Mechanism-based Inhibition of CYP1A1 and CYP3A4 by the

Regular Article

Mechanism-based Inhibition of CYP1A1 and CYP3A4by the Furanocoumarin Chalepensin

Yune-Fang UENG1,2,3,*, Chien-Chih CHEN1,4, Hiroshi YAMAZAKI5, Kazuma KIYOTANI6,Yu-Ping CHANG1, Wei-Shang LO1, Ding-Tzai LI7 and Pei-Lun TSAI7

1Laboratory of Drug Metabolism, National Research Institute of Chinese Medicine, Taipei, Taiwan, ROC2Institute of Medical Science, Taipei Medical University, Taipei, Taiwan, ROC

3Department of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC4Department of Biotechnology, Hungkuang University, Taichung County, Taiwan, ROC

5Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Tokyo, Japan6Laboratories for Pharmacogenetics, Genotyping Development, and Medical Informatics,

RIKEN Center for Genomic Medicine, Yokohama, Japan7Mass Solution Technology Co., Ltd., New Taipei City, Taiwan, ROC

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: The cytochrome P450 (P450, CYP) 2A6 inhibitor chalepensin was found to inhibit humanCYP1A1, CYP1A2, CYP2A13, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 to different extents. CYP1A1and CYP3A4 underwent pronounced mechanism-based inactivation by chalepensin and had the smallestIC50 ratios of inhibition with NADPH-fortified pre-incubation (IC50(+)) to that without pre-incubation (IC50(¹)).CYP2E1 had the least susceptibility to mechanism-based inactivation. This inactivation of CYP1A1 andCYP3A4 exhibited time-dependence, led to a loss of spectrophotometrically detected P450, and couldnot be fully recovered by dialysis. Pre-incubation with chalepensin did not affect NADPH-P450 reductaseactivity. Cytosol-supported glutathione conjugation protected CYP3A4 but not CYP1A1 against the inacti-vation by chalepensin. Cytosolic decomposition of chalepensin may contribute partially to the protection.The high epoxidation activities of CYP1A1, CYP2A6, and CYP3A4 were in agreement with their pro-nounced susceptibilities to mechanism-based inactivation by chalepensin. Considering both the IC50 valuesand inactivation kinetic parameters, the threshold concentrations of chalepensin for potential druginteractions through inhibition of CYP2A6 and CYP3A4 were estimated to be consistently low. Theseresults demonstrate that chalepensin inhibits multiple P450s and that epoxidation activity is crucial for thepotential drug interaction through mechanism-based inhibition.

Keywords: chalepensin; cytochrome P450; mechanism-based inhibition; epoxidation; hydroxylation

Introduction

The microsomal cytochrome P450 (P450, CYP)-dependentmonooxygenase system plays a major role in the oxidative metabo-lism of a variety of marketed drugs and the chemical constituentsof herbal medicines. Oxidations performed by this membrane-bound monooxygenase system require two electrons suppliedfrom a flavoprotein NADPH-P450 reductase (CPR) to a memberof the P450 hemoprotein family.1) CYP1A2, CYP2A6, CYP2C,CYP2D6, CYP2E1, and CYP3A4 constitute about 73% of the totalP450 content in human liver microsomes.2) CYP3A4 appears to be

the most abundant P450 form in human livers and it oxidizes about46% of marketed drugs.3) CYP1A1 and CYP2A13 are essentiallyextrahepatic P450 isoforms.1,4) CYP1A1 and CYP1A2 have about70% amino acid sequence identity, and CYP1A1 is of particularnote because of its importance in the detoxification and metabolicactivation of the mutagenicities of polycyclic aromatic hydro-carbons, such as the air pollutant benzo(a)pyrene.1,5) CYP2A6and CYP2A13 have about 94% amino acid sequence identity.CYP2A6 is mainly expressed in the liver, whereas CYP2A13is predominantly expressed in the respiratory tract.4) AlthoughCYP1A2, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 oxidize

Received October 2, 2012; Accepted December 1, 2012J-STAGE Advance Published Date: December 18, 2012, doi:10.2133/dmpk.DMPK-12-RG-113*To whom correspondence should be addressed: Yune-Fang UENG, Ph.D., National Research Institute of Chinese Medicine, 155-1, Li-NongStreet, Sec. 2, Taipei 112, Taiwan, ROC. Tel. ©886-2-28201999 ext. 6351, Fax. ©886-2-28264266, E-mail: [email protected] work was supported by grants NSC97-2320-B-077-009-MY3, NSC99-2918-I-077-001 (short-term study abroad research program), andNSC101-2320-B-077-001-MY3 from the National Science Council and a grant from the National Research Institute of Chinese Medicine, Taiwan.

Drug Metab. Pharmacokinet. 28 (3): 229–238 (2013). Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)

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http://dx.doi.org/10.2133/dmpk.DMPK-12-RG-113

more than 70% of marketed drugs,3) CYP2A6 participates in theoxidation of several important drugs, including halothane, tegafur,and losigamone.6) CYP2A13 exhibited only 12% of the coumarin7-hydroxylation activity of CYP2A6 but had 75 times its 4-(meth-ylnitrosamino)-1-(3-pyridyl)-1-butanone oxidation activity. It isknown that CYP2A13 participates in the oxidation of drugs suchas theophylline, phenacetin, and chlorzoxazone.7,8) The activitiesof CYP2A13 could be comparable to or even greater than thoseof the hepatic P450s mainly involved in the oxidation of thesedrug substrates. CYP2A6 and CYP2A13 show different levels ofsusceptibility to inhibition by isothiocyanates such as tert-butylisocyanate.9) The differences in the selectivities with respect tosubstrates and inhibitors of these two CYP2A members can havesignificant effects in terms of biological responses.

Clinically relevant drug interactions are frequently attributedto functional alterations in P450s, such as the mechanism-basedinactivation of CYP3A4 by grapefruit furanocoumarins.10,11)

Mechanism-based inhibitors are oxidized by a P450 to generatean active metabolite that irreversibly inactivates the P450. Becausethe formation of metabolite-modified P450 irreversibly inactivatesP450 function, mechanism-based inhibition is generally thoughtto have a greater effect than reversible inhibition does. Sekiguchiet al.12) reported a high-throughput screening method that canpredict the change in the area under the plasma drug concentra-tion versus time curve (AUC) caused by the mechanism-basedinactivation of P450s. This prediction was based on the IC50

inhibition values with NADPH-fortified pre-incubation (IC50(+))and without NADPH-fortified pre-incubation (IC50(¹)). A dou-bling of the AUC was considered to indicate a high risk ofdrug interaction. Mayhew et al.13) proposed another method forassessing in vivo inhibition using the inactivation kinetic param-eters [kinact (maximal inactivation rate constant) and KI (concen-tration required for half-maximal inactivation)] obtained fromin vitro studies. The results obtained using these methods showeda good correlation with known drug interaction events with a lowincidence of false-negative results.

