Projets de recherche

Code: 14B26CR

Aside from human endogenous erythropoietin (EPO), its recombinant analogues (e.g. Epoetins alfa/beta/delta/omega, Darbepoetin alfa) and EPO-mimetics (e.g. Peginesatide), which stimulate erythropoieses through the EPO-receptor pathway, formation of erythrocytes can also be stimulated via the so called €activin-receptor type IIA signal transduction pathwayÅ: as soon as ligands, which interact with activin-receptor type IIA (€ActRIIA ligandsÅ) are removed by so-called ActRIIA ligand traps in a targeted way, erythropoiesis is also stimulated. ActRIIA ligands belong in particular to the transforming growth factor-beta (TGF-‚) superfamily (e.g. activin).
Sotatercept (ACE-011, ActRIIA-IgGI), a fusion protein consisting of the extracellular domain of ActRIIA receptor and the Fc-part of human immunoglobulin G1 (IgG1), is capable of acting as ActRIIA ligand trap, and was primarily developed as pharmaceutical for enhancing bone mineralisation in order to revert osteoporosis. However, aside from increasing bone mineral density, it was discovered that Sotatercept also stimulates erythropoiesis in a dose dependant manner leading to an increase in red blood cell counts. Sotatercept is currently intensely investigated in several phase II clinical trials.

Main Findings:

Two methods for the detection of Sotatercept in serum and urine samples were successfully developed. One method uses covalent immobilization of the capture antibody on agarose beads. It requires 10 µg of a polyclonal goat anti-ACVR2A antibody per sample (AF340 from R&D Systems) but needs double-blotting in case multiplexed detection of Sotatercept together with epoetins is necessary. The other method applies commercial magnetic beads coated with an antibody (sheep anti-rabbit) directed against the capture antibody. After incubation of the serum samples, the complex consisting of Soatercept and the capture antibody (a polyclonal rabbit antibody, 10257-T52 from Sino Biological) is bound by the anti-rabbit antibodies on the beads. After Western blotting, Soatercept is detected with the biotinylated version of the capture antibody of the other method (BAF340 from R&D Systems) and streptavidin-HRP. It omits incubation with a secondary antibody and thus is simpler, faster, and cheaper (only ca. 1.5 µg of capture antibody are required) The second protocol is compatible with single-blotting if the biotinylated clone AE7A5 antibody is used for the simultaneous detection of epoetins.

Code: 14B27CR 

Currently, comprehensive initial testing for erythropoiesis stimulating agents (ESAs) is done by electrophoretic methods (IEF-, SDS-, SAR-PAGE) according to WADA TD2013EPO and the forthcoming TD2014EPO. However, these protocols only comprise endogenous and recombinant erythropoietins and their analogues (e.g. darbepoetin alfa). ESAs, which lack the primary structure of EPO and in particular the first 26 amino acids of the N-terminus, cannot be detected due to non-interaction with the monoclonal anti-EPO antibody used for Western blotting (clone AE7A5). Peginesatide (Omontys; formerly known as Hematide) is a so-called EPO-mimetic peptide, which is structurally unrelated to the amino acid sequence of EPO. Two methods (SDS-PAGE, electrospray mass spectrometry) were developed and published in 2011 in order to overcome this situation. However, both methods are unable to simultaneously detect epoetins (EPOs) - the majority of misused ESAs. By modifying Sarcosyl (SAR)-PAGE - one of the main electrophoretic methods used in EPO anti-doping testing - peginesatide will now also become part of routine ESA doping testing with SAR-PAGE. Despite Omontys was recalled in 2013, misuse by athletes cannot be excluded.

Main Findings: 

An electrophoretic method was successfully developed in order to get Peginesatide, a PEGylated EPO-mimetic peptide dimer, migrate in SAR-PAGE, which is one of the main methods used in EPO-testing. Due to its short amino acid chain and the two PEG-groups, sarcosyl cannot sufficiently solubilize Peginesatide for running it on SAR-PAGE. Upon replacement of 30% of the SAR in the running buffer by SDS (resulting in a final composition of 0.03% SDS and 0.07% SAR), Peginesatide migrates into the stacking gel without significantly broadening the CERA-band. With the optimized "SDS-modulated" SAR-PAGE method (mSAR-PAGE), Peginesatide can also be differentiated from CERA, since both bands are well separated from each other.

