Projets de recherche

Code: 11C2SO

Endothelin receptors antagonists (ERA), such as bosentan and ambrisentan, are a class of vasoactive drugs that have been developed for the treatment of pulmonary arterial hypertension. It has been anecdotally reported that ERA is frequently used among top-level athletes to counteract exercise-induced rise in pulmonary vascular pressures and increase exercise performance. Yet, the effects of ERA on exercise capacity in healthy humans are puzzling, with the drugs not included in the current Prohibited List, since the ergogenic potential is yet to be fully understood and determined. Furthermore, the urinary excretion of ERA metabolites following administration has not been studied systematically at rest and during exercise in athletes, as a way to detect its intake if performance-enhancing potential is confirmed. In the planned study ERA will be administered in newly approved doses for 8 weeks in order to assess the presumed doping potential for both male and female athletes, and to monitor serum and urinary ERA excretion dynamics after single- and multiple-dose administration. The possible effects of prolonged ERA administration in higher doses on exercise performance may be relevant, if further confirmed, in terms of their possible fraudulent utilization to influence exercise performance in sports, raising the difficult question of whether, particularly in some circumstances, the ERA might be considered as prohibited substances in athletes. 

Main Findings: 

Endothelin receptors antagonists (ERA) are a class of vasoactive drugs that have been developed for the treatment of pulmonary arterial hypertension (PAH). The use of ERA among athletes to counteract exercise-induced rise in pulmonary vascular pressures and increase exercise performance is rather unexplored and data on efficacy and safety are limited. In particular, an increase of maximal oxygen consumption after ERA administration can be regarded as critical and should be evaluated.  
The present study evaluated the effects of orally administered selective (bosentan) and non-selective ERA (ambrisentan) on aerobic and anaerobic performance, serum and urine biochemical outcomes, and the occurrence of adverse events during the intervention in healthy humans. Thirty male and female volunteers were randomized in a double-blind design to receive bosentan (250 mg daily), ambrisentan (10 mg per day) or placebo by oral administration for 8 weeks. Treatment with two different oral doses of ERA for 8 weeks had no major effect on body composition, strength, aerobic and anaerobic performance indicators and hormone profiles in physically active men and women as compared to placebo (p > 0.05). Serum hepatic enzymes increased significantly from before to after administration in both ERA groups (P < 0.001).  
The proportion of participants who reported minor adverse events was similar in all intervention groups. These preliminary data suggest that 8-week oral administration of ERA is ineffective for exercise performance enhancement in healthy men and women, with significant effect on liver enzyme efflux and minimal incidence of reported side-effects.

Code: 11A16CA

New steroids openly appear on the market in products labelled with a rather confusing nomenclature. Once characterized, pharmaceutical grade products not being available, knowledge of the biotransformation pathways essential to an efficient detection of utilization by athletes is difficult to gain since administration to human volunteers should be restricted to the minimum.  
The alternative is a reliable in vitro model.  Human hepatocytes, fresh or cryopreserved are now available commercially. We have successfully produced and identified phase I metabolites from incubations of human hepatocytes with different steroids, such as 17-methyldrostanolone and desoxymethyltestosterone (DMT). 
The aim of this project is to produce in vitro from human hepatocytes and liver fractions the metabolites of two steroids, the 17-methylated derivatives of stenbolone and its isomer methenolone. The principal metabolites will be synthesized and characterized by NMR and mass spectrometry. 
The characterization of metabolites will enable the identification of markers of utilization to be incorporated in routine testing methods. The approach for the chemical synthesis of metabolites will be shared with NMI insuring the distribution to other doping control laboratories. Improving the knowledge of steroid biotransformation is a further benefit from these studies. 

