analysis of mechanism-based inhibition of cyp 3a4 by a...

1521-009X/44/10/1608–1616$25.00 http://dx.doi.org/10.1124/dmd.116.071654DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:1608–1616, October 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Analysis of Mechanism-Based Inhibition of CYP 3A4 by a Series ofFluoroquinolone Antibacterial Agents s

Akiko Watanabe, Hideo Takakusa, Takako Kimura, Shin-ichi Inoue, Hiroyuki Kusuhara,and Osamu Ando

Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo, Japan (A.W., H.T., S.I., O.A.);Structural Biology Group, Biological Research Department, Daiichi Sankyo RD Novare Co., Ltd., Tokyo, Japan (T.K.); and

Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan (H.K.)

Received May 19, 2016; accepted July 27, 2016

ABSTRACT

A series of fluoroquinolone compounds (compounds 1–9), whichcontain a common quinolone scaffold, inactivated the metabolicactivity of CYP3A. The purpose of this study was to identifymechanism-based inhibition (MBI) among these fluoroquinolonecompounds by metabolite profiling to elucidate the association ofthe substructure and MBI potential. Reversibility of MBI afterincubation with potassium ferricyanide differed among the testcompounds. Representative quasi-irreversible inhibitors form ametabolite-intermediate (MI) complex with the heme of CYP3A4according to absorption analysis. Metabolite profiling identified thecyclopropane ring-opened metabolites from representative irre-versible inhibitors, suggesting irreversible binding of the carbon-centered radical species with CYP3A4. On the other hand, the oximeform of representative quasi-irreversible inhibitors was identified,

suggesting generation of a nitroso intermediate that could form theMI complex. Metabolites of compound 10 with a methyl group atthe carbon atom at the root of the amine moiety of compound8 include the oxime form, but compound 10 did not show quasi-irreversible inhibition. The docking study with CYP3A4 suggestedthat a methyl moiety introduced at the carbon atom at the root ofthe primary amine disrupts formation of the MI complex betweenthe heme and the nitroso intermediate because of steric hindrance.This study identified substructures of fluoroquinolone compoundsassociated with the MBI mechanism; introduction of substitutedgroups inducing steric hindrance with the heme of P450 canprevent formation of an MI complex. Our series of experimentsmay be broadly applicable to prevention of MBI at the drugdiscovery stage.

Introduction

Some drug–drug interactions (DDIs) involve time-dependent in-hibition (TDI) of drug-metabolizing enzymes in which a reactiveintermediate generated by a metabolic enzyme interacts with the enzymequasi-irreversibly or irreversibly, thereby inactivating the enzyme. TheTDI phenomenon, which involves enzymatic activity loss induced byincubating enzymes with inhibitors before the addition of substrates, isused in kinetic experiments. Mechanism-based inhibition (MBI) refersto a subset of TDI focusing on a chemical mechanism in which reactiveintermediates lead to the inactivation of enzymes. A prior study pointedout that the distinction between TDI and MBI must be appreciated(Grimm et al., 2009). In this report, we focus on the mechanism ofenzyme inactivation caused by reactive intermediates, and we thus usethe termMBI. Cytochrome P450 (P450) is an enzyme that is responsiblefor the metabolism of many drugs in human (Guengerich, 2001). Thus,MBI of P450 may cause a clinically severe DDI because the enzymaticactivity is recovered only by synthesis of a new enzyme; thus, the

inhibition continues even after the inhibitor is eliminated from the body.Indeed, many drugs have been withdrawn from the market becauseof P450-related DDIs (Wienkers and Heath, 2005). Pharmaceuticalcompanies have attempted to develop in vitro test systems to attenuatethe MBI potential of drug candidates for P450 at the early stage of drugdiscovery (Watanabe et al., 2007; Grime et al., 2009; Grimm et al., 2009;Zientek et al., 2010; Yates et al., 2012).MBI of P450 involves the irreversible or quasi-irreversible binding of

a reactive intermediate metabolite to the metabolizing enzyme (Linand Lu, 1998). The irreversible inhibition is caused by covalent bindingof reactive intermediates to the heme or apoprotein of the active site ofP450, whereas the quasi-irreversible inhibition is caused by formation ofa stable metabolite-intermediate (MI) complex with the ferrous formof the heme iron atom (Ullrich and Schnabel, 1973). A large numberof compounds including methylenedioxybenzenes, alkylamines, andhydrazines have been reported to form MI complexes (Murray, 1997;Lin and Lu, 1998; Orr et al., 2012). The MI complexes can dissociateafter treatment with potassium ferricyanide, which oxidizes iron tothe ferric form and recovers the enzymatic activity (Buening andFranklin, 1976; Muakkassah et al., 1982); on the basis of that mecha-nism, we established an assay to distinguish between irreversible and

dx.doi.org/10.1124/dmd.116.071654.s This article has supplemental material available at dmd.aspetjournals.org.

