Metabolic Drug-Drug Interaction Potential of Macrolactin A and 7-O-Succinyl Macrolactin A Assessed by Evaluating Cytochrome P450 Inhibition and Induction and UDP-Glucuronosyltransferase Inhibition In Vitro

Abstract

Macrolactin A (MA) and 7-O-succinyl macrolactin A (SMA), polyene macrolides containing a 24-membered lactone ring, show antibiotic effects superior to those of teicoplanin against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. MA and SMA are currently being evaluated as antitumor agents in preclinical studies in Korea. We evaluated the potential of MA and SMA for the inhibition or induction of human liver cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGTs) in vitro to assess their safety as new molecular entities. We demonstrated that MA and SMA are potent competitive inhibitors of CYP2C9, with K_i values of 4.06 μM and 10.6 μM, respectively. MA and SMA also weakly inhibited UGT1A1 activity, with K_i values of 40.1 μM and 65.3 μM, respectively. However, these macrolactins showed no time-dependent inactivation of the nine CYPs studied. In addition, MA and SMA did not induce CYP1A2, CYP2B6, or CYP3A4/5. On the basis of an 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 the inhibition 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 of MA with CYP2C9-targeted drugs, our findings offer a clearer understanding and prediction of drug-drug interactions for the safe use of MA and SMA in clinical practice.

INTRODUCTION

Macrolactins are polyene macrolides containing a 24-membered lactone ring. First isolated from a deep-sea marine bacterium, macrolactins are mostly secondary metabolites of marine microorganisms (1, 2). At least 18 isolated macrolactins have been reported, including some recent discoveries (2, 3). Five of these macrolactins were generated by Bacillus polyfermenticus KJS-2 (BP-2) and were identified as macrolactin A (MA), 7-O-malonyl macrolactin A, 7-O-succinyl macrolactin A (SMA), macrolactin E, and macrolactin F (4). MA was also isolated from Bacillus subsp. sunhua in the soil of a potato cultivation area (5) and was produced by a soil Streptomyces species (6) and by Bacillus amyloliquefaciens FZB42 (7). Because of their unreliable supply from cell culture, structural uniqueness, and broad therapeutic potential, MA and macrolactin analogs have been attractive targets for asymmetric syntheses (8). Indeed, macrolactins A and E have been chemically synthesized (9–11).

Both MA and SMA show antibiotic effects against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus (4, 12). The MICs of MA and SMA against methicillin-resistant Staphylococcus aureus are 2 and <0.25 μg/ml, respectively, which are superior to that of teicoplanin (4). MA and SMA also exhibited excellent antibacterial activities on intestinal vancomycin-resistant enterococci colonization in mice (4). MA has a broad spectrum of activity, with significant antiviral and cancer cell cytotoxic properties, including inhibition of B16-F10 murine melanoma 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 antimetastatic effects, anti-inflammatory activity, and antiangiogenesis activity (13). MA and SMA are currently being evaluated in preclinical studies as anti-macular degeneration and antitumor agents at Daewoo Pharmaceutical Company (Gimhae, Republic of Korea). Despite the excellent pharmacological properties of MA and SMA, to date there is no information regarding the drug-drug interactions of MA and SMA mediated through cytochrome P450 (CYP) isoforms or UDP-glucuronosyltransferase (UGT) isoforms.

The drug-drug interactions owing to the inhibition of CYPs and UGTs should be considered in the development of new chemical entities; this is an important concern in drug discovery and development research and in the evaluation of patient safety in clinical practice (14, 15). Most drug-drug interactions are mediated primarily by the inhibition of CYPs and UGTs expressed in human liver microsomes and, to a lesser extent, the induction of these drug-metabolizing enzymes. Drug-drug interactions are major causes of the adverse effects leading to the abandonment of promising new drugs. Thus, the evaluation of potential CYP inhibition and induction and UGT inhibition is essential for assessing the safety of a drug (16, 17, 18). To our knowledge, no previous report has evaluated the drug-drug interactions of MA and SMA.

In this study, we evaluated whether MA and SMA were competitive inhibitors or time-dependent inactivators of CYP enzymes in vitro using human liver microsomes. We also investigated the ability of MA and SMA to induce the major CYP enzymes in vitro using human hepatocytes and to inhibit UGT enzymes in human liver microsomes. These findings regarding the potential for drug-drug interactions with MA and SMA as inhibitors or inducers of CYPs and/or UGTs provide important information for the development of MA and SMA as new drug entities.

