Untitled record

Androgenic pharmaceutical compounds with weak estrogenic effects

Androgenic Pharmaceuticals With low Estrogenic Effect

Full description

Background of the invention

The present invention provides new methods of administering and dosing dehydroepiandrosterone (DHEA) to take advantage of positive androgen effects (e.g. in flaps of the vaginal propria and/or intramuscular muscle layers), without causing unwanted estrogenic effects. In addition to DHEA, other sex steroids may be used (eg, dehydroepiandrosterone-sulfate, androst-5-ene-3.beta.,17.beta.-diol, and 4-androstene-3,17-dione).

Several hormone-related treatments are known. For example, many of them deliver sex steroids such as estrogen or systemically and/or target tissue. In addition to the direct administration of androgen and/or estrogen hormones, substances producing sex steroids that can be converted to estrogen and/or androgen in a tissue are used in many cases. Both androgen and estrogen hormones may be beneficial in some cases and harmful in others, depending, among other things, on the target tissue, the specific needs of the patient, and the extent to which the non-target tissue may be affected. Some treatments, although targeted, may have undesirable activity elsewhere in the body (such as topical administration of the pharmaceutical agent nevertheless increasing the presence of the pharmaceutical product or one of its metabolites in the body's systems). Also, the mechanism of action is not fully understood, especially the relative contributions of androgen and estrogen hormones

General description of the invention

It is the purpose of the present invention to use certain doses, formulations and methods of administration to better achieve the beneficial effects of sex steroids and avoid their undesirable side effects.

In one aspect, the invention provides a method for treating and/or reducing the possibility of vaginal diseases or conditions associated with hormonal imbalance in postmenopausal women, wherein the said method includes administering a sex steroid-producing substance selected from the group including:

dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androst-5-ene-3.beta.,17.beta.-diol, and 4-androsten-3,17-dione

To a patient in need of such treatment, a sex steroid-producing substance is administered in a therapeutic amount that increases the level of circulating androgen metabolites without increasing the level of estradiol above the values found in a normal woman who no longer menstruates.

On the other hand, the invention provides a method for treating and/or reducing the possibility of developing symptoms or diseases resulting from menopause, in women of postmenopausal age, where the aforementioned method includes administering a substance producing sex steroids selected from the group that includes:

dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androst-5-ene-3.beta.,17.beta.-diol, and 4-androsten-3,17-dione

To a patient in need of such treatment, a substance producing sex steroids is given in a therapeutic amount that increases the level of circulating androgen metabolites without increasing the level of circulating estradiol above the values found in a normal woman who no longer menstruates to avoid the risk of breast and uterine cancer. .

On the other hand, the invention provides a method for treating and/or reducing the possibility of developing symptoms or diseases resulting from menopause, in postmenopausal women, where the aforementioned method includes administering a substance producing sex steroids selected from the group containing: :

dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androst-5-ene-3.beta.,17.beta.-diol, and 4-androsten-3,17-dione

To a patient in need of such treatment, a sex steroid-producing substance is administered in a therapeutic amount that increases the level of circulating androgen metabolites and also includes the administration, as part of combination therapy, of a therapeutically effective amount of a selective estrogen receptor modulator to avoid the risk of breast and uterine cancer. Which usually affects postmenopausal women and to prevent bone loss, fat accumulation, and type 2 diabetes.

In another aspect, the invention provides a method for treating vaginal conditions affecting the lamina propria or intramuscular layers comprising intravaginal administration of DHEA at a daily dose of 3 to 13 mg.

In another aspect, the invention provides a pharmaceutical composition comprising a sex steroid producing substance selected from the group including:

dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androst-5-ene-3.beta.,17.beta.-diol, and 4-androsten-3,17-dione

It also includes an excipient, diluent or pharmaceutically acceptable carrier selected from the triglycerides group of saturated fatty acids C12-C18 containing varying amounts of the corresponding partial glycerides (solid fats, Witepsol), butter, and triglycerides. The mixture contains:

oleic, palmitic, and stearic acids (cocoa butter), partially hydrogenated cottonseed oil (Cotomar), hydrogenated fatty alcohols and esters (Dehydag Base I, Base II or Base III may also contain glycerides of C12-saturated fatty acids 16), triglycerides derived from palm, palm kernel, and coconut oils containing glyceryl monostearate and polyoxyl stearate (Fattibase), Hexaride Base 95, and higher soluble fractions of coconut and palm kernel oil (Hydrokote), oils Hydrogenated and rearranged vegetable (S-70-XX95 and S-070-XXA), eutectic mixtures of mono-, di-, and triglycerides derived from natural vegetable oils (Suppocire), triglycerides, and Tween 61 , triglyceride compounds derived from coconut oil (Wecobee), theobroma oil, semi-synthetic glycerides (Japocire, Ovucire), and a mixture of mono-, di-, and triglycerides. Triglycerides derived from saturated fatty acids (Massa Estarinum) and combinations of the above (see Allen et al. 2008). This invention includes carriers comprising fluids in which DHEA and other product substances are soluble.

In another aspect, the invention provides a vaginal suppository comprising 0.25-2.00 percent, more specifically 0.5 percent DHEA, by weight relative to the total weight of the suppository, of DHEA, and also comprising a lipophilic excipient. A particularly suitable excipient is witepsol H-15.

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

In addition to other forms of administering product materials, the invention provides vaginal suppositories and vaginal creams formulated using preferred excipients and preferred concentrations of the product.

Vaginal administration is preferred because the topical action produces desired androgen effects on the desired vaginal layers in much lower doses than if administered via another route. Doses can also be determined by other methods of administration by changing previous doses and concentrations to create a known difference between methods of administration. The resident physician can change doses appropriately according to the individual patient's response.

In preferred embodiments, the sex steroid producing substance is DHEA.

Brief explanation of the drawings

Figure 1 shows serum levels of DHEA and 5-Diol on day 1 or day 7 in a population of women aged 40-75 who had missed periods after daily administration of vaginal suppositories containing 0%, 0.5%, 1.0%. Or 1.8% of DHEA. Data are expressed as mean SEM (n = 9 or 10).

Figure 2 shows serum levels of Testo and DHT on day 1 or day 7 in a population of women aged 40-75 who had missed periods after daily administration of vaginal suppositories containing 0%, 0.5%, 1.0% or 1.8% DHEA. (n=8). Data are expressed as mean SEM (n = 8 to 9). Testo levels from one patient in the placebo group were excluded due to idiopathic Testo levels not seen in any other steroid.

Figure 3 shows serum levels of E1 and E2 on day 1 or day 7 in a population of women aged 40-75 who had missed periods after daily administration of vaginal suppositories containing 0%, 0.5%, 1.0% or 1.8% DHEA. . Data are expressed as mean SEM (n = 9 or 10).

Figure 4 shows serum levels of E1-S and DHEA-S on day 1 or day 7 in a population of women aged 40-75 who had missed periods after daily administration of vaginal suppositories containing 0%, 0.5%, 1.0 % or 1.8% of DHEA. Data are expressed as mean SEM (n = 9 or 10).

Figure 5 shows serum levels of 4-Dione and ADT-G on day 1 or day 7 in a population of women aged 40-75 who had missed periods after daily administration of vaginal suppositories containing 0%, 0.5%, 1.0% or 1.8% of DHEA. Data are expressed as mean SEM (n = 9 or 10).

Figure 6 shows serum levels of 3alpha-diol -3G and 3alpha-diol -17G on day 1 or day 7 in a population of women aged 40-75 who stopped menstruating after one day of administration of 0% vaginal suppositories. 0.5%, 1.0% or 1.8% of DHEA. Data are expressed as mean SEM (n = 9 or 10).

Figure 7 shows the average twenty-four-hour serum concentration (AUC0/24 hours) of DHEA, 5-diol, DHEA-S, 4-Dione, Testo and DHT measured on day 1 or day 7 one day after administration of the vaginal suppositories containing 0%, 0.5%, 1.0% or 1.8% of DHEA. Data are expressed as mean SEM (n = 8 to 10). Testo levels were excluded from one patient in the placebo group (n=8 in that group). Serum steroid concentrations measured in a number of women aged 30-35 who did not menstruate were added as a reference. Data are expressed as mean (n = 47) while the 5th and 95th percentiles are indicated (dashed lines). *, p less than 0.05, **, p less than 0.01, trial (day 7) versus placebo satisfaction treatment (day 7).

Figure 8 shows the average twenty-four-hour serum concentration (AUC0/24 hours) of ADT-G, 3α-diol -3G, 3α-diol -17G, E1, E2 and E1-S measured on day 1 or day 7 after daily administration of the suppositories. Vaginal suppositories containing 0%, 0.5%, 1.0% or 1.8% of DHEA. Data are expressed as mean SEM (n = 9 or 10). Serum steroid concentrations measured in a number of women aged 30-35 who did not menstruate were added as a reference. Data are expressed as mean (n = 47) while the 5th and 95th doses are indicated (dashed lines). *, p less than 0.05, **, p less than 0.01, trial (day 7) versus placebo satisfaction treatment (day 7).

Figure 9 shows changes in serum levels of total androgen metabolites ADT-G, 3alpha-diol-17G in postmenopausal women with vaginal atrophy after intravaginal administration of excessive doses of DHEA. Data are expressed as percentages of serum levels of the same steroid metabolites observed in young premenopausal women (age 30-35). The conversion level is obtained by dividing the total serum levels of ADT-G, 3α-diol-3G and 3α-diol-17G in women receiving DHEA doses of 0.5%, 1.0% and 1.8% by the values found in premenopausal women. (Data taken from Labrie et al., 2006). Changes in serum DHEA level compared to normal premenopausal women are also indicated in comparison to indicate the effectiveness of the conversion (0 --- 0). .............; and -basal levels of androgen metabolites and DHEA, respectively.

Figure 10 shows the maturity index (a) and vaginal pH (b) measured on day 1 and day 7 in a number of women aged 40-75 who had missed periods after daily administration of vaginal suppositories containing 0%, 0.5 %, 1.0% or 1.8% of DHEA.

Data are expressed as mean SEM (n = 9 or 10). *, p less than 0.05, **, p less than 0.01, data on day 7 versus data on day 1.

Figure 11 shows the survival time of dehydroepiandrosterone (DHEA) (A) and androst-5-ene-3.beta.,17.beta.-diol (5-diol) (B) in the serum after a single oral dose of two DHEA capsules. The amount of each is 50 mg, or apply 4 g of 10% DHEA cream or gel for postmenopausal women.

Figure 12 shows the survival time of androstenedione (4-dione) (A) and testosterone (B) in the serum after administering a single oral dose of two 50 mg DHEA capsules or applying 4 g of 10% DHEA cream or gel to women who At the age after menopause.

Figure 13 shows the time period for the survival of estrone (E.sub.1) (A) and 17.beta.-estradiol (E.sub.2) (B) in the serum after a single oral dose of two 50 DHEA capsules. mg or apply 4 g of 10% DHEA cream or gel for postmenopausal women.

Figure 14 shows the survival time of dehydroepiandrosterone sulfate (DHEA-S) (A) and estrone sulfate (E.sub.1-S) (B) in the serum after a single oral dose of two 50 mg DHEA capsules or administered as a single dose. 4 g of 10% DHEA cream or gel for postmenopausal women.

Figure 15 shows the time period for the survival of androsterone glucuronide (ADT-G) (A) and androstone 3.alpha.,17.beta.-diol-glucuronide (3.alpha.-diol-G) (B) in the serum after daily dose administration. Orally, take two 50 mg DHEA capsules, or apply 4 g of 10% DHEA cream or gel for postmenopausal women.

Figure 16 shows the survival time of dehydroepiandrosterone (DHEA) (A) and andros-5-ene-3.beta.,17.beta.-diol (5-diol) (B) in the serum after a daily oral dose of two capsules. Each 50 mg of DHEA or 4 g of 10% DHEA cream or gel for postmenopausal women. Measurements were performed on day 14 of dosing.

Figure 17 shows the survival time of androstenedione (4-dione) (A) and testosterone (B) in the serum after daily oral administration of two 50 mg DHEA capsules or application of 4 g of 10% DHEA cream or gel to women who At the age after menopause. Measurements were performed on day 14 of dosing.

Figure 18 shows the time period for the survival of estrone (E1) (a) and estradiol (E2) in the serum after daily oral administration of two 50 mg DHEA capsules or application of 4 g of 10% DHEA cream or gel to women of age Menopause in postmenopausal women. Measurements were performed on day 14 of dosing.

Figure 19 shows the survival time of dehydroepiandrosterone sulfate (DHEA-S) (A) and estrone sulfate (E.sub.1-S) (B) in serum after daily oral administration of two 50 mg capsules of DHEA or application. 4 g of 10% DHEA cream or gel for postmenopausal women. Measurements were performed on day 14 of dosing.

Figure 20 shows a period of time for the survival of:

androsterone glucuronide (ADT-G) (A) and androstene-3.alpha.,17.beta.-diol-G (3.alpha.-diol-G) (B)

After giving an oral daily dose of two 50 mg capsules of DHEA or applying 4 g of 10% DHEA cream or gel to postmenopausal women. Measurements were performed on day 14 of dosing.

Figure 21 shows the ratios of 24-hour AUC0 values for DHEA and its metabolites on the fourteenth day of dosing compared to the baseline values before treatment. The corresponding numerical values can be found in Table 5.

Figure 22 shows the effect of DHEA (Prasterone) doses administered daily at 0.0%, 0.25%, 0.5% and 1.0% intravaginally for 2, 4, 8 and 12 weeks based on the percentage of parabasal vaginal cells in postmenopausal women. The cycle in postmenopausal women. Data are expressed as mean SEM.

Figure 23 shows the effect of DHEA (Prasterone) doses given daily at 0.0%, 0.25%, 0.5% and 1.0% intravaginally for 2, 4, 8 and 12 weeks based on the percentage of superficial vaginal cells in postmenopausal women. . Data are expressed as mean SEM.

Figure 24 shows the effect of DHEA (Prasterone) doses given daily at 0.0%, 0.25%, 0.5% and 1.0% intravaginally for 2, 4, 8 and 12 weeks on the basis of vaginal pH in postmenopausal women. . Data are expressed as mean SEM.

Figure 25 shows the effect of DHEA (Prasterone) doses given daily at 0.0%, 0.25%, 0.5% and 1.0% intravaginally for 2, 4, 8 and 12 weeks based on the change in the severity of vaginal atrophy symptoms experienced by women themselves. As the most annoying thing. Values are compared to day 1 and values are expressed as mean SEM.

Figure 26 shows the effect of DHEA (Prasterone) doses given daily at 0.0%, 0.25%, 0.5% and 1.0% intravaginally for 2, 4, 8 and 12 weeks based on the change in vaginal discharge assessed on vaginal examination. Data are expressed as mean SEM.

