Metabolic Drug-Drug Interaction Potential of Macrolactin A and7-O-Succinyl Macrolactin A Assessed by Evaluating Cytochrome P450Inhibition and Induction and UDP-Glucuronosyltransferase InhibitionIn Vitro

Soo Hyeon Bae,a Min Jo Kwon,a Jung Bae Park,a Doyun Kim,a Dong-Hee Kim,b Jae-Seon Kang,c Chun-Gyu Kim,d Euichaul Oh,a

Soo Kyung Baea

College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, Bucheon, Republic of Koreaa; Research andDevelopment Center, Daewoo Pharmaceutical Co., Ltd., Busan, Republic of Koreab; Department of Pharmacy, Kyungsung University, Busan, Republic of Koreac;Department of Pharmaceutical Engineering, Inje University, Gimhae, Gyeongnam, Republic of Koread

Macrolactin A (MA) and 7-O-succinyl macrolactin A (SMA), polyene macrolides containing a 24-membered lactone ring, showantibiotic effects superior to those of teicoplanin against vancomycin-resistant enterococci and methicillin-resistant Staphylo-coccus aureus. MA and SMA are currently being evaluated as antitumor agents in preclinical studies in Korea. We evaluated thepotential of MA and SMA for the inhibition or induction of human liver cytochrome P450 (CYP) enzymes and UDP-glucurono-syltransferases (UGTs) in vitro to assess their safety as new molecular entities. We demonstrated that MA and SMA are potentcompetitive inhibitors of CYP2C9, with Ki values of 4.06 �M and 10.6 �M, respectively. MA and SMA also weakly inhibitedUGT1A1 activity, with Ki values of 40.1 �M and 65.3 �M, respectively. However, these macrolactins showed no time-dependentinactivation of the nine CYPs studied. In addition, MA and SMA did not induce CYP1A2, CYP2B6, or CYP3A4/5. On the basis ofan in vitro-in vivo extrapolation, our data strongly suggested that MA and SMA are unlikely to cause clinically significant drug-drug interactions mediated via inhibition or induction of most of the CYPs involved in drug metabolism in vivo, except for theinhibition of CYP2C9 by MA. Similarly, MA and SMA are unlikely to inhibit the activity of UGT1A1, UGT1A4, UGT1A6,UGT1A9, and UGT2B7 enzymes in vivo. Although further investigations will be required to clarify the in vivo interactions ofMA with CYP2C9-targeted drugs, our findings offer a clearer understanding and prediction of drug-drug interactions for thesafe use of MA and SMA in clinical practice.

Macrolactins are polyene macrolides containing a 24-mem-bered lactone ring. First isolated from a deep-sea marine

bacterium, macrolactins are mostly secondary metabolites of ma-rine microorganisms (1, 2). At least 18 isolated macrolactins havebeen reported, including some recent discoveries (2, 3). Five ofthese macrolactins were generated by Bacillus polyfermenticusKJS-2 (BP-2) and were identified as macrolactin A (MA), 7-O-malonyl macrolactin A, 7-O-succinyl macrolactin A (SMA), ma-crolactin E, and macrolactin F (4). MA was also isolated fromBacillus subsp. sunhua in the soil of a potato cultivation area (5)and was produced by a soil Streptomyces species (6) and by Bacillusamyloliquefaciens FZB42 (7). Because of their unreliable supplyfrom cell culture, structural uniqueness, and broad therapeuticpotential, MA and macrolactin analogs have been attractive tar-gets for asymmetric syntheses (8). Indeed, macrolactins A and Ehave been chemically synthesized (9–11).

Both MA and SMA show antibiotic effects against vancomy-cin-resistant enterococci and methicillin-resistant Staphylococcusaureus (4, 12). The MICs of MA and SMA against methicillin-resistant Staphylococcus aureus are 2 and �0.25 �g/ml, respec-tively, which are superior to that of teicoplanin (4). MA and SMAalso exhibited excellent antibacterial activities on intestinal van-comycin-resistant enterococci colonization in mice (4). MA has abroad spectrum of activity, with significant antiviral and cancercell cytotoxic properties, including inhibition of B16-F10 murinemelanoma cell replication and mammalian herpes simplex viruses(1, 8). MA has been shown to protect lymphoblast cells against

HIV by inhibiting viral replication (1). SMA also exhibits antime-tastatic effects, anti-inflammatory activity, and antiangiogenesisactivity (13). MA and SMA are currently being evaluated in pre-clinical studies as anti-macular degeneration and antitumoragents at Daewoo Pharmaceutical Company (Gimhae, Republicof Korea). Despite the excellent pharmacological properties ofMA and SMA, to date there is no information regarding the drug-drug interactions of MA and SMA mediated through cytochromeP450 (CYP) isoforms or UDP-glucuronosyltransferase (UGT)isoforms.

The drug-drug interactions owing to the inhibition of CYPsand UGTs should be considered in the development of new chem-ical entities; this is an important concern in drug discovery anddevelopment research and in the evaluation of patient safety inclinical practice (14, 15). Most drug-drug interactions are medi-ated primarily by the inhibition of CYPs and UGTs expressed inhuman liver microsomes and, to a lesser extent, the induction ofthese drug-metabolizing enzymes. Drug-drug interactions are

Received 6 January 2014 Returned for modification 15 March 2014Accepted 25 May 2014

Published ahead of print 2 June 2014

Address correspondence to Soo Kyung Bae, basesk@catholic.ac.kr.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.00018-14

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major causes of the adverse effects leading to the abandonment ofpromising new drugs. Thus, the evaluation of potential CYP inhi-bition and induction and UGT inhibition is essential for assessingthe safety of a drug (16, 17, 18). To our knowledge, no previousreport has evaluated the drug-drug interactions of MA and SMA.

In this study, we evaluated whether MA and SMA were com-petitive inhibitors or time-dependent inactivators of CYP en-zymes in vitro using human liver microsomes. We also investi-gated the ability of MA and SMA to induce the major CYPenzymes in vitro using human hepatocytes and to inhibit UGTenzymes in human liver microsomes. These findings regarding thepotential for drug-drug interactions with MA and SMA as inhib-itors or inducers of CYPs and/or UGTs provide important infor-mation for the development of MA and SMA as new drug entities.