Main Findings:

We have synthesized and characterized two designer steroids, 17α-methylmethenolone and 17α-methylstenbolone; the latter is proposed on the internet and two groups have reported different and contradictory results.  Incubations with fresh hepatocytes and S9 liver fractions were carried out. Structures were proposed for the main and more relevant metabolites from the GC-MS analysis of their TMS-ether, TMS-enol and TMS-ether (TMS-d9) derivatives in agreement with literature published for the non-methylated analogs methenolone and stenbolone (1-3).  A GC-MS/MS SRM method was developed and permitted the detection of these metabolites in an athlete’s sample in which methylstenbolone was detected and also in a reference urine sample provided by WAADS in 2014.   
In vitro metabolites of 17α-methylmethenolone 1 
Three main metabolites 6,17β-dihydroxy-1,17α-dimethyl-5α-androst-1-en-3-one (M2), 
16,17β-dihydroxy-1,17α-dimethyl-5α-androst-1-en-3-one (M3) and 2,17β-dihydroxy-1,17α-dimethyl-5α-androst-1-en-3-one (M4)  were produced from the incubation of 1 with cryopreserved and fresh hepatocytes or S9 fractions, along with a trace of the 3α-OH metabolite (M1, 3α-SDH).   In vitro metabolites of 17α-methylstenbolone 2 
The incubations of methylstenbolone with hepatocytes and S9 fractions afforded several metabolites.  Three, along with the reduced 3α-OH M1 were more important and expected to be present in human urine: 17β, 18-dihydroxy-2,17α-dimethyl-5α-androst-1-en-3-one (M2), 6,17β-dihydroxy-2,17α-dimethyl-5α-androst-1-en-3-one (M3), 16,17β-dihydroxy-2,17α-dimethyl-5α-androst-1-en-3-one (M4)

Code: 11A18SS

Clenbuterol is a well known beta-agonist which is being used in animal husbandry and sports for growth promoting purposes. Lately adverse analytical findings in athletes were claimed to be due to contaminated meat. This project is aimed at finding a strategy and marker to distinguish Clenbuterol in urine from athletes due to meat consumption or illegal use of Clenbuterol containing supplement or preparations. 
The hypothesis is that Clenbuterol, a racemic (1:1) mixture of stereoisomers as a pure compound, is present in another composition, 3:1, in meat due to differences in pharmacokinetics and pharmacodynamics. Ingestion of this different ratio from meat can lead to a different ratio in athletes urine. If this is the case then it is possible to discriminate consumption of contaminated meat from illegal administration of Clenbuterol (supplements).  For this, methods of analysis have to be developed to separate the stereoisomers in supplements, meat and urine.   
Extraction of Clenbuterol from meat must be optimised because the incidence of positive Clenbuterol findings in residue control is very low. And several poisoning cases and positive doping cases have been found. So there is a possible underestimation of Clenbuterol residues in meat due to possible bound residues or incompete extraction. 
Information on levels of the different isomers in meats has to be collected. As also quantification of stereoisomers in preparations and urine samples from controlled studies or from inadverted use from eg. tourists. 
After that a proposal, workflow, how to use these tools in doping control must be developed. 

Main Findings:

WADA funded project 11A18SS was written to show a proof of principle that it is possible to discriminate adverse analytical findings by clenbuterol illegal use from consumption of contaminated meat. 
Tools for this proof of principle were developed. 
Sensitive and selective analytical methods were developed using UHPLC-MS/MS and SFC-MS/MS for meat, bovine and human urine and preparations. 
Incurred meat with clenbuterol was produced. 
The tools were used on real samples provided by anti-doping laboratories. 
It was shown that meat from an animal treated with clenbuterol contained a different S/R ratio for clenbuterol. The meat was R-enantiomer enriched. 
Pharmaceutical preparations showed S/R ratio around 1. 
From the small number of urine samples from Mexico, presumed to be contaminated via meat the S/R clenbuterol ratio were lower than the S/R clenbuterol ratio from humans having consumed clenbuterol in an administration trial. 

Code: 11B16TP

Although autologous blood transfusions are a prohibited method, there is at present no unequivocal test and it seems from recent scandals that the technique remains a significant problem in various sports. During the past months and years, several research attempts to develop a valid detection method have been performed but so far have failed to identify a definite, unequivocal test. Whereas a method for detection of homologous blood transfusion was established in 2002, the most contemporary (indirect) attempt to detect autologous transfusions is the Athlete Biological Passport. 
In this one-year project, our group focuses on the detection of autologous blood transfusion by a detailed analysis of red blood cell density fractions which could be altered in the time course after transfusion. In this context, the so-called ‘neocytolysis’ as a presumed selective destruction of young red blood cells (neocytes) was described as a process to possibly down-regulate hemoglobin mass when it is “excessive” or maladaptive for the environment and was seen as potentially important in blood doping settings. Several researchers assume that this regulation of red cells can be expressed when measuring their main density fractions. 
In a pilot study, as an unexpected, but compelling and promising finding, our group was able to show that the transfusion of autologous blood leads to a distinct shift in the red cell density fractions with an increase of red cells of a high density. Flow cytometry with measurement of red blood cell surface markers shall be used to further evaluate these shifts and to allow identification of a certain set of markers being specific for autologous transfusion. This project will consist of a cross-sectional and longitudinal part where multiple transfusions and a control group shall be included to test for reproducibility and specificity of the methods. 

