Projets de recherche

Code: 24T04CR

Unfortunately, the mass difference between rEPO and uEPO/bEPO is not large enough to result in a clear separation on SAR- and SDS-PAGE. Instead, a “mixed” band containing both compounds is frequently observed. The lower the rEPO concentration becomes, the fainter the area above the endogenous band becomes (“diffuse or faint area” according to TD2024EPO, p. 17). In case of rEPO-doping, this faint area must extend beyond the cut-off line defined by the band apex of Epoetin-δ (Dynepo) or Epoetin-α.

The weaker the area becomes (e.g. due to intravenous rEPO microdosing), the more difficult the band profile is to interpret. So far, the interpretation relies solely on the experience of the analyst and second opinion provider(s) and thus has been challenged by the athletes’ attorneys in the past. The aim of this project is to define a criterion that enables an objective assessment of the diffuse area. Since all WADA labs use GASepo platform for image analysis, the criterion will be developed using this software. Two approaches will be evaluated: (1) the ratio of relative pixel volumes above the rEPO migration cut-off line (“Dynepo-line”), and (2) the differentiation of the band side plot.

The evaluation will be first done with urine samples after SAR-PAGE analysis. If successful, studies on serum/plasma and possibly DBS will be performed immediately. As part of this project, a revision of GASepo will be implemented to enable an automated cutting of bands along the “Dynepo” line. The project consists of three phases, namely (1) the intralaboratory phase phase (including a GASepo revision) performed by our lab leading to the proposal of threshold criteria for "AAF", "ATF" and "negative findings", (2) the iterlaboratory validation phase performed by 4 WADA accredited laboratories, and (3) the blinded test samples phase

Code: 241C18AM

Tapentadol and dihydrocodeine are potent narcotic analgesics. They are currently on WADA's Monitoring Programme as there is concern that they may be used as replacement drugs for the recently prohibited tramadol, to confer a performance advantage by reducing exertional pain and allowing an athlete to work even harder. The drugs' centrally mediated side effects may also present a risk to athletes by reducing motor control which may increase risk of injury to the athlete and their competitors.

This project will employ an experimental design that identifies whether these narcotics allow

athletes to work harder by reducing pain, and thus allow for a better performance. It will also assess whether they impair a rider's stability and control of their bicycle. Therefore, the main outcome of this project will be to provide robust experimental evidence to inform whether the in-competition use of tapentadol and dihydrocodeine should be regulated due to potential ergogenicity and athlete safety.

Twenty-one highly trained cyclists will complete a randomised, controlled, double-blinded crossover study design. Participants will ingest tapentadol, dihydrocodeine, or a placebo and complete an assessment of their balance/stability during intense cycling followed by a laboratory cycling task that replicates the time/intensity demands of professional road cycling. Together, the cycling tasks will involve fixed-intensity and self-paced time trial cycling, amounting to approximately 1.5 hours of hard cycling - a duration and intensity that is representative of the context in which narcotic analgesics are purportedly taken to enhance performance. Exercise performance (completion time), perceptual responses (perceived pain and effort) and participant's control of their bike (lateral movement) will be compared between conditions, with results used to inform consultation with WADA regarding the S7 Narcotics category of the Prohibited List.

Code: 241C07MT

With the ongoing development of new drugs, the addition of newly developed substances to the World Anti-Doping Agency's Prohibited List has been and continues to be a critical part of anti-doping research. Also, the knowledge of the metabolism of such new compounds is an important tool to identify suspicious samples in routine doping controls. In recent years, ryanodine channel complex stabilizers (Rycals) have received growing attention in the anti doping community due to their potential performance-enhancing effects, by stabilizing the calcium channels which are located in the skeletal muscle. To date, the compounds S107, JTV-519, ARM036 and ARM210 have been investigated for such properties and hence are potential doping agents. While the metabolic behavior of S107 has been investigated, little is known on the metabolism of JTV 519, ARM036 and ARM210. Additionally, the ARM 036 and ARM 210 are either of limited availability or not commercially available.

