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Evaluation of 3-O-Methylfluorescein as a Selective Fluorometric Substrate for CYP2C19 in Human Liver Microsomes

DMPK and Pharmaceutics Department, and Drug and Biomaterial Research and Development, Genzyme Corporation, Waltham, Massachusetts (S.S., J.S., T.J.O., H.L.); and Department of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Health Sciences, Boston, Massachusetts (D.A.W.)

(Received December 21, 2006; accepted February 27, 2007)

	Abstract

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Cytochrome P450 (P450) fluorometric high-throughput inhibition assays have been widely used for drug-drug interaction screening particularly at the preclinical drug discovery stages. Many fluorometric substrates have been investigated for their selectivity, but most are found to be catalyzed by multiple P450 isozymes, limiting their utility. In this study, 3-O-methylfluorescein (OMF) was examined as a selective fluorescence substrate for CYP2C19 in human liver microsomes (HLMs). The kinetic studies of OMF O-demethylation in HLMs using a liquid chromatography/mass spectrometry method exhibited two-enzyme kinetics with apparent K_m and V_max values of 1.14 ± 0.90 µM and 11.3 ± 4.6 pmol/mg/min, respectively, for the high affinity component(s) and 57.0 ± 6.4 µM and 258 ± 6 pmol/mg/min, respectively, for the low affinity component(s). Studies utilizing cDNA-expressed individual P450 isoforms and P450-selective chemical inhibitors showed that OMF O-demethylation to fluorescein was selective for CYP2C19 at substrate concentrations 1 µM. At substrate concentrations 10 µM, other P450 isozymes were found to catalyze OMF O-demethylation. In HLMs, analysis of the two-enzyme kinetics in the presence of P450 isozyme-selective chemical inhibitors (ticlopidine for CYP2C19, sulfaphenazole for CYP2C9, and furafylline for CYP1A2) indicated that CYP2C19 was the high affinity component and CYP2C9 was the low affinity component. Based on these findings, a fluorometric assay was developed using 1 µM OMF and 2 µM sulfaphenazole for probing CYP2C19-mediated inhibition in HLMs. The IC₅₀ data of 13 substrates obtained from the fluorometric assay developed in this study correlated well with that reported in the literature using nonfluorescence assays.

The substrate typically used in a fluorometric inhibition assay for CYP2C19 is 3-cyano-7-ethoxycoumarin. However, 3-cyano-7-ethoxycoumarin also exhibits catalytic activity toward CYP1A1, CYP1A2, CYP1B1, CYP2B6, and CYP2E1 (Stresser et al., 2002; Ghosal et al., 2003) and is therefore unsuitable as a selective substrate for CYP2C19. The purpose of this study was to characterize the enzyme kinetics and the specificity of O-demethylation reaction of 3-O-methylfluorescein (OMF) in both cDNA-expressed P450 isozyme systems and HLMs. Based on these findings, a fluorometric assay using OMF was developed as a selective substrate for probing CYP2C19-mediated DDIs.

	Materials and Methods

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Materials. OMF, (+)-N-3-benzyl-nirvanol, and cDNA-expressed human P450s CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 were purchased from BD Biosciences (Woburn, MA). Pooled HLMs (n = 20 donors of mixed gender) at 20 mg/ml protein concentration were purchased from Xenotech LLC (Lenexa, KS). Acetonitrile and water (high-performance liquid chromatography grade) were obtained from EMD Chemicals Inc. (Gibbstown, NJ). Magnesium chloride hexahydrate was obtained from JT Baker (Phillipsburg, NJ). Amitriptyline, felbamate, fluvoxamine maleate, fluorescein (FL) sodium salt, fluoxetine, furafylline, glucose 6-phosphate, NADP⁺, glucose-6-phosphate dehydrogenase, imipramine, ketoconazole, labetalol hydrochloride, lansoprazole, omeprazole, progesterone, quinidine, sulfaphenazole, ticlopidine, tranylcypromine, and all the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Linearity and Enzyme Kinetics of OMF O-Demethylation by CYP2C19 in HLMs. The HLM incubations were carried out in a total volume of 200 µl. The reaction mixtures containing NADPH-regenerating system (1.3 mM NADP⁺, 3.3 mM glucose 6-phosphate, 0.4 U/ml glucose-6-phosphate dehydrogenase, and 3.3 mM magnesium chloride) in 50 mM potassium phosphate buffer (pH 7.4) were preincubated at 37°C for 10 min. In the enzyme kinetic experiments, 2 µl of OMF in acetonitrile stock solutions (50-12,000 µM) was added into the reaction mixtures. The reactions were initiated by the addition of HLMs (0.5 mg/ml final protein concentration) followed by incubation at 37°C. Before running the enzyme kinetics for the formation of FL (OMF O-demethylation metabolite), the linearity of the FL formation was determined by incubating 0.5 µM OMF with varying amounts of microsomal protein (0.3-2.35 mg/ml). The time dependence of FL formation was also investigated by incubating 0.5 µM OMF with 0.5 mg/ml microsomal protein followed by sample drawing at 5, 10, 15, 30, 45, and 60 min. The optimal protein concentration and incubation time were chosen to be 0.5 mg/ml and 30 min, respectively, for the enzyme kinetic experiments. The reactions were terminated by the addition of 600 µl of ice-cold 50% v/v acetonitrile/water containing 0.4 µM labetalol as an internal standard for liquid chromatography/mass spectrometry (LC/MS) analysis. The reaction mixtures were then centrifuged in Heraeus Biofuge Pico (Kendro Laboratory Products, Newtown, CT) at 14,443g for 5 min, and the resultant supernatant (20 µl) was analyzed by LC/MS analysis. The kinetic parameters were calculated from untransformed data by nonlinear regression using Prism software (GraphPad Software Inc., San Diego, CA).

