Projets de recherche

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.

Code: 21D06RV

The steroid profile is used to detect administration of testosterone and other endogenous anabolic androgenic steroids. Metabolites included in the steroid profile have both gonadal and adrenal origin. Glucocorticosteroids (GC) inhibit the hypothalamic-pituitary-adrenal axis, and reduce the adrenal steroid production. For that reason, an effect on the urinary steroid profile might be expected. 

In a previous project, we evaluated the effect of single oral or intramuscular administration of GC on the steroid profile. A significant decrease in the excretion rates of steroid profile metabolites was observed after GC administration that was found to be associated with the dose. However, the ratios between metabolites evaluated in the athlete’s biological passport were not significantly altered, although all volunteers receiving the highest intramuscular dose showed a clear decrease in some of the ratios. Given the significant decrease in the excretion rates of steroid profile metabolites after a single dose and the alterations of some of the ratios, the question of what would happen after repeated administration of GC arose.

 The aim of the present project is to evaluate the effect of repeated oral doses of dexamethasone (DEX) or methylprednisolone (MP) on the steroid profile. Repeated oral doses of DEX or MP will be administered to eight healthy volunteers. The steroid profile will be measured by GC-MS/MS in urines collected before and after administration. The excretion rates of the metabolites will be calculated. In addition the adaptive module will be applied: samples collected before administration will be used to establish the reference ranges for each volunteer, and post-administration samples will be individually compared with these reference ranges.

Main findings

The steroid profile (SP) is a powerful tool to detect the misuse of endogenous anabolic androgenic steroids in sports. The SP consists of the longitudinal monitoring of concentrations of testosterone (T), its related metabolites (epitestosterone, E; androsterone, A; etiocholanolone, Etio; 5α-androstane-3α,17β-diol, 5aAdiol; and 5β-androstane-3α,17β-diol, 5bAdiol), and the ratios between them (T/E, 5aAdiol/E, A/T, A/Etio and 5aAdiol/5bAdiol). Alterations on the SP cannot only be associated with doping practices, but also with the presence of confounding factors. Glucocorticoids (GC) could be a confounding factor to the SP since they inhibit the HPA axis, and the SP metabolites have partial adrenal origin. In fact, in a study conducted by our group, GC treatments involving single-dose systemic administration produced a reduction of the excretion rates of the SP metabolites, as well as a decrease of the A/T and 5aAdiol/E ratios. However, the reduction in the SP ratios did not result in atypical profiles. Considering these results, the question regarding the potential effect of repeated GC administrations on the SP arose.

In this study, the impact of multiple oral administrations of GCs on the SP was evaluated in two studies, using two of the GCs most detected in sports drug testing: methylprednisolone (MP) and dexamethasone (DEX) (n=8 volunteers each study). In MP study, 12 mg of MP were administered per day during 3 consecutive days. In the DEX study, 2 mg of DEX were administered every 12 h for 5 consecutive days. Urine samples were collected before, during and after the GC treatments, and the SP was measured in all samples using gas chromatography-tandem mass spectrometry. The multiple dose oral administration of GCs resulted in a treatment-dependent reduction of the excretion rates of some SP metabolites (5aAdiol, A and Etio) and the SP ratios A/T and 5aAdiol/E. The T/E ratio was not significantly affected.

Overall, although the consumption of GC could result in atypical profiles for A/T and 5aAdiol/E, according to the cost/benefit assessment, GC should not be considered a confounding factor to the SP since misunderstandings in the evaluation of the SP would only take place in very specific situations and, in those cases, the analysis by GC/C/IRMS of the sample triggering the atypical profile would demonstrate the endogenous origin of the SP metabolites.

Code: 21C19EI

Our research hypothesis is that combined intake of diosgenin and ecdysterone is an effective strategy to induce anabolic responses resulting in the enhancement of performance, in particular strength and speed. Therefore, we want to test the dose-dependent anabolic activity of different combinations of ecdysterone and diosgenin in our well-established in vitro test system for anabolic activity. Diosgenin and ecdysterone will be tested dose-dependently as single compounds and in combinations in comparison to other anabolic substances (testosterone, estradiol and IGF1). 

