mFAAH (IC50 = 1.8 μM); rFAAH (IC50 = 1.4 μM); hFAAH (IC50 = 2.4 μM)

Effect of Biochanin A upon FAAH-2 activity [1]
FAAH-2 is a second N-acylethanolamine-hydrolyzing enzyme sharing a ∼20% sequence identity with FAAH, and which is found in primates, but not in rats or mice (Wei et al., 2006). The effect of biochanin A towards the activity of FAAH-2 was investigated using 16 nM [3H]oleoylethanolamine as substrate. The FAAH-transfected membranes were also tested for comparative purposes (Figure 3). As expected, FAAH was inhibited by biochanin A with a pI50 value of 6.21 ± 0.02, corresponding to an IC50 value of 0.62 µM. In these experiments, the contribution to the total activity by the cells per se (i.e. the mock transfectants) was very low. The catalytic activity of the membranes from FAAH-2-transfected cells, however, was also low. Hence, at the higher protein concentration needed (compared to the situation for FAAH), there was a significant hydrolysis of [3H]OEA in the mock-transfected cell membranes. This basal hydrolysis was sensitive to biochanin A and so presumably represents the low level of FAAH native to the cells. The difference between the mock-transfected and the FAAH-transfected cells, used as an estimate of FAAH-2 activity, was not inhibited by biochanin A. Thus, this compound is selective for FAAH versus FAAH-2.

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Interaction of Biochanin A, daidzein, formononetin and genistein with CB receptors [1]
The ability of the four isoflavones to inhibit the binding of the CB receptor agonist ligand [3H]CP 55940 to brain CB1 receptors and recombinant CB2 receptors is shown in Figure 5. Although the compounds could produce significant reductions in the specific binding of [3H]CP 55940 to both receptors, these effects were modest. Thus, the maximum inhibition found was 27 ± 7 and 33 ± 5% for brain CB1 receptors and recombinant CB2 receptors, respectively, produced in both cases with 30 µM biochanin A.

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Biochanin A inhibits hydrolysis of 0.5 μM AEA by mouse, rat, and human FAAH with IC50 values of 1.8, 1.4, and 2.4 μM, respectively. Biochanin A inhibits FAAH at a pIC50 of 6.21±0.02 and an IC50 of 0.62 μM. Biochanin A inhibits URB597-sensitive tritium retention at high nanomolar to low micromolar concentrations. Experiments were conducted with human FAAH and 0.5 μM [3H]AEA, and the assay conditions improved utilization. Biochanin A, Genistein, Formononetin, and Daidzein inhibited activity in the low micromolar range with IC50 values of 6.0, 8.4, 12, and 30 μM, respectively [1].

Behavioural effects of i.v. Biochanin A alone or in combination with AEA [1]
The compounds were given either together with different doses of AEA. As expected, AEA produced dose-dependent effects upon all four components of the tetrad test (locomotion, nociception, body temperature and ring immobility). Biochanin A (15 mg·kg−1 i.v.) was without effects on its own, but significantly potentiated the effects of AEA (10 mg·kg−1 i.v.) (Figure 7). The AEA levels in the brains of the Biochanin A-treated animals were not affected (Figure 8). Levels of other N-acylethanolamines were not measured. Daidzein (10 mg·kg−1 i.p.) was also tested. In this case, however, there was a higher level of behavioural response to AEA alone, and no significant potentiation could be seen, although the combination of AEA + daidzein produced results similar to those seen with biochanin + AEA (data not shown). The experiments were undertaken on different occasions (chronologically, daidzein was the first, and the results with this compound led to the choice of a different route of administration for biochanin A).

