Projets de recherche

Code: 09D3PB

In the last years the WADA has founded studies of our groups aiming to develop in vitro test systems for the structure independent identification of anabolic substances. Our test system is a stable transfected yeast transactivation system for the identification of substances with affinity to the androgen receptor. Using this test system we could identify and characterise several designer steroids. In the last funding period we further characterized SC and started with the construction of a new reporter gene system in Schizosaccharomyces pombe. SC was able to detect anabolic steroids and their metabolites with a high specificity and sensitivity in urine of abusers (Zierau et al. 2008). Even selective androgen receptor modulators (SARMs) could be detected with SC. In excretion studies with Methyltestosterone in close cooperation with the doping control lab cologne SC was able to detect 1-Testosterone abuse up to 307 hours (GCMS detection limit was 118 hours). Treatment of the urine (concentration, purification) further increases the sensitivity of SC. Using new reporter gene plasmids we could reduce the duration time of the test from 2 days down to 18 h. In addition SP was successfully generated and is now ready to be further characterized. Reaching these milestones, our future aim is to use the SC to supplement GCMS techniques in routine doping analytics. Therefore we want to develop a standard routine procedure protocol to use the system in routine analysis. We also want to further enhance the sensitivity of the system by validation the newly generated SP system. In addition our SC system will be used to identify new long-term metabolites of anabolic steroids. So SC will in addition further improve the sensitivity of the GCMS detection systems.

Main findings

The results of this research Project present biologic and pharmacological “in vitro” and “in vivo” evidence that increases of EPO secretion during limited oxygen availability may be affected by extracellular adenosine generation and signaling. Therefore, we suggest that adenosine may be a doping agent “in vivo” since bone marrow is an hypoxic tissue. The A3 receptor does not appear to be involved in these direct effects of adenosine in HeL023 differentiation. On the contrary, the A3 receptor appears to be the mediator for the increase in EPO production induced by Adenosine.

The molecular signaling induced by Adenosine to increase EPO release by A3 receptors promotes HIF-1 expression. In particular, Adenosine can affect hematopoiesis at three levels: 1) increasing EPO concentration; 2) increasing ET-differentiation and 3) impairing MK-commitment. Adenosine, through A1 and A3 receptors, improves ET differentiation, accelerating the early commitment signaling. Adenosine may impair MK differentiation tuning PM fate toward the ET commitment. The mechanism appeared to involve the distal molecular effectors of EPO signaling pathway. Moreover, Adenocard increases EPO blood levels. Even if the increase was of modest entity, it is possible that adenosine, given as a unique bolo, has been rapidly degraded in few hours “in vivo”. We suggest that a chronic Adenocard treatment may generate a greater EPO increase with relevant physiological effects on PM cells

Code: 09E12RM

The fatigue that accompanies prolonged exercise in most often ascribed to events occurring in the periphery (glycogen depletion, dehydration). Recent evidence, however, suggests that the central nervous system plays a critical role, especially during exercise in the heat. This is an important consideration, as many major championships take place during the summer, often in countries with warm climates.

Many stimulants acting on the CNS are included in the WADA Prohibited List; they can enhance performance and may pose a risk to health. Pharmacological manipulation of the CNS function can influence mood, the sensation of effort and/or thermal stress, cognitive function and tolerance to pain and discomfort. Drugs that manipulate brain neurotransmitters, in particular serotonin, dopamine and noradrenaline, have been shown to influence exercise performance, but many of these agents are not currently included on the Prohibited List. Many of these drugs are used in the treatment and management of a wide variety of psychiatric disorders, so the pharmaceutical industry is constantly producing novel, more selective and more powerful agents, with the potential for marked effects on exercise performance. Athletes may have legitimate reasons for their use (under Therapeutic Use Exemption; TUE), but the inappropriate use of these agents by athletes to enhance performance is a distinct possibility.

These drugs, which are readily accessible over the internet, may also pose a risk to health as the normal limits to body temperature may be exceeded. At least one high profile death during the Tour de France has been ascribed to hyperthermia consequent upon amphetamine use.

This project will investigate the performance effects of drugs affecting the CNS, and will also consider the various factors (gender, environment etc) that may influence their efficacy. These findings will help determine the suitability of this class of drugs for future inclusion on the Prohibited List.

Main findings

Stimulant medications enhance the activity of the central and peripheral nervous systems, producing a variety of effects including increased alertness, motivation and arousal, elevated heart rate and blood pressure, altered mood and the perception of a diminished requirement for food and sleep. Since these responses are likely to influence athletic performance, the aim of this project was to examine evidence that pharmacological manipulation of the central nervous system can produce improvements in exercise performance. Anecdotal reports, evidence from screening procedures used by WADA-accredited laboratories, as well as TUE application statistics, provide some evidence that athletes may be increasingly turning drugs of this nature. Of course, athletes may have legitimate reasons for the use of these drugs, but these agents may also be used with the sole aim to enhance performance. Of particular concern would be medications employed in the treatment of attention deficit hyperactivity disorder (ADHD) and narcolepsy.

