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Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania
(Received February 23, 2006; accepted May 19, 2006)
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Abstract |
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-naphthoflavone, clotrimazole, ketoconazole, miconazole, nicardipine, and verapamil significantly inhibited human liver microsomal esterase activity, with apparent IC50 values of 18.0, 20.5, 6.5, 15.0, 19.4, and 5.4 µM, respectively. All of these showed 
20% inhibition of human liver microsomal esterase activity at concentrations typically used for P450 reaction phenotyping studies, with clotrimazole, miconazole, nicardipine, and verapamil showing >60% inhibition. Unlike the chemical inhibitors, no inhibition of human liver microsomal esterase activity was observed in the presence of mAb to CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. These results suggest that P450 chemical inhibitors are capable of inhibiting human liver microsomal esterase activity and should not be used to assess the role of P450 enzymes in the biotransformation of esters. The lack of inhibition of human liver microsomal esterase activity by P450-specific monoclonal antibodies suggests that they may be used to assess the role of P450 enzymes in the biotransformation of esters. Additional experiments to assess the contribution of oxidative enzymes in the metabolism of esters may include incubations in the presence and absence of ß-nicotinamide adenine dinucleotide 2'-phosphate reduced.
; Ortiz de Montellano, 1999). Human liver microsomes contain more than 15 different P450 isoenzymes, including CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7, that have the potential to biotransform xenobiotics and/or endogenous substrates (Parkinson, 1996; Madan et al., 2002). Due to broad substrate specificity of P450 enzymes, it is possible for more than one enzyme to be involved in the metabolism of a single compound. It is also possible for one P450 enzyme to catalyze two or more metabolic pathways for the same drug. In vitro methods have been established to determine which P450 isoform(s) are involved in the metabolism of a specific drug. This process, also referred to as P450 reaction phenotyping, integrates data obtained from native human liver microsomes, intact cell models, recombinant P450s, and inhibition studies with P450-selective chemical inhibitors and specific antibodies (Parkinson, 1996; Rodrigues, 1999). Reaction phenotyping studies can establish whether a particular P450 enzyme is involved in the biotransformation of a drug, as well as to what extent the P450 enzyme contributes to the metabolism of that drug. Once it has been determined, by in vitro methods, which P450 enzyme(s) are involved in the metabolism of a specific drug, then clinical studies can be designed to address potential issues such as drug-drug interactions and genetic polymorphisms, which could affect pharmacokinetics, efficacy, or toxicity.
Although P450 enzymes play a prominent role in drug metabolism, other enzymes, such as esterases, can also be important. Mammalian carboxylesterases are located in the endoplasmic reticulum and cytosol of many tissues where they hydrolyze ester- and amide-containing chemicals and drugs to their respective free acids. They contribute to the detoxification or the metabolic activation of exogenous compounds such as drugs, environmental toxicants, and carcinogens as well as endogenous compounds (Satoh and Hosokawa, 1998; Satoh et al., 2002). In general, the types of esterases involved in the metabolism of exogenous compounds include carboxylesterases, arylesterases, and cholinesterases, whereas endogenous compounds are mainly hydrolyzed by lipases, acetylesterases, and acetylcholinesterases (Williams, 1985). However, the metabolism of ester-containing compounds may not be exclusively catalyzed by hydrolase enzymes. This was shown by Guengerich (Guengerich, 1987; Guengerich et al., 1988), who demonstrated the P450-catalyzed oxidative ester cleavage of 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester. The product, 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester, was formed in the presence of substrate, enzymatically active microsomes, and an NADPH-generating system. The formation of these products was observed with five different P450 enzymes; however, the extent of formation varied with the enzyme (Guengerich, 1987; Guengerich et al., 1988).
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Materials and Methods |
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). All other chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO), Fisher Scientific International Inc. (Hampton, NH), or Aldrich Chemical Co. (Milwaukee, WI).
