Projets de recherche

Code: 13D14MM 

In the doping control field, the concept of urinary steroid profiling was first introduced in the '80s by Donike et al. to reveal the misuse of testosterone and its precursors. The kinetics of excretion in urine of the target compounds that are part of the steroid profile may be affected by various factors, primarily among them sex, age, ethnicity, physical activity, diet, alcohol consumption, as well as other physiological or pathological conditions. We are focusing our attention on another possible cause of alteration of the urinary steroid profile, that is on the consequences of drug-drug interactions. Examples of agents affecting the urinary steroid profile that are included in the WADA list of banned substances and methods are mainly represented by the masking agent probenecid and by the whole class of the aromatase inhibitors. In addition to those drugs, several classes of compounds not included in the WADA list have been reported to play a significant role on testosterone synthesis and metabolism. An example is represented by the antifungal ketoconazole that acts as inhibitor of the cytochrome P-450-dependent enzymes involved in the hydroxylation during steroid hormone synthesis and in the oxidative/reductive reactions during steroid hormone phase I metabolism. 
Even if the effects of ketoconazole on the steroids hormone synthesis are well known, very little information is available about its impact on the effectiveness of the screening and confirmation strategies routinely followed by the WADA-accredited antidoping laboratories to detect the abuse of androgenic anabolic steroids that are normally present in the human body. This project aims to evaluate the alterations caused by the intake of azole antifungal drugs and to verify whether these effects could in some way affect the reliability and the accuracy of the analytical strategies currently followed to detect doping by testosterone and precursors.

Main Findings: 

The objective of this project was to investigate the effects of azole antifungals administration on the strategies currently adopted by the anti-doping laboratories to report an adverse analytical finding for testosterone related steroids. More specifically, we have considered the effects of the administration of miconazole and fluconazole by different routes and doses on the physiological circadian fluctuation of both the parameters reported in the TD2016EAAS and the endogenous reference compounds reported in the TD2016IRMS. In the first part of this project, the circadian fluctuations of the parameters of the steroid profile selected were evaluated in four male Caucasian subjects for at least five days, and basal ranges of each parameter in each subject were established. These basal ranges were then utilized to evaluate the potential effects of azole antifungals administration on the determination of the parameters selected to detect doping by testosterone related compounds. Finally analytical methods to screen and confirm azole antifungals in human urine were set up and validated. 
The results obtained show that both miconazole and fluconazole are able to alter significantly the key parameters of the steroid profile. More in details, the imidazole antifungal miconazole causes after oral and buccal administration (i) a significant increase of the 5α-androstan-3α,17β-diol/5β-androstan-3α,17β-diol ratio, (ii) a significant decrease of androsterone, etiocholanolone, androsterone/etiocholanolone ratio, androsterone/testosterone ratio, and 5α-androstan-3α,17β-diol/epitestosterone and (iv) a moderate decrease of the 5β-androstan-3α,17β-diol and 5α-androstan-3α,17β-diol. Limited effects were instead registered after dermal intake. These finding can be primarily explained by the ability of miconazole and its metabolites in altering the kinetic/efficacy of de-glucuronidation of the endogenous steroids by β-glucuronidase. The oral intake of the triazole antifungal fluconazole, instead, increase the urinary levels of androsterone, etiocholanolone, androsterone/etiocholanolone ratio, androsterone/testosterone ratio, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, 5α-androstan-3α,17β-diol/5β-androstan-3α,17β-diol ratio, and 5α-androstan-3α,17β-diol/epitestosterone ratio. This finding is not linked to the efficacy of the enzymatic hydrolysis by β-glucuronidase, but might be explaining considering the putative anti-aromatase activity of the triazole antifungal agents. 
These evidences underline the importance to screen for the azole antifungals in the athlete urines collected in occasion of doping control test, to reduce the risk of reporting uncorrected results.

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.

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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.

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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.