New potential biomarkers for mesterolone misuse in human urine by liquid chromatography quadrupole time-of-flight mass spectrometry.

In this paper, mesterolone metabolic profiles were investigated carefully. Mesterolone was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry (LC-QTOFMS) for the first time. Liquid-liquid extraction was applied to processing urine samples, and dilute-shoot analyses of intact metabolites were also presented. In LC-QTOFMS analysis, chromatographic peaks for potential metabolites were hunt down by using the theoretical [M-H](-) as target ions in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Ten metabolites including seven new sulfate and three glucuronide conjugates were found for mesterolone. Because of no useful fragment ion for structural elucidation, gas chromatography-mass spectrometry instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after solvolysis. Thus, their potential structures were proposed particularly by a combined MS approach. All the metabolites were also evaluated in terms of how long they could be detected, and S1 (1α-methyl-5α-androst-3-one-17ß-sulfate) together with S2 (1α-methyl-5α-androst-17-one-3ß-sulfate) was detected up to 9 days after oral administration, which could be the new potential biomarkers for mesterolone misuse.

Effects of topically applied antiandrogenic compounds on sebaceous glands of hamster ears and flank organs.

Growth of sebaceous glands in the ears and flank organs of castrated male hamsters is dependent on androgen substitution. Taking this for granted, a study was done to compare the effects of topical antiandrogenic treatment in vivo on the morphology and size of sebaceous glands with the concomitant changes in in vitro metabolism of 3H-testosterone. The role of dihydrotestosterone in sebaceous gland stimulation was thereby investigated. Topical treatment was carried out with the androgen antagonist 17 alpha-propylmesterolone (PM), with 4-androsten-3-one-17 beta-carboxylic acid (17 beta-C), and 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA), both described as specific 4-steroid-5 alpha-reductase inhibitors, and with progesterone (PRO), which is an androgen receptor antagonist with 5 alpha-reductase inhibiting properties. Regrowth of sebaceous glands after castration and substitution with testosterone propionate or dihydrotestosterone could be inhibited by topical PM and PRO. This occurred irrespective of the influence on testosterone metabolism and irrespective of the mode of substitution. 4-MA, on the other hand, while exhibiting strong 5 alpha-reductase inhibition in vitro, was ineffective in reducing sebaceous gland sizes in vivo. The compound 17 beta-C was ineffective in every respect. In no case were systemic antiandrogenic effects on prostates and seminal vesicles observed. Our results support the view that the DHT formation rate has no regulatory function for growth of sebaceous glands in hamsters and that PM and PRO counteract the androgenic stimulus by their competitive antagonistic binding to the androgen receptor, but not by their influence on testosterone metabolism.

Sex hormones and antiandrogens influence in vitro growth of dermal papilla cells and outer root sheath keratinocytes of human hair follicles.

Anagen hair bulb papillae, interfollicular dermal fibroblasts, and interfollicular keratinocytes isolated from fronto-parietal scalp biopsies as well as outer root sheath keratinocytes from plucked anagen hairs were separately grown in subculture for 14 d. The effect of different concentrations (2.4 nM-17.3 microM) of testosterone, dihydrotestosterone, and the antiandrogens cyproterone acetate or 17 alpha-propylmesterolone on growth behavior of the mesenchymal and epithelial cell types of the hair follicle were comparatively studied by means of growth curves, cell doubling times, and 3H-thymidine incorporation. For control, all cell lines were subcultured in hormone-free medium. Testosterone and dihydrotestosterone (345 nM) significantly reduced proliferation of papilla cells compared with dermal fibroblasts (p < 0.01) and outer root sheath keratinocytes compared with interfollicular keratinocytes (p < 0.01), as well as compared with cells cultured in control medium. Low concentrations of 17 beta-estradiol were ineffective, whereas doses of 180 nM 17 beta-estradiol increased the growth velocities of all cell types, especially of papilla cells, compared with dermal fibroblasts. Low doses of either cyproterone acetate (24 nM) or 17 alpha-propylmesterolone (29 nM) induced a growth enhancement, especially of papilla cells and outer root sheath keratinocytes, whereas high doses of cyproterone (1.20 microM) and 17 alpha-propylmesterolone (1.45 microM) had opposite effects. These changes were significant between papilla cells and dermal fibroblasts as well as between outer root sheath keratinocytes and interfollicular keratinocytes. Applying increasing doses of androgens to cyproterone acetate (24 nM)- or 17 alpha-propylmesterolone (29 nM)-containing media neutralized the growth-stimulating effect of antiandrogens, particularly in papilla cells and outer root sheath keratinocytes. However, minor differences between testosterone and dihydrotestosterone effects on cell growth were found. The data clearly demonstrate that the changes of in vitro growth of hair follicle cells depend on the concentrations of androgens and antiandrogens, as higher doses of both antiandrogens tested retarded the cell proliferation similar to testosterone or dihydrotestosterone. The papilla cells and outer root sheath keratinocytes reacted more sensitively to the hormones tested, thereby confirming the concept of a distinct androgen sensitivity of these specialized hair follicle cells.