MATERIALS AND METHODSChemicals and reagents. Macrolactin A (MA) [(3Z,5E,8R,9E,11Z,14S,16S,17E,19E,24R)-8,14,16-trihydroxy-24-methyl-1-oxacyclotetracosa-3,5,9,11,17,19-hexaen-2-one] (Fig. 1) and 7-O-succinyl macrolactin A(SMA) (4-[{(3Z,5E,8S,9E,11Z,14S,16R,17E,19E,24R)-14,16-dihydroxy-24-methyl-2-oxo-1-oxacyclotetracosa-3,5,9,11,17,19-hexaen-8-yl}oxy]-4-oxobutanoic acid) (Fig. 1) were donated by Daewoo PharmaceuticalCompany (Gimhae, Republic of Korea). �-NADP, glucose 6-phosphate,glucose-6-phosphate dehydrogenase, MgCl2, alamethicin, uridine 5=-diphosphoglucuronic acid trisodium salt (UDPGA), Trizma base, Trizmahydrochloride, acetaminophen, amodiaquine, chlorzoxazone, coumarin,dextromethorphan, dimethyl sulfoxide (DMSO), fetal bovine serum,L-glutamine, �-estradiol, trifluoperazine dihydrochloride, serotoninhydrochloride, 3=-azido-3=-deoxythymidine (zidovudine), hecogenin,1-naphthol, niflumic acid, chlorpropamide, phenytoin, theophylline,phenacetin, tolbutamide, 3-methylcholanthrene, rifampin, rosiglitazone,potassium fluoride, formic acid, and Williams’ medium E were purchasedfrom Sigma-Aldrich Corp. (St. Louis, MO, USA). Cryopreserved humanhepatocytes, pooled human liver microsomes (50 male and female donors),collagen I Cellware, a high-viability cryohepatocyte recovery kit, hepatocyteculture medium, 4-hydroxytolbutamide, and 6-hydroxybupropion werepurchased from BD Gentest (Woburn, MA, USA). Bupropion, efavirenz,6-hydroxybupropion, 7-hydroxycoumarin, 4=-hydroxymephenytoin, 1=-hy-droxymidazolam, p-hydroxyrosiglitazone, 6�-hydroxytestosterone, S-me-phenytoin, midazolam, nilotinib, and propofol were purchased from To-ronto Research Chemicals (North York, ON, Canada). All solvents wereof high-performance liquid chromatography (HPLC) grade and were ob-tained from Burdick & Jackson Co. (Morristown, NJ, USA). Other chem-icals were the highest quality available.

Screening for competitive inhibition of the activity of cytochromeP450 by MA and SMA. The inhibitory effects of MA and SMA on nine CYPisozymes were tested in pooled human liver microsomes. Phenacetin O-de-ethylase, coumarin 7-hydroxylase, bupropion hydroxylase, rosiglitazone p-hydroxylase, tolbutamide 4-hydroxylase, S-mephenytoin 4-hydroxylase,dextromethorphan O-demethylase, chlorzoxazone 6-hydroxylase, and mida-

zolam 1=-hydroxylase activities were determined as probes for CYP1A2,CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, andCYP3A activities, respectively, in human liver microsomes as described pre-viously (19), with slight modifications of the methods for the cocktail incu-bation and tandem mass spectrometry. The test compounds MA and SMAand all the substrates were dissolved in methanol and serially diluted withmethanol to the required concentrations. The methanol in these solutions(except coumarin, for solubility reasons) was then evaporated under reducedpressure using an AES2010 SpeedVac (Thermo Electron Co., Waltham, MA,USA) to minimize the toxicity of the solutions. Because it has low solubility in0.1 M phosphate buffer (pH 7.4), coumarin dissolved in methanol was addeddirectly to the reaction tube (final methanol concentration, 0.5%).

Briefly, the incubation mixtures containing the pooled human livermicrosomes (final concentrations, 0.25 mg/ml), each P450-selective sub-strate, and an NADPH-generating system (1.3 mM NADP�, 3.3 mM glu-cose 6-phosphate, 3.3 mM MgCl2, and 0.4 unit/ml glucose-6-phosphatedehydrogenase) were preincubated for 5 min at 37°C. The reaction wasinitiated by addition of an aliquot of MA or SMA (concentration range,0 –50 �M) and incubated for 15 min at 37°C in a shaking water bath.When SMA as an inhibitor was incubated, a 10-�l aliquot of 1 M potas-sium phosphate (KF) in 0.1 M phosphate buffer (pH 7.4) was addedbefore the incubation to inhibit the esterase activity because SMA is rap-idly hydrolyzed into MA by esterases (19, 20). The reaction was stopped byaddition of 50 �l of ice-cold acetonitrile, which contained 2 �M chlor-propamide as an internal standard. The incubation mixtures were centri-fuged (13,000 � g, 15 min, 4°C), and aliquots of the supernatants wereinjected into a liquid chromatography-tandem mass spectrometry(LC-MS/MS) system. According to the U.S. FDA 2006 draft guidance forindustry (17) for in vitro drug-drug interaction experimental design, amaximal concentration of the investigational drug can be established as10-fold the average plasma concentration. The maximum concentrationsof drug in plasma (Cmax) were reported to be 1.36 �g/ml (3.38 ��) forMA and 0.324 �g/ml (0.645 ��) after oral administration of MA or SMA(21), respectively, at a dose of 50 mg/kg, which appeared to be a supraef-fective dose in a mouse model (preliminary data from our laboratory notshown); thus, the maximal concentration of MA or SMA used in thisstudy was 50 �M.

All incubations were performed in triplicate, and mean values wereused for analysis. Additionally, identical parallel incubation samples con-taining well-known reversible CYP inhibitors were included as positivecontrols. Concentrations of P450-selective substrates close to their re-ported Km values were used (Table 1) (19, 22).

Ki determination for inhibition of CYP2C9 by MA and SMA. On thebasis of the 50% inhibitory concentrations (IC50s), experiments to deter-mine the Ki values of MA and SMA for CYP2C9 were conducted. Briefly,tolbutamide (CYP2C9 substrate) was incubated with MA (0 to 50 �M),SMA (0 to 50 �M), or sulfaphenazole (0 to 2 �M), a well-known inhibitorof CYP2C9. The concentrations of tolbutamide (50, 100, and 150 �M)were near the Km value. Other procedures were similar to those of thereversible inhibition studies. The reaction rates were linear with respect tothe incubation time and the microsomal protein content under theseconditions. All incubations were performed in triplicate, and mean valueswere used for analysis.

Time-dependent inactivation of the activities of nine cytochromeP450s by MA and SMA as determined using IC50 shift assays. The IC50

shift assay is one of most efficient and convenient methods for evaluatingthe time-dependent inhibitory effects of MA and SMA. Changes in theenzymatic activity are usually detected with and without preincubation ofthe test compound for a defined period. A change in the IC50 to a lowervalue (“shift”) following preincubation indicates time-dependent inacti-vation (23).

Pooled human liver microsomes (0.25 mg/ml) were incubated withMA or SMA (0 to 50 �M) in the absence or presence of an NADPH-generating system for 30 min at 37°C (i.e., the “inactivation incubation”).After inactivation incubation, aliquots (10 �l) were transferred to fresh

FIG 1 Chemical structures of macrolactin A (MA) (A) and 7-O-succinyl ma-crolactin A (SMA) (B).

