h/ml) E2 (pg.h/ml) DHEA-S (µg.h/ml) E1-S (pg.h/ml) ADT-G (ng.h/ml) Za-diolG (ng.h/ml) 2 capsules 50 mg 1st day of dosing 153 (19) 19.0 (20) 40.2 (39) 9.47 (31) 745 (30) 136(20) 108 (20) 4.81 (39) 4112(24) 259 (44) 14th day of dosing 144 (26) 20.4 (25) 43.6 (27) 9.72 (23) 910 (23) 165 (25) 95.0(16) 7.44 (36) 5607(28) 453 (41) 14th day/ 1st day 0.94 1.07 1.08 1.03 1.22 1.21 0.88 1.55 1.36 1.75

g 10%

1st day of dosing 107 (33) 13.7(31) 12.3 (43) 8.35 (16) 680 (48) 147 (50) 10.7(45) 4.83 (58) 404 (62) 50.6 (93) 14th day of dosing 273 (36) 39.7 (31) 22.8 (33) 8.77(16) 847 (22) 175 (27) 19.9(34) 7.96 (39) 977 (66) 114(104) 14th day/ 1st day 2.55 2.90 1.85 1.00 1.24 1.19 1.86 1.65 2.42 2.25 4 g 10% g 1st day of dosing spruce 101 (49) 10.3 (55) 13.3 (45) 8.76(16) 620 (31) 214(137) 11.2 (35) 5.53 (84) 254 (30) 38.3 (86) 14th day of dosing 188 (30) 27.2 (32) 21.3 (51) 8.04 (22) 785 (40) 152 (24) 18.6(34) 9.11(106) 455 (23) 60.3 (85) 14th day/ 1st day 1.86 2.64 1.60 0.96 1.27 0.71 1.66 1.65 1.79 1.57

The values in parentheses indicate the percentage coefficient of variation. DHEA was administered orally (2 x 50 mg capsules) or by skin application of 4 g of 10% DHEA cream or gel.

- 28 041509

As can be clearly seen in Table 5 and FIG. 21, there was no significant change in AUC0-244 values of serum E _and E ₂ or testosterone measured on day 14 of dosing compared to pre-dose levels. However, significant increases were observed for the other two steroids. So, after daily oral administration of 100 mg DHEA for 2 weeks, the area under the curve of DHEA concentration, measured during 24 hours after administration of this steroid, increased by 167% relative to the pre-treatment value, while for 5-diol, 4-dione, DHEA -S, E _r S, ADT-G, and Za-diol-G showed respective increases of 138%, 238%, 873%, 60%, 1820%, and 874%.

Table 5. AUC0-244 values of DHEA and its metabolites before treatment and on day 14

Steroid DHEA (ng.h/ml) 5-diol (ng.h/ml) 4-dione (ng.h/ml) Testosterone (ng.h/ml) E1 (pg.h/ml) E2 (pg.h/ml) DHEA-S (µg.h/ml) E1-S (pg.h/ml) ADT-G (ng.h/ml) Za-diolG (ng.h/ml) Background (before processing) 53.9 8.56 12.9 8.72 717 135 9.76 4.64 292 46.6 (A) 2 x 50 mg capsules 14th day of dosing 144 20.4 43.6 9.72 910 165 95.0 7.44 5607 453 14th day / 1st day 2.67 2.38 3.38 1,P 1.27 1.22 9.73 1.60 19.2 9.74 (C) 4 g 10% cream 14th day of dosing 5.06 4.63 1.77 1.01 1.18 1.30 2.04 1.72 3.34 2.45 14th day/ 1st day 1.86 2.64 1.60 0.96 1.27 0.71 1.66 1.65 1.79 1.57 (C) 4 g 10% gel 14th day of dosing 188 27.2 21.3 8.04 785 152 18.6 9.11 455 60.3 14th day/ 1st day 3.49 3.18 1.65 092 1.09 1.13 1.91 1.96 1.56 1.30

DHEA was administered orally or transdermally via cream or gel. Background values for AUCo-244 were calculated by multiplying pretreatment serum steroid levels including screening by 24 hours.

With the exception of DHEA and 5-diol, lower increases were observed after administration of DHEA cream or gel. Indeed, after application of the DHEA cream, the AUC0.244 value of DHEA-S only increased by 104%, while the AUC0-244 values of 4-dione, Ei-S, ADT-G and Za-diol-G increased by 77, 72 , 234 and 145% higher than control, respectively. On the other hand, the AUC values for DHEA and 5diol increased by 406% and 363%, respectively (Table 5, Figure 21). Comparable but slightly lower increases were observed with DHEA gel, where the AUC0-244 values of serum 4-dione, DHEA-S, Ei-S, ADT-G and Za-diol-G were increased by 65, 91, 96, 56 and 30 % higher than control, while the values of AuCo.24h for DHEA and 5-diol increased by 249 and 238%, respectively.

Recent discoveries by the authors of this invention (Labrie, Belanger et al., 2007b) have shown that the changes in serum DHEA observed after the administration of exogenous DHEA are at least 100% overestimated of the true changes in the formation of sex steroid hormones. In support of these data, FIG. 21 shows that after administration of DHEA via cream or gel, changes in serum DHEA are a marked overestimation of changes in serum levels of all measured steroids except for 5-diol, a direct metabolite of DHEA. For DHEA cream, changes in AUC0-244 values for serum 4-dione, DHEA-S, E _r S, ADT-G, and Za-diol-G are only 77, 104, 72, 234, and 145% compared to a 406% increase relative to pre-treatment levels observed for serum DHEA.

