Meclizine, a pregnane X receptor agonist, is a direct inhibitor and mechanism-based inactivator of human cytochrome P450 3A

Biochemical Pharmacology 97 (2015) 320–330

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Meclizine, a pregnane X receptor agonist, is a direct inhibitor and mechanism-based inactivator of human cytochrome P450 3A Winnie Yin Bing Fooa , Hwee Ying Taya , Eric Chun Yong Chana , Aik Jiang Laua,b,* a b

Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 June 2015 Accepted 29 July 2015 Available online 31 July 2015

Meclizine is an agonist of human pregnane X receptor (PXR). It increases CYP3A4 mRNA expression, but decreases CYP3A-catalyzed testosterone 6b-hydroxylation in primary cultures of human hepatocytes, as assessed at 24 h after the last dose of meclizine. Therefore, the hypothesis to be tested is that meclizine inactivates human CYP3A enzymes. Our findings indicated that meclizine directly inhibited testosterone 6b-hydroxylation catalyzed by human liver microsomes, recombinant CYP3A4, and recombinant CYP3A5. The inhibition of human liver microsomal testosterone 6b-hydroxylation by meclizine occurred by a mixed mode and with an apparent Ki of 31
6 mM. Preincubation of meclizine with human liver microsomes and NADPH resulted in a time- and concentration-dependent decrease in testosterone 6bhydroxylation. The extent of inactivation required the presence of NADPH, was unaffected by nucleophilic trapping agents or reactive oxygen species scavengers, attenuated by a CYP3A substrate, and not reversed by dialysis. Meclizine selectively inactivated CYP3A4, but not CYP3A5. In contrast to meclizine, which has a di-substituted piperazine ring, norchlorcyclizine, which is a N-debenzylated meclizine metabolite with a mono-substituted piperazine ring, did not inactivate but directly inhibited hepatic microsomal CYP3A activity. In conclusion, meclizine inhibited human CYP3A enzymes by both direct inhibition and mechanism-based inactivation. In contrast, norchlorcyclizine is a direct inhibitor but not a mechanism-based inactivator. Furthermore, a PXR agonist may also be an inhibitor of a PXRregulated enzyme, thereby giving rise to opposing effects on the functional activity of the enzyme and indicating the importance of measuring the catalytic activity of nuclear receptor-regulated enzymes. ã 2015 Elsevier Inc. All rights reserved.

Chemical compounds studied in this article: Meclizine dihydrochloride monohydrate (PubChem CID: 173612) Norchlorcyclizine (PubChem CID: 9340) 6b-Hydroxytestosterone (PubChem CID: 65543) Testosterone (PubChem CID: 6013) Prednisolone (PubChem CID: 5755) Ketoconazole (PubChem CID: 456201) Erythromycin (PubChem CID: 12560) Glutathione (PubChem CID: 124886) N-acetylcysteine (PubChem CID: 12035) Keywords: CYP3A Inhibition Mechanism-based inactivation Meclizine Norchlorcyclizine Pregnane X receptor

1. Introduction Meclizine, which is a piperazine-type first-generation histamine H1 antagonist (Fig. 1A), is commonly used for the treatment and prevention of nausea and vomiting in various conditions, including vertigo and motion sickness [1]. Animal studies have shown that meclizine have other pharmacological effects; for example,

Abbreviations: Ki, inhibition constant or equilibrium dissociation constant for the enzyme–inhibitor complex; KI, inactivator concentration at half-maximal rate of inactivation; kinactivation, maximal inactivation rate constant; Km, Michaelis– Menten constant; kobs, observed first-order rate constant for inactivation; MS, mass spectrometry; t1/2, time required for half of the enzyme molecules to be inactivated; PXR, pregnane X receptor; UPLC, ultra-high performance liquid chromatography. * Corresponding author at: Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, 117543, Singapore. Fax: +65 6779 1554. E-mail addresses: [email protected] (W.Y.B. Foo), [email protected] (H.Y. Tay), [email protected] (E.C.Y. Chan), [email protected] (A.J. Lau). http://dx.doi.org/10.1016/j.bcp.2015.07.036 0006-2952/ ã 2015 Elsevier Inc. All rights reserved.

neuroprotection in Huntington’s disease models [2], cardioprotection and neuroprotection against ischemia-reperfusion injury [3], inhibition of mitochondrial respiration and energy metabolism [4], and promotion of skeletal growth in transgenic mice with achondroplasia [5]. The parent drug is excreted unchanged or eliminated as various metabolites in human feces and urine [6]. Among the ten metabolites identified, norchlorcyclizine, which is a Ndebenzylated metabolite, is one of the major metabolites detected in human urine (Fig.1B) [6]. The enzymes responsible for catalyzing the formation of those metabolites were not investigated in that study. In a separate study, meclizine was reported to be metabolized predominantly by cytochrome P450 2D6 (CYP2D6), but the structure of the metabolite of the CYP2D6-catalyzed oxidative reaction was not elucidated [1]. Despite the long history in the therapeutic use of meclizine, human pharmacokinetic and metabolism studies on meclizine reported in the literature are sparse. Cytochrome P450 3A (CYP3A) is the most abundant cytochrome P450 enzymes expressed in human liver, accounting for

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Fig. 1. Chemical structure of (A) meclizine and (B) norchlorcyclizine.

approximately 30% of the total hepatic cytochrome P450 enzymes [7,8]. Isoforms of CYP3A include CYP3A4 and CYP3A5, which are expressed predominantly in the liver and gastrointestinal tract [8]. CYP3A induction is regulated by several members of the superfamily of nuclear receptors, including pregnane X receptor (PXR) [9]. CYP3A enzymes catalyze the oxidative metabolism of many endogenous and exogenous substances. In fact, CYP3A4 metabolizes the largest proportion of drugs (>30%) compared to other cytochrome P450 enzymes [10]. Induction and inhibition of CYP3A enzymes are mechanisms underlying many of the pharmacokinetic-type of drug–drug interactions, which may alter drug efficacy and/or toxicity [11]. Previously, meclizine was identified as an agonist of human PXR [12]. It increased CYP3A4 mRNA expression, but decreased CYP3Acatalyzed testosterone 6b-hydroxylation in primary cultures of human hepatocytes [12]. The decrease in CYP3A activity was still evident at 24 h after the last dose of meclizine, and the hepatocytes were washed at the end of the treatment period prior to the addition of testosterone substrate. Therefore, the hypothesis to be tested is that meclizine inhibits the catalytic activity of human CYP3A enzymes. The current study was designed to determine whether meclizine is a direct inhibitor of CYP3A and to characterize the enzyme kinetics and mechanism of CYP3A inhibition. We also determined whether meclizine is a mechanism-based inactivator of CYP3A and characterized the kinetics of inactivation. Piperazine ring has been implicated in mechanism-based inactivation [13]. Norchlorcyclizine has a mono-substituted piperazine ring, whereas meclizine has a di-substituted piperazine ring (Fig. 1). Therefore, we investigated whether the mono-substituted norchlorcyclizine, which is a N-debenzylated metabolite of meclizine present in the largest quantity in human urine [6], plays a role in the inactivation of human liver microsomal CYP3A by meclizine. Overall, our findings indicate that meclizine, which is a PXR agonist [12], can inhibit and inactivate CYP3A, thereby leading to opposing effects on the functional activity of the enzyme and indicating the importance of measuring not only the mRNA and protein expression, but also the catalytic activity of nuclear receptor-regulated enzymes. 2. Materials and methods 2.1. Chemicals and reagents Meclizine dihydrochloride monohydrate and norchlorcyclizine [1-[(4-chloro)phenylmethyl]-piperazine] were purchased directly from Toronto Research Chemicals Inc. (North York, ON, Canada).

