Metabotropic glutamate receptor 7 (mGluR7) remains the most elusive of the eight known mGluRs primarily because of the limited availability of tool compounds to interrogate its potential therapeutic utility. The discovery of N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082) as the first orally active, brain-penetrable, mGluR7-selective allosteric agonist by Mitsukawa and colleagues (Proc Natl Acad Sci USA 102:18712-18717, 2005) provides a means to investigate this receptor system directly. AMN082 demonstrates mGluR7 agonist activity in vitro and interestingly has a behavioral profile that supports utility across a broad spectrum of psychiatric disorders including anxiety and depression. The present studies were conducted to extend the in vitro and in vivo characterization of AMN082 by evaluating its pharmacokinetic and metabolite profile. Profiling of AMN082 in rat liver microsomes revealed rapid metabolism (t(1/2) < 1 min) to a major metabolite, N-benzhydrylethane-1,2-diamine (Met-1). In vitro selectivity profiling of Met-1 demonstrated physiologically relevant transporter binding affinity at serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET) (323, 3020, and 3410 nM, respectively); whereas the parent compound AMN082 had appreciable affinity at NET (1385 nM). AMN082 produced antidepressant-like activity and receptor occupancy at SERT up to 4 h postdose, a time point at which AMN082 is significantly reduced in brain and plasma while the concentration of Met-1 continues to increase in brain. Acute Met-1 administration produced antidepressant-like activity as would be expected from its in vitro profile as a mixed SERT, NET, DAT inhibitor. Taken together, these data suggest that the reported in vivo actions of AMN082 should be interpreted with caution, because they may involve other mechanisms in addition to mGluR7.

Alpha-7 nicotinic acetylcholine receptor (alpha 7 nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment. We report a series of novel, potent small molecule agonists (4-18) of the alpha 7 nACh R deriving from our continuing efforts in the areas of Alzheimer's disease and schizophrenia. One of the compounds of the series containing a urea moiety (16) was further shown to be a selective agonist of the alpha 7 nAChR with excellent in vitro and in vivo profiles, brain penetration, and oral bioavailability and demonstrated in vivo efficacy in multiple behavioral cognition models. Structural modifications leading to the improved selectivity profile and the biological evaluation of this series of compounds are discussed.

In functional assay assessments using the five muscarinic receptor subtypes, a second generation of muscarinic M(1)-preferring receptor agonists [AC-42 (1), AC-260584 (2), 77-LH-28-1 (3) and LY-593039 (4)] was shown to have higher selectivity for muscarinic M(1) over M(3) receptor as compared to historical agonists [talsaclidine (8), sabcomeline (10), xanomeline (11), WAY-132983 (12), cevimeline (9) and NGX-267 (6)]. Another striking difference of these more recent compounds is their affinities for the dopamine D(2) and 5-HT(2B) receptors. Taken together, these results suggest that the newer compounds may have a greater clinical safety profile, especially with regard to muscarinic M(3) receptor-mediated events, than the historical agonists, but their affinities for other receptors may still compromise their use to validate the therapeutic potential of muscarinic M(1) receptor agonists.

The alpha 7 nicotinic acetylcholine receptor (nAChR) has been implicated in Alzheimer's disease and schizophrenia, leading to efforts targeted toward discovering agonists and positive allosteric modulators (PAMs) of this receptor. In a Ca2+ flux fluorometric imaging plate reader assay, SB-206553 ( 3,5-dihydro-5-methyl-N-3-pyridinylbenzo[1,2-b:4,5-b']-di pyrrole-1(2H)-carboxamide),a compound known as a 5-hydroxytryptamine(2B/2C) receptor antagonist, produced an 8-fold potentiation of the evoked calcium signal in the presence of an EC20 concentration of nicotine and a corresponding EC50 of 1.5 mu M for potentiation of EC20 nicotine responses in GH4C1 cells expressing the alpha 7 receptor. SB-206553 was devoid of direct alpha 7 receptor agonist activity and selective against other nicotinic receptors. Confirmation of the PAM activity of SB-206553 on the alpha 7 nAChR was obtained in patch-clamp electrophysiological experiments in GH4C1 cells, where it failed to evoke any detectable currents when applied alone, yet dramatically potentiated the currents evoked by an EC20 (17 mu M) and EC100 (124 mu M) of acetylcholine (ACh). Native nicotinic receptors in CA1 stratum radiatum interneurons of rat hippocampal slices could also be activated by ACh (200 mu M), an effect that was entirely blocked by the alpha 7-selective antagonist methyllycaconitine (MLA). These ACh currents were potentiated by SB-206553, which increased the area of the current response significantly, resulting in a 40-fold enhancement at 100 mu M. In behavioral experiments in rats, SB-206553 reversed an MK-801 (dizocilpine maleate)-induced deficit in the prepulse inhibition of acoustic startle response, an effect attenuated in the presence of MLA. This latter observation provides further evidence in support of the potential therapeutic utility of alpha 7 nAChR PAMs in schizophrenia.