Figure 27 shows the effect of DHEA (Prasterone) doses given daily at 0.0%, 0.25%, 0.5% and 1.0% intravaginally for 2, 4, 8 and 12 weeks based on the change in vaginal color on vaginal examination. Data are expressed as mean SEM.

Figure 28 shows the effect of DHEA (Prasterone) doses given daily at 0.0%, 0.25%, 0.5% and 1.0% intravaginally for 2, 4, 8 and 12 weeks based on the change in consistency of the vaginal epithelium on vaginal examination. Data are expressed as mean SEM.

Figure 29 shows the effect of daily doses of DHEA (Prasterone) administered at 0.0%, 0.25%, 0.5% and 1.0% intravaginally for 2, 4, 8 and 12 weeks based on the change in vaginal epithelium thickness assessed on vaginal examination. Data are expressed as mean SEM.

Figure 30 shows the average twenty-four-hour serum concentrations (AUG)-24h/24) of DHEA, 5-Diol, DHEA-S, E1, E2 and E1-S measured on days 1 and 7 one day after vaginal ovalbumin administration. Contains 0.5% DHEA. Data are expressed as mean SEM (n = 10). Serum steroid concentrations measured in a number of women who did not stop menstruating and aged 30-35 (n = 47), as well as in a number of women who did not menstruate and aged 55-65 (n = 369), were added as reference data and were expressed as the average of the two percentages. 5 and 95 (dashed lines). *, p less than 0.05, **, p less than 0.01, trial versus baseline value. (Data taken from Labrie, Cusan et al. 2008).

Figure 31 shows the average twenty-four-hour serum concentrations (AUC0/24 hours) of 4-Dione, testosterone, DHT ADT-G, 3.alpha.-diol-3G and 3.alpha.-diol-17G measured on days 1 and 7 days after administration of vaginal oval containing 0.5% DHEA. Data are expressed as mean SEM (n = 10). Serum steroid concentrations measured in a number of women who did not menstruate and were aged 30-35 (n = 47) and in a number of women who did not menstruate and aged 55-65 (n = 369) were added as reference data and were expressed as the average of the two percentages 5 and 95 (dashed lines). *, p less than 0.05, trial versus baseline value. (Data taken from Labrie, Cusan et al. 2008).

Detailed description

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Vaginal dryness is found in 75% of postmenopausal women (Wines and Willsteed 2001; NAMS 2007). For a variety of reasons, especially fear of complications from estrogen, 20 to 25% of women with symptoms of vaginal atrophy seek medical treatment (Pandit and Ouslander 1997; NAMS 2007). Therefore, there is a clear medical need and great opportunity to improve the lives of a large group of women who have been left with vaginal atrophy for much of their lives. It can be said that although hot flashes decrease on their own over time, the symptoms of vaginal atrophy, vaginal dryness, vaginal and penile irritation/itching, and dyspareunia become more severe over time if not treated.

Based on the well-known fact that estrogen secretion by the ovaries stops at menopause, systemic and topical estrogen hormones have until now been the only means of treating vaginal atrophy. However, systemic estrogen + and 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; 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), and 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. al. 2002; Lyytinen, Pukkala et al. 2006) as well as endometrial cancer (estrogens alone) (Gambrell, Massey et al. 1980; Persson, Adami et al. 1989; Voigt, Weiss et al. 1991; Jick, Walker et al. 1993; Grady, Gebretsadik et al. 1995; Beral, Bull et al. 2005). Awareness following the Women's Health Initiative Study (Rossouw, Anderson et al. 2002) had the greatest impact in raising doubts about the safety of available treatments for menopausal symptoms (Archer 2007).

Although intravaginal formulations have been developed to avoid systemic estrogen exposure, several studies agree that all intravaginal estrogen formulations result in relatively high serum estrogen levels measured directly or through their systemic effects (Englund and Johansson 1978; Rigg, Hermann et al. 1978; . 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 serum estrogen levels clearly indicate that the use of intravaginally administered estrogen formulations is also likely to be associated with an increased risk of breast and uterine cancer (Kvorning and Jensen 1986; Mattson, Culberg et al. 1989). ; Rosenberg, Magnusson et al. 2006; Official concerns have arisen about the stimulant effects of vaginal estrogen formulations on the endometrium (NAMS 2007).

For most previous measurements of serum estradiol (E2) levels after radioimmunoassays of intravaginal administration of estrogen hormones, a technique that lacks specificity, precision, reliability and sensitivity (Rinaldi, Dechaud et al. 2001), we measured serum estrogen hormones Using GLP-validated mass spectrometry experiments (Good Laboratory Practice) after intravaginal administration of the two most commonly used estrogen formulations (Labrie, Cusan et al. 2008). This study explicitly demonstrates that both an E2 pill (25 ng of E2/day) and conjugated estrogen cream (1 g of 0.625 mg of conjugated estrogen/day), after a week of daily administration, result in an approximately 5-fold increase in E2. Serum in postmenopausal women. These data suggest that the effects of estrogen hormones applied topically to the vagina are likely to be confined to the vagina and the systemic effect is as previously predicted (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 previous safety concerns about estrogen hormones given systemically and topically, recent data clearly indicate that women not only suffer from a lack of estrogen hormones at the time of menopause, but that they are also permanently deprived, in their early thirties, of the androgen hormones that form tissue. Specific peripheral targeting by converting dehydroepiandrosterone (DHEA) into androgen and/or estrogen hormones (Labrie, Belanger et al. 1988; Labrie 1991; Labrie, Luu-The et al. 2003; Labrie, Luu-The et al. 2005). In fact, serum DHEA and DHEA sulfate consistently reduce the peak observed at age 30 (Orentreich, Brind et al. 1984; Labrie, Belanger et al. 1997; Labrie, Luu-The et al. 2003) to a value of 60% of menopause (Labrie, Belanger et al. 2006).

Regarding the role of androgen hormones in women, it is important to mention that women secrete an amount of androgen hormones up to 50% as observed in men (Labrie, Belanger et al. 1997; Labrie, Luu-The et al. 2005). Because serum DHEA is the predominant source of androgen hormones that perform a number of physiological roles in women (Labrie, Luu-The et al. 2003; Labrie 2007), the 60% reduction of circulating DHEA already present at the time of menopause results in a corresponding 60% reduction of total androgen concentration (Labrie, Belanger et al. 2006), leading to potential signs and symptoms of androgen deficiency in bone. , intramuscular muscles, skin, mammary gland, vagina, and brain, and also influence the metabolism of glucose metabolite, insulin, and lipids (Labrie, Luu-The et al. 2003; Labrie 2007). Among androgen target tissues, recent data have demonstrated that the vagina is sensitized to androgen hormones after DHEA administration in the rat, where it has beneficial effects not only on the superficial epithelial layer of the vagina but also on collagen fibers in the genus propria and on intramuscular muscles (Berger, El -Alfy et al. 2005).

Based on data confirmed by clinical (Labrie, Diamond et al. 1997; Labrie, Cusan et al. 2008) and pre-clinical (Sourla, Flamand et al. 1998; Berger, El-Alfy et al. 2005) demonstrating the beneficial effects on the vagina of DHEA administered transdermally or topically, the present clinical trial is a prospective, randomized, and placebo-controlled study of the effect of three doses of DHEA administered daily intravaginally for 12 weeks on the basis of changes in superficial and parabasal cells. , vaginal pH and the most bothersome symptoms of vaginal atrophy as the main purposes. The data clearly indicate that DHEA, which is given topically, is very effective and rapid in correcting all signs and symptoms of vaginal atrophy, as an effect approaching the maximum is achieved two weeks after giving a dose of DHEA. There are no significant changes in estrogen or androgen hormones in the serum, while all Other steroids remain constant or remain in the proportions found in a normal woman who no longer menstruates.

When DHEA is administered topically into the vagina, the beneficial effects of the estrogen and androgen hormones in the vagina are achieved without significant release of estradiol or testosterone into the blood (Labrie, Cusan et al. J. Ster. Biochem. Mol. Biol. In press). When androgen and/or estrogen hormones are formed from DHEA according to the internal secretion process, no tissue response is expected because the response depends on the activity of enzymatic functioning that is specifically present in each cell of the tissue. Thus, it cannot be predicted, from the androgen and estrogen hormones that are produced from DHEA in one tissue, the extent to which similar androgen and estrogen hormones are produced in another tissue.

The results of the ERC-210 clinical trial (Example 3) demonstrate, for the first time, that topical administration of DHEA as hormone-producing replacement therapy (HPRT) is highly effective and rapid in correcting the symptoms and signs of vaginal atrophy in postmenopausal women.

Most importantly, this can be achieved at a dose (0.5%) of DHEA that does not lead to an increase in the levels of active estrogen or androgen hormones in the serum and without the slightest or no change in serum DHEA, while any metabolites remain in the proportions found in a normal woman who did not She is menstruating (Labrie, Cusan et al. 2008).

Although 75% of postmenopausal women suffer from vaginal atrophy (Wines and Willsteed 2001; NAMS 2007), and thus their quality of life is affected for a significant period of time, only 20% seek treatment. (Pandit and Ouslander 1997), primarily due to the fear of breast cancer associated with increased levels of estrogen hormones in the blood. Because the secretion of estrogen in the systemic cycle is exclusively linked to the ovaries and stops with menopause, giving estrogen hormones to postmenopausal women does not seem normal from a physiological standpoint. After WHI, the scientific challenge is to explore hormonal replacement therapies and formulations that give all the benefits of estrogens while improving their quality of life, minimizing risks and maximizing benefits (Archer 2007). Because non-estrogen therapy has not been proven effective (Nelson, Vesco et al. 2006; Suckling, Lethaby et al. 2006), women and their doctors are not aware of any safe treatment for vaginal atrophy.

Several forms of estrogen are known to be an effective treatment for vulvovaginal atrophy (Pandit and Ouslander 1997; Utian, Shoupe et al. 2001). In fact, a transvaginally administered E2 tablet has been shown to be as effective as an E2 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 differs by a 5-fold increase in serum E2 as measured spectrophotometrically after treatment with intravaginal E2 or conjugated estrogen hormones (Labrie, Cusan et al. 2008). These recent data confirm changes in serum estrogens from a large body of studies that all intravaginally administered estrogen formulations result in elevated serum estrogen concentrations as measured by radioimmunoassays or by 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 frequently reported adverse effects with vaginal estrogen are vaginal bleeding and breast pain that are secondary to increased serum estrogen (Suckling, Lethaby et al. 2006). These side effects have been reported for the E2 ring, conjugated estrogen cream as well as the E2 tablet (Ayton, Darling et al. 1996; Weisberg, Ayton et al. 2005). In light of the above, there are also concerns about the stimulating effects of vaginal estrogens on the endometrium (NAMS 2007). Uterine bleeding, breast pain, and perineal pain were reported in 9% of those taking the vaginal tablet for 24 weeks while 34% complained of the same symptoms in the vaginal estrogen conjugated cream group (Rioux, Devlin et al. 2000). Suckling, Lethaby et al. 2006 reported no difference between different vaginal estrogen preparations.

It is known that atrophic vaginitis in postmenopausal women may be increased in severity or induced by the use of aromatase inhibitors to treat breast cancer. In fact, these drugs have benefits in the treatment of breast cancer by reducing 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 in which seven women with breast cancer patients were treated with Vagifem, which contains aromatase inhibitors at a daily dose of 25 mcg for two weeks and then twice weekly, serum E2 increased from an average of 3 pmol/L to 72 pmol/L. litres, over 2 weeks (range 3 to 232) (Kendall, Dowsett et al. 2006). Serum E2 levels generally decreased thereafter to levels of 40 pmol/L or less although levels of 137 and 219 pmol/L were present at weeks 7-10. The patient who received Premarin cream had serum E2 levels of 83 pmol/L in two weeks. It should be noted that the blood samples for E2 measurement were taken at the time of the patient's visit, which is not the same time as the highest serum E2 levels after Vagifem administration. Therefore, it is likely that the ratios in (Kendall, Dowsett et al. 2006) underestimate, to an unknown extent, the true elevation of serum E2 after intravaginal administration of Vagifem tablets or Premarin cream. The researchers concluded that the use of Vagifem with aromatase inhibitors is prohibited. These results in women with breast cancer treated with aromatase inhibitors raise a serious issue about the use of any estrogen administered vaginally as well as orally or transdermally in postmenopausal women. Elevation of serum E2 after treatment with many vaginal estrogen preparations leading to an increased risk of breast cancer is a well-known problem (Rosenberg, Magnusson et al. 2006). Although the study had a small number of events and a short follow-up period (4.7% of the subgroup of 1472 women) did not show a statistically significant difference in disease-free survival in the subgroup of women who used vaginal estrogen (Dew, Wren et al. 2003), it does not seem reasonable or acceptable to increase serum E2 levels during treatment for breast cancer when the goal of treatment with aromatase inhibitors is specifically to maximally inhibit E2 biosynthesis.

In a recent study using Vagifem, the E2 tablet, when administered in a dose of 25 μg, was shown to bring the range of serum E2 levels up to 80 pmol/L with values <50 pmol/L during and after 14 days (Kvorning and Jensen 1986). . In a more recent study using Vagifem, the median and maximum range of 24-hour serum E2 concentrations were measured at 180 99 pmol/L and 84 pmol/L for the 25 mcg dose while the values were 81 62 pmol/L and 40 pmol/L. L, respectively, are present at a dose of 10 μg (Notelovitz, Funk et al. 2002). Other estrogen tablets and creams administered vaginally have resulted in higher serum estrogen levels (Schiff, Tulchinsky et al. 1977; Rioux, Devlin et al. 2000).

With 10 μg and 25 μg E2 tablets administered vaginally, serum E2 has been shown to increase values by about 90 and 160 pmol/L, respectively, from basal values of about 35 pmol/L (Nilsson and Heimer 1992 ). Serum E2 containing Vagifem was reported at a maximum plasma concentration of 51.34 pg/mL on day 1, with this value practically unchanged on days 14 (47.21 pg/mL) and 84 (49.27 pg/mL) (Vagifem, Physician Package Insert 1999).

In another study, after 52 weeks of treatment with 25 mcg of vagifem, serum E2 levels were shown to remain stable with a range from 10.3-21.5 pg/ml to 9.9 pg/ml (Bachmann, Lobo et al. 2008). These data can be explained by stating that the blood samples were likely taken 3 or 4 days after vagifem was applied. It is also important to note that high pre-treatment serum E2 levels are likely to be associated with a loss of specificity in the immunological trials used, as spectrophotometric serum E2 levels in postmenopausal women are reduced by two or more times (Labrie, Belanger et al. al. 2006).

