Projets de recherche

Code: 18B09CR

Chapter S4 of WADA’s Prohibited List 2018 (“Hormone and metabolic modulators”) lists myostatin inhibitors under sub-chapter 4 (“Agents modifying myostatin function(s)”). Follistatin (FST) suppresses signaling of myostatin and subsequently leads to an increase in muscle mass and loss of body fat. FST is a secreted glycoprotein, which can be found in many tissues and organs (e.g. pituitary, bone marrow, ovary, kidney, liver, blood vessels). Due to alternative splicing, three FST-isoforms exist (FS-288, FS-300, and FS-315). The isoform with 315 amino acids (FS-315) is the dominant one. FS-315 can also be detected in blood. Typical concentrations in serum and plasma are in the range of 2-3 ng/mL.  

So far, no approved follistatin pharmaceuticals are available.  Nevertheless, follistatins can be bought from many internet providers for “research purposes”. Their products are labelled either “Follistatin”, “Follistatin 344”, or “Follistatin 315”. Most of these proteins are expressed in E. coli and hence lack the characteristic glycosylation of human endogenous follistatins. This fact will be exploited in order to detect doping with follistatins. After immunoaffinity purification (serum/plasma, urine), FST will be separated by electrophoresis (SDS-, SAR-, or IEF-PAGE) and detected by Western blotting. Due to the missing glycosylation, “black market” FSTs will not only differ in molecular mass but also isoelectric point (pI) from the endogenous FSTs.

Main Findings:

Follistatin (FS), a myostatin-inhibiting protein, is prohibited according to chapter S4 of the "WADA Prohibited List 2022". While currently no approved pharmaceutical formulations of Follistatin are available, Follistatin can be bought on the black market. Most of the products are labelled "Follistatin 344" (FS344), few "Follistatin 315". A study on FS344 black market products was performed and an electrophoretic detection method for serum and urine developed. While only 9 of the 17 tested products actually contained Follistatin, in some of the others growth promoting peptides were found (e.g. MGF, GHRP-2). Suprisingly, all nine products contained His-tagged FS344 and a high degree of its oligomoers. The detection method is based on immunomagnetic purification followed by SDS-PAGE and Western bloting with a monoclonal anti-His antibody. Alternatively, a monoclonal anti-FS antibody can be used. For immunoprecipitation (IP), a polyclonal anti-Follistatin antibody is applied. An evaluation of suitable antibodies for IP and immunoblotting is also presented. Furthermore, practicall all currently available Follistatin standards were investigated. The detection limit of the method for back market FS344 in urine is ca 0.1 ng/mL for 10 mL. For a sample volume of 100 μL, an LOD of 5 ng/mL could be achieved for serum. Due to the presence of His-tags an unambiguous differentiation from endogenous Follistatin is possible.

Code: 18C02MT 

Testosterone misuse still remains a challenging task for doping control laboratories as this steroid is produced naturally by each and everybody. As it is detectable in all urine samples, concentration-based thresholds have been established to uncover testosterone administration. As soon as these thresholds are exceeded, samples are forwarded to isotope ratio mass spectrometry determinations (IRMS) to elucidate the steroid´s source and to unambiguously differentiate between naturally elevated testosterone concentrations and doping.

Recently a promising new target analyte for IRMS determinations was reported, epiandrosterone (EPIA). This steroid enables to prolong the detection of a single testosterone or testosterone prohormone administration from 24 h to more than 100 h using IRMS. Unfortunately, EPIA is only excreted into urine in it sulfoconjugated form while all other steroids routinely employed in IRMS are excreted glucuronidated. To investigate EPIA an additional time consuming step in sample preparation is inevitable.