Chalepensin is a furanocoumarin compound with a dimethylallyl side chain on the pyran-2-one moiety. It is present in severalherbs, especially those of the Rutaceae family, including Rutagraveolens (rue), Ruta chalepensis, and Stauranthus perfora-tus.14,15) Some of these herbs are used in America, Asia and Europefor the treatment of headache, wounds, and rheumatic disorders.16)

The chalepensin contents in the extracts of rue stem, leaves,and flowers are 13%, 7%, and 11%, respectively.15) Chalepensinexhibits a variety of pharmacological/toxicological activities, in-cluding cytotoxicity in several cancer cell lines and anti-fertilitytoxicity.14,17) Our previous report demonstrated that chalepensinis a mechanism-based inhibitor of CYP2A6.18) The inactivationof CYP2A6 was suggested to be through the epoxidation at theallyl side chain (Fig. 1). The epoxide intermediate generated byCYP2A6 was presumably detected as dihydrodiol and could beeliminated through glutathione conjugation, which protectedCYP2A6 against this mechanism-based inactivation. However,the inhibitory effects of chalepensin on P450s other than CYP2A6have not been reported.

To investigate the inhibitory effects of chalepensin on differenthuman P450s, the activities of the main hepatic P450s CYP1A2,CYP2C9, CYP2D6, CYP2E1, and CYP3A4 were determined; theextrahepatic CYP1A1 and CYP2A13 were studied for comparison.To compare the relative potential risk of drug interaction, we used

both high-throughput screening and inactivation kinetic methodsto estimate the threshold concentration of chalepensin required toproduce a doubling of the AUC through inhibition of P450s.

Materials and Methods

Chemicals and enzymes: Chalepensin was isolated andpurified from the ethanol extract of the aerial part of Rutagraveolens L., as described previously.18) The purity of chalepensinwas greater than 99% according to nuclear magnetic resonance(NMR) and high-performance liquid chromatography (HPLC)analyses. Bovine serum albumin (BSA), chlorzoxazone, coumarin,glucose-6-phosphate, glucose-6-phosphate dehydrogenase, 7-hy-droxycoumarin, ¢-nicotinamide adenine dinucleotide phosphate(NADP+) sodium salt, the reduced form of NADP+ (NADPH), andnifedipine were purchased from Sigma-Aldrich (St. Louis, MO).

Preparation of bacterial membranes expressing humanP450s and mouse liver cytosol: The expression constructs ofCYP1A1, CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, andCYP3A4 were generously provided by Dr. F. Peter Guengerich(Nashville, TN).19) The CYP2A13 construct was prepared as de-scribed previously.20) These bicistronic human constructs consistedof the coding sequence of a P450 followed by that of NADPH-P450 reductase (CPR) and were transformed to Escherichia coliDH5¡ by electroporation (Gene Pulser II, Bio-Rad Laboratories,Hercules, CA). Bacterial membranes were prepared19,21) and storedat ¹75°C until use. BD-Gentest supersomes expressing CYP2B6(+cytochrome b5) and CYP2C19 were purchased from BDBiosciences (San Jose, CA). The O-deethylation activities ofCYP1A1 toward 7-ethoxyresorufin and 7-ethoxycoumarin were13.9 « 2.1, and 6.95 « 0.87 nmol/min/nmol P450, respectively.The 7-methoxyresorufin O-demethylation and 7-ethoxyresorufin

Fig. 1. MS/MS spectra of protonated hydroxychalepensin and chalepensindihydrodiol, recorded with an Exactive Orbitrap LC-MS/MS instrumentChalepensin oxidation by CYP2A6 was determined using 20 pmol P450/ml.After incubation, oxidation products were subjected to LC-MS/MS analysis.The exact m/z values of the fragments indicated in the MS/MS spectra weredetermined as described in Materials and Methods.

Yune-Fang UENG, et al.230

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O-deethylation activities of CYP1A2 were 4.64 « 1.02 and 1.41 «0.16 nmol/min/nmol P450, respectively. The coumarin 7-hydro-xylation activities of CYP2A6 and CYP2A13 were 7.22 « 0.81and 0.48 « 0.03 nmol/min/nmol P450, respectively. The oxida-tion activities of CYP2C9 (tolbutamide hydroxylation), CYP2D6(dextromethorphan O-demethylation), CYP2E1 (chlorzoxazonehydroxylation), and CYP3A4 (nifedipine oxidation) and were5.08 « 0.33, 26.8 « 1.9, 40.8 « 6.1, and 3.88 « 0.46 nmol/min/nmol P450, respectively. The supersomes expressing CYP2B6 andCYP2C19 had 7-ethoxy-4-trifluoro-methyl-coumarin deethylaseand mephenytoin oxidation activities of 6.7 and 7.1 nmol/min/nmol P450 (data provided by BD Biosciences), respectively. MaleC57BL/6JNarl mice (5 weeks old, weight 17–20 g) were purchasedfrom the National Laboratory Animal Center (Taipei, Taiwan).All experimental protocols involving animals were reviewed andapproved by the Institutional Animal Care and Use Committeeof the National Research Institute of Chinese Medicine. Mouseliver cytosol was prepared by differential centrifugation at 4°Cfollowing the methods of Alvares and Mannering.22)

Determination of P450 and glutathione S-transferase activ-ities: CPR activity was determined using cytochrome c as asubstrate following the method of Phillips and Langdon.23) P450activities were determined in the assays using 20 nM P450. 7-Ethoxyresorufin O-deethylation (EROD) and 7-methoxyresorufinO-demethylation activities were determined by measuring thefluorescence of resorufin.24) 7-Ethoxycoumarin O-deethylation(ECOD) activity was determined following the method of Greenleeand Poland.25) Coumarin 7-hydroxylation and dextromethorphanO-demethylation activities were determined using an HPLC sys-tem equipped with a fluorescence detector.26,27) Oxidation activitiestoward nifedipine, tolbutamide, and chlorzoxazone were deter-mined using an HPLC system equipped with a UV detector.28–30)

Substrate concentrations were 2 µM 7-ethoxyresorufin, 20 µM cou-marin, 0.5mM chlorzoxazone, 200 µM dextromethorphan, 20 µM7-methoxyresorufin, 200µM nifedipine, and 2.5mM tolbutamide.Glutathione S-transferase activity was determined using 1-chloro-2,4-dinitrobenzene as a substrate.31) The cytosolic protein concen-tration was determined by the method of Lowry et al.32)

Determination of IC50 values: Chalepensin was dissolvedin methanol and the final concentration of methanol was less than1% during the determination of monooxygenase activity. Thecontrol incubation contained the same concentration of methanol.Activities were determined in incubations containing increasingconcentrations of chalepensin with or without NADPH-fortifiedpre-incubation of P450s with chalepensin at 37°C for 10min asdescribed previously.18) The concentrations of chalepensin requiredfor 50% inhibition of catalytic activities without pre-incubation(IC50(¹)) or with pre-incubation (IC50(+)) were calculated by curvefitting (Grafit, Erithacus Software Ltd., Staines, UK).

