Projets de recherche

Code: 14D06RV

The misuse of testosterone and other endogenous anabolic androgenic steroids is detected through alterations in the urinary steroid profile. The steroid profile, composed of concentrations and ratios of endogenous steroid hormones, has been implemented by WADA in the athlete´s biological passport. 
Metabolites included in the steroid profile have both gonadal and adrenal origin. Administration of glucocorticosteroids inhibits the hypothalamic-pituitary-adrenal axis by negative feedback, and reduces the adrenal steroid production. Due to significant adrenal origin of some of the metabolites included in the steroid profile (androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, and 5β-androstane-3α,17β-diol and epitestosterone), it might be expected that reduction in the production of androgens by adrenal glands will have an effect on the urinary steroid profile, mainly in women where the relative importance of androgens generated in the adrenal cortex is greater. Due to the wide use of glucocorticosteroids in sports, its effect on the steroid profile deserves to be studied. 
The aim of the project is to investigate the impact of the administration of glucocorticosteroids by systemic routes on the parameters of the steroid profile in healthy volunteers. The effect of single systemic doses (intramuscular or oral doses) of synthetic glucocorticosteroids on the different parameters of the steroid profile will be evaluated in men and women. 

Main Findings: 

The steroid profile is a powerful tool to detect the misuse of endogenous anabolic androgenic steroids (EAAS) in sports. Glucocorticoids (GCs), which are only prohibited in competition using systemic administration routes, inhibit the hypothalamic-pituitary-adrenal axis. Due to the partial adrenal origin of the compounds included in the steroid profile, the administration of GCs might affect their excretion in urine and, therefore, modify the steroid profile. The aim of the present work was to investigate if GCs administered by either systemic or local routes could affect the steroid profile. 
Three of the most frequently detected GCs in sports (prednisolone, betamethasone and triamcinolone acetonide) were administered to healthy male and female volunteers (n=40) using different administration routes (topical, oral and intramuscular administrations at different doses). In total, 66 administration studies were performed. Urine samples were collected before and after GCs administration. The steroid profile (testosterone T, epitestosterone E, androsterone A, etiocholanolone Etio, 5α-androstane-3α,17β-diol 5αAdiol and 5β-androstane-3α,17β-diol 5βAdiol) was measured by gas chromatography-mass spectrometry.
The excretion rates of the steroid profile metabolites decreased after systemic GC administration (oral and intramuscular administrations). This excretion decrease was found to be associated with the dose and the administration route. However, the ratios evaluated on the steroid profile module of the athlete's biological passport model were not altered.
The results obtained show that GC administration does not distort the establishment of normal ranges of T/E, 5αAdiol/5βAdiol, A/T, A/Etio or 5αAdiol/E ratios. Therefore, GCs administration does not need to be considered a confounding factor in the steroid profile evaluation

Code: 14D04MT 

Growth homone releasing hormones (GHRH) induce growth hormone (hGH) secretion and are prohibited as performance enhancing agents as they act as classical releasing factors of hGH. The availability and the effects are extensively discussed in several internet blogs and black-market platforms. Three synthetic analogues to GHRH (Geref, Tesamorelin and CJC-1295) have been shown to be available to athletes aiming at undermining the doping control system, whereby Geref and Tesamorelin are approved as therapeutics and CJC-1295 has been in clinical trials. Generally, only limited pharmacokinetic data are available for all three peptide-based drugs and their metabolism, particularly their renal elimination after standard therapeutic administration, is largely unknown. This hinders an effective analysis in blood or urine samples for these compounds due to the lack knowledge about relevant metabolites to screen for.

The main parts of the planned project are thus the characterization of metabolites of all GHRH analogues in a rat model (in-vivo) and human blood specimens (in-vitro). Subsequently, effective target peptides (intact drug or derived metabolites) will be evaluated in different biological fluids allowing to establish an initial test method as well as confirmatory procedure inclusive of all relevant target analytes.

