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Résumé

Comprendre et prévenir le dopage chez les jeunes sportifs en Espagne et en France. Se doper ou pas : approche comparative des processus de recours au dopage et de lutte contre le dopage chez les jeunes sportifs en Espagne et en France » est une étude vise à identifier le rapport que les jeunes peuvent entretenir avec le dopage dans leur accession à la performance et leur recherche de résultats sportifs. Elle portera sur une analyse contemporaine des problèmes de dopage de 1975 à nos jours à travers l’exemple de trois sports (l’athlétisme, le basket-ball et le cyclisme) dans deux pays européens ou cette question semble avoir été traitée différemment. Trois niveaux d’études seront utilisés et articulés entre eux :

1. Un niveau historique

2. Un niveau juridique

3. Un niveau psychosocial.

Ces trois niveaux ne sont pas dissociables c’est en effet dans l’articulation des trois niveaux que se joue la compréhension du recours au dopage ou, contraire l’apprentissage de comportements « normaux ». Il ne s’agit donc pas seulement de produire de la connaissance dans chacun de ces niveaux mais également à observer comment ces différents niveaux interagissent et permettent de comprendre le recours aux comportements dopants ou non des jeunes sportifs. La finalité de ce travail s’inscrit donc pleinement dans la première catégorie de projets définis par l’AMA-WADA, à savoir : « Renforcement de la connaissance des causes des comportements de dopage et des facteurs influençant ces comportements » et même sur les risques et les facteurs de prévention du dopage. Il est évident cependant que seront également évalués les programmes d’intervention existants et analysés tout à la fois des modèles de comportements et des distinctions culturelles.

Publications connexes

Se doper ou pas : les jeunes sportifs face à la tentation du dopage

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

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

Code: 11C7MT 

AICAR has been shown to exhibit performance enhancing effects after oral administration due to the stimulation of fat utilization and increased production of mitochondria in laboratory mice after a 4-weeks treatment with AICAR. The drug candidate has not yet received clinical approval and currently undergoes phase-II clinical trials. In urine of healthy humans considerable amounts (up to 7500 ng/mL) were present due to the natural occurrence of AICAR as a (by-) product of purine biosynthesis. Normal (log-transformed) distribution of AICAR excretion into urine was observed, if corrected to creatinine rather than to specific gravity; however, the occurrence of elevated urinary levels (> 3500 ng/mg) after an illicit administration were assumed to last for several hours only, and the detection window offers only limited options for effective doping controls. 
Circulating AICAR is dedicated to enter the cells with a simultaneous biotransformation from AICAR-riboside to AICAR-ribtotide (phosphorylation at position 5`). This conversion occurs in considerable extent also in erythrocytes. After exposure of erythrocytes to AICAR, the red blood cells contain a significantly elevated amount of AICAR-ribotide and this level is described to be remain stable for approximately 10 days. In the present study it is planned to investigate whether the determination of AICAR-ribotide in red blood cells can serve as potential long-term marker for an illicit AICAR administration. Riboside and ribotide concentrations in plasma and red blood cells of healthy volunteers will be determined by LC-MS/MS and quantified by means of isotope dilution mass spectrometry. Subsequently, reference values will be statistically evaluated. 
Preliminary experiments have shown that in-vitro experiments (incubating AICAR reference substance in whole blood) induce immediate biotransformation of the riboside to the corresponding ribotide within the erythrocytes. After lysis the elevated ribotide levels can be determined and allow for the development of a pharmacokinetic model for in-vivo conditions.

Main Findings: 

