Projets de recherche

Code: 15C17MM

Vaptans are a relatively new class of compounds with diuretic suitable for publication on WADA's website effects. They increase water excretion by inhibiting its reabsorption in the renal collecting ducts. For these reasons, vaptans have been included by the World Anti-Doping Agency in the section S5 €Diuretics and Masking agentsÅ of the list of substances and methods that are prohibited in sport. Previous investigators have reported that this class of compounds is extensively cleared by hepatic metabolism via oxidative enzymes. Consequently, the most efficacy strategy to detect them in urine samples could not be achieved by simply targeting the drug itself: the selection of one or more diagnostic metabolites is necessary. Very few data are reported in literature on the metabolic profile of these compounds in humans; at the same time, the influence of physiological and environmental factors on their excretion profile is also almost completely unexplored yet.
This project aims to define the phase I and phase II metabolic reactions and to characterize the enzymatic isoforms involved in the biotransformation pathways of vaptans. Alterations of the metabolic profile of vaptans provoked by physiological (i.e. sex, genetic polymorphism) and environmental (drug-drug interactions) factors, will be evaluated to obtain information on the impact of these factors on the biotransformation pathways detected. Finally, the possibility of obtaining sufficient amounts of vaptans metabolites by enzyme-assisted synthesis will also be explored. Human liver microsomes will be the source of the isoenzymes involved in the phase I metabolism of vaptans; the metabolites once formed will be isolated by HPLC, characterized by MS and used as reference materials to set up and validate efficacy analytical procedures to detect vaptans in urine

Main Findings:

This research project focused on the characterization of the main biotransformation pathways of vaptans with the aim to identify class-specific metabolic pathways of vaptans and select the most appropriate marker(s) of intake. 
In the first part of this project, the in vitro metabolism protocols using either pooled human liver microsomes or recombinant human CYP and UGT isoenzymes were optimized and validated in order to obtain a good correlation with the metabolism reported in humans. The optimized in vitro protocols were subsequently used to evaluate the effects of physiological (gender and genetic polymorphism) and environmental (drug-drug interactions) factors on the metabolic profile of vaptans.
Lxivaptan, mozavaptan and tolvaptan were extensively biotransformed (tolvaptan>lixivaptan>mozavaptan) mainly by CYP3A subfamily and to a lesser degree by CYP2C19 and 2D6 enzymatic isoforms to 13, 4 and 20 metabolic products respectively. Conivaptan was moderately biotransformed by CYP3A subfamily to 2 hydroxylated metabolites due to its well-known inhibitory activity against the CYP3A4 enzymatic isoform. The phase-I biotransformation pathways include hydroxylation in different positions, carboxylation, dehydrogenation, hydrogenation, N-dealkylation, isomerization and combinations of them. Most of the above-mentioned phase I metabolites once formed undergo an extensive glucuronidation by UGT2B isoforms. 
Concerning the effects of physiological an environmental factors, the results obtained showed that (i) no appreciable gender differences were registered in our experimental conditions for all the compounds under investigation; (ii) significant differences were instead registered depending on the specific allelic variant used. More in details, in the presence of the allelic variant CYP3A5 *1*1, the formation of the   phase I metabolites of second step appeared to be faster if compared to the other CYP3A5 allelic variants (CYP3A5 *3*3 and CYP3A5 *1*3) and finally (iii) the levels of all the metabolites were significantly reduced in the presence of the imidazole antifungals tested. On the contrary, in the presence of the steroidal progestins the reduction was less evident, and observed only at the highest concentrations studied.
On the basis of the above observations the most appropriate merkers of intake for conivaptan is the parent compound; the parent compound and the hydroxylated metabolites for lixivaptan; the parent compound and the de-methylated metabolite for mozavaptan; and finally the parent compound and the hydroylated and N-dealkilated hydroxylated metabolites for tolvaptan. These analytes were easily added to the LC-MS/MS analytical procedure currently used in our laboratory to detect more than 150 prohibited compounds.

