Involvement Of CYP2B6 In The Biotransformation Of Propofol By Human Liver Microsomes

IntroductionPropofol, a short - acting intravenous anesthetic, is commonly used in the induction and maintenance of anesthesia or in the sedation of patients. Since no analgesia or muscle relaxation function, propofol has been frequently used in combination with other agents such as opioids or other anesthetics. Recently, it was found that propofol shown to have drug interaction with those compounds and shown the individual variation of pharmacodynamics in clinic. Since inhibition of the metabolism of simultaneously administered drugs is often observed when both agents are metabolized by the same cytochrome P450 (CYP) isoforms and the hydroxylation accounts for about 40% of the total dose of propofol by CYP, it is important to elucidate the CYP isoforms which are involved in the metabolism of propofol to predict the pharmacokinetics and pharmacodynamics.The cytochrome P450 comprise a superfamily of hemoproteins catalyzing the oxidative metabolism of exogenous chemicals and endogenous substances. It's mainly expressed in the endocytoplasmic reticulum of liver cell. Because the biotransformation catalyzed by CYP is the main metabolism route of drugs, CYP is also called hepatic - drug enzyme. Recently, when studying the pharmacokinetics of propofol, it was found that CYP2B isoform was potentially involved in the propofol hydroxylation by rat liver microsomes. And court proposed that CYP2B11 was the principal hepatic enzyme for propofol hydroxylase in dogs. Since CYP2B6 is the only isoform with high activity in the CYP2B subfamily in human, it may be the major enzyme that catalyzes the hydroxylation of propofol in the human liver. In this study, we prepared a bank of human liver micro-somes (HLMs) derived from 12 donors for incubation studies of propofol and de-termine the rates of propofol metabolism in HLMs. Then we used two kinds of selective chemical inhibitor of CYP2B6 to ascertain and evaluate the role of CYP2B6 in the metabolism of propofol in human, and it maybe supply an explanation for the individual variation of pharmacodynamics of propofol in clinic.Materials and Methods1. Specimens12 specimens were obtained from patients (5 males and 7 females) with liver disease who underwent partial hepatectomy operation in the first affiliated hospital of China Medical University between October 2004 and July 2005.2. ReagentsPure propofol was provided by Libang Co. (Xi'an, China). Nicotinamide adenine dinucleotide phosphate (NADP+) was purchased from Amresco Co. Glucose - 6 - phosphate ( G6P) , glucose - 6 - phosphate dehydrogenase (G6PDH), orphenadrine, thioTEPA, Folin phenol, bovine serum albumin, thymol were purchased from Sigma Co. Acetonitrile and methanol ( chromato-graphic grade) were obtained from Shield Co. (Tianjin, China). Other chemicals and organic solvents were of the highest grade commercially available.3. Experimental Methods1) Preparation of human liver microsomesMicrosomes were prepared from liver tissues by differential centrifugation as described previously and determine the protein concentration by a modification of Lowry' s method using bovine serum albumin as standard.2) Incubation conditionThe incubation mixture consisted of 50 jxl of human liver microsomes containing 0. 05 mg protein, 100 jxl of NADPH - regenerating system (0. 5 mM NADP+ , 5 mM G6P, 2 unit -ml"1 G6PDH, and 6 mM MgCl2) , and 100 pi of propofol working solution. G6PDH was replaced by steamed water in control team. Propofol was initially prepared in dimethyl sulfoxide solution, and the final concentration in the incubation mixture was adjusted to 20 jxM. The mixture was incubated at 37 °C in individual tubes for 0, 5, 10, 15 and 30 min, respec-tively. Reactions were started by adding propofol working solution, then terminated by the addition of 250 jxl of cool - acetonitrile containing thymol (2.5 [xg ? ml ~ ) as the internal standard to each tube and cooled on ice. In two specific CYP2B6 - inhibition study team, before adding propofol working solution to start the incubation reaction, microsomes (0.05 mg protein) and NADPH -regenerating system were preincubated with orphenadrine (50 yM ) for 15 min or thio-TEPA (10 jxM ) for 3 min.3 ) High performance liquid chromatography ( HPLC) HPLC was obtained from HP Co. (model 1100) , consisted of a quarter -head pump (Model G1314A) serially connected with a 250 x4. 6 mm ID reverse - phase ODS column with ultraviolet absorbance detector ( Model G1314A). The UV detector was set with an absorbance wavelength of 270 nm. The HPLC mobile phase consisted of 50% acetonitrile, 40% water, and 10% methanol with a flow rate of 1. 2 ml ? min ~ . The limit of detection of propofol was 0.05 (xM. The inject sample volumn was 50 jxl.ResultsThere was an inverse linear correlation between incubation time and the logarithm of the concentration of propofol remaining in the incubation mixture, which is indicative of first - order kinetics. The interindividual variation in the rate constants for propofol metabolism by human liver microsomes in the twelve individuals was no more than 3 -fold (median, 3.9 nmol ? min"1 ? mg"1 protein;range, 1.96 ~ 5. 97 nmol ? min ~ ? mg ~ protein) . In the control team, incubations performed without NADPH - regenerating system decreased the rate constant by 79%.In the specific CYP2B6 - inhibition study, the mean rate constants for propofol metabolism by liver microsomes preincubated with orphenadrine and thioTEPA were 2. 44 nmol ? min"1 ? mg"1 protein (2. 16, 2. 71) and 2. 23 nmol ? min"1 ? mg"1 protein (2.03, 2.43) , respectively, which were significantly lower than the control values without inhibitors (P <0.01). The propofol hydroxylase activities were inhibited about 37.5% and 42.7% by orphenadrineand thioTEPA, repectively.ConclusionCYP2B6 is predominantly involved in the oxidation of propofol by human liver microsomes. It may be one of explantation of the individual variation of pharmacodynamics of propofol in clinic.