The Acute Antiallodynic Effect of Tolperisone in Rat Neuropathic Pain and Evaluation of Its Mechanism of Action

Abstract

Current treatment approaches to manage neuropathic pain have a slow onset and their use is largely hampered by side-effects, thus there is a significant need for finding new medications. Tolperisone, a centrally acting muscle relaxant with a favorable side effect profile, has been reported to affect ion channels, which are targets for current first-line medications in neuropathic pain. Our aim was to explore its antinociceptive potency in rats developing neuropathic pain evoked by partial sciatic nerve ligation and the mechanisms involved. Acute oral tolperisone restores both the decreased paw pressure threshold and the elevated glutamate level in cerebrospinal fluid in neuropathic rats. These effects were comparable to those of pregabalin, a first-line medication in neuropathy. Tolperisone also inhibits release of glutamate from rat brain synaptosomes primarily by blockade of voltage-dependent sodium channels, although inhibition of calcium channels may also be involved at higher concentrations. However, pregabalin fails to affect glutamate release under our present conditions, indicating a different mechanism of action. These results lay the foundation of the avenue for repurposing tolperisone as an analgesic drug to relieve neuropathic pain.

Keywords: CSF glutamate content; neuronal glutamate release; neuropathic pain; pregabalin; synaptosome; tolperisone.

Figure 1

The antiallodynic effect of tolperisone (left panels) and pregabalin (right panels) following acute per os treatment (25, 50, 100 mg/kg). Graphs show the means of PPT ± S.E.M. in grams of animals’ left (healthy, L) and right (operated, R) paws before (baseline) and after treatment (60 min; 120 min; 180 min) with either tolperisone or pregabalin. Asterisks mark the significant differences compared to left (healthy) paws or vehicle treated group (one-way ANOVA, F(9, 104) = 16.17 (tolperisone 0′), F(9, 104) = 5.182 (tolperisone 60′), F(9, 92) = 3.354 (tolperisone 120′), F(9, 92) = 3.028 (tolperisone 180′), F(9, 82) = 11.15 (pregabalin 0′), F(9, 82) = 5.955 (pregabalin 60′), F(9, 82) = 5.464 (pregabalin 120′), F(9, 82) = 6.234 (pregabalin 180′), Newman–Keuls post-hoc test; ****: p < 0.0001; ***: p < 0.001; **: p < 0.01; *: p < 0.05; ns: non-significant). In each treatment group 6–16 animals were used.

Figure 2

Glutamate content in CSF obtained from pSNL rats 14 days after surgery. Rats were treated with 25, 50, and 100 mg/kg dose of tolperisone (A) or pregabalin (B) per os or vehicle (A,B), and CSF samples were taken 3 h after treatment. Columns represent the mean of amino acid content ± S.E.M. in µM in the indicated groups. Asterisks mark the significant differences compared to vehicle treated group (one-way ANOVA, F(4, 54) = 6.774 (A), F(4, 50) = 8.478 (B), Tukey post-hoc test; ***: p < 0.001; **: p < 0.01; *: p < 0.05; ns: non-significant). In each treatment group 6–18 animals were used.

Figure 3

Effect of tolperisone on glutamate release from rat brain synaptosomes evoked by 1 mM 4-aminopyridine. Tolperisone was administered as a pretreatment 20 min prior to stimulation. Concentration of released glutamate was measured 6 min after stimulation. All data points were normalized using the unstimulated, baseline release and presented as % of the stimulated glutamate release in the absence of test compounds (black bar). All columns represent mean of glutamate release ± S.E.M. in % in the indicated groups. Asterisks mark the significant differences compared to stimulated glutamate release in the absence of test compounds (one-way ANOVA, F(4, 44) = 33.63, Tukey post-hoc test; ****: p < 0.0001; ns: non-significant). In each treatment group 4–30 parallel experiments were used.

Figure 4

Effect of tolperisone, sodium channel blockers (TTX—tetrodotoxin; CBZ—carbamazepine; LID—lidocaine), and calcium channel blockers (VER—verapamil; ω-CTX—ω-conotoxin; PRG—pregabalin) on glutamate release from rat brain synaptosomes evoked by 1 mM 4-aminopyridine. Tolperisone and channel blockers were administered as a pretreatment 20 min prior to stimulation. Concentration of released glutamate was measured 6 min after stimulation. All data points were normalized using the unstimulated, baseline release and presented as % of the stimulated glutamate release in the absence of test compounds (black bar). All columns represent mean of glutamate release ± S.E.M. in % in the indicated groups. Asterisks mark the significant differences compared to stimulated glutamate release in the absence of test compounds (one-way ANOVA, F(7, 50) = 16.83, Tukey post-hoc test; ****: p < 0.0001; ***: p < 0.001; ns: non-significant). In each treatment group 4–30 parallel experiments were used.

Figure 5

Effect of tolperisone, sodium channel blockers (TTX—tetrodotoxin; CBZ—carbamazepine; LID—lidocaine), and calcium channel blockers (VER—verapamil; ω-CTX—ω-conotoxin; PRG—pregabalin) on glutamate release from rat brain synaptosomes evoked by 33 mM potassium chloride. Tolperisone and channel blockers were administered as a pretreatment 20 min prior to stimulation. Concentration of released glutamate was measured 6 min after stimulation. All data points were normalized using the unstimulated, baseline release and presented as % of the stimulated glutamate release in the absence of test compounds (black bar). All columns represent mean of glutamate release ± S.E.M. in % in the indicated groups. Asterisks mark the significant differences compared to stimulated glutamate release in the absence of test compounds (one-way ANOVA, F(7, 40) = 3.599, Tukey post-hoc test; *: p < 0.05; **: p < 0.01; ns: non-significant). In each treatment group 4–19 parallel experiments were used.