Incubation with cDNA-Expressed Human P450 Isozymes. CYP2C19 selectivity for the OMF substrate was shown by performing incubations with 11 cDNA-expressed human P450 isozymes, namely, CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5. The reaction mixtures were prepared as described previously in the HLM incubation assay except that the OMF concentrations used were 1 and 40 µM, and the HLMs were substituted with each of the 11 cDNA-expressed P450 isozymes. For the P450 isozyme whose activity was derived from the same marker substrate used by the two suppliers (cDNA-expressed P450 isozymes from BD Biosciences versus HLMs from Xenotech LLC), the concentration of cDNA-expressed P450 isozyme in the incubations was chosen to have activity approximate to that of HLM incubations (Table 1). All the other isozyme concentrations were chosen to reflect the relative abundance of P450s present in human liver (Williams, 2002) (Table 2).

Inhibition of P450 Isozymes by Selective Chemical Inhibitors. The involvement of CYP2C9, CYP2C19, and CYP1A2 in OMF O-demethylation was evaluated using CYP2C19-, CYP2C9-, and CYP1A2-selective inhibitors. The incubations were performed by preincubating HLMs with the NADPH-regenerating system in 50 mM potassium phosphate buffer (pH 7.4) and 2 µM sulfaphenazole (CYP2C9 competitive selective inhibitor), 10 µM furafylline (CYP1A2 mechanism-based selective inhibitor), or 5 µM(+)-N-3-benzyl-nirvanol (CYP2C19 competitive selective inhibitor) at 37°C for 10 min. To remove the activity of CYP2C19, 100 µM ticlopidine was preincubated with the HLMs and NADPH-regenerating system for 30 min at 37°C. All the reactions were initiated by the addition of OMF in acetonitrile stock solutions to give final concentrations ranging from 0.5 to 120 µM. In addition to the concentration ranges of OMF, incubations of OMF at 1 and 10 µMinthe presence or absence of CYP2C19-, CYP2C9-, and CYP1A2-selective inhibitors, either alone or in combination, were conducted to further identify the enzymes involved in the O-demethylation reaction. The reactions were terminated as described previously in the HLM incubation experiment and analyzed by LC/MS.

LC/MS Analysis. The LC/MS system comprised an Agilent 1100 series binary pump (Agilent Technologies Inc., Santa Clara, CA), a CTC Leap PAL autosampler (Leap Technologies, Carrboro, NC), and either an API3000 or API4000 triple quadrupole mass spectrometer (Applied Biosystems/Sciex, Foster City, CA) equipped with a Valco valve (VICI Valco Instruments, Houston, TX) and an electrospray ionization interface operated in positive ion detection mode. The LC/MS system was controlled by Analyst software version 1.4.1 (Applied Biosystems/Sciex). The high-performance liquid chromatography column used was a YMC ODS-AQ (3 µm, 120 Å, 4.6 x 50 mm, Waters Corporation, Milford, MA). The linear gradient elution was performed using the following mobile phase systems at a flow rate of 0.5 ml/min; solvent A contained 5 mM ammonium acetate buffer (pH 4.5), and solvent B was acetonitrile. The gradient program initiated at 30% B ramping to 90% B over 4 min and then held for 2 min before returning to the initial starting conditions. The LC eluent for the first 1.5 min was diverted to waste. The samples were analyzed for FL formation by LC/MS. The mass spectrometer was operated in Q1 multiple ion mode at 332.90 m/z for FL and at 328.90 m/z for labetalol (internal standard). The standard curve was prepared by spiking 10 µl of FL solution at concentrations ranging from 0.39 to 100 µM into 190 µl of blank incubation mixture without the NADPH-regenerating system components and then immediately quenching the reaction by addition of 600 µl of ice-cold 50% v/v acetonitrile/water containing 0.4 µM labetalol as internal standard. The standard solutions were then treated as described previously in the HLM incubation assay and analyzed by LC/MS.