Mechanistic studies will provide more evidence which molecular pathways are involved in its anabolic activity. Comparison with the recently characterised plant-derived anabolic substance ecdysterone in our in vitro test system will provide first information regarding its potential anabolic potency if abused orally in athletes. Ecdysterone was first identified to be anabolic in this test system, which was later confirmed in a human training study [10]. Taken together, the results of our investigations will be part in the pharmacological characterisation of these substances. This knowledge will be helpful to verify and justify the already existing limits of the combinations of ecdysterone and diosgenin for positive testing, to get additional information about side effects and to improve already existing doping tests for these substances. 

Study 1: Binding affinity of ecdysterone and diosgenin combination to the androgen and estrogen receptor.
In our yeast transactivation system, we will examine the binding affinity of ecdysterone and diosgenin combination to the androgen and estrogen receptor in comparison to testosterone, ecdysterone and diosgenin separately. We will determine the respective Michaelis constant (MC) of the combined substances.

Study 2: Anabolic potency of ecdysterone and diosgenin combination
In our C2C12 cell culture system, we will examine dose-dependent anabolic effects of ecdysterone and diosgenin combination in comparison to testosterone, IGF1 and ecdysterone and diosgenin. This could allow to estimate the anabolic potency of a combination of phytosteroids in comparison to the other anabolic substances.

Study 3: Mechanisms of action
In coincubation experiments with anti-estrogens, antiandrogens, glucocorticoids and antagonists to the IGF-1 receptor (like Losartan), we will determine signal transduction pathways mainly involved in the anabolic activity of this combinations. 

Main Findings

For decades, plant-derived phytosteroids like ecdysterone (Ecdy) have been used to enhance physical performance, especially in terms of strength, due to their anabolic properties. Recent findings indicate that Ecdy is increasingly being used in combination with other phytosteroids such as diosgenin (Dio). However, there is limited data available on the combinatorial effects of phytosteroids. Our study aimed to investigate the anabolic effects and underlying molecular mechanisms of various combinations of Dio and Ecdy in C2C12 myotubes in a dose-dependent manner.

In differentiated C2C12 cells, dose-dependent effects of Dio and Ecdy and combinations (ranging from 2 µM to 0.1 nM) on myotube size were investigated. In addition, the mRNA expression of genes associated with muscle hypertrophy, such as IGF-1 and PIK3R1, was analyzed by RT PCR. To delve into the molecular mechanisms, we co-incubated Dio with inhibitors of the estrogen receptor (ER) and androgen receptor (AR), along with dexamethasone (DEX), and evaluated myotube diameter. Moreover, the interaction of Dio with the AR and the ER was examined in a yeast transactivation assay.

Different combinations of Dio and Ecdy had positive effects on the myotube diameter. Combinations of Dio and Ecdy also affected mRNA expression of IGF-1 and PIK3R1. Contrary to the ER inhibitor ZK 191703, the AR inhibitor Flutamide, and DEX treatment effectively counteracted Dio induced myotube diameter growth. Dio did not exhibit agonistic activity in the AR and ER yeast transactivation assays, but it displayed antagonistic effects on the AR rather than the ER. The full mechanism affecting myotube diameter needs to be further elucidated.

Our in vitro findings provide evidence that combinations of Dio and Ecdy display anabolic activity in C2C12 cells in an additive manner. Dio has anti-androgenic activity in the yeast cell model, while Ecdy has anti-oestrogenic activity. Moreover, our data suggest that activation of the PI3K/Akt/mTOR pathway is essential for Dio effects on myotubule diameter. Considering that these results were obtained in vitro in myoblast cells (C2C12) from rodents, future studies are needed to investigate these effects in human training interventions to conclude on any anabolic effect in human.

