Projets de recherche

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.

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.

Code: 23A03JP

Sulfated phase II metabolites have gained great interest as long term metabolites of many anabolic steroids. The direct analysis of those sulfates using LC/MSMS has been extensively used as the research tool to finding new long-term metabolites. However, sulfates are known to be potentially unstable, and very particularly at acidic pH where catalysis occurs. Urine samples normally have acidic pH and the stability of those precious long-term metabolites in the biological matrix needs to be investigated. The preparation of reference urine samples to be used within the EQAS quality Assessment scheme has led us to obtaining shocking evidence on the instability of some of the analytes of interest in routine doping control. In particular, some long-term sulfated phase II steroid metabolites. As a good example, results obtained when assaying the stability of a excretion study of danazol, 7 days at different temperatures showed a huge increase in the concentration of the two metabolites (ethisterone and 2-OH-methyl-ethisterone). This increase is attributed to the hydrolysis of their sulfated phase II counterparts. The aim of the present project is the study of the stability of sulfated phase II metabolites using available excretion studies of danazol, mesterolone, clostebol and drostanolone, all having known sulfated long-term metabolites, as proof of concept. The idea is developing conditions to keep those sulfates stable for sample preparation and/or establish the conditions for a complete hydrolysis in order to target the analysis of the resulting compounds.

Code: DBS23SP06CB

Over the ten past years, more and more strategies based on dried blood spot sampling for antidoping have been developed for different classes of substances. Among the ones focusing on small peptides detection (<2kDA), few allow the simultaneous analysis of a large panel of peptides such as GHRP, GHS or GnRH as indicated in the list of prohibited substances from the World Anti-doping Agency (WADA), and the most complete one has been proposed by Lange et al. 2020. The main objective of this present project is first to implement a multi-peptides analysis strategy for the extraction and detection of small peptides and evaluate the impact of the device used for spotting. Performances between cellulose and polymeric devices will be assessed. The influence of the size of the spot will be also assessed to determine if a larger volume of the spot can help to improve LODs. The instrumentation used for such application will also be evaluated to determine which technology is fit for purposes between LC-HRMS instruments and triple quadrupole.

Code: DBS23HIF01MO

Recombinant protein regents of the erythroid growth factor erythropoietin (EPO) have been widely used for anemia treatment, but they have been frequently employed for hematopoietic doping to improve endurance performance. In addition to EPO reagents, five types of small compounds activating hypoxia-inducible transcription factors (HIFs), which induce endogenous EPO production, have been launched in Japan as anemia medications since 2019. Given that HIF-activating agents (HIF-AAs) are administered orally, there is concern that they are used more easily for hematopoietic doping than EPO reagents, which are administered as injections. Although doping detection systems of EPO reagents have already been established, reliable detection methods of HIF-AAs should be urgently established. The applicants at Tohoku University are in charge of the genetic analysis for WADA and have demonstrated the molecular mechanisms by which HIF-AAs induce EPO production, whereas those at LSI Medience Corporation (LSIM) have experience managing anti-doping control tests in Tokyo 2020 Olympic and Paralympic Games with a WADA-accredited highly sensitive LC-MS/MS analyzer. Additionally, the applicants at LSIM recently found differences in the detection efficiency of trimetazidine between dried blood spot (DBS) samples from fingertips and brachial capillaries. Furthermore, the applicants previously optimized detection methods of HIF-AAs in the blood and urine of mice and found that each HIF-AA exhibits specific pharmacodynamics and pharmacokinetics after oral administration. Notably, HIF-AAs were commonly cleared within 48 hours after administration in mice. These data obtained by the applicants suggest that highly sensitive detection methods of the doping use of HIF-AAs should be established to detect them in small amounts of matrix for a long time after administration. Therefore, in this project, the applicants at Tohoku University and LSIM collaboratively aim to develop methods for highly sensitive and efficient detection of HIF-AAs in DBS and urine samples from humans. To elucidate the pharmacokinetics of various HIF-AAs in humans, the applicants will measure the concentrations of five types of HIF-AAs (daprodustat, enarodustat, molidustat, roxadustat, and vadadustat) in DBS and urine samples from healthy volunteers administered HIF-AAs. DBS samples will be obtained from fingertips and brachial capillaries to identify the differences in the detection efficiencies of HIF-AAs between the sampling sites. Additionally, DBS and urine samples from a total of 100 renal anemia patients treated with HIF-AAs will be analyzed. The stability of HIF-AAs in DBS and urine samples for up to six months will also be evaluated as well as the minimum required performance levels (MRPL) of HIF-AAs. The anticipated outcomes of studying the pharmacokinetics of five types of HIF-AAs in human samples are directly applicable for the development of anti-doping tests of HIF-AAs.