The present series of studies provide clear evidence to support the inclusion of Bupropion on future Prohibited Lists. The findings of the studies presented here, along with our previously published work, demonstrate that bupropion can produce a significant, measureable and consistent improvement in exercise performance in warm conditions when taken at the maximal therapeutic dose (+7-9% vs a placebo condition). This response is apparent in both male and female participants, and does not appear to be influenced by pre-exercise nutritional intake. Increased exercise performance following administration of bupropion was associated with the attainment of higher core temperatures and heart rates during the later stages of exercise. This occurs without any apparent change in the subjects’ perceived exertion or thermal stress. It is distinctly possible that the use of bupropion and methylphenidate by athletes competing in warm conditions will increase susceptibility to heat illness and potentially fatal heat stroke.

Contrary to the findings of a recently published report, ingestion of tyrosine (the precursor of dopamine) produced no effect on physical performance during an exhaustive cycle test; this response may be due to rate limiting steps in catecholamine synthesis from tyrosine. The administration of the Parkinson’s drug, Sinemet (containing L-Dopa), also failed to significantly influence exercise performance, but performance may have been limited by side effects reported by some participants. S-adenosylmethionine (SAM) has become popular as an alternative therapy for the treatment/management of depression and other related disorders. Despite some effects on the physiological response to exercise, there was no evidence of a benefit to exercise performance following a 7 days of administration.

In conclusion, it appears that bupropion can produce a measureable and consistent improvement in exercise performance in warm conditions when taken at the maximal therapeutic dose. With this in mind, inclusion of bupropion on future Prohibited Lists appears to be warranted. Substances that alter catecholamine production do not appear to consistently influence performance, but further investigation is justified.

Code: 09A05DP 

This project aims at four goals: 
A. Interpretation and evaluation of precursor ion scanning chromatograms (urine profile recognition) 
In the field of anti-doping control, chromatograms from target steroid-analysis are generally evaluated and interpreted visually by the analyst which is trained for this purpose. 
A similar approach will be adopted for evaluating the precursor scanning chromatograms. To get acquainted with the chromatograms, around 50 blank samples will be analysed.  In a second step precursor ion scan chromatograms obtained from controlled administration studies (e.g. methyltestosterone, methanedienone,…) and from adverse analytical findings from routine target screening will also be investigated to get acquainted with positive samples. The applicability of instrument software for this purpose will be evaluated.  
B. Application to real samples
Urine-samples will be analysed. These samples will include all out of competition samples and both out of competition samples and in competition samples   
C. Study of the influence of different population factors in the detectability of different markers for several anabolic steroids 
A single dose of the previously studied steroids (stanozolol and methyltestosterone) will be administered to six volunteers belonging to different population groups. Urine will be collected for three weeks and a method including all feasible markers will be applied. The best marker(s) will be selected based on the results obtained. This procedure will also be followed if promising new markers are found in the re-evaluated steroids. 
D. Re-evaluation of the metabolism of additional steroids by LC-MS/MS looking for alternative markers for the detection of steroid misuse 
Three or four additional steroids will be re-evaluated via the analysis by LC-MS. These steroids will be selected based on the availability of excretion studies in both laboratories.  
Appropriate precursor scan and/or neutral loss scan methods will be applied for each steroid depending on their structure. Feasible metabolites will be characterized by MS techniques. The metabolic nature of these metabolites will be determined, if necessary, by the analysis of chimeric mouse urine after the drug administration. A full excretion study will be analyzed in order to determine the long term metabolites. 

Main Findings: 

Anabolic steroids with a 3-keto-4-ene structure have in common that they fragment by LC-ESI-MS/MS at high collision energy in tree common ions: methyltropylium (m/z 105), tropylium (m/z 91) and phenyl ion (m/z 77). The use of a precursor ion scan method for these 3 ions would allow to detect new/unknown AAS and metabolites with 3-keto-4-ene structures. 
In this project, the potential of approaches based on precursor ion scan for doping control purposes have been evaluated in two scenarios: (i) routine application and (ii) metabolic studies of AAS.
In the routine application, the potential use of the approach in two laboratories (Gent and Barcelona) was explored. The analysis of a common set of 12 samples allowed for the confirmation of the suitability of the method for the detection of positive samples for known steroids. The analysis of this common set of samples also revealed several limitations in the harmonization between laboratories regarding relative retention times and the precursor ions observed. 
During the whole project, a total of 1911 routine-samples were analyzed using the established precursor scan protocol by the two laboratories. These samples covered both samples from athletes from high-risk sports (strength sports) and urines samples from athletes who tested positive for known steroids. Seven suspicious samples were detected. Despite the application of several analytical strategies such as product ion scan in both positive and negative ionization mode with accurate mass measurements and GC-MS, none of them could be clearly assigned to belong to the AAS family. Regarding metabolic studies, the method allowed for the detection of several metabolites for boldione, 4-chloro-methanedienone and clostebol. Several previously unreported metabolites were detected and characterized.  
Finally, the effect of population factors in the detectability of long-term metabolites was evaluated in two different scenarios: stanozolol and clostebol. Results showed that both 17-epistanozolol-N-Glucuroinide and 4-chloro-5-androst-3β-ol-17-one 3β-sulfate are the long-term metabolites for stanozolol and clostebol irrespective of the ethnicity of the volunteer. 

