Projets de recherche

Dr. Andrew Levy, UK

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Code: 13D08AR 

Our research program encompasses projects designed to investigate how the use of drugs (emergency pills and androgens) and genetic variation interfere with the Athletes Biological Passport (ABP). ABP is essential to detect doping with anabolic agents, especially with the decreasing doses evidently used by some athletes. However, there are many factors that may contribute to the inter-individual variability in steroid profiles, i.e. drug use, diseases, genetic variation etc. Preliminary results indicate that the use of an emergency pill influences the steroid profile and confounds the test interpretation. We will therefore investigate how the use of emergency pills affects the excretion of steroids in females. We and others have shown that UGT2B17 genetic polymorphism has a large impact on testosterone doping test. We will continue to evaluate the use of genetic testing of UGT2B17 as well as other polymorphisms in sport samples. Finally we will study how androgens affects the haematological module. 
It is of great importance that the athletes ABP will be able to compensate for all possible variability in longitudinal steroid profiles. More knowledge is therefore needed about how drug use and genetic variation may affect the ABP and hence outcome of doping tests.

Main Findings: 

Study 1: We show that DNA extracted from urine samples is not good enough for individual genetic testing in anti-doping work. We showed that UGT2B17 ins/ins men exert higher T/E than ins/ins females and that some del/del athletes (particularly men in power sports) had T/E ˃0.4. The CYP17A1, and UGT2B7 SNPs investigated could not explain the large inter-individual variation in urinary concentration of epitestosterone. 
Study 2: We have shown that the ABP ratios various randomly throughout the menstrual cycle, epitestosterone being the only metabolite that are significantly altered. The administration of an EC decreased the urinary concentrations of epitestosterone 24 hours after the intake, and it is possible that a use of EC could result in atypical finding in the ABP. 
Study 3: We conclude that testosterone but not nandrolone mediates significant effects on the hematocrit profile and gene expression of the EPO gene in blood. Moreover, testosterone increases the serum levels of the future ABP biomarker P-III-NP. It may be of importance to know how the different modules interact for the interpretation of the ABP results. We will continue to study these biomarkers in relation to each other, both at baseline and after the administration of different doping agents.  

Code: 13B08RG 

Growth hormone secretagogues (GHS) are molecules that stimulate the secretion of human growth hormone from the pituitary. They have proven to be potent agonists of hGH secretion reaching circulating hGH values that are even not easily achievable with rhGH administrations. As such, they are the prime candidate pharmaceuticals to replace rhGH abuse. GHS display large structural diversity and many molecules are under development by the pharmaceutical industry. In order to address the entire family of known and upcoming molecules it can be targeted what all GHS have in common: the interaction with the GHSR1a receptor. Based on this premise we developed and validated a competition assay using a radiotracer, 125I-ghrelin. Given the difficulties for testing radioactivity at some antidoping laboratories, and the recent implementation of chemiluminescent tube-based immunoassays for hGH we have studied the potential replacement of the radiotracer by a chemiluminescent one. Accordingly, the development of a method using biotinylated ghrelin and streptavidin labeled with a chemiluminescent tag could be effective to achieve this purpose. 
The main objectives of this project were: - Obtention of appropriate chemiluminescent tags bound to strep traviding and synthesis, purification, and charactesation of streptavidin conjugated to acridinium, not commercially available.
- Set-up of a competition assay protocol employing different chemiluminescent tags.
- Comparison of the radiotracer and the chemiluminescent tracers.
- Assessment of the chemiluminescent competition assay in urine samples.

Main findings:

