Short Communication Rat Cytochrome P450 2C11 in Liver Microsomes Involved in Oxidation of Anesthetic Agent Propofol and Deactivated by Prior Treatment with Propofol

Propofol (2,6-diisopropylphenol) is a widely-used anesthetic agent attributable to its rapid biotransformation. Liver microsomal cytochrome P450 (P450) isoforms involved in the biotransformation of propofol in rats and the effects of propofol in vivo on P450 levels in rats were investigated. Of six cDNA-expressed rat P450 isoforms tested, CYP2B1 and CYP2C11 had high catalytic activities from 5 M and 20 M propofol concentrations, respectively. Rates of propofol metabolism, at a substrate concentration of 20 M based on the reported human blood concentration, were decreased by intraperitoneal treatment of propofol with male rats, in contrast to a strong induction by phenobarbital. Single intravenously administered propofol (10 mg/kg) caused the decrease of total P450 and CYP2C contents and activities of testosterone 16 -hydroxylation and propofol metabolism in liver microsomes from male rats. The suppressive effects were caused by administered propofol (10 mg/kg) twice every 4 h on CYP2B activities such as testosterone 16 -hydroxylation or pentoxyresorufin O-depentylation, in addition to the strong suppression of CYP2C function by the single propofol treatment. These results suggest that CYP2C11, presumably deactivated by propofol, has an important role in propofol metabolism in rat liver microsomes. Repeated administration of propofol could markedly decrease the biotransformation of propofol via P450 deactivation. Propofol (2,6-diisopropylphenol) is administered as a bolus for the induction of anesthesia and as an infusion for maintenance of anesthesia or for sedation (Bryson et al., 1995). One of the major advantages of this drug over other injectable anesthetic agents is the rapid and complete recovery that occurs even after relatively prolonged intravenous infusions (Mandsager et al., 1995). This property is attributable to rapid and extensive biotransformation of the parent compound, primarily by the liver. The relative contribution of individual metabolic pathways has been found to vary among animal species and humans (Simons et al., 1991; Sneyd et al., 1994). Although there are several reports of propofol pharmacokinetics or drug interactions in humans (Guitton et al., 1998; McKillop et al., 1998; Hamaoka et al., 1999; Court et al., 2001; Oda et al., 2001; Tanaka et al., 2004), the precise roles of cytochrome P450 (P450) isoforms in the propofol disposition are still unknown. Moreover, there is no report of the effects of propofol on P450 induction or deactivation as a determinant factor of the pharmacodynamic and/or pharmacokinetics of propofol. There has been reported a significant association between receiving a long-term and high-dose propofol infusion and developing progressive myocardial failure (Bray, 1998). In the present study, the roles of rat P450 isoforms involved in propofol metabolism were investigated with recombinant rat P450 isoforms and rat liver microsomes, mainly at a substrate concentration of 20 M, based on the human plasma concentration (McKillop et al., 1998). The effects of P450 deactivation by propofol on the activities of propofol oxidation in rat liver microsomes were also investigated. Materials and Methods Chemicals. Propofol (Diprivan injectable emulsion) was purchased from AstraZeneca (Osaka, Japan). As an in vitro substrate source, propofol (207854-8) was also obtained from Sigma-Aldrich (St. Louis, MO). The other chemicals and reagents used were obtained in the highest grade available commercially. Enzyme Preparations. Male and female Wistar rats (7 weeks old) were obtained from Japan SLC (Hamamatsu, Japan). Male rats were treated intraperitoneally with typical P450 inducers including -naphthoflavone (50 mg/ kg, CYP1A), phenobarbital (80 mg/kg, CYP2B and CYP3A), and dexamethasone (50 mg/kg, CYP3A) daily for 3 days (Yamazaki et al., 2001a,b). Some rats were treated with propofol (10 mg/kg) via intraperitoneal administration three times in a half-day. In separate experiments, propofol (10 mg/kg) was administered intravenously to male and female rats once or twice every 4 h. This interval (of 4 h) was based on 10 times as long as the reported half-life of propofol ( 20 min at 10 mg/kg/h) (Hamaoka et al., 1999). Liver microsomes from these rats were prepared 4 h after the final treatment of propofol. These studies were approved by the Committee on the Care and Use of Laboratory Animals for Showa Pharmaceutical University. Recombinant rat P450 isoforms expressed in microsomes of insect cells (Supersomes) were obtained from BD Gentest (Woburn, MA). Catalytic activities by those P450 enzymes are provided in the data sheets by the manufacturer. Enzyme Assays. Disappearance rates of propofol were determined according to the high-performance liquid chromatography method described previously (Dowrie et al., 1996) with minor modifications. In brief, the typical incubation mixture of a total volume of 0.25 ml contained microsomal protein (0.025 mg) or recombinant P450 (8 pmol), 20 M propofol, and an NADPHgenerating system in 0.1 M potassium phosphate buffer (pH 7.4) unless This work was supported in part by the Ministry of Education, Science, Sports and Culture of Japan, The Research Foundation for Pharmaceutical Sciences, and Japan Research Foundation for Clinical Pharmacology. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.106.011627. ABBREVIATIONS: P450, cytochrome P450; ANOVA, analysis of variance. 0090-9556/06/3411-1803–1805$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 11 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 11627/3146384 DMD 34:1803–1805, 2006 Printed in U.S.A. 1803 at A PE T Jornals on A ril 6, 2017 dm d.aspurnals.org D ow nladed from specified. The principal propofol concentration of 20 M was chosen because of the blood concentration in humans (McKillop et al., 1998). Propofol protein binding ( 90%) was not considered in our in vitro work. Incubations were carried out for 10 min at 37°C. The reaction was terminated by adding 4.0 ml of pentane containing 0.1 g of 2-tert-butyl-6-methylphenol/ml. After vortex mixing, the tubes were centrifuged at 1200g for 10 min. The organic phase was transferred to a clean tube and evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was dissolved in 0.1 ml of mobile phase. The liquid chromatography system consisted of a pump and an electrochemical detector (L-ECD-6A; Shimadzu, Kyoto, Japan) using an analytical C18 reversed-phase column (150 4.6 mm). The mobile phase was acetonitrile/ 0.028 M phosphate buffer (pH 2.8) (60:40 v/v), at a flow rate of 1.5 ml/min. Activities of ethoxyresorufin O-deethylation (CYP1A), pentoxyresorufin O-depentylation (CYP2B), and testosterone 16 -, 16 -, and 6 -hydroxylation (CYP2B, CYP2C, and CYP3A, respectively) were determined as described previously (Yamazaki et al., 2001a,b). The concentrations of total P450 (Omura and Sato, 1964), CYP2C (Shimada et al., 1994), and NADPH-P450 reductase (EC 1.6.2.4) (Parikh et al., 1997) were determined as described previously. Statistical Analysis. Statistical analysis was carried out using the computer program Instat (GraphPad Software, San Diego, CA). One-way ANOVA with Dunnett’s post hoc test was performed for analysis of repeated intravenous administrations of propofol (p 0.05). Results and Discussion Six recombinant rat P450 isoforms were used to determine which P450 isoforms were active in catalyzing the propofol metabolism (Table 1). Based on the reported plasma concentrations of propofol (20 M) after treatment in humans (McKillop et al., 1998), substrate concentrations of 5 and 20 M were used in this study. CYP2C11 and CYP2B1 showed high activities of propofol metabolism at both substrate concentrations. However, CYP1A2, CYP2D1, CYP2E1, and CYP3A2 showed low activities under the present conditions. Among typical P450 inducers administered intraperitoneally to male rats, phenobarbital caused the most induction of propofol metabolism when 20 M propofol was used as a substrate (Fig. 1A). In contrast, significantly decreased propofol metabolism by propofol treatment itself (10 mg/kg) was observed. To examine in detail whether propofol decreased propofol biotransformation, rats were treated intravenously once or twice with propofol (10 mg/kg every 4 h). Rates of propofol metabolism in male rats were also decreased by an intravenous propofol treatment in liver microsomes from rats (Fig. 1B). This suppression of the oxidative metabolism was dependent on the repeated intravenous propofol treatments. In contrast, liver microsomes from female rats had low and unaffected propofol metabolism in the propofol treatments. These results suggested that constitutive male-specific CYP2C11 and inducible CYP2B isoforms had important roles for propofol metabolism in rat liver microsomes. Because we used limited rat recombinant P450 isoforms (Table 1), it should be mentioned that other major CYP2C (but not female-specific CYP2C12) or CYP2B isoforms might be expected to contribute to propofol metabolism in rats. Accordingly, inhibitory effects of propofol on CYP2B and CYP2C activities, but not CYP3A, were seen when propofol and testosterone were coincubated with rat liver microsomes (Table 2). Intravenously administered propofol significantly decreased total P450 and CYP2C contents in male rat liver microsomes (Fig. 2A). Propofol also decreased testosterone 16 -hydroxylation activities (CYP2C) in male rat liver microsomes (Fig. 2B), consistent with the immunochemical results. Repeatedly administered propofol (10 mg/kg twice every 4 h) also significantly decreased testosterone 16 -hydroxylation and pentoxyresorufin O-depentylation activities (CYP2B) in male rat liver microsomes. A similar decrease of ethoxyresorufin O-deethylation activities was seen. However, there were no changes with regard to the NADPH-P450 reductase levels or CYP3A-mediated testosterone 6 -hydroxylation activities in rat liver TABLE 2 Inhibitory effects of propofol on testosterone 16 -, 16 -, and 6 -hydroxylation (CYP2C, CYP2B, and CYP3A, respectively) in liver microsomes from untreated male rats Testosterone (50 M) was incubated at 37°C for 10 min with male rat liver microsomes in the absence or presence of propofol (20 M). Data are means S.D. from three untreated male rats. Hydroxylation Testosterone Hydroxylation (%) Without Propofol With Propofol