For androgens, it is now well established that uriding glucuronosyltransferase 2B7 (UGT 2B7),

-29 041509

UGT 2B15 and UGT 2B17 are the three enzymes responsible for the glucuronidation of all androgens and their metabolites in humans (Belanger et al., 2003). This recent completion of the identification and characterization of all UDT-glucuronosyltransferases makes possible the use of glucuronide derivatives of androgens as markers of total androgenic activity in both women and men (Labrie, Belanger et al., 2006; Labrie, Belanger, et al, 2007b and Swanson et al., 2007). Thus, since all androgens are metabolized to ADT-G and 3α-diol-G, this percent efficiency estimate of percutaneous DHEA in converting to active androgens is therefore estimated to be 52% when the changes in ADT-G and 3α-diol-G are added. (weighted value 211% vs 406% DHEA change). Similarly, after administration of the DHEA gel, the 249% increase in serum DHEA translates into increases of only 65%, 91%, 96%, 56%, and 30% in AUC ₀ values. ₂ 4 _M serum 4-dione, DHEA-S, E1-S1, ADT-G and 3α-diol-G, respectively.

Since high levels of glucuronidation in the gut and liver explain the high serum levels of ADT-G and 3α-diol-G (Belanger et al., 2003) after oral DHEA, the relatively small increase in serum E1-S (60%) compared to the increase in 167 The % serum DHEA after oral DHEA administration indicates a 36% relative efficiency in conversion to estrogens. As shown previously (Labrie, Belanger et al., 1997; Labrie, Belanger et al., 2006; Labrie, Belanger, et al., 2007b), these results indicate that DHEA administered to postmenopausal women is predominantly converted to androgens. rather than estrogen. Discussion

The results presented clearly show that during prolonged treatment with DHEA cream or gel, the concentration of all steroids quickly reaches a plateau with no detectable change in the serum concentration of any of the steroids measured during daily application of DHEA to the skin. Thus, from 24 hours after the first transdermal administration of DHEA, the concentration of all steroids remains at the same level, indicating that daily application of DHEA to the skin maintains constant serum levels of DHEA and all its metabolites. In postmenopausal women, the circadian variation in serum DHEA is already known to be relatively small compared to the situation in normal menstruating premenopausal women (Lui and Laughlin, 1990).

The presented results also show that after daily oral administration of DHEA, there is no significant accumulation of DHEA or its metabolites. In addition, the metabolism of DHEA after oral or transdermal administration is quantitatively similar, with qualitative differences attributed to enterohepatic metabolism after oral administration.

Higher AUC ₀ values. _24m serum DHEA-S, ADT-G and 3α-diol-G combined with lower AUC ₀ values. _24m DHEA and 5-diol after oral as opposed to transdermal administration show that gastrointestinal metabolism and/or liver first pass not only results in higher levels of DHEA to DHEA-S conversion due to DHEA sulfotransferase activity (Luu -The et al., 1995), but also to increased DHEA metabolism to androgens and their inactivation due to hepatic glucuronosyltransferase activity (Belanger et al., 2003; Turgeon et al., 2001 and Hum et al., 1999). Indeed, as shown in Table. 1, exposure to DHEA at 144 ng-h/ml (AUC _{0 .24m} ) on day 14 of oral administration of 100 mg DHEA results in AUC ₀ values. _24m 5607 ng-h/ml and 453 ng-h/ml for ADT-G and 3α-diol-G, respectively. On the other hand, after transdermal administration of 10% DHEA cream, the AUC values for DHEA, ADT-G and 3α-diol-G are 273, 977 and 114 ng-h/ml, respectively. Thus, after oral administration, 1 ng-h/mL DHEA corresponds to an AUC value of 42.1 ng-h/mL for the combination of two androgen metabolites (ADT-G+3α-diol-G), whereas after cream application, exposure 1 ng-h/ml DHEA corresponds to 4.0 ng-h/ml for this sum of two androgen metabolites. These data indicate that oral administration of DHEA results in approximately 10 times higher conversion of DHEA to ADT-G and 3α-diol-G than transdermal administration, at least at the doses used. When performing the same calculations for the results obtained after the introduction of DHEA via gel, exposure to 1 ng-h/ml DHEA is accompanied by the value of AUC ₀ . _24m 2.7 ng-h/ml for ADT-G+3α-diolG, indicating an even higher ratio between oral and transdermal DHEA.

As shown in Table. 4 while the AUC value _{is 0} . _24m DHEA 1 ng-h/ml results in an AUC ₀ value. _24m 660 ng-h/ml for DHEA-S after oral administration of DHEA, corresponding values of 73 and 99 ng-h/l are observed after application of the precursor steroid via cream or gel. Thus, there is a 6.7-9.0 times higher amount of DHEA-S in the circulation after the same exposure to circulating DHEA (serum AUC _0.24m ) after _oral administration compared to transdermal administration of DHEA under the conditions tested. These results show a comparable effect of GI and liver passage of DHEA on serum levels of DHEA-S, ADT-G, and 3α-diol-G.

Although a lower difference can be seen, relatively higher serum 4-dione levels are observed after oral DHEA versus transdermal steroid administration.

- 30 041509 predecessor. Thus, after oral administration of DHEA, an AUC value of _0-244 1 _ng -h/ml leads to an AUC value of _0-244 0.3 ng-h/ml of 4-dione, while values of 0.08 and 0.11 ng-h/ml are observed after administration of DHEA via cream and gel, respectively. As measured in the bloodstream, the conversion of DHEA to 4-dione is therefore 2.70-3.76 times higher after oral administration compared to transdermal administration of DHEA.

