Human cytochrome P450 mono-oxygenase system is suppressed by propofol

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1 British Journal of Anaesthesia 1995; 74: Human cytochrome P450 mono-oxygenase system is suppressed by propofol T. L. CHEN, T. H. UENG, S. H. CHEN, P. H. LEE, S. Z. FAN AND C. C. LIU Summary We have studied the effect of propofol on the cytochrome P450-dependent mono-oxygenase system in human liver microsomes by assaying mono-oxygenase activities toward specific cytochrome P450 isoform test substrates, aniline, 7- ethoxycoumarin, benzphetamine and benzo(a) pyrene. Propofol inhibited benzo(a)pyrene hydroxylation to a greater extent than the oxidative metabolism of the other test substrates, even at 0.05 mmol litre" 1. The degrees of inhibition of benzphetamine A/-demethylation and 7-ethoxycoumarin O-de-ethylation were similar, while aniline hydroxylation was least affected by propofol. Spectral analysis showed that propofol competed with carbon monoxide for binding to the haem moiety of haemoprotein in the P450 enzyme. The variable inhibition observed may be caused by the differential binding of propofol to P450 isoforms. Propofol mmol litre" 1 exhibited a concentration-dependent inhibitory effect on human cytochrome P450 2E1, 2B1 and 1A1. These inhibitory actions of propofol on human liver microsomal enzymes in vitro suggest that potential drug interactions may exist between propofol and other drugs administered clinically. (Br. J. Anaesth. 1995; 74: ) Key words Anaesthetics i.v., propofol. Enzymes, cytochrome P450. Liver, microsomes. Propofol is used widely for induction and maintenance of anaesthesia [1, 2]; its high lipophilicity, rapid onset of action and predictable duration of action make it suitable for a wide range of surgical procedures [3,4]. The principal metabolites of propofol are inactive glucuronide conjugates of alkyl phenol and the corresponding quinol, 2,6- diisopropyl-l,4-quinol, which is produced by the cytochrome P450-dependent mono-oxygenase system [5]. Co-administration of other agents could theoretically affect the pharmacokinetic properties of propofol. Continuous infusion of propofol for sedation in the intensive care unit [6] might impair the intrinsic clearance of other P450 substrates, such as propranolol [7]. In addition, the enzymatic degradation of alfentanil and sufentanil was inhibited by propofol in liver microsomes [8]. The purpose of this study was to investigate the interaction between propofol and the human cytochrome P450 mono-oxygenase system. We have examined the effects of propofol on mono-oxygenase activities toward aniline, benzphetamine and benzo(a)pyrene, which are marker substrates for cytochrome P450 isoforms 2E1, 2B1 and 1A1 [9], respectively. The rationale for choosing these P450 substrates was that cytochrome P450 2E1 has been reported to be the major enzyme metabolizing sevoflurane and isoflurane [10]. Cytochrome P450 2B1 activity, induced in phenobarbitone-treated rats, could be inhibited by propofol [11]. Cytochrome P450 1A1 is also involved in induction and metabolism of mammalian carcinogens [12]. The interaction of propofol with specific isoforms of human liver cytochrome P450 remains to be determined. Materials and methods SPECIMENS AND SUBJECTS After obtaining informed consent and local Ethics Committee approval, human liver samples were obtained from the Department of Surgery, National Taiwan University Hospital. Two specimens were from kidney donors and three from normal livers in patients with hepatocellular carcinoma. Exclusion criteria included liver function abnormality. All liver tissues were frozen immediately in liquid nitrogen and stored at 80 C. The time between removal and freezing did not exceed 10 min. Liver tissue was homogenized in ice cold 1.15% KC1 (w/v) with a Teflon homogenizer. The homogenate was centrifuged at 9000 g for 20 min at 4 C. The supernatant was centrifuged further at for 60 min at 4 C. The precipitate was rehomogenized in 1.15% KC1 and centrifuged at for 60 min to obtain the microsomal pellet [13]. This pellet was resuspended in 0.1 mol litre" 1 of potassium phos- TA-LIANG CHEN*, MD, SHOU-ZEN FAN, MD, CHIEN-CHIANG LIU, MD, PHD, (Department of Anesthesiology); PO-HUANG LEE, MD, PHD (Department of Surgery); National Taiwan University Hospital; TZUU-HUEI UENG, PHD, SUNG-HO CHEN, BS (Graduate Institute of Toxicology, College of Medicine); National Taiwan University, Taipei, Taiwan, Republic of China. Accepted for publication: October 19, * Address for correspondence: Department of Anesthesiology, National Taiwan University Hospital, 7 Chung-Shan S. Road, Taipei, Taiwan, 100, Republic of China.