Main Findings:

The most important result of this project was the transfusion of autologous blood, predominantly with long-term storage for 7 weeks, lead to distinct changes of the RBC population shortly after transfusion. These effects were not detectable in full blood but in RBC samples from high density band established by sel-forming Percll gradients based on several parameters. However, the time window for detection of thse changes seems to be short, possibly being limited to a maximum of 24 up to 48/72 hours depending on the tested parameter. Systemic reactions to autologous transfusions were supported by the analysis of classical markers such as serum ferritin and hematology measurements showing significant changes which support the high value of serum ferritin as an important marker of autologous transfusion. However, we were unable to demonstrate any significant effect of autologous transfusion n the RBC surface marker profile in full blood or one of the three RBC subpopulations. In summary, we believe that the methods will be difficult to implement in the routine of any antidoping laboratory. Because of the elaborate preparations and short detection window (perhaps only valid for long stored blood). This project unfortunaly may not reveal a direct test for autologous blood doping at this stage. Nevertheless, the results are very interesting from a physiological point of view and will be discussed in the next paragraphs. In the last section, we will further comment on possible applications for on-going and future research in the field. 

Code: 11B10JP

All humans express antibodies against N-glycolyl-neuraminic acid (Neu5Gc), a sialic acidhumans cannot produce that is present in glycoproteins from animal sources. Small amounts ofNeu5Gc can metabolically be incorporated to human proteins resulting in the development of an immune response. 
Neu5Gc is present in recombinant glycoproteins (e.g. rEPO and analogues) since they are expressed in non-human cell lines. It has been proven that mice receiving injections of a recombinant glycoprotein, containing Neu5Gc, immediately produce antibodies against it. 
The hypothesis is that the concentration of antibodies against Neu5Gc must be much much higher in subjects treated with recombinant proteins (e.g. rEPO) than in the general population whre the exposure to Neu5Gc happens through a complex mechanism from the diet. 
The main objectives of the project are: 1- Developing a test to quantify antibodies against Neu5Gc circulating in human blood (serum or plasma) based on what has already been described in the literature.
2- Test human blood samples from healthy individuals and from patients being treated with rEPO.
3- Refine the analytical test in order to increase specificity and minimise background by testing different Neu5Gc containing antigens and approaches (inhibition).
4- Validate the test for anti-doping purposes establishing thresholds to ascertain the illegal exposure to recombinant glycoproteins as well as studying the pharmacokinetics of antibody production.

Main Findings: 

Humans do not possess the enzymatic capability of producing the sialic acid N-glycolyl-neuraminic acid (Neu5Gc). It has been described that, as Neu5Gc is present in animal products, small amounts of Neu5Gc can metabolically be incorporated into human proteins resulting in the development of an immune response. Anti-Neu5Gc antibodies (with different linkages to other monosaccharides in glycans) have been described as measurable in almost all human beings. We hypothesised that, as Neu5Gc is present in recombinant glycoproteins (e.g. rEPO and analogues), expressed in non-human cell lines, treatment with these substances should give rise to the development of a higher title of specific antibodies. Following the findings published in the literature, the overall aim of the project was the detection of circulating antibodies raised specifically against Neu5Gc, with the α2,3-linkage to Galactose found in recombinant EPO and analogues. 
Following the project plan, a specific antigen Neu5Gc-α2,3-Gal-β1,4-GlcNAc was synthesised linked to biotin (through an hexaethylenglycol bridge) or to KLH. 
Serum samples were obtained from patients under chronic NESP treatment as well as healthy untreated volunteers. Additionally samples were also obtained from volunteers participating in a clinical trial receiving rEPO treatment for 3 weeks. 
Detecting particular human antibodies in serum samples has proven to be very challenging as the concentration of total IgG is huge and non-specific binding is a major drawback. 
Different antigens were tested including Neu5Gc or Neu5Ac linked to a polyacrylamide (as described in the literature), the whole rEPO and our specific rEPO trisaccharide. 
After optimising all ELISA parameters, and being able to minimise background, small differences in anti-trisaccharide titre could be observed between patients treated with NESP and healthy volunteers. However those mean differences would not allow setting a cut-off to differentiate between them. However, when those tests were applied to samples from healthy volunteers submitted to rEPO treatment (e.g. resembling doping cases) there was no measurable increase in antibody titres. 
In conclusion, despite the many reports in the literature suggesting that circulating anti-Neu5Gc antibodies are ubiquitous, those measurements could not be consistently reproduced nor a clear titre of Anti-Neu5Gc antibodies could be detected either in patients using a rEPO analogue chronically nor in volunteers submitted to rEPO treatment. 

