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. 2024 Jun 26:17:2267-2278.
doi: 10.2147/JPR.S467535. eCollection 2024.

Kv7 Channel Activators Flupirtine and ML213 Alleviate Neuropathic Pain Behavior in the Streptozotocin Rat Model of Diabetic Neuropathy

Ashraf Ibrahim Ahmed  1 , Salma Al-Nuaimi  1 , Ayman Mustafa  1 , Asad Zeidan  1 , Abdelali Agouni  2 , Laiche Djouhri  1
Affiliations

Kv7 Channel Activators Flupirtine and ML213 Alleviate Neuropathic Pain Behavior in the Streptozotocin Rat Model of Diabetic Neuropathy

Ashraf Ibrahim Ahmed et al. J Pain Res. .

Abstract

Background & objective: Chronic peripheral neuropathic pain (PNP) is a debilitating condition that is associated with many types of injury/diseases, including diabetes mellitus. Patients with longstanding diabetes develop diabetic PNP (DPNP), which is resilient to currently available drugs. The underlying molecular mechanisms of DPNP are still illusive, but Kv7 channels that have been implicated in the pathogenesis of various types of chronic pain are likely to be involved. Indeed, using the streptozotocin (STZ) rat model of DPNP, we have previously shown that Kv7 activation with their non-selective activator retigabine attenuated neuropathic pain behavior suggesting that these channels are implicated in DPNP pathogenesis. Here, we evaluated, in the same STZ model, whether the more potent and more selective Kv7 channel openers flupirtine and ML213 attenuate STZ-induced pain hypersensitivity.

Methods: Male Sprague Dawley rats (250-300 g) were used. The STZ model involved a single injection of STZ (60 mg/kg, i.p.). Behavioral testing for mechanical and heat pain sensitivity was performed using a dynamic plantar aesthesiometer and Hargreaves analgesiometer, respectively.

Results: STZ rats exhibited behavioral signs of mechanical and heat hypersensitivity as indicated by significant decreases in the mean paw withdrawal threshold (PWT) and mean paw withdrawal latency (PWL), respectively, at 35 days post-STZ treatment. Single injections of flupirtine (10 mg/kg, i.p.) and ML213 (5 mg/kg, i.p.) to STZ rats (35-days after STZ treatment) caused significant increases in the mean PWT, but not PWL, indicating attenuation of mechanical, but not heat hypersensitivity. Both flupirtine and ML213 were as effective as the positive control gabapentin (10/kg, i.p.), and their anti-allodynic effects were prevented by the Kv7 channel-specific blocker XE991 (3 mg/kg, i.p.).

Conclusion: The findings suggest that Kv7 channels are involved in the mechanisms of mechanical but not heat hypersensitivity associated with DPNP, and that their activation may prove to be effective in alleviating DPNP symptoms.

Keywords: Kv7 channels; chronic pain; diabetic neuropathy; ion channels; rat model of pain.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
A schematic representation of the experimental design and the timeline of the behavioral pain testing. Of the 66 male SD (Sprague Dawley) rats used, 54 were STZ rats (received a single injection of 60 mg/kg, i.p.) and the remaining 12 rats were naïve (not treated with STZ). All the rats were acclimatized/habituated for 5–7 consecutive days to the procedure room and the testing chambers. After acclimatization, the rats were randomly divided into STZ and naïve rats. The STZ rats were divided into six groups: (1) FL (flupirtine group, n=10), (2) ML (ML213 group, n =10 rats), (3) GP (gabapentin, positive control group, n=10), (4) E & FL (EX991 plus flupirtine, n=8), (5) E & ML (EX991 plus ML213, n=8) and (6) Veh (vehicle control group (n=8). Pain behavioral testing was conducted before STZ treatment (baseline pain testing) and 21, 28 and 35 days post-STZ as well as 1–24 hours post-drug (FL and ML) at 35-days post-STZ. For the naïve rats, pain behavioral testing was conducted before (pe-drug) and after (post-drug) FL and ML administration.
Figure 2
Figure 2
Behavioral signs of STZ-induced mechanical and heat hypersensitivity. Mechanical pain sentivity (A) and heat pain sensitivity (B) The data are presented as mean ± SEM. Each dot per column represents one rat. Figure 2A shows that STZ treatment caused highly significant (p<0.01) decreases in the mean PWL at all the time points tested (21, 28 and 35 days (D) post-STZ), indicating that STZ rats developed heat hypersensitivity at these time points. In contrast, there was a significant (p<0.01) decrease in the mean PWT only at 35 days (D) pos-STZ (Figure 2B), indicting a delay in the development of mechanical hypersensitivity. Note that comparisons were between pre-STZ values (before STZ treatment) and post-STZ values at different time points. Statistical tests were made with one-way ANOVA test followed with a Tukey’s multiple comparison (post hoc) test. The level of statistical significance is indicated with asterisks (**p < 0.01).
Figure 3
Figure 3
Effects of flupirtine, ML213 and gabapentin on STZ-induced mechanical and heat hypersensitivity. Compared to vehicle, a single injection of flupirtine at a dose of 10 mg/kg and a single injection of ML213 at a dose of 5 mg/kg significantly reversed the STZ-induced decreases in the mean PWT at 1–2 h, but not, at 24 h post-drug treatment (A), indicating that these Kv7 activators attenuated STZ-induced mechanical hypersensitivity. The effects of both flupirtine and ML213 were similar to those of gabapentin (10 mg/kg), which was used as the positive control. However, unlike gabapentin which also attenuated STZ-induced heat hypersensitivity (B), both flupirtine and ML213 given at the same doses mentioned above had no significant effect on STZ-induced decrease in PWL indicating that the heat hypersensitivity in STZ rats was not affected by these kv7 channel agonists (B). Note that comparisons were between the PWT and PWL values at the two time points after drug administration (post-drug) and those of the vehicle before drug treatment 35 days after STZ. Statistical tests were made with one-way ANOVA and Tukey’s multiple comparison test. The level of statistical significance is indicated with asterisks as follows: **p < 0.01; * p<0.05.
Figure 4
Figure 4
Effects of XE991 on the anti-allodynic effects of flupirtine and ML213 in STZ rats. STZ treatment caused significant decreases in the mean PWT (A) and mean PWL (B) 35 days post-treatment in all five groups of rats (vehicle, flupirtine, ML213, XE991 plus flupirtine and XE991 plus ML213) indicating that all rat groups developed mechanical (A) and heat (B) hypersensitivity. Compared with vehicle (n = 8 rats), single injections of flupirtine (10 mg/kg, n=8 rats) and ML213 (5 mg/kg, n=8 rats) significantly reduced mechanical hypersensitivity at 1–2 h post-drug treatment (A). These anti-allodynic effects of flupirtine and ML213 were prevented/blocked by pretreatment with the Kv7 channel antagonist XE991 (3mg/kg, n=8 rats) indicating that the effects of flupirtine and ML213 were mediated by Kv7 channels. Statistical tests were made with one-way repeated measures ANOVA, followed with Tukey’s post-hoc test. The level of statistical significance is indicated with asterisks as follows: **p < 0.01; * p<0.05.
Figure 5
Figure 5
Effects of flupirtine and ML213 on pain sensitivity in naïve rats. Injections of flupirtine and ML213 at the same dose of 10 mg/kg and 5 mg/kg respectively into naive rats caused no significant changes in the mean PWT (A) or PWL (B) indicating they had no obvious effects on pain sensitivity on normal rats. Statistical tests were made with one--way repeated measures ANOVA.
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