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incubation tubes (final volume, 100 �l) containing an NADPH-generat-ing system and each P450-selective substrate cocktail set. When SMA wasstudied, a 10-�l aliquot of 1 M KF was added to both the inactivation andincubation mixtures. After the inactivation incubation, a 10-�l aliquot ofeach inactivation mixture was added to the cocktail substrate in 50 mMphosphate buffer (final concentration of human liver microsomes, 0.025mg/ml). After 5 min, the NADPH-regenerating system was added. Thereaction mixture was incubated for 15 min. The reaction was stopped byaddition of 50 �l of cold acetonitrile containing 100 nM chlorpropamideas an internal standard. The mixtures were centrifuged (13,000 � g, 15min, 4°C), and aliquots of the supernatants were injected into an LC-MS/MS system. All incubations were performed in triplicate, and meanvalues were used for analysis.

Inductive effects of MA and SMA on the activity of cytochromeP450s in cryopreserved human hepatocytes. Three different sources ofcryopreserved human hepatocytes (lot numbers HH304, HH311, andHH318; BD Biosciences, Woburn, MA, USA) were thawed using a cryo-preserved hepatocyte purification kit, seeded on collagen I culture dishesat a density of 0.7 � 106 cells/ml in hepatocyte culture medium, andcultivated at 37°C in a humidified incubator with 5% CO2 for 24 h. Thehepatocyte cultures were pretreated for two consecutive days with hepa-tocyte culture medium containing MA (0.1, 1, and 10 �M), SMA (0.1, 1,and 10 �M), solvent controls, or prototypical inducers. Solvent controlswere treated with vehicle (0.1% DMSO) as a negative control. As proto-typical inducers, 20 �M rifampin (positive control for CYP3A4/5), 500�M phenobarbital (positive control for CYP2B6), and 1 �M 3-methyl-cholanthrene (positive control for CYP1A2) were used. At 24 h after thefinal treatment, the hepatocyte cultures were incubated with Williams’ Ebuffer containing 100 �M phenacetin, 5 �M midazolam, and 50 �Mbupropion for 1 h. Then, 20-�l aliquots of the incubation mixtures weretransferred to 1.5-ml microcentrifuge tubes, 180 �l of acetonitrile wasadded to each tube, and the mixtures were centrifuged (13,000 � g, 15min, 4°C). Aliquots of the supernatants were injected into an LC-MS/MSsystem for CYP1A2 (phenacetin O-deethylation), CYP2B6 (bupropionhydroxylation), and CYP3A4/5 (midazolam 1=-hydroxylation) activitymeasurements. All experiments were conducted in triplicate.

Screening for reversible inhibition of the activity of UGT isoformsby MA and SMA. The inhibitory effects of MA and SMA on UGT1A1,UGT1A4, UGT1A6, UGT1A9, and UGT2B7 activities were evaluated. Thesubstrates and their concentrations used are listed in Table 1. Incubationmixtures containing 100 mM Tris buffer, pooled human liver microsomes(final concentration, 0.25 mg/ml), 25 �g/ml alamethicin, 5 mM MgCl2,UGT-selective substrates, and MA or SMA (concentration range, 0 to 500�M) were preincubated on ice for 30 min to allow alamethicin pore for-mation. The reaction was initiated by addition of 5 mM UDPGA, followedby incubation for 60 min (30 min for UGT1A9) at 37°C in a shaking water

bath. The reaction was stopped by addition of 50 �l of ice-cold acetonitrilecontaining 2 �M chlorpropamide as an internal standard. The incubationmixtures were centrifuged (13,000 � g, 15 min, 4°C), and aliquots of thesupernatants were injected into an LC-MS/MS system. All incubationswere performed in triplicate, and mean values were used for analysis.Known potent inhibitors were included as positive controls to evaluatethe suitability of these experiments and to compare their IC50s with thoseof MA and SMA. Nilotinib, hecogenin, 1-naphthol, niflumic acid, andefavirenz were used as well-known inhibitors for UGT1A1, UGT1A4,UGT1A6, UGT1A9, and UGT2B7, respectively. All substrates and inhib-itors used as positive controls were selected according to published reports(24–29). On the basis of the observed potency, experiments to determinethe Ki values of MA and SMA for UGT1A1 were conducted. Briefly, �-es-tradiol (a UGT1A1 substrate) was incubated with MA (0 to 200 �M),SMA (0 to 200 �M), or nilotinib (0 to 5 �M), a well-known inhibitor (29).Other procedures were similar to those for the reversible inhibition stud-ies. All incubations were performed in triplicate, and the mean values wereused for analysis.

LC-MS/MS analysis of the metabolites from nine probe CYP sub-strates and five UGT substrates to evaluate in vitro CYP and UGT inhi-bition. Metabolites of nine P450-selective substrates were analyzed usinga tandem quadrupole mass spectrometer (QTrap 5500 LC-MS/MS sys-tem; Applied Biosystems, Foster City, CA) equipped with an electrosprayionization interface, as reported previously (19).

For UGT inhibition assays, samples were analyzed using a tandemquadrupole mass spectrometer (QTrap 3200 LC-MS/MS system; ABSciex) and an Agilent 1260 series high-performance liquid chromatogra-

TABLE 2 IC50 values of MA and SMA for each CYP isozyme in humanliver microsomesa

Isozyme Reaction

IC50 (�M) forb:

MA SMA

CYP1A2 Phenacetin O-deethylation �50 �50CYP2A6 Coumarin 7-hydroxylation �50 �50CYP2B6 Bupropion hydroxylation �50 �50CYP2C8 Rosiglitazone p-hydroxylation 26.4 20.5CYP2C9 Tolbutamide 4-hydroxylation 9.05 15.4CYP2C19 S-Mephenytoin 4=-hydroxylation 25.9 39.5CYP2D6 Dextromethorphan O-demethylation �50 �50CYP2E1 Chlorzoxazone 6-hydroxylation �50 �50CYP3A Midazolam 1=-hydroxylation �50 �50a The assay conditions are described in Materials and Methods.b Data are expressed as the means of triplicate determinations.

TABLE 1 Substrates, their metabolites, and their LC-MS/MS conditions for human CYP and UGT assays

Isozyme Substrate Concn (�M) Metabolite Transition (m/z) Mode CEa (eV)

CYP1A2 Phenacetin 50 Acetaminophen 152 ¡ 110 � 21CYP2A6 Coumarin 5 7-Hydroxycoumarin 163 ¡ 107 � 30CYP2B6 Bupropion 50 6-Hydroxy bupropion 256 ¡ 238 � 19CYP2C8 Rosiglitazone 1 p-Hydroxyrosiglitazone 374 ¡ 151 � 33CYP2C9 Tolbutamide 100 4-Hydroxytolbutamide 287 ¡ 89 � 42CYP2C19 S-Mephenytoin 100 4=-Hydroxymephenytoin 230 ¡ 150 � 22CYP2D6 Dextromethorphan 5 Dextrorphan 258 ¡ 157 � 60CYP2E1 Chlorzoxazone 50 6-Hydroxychlorzoxazone 184 ¡ 120 25CYP3A Midazolam 5 1=-Hydroxymidazolam 342 ¡ 203 � 25UGT1A1 �-Estradiol 10 �-Estradiol 3-glucuronide 447 ¡ 271 52UGT1A4 Trifluoperazine 40 Trifluoperazine N-glucuronide 584 ¡ 408 � 22UGT1A6 Serotonin 4,000 Serotonin O-glucuronide 353 ¡ 160 � 33UGT1A9 Propofol 100 Propofol O-glucuronide 353.1 ¡ 177.1 34UGT2B7 Zidovudine 100 Zidovudine 5=-glucuronide 442 ¡ 125 30a CE, collision energy.

