Bryan G. Johnson

Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized Phase 2 clinical trial

Schizophrenia is a chronic, complex and heterogeneous mental disorder, with pathological features of disrupted neuronal excitability and plasticity within limbic structures of the brain. These pathological features manifest behaviorally as positive symptoms (including hallucinations, delusions and thought disorder), negative symptoms (such as social withdrawal, apathy and emotional blunting) and other psychopathological symptoms (such as psychomotor retardation, lack of insight, poor attention and impulse control). Altered glutamate neurotransmission has for decades been linked to schizophrenia, but all commonly prescribed antipsychotics act on dopamine receptors. LY404039 is a selective agonist for metabotropic glutamate 2/3 (mGlu2/3) receptors and has shown antipsychotic potential in animal studies. With data from rodents, we provide new evidence that mGlu2/3 receptor agonists work by a distinct mechanism different from that of olanzapine. To clinically test this mechanism, an oral prodrug of LY404039 (LY2140023) was evaluated in schizophrenic patients with olanzapine as an active control in a randomized, three-armed, double-blind, placebo-controlled study. Treatment with LY2140023, like treatment with olanzapine, was safe and well-tolerated; treated patients showed statistically significant improvements in both positive and negative symptoms of schizophrenia compared to placebo (P < 0.001 at week 4). Notably, patients treated with LY2140023 did not differ from placebo-treated patients with respect to prolactin elevation, extrapyramidal symptoms or weight gain. These data suggest that mGlu2/3 receptor agonists have antipsychotic properties and may provide a new alternative for the treatment of schizophrenia.

LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors

The in vitro pharmacology of a structurally novel compound, LY341495, was investigated at human recombinant metabotropic glutamate (mGlu) receptor subtypes expressed in non-neuronal (RGT, rat glutamate transporter) cells. LY341495 was a nanomolar potent antagonist of 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD)-induced inhibition of forskolin-stimulated cAMP formation at mGlu2 and mGlu3 receptors (respective IC50S of 0.021 and 0.014 microM). At group I mGlu receptor expressing cells, LY341495 was micromolar potent in antagonizing quisqualate-induced phosphoinositide (PI) hydrolysis, with IC50 values of 7.8 and 8.2 microM for mGlu1a and mGlu5a receptors, respectively. Among the human group III mGlu receptors, the most potent inhibition of L-2-amino-4-phosphonobutyric acid (L-AP4) responses was seen for LY341495 at mGlu8, with an IC50 of 0.17 microM. LY341495 was less potent at mGlu7 (IC50 = 0.99 microM) and least potent at mGlu4 (IC50 = 22 microM). Binding studies in rat brain membranes also demonstrated nanomolar potent group II mGlu receptor affinity for LY341495, with no appreciable displacement of ionotropic glutamate receptor ligand binding. Thus, LY341495 has a unique range of selectivity across the mGlu receptor subtypes with a potency order of mGlu3 > or = mGlu2 > mGlu8 > mGlu7 >> mGlu1a = mGlu5a > mGlu4. In particular, LY341495 is the most potent antagonist yet reported at mGlu2, 3 and 8 receptors. Thus, it represents a novel pharmacological agent for elucidating the function of mGlu receptors in experimental systems.

3,5-DIHYDROXYPHENYLGLYCINE IS A HIGHLY SELECTIVE AGONIST FOR PHOSPHOINOSITIDE-LINKED METABOTROPIC GLUTAMATE RECEPTORS IN THE RAT HIPPOCAMPUS

Abstract: Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G protein‐coupled glutamate receptors that are linked to multiple second messenger systems in the CNS. In this study the selectivity of mGluR agonists for different mGluR second messenger effects was characterized in slices of the rat hippocampus. The mGluR agonists (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid and (2S,3S,4S)α‐(carboxycyclopropyl)glycine produced multiple effects on second messengers that included enhanced phosphoinositide hydrolysis in both adult and neonatal rat hippocampus, inhibition of forskolin‐stimulated cyclic AMP (cAMP) formation in adult tissue, and increases in basal cAMP formation in the neonatal hippocampus. In contrast, 3,5‐dihydroxyphenylglycine was potent and effective in increasing phosphoinositide hydrolysis in both adult and neonatal hippocampus but unlike the other mGluR agonists did not inhibit forskolin‐stimulated cAMP formation (in the adult) or substantially enhance basal cAMP formation (in the neonate). Thus, in the rat hippocampus mGluR agonist‐mediated increases or decreases in cAMP formation are not secondary to mGluR‐mediated changes in phosphoinositide hydrolysis. Furthermore, 3,5‐dihydroxyphenylglycine can be used to activate subpopulations of mGluRs coupled to phosphoinositide hydrolysis with minimal effects on cAMP‐mGluR second messenger systems.

