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Ketoconazole Inhibits the Cellular Uptake of Anandamide via Inhibition of FAAH at Pharmacologically Relevant Concentrations
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 1, e87542- p.Article in journal (Refereed) Published
Abstract [en]

Background: The 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 Findings: The 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 mu M, 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 mu M. Conclusions/Significance: The 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.

Place, publisher, year, edition, pages
2014. Vol. 9, no 1, e87542- p.
National Category
Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:umu:diva-86616DOI: 10.1371/journal.pone.0087542ISI: 000330288000212OAI: oai:DiVA.org:umu-86616DiVA: diva2:714883
Available from: 2014-04-29 Created: 2014-03-03 Last updated: 2017-12-05Bibliographically approved
In thesis
1. The endocannabinoid system: a translational study from Achilles tendinosis to cyclooxygenase
Open this publication in new window or tab >>The endocannabinoid system: a translational study from Achilles tendinosis to cyclooxygenase
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The endogenous cannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG) exert their effect by activating cannabinoid receptors (CB). These receptors mediate a broad range of physiological functions such as beneficial effects in pain and inflammation, although little is known about the expression of CB receptors in human pain conditions. AEA and 2-AG are short- lived molecules due to their rapid cellular accumulation and metabolism. The enzymes primarily responsible for their degradation are fatty acid amide hydrolase (FAAH) for AEA and monoacylglycerol lipase (MGL) for 2-AG. Inhibition of endocannabinoid metabolism is a potential approach for drug development, and there is a need for the identification of novel compounds with inhibitory effects upon FAAH and MGL.

In Paper I of this thesis, the expression of CB1 receptors in human Achilles tendon was examined. We found expression of CB1 receptors in tenocytes, blood vessel wall as well as in the perineurium of the nerve. A semi-quantitative analysis showed an increase of CB1 receptors in painful human Achilles tendinosis.

In papers II and III, termination of AEA signalling was investigated via inhibition of FAAH. In Paper II, Flu-AM1, an analogue of flurbiprofen, was investigated. The compound inhibited both FAAH and the oxygenation of 2-AG by cyclooxygenase-2. In Paper III the antifungal compound ketoconazole was shown to inhibit the cellular uptake of AEA in HepG2, CaCo-2 and C6 cell lines in a manner consistent with inhibition of FAAH.

The role of FAAH in gating the cellular accumulation of AEA was investigated in Paper IV. FAAH has been shown to control the concentration gradient of AEA across the plasmamembrane in RBL2H3 cells, whereas no such effect is seen in other FAAH-expressing cell lines. To determine whether this effect is assay dependent or due to intrinsic differences between the cell lines, we assayed four cell lines with different levels of FAAH expression using the same methodology. We found that the sensitivity of FAAH uptake inhibition was not dependent on the expression level of FAAH, suggesting that factors other than FAAH are important for uptake.

Paper V is focused on the inhibition of MGL. Prior to this study no selective inhibitors of the enzyme had been described. Thus, we screened a number of compounds for their inhibitory effect on MGL. Troglitazone was found to be an inhibitor of MGL, although its potency was dependent upon the enzyme assay used. 

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2014. 72 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1663
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-91573 (URN)978-91-7601-089-1 (ISBN)
Public defence
2014-09-05, Hörsal E04 Unod R1, Byggnad 6E, Norrlands universitetssjukhus, Umeå, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2014-08-15 Created: 2014-08-11 Last updated: 2014-08-14Bibliographically approved

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Björklund, EmmelieJacobsson, Stig O. P.Fowler, Christopher J.

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