Projets de recherche

Code: R16R03GJ 

Beta2-agonists are among the most commonly used drugs by athletes, which is related to the high prevalence of asthma and exercise induced bronchoconstriction (EIB) in this population.
This project will determine analytical urine thresholds for two beta2-agonist asthma drugs, salmeterol and terbutaline. 
Salmeterol is used to treat asthma and is permitted in sport when taken by inhalation in accordance with the manufacturers’ recommended regimen. There is currently no urine threshold for an adverse analytical finding (AAF) and athletes can freely administer supratherapeutic doses for doping purposes with impunity. The project will provide data on urine levels of salmeterol and other compounds related to its use found after inhaled dosing at both permitted (therapeutic) and prohibited (supratherapeutic) doses over a seven day treatment period.
Urinary thresholds and decision limits are a way to avoid excessive use of beta2-agonist asthma drugs by athletes and to lessen the burden associated with therapeutic use exemption (TUE) applications. One of the most common beta2-agonists, terbutaline, is widely used in Europe but is currently prohibited unless a TUE has been granted. Terbutaline is responsible for over three quarters of AAFs associated with beta2-agonists in doping control. The project will develop a urine threshold for terbutaline to discriminate between normal therapeutic use via inhalation, high dose (supratherapeutic) inhalation and oral ingestion of terbutaline.
Both salmeterol and terbutaline are chiral compounds administered as 50:50 mixtures of two enantiomers (stereoisomers) which are molecules with non-superimposable mirror images (analogous to right and left hands). Differences in the way the body excretes enantiomers of the same drug can be used to improve discriminatory capability of urine doping detection methods – this requires the use of advanced stereoselective UPLC-MS/MS assays to distinguish between enantiomers which will be used in this project.

Main Findings: 

Background. Terbuutaline, a short acting β2-agonist, is widely used in Europe and accounts for 76% of adverse analytical findings (AAFs) of β2-agonists in doping control. Terbutaline is a chiral drug consisting of a 1:1 ratio of two enantiomers ®-terbutaline and (S)-terbutaline, and given differences in metabolism between both enantiomers, the ratio of enantiomers may afford the ability to distinguish between oral and inhaled routes of administration.

Objective. To discriminate between therapeutic inhaled dosing and both supratherapeutic inhaled administration and oral dosing of terbutaline using a chiral assya for terbutaline applied to urine.

Methods. Part Ia: The study was crossover design, with 14 well-trained men and women undertaking three intervention arms; Inhalation regimen (low dose therapeutic) consisting of 1 mg twice daily for 7 days (2 mg/day); inhalation regimen (high dose supratherapeutic) consisting of 2 mg twice daily for 7 days (4 mg/day); prohibited oral regimen consisting of 10 mg daily for 7 days; prohibited oral regimen consisting of 20 mg daily for 3 days. Urine was collected 2 h post dose on each treatment day and (R)- and (S)-terbutaline determined using a chiral UPLC-MS/MS method. Part Ib: A further investigation of 25 mg/d oral terbutaline (15 mg morning + 10 mg afternoon) was undertaken in nine trained men for 4 weeks for comparison with urine collected 1-3 hours after administration on the first day, half-way through the intervention, and the final day. Part II: The study was an acute (24 h) PK study examining urine levels and R:S terbutaline ratio following dosing with 10 mg oral or 4 mg inhaled terbutaline and 1½ h exercise in highly-trained men with asthma.  

Results. Part Ia: Racemic terbutaline urine levels were highest in the supratherapeutic inhaled group. The sulfate metabolite was the main metabolite with some evidence of enantioselective metabolism. The urine log(R:S) terbutaline enantiomer ratio demonstrated excellent diagnostic performance at 2 h post dose with the ability to distinguish between oral and inhaled dosing, with sensitivity and specificity both greater than 98% based on an arbitrary cut-off value of 0.1.  Further discrimination between therapeutic inhaled (1 mg twice daily) and supratherapeutic inhaled (2 mg twice daily) dosing, was demonstrated using a urine threshold approach set at an arbitrary 1000 ng/ml with sufficient specificity (>98%), and while sensitivity was modest at 19%, adoption of this approach could potentially disincentivize supratherapeutic inhaled doping. Part Ib: resulted in similar findings to Part 1a with respect to log(R:S) ratio, noting that maximum levels were not achieved until week 4. Part II: In the acute PK study, urine levels were higher following inhaled administration than oral but the predictive value of R:S ratio was not repeated from Part Ia. 