The new method also allows the simultneous detection of Peginesatide and all EPO-based ESAs on one blot. By duplexing the two primary antibodies (clones 11 F9 and AE7A5) followed by incubation with an HRP-labelled anit-mouse secondary antibody, which binds to both antibodies, single blotting can be applied for detection.

However, the modification of an already published method for immunoaffinity purification of Peginesatide from serum and plasma samples could not be solved so far. The published protocol used covalently immobilized clone 1G9 antibody and released bound Peginesatide by heating the bead in SDS-sample buffer. Despite double-blotting, bands from the light antibody chain remained visible on the blot. Hence, this approach was not applicable for the simultaneous detection of Peginesatide next to epoetins by single blotting. In search of a solution, three alternative methods were tested: (1) covalent immobilization of clone 1G9 antibody on agarose beads followed by acidic elution; (2) immunoprecipitation using anti-antibody coated magnetic beads; (3) immunoprecipitation using biotinylated clone 1G9 antibody and streptavidin-coated magnetic beads. However, they did not yet lead to useful results. Despite the projct has ended, we will continue trying to find a solution

Code: T14M03WS

Investigations in connection with an adverse analytical finding for the prohibited diuretic chlorazanil have raised the presumption that chlorazanil may originate from the application of the non-prohibited anti-malaria drug proguanil (ingredient e.g in medicament Malarone® from GSK). The structure of metabolites of the non-prohibited anti-malaria drug proguanil suggests that they can be converted to chlorazanil. To check this hypothesis it should be found out whether chlorazanil is a metabolite of proguanil or whether proguanil metabolites are converted in the urine to chlorazanil by chemical or bacterial influences or whether chlorazanil is a byproduct of the synthesis of proguanil. 
Furthermore parameters should be identified which allow a discrimination between the application of the non-prohibited proguanil and the prohibited chlorazanil.

Main Findings:

Chlorazanil (Ordipan, N-(4-chlorophenyl)-1,3,5-triazine-2,4-diamine) is a diuretic agent and as such prohibited in sport according to the regulations of the World Anti-Doping Agency (WADA). Despite its introduction into clinical practice in the late 1950s, the worldwide very first two adverse analytical findings were registered only in 2014, being motive for an in-depth investigation of these cases. Both individuals denied the intake of the drug; however, the athletes did declare the use of the antimalarial prophylactic agent proguanil due to temporary residences in African countries.  While no chlorazanil was found in drug formulations, the urine samples of 2 out of 4 proguanil users returned findings for chlorazanil at low ng/mL levels, similar to the adverse analytical findings in the doping control samples. Further, in the presence of formaldehyde, formic acid and related esters, 4-chlorophenyl-biguanide was found to produce chlorazanil in human urine, suggesting that the detection of the obsolete diuretic agent was indeed the result of artefact formation and not of the illicit use of a prohibited substance
A structural similarity between chlorazanil and proguanil is given but no direct metabolic relation has been reported in the scientific literature. Moreover, chlorazanil has not been confirmed as a drug impurity of proguanil. Proguanil however is metabolized in humans to N-(4-chlorophenyl)-biguanide, which represents a chemical precursor in the synthesis of chlorazanil. In the presence of formic acid, formaldehyde, or formic acid esters, N-(4-chlorophenyl)-biguanide converts to chlorazanil.  
In order to probe for potential sources of the chlorazanil detected in the doping control samples, drug formulations containing proguanil and urine samples of individuals using proguanil as antimalarial drug were subjected to liquid chromatography-high resolution/high accuracy mass spectrometry. In addition, in vitro simulations with 4-chlorophenyl-biguanide and respective reactants were conducted in urine and resulting specimens analyzed for the presence of chlorazanil.  

