WO2007054791A2 - Androgen glucuronides as markers of androgenic activity - Google Patents

Abstract

Embodiments of the invention provide methods for diagnosis of diseases and disorders related to androgen amount. This is accomplished by detecting androgen glucuronide derivatives, such as androsterone (ADT) and/or androstane-3α , 17β -diol (3α -diol) in blood or tissue. In some embodiments of the invention glucuronide derivatives (or their absence) are detected by mass spectrometry (LC-MS/MS) and/or gas chromatography - tandem mass spectrometry (GC-MS). A low amount of glucuronide derivatives in relation to the mean amount of glucuronide derivatives in a comparable sample population is indicative of androgen deficiency. Further embodiments provide methods for treating skin that exhibits signs of ageing, as well as methods for testing the efficacy of compositions for treating skin that exhibits signs of ageing.

Description

Title: Androgen Glucuronides as Markers of Androgenic Activity

Cross-Reference to Related Applications

This application claims priority to United States Provisional Patent Application No. 60/734,270, filed on November 8, 2005. That application is incorporated by reference as if fully rewritten herein..

Field of the Invention

Embodiments of the invention relate to the correlation between the presence of androgen glucuronides in the blood and the total amount of androgen activity in a person. Embodiments of the invention permit a a person, for example a clinician to obtain an accurate status of androgen activity in a person, allowing the a person, for example a clinician to adapt a regimen of hormonal supplementation that will aid in treatment of any of a number of disorders. These disorders include, for example, but are not limited to, osteoporosis, sexual dysfunction, type 2 diabetes, obesity, arteriosclerosis, hypertension, breast cancer, loss of muscular strength, physical fitness, and well-being and skin alterations. Embodiments of the invention also relate to administration and evaluation of anti-ageing skin treatments.

Background of the Invention

Despite the long series of cohort studies performed during the last 20 years, the correlation between serum testosterone and any clinical situation believed to be under androgen control in women has remained elusive. In fact, the androgens in women are reported to originate from three sources: (1) testosterone secreted directly by the ovaries; (2) testosterone secreted directly by the adrenals; and (3) most importantly, the local formation of androgens from the precursor dehydroepiandrosterone (DHEA) of adrenal origin. The androgens made in large amounts in peripheral tissues from DHEA act in the same cells where synthesis takes place and are not released in significant amounts as active androgens in the circulation. This makes the measurement of serum testosterone unreliable as a marker or androgenic activity. One area where measurement of androgens would be beneficial is in the field of ageing skin. Ageing of the skin results from the effects of intrinsic and extrinsic factors on the skin. The changes in the skin resulting from intrinsic or physiological ageing are the consequence of a genetically programmed senescence involving endogenous factors. This intrinsic ageing gives rise especially to a slowing- down of renewal of the skin cells. Histologically, the skin is thinner overall, both at the epidermal and dermal levels. The density of the fibrous macromolecules in the dermis (elastine and collagen) is reduced. In contrast, extrinsic ageing entails histopathological changes such as an excessive accumulation of elastic material in the upper dermis and degeneration of the collagen fibers.

Clinically, the signs of ageing are generally reflected by the appearance of wrinkles and fine lines, a slackening of the cutaneous and subcutaneous tissues, a loss of skin elasticity and atonia of the skin texture. The loss of firmness and tonicity of the skin, like wrinkles and fine lines, is at least partly explained by dermal and epidermal atrophy and also by a flattening of the dermoepidermal formation; the skin is thinner and more flaccid, and the epidermis reduces in thickness. In addition, the complexion of the skin is generally modified; it appears paler and yellower; this appears to be due essentially to a disorganization of the microcirculation (less hemoglobin in the papillary dermis). Another clinical sign of ageing is the dry and coarse appearance of the skin, which is due essentially to a more pronounced level of desquamation; by scattering light rays, these squamae also contribute toward a somewhat gray appearance of the complexion.

Furthermore, many colored and/or darker marks appear at the surface of the skin, and more especially on the hands, giving the skin a nonuniform appearance. In general, these marks are due to a large production of melanin in the epidermis and/or dermis of the skin. In certain cases, these marks may become cancerous. Moreover, diffuse irritations and sometimes telangiectasia occur in certain areas of the skin.

Some of these signs are more particularly associated with intrinsic or physiological ageing, i.e. age-related ageing, whereas others are more specific to extrinsic ageing, i.e. ageing generally brought about by the environment; this more particularly involves photoageing due to exposure to sunlight, light or any other radiation. Another sign of ageing of the skin that constitutes a major criterion in the degradation of the appearance of the skin is the accentuation of the weathered appearance.

The weathered appearance is characterized by a change in the visual appearance and also the touch behavior of the skin. More specifically, the skin visually takes on the appearance of cigarette paper, giving it an appearance similar to that of a sheet of papyrus. In addition, when it is gently pinched between the thumb and index finger, the skin forms numerous thin, sharp folds having the appearance of creased paper. Finally, the feel of the skin shows that its superficial portions appear to be floating on the deeper portions, giving the skin, at the very advanced stage of a weathered appearance, the look of crumpled paper. It would be desirable to provide methods for treating ageing skin, for predicting efficacy in a particular person of treatments for ageing skin, and for determining actual efficacy in a particular person of treatments for ageing skin. Brief Summary of the Invention

AU androgens, no matter their source or origin, follow the same obligatory route of inactivation and transformation into glucuronide derivatives, which all exit through the circulation. The only valid marker of androgenic activity in both pre- and postmenopausal women is the blood concentration of the androgen glucuronide derivatives measured by liquid chromatography tandem mass spectrometry. Women with true androgen deficiency involving osteoporosis, sexual dysfunction, the metabolic syndrome and a series of other clinical situations affecting women's health could benefit by having their deficiency properly diagnosed, then they could receive appropriate androgen therapy or prevention in cases of true androgen deficiency.

Embodiments of the invention provide methods for diagnosis of diseases and disorders related to androgen amount. This is accomplished by detecting androgen glucuronide derivatives, such as androsterone (ADT) and/or androstane-3α, 17β-diol (3α-diol) in blood or tissue. In some embodiments of the invention glucuronide derivatives (or their absence) are detected by mass spectrometry (LC- MS/MS) and/or gas chromatography - tandem mass spectrometry (GC-MS). A low amount of glucuronide derivatives in relation to the mean amount of glucuronide derivatives in a comparable sample population is indicative of androgen deficiency.