Code: 09B3JP

The analytical strategy of doping control relies on the availability of reference standards against which compare the results obtained after the application of any procedure. The situation with the endogenous urinary EPO standard (NIBSC 2nd international reference preparation) needs careful attention: - the urinary standard (uEPO) from NIBSC is coming to an end and according to the correspondence held with NIBSC, there are no plans to produce a new batch.
- At this stage, only 2 vials (20 IU) per laboratory and year will be provided.
- The NIBSC uEPO standard was produced by unspecific protein concentration from urine of anaemic patients. Hence it contains very high concentrations of proteins other than uEPO.
- Despite all efforts devoted so far, there has not been an appropriate characterisation of the origin of the charges responsible for its IEF behaviour.
- The comparison between any standard obtained from patients and a standard obtained from healthy volunteers has to be performed.
- An appropriate analytical standard of serum or plasma EPO is needed and has to be developed.               The technology for EPO immunopurification has already been developed and the members of the research team have first-hand experience in the field. However, its up-scaling for large volume processing (e.g. ten litres or urine per day) has to be developed tested properly. 
With this background, the aim of the project is: - developing the methodology for producing a urinary EPO standard by processing urine from patients and healthy volunteers.
- producing immunopurified urinary and serum EPO standards, available for doping control purposes.              As secondary objectives, the daily variation of their urinary EPO profiles will be studied and, using EPO from patients producing elevated concentrations of the structural characterisation of EPO will be approached.

Main Findings: 

The general aim of the project was the development of a large scale inmunopurification procedure and its application for the preparation of a relatively large scale batch of endogenous EPO standard in urine and, if feasible, plasma from healthy volunteers and patients producing large amounts of this glycoprotein.
The immunopurification procedure developed implied a sample pretreatment includingdissolution under alcaline conditions of the sediment/precipitates present in urine, followed by a thorough multi‐step filtration (first, a filtration through a triple glass fibre filter AP25 (Millipore), 2 µm AP20 (Millipore) and 0.5 µm RW06 (Millipore); second a 0.22 µm Millipak filter unit).  
Immunopurification of urinary volumes between 1.5 and 2 L was achieved using 2 mL Sepharose columns containing as much as 12 mg of anti‐EPO monoclonal antibody. Columns needed to be used at 4ºC, overnight at a 2‐3 mL/min flow rate. Recovery, during the development phase showed to be roughly around 50% for solutions of up to 80 IU/L.  
However when real urine samples were treated, it soon became evident that the urinary matrix was able to block the columns, even after substantial filtration. Tamm‐Horsfall protein, the most abundant glycoprotein in urine, could have been part of the problem. Extensive washing of the immunoaffinity columns, changing of filtering procedures as well as heating the urine samples seemed to delay the problem, but did not solve it.
On the other hand, it was impossible to find proper patients or volunteers with elevated EPO excretion. Patients from clinical hematology, pneumology or oncology resulted in depleted EPO concentrations. Finally it was decided to use urine from healthy volunteers with serious implications regarding the amount of EPO that could be reached.  Immunopurified EPO was very sensitive to preparation conditions with significant losses due to irreversible adsorption on glass or plastic vials.
Overall, recovery was below 5% and only a very small, symbolic, batch of 15 vials of 1.6 IU/vial of liophylized material could be obtained in a buffer composition compatible with electrophoretic or mass spectrometric methods.
Obtaining reasonable amounts of this glycoprotein standard, and at a reasonable cost, would imply an international approach with access to a number of high EPO excreting individuals under proper long term conditions.

