Mechanistic and pharmacological characterization of PF-04457845: a highly potent and selective fatty acid amide hydrolase inhibitor that reduces inflammatory and noninflammatory pain

Abstract

The endogenous cannabinoid (endocannabinoid) anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH). Pharmacological blockade of FAAH has emerged as a potentially attractive strategy for augmenting endocannabinoid signaling and retaining the beneficial effects of cannabinoid receptor activation, while avoiding the undesirable side effects, such as weight gain and impairments in cognition and motor control, observed with direct cannabinoid receptor 1 agonists. Here, we report the detailed mechanistic and pharmacological characterization of N-pyridazin-3-yl-4-(3-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzylidene)piperidine-1-carboxamide (PF-04457845), a highly efficacious and selective FAAH inhibitor. Mechanistic studies confirm that PF-04457845 is a time-dependent, covalent FAAH inhibitor that carbamylates FAAH's catalytic serine nucleophile. PF-04457845 inhibits human FAAH with high potency (k(inact)/K(i) = 40,300 M(-1)s(-1); IC(50) = 7.2 nM) and is exquisitely selective in vivo as determined by activity-based protein profiling. Oral administration of PF-04457845 produced potent antinociceptive effects in both inflammatory [complete Freund's adjuvant (CFA)] and noninflammatory (monosodium iodoacetate) pain models in rats, with a minimum effective dose of 0.1 mg/kg (CFA model). PF-04457845 displayed a long duration of action as a single oral administration at 1 mg/kg showed in vivo efficacy for 24 h with a concomitant near-complete inhibition of FAAH activity and maximal sustained elevation of anandamide in brain. Significantly, PF-04457845-treated mice at 10 mg/kg elicited no effect in motility, catalepsy, and body temperature. Based on its exceptional selectivity and in vivo efficacy, combined with long duration of action and optimal pharmacokinetic properties, PF-04457845 is a clinical candidate for the treatment of pain and other nervous system disorders.

Fig. 1.

Structures of PF-04457845 and other FAAH inhibitors.

Fig. 2.

Mechanism of covalent, irreversible inhibition of FAAH by PF-04457845. The catalytic triad Ser241-Ser217-Lys142 is shown.

Fig. 3.

A, progress curves for inhibition of human FAAH by PF-04457845 in the presence (5–625 nM) or absence of PF-04457845. B and C, FAAH inhibition by PF-04457845 is time-dependent. Initial rates were determined using a microplate reader as described under Materials and Methods. The concentrations of PF-04457845 were varied from 1 nM to 10 μM. Three separate experiments were performed, and the averages ± S.D. were plotted as percentage of control versus inhibitor concentration and fit to the equation, y = 100/ [1 + (x/IC₅₀)^z], using KaleidaGraph (Synergy Software, Reading, PA), where IC₅₀ is the inhibitor concentration at 50% inhibition and z is the Hill slope (the slope of the curve at its inflection point). The IC₅₀ curves are shown for inhibition of human FAAH (B) and rat FAAH (C) with preincubation times of 1, 5, 30, and 60 min.

Fig. 4.

Selectivity profiling of PF-04457845 and URB597 by competitive ABPP. Gel images of proteomes labeled with FP-rhodamine in the presence or absence of FAAH inhibitors (lane 1, DMSO; lane 2, URB597 at 100 μM; lane 3, URB597 at 10 μM; lane 4, PF-04457845 at 100 μM; lane 5, PF-04457845 at 10 μM; lane 6, DMSO) are shown. A and B, gel profiles of FP-rhodamine-labeled membrane serine hydrolases from mouse and human in brain (A) and liver (B) in the presence or absence of PF-04457845 and URB597. C and D, gel profiles of FP-rhodamine-labeled soluble serine hydrolases in brain, liver, and heart from human (C) and mouse (D) in the presence or absence of PF-04457845 and URB597. The band on the gel corresponding to the FAAH enzyme is highlighted by the black arrow. Note that URB597, not PF-04457845, blocks FP-rhodamine labeling of several soluble serine hydroases from liver and heart as well as membrane hydrolases from liver (red arrows and brackets). In all cases, fluorescent gel images are shown in grayscale.

Fig. 5.

Assessment of in vivo protein targets of alkyne analogs of PF-04457845 and URB597 by CC-ABPP. A, structures of PF-04457845yne and JP104, alkyne analogs of PF-04457845 (top) and URB597 (bottom). B and C, gel profiles of CC-ABPP studies. Brain (B) or liver (C) mice proteomes were isolated after treatment with PF-04457845yne and JP104 for 2 h at 10 mg/kg i.p., reacted with a rhodamine-azide tag under CC conditions, and analyzed in-gel fluorescent scanning (shown in grayscale). PF-04457845yne selectively labels FAAH in both brain and liver tissues as shown [∼60-kDa band is absent in FAAH(−/−) mice], which is in contrast to JP104, which labels several additional proteins in liver [protein bands present in both FAAH(+/+) and FAAH(−/−) mice]. Note that the 55-kDa protein band observed in liver proteome from PF-04457845yne-treated FAAH(−/−) mice was also detected in liver proteomes from vehicle-treated FAAH(+/+) and FAAH(−/−) mice and therefore most likely represents a background protein that cross-reacts with the azide-rhodamine tag.

Fig. 6.