Code: 14A32GJ 

Formoterol is a long acting beta2-agonist allowed for use in athletes via inhalation at doses up to a predetermined limit, and is not allowed to be administered orally. The drug is usually administered and measured as a racemic mixture consisting of an active R/R- and inactive S/S- enantiomer, each with a different time course in the body which also differs by route of administration. The inactive S/S-formoterol is preferentially eliminated in urine relative to the active R/R-formoterol. Recent work has demonstrated that the Adverse Analytical Finding (AAF) limit, currently 40 ng/ml in urine, is
difficult to reach and some authors have even questioned whether it is worth having an AAF limit for formoterol at all. To date, anti-doping strategies have not capitalised on this difference between formoterol enantiomer elimination from the body. We will use our advanced analytical technique (enantioselective LC-MS/MS) to characterise the urinary levels and ratio of both R/R- and S/S-formoterol enantiomers, as well as the glucuronide metabolite in athletes dosed with formoterol in prohibited regimens; an “acute” treatment regimen that exceeds the current allowable dose via inhalation and an oral regimen consisting of 160 μg/day for 7 days. The secondary objective is to characterise the urinary levels and ratio of formoterol enantiomers and glucuronide metabolite in athletes using repeated “chronic” inhaled rac-formoterol under permitted regimens. Study
design will be double-blinded placebo controlled with two “chronic” 7 day treatment regimens; formoterol delivered by inhalation at just below the maximum allowable dose, and approximately half of the maximum allowable dose. This project will allow doping agencies to better discriminate between permitted and prohibited formoterol dosing.

Main Findings: 

Oral dosing of beta2-agonists is known to lead to beneficial performance effects in athletes but some drugs of this class are allowed to be delivered by inhalation for use in athletes with asthma. Formoterol is a long acting beta2-agonist permitted for use in athletes at inhaled doses up to 54 μg over 24 hours, however, a threshold limit of 40 ng/ml in urine is used to control for supratherapeutic dosing. The drug is usually administered as the racemic (rac-) mixture consisting of the active (R,R)- and inactive (S,S)- enantiomers with different metabolic profiles. The primary objective of the study was to examine whether the urinaary levels and ratio of formoterol enantiomers in individuals dosed with formoterol could be used to improve the sensitivity and specificity of a urine threshold approach to ascertain whether individuals were using formoterol in a permitted manner. Urine levels were measured following prohibited regimens; an "acute" treatment regimen that exceeds the current allowable dose (54 μg in 24 hours) by administering a once-off 72 μg dose via inhalation at baseline, and an oral regimen consisting of 156 μg/day for 7 days. These levels were compared with repeated "chronic" inhaled rac-formoterol under permitted regimens (12 μg twice daily for 7 days, 24 μg twice daily for 7 days). Comparisons were made with current urine dosing threshold and decision limits (TD2019DL) to assess the utility of enantioselective ratios. Using the current TD2019DL method, none of the samples from the chronic permitted regimens exceeded the current threshold for total formoterol (40 ng/ml) supporting the existing approach. The acute inhaled and oral prohibited regimens resulted in 7/127 and 21/124 samples exceeding the threshold  (TD2019DL), and 5/127 and 13/124 exceeding the decision limit respectively. There were an increased number of specific gravity. Females demonstrated higher urine levels than males. As prediced, there were differences in enantiomer ratio between oral and inhaled administration, but receiver operating characteristic (ROC) analysis for log(R,R:S,S) formoterol enantiomer ratio showed poor diagnostic performance (ROC area = 0.716 for oral treatment) and was inferior to the current TD2019DL approach, possibly due to considerable pharmacogenetic enantionselective differences in glucoronidation metabollism. In conclusion, the current urine method (TD2019DL) based on a threshold and decision limit of 40 ng/ml and 50 ng/l respectively appears a valid approach to discriminate between oral (prohibited) and permitted inhaled dosing regimens in urine spot samples but requires further validation with regard to intra-day spot sampling variability and dosing regimens. Furthr work is required to understand the basis of the significant differences in enantioselective glucuronidation between subjects and genders, and improve the sensitivity of the current threshold. Formoterol elimination seems to be particularly sensitive to urine hydration and a greater understanding of exercise on hydration and excretion is also warranted with regard to application of TD2019DL threshold approach.

Code: 14A15RH

Over the last decades, an increasing number of pharmaceutical peptides and proteins have entered the market. Unfortunately, protein drugs are also misused by athletes to illicitly enhance their performance. As these biopharmaceuticals often are highly similar to natural endogenous proteins, reliably tracing of protein doping products in urine or blood is very challenging. Current anti-doping methods can detect prohibited protein substances, but they do not provide unambiguous information on the molecular structure of the detected species, leaving room for errors. Therefore, advanced analytics such as liquid chromatography-mass spectrometry (LC-MS), have gained popularity in doping analysis. However, LC of intact protein molecules is troublesome, and so far few suitable LC-MS methodologies for distinguishing endogenous from manufactured proteins have been developed.