It should be noted that although methods of the invention may be used to determine either extreme of androgen presence (excess or absence), most embodiments will involve determination and/or treatment of androgen deficiency. Disorders and diseases resulting from androgen deficiency include but are not limited to the metabolic syndrome (type 2 diabetes, obesity, arteriosclerosis and hypertension), sexual dysfunction, osteoporosis, breast cancer, skin atrophy as well as loss of muscular strength, physical fitness and well-being. Further embodiments of the invention provide a method for comparing systemic androgen glucuronide levels with local levels in tissue. This allows a a person, for example a clinician to determine the hormonal levels of skin or other tissue.

In some embodiments of the invention, the biological sample is human blood, human tissue, or human serum.

In further embodiments of the invention are provided methods of monitoring treatment of androgen amount in a subject. In some embodiments, the method involves comparing androgen glucuronide derivative levels in a first and a second biological sample of a subject, wherein the first biological sample is taken from the subject at an earlier point in time than a second biological sample, and wherein the second biological sample is collected following treatment of the subject for disorders related to androgen amount. In a further embodiment this method is used where a subject is being treated with skin anti-ageing compositions.

Further embodiments of the invention provide methods for monitoring progression of disorders involving androgen amount. In some embodiments, this method involves comparing androgen glucuronide derivative levels in a first and a second biological sample of a subject, wherein the first biological sample is take from the subject at an earlier point in time than a second biological sample, and wherein the increased or decreased amount of androgen glucuronide derivatives is indicative of progression of a disorder. Brief Description of the Several Views of the Drawings

Figure 1. Figure 1 depicts a schematic representation of the very important contribution of the precursor DHEA of adrenal origin to total androgenic activity in postmenopausal women with a parallel minor contribution of testosterone of ovarian and adrenal origins. By intracrine mechanisms, DHEA is transformed into testosterone and DHT in peripheral tissues and then, into the inactive metabolites ADT and 3α-diol before transformation into the water soluble glucuronide derivatives ADT-G, 3α-diol-3G and 3α-diol-17G by the glucuronosyl transferases (UGTs) 2B7, 2 Bl 5 and 2 Bl 7. These water soluble metabolites are then released in the general circulation where they can be measured. A very small proportion of the testosterone and DHT made intracellularly by the steroidogenic enzymes of the intracrine pathway diffuse into the circulation. The height of the bars is proportional to the concentration of each steroid.

Figure 2. Figure 2 depicts a lack of correlation between serum ADT-G (androsterone glucuronide) and testosterone levels in three hundred seventy-seven (377) 55-65 year-old postmenopausal women. The Spearman Correlation Coefficient value of 0.13 is indicated.

Figure 3. Figure 3 depicts a lack of correlation between serum testosterone and ADT-G levels in forty-seven (47) 30-35 year-old premenopausal women. The Spearman Correlation Coefficient value of 0.08 is indicated.

Figure 4. Figure 4 depicts a lack of correlation between serum testosterone and DHEA levels in three hundred seventy-seven (377) 55-65 year-old postmenopausal women. The Spearman Correlation Coefficient value of 0.25 is indicated. Figure 5. Figure 5 shows correlation between serum DHEA (dehydroepiandrosterone) and 5-diol (androst-5-ene-3β, 17βdiol) levels in three hundred seventy seven (377) 55-65 year-old postmenopausal women. The Spearman Correlation Coefficient value (r²) is indicated. Detailed Description of the Invention

The case-control and prospective cohort studies that analyzed the correlation between serum testosterone levels and the incidence of breast cancer, obesity, insulin resistance, sexual dysfunction or other clinical problems in women always yielded contradictory results (reviewed in Labrie, F. et al. "Endocrine and intracrine sources of androgens in women: inhibition of breast cancer and other roles of androgens and their precursor dehydroepiandrosterone" Endocrine Reviews 24, 152-182 (2003); Basson, R., "A New Model of Female Sexual Desire" Endocrine News 29, 22 (2004); Tchemof, A. & Labrie, F. "Dehydroepiandrosterone, obesity and cardiovascular disease risk. A review of human studies" European Journal of Endocrinology 151, 1-14 (2004); Shifren, J. L., et al "Transdermal testosterone treatment in women with impaired sexual function after oophorectomy" N Engl dMed 343, 682-8. (2000); Sherwin, B. B. & Gelfand, M. M. "The role of androgen in the maintenance of sexual functioning in oophorectomized women" Psychosom Med. 49, 397-409 (1987); Cameron, D. R. & Braunstein, G. D. "Androgen replacement therapy in women" Fertil Steril 82, 273-89 (2004); Garland, C. F., et al. "Sex hormones and postmenopausal breast cancer: a prospective study in an adult community" Am J Epidemiol 135, 1220-30 (1992); Leiblum, S., et al. "Vaginal atrophy in the postmenopausal women. The importance of sexual activity and hormones" JAMA 249, 2195-2198 (1983); Lipworth, L., et al. "Serum steroid hormone levels, sex hormone-binding globulin, and body mass index in the etiology of postmenopausal breast cancer" Epidemiology 7, 96-100 (1996); Davis, S. R., et al. "Testosterone enhances estradiol's effects on postmenopausal bone density and sexuality" Maturitas 21, 227-36. (1995)). Such a lack of correlation is difficult to reconcile with the well demonstrated stimulation of sexual function and the inhibitory effects of exogenous androgens on obesity and breast cancer. (Labrie et al., 2003; Basson, 2004; Shifren et al. 2000; Sherwin et al. 1987; Garland et al. 1992; Lipworth, et al. 1996; FeIs, E. "Treatment of breast cancer with testosterone propionate. A preliminary report" J Clin. Endocrinol. 4, 121-125 (1944); Segaloff, A., et al. "Hormonal therapy in cancer of the breast. 1. The effect of testosterone propionate therapy on clinical course and hormonal excretion." Cancer 4, 319-323 (1951)). This lack of consistency between the serum levels of testosterone and the effect of exogenous androgens has raised serious doubts about the validity of measurements of total and free serum testosterone as markers of androgenic activity in women. One explanation for the lack of correlation between serum testosterone and clinical parameters known to be under androgen control could be related to the recent data showing that the majority of androgens in women are made locally in peripheral target tissues from the inactive precursor dehydroepiandrosterone (DHEA) of adrenal gland origin. (Labrie et al. 2003; Labrie, F. "Intracrinology" MoI. Cell. Endocrinol. 78, Cl 13-Cl 18 (1991); Labrie, F., et al. " Physiological changes in dehydroepiandrosterone are not reflected by serum levels of active androgens and estrogens but of their metabolites: intracrinology" J Clin. Endocrinol. Metab. 82, 2403-2409 (1997)). Since androgens made locally do not originate from circulating testosterone, such data imply that measurement of the serum levels of testosterone is of questionable biological and clinical significance. In fact, the androgens testosterone and dihydrotestosterone (DHT) made in peripheral tissues from DHEA of adrenal origin exert their action locally in the same cells where synthesis takes place with only minimal release as active androgens in the circulation (Figure 1). Following their formation and availability for local intracellular action, the active androgens are inactivated and transformed in the same cells into water soluble glucuronide derivatives which diffuse quantitatively into the general circulation where they can be measured before their elimination by the kidneys (Figure 1). This recently identified mechanism of steroid formation and action has been named intracrinology. (Labrie, 1991; Labrie, C. et al. " Androgenic activity of dehydroepiandrosterone and androstenedione in the rat ventral prostate" Endocrinology 123, 1412-1417 (1988)).