The aim of this study is to investigate the potential of two other possible long term markers in IRMS excreted glucuronidated and therefore avoiding the additional step. The so called “Epidiols” are known long term metabolites of epitestosterone and might also work for other steroid administrations. In a preliminary study employing dehydroepiandrosterone both Epidiols were remarkably influenced by the steroid administration supporting the hypothesis that these new markers will improve steroid detection by IRMS. To further substantiate this finding we are going to re-analyze other excretion studies after improving and validating the already existing method for IRMS determinations of both Epidiols.

Main Findings: 

A novel IRMS method for determination of carbon isotope ratios (CIR) of 5a- and 5bEpiD was developed and fully validated in line with WADA requirements encompassing limits of detection, linear range and absence of isotopic fractionation. By means of investigations on a reference population encompassing n=72 individuals it was possible to derive referece-based decision limits, which demonstrated that the novel method is fit-for-purpose. Several administration trials were investigated to elucidate the potential of both markers. For a single oral T administration, the effect on both 5a- and 5bEpiB was less pronounced compared to traditional markers and offreed merely short detection windows. After consecutive transdermal T-Gel administrations, especially 5bEpiD was significantly influenced while 5aEpiD shows similar CIR,s as 5a- and 5bDIOL. Unfortunately, the first sample after cessation of T-Gel administration was collected after 5 days, and here all CIR values had returnedd to natural abundance. The administration of a single oral dose of androstenedione resulted in a prolonged depletion of CIRs for 5a- and 5bEpiD compared to other target compunds, but the influence was less pronounced, i.e. the CIR values were not found to be influenced beyond the established reference-based decision limits.

While a direct application of 5a- and 5bEpiD as long-term markers for steroid administrations appears to offer limited added value, these additional target compounds may be beneficial in detecting multiple transdermal T-Gel administration even if so called low-doses are applied. Moreover, they could support the differentiation between single administrations (including inadvertent exposure) and multiple and therefore intended administrations. Further research will be necessary to elucidate this potential of 5a- and 5bEpiD.

Code: 18A03MT 

Ghrelin and Ghrelin mimetics belong to the prohibited substances according to the current WADA Prohibited List. After administration, they induce the secretion of growth hormone into the circulation and are considered as performance enhancing accordingly. While Ghrelin itself is an endogenous peptide hormone, Ghrelin mimetics (such as Capromorelin, Macimorelin and Tabimorelin) are low molecular mass synthetic peptides (or peptide similars) which act as agonists at the ghrelin receptor. Although prohibited, efficient detection methods for these drugs are scarce. Thus, it is planned to develop analytical methods to determine the potential misuse of the new hGhrelin mimetics (Capromorelin, Macimorelin and Tabimorelin) in doping control samples. This will include the investigation of efficient extraction procedures, metabolism studies (in-vitro/ in-vivo) and identification of reliable target analytes in the respective matrix. Results will be validated and published.

Main Findings: 

Analytical methods to determine the potential misuse of the ghrelin mimetics capromorelin (CP-424,391), macimorelin (macrilen, EP-01572), and tabimorelin (NN703) in sports were developed. Therefore, different extraction strategies, i.e. solid-phase extraction, protein precipitation, as well as a ‘dilute-and-inject’ approach, from urine and EDTA-plasma were assessed and comprehensive in vitro/vivo experiments were conducted, enabling the identification of reliable target analytes by means of high resolution mass spectrometry. The drugs’ biotransformation led to the preliminary identification of 51 metabolites of capromorelin, 12 metabolites of macimorelin, and 13 metabolites of tabimorelin. Seven major metabolites detected in rat urine samples collected post-administration of 0.5-1.0 mg of a single oral dose underwent in-depth characterization, facilitating their implementation into future confirmatory test methods. In particular, two macimorelin metabolites exhibiting considerable abundances in post-administration rat urine samples were detected, which might contribute to an improved sensitivity, specificity, and detection window in case of human sports drug testing programs. Further, the intact drugs were implemented into World Anti-Doping Agency (WADA)-compliant initial testing (LOD: 0.02-0.60 ng/mL) and confirmation procedures (LOI: 0.18-0.89 ng/mL) for human urine and blood matrices. The obtained results allow extending the test spectrum of doping agents in multi-target screening assays for growth hormone-releasing factors from human urine.

