Projets de recherche

Code: 13C31JC

With the increased capacity to detect the drugs and chemicals used for doping to enhancing performance, more instances of blood transfusion (allogeneic or autologous), termed "blood doping", have been found in different athletic competition. While blood doping methods was prohibited by the International Olympic Committee in the 1980s, there is a lack of direct and reliable methods to detect blood doping, especially from their own blood (autologous blood transfusion (ABT)). Therefore, there is an urgent need for the development of better and novel methods to detect ABT and other blood doping. These blood doping efforts are to increase the number of red blood cells (RBC) to better the oxygen-carrying capacity and athletic performance. We have discovered and characterized the large amount of genetic information in the red blood cells (RBC) that are usually used for the blood doping. We plan to apply genomic technology and advanced bioinformatics to global analyze the changes of the genetic materials of RBC during in vitro storage to identify "gene signatures" of blood storages. These gene signatures will be validated and used to identify individuals who may have received their own stored blood to enhance their athletic performance.

Main Findings:

With the increased capacity to detect the drugs and chemicals used for doping to enhance performance, more instances of blood transfusion (allogeneic or autologous), termed “blood doping”, have been found in different athletic competitions. While blood doping methods were prohibited by the International Olympic Committee in the 1980s, there is a lack of direct and reliable methods to detect autologous blood transfusions (ABT). Therefore, there is an urgent need for the development of better and novel methods to detect ABT and other forms of blood doping. We have previously discovered the presence of large amount of microRNAs in the human mature RBC. These genetic materials offer an unique window into the development history and environmental exposure of the RBC, the main cell types used for blood doping. In our previous works, we have found that the genomic analysis of the RBC microRNAs in sickle cell diseases offer important insights into the heterogeneity of the anemia severity and malaria resistance that are caused by the elevated miR-144 and miR-451, respectively. In this project, we will perform global microRNA analysis of the RBC during storage to identify a unique gene signature that is found only in the stored RBC. This signature can then be used to analyze the blood of athletes to detect the possibility of blood doping based on the presence of these signatures of stored RBC. To achieve this scientific goal, we first used the high throughput sequencing to comprehensively analyze all the long and short-sized RNAs in the fresh RBCs before placed under storage. In addition, we used a state-of-the-art profiling procedures to analyze the changes in the RBC microRNA during storages. We have identified and validated several microRNAs that are specifically found in the stored red cells that can be used for detecting blood doping. We will continue to seek support to further investigate the basis of this “storage gene signature” and validate their ability to detect blood doping in additional samples and real volunteers who have received ABT.

Code: 13D24GJ

Oral dosing of salbutamol is known to lead to beneficial performance effects in athletes and is banned, however, it is allowed to be delivered by inhalation for use in athletes with asthma. The drug is usually used as the racemic mixture consisting of active R- and inactive S-enantiomers (non-superimposable mirror image molecules) which have a different time course in the blood and urine. 
These differences are further amplified by whether the drug is taken orally or by inhalation. To date, anti-doping strategies have not capitalised on this difference in how enantiomers are eliminated from the body. Furthermore, studies have not adequately investigated the effects of repeated dosing of both inhaled and oral salbutamol over several days on urine levels in a doping control context. We will apply our advanced analytical technique that can measure both R- and S-salbutamol in urine, to samples from patients treated with either oral or inhaled therapy over the course of a week. The project will validate the benefits, namely increased sensitivity and reduced false positives and false negatives, of measuring R- and S-salbutamol enantiomers. This will allow to better discriminate between oral and inhaled salbutamol.

Main Findings: 

Background: Salbutamol is a chiral drug, consisting of non-superimposable mirror image molecules called enantiomers (R- and S- salbutamol). Salbutamol is usually administered as a 1:1 racemic mixture of these enantiomers. The body metabolises and eliminates R- and S- salbutamol differently over time (called enantioselective pharmacokinetics).  

Objectives: The primary objective of this project was to improve discrimination between prohibited oral and permitted inhaled dosing by using ratios of salbutamol enantiomers in spot urine samples collected over one week of treatment duration with high doses. 