6b-Hydroxytestosterone (Cayman Chemical Co., Ann Arbor, MI, U. S.A.) was purchased from iDNA Biotechnology Pte. Ltd., Singapore. Testosterone, prednisolone, NADPH, ketoconazole, erythromycin, glutathione, N-acetylcysteine, catalase, superoxide dismutase, and DMSO were bought from Sigma–Aldrich Pte., Ltd., Singapore. All other commercially available chemicals were of analytical or high performance liquid chromatographic grade. 2.2. Human liver microsomes and recombinant enzymes Human liver microsomes (pooled from 50 individual donors; catalog #452156; lot 88114), human recombinant CYP3A4 (catalog #456202), human recombinant CYP3A5 (catalog #456256), and control insect cell microsomes co-expressing NADPH-cytochrome P450 reductase and cytochrome b5 (catalog #456244) were purchased from BD Gentest (Woburn, MA, U.S.A.) via Bio Laboratories Pte., Ltd., Singapore. 2.3. Testosterone 6b-hydroxylation assay Unless specified otherwise, each standard 200 ml incubation mixture contained potassium phosphate buffer (100 mM, pH 7.4), NADPH (1 mM), testosterone (40 mM for human liver microsomal experiments or 20 mM for recombinant CYP3A4 and CYP3A5 experiments), and human liver microsomes (60 mg protein), recombinant CYP3A4 (2 pmol), or recombinant CYP3A5 (2 pmol). Each incubation mixture was pre-warmed for 3 min at 37  C in a shaking water bath. Enzymatic reaction was initiated by adding NADPH and terminated 10 min later by adding 200 ml icecold acetonitrile containing prednisolone (1 mM final concentration; internal standard). Each sample was mixed on a vortex, placed immediately in an ice bath, and centrifuged at 16,000  g for 15 min at 4  C. The supernatant was then transferred to a 96-well microplate for analysis of 6b-hydroxytestosterone and prednisolone by UPLC–MS–MS. To construct a calibration curve for each experiment, 6b-hydroxytestosterone (final concentrations of 0.1– 20 mM in 0.1% v/v DMSO) was freshly prepared from stock solutions (0.1–20 mM in DMSO), added to the standard incubation mixture but without enzymes or NADPH, and subjected to the same procedures as described above. Enzyme kinetic analysis of testosterone 6b-hydroxylation was performed at substrate concentrations ranging from 6.25 to 400 mM. In the human liver microsomal and recombinant CYP3A5 experiments, the values of Vmax and apparent Km were estimated from nonlinear leastsquares regression analysis (GraphPad Prism 6.0; Graphpad

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Software Inc., San Diego, CA, U.S.A.) of the rate of enzyme activity (V) and substrate concentration (S) data using the Michaelis– Menten model: V¼

V max ½S K m þ ½S

In the recombinant CYP3A4 experiment, the values of Vmax and apparent Km were estimated using the substrate inhibition model: V¼

V max ½S

reaction in the secondary incubation mixture was incubated for 10 min at 37  C and terminated by adding 200 ml ice-cold acetonitrile containing prednisolone (1 mM final concentration; internal standard). Ketoconazole (0.05 mM; negative control), which is a known direct inhibitor and not a mechanism-based inactivator [14], and erythromycin (50 mM; positive control), which is a mechanism-based inactivator of CYP3A [16], were used as controls. The observed first order rate constant for inactivation (kobs) was calculated by the equation: At ¼ A0 ekobs t

K m þ ½S þ ½S2 =K s

where Ks represents the equilibrium dissociation constant between the substrate and the binding site of the enzyme. Based on visual inspection and various measures of goodness of fit, including r2, absolute sum of squares and Sy.x, the substrate inhibition model was better than the Michaelis–Menten model in fitting the CYP3A4 data.

where At is the enzyme activity at time t and A0 is the enzyme activity at 0 min. The maximal inactivation rate constant (kinactivation) and half-maximal inactivation concentration (KI) were calculated by the following equation [17] using nonlinear least-squares regression (GraphPad Prism 6.0; Graphpad Software Inc., San Diego, CA, U.S.A.):

2.4. Enzyme inhibition experiments

kobs ¼

The inhibition experiments were conducted in the presence of varying concentrations of meclizine (0.1–300 mM), norchlorcyclizine (1–300 mM), or methanol (0.5% v/v; vehicle), as described in each figure legend. Ketoconazole (1 mM), which is a direct inhibitor of CYP3A [14], was used as a positive control. In each incubation mixture, the final concentration of methanol was 0.5% v/v, which did not affect CYP3A-catalyzed testosterone 6b-hydroxylation activity [15]. Enzymatic reaction was initiated by adding NADPH. The half-maximal inhibitory concentration (IC50) was determined from a concentration–response curve using the equation:

where I is the inactivator concentration. The time required for half of the enzyme molecules to be inactivated (t1/2) was determined by the equation:

Effect ¼ E0 þ

Emax  E0 1 þ 10½ðlog IC50 log½IÞHill

Slope

where I is the inhibitor concentration, E0 is the minimum effect, and Emax is the maximum effect (GraphPad Prism 6.0; Graphpad Software Inc., San Diego, CA, U.S.A.). To characterize the enzyme kinetics of CYP3A inhibition, multiple concentrations of meclizine (0, 7.5, 30, and 75 mM) and testosterone (20, 40, 80, and 160 mM) were used. The apparent Ki value (equilibrium dissociation constant for the enzyme–inhibitor complex) and mode of inhibition were determined by nonlinear least-squares regression analysis of the metabolite formation data collected at various substrate and inhibitor concentrations, using equations for competitive, noncompetitive, uncompetitive, and mixed model inhibition (GraphPad Prism 6.0; Graphpad Software Inc., San Diego, CA, U.S.A.). The Akaike information criterion was used as a measure of goodness of fit. The mode of inhibition was further verified by visual inspection of the Lineweaver–Burk plot of the enzyme kinetic data. 2.5. Enzyme inactivation experiments Primary incubation mixture (200 ml) containing potassium phosphate buffer (100 mM, pH 7.4), meclizine (15, 30, 60, or 120 mM), norchlorcyclizine (120, 240, or 360 mM), or methanol (0.25% v/v; vehicle) in the presence or absence of NADPH (1 mM) was pre-warmed for 2 min at 37  C in a shaking water bath. Enzymatic reaction was initiated by adding human liver microsomes (100 mg protein), recombinant CYP3A4 (5 pmol), or recombinant CYP3A5 (5 pmol) and preincubated for various time (as specified in the figure legends). Subsequently, an aliquot (10 ml) of the primary incubation mixture was transferred to 190 ml of pre-warmed (for 3 min at 37  C) secondary incubation mixture (total volume of 200 ml) containing potassium phosphate buffer, testosterone (200 mM), and NADPH (1 mM). The enzymatic