Code: 11D16GF

In late 2008, several synthetic cannabinoids were detected in herbal smoking mixtures, commonly called "spice". Today a great variety of such synthetic cannabinoid receptor agonists is known and it expected that further substances will appear. Many European countries have banned these substances, and the World Anti-Doping Agency (WADA) prohibited synthetic cannabinoids in-competition in 2010. To allow an adequat detection of synthetic cannabinoids in a urine sample, knowledge of metabolism is a key issue. Performing human clinical studies is however difficult, due to the lack of a toxicological profile. 
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 complement of enzymes and cofactors. In the proposed project, an in vitro model using cryopreserved human hepatocytes will be used to investigate the metabolism of selected synthetic cannabinoids. The doping control laboratory in Seibersdorf is currently involved in a Wada-funded project testing and optimising the use of commercial available cryopreserved hepatocytes as an in vitro model for doping substances. The optimised model will be applied on the substances in the current study, and the main metabolites will be characterized by various mass spectrometric methods. A verification of the postulated metabolism from the in vitro studies is allowed by the investigation of in vivo urinary metabolic profiles in samples from drug drivers, supplied by the Vienna Police Department. 
Finally, the incubation extracts will be evaluated as a potential source of reference material in doping control, as access to such material is of major importance in an unequivocal identification of metabolites in a doping control sample. 

Main Findings:

Aim of the project was to use cryopreserved hepatocytes to study the metabolism and characterize metabolites of selected synthetic cannabinoids dominant on the market today. Due to the continuous emergence of new “spice” compounds on the marked, a rapid investigation of the metabolism is of major importance, especially since some synthetic cannabinoids are already reported not to be excreted unchanged in human urine [1]. 
Four of the most prevalent compounds in positive forensic samples are reported to be JWH-122, JWH-210, AM-2201 and JWH-081 [2]. Thus, these four compounds were selected for the studies on the human metabolism of cannabinoids, in addition to JWH-018 as a model compound. 
The results from the study suggest monohydroxylation as a major metabolic pathway for all five compounds investigated. Additionally, a major JWH 018 metabolite is carboxylated, while one of the major JWH-210 metabolites is dihydroxylated. Furthermore, additionally abundant metabolites for AM-2201 included one dihydroxylated, dihydrodiol, one desfluoropentylated, and defluorinated metabolites. Regarding JWH-081, a major dihydrodiol metabolite was identified, in addition to the abundant monohydroxylated compounds. Hence, even though similar metabolic pathways are observed for the five investigated compounds, differences are also present. Although varied conjugation rates were observed, the majority of the main metabolites were excreted as glucuronide conjugates. 
Incubations with hepatocytes seem to represent a very useful model for identifying and predicting potential major urinary metabolites. Looking at the model compound JWH-018, the metabolites generated in the incubation mixtures were in good correspondence with reported major metabolites excreted in urine. Furthermore, the JWH-210 hepatocyte extracts resembled the metabolic profile observed in a forensic urine sample. Altogether, taking information from our experiments and available literature into account, it seems that the metabolites generated in human hepatocyte incubation extracts reflect the prevailing human metabolism.