Results: As predicted, the S:R ratio was greater in oral versus inhaled dosing but the difference was not a reliable overall determinant of route of salbutamol administration, with significant intra- and interpatient variability. Enantiomer ratios offered modest improvement over the current urine threshold in diagnostic capability. During the trial, several subjects exceeded the current urine threshold of 1000 ng/ml after administering salbutamol via inhalation in a permitted manner, both in one week of treatment (800 microgram daily), as well as after an acute dosing regimen within 24 hours (2x 800 micrograms 12 hours apart). Some individuals appeared to have a higher likelihood of exceeding the threshold and more work is required to understand these determinants. A secondary finding of the study was that use of urine specific gravity (USG) normalisation to 1.020 for all samples to account for hydration improved diagnostic performance (mainly sensitivity), compared to both correction for concentrated samples only (as per TD2018DL) and uncorrected concentration. 

Conclusions: Salbutamol S:R ratio is different between oral and inhaled delivery, but the ratio only provided a modest increase in diagnostic performance over existing approaches. The 1000 ng/ml urine threshold is an imperfect classifier of prohibited dosing with potential for some individuals to exceed the threshold using permitted inhalation high and repetitive dosing. USG normalisation for all samples improves the performance of the salbutamol urine threshold 

Code: 13D21DS 

Among the misuse of erythropoietic stimulating agents, the application of substances that induce EPO gene expression by stabilizing HIF 1 as cobalt (II) ions or carbon monoxide inhalation seems to be widely distributed. For athletes, the advantages of the misuse of both substances are low costs, high erythropoietic effectiveness, and no risk of detection, because they are not yet included into the prohibited list of WADA.
The aim of this project is to demonstrate significant effects of cobalt and CO-inhalation on the erythropoietic system which can be detected by the Athlete Biological Bloodpass and direct methods. Special aims are: 1. to monitor the physiological effects of different doses of cobalt and carbon monoxide on plasma-EPO, Hb-mass, and aerobic performance, 2. to prove if the effects of cobalt and CO can be detected by the recently developed Bayesian probabilistic inference techniques, which are the basis for the athletes biological passport, 3. to develop and evaluate direct detection methods for cobalt-misuse, and 4. to evaluate whether the endogenously produced EPO after stimulation by cobalt and CO differs from normal native EPO. 
In a first study the optimal dosage of cobalt and carbon monoxide on endogenous EPO production will be determined. In a second step, application of either cobalt or CO for 3 weeks will provide data on (1.) performance effects of both drugs, (2.) stimulation of endogenous EPO, (3.) changes of hemoglobin mass, and (4) changes of hematological parameters which will be analyzed by the statistics of the athlete biological passport. Cobalt concentration and EPO isoforms will be analyzed in urine and/or blood samples. We expect to develop methods which can be used for the detection of endogenous stimulation of erythropoietin by cobalt and carbon monoxide.

Main Findings: 

Introduction: From the mid 40ies until the late 70ies of the last century cobaltous ions (Co++) were used as an effective drug to treat anemia of different origins. Due to severe side effects this kind of treatment was replaced by other erythropoietic stimulating agents like recombinant human erythropoietin. Co++ acts similarly as a hypoxic stimulus; it stabilizes HIF-1α and HIF-2α and thereby increases the endogenous erythropoietin production. Because of its possible performance enhancing effect Co++ has been added to WADA’s list of prohibited substances and methods. Co++ can be easily purchased and it is recommended from suppliers of nutritional supplements to athletes to boost their performance. The aim of this project was to investigate the effects of low dose Co++ ingestion and to determine the minimum oral dosage which is necessary to increase erythropoietic processes.
Methods: Three sub-studies were carried out: I. Single Co++ dosages between 1mg and 10mg; II. Co++ administration for 5 days with the lowest dosage found to be effective in study I; III. 3-week Co++ administration with the lowest effective dosage (5 mg/day). We determined plasma [EPO], all parameters used for the athlete’s biological passport (ABP) as well as in study III hemoglobin mass and performance. In total, 63 male recreational athletes participated at the studies which were conducted in a double blind design.
Results and discussion: Study I: Single dosages until 2mg Co++ had no erythropoietic effect. Plasma [EPO] increased 5h after the 5mg administration by 18 ±14% (p<0.05) and until 7h following the 10mg Co++ administration by 41 ±15% (p<0.001). Study II: Following the 5-day Co++ application 5mg showed no significant effects on any parameter while 10mg increased [EPO] by 28 ±26% (p<0.05) and the immature reticulocyte fraction by 50 ±21% (p<0.001). [Ferritin] decreased in the 10mg group by 22 ±17ng/ml, p<0.01). Study III: During the 3-week application period plasma [EPO] increased by 30 ±39% (p<0.001) and total hemoglobin mass by 17.1 ±16.8g (p<0.001) while Ferritin decreased by 17 ±18 ng/ml (p<0.01). Although VO2max was not affected, submaximal performance (+11 ±xy Watts at 2 mmol/l lactate, p<0.05) and time until exhaustion (35 ±xy sec., p<0.05) was slightly improved after the 3-week administration period.
Discussion: Co++ administration using dosages 2-5-times above the recommendations of suppliers of nutritional supplements and ~ 2-times above the amount which is considered to be safe and without any side effects in case of life-long daily administration show measurable erythropoietic and performance-enhancing effects. We therefore recommend to install threshold limits for Co++ concentrations in urine and in blood to deter and detect blood manipulation by Co++ administration.