The results of the table. 5 show that AUC _0-24m DHEA increased by 167% over control after daily oral administration of 100 mg DHEA compared to baseline levels before treatment, while daily transdermal administration of 4 g of 10% DHEA cream and gel increased serum DHEA levels by 406 and 249%, respectively. Since 400 mg DHEA was applied to the skin compared to 100 mg for the oral route, and assuming linearity, the data presented indicate that the oral route is 2.9 and 4.8 times more effective than the drug used for DHEA cream or gel, respectively.

In one study, also performed in postmenopausal women, oral administration of 150 mg and 300 mg of micronized colloidal milled DHEA resulted in peak serum levels of DHEA-S, DHEA, and testosterone of approximately 1.5, 15, and 2.75 ng/mL post-dose. 300 mg and 10 µg/ml, 12 µg/ml and 1.6 ng/ml after a dose of 150 mg DHEA, respectively (Buster et al., 1992). A study of these early results shows that a 20-fold increase in serum DHEA-S resulted in only a 6.9-fold increase in serum testosterone, while serum DHEA increased 11.6-fold. In addition, correcting measured serum testosterone values to one third to account for two thirds of non-specific binding in radioimmunoassay, these serum testosterone levels remain within physiological levels for up to 12 hours after a 150 mg DHEA dose (Buster et al., 1992).

Similar differences observed between oral and transdermal routes for serum DHEA are observed for 5-diol, which is converted directly from DHEA by 17β hydroxysteroid dehydrogenase (Labrie, Luu-The, et al., 2000). Indeed, the AUC _o _ ₂₄₄ of 5-diol increases by approximately 138% above control after oral administration of 100 mg DHEA, and increases of 363 and 218% are measured after application of 400 mg DHEA cream and gel, respectively.

As mentioned above, humans are unique, with some other primates, in that they have adrenal glands that secrete large amounts of the precursor steroids DHEA and DHEA-S, which are converted to 4-dione and then to active androgens and/or estrogens in peripheral intracrine tissues (Labrie, 1991; Labrie, Belanger, et al., 1995; Labrie et al., 1996; Labrie, Luu-The, et al., 1997; Simpson, 2000; Labrie, Luu-the, et al. , 2005; Labrie, Poulin, et al., 2006 and Labrie, Belanger, et al., 1998). It is also surprising that man, along with the presence of very complex endocrine and paracrine systems, is generously endowed with the formation of sex steroid hormones in peripheral tissues (Labrie, 1991) and (Belanger, et al., 1998). Indeed, while the ovaries and testicles are the only sources of androgens and estrogens in lower mammals, the situation is very different in humans and higher primates, where active sex steroid hormones are synthesized largely or entirely locally in peripheral tissues, thus providing target tissues with controls, which correct the formation and metabolism of sex steroid hormones for local needs.

Adrenal secretion of DHEA and DHEA-S increases during physiological puberty in children aged 6-8 years, and circulating DHEA-S peaks between age 20 and 30 years. Thereafter, serum levels of DHEA and DHEA-S markedly decrease (Belanger et al., 1994). In fact, at age 70 years, serum DHEA-S levels decrease to approximately 20% of their maximum values, while they may have a decrease of 95% at age 85-90 years (Belanger et al., 1994) and (Migeon et al. ., 1957). This 70-95% decrease in adrenal DHEA and DHEA-S production during aging results in a dramatic decrease in androgen and estrogen production in peripheral target tissues (Labrie, Belanger et al., 2006). Such a pronounced decrease in the formation of sex steroid hormones in peripheral tissues may probably be involved in the pathogenesis of a number of diseases associated with aging.

As mentioned earlier, the transformation (transformation) of DHEA and DHEA-S into active androgens and/or estrogens in peripheral target tissues depends on the level of expression of various steroidogenic and metabolic enzymes in each cell type (Labrie, 1991). The elucidation of the structure of most of these tissue-specific genes, which encode the steroidogenic enzymes responsible for the conversion of DHEA and DHEA-S to androgens and/or estrogens, has allowed rapid progress in this area (Labrie, Belanger et al., 1995; Labrie, Luu-The et al. al., 1997; Labrie, Simard et al., 1996; Labrie, Luu-The et al., 2005; Labrie, Poulin et al., Labrie, Luu-The, et al., 2000; Labrie, Sugimoto, et al. ., 1992, Labrie, Simard et al., 1992; Luu-The, et al., 1995; and Labrie, Durocher et al., 1995).

The results showing the presence of relatively high levels of androgen metabolites in healthy women (Labrie, Belanger, et al., 1997; Labrie, Belanger et al., 2006; Labrie, and Belanger, et al., 2007b) strongly suggest that androgens play the main physiological role, but still

- 31 041509 an underestimated role in women. The 44.5% decrease that occurs in serum DHEA from age 20-30 to age 40-50 in women (Belanger et al., 2006) could reasonably explain the bone loss that precedes menopause. Indeed, age-related bone loss (osteoporosis) has been reported to begin in the forties, and changes in bone metabolism have been found well before menopause (Mazess 1982; Riggs, et al., 1981, and Johnston et al., 1985). Consistent with these findings, bone density was lower at all sites tested in women classified as perimenopausal (perimenopausal) (Steinberg, et al., 1989). Consistent with these findings, changes in adrenal androgen precursor secretion precede by 10–20 years the decrease in ovarian estrogen secretion, which abruptly ceases at menopause (Labrie, Belanger, et al., 2006).

It is important to understand that there is not only a 50% decrease in serum DHEA and DHEA-S between age 21 and age 50, but also a similar decrease in serum testosterone (Zumoff et al., 1995). Such data might well suggest that hormone replacement therapy using androgens or their precursor(s) should be started early in menopause to compensate for this early decrease in adrenal androgen precursor secretion and a parallel decrease in serum testosterone (Labrie, 2006).

