Heterocycle-functional steroidal derivatives: Design, synthesis, bioevaluation and SARs of steroidal pyrazolo[1,5-a]pyrimidines as potential ALK inhibitors.

Two series of heterocyclic steroidal pyrazolo[1,5-a]pyrimidines derived from dehydroepiandrosterone (DHEA) and epiandrosterone (EPIA) were designed and synthesized, and these compounds were screened for their potential antiproliferation activities. The preliminary bioassay indicated that some of target compounds exhibited significantly good antiproliferation activities against human melanoma cell line (A875) and human hepatocellular carcinoma (Huh-7) cell lines compared with 5-fluorouracil (5-FU), and some of which present good antiproliferative activities as potential ALK inhibitors. The detailed analysis of structure-activity relationships (SARs) based on the inhibition activities, kinase assay, and molecular docking demonstrated that the antiproliferation activities of these steroidal pyrazolo[1,5-a]pyrimidine might be affected by the ß-hydroxyl group of steroidal scaffold and the N atom of pyridine heterocycle. Especially, compound 4c has certain inhibitory effects on the tyrosine protein kinases ALK, CDK2/CyclinE1, FAK, CDK5/P35, CDK9/CyclinT1, CDK5/P25, PIM2, CDK2/CyclinA2, CDK1/CyclinB1, etc., and which displayed highest inhibitory effect on the kinases of ALK with inhibition rate 40.63 % at the concentration of 10 µM, which induced cell death in A875 cells at least partly (initially), by apoptosis.

Involvement of porcine and human carbonyl reductases in the metabolism of epiandrosterone, 11-oxygenated steroids, neurosteroids, and corticosteroids.

Porcine carbonyl reductases (pCBR1 and pCBR-N1) and aldo-keto reductases (pAKR1C1 and pAKR1C4) exhibit hydroxysteroid dehydrogenase (HSD) activity. However, their roles in the metabolism of porcine-specific androgens (19-nortestosterone and epiandrosterone), 11-oxygenated androgens, neurosteroids, and corticosteroids remain unclear. Here, we compared the steroid specificity of the four recombinant enzymes by kinetic and product analyses. In C18/C19-steroids,11-keto- and 11ß-hydroxy-5α-androstane-3,17-diones were reduced by all the enzymes, whereas 5α-dihydronandrolone (19-nortestosterone metabolite) and 11-ketodihydrotestosterone were reduced by pCBR1, pCBR-N1, and pAKR1C1, of which pCBR1 exhibited the lowest (submicromolar) Km values. Product analysis showed that pCBR1 and pCBR-N1 function as 3α/ß-HSDs, in contrast to pAKR1C1 and pAKR1C4 (acting as 3ß-HSD and 3α-HSD, respectively). Additionally, 17ß-HSD activity was observed in pCBR1 and pCBR-N1 (toward epiandrosterone and its 11-oxygenated derivatives) and in pAKR1C1 (toward androsterone, 4-androstene-3,17-dione and their 11-oxygenated derivatives). The four enzymes also showed different substrate specificity for 3-keto-5α/ß-dihydro-C21-steroids, including GABAergic neurosteroid precursors and corticosteroid metabolites. 5ß-Dihydroprogesterone was reduced by all the enzymes, whereas 5α-dihydroprogesterone was reduced only by pCBR1, and 5α/ß-dihydrodeoxycorticosterones by pCBR1 and pCBR-N1. The two pCBRs also reduced the 5α/ß-dihydro-metabolites of cortisol, 11-deoxycortisol, cortisone, and corticosterone. pCBR1 exhibited lower Km values (0.3-2.9 µM) for the 3-keto-C21-steroids than pCBR-N1 (Km=10-36 µM). The reduced products of the 3-keto-C21-steroids by pCBR1 and pCBR-N1 were their 3α-hydroxy-metabolites. Finally, we found that human CBR1 has similar substrate specificity for the C18/C19/C21-steroids to pCBR-N1. Based on these results, it was concluded that porcine and human CBRs can be involved in the metabolism of the aforementioned steroids as 3α/ß,17ß-HSDs.

