Projets de recherche

Code: 10D20VB

The higher prevalence of asthma and shortness of breath during exercise among elite athletes has resulted in increases use of anti-asthmatic medication among elite athletes. Studies of therapeutic doses of beta-agonists have in general not shown effect on the cardio-respiratory capacity. But theses doses are probably not the case in prohibited use of medication among athletes. The last international doping cases have shown significantly high intake of beta2-agonist, indicating either above therapeutic level of inhaled use, or systemic use of ordinary used anti-asthma medication. If intake of high doses of beta-agonist leave the aerobic capacity, muscular power or recovery is unchanged, ordinary used beta-agonist probably should be removed entirely from the prohibited list. Before this substantial change of the WADA code, more research is needed.

The cardiovascular effect of high doses of beta-agonists, with better overall performance, oxygen uptake and oxygen kinetics, has never been thoroughly studied, which is of importance as doping doses seldom are within therapeutic level.

Purpose: To examine whether high doses of salbutamol and Terbutaline is a class effect of beta2-agonists, different from therapeutic doses. Animal models have shown that salbutamol, in higher doses than normal therapeutic asthma doses, increased the contraction of skeletal muscles and keep a persistent effort. Furthermore, another animal model has recently shown increased force and recovery of the skeletal muscles after Salbutamol. Based on the knowledge of therapeutic doses beta2-agonists seems to be unimportant, but illegal use of beta2- agonist probably would be in higher doses which could be of substantial importance.

Purpose: To examine whether Salbutamol and Terbutaline in a human in vivo model similar to the Rat model can show the same positive effect on peripheral leg muscles given as a single high dose prior to exercise and continuously over 2 weeks.

Main Findings

Our purpose was to investigate effects of high dose beta2-agonists on aerobic and anaerobic exercise performance along with muscular effects on contractile force, metabolism, and ion handling in healthy trained men. To investigate these purposes, we conducted seven experiments.

First four experiments investigated the effects of high dose inhaled terbutaline (15-20 mg) on exercise performance and sprinting peak and mean power, as well as on muscle metabolism, contractile properties, and ion handling. Our data showed that terbutaline increased contractile force and enhanced sprinting peak and mean power, but had no effect on time-trial performance or on endurance performance. In skeletal muscles, terbutaline improved Ca2+ handling and counteracted exercise-induced reductions in Na+/K+-ATPase Vmax. Furthermore, terbutaline elevated rate of glycolysis. Thus, performance-enhancing effects of high dose terbutaline may be attributed to effects on skeletal muscles through enhanced Ca2+ handling and elevated glycolytic activity.

In the latter three experiments, we investigated the effects of acute and short-term administration of oral salbutamol (8 mg) and terbutaline (20-30 mg) on exercise performance, muscle contractile properties, and sprinting peak and mean power. Our data showed an acute enhancing effect of terbutaline on contractile properties of m. quadriceps. Four-week administration of oral terbutaline (2∙10-15 mg/d) elicited muscle hypertrophy, reduced fat mass and increased contractile force. Acute and two-week administration of oral salbutamol enhanced sprinting peak power, but had no effect on endurance performance.

In conclusion, our data supports the restriction of high dose inhaled terbutaline and oral beta2-agonists in competitive sports in and out of competition.

Code: 10D12GF

In doping controls, knowledge of steroid metabolism is a key issue to ensure adequate detection of steroids in a urine sample. Metabolism studies are usually performed in vivo by collecting urine samples over a certain period of time after administration of the steroid. However, with new designer steroids on the marked lacking a toxicological profile, this approach has become difficult. Moreover, metabolites may be present at low concentrations in urine, making detection and characterization difficult.

In vitro metabolism studies is an alternative to excretion studies in humans, and human hepatocytes are recognized to be a very close model to the human liver with intact cell membranes and the full compliment of enzymes and cofactors. The limited availability of fresh human hepatocytes has long been the major disadvantage using hepatocytes as a model. The progress in cryopreservation techniques, however, has resulted in an increased accessibility and human cryopreserved hepatocytes are now commercially available.

