Characterization of CYPs and UGTs Involved in Human Liver Microsomal Metabolism of Osthenol.

Pil Cho,Taeho Lee,Geunhyung Jo,Yun Jin,Tae Jeong,Sangkyu Lee,Doohyun Lee,Sanjita Paudel

doi:10.3390/pharmaceutics10030141

Pil Cho, Taeho Lee + Show 6 more

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https://doi.org/10.3390/pharmaceutics10030141

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Journal: Pharmaceutics	Publication Date: Aug 30, 2018
Citations: 5	License type: CC BY 4.0

Affiliation: Kyungpook National University, Yeungnam University

Abstract

Osthenol is a prenylated coumarin isolated from the root of Angelica koreana and Angelica dahurica, and is an O-demethylated metabolite of osthole in vivo. Its various pharmacological effects have been reported previously. The metabolic pathway of osthenol was partially confirmed in rat osthole studies, and 11 metabolic products were identified in rat urine. However, the metabolic pathway of osthenol in human liver microsomes (HLM) has not been reported. In this study, we elucidated the structure of generated metabolites using a high-resolution quadrupole-orbitrap mass spectrometer (HR-MS/MS) and characterized the major human cytochrome P450 (CYP) and uridine 5′-diphospho-glucuronosyltransferase (UGT) isozymes involved in osthenol metabolism in human liver microsomes (HLMs). We identified seven metabolites (M1-M7) in HLMs after incubation in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and uridine 5′-diphosphoglucuronic acid (UDPGA). As a result, we demonstrated that osthenol is metabolized to five mono-hydroxyl metabolites (M1-M5) by CYP2D6, 1A2, and 3A4, respectively, a 7-O-glucuronide conjugate (M6) by UGT1A9, and a hydroxyl-glucuronide (M7) from M5 by UGT1A3 in HLMs. We also found that glucuronidation is the dominant metabolic pathway of osthenol in HLMs.

Highlights

Osthenol (7-hydroxy-8-(3-methyl-2-butenyl)-2H-1-benzopyran-2-one, Figure 1a) is a prenylated coumarin isolated from the root of Angelica koreana and Angelica dahurica [1,2]
To investigate the uridine -diphospho-glucuronosyltransferase (UGT) isoform associated with glucuronidation from the hydroxylated osthenol, 10 μM osthenol was combined with 5 pmoles human recombinant cytochrome P450 (CYP) 1A1 and 1A2 in 0.1 M Kpi buffer
Osthenol was detected at m/z 229 at 13.7 min in negative mode, which showed a stronger intensity compared to the positive mode

Summary

Introduction

Osthenol (7-hydroxy-8-(3-methyl-2-butenyl)-2H-1-benzopyran-2-one, Figure 1a) is a prenylated coumarin isolated from the root of Angelica koreana and Angelica dahurica [1,2]. The reaction mixture consisted of 5 pmoles of human recombinant cDNA-expressed CYP 2.3. Human recombinant The reaction mixture consisted of 5 pmoles of human recombinant cDNA-expressed CYP isoforms (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5) and 20 μM osthenol with NGS system in a 200 μL reaction volume in the presence of 0.1 M Kpi buffer (pH 7.4). Osthenol (10 μM) was pre-incubated in a 200 μL reaction mixture containing 0.1 mg/mL human recombinant UGT isoforms and 0.5 mg/mL alamethicin at 37 ◦C for 5 min, and kept on ice for 15 min to activate the enzymes. 0.1 M Kpi buffer (pH 7.4) was added to the mixture and incubated at 37 ◦C for 5 min. The reaction was initiated by the addition of 10 mM UDPGA and NGS solution and incubated for 60 min at 37 ◦C. The analysis of osthenol metabolites was as described in the above section

Reaction Phenotyping for M7

Instruments

Identification of Phase I and Phase II Metabolites of Osthenol in HLMs

Elucidation of Metabolite Structure

C13 H13 O3