In this project, we describe the synthesis of ARM 036 and ARM 210 using a multi step approach. After synthesis, the Rycals S107, JTV 519, ARM 036 and ARM 210 are metabolized in vitro using human liver preparations. The samples are analyzed by means of liquid chromatography (LC) coupled with to a high resolution mass spectrometer (HRMS) to identify possible transformation products. These possible metabolites are then analyzed by tandem mass spectrometry (MS/MS) to gain further information on their possible structures. Suitable metabolites are subjected to chemical synthesis in order to confirm their proposed structures. Precursor compounds as well as identified metabolites are potential targets for doping control analysis and are therefore of particular interest for anti doping research

Code: 241A15MT

In 2024, the Glucagon-like-peptide-1 (GLP-1) analog Semaglutide has been included in the WADA monitoring program. While the underlying idea for the development of this peptide-based drug was the improved therapy of diabetes mellitus type II, the resulting anti-obesity properties cause an alarming run in the non-diabetic community (incl. athletes) for Semaglutide-containing drugs like Ozempic or Wegovy, presumably used for weight management purposes. The drug is administered once a week by subcutaneous injection of 0.25 to 2.5 mg and owns a half-life of approx. 8 days. In contrast to other peptide-based drugs, circulating time and levels are comparably high, thus, the analysis of blood and DBS samples by means of LC-MS appears sensible and advantageous. Due to the extensive metabolism and low renal clearance, neglectable levels of intact Semaglutide are excreted, and relatively non-characteristic metabolites appear in urine only.

Besides Semaglutide, also other GLP-1 receptor agonists are available (e.g. Liraglutide, Lixisenatid and Tirzepatide) as approved medications with comparable biological effects. Noteworthy, the most important analogs Semaglutide, Liraglutide and Tirzepatide share an incorporation of artificially modified lysine residues, which enables the aimed action profile after application. This modification facilitates the analysis by LC-MS considerably due to the explicitly unnatural property of these structures. In this study, it is planned to include several of these GLP-1 analogs into one testing procedure for blood and DBS samples. After successful development, also the analysis of post administration samples with paired blood/DBS and urine-samples from volunteers treated with different GLP-1 receptor agonists is planned in order to enable the comparison of plasma/whole blood and urine levels.

Code: 241A14MT

Kisspeptins play an important role in human reproductive health by regulating the pituitary secretion of gonadotropin-releasing hormone (GnRH). This renders them therapeutically interesting for the treatment of hypogonadism or anovulation, as well as hormone-dependent cancers. However, the stimulation of GnRH release (and thus indirect stimulation of gonadal testosterone synthesis) also harbors the potential for misuse for doping purposes, which is why Kisspeptins have been introduced to the WADA Prohibited List in 2024.

Within this research project, we aim to develop a liquid chromatography-mass spectrometry (LC-MS) based method to detect the potentially endogenously occurring peptide hormone Kisspeptin-54, its fragments Kisspeptin-14, Kisspeptin-13 and Kisspeptin-10, as well as the synthetic Kisspeptin receptor 1 (KISSR1) agonist TAK-448 in human urine and serum.

Following method optimization and validation, a suitable number of urine and serum samples will be analyzed as a reference population, to investigate tentative endogenous Kisspeptin levels and to assess a possible need for threshold levels in doping control analysis. Furthermore, metabolism studies will be included, employing suitable in vitro approaches. The resulting data will be evaluated with regard to specific peptide metabolites, that can help to improve the method’s detection capacity for authentic samples following Kisspeptin administration

Code: 241A04MT

Already in 2006 the synthesis of (17α,20E)‐17,20‐[(1‐methoxyethylidene) bis (oxy)]‐3‐oxo‐19‐norpregna‐4,20‐diene‐21‐carboxylic acid methyl ester (YK-11) was published and investigations into its pharmacological properties demonstrated the potential of YK-11 as a selective androgen receptor modulator (SARM). Albeit YK-11 has not been approved for humans until now, it was first detected in a black-market product in 2017 and several times thereafter. According to the Dietary Supplement Label Database issued by the National Institutes of Health, at least 6 different preparations are currently available in the United States alone claiming to contain YK-11.