In the proposed project, the use of cryopreserved human hepatocytes to investigate metabolism of doping agents will be evaluated. Emphasis will be put on anabolic steroids, and the developed model will be tested on two steroids with a well known metabolic profile and one designer steroid with an unknown profile. After hepatocyte incubation and a clean-up procedure, analysis will be performed with liquid chromatography and gas chromatography, combined with mass spectrometry. The in vitro biotransformations and metabolic profiles will be studied in detailed, including the correlation to known in vivo urinary profiles, if available.

Main Findings

The present study deals with the usefulness of cryopreserved hepatocytes to access metabolic mixtures of doping substances. The described in vitro methods are simple systems which are fairly easy and fast to perform. The in vitro hepatocyte metabolism has proven to be close to the human metabolism. It does, however, not reflect the whole complexity of human metabolism, particularly in the light of long term metabolites and phase 2 metabolism.

Despite this, incubations with hepatocytes represent a very useful model for identifying and predicting potential metabolites. Major advantages are amongst others:

- A fast access to a metabolic mixture, which can directly be used for detection strategies of doping substances.

- New non-approved substances with unknown toxicological profiles can be used as substates resulting in metabolites as markers for screening and confirmation purposes.

- No ethical approval is necessary

- Easy detection of possible metabolites due to limited matrix interference of the incubation mixture. Compared to microsomes, the in vivo situation is better reflected with hepatocytes, taking into account drug transport. Furthermore, cryopreserved hepatocytes have several advantages over fresh hepatocytes, first of all a better availability. In addition, cells from several donors can easily be pooled for experiments. Drugs that are metabolized to a high extent in the liver tend to show higher interindividual differences and sometimes genetic polymorphism. Hence, a pooled experiment can better reflect the metabolic picture of the average population.

In this study, human hepatocyte incubations have led to biotransformations generating major metabolites of androstendione and metandienone reported in vivo in humans. Additionally, several metabolites of the synthetic steroid norbolethone were detected, which of only two have been previously described.

Code: 10D1PD 

The classical methodology to detect anabolic steroids or other anabolic substances in doping analytics is GCMS and LCMS. However, the example of THG has demonstrated that even substances with a chemical structure typical for this class of substances are sometimes not identified during routine screening if their exact chemical structure is unknown. Moreover pharmaceutical companies are developing non steroidal androgen receptor modulators (SARMs) which have a complete different chemical structure and metabolism than classical anabolic steroids. Therefore indirect detection techniques may be very helpful in the future to supplement the direct detection of anabolic substance abuse. From the classical anabolic steroids, including Testosterone, it is known that exogenous administration of these substances directly affect the production of endogenous hormones via feedback mechanisms on the hypothalamic pituitary gland axis (Mahabadi et al. 2009). This concept is so efficient that treatment with low testosterone doses is a well established pharmaceutical concept for male contraception (Gu et al. 2009, Misro et al. 2009). The simultaneous detection of ACTH, TSH, PRL, FSH, and LH in comparison to inhibin, estradiol, thyroxin and testosterone levels and indicators for anabolic activity, such as IGF-1 and myostatin, could be a supportive strategy to detect anabolic substance misuse, including SARMs. 

Main Findings: 

The classical methodology to detect anabolic steroids or other anabolic substances in doping analytics is GCMS and LCMS. However, the example of THG has demonstrated that even substances with a chemical structure typical for this class of substances are sometimes not identified during routine screening if their exact chemical structure is unknown. Moreover pharmaceutical companies are developing non steroidal androgen receptor modulators (SARMs) which have a complete different chemical structure and metabolism than classical anabolic steroids. Therefore indirect detection techniques may be very helpful in the future to supplement the direct detection of anabolic substance abuse. From the classical anabolic steroids, including Testosterone, it is known that exogenous administration of these substances directly affect the production of endogenous hormones via feedback mechanisms on the hypothalamic pituitary gland axis (Mahabadi et al. 2009). This concept is so efficient that treatment with low testosterone doses is a well established pharmaceutical concept for male contraception (Gu et al. 2009, Misro et al. 2009). The simultaneous detection of ACTH, TSH, PRL, FSH, and LH in comparison to inhibin, estradiol, thyroxin and testosterone levels and indicators for anabolic activity, such as IGF-1 and myostatin, could be a supportive strategy to detect anabolic substance misuse, including SARMs

Code: 10C2BC

Already in 1990, rHuEpo was banned by the American Medical Association and the International Olympic Committee (IOC). However, the abuse of rHuEpo continues and a sensitive and robust detection test is needed. The current method directly measures urinary rHuEpo, and is based on differences in glycosylation between rHuEpo and endogenous Epo. However, this method is expensive and has low sensitivity.

