Use of human liver microsomes to assess flavin-containing monooxygenase (FMO) metabolism in vitro

ISSX NA 2023 -- Flavin-containing monooxygenases (FMOs) are a class of enzymes that oxidize a wide variety of xenobiotics, particularly by oxygenation of nucleophilic nitrogen and sulfur heteroatoms. An analysis of the metabolism of 860 drugs showed that FMOs contribute to 2% of all oxidoreductive reactions, more than any other non-cytochrome P450 (CYP) enzyme. There are five functional mammalian isoforms of FMO (FMO1-5). FMO3 is regarded as having the greatest role in drug metabolism and is predominantly expressed in the liver. FMO1 has the broadest substrate acceptance and is predominantly expressed in the kidney and lung. Human liver microsomes (HLM) are often used for in vitro drug testing; however, a standardized method for quantifying and verifying specific FMO activity in HLM is lacking. Here we describe the development of itopride as an FMO-specific probe substrate in HLM and optimization of incubation conditions. Additionally, since FMOs are sensitive to high temperatures in the absence of nicotinamide adenine dinucleotide phosphate (NADPH), a selective heat-inactivation method was investigated for separating FMO and CYP activity. The conclusions from this work serve as a verification for the measurement of FMO metabolism in vitro.