Code: 09E24XD

Salbutamol belongs to the class of beta-2-agonists, and its use is prohibited by the WADA if administered for reasons different than the pharmacological treatment of respiratory diseases (i.e. asthma , broncospasm).  At present, discrimination between the therapeutic and illicit use of salbutamol is based on concentration thresholds, being the urinary concentration of non-sulfated drug mainly, but not exclusively, related to the administered dose and to the route of administration.  Small differences in metabolism can be observed due to the difficulty of avoiding a partial oral administration during inhalation administration where a fraction of the dose is swallowed. It appears that investigation of the location of the majority of salbutamol metabolism is of great interest and may add additional knowledge for better discrimination of salbutamol route of administration. 
It seems adequate in terms of analytical and metabolic reasons to develop a LC/MS/MS method for the simultaneous detection of the free, glucuronoconjugated and sulphated forms of salbutamol. In addition, an evaluation of the spontaneous urinary salbutamol hydrolysis could be estimated (stability test).  The combination of chiral liquid chromatography coupled to mass spectrometry will add a dimension to salbutamol metabolism assessment.  The determination of the well accepted S/R ratio of the free + glucuronated salbutamol to differentiate oral vs. inhaled administation would be complemented by the determination of the S/R ratio of the sulphated fraction. The determination of the sulphated fraction will offer the possibility to better understand and discriminate between different routes of administration. 
The genotypic characterization of selected sulfotransferases (SULT’s) of the individuals participating to the study (coming from two different ethnic groups) will also be performed, in order to correlate the genotypic expression of the sulfotransferases (SULT), that are the enzymes responsible for salbutamol metabolism with the phenotypic data obtained from the experiments described in the previous step. 

Main Findings: 

Salbutamol is one of the most used β2-adrenoceptor agonists by athletes for relieving bronchospasm and for prevention of exercise-induced-asthma. Inhalation is the preferred route of administration, but also the oral route is recommended, in particular for populations where inhalation presents practical problems (i.e. young children). Since salbutamol, similar to other β2-adrenoceptor agonists, produces an anabolic effect when sufficiently high doses are administered orally, its use is approved by the World Anti-Doping Agency (WADA) only after inhaled administration. Currently, sport authorities monitor salbutamol use in and out of competition: urine concentrations greater than the Decision Limit of 1200 ng/mL (free + glucuronated salbutamol) are considered adverse analytical finding unless the non- inhaled administration could be excluded by other means. Previous studies have demonstrated that after oral administration, the majority of salbutamol is found in the urine either as the parent compound (24-33%) or as conjugated sulfate metabolite (48%).On the contrary, no significant biotransformation occurs in the lungs, thus the percentage of salbutamol metabolite depends mainly on the percentage of the dose that is swallowed and absorbed from the gastrointestinal tract.
Salbutamol is administered as a mixture of two enantiomers: S(+)-and R(-)-salbutamol and enantioselective disposition studies have demonstrated that after oral administration the enantiomers are conjugated at a different rate by the body tissues. The active R(-) enantiomer undergoes a higher rate of sulfation, and therefore, after oral intake the non metabolized S(+) enantiomer is excreted in a greater level than the non metabolized R(-) enantiomer. For the above reasons the ratio between S(+) and R(-) of the unchanged salbutamol was proposed as marker of oral administration.
Based on the above evidence, an analytical method involving a solid-phase clean-up procedure followed by a chiral HPLC separation and a mass spectrometric detection to quantify separately the enantiomers of salbutamol and it sulphate conjugates has been developed and validated according to ISO17025 and WADA requirements.
The urinary results confirmed that after inhalation the enantiomeric ratio between S(+) and R(-) of the non metabolized and of the metabolized salbutamol strongly depends on the percentage of the dose that is swallowed.  The sulphotransferases (SULT’s) responsible for the conjugation of salbutamol in different tissues, and for the different excretion of S(+) and R(-) enantiomers depending on the route of administration, are polymorphic and then large differences between individuals may be expected. In some specific cases, this may be the reason for reaching elevated concentrations of salbutamol after inhaled therapeutic administrations (permitted). To access to the information of the genotyping of this SULT’s may be crucial for the correct interpretation of the phenotypic data. To obtain genomic DNA from blood or saliva samples is a common practice in forensic investigations. In doping analysis urine still being the unique biological specimen available in more than 80-90% of the cases. Then it demonstrated the feasibility of obtaining genomic DNA from urine samples of the adequate quality for the genotyping of the sulfotranferases of interest.  A method for the extraction and amplification of genomic DNA form blood, urine and saliva samples was developed and validated, permitting to obtain genomic DNA of adequate quality starting from 2-5 μL of serum, 1-10 mL of urine or 2-5 μL of saliva respectively.