In addition to the above-identified major issue about the biological significance of serum testosterone which does not take into account the large amount of androgens made in peripheral tissues, it should be mentioned that the radioimmunoassays generally used to measure serum sex steroids have limited specificity. (Dowsett, M. & Folkerd, E. "Deficits in plasma oestradiol measurement in studies and management of breast cancer" Breast Cancer Res 7, 1-4 (2005); McShane, L. M., et al. "Reliability and validity of serum sex hormone measurements" Cancer Epidemiol Biomarkers Prey 5, 923-8 (1996); Rinaldi, S. et al. "Reliability and validity of commercially available, direct radioimmunoassays for measurement of blood androgens and estrogens in postmenopausal women" Cancer Epidemiol Biomarkers Prey 10, 757-65 (2001); Dorgan, J. F., et al. "Measurement of steroid sex hormones in serum: a comparison of radioimmunoassay and mass spectrometry" Steroids 67, 151-8 (2002)).

There remains a need in the art to make available a reliable marker of androgenic activity in women. Access to a precise and valid marker of androgenic activity in order to assess with confidence the role of androgens in a series of problems particularly frequent after menopause. These problems include, for example, but are not limited to, the metabolic syndrome (type 2 diabetes, obesity, arteriosclerosis and hypertension), sexual dysfunction, osteoporosis, breast cancer, skin atrophy as well as loss of muscular strength, physical fitness and well-being. Accurate assessment will help a a person, for example a clinician to provide an adequate hormonal supplement to treat these and other androgen-related conditions.

We have found that blood concentration of androgen glucuronide derivatives is a valid marker of androgenic activity in women. We have used liquid chromatography - mass spectrometry (LC-MS/MS) and gas chromatography - tandem mass spectrometry (GC-MS) to measure nine androgens, their precursors and metabolites in 377 postmenopausal women in good health aged 55 to 65 years. We have used this data to analyze the correlation between serum testosterone and what we have found to be the true markers of total androgenic activity, namely the glucuronide derivatives of androsterone (ADT) and androstane-3α, 17β-diol (3α-diol), the obligatory route of elimination of androgens. We have compared with data obtained in 47 normal 30 to 35 year-old normally cycling women.

Our data clearly shows that measurement of total androgenic activity reflected by the serum levels of ADT-G and 3α-diol-G is more effective than measurement of the serum levels of any other steroid, including testosterone. This allows us to accurately assess the role of androgens in a long series of problems affecting women's health, particularly those occurring after menopause. It further allows use of androgen therapy in cases of true androgen deficiency.

We have found that serum testosterone shows no correlation with total androgen metabolites. The serum levels of androgens as well as their precursors and glucuronide derivatives measured by mass spectrometry in 377 healthy postmenopausal women aged 55 to 65 years are shown in Table 1. For comparison, the values found in normal cycling 30 to 35 year-old women are shown in the same table. As can be seen in Figure 2 and Table 2, no useful correlation is found between serum testosterone and ADT-G (r = 0.13). ADT-G by itself accounts for about 93% of the obligatory metabolites of androgen elimination. An even lower correlation is observed between serum testosterone and the serum levels of the two other androgen glucuronides, namely 3α-diol-3G (androstane-3α, 17β-diol-3G) (r =

0.07) and 3α-diol-17G (r = 0.05) (Table 2). A similar lack of correlation between testosterone and ADT-G (r = 0.08) is seen in normal cycling 30- to 35-years old women (Figure 3). A somewhat better but still poor correlation is observed between serum testosterone and DHEA, the main source of androgens in women, with a r value of 0.25 (Figure 4) or between DHEA and ADT-G with a r value of 0.43, (Table 2).

In fact, testosterone is, among all the steroids measured, the one showing the lowest correlation with the three glucuronide derivatives of androgens. This suggests that variable rates of secretion of testosterone by the ovary and/or adrenal could be responsible for the lack of correlation of ADT-G and 3α-diol-G with serum testosterone, which is the sum of testosterone of ovarian and adrenal origins secreted directly into the blood plus testosterone diffusing from the peripheral tissues following peripheral transformation of DHEA into androgens (Figure 1). It is also possible that the low level of diffusion of peripheral tissue-made testosterone could be highly variable and explain, at least partially, the lack of correlation. Better correlations are observed, however between serum DHEA and its 17o>reduced