ABBREVIATIONS: DC-159a, (+)-7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid hemihydrate; DDI, drug–drug interaction; DK-507k, 7-[(7S)-7-amino-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-4-oxoquinoline-3-carboxylic acid; DX-619, 7-[(3R)-3-(1-aminocyclopropyl)pyrrolidin-1-yl]-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-4-oxoquinoline-3-carboxylic acid; HLM, human liver microsome; LC, liquid chromatography;m/z, mass-to-charge ratio;MBI, mechanism-based inhibition; MI, metabolite-intermediate; MS/MS, tandem mass spectrometry; P450, cytochrome P450; PDB, Protein Data Bank;Rt, retention time; TDI, time-dependent inhibition.

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quasi-irreversible inhibition in our previous report (Watanabe et al.,2007). Furthermore, incubation with potassium ferricyanide has alsobeen applied to identification of metabolites that form an MI complex. Anitroso intermediate generated from lapatinib, which can cause quasi-irreversible inhibition of CYP3A4, increased in abundance after theaddition of potassium ferricyanide to the reaction solution of recombinantCYP3A4 and lapatinib (Barbara et al., 2013). Identification of the MBImechanism will also be useful for analysis of the risk of drug-inducedtoxicities caused by covalent binding of reactive intermediates to proteinsand lipids, in addition to DDIs (Zhou et al., 2004; Fontana et al., 2005;Kalgutkar et al., 2005; Walgren et al., 2005; Takakusa et al., 2011).We developed novel fluoroquinolone antibacterial agents, compound

1 (also known as DX-619 or 7-[(3R)-3-(1-aminocyclopropyl)pyrrolidin-1-yl]-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-4-oxoquinoline-3-carboxylic acid) and compound 8 (also known as DK-507k or7-[(7S)-7-amino-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-4-oxoquinoline-3-carboxylic acid), at theclinical stage (Otani et al., 2003; Fujikawa et al., 2005). These compoundsshow significant MBI potential toward CYP3A (Imamura et al., 2013;Odagiri et al., 2013). Indeed, on phase I samples, it was shown that DX-619causes a significant reduction in apparent 6b-hydroxycortisol formationclearance, an index of CYP3A4 activity in the liver, during DX-619administration (Imamura et al., 2013). Because CYP3A is responsiblefor most of the P450-mediated drug metabolism (Wienkers and Heath,2005), DX-619 will cause moderate DDIs with various CYP3Asubstrate drugs. Compounds 1 and 8 have the same scaffold except forsubstructures of positions C6 and C7 in the quinolone ring (Table 1).Compound 10 (also known as DC-159a [(+)-7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid hemihydrate]), in which a methyl group was introduced at thecarbon atom at the root of the C7-amino moiety of compound 8, wasdesigned for prevention of MBI and actually turned out to be MBInegative, sustaining the pharmacological activity (Hoshino et al.,2008; Odagiri et al., 2013). This study aimed to clarify the MBImechanism caused by our fluoroquinolones and to then investigate theeffect of the methyl group introduced into compound 10 on preventionof MBI. In addition, we analyzed a series of fluoroquinoloneantibacterial candidates containing a common quinolone scaffold(Table 1) to determine the possible association of the substructurewith MBI potential.

Materials and Methods

Materials. All of the tested fluoroquinolone compounds shown in Table 1 weresynthesized by Daiichi Sankyo Co., Ltd. (Tokyo, Japan). The synthesis method ofcompound 10 as a representative fluoroquinolone compound was described in aprevious report (Odagiri et al., 2013). Midazolam maleate salt was purchased fromSigma-Aldrich (St. Louis,MO). 19-Hydroxymidazolamwas purchased fromUltrafine(Manchester, UK); 0.5 M potassium phosphate buffer, [13C3]hydroxymidazolam,NADPH Regenerating System Solution A and B, and recombinant human CYP3A4Supersomes containing P450 reductase and cytochrome b5 were acquired fromCorning (Woburn,MA). Potassium ferricyanide was purchased fromKanto ChemicalCo. Inc. (Tokyo, Japan). Fifty mixed-sex, donor-pooled human liver microsomes(HLMs) were acquired from XenoTech LLC (Lenexa, KS). All other reagents andsolvents were of the highest grade commercially available.

Screening for Reversibility of MBI of CYP3A Using HLMs. The screeningwas performed as described previously (Watanabe et al., 2007).

Reversibility of MBI Using Recombinant Human CYP3A4 Supersomes.A total of 270 ml preincubation solution contained 10 pmol/ml recombinantCYP3A4 Supersomes in 0.1 M potassium phosphate buffer with or without testcompounds (final concentration of 10, 30, or 100 mM). The final solventconcentration in the preincubation solutions was 1% (v/v) dimethylsulfoxide.Preincubation reactions were initiated by the addition of 30 ml of an

NADPH-generating system consisting of NADPH Regenerating System Solu-tions A and B. After a 0-minute or 30-minute preincubation, 50 ml of eachpreincubation solution was added to 50 ml of the solutions containing 0.1 Msodium phosphate buffer with or without 2 mM potassium ferricyanide and wasthen incubated for 10 minutes. After the 10-minute reaction, each reactionmixture was diluted 5-fold with incubation solution containing 0.1 M potassiumphosphate buffer and 25 mM midazolam as a substrate [final concentration1% (v/v) acetonitrile]. At the end of the 10-minute incubation reactions, 100-mlaliquots of each incubation solution were added to the mixture of 50 mlacetonitrile containing 2 mM [13C3]hydroxymidazolam as an internal standardand 100 ml methanol. The samples were centrifuged at 2000g for 3 minutes,and the supernatants were transferred to other plates. A standard curve of

TABLE 1

Structures of fluoroquinolone compounds tested in this study

Substructures of positions C1–C5 in a quinolone scaffold are common among all compounds.Each substructure of positions C6–C8 is shown below.