The results submitted in this project showed the development of an efficient chemiluminescent method performed in three different chemiluminescent tags: acridinium (AC), alkaline phosphatase (AP) and horseradish peroxidase (HRP). This method, based in the interaction between biotinylated ghrelin and streptavidin labeled with a chemiluminescent tag, was evaluated following different steps. After evaluating biotinylated ghrelin in a competition binding assay, the Ki obtained was compared with the Ki values from competition assays performed with ghrelin without any tag, and no interference in the interaction with the receptor was showed. Biotinylated ghrelin appeared then as a suitable ligand. 
Subsequently, the optimum time of addition of streptavidin was tested to avoid any effect on the recognition of biotinylated ghrelin by the receptor,and it was determined its addition 40 min after of the starting of the competition assay. After synthesis and purification of streptavidin labeled with AC, the protocol was optimized for this and the other two chemiluminescent tags (streptavidin labelled with alkaline phosphatase AP and horseradish peroxidase HRP, respectively) through the evaluation of several conditions as: number of washes, membranes obtained from different amount of cells, alternative binding buffers, amount of ST per sample, presence or absence of detergent and process changes of adding of the chemiluminescent substrates, among others. The optimal protocols for each label were tested in a competition assay with different amounts of GHRP-2 in binding buffer and urine and the results showed the best sensitivity with HRP tag. In fact, the presence of GHRP-2 in urine samples obtained 1.5 hours after intravenous treatment with 100 μg of GHRP-2 from in a clinical trial with sedentary health volunteers treated was detected. 
In conclusion, the results shown in this project demonstrate the development of a chemiluminescent method based on interaction between biotinylated ghrelin and the GHSR1a receptor, which is more sensitive than other non-radioactive methods described so far but has less sensitivity than the radioactive method described previously by our own group.  

Code: 13D25MT 

The misuse of recombinant human insulin in sport has frequently been mentioned by confessing athletes but is not detectable with currently available methodologies. This is mainly due to the fact that there are no measurable differences to the endogenously produced hormone. Several attempts to establish diagnostic marker ratios to other endogenously produced hormones (C-peptide) failed due to unstable or highly variable conditions in the living organism. With the present study we aim to establish potential marker metabolites, which will uncover a surreptitious insulin application. Pilot studies demonstrated that there are substantial differences in the degradation/metabolism of insulin between the endogenously secreted and the recombinant and injected insulin. It is assumed, that these differences are due to the exposition of insulin to specific enzymes after subcutaneous or intramuscular administration. 
Several urinary metabolites were identified in earlier studies by this research group and an extension of this approach with most modern analytical instruments is planned, potentially revealing new indicative and diagnostic metabolites. Therefore, purification of target compounds from urine followed by the determination of insulin metabolite profiles by LC-MS/MS is planned, which might serve as diagnostic tool to uncover the misuse of recombinant human insulin. 

Main Findings: 

This study was conducted to explore the metabolic fate of subcutaneously administered recombinant human insulin. Due to the exposure to endogenous proteases in the subcutaneous tissue, a minor amount of the bioactive peptide hormone is cleaved to its truncated metabolite DesB30 human insulin, which still owns the full biological activity. In addition to that, DesB30 is a known by-product of recombinant insulin preparations (at ~ 1%) and, thus, it can enter the circulation from the injection site.  Post administration of recombinant human insulin, circulating DesB30 was identified in plasma samples of diabetic patients (type II). No specimen obtained from healthy volunteers with endogenous insulin analyzed in the context of this study contained DesB30, not even when the production of insulin was stimulated by ingestion of a concentrated glucose solution (OGTT).  The analysis of athletes’ routine doping control samples yielded analytical findings of trace amounts (=LOD) of DesB30 HI in 3 out of 64 samples. The additional monitoring of the respective C-peptide levels yielded inconspicuous results for all athlete samples but, conversely, suppressed C-peptide levels in diabetic individuals after HI administration.  Thus, the detection of DesB30 in athlete blood samples in combination with C-peptide levels represents a potential combination of markers for the misuse of recombinant human insulin in sports. Due to degradation processes, urine specimens were not found suitable for this approach. Furthermore, special care regarding the storage and sampling conditions are crucial for the collected plasma samples and degradation processes must be avoided by applying appropriate conditions during the whole post-sampling period until analysis. This is important for both inhibiting the degradation of HI to DesB30 HI on the one hand and stabilizing the C-peptide of the other hand. 

