Fatty acid amide hydrolase-dependent generation of antinociceptive drug metabolites acting on TRPV1 in the brain

Abstract

The discovery that paracetamol is metabolized to the potent TRPV1 activator N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404) and that this metabolite contributes to paracetamol's antinociceptive effect in rodents via activation of TRPV1 in the central nervous system (CNS) has provided a potential strategy for developing novel analgesics. Here we validated this strategy by examining the metabolism and antinociceptive activity of the de-acetylated paracetamol metabolite 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA), both of which may undergo a fatty acid amide hydrolase (FAAH)-dependent biotransformation to potent TRPV1 activators in the brain. Systemic administration of 4-aminophenol and HMBA led to a dose-dependent formation of AM404 plus N-(4-hydroxyphenyl)-9Z-octadecenamide (HPODA) and arvanil plus olvanil in the mouse brain, respectively. The order of potency of these lipid metabolites as TRPV1 activators was arvanil = olvanil>>AM404> HPODA. Both 4-aminophenol and HMBA displayed antinociceptive activity in various rodent pain tests. The formation of AM404, arvanil and olvanil, but not HPODA, and the antinociceptive effects of 4-aminophenol and HMBA were substantially reduced or disappeared in FAAH null mice. The activity of 4-aminophenol in the mouse formalin, von Frey and tail immersion tests was also lost in TRPV1 null mice. Intracerebroventricular injection of the TRPV1 blocker capsazepine eliminated the antinociceptive effects of 4-aminophenol and HMBA in the mouse formalin test. In the rat, pharmacological inhibition of FAAH, TRPV1, cannabinoid CB1 receptors and spinal 5-HT3 or 5-HT1A receptors, and chemical deletion of bulbospinal serotonergic pathways prevented the antinociceptive action of 4-aminophenol. Thus, the pharmacological profile of 4-aminophenol was identical to that previously reported for paracetamol, supporting our suggestion that this drug metabolite contributes to paracetamol's analgesic activity via activation of bulbospinal pathways. Our findings demonstrate that it is possible to construct novel antinociceptive drugs based on fatty acid conjugation as a metabolic pathway for the generation of TRPV1 modulators in the CNS.

Figure 1. Production of TRPV1 active drug metabolites in the mouse brain from 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA).

Formation of arvanil and olvanil in rat brain homogenates incubated with HMBA (100 µM) for 60 min (A; n = 6). These N-acylamines were not detected in brain homogenates exposed to vehicle only. TRPV1-dependent vasorelaxation evoked by AM404 (n = 6), N-(4-hydroxyphenyl)-9Z-octadecenamide (HPODA; n = 9), arvanil (n = 12) and olvanil (n = 7) in rat mesenteric arterial segments precontracted with phenylephrine (B). Content of AM404 (C and E) and HPODA (D and F) in the brain 20 min after intraperitoneal injection of 4-aminophenol at doses of 30 mg/kg (C and D; n = 5) and 100 mg/kg (E; n = 6–12 and F; n = 6) in different FAAH genotypes. Contents of arvanil (G; n = 6) and olvanil (H; n = 6) in the brain 20 min after intraperitoneal injection of HMBA at a dose of 300 mg/kg in different FAAH genotypes. Values are expressed as mean ± SE. *p<0.05, **p<0.01 and ***p<0.001 when compared to wild-type littermates, using Mann-Whitney U test (A, C, D, G and H) or Kruskal-Wallis one-way ANOVA followed by Dunn’s multiple comparisons test (E and F).

Figure 2. Content of AM404 in the rat brain after intraperitoneal injection of 4-aminophenol (100 mg/kg).

Brain content of AM404 at different time points after 4-aminophenol administration (A; n = 6). Brain content of AM404 20 min after 4-aminophenol injection in animals pretreated with phenylmethanesulfonyl fluoride (PMSF; 10 mg/kg s.c.) 20 min before 4-aminophenol administration (B; n = 6). The AM404 content is presented as normalized peak area (nPA) per g protein. Results are expressed as mean ± SE. **p<0.01 and ***p<0.001 when compared to time zero (A) or vehicle-treated animals (B), using Kruskal-Wallis one-way ANOVA followed by Dunn’s multiple comparisons test (A) or Mann-Whitney U test (B).