Spectral analysis: The P450 concentration was determinedby the spectrophotometric method of Omura and Sato.33) Todetermine the loss of spectrophotometrically detected P450 aftermechanism-based inhibition, incubation mixtures contained 55pmol/ml CYP1A1 or 703 pmol/ml CYP3A4, 0–5µM chalepensin,and an NADPH-generating system in 75mM potassium phosphatebuffer, pH 7.4. Reactions were carried out at 37°C for 10min.After incubation, the reaction mixtures were put on ice and P450concentrations were determined.

Dialysis experiments: To determine the NADPH-dependentactivities of CPR and P450s in dialyzed samples, NADPH was

used instead of an NADPH-generating system18) in the pre-incubation of P450 with chalepensin. Thus, the remaining NADPHand chalepensin after pre-incubation could be removed by dialysisand the activities of dialyzed samples could be determined in areaction initiated by NADPH or an NADPH-generating system.Bacterial membranes expressing bicistronic human P450s (20 nM)were incubated with chalepensin in a 50mM potassium phos-phate buffer (pH 7.4) containing 200µM NADPH at 37°C for15min. Incubations containing the same volume of methanol butno chalepensin were performed as the vehicle controls. After pre-incubation, the mixture was put on ice and the activity of an aliquot(before dialysis) was determined using substrate as the initiationagent. An aliquot of 200µl was dialyzed using a minidialysisunit (10 kDa cutoff, Thermo-Pierce Biotechnology, Rockford, IL)against 120ml of 50mM potassium phosphate containing 0.1mMEDTA at 4°C. The dialysis buffer was changed 3 times at 30-minintervals. CPR and P450 activities of the dialyzed samples weredetermined as described above. To evaluate the recovery ofdialysis, the protein concentration was determined using a dye-binding assay following the instruction manual of the Bio-RadProtein assay kit (Bio-Rad Laboratories). The dialysis recoverywas 104 « 3% (mean « SEM of 12 determinations).

Characterization of the influence of glutathione conjugation:To elucidate the effects of glutathione conjugation on mechanism-based inhibition of P450s by chalepensin, P450 activities weredetermined under various pre-incubation conditions. Mouse livercytosols with a 1-chloro-2,4-dinitrobenzene conjugation activity of5.3–8.8 µmol/min/mg protein were used as a source of glutathioneS-transferase. In the presence of an NADPH-generating systemand chalepensin, P450 was pre-incubated with 5mM glutathioneand/or 1mg/ml mouse liver cytosolic proteins at 37°C for 10min.Based on the IC50 values of CYP1A1 and CYP3A4 inhibition,0.15, 0.2, and 4 µM chalepensin was added to the pre-incubationmixture for the inhibition of EROD (CYP1A1), ECOD (CYP1A1),and nifedipine oxidation (CYP3A4), respectively. As the negativecontrol experiments, heat-treated (95°C for 15min) mouse livercytosol (for the inactivation of cytosolic enzymes) or 1mg/mlBSA (for non-specific protein binding) was included in the pre-incubation. After the pre-incubation, P450 substrates were addedand their activities were determined as described above.

To examine the potential decomposition of chalepensin in thepresence of cytosol/glutathione, an NADPH-fortified incubation ofchalepensin was performed at 37°C in the absence or presence ofcytosol (1mg protein/ml) and glutathione (5mM). The concen-tration of chalepensin used in each assay was the same as describedabove. A vehicle control incubation containing the same concen-tration of methanol without chalepensin and cytosol/glutathionewas prepared. After 10min of incubation, the reaction was stoppedby heating at 98–100°C for 3min and then the reaction tubeswere immediately put on ice for 3min and mixed with a vortexmixer. NMR analysis of heat-treated chalepensin indicated thatthere was no destruction of chalepensin. P450 and an NADPH/NADPH-generating system (in potassium phosphate buffer) werethen added to this mixture to start the P450 pre-incubation (37°C,10min). To evaluate the protective effect of glutathione con-jugation, cytosol and glutathione were added again in the P450 pre-incubation mixture. After 10-min pre-incubation, P450 substrateswere added and the P450 activities were determined as describedabove.

Chalepensin hydroxylation and epoxidation determinations:

Chalepensin: Substrate and Inhibitor of Human P450s 231


The formation of chalepensin oxidation products was determinedas described previously.18) The exact m/z values of fragmentsof protonated hydroxychalepensin and chalepensin dihydrodiolwere analyzed using an Exactive Orbitrap mass spectrometer sys-tem (Thermo Fisher, Suwanee, GA) with an electrospray volt-age of 4,000V and an in-source-fragmentation voltage of 15–50V. Primarily, each 0.5-ml reaction mixture contained 10 pmolP450, 50mM potassium phosphate buffer (pH 7.4), 3 or 200µMchalepensin, and an NADPH-generating system. Reactions werecarried out at 37°C for 30min and were stopped by the addition ofperchloric acid. After centrifugation, the supernatant was injectedinto a C18 column (Thermo Biobasic-18, 5 µm, 2.1mm © 15 cm,Thermo) in a liquid chromatography/dual mass spectrometry(LC-MS/MS) system (LTQ Velos, Thermo Fisher) to determinemetabolite formation. Chalepensin epoxidation activity was deter-mined by measuring the formation of dihydrodiol, which waspresumably generated from the hydrolysis of an epoxide inter-mediate. Quantitative comparison of the metabolite formationwas calculated by determination of the peak area of fragmentationions extracted in the MS/MS system with the correct chromato-graphic retention time. Figure 1 shows the fragmentation ionsin the MS/MS spectra of protonated hydroxychalepensin andchalepensin dihydrodiol. The selective fragmentation ions forhydroxychalepensin and chalepensin dihydrodiol had m/z valuesof 227 and 271, respectively. The oxidation activity was calculatedas the peak area of metabolite/min/nmol P450.