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Effects of i.pl. URB597 and Biochanin A upon formalin-induced phosphorylation of ERK in the spinal cord [1]
The i.pl. administration of formalin into the hindpaw of pentobarbital-anaesthetized mice produced a rapid phosphorylation of ERK in L4-S1 lumbar spinal cord ipsi-, but not contralateral to the injection (Figure 6A–C), consistent with data in awake animals (Karim et al., 2001). This phosphorylation was reduced by the i.pl. administration of 100, but not 30 or 10, µg of the selective FAAH inhibitor URB597. The effect of 100 µg URB597 was significantly inhibited by concomitant administration of the CB1 receptor antagonist/inverse agonist AM251 (30 µg). (Figure 6D). Biochanin A was tested at doses of 30, 100 and 300 µg. The highest dose also reduced formalin-induced ERK phosphorylation in a manner antagonized by AM251 (Figure 6E). Thus, biochanin A behaved like URB597 after local administration to the paw.

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Test doses for biochanin A were 30, 100, and 300 μg. In a way that AM251 inhibited, the maximum dosage also decreased formalin-induced ERK phosphorylation. Therefore, upon topical application to the paw, Biochanin A exhibits behavior similar to URB597. Both URB597 (30 μg i.pl.) and Biochanin A (100 μg i.pl.) prevent the spinal cord phosphorylation of extracellular signal-regulated kinase, which is generated by injecting formalin into the plantar region of mice under anesthesia. AM251, a 30 μg i.pl. CB1 receptor antagonist/inverse agonist, considerably lessened the effects of both substances. While biochanin A (15 mg/k iv) does not raise brain AEA concentrations, it does slightly improve the tetrad test effects of 10 mg/kg iv AEA. While AEA (10 mg/kg iv) has little impact on its own, biochanin A (15 mg/kg iv) greatly increases its effect [1].

FAAH activity measurements [1]
For experiments with FAAH, rat liver homogenates, mouse brain homogenates and membranes from COS7 cells transfected with the human enzyme were used. Frozen (−80°C) livers from adult C57BL/6 mice and frozen brains (minus cerebella) from adult Wistar or Sprague-Dawley rats were thawed and homogenized in 20 mM HEPES, 1 mM MgCl2, pH 7. The homogenates were centrifuged at ∼35 000×g for 20 min at 4°C. After resuspension in buffer followed by recentrifugation and a second resuspension in buffer, the pellets were incubated at 37°C for 15 min. This incubation was undertaken in order to hydrolyse all endogenous FAAH substrates. The homogenates were 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 were then frozen at −80°C in aliquots until used for assay. FAAH was assayed in the homogenates and in the COS7 cell membranes by the method of Boldrup et al. (2004) using 0.5 µM (unless otherwise stated) [3H]AEA labelled in the ethanolamine part of the molecule. Blank values were 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 was 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 was 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 (Wei et al., 2006). When 0.5 µM [3H]AEA was used as substrate, assay conditions for rat brain and mouse liver were chosen so that <10% of added substrate was metabolized. For the human FAAH samples, <5% of the [3H]AEA was metabolized in all cases. For 16 nM [3H]OEA, a limited supply of an expensive ligand meant that optimization was not possible, and the amount of substrate utilized was higher (34 ± 1 and 0.5 ± 0.1% for FAAH and its corresponding mock-transfected, respectively; 40 ± 2 and 21 ± 0.4 for FAAH-2 and its corresponding mock-transfected respectively). However, when we ran experiments with human FAAH and 0.5 µM [3H]AEA with assay conditions giving these higher utilization rates, the activity was still inhibited by Biochanin A, genistein, formononetin and daidzein in the low micromolar range (IC50 values of 6.0, 8.4, 12 and 30 µM, respectively; data not shown).

