The Biotransformation And Stereoselective Drug Metabolism Of ABP

The biotransformation of 6-amino butyl phthalide (ABP) has been studied. First, we examined the metabolites of ABP after in-vitro metabolism by rat liver microsomes. The results demonstrate that amino acylation reaction is the major route of metabolism. In-vivo metabolism studies were carried out by analysis of metabolites in rat bile, urine and plasma with gradient HPLC method. Four metabolites were isolated and purified from rat bile and determined. The structural characterization of metabolites was also conducted by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques, and some minor metabolites were detected. Based on the study of in-vitro and in-vivo metabolism, thirteen metabolites were determined, which basically clarified the metabolic pathways of ABP. Additionally, the stereoselective aspects in in-vitro and in in-vivo metabolism have been examined.This paper includes:1. The metabolism of ABP in rat liver microsomeABP was incubated by rat liver microsome and analyzed by HPLC-DAD method. Four minor components were determined as the metabolites of ABP by comparison of their UV spectra with ABP. The sample was also analyzed by GC-MS, and three metabolites (Ml, M2 and M3) were identified. Structures of Ml and M2 were reconfirmed by comparison with synthetic standards. In-vitro metabolism studies indicate acylation to be the major metabolic pathway for ABP.2. In-vivo metabolism of ABPA gradient HPLC method was developed to determine ABP and its metabolites in rat bile, urine and plasma. Four metabolites (M4, M5, M6 and M7) were isolated and purified from the bile and characterized by MS, MS/MS and ~1HNMR. In addition, some minor metabolites were determined. In-vivo metabolism studies show that ABP undergoes extensive metabolism, and the metabolic reactions are dominated by amino acetylation, together with hydroxylation.3. Stereoselective aspects in in-vitro and in in-vivo metabolism of ABPThe stereoselective aspects in in-vitro and in in-vivo metabolism have been investigated. No distinct discrepancy was observed after incubation of the enantiomers with rat liver microsome. After administration of (+)-ABP and (-)-ABP separately, the rat bile, urine and plasma were collected and determined. It can be seen that in bile the metabolites M1, M6 and M7 formed from (+)-ABP are more than those from (-)-ABP, which shows noticeable stereoselectivity. The results obtained by analysis of urine show that (+)-ABP is mainly excreted as metabolites, while (-)-ABP as proto type. In plasma, the M1 formed from (+)-ABP is more than any other metabolites, while M4 formed from (-)- ABP is the most. In addition, the discrepancy is also present between the enantiomers and the racemate.4. Determination of ABP and its metabolites by GC-MSGC-MS technique was used for determining ABP and its metabolites. Eleven metabolites were detected and characterized by EI-MS spectrum. The hydroxylated site of M3 has not been determined according to MS spectrum.5. Determination of ABP and its metabolites by LC-MS and LC-MS/MSLC-MS and LC-MS/MS techniques were used to determine the metabolites of ABP in biological samples. Eight metabolites were detected, including 3-hydroxyl-ABP (M13), which has not been detected by other methods.