Orally active Epac inhibitor reverses mechanical allodynia and loss of intraepidermal nerve fibers in a mouse model of chemotherapy-induced peripheral neuropathy

Abstract

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect of cancer treatment that significantly compromises quality of life of cancer patients and survivors. Identification of targets for pharmacological intervention to prevent or reverse CIPN is needed. We investigated exchange protein regulated by cAMP (Epac) as a potential target. Epacs are cAMP-binding proteins known to play a pivotal role in mechanical allodynia induced by nerve injury and inflammation. We demonstrate that global Epac1-knockout (Epac1-/-) male and female mice are protected against paclitaxel-induced mechanical allodynia. In addition, spinal cord astrocyte activation and intraepidermal nerve fiber (IENF) loss are significantly reduced in Epac1-/- mice as compared to wild-type mice. Moreover, Epac1-/- mice do not develop the paclitaxel-induced deficits in mitochondrial bioenergetics in the sciatic nerve that are a hallmark of CIPN. Notably, mice with cell-specific deletion of Epac1 in Nav1.8-positive neurons (N-Epac1-/-) also show reduced paclitaxel-induced mechanical allodynia, astrocyte activation, and IENF loss, indicating that CIPN develops downstream of Epac1 activation in nociceptors. The Epac-inhibitor ESI-09 reversed established paclitaxel-induced mechanical allodynia in wild-type mice even when dosing started 10 days after completion of paclitaxel treatment. In addition, oral administration of ESI-09 suppressed spinal cord astrocyte activation in the spinal cord and protected against IENF loss. Ex vivo, ESI-09 blocked paclitaxel-induced abnormal spontaneous discharges in dorsal root ganglion neurons. Collectively, these findings implicate Epac1 in nociceptors as a novel target for treatment of CIPN. This is clinically relevant because ESI-09 has the potential to reverse a debilitating and long-lasting side effect of cancer treatment.

Figure 1. Effect of the Epac inhibitor ESI-09 on paclitaxel-induced mechanical allodynia and SNI-induced neuropathic pain

Male WT mice were treated with paclitaxel for two weeks (3 doses of 10 mg/kg i.p. per week). (A) ESI-09 (20 mg/kg) or vehicle was given by oral gavage for 6 days starting on the first day (n=5/group) or (B) 10 days after completion of paclitaxel treatment (n=11/group). Mechanical allodynia was measured over time. (C) and (D) Spared nerve injury (SNI) or sham surgery was performed on male WT mice. (C) ESI-09 (50 mg/kg) or vehicle was given by oral gavage for 6 days starting on the 3^rd day (n=7/group) after SNI or (D) 8 days after SNI (n=6/group). Mechanical allodynia was measured over time in ipsilateral and contralateral (Supplementary Figure 1) hind paw at 24 hours after ESI-09 treatment. In all panels, mechanical allodynia was quantified with von Frey Hairs using the up and down method. Data are expressed as 50% withdrawal threshold in grams and represent Mean ± SEM. Repeated measures two-way analysis of variance (ANOVA) showed a significant effect after ESI-09 administration vs vehicle group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. bsl, baseline.

Figure 2. Effect of Epac1 deletion on paclitaxel-induced mechanical allodynia

Male and female WT, Epac1−/−, and N-Epac1−/− mice were treated with paclitaxel as in Figure 1B and mechanical allodynia was measured over time. WT, n=14, Epac1−/− n=9, N-Epac1−/− n=12 mice. Data are expressed as mean ± SEM. ***p<0.001 for WT vs. N-Epac1−/− and ###p<0.001 for WT vs. Epac1−/−. bsl, baseline.