Main Findings: 

The family of growth hormone releasing hormones is prohibited in sport due to performance enhancing properties, attributed to enhanced endogenous growth hormone production and/or secretion. Most prominent candidates include the single-chain peptidic drugs/drug candidates Geref (Sermorelin), Tesamorelin, CJC-1295 and CJC-1293. Effective doping control analysis is featured by sensitive determination of the most reliable target metabolites in the respective biological fluid (urine or blood). Thus, the knowledge about the metabolism of a prohibited substance is crucial. In this study, detailed analytical information about the target peptides and their potential metabolites after in vitro experiments and literature review was obtained. Most promising analyte candidates for doping control purposes were synthesized and characterised by mass spectrometry. Several sample preparation strategies were evaluated using solid phase extraction, protein precipitation, ultrafiltration prior to immunoaffinity purification (with magnetic beads or MSIA) and liquid chromatographic-mass spectrometric detection. All developed methods showed sufficient sensitivity to cover the estimated concentrations after administration, and the implementation into existing procedures for other prohibited peptides (insulins, synacthen, etc.) is enabled. The procedures were validated for qualitative result interpretation and considered “fit for purpose” for doping control analysis.

Code: 14D02MT 

Testosterone misuse still remains a challenging task for doping control laboratories as the steroid is a naturally occurring hormone. It is detectable in all doping control urine samples, and concentration-based thresholds have been established to indicate illicit testosterone administrations. 
To facilitate testosterone excretion into urine it is metabolically converted to testosterone-glucuronide, a conjugate with substantially increased solubility in water. Depending on the genotype of an athlete, this transformation is impaired, resulting in very low concentrations of testosterone-glucuronide in urine even after administration of significant amounts of the drug. Routine strategies to disclose testosterone doping are therefore not applicable to athletes with this particular genetic constitution, and depending on the geographic/ethnic origin, up to 70 % of individuals produce these very low urinary concentrations. Despite this limitation to detect testosterone misuse by routine screening methods in so-called ‘del/del’ steroid profiles, the confirmatory analysis based on carbon isotope ratio determinations is a viable option for such samples. 
The aim of the present study will be to investigate and compare the metabolism of testosterone in both genotypes (athletes with high and very low urinary excretion of testosterone-glucuronide), i.e. to determine the metabolic fate and alternative routes of elimination. This will support the identification of potential markers and strategies to enhance sports drug testing for testosterone misuse independent from an athlete’s genotype and the corresponding individual metabolism. Deuterium-labeled testosterone will be administered, and all metabolites tagged by deuterium atom(s) will be identified unambiguously by hydrogen isotope ratio mass spectrometry. All analytes of interest will be quantified and identified by means of high-resolution and high-accuracy mass spectrometry to provide new insights into the metabolism and elimination of testosterone particularly in ‘del/del’ genotypes. 

Main Findings: 

Testosterone (T) misuse still represents a major problem in sports drug testing. Many strategies have been developed and applied to routine doping controls within the recent years to enable both identifying suspicious samples in initial testing procedures and to confirm the exogenous origin of urinary T by means of carbon isotope ratio (CIR) determinations. Depending on the tested individual’s genotype of UGT2B17, significantly different amounts of T are glucuronidated and excreted, which results in unaffected T/epitestosterone ratios after T misuse in those subjects with the deletion/deletion polymorphism (del/del). 

The aim of this study was to investigate differences in metabolic pathways of orally administered T between persons of del/del and insertion/insertion (ins/ins) genetic polymorphism. Therefore, a recently established method using hydrogen isotope ratios together with high-resolution and high-accuracy mass spectrometry was applied after administration of deuterated T to n = 4 subjects including both genotypes. Participants collected urine specimens directly before and for up to 8 days after the application. Urine aliquots were prepared to yield unconjugated, glucuronidated, and sulphoconjugated fractions of urinary steroids.

Besides the significant difference in the excretion of T-glucuronide, all measured metabolites varied rather on an individual basis than due to a genotype difference. New T metabolites (both methylated and de-methylated) were detected and investigated regarding their potential to enhance the screening for T misuse. Sulphoconjugated epiandrosterone was further identified as the biomarker allowing for a significant prolonged retrospective detection of T misuse by means of CIR determinations when compared to the currently applied sports drug testing procedures.

