Studies on the in vivo metabolism of methylstenbolone and detection of novel long term metabolites for doping control analysis.

The anabolic-androgenic steroid methylstenbolone (MSTEN; 2α,17α-dimethyl-17ß-hydroxy-5α-androst-1-en-3-one) is available as a so-called designer steroid or nutritional supplement. It is occasionally detected in doping control samples, predominantly tested and confirmed as the glucuronic acid conjugate of methylstenbolone. The absence of other meaningful metabolites reported as target analytes for sports drug testing purposes can be explained by the advertised metabolic stability of methylstenbolone. In 2013, a first investigation into the human metabolism of methylstenbolone was published, and two hydroxylated metabolites were identified as potential targets for initial testing procedures in doping controls. These metabolites were not observed in recent doping control samples that yielded adverse analytical findings for methylstenbolone, and in the light of additional data originating from a recent publication on the in vivo metabolism of methylstenbolone in the horse, revisiting the metabolic reactions in humans appeared warranted. Therefore, deuterated methylstenbolone together with hydrogen isotope ratio mass spectrometry (IRMS) in combination with high accuracy/high resolution mass spectrometry were employed. After oral administration of a single dose of 10 mg of doubly labeled methylstenbolone, urine samples were collected for 29 days. Up to 40 different deuterated methylstenbolone metabolites were detected in post-administration samples, predominantly as glucuronic acid conjugates, and all were investigated regarding their potential to prolong the detection window for doping controls. Besides methylstenbolone excreted glucuronidated, three additional metabolites were still detectable at the end of the study on day 29. The most promising candidates for inclusion into routine sports drug testing methods (2α,17α-dimethyl-5α-androst-1-ene-3ß,17ß-diol and 2α,17α-dimethyl-5α-androst-1-ene-3α,17ß-diol) were synthesized and characterized by NMR.

The synthesis of 16-androstene sulfoconjugates from primary porcine Leydig cell culture.

Increased public interest in the welfare of pigs reared for pork production has led to an enhanced effort in finding alternatives to castration for controlling the unpleasant odour and flavour from heated pork products known as boar taint. The purpose of this study was to investigate the testicular metabolism of androstenone, one of the major components of boar taint. Leydig cells were isolated from mature boars and incubated with radiolabeled androstenone for 10 min, 1 h, 4 h, 8 h, and 12 h. Steroid profiles were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS/MS). Sulfoconjugated, but not glucuronidated steroids were produced by Leydig cells. Approximately 85% of androstenone was converted into sulfoconjugated metabolites in Leydig cell incubations after 8 h. This sulfoconjugate fraction included androstenol-3-sulfate and two major sulfated forms of androstenone. Following removal of the sulfate group, these two sulfated forms of androstenone returned the parent compound androstenone, and not a hydroxylated metabolite. These findings provided direct evidence for the testicular production of sulfoconjugated forms of androstenone and androstenol in the boar. The high proportion of sulfoconjugates produced by the Leydig cells emphasizes the importance of steroid conjugation, which serves to regulate the amount of unconjugated steroid hormones available for accumulation in adipose tissue.

Metabolic study of methylstenbolone in horses using liquid chromatography-high resolution mass spectrometry and gas chromatography-mass spectrometry.