Association of genetically predicted 486 blood metabolites on the risk of Alzheimer's disease: a Mendelian randomization study.

Background: Studies have reported that metabolic disturbance exhibits in patients with Alzheimer's disease (AD). Still, the presence of definitive evidence concerning the genetic effect of metabolites on AD risk remains insufficient. A systematic exploration of the genetic association between blood metabolites and AD would contribute to the identification of new targets for AD screening and prevention. Methods: We conducted an exploratory two-sample Mendelian randomization (MR) study aiming to preliminarily identify the potential metabolites involved in AD development. A genome-wide association study (GWAS) involving 7,824 participants provided information on 486 human blood metabolites. Outcome information was obtained from a large-scale GWAS meta-analysis of AD, encompassing 21,982 cases and 41,944 controls of Europeans. The primary two-sample MR analysis utilized the inverse variance weighted (IVW) model while supplementary analyses used Weighted median (WM), MR Egger, Simple mode, and Weighted mode, followed by sensitivity analyses such as the heterogeneity test, horizontal pleiotropy test, and leave-one-out analysis. For the further identification of metabolites, replication and meta-analysis with FinnGen data, steiger test, linkage disequilibrium score regression, confounding analysis, and were conducted for further evaluation. Multivariable MR was performed to assess the direct effect of metabolites on AD. Besides, an extra replication analysis with EADB data was conducted for final evaluation of the most promising findings. Results: After rigorous genetic variant selection, IVW, complementary analysis, sensitivity analysis, replication and meta-analysis with the FinnGen data, five metabolites (epiandrosterone sulfate, X-12680, pyruvate, docosapentaenoate, and 1-stearoylglycerophosphocholine) were identified as being genetically associated with AD. MVMR analysis disclosed that genetically predicted these four known metabolites can directly influence AD independently of other metabolites. Only epiandrosterone sulfate and X-12680 remained suggestive significant associations with AD after replication analysis with the EADB data. Conclusion: By integrating genomics with metabonomics, this study furnishes evidence substantiating the genetic association of epiandrosterone sulfate and X-12680 with AD. These findings hold significance for the screening, prevention, and treatment strategies for AD.

Novel Steroidal[17,16-d]pyrimidines Derived from Epiandrosterone and Androsterone: Synthesis, Characterization and Configuration-Activity Relationships.

Two series of novel steroidal[17,16-d]pyrimidines derived from natural epiandrosterone and androsterone were designed and synthesized, and these compounds were screened for their potential anticancer activities. The preliminary bioassay indicated that some of these prepared compounds exhibited significantly good cytotoxic activities against human gastric cancer (SGC-7901), lung cancer (A549), and hepatocellular liver carcinoma (HepG2) cell lines compared with 5-fluorouracil (5-FU), epiandrosterone, and androsterone. Especially the respective pairs from epiandrosterone and androsterone showed significantly different inhibitory activities, and the possible configuration-activity relationships have also been summarized and discussed based on kinase assay and molecular docking, which indicated that the inhibition activities of these steroidal[17,16-d]pyrimidines might obviously be affected by the configuration of the hydroxyl group in the part of the steroidal scaffold.

Semi-automated serum steroid profiling with tandem mass spectrometry.

