Projets de recherche

Code: 07A14MA

Despite a committed antidoping movement, abuse of recombinant human erythropoietin (rHuEPO) continues. Clandestine use extends to the use of novel rHuEPO products in the hope these may be undetectable via the electrophoresis test which distinguishes endogenous EPO from the synthetic versions based on different electric charges on the respective molecules. Recombinant erythropoietins are the number one revenue-generating class of biological products on the market, with worldwide sales exceeding US $12 billion. The lapse of European patents in 2004 has opened the door for approval of biosimilar versions of rHuEPO, and multiple pharmaceutical companies are now embarked on the development of viable alternatives for this lucrative market. In order to maintain an effective screening process it is vital for laboratories to obtain pre-emptive information regarding the structure and detectability of these novel anaemia drugs. Several classes of products are being developed: biosimilar rHuEPOs (same molecular structure but produced using different procedures), long lasting EPOs (molecule modified to extend half-life in circulation), synthetic EPO receptor agonists (completely different molecular structure which nonetheless stimulates the EPO receptor) and EPO replacements (stimulate red cell production via non-EPO receptor pathways). The aim of this research is to validate the capacity for isoelectric focussing to discriminate between novel anaemia drugs and endogenous EPO, and where this proves impractical to develop new detection methodologies. We will enter collaborative arrangements with the pharmaceutical companies to seek access to their products prior to commercial release in order to thwart attempts to use novel drugs before an appropriate test has been introduced by antidoping authorities. The drugs will be screened at the WADA-accredited laboratory in Paris who developed the isoelectric test and are world leaders in its application to doping control.

Main Findings

An extensive network including pharmaceutical company representatives and their expert consultants was activated in order to identify and detect novel anaemia drugs. A comprehensive market appraisal and de novo interrogation of literature and websites dramatically expanded upon the initial estimate made in 2007 that around one dozen copy EPOs were available. The most recent estimate suggests this number to be in excess of 100 in 2010. A significant number of these products have been procured and analysed by an anti-doping laboratory; several unusual profiles, and in a small number of cases inexplicable characteristics, have been reported. An important emphasis of the project has been to facilitate collaboration between pharmaceutical manufacturers and the WADA. Introductions were made to different pharmaceutical and biotechnology companies which enabled WADA to commence discussions and in several cases in-depth collaboration to develop detection methods for novel ESAs considered of high interest. In 2009 the ESA industry experienced considerable upheaval following the publication of results (‘TREAT” study) indicating that administration of exogenous EPO posed previously unrecognised health risks. Subsequently it is anticipated that substantially greater research emphasis will be placed in the foreseeable future on development of novel ESA agents.

Code: 07C17JD

The project deals with the developpment of immunological methods for the screening and the confirmation analysis of tetracosactide in serum. In vitro and in vivo immunological studies will be performed to develop immunological tools and their performance will be compared to that of a spectrophysical method using a Q trap instrument. Immunoextraction from serum prior to immunological analysis will be scheduled for confirmation analysis but not for screening analysis. The aim is the specific detection of tetracosactide in serum at concentration level as low as 100 pg/ml or less in order to offer a relevant detection window.

Main Findings

Tetracosactide is a synthetic peptide which exercises the same biological effects as endogenous Adreno CorticoTropin Hormone (ACTH) produced by anterior pituitary gland. Its structure reproduces the first 24 amino acids of ACTH which is composed of 39 ones. A screening method by ELISA was developed and validated for detection of Tetracosactide in plasma. The principle of the method was to use an ELISA kit (Enzyme Immunossay Kit from Peninsula Laboratories, San Carlos, USA), that reacts with the part 1-24 of ACTH (and thus Tetracosactide), after total elimination of endogenous ACTH from the tested plasma samples. In such conditions, ELISA became specific for Tetracosactide. The sample preparation consisted in removing most of the proteins and any trace of ACTH from plasma. This was achieved by cation-exchange chromatography (CM Sephadex ®). The preanalytical conditions were shown to be very important. Only EDTA plasma samples must be analysed and if the plasma samples could not be frozen just after collection, it was essential to quickly ship them to the laboratory in refrigerated conditions. The detection limit of the method was 13 pg/mL. In parallel, a confirmation method by LC-MS/MS (Thermo) Triple Quad, ESI was developed and validated. The preparation of plasma included cation exchange chromatography (CM Sephadex ®) and solid-phase extraction (Oasis® HLB). The detection limit of the method was < 50 pg/mL.