Active androgens and estrogens synthesized in peripheral tissues exhibit their activity in the cells of their origin, and there is very little diffusion of these active sex steroid hormones, resulting in very low levels in the circulation (blood stream). Indeed, as observed previously (Labrie, Belanger et al., 1997) and confirmed in this invention, the most striking effects of DHEA administration are observed at circulating levels of glucuronide derivatives of DHT metabolites, namely ADT-G and 3α-diol-G, while while there are no significant changes or only minor changes in serum levels of testosterone, E1 or E ₂ . These active steroids are produced locally in peripheral intracrine tissues, which have suitable steroidogenic enzymes for DHT synthesis from adrenal DHEA and DHEA-S, as well as enzymes that convert DHT to the inactive metabolites ADT and 3a-diol, which are further modified by glucuronidation (Belanger et al., 2003).

In one recent study, daily oral administration of 50 mg DHEA had no significant effect on serum testosterone or DHT, while DHEA and ADT-G increased to a similar extent (80-90%) (Arlt et al., 2001). In another study, pre-dose serum levels of DHEA-S in postmenopausal women increased from 0.55 to approximately 1.4 µg/mL (Casson et al., 1998) after oral administration of 25 mg DHEA for 6 months. However, serum levels of DHEA and testosterone measured 23 hours after the last DHEA administration did not change significantly. Another study showed that a daily dose of 50 mg DHEA resulted in serum androgen levels in the premenopausal range (Buster et al., 1992).

The data presented clearly demonstrate that DHEA and DHEA-S are converted in specific peripheral intracrine tissues into active androgens and/or estrogens, which may exhibit their biological actions at the site of their synthesis with no or only little release of active steroids into the bloodstream. Thus, changes in serum levels of testosterone, E1 or _E2 cannot be used as parameters for the conversion of DHEA to androgens or estrogens (Labrie, Belanger et al., 2006). Indeed, active steroids are metabolized in the same cells in which they were synthesized and exert their effects in inactive glucuronidated and sulfated metabolites, which eventually diffuse into the extracellular space and can be measured in the circulation (Labrie, Belanger et al., 2006; Labrie, Belanger et al., 1997 and Labrie, Belanger et al., 2007). The measurement of conjugated androgen metabolites is the only approach that accurately assesses the total pool of androgens in women. It is most likely that a similar situation exists for estrogens, although an accurate assessment of the pharmacokinetics of estrogen metabolism and the identification of their metabolites still needs to be established.

Example 3

Clinical study of ERC-210.

Intravaginal DHEA, physiological treatment of vaginal atrophy.

Objects and methods.

This study is a phase III, prospective (prospective), multicenter, randomized, placebo-controlled, double-blind study with 50 subjects per study arm (200 subjects in total). Thus, two hundred postmenopausal women were randomized to receive daily vaginal ovulation with the following DHEA concentrations: 0.0, 0.25 (3.25 mg DHEA), 0.5 (6.5 mg DHEA), or 1.0% (13 mg DHEA ), administered intravaginally with a topical instrument. This study was divided into two phases, namely, screening followed by a period of treatment (treatment) with a duration of 12 weeks. Inclusion criteria (into the study) were as follows: postmenopausal women who satisfy a or b or c:

- 32 041509

a) absence of menstrual cycles for at least one year; or

b) FSH levels >40 mIU/ml (within 60 days prior to day 1) in women without a menstrual cycle for >6 months but <12 months, or hysterectomized women who were premenopausal at the time of hysterectomy; or

c) there are weeks or more (screening visit) after bilateral oophorectomy.

Women who have themselves experienced at least one moderate-severe symptom from the following symptoms:

vaginal dryness (absent, mild, moderate or severe), vaginal and / or vulvar irritation / itching (absent, mild, moderate or severe), vaginal pain associated with sexual activity (absent, mild, moderate or severe).

Women should identify which symptom is most troubling to them at the start of treatment. The change in this symptom will be observed and will serve to evaluate the effect of the treatment.

Women aged between 40 and 75 years.

Ready to participate and signed informed consent.

Women who have a low maturity index (no more than 5% of superficial cells on a vaginal smear).

Women who have a vaginal pH above 5.

Normal mammography within 9 months of study start.

Normal breast examination.

Normal PAP smear (which includes inflammatory changes) within the last 12 months (Day 1). For women undergoing a hysterectomy, the swab will consist of at least one glass slide.

Absence of previous or current drug addiction or alcoholism.

Body weight within 18.5-35 ideal body weight according to body mass index (BMI) (WHO).

No hepatic or renal disorder or condition known to affect drug or steroid metabolism.

Normal background hematology, clinical chemistry, and urinalysis.

Negative serology for HIV1/HIV2 and hepatitis B and C.

The exclusion criteria were:

undiagnosed atypical genital bleeding, previous diagnosis of cancer other than skin cancer (not melanoma), endometrial hyperplasia on screening biopsy or endometrial cancer, active or history of thromboembolic disease, significant metabolic or endocrine disease, clinically significant gastrointestinal disease liver or gallbladder, recurrent headache (migraine) not controlled by conventional therapy, diabetes mellitus not controlled by conventional therapy, significant complication from previous hormonal therapy, use of estrogen alone as an injectable drug therapy or progestin implant within 3 months before entry into study (screening visit), use of estrogen bolus or injectable progestin drug within 6 months prior to study entry, exposure to oral estrogen, progestin, or exposure to DHEA or intrauterine progestin therapy within eight weeks prior to background evaluations, vaginal hormone products (rings, creams or gels) or transdermal estrogen alone or estrogen/progestin products within 4 weeks prior to background evaluations. Patients may be subjected to washout as follows, but after the required washout period, they must complete the Vaginal Atrophy Questionnaire:

at least an eight-week washout period for prior oral therapy using estrogen, DHEA, and/or progestin;

at least a four-week washout period for previous transdermal hormone therapy;

at least a four week washout period for locally delivered hormone replacement therapy for vaginal dryness;

at least 6 months for prior injectable drug therapy using an estrogen or progestin bolus;

seven weeks or more for previous intrauterine therapy using progestin;

