Discovery of PF-04457845: A Highly Potent, Orally Bioavailable, and Selective Urea FAAH Inhibitor

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic and anti-inflammatory phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and other nervous system disorders. Herein, we report the discovery and characterization of a highly efficacious and selective FAAH inhibitor PF-04457845 (23). Compound 23 inhibits FAAH by a covalent, irreversible mechanism involving carbamylation of the active-site serine nucleophile of FAAH with high in vitro potency (k(inact)/K(i) and IC(50) values of 40300 M(-1) s(-1) and 7.2 nM, respectively, for human FAAH). Compound 23 has exquisite selectivity for FAAH relative to other members of the serine hydrolase superfamily as demonstrated by competitive activity-based protein profiling. Oral administration of 23 at 0.1 mg/kg results in efficacy comparable to that of naproxen at 10 mg/kg in a rat model of inflammatory pain. Compound 23 is being evaluated in human clinical trials.     

A role for the ventral hippocampal endocannabinoid system in fear-conditioned analgesia and fear responding in the presence of nociceptive tone in rats

The endogenous cannabinoid (endocannabinoid) system plays an important role in fear-conditioned analgesia (FCA) and expression and extinction of conditioned fear. The hippocampus has an established role in both pain and conditioned fear and is a substrate for endocannabinoid activity. This study aimed to investigate the role of the endocannabinoid system in the ventral hippocampus (vHip) in FCA and in fear responding in the presence of nociceptive tone. Fear-conditioned rats displayed significantly increased freezing and 22-kHz ultrasonic vocalisation and a reduction in formalin-evoked nociceptive behaviour (ie, FCA) upon re-exposure to a context previously paired with footshock. Tissue levels of the endocannabinoids, anandamide, and 2-arachidonoylglycerol, as well as the fatty acid amide, palmitoylethanolamide, were significantly higher in the vHip of fear-conditioned rats compared with non-fear-conditioned controls. URB597 (inhibitor of fatty acid amide hydrolase [FAAH]), administered bilaterally into the vHip, significantly enhanced FCA during the entire trial and increased fear responding in formalin-treated rats early in the trial. The URB597-induced enhancement of FCA was blocked by intra-vHip administration of the cannabinoid₁ (CB₁) receptor antagonist/inverse agonist rimonabant. Intra-vHip rimonabant alone had no effect on the expression of FCA, and URB597 did not significantly alter formalin-evoked nociceptive behaviour in non-fear-conditioned rats. These data suggest an important role for the endocannabinoid system in the vHip in FCA, whereby levels of 2-arachidonoylglycerol and the FAAH substrates palmitoylethanolamide and anandamide are increased in rats expressing FCA, and pharmacological inhibition of FAAH in the vHip enhances this form of endogenous analgesia via a CB₁ receptor-dependent mechanism.     

Cannabinoid CB1 receptors regulate neuronal TNF-α effects in experimental autoimmune encephalomyelitis

Cannabinoid CB1 receptors (CB1Rs) regulate the neurodegenerative damage of experimental autoimmune encephalomyelitis (EAE) and of multiple sclerosis (MS). The mechanism by which CB1R stimulation exerts protective effects is still unclear. Here we show that pharmacological activation of CB1Rs dampens the tumor necrosis factor α (TNFα)-mediated potentiation of striatal spontaneous glutamate-mediated excitatory postsynaptic currents (EPSCs), which is believed to cogently contribute to the inflammation-induced neurodegenerative damage observed in EAE mice. Furthermore, mice lacking CB1Rs showed a more severe clinical course and, in parallel, exacerbated alterations of sEPSC duration after induction of EAE, indicating that endogenous cannabinoids activate CB1Rs and mitigate the synaptotoxic action of TNFα in EAE. Consistently, we found that mice lacking the fatty acid amide hydrolase (FAAH), and thus expressing abnormally high brain levels of the endocannabinoid anandamide, developed a less severe EAE associated with preserved TNFα-induced sEPSC alterations.CB1Rs are important modulators of EAE pathophysiology, and might play a mechanistic role in the neurodegenerative damage of MS patients.     