In a previous study, oral and vaginal administration of 1.25 mg of Premarin increased serum E2 and estrone levels to at least 100 pg/mL and 1000 pg/mL, respectively, 24 hours after administration, with levels becoming somewhat higher after administration. Transvaginally. Serum gonadotropin levels were reduced in some cases (Englund and Johansson 1978). Similar data were reported by Rigg, Hermann et al. 1978. In a recent study, 3 months after oral or intravaginal administration of 0.625 mg of Premarin, serum E2 levels increased to 83.1 and 58.6 pg/mL, respectively (Long, Liu et al. 2006), demonstrating very significant systemic exposure. After intravaginal and oral estrogen administration, serum E2 decreased by only 36% intravaginally compared to oral administration of conjugated estrogens.

In a 12-week study using Premarin vaginal cream at a daily dose of two grams, three times weekly, 21% of women experienced bleeding after a progestogen test (Nachtigall 1995). Furthermore, 12% of these women showed increased endometrial thickness according to ultrasonography.

No increase in serum E1, E2 or E1S levels has been reported with vaginal ring use (Nachtigall 1995; Gupta, Ozel et al. 2008) although a significant increase in E1S and E2 has been observed in women over 60 years of age (Naessen, Rodriguez- Macias et al. 2001). In the EST episode group according to a recent study, serum E2 increased from 16 22 pmol/L to 49 64 pmol/L at week 24 (Weisberg, Ayton et al. 2005). On the other hand, in the Vagifem group, serum E2 increased from 15 33 pmol/L to 36 51 pmol/L. Despite this, the researchers reported that serum E2 remained the same or approached the values found in a normal woman who no longer menstruates. At week 48 after treatment with the EST ring or Vagifem, 30-32% of women complained of urinary incontinence, 36-39% of urinary urgency and 18-33% of dyspareunia (Weisberg, Ayton et al. 2005).

The three studies confirmed that the E2 ring administered vaginal ring permits a low serum E2 during a 90-day period, with the exception of a rise in serum estrogen that reaches the lower region of the ratio found in a normal woman, or 100 to 200 pmol/ml during 0.5 hours. 8 first after insertion of the ring (Holmgren, Lindskog et al. 1989; Schmidt, Andersson et al. (1994) (Baker and Jaffe 1996), that is, daily intravaginal administration of 7.5 micrograms of E2 has systemic effects represented by a significant increase in bone mineral density in the total hip and lower back after two years of treatment with intravaginal E2 ( Salminen, Saaf et al. 2007).

In light of the above, there are concerns about the stimulant effects of vaginal estrogens on the endometrium (NAMS 2007). After 12 weeks of treating 32 women with 25 mcg E2 administered intravaginally (Vagifem), one patient was found to have mild hyperplasia (Bachmann, Lobo et al. 2008). In a 24-week study of 80 women, there was one case of proliferative endometrium (Rioux, Devlin et al. 2000) In a 52-week study of 31 women, only two women developed proliferative endometrium (Mettler and Olsen 1991).

In a 12-week study using Premarin vaginal cream at a dose of 2 grams, three times weekly, 21% of women experienced bleeding after a progestogen test (Nachtigall 1995). 12% of these women showed increased endometrial thickness according to ultrasonography. The use of 0.3 mg of conjugated estrogens administered intravaginally, three times weekly, may induce endometrial proliferation, unfortunately rarely, as this was observed in only one of twenty women (Nachtigall 1995).

Sustained release of the estradiol epitope (EST epitope) induced proliferation in endometrial cells similar to 0.625 mg of Premarin cream (Ayton, Darling et al. 1996) but less than 1.25 mg of Premarin (Nachtigall 1995). In fact, both the vaginal ring (EST ring) and conjugated estrogen cream (Premarin cream) have been shown to induce endometrial cell proliferation (Nachtigall 1995; Ayton, Darling et al. 1996). Two cases of moderate endometrial cell proliferation or endometrial polyp hyperplasia were found with an E2 loop (Nachtigall 1995), while two cases of hyperplasia (one simple and one complex, with no patterns) were found with conjugated estrogen cream in the conjugated estrogen cream trial versus E2 disc (Rioux, Devlin et al. 2000). E2 vaginal tablet has been associated with endometrial hyperplasia in a manner similar to estriol vaginal tablet (Dugal, Hesla et al. 2000; Manonai, Theppisai et al. 2001) but less so than conjugated estrogen cream (Manonai, Theppisai et al. 2001).

Although serum estrogen levels are increased to a lower extent after topical vaginal application than with oral or transdermal HRT or ERT, the risk of breast cancer remains an issue and there is still uncertainty about the safety of estrogen compounds administered vaginally. (Suckling, Lethaby et al. 2006; NAMS 2007). In fact, although the increase in serum estrogen compounds is lower after vaginal administration than after oral or transdermal administration, postmenopausal levels are significantly higher for all vaginal estrogen formulations (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 without vaginal atrophy (estimated at 25% of the postmenopausal group) and The remaining 75% is for postmenopausal women with vaginal atrophy (Wines and Willsteed 2001; NAMS 2007), by secreting estrogen compounds into the systemic circulation because the systemic secretion of estrogen stops in all women at the time of menstruation. Therefore, insufficient estrogen secretion does not represent a valid explanation for the occurrence of vaginal atrophy symptoms in the majority of women after menstruation.

However, the formation of sex steroids does not stop when ovarian function stops in the postmenstrual period. Current progress in our understanding of the physiology of endocrine disorder in women demonstrates that after menopause, DHEA secretion by adrenals represents the only source of sex steroids synthesized exclusively in the target tissues (Labrie 1991). Unlike estrogen compounds of ovarian origin that are secreted into the general circulation where they can be measured, DHEA represents an inactive byproduct that is transformed in peripheral tissues at multiple rates according to the level of expression of estrogen-generating enzymes in each tissue. The process of endogenous secretion allows local tissue formation of active sex steroids with a distinct release of active steroids into the circulation (Labrie, Dupont et al. 1985; Labrie, Belanger et al. 1988; Labrie 1991; Labrie, Luu-The et al. 2005).

However, DHEA secretion decreases with age, and a 60% decrease has already been observed at the time of menstruation

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 women with and without postmenopausal symptoms is the amount of DHEA secreted by adrenals or the sensitivity of the vaginal tissue to DHEA. The difference in women's sensitivity is likely to be related, to an unknown extent, to the level of activity of the enzymatic machinery specific to each cell type (Labrie 1991; Labrie, Belanger et al. 2005). With this knowledge, DHEA is a physiological hormonal replacement therapy for postmenopausal women.

As shown in previous studies (Labrie 1991; Labrie, Luu-The et al. 2003; Labrie, Luu-The et al. 2005; Labrie, Belanger et al. 2007), supplementation with physiological amounts of exogenous DHEA allows the biosynthesis of androgen and/or estrogen only in appropriate target tissues as they include steroidogenic enzymes in the endogenous state (Labrie, Luu-The et al. 2005). Active androgen and estrogens synthesized locally from DHEA in peripheral target tissues exert their effects in the same cells where they are created. Advantageously, very little active sex steroids leak into the circulation, thus explaining the significant benefits observed in the vagina with no significant change in circulating estrogen and androgen (Labrie, Cusan et al. 2008). This local biosynthesis, action and inactivation of estrogen and androgen in target cells eliminates exposure to excess sex steroids and thus eliminates the excess risk of unwanted side effects from exposure to excessive levels of estrogen, including breast, ovarian and uterine cancer (Gambrell , Massey et al. 1980; Persson, Adami et al. 1991; 1993; Jake, 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).

The change in pH is a valid variable that reflects the beneficial effect of vaginal atrophy treatment. After 12 weeks of intravaginal treatment with 25 mcg E2, the proportion of patients who had a pH below 5.0 was 51% compared to 21% in the placebo group (Bachmann, Lobo et al. 2008). However, at baseline, 11.2% and 13% of women had a pH of less than 5.0 in the corresponding groups. In the ERC-210 clinical trial (Example 3), no patients had a pH less than 5.0 at the start of treatment and 12%, 36%, 46%, and 48% had a pH less than 5.0 at the start of 12 weeks. In the 0%, 0.5%, 0.5% and 1.0% of DHEA groups, respectively.

In the ERC-210 clinical trial (Example 3), the effect of DHEA on vaginal epithelial cell maturation was particularly rapid: 0.5% of DHEA oocytes were already observed, and 79% of the maximum effect on parabasal cells was observed at 2 weeks while Note that 48% of the maximum stimulating effect is exerted on the surface cells at the same time as the interstitial period. On the other hand, 85% of the maximum effect of 0.5 DHEA on the percentage of surface cells was achieved in a period of 4 weeks. Likewise, 63% of the maximum effect of 0.5% DHEA on the most fatigued symptoms was observed at 2 weeks and 87% was reached at 4 weeks. Furthermore, only 17.8% of women showed no change in their most bothersome symptoms at 12 weeks in the 0.5% DHEA group compared to 48.8 in the placebo group.

The effect of DHEA on parabasal cells is rapid since parabasal cells decreased to less than 20% in one month of using three doses of DHEA. The percentage effect on surface cells is also very rapid using 100% of the effect observed at week 2 with a high dose of DHEA (1%). In a study of vaginal estrogen cream or tablets, about 50% of the effect measured at week 12 was observed at 2 weeks (Rioux, Devlin et al. 2000). These data indicate that the effect of DHEA is not small and may exceed the effect of vaginal E2 and conjugated estrogen formulations.

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

In the current study, the vaginal maturity value (VMV) increased from 27.45 at baseline 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 the baseline, while the percentage of cells adjacent to the base decreased by 41.66% in the group treated with estrogen. In the same study, vaginal pH decreased from 6.74 at baseline to 5.05 (1.69 or 24% decrease in the estrogen group). The most bothersome symptoms decreased from 2.58 to 1.04 (-1.54) in the estrogen group compared with a decrease from 2.59 to 1.84 (0.75) in the placebo. These data observed with estrogen use are comparable to the 1.56 reduction in severity of the most bothersome symptoms at 12 weeks in the 0.5% DHEA group and the 0.67 reduction in the placebo group observed in the clinical trial (Exhibit No. ERC-210(3)).

At week 12, 11% of ESTring cases and 24% of Vagifem cases had persistent epithelial cell atrophy. At week 48, the corresponding values were 8% and 14% (Weisberg, Ayton et al. 2005). At week 48 of treatment with Vagifem and ESTring, 33% of women still had vaginal dryness (Weisberg, Ayton et al. 2005), on the other hand, vulvar itching remained in 15% and 20% of women after treatment with ESTring and Vagifem, respectively, while 33% and 28% still suffered from dyspareunia after treatment with ESTring and VAGIFEM, respectively. Bleeding after progestogen testing was 7% in the Vagifem group and 0% in the ESTring group.

After three months of daily administration of 0.625 mg of Premarin orally or vaginally (in cream form) a respectively 70.6% and 75% improvement in dyspareunia was observed (Long, Liu et al. 2006). In this study, it was found that 1 gram of 0.625 mg of Premarin cream represented the lowest dose for treating sexual dysfunction.

In women who received 25 μg of E2 transvaginally, dyspareunia was present in 12.4% of cases after 12 months of treatment (Simunic, Banovic et al. 2003). The success rate of topical treatment with E2 tablets was 84.5% as reported by patients and 98.1% as reported by clinicians (Simunic, Banovic et al. 2003). Bachmann et al. 1992 (Bachmann, Notelovitz et al. 1992) showed that 40 to 50% of women on oral estrogen replacement therapy showed complaints of vaginal dryness.

As previously shown after 12 hours of treatment with DHEA (Labrie, Diamond et al. 1997), the ERC-210 clinical trial (Example 3) showed no effects on endometrial tissue after 3 months of administration of the DHEA hormone product. Transvaginally as demonstrated by histological examination of endometrial biopsies obtained before and after 12 weeks of treatment. These data are consistent with the absence of aromatase activity in human endometrium (Baxendale, Reed et al. 1981; Bulun, Lin et al. 2005). These data are strongly supported by the well-known clinical observation that endometrial atrophy is a postmenopausal feature despite continuous DHEA secretion throughout life (Labrie, Luu-The et al. 2005; Labrie, Belanger et al. 2006). ). The human endometrium's absence of steroidogenic enzymes necessary to convert DHEA to estrogen compounds is consistent with the physiological role of the endometrium being exclusively active during the reproductive years when its function is controlled primarily by ovarian hormones and placental origins. There is no physiological role for the postmenopausal endometrium that would explain any continuing effect of estrogen compounds after the cessation of estrogen secretion through the ovary. Thus, the enzymes required for the synthesis of estrogen compounds from DHEA are not expressed in the endometrium, which represents a tissue completely dependent on estrogen compounds of ovarian origin.

Estrogens alone have been known to stimulate endometrial cell proliferation (Smith, Prentice et al. 1975), while progestogens administered in combination with estrogen compounds antagonize the stimulatory effect of estrogens (Feeley and Wells 2001). Since androgen receptors are expressed in human endometrium and stromal cells.

(Mertens, Heineman et al. 1996), it is important to mention that the clinical study that examined the effect of androgen compounds did not show the effect on the endometrium of a relatively high dose of testosterone in postmenopausal women (testosterone undecanoate, 40 mg every second day) (Mertens, Heineman et al. 1996). Zang, Sahlin et al. 2007). In women receiving estradiol valerate (2 mg/daily), ionization constant numbering increased by 50% at 3 months of treatment while immediate administration of testosterone decreased ionization constant numbering to 28%. Ki67 numbering increased only in the two groups receiving estrogen but decreased with the addition of testosterone in stromal cells. While testosterone does not have a stimulating effect on endometrial proliferation in women, it has been shown to produce an anti-estrogen effect in the endometrium.

While FDA guidance urges payers to establish minimum doses and exposures for both estrogen compounds and progestogen compounds, it should be recognized that although estrogen compounds are effective for correcting symptoms of vaginal atrophy and symptoms of vascular diameter change, androgen compounds do not represent physiological hormones that It allows 25% of postmenopausal women to avoid symptoms ranging from moderate to serious degrees of vaginal atrophy. These women remain relatively symptom-free throughout the postmenopausal years. Because the only source of sex steroids in postmenopausal women, in both symptomatic and asymptomatic women, is the biosynthesis of estrogen and androgen from adrenal DHEA, by the endogenous secretory mechanism. DHEA replacement is the only physiological method that allows women suffering from postmenopausal symptoms to be supplied with the missing amount of DHEA responsible for those symptoms. The method is called hormone replacement therapy (HPRT), and it should correct vaginal atrophy and symptoms of vascular diameter changes with no greater risk than postmenopausal women who do not have symptoms of vaginal atrophy due to higher exposure to DHEA and the sex steroids that are produced in the vagina. cells internally by the process of internal secretion.

