Short Communication Is 1-Aminobenzotriazole an Appropriate in Vitro Tool as a Nonspecific Cytochrome P450 Inactivator?

1-Aminobenzotriazole (1-ABT) is generally considered to be a nonselective mechanism-based inactivator of both human and nonhuman cytochrome P450 (P450) enzymes. Thus, 1-ABT is routinely used when conducting in vitro reaction phenotyping studies with new chemical entities in drug discovery to decipher P450 from non-P450-mediated metabolism. Experiments with pooled human liver microsomes (HLMs) demonstrated that carbon monoxide binding, although substantially reduced after a 30-min preincubation with 1-ABT, was still measurable. Thus, remaining activity of nine major human P450s (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) in HLMs was determined using established selective probe substrates after 30-min preincubation with either 1-ABT (1 mM), a positive control time-dependent inhibitor, or a competitive inhibitor. Whereas P450 2A6 and 3A4 activity was essentially eliminated upon 30-min pretreatment with 1-ABT, the other human P450s were less affected, with at least 20% activity remaining after pretreatment. In contrast, most of the known P450 selective timedependent inhibitors were more effective inactivators than 1-ABT at lower concentrations. A particularly interesting finding was that 1-ABT was quite ineffective at inactivating P450 2C9, with roughly 60% activity remaining after pretreatment, which suggests that 1-ABT is much less selective for certain human P450s. This collection of data clearly demonstrates that assuming 1-ABT is a nonselective P450 inhibitor in vitro is risky, and false conclusions regarding remaining metabolic activity being non-P450 mediated after 1-ABT pretreatment may be made. In vitro reaction phenotyping, where enzymes contributing to the biotransformation of new chemical entities are identified, is a routine practice in drug discovery (Williams et al., 2003; Zhang et al., 2007). This information is essential for understanding the relative contributions of metabolic pathways to overall clearance and, thus, the risk of pharmacokinetic drug-drug interactions and/or interpatient variability in drug exposure. One critical issue in conducting these in vitro studies is having the appropriate tool substrates and inhibitors of the various human cytochrome P450 (P450) enzymes. A particularly useful tool inhibitor historically has been 1-aminobenzotriazole (1-ABT), which is thought to inactivate P450 enzymes nonselectively by covalent modification of the heme prosthetic group following bioactivation (Ortiz de Montellano and Mathews, 1981). Thus, it has become a common in vitro practice to preincubate either human liver microsomes (HLMs) or hepatocytes with high concentrations of 1-ABT ( 1 mM) before the introduction of test compounds (substrate-depletion approach) to decipher P450 from non-P450 mediated-metabolism (Dalmadi et al., 2003; Williams et al., 2003; Kostrubsky et al., 2006). Although it is acknowledged that this approach is useful for determining P450-mediated metabolism, the characterization of the specific effects of 1-ABT on the major human P450 enzymes in vitro has been limited (Emoto et al., 2005). The purpose of the current research was to evaluate the use of 1-ABT as a nonselective inactivator of P450 by determining the remaining activity of the major human P450 enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) in HLMs after pretreatment with 1 mM 1-ABT. These data will hopefully serve as a caution for the assumptions commonly made when using 1-ABT for in vitro reaction phenotyping studies. Materials and Methods Chemicals. Potassium phosphate buffer, NADP , isocitric acid, isocitric dehydrogenase, magnesium chloride, 1-aminobenzotriazole, sodium dithionite, tolbutamide, diclofenac, 4 -hydroxydiclofenac, sulfaphenazole, dextromethorphan, dextrorphan, paroxetine, quinidine, phenacetin, acetaminophen, furafylline, -naphthoflavone, midazolam, 1 -hydroxymidazolam, erythromycin, ketoconazole, bupropion, ticlopidine, clotrimazole, paclitaxel (Taxol), amiodarone, (S)-mephenytoin, 4 -hydroxymephenytoin, ( )-N-3-benzyl nirvanol, coumarin, 7-hydroxycoumarin, 8-methoxypsoralen, tranylcypromine, chlorzoxazone, 6-hydroxychlorzoxazone, and diethyldithiocarbamate were all purchased from Sigma-Aldrich (St. Louis, MO). Montelukast was purchased from Sequoia Research Products (Pangbourne, UK). Pooled HLMs (pool of 50 donors), 6 -hydroxypaclitaxel, and hydroxybupropion were purchased from BD Biosciences (San Jose, CA), whereas tienilic acid was available in the