Code: ISF15E01PC

Skeletal muscle size is dynamic and responsive several stimuli, among which exercise. However, while resistance exercise results in hypertrophy and increased force generation, endurance exercise leads to increased expression of the peroxisome proliferator-activated receptor (PPAR)-€ coactivator-1Å (PGC-1Å) and increased number of mitochondria, with little changes in the skeletal muscle mass. Both types of exercise stimulate the proliferation of satellite cells, muscle-specific stem cells that will subsequently differentiate and fuse with existing myofibers. Finally, exercise also increases circulating microRNAs (miRs), short noncoding RNAs that post-transcriptionally modulate gene expression. Some miRs (myomiRs: miR-1, miR-133a, miR-133b, miR-206) are abundant in the skeletal muscle and appear modulated in muscle atrophy, hypertrophy and myogenesis.
Autologous transplantation of myogenic stem cells has been proposed as the next generation doping strategy. The number of myogenic precursors may be increased both 'artificially' by autologous implantation, or physiologically, by regular exercise. However, while all myogenic precursors are increased by the implantation, exercise mainly leads to satellite cell accumulation.
The target of the study is to define if autologous myogenic stem cell implantation may effectively impinge on muscle mass and function and to clarify if circulating myomiR levels can be useful as biomarkers of myogenic stem cell abundance and type. The study will be developed by using transgenic mice hyperexpressing PGC-1Å specifically in the skeletal muscle. These mice mimic the condition of endurance-trained subjects, being characterized by high exercise performance. Briefly, circulating myomiRs will be evaluated in these mice, and correlated with the abundance and type of myogenic stem cells. Subsequently, myogenic stem cells isolated from wild-type and transgenic animals will be implanted into wild-type recipients, to test the effectiveness of the procedure in improving muscle mass/function. The results obtained with the transgenic mice will be validated on human volunteers.

Main Findings:

Skeletal muscle size is dynamic and responsive to several stimuli, among which exercise. However, while resistance exercise results in hypertrophy and increased force generation, endurance exercise leads to increased expression of the peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α) and increased number of mitochondria, with little changes in the skeletal muscle mass. Both types of exercise stimulate the proliferation of satellite cells, muscle-specific stem cells, that will subsequently differentiate and fuse with existing myofibers. Finally, exercise also increases circulating microRNAs (miRs), short noncoding RNAs that post-transcriptionally modulate gene expression. Some miRs are modulated in muscle atrophy, hypertrophy and myogenesis.

Autologous transplantation of myogenic stem cells has been proposed as the next generation doping strategy. The number of myogenic precursors may be increased both 'artificially', by autologous implantation, or physiologically, by regular exercise. The target of the study has been to define if: 1)autologous myogenic stem cell implantation may effectively impinge on muscle mass and function; 2) circulating miR levels can be useful as biomarkers of myogenic stem cell abundance and type. The study has been developed by using transgenic mice hyperexpressing PGC-1α specifically in the skeletal muscle (MCK-PGC-1α). These mice are characterized by a high amount of myogenic stem cells compared to wild-type (WT) animals, and mimick the condition of endurance-trained subjects, being characterized by high exercise capacity.

The effects of the infusion of myogenic precursors isolated from WT or MCK-PGC-1α mice into Tibialis anterior (TA) muscles of WT hosts, either undergoing muscle damage or not, have been evaluated. Transplantation of myogenic precursors isolated from WT animals does not alter TA morphology and metabolism. Surprisingly, WT cell injection after muscle damage is able to restore the physiological muscle phenotype. Interestingly, when myogenic cells deriving from MCK-PGC-1α animals are infused, a shift towards the oxidative phenotype in both damaged and undamaged muscles can be observed. Histological analyses put in evidence small and oxidative fibers, while molecular markers show an increase in mRNA or protein expression levels of many markers related to myogenesis (Pax-7, Myogenin, embryonal Myosin) and mitochondria (PGC-1α, Tom20). These results suggest that the engraftment of WT myogenic precursors improve muscle regeneration after severe injury. In addition, PGC-1α overexpression might favor myogenic differentiation and affect regeneration by converting the canonical mixed phenotype of a muscle into the oxidative one, endowed with increased exercise capacity. These findings, potentially relevant to regenerative medicine aimed to improve several diseases, may also open the way to a non-ethical use as doping strategy.