- 33 041509 six months or more for prior progestin implants and estrogen-only injectable drug therapy, prior androgen or anabolic steroid treatment within 3 months prior to screening visit, oral corticosteroid treatment within six weeks of study start, continued use is not allowed corticosteroids (intermittent nasal spray or topical application to the skin, eyes or ears is allowed), heart failure or evidence of coronary heart disease, hypertension equal to or greater than 160/95 mm Hg. column or not controlled by standard therapy, confirmed clinically significant depression or confirmed history of major psychiatric disorder, administration of any investigational drug within 30 days of screening visit, clinically relevant atypical serum biochemistry or hematology, background cervical cytology showing low-grade squamous epithelial lesions ( LGISIL) or worse, smoking more than 10 cigarettes a day, drugs that interfere with estrogen metabolism (eg, ketoconazole, inhibitors of steroid formation or action),

SERM or drug that interacts with steroid receptors, known or palpable presence of uterine fibroids on gynecological examination, bleeding disorder, or anticoagulant drug therapy.

Laboratory tests.

Routine laboratory tests, namely hematology (including CBC and coagulation), blood chemistry, and urinalysis, were performed at all visits. Serum FSH was to be measured only in women who had not had a menstrual cycle for >6 months but <12 months, or who were premenopausal at the time of hysterectomy. Serum levels of steroids DHEA, DHEA-S, androst-5-en-3b,17b-diol (5-diol), dihydrotestosterone (DHT), testosterone (dough), androstenedione (4-dione), estrone (E1), estradiol ( E ₂ ), Ei-S, androsterone glucuronide (ADTG), androstan-3α,17β-diol-3G (3α-diol-3G) and 3α-diol-17G were measured at the Laboratory of Molecular Endocrinology, CHUL Research Center by mass spectrometry as described (Labrie, Belanger et al., 2006; Labrie, Belanger et al., 2007; Labrie, Cusan et al., 2008).

Vaginal pH and vaginal cytology.

For Maturity Index and Papanicolaou (PAP) smear, all specimens were examined by a cytopathologist (Dr. Robert Dube, Department of cytology-pathology, Enfant-Jesus Hospital, Quebec City, Canada), who was not aware of the treatment regimens. A 100 cell count was performed to classify cells as superficial (S), intermediate (I), and parabasal (P) squamous cell types (Meisels 1967; Wied 1993).

Vaginal swabs were obtained by scraping the middle third of the lateral wall of the vagina with the rounded end of an Ayre spatula. This material was then applied to a glass slide and immediately fixed with Spray-Cyte. These samples were sent to the central laboratory to determine the maturity index.

Vaginal pH was measured by applying a pH test strip directly to the side wall of the vagina with tweezers. For a Pap smear, if not performed within the past 12 months, specimens were obtained from the endocervix (cervical canal mucosa), exocervix, and vaginal fornix and immediately fixed with cytospray. These samples were collected with an Ayre spatula.

endometrial biopsy.

Endometrial biopsy was performed at screening and at month 3 at the end of the study. All biopsies were examined by the same central laboratory pathologist (Dr. Robert Dube, Department of cytology-pathology, Enfant-Jesus Hospital, Quebec City, Canada).

Vaginal examination.

At the same time intervals of 2, 4, 8, and 12 weeks, the gynecologist or physician in charge of this study at each site performed a vaginal examination to assess severity (absent, mild, moderate, or severe, analyzed using values of 0, 1 , 2 and 3, respectively) on the main symptoms of vaginal atrophy, namely, vaginal secretions, vaginal color, integrity of the vaginal epithelium, and thickness of the surface of the vaginal epithelium. As can be seen in FIG. 26-29, there was a time-dependent, dose-dependent and statistically significant improvement in all four features of vaginal atrophy. Indeed, the beneficial effects observed by the gynecologist and/or physician are nearly consistent with those reported by women in relation to their most distressing symptoms.

Vaginal examination was performed at screening and then at day 1 and at weeks 2, 4, 8, and 12. Vaginal secretions, vaginal color, vaginal epithelial integrity, and vaginal surface thickness

- 34 041509 epithelium was assessed according to the following severity: absent, mild, moderate or severe. These definitions of severity were as follows.

a) Vaginal secretions.

No atrophy: normal clear secretions marked on the vaginal walls.

Light: surface coverage of secrets, difficulty inserting a mirror.

Moderate: insufficient secretion that does not cover the entire vault of the vagina, lubrication may be required when inserting a speculum to prevent pain.

Severe: no secretion, inflamed vagina with marked ulceration, requires lubrication when inserting a speculum to prevent pain.

b) Integrity of the vaginal epithelium.

Absence of atrophy: normal.

Mild: The vaginal surface bleeds on scraping.

Moderate: The vaginal surface bleeds on light contact.

Severe: The vaginal surface has capillary hemorrhages (petechiae) before contact and bleeds on light contact.

c) The thickness of the vaginal epithelial surface.

Absence of atrophy: wrinkling and elasticity of the vaginal vault.

Mild: Slight folding with some elasticity of the vaginal vault noted.

Moderate: smooth, some elasticity of the vault of the vagina.

Severe: smooth, lacking elasticity, compression of the upper 1/3 of the vagina, or loss of vaginal tone (bladder hernia and rectocele (rectal prolapse)).

d) The color of the vagina.

No atrophy: pink.

