Projets de recherche

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Description à venir.

Karine Corrion, France

Dr. Andrew Levy, UK

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Code: 13D08AR 

Our research program encompasses projects designed to investigate how the use of drugs (emergency pills and androgens) and genetic variation interfere with the Athletes Biological Passport (ABP). ABP is essential to detect doping with anabolic agents, especially with the decreasing doses evidently used by some athletes. However, there are many factors that may contribute to the inter-individual variability in steroid profiles, i.e. drug use, diseases, genetic variation etc. Preliminary results indicate that the use of an emergency pill influences the steroid profile and confounds the test interpretation. We will therefore investigate how the use of emergency pills affects the excretion of steroids in females. We and others have shown that UGT2B17 genetic polymorphism has a large impact on testosterone doping test. We will continue to evaluate the use of genetic testing of UGT2B17 as well as other polymorphisms in sport samples. Finally we will study how androgens affects the haematological module. 
It is of great importance that the athletes ABP will be able to compensate for all possible variability in longitudinal steroid profiles. More knowledge is therefore needed about how drug use and genetic variation may affect the ABP and hence outcome of doping tests.

Main Findings: 

Study 1: We show that DNA extracted from urine samples is not good enough for individual genetic testing in anti-doping work. We showed that UGT2B17 ins/ins men exert higher T/E than ins/ins females and that some del/del athletes (particularly men in power sports) had T/E ˃0.4. The CYP17A1, and UGT2B7 SNPs investigated could not explain the large inter-individual variation in urinary concentration of epitestosterone. 
Study 2: We have shown that the ABP ratios various randomly throughout the menstrual cycle, epitestosterone being the only metabolite that are significantly altered. The administration of an EC decreased the urinary concentrations of epitestosterone 24 hours after the intake, and it is possible that a use of EC could result in atypical finding in the ABP. 
Study 3: We conclude that testosterone but not nandrolone mediates significant effects on the hematocrit profile and gene expression of the EPO gene in blood. Moreover, testosterone increases the serum levels of the future ABP biomarker P-III-NP. It may be of importance to know how the different modules interact for the interpretation of the ABP results. We will continue to study these biomarkers in relation to each other, both at baseline and after the administration of different doping agents.  

Code: 13B08RG 

Growth hormone secretagogues (GHS) are molecules that stimulate the secretion of human growth hormone from the pituitary. They have proven to be potent agonists of hGH secretion reaching circulating hGH values that are even not easily achievable with rhGH administrations. As such, they are the prime candidate pharmaceuticals to replace rhGH abuse. GHS display large structural diversity and many molecules are under development by the pharmaceutical industry. In order to address the entire family of known and upcoming molecules it can be targeted what all GHS have in common: the interaction with the GHSR1a receptor. Based on this premise we developed and validated a competition assay using a radiotracer, 125I-ghrelin. Given the difficulties for testing radioactivity at some antidoping laboratories, and the recent implementation of chemiluminescent tube-based immunoassays for hGH we have studied the potential replacement of the radiotracer by a chemiluminescent one. Accordingly, the development of a method using biotinylated ghrelin and streptavidin labeled with a chemiluminescent tag could be effective to achieve this purpose. 
The main objectives of this project were: - Obtention of appropriate chemiluminescent tags bound to strep traviding and synthesis, purification, and charactesation of streptavidin conjugated to acridinium, not commercially available.
- Set-up of a competition assay protocol employing different chemiluminescent tags.
- Comparison of the radiotracer and the chemiluminescent tracers.
- Assessment of the chemiluminescent competition assay in urine samples.