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phy (HPLC) system with a reversed-phase column (Poroshell 120 C18, 50mm � 4.6 mm inside diameter [i.d.], 2.7-�m particle size; Agilent) main-tained at 40°C.

The single-reaction monitoring mode with specific precursor/production transitions was used for quantification. Table 1 lists the mass transi-tions for the metabolites of nine CYP- and five UGT-selective substratesand their collision energies. Peak areas for all analytes were integratedautomatically using Analyst software (version 1.5.2; Applied Biosystems).

Data analysis. For reversible inhibition and mechanism-based inhibi-tion screening, both P450-mediated activities and UGT activities, deter-mined using known probe substrates, in the presence of MA or SMA areexpressed as percentages of the corresponding control values (0 �M MAor 0 �M SMA). A graph of the percentage of control activity versus theconcentration of the test inhibitor was used to fit the data. The concen-tration required to inhibit enzyme activity by 50% (IC50) was calculatedusing nonlinear curve fitting with SigmaPlot (version 8.0; Systat Software,Inc.).

The apparent kinetic parameters for the inhibitory potential (Ki val-ues) were estimated from the fitted curves using WinNonlin software(version 4.0; Pharsight, Mountain View, CA). The inhibition data werefitted to different models of enzyme inhibition (competitive, noncompet-itive, uncompetitive, or mixed) by nonlinear least-squares regressionanalysis (WinNonlin software). The most appropriate inhibition modelwas selected based on the goodness-of-fit criterion following a visual in-spection of the data, the coefficient of determination (R2), and the cor-rected Akaike information criterion. For visual inspection, data are pre-sented as Dixon plots.

The fold induction of the enzyme activity in samples that containedthe CYP inducers was determined by comparing the metabolite formed ineach sample with that of the vehicle control sample (cells incubated with 0.1%DMSO only). This comparison was made by dividing the peak area ratio(analyte divided by internal standard) on the LC-MS/MS chromatogram ofthe respective metabolites formed in each sample after a 60-min incubationby the peak area ratio of the vehicle control at the same time point.

FIG 2 IC50 curves of MA for human P450 activities using the cocktail substrate, including CYP1A2 for phenacetin O-deethylase (A), CYP2A6 for coumarin7-hydroxylase (B), CYP2B6 for bupropion hydroxylase (C), CYP2C8 for rosiglitazone p-hydroxylase (D), CYP2C9 for tolbutamide 4-hydroxylase (E), CYP2C19for S-mephenytoin 4-hydroxylase (F), CYP2D6 for dextromethorphan O-demethylase (G), CYP2E1 for chlorzoxazone 6-hydroxylase (H), and CYP3A4/5 formidazolam 1=-hydroxylase (I). Data are the means standard deviations from triplicate determinations. The dashed lines represent the best fit to the data usingnonlinear regression.

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RESULTSScreening for reversible inhibition on the activities of cyto-chrome P450s by MA and SMA. The inhibitory effects of MA andSMA on the activities of nine CYP isozymes (CYP1A2, CYP2A6,CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, andCYP3A4/5) are shown in Table 2 and Fig. 2 and 3. The IC50s for thepositive controls used in the reversible inhibition studies were ingood agreement with published values to an acceptable degree ofaccuracy (19, 30, 31).

Of the nine P450 isoforms tested, CYP2C9-catalyzed tolbut-amide hydroxylation was most strongly inhibited by MA andSMA, with IC50s of 9.05 and 15.4 �M, respectively (Table 2). MAand SMA also showed weak inhibition of CYP2C8 and CYP2C19,with IC50s of 26.4 �M for MA and 20.5 �M for SMA and 25.9 �M

for MA and 39.5 �M for SMA, respectively. No inhibition wasapparent for the other CYPs tested (Table 2); the remaining activ-ities at the highest concentration tested (50 �M) were �80%.

Ki determination for inhibition of CYP2C9 by MA and SMA.On the basis of the IC50s, to characterize the type of reversibleinhibition of CYP2C9 by MA and SMA, enzyme kinetic assayswere conducted by varying the concentrations of MA or SMA andthe CYP2C9 probe substrate tolbutamide. Additionally, identicalparallel incubation samples containing a known potent inhibitorof CYP2C9, sulfaphenazole, were included as positive controls.Representative Dixon plots for the inhibition of CYP2C9 by MA andSMA and the positive-control sulfaphenazole in human liver micro-somes are shown in Fig. 4. Both MA and SMA inhibited CYP2C9 withKi values of 4.06 �M and 10.6 �M, respectively. The Ki value for the

FIG 3 IC50 curves of SMA for human P450 activities using the cocktail substrate, including CYP1A2 for phenacetin O-deethylase (A), CYP2A6 for coumarin7-hydroxylase (B), CYP2B6 for bupropion hydroxylase (C), CYP2C8 for rosiglitazone p-hydroxylase (D), CYP2C9 for tolbutamide 4-hydroxylase (E), CYP2C19for S-mephenytoin 4-hydroxylase (F), CYP2D6 for dextromethorphan O-demethylase (G), CYP2E1 for chlorzoxazone 6-hydroxylase (H), and CYP3A4/5 formidazolam 1=-hydroxylase (I). Data are the means standard deviations from triplicate determinations. The dashed lines represent the best fit to the data usingnonlinear regression.

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positive control, sulfaphenazole, against CYP2C9 was 0.269 �M,which was within the accepted range of Ki values based on the U.S.FDA 2006 draft guidance (17). A visual inspection of the Dixon plotsand further analysis of the enzyme inhibition modes suggested thatthe inhibition data for MA, SMA, and sulfaphenazole all fit well withcompetitive inhibition (Fig. 4). The potency of MA for inhibition ofCYP2C9 was 2.61-fold higher than that of SMA.

Time-dependent inhibitory effects of MA and SMA on theactivity of nine cytochrome P450s evaluated using an IC50 shiftassay. A shift in the inhibition curve toward a lower IC50 followinga 30-min preincubation in the presence of NADPH is an indicatorof time-dependent inhibition. However, after a 30-min preincu-bation of MA or SMA with human liver microsomes in the pres-ence of NADPH, no shifts in the IC50s were apparent for inhibi-

tion of the nine CYPs (data not shown). These results suggestedthat MA and SMA are not time-dependent inhibitors.