metabolite androstenediol (5-dio]) (r=0.69) (Figure 5), DHEA and androstenedione (r = 0.63) as well as between DHEA and its sulphated metabolite DHEA-S (r = 0.60) (Table 2). The only means of assessing androgenic activity in specific tissues is the direct measurement of the intratissular concentration of the active androgens. Such measurements are not possible in the human except under exceptional circumstances, such as in samples of tissue obtained at surgery. (Poortman, J., et al. "Subcellular distribution of androgens and oestrogens in target tissue" J. Steroid Biochem. 19, 939-945 (1983); Labrie, F., et al. "A. in Important Advances in Oncology" (eds. de Vita, V. T., Hellman, S. & Rosenberg^', S. A.) 193-217 (J.B. Lippincott, Philadelphia, 1985); Belanger, B. et al. "Comparison of residual C-19 steroids in plasma and prostatic tissue of human, rat and guinea pig after castration: unique importance of extratesticular androgens in men" J. Steroid Biochem. 32, 695- 698 (1989)). Although it does not permit the assessment of androgenic activity in specific tissues, the assay by validated mass spectrometry techniques of the glucuronide derivatives of ADT and 3α-diol permits an accurate measure of total androgenic activity in the whole organism. In fact, it is now well established that UGT 2 B7, UGT 2 B 15 and UGT 2 B17 are the three enzymes responsible for the glucuronidation of all androgens and their metabolites in the human. (Belanger, A., et al. "Inactivation of androgens by UDP-glucuronosyltransferase enzymes in humans" Trends Endocrinol Metab 14, 473-479 (2003)). Identification and characterization of all the human UDP-glucuronosyl transferases makes possible the use of the glucuronide derivatives of androgens as markers of total androgenic activity in both women and men. ADT-S is also a metabolite excreted in large amounts, but this steroid is exclusively of adrenal origin and does not reflect androgenic activity in peripheral tissues

It is thus remarkable that man, in addition to possessing very sophisticated endocrine and paracrine systems, has largely vested in sex steroid formation in peripheral tissues. (Labrie, 1991; Labrie, 1998; Labrie 1985; Belanger 1985; Labrie, F., et al. "Physiological changes in dehydroepiandrosterone are not reflected by serum levels of active androgens and estrogens but of their metabolites: intracrinology" J CHn Endocrinol Metab 82, 2403-9. (1997); Zwieker, H. & Rittmaster, R. S. "Androsterone sulfate: physiology and clinical significance in hirsute women" J Clin. Endocrinol. Metab. 76, 112-6 (1993)). The level of transformation of the adrenal precursor steroid DHEA into androgens and/or estrogens in peripheral target tissues thus depends upon the level of expression of the various steroidogenic enzymes in each cell of each of these tissues. (Labrie, 2003; Labrie, 1991). This situation of a high secretion rate of adrenal precursor sex steroids by the adrenals in men and women is thus completely different from all animal models used in the laboratory, namely rats, mice, guinea pigs, and all others (except monkeys), where the secretion of sex steroids takes place exclusively in the gonads and the adrenals do not secrete significant amounts of DHEA. (Belanger, 1989). The classical concept of androgen and estrogen secretion in women assumed that all sex steroids had to be transported by the general circulation following secretion by the ovaries before reaching the target tissues. According to this classical concept, it was erroneously believed that the active steroids could be measured directly in the circulation, thus providing a potentially easily accessible measure of the general exposure to sex steroids.

In fact, this concept is valid only for the animal species lower than primates but it does not apply to the human, especially in post menopausal women where all estrogens and almost all androgens are made locally from DHEA in the peripheral tissues which possess the enzymes required to synthesize active sex steroids. This local biosynthesis and action of androgens in target tissues eliminates the exposure of other tissues to androgens and minimizes the risks of undesirable masculinizing or other androgen-related side effects. The same applies to estrogens, although we feel that a reliable parameter of total estrogen secretion (comparable to the glucuronides identified for androgens) has yet to be determined.

The present data show that the best and perhaps only valid means of assessing androgenic activity in women is to measure ADT-G, the metabolite that accounts for 93% of total androgen glucuronide derivatives. ADT-G and 3α-diol-G may also be measured. Measurements may be conducted, for example, by a validated liquid chromatography tandem mass spectrometry (LC/MS-MS) technique. This may replace measurement of serum testosterone. This strategy can identify the cases of true androgen deficiency and allows the possibility of an appropriate androgen therapy or prevention regimen. In human skin, as with many peripheral tissues, androgens are in large part made locally from the inactive precursor dehydroepiandrosterone. The skin is an androgen-sensitive and estrogen-sensitive tissue that is able to make sex hormones locally and control the intracellular levels of sex steroids according to local needs. Using the methods taught herein, one may examine skin samples (for example, tissue biopsies or blisters) to determine intratissular quantification of either active androgens or glucuronide derivatives to precisely assess androgen activity.

Where both active androgen levels and glucuronide levels are measured, the differential between the two may be used to assess the amount of active androgens formed locally by peripheral tissues. This allows extrapolation of the hormonal status of peripheral tissue. One peripheral tissue is skin.

Information obtained through practice of the methods of glucuronide detection as taught herein may be used, for example, to determine an effective amount of a therapeutic composition that may be administered to treat an androgen-related disorder. Compositions may be administered, for example, as a local supplement. Suitable supplements include, for example, but are not limited to, those reported in United States Patent No. 5,843,932, to Labrie. Other compositions that may be used include DHEA, DHEA precursors, and DHEA derivatives, as described below. One skilled in the art will recognize that an "effective amount" of a given therapeutic composition is an amount necessary to obtain a desired result given such factors as the type and severity of the disorder, the age and body type of a patient, and other factors. One skilled in the art will further recognize that a "therapeutic composition" is a composition known to have a desired effect for treatment of some disorder. One method of local supplementation involves topical application of an active androgen or an androgen precursor such as DHEA and its derivatives. Since all metabolic enzymes necessary to transform active androgens to their inactive forms are in the skin, use of topical application offers focused activity in the skin and avoids systemic hormonal effects.

In another embodiment, biologically active compounds that prevent inactivation of local androgens into glucuronide derivatives are applied to an area in need to treatment to increase local androgen activity. These glucuronide inhibitors may be applied in combination with active androgens. Principles reported herein may also be applied to measurement of systemic estrogen. A precise evaluation of androgen and estrogen activity can help a a person, for example a clinician to propose adequate estrogen treatment, androgen treatment, or a combination treatment with estrogens, androgens, and/or their precursors. Measurement of systemic or local androgen or estrogen activity in skin allows treatment of skin disorders including, for example, but not limited to, dryness, skin pigmentation, ageing, atrophy, wrinkles, physical properties (elasticity), desquamation, hair growth, and sebum production.

