Probucol is anti-hyperalgesic in a mouse peripheral nerve injury model of neuropathic pain

Abstract

2,6-di-tert-butylphenol (2,6-DTBP) ameliorates mechanical allodynia and thermal hyperalgesia produced by partial sciatic nerve ligation in mice, and selectively inhibits HCN1 channel gating. We hypothesized that the clinically utilized non-anesthetic dimerized congener of 2,6-DTBP, probucol (2,6-di-tert-butyl-4-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)sulfanylpropan-2-ylsulfanyl]phenol), would relieve the neuropathic phenotype that results from peripheral nerve damage, and that the anti-hyperalgesic efficacy in vivo would correlate with HCN1 channel inhibition in vitro. A single oral dose of probucol (800 mg/kg) relieved mechanical allodynia and thermal hyperalgesia in a mouse spared-nerve injury neuropathic pain model. While the low aqueous solubility of probucol precluded assessment of its possible interaction with HCN1 channels, our results, in conjunction with recent data demonstrating that probucol reduces lipopolysaccharide-induced mechanical allodynia and thermal hyperalgesia, support the testing/development of probucol as a non-opioid, oral antihyperalgesic albeit one of unknown mechanistic action.

Keywords: Alkylphenol; Anti-hyperalgesia; HCN1; Ion channel; Spared nerve injury.

Fig. 1

Probucol attenuates mechanical and thermal hyperalgesia. A. Structures as indicated. B. Representative LC-MS/MS probucol chromatogram. C. Probucol extraction and detection from spiked samples is quantitative. D-F. Groups of mice (n = 9/ treatment group) underwent surgery (Day 0), behavioral testing (Day 7, pre-probucol) and, following oral administration of probucol, testing 24 hrs later (Day 8). D. Probucol had no effect on time on rotarod but significantly relieved mechanical allodynia and thermal hyperalgesia (E) (mechanical: p = 0.0446 comparing SNI probucol treated vs. SNI vehicle treated at the 8 day timepoint, t = 2.2054, df = 14 as indicated by *; and thermal: p = 0.0096 comparing SNI probucol treated vs. SNI vehicle treated at the 8 day timepoint, t = 3.0000, df = 14 as indicated by **). F. Antihyperalgesic efficacy was independent of whole-blood concentration. Lines are linear regressions. The plus-probucol sham outlier at 1.6 g in the von Frey data was excluded from the regression.

Fig. 2

Probucol solubility is too low to permit examination of acute effects on HCN1 channel function in vitro.A. Following probucol dispersal at 33 µM (nominal), saline and DMEM (±FBS) samples were equilibrated for 6 days at 37 °C and concentration measured (per Methods). B. Representative whole cell recordings of HCN1 channels obtained from stably-transfected HEK293 cells following incubation in the absence or presence of probucol (33 μM nominal) in DMEM-10% FBS for 6 days. Records (left) are at voltages between −15 mV and −125 mV (scale bars: 1nA, 500 ms). Records (right) are tail currents at 0 mV (scale bars: 100 pA, 50 ms). Sweeps in red are those obtained with activation at −65 mV. C. Normalized activation curves constructed from the cells in A plus a vehicle control; smooth lines are fits of the Boltzmann equation. D. V_1/2 as function of days of exposure to DMEM-10% FBS supplemented with either 1% EtOH or 1% EtOH plus probucol (33 μM nominal). E. V_1/2 vs. maximal tail current amplitude. F. Photographs of HEK cells stably-transfected with HCN1 following 6 days of culture in basal media or basal media supplemented with 1% EtOH ± probucol (33 μM nominal). Arrowheads highlight small bright and dark flecks in the probucol-treated panel. (G) Tl⁺ flux as a function of pre-equilibration of LUVs. (H) Dynamic light scattering intensity as a function of particle size. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)