MATERIALS AND METHODS

Chemicals 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 Pharmaceutical Company (Gimhae, Republic of Korea). β-NADP, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, MgCl₂, alamethicin, uridine 5′-diphosphoglucuronic acid trisodium salt (UDPGA), Trizma base, Trizma hydrochloride, acetaminophen, amodiaquine, chlorzoxazone, coumarin, dextromethorphan, dimethyl sulfoxide (DMSO), fetal bovine serum, l-glutamine, β-estradiol, trifluoperazine dihydrochloride, serotonin hydrochloride, 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 purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Cryopreserved human hepatocytes, pooled human liver microsomes (50 male and female donors), collagen I Cellware, a high-viability cryohepatocyte recovery kit, hepatocyte culture medium, 4-hydroxytolbutamide, and 6-hydroxybupropion were purchased from BD Gentest (Woburn, MA, USA). Bupropion, efavirenz, 6-hydroxybupropion, 7-hydroxycoumarin, 4′-hydroxymephenytoin, 1′-hydroxymidazolam, p-hydroxyrosiglitazone, 6β-hydroxytestosterone, S-mephenytoin, midazolam, nilotinib, and propofol were purchased from Toronto Research Chemicals (North York, ON, Canada). All solvents were of high-performance liquid chromatography (HPLC) grade and were obtained from Burdick & Jackson Co. (Morristown, NJ, USA). Other chemicals were the highest quality available.

FIG 1.

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

Screening for competitive inhibition of the activity of cytochrome P450 by MA and SMA.

The inhibitory effects of MA and SMA on nine CYP isozymes were tested in pooled human liver microsomes. Phenacetin O-deethylase, coumarin 7-hydroxylase, bupropion hydroxylase, rosiglitazone p-hydroxylase, tolbutamide 4-hydroxylase, S-mephenytoin 4-hydroxylase, dextromethorphan O-demethylase, chlorzoxazone 6-hydroxylase, and midazolam 1′-hydroxylase activities were determined as probes for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A activities, respectively, in human liver microsomes as described previously (19), with slight modifications of the methods for the cocktail incubation and tandem mass spectrometry. The test compounds MA and SMA and all the substrates were dissolved in methanol and serially diluted with methanol to the required concentrations. The methanol in these solutions (except coumarin, for solubility reasons) was then evaporated under reduced pressure using an AES2010 SpeedVac (Thermo Electron Co., Waltham, MA, USA) to minimize the toxicity of the solutions. Because it has low solubility in 0.1 M phosphate buffer (pH 7.4), coumarin dissolved in methanol was added directly to the reaction tube (final methanol concentration, 0.5%).

Briefly, the incubation mixtures containing the pooled human liver microsomes (final concentrations, 0.25 mg/ml), each P450-selective substrate, and an NADPH-generating system (1.3 mM NADP⁺, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl₂, and 0.4 unit/ml glucose-6-phosphate dehydrogenase) were preincubated for 5 min at 37°C. The reaction was initiated by addition of an aliquot of MA or SMA (concentration range, 0–50 μM) and incubated for 15 min at 37°C in a shaking water bath. When SMA as an inhibitor was incubated, a 10-μl aliquot of 1 M potassium phosphate (KF) in 0.1 M phosphate buffer (pH 7.4) was added before the incubation to inhibit the esterase activity because SMA is rapidly hydrolyzed into MA by esterases (19, 20). The reaction was stopped by addition of 50 μl of ice-cold acetonitrile, which contained 2 μM chlorpropamide as an internal standard. The incubation mixtures were centrifuged (13,000 × g, 15 min, 4°C), and aliquots of the supernatants were injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. According to the U.S. FDA 2006 draft guidance for industry (17) for in vitro drug-drug interaction experimental design, a maximal concentration of the investigational drug can be established as 10-fold the average plasma concentration. The maximum concentrations of drug in plasma (C_max) were reported to be 1.36 μg/ml (3.38 μM) for MA and 0.324 μg/ml (0.645 μM) after oral administration of MA or SMA (21), respectively, at a dose of 50 mg/kg, which appeared to be a supraeffective dose in a mouse model (preliminary data from our laboratory not shown); thus, the maximal concentration of MA or SMA used in this study was 50 μM.

All incubations were performed in triplicate, and mean values were used for analysis. Additionally, identical parallel incubation samples containing well-known reversible CYP inhibitors were included as positive controls. Concentrations of P450-selective substrates close to their reported K_m values were used (Table 1) (19, 22).

TABLE 1.