In further embodiments, methods are provided to prevent or alleviate signs of ageing skin by maintaining in a patient's skin an optimal level of a compound used to prevent or alleviate signs of ageing skin. Compounds useful in these embodiments include but are not limited to DHEA, DHEA precursors, and DHEA derivatives, as reported below. Inhibitors of the glucuronidation enzymes (UGT) which inactivate androgens into glucuronides derivative, may also be used, in particular topically, after determination of the levels of glucuronide derivatives according to the invention. An optimal level of a compound may be provided, for example, by comparing androgen glucuronide derivative levels in a first and a second biological sample of a subject, wherein the first biological sample is taken from the subject at an earlier point in time than a second biological sample, and wherein the second biological sample is collected following treatment of the subject with skin anti-ageing compounds. Following comparison of the results, the amount of compound administered to the patient may be changed to optimize the results obtained. Multiple iterations of this process may be performed.

In a further embodiment, a method is provided to test the efficacy of an anti-ageing product in a selected population of test subjects. In this embodiment, androgen glucuronide derivative levels are determined in first and a second biological samples of a plurality of subjects in a selected population, wherein a first biological sample is taken from each subject at an earlier point in time than a second biological sample, and wherein a second biological sample is collected from each following treatment of the subject with a skin anti-ageing composition. Glucuronide derivative levels in the first and second samples for the test subjects may then be compared, either individually or collectively, to determine if the skin anti-ageing composition had the desired effect. Other indicia of a successful anti-ageing treatment may also be reviewed in the subjects to determine if the skin anti-ageing compositions had the desired effect. Such indicia may include, for example, but are not limited to, skin dryness, pigmentation, atrophy, elasticity, wrinkles, desquamation, hair growth, and sebum production.

In a further embodiment, a method is provided to prognosticate the likelihood that a subject will benefit from treatment with a composition (for example a cosmetic composition, which may be, for example, an oral composition, a topical composition, or a combination of the two) for preventing or alleviating the signs of ageing. In this method, androgen glucuronide derivative levels in a biological sample of a subject are determined as provided herein. One skilled in the art may then compare the patient's androgen level with an optimal and/or threshold androgen level calculated based on factors known to those skilled in the art. Such factors could include, for example, but are not limited to, age and physical condition of a subject.

If the subject's androgen level is near, at, or above the optimal and/or desired level, then a a person, for example a clinician may predict that further treatment with androgen-related anti-ageing compounds (including, for example, but not limited to DHEA, DHEA precursors, and DHEA derivatives) would be of little benefit. If the subject's androgen level is below the threshold amount (including if the androgen level is zero), then it is likely that the subject would benefit from administration of the treatment. Compositions useful for treatment methods described herein include, for example, but are not limited to, DHEA, DHEA precursors, and DHEA derivatives. DHEA, or dehydroepiandrosterone, is a natural steroid produced essentially by the adrenal glands. It is known for its anti-ageing properties, associated with its ability to promote epidermal keratinization (JP-07 196 467) and to combat osteoporosis (US-5 824 671), or alternatively in the treatment of dry skin, on account of its ability to increase the endogenous production and secretion of sebum and to strengthen the skin's barrier effect (US-4496 556). DHEA is also described in the treatment of obesity and diabetes (WO 97/13500). It has also been proposed to use DHEA sulfate against alopecia (JP-60 142 908) and to treat various signs of ageing such as wrinkles, loss of radiance of the skin and slackening of the skin (EP-O 723 775).

The DHEA that may be used according to the invention is available, for example, from the companies Sigma and Akzo Nobel. The expression "DHEA precursors" means its immediate biological precursors or substrates, and also its chemical precursors. Examples of biological precursors are cholesterol, pregnenolone, 17α-hydroxy pregnenolone, 5-

androstenediol, DHEA sulfate, 17α-hydroxy pregnenolone sulfate and 5- androstenediol sulfate, this list not intending to be limiting. Examples of chemical precursors are sapogenins such as diosgenin (or spirost-5-ene-3-β-ol), hecogenin,

smilagenin and sarsapogenin, and also natural extracts containing them, in particular fenugrec and extracts of Dioscorea plants such as wild yam root, this list not intended to be limiting.

The expression "DHEA derivatives" means both its metabolic derivatives and its chemical derivatives. Metabolic derivatives that may be mentioned especially include 5-androstene-3β-17β-diol (or adiol), 5-androstene-3β-17β-diol

sulfate and 4-androstene-3,17-dione, this list not intended to be limiting. Chemical derivatives that may be mentioned especially include salts, in particular water-soluble salts, such as DHEA sulfate. Mention may also be made of esters, such as the hydroxycarboxylic acid esters of DHEA described in US-5 736 537 or other esters such as DHEA salicylate, acetate, valerate or enanthate.

Among the metabolites of DHEA, one that may be useful in preferred treatment embodiments is 7α-hydroxy-DHEA. It has indeed been demonstrated that this metabolite, which does not possess the hormonal activity of DHEA, made it possible to increase the proliferation of the fibroblasts and the viability of the human keratinocytes and had anti-free radical effects (WO 98/40074). It has also been demonstrated, on rats (WO 00/28996), that 7α-hydroxy-DHEA increased the thickness of the dermis and the elastin and collagen content of the skin. This metabolite of DHEA may be particularly useful for preventing and/or treating the harmful effects of UV radiation on the skin, for controlling wrinkles and for increasing skin firmness and tone.

7α-Hydroxy-DHEA is, with 5-androstene-3β,17β-diol, a major

metabolite of DHEA, which is obtained by the action of 7α-hydroxylase on DHEA.

Among the minor metabolites of DHEA, there may be mentioned 7β-hydroxy-DHEA,

which is obtained by the action of 7β-hydroxylase on DHEA and 7-keto-DHEA,

which is itself a metabolite of 7β-hydroxy-DHEA.

In a further embodiment of the invention, tests reported herein may be used as a precise diagnostic to assess the role of nutritional bioactives (phytohormones). This allows one skilled in the art to propose lower or higher hormonal dose intake. This proposal may be made, for example, for skin and beauty purposes.