Protein profiling, or proteomics, provides a novel and powerful method for characterizing the complete catalogue of proteins expressed in a biological system. The aim of the current project is to study the impact of more prolonged rHuEpo exposure on serum proteomics in healthy human subjects, in order to detect novel biomarkers for Epo abuse.

The study will have a randomized double-blinded design. A total of 4 experimental groups will be included in the study; 1. rHuEpo treatment, 2. rHuEpo + exercise, 3. Placebo, 4. Placebo + exercise. A total of 12 healthy inactive male subjects will be included in each group. The treatment period will be 12 weeks followed by a 3 week washout period. rHuEpo will be administrated s.c. at a dose of 5000 IU (~60IU/kg) every second day for the first 2 weeks, on three consecutive days during week 3, and once weekly during weeks 4-12. All subjects will be supplemented by 100 mg iron orally/week. The exercise will consist of 1½h biking at 60-75% of VO2max 3 times per week.

Proteomics will be performed on serum samples collected before treatment and on days 16, 84 (end of treatment) and 105 (end of washout period). It is the hope that the current study will identify novel biomarkers that are specific for rHuEpo treatment, and not effected by exercise itself, that can be used in future anti doping test.

Main Findings

RESULTS: a total 125 spots were identified in the serum 2DE gels, here of was 80 observed in all gels in the current study, isoforms of 6 proteins changed significant during the intervention and washout period in response to ESA treatment. When comparing all 4 experimental groups, isoforms of 2 proteins (sertransferrin and haptoglobin, respectively) showed a significant response to ESA treatment. Hereof, one spot (g), containing haptoglobin, showed a significant lower intensity in all subjects in the training-Epo group during the ESA treatment period, and an increase in intensity during the washout period. Thus, this isoform of haptoglobin could be a potential new anti-doping marker.

CONCLUSION: It is possible by a proteomic approach to identify serum protein isoforms that change significantly in response to Epo abuse without being affected by endurance training. Especially spot G seems promising as a novel biomarker for EPO abuse, since the level of this haptoglobin isoform was significantly decreased in all subjects during the treatment period, and increased again in all subject during the washout period.

Code: 10C19TF

The search for accurate and effective indicators for doping in sport requires the use of many different methods and tools. Traditionally, such tests have relied on the detection of suspected doping agents themselves. However, modern genetic methods have become available that promise not only to improve traditional methods to detect doping substances, but also to provide completely new methods to identify doping by detecting changes in the ways that genes are expressed in doped cells and tissues. WADA has established an extensive research program toward this aim and a number of WADA-supported laboratories have examined the effects of a variety of agents on gene expression such as anabolic steroids, growth and related factors and agents that affect oxygen delivery to tissues. However, detecting such changes is complicated by the fact that the various studies all use different materials and experimental methods.

To identify key changes that identify with scientific and legal certainty the effects of a doping substance and to distinguish useful signals from others caused by training, nutrition, ethnic background, gender, age, etc., it is important to compare the results from many independent studies by using the most powerful modern informatics and computational methods. We have established a WADA-supported informatics facility in this laboratory and have used it to obtain preliminary proof that simultaneous comparative analysis of several independent studies can tentatively identify genes or sets of genes that are altered by exposure to doping agents and that may provide new genetic signatures of doping. We now propose to collaborate with other WADA investigators to expand these initial comparative analyses to a larger set of independent WADA research studies and to published studies in the general biomedical literature. We will include studies related to growth factors and muscle function, oxygen delivery, anabolic steroids and modulators of gene expression.

Main Findings

These two related studies were designed to test the concept that exposure of humans to doping agents disturbs the normal expression of many of the 20,000 genes in human cells and whether those changes can be used as a rigorous proof or a “signature” of exposure to specific doping agents. These kinds of studies are made possible by modern genetic techniques that allow an estimate of the extent of expression of all human genes on a single square inch silicon chip. Our studies also were designed to determine if several studies carried out under different conditions in different laboratories could easily be analyzed even though they used slightly different techniques and methods of analysis. This method of analysis of multiple and slightly different sets of data from different studies is called “meta-analysis” and examples of meta-analysis have been successful in many other kinds of research. We therefore carried out meta-analysis of three independent studies of the effects on gene expression in blood samples from athletes of administration of human growth hormone (HGH) and three separate studies of the effects of erythropoietin (Epo) or hypoxia in the blood cells of human athletes and in mice.