Substances producing sex steroids according to the invention are preferably given in a dose range (1) between 0.5 to 100 mg per day, (preferably from 3 to 50 mg per day, and preferably from 3 to 13 mg per day), when administered intravaginally; (2) in a dose range of 15 to 200 mg daily (preferably 30 to 100 mg daily), when administered to the skin; (3) in a dose range of 10 to 200 mg daily (preferably 25 mg to 100 mg daily), e.g. 75 mg daily, when administered orally; or (4) in a dose range between 1.0 to 25 mg daily (preferably 3.25 to 20 mg daily), when administered non-intestinally (i.e. intramuscular, subcutaneous).

In a pharmaceutical composition for vaginal administration, DHEA or other product is preferably present at a concentration between 0.1 and 10% by weight relative to the total weight of the composition and preferably between 0.2 and 3.0% by weight and especially between 0.25 and 2.0%. For example, 1.3 milliliters (ml) of vaginal suppositories containing 0.5% DHEA (by weight of the total formulation) were given once daily, preferably providing 6.5 mg/day of DHEA. Larger or smaller suppositories, with different concentrations, can be used, while the dosage is kept within the desired range.

In a pharmaceutical composition for intradermal administration, DHEA or other product substances are preferably present in a concentration between 0.1 and 10% by weight relative to the total weight of the composition, preferably between 0.2 and 2.0%, and especially between 0.3 and 1.5%. .

In a pharmaceutical composition for oral administration, DHEA or other product is preferably present in a concentration between 5 and 98% by weight relative to the total weight of the composition and preferably between 10 and 50%, and especially between 15 and 40%.

In a pharmaceutical formulation for non-enteral administration (i.e. intramuscular or subcutaneous), DHEA or the other product is preferably present at a concentration between 0.2 mg/ml and 25 mg/ml, and preferably between 0.65 and 15 mg/ml. ml, especially between 2 mg/ml and 10 mg/ml.

Example of the efficiency of the invention:

Example No. (1):

ERC-213 clinical trial

Bioavailability of DHEA after administration of vaginal suppositories in postmenopausal women with stage I vaginal atrophy, in a randomized, controlled, satisfactory dose study

Pharmacokinetics and topical effect of daily administration of DHEA suppositories for a week

The primary objective of this study was to evaluate the systemic bioavailability of DHEA and its metabolites after daily intravaginal administration of suppositories at four different DHEA concentrations. This study was a randomized, controlled-dose, randomized controlled trial with 10 subjects per group. Therefore, 40 postmenopausal women were randomly assigned to receive a daily dose of one suppository of the following concentrations: 0.0%, 0.5% (6.5 mg DHEA/suppository), 1.0% (13 mg DHEA/suppository), or 1.8%. (23.4 mg DHEA/suppositories).

Maturity index and vaginal pH were measured pre-treatment and 7 days after treatment to provide an explanation of the topical effect of DHEA during the short treatment period.

As shown in Figure 1B, Table 1 and Table 2, daily intravaginal administration of 1.3 ml of a suppository containing 0.5%, 1.0% and 1.8% DHEA resulted in a sustained increase of serum DHEA. AUC values from 0 to 24 hours are 24.8±4.8 ng/hour/mL, 56.2±8.9 ng/hour/mL (P<0.05), 76.02±10.3 ng/hour/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% compared to the control sample when doses of 0.5%, 1.0% and 1.8% of DHEA were administered respectively. As observed for all other steroids, similar AUC values were observed from 0 to 24 h on days 1 and 7.

In fact, the average serum value of 4.76 ± 0.42 ng/ml of DHEA after treatment with the highest value (Table 2) was similar to the value of 4.47 ± 2.19 ng/ml in (47) normal women aged 30 to 35. year before menopause (Labrie, Belanger et al. 2006). The survival of serum DHEA after any of the doses of DHEA used within the limits in premenopausal women is well illustrated in Figure (7a).

As previously observed after oral or dermal administration of DHEA (Labrie, Belanger et al. 2007), serum 5-Diol concentration follows a pattern that is not possible for DHEA, although lower concentrations can be observed. In fact, the AUC ranged from 0 to 24 hours from doses of 5.60±0.60 ng/hour/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 0.5%, 1.0% and 1.8 DHEA, respectively (1D, Table 1). These changes correspond to increases of 75%, 147%, and 476% over the comparison sample. Only 1.8% of the DHEA dose caused increases in serum 5-Diol concentration that exceeded values found in normal premenopausal women (Figure 7B) during the 24 hours after vaginal administration of DHEA on day 7.

The AUC value from zero to 24 hours of serum Testo showed no changes at the 0.5% dose (2.79 0.30 ng/hour/ml versus 2.58 0.33 ng/hour/ml in the group receiving placebo) (Figure 2B) ). At doses of 1.0% and 1.8%, AUC values from 0 to 24 hours were 4.54 ± 0.91 ng/h/ml (P < 0.05) and 5.97 ± 0.69 ng/ml (P < 0.01) in Table 1. ). These values translate to mean serum Testo levels of 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 when using the highest dose of 1.8% DHEA, Testo serum levels remained within the normal range for premenopausal women at 0.18 0.07 ng/ml (0.06 0.31, 5 to 95%) (Labrie, Belanger et al. 2006) ( Figure 7 e). On the other hand, the dose of 1.0% (0.18 0.07 ng/ml) corresponds to the values found in women before menopause, specifically 0.19 0.4 (Figure 7 e).

In Figure 3c and (d), serum DHT increased the AUC value from 0 to 24 hours from 0.58 ± 0.07 ng/h/ml in the sativa group on day 7 to 0.93 ± 0.11 (NS), and 1.31 ± 0.26 (NS). P < 0.05) and 1.93 ± 0.23 (P < 0.01) ng/h/ml in 0.5%, 1.0% and 1.8% of DHEA groups, respectively (Table 2). These values correspond to average 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, the highest DHEA dose was achieved, and normal serum DHT levels of 0.07 0.03 ng were observed. /ml in premenopausal women (Labrie, Belanger et al. 2006) (Figure 7 f).

Mean serum E1 concentrations were measured at 12.6±1.41 ng/ml in the placebo group on day 7 (Table 2) while there was no significant change in the 0.5% DHEA dose condition (15.4±2.04 ng/ml). / 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 the 1.0% and 1.5% DHEA dose, respectively. The AUC values from zero to 24 hours are shown in Figure No. (3b) and this is explained in Table No. (1).

Mean serum E2 concentrations were measured at 2.77 pg/ml and 4.04–0.69 pg/ml (NS) in the sativa and 0.5% DHEA groups, respectively (Table 2). Median E2 concentrations of 6.01 ± 1.31 pg/ml (P < 0.05) and 5.68 ± 0.84 pg/ml (P < 0.05) were reached at day 7 in women receiving the 1.0% and 1.8% DHEA doses as a result of absolute increases 3.18 and 2.85 pg/ml compared to placebo, respectively. Data were compared with serum E1-S concentrations with mean serum levels of 0.12, 0.02 ng/ml, and 0.13 ng/ml (NS) in the groups receiving NS and 0.5% DHEA, respectively (Table No. (2)). Values of 0.18–0.03 ng/ml 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) compared to the placebo group.

As shown in Figures 4b and 4d, a comparable pattern is observed for both E1-S and DHEA-S. The AUC value from 0 to 24 for serum DHEA-S concentration was measured at 8.35 ± 2.22 ng/h/ml in the group that received placebo and 13.3 ± 3.16 ng/h/ml in the group that received 0.5% DHEA (NS). ). With the two highest doses of DHEA, AUC values from 0 to 24 were measured at 16.5 ± 2.71 ng/h/ml (NS) and 19.3 ± 3.59 ng/h/ml (P < 0.05), respectively (Figure 4D, Table No. (1)). All DHEA values for all DHEA doses remain below the serum DHEA-S levels observed in premenopausal women, which show a mean of 1.27 0.62 ng/ml (7g).

As shown in Figure 5B, the AUC value from 0 to 24 hours of serum 4-dione after DHEA administration on day 7 was measured at 0.80 6.34 and 0.84 ng/h/ml (SN) 8.71 for the group. The treatment group received 0.5% DEAH at a concentration of 0.5%, respectively. At higher DHEA doses, the 0- to 24-hour AUC values of 4-dione increased slightly to 11.1±1.51 (P<0.01) and 11.9±0.81 (P<0.01) ng/hour/mL, respectively. As can be seen in Figure 7D and Table 2, all serum 4-dione values remained below the average serum 4-dione concentrations observed in normal premenopausal women. In fact, the highest DHEA dose resulted in mean serum 4-dione concentrations of 0.50–0.03 ng/ml while the mean value in menstruating women aged 30 to 35 years was 0.96–0.35 ng/ml (Labrie, Belanger et al . 2006) (Appendix 2), thus only reaching 50% of the serum Dion levels observed in premenopausal women.

When taking into account the critical role of serum levels of ADT-G, 3α- diol - 3G, and 3α- diol - 17G (Labrie, Belanger et al. 2006), it is preferable in (5d) and Table (2) that an increase is observed. ADT-G increased from a mean value 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 for both DHEA groups). Similar changes can be observed for the small androgen metabolites 3a- diol - 17G and 3a diol - 17G (6b, 6d, 8b, and 8c, Table 1 and Table 2). It is important to point out, as shown in Figure 8, that even at the higher DHEA doses used, the average serum levels of ADT - G 3a diol - 3G and 3a- diol - 17G remained 36%, 11%, and 6%. Lower than the average serum levels found in women before menopause.

As shown in Table 2, the amount of glucuronides metabolites measured in a 24-hour period on day 7 to administer 1.3 mL of suppository containing 1.8% DHEA (23.4 mg DHEA) was 28.2 ng/mL while The average serum concentration of the same metabolite in premenopausal women aged 30 to 35 years was about 42.8 ng/ml (Labrie, Belanger et al. 2006) (Appendix 2). Therefore, the highest DHEA dose used results in only 65.7% of the corresponding value for total androgen metabolites found in normal young menstruating women. On the other hand, 0.5% and 1.0% doses of DHEA lead to amounts of androgen metabolites of 21.02 ng and 21.53 ng/ml, respectively, thus corresponding to only 49.0% and 50.2% of the values observed in women who had Menopause stage (Figure 9). We previously found that daily oral administration of 100 mg DHEA results in 74% of levels found in premenopausal women (Labrie, Belanger et al. 2007). We have previously observed that after oral or transdermal administration of DHEA the changes of serum DHEA are approximately 100% an overassessment of changes in steroid composition that is reflected by changes in ADT-G, 3a-diol-3G, and 3a-diol-17G (Labrie , Belanger et al. 2007). As shown in Figure 9, mean serum DHEA levels went from 23% of the value observed in premenopausal women in the placebo group to 52%, 71%, and 106% in women receiving DHEA doses. At concentrations of 0.5%, 1.0%, and 1.8%, respectively. The data from Figure 9 indicate that the changes in serum DHEA after intravaginal DHEA administration are also an overestimation of changes in androgen composition and likely also in estrogen formation as indicated by the smaller changes in serum E1-S (Table No. (1)). In fact, for 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% overestimate). At the highest DHEA dose, serum androgen metabolites increased by 47.1% while serum DHEA increased by 83.5 (77% overestimation).

Table No. (1):

Area under the curve values (AUC 0 to 24 hours) for DHEA and eleven metabolites on days 1 and 7 for daily vaginal administration of DHEA Suppositories to postmenopausal women aged 40 to 75 years with vaginal atrophy.

DHEA

5-diol

TESTO

DHT

E1

E2

group

the value

nanograms/hour/ml

Today 7 ng/hour/ml

Day 1

nanograms/hour/ml

Today 7 ng/hour/ml

Day 1

nanograms/hour/ml

Day 7

nanograms/hour/ml

Day 1

nanograms/hour/ml

Today 7 ng/hour/ml

Today 1 pg/hour/ml

Today 7 pg/hour/ml

Today 1 pg/hour/ml

Today 7 pg/hour/ml

Satisfaction therapy

middle

SEM

24.47

4.80

24.82

4.77

5.55

0.59

5.60

0.60

2.71a

0.34

2.58 a

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%

middle

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%

middle

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

81.47

DHEA 1.8%

middle

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

E1-S

DHEA-S

4-dione

ADT-G

3 α-diol 3G

3 α-diol 17G

Day: 1 ng/hour/ml

Today 7 ng/hour/ml

Day: 1 mcg/hour/ml

Today 7 mcg/hour/ml

Day 1

nanograms/hour/ml

Today 7 ng/hour/ml

Day: 1 ng/hour/ml

Today 7 ng/hour/ml

Day: 1 ng/hour/ml

Today 7 ng/hour/ml

Day 1

nanograms/hour/ml

Day 7

nanograms/hour/ml

treatment

Satisfying

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

69.54

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

1.11

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: Data for one patient were excluded

Table No. (2): Mean serum steroid levels of DHEA and eleven metabolites on days 1 and 7 of daily vaginal administration of DHEA suppositories to postmenopausal women aged 40 to 75 years with vaginal atrophy.

The values were obtained by dividing the AUC values from 0 to 24 hours measured on days 1 and 7 by 24 and thus yield the average serum concentration of each steroid in a 24-hour period. Serum steroid concentrations in premenopausal women aged 30 to 75 years were added as a reference.