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

Isozyme	Substrate	Concn (μM)	Metabolite	Transition (m/z)	Mode	CEa (eV)
CYP1A2	Phenacetin	50	Acetaminophen	152 → 110	+	21
CYP2A6	Coumarin	5	7-Hydroxycoumarin	163 → 107	+	30
CYP2B6	Bupropion	50	6-Hydroxy bupropion	256 → 238	+	19
CYP2C8	Rosiglitazone	1	p-Hydroxyrosiglitazone	374 → 151	+	33
CYP2C9	Tolbutamide	100	4-Hydroxytolbutamide	287 → 89	+	42
CYP2C19	S-Mephenytoin	100	4′-Hydroxymephenytoin	230 → 150	+	22
CYP2D6	Dextromethorphan	5	Dextrorphan	258 → 157	+	60
CYP2E1	Chlorzoxazone	50	6-Hydroxychlorzoxazone	184 → 120	−	25
CYP3A	Midazolam	5	1′-Hydroxymidazolam	342 → 203	+	25
UGT1A1	β-Estradiol	10	β-Estradiol 3-glucuronide	447 → 271	−	52
UGT1A4	Trifluoperazine	40	Trifluoperazine N-glucuronide	584 → 408	+	22
UGT1A6	Serotonin	4,000	Serotonin O-glucuronide	353 → 160	+	33
UGT1A9	Propofol	100	Propofol O-glucuronide	353.1 → 177.1	−	34
UGT2B7	Zidovudine	100	Zidovudine 5′-glucuronide	442 → 125	−	30

^aCE, collision energy.

K_i determination for inhibition of CYP2C9 by MA and SMA.

On the basis of the 50% inhibitory concentrations (IC₅₀s), experiments to determine the K_i values of MA and SMA for CYP2C9 were conducted. Briefly, tolbutamide (CYP2C9 substrate) was incubated with MA (0 to 50 μM), SMA (0 to 50 μM), or sulfaphenazole (0 to 2 μM), a well-known inhibitor of CYP2C9. The concentrations of tolbutamide (50, 100, and 150 μM) were near the K_m value. Other procedures were similar to those of the reversible inhibition studies. The reaction rates were linear with respect to the incubation time and the microsomal protein content under these conditions. All incubations were performed in triplicate, and mean values were used for analysis.

Time-dependent inactivation of the activities of nine cytochrome P450s by MA and SMA as determined using IC₅₀ shift assays.

The IC₅₀ shift assay is one of most efficient and convenient methods for evaluating the time-dependent inhibitory effects of MA and SMA. Changes in the enzymatic activity are usually detected with and without preincubation of the test compound for a defined period. A change in the IC₅₀ to a lower value (“shift”) following preincubation indicates time-dependent inactivation (23).

Pooled human liver microsomes (0.25 mg/ml) were incubated with MA or SMA (0 to 50 μM) 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 μl) were transferred to fresh incubation tubes (final volume, 100 μl) containing an NADPH-generating system and each P450-selective substrate cocktail set. When SMA was studied, a 10-μl aliquot of 1 M KF was added to both the inactivation and incubation mixtures. After the inactivation incubation, a 10-μl aliquot of each inactivation mixture was added to the cocktail substrate in 50 mM phosphate buffer (final concentration of human liver microsomes, 0.025 mg/ml). After 5 min, the NADPH-regenerating system was added. The reaction mixture was incubated for 15 min. The reaction was stopped by addition of 50 μl of cold acetonitrile containing 100 nM chlorpropamide as an internal standard. The mixtures were centrifuged (13,000 × g, 15 min, 4°C), and aliquots of the supernatants were injected into an LC-MS/MS system. All incubations were performed in triplicate, and mean values were used for analysis.

Inductive effects of MA and SMA on the activity of cytochrome P450s in cryopreserved human hepatocytes.

Three different sources of cryopreserved human hepatocytes (lot numbers HH304, HH311, and HH318; BD Biosciences, Woburn, MA, USA) were thawed using a cryopreserved hepatocyte purification kit, seeded on collagen I culture dishes at a density of 0.7 × 10⁶ cells/ml in hepatocyte culture medium, and cultivated at 37°C in a humidified incubator with 5% CO₂ for 24 h. The hepatocyte cultures were pretreated for two consecutive days with hepatocyte culture medium containing MA (0.1, 1, and 10 μM), SMA (0.1, 1, and 10 μM), solvent controls, or prototypical inducers. Solvent controls were treated with vehicle (0.1% DMSO) as a negative control. As prototypical inducers, 20 μM rifampin (positive control for CYP3A4/5), 500 μM phenobarbital (positive control for CYP2B6), and 1 μM 3-methylcholanthrene (positive control for CYP1A2) were used. At 24 h after the final treatment, the hepatocyte cultures were incubated with Williams' E buffer containing 100 μM phenacetin, 5 μM midazolam, and 50 μM bupropion for 1 h. Then, 20-μl aliquots of the incubation mixtures were transferred to 1.5-ml microcentrifuge tubes, 180 μl of acetonitrile was added to each tube, and the mixtures were centrifuged (13,000 × g, 15 min, 4°C). Aliquots of the supernatants were injected into an LC-MS/MS system for CYP1A2 (phenacetin O-deethylation), CYP2B6 (bupropion hydroxylation), and CYP3A4/5 (midazolam 1′-hydroxylation) activity measurements. All experiments were conducted in triplicate.