Inductive effect of MA and SMA on the activities of cyto-chrome P450s in cryopreserved human hepatocytes. To evaluatethe abilities of MA and SMA to induce the expression of CYPenzymes, cryopreserved human hepatocytes were used. In accor-dance with the U.S. FDA 2012 recommendations for drug inter-action studies (18), three different donors were used, and the po-tential for the induction of CYP1A2, CYP2B6, and CYP3A wasevaluated. Generally, the cultured hepatocytes were characteristi-cally cuboidal and contained intact cell membranes and granularcytoplasm with one or two centrally located nuclei during thetreatment period. The (fold) increases above DMSO (negativecontrol) are listed in Table 3. As expected, all preparations of hu-

FIG 4 Dixon plots to determine Ki values for CYP2C9 of MA (A), SMA (B) and sulfaphenazole (C). The concentrations of tolbutamide were 50, 100, and 150�M, respectively. v represents the formation rate of tolbutamide hydroxylation (nmol/min/mg protein). Data are the mean values from triplicate determinations.The solid lines for MA, SMA, and sulfaphenazole fit well to all competitive inhibition types.

TABLE 3 Induction potential of MA and SMA on activities of CYP2A1, CYP2C9, and CYP3A4 in three different lots of cryopreserved humanhepatocytes

Enzyme and lot no.

Fold induction enzyme activitya (treated/vehicleb control) for:

MA at enzyme concn(�M) of:

SMA at enzyme concn(�M) of:

3-Methylcholanthreneat enzyme concn of1 �M

Phenobarbital atenzyme concnof 500 �M

Rifampin at enzymeconcn of 20 �M0.1 1 10 0.1 1 10

CYP1A2HH304 1.01 1.02 0.951 1.12 1.06 1.24 7.29HH311 0.988 0.811 0.847 0.868 0.881 0.862 5.51HH318 1.38 1.33 1.23 1.01 1.00 0.950 13.3

CYP2B6HH304 1.46 1.55 1.96 1.30 1.26 1.41 9.46HH311 0.978 1.01 1.10 0.963 0.865 1.00 6.12HH318 0.977 0.987 1.06 0.901 0.852 1.08 6.49

CYP3A4/5HH304 0.957 0.837 0.858 0.937 0.932 0.753 5.18HH311 0.841 0.900 0.842 0.945 0.868 0.944 7.95HH318 0.987 0.819 0.805 0.971 0.847 0.702 5.44

a The assay conditions are described in Materials and Methods. The fold increased enzyme activity in each CYP isoform is the mean from three independent experiments.b Vehicle, 0.1% (vol/vol) DMSO, negative control.

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man hepatocytes produced marked elevations after treatmentwith prototypical CYP inducers (positive controls). Treatmentwith 1 �M 3-methylcholanthrene induced CYP1A2 activity (8.71-fold 4.10-fold), and treatment with 500 �M phenobarbital pro-duced an increase in CYP2B6 activity (7.36-fold 1.83-fold) inthe three donors compared with that in the vehicle control (0.1%DMSO). Induction of CYP3A4/5 activity (6.20-fold 1.53-fold)was observed following incubation with 20 �M rifampin. Induc-tion of CYP2B6 was also observed following incubation with 20�M rifampin, and comparable mean fold increases in CYP3A4/5above that produced by phenobarbital were seen (data notshown). Treatment with MA or SMA at 0.1, 1, and 10 �M hadlittle or no effect on CYP1A2, CYP2B6, and CYP3A4/5 activities(Table 3). Although MA or SMA increased induction by 0.811- to1.38-fold for CYP1A2, 0.852- to 1.96-fold for CYP2B6, and 0.702-to 0.987-fold for CYP3A4/5 compared with that for the vehiclecontrol (0.1% DMSO), the increases in enzyme activity were lessthan 20% of the positive-control activity, which is the lower limit

indicating significantly induced activity. Thus, these results dem-onstrated that MA and SMA did not significantly induce CYPisozyme activity.

Screening for competitive inhibition of MA and SMA on theactivities of five UGT isoforms. The inhibitory effects of MA andSMA on the activities of five UGT isozymes (UGT1A1, UGT1A4,UGT1A6, UGT1A9, and UGT2B7) are shown in Table 4 and Fig. 5and 6.

MA and SMA showed only weak inhibitory effects on the fiveUGTs. MA weakly inhibited UGT1A1 and UGT1A9 activities withIC50s of 36.0 �M and 89.8 �M, respectively (Table 4). SMA alsoslightly inhibited UGT1A1, with an IC50 of 53.9 �M. No inhibi-tion was apparent for the other UGTs tested (Table 4). The IC50sof the positive controls were as follows: 0.977 �M nilotinib forUGT1A1, 77.1 �M hecogenin for UGT1A4, 138 �M 1-naphtholfor UGT1A6, 0.288 �M niflumic acid for UGT1A9, and 41.8 �Mefavirenz for UGT2B7, respectively (data not shown).

Further analysis of MA and SMA inhibition kinetics revealed thatthe drugs had Ki values of 40.6 �M and 58.7 �M, respectively, inhuman liver microsomes (Fig. 7). On the basis of the Dixon plots, MAand SMA exhibited competitive inhibition against UGT1A1-cata-lyzed �-estradiol 3-glucuronidation. Nilotinib strongly inhibitedUGT1A1, which exhibited a Ki of 0.658 �M in a competitive manner,and to a greater degree than MA and SMA.

DISCUSSION

Unexpected drug-drug interactions are a major cause of adverseeffects leading to the regulatory withdrawal of promising newdrugs. Many of these interactions involve inhibition and, to alesser extent, induction of drug-metabolizing enzymes such asCYP and UGT isozymes. Consequently, assessment of the poten-

TABLE 4 IC50 values of MA and SMA for each UGT isozyme in humanliver microsomesa

Isozyme Reaction

IC50 (�M) forb:

MA SMA

UGT1A1 �-Estradiol 3-glucuronidation 36.0 53.9UGT1A4 Trifluoperazine N-glucuronidation �500 �500UGT1A6 Serotonin O-glucuronidation �500 �500UGT1A9 Propofol O-glucuronidation 89.8 �500UGT2B7 Zidovudine 5=-glucuronidation �500 �500a The assay conditions are described in Materials and Methods.b Data are expressed as the means of triplicate determinations.

FIG 5 IC50 curves of MA for human UGT activities using UGT1A1 for �-estradiol 3-glucuronidase (A), UGT1A4 for trifluoperazine N-glucuronidase (B),UGT1A6 for serotonin O-glucuronidase (C), UGT1A9 for propofol O-glucuronidase (D), and UGT2B7 for zidovudine 5=-glucuronidation (E). Data are themeans standard deviations from triplicate determinations. The dashed lines represent the best fit to the data using nonlinear regression.

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tial for drug-metabolizing enzyme inhibition or induction is anessential step in the safety evaluation of new drugs and herbalsupplements (16–18). In the present study, the inhibitory andinductive effects of MA and SMA on nine CYP isozymes and fiveUGT isozymes were evaluated in vitro to assess the potential of MAand SMA to cause drug-drug interactions with other concomi-tantly administered drugs.

