Joseph P. Tizzano

Metabotropic glutamate 2 receptor potentiators: receptor modulation, frequency-dependent synaptic activity, and efficacy in preclinical anxiety and psychosis model(s)

RationaleTo increase subtype selectivity and provide a novel means to alter receptor function, we discovered and characterization potentiators for the metabotropic glutamate 2 receptor (mGlu2).Methods and resultsA class of 3-pyridylmethylsulfonamides (e.g., 3-MPPTS; 2,2,2-trifluoro-N-[3-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide) were found to be potent, subtype-selective potentiators of human and rat mGlu2. The sulfonamides increased agonist potency in functional assays but did not displace orthosteric radiolabeled antagonist or agonist binding to cloned mGlu2 receptors. Rather, the modulators increased the affinity of most of the orthosteric agonists including glutamate, DCG-IV (2S,2′R,3′R)-2-(2′,3′-dicarboxylcyclopropyl)glycine), and LY354740 (1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-bicaroxylate monohydrate). In striatal brain slices, LY354740 inhibited evoked excitatory postsynaptic potentials (EPSPs) equally well following either a low- (0.06 Hz) or high (4 Hz)-frequency stimulation of corticostriatal afferents. In contrast, the mGlu2 potentiator cyPPTS (2,2,2-trifluoro-N-[3-(cyclopentyloxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide) inhibited striatal EPSPs only at higher frequencies of stimulation (2 and 4 Hz). Several sulfonamides including 4-MPPTS, 4-APPES (N-[4-(4-carboxamidophenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride monohydrate) and/or CBiPES N-[4′-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride) were tested in mGlu2/3 agonist-sensitive rodent model(s) of anxiety and psychosis. As seen with LY354740, both 4-MPPTS and 4-APPES were efficacious in a rat fear-potentiated startle paradigm. Likewise in mice, CBiPES attenuated a stress-induced hyperthermia and PCP-induced hyperlocomotor activity. Furthermore, CBiPES mediated alteration in PCP-induced hyperlocomotor activity was sensitive to mGlu2/3 antagonist pretreatment.ConclusionsTaken together, the data indicate mGlu2 receptor potentiators have a unique use-dependent effect on presynaptic glutamate release, and show efficacy in several mGlu2/3-sensitive animal models of psychiatric disorders.

Induction or protection of limbic seizures in mice by mGluR subtype selective agonists

The behavioral consequences of metabotropic glutamate receptor (mGluR) activation were investigated following intracerebral administration of the mGluR selective agonists (RS)3,5-dihydroxyphenyl-glycine (3,5-DHPG), (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD), (1R,3S)-1-aminocyclopentane-1,3-dicarboxylate (1R,3S-ACPD), L-2-amino-4-phosphonobutyrate (L-AP4), L-serine-O-phosphate (L-SOP) and (2S,3S,4S)alpha-(carboxycyclopropyl)glycine (L-CCGI) into the thalamus in mice. Injections of 3,5-DHPG, 1S,3R-ACPD and L-CCGI produced dose-dependent increases in limbic seizures with a potency order of 3,5-DHPG = 1S,3R-ACPD > L-CCGI. This effect of 1S,3R-ACPD was stereoselective, since the inactive isomer (1R,3S-ACPD) did not elicit seizure activity. Limbic seizures induced by the phosphoinositide-coupled mGluR subtype selective agonist 3,5-DHPG were attenuated by the mGluR antagonist L-2-amino-3-phosphonopropanoic acid (L-AP3) and dantrolene, inhibitors of mGluR-mediated intracellular calcium mobilization. Interestingly, L-AP4, L-SOP and low doses of L-CCGI also protected against 3,5-DHPG seizures. These data indicate that mGluR agonist-induced limbic seizures in mice are mediated by activation of phosphoinositide-coupled mGluRs. Furthermore, these seizures can be protected against by activation of mGluRs that are negatively-linked to cAMP formation.

