Title page Regulation of Inflammatory Pain by Inhibition of Fatty Acid Amide Hydrolase (FAAH)

Abstract Although cannabinoids are efficacious in laboratory animal models of inflammatory pain, their established cannabimimetic actions diminish enthusiasm for their therapeutic development. Conversely, fatty acid amide hydrolase (FAAH), the chief catabolic enzyme regulating the endogenous cannabinoid N-arachidonoylethanolamine (anandamide), has emerged as an attractive target to treat pain and other conditions. Here, we tested WIN55,212-2, a cannabinoid receptor agonist, as well as genetic deletion or pharmacological inhibition of FAAH in the lipopolysaccharide (LPS) mouse model of inflammatory pain. WIN55,212 significantly reduced edema and hotplate hyperalgesia caused by LPS infusion into the hind paws, though the mice also displayed analgesia and other CNS effects. FAAH (-/-) mice exhibited reduced paw edema and hyperalgesia in this model, without apparent cannabimimetic effects. Transgenic mice expressing FAAH exclusively on neurons continued to display the anti-edematous, but not the anti-hyperalgesic, phenotype. The CB 2 receptor antagonist, SR144528, blocked this non-neuronal, anti-inflammatory phenotype, and the CB 1 receptor antagonist, rimonabant, blocked the anti-hyperalgesic phenotype. The FAAH inhibitor, URB597 attenuated the development of LPS-induced paw edema and reversed LPS-induced hyperalgesia through respective CB 2 and CB 1 receptor mechanisms of action. However, the TRPV1 receptor antagonist, capsazepine, did not affect either the anti-hyperalgesic or antiedematous effects of URB597. Finally, URB597 attenuated levels of the pro-inflammatory cytokines IL-1β and TNF-α in LPS-treated paws. These findings demonstrate that simultaneous elevations in non-neuronal and neuronal endocannabinoid signaling are possible through inhibition of a single enzymatic target, thereby offering a potentially powerful strategy to treat chronic inflammatory pain syndromes that operate at multiple levels of anatomical integration

<span style="font-size: 20.5pt;mso-bidi-font-size:14.5pt;font-family:"Times New Roman","serif"">D₂-dopamine receptor and α₂,adrenoreceptor-mediated analgesic response of quercetin </span>

