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Publications connexes

• Reviewing research into attitudes towards doping in sport: Time to take stock
• Doping in sport: A review of medical practitioners’ knowledge, attitudes and beliefs
• Reviewing Coaches' Knowledge, Attitudes and Beliefs regarding Doping in Sport

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Code: 06B3MT

Growth hormone (GH) therapy has become a frequently employed strategy to fight dwarfism, and with the advent of biotechnologically produced recombinant GH, purity and sufficient amounts have been guaranteed. However, the need to prepare recombinant peptide hormones and repetitively inject drugs has led to gene therapy approaches that aim to generate GH-producing cells. Early attempts were based on myoblasts and fibroblasts modified to produce GH as growth hormone gene therapy does not necessarily need to be pituitary-specific because targets of GH are peripheral organs. The abuse of GH in sports has been a serious issue since years, and first assays enabling the discrimination between natural isoforms and recombinant GH have been established using a so-called differential immunoassay approach. More detailed information on GH isoforms used for the determination of GH administration is obtained using proteomics approaches employing 2D gel electrophoresis followed by mass spectrometric identification and characterization of GH and its fragments. In order to provide fundamental information for new methods complementing the commonly accepted assay and to provide basics for another option to cope with the problem of GH misuse, which includes administration of recombinant GH as well as gene therapy, GH isoforms shall be isolated of from human plasma followed by 2D gel electrophoresis, visualization, and subsequent mass spectrometric identification. Using this strategy, GH isoforms can be considered individually in terms of a profile, and more information on the variability and stability of single items is obtained. Owing to the well known fact that endogenously produced GH is down-regulated upon GH administration, the considerable change in GH isoform profiles should be detected using proteomics technology. Moreover, the fact that future GH gene therapy may include cells different from the pituitary, significantly different isoform profiles are possible and should principally be detectable employing the technology established in this project.

Main Findings

Growth hormone is widely abused by athletes for its anabolic and lipolytic as well as growth promoting effects. The presented method is capable of discriminating endogenous and recombinant growth hormone in plasma or serum samples if the concentration is high enough to detect endogenous isoforms which is the case for samples with a normalized spot volume of the main 22 kDa spot of >0.52. The capability to detect and visualize discrete isforms of hGH represents an important advantage for confirmatory HG analyses in sports drug testing and could compliment currently employed assays to reveal GH misuse. While endogenous samples show two or four isoforms on the blots, samples containing recombinant growth hormone lead to detection of one spot only. In addition to the volume limit, the order of appearance is considered and the detection of a more acidic 22 kDa spot, which is phosphorylated in endogenous samples but may be an artefact from protein production in samples containing recombinant growth hormone. The suggested discrimination limit of 0.52 may be corrected to even lower values if larger samples populations are analyzed. Additionally all growth hormone variants that are detected in the doping control method were identified by mass spectrometry approaches. This makes the doping control method even more powerful and allows a better evaluation of results.

Code: 06B17HM

Doping analysis is always in the misery of ethics, minimal invasive tissue collection and the establishment of sensitive, valid and reliable techniques to detect illegal use of forbidden substances in sports. Until now GC MS is the technique of choice to quantify the banned substances or their metabolites. But in the last years, modern techniques of molecular biology are in development to become alternative or supplemental choice to elucidate the abuse of diverse doping substances. Therefore, the combination of diversified screening of gene expression by high through put gene array analysis, evaluation of array data by quantitative RT PCR, information transfer to customized cheap gene array and the verification by established methods such as GC MS would be a useful synergy in the fight against doping. The following research application includes several subprojects. In appreciation of ethic recommendations of tissue collection, buccal epithelium, lymphocytes and leukocytes could serve as minimal invasive RNA source for the proposed comparative molecular biology analysis. The main focus is located on the evaluation of a fast and reproducible method to isolate sufficient amounts of intact total RNA out of the tissues by RNA-stabilizing chemical extraction techniques. The second step is to screen the two tissues for possible side effects by physical activity. Therefore, baseline results should be generated by a group of control sportmen. Following, three additional populations, asthmatics, anti aging patients (and/or patients with testicular hypofunction) and bodybuilders, should be tested for either the use of inhalative beta-2-agonists or anabolic steroids by gene array, RT PCR and GC MS in comparison to the control population. The main objective of the present study is to evaluate an innovative valid long term screening technique to elucidate candidate genes which are modulated by illegal substance and not by factors such as physical activity or circadian rhythm.