LY354740 : a metabotropic glutamate receptor agonist which ameliorates symptoms of nicotine withdrawal in rats

LY354740 is a conformationally constrained analog of glutamate with high selectivity and nanomolar agonist activity at Group II metabotropic glutamate receptors (mGluRs). This orally active compound is a new drug candidate which is being developed for the treatment of anxiety. In this study, LY354740 was investigated in a model of nicotine withdrawal using the acoustic startle reflex (sensorimotor reactivity) in rats. Nicotine (6 mg/kg/day) was administered for 12 days subcutaneously by osmotic minipumps. After 12 days the pumps were removed and the animals were allowed to go through spontaneous withdrawal. Cessation of chronic nicotine exposure led to increased startle responding for 4 days following withdrawal. Treatment with LY354740 (0.0001-0.1 mg/kg, i.p.; 0.03-3 mg/kg, oral) produced a dose-dependent attenuation of the enhanced auditory startle responding following withdrawal of nicotine with intraperitoneal and oral ED50 values of 0.003 mg/kg and 0.7 mg/kg, respectively. These effects were stereoselective since the (-)-enantiomer of LY354740, LY366563, was without effect in this model. LY354740 produced no changes in the sensorimotor reactivity of rats not exposed to nicotine at oral doses up to 10 mg/kg. These data support the functional role of mGluR agonists in nicotine withdrawal and indicate that LY354740 may be efficacious in reducing the symptoms associated with nicotine withdrawal during smoking cessation in humans.

Nicotine withdrawal: a behavioral assessment using schedule controlled responding, locomotor activity, and sensorimotor reactivity

Three different behavioral measures were used to assess the effects of abrupt cessation of chronic nicotine treatment. Nicotine (0, 3, or 6 mg/kg per day) was continuously administered for 12 days in rats by surgically implanting Alzet osmotic mini-pumps subcutaneously. Experiment 1 employed a light/dark discrimination task. There were no significant effects on number of responses or percent correct responding either during nicotine administration, or following cessation of nicotine. Experiment 2 examined ambulatory (locomotor) and nonambulatory activity. Chronic nicotine administration produced significant dose-dependent increases in both ambulatory and nonambulatory activity during the first 3 days of exposure. However, no significant alterations were seen in activity levels following nicotine cessation. Experiment 3 examined sensorimotor reactivity using the auditory startle response. During nicotine withdrawal, significant increases were seen in startle amplitude in both nicotine groups for 4 days. Nicotine (0.4 mg/kg, IP) administered before startle testing during the withdrawal phase attenuated the increased reactivity seen during nicotine cessation. These studies indicate that 1) rats display increased sensorimotor reactivity after cessation of chronic nicotine exposure, and 2) the expression of nicotine dependence and withdrawal is dependent on the behavioral task employed.

In vitro and in vivo antagonism of AMPA receptor activation by (3s,4ar,6r,8ar)-6-[2-(1(2)h-tetrazole-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid

The in vitro and in vivo pharmacology of a structurally novel competitive antagonist for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of excitatory amino acid receptors is described. LY215490, (+/-)(6-(2-(1-H-tetrazol-5-yl)ethyl) decahydroisoquinoline-3-carboxylic acid), was shown to displace selectively 3H-AMPA and 3H-6-cyano-7-nitro- quinoxaline-2,3-dione (3H-CNQX) binding to rat brain membranes. LY215490 potently antagonized quisqualate-and AMPA-induced depolarizations of rat cortical slices in a competitive manner, while requiring higher concentrations to antagonize the effects of N-methyl-D-aspartate (NMDA) and kainate. In slices of rat hippocampus, LY215490 also selectively antagonized AMPA-evoked release of 3H-norepinephrine. These AMPA receptor activities were due to the (-) isomer of the compound. (3S,4aR,6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl] decahydroisoquinoline-3-carboxylic acid (LY293558). LY215490 was centrally active following parenteral administration in mice as demonstrated by protection versus maximal electroshock seizures and decreases in spontaneous motor activity. LY215490 (its active isomer being LY293558) represents a novel pharmacological agent for in vitro and in vivo studies of AMPA receptor function in the CNS.