Targeting the endocannabinoid system in the amygdala kindling model of temporal lobe epilepsy in mice

The endocannabinoid system can be considered as a putative target to affect ictogenesis as well as the generation of a hyperexcitable epileptic network. Therefore, we evaluated the effect of a CB1 receptor agonist (WIN55.212-2) and of an inhibitor of the enzymatic degradation of the endocannabinoid anandamide (fatty acid hydrolase inhibitor URB597) in the amygdala kindling model of temporal lobe epilepsy. Only minor effects on seizure thresholds and seizure parameters without a clear dose-dependency were observed in fully kindled mice. When evaluating the impact on kindling acquisition, WIN55.212-2 significantly delayed the progression of seizure severity. In contrast, URB597 did not affect the development of seizures in the kindling paradigm. Analysis of cell proliferation and neurogenesis during the kindling process revealed that URB597 significantly reduced the number of newborn neurons. These data give first evidence that CB1-receptor activation might render a disease-modifying approach. Future studies are necessary that further analyze the role of CB1 receptors and to confirm the efficacy of CB1-receptor agonists in other models of chronic epilepsy.     

Measuring Endocannabinoid Hydrolysis: Refining our Tools and Understanding

Endocannabinoids (eCBs) are lipid transmitters that are released from membrane precursors in response to specific stimuli, activate cannabinoid receptors—the molecular targets of compounds produced by Cannabis sativa—and are then rapidly inactivated by uptake and enzymatic hydrolysis. This signaling system is implicated in a wide range of biological processes, including pain sensation, immunomodulation, appetite regulation, development, and cognitive and emotional states. The balance between eCB release and inactivation determines the extent of eCB accumulation, with enzymatic hydrolysis functioning as an important limiting step. Pharmacological inhibition of eCB-hydrolyzing enzymes offers great therapeutic and experimental promise for enhancing this ubiquitous signaling system only where and when these transmitters are naturally produced. The following mini-review summarizes the latest developments concerning eCB-hydrolyzing enzymes, with an emphasis on the techniques used to measure their activities and how these have helped increase our understanding of the role that eCBs play in regulating fundamental biological functions.     

Modulation of the endocannabinoid system: Neuroprotection or neurotoxicity?

There is now a large volume of data indicating that compounds activating cannabinoid CB₁ receptors, either directly or indirectly by preventing the breakdown of endogenous cannabinoids, can protect against neuronal damage produced by a variety of neuronal “insults”. Given that such neurodegenerative stimuli result in increased endocannabinoid levels and that animals with genetic deletions of CB₁ receptors are more susceptible to the deleterious effects of such stimuli, a case can be made for an endogenous neuroprotective role of endocannabinoids. However, this is an oversimplification of the current literature, since (a) compounds released together with the endocannabinoids can contribute to the neuroprotective effect; (b) other proteins, such as TASK-1 and PPARα, are involved; (c) the CB₁ receptor antagonist/inverse agonist rimonabant has also been reported to have neuroprotective properties in a number of animal models of neurodegenerative disorders. Furthermore, the CB₂ receptor located on peripheral immune cells and activated microglia are potential targets for novel therapies. In terms of the clinical usefulness of targeting the endocannabinoid system for the treatment of neurodegenerative disorders, data are emerging, but important factors to be considered are windows of opportunity (for acute situations such as trauma and ischemia) and the functionality of the target receptors (for chronic neurodegenerative disorders such as Alzheimer's disease).     

Interactions of cannabidiol with endocannabinoid signalling in hippocampal tissue

The phytocannabinoid cannabidiol (CBD) possesses no psychotropic activity amid potentially beneficial therapeutic applications. We here characterized interactions between CBD (1 microM) and the endocannabinoid system in cultured rat hippocampal cells. The CBD-induced Ca2+ rise observed in neurons and glia was markedly reduced in the presence of the endogenous cannabinoid anandamide in neurons, with no alteration seen in glia. Neuronal CBD responses were even more reduced in the presence of the more abundant endocannabinoid 2-arachidonyl glycerol, this action was maintained in the presence of the CB1 receptor antagonist AM281 (100 nM). Neuronal CBD responses were also reduced by pre-exposure to glutamate, expected to increase endocannabinoid levels by increasing in [Ca2+]i. Application of AM281 at 1 microM elevated CBD-induced Ca2+ responses in both cell types, further confirming our finding that endocannabinoid-mediated signalling is negatively coupled to the action of CBD. However, upregulation of endogenous levels of endocannabinoids via inhibition of endocannabinoid hydrolysis (with URB597 and MAFP) could not be achieved under resting conditions. Because delta9-tetrahydrocannabinol did not mimic the endocannabinoid actions, and pertussis toxin treatment had no effect on CBD responses, we propose that the effects of AM281 were mediated via a constitutively active signalling pathway independent of CB1 signalling. Instead, signalling via G(q/11) and phospholipase C appears to be negatively coupled to CBD-induced Ca2+ responses, as the inhibitor U73122 enhanced CBD responses. Our data highlight the interaction between exogenous and endogenous cannabinoid signalling, and provide evidence for the presence of an additional pharmacological target, sensitive to endocannabinoids and to AM281.     

