Pharmacologic Characterization of JNJ-42226314, [1-(4-Fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone, a Reversible, Selective, and Potent Monoacylglycerol Lipase Inhibitor.

The serine hydrolase monoacylglycerol lipase (MAGL) is the rate-limiting enzyme responsible for the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) into arachidonic acid and glycerol. Inhibition of 2-AG degradation leads to elevation of 2-AG, the most abundant endogenous agonist of the cannabinoid receptors (CBs) CB1 and CB2. Activation of these receptors has demonstrated beneficial effects on mood, appetite, pain, and inflammation. Therefore, MAGL inhibitors have the potential to produce therapeutic effects in a vast array of complex human diseases. The present report describes the pharmacologic characterization of [1-(4-fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone (JNJ-42226314), a reversible and highly selective MAGL inhibitor. JNJ-42226314 inhibits MAGL in a competitive mode with respect to the 2-AG substrate. In rodent brain, the compound time- and dose-dependently bound to MAGL, indirectly led to CB1 occupancy by raising 2-AG levels, and raised norepinephrine levels in cortex. In vivo, the compound exhibited antinociceptive efficacy in both the rat complete Freund's adjuvant-induced radiant heat hypersensitivity and chronic constriction injury-induced cold hypersensitivity models of inflammatory and neuropathic pain, respectively. Though 30 mg/kg induced hippocampal synaptic depression, altered sleep onset, and decreased electroencephalogram gamma power, 3 mg/kg still provided approximately 80% enzyme occupancy, significantly increased 2-AG and norepinephrine levels, and produced neuropathic antinociception without synaptic depression or decreased gamma power. Thus, it is anticipated that the profile exhibited by this compound will allow for precise modulation of 2-AG levels in vivo, supporting potential therapeutic application in several central nervous system disorders. SIGNIFICANCE STATEMENT: Potentiation of endocannabinoid signaling activity via inhibition of the serine hydrolase monoacylglycerol lipase (MAGL) is an appealing strategy in the development of treatments for several disorders, including ones related to mood, pain, and inflammation. [1-(4-Fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone is presented in this report to be a novel, potent, selective, and reversible noncovalent MAGL inhibitor that demonstrates dose-dependent enhancement of the major endocannabinoid 2-arachidonoylglycerol as well as efficacy in models of neuropathic and inflammatory pain.

In vivo Characterization of a Selective, Orally Available, and Brain Penetrant Small Molecule GPR139 Agonist.

Recently, our group along with another demonstrated that GPR139 can be activated by L-phenylalanine (L-Phe) and L-tryptophan (L-Trp) at physiologically relevant concentrations. GPR139 is discretely expressed in brain, with highest expression in medial habenula. Not only are the endogenous ligands catecholamine/serotonin precursors, but GPR139 expressing areas can directly/indirectly regulate the activity of catecholamine/serotonin neurons. Thus, GPR139 appears expressed in an interconnected circuit involved in mood, motivation, and anxiety. The aim of this study was to characterize a selective and brain penetrant GPR139 agonist (JNJ-63533054) in relevant in vivo models. JNJ-63533054 was tested for its effect on c-fos activation in the habenula and dorsal striatum. In vivo microdialysis experiments were performed in freely moving rats to measure basal levels of serotonin or dopamine (DA) in prefrontal cortex (mPFC) and nucleus accumbens (NAc). Finally, the compound was profiled in behavioral models of anxiety, despair, and anhedonia. The agonist (10-30 mg/kg, p.o.) did not alter c-fos expression in medial habenula or dorsal striatum nor neurotransmitter levels in mPFC or NAc. JNJ-63533054 (10 mg/kg p.o.) produced an anhedonic-like effect on urine sniffing, but had no significant effect in tail suspension, with no interaction with imipramine, no effect on naloxone place aversion, and no effect on learned helplessness. In the marble burying test, the agonist (10 mg/kg p.o.) produced a small anxiolytic-like effect, with no interaction with fluoxetine, and no effect in elevated plus maze (EPM). Despite GPR139 high expression in medial habenula, an area with connections to limbic and catecholaminergic/serotoninergic areas, the GPR139 agonist had no effect on c-fos in medial habenula. It did not alter catecholamine/serotonin levels and had a mostly silent signal in in vivo models commonly associated with these pathways. The physiological function of GPR139 remains elusive.

