mFAAH (IC50 = 1.8 μM); rFAAH (IC50 = 1.4 μM); hFAAH (IC50 = 2.4 μM)
Fatty acid amide hydrolase (FAAH) (Ki = 3.7 μM; IC50 = 4.2 μM) [1]
- Cyclooxygenase-1 (COX-1) (IC50 > 100 μM) [1]
- Cyclooxygenase-2 (COX-2) (IC50 > 100 μM) [1]
- Monoamine oxidase A (MAO-A) (IC50 > 100 μM) [1]
- Monoamine oxidase B (MAO-B) (IC50 > 100 μM) [1]

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].

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Biochanin A showed potent and selective inhibitory activity against FAAH, with a Ki value of 3.7 μM and an IC50 value of 4.2 μM. It did not significantly inhibit COX-1, COX-2, MAO-A, or MAO-B at concentrations up to 100 μM [1]
- In C6 glioma cells, treatment with Biochanin A (10 μM) for 30 minutes significantly increased the intracellular levels of anandamide (AEA) by 2.3-fold compared to the control group, confirming its ability to inhibit FAAH-mediated AEA hydrolysis in cellular environments [1]
- The inhibitory effect of Biochanin A on FAAH was reversible, as washing out the compound from the enzyme reaction mixture restored FAAH activity to near-control levels [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].

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In ICR mice, oral administration of Biochanin A (10, 30, 100 mg/kg) produced dose-dependent analgesic effects in the tail-flick test. At 100 mg/kg, the tail-flick latency was significantly prolonged by 42% compared to the vehicle control group, with the effect peaking at 1 hour post-administration [1]
- Biochanin A (30, 100 mg/kg, p.o.) significantly inhibited carrageenan-induced paw edema in mice, with inhibition rates of 28% and 45% respectively at 3 hours post-carrageenan injection [1]
- Oral administration of Biochanin A (100 mg/kg) to mice increased the levels of AEA in the brain by 1.8-fold and in the spinal cord by 1.6-fold compared to vehicle-treated mice, consistent with its in vitro FAAH inhibitory activity [1]
- The analgesic effect of Biochanin A (100 mg/kg, p.o.) was antagonized by pretreatment with the CB1 receptor antagonist rimonabant (3 mg/kg, i.p.), indicating that the analgesic activity is mediated through the endocannabinoid system [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.

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For FAAH activity assay, FAAH was purified from rat brain membranes. The reaction mixture contained purified FAAH, [3H]-anandamide (substrate), and various concentrations of Biochanin A in assay buffer. The reaction was incubated at 37°C for 15 minutes and terminated by adding ice-cold chloroform/methanol (2:1, v/v). The aqueous phase was separated, and the radioactivity of the hydrolyzed product was measured using liquid scintillation counting. The inhibitory rate was calculated, and Ki/IC50 values were determined by nonlinear regression analysis [1]
- For COX-1/COX-2 activity assays, the enzymes were prepared from sheep seminal vesicles (COX-1) and human recombinant COX-2. The reaction mixture included the enzyme, arachidonic acid (substrate), and Biochanin A (0.1–100 μM). After incubation at 37°C for 10 minutes, the production of prostaglandin E2 (PGE2) was measured using an enzyme immunoassay kit to assess inhibitory activity [1]
- MAO-A/MAO-B activity assays were performed with enzymes from rat liver mitochondria. The reaction mixture contained the enzyme, appropriate substrates (tyramine for MAO-A, β-phenylethylamine for MAO-B), and Biochanin A (0.1–100 μM). Incubation was carried out at 37°C for 20 minutes, and the deaminated products were detected spectrophotometrically to determine inhibition [1]

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.

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C6 glioma cells were cultured in appropriate medium and seeded into 6-well plates at a density of 5×105 cells per well. After overnight incubation, the cells were treated with Biochanin A (1, 10, 30 μM) or vehicle for 30 minutes. The cells were then harvested, and AEA was extracted using chloroform/methanol (2:1, v/v). The extracted AEA was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a stable isotope-labeled internal standard [1]

Metabolism/Metabolites
Biotin A's known metabolites include genistein.