Main Findings

The current test results demonstrate that gene array analytics has not yet reached the aspired quality level to guarantee a constant reproducibility over time–particularly when involving batch removal. In addition, the low level of reproducibility may lead to the discrepancy between gene array and RT-PCR results. Realtime RT-PCR revealed high reproducibility. Results were subject to intense biological variation in relation to exercise load, time and hormone substitution. Additionally, hormone analyses from the saliva showed a potential method for direct evidence.

Future plans include: Expanded cluster analyses or new mathematical models possibly elicit further target genes that have to be validated via RT-PCR.

Code: 06A03AM

A method is proposed which will enable specific detection of adminstered recombinant erythropoietin (EPO) and ARANESP™ (Darbepoetin alfa) in urine, or other biological fluid such as blood, without regard for levels of EPO activity. This method is specific for recombinant erythropoietin and will not be subject to interference by endogenous erythropoietin. This method is based on the immunochemical detection of recombinant erythropoietin and ARANESP™ by specific detection of carbohydrate moieties which are unique to recombinant erythropoietin and ARANESP™ and are absent from the endogenous glycoprotein. This method is feasible due to the fact that the glycosylation of recombinant glycoproteins is dependent on the cell line in which the protein is expressed and the culture conditions. For these reasons, although possible, it is not necessary to determine levels of EPO activity to detect administration of rHuEPO to the athlete. This method will be rapid, cheap, and more specific than current methods in use. Glycozyme, Inc. in discussions with two different companies to produce the ELISA kit which could also be used to monitor the dosage of legitimate patients.

Main Findings

The immediate goal of this work was to obtain one or more monoclonal antibodies against the carbohydrate moiety of rHuEPO. The clones from the fusion screen are now monoclonal. The initial screen was against EPO and DG-EPO in which they did not bind DG-EPO as well as EPO by an amount that roughly correlates with the sialic acid content. The supernatants from five clones can be almost completely inhibited(at the concentrations tested) from binding EPO by Maackia amurensis lectin which specifically binds Nacetlyneuraminic acid α(2-3) galactose which is present on rHuEPO produced in CHO cells. (CHO cells do not have the enzymatic machinery to produce Nacetylneuraminic acid α(2-6) galactose.) The EPO molecule has been shown to have the carbohydrate chains on one side of the conformation with the antennary chains protruding so it is unlikely that the lectin binding the terminal sugars interfere with the protein chain. Finally, the supernatants from the twelve clones tested bind the glycopeptides GP-8 and GP-9 which were linked to KLH. This is a very important observation for several reasons. Our original rabbit anti-EPO antibody was obtained from a rabbit immunized with rHuEPO obtained from Amgen. This polyclonal antibody was shown to bind N-acetylneuraminyl α(23)galactose and galactosyl ß(1-4) N-acetylglucosamine (lactosamine) (Pazur, et. al., 2000). The rabbit antibody was shown to bind GP-9. The glycopeptides GP-8, GP-9 and GP-10 were obtained by treatment of the rHuEPO with Glu-c. Further, the rHuEPO from which the glycopeptides were isolated was obtained from another company which, according to some, would be considered a biosimilar rHuEPO even though it is still produced in CHO cells. So there then is indirect evidence that the monoclonal antibodies may bind one of the two antigenic determinants of the rabbit antibody. These results are very encouraging but these are still screening tests so now the antibodies must be produce in sufficient quantities to further characterize the carbohydrate binding specificity.

