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

Highlights

• The metabolites of xanthone were identified using LCMSⁿ-ESI-IT-TOF and NMR.

• The pathways of xanthone metabolism were elucidated in human liver microsomes.

• The CYP450 isoforms responsible for the metabolism of the xanthone were identified.

Abstract

1-Hydroxyl-2,3,5-trimethoxyxanthone (HM-1) is one of the main constituents extracted from Halenia elliptica D. Don, which is a traditionally used Tibetan medicinal plant. The aim of this study was to illustrate the proposed metabolic pathways of HM-1 and identify which cytochrome P450 (CYP450) isoforms involved in its metabolism by using pooled human liver microsomes (HLMs) and recombinant CYP450 isoforms with selective chemical inhibitors. Metabolites were identified by high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMSⁿ-ESI-IT-TOF) and nuclear magnetic resonance spectroscopy (hydrogen-1 NMR and carbon-13 NMR). Three metabolites (M1–M3) were identified, which demonstrated that demethylation and hydroxylation were the major Phase I metabolic reactions for HM-1 in HLMs. The structure of another metabolite (M4) was still unclear. The enzymatic kinetics of M1 (K_m = 23.19 ± 14.20 μM) and M2 (K_m = 32.06 ± 17.09 μM) exhibited substrate inhibition; whereas, the formation of M3 (K_m = 5.73 ± 0.70 μM) and M4 (K_m = 16.43 ± 5.12 μM) displayed Michaelis–Menten kinetics. The intrinsic clearance (V_max/K_m) of M3 was highest among these metabolites, suggesting that M3 was the major metabolite of HM-1. Moreover, CYP3A4 and CYP2C8 were the primary CYP450 isoform responsible for the metabolism of HM-1. CYP1A2, CYP2A6, CYP2B6, CYP2C9 and CYP2C19 were also involved in HM-1 metabolism, especially in the formation of M3. This study finally provides evidence of substrate inhibition and metabolism-based drug–drug interaction for the medicinal preparations containing HM-1 used in clinic.

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.