Inhibition of acid-sensing ion channels by diminazene and APETx2 evoke partial and highly variable antihyperalgesia in a rat model of inflammatory pain

Abstract

Background and purpose: Acid-sensing ion channels (ASICs) are primary acid sensors in mammals, with the ASIC1b and ASIC3 subtypes being involved in peripheral nociception. The antiprotozoal drug diminazene is a moderately potent ASIC inhibitor, but its analgesic activity has not been assessed.

Experimental approach: We determined the ASIC subtype selectivity of diminazene and the mechanism by which it inhibits ASICs using voltage-clamp electrophysiology of Xenopus oocytes expressing ASICs 1-3. Its peripheral analgesic activity was then assessed relative to APETx2, an ASIC3 inhibitor, and morphine, in a Freund's complete adjuvant (FCA)-induced rat model of inflammatory pain.

Key results: Diminazene inhibited homomeric rat ASICs with IC₅₀ values of ~200-800 nM, via an open channel and subtype-dependent mechanism. In rats with FCA-induced inflammatory pain in one hindpaw, diminazene and APETx2 evoked more potent peripheral antihyperalgesia than morphine, but the effect was partial for APETx2. APETx2 potentiated rat ASIC1b at concentrations 30-fold to 100-fold higher than the concentration inhibiting ASIC3, which may have implications for its use in in vivo experiments.

Conclusions and implications: Diminazene and APETx2 are moderately potent ASIC inhibitors, both inducing peripheral antihyperalgesia in a rat model of chronic inflammatory pain. APETx2 has a more complex ASIC pharmacology, which must be considered when it is used as a supposedly selective ASIC3 inhibitor in vivo. Our use of outbred rats revealed responders and non-responders when ASIC inhibition was used to alleviate inflammatory pain, which is aligned with the concept of number-needed-to-treat in human clinical studies.

Linked articles: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Figure 1

Diminazene inhibits ASICs via open channel block. (A–D) Concentration–effect curves comparing the inhibitory effect of diminazene on rat (r)ASIC channels expressed in Xenopus laevis oocytes when applied during conditioning alone (cond.), during the low pH stimulus alone (stim.) and during both conditioning and pH stimulus (cond. & stim.) (all n = 5 except ASIC3 cond. & stim. n = 8). (E) Voltage dependence of the inhibitory effect of diminazene (100 nM and 1 μM) applied during the pH 6 stimulus to rASIC1a (−90, −60 and −30 mV, n = 9; 0 mV, n = 8), rASIC1b (−90 and −30 mV, n = 6; −60 and 0 mV, n = 7), rASIC2a (n = 6) and rASIC3 (n = 7). (F) Concentration–effect curves for diminazene applied during both conditioning time and pH stimulus showing the subtype selectivity at ASIC1a (n = 5), 1b (n = 5), 2a (n = 5) and 3 (n = 8). (G) Summary of the pIC₅₀ values for each condition and each channel subtype tested. Statistical comparison of logIC₅₀ values was carried out using an extra sum‐of squares F test in Graphpad Prism 7 when fitting log[inhibitor] versus normalized response curves; statistical analysis of the rASIC2a IC₅₀ was not possible due to lack of an accurate IC₅₀ value from the incomplete concentration–effect curve.

Figure 2

Diminazene (DA) affects the gating of ASIC1a but not other subtypes. (A) Current traces showing concentration‐dependent inhibitory effect of diminazene on rat ASIC subtypes when applied during low pH stimulation. (B) Same traces as in (A) scaled (i.e. normalized to peak current amplitude) to illustrate the significant effect of diminazene (1 and 10 μM) on the activation and desensitization kinetics of ASIC1a but not ASIC1b. Note the traces in the presence of 10 μM diminazene are stepped due to scaling up very small currents. (C) Quantitation of the effect of diminazene on the activation and desensitization of ASIC homomeric channels presented as rise and decay times (10–90%). ASIC1a, 1b and 3 n = 5; ASIC2a n = 7, * P < 0.05, significantly different from control: paired t‐test.

Figure 3

Diminazene (DA) has highly variable antihyperalgesic activity in the FCA rat model of unilateral inflammatory pain. Mean (±SEM) paw pressure thresholds (PPT values), mean (±SEM) change in paw pressure thresholds (ΔPPTs) and mean (±SEM) change in ΔPPT area under the curve values (ΔPPT AUCs) for the ipsilateral (A, C, E) and contralateral (B, D, F) hindpaws of FCA rats following administration of single 100 μL intra‐plantar bolus doses of saline (vehicle control) (n = 15), morphine (Mor) at 700 nmol (n = 27), diminazene at 1.9 nmol (n = 15), 5.8 nmol (n = 13), 19.4 nmol (n = 16), 58.2 nmol (n = 17), 194 nmol (n = 16) and 582 nmol (n = 14). PPTs were measured at time 0 (pre‐dosing) and at 30, 45, 60, 75, 90, 120 and 180 min post‐dosing. The dotted line in (A) indicates the PPT from the contralateral hindpaw. *P < 0.05, significantly different from vehicle; one‐way ANOVA.

