Pharmacological and pharmacokinetic properties of JNJ-40411813, a positive allosteric modulator of the mGlu2 receptor

Compounds modulating metabotropic glutamate type 2 (mGlu2) receptor activity may have therapeutic benefits in treating psychiatric disorders like schizophrenia and anxiety. The pharmacological and pharmacokinetic properties of a novel mGlu2 receptor-positive allosteric modulator (PAM), 1-butyl-3-chloro-4-(4-phenyl-1-piperidinyl)-2(1H)-pyridinone (JNJ-40411813/ADX71149) are described here. JNJ-40411813 acts as a PAM at the cloned mGlu2 receptor: EC50 = 147 ± 42 nmol/L in a [35S]GTPγS binding assay with human metabotropic glutamate type 2 (hmGlu2) CHO cells and EC50 = 64 ± 29 nmol/L in a Ca2+ mobilization assay with hmGlu2 Gα16 cotransfected HEK293 cells. [35S]GTPγS autoradiography on rat brain slices confirmed PAM activity of JNJ-40411813 on native mGlu2 receptor. JNJ-40411813 displaced [3H]JNJ-40068782 and [3H]JNJ-46281222 (mGlu2 receptor PAMs), while it failed to displace [3H]LY341495 (a competitive mGlu2/3 receptor antagonist). In rats, JNJ-40411813 showed ex vivo mGlu2 receptor occupancy using [3H]JNJ-46281222 with ED50 of 16 mg/kg (p.o.). PK-PD modeling using the same radioligand resulted in an EC50 of 1032 ng/mL. While JNJ-40411813 demonstrated moderate affinity for human 5HT2A receptor in vitro (Kb = 1.1 μmol/L), higher than expected 5HT2A occupancy was observed in vivo (in rats, ED50 = 17 mg/kg p.o.) due to a metabolite. JNJ-40411813 dose dependently suppressed REM sleep (LAD, 3 mg/kg p.o.), and promoted and consolidated deep sleep. In fed rats, JNJ-40411813 (10 mg/kg p.o.) was rapidly absorbed (Cmax 938 ng/mL at 0.5 h) with an absolute oral bioavailability of 31%. Collectively, our data show that JNJ-40411813 is an interesting candidate to explore the therapeutic potential of mGlu2 PAMs, in in vivo rodents experiments as well as in clinical studies.