Common Scaffold

Position

C-6 C-7 C-8

Compound 1 —H —O—

Compound 2 —H —O—

Compound 3 —F —O—


Compound 5 —F




Compound 9 —F


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19-hydroxymidazolam was constructed to determine its concentration in thesamples. The concentration range of the standard curve was 8–1000 nM. Allsamples were analyzed by liquid chromatography (LC)–tandem mass spectrom-etry (MS/MS) using a Waters Acquity UPLC and TQD system (Waters,Manchester, UK). Chromatographic separation was performed on a SunFireC18 column (3.5-mm particle size, 2.1 � 100 mm; Waters). The massspectrometer was operated in positive electrospray ionization mode. The mass-to-charge ratio (m/z) (precursor → product) values of 19-hydroxymidazolam and[13C3]hydroxymidazolam were 342 → 324 and 345 → 327, respectively.Concentrations of 19-hydroxymidazolam in the samples were calculated usingMassLynx software (version 4.1; Waters). The percentage of metabolic activity[% of control(0 minutes) and % of control(30 minutes)] was obtained as follows and isdetailed in a previous report (Watanabe et al., 2007).

% of controlð0 minutesÞ ¼ vð0 minutes;þ inhibitorÞvð0 minutes;2 inhibitorÞ

� 100

% of controlð30 minutesÞ ¼ vð30 minutes;þ inhibitorÞvð30 minutes;2 inhibitorÞ

� 100

v(0 minutes, 6inhibitor) and v(30 minutes, 6inhibitor) indicate the metabolic activity after0-minute or 30-minute preincubation with (+) or without (2) an inhibitor,respectively.

Determination of KI and kinact values. A total of 180 ml preincubationsolution contained 10 pmol/ml recombinant CYP3A4 Supersomes in 0.1 Mpotassium phosphate buffer with or without test compounds (five or sixconcentrations per test compound). The preincubation reactions were initiatedby the addition of 20 ml of an NADPH-generating system. After a 0-, 2-, or5-minute preincubation for compound 1 and 0-, 5-, or 15-minute preincubation forcompound 6, 20ml of each preincubation mixture was added to 180ml incubationsolution containing 0.1 M potassium phosphate buffer and 25 mM midazolam.After the 10-minute incubation reactions, samples were prepared for LC-MS/MSanalysis and concentrations of 19-hydroxymidazolam in the samples andpercentages of control values for each preincubation time with each inhibitorconcentration were determined as described above. The natural logarithm of thepercentage of the control was plotted against the preincubation times for eachconcentration of a test compound. The slope from the linear regression analysisprovided the observed inactivation rate constant (kobs) for each concentration; kobsand the inhibitor concentration (I) were fitted into the following expression byusing Phoenix WinNonlin 6.1 software (Certara G.K., Princeton, NJ).

kobs ¼ kinact � I

KI þ I

Absorption Analysis for MI Complex Formation. A total of 180ml reactionmixture containing 100 pmol/ml recombinant human CYP3A4 Supersomes,50 mM test compound [final concentration 1% (v/v) dimethylsulfoxide], and0.1 M potassium phosphate buffer was transferred to the microplate well andpreincubated at 37�C for 5 minutes. For the reference well, the solvent was addedto the reaction mixture in place of the test compound. The reaction was initializedby adding 20 ml of the NADPH-generating system to the reaction mixture.Absorbance at 455 nm and 490 nmwas monitored for 20 minutes at 37�C using aVersaMax microplate reader (Molecular Devices, Sunnyvale, CA) controlled bySoftMax Pro software (version 5.4.6; Molecular Devices). Using the absorbancedata at 455 nm and 490 nm, the absorbance difference between 455 nm and490 nm was calculated.

Structural Elucidation of Metabolites of Test Compounds afterIncubation with Recombinant Human CYP3A4 Supersomes. Five microli-ters of a test compound solution (5mM in dimethylsulfoxide) was added to 445mlincubation solution consisting of 100 pmol/ml recombinant human CYP3A4 and0.1 M potassium phosphate buffer. For the 0-minute incubation sample, 500 mlacetonitrile was added to the incubationmixture, followed by the addition of 50mlof the NADPH-generating system. For the 30-minute incubation sample, 50 ml ofthe NADPH-generating system was added to the incubation mixture, and themixture was then incubated at 37�C for 30 minutes. Afterward, the reaction wasterminated by the addition of 500 ml acetonitrile. Incubation samples werecentrifuged at 9000g for 3 minutes, and the supernatants were concentrated usinga centrifugal evaporator. The concentrated samples were analyzed by means of a