t1=2 ¼

kinactivation ½I K I þ ½I

ln 2 kinactivation

2.6. Effects of exogenous nucleophilic trapping agents and scavengers of reactive oxygen species An exogenous nucleophilic trapping agent (5 or 10 mM GSH or N-acetylcysteine) or a scavenger of reactive oxygen species (1000 U or 2000 U catalase or 500 U or 1000 U superoxide dismutase) was preincubated with meclizine (120 mM), human liver microsomes (100 mg protein), and NADPH (1 mM) in the primary incubation mixture (200 ml). The control experiments include primary incubation mixture without both the trapping agent/scavenger and meclizine and that without the trapping agent/scavenger only. Each standard incubation mixture was subjected to the same procedures as described under Section 2.5. The residual CYP3A4 activity was determined by the testosterone 6b-hydroxylation assay. 2.7. Effects of a competing CYP3A substrate Testosterone was included in the primary incubation mixture (200 ml). Final concentrations of testosterone were 15, 30, 60 and 120 mM, corresponding to a molar ratio of testosterone to meclizine of 0.125, 0.25, 0.5, and 1, respectively. The control experiments include primary incubation mixture without both testosterone and meclizine and that without testosterone or meclizine only. Each standard incubation mixture was subjected to the same procedures as described under Section 2.5. The residual CYP3A4 activity was determined by the testosterone 6b-hydroxylation assay. 2.8. Dialysis experiment Primary incubation mixture (200 ml) containing meclizine (120 mM) or methanol (0.25% v/v; vehicle), NADPH (1 mM), and potassium phosphate buffer (100 mM, pH 7.4) was pre-warmed for 2 min at 37  C in a shaking water bath. Enzymatic reaction was initiated by adding human liver microsomes (100 mg protein), and the mixture was preincubated for 0 or 30 min. Subsequently, an aliquot (10 ml) of primary incubation mixture was transferred to

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190 ml of prewarmed (for 3 min at 37  C) secondary incubation mixture (200 ml) containing potassium phosphate buffer, testosterone (200 mM), and NADPH (1 mM). Simultaneously, another aliquot (100 ml) of the primary incubation mixture was transferred to a Slide-A-Lyzer mini dialysis device (0.1 ml) with a molecular weight cutoff of 10,000 (Pierce Chemical Co., Rockford, IL, U.S.A.) and placed in a beaker filled with 500 ml of ice-cold potassium phosphate buffer (100 mM, pH 7.4). Dialysis was performed on ice for 4 h with constant gentle stirring. Subsequently, 10 ml of the dialyzed mixture was transferred to the secondary incubation mixture and the residual CYP3A4 activity was determined by the testosterone 6b-hydroxylation assay. 2.9. Partition ratio determination Primary incubation mixture (200 ml) containing meclizine (0.1– 100 mM) or methanol (0.25% v/v; vehicle), NADPH (1 mM), and potassium phosphate buffer (100 mM, pH 7.4) was pre-warmed for 2 min at 37  C in a shaking water bath. Enzymatic reaction was initiated by adding recombinant CYP3A4 (1 pmol/ml), and the mixture was preincubated for 0 or 30 min. Each standard incubation mixture was subjected to the same procedures as described under Section 2.5. The residual CYP3A4 activity was determined by the testosterone 6b-hydroxylation assay. 2.10. Quantification of 6b-hydroxytestosterone by UPLC–MS–MS The amount of 6b-hydroxytestosterone and prednisolone (internal standard) was quantified using published UPLC–MS– MS methods [18,19], but with modifications. The UPLC–MS–MS system consisted of an ACQUITY UPLC system (Waters, Milford, MA, U.S.A.) (distributed by Waters Pacific Pte., Ltd., Singapore) interfaced with a hybrid quadrupole linear ion-trap mass spectrometer equipped with a TurboIonSpray electrospray ionization (ESI) source (QTRAP 3200; Applied Biosystems, Foster City, CA, U.S.A.) (distributed by Applied Biosystems Sciex, Singapore). The UPLC and MS systems were controlled by Analyst 1.4.2 software (Applied Biosystems). Chromatography separation was achieved using a Waters ACQUITY UPLC BEH C18 column (2.1  50 mm i.d., 1.7 mm). The mobile phases were 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The elution conditions were optimized as follows: isocratic at 20% B (0.00– 1.00 min), linear gradient from 20% to 70% B (1.01–3.40 min), isocratic at 95% B (3.41–3.99 min), and isocratic at 20% B (4.00– 4.50 min). Column and sample temperatures were set at 45  C and 4  C, respectively. Flow rate was 0.5 ml/min and the injection volume was 5 ml. UPLC effluent was introduced directly into the mass spectrometer interface from 1.5 to 3.5 min. 6b-Hydroxytestosterone and prednisolone were analyzed using positive ESI mode and multiple reaction monitoring transitions of mass-to-charge (m/z) ratios from 305 to 269 and from 361 to 343, respectively. The MS source conditions were as follows: curtain gas, 20 psi; collision gas, medium; ion spray voltage, 5500 V; temperature, 550  C; ion source gas 1, 40 psi; and ion source gas 2, 45 psi. The compounddependent MS parameters for 6b-hydroxytestosterone were as follows: entrance potential, 6 V; collision energy, 20 V; collision cell entrance potential, 20 V; collision cell exit potential, 3 V; declustering potential, 52 V; and dwell time, 250 ms. The compound-dependent MS parameters for prednisolone were as follows: entrance potential, 3 V; collision energy, 15 V; collision cell entrance potential, 22 V; collision cell exit potential, 3 V; declustering potential, 52 V; and dwell time, 250 ms. A calibration curve was constructed using weighted (1/x2) linear least-squares regression analysis of the peak area ratio (6b-hydroxytestosterone to prednisolone) versus amount of the metabolite in the incubation mixture.

