Carl D. Overshiner

N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a Novel Metabotropic Glutamate 2 Potentiator with Potential Anxiolytic/Antidepressant Properties: In Vivo Profiling Suggests a Link between Behavioral and Central Nervous System Neurochemical Changes

The normalization of excessive glutamatergic neurotransmission through the activation of metabotropic glutamate 2 (mGlu2) receptors may have therapeutic potential in a variety of psychiatric disorders, including anxiety/depression and schizophrenia. Here, we characterize the pharmacological properties of N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a structurally novel, potent, and selective allosteric potentiator of human and rat mGlu2 receptors (EC50 = 23 and 13 nM, respectively). THIIC produced anxiolytic-like efficacy in the rat stress-induced hyperthermia assay and the mouse stress-induced elevation of cerebellar cGMP and marble-burying assays. THIIC also produced robust activity in three assays that detect antidepressant-like activity, including the mouse forced-swim test, the rat differential reinforcement of low rate 72-s assay, and the rat dominant-submissive test, with a maximal response similar to that of imipramine. Effects of THIIC in the forced-swim test and marble burying were deleted in mGlu2 receptor null mice. Analysis of sleep electroencephalogram (EEG) showed that THIIC had a sleep-promoting profile with increased non-rapid eye movement (REM) and decreased REM sleep. THIIC also decreased the dark phase increase in extracellular histamine in the medial prefrontal cortex and decreased levels of the histamine metabolite tele-methylhistamine (t-MeHA) in rat cerebrospinal fluid. Collectively, these results indicate that the novel mGlu2-positive allosteric modulator THIIC has robust activity in models used to predict anxiolytic/antidepressant efficacy, substantiating, at least with this molecule, differentiation in the biological impact of mGlu2 potentiation versus mGlu2/3 orthosteric agonism. In addition, we provide evidence that sleep EEG and CSF t-MeHA might function as viable biomarker approaches to facilitate the translational development of THIIC and other mGlu2 potentiators.

M1 and M2 Muscarinic Receptor Subtypes Regulate Antidepressant-Like Effects of the Rapidly Acting Antidepressant Scopolamine

Scopolamine produces rapid and significant symptom improvement in patients with depression, and most notably in patients who do not respond to current antidepressant treatments. Scopolamine is a nonselective muscarinic acetylcholine receptor antagonist, and it is not known which one or more of the five receptor subtypes in the muscarinic family are mediating these therapeutic effects. We used the mouse forced-swim test, an antidepressant detecting assay, in wild-type and transgenic mice in which each muscarinic receptor subtype had been genetically deleted to define the relevant receptor subtypes. Only the M1 and M2 knockout (KO) mice had a blunted response to scopolamine in the forced-swim assay. In contrast, the effects of the tricyclic antidepressant imipramine were not significantly altered by gene deletion of any of the five muscarinic receptors. The muscarinic antagonists biperiden, pirenzepine, and VU0255035 (N-[3-oxo-3-[4-(4-pyridinyl)-1-piper azinyl]propyl]-2,1,3-benzothiadiazole-4-sulfonamide) with selectivity for M1 over M2 receptors also demonstrated activity in the forced-swim test, which was attenuated in M1 but not M2 receptor KO mice. An antagonist with selectivity of M2 over M1 receptors (SCH226206 [(2-amino-3-methyl-phenyl)-[4-[4-[[4-(3 chlorophenyl)sulfonylphenyl]methyl]-1-piperidyl]-1-piperidyl]methanone]) was also active in the forced-swim assay, and the effects were deleted in M2−/− mice. Brain exposure and locomotor activity in the KO mice demonstrated that these behavioral effects of scopolamine are pharmacodynamic in nature. These data establish muscarinic M1 and M2 receptors as sufficient to generate behavioral effects consistent with an antidepressant phenotype and therefore as potential targets in the antidepressant effects of scopolamine.