LTQ-Orbitrap XL (Thermo Fisher Scientific, San Jose, CA) equipped with anAcquity UPLC PDA system (Waters). Solvents A and B were based on H2Ocontaining 0.1% (v/v) trifluoroacetic acid and acetonitrile (LC–mass spectrometrygrade) with 0.1% (v/v) trifluoroacetic acid, respectively. In hydrogen-deuteriumexchange experiments, D2O with 0.1% (v/v) trifluoroacetic acid was used assolvent A. Analyte separation was achieved using an Acquity UPLC BEH C18column (100� 2.1 mm, 1.7-mm particle size) at a flow rate of 0.5 ml/min under alinear gradient from 10% to 70%B (0–8 minutes). The data were acquired in full-scan and MS/MS modes. Mass spectrometry conditions were as follows:electrospray voltage, 4.2 kV; capillary temperature, 275�C; sheath gas flow rate,30 arbitrary units; auxiliary gas flow rate, 15 arbitrary units; resolution, 30,000 forfull-scan mode and 7500 for MS/MS mode; ionization, electrospray ionization inpositive ion mode; activation type for MS/MS, higher-energy collision dissoci-ation; and collision energy for MS/MS, 40% and 50%.

CYP3A4 Docking of Compound 6 and Its Nitroso Metabolite. Moleculardocking of compound 6 and its nitroso metabolite to CYP3A4 was performed usingthe crystal structures of CYP3A4 [ProteinData Bank (PDB) code 2V0M (Ekroos andSjögren, 2006) for compound 6 and PDB code 4I4G (Sevrioukova and Poulos, 2013)for the nitroso metabolite of compound 6]. The substrates were prepared using theLigPrep module of Maestro (version 3.5; Schrödinger, LLC, New York, NY).Starting with two-dimensional structures, LigPrep produces a three-dimensionalstructure with ionization states at pH 7. In compound 6, a formal charge of the aminegroup, which is an iron-coordinating moiety, was manually modified to 0. Dockingwas carried out using theGlide dockingmodule ofMaestro (version 6.8; Schrödinger,LLC) with the positional constraint on the reference crystal structures [CambridgeStructural Database code CICNEH (Munro et al., 1999) for compound 6 and PDBcode 4M4A (Yi et al., 2013) for the nitrosometabolite of compound 6] to keep typicaliron-coordinate geometry, because Glide did not recognize nitrogen atoms of the-NH2 or nitroso moiety as iron coordinators. After Glide docking, methylatedcompounds were manually modeled by adding a methyl group to the representativedocking models of each compound.

Results

Reversibility of MBI for CYP3A. To distinguish between quasi-irreversible and irreversible binding to CYP3A by fluoroquinolones, thescreening of MBI reversibility was performed in HLMs using thesecompounds (Supplemental Table 1). The activity of 19-hydroxymidazolamformation from midazolam by HLMs was monitored as the CYP3Aactivity. The enzymatic activity of CYP3A inactivated after 30-minutepreincubation with the compound containing cyclopropylamine (com-pounds 1–5) in the pyrrolidine ring of the 79 position of fluoroquinolonedid not recover after oxidation with potassium ferricyanide. Thesecompounds were assumed to be irreversible inhibitors. In contrast,enzymatic activity that was reduced after 30-minute preincubationwith the compound containing an amine moiety (compounds 6–9) inthe ring form of the 79 position of fluoroquinolone tended to recoverafter oxidation with potassium ferricyanide. It was shown that thesecompounds could bind to CYP3A quasi-irreversibly. Unlike compounds1–9, the decrease in enzymatic activity after 30-minute preincubationwith compound 10 was very low.The assay of MBI reversibility was also performed in recombinant

human CYP3A4 Supersomes by using these compounds. Enzymaticactivities reduced by 30-minute preincubation with each of compounds6–9 were restored more than 20% with the addition of potassiumferricyanide and were close to the activity after 0-minute preincubation;however, those with each of compounds 1–5 were not fully restored,although the percentage of control data of compounds 3–5 after30-minute preincubation followed by incubation with potassium ferricy-anide was statistically higher than that without potassium ferricyanide(Fig. 1). This study clearly distinguished between compounds 1–5 asirreversible inhibitors and compounds 6–9 as quasi-irreversible inhibi-tors. CYP3A4 was shown to be one of the CYP3A isozymes responsiblefor MBI observed in HLMs. On the basis of this result, recombinanthuman CYP3A4 Supersomes were used in the following experiments.

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KI and kinact values of compounds 1 and 6 as representative irreversibleand quasi-irreversible inhibitors, respectively, were obtained forrecombinant CYP3A4 Supersomes (Supplemental Fig. 1; SupplementalTable 2). These two compounds showed different characteristicsindicated as kinetic parameters, with higher KI (395 6 47 mM) andhigher kinact (0.4596 0.024min21) for compound 1 and lowerKI (7.2161.38 mM) and lower kinact (0.190 6 0.009 min21) for compound 6.Absorption Analysis for MI Complex Formation. To determine

whether inhibition of CYP3A4 enzymatic activity by the fluoroquinolonecompounds assumed to be quasi-irreversible inhibitors occurs via theformation of an MI complex, absorbance at 455 nm and 490 nm wasmonitored for 20 minutes after the addition of the NADPH-generatingsystem in the reaction mixture containing recombinant CYP3A4 Supersomesand each test compound. The absorbance difference between 455 nmand 490 nm was plotted against time (Fig. 2). Compounds 6 and 8 wereused as representative quasi-irreversible inhibitors of CYP3A4, andcompound 10 was used as a noninhibitor or weak inhibitor. The absorbancedifference increased in a time-dependent manner and reached a plateauafter approximately 15minutes for incubation with compound 6 or 8, butthe increase was not observed with compound 10.Structural Elucidation of Metabolites of Irreversible Inhibitors after