Screening for reversible inhibition of the activity of UGT isoforms by MA and SMA.

The inhibitory effects of MA and SMA on UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 activities were evaluated. The substrates and their concentrations used are listed in Table 1. Incubation mixtures containing 100 mM Tris buffer, pooled human liver microsomes (final concentration, 0.25 mg/ml), 25 μg/ml alamethicin, 5 mM MgCl₂, UGT-selective substrates, and MA or SMA (concentration range, 0 to 500 μM) were preincubated on ice for 30 min to allow alamethicin pore formation. The reaction was initiated by addition of 5 mM UDPGA, followed by incubation for 60 min (30 min for UGT1A9) at 37°C in a shaking water bath. The reaction was stopped by addition of 50 μl of ice-cold acetonitrile containing 2 μM chlorpropamide as an internal standard. The incubation mixtures were centrifuged (13,000 × g, 15 min, 4°C), and aliquots of the supernatants were injected into an LC-MS/MS system. All incubations were performed in triplicate, and mean values were used for analysis. Known potent inhibitors were included as positive controls to evaluate the suitability of these experiments and to compare their IC₅₀s with those of MA and SMA. Nilotinib, hecogenin, 1-naphthol, niflumic acid, and efavirenz were used as well-known inhibitors for UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7, respectively. All substrates and inhibitors used as positive controls were selected according to published reports (24–29). On the basis of the observed potency, experiments to determine the K_i values of MA and SMA for UGT1A1 were conducted. Briefly, β-estradiol (a UGT1A1 substrate) was incubated with MA (0 to 200 μM), SMA (0 to 200 μM), or nilotinib (0 to 5 μM), a well-known inhibitor (29). Other procedures were similar to those for the reversible inhibition studies. All incubations were performed in triplicate, and the mean values were used for analysis.

LC-MS/MS analysis of the metabolites from nine probe CYP substrates and five UGT substrates to evaluate in vitro CYP and UGT inhibition.

Metabolites of nine P450-selective substrates were analyzed using a tandem quadrupole mass spectrometer (QTrap 5500 LC-MS/MS system; Applied Biosystems, Foster City, CA) equipped with an electrospray ionization interface, as reported previously (19).

For UGT inhibition assays, samples were analyzed using a tandem quadrupole mass spectrometer (QTrap 3200 LC-MS/MS system; AB Sciex) and an Agilent 1260 series high-performance liquid chromatography (HPLC) system with a reversed-phase column (Poroshell 120 C18, 50 mm × 4.6 mm inside diameter [i.d.], 2.7-μm particle size; Agilent) maintained at 40°C.

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

Data analysis.

For reversible inhibition and mechanism-based inhibition screening, both P450-mediated activities and UGT activities, determined using known probe substrates, in the presence of MA or SMA are expressed as percentages of the corresponding control values (0 μM MA or 0 μM SMA). A graph of the percentage of control activity versus the concentration of the test inhibitor was used to fit the data. The concentration required to inhibit enzyme activity by 50% (IC₅₀) was calculated using nonlinear curve fitting with SigmaPlot (version 8.0; Systat Software, Inc.).

The apparent kinetic parameters for the inhibitory potential (K_i values) were estimated from the fitted curves using WinNonlin software (version 4.0; Pharsight, Mountain View, CA). The inhibition data were fitted to different models of enzyme inhibition (competitive, noncompetitive, uncompetitive, or mixed) by nonlinear least-squares regression analysis (WinNonlin software). The most appropriate inhibition model was selected based on the goodness-of-fit criterion following a visual inspection of the data, the coefficient of determination (R²), and the corrected Akaike information criterion. For visual inspection, data are presented as Dixon plots.

The fold induction of the enzyme activity in samples that contained the CYP inducers was determined by comparing the metabolite formed in each 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 of the respective metabolites formed in each sample after a 60-min incubation by the peak area ratio of the vehicle control at the same time point.

RESULTS

Screening for reversible inhibition on the activities of cytochrome P450s by MA and SMA.