Main findings:

The results submitted in this project showed the development of an efficient chemiluminescent method performed in three different chemiluminescent tags: acridinium (AC), alkaline phosphatase (AP) and horseradish peroxidase (HRP). This method, based in the interaction between biotinylated ghrelin and streptavidin labeled with a chemiluminescent tag, was evaluated following different steps. After evaluating biotinylated ghrelin in a competition binding assay, the Ki obtained was compared with the Ki values from competition assays performed with ghrelin without any tag, and no interference in the interaction with the receptor was showed. Biotinylated ghrelin appeared then as a suitable ligand. 
Subsequently, the optimum time of addition of streptavidin was tested to avoid any effect on the recognition of biotinylated ghrelin by the receptor,and it was determined its addition 40 min after of the starting of the competition assay. After synthesis and purification of streptavidin labeled with AC, the protocol was optimized for this and the other two chemiluminescent tags (streptavidin labelled with alkaline phosphatase AP and horseradish peroxidase HRP, respectively) through the evaluation of several conditions as: number of washes, membranes obtained from different amount of cells, alternative binding buffers, amount of ST per sample, presence or absence of detergent and process changes of adding of the chemiluminescent substrates, among others. The optimal protocols for each label were tested in a competition assay with different amounts of GHRP-2 in binding buffer and urine and the results showed the best sensitivity with HRP tag. In fact, the presence of GHRP-2 in urine samples obtained 1.5 hours after intravenous treatment with 100 μg of GHRP-2 from in a clinical trial with sedentary health volunteers treated was detected. 
In conclusion, the results shown in this project demonstrate the development of a chemiluminescent method based on interaction between biotinylated ghrelin and the GHSR1a receptor, which is more sensitive than other non-radioactive methods described so far but has less sensitivity than the radioactive method described previously by our own group.  

Code: 13D25MT 

The misuse of recombinant human insulin in sport has frequently been mentioned by confessing athletes but is not detectable with currently available methodologies. This is mainly due to the fact that there are no measurable differences to the endogenously produced hormone. Several attempts to establish diagnostic marker ratios to other endogenously produced hormones (C-peptide) failed due to unstable or highly variable conditions in the living organism. With the present study we aim to establish potential marker metabolites, which will uncover a surreptitious insulin application. Pilot studies demonstrated that there are substantial differences in the degradation/metabolism of insulin between the endogenously secreted and the recombinant and injected insulin. It is assumed, that these differences are due to the exposition of insulin to specific enzymes after subcutaneous or intramuscular administration. 
Several urinary metabolites were identified in earlier studies by this research group and an extension of this approach with most modern analytical instruments is planned, potentially revealing new indicative and diagnostic metabolites. Therefore, purification of target compounds from urine followed by the determination of insulin metabolite profiles by LC-MS/MS is planned, which might serve as diagnostic tool to uncover the misuse of recombinant human insulin. 

Main Findings: 

This study was conducted to explore the metabolic fate of subcutaneously administered recombinant human insulin. Due to the exposure to endogenous proteases in the subcutaneous tissue, a minor amount of the bioactive peptide hormone is cleaved to its truncated metabolite DesB30 human insulin, which still owns the full biological activity. In addition to that, DesB30 is a known by-product of recombinant insulin preparations (at ~ 1%) and, thus, it can enter the circulation from the injection site.  Post administration of recombinant human insulin, circulating DesB30 was identified in plasma samples of diabetic patients (type II). No specimen obtained from healthy volunteers with endogenous insulin analyzed in the context of this study contained DesB30, not even when the production of insulin was stimulated by ingestion of a concentrated glucose solution (OGTT).  The analysis of athletes’ routine doping control samples yielded analytical findings of trace amounts (=LOD) of DesB30 HI in 3 out of 64 samples. The additional monitoring of the respective C-peptide levels yielded inconspicuous results for all athlete samples but, conversely, suppressed C-peptide levels in diabetic individuals after HI administration.  Thus, the detection of DesB30 in athlete blood samples in combination with C-peptide levels represents a potential combination of markers for the misuse of recombinant human insulin in sports. Due to degradation processes, urine specimens were not found suitable for this approach. Furthermore, special care regarding the storage and sampling conditions are crucial for the collected plasma samples and degradation processes must be avoided by applying appropriate conditions during the whole post-sampling period until analysis. This is important for both inhibiting the degradation of HI to DesB30 HI on the one hand and stabilizing the C-peptide of the other hand. 