Incubation with Recombinant CYP3A4 Supersomes. To gain mech-anistic understanding of the irreversible inhibition of fluoroquinolonecompounds, we performed a structural analysis by LC-MS/MS ofmetabolites after incubation of compound 1, a representative irreversible

inhibitor, with recombinant CYP3A4 Supersomes. Using full-scan condi-tions, fourmetabolites related to oxidation reactions of the cyclopropylaminemoiety of compound 1 [cpd1-M1 [retention time (Rt) = 2.91 minutes, m/z418.1772], cpd1-M2 (Rt = 3.58 minutes, m/z 418.1771), cpd1-M3 (Rt =4.41 minutes, m/z 419.1611), and cpd1-M4 (Rt = 5.26 minutes, m/z416.1615)] were detected as shown in the mass chromatograms (Fig. 3A).The mass spectrometric data are summarized in Supplemental Table 3. Themolecular composition was estimated to be C21H24N3O5F for cpd1-M1 andcpd1-M2, C21H23N2O6F for cpd1-M3, and C21H22N3O5F for cpd1-M4 byaccuratemassmeasurements.Hydrogen-deuteriumexchangemeasurementsusing D2O as an eluent revealed the molecular ions [M + D]+ at m/z423.2079 (cpd1-M1),m/z 423.2065 (cpd1-M2), m/z 422.1797 (cpd1-M3),and m/z 418.1734 (cpd1-M4), indicating that the numbers of exchangeableprotons of the metabolites were 4, 4, 2, and 1, respectively. The proposedstructure of each metabolite based on the MS/MS fragmentation patterns,molecular compositions, and number of exchangeable protons are shownin Fig. 3B. cpd1-M1 and cpd1-M2 were found to be oxidative (+O)metabolites according to the estimated molecular composition. Theproduct ions (m/z 305, m/z 287, and m/z 229), which correspond to thequinolone moiety of cpd1-M1 and cpd1-M2, were identical to those ofthe unchanged form, pointing to the monooxygenation in the pyrrolidinylcyclopropylamine side chain. In addition, the increase (+1) of theexchangeable protons in cpd1-M1 and cpd1-M2 indicated hydroxylationon carbon atoms, notN-oxidation or hydroxylamine formation. cpd1-M3was proposed to be the hydroxyethyl carbonyl form in the side chain onthe basis of its molecular compositional change (+2O-N-H), fragmenta-tion pattern, and number of exchangeable protons (n = 2). cpd1-M4 wasproposed to be the dihydroisoxazole form in the side chain based on itsmolecular compositional change (+O-2H), fragmentation pattern, andnumber of exchangeable protons (n = 1).Structural Elucidation of Metabolites of Quasi-Irreversible

Inhibitors after Incubation with Recombinant CYP3A4 Supersomes.To gain mechanistic understanding of quasi-irreversible inhibition offluoroquinolone compounds, we carried out a structural analysis byLC-MS/MSofmetabolites after incubation of compound 6, a representativequasi-irreversible inhibitor, with recombinant CYP3A4 Supersomes.The full-scan analysis detected five metabolites related to oxidationreactions of the amino azaspiro[4.4]nonan moiety of compound 6[cpd6-M1 (Rt = 3.45 minutes, m/z 450.1834), cpd6-M2 (Rt =3.88 minutes, m/z 450.1833), cpd6-M3 (Rt = 6.50 minutes, m/z448.1677), cpd6-M4 (Rt = 6.50 minutes,m/z 435.1729), and cpd6-M5(Rt = 7.16 minutes, m/z 433.1569)], which were detected as shown inthe mass chromatograms (Fig. 4A). The mass spectrometric data aresummarized in Supplemental Table 3. The molecular compositionwas estimated to be C22H25N3O5F2 for cpd6-M1 and cpd6-M2,C22H23N3O5F2 for cpd6-M3, C22H24N2O5F2 for cpd6-M4, andC22H22N2O5F2 for cpd6-M5 by accurate mass measurements.Hydrogen-deuterium exchange measurements using D2O as an eluentrevealed the molecular ions [M + D]+ at m/z 455.2144 (cpd6-M1),m/z

Fig. 2. Absorbance difference between 455 and 490 nm of CYP3A4 incubated withcompound 6 (circles), 8 (squares), or 10 (triangles) for 20 minutes after the additionof the NADPH-generating system. Each symbol represents the mean 6 S.D. oftriplicate experiments. *P , 0.01.

