Periodic Reporting for period 2 - MET-A-FOR (Metabolomic analysis for the forensic detection of drugs of abuse in performance and food producing animals) Reporting period: 2017-01-01 to 2018-12-31 Summary of the context and overall objectives of the project Forensic detection of the illicit use of drugs in performance and food-producing animals is experiencing a growing threat in the form of new compounds emerging from human drug development and clinical applications. One such class of compounds are known as Selective Androgen Receptor Modulators (SARMs) which are therapeutic agents with anabolic activity. From a therapeutic perspective SARM-like compounds hold advantages over existing steroids used for the treatment of human conditions as they demonstrate full anabolic activity in target tissues such as bone and muscle but reduced androgenic activity on other organs such as liver, prostate and cardiovascular tissue, thereby eliminating undesirable effects typically associated with conventional anabolic androgenic compounds. Oral bioavailability facilitates ease of administration to animals whilst a short half-life and rapid metabolism and elimination from the body makes detection analysis extremely challenging. Ease of availability, simplicity of use, advantageous biological effects and short detection windows are key features increasing the potential for SARM misuse, and consequently they are widely recognised as drugs of abuse in both human and animal (e.g. equine and canine) sports, and as emerging candidates for illicit use in food-producing species. Although many SARM compounds are currently undergoing evaluation in various studies, as yet none are approved for pharmaceutical use, there is widespread SARM availability via black- and grey-market sources. SARMs have gained particular popularity in professional sports and are banned by the World Anti-Doping Agency (WADA), the International Agreement on Breeding, Racing and Wagering (IABRW) and Fédération Equestre Internationale (International Equestrian Federation, FEI), with many reports of positive findings from routine testing. The potential for SARMs to be further adopted for use in food-producing animals (e.g. in cattle livestock) to increase muscle growth and reduce fat mass also remains a distinct threat. Advanced and reliable screening and confirmatory analytical assays are therefore required to better detect SARM use for doping practices in sport and monitor for potential misuse in stock farming. The MET-A-FOR project has focused on the development and practical advancement of forensic testing for drugs of abuse based on drug and metabolite profiling through the completion of interconnecting research projects focused on better understanding the metabolism of SARMs, their physiological impact in vivo, and development of sensitive multi-analyte mass spectrometry based analysis. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Work performed on the MET-A-FOR project has assessed the ability of metabolism studies using in vitro incubation methodologies to rapidly predict patterns of in vivo metabolism of SARM compounds. The aim of in vitro approaches is to accurately reflect the metabolite profile of illegally administered drugs in the absence of extensive elimination and pharmacokinetic studies and thereby facilitate rapid development of mass spectrometry based methods to more quickly detect new and emerging designer drug abuse as evidence arises that they are being used. Work on the project has prioritised key classes/types of SARM compounds (both from reputable sources but also unverifiable (e.g. web-based) vendors) based on their current availability and on the likely level of risk of compounds to actually be used illegally in sport and food animals - work has also examined new SARM compounds emerging from clinical focused pharmaceutical drug development. Liver/microsomal preparations/fractions from different species have been used in conjunction with LC-MS analysis to profile and identify phase 1 and 2 metabolites of SARMs to help predict and compare to actual in vivo drug metabolism. Various methods (LC-MS and 2D HR-NMR) were applied to the profiling of metabolomic responses to SARMs in urine/plasma (and tissues) and to identify key signatures (metabolites) of SARM compound administration. Proteomic analyses of target tissue (muscle/liver) responses to SARMs has additionally revealed new information on the physiological affects of these compounds in vivo. The project has focused on the extensive development and optimisation of sample extraction, liquid chromatography and mass spectrometry analysis that can be applied to the analytical detection of SARMs of interest in various test matrices across a range of relevant target species (e.g. equine, bovine, canine, murine, human), with a particular emphasis on urine/blood of bovine/equine origin. Fit-for-purpose sample extraction and UHPLC-MS/MS-based analytical methods have thereby been developed within the project for the screening of 15 SARM compounds under commercially offered or regulatory control testing environments. The developed methods have been validated in accordance with relevant legislation such as the EU Commission Decision 2002/657/EC criteria and Community Reference Laboratories Residues (CRLs) guidelines - with an analytical LC-MS run-time of between 12-14 minutes, a total of 50 samples can be analysed in a single day using these high-throughput assays. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The MET-A-FOR project has applied current and emerging technological developments in the field of mass spectrometry and metabolomic profiling to try to develop new methodologies which can improve the analytical screening for misuse of emerging drugs such as SARMs. Using combined academic and industrial beneficiary expertise, project outputs have resulted in innovative multi-residue UPLC-MS/MS screening assays for key SARM compounds with different physicochemical properties, chosen based upon their reported use in human and animals. The developed methods have been validated in various matrices (urine/plasma/muscle) from a range of species (equine, canine, human, bovine and murine) and employed to screen for SARM residue presence in racing animals (equine and canine), amateur and elite athletes, as well as livestock (bovine) animals. These rapid and cost effective assays can be employed for analytical screening purposes to determine the potential level of SARMs abuse ensuring consumer safety and fair play in animal and human performance sports. The knowledge, advanced analytical techniques and skilled researchers trained within MET-A-FOR will increase monitoring controls for drug misuse helping to maintain animal welfare standards (sport and livestock) within Europe in compliance with respective regulations and that the integrity of our performance sport and food production animals are of the highest level. Project Logo