chemical bank at Pfizer Global Research and Development. All other chemicals were obtained from commercial sources and were of the highest purity available. P450 Time-Dependent Inhibition Assays. Remaining enzyme activity using selective cytochrome P450 probe substrates was determined after a primary incubation in HLMs with 1-ABT (1 mM), a positive control timedependent inhibitor (TDI), and a competitive inhibitor (0.05% total organic v/v) in triplicate for nine major human P450 enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4). Incubations were conducted in a 96-well plate format at 37°C using an Eppendorf MTP ThermoMixer (Eppendorf AG, Hamburg, Germany) and consisted of HLMs (1 mg/ml primary incubation, 0.05 mg/ml secondary incubation) in 100 mM potassium phosphate buffer, pH 7.4, with a NADPH-regenerating system (3 mM MgCl2, 1 mM, NADP , Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.024075. ABBREVIATIONS: P450, cytochrome P450; 1-ABT, 1-aminobenzotriazole; HLM, human liver microsomes; TDI, time-dependent inhibitor; LC/MS/MS, liquid chromatography/tandem mass spectrometry; CO, carbon monoxide. 0090-9556/09/3701-10–13$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 1 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 24075/3420483 DMD 37:10–13, 2009 Printed in U.S.A. 10 at A PE T Jornals on A ril 6, 2017 dm d.aspurnals.org D ow nladed from 5 mM isocitric acid, and 1 unit/ml isocitrate dehydrogenase). At selected times (0 and 30 min), 15l aliquots were taken out of the primary incubation mixture and diluted 20-fold into a secondary incubation mixture (285 l) containing the selective probe substrate (concentration 5 times the published apparent Km), to measure residual activity. Substrate concentrations and secondary incubation times for each P450 probe substrate were as follows: phenacetin (150 M, 30 min), coumarin (10 M, 6 min), bupropion (350 M, 20 min), paclitaxel (50 M, 15 min), diclofenac (25 M, 5 min), (S)mephenytoin (250 M, 45 min), dextromethorphan (20 M, 20 min), chlorzoxazone (300 M, 20 min), and midazolam (10 M, 6.5 min). Secondary incubations were quenched with an equal volume of cold acetonitrile (300 l) containing internal standard (0.2 M tolbutamide). Plates were centrifuged at 3000g for 10 min, supernatant was transferred to a separate analytical plate, and metabolite formation was analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) as described below. After treatment with 1-aminobenzotriazole, positive control TDI, and competitive inhibitor, the percentage of remaining P450 activity was calculated by comparing the metabolite formation after 0and 30-min preincubation with solvent control incubations without inhibitor. Data were plotted for visualization using GraphPad Prism (GraphPad Software Inc., San Diego, CA) software. Spectrophotometric Analysis. Reduced carbon monoxide (CO) difference spectra were recorded on a Hitachi (Danbury, CT) U3300 dual-beam spectrophotometer, and data were analyzed using UV Solutions 1.2 software (Hitachi). In brief, a 5-ml incubation (containing 1 mg/ml HLMs) and 1 mM 1-ABT in 100 mM potassium phosphate buffer (pH 7.4) was prepared, and 2100l aliquots were taken at 0 ( NADPH) and 30 min after initiation of incubation with 1 mM NADPH at 37°C. Aliquots were reduced by addition of sodium dithionite and then evenly divided between two 1 ml, 1-cm path length cuvettes. A baseline was recorded, and then carbon monoxide was gently bubbled into the sample cuvette (approximately 1 bubble per second for 1 min). Next, absorbance was scanned from 400 to 600 nm, and peak absorbance at 450 nm was determined. Control studies included incubations in the presence of NADPH without 1-ABT to determine loss of CO binding over the 30-min incubation period. Percentage of loss in CO-binding was roughly determined by difference in the peak absorbance observed at 450 nm between incubations (corrected for the drift below 0 absorbance units in baseline). LC/MS/MS Analysis. Metabolites were analyzed on a PE Sciex API-3000 (Applied Biosystems, Foster City, CA) triple-quadrupole instrument, with the exception of 4 -hydroxymephenytoin, which was analyzed on a PE Sciex API-4000. The mass spectrometer was equipped with an electrospray ionization interface connected in line with a Shimadzu LC20AD (Shimadzu Scientific Instruments, Columbia, MD) pump and a Leap Technologies CTC PAL (Carrboro, NC) autosampler. A 10l aliquot of the sample was injected onto the column and eluted using a programmed flow rate from 0.4 to 0.6 ml/min. All chromatographic separations were carried out