The inhibitory effects of MA and SMA on the activities of nine CYP isozymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5) are shown in Table 2 and Fig. 2 and 3. The IC₅₀s for the positive controls used in the reversible inhibition studies were in good agreement with published values to an acceptable degree of accuracy (19, 30, 31).

TABLE 2.

IC₅₀ values of MA and SMA for each CYP isozyme in human liver microsomes^a

Isozyme	Reaction	IC50 (μM) forb:
		MA	SMA
CYP1A2	Phenacetin O-deethylation	>50	>50
CYP2A6	Coumarin 7-hydroxylation	>50	>50
CYP2B6	Bupropion hydroxylation	>50	>50
CYP2C8	Rosiglitazone p-hydroxylation	26.4	20.5
CYP2C9	Tolbutamide 4-hydroxylation	9.05	15.4
CYP2C19	S-Mephenytoin 4′-hydroxylation	25.9	39.5
CYP2D6	Dextromethorphan O-demethylation	>50	>50
CYP2E1	Chlorzoxazone 6-hydroxylation	>50	>50
CYP3A	Midazolam 1′-hydroxylation	>50	>50

^aThe assay conditions are described in Materials and Methods.

^bData are expressed as the means of triplicate determinations.

FIG 2.

IC₅₀ curves of MA for human P450 activities using the cocktail substrate, including CYP1A2 for phenacetin O-deethylase (A), CYP2A6 for coumarin 7-hydroxylase (B), CYP2B6 for bupropion hydroxylase (C), CYP2C8 for rosiglitazone p-hydroxylase (D), CYP2C9 for tolbutamide 4-hydroxylase (E), CYP2C19 for S-mephenytoin 4-hydroxylase (F), CYP2D6 for dextromethorphan O-demethylase (G), CYP2E1 for chlorzoxazone 6-hydroxylase (H), and CYP3A4/5 for midazolam 1′-hydroxylase (I). Data are the means ± standard deviations from triplicate determinations. The dashed lines represent the best fit to the data using nonlinear regression.

FIG 3.

IC₅₀ curves of SMA for human P450 activities using the cocktail substrate, including CYP1A2 for phenacetin O-deethylase (A), CYP2A6 for coumarin 7-hydroxylase (B), CYP2B6 for bupropion hydroxylase (C), CYP2C8 for rosiglitazone p-hydroxylase (D), CYP2C9 for tolbutamide 4-hydroxylase (E), CYP2C19 for S-mephenytoin 4-hydroxylase (F), CYP2D6 for dextromethorphan O-demethylase (G), CYP2E1 for chlorzoxazone 6-hydroxylase (H), and CYP3A4/5 for midazolam 1′-hydroxylase (I). Data are the means ± standard deviations from triplicate determinations. The dashed lines represent the best fit to the data using nonlinear regression.

Of the nine P450 isoforms tested, CYP2C9-catalyzed tolbutamide hydroxylation was most strongly inhibited by MA and SMA, with IC₅₀s of 9.05 and 15.4 μM, respectively (Table 2). MA and SMA also showed weak inhibition of CYP2C8 and CYP2C19, with IC₅₀s of 26.4 μM for MA and 20.5 μM for SMA and 25.9 μM for MA and 39.5 μM for SMA, respectively. No inhibition was apparent for the other CYPs tested (Table 2); the remaining activities at the highest concentration tested (50 μM) were >80%.

K_i determination for inhibition of CYP2C9 by MA and SMA.

On the basis of the IC₅₀s, to characterize the type of reversible inhibition of CYP2C9 by MA and SMA, enzyme kinetic assays were conducted by varying the concentrations of MA or SMA and the CYP2C9 probe substrate tolbutamide. Additionally, identical parallel incubation samples containing a known potent inhibitor of CYP2C9, sulfaphenazole, were included as positive controls. Representative Dixon plots for the inhibition of CYP2C9 by MA and SMA and the positive-control sulfaphenazole in human liver microsomes are shown in Fig. 4. Both MA and SMA inhibited CYP2C9 with K_i values of 4.06 μM and 10.6 μM, respectively. The K_i value for the positive control, sulfaphenazole, against CYP2C9 was 0.269 μM, which was within the accepted range of K_i values based on the U.S. FDA 2006 draft guidance (17). A visual inspection of the Dixon plots and further analysis of the enzyme inhibition modes suggested that the inhibition data for MA, SMA, and sulfaphenazole all fit well with competitive inhibition (Fig. 4). The potency of MA for inhibition of CYP2C9 was 2.61-fold higher than that of SMA.

FIG 4.