Code: 13D23SS

Several elite athletes were tested positive for clenbuterol and claimed that these findings were caused by the consumption of meat containing clenbuterol. From several studies and EU monitoring programs it is shown that clenbuterol may be present in meat and after consumption of meat concentrations of clenbuterol in urine can be found. To discriminate between clenbuterol administrated via a pharmaceutical preparation or by ingestion of contaminated meat products research was started in 2012 (WADA 11A18SS). The focus of this study was to determine if there was a difference between the ratio of the two enantiomers (left- and right-- hand form of clenbuterol) in meat, and if after consumption of meat there was a difference between the ratio of the two enantiomers in urine when compared to the ratio in urine after illegal oral administration of preparations to humans. 
In 11A18SS the proof is given that the hypothesis is feasible and the analytical methods are in place and capable to detect the differences.  In the proposed project the focus will be on establishing a relation between the consumption of contaminated meat and a change in ratio of clenbuterol enantiomers in urine via a controlled experiment.  The project will result in a decision model that can be used to assess the source of an adverse analytical finding for clenbuterol.  A new technique focusing on untargeted analyses will also be tested on the acquired samples. This will take into account if there are any other changes in metabolic profiles after both ways of ingestion and will try to discriminate on this basis. This technique is known as metabolomics. 

Main Findings:

The aim of the project was to confirm the proof of principle from a former project with controlled human trials and to build a decision model to be used in sports doping analysis to distinguish AAF for contaminated meat from pharmaceutical preparations. Meat and liver were collected from an animal experiment.  The meat and liver were used for a controlled human trial in which these tissues were consumed by volunteers. Other volunteers ingested a pharmaceutical preparation containing clenbuterol (racemic mixture) or enantio pure clenbuterol. Urine samples of the volunteers were collected for over a week after consumption. All urine samples were analysed using the methods developed in 11A18SS project. The urine samples were also used for untargeted profiling experiments.  
In meat from the animal experiment had S/R clenbuterol ratio of around 1 and 0.9 depending on the animal. This shows that the enrichment of R-clenbuterol in meat of treated animals is not a process that is stable, reproducible and comparable for individual animals and maybe also depends on the concentration administered to the animal. For bovine liver no information on the ratio was available before. Data from bovine liver showed that in liver the S-enantiomer is either enriched or R-clenbuterol very depleted. This is the opposite of the ratio in veal meat. This means that consumption of liver with incurred residues can possibly lead to an opposite ratio in human urine compared to the consumption of veal meat. It was also shown that preparation (cooking and baking) of the incurred meat and liver had little to no effect on the ratio determined prior to preparation.  
In the administration studies a distinction based on the proportion of S-clenbuterol in the human urine samples was possible between those receiving liver (padmin=0.635±0.004) from the other two groups, Spiropent® tablet (padmin=0.499±0.001) respectively meat (padmin=0.509±0.006). A distinction between the volunteers receiving meat and Spiropent® tablets cannot be made based on the enantiomeric composition, due to the reason that the ingested proportions of S-clenbuterol are too close to each other.  
The analysis focusing on potential metabolites of clenbuterol in a targeted non-targeted design predicted a high theoretical number of metabolites/biomarkers. Unfortunately, not many of the predicted metabolites were found in the urine samples of the volunteers. Based on the outcome of the analysis and the statistical processing there is no grouping of the compounds possible at present. There is no underlying mechanism found what could be used to separate the different treatment groups. So it was concluded that it is not possible to use clenbuterol metabolites to discriminate between intentional and unintentional intake of clenbuterol using non-enantiomeric separation.  
Overall it seemed that inter individual differences between elimination kinetics in bovine animals are present. This made setting an absolute threshold or guideline for discrimination not possible. The method developed in the project can be used when there are adverse analytical findings for clenbuterol to obtain additional information. When the S proportion clenbuterol in human urine resembles 0.5 (the S proportion in a pharmaceutical preparation) this is not definite proof of illegal use. However, if the S-proportion is higher than 0.59 this means that in 95% of the cases this is not due to administration of clenbuterol in (racemic) tablet forms.

