Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes Kinetics and sensitivity to cytochrome P450 2D inhibitors Annalise Di Marco 1 , Dan Yao 2 and Ralph Laufer 1 1 Department of Pharmacology, Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Merck Sharp and Dohme Research Laboratories, Rome, Italy; 2 Labeled Compound Synthesis, Department of Drug Metabolism, Merck Research Laboratories, Rahway, NJ, USA Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhi- bition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP sub- strates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhib- ited saturable kinetics in rat microsomes and hepatocytes, with apparent K m and V max values of 2.1 vs. 2.8 l M and 0.74 nmolÆmin )1 per mg microsomal protein vs. 0.11 nmolÆmin )1 per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC 50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2–3-fold lower IC 50 in hepatocytes than in microsomes. The cell- associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracel- lular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport. Keywords: CYP2D; cytochrome P450; hepatocytes; micro- somes. The pharmacokinetic and toxicokinetic properties of phar- maceuticals depend in great part on their biotransformation by drug-metabolizing enzymes. The main drug-metaboli- zing system in mammals is cytochrome P450 (CYP), a family of microsomal isozymes present predominantly in the liver. Multiple CYPs catalyze the oxidation of chemicals of endogenous and exogenous origin, including drugs, steroids, prostanoids, eicosanoids, fatty acids, and environ- mental toxins [1]. If a drug that is metabolized by a particular CYP isozyme is coadministered with an inhibitor of that same enzyme, changes in its pharmacokinetics can occur, which can give rise to adverse effects [2–5]. It is therefore important to predict and prevent the occurrence of clearance changes caused by metabolic inhibition. During the drug discovery process, it has become routine practice in the pharmaceutical industry to assess CYP inhibition potential of drug candidates in order to exclude potent inhibitors from further development [6–8]. The extent of metabolic interaction between two drugs depends on their relative K m and K i values and concentra- tions at the site of metabolism [3]. In recent years, substantial progress has been made in the development of in vitro screening methods to quantitatively determine kinetic parameters of CYP inhibition. Using either recom- binant CYP proteins or liver microsomes, together with appropriate probe substrates, these assays can be used to measure K i values for competitive CYP inhibitors [7,9,10]. It is not entirely clear, however, whether these systems accurately and quantitatively reflect drug interactions that occur in vivo. One possible drawback of recombinant enzymes is that inhibitory potency may depend on inter- actions with multiple CYPs present in the microsomal, but not recombinant, systems. The intracellular concentration of drugs (substrates and inhibitors) that is available for interacting with a particular CYP may also depend on Correspondence to R. Laufer, IRBM P. Angeletti, Via Pontina km 30,600, 00040 Pomezia (Roma), Italy. Fax: + 39 0691093 654, Tel.: + 39 0691093 440, E-mail: ralph_laufer@merck.com Abbreviation: CYP, cytochrome P450. (Received 5 June 2003, revised 11 July 2003, accepted 22 July 2003) Eur. J. Biochem. 270, 3768–3777 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03763.x processes lacking in microsomes, such as drug transport across the plasma membrane, metabolism by cytosolic enzymes, and binding to intracellular proteins. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. Isolated hepatocytes have been used extensively to study drug metabolism, cytotoxicity, and induction of drug-metaboli- zing enzymes [11–15]. However, there are few reports of CYP inhibition studies using this system (see for example [13,16–18]), probably because of the technical challenge posed by the lower specific activity of CYP in cultured cells relative to microsomal preparations. The objective of this study was to compare the inhibitory potencies of CYP inhibitors in microsomes and hepatocytes. We developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Materials and methods Materials [O-methyl- 14 C]Dextromethorphan (61 mCiÆmmol )1 ) was synthesized at Merck Research Laboratories, Rahway, NJ,USA.[ 3 H]Quinine and [ 3 H]quinidine were purchased from American Radiolabeled Chemicals. [ 3 H]Taurocholic acid was from Perkin–Elmer Life Sciences, and [ 14 C]for- maldehyde and [ 14 C]formic acid were from Amersham Biosciences. Cell culture media were purchased from Gibco- BRL, and chemicals from Sigma. 