Code: 09E26PV 

Anabolic steroids are amongst the most misused substances in doping control and are intensively metabolized in humans. Adequate screening for misuse of these substances therefore relies on the detection of metabolites in urine samples collected from athletes. 
Most of the studies investigating the metabolism of pharmaceutically available steroids were performed in the 1980’s via gas chromatography mass spectrometry (GC-MS). This research resulted in the selection of appropriate metabolites for the detection of steroid misuse. Over the years the selection of metabolites was further elaborated to include several metabolites that can result in prolonged detection times. Recent reinvestigation of metabolism via LC-MS resulted in more appropriate metabolites for this technology, than those detected via GC-MS. 
Over the last decade, a high number of non-pharmaceutical steroids have been introduced on the market as “nutritional supplements” or as black market designer steroids, while most of the pharmaceutical preparations have been retracted.  For both types, approval of administration by an Ethical Committee has become almost impossible. The UPA+/+SCID mouse model with humanized liver has proven to be the best appropriate model to mimic human steroid metabolism in the liver and application of this model to the metabolism of methandienone and methyltestosterone has contributed to the detection of new and long-term metabolites detectable via liquid chromatography tandem mass spectrometry (LC-MSn). 
The current project would (re)investigate metabolism of several groups of steroids: 1. Substances currently listed on the prohibited list, but with limited knowledge on metabolism
2. Substances currently not explicitly listed.

Main Findings: 

The aim of the project was to study the metabolism of different steroid compounds by means of a humanized chimeric mouse model. This uPA+/+-SCID mouse model has its liver transplanted with functional human hepatocytes. In the past the chimeric mouse model has proven to be a valuable tool in elucidating the urinary steroid metabolism, especially for those steroids for which it is difficult to obtain ethical approval for human excretion studies.

In this project the metabolism of 7 steroid compounds was investigated:
* methyl-1-testosterone
* oxabolone
* prostanozol
* 6-bromo-androstenedione
* 3α-androstanol
* 17-methyldrostanolone
* 4-chloro-17-methylandrostenediol (promagnon and methylclostebol)

The metabolism of these 7 steroids was investigated by use of administration studies to the chimeric mouse model. The analyses of the mouse urine samples were performed on a combination of GC-MS and LC-MS/MS instruments. Comparing the pre- and post-administration
mouse urine samples allowed us to obtain a urinary metabolic profile for all of the steroids. From all the detected metabolites, the most appropriate markers were selected based on their usefulness as target markers for steroid abuse. Those metabolites were implemented where possible in our routine screening methods for anti-doping screening, making our methods even more complete and comprehensive. Additionally, within the scope of the project, also the in vitro technique of human liver microsomes was optimized to assist this research.

In the future this promising mouse model will be used to further encourage the fight against doping by evaluating some prohormones and food supplements based on the urinary results
of the chimeric mice.

Code: 09A13MF 

Autologous blood transfusion is banned; however currently no test exists to detect this form of blood doping. Anecdotal reports suggest that elite athletes have been using autologous transfusions since at least the 1980s, and the Operacion Puerto scandal in Spain has revealed that some athletes continue to utilise transfusions. Blood must be withdrawn, then stored for several weeks prior to reinfusion (in the meantime the body replenishes the depleted blood so that there will be a corresponding boost in the total quantity of blood cells in circulation when the blood is finally reinfused). Blood is either refrigerated or frozen in specially designed blood storage bags. 
Whilst the blood is in contact with the storage bag, it is known that there is an exchange of material between the plastic storage bag and the blood. Since the storage bags are man-made, the synthetic compounds that find their way into the blood are not normally found in the body. 
Their presence in a doping control sample may therefore be used as a diagnostic test for the prior use of autologous transfusion.  This project will investigate whether the presence of plastic compounds in blood samples are sufficiently distinct to enable their use as a diagnostic. Pending the successful outcome from this first stage, we will extend our studies to document the persistence of these markers in volunteers after they have received blood transfusion to establish the likely window of detection associated with this type of testing. 

Main Findings: 

Two approaches were investigated to detect the presence of DEHP in reinfused red blood cells (RBC). The first approach was indirect and based on the hypothesis that DEHP was antigenic and that development of a suitable antibody could allow the flow cytometric detection of DEHP-contaminated cells. Substantial efforts were made to develop a monoclonal antibody against DEHP, however these attempts were unsuccessful. Because of the lack of these antibodies, this component of the research had to be abandoned. 
The second approach was direct and turned on the ability of GC-MS to detect trace amounts of DEHP in lysed RBC membranes. Provided that rigorous measures were taken to avoid contamination during sample preparation and analysis, we demonstrated that the DEHP levels found in the membranes of stored RBC could be easily distinguished from background levels. However DEHP levels in RBC fell rapidly once the cells had been reinfused into circulation.