FD Fluorometric Assay. Fluorescein diacetate (FD) is a model fluorogenic substrate for measuring esterase activity. In the presence of microsomal esterases, FD is hydrolyzed to the fluorescent product fluorescein through a two-step process referred to as fluorochromasia (Rotman and Papermaster, 1965; Dive et al., 1987) (Fig. 1). Fluorescein formation was measured using a fluorometer (SPECTRAmax Gemini plate reader; Molecular Devices Corp, Sunnyvale, CA) at excitation and emission set at 490 and 520 nM, respectively.
Determination of the Km for FD Hydrolysis by Human Liver Microsomes. Experiments were conducted in 96-well flat bottom plates in a volume of 0.2 ml, containing 0.198 ml of potassium phosphate buffer (0.1 M, pH 7.4) and 1 µl of pooled human liver microsomes (5 µg/ml microsomal protein). The plates were preincubated at 37°C in a fluorometer for 3 min. Reactions were initiated by the addition of 1 µl of FD (0.78–100 µM final concentration), and the reaction product (fluorescein) was measured for 10 min at 9-s intervals. Control incubations also were performed in the absence of protein and FD. The percent solvent from the FD stock solution did not exceed 0.5% in the incubations. The Km range was estimated from both a Lineweaver-Burke plot (1/Vi versus 1/[S]) of the FD hydrolysis data and a Michaelis-Menten plot (Vi versus [S]), where Vi was determined from the linear portion of the product formation versus time curve, and [S] represented the substrate concentration.
Initial Screen for Inhibition of Human Liver Microsomal Esterase Activity. Pooled human liver microsomes were incubated with FD at a concentration close to the apparent Km (30 µM) in the presence and absence of P450 chemical inhibitors. Initial incubations were performed in 96-well flat bottom plates containing 0.198 ml of potassium phosphate buffer (0.1 M, pH 7.4), 1 µl of P450 chemical inhibitor (100 µM final concentration), and 1 µl of pooled human liver microsomes (5 µg/ml microsomal protein). The plates were analyzed using the FD assay as described above. The percent solvent from FD and the inhibitors did not exceed 0.5% in the incubations. The percent inhibition was estimated comparing the velocities of the fluorescein product formation in the presence and absence of inhibitor.
Determination of IC50 Values for P450-Specific Inhibitors. If, from the initial screen, a specific inhibitor inhibited the hydrolysis of FD, then studies were conducted to characterize the apparent IC50. Incubations were performed in 96-well flat bottom plates containing 0.198 ml of potassium phosphate buffer (0.1 M, pH 7.4), 1 µl of P450 chemical inhibitor (0.024–200 µM final concentration), and 1 µl of pooled human liver microsomes (5 µg/ml microsomal protein). The plates were analyzed in the FD assay as described above. The percent solvent from FD and the inhibitors did not exceed 0.5% in the incubations. The percent activity remaining of FD hydrolysis was plotted against the range of inhibitor concentrations on a semi-log scale. The IC50 value was determined by fitting the equation (max – min)/(1 + (X/IC50)n) + min (where max is the maximum inhibition, min is the minimum inhibition, X is the inhibitor concentration, and n is a unitless number) to the percent activity remaining versus the log of the inhibitor concentration data (Sai et al., 2000).
P450-Specific Monoclonal Antibody Incubations in Human Liver Microsomes. Incubations with pooled human liver microsomes were conducted to determine the inhibitory potential of specific mAbs against CYP1A2, 2C8, 2C9, 2C19, CYP2D6, CYP3A4, or control IgG on human liver microsomal esterase activity. A 0.5-mg protein concentration of ascites containing the inhibitory mAb or control IgG was added to 0.180 ml of potassium phosphate buffer (0.1 M, pH 7.4) containing 1 mg/ml liver microsomes (Mei et al., 1999). The mixture was vortexed and preincubated for 15 min on ice (5 min at room temperature for the mAb to CYP2D6). A 5 µg/ml microsomal protein aliquot of the preincubation mixture was then removed and assayed by the FD esterase activity assay as described above. The percent solvent from FD did not exceed 0.5% in the incubations. A separate 0.25 mg/ml microsomal protein aliquot of the preincubation mixture was assayed for P450 activity.