Code: 14C06FM 

No method of direct detection of autologous blood transfusion (ABT) is currently available in sport. The implementation of the Athlete's Biological Passport has increased the search for new biomarkers or new techniques as 'omics' technologies to detect ABT. Otherwise both the phases of blood withdrawal and blood reinfusion might be accompanied by changes of different parameters sensitive to the oxygen availability with generation of integrated molecular profiles. 
We hypothesized that among the potential sensitive parameters, two biomarkers might show changes of interest during ABT: microRNA, small non coding RNAs that regulate gene expression and fetal haemoglobin (HbF), the main oxygen transport protein in the human fetus and still present at low concentration in red blood cells of healthy adults. The objective of the project is to test this hypothesis during three experimental phases by means of 6 tasks: i) the determination of possible changes of HbF and miRNA pathways following blood withdrawal and re-infusion and the identification of proteins and miRNAs to be candidate as molecular markers of ABT (Phase-1-TASK 1); ii) the determination of the reproducibility of HbF and selected microRNA patterns with respect to age, sex and training status. (Phase-2-TASK 2); iii) the determination of the effects on these molecular markers of the exposure to acute exercise and altitude (Phase-2-TASKS 3-4); iv) the development of protocols, assays and platforms suitable for applications in the field of anti-doping (Phase-3-TASK 5-6). When the hypothesis was confirmed, the information of the microRNA profile and of HbF levels should be reported in the Athlete’s Biological Passport to allow the detection of ABT in presence of deep changes, even after combined analysis, of the biomarkers.

Main Findings: 

The lack of a method for directly detecting ABT stimulates the search for new biomarkers and the use of new technologies. This project aimed to identify novel molecular markers useful in the anti-doping field to detect ABT. The hypothesis was that both the phases of ABT, blood withdrawal and reinfusion, might be accompanied by changes in different parameters sensitive to oxygen availability with the generation of integrated molecular profiles. The study focused on changes induced by ABT in: i) HbF, the primary oxygen transport protein in the human fetus and still present at low concentrations in RBC of healthy adults and never tested for anti-doping purposes, and ii) microRNA (miRNAs), small non-coding RNAs that regulate gene expression and are related to erythroid differentiation, HbF production and transcriptional regulation of γ-globin genes.Twenty-four healthy trained male subjects were enrolled and randomized into Transfusion (T) and a Control (C) groups. The T subjects underwent nine seriated blood samples before and after the procedures of withdrawal, and their blood was refrigerated (4°±2°C) or cryopreserved (-80°C). They also received reinfusions. Blood samples were obtained for analysis from the C subjects at the same time points. For the entire population, we analyzed traditional hematological parameters, physical performance assessed by a treadmill test, HbF and other Hgb forms. Microarray analysis of a set HbF-related miRNAs was also performed for 6 subjects of the T group and for a pool of C group blood samples at selected time points.  
For the T group, a moderate change in traditional hematological parameters, particularly following blood withdrawal, and increased performance following reinfusion were observed.  
HbF showed a marked increase in the T group following withdrawal and a decrease after blood reinfusion. Moreover, HbA1c, another form of Hb that can be robustly measured and whose values correlated with HbF values, was found to be stable in the C group. We noted a continuously decreasing trend in HbA1c both after blood withdrawal and reinfusion in the T group. This unusual consistent trend in the T group resulted in a significantly lower level of HbA1c for the parameter compared to baseline values, with abnormally low values in 42% of the transfused subjects. HbF-related miRNA analysis performed in a subgroup of T subjects revealed a pool of 12 miRNAs associated with HbF and regulating transcriptional repressors that were differently expressed in the two phases of the intervention in 5 out of 6 subjects and stable in the pool of control subjects. Notably, among the 5 T subjects with overexpressed miRNAs, 3 individuals exposed an HbA1c value below the cut-off value and, one subject has an HbA1c value roughly equal to the cut-off level.  
In conclusion, this study supports the hypothesis that ABT evokes changes of potential interest in the anti-doping field in sensors of relative hypoxia-hyperoxia. In particular, HbA1c, a biomarker largely used in metabolic diseases and never before tested for anti-doping purposes, showed an interesting pattern following ABT. Similarly, miRNA expression was found to be modified following ABT with person-to-person variability. However, the selective changes in T-group individuals supports the concept that miRNAs analysis might be useful for the detection of ABT. This framework of integrated parameters, which enabled us to recognize a relevant group of transfused subjects, requires further studies.