Introduction
Among the metabotropic glutamate (mGlu) receptors, mGlu2, an inhibitory presynaptic autoreceptor, has emerged as a novel therapeutic target for the treatment of psychiatric disorders including schizophrenia, depression and anxiety, which are characterized by glutamatergic dysfunction Riaza Bermudo-Soriano et al. 2012;Sanacora et al. 2012).
Testing of selective mGlu2/3 receptor agonists in animal studies involving N-methyl-D-aspartate (NMDA) receptor antagonists like phencyclidine (PCP) provided early evidence that mGlu2/3 receptors may represent a novel target for schizophrenia treatment. Both LY354740 and LY379268, potent orthosteric mGlu2/3 agonists, inhibit PCP-evoked increases in glutamate levels and PCPinduced hyperlocomotion in rats (Moghaddam and Adams 1998;Cartmell et al. 1999). A prodrug of another mGlu2/3 receptor agonist, LY404039, improved positive and negative symptoms and was well tolerated in patients with schizophrenia (Patil et al. 2007;Mezler et al. 2010). However, the improvements in schizophrenia-related symptoms were not confirmed in subsequent follow-up trials (Kinon et al. 2011;Stauffer et al. 2013), and hence it is questioned whether only particular symptoms or disease stages exhibit a glutamatergic-based origin (Goff and Coyle, 2001;Marsman et al., 2013), and whether earlier results can be generalized to the broader population of patients with schizophrenia or whether these are specific only to subpopulations of patients.
Activation of mGlu2/3 receptors also results in anxiolytic effects in preclinical models (Rorick-Kehn et al. 2007). In healthy volunteers, LY354740 ameliorated fearpotentiated startle and panic induction after CO 2 challenge . LY544344, the prodrug of LY354740, showed efficacy in the treatment of patients with generalized anxiety disorder (Dunayevich et al. 2008).
It has been suggested that mGlu2 but not mGlu3 receptor mediates the actions of the mGlu2/3 receptor orthosteric agonists LY379268 and LY404039 in mouse models predictive of antipsychotic activity (Fell et al. 2008). All agonists identified so far, however, lack mGlu2 receptor subtype selectivity and also act on the mGlu3 receptor (Fell et al. 2008;Woolley et al. 2008). Treatment with mGlu2/3 receptor agonists may also have potential limitations in terms of tolerance development (Galici et al. 2005). mGlu2-positive allosteric modulators (PAMs) are therefore developed as an alternative, as they increase the endogenous mGlu2 receptor signaling, have greater selectivity than orthosteric agonists, and may maintain activity based on local, transient, and temporal release of glutamate, possibly reducing the risk of tolerance. Preclinical proof-of-concept studies evaluating the mGlu2 receptor PAMs LY487379 and BINA in models predictive of anxiolytic or antipsychotic activity have demonstrated that PAMs can mimic the effects of direct agonists (Johnson et al., 2003(Johnson et al., , 2005Galici et al. 2005. The structurally novel allosteric potentiator THIIC (also known as LY2607540) also showed activity in models predicting anxiolytic or antidepressant effects (Fell et al. 2011).
Recently, the first clinical data with mGlu2 receptor PAMs were disclosed: AZD8529, tested as monotherapy in a phase 2 schizophrenia trial, was not effective, while the active comparator, risperidone, demonstrated activity (Litman et al. 2014). In an exploratory phase 2a study in schizophrenia, the mGlu2 PAM JNJ-40411813/ADX71149 (1-butyl-3-chloro-4-(4-phenyl-1-piperidinyl)-2(1H)-pyridinone) met the primary objectives of safety and tolerability. Moreover, patients treated with antipsychotics who experienced residual negative symptoms were identified as a subgroup of patients who may benefit from add-on treatment with JNJ-40411813 Kent et al. 2013). In the adjunctive treatment of major depressive disorder with significant anxious features, administration of JNJ-40411813 in the dose range tested did not appear to have a clinically significant impact on symptoms (Kent et al. 2014). The clinical results obtained with JNJ-40411813 may greatly increase our understanding of the potential of drugs modulating the mGlu2 receptor and further clinical studies, including in additional indications, are awaited.
Here, we describe the pharmacological and pharmacokinetic (PK) properties of JNJ-40411813, including receptor occupancy and effects on sleep-wake architecture in rats. The activities of JNJ-40411813 in behavioral in vivo models predictive of antipsychotic efficacy are described elsewhere (Lavreysen et al. submitted).

In vitro pharmacology
Functional mGlu2 assays [ 35 S]GTPcS binding was performed using membranes from Chinese hamster ovary (CHO) cells expressing the rat or human mGlu2 (hmGlu2) receptor. Ca 2+ assays were performed in G a16 -HEK 293 cells expressing the hmGlu2 receptor. The procedures for both these assays are described in Lavreysen et al. 2013.
[ 35 S]GTPcS binding to rat brain sections Autoradiography of agonist-stimulated [ 35 S]GTPcS binding in rat brain sections was performed as previously described (Lavreysen et al. 2013). The mGlu2 specificity of the assay was assessed by measuring [ 35 S]GTPcS binding in wild-type and mGlu2 knockout mice brain sections in parallel. Displacement studies, on both membranes prepared from hmGlu2-expressing CHO cells as well as rat cortex tissue, were performed using 4 nmol/L of [ 3 H]JNJ-46281222 (a specific mGlu2 radioligand with K D~3 nmol/L in membranes from hmGlu2 receptor transfected CHO cells and rat cortex membranes manuscript in preparation). Assay mixtures were incubated for 60 min at room temperature in 0.5 mL containing 75 lg (hmGlu2 CHO or cortex) membrane protein. Nonspecific binding (about 30% of total binding) was estimated in the presence of 10 lmol/L of JNJ-42341806. Filtration was performed using Unifilter-96 GF/C filters presoaked in 0.1% PEI and a 40-well manifold or 96-well Brandell harvester 96. After the addition of scintillation liquid, radioactivity on the filters was measured in a Microplate Scintillation and Luminescence Counter or Liquid Scintillation Analyzer (Perkin Elmer). Inhibition curves plotting percentage of total binding versus log concentrations of JNJ-40411813 were generated using GraphPad Prism (version 4.02) and analyzed using nonlinear regression analysis.

