Effect of MPX-004 and MPX-007 on glutamate/glycine-induced currents in <i>Xenopus</i> oocytes expressing GluN1 and GluN2A, B, C, or D.

<p>Oocytes were exposed to MPX-004 or MPX-007 at concentrations from 10 nM to 10 μM as indicated. Inhibition curves were generated using GraphPad Prism and IC₅₀ values were generated using CCD Vault. The IC₅₀s for inhibition of GluN2A-mediated currents were 198 ± 17 and 143 ± 10 nM for MPX-004 and MPX-007, respectively. Each data point is a mean (± standard deviation) of data from 4–12 oocytes.</p

Effects of MPX-004 and MPX-007 on NMDA/glycine-induced currents of rat cortical neurons in primary culture.

<p>Cortical neurons were maintained in primary culture for 13–15 days and then examined using whole-cell manual patch clamp for current evoked by NMDA (100 μM) + glycine (10 μM) applied for 4 seconds during 10 second pulses to +20 or +40 mV from a holding potential of -70 mV. Inhibition of NMDA-activated currents was quantified during exposure to 10 μM MPX-007, MPX-004, Ro 25–6981, or a combination of Ro 25–6981 + MPX-004. Currents were blocked ~25–30% by either MPX-007 or MPX-004 alone, ~70% by Ro 25–6981 alone, and ~85% by Ro 25–6981 plus MPX-004.</p

Synthetic schemes for compounds 2–10.

<p>Synthetic schemes for compounds 2–10.</p

Effect of MPX-004 on isolated NMDA receptor-mediated fEPSPs in rat hippocampal CA1 stratum radiatum in response to stimulation of Schaffer collateral input.

<p>Slices were exposed to MPX-004 from 100 nM to 30 μM in half log concentration increments as well as to 50 μM. Right panel- Time course for inhibition of fEPSPs after application of different concentrations of MPX-004. Left panel- Percent inhibition at 40 min after application for each MPX-004 concentration. Maximum inhibition was approximately 60% of the fEPSPs at 30 or 50 μM. The IC₅₀ of MPX-004 for inhibition of fEPSPs was 3.4 μM. Curves in the right panel and data point in the left panel are a mean (± SEM) of 4, 5, 6, 8, 6, 5 and 2 slices obtained from 2, 2, 3, 3, 2, 2 and 1 rats exposed to 0.1, 0.3, 1, 3, 10, 30 and 50 μM MPX-004, respectively. In separate experiments, a highly selective GluN2B NAM inhibited approximately 40% of the fEPSP (data not shown).</p

Concentration-response of TCN-201, MPX-004, and MPX-007 inhibition of Ca²⁺ responses mediated by GluN2A expressed in HEK cells.

<p>Cells were stimulated with glutamate and glycine (3 μM each) in the presence of compounds at a range of concentrations. Curves for inhibition of the Ca²⁺ response in GluN2A-expressing cells were derived from fits to the Hill equation using GraphPad Prism (v6.00 for Mac, GraphPad Software, La Jolla California USA). Whereas MPX-004 and MPX-007 achieve full inhibition of the GluN2A Ca²⁺ response by ~ 3 μM, TCN-201 never inhibits more than ~40% of the response. Each data point is a mean (± standard deviation) of data from 20–86 experiments).</p

The structures of TCN-201, MPX-004 and MPX-007.

<p>The values are IC₅₀s for inhibition of Ca²⁺ responses mediated by GluN2A receptors expressed in HEK cells.</p

Treatment with HC-070, a potent inhibitor of TRPC4 and TRPC5, leads to anxiolytic and antidepressant effects in mice

