Research projects

Code: 11C21BG

This project seeks to build on the very important findings of a project funded previously by the Australian Government’s Anti-Doping Research Program (ADRP), into the potential application of transcriptional profiles derived from peripheral blood mononuclear cells (PBMCs -lymphocytes & monocytes) for the detection of rhGH. That project identified sets of genes that were consistently either “up” or “down” regulated in mRNA expression, changes that were evident at least 21 days after the final administration of rhGH. The 21 day time period is an extremely important finding given that the current detection “window” with the WADA-approved test for rhGH is only 36 hours (Holt & Sonksen, 2008). The findings of the previous ADRP-funded study clearly provide the foundation for the development of a new, more sensitive test for the detection rhGH, a test with a vastly improved detection “window’. 
We anticipate that the proposed project will result in the development of a new, more sensitive test for rhGH based on alterations in gene expression assessed at either the transcriptional and/or proteomic level. A cell’s transcriptional “profile” is obtained by measurement of (potentially, genome-wide) genespecific mRNA levels via use of oligonucleotide microarray technology. Subsequent quantitative RT-PCR analyses allow verification of alterations in the expression of candidate genes. A cells “translational”/proteomic profile can be assessed via the techniques of SDS-PAGE and western blotting. These technologies will allow the identification of gene “subsets” whose activity at a specific time-point, is either up-regulated, down-regulated or remains unchanged following a specific intervention. The set of genes responding transcriptionally or translationally to a specific intervention and the temporal extent of such changes potentially provide a set of unique indicators that are characteristic of that intervention. 

Main Findings: 

This project has been carried out with the support of The World Anti-Doping Agency. The investigation aimed at pinpointing a set of differentially expressed mRNA genes associated specifically to the uptake of recombinant growth hormone. 
Particular attentions of this project focused on the determination of exercise related induce gene expression and to compare whether such expression profile shared similarities with rhGH related expression profile. In other words, this project aimed at finding the unique and specific rhGH related gene expression signature eliminating possible additional unrelated expression variables that different types of exercise induce. 
Three types of exercise regimen (endurance, resistance and anaerobic exercises) were analysed in untrained participants and temporal PBMC extracted cell gene expression profiling using microarray technology was determined for each type of exercise. In addition, mRNA expression profiling was performed on trained individuals carefully sub-grouped in the same types of exercise mentioned previously. 
Our analysis used a series of state of the art bio-informatics tools aimed at reducing background noise, batch effects between microarrays, false positives and any other un-necessary variables to aim at narrowing the candidate genes at the highest significant relevance to our study and conclusions. 
The main results obtained in this document are reporting the PBMC’s temporal expression profiling from: I. participants administered with rhGH;
II. untrained participants undertaking three types of exercise. In addition, non-temporal expression profiling at rest was evaluated on trained participants grouped in the same types of exercise (endurance, resistance and anaerobic).The comparison of all the different profiles has shown that:I. 47 genes are specific to rhGH and unrelated with any of the three exercise related gene expression in untrained individuals.
II. 2 genes specific to rhGH are seen to be similar to both the Trained and Untrained expression profiles. These two genes consisted of the interferon gamma and the tubulin folding cofactor C.Of note, this two candidate genes were observed specifically in the resistance groups. Finally, these two genes showed fold gene expression levels that are, and across all time points, lower in rhGH related PBMCs than the fold expression values of the resistance groups. 
The common limitations of this present study are: - Batch effect related to the analysis of the rhGH related study that required the action of the use of a bio-informatic batch effect removal tool.
- Possible dosage of the rhGH that was administered to reflect a more pronounced expression profile.

Code: 11A26CS

Determining the origin of anabolic steroids that are also produced endogenously in the human body, is a major issue in doping control. Increasing amounts of anabolic steroid preparations of nandrolone, boldenone and testosterone preparations, both in a ready-to-use form and as bulk material, have been confiscated by the Norwegian Custom Authorities and the Norwegian Police. 
In rare circumstances, nandrolone and boldenone metabolites may be produced naturally in humans and animals. Hence, the measurement of the 13C‰ value of nandrolone, boldenone and testosterone of synthetic origin is important for their comparison with the nandrolone and boldenone metabolites, and testosterone endogeneously produced. As a result of this, IRMS is required for a definitive assignments of the origin of nandrolone, boldenone and testosterone found in urine. 
The aim of the project is to determine the 13C/12C ratios of nandrolone, boldenone and testosterone available on the Scandinavian black market. The success of the established doping test is depending on a significant difference in 13C‰ values between the administered product and the endogenously produced steroids. A comprehensive knowledge of these data with a global perspective is essential for doping analysis of certain anabolic-androgenic steroids. 
In addition, the confiscated testosterone preparations will also be investigated for the content of other possible masking agents like epitestosterone. 

Main Findings:

There have been a substantial number of seized doping preparations, including preparations of nandrolone, boldenone and testosterone, in Norway lately. Determining the origin of anabolic androgenic steroids (AAS) that are also produced endogenously in the human body, is a major issue in doping control. The aim of this study was to use GC-C-IRMS to determine the δ13C‰ values of nandrolone, boldenone and testosterone in seized preparations, and to what extent these products have been designed to mimic the endogenous δ13C‰ values, and hence prevent positive GC-C-IRMS findings. Out of 55 products, one nandrolone and five testosterone products (11%) were found to display δC‰ values between -24‰ and -26‰. In addition one nandrolone and eight testosterone products (16%) displayed δC‰ values of -27‰, which is close to the upper reference range (-26‰) reported for urinary reference metabolites. None of the investigated products contained epitestosterone. This study shows that there are AAS available with δC‰ values near and within the endogenous urinary reference range in Norway. 

