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Characterization of human liver cytochrome P450 enzymes involved in the metabolism of rutaecarpine

https://doi.org/10.1016/j.jpba.2005.10.039Get rights and content

Abstract

Rutaecarpine has recently been characterized to have an anti-inflammatory activity through cyclooxygenase-2 inhibition. The incubation of rutaecarpine with human liver microsomes in the presence of NADPH generated six isobaric mono-hydroxylated metabolites. The specific cytochrome P450 (CYP) isozymes responsible for rutaecarpine metabolites were identified using the combination of chemical inhibition, immuno-inhibition and metabolism by cDNA expressed CYP enzymes. The results suggested that CYP3A4 might play major roles in the metabolism of rutaecarpine in human liver microsomes. The production of M1, M2, M3, M4 and M6 formed in human liver microsomes was inhibited by ketoconazole, a selective CYP3A4 inhibitor, and anti-CYP3A4 antibody. CYP1A2 and CYP2C9 played minor roles in the metabolism of rutaecarpine. These results were confirmed in microsomes derived from cDNA expressed lymphoblastoid cells. CYP3A4 microsome clearly formed M1, M2, M3 and M6. CYP1A2 and CYP2C9 microsomes comparably formed M5.

Introduction

Rutaecarpine (8,13-dihydro-7H-indolo [2′,3′:3,4]pyrido[2,1-b]quinazolin-5-one) is an alkaloid originally isolated from the unripe fruit of Evodia rutaecarpa, which has traditionally been used in treatment of gastrointestinal disorders. In addition, rutaecarpine has recently been characterized to have an anti-inflammatory activity through cyclooxygenase-2 inhibition [1]. To develop rutaecarpine as an anti-inflammatory agent, total synthesis of rutaecarpine has successfully been established in our group [2].

Rutaecarpine was found to be metabolized by cytochrome P450 (CYP) in rat liver microsomes [3]. From the study, rutaecarpine was metabolized to nine different metabolites—i.e., 5-mono-hydroxylated and 4-di-hydroxylated rutaecarpine. In addition, 3-methylcholanthrene- and phenobarbital-induced microsomes greatly increased the formation of mono-hydroxylated metabolites [4]. Most recently, structures of four mono-hydroxylated metabolites, formed in untreated and 3-methylcholanthrene-induced rat liver microsomes, were identified by mass, UV absorbance and 1H NMR spectra with those of authentic synthetic standards [5].

Moreover, rutaecarpine showed the modulatory effects on CYP1A1 and 1A2 in human or mouse liver and kidney [6], [7], [8], [9] and CYP2B in rat liver [4]. Recently, rutaecarpine identified as a component from Evodia fruit extract showed a mechanism-based inactivation on CYP3A4 activity [10].

Although several previous studies reported that rutaecarpine was metabolized by rat liver microsomes, possibly by CYP1A and 2B, metabolism of rutaecarpine by human liver microsomes has not yet been studied. The purposes of the present study were to know the metabolite formation from rutaecarpine in human liver microsomes and to characterize the major human CYP isozyme(s) involved in rutaecarpine metabolism, because these data would be crucial in early development of new drug candidates.

Section snippets

Chemicals

Rutaecarpine (purity, >99.8%) used in this study was chemically synthesized in our group [3]. Pooled human liver microsomes, human B-lymphoblastoid-derived CYP containing microsomes and rabbit polyclonal anti-human CYP antibodies were obtained from Gentest (Woburn, MA). Glucose 6-phosphate, β-NADP+, glucose 6-phosphate dehydrogenase, furafylline, diethyldithiocarbamate and ammonium formate were obtained from Sigma Chemical Co. (St. Louis, MO). Sulfaphenazole and ketoconazole were obtained from

Results

In previous studies, rutaecarpine was found to be metabolized in rat liver microsomes in CYP-dependent manners [3], [4], [5]. Representative extracted ion chromatograms obtained from the incubation of rat and human liver microsomes with rutaecarpine are shown in Fig. 1. The incubation of rutaecarpine with human liver microsomes in the presence of NGS generated six isobaric mono-hydroxylated metabolites. Six metabolites showed retention times of 9.2, 9.8, 10.9, 11.8, 13.0 and 13.6 min,

Discussion

From several previous studies, rutaecarpine was found to be metabolized by CYP-dependent manners in rat liver microsomes [3], [4], [5]. The present study demonstrated for the first time that rutaecarpine could be metabolized by CYP enzymes in human liver microsomes. The results indicated that rutaecarpine could be metabolized to six mono-hydroxylated metabolites in control rat and pooled human liver microsomes (Fig. 1). The same metabolites were identified in both microsomes, although the rate

Acknowledgments

This study was supported by a grant from Korea Research Foundation supporting the Institute for Drug Research, Yeungnam University (2004-005-E00004).

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