Binding Mode and Selectivity of Steroids towards Glucose-6-phosphate Dehydrogenase from the Pathogen Trypanosoma cruzi.

Glucose-6-phosphate dehydrogenase (G6PDH) plays a housekeeping role in cell metabolism by generating reducing power (NADPH) and fueling the production of nucleotide precursors (ribose-5-phosphate). Based on its indispensability for pathogenic parasites from the genus Trypanosoma, G6PDH is considered a drug target candidate. Several steroid-like scaffolds were previously reported to target the activity of G6PDH. Epiandrosterone (EA) is an uncompetitive inhibitor of trypanosomal G6PDH for which its binding site to the enzyme remains unknown. Molecular simulation studies with the structure of Trypanosoma cruzi G6PDH revealed that EA binds in a pocket close to the G6P binding-site and protrudes into the active site blocking the interaction between substrates and hence catalysis. Site directed mutagenesis revealed the important steroid-stabilizing effect of residues (L80, K83 and K84) located on helix α-1 of T. cruzi G6PDH. The higher affinity and potency of 16α-Br EA by T. cruzi G6PDH is explained by the formation of a halogen bond with the hydrogen from the terminal amide of the NADP+-nicotinamide. At variance with the human enzyme, the inclusion of a 21-hydroxypregnane-20-one moiety to a 3ß-substituted steroid is detrimental for T. cruzi G6PDH inhibition. The species-specificity of certain steroid derivatives towards the parasite G6PDH and the corresponding biochemically validated binding models disclosed in this work may prove valuable for the development of selective inhibitors against the pathogen's enzyme.

Prohormone supplement 3ß-hydroxy-5α-androst-1-en-17-one enhances resistance training gains but impairs user health.

Prohormone supplements (PS) are recognized not to impart anabolic or ergogenic effects in men, but the research supporting these conclusions is dated. The Anabolic Steroid Control Act was amended in 2004 to classify androstenedione and 17 additional anabolic compounds as controlled substances. The viability of PS that entered the market after that time have not been evaluated. Seventeen resistance-trained men (23 ± 1 yr; 13.1 ± 1.5% body fat) were randomly assigned to receive either 330 mg/day of 3ß-hydroxy-5α-androst-1-en-17-one (Prohormone; n = 9) or sugar (Placebo; n = 8) per os and complete a 4-wk (16 session) structured resistance-training program. Body composition, muscular strength, circulating lipids, and markers of liver and kidney dysfunction were assessed at study onset and termination. Prohormone increased lean body mass by 6.3 ± 1.2%, decreased fat body mass by 24.6 ± 7.1%, and increased their back squat one repetition maximum and competition total by 14.3 ± 1.5 and 12.8 ± 1.1%, respectively. These improvements exceeded (P < 0.05) Placebo, which increased lean body mass by 0.5 ± 0.8%, reduced fat body mass by 9.5 ± 3.6%, and increased back squat one repetition maximum and competition total by 5.7 ± 1.7 and 5.9 ± 1.7%, respectively. Prohormone also experienced multiple adverse effects. These included a 38.7 ± 4.0% reduction in HDL (P < 0.01), a 32.8 ± 15.05% elevation in LDL (P < 0.01), and elevations of 120.0 ± 22.6 and 77.4 ± 12.0% in LDL-to-HDL and cholesterol-to-HDL ratios, respectively (both P < 0.01). Prohormone also exhibited elevations in serum creatinine (19.6 ± 4.3%; P < 0.01) and aspartate transaminase (113.8 ± 61.1%; P = 0.05), as well as reductions in serum albumin (5.1 ± 1.9%; P = 0.04), alkaline phosphatase (16.4 ± 4.7%; P = 0.04), and glomerular filtration rate (18.0 ± 3.3%; P = 0.04). None of these values changed (all P > 0.05) in Placebo. The oral PS 3ß-hydroxy-5α-androst-1-en-17-one improves body composition and muscular strength. However, these changes come at a significant cost. Cardiovascular health and liver function are particularly compromised. Given these findings, we feel the harm associated with this particular PS outweighs any potential benefit.

Synthesis and preliminary bioevaluation of 99mTc(CO)3-17α-triazolylandrost-4-ene-3-one derivative prepared via click chemistry route.