Code: 11A13CG

Anabolic androgenic steroids (AAS) are the most frequent abused substances in sports and are included in the World Antidoping Agency (WADA) List of prohibited substances. AAS are extensively metabolized in the liver and their target tissues. The metabolic pathways are divided into phase-I and phase-II reactions. Phase-I reactions involve oxidation, hydrolysis and reduction, which introduce new functional groups for the subsequent phase-II reactions (i.e. conjugation).

For the endogenous androgens and for exogenous AAS the main phase-II reactions are conjugation with glucuronic acid (glucuronidation) or with a sulfo-moiety (sulphatation). The screening methods used by the Antidoping Laboratories usually focus on those metabolites, that are excreted unconjugated or as glucuronides into the urine. Extraction of the gluco- deconjugated steroids from the matrix and concentration of the analytes is performed by liquid-liquid extraction (with diethylether or tertbutylmethylether) or solid phase extraction followed by mass spectrometric detection either by liquid chromatography mass spectrometry or gas chromatography mass spectrometry. By using this initial screening extraction protocol the Antidoping Laboratories substantially ignore the sulfo-conjugated part of AAS. Nevertheless there are AAS for which the longer detected metabolite is a sulfo-conjugate. Sulfo-conjugated AAS are relatively easy to be detected directly since the development of instrumentation providing interfacing of liquid chromatographic (LC) separation to mass spectrometric (MS) detection, especially via electrospray ionisation (ESI), has opened up broad possibilities for the direct analysis of sulfo-conjugated substances. Moreover, sulfo-conjugated substances can be extracted from urine using ethyl acetate, instead of diethylether, as extraction solvent. The objectives of this project will be:

a) To develop a screening method for the already known sulfo-conjugated metabolites of AAS, and

b) To investigate the existence of not yet reported sulfo-conjugated metabolites of AAS, that can improve detectability and identification in either initial screening protocol or confirmation methods.

Main Findings

Anabolic steroids such as oxandrolone, madol, formebolone, methenolone, 17- methylnandrolone and mesterolone were tested for the existence of sulfo-conjugated metabolites. Metabolic samples from long-term excretion studies were tested for any sulphate metabolite and where any sulphate metabolite was found, an evaluation of its retrospectivity was performed in comparison with their free and gluco-conugated metabolites used for their monitoring in GC/HRMS analysis. In most cases where new metabolites were found, a detailed characterization of their structures based on mass spectrometry techniques was also performed. Additionally, spotted metabolic samples for oxymetholone, drostanolone, norethandrolone, danazol, clostebol, methandriol, calusterone, furazabol, fluoxymesterone, oxymesterone, boldenone, mesterolone, methandienone, methyltestosterone, oral turinabol, methenolone, and tibolone that include the known, up to that time, anabolic steroids with sulphates metabolites, as well as other AASs with unknown sulphate metabolism were tested in order to develop a new screening method for sulphate metabolites. Samples from the above listed steroids were extracted and analyzed using a screening method based on alkaline extraction with ethylacetate and LC/QTOF analysis in a negative mode. Potential sulfate metabolites of these steroids were drawn and the molecular ions were calculated and extracted using the instrument software.

Conclusions

The investigation of sulfo-conjugated metabolites of methenolone and mesterolone led to the discovery of new metabolites of at least equal or better retrospectivity compared to the already known gluco-conjugated metabolites detected by GCMS. Furthermore, a sulfo-conjugated long-term metabolite of 17-methylnandrolone was discovered (unpublished results). The analysis of madol, formebolone and oxandrolone didn’t lead to any new (sulfo-) metabolite, at least to a concentration level that would be detectable by the technology used in this study. A screening method for sulfo-conjugated metabolites was developed for several anabolic steroids based on literature data, using accurate mass measurements for the extracted ion chromatograms and leading to a number of new sulfo-conjugated metabolites. Their structures, as well as their retrospectivity for the monitoring of their parent compounds abuse for doping control purposes were not evaluated in the framework of this project.