Fig. 1. Reversibility of MBI of CYP3A4 by fluoroquinolonecompounds in recombinant human CYP3A4 Supersomes.The percentage of control data after 0-minute preincubationfollowed by incubation with (dark gray) or without (black)potassium ferricyanide and that after 30-minute preincubationfollowed by incubation with (white) or without (light gray)potassium ferricyanide were obtained. Concentrations of the testcompounds were as follows: 100 mM for compounds 1, 3, 7, 8,and 10; 30 mM for compounds 2, 4, 5, and 9; and 10 mM forcompound 6. Each bar represents the mean 6 S.D. of triplicateexperiments. *P , 0.01; **P , 0.001; ***P , 0.0001. NS, notsignificant.


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455.2142 (cpd6-M2), m/z 451.1863 (cpd6-M3), m/z 438.1912 (cpd6-M4), and m/z 435.1690 (cpd6-M5), indicating that the numbers ofexchangeable protons of the metabolites were 4, 4, 2, 2, and 1,

respectively. The proposed structure of each metabolite based on theMS/MS fragmentation patterns, molecular compositions, and num-bers of exchangeable protons are shown in Fig. 4B. cpd6-M1 and

Fig. 3. (A) Extracted ion chromatograms of the metabolites in the samples after 30-minute incubation of compound 1 with recombinant CYP3A4 Supersomes in thepresence of the NADPH-generating system. (B) The structure of compound 1 and proposed structures of the metabolites with the fragmentation schemes.

Fig. 4. (A) Extracted ion chromatograms of the metabolites in the samples after 30-minute incubation of compound 6 with recombinant CYP3A4 Supersomes in thepresence of the NADPH-generating system. (B) The structure of compound 6 and proposed structures of the metabolites with the fragmentation schemes.


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cpd6-M2 were found to be oxidative (+O) metabolites according to theestimated molecular composition. The product ion (m/z 279), whichcorresponds to the quinolone moiety of cpd6-M1 and cpd6-M2, wasidentical to that of the unchanged form, pointing to monooxygenation inthe amino azaspiro[4.4]nonan side chain. In addition, the increase (+1) ofthe exchangeable protons in cpd6-M1 and cpd6-M2 indicated hydroxy-lation on carbon atoms, not N-oxidation or hydroxylamine forma-tion. cpd6-M3 was found to be the oxime form in the side chain on thebasis of its molecular compositional change (+O-2H), fragmentationpattern, and number of exchangeable protons (n = 2). cpd6-M4 andcpd6-M5 were proposed to be the hydroxyl form and keto form viaoxidative deamination in the side chain based their molecularcompositional changes +O-N-H and +O-N-3H, respectively. Theseproposed metabolite structures were consistent with the fragmentationpatterns and the number of exchangeable protons of cpd6-M4 andcpd6-M5.In addition, we analyzed metabolites after incubation of compound

10, which has the methyl moiety in the side chain but does not causequasi-irreversible inhibition for CYP3A4, with recombinant CYP3A4Supersomes. The full-scan analysis detected four metabolites related tooxidation reactions of the amino azaspiro[2.4]heptan moiety of com-pound 10 [cpd10-M1 (Rt = 3.97 minutes, m/z 434.1514), cpd10-M2(Rt = 4.09 minutes, m/z 436.1674), cpd10-M3 (Rt = 5.86 minutes, m/z466.1415), and cpd10-M4 (Rt = 6.88 minutes, m/z 450.1470)], whichwere detected as shown in mass chromatograms (Fig. 5A). The massspectrometric data are summarized in Supplemental Table 3. Themolecular compositions were estimated to be C21H22N3O5F2 for

cpd10-M1, C21H24N3O5F2 for cpd10-M2, C21H22N3O7F2 for cpd10-M3, and C21H22N3O6F2 for cpd10-M4 by accurate mass measurements.Hydrogen-deuterium exchange measurements using D2O as an eluentrevealed the molecular ions [M + D]+ at m/z 436.1653 (cpd10-M1), m/z440.1935 (cpd10-M2), m/z 469.1617 (cpd10-M3), and m/z 452.1601(cpd10-M4), indicating that the numbers of exchangeable protons of themetaboliteswere 1, 3, 2, and 1, respectively. The proposed structure of eachmetabolite according to the MS/MS fragmentation patterns, molecularcompositions, and numbers of exchangeable protons is shown in Fig. 5B.cpd10-M1, cpd10-M2, and cpd10-M4 were found to be the nitroso form,hydroxylamine form, and nitro form in the side chain according to itsmolecular compositional changes +O-2H, +O, and +2O-2H, respectively.These proposed metabolite structures were consistent with the fragmenta-tion patterns and the number of exchangeable protons. On the basis of itsmolecular compositional change (+2O-2H), cpd10-M3 was found to be anoxidative metabolite of the amino azaspiro[2.4]heptan side chain of thenitro form (cpd10-M4). It was shown that compound 10 is alsometabolizednear the amino moiety in the side chain by CYP3A4.CYP3A4 Docking of Compound 6 and Its Nitroso Metabolite. To

study the effect of a methyl group introduced at the carbon atom at theroot of the C7-amino moiety of the quasi-irreversible inhibitors forbinding to CYP3A4, molecular docking of compound 6 as a represen-tative quasi-irreversible inhibitor and its nitroso metabolite to CYP3A4was performed using the crystal structure of CYP3A4 (PDB code 2V0Mand 4I4G for each; Ekroos and Sjögren, 2006; Sevrioukova and Poulos,2013) as shown in Figs. 6 and 7.We obtained the possible docking modeof compound 6 and its nitroso metabolite against CYP3A4, which

Fig. 5. (A) Extracted ion chromatograms of the metabolites in the samples after 30-minute incubation of compound 10 with recombinant CYP3A4 Supersomes in thepresence of NADPH. (B) The structure of compound 10 and proposed structures of the metabolites with the fragmentation schemes.