Methylstenbolone (2,17α-dimethyl-5α-androst-1-en-17ß-ol-3-one) is a synthetic anabolic and androgenic steroid (AAS) sold as an oral 'nutritional supplement' under the brand names 'Ultradrol', 'M-Sten' and 'Methyl-Sten'. Like other AASs, methylstenbolone is a prohibited substance in both human and equine sports. This paper describes the studies of the in vitro and in vivo metabolism of methylstenbolone in horses using LC/HRMS, GC/MS and GC/MS/MS. Phase I in vitro metabolic study of methylstenbolone was performed using homogenised horse liver. Hydroxylation was the only biotransformation observed. Six in vitro metabolites were detected including four mono-hydroxylated metabolites, namely 16α/ß-hydroxymethylstenbolone (M1a, M1b), 20-hydroxymethylstenbolone (M1c), 6-hydroxymethylstenbolone (M1d), and two dihydroxylated methylstenbolone metabolites (M2c-M2d). An in vivo experiment was carried out using two retired thoroughbred geldings. Each horse was administered with 100 mg methylstenbolone supplement by stomach tubing daily for three consecutive days. Methylstenbolone and 14 metabolites were detected in the post-administration urine samples. The proposed in vivo metabolites included 16α/ß-hydroxymethylstenbolone (M1a, M1b), 20-hydroxymethylstenbolone (M1c), two dihydroxylated methylstenbolone (M2a, M2b), 17-epi-methylstenbolone (M3), methasterone (M4), 2,17-dimethylandrostane-16,17-diol-3-one (M5), dihydroxylated and reduced methylstenbolone (M6), 2α,17α-dimethylandrostane-3α,17ß-diol (M7), 2,17-dimethylandrostane-3,16,17-triol (M8a-M8c) and 2,17-dimethylandrostane-2,3,16,17-tetraol (M9), formed from hydroxylation, reduction and epimerisation. Methylstenbolone and ten of its metabolites could be detected in post-administration plasma samples. The highest concentration of methylstenbolone detected in urine was about 10-36 ng/mL at 3-4 h after the last administration, while the maximum concentration in plasma was about 0.4-0.7 ng/mL at 1 h after the last administration. For controlling the misuse of methylstenbolone, M8c and M9 gave the longest detection time in urine, while M4, M5 and M6 were the longest detecting analytes in plasma. They could be detected for up to 5 and 4.5 days respectively in urine and plasma. Apart from 16α/ß-hydroxymethylstenbolone (M1a, M1b), the methylstenbolone metabolites reported herein have never been reported before.

Genome shuffling of Colletotrichum lini for improving 3ß,7α,15α-trihydroxy-5-androsten-17-one production from dehydroepiandrosterone.

3ß,7α,15α-Trihydroxy-5-androsten-17-one (7α,15α-diOH-DHEA) is a key intermediate of the novel oral contraceptive Yasmin. It can be catalyzed from dehydroepiandrosterone (DHEA) through Colletotrichum lini. Improvement of 7α,15α-diOH-DHEA production was performed through recursive protoplast fusion of C. lini ST in a genome shuffling format. 7α,15α-diOH-DHEA yield of the best performing recombinant C. lini ST-F307 reached 6.08 g/L from 10 g/L DHEA, and this was 94.9% higher than that of the initial C. lini ST strain. Through optimized conditions, the 7α,15α-diOH-DHEA yield was increased to 9.32 g/L from 12 g/L DHEA, with 1.5% ethanol as cosolvent. This is the highest reported substrate concentration and 7α,15α-diOH-DHEA production with one-step substrate addition. Moreover, C. lini ST-F307 showed high P450 enzyme activity and gene transcript levels of several cytochrome P450s, and this might contribute to the enhancement of 7α,15α-diOH-DHEA production. Genome shuffling was an efficient approach to breed high-yield strains.

Enhanced 3ß,7α,15α-Trihydroxy-5-Androsten-17-One Production from Dehydroepiandrosterone by Colletotrichum lini ST-1 Resting Cells with Tween-80.

