Projets de recherche

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Doping occurs in a complex and dynamic environment. Often, the sporting system in which doping occurs comprises multiple hierarchical levels that is challenging to fully elucidate. To date, research in doping has typically focused on athletes and athlete support personnel at the sharp end of this hierarchy rather than the broader systemic factors that contribute to doping. To understand and optimise the behaviour of complex systems (i.e., the anti-doping system), the application of contemporary systems thinking-based methods has been recommended. Such methods allow for analytical modelling to be conducted to understand and identify the numerous interacting factors that influence system behaviour. This enables the identification of opportunities to intervene and optimise the overall system rather than its component parts.

Code: 21B07CR

All detection methods for ERA-doping are currently based on electrophoretic separation and Western blotting and due to its high sensitivity apply the same primary antibody (clone AE7A5 anti-EPO

antibody). Upon initiative of the grant applicant, a biotinylated version of the antibody was generated in 2018, which led to further increase in sensitivity and simplification of the method. The antibody

also became part of WADA TD2021EPO. One drawback of clone AE7A5 antibody is that it non-specifically binds to some proteins, which are present in human urine and blood. The non-specific

binding is caused by the fact, that the peptide used for generating the antibody contained two errors in the amino acid sequence. Hence, all doping control samples have to be first immunoaffinity-purified before electrophoresis. For this purpose, a different EPO antibody has to be used. To ensure better selectivity both the ISL [11] and the TD2021EPO, even if not totally mandatory, recommend that the CP differs from the ITP. Since no alternative to clone AE7A5 antibody is known so far, laboratories have to establish two different immunoaffinity purification procedures for ITP and CP. On the other hand, there are ca. 150 anti-human EPO antibodies commercially available, which have largely not been compared with clone AE7A5 antibody regarding sensitivity and selectivity. If an alternative antibody could be found, this would not only simplify EPO testing but would also generate more flexibility for the ITP and CP. Aim of the proposed project is a large scale evaluation of commercial anti-human EPO antibodies. About 100 antibodies will be tested regarding their sensitivity and selectivity in comparison with clone AE7A5 EPO antibody.

Code: 21T07NN

Detection of blood doping remain a challenge, and novel detection methods are warranted. In the present project, the aim is to evaluate the potential of changes in red blood cell proteins for detection of recombinant human erythropoietin (rhEpo) misuse at sea level and at altitude in males and females. The red cell protein content is expected to change with rhEpo treatment, as the transcription of genes coding for red cell proteins change during blood doping practices. Indeed, changes in a few proteins of red blood cells are known to change with rhEpo misuse. However, whether the changes in these proteins are sensitive and specific to rhEpo misuse and whether more sensitive and more specific proteins exist remains unknown. Additionally, it remains unknown whether hypoxic exposure or gender are confounding factors to these changes. To answer these questions, the present project will investigate more than 1000 different red blood cell proteins using mass spectrometry and identify the most sensitive and specific ones. Further, the present project will develop and validate a model for rhEpo detection based on the detected proteins. Our goal is to develop a ready-to-use robust method that will allow precise quantification of our novel biomarkers within approximately 4 h, from receiving the sample to the result.

Code: 21C20VB

This project aims to determine whether new-generation ultra long-acting beta2-agonist (U-LABA) formulations containing vilanterol and indacaterol can enhance performance when inhaled at therapeutic or supratherapeutic doses in highly-trained males and females. The project will also examine whether U-LABA can induce muscle hypertrophy when used chronically.

Objectives: This project will investigate the potential performance-enhancing effects of vilanterol and indacaterol at therapeutic and supratherapeutic inhaled doses. THe formulations tested are Relvar® (22 µg/dose vilanterol+fluticasone 55 µg/dose) and Atectura® (indacaterol 125 µg/dose+mometasone 62 µg/dose) as these are U-LABA formulations of relevance for asthma treatment.

Code: 22E05MT

Gene doping has been classified as a prohibited method in the WADA prohibited list for many years. As gene therapeutic approaches improve and concerns regarding their safe clinical application decline, concerns about their illicit use in elite sports are rising sharply. The outlined project aims at the evaluation of a novel gene doping detection approach emplying high multiplex MALD-TOF mass spectrometry (MS). As preliminary work, a prototype panel able of the multiplex detection of transgenic expression of seven relevant gene doping targets (EPO, FST, GH-1, IGF-1 MSTN(-Propeptide), VEGF-A, VEGF-D) has been designed and pre-tested for its general specificity and applicability. Central aim of the proposed project is the in-depth validation of the prototype panel with regard to its specificity, sensitivity, and reproducibility. Futhermore, suitable reference material will be designed and tested taking advantage of the single-base extension and detection technique of the proposed chemistry and detection method. In a final step, the optimized and validated panel and reference material will be tested for its applicability to routine testine procedures with a first set of 1000 (doping control) specimens consented for research purposes. If successful, the described high multiplex gene doping detection panel and MALDI-TOF MS detection approach would allow a time- and cost-effective detection of multiple gene doping relevant targets at once and could thus expand the range of available tests for gene doping detection.

