Licoricidin isolated from Glycyrrhiza uralensis is known to have anticancer, anti-nephritic, anti-Helicobacter pylori,and antibacterial effects. In this study, a cocktail probe assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to investigate the modulating effect of licoricidin on cytochrome P450 (CYP) enzymes in human livermicrosomes. When licoricidin was incubated at 0-25 μM with CYP probes for 60 min at 37oC, it showed potent inhibitoryeffects on CYP2B6-catalyzed bupropion hydroxylation and CYP2C9-catalyzed diclofenac 4’-hydroxylation with half maximalinhibitory concentration (IC50) values of 3.4 and 4.0 μM, respectively. The inhibition mode of licoricidin was revealed as competitive,dose-dependent, and non-time-dependent, and following the pattern of Lineweaver-Burk plots. The inhibitory effect oflicoricidin has been confirmed in human recombinant cDNA-expressed CYP2B6 and 2C9 with IC50 values of 4.5 and 0.73 μM,respectively. In conclusion, this study has shown the potent inhibitory effect of licoricidin on CYP2B6 and CYP2C9 activitycould be important for predicting potential herb-drug interactions with substrates that mainly undergo CYP2B- and CYP2C9-mediated metabolism.
Honokiol and magnolol, the major bioactive neolignans of magnolia officinalis, are the most important constituents ofthe crude drug prescriptions that are used in the therapy of neuroses and various nervous disorders. There have been limitedreports on the effects of neolignoid compounds on human cytochrome P450 activity. Therefore, the inhibitory effects of honokioland magnolol on seven human cytochrome P450 s were evaluated in human liver microsomes. Honokiol and magnololshowed the most potent inhibition of CYP1A2-mediated phenacetin O-deethylase activity (IC50 values of 3.5 and 5.4 mM,respectively) among the seven P450s tested. These in vitro data indicate that neolignan compounds can inhibit the activity ofCYP1A2 and suggest that these compounds should be examined for potential pharmacokinetic drug interactions in vivo.
Mollugin isolated from Rubia cordifolia is known to have anti-inflammatory, anti-cancer, and anti-viral activities. Inthe present study, a cocktail probe assay and LC-MS/MS were used to investigate the modulating effect of mollugin on cytochromeP450 (CYP) enzymes in male ICR mice. After mollugin was orally administrated to mice at the 20, 40, or 80 mg/kg for3 days, the activities of CYP in hepatic S-9 fractions were investigated. Unlike the selective inhibitory effect of mollugin onCYP1A2-catalyzed phenacetin O-deethylation in vitro, mollugin only significantly inhibited the activity of CYP2E1-catalyzedchlorzoxazone 6-hydroxylase in vivo. The activities of other CYPs were only slightly altered by mollugin. The results of thisstudy suggest that mollugin might cause herb-drug interactions via the selective inhibition of CYP2E1 in vivo.
Ginseng, a traditional herbal drug, has been used in Eastern Asia for more than 2000 years. Various ginsenosides, which are the major bioactive components of ginseng products, have been shown to exert numerous beneficial effects on the human body when co-administered with drugs. However, this may give rise to ginsenoside-drug interactions, which is an important research consideration. In this study, acassette assay was performed the inhibitory effects of 12 ginsenosides on seven cytochrome P450 (CYP) isoforms in human liver microsomes (HLMs) using LC-MS/MS to predict the herb-drug interaction. After incubation of the 12 ginsenosides with seven cocktail CYP probes, the generated specific metabolites were quantified by LC-MS/MS to determine their activities. Ginsenoside Rb1 and F2 showed strong selective inhibitory effect on CYP2C9-catalyzed diclofenac 4′-hydroxylation and CYP2B6-catalyzed bupropion hydroxylation, respectively. Ginsenosides Rd showed weak inhibitory effect on the activities of CYP2B6, 2C9, 2C19, 2D6, 3A4, and compound K, while ginsenoside Rg3 showed weak inhibitory effects on CYP2B6. Other ginsenosides, Rc, Rf, Rg1, Rh1, Rf, and Re did not show significant inhibitory effects on the activities of the seven CYPs in HLM. Owing to the poor absorption of ginsenosides after oral administration in vivo, ginsenosides may not have significant side effects caused by interaction with other drugs.
