Background <p>Anti-obesity medications are recommended for patients who do not achieve and maintain weight loss despite lifestyle interventions. S-309309 is a novel oral inhibitor of monoacylglycerol O-acyltransferase 2 being developed as a treatment for obesity.</p> Objective <p>The objective of the study was to investigate the safety, clinical pharmacology, pharmacokinetics and pharmacodynamic biomarker of S-309309.</p> Methods <p>A phase I, single-center, two-part, randomized, double-blind, placebo-controlled study of S-309309 following oral administration of a single-ascending dose (part 1) and a multiple dose (part 2) in healthy adults with or without obesity was conducted. We also assessed the effect of food on the pharmacokinetics of S-309309 and the effect of S-309309 on electrocardiogram parameters, the pharmacokinetics of midazolam (a cytochrome P450 3A substrate), and the pharmacodynamic biomarker of monoacylglycerol O-acyltransferase 2 inhibition.</p> Results <p>In part 1 (<i>N</i> = 50), a single-ascending dose of S-309309 in healthy adults demonstrated dose proportionality and comparable exposure of S-309309 between the fasted and fed states. In part 2 (<i>N</i> = 24), no clinically meaningful difference was observed in the pharmacokinetics of multiple doses between healthy adults with or without obesity. S-309309 did not affect the pharmacokinetics of the cytochrome P450 3A substrate. The pharmacodynamic biomarker of monoacylglycerol O-acyltransferase 2 inhibition, dicarboxylic acid (18:1), was significantly increased after S-309309 administration in healthy adults with or without obesity. Overall, S-309309 demonstrated acceptable safety and tolerability without any serious adverse events or discontinuations because of adverse events, and did not have a clinically relevant effect on the heart rate or cardiac conduction. An effect on the placebo-corrected change-from-baseline corrected QT interval, corrected for heart rate using the Fridericia method, exceeding 10 ms can be excluded.</p> Conclusions <p>S-309309 was well tolerated as single-dose (up to 300 mg) and multiple-dose (50 mg once daily for 14 days) oral administration. The pharmacokinetic characteristics remained unaffected by obesity and food intake. S-309309 did not affect the pharmacokinetics of the cytochrome P450 3A substrate. Overall, S-309309 had an acceptable safety profile and favorable pharmacokinetic and pharmacodynamic characteristics.</p> Clinical Trial Registration <p>NCT05247970, date of registration: 8 February, 2022.</p>