Kinetic analyses and determination of partition ratio: Theinactivation rates (Kapp) under different chalepensin concentrationswere determined as the slopes calculated by linear regressionanalysis of the plot of the natural logarithm percent of remainingactivity versus pre-incubation time. The maximal inactivation rateconstant (kinact) of chalepensin and the chalepensin concentrationrequired for half-maximal inactivation (KI) were estimated usingnonlinear regression according to the equation Kapp = (kinact0[I])/(KI + [I]), with the initial values calculated from the doublereciprocal plot of inactivation rate versus the concentration ofinhibitor chalepensin, [I].34) The partition ratio of a mechanism-based inhibitor is the ratio of the amount of inhibitor released as theoxidation product to the amount covalently bound to the enzyme.34)

The partition ratio was estimated using the enzyme titration method,in which bacterial membrane expressing CYP1A1 and CYP3A4(20 pmol P450/ml) was pre-incubated with increasing concentra-tions of chalepensin for 30min in the presence of an NADPH-generating system to generate extensive inhibition. The remainingactivity was plotted against the molar ratio of chalepensin to P450.The turnover number (partition ratio + 1) was estimated as theintersection with the x-axis of lines established by regression of thelinear portions at low and high molar ratios of chalepensin to P450.

Threshold concentration estimation: According to the high-throughput screening method12) (Eq. 1) and the inactivation kineticmethod13) (Eq. 2), the threshold concentration required to producea doubling of the AUC was estimated. Because of variations inthe estimated degradation rate constant kdeg determined in differ-ent human samples and determination systems,35) the constantsobtained from a single determination system of cultured humanliver slices were used.36) The kdeg values of CYP1A2, CYP2A6,CYP2C9, CYP2D6, CYP2E1, and CYP3A4 were 0.000321,0.000444, 0.000111, 0.000165, 0.000428, and 0.000146min¹1,respectively.

AUCi=AUC ¼ f1þ ð1þ ½1þ ðIC50ð�Þ=IC50ðþÞ=kdeg � tÞ� ðln 2=ð1þ IC50ðþÞ=IC50ð�ÞÞÞ�Þ� ½I�=IC50ð�Þg=ð1þ ½I�=IC50ð�ÞÞ ð1Þ

AUCi=AUC ¼ ½kdeg þ ðkinact � ½I�=ðKI þ ½I�ÞÞ�=kdeg ð2Þwhere AUCi/AUC is the predicted AUC ratio of in vivo exposureof a P450-metabolized drug with the co-administration of theinhibitor i to that without the inhibitor.

Statistical analyses: The statistical significance of the differ-ence between two different incubation conditions was evaluatedusing Student’s t test. A p value of less than 0.05 was consideredto be statistically significant. The linear relationship betweenpairs of variables was analyzed using Sigma Plot software (10.0,SPSS Inc.).

Results

Inhibition of P450 activities by chalepensin in the absenceand presence of NADPH-fortified pre-incubation: WithoutNADPH-fortified pre-incubation, CYP2A13 and CYP2D6 werethe isoforms most sensitive to inhibition by chalepensin withIC50(¹) values less than 2µM (Table 1). CYP1A1, CYP1A2, andCYP2C9 were inhibited to a lesser extent and had IC50(¹) values inthe range of 3–9µM. Using the same substrate (7-ethoxyresorufin),the IC50(¹) values for CYP1A1 and CYP1A2 inhibition werealmost identical. CYP2E1 and CYP3A4 exhibited relatively lowsusceptibilities to inhibition and had IC50(¹) values higher than60µM. The inhibition of CYP2A6 as well as CYP1A1/CYP3A4was markedly increased by NADPH-fortified pre-incubationwith chalepensin. The IC50(+)/IC50(¹) ratios of CYP1A1 andCYP3A4 were comparable to that of the CYP2A6, which isknown to undergo mechanism-based inhibition in the presenceof chalepensin.17) The inhibition of CYP2A13, CYP2C9, andCYP2D6 exhibited moderate enhancement as a result of pre-incubation and had IC50(+)/IC50(¹) ratios in the range of 0.3–0.4. Incontrast, the IC50(+) and IC50(¹) values for inhibition of CYP2E1had a difference of only 12%, suggesting the absence of obviousmetabolic inactivation of CYP2E1 by chalepensin.

The destruction of spectrally detected P450 and the influenceof dialysis: Because CYP1A1 and CYP3A4 inhibition had thesmallest IC50(+)/IC50(¹) ratios, P450 destruction and the influence ofdialysis were studied for these two P450s. The NADPH-fortifiedpre-incubation of P450 with chalepensin for 10min resulted ina reduction in spectrophotometrically detected P450 because ofthe chalepensin-mediated inactivation of CYP1A1 (Fig. 2A) and

Table 1. IC50 values for human cytochrome P450 inhibition by chalepensin

P450 Substrate IC50(¹), µM IC50(+), µM IC50(+)/IC50(¹)

1A1 7-Ethoxyresorufin 2.86 « 0.09 0.21 « 0.02 0.091A2 7-Ethoxyresorufin 3.01 « 0.18 2.09 « 0.09 0.691A2 7-Methoxyresorufin 8.56 « 0.69 5.34 « 0.81 0.622A6 Coumarin 82.3 « 8.3 1.38 « 0.13 0.02a

2A13 Coumarin 0.21 « 0.02 0.07 « 0.01 0.302C9 Tolbutamide 6.58 « 3.03 2.25 « 0.41 0.342D6 Dextromethorphan 1.67 « 0.12 0.61 « 0.03 0.372E1 Chlorzoxazone 61.1 « 8.7 53.6 « 7.1 0.883A4 Nifedipine 61.7 « 2.0 2.48 « 0.17 0.04

Activities of bacterial membranes expressing human P450 were determinedusing 20 pmol P450/ml in the assays. The IC50(¹) and IC50(+) values were deter-mined for P450 inhibition without and with NADPH-fortified pre-incubation,respectively.aIC50 values reported previously.17)



CYP3A4 (Fig. 2B). These reductions were concentration-depend-ent. To examine the influence of dialysis and the change of CPRreduction activity, bacterial membranes bicistronically expressingP450s and CPR were incubated with 0, 1.5, and 5µM chalepensinin the presence of NADPH. After this oxidative inactivation,an aliquot of reaction mixture was dialyzed to remove freechalepensin and the remaining NADPH. P450 and CPR activitiesof samples before and after dialysis were determined as describedin Materials and Methods. For CYP1A1 inhibition, the 98%decrease of activity by chalepensin (1.5, and 5µM) was reduced to60% after dialysis (Fig. 2C). However, for CYP2A6 inhibition,the decrease of activity could not be recovered after dialysis. ForCYP3A4 inhibition, the decrease of activity by 1.5 µM chalepensincould not be recovered after dialysis, whereas a 59% decrease ofactivity by 5 µM chalepensin was reduced to 31% by dialysis.Without pre-incubation, the cytochrome c reduction activity ofCPR was not affected by 5µM chalepensin (data not shown). Withpre-incubation with chalepensin, dialysis did not generate a changegreater than 20% in CPR activity (Fig. 2D).