In this project, we will develop a highly selective and generic analytical platform for the unambiguous characterization of prohibited protein substances in biofluids, like urine and blood. To this end, capillary electrophoresis (CE) will be combined with high-resolution MS. In contrast to LC, CE can provide highly efficient separations of intact protein species. High-end MS detection will yield accurate molecular masses of the separated proteins and their isoforms. This way, CE-MS will allow consistent discrimination of banned protein species from endogenous proteins. In order to meet the requirement to measure very low protein concentrations, a novel ultrasensitive interface will be employed for coupling CE and MS. Moreover, for sample pretreatment, advanced affinity extraction and in-capillary preconcentration strategies will be exploited to achieve very low detection limits. Ultimate goal is to aid the fight against doping by delivering a new reliable method for high-throughput assessment of prohibited performance- enhancing proteins.      

Main findings: 

Results. Major efforts have been put in developing a robust and sensitive capillary electrophoresis method to allow hGH isoform separation and to live up to the requirements for doping analysis. Separation of the two main hGH isoforms can be achieved using a volatile alkaline background electrolyte (BGE). Evaporation of volatile BGE components, leading to severe method instability, could be circumvented by the use of a mineral oil overlay. The nature and order of conditioning showed to have a big impact on the migration time (MT) stability. After verifying several combinations, MT RSDs for the electroosmotic flow and analyte remained constant and were below 0.3% and 0.5%, respectively, for 48 consecutive injections. 
High resolution mass spectrometry (MS) in combination with CE separation enabled structural characterization of endogenous hGH. Next to the differentiation of the 20 kDa and 22 kDa isoform, two known deamidated forms of asparagine were observed. Furthermore, two truncated forms, resulting from the loss of the first two amino acids from the N-terminus, were distinguished. All these many other modifications are, most probably, formed during the long lifetime of this standard (over 30 years). Interestingly, the hGH isoform ratio observed in commercial human standards found with CE-MS (1:15-25) correlated well with reported values (~1:20). 
The CE method enabled for the first time to assess the affinity of commercial hGH antibodies on an individual isoform level. Careful optimization of the binding and elution steps revealed a good recovery for the 22 kDa isoform using one mAb. However, in all other cases low recovery was obtained. This raises the question whether current mAbs are able to accurately distinguish hGH isoform and should be used for sample clean-up and biochemical assays. Division of BioAnalytical Chemistry WADA final report 
Conclusions. A simple and robust CE method has been developed that can separate the two main isoforms of hGH under MS compatible conditions. The hyphenation with MS detection ensures detailed characterization of hGH isoforms. It is the first time that unambiguous assignment of the 20 and 22 kDa variant was made possible showing a clear improvement over current methodologies. Unfortunately, the current commercial interfaces do not provide the sensitivity or robustness without further optimization. The developed CE method did indicate that using the available commercial antibodies raised against the two main isoforms of hGH do not show the same affinity.

Code: 14D16JB

Glucocorticoids are widely used among athletes in treatment of acute inflammation and in chronic inflammatory diseases such as asthma. However, glucocorticoids may be misused to increase recovery and performance during competition, and several former Tour de France cyclists have admitted use of glucocorticoids as performance-enhancing agents during races. The current prohibited list allows glucocorticoids to be applied as a skin cream or inhaled from an inhalator, whereas injections and intake of pills are prohibited. In order to distinguish between prohibited misuse and
therapeutic use, and to establish urinary thresholds, it is necessary to investigate differences in urine concentrations between the different administration forms. The aim of the current study is thus to investigate differences in the urine concentration of the two most common glucocorticoids following different routes of administration during exercise.

Main Findings: 

