Identification of human in vitro metabolites of the haemoglobin S polymerization inhibitor voxelotor for doping control purposes

Voxelotor (GBT440) is a haemoglobin S polymerization inhibitor used to treat anaemia in sickle cell disease. Due to an increase of arterial oxygen saturation as well as serum erythropoietin and haemoglobin, the World Anti-Doping Agency included voxelotor in the list of prohibited substances and methods in 2023. The objective of the present study was to identify and characterize metabolites of voxelotor to detect a potential misuse by athletes. The biotransformation was studied in vitro using the human hepatocellular cell line HepG2 and pooled human liver microsomes. The metabolites were analysed using high-performance liquid chromatography (high-resolution) mass spectrometry. In total, three phase I metabolites and six phase II metabolites (resulting from glucuro-conjugation and O-methylation) were formed by the HepG2 cells in a time-dependent manner, and two phase I metabolites were generated by the liver microsomes, among them one also found in the HepG2 incubations. A reduced metabolite and the glucuro-conjugate of a reduced metabolite were the most abundant formed by HepG2 cells. In addition, metabolites resulting from mono-hydroxylation, reduction and O-methylation in different combinations were identified. Voxelotor was also found as glucuro-conjugate with a low abundance. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and, consequently, a detection of an abuse in an athlete urine sample might be possible

Medaka embryos as a model for metabolism of anabolic steroids

In anti-doping science, the knowledge of drug metabolism is a prerequisite to identify analytical targets for the detection of misused prohibited substances. As the most obvious way to study xenobiotic metabolism, the administration to human volunteers, faces ethical concerns, there is a need for model systems. In the present study, we investigated whether Oryzias latipes (medaka) embryos might be an alternative, non-animal test model to study human-like metabolism. In the present study, we exposed medaka embryos at the morula stage to the anabolic steroid metandienone (10 µM or 50 µM) for a period of 2 or 8 days. According to the fish embryo toxicity test (OECD test), we assessed the developmental status of the embryos. We further investigated metandienone metabolites by high-performance liquid chromatography- and gas chromatography-mass spectrometry. Medaka embryos produced three mono-hydroxylated and one reduced metabolite known from human biotransformation. Developmental malformations were observed for the exposition to 50 µM metandienone, while a significant elevation of the heart beat was also present in those individuals exposed to the lower dose for 8 days. The present study demonstrates that the medaka embryo represents a promising model to study human-like metabolism. Moreover, the judgement of developmental parameters of the fish embryos enables for the simultaneous assessment of toxicity

Androgenic properties of the dietary supplement 5α‑hydroxy‑laxogenin

Dietary supplements sold for anabolic benefits or performance enhancement often contain substances, which are nonapproved and might lack quality controls. With regard to athletes, the inclusion of substances or methods in the prohibited list of the World Anti-Doping Agency is based on medical or scientific evidence. 5α-hydroxy-laxogenin is a synthetic spirostane-type steroid, which is contained in dietary supplements and advertised as anabolic agent. To date, evidence is missing on anabolic or androgenic activity of 5α-hydroxy-laxogenin. We investigated its androgenic potential in two in vitro bioassays. While no activity was observed in the yeast androgen screen, 5α-hydroxy-laxogenin was able to trans-activate the androgen receptor in human prostate cells in a dose-dependent manner. Interestingly, a biphasic response was observed with antagonistic properties at lower concentrations and agonistic effects at higher concentrations tested. The demonstrated androgenic properties of the higher concentrations demonstrate that further investigations should focus on the safety as well as on potential anabolic effects of 5α-hydroxy-laxogenin. This is of interest with regard to abuse for doping purposes

Agreement of steroid profiles in Athlete Biological Passport residues and corresponding serum samples

The steroid module of the Athlete Biological Passport (ABP) is based on the analysis of six endogenous steroids in urine samples and a Bayesian statistical approach. However, the urinary steroid concentrations may be affected by confounders like microbial degradation, possible co-administration of diuretics as masking agents, insufficient conjugate hydrolysis or UGT2B17 gene polymorphisms affecting glucuronidation. Therefore, it can be helpful to use other matrices (ABP blood and serum samples) to quantify steroids and thereby support noticeable deviations in the Athlete Biological Passport, for example, abnormally increased urinary testosterone/epitestosterone (T/E) ratios. Aim of the study was to investigate the feasibility to re-use plasma obtained from athlete ABP blood samples for measuring a steroid profile. Therefore, testosterone, androstenedione, cortisol and cortisone were quantified in 36 intra-individual matching ABP blood and serum samples. The steroid levels measured in both matrices showed a high agreement indicating a good stability uninfluenced by storage temperature and duration. Our results pointed out the possibility to expand the athlete ABP blood analysis for steroid profiling