Selectivity assays mGlu receptor panel
Ca 2+ assays with human mGlu1, 3, 5, 7, or 8 receptorexpressing HEK 293 cells were performed as reported in Lavreysen et al. (2013), except for a slight change in the procedure for mGlu5: cells expressing the human mGlu5 receptor were seeded at 40,000 cells/well in MW384. Twenty-four hours after seeding, cells were incubated for 90 min in Ca 2+ assay kit (Molecular Devices) dissolved in saline PBS supplemented with 5 mmol/L probenecid, pH 7.4 (f.c. 2.5 mmol/L probenecid as loading buffer was added on the cell layer without removal of medium) before measurements.
Measurement of [ 35 S]GTPcS binding to membranes from CHO cells expressing the rat mGlu6 receptor and membranes from L929sA cells expressing the human mGlu4 receptor were conducted as described in Lavreysen et al. 2013.

Additional selectivity assays
The effect of JNJ-40411813 on [ 3 H]ketanserin binding to human 5HT 2A receptors expressed in NIH3T3 cells was evaluated; JNJ-40411813 was further assessed for activity at the D 2 receptor using the [ 35 S]GTPcS assays performed in CHO cells stably expressing the human D 2L receptor. Membranes containing CHO cells and JNJ-40411813 were diluted in assay buffer containing 50 mmol/L Tris, pH 7.4, 100 mmol/L NaCl, 1 mmol/L ethylene glycol tetraacetic acid (EGTA), 3 mmol/L MgCl 2 , 10 lmol/L GDP, 10 lmol/L Dithiothreitol (DTT). The assay buffer used for diluting the membranes additionally contained 10 lg/ mL saponin. The procedure followed for the [ 35 S]GTPcS assay was similar to that used for CHO cells expressing the hmGlu2 receptor. [ 35 S]GTPcS was used at a final concentration of 0.25 nmol/L, and 10 lmol/L dopamine was used as a positive control.
JNJ-40411813 was also tested at a concentration of 10 lmol/L by CEREP (Celle L'Evescault, France) for its inhibition of radioligand binding to a battery of neurotransmitter and peptide receptors, ion channels, and transporters. Functional 5HT 2A receptor assays were also performed at CEREP. These studies were conducted as per standard protocols.
In vivo pharmacology mGlu2 and 5HT 2A receptor occupancy Ex vivo mGlu2 occupancy studies were performed using [ 3 H]JNJ-46281222 manuscript in preparation). A dose-response experiment was performed to measure the ED 50 (dose at which there is 50% receptor occupancy) for mGlu2 occupancy 1 h following JNJ-40411813 (p.o.) administration. After sacrifice, rat brains were immediately removed from the skull and rapidly frozen. The mGlu2 occupancy was measured in the striatum from individual rats as follows: brain sections (20 lm thick) were incubated for 10 min with 1 nmol/L [ 3 H] JNJ-46281222 in 50 mmol/L Tris-HCl, pH 7.4 containing 2 mmol/L MgCl 2 , 2 mmol/L CaCl 2 , and 0.3% Bovine serum albumin (BSA). The sections were washed and dried under a stream of cold air and exposed in a b-imager-2000 (Biospace Lab, Paris, France) for 4 h. Radioactivity from striatal tissue was quantified using the Beta Vision+ program (Biospace Lab). Nonspecific binding was measured on adjacent sections in presence of 10 lmol/L JNJ-42341806. Specific binding was calculated as the difference between total binding and nonspecific binding. Percentage of receptor occupancy by the drug corresponded to 100% minus the percentage of receptor labeling. For the determination of ED 50 values, the percentage of receptor occupancy was plotted against dosage and the sigmoidal log dose-effect curve of best fit was calculated by nonlinear regression analysis using the GraphPad Prism program (version 4.02).
A time-course occupancy experiment was performed after s.c. (2.5 and 10 mg/kg) and p.o. (5 and 20 mg/kg) administration of JNJ-40411813 in male Wistar rats. The animals were sacrificed at specific time points (0.5, 1, 2, 4, 8, and 24 h after drug administration). Brains were processed as described above. To enable PK-PD modeling, levels of JNJ-40411813 were also analyzed in brain and plasma of each individual rat (PK analysis section). Data were analyzed using a relative maximal effect (E max ) model.
To evaluate 5HT 2A receptor occupancy, male Sprague-Dawley rats were treated with vehicle or increasing doses of JNJ-40411813 (s.c. or p.o.); the 5HT 2A receptor radioligand [ 3 H]MDL-100907 (10 lCi/animal) was injected intravenously (i.v.) 30 min before sacrifice. Brains were immediately dissected from the skull and rapidly frozen in dry ice-cooled 2-methylbutane (À40°C). Sections (20 lm thickness) were cut using a Leica CM 3050S cryostat-microtome (Leica, Brussels, Belgium), and thawmounted on microscope slides (SuperFrost Plus, Labo-Nord, France). Three frontal cortical sections and one cerebellum section were collected per glass slide. Brain sections were loaded in a b-imager-2000 (Biospace Lab) for 8 h and radioactivity emerging from the delineated brain area was quantified using the Beta Vision + program (Biospace Lab). The specific binding was calculated as the difference between the total binding in the frontal cortex and the cerebellum. The specific binding of [ 3 H] MDL-100907 in frontal cortex of drug-treated rats was expressed as the percentage of specific binding in vehicletreated rats. Percentage occupancy of the drug corresponded to 100% minus the percentage labeling in the treated animal. For the determination of ED 50 values, the percentage of receptor occupancy was plotted against dose and the sigmoidal log dose-effect curve of best fit was calculated by nonlinear regression analysis using the GraphPad Prism software (version 4.02).