While the pharmacokinetics of methylprednisolone is well established in untrained individuals at rest, no studies have, to our best knowledge, investigated the pharmacokinetics of methylprednisolone during simulated competitive exercise applicable to real life sport settings in highly trained individuals. In a randomized open-label crossover study, we investigated urine pharmacokinetics of methylprednisolone after oral, intramuscuar (vastus lateralis and erector spinae muscles), and intravenous administration (16 mg for each route) in conjuction with exercise in 16 endurance-trained men [aged 25±4 years with a VO2max of 63±4 (mean±SD)]. After administration of methylprednisolone, subjects performed 3 hours of cycling exercise at 55-60% VO2max. Urine samples were collected prior and 0-25 hours following administration and were analysed for concentrations of unchanged methyprednisolone using HPLC-MS/MS. Urine excretion rate of unchanged methylprednisolone followed an exponential decline for the intravenous and intramuscular injection routes, peaking withine the first 1½ hours and reaching values close to zero 12-24 hours following injection. For the oral route, excretion rate peaked 1½-3 hours after ingestion, reaching values close to zero 12-24 hours following ingestion. Urine concentrations of methylprednisolone displayed substantial inter-individual variability. Following intravenous or intramuscular injection, urine concentrations peaked 1½ and 3 hours following administration and declined rapidly, displaying low concentrations 8-12 hours following injection. Urine concentrations were lower following oral ingestion during the firsst 1½ hours after administration, but slightly higher than the other routes 8-24 hours afer administration. These observations indicate that urine spot sampling cannot discriminate different systemic routes of methylprednisolone administration based on the concentrations of unchanged methylprednisolone.

Code: 14A34XD

Formestane is an anti-estrogenic drug used on the treatment of breast cancer. In humans, estrogens are strong pituitary inhibitors of gonadotrophins releasing factors. The inhibition of the estrogens synthesis produces an increase of luteinizing hormone (LH) and then a net increase of testosterone production is expected. In addition the combined administration with testosterone will reduce the side effects linked to aromatization like gynecomastia. For these reasons, anti-estrogenic substances were included in 2004 in the World Anti Doping Agency (WADA) List of Prohibited substances. 
The analytical methodologies developed so far are based GC/MS or LC/MS, targeting formestane itself. Traces of formestane can be produced endogenously and detected in urine samples in low concentrations(0.5-20 ng/mL) and thus, since 2011, it is mandatory according to WADA rules to perform a confirmation based on isotope ratio mass spectrometry (IRMS) in order to assess the synthetic origin of formestane before releasing an adverse analytical finding. 
The IRMS developed methods require two consecutive liquid chromatographic purifications (HPLC) before obtaining extracts of adequate purity, and not all laboratories are currently prepared to perform such IRMS analyses.  The metabolism of formestane analysis has been extensively described in a single male volunteer. It appears that among the high number of metabolites described 4a-hydroxy-epiandrosterone has a longer detection window. A metabolic approach based on the detection of specific metabolites of formestane may avoid the use of IRMS for the confirmation of formestane intake, thus reducing the cost and complexity of the analyses. Simultaneously in the case IRMS should still needed, the development of a method for the detection of long term metabolites, excreted for a longer time compared to formestane and in larger amounts in the elimination phase, will certainly improve the detection capacity of formestane.

Main Findings:

Formestane confirmation by IRMS is not an easy task and the method is not yet implemented in all WADA accredited laboratories. IRMS confirmations are time consuming and generate additional cost to the Testing Authorities.  The aim of this work was to improve the knowledge on formestane detection and on its metabolism in order to find specific biomarkers that may reduce to the minimum or even discard the need of IRMS. To do so, oral and transdermal excretion studies were performed and the metabolism of formestane was studied. The first observation is that if no specific method is applied (MS/MS) the estimation of formestane concentration in urine is overestimated, its detection may interfere with the detection of 2-hydroxyandrostenedione. The 4-hydroxylation of steroids is residual while the 2- hydroxylation that is much more important.  The main specific metabolites of formestane (4OH-AED), detected in different proportions depending on the administration route, are 4αOH-epiandrosterone (4OHEA) and 4αOH-androsterone (4OH-A). These metabolites have not been detected in urine samples so far. Their endogenous origin can be most probably discarded, masking their detection specific to trace back a formestane administration. The detection of these metabolites and some metabolic ratios (4OH-EA/4OH-AED, 4OHA/4OH-AED and 4OH-EA/4OH-A) may allow to detect the exogenous administration of formestane and to discriminate the route of administration without the need of using IRMS.
Formestane confirmation by IRMS is not an easy task and the method is not yet implemented in all WADA accredited laboratories. IRMS confirmations are time consuming and generate additional cost to the Testing Authorities.  The aim of this work was to improve the knowledge on formestane detection and on its metabolism in order to find specific biomarkers that may reduce to the minimum or even discard the need of IRMS. To do so, oral and transdermal excretion studies were performed and the metabolism of formestane was studied. The first observation is that if no specific method is applied (MS/MS) the estimation of formestane concentration in urine is overestimated, its detection may interfere with the detection of 2-hydroxyandrostenedione. The 4-hydroxylation of steroids is residual while the 2- hydroxylation that is much more important.
The main specific metabolites of formestane (4OH-AED), detected in different
proportions depending on the administration route, are 4αOH-epiandrosterone (4OHEA) and 4αOH-androsterone (4OH-A). These metabolites have not been detected in urine samples so far. Their endogenous origin can be most probably discarded, masking their detection specific to trace back a formestane administration. The detection of these metabolites and some metabolic ratios (4OH-EA/4OH-AED, 4OHA/4OH-AED and 4OH-EA/4OH-A) may allow to detect the exogenous administration of formestane and to discriminate the route of administration without the need of using IRMS.

