**3. Different molecular targets of AM404**

#### **3.1. COX enzyme**

The first historical hypothesis for the action of paracetamol, proposed by Flower and Vane, was the inhibition of COX [16]. In cell cultures, inhibition of COX by paracetamol was observed in different cell types, brain slices, or homogenates [16–18] with conflicting results [19]. Paracetamol seems to have only a weak inhibitory effect on prostaglandin production in cell culture, with IC50 values mostly around 100 μM [20]. In animals, paracetamol reduced prostaglandin in cerebrospinal fluid [21], the spinal cord [22] and the brain [23, 24]. Interestingly, AM404 was shown to be an inhibitor of COX on isolated COX-1 and COX-2 and in LPSinduced prostaglandin E2 formation in RAW264.7 macrophages [2].

However, an orally administered analgesic dose of paracetamol (200 mg/kg) in mice did not affect brain prostaglandin E<sup>2</sup> (PGE<sup>2</sup> ) content, while a high intraperitoneal dose (300 mg/kg), which impairs mice locomotor activity, reduced the content of prostanoid levels in the brain (PGE<sup>2</sup> ), kidneys (PGE<sup>2</sup> ) and blood (thromboxane B<sup>2</sup> ) [7]. Paracetamol has a different pharmacological profile from that of the competitive COX inhibitor ibuprofen. In a context of noninflammatory pain, ibuprofen did not reduce pain, whereas paracetamol did, as observed in the first phase of formalin tests, tail immersion and von Frey tests in mice [7]. Altogether, these results indicate that the analgesic action of paracetamol cannot be attributed to inhibition of COX. Furthermore, the inhibitory effect of paracetamol on COX observed by some authors seems more closely related to its hypothermic/antipyretic effects than to its analgesic action [21, 23].

Further studies are needed before the involvement of COX can be fully ruled out. A study showing that PGs measured in mice after administration of 200 mg of paracetamol were not decreased was performed with naive animals [7]. In a neuroinflammatory context such as chronic pain, in which PGs contribute to the maintenance of the process, it is possible that repeated administration of paracetamol could induce an inhibition of COX and that such a mechanism could be involved in the analgesic action of paracetamol.

#### **3.2. CB1 receptor**

AM404 is able to indirectly activate the cannabinoid receptor CB1 by inhibiting the degradation [25] and reuptake [26, 27] of anandamide. Involvement of this receptor in the action of paracetamol was confirmed by a study showing that CB1 knockout mice and rats pretreated with a specific CB1 antagonist (AM251) were insensitive to paracetamol [5, 28]. Corroborating these results, we showed that the analgesic effect of *p*-aminophenol was also suppressed by AM251 [6]. Interestingly, it was shown in a neuropathic rat pain model that the synergic or additive antinociception of paracetamol with gabapentin, memantine, or tramadol was attenuated by pretreatment with AM251 [29]. In the same study, the intrinsic analgesic effect of gabapentine, memantine, or tramadol was not affected by CB1 receptor antagonist.

The involvement of CB1 receptor seems independent of the potential inhibitory effect of AM404 on cannabinoid reuptake because the overall brain content of endocannabinoids (anandamide, 2-arachidonoylglycerol and palmitoylethanolamide) was not affected by an administration of paracetamol [7] or *p*-aminophenol [6] in mice or in rats. In addition, paracetamol does not bind directly CB1 receptors [5]. Thus, the relationship between paracetamol and CB1 remains to be elucidated.

## **3.3. TRPV1 receptor**

**3. Different molecular targets of AM404**

210 Pain Relief - From Analgesics to Alternative Therapies

(PGE<sup>2</sup>

) and blood (thromboxane B<sup>2</sup>

The first historical hypothesis for the action of paracetamol, proposed by Flower and Vane, was the inhibition of COX [16]. In cell cultures, inhibition of COX by paracetamol was observed in different cell types, brain slices, or homogenates [16–18] with conflicting results [19]. Paracetamol seems to have only a weak inhibitory effect on prostaglandin production in cell culture, with IC50 values mostly around 100 μM [20]. In animals, paracetamol reduced prostaglandin in cerebrospinal fluid [21], the spinal cord [22] and the brain [23, 24]. Interestingly, AM404 was shown to be an inhibitor of COX on isolated COX-1 and COX-2 and in LPS-

**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.

 formation in RAW264.7 macrophages [2]. However, an orally administered analgesic dose of paracetamol (200 mg/kg) in mice did not

which impairs mice locomotor activity, reduced the content of prostanoid levels in the brain

logical profile from that of the competitive COX inhibitor ibuprofen. In a context of noninflammatory pain, ibuprofen did not reduce pain, whereas paracetamol did, as observed in the first phase of formalin tests, tail immersion and von Frey tests in mice [7]. Altogether, these results indicate that the analgesic action of paracetamol cannot be attributed to inhibition of COX. Furthermore, the inhibitory effect of paracetamol on COX observed by some authors seems more closely related to its hypothermic/antipyretic effects than to its analgesic action [21, 23]. Further studies are needed before the involvement of COX can be fully ruled out. A study showing that PGs measured in mice after administration of 200 mg of paracetamol were not

) content, while a high intraperitoneal dose (300 mg/kg),

) [7]. Paracetamol has a different pharmaco-

**3.1. COX enzyme**

induced prostaglandin E2

(PGE<sup>2</sup>

affect brain prostaglandin E<sup>2</sup>

), kidneys (PGE<sup>2</sup>

Subsequent studies have shown that AM404 is also a potent activator of the capsaicin receptor TRPV1, as reported in patch-clamp experiments [30, 31]. Interestingly, local injection of AM404 in the paw of mice resulted in pain behavior (licking and lifting of the injected paw), a behavior not found in TRPV1−/− mice [7].

The contribution of TRPV1 to the action of paracetamol has been explored by both genetic and pharmacological approaches to inhibit it. Results showed that a genetic inactivation of TRPV1 abolished the antinociceptive effects of paracetamol in the mouse formalin, von Frey and tail immersion tests [7]. Pharmacological blockade of TRPV1 by capsazepine in rats also suppressed the analgesic effect of paracetamol [7]. Observations made on paracetamol can be extended to *p*-aminophenol, since pretreatment with capsazepine in rats or administration in TRPV1−/− mice prevented the antinociceptive effect of *p*-aminophenol [6]. Further, the analgesic effect of the intracerebroventricular injection of AM404 was lost in TRPV1−/− mice [7]. In a calcium imaging experiment, human embryonic kidney (HEK) cells, which constitutively expressed FAAH, were transfected with TRPV1. AM404 induced intracellular calcium mobilization [30]. This response was not observed in cells pretreated with capsazepine or in cells that were not transfected with TRPV1. In agreement with the previous results, bath application of *p*-aminophenol also induced an increase in intracellular calcium, smaller and slower than that of AM404. The calcium increase induced by *p*-aminophenol was abolished in cells either pretreated with capsazepine or not transfected with TRPV1 [30]. The effect of TRPV1 was due to metabolization of *p*-aminophenol into AM404 because *p*-aminophenol-induced calcium mobilization was lost in cells pretreated with an FAAH inhibitor.

To accurately establish the location of the involvement of TRPV1 in paracetamol action, systemic administration of paracetamol was challenged with the selective blockade of TRPV1 in the brain. Injection of capsazepine into the lateral ventricle of mice abolished the antinociceptive effects of paracetamol [7]. Similarly, the antinociceptive activity of *p*-aminophenol was also lost in mice intracerebroventricularly preinjected with capsazepine [6]. Collectively, these findings identify brain TRPV1 as an important effector of paracetamol.