Immunohistochemical distribution of the cannabinoid receptor 1 and fatty acid amide hydrolase in the dog claustrum

Cannabinoid receptor 1 (CB1R) and fatty acid amide hydrolase (FAAH) are part of the endocannabinoid system (ECB) which exerts a neuromodulatory activity on different brain functions and plays a key role in neurogenesis. Although many studies have reported FAAH and CB1R expression in the brain of different animal species, to the best of our knowledge they have never been described in the canine claustrum. Claustrum samples, obtained from necropsy of four neurologically normal dogs, were formalin fixed for paraffin embedding. Sections were either stained for morpho-histological analysis or immunostained for CB1R and FAAH. Analysis of adjacent sections incubated with the two antisera showed a complementary labeling pattern in the claustrum, with CB1R antibody staining fibers while anti-FAAH antibody stained cell bodies and the proximal portion of dendrites; this particular anatomical relationship suggests a retrograde endocannabinoid action via CB1R. CB1R and FAAH complementary immunostaining and their cellular localization reported here provide the first anatomical evidence for existence of the ECB in the dog claustrum.     

Mapping human brain fatty acid amide hydrolase activity with PET

Endocannabinoid tone has recently been implicated in a number of prevalent neuropsychiatric conditions. [¹¹C]CURB is the first available positron emission tomography (PET) radiotracer for imaging fatty acid amide hydrolase (FAAH), the enzyme which metabolizes the prominent endocannabinoid anandamide. Here, we sought to determine the most suitable kinetic modeling approach for quantifying [¹¹C]CURB that binds selectively to FAAH. Six healthy volunteers were scanned with arterial blood sampling for 90 minutes. Kinetic parameters were estimated regionally using a one-tissue compartment model (TCM), a 2-TCM with and without irreversible trapping, and an irreversible 3-TCM. The 2-TCM with irreversible trapping provided the best identifiability of PET outcome measures among the approaches studied (coefficient of variation (COV) of the net influx constant K_i and the composite parameter λk₃ (λ=K₁/k₂) <5%, and COV(k₃)<10%). Reducing scan time to 60 minutes did not compromise the identifiability of rate constants. Arterial spin labeling measures of regional cerebral blood flow were only slightly correlated with K_i, but not with k₃ or λk₃. Our data suggest that λk₃ is sensitive to changes in FAAH activity, therefore, optimal for PET quantification of FAAH activities with [¹¹C]CURB. Simulations showed that [¹¹C]CURB binding in healthy subjects is far from a flow-limited uptake.     

Oleamide: A Fatty Acid Amide Signaling Molecule in the Cardiovascular System?

Oleamide (cis‐9,10‐octadecenoamide), a fatty acid primary amide discovered in the cerebrospinal fluid of sleep‐deprived cats, has a variety of actions that give it potential as a signaling molecule, although these actions have not been extensively investigated in the cardiovascular system. The synthetic pathway probably involves synthesis of oleoylglycine and then conversion to oleamide by peptidylglycine α?amidating monooxygenase (PAM); breakdown of oleamide is by fatty acid amide hydrolase (FAAH). Oleamide interacts with voltage‐gated Na⁺ channels and allosterically with GABA_A and 5‐HT₇ receptors as well as having cannabinoid‐like actions. The latter have been suggested to be due to potentiation of the effects of endocannabinoids such as anandamide by inhibiting FAAH‐mediated hydrolysis. This might underlie an “entourage effect” whereby co‐released endogenous nonagonist congeners of endocannabinoids protect the active molecule from hydrolysis by FAAH. However, oleamide has direct agonist actions at CB₁ cannabinoid receptors and also activates the TRPV1 vanilloid receptor. Other actions include inhibition of gap‐junctional communication, and this might give oleamide a role in myocardial development. Many of these actions are absent from the trans isomer of 9,10‐octadecenoamide. One of the most potent actions of oleamide is vasodilation. In rat small mesenteric artery the response does not involve CB₁ cannabinoid receptors but another pertussis toxin‐sensitive, G protein–coupled receptor, as yet unidentified. This receptor is sensitive to rimonabant and O‐1918, an antagonist at the putative “abnormal‐cannabidiol” or endothelial “anandamide” receptors. Vasodilation is mediated by endothelium‐derived nitric oxide, endothelium‐dependent hyperpolarization, and also through activation of TRPV1 receptors. A physiological role for oleamide in the heart and circulation has yet to be demonstrated, as has production by cells of the cardiovascular system, but this molecule has a range of actions that could give it considerable modulatory power.