Code: 14C27DE 

The ABP was proposed by WADA nearly a decade ago to longitudinally monitor and define an athlete’s individual blood variables in an attempt to indirectly detect doping. The ABP relies on the monitoring of blood variables sensitive to the administration of performance enhancing drugs (PEDs) to identify abnormalities in an athlete’s profile that cannot be explained by a normal physiological or pathological condition. When reviewing irregularities in an athlete’s blood profile, the experts must consider the effect confounding factors such as physical exercise have on the ABP. Indeed, studies have shown decreases in the absolute blood volume and increases in hemoglobin (HGB) concentration when subjects were acutely dehydrated. The ability to characterize an athlete’s hydration status at the time of blood collection would assist experts when reviewing irregularities in the ABP. The most widely used indicator of hydration status is osmolality, a measurement of the electrolyte-water balance in the body. Albumin is the most abundant protein in plasma and is largely responsible for attracting water into the circulatory system. Elevated albumin is typically a sign of dehydration. As lactate and other metabolites accumulate in working muscles, plasma water will be pulled to the working muscles thereby reducing plasma volume and elevating albumin concentration. Interestingly, increases in blood lactate correlate to decreases in plasma volume after maximal exercise. Therefore, it may be possible to indirectly assess relative plasma volume by measuring blood lactate. The purpose of the present study is to compare the changes in serum albumin, osmolality and lactate, potential markers of fluid balance, in the context of the ABP when athletes are subjected to cycling trials of varying levels of dehydration and exercise intensity. The inclusion of additional biomarkers in the ABP responsive to whole body hydration will strengthen the sensitivity of the ABP.

Main Findings: 

This study aimed to identify markers of dehydration not currently accounted for in the hematological passport, specifically serum albumin and serum osmolality, in addition to understanding the effect of exercise-induced dehydration on the current markers of the hematological passport (hemoglobin, reticulocyte%, OFF-score, and ABPS).  Twelve subjects underwent multiple controlled exercise trials designed to induce varying levels of dehydration.  Pre-exercise blood samples were collected to establish baseline values for individual passports.  During exercise interventions, blood samples were collected before the start of exercise and immediately following exercise at 10-minute, 1-hour, 2-hour, and 24-hour time points.  Plasma volume (PV) decreases, as calculated using the Dill and Costill method, caused by dehydration-inducing exercise resulted in significant increases in hematological parameters (hemoglobin ([Hb]), hematocrit (Hct), serum albumin concentration (ALB), serum osmolality (Osm), and calculated OFF-Hr score) at varying time points following exercise.  These changes (increases) in ALB were found to be highly correlated with changes in [Hb] (r = 0.784) and PV shifts (r = 0.809), while no correlation was identified between Osm and [Hb] or PV shifts.  Additionally, the effect of exercise-induced dehydration was analyzed for each individual in the context of the Athlete Biological Passport.  One case existed where the dehydration-induced increase in [Hb] triggered an atypical finding at 95% specificity, however no instances occurred at 99% specificity where increases in hematological variables or the athlete biological passport score (ABPS) exceeded the individual’s upper threshold.  