7α,15α-diOH-DHEA is a key precursor of the novel oral contraceptive Yasmin. Colletotrichum lini could catalyze dehydroepiandrosterone (DHEA) at the 7α and 15α positions. In this work, C. lini resting cells were applied in the bioconversion of DHEA to 7α,15α-diOH-DHEA. In the presence of 2 % (w/v) Tween-80, the conversion efficiency of DHEA increased drastically. The DHEA conversion and the 7α,15α-diOH-DHEA yield increased by 34.6 and 87.0 %, respectively, at the DHEA concentration of 10 g/L. Furthermore, the effects of Tween-80 on substrate solubility and C. lini physiological properties were studied. Results showed that the DHEA solubility with 2 % Tween-80 increased by 7.8 times. Meanwhile, the mycelia were integrated and full in the presence of 2 % Tween-80. The analysis on fatty acid profile of the C. lini cell membrane indicated that Tween-80 increases the content of unsaturated fatty acid. All above results suggested that the enhanced product yield caused by Tween-80 was mainly associated with easier substrate-molecule transportation across the cell membrane of C. lini.

Structures of human steroidogenic cytochrome P450 17A1 with substrates.

The human cytochrome P450 17A1 (CYP17A1) enzyme operates at a key juncture of human steroidogenesis, controlling the levels of mineralocorticoids influencing blood pressure, glucocorticoids involved in immune and stress responses, and androgens and estrogens involved in development and homeostasis of reproductive tissues. Understanding CYP17A1 multifunctional biochemistry is thus integral to treating prostate and breast cancer, subfertility, blood pressure, and other diseases. CYP17A1 structures with all four physiologically relevant steroid substrates suggest answers to four fundamental aspects of CYP17A1 function. First, all substrates bind in a similar overall orientation, rising ∼60° with respect to the heme. Second, both hydroxylase substrates pregnenolone and progesterone hydrogen bond to Asn(202) in orientations consistent with production of 17α-hydroxy major metabolites, but functional and structural evidence for an A105L mutation suggests that a minor conformation may yield the minor 16α-hydroxyprogesterone metabolite. Third, substrate specificity of the subsequent 17,20-lyase reaction may be explained by variation in substrate height above the heme. Although 17α-hydroxyprogesterone is only observed farther from the catalytic iron, 17α-hydroxypregnenolone is also observed closer to the heme. In conjunction with spectroscopic evidence, this suggests that only 17α-hydroxypregnenolone approaches and interacts with the proximal oxygen of the catalytic iron-peroxy intermediate, yielding efficient production of dehydroepiandrosterone as the key intermediate in human testosterone and estrogen synthesis. Fourth, differential positioning of 17α-hydroxypregnenolone offers a mechanism whereby allosteric binding of cytochrome b5 might selectively enhance the lyase reaction. In aggregate, these structures provide a structural basis for understanding multiple key reactions at the heart of human steroidogenesis.

Enhanced biotransformation of dehydroepiandrosterone to 3ß,7α,15α-trihydroxy-5-androsten-17-one with Gibberella intermedia CA3-1 by natural oils addition.

Dihydroxylation of dehydroepiandrosterone (DHEA) is an essential step in the synthesis of many important pharmaceutical intermediates. However, the solution to the problem of low biohydroxylation conversion in the biotransformation of DHEA has yet to be found. The effects of natural oils on the course of dihydroxylation of DHEA to 3ß,7α,15α-trihydroxy-5-androsten-17-one (7α,15α-diOH-DHEA) were studied. With rapeseed oil (2 %, v/v) addition, the bioconversion efficiency was improved, and the 7α,15α-diOH-DHEA yield was increased by 40.8 % compared with that of the control at DHEA concentration of 8.0 g/L. Meantime, the ratio of 7α,15α-diOH-DHEA to 7α-OH-DHEA was also increased by 4.5 times in the rapeseed oil-containing system. To explain the mechanism underlying the increase of 7α,15α-diOH-DHEA yield, the effects of rapeseed oil on the pH of the bioconversion system, the cell growth and integrity of Gibberella intermedia CA3-1, as well as the membrane composition were systematically studied. The addition of rapeseed oil enhanced the substrate dispersion and maintained the pH of the system during bioconversion. Cells grew better with favorable integrity. The fatty acid profile of G. intermedia cells revealed that rapeseed oil changed the cell membrane composition and improved cell membrane permeability for lipophilic substrates.

