title: In vitro metabolism of BYZX in human liver microsomes and the structural elucidation of metabolite by liquid chromatography-mass spectrometry method.
abstract: In vitro phase I metabolism of BYZX, a novel central-acting cholinesterase inhibitor for the treatment of the symptoms of Alzheimer's disease, was studied in human liver microsomes (HLM) and the metabolite formation pathways were investigated by chemical inhibition experiments and correlation analysis. The residual concentration of substrate and the metabolite formed in incubate were determined by HPLC method. The calibration curves of BYZX were linear over the concentration range from 5.07 microM to 200.74 microM. The relative standard deviations of within day and between day were less than 5% (n=5). The limit of detection (LOD) was 0.18 microg/mL (S/N=3) and the limit of quantification (LOQ) was 0.55 microg/mL (R.S.D.=5.2%, n=5). The determination recoveries of BYZX were in the range of 98.2-104.8%. The apparent K(m) of BYZX in HLM was 53.25+/-17.2 microM, the V(max) was 0.94+/-0.77 microM/min/mg protein, and the intrinsic clearance value (Cl(int)) was 0.018+/-0.02 mL/min/mg protein. Ketoconazole and cyclosporin A were the most potent inhibitors on BYZX metabolism in HLM with IC(50) being 0.89 microM and 18.17 microM, respectively. And the inhibition constant (K(i)) of ketoconazole was 0.42 microM. The metabolite of BYZX was N-des-ethyl-BYZX elucidated by LC-MS-MS. The results demonstrated that the developed HPLC method was reliability, simple technique, and was applicable to be used for the researches of in vitro metabolism of BYZX. CYP3A4 was the major isozyme responsible for BYZX metabolism; N-dealkylation was the major metabolic pathway of BYZX. The predominant metabolite of BYZX was N-des-ethyl-BYZX detected in vitro phase I metabolism in HLM.
authors: College of Pharmaceutical Sciences, Zhejiang University, 388 Yuhangtang Road, Hangzhou, Zhejiang 310058, PR China.
authors: Zong-Ling
authors: ZL
authors: Xia
authors: Jing-Yan
authors: JY
authors: Ying
authors: Rong
authors: R
authors: Sheng
authors: Su
authors: S
authors: Zeng
authors: Yong-Zhou
authors: YZ
authors: Hu
authors: Tong-Wei
authors: TW
authors: Yao
mesh_terms: D001681:Biological Assay; D002800:Cholinesterase Inhibitors / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007192:Indenes / Q000737:chemistry / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000378:metabolism*; D015203:Reproducibility of Results; D012996:Solutions
chemical_list: C524572:BYZX compound; D002800:Cholinesterase Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007192:Indenes; D012996:Solutions