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Formalin-induced phosphorylation of extracellular signal-regulated kinase (ERK) The method was based upon that of Karim et al. (2001). Male adult C57BL/6 mice (body weight ∼22 g) were anaesthetized with an i.p. injection of 0.075 mL av 100 mg·mL−1 pentobarbital. Upon anaesthesia, the animals were injected with test compounds (URB597, Biochanin A ± AM251) or vehicle (3% Tween-80 in physiological saline) into the right rear paw (injection volume 20 µL). This vehicle was chosen because it has been used by others investigating the effects of i.pl. URB597 in pain models (Jhaveri et al., 2006;Sagar et al., 2008), thereby allowing comparison of active doses. Ten minutes later, the animals were injected with formalin (10 µL of a 3% solution dissolved in saline) into the same paw. Five minutes (unless otherwise stated) after the formalin injection, the mice were perfused transcardially with warm saline (37°C, 0.9% NaCl), followed by 300 mL of ice-cold 4% paraformaldehyde solution. A 5 min time was used because the pERK response to formalin was fully developed at this time-point (see Results). Spinal cords were dissected and placed in Falcon tubes containing ice-cold 4% paraformaldehyde solution. After 4 h at 4°C, the samples were transferred to Falcon tubes containing 30% sucrose in 0.1 M phosphate buffer and kept at 4°C, the cryoprotection solution being exchanged until the spinal cord samples were saturated. The lumbar enlargement was localized and dissected out, and a nick was made on the contralateral side to allow identification under the microscope. The sample was placed in a cryomold and affixed on dry ice using OCT compound. Coronal sections (30 µM) were cut using a freezing sliding cryostat, and the sections were transferred to 24-well culture plates containing 0.1 M phosphate buffer. After washing with phosphate buffer (twice) and PBS (once) at room temperature, the sections were incubated for 30 min at room temperature with peroxidase suppression solution (10% MeOH, 0.3% H2O2 in 0.1 M PBS). After three washes with PBS at room temperature, 1% normal goat serum in 0.1 M PBS contaning 0.02% (v/v, final concentration) Triton X-100 (1% NGST) was added. The primary pERK R6 polyclonal antibody (1:500, in NGST) was added, and the sections were incubated for 72 h at 4°C before being washed three times at RT with 1% NGST. The secondary antibody (biotinylated anti-rabbit IgG, BA-1000, diluted 1:100 in 1% NGST) was incubated with the slices for 2 h at room temperature, the plates being covered with aluminium foil. The samples were washed three times with 1% NGST prior to addition of ExtrAvidin peroxidise (1:1000) in 1% NGST and incubation for 1 h at room temperature. After two washes with PBS and one wash with phosphate buffer at room temperature, the samples were stained using the DAB kit. The sections were mounted on slides, dehydrated, dried overnight and viewed under the microscope. Four sections from each animal were analysed under the microscope by an investigator who was blind to the treatment given to the animal in question, and the mean score from the four sections was calculated.

URB597-sensitive accumulation of tritium in RBL2H3 cell membranes following incubation of cells in suspension with [3H]AEA [1]
RBL2H3 cells were cultured in minimum essential medium with Earl's salts, 15% fetal bovine serum and 100 U·mL−1 penicillin + 100 µg·mL−1 streptomycin (‘medium’). After centrifugation and resuspension in medium in Eppendorf tubes (2 × 105 cells per tube; 5 × 105 cells·mL−1), the cells were pre-incubated with either the test compounds or URB597 for 10 min at 37°C. [3H]AEA (assay concentration 100 nM; substrate labelled in the arachidonoyl part of the molecule) in medium was added and the cells were incubated for a further 10 min (final assay volume 200 µL). After incubation, the cells were sedimented using a microcentrifuge (1 min, 1000×g), then washed twice with ice cold medium, and the tritium retained by the cell membranes was determined by rapid filtration and washing with deionized water as described by Thors et al. (2008). The URB597-sensitive accumulation of tritium was defined as the radioactivity seen for the test compounds or vehicle control minus the corresponding radioactivity for the cells treated with 100 nM URB597.

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Radioligand binding experiments [1]
The ability of the isoflavones to interact with CB1 and CB2 receptors was assessed using mouse brain membranes as a source of the former, and membranes from CHO cells transfected with the human CB2 receptor as a source of the latter. A standard filtration assay was used (for details, see Ross et al., 1999; Thomas et al., 2005), and the concentration of radioligand ([3H]CP 55940) was 0.7 nM. Non-specific binding was determined in the presence of 1 µM of unlabelled CP 55940.