1400-1404<span style="font-size: 14.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">Quercetin, a biof1avonoid (100-300 <span style="font-size:14.0pt;mso-bidi-font-size: 8.0pt;font-family:" arial","sans-serif";mso-bidi-font-style:italic"="">mg/kg) produced dose dependent increase in tail-flick latency, the analgesic effect being sensitive to reversal by naloxone <span style="font-size:14.0pt; mso-bidi-font-size:8.0pt;font-family:" arial","sans-serif""="">(1 mg/kg). Prior treatment with haloperidol <span style="font-size:14.0pt; mso-bidi-font-size:8.0pt;font-family:" arial","sans-serif""="">(1 mg/kg),D1/D2 receptor antagonist <span style="font-size: 14.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">haloperidol, sulpiride (50 mg/kg), a selective D2 receptor antagonist, yohimbine (5 <span style="font-size:14.0pt;mso-bidi-font-size:8.0pt;font-family: " arial","sans-serif";mso-bidi-font-style:italic"="">mg/kg), a <span style="font-size:20.5pt;mso-bidi-font-size:14.5pt;font-family: " times="" new="" roman","serif""="">α2<span style="font-size:14.0pt; mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">-adrenoreceptor  antagonist but not by SCH 23390 a, selective D1<span style="font-size:11.5pt;mso-bidi-font-size: 5.5pt;font-family:" times="" new="" roman","serif""=""> <span style="font-size: 14.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">receptor antagonist blocked this response. Apomorphine <span style="font-size: 14.0pt;mso-bidi-font-size:8.0pt;font-family:" arial","sans-serif""="">(1 mg/kg) a mixed <span style="font-size:12.0pt;mso-bidi-font-size:6.0pt;font-family: " times="" new="" roman","serif""="">D1/D2 <span style="font-size:14.0pt;mso-bidi-font-size:8.0pt;line-height:115%; font-family:" times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";="" mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">dopamine receptor agonist, and quinpirole (0.5 <span style="font-size:14.0pt; mso-bidi-font-size:8.0pt;line-height:115%;font-family:" arial","sans-serif";="" mso-fareast-font-family:"times="" new="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"="">mg/kg), <span style="font-size:14.0pt; mso-bidi-font-size:8.0pt;line-height:115%;font-family:" times="" new="" roman","serif";="" mso-fareast-font-family:"times="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"="">a selective D<span style="font-size: 12.0pt;mso-bidi-font-size:6.0pt;line-height:115%;font-family:" times="" new="" roman","serif";="" mso-fareast-font-family:"times="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"="">2<span style="font-size:12.0pt; mso-bidi-font-size:6.0pt;line-height:115%;font-family:" times="" new="" roman","serif";="" mso-fareast-font-family:"times="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"=""> <span style="font-size:14.0pt; mso-bidi-font-size:8.0pt;line-height:115%;font-family:" times="" new="" roman","serif";="" mso-fareast-font-family:"times="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"="">receptor agonist also produced antinociception, that was reversed by haloperidol <span style="font-size:13.5pt; mso-bidi-font-size:7.5pt;line-height:115%;font-family:" arial","sans-serif";="" mso-fareast-font-family:"times="" new="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"="">(1 <span style="font-size:14.0pt; mso-bidi-font-size:8.0pt;line-height:115%;font-family:" arial","sans-serif";="" mso-fareast-font-family:"times="" new="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa;mso-bidi-font-style:italic"="">mg/kg), <span style="font-size:14.0pt;mso-bidi-font-size:8.0pt;line-height:115%;font-family: " times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";mso-ansi-language:="" en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">sulpiride (50 <span style="font-size:14.0pt;mso-bidi-font-size:8.0pt;line-height:115%;font-family: " arial","sans-serif";mso-fareast-font-family:"times="" new="" roman";mso-ansi-language:="" en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa;mso-bidi-font-style:="" italic"="">mg/kg), <span style="font-size:14.0pt;mso-bidi-font-size: 