Projets de recherche

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

Code: 06C05WS

Boldenone is a well knownanabolic steroid mainly used in cattle mast or equine doping. Although its not clinical approved for humans being, some athletes have been positive for boldenone abuse in the recent years and have been punished for their delinquency. More than then years ago the suspicion raised that some individuals have the ability to produce trace amounts of boldenone endogenously. This suspicion have never cleaarly been confirmed because there never was the ability to distinguis between endogenous or exogenous boldenone. The method of choice in this situatiopn is the measurement of the ratio of the two stable carbon isotope 12C and 13C by gas chromatograohy/combustion/isotope ratio mass spectrmetry (GC/C/IRMS). The method is employed routinely to doscriminate endogenous from exogenous testosterone. Unfortunately it requires minimum amounts of carbon that are corresponding to about 10 to 100 ng per compound. So there was not enough boldenone in the specimens for valid measurement. Nowadays it is possible to measure the C13/C12-ratio of trace amounts of anabolic steroids, if a high grade of purification with a concomitant high recovery is achieved. The aim of this project will be development of a suitable method belonging to these requirements.

Main findings

The described method enables the measurement of d13C values of urine samples containing low amounts of Bo and BM1. The LOD so far is 2 ng/mL, by using larger specimens it might be even lower. With this tool it is possible to elucidate the origin of these steroids and to distinguish between an endogenous Bo-production and an intake of prohibited substances. As there is the possibility for artificial Bo with δ13C values close to the endogenous ones, this is not a clear proof for endogenous Bo-production, but it can be taken as a hint for this kind of abnormal steroid metabolism. Further preparations will have to be analysed in order to minimize the chance of Bo-preparations with a d13C values near to the ERC.

Code: T06C01AF

Sildenafil (Viagra®) has been reported to improve exercise performance at high altitude (>3,800 m) in a subset of athletes by greater than 35%. At altitude, exercise capacity is reduced because lower oxygen pressures result in decreased delivery of oxygen to the tissues. Altitude induced performance decrements can be exacerbated by the constriction of lung vessels (pulmonary hypertension) that can occur in some individuals causing reduced cardiac output (blood pumped from the heart per minute) and further impaired gas exchange in the lungs. Sildenafil is thought to improve performance at altitude by relaxing the pulmonary vessels thereby reducing pulmonary arterial pressure, increasing cardiac output, improving gas exchange, and allowing enhanced oxygen delivery during exercise. It is currently unknown whether sildenafil could improve performance at more moderate elevations relevant to Olympic competition. Therefore, the primary objective of this investigation is to determine whether sildenafil enhances athletic performance in men and women at moderate elevations. In part one, competitive male and female cyclists will undergo two cycling exercise tests (each with a set workload and time trial component) while breathing hypoxic gas simulating an altitude of ≈3,900 m following ingestion of either placebo or sildenafil. Subjects who demonstrate drug induced benefits in time trial performance at 3,900 m will then perform similar paired exercise performance tests at 1,500 m, 2,100 m, and 2,700 m to determine whether the benefits are also present at lower elevations. In study 2, athletes will be flown to an elevation of 2,200 m. On days 4 and 5, they will perform 2 exercise tests (one each day) with either placebo or sildenafil. On day 7, subjects will be driven to 4,300 m where they will again undergo paired performance tests on days 4 and 5. Thus, the two studies will determine the efficacy of sildenafil as an ergogenic aid at moderate elevation both acutely and in partially acclimatized athletes.

Main Findings

Sildenafil had little influence on cardiovascular hemodynamics for either gender at MA or HA, but did result in higher SaO2 values compared to placebo during steady state and time trial exercise in men at HA only. Sildenafil did not affect Wpeak or 6-km time trial performance in either gender at HA or 15-km time trial performance in either gender at MA or HA.

Conclusions:

The efficacy of sildenafil during exercise at altitude is integrally related to its ability to improve oxygen delivery by increasing SaO2 and/or cardiac output. The magnitude of these effects appears to be dictated by the severity of the altitude and associated hypoxia as well as individual susceptibility to hypoxic pulmonary vasoconstriction. Sildenafil is unlikely to exert beneficial effects in oxygen delivery or exercise performance at altitudes < 4000 m for the vast majority of the endurance trained men or women.

Publications

Kressler, J, Stoutenberg M, Roos B, Friedlander AL, Viskochil R, Jacobs KA. Sildenafil does not improve exercise performance during acute hypoxia in trained men or women. Medicine and Science in Sports and Exercise (Supplement), 41: S130, 2009.

Jacobs KA, Stoutenberg M, Kressler J, Roos B, Friedlander AL. Trained women demonstrate greater preservation of peak exercise capacity during acute hypoxia than trained men. Medicine and Science in Sports and Exercise (Supplement), 41: S374, 2009.