Objectives: Highly selective and sensitive multi-analyte methods for the analysis of steroids are attractive for the diagnosis of endocrine diseases. Commercially available kits are increasingly used for this purpose. These methods involve laborious solid phase extraction, and the respective panels of target analytes are incomplete. We wanted to investigate whether an improvement of kit solutions is possible by introducing automated on-line solid phase extraction (SPE) and combining originally separate analyte panels. Methods: Sample preparation was performed using automated on-line SPE on a high-pressure stable extraction column. Chromatographic separation, including isobaric compounds, was achieved using a 0.25 mM ammonium fluoride-methanol gradient on a small particle size biphenyl column. Standard compounds and internal standard mixtures of two panels of a commercially available kit were combined to achieve an optimized and straightforward detection of 15 endogenous steroids. Validation was performed according to the European Medicines Agency (EMA) guidelines with slight modifications. Results: Validation was successfully performed for all steroids over a clinically relevant calibration range. Deviations of intra- and inter-assay accuracy and precision results passed the criteria and no relevant matrix effects were detected due to highly effective sample preparation. External quality assessment samples showed the applicability as a routine diagnostic method, which was affirmed by the analyses of anonymized clinical samples. Conclusions: It was found possible to complement a commercially available kit for quantitative serum steroid profiling based on isotope dilution LC-MS/MS by implementing automated on-line SPE, thereby improving the practicality and robustness of the measurement procedure.

D-chiro-inositol, an aromatase down-modulator, increases androgens and reduces estrogens in male volunteers: a pilot study.

BACKGROUND: Androgen deficiency affects men in the adulthood, causing several harmful effects at the reproductive and behavioural levels. Since aromatase is an enzyme that catalyses the conversion of androgens to estrogens, and it is responsible for an adequate balance of both sex hormones in males and females, the administration of molecules acting as down modulators may contribute to restore an abnormal enzymatic activity. A prospective pilot study was carried out to investigate the effect of D-chiro-inositol, a putative aromatase down-modulator, on serum levels of testosterone, estradiol, estrone, dehydroepiandrosterone and epiandrosterone from a group of adult male volunteers. Glucose, insulin, follicle-stimulating hormone, luteinizing hormone, inhibin B, D-chiro-inositol and myo-inositol serum levels were also measured. RESULTS: Male volunteers were selected according to age and body mass index. Subjects with altered glycemia and/or hormonal status, due to advanced age or abnormal weight, were enrolled in the study. Each of the 10 volunteers enrolled took oral D-chiro-inositol (1 g/day) for 1 month. Serum assays of selected markers were performed at baseline (control) and after treatment. D-chiro-inositol administration was associated to reduced serum levels of estrone (- 85.0%) and estradiol (- 14.4%), and increased serum levels of testosterone (+ 23.4%) and dehydroepiandrosterone (+ 13.8%). In addition, epiandrosterone levels were higher (+39%) after treatment. On the other hand, follicle-stimulating hormone, luteinizing hormone and inhibin B did not change. A trend toward a decrease of glycemia, insulinemia and Homeostatic Model Assessment index was observed after D-chiro-inositol treatment, although differences did not reach statistical significance. D-chiro-inositol treatment did not cause any noticeable adverse effect. CONCLUSIONS: Increased androgens and decreased estrogens seem to confirm that D-chiro-inositol acts as an aromatase down-modulator, but with a still unknown mechanism of action. This pilot study opens up new perspectives of research and therapeutic applications for D-chiro-inositol at different dosages and length of treatment. Authorization number 005/2020 released by the Local Ethics Committee of Alma Res Fertility Center, Rome. TRIAL REGISTRATION NUMBER: NCT04615767 (registry: ClinicalTrials.gov) Date of registration: November 3, 2020.