Code: 09E18GW 

In 2002 The International Olympic Committee (IOC) established the requirement for athletes to present evidence of current asthma, exercise induced asthma (EIA), exercise-induced bronchoconstriction (EIB) or airway hyperresponsiveness (AHR) through the therapeutic use exemptions (TUE) process. The World Anti-Doping Agency (WADA) introduced the IOC policy on inhaled short acting 2-agonists in January, 2009. The inclusion of inhaled short acting 2-agonists is based upon health concerns rather than anti-doping concerns; however, evidence is only available for the performance enhancing effect of inhaled short acting 2-agonists in endurance sports at low doses. 
The initial aim of this study is to examine the impact of inhaled short acting 2-agonists on team game performance and examine higher doses that remain within The WADA anti-doping upper limit. Hydration status has recently been used in the defence of a positive anti-doping test.  This has highlighted the lack of knowledge associated with potential confounding factors affecting urine analysis for short acting 2-agonists. 
The second aim of this study is to examine the impact of gender, race and hydration status on urine concentrations of short acting 2-agonists at varying doses. The results of these studies will improve our understanding of the impact of inhaled short acting 2-agonists on performance, support The WADA in the implementation of regulations on the use of inhaled short acting 2-agonist and assist in the resolution of contested doping violations. 

Main Findings: 

Part A: Seven male runners (mean + SD; age 22.4 + 4.3 years; height 179.7 + 7.0 cm; body mass 76.6 + 8.6 kg) completed 6, 5 km running time-trials (3 in a temperate environment: 20OC, 40% RH; and 3 in a hot environment: 30OC, 40% RH) following the inhalation of 800 μg or 1600 μg of Salbutamol, or a placebo.  
This is the first study to examine the impact of inhaled salbutamol at a dose 1600 µg versus 800 µg and placebo on time-trial endurance running performance. Furthermore, this study is the first to examine the pharmacokinetics of inhaled salbutamol at a dose of 1600 µg and 800 µg following a competitive endurance performance in temperate (20oC; 40% RH) and hot (30oC; 40% RH) environments. Results demonstrate no significant effect of Salbutamol on 5 km running time-trial performance in temperate or hot environments. Urine concentration of Salbutamol was below the WADA upper limit (1000 ug.ml-1) in all participants across all trials with the exception of one participant in the 1600 μg, hot trial (below the decision limit). The results of this study suggest that the current WADA guidelines, which allows athletes to inhale up to 1600 µg of Salbutamol is sufficient to avoid pharmaceutical induced performance enhancement. However, such high doses not only suggest poor management of asthma but also mean that an athlete may be at risk of contravening the current urinary threshold, particularly in hot environments. 
Part B: Eighteen male and 14 female athletes (9 Caucasian males, 9 Caucasian Females, 2 Afro-Caribbean males, 2 Afro-Caribbean females, 6 Asian [Indian sub-continent] males and 4 Asian females) were recruited for this study. Participants were required to exercise in a hot, controlled environment (35oC, 40% relative humidity) at a self-selected pace until a target weight loss (2% or 5%) was achieved in the following trials: (i) 2% reduction in in body mass (BM), following inhalation of 800μg short acting β2-agonist; (ii) 2% reduction in in BM, following inhalation of 1600μg short acting β2-agonist; (iii) 5% reduction in in BM, following inhalation of 800μg short acting β2-agonist; (iv) 5% reduction in in BM, following inhalation of 1600μg short acting β2-agonist. The results of this study demonstrate that following the inhalation of 1600 µg it is possible to present with urine Salbutamol concentrations above the current WADA limit (1000 ng.ml-1) and decision limit (1200 ng.ml-1) for salbutamol resulting in an adverse analytical finding (AAF; WADA, 2010) and warrant further investigation. There were no differences according to sex or ethnic origin however; a large inter-individual variation existed. In conclusion, a BM loss greater than 2% concomitant to the acute inhalation of 1600 µg of Salbutamol may result in a urine concentration above the current WADA upper limit and decision limit leading to a positive test finding. This finding is independent of gender or ethnic origin. Hydration status per se is a critical factor in relation to doping control. The results of this study suggest that WADA consider the role of normalising drug concentrations to urine specific gravity in an attempt to negate the impact of hydration status on doping control tests. Data from this study will assist WADA in the implementation of regulations on the use of inhaled short acting β2-agonist and assist in the resolution of contested doping violations. 
Part C: Seven male (mean + SD; age 23.1 + 3.9 years; weight 72.9 + 4.3 kg; height 177.0 + 4.7 cm) and six female (21.3 + 1.4 years; 63.9 + 5.8 kg; height 162.3 + 4.7 cm) football players completed a 52 minute football specific running protocol followed by twelve, 17.5 m sprints on three occasions following the inhalation of 800 μg or 1600 μg of Salbutamol, or a placebo. 
This is the first study to examine the impact of inhaled salbutamol at a dose of 1600 µg versus 800 µg and a placebo on simulated association football (soccer) and multiple sprint performance in male and female players. Furthermore, this study is the first to examine the pharmacokinetics of inhaled salbutamol at a dose 1600 µg and 800 µg following a simulated association football performance in male and female players. Results demonstrated no significant effect of high dose (up to 1600 µg) Salbutamol on association football (soccer) specific performance or multi-sprint performance in male or female players. Following inhalation of 1600 µg of Salbutamol, Five players (2 male; 3 female) presented with concentrations of Salbutamol in urine above the WADA upper limit (1000 ng.ml-1) with three players (1 male; 2 female) attaining a concentration above the decision limit (1200 ng.ml-1). The results of this study suggest that the current WADA guidelines, which allows athletes to inhale up to 1600 µg of Salbutamol is sufficient to avoid pharmaceutical induced performance enhancement in association football (soccer) in male and female players. However, inhalation of 1600 µg may result in a urine concentration above the current WADA upper limit and decision limit. 

