Projets de recherche

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.

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Jiri Mudrak, République Tchèque

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Code: 14B26CR

Aside from human endogenous erythropoietin (EPO), its recombinant analogues (e.g. Epoetins alfa/beta/delta/omega, Darbepoetin alfa) and EPO-mimetics (e.g. Peginesatide), which stimulate erythropoieses through the EPO-receptor pathway, formation of erythrocytes can also be stimulated via the so called €activin-receptor type IIA signal transduction pathwayÅ: as soon as ligands, which interact with activin-receptor type IIA (€ActRIIA ligandsÅ) are removed by so-called ActRIIA ligand traps in a targeted way, erythropoiesis is also stimulated. ActRIIA ligands belong in particular to the transforming growth factor-beta (TGF-‚) superfamily (e.g. activin).
Sotatercept (ACE-011, ActRIIA-IgGI), a fusion protein consisting of the extracellular domain of ActRIIA receptor and the Fc-part of human immunoglobulin G1 (IgG1), is capable of acting as ActRIIA ligand trap, and was primarily developed as pharmaceutical for enhancing bone mineralisation in order to revert osteoporosis. However, aside from increasing bone mineral density, it was discovered that Sotatercept also stimulates erythropoiesis in a dose dependant manner leading to an increase in red blood cell counts. Sotatercept is currently intensely investigated in several phase II clinical trials.

Main Findings:

Two methods for the detection of Sotatercept in serum and urine samples were successfully developed. One method uses covalent immobilization of the capture antibody on agarose beads. It requires 10 µg of a polyclonal goat anti-ACVR2A antibody per sample (AF340 from R&D Systems) but needs double-blotting in case multiplexed detection of Sotatercept together with epoetins is necessary. The other method applies commercial magnetic beads coated with an antibody (sheep anti-rabbit) directed against the capture antibody. After incubation of the serum samples, the complex consisting of Soatercept and the capture antibody (a polyclonal rabbit antibody, 10257-T52 from Sino Biological) is bound by the anti-rabbit antibodies on the beads. After Western blotting, Soatercept is detected with the biotinylated version of the capture antibody of the other method (BAF340 from R&D Systems) and streptavidin-HRP. It omits incubation with a secondary antibody and thus is simpler, faster, and cheaper (only ca. 1.5 µg of capture antibody are required) The second protocol is compatible with single-blotting if the biotinylated clone AE7A5 antibody is used for the simultaneous detection of epoetins.

Code: 14B27CR 

Currently, comprehensive initial testing for erythropoiesis stimulating agents (ESAs) is done by electrophoretic methods (IEF-, SDS-, SAR-PAGE) according to WADA TD2013EPO and the forthcoming TD2014EPO. However, these protocols only comprise endogenous and recombinant erythropoietins and their analogues (e.g. darbepoetin alfa). ESAs, which lack the primary structure of EPO and in particular the first 26 amino acids of the N-terminus, cannot be detected due to non-interaction with the monoclonal anti-EPO antibody used for Western blotting (clone AE7A5). Peginesatide (Omontys; formerly known as Hematide) is a so-called EPO-mimetic peptide, which is structurally unrelated to the amino acid sequence of EPO. Two methods (SDS-PAGE, electrospray mass spectrometry) were developed and published in 2011 in order to overcome this situation. However, both methods are unable to simultaneously detect epoetins (EPOs) - the majority of misused ESAs. By modifying Sarcosyl (SAR)-PAGE - one of the main electrophoretic methods used in EPO anti-doping testing - peginesatide will now also become part of routine ESA doping testing with SAR-PAGE. Despite Omontys was recalled in 2013, misuse by athletes cannot be excluded.

Main Findings: 

An electrophoretic method was successfully developed in order to get Peginesatide, a PEGylated EPO-mimetic peptide dimer, migrate in SAR-PAGE, which is one of the main methods used in EPO-testing. Due to its short amino acid chain and the two PEG-groups, sarcosyl cannot sufficiently solubilize Peginesatide for running it on SAR-PAGE. Upon replacement of 30% of the SAR in the running buffer by SDS (resulting in a final composition of 0.03% SDS and 0.07% SAR), Peginesatide migrates into the stacking gel without significantly broadening the CERA-band. With the optimized "SDS-modulated" SAR-PAGE method (mSAR-PAGE), Peginesatide can also be differentiated from CERA, since both bands are well separated from each other.