8.0pt;line-height:115%;font-family:" times="" new="" roman","serif";mso-fareast-font-family:="" "times="" roman";mso-ansi-language:en-us;mso-fareast-language:en-us;="" mso-bidi-language:ar-sa"="">but not by yohimbine (5 <span style="font-size: 14.0pt;mso-bidi-font-size:8.0pt;line-height:115%;font-family:" arial","sans-serif";="" mso-fareast-font-family:"times="" new="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa;mso-bidi-font-style:italic"="">mg/kg). <span style="font-size:14.0pt;mso-bidi-font-size:8.0pt;line-height:115%;font-family: " times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";mso-ansi-language:="" en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">The antinociceptive action of quercetin (200 <span style="font-size:14.0pt;mso-bidi-font-size: 8.0pt;line-height:115%;font-family:" arial","sans-serif";mso-fareast-font-family:="" "times="" new="" roman";mso-ansi-language:en-us;mso-fareast-language:en-us;="" mso-bidi-language:ar-sa;mso-bidi-font-style:italic"="">mg/kg) <span style="font-size:14.0pt;mso-bidi-font-size:8.0pt;line-height:115%;font-family: " times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";mso-ansi-language:="" en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">was potentiated by D2<span style="font-size:11.5pt;mso-bidi-font-size:5.5pt;line-height:115%;font-family: " arial","sans-serif";mso-fareast-font-family:"times="" new="" roman";mso-ansi-language:="" en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"=""> <span style="font-size:14.0pt;mso-bidi-font-size:8.0pt;line-height:115%;font-family: " times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";mso-ansi-language:="" en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">agonist quinpirole (0.2 mg/kg). Dopamine D1<span style="font-size:11.5pt; mso-bidi-font-size:5.5pt;line-height:115%;font-family:" arial","sans-serif";="" mso-fareast-font-family:"times="" new="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"=""> <span style="font-size:14.0pt; mso-bidi-font-size:8.0pt;line-height:115%;font-family:" times="" new="" roman","serif";="" mso-fareast-font-family:"times="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"="">receptor agonist SKF38393 (10 and 15 <span style="font-size:14.0pt;mso-bidi-font-size:8.0pt;line-height:115%;font-family: " arial","sans-serif";mso-fareast-font-family:"times="" new="" roman";mso-ansi-language:="" en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa;mso-bidi-font-style:="" italic"="">mg/kg) <span style="font-size:14.0pt;mso-bidi-font-size: 8.0pt;line-height:115%;font-family:" times="" new="" roman","serif";mso-fareast-font-family:="" "times="" roman";mso-ansi-language:en-us;mso-fareast-language:en-us;="" mso-bidi-language:ar-sa"="">failed to alter the antinociceptive effect of quercetin (200 mg/kg). Quercetin (200 mg/kg) reversed reserpine (2 mg/kg-4 hr) induced hyperalgesia, which was reversed by sulpiride but not by yohimbine. Thus, a role of dopamine D<span style="font-size:11.5pt;mso-bidi-font-size: 5.5pt;line-height:115%;font-family:" arial","sans-serif";mso-fareast-font-family:="" "times="" new="" roman";mso-ansi-language:en-us;mso-fareast-language:en-us;="" mso-bidi-language:ar-sa"="">2 <span style="font-size:14.0pt; mso-bidi-font-size:8.0pt;line-height:115%;font-family:" times="" new="" roman","serif";="" mso-fareast-font-family:"times="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"="">and <span style="font-size:20.5pt; mso-bidi-font-size:14.5pt;line-height:115%;font-family:" times="" new="" roman","serif";="" mso-fareast-font-family:"times="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"=""> α2 <span style="font-size:12.0pt;mso-bidi-font-size:6.0pt;line-height:115%;font-family: HiddenHorzOCR;mso-hansi-font-family:" times="" new="" roman";mso-bidi-font-family:="" hiddenhorzocr;mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:="" ar-sa"="">adrenoreceptors <span style="font-size:14.0pt;mso-bidi-font-size: 8.0pt;line-height:115%;font-family:" times="" new="" roman","serif";mso-fareast-font-family:="" "times="" roman";mso-ansi-language:en-us;mso-fareast-language:en-us;="" mso-bidi-language:ar-sa"="">is postulated in the antinoeiceptive action of quercetin.</span

