Projets de recherche

Code: 17C02MP

Increasing numbers of adverse analytical findings were reported in the recent years due to the misuse of the anabolic androgenic steroid dehydrochloromethyltestosterone (DHCMT). Once developed in the former GDR for misuse in sports it regained enormous relevance especially in the samples from Bejing and London Olympic games. Several adverse analytical findings were reported after the samples had been retested for the newly reported long-term metabolite of DHCMT. At present, the use of post administration urines instead of purified reference material has been accepted in confirmatory analyses. Very recently the currently used metabolites have been questioned in the literature. Thus a controlled administration trial in humans will be performed to provide further evidence for tracing back the long term metabolites 20ξOH-NorTHCMT and 20βOH-NorDHCMT to a DHCMT administration. Furthermore, the utilisation of in-vitro experiments will further broaden the scientific insights into metabolic pathways that lead to the generation of these metabolites.

Main Findings

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol®. It is prohibited in sports at all times. Even if discontinued as pharmaceutical in 1994, there are several adverse analytical findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, a single oral dose of DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail. Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite. The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17β-methyl-18-nor-androsta-4,13-dien-3β-ol. It may serve as additional confirmatory metabolite. To improve tracing of cheating athletes, it is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general.

Code: 17A24XD

The anabolic androgenic steroids (AAS) are prohibited in sports. They are included in the 2017 list of the World Anti-Doping Agency (WADA) as class S1. In the last years the anabolic agents accounted for most of the adverse analytical findings (AAF) in doping control (e.g. in 2015 50% of all ADAMS reported AAF). AAS undergo extensive metabolization, thus, urinary detection of a prohibited administration is mainly based on the detection of metabolites. As some steroids also occur naturally in the body, their uncoverage generally uses specific ratios, such as testosterone/epitestosterone (T/EpiT), androsterone/etiocholanolone (And/Etio), And/T, and 5α-/5β-androstane-3α,17β-diol (Adiol/Bdiol), that proved to be very stable in humans. Confirmation of the results generally require isotope ratio mass spectrometry. As confirmation is very elaborate and cost intense some minor metabolites came into the focus of anti-doping scientists to increase the efficiency of screening procedures. For the improved detection of an exogenous administration of androstenedione the usefulness of the A- or B-ring hydroxylated metabolites 4-hydroxy-androstenedione, 6z-hydroxy-androstenedione was reported. As already published 2β- and 15β-hydroxylation also occurs in testosterone metabolism with ~10% and 4% of the rate of the most dominant hepatic microsomal 6β-hydroxylation. Furthermore, it was demonstrated that the use of 2- and 4-hydroxyandrostenedione may serve as long term marker of an androstenedione administration. The metabolic generation could be confirmed by in-vitro experiments upon incubation with CYP1A2 and CYP1B1. No reports on the metabolic hydroxylation of androgens by CYP1A2 or by CYP1B1 are found in literature so far. The objective of the project is to further investigate the suitability of A-ring hydroxylation for long-term detection of endogenously occurring androgens and to extent the preliminary investigations to other prohibited steroids.

Main Findings

The hydroxylation pathway in vivo and/or in vitro studies for Testosterone (T), 4-androstenedione (AED), 19-norandrostenediol (NAD) and Methyltestosterone (MT) was investigated. Samples collected after the administration of T showed the presence of 6-OH-T according to an oral administration, but its concentration gave no additional information different to that already known (parameters of the steroid profile). On the other hand, the formation in vivo and in vitro of hydroxylated metabolites of AED, NAD and MT in position 4 allowed the formation of Formestane, Oxabolone and Oxymesterone respectively. Although the formation of these metabolites were significant (in some cases) and easily detectable, any of them showed a detection window longer that those metabolites known for AED (T/E ratio and others steroid profile parameters), NAD (19-norandrosterone, and 19-noretiocholanolone) and MT (5α and 5β reduced metabolites). Nevertheless, a special attention have to be paid when a sample with these compounds is reported, because under these circumstances a concomitant abuse of AED-formestane, NAD-oxabolone or MT-oxymetholone could be misinterpreted reported by the laboratory. Only for AED the 2-hydroxylation demonstrated an added value extending the suspicion of an AED administration based on an extended steroid profile.