Human cytochrome P450 17A1 conformational selection: modulation by ligand and cytochrome b5.

Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bifunctional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, and binding of the soluble domain of cytochrome b5 (b5). Notably, titration of b5 to CYP17A1·pregnenolone induced a set of conformational states closely resembling those of CYP17A1·17α-hydroxypregnenolone without b5, providing structural evidence consistent with the reported ability of b5 to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating that this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.

Metabolism of methylstenbolone studied with human liver microsomes and the uPA⁺/⁺-SCID chimeric mouse model.

Anti-doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono-hydroxylated derivatives (U1-U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di-hydroxylated metabolites (U11-U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC-MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method.

5α-Androst-16-en-3α-ol ß-D-glucuronide, precursor of 5α-androst-16-en-3α-ol in human sweat.

5α-Androst-16-en-3α-ol (α-androstenol) is an important contributor to human axilla sweat odor. It is assumed that α-andostenol is excreted from the apocrine glands via a H2 O-soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H2 O-soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α-androstenol, ß-androstenol sulfates, 5α-androsta-5,16-dien-3ß-ol (ß-androstadienol) sulfate, α-androstenol ß-glucuronide, α-androstenol α-glucuronide, ß-androstadienol ß-glucuronide, and α-androstenol ß-glucuronide furanose. The occurrence of α-androstenol ß-glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative-ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α-androstenol was observed after incubation of the sterile human sweat or α-androstenol ß-glucuronide with a commercial glucuronidase enzyme, the urine-isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have ß-glucuronidase activities. We demonstrated that if α- and ß-androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H2 O-soluble precursor of α-androstenol in apocrine secretion should be a ß-glucuronide.

The association between salivary hormone levels and children's inpatient aggression: a pilot study.

Aggression is a common management problem for child psychiatry hospital units. We describe an exploratory study with the primary objective of establishing the feasibility of linking salivary concentrations of three hormones (testosterone, dehydroepiandrosterone [DHEA], and cortisol) with aggression. Between May 2011 and November 2011, we recruited 17 psychiatrically hospitalized boys (age 7-9 years). We administered the Brief Rating of Aggression by Children and Adolescents (BRACHA) and Predatory-Affective Aggression Scale (PAAS) upon admission. Saliva samples were collected from the participants during a 24-h period shortly after admission: immediately upon awakening, 30 min later, and again between 3:45 and 7:45 P.M. Nursing staff recorded Overt Aggression Scale ratings twice a day during hospitalization to quantify aggressive behavior. The salivary cortisol concentrations obtained from aggressive boys 30 min after awakening trended higher than levels from the non-aggressive boys (p = 0.06), were correlated with the number of aggressive incidents (p = 0.04), and trended toward correlation with BRACHA scores (p = 0.06). The aggressive boys also showed greater morning-to-evening declines in cortisol levels (p = 0.05). Awakening levels of DHEA and testosterone were correlated with the severity of the nearest aggressive incident (p < 0.05 for both). The BRACHA scores of the aggressive boys were significantly higher than scores of the non-aggressive boys (p < 0.001). Our data demonstrate the feasibility of collecting saliva from children on an inpatient psychiatric unit, affirm the utility of the BRACHA in predicting aggressive behavior, and suggest links between salivary hormones and aggression by children who undergo psychiatric hospitalization.

Detection of designer steroid methylstenbolone in "nutritional supplement" using gas chromatography and tandem mass spectrometry: elucidation of its urinary metabolites.