Conclusion. The urine enantiomer ratio method shows some promise to discriminate between permitted inhalation and oral dosing of terbutaline in sport, and if combined with a rac-terbutaline threshold for supretherapeutic inhaled dosing, could pave the way for terbutaline in sport without the need for TUEs. Further work is required to investigate why this approach did not work in an acute single dosing scenario.

Code: ISF16D20FB 

The Athlete Biological Passport (ABP) represents an integral part of the anti-doping analyses. In the fight against doping laboratories rely on monitoring blood and steroid profile data to set up long-term, individualized profiles of athletes. To uncover a prohibited administration of pseudoendogenous steroids (class S1.1 of the WADA “List of Prohibited Substances and Methods”) laboratories monitor concentrations and ratios of various endogenously produced steroidal hormones, their precursors, and metabolites since almost 25 years. Several studies reported only very small naturally occurring intra-individual variations of urinary endogenous steroid ratios like testosterone/epitestosterone (T/EpiT),
androsterone/etiocholanolone (And/Etio), And/T, and
5α-/5β-androstane-3α,17β-diol (Adiol/Bdiol), that have been shown to be stable over months and even years in adult humans. In case of a misuse of pseudoendogenous steroids as doping substances, these ratios are altered. Thus, the ABP is used to uncover a prohibited administration of class S1.1 steroids.
However, it was shown recently that some other (non-prohibited) drugs may also influence the urinary steroid profile.
In this project we focus our attention on the class of non steroidal anti inflammatory drugs (NSAIDs), that are frequently used as pain killers and antiphlogistics. Specifically, NSAIDs have been shown to inhibit the steroid metabolizing aldo-keto-reductases (AKR) 1C, namely the 3α-hydroxysteroid dehydrogenase (AKR1C2) and the 17β-hydroxysteroid dehydrogenase (AKR1C3).
As no scientific data on the influence of these inhibitory NSAIDs on urinary steroids are available from literature, the project aims in closing this gap.

Code: 16C02CR 

The aim of the proposed project is the determination of reference distributions for cobalt in urine of elite athletes. Two cohorts will be compared: samples from athletes performing in endurance sports (i.e. sports prone to misuse of erythropoiesis stimulating agents), and samples from non-endurance sports. The reference distributions will help defining thresholds of cobalt in urine for doping control purposes, above which erythropoiesis was illegally stimulated by cobalt according to chapter S2.2 “Hypoxia-inducible factor (HIF) stabilizers” of the WADA Prohibited List 2016.[3]  
In order to stimulate erythropoiesis, athletes have to apply Co2+ ions (e.g. CoCl2) at relatively high doses, which results in urinary concentrations, which are clearly distinguishable from cobalt in negative controls and after e.g. cobalamin administration. In total, ca. 600 urine samples (females/males equally distributed) will be analysed by ICP-MS as well as regarding their statistical properties (distribution type, outliers). 

Main Findings: 

The first part of the project (“Establishing doping-related reference distributions for cobalt in human urine”) studied cobalt concentrations in urine samples of athletes. In total, 894 samples were collected worldwide and analysed by ICP-MS. About half of the athletes were from endurance sports (frequency of ESA-testing 30% or above according to WADA TD2014SSA), and about half from non-endurance sports (ESA-frequency < 30%). Furthermore, approx. 50% of the samples were from female, approx. 50% from male athletes. Non-parametric statistical analyses revealed for endurance-sports a median Co-concentration of 0.5 (0.8) μg/L (without and with specific gravity correction) for females (n=220) and 0.4 (0.6) μg/L for males (n=215). For non-endurance sports, the medians for females and males were 0.4 (0.6) μg/L (n=232) and 0.4 (0.4) μg/L (n=227), respectively. Based on the 5th and 95th percentiles the range of Co-values was 0.1 (0.2)-2.1 (2.4) μg/L for non-endurance and 0.1 (0.3)-4.9 (8.9) μg/L for endurance sports. A significant difference between the medians of Co measured in endurance and non-endurance samples (without or with SG correction) as well as the medians of Co in male and female samples (without or with SG correction) was found (p < 0.01). Seventeen samples (w/o specific gravity correction; all IC, 15 from endurance sports) showed Co-concentrations above 10 μg/L with a highest observed value of 948.0 (653.8) μg/L. Two of the 17 athletes declared cobalamin intake. Their values were 55.1 (78.7) and 10.5 (9.5) μg/L, respectively. In order to define possible decision limits (DL) at the 99.99% level, data were log-transformed and “outliers” removed to achieve normal distribution. Different DLs were obtained for males and females, as well as endurance and non-endurance sports and for data without and with SG correction.

