Ketoconazole Inhibits the Cellular Uptake of Anandamide via Inhibition of FAAH at Pharmacologically Relevant Concentrations

Emmelie Björklund,Therése N L Larsson,Christopher J Fowler,Stig O P Jacobsson,Andrea Cignarella

doi:10.1371/journal.pone.0087542

Abstract

BackgroundThe antifungal compound ketoconazole has, in addition to its ability to interfere with fungal ergosterol synthesis, effects upon other enzymes including human CYP3A4, CYP17, lipoxygenase and thromboxane synthetase. In the present study, we have investigated whether ketoconazole affects the cellular uptake and hydrolysis of the endogenous cannabinoid receptor ligand anandamide (AEA).Methodology/Principal FindingsThe effects of ketoconazole upon endocannabinoid uptake were investigated using HepG2, CaCo2, PC-3 and C6 cell lines. Fatty acid amide hydrolase (FAAH) activity was measured in HepG2 cell lysates and in intact C6 cells. Ketoconazole inhibited the uptake of AEA by HepG2 cells and CaCo2 cells with IC50 values of 17 and 18 µM, respectively. In contrast, it had modest effects upon AEA uptake in PC-3 cells, which have a low expression of FAAH. In cell-free HepG2 lysates, ketoconazole inhibited FAAH activity with an IC50 value (for the inhibitable component) of 34 µM.Conclusions/SignificanceThe present study indicates that ketoconazole can inhibit the cellular uptake of AEA at pharmacologically relevant concentrations, primarily due to its effects upon FAAH. Ketoconazole may be useful as a template for the design of dual-action FAAH/CYP17 inhibitors as a novel strategy for the treatment of prostate cancer.

Highlights

The endocannabinoid system, comprising the cannabinoid CB receptors, their endogenous ligands arachidonoylethanolamide and 2-arachidonoylglycerol (2-AG), and their synthetic and metabolic enzymes, are involved in a variety of regulatory pathways including the control of pain, appetite, reproduction, bone turnover and control of cancer [1,2,3,4,5]
AEA is removed from the extracellular space by a process of cellular uptake followed by enzymatic metabolism, primarily to arachidonic acid via the hydrolytic enzyme fatty acid amide hydrolase
In the present study we have demonstrated that ketoconazole inhibits the uptake of the endocannabinoid AEA into a variety of cell lines, and that this effect can be most explanined by the ability of the compound to inhibit Fatty acid amide hydrolase (FAAH)

Summary

Introduction

The endocannabinoid system, comprising the cannabinoid CB receptors, their endogenous ligands arachidonoylethanolamide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), and their synthetic and metabolic enzymes, are involved in a variety of regulatory pathways including the control of pain, appetite, reproduction, bone turnover and control of cancer [1,2,3,4,5]. AEA is removed from the extracellular space by a process of cellular uptake followed by enzymatic metabolism, primarily to arachidonic acid via the hydrolytic enzyme fatty acid amide hydrolase. In addition to FAAH, AEA can act as a substrate for other enzymes, including cyclooxygenase-2 and lipoxygenases [12], and evidence is accruing to suggest that such pathways may have important pathophysiological relevance [13,14]. We have investigated whether ketoconazole affects the cellular uptake and hydrolysis of the endogenous cannabinoid receptor ligand anandamide (AEA)

Methods

Results

Conclusion