This study resulted in two key findings: 1) a strong correlation was identified between percentage changes in albumin and hemoglobin (and, thus, plasma volume) following dehydrating and euhydrated exercise; and 2) dehydration-inducing exercise resulted in one atypical finding at 95% specificity and many other unusual profiles when utilizing the Athlete Biological Passport program

Code: 14A18XD 

The detection of the exogenous administration of synthetic androgens (the so called “pseudo-endogenous” steroids) having the same chemical structure of the compounds produced endogenously (i.e testosterone, 5α-dihydrotestosterone or androstenedione) is primarily based on the alterations of the urinary endogenous steroid profiles. 
A Bayesian approach and adaptive model has been adopted by WADA for the management of the steroid profiles and all the parameters obtained by the Accredited Laboratories will be collected starting 1st January 2014 in a global database integrated in the Athletes Biological Passport (ABP), permitting to establish the individual reference ranges of every athlete. 
The detection capacity of the steroid profile can be improved by the inclusion of the urinary hydroxylated androgen metabolites excreted in lower amounts but whose diagnostic values become more significant after the administration of endogenous steroids. The additional inclusion of the isotope ratio mass spectrometric (IRMS) data has demonstrated to improve the statistical discrimination between basal samples and samples obtained after controlled administration of testosterone. 
Finally, the use of IRMS is a very powerful tool for the detection of pseudoendogenous steroids if the starting material for their synthesis has a 13C composition different from the endogenously produced molecule. The detection of formulations able to circumvent their detection by IRMS already occurred since the criteria are based on population data and not on athlete’s previous data. 
The main goal of this project is to establish the long term variability of the IRMS delta values of both target (TC) and endogenous reference compounds (ERC), and of the steroid profile parameters including hydroxylated androgens metabolites in order to improve the evaluation of the longitudinal profiles. This should permit to enlarge the detection capacity and to potentially detect the abuse of preparation.

Main Findings: 

There is a definite need of finding more efficient ways to detect the exogenous administration of pseudoendogenous steroids. Although the implementation of the ABP has been a clear step forward, there is still a gap between our capacity to suspect and to confirm the abuse of these substances. The use or more specific markers of the urinary steroid profile like some hydroxylated steroids has been investigated, although their implementation is not easy and would require big efforts to harmonize their detection and quantification as was done for the current markers of the ABP.
The potential inclusion of the IRMS data in the ABP as a direct evidence of doping by pseudoendogenous steroids has been evaluated. As for the ABP markers, instead of comparing the obtained data with reference population ranges (present approach as a confirmation strategy), we suggest to incorporate these data to the ABP and to evaluate the values to the reference data produced by every single athlete.

To reach this goal, the first step was to investigate the stability of the IRMS data in both healthy volunteers and athletes. 

The variability of the IRMS data in a short, medium and long term period in 8 males and 6 females volunteers when compared to data of athletes submitted to investigations by the respective NADO due to atypical steroid profiles, showed that the variability of the individuals’ absolute delta values of the parameters studied are at least one half lower than the population ones and  much lower than the markers of the steroid module of the ABP. The data obtained from real samples of athletes showing atypical steroid profiles, show a variability comparable to the non-athletes volunteers, demonstrating that sports practice and the use dietary supplements do not influence the delta values of the endogenously produce steroids. These IRMS values depend mainly on the individuals’ diet and metabolism. 

This would allow defining individual reference ranges much narrow than the currently applied ones. This would permit (1) to extend the detection window of the pseudoendogenous administration and (2) to potentially detect the use of steroids from pharmaceutical preparations showing delta values close to the endogenous values.

The results of the present project, suggest that the IRMS data obtained with the procedures already in place in the WADA accredited Laboratories, can be used in a more performing way. If the data instead of being only used during the confirmation evaluation of an atypical steroid profile would be evaluated longitudinally, the information obtained would permit to enlarge the detection window of pseudoendogenous steroids abuse and the potential detection of pharmaceutical preparations showing delta values in the endogenous region.
We are not proposing the implementation of a new method but the better exploitation of the data already obtained. By doing so, the gap between the ability of detecting suspicious steroid profiles and the capacity of confirming them by IRMS will be drastically reduced.
There is a definite need of finding more efficient ways to detect the exogenous administration of pseudoendogenous steroids. Although the implementation of the ABP has been a clear step forward, there is still a gap between our capacity to suspect and to confirm the abuse of these substances. The use or more specific markers of the urinary steroid profile like some hydroxylated steroids has been investigated, although their implementation is not easy and would require big efforts to harmonize their detection and quantification as was done for the current markers of the ABP.
The potential inclusion of the IRMS data in the ABP as a direct evidence of doping by pseudoendogenous steroids has been evaluated. As for the ABP markers, instead of comparing the obtained data with reference population ranges (present approach as a confirmation strategy), we suggest to incorporate these data to the ABP and to evaluate the values to the reference data produced by every single athlete.

To reach this goal, the first step was to investigate the stability of the IRMS data in both healthy volunteers and athletes. 