Sleep-wake architecture in rats
The effect of oral administration of JNJ-40411813 on sleep-wake organization in rats was performed as described earlier (Ahnaou et al. 2009). In brief, male Sprague-Dawley rats were surgically implanted with electrodes for recording the cortical electroencephalogram (EEG), electrooculogram (EOG) and electromyogram (EMG). Recordings were performed for 20 h after p.o. administration of saline (n = 32) at the acrophase of sleep, followed by recordings for the same duration after acute p.o. administration of JNJ-40411813 (3, 10, and 30 mg/kg) and of vehicle (control) (n = 8 for each condition). Sleep-wake states and related variables were analyzed over 20 continuous hours using discriminative analysis based on 5 EEG frequency domain values (

Pharmacokinetic analysis
Male Sprague-Dawley rats were administered a single i.v.
Similarly, brain and plasma levels of JNJ-40411813 were analyzed in a time-course mGlu2 occupancy study and correlated with the percent occupancy of JNJ-40411813 in the same individual animal. An additional step of homogenization of brain tissue in nine parts distilled water was performed.

In vitro metabolism and identification
For metabolite identification purposes, JNJ-40411813 was incubated with human, rat, or mouse liver microsomes for 60 min at a substrate concentration of 5 lmol/L; and with human hepatocytes for 60 min at substrate concentrations of 5 and 50 lmol/L. Control incubations involved quenching with DMSO immediately after the addition of compound. Data were acquired on a Waters Acquity UPLC coupled with a QToF Premier mass spectrometer (Waters Corporation, Milford MA).
In combination with the recombinant CYP identification study (data not shown), inspection of the data suggested that four of the metabolites were accessible for NMR characterization. Scaled up incubations at 50 mmol/L in recombinant human (rh) CYP2D6 and 100 mmol/L in rhCYP3A4 were extracted and submitted for metabolite isolation and analysis by NMR.

In vivo excretion pathways
Rats were dosed with JNJ-40411813 (10 mg/kg, p.o.), urine samples were collected over 3 separate periods (0-2, 2-7, and 7-24 h post dose) and analyzed for drug-related material by LC/MS. For data interpretation, the terms major and minor are relative to each other, based on ion currents and should not be used as a formal quantification measure.

