Projets de recherche

Code: 23C07KD

Tesamorelin is a synthetic growth hormone-releasing hormone (GHRH) analogue, used for the reduction of excess abdominal fat in HIV-infected adults. As a GHRH analogue, it triggers the secretion of growth hormone (GH), which in turn increases serum insulin-like growth factor 1 (IGF-1) levels. The latter exerts an anabolic (growth) effect on tissues throughout the human body. Since this anabolic effect is evidently beneficial for many athletes, the GHRH – GH –IGF-1 axis is a common target in sports doping. All three compounds, as well as their analogues, are banned by the World Anti-Doping Agency (WADA) at all times. Besides curating the list of prohibited compounds, WADA also provides doping control laboratories with a set of minimum required performance levels (MRPL) for these compounds. These MRPLs indicate the sensitivity that a detection method should achieve for a certain compound and are commonly based on administration studies. Since doping often corresponds to the illicit use of prescription medicine, administration studies for many prohibited compounds have only been performed in the context of a clinical trial involving a specific patient population. This research project aims to perform a Tesamorelin administration study on six healthy volunteers. This will allow for a re-evaluation of the current MRPL using a study population more representative of healthy athletes, as well as generate insight into the urinary excretion pattern of Tesamorelin. Both are important factors for the anti-doping community in evaluating the performance of detection methods. For the second objective of this project, the influence of Tesamorelin administration on serum IGF-1 levels will be monitored to investigate the possibility of using IGF-1 as a long-term marker for GHRH doping. Also P-III-NP levels will be assessed to evaluate if the IGF-1/P-III-NP ratio (as will be applied in the endocrine module of the athlete biological passport) is a stronger marker than IGF-1 alone to detect Tesamorelin abuse and GH secretagogues in general in sport.

Code: T23A05GG

The project deal with the synthesis and analytical characterization of an epimeric long-term metabolite of the anabolic steroid Dehydrochloromethyltestosterone (4a-chloro-18-nor-17b-hydroxymethyl-17a-methyl-5a-androst-13-en-3a-ol)

Code: T23A03GG

The project deal with the synthesis and analytical characterization of a long term metabolite of the anabolic steroid oxymesterone (17β-methyl-4,17α-dihydroxy-androst-4-en-3-one)

Code: T23A02GG

The project deal with the synthesis and analytical characterization of the glucuronide phase II metabolite of the stimulant Adrafinil (Diphenylmethyl)sulfinyl]-N-hydroxyacetamide)

Code: T23A01GG

The project deal with the synthesis and analytical characterization of the glucuronide phase II metabolite of the anabolic agent Andarine (2S)-3-(4-acetamidophenoxy)-2-hydroxy-2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide)

Code: 23E05FD

Several polymorphisms/variants of the human EPO gene could be the cause of confounding effects in the application of the analytical methods that are routinely applied for the detection of the abuse of recombinant erythropoietin (recEPO). The most known of them is the c.577del (rs369859204) variant, a single nucleotide deletion in the position 577 of the human EPO gene exon 5. The deletion causes a shifting of the sequence within the terminal exon 5 with consequent loss of the stop codon. At translational level, this results in the formation of a longer mature protein with a higher molecular weight of 3.3 kDa. In doping control, this results in the visualization of a “double band” during the application of the routinary SAR-Page method for the detection of the abuse of recEPO. The double band may be then erroneously interpreted as a false positive sample. As stated in the WADA technical document TDEPO2022, it is suggested to screen for the presence of the c.577del variant whenever a suspected double band is detected by the SAR-Page analysis, in the final aim to avoid the possibility of a false positive finding. Moreover, at least 12 other polymorphisms/variants have been described in the literature that are potentially capable of generating confounding effects in the interpretations of the antidoping analyzes. By following the input given by WADA TDEPO2022, this project involves the development and validation of a genetic test, based on the genomic DNA Sanger sequencing method, that is capable to identify the c.577del variant and all the other polymorphisms/variants present on the human EPO gene (namely: rs1554393458, rs1554393463, rs1419397684, rs773895305, rs763035217, rs1562901775, rs71517124, rs1562902091, rs893404064, rs752755372, rs951322017, rs369859204), and the study of their incidence in determining confounding effects in the interpretation of the routinary tests performed for the purpose of detecting the abuse of recombinant EPO by athletes.

Code: 23D05SV

The aim of this study is to use artificial intelligence (AI) to evaluate blood smears as a potential matrix to detect doping with recombinant EPO (rEPO). Previous publications reported changes in the Red blood Cell Morphology after rEPO administration (e.g. Macrocytes, Stomatocytes). During these times automated haematology analysers and manual microscopy were used for the estimation of these parameters. Recently, digital Morphology is a developing field in Haematology which enables the automated analysis of blood smears by artificial intelligence. With this technology it is possible to evaluate morphological changes with an increased precision, based on a higher number of cells and to discover even minor changes in cellular shapes. AI and deep learning is capable of revealing new insights which conventional approaches were lacking so far, like predicting molecular changes on cytomorphology. Therefore, the goal of this study is to identify relevant changes in cell morphology during rEPO administration which are not addressed using current state of the art techniques. In the long term, a blood smear-based athlete blood passport providing an individual erythrocyte signature might be a prospective application of monitoring athletes by using artificial intelligence based on this postulated deep learning model.

