12 research outputs found
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 (IC<sub>50</sub>) 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
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 IC<sub>50</sub> 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 IC<sub>50</sub> values were 46.0 ± 3.9 nM and 32.5 ± 1.8 nM, respectively (n = 8). Error bars represent the standard deviation.</p
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<sub>ss</sub> = volume of distribution at steady state; MRT<sub>disp</sub> = mean residence time of drug molecules after intravascular administration; T<sub>1/2</sub> = 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<sub>PL</sub> = concentration in plasma, C<sub>BR</sub> = concentration in brain, K<sub>P,BR</sub> = partitioning coefficient between brain and plasma.</p
HC-070 reduces marble burying behavior.
<p>(A) Mice were administered vehicle or 1, 3 or 10 mg/kg HC-070 orally, 60 minutes prior to testing. The positive control, 10 mg/kg zimelidine, was administered IP 45 minutes prior to testing. At 1, 3, and 10 mg/kg, HC-070 significantly decreased the number of buried marbles compared to vehicle (p <0.05 *, p<0.01**, Dunnett’s post-hoc test following one-way ANOVA, n = 10/group). Zimelidine also decreased the number of buried marbles (p < 0.05, t-test, n = 10/group). Each animal is represented on the graph and the horizontal lines represent the mean. (B) Average plasma exposures from animals that completed the test. Error bars are 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
HC-070 inhibition of heterologously expressed mouse TRPC4 and TRPC5 channels.
<p>(A) Average currents at -80 mV (filled circles) and +80 mV (open squares) during the application of varying concentrations of HC-070 (blue bars). The TRPC5 current was activated by application of 80 μM lanthanum chloride (LaCl<sub>3</sub>) (gray bar), and 50 μM 2-aminoethyl-diphenyl-borinate (2-APB) (red bar) was used to completely block the TRPC5 current. Dotted lines depict the estimated unblocked current, to account for channel rundown (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191225#sec015" target="_blank">Methods</a>). (B) TRPC5 current-voltage curves recorded at the times in A shown by lower case letters (a, b, c). The steps at ±80 mV flanking the ramp, used to calculate the averages in A, are not shown. (C) Dose-response curves of TRPC4 and TRPC5 channel inhibition by HC-070. Symbols are the average % inhibition recorded from 3–13 cells; error bars are the standard error of the mean (SEM; in some cases smaller than the symbol). Solid lines are fits of the Hill equation to the points. Mouse TRPC5 channels activated by 80 μM LaCl<sub>3</sub> are shown by filled circles (fit parameters IC<sub>50</sub> = 0.52 nM, Hill coefficient = 1.31). Mouse TRPC4 (mTRPC4) channels activated by carbachol stimulation of human muscarinic 2 receptor (M2R) are shown in crosses (fit parameters IC<sub>50</sub> = 1.8 nM, Hill coefficient = 1.27), and mTRPC5 channels activated by carbachol stimulation of the human muscarinic type 1 (M1R) is shown in open squares (fit parameters IC<sub>50</sub> = 3.4 nM, Hill coefficient = 0.91).</p
Effects of HC-070 on CSD-induced fear hyper-reactivity.
<p>HC-070 reduces the increased capacity for fear memory in mice exposed to chronic social stress on days 1–15. (A) Day 16: Without drug administration, when placed in a relatively unfamiliar arena (context) without tone or electroshock, mice that had been exposed to chronic social defeat (CSD) tended to show more freezing—a fear behavior—than did control mice (CON). (B) Day 17: Without drug administration, mice were placed back in the same context and exposed to 6 pairings of a 20 s tone conditioned stimulus (CS) that announced a 2 s electroshock unconditioned stimulus (US). CSD mice acquired more freezing to the CS than did CON mice. The left panel presents the fear learning curve using the average freezing during CS-US trials 1–2, 3–4 and 5–6, and the right panel presents the average freezing across all six CS-US trials. Immediately after this CS-US conditioning session, CSD and CON mice received either vehicle or 1 mg/kg HC-070 orally. (C) Day 18: Mice received vehicle or 1 mg/kg HC-070 orally and 1 hour later were placed in the same context in which CS-US conditioning took place the previous day, and a 21-min test of context fear memory was conducted, i.e. in the absence of the CS (and US). The left panel presents the context-fear expression curve using the average freezing per 3-min block. The right panel presents the average context-fear expression across all 21 min. HC-070 significantly reduced the context fear memory in CSD mice, as indicated by these mice showing freezing levels similar to those of CON mice and lower than those of CSD-VEH mice. (D) Day 18: Immediately after the context fear memory test, the tone-CS fear memory test was conducted, comprising 12 trials of 30-s CS separated by 90-s inter-trial intervals. The fear expression curve to the CS is presented using the average freezing per pair of consecutive CS trials. HC-070 tended to reduce the CS fear memory in CSD mice, as indicated by the increased rate at which their freezing level during the CS attenuated compared with CSD-VEH mice, i.e. faster extinction learning. (E) Day 18: In the tone CS memory test presented in (D), freezing was also measured in the inter-trial intervals (ITIs) between CS presentations. The left panel presents the fear expression curve in ITIs between CSs using the average freezing per pair of consecutive ITIs. The right panel presents the average freezing across all 12 ITIs. Here also, HC-070 reduced fear memory in CSD mice, as indicated by the increased rate at which their freezing level during the ITIs attenuated compared with CSD-VEH mice, i.e. faster extinction learning.</p
HC-070 attenuates CCK-4 induced EPSCs recorded from the basolateral amygdala in a slice preparation.
<p>(A) Representative traces before and after CCK-4 application in the presence of vehicle. (B) Representative traces before and after CCK-4 application in the presence or absence of HC-070. (C) Quantitation of the results (n = 8). HC-070 was pre-incubated with the slice. CCK-4 was applied at the time indicated. Error bars represent the SEM (* p < 0.05, ***p <0.001, Tukey’s multiple comparisons test following two-way analysis of variance (ANOVA)). The holding potential was -70 mV.</p
HC-070 reduces time of immobility in a tail suspension test but does not impact locomotor activity.
<p>(A) Mice were administered vehicle or 0.3, 1, 3 or 10 mg/kg HC-070 PO 60 minutes prior to testing. The positive control, 8 mg/kg desipramine, was also administered orally. All doses of HC-070 reduced the time of immobility (p <0.05 *, p<0.01**, p < 0.001 *** Dunnett’s post-hoc test following one-way ANOVA, n = 13–16 group), as did desipramine (p <0.0001, t-test) Each animal is shown on the graph and the horizontal lines represent the mean. (B) Associated exposures. Error bars are the standard deviation. (C) Locomotor activity was not impacted. After 90 minutes of habituation to an activity chamber, mice were administered vehicle or 0.1, 3 or 10 mg/kg HC-070 PO. Activity was recorded for another 60 minutes. There was no effect of dose (2-way ANOVA followed by a Tukey’s multiple comparison’s test, p>0.5 for all treatments; n = 8/group).</p
HC-070 inhibits recombinantly expressed TRPC4 and TRPC5, as well as TRPC1-containing heteromultimers, in whole-cell manual patch clamp.
<p>HC-070 inhibits recombinantly expressed TRPC4 and TRPC5, as well as TRPC1-containing heteromultimers, in whole-cell manual patch clamp.</p