Code: 13D31PV

Although recent advances of steroid profiling have shown to substantially increase the detection sensitivity, still, the prevalent of misuse is larger than what doping control laboratories can detect. In particular the introduction of slow-release preparations such as T-gels and steroid patches poses anti-doping analysts for greater challenges as the metabolic footprint of misuse with these preparations is very small. 
In 2009, Bayer introduced a long-acting T undecanoate depot Nebido® for therapeutic treatment of hypogonadism in males. Nebido® should be administered only once in two or three months, which is a much longer release period compared to other long-term T-formulations (e.g. Sustanon®). A dose of 1000mg T undecanoate should be injected in the muscle. It is claimed that the insufficient T-levels are restored and do not exceed normal serum levels and likewise cause less unwanted side-effects. 
Also, it has been shown that such high T undecanoate doses can lead to elevated hematocrit levels. Moreover, sustained elevated T-levels can exert a beneficial anabolic effect on micro-damaged muscle tissue after long exercise to enhance recovery.  Interesting features for endurance athletes, for whom the biological passport is a well-established tool to screen and record their doping test results. In this light, it is assumed that described effects can be noticed in both the steroidal and blood module of the biological passport and help to find Nebido® misuse in this category of athletes.  This study aims to investigate the direct detectability of T-undecanoate as administered with Nebido® in blood as well as the influence on various blood and steroid parameters as recorded in the biological passport. 

Main Findings:

Today the steroid passport is a very performant tool to detect abnormal variations in steroid profile markers. Although recent advances of current methods for endogenous steroids have shown to substantially increase the detection sensitivity, the prevalent of misuse is far larger than what doping control laboratories can detect. Particularly, slow-release preparations pose anti-doping analysts for great challenges as the metabolic footprint of misuse with these preparations can be very small. Nebido (Bayer) is one of such long-acting slow release T preparations, containing T undecanoate, that is used for therapeutic treatment of hypogonadism in males. In sports, slow releasing T formulation are applied to rapid repair of micro damage to muscles after exercise for faster recovery. In addition, it has been shown that such high T doses can lead to elevated haematocrit levels. Moreover, sustained elevated T-levels can exert a beneficial anabolic effect on micro-damaged muscle tissue after long exercise to enhance recovery. Both features are interesting for endurance athletes, whereas T used to be related to cheating power athletes for its anabolic effects.  
The T/E ratio was clearly altered by the use of Nebido as slow release T formulation and remain detectable using longitudinal following during 3 months. Androstanediol  ratio and androsterone over etiocholanolone remain mostly unaltered, despite elevated concentrations. Minor steroid metabolites did not contribute to longer or better detection as likely the major pathways were not saturated. As a result the steroidomic model showed similar sensitivities as those observed for longitudinal evaluation of the T/E ratio.  
A very sensitive method for the detection of T in saliva was developed and applied to the oral fluid samples collected after Nebido. Although, salivary T concentrations increased to 7 fold and detectable using population based reference ranges, the basal values were restored after 14 days. Oral fluid analysis of T did not provide any added value for doping control. Hence, the pharmacokinetics of salivary T are much different compared with the observations for T-gel, also a slow-release formulation. 
Blood passports were created after the Nebido T supplementation and results demonstrated that the blood markers were significantly altered but did not results in atypical passport findings. The reticulocyte production was stimulated in the month after Nebido injection, after which a suppressive effect was observed in the second month. By this time haemoglobin was formed to give rise to an elevated plateau in the blood passport that remained below the upper individual limit. Also the off-score showed the alterations that was typical for bone marrow stimulation. 

Code: 13A08MT

The necessities generated by the high standards and quality requirements of modern sports drug testing entails numerous challenges for doping control authorities as well as accredited laboratories. Among these challenges, several prominent aspects can be approached and solved by collecting and, when indicated, testing an additional/ a complementary specimen referred to as dried blood spot (DBS). 1. An important element of efficient doping controls is the frequent and unpredictable sampling of athletes, especially out-of-competition and concerning e.g. anabolic agents, erythropoiesis-stimulating agents, etc. Here, costs associated with sample collection and analysis are particularly limiting factors.
2. With regard to in-competition controls, the differentiation whether a substance such as a stimulant or cannabinoid (i.e. not prohibited at all times) was present in the athlete's blood at pharmacologically relevant concentrations or not is required. From urine analyses, conclusions as to the blood concentration of a drug is a complex issue and has been subject of numerous studies and discussions in the past.2-5
3. The issue of instable compounds can be solved as e.g. in case of Synacthen, a substance that was confiscated in the Spanish Fuentes scandal and admittedly misused by several athletes. Due to its limited stability in blood and urine, its analysis was hampered despite the availability of analytical methods. DBS collection and analysis is considered as a valuable means to improve currently conducted doping control strategies as it addresses all of the above mentioned aspects. DBS are substantially cheaper than conventional doping control samples and thus larger collectives can be processed; drug concentrations at the time of competition can be determined; and instable analytes can be conserved. As a result, both relevant aspects of doping controls, i.e. deterrence from doping and protecion of honest athletes will be considerably strengthened. 