Code: 21T04MS 

Blood steroid profiling using ultra-high performance liquid chromatography (UHPLC)-MS/MS has been proposed as a potential additional evidence to support the scenario of an endogenous prohibited substance administration. Moreover, the blood matrix is more informative for the correlation between hormone concentration and the physiological responses. Serum steroid profiling has been proven particularly useful for T detection in female subjects in whom menstrual fluctuations may lead to a great source of variation for urinary biomarkers disrupting the sensitivity of their longitudinal monitoring. In the clinical context, profiling of a panel of steroids represent also a great asset for the characterization of possible alterations of steroid metabolism in endocrine syndromes.

A primary drawback of this approach based on blood matrix, principally in the anti-doping context, is that it requires invasive venous blood sampling. Specialized health-care personnel usually perform this procedure and samples have to be transported under specific conditions in a short time window involving important logistic costs. To reduce the large expenses associated with the collection and shipment of this matrix, dried blood spots (DBS) with the transfer of a limited volume of capillary blood onto a filter paper or similar matrix offer a convenient and more affordable alternative. The applicability of DBS in the anti-doping context has been investigated in various studies for either direct detection of prohibited substances or indirection detection through potential biomarkers . In particular, a method using microsampling and GC-MS/MS was recently developed for the quantification of T and eight synthetic anabolic androgenic steroid (AAS). However, this method was limited to the quantification of only one endogenous AAS (EAAS) and could therefore be hardly applied in the clinical context for the monitoring of steroidogenesis disorders.

Main Findings

Additional biomarkers and alternative matrices need to be investigated to improve the detection capability for doping practices with testosterone (T), especially in female athletes. The blood steroid profile consisting of the longitudinal monitoring of serum T and DHT concentrations was proposed as a complementary approach, demonstrating a high sensitivity for the detection of T gel administration in women. A primary drawback of the application of serum steroid profiling is that it requires invasive venous blood sampling and sample collection by a trained phlebotomist. Moreover, these biological specimens have to be transported under cooled temperature conditions within a short timeframe, which all increase the total costs of sample collection. Dried blood spots (DBS), which are based on the transfer of a limited volume of capillary blood onto a filter paper or similar matrix, could tackle these obstacles offering a convenient and more affordable alternative. This process benefits from minimal invasiveness, simplicity of sample collection, facilitated transport and storage conditions, and reduced costs that could allow for more frequent sampling for anti-doping programs. In this study, we developed and validated a UHPLC-MS/MS method for the simultaneous determination of eleven free and eight conjugated steroids in DBS matrix. This method was then used for the analysis of DBS samples collected in 14 healthy women during a normal menstrual cycle (control phase) followed by a 28days testosterone gel treatment (treatment phase). Results were compared with those obtained from serum matrix. The analysis of women samples demonstrated a high correlation between DBS and serum concentrations for most compounds in control phase. In treatment phase, higher testosterone concentrations were observed in capillary than in venous DBS, suggesting a possible interference resulting from testosterone contamination on finger(s) used for gel application. Steroid profiling in capillary DBS represents a simple and efficient strategy for monitoring endogenous steroid concentrations and their fluctuation in clinical context of steroid-related disorders, or for the detection of testosterone abuse in anti-doping. Ultimately, DBS may be used as screening method allowing for more frequent sampling and for targeting blood (serum) and urine sample collection that would be used for a full steroid profile and for GC/C/IRMS analysis. The increased sampling would provide a better estimation of natural baseline variability of a given athlete and would provide a better resolution of a doping picture.

Salamin O, Nicoli R, Xu C, Boccard J, Rudaz S, Pitteloud N, Saugy M, Kuuranne T. Steroid profiling by UHPLC-MS/MS in dried blood spots collected from healthy women with and without testosterone gel administration. J Pharm Biomed Anal 204, 2021, 114280, https://doi.org/10.1016/j.jpba.2021.114280

Code: 21E06MT

The rapid evolution of new medical/pharmaceutical methods extremely promotes the probability that those scientific progresses are illicitly used in order to achieve performing enhancing effects in sporting competitions. Especially the development of the CRISPR/Cas (Clustered regularly interspaced short palindromic repeat/CRISPR associated) technique for simple and precise gene editing of specific DNA sequences has drastically increased the potential of misuse in recent years. The application of the CRISPR/Cas method is classified as gene doping in the Prohibited List and is banned at all times (inand out- of competition) by the World Anti-Doping Agency. 

Therefore, the aim of this research project is the development of complementary detection methods to uncover the illicit attempt of gene doping with CRISPR/Cas by targeting the so called single guide RNA (sgRNA) as marker analyte in plasma/serum samples. Two different approaches are planned to be utilized for the detection of sgRNA, namely a direct approach employing either a gel-based detection or an identification procedure of specific xenobiotic RNA sequence segments (which will be assessed by means of SHERLOCK / specific high-sensitive enzymatic reporter unlocking), along with an indirect approach, which will be accomplished via the analysis of distinct chemical RNA modifications by means of HPLC-MS/MS. Furthermore, an administration study employing a mouse model will be conducted in order to demonstrate the proof-of-concept.