It should be recognized that although the examples herein are directed to female androgen testing, embodiments of the invention may also be directed to testing in males.

Using the testing methods of embodiments of the invention, it is possible to diagnose subjects at early stages of androgen-related disease. It is further possible to recognize subjects at risk of developing androgen-related disease. As used herein, an androgen is a compound (or one of its metabolites) having a Ki value for the human androgen receptor of less than about 2x10^"8M and an androgen receptor-mediated inhibitory effect on the growth of human breast cancer ZR-75-1 cells which reaches half-maximal value at a concentration below 10 nanomoles per liter or a compound (or one of its metabolites) which positively responds to the screening method described in United States Patent Application Publication No. 20060045847, entitled "Method for determination of anabolic activity."

EXAMPLE 1 - Assessment of Androgenic Activity Subjects: Three hundred seventy seven (377) healthy postmenopausal women aged 55 to 65 years and 47 premenopausal 30- to 35 -year old women participated in this study after approval and having given written informed consent. No subject had taken hormone replacement therapy during the previous six months. No subject was suffering from an endocrine disorder, and none was under treatment with lipid- or glucose-lowering agents. There was no active or history of thromboembolic disease, significant metabolic or endocrine disease and no clinically significant gastrointestinal, liver or gallbladder disease. There was no migraine and no diabetes mellitus not controlled by conventional therapy. There was no corticosteroid treatment within six weeks of study entry as well as treatment with β- carotenoid, retinoic acid, hydroquinone, cc-hydroxyacid (including inhaled, topical,

oral).

There was no hypertension equal to or above 160/95 mm Hg or not controlled by standard therapy as well as no confirmed clinically significant depression or confirmed severe psychiatric disturbance. There was no administration of any investigational drug within 30 days of screening visit or previous treatment with androgens or anabolic steroids within 6 months prior to the screening visit. There was no exposure to or use of antidepressants, antipsychotics, narcotic and analgesics, within 30 days prior to enrollment. Smoking any number of cigarettes was an exclusion criteria.

There was no former or present narcotic addiction or alcoholism. The body weight ranged between 18.5 and 29.9 of ideal body weight according to Body Mass Index (BMI). There was no hepatic or renal impairment or condition known to affect drug or steroid metabolism. All subjects had a medical history, complete physical examination, serum biochemistry as well as complete blood and urinalysis. Blood sampling was performed under fasting conditions between 0800 and 1030 h.

Materials and methods - Quantitation of Steroids in Human Serum: Human Blood Sample Collection for Measurement of DHEA and Related Steroids: The serum steroid levels (DHEA, DHEA-S, 5-diol, DHT, testosterone, 4-dione, ADT- G, 3α-diol-G and 3α-diol-17G were measured at the Laboratory of Molecular Endocrinology, CHUL Research Center. I. Analysis of Non-Conjugated Steroids

Preparation of Calibration Curve of Standard Samples: Dehydroepiandrosterone (DHEA), androst-5-ene-3β,17β-diol(5-diol), androstenedione (4-dione), testosterone (testo), and dihydrotestosterene (DHT), were analyzed by GC-MS. On each day of analysis, calibration standards ranging from 0.2 to 20 ng/mL for DHEA and 5-diol, from 0.05 to 10 ng/mL for 4-dione and testo and from 0.02 to 4 ng/mL for DHT were prepared using charcoal-adsorbed human serum. Extraction of Steroids from Human Serum: Briefly, 500 μL of a 0.5

M sodium acetate solution was added to each tube (1.0 mL for calibration standards).

A methanolic solution (50 μL), containing a mixture of deuterated steroid internal standards, was then added to each tube. Aliquots of 0.75mL of study samples (0.25 mL for calibration standards) were added and tubes were vortexed for ca 1 min.

1-Chlorobutane (3 mL) was then added to each tube and mixed. After centrifugation, the organic extracts were collected and purified on LC-Si SPE columns. Columns and the adsorbed material were washed with ethyl acetate :hexane (ca 6 mL; 1 :9; v:v). The analytes of interest were then eluted using 4 mL ethyl acetate:hexane (50:50; v:v), which was evaporated at 50°C. The dried residue was reconstituted in 0.5 mL ethyl acetate and vortexed for ca 15 sec.

An aliquot of lOOμL was transferred to a glass tube for the assay of 4-

dione and the remaining extract was kept in the tube for the assay of DHEA, 5-diol, testo and DHT. Both extracts were evaporated at 50°C. Derivatization of DHEA. 5-diol, testo and DHT:

Pentafluorobenzoylchloride in ethyl acetate (50 μ.L; 1/10; w/v) and pyridine in ethyl

acetate (500 μL; 1 :99; v:v) were added to the dried residue of DHEA, 5-diol, testo,

DHT, El and E2 and the samples were incubated for ca 30 min at 60°C. After evaporation of the reagent mixture, a solution of 0.5 M NaHCO₃ (1.0 mL) was added to the tubes, which were then left to react for 15 rain at room temperature. Hexane (2 mL) was then added to the tubes which are vortexed for ca 2 rain and left at room temperature for ca 10 min. The organic phase was evaporated at 50°C and the final extract reconstituted in 50 μL isooctane and then transferred into a conical vial for

injection into the GC/MS. Derivatization of 4-dione: A solution of 1 mg pentafluoroben2ylhydroxylamine/niL pyridine (100 μL) was added to the 4-dione extract and the tubes were incubated for ca 30 min at 60⁰C. After derivatization, the tubes were left to cool at room temperature for ca 5 min and hexane (3mL) was added to the samples. The mixtures were vortexed for ca 5 sec and then evaporated at 50⁰C. The final extract was reconstituted in 50 μL isooctane and then transferred into a conical vial for injection into the GC/MS system.

Analysis by GC/MS: The GC/MS system for the analysis of DHEA,

5-diol, 4-dione, testo and DHT used a 50% phenyl-methyl polysiloxane (DB- 17HT) capillary column (30mx0.25mm internal diameter, 0.15 μm film thickness) with helium as the carrier gas. The analytes and IS were detected using a HP5973 quadrupole mass spectrometer equipped with a chemical ionization source.

