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. 2022 Apr;63(4):1003-1015.
doi: 10.1111/epi.17190. Epub 2022 Feb 18.

Inhibition of TRPC3 channels by a novel pyrazole compound confers antiseizure effects

Marwa M Nagib  1 , Sicheng Zhang  1 , Nelufar Yasmen  1 , Lexiao Li  1 , Ruida Hou  1 , Ying Yu  1 , Vijay K Boda  1 , Zhongzhi Wu  1 , Wei Li  1 , Jianxiong Jiang  1
Affiliations

Inhibition of TRPC3 channels by a novel pyrazole compound confers antiseizure effects

Marwa M Nagib et al. Epilepsia. 2022 Apr.

Abstract

Objective: As a key member of the transient receptor potential (TRP) superfamily, TRP canonical 3 (TRPC3) regulates calcium homeostasis and contributes to neuronal excitability. Ablation of TRPC3 lessens pilocarpine-induced seizures in mice, suggesting that TRPC3 inhibition might represent a novel antiseizure strategy. Among current TRPC3 inhibitors, pyrazole 3 (Pyr3) is most selective and potent. However, Pyr3 only provides limited benefits in pilocarpine-treated mice, likely due to its low metabolic stability and potential toxicity. We recently reported a modified pyrazole compound 20 (or JW-65) that has improved stability and safety. The objective of this study was to explore the effects of TRPC3 inhibition by our current lead compound JW-65 on seizure susceptibility.

Methods: We first examined the pharmacokinetic properties including plasma half-life and brain to plasma ratio of JW-65 after systemic administration in mice. We then investigated the effects of TRPC3 inhibition by JW-65 on behavioral and electrographic seizures in mice treated with pilocarpine. To ensure our findings are not model specific, we assessed the susceptibility of JW-65-treated mice to pentylenetetrazole (PTZ)-induced seizures with phenytoin as a comparator.

Results: JW-65 showed adequate half-life and brain penetration in mice, justifying its use for central nervous system conditions. Systemic treatment with JW-65 before pilocarpine injection in mice markedly impaired the initiation of behavioral seizures. This antiseizure action was recapitulated when JW-65 was administered after pilocarpine-induced behavioral seizures were well established and was confirmed by time-locked electroencephalographic monitoring and synchronized video. Moreover, JW-65-treated mice showed substantially decreased susceptibility to PTZ-induced seizures in a dose-dependent manner.

Significance: These results suggest that pharmacological inhibition of the TRPC3 channels by our novel compound JW-65 might represent a new antiseizure strategy engaging a previously undrugged mechanism of action. Hence, this proof-of-concept study establishes TRPC3 as a novel feasible therapeutic target for the treatment of some forms of epilepsy.

Keywords: calcium channels; pentylenetetrazole; pilocarpine; seizure; status epilepticus; transient receptor potential channels.

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Conflict of interest statement

Conflict of Interest

None of the authors have any conflict of interest to disclose. The authors confirm that they have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Figures

