Mechanism of Action of Panaxytriol on Midazolam 1′-Hydroxylation and 4-Hydroxylation Mediated by CYP3A in Liver Microsomes and Rat Primary Hepatocytes

Abstract

In our previous study, panaxytriol (PXT) was shown to enhance midazolam (MDZ) 1′-hydroxylation significantly but to inhibit MDZ 4-hydroxylation. To explore the underlying mechanism, we investigated the effects of PXT on cytochrome P450 3A (CYP3A)-mediated MDZ metabolic pathways using rat liver microsomes (RLM), human liver microsomes (HLM), and rat primary hepatocytes. In the presence of PXT, the V max of 4-OH MDZ decreased from 0.72 to 0.51 nmol/min·mg pro in RLM and from 0.32 to 0.12 nmol/ min·mg pro in HLM, and the K m value increased from 5.12 to 7.26 µM in RLM and from 27.87 to 32.80 µM in HLM. But the presence of PXT reduced the K m and increased the V max values of MDZ 1′-hydroxylation in RLM and HLM. Interestingly, the differential effect of PXT on MDZ 4-hydroxylation and 1′-hydroxylation was also observed in primary rat hepatocytes after 45-min culture. PXT did not affect the expression levels of CYP3A1/2 mRNA in rat hepatocytes. With extension of the culture time to 6 h, however, PXT significantly inhibited both MDZ 4-hydroxylation and 1′-hydroxylation, and the expression level of CYP3A1/2 mRNA was decreased to 87% and 80% (CYP3A1) and to 89% and 85% (CYP3A2) of those in controls in the presence of PXT 4.0 and 8.0 µg/mL, respectively. These results suggest that PXT could activate MDZ 1′-hydroxylation but inhibit MDZ 4-hydroxylation by changing the CYP3A enzyme affinity and metabolic rate after a shortterm intervention. However, long-term treatment with PXT could inhibit both the 4-hydroxylation and 1′-hydroxylation of MDZ by downregulating CYP3A1/2 mRNA expression.Key words panaxytriol; midazolam hydroxylation metabolism; cytochrome P450 3A; differential effect mechanism Cytochrome P450 (CYP450) is a superfamily of hemeproteins that involved in the biotransformation of the majority of clinical drugs. The inhibition or induction effect of clinical drugs on CYP450 is considered as one of the important factors leading to potential drug-drug interactions.1-3) CYP3A4 is one of the primary CYP450 enzymes in human liver and catalyzes the metabolism of more than 50% of clinical drugs including terfenadine, theophylline and astemizole, etc. Midazolam (MDZ) is the specific substrate of CYP3A4 enzyme and is mainly metabolized to 4-hydroxymidazolam (4-OH MDZ) and 1′-hydroxymidazolam (1′-OH MDZ) by combining with the two different sites of CYP3A4 enzyme active center.4,5) Therefore, the formation of the two metabolites are often simultaneously quantified for the assessment of CYP3A4 activity.Traditional Chinese medicine is widely used in clinical practice in China because of the advantages of good curative effect and less adverse reaction. The incidence of Chinese medicine and chemical medicine interactions significantly increased as the widely using of Chinese medicine, and the change of CYP450 enzyme activity is the principal cause resulting in drug metabolic interactions. 6,7) As a well known study, salvia extract exhibited a marked inhibition effect on the expression of CYP3A4 and CYP1A2 mRNA in HepG2, and high...

Midazolam as a Probe for Drug–Drug Interactions Mediated by CYP3A4: Homotropic Allosteric Mechanism of Site-Specific Hydroxylation

“…Importantly, a similar increase in the 4OH percentage in MDZ hydroxylation was observed in the presence of some other CYP3A4 substrates in the reaction mixture, such as carbamazepine, fluconazole, and testosterone, , while the opposite effect on the 1OH:4OH ratio in CYP3A4 MDZ hydroxylation is observed in the presence of panaxytriol . In addition, acceleration of MDZ hydroxylation by CYP3A4 was reported in the presence of erlotinib and ANF, also indicating the sensitivity of MDZ to allosteric interactions with various drugs.…”

“…Importantly, a similar increase in the 4OH percentage in MDZ hydroxylation was observed in the presence of some other CYP3A4 substrates in the reaction mixture, such as carbamazepine, 17 fluconazole, 15 and testosterone, 41,42 while the opposite effect on the 1OH:4OH ratio in CYP3A4 MDZ hydroxylation is observed in the presence of panaxytriol. 43 In addition, acceleration of MDZ hydroxylation by CYP3A4 was reported in the presence of erlotinib 44 and ANF, 45 also indicating the sensitivity of MDZ to allosteric interactions with various drugs. As some of these drugs (e.g., steroids and ANF) are known to bind with high affinity to the allosteric site formed by the F−F′ and G−G′ loops of CYP3A4 and lipid headgroups of the membrane bilayer, 4,7,40,46−48 we hypothesized that MDZ can also act as a homotropic effector that interacts with the same allosteric pocket.…”

Evaluation of the inhibition potential of plumbagin against cytochrome P450 using LC-MS/MS and cocktail approach

“…In this report, however, the unusual effects of plumbagin on CYP3A2 could be explained using an atypical kinetics model. Atypical Michaelis–Menten kinetics including substrate inhibition, partial inhibition, activation, auto-activation and biphasic metabolism has been demonstrated for some CYP3A-mediated reactions 42 43 . In this study, CYP3A2 displayed auto-activation kinetics.…”

“…Multisite hypothesis was a possible mechanistic model of atypical kinetics. CYP3A enzyme except for two or more midazolam-binding sites contains an activator binding site which is closely related to the substrate binding sites 42 . Plumbagin might lead to metabolic activation by binding to the activator binding sites.…”

Effect of panaxytriol on cytochrome P450 3A4 via the pregnane X receptor regulatory pathway

“…A previous study showed that panaxytriol exhibited a differential effect on the hydroxylation metabolism of midazolam mediated by CYP3A4 in human and rat liver microsomes, promoting 1‐hydroxylation metabolism of midazolam and inhibiting its 4‐hydroxylation metabolism (Xia et al, ; Zeng, He, Xia, Zhang, & Xiong, ). PXT inhibited the expression of CYP3A1/2 mRNA in the primary hepatocytes of rats (He et al, ), implying a potential mechanism regulating the expression of CYP3A via the nuclear receptor pathway. However, whether PXT has any impact on the expression of CYP3A4 in human and leads to drug–drug metabolic interactions remains unclear.…”