The influence of glutathione conjugation: Mouse livercytosols were used as a source of glutathione S-transferase.However, the presence of cytosol in the NADPH-fortified P450pre-incubation with chalepensin enhanced chalepensin-mediateddecrease of CYP1A1-catalyzed EROD activity by 25%. Neither

BSA nor heat-denatured cytosol was the cause of this enhance-ment (Fig. 3A). To examine the decrease in EROD metabolitegeneration in the presence of cytosol, CYP1A1 was pre-incubatedwith cytosol but not chalepensin in the presence of NADPH andthen the substrate 7-ethoxyresorufin was added to determine theEROD activity. The presence of cytosol caused a 29% decreaseof EROD activity (data not shown), which was similar to the levelof inhibition enhancement by cytosol. This effect of cytosol couldbe caused by the further decomposition of metabolite resorufin bycytosolic enzyme(s) such as NADPH-quinone oxidoreductase.37)

The decrease of EROD activity resulting from pre-incubationwith chalepensin and cytosol was diminished by the addition ofglutathione and the activity was restored to a level exhibiting nosignificant difference with the inhibition by chalepensin withoutcytosol. To clarify the protective role of glutathione conjugation,7-ethoxycoumarin, another substrate of CYP1A1, was studied. Ourdeterminations showed that the presence of cytosol did not affectthe inhibition of ECOD by chalepensin (Fig. 3A). However, the

Fig. 2. The destruction of spectrally detected P450 and the influence ofdialysisPanels (A) and (B) show the loss of spectrophotometrically detected P450 byNADPH-fortified pre-incubation with chalepensin in recombinant CYP1A1 andCYP3A4 systems, respectively. Chalepensin was dissolved in methanol and anequal volume of methanol was added in the control incubation. Data representthe means of duplicated determinations. Panels (C) and (D) show the remainingP450 and NADPH-P450 reductase (CPR) activities, respectively, of chalepensin-inactivated bicistronic P450 systems before and after dialysis. Bacterial mem-branes expressing CYP1A1, CYP2A6, and CYP3A4 followed by CPR wereincubated with or without chalepensin at 37°C for 15min in the presence of200µM NADPH. Incubation without chalepensin was performed as the control.After the inactivation incubation, P450 and CPR activities of aliquots of samplesbefore and after dialysis were determined. Results are shown as the percentageof control activity and the data are the means and mean « SEM of duplicatedand 3 determinations, respectively.

Fig. 3. Differential protective effects of glutathione (GSH) conjugationagainst chalepensin-mediated mechanism-based inhibition of CYP1A1 andCYP3A4Mouse liver cytosol (1mg/ml) and GSH (5mM) were used in the assays tosupport glutathione conjugation. Panel (A) shows the effect of GSH conjugationon the mechanism-based inhibition of CYP1A1 and CYP3A4 by 0.15 and 4 µMchalepensin, respectively. In the presence of an NADPH-generating system,CYP1A1 and CYP3A4 were pre-incubated with chalepensin and co-incubatedwith various substances, as indicated. The influence of bovine serum albumin(BSA, 1mg/ml) and heat-denatured cytosol (H) were studied as the negativecontrols. Each bar represents the mean « SEM of 3–5 determinations. Panel(B) shows the effects of chalepensin and cytosol/GSH-treated chalepensinon CYP1A1 and CYP3A4 activities. Chalepensin was pre-incubated in anNADPH-containing buffer with or without cytosol/GSH. The control incubationcontained the same concentration of methanol without chalepensin. Aftertreatment, the reactions were stopped by heating and then P450 was pre-incubated with this mixture and P450 activities were determined. In the assaylabeled [cytosol/GSH]post, P450 was pre-incubated with the cytosol/GSH-treatedchalepensin, cytosol, and GSH. Each bar represents the mean « SEM of 4–5determinations. *Values significantly different from the incubation includingchalepensin but without glutathione or mouse liver cytosol, p < 0.05.



decrease of ECOD activity by chalepensin could not be recoveredby the presence of cytosol/glutathione in the pre-incubation mix-ture. The presence of BSA, glutathione, or cytosol had no effecton CYP3A4 inactivation by chalepensin (Fig. 3A). However, thepresence of cytosol and glutathione protected CYP3A4 againstmechanism-based inactivation by chalepensin, with the nifedipineactivity being restored to 90% of the activity in the absence ofchalepensin. In contrast, heat-denatured cytosol and glutathionedid not have this protective effect. These results suggest thatglutathione conjugation has a protective effect against chalepensin-mediated inactivation of CYP3A4, presumably catalyzed bycytosol/glutathione.

To examine the potential decomposition of chalepensin in thepresence of cytosol/glutathione (a possible mechanism for theabove-mentioned protective effect), the NADPH-fortified cytosol/glutathione treatment of chalepensin was performed and the pre-treatment was stopped by heating. After the pre-incubation of P450with the pre-treated vehicle control or chalepensin, P450 activitywas determined. In the vehicle-control, the ECOD and nifedipineoxidation activities of CYP1A1 and CYP3A4 were 10.9 « 0.7and 3.14 « 0.10 nmol/min/nmol P450, respectively. The pre-treatment process (without cytosol/glutathione) did not affect thebasal activities of P450s and the inhibitory effect of chalepensin(Fig. 3B). Determination of CYP1A1-catalyzed ECOD activityshowed that there was no reduction of chalepensin-mediatedinhibition by pre-treatment with cytosol. Unexpectedly, there was11% enhancement of the inhibition by cytosol/glutathione pre-treatment. In CYP3A4 assay system, there was no significantdifference in the decreases of CYP3A4 activity by chalepensin orby cytosol-treated chalepensin. However, the cytosol/glutathione-treatment reduced the inhibitory effect of chalepensin by 12%.Although the reduction by this pre-treatment was mild, cytosolicdecomposition (with NADPH and glutathione) might participatepartially in the reduction of CYP3A4 inhibition when cytosol/glutathione was present in the P450-pre-incubation mixture. Thedecrease of CYP3A4 activity by pre-incubation of P450 withcytosol/glutathione-treated chalepensin was almost fully preventedby the addition of active cytosol/glutathione, indicating the maincontribution of glutathione conjugation to the protective effect.

