Störmer E, Roots I, Brockmöller J
Humboldt-University Berlin, Institute of Clinical Pharmacology, Schumannstrasse 20/21, 10098 Berlin, Germany.
Br J Clin Pharmacol. 2000 Dec;50(6):553-61. doi: 10.1046/j.1365-2125.2000.00296.x.
The role of flavin containing monooxygenases (FMO) on the disposition of many drugs has been insufficiently explored. In vitro and in vivo tests are required to study FMO activity in humans. Benzydamine (BZD) N-oxidation was evaluated as an index reaction for FMO as was the impact of genetic polymorphisms of FMO3 on activity.
BZD was incubated with human liver microsomes (HLM) and recombinant enzymes. Human liver samples were genotyped using PCR-RFLP.
BZD N-oxide formation rates in HLM followed Michaelis-Menten kinetics (mean Km = 64.0 microM, mean Vmax = 6.9 nmol mg-1 protein min-1; n = 35). N-benzylimidazole, a nonspecific CYP inhibitor, and various CYP isoform selective inhibitors did not affect BZD N-oxidation. In contrast, formation of BZD N-oxide was almost abolished by heat treatment of microsomes in the absence of NADPH and strongly inhibited by methimazole, a competitive FMO inhibitor. Recombinant FMO3 and FMO1 (which is not expressed in human liver), but not FMO5, showed BZD N-oxidase activity. Respective Km values for FMO3 and FMO1 were 40.4 microM and 23.6 microM, and respective Vmax values for FMO3 and FMO1 were 29.1 and 40.8 nmol mg-1 protein min-1. Human liver samples (n = 35) were analysed for six known FMO3 polymorphisms. The variants I66M, P135L and E305X were not detected. Samples homozygous for the K158 variant showed significantly reduced Vmax values (median 2.7 nmol mg-1 protein min-1) compared to the carriers of at least one wild type allele (median 6.2 nmol mg-1 protein min-1) (P < 0.05, Mann-Whitney-U-test). The V257M and E308G substitutions had no effect on enzyme activity.
BZD N-oxidation in human liver is mainly catalysed by FMO3 and enzyme activity is affected by FMO3 genotype. BZD may be used as a model substrate for human liver FMO3 activity in vitro and may be further developed as an in vivo probe reflecting FMO3 activity.
含黄素单加氧酶（FMO）在多种药物处置过程中的作用尚未得到充分研究。需要进行体外和体内试验来研究人体中的FMO活性。评估了苄达明（BZD）N-氧化作为FMO的指标反应以及FMO3基因多态性对活性的影响。
将BZD与人肝微粒体（HLM）和重组酶一起孵育。使用PCR-RFLP对人肝样本进行基因分型。
HLM中BZD N-氧化物的形成速率遵循米氏动力学（平均Km = 64.0 microM，平均Vmax = 6.9 nmol mg-1蛋白质min-1；n = 35）。非特异性CYP抑制剂N-苄基咪唑和各种CYP同工型选择性抑制剂不影响BZD N-氧化。相反，在没有NADPH的情况下，微粒体经热处理后BZD N-氧化物的形成几乎被消除，并且受到竞争性FMO抑制剂甲巯咪唑的强烈抑制。重组FMO3和FMO1（在人肝中不表达）显示出BZD N-氧化酶活性，但FMO5没有。FMO3和FMO1的各自Km值分别为40.4 microM和23.6 microM，FMO3和FMO1的各自Vmax值分别为29.1和40.8 nmol mg-1蛋白质min-1。对35份人肝样本分析了六种已知的FMO3多态性。未检测到I66M、P135L和E305X变体。与至少携带一个野生型等位基因的样本（中位数6.2 nmol mg-1蛋白质min-1）相比，K158变体纯合的样本显示Vmax值显著降低（中位数2.7 nmol mg-1蛋白质min-1）（P < 0.05，曼-惠特尼U检验）。V257M和E308G替换对酶活性没有影响。
人肝中BZD N-氧化主要由FMO3催化，且酶活性受FMO3基因型影响。BZD可作为体外人肝FMO3活性的模型底物，并可进一步开发为反映FMO3活性的体内探针。