P450 Activity. To evaluate the activity of P450 after incubation with the mAbs, incubation mixtures contained 50 µl of human liver microsomal protein from the preincubation mixture (0.25 mg/ml), potassium phosphate buffer (0.1 M, pH 7.4), and the appropriate P450 marker substrate [phenacetin (100 µM) for CYP1A2, taxol (100 µM) for CYP2C8, diclofenac (100 µM) for CYP2C9, (S)-mephenytoin (400 µM) for CYP2C19, dextromethorphan (50 µM) for CYP2D6, or testosterone (250 µM) for CYP3A4] in a 0.2-ml final volume. The reaction was initiated with 1 mM NADPH after a 3-min preincubation at 37°C in a shaking water bath and was allowed to proceed for 10 to 30 min depending on the P450 tested. The reactions were terminated with the addition of 2 volumes of acetonitrile containing the appropriate internal standard, and the samples were vortexed and centrifuged (3000 rpm for 10 min). The supernatant was diluted with an equal volume of 0.1% formic acid in water and analyzed by liquid chromatography/tandem mass spectrometry. The samples were analyzed using a PerkinElmer high-performance liquid chromatography system (PerkinElmer Life and Analytical Sciences, Boston, MA) coupled with a Sciex API 2000 triple quadrupole mass spectrometer (MDS Sciex, San Francisco, CA). Percentage of control enzyme activity values were obtained by comparison of samples in the presence of each mAb versus control IgG. Conditions for liquid chromatography/tandem mass spectrometry analysis of phenacetin, taxol, diclofenac, mephenytoin, and testosterone were described by Lu et al. (2003). CYP2D6 activity was measured by dextromethorphan O-demethylation. Dextromethorphan and its metabolite (dextrorphan) were separated on a BDS Hypersil C8 column (5-µm particle, 2.0 x 50 mm; Thermo Electron Corporation, Waltham, MA) with a mobile phase consisting of solvent A (90:10 water/methanol with 0.05% formic acid in water) and solvent B (10:90 water/methanol with 0.05% formic acid in water). Samples were eluted with a 2.0-min linear gradient from a 0 to 50% solvent B (flow rate at 1.5 ml/min). Metabolite and internal standard were identified using an atmospheric pressure chemical ionization ion source in the positive ion mode [dextrorphan m/z 258.1 (MH+) 
157.1 (collision energy 53 V, dwell time 150 ms); levallorphan (IS), m/z 284.1 (MH+) 199.1 (collision energy 38 V, dwell time 150 ms].
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Results |
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P450 Chemical Inhibitor Incubations with Pooled Human Liver Microsomes. Of the P450 inhibitors studied at 100 µM (Table 1), only -naphthoflavone, clotrimazole, ketoconazole, miconazole, nicardipine, and verapamil inhibited human liver microsomal esterase to any appreciable extent. Further characterization of the inhibition potency of -naphthoflavone, clotrimazole, ketoconazole, miconazole, nicardipine, and verapamil on human liver microsomal esterase activity yielded apparent IC50 values of 18.0, 20.5, 6.5, 15.0, 19.4, and 5.4 µM, respectively (Table 1 and Fig. 3). -Naphthoflavone and ketoconazole IC50 values were >3-fold higher than concentrations typically used for P450 reaction phenotyping studies. Conversely, the IC50 values for clotrimazole, miconazole, nicardipine, and verapamil were approximately 3- to 20-fold lower than concentrations typically used for P450 reaction phenotyping studies.