Behavioural effects of Biochanin A [1]
ICR mice (Harlan Laboratories, Indianapolis, IN, USA) were 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) (for details, see Pertwee, 1972; Martin et al., 1991; Burston et al., 2008). AEA and Biochanin A were 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−1 body weight). The degree of antinociception is expressed as percentage of maximum possible effect (%MPE), defined as [(test – control time)/(10 – control time)]× 100.

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Quantification of AEA levels in the brain [1]
The mice above treated with vehicle rather than AEA were killed by decapitation 1 h after the injection of the isoflavones. Brains were collected and immersed in liquid nitrogen to ensure rapid cooling. Following extraction of lipids with methanol/chloroform (Hardison et al., 2006), AEA levels were determined by liquid chromatography/mass spectrometry analysis (Kingsley and Marnett, 2003).

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10 mg·kg-1 i.v. AEA.

Mice

Metabolism / Metabolites
Biochanin a has known human metabolites that include Genistein.

Biochamin Repellency AVIAN Red-winged blackbird Oral R50 (Repellency 50) > 1 %
Biochamin Toxicity AVIAN Red-winged blackbird Oral LD50 (Lethal Dose 50) > 100 mg/kg

[1]. Biochanin A, a naturally occurring inhibitor of fatty acid amide hydrolase. Br J Pharmacol. 2010 Jun;160(3):549-60.

Biochanin A is a member of the class of 7-hydroxyisoflavones that is 7-hydroxyisoflavone which is substituted by an additional hydroxy group at position 5 and a methoxy group at position 4'. A phytoestrogen, it has putative benefits in dietary cancer prophylaxis. It has a role as a phytoestrogen, a plant metabolite, an EC 3.5.1.99 (fatty acid amide hydrolase) inhibitor, a tyrosine kinase inhibitor and an antineoplastic agent. It is a member of 7-hydroxyisoflavones and a member of 4'-methoxyisoflavones. It is a conjugate acid of a biochanin A(1-).
Biochanin A is under investigation in clinical trial NCT02174666 (Isoflavone Treatment for Postmenopausal Osteopenia.).
biochanin A has been reported in Dalbergia nigrescens, Euchresta formosana, and other organisms with data available.
The phytoestrogen biochanin A is an isoflavone derivative isolated from red clover Trifolium pratense with anticarcinogenic properties. Treating MCF-7 human breast carcinoma cells with biochanin A alone caused the accumulation of CYP1A1 mRNA and an increase in CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity in a dose dependent manner. Biochanin A may be a natural ligand to bind on aryl hydrocarbon receptor acting as an antagonist/agonist of the pathway. (A7920). Biochanin A suppress nuclear factor-kappaB-driven interleukin-6 (IL6) expression. In addition to its physiologic immune function as an acute stress cytokine, sustained elevated expression levels of IL6 promote chronic inflammatory disorders, aging frailty, and tumorigenesis. (A7921). Biochanin A induces a decrease in invasive activity of U87MG cells in a dose-related manner. (A7922). Biochanin A activates peroxisome proliferator-activated receptors (PPAR) PPARalpha, PPARgamma, and adipocyte differentiation in vitro of 3T3-L1 preadipocytes, suggesting potential value of isoflavones, especially biochanin A and their parent botanicals, as antidiabetic agents and for use in regulating lipid metabolism. (A7923).

