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Potential inhibitory effect of 4-hydroxytoremifene on CYP2J2 activities in human liver microsomes.

INTRODUCTION

Cytochrome P450 2J2 (CYP2J2) is the exclusive member of the human 2J subfamily [15]. Unlike other P450 isozymes which were mainly found on the human liver tissue, CYP2J2 is predominantly expressed in extrahepatic tissues including the heart, lung, kidney, brain, skeletal muscle, and small intestine [26,3]. In enzymology, CYP2J2 was initially defined as a monooxygenase enzyme that catalyzes epoxide formation of some endogenous substrates including arachidonic acid [22] and linoleic acid [7]. Several studies have implicated CYP2J2 in the pathological development of human cancers for solid tumors. For example, CYP2J2, which is up-regulated in human tumors and hematologic malignant diseases [1], promotes human cancer metastasis [4] and the neoplastic phenotype of carcinoma cells [4]. Some terfenadine-related CYP2J2 inhibitors exhibited strong activity against human cancers in vitro and in vivo [2]. These findings suggest that the effective inhibitor of CYP2J2 enzyme may represent a new approach for the treatment of human cancers.

To date, several compounds have been reported CYP2J2 inhibitor. Danazol [11], decursin [14], hydroxyebastine [24], thelephoric acid [21], telmisartan [17], TSAHC [11] inhibited CYP2J2-mediated astemizole O-demethylation activity with [IC.sub.50] values of 0.02, 6.95, 1.50, 3.23, 0.42, and 2.60 [micro]M, respectively. Marketed drugs [10,11,12,17] and natural products [23,14] were screened for the inhibitory potential on CYP2J2 activities to find strong CYP2J2 inhibitor. Several studies found an inhibitory effect of [alpha]-naphthoflavone, ketoconazole, troglitazone, tranylcypromine, ebastine, and terfenadine on the rate of astemizole O-demethylation in human small intestinal microsomes and recombinant CYP2J2 isoform (Matsumoto et al., 2003; Liu et al., 2006). However, little data is available on the inhibitors of CYP2J2 enzyme to date.

In this study, we screened six tamoxifen analotues using astemizole as a CYP2J2 probe substrate in human liver microsomes to identify a novel and poten CYP2J2 inhibitor,. The inhibition constant (Ki value) and mechanism were elucidated for compounds which shown strong inhibitory potential.

MATERIALS AND METHODS

Materials:

Astemizole (AST), O-desmethyl astemizole (DAST), and hydroxyebastine (HEB) were purchased from Toronto Research Chemicals (North York, Canada). Glucose-6-phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PDH), and pnicotinamide adenine dinucleotide phosphate (NADP+), tamoxifen (TAM), 4'-hydroxytamoxifen (HTAM), toremifene (TOR), 4'-hydroxytoremifene (HTOR), and mebendazole (MBZ, internal standard, IS) were obtained from Sigma-Aldrich (St. Louis, MO). GSK5182 and 4'-hydroxy-GSK5182 (HGSK5182) were synthesized by the Daegu-Gyeongbuk Medical Innovation Foundation (Daegu, Korea) (Fig.1). Pooled human liver microsomes (HLMs) were purchased from XenoTech (Kansas, USA). Solvents were high performance liquid chromatography (HPLC) grade, and the other reagents and chemicals were of analytical grade ([greater than or equal to] 98%).

CYP2J2 inhibitor screening:

All incubations were performed in triplicate, and the data are presented as average values. The inhibitory potential of six tamoxifen analogues on CYP2J2-mediated AST O-demethylation activity was determined using pooled HLMs in the absence and presence of test compounds.

In brief, a final 100 [micro]L of incubation mixtures containing pooled HLMs (0.1 mg/ml), AST (1 [micro]M) and inhibitor (HEB (positive control), TAM, HTAM, TOR, HTOR, GSK5182, or HGSK5182 (5 [micro]M)) were pre-incubated for 5 min at 37[degrees]C. The reaction was initiated by the addition of NADPH-generating system (containing 1.3 mM [NADP.sup.+], 3.3 mM G6P, 3.3 mM Mg[Cl.sub.2], and 500 unit/ml G6PDH). After 20 min incubation time, the reactions were terminated by the addition of 50 [micro]l of ice-cold methanol containing 150 nM MBZ into the mixtures. After mixing and centrifugation at 14,000 g for 5 min at 4[degrees]C, a 100 [micro]l aliquot of the supernatant was transferred into the autosampler vial and 1 [micro]l of each samples were analyzed by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously [24]. To determine the inhibitory potentials ([K.sub.i] values) of HTOR for CYP2J2-mediated AST O-demethylation activity in HLMs, HTOR (0-50 [micro]M) was added to reaction mixtures containing different concentrations of AST (0.2, 1, and 5 [micro]M). The reaction rates were linear with incubation time and microsomal protein amount under these conditions.