Figure 3. Impact of Epac1 inhibition or deletion on paclitaxel-induced astrocyte activation

(A) Effect of ESI-09 on paclitaxel-induced GFAP activation in lumbar spinal cord dorsal horn. WT mice (n=4/group) were treated with paclitaxel ± ESI-09 as in Figure 1B. 24 hours after the last dose of ESI-09, lumbar spinal cord was collected and immuno stained with GFAP (green). A representative example is shown from each group, vehicle alone (veh+veh), paclitaxel-treated (Taxol+veh), ESI-09 + vehicle-treated (veh+ESI-09), and paclitaxel + ESI-09 treated (Taxol+ESI-09). (B) Bar graph shows the quantitation in approximately 15 to 20 dorsal horn sections. The positive percentage of naïve wild-type mice was set as 100%. (C) Spinal cord dorsal horn GFAP activation in ± paclitaxel treated WT, Epac1^−/−, and N-Epac1^−/−. n=3–4 mice/group. Mice were treated with paclitaxel as in Figure 1B and 18 days after the treatment completion lumbar spinal cord were stained with GFAP as in panel (A). A representative image is shown for each group. (D) Bar graph shows the quantitation in approximately 15 to 20 dorsal horn sections. The positive percentage of naïve wild-type mice was set as 100%. 2-way-ANOVA, Data are expressed as means ± SEM. **p<0.01. Scale bar indicates 50 μm.

Figure 4. Impact of Epac1 inhibition or deletion on paclitaxel-induced IENF loss

(A) Effect of ESI-09 on paclitaxel-induced IENFs retraction. Mice (n=4 per group) were treated with paclitaxel ± ESI-09 as in Figure 1B. Hind paw biopsies were obtained a day after the last dose of ESI-09, fixed, and stained with pan-neuronal marker PGP9.5 for IENFs (red) and collagen (green). Representative images from each group are showed, white arrows denote IENF (red) crossing the basement membrane (in green). Vehicle alone (veh+veh), ESI-09 + vehicle-treated (veh+ ESI-09), paclitaxel-treated (Taxol+veh), and paclitaxel + ESI-09 treated (Taxol + ESI-09). (B) Bar graph showing quantitation of data from all paws. IENF density is number of nerve fibers crossing the basement membrane/length of the basement membrane (mm). *p<0.05, **p<0.01. (C) Paclitaxel-induced IENFs retraction in WT (n=4), (Epac1^−/−) (n=3), (N-Epac1) (n=4) and their naïve mice (n=3/group). Mice were treated with paclitaxel as in Figure 1B. Hind paw biopsies were obtained 18 days after the completion of the paclitaxel treatment and stained for IENFs as in panel (A). A representative image from each group is shown. (D) Bar graph showing quantitation of data from all paws. **p<0.01.

Figure 5. Epac1 knockout protects mitochondrial bioenergetics and contents in the tibial nerve of taxol-treated mice

WT and Epac1−/− mice were treated with paclitaxel for two weeks (3 doses of 10 mg/kg i.p. per week) and tibial nerves were obtained 18 days after the completion of the paclitaxel treatment. Oxygen consumption rate (OCR) was measured in the tibial nerves using the Seahorse XFe 24. Two-way ANOVA revealed a signification interaction (P<0.05) for baseline respiration, ATP-coupled respiration, and maximal respiratory capacity (MRC). Tukey post-hoc analysis revealed significant differences between groups: *P<0.05. n=12 mice/group.

Figure 6. Impact of Epac inhibition on paclitaxel-induced spontaneous discharges by DRG neurons in rats

DRG neuron with spontaneous action potential (SA) isolated from segments at day 7 after paclitaxel treatment show frequency of SA was both reduced by bath application of ESI-09 (1 μM) and CE3F4 (2.5 μM). Panel 6A shows a representative recording of trace baseline, after administration of ESI-09 (1 μM) and after washout. Panel 6B shows a representative recording of trace baseline, after administration of CE3F4 (2.5 μM) and after washout. Bar graph C summarizes ESI-09 results obtained in 9 cells from 6 paclitaxel-treated animals. ***p <0.001. Bar graph D summarizes CE3F4 results obtained in 8 cells from 7 paclitaxel-treated animals. **p <0.01.