Main Findings: 

A drop of whole blood dried on filter paper (Dried Blood Spots, DBS) represents an aspiring technique for minimal-invasive sample collection in a multitude of analytical disciplines, e.g., therapeutic drug monitoring, preclinical drug development and diagnostic analysis of metabolic disorders in newborns. DBS sampling is characterized by cost-effectiveness, straightforwardness, robustness and facilitated storage and shipment conditions. 
The present investigation was conducted to highlight the opportunities arising from the implementation of DBS as complementary matrix in doping control programs. Being frequently abused, three model compounds were chosen to represent the classes of anabolic agents (stanozolol and dehydrochloromethyltestosterone) and stimulants (pseudoephedrine). A quantitative method was developed and validated for the detection of the target analytes from DBS using liquid chromatography coupled to high resolution/ high accuracy tandem mass spectrometry. The imprecision of the assay amounted to < 8% for intraday and < 18% for day-to-day measurements. Highly purified DBS sample extracts exhibited no ion suppression effects due to interfering matrix components and provided limits of detection of 20 pg/mL for stanozolol and 0.8 ng/mL for DHCMT and pseudoephedrine, respectively, notwithstanding an overall recovery of 26%. Deuterium-labeled internal standards were used to yield reliable quantitative results (accuracy 84-125%). Stability of the analytes was shown for at least 28 days at room temperature.  
Proof-of-principle for the method presented was substantiated by means of the analysis of authentic specimens obtained from administration studies with stanozolol, DHCMT and pseudoephedrine. The results provided, to our best knowledge, unprecedented detection windows for the tested anabolic agents accomplished by DBS sampling to support out-of-competition control efforts for the tested anabolic agents. Furthermore, the unambiguous proof of pharmacologically relevant blood concentrations at given urinary analyte levels are noteworthy for the improvement of in-competition controls, e.g., with regard to stimulant analysis.

Code: 13C35CC 

Stimulants are banned in competition by the World Anti-Doping Agency, except for nicotine. Psychoactive substance as nicotine is widely reported to increase alertness, improve coordination and enhance cognitive performance.
In healthy humans nicotine administration increases heart rate and blood pressure. Plasma nicotine concentrations improve anaerobic energy production and suggest a nicotine-induced sympathetic stimulation of the heart. Nicotine has been placed on WADA's 2013 Monitoring Program in order
to detect potential patterns of abuse. Smokeless tobacco is a blanket term that refers to a number of tobacco products that are used by means other than smoking. Snus is a smokeless tobacco consumed orally, which has a long tradition of use that, is widespread in North America and Scandinavian countries. It is typically placed between the upper lip and the gum, toward the front of the mouth, for approximately 30min before it is discarded. Recently, some studies on snus in competitive events showed a high use among athletes mostly in a winter sporting environment.  The aim of this study is to investigate the snus effects on sport performance. In the first year we will investigate the effects of snus in an aerobic performance by an exercise intensity nearly 80% VO2max until exhaustion correlated with the perception of effort and time to exhaustion along with a range of physiological parameters. In the second and third year the aim is to investigate the effect of snus on high-intensity exercise by on a, short-term high-intensity cycling test, (Wingate) using trained athletes. If results will show a snus effect of increasing time to exhaustion, reduced perception of effort and increase anaerobic performance, an informed decision on the potential doping of nicotine will be done. WADA could consider to include nicotine in Prohibited List of Substances and Methods, prohibited in-competition

Main Findings: 

Snus is a smokeless, orally consumed tobacco, traditionally produced and used in Sweden and in Scandinavian countries. Analytical chemistry studies showed an increase of nicotine use in athletes practicing different sports. Nicotine has also several physiological effects on heart and on the vascular system. This effect influence sport performances and exercise capacity.

