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AM404 also known as N-arachidonoylphenolamine is an active metabolite of paracetamol (acetaminophen) responsible for all or part of its analgesic action.

History
Together with a number of other anandamide analogs, AM404 was first synthesized in 1996 in a research group led by Alexandros Makriyannis at the University of Connecticut. It was already known at the time that the endogenous cannabinoid anandamide, which had been discovered in 1992, mimics the pharmacological effects of Δ9-tetrahydrocannabinol (THC)&mdash;the psychotropic substance contained in hashish and marijuana. This was a strong motivation for studying the structure-activity relationship of related compounds. The study determined however that AM404 exhibits only a weak affinity for both types of cannabinoid receptors&mdash;an order of magnitude less than anandamide.

The importance of AM404 was first observed in an article that appeared in the journal Science in 1997. Like other neural modulators, extracellular anandamide was thought to be rapidly inactivated. The pathway was hypothesized to be hydrolysis to arachidonic acid and ethanolamine, catalyzed by a membrane-bound fatty acid amide hydrolase (FAAH), which is highly expressed in the brain and liver tissues. Low FAAH activity found in brain plasma membranes indicated however that this enzyme may be intracellular. Lineweaver-Burke analysis had already identified a high-affinity component for anandamide accumulation in astrocytes, suggesting an uptake carrier was involved. Since a primary criterion for demonstrating carrier-mediated transport is pharmacological inhibition,

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researchers from the Neurosciences Institute of San Diego, in collaboration colleagues from the University of Naples and the University of Connecticut, were looking to identify inhibitors of the putative anandamide transport (AMT). Amongst the compounds they tested, AM404 turned out to be the strongest inhibitor. In vitro, a concentration of anandamide that was well below the threshold when applied alone produced an almost maximal effect when applied together with AM404. In mice, intravenous coadministration of AM404 and anandamide significantly increased the analgesic effect of the latter in the hot plate test.

A more extensive report comparing the inhibitory effect of AM404 with 33 other analogues was published in PNAS in 1999. AM404 was still the strongest inhibitor in this study. A year later, Massimiliano Beltramo and Daniele Piomelli reported that AM404 has a similar inhibitory effect on the uptake of 2-arachidonoylglycerol (2-AG)&mdash;another cannabinoid agonist that had been found to be endogenous in 1995.

Simultaneously with these discoveries a parallel line of events took place. After the first vanilloid receptor (VR1, later renamed TRPV1) was discovered in 1997, a join research group of Peter Zygmunt and Edward Högestätt (University of Lund) together with David Julius (UCSF) and Vincenzo Di Marzo (Italian National Research Council) published a paper in Nature suggesting that anandamide was a strong agonist for VR1. This paper stirred a fair amount of controversy.

A far more striking discovery was made in the same year. A collaboration betwe

In the same year the group coordinated by Vincenzo Di Marzo discovered that several AMT inhibitors, including AM404 directly activated the vanilloid receptor TRPV1 at much lower concentrations than those needed to inhibit anandamide uptake. (TRPV1 had been discovered in 1997, and given the initial name capsaicin receptor. )

Relationship with paracetamol
The connection between AM404 and paracetamol was not established until 2005 when it was shown that paracetamol, following deacetylation to its primary amine, is conjugated with arachidonic acid in the brain and spinal column to form AM404. Mice lacking the fatty acid amide hydrolase cannot metabolize this.

An article that appeared in the journal Pain in 2008 provided definitive evidence that cannabinoids are necessary for paracetamol to exert its analgesic action. AM251, a CB1 receptor antagonist, blocks the analgesic action of paracetamol, and paracetamol also has no analgesic effect in knockout mice lacking the CB1 receptor.

Pharmacology
AM404 was originally reported to be an endogenous cannabinoid reuptake inhibitor, preventing the transport of anandamide and other related compounds back from the synaptic cleft, much in the same way that common SSRI antidepressants prevent the reuptake of serotonin. Recent work on the mechanism of AM404 has suggested that the inhibition of fatty acid amide hydrolase (FAAH) by AM404 is likely responsible for all of its attributed "reuptake" properties, since intracellular FAAH hydrolysis of anandamide changes the intra/extracellular anandamide equilibrium.

AM404 is also TRPV1 agonist, and also prevents cyclooxygenase COX-1 and COX-2 and prostaglandin synthesis. AM404 is thought to induce its analgesic action through its activity on the cannabinoid, COX and TRPV systems, all of which are present in pain and thermoregulatory pathways.