Statistics
In rats, value of vigilance states expressed as means AE SEM were analyzed by a mixed-model anaylsis of variance (ANOVA) to assess the influence of JNJ-40411813 on vigilance states and related variables. For all analyses, the significance level was set at P < 0.05. The effect of JNJ-40411813 was also confirmed on the rat mGlu2 receptor as it potentiated the effect of glutamate with an EC 50 of 370 AE 120 nmol/L (Eff curve was 479 AE 33%; n = 4). In stably transfected HEK293 cells expressing the hmGlu2 receptor, JNJ-40411813 potentiated glutamate-induced Ca 2+ signaling with an EC 50 of 64 AE 29 nmol/L (n = 33). JNJ-40411813 did not demonstrate mGlu2 receptor agonist activity up to 100 nmol/L (EC 50 was 1843 AE 905 nmol/L; n = 25).

In vitro
Although application of 10 lmol/L glutamate alone did not increase the [ 35 S]GTPcS signal significantly (0-5% over basal), coaddition of JNJ-40411813 clearly potentiated its response in brain regions known to express the mGlu2 receptor, more specifically in the cortical regions, striatum, and the hippocampus (Fig. 3A). In the cortex, JNJ-40411813 alone stimulated basal binding with an EC 50 of 48 lmol/L and a maximal effect of~220%. When combined with 10 lmol/L glutamate, JNJ-40411813 stimulated [ 35 S]GTPcS binding with an EC 50 of 7.3 lmol/L and a maximal effect of~380% (Fig. 3B). These effects were absent in mGlu2 KO mice ( Fig. 4A and B), indicating that they are exclusively mGlu2 receptor mediated.
Radioligand binding to hmGlu2 CHO membranes or rat cortical membranes JNJ-40411813 at 10 lmol/L did not displace binding of the orthosteric mGlu2 receptor antagonist [ 3 H]LY341495, indicating that JNJ-40411813 does not bind to the orthosteric receptor site (Fig. 5A). JNJ-40411813 displaced binding of [ 3 H]JNJ-40068782, a PAM acting on the mGlu2 receptor, with an IC 50 of 68 AE 29 nmol/L (n = 11) in CHO cells (Fig. 5B) and 83 AE 28 nmol/L (n = 2; data not shown) in membranes prepared from rat cortex. Binding inhibition was also confirmed using [ 3 H]JNJ-46281222, a novel radioligand that allowed the evaluation of ex vivo occupancy after systemic administration of JNJ-40411813. A summary of EC 50 and K i values is shown in Table 1.   (Table 2).
Additional studies conducted at CEREP indicated that JNJ-40411813 (up to 10 lmol/L) inhibited binding at the 5HT 2A receptor (93% inhibition), but did not produce a significant (>50%) binding inhibition of any of the other targets investigated. JNJ-40411813 inhibited serotonininduced Ca 2+ signaling at the human 5HT 2A receptor, with a K b value of 1.1 lmol/L and E max of 70%, indicating that it acts as a weak 5HT 2A antagonist.

In vivo mGlu2 and 5HT 2A receptor occupancy
In a dose-response experiment, JNJ-40411813 exhibited mGlu2 receptor occupancy with an ED 50 of 16 mg/kg 1 h after p.o. dosing (Fig. 6A). In a time-course occupancy experiment, JNJ-40411813 demonstrated maximum occupancy of rat brain mGlu2 receptors 0.5-1 h after s.c. (10 mg/kg) or p.o. (20 mg/kg) dosing. A decrease in mGlu2 receptor occupancy was observed over time, with about 50% occupancy detected up to 4 h after s.c. (10 mg/kg) dosing (Fig. 6B). Dosing at different routes and time points allowed refined PK-PD modeling; since plasma-occupancy relationships obtained after s.c. and p.o. dosing were overlapping (data not shown), data from both treatment groups were pooled to give a better estimation of the PK/PD parameters. Based on the collective plasma-occupancy relationship, the estimated concentration observed at 50% of the maximum response was 1032 ng/mL for plasma (Fig. 6C). JNJ-40411813 occupied the 5HT 2A receptor with an ED 50 of 11 mg/kg after s.c. and 17 mg/kg after oral p.o. administration (Fig. 7).  When restricted to the first 4 h of the recording session (Fig. 8B), the mixed-model ANOVA revealed that JNJ-40411813 dose dependently reduced the time spent in REM sleep (treatment 9 time interaction, F[9, 212] = 2.16, P < 0.05) and light sleep (treatment 9 time interaction, F[9, 212] = 3.44, P < 0.005), while it increased the time spent in deep sleep relative to control (treatment 9 time interaction, F[9, 212] = 3.45, P < 0.005). A lowest active dose (LAD) of 3 mg/kg p.o. was noted.