IC₅₀ Determination by Fluorometric Assay. The FL fluorescence detection sensitivity was evaluated by spiking FL into the blank incubation mixture at a final concentration range of 0.32 nM to 2 µM. The blank incubation mixture contained preincubated microsomal protein with NADPH-regenerating system at 37°C for 30 min. The incubation was terminated by the addition of 75 µl of ice-cold 2 N sodium hydroxide in water, followed by the addition of 2 µl of OMF in acetonitrile stock solution to give 1 µM final concentration. Fluorescent signals from the blank incubation mixture without FL were then acquired and subtracted from the signal of the blank incubation mixture spiked with FL. The IC₅₀ fluorometric assay was conducted in black Costar 96-well plates (Corning Incorporated, Corning, NY) at a total incubation volume of 200 µl. The 96-well reaction plate layout is shown in Fig. 1. The reaction mixtures in the wells from columns 3 to 12 consisted of NADPH-regenerating system in 50 mM potassium phosphate buffer (pH 7.4), 0.5 mg/ml microsomal protein, 2 µM sulfaphenazole, and 13 substrates (0.75 µl from acetonitrile stock solution) serially diluted 3-fold with the final concentration range of 0.045 to 100 µM for ketoconazole and quinidine, 0.041 to 90 µM for tranylcypromine, 0.036 to 80 µM for omeprazole, 0.022 to 50 µM for probenecid, 0.013 to 30 µM for lansoprazole, 0.0022 to 5 µM for fluvoxamine, 0.17 to 125 µM for felbamate, 0.069 to 150 µM for imipramine, 0.006 to 12.5 µM for amitriptyline, (+)-3-N-benzyl-nirvanol, and progesterone, and 0.0011 to 2.5 µM for fluoxetine. The blank incubations in wells A1 through D1 contained 0.5 mg/ml microsomal protein in 50 mM potassium phosphate buffer (pH 7.4), NADPH-regenerating system, CYP2C9-selective chemical inhibitor (i.e., 2 µM sulfaphenazole), and CYP2C19 chemical selective inhibitor (i.e., 100 µM ticlopidine). The control incubations without inhibitors in wells E1 through F1 contained only NADPH-regenerating system and 0.5 mg/ml microsomal protein in 50 mM potassium phosphate buffer (pH 7.4). The control incubations without OMF in wells G1 through H1 contained the same components as the control incubations without inhibitors except for the addition of OMF substrate after reaction termination. The control incubations in the presence of 2 µM sulfaphenazole were conducted in wells A2 through H2. The 96-well plate was preincubated at 37°C for 30 min. The reactions were initiated by the addition of 1 µM OMF and incubated at 37°C for 30 min. For the IC₅₀ experiments with imipramine, amitriptyline, (+)-3-N-benzyl-nirvanol, progesterone, fluvoxamine, and fluoxetine, the substrates were preincubated along with ticlopidine. The rest of the substrates were added with OMF to initiate the reaction. The reactions were terminated by the addition of 75 µl of ice-cold 2 N sodium hydroxide in water. The fluorescence signals were measured at 30 min after reaction termination to obtain sufficient signal above background. The fluorescence signal was measured using CytoFluor 4000 TC Fluorescence Multi-Well Plate Reader (Applied Biosystems) with excitation filter at 485 nm (bandwidth 20 nm) and emission filter at 530 nm (bandwidth 25 nm). The IC₅₀ values were calculated as described by eq. 1:

(1)

View larger version (29K): [in this window] [in a new window]   FIG. 1. Ninety-six-well plate layout for IC50 determination toward CYP2C19 using OMF as substrate in HLM incubations.

View larger version (14K): [in this window] [in a new window]   FIG. 2. Effect of time (A) and effect of HLM protein concentration (B) to the rate of formation of OMF O-demethylation product, FL, in HLM incubations.

(2)

(3)

where S = concentration of the substrate, V = rate of the product formation, K_m = Michaelis-Menten constant expressing the substrate concentration at half of V_max, V_max1 = maximum rate of the product formation of the reaction catalyzed by enzyme 1, V_max2 = maximum rate of the product formation of the reaction catalyzed by enzyme 2, K_m1 = Michaelis-Menten constant expressing the substrate concentration at half of V_max1, and K_m2 = Michaelis-Menten constant expressing the substrate concentration at half of V_max2.

