Projets de recherche

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

Code: 06B14TK

Numerous genetic and/or pharmacological strategies exist that can increase muscle mass and strength. Many were developed to treat muscle diseases such as Duchenne’s muscular dystrophy (DMD). Importantly, these strategies have great potential to be abused by elite athletes seeking to gain a (unfair) competitive advantage, since they are, a) currently available and b) not detectable by current anti-doping protocols.

Prime amongst these are strategies for modulating the growth/development factor myostatin (GDF8); a negative regulator of muscle mass. We described an antibody-based blockade strategy to increase muscle strength and reduce muscle damage in the mouse model of DMD (Bogdanovch et al. 2002 Nature). Other blocking strategies have been described since our initial report, including a propeptide-based strategy (Bogdanovch et al. 2005 FASEB J). Of immediate concern regarding doping is the development of the myo-29 “humanized” myostatin-blocking antibodies by Wyeth, that are currently in human Phase II trials.

Currently, tests do not exist to detect myostatin blockade based ‘doping’ with antibodies, propeptides, or other reagents, underscores the need for rapidly developing a test to detect ‘doping’ not just the currently described reagents but also those that would be generated in the near future, including the use of gene doping approaches RNAi.

Thus, the challenge is to develop a standardized detection test that would be specific, sensitive and standardized enough to hold up to legal challenges that anti-doping agencies would almost certainly face by athletes caught using these tests. These challenges are not insurmountable; we propose to develop a robust and standardized assay for ‘total myostatin activity’ to serve this need.

Main Findings

The overall aims of this project are to develop tests to detect myostatin based 'doping' (blockade) and precluding abuse by elite athletes seeking to gain a (unfair) competitive advantage using myostatin-based doping. We have made excellent progress toward completion of this project and have cloned and developed the WADA-myostatin CAGA assay to serve as doping detection tests for this purpose. This luciferase-based assay emits light proportional to total myostatin activity and is used widely in research and industrial labs. We have developed two assays one in the C2C12 muscle cell line and one in the easier to grow HEK cells, hence it would be easy and efficient to transfer to WADA testing laboratories worldwide. It is important to point out that in myostatin-blockade strategies relevant for doping myostatin levels themselves are not altered rather the ability to activate the receptor or ‘activity’ is altered. Hence for detection of myostatin blockade in athletes the activity-based assay we developed i.e. the “WADA myostatin CAGA assay” will detect doping, however, assays based on detecting the molecule itself will not be useful, irrespective of their sensitivity. Technical analyses of the WADA-myostatin CAGA assay have been completed in our laboratory and the test is robust and valid based on the Z’ scores we obtained. We have also treated wild type mice and mdx mice with different myostatin inhibitors to obtain serum for validation of the myostatin-doping test if needed. A set of standards has been generated that are suitable for distribution to WADA laboratories worldwide. Given that our results demonstrate that the WADA-myostatin CAGA assay is sensitive, reproducible and robust we believe that the next logical steps would entail single-and double-blind testing of the assay with serum samples from athletes to develop normograms and developing standard operating procedures (SOP’s) for incorporation into current WADA testing protocols.

Code: R06D36WS 

Since midyear 2005 we detect with new mass spectrometric techniques in doping control samples a new unknown metandienone metabolite. First studies indicate that this metabolite can be detected for a longer time period than all other metandienone metabolites. The aim of the study is 1: to identify the structure of this unknown metandienone metabolite, 2: to investigate the excretion kinetics in comparison to other metandienone metabolites, 3: to synthesize reference substances and 4: to implement this substance in existing screening procedures for the detection of doping substances. The analysis of long-term metabolites can improve the fight against doping in the out of competition period in general, because misuse of anabolic steroids can be detected for a longer time.

Main Findings: 

Anabolic-androgenic steroids have been one of the most frequently detected drugs in amateur and professional sport. Doping control laboratories have developed numerous assays enabling the determination of administered drugs and/or their metabolic products that allow retrospectives with respect to pharmacokinetics and excretion profiles of steroids and their metabolites. A new metabolite generated from metandienone has been identified as 17a-methyl- 17b-hydroxymethyl-androst-1,4,13-trien-3-one in excretion study urine samples providing a valuable tool for the long-term detection of metandienone abuse by athletes in sports drug testing. The metabolite was characterized using gas chromatography - (tandem) mass spectrometry, liquid chromatography – tandem mass spectrometry and liquid chromatography – high resolution/high accuracy (tandem) mass spectrometry by characteristic fragmentation patterns representing the intact 3-keto-1,4-diene structure in combination with typical fragment ions substantiating the proposed C/D-ring structure of the steroid metabolite. In addition, structure confirmation was obtained by the analysis of excretion study urine specimens obtained after administration of 17-CD3-labeled Metandienone providing the deuterated analogue to the newly identified metabolite. 17a-Methyl-17bhydroxymethyl-androst-1,4,13-trien-3-one was determined in Metandienone administration study urine specimens up to 19 days after application of a single dose of 5 mg, hence providing a detection period extended by more than one week compared to commonly employed strategies.