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2.11. Statistical analysis Data were analyzed by one- or two-way ANOVA and, where appropriate, was followed by the Student Newman–Keuls multiple comparison test (GraphPad Prism 3.0, GraphPad Software, Inc., La Jolla, CA, U.S.A. and SigmaPlot 11.0, Systat Software, Inc., San Jose, CA, U.S.A.). The level of statistical significance was set a priori at p < 0.05. 3. Results 3.1. Validation of the UPLC–MS–MS method The blank sample (incubations containing heat-inactivated microsomes but without analyte) did not display peaks at the m/z transition corresponding to 6b-hydroxytestosterone or prednisolone (internal standard), thereby demonstrating specificity and lack of interference by the matrix. In quality control samples containing low (0.2 mM), medium (2 mM), or high (20 mM) concentrations of analyte, the peak area ratio of the analyte to the internal standard in incubations containing heat-inactivated human liver microsomes was not significantly different (p > 0.05) from that obtained in incubations without enzyme, indicating that the matrix did not interfere with the magnitude of the detector response. The lower limit of quantification for 6bhydroxytestosterone was 10 pmol with a signal/noise ratio of 5:1, accuracy of
20%, and precision of
20%. The calibration curve was linear from 10 to 4000 pmol, as assessed by the coefficient of determination (r2 > 0.99) and visual inspection of the regression line. The measured concentration of each standard was within 15% of the nominal concentration. Determination of intraday (n = 6) and interday (n = 3) precision and accuracy of low (0.15 mM), medium (1.5 mM), and high (15 mM) analyte concentration levels showed a% CV of <14.0% and a% bias of <14.7%, respectively. 3.2. Optimization of the testosterone 6b-hydroxylation assay The testosterone 6b-hydroxylation assay was linear with respect to the amount of enzyme (20–100 mg of human liver microsomes; 1–5 pmol of recombinant CYP3A4; and 1–5 pmol of recombinant CYP3A5) and incubation time (up to 15 min for human liver microsomes and recombinant CYP3A5; up to 10 min for recombinant CYP3A4). Unless otherwise specified, the subsequent testosterone 6b-hydroxylation assay in the direct inhibition experiments was performed with 60 mg of human liver microsomes, 2 pmol of recombinant CYP3A4 or CYP3A5, and incubation time of 10 min. 3.3. Concentration–response relationship on the inhibition of human liver microsomal-, CYP3A4-, and CYP3A5-catalyzed testosterone 6bhydroxylation by meclizine To determine whether meclizine directly inhibits microsomal CYP3A, recombinant CYP3A4 or CYP3A5, meclizine or 0.5% v/v methanol (vehicle) was incubated with testosterone and human liver microsomes, recombinant CYP3A4 or CYP3A5. Meclizine (100 mM) decreased microsomal-, CYP3A4-, and CYP3A5-catalyzed testosterone 6b-hydroxylation by 46%, 88%, and 67%, respectively. Comparatively, ketoconazole (1 mM), which is a potent direct CYP3A inhibitor [14], decreased testosterone 6b-hydroxylation catalyzed by human liver microsomes (96%), CYP3A4 (97%), and CYP3A5 (90%). As shown in Fig. 2, meclizine decreased testosterone 6b-hydroxylation in a log-linear manner at meclizine concentrations of 1–60 mM (human liver microsomes), 1–10 mM (CYP3A4), and 3–60 mM (CYP3A5). Meclizine exhibited a more

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Fig. 2. Concentration–response relationship on the inhibition of human liver microsomal-, CYP3A4-, and CYP3A5-catalyzed testosterone 6b-hydroxylation by meclizine. Pooled human liver microsomes (60 mg protein), recombinant CYP3A4 (2 pmol), or recombinant CYP3A5 (2 pmol) was incubated with NADPH (1 mM), testosterone (40 mM for microsomal experiment; 20 mM for CYP3A4 and CYP3A5 experiments), and meclizine (1, 3, 10, 30, 60, 100, or 300 mM for microsomal experiment; 0.1, 0.3, 1, 3, 10, 30, 60, or 100 mM for CYP3A4 experiment; and 0.3, 1, 3, 10, 30, 60, 100, or 300 mM for CYP3A5 experiment) or methanol (0.5% v/ v; vehicle) at 37  C for 10 min. The amount of 6b-hydroxytestosterone was quantified by UPLC–MS–MS as described under Section 2. Data are normalized to the enzymatic activity of the respective vehicle-treated control group and expressed as mean
S.E.M. for three or four independent experiments. Testosterone 6b-hydroxylation in the vehicle-treated control group was 2625
108 pmol/ min/mg protein, 46
1 pmol/min/pmol CYP3A4, and 48
2 pmol/min/pmol CYP3A5 in incubations containing human liver microsomes, CYP3A4, and CYP3A5, respectively. * Significantly different from the vehicle-treated control group (p < 0.05).

potent and complete inhibition of recombinant CYP3A4 than recombinant CYP3A5 or microsomal CYP3A, as shown by the leftward shift in the CYP3A4 dose–response curve and the decrease in the bottom plateau of the curve. The IC50 values for meclizine inhibition of testosterone 6b-hydroxylation catalyzed by human liver microsomes, CYP3A4, and CYP3A5 were 9
1, 3
0.2, and 12
1 mM, respectively. 3.4. Enzyme kinetic analysis of the inhibition of human liver microsomal CYP3A-catalyzed testosterone 6b-hydroxylation by meclizine The apparent Km values were 32
2 mM, 38
2 mM, and 19
3 mM, whereas the Vmax values were 3259
346 pmol/min/ mg protein, 152
4 pmol/min/pmol CYP3A4, and 77
4 pmol/min/ pmol CYP3A5 for testosterone 6b-hydroxylation catalyzed by human liver microsomes, recombinant CYP3A4, and recombinant CYP3A5, respectively. These values were comparable with the values in the literature [20,21]. To determine the apparent Ki and mode of inhibition of testosterone 6b-hydroxylation catalyzed by human liver microsomes, enzyme kinetic experiments were performed with multiple inhibitor concentrations (0, 7.5, 30, and 75 mM) and multiple substrate concentrations (20, 40, 80, and 160 mM). Nonlinear least-squares regression analysis and Lineweaver–Burk plot of the enzyme kinetic data indicated that meclizine inhibited CYP3A-mediated testosterone 6b-hydroxylation by a mixed mode of inhibition (Fig. 3). The apparent Ki value was 31
6 mM. As indicated above, the apparent Km value was 32
2 mM for hepatic microsomal CYP3A-mediated testosterone 6b-hydroxylation. Therefore, the relative inhibition potency was 1.0, as calculated by the ratio of apparent Ki to apparent Km.

Fig. 3. Lineweaver–Burk plot for the inhibition of human liver microsomal CYP3Acatalyzed testosterone 6b-hydroxylation by meclizine. Pooled human liver microsomes (60 mg protein) were incubated with testosterone (20, 40, 80, or 160 mM) and meclizine (0, 7.5, 30, or 75 mM) at 37  C for 10 min. The amount of 6bhydroxytestosterone was quantified by UPLC–MS–MS as described under Section 2. Data are expressed as mean
S.E.M. for three independent experiments.