Figure 3. Effects of 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA) on spontaneous locomotor activity in the mouse.

Locomotor activity was assayed by counting the number of crossings of light beams in actimetry boxes over a 15 min period. Doses of 4-aminophenol (A; n = 4) and HMBA (B; n = 6) below 100 mg/kg and 200 m/kg, respectively, failed to impair locomotor activity. 4-Aminophenol and HMBA were injected by the intraperitoneal route 10 min before the test. *p<0.05, **p<0.01 and ***p<0.001 when compared to vehicle injection, using Kruskal-Wallis one-way ANOVA followed by Dunn’s multiple comparisons test.

Figure 4. The antinociceptive effect of 4-aminophenol is dependent on fatty acid amide hydrolase (FAAH) in mouse.

The antinociceptive effect of 4-aminophenol, given at a dose that did not affect spontaneous locomotor activity (Fig. 3), was lost in FAAH^−/− mice as compared to their wild-type littermates. The effect of 4-aminophenol at a dose of 30 mg/kg (i.p.) was assessed in the formalin test in C56BL/6 wild-type mice (A; n = 8) and in the formalin (B; n = 6–9), von Frey (C; n = 6) and tail immersion (D; n = 6) tests in FAAH^−/− mice and their wild-type littermates. 4-Aminophenol was injected by the intraperitoneal route 10 min before the tests. *p<0.05, **p<0.01 and ***p<0.001 when compared to vehicle injection, using Mann-Whitney U test.

Figure 5. The antinociceptive effect of 4-aminophenol is dependent on fatty acid amide hydrolase (FAAH) in rats.

4-Aminophenol at a dose of 100 mg/kg significantly reduced the first and second phases of the formalin test (A; n = 7–8). Both the 30 mg/kg and the 100 mg/kg doses of 4-aminophenol increased the withdrawal threshold in the paw pressure test (B; n = 8). Pre-treatment with phenylmethanesulfonyl fluoride (PMSF; 10 mg/kg) substantially reduced or prevented the antinociceptive effect of 4-aminophenol (100 mg/kg) in the formalin (C; n = 10–14) and paw pressure (D; n = 7–11) tests. The different doses of 4-aminophenol were injected by the intraperitoneal route 10 min before the tests. PMSF was injected subcutaneously 20 min before 4-aminophenol administration. *p<0.05, **p<0.01 and ***p<0.001 when compared to vehicle injection, using Kruskal-Wallis one-way ANOVA followed by Dunn’s test (A), repeated measures two-way ANOVA followed by Dunnetts (B) or Sidak’s (D) multiple comparisons tests, or Mann-Whitney U test (C).

Figure 6. Effects of 4-hydroxy-3-methoxybenzylamine (HMBA) on nociception in the formalin test in mice.

At a dose that did not affect spontaneous locomotion (Fig. 3B), HMBA (100 mg/kg) inhibited nocifensive behaviour in both phases of the formalin test in C57BL/6 wild-type mice (A; n = 5–7). The antinociceptive effect of HMBA disappeared in FAAH^−/− mice as compared to their wild-type littermates (B; n = 4–8), whereas dipyrone (30 mg/kg) produced similar antinociception in FAAH^+/+ and FAAH^−/− mice (C; n = 5–7). HMBA and dipyrone were injected by the intraperitoneal route 10 min before the tests. *p<0.05 and **p<0.01 when compared to vehicle injection, using Mann-Whitney U test.

Figure 7. The antinociceptive effect of 4-aminophenol is dependent on TRPV1 in the mouse.

The antinociceptive effect of 4-aminophenol (30 mg/kg) in the mouse formalin (A; n = 5–6), von Frey (B; n = 6) and tail immersion (C; n = 6) tests disappeared in TRPV1^−/− mice as compared to their wild-type littermates. 4-Aminophenol was injected by the intraperitoneal route 10 min before the tests. **p<0.01 when compared to vehicle injection, using Mann-Whitney U test.

Figure 8. The antinociceptive effect of 4-aminophenol is dependent on TRPV1 in the rat.