Light: lighter in color.

Moderate: paler in color.

Severe: clear, or colorless, or inflamed.

Statistics.

Summary tabulations will be made that represent the series of observations, mean or geometric mean as appropriate, standard deviation, standard error of the mean, 95% two-sided confidence interval (CI), median, minimum and maximum for continuous variables, and count and percent per category for categorical data. Statistical analyzes are performed at a two-sided significance level of 0.05 unless otherwise indicated. These summation categories will primarily consist of dose levels of DHEA treatments, 0% placebo, 0.25%, 0.5%, and 1.0% DHEA.

The primary endpoints for analysis will consist of the following.

A statistically significant improvement in a moderate-severe symptom identified by the female subject as her most distressing symptom. This symptom severity is based on symptoms of increasing severity: none, mild, moderate, or severe. These estimates will be analyzed using the values 0, 1, 2 and 3, respectively; all subjects must have at least one underlying symptom that is classified as 2 or 3. Symptoms of interest are vaginal dryness, vaginal and/or vulvar irritation/itching, and vaginal pain associated with sexual activity.

A statistically significant decrease in the number of parabasal cells and a statistically significant increase in the number of superficial cells. This data is measured as a percentage. The maturity value will also be calculated.

Statistically significant decrease in vaginal pH.

Population analysis.

The Intent-to-Treat (ITT) population will consist of treated subjects with a baseline assessment and at least one post-baseline efficacy assessment. Subjects who received the wrong treatment will be analyzed as randomized. This population analysis should be considered as the primary population analysis. Subjects in this population who are devoid of post-background observations will have this latter value advanced for efficacy analyses.

The Per Protocol Population (PP) consists of a subset of this treated population that completes this study at a time point of 12 weeks without violations of the main protocol that are considered to violate the efficacy data. Major protocol violations will be identified prior to blind study discovery, based on review of data sheets and monitoring of reports of protocol deviations. Subjects in the PP population must receive at least 90% of the required number of study treatment applications in the protocol-specified duration for that subject, based on that subject's diary data. Subjects in the PP population should strictly adhere to the visit window pattern of ±3 days for day 14 and ±7 days for weeks 4, 8, and 12. Subjects who received incorrect treatment but for whom the treatment received may be unambiguous

- 35 041509 tentatively confirmed to be analyzed in the PP population as treated, provided that they do not have other disorders that compromise these data. The PP population will be the maintenance population for efficacy data analysis.

The reliability population will be defined as all subjects who receive administration of any test product (DHEA at any dose or placebo) and who have available reliability information. All reliability data analyzes will be based on this population. The analysis will be based on the actual treatment received.

Evaluation of efficiency.

Efficacy analyzes will be performed primarily on the ITT population and the Per-Protocol population will provide supportive efficacy analyses. The aim of this primary study is to evaluate the dose-response response of vaginal mucosal parameters to local action of DHEA in postmenopausal women suffering from vaginal atrophy, in particular, to determine the minimum dose of DHEA that produces the maximum effect on the vaginal mucosa. These co-primary endpoints for achieving this goal are a decrease in parabasal cells, a decrease in vaginal pH, an increase in superficial cells (together, these endpoints will be referred to as physiological parameters), and the subject's self-reported most distressing symptom, including vaginal dryness, vaginal and/or or vulvar pruritus/irritation and vaginal pain associated with sexual activity (together, these endpoints will be referred to as symptom scores). In addition to these primary endpoints, the maturity value will also be calculated. Subject-reported symptom scores will be as follows: no symptom, mild, moderate, or severe, using analysis values of 0, 1, 2, or 3, respectively. All endpoints must show statistically significant effects relative to placebo, and therefore no statistical adjustment is required for multiple endpoints.

The primary time point for analysis will be the 12-week evaluation, with additional data submissions for 2, 4, and 8 weeks. The change from background to score after background will be used for analysis, as well as the difference from placebo.

Results.

Since parabasal cells are usually the predominant category in the vaginal smear of postmenopausal women suffering from at least one moderate to severe symptom of vaginal atrophy, FIG. 22 and in table. 6 it can be seen that already at 2 weeks of treatment, the lowest dose of DHEA (0.25%) reduced % of parabasal cells by 29.5±0.51% from 56.0 to 26.5%, while reductions of 37.8± 0.46% and 36.6% were observed, respectively, with doses of 0.5 and 1.0% DHEA at the same time interval. At the standard duration of 12 weeks of treatment, reductions of 39.5±0.57% (p<0.000001), 45.6±0.55% (p<0.000001) and 45.2±0.53% (p<0.000001) and 45.2±0.53% (p <0.000001) with DHEA doses of 0.25, 0.5 and 1.0% DHEA, respectively, while no significant effect was observed in the placebo group at any time interval.

While no significant effect was observed at 12 weeks in the placebo group on % change in surface cells (Table 6), increases of 3.96±0.10% (p=0.0002), 6.71± 0.14% (p=0.00001) and 5.92±0.12% (p=0.00001) in the 0.25, 0.5 and 1.0% DHEA groups, respectively. It can also be seen that at a dose of 0.5% DHEA, 48.0%, the maximum effect was achieved at 2 weeks, while at 4 and 8 weeks, the maximum effect of 84.8 and 99.0% was achieved. At a dose of 1.0% DHEA, the maximum effect was achieved already at 2 weeks. Fig. 23 illustrates the absolute values of % surface cells at various DHEA doses and time intervals.