Kinetic analyses and partition ratios of the mechanism-based inhibition of CYP1A1 and CYP3A4 by chalepensin:NADPH-fortified pre-incubation of CYP1A1 and CYP3A4 withchalepensin induced a time-dependent inactivation of EROD andnifedipine oxidation activities, respectively (Figs. 4A and 5A).Non-linear regression analysis of the inactivation of CYP1A1generated a kinact of 0.400 « 0.033min¹1 and an apparent KI of0.64 « 0.21 µM (r = 0.971) (Fig. 4B), which were very close tothe values (kinact: 0.384min¹1; apparent KI: 0.56 µM, r = 0.984)generated by linear regression analyses of the double recipro-cal plots of the CYP1A1 inactivation rate and the chalepensinconcentration (Fig. 4C). The KI value for CYP3A4 inhibition[3.28 « 1.05 µM (r = 0.967) using non-linear regression analysis(Figs. 5B) and 5.68 µM (r = 0.980) using linear regression ana-lysis (Fig. 5C)] was higher than that for CYP1A1 inhibition,indicating that a higher concentration of chalepensin was requiredto achieve half-maximal inactivation of CYP3A4. The maximalinactivation rate constant (kinact) of CYP3A4 inhibition (0.164 «0.015min¹1 using nonlinear regression analysis and 0.203min¹1

using linear regression analysis) was lower than that for CYP1A1inhibition.

Fig. 4. Time-dependent enhancement of the inhibitory effect of chalepen-sin on CYP1A1 activity(A) The remaining activity is shown as the percentage of the activity at time0 of the vehicle control incubation. (B) Plot of inactivation rate (Kapp) versuschalepensin concentration. (C) Double-reciprocal plot of the relationshipbetween inactivation rate and chalepensin concentration. (D) Plot of remainingactivity versus the molar ratio of chalepensin to CYP1A1. Data represent themean and mean « SEM value of two and three separate experiments withduplicates, respectively.

Fig. 5. Time-dependent enhancement of the inhibitory effect of chalepen-sin on CYP3A4 activity(A) The remaining activity is shown as the percentage of the activity at time0 of the vehicle control incubation. (B) Plot of inactivation rate (Kapp) versuschalepensin concentration. (C) Double-reciprocal plot of the relationshipbetween inactivation rate and chalepensin concentration. (D) Plot of remainingactivity versus the molar ratio of chalepensin to CYP3A4. Data represent themean and mean « SEM of two and three separate experiments with duplicates,respectively.



To ensure complete inactivation, CYP1A1 and CYP3A4 werepre-incubated with chalepensin and an NADPH-generating systemfor 30min and then their activities were determined. Titration ofthe remaining activity when using increasing concentrations ofchalepensin generated turnover numbers (partition ratio +1) of 33and 213 for the inactivation by chalepensin of CYP1A1 (Fig. 4D)and CYP3A4 (Fig. 5D), respectively. These results indicate that 33and 213 molecules of chalepensin were oxidized by one moleculeof CYP1A1 and CYP3A4, respectively, for every molecule ofenzyme inactivated. The partition ratios of CYP1A1 and CYP3A4inactivation are therefore 32 and 212, respectively.

Estimation of threshold concentration of chalepensin fordrug interaction resulting from hepatic P450 inhibition: Theconcentration of chalepensin that doubles the AUC of the substrateconcentration versus time curve was defined as the threshold con-centration for drug interaction and was estimated using the high-throughput-screening method (using IC50 values) and the inactiva-tion kinetic method (using inhibitory kinetic parameters) proposedby Sekiguchi et al.12) and Mayhew et al.,13) respectively. Relativelylow chalepensin threshold concentrations in the range of 2.0–5.3 nM were estimated for the inhibition of CYP2C9, CYP2D6,and CYP3A4 (Table 2). For CYP2A6 inhibition, the thresholdconcentration was 10.6 nM. For CYP1A2, the threshold concen-trations were 23.6 and 50.2 nM for the substrates 7-ethoxyresorufinand 7-methoxyresorufin, respectively. However, the threshold con-centration for CYP2E1 inhibition was as high as 2 µM. Amongthese human hepatic P450s, CYP2A6 and CYP3A4 were mostaffected by metabolic inactivation, i.e., they had the greatestdecrease of IC50 value on pre-incubation with chalepensin. Thus,the threshold concentrations of chalepensin for the inactivation ofCYP2A6 and CYP3A4 were further estimated using the inactiva-tion kinetic method proposed by Mayhew et al.13) Using thismethod, the estimated threshold concentrations of chalepensin forCYP2A6 and CYP3A4 inhibition were 7.1 and 2.9 nM, respec-tively (Table 2); these values are very close to those estimatedusing the high-throughput screening method.

Chalepensin oxidation activities of recombinant P450enzymes: Based on the hepatic (1.4 µM) and total plasmaconcentrations (2.8 µM) of chalepensin determined in our pre-vious mouse study (in which mice were treated with 10mg/kgchalepensin18)) and the IC50(+) values (0.1–54µM) of P450s deter-

mined in this study, chalepensin hydroxylation and epoxidationactivities were determined using 3 and 200 µM chalepensin. Theformation of hydroxylation and epoxidation metabolites by P450swas confirmed by chromatograms and MS/MS spectra of LC-MSanalysis. A difference in oxidation activities for 3 and 200 µMchalepensin of less than 20% was considered to be insignificant.At 3 µM chalepensin, CYP2A6 had the highest chalepensin hy-droxylation and epoxidation activities. P450 isoforms includingCYP1A1, CYP2A13, CYP2C9, CYP2C19, CYP2D6, CYP2E1,and CYP3A4 had moderate chalepensin hydroxylation activities,whereas the activities of CYP1A2 and CYP2B6 were low. At 3 µMchalepensin, CYP1A1, CYP2A13, CYP2C9, CYP2D6, CYP2E1,and CYP3A4 had moderate chalepensin epoxidation activitieslower than that of CYP2A6, whereas the epoxidation activities ofCYP1A2, CYP2B6, and CYP2C19 were below the detection limit(Fig. 6). For a chalepensin concentration of 200µM, CYP1A1,CYP2C9, CYP2C19, CYP2E1, and CYP3A4 had chalepensinhydroxylation activities higher than that of CYP2A6. CYP1A2,CYP2A13, CYP2B6, and CYP2D6 had detectable but relativelylow chalepensin hydroxylation activities. At 200 µM chalepensin,CYP2A6 had the highest chalepensin epoxidation activity, andCYP1A1 and CYP3A4 had slightly lower epoxidation activities.CYP2A13, CYP2C9, CYP2D6, and CYP2E1 had moderate epoxi-dation activities at 200 µM chalepensin, whereas CYP1A2,CYP2B6 and CYP2C19 activities were relatively low or belowthe detection limit.