	Results

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Kinetics Determination of OMF O-Demethylation in HLMs. From Fig. 2, the kinetics for the formation of FL was optimized with respect to incubation time at 30 min and HLM protein concentration at 0.5 mg/ml. In the substrate-velocity analysis, the Eadie-Hofstee plot exhibited biphasic kinetic characteristic (Fig. 3 insert) over the OMF concentration range 0.5 to 120 µM, suggesting more than one enzyme was involved in OMF O-demethylation (Tracy and Hummel, 2004). A comparison between nonlinear data fitting for Michaelis-Menten single-enzyme and two-enzyme plots was performed, and the F test (P < 0.05) indicated a significantly better fit with the two-enzyme model (eq. 3). Using this equation, the kinetic parameters determined for OMF O-demethylation by HLMs were K_m1 = 1.14 ± 0.90 µM and V_max1 = 11.3 ± 4.6 pmol/mg/min for the high affinity enzyme(s) and K_m2 = 57.0 ± 6.4 µM and V_max2 = 258 ± 6 pmol/mg/min for the low affinity enzyme(s).

View larger version (14K): [in this window] [in a new window]   FIG. 3. Kinetic analysis of OMF O-demethylation: Michaelis-Menten plot with nonlinear regression for a two-enzyme system, and Eadie-Hofstee plot of transformed data from FL formation (inset).

View larger version (17K): [in this window] [in a new window]   FIG. 4. The OMF O-demethylation activities of expressed P450 isozymes at OMF concentrations of 1 µM (A) and 40 µM (B).

View larger version (19K): [in this window] [in a new window]   FIG. 5. Michaelis-Menten plots and Eadie-Hofstee plots (inset) for FL formation in the presence of CYP2C19 inhibitors: 100 µM ticlopidine (a mechanism-based inhibitor) (A) and 5 µM(+)-N-3-benzyl-nirvanol (a competitive inhibitor) (B) in HLM incubations.

P450 Isozyme-Selective Chemical Inhibition Studies. The effect of P450 isozyme-selective chemical inhibitors ticlopidine and (+)-N-3-benzyl-nirvanol (for CYP2C19), sulfaphenazole (for CYP2C9), and furafylline (for CYP1A2) toward O-demethylation of OMF by HLMs over the concentration range 0.5 to 120 µM OMF in HLMs are shown in Figs. 5, 6, and 7, respectively, and their associated kinetic parameters are summarized in Table 3. When (S)-mephenytoin was added to preincubated HLMs, NADPH-regenerating system, and ticlopidine reaction mixture, >95% inhibition was observed for the formation of (S)-4'-hydroxy-mephenytoin, a reaction catalyzed by CYP2C19 (data not shown). Therefore, the HLM preincubation with ticlopidine effectively removed CYP2C19 activity. When ticlopidine was added to inhibit the contribution of CYP2C19, the Eadie-Hofstee plot for OMF O-demethylation exhibited single-like enzyme characteristics over the concentration range 0.5 to 120 µM OMF (Fig. 5A). The apparent kinetic parameters obtained from the Michaelis-Menten single-enzyme model were K_m = 103.9 ± 32.6 µM and V_max = 143 ± 25 pmol/mg/min. Similarly, when the CYP2C19 competitive selective chemical inhibitor (+)-N-3-benzyl-nirvanol was added to the incubation, single-like enzyme characteristics were observed (Fig. 5B). The apparent kinetic parameters obtained from the Michaelis-Menten single-enzyme model were K_m = 92.26 ± 8.09 µM and V_max = 442 ± 20 pmol/mg/min. In the presence of sulfaphenazole, the Eadie-Hofstee plot exhibited single-like enzyme characteristics over the OMF concentration range 0.5 to 10 µM with apparent K_m of 3.97 ± 0.28 µM and V_max of 22.8 ± 0.7 pmol/mg/min (Fig. 6). The apparent kinetic parameters obtained for OMF O-demethylation in the presence of furafylline were K_m1 = 1.74 ± 1.79 µM and V_max1 = 8.1 ± 4.0 pmol/mg/min for high affinity enzyme(s) and K_m2 = 98.9 ± 43.4 µM and V_max2 = 284 ± 41 pmol/mg/min for low affinity enzyme(s) (Fig. 7).

View larger version (19K): [in this window] [in a new window]   FIG. 6. Michaelis-Menten plots and Eadie-Hofstee plots (inset) for FL formation in the presence of 2 µM sulfaphenazole (CYP2C9 inhibitor) in HLM incubations.

View larger version (18K): [in this window] [in a new window]   FIG. 7. Michaelis-Menten plots and Eadie-Hofstee plots (inset) for FL formation in the presence of 10 µM furafylline (CYP1A2 inhibitor) in HLM incubations.

View larger version (27K): [in this window] [in a new window]   FIG. 8. The effect of ticlopidine, sulfaphenazole, and furafylline on the O-demethylation of OMF at 1 and 10 µM OMF in HLM incubations.

