Projets de recherche

Code: 252C04GH

6β-chlorotestosterone (6β-Cl-T) is a new steroid-like compound reported in the blank market in recent years. Metabolic profile of 6β-Cl-T in human urine after an oral dose of 40 mg was studied in our previous report. Ten new metabolites including four Phase I metabolites and six Phase II metabolites were characterized and potentially identified. Furthermore, the influence on steroidal profile after administration of 6β-Cl-T was evaluated by GC-MS/MS. Results showed that 6β-Cl-T could transform to T in one male volunteer, so as to cause an AAF for T and its metabolites. In order to figure out the origin of T in this male volunteer, GC-C-IRMS was applied in the two samples at abnormal high T/E ratio values of 6.86 and 5.0 (> 4). In total, δ13C values were determined in 5 target compounds (TCs), which were T, 5αAdiol, 5βAdiol, A, and Etio. However there was no significant change in increasing testosterone levels for women individuals and UGT2B17 gene deletion (del/del) male individuals, which might cause false negative results in these people.

The current application is a follow-up study of our previous project, and the objectives of this research are to (1) find the dechlorinated metabolites (biomarkers) and conversion mechanism from 6β-Cl-T to T, aiming to mitigate the drawback of conventional ABP strategies failing to be effective for UGT2B17 gene deletion (del/del) population after medication of 6β-Cl-T, (2) employ biological methodologies for the synthesis, purification, and enrichment of the principal metabolite(s) of 6β-Cl-T, and (3) chemical synthesis of metabolites, and finally distribute metabolite(s) to WADA-accredited doping control laboratories.

Code: 252C03BL

In steroid hormone testing, the biological passport indicators of athletes, which serve as longitudinal monitoring parameters, are susceptible to interference from various factors, and 5α-reductase inhibitors represent one such co-founding factor. Given that other 5α-reductase inhibitors, such as finasteride and dutasteride, are already monitored for their potential impact on steroid detection in anti-doping, comprehensive metabolic studies of epristeride are crucial to understand its impact for doping control purposes. Epristeride, a novel non-competitive inhibitor of type II 5α-reductase, has emerged as a potential therapeutic alternative for benign prostatic hyperplasia. This study aims to investigate the metabolic characteristics of Epristeride and elucidate its metabolic pathways in humans, investigating its impact on the steroid indicators of athletes' biological passports, examining the differences in effects among populations with different genotypes, and comparing the variations in effects between the Chinese population and the Caucasian population. Thereby providing a scientific basis for assessing its potential risks to athletes and doping control.

Code: 252A09MT

Many substances (also some of those belonging to the WADA Prohibited List) tend to enrich (or deplete) in red blood cells and, thus, will show significantly deviating concentrations in whole blood (such as DBS) and plasma (or serum) samples. For an accurate evaluation and interpretation of analytical results obtained from DBS analyses, the information about the RBC-to-plasma-ratio for each substance is crucial, not only in the context of quantitative result management. The RBC-to-plasma-ratio can be characterized by a simple and straight forward in-vitro approach with incubating the substances of interest in whole blood, followed by the separate analysis of the cells and the plasma fractions. With those experiments, the partitioning of nearly all prohibited substances between erythrocytes and blood plasma can be determined. These data will be of substantial importance for future result management processes where concentrations of MRL- and threshold-substances in DBS are to be evaluated.

Code: 25T03MP

Ecdysterone was demonstrated to exhibit anabolic properties in-vitro and in-vivo (doi10.3390/nu16091382). Thus, ecdysterone supplements are gaining more and more attention in the sports community. In humans, a study funded by WADA (WADA15C18MP, doi10.1007/s00204-019-02490-x) already demonstrated performance enhancement in young man.

To further substantiate the effects and to support an inclusion of ecdysterone in the Prohibited List, the ListEAG suggested to perform additional experiments. A double-blind placebo-controlled intervention trial in humans will be conducted with ecdysterone supplementation in combination with a structured supervised resistance training over 12 weeks in resistance-trained males. Based on previous findings, the study will focus on resistance training and anabolic effects of ecdysterone. A standardized test battery for muscle strength will be used to evaluate potential performance enhancement based on the recommendations of NSCA recommendations with additional on individual muscle thickness etc.