Azolyl steroids are known to manifest antiprostate cancer and antiandrogenic activities. These azolyl steroids have been shown to express affinity toward androgen receptors (ARs) overexpressed on LNCaP (human prostate adenocarcinoma) cell line. Hence, suitably derivatized azolyl steroids can be envisaged as potential vectors for targeting overexpression of ARs in prostate cancer. In the present study, testosterone has been derivatized to 17α-azidoandrost-4-ene-3-one using microwave-mediated azidation of the mesylate. Subsequently, a facile one-pot Cu(I)-catalyzed Click reaction was carried out to synthesize (99m)Tc(CO)(3)-labeled 17α-triazolylandrost-4-ene-3-one, which was characterized by HPLC. The chemical characterization of (99m)Tc(CO)(3)-17α-triazolylandrost-4-ene-3-one was carried out by preparing its corresponding rhenium complex using [NEt(4)](2)[Re(CO)(3)Br(3)] precursor. The radiolabeled complex could be prepared in >95% radiochemical yield as determined by HPLC. In vitro studies of (99m)Tc(CO)(3)-17α-triazolylandrost-4-ene-3-one complex in LNCaP cell lines overexpressing ARs showed binding of 4.95%±1.2%, with inhibition of 8%±0.9%. In vivo biodistribution studies in male Wistar rats have shown uptake in the prostate to the extent of 0.48%±0.19% injected dose/g at 1 hpi and retention therein till 3 hpi. The present study demonstrates a novel and facile one-pot reaction for preparation of (99m)Tc-labeled 17α-triazolylandrost-4-ene-3-one complex using Click chemistry. The corresponding Re-analog has been prepared for purpose of comparative characterization with the (99m)Tc-labeled complex. The radiosynthetic strategy described in this article can be further extended toward preparation of radiolabeled complexes of other triazolyl steroidal derivatives.

Seized designer supplement named "1-Androsterone": identification as 3ß-hydroxy-5α-androst-1-en-17-one and its urinary elimination.

New analogues of androgens that had never been available as approved drugs are marketed as "dietary supplement" recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids. In the present study, we report the detection of a steroid in a product seized by the State Bureau of Criminal Investigation Schleswig-Holstein, Germany. The product "1-Androsterone" of the brand name "Advanced Muscle Science" was labeled to contain 100mg of "1-Androstene-3b-ol,17-one" per capsule. The product was analyzed underivatized and as bis-TMS derivative by GC-MS. The steroid was identified by comparison with chemically synthesized 3ß-hydroxy-5α-androst-1-en-17-one, prepared by reduction of 5α-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by nuclear magnetic resonance. Semi-quantitation revealed an amount of 3ß-hydroxy-5α-androst-1-en-17-one in the capsules as labeled. Following oral administration to a male volunteer, the main urinary metabolites were monitored. 1-Testosterone (17ß-hydroxy-5α-androst-1-en-3-one), 1-androstenedione (5α-androst-1-ene-3,17-dione), 3α-hydroxy-5α-androst-1-en-17-one, 5α-androst-1-ene-3α,17ß-diol, and 5α-androst-1-ene-3ß,17ß-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common steroids of the urinary steroid profile were altered after the administration of "1-Androsterone". Especially the ratios of androsterone/etiocholanolone and 5α-/5ß-androstane-3α,17ß-diol and the concentration of 5α-dihydrotestosterone were influenced. 3α-Hydroxy-5α-androst-1-en-17-one appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to nine days after a single administration of one capsule.

The uPA(+/+)-SCID mouse with humanized liver as a model for in vivo metabolism of 4-androstene-3,17-dione.

The metabolism in primary human hepatocyte cultures often deviates from that in clinical studies, which in turn are hampered by ethical constraints. Here the use of urokinase-type plasminogen activator-severe combined immunodeficiency [uPA(+/+)-SCID] mice transplanted with human hepatocytes was investigated as a model for in vivo metabolic studies. The urinary excretion profile after oral administration of 4-androstene-3,17-dione (AD) in chimeric mice was investigated by using gas chromatography-mass spectrometry detection and was compared with previously reported metabolites of AD in humans and cell cultures. Chimeric mice exhibited an AD metabolic profile similar to that of humans, showing androsterone and etiocholanolone as major metabolites. Several hydroxylated steroids were detected as minor metabolites in the chimeric mice compared with hepatocyte cultures. A significant correlation between the extent of liver replacement and the relative abundances of human-type metabolites was established. The results for AD showed that humanized liver uPA-SCID mice can serve as an alternative model for in vivo metabolism studies in humans. In the future, this model could possibly be used for other steroids or pharmaceutical compounds.