AICAR (an adenosine monophosphate-activated protein kinase (AMPK) activator, 5-amino-4-imidazolecarboxyamide ribonucleoside, Acadesine) belongs to the class of metabolic modulators of the WADA Prohibited List and has been banned since 2009 due to its performance-enhancing capabilities. Confiscated products from team managers and anecdotal evidence have outlined the need for adequate test methods, which have been under development from urine in particular. Main issue with this emerging drug is its natural occurrence in every individual at varying concentrations.  
In the present project, the utility of erythrocytes concerning the detection of AICAR abuse was evaluated. AICAR possesses the ability to penetrate the erythrocyte membrane. After entering the red blood cell cytosol, it is converted into the corresponding phosphate, referred to as AICAR ribotide. Due to the phosphorylation, it is conserved within the erythrocyte and its concentration remains elevated for up to 10 days upon administration of therapeutic amounts of AICAR. Hence, the quantitative analysis of AICAR ribotide levels in erythrocytes was considered as a helpful tool to determine unnaturally high levels. Consequently, an assay employing a stable-isotope labelled internal standard (15N4-AICAR ribotide) was developed to allow for the quantification of the target analyte from 10 μL of erythrocytes. A total of 99 blood samples (49 male and 50 female non-elite athletes) was assayed to establish a reference population-derived ‘normal’ value for AICAR ribotide, which indicates that concentrations higher than 920 ng/mL are not in agreement with naturally circulating AICAR levels. In order to simulate elevated plasma concentrations of AICAR following oral or intravenous administration, in vitro incubations were conducted at 500, 1000, and 10,000 ng/mL in agreement with literature data concerning clinical trials. A rapid uptake of AICAR into the erythrocytes and conversion to the ribotide was observed, yielding concentrations significantly higher than 920 ng/mL within 15 min. Hence, the quantitation of AICAR ribotide in red blood cells derived either from athlete biological passport samples or complementary collected specimens (such as dried blood spots, DBS) can be a viable tool to indicate and eventually prove the illicit intake of AICAR.  

Code: 11C21BG

This project seeks to build on the very important findings of a project funded previously by the Australian Government’s Anti-Doping Research Program (ADRP), into the potential application of transcriptional profiles derived from peripheral blood mononuclear cells (PBMCs -lymphocytes & monocytes) for the detection of rhGH. That project identified sets of genes that were consistently either “up” or “down” regulated in mRNA expression, changes that were evident at least 21 days after the final administration of rhGH. The 21 day time period is an extremely important finding given that the current detection “window” with the WADA-approved test for rhGH is only 36 hours (Holt & Sonksen, 2008). The findings of the previous ADRP-funded study clearly provide the foundation for the development of a new, more sensitive test for the detection rhGH, a test with a vastly improved detection “window’. 
We anticipate that the proposed project will result in the development of a new, more sensitive test for rhGH based on alterations in gene expression assessed at either the transcriptional and/or proteomic level. A cell’s transcriptional “profile” is obtained by measurement of (potentially, genome-wide) genespecific mRNA levels via use of oligonucleotide microarray technology. Subsequent quantitative RT-PCR analyses allow verification of alterations in the expression of candidate genes. A cells “translational”/proteomic profile can be assessed via the techniques of SDS-PAGE and western blotting. These technologies will allow the identification of gene “subsets” whose activity at a specific time-point, is either up-regulated, down-regulated or remains unchanged following a specific intervention. The set of genes responding transcriptionally or translationally to a specific intervention and the temporal extent of such changes potentially provide a set of unique indicators that are characteristic of that intervention. 

Main Findings: 

This project has been carried out with the support of The World Anti-Doping Agency. The investigation aimed at pinpointing a set of differentially expressed mRNA genes associated specifically to the uptake of recombinant growth hormone. 
Particular attentions of this project focused on the determination of exercise related induce gene expression and to compare whether such expression profile shared similarities with rhGH related expression profile. In other words, this project aimed at finding the unique and specific rhGH related gene expression signature eliminating possible additional unrelated expression variables that different types of exercise induce. 
Three types of exercise regimen (endurance, resistance and anaerobic exercises) were analysed in untrained participants and temporal PBMC extracted cell gene expression profiling using microarray technology was determined for each type of exercise. In addition, mRNA expression profiling was performed on trained individuals carefully sub-grouped in the same types of exercise mentioned previously. 
Our analysis used a series of state of the art bio-informatics tools aimed at reducing background noise, batch effects between microarrays, false positives and any other un-necessary variables to aim at narrowing the candidate genes at the highest significant relevance to our study and conclusions. 
The main results obtained in this document are reporting the PBMC’s temporal expression profiling from: I. participants administered with rhGH;
II. untrained participants undertaking three types of exercise. In addition, non-temporal expression profiling at rest was evaluated on trained participants grouped in the same types of exercise (endurance, resistance and anaerobic).The comparison of all the different profiles has shown that:I. 47 genes are specific to rhGH and unrelated with any of the three exercise related gene expression in untrained individuals.
II. 2 genes specific to rhGH are seen to be similar to both the Trained and Untrained expression profiles. These two genes consisted of the interferon gamma and the tubulin folding cofactor C.Of note, this two candidate genes were observed specifically in the resistance groups. Finally, these two genes showed fold gene expression levels that are, and across all time points, lower in rhGH related PBMCs than the fold expression values of the resistance groups. 
The common limitations of this present study are: - Batch effect related to the analysis of the rhGH related study that required the action of the use of a bio-informatic batch effect removal tool.
- Possible dosage of the rhGH that was administered to reflect a more pronounced expression profile.