IC₅₀ Determination for CYP2C19 in HLMs. The FL standard curve constructed with blank incubation mixture spiked with standard solutions of FL was linear over the concentration range (0.32 nM-2 µM) with r² = 0.9987. Thirteen substrates were selected to evaluate OMF O-demethylation for use in IC₅₀ determinations in HLMs. The substrates were added to HLM incubation mixtures containing 1 µM OMF. The IC₅₀ values of the substrates were determined and compared with literature values obtained by LC/MS method using (S)-mephenytoin as a substrate in HLMs. The results are summarized in Table 4.

	Discussion

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At 1 µM OMF, greater selectivity of CYP2C19 toward O-demethylation of OMF was observed in a panel of 11 cDNA-expressed human P450 isozymes (see Fig. 4A). These results agreed well with informal reports (D. M. Stresser, personal communication) that CYP2C19, CYP1A1, and, to a lesser degree, CYP1A2 and CYP2C9 possessed OMF O-demethylase activities at similar experimental conditions. Because CYP1A1 is detected primarily in extrahepatic tissues under induced conditions and not present in significant amounts in HLMs (Williams, 2002), it should not have contributed to OMF O-demethylase activity in the HLM experiments. When OMF concentration was increased to 40 µM, multiple enzymes significantly contributed to the O-demethylation of OMF (Fig. 4B). As observed in Fig. 4B, the magnitude of this increased activity differed between individual P450 isozymes: 1.7-fold for CYP2C19, 10-fold for CYP2C9, 36-fold for CYP1A2, and 41-fold for CYP2C8. Based on the Michaelis-Menten enzyme kinetics and the observed substrate concentration-isozyme activity relationship, CYP2C19 appeared to be the high affinity component, whereas CYP2C9, CYP1A2, and CYP2C8 could be considered the low affinity components in HLMs.

In HLM incubations, a multiple-enzyme kinetics profile was observed as shown by the atypical biphasic in the Eadie-Hofstee plot (Fig. 3). The finding of multiple enzyme involvement in the O-demethylation reaction of OMF in HLMs was consistent with the data from the cDNA-expressed human P450 isozyme incubations. Studies with P450 isozyme-selective chemical inhibitors in HLMs showed that CYP2C19 and CYP2C9 were the two major enzymes catalyzing the O-demethylation reaction, contributing to 86% activity at 1 µM and 73% activity at 10 µM OMF (Fig. 8). The percentage activity inhibition by sulfaphenazole was greater at higher OMF concentrations, indicating CYP2C9 contributed more activity at higher OMF levels. These findings suggest CYP2C9 is the low affinity or the high K_m component of the characterized two-enzyme kinetics. When furafylline was added to HLMs, the nonlinearity in Eadie-Hofstee plot (Fig. 7) and the enzyme kinetics (Table 3) remained almost the same as without furafylline. Only 10% inhibitory effect was observed when furafylline was added alone to 1 or 10 µM OMF, and no significant additional inhibitory effect was seen when furafylline was coincubated with either ticlopidine, sulfaphenazole, or both. These data indicate that CYP2C9, rather than CYP1A2, was the low affinity component of the multiple-enzyme kinetics in catalyzing OMF O-demethylation in HLM incubations.

Kinetic parameters for OMF O-demethylation in the absence and presence of isozyme-selective chemical inhibitors for CYP2C19, CYP2C9, and CYP1A2 in HLMs revealed K_m of the high affinity P450 (i.e., CYP2C19) was approximately 1 µM. The K_m1 and V_max1 for the high affinity enzyme obtained from the sulfaphenazole experiment was higher than the parameters obtained from the experiments performed in the absence of chemical inhibitors (Table 3). The use of a different lot of HLMs in the sulfaphenazole experiment may have attributed to this discrepancy. The V_max2 for the low affinity enzyme obtained from the ticlopidine experiment was lower than V_max2 obtained from those experiments in the absence or presence of other chemical inhibitors (Table 3). The decrease of V_max2 was more likely caused by ticlopidine, a mechanism-based CYP2C19 chemical inhibitor (Ha-Duong et al., 2001). Because the mixture had to be preincubated for 30 min before initiating OMF O-demethylation reaction, some enzyme activity could be lost during the longer incubation time. Another reason, probably more likely, was as a result of the selectivity of ticlopidine. Ko et al. (2000) found that at 100 µM ticlopidine also inhibited both CYP1A2 and CYP2C9 activities in HLMs. Because CYP2C9 was involved in the OMF O-demethylation in HLM incubations, it was expected that V_max would decrease. When (+)-N-3-benzyl-nirvanol, a more selective competitive inhibitor of CYP2C19, was used in the inhibition study as shown in Table 3, V_max2 did not decrease.