Vaginal pH decreased at 12 weeks by 0.47 ± 0.11 from 6.52 ± 0.13 pH units in the placebo group (Table 6, Fig. 24), while decreases of 1.12 ± 0.11 were observed (р=0.0005) from 6.49±0.12, 1.35±0.3 from 6.56±0.13, 1.35±0.13 from pH units 6.56±0.13 units pH and 1.39±0.14 from 6.34±0.3 pH units in the 0.25, 0.50 and 1.0% DHEA treated groups, respectively (Table 7). In the same table, it can be seen that at a dose of 0.5% DHEA, the maximum effect on pH 70.6 and 94.1% (reduction of 1.36 pH units) was achieved at 2 and 4 weeks of treatment, respectively. On the other hand, the low dose of 0.25% DHEA produced only 83.0% of the maximum effect of the 0.5% DHEA dose at 12 weeks. No significant difference was observed in pH change between 0.50 and 1.0% DHEA doses at 4, 8 and 12 weeks (Table 7). Fig. 24 illustrates absolute pH values at various DHEA doses and time intervals.

All women were required at entry into the study to have one or more of the following symptoms of vaginal atrophy, self-reported as moderate to severe: vaginal dryness, vaginal or vulvar irritation/itching, or vaginal pain with sexual activity. Women's self-identified symptoms reported as absent, moderate, or severe were analyzed using scores of 0, 1, 2, and 3, respectively. As shown in Table. 8, at a 12-week interval, the severity of the most disturbing symptom decreased by 0.67 ± 0.15 in the placebo group, 1.27 ± 0.16 in the 0.25% DHEA group (p = 0.004 vs. placebo), 1.56 ± 0.15 in the group receiving 0.5% DHEA (p<0.0001 vs. placebo) and 1.37±0.14 in the group receiving the higher 1.0%

- 36041509 dose of DHEA (p=0.0008 vs. placebo). Fig. 25 illustrates the degree of involvement of the most disturbing symptom at different doses of DHEA and time intervals. Vaginal dryness, pain associated with sexual activity, and vaginal and/or vulvar irritation/itching were identified in the background as the most disturbing symptom. In the placebo group, vaginal dryness accounted for the % improvement reported by participants.

As shown in FIG. 25, the improvement in this most disturbing symptom was already highly significant different (p=0.004) at the 0.25% DHEA dose. The percentage of women with no change or worsening scores 1 at 12 weeks ranged from 53.5% in the placebo group to 27.5%, 17.8, and 19.6% in the 0.25, 0.5, and 1 groups .0%, respectively (Table 9). Improvements in grade 2 or 3 severity were seen in 21.8% of women treated with placebo, while 50.0%, 53.3% and 47.9% of women who received 0.25%, 0.5% and 1.0% DHEA preparations reported such an important improvement.

Only 4.6% of women showed a reduction from severe to none in the placebo group compared to 7.5%, 20%, and 10.9% in the same DHEA-treated groups.

At the same time intervals of 2, 4, 8, and 12 weeks, the gynecologist or clinician responsible for this clinical study performed a vaginal examination at each point in the study to assess severity (none, mild, moderate, or severe, analyzed using values of 0 , 1, 2, and 3, respectively) on the main features of vaginal atrophy, namely, vaginal secretions, vaginal color, vaginal epithelial integrity, and vaginal epithelial surface thickness. As can be seen in FIG. 26-29 observed time dependent and dose dependent as well as highly statistically significant improvement in all four symptoms of vaginal atrophy. Indeed, the beneficial effects observed by the gynecologist or physician are almost consistent with these self-reported effects on women's most troubling symptoms, as well as effects on vaginal parabasal and superficial cells and pH, which are objective parameters of DHEA action.

Fig. 30 and 31 illustrate 24 hour mean (calculated from AUC _o _ ₂₄₄ values measured on days 1 and 7 of treatment) serum steroid levels of DHEA and eleven of its metabolites taken from a recent study (Labrie, Cusan et al., 2008). It can be seen that only serum DHEA and 5diol (and 4-dione on day 1) increase significantly, but within the range of values found in postmenopausal women (Labrie, Belanger et al., 2006). There is no significant effect on serum estrogens ( _Eb E2 and U), as well as on serum androgens (dough and DHT).

Table 6 Change since day 1 in % of parabasal and superficial cells during topical treatment with increasing doses of DHEA*

2 weeks 4 weeks 8 weeks 12 weeks Parabasal cells 0.0% DHEA + 3.6 ±0.32 + 0.02 ± 0.32 -1.17+0.37 + 1.04 ±0.35 0.25% -29.5±0.51 -38.4±0.51 - 40.3 ± 0.55 -39.5±0.57 0.50% - 37.8 ± 0.46 - 43.4 ± 0.50 -47.8±0.49 -45.6±0.55 1.0% -36.6±0.50 -42.5±0.51 -43.7±0.50 -45.2±0.53 Surface cells 0.0% DHEA 0.10±0.03 0.37 ± 0.03 0.40±0.03 0.53±0.05 0.25% 2.32±0.07 3.38±0.08 3.42 ± 0.09 3.96±0.10 0.50% 3.22±0.05 5.69±0.09 6.64±0.11 6.71±0.14 1.0% 6.26+0.16 6.64 + 0.14 6.88+0.16 5.92±0.12

* mean±SEM.

Table 7. Change since day 1 in vaginal pH during topical ___________treatment with increasing doses of DHEA* .__________

Dose of DHEA 2 weeks 4 weeks 8 weeks 12 weeks 0.0% DHEA -0.23±0.08 -0.37±0.09 -0.51±0.10 -0.47+0.11 0.25% -0.76±0.12 -0.93 + 0.12 -1.09+0.10 -1.12 + 0.11 0.50% -0.96 + 0.14 -1.28 + 0.12 -1.36 + 0.12 -1.35+0.13 1.0% -1.13±0.12 -1.30±0.12 -1.41+0.12 -1.39+0.14

* mean±SEM.

