e primary afferent input to the 
dorsal horn of the spinal cord following formalin injection. However, we cannot exclude an interaction with TRPV1 on central projections of primary afferents terminating in the trigeminal or solitary tract nuclei. Taken together, our 1820332 findings indicate that activation of TRPV1 in brain plays a key role in the antinociceptive effects of 4aminophenol and HMBA and suggest that lipid metabolites, such as AM404 and arvanil, mediate these actions. As shown previously, intracerebroventricular injection of AM404 can reproduce the TRPV1-dependent antinociceptive effect of oral paracetamol in the mouse formalin test, although the duration of action of AM404 was relatively short, probably reflecting rapid elimination of AM404 in the absence of its precursor 4-aminophenol. PF-562271 site arvanil given by the same route also possesses antinociceptive activity, as shown in the mouse tailflick test. However, the brain contents of arvanil and olvanil were much lower than the content of AM404 and HPODA after systemic administration of HMBA and 4aminophenol, respectively. This does not necessarily mean that arvanil and olvanil produced less TRPV1 activation than AM404 and HPODA, because the final responses also depend on the potencies of the compounds. Interestingly, arvanil was shown to be equipotent with the ultrapotent TRPV1 activator resiniferatoxin in functional assays and radioligand binding studies of rodent TRPV1. We have identified two previous studies that directly compare the TRPV1 activity of AM404 with that of arvanil or olvanil. One study reported that arvanil was 933 times Analgesic TRPV1 Active Drug Metabolites in Brain more potent than AM404 in a 45Ca2+ uptake assay, using CHO cells expressing the rat orthologue of TRPV1. In another study, arvanil and olvanil were approximately 50 times more potent than AM404 in HEK293 cells expressing the human TRPV1, using fluorometric calcium imaging. In the present study, we found that arvanil and olvanil were.100 times more potent than AM404 and.300 times more potent than HPODA as TRPV1 activators. Taking these potency differences into account, it is likely that the amounts of AM404 plus HPODA and arvanil plus olvanil formed after 4-aminophenol and HMBA administration, respectively, produced comparable TRPV1 activation in the mouse brain. Indeed, these doses of HMBA and 4-aminophenol produced equivalent antinociception in the mouse formalin test. The 10877822 contribution of HPODA to the antinociceptive effect of 4AP is, however, less clear. This N-acylamine was not only less potent than AM404 as a TRPV1 activator, but its level in the mouse brain after 4-aminophenol administration was approximately one fourth of that of AM404. More importantly, while the antinociceptive activity of 4-aminophenol disappeared in FAAH2/2 mice, the brain content of HPODA after this dose of 4-aminophenol was 3 times larger in these mice than in their wildtype littermates. Thus, in contrast to the oleic acid derivative of HMBA, the corresponding derivative of 4-aminophenol seems to be generated mainly by a FAAH-independent biosynthetic pathway, although FAAH may mediate the degradation of both these N-acylamines. These findings indicate not only quantitative differences, but also qualitative differences in the fatty acid conjugation of 4-aminophenol and HMBA. AM404 was originally introduced as a selective inhibitor of cellular anandamide uptake and degradation. However, the potency of AM404 as an inhibitor of anan