Code: T06C05JS 

Non invasing imaging of gene expression will have a great potential usefulness in medecine (identify anomalous expression of gemes related to diseases, verify the result of gene therapy applications, etc) as an alternative to invasive biopsy approaches. Positron emission tomography (PET) and single photon emission computerized tomography (SPECT) technologies appear as the most sensitive imaging tools. A very important field of application of imaging tools. A very important field of application of imaing gene expression will be the prevention of the prohibited misuse of gene therapy in athletes. Some of the more studied approaches for imaging gene expression have been the the imaging of reported genes, introduced purposely through a gene transfer process, or the detection od the expressed protein when ir is an enzyme or a receptor. Neither of these characteristics take place in doped athletes, where the hormone (EPO, IGF-I, GH) is not an enzyme/receptor and where the practice is purposely attempted to be masked. An alternative of wider applicability for imaging gene expression is the detection of the actual outcome of transfection, that is, the mRNA being formed in unusual tissues after the gene transfer process. This approach is independent of the construct and the vector used for gene transfer and is applicable to any gene transfected to tissues not usually expressing the protein, such is muscle for EPO. The imaging of mRNA may be carried out by hybridization in the tissues of the mRNA molecules with suitable antisens oligonucleotides probes labeled for PET or SPECT detection. Some of the common problems when designing oligonucleotides for this purpose is the difficulty the may have in entering inro the cell in vivoand the stability of the mRNA because of the potential activation of RNAse H by the probe. In rhis regards, the recently developed Peptide Nucleic Acids (PNAs)  appear as one of the best alternatives to other obligonucleotides such as phosporothioates, 2'-O-methyl RNAs or 2'-fluoro-arabino nucleic acids. PNAs are expected to have high stability, strong hybridizinf properties, appropriate pharmacokinetic characteristics and the possibility to incorporate cell-penetrating peptides to allow cell entrance and accommodate positron or single photon emitting atoms (EG. 18f 11C, 123I) by relatively simple chemical treatments. In spite of all these advantages , the field of imaging gene expression by hybridization of mRNA by labeled obligonucleotides and analogs is not yet completely developed, and it is considered that a pilot project in animals (mice) is required before a more definitive effort be carried out for doping prevention. Accordingly, the pilot project proposed, addressed to image the presence of transfected EPO genes into muscle of mise, will follow the following steps: a) selection of target EPO-mRNA sequences suitable for hybridization b) synthesis of the anisense PNAs incorporating cell-penetrating peptides c) in vitro verification of the stability and hibridizinf properties of synthesized PNAs. d) labeling pf PNAs with PET ans SPECT emission atoms. E) transfer of EPO genes into mice muscle by electroporation. f) verification of successful EPO gene transfer and expression in trasfected mice g) imaging og gene expression in vivo by PET and/or SPECT after labeled-PNAs administration. h) conclusions and recommendations for futher steps.

Main Findings: 

The project IMAGENE (Non invasive molecular imaging of gene expression useful for doping control: Pilot study in animals after erythropoietin gene transfer) was approved by WADA on October 2004 for an initial period of one year. The results of this first period were presented to the WADA Committe on Gene Doping in Stockolm on December 2005. As a consequence of the recommendations of the meeting, an extension of the project was approved on March 2006. In this final report, an update of the overall project is presented. The hypothesis underlying the project was based on several assumptions. The first was that most of gene transfer processes produce the expression of an mRNA for the target hormone-protein in unusual cells or tissues. The second was that these mRNA molecules will hybridize with suitable antisense modified oligonucleotides such as PNAs introduced in the tissues expressing the ectopic hormone-protein. The third was that if a radiolabel of appropriate energy is associated to the modified oligonucleotides, the detection of the unusual hybridization may be carried out non-invasively from the outside of the body by suitable imaging technologies. Thus, the global objective of the project was to develop a pilot study in animals (mice) to verify the hypothesis for further potential extrapolation of the approach to humans in the future. The specific objectives were as follows a) to develop modified oligonucleotides (peptide nucleic acids (PNAs) with cell penetration properties (Tat-PNAs) to hybridize mRNA expressing transfected EPO. b) to label the Tat-PNAs with radiochemicals suitable for radiological external detection. c) to evaluate in vivo the imaging capability (PET/SPECT) in animals expressing EPO in muscle after a gene transfer process d) to develop recommendations for the development of similar procedures for the detection of EPO and other gene transferred doping hormone-proteins in humans. All the objectives of the IMAGENE project (out of PET studies) were accomplished

Code: 06C04WS

Metandienone 18-nor-17β-hydroxymethyl-17α-methyl-androsta-1,4,13-trien-3-one was recently identified as a long-term metabolite in human urine. As the metabolic fate of the D-Ring of other 17-methylated steroids was found to be similar up to now, those steroids may also yield analogue metabolites. The project aims to investigate the excretion of 18-nor-17-hydroxymethyl-17-methyl-androstane derivatives in human urines after the application of different 17-methyl steroids. The structures of the respective metabolites will be confirmed by comparison with reference substances. These compounds will be synthesized and characterized by means of mass spectrometric and NMR techniques. Additionally the kinetics of the excretion will be investigated in order to determine whether these compounds are also suitable as long-term metabolites in doping control.