Discussion

Liver contains high levels of P450s, and hepatic P450s aregenerally the first targets for the evaluation of drug interactions.In our studies of the main hepatic P450s, without pre-incubation,CYP1A2, CYP2C9, and CYP2D6 showed higher susceptibilitiesto the inhibition by chalepensin than CYP2E1 and CYP3A4 did.However, after NADPH-fortified pre-incubation, the decreasesof IC50 values that occurred in the CYP1A2, CYP2C9, CYP2D6,and CYP2E1 systems were not as great as that in the CYP3A4system, indicating potent enhancement of CYP3A4 inhibition

Table 2. Estimated concentration of chalepensin required to double theAUC of the substrate versus time curve through inhibition of the mainhuman hepatic cytochrome P450s

P450 SubstrateThreshold concentration, nM

High-throughputscreening method

Inactivationkinetic method

1A2 7-Ethoxyresorufin 23.6 —a

1A2 7-Methoxyresorufin 50.2 —

2A6 Coumarin 10.6 7.12C9 Tolbutamide 4.6 —

2D6 Dextromethorphan 2.0 —

2E1 Chlorzoxazone 2,045 —

3A4 Nifedipine 5.3 2.9

The threshold concentration of chalepensin required to cause a 2-fold increase ofthe AUC was estimated using the high-throughput screening method11) and theinactivation kinetic method.12) Chalepensin caused pronounced mechanism-based inhibition of CYP2A6 and CYP3A4 (Table 1). The inactivation kineticparameters of CYP2A6 and CYP3A4 were obtained from previous work17) andthe current work, respectively.aEm-dashes indicate that values were not determined.

Fig. 6. Chalepensin hydroxylation (A) and epoxidation (B) by human P450enzymesChalepensin oxidation activity was determined using 3 and 200 µM chalepensinin a reaction mixture containing 20 pmol P450/ml, as described in Materials andMethods. Data represent the mean « SEM of 3–4 determinations.



by an NADPH-fortified metabolic reaction. The decrease of IC50

on pre-incubation for CYP3A4 inhibition was comparable to thatfor CYP2A6 inhibition.18) These findings reveal the differentialsusceptibilities of hepatic P450s to chalepensin-mediated mecha-nism-based inhibition; CYP2A6 and CYP3A4 appeared to havethe highest susceptibility. Chalepensin-mediated inactivation didnot affect the reduction activity of CPR in CYP2A6 and CYP3A4systems, indicating that CPR-mediated electron transfer remainednormal when P450 activity was impaired. Similarly to CYP2A6inactivation, CYP3A4 inactivation caused a reduction in spec-trophotometrically detected P450. The efficiency (kinact/KI) of in-activation of CYP3A4 was similar to that of CYP2A6. AlthoughCYP3A4 had a higher IC50(+) value, a lower chalepensin epoxi-dation activity, and a higher partition ratio of chalepensin to P450compared with CYP2A6,17) the expression level of CYP3A4 ismore than 3-fold that of CYP2A6 in human liver samples2) and thenumerous drug substrates of CYP3A4 suggest that chalepensin-mediated drug interaction through mechanism-based inhibition ofCYP3A4 cannot be discounted.

The inhibition of extrahepatic CYP1A1 and CYP2A13 wascompared to their corresponding hepatic subfamily members,CYP1A2 and CYP2A6, respectively. Without pre-incubation, theIC50(¹) values for CYP1A1 and CYP1A2 inhibition were verysimilar. The influence of metabolism was not obvious in CYP1A2inhibition. However, after pre-incubation, the IC50(+) value forCYP1A1 inhibition dramatically decreased without affecting thereduction activity of CPR. The chalepensin hydroxylation andepoxidation activities of CYP1A1 were higher than those ofCYP1A2. In contrast to the selective mechanism-based inhibitionof CYP1A2 by furafylline,38) mechanism-based inhibition bychalepensin appeared to have an obvious preference for CYP1A1rather than CYP1A2. For CYP2A members, our determinationrevealed that the coumarin 7-hydroxylation activity of CYP2A13was 10% of that of CYP2A6. This result was consistent withthe CYP2A activities reported by von Weymarn et al.9) Thechalepensin hydroxylation and epoxidation activities of CYP2A13were less than 28% of those of CYP2A6 at both 3 and 200µMchalepensin, a result that was consistent with the lower coumarin7-hydroxylation activity of CYP2A13. The IC50(¹) value forCYP2A13 inhibition was much lower than that for CYP2A6inhibition, indicating potent inhibition of CYP2A13 by chalepen-sin. However, pre-incubation only moderately enhanced the inhi-bition of CYP2A13. The absence of a spectacular increase ininhibition enhancement on pre-incubation, in contrast to that ofCYP2A6, was in agreement with the relatively low epoxidationactivity of CYP2A13. For an incubation time of 30min, thechalepensin oxidation activities of P450s, such as CYP1A1,CYP2A6, and CYP3A4, might be underestimated becausemechanism-based inhibition could occur during this time. Agreater decrease of IC50 value after pre-incubation was evidentin CYP1A1, CYP2A6, and CYP3A4 with relatively higherepoxidation activities determined, whereas the IC50(+) values werenot consistently lower than those for the inhibition of other P450s.In the determination of epoxidation activity, the generation offree epoxide metabolite (hydrolyzed) was detected. However, thegenerated epoxide could bind to P450, which was not detected inthe assay. The mechanism-based inhibition could be affected byother factors, such as the efficient binding of chalepensin epoxideto P450, the susceptibility of P450 to this binding, and the kineticparameters of P450s toward their respective marker substrates.

These factors all contribute to the differential IC50(+) values ofP450s.