3.5. Inactivation of human liver microsomal CYP3A by meclizine To investigate whether meclizine inactivates CYP3A, meclizine was preincubated with human liver microsomes and NADPH at 37  C for 30 min prior to transferring an aliquot of the primary incubation mixture into a secondary incubation mixture for the conduct of the testosterone 6b-hydroxylation assay. As shown in Fig. 4A, preincubation of meclizine with microsomes and NADPH decreased testosterone 6b-hydroxylation by 70% as compared to the vehicle-treated control group, whereas erythromycin (50 mM), a mechanism-based inactivator of CYP3A [16], decreased it by 75%. In contrast, ketoconazole (0.05 mM), which is a known direct inhibitor and not a mechanism-based inactivator [14], did not show inactivation of microsomal CYP3A. 3.6. Kinetics of the inactivation of human liver microsomal CYP3A by meclizine To characterize in detail the kinetics of inactivation, varying concentrations of meclizine was preincubated with human liver microsomes and NADPH for varying lengths of time. As shown in Fig. 4B, testosterone 6b-hydroxylation declined in a time- and concentration-dependent manner when the microsomes were preincubated with meclizine in the presence of NADPH for up to 6 min. Enzyme kinetic analysis of CYP3A inactivation by meclizine was conducted by the non-linear regression plot of kobs versus meclizine concentration (Fig. 4C) and the Kitz–Wilson plot (Fig. 4D). The rate constant for maximal inactivation at saturation (kinactivation) was 0.15 min1. The time required for half of the enzymes to be inactivated (t1/2) was 4.6 min. The concentration of meclizine required to produce half the maximal rate of CYP3A inactivation (KI) was 83
14 mM. The ratio of kinactivation to KI, which is used to assess the efficiency of enzyme inactivation [22], was 0.002 min1 mM1. 3.7. NADPH-dependency in the inactivation of human liver microsomal CYP3A by meclizine To assess whether the inactivation of CYP3A by meclizine required NADPH, meclizine was preincubated with human liver microsomes at 37  C for varying length of time in the presence or absence of NADPH. In the absence of NADPH, meclizine decreased

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Fig. 4. Time- and concentration-dependent inactivation of human liver microsomal CYP3A by meclizine. (A) Pooled human liver microsomes (100 mg protein) were preincubated with NADPH (1 mM) and meclizine (120 mM), ketoconazole (0.05 mM), erythromycin (50 mM), or methanol (0.25% v/v; vehicle) at 37  C for 0 or 30 min. (B) Pooled human liver microsomes (100 mg protein) were preincubated with NADPH (1 mM) and meclizine (15, 30, 60, or 120 mM) or methanol (0.25% v/v; vehicle) at 37  C for 0, 2, 4, or 6 min. An aliquot (10 ml) of the primary incubation mixture was transferred to a secondary incubation mixture containing testosterone (200 mM) and NADPH (1 mM). Testosterone 6b-hydroxylation was determined as described under Section 2. Data are normalized to the enzymatic activity of the vehicle-treated control group that was not subjected to preincubation and expressed as mean
S.E.M. for three to four independent experiments. Testosterone 6b-hydroxylation in the vehicle-treated control group that was not subjected to preincubation was (A) 4282
676 pmol/min/mg protein and (B) 5469
239 pmol/min/mg protein. * Significantly different from the vehicle-treated control group subjected to 30 min of preincubation and the same treatment group that was not subjected to preincubation (p < 0.05). (C) A nonlinear regression plot of the observed inactivation rate constant (kobs) versus meclizine concentration. (D) A Kitz–Wilson plot of 1/kobs versus the inverse of meclizine concentration.

testosterone 6b-hydroxylation from 76
7% to 49
7% of the vehicle-treated control at 0 and 7.5 min of preincubation, respectively, but it did not further decrease CYP3A activity after 7.5 min preincubation (Fig. 5). In the presence of NADPH, meclizine decreased testosterone 6b-hydroxylation from 73
6% to 42
5% of the vehicle-treated control at 0 and 7.5 min of preincubation, respectively, and to 14
2% of the vehicle-treated control after 45 min of preincubation (Fig. 5). Given that the inactivation involved both NADPH and NADPH-dependent components (Fig. 5), the enzymatic reaction was initiated with enzymes instead of NADPH in subsequent inactivation experiments. 3.8. Lack of an effect by exogenous nucleophilic trapping agents and scavengers of reactive oxygen species on meclizine inactivation of human liver microsomal CYP3A To evaluate whether CYP3A inactivation by meclizine was confined to the active site, the drug or 0.25% v/v methanol (vehicle) was preincubated with human liver microsomes and NADPH at 37  C for 30 min in the presence or absence of nucleophilic trapping agents (glutathione and N-acetylcysteine) or scavengers of reactive oxygen species (catalase and superoxide dismutase). As shown in Fig. 6A, meclizine alone decreased testosterone 6b-hydroxylation to 32
9% of the vehicle-treated control after 30 min of preincubation, but the addition of glutathione (5 and 10 mM)

Fig. 5. Effect of NADPH on inactivation of human liver microsomal CYP3A by meclizine. Pooled human liver microsomes (100 mg protein) were preincubated with meclizine (0 or 120 mM) or methanol (0.25% v/v; vehicle) in the presence or absence of NADPH (1 mM) at 37  C for 0, 7.5, 15, 30, or 45 min. An aliquot (10 ml) of the primary incubation mixture was transferred to a secondary incubation mixture containing testosterone (200 mM) and NADPH (1 mM). Testosterone 6b-hydroxylation was determined as described under Section 2. Data are normalized to the enzymatic activity of the vehicle-treated control group that was not subjected to preincubation and expressed as mean
S.E.M. for four independent experiments. Testosterone 6b-hydroxylation in the vehicle-treated control group that was not subjected to preincubation was 5705
336 pmol/min/mg protein.

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Fig. 6. Effect of nucleophilic trapping agents, scavengers of reactive oxygen species, and a CYP3A substrate on inactivation of human liver microsomal CYP3A by meclizine. Pooled human liver microsomes (100 mg protein) were preincubated with NADPH (1 mM), meclizine (120 mM) or methanol (0.25% v/v; vehicle), and (A) glutathione (5 or 10 mM), N-acetylcysteine (5 or 10 mM), (B) catalase (1000 or 2000 U), superoxide dismutase (500 or 1000 U), or (C) testosterone (15, 30, 60 and 120 mM) at 37  C for 0 or 30 min. An aliquot (10 ml) of the primary incubation mixture was transferred to a secondary incubation mixture containing testosterone (200 mM) and NADPH (1 mM). Testosterone 6b-hydroxylation was determined as described under Section 2. Data are normalized to the enzymatic activity of the vehicle-treated control group that was not subjected to preincubation and expressed as mean
S.E.M. for three or four independent experiments. Testosterone 6b-hydroxylation activity in the vehicle-treated control group that was not subjected to preincubation was (A) 6940
285, (B) 6360
103, and (C) 8553
582 pmol/min/mg protein. * Significantly different from the vehicle-treated control group subjected to 30 min of preincubation and the same treatment group that was not subjected to preincubation (p < 0.05).