Dixon plots to determine K_i values for CYP2C9 of MA (A), SMA (B) and sulfaphenazole (C). The concentrations of tolbutamide were 50, 100, and 150 μM, 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.

Time-dependent inhibitory effects of MA and SMA on the activity of nine cytochrome P450s evaluated using an IC₅₀ shift assay.

A shift in the inhibition curve toward a lower IC₅₀ following a 30-min preincubation in the presence of NADPH is an indicator of time-dependent inhibition. However, after a 30-min preincubation of MA or SMA with human liver microsomes in the presence of NADPH, no shifts in the IC₅₀s were apparent for inhibition of the nine CYPs (data not shown). These results suggested that MA and SMA are not time-dependent inhibitors.

Inductive effect of MA and SMA on the activities of cytochrome P450s in cryopreserved human hepatocytes.

To evaluate the abilities of MA and SMA to induce the expression of CYP enzymes, cryopreserved human hepatocytes were used. In accordance with the U.S. FDA 2012 recommendations for drug interaction studies (18), three different donors were used, and the potential for the induction of CYP1A2, CYP2B6, and CYP3A was evaluated. Generally, the cultured hepatocytes were characteristically cuboidal and contained intact cell membranes and granular cytoplasm with one or two centrally located nuclei during the treatment period. The (fold) increases above DMSO (negative control) are listed in Table 3. As expected, all preparations of human hepatocytes produced marked elevations after treatment with prototypical CYP inducers (positive controls). Treatment with 1 μM 3-methylcholanthrene induced CYP1A2 activity (8.71-fold ± 4.10-fold), and treatment with 500 μM phenobarbital produced an increase in CYP2B6 activity (7.36-fold ± 1.83-fold) in the 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 μM rifampin. Induction of CYP2B6 was also observed following incubation with 20 μM rifampin, and comparable mean fold increases in CYP3A4/5 above that produced by phenobarbital were seen (data not shown). Treatment with MA or SMA at 0.1, 1, and 10 μM had little or no effect on CYP1A2, CYP2B6, and CYP3A4/5 activities (Table 3). Although MA or SMA increased induction by 0.811- to 1.38-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 vehicle control (0.1% DMSO), the increases in enzyme activity were less than 20% of the positive-control activity, which is the lower limit indicating significantly induced activity. Thus, these results demonstrated that MA and SMA did not significantly induce CYP isozyme activity.

TABLE 3.

Induction potential of MA and SMA on activities of CYP2A1, CYP2C9, and CYP3A4 in three different lots of cryopreserved human hepatocytes

Enzyme and lot no.	Fold induction enzyme activitya (treated/vehicleb control) for:
	MA at enzyme concn (μM) of:			SMA at enzyme concn (μM) of:			3-Methylcholanthrene at enzyme concn of 1 μM	Phenobarbital at enzyme concn of 500 μM	Rifampin at enzyme concn of 20 μM
	0.1	1	10	0.1	1	10
CYP1A2
HH304	1.01	1.02	0.951	1.12	1.06	1.24	7.29
HH311	0.988	0.811	0.847	0.868	0.881	0.862	5.51
HH318	1.38	1.33	1.23	1.01	1.00	0.950	13.3
CYP2B6
HH304	1.46	1.55	1.96	1.30	1.26	1.41		9.46
HH311	0.978	1.01	1.10	0.963	0.865	1.00		6.12
HH318	0.977	0.987	1.06	0.901	0.852	1.08		6.49
CYP3A4/5
HH304	0.957	0.837	0.858	0.937	0.932	0.753			5.18
HH311	0.841	0.900	0.842	0.945	0.868	0.944			7.95
HH318	0.987	0.819	0.805	0.971	0.847	0.702			5.44

^aThe assay conditions are described in Materials and Methods. The fold increased enzyme activity in each CYP isoform is the mean from three independent experiments.

^bVehicle, 0.1% (vol/vol) DMSO, negative control.

Screening for competitive inhibition of MA and SMA on the activities of five UGT isoforms.

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

TABLE 4.

IC₅₀ values of MA and SMA for each UGT isozyme in human liver microsomes^a

Isozyme	Reaction	IC50 (μM) forb:
		MA	SMA
UGT1A1	β-Estradiol 3-glucuronidation	36.0	53.9
UGT1A4	Trifluoperazine N-glucuronidation	>500	>500
UGT1A6	Serotonin O-glucuronidation	>500	>500
UGT1A9	Propofol O-glucuronidation	89.8	>500
UGT2B7	Zidovudine 5′-glucuronidation	>500	>500

^aThe assay conditions are described in Materials and Methods.

^bData are expressed as the means of triplicate determinations.

FIG 5.

