Application Of Radiolabeling Technology In Phase Ⅰ Clinical Trail Of HR011303

HR011303 is a highly selective URAT1 inhibitor,which is currently in phase III clinical trial in China.It is clinically intended to treat gout.In this research,the radioisotope labeling technique was used to study the radio-pharmacokinetics,mass balance and biotransformation of[¹⁴C]HR011303 in 6 healthy Chinese male subjects.Through in vitro experiments combined with literature reports,the reasons that may cause the species differences in the excretion of HR011303-related substances were analyzed,and the distribution and excretion mechanisms in rats and humans were further speculated.After oral administration of[¹⁴C]HR011303 suspension to 6 subjects,HR011303was rapidly absorbed.The median T_max of total radioactivity in plasma was 1.50 h and the geometric mean C_max was 1730 ng eq./m L.The geometric mean AUC_0-∞of total radioactivity was 16100 h×ng eq./m L and the arithmetic mean t_1/2 was 24.2 h.Within0-216 h after administration,the cumulative total radioactive dose recoveries in urine and feces from male subjects was 91.75%.The cumulative radioactive dose recoveries accounted for 81.50%in urine and 10.26%in feces,indicating that urinary was the main excretion route of[¹⁴C]HR011303 in humans.Using radioactive detector combined with high-resolution mass spectrometry（HR-MS）,6 metabolites were detected in plasma,urine and feces.HR011303 accounted for 87.93%of the total radioactivity in plasma and M293 accounted for 3.46%.In urine,M5 accounted for 51.57%,M5/2 accounted for 18.06%and HR011303 accounted for4.62%of the dose.Three metabolites were detected in feces.HR011303 accounting for4.24%of the dose.M424 accounted for 1.19%,M394 accounted for 0.69%and M408accounted for 0.58%of the dose.The results of previous animal experiments showed that mean cumulative radioactive dose recovery in feces was 87.2%over 168 h post-dose.In bile duct cannulation rats,cumulative radioactive dose recovery in bile was 70.9%over 48 h post-dose.Drug-related substances were mainly efflux into bile and finally excreted through feces.There are species differences in the excretion route of[¹⁴C]HR011303related substances in humans and rats.M5 was the major metabolite of HR011303 in both humans and rats.Results of microsomes incubation study showed HR011303 could be metabolized to M5 in human liver microsomes and human kidney microsomes.Whereas HR011303 can only be metabolized to M5 in rat liver microsomes and cannot undergoing metabolism in rat kidney microsomes.The results of uptake transporter study showed that HR011303 has high permeability,and M5 can be taken up through organic anion-transporting polypeptide（OATP）1B1,OATP1B3 and organic anion transporter（OAT3）.Multidrug resistance protein（MRP）3/Mrp3 and MRP2/Mrp2 possibly play a decisive role in drug disposition.MRP3/Mrp3 can mediate the efflux of compounds in hepatocytes into the blood and further excrete through the kidney,while MRP2/Mrp2 is mainly responsible for compounds from hepatocytes into bile.In addition,the expression of MRP3/Mrp3in human hepatocytes is significantly higher than that in rat hepatocytes,while the expression of MRP2/Mrp2 in rat hepatocytes is obviously higher than that in human hepatocytes.Experiments with membrane vesicles overexpressing MRP3,MRP2 and Mrp2 showed that M5 is a substrate of MRP3,MRP2 and Mrp2.Sandwich-cultured hepatocytes were used to study the bile efflux of HR011303 and M5 after HR011303was present alone.Inhibitors were used to investigate the contribution of different transporters to the efflux process.When HR011303 was present alone,it could be metabolized to M5 in sandwich-cultured rat hepatocytes（SCRH）and sandwich-cultured human hepatocytes（SCHH）.In SCRH,M5 excreted into bile with a bile efflux index（BEI）of 23.44%.MK571,the inhibitor of MRP2/Mrp2,can significantly reduce the bile efflux of M5 with the BEI decreased to 12.25%.In SCRH,the BEI values of HR011303 and M5 were 7.49%and 4.11%respectively,indicating that neither HR011303 nor M5 was obviously excreted into bile.In conclusion,this study investigated the pharmacokinetics and mass balance of[¹⁴C]HR011303 in healthy Chinese male subjects,identified the main metabolites in plasma,urine and feces,and further explored the possible reasons for excretion species differences of HR011303-related substances.