RéSUMé: CONTEXTE: L'insuffisance en androgènes affecte les hommes à l'âge adulte, causant plusieurs effets nocifs aux niveaux reproductif et comportemental. Puisque l'aromatase est. une enzyme qui catalyse la conversion des androgènes en œstrogènes, et qu'elle est. responsable d'un équilibre adéquat des hormones sexuelles chez les hommes et les femmes, l'administration de molécules agissant comme freinateurs peut contribuer à restaurer une activité enzymatique anormale. Une étude prospective pilote a été menée pour étudier l'effet du D-chiro-inositol, un potentiel freinateur de l'aromatase, sur les taux sériques de testostérone, estradiol, estrone, déhydroépiandrostérone et d'épiandrostérone d'un groupe d'hommes adultes volontaires. Le glucose, l'insuline, l'hormone folliculostimulante, l'hormone lutéinisante, l'inhibine B, le D-chiro-inositol et les taux sériques de myo-inositol ont également été mesurés. RéSULTATS: Les hommes volontaires ont été sélectionnés selon l'âge et l'indice de masse corporelle. Les hommes qui présentaient une glycémie et/ou un statut hormonal altérés en raison d'un âge avancé ou d'un poids anormal, ont été inclus dans l'étude. Chacun des 10 volontaires enrôlés a pris du D-chiro-inositol (1 g/jour) par voie orale pendant un mois. Des dosages sériques des marqueurs sélectionnés ont été réalisés avant (témoin) et après le traitement. L'administration de D-chiro-inositol a été associée à une réduction des taux sériques de l'estrone (− 85.0%) et de l'estradiol (− 14,4%), et a une augmentation des taux sériques de testostérone (+ 23,4%) et de déhydroépiandrostérone (+ 13,8%). En outre, les taux d'épiandrostérone étaient plus élevés (39%) après le traitement. D'autre part, les taux d'hormone folliculostimulante, d'hormone lutéinisante et d'inhibine B n'ont pas été modifiés. Une tendance à la diminution de la glycémie, de l'insulinémie et de l'indice d'évaluation du modèle homéostatique a été observée après traitement par D-chiro-inositol, bien que les différences n'aient pas atteint une signification statistique. Le traitement par D-chiro-inositol n'a causé aucun effet indésirable notable. CONCLUSIONS: L'augmentation des androgènes et la diminution des œstrogènes semblent confirmer que le D-chiro-inositol agit comme un freinateur de l'aromatase, mais avec un mécanisme d'action encore inconnu. Cette étude pilote ouvre de nouvelles perspectives de recherche et d'applications thérapeutiques pour le D-chiro-inositol à différents dosages et durées de traitement.

Causal Effects of Genetically Determined Metabolites on Risk of Polycystic Ovary Syndrome: A Mendelian Randomization Study.

Background: Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder that is influenced by both genetic and environmental factors. However, the etiology of PCOS remains unclear. Methods: We conducted a two-sample Mendelian randomization (MR) analysis to assess the causal effects of genetically determined metabolites (GDMs) on the risk of PCOS. We used summary level data of a genome-wide association study (GWAS) on 486 metabolites (n = 7,824) as exposure and a PCOS GWAS consisting of 4,138 cases and 20,129 controls as the outcome. Both datasets were obtained from publicly published databases. For each metabolite, a genetic instrumental variable was generated to assess the relationship between the metabolite and PCOS. For MR analysis, we primarily used the standard inverse variance weighted (IVW) method, while three additional methods-the MR-Egger, weighted median, and MR-PRESSO (pleiotropy residual sum and outlier) methods-were performed as sensitivity analyses. Results: Using genetic variants as predictors, we observed a robust relationship between epiandrosterone sulfate (EPIA-S) and PCOS (PIVW = 0.0186, PMR-Egger = 0.0111; PWeighted-median = 0.0154, and PMR-PRESSO = 0.0290). Similarly, 3-dehydrocarnitine, 4-hydroxyhippurate, hexadecanedioate, and ß-hydroxyisovalerate may also have causal effects on PCOS development. Conclusions: We identified metabolites that might have causal effects on PCOS development. Our study emphasizes the role of genetic factors underlying the causal relationships between metabolites and PCOS and provides novel insights through the integration of metabolomics and genomics to better understand the mechanisms involved in human disease pathogenesis.

Evaluation of epiandrosterone as a long-term marker of testosterone use.