As expected, we found that the genes that are expressed incorrectly after exposure to HGH and Epo are different from each other. However, we also discovered that the separate studies identified only a small number of genes that were disturbed in the same way in all three studies, leading to a conclusion that those genes are probably not specific “signatures” for exposure to HGH or Epo. We have concluded that these results could mean that these drugs do not cause significant changes in gene expression in blood cells. We prefer an explanation that the three separate HGH studies and the diverse Epo/hypoxia differed in many details that introduced too many slight differences in the timing of testing, dosages, methods of tissue preparation and methods of analysis that probably have hidden the gene expressions shared by the different studies. We are impressed that in other very recent studies supported by WADA in which one laboratory has carried out very careful studies of humans exposed to Epo, reproducible changes have been found that consistent with a genetic “signature” for exposure to Epo, demonstrating that extensive and well controlled single studies under some conditions can be an effective approach to identifying the genetic changes in blood samples or other tissues. We propose that a reasonable next step is to carry out a similar study with HGH exposure and compare results from such a study with those obtained in our meta-analysis. We are confident the overall concept of genetic signatures of doping is a correct and robust one and will add important new tools to the prevention and detection of doping.

Code: 10A15MP

As per list of the World Anti-Doping Agency (WADA) 1-Androstenedione is classified as “exogenous anabolic androgenic steroid” and is therefore prohibited in sports. Recently it appears as ingredient of products of the dietary supplement market as so-called prohormone of 1-Testosterone.

Only very limited information is available on its effects and metabolism. It is very likely that the endogenous steroids 17-hydroxy-5-androstane-3-one (5-DHT), 3-hydroxy-5-androstane-17-one (Androsterone), and 5-androstane-3,17-diol occur as metabolites. Altered steroid profiles are therefore expected, especially the ratios of 5/5-androstane-3,17-diol (ADIOL/BDIOL) and androsterone/etiocholanolone (AND/ETIO) may be influenced. A concentration of 5-DHT>21 ng/mL (adjusted for specific gravity) together with ADIOL/BDIOL>1.5, AND/ETIO>2.9, DHT/ETIO>8.2 and DHT/EpiT>0.73 is considered as indicative for the administration of 5-DHT in males. Thus, a potential misinterpretation of the results obtained after 1-Androstenedione administration has to be taken into account.

Thus, the project aims in monitoring the influences on urinary steroid profiles. An identification of other urinary metabolites as well as the evaluation of their time courses will be part of the project as well.

Main Findings

New analogs of androgens that had never been available as approved drugs are marketed as “dietary supplement” recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids.

Recently so-called prohormones of 1-testosterone (17β-hydroxy-5α-androst-1-en-3-one), namely 1-androstenedione (5α-androst-1-ene-3,17-dione, 1-AD) and 1-androstenediol (5α-androst-1-ene-3β,17β-diol) are advertised as well. Only very limited information is available on their effects and metabolism.

Within this project the urinary metabolism of 1-androstenedione was investigated and the main metabolites were monitored following administration of a single oral dose of 50 mg to six male volunteers. 1-Testosterone, 3α-hydroxy-5α-androst-1-en-17-one (1-DHA), 3β-hydroxy-5α-androst-1-en-17-one (1-DHEA), 5α-androst-1-ene-3α,17β- and -3β,17β-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common urinary steroid profile ratios were altered after the administration. Especially the ratios of androsterone/etiocholanolone and 5α-/5β-androstane-3α,17β-diol and the excretion rate of androsterone were increased for about 2 days post administration. 1-DHA appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to ten days after single administration. It was synthesized within this project and characterized by MS and NMR.