and N-acetylcysteine (5 and 10 mM) did not attenuate the inactivation by meclizine. Similarly, catalase (1000 and 2000 U) and superoxide dismutase (500 and 1000 U) did not protect CYP3A against the inactivation by meclizine (Fig. 6B). 3.9. Attenuation of meclizine inactivation of human liver microsomal CYP3A by a CYP3A substrate To investigate whether CYP3A inactivation by meclizine was protected by the presence of a competing CYP3A substrate, varying concentrations of a CYP3A substrate were preincubated with meclizine or 0.25% v/v methanol (vehicle), human liver microsomes, and NADPH at 37  C for 30 min. We selected testosterone as the CYP3A substrate because CYP3A has a large active site with multiple substrate-binding sites [23]. This approach of using testosterone as a competing substrate was used in other published studies [18,24–26], and the selected testosterone concentrations did not affect the subsequent testosterone 6b-hydroxylation assay due to the 20 dilution of the primary incubation mixture (data not shown). As shown in Fig. 6C, meclizine decreased the activity to 19
2% of the vehicletreated control after 30 min of preincubation, and increasing concentrations of testosterone attenuated the extent of the inactivation. Testosterone at concentrations of 15, 30, 60, and 120 mM corresponded to a molar ratio of testosterone to meclizine of 0.125, 0.25, 0.5, and 1, respectively. At a molar ratio of 0.125 and 0.25, the activity was increased to 36
4% and 36
3% of the vehicle-treated control, respectively. When the molar ratio was increased to 0.5 and 1, the activity was restored to that of the vehicle-treated control and the meclizine-treated group that was not subjected to preincubation.

3.10. Irreversible inactivation of human liver microsomal CYP3A by meclizine To examine whether the inactivation of human liver microsomal CYP3A by meclizine was reversible, the drug or 0.25% v/v methanol (vehicle) was preincubated with microsomes and NADPH at 37  C for 30 min before an aliquot was subjected to dialysis at 4  C for 4 h. These samples were also compared with samples that were not dialyzed. Fig. 7 shows that dialysis did not affect the magnitude of the inactivation of human liver microsomal CYP3A-catalyzed testosterone 6b-hydroxylation by meclizine. 3.11. Inactivation of recombinant CYP3A4 and CYP3A5 by meclizine To investigate which CYP3A enzyme plays a role in the inactivation of CYP3A by meclizine, enzyme inactivation experiments were performed using recombinant CYP3A4 and CYP3A5 in the presence or absence of NADPH. Preincubation of 0.25% v/v methanol (vehicle) with the recombinant enzymes for 15 min decreased testosterone 6b-hydroxylation by 7% in the absence of NADPH and 33% in the presence of NADPH. This is possibly due to NADPH-induced generation of reactive oxygen species, which have destructive effects on the enzymes [27]. Therefore, data in the recombinant enzyme experiments were normalized to the enzymatic activity of the respective vehicle-treated control group according to the length of preincubation time and the presence or absence of NADPH. In the absence of NADPH, meclizine decreased the catalytic activity to 38
4% of the vehicle-treated control, whereas in the presence of NADPH, meclizine decreased the activity to 11
2% of the vehicle-treated control after 15 min of preincubation (Fig. 8A). In contrast, meclizine did not decrease

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activity versus molar ratios of meclizine to CYP3A4. The turnover

Fig. 7. Effect of dialysis on inactivation of human liver microsomal CYP3A by meclizine. Pooled human liver microsomes (100 mg protein) were preincubated with NADPH (1 mM) and meclizine (120 mM) or methanol (0.25% v/v; vehicle) at 37  C for 0 or 30 min. An aliquot (10 ml) of the primary incubation mixture was transferred directly to a secondary incubation mixture containing testosterone (200 mM) and NADPH (1 mM), while an aliquot (100 ml) was subjected to 4 h of dialysis on ice before an aliquot (10 ml) of the dialyzed sample was transferred to the secondary incubation mixture. Testosterone 6b-hydroxylation was determined as described under Section 2. Data are normalized to the enzymatic activity of the nondialyzed vehicle-treated control group and expressed as mean
S.E.M. for three independent experiments. Testosterone 6b-hydroxylation in the non-dialyzed vehicle-treated control group was 4400
314 pmol/min/mg protein.

testosterone 6b-hydroxylation catalyzed by recombinant CYP3A5 enzymes after 15 min of preincubation (Fig. 8B). 3.12. Partition ratio for the inactivation of recombinant CYP3A4 by meclizine Given that meclizine inactivated CYP3A4 but not CYP3A5, we next determined the partition ratio for the inactivation of recombinant CYP3A4 by meclizine. A titration method was used for determining partition ratio, which is defined as the number of inactivator molecules metabolized per molecule of enzyme inactivated, and it is used to measure efficiency for inactivation [17]. Varying concentrations of meclizine (0–150 mM) were preincubated with CYP3A4 and NADPH for 30 min to ensure that the reaction was complete. Shown in Fig. 9 is a plot of CYP3A4

Fig. 9. Partition ratio for the inactivation of recombinant CYP3A4 by meclizine. Recombinant CYP3A4 (200 pmol) was preincubated with NADPH (1 mM) and meclizine (0.1, 0.3, 1, 3, 10, 30, 60, or 100 mM) or methanol (0.25% v/v; vehicle) at 37  C for 0 or 30 min. An aliquot (10 ml) of the primary incubation mixture was transferred to a secondary incubation mixture containing testosterone (200 mM) and NADPH (1 mM). Testosterone 6b-hydroxylation and partition ratio were determined as described under Section 2. Data are normalized to the enzymatic activity of the vehicle-treated control group that was not subjected to preincubation and expressed as mean
S.E.M. for five independent experiments.

number was 11.5, as obtained by extrapolating the intercept of the two linear regression lines to the x-axis. The partition ratio for CYP3A4 inactivation by meclizine was 10.5, as calculated by subtracting the turnover number by 1, assuming 1:1 stoichiometry between meclizine and recombinant CYP3A4. 3.13. Effect of a meclizine metabolite, norchlorcyclizine, on human liver microsomal CYP3A-mediated testosterone 6b-hydroxylation activity Meclizine was reported to undergo N-debenzylation to give the norchlorcyclizine metabolite in humans [6]. Similar to meclizine, norchlorcyclizine has a piperazine ring. It is not known whether this metabolite is responsible for the inactivation of CYP3A by the

Fig. 8. Effect of NADPH on inactivation of recombinant CYP3A4 and CYP3A5 by meclizine. (A) Recombinant CYP3A4 (5 pmol) or (B) CYP3A5 (5 pmol) was preincubated with meclizine (120 mM) or methanol (0.25% v/v; vehicle) in the presence or absence of NADPH (1 mM) at 37  C for 0 or 15 min. An aliquot (10 ml) of the primary incubation mixture was transferred to a secondary incubation mixture containing testosterone (200 mM) and NADPH (1 mM). Testosterone 6b-hydroxylation was determined as described under Section 2. Data are normalized to the enzymatic activity of the respective vehicle-treated control group (according to the preincubation time and the presence/absence of NADPH). Data are expressed as mean
S.E.M. for three independent experiments. * Significantly different from the same treatment group that did not contain NADPH and was not subjected to preincubation (p < 0.05).