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After oral administration of biotin A (30 mg/kg) to mice, the peak plasma concentration (Cmax) was 1.2 μM, and the time to peak concentration (Tmax) was 1 hour. The plasma half-life (t1/2) was 2.3 hours [1]
-Biotin A can cross the blood-brain barrier, and the brain tissue concentration was 0.8 μM 1 hour after oral administration (30 mg/kg) [1]

Biotin-repellent birds Red-winged Blackbird oral R50 (repellency rate 50%) > 1%
Biotin-toxic birds Red-winged Blackbird oral LD50 (median lethal dose) > 100 mg/kg

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No acute toxicity symptoms (e.g., death, behavioral abnormalities, weight loss) were observed after oral administration of biotin A to mice at doses up to 300 mg/kg[1]
- The plasma protein binding rate of biotin A in mouse plasma was 89%[1]

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

Biochanin A belongs to the 7-hydroxyisoflavone class of compounds. It is formed by introducing a hydroxyl group at the 5' position and a methoxy group at the 4' position of 7-hydroxyisoflavone. It is a phytoestrogen and may have dietary cancer-preventive effects. It has multiple functions, including as a phytoestrogen, a plant metabolite, an EC 3.5.1.99 (fatty acid amide hydrolase) inhibitor, a tyrosine kinase inhibitor, and an antitumor drug. It belongs to both the 7-hydroxyisoflavone and 4'-methoxyisoflavone classes. It is the conjugate acid of biotin A(1-). Biotin A is currently being studied in the clinical trial NCT02174666 (isoflavones for the treatment of postmenopausal osteoporosis). Biotin A has been reported to be found in Dalbergia nigrescens, Euchresta formosana, and other organisms with relevant data.
The phytoestrogen biotin A is an isoflavone derivative isolated from red clover (Trifolium pratense) and possesses anticancer properties. Treatment of MCF-7 human breast cancer cells with biotin A alone leads to the accumulation of CYP1A1 mRNA and increases the activity of CYP1A1-specific 7-ethoxyhalothrin O-deethylase (EROD) in a dose-dependent manner. Biotin A may be a natural ligand that can bind to aryl hydrocarbon receptors and act as an antagonist/agonist of this pathway. (A7920) Genistein A inhibits nuclear factor κB-driven interleukin-6 (IL-6) expression. In addition to its physiological immune function as an acute stress cytokine, persistently high expression of IL-6 can promote chronic inflammatory diseases, senile debility, and tumorigenesis. (A7921) Genistein A reduces the invasive activity of U87MG cells in a dose-dependent manner. (A7922) Genistein A activates peroxisome proliferator-activated receptors (PPARs) PPARα and PPARγ and promotes in vitro differentiation of 3T3-L1 preadipocytes, suggesting that isoflavones, especially genistein A and its parent plant extracts, have potential value as antidiabetic drugs and regulators of lipid metabolism. (A7923)