Code: 11D13MG

Performance-enhancing aids are not new to athletes. Owing to fame, honor and economic benefits arising from success in competitions, there is rather a long history of cheating in sports.  After the usage of erythropoietin could effectively been proven, lots of athletes turned to an older doping procedure: autologous blood doping to increase the number of circulating red blood cells (RBCs). This increases the oxygen delivery through the blood to the working muscle which profoundly enhances an athletes´ performance. The International Olympic Committee (IOC) forbade blood boosting after the 1984 Olympics, despite the fact that no methods had been devised for unequivocal detection. Valid and established methods for the detection of autologous blood doping are still missing but our recent findings indicate that the RBC flexibility represents a useful marker to detect blood doping. The deformability is an essential feature enabling RBCs to access and transit the capillary bed and to deliver oxygen to the muscle cells due to the fact that the vessel diameter of the capillary is smaller than the resting cell diameter. 
The aim of the presented study is the investigation of RBCs flexibility in male and female endurance athletes and male and female non-athletes during a one-year period. Thereby, a variety of parameters, known to influence RBC deformability like L-arginine and NO levels, are planned to be investigated. Also, the influence of the female hormone cycle on the RBC deformability should be investigated. Furthermore, the influence of competition and training periods and the influence of the ethnic background on RBCs flexibility will be evaluated. The findings obtained with this study will serve as a basis for further investigations and the development of a routinely applicable detection method to detect autologous blood doping.

Main Findings:

Autologous blood doping is the practice of boosting the number of red blood cells (RBC) in order to improve aerobic capacity and thus athletic performance and endurance. RBC deformability, the ability of RBC to change their shape under a given level of applied shear stress, is an important cell property to ensure oxygen and nutrient supply to the working tissue in the microcirculation. Thus, RBC deformability may also determine performance capacity. Hypothermic blood storage decreases RBC deformability, thus, monitoring RBC deformability of elite athletes during the course of the year may be suitable to detect for autologous blood doping.  
But first it was necessary to test for factors that possibly influence deformability in endurance athletes. Among these were age, ethnicity, gender, estrogen levels and training volume.  
The major results revealed increasing RBC deformability from pre-puberty to adulthood. Further, female athletes who used hormonal contraceptives and who thus showed reduced estrogen levels showed reduced RBC deformability compared to female athletes without hormonal contraception (-HC). An acute response of RBC deformability to increasing estrogen levels was not detected concluding a chronic impact of estrogen on RBC deformability. Both, male and female athletes (-HC) showed higher deformability values compared to respective non-athlete control groups, indicating a correlation of endurance exercise and RBC deformability. RBC deformability did not differ between male athletes and female athletes – HC but deformability values were significantly higher in male athletes compared to female athletes + HC. Female Asian and black athletes (all – HC) showed reduced RBC deformability compared to white female athletes – HC. Also, male Asian and black athletes showed reduced RBC deformability compared to white male athletes.  
The recent data indicated that RBC deformability is influenced by some variables such as gender or ethnicity. According to the recent data it is suggested to monitor individual RBC deformability values longitudinally to detect abnormalities possibly occurring after autologous blood doping.

Code: 11C9RS

Aim 1: Assay optimization

We will evaluate a combined assay format that is based on real-time PCR and ITC readout with a nested PCR pre-step to improve sensitivity. The current single-step qPCR assay has a sensitivity of 3 copies for the macaque Epo cDNA in the presence of 500ng - 1 OOOng macaque gDNA. A qPCR assay has been developed to the homologous locus in the human Epo cDNA with a sensitivity of 10 copies in 500ng human gDNA. A nested PCR assay has been developed for the human Epo cDNA with a sensitivity of 10 copies in 1250ng of human DNA. We will evaluate the enhanced sensitivity of nested PCR combined with the automation and rapid results of qPCR with the aim of reaching a sensitivity of less than 5 copies in 500ng genomic DNA. We will also examine the feasibility of utilizing the ITC assay format with the nested+qPCR approach. Once the assay is developed and tested using plasmids and virions spiked into gDNA and human blood, we will evaluate samples obtained directly from transduced macaques, and macaque PBLs spiked into na"fve human blood. The SV40 polyA site, which is not present in the human genome, but will be present in the rAAV vector used here, is an ideal target since there is no competition with endogenous sequences. The SV40 assay was previously developed [1Jfor the macaque studies and is considered to have the best sensitivity; it will serve as a reference for optimizing the hEPO Exon3-4 assay. Controls (ITC format) will be developed for each assay.