Figure 4

APETx2 has potent but highly variable antihyperalgesic activity in the FCA rat model of unilateral inflammatory pain. Mean (±SEM) paw pressure thresholds (PPT values), mean (±SEM) change in paw pressure thresholds (ΔPPTs) and mean (±SEM) change in PPT area under the curve (ΔPPT AUCs) for the ipsilateral (A, C, E) and contralateral (B, D, F) hindpaws of FCA rats following administration of single 100 μL intra‐plantar bolus doses of 0.05% RSA (vehicle control) (n = 10), morphine (Mor) at 700 nmol (n = 16), APETx2 at 0.03 pmol (n = 10), 0.1 pmol (n = 15), 1.1 pmol (n = 15), 3.3 pmol (n = 15) and 11 pmol (n = 16). PPTs were measured at time 0 (pre‐dosing) and at 30, 45, 60, 75, 90, 120 and 180 min post‐dosing. The dotted line in (A) indicates the PPT from the contralateral hindpaw. *P < 0.05, significantly different from vehicle; one‐way ANOVA.

Figure 5

Diminazene (DA) has potent but heterogeneous antihyperalgesic activity in the FCA rat model of unilateral inflammatory pain. Rats were classified as responders/high responders (A, C, E) or non‐responders/low responders (B, D, F) based on examination of ΔPPT AUC values for the individual ipsilateral hindpaws. ΔPPT AUC values of responders given morphine (Mor) at 700 nmol (n = 17) or diminazene at 5.8 nmol (n = 8), 19.4 nmol (n = 6), 58.2 nmol (n = 4), 194 nmol (n = 7) and 582 nmol (n = 6) were significantly higher than the corresponding ΔPPT AUC values evoked by vehicle (n = 15) (*P < 0.05 one‐way ANOVA). Diminazene at 1.9 nmol (n = 5) was not significantly different from vehicle. There was no significant difference between ΔPPT AUC values of non‐responders/low responders given morphine (n = 10) or diminazene at 1.9 nmol (n = 10), 5.8 nmol (n = 5), 19.4 nmol (n = 10), 58.2 nmol (n = 13), 194 nmol (n = 9) and 582 nmol (n = 8) when compared to the ΔPPT AUC of vehicle. The dotted line in (A) indicates the PPT from the contralateral hindpaw.

Figure 6

APETx2 has potent partial and heterogeneous antihyperalgesic activity in the FCA rat model of unilateral inflammatory pain. Rats were classified as responders/high responders (A, C, E) or non‐responders/low responders (B, D, F) based on examination of ΔPPT AUC values for the individual ipsilateral hindpaws. The ΔPPT AUC values of responders given morphine (Mor) at 700 nmol (n = 13) or APETx2 at 0.1 pmol (n = 6) and 1.1 pmol (n = 9) were significantly higher than the corresponding ΔPPT AUC values evoked by vehicle (n = 10). *P < 0.05, one‐way ANOVA. APETx2 at 0.03 pmol (n = 6), 3.3 pmol (n = 8) and at 11 pmol (n = 6) were not significantly different from vehicle. The dotted line in (A) indicates the PPT from the contralateral hindpaw.

Figure 7

APETx2 potentiates the response of rASIC1b and rASIC2a (when applied during the conditioning pH) but not that of rASIC1a, expressed in Xenopus oocytes. (A) Traces showing the minor inhibitory effect on rASIC1a but rapid and reversible potentiating effect of APETx2 (10 μM) on rat ASIC1b and ASIC2a. (B) Traces showing the concentration‐dependent effect of APETx2 on ASIC1b and ASIC2a. (C) Concentration–response curves of APETx2 on homomeric rat ASIC1a (n = 5), ASIC1b (n = 5), ASIC2a (n = 5) and ASIC3 (n = 6). Curves were fitted using the log[inhibitor] versus normalized response equation in GraphPad 7.0. (D) Quantification of the effect of APETx2 on the activation and desensitization of rASIC1b (n = 5) and rASIC2a (n = 7) whole‐cell currents recorded from Xenopus oocytes [presented as rise and decay times (10–90%)], *P < 0.05, significantly different from control; paired t‐test.