Pregabalin-Tolperisone Combination to Treat Neuropathic Pain: Improved Analgesia and Reduced Side Effects in Rats

Abstract

The current treatment of neuropathic pain (NP) is unsatisfactory; therefore, effective novel agents or combination-based analgesic therapies are needed. Herein, oral tolperisone, pregabalin, and duloxetine were tested for their antinociceptive effect against rat partial sciatic nerve ligation (pSNL)-induced tactile allodynia described by a decrease in the paw withdrawal threshold (PWT) measured by a dynamic plantar aesthesiometer. On day 7 after the operation, PWTs were assessed at 60, 120, and 180 min post-treatment. Chronic treatment was continued for 2 weeks, and again, PWTs were measured on day 14 and 21. None of the test compounds produced an acute antiallodynic effect. In contrast, after chronic treatment, tolperisone and pregabalin alleviated allodynia. In other experiments, on day 14, the acute antiallodynic effect of the tolperisone/pregabalin or duloxetine combination was measured. As a novel finding, a single dose of the tolperisone/pregabalin combination could remarkably alleviate allodynia acutely. It also restored the neuropathy-induced elevated CSF glutamate content. Furthermore, the combination is devoid of adverse effects related to motor and gastrointestinal transit functions. Tolperisone and pregabalin target voltage-gated sodium and calcium channels, respectively. The dual blockade effect of the combination might explain its advantageous acute analgesic effect in the present work.

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

Figure A1

Blood glucose level (panel (a)), left and right PWT of age-matched and diabetic animals (panel (b)), the body weight of age-matched and diabetic animals (panel (c)), the body weight of age and weight-matched, and diabetic animals at 9 weeks (panel (d)), left and right PWT of age and weight-matched animals at 9 weeks (panel (e)). Data are shown as mean ± S.E.M. of 6 age-matched, 6 weight-matched, and 20 diabetic animals. * p < 0.05 statistically significant compared to the age-matched animals. Data were analyzed by unpaired t-test for panels (a–c) and (e) and one-way ANOVA followed by Dunnett’s post-hoc test, panel (d).

Figure A2

The antiallodynic effect of carbamazepine on pSNL evoked allodynia. The PWTs were measured on day 7 prior to and after a single treatment (panel (a)), on day 14 (1 week treatment) (panel (b)), on day 21 (2 weeks treatment) (panel (c)), and on day 14 after a single treatment (panel (d)) after the operation. Tactile allodynia was measured by DPA at 60, 120, and 180 min after oral treatment. Data are shown as the mean ± SEM of 6–7 animals (panels (a–c)) and 4–13 animals (panel (d)) in each group. p > 0.05, statistically not significant: treated groups versus the vehicle right (operated) paw at the indicated time points after treatment (two-way ANOVA followed by Dunnett’s post-hoc test). Baseline (b.l.): was measured before the first treatment. Single treatment: was measured after acute administration on day 7 after the operation.

Figure 1

The antiallodynic effect of tolperisone on pSNL evoked allodynia. The PWTs were measured postoperatively on day 7 prior to and after a single treatment (panel (a)), on day 14 after 1 week of chronic treatment (panel (b)), and on day 21 after 2 weeks of chronic treatment (panel (c)). Tactile allodynia was measured by DPA at 60, 120, and 180 min after oral treatment. Data are shown as the mean ± SEM of 8–13 animals per group. * p < 0.05 statistically significant compared to the vehicle-treated group at the indicated time points after treatment (two-way ANOVA followed by Dunnett’s post-hoc test). Baseline (b.l.): was measured before the first treatment. Single treatment: was measured after acute administration on day 7 after the operation.

Figure 2

The antiallodynic effect of pregabalin on pSNL evoked allodynia. The PWTs were measured on day 7 prior to and after a single treatment (panel (a)), on day 14 (1 week treatment) (panel (b)), and on day 21 (2 weeks treatment) (panel (c)) after the operation. Tactile allodynia was measured by DPA at 60, 120, and 180 min after oral treatment. Data are shown as the mean ± SEM of 6–13 animals per group. * p < 0.05, statistically significant compared to the vehicle right (operated) paw at the indicated time points after treatment (two-way ANOVA followed by Dunnett’s post-hoc test). Baseline (b.l.): was measured before the first treatment. Single treatment: was measured after acute administration on day 7 after the operation.

Figure 3

The antiallodynic effect of duloxetine on pSNL evoked allodynia. The PWTs were measured on day 7 prior to and after a single treatment (panel (a)), on day 14 (1 week treatment) (panel (b)), and on day 21 (2 weeks treatment) (panel (c)) after the operation. Tactile allodynia was measured by DPA at 60, 120, and 180 min after oral treatment. Data are shown as the mean ± SEM of 5–7 animals per group. p < 0.05, statistically significant compared to the vehicle right (operated) paw at the indicated time points after treatment (two-way ANOVA followed by Dunnett’s post-hoc test). Baseline (b.l.): was measured before the first treatment. Single treatment: was measured after acute administration on day 7 after the operation.