IC₅₀ curves of MA for human UGT activities using UGT1A1 for β-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 the means ± standard deviations from triplicate determinations. The dashed lines represent the best fit to the data using nonlinear regression.

FIG 6.

IC₅₀ curves of SMA for human UGT activities using UGT1A1 for β-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 the means ± standard deviations from triplicate determinations. The dashed lines represent the best fit to the data using nonlinear regression.

MA and SMA showed only weak inhibitory effects on the five UGTs. MA weakly inhibited UGT1A1 and UGT1A9 activities with IC₅₀s of 36.0 μM and 89.8 μM, respectively (Table 4). SMA also slightly inhibited UGT1A1, with an IC₅₀ of 53.9 μM. No inhibition was apparent for the other UGTs tested (Table 4). The IC₅₀s of the positive controls were as follows: 0.977 μM nilotinib for UGT1A1, 77.1 μM hecogenin for UGT1A4, 138 μM 1-naphthol for UGT1A6, 0.288 μM niflumic acid for UGT1A9, and 41.8 μM efavirenz for UGT2B7, respectively (data not shown).

Further analysis of MA and SMA inhibition kinetics revealed that the drugs had K_i values of 40.6 μM and 58.7 μM, respectively, in human liver microsomes (Fig. 7). On the basis of the Dixon plots, MA and SMA exhibited competitive inhibition against UGT1A1-catalyzed β-estradiol 3-glucuronidation. Nilotinib strongly inhibited UGT1A1, which exhibited a K_i of 0.658 μM in a competitive manner, and to a greater degree than MA and SMA.

FIG 7.

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

DISCUSSION

Unexpected drug-drug interactions are a major cause of adverse effects leading to the regulatory withdrawal of promising new drugs. Many of these interactions involve inhibition and, to a lesser extent, induction of drug-metabolizing enzymes such as CYP and UGT isozymes. Consequently, assessment of the potential for drug-metabolizing enzyme inhibition or induction is an essential step in the safety evaluation of new drugs and herbal supplements (16–18). In the present study, the inhibitory and inductive effects of MA and SMA on nine CYP isozymes and five UGT isozymes were evaluated in vitro to assess the potential of MA and SMA to cause drug-drug interactions with other concomitantly administered drugs.

In high-throughput screening evaluating the activity of the nine CYPs, MA and SMA inhibited the activities of CYP2C8, CYP2C9, and CYP2C19, with IC₅₀s for MA of 26.4, 9.05, and 25.9 μM, respectively, and for SMA of 15.4, 20.5, and 39.5 μM, respectively. Other CYP isoforms were negligibly inhibited. MA and SMA inhibited CYP2C9 in a competitive manner, with K_i values of 4.06 μM and 10.6 μM, respectively. Preincubation of MA or SMA with human liver microsomes and an NADPH-generating system did not alter the inhibition potencies against the nine CYPs, suggesting that neither MA nor SMA is a time-dependent inactivator. Thus, it appears that the catalytic activities of CYPs are little changed after long-term administration of MA and SMA.

The in vitro inhibition potency alone does not dictate the likelihood of pharmacokinetic drug interactions because the in vivo concentration of the inhibitor must also be considered. In humans, the prediction of in vivo drug-drug interactions mediated via reversible P450 inhibition typically relies on the use of the C_max/K_i ratio. A ratio of >1 generally suggests that in vivo CYP inhibition is likely, whereas ratios between 0.1 and 1 indicate a lower likelihood for interactions, and ratios of <0.1 indicate that the likelihood of an in vivo interaction is remote (16, 17). The maximum concentrations in plasma (C_max) were reported to be 1.36 μg/ml (3.38 μM) for MA and 0.324 μg/ml (0.645 μM) after oral administration of MA or SMA (21), respectively, at a dose of 50 mg/kg, which appeared to be a supraeffective dose in a mouse model (preliminary data from our laboratory not shown). The C_max/K_i ratios for CYP2C9 inhibition by MA and SMA were estimated to be 0.833 and 0.0608, respectively. Thus, SMA is not anticipated to exhibit in vivo inhibition of CYP2C9. However, given the higher ratio for MA (0.833), in vivo interactions of CYP2C9-targeted drugs mediated through reversible inhibition are considered possible. Thus, we cannot exclude the possibility of an inhibition in the potency of CYP2C9 by MA in vivo. For competitive or uncompetitive inhibition, the IC₅₀/2, not the IC₅₀, might substitute for the K_i. Given the IC₅₀s of MA for inhibition of CYP2C8 and CYP2C19, the C_max/K_i ratios for CYP2C8 and CYP2C19 inhibition by MA were calculated to be 0.26 and 0.30, respectively. It should be noted that C_max represents the maximum concentration of the inhibitor attained in plasma in vivo using the highest recommended therapeutic dose to predict possible in vivo drug-drug interactions. Considering that the minimal effective dose in mice was 5 mg/kg (preliminary data from our laboratory not shown), the actual values of C_max/K_i in humans may be less than those presented.