- 37 041509

Table 8. Change since day 1 in the most distressing symptoms of vaginal atrophy during topical treatment with increasing doses of DHEA*

Dose of DHEA 2 weeks 4 weeks 8 weeks 12 weeks 0.0% DHEA -0.49±0.16 -0.79±0.16 -0.61±0.16 -0.67±0.15 0.25% -0.70±0.17 -1.11±0.16 -1.19±0.16 -1.27±0.16 0.50% -0.98±0.15 -1.36±0.15 -1.37±0.17 -1.56±0.15 1.0% -1.00±0.15 -1.29±0.14 -1.38±0.17 -1.37±0.14

* mean±SEM.

Table 9. Change since day 1 in most disturbing symptoms _____ at 12 weeks of treatment 0 (placebo), 0.25, 0.50, and 1.0% DHEA. ,

Category change -3 -2 -1 0 +1 Doses % women 0.0% DHEA 4.65 16.3 25.6 48.8 4.65 0.25% 7.50 42.5 22.5 25.0 2.50 0.50% 20.0 33.3 28.9 17.8 0.0 1.0%" 10.9 37.0 32.6 17.4 2.17

* mean±SEM.

The change from one category (severe^moderate^mild^none) is taken as -1, while the change to 2 categories was -2, and so on.

Examples of pharmaceutical compositions.

Below are, by way of example, and not limitation, several pharmaceutical compositions using the preferred active sex steroid precursor DHEA. The concentration of the active ingredient may vary over a wide range, as discussed here. The amounts and types of other ingredients that may be included are well known in the art.

Example A Vaginal or oral tablet

Ingredient Weight in % (based on the weight of the entire composition) DHEA 5.0 Gelatin 6.5 Lactose 70.5 Starch 18.0

Example B. 1.3 ml - Vaginal suppository.

Ingredient Weight in % (based on the weight of the entire composition) DHEA 0.50 Whitepsol H-15base 99.50

DHEA suppositories were prepared using Whitepsol H-15 base (available from Medisca, Montreal, Canada) as the base. Any other lipophilic base may be used such as but not limited to Butter, Cocoa Butter, Cotomar, Dehydag Base, Fattibase, Hexarcde Base 95, Hydrokote, Suppocire, Wecobee, Cocoa Butter, Japocire, Ovucire, Massa Estarinum or other combinations previous components.

Example C Vaginal or topical cream

Ingredient Weight in % (based on the weight of the entire composition) DHEA 1.0 Emulsifying wax 18.0 Light mineral oil 12.0 benzyl alcohol 1.0 Ethanol 95% USP 34.0 Purified water, USP 34.0

Vaginal or oral gelatin capsule.

Other sex steroid precursors may be used in place of DHEA in the above formulations. Several precursors may be included, in which case the combined wt. % is preferably the wt. % for the individual precursor indicated in the examples above.

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Claims (9)

This invention has been described in the form of preferred embodiments and examples, but is not limited to them. Those skilled in the art will appreciate the broader applicability and scope of this invention, which is limited only by the following claims. CLAIM 1. A method of treating or preventing, in postmenopausal women who are not taking progestin and/or estrogen, symptoms or diseases selected from the group consisting of vaginal atrophy, hypogonadism, skin atrophy, collagenosis, urinary incontinence, vasomotor symptoms, hot flashes, insulin resistance, fatigue, loss of energy, including the introduction of: a) a therapeutic amount of a sex steroid precursor selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-en-33,173-diol and 4-androsten-3,17-dione which increases the level of circulating androgen metabolites, provided that a therapeutically effective amount is 25, 50, or 100 mg/day intravaginally, 15-200 mg/day when administered by skin application, 10-200 mg/day orally, or 1.0-25 mg/day parenteral administration, and b) administering a therapeutically effective amount of a selective estrogen receptor modulator. 2. The method of claim 1, wherein the selective estrogen receptor modulator has the following chemical structure: Acolbifene (EM-652-HCl; EM-1538) 3. The method of claim 1, wherein the symptoms or diseases due to menopause are selected from the group of diseases consisting of vaginal atrophy, urinary incontinence, vasomotor symptoms, and hot flashes. 4. A method of treating or preventing vaginal diseases or symptoms in postmenopausal women who are not receiving progestin and/or estrogen selected from the group consisting of vaginal itching, vaginal atrophy, atopic vaginitis, dyspareunia, vaginal bleeding during sexual activity, loss of collagen compactness fibers of the vaginal wall and low thickness of the muscle plate of the mucous membrane of the vaginal wall in a patient, including the introduction of: a) a therapeutically effective amount of a sex steroid precursor selected from the group consisting of dehydroepiandrosterone, dehydroepianthrosterone sulfate, androst5-en-3p,173-diol and 4-androsten-3,17-dione which increases the level of circulating androgen metabolites, provided that the therapeutically effective amount is 25, 50 or 100 mg/day intravaginally, 15-200 mg/day when administered by application to the skin, 10200 mg/day orally or 1.0-25 mg/day when parenteral administration, and b) administering a therapeutically effective amount of a selective estrogen receptor modulator. 5. The method of claim 4, wherein the selective estrogen receptor modulator has the following chemical structure: Acolbifene (EM-652-HCl; EM-1538) 6. The method of claim 4, wherein the vaginal diseases are selected from the group of diseases consisting of vaginal pruritus, vaginal atrophy, atopic vaginitis, and dyspareunia. 7. The method of claim 1 or 4, wherein the sex steroid precursor is administered by application to the skin. 8. The method according to claim 1 or 4, wherein the sex steroid precursor is administered intravaginally. 9. The method of claim 1 or 4, wherein the sex steroid precursor is orally administered. -