As for the second aim of the study, circulating miR profile is comparable between WT and MCK-PGC-1α mice. Such result could be explained by the observation that MCK-PGC-1α mice in resting condition do not display a specific phenotype: the enhanced capacity of the skeletal muscle system, indeed, becomes evident upon stimulation, for example by exercise. Indeed, circulating levels of miR-21-5p and miR-181-5p are increased in exercised WT animals while not in the MCK-PGC-1α mice allowing to hypothesize that stress conditions up-regulate these miRs in WT animals, while the oxidative phenotype and the abundance of myogenic precursors occurring in MCK-PGC-1α mice likely improve the management of stressing stimuli. These results have been compared with those obtained in human volunteers practicing combined exercise for 3 months. Despite the small number of subjects included in the study, a trend towards increase can be envisaged for miR-27-3p, miR-133a-3p and miR-181-5p. This latter, in particular, follows the same trend in both exercised WT mice and volunteers. Despite the lack of robustness, these data suggest that circulating levels of selected miRs can be up-regulated by exercise, and that such modulation can be prevented by genetic manipulations able to improve myogenic precursor abundance and muscle oxidative phenotype. In particular, it could be proposed that low circulating levels of miR-181-5p in athletes suggest a shift of muscle metabolism from glycolitic to oxidative; this might reflect the infusion of autologous stem cells, either untreated or genetically modified. In this regard, the detection of low miR-181-5p levels in the circulation could be useful for a first screening, identifying subjects deserving more accurate analysis

Code: 15A17MT 

A variety of prohibited peptide hormones potentially enhancing athletic performance is still lacking sufficient detection methods in accredited anti-doping laboratories. This is mainly due to a yet missing adequate analytical methodology, which is crucial for detection of these very efficient (and therefore low-dosed) therapeutics and drug candidates in biological fluids (urine / blood).

Examples for new peptide-based compounds, which are explicitly mentioned on the Prohibited List, are Corticorelin (CRH) and Mechano Growth Factors (MGFs). Other peptidic compounds (e.g. Thymosin beta-4) share the same status, but are not named on the list so far although their misuse has been reported. They all are available as approved drugs (CRH) or at least subject of different clinical trials (Thymosin beta-4, MGF with different analogues).
Essential requirement for the mass spectrometric detection is the appropriate extraction of the target peptides from biological matrices to meet the required limits of detection in the low pg/mL range. This will be achieved by applying the established coated magnetic beads technology (which has been successfully applied to other peptide hormones such as insulin analogues, CJC-1295, Tesamorelin, Geref, Synacthen, etc.) with respective antibodies. The identification of isolated and enriched analytes will be realized after liquid chromatographic separation by means of high resolution mass spectrometry. Due to the unknown metabolic fate of the peptides after administration to humans, in-vitro experiments are planned, which will help to characterize amino acid sequences serving as additional analytical targets in routine doping controls. The method will be validated and the implementation into existing screening procedures (if possible) for peptides will be realized.

Main Findings: 

While the analysis of small prohibited peptides < 2 kDa is well established in doping control laboratories worldwide, the detection of peptides > 2 kDa still remains challenging due to low analyte concentrations, unstable target peptides, an unknown metabolism and a complex sample preparation. However, recent developments in analytical chemistry can help to overcome these issues and facilitate the development of mass spectrometric initial testing procedures for large peptide hormones. 

The aim of this research project was to optimize current methodologies for the detection of peptide-based drugs > 2 kDa. For that purpose, new metabolites were identified by using in-vitro experiments and the sample preparation procedure was additionally modified in order to implement further prohibited peptides. For several IGF-I and MGF analogues as well as CRH, relevant metabolites were identified, comprehensively characterized, and valid analytical procedures were developed. All metabolites comprised N- or C-terminal truncated amino acid sequences and resulted from exoproteolytic processes. In principle, the developed assays are applicable to both urine and blood (plasma) specimens, but as the renal clearance of some peptides/metabolites remains to be elucidated, blood represents the preferred biological matrix.

Code: 15B07MT 

Fc-fusion based drugs are an emerging class of pharmaceuticals which already found their way into competitive sports: EPO-Fc is an erythropoiesis-stimulating agent where the attachment of two immunoglobulin Fc domains to the protein results in a prolonged therapeutic activity due to an increased plasma half-life and enables administration via inhalation. While EPO-Fc can be simultaneously detected with other recombinant erythropoietins by routine doping control assays, there are currently no tests for other doping-relevant Fc-fusion proteins such as the myostatin inhibitor ActRIIB-Fc (ACE031) or the cytokine GDF15/Fc, which is a member of the transforming growth factor beta (TGFβ) superfamily. 
The TGFβ superfamily includes several growth factors which are promising therapeutic targets for metabolic disorders and muscular dystrophies. Both TGF-β inhibitors and the cytokines themselves have been pursued as drug candidates. Within this study, a proteomics-based detection assay for emerging Fc-fusion proteins relevant as performance-enhancing agents in sports will be developed. ActRIIB-Fc (ACE-031), a fusion protein composed of the extracellular domain of the human activin receptor type IIB (ActRIIB) and the Fc domain of human immunoglobulin G (IgG), which was found to significantly increase muscle mass and function by inhibition of myostatin and other ligands of ActRIIB, will be used as model compound in addition to GDF15/Fc, a fusion protein of the TGFβ cytokine growth/differentiation factor 15 (GDF15) and the immunoglobulin Fc fragment. Different peptides, mutants and constructs of GDF15 are currently tested as pharmaceuticals for the treatment of different age-related and metabolic disorders. 
The proactive development detection assays for therapeutic Fc-fusion proteins, TGFβ cytokines and TGFβ inhibitors is of great interest as several drugs of these categories are already available on the black market as well as for research purposes. 