Code: 21C11CR

Chapter S4 of WADA’s Prohibited List 2021 (“Hormone and Metabolic Modulators”) lists ACE-031 under sub-chapter 3 (“Agents preventing activating receptor IIB activation, Activin receptor IIB competitors – Decoy activating receptors”) as prohibited substance. ACE-031 (Ramatercept) is a soluble fusion protein, which blocks activin receptor type II B (ActRIIB) ligands including myostatin. So far, no approved ACE-031 pharmaceuticals are available. On the other hand, ACE-031 is sold on the black market. However, the administration of black market ACE-031 to human test persons will be ethically not justifiable. For that reason we plan a study with rats. Page 2/7 The test animals will receive black market ACE-031 at a dosage, which can be clearly detected in serum (10 mg/kg BW). After 24, 48 and 168 hours, serum and urine will be collected and tested for ACE-031 by electrophoresis and Western blotting. In addition, the metabolism of black market ACE-031 will be investigated in human microsomes as the metabolites in rat may be different from those in humans. The study will help to clarify (1) how long black market ACE-031 is detectable in blood, (2) if it can also be observed in urine, and (3) if metabolites are different in rats and humans. We have already developed a method for the detection of black market ACE-031 after electrophoretic separation and Western blotting (SDS-, SAR-PAGE).

Main Findings

Chapter S4 of WADA’s Prohibited List 2024 (“Hormone and Metabolic Modulators”) lists ACE-031 under sub-chapter S4.3 (“Agents preventing activating receptor IIB activation, Activin receptor IIB competitors such as decoy activin receptors (e.g. ACE-031)”) as prohibited substance. Currently, ACE-031 is only available on the black market. Since administration of black market ACE-031 to humans is ethically not justifiable, a study with rats was performed.

Aims of the project were:

- the characterization of 14 black market ACE-031 products by SDS-PAGE followed by Coomassie staining or Western blotting using ACVR2B-, follistatin- and His-tag-specific antibodies

- an investigation of the electrophoretic detectability of black market ACE-031 in rat serum and urine after circulation in blood for 24, 48, and 168 hours

- an in vitro metabolism study of ACE-031 using human and rat liver microsomes

Results:

Of the 14 tested ACE-031 products, 13 contained Coomassie-stainable proteins. Western blotting revealed that 12 products were indeed ACVR2B-related proteins. One product was mislabelled and contained black market follistatin instead. It could be further shown that on all 12 ACE-031 products His-tags were present as already demonstrated for black market follistatin.

After a single dose administration of black market ACE-031 (10 mg/kg body weight) to rats, the protein was detectable in all serum samples after 24 hours. While it remained traceable in the majority of the 48 h samples, it was undetectable after 7 days (168 h). No ACE-031 was found in the urine samples at all three time-points.

In order to reveal possible differences in the metabolism of ACE-031 between humans and rats, ACE-031 was incubated with human and rat liver microsomes (5, 60, 120, 300 min, 24 hours). The protein proved quite stable - only after 24 hours degradation of the main band was observed.

Conclusions:

Black market ACE-031 can be detected by SDS-PAGE and immunoblotting with a monoclonal ACVR2B-specific antibody. Additionally, it contains immunoreactive His-tags. For extraction of ACE-031 from rat serum and urine samples, a polyclonal ACVR2B-antibody linked to magnetic beads was used. After single dose administration, the protein remained detectable for at least 48 hours in most rat serum samples. No signals were obtained on Western blots after 168 hours in serum and in all urine samples.

Code: 21C09MP

The administration of anabolic androgenic steroids (AAS) is prohibited as doping in sports. Anti-doping analysis tries to target long-term metabolites of AAS to extend the detection windows. For several 17-methylsteroids the detection of 17β-hydroxymethyl-17α-methyl-13-enes demonstrated superior detection times over other metabolites monitored so far. In dehydrochloromethyltestosterone (DHCMT), active pharmaceutical ingredient of Oral Turinabol, four metabolites with this structure have been reported so far, (Sobolevsky “I”, “M2”, “M3”, and “M4”). The integration of these metabolites into initial testing procedures resulted in a strong increase of adverse analytical findings in doping control, that are currently considered to trace back an administration of DHCMT. Compared to DHCMT methylclostebol (chloromethyltestosterone, ClMT) lacks the 1(2) double bond in the A-ring. Based on common knowledge on steroid metabolism it is expected that "M3" may also be excreted after methylclostebol administration, while the other metabolites might be better suited for discrimination of the administered parent drug. The project therefore aims to investigate the excretion of the above mentioned metabolites after methylclostebol administration and to monitor their excretion kinetics. Integration of the “classical” methylclostebol metabolites will complement the investigation. A comparison with the data obtained for DHCMT is also planned.