16alpha-Bromo-epiandrosterone therapy modulates experimental feline immunodeficiency virus viremia: initial enhancement leading to long-term suppression.

16alpha-Bromo-epiandrosterone (epiBr), a synthetic derivative of the natural hormone dehyroepiandrosterone (DHEA), was evaluated for its effects on feline immunodeficiency virus (FIV) infection in experimental cats. The rationale for this study was based on the ability of DHEA to significantly reduce the mortality to viral infections in mice. DHEA and epiBr also have demonstrable in vitro anti-viral activity for both HIV-1 and FIV. Preliminary pharmacokinetic studies in cats demonstrated that subcutaneously injected epiBr was rapidly absorbed, completely metabolized, and nontoxic. Metabolites were excreted in both urine and feces, with the latter having the most complex pattern of breakdown products. Cats were then divided into four groups; two groups were infected with FIV and two uninfected. Two groups, one infected and one uninfected were treated on 5 consecutive days of weeks 0, 4, 8, 12 and 16 with epiBr. The remaining two groups were mock treated with the drug vehicle alone. Treatment started 1 week prior to infection and extended for 4 weeks after infection. Cats were observed for 20 weeks post-FIV infection. Infected cats had identical decreases in blood neutrophil and lymphocyte counts following, regardless of whether they were treated with epiBr or vehicle alone. The CD4/CD8 T-cell ratio was decreased following FIV exposure, but was significantly more decreased for the epiBr treated animals from week 2 post-infection onward. CD4+ T cells were decreased in FIV-infected cats treated with epiBr compared to their untreated cohort, while CD8+ T cells tended to be higher in treated animals. FIV infected cats that were treated with epiBr had over one-log higher virus loads at week 2 post-infection than non-epiBr treated cohorts. In spite of this enhanced initial viremia, the subsequent levels of virus in the blood were significantly lower in epiBr treated versus untreated animals. EpiBr treated cats had significantly higher FIV-p24 antibody responses than control cats receiving vehicle alone, although primary and secondary antibody responses to a T-cell dependent non-FIV antigen, keyhole limpet hemocyanin (KLH), were unaffected. EpiBr treatment significantly decreased the expected FIV-induced suppression of IL-12 p40 mRNA levels in peripheral blood mononuclear cells (PBMCs) observed at weeks 4, 5, 8, 9 and 16 post-infection, but had no influence on FIV-induced changes in IL-4, IL-6, IL-10, IFN-gamma, MIP-1alpha and RANTES.

[Transport of steroid hormones by bovine serum albumin as carrier].

The affinities of bovine serum albumin (BSA) with six kinds of steroid hormones (alpha-estradiol, ethynylestradiol, progesterone, androsterone, dehydroisoandrosterone and testosterone) were studied, and BSA was employed as a carrier to transport these hormones through the liquid membrane system, which consists of a hexane sauce phase (I), a BSA solution phase (1/15 phosphate buffer) and a hexane receiving phase (II). These steroid hormones dissolved in hexane were extensively transferred into a water phase containing BSA compared to the case of control (without added BSA). This suggests that these hormones are bound to BSA in a water phase. The ratio of hormone, BSA-binding form/free-form, is in the following order: ethynylestradiol greater than androsterone greater than testosterone greater than progesterone greater than dehydroisoandrosterone greater than alpha-estradiol in the water phase at the equilibrium conditions. The initial rate of hormone transfer from the hexane phase (I) to the water phase is followed by the first-order kinetics, and BSA induces an increase in the transfer rate except for the case of alpha-estradiol and ethynylestradiol. On the other hand, as for the increase rate of hormone concentration in the hexane phase (II), BSA makes the rate of transfer of progesterone faster than that of control, but it makes the rate of transfer of dehydroisoandrosterone more slowly. The results show the transport behaviors of steroid hormones across a water membrane are governed primarily by the partition coefficient between water and hexane phase and the addition of BSA accelerates the transport of hormones in the case of lower partition coefficients.