Code: 06A09FL

At the present time, anti-doping control for recombinant human erythropoietin (rHuEPO) relies on isoelectric focusing of ultra-filtered urine and double blotting of this hormone. This method results in an image of the isoelectric pattern of erythropoietin (EPO) present in urine and allows to differentiate between natural and recombinant hormone. Indeed, some of the isoforms composing the isoelectric profiles of natural urinary EPO are collocated with the isoforms of rHuEPO but others, more intense, present more acidic pI. This difference enables the detection of rHuEPO in urine but till now has no structural explanation. It is important to explore this structural aspect both from a fundamental scientific and anti-doping analytical points of views. We have undertaken investigations on this topic by studying the role of sialic acid residues in the isoelectric profiles of recombinant and natural urinary EPO. Our preliminary results show that in addition to sialic acid. Some other unidentified residues are responsible for the more acidic properties of natural hormone since after desialylation, the two hormones present different isolelectric patterns. This observation is very important since apparently, some isoforms corresponding to molecules devoid (or with very few) of these additional structures are observed in the asialopattern of rHuEPO but not in that of natural urinary hormone. The presence of these isoforms in a asialo pattern would thus constitute an absolute demonstration of the presence of recombinant hormone. It is thus envisaged to develop a confirmation test based on analysis of the asialopattern EPO. However, an essential condition to these investigations is obtaining specific antibodies well-recognizing the asialo-erythropoietin molecules and we have planed to produce such antibodies.

Main Findings

The objectives of this first part of the project was to obtain antibodies to asialo EPO in order to investigate the asialo EPO patterns obtained in various situations (negative, positive, unstable, atypical EPO profiles from urine samples) and from various recombinant drugs (Epoetin alfa, beta, omega, delta, Darbepoetin alfa). These antibodies would be used as primary antibodies in immunoblot following isoelectric focusing (IEF) of desialylated EPO. Immunization of two rabbits produced antiserum with very low titres that did not give any results on immunoblots. Immunization of 3 mice gave rise to better results and the most reactive mouse, after an additional injection of immunogen, was chosen for the fusion step. Sera and hybridoma (after purification of immunoglobulins by protein G affinity chromatography) were tested on immunoblot experiments. Whereas good results were obtained by dot blot, no results were obtained after IEF of desialylated Eprex. It is probable that the tested antibodies have conformational epitopes that are lost during IEF with urea. Since urea is necessary to perform IEF of EPO, the project was suspended.

Code: R06C01MS

Natural variability of endogenous substances in the body complicates the evaluation of the evidence with indirect markers of doping. Currently, reference ranges of markers of doping are derived from the analysis of data obtained from a large number of athletes. Unfortunately, such population based thresholds induce a lack of sensitivity for many markers, because they do not take into account the large heterogeneity that exists among professional athletes. For example, when the hematocrit is used as an indirect marker of blood doping, a threshold at 50% lets a margin large enough to athletes with naturally low levels (ex: 39%) to abuse from rhEPO or blood transfusions. In the same way, a limit at 4.0 or 6.0 for the ratio of testosterone over epitestosterone lets a margin large enough to the majority of male athletes to monitor their steroid profiles below this limit via the intake of exogenous testosterone. It has been shown recently that the combination of subject-based information, such as ethnical origin, gender, age or previous individual readings of the marker, with traditional population-based data can significantly enhance the sensitivity of most markers of doping (T/E, haemoglobin, ABPS,…). The approach used to integrate these different sources of information is based on Bayesian inference methods, and shares strong similarities with state-of-the-art approaches for the early detection of cancer. The aim of this project is to make this approach available to the anti-doping authorities for better decision making with any marker of doping.

Main Findings

The application called “Athlete Biological Passport Software” (ABP software) has been created. The ABP software is made freely available to any antidoping organization. The software has been distributed to some anti-doping organizations (sports federations, national anti-doping agencies, WADA accredited laboratories) and to the experts in charge of the evaluation of the haematological profiles during the ABP pilot project. The ABP software is divided into five main sections, with each section accessible through a dedicated data sheet: Athlete, Haematology, Endocrinology, Models, Results.

Publications

1. A forensic approach to the interpretation of blood doping markers. Sottas PE, Robinson N, Niggli O, Saugy M. Law, Probability and Risk 7:191-210 2008.

2. Les marqueurs indirects du dopage sanguin. Sottas PE, Robinson N, Saugy M. Revue Francophone des Laboratoires 401 : 27-38 2008.

3. The Athlete’s biological passport and indirect markers of blood doping. Sottas PE, Robinson N, Saugy M., In press in the Handbook of Experimental Pharmacology