To facilitate comparison, the effects of dialysis were studiedusing the same concentrations of chalepensin for CYP1A1,CYP2A6, and CYP3A4 inactivation. The inactivation of CYP2A6by chalepensin could not be recovered at all, which can be attributedto its high epoxidation activity. In contrast, with pre-incubation,there was an approximately 40% reduction of the decrease ofCYP1A1 activity after dialysis. Because of the low IC50(+) valuesof chalepensin for CYP1A1 inhibition, the metabolic inactivationafter pre-incubation with 1.5 and 5µM chalepensin is likely satu-rated, and the partial recovery on dialysis might result from therecovery from reversible inhibition caused by excess chalepensin.For CYP3A4, dialysis did not reverse the inhibition by 1.5 µM,but partially reduced the inhibitory effect of 5 µM chalepensin.The inhibition of both CYP3A4 and CYP2A6 by chalepensinhad IC50(+) values <5µM and the values were 7–12 times higherthan that of CYP1A1. However, the inhibition of CYP2A6 by 5µMchalepensin could not be reduced by dialysis at all. Althoughwe could not exclude the possibility that the binding of epoxidemetabolite to CYP3A4 could be saturated in the presence of 5 µMchalepensin, the partition ratio for the covalent modification ofCYP3A4 was higher than that of CYP2A6, suggesting that thebinding of chalepensin epoxide to CYP2A6 could be more efficientthan to CYP3A4. This might explain the partial recovery thatoccurred for the higher concentration of chalepensin when therewas no recovery at the lower concentration. Compared to CYP2A6,the epoxidation activity of CYP3A4 was relatively low. Thus,the 15-min pre-incubation with 5 µM chalepensin might not havebeen long enough to achieve complete inactivation. Other factors,including the kinetic behavior of CYP3A4-catalyzed chalepensinoxidation, need further investigation.

Because of interference from cytosolic NADPH-dependentreaction(s), 7-ethoxyresorufin was not a perfect substrate forinvestigating the protection against chalepensin-mediated inacti-vation of P450 resulting from glutathione conjugation when mouseliver cytosol was used as a source of glutathione S-transferase.7-Ethoxycoumarin appeared to be a better substrate. The decreaseof ECOD activity of CYP1A1 by chalepensin could not bediminished by the addition of cytosol and glutathione in the pre-incubation mixture. In contrast, the inactivation of CYP2A618) andCYP3A4 could be prevented by glutathione conjugation, presum-ably supported by the addition of cytosol and glutathione. Theefficiency (kinact/KI) of inactivation of CYP1A1 was higher thanthose of CYP2A6 and CYP3A4, and the partition ratio of CYP1A1inactivation was lower than those of CYP2A6 and CYP3A4.Compared with CYP2A6 and CYP3A4, the epoxide intermediateformed by CYP1A1 seems to diffuse slowly from the active site ofP450 hemoprotein before the inactivation event. Results of cytosol/glutathione-pre-treatment of chalepensin suggested that cytosolicdecomposition might also contribute partially to the reduction ofinhibition in the CYP3A4 system. However, this pre-treatmentdid not reduce the inhibitory effect of chalepensin on CYP1A1.The cytosol- and cytosol/glutathione-pre-treatment of chalepensincould not reduce the inhibitory effect of chalepensin on CYP2A6,either (data not shown). The decreases of CYP2A6 and CYP3A4activities by cytosol/glutathione-pre-treated chalepensin could bereduced by the presence of active cytosol/glutathione in P450-pre-incubation. Glutathione conjugation made the main contribution tothe protection against inhibition and factor(s) causing the differ-



ential influence of pre-treatment of chalepensin with cytosol/glutathione need further investigation.

In this study, we attempted to compare the potential risk of druginteraction resulting from inhibition of different P450s. Thus, thethreshold concentration of chalepensin required to evoke a dou-bling of the AUC for the substrate concentration versus time curvewas estimated. Using the IC50(+) and IC50(¹) values for the calcu-lation, the threshold concentration of chalepensin for CYP2E1inhibition was the highest among P450s, indicating the least poten-tial to cause drug interaction with CYP2E1 substrates. Althoughthe estimated threshold concentrations for CYP2C9 and CYP2D6were as low as that for CYP3A4, pre-incubation did not resultin potent enhancement of the inhibition of CYP2C9 and CYP2D6.For these two P450s, the low threshold concentrations may bedue to their low IC50(¹) values. Using high-throughput screening12)

and inactivation kinetic13) methods, our calculations showed thatthe threshold concentrations for CYP2A6 and CYP3A4 inhibitionwere consistently low. 8-Methoxypsoralen has been reported topharmacokinetically interact with coumarin in humans.39) Usingthe inactivation kinetic method with reported parameters,26) wecalculated the threshold concentration of 8-methoxypsoralen for theinhibition of recombinant CYP2A6. The threshold concentrationof 8-methoxypsoralen was estimated to be 0.4 nM, which wasmuch lower than its therapeutic plasma concentration.40) Usingthe inactivation parameters of CYP3A4 inhibition,41) we estimatedthe threshold concentrations of bergamottin and 6A,7A-dihydroxy-bergamottin to be 2.0 nM and 0.05 nM, respectively. The plasmaconcentration of bergamottin could be greater than the thresholdconcentration, and thus bergamottin likely contributes, at least inpart, to grapefruit juice-mediated drug interactions in humans.42)

These estimates suggest a good agreement with the potential riskof drug interaction. Our estimate of the threshold concentrationof chalepensin for CYP3A4 inhibition was lower than that forCYP2A6 inhibition, suggesting a risk of evoking drug interactionthrough CYP3A4 inhibition by chalepensin. Using the inactivationparameters of CYP3A4 inhibition, the threshold concentration ofchalepensin was found to be comparable to that of bergamottin,but higher than that of 6A,7A-dihydroxybergamottin. However, theestimation of threshold concentrations was based on the assump-tion that hepatic P450 only was affected, and that the synthesisand degradation of P450 were not affected by the inhibitor. Forthe prediction of AUC changes, the unbound plasma concentrationof chalepensin in humans was not available, and the kdeg valuesobtained in an in vitro system may not be the same as those inhumans in vivo. The clinical consequences of P450 inactivationdepend on several other factors, including the exposure amount,drug bioavailability, conjugation and export metabolism, the tissuedistribution of drugs, and the tissue distribution and polymorphicexpression of P450s in individuals. Thus, clinical impacts can bevariable, and their prediction needs further human in vivo studies.

In summary, our results demonstrate that multiple P450 en-zymes exhibit chalepensin hydroxylation and epoxidation activ-ities. In addition to the inhibition of CYP2A6, chalepensin causedmechanism-based inhibition of CYP1A1 and CYP3A4 withoutaffecting CPR activity. The epoxidation activities of P450s appar-ently play a key role in evaluating the susceptibility of P450sto chalepensin-mediated mechanism-based inhibition.

Acknowledgments: We appreciated the excellent technicalassistance of Mr. Chin-Hauer Yang.

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