The Rapidly Acting Antidepressant Ketamine and the mGlu2/3 Receptor Antagonist LY341495 Rapidly Engage Dopaminergic Mood Circuits

Ketamine is a rapidly acting antidepressant in patients with treatment-resistant depression (TRD). Although the mechanisms underlying these effects are not fully established, inquiry to date has focused on the triggering of synaptogenesis transduction pathways via glutamatergic mechanisms. Preclinical data suggest that blockade of metabotropic glutamate (mGlu2/3) receptors shares many overlapping features and mechanisms with ketamine and may also provide rapid efficacy for TRD patients. Central dopamine circuitry is recognized as an end target for mood regulation and hedonic valuation and yet has been largely neglected in mechanistic studies of antidepressant-relevant effects of ketamine. Herein, we evaluated the changes in dopaminergic neurotransmission after acute administration of ketamine and the mGlu2/3 receptor antagonist LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid ] in preclinical models using electrophysiologic, neurochemical, and behavioral endpoints. When given acutely, both ketamine and LY341495, but not the selective serotonin reuptake inhibitor (SSRI) citalopram, increased the number of spontaneously active dopamine neurons in the ventral tegmental area (VTA), increased extracellular levels of dopamine in the nucleus accumbens and prefrontal cortex, and enhanced the locomotor stimulatory effects of the dopamine D2/3 receptor agonist quinpirole. Further, both ketamine and LY341495 reduced immobility time in the tail-suspension assay in CD1 mice, which are relatively resistant to SSRI antidepressants. Both the VTA neuronal activation and the antidepressant phenotype induced by ketamine and LY341495 were attenuated by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo- (9CI)-benzo[f]quinoxaline-7-sulfonamide, indicating AMPA-dependent effects. These findings provide another overlapping mechanism of action of ketamine and mGlu2/3 receptor antagonism that differentiates them from conventional antidepressants and thus support the potential rapidly acting antidepressant actions of mGlu2/3 receptor antagonism in patients.

In vivo occupancy of dopamine D3 receptors by antagonists produces neurochemical and behavioral effects of potential relevance to attention-deficit-hyperactivity disorder.

Dopamine D3 receptors have eluded definitive linkage to neurologic and psychiatric disorders since their cloning over 20 years ago. We report a new method that does not employ a radiolabel for simultaneously defining in vivo receptor occupancy of D3 and D2 receptors in rat brain after systemic dosing using the tracer epidepride (N-[[(2S)-1-ethylpyrrolidin-2-yl]methyl]-5-iodo-2,3-dimethoxybenzamide). Decreases in epidepride binding in lobule 9 of cerebellum (rich in D3 receptors) were compared with nonspecific binding in the lateral cerebellum. The in vivo occupancy of the dopamine D3 receptors was dose dependently increased by SB-277011A (trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarboxamide) and U99194 (2,3-dihydro-5,6-dimethoxy- N,N-dipropyl-1H-inden-2-amine). Both antagonists increased extracellular levels of acetylcholine (ACh) in the medial prefrontal cortex of rats and modified brain-tissue levels of ACh and choline. Consistent with these findings, the D3 receptor antagonists enhanced the acquisition of learning of rats either alone or in the presence of the norepinephrine uptake blocker reboxetine as with the attention-deficit–hyperactivity disorder (ADHD) drug methylphenidate. Like reboxetine, the D3 receptor antagonists also prevented deficits induced by scopolamine in object recognition memory of rats. Mice in which the dopamine transporter (DAT) has been deleted exhibit hyperactivity that is normalized by compounds that are effective in the treatment of ADHD. Both D3 receptor antagonists decreased the hyperactivity of DAT−/− mice without affecting the activity of wild type controls. The present findings indicate that dopamine D3 receptor antagonists engender cognition-enhancing and hyperactivity-dampening effects. Thus, D3 receptor blockade could be considered as a novel treatment approach for cognitive deficits and hyperactivity syndromes, including those observed in ADHD.