96-well Oasis TM HLB extraction plates and vacuum mannifold were purchased from Waters. Preparation of rat liver microsomes Liver microsomes were prepared from male Sprague– Dawley rats. Livers were homogenized in 1.15% (w/v) KCl, and the homogenate was centrifuged at 9000 g for 30 min. The S-9 supernatant was centrifuged at 130 000 g for 1 h. The microsomal pellet was washed, resuspended in 0.15 M Tris/HCl, pH 7.4, at a protein concentration of 10 mgÆmL )1 and kept at )80 °C. Isolation of rat hepatocytes All animal care and experimental procedures were in accordance with national and company guidelines. Male Sprague–Dawley rats weighing 250 g were subjected to terminal anaesthesia using sodium pentobarbital. Rat hepatocytes were isolated by a two-step collagenase per- fusion method [19]. Cells were frozen in L15 medium containing 10% fetal calf serum and 15% dimethyl sulfoxide following the protocol described by Guyomard et al.[20] and kept in liquid nitrogen until use. After quick thawing at 37 °C, cells were loaded on L15 medium containing 0.75 M glucose [21] and centrifuged for 1 min at 300 g.Viable hepatocytes were separated by centrifugation over 30% Percoll solution for 3 min at 350 g. Cell viability was determined by Trypan Blue exclusion before freezing and after thawing and was consistently greater than 90%. The cells were resuspended in William’s Medium E containing GlutaMAX TM (Ala-Glu), 5 lgÆmL )1 insulin, 1 l M dexa- methasone, and penicillin/streptomycin, and seeded on collagen-precoated 24-well culture plates at a density of 100 000 cells per well. Cultures were maintained at 37 °Cin a humidified atmosphere of 5% CO 2 . Four hours after plating, the medium was changed as described below. Separation of [ O-methyl - 14 C]dextromethorphan from CYP2D-mediated demethylation products The CYP2D assay described in this study is based on a modification of procedures described previously for deter- mining the activity of various CYP isozymes, including CYP2D6, in hepatic microsomes [22,23]. CYP-mediated demethylation of substrates which have the leaving methyl group radiolabelled with 14 C, yields [ 14 C]formaldehyde as product, which can be isolated using reversed-phase (C8) extraction cartridges [24]. We adapted this method to 96-well format, and modified the solid-phase matrix using Oasis extraction plates. Solid-phase extraction was per- formed using a vacuum mannifold according to the instructions of the manufacturer. When the radiolabelled substrate [O-methyl- 14 C]dextromethorphan, dissolved in either microsomal assay buffer or cell incubation medium, was applied to 96-well Oasis plates, over 99.7% of radioactivity was retained on the extraction plate, and could be recovered by elution with methanol. In contrast, [ 14 C]formaldehyde and [ 14 C]formic acid, the products of CYP-mediated oxidation of [O-methyl- 14 C]dextromethor- phan, were quantitatively recovered in the combined void volume and aqueous washing of Oasis extraction plates. Microsomal CYP2D assays Microsomal incubations were performed in 96-well conical plates (Corning). They contained, in a final volume of 100 lL, 0.1 M potassium phosphate buffer, pH 7.4, 1 l M [O-methyl- 14 C]dextromethorphan (% 15 000 d.p.m. per assay), rat liver microsomes (3 lg), and NADPH- regenerating system (1 m M NADP, 5 m M glucose-6- phosphate, 3 m M MgCl 2 ,4UÆmL )1 glucose-6-phosphate dehydrogenase). After preincubation for 10 min at 37 °Cin the presence or absence of test compounds, reactions were started by the addition of the NADPH-regenerating system. After 15 min, reactions were stopped by the addition of 10 lL1 M HCl. Plates were centrifuged at 1100 g for 5 min using a microplate rotor, and supernatants loaded on 30-mg 96-well Waters Oasis extraction plates. The flow-through was collected and plates were washed twice with 200 lL water. Aliquots of the combined aqueous eluates were counted in a Packard TopCount scintillation counter using 24-well scintillation plates. Product formation was totally dependent on the presence of NADPH and was linear with time for up to 20 min, and with microsomal protein concentrationupto1mgÆmL )1 (data not shown). Hepatocyte CYP2D assays CYP2D assays in hepatocytes were performed at 37 °Cin a humidified atmosphere of 5% CO 2 in 24-well culture Ó FEBS 2003 CYP2D-mediated drug interactions (Eur. J. Biochem. 