High-resolution quadrupole-Orbitrap mass spectrometry (HRMS), with high-resolution (> 10,000 at full-width at half-maximum) and accurate mass (< 5 ppm deviation) capabilities, plays an important role in the structural elucidation of drug metabolites in the pharmaceutical industry. ML106, a derivative of imidazobenzimidazole, decreased melanin content and tyrosinase activity in a dose-dependent manner. Here, we investigated the phase 1 metabolic pathway of ML106 using HRMS in human liver microsomes (HLMs) and recombinant cDNA-expressed cytochrome P450 (CYP). After the incubation of ML106 with pooled HLMs and recombinant cDNA-expressed CYP in the presence of NADPH, five phase 1 metabolites, including three mono-hydroxylated metabolites (M1-3) and two di-hydroxylated metabolites (M4 and M5), were investigated. The metabolite structures were postulated by the elucidation of protonated mass spectra using HRMS. The CYP isoforms related to the hydroxylation of ML106 were studied after incubation with recombinant cDNA-expressed CYP. Here, we identified the phase 1 metabolic pathway of ML106 induced by CYP in HLMs.
Cytochrome P450 2J2 (CYP2J2) is a member of the cytochrome P450 superfamily, and is known to be arachidonic acid epoxygenase that mediates the formation of four bioactive regioisomers of epoxyeicosatrienoic acids (EETs). CYP2J2 is also involved in the metabolism of drugs such as albendazole, astemizole, danazol, ebastine, and terfenadine. CYP2J2 is highly expressed in the heart and cancer tissues. In this study, the inhibitory potential of ten natural products against CYP2J2 activity was evaluated using human liver microsomes and tandem mass spectrometry. Among them, bilobetin, which is a kind of biflavo-noid, exhibits a strong inhibitory effect against the CYP2J2-mediated astemizole O-demethylation (IC 50 = 0.73 µM) and terfena- dine hydroxylation (IC 50 = 0.89 µM). This result suggests that bilobetin can be used as strong CYP2J2 inhibitor in drug metabolism study.
DC23, a triazolothione resorcinol analogue, is known to inhibit heat shock protein 90 and pyruvate dehydrogenase kinase which are up-regulated in cancer and diabetes, respectively. This study was performed to elucidate the metabolism of DC23 in human liver microsomes (HLMs). HLMs incubated with DC23 in the presence of uridine 5’-diphosphoglucuronic acid (UDPGA) and/or β-nicotinamide adenine dinucleotide phosphate (NADPH) resulted in the formation of four metabolites, M1- M4. M1 was identified as DC23-N-Oxide, on the basis of LC-MS/MS analysis. DC23 was further metabolized to its glucuronide conjugates (M2, M3, and M4). In vitro metabolic stability studies conducted with DC23 in HLMs revealed significant glucuron- ide conjugation with a t 1/2 value of 1.3 min. The inhibitory potency of DC23 on five human cytochrome P450s was also investi- gated in HLMs. In these experiments, DC23 inhibited CYP2C9-mediated tolbutamide hydroxylase activity with an IC 50 value of 8.7 µM, which could have implications for drug interactions.
Ginseng (Panax ginseng Meyer) is a well-known health functional food used as a traditional herbal drug in Asian countries owing to its diverse pharmacological effects. Herb-drug interactions may cause unexpected side effects of co-administered drugs by the alteration of pharmacokinetics through effects on cytochrome P450 activity. In this study, we investigated the herb-drug interactions between Korean red ginseng extract (KRG) and five CYP-specific probes in mice. The pharmacokinetics of KRG extract induced-drug interactions were studied by cassette dosing of five CYP substrates for CYP1A, 2B, 2C, 2D, and 3A and the LC-MS/MS analysis of the blood concentration of metabolites of each of the five probes. The linearity, precision, and accuracy of the quantification method of the five metabolites were successfully confirmed. The plasma concentrations of five metabolites after co-administration of different doses of the KRG extract (0, 0.5, 1, and 2 g/kg) were quantified by LC-MS/MS and dose-dependent pharmacokinetic parameters were determined. The pharmacokinetic parameters of the five metabolites were not significantly altered by the dose of the KRG extract. In conclusion, the single co-administration of KRG extract up to 2 g/kg in vivo did not cause any significant herb-drug interactions linked to the modulation of CYP activity.