Code: 13D23SS

Several elite athletes were tested positive for clenbuterol and claimed that these findings were caused by the consumption of meat containing clenbuterol. From several studies and EU monitoring programs it is shown that clenbuterol may be present in meat and after consumption of meat concentrations of clenbuterol in urine can be found. To discriminate between clenbuterol administrated via a pharmaceutical preparation or by ingestion of contaminated meat products research was started in 2012 (WADA 11A18SS). The focus of this study was to determine if there was a difference between the ratio of the two enantiomers (left- and right-- hand form of clenbuterol) in meat, and if after consumption of meat there was a difference between the ratio of the two enantiomers in urine when compared to the ratio in urine after illegal oral administration of preparations to humans. 
In 11A18SS the proof is given that the hypothesis is feasible and the analytical methods are in place and capable to detect the differences.  In the proposed project the focus will be on establishing a relation between the consumption of contaminated meat and a change in ratio of clenbuterol enantiomers in urine via a controlled experiment.  The project will result in a decision model that can be used to assess the source of an adverse analytical finding for clenbuterol.  A new technique focusing on untargeted analyses will also be tested on the acquired samples. This will take into account if there are any other changes in metabolic profiles after both ways of ingestion and will try to discriminate on this basis. This technique is known as metabolomics. 

Main Findings:

The aim of the project was to confirm the proof of principle from a former project with controlled human trials and to build a decision model to be used in sports doping analysis to distinguish AAF for contaminated meat from pharmaceutical preparations. Meat and liver were collected from an animal experiment.  The meat and liver were used for a controlled human trial in which these tissues were consumed by volunteers. Other volunteers ingested a pharmaceutical preparation containing clenbuterol (racemic mixture) or enantio pure clenbuterol. Urine samples of the volunteers were collected for over a week after consumption. All urine samples were analysed using the methods developed in 11A18SS project. The urine samples were also used for untargeted profiling experiments.  
In meat from the animal experiment had S/R clenbuterol ratio of around 1 and 0.9 depending on the animal. This shows that the enrichment of R-clenbuterol in meat of treated animals is not a process that is stable, reproducible and comparable for individual animals and maybe also depends on the concentration administered to the animal. For bovine liver no information on the ratio was available before. Data from bovine liver showed that in liver the S-enantiomer is either enriched or R-clenbuterol very depleted. This is the opposite of the ratio in veal meat. This means that consumption of liver with incurred residues can possibly lead to an opposite ratio in human urine compared to the consumption of veal meat. It was also shown that preparation (cooking and baking) of the incurred meat and liver had little to no effect on the ratio determined prior to preparation.  
In the administration studies a distinction based on the proportion of S-clenbuterol in the human urine samples was possible between those receiving liver (padmin=0.635±0.004) from the other two groups, Spiropent® tablet (padmin=0.499±0.001) respectively meat (padmin=0.509±0.006). A distinction between the volunteers receiving meat and Spiropent® tablets cannot be made based on the enantiomeric composition, due to the reason that the ingested proportions of S-clenbuterol are too close to each other.  
The analysis focusing on potential metabolites of clenbuterol in a targeted non-targeted design predicted a high theoretical number of metabolites/biomarkers. Unfortunately, not many of the predicted metabolites were found in the urine samples of the volunteers. Based on the outcome of the analysis and the statistical processing there is no grouping of the compounds possible at present. There is no underlying mechanism found what could be used to separate the different treatment groups. So it was concluded that it is not possible to use clenbuterol metabolites to discriminate between intentional and unintentional intake of clenbuterol using non-enantiomeric separation.  
Overall it seemed that inter individual differences between elimination kinetics in bovine animals are present. This made setting an absolute threshold or guideline for discrimination not possible. The method developed in the project can be used when there are adverse analytical findings for clenbuterol to obtain additional information. When the S proportion clenbuterol in human urine resembles 0.5 (the S proportion in a pharmaceutical preparation) this is not definite proof of illegal use. However, if the S-proportion is higher than 0.59 this means that in 95% of the cases this is not due to administration of clenbuterol in (racemic) tablet forms.