270) 3769 plates containing 100 000 cells per well, unless indicated otherwise. Four hours after plating, cells were incubated in 500 lL cell incubation medium {hepatocyte culture medium (HCM [25]), supplemented with ITS + (Colla- borative Research, Bedford, MA, USA) and 10 m M sodium formate, which suppresses the formation of 14 CO 2 from [ 14 C]formate in rat hepatocytes [26]}. Plates were preincubated for 10 min with CYP inhibitors or vehicle [0.5% (v/v) dimethyl sulfoxide], before addition of 1 l M [O-methyl- 14 C]dextromethorphan (% 80 000 d.p.m. per assay). Reactions were stopped after 15 min by addition of 50 lL1 M HCl, and cell lysates were centrifuged in a tabletop centrifuge at high speed for 10 min. The supernatants were loaded on 30-mg 96-well Waters Oasis extraction plates and processed as described above for the microsomal assays, except that extraction plates were washed three times with 250 lLwater. Uptake of drugs into rat hepatocytes Uptake of radiolabelled quinine, quinidine, and taurocholic acid into rat hepatocytes was determined at 37 °Cin250 lL per well of a solution containing 116 m M NaCl, 5.3 m M KCl, 1.1 m M KH 2 PO 4 ,0.8m M MgSO 4 ,1.8m M CaCl 2 , 10 m M glucose, and 10 m M Hepes, pH 7.4. Some experi- ments were performed in sodium-free buffer containing choline chloride instead of NaCl. Incubations with 5 l M [ 3 H]quinine or [ 3 H]quinidine were carried out for 1, 2, 3, 5, and 10 min in the presence or absence of 2 l M carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Incubations with 1 l M [ 3 H]taurocholic acid were performed for 20, 40, 60, 120, and 300 s in the presence or absence of extracellular Na + . Plates were then washed 3 times with 1 mL ice-cold buffer, cells were lysed with 0.1 M NaOH, and radioactivity was determined by scintillation counting. Cell-associated radioactivity for [ 3 H]quinine and [ 3 H]quinidine reached steady-state levels after 10 min (data not shown). Results were corrected for radioactivity associated with cells at time zero, and expressed as cell/medium concentration ratio (C/M) at steady state, using an estimated intracellular volume of 4 lLÆ(10 6 cells) )1 [27]. [ 3 H]Taurocholate uptake was linear for up to 2 min (data not shown). Uptake clearance was calculated by dividing the initial uptake velocity by the substrate concentration. Determination of drug binding to hepatic proteins For the determination of the liver tissue binding of [ 3 H]quinine and [ 3 H]quinidine, rat liver was homogenized in 0.1 M potassium phosphate buffer and dialyzed against the same buffer for 12 h at 4 °C to remove coenzymes. The compounds were mixed with tissue homogenates (10, 20 and 30%, w/v) or rat liver microsomes (0.03 mgÆmL )1 ) at concentrations of 1 or 10 l M , and incubated at 37 °Cfor 30 min. Reaction tubes were then centrifuged in a tabletop centrifuge for 20 min at high speed, and the supernatants were loaded on Centrifree ultrafiltration devices (Millipore) to separate the unbound fractions. Non-specific adsorption of [ 3 H]quinine to the filters was prevented by precoating using unlabelled quinine (1 m M ). The fraction not bound to liver proteins (f u ) was calculated according to the following equation [28]: f u ¼ C f =½C f þð100=n  C b Þ ð1Þ where C f is unbound drug in ultrafiltrate, C b is bound drug, and n is the percentage of liver homogenate. Biochemical assays Protein was determined by the Bradford assay (Bio-Rad) using BSA as standard. Lactate dehydrogenase activity was determined in hepatocyte cell suspensions before plating, and in monolayers 4 h after plating, using a colorimetric method (Cytotoxicity detection kit; Roche Diagnostics). ATP content of cell monolayers was determined after cell extraction with 1.7% (w/v) trichloroacetic acid using luciferase/luciferin reagent (Sigma) and luminescent pro- duct detection. The intracellular concentration of ATP was calculated considering an intracellular volume of 4 lLÆ(10 6 cells) )1 [27]. Statistical methods Curve fitting was performed by nonlinear regression according to the Levenberg-Marquardt algorithm, using KALEIDAGRAPH TM 3.52 (Synergy Software, Reading, PA, USA). Statistical significance was assessed using a two- tailed Student’s t test. Results Viability, metabolic and transport activities of cryopreserved rat hepatocytes To assessthe metabolic state ofhepatocytes used inthis study, we determined cell-attachment efficiency, ATP content, and Na + -dependent taurocholate transport, a typical differenti- ated hepatocyte function mediated by the sodium taurocho- late cotransporting polypeptide (NTCP) [29]. The efficiency of cell attachment, determined by measuring cellular lactate dehydrogenase activities before and after plating, was 70 ± 6% (n ¼ 2). Intracellular ATP concentrations were 2.3 ± 0.4 m M (mean ± SEM, n ¼ 3), which is in close agreement with previously reported values (2.4 m M [30]). Cells transported [ 14 C]taurocholate with an uptake clearance of 24 ± 2 lLÆmin )1 per mg cellular protein (n ¼ 2). In the absence of extracellular Na + , uptake clearance was reduced sevenfold. These values are similar to those previously reported for Na + –taurocholate cotransport in rat hepato- cytes (V max /K m ¼ 17.5 lLÆmin )1 Æmg )1 [29]). Dextromethorphan O-demethylation in rat hepatocytes and microsomes When [O-methyl- 14 C]dextromethorphan was incubated with rat hepatocytes, radiolabelled reaction product(s) were produced in a time-dependent and cell-concentration- dependent manner (Fig. 1). The reaction products were not retained by Oasis TM polymeric reversed-phase sorbent, similarly to standard [ 14 C]formaldehyde and [ 14 C]formate (and unlike the substrate [O-methyl- 14 C]dextromethor- phan). Metabolite formation from [O-methyl- 14 C]dextro- methorphan in rat hepatocytes increased with substrate concentration in a saturable manner (Fig. 2A). The reaction 3770 A. Di Marco et al.