Aim 2: Assay validation

New or revised clinical diagnostic methods must be validated: defined as the process by which it is established, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical application. Components of validation involve quality control (QC) and quality assurance (QA). We will perform analytical validation of the human Epo assay to ensure specificity, sensitivity, and ruggedness (multiple labs, multiple operators, repeats and replicates). This will involve drafting test records, along with assay specifications, reagent preparation logs, and a validation plan, and executing the assay(s) several times with several replicates in Florida and Mainz. Data will be captured and evaluated statistically to determine the assay performance criteria. For pre-clinical performance validation, we will evaluate spiked (using plasmid DNA) human blood samples with respect to: sex, age, race, medical history (if available), exercise status, general health, and prescription drugs (if known). The rate of false positives and false negatives will be established by testing a statistically significant number of samples. We will screen up to 100 blood samples (50 for each lab in Florida and Mainz) for false positives in order to get an idea of the specificity of our procedures. The Simon Lab will contribute 100 human samples (Germans), and if available, substitute some of the German samples with samples of other ethnicities.

The assay validation activities support the documentation for eventual regulatory submission and eventual manufacture of an in vitro diagnostic test (IVD) kit. Ultimately, data from the assay development, analytical validation, and pre-clinical performance validation studies will be documented along with a description of each kit (with all required components) in a form suitable for regulatory submission.

As for regulatory capability and expertise, in addition to his faculty appointment and research activities described in this proposal conducted in the Department of Molecular Genetics and Microbiology, Dr. Snyder is the Director of the Center of Excellence for Regenerative Health Biotechnology at University of Florida (UF CERHB, http://www.cerhb.ufl.edu/ ). Established in 2003, the UF CERHB is committed to translational research in many different biotechnological fields including viral vectors for gene therapy. UF CERHB's biopharmaceutical development operation, offers a wide range of cGMP-compliant biopharmaceutical manufacturing and testing services to the biotechnology industry and to biomedical research institutions. Furthermore, Dr. Moullier runs a similar operation in Nantes called ATLANTIC BIO GMP (ABG). Resources are available (on a fee-for-service basis) at UF CERHB and ABG, Nantes for anti-doping PCR assay kit manufacturing.

Main Findings

It is crucial to develop reliable test methods for the detection of gene doping, given that some members of the sports community are seeking for gene transfer technology to improve exercise performance. Based on previous work we established a ´nested real-time PCR´ assay. This is an assay based on the PCR technology which is used in criminology and well-known in the court of law for the identification of genetic material in samples from sites of crime. We adapted this technique to detect single erythropoietin (EPO) DNA molecules that had been introduced into the body by means of gene transfer. Due to its high sensitivity, our technique is principally able to detect genetic modifications of certain parts of the body in conventional blood samples. Here we aimed at providing evidence of the high sensitivity and specificity of our refined procedure that now enables quantification of the artificial DNA in the blood stream.

Through an inter-laboratory study between Nantes (France), Gainesville (Florida, USA), and Mainz (Germany) two nested real-time assays were validated and utilized for an in vitro blinded study. Both assays showed unique sensitivity and specificity in a large dynamic range. According to the nested assay protocol the detection of ~1 copy of circular transgene molecule in a background of 500 ng gDNA is possible reliably. In a blinded in-vitro study the reliability of the assays to detect between 1000 – 2 copies of transgene molecules and non-template control was proven.

To validate the suitability of the nested real-time assays for the detection of gene doping a non-human primate study was conducted. Two macaques were injected intramuscularly with a recombinant adeno-associated virus serotype 8 (rAAV8) vector harboring human EPO cDNA sequence. One macaque served as non-injected control. The vector was promoterless to avoid transgene expression. Following the injection of 2.5 x 1011 viral genomes/kg, the transgene molecules were detectable 8-14 weeks.

The high sensitivity of the nested qPCR assay along with the specificity for transgene detection is essential for gene doping surveillance. The assay can be adapted to other gene doping candidate genes.