For ghrelin and its desacylated analog, a quantitative test method with an LOD of 20 pg/mL and an LOQ of 100 pg/mL was developed and subsequently, a representative population of athletes’ blood samples was analyzed. The intact (non-digested) ghrelin exhibited poor chromatographic properties due to the presence of 7 basic amino acids in the sequence and, therefore, the approach was designed to include an enzymatic hydrolysis step which yielded the tryptic peptide t1 with 11 amino acids and only one Arg residue at the C-terminus. In accordance with literature data, the analyzed plasma samples from professional athletes yielded concentrations between 26 and 177 pg/mL, which is significantly lower than concentrations reported for ghrelin post-administration plasma samples that reached peak levels of 17500 pg/mL in clinical studies.

Code: 18A16AK 

In anti-doping control, the detection of steroid conjugates is of increasing relevance. The wide detection window of certain conjugates in combination with efficient and highly sensitive LC-MS technology enables a prolonged traceability of hormone misuse. The reliable quantitation of steroid conjugates requires isotope-labelled reference materials, which are only available for a minority of doping relevant conjugates. An approach to generate steroid sulfates is the in vitro incubation with human liver S9 fraction.

The objective of the present study is to produce isotope-labelled steroid sulfates in vitro. This will be accomplished by the incubation of relevant steroids or their metabolites with liver S9 fraction and 34S-sodium sulfate. The in vitro generated reference material can be directly used for dilute-and-shoot quantification of the respective steroid sulfates in human urine samples. Moreover, the established approach could be rapidly expanded to comparable steroids and methods or materials may be shared with other WADA accredited laboratories.

Main Findings: 

The project aimed at the generation of 34S-labelled epiandrosterone sulfate as internal standard for doping routine analysis. This in vitro-synthesis should be accomplished by incubation of epiandrosterone with liver S9 fraction and 34S-labelled sodium sulfate as co-factor. Results: We introduced a modification of a previously described protocol which replaces phosphoadenosin-5’-phosphosulfate by sodium sulfate and adenosine-5’-triphosphate in the S9 fraction incubation. By using isotope-labelled sodium sulfate (Na234SO4), we generated 34S-labelled epiandrosterone-sulfate with a purity of ≥99.9 %. With this purity, the in vitro generated steroid sulfate fulfills the requirement for an internal standard for quantitation purposes. The sulfonation rate was found to be very low (13 %) and attempts to increase the reaction yield were not successful. 
Conclusions: In conclusion, we successfully introduced a protocol to generate isotope-labelled epiandrosterone sulfate with purity. Due to the low percentage of the epiandrosterone sulfonated by the S9 fraction, we were not able to provide the intented amount of min. 5 mg epiandrosterone-34SO4. Future plans: As the modified protocol was proven to generate 34S-isotope-labelled epiandrosterone, it is possible to expand investigations on comparable steroids. Future investigations should focus on the optimization of the sulonation rate of the subsrate.

Code: 18B04AM 

The increasing number of biosimilars of the first generation EPOs (epoetin alfa and beta) produced all over the world raise the question of their detection. Due to small structural changes, for some EPO Biosimilars the identification criteria edicted by WADA for rEPO may not always be reached, which would render their use by athletes undetectable. Preliminary results have identified Hemax and Epotin authorized in Algeria and Jimaixin authorized in China as rEPOs with apparent molecular weights lower than the original epoetin alfa (Eprex) and closer to endogenous EPO. Thus identification using SDS/SAR-PAGE method could be problematic while their IEF profiles always remain very basic and distinct from endogenous EPO. To go further, evaluating detection from samples resulting of a real administration in healthy subjects is needed.