DHEA

5-diol

TESTO

DHT

E1

E2

group

the value

Day: 1 ng/hour/ml

Today 7 ng/hour/ml

Day: 1 mcg/hour/ml

Today 7 mcg/hour/ml

Day 1

nanograms/hour/ml

Today 7 ng/hour/ml

Day: 1 ng/hour/ml

Today 7 ng/hour/ml

Day: 1 ng/hour/ml

Today 7 ng/hour/ml

Day 1

nanograms/hour/ml

Day 7

nanograms/hour/ml

Placebo therapy

middle

SEM

1.02

0.20

1.03

0.20

0.23

0.02

0.23

0.02

0.11*

0.01

0.11

0.01

0.026

0.003

0.024

0.003

12.73

1.44

12.58

1.41

2.90

0.32

2.77

0.29

DHEA 0.5%

middle

SEM

2.73

0.33

2.34

0.37

0.45

0.04

0.41

0.05

0.12

0.01

0.12

0.01

0.038

0.004

0.039

0.004

14.02

1.5a

15.40

2 04

3.66

0.47

4.04

0.69

DHEA 1.0%

middle

SEM

3.12

0.28

3.18

0.43

0.50

0.07

0.58

0.08

0.16

0.03

0.19

0.04

0.046

0.010

0.055

0.011

17.42

2.95

24.11

3.64

4.23

0.96

6.01

1.31

DHEA 1.8%

middle

SEM

5.15

0.39

4.78

0.42

0.79

0.04

0.88

0.07

0.21

0.03

0.25

0.03

0.068

0.008

0.081

0.010

18.07

1.57

25.04

2 35

3.74

0.49

5.68

0.84

Premenopausal women aged 30 to 35 (n=47)

middle

standard deviation

Mediator

5 to 95%

(maximum - minimum value)

4.47

2.19

4.14

1.53-9.14

)1.41 -10.37(

0.49

0.20

0.44

0 25 - 0 .34

(0.25 - 0.96(

0.10

0.07

0.17

0.06 -0.31 )0.05-0.32)

0.07

0.03

0.07

0.03-0.14

)0.03-0.17(

53.96

23.28

4947

23.74 - 87 46

(10.27-123.50(

82.05

42.-9

71.38

22.00-159.97

(17.71 -101.14(

The group receiving placebo treatment

E1-S

DHEA-S

4-dione

ADT-G

3 α-diol 3G

3 α-diol 17G

the value

Day: 1 ng/hour/ml

Today 7 ng/hour/ml

Day: 1 mcg/hour/ml

Today 7 mcg/hour/ml

Day 1

nanograms/hour/ml

Today 7 ng/hour/ml

Day: 1 ng/hour/ml

Today 7 ng/hour/ml

Day: 1 ng/hour/ml

Today 7 ng/hour/ml

Day 1

nanograms/hour/ml

Day 7

nanograms/hour/ml

0.13

0.03

0.12

0.02

0.35

0.10

0.35

0.09

0.26

0.26

0.03

7.36

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%

middle

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

02-

DHEA 1.0%

middle

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 J8

0.71

0.10

0.84

0.14

0.87

0.14

1.04

0.20

DHEA 1.8%

middle

SEM

0.18

0.03

0.25

0.05

0.62

0.11

0.81

0.11

0.44

0.03

0.50

0.03

21.28

2.20

25.74

2.88

0.85

0.10

1.08

0.14

0.92

0.11

1.34

0.18

Premenopausal women aged 30 to 35 (n=47)

middle

standard deviation

Mediator

5 to 95%

(maximum - minimum value)

1.19

0.93

0.87

0.31 -3.50

(0.21- 4.40)

1.27

0.62

1.04

0.56-2.65 )045-2.71(

0.96

0.35

0.92

0.45 - 1.64 (0.31 -1.77)

40.21

29.31

31.62

12.17- 118.2

)5.86-132.0)

1.21

0.63

1.06

0.26-2.78 (0.25-4.33)

1.43

0.93

1.35

0.25 - 2.56

(0.25- 5.71)

a Data for one patient were excluded. (Labrie, Belanger et al. 2006)

However, as shown in Table 3, it should be mentioned that there is a tendency to lower pretreatment values for many steroids in the group receiving placebo.

This is particularly associated with lower values in the placebo group for DHEA, DHEA-S, 4-dione, Testo, DHT, E2, ADT-G, and 3α-diol 17G.

Because all mean serum steroid values observed after 0.5% and 1.0% DHEA doses remain within or below values found in normal premenopausal women, no attempt is made to correct this apparent deviation. It is important to note that the average 24-hour serum levels of each steroid measured on day 7 of daily administration of the 0.5% DHEA suppositories closely correspond to the values measured in women aged 55 to 65 years, whereas the 0.5% DHEA suppositories 0.1% results in values within the range observed for normal women aged 55 to 65 years (Labrie, Belanger et al. 2006).

Because androgen metabolites represent the most reliable measure of the conversion of exogenous DHEA to androgenic compounds, the present data indicate that even at the highest doses of DHEA used in the current study meet the FDA requirements for serum levels that remain within the normal range in Premenopausal women.

Table No. (3): Data for basal serum steroid levels on days 1 and 7 of daily administration of increasing doses of DHEA into the vagina are expressed in nanograms/ml except for E1 and E2 (in picograms/ml) and DHEA-S (micrograms/ml). ).

steroid

Placebo therapy

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-dione

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

E

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

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

E1-S

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

3α-diol - 3G

Day 1

0.50 a

0.61 0.07

0.71 0.14

0.58 0.05

Day 7

0.50 a

0.75 0.11

0.96 0.25

0.96 0.15

3α-diol - 17G

Day 1

0.50 a

0.84 0.16

0.85 0.22

0.65 0.06

Day 7

0.50 a

0.95 0.16

1.18 0.30

1.27 0.19

a: Steroid levels were below the limit of quantitation for all cases (limit of quantification = 0.5 ng/ml).

After just a week of daily administration of DHEA suppositories, the maturation index increased by 107% (P< 0.01), 75% (P< 0.05), and 150% (P< 0.01) in 0.5%, 1.0%, and 1.8%. for the groups receiving DHEA, respectively (Figure 10a). No changes were observed in the group receiving placebo between days 1 and 7. On the other hand, the vaginal pH decreased from 6.29 ± 0.21 to 5.75 ± 0.27 (P < 0.05), 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 the groups that Receive DHEA at concentrations of 0.5%, 1.0%, and 1.8% (Figure 10B). There were no changes in vaginal pH in the group receiving placebo.

Example No. (2):

Bioavailability and metabolism of Dehydroepiandrosterone and its transdermal administration in postmenopausal women.

1- Introduction

Humans, and other primates, represent a unique animal species in that they secrete large amounts of inactive DHEA-producing steroids, particularly DHEA-S, which are converted to active androgen and/or estrogen 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., 2006 and Simpson 2000). In fact, plasma DHEA-S levels in adult men and women are 100 to 500 times higher than testosterone levels and 1,000 to 10,000 times higher than estradiol levels, thus leading to a large store of the essential substance that is converted into androgens and/or estrogens in peripheral secretory tissues that involve the enzymatic mechanisms necessary to convert DHEA into active sex steroids (Labrie 1991, and Labrie, Luu-The et al., 2005) In fact, the term endocytosis was first coined in 1988 (Labrie, Belanger et al., 1988) to describe the synthesis of potent steroids prepared in the same cells as showing their effect with no release into the extracellular space and macrocirculation or the presence of Release a little before it becomes ineffective (Labrie, 1991).

The marked decrease in DEHA-S formation by adrenaline during aging ( Belanger et al., 1994 ; Vermeulen and Verdonck, 1976 ; and Migeon et al., 1957 ) results in a significant reduction in the formation of androgens and estrogens in the target peripheral tissues, namely A condition that can be 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). Furthermore, the benefits of DHEA administered to postmenopausal women should receive more attention, especially those related to bone, skin, vaginal, glucose, insulin metabolism, and fat mass, which develop after oral administration. (Villareal and Holloszy, 2004; Baulieu et al., 2000; Morales, et al. 1994; and Kawano et al. 2003) and transepidermally (Diamond et al., 1996 and Labrie Diamond et al. 1997). This becomes of particular importance to obtain more specific knowledge about bioavailability, pharmacokinetics and metabolism of DHEA after following these two routes of administration.

Because the use of a pharmacological dose of 2-week transdermal DHEA has already been demonstrated, these measurements of serum testosterone (testo) and estradiol (E2) do not provide a reliable assessment of the actual pool of androgens and estrogens within cells (Labrie, Belanger et al. (1997; Labrie, Belanger et al., 2006 and Labrie, Belanger, et al., 2007b) and we compared serum levels of DHEA and 9 steroids known to be closely related to active androgens and estrogens and their metabolites. Detailed analysis of 24-h changes of serum steroid levels was performed on day 1 and after 2 weeks of daily administration of DHEA through oral as well as transdermal use with DEAH cream or gel.

2- Cases and methods:

Thirty-six healthy women aged 60 to 70 years participated in the study after IRB approval as well as their consent informed in writing. Body weight was 20% of normal body weight according to Metropolitan Life tables.

No case suffered from a significant metabolic or endocrine disorder, coronary disease, or high blood pressure. No woman had been treated with androgens or anabolic steroids in the 6 months before screening. All participants had a medical history, comprehensive physical examination, serum biochemical features including lipids, comprehensive blood examination, urinalysis, and detailed serum hormone determinations during the screening phase of the protocol.

3- Study design, treatment and measurements

This study was an open, randomized trial with 12 cases per group. After written informed consent was obtained and women were determined to be suitable for the trial, each case was randomly assigned to receive DHEA by cream, gel, or orally. Cases received daily before breakfast for 14 days in the research clinic either 4 grams of DHEA gel at a concentration of 10% DHEA or 4 grams of cream of DHEA at a concentration of 10% over a total area of 30 cm by 30 cm of the thigh or 50 mg capsules of DHEA orally before breakfast.

Blood samples were taken at 00:08 to 00:09 at screening and before DHEA administration, on day 1 of dosing, as well as on days 2, 4, 7, 10, and 14. On days 1 and 14, blood samples were obtained at half an hour, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, and 24 hours after DHEA administration.

4- Serum steroid analysis:

Measured:

DHEA, DHEA-S, androst-5-ene-3.beta.,17.beta.-diol (5-diol), testosterone, androstenedione (4-dione), 17.beta.-estradiol (E.sub.2) ), estrone (E.sub.1), estrone sulfate (E.sub.1-S), androsterone glucuronide (ADT-G), and androstane-3*,17.beta.-diol glucuronide (3 .alpha.- diol-G)

This is done by gas chromatograph/mass spectrometer:

(DHEA, 5-diol, 4-dione, testosterone, E.sub.1 and E.sub.2) using electron impact or chemical ionization and by liquid chromatograph/tandem mass spectrometry (DHEA-S, E1-S). S, ADT-G, 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 the serum concentration of each steroid between 0 and 24 hours (AUC of 0 24 hours) was measured. Areas under the curves were calculated by the linear trapezoid method (independent model). The relative bioavailability of DHEA gel, DHEA cream, and DHEA capsules was based on the mean difference in the logarithm of transformed AUC values. All calculations were done using SAS software (SAS Institute, Cary, NC, USA).

6- Results

Oral administration of two 50 mg DHEA capsules resulted in an increase of serum DHEA from 0.3 2.3 ng/ml to a maximum value of 2.5 15.6 ng/ml at 1 h with a continued decline thereafter to 5.7 ng/ml at 6 h followed by a steady state. Relative up to 24 hours (Figure 11a). When 4 g of 10% DHEA gel or cream was applied to a 30 cm area of thigh skin, serum DHEA levels only started to increase at 12 h, reaching values of 2.0 8.2 and 1.2 8.0 nmol/L, respectively at 24 h. hour (Figure 11a). There was no significant difference between the cream and gel in terms of serum levels of DHEA at any of the time intervals studied up to 24 hours after the first application of the steroid product to the skin.

When 5-Diol was measured in serum after oral administration of DHEA, the concentration of 5-Diol increased from the pre-treatment concentration of 0.31 0.03 ng/ml to a maximum value of 1.19 0.13 ng/ml at 1 hour with a slow and continuous decline thereafter to reach to 0.79 0.05 ng/ml at 24 hours (Figure 11b). It can be seen that in the same figure, serum 5-Diol levels increased more slowly after transdermal administration of DHEA with the cream or gel to reach statistically significantly different values of 1.00 - 0.14 ng/ml for the cream and 0.72 - 0.14 ng/ml for the gel. 24 hours.

After oral administration of DHEA, serum 4-dione increased from 0.6 0.1 ng/ml to a maximum value of 9.5 2.2 ng/ml at 1 h and was followed by a rapid decline to values at which it remained at a relative steady state of approximately 1.2 ng/ml between 8 and 24 hours (Figure 12a). After administration of DHEA on the other hand by cream or gel, the first increase of serum 4-dione was observed only at 24 h with values of 0.9 0.1 and 0.8 0.1 ng/ml for cream and gel, respectively.

A comparative pattern was observed for serum testosterone. In fact, after oral administration of 50 mg DHEA capsules, 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 hours and was followed by a relative stability thereafter until 24 hours (Figure 12B). When DHEA was used as a cream or gel, the first increase was observed at 24 hours and was approximately 0.45 ng/ml. As observed in Figures 13a and 13b, the first administration of DHEA by the oral or transdermal route had no statistical effect on serum levels of E1 or E2 during the first 24 hours.

On the other hand, serum DHEA-S followed a similar pattern, although slightly delayed compared to DHEA and 5-Diol after oral administration with two 50 mg DHEA capsules (Figure 14a). Serum DHEA-S therefore increased from 0.4 ± 0.1 μg/ml to 7.7 ± 1.0 μg/ml at 1 hour to a maximum value of 8.4 ± 0.6 μg/ml at 2 hours with a continuous decrease to 2.7 ± 0.3 μg/ml at 24 hours. No change in serum DHEA-S was observed during the first 24 hours after administration of DHEA in cream or gel form. On the other hand, serum E1-S does not change significantly during the first 24 h after the first 24 h.

Serum ADT-G, a major metabolite of androgens, increased from 3 14 ng/ml to 150 760 ng/ml at 1 h and 140 790 ng/ml at 2 h and then decreased continuously to 5 92 ng/ml. at 12 hours and 70 5 ng/ml at 24 hours (Figure 15a). On the other hand, it increased from 2.2 0.5 ng/ml to 14.5 2.0 ng/ml at 2 hours (Figure 15b). Therefore however the observed reduction of 3 α- diol - G was slower than that of ADT-G, and only a 40% reduction was observed between 2 and 24 hours after oral DHEA administration. After applying 4 grams of 10% DHEA to the skin, there was no significant change in serum ADT-G or 3α diol G up to 24 hours (Figure 15b).

When measurements of the same kinetic parameters were repeated on day 14 for daily dosing, it could be seen that administration of two 50 mg DHEA capsules led to a pre-dosing value of 0.4 4.2 ng/ml, to a maximum concentration of 4.4 14.8 ng DHEA/ml at 1 hour. This was followed by a continuous decrease after that to 4.5 0.4 ng/ml at 24 hours (Figure 16a). On the other hand, when DHEA was administered by cream or gel, no distinct changes were observed during the 24-hour period and serum DHEA remained between 10 ng/ml and 15 ng/ml after cream application, and between 7 ng/ml and 11 ng. /ml after applying the gel.

Similarly, when serum 5-Diol was measured on day 14, the serum concentration of this steroid increased from 0.46 ± 0.04 ng/ml to 1.37 ± 0.21 ng/ml at 1 h with a slow decline thereafter to 0.64 ± 0.06 ng/ml at 24 h. (Figure 16b). As observed for DHEA, serum Diol remained nearly constant during the 24-hour period at approximately 1.5–1.9 ng/ml following cream application and 1.0–1.3 ng/ml following gel application.

When serum 4-dione was measured on day 14 of dosing, the serum concentration of this steroid increased from 1.3 0.2 ng/ml to a maximum value of 1.7 9.8 ng/ml and was followed by a rapid decrease to 0.1 1.5 ng/ml at 6 hours with a value of 0.1 1.2. ng/ml at 24 hours (Figure 17a). After applying DHEA to the skin as a cream or gel, there was no significant increase of serum 4-dione to about 2.5 ng/ml at 2 hours with values that then remained relatively stable at 1.0–1.6 ng/ml up to 24 hours (Figure 17A). )).

