In vitro SR9011 metabolism using 3D cell culture and tandem mass spectrometry

Code: 241C03JFN

The REV-ERB alpha and beta agonist SR9011 was initially synthesized to explore the relations between sleep disorders and circadian rhythm in rodent models. In parallel, studies highlighted that SR9011 ameliorates muscular growth and affects mitochondrial composition, making this compound attractive to athletes who desire to improve their sports performance. Consequently, SR9011 was placed on the WADA prohibited substance list in 2018. However, only the compound was included because of poorly understood and characterized metabolism. Since January 2024, some metabolites of SR9011 have been included in the WADA list of banned substances. So far, two publications have suggested structures some metabolites, although their structure must be re-examined. The first objective of this project is to adapt our previous protocol using human subcellular liver fractions to spheroid cell culture to corroborate the presence of oxidative metabolites. By this, we aimed to refine our previously suggested metabolites to reinforce those susceptible to be detected in urine samples. The spheroid which are terminally differentiated hepatic cells derived from a human hepatic progenitor cell line will be used to obtain metabolites. To achieve metabolites generation, spheroid and SR9011 will be incubated in a cell culture medium. In parallel, considering the phase II metabolism of SR9011 has not been addressed to date, the investigation of glucuronide conjugates on SR9011 will constitute the second objective. Finally, since WADA has included four oxidative metabolites in the list of banned substances and considering that no reference materials are currently available, the third objective of this proposal consists of adapting small-scale synthesis routes to large-scale production of those metabolites in readiness to provide enough reference material to worldwide anti-doping laboratories.