Code: ISF17D04SI

The original idea of “athlete`s performance passport” or monitoring
individual performances for better informed decisions on doping
testing has been presented by Schumacher and Pottgiesser. The main objective of an “athlete`s performance passport” in sport is to distinguish between consistent and unexpectedly disproportionate performances. Excellent performance itself is not a proof of any wrongdoing or doping. However, through longitudinal monitoring, inconsistently excellent performance could be a warning sign that need further attention from anti-doping authorities.The purpose of this project is to establish framework for the longitudinal performance monitoring and identification criteria of athletes with outline performance in middle- and long distance
runners population.

Code: 17D09RV 

The detection of endogenous anabolic steroids (EAS) abuse is currently performed using the steroid profile. The steroid profile is composed of concentrations of testosterone and related endogenous metabolites excreted as glucurono-conjugates, and ratios between them, being Testosteone/Epitestosterone ratio the most important one. These parameters are monitored for each individual to define the individual basal ranges, and changes on them reveal the use of EAS. Suspicious samples are confirmed by carbon isotope ratio mass spectrometry to demonstrate the exogenous origin of testosterone and metabolites. The steroid profile is a powerful tool to detect EAS misuse, however improvements are needed to prolong detection windows. Testosterone and metabolites are also excreted as sulfates in urine. The sulfate fraction has not been comprehensively evaluated for the detection of EAS misuse. The objective of the project will be to evaluate the sulfate fraction of testosterone metabolites to look for new biomarkers to prolong the detectability of the misuse of EAS. First, a comprehensive methodology to quantify endogenous steroid sulfates based on their direct analysis by liquid chromatography-tandem mass spectrometry will be optimized and validated. Second, steroid sulfates will be quantified in urines obtained from healthy population to define normal population ranges. Finally, steroid sulfates will be quantified in urines collected after administration of testosterone to healthy volunteers by different routes. Evaluation of sulfate metabolites as markers of EAS administration will be performed by comparison of the excretion profiles of testosterone and metabolites excreted as sulfates with the excretion profiles of metabolites included in the conventional steroid profile. The successful outcome of the project will be directly applicable to sports drug testing by improving the detection of EAS misuse.

Main Findings: 

The objective of the project was a comprehensive evaluation of the sulfate fraction of testosterone (T) metabolites to look for new biomarkers to prolong the detectability of the misuse of endogenous anabolic steroids. First of all, an analytical method was developed and validated to quantify fourteen T related metabolites conjugated with sulfate, based on a mixed-mode solid pahse extraction and the direct measurement of sulfate metabolites by LC-MS/MS. The concentrations of sulfate metabolites in healthy volunteers, including Caucasion and Asian volunteers, were measured.

The usefulness of sulfate metabolites to detect oral T misuse was evaluated after administration of a single oral dose to five Caucasian male volunteers. Using individual threshold limits epiandrosterone sulfate (epiA-S)  improved the detection times (DTs) with respect to T/epitestosterone (E) ration in all five volunteers. Androsterone (A), etiocholanolen (Etio) and two androstanediol sulfates also improved DTs for some volunteers. The most promising results were obtained using ratios between sulfates of epiA, A or androstandediol 1 and E, and also sulfates of epiA or androstanediol 1 and dehydroandrosterone (DHA). These ratios prolonged the DT of oral T administration, in some cases several days after administration, and therefore significantly improving the retorspectivity compared to sulfate concentrations or to the conventionsl T/E ratio.

Sulfate metabolites were also evaluated after a single intramuscular (IM) injection of T to six Caucasian and six Asian helthy male volunteers. Principal component analysis (PCA) was used to obtain the most useful markers for discrimination between pre- and post-administration samples. For Caucasian volunteers, a separation between pre- and post-administration samples was observed in PCA, whereas for Asian no separation was obtained. Seventeen ratios between sulfate metabolites were selected and further considered. DTs of each ratio were evaluated using individual thresolds for each volunteer, and the best results were obtained using rations involving T and E sulfates in the denominator. The best marker was the ratio A-S/T-S which prolonged the DTs with respect to T/E ration in all Caucasian volunteers and in two Asian volunteers. Other ratios A-S or Etio-S and E-S, and, sulfates of Etio, DHA or epiA and T-S were also found adequate.