Code: 14D05KC 

It is generally accepted in the sporting world that dehydroepiandrosterone (DHEA) use enhances physical performance, and DHEA therefore figures on the World Anti-Doping Agency (WADA) list of prohibited substances in sports, both in and out of competition. DHEA is a weak androgen that needs conversion to more potent testosterone, and the assumption is that athletes expect a significant increase in circulating testosterone through exogenous DHEA administration, with a subsequent improvement in performance. The literature on the ergogenic effects of DHEA intake is nevertheless very scant, and to our knowledge none of the studies has presented evidence that DHEA use improves athletic performance. However, this does not mean that an effect should be ruled out as these studies were focused on young male athletes, and no study has yet investigated DHEA effects in female athletes. Most of the time, the urine concentrations of endogenous DHEA are lower than 100 ng/mL in athletes, but in some cases concentrations may reach 200 ng/mL. It was recently demonstrated that exogenous DHEA administration impacted not only DHEA itself, but also the two metabolites, 5α-androstane-3α,17β-diol and5β-androstane-3α,17β-diol, both of which are included in the steroidal module of the Athlete Biological Passport. However, there are no studies on how DHEA affects the other steroids included in this steroidal module. 
We therefore propose to more fully investigate DHEA action during exercise, with a particular focus on: 1) the ergogenic impact of this drug in men and women during supramaximal and maximal exercise, using validated laboratory and field tests;
2) the metabolic and endocrine responses after DHEA administration;
3) the concentrations of urine steroids after physical exercise, in order to determine the metabolic signature of DHEA use as part of the steroidal module of the Athlete Biological Passport.

Main Findings: 

We investigated the ergogenic, metabolic and endocrine effects of short-term DHEA administration in young healthy women. In parallel, we determined the impact of DHEA on food intake, body composition and cytokines as well as on urine steroid concentrations in order to find the metabolic signature of exogenous DHEA as part of the Athlete Biological Passport. First, we showed that short-term DHEA intake (100 mg per day for 28 days) markedly increased blood DHEA, DHEA-S, androstenedione, testosterone, DHT and estrone in recreationally-trained women. No changes were found in the other parameters investigated, i.e., cortisol, IGF1, SHBG, free, blood glucose, liver transaminases and lipid status. Curiously, the marked increase in blood testosterone (10 fold) and DHT (5 fold) induced by DHEA administration did not induce a significant improvement in supramaximal or maximal performance. Moreover, there was no significant difference in body composition, food intake, cytokines and mood. Regarding the urine analyses for the potential metabolic signature of DHEA, it appears that short-term DHEA administration in women significantly impacted up to 24 hours after the last administration urine concentrations of etiocholanolone, 7 keto DHEA, 7β-OH DHEA, 3a-5cyclo DHEA, 5a-Aane, 5β- Aane. In parallel, we found after DHEA treatment a transient classical increase in testosterone, coupled to an increase in epitestosterone, never previously reported. Last, we noted high significant correlations between urine and saliva concentrations for both DHEA and testosterone up to 24 hours after the last DHEA administration. In conclusion, these data indicate that our short-term DHEA administration did not improve performance or have an anabolic effect in young female recreationally trained athletes, despite the striking increase in androgenic hormones. Further studies are specifically needed to determine whether a higher daily dosage administered in small doses over the course of the day and/or longer DHEA treatment would generate an ergogenic effect in young female athletes during such anaerobic exercise.

Code: 14B03MT 

In the past years, the number of reports on novel and innovative pharmaceuticals in relation to erythropoietin mimetic agents (EMA) increased considerably. The future perspective, to generate structures with a prolonged effect in patients by retaining the benefits of erythropoietin, led to significant changes in the molecular structure in emerging drug candidates. As a consequence for doping controls, the detection of an illicit intake of such compounds by athletes has continued to represent a complex analytical challenge.

The use of monoclonal antibodies for every variation and new structure is possible; however, anti-doping laboratories can profit from one single feature of all EMA – the affinity for the erythropoietin receptor (EPOR).
1. A modern cost- and time-effective tool ensuring low consumption of sample material and consumables particularly in peptide analytics, is the application of antibody-coated magnetic beads. A substitution of the antibody with the EPOR provides the platform for a new type of easy-to-use screening assay.
2. With regard to their widespread nature (i.e. from all forms of recombinant Epoetins to pegylated dimerized peptide molecules such as Peginesatide), an EMA-enriched extract is obtained that can be subjected to established as well as future detection and identification methods for prohibited erythropoiesis-stimulating and EPOR activating agents.
3. Due to the unification of urine pre-purification steps, cost intensive processes can be diminished and simplified; enabling at the same time a faster screening procedure.