Figure 3

The antiallodynic effect of duloxetine on pSNL evoked allodynia. The PWTs were measured on day 7 prior to and after a single treatment (panel (a)), on day 14 (1 week treatment) (panel (b)), and on day 21 (2 weeks treatment) (panel (c)) after the operation. Tactile allodynia was measured by DPA at 60, 120, and 180 min after oral treatment. Data are shown as the mean ± SEM of 5–7 animals per group. p < 0.05, statistically significant compared to the vehicle right (operated) paw at the indicated time points after treatment (two-way ANOVA followed by Dunnett’s post-hoc test). Baseline (b.l.): was measured before the first treatment. Single treatment: was measured after acute administration on day 7 after the operation.

Figure 4

The acute antiallodynic effect of tolperisone (panel (a)), pregabalin (panel (b)), a combination of tolperisone and pregabalin (panel (c)), duloxetine (panel (d)), and a combination of tolperisone and duloxetine (e) on the pSNL evoked allodynia. The PWT was measured on day 14 after the operation. Tactile allodynia was measured by DPA at 60, 120, and 180 min after acute oral treatment. Data are shown as the mean ± SEM of 6–8 animals (panel (a)), 8 animals (panel (b)), 5–8 animals (panel (c)), 4–5 animals (panel (d)), and 5–8 animals (panel (e)) per group. * p < 0.05, statistically significant compared to the vehicle right (operated) paw at the indicated time points after treatment (two-way ANOVA followed by Dunnett’s post-hoc test). Baseline (b.l.): was measured before the treatment.

Figure 4

The acute antiallodynic effect of tolperisone (panel (a)), pregabalin (panel (b)), a combination of tolperisone and pregabalin (panel (c)), duloxetine (panel (d)), and a combination of tolperisone and duloxetine (e) on the pSNL evoked allodynia. The PWT was measured on day 14 after the operation. Tactile allodynia was measured by DPA at 60, 120, and 180 min after acute oral treatment. Data are shown as the mean ± SEM of 6–8 animals (panel (a)), 8 animals (panel (b)), 5–8 animals (panel (c)), 4–5 animals (panel (d)), and 5–8 animals (panel (e)) per group. * p < 0.05, statistically significant compared to the vehicle right (operated) paw at the indicated time points after treatment (two-way ANOVA followed by Dunnett’s post-hoc test). Baseline (b.l.): was measured before the treatment.

Figure 5

The acute antiallodynic effect of tolperisone and pregabalin on type 1 diabetes evoked tactile allodynia. The left and right PWT was measured in week 9 after diabetes induction. Tactile allodynia was measured by DPA at 60 and 120 min after acute oral treatment. Data are shown as the mean ± SEM of 3–5 animals per group. * p < 0.05, statistically significant compared to the vehicle-treated groups (one-way ANOVA followed by Dunnett’s post-hoc test). The baseline was measured before treatment.

Figure 6

CSF glutamate content of mono-neuropathic (pSNL) and sham operated rats after acute treatment with orally administered tolperisone (25 mg/kg), pregabalin (25 mg/kg), or their combination (both at 25 mg/kg) on day 14 after pSNL operation. Data are shown as mean ± SEM of n = 4–21 animals per group. * p < 0.05 vs. other groups (one-way ANOVA followed by Dunnett’s post-hoc test).

Figure 7

Effect of tolperisone (100 μM), pregabalin (250 μM), or their combination on glutamate release from rat brain synaptosomes induced by 1 mM 4-aminopyridine. Drugs were administered as a pretreatment 20 min prior to stimulation. The 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 (gray bar). All columns show the mean of glutamate release ± SEM in % in the indicated groups. * p < 0.05 vs. stimulated glutamate release by 1 mM 4-aminopyridine alone and treatment groups (one-way ANOVA followed by Dunnett’s post-hoc test). In each treatment group, 4–13 parallel experiments were used.

Figure 8

Effect of acute oral administration of tolperisone (100 and 150 mg/kg), pregabalin (25, 50, and 100 mg/kg), and the combination of tolperisone and pregabalin (both at 25 mg/kg) or vehicle at 60 and 120 min on motor coordination and balance of animals. Columns show the time latency in the rotarod assay. Data are shown as the mean ± SEM of 5–19 animals per group, measured at the peak effect of test compounds. * p < 0.05 statistically significant compared to the vehicle (one-way ANOVA followed by Dunnett’s post-hoc test).

Figure 9

Effect of acute oral administration of tolperisone (25 and 50 mg/kg), pregabalin (25 and 50 mg/kg), and the tolperisone and pregabalin combination (both at 25 mg/kg) on the GI transit of naïve animals 30 min after a charcoal meal. Columns represent the charcoal travel (%) in the charcoal meal test. Data are shown as the mean ± SEM of 5–6 animals per group, measured at the peak effect of test compounds. * p < 0.05 statistically significant compared to the vehicle (one-way ANOVA followed by Dunnett’s post-hoc test).

Scheme 1

Experimental schedule 1.