OMF was used at 1 µM in the developed fluorometric assay because this concentration was selective toward OMF O-demethylation and also similar to the K_m of CYP2C19. Sulfaphenazole (2 µM) was added to the incubation mixture to minimize the effect of CYP2C9 activity. For 13 substrates in which the CYP2C19 inhibition profile was characterized (Table 4), the IC₅₀ data obtained from the fluorometric assay reported in this study correlated well with those reported in the literature using LC/MS with (S)-mephenytoin as a chemical substrate. Although the HLM incubation mixture in the chemical substrate approach did not contain sulfaphenazole (a CYP2C9 inhibitor), IC₅₀ values obtained from this fluorometric assay were similar to those obtained from the chemical substrate approach. Ko et al. (1998) conducted experiments to investigate (S)-mephenytoin N-demethylation by CYP2C9 in HLMs. They found that CYP2C9 was one of two enzyme components with apparent K_m of 174 µM and V_max of 170.5 pmol/min/mg protein toward (S)-mephenytoin N-demethylation, a minor route of (S)-mephenytoin metabolism in HLMs compared with 4'-hydroxylation of (S)-mephenytoin. Because most of the data shown in Table 4 were conducted at lower concentration of (S)-mephenytoin (i.e., 30 µM, K_m of CYP2C19 for 4'-hydroxylation of (S)-mephenytoin), the contribution of (S)-mephenytoin by CYP2C9 (a nontarget enzyme) would be a minor component. The IC₅₀ values determined for the 13 substrates ranged from 0.47 to 108.7 µM, which shows the wide dynamic range of the assay. There are a number of reports on the application of fluorescence assays for P450-mediated drug inhibition studies in HLMs: coumarin for CYP2A6 (Donato et al., 2004), 3-[2-(N,N-diethyl-N-methylammonium)-ethyl]-7-methoxy-4-methylcoumarin and 7-methoxy-4-(aminomethyl)-coumarin (MAMC) for CYP2D6 (Onderwater et al., 1999; Venhorst et al., 2000a,b; Chauret et al., 2001; Yamamoto et al., 2003), 9-N-(methylamino)acridine for CYP1A1 and CYP2D6 (Mayer et al., 2007), 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5H)-one for CYP3A4 (Chauret et al., 1999; Nicoll-Griffith et al., 2004), and dibenzylfluorescein for CYP3A4 (Ghosal et al., 2003). Interestingly, the use of MAMC as a selective substrate for probing CYP2D6 inhibition in HLMs involved the addition furafylline (selective CYP1A2 inhibitor) in the incubation (Venhorst et al., 2000b), similar to our approach that required the addition of sulfaphenazole (selective CYP2C9 inhibitor) in the incubation. Venhorst (2000b) reported MAMC O-methylation was catalyzed mainly by CYP2D6 and to a small extent by CYP1A2. Addition of furafylline completely eliminated the contribution of CYP1A2 toward MAMC O-methylation. To our knowledge, this study represents the first reported utility of OMF in evaluating CYP2C19-mediated drug inhibition in HLMs.

	Acknowledgments

 
We thank Dr. David Stresser at BD Science for scientific discussion and advice before initiating this research study.

	Footnotes

 
doi:10.1124/dmd.106.014472.

ABBREVIATIONS: DDI, drug-drug interaction; P450, cytochrome P450; HLM, human liver microsome; OMF, 3-O-methylfluorescein; FL, fluorescein; LC/MS, liquid chromatography/mass spectrometry; MAMC, 7-methoxy-4-(aminomethyl)-coumarin.

Address correspondence to: Hanlan Liu, DMPK and Pharmaceutics Department, Drug and Biomaterial R&D, Genzyme Corporation, 153 2nd Avenue, Waltham, MA 02451. E-mail: hanlan.liu{at}genzyme.com

	References

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Andersson TB, Bredberg E, Ericsson H, and Sjöberg H (2004) An evaluation of the in vitro metabolism data for predicting the clearance and drug-drug interaction potential of CYP2C9 substrates. Drug Metab Dispos 32: 715-721.[Abstract/Free Full Text]

Atkinson A, Kenny JR, and Grime K (2005) Automated assessment of time-dependent inhibition of human cytochrome P450 enzymes using liquid chromatography-tandem mass spectrometry analysis. Drug Metab Dispos 33: 1637-1647.[Abstract/Free Full Text]

Bapiro TE, Egnell A, Hasler JA, and Masimirembwa CM (2001) Application of higher throughput screening (HTS) inhibition assays to evaluate the interaction of antiparasitic drugs with cytochrome P450S. Drug Metab Dispos 29: 30-35.[Abstract/Free Full Text]

Chauret N, Dobbs B, Lackman RL, Bateman K, Nicoll-Griffith DA, Stresser DM, Ackermann JM, Turner SD, Miller VP, and Crespi CL (2001) The use of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) as a specific CYP2D6 probe in human liver microsomes. Drug Metab Dispos 29: 1196-1200.[Abstract/Free Full Text]