Serum testosterone increased on day 14 of dosing after oral administration of 100 mg of DHEA from 0.31 0.04 ng/ml to a maximum value of 0.83 0.11 ng/ml at 1 h and was followed by a continuous decrease to a value of 0.37 0.04 ng/ml at 24 h (Figure No. (17B)). After applying DHEA as a cream or gel, serum levels of testosterone remained unchanged during the 24-hour period at approximately 0.3 ng/ml, and this value was not statistically significantly different from the pre-treatment value. Remarkably, on day 1, there was no significant change in serum levels of E1 (Figure 18a) or E2 (Figure 18e) during the 24 hours followed by the 14th daily administration of DHEA by oral or transdermal administration.

Serum DHEA-S increased from the pre-dosing level of 1.95 ± 0.15 μg/ml to 8.3 ± 0.4 μg/ml at 1 hour and then decreased continuously to 2.6 ± 0.3 μg/ml at 24 hours (Figure 19A). No significant changes of serum DHEA-S were observed after application of DHEA to the skin. On the other hand, serum E1-S did not change during 24 hours after the 14th daily administration of DHEA by oral or transdermal administration (Figure 19B).

While serum ADT-G started on day 14 at a higher level than on day 1, it increased rapidly from 1,66 ng/ml to 105,996 ng/ml at 1 h and then decreased continuously to 116 ng/ml at 12 h. and 91 15 ng/ml at 24 hours (Figure (20a)). There was no significant change in serum ADT-G levels after application of DHEA to the skin. On the other hand, serum 3α diol G increased from 12 2.5 ng/ml to 29.4 5.5 ng/ml at 2 hours, decreasing slowly after that and reaching 13 3.0 ng/ml at 24 hours after the 14th time of daily oral administration of 100 mg of DHEA. No significant changes in serum 3α diol G were observed after transdermal administration of DHEA (Figure 20B).

To obtain a more accurate measure of the accumulation of DHEA and its metabolites, we compared the areas under the curve of serum steroid concentrations (AUC values from 0 to 24) measured on days 1 and 14 of dosing. As expected from Figure No. (11), Figure No. (12), Figure No. (13), Figure No. (14), Figure No. (15), Figure No. (16), Figure No. (17), and Figure No. (18). ), Figure No. (19), and Figure No. (20), the AUC values were similar from zero to 24 hours for all steroids, except that the estrogen (E1-S) and androgen (ADT-G and 3α diol - G) metabolites, as a result of some accumulation Those steroids, so on the first day and day 14 to give DHEA orally (Table No. (4)). On the other hand, following on-the-skin administration of DHEA, and because DHEA is absorbed more slowly following administration in cream or gel form, 155% or 86% of higher AUC0-24 h values for DHEA were observed on day 14 compared to day 1 of Give the dose in order. Higher values were also observed for each of the other steroids, except for E1, E2, and testosterone which did not show this.

Table 4:

AUC0-24 h values measured on day 1 and day 14 of dose administration, in addition to their percentages.

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)

3α-diol -

G(ng h/ml)

2 50 mg capsule

First sampling

153 (19 )

19.0 (20)

40.2(39)

9.47 (31)

754 (30)

136 (20)

108 (20)

4.81 (39)

4112(24)

259 (44)

Sampling XIV

144 (26)

20.4 (25)

34.6 (27)

9.72 (23)

910 (23)

165 (25)

95.0 (16)

7.44 (36)

5607 (28)

453 (41)

Fourteenth/first

0.94

1.07

1.07

1.08

1.22

1.21

0.88

1.55

1.36

1.75

4 g of 10% cream

First sampling

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)

Sampling XIV

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)

Fourteenth/first

2.55

2.90

1.85

1.00

1.24

1.19

1.86

1.65

2.42

2.25

4 g of 10% gel

First sampling

101 (49)

103 (55)

13.3 (45)

8.76 (11)

620 (31)

214 (137)

11.2(35)

5.53(84)

254 (30)

38.3 (86)

Sampling XIV

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)

Fourteenth/first

1.86

2.64

1.60

0.96

1.27

0.71

1.66

1.65

1.79

1.57

Values in parentheses represent the percentage coefficient of variation. DHEA was given orally (2 50 mg capsules) or following skin application of 4 g of 10% DHEA cream or 4 g of 10% gel.

As can be clearly observed in Table 5 and Figure 21, there was no significant change in the AUC0-24 h values for serum E1, E2, or testosterone measured on day 14 post-dosing compared to pre-dosing levels. But significant increases have been observed in all of the other steroids. Thus, following oral administration of 100 mg of DHEA daily for two weeks, the area under the DHEA concentration curve measured during the 24 hours following steroid administration increased by 167% over the initial treatment value while an increase in both 5-diol and , 4-Dione, DHEA-S, E1-S, ADT-G and 3-alpha-diol-G were 138%, 238%, 873%, 60%, 182% and 874%, respectively.

Table 5 Initial treatment and AUC0-24 h values for DHEA and its metabolite metabolites at 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)

3α-diol -

G(ng h/ml)

Basal

(initial treatment)

53.9

8.56

12.9

8.72

717

135

9.76

4.64

929

46.6

(a) 2 50 mg capsules

Day fourteen

144

20.4

43.6

9.72

910

165

95.0

7.44

5607

453

Fourteenth / basal

2.67

2.38

3.38

1.11

1.27

1.22

9.73

1.60

19.2

9.74

(b) 4 g of 10% cream

Day fourteen

273

39.7

22.8

8.77

847

175

19.9

7.96

977

114

Fourteenth / basal

5.06

4.63

1.77

1.01

1.18

1.30

2.04

1.72

3.34

2.45

(c) 4 g of 10% gel

Day fourteen

188

27.2

21.3

8.04

785

152

18.6

9.11

455

60.3

Fourteenth / basal

3.49

3.18

1.65

092

1.09

1.13

1.91

1.96

1.56

1.30

DHEA is given orally or through the skin in cream or gel form. Basal AUC0-24 h values were calculated by multiplying basal serum steroid levels for initial treatment including screening within 24 hours.

With the exception of DHEA and 5-Diol, smaller increases have been observed following administration of DHEA cream or gel. In fact, following DHEA cream application, the AUC0-24 h value for DHEA-S increased by only 104% while the AUC0-24 h values for 4-Dione, E1-S, ADT-G, and 3 alpha-diol G increased by 77%, And 72%, 234%, and 145% for the standard sample, respectively. On the other hand, the AUC values of DHEA and 5-Diol increased by 406% and 363%, respectively (Table 5, Figure 21). Similar but smaller increases were observed for DHEA gel, where AUC values 0-24 h for 4-Dione, DHEA-S, E1-S, ADT-G, and 3 alpha-diol G increased by 65%, 91%, 96%, and 56%. And 30% over the standard sample, while the AUC0-24 h values for DHEA and 5-Diol increased by 249% and 238%, respectively.

Our recent results (Labrie, Belanger et al., 2007b) demonstrated that the changes in serum DHEA observed following exogenous DHEA administration amounted to an almost 100% overestimation of the true changes in sex steroid composition. In support of these data, Figure 21 shows that following administration of DHEA in cream or gel form, changes in serum DHEA were characterized as overestimates of changes in serum levels of all steroids measured except for 5-Diol, the immediate metabolite of DHEA. For DHEA cream, the changes in AUC values 0-24 h for serum 4-Dione, DHEA-S, E1-S, ADT-G, and 3-alpha-diol were only 77%, 104%, 72%, 234%, and 145%. Compared to a 406% increase over initial treatment levels of serum DHEA.

For androgenic hormones, it is well established that uridine glucuronosyl transferase 2B7 (UGT 2B7), UGT 2B15, UGT 2B17 are the three enzymes responsible for the glucuronidation of all androgen hormones and their metabolites in the human body (Belanger et al. 2003). This recent recognition of the identification of all human UDT-glucuronosyl transferases makes it possible to use glucuronidation derivatives of androgen hormones as markers of full androgen activity in both women and men (Labrie, Belanger et al., 2006; Labrie, Belanger). , et al, 2007b and Swanson et al 2007). Accordingly, given the metabolite metabolism of all the androgen hormones in ADT-G and 3 alpha-diol-G, the percentage efficiency of transdermal DHEA to convert to active androgen hormones is estimated at 52% when adding the changes in ADT-G and

<img file="SA3067B1_D0001.tif" />

(Weighted value of 221% compared to DHEA changes of 406%). Likewise, following administration of DHEA gel, the 249% increase of serum DHEA translates into increases of only 65%, 91%, 96%, 56%, and 30% in AUC0-24 h values for the 4-Dione, DHEA-S, and E1-dione hormones. S1, ADT-G, and 3α-diol-G serology, in order.

Because an elevated level of glucuronidation in the intestine and liver explains the elevated serum level of ADT-G and 3α-diol-G (Belanger et al. 2003) following oral DHEA administration, the relatively small increase in serum E1-S (60 %) compared to the 167% increase of serum DHEA after oral DHEA administration indicates a 36% relative efficacy of conversion to estrogen. As previously shown (Labrie, Belanger et al., 1997; Labrie, Belanger et al., 2006; Labrie, Belanger, et al, 2007b), current data indicate that DHEA administered to menopausal women is often Converting it to androgen hormones instead of estrogen hormones.

discussion

The present data clearly show that during chronic treatment with DHEA in cream or gel form, the concentration of each steroid quickly reaches a level that does not entail a detectable change in the serum concentration of any of the steroids measured during daily application of DHEA to the skin. Accordingly, starting 24 hours after the first administration of DHEA on the skin, the concentration of all steroids remains at the same level, which thus indicates that daily application of DHEA to the skin maintains constant serum levels of DHEA and all metabolites. His own. In menopausal women, it is already known that the variation in the circadian rhythm of serum DHEA is relatively small compared to the situation in non-menopausal women with normal menstruation (Lui and Laughlin, 1990).

The present data also show that following daily oral administration of DHEA, there is no significant accumulation of DHEA or its metabolites. Furthermore, the metabolism of DHEA metabolite following oral or percutaneous administration is quantitatively similar, and quantitative differences are explained by entero-hepatic metabolism following oral administration.

The higher AUC0-24 h values for serum DHEA-S, ADT-G, and 3-alpha-diol-G combined with the lower AUC0-24 h values for DHEA and 5-Diol following oral administration relative to dermal administration indicate However, metabolite metabolism through the gastrointestinal tract and/or the hepatic pathway leads to a high level of conversion of DHEA to DHEA-S through DHEA-SULFOTRANSFERASE activity (Luu-The et al., 1995) in addition to the metabolite DHEA being further metabolized into androgen hormones and not being activated through the activity of liver glucuronosyltransferases (Belanger et al. 2003; Turgeon et al., 2001 and Hum et al., 1999). In fact, as shown in Table 1, DHEA exposure of 144 ng h/mL (AUC0-24 h) on day 14 of oral administration of 100 mg DHEA results in AUC0-24 h values of 5607 ng h/ml and 453 ng h/ml for ADT-G and 3α-diol-G, respectively. On the other hand, after on-the-skin administration of 10% DHEA cream, the AUC values for DHEA, ADT-G, and 3α-diol-G are 273 ng/ml, 977 ng/ml, and 114 nano g/h/ml, respectively. Therefore, after oral administration, 1 ng h/ml of DHEA corresponds to an AUC value of 42.1 ng h/ml for the combination of the two androgen metabolisms (ADT-G + 3 alpha-diol -G) while following application of DHEA cream, Exposure to 1 ng h/ml of DHEA corresponds to 4 ng h/ml for the total metabolite of androgenic hormones. Such data indicate that oral administration of DHEA results in approximately ten times greater levels of conversion of DHEA to ADT-G and 3-alpha-diol-G than intradermal administration, at least at the doses that were used. When the same calculations are made for the data obtained after administration of DHEA in gel form, a DHEA exposure of 1 ng h/ml is associated with an AUC0-24 value of 2.7 ng h/ml for ADT-G + 3 alpha-diol -G. , which thus indicates a higher ratio between oral and dermal administration of DHEA.

As shown in Table 4, while an AUC0-24 h value for DHEA of 1 ng h/ml leads to a ng h/ml value of 660 ng h/ml for DHEA-S following oral administration of DHEA, it was observed Corresponding values of 73 ng/ml and 99 ng/ml after using the steroid substance produced in the form of a cream or gel. Therefore there is a multiple of 6.7-9 times the amount of DHEA-S in the circulation following the same exposure to DHEA in the circulation (serum AUC value 0-24 h) after oral administration of DHEA relative to the dermal route under the test conditions. The current data show a similar effect of the passage of DHEA through the intestinal tract and the liver on the serum hormones DHEA-S, ADT-G, and 3-alpha-diol-G.

Although a smaller difference was observed, relatively higher levels of serum 4-Dione were observed after oral administration of DHEA compared to intradermal administration of the steroid product. Therefore, for oral administration of DHEA, a value of 1 ng h/ml AUC0-24 h for DHEA results in an AUC0-24 h value for 4-Dione of 0.3 ng h/ml while values of 0.08 ng h/ml have been observed. ml and 0.11 ng h/ml after administration of DHEA as cream and gel, respectively. As measured in the circulation, the conversion of DHEA to 4-Dione is 2.70-3.76 times higher than that of oral administration of DHEA compared to the transdermal route.

The data in Table 4 show that the AUC0-24 h value of DHEA increased by 167% over the standard sample following daily oral administration of 100 mg of DHEA compared to basal levels of initial treatment while daily intradermal administration of 4 g of 10 % DHEA cream increased serum DHEA levels by 406% and 249%, respectively. Given 400 mg of DHEA applied to the skin compared to 100 mg orally, and assuming linearity, the current data indicate that the oral route is 2.9 to 4.8 times more effective than the formulation used for DHEA cream and gel, respectively.

In a study also performed in postmenopausal women, oral administration of 150 to 300 mg of micronized DHEA maximized serum DHEA-S, DHEA, and testosterone levels of 1.5 mg/mL, 15 ng/mL, and 2.75 nM. g/ml after a dose of 300 mg, 10 μg/ml, 12 μg/ml, and 1.6 ng/ml after a DHEA dose of 150 mg, respectively (Buster et al. 1992). Examination of these early results showed that a 20-fold increase in serum DHEA-S resulted in only a 6.9-fold increase in testosterone while serum DHEA increased by 11.6-fold. Furthermore, when measured serum testosterone values were adjusted to one-third to account for non-specific two-thirds binding in the radioimmunoassay, serum testosterone levels remained within the range of physiological levels during the 12 hours following administration of a 150 mg DHEA dose (Buster et al. 1992). .