2 Suppression of cytochrome P450 by propofol 559 phate buffer at ph 7.4 for assay of mono-oxygenase activities. Microsomal protein was assayed by the method of Lowry and colleagues [14] using bovine serum albumin (BSA) as standard. MONO-OXYGENASE ASSAYS Pure propofol (ICI Pharmaceutica, Zeneca, UK) was diluted in methanol and mixed with the microsomal suspension before being added to the assay system. An equal volume of methanol was added to each assay as a solvent control. Benzo(a)- pyrene hydroxylation activity for aryl hydrocarbon hydroxylase was determined by measuring the formation of phenolic metabolites according to the method of Nebert and Gelboin [12]. The microsomal incubation was performed in the dark room with 2 ml of microsomal protein (2 mg ml" 1 ) in phosphate buffer containing (mmol litre" 1 ): NADPH 1.05, MgCl 2 2.9, KH 2 PO 4 -K 2 HPO (ph 7.4) and BSA 0.2 mg ml" 1. Propofol was introduced into the reaction vials at various concentrations and was gently mixed with microsomal suspensions before the substrate, benzo(a)pyrene 1 mmol litre" 1 was added. Incubation was performed at 37 C for 10 min and terminated by adding acetone. The fluorescent metabolite was extracted sequentially by n-hexane and NaOH and measured by spectrofluorimetry (Hitachi F-3010). 7-Ethoxycoumarin O- de-ethylation activity was assayed by measuring the formation of thefluorescentmetabolite of 7-hydroxycoumarin using the method of Greenlee and Poland [15] by incubating the mixture of microsomes and propofol with MgCl 2 5 mmol litre" 1, NADPH 0.5 mmol litre" 1, NADH 0.5 mmol litre" 1, BSA 2 mg and 7-ethoxycoumarin 0.5 mmol litre" 1 in KH 2 PO 4 - K 2 HPO 4 buffer 60 mmol litre" 1 (ph 7.4). The reaction was terminated with trichloroacetic acid and the fluorescent metabolite was extracted by chloroform and NaOH. Aniline hydroxylation activity was determined by measuring the formation of p-aminophenol from aniline according to the method of Imai, Ito and Sato [16]. The incubation system contained NADP 0.1 mmol litre" 1, glucose-6-phosphate 1 mmol litre" 1, glucose-6-phosphate dehydrogenase 2.8 u. ml" 1 in Tris buffer mmol litre" 1 aniline hydrochloride 0.5 mmol litre" 1. After incubation, phenol solution (phenol/naoh 0.2 mmol litre" 1 = 1/40) was added to produce the metabolite which was measured spectrophotometrically (Hitachi 557) at 630 run. Benzphetamine demethylation activity was determined by measuring the formation of formaldehyde using Nash's reagent [17] after incubation with NADP 0.4 mmol litre" 1, semicarbazide HC1 8 mmol llitre" 1, glucose-6-phosphate 4 mmol litre" 1, glucose-6-phosphate dehydrogenase 2 u. and benzphetamine 2 mmol litre" 1 in KH 2 PO 4 -K 2 HPO 4 buffer 8 mmol litre" 1 (ph 7.4). Because methanol interferes with formaldehyde formation, propofol was diluted directly into microsomes in the assay of benzphetamine iv-demethylation. Microsomal P450 content was determined by the carbon monoxide difference spectrum, as described by Omura and Sato [18]. In order to determine the effect of propofol on the binding of carbon monoxide to the cytochrome P450 haem moiety, propofol 1 mmol litre" 1, NADPH, or both, were added to the liver microsomes and the mixture incubated at 37 C for 10 min before assaying the P450 content by perfusion of microsomal suspension with carbon monoxide. Statistical analysis was performed by Student's t test for comparison of the propofol and control groups, and two-way ANOVA for various propofol concentrations with the control group. P < 0.05 was considered statistically significant. Results An initial study was carried out to determine the effect of propofol on mono-oxygenase activities using microsomes prepared from a single liver specimen. Microsomes in this study were prepared from three different regions of this normal liver and duplicated in each assay. Figure 1 shows that the addition of propofol mmol litre" 1 to the mono-oxygenase assay system caused a concentration-dependent decrease in aniline hydroxylase and 7- ethoxycoumarin O-de-ethylase activities. Propofol caused similar concentration-dependent decreases in mono-oxygenase activities toward other test substrates, benzo(a)pyrene and benzphetamine (fig. 1). Benzo(a)pyrene hydroxylase activity appeared to be most sensitive to propofol. The decreases in enzyme activity were apparent and approached maximal levels at propofol mmol litre" 1. Microsomes were prepared from five liver specimens and mono-oxygenases activities of these microsomes were determined in the presence of Propofol (mmol litre" 1 ) Figure 1 Concentration-dependent inhibition of mono-oxygenase activities towards the test substrates aniline hydroxylase (A) 5 7- ethoxycoumarin O-de-ethylase (A)> benzo(a)pyrene hydroxylase (O) and benzphetamine demethylase ( ) by propofol. Microsomes were prepared from three different regions of the liver sample and duplicated in each enzyme assay. Data are mean (SEM) percentage of control. Significant differences: *compared with control; fcompared with propofol 0.5 mmol litre"