Code: 11B1JB

Operational Technologies Corp. (OpTech) has already demonstrated and published (BrunoJ.G., et al. J. Biomolec. Techniques, In Press, 2011) the ability to develop aptamers (surrogate antibodies composed of DNA) which can discriminate synthetic (recombinant) from natural human growth hormone (hGH) with initial funding from WADA. The aptamers developed by OpTech detect minor amino acid modifications and differences added by E. coli host cells as noted by Hepner et al. (2005 and 2006) to research grade synthetic hGH. Under this follow on grant proposal, OpTech will develop a new set of discriminatory aptamers against one or more pharmaceutical grade recombinant hGH targets (Genotropin, Norditropin, etc.). OpTech has recently demonstrated ultrasensitive (sub-picogram/ml) detection of hGH with some of its original hGH aptamers in an aptamer-magnetic bead sandwich electrochemiluminescence (ECL) format. ECL is already used by Roche Diagnostics for ultrasensitive detection of numerous clinical analytes. Therefore, OpTech will develop several combinations of capture and reporter aptamers to detect pharmaceutical hGH at sub-pg levels. Finally, because ECL is a laboratory-bound technique, OpTech will investigate the potential for presumptive field testing for synthetic hGH in aptamer-based lateral flow test strips using blood or urine samples for preliminary screening of athletes. To enhance sensitivity, OpTech will explore the use of quantum dots (QDs) and fluorescent nanoparticles (FNPs) along with a UV penlight to enhance visual detection of synthetic hGH in lateral flow test strips. Disposable low pressure size-exclusion chromatography columns may be needed to remove creatinine and urea in serum and urine prior to aptamer-based assays. These substances appear to denature aptamer secondary and tertiary structures which are critical to proper folding and binding of aptamers to hGH and other targets. This is anticipated to be a two year project. 

Main Findings: 

Operational Technologies Corp. (OpTech) was unable to obtain Genotropin or Norditropin from Pfizer, Novo Nordisk, or other sources.  However, OpTech developed DNA aptamers against the oxidized peptide regions of Genotropin defined by Hepner et al. (2005 and 2006) and thought to be potential sites for discrimination of recombinant vs. natural hGH in ~ 2% of Genotropin molecules. OpTech screened these aptamers against research-grade rhGH and natural pituitary hGH and identified several candidate aptamers (e.g., Hep 5-6R, Hep 6-6R and Hep 6-12R) which may be able to discriminate Genotropin from natural hGH.  However, without access to authentic Genotropin, OpTech could not test this hypothesis fully.  Therefore, OpTech elected to use the remaining funds to explore new and potentially even better approaches to detecting hGH abuse in athletes with aptamer-based assays.  These new approaches were: 1) Development of aptamers to bind human IGF-1 and PIIINP which are both long-lived serum biomarkers of rhGh use.  This work resulted in development of a preliminary enzyme-linked IGF-1 aptamer-magnetic bead sandwich assay which functioned in pure human serum with a lower limit of detection of ~ 30 ng/mL and linear detection to > 1,000 ng/mL.  The top-ranked IGF-1 aptamer candidates (3F and 25R) may bind a region of IGF-1 that is accessible even when IGF-1 is bound by its binding proteins in serum.  Unfortunately, these IGF-1 aptamers also demonstrated significant cross-reactivity with Brain Natriuretic Peptide (BNP), bone collagen Helical Peptide (HP) and rhGH, but much lower affinity for C-telopeptide (CTx) or N-telopeptide (NTx) of bone collagen.  Preliminary data also indicated that development of a PIIINP aptamer assay with linear quantification in the high to mid-ng/mL range was possible using at least 3 different aptamer candidates identified during ELISA-like screening (ELASA).
2) Development of aptamers against specific peptide regions of the 20kD and 22kD isoforms of hGH to emulate the current ratiometric immunoassay.  OpTech developed aptamers against the 15-amino acid region (EEAYIPKEQKYSFLQ) of 22kD hGH which is not present in 20 kD hGH and also developed aptamers against the fused flanking regions (DTYQEFNPQTSL) resulting from alternative splicing to enable discrimination of unique regions on each isoform of hGH.  Two lead candidates designated “Splice 20-14R and Splice 22-5F” were identified for potential future ratiometric assay development.Both approaches appear promising and, if fully developed, would have the advantage of overcoming lot-to-lot variability associated with antibodies and immunoassays, because aptamer DNA synthesis from known sequences is a very high fidelity process.