(Eur. J. Biochem. 270) Ó FEBS 2003 rate as a function of substrate concentration was fitted to the Hill equation: v ¼ V max  S n S n 50 þ S n ð2Þ where v and V max are the observed and maximal rates of metabolism, S 50 is the substrate concentration at half V max , and n is the Hill coefficient. The values obtained were S 50 ¼ 2.80 ± 0.01 l M , V max ¼ 0.11 ± 0.01 nmolÆmin )1 per mg cellular protein, and n ¼ 0.82 ± 0.01. An Eadie–Hofstee plot of these data was monotonous, with slight deviation from linearity (Fig. 2A, inset). For comparison, we also determined the kinetics of dextromethorphan O-demethylation in rat microsomes (Fig. 2B). Fitted kinetic constants were S 50 ¼ 2.10 ± 0.01 l M , V max ¼ 0.74 ± 0.01 nmolÆmin )1 per mg micro- somal protein, and n ¼ 0.88 ± 0.01. Also in this case, the Eadie–Hofstee plot of these data was monotonous, with slight deviation from linearity (Fig. 2B, inset). We next examined the effect of isoform-specific CYP inhibitors on dextromethorphan O-demethylation. As shown in Fig. 3, the reaction in rat hepatocytes was inhibited by quinine, which is a known inhibitor of rat CYP2D [31–33], but not by a-naphthoflavone (inhibitor of Fig. 1. Time-dependent and cell-concentration-dependent demethylation of [O-methyl- 14 C]dextromethorphan in rat hepatocytes. Substrate was incubated with 100 000 cells (circles) or 300 000 cells (squares) and product formation was determined at the indicated times. Results are mean ± deviation from duplicate experiments. Fig. 2. Kinetics of [O-methyl- 14 C]dextromethorphan demethylation in rat hepatocytes (A) and rat liver microsomes (B). Data were fitted to the Hill equation as described in Results. Each point is the mean ± deviation from duplicate experiments. Insets: Eadie–Hofstee plots of the data. Fig. 3. Effect of CYP inhibitors on [O-methyl- 14 C]dextromethorphan demethylase activity in rat hepatocytes. Results are expressed as per- centage enzymatic activity relative to that of the vehicle control. Inhibitors used were: 1 l M a-naphthoflavone (ANF), 10 l M sulfa- phenazole (SPZ), 10 l M quinine (QUIN), and 10 l M troleandomycin (TAO). Results are mean ± deviation from duplicate experiments. Ó FEBS 2003 CYP2D-mediated drug interactions (Eur. J. Biochem. 270) 3771 rat CYP1A1/2 [34]), sulfaphenazole (rat CYP2C11 [35]), and troleandomycin (rat CYP3A [36]). The selected inhi- bitor concentrations were based on the above literature references. Effect of quinine and quinidine on dextromethorphan O-demethylation A characteristic feature of rat CYP2D enzymes is that, in contrast with the human enzyme, they are inhibited by quinine more potently than by quinidine [17,31,37]. As shown in Fig. 4A, quinine was a more potent inhibitor than quinidine of [O-methyl- 14 C]dextromethorphan O-demethy- lation in rat hepatocytes. Inhibition curves were fitted to a four-parameter logistic model: Y ¼ 1 1 þðx=IC 50 Þ n ð3Þ where Y is the fraction of enzyme activity relative to no-inhibitor controls, X is the concentration of inhi- bitor, IC 50 the concentration for half-maximal inhibi- tion, and n the slope factor. The results of the fitting are summarized in Table 1. Quinine and quinidine had IC 50 values of 0.9 and 4.7 l M , respectively. The slope factors were 0.57 and 0.64, respectively, suggesting interaction with more than one enzyme or binding site. Inhibition curves were also fitted to a two-site inhibition model (Fig. 4): Y ¼ A 1 þðx=IC 50À1 Þ þ 1 À A 1 þðx=IC 50À2 Þ ð4Þ where Y is the fraction of enzyme activity relative to no-inhibitor controls, A is the fraction of enzymes with IC 50-1 , and 1 ) A the fraction of enzymes with IC 50-2 .As shown in Table 1, correlation coefficients (r) for the nonlinear regression curve fits using the two-enzyme model were slightly higher than those for the logistic fits. Approximately 40% of the enzymatic activity in rat hepatocytes was inhibited by quinine and quinidine with high affinity (IC 50-1 0.06 and 0.51 l M , respectively), and Fig. 4. Effect of quinine and quinidine on [O-methyl- 14 C]dextromethorphan demethylase activity. (A) Rat hepatocytes; (B) rat liver microsomes. Enzymatic activity was determined in the presence of quinine (circles) or quinidine (squares), and results were expressed as percentage of control activity in the absence of inhibitor. Data