Molecule

Description

Neurological Disease

Description

LY404039 is a potent agonist of recombinant human mGlu2, mGlu3 receptors and rat neurons expressing native mGlu2/3 receptors with K_i of 149 nM, 92 nM and 88 nM, respectively.

Targets

IC₅₀

In Vitro

LY404039 exhibits low binding affinity to group III mGlu receptors including mGlu6, mGlu7 and mGlu8 with a K_i value more than 5 μM. LY404039 shows little affinity for ionotropic glutamate receptors, glutamate transporter subtypes, monoamine and other receptors. LY404039 potently inhibits forskolin-stimulated cAMP formation in cells expressing human mGlu2 and mGlu3 receptors. LY404039 suppresses electrically evoked excitatory activity in the striatum, and serotonin-induced L-glutamate release in the prefrontal cortex. LY404039 could modulate glutamatergic activity in limbic and forebrain areas relevant to psychiatric disorders and may be devoid of negative side effects associated with current antipsychotics and anxiolytics. ^[1]

In Vivo

LY404039 demonstrates higher plasma exposure and better oral bioavailability. LY404039 may be valuable in the treatment of neuropsychiatric disorders, including anxiety and psychosis. ^[1] In wild-type animals, LY404039 significantly reverses d-amphetamine(AMP)-induced increase in ambulations, distance traveled, and reduced time spent at rest. LY404039 reverses phencyclidine (PCP)-evoked behaviors at 10 mg/kg. The antipsychotic-like effects of LY404039 on PCP and AMP-evoked behavioral activation are absent in mGlu2 and mGlu2/3 but not in mGlu3 receptor-deficient mice. In contrast, clozapine and risperidone inhibit PCP-evoked behaviors in both wild-type and mGlu2/3 receptor-deficient mice. ^[2] LY404039 reduces responding on the EtOH in the pavlovian spontaneous recovery (PSR) test and reduces the expression of an alcohol deprivation effect (ADE) during relapse, but does not affect EtOH responding under maintenance conditions. LY404039 inhibits the expression of alcohol seeking and relapse behavior without altering alcohol self-administration behavior. ^[3] Moreover, LY404039 attenuates amphetamine- and phencyclidine-induced hyperlocomotion. LY404039 could inhibit conditioned avoidance responding and also reduces fear-potentiated startle in rats and marble burying in mice. Importantly, LY404039 does not produce sedative effects or motor impairment in the conditioned avoidance task. LY404039 also increases dopamine and serotonin release/turnover in the prefrontal cortex. ^[4]

Clinical Trials

LY2140023, an oral prodrug of LY404039, is currently in Phase III clinical trial in patients with schizophrenia.

Features

LY2140023 is being investigated as an exciting new medicine that may herald the arrival of third-generation antipsychotic drugs.

Receptor Binding Assays

LY404039 is dissolved in DMSO to make a 10 mM stock solution. Cell lines expressing human mGlu2, mGlu3, mGlu1a, mGlu5a, mGlu4a, mGlu6, mGlu7a, and mGlu8 receptors are derived and cultured in DMEM with 5% dialyzed fetal bovine serum, 1 mM glutamine, 1 mM sodium pyruvate, 50 mg/mL Geneticin, and 0.2 mg/mL hygromycin B. Confluent cultures are passaged weekly. These cells are referred to as rat glutamate transporter (RGT) cells. RGT cells are actually AV12-664 cells, which have been stably transfected with a glutamate transporter (GLAST) to prevent the accumulation of L-glutamate in the cell media. Membranes from mGlu receptor-expressing RGT cells are prepared by harvesting adherent cells from confluent T-150 flasks with a cell scraper. Cells and media are then placed in 50-mL conical tubes and centrifuged at 10³ g for 5 min at 4 °C. The supernatant is removed, and the pellet is frozen at –10 °C until use. Frozen pellets are routinely thawed at room temperature for about 10 min when required. Washed cell membranes are prepared by adding 20 mL of 10 mM potassium phosphate buffer, pH 7.6 and 100 mM potassium bromide, at 5 °C and homogenizing with a tissuemizer for 15 s at 90% output. The homogenate is centrifuged at 4.8 × 10³ g at 5 °C for 10 min. This last step is repeated twice, the final pellet is resuspended in 10 mL of the same buffer and reserved on ice until initiating the binding assay. [³H]LY341495 Binding: Group II mGlu Receptors: [³H]LY341495 binding is assayed in a reaction mixture containing 10 mM potassium phosphate, pH 7.6, 100 mM potassium bromide, and 1nM [³H]LY341495 (final volume, 500 mL). The incubation is initiated by the addition of the membrane suspension (15 mg of membrane protein) and kept on ice for 30 min. Incubation is terminated by rapid filtration with a cell harvester through glass fiber Whatman GF/B filters prewet with the same potassium phosphate buffer assay buffer at 4 °C. The filters are washed 5 times with 1 mL of buffer. Filter sections are transferred to minivials, and 5 mL liquid scintillation cocktail is added to each vial. Vials are allowed to set for several hours before counting on a liquid scintillation counter. Protein concentrations are quantified by microassay. Nonspecific binding is determined in the presence of 1 mM L-glutamate. [³H]LY341495 Binding: Group III mGlu Receptors: Membranes from cells expressing recombinant human group III mGlu receptors are prepared the same as described for group II mGlu receptor membranes. To start the reaction, washed tissue (0.05–0.20 mg of protein) is added to 10 nM [³H]LY341495 and LY404039 in assay buffer. The final assay volume is 0.5 mL. Nonspecific binding is defined with 1 mM L-glutamate or 1 mM L-serine-O-phosphate (for mGlu7a). Assay plates are incubated for 45 min on ice, and the reaction is terminated by rapid filtration (GF/B filters) and washed twice with 1 mL of ice-cold assay buffer. Filters are placed in minivials, scintillation cocktail is added and counting is done in a liquid scintillation counter. Protein concentration is determined using the microassay. [³H]LY341495 Binding to Rat Brain Membranes: Brain tissue is obtained by decapitating adult male Sprague-Dawley rats (150–250g). The forebrain (cortex, striatum, and hippocampus) is used; the brain tissue is homogenized in 30 mM Tris-HCl buffer, pH 7.6 and 2.5 mM CaCl₂, at 5 °C and washed 3 times by centrifugation. The material is incubated at 37 °C for 30 min followed by 3 more washes, finally resuspended in 10 volumes of buffer and frozen at –20 °C. Frozen pellets of rat brain homogenate are thawed on the day of assay and washed 3 times with ice-cold assay buffer (10 mM potassium phosphate containing 100 mM potassium bromide, pH 7.6). The tissue (0.02–0.06 mg of protein) is added to deep-well polypropylene microtiter plates, which contain 1 nM [³H]LY341495 and LY404039 in assay buffer. The final assay volume is 0.5 mL. Nonspecific binding is defined with 1 mM L-glutamate. Assay plates are incubated for 30 minutes on ice, and bound and free radioligands are separated by rapid filtration with 5 × 1 mL of ice-cold assay buffer using Whatman GF/B filters. Protein concentration is determined using the microassay.

Animal Models

Separate mGlu2 and mGlu3 receptor knockout mice are established.

Formulation

Dissolved in 0.9% saline and the pH is adjusted to 6.0 with 1 M NaOH

Doses

10 mg/kg

Administration

Administered via i.p.