The objectives of this project are:
- to analyze blood and urine samples using IEF and SAR-PAGE methods according to the TD2014EPO and to compare identification capacities and establish the window of detection for each rEPO tested.
- to test complementary strategies to improve detection of EPO biosimilars: neuraminidase treatment that has been shown to improve the migration distance between rEPO and endogenous EPO as well as a 2-D separation gel approach (mixing IEF and SDS separation) will be tested by AFLD on samples that show a problematic rEPO identification.

In addition specific glycosylations of each Biosimilar will be characterized by LC-MS coupled to fluorescence.

Main Findings: 

Recombinant erythropoietin (rEPO) biosimilars are generic epoetin drugs developed following an expired patent. All licensed EPO biosimilars shall demonstrate the same saferty and efficacy for therapeutic use as the original drug but small structural differences compared to the reference product due to some variations in the production process can be accepted. After analyzing various EPO-biosimilars, three different kinds were selected for further characterization due to their slightly lower apparent molecular weight (MW) compared to the original epoetin alpha drug Eprex®.  Jimaixin™ authorized in China, and Hemax® and Epotin™ authorized in Algeria.

The aims of this research were:
i) to study the electrophorectic profiles obtained by IEF and SDS-PAGE of the three Biosimilars spiked in urine and plasma and to evaluate their identification following WADA's criterai (TD2014EPO),
ii) to test complementary strategies to improve detection of EPO biosimilars using a two-dimensional electrophoresis approach (SDS separation following IEF) and a neuraminidase treatment of the sample shown to increase the separation between recombinant and endogenous EPO by SDS-PAGE,
iii) to evaluate by mass spectrometry the specific N-glycosylation pattern of each biosimilar and compare with the original rEPO Eprex®. 

Experiments with spiked urine and plasma samples showed that samples spiked witht he Biosimilars were more challenging to identify for rEPO compared to Eprex in particular at low amount. Differences were seen according to the method of identification used and the biosimilar spiked. Epotin and Jimaixin were more difficult to identify by IEF compared to SDS-PAGE while it was the opposite for Hemax. The SDS-PAGE method applied to urine samples had the higher identificaiton rate considering the three biosimilars. Two-dimensional electrophoresis experiments did not improve the detection. This analysis proved complex to perform and no clear criteria could be used to identify rEPO.

Samples pre-treated with Neuraminidase gave promising results. Using the SDS-PAGE, the EPO bands were slightly broader compard to untreated samples. Neuraminidase-treated Dynepo could be used as a sparation marker between endogoenous and exogenous signals and this could improve the identification of low doses of biosimilars. By IEF, an interesting pattern for the biosimilars treated with neuraminidase was found. Using a 2-10 pH gradient gel was necessary to detect three thin additional bands inserted between the main EPO isoforms. This characteristic was observed for the three biosimilars while these bands were totally absent in non-spiked samples.

N-glycosylations of Eprex® and the biosimilars were identified by MALDI-TOF and adundance of the various glycan forms were compared.
All three biosimilars were enriched in bi and tri antennae forms while a decrease was observed in particular for the main glycan forms of Eprex® (tetraantennae tri- and tetra-sialylated forms). Hemax had the glycan profile the closest to Eprex® while Epotin™ and Jimaixin™ presented more loss of sialic acids. These results on N-glycan were in good agreement with the presence of additional basic isoforms in their IEF-PAGE profile.

In conclusion, even if these biosimilars may present some challenges at low concentration, current methods used in the anti-doping laboratories and if necessary an easy-to-implement neuraminidase pretreatment of the samples can assure detection of these compounds

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

Ce document n'est actuellement disponible qu'en anglais

Code: ISF17SC01KS

Serum samples from 35 individuals (25 males, 10 females) who were administered Human Growth Hormone (hGH) or placebo for 3 weeks and followed for a total of 4 weeks pre-administration, the 3-week administration period and a further 6 weeks post-administration will be analyzed on the SOMAscan biomarker discovery platform for the detection of potentially novel biomarkers of hGH abuse.