represent mean ± SEM from three to five separate experiments. Curves were fitted to a two-site inhibition model as described in Results. Table 1. Kinetic parameters for inhibition of [O-methyl- 14 C]dextromethorphan demethylation by quinine and quinidine in rat liver microsomes and rat hepatocytes. Inhibition data (Fig. 4) were fitted to a four-parameter logistic model or a two-site inhibition model as described in the text. n,slope factor; A, fraction of high-affinity sites; IC 50 , concentration that produces 50% inhibition; IC 50-1 ,IC 50 for high-affinity sites; IC 50-2 ,IC 50 for low- affinity sites; r, correlation coefficient of the nonlinear regression curve fit. Results are parameter values (± SEM), as calculated by the curve-fitting software. Inhibitor Enzyme source Fit type 4-parameter logistic 2 enzymes r IC 50 nrAIC 50-1 IC 50-2 Quinine Hepatocytes 0.9912 0.9 ± 0.16 0.57 ± 0.05 0.9981 0.40 ± 0.04 0.06 ± 0.02 5.0 ± 1.0 Quinidine Hepatocytes 0.9956 4.7 ± 0.51 0.64 ± 0.04 0.9980 0.41 ± 0.07 0.51 ± 0.18 19.0 ± 4.7 Quinine Microsomes 0.9954 1.7 ± 0.21 0.53 ± 0.03 0.9986 0.45 ± 0.03 0.13 ± 0.03 12.6 ± 2.1 Quinidine Microsomes 0.9980 15.0 ± 0.9 0.72 ± 0.03 0.9976 0.45 ± 0.14 3.3 ± 1.5 48.9 ± 20.4 3772 A. Di Marco et al.(Eur. J. Biochem. 270) Ó FEBS 2003 about 60% with lower affinity (IC 50-2 5 and 19 l M , respectively). Also in rat liver microsomes quinine had a lower IC 50 than quinidine, and both compounds exhi- bited slope factors smaller than unity (Fig. 4B and Table 1). The IC 50 values for quinine and quinidine in rat liver microsomes were twofold and threefold higher than in hepatocytes, but this difference was statistically significant only for quinidine (P < 0.01). When data were fitted to a two-site inhibition model, relative ratios of high-affinity and low-affinity binding sites in rat liver microsomes were similar to those in hepatocytes. Also in this case, correlation coefficients for the two-enzyme model curve fits were slightly better than those for the logistic fits (Table 1). IC 50 values of quinine and quinidine for both high-affinity and low-affinity binding sites in rat liver microsomes were between twofold and threefold higher than the corresponding values in rat hepatocytes (Table 1), but these differences were not statistically significant (P > 0.05). Even though the differences in IC 50 values between microsomes and hepatocytes were small and for the most part not significant, there appeared to be a trend towards lower IC 50 values in intact hepatocytes. This may be due to differences between the concentrations of free drug available for enzyme inhibition in the two systems. To test this hypothesis, we measured the total concentration of quinine and quinidine in rat hepatocytes, as well as their free (non- protein-bound) fractions in both microsomes and hepatic tissue. Both quinine and quinidine accumulated in rat hepatocytes and reached steady-state concentrations that were 64-fold and 75-fold higher than their extracellular concentrations, respectively (Table 2). More than 50% of the accumulation of quinine and quinidine was inhibited by ATP depletion using 2 l M carbonyl cyanide p-trifluoro- methoxyphenylhydrazone, suggesting that it was mediated by active drug transport into the hepatocytes (data not shown). Cell-associated drugs can bind to tissue proteins, and only the free fraction may be available for interaction with microsomal CYP2D. Radiolabelled quinine and quinidine bound extensively to proteins in rat liver homo- genates, with free fractions between 0.03 and 0.06 (Table 2). In contrast, free fractions of both compounds were close to unity in the rat liver microsome incubation system, consis- tent with the very low concentration of microsomal protein (30 lgÆmL )1 ) used in the assay (data not shown). Thus, free concentrations of quinine and quinidine inside rat hepato- cytes may not equal those in the extracellular medium, and IC 50 should be corrected by a factor that takes into account cellular accumulation and protein binding. The ratio between the intracellular concentration of free drug ([I] cell, free ) and that of total drug added to the hepatocyte culture medium ([I] medium ) is given by: ½I cell;free ½I medium ¼ f u;cell xC=M ð5Þ However, f u,cell , the free fraction of drug within the hepatocyte cytoplasm, cannot be measured experiment- ally. If this value were similar to the free fraction in liver homogenate (i.e. f u,cell ¼ f u,tissue ), then free drug concentrations inside hepatocytes would be 2–3-fold higher than that added to the culture medium. Inhibition of CYP2D activity We next investigated the effects of several drugs on [O-methyl- 14 C]dextromethorphan O-demethylation in rat microsomes and hepatocytes. As depicted in Fig. 5A and summarized in Table 3, pyrilamine, propafenone, terfena- dine, verapamil