Code: 09C10PD

In the last years the WADA has founded studies of our groups aiming to develop in vitro test systems for the structure independent identification of anabolic substances. Our test system is a stable transfected yeast transactivation system for the identification of substances with affinity to the androgen receptor. Using this test system we could identify and characterise several designer steroids. In the last funding period we further characterized SC and started with the construction of a new reporter gene system in Schizosaccharomyces pombe. SC was able to detect anabolic steroids and their metabolites with a high specificity and sensitivity in urine of abusers (Zierau et al. 2008). Even selective androgen receptor modulators (SARMs) could be detected with SC. In excretion studies with Methyltestosterone in close cooperation with the doping control lab cologne SC was able to detect 1-Testosterone abuse up to 307 hours (GCMS detection limit was 118 hours). Treatment of the urine (concentration, purification) further increases the sensitivity of SC. Using new reporter gene plasmids we could reduce the duration time of the test from 2 days down to 18 h. In addition SP was successfully generated and is now ready to be further characterized. Reaching these milestones, our future aim is to use the SC to supplement GCMS techniques in routine doping analytics. Therefore we want to develop a standard routine procedure protocol to use the system in routine analysis. We also want to further enhance the sensitivity of the system by validation the newly generated SP system. In addition our SC system will be used to identify new long-term metabolites of anabolic steroids. So SC will in addition further improve the sensitivity of the GCMS detection systems.

Main findings

The classical methodology to detect anabolic steroids or other anabolic substances in anti-doping analytics is MS. However, in particular the example of THG has demonstrated that even substances with a chemical structure typical for this class of substances are sometimes not detected during routine screening by MS, if their exact chemical structure is unknown. Moreover a great number of substances have been developed since the fifties and nowadays many pharmaceutical companies are working on non-steroidal androgen receptor modulators (SARMs) which have a completely different chemical structure and metabolism than classical anabolic steroids. In 2005 and 2006 WADA has funded pilot studies of our groups aiming to develop in vitro test systems for the structure independent identification of anabolic substances.

In these funding periods we have successfully established and validated the use of yeast reporter gene systems (SC) for the detection of substances with the ability to bind to the androgen receptor in urine. Besides that we could demonstrate that SC is able to detect anabolic steroids and SARMS in urine. Samples from athletes abusing anabolic steroids were successfully uncovered using SC. The primary aim of our ongoing (LIVE) project was to use the SC system to supplement MS techniques in routine anti-doping analytics. The system is easy to perform, without high tech equipment, cheap and the results are conclusive. Therefore it is most suitable to be used as a pre-screening system to identify the misuseof anabolic steroids, independent of the chemical structure, especially in training controls. Similar systems are successfully in use to identify anabolic misuse in other fields as for example application for enhancement of meat production in livestock. For this we aimed especially to finish the construction of SP and to validate SP in comparison to SC. The “new” SC and SP yeast construction had been finalized and the characterized, resulting in a backup or complementary system for the already established SC system by the SP. Overall the “new” systems are a bit less sensitive and little higher operating expense make them less efficient compared to the “old” SC system.

The next aim was the use and validation of the SC system in further excretion studies with problematic steroids in comparison to MS. A number of different substances have been analysed and in parts results of this of the project part have already been published. Summarizing this, the obtained results are encouraging, but seem to depend on the substance and their specific metabolism. This information is very helpful to further characterise the advantages but also limitations of SC.

Another aim of this founding period was to validate whether the SC system is capable to detect SARM abusing, which was carried out as an animal experiment. Animal experiments with SARMs were performed and post administration urines analysed with the SC. The results indicate that SC is also able to detect such substances in the urines of excretion studies.

A very important aim was the demonstration of the capability of the SC to identify longterm metabolites. This highlight of our so far conducted experiments was the general test whether the SC is a suitable bio detector to identify stable metabolites in the urine for further structure analysis. The presented results demonstrate that that we succeed in this proof of the principle. We are optimistic that we will be able to succeed in identifying a number of long-term metabolites structures in the future using a larger preparative experimental scale.