Main Findings

Following the administration of Bolasterone, Methandriol, 17-Methyl-19-nortestosterone, Mibolerone, Oxandrolone and Stanozolol no analogous metabolites were detected. Synthesis of 17α-hydroxymethyl-17β-methyl-18-norandrosta-1,4,13-trien-3-one from androst4-ene-3,17-dione and its NMR confirmation were successfully performed, allowing the confirmation of the stereochemistry at C-17. Chemical synthesis of 17β-hydroxymethyl-17αmethyl-18-norandrosta-1,4,13-trien-3-one as test compound failed up to now. Hence, no reference material for the new metabolites is available.

Code: R06A03MS

In spring 2005, 5 WADA-accredited laboratories participated in a quality test for homologous blood transfusion detection organized and funded by the Lausanne laboratory. Since then, other laboratories have implemented the methodology to detect homologous blood transfusion. Another test to check educational levels of all laboratories using this methodology should be organized. As in 2005, blood samples will be preapred by the Transfusion Medicine Unit. This Unit is ISO17025 accredited, has very strong links with the Red Cross Blood Bank of the University Hospital and is directed by a physician deeply engaged in research concerning blood transfusion and internationally recognized for their scientific knowledge. This sample preparation is totally outside of any anti-doping laboratory, permitting to have a double-blind study. The Lausanne laboratory role is to send the samples to all WADA-accredited laboratories, to collect and to analyze all data furnished by the laboratories. A final report in collaboration with WADA will be written by the Lausanne laboratory.

Main Findings

As part of the WADA educational test, during the week of August 6, 2007 five (5) stabilised blood samples were distributed to the eight (8) WADA-accredited laboratories which had indicated their capability to perform homologous blood transfusion anti-doping analyses. The laboratories were asked to submit the results of this educational test. By December 7, 2007 all answers were received. All laboratories correctly applied the flow cytometry methodology to analyse the samples.

Code: 06A06JS

Homologous blood transfusion is at present detectable by flow cytometry methodology applied to blood samples. Nevertheless, blood sampling for antidoping control is carried out only in some selected situations and in no way can be considered a universal screening approach. The existence of a methodology applicable to urine to suspect of potential blood transfusion will be of enormous benefit for the fight against doping. In the other hand, autologous blood transfusion remains elusive to detection. However, if evidence of blood transfusion of any type could be obtained but signs of homologous transfusion be absent, the conclusion of autologous blood transfusion would be a direct outcome. The present project aims at developing screening and confirmatory methods able to detect a blood or red blood cells transfusion process to a given individual. The screening approach is based on the search in urine of plasticizers known to be leaked from blood bags towards the liquid and solid portions of bags contents and to be incorporated to donor’s body after transfusion. The presence of unusually high amounts of metabolites of plasticizers in urine would alert on the use of a potential prohibited method. Subsequent blood analysis could confirm the transfusion process by different approaches. As an extension of the screening procedure, unchanged plasticizer in plasma and red cells membranes would confirm the screening finding. Also residues of solvents used for cryogenic storage of red blood cell will be investigated for that kind of blood preservation. Additionally, blood analysis will allow to ascertain the presence of aged red blood cells deteriorated during the blood storage process. Accordingly, several markers known to be related to red cells aging will be studied by flow cytometry of red blood cells and those more sensitive be proposed as physiological confirmatory measurements. In conjunction with the present flow cytometry method to detect homologous blood transfusion, discrimination of type of transfusion appears feasible. In summary, screening and confirmatory methodologies of general application to detect blood transfusion appear feasible to be obtained through the present project.

Main Findings

Results recently obtained in the project suggest us that DEHP metabolites could be specific markers for the detection of transfusions (homologous or autologous). An extraction and detection method has been developed for quantitative determination of DEHP metabolites in urine. Liquid-liquid extraction has been used and the compounds were unequivocally and efficiently detected by UPLC MS/MS in very widely concentration range. Much higher concentrations of DEHP metabolites have been detected in samples of transfused individuals compared to the general population. The majority of the samples analyzed from sportsmen behave as non exposed subjects. In summary, the screening methodology proposed and the results obtained indicate that the procedure, to be carried out primarily in urine, could be applicable to all urine samples submitted to doping control. Thus, the approach is a promising tool for detecting of the use of blood transfusions (autologous, homologous) in sportsmen, being a fast and efficient method.

Publications

1. Monfort N., Ventura R., Latorre A., Belalcazar V., López M., Segura J., Phthalate exposure: new clue to suspect illicit blood transfusion, Recent advances in Doping Analysis, 2010, 17:147

2. Monfort N., Ventura R., Latorre A., Belelcazar V., López M., Segura J., Urinary di-(2ethylhexyl)phthalate metabolites in athletes as screening measure for illicit blood doping: a comparison study with patients receiving blood transfusion, Transfusion, 2010, 50:145-149