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Background and purpose: Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide (AEA), are effective in a number of animal models of pain. Here, we investigated a series of isoflavones with respect to their abilities to inhibit FAAH.
Experimental approach: In vitro assays of FAAH activity and affinity for CB receptors were used to characterize key compounds. In vivo assays used were biochemical responses to formalin in anaesthetized mice and the 'tetrad' test for central CB receptor activation.
Key results: Of the compounds tested, biochanin A was adjudged to be the most promising. Biochanin A inhibited the hydrolysis of 0.5 microM AEA by mouse, rat and human FAAH with IC(50) values of 1.8, 1.4 and 2.4 microM respectively. The compound did not interact to any major extent with CB(1) or CB(2) receptors, nor with FAAH-2. In anaesthetized mice, URB597 (30 microg i.pl.) and biochanin A (100 microg i.pl.) both inhibited the spinal phosphorylation of extracellular signal-regulated kinase produced by the intraplantar injection of formalin. The effects of both compounds were significantly reduced by the CB(1) receptor antagonist/inverse agonist AM251 (30 microg i.pl.). Biochanin A (15 mg.kg(-1) i.v.) did not increase brain AEA concentrations, but produced a modest potentiation of the effects of 10 mg.kg(-1) i.v. AEA in the tetrad test.
Conclusions and implications: It is concluded that biochanin A, in addition to its other biochemical properties, inhibits FAAH both in vitro and peripherally in vivo.[1]

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In the present study, the ability of a series of isoflavones related to genistein and daidzein has been investigated with respect to their effects upon FAAH. Of the compounds tested, the isoflavone biochanin A was chosen for further study. Biochanin A was a mixed-type inhibitor of the rat FAAH, and showed similar low micromolar potencies towards rat, mouse and recombinant human FAAH. In intact RBL2H3 cells, 1 µM biochanin A produced a large (∼75%) decrease in the URB597-sensitive accumulation of tritium label following incubation of the cells with [3H]AEA (labelled in the arachidonoyl portion of the molecule), consistent with inhibition of FAAH in these cells. In contrast, biochanin A had modest effects upon the binding of [3H]CP 55940 to CB1 and CB2 receptors.

In the formalin model of persistent pain, the behavioural responses (such as flinching, licking and biting of the paw) to the i.pl. injection of formalin are relatively rapid and follow two phases: an initial phase within seconds of the injection due to direct stimulation of nociceptors followed, after a short lag, by a second phase, which is the result of a central nociceptive sensitization. The second phase of the formalin test is sensitive to FAAH inhibitors (Lichtman et al., 2004a; Maione et al., 2007; Sit et al., 2007) and to genetic ablation of the enzyme (Lichtman et al., 2004b). There is good evidence that the expression of pERK in L4-S1 lumbar spinal cord is a useful marker of the activation of central nociceptive sensitization, such as is seen after the administration of formalin to the hindpaw (review, see Gao and Ji, 2009). Thus, pERK expression is induced by noxious, but not by innocuous, stimuli to the hindpaws of anaesthetized rats (Ji et al., 1999), and in formalin-treated mice and rats, the expression pattern follows the same time-course as the second phase of nocifensive behaviour (Ji et al., 1999; Karim et al., 2001). Importantly, the second phase of nocifensive behaviour in both species is blocked by PD98059, a MEK kinase inhibitor that prevents the phosphorylation of ERK (Ji et al., 1999; Karim et al., 2001). In spinal cord slices, the CB1 receptor agonist arachidonoyl-2′-chlorethylamide reduces the pERK expression produced by capsaicin (Kawasaki et al., 2006). The latter finding would suggest that this pathway may be responsive to an indirect activation of CB1 receptors secondary to FAAH inhibition. We found that the formalin-induced phosphorylation of spinal ERK ipsi-, but not contralaterally to the injection, was indeed inhibited by URB597 in the mouse strain used (C57BL/6, the strain used by Karim et al., 2001). The effective dose of URB597 is in the same range as found previously to produce effects in other pain models, albeit in the rat rather than the mouse (Jhaveri et al., 2006; Desroches et al., 2008; Sagar et al., 2008). AM251 given concomitantly produced a large reduction of the effect of URB597, indicating that the response to URB597 is mediated at least in part by CB1 receptors. Biochanin A produced the same pattern of AM251-sensitive inhibition of spinal ERK phosphorylation as seen with URB597, consistent with a local FAAH inhibition in vivo. The small difference in the relative potencies of biochanin A and URB597 (as compared with the large difference in vitro, where URB597 inhibits FAAH in the low nanomolar range; Kathuria et al., 2003) may reflect the reduced local pH of the inflamed tissue (Häbler, 1929), because URB597 inhibits FAAH in intact cells in a pH-sensitive manner (Paylor et al., 2006).