In high-throughput screening evaluating the activity of thenine CYPs, MA and SMA inhibited the activities of CYP2C8,

CYP2C9, and CYP2C19, with IC50s for MA of 26.4, 9.05, and25.9 �M, respectively, and for SMA of 15.4, 20.5, and 39.5 �M,respectively. Other CYP isoforms were negligibly inhibited.MA and SMA inhibited CYP2C9 in a competitive manner, withKi values of 4.06 �M and 10.6 �M, respectively. Preincubationof MA or SMA with human liver microsomes and an NADPH-generating system did not alter the inhibition potencies againstthe nine CYPs, suggesting that neither MA nor SMA is a time-dependent inactivator. Thus, it appears that the catalytic activ-

FIG 6 IC50 curves of SMA for human UGT activities using UGT1A1 for �-estradiol 3-glucuronidase (A), UGT1A4 for trifluoperazine N-glucuronidase(B), UGT1A6 for serotonin O-glucuronidase (C), UGT1A9 for propofol O-glucuronidase (D), and UGT2B7 for zidovudine 5=-glucuronidation (E). Dataare the means standard deviations from triplicate determinations. The dashed lines represent the best fit to the data using nonlinear regression.

FIG 7 Dixon plots to determine Ki values for UGT1A1 of MA (A), SMA (B), and nilotinib (C). The concentrations of �-estradiol were 5, 10, and 20 �M,respectively. v represents the formation rate of �-estradiol-3-glucuronide (pmol/min/mg protein). Data are the mean values of triplicate determinations. Thesolid lines for MA, SMA, and nilotinib fit well to all competitive inhibition types.

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ities of CYPs are little changed after long-term administrationof MA and SMA.

The in vitro inhibition potency alone does not dictate the like-lihood of pharmacokinetic drug interactions because the in vivoconcentration of the inhibitor must also be considered. In hu-mans, the prediction of in vivo drug-drug interactions mediatedvia reversible P450 inhibition typically relies on the use of theCmax/Ki ratio. A ratio of �1 generally suggests that in vivo CYPinhibition is likely, whereas ratios between 0.1 and 1 indicate alower likelihood for interactions, and ratios of �0.1 indicate thatthe likelihood of an in vivo interaction is remote (16, 17). Themaximum concentrations in plasma (Cmax) were reported to be1.36 �g/ml (3.38 ��) for MA and 0.324 �g/ml (0.645 ��) afteroral administration of MA or SMA (21), respectively, at a dose of50 mg/kg, which appeared to be a supraeffective dose in a mousemodel (preliminary data from our laboratory not shown). TheCmax/Ki ratios for CYP2C9 inhibition by MA and SMA were esti-mated to be 0.833 and 0.0608, respectively. Thus, SMA is notanticipated to exhibit in vivo inhibition of CYP2C9. However,given the higher ratio for MA (0.833), in vivo interactions ofCYP2C9-targeted drugs mediated through reversible inhibitionare considered possible. Thus, we cannot exclude the possibility ofan inhibition in the potency of CYP2C9 by MA in vivo. For com-petitive or uncompetitive inhibition, the IC50/2, not the IC50,might substitute for the Ki. Given the IC50s of MA for inhibition ofCYP2C8 and CYP2C19, the Cmax/Ki ratios for CYP2C8 andCYP2C19 inhibition by MA were calculated to be 0.26 and 0.30,respectively. It should be noted that Cmax represents the maxi-mum concentration of the inhibitor attained in plasma in vivousing the highest recommended therapeutic dose to predict pos-sible in vivo drug-drug interactions. Considering that the minimaleffective dose in mice was 5 mg/kg (preliminary data from ourlaboratory not shown), the actual values of Cmax/Ki in humansmay be less than those presented.

In addition to inhibition studies of CYP isozymes, inductionstudies are also essential to estimate drug-drug interactions. Theinduction of drug-metabolizing CYP enzymes might reduce theconcentrations of concomitant drugs such that plasma drug con-centrations may be too low for pharmacological effects. Reduc-tions in the plasma concentrations of antibacterial and antibioticdrugs may create serious problems, such as bacterial drug resis-tance. The U.S. FDA 2012 guidance (18) recommends thatCYP1A2, CYP2B6, and CYP3A be initially evaluated in vitro. TheU.S. FDA 2006 draft guidance for industry (17) states that a drugthat produces a change of �40% of the positive control in vitro canbe considered an enzyme inducer, and in vivo evaluation is war-ranted. The European Medicines Agency 2010 draft guideline (16)indicates that for an investigational drug to be ruled out as anenzyme inducer, any increase in enzyme activity must be �20% ofthe positive-control activity. In the present study, MA and SMAtreatments showed no significant induction of CYP1A2, CYP2B6,or CYP3A4/5 activity in cultured human hepatocytes under con-ditions in which the positive controls exerted their anticipatedinductive effects. Even at the highest concentration tested (10�M), MA and SMA were only 12.6% and 13.3% as effective asomeprazole in inducing CYP1A2 activity, 18.3% and 16.0% aseffective as phenobarbital in inducing CYP2B6 activity, and 14.0%and 13.1% as effective as rifampin in inducing CYP3A4/5 activity,respectively, indicating that MA and SMA are unlikely to produceclinically significant CYP enzyme induction. However, there is a

limitation to our conclusions. We evaluated the induction in en-zyme activities for the CYPs, whereas the FDA recently stated thatalterations in the mRNA level for the target gene should be used asan endpoint for evaluating the potential of investigational drugs asenzymes (18). It was reported (32) that the CYP3A4 mRNA ex-pression level is a more reliable marker than the CYP3A4/5 activityfor detecting the induction of drug-metabolizing enzymes.

Recently, the role of UGTs in the inactivation and eliminationof many drugs has drawn attention. Thus, an evaluation of thepotential of a new compound to inhibit UGT isozymes has be-come crucial in new drug development (28, 33, 34). Of the 18 UGTproteins significantly expressed in liver, the current evidence sug-gests that UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, andUGT2B15 are of the greatest importance in the hepatic metabo-lism of drugs, although UGT1A3, UGT2B4, and UGT2B10 alsocontribute to drug glucuronidation (34, 35). The five UGT isoen-zymes (UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7)used in the present study were chosen because of the lack of anauthentic S-oxazepam and its glucuronide standard, a typical sub-strate of UGT2B15 (36). MA and SMA showed only weak inhibi-tory effects on the five UGTs. Of the tested UGTs, UGT1A1 activ-ity was weakly inhibited by MA and SMA, with IC50 (Ki) values of36.0 (40.6) �M and 53.9 (58.7) �M, respectively. UGT1A9 wasalso slightly inhibited by MA. No inhibition was apparent for theother UGTs tested. With regard to the inhibitory potentials ofUGT1A1 by MA and SMA, their Cmax/Ki ratios were calculated tobe �0.1. The results of an in vitro-in vivo extrapolation indicatedthat MA and SMA will not produce clinically significant inhibitionof UGT1A1.