and ketoconazole inhibited the reaction in intact hepatocytes with IC 50 values in the micromolar range. Slope factors (determined by fitting the data to a four- parameter logistic equation) ranged from 0.5 (pyrilamine) to 1.1 (propafenone). In rat liver microsomes, IC 50 values for pyrilamine, propafenone and verapamil were 2–3-fold higher than in hepatocytes, ketoconazole had comparable IC 50 values, and terfenadine a slightly lower IC 50 than in hepatocytes (Fig. 5B and Table 3). This difference between microsomes and hepatocytes was statistically significant only for verapamil (P < 0.01). Slope factors for all compounds were very similar to those obtained in hepatocytes (Table 3). It was not possible to resolve the curves for these compounds into two distinct components using a two-site inhibition model (data not shown). Discussion Even though hepatic microsomes represent the most widely used in vitro system for the study of potential drug interactions, it has been reported that concentrative uptake of some CYP inhibitors into the liver can cause drug interactions in vivo that are more pronounced than those predicted by inhibitory potency in a microsomal system [5,28,38,39]. Freshly isolated and cryopreserved hepatocytes are an important experimental tool for the evaluation of drug metabolism, hepatotoxicity and induction of drug- metabolizing enzymes [11–15]. The purpose of the present study was to use this system to determine the inhibitory potencies of a series of CYP2D inhibitors and to compare the results with those obtained in liver microsomes. To this end, we developed a sensitive assay method suitable for rapidly assessing the potential of chemical compounds to inhibit CYP2D enzymes in both systems. CYP-catalyzed demethylation of substrates which had the leaving methyl group radiolabelled with 14 C, yielding [ 14 C]formaldehyde as product, has been previously used Table 2. Accumulation in rat hepatocytes and hepatic protein binding of quinine and quinidine. Accumulation of quinine and quinidine (5 l M ) in rat hepatocytes is expressed as the steady state ratio (C/M) between cell associated and extracellular drug concentrations. Binding to rat hepatic proteins was determined at two drug concentrations, 1 and 10 l M , and results were expressed as fraction of free drug, f u . To calculate f u · C/M, the f u for the two drug concentrations was averaged. Compound C/M f u f u · C/M1 l M 10 l M Quinine 64 0.037 0.056 3.0 Quinidine 75 0.028 0.031 2.2 Ó FEBS 2003 CYP2D-mediated drug interactions (Eur. J. Biochem. 270) 3773 to assay the activity of various CYP isoforms in liver microsomes [23,24,40,41]. A related method is used to determine CYP3A4 activity in human subjects in vivo.The so-called erythromycin breath test measures the disposition in the breath of 14 CO 2 formed from further oxidation of [ 14 C]formaldehyde, the product of CYP3A4-catalyzed N-demethylation of [N-methyl- 14 C]erythromycin [42]. Even though formation of 14 C-labelled formaldehyde, formate and CO 2 from CYP-mediated N-demethylation of amino- pyrine in isolated hepatocytes was described over 25 years ago [43], aminopyrine is not suitable as a substrate for assaying the activity of specific CYPs, because its N-demethylation is mediated by multiple CYP isozymes [44]. In contrast, [O-methyl- 14 C]dextromethorphan can be used to specifically determine CYP2D activities in rat hepatocytes. The present experiments using isoform-select- ive CYP inhibitors show that the demethylation of [O-methyl- 14 C]dextromethorphan in rat hepatocytes was mediated by enzymes of the CYP2D family. The reaction was inhibited by the CYP2D inhibitor quinine but not by specific inhibitors of rat CYP1A1/2, CYP2C11 and CYP3A. In addition, the more potent inhibition by quinine relative to quinidine is a characteristic feature of rat CYP2D enzymes. Except for CYP1A and CYP2B, for which cell-based fluorimetric assays have been reported [45,46], non-HPLC assays suitable for determining CYP inhibition in intact hepatocytes have not been described to date. The present method can be used for relatively high throughput screening of CYP2D inhibitors, because of the possibility of carrying out reactions using as few as 100 000 cells attached to the wells of 24-well culture plates and processing the reaction products in 96-well solid-phase extraction plates. Compared with conventional methods for measuring CYP activity in intact hepatocytes, which entail preparation of microsomes and HPLC separation of reaction products, the new CYP2D assay procedure described here has the advantage of greatly improved simplicity, speed and sensitivity. The latter factor is particularly important for assessing CYP inhibition, because competitive inhibition assays should be performed using substrate concentrations