Sleep-wake architecture in rats
A detailed analysis of sleep variables showed that the decreased time spent in light sleep and REM sleep was derived from the decrease in the number of episodes and the mean duration of these vigilance states. The enhanced time spent in deep sleep resulted from an increase in the mean duration, whereas the number of periods of this vigilance state remained unchanged (data not shown). Examination of total number of state transitions indicated that JNJ-40411813 consistently reduced switches from deep sleep to passive waking ("treatment 9 time" interaction: F [9,212] = 2.6, P < 0.05), and from REM sleep to active waking ("treatment" F[3, 28] = 4.9, P < 0.05), "time" F[3, 212] = 0.9, P = 0.46); however, the ANOVA interaction was not significant ("treatment 9 time"; F[9, 212] = 1.3, P = 0.19) (Fig. 8C). The potent enhancing effect on deep sleep duration was consistent with marked reduction of transitions from light sleep to deep sleep stages ("treatment 9 time" interaction: F[9, 212] = 3.2, P < 0.005) suggesting a direct effect of JNJ-40411813 on the mechanism of initiation and maintenance of stable deep sleep episodes (Fig. 8D). Concomitant to changes in REM sleep state, JNJ-40411813 consistently lengthened the REM sleep onset latency (Fig. 8A right bottom upper panel).

Pharmacokinetics of JNJ-40411813
The PK analysis revealed that JNJ-40411813, after a single i.v dose of 2.5 mg, demonstrated a mean plasma exposure (AUC 0-∞ ) of 1833 AE 90 ng.h/mL. The mean plasma clearance (CL) was 1.4 AE 0.1 L/h/kg, and the mean volume of distribution (V dz ) was 2.3 AE 0.2 L/kg indicating distribution of the compound to other tissues. Plasma levels were below the limit of quantification at the 24-h time point (Fig. 9). JNJ-40411813 was rapidly absorbed following a single p.o. administration to fed rats at a dose of 10 mg/ kg, with a C max of 938 ng/mL at 0.5 h post dose. The mean exposure (AUC 0-∞ ) was 2250 AE 417 ng.hour/mL, resulting in an absolute oral bioavailability of 31%. Plasma levels decreased with a mean half-life (t 1/2 [2-7 h]) of 2.3 AE 0.5 h.
Mean plasma concentration-time profiles were comparable after single p.o. dosing at 2.5, 5, 10, and 20 mg/kg, and C max and AUC 0-∞ values increased proportionally across the dose range tested (data not shown).

Metabolism of JNJ-40411813
JNJ-40411813 forms a variety of oxidative metabolites in human, rat, and mouse liver microsomes. The major metabolites excreted in rat urine were consistent with the in vitro findings. One of the JNJ-40411813 metabolites (JNJ-42159052; Fig. 10

