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. 2023 Apr 14;9(4):e15423.
doi: 10.1016/j.heliyon.2023.e15423. eCollection 2023 Apr.

Reduced pain sensitivity of episodic pain syndrome model mice carrying a Nav1.9 mutation by ANP-230, a novel sodium channel blocker

Hiroko Okuda  1   2 , Sumiko Inoue  1 , Yoshihiro Oyamada  1   3 , Akio Koizumi  1   4 , Shohab Youssefian  1   5
Affiliations

Reduced pain sensitivity of episodic pain syndrome model mice carrying a Nav1.9 mutation by ANP-230, a novel sodium channel blocker

Hiroko Okuda et al. Heliyon. .

Abstract

The sodium channel Nav1.9 is expressed in the sensory neurons of small diameter dorsal root ganglia that transmit pain signals, and gain-of-function Nav1.9 mutations have been associated with both painful and painless disorders. We initially determined that some Nav1.9 mutations are responsible for familial episodic pain syndrome observed in the Japanese population. We therefore generated model mice harboring one of the more painful Japanese mutations, R222S, and determined that dorsal root ganglia hyperexcitability was the cause of the associated pain. ANP-230 is a novel non-opioid drug with strong inhibitory effects on Nav1.7, 1.8 and 1.9, and is currently under clinical trials for patients suffering from familial episodic pain syndrome. However, little is known about its mechanism of action and effects on pain sensitivity. In this study, we therefore investigated the inhibitory effects of ANP-230 on the hypersensitivity of Nav1.9 p.R222S mutant model mouse to pain. In behavioral tests, ANP-230 reduced the pain response of the mice, particularly to heat or mechanical stimuli, in a concentration- and time-dependent manner. Furthermore, ANP-230 suppressed the repetitive firing of dorsal root ganglion neurons of these mutant mice. Our results clearly demonstrate that ANP-230 is an effective analgesic for familial episodic pain syndrome resulting from DRG neuron hyperexcitability, and that such analgesic effects are likely to be of clinical significance.

Keywords: Behavioral experiments; Electrophysiology; Episodic pain syndrome; Nav1.9 voltage-gated sodium channel; Non-opioid analgesics; Pain model mice; Pharmacology; Sodium channel blocker.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
The analgesic effects of ANP-230 on R222S mice. Reduced pain behavior by oral administration of ANP-230. (A) Dose-dependent inhibition of the hyper-thermal sensitivity of R222S and WT mice before and after 1 h ANP-230 (ANP) administration (R222S, n = 9–10; WT, n = 8–9). (B) Inhibition of the hypo-thermal sensitivity of R222S mice but not WT mice at each dose of ANP-230 (WT, n = 9–10; R222S, n = 9–11). Pregabalin (PGB) had an inhibitory effect on the hypo-thermal sensitivity of both mice groups but not on their hyper-thermal sensitivity. (C) Dose-dependent inhibition of the mechanosensitivity of R222S mice but not WT mice before and after ANP-230 administration (R222S, n = 9–11; WT, n = 10). (D) Reduction of the hyper-mechanosensitivity of R222S mice by repetitive administration of low dose (3 mg/kg) ANP-230 (R222S, n = 8–9; WT, n = 11). (E) Scheme showing the effects of the first and second cold periods on the mechanosensitivity of non treated WT and R222S groups, and of the time course administration of vehicle or ANP-230 on the mechanosensitivity of R222S mice. Two cold exposure periods were set to ensure complete cold exposure conditions. (F) Time course of ANP-230 (30 mg/kg) on the mechanosensitivity of R222S mice. No significant differences were observed between the first (beige) and second (orange) mechanosensitivity tests in R222S and WT mice (R222S, n = 5; WT, n = 6). The duration of the analgesic effects of ANP-230 on R222S mice (n = 5–7) continued for at least 3 h but not up to 6 h. In Figs. A to D, the white and blue bars represent the before and after vehicle or drug administration, respectively (*p < 0.05, †p < 0.01 comparing before and after vehicle or drug administration; One way ANOVA), and in Fig. F the white and blue bars represent the vehicle or ANP-230 administration, respectively in the treated group (*p < 0.05, †p < 0.01 as compared to vehicle and drugs in treated group; two-sided Student's t-test). Data are presented as mean ± S.E.M. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2
Fig. 2
ANP-230 reduces the hyperexcitability of small DRG of R222S mice. (A) Representative traces of the action potential (AP) firing before and after administration of 10 and 30 μM ANP-230, recorded from small DRG neurons (<25 μm) in R222S and WT at 210 pA during 500 ms. The decline of repetitive APs of R222S mice, but not in WT mice, by administration of ANP-230. Dashed lines represent the 0 mV in A and C. (B) Dose-dependent effects of ANP-230 on firing probability of DRG neurons of R222S (upper panel; Before, n = 16; ANP-230 10 μM, n = 7; ANP-230 30 μM, n = 9), but not in DRG neurons of WT (lower panel; Before, n = 14; ANP-230 10 μM, n = 7; ANP-230 30 μM, n = 7). The 500-ms-step current injections ranged from 10 to 210 pA. Data are presented as mean ± S.E.M. (*p < 0.05,†p < 0.01 in comparisons of before and after ANP-230 administration at each dose; two-sided Student's t-test). (C) The firing frequency partially reversed to hyperexcitable conditions by washout. Each trace is a representative trace recording at an input current of 135 pA before, after 30 μM ANP-230 administration, and after wash conditions. (D) The effect of ANP-230 on the firing frequency of R222S mice (n = 3) was significantly cleared by the wash conditions. (E) Shift in the resting membrane potential (RMP) of R222S and WT mice in response to ANP-230. The recovery of the polarized RMP towards hyperpolarization was significant in R222S mice (n = 6) at a high ANP-230 dose, but there was no shift in WT mice at either dose (n = 6–7). (F) Recovery of the depolarized current threshold of R222S mice by both doses of ANP-230 (R222S, n = 7–8; WT, n = 7–6). (G) Changes in input impedance in R222S and WT mice. There were no significant changes in input impedance following administration of ANP-230 at either dose (R222S, n = 4–5; WT, n = 5). Before ANP-230 administration is represented by black line traces, white dots and bars, and after 10 and 30 μM ANP-230 administration represented by blue and deep blue line traces, dots and bars, respectively. *p < 0.05, †p < 0.01 in comparisons of before and after administration or wash; two-sided Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3
Fig. 3
Effects of ANP-230 on the accumulation of Na+ currents in R222S mice. (A) Representative traces of AP firing before and after administration of ANP-230 at current threshold. The lower traces represent the expanded view of each 1st AP. Dashed lines represent the 0 mV. (B) The high ANP-230 dose significantly reduced the after hyperpolarization (AHP) of the 1st spike at current threshold in R222S mice (R222S, n = 6–7). (C) The high dose of ANP-230 significantly reduced the time constants of the AHP decay of the 1st spike (tau) in R222S mice (R222S, n = 5–6). (D) The recovery of depolarized voltage level during input currents at threshold current were significant at both ANP-230 doses in R222S (R222S, n = 5–7) but not in WT (WT, n = 4–7) mice. (E) No significant changes in the AHP of stimulus end to ANP-230 were observed in either R222S or WT mice. Color representations follow those in legend of Fig. 2. *p < 0.05, †p < 0.01 in comparisons of before and after administration or wash; two-sided Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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