Identification and evaluation of long-term markers is crucial in prolonging the detection window for anabolic steroid abuse in sport. Recently, sulfoconjugated epiandrosterone was identified as a potential long-term marker for the abuse of certain endogenous anabolic agents, including testosterone, which continues to be widely used as a performance enhancing agent in sport. To evaluate the applicability of epiandrosterone sulfate as a marker for testosterone use, administration studies were conducted with multiple modes of testosterone administration - transdermal, intramuscular, and subcutaneous. A modified sample preparation method was used to collect both glucuronidated and sulfoconjugated analytes of interest. Carbon isotope ratio measurements from the administration studies are presented here. Epiandrosterone was less effective than the conventionally used target compounds for detection of the low dose application (transdermal gel). With intramuscular administration, epiandrosterone was more diagnostic than with transdermal administration, but it did not prolong the detection window more than the conventional target compounds. With subcutaneous administration, the doses administered to the subjects were varied and the effect on the epiandrosterone values was dependent on the magnitude of the dose administered. Epiandrosterone does not appear to be a useful marker in the detection of low dose testosterone administration. It is responsive to higher dose administration, but it does not provide an extension of the detection window relative to conventional target compounds.

Development and validation of a fast gas chromatography combustion isotope ratio mass spectrometry method for the detection of epiandrosterone sulfate in urine.

In doping control, to confirm the exogenous origin of exogenously administered anabolic androgenic steroids (AAS), a gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) analysis is performed. Recently published work suggests that epiandrosterone sulfate (EpiAS) is a promising IRMS target compound for the detection of AAS, capable of prolonging the detection window. However, EpiAS is only excreted in urine in its sulfoconjugated form, while all other IRMS target compounds are excreted glucuronidated, meaning that EpiAS cannot be incorporated in the existing IRMS methods. A separate extensive sample preparation needs to be performed on this compound with a different hydrolysis and extraction procedure and a different liquid chromatography (LC) clean-up. The current work presents a new, fast, and easy to implement EpiAS IRMS method. The approach was based on the direct GC analysis of non-hydrolyzed EpiAS, making the solid phase extraction, hydrolysis, and acetylation step redundant. Sample preparation consisted of a simple liquid-liquid extraction, followed by LC fraction collection. A population study was performed to check compliance with the criteria drafted by the World Anti-Doping Agency (WADA). To verify the applicability of the developed approach, the method was applied to the samples of four administration studies (i.e. dehydroepiandrosterone (DHEA), testosterone gel (T gel), androstenedione (ADION), and intramuscular testosterone undecanoate. In contrast to previously published data, the strength of EpiAS as the target compound and the prolongation of the detection window in comparison with the conventional IRMS target compounds was less pronounced.

Synthesis and bioevaluation of novel steroidal isatin conjugates derived from epiandrosterone/androsterone.

Steroids are classes of natural products widely distributed in nature, which have been demonstrated to exhibit broad biological functions, and have also attracted increasing interest from bioorganic and pharmaceutical researches. In order to develop novel chemical entities as potential cytotoxic agents, a series of steroidal isatin conjugations derived from epiandrosterone and androsterone were efficiently prepared and characterized, and all these obtained compounds were screened for their potential cytotoxic activities. The preliminary bioassay indicated that most of the newly synthesized compounds exhibited good cytotoxic activities against human gastric cancer (SGC-7901), melanoma (A875), and hepatocellular liver carcinoma (HepG2) cell lines compared with 5-fluorouracil (5-FU), which might be considered as promising scaffold for further development of potential anticancer agents.

Synthesis and Biological Activity of Several Modified 5α-Androstanolone Derivatives.

Several new N-containing epiandrosterone derivatives modified by phenylacetic acid chloride were synthesized for biological activity studies. Compounds with antiviral activity were discovered among them and 3ß-hydroxy-1'-aryl-3'-methyl-5'-androstano[17,16-d]pyrazolines prepared by us earlier.

Transport of steroid 3-sulfates and steroid 17-sulfates by the sodium-dependent organic anion transporter SOAT (SLC10A6).