The enrichment and isolation of low-concentrated proteins from complex matrices such as human urine is a valuable workup for further analysis and optimizes the exploitation of sample volumes provided. By using EPOR-coated magnetic nanoparticles, a pre-selection of potent erythropoiesis stimulating agents and potential non-hematological EPOR activators is accomplished, facilitating and expanding initial testing procedures for protein and peptide drugs.

Main Findings: 

For several years, the number of reports about developments regarding novel and innovative pharmaceuticals concerning erythropoietin (EPO) receptor-activating compounds and EPO mimetic agents (EMA) increased considerably. This class of compounds includes, amongst others, biotechnologically derived forms of erythropoietin and their modified derivatives (Darbepoetin alfa, C.E.R.A.). The latter were developed to improve the pharmacokinetic characteristics by simultaneously maintaining their intrinsic activity. The development of small EPO-mimetic peptides (EMPs), which share no sequence homology with the primary structure of erythropoietin, introduced additional opportunities for the treatment of anemia. Contrary to the therapeutic benefits of expanding the group of EMA, these erythropoiesis-stimulating and endurance performance-enhancing drugs represent a new analytical challenge for doping control laboratories. The increasing complexity of the class of compounds has amplified the relevance of comprehensive and flexible detection methods and thus the development of a generic assay for the detection of new erythropoietin receptor (EPOR)-activating agents was established, exploiting the common feature of these compounds – their ability to bind to the EPOR. The application of magnetic beads equipped with a modified form of a monoclonal antibody proved to provide and effective sample extraction/preparation tool, ensuring reproducible results and low sample consumption. An EPOR-IgG1 fusion protein was coupled to Protein A/G-coated magnetic beads, providing the basis for the developed straight-forward receptor-affinity purification strategy for EMA. The receptor/ligand-enriched extract obtained from (doping control) urine samples is subsequently analyzed by means of established gel electrophoretic (SAR-PAGE or IEF) analytical methods or, complementary, by bottom-up liquid chromatography – (tandem) mass spectrometry. Proof-of-concept data were obtained from native and/or spiked urine specimens containing different ESAs, including darbepoetin alpha and C.E.R.A. as well as the PEGylated dimerized EMP molecule peginesatide. The use of a generic pre-purification protocol to isolate ESAs from human urine can improve the comprehensiveness of routine doping controls and allows to accelerate sample analyses while reducing costs associated with sample preparation steps. Overall, the principle of target protein isolation and enrichment by receptor-affinity purification has been shown to represent a valuable contribution to modern doping control analytical strategies, which warrants further investigation regarding the breadth of drugs possibly covered with this methodology.

Code: 14C35JP

Blood transfusion is a prohibited method in sport. The currently applied method to detect homologous blood transfusion is based on the detection of cell surface blood group antigens that would be different between donors. There is no method available to detect autologous blood transfusion. However, there is evidence that changes occur in red blood cells (RBCs) stored ex-vivo (e.g. in a blood bag or as a frozen concentrate) that do not occur in a normal RBC population. The present proposal intends to empirically select antibodies from a large phage display antibody library able to recognize those specific changes in stored RBCs. Using the novel technology of single cell selection, antibodies recognizing only stored cells will be selected and against antigens broadly occurring in stored cells and that do not show in naturally aging RBCs. Thus, the objectives of the project are:

1.- Selection of an antibody(s) clone from a phage display Library able to specifically identify red blood cells which have been stored in a blood bag or frozen and reconstituted as red blood cell concentrate.

2.- Study the kinetic evolution of the detected antigen along storage period or blood manipulation.

3.- Produce recombinant antibody formats containing the proper tag for optimal use for cell sorting technologies (MACS, FACS)

4.- Development of a Magnetic-activated cell sorting (MACS) procedure for single cell detection.

5.- Adaptation of the methodology to a Flow-Cytometry platform for higher throughput.

Main Findings: 

AIMS
The main objective of the project was to the develop of antibodies able to recognize changes in red blood cells (RBCs) related to ex-vivo storage to unambiguously identify the use of blood transfusion, both autologous blood transfusion (ABT) or homologous blood transfusion (HBT).