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P450 Monoclonal Antibody Incubations with Pooled Human Liver Microsomes. Studies were conducted with mAbs to CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4 at concentrations used for reaction phenotyping studies. No inhibition of human liver microsomal esterase activity was observed for any of the mAbs relative to control incubations in the presence of control IgG. The mAbs did show 70% inhibition of their respective P450 activity compared with control IgG, consistent with previously observed data (unpublished data; Table 2).
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Discussion |
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; Ashour et al., 1987; Hosokawa et al., 1988). Recently, Zhu et al. (2000) studied rat and human hepatocytes to examine the mechanism of carboxylesterase induction by P450 inducers including dexamethasone. Based on their findings, they proposed that regulation of carboxylesterase gene expression by dexamethasone was due to an alteration in transcriptional rate and/or mRNA stability. The glucocorticoid receptor and the pregnane X receptor (PXR) are known to mediate dexamethasone induction, with the glucocorticoid receptor requiring nanomolar levels and PXR requiring micromolar levels for activation. Zhu et al. (2000) suggested that the dexamethasone induction of hCE-1 and hCE-2 (the two major human liver isozymes) was mediated by PXR as a result of observed hCE-1 and hCE-2 induction only when dexamethasone was at micromolar levels.
In addition to the liver, the expression of hCE-1 and hCE-2 has been observed in small intestine, colon, testis, kidney, spleen, and heart (Satoh et al., 2002). Both hCE-1 and hCE-2 are important for systemic clearance of esters from blood through the liver. In many instances they catalyze the metabolism of the same substrates with different efficiencies, presumably due to the affinity of different structural features within the active site (Satoh et al., 2002). Recent studies of esterases have provided evidence for multiple forms of the enzymes. Sequences of newly identified carboxylesterase isozymes showing high homology with human liver carboxylesterases also showed high substrate-specificity similarity. This led to the proposal of Satoh and Hosokawa (1998) to classify carboxylesterases into four families: CES1, CES2, CES3, and CES4. The CES1 family includes the major forms of carboxylesterase isozymes and has been divided into subfamilies (i.e., CES1A) and sub-subfamilies (i.e., CES1A1). CES1A1 includes the major form of human carboxylesterases. CES1B includes esterases which catalyze long-chain acyl-coenzyme A hydrolysis and CES1C are secretory-type esterases (Satoh and Hosokawa, 1998; Satoh et al., 2002). hCE-1 and hCE-2 belong to the CES1 and CES2 classes of carboxylesterases, respectively. As well, more general esterase classification systems have been proposed based on substrate and inhibitor specificity or interactions with organophosphorus insecticides (Williams, 1985; McCracken et al., 1993).
The role of esterases in the metabolism of compounds is becoming increasingly important as, during the discovery of new drugs, candidate molecules are being synthesized which may contain an ester function. However, due to the nonselective substrate and inhibition specificity of esterases, phenotyping of specific esterases involved in the metabolism of a given compound is difficult.
Esterases have been reported to exhibit clinically relevant polymorphisms, which can lead to variable clearance of drugs. Cholinesterase, or butyrylcholinesterase, is abundant in human plasma and serum and hydrolyzes the muscle relaxant succinylcholine to succinylmonocholine and choline (Lockridge, 1990; Daly et al., 1993). Succinylcholine was introduced for use in 1951 having a quick onset of action resulting in complete paralysis and a rapid recovery free of toxic side effects. In some patients, however, paralysis lasted for hours instead of minutes, which often required life-saving intervention. For detailed discussion of the biochemistry and pharmacogenomics of cholinesterase, please refer to Lockridge (1990), Darvesh et al. (2003), and Kalow (2004).
Another esterase identified as having genetic variants is paraoxonase-1 (PON1). PON1 is a serum enzyme that catalyzes the hydrolysis of organophosphate esters, carbamates, and aromatic carboxylic acid esters. For a detailed discussion of the pharmacogenomics and catalytic efficiency of PON1, refer to Costa et al. (2003).