Disinhibitory effects of LY354740, a new mGluR2 agonist, on behaviors suppressed by electric shock in rats and pigeons

Compounds which affect glutamate transmission are reported as anxiolytic in a number of animal models. In the present studies, we evaluated the anxiolytic effect of a new mGluR2 agonist, LY354740, in rats and pigeons. LY354740 was evaluated in three different rat punished responding assays, using different levels of shock intensity. LY354740 and its racemate, LY314582, were generally ineffective at increasing rates of responding suppressed by electric shock. However, both compounds did increase responding during time‐out periods between scheduled components, and both increased responding during a punishment extinction assay. LY354740 was also evaluated in two separate pigeon punished responding procedures. LY354740 was ineffective at increasing punished responding under one schedule, but increased punished responding in the second assay, when shock was not delivered coincident with reinforcement. Clearly, LY354740 was effective in attenuating suppressed responding in some cases, but not all. LY354740 appears to produce a disinhibitory effect on behaviors with low baseline rates of responding (in rats), and a disinhibitory effect on punished behaviors in schedules with less stringent punishment conditions (in rats and pigeons). These results suggest that LY354740 may be useful as a clinically effective anxiolytic. Drug Dev. Res. 47:37–44, 1999.

Pharmacological profile of LY301317, a potent and selective 5-HT1A agonist

LY301317 ((4r)‐(−)‐4‐(dipropylamino)‐6‐(5‐oxazolinyl)‐1,3,4,5‐tetrahydrobenz[c,d]indole) has high affinity for the 5‐HT1A receptor and weak affinity for the 5‐HT1D and histamine‐H1 receptors. No significant affinity was found for the other amine receptors studied. In rats, LY301317 produced potent in vivo effects that are characteristic of compounds with agonist activity at the 5‐HT1A receptor, such as an increase in serum corticosterone concentration, a reduction in 5‐HIAA concentration in brain tissue, induction of flat body posture and lower lip retraction (components of a serotonin syndrome), and a decrease of core body temperature. In pigeons trained to discriminate 8‐hydroxy‐2‐(di‐n‐propylamino)‐tetralin (8‐OH‐DPAT) from saline, full generalization to LY301317 was observed. LY301317 increased punished responding in both the pigeon and rat conflict tests for anxiolytic activity. LY301317 reduced immobility in the rat forced swim model used to indicate potential antidepressant activity. In pigeons, LY301317 blocked emesis induced by a chemical (ditolyguanidine), by a 5‐HT3 agonist (m‐(chlorophenyl)‐biguanide), and by an oncolytic agent (cisplatin), as well as vomiting induced by conditioning to environmental stimuli (a model of anticipatory nausea and vomiting). In addition, LY301317 blocked cisplatin‐ and ipecac‐induced vomiting in the dog and motion‐induced emesis in the cat. It was concluded that LY301317 is an orally active, potent, and selective agonist for the 5‐HT1A receptor with potential clinical utility as an anxiolytic, an antidepressant, and a broad‐spectrum antiemetic. Drug Dev. Res. 40:17–34, 1997.

Comparative Effects of LY3020371, a Potent and Selective mGlu2/3 Receptor Antagonist, and Ketamine, a Non-Competitive NMDA Receptor Antagonist, in Rodents: Evidence Supporting Use for the Treatment of Depression

The ability of the N-methyl-d-aspartate receptor antagonist ketamine to alleviate symptoms in patients suffering from treatment-resistant depression (TRD) is well documented. In this paper, we directly compare in vivo biologic responses in rodents elicited by a recently discovered metabotropic glutamate (mGlu) 2/3 receptor antagonist 2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY3020371) with those produced by ketamine. Both LY3020371 and ketamine increased the number of spontaneously active dopamine cells in the ventral tegmental area of anesthetized rats, increased O2 in the anterior cingulate cortex, promoted wakefulness, enhanced the efflux of biogenic amines in the prefrontal cortex, and produced antidepressant-related behavioral effects in rodent models. The ability of LY3020371 to produce antidepressant-like effects in the forced-swim assay in rats was associated with cerebrospinal fluid (CSF) drug levels that matched concentrations required for functional antagonist activity in native rat brain tissue preparations. Metabolomic pathway analyses from analytes recovered from rat CSF and hippocampus demonstrated that both LY3020371 and ketamine activated common pathways involving GRIA2 and ADORA1. A diester analog of LY3020371 [bis(((isopropoxycarbonyl)oxy)-methyl) (1S,2R,3S,4S,5R,6R)-2-amino-3-(((3,4-difluorophenyl)thio)methyl)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylate (LY3027788)] was an effective oral prodrug; when given orally, it recapitulated effects of intravenous doses of LY3020371 in the forced-swim and wake-promotion assays, and augmented the antidepressant-like effects of fluoxetine or citalopram without altering plasma or brain levels of these compounds. The broad overlap of biologic responses produced by LY3020371 and ketamine supports the hypothesis that mGlu2/3 receptor blockade might be a novel therapeutic approach for the treatment of TRD patients. LY3020371 and LY3027788 represent molecules that are ready for clinical tests of this hypothesis.