Code: 11D3JD 

Stimulants are banned in competition by the World Anti-Doping Agency, except for a small number of therapeutic agents subject to monitoring, and a large number of compounds have been placed on the prohibited list. However, a number of agents available as over the counter medicines for therapeutic uses, although subject to monitoring, may enhance performance. In addition, a large number of other unlisted stimulants are available, some of which appear in athlete’s samples. There is only very limited information on the relative potencies of even monitored agents as stimulants. For some agents even the mode of action is not well understood. This project seeks to supply that information. In this project, the stimulant actions of a wide range of compounds, prohibited, specified, monitored or unlisted, will be investigated in pharmacological and physiological bioassays in an animal model. This study will provide data giving direct measurement of the relative potencies of a wide range of stimulants at a number of biomarkers of stimulant action. The results will allow informed decisions on the doping potential of monitored substances relative to prohibited or unlisted substances. The wider implications are that these bioassays can be quickly performed to give robust information on any new compound appearing in athlete samples or to decide on the acceptable agents and doses for approved therapeutic uses. Generation and dissemination of detailed pharmacological information is essential to prevent doping in sport and to provide alternative therapies. 

Main Findings:

1. Bupropion is a stronger noradrenaline reuptake blocker than previously reported. It is likely to have both peripheral cardiovascular stimulant actions and central behavioural actions in doses used clinically.
2. The majority of direct adrenoceptor stimulants investigated in this study showed relatively high potency as β-adrenoceptor agonists in producing a tachycardia. Although β1-adrenoceptor stimulation may be of limited benefit in sport (see Davis et al., 2008), this merits further consideration, especially if some of these agents are found to have additional  β2-adrenoceptor agonist actions.
3. Cathine is likely to be an α2-adrenoceptor agonist, and this merits further study, as central clonidine-like actions as well as peripheral actions may be important for this agent.
4. Selective actions at α1D-adrenoceptors occur for a number of agents. It is unclear if this action is of benefit in enhancing performance. This merits further investigation.
5. Locomotor studies by telemetry are a useful method to study central stimulant actions. Both bupropion and modafinil were found to have central stimulant actions in doses employed.
6. A large amount of data have been obtained on the cardiovascular effects of 12 receptor stimulants, and on the peripheral and central effects of 3 monoamine transporter blockers.  On publication, this will greatly increase the literature on the pharmacology of these agents.  On acceptance for publication, copies of these papers will be forwarded to WADA.

Code: 11A6SD 

The aim of the project is to provide an isotopically labelled internal standard of a key testosterone metabolite. These will facilitate high accuracy measurement of this analyte in human urine thereby contributing to WADAs implementation of the Athletes Passport. The synthesis of d4-epitestosterone-17-b-glucuronide will be performed using a combination of literature precedents and chemistry routinely used by the Chemical Reference Materials team at NMI Australia. This material will be purified to the highest level possible using chromatographic and recrystallisation techniques. The material will then be fully characterised to afford fit for purpose status for use as an internal standard. The identity will be confirmed using a range of spectroscopic techniques including nuclear magnetic resonance, infrared spectroscopy and mass spectrometry. This will be complimented by analysis using a range of traditional techniques (HPLC with ELS detection, Karl Fischer moisture analysis and thermogravimetric analysis) to determine the chemical purity of the material. The isotopic purity will be determined using mass spectrometry on either the deglucuronidated steroid (GC-MS) or direct analysis of the parent material (LC-MS). 
Fully certified material will be packaged in 1 mg lots and made available to all WADA accredited laboratories. 

Main Findings: 

This summarizes the synthesis of 1,16,16,17-d4-epitestosterone-17-O--glucuronic acid (1) using a combination of literature precedents and chemistry used previously by the Chemical Reference Materials team.  The material has undergone extensive purification to minimise impurities of similar structure.   
HPLC analysis with photodiode array detection (PDA) confirmed an organic purity in excess of 99% mass fraction.  The overall purity value was lower (~93% mass fraction) than the idealised >99% mass fraction, which can be attributed to significant mass fractions of occluded water.  This is typical for glucuronic acids of this type and no attempt was made to remove the water because of concerns that the inevitable re-absorption of moisture would introduce stability issues.The isotopic purity of each material has been determined using GC-MS analysis of the de-glucuronidated steroid entity, confirming high levels of deuteration, with essentially zero non-deuterated (d0) present, making the isotopically labelled internal standard fit for its intended purpose.   The structure of the 1,16,16,17-d4-epitestosterone-17-O--glucuronic acid (1) has been unequivocally determined using a combination of spectroscopic techniques . Direct comparison with the spectroscopic data obtained for a fully certified sample of non-deuterated epitestosterone-17-O--glucuronic acid confirmed the regio- and stereochemistry. Further evidence was provided by co-elution studies of the parent compound and/or the liberated steroid moiety, with the fully certified native counterparts.
 Mass spectrometry
 Infra-Red spectroscopy
1H / 2H / 13C Nuclear Magnetic Resonance spectroscopy
1,16,16,17-d4-Epitestosterone-17-O--glucuronic acid (1) was assessed for chemical purity using a complementary range of analytical techniques to determine the mass fraction of the main component. Full characterisation and certification of this material has been performed and each material is currently available to the Australian Sports Drug Testing Laboratory (ASDTL) and other sports doping control laboratories around the world. 

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.