Activity-based Detection of HIF stabilizers: a Future-proof Complementing Method in Doping Control?

Small-molecule HIF stabilizers are being misused in sports to ‘artificially’ increase red blood cells. WADA banned their use, but the structural variety in this relatively new class of performance enhancing drugs and the rapid pace at which new drug candidates are emerging hampers their detection in biological fluids, as many currently used techniques target specific structures – a problem also encountered with NPS. Therefore, a future-proof strategy was envisaged, capable of detecting ANY HIF stabilizer (known AND unknown ones). A previously developed cell-based assay, monitoring the upstream mechanism of HIF1 activation (heterodimerization of HIF1α with HIF1β), was used to detect HIF stabilizers in spiked urine samples based on their HIF stabilizing activity. The HIF1 bioassay proved to be universal, detecting every HIF stabilizer tested so far (including enarodustat, IOX2, JNJ-42041935, etc.). Using roxadustat as a prototype compound, sensitivity in known urine matrices was determined and ranged from 1-25 ng/mL. A limit-of-detection in blind-coded roxadustat-spiked urine matrices was determined to evaluate routine applicability of this activity-based detection method. Proof of concept was established that HIF stabilizers can be detected in spiked urine samples by a cell-based assay format through the measurement of increased HIF activation.