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Fig. 10. Effect of norchlorcyclizine on human liver microsomal CYP3A activity. (A) Pooled human liver microsomes (100 mg protein) were preincubated with NADPH (1 mM) and norchlorcyclizine (120, 240, or 360 mM), meclizine (120 mM), ketoconazole (0.05 mM), erythromycin (50 mM), or methanol (0.25% v/v; vehicle) at 37  C for 0 or 30 min. An aliquot (10 ml) of the primary incubation mixture was transferred to a secondary incubation mixture containing testosterone (200 mM) and NADPH (1 mM). Testosterone 6bhydroxylation was determined as described under Section 2. Data are normalized to the enzymatic activity of the vehicle-treated control group that was not subjected to preincubation and expressed as mean
S.E.M. for three independent experiments. Testosterone 6b-hydroxylation in the vehicle-treated control group that was not subjected to preincubation was 8013
378 pmol/min/mg protein. * Significantly different from the vehicle-treated control group subjected to 30 min of preincubation and the same treatment group that was not subjected to preincubation (p < 0.05). (B) Pooled human liver microsomes (60 mg protein) were incubated with NADPH (1 mM), testosterone (40 mM), and norchlorcyclizine (1, 3, 10, 30, 60, 100, or 300 mM) or methanol (0.5% v/v; vehicle) at 37  C for 10 min. The amount of 6b-hydroxytestosterone was quantified by UPLC–MS–MS as described under Section 2. Data are normalized to the enzymatic activity of the vehicle-treated control group and expressed as mean
S.E.M. for four independent experiments. Testosterone 6b-hydroxylation in the vehicle-treated control group was 4444
306 pmol/min/mg protein. * Significantly different from the vehicle-treated control group (p < 0.05).

parent drug. Therefore, we investigated whether norchlorcyclizine is an inactivator of human CYP3A. Norchlorcyclizine, at concentrations of 120, 240, and 360 mM, did not decrease liver microsomal testosterone 6b-hydroxylation after 30 min of preincubation (Fig. 10A). In the same experiment, meclizine (120 mM) decreased the catalytic activity to 29
4% of the vehicle-treated control and erythromycin (50 mM), which is a mechanism-based inactivator of CYP3A [16], decreased the activity to 20
2%, whereas ketoconazole (0.05 mM), which is a known direct inhibitor and not a mechanism-based inactivator [14], had no effect. To compare with the parent drug, we investigated whether norchlorcyclizine directly inhibits human liver microsomal CYP3Acatalyzed testosterone 6b-hydroxylation activity. Norchlorcyclizine (100 mM) and ketoconazole (1 mM; positive control) [14] decreased CYP3A activity by 78% and 96%, respectively (data not shown). Concentration–response experiments indicated that norchlorcyclizine (0–300 mM) directly inhibited microsomal CYP3A activity in a log-linear manner at concentrations of 10– 100 mM and yielded an IC50 of 34
4 mM (Fig. 10B). 4. Discussion A conclusion in the present study is that meclizine directly inhibits human liver microsomal CYP3A-catalyzed testosterone 6b-hydroxylation by a mixed mode. Based on our enzyme kinetics experiments, the apparent Ki (31
6 mM) was similar to apparent Km (32
2 mM), indicating that the inhibition is likely to occur at a 1:1 molar ratio of meclizine to testosterone. As analyzed using recombinant enzymes, the drug directly inhibited both CYP3A4 and CYP3A5 isoforms. Previously, norchlorcyclizine, a Ndebenzylated metabolite of meclizine, was detected in human urine after ingestion of meclizine by human volunteers [6]. Similar to the parent drug, norchlorcyclizine is also a direct inhibitor of liver microsomal CYP3A. It is of interest to note that other

piperazine-type antihistamines, e.g., chlorcyclizine [28] and cetirizine [29], can also be metabolized to norchlorcyclizine. Another major conclusion is that meclizine is an irreversible mechanism-based inactivator of CYP3A. This conclusion is based on various experimental findings that the inactivation of CYP3A by meclizine was: (1) time- and concentration-dependent; (2) required NADPH (at longer time range); (3) not affected by nucleophilic trapping agents (glutathione and N-acetylcysteine) or scavengers of reactive oxygen species (catalase and superoxide dismutase); (4) protected by a competing CYP3A substrate (testosterone); (5) not reversed by dialysis; and (6) exhibited saturable kinetics. Therefore, meclizine fulfilled the criteria for mechanism-based inactivation [17]. The kinactivation value for the inactivation of human liver microsomal CYP3A by meclizine was similar to that for other inactivators, e.g., raloxifene [30], an order of magnitude greater than that for lapatinib [18], and an order of magnitude lesser than that for nicardipine [31]. The potency of CYP3A inactivation (KI) by meclizine was lesser than other drugs, e.g., erythromycin [32]. Norchlorcyclizine has a mono-substituted piperazine ring (Fig. 1B). Unlike meclizine, which has a di-substituted piperazine ring (Fig. 1A), norchorcyclizine is not a mechanism-based inactivator and not responsible for the inactivation of human liver microsomal CYP3A by meclizine. This novel finding suggests that the di-substituted piperazine ring or the benzyl substituent, which is present in meclizine but not norchlorcyclizine, may be implicated in the mechanism-based inactivation. It has been reported that hydroxylation of the carbon atom adjacent to a piperazine nitrogen could lead to generation of a reactive iminium intermediate that binds directly to CYP3A or undergo further NADPH-dependent metabolism to inactivate CYP3A [13]. The benzyl group has also been reported to form reactive intermediates and cause CYP inactivation [33]. Recently, two di-substituted piperazine chemicals (SCH66712 and EMTPP) were reported to