Effect of cyclooxygenase-2 (COX-2) inhibitors in various animal models (bicuculline, picrotoxin, maximal electroshock-induced convulsions) of epilepsy with possible mechanism of action

286-291Enzyme cyclooxygenase (COX) is reported to play a significant role in neurodegeneration and may play a significant role in the pathogenesis of epilepsy. Bicuculline (4 mg/kg; ip), picrotoxin (8 mg/kg; ip) and electroshock (60 mA for 0.2 sec) significantly induced convulsions in male Laka mice. COX-inhibitors viz. nimesulide (2.5 mg/kg; ip) and rofecoxib (2 mg/kg, ip) administered 45 minutes prior to an epileptic challenge prolonged mean onset time of convulsions, decreased duration of clonus and decreased % mortality rate against bicuculline- and picrotoxin-induced convulsions in mice. COX-2 inhibitors were ineffective towards maximal electroshock-induced convulsions. Nimesulide (1 mg/kg) and rofecoxib (1 mg/kg) also enhanced the effect of subprotective dose of muscimol against picrotoxin-induced convulsions. The result of the present study strongly suggests for a possible role of cyclooxygenase isoenzymes particularly, COX-2 in the pathophysiology of epilepsy and its GABAergic modulation

FK506 as effective adjunct to L-dopa in reserpine-induced catalepsy in rats

1264-1268Reserpine-induced catalepsy is a widely accepted animal model of Parkinson's disease. In the present study reserpine (2.5mg/kg, ip) 20 hr and alpha-mehyl- para-tyrosine (AMPT; 200 mg/kg, ip), one hour before the experiment induced significant catalepsy in rats as assessed by bar test. There was a significant increase in the time spent on the bar in bar test as compared to the control untreated rats. L-dopa (100 mg/kg, ip) and carbidopa (10 mg/kg, ip) combination, a conventional therapy was less effective in reversing reserpine- induced catalepsy. Pretreatment with FK506, a neuroprotectant (0.5-2 mg/kg, po) not only dose dependently reduced the catalepsy in reserpine-treated rats but a lower dose 1mg/kg) potentiated the motor stimulant actions of sub threshold dose of  L-dopa(100 mg/k g, ip) and carbidopa (10mg/kg, ip) combination. Anticataleptic effect of FK506 was blocked dose dependently by specific D2 receptor blocker sulpiride (25-100 mg/kg, ip ). In conclusion, the findings of the present study suggest that FK506 has , an indirect modulatory action on the dopamine D2 receptors. FK506 being a neuroprotectant, could be used as an effective adjunet to L-dopa for the treatment of neuroleptic- induced extrapyramidal side effects

Antinociceptive action of FK506 in mice

1405-1409<span style="font-size: 14.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">Immunophilins are abundantly present in the brain as compared to the immune system. Immunophilin-binding agents like FKS06 are known to inactivate neuronal nitric oxide synthase (nNOS) by inhibiting calcineurin and decrease the <span style="font-size: 14.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">production of nitric oxide. Nitric oxide is involved in the mediation of nociception at the spinal level. In the present study, the effect of FKS06 on the tail flick response in mice and the possible involvement of NO-L-arginine pathway in this <span style="font-size: 14.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">paradigm was evaluated. <span style="font-size:14.0pt;mso-bidi-font-size:8.0pt; font-family:" arial","sans-serif";mso-bidi-font-style:italic"="">FKS06 (0.5, 1 and 3 mg/kg, ip) produced a significant antinociception in the tail flick test. Nitric <span style="font-size: 14.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">oxide synthase (NOS) inhibitor L-NAME significantly and dose dependently (10-40 mg/kg, ip) potentiated the FKS06 (0.5 mg/kg)-induced antinocieeption. On the other hand, NOS substrate L-arginine (100, 200 and 400 mg/kg) inhibited the <span style="font-size:14.0pt;mso-bidi-font-size:8.0pt;line-height:115%; font-family:" times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";="" mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">FKS06-induced antinociception in a dose-dependent manner. Concomitant administration of L-NAME (20 and 40 mg/kg) with L-arginine (200 mg/kg) blocked the inhibition exerted by L-arginine on the FKS06-induced antinociception. Thus, it was concluded that NO- L-arginine pathway may be involved in the FKS06-induced antinociception in tail flick test.</span

Synergy between Enzyme Inhibitors of Fatty Acid Amide Hydrolase and Cyclooxygenase in Visceral Nociception

The present study investigated whether inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide catabolism, produces antinociception in the acetic acid-induced abdominal stretching model of visceral nociception. Genetic deletion or pharmacological inhibition of FAAH reduced acetic acid-induced abdominal stretching. Transgenic mice that express FAAH exclusively in the nervous system displayed the antinociceptive phenotype, indicating the involvement of peripheral fatty acid amides. The cannabinoid receptor 1 (CB1) receptor antagonist, rimonabant, but not the cannabinoid receptor 2 (CB2) receptor antagonist, SR144528, blocked the antinociceptive phenotype of FAAH(-/-) mice and the analgesic effects of URB597 (3′-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) or OL-135 (1-oxo-1[5-(2-pyridyl)-2-yl]-7-phenyl heptane), respective irreversible and reversible FAAH inhibitors, administered to C57BL/6 mice. The opioid receptor antagonist, naltrexone, did not block the analgesic effects of either FAAH inhibitor. URB597, ED50 [95% confidence interval (CI) = 2.1 (1.5-2.9) mg/kg], and the nonselective cyclooxygenase inhibitor, diclofenac sodium [ED50 (95% CI) = 9.8 (8.2-11.7) mg/kg], dose-dependently inhibited acetic acid-induced abdominal stretching. Combinations of URB597 and diclofenac yielded synergistic analgesic interactions according to isobolographic analysis. It is important that FAAH(-/-) mice and URB597-treated mice displayed significant reductions in the severity of gastric irritation caused by diclofenac. URB597 lost its gastroprotective effects in CB1(-/-) mice, whereas it maintained its efficacy in CB2(-/-) mice, indicating a CB1 mechanism of action. Taken together, the results of the present study suggest that FAAH represents a promising target for the treatment of visceral pain, and a combination of FAAH inhibitors and NSAIDs may have great utility to treat visceral pain, with reduced gastric toxicity