Discussion
Modulation of mGlu2/3 receptor activity has emerged as a novel potential therapeutic strategy for treating psychiatric and neurological disorders (Niswender and Conn 2010). Extensive research conducted on either direct agonist or allosteric modulator approaches to regulate mGlu2 receptor function is aimed to provide benefit in the treatment of these disorders (Trabanco et al. 2011;Trabanco and Cid 2013).
We showed that JNJ-40411813 exhibits mGlu2 PAM activity, both at cloned mGlu2 receptors as well as native receptors in the brain tissue. JNJ-40411813 at higher concentrations also induced modest activation of [ 35 S]GTPcS binding in absence of glutamate, suggesting that the The pharmacological read-out used to detect each activity is indicated in brackets. compound has some intrinsic agonist efficacy. However, the possibility that this is, in fact, a modulation of endogenous, albeit low levels of glutamate cannot be excluded. JNJ-40411813 also increased the glutamate-induced [ 35 S] GTPcS signal in brain regions known to express the mGlu2 receptor (specifically in cortical regions, striatum, and hippocampus). Unlike agonists that bind directly to the orthosteric binding site of the receptor, PAMs exhibit a unique mechanism of action wherein they bind to an allosteric binding site that is distant from the agonist binding site (Niswender and Conn 2010). JNJ-40411813 displaced [ 3 H]JNJ-40068782 and [ 3 H]JNJ-46281222, both PAMs of the mGlu2 receptor, but failed to displace [ 3 H]LY341495, a competitive mGlu2/3 receptor antagonist, thus, confirming that it binds to an allosteric site. Furthermore, in presence of JNJ-40411813, the binding affinity of the mGlu2/3 receptor agonist DCG-IV was increased, without affecting the number of binding sites, thus confirming earlier observations (Lavreysen et al. 2013).
JNJ-40411813 demonstrated high selectivity for the mGlu2 receptor with no agonist, and antagonist activity for other mGlu receptors. Additionally, we did not find appreciable mGlu3 PAM activity (EC 50 for mGlu3 PAM activity >150-fold EC 50 for mGlu2 PAM activity), indicating that JNJ-40411813 acts as a selective mGlu2 PAM. JNJ-40411813 inhibited 5HT 2A receptor function in vitro, although at~10-fold higher concentrations compared with the mGlu2 receptor. JNJ-40411813 did not activate the D 2 receptor (data not shown), indicating that JNJ-40411813 does not act via the dopaminergic system.
To our knowledge, this is the first time that direct rodent mGlu2 target engagement studies are reported. With use of the novel mGlu2-specific radioligand mGlu2 receptor after systemic administration, an important finding that permits the linking of receptor occupancy levels with efficacy in in vivo pharmacodynamic readouts or animal models of disease. PK-PD modeling resulted in an EC 50 value of 1032 ng/mL. Interestingly, although there seemed to be a good relationship between JNJ-40411813 plasma concentrations and receptor binding to the mGlu2 receptor, JNJ-40411813 unexpectedly also bound 5HT 2A receptors to the same level at similar doses, which could not be explained by the moderate in vitro 5HT 2A potency of JNJ-40411813. Further experiments revealed that JNJ-40411813 exhibited a longer duration of action for 5HT 2A versus mGlu2 receptor occupancy (data not shown). We found that in rats, JNJ-40411813 is metabolized to a variety of oxidative metabolites, including the metabolite JNJ-42159052, a relatively potent 5HT 2A receptor antagonist, which in all likelihood, contributes to and explains the relatively high 5HT 2A receptor binding in rats. These findings suggest that both mGlu2 receptor activity and 5HT 2A receptor antagonism can contribute to the preclinical in vivo activity profile of JNJ-40411813. Importantly, because this metabolite is not found to the same extent in humans, it is believed to be clinically irrelevant .
JNJ-40411813 significantly suppressed REM and promoted deep sleep in rats. Assessment of sleep-wake cycle is an accurate and validated tool that is sensitive to centrally active drugs. Glutamate and serotonin signaling are the key neurochemical components of sleep-promoting and arousal centers in the brain. Glutamate release shows rhythmic fluctuations reaching maximal levels during waking and REM sleep in cortical areas such as the rostromedial medulla, orbitofrontal, and prefrontal cortex (Kodama et al. 1998;Lopez-Rodriguez et al. 2007). The mGlu2 receptors are localized predominantly in presynaptic terminals of glutamate neurons, where they inhibit the release of glutamate. Therefore, it is not surprising that pharmacologic modulation of mGlu2 receptor would be effective in modulating vigilance states.