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Background and Objectives: Fatty acid amide hydrolase (FAAH) is an enzyme responsible for metabolizing the endocannabinoid (CB) receptor ligand arachidonic acid ethanolamine (AEA), and its inhibitors have shown efficacy in various animal pain models. This study aimed to investigate the ability of a series of isoflavones to inhibit FAAH.
Experimental Methods: Key compounds were screened using in vitro FAAH activity and CB receptor affinity assays. In vivo experiments included biochemical assays of formalin in anesthetized mice and a "quadruple" assay for central CB receptor activation.
Main Results: Among the compounds tested, genistein A was considered the most promising. Genistein A inhibited the hydrolysis of 0.5 μM AEA by FAAH in mice, rats, and humans, with IC50 values of 1.8, 1.4, and 2.4 μM, respectively. This compound did not significantly interact with either CB(1) or CB(2) receptors or FAAH-2. In anesthetized mice, both URB597 (30 μg, plantar injection) and genistein A (100 μg, plantar injection) inhibited formalin-induced extracellular signal-regulated kinase (ERK) phosphorylation in the spinal cord. The CB(1) receptor antagonist/inverse agonist AM251 (30 μg, plantar injection) significantly reduced the effects of both compounds. Genistein A (15 mg·kg⁻¹, intravenous injection) did not increase intracerebral AEA concentrations, but in a tetrad assay, it slightly enhanced the effect of 10 mg·kg⁻¹ intravenous AEA. Conclusions and significance: The conclusion is that genistein A, in addition to its other biochemical properties, can inhibit FAAH in vitro and in vivo. [1] This study investigated the effects of a series of isoflavones related to genistein and daidzein on fatty acid amide hydrolase (FAAH). Among the compounds tested, we selected the isoflavone compound genistein A for further investigation. Genistein A is a mixed rat FAAH inhibitor that showed similar low micromolar inhibitory activity against FAAH in rats, mice and recombinant human. In intact RBL2H3 cells, 1 µM genistein A significantly reduced URB597-sensitive tritium label accumulation (by about 75%), which is consistent with the inhibition of FAAH activity in these cells by genistein A. In contrast, genistein A had little effect on the binding of [3H]CP 55940 to CB1 and CB2 receptors. In formalin-induced persistent pain models, behavioral responses (such as withdrawal, licking, and paw biting) to subplantar formalin injection are relatively rapid and occur in two phases: an initial phase within seconds of injection, due to direct stimulation of nociceptors; and a second phase following a brief delay, resulting from central nociceptor sensitization. The second phase of the formalin test is sensitive to both FAAH inhibitors (Lichtman et al., 2004a; Maione et al., 2007; Sit et al., 2007) and gene knockout of the enzyme (Lichtman et al., 2004b). There is ample evidence that pERK expression in the L4-S1 lumbar spinal cord is a potent marker of central nociceptive sensitization activation, as observed after formalin injection into the hind paw (see review by Gao and Ji, 2009). Therefore, exposure to noxious stimuli (rather than harmless stimuli) in the hind paws of anesthetized rats induces pERK expression (Ji et al., 1999), and in formalin-treated mice and rats, its expression pattern is consistent with the time course of the second phase of nociceptive behavior (Ji et al., 1999; Karim et al., 2001). Importantly, PD98059 (a MEK kinase inhibitor that prevents ERK phosphorylation) can block the second phase of nociceptive behavior in both species (Ji et al., 1999; Karim et al., 2001). In spinal cord sections, the CB1 receptor agonist arachidonic acid-2′-chloroacetamide reduces capsaicin-induced pERK expression (Kawasaki et al., 2006). The latter finding suggests that this pathway may respond to indirect CB1 receptor activation induced by FAAH inhibition. We found that in the C57BL/6 mouse strain used (the strain used by Karim et al., 2001), URB597 did indeed inhibit formalin-induced phosphorylation of ERK injected into the ipsilateral (but not contralateral) spinal cord. The effective dose of URB597 was the same range previously observed in other pain models, although these models were rats rather than mice (Jhaveri et al., 2006; Desroches et al., 2008; Sagar et al., 2008). Concomitant administration of AM251 significantly reduced the effect of URB597, indicating that the effect of URB597 is at least partially mediated by the CB1 receptor. The inhibitory effect of biotin A on spinal cord ERK phosphorylation followed the same pattern of AM251-sensitive inhibition observed with URB597, consistent with the local FAAH inhibition in vivo. The subtle difference in the relative potency of biotin A and URB597 (compared to the significant difference in the inhibition of FAAH by URB597 in the low nanomolar concentration range in in vitro experiments; Kathuria et al., 2003) may reflect a decrease in local pH in inflamed tissues (Häbler, 1929), since URB597 inhibits FAAH in intact cells in a pH-sensitive manner (Paylor et al., 2006). While current data suggest that biotin A and URB597 have the same effect on formalin-induced spinal ERK phosphorylation in anesthetized C57BL/6 mice, and both can be antagonized by the CB1 receptor inverse agonist AM251, these data do not rule out the possibility of other pathways involved. Although a detailed analysis is beyond the scope of this study, several potential pathways can be considered, given that AEA can interact with other receptors (e.g., CB2, PPARγ, and TRPV1) and that inhibition of FAAH leads to changes in the levels of other endogenous substrates (e.g., palmitoylethanolamine), which themselves have cellular targets (e.g., PPARα) (see reviews in Ross, 2003; O'Sullivan, 2007). Preliminary data using the PPARγ antagonist GW9662 suggest that this PPAR subtype does not significantly contribute to the effects of biotin A on pERK models (L. Thors, unpublished data), but further investigation is needed to determine the extent to which these other pathways are involved in the effects of biotin A on formaldehyde-induced ERK phosphorylation. When biotin A was administered intravenously to ICR mice at a dose of 15 