17 alpha-Propylmesterolone (SH 434): an antiandrogenic sebosuppressive substance not influencing circulating testosterone concentrations. Experimental studies in Syrian hamsters.

17 alpha-Propylmesterolone is a new synthetic 5 alpha-reduced steroid with a propyl group in C-17 position and a methyl group in the A ring. The antiandrogenic action of 17 alpha-propylmesterolone on the sebaceous glands, testes weights and plasma testosterone concentrations were examined in the animal model of the Syrian hamster. The substance was given systemically (3, 5 or 10 mg/kg) and topically (3 or 5 mg/kg). 17 alpha-propylmesterolone reduced both sebaceous gland size and sebogenesis significantly in a dose-dependent manner. The topical administration was more effective than the systemic treatment. There was no influence of 17 alpha-propylmesterolone on testosterone concentration in plasma or testes weights although a diminished capacity or absence of free cytoplasmatic androgen receptor sites was detected in the sebaceous glands of the systematically or topically treated Syrian hamster. 17 alpha-Propylmesterolone exerts a potent topical sebosuppressive effect in the animal model. These findings should give rise to human studies and clinical trials.

Topical anti-androgenicity of a new 4-azasteroid in the hamster.

The topical anti-androgenic activity of L-651,580 (methyl 3-oxo-4-methyl-4-aza-5 alpha-androst-1-ene-17 beta-carboxylate) was established in a series of for experiments using castrated male hamsters. During each 21-day experiment, the animals received a daily subcutaneous injection of 40 micrograms testosterone propionate or 20 micrograms dihydrotestosterone propionate. Test compound in 25 microliters of gel was applied daily to the left flank organ. Compounds assayed included L-651,580, WIN 17,665 (17 alpha-propyltestosterone), and SH-434 (17 beta-hydroxy-1 alpha-methyl-17 alpha-propyl-5 alpha-androstan-3-one). Endpoints were flank organ area, sebaceous gland area, and prostate weight. Very similar results were obtained with L-651,580 and WIN 17,665. Daily doses of 0.25 mg or more of either compound usually produced a significant reduction in the areas of treated flank organs and sebaceous glands underlying treated flank organs. Neither compound caused significant changes in the area of the contralateral flank organs and sebaceous glands, which indicated they possess little or no systemic activity at topically effective treatment levels. In direct comparisons, SH-434 was less anti-androgenic than L-651,580 or WIN 17,665, although in one experiment, 0.5 mg/d of SH-434 significantly reduced the area of treated flank organs and sebaceous glands. Neither WIN 17,665 nor SH-434 caused a change in prostate weight; however, in one of four tests, a significant decrease was induced by the 0.5 mg/d level of L-651,580. The results of these experiments show that the topical anti-androgenicity of L-651,580 compares very favorably with that of WIN 17,665 and SH-434. They also indicate that the topical administration of effective dosage levels of L-651,580 causes few, if any, systemic effects.

Determination of 17 beta-hydroxy-1 alpha-methyl-17 alpha-propyl-5-alpha-androstan-3-one in plasma by gas chromatography-mass spectrometry with single-ion detection.

The topical anti-androgen 17 beta-hydroxy-1 alpha-methyl-17 alpha-propyl-5 alpha-androstan-3-one is determined in plasma samples by extracting with ether and subsequent mass fragmentography with single-ion detection at m/z 303. 17 beta-Hydroxy-1 alpha-methyl-17 alpha-pentyl-5 alpha-androstan-3-one, added to the samples before extraction, is used as the internal standard. Reproducibility was calculated to be +/- 5.9% at the 5-ng/mL level and 0.4% at the 20-ng/mL level. The limit of detection is approximately 1 ng/mL. Total gas chromatography-mass spectrometry analysis time is approximately 10 min/sample.

[An effective topical antiandrogen--17 alpha-propylmesterolone--in acne]. / Ein wirksames topisches Antiandrogen--17 alpha-Propylmesterolon--bei Akne.

17 alpha-Propylmesterolon, a new topically active antiandrogen, was administered to 25 patients with papulopustular or comedo acne and severe seborrhoea. The patients were supplied with a 3% solution of 17 alpha-propylmesterolone in 70% alcohol and were advised to apply it to the face twice daily for a mean duration of 14 weeks. Clinical follow-ups and thin-layer-chromatography determination of the sebum secretion rate (SER) and the lipid fractions were performed monthly. At the end of the treatment period 72% of the patients had improved. Comedo acne was reduced by 2 grades, papulopustular acne by 1.8 grade. The mean reduction of seborrhoea was 2 grades. SER was reduced in 62.5% of the patients to 76.4 +/- 8.8% of pretreatment values. In addition to sebaceous gland lipids, epidermal lipids were also inhibited effectively. This finding is in accordance with the marked reduction of comedones. Thus, in addition to sebaceous gland inhibition, epidermal structures may also be the target of the antiandrogen.

Action of steroids on LH-RH provoked gonadotropin release.

