In vitro effects of 95% khat ethanol extract (KEE) on human recombinant cytochrome P450 (CYP)1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5

Abstract

Objectives Khat, a natural amphetamine-like psychostimulant plant, are widely consumed globally. Concurrent intake of khat and xenobiotics may lead to herb-drug interactions and adverse drug reactions (ADRs). This study is a continuation of our previous study, targeted to evaluate the in vitro inhibitory effects of khat ethanol extract (KEE) on human cytochrome (CYP) 1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, and CYP3A5, major human drug metabolizing enzymes. Methods In vitro fluorescence enzyme assays were employed to assess CYPs inhibition with the presence and absence of various KEE concentrations. Results KEE reversibly inhibited CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 but not CYP1A2 with IC50 values of 25.5, 99, 4.5, 21, 27, 17, and 10 μg/mL respectively. No irreversible inhibition of KEE on all the eight CYPs were identified. The Ki values of CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 were 20.9, 85, 4.8, 18.3, 59.3, 3, and 21.7 μg/mL, respectively. KEE inhibited CYP2B6 via competitive or mixed inhibition; CYP2E1 via un-competitive or mixed inhibition; while CYP2A6, CYP2C8, CYP2C19, CYP2J2 and CYP3A5 via non-competitive or mixed inhibition. Conclusions Caution should be taken by khat users who are on medications metabolized by CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, and CYP3A5.

Protein-Ligand Identification andIn VitroInhibitory Effects of Cathine on 11 Major Human Drug Metabolizing Cytochrome P450s

“…25 Besides, the subsequent studies in our laboratory found that khat ethanol extracts inhibited most of the major drug metabolizing CYPs including CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 except CYP1A2. 26 On the other hand, our ongoing investigations demonstrated that the CYPs were inhibited by cathinone differently from that of khat extracts (unpublished data). A study by Bedada et al 35 using human volunteers on one week of daily khat use (400 g) showed khat significantly inhibited CYP2D6, marginally inhibited CYP3A4, CYP2C19 and CYP1A2.…”

“…59 The current study utilizes high-throughput fluorescencebased assays for detection of enzyme-drug interactions. 26,60 In spite of that, a number of caveats exist in this in vitro study using recombinant enzyme preparations as compared to human liver microsomes or hepatocytes. In real-life, owing to the different non-specific binding, accessory proteins or proteinprotein interactions, the enzyme matrix itself may contribute to prominent variations in IC 50 values from one in vitro system to the other 61 which could not be investigated using the current in vitro methods.…”

“…Time curves were plotted afterwards to define the incubation time for single CYP assays namely 60 minutes for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and 120 minutes for CYP3A5, respectively, as determined previously. 25,26…”

“…Time curves were plotted afterwards to define the incubation time for single CYP assays namely 60 minutes for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and 120 minutes for CYP3A5, respectively, as determined previously. 25,26 The inhibitory effects of cathine on human CYP enzymes activities were determined using Vivid ® CYP450 Screening Kits 27 The CYP enzyme specific substrates added were as follows: Vivid ® BOMCC (7-benzyloxymethyloxy-3-cyanocoumarin) for CYP2B6, CYP2C9, CYP3A4 and CYP3A5; Vivid ® CC (3-cyanocoumarin) for CYP2A6; Vivid ® DBOMF (dibenzylmethylfluorescein) for CYP2C8; Vivid ® EOMCC (ethoxymethyloxy-3-cyanocoumarin) for CYP1A2, CYP2C19, CYP2D6 and CYP2E1; Vivid ® MOBFC (7-p-methoxybenzyloxy-4-trifluorocoumarin) for CYP2J2; and 0.03 mM NADP + . The mixture was shaken at room temperature, 60 minutes for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and 120 minutes for CYP3A5.…”

“…Ensuing this study, it was found that KEE also inhibited other major human drug metabolizing CYP isoforms namely CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 except CYP1A2 in vitro. 26 Our ongoing studies, hence, are investigating modulatory effects of main active compounds of khat on the major drug metabolizing CYPs in vitro . The current study aimed to follow up on the in vitro inhibitory effects and mode of inhibitions of cathine, which is the second main active component present in khat plant, on several major drug metabolizing CYPs namely CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP3A5.…”