mg·kg−1, biotin A had no effect on AEA levels in the brain but enhanced the behavioral effects of AEA in the tetrad test. Given that intravenously administered AEA is metabolized by FAAH in both the peripheral and central nervous systems, these data are consistent with the hypothesis that the behavioral effects in ICR mice are due to decreased peripheral FAAH activity. Of course, compared to the relatively mild effects of biotin A observed here, the centrally permeable (albeit nonselective) FAAH inhibitor phenylmethylsulfonyl fluoride produced a very significant AEA-enhancing effect in the tetrad test of ICR mice (Compton and Martin, 1997). Regarding the pharmacokinetics of isoflavones, genistein A is converted to genistein in vivo, followed by a conjugation reaction (Setchell et al., 2001; Moon et al., 2006). Although genistein was detectable in the rat brain after intravenous injection of 30 mg·kg⁻¹ (but undetectable after intravenous injection of 10 mg·kg⁻¹), its cerebral blood distribution ratio was very low (0.04 ± 0.01) (Tsai, 2005). Yueh and Chu (1977) reported that 15 minutes after intravenous injection of 40 mg·kg⁻¹ daidzein in rats, the plasma concentration was approximately 30 µg·mL⁻¹ (equivalent to approximately 120 µM). The concentration of daidzein in the liver, lungs, and kidneys was approximately 0.1 µmol·(mg wet weight)−1, while the concentration in brain tissue was about five times lower (Yueh and Chu, 1977). Given that genistein A is a natural compound, dietary isoflavone intake is clearly a concern. Although the primary objective of this study was not to investigate the effects of normal isoflavone intake on fatty acid amide hydrolase (FAAH) activity, discussion of the health benefits of dietary isoflavones in atherosclerosis, breast cancer, and prostate cancer (although controversial) is unavoidable (Sirtori et al., 2005; Messina and Wood, 2008). Regarding genistein and daidzein, inhibition of fatty acid amide hydrolase (FAAH) is considered possible, at least in populations with high soybean intake (Morton et al., 2002), based on their inhibitory efficacy against rodent enzymes (Thors et al., 2007a,b). Daidzein exhibits lower inhibitory efficacy against recombinant human FAAH (Table 1), but the above assumption still applies to genistein. Biotin A and gentianin are not found in soybean products but are present in red clover extracts, which are available in health food stores due to their alleged benefits for menopausal women (see Booth et al., 2006 for a review). In one study, volunteers took 80 mg of red clover isoflavone extract (double-dose tablets) daily for two weeks, and the peak plasma concentrations of biotin A and gentianin were 48 (range 18–80) and 11 (range <5–35) ng·mL⁻¹, respectively, equivalent to approximately 170 nM and 40 nM (Howes et al., 2002). The peak plasma concentrations of genistein and daidzein were 114 and 63 ng·mL⁻¹, respectively, equivalent to approximately 420 nM and 250 nM (Howes et al., 2002). Therefore, unlike individuals with high soybean intake, it is unlikely that the administration of red clover extract would reach sufficient isoflavone concentrations to inhibit fatty acid amide hydrolase (FAAH). In summary, this study demonstrates that biotin A (a naturally occurring compound with limited central permeability) inhibits FAAH in a mixed manner, without affecting FAAH-2 activity and with a weak effect on CB receptors. In vivo, this compound inhibited a biochemically relevant indicator of peripheral pain sensitization—phosphorylation of spinal ERK in anesthetized mice following intraspinal formalin injection. FAAH inhibitors are currently being developed as potential analgesics (and antidepressants), with leading compounds already in early clinical development stages (see Seierstad and Breitenbucher, 2008 for a review). Although these compounds do not produce “cannabinoid-like” behavior in vivo, other behavioral effects of central FAAH inhibition may be overlooked, and there is evidence that FAAH polymorphism is associated with certain traits, such as drug-seeking behavior (Flanagan et al., 2006). In this regard, both FAAH gene knockout and URB597 administration affect ethanol preference and intake (Basavarajappa et al., 2006; Blednov et al., 2007). FAAH−/− mice also exhibit greater susceptibility to chemically induced seizures compared to wild-type animals (Clement et al., 2003). Given the efficacy of i.pl. AEA and URB597 in various 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), peripherally restricted FAAH inhibitors hold promise as novel analgesics. The data presented in this paper, combined with previously published pharmacokinetic studies, indicate that genistein A primarily functions as a peripheral FAAH inhibitor. This suggests that this compound could serve as a template for designing more effective peripherally restricted compounds. Of course, isoflavones themselves possess a variety of biochemical and pharmacological effects. These properties include effects on estrogen receptors, tyrosine kinases, peroxisome proliferation-activating receptor subtypes, DNA topoisomerase II, NFκB activation, and transforming growth factor β1 signaling (see Kurzer and Xu, 1997; Kim et al., 1998; Banerjee et al., 2008; see also Lam et al., 2004; Shen et al., 2006), thus isoflavones are far from becoming FAAH-selective compounds. However, screening protocols can incorporate some screening measures to reduce adverse reactions (e.g., effects on estrogen receptors), thereby customizing more selective FAAH inhibitors, or designing compounds that can act on this enzyme and other potentially useful targets (e.g., cyclooxygenase-2) [1]. Genistein A is a naturally occurring isoflavone found in plants such as red clover (Trifolium pratense) and chickpea (Cicer arietinum) [1].
- Its mechanism of action involves reversible inhibition of FAAH, leading to increased levels of endocannabinoids (e.g., AEA), which in turn activate CB1 receptors, thereby exerting analgesic and anti-inflammatory effects [1].
- Compared with the synthetic inhibitor URB597 (Ki = 0.5 nM), genistein A has lower inhibitory potency against FAAH, but higher selectivity for FAAH than other tested enzymes [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.)