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Biochanin A acts as a peripheral inhibitor of fatty acid amide hydrolase
Umeå University, Faculty of Medicine, Pharmacology and Clinical Neuroscience, Pharmacology. (Christopher Fowler)
Department of Pharmacology and Toxicology, Virginia Commenwealth University, Richmond, Virginia.
Department of Anesthesiology, Washington University, School of Medicine, St. Louis, Missouri.
Department of Chemistry and Skaggs Institute for Chemical Biology, La Jolla, California.
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(English)Manuscript (Other academic)
Abstract [en]

Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide, are effective in animal models of inflammatory, cancer, visceral and neuropathic pain. No peripherallyrestricted FAAH inhibitors have yet been reported. The isoflavones are a class of naturallyoccurring compounds that show little brain penetration, and two of these, genistein and daidzein, inhibit FAAH at low micromolar concentrations. Here, we report that the related isoflavone biochanin A inhibits the hydrolysis of 0.5 μM anandamide by mouse, rat and human FAAH with IC50 values of 1.8, 1.4, and 2.4 μM, respectively. The inhibition of rat FAAH by biochanin A was mixed type in nature, with Ki slope and Ki intercept values of 1.1 and 8.2 μM, respectively. Anandamide hydrolysis was also inhibited in intact basophilic leukaemia cells at sub- to low- micromolar concentrations. The compound did not interact to any major extent with CB1 or CB2 receptors. In vivo, biochanin A (10 mg/kg i.v.) did not increase brain anandamide concentrations, but produced a modest potentiation of the effects of 10 mg/kg i.v. anandamide in the tetrad test. In anaesthetized mice, URB597 (30 μg/paw) and biochanin A (100 μg/paw) both inhibited the spinal phosphorylation of extracellular receptor kinase produced by the intraplantar injection of formalin. The effects of both compounds were significantly reduced by the CB1 receptor antagonist/inverse agonist AM251 (30 μg/paw). It is concluded that biochanin A may be useful as a template for the design of novel, peripherally-acting FAAH inhibitors.

Keyword [en]
fatty acid amide hydrolase; inflammatory pain; cannabinoid; isoflavones, biochanin A; anandamide
National Category
Pharmacology and Toxicology
URN: urn:nbn:se:umu:diva-22172OAI: diva2:213032
Available from: 2009-04-28 Created: 2009-04-27 Last updated: 2010-01-14Bibliographically approved
In thesis
1. The cellular processing of the endocannabinoid anandamide and its pharmacological manipulation
Open this publication in new window or tab >>The cellular processing of the endocannabinoid anandamide and its pharmacological manipulation
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG) exert most of their actions by binding to cannabinoid receptors. The effects of the endocannabinoids are short-lived due to rapid cellular accumulation and metabolism, for AEA, primarily by the enzymes fatty acid amide hydrolase (FAAH). This has led to the hypothesis that by inhibition of the cellular processing of AEA, beneficial effects in conditions such as pain and inflammation can be enhanced. The overall aim of the present thesis has been to examine the mechanisms involved in the cellular processing of AEA and how they can be influenced pharmacologically by both synthetic natural compounds.

Liposomes, artificial membranes, were used in paper I to study the membrane retention of AEA. The AEA retention mimicked the early properties of AEA accumulation, such as temperature-dependency and saturability.

In paper II, FAAH was blocked by a selective inhibitor, URB597, and reduced the accumulation of AEA into RBL2H3 basophilic leukaemia cells by approximately half. Treating intact cells with the tyrosine kinase inhibitor genistein, an isoflavone found in soy plants and known to disrupt caveolae-related endocytosis, reduced the AEA accumulation by half, but in combination with URB597 no further decrease was seen. Further on, the effects of genistein upon uptake were secondary to inhibition of FAAH. The ability to inhibit the accumulation and metabolism of AEA was shared by several flavonoids (shown in paper III). In paper IV, the isoflavone biochanin A and URB597 had effects in vivo, in a model of persistent pain, effects decreased by the cannabinoid receptor 1 antagonist AM251.

In paper VI, the cellular processing of the endocannabinoid metabolites following degradation was examined, a mechanism poorly understood. It was found that nitric oxide (NO) donors significantly increased the retention of tritium in cell membranes following incubation with either tritiated AEA or 2-AG. Further experiments revealed that the effect of NO donors mainly involves the arachidonate part of the molecules. Inhibition of FAAH completely reduced the effect of NO donors in cells with a large FAAH component, indicating that the effects were downstream of the enzyme.

These results suggest that the cellular processing of endocannabinoids can be affected in a manner of different ways by pharmacological manipulation in vitro and that naturally occurring flavonoid compounds can interact with the endocannabinoid system.

Place, publisher, year, edition, pages
Umeå: Farmakologi och klinisk neurovetenskap, 2009. 80 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1247
Endocannabinoid, anandamide, cellular processing, pain, flavonoids, fatty acid amide hydrolase
National Category
Pharmacology and Toxicology
Research subject
Medical Pharmacology
urn:nbn:se:umu:diva-22221 (URN)978-91-7264-732-9 (ISBN)
Farmakologi, 901 87, Umeå
Public defence
2009-05-22, Sal E04, byggnad 6E, Umeå universitetssjukhus, 901 89 Umeå, Umeå, 09:00 (English)
Available from: 2009-04-30 Created: 2009-04-28 Last updated: 2010-01-18Bibliographically approved

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