While the present data show that in anaesthetized C57BL/6 mice, biochanin A and URB597 produced the same effects upon spinal cord ERK phosphorylation in response to formalin, and that both are antagonized by the CB1 receptor inverse agonist AM251, the data do not rule out the involvement of other pathways. Although a detailed treatment is outside the scope of the present investigation, several potential pathways can be considered, given both the ability of AEA to interact with other receptors (such as CB2, PPARγ and TRPV1), and that the inhibition of FAAH will produce changes in the levels of other endogenous substrates such as palmitoylethanolamide, which have their own cellular targets, such as PPARα (for reviews, see Ross, 2003; O'Sullivan, 2007). Preliminary data with the PPARγ antagonist GW9662 suggest that this PPAR isoform does not contribute to any obvious extent in the effects of biochanin A in the pERK model (L. Thors, unpubl. data), but further studies are needed to determine the extent to which these other pathways can contribute to the effects of biochanin A on ERK phosphorylation in response to formalin.

When given i.v. to ICR mice at a dose of 15 mg·kg−1, biochanin A was without effect on brain levels of AEA, but produced a potentiation of the behavioural effects of AEA in the tetrad test. Given that i.v. administered AEA will be subject to both peripheral and central metabolism by FAAH, the data are consistent with the hypothesis that the behavioural effects in the ICR mice are due to a reduced peripheral FAAH activity. Certainly, the centrally permeable (albeit non-selective) FAAH inhibitor phenylmethylsulphonyl fluoride produces a very robust potentiation of AEA in the tetrad test in ICR mice (Compton and Martin, 1997), compared to the more modest effects of biochanin A seen here. With respect to pharmacokinetic studies of isoflavones, biochanin A is converted to genistein followed by conjugation in vivo (Setchell et al., 2001; Moon et al., 2006), and although genistein can be detected in the rat brain after administration of 30 mg·kg−1 i.v. (but not after 10 mg·kg−1 i.v.), the brain-to-blood-distribution ratio is very low (0.04 ± 0.01) (Tsai, 2005). Yueh and Chu (1977) reported that 15 min after i.v. administration of 40 mg·kg−1 of daidzein to rats, plasma concentrations were ∼30 µg·mL−1 (corresponding to ∼120 µM). The liver, lung and kidney concentrations of daidzein were ∼0.1 µmol·(mg wet weight)−1, while the brain concentration was about fivefold lower (Yueh and Chu, 1977).

Given that biochanin A is a naturally occurring compound, an obvious issue is dietary isoflavone consumption. Although the study was not primarily aimed at considering the effects of normal consumption of isoflavones upon FAAH activity, discussion of this is unavoidable given the suggested (and questioned) health benefits of dietary isoflavones in atherosclerosis, and breast and prostate cancer (Sirtori et al., 2005; Messina and Wood, 2008). In the case of genistein and daidzein, inhibition of FAAH was deemed possible, at least in individuals with a high soy intake (Morton et al., 2002), on the basis of the potencies towards the rodent enzyme (Thors et al., 2007a,b;). Daidzein is less potent towards the recombinant human FAAH (Table 1), but the suggestion still holds for genistein. Biochanin A and formononetin are not present in soy products, but are found in red clover extracts, which are available in health food stores for their purported effects in menopause (review see Booth et al., 2006). In a study where volunteers were given 80 mg of red clover isoflavone extracts (a double-strength tablet) per day for 2 weeks, maximum plasma concentrations of biochanin A and formononetin were 48 (range 18–80) and 11 (range <5–35) ng·mL−1, respectively, corresponding to ∼170 and ∼40 nM, respectively (Howes et al., 2002). The peak plasma genistein and daidzein concentrations were 114 and 63 ng·mL−1, respectively, corresponding to ∼420 and ∼250 nM, respectively (Howes et al., 2002). Thus, in contrast to the situation for individuals with a high soy intake, it is rather unlikely that sufficiently high isoflavone concentrations are achieved for inhibition of FAAH to occur after ingestion of red clover extracts.

