Projets de recherche

Code: 11C25FB 

Blood doping is aimed to illicitly increase the oxygen delivery to tissues to improve sport performance, especially in endurance sports/disciplines. At present, WADA-accredited antidoping laboratories can count on several analytical strategies to detect most if not all the different forms of blood doping: these detection strategies may be either “direct” (aimed to detect the “non endogenous” substances administered to obtain the blood-doping effect), and “indirect (where the longitudinal stability of a panel of hematological and/or biochemical parameters is regularly monitored to spot any significant deviation from the individual physiological ranges). 
To the best of our knowledge, the only “masking strategy” for blood doping that has been considered so far is the practice of “blood dilution”. This strategy is performed before a doping control test, and it is obtained by intravenous infusions of physiologic solutions (with or without the addition of modified polysaccharides, used also as plasma volume expanders). This practice is aimed both to dilute specific target analytes, making therefore more problematic their detection by the WADA laboratories, as well as to reduce the value of some diagnostic haematological parameters that are considered in the framework of longitudinal monitoring of the athletes. 
We postulate that an additional, and in principle very effective, masking strategy, would be the administration of supramolecular structures, and primarily among them phospholipidic liposomes, with the objective to reduce the free concentration of circulating peptides, polypeptides and proteins that are analytical targets of both “direct” and “indirect” methods of detection. 
The present project is aimed to verify the effectiveness of this potential masking strategy, and to develop specific, selective, and suitable analytical methods to ensure its detection by the WADA-accredited laboratories. 

Main Findings: 

The detection, and ideally quantification, of doping agents that still remain “invisible” to the anti-doping laboratories are one of the most urgent challenges for the anti-doping community. To be “invisible”, a doping agent needs to satisfy one or more of the following conditions: (i) to be still unknown; (ii) to be identical to an endogenous substance, (iii) to be present in the biological fluids in a concentration smaller than the limit of detection of the available analytical methods, and/or (iv) to be masked by masking agents that are themselves unknown. This research project has specifically addressed this last point. 
In the last few years, novel and potentially more effective masking agents have been considered in sport doping. Among them, drug delivery systems (DDS) seem to be particularly attractive to cheaters. DDS may indeed be used to alter the absorption, release, distribution and excretion profile of prohibited drugs, making their detection by the WADA-accredited laboratories more problematic. 
In this study we have considered a particular class of prohibited drugs vehiculated by drug delivery systems, and, specifically liposomial encapsulated hemoglobins (LEHs). Basically, LEHs, tha are also defined “hemosomes”, are a new form of hemoglobin-based oxygen carriers (HBOCs) that mimic the oxygen diffusivity of erythrocytes, so that they may be considered “artificial red blood cells” rather than “artificial hemoglobins”. Besides their multiple, potential clinical applications, and their extremely promising therapeutic utility, LEHs could be misused as a sport doping practice, since they can increase the oxygen carrying capacity of blood, with the consequent improvement of sport performance, especially in endurance disciplines. Although LEH are not commercially available yet, their preparation is relatively straightforward, and this may increase the interest of those seeking an “invisible” way to take advantage of “blood doping”. 
The main results of the present research project can be summarized as follows: I. Liposome-encapsulated haemoglobins (LEHs) are stable enough to be illicitly used as performance-enhancing drugs.
II. LEHs are “challenging” to detect with current methods for the initial testing analysis are applied.
III. Direct detection of LEHs in blood can be achieved by targeting the intact liposome-hemoglobin complex by flow cytofluorimetry with double coloring. The performance of the methods is further improved by using coated microbeads.
IV. Indirect detection of the intake of LEHs and/or of other liposome vehiculated drugs can be performed by HPLC-MS/MS using a aqueous normal phase, by monitoring the entire phospholipid and sphingomyelin profile in urine. The above results confirm what already reported in a previous research project also funded by the WADA (project code 09D9FB), i.e. that liposomes could represent an effective form of “doping delivery systems”, with potential masking effects. Efficient detection of LEHs requires upgrade of routine detection methods to identify their intake either in blood (by flow cytofluorimetry based techniques) and/or in urine (by HPLC-MS/MS based techniques).

Code: 11A22OP 

Testosterone misuse is the most detected doping offence in screening analysis by means of the ratio between testosterone and epitestosterone (T/E).

Recently, four testosterone metabolites have been reported in our laboratory after alkaline treatment of the sample. In a previous WADA project (reference 10A16OP), we have shown that the detection of these metabolites and the ratio between them is useful for the detection of testosterone misuse after oral and topic administration. Therefore, the addition of these metabolites into screening methods seems to be a promising complement to T/E.

