Deoxyschizandrin, a Naturally Occurring Lignan, Is a Specific Probe Substrate of Human Cytochrome P450 3A

Abstract

To accurately predict the modifications done during metabolic processes by cytochrome P450 (P450) 3A enzyme, selecting substrates that best represent a broad range of substrate substitutions and that follow the Michaelis-Menten kinetic properties is highly necessary. In the present study, the oxidative pathways of deoxyschizandrin (DS), the most abundant lignan in Fructus Schisandrae fruit extract, were characterized with liver microsomes from human (HLM) and rat (RLM). Only one monohydroxylated metabolite 7(S)-hydroxylated metabolite (isoschizandrin, ISZ), was identified using liquid chromatography-mass spectrometry and nuclear magnetic resonance techniques. CYP3A4 and CYP3A5 were found to be the major isoforms involved in the monohydroxylation of DS. Also, the kinetic studies showed that DS hydroxylation obeyed MichaelisMenten kinetics both in HLM and in RLM. However, the subsequent metabolism of ISZ was nearly nonexistent when DS was present. More importantly, the interactions between DS and three well characterized CYP3A probe substrates, testosterone (TST), midazolam (MDZ), and nifedipine (NIF), were studied. TST and MDZ were shown to compete with DS for the mutual binding site, causing K m to be increased. The presence of DS also lowered the binding affinities for MDZ and TST. However, DS showed only slight inhibitory effects on nifedipine (NIF) oxidation even though NIF was able to inhibit DS hydroxylation in a noncompetitive fashion. These results show that DS is a good representative substrate of MDZ and TST primarily due to their shared, large binding regions on CYP3A. Therefore, DS is an attractive candidate as a novel CYP3A probe substrate for predicting the metabolic modifications in CYP3A activity.

Gomisin A is a Novel Isoform-Specific Probe for the Selective Sensing of Human Cytochrome P450 3A4 in Liver Microsomes and Living Cells

“…In liver microsomes from two individual donors, the CYP3A4 protein contents were almost equal, but the CYP3A5 level was comparable. The inhibitory potentials of GA increased with the decrease of the CYP3A5 protein levels for the four typical co-substrates of CYP3A4/5: midazolam, nifedipine, testosterone, and deoxyschizandrin [17]. Specifically, when the contribution of CYP3A5 increased for the substrate metabolism, the inhibitory efficiency of GA decreased and vice versa.…”

“…With respect to the structure of ligands, it is worth noting that the dibenzocyclooctadiene lignans with six methoxy groups on two benzene rings is the most potent co-substrate of CYP3A4 and CYP3A5, such as deoxyschizandrin 7-hydroxylation [17]. In contrast, the lignans preferred to be selectively metabolized by CYP3A4, but not CYP3A5, in cases when the C-7 of the lignans was hydroxylated, such as GA 8-hydroxylation and schizandrin 8-hydroxylation [18].…”

“…Then, a cell-based assay was used to study the CYP3A4 catalytic activity, where HepG2 cells (1×10 6 / well) were cultured in 6-well plates for 2 days and treated for another 72 h with 10 μM of rifampin or with the 0.1% DMSO vehicle (control). Deoxyschizandrin was used as a control substrate; deoxyschizandrin was metabolized by both CYP3A4 and CYP3A5 [17]. Two milliliters of incubation medium containing 20 μM of GA or 20 μM of deoxyschizandrin was added concomitantly to each well and incubated for 60 min at 37°C.…”

“…Recently, we also found that CYP3A played a key role in the biotransformation of natural lignans (such as schizandrin and deoxyschizandrin) with the dibenzocyclooctadiene skeleton from Fructus Schisandrae Chinensis [17,18]. Notably, these naturally occurring lignans also have a rigid polycyclic skeleton, but they are non-steroidal compounds.…”

Metabolism profiling of amino-noscapine

“…The experiment performed by Fang et al evaluated the metabolic behavior of drug candidate corynoline (Fang et al 2011). In vitro phase I incubation system was employed to find the probe efficiency of deoxyschizandrin as cytochrome P450 3A (Wu et al 2014). The experiment performed by Zhu et al (2012) also showed the successful application of in vitro incubation mixture in the phase II metabolic profiling.…”