Novel bicyclo[3.1.0]hexane analogs as antagonists of metabotropic glutamate 2/3 receptors for the treatment of depression

Negative modulators of metabotropic glutamate 2 & 3 receptors demonstrate antidepressant-like activity in animal models and hold promise as novel therapeutic agents for the treatment of major depressive disorder. Herein we describe our efforts to prepare and optimize a series of conformationally constrained 3,4-disubstituted bicyclo[3.1.0]hexane glutamic acid analogs as orthosteric (glutamate site) mGlu2/3 receptor antagonists. This work led to the discovery of a highly potent and efficacious tool compound 18 (hmGlu2 IC50 46±14.2nM, hmGlu3 IC50=46.1±36.2nM). Compound 18 showed activity in the mouse forced swim test with a minimal effective dose (MED) of 1mg/kg ip. While in rat EEG studies it exhibited wake promoting effects at 3 and 10mg/kg ip without any significant effects on locomotor activity. Compound 18 thus represents a novel tool molecule for studying the impact of blocking mGlu2/3 receptors both in vitro and in vivo.

Behavioral Effects of a Novel Benzofuranyl-Piperazine Serotonin-2C Receptor Agonist Suggest a Potential Therapeutic Application in the Treatment of Obsessive–Compulsive Disorder

Selective serotonin reuptake inhibitors (SSRIs) are the only effective pharmacological treatments for obsessive–compulsive disorder (OCD). Nonetheless, their generally limited efficacy, side-effects, and delayed onset of action require improved medications for this highly prevalent disorder. Preclinical and clinical findings have suggested serotonin2C (5-HT2C) receptors as a potential drug target. Data in rats and mice are presented here on the effects of a novel 5-HT2C receptor agonist ((3S)-3-Methyl-1-[4-(trifluoromethyl)-7-benzofuranyl]-piperazine) (CPD 1) with high potency and full efficacy at 5-HT2C receptors and less potency and partial agonism at 5-HT2A and 5-HT2B receptors. Effects of CPD 1 on consummatory (schedule-induced polydipsia in rats) and non-consummatory behaviors (marble-burying and nestlet-shredding in mice) that are repetitive and non-habituating were studied. We also evaluated the effects of CPD 1 in rats with isoproterenol- and deprivation-induced drinking in rats to compare with the polydipsia studies. The SSRIs, fluoxetine, and chlomipramine decreased the high rates of drinking in rats engendered by a schedule of intermittent food delivery (schedule-induced polydipsia). The effects of fluoxetine, but not of d-amphetamine, were prevented by the selective 5-HT2C receptor antagonist SB242084. The 5-HT2C receptor agonists Ro 60-0175 and CPD 1 also decreased drinking, but unlike the SSRIs and Ro 60-0175, CPD 1 dose-dependently decreased excessive drinking without affecting lever press responses that produced food. The effects of CPD 1 were prevented by SB242084. CPD 1 also suppressed drinking induced by isoproterenol and by water deprivation without affecting normative drinking behavior. CPD 1, like fluoxetine, also suppressed marble-burying and nestlet-shredding in mice at doses that did not affect rotarod performance or locomotor activity. The behavioral specificity of effects of CPD 1 against repetitive and excessive behaviors suggests a potential therapeutic application in OCD.