Kinase experiment:

For experiments with FAAH, rat liver homogenates, mouse brain homogenates and membranes from COS7 cells transfected with the human enzyme are used. Frozen (–80℃) livers from adult C57BL/6 mice and frozen brains (minus cerebella) from adult Wistar or Sprague-Dawley rats are thawed and homogenized in 20 mM HEPES, 1 mM MgCl2, pH 7. The homogenates are centrifuged at ~35000×g for 20 min at 4℃. After resuspension in buffer followed by recentrifugation and a second resuspension in buffer, the pellets are incubated at 37℃ for 15 min. This incubation is undertaken in order to hydrolyse all endogenous FAAH substrates. The homogenates are then centrifuged as above, recentrifuged and resuspended in 50 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA and 3 mM MgCl2. The homogenates are then frozen at –80℃ in aliquots until used for assay. FAAH is assayed in the homogenates and in the COS7 cell membranes using 0.5 μM (unless otherwise stated) [3H]AEA labelled in the ethanolamine part of the molecule. Blank values are obtained by the use of buffer rather than homogenate. In the experiments comparing effects of Biochanin A upon FAAH and FAAH-2, the same assay is used but with 16 nM [3H]oleoylethanolamide ([3H]OEA) as substrate and with an incubation phase at room temperature. The choice of OEA rather than AEA for FAAH-2 is motivated by the relative rates of hydrolysis: OEA is metabolized four times faster than AEA by FAAH-2, whereas for FAAH the rate of hydrolysis of OEA is about a third of that for AEA. When 0.5 μM [3H]AEA is used as substrate, assay conditions for rat brain and mouse liver are chosen so that

Animal experiment:

Mice[1] ICR mice are used for the behavioural tests measuring spontaneous activity (over a 10 min testing period), rectal temperature, ring immobility (over a 5 min testing period) and nociceptive threshold (tail flick tests). AEA and Biochanin A are dissolved in a vehicle consisting of ethanol, Emulphor-620 and physiological saline in a ratio of 1:1:18 v/v, and administered i.v. to the animals via the tail vein (injection volume 10 μL/g body weight). The degree of antinociception is expressed as percentage of maximum possible effect (%MPE), defined as [(test-control time)/(10-control time)]×100.

IC50: 1.8, 1.4 and 2.4 μM for mouse, rat and human FAAH, respectively

Biochanin A is a natural inhibitor of fatty acid amide hydrolase. Inhibitors of fatty acid amide hydrolase (FAAH), which responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide (AEA), are effective in a number of animal models of pain.

In vitro: Biochanin A was found to be the most promising candidate. Biochanin A inhibited the hydrolysis of 0.5 mM AEA by mouse, rat and human FAAH with IC50 values of 1.8, 1.4 and 2.4 μM respectively. Biochanin A was found to not interact to any extent with CB1 or CB2 receptors, nor with FAAH-2 [1].

In vivo: In anaesthetized mice, both URB597 and biochanin A inhibited the spinal phosphorylation of extracellular signal-regulated kinase caused by the intraplantar injection of formalin. The effects of both URB597 and biochanin A were found to be significantly reduced by the CB1 receptor antagonist/inverse agonist AM251. In addition, in the tetrad test, biochanin A did not increase brain AEA concentrations, but produced a modest potentiation of the effects of 10 mg·kg-1 i.v. AEA [1].

Clinical trial: N/A

Reference:
[1] Thors L,Burston JJ,Alter BJ,McKinney MK,Cravatt BF,Ross RA,Pertwee RG,Gereau RW 4th,Wiley JL,Fowler CJ.  Biochanin A, a naturally occurring inhibitor of fatty acid amide hydrolase. Br J Pharmacol.2010 Jun;160(3):549-60.