Code: 06D04MF

The urinary testosterone/epitestosterone (T/E) ratio determination, based on quantification of testosterone glucuronide and epitestosterone glucuronide in urine, is a major tool in attempts to expose illegal use of exogenous testosterone by sportsmen. Nonetheless, the metabolic processes that determine the urinary concentration of testosterone and epitestosterone are complex and not fully understood. In particular, little is currently known how the T/E ratio is affected by genetic variations in genes that encode enzymes and transporters that are involved in the metabolism and secretion of these steroids. It is important to gain deeper knowledge on the metabolism of both testosterone and epitestosterone since changes in either system would affect the all-important T/E ratio. In addition, it is essential to further develop the doping test methods for the detection of anabolic steroids abuse in order to make them less sensitive to genetic polymorphism among the athletes. Glucuronidation is the major conjugation pathway of anabolic steroids in humans and changes in the activity or the expression level of the UDP-glucuronosyltransferases (UGTs) that are directly involved in testosterone or epitestosterone glucuronidation could significantly change the T/E ratio. A part of the proposed research will therefore be dedicated to close examination of the glucuronidation of anabolic steroids by the human UGTs, including possible effects of genetic variability (polymorphism) on the glucuronidation of testosterone and epitestosterone. The second major section of the proposed research will concentrate on the urinary concentrations of several steroid metabolites in order to provide a better picture of possible abuse of testosterone or its prohormones, even by athletes that carry mutations within their UGTs, or in other proteins that take part in the metabolism and secretion of testosterone and/or epitestosterone. The combined results are expected to provide valuable information that could significantly improve the fight against doping, while lowering the risk of “false positives”.

Main Findings

The main conclusions of our studies within this WADA supported project are the following:

1. Examination of all the 19 human UGTs of subfamilies 1A, 2A and 2B revealed that UGT2B17 is by far the main contributor to testosterone glucuronidation in man. This result provides the explanation for the previously observed effect of UGT2B17 deletion on the T/E.

2. Examination of all the 19 human UGTs of subfamilies 1A, 2A and 2B revealed that UGT2B7 is by far the main contributor to epitestosterone glucuronidation in man.

3. Both UGT2B7 and UGT2B17 are the major contributors to the glucuronidation of androsterone as well as etiocholanolone, even if UGT2B17 has significantly higher affinity and higher turnover rate with etiocholanolone.

4. UGT2B7 is the main contributor to the glucuronidation of 5α-diol on the 3-OH, whereas UGT2B15 and UGT2B17 are mainly responsible for its glucuronidation on the 17-OH.

5. UGT2B17 is the main contributor to the glucuronidation of 5β-diol, primarily on the 17-OH.

6. UGT2A1 has the capacity for glucuronidating testosterone, epitestosterone, and etiocholanolone at high rate, but it probably does not contribute noticeably to the level of androgen glucuronides in the urine due to its limited expression.

7. Low levels of urinary testosterone glucuronide concentration are not compensated for by higher level of testosterone sulfate.

8. Low levels of urinary epitestosterone glucuronide, not high levels of testosterone glucuronide, are the main reason for high T/E in athletes that have not used exogenous testosterone.

9. In an effort to develop a system for detecting possible abuse of exogenous testosterone by sportsmen that will not be dependent on the activity of UGT2B17, we suggest to concentrate on 3-glucuronide of 5α-diol. Another possible target molecule from this point of view is androsterone glucuronide.

Publications

Taina Sten, Ingo Bichlmaier, Tiia Kuuranne, Antti Leinonen, Jari Yli-Kauhaluoma, Moshe Finel. UDP-Glucuronosyltransferases (UGTs) 2B7 and UGT2B17 Display Converse Specificity in Testosterone and Epitestosterone Glucuronidation, whereas UGT2A1 Conjugates Both Androgens Similarly. Drug Metabol Disp (2009) 37:417–423

Code: R06B02AE

The research group of M. Bidlingmaier, C. Strasburger and Z. Wu has demonstrated the detection of administered recombinant growth hormone (hGH) based on the "differential immunoassay approach". This approach is based on the measurement of the GH concentration in human serum samples by 2 different assays employing specific monoclonal antibodies. This test has been implemented in WADA accredited laboratories as research test system based on ELISA technique. The aim of this project is to adapt this test to the chemiluminescence technique which is much more sensitive and robust than other detection systems, to optimize the functional assay sensitivity and to determine the technical assay characteristics. The successful development is of crucial importance for the availability of commercial test kits.

Main Findings

1. The assay technical characteristics (Sensitivity, intra- and inter-assay variability, accuracy, etc) are significantly improved with respect to the previous assay platform. This allows for quantification of almost all samples analysed.

2. Four different reference populations were considered for the determination of assay cut-off ratios. These populations included i) samples from a demographic study on elite athletes (5 major ethnic groups, i.e. Caucasians, East Asians, Australian aborigines, Africans and other ethnicities; from 12 different countries and 10 major sports disciplines); ii) samples from baseline measurements of a GH-administration study on recreational athletes; iii) samples from a blood bank (all Caucasians) and iv) samples from elite athletes (ethnicity unknown).