In 2018 the human metabolism of YK-11 was investigated and two main urinary metabolites were detected suitable to be implemented into routine doping control procedures. YK-11 itself was not detected in urine, presumably due to the fact that it undergoes fast metabolism in humans. As the parent compound YK-11 will not be found in urine after administration, doping controls can only rely on the identified urinary metabolites. Both metabolites were synthesized in order to elucidate their chemical structure, but no reference material at a larger scale was synthesized at that time suitable to be provided to all doping control laboratories worldwide.

In order to enable all laboratories to implement the urinary metabolites of YK-11 into their screening procedures, the production of a suitable reference material of both YK-11 metabolites will be inevitable. Therefore, the aim of this research project is to reinvestigate the different potential routes of chemical synthesis, optimize these and produce reference material of both YK-11 metabolites in order to distribute this to all accredited doping control laboratories.

Code: 241A02MT

In sports drug testing, isotope ratio mass spectrometry is employed to enable the differentiation between endogenous and exogenous sources of urinary steroids, i.e. steroids produced inside the body or administered. Testosterone or testosterone-prohormones may be used by athlete for performance enhancement. As these steroids are endogenous, their presence alone in human urine is not suspicious. Only if urinary concentrations may exceed individual thresholds, a confirmation of the exogenous source becomes necessary and carbon isotope ratios proved to be the method of choice here.

Sample preparation protocols for isotope ratio mass spectrometry-based method are complicated and time-consuming as the purity of analytes is crucial and a prerequisite for valid isotope ratio determinations. Currently applied sample preparations encompass solid and liquid-liquid extraction steps to pre-purify and concentrate urine samples prior to the final clean-up step employing high performance liquid chromatography and fraction collection. This powerful tool for sample clean-up comes along with relatively long run-times per sample of more than 45 min and it may be necessary to use even a two-fold separation here.

This research project aims for improving this crucial step in sample preparation and shorten the run-times per sample by employing supercritical fluid chromatography coupled to a novel fraction collection tool. In supercritical fluid chromatography, pressurized carbon dioxide is used as solvent offering unique features for chromatographic separation not directly comparable to the commonly applied mixtures of water and organic solvent. Therefore, a novel sample preparation method suitable for isotope ratio mass spectrometry will be developed and validated within this project investigating the benefits of supercritical fluid chromatography in sports drug testing. To evaluate the new method and facilitate its implementation into routine doping controls, reference population-based values will be determined and compared to existing routine applications.

Code: 23A20MP

GC-C-IRMS is used to confirm the exogenous origin of endogenous anabolic steroids. Samples are analyzed with a fast initial testing procedure to isolate suspicious samples and, afterwards, the suspicious samples are analyzed with an IRMS confirmatory method. High throughput IRMS will allow a substantial expansion of CIR analyses, enabling a considerable increase in doping violation detections and the set-up of an efficient CIR ABP module. High throughput IRMS is also highly desirable in those cases where a large number of samples needs to be tested on IRMS in relative short period of time e.g., preseason/regular season testing and Olympic Games.

Code: 23B02CR

Chapter S2 of WADA’s Prohibited List 2023 (“Peptide hormones, growth factors, related substances, and mimetics”) lists Erythropoietin Receptor Agonists (ERAs) and Transforming growth factor beta (TGF-ß) signalling inhibitors (luspatercept, sotatercept) under chapter 1 (“Erythropoietins (EPO) and agents affecting erythropoiesis”) as prohibited substances. Currently, SAR- and SDS-PAGE are the most frequently applied techniques for the initial testing and confirmation procedures (ITP, CP) for ERAs in WADA accredited laboratories worldwide (TD2022EPO). While electrophoretic detection methods for TGF-ß signalling inhibitors were also developed, they are not yet part of the technical document. In 2019 and 2020, the first luspatercept-based pharmaceutical (Reblozyl®, “luspatercept-aamt”) was approved by FDA (USA) and EMA (Europe).