Besides the cases already tested, the measurement of T/E has also additional limitations like (i) its use in samples with low basal T/E or (ii) it can be affected by external factors like alcohol intake. Most of these external factors are related with the glucuronidation process. Since the basic released metabolites are not glucuronidated, they can be less affected by these factors providing a complementary tool for testosterone misuse detection. The knowledge about the phase II metabolic pathway which is associated with the presence of these metabolites can provide useful information for the antidoping control field. Among other adbantages, knowing the phase II metabolite would theoretically allow for including these metabolites in already existing screening procedures.

Therefore, the goal of this follow-up project is to evaluate of the usefulness of the quantitative detection of the metabolites released after basic treatment for the detection of testosterone misuse. In order to achieve this main goal, the project will be divided in three specific objectives: (i) detection of testosterone misuse in population with low basal T/E values, (ii) study of the effect of several factors which can vary the steroidal profile in the values of these metabolites and  (iii) elucidation of the phase II metabolic pathway related with the occurrence of these metabolites.

Main Findings: 

Recently, four new testosterone metabolites (Δ1-AED, Δ6-AED, Δ6-T and Δ15-AD) have been reported in our laboratory after alkaline treatment of the urine. These metabolites and the ratio between them were found to be useful for the detection of oral testosterone misuse. Therefore, the addition of these metabolites into screening methods seemed to be a promising complement to the steroid profile.

The goal of this follow-up project was to evaluate the usefulness of the detection of these metabolites for doping control. For this purpose, the project was divided in three specific objectives: (i) elucidation of the metabolic pathway responsible of the occurrence of these metabolites, (ii) study of the effect of several factors which can vary the steroidal  profile in the values of these metabolites and (iii) detection of testosterone misuse by these metabolites.

We demonstrated that the studied metabolites are produced from the degradation of cysteine conjugates. The formation of these metabolites implies an unreported metabolic biotransformation: 6,7-dehydrogenation as phase I metabolism followed by conjugation with glutathione and subsequent transformation to cysteine conjugates. The postulated pathway was supported by studies with human hepatocyte cells systems. Analogously to testosterone, this pathway might also be present in other steroids, opening the possibility of targeting additional biomarkers. This fact was confirmed by the detection of 24 boldione metabolites (11 conjugates with cysteine and 13 conjugated with N-acetylcysteine).

The influence of several factors in the excretion of the studied metabolites was evaluated. Degradation, freeze/thaw cycles and infradian variability studies showed moderate variations (below 40%) for these metabolites. UGT2B17 polymorphism does not influence the excretion of cysteinyl compounds whereas the intake of exogenous substances (alcohol or 5α-reductase inhibitors) dramatically affects their excretion. During pregnancy only the excretion profile of Δ1-AED increased. Overall, the presented data describes the stability of the urinary cysteinyl steroids under the influence of many factors, proving their potential as suitable parameters to be included in the steroid profile.

The usefulness of cysteinyl markers for the detection of T im and T gel misuse was studied. Among cysteinyl metabolites Δ1-AED/Δ15-AD was shown to be the best marker after T im allowing the detection in all studied cases. The use of this marker also allowed the detection of T gel misuse in almost all volunteers with T/E around 1. Worse results were observed for the detection of T gel misuse in volunteers with T/E below 0.2. Anyway, the use of cysteinyl markers did not increase the detection capabilities of the current steroid profile questioning their usefulness for doping control analysis.

Code: R11B01JS

The research group of N. Leuenberger. J. Saugy, R. B. Mortensen, P. J. Schatz. S. Giraud and M. Saugy has demonstrated the detection or Hema tide™ (OMONTYS.., in anti-doping samples. This approach 1s based on the measurement of pegmesatlde in human serum/plasma samples by an assay and western blot employing specific monoclonal antibodies. This test has been implemented in WADA accredited laboratories as research test system based on ELISA (Enzyme-Linked lmmunoSorbent Assay) technique (screening method) and western blot (confirmation method}.

Actually the coated antibody of the assay is fresh coated directly before the measurement. Between different antibody batches there are variations in the geherated data of the ELISA The aim of this project Is to identfly a method to ensure antibody batches which result m reproducible data. Diflerent methods of antibody production, coating and storage of the antibody and the coated rnicroplate will be tested to establish a safe verilication procedure to detect ?g1nesatide in human blood samples.

The successful development of a stable and functional coated antl-peginesatide antibody is ot

crucial importance for the availability of commercial test kits.