Main findings

A total of thirty healthy athletes participated in a controlled, interventional trial in which they received either rEPO or a placebo over a three-week period. Each participant provided 13 blood samples across an 11-week timeframe, encompassing pre-administration, administration, and post-administration phases. PBS were prepared and digitized at each time point for subsequent analysis.Hematological parameters, including hemoglobin concentration, hematocrit, and reticulocyte indices, showed distinct temporal patterns between the rEPO-treated and placebo groups. Digitized PBS were analysed using a customized deep learning pipeline based on the Haemorasis pipeline, specifically adapted for this study. For the binary classification task comparing PBS samples obtained after iron supplementation but without/before rEPO to those obtained at the peak of reticulocyte response following rEPO administration, the model achieved an area under the receiver operating characteristic curve (AUC) of 0.79 for the entire cohort. Subgroup analyses yielded an AUC of 0.72 for male participants and 0.66 for female participants. These preliminary findings suggest that AI-driven morphological analysis of PBS may serve as a novel and complementary approach for the detection of rEPO doping. However, further validation in larger and more diverse cohorts is necessary to confirm these results and refine the model for potential application in anti-doping efforts.

Code: 23C03MT

Stimulants are substances with wake-promoting effects, whose misuse in sports is only prohibited in-competition. The high analytical sensitivity of anti-doping laboratories allows for comprehensive screening procedures, which cannot only reveal the misuse of stimulants, but also unintended doping due to the use of contaminated/adulterated nutritional supplements. Therefore, stimulants constantly account for more than 10% of all adverse analytical findings (AAF) reported by the World Anti-Doping Agency (WADA). Due to the easy detectability of prohibited stimulants, especially novel drugs such as flmodafinil and fladrafinil have an enormous potential for being misused in sports. These bisfluoro analogs of modafinil and its pro-drug adrafinil are supposed to be significantly more effective than the original compounds, but currently not explicitly mentioned in the WADA Prohibited List. Consequently, the aim of this research project is to investigate the urinary metabolism of flmodafinil and fladrafinil, as well as their elimination behavior in urine and blood. The acquired data will lay the foundation for the implementation of both drugs as well as their metabolites into doping control routine procedures. Moreover, a retrospective evaluation of in-competition routine samples will be conducted to determine the prevalence of these stimulants in sports.

Code: 23A13MT

Members of the transforming growth factor beta (TGF-β) superfamily such as myostatin (MSTN), activin A, and GDF-11, are dimeric cytokines signaling through activin receptors. They play important regulative roles in different biological processes as for example the formation of muscle and red blood cells. Therefore, inhibitors of the activin receptor signaling pathways (IASPs) can be considered as potential performance-enhancing agents in sports, which are included in sections S2 (“Peptide hormones, growth factors, related substances and mimetics”) and S4 (“Hormone and metabolic modulators”) of the WADA Prohibited List. They comprise targeted (e.g. anti-MSTN antibodies) as well as multi-targeting approaches (e.g. receptor competitors and anti-ActRII antibodies), and for several drug candidates, doping control detection methods based on electrophoretic techniques and Western blotting or LC-HRMS/MS have been proactively developed within the last years. This is of utmost importance, as, even without clinical approval, reference material for such emerging drugs is often available for research purposes and/or distributed on the black market. Therefore, the aim of this research project is to develop a combined mass spectrometric detection assay for the novel anti-activin A antibody Garetosmab and other IASPs such as Sotatercept, Domagrozumab, and Luspatercept. A combined extraction of the target analytes from plasma/serum can be achieved by using NHS magnetic sepharose beads coupled to different TGF-β cytokines such as activin A, MSTN, and GDF-11. Subsequent LC-HRMS/MS analysis will not only allow for an unambiguous identification of the target analytes based on the amino acid level, but also retrospective sample evaluation for the presence of novel IASPs binding to one of the TGF-β cytokines employed for affinity purification.

Code: 23A07MT

MENT (7α-methyl-19-nortestosterone) is not explicitly listed on the WADA´s Prohibited List, albeit it falls under section S1 Anabolic Agents. Developed in the 1960´s, its anabolic properties were found to be ten-times that of testosterone, the main endogenous anabolic androgenic hormone. Considering its fast-metabolic clearance rate, MENT seemed a suitable candidate for male hormonal contraception and hormonal replacement therapies. Despite promising results obtained in the 1990´s, MENT failed to receive full clinical approval due to ascertained side effects. Nevertheless, it is still easily available via internet-based providers and an interesting alternative for cheating athletes due to its described properties. Human urinary metabolites of MENT have only been described once in the context of sports drug testing and 3 metabolites were identified, detectable at high urinary concentrations for only 14 hours. Considering the human metabolism of steroids in general, it can be expected that metabolites are formed and excreted into urine for a longer time period, most probably at much lower concentrations. Aim of this research project will be the re-investigation of the human MENT metabolism employing an administration trial with deuterated MENT to enable the sensitive detection of all formed metabolites by hydrogen isotope ratio mass spectrometry. This method was already enrolled in several successful research projects on metabolite detection. All metabolites detected will be further investigated by high resolution/high accuracy mass spectrometry in order to potentially elucidate their chemical structural. In a second administration trial, samples will be collected from three volunteers after the administration of unlabeled MENT to confirm the findings of the deuterated excretion study and to enable consideration of inter-individual differences in steroid metabolism to identify those metabolites most suitable for drug testing. Promising metabolites will then be in-cooperated into existing sports drug testing methods.