In addition to inhibition studies of CYP isozymes, induction studies are also essential to estimate drug-drug interactions. The induction of drug-metabolizing CYP enzymes might reduce the concentrations of concomitant drugs such that plasma drug concentrations may be too low for pharmacological effects. Reductions in the plasma concentrations of antibacterial and antibiotic drugs may create serious problems, such as bacterial drug resistance. The U.S. FDA 2012 guidance (18) recommends that CYP1A2, CYP2B6, and CYP3A be initially evaluated in vitro. The U.S. FDA 2006 draft guidance for industry (17) states that a drug that produces a change of ≥40% of the positive control in vitro can be considered an enzyme inducer, and in vivo evaluation is warranted. The European Medicines Agency 2010 draft guideline (16) indicates that for an investigational drug to be ruled out as an enzyme inducer, any increase in enzyme activity must be <20% of the positive-control activity. In the present study, MA and SMA treatments showed no significant induction of CYP1A2, CYP2B6, or CYP3A4/5 activity in cultured human hepatocytes under conditions in which the positive controls exerted their anticipated inductive effects. Even at the highest concentration tested (10 μM), MA and SMA were only 12.6% and 13.3% as effective as omeprazole in inducing CYP1A2 activity, 18.3% and 16.0% as effective 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 produce clinically significant CYP enzyme induction. However, there is a limitation to our conclusions. We evaluated the induction in enzyme activities for the CYPs, whereas the FDA recently stated that alterations in the mRNA level for the target gene should be used as an endpoint for evaluating the potential of investigational drugs as enzymes (18). It was reported (32) that the CYP3A4 mRNA expression level is a more reliable marker than the CYP3A4/5 activity for detecting the induction of drug-metabolizing enzymes.

Recently, the role of UGTs in the inactivation and elimination of many drugs has drawn attention. Thus, an evaluation of the potential of a new compound to inhibit UGT isozymes has become crucial in new drug development (28, 33, 34). Of the 18 UGT proteins significantly expressed in liver, the current evidence suggests that UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 are of the greatest importance in the hepatic metabolism of drugs, although UGT1A3, UGT2B4, and UGT2B10 also contribute to drug glucuronidation (34, 35). The five UGT isoenzymes (UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) used in the present study were chosen because of the lack of an authentic S-oxazepam and its glucuronide standard, a typical substrate of UGT2B15 (36). MA and SMA showed only weak inhibitory effects on the five UGTs. Of the tested UGTs, UGT1A1 activity was weakly inhibited by MA and SMA, with IC₅₀ (K_i) values of 36.0 (40.6) μM and 53.9 (58.7) μM, respectively. UGT1A9 was also slightly inhibited by MA. No inhibition was apparent for the other UGTs tested. With regard to the inhibitory potentials of UGT1A1 by MA and SMA, their C_max/K_i ratios were calculated to be <0.1. The results of an in vitro-in vivo extrapolation indicated that MA and SMA will not produce clinically significant inhibition of UGT1A1.

To our knowledge, there are no reports of in vitro drug-drug interactions with MA and SMA mediated via CYP or UGT isozymes. In this study, we demonstrated that MA and SMA are potent competitive inhibitors of CYP2C9 in vitro, with K_i values of 4.06 μM and 10.6 μM. MA and SMA also weakly inhibited UGT1A1 activity in vitro, with K_i values of 40.6 μM and 58.7 μM, respectively. In addition, the results of the present study showed that MA and SMA should not produce clinically significant induction of CYP isozymes. On the basis of the in vitro-in vivo extrapolation, the data presented in this work strongly suggested that, except for the inhibition of CYP2C9 by MA, MA and SMA are unlikely to cause clinically significant metabolic drug-drug interactions mediated via inhibition or induction of most of the P450 enzymes involved in drug metabolism in vivo. Similarly, MA and SMA will be unlikely to inhibit the in vivo enzymatic activity of UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7. In addition, CYP2C9 is a polymorphically expressed enzyme, and the CYP2C9*3 variant, in particular, exhibits substantially reduced substrate turnover, which may further confound the predictions of the drug-drug interaction potential (37). Further investigations will 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 of drug-drug interactions for the safe and effective use of MA and SMA in clinical practice.