Marsdenia tenacissima extract inhibits gefitinib metabolism in vitro by interfering with human hepatic CYP3A4 and CYP2D6 enzymes

Ethnopharmacological relevance

The stem of Marsdenia tenacissima (Roxb.) Wight et Arn. is mainly produced in Yunnan China and has long been used as a medicine to treat cancer in China. Xiao-Ai-Ping injection, the water-soluble part of the stem of Marsdenia tenacissima, is administrated as an anti-cancer agent in clinics for decades. Our previous study showed that Marsdenia tenacissima extract (MTE) restored gefitinib sensitivity in gefitinib-resistant non-small cell lung cancer (NSCLC) cells, but the mechanism involved is unknown. Gefitinib undergoes hepatic metabolism predominantly through human cytochrome P450 (CYP) 3A4 and CYP2D6 enzymes. This study aims to evaluate whether MTE interferes with gefitinib metabolism via human hepatic P450 enzymes.

Material and methods

A cocktail-substrate assay was used to test the effect of MTE on major CYP enzyme activities by incubation of pooled human liver microsomes with specific substrate probes of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in the absence and presence of MTE. Recombinant human CYP450 enzymes were used to predict in vitro gefitinib metabolic clearance in the absence and presence of MTE. The metabolites of the substrate probes and gefitinib were detected by high-performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS). Human hepatoma HepG2 cells were used to investigate the effect of gefitinib alone or in combination with MTE on CYP3A4 and CYP2D6 mRNA and protein expression.

Results

The cocktail-substrate assay showed that MTE inhibited CYP450 activities in human liver microsomes with the inhibition rate of 3A4>2C9>2C19>1A2>2D6. The co-administration of MTE with gefitinib significantly decreased the in vitro intrinsic clearance (Cl_int) of gefitinib by 2.6 and 4.0-fold for CYP2D6 and CYP3A4, respectively, but did not affect other CYP450s. CYP2D6 and CYP3A4 mRNA and protein expression in human hepatoma HepG2 cells were greatly reduced in the combined gefitinib and MTE treatment.

Conclusion

We demonstrate that MTE inhibits gefitinib metabolism by interfering with CYP3A4 and CYP2D6. Meanwhile, MTE combined with gefitinib down-regulates the mRNA and protein expression of CYP3A4 and CYP2D6 in the HepG2 cells. Thus, these data suggest that MTE is a promising herbal medicine to enhance gefitinib efficacy through improving its metabolic stability.