Code: 13D22RV 

Glucocorticosteroids are widely used in sports medicine for the treatment of conditions such as asthma and acute injuries. The use of glucocorticosteroids by oral, intravenous, intramuscular and rectal routes is forbidden, and their use by other routes is allowed. Since some glucocorticosteroids are marketed in different administration forms, the distinction between different routes of administration through the analysis of urine samples is needed. A reporting level of 30 ng/mL for glucocorticosteroids and their metabolites has been established by WADA to detect corticosteroid misuse. Unfortunately, this reporting level is not suitable to distinguish therapeutic use from forbidden administration for all corticosteroids and the investigation of more effective criteria of discrimination is needed. 
The aim of the project is to elaborate analytical strategies to be used in the effective differentiation between legal and forbidden administration routes for synthetic glucocorticosteroids. A comprehensive study of the metabolic pathways of corticosteroids will be performed using liquid chromatography coupled to tandem mass spectrometry. Both phase I (including saturated A-ring and acidic metabolites) and phase II (including conjugates with sulphate and cysteine) metabolism will be studied in several excretion studies already available in our lab for triamcinolone acetonide, betamethasone, prednisolone, methylprednisolone and budesonide. 
Differences between responses of every metabolite detected in the different administration routes will be evaluated. Potential markers will be selected based on these results. Selectivity and sensitivity of these potential markers will be compared with current reporting level. 

Main Findings: 

The objective of the project was to evaluate the current WADA criterion to detect glucocorticoid (GC) misuse.  The urinary profiles of budesonide (BUD), triamcinolone acetonide (TA) and betamethasone (BET) were studied in clinical studies were they were administered by allowed and forbidden administration routes in order to look for the best discrimination markers for each GC. 

For BUD, the metabolite 6β-hydroxy-BUD was confirmed to be the best the marker to detect BUD misuse. However, high 6β-hydroxy-BUD concentrations were found after high-dose inhaled treatments and, for this reason, a reporting level of 40 ng/mL is proposed to discriminate allowed from forbidden administrations. 
For TA, a reporting level of 30 ng/mL of TA did not allow the detection of intramuscular administration. A reporting level of 5 ng/mL for TA was proposed to detect intramuscular use, and to discriminate topical and intranasal from intramuscular administrations. Concentrations obtained after intraarticular and periarticular administrations, which are currently regarded as allowed administration routes, need be taken into account before proposing a definitive reporting level to discriminate allowed from forbidden administrations of TA. 

For BET, a reporting level of 40 ng/mL for BET is proposed to discriminate topical and intranasal from oral and intramuscular administrations. However, concentration values after intraarticular and periarticular administrations are similar to those obtained after intramuscular administrations. 

The results of the present project demonstrate the need of studying the metabolism of each GC after allowed and prohibited administration routes in order to establish the best discrimination criterion for each GC.

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).

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Karine Corrion, France