The α-7 nicotinic receptor is an important neuronal subtype of ligand gated ion channels. It forms a pentameric homomer that is activated by acetylcholine or nicotine to evoke rapidly activating and desensitizing currents. Activation of α-7 receptors has been implicated as a therapeutic strategy in schizophrenia and Alzheimer disease. Small molecule positive allosteric modulators (PAM's) have been shown to enhance α-7 currents, and are classified as type 1 or type 2 PAMs. The type 1 PAMs enhance the current amplitude but do not alter desensitization, while the type 2 PAMs enhance the current amplitude but also slow the desensitization of the receptor. In order to understand the mechanism of action of the type 1 and type 2 PAM's, we tested three modulators on the QPatch automated patch-clamp system using GH4C1 cells stably expressing the rat α-7 receptor. We also explored the effect of repeated applications of these agents on their modulatory activity. Consistent with previously published data, PNU-120596 showed type 2 PAM activity accompanied with a decreasing magnitude of potentiation with repeated applications. Estimated EC50 values for PNU-120596 were stable with repeated compound application. NS-1738 produced a type 1 PAM activity, and a cumulatively increased potentiation following repeated applications, accompanied by an approximate 3-fold increase in EC50. In contrast, SB-206553 had similar potency and effects on the potentiation with repeated application. These results indicate that the different α-7 receptor PAMs have different rates of activation and desensitization, in addition to their type 1 or 2 effects on receptor desensitization.

A common historical strategy to the discovery of nicotinic receptor ligands has involved the use of radioligand-binding assays for ligand identification in combination with two-electrode voltage clamp in Xenopus oocytes for electrophysiological characterization. More recently, higher-throughput methodologies have replaced these approaches to accommodate screening of large compound libraries and to provide increased capacity for electrophysiological profiling in mammalian cell lines. We, and others, have implemented cell-based screening assays using the fluorometric imaging plate reader (FLIPR) for primary and lead optimization screening of nicotinic receptor agonists and positive allosteric modulators (PAMs). Using GH4C1 cells expressing the rat alpha7 nicotinic receptor, both acetylcholine and nicotine produced concentration-dependent elevations of intracellular calcium with EC(50) values of 5.5 and 1.6 microM, respectively. PAM activity was robustly detected using the FLIPR assay; for example, the known alpha7 receptor PAM 5-hydroxyindole failed to directly activate the receptor but produced a leftward shift of the nicotine concentration-response curve in combination with a potentiation of the maximum evoked response to nicotine. Electrophysiological confirmation of agonist activity was achieved using the Dynaflow rapid perfusion system and patch clamp in the same GH4C1 cell expression system. Estimated EC(50) values for acetylcholine-evoked currents in GH4C1/alpha7 cells were 55 and 576 microM for area-under-the-curve (AUC) and maximum peak height calculations, respectively. Similarly, PAM activity was confirmed using electrophysiological recordings while also allowing for the mechanistic discrimination of compounds, not possible using the FLIPR assay. Specifically, PAMs capable of slowing the rapid desensitization of alpha7 receptors to different extents were discernable in these studies. Further improvements in the capacity to screen compounds using electrophysiology has been achieved by implementation of high-throughput gigaohm quality recording systems such as the QPatch and PatchXpress where agonist EC(50) values are highly comparable to those obtained using conventional manual patch clamp.