The new method also allows the simultneous detection of Peginesatide and all EPO-based ESAs on one blot. By duplexing the two primary antibodies (clones 11 F9 and AE7A5) followed by incubation with an HRP-labelled anit-mouse secondary antibody, which binds to both antibodies, single blotting can be applied for detection.

However, the modification of an already published method for immunoaffinity purification of Peginesatide from serum and plasma samples could not be solved so far. The published protocol used covalently immobilized clone 1G9 antibody and released bound Peginesatide by heating the bead in SDS-sample buffer. Despite double-blotting, bands from the light antibody chain remained visible on the blot. Hence, this approach was not applicable for the simultaneous detection of Peginesatide next to epoetins by single blotting. In search of a solution, three alternative methods were tested: (1) covalent immobilization of clone 1G9 antibody on agarose beads followed by acidic elution; (2) immunoprecipitation using anti-antibody coated magnetic beads; (3) immunoprecipitation using biotinylated clone 1G9 antibody and streptavidin-coated magnetic beads. However, they did not yet lead to useful results. Despite the projct has ended, we will continue trying to find a solution

Code: T14M03WS

Investigations in connection with an adverse analytical finding for the prohibited diuretic chlorazanil have raised the presumption that chlorazanil may originate from the application of the non-prohibited anti-malaria drug proguanil (ingredient e.g in medicament Malarone® from GSK). The structure of metabolites of the non-prohibited anti-malaria drug proguanil suggests that they can be converted to chlorazanil. To check this hypothesis it should be found out whether chlorazanil is a metabolite of proguanil or whether proguanil metabolites are converted in the urine to chlorazanil by chemical or bacterial influences or whether chlorazanil is a byproduct of the synthesis of proguanil. 
Furthermore parameters should be identified which allow a discrimination between the application of the non-prohibited proguanil and the prohibited chlorazanil.

Main Findings:

Chlorazanil (Ordipan, N-(4-chlorophenyl)-1,3,5-triazine-2,4-diamine) is a diuretic agent and as such prohibited in sport according to the regulations of the World Anti-Doping Agency (WADA). Despite its introduction into clinical practice in the late 1950s, the worldwide very first two adverse analytical findings were registered only in 2014, being motive for an in-depth investigation of these cases. Both individuals denied the intake of the drug; however, the athletes did declare the use of the antimalarial prophylactic agent proguanil due to temporary residences in African countries.  While no chlorazanil was found in drug formulations, the urine samples of 2 out of 4 proguanil users returned findings for chlorazanil at low ng/mL levels, similar to the adverse analytical findings in the doping control samples. Further, in the presence of formaldehyde, formic acid and related esters, 4-chlorophenyl-biguanide was found to produce chlorazanil in human urine, suggesting that the detection of the obsolete diuretic agent was indeed the result of artefact formation and not of the illicit use of a prohibited substance
A structural similarity between chlorazanil and proguanil is given but no direct metabolic relation has been reported in the scientific literature. Moreover, chlorazanil has not been confirmed as a drug impurity of proguanil. Proguanil however is metabolized in humans to N-(4-chlorophenyl)-biguanide, which represents a chemical precursor in the synthesis of chlorazanil. In the presence of formic acid, formaldehyde, or formic acid esters, N-(4-chlorophenyl)-biguanide converts to chlorazanil.  
In order to probe for potential sources of the chlorazanil detected in the doping control samples, drug formulations containing proguanil and urine samples of individuals using proguanil as antimalarial drug were subjected to liquid chromatography-high resolution/high accuracy mass spectrometry. In addition, in vitro simulations with 4-chlorophenyl-biguanide and respective reactants were conducted in urine and resulting specimens analyzed for the presence of chlorazanil.  