The anxiolytic action of mGlu2/3 receptor agonist, LY354740, in the fear-potentiated startle model in rats is mechanistically distinct from diazepam.

The fear-potentiated startle paradigm has been characterized for drugs that act via ionotropic (NMDA and AMPA/kainate receptor) glutamate receptor mechanisms. Previous studies have shown that the potent systemically active mGlu2/3 receptor agonist, LY354740, effectively reduced the expression of fear-potentiated startle responses in rats. The present study examined the effects of LY354740 in a pre- versus post-fear conditioning paradigm and compared the effects to diazepam. Diazepam (0.3, 0.6, and 1.0 mg/kg ip) attenuated both pre- and post-fear conditioning startle responses in a dose-related manner. In contrast, LY354740 (0.03, 0.3, and 3.0 mg/kg ip) did not disrupt preconditioning startle responses at doses that attenuated post-fear conditioning responses. The benzodiazepine antagonist, flumazenil, at a dose (2 mg/kg sc) that did not alter fear-potentiated startle per se, selectively reversed suppression of fear responses to diazepam (0.6 mg/kg ip) while not affecting fear suppression induced by LY354740 (0.3 mg/kg ip). At a dose of 1 mg/kg ip, the mGlu2/3 receptor antagonist, LY341495, did not disrupt fear-enhanced startle per se, but completely reversed the postconditioning anxiolytic effects of LY354740 in this model. This dose of LY341495 had no effect on fear suppression by diazepam. These results demonstrate that fear suppression by diazepam and LY354740 involves different neuronal mechanisms. While diazepam acts via the facilitation of GABAergic transmission, LY354740 induces its actions via the glutamatergic system, specifically mGlu2/3 receptor activation. Furthermore, in contrast to disruption of fear conditioning as well as fear suppression by diazepam, LY354740 had selective effects on fear expression, suggesting anxiolytic actions without the associated memory impairment.

Intracerebral 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) produces limbic seizures that are not blocked by ionotropic glutamate receptor antagonists

The functional role of metabotropic glutamate receptor (mGluR) activation was investigated following intracerebral administration of 1S,3R-ACPD in mice. Injections of 1S,3R-ACPD (50-800 nmol in 5 microliters) into the thalamus produced a dose-dependent increase in limbic seizures. These effects were stereoselective since 1R,3S-ACPD, did not elicit seizure activity. Pharmacologically, limbic seizures were attenuated by the mGluR partial agonist/antagonist L-2-amino-3-phosphonopropionate (L-AP3) and dantrolene, an inhibitor of intracellular calcium mobilization, but not by D-AP3 or ionotropic glutamate receptor antagonists (MK-801 or GYKI-52466). Thus, activation of mGluRs by 1S,3R-ACPD in mice, induces limbic seizures that may involve the mobilization of intracellular calcium stores.

Inhibition of group I metabotropic glutamate receptor responses in vivo in rats by a new generation of carboxyphenylglycine-like amino acid antagonists

A series of novel group I metabotropic glutamate receptor (mGlu) antagonists have been designed on the basis of the 4-carboxyphenylglycine pharmacophore. The compounds are either mGlu1 receptor selective or equipotent for both mGlu1 and mGlu5 receptors and have IC(50) values ranging from 1 to 30 microM determined by phosphoinositide hydrolysis (PI) assay in vitro. All the compounds produced dose-dependent inhibition of group I mGlu receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG)-induced limbic seizure responses in mice with ED(50) values ranging from 9 nmol for LY393053 to 138 nmol for LY339840 after intracerebroventricular injection and were more potent than the mGlu1 receptor antagonist 1-aminoindan-1,5-dicarboxylic acid (ED(50)=477 nmol). Further antagonist actions were also demonstrated in a model of (RS)-DHPG-induced PI hydrolysis in vivo such that LY367385 and the active cis isomer of LY393053 produced dose-dependent inhibition of PI responses in both cerebellum and hippocampus. Cis LY393053 also inhibited hippocampal PI responses when administered intraperitoneally at a dose of 30 mg/kg. These compounds define a new series of group I mGlu receptor antagonists which may serve as useful experimental tools.