II. Analysis of the Conjugated Steroids - ADT-G, 3α-Dinl-3G and 3α-

Diol-17G Preparation of Calibration Curve of Standard Samples: Androsterone glucuronide (ADT-G), androstane-3α, 17β-Diol-3glucuronide (3α-Diol-3G) and

androstane-3α, 17β-Diol-17glucuronide (3α-Diol-17G) were analyzed by a

LC/MS/MS method using TurboIonSpray. On each day of analysis, calibration standards ranging from 2 to 200 ng/niL for ADT-G and from 0.50 to 50 ng/mL for 3α-Diol-3G and 3α-Diol-17G were prepared using a mixture of charcoal-adsorbed

serumrwater (l:l;v:v).

Extraction from Human Serum: Briefly, 500 μL of serum sample was

transferred to each tube. Water (500 μL) was added and the tubes are then vortexed.

A methanolic solution (100 μL) containing the deuterated steroid internal standard was then added to each tube. A solution OfNaH₂PO₄/ citric acid buffer (1.5mL) was added and the tubes were vortexed again. Samples were transferred to the C- 18 SPE columns. Each column was then washed with water and a solution of methanol: water (50:50;v:v). The analytes of interest were then eluted using a solution (4 mL) of methanol: water (80:20;v:v), containing 10 mM ammonium acetate. The eluates were evaporated at 45°C and the dried residue was reconstituted in a solution (100 μL) of methanol:water (50:50;v:v) containing 0.01% acetic acid prior to analysis.

Analysis bv LC-MS/MS: The HPLC system used a 150 x 4.6-mm, 4- μm particle size Synergy Hydro-RP column at a flow rate of 1.0 niL/min. ADT-G,

3α-Diol-3G and 3α-Diol-17G was detected using a Sciex AP13000 triple quadrupole mass spectrometer, equipped with TurboIonSpray™.

III. Analysis of the Conjugated Steroid DHEA-S

Preparation of Calibration Curve Standard Samples: Dehydroepiandrosterone-sulfate (DHEA-S) was analyzed by a LC/MS/MS method using Turbolonspray. On each day of analysis, calibration standards ranging from 0.075 to 10 μ/mL were prepared using PBSxharcoal adsorbed serum (l:l;v:v).

Extraction from Human Serum: Briefly, 100 μL of the serum sample

is transferred to individual tubes and 2 mL of PBS buffer was added. A methanolic solution (50 μL) containing the deuterated steroid internal standard was then added to

each tube. Samples were transferred on Oasis HLB SPE columns and each column was washed with water and methanol:water (10:90;v:v). The analytes of interest were then eluted with 4 mL of methanol. Methanol was evaporated at 35⁰C and the dried residue reconstituted in 125μL of methanol: water (50:50;v:v) and then filtered on 0.2μm nylon filter. Part of this solution (20 μL) was diluted in 0.5 mL of methanol: water (50:50;v:v) containing 5 mM ammonium acetate and 0.01% acetic acid for the DHEA-S analysis.

Analysis by LC-MS/MS: For DHEA-S analysis, the HPLC system used a 100 x 3.2-mm, 5-μm particle size, Phenomenex Columbus Cl 8 column at a flow rate of 0.5 mL/min. DHEA-S was detected using a Sciex API 300 or 3000 triple quadrupole mass spectrometer, respectively, equipped with Turbolonspray™. IV. Statistical Analysis

The distribution characteristics of the serum levels are presented in

Table 1 for all measured androgen metabolites. Some values are graphically presented on Figures 1 to 4. For the women aged 55 to 65 years, the distributions of

DHEA, 4-dione and testo were fitted to lognormal distributions. However, for 5-diol,

DHT, 3α-Diol-3G, 3α-Diol-17G, DHEA-S and ADT-G, a normal kernel density estimate was used to represent the distribution. For the women aged 30 to 35 years, distributions were fitted to lognormal distributions except for 3α-diol-17G where a normal kernel density estimate was used. The closeness of the relationship between any two circulating and two circulating androgens was estimated by the Spearman correlation coefficient (Table 2).

The disclosure of each publication, patent, and patent application cited or described in this document is hereby incorporated by reference herein, in its entirety.

Various modifications of the invention in addition to those shown and described herein will be apparent to one skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Table 1. SERUM STEROID LEVELS IN POST-MENOPAUSAL WOMEN AGED 55 TO 65 YEARS AS WELL AS IN NORMAL

CYCLING WOMEN AGED 30 TO 35 YEARS

*: Androstane-3α, 17β-diol-3G ²: Androstane-3α, 17β-diol-17G

Table 2. SPEARMAN CORRELATION COEFFICIENTS (r2) FOR STEROID LEVELS IN POST-MENOPAUSAL

WOMEN AGED 55 TO 65 YEARS AS WELL AS IN NORMAL CYCLING WOMEN AGED 30 TO 35 YEARS

-4

¹I Androstane-3α, 17β-diol-3G ²: Androstane-3α, 17β-diol-17G

Claims

We claim:

1. A method for determining the presence or amount of androgens in a test sample comprising detecting the presence or amount of at least one glucuronide selected from the group consisting of androsterone-glucuronide and androstane-3α, 17β-diol-3G (3α-diol-3G), wherein the presence or amount of the glucuronide is related to the presence or amount of androgens in the test sample.

2. The method of claim 1, wherein said glucuronide is androsterone- glucuronide.

3. The method of claim 1, wherein the presence or amount of said glucuronide is detected by a member of the group consisting of liquid chromatography-mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry.

4. The method of claim 1, wherein said test sample is a biological sample selected from the group consisting of blood, serum, and tissue.

5. A method for diagnosing an androgen-deficiency disorder, comprising detecting in a test sample the absence or amount of at least one glucuronide selected from the group consisting of androsterone-glucuronide and androstane-3α, 17β-diol- 3G (3α-diol-3G), wherein the amount or absence of the glucuronide in said test sample is indicative of the presence of an androgen-deficiency disorder.

6. The method of claim 5, wherein said glucuronide is androsterone- glucuronide.

7. The method of claim 5, wherein the presence or amount of said glucuronide is detected by a member of the group consisting of liquid chromatography-mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry.

8. The method of claim 5, wherein said test sample is a biological sample selected from the group consisting of blood, serum, and tissue.