3 560 British Journal of Anaesthesia Table 1 Catalytic activity data (mean (SEM)) of mono-oxygenases in five human liver microsomal preparations preincubated with propofol. Significant differences {P < 0.05): *compared with control; t com P are d with propofol 0.5 mmol litre" 1 Propofol (mmol litre ') Assay Control Aniline hydroxylation (nmol p-aminophenol/min/mg protein) 7-Ethoxycoumarin O-de-ethylation (nmol 7-hydroxycoumarin/min/mg protein Benzphetamine N-demethylation (nmol formaldehyde/min/mg protein) Benzo(a)pyrene hydroxylation (pmol 3-hydroxybenzo(a)pyrene/min/mg protein) (0.023) (0.019)* 0.280(0.012) 0.159(0.019)* 1.462(0.121) 0.847(0.026)* 26.03(2.14) 7.54(1.68)* (0.017)* 0.139(0.024)* (0.035)*+ 4.68(1.02)*+ Table 2 Mean (SEM) data for cytochrome P450 content of five human liver microsomal preparations. Propofol 1 mmol litre" 1, NADPH, or both, were added and incubated at 37 C for 10 min before assay of P450 content using the carbon monoxide difference spectrum method of Omura and Sato [18]. *P < 0.05 compared with control Incubation Control + Propofol + Propofol, NADPH Cytochrome P450 content (nmol/mg protein) 0.270(0.012) (0.015)* 0.210(0.016)* propofol 0.5 and 1.0 mmol litre" 1. Table 1 shows that addition of propofol 0.5 mmol litre" 1 caused 30%, 43%, 42% and 71% decreases in aniline hydroxylase, 7-ethoxycoumarin O-de-ethylase, benzphetamine iv-demethylase and benzo(a)pyrene hydroxylase activities, respectively (P < 0.05). Addition of propofol 1.0 mmol litre" 1 caused 35%, 50%, 67% and 82% decreases in mono-oxygenases activities toward aniline, 7-ethoxycoumarin, benzphetamine and benzo(a)pyrene, respectively. Propofol 1.0 mmol litre" 1 tended to yield a greater inhibition than 0.5 mmol litre" 1 (table 1). Preincubation of liver microsomes with propofol 1.0 mmol litre" 1 at 37 C for 10 min before assay was found to cause an 18% decrease in P450 content (P < 0.05). A 22 % decrease in P450 content was observed with preincubation in the presence of propofol 1.0 mmol litre" 1 nd NADPH (P < 0.05) (table 2). Discussion Studies of propofol interactions with other drugs are of clinical interest because other drugs such as phenobarbitone, benzodiazepines, antibiotics or opioids [8] act as substrates for cytochrome P450 and their pharmacodynamic actions may be modified clinically. Marked species difference was observed in a comparison of metabolism and intrinsic clearance of propofol in different animal models [19] and these species differences prompted us to study the interaction of propofol and drug metabolizing enzymes using human liver microsomes. We assessed the effects of propofol in concentrations of mmol litre" 1 in human liver; the lower concentrations of mmol litre" 1 are of clinical relevance, but the pattern of inhibition by propofol in these concentrations was similar to 1.0 mmol litre" 1. The present report is the first to show that propofol inhibited P450 and a variety of monooxygenases in human liver microsomes. Baker, Chadam and Ronnergerg [11] reported that propofol inhibited aniline hydroxylase and benzphetamine N- demethylase activities in rat liver microsomes, but the effects on other substrates were not reported. Comparison of the inhibitory effects of propofol on mono-oxygenase activities in rats and humans revealed marked species variations. In rats, although propofol inhibited metabolism of benzphetamine which was catalysed mainly by cytochrome P450 2B1, the inhibition seemed to be more obvious in phenobarbitone-treated rats [11]. In human liver, propofol showed a greater degree of inhibition of benzophetamine iv-demethylation than in untreated rats. The inhibitory effect of propofol on cytochrome P450 2B1 of human liver was also demonstrated by a similar degree of inhibition of 7-ethoxycoumarin O-de-ethylase activity as benzphetamine N- demethylase. This species variation indicates that cytochrome P450 2B1 in human liver is more sensitive to propofol than untreated rats. In contrast with P450 2B1, the present studies of aniline hydroxylation showed that propofol was a less potent inhibitor of human P450 2E1. In concentrations of mmol litre" 1, propofol exhibited a similar