Recovery measures and mechanistic investigations are also added to complement the study. Nutrition will be monitored and all groups are explicitly checked for sufficient protein intake.

Code: 25T242D01CW

The present project will conduct an integrated study on Endogenous Anabolic Androgenic Steroids (EAAS) metabolism in the Chinese population. The population distribution of UGT2B17 gene polymorphism, the long-term comparison of EAAS metabolism among individuals with different UGT2B17 gene deletion polymorphisms, and testosterone intervention experiment are the main focuses of this project. Through population studies, long-term research, and interventional trials, the metabolic profile characteristics of testosterone in the Chinese population will be illustrated. The metabolic characteristics of populations having different UGT2B17 genotypes will be highlighted.

Especially focusing on the long-term study data, which can reflect the environmental influence on urine EAAS and provide a data reference for the optimization of biological passports in the future. By correlating blood steroid profiles, urinary steroid profiles, and stable carbon isotope ratio fingerprint features, basic data and case analyses will be provided for EAAS substance detection in the UGT2B17 del/del population. A comprehensive detection strategy for EAAS, including the combined application of BSP, SP and GC/C/IRMS will be discussed in the project. The influencing factors of diet, region, and other factors on EAAS metabolism in the Chinese population will also be studied. ‌Although DBS analysis is not included in this project, the samples will be collected for subsequent investigation.

Code: 25T04MT

A major concern both for athletes and result management authorities is the possibility of nutritional supplement/dietary product contamination with minute amounts of anabolic agents leading to adverse-analytical findings (AAFs) in doping controls. The analytical sensitivity of anti-doping laboratories has been optimized to allow for utmost retrospectivity in sports drug testing, but the problem arising from such a performance is that also trace amounts introduced into an athlete’s organism by contaminated supplements or food are detected.

To support the process of fair and comprehensive case management, in-depth investigations into the elimination profiles and metabolite ratios of micro-dosed LGD-4033 were conducted in 2020/21. For that purpose, single- and multi-micro-dose excretion studies at 1, 10 and 50 μg LGD-4033 were conducted, and the collected samples analyzed by enzymatic hydrolysis, solid-phase extraction (SPE) and LC-HRMS/MS. A total of 15 phase I metabolites was detected and the ratios of LGD-4033 and the epimer M1 as well as the metabolites M5-a and M5-b were found to shift over time. The combined evaluation of the LGD-4033/metabolite concentrations and ratios can be utilized to estimate whether a minute amount of the drug was recently ingested or a larger, pharmacologically relevant amount of the doping agent was administered several days/weeks ago and thus evaluate the plausibility of potential contamination scenarios.

Within this follow-up research project, the available micro-dose administration study samples will be re-analyzed to quantify the main long-term metabolite of LGD-4033 (M5-a) by using meanwhile available certified reference material.

Code: 25T02AM

Despite suspected high prevalence of Growth Hormone (GH) doping, detection of GH doping is complex. The indirect method based on two biomarkers increased by GH (growth factor IGF-I and procollagen propeptide P-III-NP) has recently been integrated in the endocrine module from the Athlete biological passport (ABP) to better identify doping with GH using a P-III-NP assay for Centaur Analyzer and IGF-I by top-down mass spectrometry.

However Centaur analyzer and its P-III-NP assay are to be ended soon (2027) and need to be replaced. Siemens proposes a new Atellica analyzer with a test for P-III-NP that is described as highly similar to Centaur results in the normal to pathological range, but this need further confirmation in the athletes population range The aims of the project are to evaluate and confirm this new technical option for P-III-NP quantification in the coming years.

1- Comparison of P-III-NP assays performed on two Siemens analyzers and impact of storage : 200 serum samples previously analyzed for endocrine passport will be reanalyzed for P-III-NP in parallel on Advia Centaur and on the new Siemens analyzer Atellica in order to evaluate the similarity of results in an athletes population.

This will also allow a direct comparison to initial values to confirm storage conditions (-80°C) are appropriate for reanalysis;

2- Impact of various degrees of hemolysis on P-III-NP and IGF-I by adding free hemoglobin to clear sample and checking the quantification of both markers.