Main findings

The antibodies mAb anti-PEG WADA1A8 and mAb anti-Hematide WADA11F9 of the HematideTM ELISA generate in sandwich with the analyte HematideTM well differentia-ble values. The labs of Paris and Lausanne seem to have problems with the stability of the mAb anti-PEG WADA1A8 antibody. Their results are often not reproducible.

The results of this project cannot trace back these problems to the antibody. The phys-ical QC showed no changing in the structure of the protein. The antibody WADA1A8 shows a stable structure over the time of 12 months. However, the manual preparation showed the same variability in our lab when fresh prepared plates were used. If pa-rameters are fixed (see section 4.3.3) these variability cannot be find. The reason of the problems evolve from the suboptimal conditions for the ELISA and not researched backgrounds for the analyte HematideTM and the both antibodies.

The PEG linker and the anti-PEG antibody are sensitive for detergents because of homologous structures to polyethylene-glycol. In the published assay system [N. Leuenberger et al., Methods for detection and confirmation of Hema-tideTM/peginesatide in anti-doping samples, Forensic Sci. Int.(2011)] Tween 20 is used in several assay steps. Also in cell culture it is common that detergents like Kolli-phor (also known as P188/Pluronic) are added. This could influence the anti-PEG an-tibody already in cell cultivation. This omnipresent application can result in complica-tions of the ELISA. In additional experiments (section 4.3.4) the effect of the replace-ment of Tween 20 against CHAPS shows a significant improvement of the assay sys-tem.

Another possibility to improve the assay is to change the antibodies in the ELISA sandwich. It would be of advantage to use the analyte specific antibody anti-Hematide (WADA11F9) as immobilized captor-antibody. The actual coated antibody anti-PEG antibody (WADA1A8) could be engaged by polyethylene-glycol homologous struc-tures. The assay could be more specific and more stable by this change.

Also critical for the test system is the performance. The microplates should not be coated freshly by the operator. The ELISA should be as easy as possible in practice. A lot of factors are unknown yet. The influence of temperature variation, the endpoints of binding are not determined. The analyte HematideTM has to be analyzed for behav-ior.

After these analysis and optimization of the assay system there are good perspectives to create a well working, reproducible ELISA which is user-friendly and possible to produce commercially.

Code: R10M1MA

There is a consensus in the international community that action must be taken against those behind-the-scene individuals who propagate and facilitate doping in sport. The first step must be to identify those responsible. Law enforcement agencies have used Social Network Analysis (SNA) to tease apart and identify key figures in complex investigations concerning drug trafficking, counter terrorism, money laundering, organised crime and people smuggling. It is proposed to apply SNA to study the social affiliations of convicted blood dopers. This will provide a baseline from which to gain a more sophisticated understanding of the blood doping environment.

Social Network Analysis requires three successive phases of activity. First, information about the links and relationships between the entities under investigation are extracted from existing data (e.g., newspaper reports, internet sites, public databases). Next a structural analysis is conducted to identify central members, subgroups and patterns of interaction between the parties. Finally, those relationships and associations are visualised using a software program that spatially distributes individuals according to their social interactions (i.e., individuals with strong ties are plotted closest together).

Main findings

The use of banned blood transfusions by athletes erodes the integrity of elite sport. Athletes are lured by the substantial performance advantage bestowed by blood doping, together with the realisation that its use cannot be detected by doping controls. Rogue doctors and transfusion technicians have set up extensive and sophisticated doping networks to meet this demand and the covert provision of specialist advice and equipment has proven to be a lucrative trade.

Novel strategies are required to identify participants in blood doping networks. One avenue to gather evidence about networks, and indirectly the athletes who are utilising such facilities, is to obtain eyewitness testimony via investigative interviews. For example, with regard to the transfusion networks that have so far been discovered, invariably some peripheral teammates and support staff of the doped athletes were aware of the existence of the network. However for this interview-based approach to be cost effective, there must be some way to flag persons of interest for interviews amongst the pool of several thousand athletes and support staff.

This study evaluated whether network analysis is an effective method for targeting interviewees for investigations into blood doping networks. Specifically, whether it was possible to identify and rank teammates and staffers in terms of their closeness to riders who had been implicated in blood doping practices.

The study determined that network analysis was capable of prioritising individuals based on the premise that individuals with the greatest exposure to doped athletes would have the highest likelihood of possessing relevant information. These findings have implications for antidoping authorities who seek to rationalise their allocation of scarce investigatory resources. This study makes recommendations for how network analysis can be incorporated into investigative operations.

