Development of analytical methods for the detection of HIF-activating agents in dried blood spot samples

Code: DBS23HIF01MO

Recombinant protein regents of the erythroid growth factor erythropoietin (EPO) have been widely used for anemia treatment, but they have been frequently employed for hematopoietic doping to improve endurance performance. In addition to EPO reagents, five types of small compounds activating hypoxia-inducible transcription factors (HIFs), which induce endogenous EPO production, have been launched in Japan as anemia medications since 2019. Given that HIF-activating agents (HIF-AAs) are administered orally, there is concern that they are used more easily for hematopoietic doping than EPO reagents, which are administered as injections. Although doping detection systems of EPO reagents have already been established, reliable detection methods of HIF-AAs should be urgently established. The applicants at Tohoku University are in charge of the genetic analysis for WADA and have demonstrated the molecular mechanisms by which HIF-AAs induce EPO production, whereas those at LSI Medience Corporation (LSIM) have experience managing anti-doping control tests in Tokyo 2020 Olympic and Paralympic Games with a WADA-accredited highly sensitive LC-MS/MS analyzer. Additionally, the applicants at LSIM recently found differences in the detection efficiency of trimetazidine between dried blood spot (DBS) samples from fingertips and brachial capillaries. Furthermore, the applicants previously optimized detection methods of HIF-AAs in the blood and urine of mice and found that each HIF-AA exhibits specific pharmacodynamics and pharmacokinetics after oral administration. Notably, HIF-AAs were commonly cleared within 48 hours after administration in mice. These data obtained by the applicants suggest that highly sensitive detection methods of the doping use of HIF-AAs should be established to detect them in small amounts of matrix for a long time after administration. Therefore, in this project, the applicants at Tohoku University and LSIM collaboratively aim to develop methods for highly sensitive and efficient detection of HIF-AAs in DBS and urine samples from humans. To elucidate the pharmacokinetics of various HIF-AAs in humans, the applicants will measure the concentrations of five types of HIF-AAs (daprodustat, enarodustat, molidustat, roxadustat, and vadadustat) in DBS and urine samples from healthy volunteers administered HIF-AAs. DBS samples will be obtained from fingertips and brachial capillaries to identify the differences in the detection efficiencies of HIF-AAs between the sampling sites. Additionally, DBS and urine samples from a total of 100 renal anemia patients treated with HIF-AAs will be analyzed. The stability of HIF-AAs in DBS and urine samples for up to six months will also be evaluated as well as the minimum required performance levels (MRPL) of HIF-AAs. The anticipated outcomes of studying the pharmacokinetics of five types of HIF-AAs in human samples are directly applicable for the development of anti-doping tests of HIF-AAs.