| Objective: To study the Stereoselectivity in trans-tramadol [( ±)-trans T] metabolism and trans-O-demethyltramadol [( ±)-Ml] formation in rat liver microsomes in vitro.Methods: SD rats, male, were used to prepare the rat liver microsomes by using differential ultracentrifugation. At different concentrations, (±)-trans T and its enantiomers were separately incubated with rat liver microsomes in vitro alone, or with quinine and quinidine (CYP-selective inhibitors), or with other drugs such as dextromethorphan, prorafenone and fluoxetine. After incubating for 90 min at 37 °C with constant shaking, the incubation products were removed out and extracted with ethyl acetate. The concentrations of (±)-trans T enantiomers and (±)-Ml enantiomers in incubation were determined by high performance capillary electrolphoresis (HPCE). For (±)-trans T metabolism, the metabolic rates of (±)-trans T enantiomers were obtained by dividing their depleted concentrations with the incubation time. For (±)-Ml formation, the formation rates of (±)-Ml enantiomers wereobtained by dividing their concentrations in the incubation with the incubation time. For (±)-Ml enantiomers formation, the enzyme kinetics parameters, including K,,, (apparent Michaelis-Menten constant), Vmax (maximal velocity) and Clint (instrinsic clerarance), were assayed by using enzyme kinetic analysis method. In the inhibitory experiments, Dixon was used to determine Kf values of quinine and quinidine on the formation of ( ± )-Ml enantiomers. In the study of coincubating (±)-trans T enantiomers with dextromethor-phan, prorafenone or fluoxetine, the metabolic rates of (±)-trans T enantiomers, the formation rates of (±)-Ml enantiomers and the inhibitions were calculated.Results: When each enantiomer of (±)-trans T was incubated separately with rat liver microsomes, it was observed that (±)-Ml was formed from (±)-trans T, whereas (-)-Ml was produced from (-)-trans T. There was no chiral inversion between the enantiomers of (±)-trans T. Stereoselectivity in (±)-trans T metabolism and (±)-Ml formation were found. The metabolic rate of (±)-trans T exhibited a significant lower value than that of (-)-trans T ( 19.75 ± 2.53 pmol·min1·rag'1 vs 24.19 ± 1.74 pmol·minˉ1·mgˉ1 ), and the formation rate of (±)-Ml exhibited a significant lower value than that of (-)-Ml (6.40 ± 1.24 pmol·minˉ1·mgˉ1 vs 12.67± 1.05 pmol·minˉ1·mgˉ1). The formation of (±)-Ml enantiomers was found to fit the single-enzyme Michaelis-Menten model. For (±)- Ml, the enzyme kinetics parameters of Km , Vmax and Clint were 38.56 ± 4.47 U mol·I/1, 29.57 ± 4.08 pmol·mmˉ 1·mgˉ1 and 0.77±0.11 mL·minˉ1·gˉ1 respectively. For (-)-Ml, they were 38.29 ± 4.44 u mol·I/1, 52.88 ± 3.21 pmol·minˉ1·mgˉ1 and 1.39±0.07 mL·minˉ1·gˉ1 respectively. The formation of (±)-Ml enantiomers had the same Km, but (±)-Ml formation had lower Vmax and Clint than (-)-Ml formation. This fact indicated that (±)-Ml enantiomers formation were catalysed by the same cytochrome P450 isoenzyme at different rates, and the isoenzyme yielded higher enzymatic efficacy for (-)-Ml formation than for (±)-Ml formation.Compared with the results when (±)-trans T enantiomers were used as the substrates, the metabolic rates of (± }-trans T enantiomers metabolism and the formation rates of (±)-Ml enantiomers formation decreased when the racemate was used as the substrate. The metabolic rate of (±}-trans T decreased more than that of (-)-trans T (19.73% vs 4.76%); and the ratio of (-)/(±)-trans T increased from 1.24 ±0.10 to 1.47±0.16 obviously (p<0.05). The formation rate of (±)-Ml decreased more than that of (-)-Ml (60.15% vs 9.54%); and the ratio of (-)/(±)-Ml increased from 2.02±0.24 to 4.71±1.05 (p<0.01). It was also demonstrated from the enzyme kinetics assay results that the Km values for both (±)-Ml enantiomers formation increased, but the Vmax increased slightly for (±)-Ml formation, and decreasedobviously for (-)-Ml formation. In the inhibitory ex...
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