The second part (“Reference values for cobalt in serum and urine after cobalamin /vitamin B12) administration”) investigated Co-concentrations in urine and serum samples after application of cobalamin (50/1000 μg cyanocobalamin oral, 1000 μg cyanocobalamin intramuscular, 1000 μg hydroxylcobalamin intramuscular). Only hydroxylcobalamin led to an increase in urinary and serum Co-concentrations. Maximum values were 11.7 (9.8) and 3.9 μg/L, respectively. No influence of cobalamin on ABP-blood parameters (HGB, Ret%) was found. However, the blood drawing system needs to be carefully selected and validated in order to not contaminate serum samples with cobalt as observed with the “butterfly needle system”.

In case a threshold for Co in athletes' samples is defined in the future, it will be necessary to remove cobalamin in confirmation samples as recently shown by Knoop et al. (Rapid Commun Mass Spectrom. 2020;34(7):e8649).

Code: ISF16D12JS

The steroidal module of the Athlete Biological Passport has now been in use for 24 months. The biomarkers used are testosterone, four of its metabolites and epitestosterone. Using these, five steroid ratios are calculated and monitored in the adaptive model. An atypical passport finding will be generated if any of these ratios are outside the individual reference limits, and it is the task of the Athlete Passport Management Unit to evaluate whether further confirmation (i.e. IRMS or follow up testing) is needed. With the exception of the extensively studied T/E ratio, the impact of doping or other confounding factors on the other biomarkers needs further investigation. The largest confounders of the steroid biomarkers are genetic factors, bacterial contamination, alcohol and certain non-prohibited drugs. These confounders are well known and the adaptive model compensates for these, or, in the case of alcohol and bacterial contamination, this is reported by the laboratory. However, it is evident after two years of monitoring steroid ratios in the biological passport that there is still large unexplained variation in the ratios. The origin and extent of this variation in actual doping test results and longitudinal profiles in athletes have not been investigated to date. 
This will be a retrospective study analyzing the variation in steroid profiles (single samples) for > 6000 urine samples, as well as longitudinal profiles (passports) in athletes with >10 samples in their steroidal passport (n ≈ 200), from the beginning of 2014 to date. In order to improve the future interpretation of steroid profiles, we will study the behavior of the biomarkers in e.g. different types of sport, in/out of competition samples, seasonal variations, in athletes using non-prohibited drugs as well as in reported AAFs for prohibited drugs (anabolic agents and/or hormones and metabolic modulators).

Main Findings: 

The steroid module of the Athlete Biological Passport has now been in use for about four years. Its usefulness has been proven when detecting doping with endogenous steroids, e.g. testosterone, especially in individuals with naturally low T/E ratios due to the UGT2B17 deletion polymorphism, with an increased number of positive IRMS analyses in samples with T/E < 4.0. However, the steroid profiles are complicated to interpret and there is still much variation in the five ratios that is difficult to explain. A large systematic study of natural variation and confounders of the steroid ratios and concentrations is needed in order to provide the scientists evaluating the passport with sharper tools, not only to select the profiles suspicious of doping, but also to be able to reject and not spend unnecessary time and resources on profiles showing atypical results due to natural causes. The ultimate goal is to be able to proceed with a passport case, where the steroidal passport is the only evidence of doping.

In this study, we used over 11 000 steroid profiles from Swedish and Norwegian athletes to determine both the inter- and intra-individual variations of all steroids and ratios in the steroidal passport. Furthermore, we investigated if these variations could be associated with factors such as gender, age, type of sport, collection time of day and time of year as well as if the sample was taken in competition or out of competition.

We show that there are factors reported in today’s doping tests that significantly affect the steroid profiles. There were large interindividual variation in the steroid profiles and only part of this variation, up to 16 %, could be explained by the factors studied. The factors with the largest influence on the steroid profile was what type of sport classification the athlete belonged to and if the urine was collected In or Out of Competition. For women all steroids showed higher levels when collected IC than OOC except for 5βAdiol. T/E, A/Etio and 5αAdiol/5βAdiol show higher levels in competition whereas A/T and 5αAdiol/E are lower. For men all ratios but A/T were affected but to a lesser extent. There were also significant differences based on what time of day and time of year the urine sample was collected.