These data will help to confirm that the new technical options for P-III-NP detection are fit-for-purpose and will increase knowledge on how variations in quality of serum impact endocrine passport.

Code: 25T01RV

Glucocorticoids (GC) are forbidden in sports competitions when used by injectable, oral or rectal routes. They are allowed by other routes for therapeutic purposes. Since most GCs are marketed in different administration forms, the distinction between routes of administration is needed to ensure safe treatments by allowed routes of administration and to detect the use for doping purposes during competitions. Based on scientific data available, compound-specific reporting levels were established for some GC and washout periods after some administration routes.

There is no data in literature regarding concentrations of GCs in urine after intravenous administration. The objective of the present project will be to perform administration studies with dexamethasone (DEX) and methylprednisolone (MP) using intra-venous (IV) administration, to evaluate the suitability of the reporting levels already defined for DEX and MP to detect IV administration and to define the washout periods after IV administration to avoid fase adverse analytical findings due to treatments close to competitions.

Code: 242D03OS

Detecting doping with endogenous substances is challenging due to difficulties in distinguishing between natural production and external sources. Testosterone (T), particularly when administered transdermally, exemplifies this challenge. Recent advances in blood steroid profiling, specifically measuring T and its ratio to androstenedione (A4), have improved detection, particularly in females and individuals with UGT2B17 enzyme deletion. However, current methods and interpretation still require complementary biomarkers to enhance sensitivity and selectivity.

Sulfated steroids, key players in human steroid metabolism, offer potential in this regard. Unlike free steroids, these hydrophilic, conjugated forms are stored in higher concentrations in bodily fluids, providing a reactive reservoir that can be converted back into active steroids to maintain homeostasis. Sulfo-conjugation, a process

mediated by sulfotransferase enzymes (SULT), is a significant metabolic pathway for testosterone and its precursors & metabolites. This makes sulfated steroids, which have a prolonged elimination time compared to free testosterone, ideal long-term biomarkers for targeting exogenous testosterone administration.

In this study, we aim to evaluate the sensitivity of endogenous sulfated steroids in serum and urine for detecting transdermal testosterone use in healthy women. We will analyze samples from two prior clinical trials involving topical testosterone administration. The first trial involved 14 women undergoing 28 days of T gel administration, with serum and urine samples collected every two days for 12 weeks. The second study included 48 women randomized to 10 weeks of daily testosterone cream or placebo treatment with urine and serum samples collected before and after treatment. Urine and serum samples will be analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify steroid sulfates and their conjugated metabolites. Profiles of top candidates will be developed to improve testosterone detection methods.

Code: 242C12EI

The popularity of using plant steroids to improve physical health and performance is on the rise. One such plant steroid is diosgenin (DIO), primarily found in fenugreek (DOI: 10.9755/ejfa.2016-01-086). In vitro studies on adipocytes have demonstrated that glucose and fat metabolism are beneficially influenced by DIO (DOI: 10.1002/mnfr.200900609). These findings are further supported by animal studies (DOI: 10.1155/2019/7213913; DOI: 10.1016/j.jsbmb.2014.02.020). A recent review also supports these observations, indicating that chronic application of fenugreek can significantly reduce fat mass (DOI: 10.1055/a-2048-5925). Additionally, improvement of endurance performance was observed in mice (DOI:10.3177/jnsv.52.287). However, there is a significant lack of data available on the effects of DIO on endurance performance in humans. In a two-arm randomized controlled double-blind crossover trial the effects of 500mg DIO will be tested against a placebo group. A total of 15 endurance-experienced participants will consume a dietary supplement rich in DIO or a placebo product over 14 days. The initial application phase will be followed by a 2-week wash-out period, and then a second application phase. Before and after both application phases, participants' performance will assessed through isometric mid-thigh pull, cycling-ergometer step and ramp test. Thereby, maximal fat oxidation, lactate threshold and maximal oxygen uptake will be analyzed via spiroergometric and lactate data to determine corresponding metabolic profiling & energy metabolism. Training sessions will be monitored throughout the study period to ensure participants maintain their usual training regime. In addition, the metabolism of glucose in C2C12 cells after treatment with DIO will be investigated in our established cell culture system.