LY354740 is a Potent and Highly Selective Group II Metabotropic Glutamate Receptor Agonist in Cells Expressing Human Glutamate Receptors

The novel compound LY354740 is a conformationally constrained analog of glutamate, which was designed for interaction at metabotropic glutamate (mGlu) receptors. In this paper the selectivity of LY354740 for recombinant human mGlu receptor subtypes expressed in non-neuronal (RGT) cells is described. At human mGlu2 receptors, LY354740 produced > 90% suppression of forskolin-stimulated cAMP formation with an EC50 of 5.1 +/- 0.3 nM. LY354740 was six-fold less potent in activating human mGlu3 receptors (EC50 = 24.3 +/- 0.5 nM). LY354740 inhibition of forskolin-stimulated cAMP formation in human mGlu2 receptor-expressing cells was blocked by competitive mGlu receptor antagonists, including (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) and LY307452 ((2S,4S)-2-amino-4-(4,4-diphenylbut-1-yl)-pentane-1,5-dioic acid). LY354740 had no agonist or antagonist activities at cells expressing human mGlu4 or mGlu7 (group III mGlu receptors) (EC50 > 100,000 nM). When tested at group I phosphoinositide-coupled human mGlu receptors (mGlu1a and mGlu5a), LY354740 did not activate or inhibit mGlu receptor agonist-evoked phosphoinositide hydrolysis at up to 100,000 nM. Electrophysiological experiments also demonstrated that LY354740 also had no appreciable activity in cells expressing human recombinant AMPA (GluR4) and kainate (GluR6) receptors. Thus, LY354740 is a highly potent, efficacious and selective group II (mGlu2/3) receptor agonist, useful to explore the functions of these receptors in situ.

Excitatory amino acid agonist-antagonist interactions at 2-amino-4-phosphonobutyric acid-sensitive quisqualate receptors coupled to phosphoinositide hydrolysis in slices of rat hippocampus.

Abstract: Studies were carried out to define the relative affinities and intrinsic activities of excitatory amino acid agonists that activate receptor sites coupled to phosphoinositide hydrolysis in brain. Slices of rat hippocampus were prelabeled with myo‐[3H]inositol, and agonist stimulation was indexed by measuring the accumulation of [3H]inositol monophosphate ([3H]IP) in the presence of Li+. It was observed that ibotenic (IBO) and quisqualic (QUIS) acids both elicit highly significant, concentration‐dependent stimulation of phosphoinositide hydrolysis. Whereas maximal stimulation by IBO (10−3M) was four‐ to fivefold over basal values, the maximal effect of QUIS (10−4M) was less (about twofold). Based on the relative concentrations required for 50% maximal stimulation, QUIS was 20 times more potent than IBO. Stimulation of phosphoinositide hydrolysis by either IBO or QUIS was additive to the effects of nonexcitatory amino acid agonists (carbachol and norepinephrine) in this tissue. However, the stimulatory effects of IBO plus QUIS were not additive. At ≥ 10−4M, QUIS significantly inhibited phosphoinositide hydrolysis by a maximal stimulatory concentration of IBO (10−3M) to a level observed with QUIS alone. Other excitatory amino acid agonists, including kainate, N‐methyl‐d‐aspartate, and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), had no stimulatory effects at concentrations as high as 10−3M. The d,l or l forms of 2‐amino‐4‐phosphonobutyric acid (AP4), but not d‐AP4, significantly enhanced [3H]IP levels to ∼135% of basal values. d,l‐AP4 and l‐AP4 also inhibited stimulation by either IBO or QUIS, but relative inhibition of QUIS effects was quantitatively less than that of IBO effects. These studies indicate that IBO represents a full agonist at these “AP4‐sensitive QUIS receptors” coupled to phosphoinositide hydrolysis in brain. QUIS is a partial agonist with the highest affinity for the receptor, and l‐AP4 represents a partial agonist with relatively weak intrinsic activity. Furthermore, this receptor effect is not mediated at “QUIS” receptor sites that are activated by AMPA.

Inhibition of cyclic AMP formation by a selective metabotropic glutamate receptor agonist.