In conclusion, the present study has demonstrated that biochanin A, a naturally occuring compound with limited central penetration, inhibits FAAH in a mixed-type manner, does not affect FAAH-2 activity and has only modest effects upon CB receptors. In vivo, the compound can inhibit a biochemical correlate of peripheral pain sensitization, spinal cord ERK phosphorylation following i.pl. formalin treatment to anaesthetized mice. FAAH inhibitors are currently under drug development as potential analgesic (and antidepressant) agents, and the leading compounds are in early clinical development (review, see Seierstad and Breitenbucher, 2008). Although these compounds do not produce ‘cannabinoid-like’ behaviours in vivo, other behavioural effects of central FAAH inhibition may be overlooked, and there is evidence that polymorphisms of FAAH are associated with certain traits, such as drug-seeking behaviour (Flanagan et al., 2006). In this respect, both genetic ablation of FAAH and the administration of URB597 affect ethanol preference and consumption (Basavarajappa et al., 2006; Blednov et al., 2007). FAAH−/− mice also show an increased susceptibililty to chemically induced seizures compared to wild-type animals (Clement et al., 2003). Given that i.pl. administration of AEA and URB597 produced effects in several pain models (Calignano et al., 2001; Sokal et al., 2003; Guindon et al., 2006; Jhaveri et al., 2006; Desroches et al., 2008; Khasabova et al., 2008; Sagar et al., 2008), a case can be made for the identification of peripherally restricted FAAH inhibitors as novel analgesic agents. The data presented here, together with previously published pharmacokinetic studies, suggest that biochanin A acts primarily as a peripheral inhibitor of FAAH. This raises the possibility that the compound may be useful as a template for the design of more potent, peripherally restricted compounds. Of course, the isoflavones are a class of compounds with a variety of biochemical and pharmacological effects. Such properties include actions upon oestrogen receptors, tyrosine kinase, peroxisome proliferator-activated receptor isoforms, DNA topoisomerase II, NFκB activation and transforming growth factor β1 signalling, among others (reviews, see Kurzer and Xu, 1997; Kim et al., 1998; Banerjee et al., 2008; see also Lam et al., 2004; Shen et al., 2006), and thus the isoflavones are certainly a long way from being FAAH-selective compounds. However, a screening programme could incorporate screens to reduce unwanted actions (such as effects upon oestrogen receptors) to tailor more selective FAAH inhibitors, or to design compounds with actions upon this enzyme and other potentially useful targets, such as, for example, cyclooxygenase-2 [1].

C16H12O5

284.26

284.068

C, 67.60; H, 4.26; O, 28.14

491-80-5

5280373

White to light yellow solid powder

1.4±0.1 g/cm3

518.6±50.0 °C at 760 mmHg

210-213 °C(lit.)

198.3±23.6 °C

0.0±1.4 mmHg at 25°C

1.669

3.14

2

5

2

21

424

0

COC1=CC=C(C=C1)C2=COC3=CC(=CC(=C3C2=O)O)O

WUADCCWRTIWANL-UHFFFAOYSA-N

InChI=1S/C16H12O5/c1-20-11-4-2-9(3-5-11)12-8-21-14-7-10(17)6-13(18)15(14)16(12)19/h2-8,17-18H,1H3

5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.79 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.79 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)