The variability of the IRMS data in a short, medium and long term period in 8 males and 6 females volunteers when compared to data of athletes submitted to investigations by the respective NADO due to atypical steroid profiles, showed that the variability of the individuals’ absolute delta values of the parameters studied are at least one half lower than the population ones and  much lower than the markers of the steroid module of the ABP. The data obtained from real samples of athletes showing atypical steroid profiles, show a variability comparable to the non-athletes volunteers, demonstrating that sports practice and the use dietary supplements do not influence the delta values of the endogenously produce steroids. These IRMS values depend mainly on the individuals’ diet and metabolism. 

This would allow defining individual reference ranges much narrow than the currently applied ones. This would permit (1) to extend the detection window of the pseudoendogenous administration and (2) to potentially detect the use of steroids from pharmaceutical preparations showing delta values close to the endogenous values.

The results of the present project, suggest that the IRMS data obtained with the procedures already in place in the WADA accredited Laboratories, can be used in a more performing way. If the data instead of being only used during the confirmation evaluation of an atypical steroid profile would be evaluated longitudinally, the information obtained would permit to enlarge the detection window of pseudoendogenous steroids abuse and the potential detection of pharmaceutical preparations showing delta values in the endogenous region.
We are not proposing the implementation of a new method but the better exploitation of the data already obtained. By doing so, the gap between the ability of detecting suspicious steroid profiles and the capacity of confirming them by IRMS will be drastically reduced.

Code: 14B14ZW

Detection of insulin or insulin analogue doping is still a challenge for doping analysis laboratories. Until now no screening method is available providing the fast and reliable detection of doping with insulin analogue. The insulin analogues are modified insulin preparations to show faster acting or long acting effects compared to regular insulin, which are therefore broadly used in clinical therapy for diabetes and might also be intensively misused in sport and body building. The insulin analogues have amino acid sequences different to regular insulin. Such difference could be recognized by specially selected monoclonal antibodies, which have their binding site or epitopes involved in the positions where amino acid sequences are altered in insulin analogues.  
In this project, we will at first generate and select two monoclonal antibodies which bind all insulin analogues significantly differently. Thereafter a differential immunoassay based on immune-PCR Imperacer Technologie from Chimera Biotech will be constructed with these 2 antibodies. The immune-PCR is the ultra sensitive immunoassay using antibody-DNA conjugate. The DNA fragment can specifically amplify millions fold and the signal will be directly read out in a real-time PCR machine.  An insulin analogue is detected if the insulin concentrations determined by the two assays are significantly differently. This ultrasensitive real-time immune-PCR duplex assay could directly use small amount of urine samples for doping control. Furthermore, efforts will be made to produce specific monoclonal antibodies to each insulin analogue. Each monoclonal antibody will only bind one insulin analogue but not regular insulin or other analogues. 
Differential immunoassays, especially multiplex immune-PCR constructed with these antibodies will be able to identify quickly and unequivocally which insulin analogues are misused. 

Main Findings: 

In this research project, high affinity anti-insulin monoclonal antibodies (mAbs) including several regular insulin specific mAbs and glargine specific mAbs were produced and they were carefully examined for their binding to insulin and insulin analogues. More than 30 best hybridomas were chosen to be cloned with limited dilution. The antibodies were produced in protein free medium, purified with affinity column and biotinylated. The mAb pairs for the sandwich immunoassays were identified through examining more than 900 antibody combinations. A permissive insulin sandwich assay using mAb 5E10 and a regular insulin preferential assay using mAb 7F3 were constructed. A glargine specific sandwich assay with mAb 6E8 was also established. These three assays are ultra-sensitive (limit of detection <1 pg/ml) and together they can identify any insulin analogue in urine and serum directly with only a few hundred microliters of samples. In a pilot study, these assays showed to be able to identify low amount of insulin analogues in the urine samples of the diabetic patients treated insulin analogues alone or combined with regular insulin preparations. 

Furthermore, a regular insulin specific sandwich assay was also constructed with mAb 1B7. This assay together with the permissive insulin assay can identify insulin analogues, bovine and porcine insulin. Due to its high specificity but moderate sensitivity, this assay would be more suitable for the confirmation assay. Additionally, a universal ultrasensitive Immuno-PCR assay format using real-time PCR cycler was constructed, which can easily be converted to the ultra-sensitive duplex assays