**2. Paracetamol, a prodrug of which AM404 is the active metabolite**

In 2005, Högestätt et al. [2] showed that paracetamol, following its hepatic deacetylation to *p*-aminophenol, is metabolized in the brain by the fatty acid amide hydrolase (FAAH) enzyme to form AM404 (N-(4-Hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide) (**Figure 1**).

After administration of deuterium-labeled paracetamol in rats, they detected deuteriumlabeled AM404 and *p*-aminophenol in the brain. They further showed that formation of *p*-aminophenol was present in all tissues, with highest levels in the liver and that AM404 was mainly found in the brain. The latter results were confirmed in a recent study [3].

Incubation of brain homogenate with *p*-aminophenol *in vitro* but not with paracetamol (except at high doses) leads to the formation of AM404 [2]. This is not the case if brain

 **Figure 1.** Metabolization of paracetamol into AM404. AM404: N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide. FAAH, fatty acid amide hydrolase.

homogenate is boiled, pretreated with PMSF (a broad-spectrum protease, esterase and amidase inhibitor [4]) or if brain homogenate comes from FAAH−/− mice. Incubation of isolated FAAH with *p*-aminophenol and arachidonic acid leads to the formation of AM404. *In vivo*, paracetamol does not produce AM404 in the brains of rats pretreated with PMSF or in FAAH−/− mice.

on the inhibition of cyclooxygenases and/or the activation of the serotonergic system [1], we show that the endocannabinoid and vanilloid systems and the T-type calcium-channel Cav3.2 are emerging as new targets of its action *via* complex metabolic and neuronal pathways.

In 2005, Högestätt et al. [2] showed that paracetamol, following its hepatic deacetylation to *p*-aminophenol, is metabolized in the brain by the fatty acid amide hydrolase (FAAH) enzyme

After administration of deuterium-labeled paracetamol in rats, they detected deuteriumlabeled AM404 and *p*-aminophenol in the brain. They further showed that formation of *p*-aminophenol was present in all tissues, with highest levels in the liver and that AM404 was

Incubation of brain homogenate with *p*-aminophenol *in vitro* but not with paracetamol (except at high doses) leads to the formation of AM404 [2]. This is not the case if brain

 **Figure 1.** Metabolization of paracetamol into AM404. AM404: N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide.

FAAH, fatty acid amide hydrolase.

**2. Paracetamol, a prodrug of which AM404 is the active metabolite**

208 Pain Relief - From Analgesics to Alternative Therapies

to form AM404 (N-(4-Hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide) (**Figure 1**).

mainly found in the brain. The latter results were confirmed in a recent study [3].

We speculated that this metabolic pathway was involved in its analgesic action and decided, therefore, to investigate the analgesic effect of paracetamol metabolites. Systemic administration of *p*-aminophenol or intracerebroventricular injection of AM404 produced an analgesic effect in animals.

We then investigated the involvement of FAAH in the action of paracetamol using mice deleted for the FAAH gene (genetic strategy) and systemic administration of PMSF or URB597, nonspecific and specific FAAH inhibitors, respectively, (pharmacological strategy) to inhibit the FAAH enzyme. Both strategies resulted in the abolition of paracetamol-induced (1) brain synthesis of AM404 and (2) analgesic action [5]. Likewise, the analgesic effect and brain formation of AM404 induced by *p*-aminophenol were decreased in FAAH−/− mice and in rats pretreated with PMSF [6].

The involvement of FAAH in the action of paracetamol was observed in different pain tests (paw pressure, von Frey, tail immersion and formalin tests) and modalities (thermal, mechanical and chemical *stimuli*) [5–7]. However, the experiments were conducted in naive animals, in a context far removed, therefore, from the clinical setting, in which paracetamol is used for pathological pain, notably nociceptive pain [8, 9]. Thus, the involvement of FAAH in the action of paracetamol was studied in a more relevant clinical context using an inflammatory mouse model submitted to thermal and mechanical *stimuli* to assess allodynia and hyperalgesia. The anti-allodynic and anti-hyperalgesic effects of paracetamol observed in this model were lost in FAAH−/− mice [10], which lend further weight to the involvement of the FAAH in inflammation.

Although it is now generally acknowledged that the action of paracetamol is central rather than peripheral, opinions still differ [11, 12]. FAAH is a ubiquitous enzyme [4]. Some authors detected AM404 in blood after paracetamol administration [13]. We investigated the peripheral versus central involvement of FAAH in the action of paracetamol studying its effect with an FAAH inhibitor that readily crosses the blood-brain barrier, URB597 and a peripherally restricted FAAH inhibitor, URB937 [14, 15] (**Figure 2A**).

The fact that the analgesic action of paracetamol is maintained after URB937 administration and lost after URB597 treatment [10] shows that only brain and not peripheral, FAAH is involved and thereby confirms the central action of paracetamol. As a counterproof, the peripherally restricted FAAH inhibitor URB937 was intracerebroventricularly injected and challenged with paracetamol (**Figure 2B**). A supra-spinal injection of URB937 in mice prior to paracetamol reversed its analgesic actions.

All these results show that supra-spinal FAAH is required for the desired effect of the paracetamol.

**Figure 2.** Pharmacological strategies to block central and/or peripheral FAAH. (A) Global or peripheral FAAH was inhibited by a systemic injection of URB597 (a brain permeant compound) or URB937 (a peripherally restricted FAAH inhibitor), respectively. (B) URB937 was supraspinally injected to specifically inhibit brain FAAH.