The use of "nutritional supplements" containing unapproved substances has become a regular practice in amateur and professional athletes. This represents a dangerous habit for their health once no data about toxicological or pharmacological effects of these supplements are available. Most of them are freely commercialized online and any person can buy them without medical surveillance. Usually, the steroids intentionally added to the "nutritional supplements" are testosterone analogues with some structural modifications. In this study, the analyzed product was bought online and a new anabolic steroid known as methylstenbolone (2,17α-dimethyl-17ß-hydroxy-5α-androst-1-en-3-one) was detected, as described on label. Generally, anabolic steroids are extensively metabolized, thus in-depth knowledge of their metabolism is mandatory for doping control purposes. For this reason, a human excretion study was carried out with four volunteers after a single oral dose to determine the urinary metabolites of the steroid. Urine samples were submitted to enzymatic hydrolysis of glucuconjugated metabolites followed by liquid-liquid extraction and analysis of the trimethylsilyl derivatives by gas chromatography coupled to tandem mass spectrometry. Mass spectrometric data allowed the proposal of two plausible metabolites: 2,17α-dimethyl-16ξ,17ß-dihydroxy-5α-androst-1-en-3-one (S1), 2,17α-dimethyl-3α,16ξ,17ß-trihydroxy-5α-androst-1-ene (S2). Their electron impact mass spectra are compatible with 16-hydroxylated steroids O-TMS derivatives presenting diagnostic ions such as m/z 231 and m/z 218. These metabolites were detectable after one week post administration while unchanged methylstenbolone was only detectable in a brief period of 45 h.

[Breeding of high 3beta,7alpha,15alpha-trihydroxy-5-androsten-17-one transforming strains and their conversion process optimization].

In order to improve transformation efficiency of dehydroepiandrosterone (DHEA) into 3beta,7alpha,15alpha-trihydroxy-5-androsten-17-one (7alpha,15alpha-diOH-DHEA) by Gibberella intermedia CA3-1, we investigated the strains breeding and their conversion process optimization. G. intermedia CA3-1 strains were treated with 0.12 mg/mL 1-methyl-3-nitro-1-nitroso-guanidin (NTG) for 30 min and chosen by 350 micromol/L minimum inhibitory concentration ketoconazole resistance marker. The high production strain named M-10 with a good genetic stability was selected and the product molar yield achieved to 70.2%, which was 20% higher than that of original strain. Under the improved conversion process with the DHEA concentration of 5 g/L, the product molar yield of the mutant M-10 reached 75.6%, which was improved by 31.3% than that of original strain.

Mass spectrometric description of novel oxymetholone and desoxymethyltestosterone metabolites identified in human urine and their importance for doping control.

The metabolism of two anabolic steroids - oxymetholone and desoxymethyltestosterone - was reinvestigated to identify new targets potentially valuable for the antidoping analysis. Excretion urine samples from the laboratory reference collection were used in this study. Following fractionation of the urinary extract by means of high performance liquid chromatography (HPLC), each fraction was subjected to gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis after trimethylsilylation. About 20 metabolites were found for desoxymethyltestosterone and more than 40 for oxymetholone, with many of them being isomeric compounds. In addition to the well-known reduced and hydroxylated metabolites, 18-nor-17,17-dimethyl and 18-nor-17-hydroxymethyl-17-methyl steroids were also identified. Having evaluated all the metabolites in terms of how long they could be detected, we suggest that 18-nor-2ξ,17ß-hydroxymethyl-17α-methyl-5α-androst-13-en-3α-ol is an important marker of oxymetholone abuse. In case of desoxymethyltestosterone, better detectability could be achieved if 18-nor-17,17-dimethyl-5α-androst-13-en-2ξ,3α-diol is monitored. These novel metabolites could be detected using GC-MS/MS at least for 14 days after administration of these anabolic steroids compared to 5-7 days for previously reported metabolites.

Characterization of desoxymethyltestosterone main urinary metabolite produced from cultures of human fresh hepatocytes.