Last but not least our aim was to develop a standard operation procedure (SOP) to use SC system in routine anti-doping analysis. In our focused parallel analysis in Cologne and Dresden we have been able to acquire a SOP working in both laboratories with the identical efficiency. The simultaneously analyzed blinded urine samples impressively demonstrated that the results do not vary in quality even if performed in different laboratories. These results confirm the stability as well as the reliability of the SC assay and demonstrate its value as a screening assay in routine anti-doping analysis.

Code: 09D9FB

The project was developed using the observation that “Liposom Forte®”, a pharmaceutical-grade product containing “empty” liposomes, was reportedly used by athletes. Liposom Forte® was found stored together with banned and non-banned drugs during investigations carried out by Italian legal authorities.

Due to the negligible direct effect on the enhancement of performance of “Liposom Forte®” and similar pharmaceutical products containing empty liposomes, we postulate these products could be used as masking agents to make detection of other forbidden drugs more difficult. Liposomes can be used as masking agents by following one or more of the following hypothesized strategies: i) Injection immediately after being mixed with steroids to produce “home-made” slow/sustained release preparations;

ii) Injection as such (“empty”), before an “expected” anti-doping test to promote interaction with steroids/metabolites circulating in the organism and altering the excretion profile;

iii) Direct addition to the sample collected for anti-doping tests to reduce the concentration of “free” (i.e. not bound to liposomes) steroids/metabolites and reducing the efficacy of the laboratory analytical procedures used for detection. We have preliminarily shown that an interaction between liposomes and androgenic anabolic steroids can cause a reduced efficacy of the analytical procedures (normally based on GC/MS analysis of the corresponding TMS-derivatives after enzymatic hydrolysis) followed by anti-doping laboratories. We have also preliminarily demonstrated that direct addition of liposomes to urine samples containing steroids causes a masking effect: the amount of steroid detected in the samples was significantly reduced after liposome addition.

We plan to continue the present research considering the following aspects: i) More thorough evaluation of the binding ability and masking potential of liposomes in vitro.

ii) Assessment of the masking potential of liposomes in vivo.

iii) Characterize the physico-chemical properties of liposomes and improve analytical methods used to detect the presence of liposomes in biological samples

Main Findings

One of the hottest topics in antidoping research is the study of the so called masking agents, i. e. substances or methods capable of “hiding” other forbidden substances, thus reducing the efficacy of the experimental strategies used to detect the abuse of doping agents by the analysis of biological fluids. The class of masking agents was originally limited to substances with diuretic effects, but it has been progressively expanding, now comprising also agents that can interfere with either the pharmacokinetics of other banned substances and/or with the analytical procedures normally applied by the WADA-accredited anti-doping laboratories for the detection and, where required, the quantitative determination of the concentration, in biological fluids, of other banned substances.

To the best of our knowledge, this research project is the first one to specifically consider the possible relevance, as masking agents in sport doping, of liposomes, a class of supramolecular structures constituted by phospholipids extensively studied in the pharmaceutical field for their properties as drug delivery systems. More specifically, we have focused our attention on the potential masking effects of liposomes on anabolic androgenic steroid (AAS), to date the most widely abused class of prohibited substances in sports doping.

The results we have obtained can be summarized as follows: I. Liposomes have been shown to interact with anabolic androgenic steroids, leading to a reduced analytical recovery of both the parent compound and the glucuronide metabolites.

II. The interaction occurs in a relatively wide range of experimental conditions, and it has been verified for several representative pseudo-endogenous anabolic androgenic steroids and for various liposomes, differing in charge, size and chemical composition.

III. The effect is particularly noteworthy whenever a quantitative determination of the target steroid(s) is required, as it is the case of threshold substances (i.e. 19-norandrosterone) and/or of the evaluation of the urinary steroid profile in the framework of longitudinal testing and/or of the Athlete Biological Passport.

IV. Specific countermeasures to ideally annul the observed “masking effect” include the strict monitoring and control of all quality parameters of the analytical procedure(s) followed for the analysis of AAS, with special emphasis, in the case of GC-MS based methods, on the efficacy of the derivatization step.

V. A novel analytical procedure has been designed, developed and validated, to detect and quantitate a wide variety of liposome constituents (phospholipids and sphingomyelins) in biological matrices (blood and/or urine) and in pharmaceutical products, with the aim to identify suitable markers for the detection of liposome intake by the analysis of blood and/or urine samples.

The results of the present project unearthed a new and effective class of masking agents, that go beyond those with a direct pharmacodynamic effect, that act as true and effective “doping delivery systems”, altering the pharmacokinetic properties (i.e. transport, distribution and elimination) of doping drugs.