A769662 dose-dependently blocks rat TG VGSC current amplitude.

<p>(A) Representative Na⁺ current traces in TG neurons in the presence of A769662. Currents were elicited in TG neurons with a current step protocol initiated from -80 mV to 0 mV for 25 msec from a holding potential of -80 mV. (B) Acute application of A769662 reduced the Na⁺ current amplitude in TG neurons (n = 6, **** p < 0.0001). C) Data generated was fitted to a hill equation to plot the dose response curve for percent current amplitude block by A769662. A769662 inhibited Na⁺ current amplitude with an IC₅₀ of 10 μM. D) Peak current vs time plot of the effect of A769662 on Na⁺ current amplitude. Arrows correspond to A769662 (blue) and washout (red) in A) and B).</p

A769662 exhibits local anesthetic effects in rats.

<p>(A) Administration of A769662 (30 μg, 100 μg and 300 μg) into the popliteal fossa produces a dose-dependent nerve block by reversing the paw withdrawal latency to noxious thermal heat using the Hargreaves method compared to vehicle treated rats (n = 4–7, regular two-way ANOVA, * p < 0.05 and *** p < 0.001). (B) Paw withdrawal latency plotted as a function of A769662 or PT1 dose. Dose-response curve of A769662 was generated by fitting data to the Hill equation yielding an IC₅₀ of 90 μg.</p

A769662 applied acutely to rat TG neurons blocks AP firing evoked by ramp current stimuli.

<p>(A) Representative action potential traces in response to ramp current stimuli before and after acute application of (5 sec) of A769662 (200 μM). Acute application of A769662 (n = 5) onto rat TG neurons significantly reduced numbers of action potentials (B) and reversed time-to-first action potential (AP) peak compared to control (n = 5). This effect was reversible with washout (n = 5). Differences in the mean numbers of action potentials among groups were analyzed by comparing the slopes and intercepts generated from linear regression Comparisons among groups for time to first spike were performed by two-way ANOVA. Colored stars denote significant effects compared to control group. * p < 0.05, ** p < 0.01 and *** p < 0.001.</p

A769662 blocks hNa_v1.7 currents in HEK cells.

<p>(A) A step from -80 mV to 0 mV for 25 ms was used to elicit currents in hNa_v1.7-transfected HEK cells with or without A769662 application. (B) Acute application of A769662 (200 μM) reduced the Na⁺ current amplitude in hNa_v1.7-transfected HEK cells (n = 10, one-way ANOVA, **** p < 0.0001 C) Peak current vs time plot of the effect of A769662 on hNa_v1.7 currents. Arrows correspond to A769662 (blue) and washout (red) in A) and B).</p

Lack of use-dependent effects of A769662 on rat TG neurons.

<p>30 repetitive 25 ms depolarizing pulses of -10 mV were applied from a holding potential of -80 mV at 0.5Hz (A) and 5Hz (B) in the absence (●) and presence (■) of A769662 (200 μM). Peak current amplitude at each pulse was normalized by the peak current amplitude of the first pulse under each condition and plotted vs the pulse number. (C) Fraction of current at the 30^th depolarizing pulse at 0.5 Hz and 5 Hz in the absence and presence of A769662 (n = 5–15, two-way ANOVA, * p < 0.05, *** p <0.001).</p

PT1 and resveratrol inhibit NGF-induced hyperexcitability but have no acute effect on action potential firing.

<p>(A) Patch clamp analysis of rat TG neurons cultured in the presence of NGF (50 ng/ml, n = 11) show an increase in the number of ramp evoked action potentials compared to control (n = 8) which is reversed by PT1 (100 μM; 1 hr, n = 11) and resveratrol (200 μM, 1 hr, n = 6). Colored stars denote significant effects compared to NGF group. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. (B) and (C) Acute application of PT1 (30 μM, n = 10; 100 μM, n = 8) demonstrates no influence on action potential firing compared to control. The number of ramp current evoked action potentials was normalized to control. (D) Acute application of other AMPK activators (resveratrol, 200 μM, n = 6; metformin, 20 mM, n = 6) does not block VGSCs.</p

A769662 showed no binding at the local anesthetic binding site.