Desoxymethyltestosterone (DMT; 17ß-hydroxy-17α-methyl-5α-androst-2-ene) is a designer steroid present in hormonal supplements distributed illegally as such or in combination with other steroids, for self-administration. It figures on the list of substances prohibited in sports and its detection in athlete's urine samples is based upon the presence of the parent compound or the main urinary metabolite, which has not been characterized yet. Following its isolation from cultures of human fresh hepatocytes and S9 fractions of liver homogenates, we were able to identify this metabolite as being 17α-methyl-2ß,3α,17ß-trihydroxy-5α-androstane. Other minor metabolites were also characterized. The production, isolation, NMR, mass spectral analyses and chemical synthesis are presented.

Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001.

Cytochrome P450 17A1 (also known as CYP17A1 and cytochrome P450c17) catalyses the biosynthesis of androgens in humans. As prostate cancer cells proliferate in response to androgen steroids, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer, but drug development has been hampered by lack of information regarding the structure of CYP17A1. Here we report X-ray crystal structures of CYP17A1, which were obtained in the presence of either abiraterone, a first-in-class steroidal inhibitor recently approved by the US Food and Drug Administration for late-stage prostate cancer, or TOK-001, an inhibitor that is currently undergoing clinical trials. Both of these inhibitors bind the haem iron, forming a 60° angle above the haem plane and packing against the central I helix with the 3ß-OH interacting with aspargine 202 in the F helix. Notably, this binding mode differs substantially from those that are predicted by homology models and from steroids in other cytochrome P450 enzymes with known structures, and some features of this binding mode are more similar to steroid receptors. Whereas the overall structure of CYP17A1 provides a rationale for understanding many mutations that are found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate a better understanding of the enzyme's dual hydroxylase and lyase catalytic capabilities and assist in rational drug design. Specifically, structure-based design is expected to aid development of inhibitors that bind only CYP17A1 and solely inhibit its androgen-generating lyase activity to improve treatment of prostate and other hormone-responsive cancers.

Pharmacology and immune modulating properties of 5-androstene-3ß,7ß,17ß-triol, a DHEA metabolite in the human metabolome.

Androst-5-ene-3ß,7ß,17ß-triol (ßAET) is an anti-inflammatory metabolite of DHEA that is found naturally in humans, but in rodents only after exogenous DHEA administration. Unlike DHEA, C-7-oxidized DHEA metabolites cannot be metabolized into potent androgens or estrogens, and are not peroxisome proliferators in rodents. The objective of our current studies was to characterize the pharmacology of ßAET to enable clinical trials in humans. The pharmacology of ßAET was characterized by pharmacokinetics, drug metabolism, nuclear hormone receptor interactions, androgenicity, estrogenicity, and systemic toxicity studies. ßAET's acute anti-inflammatory activity and immune modulating characteristics were measured in vitro in RAW264.7 cells and in vivo in murine models with parenteral administration. ßAET was rapidly metabolized and cleared from circulation in mice and monkeys. ßAET was weakly androgenic and estrogenic in immature rodents, but not bound by androgen, estrogen, progesterone, or glucocorticoid nuclear hormone receptors. ßAET did not induce peroxisome proliferation, nor was it systemically toxic or trophic for sex hormone responsive tissues in mature rats and monkeys. ßAET significantly attenuated acute inflammation both in vitro and in vivo, augmented immune responses in adult mice, and reversed immune senescence in aged mice. ßAET may contribute to the anti-inflammatory activity in rodents attributed to DHEA. Unlike DHEA, ßAET's anti-inflammatory activity cannot be ascribed to activation of PPARs, androgen, or estrogen nuclear hormone receptors. Exogenous ßAET is unlikely to produce untoward toxicity or hormonal perturbations in humans.

Novel components of the human metabolome: the identification, characterization and anti-inflammatory activity of two 5-androstene tetrols.