The Triple Uptake Inhibitor (1R,5S)-(+)-1-(3,4-Dichlorophenyl)-3-azabicyclo[3.1.0] Hexane Hydrochloride (DOV 21947) Reduces Body Weight and Plasma Triglycerides in Rodent Models of Diet-Induced Obesity

Selective inhibitors of biogenic amine (e.g., serotonin, norepinephrine, and dopamine) uptake exhibit varying degrees of safety and efficacy as antiobesity agents. Moreover, preclinical findings suggest that the combined inhibition of monoamine neurotransmitter transporters synergistically enhances antiobesity activity. (1R,5S)-(+)-1-(3,4-Dichlorophenyl)-3-azabicyclo-[3.1.0] hexane hydrochloride (DOV 21947) inhibits norepinephrine, 5-hydroxytryptamine, and dopamine uptake, and it reduces body weight in rodent models of diet-induced obesity (DIO). DIO rats treated orally with DOV 21947 for 1 to 24 days showed significantly lower body weights than vehicle-treated DIO rats. The decrease in body weight resulted specifically from a loss of retroperitoneal and mesenteric depots of white adipose tissue. DOV 21947 also reduced daily food intake in DIO rats, but consumption returned to control levels after 11 days of treatment. With the exception of a decrease in triglyceride levels, blood chemistry was unaltered after 24 days of DOV 21947 treatments. DOV 21947 had no effect on motor activity. Although DOV 21947 increased respiratory rate and decreased the tidal volume of normal rats, it did not alter the minute volume. In addition, DOV 21947 did not significantly affect blood pressure, heart rate, electrocardiographic indices or body temperature in telemeterized dogs. However, it caused a sustained, but reversible reduction in the rate of body weight gain for as long as 6 months in normal rats, and for up to 1 year in normal dogs. In summary, DOV 21947 is effective in causing a sustained and selective reduction in fat content and triglyceride levels in animal models of obesity without significantly altering vital organ function.

Blockade of the second messenger functions of the glutamate metabotropic receptor is associated with degenerative changes in the retina and brain of immature rodents

Activation of metabotropic glutamate receptors (mGluR) by Glu or related mGluR agonists triggers phosphoinositide (PI) hydrolysis, intracellular Ca2+ mobilization and protein kinase C activation. These mGluR agonist-stimulated events are inhibited strongly by 2-amino-3-phosphono-L-propionic acid (L-AP3) and L-aspartate-beta-hydroxamate (L-A beta H), and much more weakly by D-AP3 and L-serine-O-phosphate (L-SOP). Daily s.c. administration of DL-AP3 subchronically to infant rodents causes the developing retina and optic nerves to degenerate. In the present study, we describe the evolution of the cytopathological reaction in the developing rodent retina following DL-AP3 treatment and show that DL-AP3 can induce similar cytopathological changes in several regions of the immature rodent brain. In addition, we show that the retinotoxic action of DL-AP3 is mimicked by L-A beta H but not by L-SOP, and that L-AP3 is a much stronger retinotoxin that D-AP3. These observations suggest a possible mechanistic link between the PI-hydrolysis blocking action and retinotoxic action. Our findings are consistent with the hypothesis that under normal physiological circumstances, the Glu metabotropic receptor through its PI-hydrolysis-linked second messenger functions provides vitally important support for developing neurons, and that disruption of this support can cause widespread neuronal degeneration.