<div><p>Background</p><p>Forty million adults in the US suffer from anxiety disorders, making these the most common forms of mental illness. Transient receptor potential channel canonical subfamily (TRPC) members 4 and 5 are non-selective cation channels highly expressed in regions of the cortex and amygdala, areas thought to be important in regulating anxiety. Previous work with null mice suggests that inhibition of TRPC4 and TRPC5 may have anxiolytic effects.</p><p>HC-070 <i>in vitro</i></p><p>To assess the potential of TRPC4/5 inhibitors as an avenue for treatment, we invented a highly potent, small molecule antagonist of TRPC4 and TRPC5 which we call HC-070. HC-070 inhibits recombinant TRPC4 and TRPC5 homomultimers in heterologous expression systems with nanomolar potency. It also inhibits TRPC1/5 and TRPC1/4 heteromultimers with similar potency and reduces responses evoked by cholecystokinin tetrapeptide (CCK-4) in the amygdala. The compound is >400-fold selective over a wide range of molecular targets including ion channels, receptors, and kinases.</p><p>HC-070 <i>in vivo</i></p><p>Upon oral dosing in mice, HC-070 achieves exposure levels in the brain and plasma deemed sufficient to test behavioral activity. Treatment with HC-070 attenuates the anxiogenic effect of CCK-4 in the elevated plus maze (EPM). The compound recapitulates the phenotype observed in both null TRPC4 and TRPC5 mice in a standard EPM. Anxiolytic and anti-depressant effects of HC-070 are also observed in pharmacological in vivo tests including marble burying, tail suspension and forced swim. Furthermore, HC-070 ameliorates the increased fear memory induced by chronic social stress. A careful evaluation of the pharmacokinetic-pharmacodynamic relationship reveals that substantial efficacy is observed at unbound brain levels similar to, or even lower than, the 50% inhibitory concentration (IC₅₀) recorded in vitro, increasing confidence that the observed effects are indeed mediated by TRPC4 and/or TRPC5 inhibition. Together, this experimental data set introduces a novel, high quality, small molecule antagonist of TRPC4 and TRPC5 containing channels and supports the targeting of TRPC4 and TRPC5 channels as a new mechanism of action for the treatment of psychiatric symptoms.</p></div

Pharmacokinetic properties of HC-070.

<p>PK profiles of HC-070 after intravenous (A) and oral (B) administration in C57BL/6 mice. Plasma concentrations were determined by LC-MS/MS. Points represent the individual concentrations at the times indicated. Lines represent mean exposure (n = 12 mice/arm, n = 3 data points per time point). (C) Summary of PK properties. CL = clearance; V_ss = volume of distribution at steady state; MRT_disp = mean residence time of drug molecules after intravascular administration; T_1/2 = half-life. (D) Plasma and brain concentrations measured 2 hours after intravenous or oral administration of 1 or 10 mg/kg HC-070, respectively. C_PL = concentration in plasma, C_BR = concentration in brain, K_P,BR = partitioning coefficient between brain and plasma.</p

Structures and activity in fluorometric assays of HC-070 and HC-608.

<p>The chemical structures of (A) HC-070 and (B) HC-608 (Pico145). (C) Inhibition of hTRPC5 by HC-070 and HC-608 in indicator-assisted calcium influx analysis. Concentrations tested ranged from 1 picomolar (pM) to 1 μM. Each data point represents the average of 8 measurements from a 384-well plate. Error bars show the standard deviation. The IC₅₀ values were 9.3 ± 0.9 and 6.2 ± 0.5 nanomolar (nM), respectively. (D) Inhibition of hTRPC4 by HC-070 and HC-608 over the same range of concentrations. The IC₅₀ values were 46.0 ± 3.9 nM and 32.5 ± 1.8 nM, respectively (n = 8). Error bars represent the standard deviation.</p

HC-070 decreases anxiety in a standard EPM.

<p>(A) Mice were administered vehicle or 0.3, 1 or 3 mg/kg HC-070 orally 60 minutes prior to EPM. The positive control, 1.5 mg/kg diazepam, was administered IP 30 minutes prior to testing. At 3 mg/kg, HC-070 significantly increased the number of open arm entries compared to the oral control (** p<0.01, Dunnett’s post-hoc test following one-way ANOVA). The positive control, 1.5 mg/kg diazepam, also increased open arm entries (* p <0.05, t-test). Animals that jumped or fell off the EPM during the test were excluded, such that the sample size (n) differed between groups. Each animal is shown on the graph (circles) and the horizontal lines represent the mean. (B) Average plasma and brain exposures from satellite animals dosed with 0.3, 1, or 3 mg/kg HC-070, 60 minutes post dosing. Error bars show standard deviation.</p