that are not much higher than the K m . The concentration of dextromethorphan Table 3. Kinetic parameters for inhibition of [O-methyl- 14 C]dextromethorphan demethylation by CYP2D inhibitors in rat liver microsomes and rat hepatocytes. Inhibition data (Fig. 5) were fitted to a four-parameter logistic model. n,slopefactor;IC 50 , concentration that produces 50% inhibition. Results are parameter values (± SEM), as calculated by the curve-fitting software. Inhibitor IC 50 (l M ) n Hepatocytes Microsomes Hepatocytes Microsomes Pyrilamine 1.3 ± 0.3 2.6 ± 0.4 0.49 ± 0.05 0.49 ± 0.03 Propafenone 1.9 ± 0.2 4.4 ± 1.2 1.12 ± 0.10 0.95 ± 0.21 Verapamil 3.6 ± 0.3 11.1 ± 0.9 0.78 ± 0.04 0.95 ± 0.06 Ketoconazole 0.7 ± 0.1 0.6 ± 0.1 0.69 ± 0.05 0.83 ± 0.09 Terfenadine 3.8 ± 0.6 2.3 ± 0.5 0.81 ± 0.09 0.79 ± 0.11 Fig. 5. Effect of CYP2D inhibitors on [O-methyl- 14 C]dextromethorphan demethylase activity. (A) Rat hepatocytes; (B) rat liver microsomes. Enzymatic activity was determined in the presence of pyrilamine (d), propafenone (j),verapamil(m), ketoconazole (s), or terfenadine (h). Results were expressed as percentage of control activity in the absence of inhibitor. Data represent mean ± SEM from three to four experiments. Curves were fitted to a four-parameter logistic inhibition model as described in Results. 3774 A. Di Marco et al.(Eur. J. Biochem. 270) Ó FEBS 2003 (1 l M ) used in the present hepatocyte assay fulfils this requirement. Even though we validated the assay for rat CYP2D only, the general method of measuring the radiolabelled products of CYP-mediated dealkylation reac- tions should be easily adaptable to other CYP isoforms and hepatocytes of other species, including humans, using appropriate probe substrates, such as [O-ethyl- 14 C]phenace- tin [40], [O-methyl- 14 C]naproxen [24], [N-methyl- 14 C]eryth- romycin [23,41] and [N-methyl- 14 C]diazepam [24]. Dextromethorphan O-demethylation in rat liver micro- somes and hepatocytes has previously been studied using nonradiometric methods, and it was reported that this reaction is mediated by multiple enzyme systems. In rat liver microsomes, O-demethylation of unlabelled dextromethor- phan is mediated by high-affinity and low-affinity enzyme systems, with apparent K m values of 1–3 l M and 43–158 l M , respectively [18,37]. In rat hepatocytes, O-demethylation of unlabelled dextromethorphan was reported to display sigmoidal kinetics, with an S 50 value of 13 l M and a Hill coefficient of 2.4. The rat CYP2D family comprises six members, denominated CYP2D1-5 and CYP2D18 [47,48]. Dextromethorphan O-demethylation is catalyzed by cDNA-expressed rat CYP2D2 but not CYP2D1 [49]. Indirect evidence suggests that other CYP2D isoforms can catalyze this reaction. Dextromethorphan interacts with multiple CYP2D isoforms, as it was shown to inhibit the metabolism of 7-methoxy-4-(aminomethyl)coumarin by CYP2D1, CYP2D2, CYP2D3 and CYP2D4 with IC 50 values of 264, 5.6, 18.6, and 136 l M , respectively [50]. These results suggest that the high-affinity component of the reaction is mediated by CYP2D2 with a possible contribu- tion of CYP2D3, while the low-affinity component may be mediated by CYP2D1 and/or CYP2D4. In this study, [O-methyl- 14 C]dextromethorphan demethylation in rat liver microsomes and hepatocytes exhibited apparent K m values of 2.1 and 2.8 l M ,andV max values of 0.74 nmolÆmin )1 per mg microsomal protein vs. 0.11 nmolÆmin )1 per mg cellular protein, respectively, with Hill coefficients close to unity and Eadie–Hofstee plots that deviated only slightly from linearity. The apparent microsomal K m and V max values are comparable to those previously reported [18,37] for the high-affinity component of dextromethorphan O-demethy- lation in rat liver microsomes (K m ¼ 1.1–2.5 l M ; V max ¼ 0.42–0.85 nmolÆmin )1 Æmg )1 ).Itislikelythatmark- edly biphasic kinetics were not observed in the present experiments because the [O-methyl- 14 C]dextromethorphan concentrations used did not exceed 25 l M (microsomes) and 100 l M (hepatocytes), which is close to the apparent K m of the low-affinity component reported in rat liver microsomes. In contrast, kinetic studies with unlabelled dextromethor- phan were performed using substrate concentrations up to 500–600 l M [18,37]. Thus, under the present conditions, [O-methyl- 14 C]dextromethorphan O-demethylation was pri- marily mediated by CYP2D isoforms with high substrate affinity. The hypothesis that [O-methyl- 14 C]dextromethorphan O-demethylation in rat liver microsomes and hepatocytes is mediated by high-affinity CYP2D isoforms including CYP2D2 and possibly CYP2D3 is supported by the inhibition profile of quinine and quinidine. In both micro- somes and hepatocytes, these compounds inhibited the reaction in a biphasic manner, suggesting interaction with at least two enzyme