Because it has been shown that P450 enzymes, in addition to esterases, catalyze the cleavage of ester-containing compounds, it becomes important to understand the involvement of these different enzyme families in the metabolism of a drug candidate. The determination of which enzyme family is involved may have an impact on the development of a drug candidate, such as potential for drug-drug interactions and the potential for polymorphic disposition. For many pharmaceutical drug programs, routine screening of compounds occurs in the discovery stage, and it is possible to generate large numbers of esters with -hydrogens, which may undergo P450-mediated cleavage. The present study demonstrated the potential of P450-selective and -nonselective inhibitors to inhibit human liver microsomal esterase activity.
FD has been used by a number of laboratories as a model substrate for esterases, including those bound to microsomes. In Bort et al. (1996), a 10 µM concentration of FD was used to measure esterase activity of sera and liver fractions including S9, cytosol, and microsomes. Breeuwer et al. (1995) stated that FD is hydrolyzed by intracellular esterases after transport into cells. They also stated that apparently most cells, whether mammalian, yeast, or bacteria, could hydrolyze FD. In this study, we have experimentally tested for the formation of the fluorescein product of fluorescein diacetate hydrolysis and only observed this to occur at any reasonable rate in the presence of the human liver microsomes. This provides evidence that fluorescein diacetate is a model substrate for liver microsomal esterases. The number of esterases for which it is a substrate and their identities, however, is unknown at this time.
Using FD to measure human liver microsomal esterase activity, -naphthoflavone, clotrimazole ketoconazole, miconazole, nicardipine, and verapamil significantly inhibited the formation of the fluorescein product. The apparent IC50 values of esterase activity for -naphthoflavone and ketoconazole were 18 and 6.5 µM, respectively (Fig. 3). These values were >3-fold higher than the concentrations typically used for P450 reaction phenotyping studies. As such, it can be expected that significant esterase inhibition will occur. At concentrations typically used for reaction phenotyping, clotrimazole, miconazole, nicardipine, and verapamil could be expected to inhibit human liver microsomal esterase by >20% (data not shown). In contrast, mAbs to CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4 showed no inhibition of human liver microsomal esterase activity at levels that almost completely (70%) inhibited their respective P450 enzymes.
Much attention is directed toward the P450 metabolism of drugs, and as a result, many other biotransformation enzymes, such as esterases, often get overlooked. Esterases, however, are responsible for the metabolism of a number of endogenous and exogenous compounds. They can be induced, inhibited, and are subject to genetic polymorphisms, which can have clinical implications for the development of a drug. The results from the current study suggest that P450 chemical inhibitors should not be used to assess the role of P450 enzymes in the biotransformation of esters. Their potential to inhibit human liver microsomal esterase activity may result in an overestimation of the contribution of P450 enzymes in the metabolism of esters leading to a misinterpretation of potential drug-drug interactions. In contrast, P450 mAbs may be a useful tool to determine the contribution of P450 enzymes on the metabolism of esters as they were shown to have no effect on human liver microsomal esterase activity. Additional experiments to assess the contribution of oxidative enzymes in the metabolism of esters may include incubations in the presence and absence of ß-nicotinamide adenine dinucleotide 2'-phosphate reduced (ß-NADPH).
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Footnotes |
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ABBREVIATIONS: P450, cytochrome P450; mAbs, monoclonal antibodies; FD, fluorescein diacetate; CES, carboxylesterase; PXR, pregnane X receptor; CES, carboxylesterase; PON1, paraoxonase-1.
1 Current affiliation: Department of Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Inc., King of Prussia, PA. 
Address correspondence to: Stacey L. Polsky-Fisher, M.S., Department of Drug Metabolism, WP75B-200, Merck Research Laboratories, West Point, PA 19486. E-mail: stacey_polsky{at}merck.com
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–) and ketoconazole (–
–) are shown with apparent IC50 values of 18.0 and 6.5 µM, respectively.