Chauret N, Tremblay N, Lackman RL, Gauthier JY, Silva JM, Marois J, Yergey JA, and Nicoll-Griffith DA (1999) Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe. Anal Biochem 276: 215-226.[CrossRef][Medline]

Cohen LH, Remley MJ, Raunig D, and Vaz AND (2003) In vitro drug interaction of cytochrome P450: an evaluation of fluorogenic to conventional substrates. Drug Metab Dispos 31: 1005-1015.[Abstract/Free Full Text]

Crespi CL, Miller VP, and Penman BW (1997) Microtiter plate assays for inhibition of human drug metabolizing cytochromes P450. Anal Biochem 248: 188-190.[CrossRef][Medline]

Dierks EA, Stams KR, Lim H, Cornelius G, Zhang H, and Ball SE (2001) A method for the simultaneous evaluation of the activities of seven major human drug-metabolizing cytochrome P450s using an in vitro cocktail of probe substrates and fast gradient liquid chromatography tandem mass spectrometry. Drug Metab Dispos 29: 23-29.[Abstract/Free Full Text]

Donahue SR, Flockhart DA, Abernethy DR, and Ko JW (1997) Ticlopidine inhibition of phenytoin metabolism mediated by potent inhibition of CYP2C19. Clin Pharmacol Ther 62: 572-577.[CrossRef][Medline]

Donato MT, Jiménez N, Castell JV, and Gómez-Lechón J (2004) Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metab Dispos 32: 699-706.[Abstract/Free Full Text]

Ghosal A, Hapangama N, Yuan Y, Lu X, Horne D, Patrick JE, and Zbaida S (2003) Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes. Biopharm Drug Dispos 24: 375-384.[CrossRef][Medline]

Glue P, Banfield CR, Perhach JL, Mather GG, Racha JK, and Levy RH (1997) Pharmacokinetic interaction with felbamate. Clin Pharmacokinet 33: 214-224.[Medline]

Ha-Duong N, Dijols S, Macherey A, Goldstein JA, Dansette PM, and Mansuy D (2001) Ticlopidine as a selective mechanism-based inhibitor of human cytochrome P450 2C19. Biochemistry 40: 12112-12122.[CrossRef][Medline]

Ito K, Iwatsubo T, Kanamitsu S, Ueda K, Suzuki H, and Sugiyama Y (1998) Prediction of pharmacokinetic alterations caused by drug-drug interaction: metabolic interaction in the liver. Pharmacol Rev 50: 387-411.[Abstract/Free Full Text]

Jurima-Romet M, Crawford K, Cyr T, and Inaba T (1994) In vitro inhibition by macrolide antibiotics and azole antifungals. Drug Metab Dispos 22: 849-857.[Abstract]

Ko JW, Desta Z, and Flockhart DA (1998) Evaluation of omeprazole and lansoprazole as inhibitors of cytochrome P450 isoforms. Drug Metab Dispos 26: 775-778.[Abstract/Free Full Text]

Ko JW, Desta Z, Soukhova NV, Tracy T, and Flockhart DA (2000) In vitro inhibition of the cytochrome P450 (CYP) system by the antiplatelet drug ticlopidine: potent effect on CYP2C19 and CYP2D6. Br J Clin Pharmacol 49: 343-351.[CrossRef][Medline]

Ko JW, Sukhova N, Thacker D, Chen P, and Flockhart DA (1997) Evaluation of omeprazole and lansoprazole as inhibitors of cytochrome P450 isoforms. Drug Metab Dispos 25: 853-862.[Abstract/Free Full Text]

Kobayashi K, Yamamoto T, Chiba K, Tani M, Ishizaki T, and Kuroiwa Y (1995) The effects of selective serotonin reuptake inhibitors and their metabolites on S-mephenytoin 4'-hydroxylase activity in human liver microsomes. Br J Clin Pharmacol 40: 481-485.[Medline]

Li X, Andersson T, Ahlstrom M, and Weidolf L (2004) Comparison of inhibitory effects of the proton pump-inhibiting drugs omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole on human cytochrome P450 activities. Drug Metab Dispos 32: 821-827.[Abstract/Free Full Text]

Lin JH and Lu AYH (1998) Inhibition and induction of cytochrome P450 and the clinical implications. Clin Pharmacokinet 35: 361-390.[CrossRef][Medline]

Mayer RT, Dolence EK, and Mayer GE (2007) A real-time fluorescence assay for measuring N-dealkylation. Drug Metab Dispos 35: 103-109.[Abstract/Free Full Text]

Michalets EL (1998) Update: clinically significant cytochrome P-450 drug interactions. Pharmacotherapy 18: 84-112.[Medline]