If these significant changes are relevant when longitudinally monitoring athletes in the steroidal module of the ABP, must be further evaluated.

Code: 16E12HH 

Gene doping represents a threat to the integrity of sport and the suitable for publication on WADA's website health of athletes. The anti-doping community has been focusing efforts on developing a test for its detection. The current methodology to detect doping genes in an athletes’ blood uses the polymerase chain reaction (PCR) that targets unique sequences in a doping gene, which correspond to exon-exon junctions in the intronless transgene. These so-called real-time PCR assays detect unique sequences in the complementary DNA (cDNA) for human erythropoietin (EPO) and other doping genes such as insulin-like growth factor-1, growth hormone, growth hormone releasing hormone and follistatin. 
As the sequences of cDNA of Epo and other doping genes are known, it is relatively easy to aggravate these tests, which will then result in a false-negative result. Recently, we developed a new gene doping detection assay that will overcome this problem. The test is based on targeted sequencing of doping genes with potential to detect any doping gene in any context with a very high sensitivity. Using an in-house designed next generation sequencing assay, we developed a gene doping detection assay for cDNA of EPO which targets all potential exon-exon junctions of all possible EPO-transcripts. 
We propose to evaluate and further develop a multiplex ‘gene doping detection panel’ which targets genes for, among others, insulin-like growth factor-1, growth hormone, growth hormone releasing hormone and follistatin. The panel allows simultaneous detection of several ‘sport-specific’ genes in one sample, reducing the test’s cost and turn-around-time. This research is crucial in the development of a reliable routine method for detection of gene doping that may be potentially used in all sports. 

Main Findings: 

The main aim of the project was to evaluate and further develop a next generation sequencing-based multiplex ‘gene doping detection panel’. First we developed probes for the detection by sequencing of Erythropoietin, Insulin-like growth factor and Growth Hormone. Second, we evaluated the developed probes in the multiplex sequencing of these genes. Finally, we optimized and fine-tuned the developed method and determined the sensitivity of the developed method.

Our results show that, using the developed probes for next generation sequencing, we were able to simultaneously detect plasmid-derived cDNA copies of Erythropoietin, Insulin-like growth factor and Growth Hormone in a background of genomic DNA with 100% specificity. We were able to detect EPO GH1, GH2, IGF1 and IGF2 cDNA in concentrations below 0.01 percent gDNA at all exon-exon boundaries. For quantification of the amount of cDNA we spiked a GFP plasmid into the samples and found stable numbers of GFP across samples, enabling quantification of gene-doping cDNA levels.

Code: ISF16D21LE 

The administration of recombinant human growth hormone (rhGH) and/or small peptides, i.e. GH releasing factors (GHRF) that stimulate the endogenous production of GH have increased recently as a result of the availability and lack of sensitive tests. Two independent immunoassay methods are currently being employed to detect rhGH doping as well as mass-spectrometric approaches to find GHRFs.  
Our research proposal encompasses projects designed to investigate how the use of rhGH and GHRFs affect the traditional markers as well as putative markers such as miRNA. We will conduct a study in healthy male volunteers that will be given rhGH (Somatropin – two different doses) daily for one week. Some participants will be given two doses of sermorelin (Geref), a GHR peptide, for one week. Urine and serum samples will be collected several times prior to the administration in order to study the different markers longitudinally. Moreover, the steroid profile will be monitored in relation to hrGH/GHRP administration in order to see how the biomarkers of the different ABP modules interact. The use of small GH-releasing peptides is difficult to study in controlled settings since small peptides are not available as traditional drugs. Here we will use samples from patients, both men and women, self-reporting doping with peptides (as well as other doping agents) in order to see which peptides can be identified with the different approaches. 

