Heterotropic Activation of Cytochrome P450 3A4 by Perillyl Alcohol

<b>Background/Objectives</b>: Perillyl alcohol (POH), a monoterpene natural product derived from the essential oils of plants such as perilla (<i>Perilla frutescens</i>), is currently in phase I and II clinical trials as a chemotherapeutic agent. In this study, we investigated the effect of POH on cytochrome P450 (CYP) activity for evaluating POH-drug interaction potential. <b>Methods</b>: The investigation was conducted using pooled human liver microsomes (HLMs), recombinant CYP3A4 (rCYP3A4) enzymes, and human pluripotent stem cell-derived hepatic organoids (hHOs) employing liquid chromatography-tandem mass spectrometry. <b>Results</b>: POH inhibited the activities of CYP2A6 and CYP2B6 with K_i of 6.35 and 3.78 μM, respectively, whereas it stimulated CYP3A4 activity in pooled HLMs incubated with midazolam (MDZ). In a direct CYP inhibition assay using HLMs, activities of CYP2C9, CYP2C19, and CYP2E1 were also inhibited by POH, with IC₅₀ values greater than 50 μM, but those of CYP1A2, CYP2C8, CYP2D6, and CYP3A4 (testosterone) were not significantly inhibited. In pooled HLMs, the V_max/K_m value of 1'-hydroxy MDZ, but not that of 4-hydroxy MDZ, was increased 2.7-fold by 100 μM POH compared with that in the absence of POH. Moreover, stimulation of MDZ 1'-hydroxylation by CYP3A4 was observed in hHOs and rCYP3A4 with cytochrome <i>b</i>₅ but not rCYP3A4 without cytochrome <i>b</i>₅. Furthermore, activation of CYP3A4-mediated metabolism by POH was observed in HLMs incubated with fimasartan but not atorvastatin, buspirone, donepezil, nifedipine, or tadalafil, suggesting a substrate-dependent activation of CYP3A4 by POH. <b>Conclusions</b>: POH inhibits CYP2A6 and CYP2B6, but it activates CYP3A4. These findings underscore the need for further evaluation of the interactions of clinical drugs with POH.