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maintain typical iron-coordinate geometry, as shown in Fig. 6A andSupplemental Fig. 5, and Fig. 7A and Supplemental Fig. 6, respectively.The distance between amine nitrogen and the heme iron was 2.25 Å forcompound 6, and the angle u1 (C-N-Fe) was 110.0� (Fig. 6A). Thedistance between amine nitrogen and the heme iron was 2.35 Å for thenitroso metabolite, and the angle u2 (C-N-Fe) was 130.0� (Fig. 7A). Itwas assumed that these docking models were plausible especiallyaround the heme iron and these structures were used for further analysis.Methyl moieties manually added to these docking models indicated thatthere would be a severe steric clash between addedmethyls and hemes asshown in Figs. 6B and 7B.

Discussion

In this study, we focused on the effect of substructures of a series offluoroquinolone antibacterial compounds with a common scaffold onthe MBI potential toward CYP3A. To obtain information on thebinding mechanism of the fluoroquinolone compounds, we performedMBI reversibility screening using HLMs established in our previousstudy (Watanabe et al., 2007) and we examined the reversibility usingrecombinant human CYP3A4 Supersomes. The fluoroquinolonecompounds (compounds 1–9) were classified into irreversible andquasi-irreversible inhibitors (compounds 1–5 and compounds 6–9,respectively). The classification appears to depend on substructures ofthe 79 position of the quinolone ring, the pyrrolidine ring bearingcyclopropylamine (compounds 1–5), and the pyrrolidine or azaspiroring bearing a primary amine (compounds 6–9). Compound 10, which

contains a methyl group at the carbon atom at the root of theprimary amine of compound 8, did not show the inhibitory effect inboth matrices. MBI could be reproduced in recombinant humanCYP3A4 Supersomes. In the subsequent experiments clarifyingthe detailed MBI mechanism of these compounds, we used CYP3A4Supersomes.Absorption analysis of the reactionmixtures of recombinant CYP3A4

Supersomes with these quasi-irreversible inhibitors was performed toconfirm MI complex formation. This assay is based on previous reportsshowing that the absorbance difference between 455 and 490 nmincreases in a time-dependent manner and reaches a plateau afterincubation of P450 with compounds forming an MI complex (Ullrichand Schnabel, 1973; Buening and Franklin, 1976; Franklin, 1991). Theabsorbance difference increased with incubation time in the presence ofcompound 6 or 8 but not compound 10 (Fig. 2). This result indicates thatcompounds 6 and 8 cause quasi-irreversible inhibition via MI complexformation with CYP3A4, and compound 10 does not. The order of theabsorbance difference was consistent with that of CYP3A4 inhibitionafter 30-minute preincubation with these compounds (Fig. 1).The structural analysis of metabolites after incubation of irreversible

and quasi-irreversible inhibitors with recombinant human CYP3A4Supersomes was performed to study the mechanistic difference betweenirreversible and quasi-irreversible inhibition by the fluoroquinolonecompounds from the viewpoint of drug metabolism. The proposedmetabolic pathways for compounds 1 and 6 are shown in Figs. 8 and 9,respectively. In the case of compound 1, cpd1-M3 was found to be thehydroxyethyl carbonyl form,which indicates that compound 1 undergoes

Fig. 7. (A) Docking model of the nitroso metabolite ofcompound 6 and CYP3A4. The heme moiety is also shown.(B) The manually edited model of the nitroso metabolite ofcompound 6 indicates that an additional methyl group (redcircle) would have a severe clash with heme (shown in aspace-filling model with Van der Waals radii).

Fig. 6. (A) Docking model of compound 6 and CYP3A4.The structure of CYP3A4 is shown as a ribbon model and asolvent-accessible surface in white. The compound andheme are presented as a tube model. (B) The manuallyedited model of compound 6 indicates that an additionalmethyl group (red circle) would have a severe clash withheme (shown in the space-filling model with Van der Waalsradii).


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ring-opening metabolism. In line with the existing literature on themetabolism of cyclopropylamines (Cerny and Hanzlik, 2005, 2006),cpd1-M3 formation can be explained by the hydrogen abstraction fromthe primary amine group of compound 1, followed by ring opening of theaminyl radical to form carbon-centered radical species, hydroxylation,and hydrolysis of the imine into a keto form. The radical intermediates arelikely to irreversiblymodify CYP3A4 and to cause irreversible inhibition.A precursor of cpd1-M3, the hydroxylated imine intermediate, waspresumed to be oxidized into the dihydroisoxazole form (cpd1-M4). Thesame ring-opening metabolites of cyclopropylamines were also detectedafter incubation of the other irreversible inhibitors, compound 2 or 5, withrecombinant CYP3A4 (Supplemental Figs. 2 and 3).In contrast with irreversible inhibitors, ring-opened metabolites