The data obtained in the project provide a comprehensive insight bout the usefulness of endogenous sulfate metabolites as biomarkers for the detection of oral and IM T misuse. They can drastically increase the DTs with respect to the conventional T/E ratio, especially after oral T administration and, according to our results, its inclusion in the steroid profile is strongly recommended.

Code: 17C09DL

The present proposal builds up on a previous project funded by the WADA entitled: Tramadol and sport: Effects on physical and sustained attention performance during cycling exercise. The aim of this project is to investigate further the effect of tramadol on cycling performance at the physical, cognitive and brain levels by: 1) addressing the potential moderator effect of physical fatigue; 2) testing the effect of the pain-killer on intense short anaerobic efforts (as final sprint); 3) studying the effect of the combination of tramadol and caffeine. This is particularly relevant as it appears from anecdotal reports that pro-cyclists are consuming a combination of the two drugs in order to improve performance.
The project consists of a placebo-controlled, double blind experiment. A single dose of 100mg of tramadol, 6mg/kg of caffeine, and the combination of both, versus placebo, will be administered to participants (in separate sessions). They will then complete a 40-min cycling submaximal exercise (at 60% VO2max). A 20-min indoor Time-Trial (TT) will follow this. Participants will complete a visual discrimination task while cycling both during the submaximal and TT tests. After that, participants will undertake three 30-sec Wingate tests. The Psychomotor Vigilance Task will be performed prior to and
after the exercise phase. Electroencephalography will be continuously recorded throughout the cycling exercise and at rest. We will also obtain measures of subjective performance such as perceived effort and mental fatigue. Subjects will complete the Profile of mood state questionnaire before and after every session.
We will investigate the effect of tramadol, paracetamol and the combination of both vs. placebo on physiological and subjective parameters related to cycling performance (at the physical, cognitive and brain levels) at constant load, during a self-paced indoor time trial and during short maximal intensity efforts.

Main Findings:

The use of tramadol, a weak opioid, is a controversial current topic of debate in cycling. However, little is known about is potnetial ergogonic and cognitive (harmful) effects. In this project, we aimed at providing novel empirical evidence on this issue. As control conditions we included placebo and paracetamol, the other mild analgesic commonly used in cylcing. A randomized, double-blind, placedbo-controlled trial (EudraCT number: 2018-000388-10) design study was performed. 20 participants completed a 40-min constant work-rate at 60% of the VO2max followed by a 20-min time trial (TT) on a cycle-ergometer after ingesting 100 mg tramadol, 1.5 g paracemtamol, or placebo (in seprate days/sessions). Participants completed the Psychomotor Vigilance Task (PVT) before substance administration and ten minutes before starting the warm-up, and the Sustained Attnetion to Response Task (SART) during the 60 min of exercise. Electroencephalography (EEG), heart rate (HR), and power output were recorded throughout the sessions. The rating of perceived effort (RPE; by means of he Borg scale) was measured at the end of the 40-min constant work-rate and the 20-min TT.

The PVT (baseline-corrected) results showed that participants were faster in the tramadol condition than in the paracetamol and placebo conditions. The average power output during the 20-min TT only revealed greater performance in the tramadol than in the paracetamol condition. Higher HR values were reported in the tramadol condition than in the paracetamol and placebo conditions. RPE findings evidenced lower values in hte tramadol condition than the other conditions in the 40-min constant work while no differences were observed in the 20-min TT. Cognitive performance in the SART did not differ between substances in the 20-min TT. During the 40-min constant work-rate, better accuracy was reported in the tramadol condition than in the placebo condition, accompanied by slower RT. The speed-accuracy trade off could be a sign of improved congitive control (i.e., better ability to inhibit unwanted responses at the expense of being slower). The analysis of EEG tonic spectral power revearled increased power in the tramadol condition than in the other conditions at rest, while no differences in EEG oscillatory activity (either tonic or event-related) were found during the exercise.

In sum, the results of this clinical trial sugest that neither tramadol nor paracetamol (vs. placebo) seem to improve exercise performance or impair the ability to stay focused during a pre-loaded 20-min TT.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

 Chan, Hong Kong, Chine

Description à venir.

Ce document n'est actuellement disponible qu'en anglais.