Abstract: It is well documented that the effects of excitatory ammo acid (EAA) agonists on phosphoinositide hydrolysis involve a GTP‐binding protein‐linked or “metabotropic’ receptor mechanism. The mechanisms by which EAAs alter cyclic AMP levels in brain slices, however, are not yet clear. In this study, the selective metabotropic EAA agonist trans‐(±)‐l‐aminocyclopentane‐l,3‐dicarboxylic acid and its isomers were examined for effects on basal and forskolin‐stimulated cyclic AMP formation in slices of the rat hippocampus. Trans‐(±)‐l‐Aminocyclopentane‐l,3‐dicarboxylic acid had little effect on basal cyclic AMP but inhibited forskolin‐stimulated cyclic AMP formation in a biphasic manner. The 1S,3R isomer of 1‐aminocy‐clopentane‐l,3‐dicarboxylic acid produced potent but only partial (~50%) inhibition of forskolin‐stimulated cyclic AMP formation. 1R,3S–l‐Aminocyclopentane‐l,3‐dicarboxylic acid fully inhibited forskolin‐stimulated cyclic AMP but with lower potency than the IS,3R isomer. These results show that in addition to the formation of phosphoinositide‐derived second messengers, the cellular consequences of selectively activating hippocampal metabotropic EAA receptors include an alteration of cellular cyclic AMP levels.

Metabotropic Glutamate Receptors in the Control of Mood Disorders

Current treatments for depression are less than optimal in terms of onset of action, response and remission rates, and side-effect profiles. Glutamate is the major excitatory neurotransmitter controlling synaptic excitability and plasticity in most brain circuits, including limbic pathways involved in depression. Thus, drugs that target glutamate neuronal transmission offer novel approaches to treat depression. Recently, the NMDA receptor antagonist ketamine has demonstrated clinical efficacy in a randomized clinical trial of depressed patients. Metabotropic glutamate (mGlu) receptors function to regulate glutamate neuronal transmission by altering the release of neurotransmitter or modulating the post-synaptic responses to glutamate. Accumulating evidence from biochemical and behavioral studies support the idea that the regulation of glutamatergic neurotransmission via mGlu receptors is linked to mood disorders and that these receptors may serve as novel targets for the discovery of small molecule modulators with unique antidepressant properties. For example, mGlu receptor modulation can facilitate neuronal stem cell proliferation (neurogenesis) and the release of neurotransmitters that are associated with treatment response to depression in humans (serotonin, norepinephrine, dopamine). In particular, compounds that antagonize mGlu2, mGlu3 and/or mGlu5 receptors (e.g. LY341495, MSG0039, MPEP) have been linked to the above pharmacology and have also shown in vivo activity in animal models predictive of antidepressant efficacy such as the forced-swim test. The in vivo actions of these agents can be antagonized by compounds that block AMPA receptors, suggesting that their actions are direct downstream consequences of the enhancement of glutamate neuronal transmission in brain regions involved in depression. These data provide new approaches to finding mechanistically distinct drugs for depression that may have advantages over current therapies for some patients. Moreover, since the mood disorders encompase a non-homogenous set of symptoms, comorbid disorders, and potential etiologies, the rich arsensel that exists within the mGlu receptor families provides an opportunity for both broad and customized therapeutics.

Inhibition of Excitatory Amino Acid-Stimulated Phosphoinositide Hydrolysis in the Neonatal Rat Hippocampus by 2-Amino-3-Phosphonopropionate

Abstract: The effects of excitatory amino acid agonists and α‐amino‐ω‐phosphonocarboxylic acid antagonists on phosphoinositide hydrolysis in hippocampal slices of the 7‐day neonatal rat were examined. Significant stimulation of [3H]inositol monophosphate formation was observed with ibotenate, quisqualate, l‐glutamate, l‐aspartate, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, l‐homocys‐teate, and kainate. N‐Methyl‐D‐aspartate had no effect. Of these agonists, ibotenate and quisqualate were the most potent and efficacious. Stimulations by ibotenate and quisqualate were partially inhibited by l‐2‐amino‐4‐phosphonobutyrate (10−3M), but this antagonist had no effect on l‐glutamate, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoIepropionic acid, or kainate. At 10−3M, D,L‐2‐amino‐3‐phosphonopropionate completely inhibited ibotenate and quisqualate stimulations, partially inhibited l‐glutamate stimulation, and had no effect on α ‐ amino ‐3‐ hydroxy ‐5‐ methyl ‐4‐ isoxazolepropionic acid‐, kainate‐, or carbachol‐induced [3H]inositol monophosphate formation. Concentration‐effect experiments showed D,L‐2‐amino‐3‐phosphonopropionate to be five times more potent as an antagonist of ibotenate‐stimulated phosphoinositide hydrolysis than L‐2‐amino‐4‐phosphonobuty‐rate. Thus in the neonatal rat hippocampus, like in the adult rat brain, D,L‐2‐amino‐3‐phosphonopropionate is a selective and relatively potent inhibitor of excitatory amino acid‐stimulated phosphoinositide hydrolysis. Because this glutamate receptor is uniquely sensitive to D,L‐2‐amino‐3‐phosphono‐propionate, these studies provide further pharmacological evidence for the existence of a novel excitatory amino acid receptor subtype that is coupled to phosphoinositide hydrolysis in brain.