Main Findings: 

Cytokines of the TGF-β superfamily as well as their inhibitors are currently being evaluated as potential protein drugs for the therapy of a variety of diseases such as metabolic disorders and muscular dystrophies. The aim of this research project was to develop detection assays for two emerging Fc-fusion proteins potentially relevant as performance-enhancing agents in sports: The TGF-β cytokine GDF-15/Fc and an ActRIIB-Fc fusion protein related to the TGF-β/myostatin inhibitor ACE-031. Two complementary, multiplexed detection assays for ActRIIB-Fc were developed by using affinity purification in combination with either proteolytic digestion and LC-HRMS, or Western blotting. Both approaches can readily be modified to include other ActRII-Fc fusion proteins such as Luspatercept (modified ActRIIB-Fc). A central and critical aspect of the test methods is the ability of all target analytes to bind to activing-receptor ligands; in case of the model compound GDF-15/Fc, the available reference materials were found to be of substandard quality and w provided as multimer, which significantly interfered with affinity purification process due to the elimination of its binding capability.  Consequently, this analyte was not included into the test method validation process but the analyte’s characteristics and its potential implementation into flexible and expandable assays was elucidated. Overall, the developed assays proved fit-for-purpose and will expand the range of available test methods for emerging protein therapeutics, particularly those of higher molecular mass.

Code: 15A22MT 

The number of samples and target analytes is constantly increasing, necessitating all analytical methods to be optimized with regards to speed and costs effectiveness to follow the challenges of the modern anti-doping fight. Low molecular mass peptidic drugs (< 2kDa) have become more and more prominent on the Internet and in black markets, and their misuse in elite sport has been proven in the past.

The analysis of these new compounds is possible and methods are installed in the accredited anti-doping laboratories worldwide.  Unfortunately, these methods are largely designed to cover small peptidic drugs only in exclusive categories or after dedicated sample preparation as shown for instance for growth hormone releasing peptides, gonadorelins, desmopressin, TB-500, AOD-9604 and ARA-290. This is mainly due to the required sensitivity of these methods that enables the detection of the drugs in very low concentrations (sub-ng/mL). Within this study, a generic sample preparation strategy (dilute and inject) which is valid for several classes of compounds (diuretics, stimulants, anabolic agents etc.), will be evaluated for the detection of low molecular mass peptide hormones in urine even in the sub-ng/mL-levels. Therefore, several types of mass spectrometers and liquid chromatography methodologies will be assessed to find the best option for the aimed combination. This will enable a comprehensive multi-target screening within one analysis.

Main Findings: 

The number of small peptides prohibited in sports is steadily increasing, as well as the number of samples that have to be analyzed in sports drug testing programs. Consequently, fast, comprehensive, sensitive, and reliable new methods are needed to accordingly cover all these challenges. “Dilute-and-inject” approaches for urine analysis by liquid chromatography/mass spectrometry represent the most simplified way with a minimum of sample preparation workload. But due to the missing pre-concentration and purification steps, sophisticated chromatographic and mass spectrometric conditions are required to enable sensitive initial testing procedures. All of these aspects were taken into consideration when developing this new screening procedure by direct urine injection with online pre-concentration using a trapping column, liquid chromatographic separation, and subsequent detection by means of ion mobility coupled to high resolution mass spectrometry. 
Additionally, the in-vitro metabolism of selected model peptides such as ARA-290 and GHRP-3 was evaluated and new metabolites were identified by using different strategies as for example incubation with plasma, serum, human liver microsomes, and specific enzymes (e.g. amidases).

Ce guide de recherche en sciences sociales, qui comprend un questionnaire standard, est conçu pour aider les organisations antidopage (OAD) à mesurer les croyances et les comportements des sportifs relativement au dopage ainsi qu’à évaluer l’efficacité des programmes antidopage.