<p>(<b>A, B</b>) Nav1.7 current traces recorded under control (solid line), presence of 200μM A769662 (dash line), and washout (dot lines) conditions respectively from a cell expressing WT Nav1.7 channels (<b>A</b>) or a cell expressing F1737A/Y1744A mutant channels (<b>B</b>); (<b>C,D</b>) 200μM A769662 reduced Nav1.7 sodium channel currents from cells expressing WT channels (<b>C</b>) and cells expressing F1737A/Y1744A mutant channels (<b>D</b>), and washing out could partially reverse the inhibition effects for both WT and F1737A/Y1744A mutant channels.</p

The Anti-Diabetic Drug Metformin Protects against Chemotherapy-Induced Peripheral Neuropathy in a Mouse Model

<div><p>Chemotherapy-induced peripheral neuropathy (CIPN) characterized by loss of sensory sensitivity and pain in hands and feet is the major dose-limiting toxicity of many chemotherapeutics. At present, there are no FDA-approved treatments for CIPN. The anti-diabetic drug metformin is the most widely used prescription drug in the world and improves glycemic control in diabetes patients. There is some evidence that metformin enhances the efficacy of cancer treatment. The aim of this study was to test the hypothesis that metformin protects against chemotherapy-induced neuropathic pain and sensory deficits. Mice were treated with cisplatin together with metformin or saline. Cisplatin induced increased sensitivity to mechanical stimulation (mechanical allodynia) as measured using the von Frey test. Co-administration of metformin almost completely prevented the cisplatin-induced mechanical allodynia. Co-administration of metformin also prevented paclitaxel-induced mechanical allodynia. The capacity of the mice to detect an adhesive patch on their hind paw was used as a novel indicator of chemotherapy-induced sensory deficits. Co-administration of metformin prevented the cisplatin-induced increase in latency to detect the adhesive patch indicating that metformin prevents sensory deficits as well. Moreover, metformin prevented the reduction in density of intra-epidermal nerve fibers (IENFs) in the paw that develops as a result of cisplatin treatment. We conclude that metformin protects against pain and loss of tactile function in a mouse model of CIPN. The finding that metformin reduces loss of peripheral nerve endings indicates that mechanism underlying the beneficial effects of metformin includes a neuroprotective activity. Because metformin is widely used for treatment of type II diabetes, has a broad safety profile, and is currently being tested as an adjuvant drug in cancer treatment, clinical translation of these findings could be rapidly achieved.</p></div

Effect of metformin treatment on cisplatin-induced sensory deficits.

<p>A). Mice (n = 6–10/group) were treated with cisplatin and metformin as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100701#pone-0100701-g001" target="_blank">Figure 1</a>. The time to respond to an adhesive patch on the hind paw was monitored. B). Effect of delayed metformin on cisplatin induced sensory deficits (n = 7–8/group). C). Effect of cisplatin treatment on rotarod performance. Mice (n = 6/group) were treated with cisplatin and their performance on the rotarod was monitored as an index of motor coordination. E). Mice (n = 7/group) were treated with lidocaine (5 µL, 4% in saline) or saline and 10 mins. later, the time to respond to an adhesive patch on the hind paw was measured. * p<0.05, ** p<0.01 vs. Saline+Saline; # p<0.05 vs. Saline+Cisplatin.</p

Effect of metformin on cisplatin- or paclitaxel-induced mechanical allodynia in mice.

<p>A). Treatment schedule; X: injection; O: no treatment. B) Mice (n = 10–12/group) were treated with cisplatin (cumulative dose 23 mg/kg i.p.) and metformin (200 mg/kg/dose i.p.) as depicted in panel A. Mechanical allodynia was quantified with von Frey hairs using the up and down method. C). Effect of delayed metformin treatment on cisplatin-induced mechanical allodynia. Mice (n = 8/group) received i.p. injections with cisplatin (cumulative dose 23 mg/kg i.p.) and delayed metformin (200 mg/kg/dose i.p.) as depicted in panel A and mechanical allodynia was monitored. D). Mice (n = 4–7/group) were treated with paclitaxel (10 mg/kg/every other day, i.p. for two weeks) and metformin (200 mg/kg i.p. daily from one day before until one day after paclitaxel) and mechanical allodynia was measured. E). Change in body weight after cisplatin and metformin treatment. ** p<0.01 vs. Saline+Saline group; ## p<0.01 vs. Saline+Cisplatin; && p<0.01 vs. Saline+Paclitaxel.</p