Two natural 5-androstene steroid tetrols, androst-5-ene-3ß,7ß,16α,17ß-tetrol (HE3177) and androst-5-ene-3α,7ß,16α,17ß-tetrol (HE3413), were discovered in human plasma and urine. These compounds had significant aqueous solubility, did not bind or transactivate steroid-binding nuclear hormone receptors, and were not immunosuppressive in murine mixed-lymphocyte studies. Both compounds appear to be metabolic end products, as they were resistant to primary and secondary metabolism. Both were orally bioavailable, and were very well tolerated in a two-week dose-intensive toxicity study in mice. Anti-inflammatory properties were found with exogenous administration of these compounds in rodent disease models of multiple sclerosis, lung injury, chronic prostatitis, and colitis.

Population based evaluation of a multi-parametric steroid profiling on administered endogenous steroids in single low dose.

Steroid profiling provides valuable information to detect doping with endogenous steroids. Apart from the traditionally monitored steroids, minor metabolites can play an important role to increase the specificity and efficiency of current detection methods. The applicability of several minor steroid metabolites was tested on administration studies with low doses of oral testosterone (T), T gel, dihydrotestosterone (DHT) gel and oral dehydroepiandrosterone (DHEA). The collected data for all monitored parameters were evaluated with the respective population based reference ranges. Besides the traditional markers T/E, T and DHT, minor metabolites 4-OH-Adion and 6α-OH-Adion were found as most sensitive metabolites to detect oral T administration. The most sensitive metabolites for the detection of DHEA were identified as 16α-OH-DHEA and 7ß-OH-DHEA but longest detection up to three days (after oral administration of 50 mg) was obtained with non-specific 5ß-steroids and its ratios. Steroids applied as a gel had longer effects on the metabolism but were generally not detectable with universal decision criteria. It can be concluded that population based reference ranges show limited overall performance in detecting misuse of small doses of natural androgens. Although some minor metabolites provide additional information for the oral testosterone and DHEA formulations, the topical administered steroids could not be detected for all volunteers using universal reference limits. Application of other population based threshold limits did not lead to longer detection times.

A 17beta-derivative of allopregnanolone is a neurosteroid antagonist at a cerebellar subpopulation of GABA A receptors with nanomolar affinity.

BACKGROUND AND PURPOSE: High-affinity, subtype-selective antagonists of the neurosteroid binding sites of GABA(A) receptors are not available. We have characterized an allopregnanolone derivative as an antagonist of cerebellar GABA(A) receptors with nanomolar affinity. EXPERIMENTAL APPROACH: Receptor binding and electrophysiological methods were used for the allosteric modulation of cerebellar GABA(A) receptors by an allopregnanolone derivative, (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3alpha-ol (HBAO). GABA(A) receptors of rat cerebellar membranes were labelled with the chloride channel blocker [(3)H]ethynylbicycloorthobenzoate (EBOB). The ionophore function of GABA(A) receptors was studied by whole-cell patch clamp electrophysiology in cultured rat cerebellar granule and cortical cells. KEY RESULTS: Partial displacement of cerebellar [(3)H]EBOB binding by nanomolar HBAO was attenuated by 0.1 mM furosemide, an antagonist of alpha(6) and beta(2-3) subunit-containing GABA(A) receptors. Displacement curves of HBAO were reshaped by 30 nM GABA and shifted to the right. However, the micromolar potency of full displacement by allopregnanolone was not affected by 0.1 mM furosemide or 30 nM GABA. The nanomolar, but not the micromolar phase of displacement of [(3)H]EBOB binding by GABA was attenuated by 100 nM HBAO. Submicromolar HBAO did not affect [(3)H]EBOB binding to cortical and hippocampal GABA(A) receptors. HBAO up to 1 microM did not affect chloride currents elicited by 0.3-10 microM GABA, while it abolished potentiation by 1 microM allopregnanolone with nanomolar potency in cerebellar but not in cortical cells. Furosemide attenuated cerebellar inhibition by 100 nM HBAO. CONCLUSIONS AND IMPLICATIONS: HBAO is a selective antagonist of allopregnanolone, a major endogenous positive modulator via neurosteroid sites of cerebellar (probably alpha(6)beta(2-3)delta) GABA(A) receptors.