RESULTS
Eight promising clones (single domain antibodies fragments, sdAb) were selected showing potential application as a screening and/or confirmation test to selectively recognise stored RBCs.
On the other hand, flow cytometry has proven to be sensitive enough to detect 0.1% cells of a target population and, using a pre-concentration technique like cell sorting, this limit of detection can be greatly increased.
Once the sdAb fragments have been selected from the phage display library, they can be optimised by modifying each position in the CDR regions to increase specificity, optimise the tag sequences with which they are expressed, etc. so that the final instrumental set-up is optimised.

CONCLUSIONS
The overall results demonstrate that the selection of antibodies from a large phage display library is a feasible approach to develop a procedure to detect surface antigens produced on RBCs through ex-vivo blood storage. These antigens can be potentially detected using flow cytometry in a minute subpopulation of RBCs in a freshly drawn blood sample

Code: 14B11MT 

Key regulator of skeletal muscle mass in developing embryonic and adult skeletal muscle is the growth factor myostatin. In animal models, both myostatin deficiency and inhibition resulted in an increased muscle mass and function due to an elevated volume and/or number of skeletal muscle fibers. Consequently, myostatin inhibitors may not only provide a therapeutic approach for the treatment of muscular diseases but also be misused as performance-enhancing agents in sports. 
The aim of the planned project is to establish a detection method for MYO-029 (Stamulumab), a recombinant human myostatin antibody which was developed by Wyeth Pharmaceuticals in 2005. MYO-029 specifically blocks myostatin signaling by preventing the interaction between the growth factor and its target receptor. Mice treated with such a neutralizing antibody showed an enhanced muscularity and a first clinical trial on humans yielded a good tolerance and safety of the drug. Within this project, two different immunological approaches will be used for the detection of MYO-029 misuse in cheating athletes. Both the western blot and ELISA will use recombinant human myostatin as bait to capture MYO-029 potentially present in plasma/serum samples. Antibody specifically bound to the immobilized antigen can then be detected by using an enzyme-coupled secondary antibody direct against its species-specific portion. Additionally, an antibody-based internal standard will be implemented by using different species-specific secondary antibodies conjugated to distinct fluorophores. Following method optimization, both approaches will be thoroughly characterized to ensure their reliability, sensitivity, and transferability. They will serve as proof of principle for the detection of myostatin inhibitors and antibody-based pharmaceuticals in doping control samples.

Main Findings:

Myostatin is the key regulator of skeletal muscle mass and inhibition of its signaling pathway can result both in an increased muscle mass and function. Within this project, two complementary detection methods for the recombinant human anti-myostatin antibody MYO-029 were developed by using immunological approaches.  
First, a qualitative western blot-based assay was set up and comprehensively characterized. It was found to be highly specific, robust, and linear from 0.1 to 1 µg/mL. The precision was successfully specified at three different concentration levels and the recovery of the affinity purification was 58%. Consequently, in consideration of the World Anti-Doping Code International Standard for Laboratories (ISL) 2015, paragraph 6.2.4.3.1.3, the assay can be considered fit-for-purpose for an application in routine doping control analysis.  
Additionally, an ELISA capable of detecting MYO-029 in human serum was developed as initial testing procedure. The conducted experiments show that a very sensitive and reproducible detection of anti-myostatin antibodies in a microtiter plate is possible. However, further optimization and characterization e.g. to reduce non-specific background signals is advisable if such approaches will be considered for routine doping controls, especially when multiplexing for different antibodies relevant for doping controls is desirable. 