Main Findings: 

It was found that the GH isoform ratio may detect rhGH intake when 1 and 4IU/day were administered for two weeks to healthy men. Using the biomarker test, none of the participants (n=9) displayed a GH2000 score above the population-based score of 9.98. However, when longitudinally monitored, the GH2000 score and its components IGF-I and P-III-NP, most of the participants showed values outside their individual calculated thresholds (mean four baseline values ±3 SD). Also, the longitudinally testing approach ws studied in four individuals in relation to 5 days GHRH(1-44) administration. It was found that monitoring of IGF-I may be useful for identifying also the intake of GHRH. Additionally, it was investigated if rhGH/GHRH administration exert an impact on ABP biomarkers. A minor increase i RET% and OFF-score after rhGH treatment was found, not resulting in any atypical passport findings. The urinary steroid profile, as well as serum concenctrations of androgens were not affected by rhGH. Moreover, the validity of putative biomarkers was assessed. It was concluded that miRNAs previously associated with GH supplementary treatment were not affected by the rhGH doses given here. Previous findings that fibronectin 1 may be a promising additive protein for detection of rhGH was confirmed.

Longitudinally monitoring of IGF-I and P-III-NP in an endocrine module may be a promising method in the future to increase the chances to detect rhGH/GHRH doping in men. The inclusion of additionaly biomarkers such as fibronectin 1 may increase the effect and/or detection window in some individuals.

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Code: ISF16E11FP 

The variable effects of anabolic androgenic steroids (AAS) on increasing skeletal muscle mass and strength has been well documented, as is the misuse of AAS in sport. Recent evidence suggest either a long- or short-term exposure to AAS might have a sustained effect on muscle morphological changes, for example, with increased muscle mass, capillary per fibre, muscle fibre size and myonuclei density, leading to improved performance. A positive correlation between the number of myonuclei and training response
following exposure to AAS in a mouse model seems to suggest a link between the formation of extra myonuclei and the extent of “muscle memory”; an idea that requires further investigation in humans.

Strength training can also increase the number of nuclei in muscle fibres. Adaptations in muscle mass by strength training are significantly enhanced in previously trained individuals despite a prolonged detraining period. Given the persistence of muscle nuclei, the use of AAS combined with training will have a greater impact on muscle hypertrophy than either training or steroid use alone. To detect the long-term effect of AAS and training (even after drugs are no longer detectable in the human system), abnormal changes in
skeletal muscle morphology illustrated by specific gene markers in response to the stimuli exist and will allow these molecular signals to be picked up by modern gene screening methods. In the proposed project, gene expression profiling of skeletal muscle in response to AAS exposure will be carried out using total RNA-seq. The molecular, histological and training response markers will be integrated for the detection of short- and long-term effects of AAS and will be incorporated into the steroid module of the Athlete Biological
Passport for improved validity and reliability.
 

Main Findings:

Fat Free Mass (FFM) of RP2-5, who ceased AAS usage ≤2 weeks prior to visit one with 19-28 weeks between visits, decreased by 3.9-4.7 kg. FFM of RP1, who ceased AAS usage 34 weeks prior to visit one with 28 weeks between visits, decreased by 0.9 kg. Fibre CSA decreased for RP1 and RP2 between visits (7566 vs 6629 µm²; 7854 vs 5677 µm²) whilst myonuclei per fibre remained similar (3.5 vs 3.4; 2.5 vs 2.6). Fibre CSA (7167 vs 7889 µm²) and myonuclei per fibre (2.6 vs 3.3) increased for RP3 between visits. Mean fibre CSA was significantly higher in RT-AS (n=17) (8160 ± 1769 µm²) compared to C (n=5) (6477 ± 1271 µm², p=0.028). There were no significant differences between C, RT (n=15), RT-AS & PREV (n=6) for myonuclei per fibre. Myonuclei per fibre and CSA for all biopsied participants (n=43) was significantly correlated (r=0.8, p<0.001). 
All whole blood samples (n=60) were subjected to RNA-Seq as they had purified total RNA that was of sufficient concentration, purity, and integrity. In comparison with RT participants who ceased AAS exposure <1 week ago (n=10), 1-2 weeks ago (n=5), 10-50 weeks ago (n=4), and >52 weeks ago (n=7), had 612, 464, 173 and 188 genes differentially expressed, respectively. RP2-5 had 33 differentially expressed genes between visits which were mainly associated with the interferon signalling pathway linked to the immune system. RNA-Seq of corresponding muscle samples is currently underway. The finding of comparable myonuclei per fibre numbers despite decrements in fibre CSA post AAS usage is consistent with the “muscle memory” mechanism. RNA-Seq identified 33 genes that could provide novel beneficial biomarkers. 
Further research, particularly the longitudinal monitoring of AAS users post usage is required to confirm these intriguing findings.