systems. Curve fitting to a logistic model or to a two-site model produced excellent fits with correlation coefficients close to unity. We preferred to analyze the data according to the two-site model for the following reasons. Slope factors for the logistic fits were significantly smaller than 1, suggesting interaction with multiple enzyme systems, or allosteric behaviour. Individual rat CYP2D isoforms display Michaelis–Menten kinetics with the substrate 7-methoxy-4-(aminomethyl)coumarin [50], and to our knowledge, allosteric kinetics has not been reported for other ligands. On the other hand, it is well known that dextromethorphan can interact with multiple CYP2D isoforms [50], and the observed biphasic inhibition kinetics most likely reflect this property. The higher-affinity component for quinine displayed IC 50 values of 0.13 l M (microsomes) and 0.06 l M (hepatocytes), whereas the lower-affinity component had IC 50 values of 12.6 l M (microsomes) and 5.0 l M (hepatocytes). These values are close to the reported IC 50 values of quinine for inhibition of CYP2D2-mediated and CYP2D3-mediated dealkylation of 7-methoxy-4-(aminomethyl)coumarin, 0.09 and 12.0 l M , respectively [50]. The high-affinity and low-affinity compo- nents of quinidine inhibition of [O-methyl- 14 C]dextrometh- orphan O-demethylation displayed IC 50 values of 3.3 l M (microsomes) and 0.51 l M (hepatocytes), vs. 48.9 l M (microsomes) and 19.0 l M (hepatocytes), respectively. Again, these values are similar to the reported IC 50 values for inhibition of CYP2D2-mediated and CYP2D3-medi- ated dealkylation of 7-methoxy-4-(aminomethyl)coumarin, 2.8 and 26.9 l M , respectively. The effect of several additional drugs on [O-methyl- 14 C]dextromethorphan demethylase activity was assessed in both rat liver microsomes and hepatocytes. Pyrilamine [51], propafenone [37] and terfenadine [52] are known to be potent rat and/or human CYP2D inhibitors, whereas verapamil was reported to be a weak (IC 50 60 l M ) inhibitor of human CYP2D6 [53]. Even though ketocon- azole has not been reported to inhibit CYP2D isoforms, it is known to be a nonspecific inhibitor of various rat CYPs, including CYP1A, CYP2C, CYP2E and CYP3A [54]. Even though some of these compounds inhibited the reaction with slope factors significantly lower than 1, suggesting inter- action with more than one enzyme, the relative contribu- tions of distinct enzymatic systems could not be resolved by curve fitting. Additional studies, using cDNA-expressed rat CYP2Ds will be needed to determine the interactions of these compounds with specific isoforms. In general, there was reasonable agreement between IC 50 values determined in microsomes vs. hepatocytes. Some inhibitors, including quinine and quinidine, displayed 2–3-fold lower IC 50 values in hepatocytes than in microsomes, but this difference was statistically significant only for quinidine and verapamil. One possible explanation for this trend is that some of the drugs accumulate to a moderate extent in hepatocytes. We found that cell-associated concentrations of quinine and quinidine were about 70-fold higher than extracellular concentrations. However, part of the cell-associated drug is probably bound to intracellular proteins and may thus not be available for interaction with CYP2D. Both compounds were found to bind extensively to proteins in homogenates from rat liver. Even though intracellular protein binding may be different from that observed in tissue homogenates, Ó FEBS 2003 CYP2D-mediated drug interactions (Eur. J. Biochem. 270) 3775 it is interesting to note that the cell/medium concentration ratio, corrected for tissue protein binding, is between 2 and 3, i.e. strikingly similar to some of the observed ratios between IC 50 values in microsomes vs. hepatocytes. In conclusion, for the CYP inhibitors investigated in this study, only slight differences in inhibitory potencies were observed between intact hepatocytes and liver microsomes. Even though some drugs can reach high intrahepatic concentra- tions [5], this effect may be partially offset by binding to intracellular proteins. 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Pharmacol. 45, 107–114. Ó FEBS 2003 CYP2D-mediated drug interactions (Eur. J. Biochem. 270) 3777 . Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes Kinetics and sensitivity to cytochrome P450 2D inhibitors Annalise Di Marco 1 , Dan Yao 2 and Ralph. Accumulation in rat hepatocytes and hepatic protein binding of quinine and quinidine. Accumulation of quinine and quinidine (5 l M ) in rat hepatocytes is expressed as the steady state ratio (C/M). lLwater. Uptake of drugs into rat hepatocytes Uptake of radiolabelled quinine, quinidine, and taurocholic acid into rat hepatocytes was determined at 37 °Cin250 lL per well of a solution containing 116