Regulation of Inflammatory Pain by Inhibition of Fatty Acid Amide Hydrolase

ABSTRACT Although cannabinoids are efficacious in laboratory animal models of inflammatory pain, their established cannabimimetic actions diminish enthusiasm for their therapeutic development. Conversely, fatty acid amide hydrolase (FAAH), the chief catabolic enzyme regulating the endogenous cannabinoid N-arachidonoylethanolamine (anandamide), has emerged as an attractive target for treating pain and other conditions. Here, we tested WIN 55212 and CB 1 mechanisms of action. However, the transient receptor potential vanilloid type 1 antagonist capsazepine did not affect either the antihyperalgesic or antiedematous effects of URB597. Finally, URB597 attenuated levels of the proinflammatory cytokines interleukin-1␤ and tumor necrosis factor ␣ in LPS-treated paws. These findings demonstrate that simultaneous elevations in non-neuronal and neuronal endocannabinoid signaling are possible through inhibition of a single enzymatic target, thereby offering a potentially powerful strategy for treating chronic inflammatory pain syndromes that operate at multiple levels of anatomical integration. Increased pain sensitivity is one of the most common and debilitating symptoms of inflammatory disorders and is caused by various mediators, including neuropeptides, eicosanoids, and cytokine

Regulation of Inflammatory Pain by Inhibition of Fatty Acid Amide Hydrolase

Although cannabinoids are efficacious in laboratory animal models of inflammatory pain, their established cannabimimetic actions diminish enthusiasm for their therapeutic development. Conversely, fatty acid amide hydrolase (FAAH), the chief catabolic enzyme regulating the endogenous cannabinoid N-arachidonoylethanolamine (anandamide), has emerged as an attractive target for treating pain and other conditions. Here, we tested WIN 55212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de)-1,4-benzoxazin-6-yl]-1-napthalenylmethanone], a cannabinoid receptor agonist, and genetic deletion or pharmacological inhibition of FAAH in the lipopolysaccharide (LPS) mouse model of inflammatory pain. WIN 55212-2 significantly reduced edema and hot-plate hyperalgesia caused by LPS infusion into the hind paws, although the mice also displayed analgesia and other central nervous system effects. FAAH(−/−) mice exhibited reduced paw edema and hyperalgesia in this model without apparent cannabimimetic effects. Transgenic mice expressing FAAH exclusively on neurons continued to display the antiedematous, but not the antihyperalgesic, phenotype. The CB2 cannabinoid receptor (CB2) antagonist SR144528 [N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide] blocked this non-neuronal, anti-inflammatory phenotype, and the CB1 cannabinoid receptor (CB1) antagonist rimonabant [SR141716, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] blocked the antihyperalgesic phenotype. The FAAH inhibitor URB597 [cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester] attenuated the development of LPS-induced paw edema and reversed LPS-induced hyperalgesia through the respective CB2 and CB1 mechanisms of action. However, the transient receptor potential vanilloid type 1 antagonist capsazepine did not affect either the antihyperalgesic or antiedematous effects of URB597. Finally, URB597 attenuated levels of the proinflammatory cytokines interleukin-1β and tumor necrosis factor α in LPS-treated paws. These findings demonstrate that simultaneous elevations in non-neuronal and neuronal endocannabinoid signaling are possible through inhibition of a single enzymatic target, thereby offering a potentially powerful strategy for treating chronic inflammatory pain syndromes that operate at multiple levels of anatomical integration