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inactivate recombinant CYP3A4 [34]. Other microsomal CYP3A mechanism-based inactivators with a di-substituted piperazine ring include imatinib [35], delavirdine [36], L-745,870 [33], and indinavir [37,38]. In the case of imatinib, it was postulated that the inactivation may be due to a reactive intermediate produced in the formation of N-desmethylimatinib [35]. Taken together with our findings, these observations suggest a role of the di-substituted piperazine ring or the N-dealkylated group of the piperazine ring in the mechanism-based inactivation of CYP3A. It is not known whether other di-substituted piperazine antihistamines (e.g., cetirizine, chlorcyclizine, and hydroxyzine) have similar effect. The time-dependent inactivation of CYP3A by meclizine involves NADPH-dependent and NADPH-independent pathways. A relatively fast NADPH-independent inactivation of CYP3A by meclizine occurred up to 7.5 min of preincubation. When the preincubation time increased beyond 7.5 min, further decrease in CYP3A activity occurred only in the presence of NADPH. This NADPH-dependent inactivation pathway is due to mechanismbased inactivation of CYP3A by meclizine, as supported by the experimental data discussed above. A possible explanation for the NADPH-independent inactivation pathway is that the parent drug may inactivate CYP3A. Two pieces of experimental evidence support the conclusion that the parent drug may be an inactivator of human CYP3A. First, without preincubation and NADPH, meclizine decreased liver microsomal testosterone 6b-hydroxylation by 25–30% of the vehicle-treated control. Second, meclizine inactivated recombinant CYP3A4 in the absence of NADPH and in a time-dependent manner. Therefore, the parent drug is directly involved in the inactivation of CYP3A4. A potential mechanism may involve meclizine undergoing spontaneous non-enzymatic ring change to form a reactive intermediate that may be responsible for the inactivation. Interestingly, delavirdine, which has a di-substituted piperazine ring similar to meclizine, also inactivated human liver microsomal CYP3A at the zero preincubation time point, as assessed by the triazolam 40 -hydroxylation assay [36]. Meclizine exhibits isoform-selective mechanism-based inactivation of human CYP3A. CYP3A4 and CYP3A5 share >80% amino acid identity and have an overlapping set of substrates and inhibitors [39]. However, some drugs are metabolized predominantly by one of the isoforms or are catalyzed by the two isoforms with different metabolic kinetics [39–41]. Only a small number of chemicals have a significant binding preference for one of the isoforms [42]. Our current findings indicate that meclizine inactivated recombinant CYP3A4 but not CYP3A5. The estimated partition ratio of meclizine for CYP3A4 inactivation was 10.5, and a value of <50 was classified as a relatively important mechanismbased inactivator of CYP3A4 [43]. The selective inactivation of CYP3A4 suggests that meclizine may be a potential chemical tool for differentiating the enzymatic functionality of CYP3A4 and CYP3A5. Our present data showing mechanism-based inactivation of human liver microsomal CYP3A and recombinant CYP3A4 by meclizine explain the previously observed decrease in CYP3A catalytic activity in human hepatocytes treated with meclizine [12]. Given that both PXR and CYP3A have a large, hydrophobic, and flexible ligand-binding domain [23,44], it is possible that an activator of PXR is also a substrate, direct inhibitor, or mechanismbased inactivator of CYP3A. Our current findings indicate for the first time that meclizine, which is a PXR agonist [12], can inhibit and inactivate CYP3A, thereby giving rise to opposing effects on the functional activity of the enzyme and indicating the importance of measuring the functional activity of nuclear receptor-regulated enzymes. Ritonavir is another chemical that has dual and opposing effects of activating PXR and inhibiting CYP3A catalytic activity [45].

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The plasma concentration of meclizine was 0.2 mM in healthy human volunteers who received 25 mg of meclizine [1]. However, the concentration of this drug in the gastrointestinal fluids is expected to be much greater. Assuming 500 ml of fluid in the gastrointestinal tract [46], a 25 mg dose of meclizine would reach 128 mM. Moreover, meclizine was reported to be predominantly concentrated in the intestine of rats [47]. These suggest that meclizine may reach concentrations that inhibit and inactivate CYP3A in the gastrointestinal tract, potentially affecting the oral bioavailability of co-administered drugs that undergo CYP3Acatalyzed biotransformation. CYP3A accounts for 70–80% of the total cytochrome P450 content in the mucosa of small intestines, and metabolism within the intestine plays an important role in determining oral bioavailability, efficacy, and toxicity of a drug [48]. Intestinal CYP3A4 has been reported to contribute to clinical drug–drug interactions and interindividual variability in oral bioavailability [49–52]. Ritonavir [53], itraconazole [50], grapefruit juice [54], and cranberry juice [55] are examples of drugs and food that inhibit intestinal CYP3A4 and affect drug levels in humans. In conclusion, meclizine, which is a PXR agonist [12], directly inhibits human liver microsomal CYP3A by a mixed mode. It is also a mechanism-based inactivator of CYP3A, and exhibits isoformselective inactivation of CYP3A4 but not CYP3A5. In contrast, norchlorcyclizine, which is a N-debenzylated metabolite of meclizine, is a direct inhibitor but not a mechanism-based inactivator of CYP3A. This suggests that a di-substituted piperazine ring or a benzyl substituent in meclizine, but not a monosubstituted piperazine ring in norchlorcyclizine, may be a determinant for mechanism-based inactivation. Furthermore, our findings indicate that a PXR activator may also be an inhibitor/inactivator of the enzyme that it regulates, and the inhibitory effect may negate the induction effect. Mechanismbased inactivation of CYP3A4 by meclizine reported in this study would explain why treatment with primary cultures of human hepatocytes with meclizine decreased CYP3A-catalyzed testosterone 6b-hydroxylation, even though it activated human PXR and increased CYP3A4 mRNA expression [12]. Therefore, it is important to measure not only the mRNA and protein expression but also the catalytic activity of a nuclear receptor-regulated enzyme in order to fully elucidate the overall effect of a nuclear receptor modulator on the target enzyme of interest. Conflict of interest None. Acknowledgement This research was supported by the National University of Singapore research grant [R-148-000-185-133]. References [1] Z. Wang, B. Lee, D. Pearce, S. Qian, Y. Wang, Q. Zhang, M.S. Chow, Meclizine metabolism and pharmacokinetics: formulation on its absorption, J. Clin. Pharmacol. 52 (2012) 1343–1349. [2] V.M. Gohil, N. Offner, J.A. Walker, S.A. Sheth, E. Fossale, J.F. Gusella, M.E. MacDonald, C. Neri, V.K. Mootha, Meclizine is neuroprotective in models of Huntington’s disease, Hum. Mol. Genet. 20 (2011) 294–300. [3] V.M. Gohil, S.A. Sheth, R. Nilsson, A.P. Wojtovich, J.H. Lee, F. Perocchi, W. Chen, C.B. Clish, C. Ayata, P.S. Brookes, V.K. Mootha, Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis, Nat. Biotechnol. 28 (2010) 249–255. [4] V.M. Gohil, L. Zhu, C.D. Baker, V. Cracan, A. Yaseen, M. Jain, C.B. Clish, P.S. Brookes, M. Bakovic, V.K. Mootha, Meclizine inhibits mitochondrial respiration through direct targeting of cytosolic phosphoethanolamine metabolism, J. Biol. Chem. 288 (2013) 35387–35395. [5] M. Matsushita, S. Hasegawa, H. Kitoh, K. Mori, B. Ohkawara, A. Yasoda, A. Masuda, N. Ishiguro, K. Ohno, Meclozine promotes longitudinal skeletal

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