Code: 14A01RB 

Because of their performance-enhancing effects anabolic androgenic steroids (AAS) have been prohibited by the International Olympic Committee since 1976. However, they still belong to the most frequently detected substances in doping tests and improvement of the detection capabilities is one major aim in anti-doping research.  This includes a better understanding of their metabolism in the human body and the discovery of long-term metabolites. In this context cytochromes P450s (P450s), who contribute to the biotransformation and subsequent renal clearance of exogenous compounds as well as to the biosynthesis of endogenous steroids, are a promising target for the investigation of the metabolic pathways of exogenous steroids like AAS. While the metabolism of xenobiotics by liver P450s is well studied, their biotransformation by adrenal P450s is nearly unknown. Consequently, our aim is to study the metabolism of AAS by P450s from the human adrenal cortex, in order to provide new reference material for doping analysis. Adrenal P450s are involved in the biosynthesis of steroid hormones and only very few cases regarding the metabolism of xenobiotics by these P450s have been described so far. Therefore, our project aims to investigate the in vitro metabolism of AAS by these P450s employing the world-wide unique set of purified proteins available in our lab. 
The applicability of discovered novel metabolites as (long-term) markers in doping tests will be examined by comparison with the urinary excretion profile in a mass spectrometry analysis. For metabolites, that turn out to be useful biomarkers, a larger scale production by genetically modified bacterial cell factories will be developed to create a basis for the supply of these metabolites to other laboratories for incorporation in routine doping analysis. 

Main Findings: 

Anabolic androgenic steroids (AAS) are frequently misused for doping purposes due to their performance enhancing effects. In the human body, they are metabolized, as all other drugs, in the liver to facilitate their clearance via the urine. The detection of AAS metabolites in the urine of an athlete by doping controls can demonstrate a doping delict. Most metabolic reactions in the liver are carried out by a group of proteins, or enzymes, called cytochromes P450. Closely related enzymes, which also belong to the cytochrome P450 group, are responsible for the synthesis of natural steroid hormones in the adrenal, in reproductive and some other tissues. The question arises whether these steroid-synthesizing enzymes can also contribute to the metabolism of AAS and produce alternative target metabolites for doping controls. The six human steroid-synthesizing enzymes (CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP19A1 and CYP21A2) were produced in genetically modified bacteria (Escherichia coli) and purified to evaluate their capability to metabolize the AAS oral-turinabol (OT), stanozolol and oxandrolone. Three of the enzymes (CYP11A1, CYP11B1 and CYP11B2) were found to metabolize OT and the structures of the emerging metabolites were elucidated by different methods (nuclear magnetic resonance spectroscopy, liquid chromatography tandem mass spectrometry). We identified five new OT metabolites (11β- hydroxy-OT, 11β,18-dihydroxy-OT, 11β-hydroxy-OT-18-aldehyde, 2-hydroxy-OT and 2,16-dihydroxy-OT) that have not been described in the literature to date and therefore represented interesting candidates as target analytes for doping controls. However, when we analyzed urine from an OT administration study, none of the compounds could be detected. This suggests that the new OT metabolites are further metabolized by additional enzymes prior to their excretion with the urine, which hampered their detection. In conclusion, the new OT metabolites cannot be used themselves to demonstrate a doping delict, but can serve as intermediates for the synthesis of their downstream metabolites, that might be excreted with the urine.

Code: 13D15GP

The use of anabolic androgenic steroids (AAS) is widespread within both competitive and recreational sports in which large, strong muscles are an advantage or the primary goal (bodybuilding). To build more muscle mass and to enhance muscle force and power, AAS are particularly effective. In this study, we want to investigate the long-term effects in the skeletal muscles that in particular are related to nuclei of the muscle fibers. Because the nucleus of a cell controls the different cellular-processes, including adaptations to training, long-term or irreversible changes in the number of nuclei in a muscle fiber will potentially give a person using AAS an advantage also after he/she stops using the drugs. Athletes caught as drug users are quarantined for at most 2 years for a first time offence, however, this should be reevaluated if the effects of AAS are shown to last many years after the athlete stops using the drugs. These research aims for a human study are directly derived from a recent animal study conducted by some of the co-investigators in this application. In this human study we will recruit males (20-40 years) with no strength training experience. The volunteers will be administrated a testosterone preparation or placebo (saline) by intramuscular injections during a 3 month period. Concomitantly they will follow a heavy strength training program. After the initial 3 months the participants will refrain from strength training for 9 months, but then re-train their muscle for 3 months without any drugs. Muscle tissue samples, measures of muscle mass and strength will be collected and assessed before and after each period. The main question is if the volunteers that received testosterone during the initial 3 months will re-build their muscle faster than the participants that received placebo.
The use of anabolic steroids has in an animal study been shown to give
long-term effects in skeletal muscle, by adding nuclei (DNA) to the muscle fibres and accelerating re-training. This means that the advantage of anabolic steroid use may be present in years after the athlete has stopped using the drugs. This study aims to test the hypothesis that the results from this animal study applies for humans.