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

Code: 14A29OP

The project has two main goals. Firstly, the project aims to evaluate the potential of 3α-glucuronide-6β-hydroxyandrosterone (6OH-A-3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio-3G) for the screening of endogenous anabolic androgenic steroid misuse. For this first purpose, a quantitative method validated during the project 12A13OP will be applied to several samples from excretion studies already available in our laboratory. Secondly, the project also aims to characterize, elucidate and synthesize the marker M170T298. For this purpose, a large range of analytical strategies will be applied e.g. different derivatization steps, an in-depth study of accurate mass and tandem mass spectra. Once elucidated, the marker will be synthesized and characterized by NMR. After the synthesis of the marker, it would be useful to develop a quantitative method and to check its potential for other administration routes of testosterone. It is intended to reach these goals in a follow-up project once the objectives of the present project are achieved.

Main findings

In previous WADA funded projects (11A9RV and 12A13OP), our research group revealed that two testosterone metabolites resistant to enzymatic hydrolysis: 3α-glucuronide-6β-hydroxyandrosterone (6OH-A-3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio-3G) were present in urine. We characterized them, synthesized them and validated a method for their quantitation. However, their potential usefulness for doping control analysis was not fully evaluated.

On the other hand, common investigations for the screening of testosterone misuse are focused on metabolites with a predicted structure. Thus, unpredicted markers remain not evaluated. In order to cover this gap, we performed a preliminary metabolomic study which showed the presence of a marker (code M170T298) which is a potential urinary marker for testosterone administration. In order to confirm this fact, the characterization, elucidation and synthesis of this marker are required.

Thus, the project has two main goals. Firstly, the project aims to evaluate the potential of 6OH-A-3G and 6OH-Etio-3G for the screening of EAAS misuse. Secondly, the project also aims to characterize, elucidate and synthesize the marker M170T298.

Regarding the first goal, the usefulness of 6OH-A-3G and 6OH-Etio-3G was tested in three scenarios: (i) oral administration, (ii) intramuscular administration and (iii) gel administration. After oral administration of testosterone undecanoate we found that four markers containing either 6OH-A-3G or 6OH-Etio-3G presented detection windows (DWs) larger for all volunteers than those obtained by T/E and comparable to those reported by using cysteinyl metabolites. After intramuscular administration, markers containing either 6OH-A-3G or 6OH-Etio-3G provided detection windows that were similar or longer than those obtained by markers currently included in the steroid profile and clearly higher than those obtained by cysteinyl markers. Finally, the administration of testosterone gel could be screened by markers containing either 6OH-A-3G or 6OH-Etio-3G reaching similar to those obtained by markers currently included in the Athlete Biological Passport.

Regarding the second goal, during this project, we have characterized, elucidated and synthesized the marker M170T298. Our research shows that this marker is 1-cyclopentenoyl glycine (1-CPG) which is probably coming from the metabolism of the cypionic acid released after the hydrolysis of testosterone cypionate. Therefore, it can be valuable to detect the misuse of testosterone cypionate. This fact has been confirmed by analysis of samples collected after testosterone cypionate administration. The determination of 1-CPG provided the longest DWs for these samples

The WADA (World Anti-Doping Agency) Education Committee is seeking to target its Social Science Research Program to areas that would have greatest return in terms of enhancing the effectiveness of anti-doping education interventions. The objective of this project was to use the Sport Drug Control Model framework (SDCM; Donovan et al., 2002) to establish priorities for future social science research by reviewing what research has been completed in each of the model’s domains and identifying where more research is needed. The primary required outcome was the list of recommendations rather than the review per se.

Recognising the pivotal role that coaches play in clean sport, the World Anti-Doping Agency
(WADA) commissioned the International Council for Coaching Excellence (ICCE) to conduct a
research project to explore the potential of High Performance Training Centres (HPCs) to
become anti-doping training providers. This report provides guidance for the development of
a sustainable, cooperative and international anti-doping training programme for high
performance coaches.

The “One Resource Kit for Teachers” project was promoted in 2014 by a group of international Organizations composed by the World Anti‐Doping Agency (WADA), the United Nations Educational, Scientific and Cultural Organization (UNESCO), the International Olympic Committee (IOC), the International Paralympic Committee (IPC), the International Council for Sport Science and Physical Education (ICSSPE) and the International Fair Play Committee (IFPC). The main goals reported in the call were to map curriculum requirements for values education across the globe and to develop and pilot a contemporary resource kit for teachers that supports the delivery of their existing curriculum requirements for values-based education through sport.