Climbazole Is a New Potent Inducer of Rat Hepatic Cytochrome P450

The Journal of Toxicological Sciences, 141 Vol.26, No.3, 141―150, 2001 CLIMBAZOLE IS A NEW POTENT INDUCER OF RAT HEPATIC CYTOCHROME P450 Yasuna KOBAYASHI1, Michiya SUZUKI2, Naomi OHSHIRO1, Takashi SUNAGAWA2, Tadanori SASAKI1, Shogo TOKUYAMA1, Toshinori YAMAMOTO1 and Takemi YOSHIDA2 1Departments of Clinical Pharmacy and 2Biochemical Toxicology, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo142-8555, Japan (Received March 19, 2001; Accepted April 25, 2001) ABSTRACT ― We examined the effect of climbazole on the induction of rat hepatic microsomal cytochrome P450 (P450), and compared the induction potency with other N-substituted azole drugs such as clorimazole. We found that climbazole is found to be a potent inducer of rat hepatic microsomal P450 as clorimazole. Induced level of P450 by climbazole was almost similar in extent to clorimazole when compared with other imidazole drugs in a dose- and time-dependent manner. Parallel to the increase in P450, climbazole increased aminopyrine and erythromycin N-demethylase, ethoxycoumarin O-deethylase, and androstenedione 16β- and 15α/6βhydroxylase activities; however, clorimazole did not induce aminopyrine N-demethylase activity irrespective of its marked increase in P450 content. Immunoblot analyses revealed that climbazole induced CYP2B1, 3A2 and 4A1. The present findings indicate that climbazole is a new potent inducer of hepatic microsomal P450 and drug-metabolizing enzymes like clorimazole, but it may have some differential mechanism(s) for these enzymes’ induction in rat liver. KEY WORDS: Climbazole, Cytochrome P450, Imidazole, Enzyme induction INTRODUCTION ducing P450 induction. Additionally, we have shown that 1-trityl- and 1-diphenylmethyl-imidazoles induces Cytochrome P450 (P450) constitutes a superfami- CYP2B, whereas 1-benzylimidazole increases both ly of hemeproteins that play a pivotal role in the metab- CYP2B and CYP1A species (Kobayashi et al., 1993). olism of a wide variety of foreign compounds including Of the induction of Phase II drug-metabolizing drugs, environmental contaminants and carcinogens enzymes, Franklin (1991) has reported that 2-substitut- (Gonzalez, 1988). N-substituted drugs and chemicals ed dipyridines dramatically increased Phase II enzyme are high potent inducers and/or inhibitors of rat hepatic activities without affecting on total P450 content. P450 (Lavrijsen et al., 1986; Rodrigues et al., 1988; Currently, Vargas et al. (1998) have reported that chem- Laignelet et al., 1989; Hostetler et al., 1989; Sabzevari ical structure requirements for the induction of Phase II et al., 1995). Among these compounds, Ritter and enzymes in the intestine are markedly different from Franklin (1987a, 1987b) have demonstrated that the that for the liver. All of these findings (Lavrijsen et al., antimycotic drug clotrimazole is the most powerful 1986; Ritter and Franklin, 1987a, 1987b; Rodrigues et inducer of rat hepatic P450. We have shown that substi- al., 1988; James, 1988; Laignelet et al., 1989; Papac tuted compound(s) having benzyl moiety bound to imi- and Franklin, 1988; Franklin, 1991, 1992; Franklin and dazole- and/or pyridine-rings at 1- and 4-position of the Moody, 1992; Cho et al., 1995; Vargas et al., 1998) rings, respectively, are the minimum structural unit for together with our reports (Matsuura et al., 1991; the induction of P450 (Matsuura et al., 1991). Our Kobayashi et al., 1992, 1993, 1994, 1995, 1996a, results suggest that aromatic moiety linked to the aryl 1996b, 2000, 2001; Yoshida et al., 1995) make us con- ring is an indispensable structural component for pro- clude that the structural requirements for the induction Correspondence : Takemi YOSHIDA Vol. 26 No. 3 142 Y. KOBAYASHI et al. of P450 enzymes by imidazole ring-containing com- CLZ was suspended in an appropriate volume of corn pounds would need to have an aromatic substitutent at oil and injected intraperitoneally into rats at doses rang- 1 position of the imidazole ring. Thus, investigation ing from 0.10 to 0.80 mmol/kg. In the time-course and elucidation of the structural feature of imidazole study, CLZ was injected intraperitoneally into rats at derivatives as P450 inducer and/or inhibitor are impor- the fixed dose of 0.60 mmol/kg, and the livers were tant for the prediction of human and animal toxicities. collected at 24, 48, 72, 96, and 120 hr. To examine the Climbazole (1-(4-chlorophenoxy)-1-(imidazole-1- induction potency of P450, all N-substituted azole yl)3,3-dimethylbutan-2-one) (CLZ) is one of the imida- antimycotic drugs and H2-antagonist cimetidine were zole-containing drugs that were initially expected to suspended in an appropriate volume of corn oil and have antimycotic and antidandruff effects. Since CLZ injected intraperitoneally into rats at the dose of 80 has both aromatic and aliphatic moieties linked to N- mg/kg/day (1 ml/kg) for 2 consecutive days. methylimidazole, we could expect that this chemical Phenobarbital sodium was dissolved in saline and would produce a potent induction of hepatic P450 and injected intraperitoneally