Similar differences observed between oral and on-the-skin administration of serum DHEA were observed for 5-Diol that was converted directly from DHEA by 17.beta.-hydroxysteroid dehydrogenase (Labrie, Luu-The, et al. 2000 ). In fact, while the AUC0-24 h value for 5-Diol increased approximately 138% over the standard sample after oral administration of 100 mg DHEA, increases of 363% and 218% were measured after application of approximately 400 mg DHEA cream and gel.

According to what was mentioned, humans are unique, in addition to some other primates, in that they contain adrenal glands that secrete large quantities of the steroids producing DHEA and DHEA-S, which are converted to 4-Dione, and then to androgen and/or hormones. Estrogen hormones are effective in intracrine peripheral intercellular tissue (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). Therefore, it is noted that humans, in addition to having endocrine disorders and very complex neighboring cells, are largely responsible for the formation of sex steroids in peripheral tissues (Labrie, 1991) and (Belanger, et al., 1998). In fact, while the ovaries and testes are the exclusive sources of secretion of androgen and estrogens in lower mammals, the situation is different in humans and higher primates, where partially or completely active sex steroids are generally synthesized locally in peripheral tissues, so target tissues with control elements are available. By adjusting the formation and metabolism of sexual steroid metabolites according to local requirements.

Adrenal secretion of DHEA and DHEA-S increases during adrenarche in children aged 6 to 8 years, and maximum values of circulating DHEA-S in the blood are reached between the ages of 20 and 30 years. Therefore, serum levels of DHEA and DHEA-S are significantly reduced (Belanger et al. 1994). In fact, by the age of 70, serum DHEA-S levels decrease to about 20% of their peak values while they can decrease by 95% between the ages of 85 and 90 years (Belanger et al. 1994) and (Migeon et al., 1957). The 70-95% decrease in the formation of DHEA and DHEA-S by the adrenal glands during aging leads to a significant decrease in the formation of androgen hormones and estrogens in the target peripheral tissues (Labrie, Belanger et al., 2006). Such a marked decrease in the formation of sex steroids in peripheral tissues may also be implicated in the origin of a series of age-related diseases.

As previously mentioned, the conversion of DHEA and DHEA-S into active androgen and/or estrogen hormones in target peripheral tissues depends on the level of genetic expression of the various steroidogenic and metabolite enzymes in each cell type (Labrie, 1991). Elucidating the structure of most of the tissue-specific genes encoding the steroidogenic enzymes responsible for converting DHEA and DHEA-S into androgen and/or estrogen hormones has led to significant progress in this field (Labrie, Belanger et al., 1995; Labrie, Luu-The et al., 1997; Labrie, Simard et al., 1996; Labrie, Luu-The et al., 2005; Labrie, Luu-The, et al 1992, Labrie, Simard et al, 1992; Luu-The, et al 1995;

Data showing relatively high levels of androgen metabolite metabolites in normal women (Labrie, Belanger, et al. 1997; Labrie, Belanger et al., 2006; Labrie, and Belanger, et al, 2007b) strongly suggest that androgen hormones play a role in It plays a major physiological role in women, but this role is not properly appreciated. The 44.5% decline in serum DHEA between 20 and 30 to 40 and 50 years of age in women (Belanger et al., 2006) could strongly explain the bone loss that precedes menopause. In fact, age-related bone loss has been reported to begin in the 40s and the bone regeneration cycle has been discovered before premenopausal women completely cease (Mazess 1982; Riggs, et al. 1981, and Johnston et al. 1985). According to these findings, bone density was lower at all sites examined in women classified as near-menopause than in premenopausal women (Steinberg, et al., 1989). According to these findings, changes in androgen secretion, a substance produced by the adrenal glands, precede by about 10-20 years the decrease in ovarian estrogen secretion that suddenly stops at menopause (Labrie, Belanger, et al. 2006).

It is important to realize that not only has a decline in serum DHEA and DHEA-S been observed by approximately 50% between years 21 and 50 but a similar decline has also been observed for serum testosterone (Zumoff et al. 1995). Such data may also confirm that hormone replacement therapy using androgen hormones or their-producing substances should be started early at menopause in order to compensate for the early decrease in the secretion of androgen-producing substances by the adrenal glands and the corresponding decrease in testosterone. Al-Musalla (Labrie, 2006).

Active androgen hormones and estrogens synthesized in the target peripheral tissues exert their activity in the cells of origin and very little diffusion of active sex steroids occurs, resulting in very low levels in the circulation. In fact, as observed previously (Labrie, Belanger et al., 1997) and confirmed in the present study, the most striking effects of DHEA administration were observed on the levels of glucuronidation derivatives of DHT metabolite metabolites in the circulation, i.e. more specifically ADT-G. and 3 alpha-diol-G, while no significant changes were observed or very slight changes were observed in the serum levels of testosterone, E1 or E2. These active steroids are produced locally in peripheral intracrine tissues that possess the appropriate steroidogenic enzymes for DHT synthesis from the adrenal substances producing DHEA and DHEA-S as well as enzymes that convert DHT to the inactive metabolite metabolites ADT and 3-alpha-diol, which are also modified by glucuronidation. (Belanger et al. 2003).

In a recent study, daily oral administration of 50 mg of DHEA did not significantly affect testosterone or serum DHT while increasing DHEA and ADT-G to a similar extent (80–90%) (Arlt et al. 2001). In another study, pre-dosing serum levels of DHEA-S in postmenopausal women increased from 0.55 mcg/ml to approximately 1.4 mcg/ml (Casson et al. 1998), after daily oral administration of 25 mg of DHEA for 6 months. However, serum DHEA and testosterone levels, measured 23 hours after the last DHEA administration, did not change significantly. Another study indicated that a daily oral dose of 50 mg of DHEA resulted in serum androgen levels in the premenopausal range (Buster et al. 1992).

The present data clearly demonstrate that DHEA and DHEA-S are converted in specific peripheral intracrine tissues into active androgen and/or estrogen hormones that can exert their bioactive effects at the site of their synthesis with no, or only little release, of active steroids into the circulation. Therefore, serum levels of testosterone, E1 or E2 cannot be used as variables to convert DHEA to androgen or estrogen hormones (Labrie, Belanger et al., 2006). In fact, active metabolite steroids are metabolized in the same cells in which they were synthesized and exert their effect in inactive metabolites to which a glucuronidated and sulfated group has been introduced that 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). Measurement of conjugated metabolites of androgen hormones is the only method that allows an accurate assessment of the total androgen hormone pool in women. It is likely that estrogens will have a similar situation, although careful evaluation of the pharmacokinetic properties of estrogen metabolism and the identification of their metabolites will need to be determined.

Example No. 3

Clinical trial Analysis Populations

Intravaginal DHEA, Physiological Treatment of Vaginal Atrophy

Topics and methods

This study is a three-phase, prospective, multicenter, randomized, placebo-controlled, double-blind trial of 50 patients per arm (for a total of 200 patients). So two hundred postmenopausal women were randomly assigned to receive an egg of the following concentrations of DHEA daily: 0%, 0.25% (3.25 mg of DHEA), 0.5% (6.5 mg of DHEA) or 1.0% (13 mg of DHEA). of DHEA) which is placed inside the vagina. The study was divided into two phases, meaning more specifically screening was followed by a 12-week treatment period.

The selection criteria were as follows:

- Postmenopausal women who fit (A), (B), or (C):

a. Absence of menstruation for at least one year Normal clear secretions noted on vaginal walls, or;

B. FSH levels greater than or equal to 40 mlU/mL (within 60 days before day 1) in women who have had no menstruation for greater than or equal to 6 months but less than 12 months, or hysterectomized women who are premenopausal women at the time of hysterectomy or;

C. Six weeks or more after the screening visit following bilateral oophorectomy.

- Women who self-identify with at least one of the following moderate to severe symptoms:

Vaginal dryness (none, mild, moderate, or severe).

Vaginal and/or vulvar irritation/itching (none, mild, moderate, or severe).

Vaginal pain associated with sex ual activity (none, mild, moderate, or severe).

Women should recognize which symptom is most bothersome to them when starting treatment. This presentation will come next, and will evaluate the effect of the treatment.

- Women aged between 40 and 75 years.

- Women who wish to participate in the study and who agree to sign a written consent.

- Women with a small maturation index (the guiding fraction is no more than 5% of the surface cells on the vaginal sample).

- Women with a vaginal pH higher than 5.

- Normal mammography within 9 months of starting the study.

- Normal breast examination.

- A normal PAP sample (including inflammatory changes) within the last 12 days (from day 1). For women who have had a hysterectomy, the PAP sample will consist of at least one slide.

- Absence of former or present narcotic addiction or alcoholism.

- A body weight in the range of 18.5 to 35 of the typical body weight according to the Body Weight Index (BMI) (WHO).

- The absence of a hepatic or renal impairment or condition known to affect the metabolism of the drug or steroid metabolite.

- Normal baseline hematology blood characteristics, clinical chemistry, and urinalysis at baseline.

- Negative serology for all negative serology of HIV1/HIV2 and hepatitis B and C.

The exclusion criteria from the study were as follows:

- Undiagnosed abnormal genital bleeding.

- Previous diagnosis of cancer, with the exception of skin cancer (non-melanoma).

- Endometrial hyperplasia with a live sample upon examination or endometrial cancer.

Active or history of thromboembolic disease.

- Marked metabolic or endocrine disease.

- Clinically significant gastrointestinal, liver, or gallbladder disease.

- Recurring migraine headaches that are not controlled by conventional treatment.

Diabetes that is not controlled with conventional treatment.

- Noticeable complications when receiving prior hormonal treatment.

- Use of estrogen-only injectable drug therapy or progestin implant within 3 months before entering the study (screening visit).

- Use of an injectable drug containing estrogen or progestin within six months before entering the study.

-Take oral estrogen, progestin, DHEA, or intrauterine progestin therapy in the 6 weeks before evaluation at baseline.

-Vaginal hormonal products (rings, cream, or gel types), transdermal estrogen alone, or estrogen/progestin products in the 4 weeks before evaluation at baseline.

Patients may bathe as follows, but must answer the Vaginal Atrophy Questionnaire after the required bathing period:

A washout period of at least 8 weeks for prior treatment with estrogen, DHEA, and/or oral progestin.

A washout period of at least 4 weeks for prior oral transdermal hormone therapy.

A washout period of at least 4 weeks for hormone replacement therapy given topically to treat vaginal dryness.

At least six months of estrogen therapy or progestin injectable drug therapy.

Eight weeks or longer for prior intrauterine progestin therapy.

Six months or longer for prior progestin implants or estrogen-alone injectable drug therapy.

- Pre-treatment with androgen hormones and anabolic steroids within 3 months before the screening visit.

- Oral corticosteroid therapy within six months of starting the study.

- Not using the permitted continuous use of corticosteroid therapy (allowing intermittent or local nasal spray on the skin, in the eyes, or in the ears).

- Heart failure or manifest coronary heart disease.

- High blood pressure that is greater than or equal to 160/95 mmHg or that is not controlled by standard therapy.

- Clinically significant depression or confirmed history of severe psychiatric disturbance.

- Taking a drug under trial within 30 days of the examination visit.

- Relevant abnormal clinical biochemical or hematological properties.

- Cytologic features of the cervix at the base line showing the presence of psoriatic limited intraepithelial disease (LGISIL) or worse.

- Smoking more than 10 cigarettes per day.

- Taking drugs that interfere with the metabolism of estrogen hormones (for example, ketoconazole, action inhibitors or steroids).

- SERMs or a drug that interacts with steroid receptors.

- The known presence of uterine fibroma or its appearance in a gynecological examination.

- Coagulation disorders or when receiving anticoagulant drug therapy.

Laboratory Tests

The usual laboratory tests, i.e. blood tests (including complete blood count and coagulation), blood chemistry, and urinalysis were performed at all visits. Serum FSH should only be measured in women who had been menstruating for more than 6 months but less than 12 months or who were premenopausal at the time of hysterectomy. Levels were measured:

DHEA, DHEA-S, androst-5-ene-3.beta.,17.beta.-diol (5-diol), dihydrotestosterone (DHT), testosterone (testo), androstenedione (4-dione), estrone (E. sub.1), estradiol (E.sub.2), E.sub.1-S, androsterone glucuronide (ADT-G), androstane-3.alpha.,17.beta.-diol-3G (3.alpha. -diol-3G) and 3.alpha.-diol-17G in serum in the Laboratory of Molecular Endocrinology, CHUL Research Center using mass spectrometry as described (Labrie, Belanger et al. 2006; Labrie, Belanger et al. . 2007; Labrie, Cusan et al. 2008).

Vaginal pH and Cytology

For maturation parameters and PAP samples, all samples were examined by the same cytopathologist (Dr. Robert Dube, Department of Cytopathology, Enfant-Jesus Hospital, Quebec City, Canada) who was blinded to the treatment regimens. 100 cells were counted to classify cells as superficial (S), intermediate (I), parabasal (P) and squamous (Meisels 1967; Wied 1993).

Vaginal specimens were obtained by peeling the middle third of the lateral wall of the vagina using the rounded end of an Ayre flat-tip knife. This material was then placed on a glass slide and immediately mounted using Spray-Cyte. The samples were then sent to the central laboratory to determine the maturity factor.

Vaginal pH was measured by placing a pH strip directly on the side wall of the vagina using forceps. For the Papanicolaou sample, if not performed in the last 12 months, samples are obtained from the endocervix, exocervix, and vaginal vault, and fixed using cytoreduction. Samples were collected using an Ayre knife.

Endometrial Biospy

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

Vaginal Examination

At the same time periods of 2, 4, 8, and 12 weeks, the gynecologist or study physician at each site performed a vaginal examination to assess the degree of risk (none, mild, moderate, or severe, using values of 0, 1, 2, and 3). In order) for the main signs of vaginal atrophy, more specifically vaginal secretions, vaginal color, integrity of the vaginal epithelium, and thickness of the surface of the vaginal epithelium. As can be seen in Figures 26 to 29, time-dependent, dose-related, and statistically significant improvement was observed for all four signs of vaginal atrophy. In fact, the beneficial effects observed by the gynecologist and/or supervising physician can be almost identical to those reported by women themselves about their most bothersome symptoms.

Vaginal examination was performed at screening and then at day 1 and weeks 2, 4, 8, and 12. Vaginal discharge, vaginal color, vaginal epithelial integrity, and vaginal epithelial surface thickness were evaluated according to the following severity scores: none, minor, moderate, or severe. The definitions of risk were as follows:

A) Vaginal secretions

No atrophy:

Normal clear discharge observed on the vaginal walls

Mild: a superficial coating of secretions, and difficulty in inserting the speculum.

Moderate: A very slight discharge that covers the entire vaginal vault and may need to be lubricated with a speculum to prevent pain.