Medaka embryos as a model for metabolism of anabolic steroids

In anti-doping science, the knowledge of drug metabolism is a prerequisite to identify analytical targets for the detection of misused prohibited substances. As the most obvious way to study xenobiotic metabolism, the administration to human volunteers, faces ethical concerns, there is a need for model systems. In the present study, we investigated whether Oryzias latipes (medaka) embryos might be an alternative, non-animal test model to study human-like metabolism. In the present study, we exposed medaka embryos at the morula stage to the anabolic steroid metandienone (10 µM or 50 µM) for a period of 2 or 8 days. According to the fish embryo toxicity test (OECD test), we assessed the developmental status of the embryos. We further investigated metandienone metabolites by high-performance liquid chromatography- and gas chromatography-mass spectrometry. Medaka embryos produced three mono-hydroxylated and one reduced metabolite known from human biotransformation. Developmental malformations were observed for the exposition to 50 µM metandienone, while a significant elevation of the heart beat was also present in those individuals exposed to the lower dose for 8 days. The present study demonstrates that the medaka embryo represents a promising model to study human-like metabolism. Moreover, the judgement of developmental parameters of the fish embryos enables for the simultaneous assessment of toxicity

Detection of 18-methyl steroids: case report on a forensic urine sample and corresponding dietary supplements

The detection of a putative 18-methyl-19-nortestosterone metabolite in a forensic bodybuilder's urine sample collected as part of a criminal proceeding has triggered a follow-up investigation. Four different dietary supplements in the possession of the suspect were examined with regard to possible precursor steroids. This led to the detection of the declared ingredient methoxydienone, which was confirmed by both, GC–MSMS and LC-HRMSMS. As neither 18-methyl-testosterone, nor 18-methyl-19-nortestosterone were detectable in the supplements, the possibility that the metabolite originates from methoxydienone was investigated. For this purpose, the metabolic fate of methoxydienone was studied in vitro using human HepG2 cells and in vivo by a single oral administration. While the 18-methyl-19-nortestosterone metabolite was not generated by HepG2 cells incubated with methoxydienone, it was observed in the urine samples collected at 2, 6, 10 and 24 h after methoxydienone administration. Moreover, the potential binding of methoxydienone as ligand to the human androgen receptor was modelled in silico in comparison with 18-methylnandrolone, for which androgen receptor activation had been shown in an in vitro approach before. In conclusion, we could ascribe the presence of the 18-methyl-19-nortestosterone metabolite in a forensic urine sample to originate from methoxydienone present in dietary supplements. Methoxydienone was observed to slowly degrade by demethylation of the methoxy substituent in liquid solutions. While no compound-specific intermediates were identified that allowed differentiation from other 18-methyl steroids, the 18-methyl-19-nortestosterone metabolite proved to be a suitable marker for reliable detection in doping analysis

Supplemental material for Influence of estrogen on individual exercise motivation and bone protection in ovariectomized rats

<p>Supplemental material for Influence of estrogen on individual exercise motivation and bone protection in ovariectomized rats by Sebastian T Müller, Annekathrin M Keiler, Kristin Kräker, Oliver Zierau, Ricardo Bernhardt in Laboratory Animals</p

B-cell homeostasis is maintained during two months of head-down tilt bed rest with or without antioxidant supplementation

Alterations of the immune system could seriously impair the ability to combat infections during future long-duration space missions. However, little is known about the effects of spaceflight on the B-cell compartment. Given the limited access to astronaut samples, we addressed this question using blood samples collected from 20 healthy male volunteers subjected to long-duration bed rest, an Earth-based analog of spaceflight. Hematopoietic progenitors, white blood cells, total lymphocytes and B-cells, four B-cell subsets, immunoglobulin isotypes, six cytokines involved in inflammation, cortisone and cortisol were quantified at five time points. Tibia microarchitecture was also studied. Moreover, we investigated the efficiency of antioxidant supplementation with a cocktail including polyphenols, omega 3, vitamin E and selenium. Our results show that circulating hematopoietic progenitors, white blood cells, total lymphocytes and B-cells, and B-cell subsets were not affected by bed rest. Cytokine quantification suggested a lower systemic inflammatory status, supported by an increase in serum cortisone, during bed rest. These data confirm the in vivo hormonal dysregulation of immunity observed in astronauts and show that bed rest does not alter B-cell homeostasis. This lack of an impact of long-term bed rest on B-cell homeostasis can, at least partially, be explained by limited bone remodeling. None of the evaluated parameters were affected by the administration of the antioxidant supplement. The non-effectiveness of the supplement may be because the diet provided to the non-supplemented and supplemented volunteers already contained sufficient antioxidants. Given the limitations of this model, further studies will be required to determine whether B-cell homeostasis is affected, especially during future deep-space exploration missions that will be of unprecedented durations