Pharmacokinetic profile of PF-04457845 in rats. After oral administration of PF-04457845 at 1 mg/kg, plasma (ng/ml) or brain (ng/g tissue) concentrations of PF-04457845 were measured at 1, 2, 4, 8, and 24 h.

Fig. 7.

Antihyperalgesic effects of PF-04457845 in the CFA model of inflammatory pain in rats. A, PF-04457845 at 0.003 to 10 mg/kg p.o. produces a reduction of mechanical allodynia (hyperalgesia) (black bars). The effect of the nonsteroidal anti-inflammatory drug naproxen (10 mg/kg p.o., hatched bar) is shown for comparison. PWTs were measured at 4 h after drug treatment and were significantly different for PF-04457845 (0.1–10 mg/kg) and naproxen compared with vehicle-treated groups. ***, p < 0.001; n = 8 rats/group. B, C, and D, PF-04457845-treated rats at 0.1 to 10 mg/kg show near-complete inhibition of FAAH activity (B), elevated AEA levels in brain tissue and blood leukocytes/plasma (C), and elevated PEA/OEA levels in brain tissue (D). All FAAH activity and NAE measurements were determined at 4 h after drug treatment and were significantly different between PF-0457845- and vehicle-treated groups (p < 0.001 for FAAH activity; p < 0.01 for NAEs; n = 3 rats/group). E, brain and plasma levels of PF-04457845 measured at 4 h after drug treatment. n = 3 rats/group. All data are expressed as means ± S.E.M.

Fig. 8.

Time course for antihyperalgesic effects of PF-04457845 (1 mg/kg p.o.) in the CFA model of inflammatory pain in rats. All data are expressed as means ± S.E.M. A, a single dose treatment of PF-04457845 (1 mg/kg p.o.) produces a reduction of mechanical allodynia at least for 24 h. ***, p < 0.001; n = 11 rats per group. B to D, at 1, 2, 4, 8, and 24 h after treatment with PF-04457845, near-complete inhibition of FAAH activity (B), elevated AEA levels in brain tissue and blood leukocytes/plasma (C), and elevated PEA/OEA levels in brain tissue (D) are found. All FAAH activity and NAE measurements were determined at the indicated times after drug treatment and were significantly different between PF-04457845- and vehicle-treated groups (p < 0.001; n = 3 rats/group). E, blockade of antihyperalgesic effects of PF-04457845 (3 mg/kg p.o.) by CB1 and CB2 antagonists (SR141716 and SR144528, respectively; 3 mg/kg i.p.; each administered 10 min before measurement of PWTs). Note that neither the CB1 nor CB2 antagonist displayed significant effects on mechanical allodynia in rats not treated with PF-04457845 (hatched bars). #, p < 0.01, for PF-04457845- versus vehicle-treated groups. **, p < 0.01, for vehicle-PF-04457845 versus CB1/CB2 antagonist-PF-04457845-treated groups. n = 8 rats/group.

Fig. 9.

Antihyperalgesic effects of PF-04457845 in the MIA model of noninflammatory pain in rats. At 14 days post-MIA injection, primary mechanical hyperalgesia of the ipsilateral knees is observed (predosing baseline, day 1) as shown by a decreases in the joint compression threshold compared with contralateral values (average contralateral values for vehicle-treated rats are illustrated by the dotted line at 400 ± 14 g; n = 10). A, PF-04457845 at 0.3 and 3 mg/kg p.o. once daily for 3 consecutive days produces a reduction of mechanical allodynia (hyperalgesia) at 2 and 4 h after drug treatment on both days 1 and 3 (gray bars for 0.3 mg/kg and black bars for 3 mg/kg). All but 0.3 mg/kg-treated rats at 4 h on day 1 were statistically significant compared with vehicle-treated groups. The effect of the treatment with the COX2 inhibitor celecoxib (30 mg/kg p.o.) twice daily for 3 consecutive days (hatched bars) is shown for comparison. Joint compression thresholds were measured at 2 and 4 h postadministration of drug treatment after 14 days after MIA injection on days 1 and 3. B, joint compression thresholds were measured in the hind knee joint contralateral to injury by MIA injection. Inhibitor treatment does not affect pain behavior in the contralateral knee joint. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n = 10 rats/group. Data are expressed as means ± S.E.M.

Fig. 10.

Assessment of cannabinoid behaviors of PF-04457845 in mice. Mice treated with PF-04457845 (1 and 10 mg/kg p.o.) at 4 h postdosing were subjected to behavioral testing for cannabinoid phenotypes, termed the “tetrad test,” which includes assessment of locomotive activity (A), thermal nociception using a tail-immersion test (B), catalepsy (C), and body temperature (D). Mice were also treated with WIN 55212-2 (WIN) (1 and 10 mg/kg i.p.), and behavioral analysis was performed 30 min postdosing. As expected, a cannabinoid receptor agonist, WIN 55212-2, caused full cannabinoid behaviors (i.e., hypomotility, thermal antinociception, catalepsy, and hypothermia). In contrast, PF-04457845 elicited no effect in locomotive activity, catalepsy, and body temperature while displaying significant antinociceptive effects in the thermal tail immersion test. Data are presented as average ± S.E.M., n = 8 mice/group. **, p < 0.01; ***, p < 0.001 for treatment groups versus their respective vehicle controls.