Previous pharmacological studies using mGlu2 receptor agonist and PAMs demonstrated that central or systemic activation of mGlu2 receptor consistently suppressed REM sleep and prolonged its onset latency in rats (Ahnaou et al. 2009;Fell et al. 2011;Siok et al. 2012;Lavreysen et al. 2013). The specific suppression effect on REM sleep was confirmed in wild type, but not in mGlu2 receptor knockout mice (Ahnaou et al. 2009). The concentration of JNJ-40411813 that achieved ≥30% mGlu2 receptor occupancy resulted in consistent inhibition of REM sleep, indicating that relatively low levels of occupancy may be sufficient for this particular effect.
The distribution of serotonin 5HT 2A receptors in the brain regions known to be important in the regulation of sleep and wake, such as the hypothalamus and thalamus, aligns with the growing body of preclinical and clinical data to support a role for 5HT 2A antagonists in promotion of deep sleep, and therefore these antagonists offer potential to address several unmet needs in insomnia pharmacotherapy (Vanover and Davis 2010;Zisapel 2012). It is likely that the functional effects of JNJ-40411813 on deep sleep observed in this study arise from an interaction with the 5HT 2A receptor via the metabolite JNJ-42159052. 5HT 2A antagonists were shown to have no significant effect on REM sleep (Morairty et al. 2008), further supporting the hypothesis that the observed reduced REM sleep is due to mGlu2 receptor activation.
The majority of hypothesized functions for deep sleep and REM sleep states, and in particular the one that received the most attention has been waking related. The   idea that both sleep states are necessary for optimal cognitive function in waking has been the basis of a number of related hypotheses in the recent scientific literature. The dual-process hypothesis proposes that deep sleep is beneficial for declarative memories, whereas REM sleep is important for consolidation of nondeclarative, procedural, and emotional memories (Ackermann and Rasch 2014). The fact that REM sleep naturally follows deep sleep supports the complementing contributions of sequential deep sleep and REM sleep to synaptic form of consolidation for stabilizing memories. Accordingly, one would expect that suppression of REM sleep impairs memory consolidation. Pharmacological suppression of REM sleep in human by administration of antidepressants (selective noradrenaline or serotonin reuptake inhibitors) did not impair memory consolidation (Rasch et al. 2009), which is in agreement with clinical observations that antidepressant treatment does not affect memory function.
JNJ-40411813 when administered at a single dose of 10 mg/kg in fed rats was absorbed rapidly, with moderate oral bioavailability (31%). This could be attributed to low water solubility of JNJ-40411813, leading to incomplete in vivo dissolution and hepatic metabolism of the drug. JNJ-40411813 (2.5 mg/kg, i.v.) was well distributed to other tissues, due to both its lipophilic property and high cell permeability which enables easy permeability across the cell membrane. The mean plasma concentration-time profile of JNJ-40411813 was linear across the dose range tested.
In conclusion, JNJ-40411813, a novel, potent, and systemically active mGlu2 PAM, showed more than 50-fold selectivity for the mGlu2 receptor over other subtypes and moderate in vitro affinity for the 5HT 2A receptor. Nevertheless, it could not explain the equipotency observed in terms of mGlu2 and 5HT 2A occupancy. Rather, JNJ-40411813 metabolite accounted for additional 5HT 2A engagement in vivo. The combined mGlu2 PAM/ 5HT 2A antagonist profile of JNJ-40411813 was highlighted by an effect on REM sleep as well as deep sleep in rats. The in vivo behavioral effects of JNJ-40411813, which are described elsewhere (Lavreysen et al. submitted) prompted us to select JNJ-40411813 for further clinical development, which is ongoing. Chikramane, Ph.D. (SIRO Clinpharm Pvt. Ltd.) provided writing assistance and Wendy Battisti (Janssen Research & Development, LLC.) provided additional editorial and scientific support for this manuscript.

Author Contributions
Lavreysen, Ahnaou, Mackie, Langlois, Drinkenburg, Pype, L€ utjens, and Le Poul participated in study design. Lavreysen, Ahnaou, Mackie, Langlois, Drinkenburg, Pype, L€ utjens, Le Poul, Trabanco, and Cid Nuñez performed data analysis and/or data interpretation. Cid Nuñez contributed to the discovery of the investigation compound. Lavreysen, Ahnaou, Mackie, Langlois, Drinkenburg, Pype, L€ utjens, Le Poul, Trabanco, and Cid Nuñez wrote or contributed to the writing of this manuscript.   of Addex Therapeutics, Switzerland. All authors met IC-MJE criteria and all those who fulfilled those criteria are listed as authors. All authors had access to the study data, provided direction and comments on the manuscript, made the final decision about where to publish these data, and approved submission to the journal.