Ciprofol (HSK3486) is a novel intravenous agent for general anesthesia. In humans, HSK3486 mainly undergoes glucuronidation to form M4 (fraction of clearance [f]: 62.6%), followed by the formation of mono-hydroxylated metabolites that further undergo glucuronidation and sulfation to produce M5-1, M5-2, M5-3, and M3 (summed f: 35.2%). However, the complete metabolic pathways of HSK3486 in humans remain unclear. In this study, by comparison with chemically synthesized reference standards， three mono-hydroxylated metabolites (M7-1, 4-hydroxylation with an unbound intrinsic clearance [CL] of 2211 μL/min/mg; M7-2, ω-hydroxylation with a CL of 600 μL/min/mg, and M7-3, (ω-1)-hydroxylation with a CL of 78.4 μL/min/mg) were identified in human liver microsomes, and CYP2B6 primarily catalyzed their formation. In humans, M7-1 was shown to undergo glucuronidation at the 4-position and 1-position by multiple UDP-glucuronosyltransferases (UGTs) to produce M5-1 and M5-3, respectively, or was metabolized to M3 by cytosolic sulfotransferases. M7-2 was glucuronidated at the ω position by UGT1A9, 2B4, and 2B7 to form M5-2. UGT1A9 predominantly catalyzed the glucuronidation of HSK3486 (M4). The CL values for M4 formation in human liver and kidney microsomes were 1028 and 3407 μL/min/mg, respectively. / extrapolation analysis suggested that renal glucuronidation contributed approximately 31.4% of the combined clearance. In addition to HSK3486 glucuronidation (M4), 4-hydroxylation (M7-1) was identified as another crucial oxidative metabolic pathway (f: 34.5%). Further attention should be paid to the impact of CYP2B6- and UGT1A9-mediated drug interactions and gene polymorphisms on the exposure and efficacy of HSK3486. This research elucidates the major oxidative metabolic pathways of HSK3486 (the formation of three mono-hydroxylated metabolites: M7-1, M7-2, M7-3) as well as definitive structures and formation pathways of these mono-hydroxylated metabolites and their glucuronides or sulfate in humans. This research also identifies major metabolizing enzymes responsible for the glucuronidation (UGT1A9) and oxidation (CYP2B6) of HSK3486 and characterizes the mechanism of extrahepatic metabolism. The above information is helpful in guiding the safe use of HSK3486 in the clinic.
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