9. The method of claim 5, wherein said androgen-deficiency disorder is selected from the group consisting of metabolic syndrome, diabetes, obesity, arteriosclerosis, hypertension, sexual dysfunction, osteoporosis, breast cancer, loss of muscular strength, and skin atrophy.

10. A method for assessing the hormonal status of a tissue sample, comprising determining the amount of active androgens produced by said tissue, wherein said amount of active androgens is determined by comparing the difference between active androgen blood measurement and inactive androgen levels.

11. The method of claim 10, wherein said inactive androgen levels are determined by detecting in a test sample the absence or amount of at least one glucuronide selected from the group consisting of androsterone-glucuronide and androstane-3α, 17β-diol-3G (3α-diol-3G), wherein the amount or absence of the glucuronide in said test sample is indicative of the inactive androgen levels in said sample.

12. The method of any of claims 1 to 11, wherein said methods are effected in vitro.

13. A method for treating an androgen-deficiency disorder, comprising obtaining a test sample from an individual having an androgen-deficiency disorder, detecting in said test sample the absence or amount of at least one glucuronide selected from the group consisting of androsterone-glucuronide and androstane-3ct, 17β-diol-3G (3α-diol-3G), wherein the amount or absence of said glucuronide in said test sample is indicative of the effective amount of a therapeutic composition necessary to treat said disorder; and treating said individual with said effective amount of said therapeutic composition.

14. The method of claim 13, wherein said androgen-deficiency disorder is selected from the group consisting of metabolic syndrome, diabetes, obesity, arteriosclerosis, hypertension, sexual dysfunction, osteoporosis, breast cancer, loss of muscular strength, and skin atrophy.

15. The method of claim 13, wherein said therapeutic composition is a phytohormone.

16. The method of claim 13, wherein said therapeutic composition is a topically applied hormone.

17. The method of claim 13, wherein said glucuronide is androsterone- glucuronide.

18. The method of claim 13, wherein the amount or absence of said glucuronide is detected by a member of the group consisting of liquid chromatography-mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry.

19. The method of claim 13, further comprising the step of comparing the amount or absence of said glucuronide in said test sample with the amount or absence of said glucuronide in a second test sample obtained from said individual at a time earlier than or a time later than said test sample was obtained from said individual.

20. The method of claim 19, wherein said androgen-deficiency disorder is one or more signs of ageing skin, and wherein said therapeutic composition comprises one or more of dehydroepiandrosterone (DHEA), DHEA precursors, and DHEA derivatives.

21. A method for predicting whether an individual in need of treatment for preventing or alleviating signs of skin ageing will benefit from administration of an anti-ageing composition, comprising obtaining a test sample from an individual in need of treatment for preventing or alleviating signs of skin ageing, detecting in said test sample the absence or amount of at least one glucuronide selected from the group consisting of androsterone-glucuronide and androstane-3α, 17β-diol-3G (3α-diol-3G), wherein the amount or absence of said glucuronide in said test sample is indicative of the benefit said individual will receive from administration of, said anti-ageing composition.

22. The method of claim 21, wherein the absence of said glucuronide or the presence of an amount of glucuronide that is lower than a threshold amount indicates that administration of said composition is likely to benefit said individual.

23. The method of claim 21, wherein said anti-ageing composition comprises at least one member of the group consisting of dehydroepiandrosterone (DHEA), DHEA derivatives, and DHEA precursors.

24. A method for testing the efficacy of a composition for anti-ageing skin treatment, comprising (a) obtaining first and second test samples from at least one individual in need of an anti-ageing skin treatment, wherein said first test sample is obtained prior to administering an amount of a composition to be tested for efficacy for anti-ageing skin treatment, and wherein said second test sample is obtained subsequent to administration of said composition; (b) detecting in each of said test samples the absence or amount of at least one glucuronide selected from the group consisting of androsterone-glucuronide and androstane-3α, 17β-diol-3G (3α-diol-3G); and (c) comparing the results of the detecting step for each test sample, wherein a greater amount of said glucuronide in said second test sample than in said first test sample indicates that said composition is effective as an anti-ageing skin treatment.

25. The method of claim 24, wherein said composition comprises at least one member of the group consisting of dehydroepiandrosterone (DHEA)₅ DHEA derivatives, and DHEA precursors.

26. The method of any of claims 21 to 25, wherein said methods are effected in vitro.

27. Use of a detected amount or absence of at least one glucuronide in a test sample for establishing a treatment of an androgen-deficiency disorder for an individual; comprising obtaining a test sample from an individual having an androgen-deficiency disorder, detecting in said test sample the absence or amount of at least one glucuronide selected from the group consisting of androsterone- glucuronide and androstane-3α, 17β-diol-3G (3α-diol-3G), wherein the amount or absence of said glucuronide in said test sample is indicative of the effective amount of a therapeutic composition necessary to treat said disorder.

28. Use of an effective amount of a therapeutic composition necessary to treat an androgen-deficiency disorder of an individual, wherein said effective amount of a therapeutic composition is determined by correlating the amount or absence of at least one glucuronide selected from the group consisting of androsterone-glucuronide and androstane-3α, 17β-diol-3G (3α-diol-3G) in a test sample of said individual with said disorder.

29. The use of claim 28 or 29, wherein said androgen-deficiency disorder is selected from the group consisting of metabolic syndrome, diabetes, obesity, arteriosclerosis, hypertension, sexual dysfunction, osteoporosis, breast cancer, loss of muscular strength, and skin atrophy.

30. The use of claim 28 or 29, wherein said therapeutic composition is a phytohormone.

31. The use of claim 28 or 29, wherein said therapeutic composition is a topically applied hormone.

32. The use of claim 28 or 29, wherein said glucuronide is androsterone- glucuronide.

33. The use of claim 28 or 29, wherein the amount or absence of said glucuronide is detected by a member of the group consisting of liquid chromatography-mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry.

34. The use of claim 28 or 29, further comprising the step of comparing the amount or absence of said glucuronide in said test sample with the amount or absence of said glucuronide in a second test sample obtained from said individual at a time earlier than or a time later than said test sample was obtained from said individual.

35. The use of claim 28 or 29, wherein said androgen-deficiency disorder is one or more signs of ageing skin, and wherein said therapeutic composition comprises one or more of dehydroepiandrosterone (DHEA), DHEA precursors, and DHEA derivatives.