CYP3A4 inhibition and induction studies coupled to parallel artificial membrane permeability assay (PAMPA) for improved prediction of in-vitro botanical-drug interactions
Type of DegreeMaster's Thesis
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The consumption of botanical dietary supplements (BDS) is a common practice among the US population. However, the potential for botanical-drug interactions exists, and their mechanisms have not been thoroughly studied. CYP3A4 is an important enzyme that contributes to the metabolism of about 60% of clinically used drugs. This study investigates the potential for CY3A4-mediated botanical-drug interactions of Lepidium meyenii Walpers (maca) root and Euterpe oleracea Mart. (açaí) berries since they are commonly used BDS that may be co-administered with CYP3A4-metabolized drugs. In an attempt to decrease the general discrepancy between in vivo and in vitro studies, the absorption profiles, particularly for passive diffusion, and metabolism profiles of plant extracts were investigated. Specifically, the parallel artificial membrane permeability assay (PAMPA) model was utilized to simulate intestinal filtration of passively diffused constituents of açaí and maca extracts. These were subsequently screened for in vitro liver CYP3A4 inhibition and induction. In the inhibition assay, midazolam was used as the probe substrate on genotyped human liver microsomes (CYP3A5 null), and the production of its 1′-substituted metabolite when co-cultured with extract treatments was monitored. In the induction assay, extract treatments were applied to human primary hepatocytes, and quantitative PCR analysis was performed to determine CYP3A4 mRNA expression. Moreover, an in-vitro LC-MS-based methodology was established to access human liver metabolites mediated by UGT or CYP enzymes. As a result, passively diffused constituents of the methanol açaí extract (IC50 of 28.03 µg/µL) demonstrated the highest inhibition potential, and, at 1.5 µg/µL, induced significant changes in CYP3A4 gene expression. The composition of this extract was further investigated using the chemometric tool Mass Profiler Professional (MPP) on liquid chromatography-mass spectroscopy (LC-MS) data. Subsequently, five compounds of interest characterized by high abundance or high permeability were extracted for further study. This included efforts in effective passive permeability determination and structural elucidation by tandem mass spectrometry (MS/MS). Furthermore, a total of 9 metabolites of compounds in methanol açaí extract were separated and annotated.