degree of inhibition (17-21 %) of the aniline hydroxylase activity of human liver. In the untreated or isoniazid-treated animal, propofol was less potent at inhibiting the isozyme of P450 2E1 [11], which was similar to the results obtained from the human liver in our study. We found that propofol inhibited the oxidative metabolism of benzo(a)pyrene and 7-ethoxycoumarin, in addition to benzphetamine and aniline. Interestingly, benzo(a)pyrene was more sensitive to inhibition than the other substrates; even with propofol 0.05 mmol litre" 1, hydroxylase was inhibited by 20% (fig. 1). With propofol concentrations of mmol litre" 1, benzo(a)pyrene hydroxylase activity was inhibited by 80%; in contrast, the degrees of inhibition of benzphetamine and aniline metabolism were about 50% and 30%, respectively (table 1). One possible explanation for this variation is that the P450 isoforms responsible for metabolism

4 Suppression of cytochrome P450 by propofol 561 of the test substrates are inhibited differentially by propofol. Aniline, benzphetamine and benzo(a) pyrene are selective substrates of P450 2E1, 2B1 and 1A1, respectively [9]. The differential effect on mono-oxygenase activities suggests that human P450 1A1 is more sensitive than 2B1 and 2E1 to the inhibitory effect of propofol. The present data also indicate that human P450 2B1 is more sensitive than 2E1 to propofol inhibition, in agreement with the previous results of Baker, Chadam and Ronnergerg [11] who reported that propofol caused a greater degree of inhibition of mono-oxygenase activities in rats pretreated with phenobarbitone than isoniazid, which are potent inducers of P450 2B1 and 2E1, respectively [9]. However, it should be noted that clinically P450 2B1 is not the major form in human, unlike rats [20], and induction by phenobarbitone cannot be produced in normal human livers. Multiple P450 isoforms are present in human liver. The present study has shown that mono-oxygenase activities toward the marker substrates for P450 isoforms 1A1, 2B1 and 2E1 were present in the liver (table 1). A series of biochemical studies such as immunoblotting, immunoinhibition and chemical inhibitor studies are required to precisely determine the proportions of the P450 isoforms in the liver microsomes. In the preliminary studies, we found that P450 proteins immunorelated to 1A1, 2B1 and 2E1 were detectable in liver microsomes, the contents of the P450 isoforms were variable (data not shown) and the degrees of variation of P450 contents were similar to those observed with the mono-oxygenase activities associated with the P450 isoforms (table 1). Kharasch and Thummel [10] also reported variation in the content of P450 2E1, the primary P450 isoform responsible for metabolism of sevoflurane, isoflurane and methoxyflurane, in human liver microsomes. Variation of human P450 content is to be expected as the enzyme system is subject to induction and inhibition by many physiological and environmental factors including age, sex, disease, diet and medication [20]. Our data showed that P450 and dependent mono-oxygenase activities were susceptible to the inhibitory effect of propofol. Variation in the contents of P450 2E1 and other isoforms may have a significant effect on the pharmacokinetics of anaesthetics and other drugs. It is difficult to identify which factors were responsible for inhibition of cytochrome P450 by propofol because of the multiplicity of the isozymes. However, we found that propofol inhibited the mono-oxygenase activities of P450 2B1 and 1A1 in a different pattern from P450 2E1. It has been hypothesized that the inhibition results from direct interaction of propofol with cytochrome P450 active sites [11]. Propofol has a structural similarity with the butylated hydroxytoluene which was a cytochrome P450 inhibitor [21,22]. Many large molecules, such as steroid hormones, also can bind and be metabolized by the phenobarbitone-induced P450 isozymes [23, 24]. Therefore, the inhibitory effect of propofol on the isozymes of 2B1 and 1A1 might be because of the spatial affinity of propofol with the active sites of P450. On the contrary, cytochrome P450 2E1 has a major affinity for small molecules such as ethanol and acetone [25]. Propofol might be too large to effectively bind to the active sites of this isozyme and therefore the inhibitory effect of propofol was less for the P450 2E1 isoform. Therefore, the interaction between substrates and cytochrome P450 binding sites appears crucial. Yet direct evidence for this interaction is lacking. 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