Hence, routine testing for TGF-ß signalling inhibitors will become necessary in the near future. For the detection of erythropoietins and TGF-ß signalling inhibitors in blood and urine immunoaffinity purification is required before electrophoretic separation. Due to significant structural differences between these compounds, three different capture antibodies have to be employed, i.e. anti-EPO, anti-activin receptor type IIA (ACVR2A), and type IIB (ACVR2B) antibodies for epoetins, sotatercept and luspatercept, respectively. A protocol for the combined immunopurification of ERAs, sotatercept and luspatercept followed by isoelectric focusing (IEF-PAGE) was published in 2019, another one for ERAs and luspatercept in combination with SDS- and SAR-PAGE in 2021. Both protocols were based on covalent immobilization of relatively large amounts of the capture antibodies on magnetic beads.

Consequently, the beads had to be re-used several times for cost-reduction. Additionally, sotatercept was not included in the protocol for SDS- and SAR-PAGE. We already developed individual protocols for capturing sotatercept and luspatercept in serum samples, which use non-covalent immobilization of very small antibody amounts on anti-antibody coated magnetic beads. Additionally, we presented a similar cost-minimized protocol for the purification of EPOs from blood and urine at the Cologne workshop in March 2023. The plan is to combine these protocols in order to simultaneously purify all three compounds from the three sample matrices (serum/plasma/urine). Another disadvantage of the 2021 protocol was, that EPOs and luspatercept could not be detected in a truly multiplexed way after electrophoretic separation and Western blotting.

The reason were interferences caused by non-specific binding of the two detection antibodies with co-eluted proteins (the protocol used the same polyclonal antibody for capture and detection of luspatercept). Hence, the membrane had to be first incubated with an anti-EPO antibody followed by re-incubation with the anti-ACVR2B antibody. Contrary to that, the proposal of this project is that (1) the target proteins will be simultaneously detected by incubating the blot membrane with a mixture of all three detection antibodies, (2) it will also include sotatercept, and (3) the protocol will also be applicable to urine (it was shown in 2022 that luspatercept is also partly excreted in urine).

Code: 23E04FP

It is well known that the use performance-enhancing substances by athletes provide an unfair advantage and doping is contrary to the ‘the spirit of sport’. However, a randomised response surveys conducted between 2011-2018 indicate that >15% of athletes have doped with banned substances. Yet in 2018, WADA reported only 2% positive samples, with hormone doping accounting for >50% of adverse analytical findings. These statistics suggests that micro-dosing, off-competition doping and cyclical (on/off)/pyramiding patterns of administering anabolic steroids (AAS) have allowed athletes to evade current detection methods, while beneficial for building muscle mass, strength and performance. In addition, drug administration studies in humans have demonstrated that testosterone dose-dependently increases satellite cell and myonuclei number, muscle fibre cross sectional area (CSA) and induces hypertrophy of muscle fibres. These benefits may remain long after cessation of AAS use and/or detraining leading a muscle atrophy, when a new stimulus (i.e., returning to training) is given, the performance is enhanced, a phenomenon known as “muscle memory”. The “muscle memory” associated with AAS could explain why androgen cycling has been so effective for doping athletes. AAS treatment in mice for 14 days has been shown to increase myonuclei number by 66%, and these are retained despite drug withdrawal and contribute to faster muscle growth in response to muscle overload (Egner et al, 2013). These findings indicate that even brief exposure to AAS may provide long-term benefits for muscle performance. Increasing understanding of the mechanisms associated with AAS muscle memory is thus vital for enhancing anti-doping detection. Other mechanisms, besides myonuclei accretion/retention, have also been implicated with the muscle memory phenomenon, and the understanding of this process is crucial for the identification of long-term biomarkers. Therefore, the aim of this research is to identify relevant biomarkers associated with past-AAS use. Building on previous work, we will generate and characterise a mouse model of AAS muscle memory and explore the associated mechanisms using transcriptomic, phosphoproteomic and metabolomic approaches. In our previous research funded by a WADA research grant (16E11FP), we have established a tissue biobank (muscle biopsies, saliva, urine, serum) from powerlifters that either do not use AAS, actively use AAS or have previously used AAS. Guided by findings in mice, we will examine the utility of these markers for identifying current and past-AAS use in our biobank of tissues. Our findings are envisaged to enhance anti-doping detection, provide guidance for appropriate punitive measures in response to positive tests, and contribute to ongoing debates regarding transgender athlete inclusion in elite competition.