The sodium-dependent organic anion transporter SOAT/Soat shows highly specific transport activity for sulfated steroids. SOAT substrates identified so far include dehydroepiandrosterone sulfate, 16α-hydroxydehydroepiandrosterone sulfate, estrone-3-sulfate, pregnenolone sulfate, 17ß-estradiol-3-sulfate, and androstenediol sulfate. Apart from these compounds, many other sulfated steroids occur in mammals. Therefore, we aimed to expand the substrate spectrum of SOAT and analyzed the SOAT-mediated transport of eight different sulfated steroids by combining in vitro transport experiments in SOAT-transfected HEK293 cells with LC-MS/MS analytics of cell lysates. In addition, we aimed to better understand the structural requirements for SOAT substrates and so selected structural pairs varying only at specific positions: 3α/3ß-sulfate, 17α/17ß-sulfate, mono-sulfate/di-sulfate, and 17α-hydroxylation. We found significant and sodium-dependent SOAT-mediated transport of 17α-hydroxypregnenolone sulfate, 17ß-estradiol-17-sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and 5α-dihydrotestosterone sulfate. However, 17ß-estradiol-3,17-disulfate was not transported by SOAT. IN CONCLUSION: SOAT substrates from the group of sulfated steroids are characterized by a planar and lipophilic steroid backbone in trans-trans-trans conformation of the rings and a negatively charged mono-sulfate group at positions 3' or 17' with flexibility for α- or ß- orientation. Furthermore, 5α-reduction, 16α-hydroxylation, and 17α-hydroxylation are acceptable for SOAT substrate recognition, whereas addition of a second negatively charged sulfate group seems to abolish substrate binding to SOAT, and so 17ß-estradiol-3,17-disulfate is not transported by SOAT.

Epiandrosterone sulfate prolongs the detectability of testosterone, 4-androstenedione, and dihydrotestosterone misuse by means of carbon isotope ratio mass spectrometry.

In the course of investigations into the metabolism of testosterone (T) by means of deuterated T and hydrogen isotope ratio mass spectrometry, a pronounced influence of the oral administration of T on sulfoconjugated steroid metabolites was observed. Especially in case of epiandrosterone sulfate (EPIA_S), the contribution of exogenous T to the urinary metabolite was traceable up to 8 days after a single oral dose of 40 mg of T. These findings initiated follow-up studies on the capability of EPIA_S to extend the detection of T and T analogue misuse by carbon isotope ratio (CIR) mass spectrometry in sports drug testing. Excretion study urine samples obtained after transdermal application of T and after oral administration of 4-androstenedione, dihydrotestosterone, and EPIA were investigated regarding urinary concentrations and CIR. With each administered steroid, EPIA_S was significantly depleted and prolonged the detectability when compared to routinely used steroidal target compounds by a factor of 2 to 5. In order to simplify the sample preparation procedure for sulfoconjugated compounds, enzymatic cleavage by Pseudomonas aeruginosa arylsulfatase was tested and implemented into CIR measurements for the first time. Further simplification was achieved by employing multidimensional gas chromatography to ensure the required peak purity for CIR determinations, instead of sample purification strategies using liquid chromatographic fractionation. Taking into account these results that demonstrate the unique and broad applicability of EPIA_S for the detection of illicit administrations of T or T-related steroids, careful consideration of how this steroid can be implemented into routine doping control analysis appears warranted. Copyright © 2017 John Wiley & Sons, Ltd.

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.

Hydroxylation of DHEA and its analogues by Absidia coerulea AM93. Can an inducible microbial hydroxylase catalyze 7α- and 7ß-hydroxylation of 5-ene and 5α-dihydro C19-steroids?

In this paper we focus on the course of 7-hydroxylation of DHEA, androstenediol, epiandrosterone, and 5α-androstan-3,17-dione by Absidia coerulea AM93. Apart from that, we present a tentative analysis of the hydroxylation of steroids in A. coerulea AM93. DHEA and androstenediol were transformed to the mixture of allyl 7-hydroxy derivatives, while EpiA and 5α-androstan-3,17-dione were converted mainly to 7α- and 7ß-alcohols accompanied by 9α- and 11α-hydroxy derivatives. On the basis of (i) time course analysis of hydroxylation of the abovementioned substrates, (ii) biotransformation with resting cells at different pH, (iii) enzyme inhibition analysis together with (iv) geometrical relationship between the C-H bond of the substrate undergoing hydroxylation and the cofactor-bound activated oxygen atom, it is postulated that the same enzyme can catalyze the oxidation of C7-Hα as well as C7-Hß bonds in 5-ene and 5α-dihydro C19-steroids. Correlations observed between the structure of the substrate and the regioselectivity of hydroxylation suggest that 7ß-hydroxylation may occur in the normal binding enzyme-substrate complex, while 7α-hydroxylation-in the reverse inverted binding complex.