Motulsky HJ and Ransnas LA (1987) Fitting curves to data using nonlinear regression: a practical and nonmathematical review. FASEB J 1: 365-374.[Abstract]

Nicoll-Griffith DA, Chauret N, Houle R, Day SH, D'Antoni M, and Silva JM (2004) Use of a benzyloxy-substituted lactone cyclooxygenase-2 inhibitor as a selective fluorescent probe for CYP3A activity in primary cultured rat and human hepatocytes. Drug Metab Dispos 32: 1509-1515.[Abstract/Free Full Text]

Onderwater RCA, Venhorst J, Commandeur JNM, and Vermeulen NPE (1999) Design, synthesis, and characterization of 7-methoxy-4-(aminomethyl)-coumarin as a novel and selective cytochrome P450 2D6 substrate suitable for high-throughput screening. Chem Res Toxicol 12: 555-559.[CrossRef][Medline]

Somogyi A and Muirhead M (1987) Pharmacokinetic interactions of cimetidine 1987. Clin Pharmacokinet 12: 321-366.[Medline]

Stresser DM, Blanchard AP, Turner SD, Erve JCL, Dandeneau AA, Miller VP, and Crespi CL (2000) Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates. Drug Metab Dispos 28: 1440-1448.[Medline]

Stresser DM, Turner SD, Blanchard AP, Miller VP, and Crespi CL (2002) Cytochrome P450 fluorometric substrates: identification of isoform-selective probes for rat CYP2D2 and human CYP3A4. Drug Metab Dispos 30: 845-852.[Abstract/Free Full Text]

Suzuki H, Kneller MB, Haining RL, Trager WF, and Rettie AE (2002) (+)-N-3-benzyl-nirvanol and (-)-N-3-benzyl-phenobarbital: new potent and selective in vitro inhibitors of CYP2C19. Drug Metab Dispos 30: 235-239.[Abstract/Free Full Text]

Tracy TS and Hummel MA (2004) Modeling kinetic data from in vitro drug metabolism enzyme experiments. Drug Metab Rev 36: 231-242.[CrossRef][Medline]

Tucker GT, Houston JB, and Huang SM (2001) Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential—toward a consensus. Pharm Res (NY) 18: 1071-1080.

Venhorst J, Onderwater RCA, Meerman JHN, Commandeur JNM, and Vermeulen NPE (2000a) Influence of N-substitution of 7-methoxy-4-(aminomethyl)-coumarin on cytochrome P450 metabolism and selectivity. Drug Metab Dispos 28: 1524-1532.[Medline]

Venhorst J, Onderwater RCA, Meerman JHN, Vermeulen NPE, and Commandeur JNM (2000b) Evaluation of a novel high-throughput assay for cytochrome P450 2D6 using 7-methoxy-4-(aminomethyl)-coumarin. Eur J Pharm Sci 12: 151-158.[CrossRef][Medline]

Walsky RL and Obach RS (2004) Validated assays for human cytochrome P450 activities. Drug Metab Dispos 32: 647-660.[Abstract/Free Full Text]

Weaver R, Graham KS, Beattie IG, and Riley RJ (2003) Cytochrome P450 inhibition using recombinant proteins and mass spectrometry/multiple reaction monitoring technology in a cassette incubation. Drug Metab Dispos 31: 955-966.[Abstract/Free Full Text]

Wienkers LC (2002) Factors confounding the successful extrapolation of in vitro CYP3A inhibition information in the in vivo condition. Eur J Pharm Sci 15: 239-242.[CrossRef][Medline]

Williams DA (2002) Drug metabolism, in Foye's Principles of Medicinal Chemistry (Lemke TL, Williams DA eds) ed 5, pp 174-233, Philadelphia, Williams & Wilkins.

Yamamoto T, Suzuki A, and Kohno Y (2003) High-throughput screening to estimate single or multiple enzymes involved in drug metabolism: microtitre plate assay using a combination of recombinant CYP2D6 and human liver microsomes. Xenobiotica 33: 823-839.[CrossRef][Medline]

Yan Z and Caldwell GW (2001) Metabolic profiling, and cytochrome P450 inhibition & induction in drug discovery. Curr Topics Med Chem 1: 403-425.[CrossRef]

Yao C, Kunze KL, Trager WF, Kharasch ED, and Levy RH (2003) Comparison of in vitro and in vivo inhibition potencies of fluvoxamine toward CYP2C19. Drug Metab Dispos 31: 565-571.[Abstract/Free Full Text]

Yu K, Yim D, Cho J, Park S, Park J, Lee K, Jang I, Yi S, Bae K, and Shin S (2001) Effect of meprazole on the pharmacokinetics of moclobemide according to the genetic polymorphism of CYP2C19. Clin Pharmacol Ther 69: 266-273.[CrossRef][Medline]

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