Code: 11A26CS

Determining the origin of anabolic steroids that are also produced endogenously in the human body, is a major issue in doping control. Increasing amounts of anabolic steroid preparations of nandrolone, boldenone and testosterone preparations, both in a ready-to-use form and as bulk material, have been confiscated by the Norwegian Custom Authorities and the Norwegian Police. 
In rare circumstances, nandrolone and boldenone metabolites may be produced naturally in humans and animals. Hence, the measurement of the 13C‰ value of nandrolone, boldenone and testosterone of synthetic origin is important for their comparison with the nandrolone and boldenone metabolites, and testosterone endogeneously produced. As a result of this, IRMS is required for a definitive assignments of the origin of nandrolone, boldenone and testosterone found in urine. 
The aim of the project is to determine the 13C/12C ratios of nandrolone, boldenone and testosterone available on the Scandinavian black market. The success of the established doping test is depending on a significant difference in 13C‰ values between the administered product and the endogenously produced steroids. A comprehensive knowledge of these data with a global perspective is essential for doping analysis of certain anabolic-androgenic steroids. 
In addition, the confiscated testosterone preparations will also be investigated for the content of other possible masking agents like epitestosterone. 

Main Findings:

There have been a substantial number of seized doping preparations, including preparations of nandrolone, boldenone and testosterone, in Norway lately. Determining the origin of anabolic androgenic steroids (AAS) that are also produced endogenously in the human body, is a major issue in doping control. The aim of this study was to use GC-C-IRMS to determine the δ13C‰ values of nandrolone, boldenone and testosterone in seized preparations, and to what extent these products have been designed to mimic the endogenous δ13C‰ values, and hence prevent positive GC-C-IRMS findings. Out of 55 products, one nandrolone and five testosterone products (11%) were found to display δC‰ values between -24‰ and -26‰. In addition one nandrolone and eight testosterone products (16%) displayed δC‰ values of -27‰, which is close to the upper reference range (-26‰) reported for urinary reference metabolites. None of the investigated products contained epitestosterone. This study shows that there are AAS available with δC‰ values near and within the endogenous urinary reference range in Norway.

Code: 11D3JD 

Stimulants are banned in competition by the World Anti-Doping Agency, except for a small number of therapeutic agents subject to monitoring, and a large number of compounds have been placed on the prohibited list. However, a number of agents available as over the counter medicines for therapeutic uses, although subject to monitoring, may enhance performance. In addition, a large number of other unlisted stimulants are available, some of which appear in athlete’s samples. There is only very limited information on the relative potencies of even monitored agents as stimulants. For some agents even the mode of action is not well understood. This project seeks to supply that information. In this project, the stimulant actions of a wide range of compounds, prohibited, specified, monitored or unlisted, will be investigated in pharmacological and physiological bioassays in an animal model. This study will provide data giving direct measurement of the relative potencies of a wide range of stimulants at a number of biomarkers of stimulant action. The results will allow informed decisions on the doping potential of monitored substances relative to prohibited or unlisted substances. The wider implications are that these bioassays can be quickly performed to give robust information on any new compound appearing in athlete samples or to decide on the acceptable agents and doses for approved therapeutic uses. Generation and dissemination of detailed pharmacological information is essential to prevent doping in sport and to provide alternative therapies. 