Deliverables. The primary aims of this study were the following:

• Identify proteins that respond to hGH treatment.

• Identify proteins that distinguish the hGH treated samples from the untreated samples.

a. The Deliverables provided to Client will include:

i. Normalized and, if applicable, calibrated data, measured in Relative Fluorescence Units (RFU), for each of the analytes organized on a per-sample basis, delivered in an Excel-compatible format;

ii. For each marker that reaches significance threshold: boxplots with longitudinal profiles (line plots) for individuals

iii. Spreadsheet (CSV) containing model statistics for all markers tested ordered by significance of fixed effect for time.

The following analyses are proposed:

Analysis 1: Identify protein biomarkers that show a time- and dose-dependent change in protein signal in response to hGH treatment. Baseline, During and Post treatment logRFU will be fit with mixed models to account for the correlated structure of the time series data and to evaluate the fixed effects of time. Pair-wise comparisons (to baseline) based on difference in least squares means within significant fixed effects will be performed to identify specific time-dependent differences.

Additional Analysis to be Provided:

Building of classification models for selection of proteins that exhibit “stable” differential expression between treatment groups and over time of study protocol.

Main findings

We reported quantitative blood circulating levels for over 1,300 proteins in serum from 35 volunteers, sampled at 22 time points. The study includes controls and three levels of doping, and samples were collected at baseline, during the doping phase and in a follow-up period. We identified 66 proteins that displayed strong association with doping. We characterized these proteins with respect to the influence of diurnal variation, sex, and genetic background of the study participants and computed multiple ROC curves for each protein, depending on the targeted period and dosage. We then built multi-variate classification models and showed that models with an AUC of up to 93.3% can be constructed using a Random Forest approach with 66 proteins, and 82.2% using glmnet.lasso with stability selection based on five protein markers alone. Furthermore, we evaluated the 66 proteins on their potential as a doping biomarker based on the requirement that they do not identify any false positive, using the percentage of doped individuals that have been identified at least once as a quality criterion. Finally, we tested the performance of selected sets of composite markers. Using 5 markers and aiming at a zero false positive rate, around 25-35% of all doped samples could be identified in a real-world scenario. In a second multi-marker analysis we evaluated the performance of six multi-marker models under more realistic conditions. Table 4 provides the top performing models, as a function of the selection criteria. The provided supporting data and plots can now be used to further develop and select the most suitable candidate markers for development of targeted markers.

Code: ISF17A15ZG

our objectives are:

- to reproduce an ABT experiment similar to what athletes are susceptible to perform (small volume of blood: 200ml) and to compare in the same assay reinfusion of refrigerated blood (stored less than one month) or reinfusion of cryopreserved blood. A group of subjects that will donate blood but will not be reinfused will also be integrated as controls.

- to focus on the characterization of small cells (RBC) and microparticles before and after ABT to identify morphological and cell surface markers changes, as well as changes in protein content by proteomic analyses of microparticles.

- to analyse the structural and antigenic properties of band 3, the major protein of the red cell membrane.

We will :

(i) determine if these techniques can efficiently identify an ABT of small volume. And if similar modifications are identified when blood was conserved refrigerated or frozen.

(ii) determine the window of detection of an ABT of small volume with these tests..

Code: ISF17A32DE

To explore the detection capability of rEPO administration from a dried blood spot.  Primarily, the immature reticulocyte surface protein CD71 will be analyzed in a longitudinal manner to evaluate changes following EPO administration.  Secondarily, we will seek to optimize direct detection of rEPO from a single or multiple dried blood spots using conventional SAR-PAGE techniques.  Importantly, the ability to detect ESA abuse from a dried blood spot, whether directly or via longitudinal means (i.e. CD71+ cells), would be critical for the future of doping control testing.