Figure 1.
Figure 1.
JW-65 is a novel, CNS-permeable, stable, and safe inhibitor of TRPC3 channels. (A) Isosteric replacement of ester with an amide linker in the head moiety and incorporation of a conformationally restricted ring (pyridone) in the tail group of Pyr3 led to the discovery of a novel TRPC3 inhibitor JW-65 (compound 20) , which is safer and metabolically more stable than Pyr3 without compromising its high potency and selectivity. (B) Pharmacokinetic study of JW-65 in adult male C57BL/6 mice after a single systemic dose (100 mg/kg, i.p.). The plasma concentration of compound quickly reached the maximum (156 ng/mL) about 15-30 min after injection and did not decline until 4 h after dosing (**P < 0.01; ****P < 0.0001, one-way ANOVA and Dunnett's multiple comparisons test). JW-65 had a plasma half-life of 3.1 h. (C) JW-65 showed a brain to plasma ratio of ~0.3 at both 1 h and 2 h after injection. Data are shown as mean +/± SEM (N = 3 mice per time point). It is noted that the mice utilized to determine the plasma half-life of JW-65 were different from those for the evaluation of its brain penetration.
Figure 2.
Figure 2.
Experimental procedures to examine the antiseizure effects of TRPC3 inhibitor JW-65 in mouse models of chemoconvulsant-induced seizures. (A) In the pre-treatment study on pilocarpine-induced seizures, C57BL/6 mice were treated by vehicle (10% DMSO, 40% PEG 300, 10% Tween 80, and 40% saline) or compound JW-65 (100 mg/kg, i.p.) for two consecutive days. On the experiment day, mice were treated by methylscopolamine and terbutaline (2 mg/kg each in saline, i.p.), and 15 min later by JW-65 (100 mg/kg, i.p.). After another 15 min, mice were treated by pilocarpine (250 mg/kg in saline, i.p.) to induce SE. Behavioral seizures were observed and classified using a modified Racine scale. (B) In the post-treatment study on pilocarpine-induced seizures, mice were first treated by methylscopolamine and terbutaline (2 mg/kg each in saline, i.p.), and 30 min later by pilocarpine (220 mg/kg in saline, i.p.) to induce seizures. After behavioral seizures (stage-2) were observed, which typically occurred ~15 min after pilocarpine injection, mice were treated by JW-65 (100 mg/kg, i.p.) once. Behavioral seizures of mice were classified, and the electrographic seizures were recorded by surface electroencephalography (EEG) recording. (C) In the post-treatment study on pentylenetetrazole (PTZ)-induced seizures, mice were first treated by vehicle or JW-65 (100 mg/kg, i.p.) once, and 30 min later by pentylenetetrazole (~80 mg/kg, s.c.). Behavioral seizures were observed for 30 min.
Figure 3.
Figure 3.
Pre-treatment with JW-65 impairs the initiation and development of pilocarpine-triggered seizures. (A) Mice were pre-treated by JW-65, followed by pilocarpine injection for seizure induction. The behavioral seizure scores were tabulated every 5 min for up to 2 h and compared (N = 13 and 11 for vehicle group and JW-65 group, respectively, ***P < 0.001, two-way ANOVA and post hoc Bonferroni test for multiple comparisons). (B) Latencies of mice to behavioral seizures (stage-2, stage-3, and stage-4) after pilocarpine administration in mice (**P < 0.01; ***P < 0.001, t-test). (C) The average seizure scores during the 2 hour-observation after pilocarpine injection (***P < 0.001, t-test). (D) The maximal seizure scores were compared between vehicle and JW-65 groups (***P < 0.001, t-test). (E) Number of mice that entered SE during the 2 hour-observation period (***P < 0.001, Fisher’s exact test). (F) Mortality of mice caused by pilocarpine-induced SE (P = 0.0983, Fisher’s exact test).
Figure 4.
Figure 4.
Post-treatment with JW-65 suppresses the progression and escalation of pilocarpine-induced SE. (A) Mice were treated by pilocarpine for seizure induction and ~15 min later by JW-65 after behavioral seizures (stage-2) were established. The behavioral seizure scores were tabulated every 5 min for up to 2 h and compared (N = 13 and 9 for vehicle and TRPC3 groups, respectively, ***P < 0.001, two-way ANOVA and post hoc Bonferroni test for multiple comparisons). (B) Latencies of animals to behavioral seizures (stage-2, P = 0.6857; stage-3, ***P < 0.001; stage-4, ***P < 0.001, t-test) after pilocarpine administration in mice. (C) The average seizure scores during the 2 hour-observation period after pilocarpine injection (***P < 0.001, t-test). (D) The maximal seizure scores were compared between JW-65 and vehicle-treated mice (***P < 0.001, t-test). (E) Number of mice that eventually experienced SE during the 2 hour-observation (***P < 0.001, Fisher’s exact test). (F) Animal mortality caused by pilocarpine-induced seizures (P = 0.1649, Fisher’s exact test).
Figure 5.
Figure 5.
TRPC3 inhibition by JW-65 reduces electrographic seizures in mouse pilocarpine model of SE. Mice were first treated by pilocarpine to induce seizures, followed by treatment with vehicle (A) or JW-65 (B). Time-locked video-EEG was used to monitor the electrographic seizures in these mice. (C) The total numbers of EEG spikes during the first hour, two hours, and four hours after pilocarpine injection were identified by DClamp 4.1 EEG analysis software and compared (N = 7, **P < 0.01; ***P < 0.001, two-way ANOVA and post hoc Bonferroni test for multiple comparisons).
Figure 6.
Figure 6.
Inhibition of TRPC3 channels reduces susceptibility of animals to PTZ-induced seizures. Adult C57BL/6 mice were first treated by vehicle, TRPC3 inhibitor JW-65 (20, or 100 mg/kg, i.p.), and 30 minutes later by pentylenetetrazole (PTZ, ~80 mg/kg, s.c.) for the induction of seizures. Conventional anticonvulsant phenytoin (40 mg/kg, i.p.) was used as a comparator in the same study. (A) Percentages of mice reaching myoclonic jerk (MJ, Left) and generalized tonic-clonic seizure (GTCS, Right) were compared between vehicle and drug-treated mice (**P < 0.01; ***P < 0.001, Mantel-Cox log-rank test). (B) Latencies to the first MJ (Left) and first GTCS (Right) were compared (*P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA and Dunnett's multiple comparisons test). Data are shown as mean + SEM (N = 11, 8, 10, and 7 for vehicle group, 20 mg/kg JW-65 group, 100 mg/kg JW-65 group, and 40 mg/kg phenytoin group, respectively).
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