In vivo Calcium Imaging Reveals That Cortisol Treatment Reduces the Number of Place Cells in Thy1-GCaMP6f Transgenic Mice.

The hippocampus, a structure essential for spatial navigation and memory undergoes anatomical and functional changes during chronic stress. Here, we investigate the effects of chronic stress on the ability of place cells to encode the neural representation of a linear track. To model physiological conditions of chronic stress on hippocampal function, transgenic mice expressing the genetically encoded calcium indicator GCaMP6f in CA1 pyramidal neurons were chronically administered with 40 µg/ml of cortisol for 8 weeks. Cortisol-treated mice exhibited symptoms typically observed during chronic stress, including diminished reward seeking behavior and reduced adrenal gland and spleen weights. In vivo imaging of hippocampal cellular activity during linear track running behavior revealed a reduced number of cells that could be recruited to encode spatial position, despite an unchanged overall number of active cells, in cortisol-treated mice. The properties of the remaining place cells that could be recruited to encode spatial information, however, was unperturbed. Bayesian decoders trained to estimate the mouse's position on the track using single neuron activity data demonstrated reduced performance in a cue richness-dependent fashion in cortisol-treated animals. The performance of decoders utilizing data from the entire neuronal ensemble was unaffected by cortisol treatment. Finally, to test the hypothesis that an antidepressant drug could prevent the effects of cortisol, we orally administered a group of mice with 10 mg/kg citalopram during cortisol administration. Citalopram prevented the cortisol-induced decrease in single-neuron decoder performance but failed to significantly prevent anhedonia and the cortisol-induced reduction in the proportion place cells. The dysfunction observed at the single-neuron level indicates that chronic stress may impair the ability of the hippocampus to encode individual neural representations of the mouse's spatial position, a function pivotal to forming an accurate navigational map of the mouse's external environment; however, the hippocampal ensemble as a whole is resilient to any cortisol-induced insults to single neuronal place cell function on the linear track.

Characterization of JNJ-42847922, a Selective Orexin-2 Receptor Antagonist, as a Clinical Candidate for the Treatment of Insomnia.

Dual orexin receptor antagonists have been shown to promote sleep in various species, including humans. Emerging research indicates that selective orexin-2 receptor (OX2R) antagonists may offer specificity and a more adequate sleep profile by preserving normal sleep architecture. Here, we characterized JNJ-42847922 ([5-(4,6-dimethyl-pyrimidin-2-yl)-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl]-(2-fluoro-6-[1,2,3]triazol-2-yl-phenyl)-methanone), a high-affinity/potent OX2R antagonist. JNJ-42847922 had an approximate 2-log selectivity ratio versus the human orexin-1 receptor. Ex vivo receptor binding studies demonstrated that JNJ-42847922 quickly occupied OX2R binding sites in the rat brain after oral administration and rapidly cleared from the brain. In rats, single oral administration of JNJ-42847922 (3-30 mg/kg) during the light phase dose dependently reduced the latency to non-rapid eye movement (NREM) sleep and prolonged NREM sleep time in the first 2 hours, whereas REM sleep was minimally affected. The reduced sleep onset and increased sleep duration were maintained upon 7-day repeated dosing (30 mg/kg) with JNJ-42847922, then all sleep parameters returned to baseline levels following discontinuation. Although the compound promoted sleep in wild-type mice, it had no effect in OX2R knockout mice, consistent with a specific OX2R-mediated sleep response. JNJ-42847922 did not increase dopamine release in rat nucleus accumbens or produce place preference in mice after subchronic conditioning, indicating that the compound lacks intrinsic motivational properties in contrast to zolpidem. In a single ascending dose study conducted in healthy subjects, JNJ-42847922 increased somnolence and displayed a favorable pharmacokinetic and safety profile for a sedative/hypnotic, thus emerging as a promising candidate for further clinical development for the treatment of insomnia.

Pharmacological characterization of a novel centrally permeable P2X7 receptor antagonist: JNJ-47965567.