To our knowledge, there are no reports of in vitro drug-druginteractions with MA and SMA mediated via CYP or UGTisozymes. In this study, we demonstrated that MA and SMA arepotent competitive inhibitors of CYP2C9 in vitro, with Ki values of4.06 �M and 10.6 �M. MA and SMA also weakly inhibitedUGT1A1 activity in vitro, with Ki values of 40.6 �M and 58.7 �M,respectively. In addition, the results of the present study showedthat MA and SMA should not produce clinically significant induc-tion of CYP isozymes. On the basis of the in vitro-in vivo extrap-olation, the data presented in this work strongly suggested that,except for the inhibition of CYP2C9 by MA, MA and SMA areunlikely to cause clinically significant metabolic drug-drug inter-actions mediated via inhibition or induction of most of the P450enzymes involved in drug metabolism in vivo. Similarly, MA andSMA will be unlikely to inhibit the in vivo enzymatic activity ofUGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7. In addi-tion, CYP2C9 is a polymorphically expressed enzyme, and theCYP2C9*3 variant, in particular, exhibits substantially reducedsubstrate turnover, which may further confound the predictionsof the drug-drug interaction potential (37). Further investigationswill be required to clarify the in vivo interactions of the CYP2C9-targeted drugs with MA and the genotype-dependent inhibition.These findings should enable an understanding and prediction ofdrug-drug interactions for the safe and effective use of MA andSMA in clinical practice.

ACKNOWLEDGMENTS

This research was supported by the Bio and Medical Technology Develop-ment Program of the National Research Foundation funded by the Ministryof Science, ICT and Future Planning (grant 2013M3A9B5075838) and theResearch Fund of The Catholic University of Korea (2013).

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We declare no conflicts of interest.

REFERENCES1. Gustafson A, Roman W, Fenical J. 1989. The macrolactins, a novel class

of antiviral and cytotoxic macrolides from a deep-sea marine bacterium. J.Am. Chem. Soc. 111:7519 –7524. http://dx.doi.org/10.1021/ja00201a036.

2. Lu X, Xu Q, Liu X, Cao X, Ni K, Jiao B. 2008. Marine drugs—macrolactins. Chem. Biodivers. 5:1669 –1674. http://dx.doi.org/10.1002/cbdv.200890155.

3. Nagao T, Adachi K, Sakai M, Nishijima M, Sano H. 2001. Novelmacrolactins as antibiotic lactones from a marine bacterium. J. Antibiot.54:333–339. http://dx.doi.org/10.7164/antibiotics.54.333.

4. Kim DH, Kim HK, Kim KM, Kim CK, Jeong MH, Ko CY, Moon KH,Kang JS. 2011. Antibacterial activities of macrolactin A and 7-O-succinylmacrolactin A from Bacillus polyfermenticus KJS-2 against vancomycin-resistant Enterococci and methicillin-resistant Staphylococcus aureus. Arch.Pharm. Res. 34:147–152. http://dx.doi.org/10.1007/s12272-011-0117-0.

5. Han JS, Cheng JH, Yoon TM, Song J, Rajkarnikar A, Kim WG, Yoo ID,Yang YY, Suh JW. 2005. Biological control agent of common crab diseaseby antagonistic strain Bacillus sp. sunhua. J. Appl. Microbiol. 99:213–221.http://dx.doi.org/10.1111/j.1365-2672.2005.02614.x.

6. Choi S, Bai D, Yu J, Shin CS. 2003. Characteristics of the squalenesynthase inhibitors produced by a Streptomyces species isolated from soils.Can. J. Microbiol. 49:663– 668. http://dx.doi.org/10.1139/w03-084.

7. Schneider K, Chen X, Vater J, Franke P, Nicholson G, Borriss R,Süssmuth RD. 2007. Macrolactin is the polyketide biosynthesis productof the pks2 cluster of Bacillus amyloliquefaciens FZB42. J. Nat. Prod. 70:1417–1423. http://dx.doi.org/10.1021/np070070k.

8. Kobayashi Y, Fukuda A, Kimachi T, Ju-ichi M, Takemoto Y. 2004.Asymmetric synthesis of macrolactin analogue. Tetrahedron Lett. 45:677–580. http://dx.doi.org/10.1016/j.tetlet.2003.11.055.

9. Smith AB, III, Ott GR. 1998. Total syntheses of ()-macrolactin A,(�)-macrolactin E, and ()-macrolactinic acid: an exercise in Stille cross-coupling chemistry. J. Am. Chem. Soc. 120:3935–3948. http://dx.doi.org/10.1021/ja980203b.

10. Marino JP, McClure MS, Holub DP, Comasseto JV, Tucci FC. 2002.Stereocontrolled synthesis of ()-macrolactin A. J. Am. Chem. Soc. 124:1664 –1668. http://dx.doi.org/10.1021/ja017177t.

11. Bonini C, Chiummiento L, Pullez M, Solladié G, Colobert F. 2004.Convergent highly stereoselective preparation of the C12–C24 fragmentof macrolactin A. J. Org. Chem. 69:5015–5022. http://dx.doi.org/10.1021/jo049556l.

12. Romero-Tabarez M, Jansen Sylla R, Lünsdorf M, Häußler H, Santosa S,Timmis DA, Molinari KN, G. 2006. 7-O-Malonyl macrolactin A, a newmacrolactin antibiotic form Bacillus subtilis active against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci, and asmall-colony variant of Burkholderia cepacia. Antimicrob. Agents Che-mother. 50:1701–1709. http://dx.doi.org/10.1128/AAC.50.5.1701-1709.2006.

13. Kim JM, Jung JW, Kim DH, Jang JS, Kim CG, Kang HE. 2013. Asimple and sensitive HPLC-UV determination of 7-O-succinyl macro-lactin A in rat plasma and urine and its application to a pharmacoki-netic study. Biomed. Chromatogr. 27:273–279. http://dx.doi.org/10.1002/bmc.2786.

14. Bjornsson TD, Callaghan JT, Einolf HJ, Fischer V, Gas L, Grimm S, KaoJ, King SP, Miwa G, Ni L, Kumar G, McLeod J, Obach RC, Roberts S,Roe A, Shah A, Snikeris F, Sullivan JT, Tweedie D, Vega JM, Walsh J,Wrighton SA. 2003. The conduct of in vitro and in vivo drug-drug inter-action studies: a pharmaceutical research and manufactures of America(PhRMA) perspective. Drug Metab. Dispos. 31:815– 832. http://dx.doi.org/10.1124/dmd.31.7.815.

15. Zhang L, Reynolds KS, Whao P, Huang SM. 2010. Drug interactionsevaluation: an integrated part of risk assessment of therapeutics. Toxicol.Appl. Pharmacol. 243:134–145. http://dx.doi.org/10.1016/j.taap.2009.12.016.

16. European Medicines Agency. 2010. Guideline on the investigation ofdrug interactions. European Medicines Agency, London, UnitedKingdom. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2010/05/WC500090112.pdf.

17. Food and Drug Administration, Center for Drug Evaluation andResearch. 2006. Guidance for industry. Drug interaction studies—study design, data analysis, implication for dosing, and labeling rec-

ommendations. Draft guidance. Food and Drug Administration,Rockville, MD.