The purpose of this study was to investigate the snus effects on aerobic performance (80% VO2max) during exercise until exhaustion and the perception of fatigue during abstinence (AC) and satiety condition (SC). We recruited 18 healthy male athletes daily snus users. The study was a double-blind cross-over design. Subjects were tested during three sessions on cycle-ergometer.  Experimental session 1 (EXP1) consisted in maximal incremental exercise test (MIT) to determine the VO2max. EXP2 and EXP3 consisted in exercise, after 25 min from snus administration, at 80% VO2max until exhaustion (TTE) under AC and SC conditions. During the tests, muscle and cerebral oxygenation was recorded by means of NIRS (near-infrared spectroscopy) and the global rating of perceived exertion (RPE). We measured, non-invasively the dynamic response of tissue O2 saturation (Ox%) on tight muscle by a vascular occlusion test (VOT).

Nicotine and cotinine analysis confirmed the different initial conditions (AC vs. SC). We found a significant increase (+13.1%) in TTE (24.1 ± 10.7 min; means ± SD during AC session; 20.9 ± 8.0 min during SC session; means ± SD, P = 0.0131). RPE values did not significantly change during exercise. We found significant differences in the oxygenation of the cerebral and muscular tissues at rest (T0) and over VOT. Interestingly, our data showed significant differences in metabolic responses (VO2; VCO2; HR; VO2/HR) and in cardiovascular responses (SV; EJT) at baseline (T0). Our sample of athletes consumed 8.1 ± 4.0 (means ± SD) daily snus portions with a value of 5.9 ± 1.9 (means ± SD) of the Fagerstrom Test for Nicotine Dependence-Smokeless Tobacco (FTNDST).

The results of this project research showed that the increase of performance (+13.1% TTE) due to snus administration could be associated to the nicotine effects in order to keep the allostatic state.  In conclusion, our study highlighted that athletes addicted to nicotine would use snus to maintain their nicotine level to “feel normal” in order to improve sport performance.

Code: 13C36AB 

Over the last decade, significant efforts by us and other researchers in the anti-doping community have led to the development of methodology for direct detection of gene doping, a new threat to the world of sport.  The methodology involves polymerase chain reaction (PCR) assays targeting intronless complementary DNA (cDNA) sequences that are present in doping genes but absent in intron-containing endogenous genes. To ensure the method for detection of erythropoietin (EPO) gene doping performs as intended when implemented in routine test laboratories, an EPO DNA reference material (RM) has been prepared and characterized in our laboratory for use as a control. This RM has been carefully designed to ensure that inadvertent cross-contamination of a tested sample with the RM will not lead to a false positive test result. We are currently optimizing and validating protocols for blood sample collection, processing and storage. This will then provide a complete protocol from sample collection through to PCR detection together with an appropriate RM for ongoing monitoring of the method performance. 
This project aims to translate the method for direct detection of gene doping into a routine test method which is implemented in three WADA-accredited laboratories. The method will be initially implemented in the Australian Sports Drug Testing Laboratory (ASDTL). The close association of the applicants' laboratory and ASDTL (both laboratories within the National Measurement Institute, Australia (NMI)) will allow efficient review of the method when conducted in a routine testing environment and enable rapid finalization of the routine test method protocol. The method will then be implemented in two additional WADA-accredited laboratories and the competency of the three laboratories demonstrated through interlaboratory analysis of test samples. 
The project is scheduled for completion early 2016 which is several months prior to the Rio de Janeiro 2016 Olympic Games.

Main Findings: 

Over the last decade, significant efforts have led to the development and validation of methodology for detecting erythropoietin gene doping together with preparation of a DNA reference material to monitor test method performance. This project aimed to translate the method into a routine test and to implement it in one WADA-accredited laboratory, the Australian Sports Drug Testing Laboratory, which is part of the National Measurement Institute of Australia. We documented the complete test for detecting erythropoietin gene doping, provided staff training and assisted the laboratory during method implementation. Following test implementation in a routine laboratory environment, we assessed staff competence in analyzing blind samples and validated the test using blood samples which were prepared to model positive and negative gene doping samples from athletes