BACKGROUND AND PURPOSE: An increasing body of evidence suggests that the purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7) in the CNS may play a key role in neuropsychiatry, neurodegeneration and chronic pain. In this study, we characterized JNJ-47965567, a centrally permeable, high-affinity, selective P2X7 antagonist. EXPERIMENTAL APPROACH: We have used a combination of in vitro assays (calcium flux, radioligand binding, electrophysiology, IL-1ß release) in both recombinant and native systems. Target engagement of JNJ-47965567 was demonstrated by ex vivo receptor binding autoradiography and in vivo blockade of Bz-ATP induced IL-1ß release in the rat brain. Finally, the efficacy of JNJ-47965567 was tested in standard models of depression, mania and neuropathic pain. KEY RESULTS: JNJ-47965567 is potent high affinity (pKi 7.9 ± 0.07), selective human P2X7 antagonist, with no significant observed speciation. In native systems, the potency of the compound to attenuate IL-1ß release was 6.7 ± 0.07 (human blood), 7.5 ± 0.07 (human monocytes) and 7.1 ± 0.1 (rat microglia). JNJ-47965567 exhibited target engagement in rat brain, with a brain EC50 of 78 ± 19 ng·mL(-1) (P2X7 receptor autoradiography) and functional block of Bz-ATP induced IL-1ß release. JNJ-47965567 (30 mg·kg(-1) ) attenuated amphetamine-induced hyperactivity and exhibited modest, yet significant efficacy in the rat model of neuropathic pain. No efficacy was observed in forced swim test. CONCLUSION AND IMPLICATIONS: JNJ-47965567 is centrally permeable, high affinity P2X7 antagonist that can be used to probe the role of central P2X7 in rodent models of CNS pathophysiology.

JNJ-39220675, a novel selective histamine H3 receptor antagonist, reduces the abuse-related effects of alcohol in rats.

RATIONALE: A few recent studies suggest that brain histamine levels and signaling via H(3) receptors play an important role in modulation of alcohol stimulation and reward in rodents. OBJECTIVE: The present study characterized the effects of a novel, selective, and brain penetrant H(3) receptor antagonist (JNJ-39220675) on the reinforcing effects of alcohol in rats. METHODS: The effect of JNJ-39220675 on alcohol intake and alcohol relapse-like behavior was evaluated in selectively bred alcohol-preferring (P) rats using the standard two-bottle choice method. The compound was also tested on operant alcohol self administration in non-dependent rats and on alcohol-induced ataxia using the rotarod apparatus. In addition, alcohol-induced dopamine release in the nucleus accumbens was tested in freely moving rats. RESULTS: Subcutaneous administration of the selective H(3) receptor antagonist dose-dependently reduced both alcohol intake and preference in alcohol-preferring rats. JNJ-39220675 also reduced alcohol preference in the same strain of rats following a 3-day alcohol deprivation. The compound significantly and dose-dependently reduced alcohol self-administration without changing saccharin self-administration in alcohol non-dependent rats. Furthermore, the compound did not change the ataxic effects of alcohol, alcohol elimination rate, nor alcohol-induced dopamine release in nucleus accumbens. CONCLUSIONS: These results indicate that blockade of H(3) receptor should be considered as a new attractive mechanism for the treatment of alcoholism.

Selective blockade of the orexin-2 receptor attenuates ethanol self-administration, place preference, and reinstatement.

RATIONALE: Orexin-1 receptor antagonists have been shown to block the reinforcing effects of drugs of abuse and food. However, whether blockade of orexin-2 receptor has similar effects has not been determined. We have recently described the in vitro and in vivo effects of JNJ-10397049, a selective and brain penetrant orexin-2 receptor antagonist. OBJECTIVE: The goal of these studies was to evaluate whether systemic administration of JNJ-10397049 blocks the rewarding effects of ethanol and reverses ethanol withdrawal in rodents. As a comparison, SB-408124, a selective orexin-1 receptor antagonist, was also evaluated. METHODS: Rats were trained to orally self-administer ethanol (8% v/v) or saccharin (0.1% v/v) under a fixed-ratio 3 schedule of reinforcement. A separate group of rats received a liquid diet of ethanol (8% v/v) and withdrawal signs were evaluated 4 h after ethanol discontinuation. In addition, ethanol-induced increases in extracellular dopamine levels in the nucleus accumbens were tested. In separate experiments, the acquisition, expression, and reinstatement of conditioned place preference (CPP) were evaluated in mice. RESULTS: Our results indicate that JNJ-10397049 (1, 3, and 10 mg/kg, sc) dose-dependently reduced ethanol self-administration without changing saccharin self-administration, dopamine levels, or withdrawal signs in rats. Treatment with JNJ-10397049 (10 mg/kg, sc) attenuated the acquisition, expression, and reinstatement of ethanol CPP and ethanol-induced hyperactivity in mice. Surprisingly, SB-408124 (3, 10 and 30 mg/kg, sc) did not have any effect in these procedures. CONCLUSIONS: Collectively, these results indicate, for the first time, that blockade of orexin-2 receptors is effective in reducing the reinforcing effects of ethanol.

In vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a selective brain penetrant small molecule antagonist of the neuropeptide Y Y(2) receptor.

RATIONALE: The lack of potent, selective, brain penetrant Y(2) receptor antagonists has hampered in vivo functional studies of this receptor. OBJECTIVE: Here, we report the in vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a novel Y(2) receptor antagonist. METHODS: The affinity of JNJ-31020028 was determined by inhibition of the PYY binding to human Y(2) receptors in KAN-Ts cells and rat Y(2) receptors in rat hippocampus. The functional activity was determined by inhibition of PYY-stimulated calcium responses in KAN-Ts cells expressing a chimeric G protein Gqi5 and in the rat vas deferens (a prototypical Y(2) bioassay). Ex vivo receptor occupancy was revealed by receptor autoradiography. JNJ-31020028 was tested in vivo with microdialysis, in anxiety models, and on corticosterone release. RESULTS: JNJ-31020028 bound with high affinity (pIC(50) = 8.07 +/- 0.05, human, and pIC(50) = 8.22 +/- 0.06, rat) and was >100-fold selective versus human Y(1), Y(4), and Y(5) receptors. JNJ-31020028 was demonstrated to be an antagonist (pK(B) = 8.04 +/- 0.13) in functional assays. JNJ-31020028 occupied Y(2) receptor binding sites (approximately 90% at 10 mg/kg) after subcutaneous administration in rats. JNJ-31020028 increased norepinephrine release in the hypothalamus, consistent with the colocalization of norepinephrine and neuropeptide Y. In a variety of anxiety models, JNJ-31020028 was found to be ineffective, although it did block stress-induced elevations in plasma corticosterone, without altering basal levels, and normalized food intake in stressed animals without affecting basal food intake. CONCLUSION: These results suggest that Y(2) receptors may not be critical for acute behaviors in rodents but may serve modulatory roles that can only be elucidated under specific situational conditions.

The effects of thioperamide on extracellular levels of glutamate and GABA in the rat prefrontal cortex.

RATIONALE: Histamine H3 receptors (H3R) are presynaptic heteroreceptors that negatively modulate the release of histamine and other neurotransmitters such as acetylcholine. Blocking H3 receptors with antagonists/inverse agonists has been shown to be procognitive and this effect has often been associated with increases in acetylcholine transmission. H3 receptors are abundantly expressed in the prefrontal cortex, an area associated with cognitive performance. While the procognitive effects of H3 receptor antagonists/inverse agonists may depend on alterations to acetylcholine or histamine release, other transmitters involved in cognitive processing such as glutamate and gamma-aminobutyric acid (GABA) may also be involved. OBJECTIVE: The purpose of the present study was to examine the effects of thioperamide, an H3 receptor antagonist, on extracellular levels of glutamate and GABA in the prefrontal cortex. MATERIALS AND METHODS: By means of in vivo microdialysis on freely moving Sprague Dawley rats, samples were collected and assayed via high-performance liquid chromatography coupled to electrochemical detection. RESULTS: Replacement of calcium with magnesium revealed that the release of GABA, but not glutamate, was calcium-dependent. Thioperamide (10-20 mg/kg) did not affect basal glutamate or GABA release. Perfusion with a high concentration of potassium (100 mM) increased GABA, but not glutamate, release and thioperamide (20 mg/kg) attenuated the effects of high potassium on GABA release. CONCLUSION: These data indicate that H3 receptors in the prefrontal cortex can enhance stimulated GABA release, but do not regulate basal levels of glutamate or GABA.