For biological characterization 1-androstenedione, and its metabolites 1-testosterone, 1-DHA, and 1-DHEA were tested in a yeast androgen assay as measure of the androgenic potential. 1-AD displayed androgenic effects itself (1/10 as active as 5α-dihydrotestosterone (DHT)) and thus may not be classified as prohormone but as active hormone. However, its metabolite 1-testosterone is able to transactivate androgen receptor driven reporter genes in the yeast androgen screen with a comparable potency as the reference androgen DHT. The two other tested compounds, 1-DHA and 1-DHEA, were about 1/100 as active as DHT.

Code: 10B12MU

We have developed immunoassays for 20K/22KGH isoforms in serum, and the detectability of GH doping was proven.

This proposal is aiming to make the ELISA kits available for the antidoping laboratories, to extend the methods to detect GH in urine samples, and to maximize the assay performance by multiplexing using flow cytometry that is common to the homologous blood transfusion test.

Main Findings

It is well known that active ingredient of pharmaceutical hGH preparation is 22KDa-hGH isoform (22KGH) and administration of GH increases 22KGH to 20KDa-hGH (20KGH) isoform ratio. Because production of those isoforms is being regulated genetically and formed as a result of splicing mRNA during expression of hGH-N gene, a basal 22KGH/20KGH level in human is very stable. We have established highly sensitive sandwich immunoassays for 22KGH and 20KGH in serum, the assay performance was validated according to WADA ISL and the methods have been implemented on 3 assay platforms, i.e., 96-hole microplate assays, multiplexed 20KGH&22KGH microsphere immunoassay for Luminex® and multiplexed immunoassay by flowcytometry. For all three of the assay platforms, the performance is confirmed to be basically same, and the LOQ for 22KGH and 20KGH are 10 and 20pg/mL serum, respectively. Essentially no age-related decrease, no sex-, ethnical- and sporting type-dependent differences of 22KGH/20KGH are observed. Advantage of the detection of GH doping by monitoring the isoform ratio would be less factors influencing the test results, simplicity of the marker to be monitored, namely, two single molecules that are well characterized in the literatures. Serum isoform ratio immediately goes up from about 10 to several thousands after single GH dose, and returned to the base level in about 32 hours. Our data represented possibility to extend the isoform selective GH test to urine samples.

Code: 10A13CG

Aims

The doping control samples are not protected by environment conditions during their transportation from the collection sites to the WADA Accredited Laboratories. The WADA Technical Document for reporting doping violations concerning endogenous steroids is issuing parameters to check for urinary microbial integrity for certain steroids. Endogenous steroids profiles of urine samples may undergo changes due to the occurrence of microorganisms that can be found in the human body or the surrounding environment, especially during their transportation in the warm periods of the year. Moreover, suspicions of tampering of doping control samples with proteases to mask the administration of peptide hormones were confirmed recently.

The Doping Control Laboratory of Athens, OAKA, was granted in 2005 by a WADA research fund to study different approaches regarding stabilization of urine doping control samples during their transportation to the Laboratories. Evaluation of results showed that the application of the stabilization mixture into urine aliquots had a lethal effect on the populations of the microorganisms tested. Moreover, when the stabilization mixture was included in urine aliquots, enzymatic digestion of rEPO and dissociation of the hCG intact molecule in the presence of proteases were inhibited. However, matrix interferences were recorded in mass spectrometric routine analytical procedures.

The current project focuses on the creation of a specially designed urine sample collection plastic container, coated in its interior surface with a suitable chemical stabilization mixture aiming at improving the quality of sport urine samples. The stabilization mixture should be effective in prohibiting degradation caused by microorganisms and proteolytic enzymes, have immediate efficiency, low cost, lack of toxicity and absence of matrix interferences in the doping control analysis. In the current study, the different components of the stabilization mixture will undergo experimental cycles trying to optimize and compromise between stabilization efficiency and analytical matrix interferences.

