Projets de recherche

Code: 13D34PV 

In the recent years, the use of performance-enhancing peptides has become a growing issue in human as well as in equine sports drug testing. A product called TB-500, claimed to increase muscle growth and tissue repair in horses and other mammals, is available on the Internet and officially distributed.  Anecdotal use not only in horse racing, but also in human sport has been documented, reaching also worldwide attention from the media. The active content has been identified as the N-terminal acetylated 17-23 fragment of thymosin beta4 (Ac-LKKTETQ) by our group.  
Detection of the intact TB-500 and several metabolites has been achieved in horse urine and plasma, but no data are available for detectability in human. It has already been demonstrated that in vitro/ex vivo studies can be used to obtain reliable indication on the metabolism of small peptides.   
The aims of the project are therefore to 1) characterize human metabolism of TB-500 by using human liver microsomes and human S9 fraction (for in vitro studies) and human plasma/serum (ex vivo studies); 2) synthesize appropriate amounts of certified reference standards of TB-500 and its most representative metabolites by solid-phase synthesis; 3) determination of the detection limits of the relevant metabolites and implementation of the metabolites into peptide-screening methods. The synthesized reference standards will be distributed to other human doping control laboratories.

Main Findings: 

A product called TB-500, claimed to increase muscle growth and tissue repair in horses and other mammals, is available on the internet and officially distributed. It is presented as “the synthetic peptide of the active region of thymosin-beta 4 (Tβ4)”, without any further qualitative description such as amino acid sequence or molecular weight.  
In this project the first goal was to characterize human metabolism of TB-500 by using human liver microsomes and human S9 fraction (for in vitro studies) and human plasma/serum (ex vivo studies). Results of this study show that TB-500 showed serial cleavage at the C-terminus, whereas acetylation of the leucine seemed to provide efficient protection of the N-terminus. Results were similar to those described by Ho et al. in the horse using horse liver homogenate as in vitro model. 
 In a second phase of the project, appropriate amounts of 3 metabolites TB-500 M(1-2), TB-500 M(1-3) and TB-500 M(1-5) observed in the first part of the project were synthesized using typical Fluorenylmethyloxycarbonyl (FMoC)-synthesis strategy. As proposed, the synthesized reference standards were distributed to other human doping control laboratories. Besides the synthesis of the metabolites, a heavy version of the TB-500 (TB-500-d3) was synthesized for use as internal standard. In a final step of the project, the LODs of the synthesized metabolites were determined and implemented into an in-house screening method for the detection of peptides in urine. LODs were 500 pg/mL for TB-500 M(1-2), 100 pg/mL for TB-500 M(1-3) and 50 pg/mL for TB-500 M(1-5).

Description à venir.

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

Description à venir.

Karine Corrion, France

Dr. Andrew Levy, UK

Ce projet de recherche a été complété et n'est actuellement disponible qu'en anglais.

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

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.