LC-MS/MS analysis:

The concentration of DAST was measured by LC-MS/MS as described previously (Yoon and Liu, 2011), using a LCMS 8040 system (Shimadzu, Tokyo, Japan) equipped with an electrospray ionization (ESI) interface to generate protonated ions [M+H]+. O-Desmethyl astemizole was separated on a reversed-phase Luna C18 column (50 x 2 mm i.d., 5 pMparticle size; Phenomenex, Torrance, CA, USA). The mobile phase (acetonitrile containing 0.1% formic acid (A) and water containing 0.1% formic acid (B)) was eluted using a HPLC system (Shimadzu, Tokyo, Japan) at a flow rate of 0.2 ml/min. The gradient was set for solvent B of 10% at initial stage, ramped into 50% at 3 min, and immediately decreased to 10% up to 6 min. The mass spectrometer was operated in positive ionization mode and calibrated using the manufacturer's calibration mixture. The operating conditions were as follows: capillary temperature, 350[degrees]C; vaporizer temperature, 300 0C; ionization voltage, 4 kV; and collision gas (argon) pressure, 1.5 mTorr. The collision energy was set to 35 and 17 eV for DAST and MBZ, respectively. Quantitation was performed by selected reaction monitoring (SRM) of the precursor ion [[M+H].sup.+] and the related product ion for DAST, using the internal standard (150 nM MBZ) to establish peak area ratios. Ions were detected by monitoring the transitions of m/z 445>204 for DAST and m/z 296>264 for IS (Table 1). The analytical data were processed by Xcalibur (version 2.1) software. Under these LC-MS/MS operating conditions, DATZ and IS were eluted at 2.50 and 3.95 min, respectively (Fig. 2). The lower limit of quantitation for the analyte was 1.0 nM.

Data analysis:

The inhibitory potential of six tamoxifen analogues on CYP2J2-mediated activities were expressed as a percentage of the corresponding control values. The [IC.sub.50] values were determined from the following equation using the WinNonlin software (Pharsight, Mountain View, CA): percentage of control activity = 100*[1-(I/(I+[IC.sub.50]))], where I is the concentration of inhibitor, and [IC.sub.50] is the inflection point on the curve [6]. The apparent kinetic parameters for the inhibitory potential ([K.sub.i]) were first estimated by graphical methods, such as Lineweaver-Burk, Dixon and secondary reciprocal plots, and were finally determined by non-linear least squares regression analysis, based on the best enzyme inhibition model [18] using the WinNonlin software. In our experiments, the inhibition data were consistently best fitted by the noncompetitive inhibition model. The models tested included pure and partial competitive inhibition, non-competitive inhibition, mixed-type inhibition, and uncompetitive inhibition [10,11].

RESULTS AND DISCUSSION

Inhibitory effects of six tamoxifen analogues (Fig. 1) were evaluated for the CYP2J2-mediated astemizole O-demethylase activity in HLMs. Hydroxyebastine (5 [micro]M), positive control, showed similar inhibitory potential compared to previsous data. 4-Hydroxytoremifene inhibited CYP2J2-catalyzed astemizole O-demethylase activity at a 5 [micro]M concentration (> 70%), whereas the other compounds showed weak or negligible inhibitory effects (< 60%) (Fig. 3). Based on these data, we underwent to test the inhibitory effect of HTOR on CYP2J2-mediated astemizole O-demethylation activity. HTOR moderately inhibited CYP2J2-catalyzed astemizole Odemethylation activity in a concentration dependent manner, with an [IC.sub.50] values of 8.4 [micro]M(Fig. 4), which are comparable to the [IC.sub.50] values of haloperidol ([IC.sub.50] = 14.5 [micro]M, [12], thelephoric acid ([IC.sub.50] = 3.23 [micro]M, and decursin ([IC.sub.50] = 6.95 [micro]M, [11].

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

As HTOR inhibited CYP2J2 enzyme activity, we sought to clarify the mechanism of its inhibition. The Lineweaver-Burk plots and secondary reciprocal plots indicated that HTOR inhibited CYP2J2 enzyme activity with an apparent [K.sub.i] value of 11.1 [micro]M(Fig. 5). However, its inhibitory potential was less potent than that of danazole [[K.sub.i] = 0.02 [micro]M, [12], telmisartan [[K.sub.i] = 0.19 [micro]M [17] and flunarizine [[K.sub.i] = 0.13 [micro]M], and decursin [[K.sub.i] = 8.34 [micro]M, [9]. The Lineweaver-Burk plot intersected above the x-axis [18,19,20], indicating that HTOR inhibited the reaction in a noncompetitive manner over the substrate range from 0.5 to 5 [micro]M astemizole (Fig. 5).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

In conclusion, we investigated 4-hydroxytoremifene as a CYP2J2 inhibitor through the evaluation of CYP2J2-mediated astemizole O-demethylase activity. HTOR inhibited CYP2J2-catalyzed astemizole Odemethylation activity with K1 value of 11.1 [micro]M in a noncompetitive inhibition mode. The present data suggest that hydroxytoremifene is a potential candidate for further evaluation for its CYP2J2 targeting anti-cancer activities. Studies are currently underway to evaluate hydroxytoremifene as a potential chemotherapeutics.

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Nguyen Minh Phuc, Zhexue Wu, Eun Young Lee and Kwang-Hyeon Liu

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, Korea

ARTICLE INFO

Article history:

Received 23 June 2015

Accepted 25 July 2015

Available online 30 August 2015

Corresponding Author: Nguyen Minh Phuc, College of Pharmacy and Research Institute of Pharmaceutical Science, Kyungpook National University, Daegu 702-701, Korea.

Tel: +82 53 950 8567; Fax: +82 53 950 8557; E-mail: dstlkh@knu.ac.kr (K.-H. Liu)
Table 1: Mass operation parameters for the analysis of
O-desmethylastemizole and mebendazole.

Compound                Transition    Polarity      Collision
                          (m/z)                    energy (eV)

O-Desmethylastemizole    445>204     [ESI.sup.+]       35
Mebendazole (IS)         296>264     [ESI.sup.+]       17
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Author:Phuc, Nguyen Minh; Wu, Zhexue; Lee, Eun Young; Liu, Kwang-Hyeon
Publication:Advances in Environmental Biology
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Date:Aug 1, 2015
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