Projets de recherche

Code: 242B02CR

Chapter S2 of WADA’s Prohibited List 2024 (“Peptide hormones, growth factors, related substances, and mimetics”) lists under sub-chapters 2 and 3 “Peptide hormones and their releasing factors” and “Growth factors and growth factor modulators” as prohibited substances. Chapter S4 “Hormone and metabolic modulators”, sub-chapter 3 (“Agents preventing activin repetor IIB activation) additionally mentions “Activin receptor IIB competitors” (e.g. ACE-031) and “Myostatin inhibitors” (e.g. follistatin, myostatin propeptide) as prohibited proteins. Most of these compounds have not been pharmaceutically approved but are available on the black market. In the past, only studies on injectable black market peptides and proteins were performed. They led to the identification of unknown compounds with doping relevance and revealed the presence of “tags”, which made them clearly distinguishable from the body's own proteins (e.g. follistatin with His-tags, myostatin propeptide with GST-tags). Recently, an increasing amount of nasal sprays containing growth promoting peptides and proteins (e.g. AOD-9694, ACE-031, CJC-1295, Follistatin 344, GDF-8) appeared on the black market. So far, no comprehensive study on these products has been performed. Hence, it is unclear whether the compounds contained in nasal sprays are detectable with the current methods or not. For example, some might contain “tag-free” proteins that pass through mucous membranes more easily but are undetectable using existing methods. Within the scope of this project, ca. 50 peptide/protein-based nasal sprays will be purchased on the black market and analyzed with high resolution mass spectrometry and gel-electrophoresis. Subsequently, their detectability with the currently applied methods will be evaluated. In case of unknown compounds, top-down and bottom-up proteomics will be applied for identification. For compounds which cannot be detected with the current methods, alternative strategies will be proposed.

Code: 241C03JFN

The REV-ERB alpha and beta agonist SR9011 was initially synthesized to explore the relations between sleep disorders and circadian rhythm in rodent models. In parallel, studies highlighted that SR9011 ameliorates muscular growth and affects mitochondrial composition, making this compound attractive to athletes who desire to improve their sports performance. Consequently, SR9011 was placed on the WADA prohibited substance list in 2018. However, only the compound was included because of poorly understood and characterized metabolism. Since January 2024, some metabolites of SR9011 have been included in the WADA list of banned substances. So far, two publications have suggested structures some metabolites, although their structure must be re-examined. The first objective of this project is to adapt our previous protocol using human subcellular liver fractions to spheroid cell culture to corroborate the presence of oxidative metabolites. By this, we aimed to refine our previously suggested metabolites to reinforce those susceptible to be detected in urine samples. The spheroid which are terminally differentiated hepatic cells derived from a human hepatic progenitor cell line will be used to obtain metabolites. To achieve metabolites generation, spheroid and SR9011 will be incubated in a cell culture medium. In parallel, considering the phase II metabolism of SR9011 has not been addressed to date, the investigation of glucuronide conjugates on SR9011 will constitute the second objective. Finally, since WADA has included four oxidative metabolites in the list of banned substances and considering that no reference materials are currently available, the third objective of this proposal consists of adapting small-scale synthesis routes to large-scale production of those metabolites in readiness to provide enough reference material to worldwide anti-doping laboratories.

Code: 242E03KC

Gene doping, which is banned by WADA, is the alteration of an athlete’s DNA in order to obtain athletic advantage. Rapid advances in genome editing technologies pose a significant future threat to fair competition and athlete safety. This project aims to address this challenge by focusing on detecting performance-enhancing genome modifications.

This research will begin with animal models to study how effectively genome editing can be achieved in tissues relevant for doping and then be detected non-invasively. From there, the research will work towards developing accurate, affordable, and scalable methods to detect genome editing in humans in the context of performance enhancement.

The project consists of three main objectives:

1. Animal studies. Using state-of-the-art genomic technologies, we will perform genome editing experiments in mice. This will help us assess the efficiency of genome editing across different tissues and identify non-invasive detection methods.
2. Human genome editing detection assay. We will create a targeted deep sequencing tool to detect edits in 20-30 human genes linked to doping. We will develop software to differentiate genuine edits from experimental errors and naturally occurring variations.
3. Validation. We will validate the assay by applying it to lab-edited primary human hematopoietic stem cells and employing a blinded computational analysis to identify genome editing events.

This pioneering research will provide crucial insights into how new genomic technologies could be used in doping and on the factors that affect editing detectability. Our goal is to create novel, cost-effective methods for detecting genome editing in humans, supporting anti-doping efforts worldwide.

Code: 242B05SV

ERA analysis has been repeatedly legally challenged. One of the criticised points is potential protein overload related to exercise induced proteinuria. Although exercise induced proteinuria has been investigated for IEF and SDS-PAGE and samples are immunopurified, no standardized data are available for the currently used SAR-PAGE method.

Although both methods apply the same measurement principle, which is basically separation by migration behaviour based on molecular weight and posttranslational modifications such as glycosylation or pegylation of the molecule, the detergent is different. In addition, the analysis method evolved with the introduction of the biotinylated version of the AE7A5 Anti-EPO antibody being now used for the analysis, and no data exist which can exclude the possibility that contents of exercise urines might cross-react with, for example, the Streptavidin-HRP complex which is used for the visualization by chemiluminescence.

Further, it has not been demonstrated whether high intensity exercise in a hypoxic environment could further affect the ERA analysis, for example by a higher excretion of erythropoietin. Therefore, it is planned to investigate urine samples from 30 athletes who are subjected to an exercise protocol on a treadmill or cycling ergometer known to induce proteinuria. In addition, urine samples from 10 athletes following an acute hypoxic exercise protocol will be collected and examined.

Code: 241E07CS

The detection of gene and cell doping in athletes is crucial to maintain fair competition and prevent the use of performance-enhancing substances that can be harmful to an athlete's health. In this project, we propose the development of all-in-one gene doping detection chip paper based on Multiplexed Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a system with on-chip target gene amplification and detection functions. This chip will facilitate the sequential execution of multiple isothermal nucleic acid amplification and CRISPR/Cas12a sensing reactions directly on small blood samples, eliminating the need for separate pre-processing steps. Our proposed all in one gene doping chip paper features three key components: a sample pad, four conjugation pads, and a test membrane. The sample pad, preloaded with dried reaction buffer, allows for simple heat treatment of whole blood, releasing transgene DNA directly onto the chip without pre-preparation step. Each conjugation pad, linked to a specific target gene, combines on-chip RPA with a Cas12a reaction. In just 15 minutes at 37°C, RPA amplifies the target gene using specific primers. Next, Cas12a cleaves a reporter molecule (biotin-FAM) in the presence of the target exogenous DNA, but leaves it intact in its absence. Finally, the test membrane, pre-coated with streptavidin and anti-anti-FAM lines, captures the reporter depending on the target presence.

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