into rats at a dose of 80 drug-metabolizing enzymes with a somewhat different mg/kg/day (0.35 mmol/kg/day) for 2 consecutive days. effect on the enzymes from other azoles having either Control animals were given an equal volume of vehicle aromatic (ex; triphenyl moiety) or aliphatic moieties alone in the same way. All animals were fasted for 24 (ex; t-butyl moiety). However, there is no report con- hr before being killed. cerned with the induction of P450 and drug-metabolizing enzymes by imidazole-containing compounds hav- Preparation of liver microsomes ing both moieties. Therefore, we employed and exam- Livers were perfused in situ with ice-cold 0.9% ined the effect of CLZ on the induction of P450 and NaCl solution. The livers were promptly removed from drug-metabolizing enzymes, and on inducible P450 the body after the perfusion, minced by scissors and species in male and female rats, together with compar- homogenized immediately with 5 volumes of 1.15% ing its ability to produce the P450 induction response (w/v) KCl solution using a Potter-Elvehjem homoge- to other azole drugs. nizer (Matsushima Electric Industrial Co., Ltd., Tokyo, Japan) with Teflon pestle. The homogenates were cen- MATERIALS AND METHODS trifuged at 9,000 g for 20 min, and the resulting super- natants were further centrifuged at 105,000 g for 60 Chemicals min. The resulting microsomal pellet was suspended in CLZ was kindly donated from Kao Co. (Tokyo, 10 mM Tris acetate buffer (pH 7.4) containing 1 mM Japan). Phenobarbital sodium and 4-chloro-1-naphthol EDTA, 20% glycerol and 400μM phenylmethylsul- were obtained from Wako Pure Chemical Industries fonyl fluoride (PMSF). Resuspended pellet was used (Tokyo, Japan). Clotrimazole, econazole, bifonazole, for determining total P450 content, aminopyrine N- sulconazole, miconazole, ketoconazole, cimetidine, and demethylase, ethoxycoumarin O-deethylase, ery- protein standard for SDS-PAGE were purchased from thromycin N-demethylase, and [14C]androstenedione Sigma Chemical (St. Louis, MO, U.S.A.). 4- 16α-, 16β-, 15α/6β-hydroxylase activities, and for [14C]Androst-4-ene-3,17-dione (56 mCi/mmol) was immunoblot analysis. purchased from Japan Isotope Association (Tokyo, Japan). All other chemicals and supplies not listed here Enzyme assays were obtained from commercially available sources. Hepatic total P450 content (about 2 mg protein/ml) was determined from a carbon monoxide Animals and treatments difference spectrum of dithionite-treated microsomes Male and female Wistar rats (170 - 220 g) were dissolved in microsome solubilization buffer (100 mM obtained from Nippon Seibutsu Zairyo Ctr. (Tokyo, potassium phosphate buffer, pH 7.3, 0.5% sodium Japan), and were used in this study. They were fed a cholate, 1 mM EDTA, 20% glycerol, and 0.4% commercial solid diet (MF, Oriental Yeast Co., Tokyo, Emulgen 911), and recorded with a HITACHI U-2000 Japan) and water ad libitum. They were housed in spectrophotometer using a difference extinction coeffe- cages at a constant temperature between 22 to 26℃ cient (450-490 nm) of 91 mM－1cm－1 as described by under a 12-hr light/dark cycle (light 08:00-20:00) and Omura and Sato (1964). Erythromycin and aminopy- had a minimum of 1 week to acclimate to these condi- rine N-demethylase activities were measured by deter- tions before experiments. In the dose-response study, mining the formaldehyde formation according to the Vol. 26 No. 3 143 Induction of cytochrome P450 by climbazole. method of Nash (1953). Ethoxycoumarin O-deethylase In vitro inhibition assay activity was assayed as described previously (He et al., CLZ was dissolved in a minimal volume of 1995; Grimm et al., 1994) with some modification. dimethyl sulfoxide (DMSO) and diluted with water. Twentyμg protein was incubated with substrate for 10 The final concentration of DMSO in the assay medium min at 37℃ and the concentration of the product (7- did not exceed 0.015%. Solution of the compound was hydroxycoumarin) was determined from its fluores- added to each assay at the final concentrations indicat- cence with an extinction wavelength of 366 nm and ed in the figure. The microsomal suspensions for ben- emission wavelength of 454 nm (Miller and Halpert, zphetamine N-demethylase, erythromycin N-demethy- 1986). Androstenedione hydroxylase activity was lase and dimethylnitrosamine N-demethylase activities assayed by the method of Graves et al. (1987). used were prepared from phenobarbital- (80 mg/kg/day Microsomal preparation (20μg) was incubated with 25 for 2 days), dexamethasone- (300 mg/kg/day for 2 μM [14C] androstenedione and 1 mM NADPH in a days) and pyridine-pretreated (100 mg/kg/day for 2 final volume of 100μl of 50 mM HEPES buffer (pH days) male rats, respectively. Each value represents the 7.6) containing 15 mM MgCl2 and 0.1 mM EDTA. percent of control benzphetamine N-demethylase (5.86 Incubations were carried out for 10 min at 37℃ and nmol/min/mg protein), erythromycin N-demethylase were quenched by the addition of 50μl of tetrahydrofu- (18.35 nmol/min/mg protein) and dimethylnitrosamine ran.

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