Main Findings

The current project focuses on the creation of a specially designed plastic urine collection container, coated in its interior surface with the previously developed in-house stabilization mixture aiming at improving the quality of sport urine samples without posing analytical interferences problems in accredited laboratories. Before implementing the chemical stabilization mixture in an industrial scale, the right application form (liquid, freeze-dried, or spray-coated) should be carefully selected and tested in pilot-scale so that it can be implemented in the doping control sampling protocol. The spray coating application form was selected as the more easily acceptable by the end user. The chemical stabilization mixture was spray coated in the interior walls of plastic urine Collection containers to simulate the doping control urine collection process. Its efficiency against steroid glucuronide degradation caused by five microorganisms (E. coli, N. simplex. E. faecalis, A. flavus, C. albicans) and enzymatic breakdown of intact hCG induced by six proteolytic enzymes (papain, pepsin, trypsin, α-chymotrypsin, bromelain and subtilisin A) was investigated during incubation experiments at 37 ºC. Also, two WADA accredited laboratories, DoCoLab (Ghent) and Laboratorio Antidoping FMSI (Rome) undertook the investigation of the stabilization mixture efficiency against rEPO degradation in the presence of proteolytic enzymes. Moreover, a systematic evaluation of eventual analytical interferences in the presence of the stabilization mixture in spray coated form was conducted by the Athens Doping Control Lab (DCLA), DoCoLab and Laboratorio Antidoping FMSI.

The addition of the chemical stabilization mixture in spray-coated form in the interior surface of plastic urine containers inhibited microbial growth and prevented steroid degradation at the end of a 7-day incubation period at 37 °C. The occurrence of the chemical stabilization mixture in spray-coated form prevented rEPO degradation by four proteases (papain, pepsin, trypsin and bromelain) during the 4-day period at room temperature. When α-chymotrypsin or subtilisin A were spiked, IEF signals were detectable at t = 0 in the basic area of the gel only in stabilized aliquots but were eliminated at t = 4 in both the stabilized and unstabilized aliquots. Regarding the degradation of intact hCG induced by proteolytic enzymes, hCG levels were higher in stabilized aliquots at the end of the 4-day incubation period at 37 °C for five out of six proteases tested (bromelain, papain, pepsin, α-chymotrypsin and trypsin). The presence of the chemical stabilization mixture in spray-coated form slowed down the degradation of intact hCG when subtilisin A was spiked but at t = 4, intact hCG levels were undetectable, irrespective of the presence or absence of the stabilization mixture. The evaluation of analytical interferences in the presence of the stabilization mixture in spray coated form, conducted by three WADA accredited labs, showed that some volatile compounds were negatively affected, depending on the urine matrix.

Code: 10A16OP

Testosterone misuse is the most detected doping offence in screening analysis. Nowadays, the ratio between testosterone and epitestosterone (T/E) is measured in the screening method. The exogenous nature of testosterone in those samples showing a T/E higher than 4 is confirmed by GC-C-IRMS. Although this approach gives satisfactory results in most of the testosterone misuse cases, there are some situations in which the value of 4 is not reached even after acknowledged testosterone administration. Those problematic situations include (i) detection of testosterone misuse in population with low basal T/E values, (ii) long term detection of testosterone misuse after oral intake and (iii) detection of testosterone misuse after topical administration.

Recently, four new testosterone metabolites have been reported in our laboratory after treatment of the sample with basic media. The detection of these metabolites in urine samples collected from a volunteer after oral testosterone administration showed that they substantially improved the detection of testosterone misuse in that individual case. Therefore, the addition of these metabolites into screening methods seems to be a promising complement to the commonly used T/E ratio.

Therefore, this project aims to check the usefulness of the quantitative detection of these metabolites for the detection of testosterone misuse in the challenging cases mentioned above: (i) detection of testosterone misuse in population with low basal T/E values, (ii) long term detection of testosterone misuse after oral intake and (iii) detection of testosterone misuse after topical administration. For this purpose, a quantitative method will be developed and validated. Afterwards, the method will be applied to samples collected after different testosterone applications.

Main Findings

Recently, some testosterone metabolites appearing in urine after alkaline treatment have been reported in our laboratory. In this project, the applicability of these metabolites for doping control purposes has been evaluated.

In the first step of the project a quantitative method for the detection of alkaline released metabolites in human urine was developed and validated. Analytical figures (linearity, precision, accuracy and limits of quantification) of the validated method were appropriate for the quantification of both basal levels present in urine of non-treated subjects and concentration levels present after testosterone administration. Besides the satisfactory figures, the method involves an easy and rapid sample treatment which favors its application to the large number of samples required for the development of the project.