Code: 10A24XD 

In extremely rare circumstances the presence of 19-norandrosterone (19-NA) in human urine can be explained by the “instability” or “activity” of the urine specimens. This is most likely due to the sample transportation and storage in the laboratory and is based on the 19- demethylation of abundant endogenous steroids in the urine samples.

Tests for the assessment of the activity of the urine samples have been established. The hypothesis is that 19-NA will be produced by 19- demethylation of androsterone, the main androgen metabolite present in the urine. Briefly, an aliquot of sample to be tested for activity will be incubated in the presence of deuterated androsterone. The formation of deuterated 19-NA will be the proof of urine activity.  

From the data collected until now, it appears that the instability of these urine samples is due to enzymatic activity expressed (from exogenous origin) in the sample. It seems unlikely that the 19-demethylation would be the result of a pure, unassisted chemical reaction. If this were the case, this phenomenon would be reproducible at any time, and is not.  The growth of microorganisms in the urine samples is not unlikely. It is reasonable to link this enzymatic activity to the presence of a microorganism growing in the urine.  The expression of aromatase is restricted to the gonads and brain in many vertebrates, from aquatic and avian species to mammals. The removal of the methyl group in the 19 position seems not linked to an aromatization process since no microorganism expresses such an enzyme.  An important demethylase enzyme present in some microorganisms is the 14-demethylase (CYP51A1). The activity of this enzyme is crucial to the life of these microorganisms since it is the responsible for the formation of ergosterol from lanosterol. The hypothesis is that CYP51 uses androsterone as “substrate” consequently producing 19-NA.

Main Findings: 

The present was focused on the hypothesis that the CYP51 (14α-demethylase) of fungal origin may be the cause of the alteration of the metabolic profile found in the so-called active urines. In these cases a concentration of 19-NA beyond the limits allowed, but with a ratio 19-NA/19-NE reversed compared to that A / E (usually the concentration of 19-NA in urine is greater) are observed.
To check if the CYP51 is able to demethylate androsterone and / or etiocholanolone, yeasts such as S. cerevisiae and C. albicans were chosen. As substrates of of the yeasts, androsterone, etiocholanolone and  androstenedione were selected as being the most probable substrates, based on their structure and on the amount present in routine anti-doping samples.
The following experiments were performed:
1. S. cerevisiae and C. albicans were incubated in the presence of androsterone, etiocholanolone, and androstenedione in culture medium and in synthetic urine (in sterile conditions). The products of fungal metabolism were found both in the supernatant and in cell lysate.
2. S. cerevisiae and C. albicans were incubated in human urine in non-sterile conditions. Alterations of the hormonal profile in urine were evaluated according to the protocols of the anti-doping laboratory of Rome, used in the screening of banned substances.
The main conclusions of the present project are:
· The demethylation process of steroids produced in the so-called active urines is not originated chemically, at least under the common laboratory conditions during the sample processing of the urine samples in doping control.
· The aromatization process that is the responsible of the in-vivo formation of 19-norandrosterone is not the responsible for the 19-demethylation ex-vivo during sample storage of the urine samples.
· In cultured C. albicans and S. cerevisiae conducted in SDB medium and in the presence of steroid hormones, any relevant reaction of demethylation was observed. By-products of fungal metabolism on these hormones have been detected.
· In human urine inoculated with the same microorganisms, changes in the hormonal profile were detected, that can be attributed to the activity of C. albicans. These observations were previously detected in culture medium under sterile conditions. Even in these experiments the formation of 19-narandrosterone and 19-noretiocholanolone was not detected.
· Although the formation of 19-norsteroids object of this study was not detected, the changes in urinary steroid profile as a result of contamination with fungi are considered to be relevant for the correct interpretation of the data in doping control.
Under the experimental conditions described, where the functional growth of fungi has been demonstrated, the transformation of androsterone and etiocolanolone to their respective 19-norderivatives, as happens in the case of active urine, was not observed.  With the current obtained knowledge, we can say that the ex-vivo process of demethylation is complex and involving several actors. The proposed test to demonstrate the “activity” of a particular sample provided that deuterated androsterone or etiocholanolone were transformed in their corresponding 19-nor deuterated products. This is not the case with the presence of the tested fungi alone. The process of demethylation implies the presence of an unsaturation in alpha with respect to the methyl group to be removed, absent in androsterone and etiocholanolone. Our experimental evidence supports the hypothesis that the process is a multistep process consisting in a first dehydrogenation of A ring, most probably in the C1 position,
followed by the multistep oxidation or the methyl group

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

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

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

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

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