Code: T07D01RB 

Many studies are being carried out to develop methods for identifying exposure to growth factors such as human growth hormone, IGF1 and other related factors with potential application in sport doping. A number of these studies are based on the hypothesis that identification of genes and their protein products abnormally expressed in tissues treated with these factors can provide definitive evidence – i.e., signatures - for exposure to such growth factors. A number of related parallel studies are being conducted under the sponsorship of WADA and an enormous amount of information is being accumulated. However, to date no concerted effort has been made to collate and compare these large data sets and results to identify factors common to these studies that would represent the markers most likely to be informative in a drug testing setting. We propose to carry out a pilot informatics study to compare results from several completed and on-going WADA-sponsored studies.

Main Findings: 

Most of the current methods to detect doping rely on the use of chemical tests to identify drugs in the body. These methods are very powerful and often highly effective. Recent advances have enabled scientists to develop entirely new and potentially even more effective kinds of tests that are based on the detection of the broad changes that drugs cause in the genes and the proteins of affected tissues. These new methods can examine the ways in which all of the 25,000 human genes are affected by exposure to drugs and how those changes in genes affect the content of the even larger number of proteins in human cells. WADA has mounted a major research effort to use these approaches to develop detection methods for doping and is supporting work in more than a dozen laboratories scattered around the world aimed at finding genetic and protein changes in blood, urine and other body fluids and tissues that can definitively identify exposure to given drugs. Very promising results are coming from this effort and potentially useful "signature" gene and protein changes are being identified. A feature of these approaches is that they produce enormous amounts of information that require unified methods of analysis that are generally beyond the computing and informatics capabilities of individual laboratories. To ensure that the data are being evaluated in a consistent and effective manner, WADA has established a centralized bioinformatics laboratory that uses the most modern computational and analytical methods to harmonize the results of all the separate research laboratories to ensure that tests coming from this approach are highly sensitive, accurate and specific. The facility has succeeded in establishing an effective infrastructure and operating procedures and is beginning to obtain and analyze data from the research laboratories. WADA is confident that this more unified and centralized approach to doping detection will lead to far more effective tests that are rigorous, accurate and sensitive and that provide an effective and fair approach to protect athletes and Sport from doping.

Code: 06B06MG

This is a highly interdisciplinary and coordinated project that is aimed at understanding the molecular modifications induced by prolonged IGF-1 gene expression in the skeletal muscle after viral mediated gene transfer in rodent animals. The project will exploit the availability of AAV vectors expressing different IGF-1 isoforms to transduce skeletal muscles, a unique system that permites the long-term evaluation of the effects induced by the growth factor in vivo. In the treated animals, the signatures of these modifications will be analyzed by detecting the presence of the delivered transgenes in serum by quantitative real-time PCR and by analyzing the modifications of the proteomic pattern in muscle by advanced proteomics and mass spectrometry. These studies will be complemented by a parallel evaluation of the proteomic modifications induced by IGF-1 gene transfer in skeletal muscle satellite cells in vitro.

The project will be divided into three tasks.

Task 1. The overall purpose of this task will be the identification of a proteomic signature of IGF-1 gene transfer in muscle cells. The project will involve the development of animal models for AAV-mediated IGF-1 gene transfer. In particular, viral vectors expressing different IGF-1 isoforms will be produced and used to inoculate the tibialis anterior and femoral quadriceps muscles of mice and rats. At different times after transduction, proteomic analysis of the transduced muscles will be performed. These studies will take advantage of state-of-the-art technology 2-DE DIGE and will have the ultimate purpose of identifying protein patterns specific to the IGF-1 expressing muscles. These studies will be paralleled by the analysis of proteomic changes in human skeletal myoblasts after ex vivo gene transfer of the IGF-1 cDNAs.

Task 2. This task is aimed at the identification of novel peptides markers that might be exploited for anti-doping purposes. In particular, the project is aimed at the indentifications, quantification and qualitative assessment of the proteins differential expressed in muscle transduced with IGF-1. Differentially expressed or modified proteins will be analyzed using advanced mass spectrometry instrumentation, including MALDI-TOF/TOF, ESI-linear quarupole ion trap and high resolution FT-MS instrumentation.

Task 3. This task is aimed at the assessment of gene doping by monitoring the presence of exogenous gene fragments in animal serum. This possibility is based on different experimental and clinical reports that indicate that muscle exercise determines signficant damage to muscle cells, with the release of intracellular content into the bloodstream. At different times after in vivo IGF-1 gene transfer, both in resting conditions and during muscle exercise, serum will be analyzed for the presence of promoter-, vectore backbone- or cDNA-specific gene fragments by TaqMan-based Real Time quantitative PCR.

Main findings

The main findings are not available due to the sensitivity of the information and results developed in this project.

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

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

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

Description à venir.

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

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

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.