using a gradient elution profile on a Zorbax 3.5m Eclipse Plus (Agilent Technologies, Santa Clara, CA) C18 2.1 50 mm column, with the exception of 4 -hydroxymephenytoin, which was separated on an Alltima (Grace Davison Discovery Science, Deerfield, IL) 3m C18 2.1 50 mm column. The source temperature was set to 400°C, and mass spectral analyses were performed using multiple reaction monitoring. Gradient profiles, ionization modes, and fragment transitions for the various metabolites are listed in Table 1. Tolbutamide was used as the internal standard for both negative and positive ionization modes; the respective transitions were 269.1/170.0 and 271.1/91.2. All data were analyzed using PE Sciex Analyst 1.4.1 software. Results and Discussion 1-ABT is often used as a nonselective inactivator of the human P450 enzymes when conducting reaction phenotyping studies in drug discovery (Williams et al., 2003), yet a full characterization of the remaining activity of the major human P450s after pretreatment with 1-ABT has yet to be assessed. In the studies described herein, we adopted an incubation condition commonly found in the literature and measured residual enzyme activity for nine of the major human P450s (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) after preincubation with 1 mM 1-ABT for 30 min. After the aforementioned preincubation conditions with 1-ABT, roughly 20% of the spectrally detectable 450 is remaining ( 80% loss), indicated by the peak absorbance differences at 450 nm (Fig. 1), which suggests that measurable enzymatic activity probably still remains. Regardless of the loss in spectrally detectable P450, it is still unknown which of the P450 enzymes are most affected by 1-ABT. As demonstrated in Table 2, all of the human P450 enzymes tested were inactivated after preincubation with 1-ABT, albeit to differing degrees, as evidenced by a lower percentage of remaining activity after preincubation with inhibitor compared with no preincubation. In addition, incubations with control TDIs resulted in loss of P450 activity after 30 min, whereas incubations with selected competitive inhibitors resulted in minor, if any, differences in enzymatic activity after 30-min preincubation (Table 2). In the cases of P450s 2A6 and 3A4, enzyme activity was essentially completely inhibited (remaining activities of 2%) after the preincubation with 1-ABT. However, for the remaining P450 enzymes, residual activity ranged between 19 and 58%. In addition, whereas 1-ABT produced an equal or greater loss of activity in comparison with the control inhibitors of P450 2C19 and 2E1 at the concentrations tested (19 and 25%, respectively), this was not observed for P450s 1A2, 2B6, 2C8, 2C9, and 2D6, where either the positive control TDI or competitive inhibitor (or both) displayed more potent inhibition of enzymatic activity than 1-ABT at lower concentrations (Table 2), perhaps arguing against the use of 1-ABT in vitro. In particular, for P450 2C9, tienilic acid as well as the competitive inhibitor sulfaphenazole were significantly more effective, with 11 and 27% remaining activity, respectively, compared with 58% with 1-ABT (Table 2). This notably weak inactivation of P450 2C9 was particularly interesting and is consistent with previous reports (Emoto et al., 2003, 2005). Prototypical substrates of P450 2C9 are carboxylic acids such as flurbiprofen, naproxen, ibuprofen, and diclofenac (Tracy et al., 1996, 1997; Klose et al., 1998), which are anionic at pH 7.4, and proposed to be involved in a key binding interaction with Arg108, a TABLE 1 Summary of LC/MS/MS analytical conditions for selective P450 probe substrate metabolites measured from secondary incubations Metabolite Electrospray Ionization Mode MS Transition m/z Gradient Profile %B(min) Acetaminophen 152/110 10(0):10(1.2):98(3.5) 7-Hydroxycoumarin 161/133 5(0):5(0.8):95(1.5) Hydroxybupropion 256/131 5(0):5(0.8):98(2.5) 6 -Hydroxypaclitaxel 870/286 5(0):5(0.8):98(2.5) 4 -Hydroxydiclofenac 310/266 5(0):5(0.3):95(2.5) 4 -Hydroxymephenytoin 233/190 5(0):5(0.3):100(3.5) Dextrorphan 258/157 10(0):10(0.8):95(2.0) 6-Hydroxychlorzoxazone 184/120 5(0):5(1.0):100(2.0) 1 -Hydroxymidazolam 342/203 10(0):10(0.8):95(2.0) a Tolbutamide was used as the internal standard for both negative and positive ionization modes; the respective transitions were m/z 269/170 and 271/91. b Mobile phases for all analyses were as follows: A, 0.1% formic acid in H2O and B, 0.1% formic acid in acetonitrile. 11 1-ABT INACTIVATION OF HUMAN P450 at A PE T Jornals on A ril 6, 2017 dm d.aspurnals.org D ow nladed from