Pathways and genes involved in steroid hormone metabolism in male pigs: a review and update.

This paper reviews state-of-the-art knowledge on steroid biosynthesis pathways in the pig and provides an updated characterization of the porcine genes involved in these pathways with particular focus on androgens, estrogens, and 16-androstenes. At least 21 different enzymes appear to be involved in these pathways in porcine tissues together with at least five cofactors. Until now, data on several porcine genes were scarce or confusing. We characterized the complete genomic and transcript sequences of the single porcine CYP11B gene. We analyzed the porcine AKR1 gene cluster and identified four AKR1C, one AKR1C like genes and one AKR1E2 gene. We provide evidence that porcine AKR1C genes are not orthologous to human AKR1C. A new nomenclature is thus needed for this gene family in the pig. Thirty-two genes are now described: transcript (30+2 characterized in this study) and genomic (complete: 18+1 and partial: 12+1) sequences are identified. However, despite increasing knowledge on steroid metabolism in the pig, there is still no explanation of why porcine testes can produce androstenone and epiandrosterone, but not dihydrotestosterone (DHT), which is also a reduced steroid.

Influences of musk administration on the doping test.

Musk is widely used as a traditional drug in Asia for the treatment of stroke, tumour, and cardiopathy with an oral dosage of 0.03-0.1 g per day. Because of the potential anabolic effect, musk preparations have been included in the list of medical products containing prohibited substances employed for doping. The application of musk pod formulation was regarded as the reason of some adverse analytical findings in the 2011 FIFA Women's World Cup. In order to investigate the influence of musk administration on the doping test, we executed a chemical analysis and excretion study. The gas chromatography/mass spectrometry (GC-MS) analysis demonstrated the diversity of steroid concentrations in musk samples. Furthermore, the δ(13)C-values of steroids from wild deer musk showed more depleted than those of domestic deer musk by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis. Because the steroids from some musk had δ(13)C-values in the range of naturally produced steroids in human body, the possible abuse of this kind of musk is very hard to be detected by isotope ratio mass spectrometry (IRMS) in doping control. Musk grains from wild and domestic deer were administrated for the excretion study respectively. Spot urine samples were collected from two male volunteers before and after 100 mg musk grains administration. The profiles and carbon isotope ratios of urinary steroids were determined by GC-MS and GC/C/IRMS. The ingestion of either wild or domestic deer musk did not lead to the adverse analytical finding of doping control in the single dosage of 100mg.

Assessment of steroidogenesis and steroidogenic enzyme functions.

There is some confusion in the literature about steroidogenesis in endocrine glands and steroidogenesis in peripheral intracrine tissues. The objective of the present review is to bring some clarifications and better understanding about steroidogenesis in these two types of tissues. Concerns about substrate specificity, kinetic constants and place of enzymes in the pathway have been discussed. The role of 17α-hydroxylase/17-20 lyase (CYP17A1) in the production of dehydroepiandrosterone and back-door pathways of dihydrotestosterone biosynthesis is also analyzed. This article is part of a Special Issue entitled "Synthesis and biological testing of steroid derivatives as inhibitors".

Influence of polyvinylpyrrolidone and hydroxypropyl - beta - cyclodextrine on solubility of dehydro epiandrosterone

PVP only does not enhance the solubility of DHEA but a combination effect will manifeste once the substance is used concurrently with HPCD. In 5,59% HPCD solution, this solubility was enhanced by 115 times and in 5,59% HPCD solution adding 0,1% PVP it was enhanced by 139 times. There is a strict interaction between its components, different evidently from the physical mixed graphic