Severe: nonexistent discharge, inflamed, ulcer noted, needing lubrication with speculum to prevent pain.

B) Vaginal Epithelial Integrity

No atrophy: normal.

Simple: the presence of blood on the surface of the vagina with scraping.

Moderate:

The presence of blood on the surface of the vagina with light contact.

Severe: The vaginal surface includes bleeding spots before contact and bleeds with light contact.

c) Vaginal epithelial surface thickness

Rugation and elasticity of the vault.

Simple: Poor rogation and some elasticity noted of vaginal vault.

Moderate: Smooth, with some elasticity to the vaginal vault.

Severe: smooth, no elasticity, constricts of the upper 1/3 of the vagina or loss of vaginal tone (cystocele and rectocele hernia).

D) Vaginal Color

No atrophy: Pink.

Simple: lighter colour.

Moderate: pale in color.

Severe: transparent in color, either colorless or fluorescent.

Statistics statistics

Summary tables will be prepared that display a number of observations, the mean or geometric mean as necessary, the standard deviation, the standard error of the mean, a two-sided 95% confidence interval (CI), the mean, minimum, and maximum for continuous variables, and the number and percentage. For each category for categorical data. Statistical analyzes will be performed at a two-sided significance level of 0.05 unless otherwise stated. The summary categories will generally consist of dose levels for DHEA treatments, 0% (satisfactory), 0.25%, 0.5%, and 1% DHEA.

The primary endpoints of the analysis will consist of the following:

The statistically significant improvement in the moderate to severe symptom that the patient identified as being most bothersome to her. The severity of the symptom is based on symptoms of increasing severity: none, mild, moderate, or severe. These estimates will be analyzed using values 0, 1, 2, and 3, respectively; All patients must have at least one symptom at the base, rated as 2 or 3. Symptoms of interest are vaginal dryness, vaginal activity and/or vulvar irritation/itching, and vaginal pain associated with sex ual.

A statistically significant decrease in cells adjacent to the base and a statistically significant increase in superficial cells. Data is measured in percentage. The maturity value will also be calculated.

A statistically significant decrease in vaginal pH.

Statistical groups

The intent-to-treat (ITT) group will consist of cases treated with an efficacy assessment at baseline and at least one efficacy assessment after baseline. Patients who may have received the wrong treatment will be randomly analyzed. This analysis group should be viewed as the main analysis group. For patients in this group who will not experience feedback beyond baseline, their last value will be pushed forward for efficacy analyses.

A per-protocol (PP) group consists of the subset of the treated group that completes the study within the 12-week time point without significant protocol violations that are perceived to compromise efficacy data. Major protocol violations will be identified before study unblinding based on review of data sheets and monitoring reports of protocol deviations. Patients in the PP group must receive at least 90% of the required number of study treatment orders in the duration specified by the protocol for that patient, based on the patient's daily data. Patients in the PP group must adhere to the visit schedule: 3 days for day 14, and 7 days for weeks 4, 8, and 12. For patients who received the wrong treatment, and for whom the treatment they received can be clearly confirmed, they will be analyzed in the PP group according to their treatment, provided that there are no other violations that spoil their data. The PP group will serve as a support group for the analysis of efficacy data.

The safety population will be defined as all female patients given either test product (DHEA at any dose or satisfactory treatment) and for whom any safety information is available. All safety data analyzes will be based on this group. The analysis will be based on the treatment actually received.

Evaluation of effectiveness

Efficacy analyzes will be performed primarily on the ITT cohort, and the PP cohort will provide supporting efficacy analyses. The primary objective of the study is to evaluate the dose-response of vaginal mucosal treatments for topical DHEA in postmenopausal women with vaginal atrophy, specifically by administering the lowest dose of DHEA that produces the maximum effect on vaginal mucus. The major combined endpoints that meet this goal are decreased in parabasal cells, decreased in vaginal pH, and increased in superficial cells (these endpoints will be referred to together as physiological parameters). The most disturbing symptom for the patient to report on herself includes: Vaginal dryness, vaginal or vulval irritation/itching, and vaginal pain at sex ual activity (and these points will be noted final together as the coefficients of the presentation degrees). In addition to these main endpoints, a maturity value will also be calculated. The women's self-reported symptom scores take the following values: none, mild, moderate or severe and are analyzed using values of 0, 1, 2 and 3, respectively. All endpoints must demonstrate statistically significant effects for the satisfactory drug, so no statistical adjustment for multiple endpoints is needed.

The main time point for analysis will be the week 12 assessment, with data further presented for weeks 2, 4 and 8. The change from baseline to post-baseline assessment will be used for analysis as well as the difference using placebo.

Results

Because parabasal cells usually represent the predominant category in the vaginal sample of postmenopausal women with at least one moderate to severe symptom of vaginal atrophy, they can be seen in Figure 22 and Table 6 as at two weeks of treatment , the lowest dose of DHEA (0.25%) decreased the percentage of parabasal cells by 29.50.51 from 56 to 26.5% while reduction percentages of 37.80.46% and 36.6%, respectively, were observed using 0.5% and 1% DHEA doses in Same time period.

In the standard period of 12 weeks of treatment, reduction percentages of 39.50.57% (p &lt; 0.000001), 45.60.55% (p &lt; 0.000001) and 45.2 0.53% (p &lt; 0.000001) were observed using 0.25% and 0.5 % and 1% of DHEA doses, respectively, while no significant effect was observed in the placebo group at any time point.

While no significant effect was observed at 12 weeks in the placebo group on percentage change in surface cells (Table 6), percentages of increase of 3.96±0.10% (p = 0.0002), 6.71±0.14% (p = 0.000001) and 5% were measured. 92 0.12% (p = 0.000001) in 0.25%, 0.5% and 1.0% of DHEA groups, respectively. It can also be observed that at 0.5% of the DHEA dose, 48% of the maximum effect was reached at 2 weeks while at weeks 4 and 8, 84.8% and 99.0% of the maximum effect was achieved. At the 1% dose of DHEA, the maximum effect was reached already in 2 weeks. Figure 23 shows the absolute values of the percentage of surface cells at different DHEA doses and time periods.

The vaginal pH decreased in the 12 weeks by 0.47, 0.11, from 6.52, 0.13 units in the placebo group (Table 6, Figure 24), while decrease rates of 0.12, 0.11 (p = 0.0005) from 6.49, 0.12 units, 1.35 were observed. 0.3 out of 6.56 0.13 pH units, 1.35 0.13 out of 6.56 0.13 pH units and 1.39 0.14 out of 6.34 0.3 pH units in 0.25%, 0.50% and 1.0% of DHEA-treated groups, respectively (Table 7). In the same table it can be noted that at 0.5% of the DHEA dose, 70.6% and 94.1% of the maximum effect on pH (a decrease 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 achieved only 83% of the maximum effect of 0.5% DHEA at weeks 12. No significant difference in pH change was observed between the 0.50% and 1.0% doses of DHEA at weeks 4, 8, and 12. (Table 7). Figure 24 shows the absolute pH values at different DHEA doses and time periods.

All women needed to have at entry into the study one or more of the following symptoms of vaginal atrophy that they self-rated as mild to severe: dryness, irritation/itching of the vagina or vulva, or vaginal pain at ual sex activity . Symptoms that women self-identified as none, mild, moderate or severe were analyzed using values 0, 1, 2 and 3, respectively. As shown in Table 8, in the 12-week period, the severity of the most bothersome symptoms was reduced by 0.67 ± 0.15 in the placebo group, 1.27 ± 0.16 in the DHEA group by 0.25% (p = 0.004 versus placebo), 1.56 ± 0.15 in The group receiving 0.5% DHEA (p &lt; 0.0001 vs. placebo) and 1.37 ± 0.14 in the group receiving the 1% higher DHEA dose (p &lt; 0.0008 vs. placebo). Figure 25 shows the degree of improvement of the most bothersome symptoms at different doses of DHEA and time periods. Vaginal dryness, vaginal pain at sex ual activity, and vaginal or vulval irritation/itching at the base line were identified as the most bothersome symptoms. In the placebo group, vaginal dryness explained the percentage of improvements observed by study participants.

As shown in Figure 25, the improvement in the most bothersome symptom was indeed significantly different (p = 0.004) at the 0.25% DHEA dose. The percentage of women with no change or worsening of 1 at 12 weeks went from 53.5% in the placebo group to 27.5%, 17.8% and 19.6% in the 0.25%, 0.5% and 1.0% groups, respectively ( Table 9). Improvements across two or three severity categories were observed in 21.8% of women treated with placebo while 50.0%, 53.3% and 47.9% of women receiving 0.25%, 0.5% and 1.0% DHEA formulations reported Such a significant improvement. Only 4.6% of women reported a reduction in severity to none in the placebo group compared to 7.5%, 20% and 10.9% in the same DHEA group. At the same time periods of 2, 4, 8 and 12 weeks, the gynecologist or physician supervising the clinical trial at each study site performed a vaginal examination to assess the degree of risk (none, mild, moderate or severe and analyzed using values 0, 1, 2 and 3, respectively) of For the main signs of vaginal atrophy, i.e. vaginal discharge, vaginal colour, integrity of the vaginal epithelium, and surface thickness of the vaginal epithelium. As can be seen in Figures 26 to 29, a time-dependent, dose-related, and statistically significant improvement was observed for all four signs of vaginal atrophy. In fact, the beneficial effects observed by the gynecologist or physician can almost be matched to those reported by women about their most bothersome symptoms as well as the effects on parabasal and superficial vaginal cells and pH which are objective parameters of the effect of DHEA.

Figures 30 and 31 show mean 24-h serum steroid levels (calculated from AUG0-24h values measured on days 1 and 7 of treatment) for DHEA and 11 of its metabolites taken from a recent study (Labrie, Cusan et al. 2008). ). It can be seen that only serum DHEA and 5-Diol (and 4-Dione on day 1) increase significantly but within the values found in postmenopausal women (Labrie, Belanger et al. 2006). Serum estrogen hormones (E1, E2 and E1S) as well as serum androgen hormones (testo and DHT) were not significantly affected.

Table 6

Change from day 1 in percentage of parabasal cells

and surface cells during topical treatment with increasing doses of DHEA*

Two weeks

4 Weeks

8 Weeks

12 week

Parabasal cells

Zero% DHEA

+ 3.6 0.32

+ 0.02 0.32

- 1.17 0.37

+ 1.04 0.35

0.25% of DHEA

-29.5 0.51

- 38.4 0.51

-40.3 0.55

- 39.5 0.57

0.50% of DHEA

- 37.8 0.46

-43.4 0.50

- 47.8 0.49

- 45.6 0.55

1.0% of DHEA

- 36.6 0.50

-42.5 0.51

- 43.7 0.50

- 45.2 0.53

Surface cells

Zero% DHEA

0.10 0.03

0.37 0.03

0.40 0.03

0.53 0.05

0.25% of DHEA

2.32 0.07

3.38 0.08

3.42 0.09

3.96 0.10

0.50% of DHEA

3.22 0.05

5.69 0.09

6.64 0.11

6.71 0.14

1.0% of DHEA

6.26 0.16

6.64 0.14

6.88 0.16

5.92 0.12

* Average SEM

Table 7: Change from day 1 in vaginal pH during topical treatment with increasing doses of DHEA*

DHEA dosage

Two weeks

4 Weeks

8 Weeks

12 week

Zero% DHEA

-0.23 0.08

-0.37 0.09

-0.51 0.10

-0.47 0.11

0.25% of DHEA

-0.76 0.12

-0.93 0.12

-1.09 0.10

-1.12 0.11

0.50% of DHEA

-0.96 + 0.14

-1.28 0.12

-1.36 0.12

-1.35 0.13

1.0% of DHEA

-1.13 0.12

-1.30 0.12

-1.41 0.12

-1.39 0.14

* Average SEM

Table 8

Change from Day 1 in the most bothersome symptom of vaginal atrophy during topical treatment with increasing doses of DHEA*

DHEA dosage

Two weeks

4 Weeks

8 Weeks

12 week

Zero% DHEA

-0.49 0.16

-0.79 0.16

-0.61 0.16

-0.67 + 0.15

0.25% of DHEA

-0.70 0.17

-1.11 0.16

-1.19 0.16

-1.27 0.16

0.50% of DHEA

-0.98 0.15

-1.36 0.15

-1.370.17

-1.56 0.15

1.0% of DHEA

-1.00 0.15

-1.29 0.14

-1.380.17

-1.37 0.14

* Average SEM

Table 9

Change from Day 1 in most bothersome symptoms in 12 weeks of treatment with 0% (placebo), 0.25%, 0.50% and 1% DHEA. A change in one category (severe -&gt; moderate -&gt; minor -&gt; none) was treated as -1 while a change in two categories was treated as -2, etc...

Change in category

-3

-2

-1

0

+1

Dosages

Percentage of women

Zero% DHEA

4.65

16.3

25.6

48.8

4.65

0.25% of DHEA

7.50

42.5

22.5

25.0

2.50

0.50% of DHEA

20.0

33.3

28.9

17.8

0.0

1.0% of DHEA

10.9

37.0

32.6

17.4

2.17

Examples of pharmaceutical formulations

Below are presented, but not limited to, various pharmaceutical formulations using DHEA as an active sex steroid product. The concentration of the active ingredient can be varied over a wide range as discussed here. The quantities and types of other components that may be included are well known in the art.

Example No. A - Vaginal or oral tablet

Component

Weight percentage

(using the weight of the total composition)

DHEA

5.0

Gelatin capsule

6.5

Lactose

70.5

Starch

18.0

Example B - vaginal or oral tablet

Component

Weight percentage

(using the weight of the total composition)

DHEA

0.50

Whitepsol H-15 base

99.50

DHEA suppositories were prepared using Whitepsol H-15 base (sold by Medisca, Montreal, Canada). Any lipophilic base can be used, including but not limited to butter, cocoa butter, Cotomar, Dehydag base, Fattibase, Hexaride Base 95, Hydrokote, Suppocire, Wecobee, theobroma oil, Japocire, Ovucire, Massa Estarinum, or other combinations of the above. .

Example No. C: Vaginal or topical cream

Component

Weight percentage

(using the weight of the total composition)

DHEA

1.0

Emulsifying wax, NF

18.0

Light mineral oil, NF

12.0

Benzyl alcohol

1.0

Percentage of Ethanol 95% USP

34.0

Purified water, USP

34.0

Vaginal or Oral Gelatin Capsule

Other sex steroid producing substances DHEA can be substituted in the above formulations. More than one product may be included, in which case the combined weight percentage is preferably the weight percentage of the single product given in the examples above.

The invention is described in terms of but is not limited to preferred embodiments and examples. Those skilled in the art will have a practical understanding of the application and broader scope of the invention that is limited by the patentable elements here.