Pharmacological and Pharmacokinetic Properties of a Structurally Novel, Potent, and Selective Metabotropic Glutamate 2/3 Receptor Agonist: In Vitro Characterization of Agonist (–)-(1R,4S,5S,6S)-4-Amino-2-sulfonylbicyclo[3.1.0]-hexane-4,6-dicarboxylic Acid (LY404039)

Group II metabotropic glutamate (mGlu) receptor agonists, including (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740) and (–)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), have demonstrated efficacy in animal models of anxiety and schizophrenia, and LY354740 decreased anxiety in human subjects. Herein, we report the in vitro pharmacological profile and pharmacokinetic properties of another potent, selective, and structurally novel mGlu2/3 receptor agonist, (–)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY404039) and provide comparisons with LY354740. Similar to LY354740, LY404039 is a nanomolar potent agonist at recombinant human mGlu2 and mGlu3 receptors (Ki = 149 and 92, respectively) and in rat neurons expressing native mGlu2/3 receptors (Ki = 88). LY404039 is highly selective for mGlu2/3 receptors, showing more than 100-fold selectivity for these receptors, versus ionotropic glutamate receptors, glutamate transporters, and other receptors targeted by known anxiolytic and antipsychotic medications. Functionally, LY404039 potently inhibited forskolin-stimulated cAMP formation in cells expressing human mGlu2 and mGlu3 receptors. Electrophysiological studies indicated that LY404039 suppressed electrically evoked excitatory activity in the striatum, and serotonin-induced l-glutamate release in the prefrontal cortex; effects reversed by LY341495. These characteristics suggest LY404039 modulates glutamatergic activity in limbic and forebrain areas relevant to psychiatric disorders; and that, similar to LY354740, it works through a mechanism that may be devoid of negative side effects associated with current antipsychotics and anxiolytics. Interestingly, despite the slightly lower potency (∼2–5-fold) of LY404039 versus LY354740 in binding, functional, and electrophysiological assays, LY404039 demonstrated higher plasma exposure and better oral bioavailability in pharmacokinetic experiments. Collectively, the current data indicate that LY404039 may be valuable in the treatment of neuropsychiatric disorders, including anxiety and psychosis.

Group III human metabotropic glutamate receptors 4, 7 and 8: Molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells

Cloning and expression in a stable mammalian cell line co-transfected with a glutamate transporter (RGT cells) were used as tools for studying the functions and pharmacological properties of group III metabotropic glutamate receptors (mGluRs). Complementary DNAs (cDNAs) encoding the human mGluR4, human mGluR7, and human mGluR8 were isolated from human cerebellum, fetal brain or retinal cDNA libraries. The human mGluR4, mGluR7 and mGluR8 receptors were 912, 915 and 908 amino acid residues long and share 67-70% amino acid similarity with each other and 42-45% similarity with the members of mGluR subgroups I and II. The human mGluR4 and mGluR7 had amino acid identity of 96% and 99.5% with rat mGluR4 and 7, respectively, whereas the human mGluR8 has 98.8% amino acid identity with the mouse mGluR8. The nucleotide and amino acid sequences in the coding region of human mGluR4 and mGluR7 were found to be identical to the previously published sequences by Flor et al. and Makoff et al. Following stable expression in RGT cells, highly significant inhibitions of forskolin stimulation of cAMP production by group III agonists were found for each receptor. The relative potencies of the group III agonist L-AP4 varied greatly between the group III clones, being mGluR8>mGluR4 >> mGluR7. The reported group II mGluR agonist L-CCG-I was a highly potent mGluR8 agonist (EC50=0.35 microM), with significant agonist activities at both mGluR4 (EC50=3.7 microM) and mGluR7 (EC50=47 microM). The antagonist potency of the purported group III mGluR antagonist MPPG also varied among the receptors being human mGluR8 >> mGluR4 = mGluR7. The expression and second messenger coupling of human group III mGluRs expressed in the RGT cell line are useful to clearly define the subtype selectivities of mGluR ligands.