In a second step, the validated method was applied to 173 urine samples and reference population concentrations were calculated. Using the software refval a concentration threshold was established for every metabolite and for the ratios calculated between them. The applicability of these thresholds was evaluated. Similarly to T/E, these population based thresholds are useful when large doses of endogenous steroids are administered like in the oral administration of testosterone. However, in small doses, these thresholds are normally not exceeded and, therefore, individual threshold values like the ones used in the Athlete Biological Passport seem to be the option of choice in order to apply these metabolites in the fight against doping.

In the last step, the applicability of these metabolites for the detection of testosterone misuse has been checked. After oral administration, the quantification of these metabolites and several ratios between them allowed for the increase on the retrospectivity. The best marker seems to be 1,4-androstadien-3,17-dione (boldione). The quantification of this marker after alkaline treatment of the urine increased between 3 and 6 times the retrospectivity of the detection of oral administration of testosterone. Ratios involving boldione after alkaline treatment of the sample have been found to be also useful for the detection of administration of single topic dose of testosterone. Although ratios involving 15-AD seem to be useful for this purpose, the absence of the reference material avoid for the quantification of this metabolite.

Although the preliminary results shown in this project seem to indicate that the inclusion of testosterone metabolites released after alkaline treatment in the Athlete Biological Passport can be useful for the detection of endogenous steroid consumption, several parameters have to be evaluated before. Among them, it is remarkable the evaluation of the potential of these metabolites as markers for testosterone administration in those subjects with low basal T/E values.

Code: 10C3JM

In this project, we will develop protein target prediction software to allow the performance-enhancing potential of a molecule to be identified from its chemical structure. This will be made freely available to WADA and to national anti-doping agencies. A Web interface, suitable for intranet deployment, will be underpinned by state-of-the-art predictive software linking molecules to the protein targets against which they are active; both on-target and off-target activities are covered equally well.

We will use machine learning methods to identify substances with potential for use as doping agents. We will primarily employ the Random Forest algorithm, in which we have particular expertise and which has given us excellent results in previous studies. By means of hybrid descriptors combining the geometrical detail of Ultrafast Shape Recognition (UFS) with the chemical information provided by MACCS descriptors, we will encompass both key aspects of molecular recognition. We will use protein target prediction to obtain the on- and off-target bioactivities of molecules with known and unknown doping potential. The profile of activities across a representative panel of protein targets is the molecule’s “bioactivity spectrum”. We will use the bioactivity spectra of known performance-enhancing molecules to predict those of compounds whose performance-enhancing potential is unknown. Thus we will classify molecules, including both licensed pharmaceuticals and other drug-like compounds, as potentially performance-enhancing or otherwise.

While the use of illegal performance-enhancing substances continues to threaten both the integrity of sporting competition and the health of athletes, our software will allow early identification of potential doping molecules. These compounds can then be prioritized for experimental testing, while no further experiments need to be conducted on those with negative in silico predictions. The use of this computational technology will massively reduce the need for animal or human experiments.

Main Findings

In this project, we will develop protein target prediction software to allow the performance-enhancing potential of a molecule to be identified from its chemical structure. This will be made freely available to WADA and to national anti-doping agencies. A Web interface, suitable for intranet deployment, will be underpinned by state-of-the-art predictive software linking molecules to the protein targets against which they are active; both on-target and off-target activities are covered equally well.

We will use machine learning methods to identify substances with potential for use as doping agents. We will primarily employ the Random Forest algorithm, in which we have particular expertise and which has given us excellent results in previous studies. By means of hybrid descriptors combining the geometrical detail of Ultrafast Shape Recognition (UFS) with the chemical information provided by MACCS descriptors, we will encompass both key aspects of molecular recognition. We will use protein target prediction to obtain the on- and off-target bioactivities of molecules with known and unknown doping potential. The profile of activities across a representative panel of protein targets is the molecule’s “bioactivity spectrum”. We will use the bioactivity spectra of known performance-enhancing molecules to predict those of compounds whose performance-enhancing potential is unknown. Thus we will classify molecules, including both licensed pharmaceuticals and other drug-like compounds, as potentially performance-enhancing or otherwise.

While the use of illegal performance-enhancing substances continues to threaten both the integrity of sporting competition and the health of athletes, our software will allow early identification of potential doping molecules. These compounds can then be prioritized for experimental testing, while no further experiments need to be conducted on those with negative in silico predictions. The use of this computational technology will massively reduce the need for animal or human experiments.