Code: 14A32GJ 

Formoterol is a long acting beta2-agonist allowed for use in athletes via inhalation at doses up to a predetermined limit, and is not allowed to be administered orally. The drug is usually administered and measured as a racemic mixture consisting of an active R/R- and inactive S/S- enantiomer, each with a different time course in the body which also differs by route of administration. The inactive S/S-formoterol is preferentially eliminated in urine relative to the active R/R-formoterol. Recent work has demonstrated that the Adverse Analytical Finding (AAF) limit, currently 40 ng/ml in urine, is
difficult to reach and some authors have even questioned whether it is worth having an AAF limit for formoterol at all. To date, anti-doping strategies have not capitalised on this difference between formoterol enantiomer elimination from the body. We will use our advanced analytical technique (enantioselective LC-MS/MS) to characterise the urinary levels and ratio of both R/R- and S/S-formoterol enantiomers, as well as the glucuronide metabolite in athletes dosed with formoterol in prohibited regimens; an “acute” treatment regimen that exceeds the current allowable dose via inhalation and an oral regimen consisting of 160 μg/day for 7 days. The secondary objective is to characterise the urinary levels and ratio of formoterol enantiomers and glucuronide metabolite in athletes using repeated “chronic” inhaled rac-formoterol under permitted regimens. Study
design will be double-blinded placebo controlled with two “chronic” 7 day treatment regimens; formoterol delivered by inhalation at just below the maximum allowable dose, and approximately half of the maximum allowable dose. This project will allow doping agencies to better discriminate between permitted and prohibited formoterol dosing.

Main Findings: 

Oral dosing of beta2-agonists is known to lead to beneficial performance effects in athletes but some drugs of this class are allowed to be delivered by inhalation for use in athletes with asthma. Formoterol is a long acting beta2-agonist permitted for use in athletes at inhaled doses up to 54 μg over 24 hours, however, a threshold limit of 40 ng/ml in urine is used to control for supratherapeutic dosing. The drug is usually administered as the racemic (rac-) mixture consisting of the active (R,R)- and inactive (S,S)- enantiomers with different metabolic profiles. The primary objective of the study was to examine whether the urinaary levels and ratio of formoterol enantiomers in individuals dosed with formoterol could be used to improve the sensitivity and specificity of a urine threshold approach to ascertain whether individuals were using formoterol in a permitted manner. Urine levels were measured following prohibited regimens; an "acute" treatment regimen that exceeds the current allowable dose (54 μg in 24 hours) by administering a once-off 72 μg dose via inhalation at baseline, and an oral regimen consisting of 156 μg/day for 7 days. These levels were compared with repeated "chronic" inhaled rac-formoterol under permitted regimens (12 μg twice daily for 7 days, 24 μg twice daily for 7 days). Comparisons were made with current urine dosing threshold and decision limits (TD2019DL) to assess the utility of enantioselective ratios. Using the current TD2019DL method, none of the samples from the chronic permitted regimens exceeded the current threshold for total formoterol (40 ng/ml) supporting the existing approach. The acute inhaled and oral prohibited regimens resulted in 7/127 and 21/124 samples exceeding the threshold  (TD2019DL), and 5/127 and 13/124 exceeding the decision limit respectively. There were an increased number of specific gravity. Females demonstrated higher urine levels than males. As prediced, there were differences in enantiomer ratio between oral and inhaled administration, but receiver operating characteristic (ROC) analysis for log(R,R:S,S) formoterol enantiomer ratio showed poor diagnostic performance (ROC area = 0.716 for oral treatment) and was inferior to the current TD2019DL approach, possibly due to considerable pharmacogenetic enantionselective differences in glucoronidation metabollism. In conclusion, the current urine method (TD2019DL) based on a threshold and decision limit of 40 ng/ml and 50 ng/l respectively appears a valid approach to discriminate between oral (prohibited) and permitted inhaled dosing regimens in urine spot samples but requires further validation with regard to intra-day spot sampling variability and dosing regimens. Furthr work is required to understand the basis of the significant differences in enantioselective glucuronidation between subjects and genders, and improve the sensitivity of the current threshold. Formoterol elimination seems to be particularly sensitive to urine hydration and a greater understanding of exercise on hydration and excretion is also warranted with regard to application of TD2019DL threshold approach.