Projets de recherche

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. 

Code: 17A20LM 

Urine samples collected worldwide for anti-doping testing are not always shipped in refrigerated conditions from collection sites to WADA accredited laboratories, in particular when several transport days are required. Commensal urethral microbiota, urinary pathogens and environmental bacteria may contaminate urine and the enzymatic activity by microbial contamination can cause severe modifications to the excreted compounds, in particular to doping-relevant peptides such as hormones and growth factors. Since delays are inevitable and sample refrigeration is not always possible and effective, sound methods for preserving and stabilizing urine samples are desirable.
This Project involves the use of Volumetric Absorptive Microsampling (VAMSTM) and Dried Urine Spot (DUS) strategies for the collection of dried microsamples processed by means of streamlined workflows to be developed ad hoc, for high-throughput LC-MS/MS analysis of several peptide hormones and growth factors (i.e. GHRP-1, GHRP-2, GHRP-6, hexarelin, alexamorelin, triptorelin, AOD9064, CJC-1293, desmopressin, TB-500, hCG and ACTH). DUS and VAMS approaches represent promising alternatives to the procedures currently performed by anti-doping laboratories, allowing analyte stabilization by water loss and the consequent broadening of their detection window. This innovative sampling produces logistics savings due to the small transported volume (by air shipments and through customs) and to the possibility to keep the specimens at room temperature, with significant implications on overall analysis cost. 
The ultimate goal of this project is to establish and validate feasible but reliable protocols for the collection of urine microvolumes, stably storable and shippable with no particular precautions. As a proof of concept, instrumental analytical methods will be developed, validated and applied for the stability assessment of several peptide hormones and grow factors in dried urine microsamples stored at different conditions. In parallel, stability will be assessed in classic fluid urine stored at temperature-controlled conditions and thorough comparisons with dried microsamples will be carried out.

Main Findings: 

•    Innovative microsampling and pretreatment procedures based on dried matrices (DUS and urine VAMS) were optimised for the application to urine specimens for anti-doping purposes. Important experimental parameters were studied and specifically optimised. 
•    Original MS, HRMS and FT-IR methods were used for peptide chemical identity confirmation; then LC-MS/MS and LC-HRMS methods were developed for the simultaneous analysis of the peptides of interest in dried urine microsamples. After suitable study of the experimental conditions, the final methods provided good, solid performance within relatively short run times.
•    The LC-MS/MS and LC-HRMS methods were validated according to current guidelines, with good results for all assays and all analytes, and with similar or better performances than those of comparable published methods.
•    The microsampling, pretreatment and analysis workflow was successfully applied to the study of peptide stability in dried matrices.
•    Mid-term stability assays were carried out, both on dried and fluid urine-based matrices, with outstanding results. In fact, DUS and urine VAMS were remarkably stable, even though they were kept at RT, with all studied peptides recovered in the 80-95% range at the end of the study period (3 months). The stability of the chosen peptides, which are known to be prone to degradation under common storage conditions, was greatly enhanced in comparison to fluid urine stored at freezing (-20°C) and ultra-freezing (-80°C) temperatures. Analyte loss in fluid urine at -80°C was regularly 5-10% larger at study end than the loss in dried matrices, with widely worse losses (up to 35% more) for fluid urine kept at -20°C.
The use of innovative microsampling media offers significantly interesting perspectives towards the development of engineered, highly reliable devices.

Code: ISF17D20AM

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 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. 

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..