The action of mesterolone (1 alpha-methyl-17-beta-hydroxy-5 alpha-androsten-3-one) mesterolone cipionate, testosterone, 17 alpha-methyl-testosterone, 17 beta-hydroxy-5 alpha-androstane-3-one, cyproterone actetate and D-norgestrel on basal and LH-RH stimulated, gonadotropin release was studied in vitro. Hemi-gland preparation of the anterior pituitary were incubated in the presence or absence of steroids and/or LH-RH. Steroid hormones were added at a concentration of 25, 5 and 1 mug per ml medium. Basal levels of LH and FSH discharge were not influenced by mesterolone, whereas inhibition of LH-RH stimulated gonadotropin release was noted at 25 mug mesterolone/ml medium. The addition of mesterolone cipionate to the incubation medium did not significantly change basal FSH levels, whereas an inhibition of LH-RH provoked FSH discharge was noted. Basal LH release was stimulated by the presence of 1 mug mesterolone cipionate per 1 ml medium. LH-RH stimulated LH release in the presence of 25, 5 and 1 mug mesterolone cipionate per 1 ml medium was indistinguishable from control levels. Basal levels of LH and FSH were not affected by testosterone, whereas an impaired gonadotropin response to LH-RH was observed in the presence of testosterone. At 1 mug testosterone per 1 ml medium only LH response to LH-RH was found. Basal gonadotropin levels in the presence of 17 alpha-methyl-testosterone were indistinguishable from control values. The only inhibition of LH-RH action was found for stimulation of LH release at 25 mug 17 alpha-methyl-testosterone/ml medium. On the other hand, 17 beta-hydroxy-5 alpha-androstane-3-one stimulated basal LH and FSH discharge, and LH-RH actions were not impaired. Results of our studies with cyproterone acetate indicate that this steroid stimulates basal pituitary gonadotropin discharge at high concentration, without blunting responses to LH-RH. No action of D-norgestrel on basal and LH-RH stimulated gonadotropin release was observed. These experiments add further evidence that steroids modulate LH-RH actions at the pituitary level. It is concluded that the fine control of gonadotropin release resides at the adenohypophysis whereas the coarse control of LH and FSH discharge is at the hypothalamus and/or other centers of the central nervous system.

[Objective assessment of therapeutic hair growth--methods, possibilities and problems]. / Objektivierung des therapeutischen Haarwachstums--Methoden, Möglichkeiten und Probleme.

With regard to hair growth induced by therapeutics, we have to consider various aspects of the activity of the hair follicle. This means that a study on the efficacy of hair growth therapeutics requires a selective investigation of different kinds of follicular activity. The decision which of the non-invasive, semi-invasive or invasive techniques available should be applied in a specific case depends on the reliability of the method in relation to the technical requirement, on the one hand, and the acceptance by the volunteer as well as the clinical type and degree of hair loss, on the other. Trichorhizogram results as the only means of evaluating the efficacy of hair growth therapeutics seem problematical, since an increase of the anagen rate does not absolutely correlate with a prolongation of the anagen phase.

[Proliferation behavior of the anagen hair bulb in androgenetic alopecia before and after local administration of 17 alpha- propylmesterolone--DNA flow cytophotometry studies]. / Proliferationsverhalten des anagenen Haarbulbus bei androgenetischer Alopezie vor und nach lokaler Applikation von 17 alpha-Propylmesterolon--DNS-impulszytophotometrische Untersuchungen.

10 male test persons with androgenetic alopecia (Hamilton stage IV) were topically treated with an alcoholic 17 alpha-propylmesterolone solution (PM, 3%) for 3 months. Before and after treatment, we took deep biopsies from the fronto-temporal scalp region. Microsurgically, we isolated anagen hair bulbs, connective tissue with epidermis, and fat from the biopsy samples in order to determine cell cycle kinetics by means of DNA-flow cytometry. By comparison, cell cycle kinetics of the pre- and post-treatment samples did not show any significant changes with regard to connective tissue and fat, whereas in anagen hair bulbs, we found a significant increase of both S-phase cells (%) and the proliferation index (%). These results imply that PM, if topically applied, enhances the proliferative activity of hair matrix cells.

Efficacy of topically applied 17 alpha-propylmesterolone in acne patients.

A new substance--17 alpha-propylmesterolone--was tested in the treatment of acne. 8 male and 5 females patients applied a 3% alcoholic solution of the substance twice daily on the face for a mean period of 13 weeks. Besides clinical controls with acne grading also the determinations of the sebum excretion rate (SER) and separation of the lipid fractions was done before onset of treatment, after 14 days and then monthly. Concomitantly, the levels of several hormones (serum testosterone, prolactine, follicle stimulating hormone, luteinizing hormone and estradiol) were determined. Clinical results were moderate to excellent in most of the patients. In two patients no therapy effects became apparent after 8 weeks. SER was decreased in all patients to values between 70 and 4% of pretreatment values. Sebaceous gland lipids and epidermal lipids were both inhibited effectively. Hormonal parameters showed no significant difference of pretreatment and posttreatment values. For the first time positive effects of a topical new antiandrogen--17 alpha-propylmesterolone--could be demonstrated. The interesting finding of decrease of dermal and epidermal lipids suggests that not only sebaceous glands but also overstimulated epidermal structures may be inhibited by this antiandrogen.