Main Findings:

1. Bupropion is a stronger noradrenaline reuptake blocker than previously reported. It is likely to have both peripheral cardiovascular stimulant actions and central behavioural actions in doses used clinically.
2. The majority of direct adrenoceptor stimulants investigated in this study showed relatively high potency as β-adrenoceptor agonists in producing a tachycardia. Although β1-adrenoceptor stimulation may be of limited benefit in sport (see Davis et al., 2008), this merits further consideration, especially if some of these agents are found to have additional  β2-adrenoceptor agonist actions.
3. Cathine is likely to be an α2-adrenoceptor agonist, and this merits further study, as central clonidine-like actions as well as peripheral actions may be important for this agent.
4. Selective actions at α1D-adrenoceptors occur for a number of agents. It is unclear if this action is of benefit in enhancing performance. This merits further investigation.
5. Locomotor studies by telemetry are a useful method to study central stimulant actions. Both bupropion and modafinil were found to have central stimulant actions in doses employed.
6. A large amount of data have been obtained on the cardiovascular effects of 12 receptor stimulants, and on the peripheral and central effects of 3 monoamine transporter blockers.  On publication, this will greatly increase the literature on the pharmacology of these agents.  On acceptance for publication, copies of these papers will be forwarded to WADA.

Code: 11A6SD 

The aim of the project is to provide an isotopically labelled internal standard of a key testosterone metabolite. These will facilitate high accuracy measurement of this analyte in human urine thereby contributing to WADAs implementation of the Athletes Passport. The synthesis of d4-epitestosterone-17-b-glucuronide will be performed using a combination of literature precedents and chemistry routinely used by the Chemical Reference Materials team at NMI Australia. This material will be purified to the highest level possible using chromatographic and recrystallisation techniques. The material will then be fully characterised to afford fit for purpose status for use as an internal standard. The identity will be confirmed using a range of spectroscopic techniques including nuclear magnetic resonance, infrared spectroscopy and mass spectrometry. This will be complimented by analysis using a range of traditional techniques (HPLC with ELS detection, Karl Fischer moisture analysis and thermogravimetric analysis) to determine the chemical purity of the material. The isotopic purity will be determined using mass spectrometry on either the deglucuronidated steroid (GC-MS) or direct analysis of the parent material (LC-MS). 
Fully certified material will be packaged in 1 mg lots and made available to all WADA accredited laboratories. 

Main Findings: 

This summarizes the synthesis of 1,16,16,17-d4-epitestosterone-17-O--glucuronic acid (1) using a combination of literature precedents and chemistry used previously by the Chemical Reference Materials team.  The material has undergone extensive purification to minimise impurities of similar structure.   
HPLC analysis with photodiode array detection (PDA) confirmed an organic purity in excess of 99% mass fraction.  The overall purity value was lower (~93% mass fraction) than the idealised >99% mass fraction, which can be attributed to significant mass fractions of occluded water.  This is typical for glucuronic acids of this type and no attempt was made to remove the water because of concerns that the inevitable re-absorption of moisture would introduce stability issues.The isotopic purity of each material has been determined using GC-MS analysis of the de-glucuronidated steroid entity, confirming high levels of deuteration, with essentially zero non-deuterated (d0) present, making the isotopically labelled internal standard fit for its intended purpose.   The structure of the 1,16,16,17-d4-epitestosterone-17-O--glucuronic acid (1) has been unequivocally determined using a combination of spectroscopic techniques . Direct comparison with the spectroscopic data obtained for a fully certified sample of non-deuterated epitestosterone-17-O--glucuronic acid confirmed the regio- and stereochemistry. Further evidence was provided by co-elution studies of the parent compound and/or the liberated steroid moiety, with the fully certified native counterparts.
 Mass spectrometry
 Infra-Red spectroscopy
1H / 2H / 13C Nuclear Magnetic Resonance spectroscopy
1,16,16,17-d4-Epitestosterone-17-O--glucuronic acid (1) was assessed for chemical purity using a complementary range of analytical techniques to determine the mass fraction of the main component. Full characterisation and certification of this material has been performed and each material is currently available to the Australian Sports Drug Testing Laboratory (ASDTL) and other sports doping control laboratories around the world.