The TRPV1 blocker capsazepine (10 mg/kg) prevented the antinociceptive effect of 4-aminophenol (100 mg/kg) in the rat formalin (A; n = 7–10) and paw pressure (B; n = 6–8) tests. Capsazepine (Cz) and 4-aminophenol were injected by the intraperitoneal route 30 min and 10 min before the tests, respectively. *p<0.05, **p<0.01 and ***p<0.001 when compared to vehicle injection, using Mann-Whitney U test (A) or repeated measures two-way ANOVA followed by Sidak’s multiple comparisons test (B).

Figure 9. TRPV1 in brain mediates the antinociceptive effects of 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA).

Intracerebroventricular injection of the TRPV1 blocker capsazepine (Cz) prevented the antinociceptive effect of 4-aminophenol (A; n = 5–7) and HMBA (B; n = 6–8) in the mouse formalin test. 4-aminophenol (30 mg/kg) and HMBA (100 mg/kg) were injected by the intraperitoneal route 10 min before the test. Capsazepine (100 nmol) was injected 5 min before the 4-aminophenol and HMBA administration. **p<0.01 when compared to vehicle injection, using Mann-Whitney U test.

Figure 10. The antinociceptive effect of 4-aminophenol is dependent on cannabinoid CB1 receptors.

The cannabinoid CB1 receptor antagonist AM251 (3 mg/kg) prevented the antinociceptive effect of 4-aminophenol (100 mg/kg) in the rat formalin (A; n = 8–11) and paw pressure (B; n = 6–7) tests. However, 4-aminophenol (100 mg/kg) did not affect the global brain levels of the endocannabinoids anandamide (C; n = 6) and 2-arachidonoylglycerol (2-AG; D; n = 6). AM251 and 4-aminophenol were injected by the intraperitoneal route 30 min and 10 min before the behavioural assays, respectively. The contents of endocannabinoids are presented as normalized peak area (nPA) per g protein. *p<0.05, **p<0.01 and ***p<0.001 when compared to vehicle injection, using Mann-Whitney U test (A), repeated measures two-way ANOVA followed by Sidak’s multiple comparisons test (B) or Kruskal-Wallis one-way ANOVA followed by Dunn’s multiple comparisons test (C and D).

Figure 11. The antinociceptive effect of 4-aminophenol is dependent on spinal serotonergic mechanisms.

Pre-treatment of rats with the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) reduced the 4-aminophenol-induced increase of the withdrawal threshold in the paw pressure test (A; n = 7–8). The serotonin receptor antagonists tropisetron (B; n = 7–8) and WAY-100635 (C; n = 6–9) prevented the antinociceptive effect of 4-aminophenol in the rat paw pressure and formalin tests, respectively. 5,7-DHT (100 µg) was administered intrathecally 7 days before the paw pressure test. 4-Aminophenol (100 mg/kg) was injected by the intraperitoneal route 10 min before the behavioural tests. Tropisetron (Trop; 500 ng) and WAY-100635 (WAY; 40 µg) were injected intrathecally 5 min before administration of 4-aminophenol. *p<0.05, **p<0.01 and ***p<0.001 when compared to vehicle injection, using repeated measures two-way ANOVA followed by Sidak’s multiple comparisons test (A and B) or Mann-Whitney U test (C).

Figure 12. Proposed mechanism by which 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA) produce antinociception.

Following passage through the blood-brain barrier, 4-aminophenol and HMBA are conjugated with free fatty acids (FFA), preferentially arachidonic acid, to yield the potent TRPV1 activators AM404 and arvanil plus olvanil, respectively, a reaction catalyzed by the enzyme fatty acid amide hydrolase (FAAH). AM404, arvanil and olvanil then activate TRPV1 in brain nuclei, regulating descending antinociceptive pathways, possibly of serotoninergic origin, thereby reducing nociceptive neurotransmission in the dorsal horn of the spinal cord. In contrast to the other lipid metabolites, HPODA is produced by a FAAH-independent pathway and it does not seem to contribute to the antinociceptive action of 4-aminophenol, consistent with HPODA being produced in smaller quantities and being a less potent TRPV1 activator than AM404.