In vitro identification of cytochrome P450 isoforms responsible for the metabolism of 1-hydroxyl-2,3,5-trimethoxy-xanthone purified from Halenia elliptica D. Don
Graphical abstract
Introduction
Halenia elliptica D. Don (H. elliptica) belongs to the Gentianaceae family. It is a Tibetan medicine traditionally used to treat liver and gall bladder diseases in China [1], [2]. “Yiganjian tablet”, an officially approved product for the treatment of hepatitis B viral infection in China, is a representative formula of H. elliptica. This formula is usually co-administrated with interferon α-2b, lamivudine and/or entecavir [3], [4], [5], [6], [7]. 1-Hydroxyl-2,3,5-trimethoxyxanthone, abbreviated as HM-1, is one of the main constituents of H. elliptica. The chemical structure of HM-1 is illustrated in Fig. 1 [8]. HM-1 has been shown to generate vasodilatation effects on rat coronary artery [9], and it also has strong antioxidant [2] and lung-protective activities [10]. 1,5-Dihydroxy-2,3-dimethoxyxanthone (HM-5), the major metabolite of HM-1 in rat liver microsomes, has also been shown to induce vasodilatation on rat coronary artery [11].
Different cytochrome P450 (CYP450) isoforms can metabolize more than one drug. Concomitant use of drugs may decrease their metabolism, and consequently increasing their concentrations in circulation. This may increase the risk of metabolism-based drug–drug interactions (DDIs) which can impose serious safety problems [12]. The FDA has recommended that potential metabolism-based DDIs mediated by specific CYP isozymes (e.g. CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5) should be investigated, due to that these are the principal types of human liver CYP450 and they are responsible for metabolizing most of clinical drugs [13]. Experimental approaches using selective chemical inhibitors in the in vitro incubation with human liver microsomes are highly recommended [14], [15].
A previous study has demonstrated that HM-1 was shown to be a potent mixed-type inhibitor of CYP1A2 and CYP2C9, but weakly inhibited CYP3A4 in a competitive mode [16]. These results provide insight into the metabolism-based DDIs of HM-1. Four metabolites were also detected in the incubation of HM-1 with rat liver microsomes, and two of them have been identified [17]. The metabolism of HM-1 in HLMs, however, has not been resolved. In the current study, CYP450 isoform-mediated metabolism was investigated to clarify the complete DDI potentials of HM-1. High performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMSn-IT-TOF) and/or hydrogen-1 and carbon-13 nuclear magnetic resonance spectroscopy (1H NMR and 13C NMR) were applied to elucidate the structures of metabolites. Specific chemical inhibitors were used to identify major CYP450 isoforms, including CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, responsible for HM-1 metabolism in pooled HLMs and recombinant human CYP450 isozymes.
Section snippets
Chemicals and materials
HM-1 (purity >99%) was produced in our lab as previously described [8]. HPLC grade acetonitrile was purchased from Fisher Chemicals (Leicester, UK). Ethyl acetate and glacial acetic acid (Beijing Chemical Works Co., Beijing, China) were analytical grade. Pooled HLMs and recombinant CYP450 isoforms were purchased from BD Biosciences (San Jose, CA, USA) and stored at −80 °C until use. Furafylline, pilocarpine, ticlopidine, quercetin, sulfaphenazole, nootkatone, quinidine, ketoconazole,
Metabolites identification by LCMSn-IT-TOF
MS data were analyzed according to the methods described in our previous study [17]. Four major metabolites (M1, M2, M3 and M4) were detected after incubation of HM-1 in pooled HLMs, as detected by HPLC-DAD (Fig. 2A). The metabolites were subsequently analyzed by on-line LCMSn-IT-TOF. The MSn data and extracted ion chromatograms (EICs) are given in Table 1 and Fig. 2B, respectively. The spectra of MS1–MS5 in positive mode and MS1 in negative mode of each metabolite obtained by LCMSn-IT-TOF were
Discussion
In the current study, HM-1 was metabolized in HLMs to form four metabolites. M3, identified as HM-5, was one of the most abundant primary metabolites. M1, M2 and M3 were characterized by LCMSn-IT-TOF alone or with NMR data, indicating that the main Phase I metabolic reactions of HM-1 were demethylation and hydroxylation in HLMs. However, the chemical structure of M4 remains unclear due to its very low production amounts, and further study is needed. In our previous study, the metabolic pathway
Conclusions
In summary, we investigated the in vitro metabolisms of HM-1. This study showed that HM-1 could be metabolized extensively by CYP450 enzymes. By mediating the oxidation reactions through demethylation and hydroxylation of HM-1, CYP3A4 and CYP2C8 appeared to be the dominant isoform, meanwhile CYP1A2 and CYP2C9 played minor roles. In addition, CYP2A6, CYP2B6 and CYP2C19 also involved in M3 formation, but with few contribution. The results indicate that potential metabolism-based DDIs maybe occur
Conflict of interest
None declared.
Acknowledgments
The authors gratefully acknowledge the financial support from National Natural Science Foundation of China (No. 30873115 and No. 81072611); National Mega-project for Innovative Drugs (2012ZX09301-002-001 and 2012ZX09301-002-006); and Special Fund of Chinese Central Government for Basic Scientific Research Operations in Commonweal Research Institutes (No. 2012CHX18). All authors would like to dedicate this work to Prof. John H.K. Yeung who passed away (July, 2012) during the preparation of this
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