were not detected with compound 6. The key metabolite of compound6 was the oxime form, cpd6-M3, which suggests the formation of anitroso intermediate. This is because alkylnitroso intermediates areknown to be generally unstable and to tautomerize to more stableoxime forms (Mansuy et al., 1977). The primary amine group in theside chain of compound 6 is likely to be oxidized to form thehydroxylamine, followed by formation of the nitroso intermediate,which appears to form an MI complex with the heme of CYP3A4, andconsequently to cause quasi-irreversible inhibition. In the case ofcompound 8, we detected the oxime and nitro form in the sidechain, which are assumed to be formed via the nitroso intermediate(Supplemental Fig. 4).Metabolite profiling suggests that oxidation of the primary amine in the

side chain is the initial step of MBI for both irreversible and quasi-irreversible inhibitors; however, the subsequent ring-opening radicalreaction was observed only for the irreversible inhibitors. Because thethree-membered ring has higher distortion energy than the five-memberedring does, the aminyl radical of the cyclopropane is presumed to be lessstable than that of cyclopentane. Therefore, the ring structure bearing aprimary amine group in quinolones seems to account for the difference inthe MBI mechanism between irreversible and quasi-irreversible bindingwith CYP3A4.To obtain direct evidence that the nitroso intermediate of compound

6 binds to the heme of CYP3A4 quasi-irreversibly, we compared theabundance of the metabolites of compound 6 in the reaction mixture ofCYP3A4 with or without the treatment with potassium ferricyanide,according to one report (Barbara et al., 2013); however, the difference inthe abundance of the metabolites could not be detected in our study (datanot shown).We assumed that the abundance of the metabolite binding toproteins nonspecifically could not be negligible compared with thatbinding to CYP3A4 heme quasi-irreversibly.

We hypothesized that compound 10 (showing no inhibitory effect onCYP3A4) was not metabolized to the reactive intermediate because ofthe steric hindrance of the methyl group introduced at the carbon atom atthe root of the primary amine group. Unexpectedly, the nitroso form ofcompound 10 (cpd10-M1) and the related metabolites generated via thenitroso form—the nitro form (cpd10-M4) and the oxidized form of thenitro form (cpd10-M3)—were also detected after incubation of com-pound 10 with recombinant CYP3A4 Supersomes (Fig. 5). The level ofthe peak area of the metabolites generated from compound 10 in themass chromatogram was comparable with that of compound 6 (data notshown), suggesting that the amount of the reactive intermediate may besufficient to inhibit CYP3A4. We speculated that the methyl group mayhinder formation of an MI complex with the heme of CYP3A4.To test this hypothesis, we conducted molecular docking studies of

compound 6 as a representative quasi-irreversible inhibitor and itsnitroso metabolite to CYP3A4. The plausible docking models ofcompound 6 and the nitroso metabolite against CYP3A4 were obtained(Figs. 6A and 7A), and the methyl moieties were then manually added tothese docking models (Figs. 6B and 7B). It was demonstrated that therewould be a severe steric clash between the addedmethyl group and hemein both docking models. Therefore, the amine nitrogen and heme ironcannot keep the optimal distance and the angle (C-N-Fe) to form an MIcomplex. A previous study reported that the potential energy ofcoordinate binding between the heme iron of myoglobin and nitricoxide depends on the distance (Negrerie et al., 2006). The potentialenergy reached the maximum at a distance of approximately 1.8 Å andthen decreased dramatically according to dissociation between the hemeiron and nitric oxide. If we apply this scheme to the binding between theheme iron of CYP3A4 and the nitroso intermediate, then the potentialenergy of binding between the heme and the nitroso intermediate may bedecreased when the distance is extended by introduction of the methylmoiety, preventing formation of the MI complex.The methyl moiety–conjugated compound, such as compound 10, is

still recognized and oxidized by CYP3A4 regardless of the sterichindrance of the methyl moiety according to the docking experiment. Itis possible that electron transfer from the heme to drugs toleratesextension of the distance between the heme and drugs.In conclusion, we elucidated the key structures that are responsible for

MBI of the fluoroquinolone antibacterial compounds, and the difference inthe ring structure bearing a primary amine group in quinolones accounts forthe difference between irreversible and quasi-irreversible inhibition ofCYP3A4. Moreover, we assume that quasi-irreversible inhibition via MIcomplex formation can be avoided after introduction of substituted groupscausing steric hindrance with the heme of P450. Our study may be widelyapplicable to the discovery of drugs free of the DDI risk via MBI.

Fig. 9. The proposed metabolic pathways for compound 6.Fig. 8. The proposed metabolic pathways for compound 1.


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Authorship ContributionsParticipated in research design: Watanabe, Kusuhara.Conducted experiments: Watanabe, Takakusa, Kimura.Performed data analysis: Watanabe, Takakusa, Kimura.Wrote or contributed to the writing of the manuscript: Watanabe, Takakusa,

Kimura, Inoue, Kusuhara, Ando.

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Address correspondence to: Akiko Watanabe, Drug Metabolism and Pharma-cokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi,Shinagawa-ku, Tokyo 140-8710, Japan. E-mail: [email protected]


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