18. Food and Drug Administration, Center for Drug Evaluation andResearch. 2012. Guidance for industry. Drug interaction studies—study design, data analysis, implication for dosing, and labeling rec-ommendations. Draft guidance. Food and Drug Administration,Rockville, MD.

19. Cho DY, Bae SH, Lee JK, Kim YW, Kim BT, Bae SK. 2014. Selectiveinhibition of cytochrome P450 2D6 by sarpogrelate and its active metab-olite, M-1, in human liver microsomes. Drug Metab. Dispos. 42:33–39.http://dx.doi.org/10.1124/dmd.113.054296

20. Clarke TA, Waskell LA. 2003. The metabolism of clopidogrel is catalyzedby human cytochrome P450 3A and is inhibited by atorvastatin. DrugMetab. Dispos. 31:53–59. http://dx.doi.org/10.1124/dmd.31.1.53.

21. Jung JW, Kim JM, Kwon MH, Kim DH, Kang HE. 2014. Pharmacoki-netics of macrolactin A and 7-O-succinyl macrolactin A in mice. Xenobi-otica 44:547–554. http://dx.doi.org/10.3109/00498254.2013.861542.

22. Yuan R, Madani S, Wei XX, Reynolds K, Huang SM. 2002. Evaluationof cytochrome P450 probe substrates commonly used by the pharmaceu-tical industry to study in vitro drug interactions. Drug Metab. Dispos.30:1311–1319. http://dx.doi.org/10.1124/dmd.30.12.1311.

23. Obach RS, Walsky RL, Venkatakrishnan K. 2007. Mechanism-basedinactivation of human cytochrome P450 enzymes and the prediction ofdrug-drug interactions. Drug Metab. Dispos. 35:246 –255. http://dx.doi.org/10.1124/dmd.106.012633.

24. Krishnaswamy S, Duan SX, von Moltke LL, Greenblatt DJ, SudmeierJ L , B a c h o v c h i n W W , C o u r t M H . 2 0 0 3 . S e r o t o n i n ( 5 -hydroxytryptamine) glucuronidation in vitro: assay development, humanliver microsome activities and species differences. Xenobiotica 33:169 –180. http://dx.doi.org/10.1080/0049825021000048809.

25. Uchaipichat V, Mackenzie PI, Elliot DJ, Miners JO. 2006. Selectivityof substrate (trifluoperazine) and inhibitor (amitriptyline, andros-terone, canrenoic acid, hecogenin, phenylbutazone, quinidine, qui-nine, and sulfinpyrazone) “probes” for human UDP-glucuronosyl-transferases. Drug Metab. Dispos. 34:449 – 456. http://dx.doi.org/10.1124/dmd.105.007369.

26. Fujiwara R, Nakajima M, Yamanaka H, Katoh M, Yokoi T. 2008.Product inhibition of UDP-glucuronosyltransferase (UGT) enzymes byUDP obfuscates the inhibitory effects of UGT substrates. Drug Metab.Dispos. 36:361–367. http://dx.doi.org/10.1124/dmd.107.018705.

27. Bélanger A, Caron P, Harvey M, Zimmerman PA, Mehlotra RK, Guil-lemette C. 2009. Glucuronidation of the antiretroviral drug efavirenz byUGT2B7 and an in vitro investigation of drug-drug interaction with zid-ovudine. Drug Metab. Dispos. 37:1793–1796. http://dx.doi.org/10.1124/dmd.109.027706.

28. Miners JO, Bowalgaha K, Elliot DJ, Baranczewski P, Knights KM. 2011.Characterization of niflumic acid as a selective inhibitor of human livermicrosomal UDP-glucuronosyltransferase 1A9: application to the reac-tion phenotyping of acetaminophen glucuronidation. Drug Metab. Dis-pos. 39:644 – 652. http://dx.doi.org/10.1124/dmd.110.037036.

29. Ai L, Zhu L, Yang L, Ge G, Cao Y, Liu Y, Fang Z, Zhang Y. 2014.Selectivity for inhibition of nilotinib on the catalytic activity of humanUDP-glucuronosyltransferases. Xenobiotica 44:320 –325. http://dx.doi.org/10.3109/00498254.2013.840750.

30. Zheng YF, Bae SH, Kwon MJ, Park JB, Choi HD, Shin WG, Bae SK.2013. Inhibitory effects of astaxanthin, �-cryptoxanthin, canthaxan-thin, lutein, and zeaxanthin on cytochrome P450 enzyme activities.Food Chem. Toxicol. 59:78 – 85. http://dx.doi.org/10.1016/j.fct.2013.04.053.

31. Han YL, Yu HL, Li D, Meng XL, Zhou ZY, Yu Q, Zhang XY, Wang FJ,Guo C. 2011. Inhibitory effects of limonin on six human cytochromeP450 enzymes and P-glycoprotein in vitro. Toxicol. In Vitro 25:1828 –1833. http://dx.doi.org/10.1016/j.tiv.2011.09.023.

32. Fahmi OA, Kish M, Boldt S, Obach RS. 2010. Cytochrome P450 3A4mRNA is a more reliable marker than CYP3A4 activity for detectingpregnane X receptor-activated induction of drug-metabolizing en-zymes. Drug Metab. Dispos. 38:1605–1611. http://dx.doi.org/10.1124/dmd.110.033126.

33. Miners JO, Mackenzie PI. 1991. Drug glucuronidation in humans. Pharma-col. Ther. 51:347–369. http://dx.doi.org/10.1016/0163-7258(91)90065-T.

34. Kiang TK, Ensom MH, Chang TK. 2005. UDP-glucuronosyltransferasesand clinical drug-drug interactions. Pharmacol. Ther. 106:97–132. http://dx.doi.org/10.1016/j.pharmthera.2004.10.013.

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35. Miners JO, Mackenzie PI, Knights KM. 2010. The prediction of drug-glucuronidation parameters in humans: UDP-glucuronosyltransferaseenzyme-selective substrate and inhibitor probes for reaction phenotypingand in vitro-in vivo extrapolation of drug clearance and drug-drug inter-action potential. Drug Metab. Rev. 42:196 –208. http://dx.doi.org/10.3109/03602530903210716.

36. Court MH, Duan SX, Guillemette C, Journault K, Krishnaswamy S, vonMoltke LL, Greenblatt DJ. 2002. Stereoselective conjugation of oxazepam

by human UDP-glucuronosyltransferases (UGTs): S-oxazepam is glucu-ronidated by UGT2B15, while R-oxazepam is glucuronidated by UGT2B7and UGT1A9. Drug Metab. Dispos. 30:1257–1265. http://dx.doi.org/10.1124/dmd.30.11.1257.

37. Kumar V, Wahlstrom JL, Rock DA, Warren CJ, Gorman LA, Tracy TS.2006. CYP2C9 inhibition: impact of prove selection and pharmacogenet-ics on in vitro inhibition profiles. Drug Metab. Dispos. 34:1966 –1975.http://dx.doi.org/10.1124/dmd.106.010926.

Bae et al.

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