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. 2019 Oct:320:113010.
doi: 10.1016/j.expneurol.2019.113010. Epub 2019 Jul 9.

The α7 nicotinic receptor silent agonist R-47 prevents and reverses paclitaxel-induced peripheral neuropathy in mice without tolerance or altering nicotine reward and withdrawal

Wisam Toma  1 , S Lauren Kyte  2 , Deniz Bagdas  3 , Asti Jackson  3 , Julie A Meade  4 , Faria Rahman  4 , Zhi-Jian Chen  5 , Egidio Del Fabbro  6 , Lucas Cantwell  7 , Abhijit Kulkarni  7 , Ganesh A Thakur  7 , Roger L Papke  8 , John W Bigbee  9 , David A Gewirtz  4 , M Imad Damaj  10
Affiliations

The α7 nicotinic receptor silent agonist R-47 prevents and reverses paclitaxel-induced peripheral neuropathy in mice without tolerance or altering nicotine reward and withdrawal

Wisam Toma et al. Exp Neurol. 2019 Oct.

Abstract

Various antitumor drugs, including paclitaxel, frequently cause chemotherapy-induced peripheral neuropathy (CIPN) that can be sustained even after therapy has been completed. The current work was designed to evaluate R-47, an α7 nAChR silent agonist, in our mouse model of CIPN. R-47 was administered to male C57BL/6J mice prior to and during paclitaxel treatment. Additionally, we tested if R-47 would alter nicotine's reward and withdrawal effects. The H460 and A549 non-small cell lung cancer (NSCLC) cell lines were exposed to R-47 for 24-72 h, and tumor-bearing NSG mice received R-47 prior to and during paclitaxel treatment. R-47 prevents and reverses paclitaxel-induced mechanical hypersensitivity in mice in an α7 nAChR-dependent manner. No tolerance develops following repeated administration of R-47, and the drug lacks intrinsic rewarding effects. Additionally, R-47 neither changes the rewarding effect of nicotine in the Conditioned Place Preference test nor enhances mecamylamine-precipitated withdrawal. Furthermore, R-47 prevents paclitaxel-mediated loss of intraepidermal nerve fibers and morphological alterations of microglia in the spinal cord. Moreover, R-47 does not increase NSCLC cell viability, colony formation, or proliferation, and does not interfere with paclitaxel-induced growth arrest, DNA fragmentation, or apoptosis. Most importantly, R-47 does not increase the growth of A549 tumors or interfere with the antitumor activity of paclitaxel in tumor-bearing mice. These studies suggest that R-47 could be a viable and efficacious approach for the prevention and treatment of CIPN that would not interfere with the antitumor activity of paclitaxel or promote lung tumor growth.

Keywords: Cancer; Mice; Neuropathy; Paclitaxel; Withdrawal; α7 nAChR.

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Conflict of interest statement

Conflicts of interest

The authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.
R-47 reverses CIPN in mice in an α7-dependent manner with no development of tolerance. A) Acute administration of R-47 at doses of 1, 5, and 10 mg/kg p.o. reverses mechanical hypersensitivity in paclitaxel-treated mice. *P < 0.0001 vs distilled water (0 mg/kg); #P < 0.0001 vs R-47 (1 mg/kg); $P < 0.0001 vs R-47 (5 mg/kg). B) Administration of MLA, an α7 nAChR antagonist, at a dose of 10 mg/kg s.c. 10 minutes before R-47 (10 mg/kg, p.o.) blocks the antinociceptive effect of R-47 in paclitaxel-treated mice. *P < 0.0001 Veh-R-47 at 180 min vs 0 min; #P < 0.0001 MLA-R-47 vs Veh-R-47 at 180 min. C) Administration of R-47 at a dose of 10 mg/kg p.o. for four days and on day 5 (challenge day) shows the antinociceptive effect in paclitaxel-treated mice. *P < 0.0001 Veh-R-47 at 180 min vs 0 min; #P < 0.0001 R-47-R-47 at 180 vs R-47-R-47 at 0 min. BL, Baseline; Veh, vehicle; Pac, paclitaxel; MLA, methyllycaconitine; n = 8 per group; data expressed as mean ± SEM.
Figure 2.
Figure 2.
R-47 prevents CIPN in mice. A) Chronic administration of R-47 at a dose of 10 mg/kg p.o. completely prevents the development of mechanical hypersensitivity. **** P < 0.0001 Veh-Veh vs Veh-Pac; #### P < 0.0001 R-47-Pac vs Veh-Pac; BL, baseline; Veh, Vehicle; Pac, Paclitaxel. n=8 per group; Data expressed as mean ± SEM. B) Mice injected with paclitaxel at a dose of 8 mg/kg i.p. for one cycle demonstrate a significant reduction of IENFs at 35 days post-paclitaxel injection. However, pre- and co-administration of R-47 at a dose of 10 mg/kg p.o. to paclitaxel-treated mice completely prevents the reduction of IENFs; *P < 0.0001 D.W.-paclitaxel vs D.W.-vehicle; #P < 0.0001 R-47-paclitaxel vs D.W.-paclitaxel. C) Immunostained sections of hind paw epidermis represent the reduction of IENF density by paclitaxel and protection by R-47. Bar represents 20 microns in all images. Images were captured under 63x magnification. IENFs, intra-epidermal nerve fibers; D.W., distilled water. Asterisks denote IENFs. n = 6 per group; data expressed as mean ± SEM.
Figure 3.
Figure 3.
R-47 prevents paclitaxel-induced alterations in dorsal horn microglia. (A) Mice injected with paclitaxel (8 mg/kg i.p. for one cycle) plus distilled water (D.W., the vehicle for R-47) reveal a significant decrease in the number of stage I microglial cells at 35 days post-paclitaxel injection, which was prevented by pre-and co-administration of R-47 at a dose of 10 mg/kg p.o. (C) Mice injected with paclitaxel plus D.W. show a significant elevation in the number of stage III microglial cells, which was prevented by pre-and co-administration of R-47. (B) There were no significant changes in the number of stage II microglial cells between the treatment groups. (D-F) Representative images of microglial stages: (D) Stage I, cells with long, thin, and highly ramified processes; (E) Stage II, cells with shorter, thickened processes with less branching; and (F) Stage III, cells with increased hypertrophic changes such as shorter, thickened processes and cell body enlargement. ****P < 0.0001 D.W.-paclitaxel vs D.W.-vehicle; ####P < 0.0001 R-47-paclitaxel vs D.W.-paclitaxel. Bar represents 10 microns in all images. Images were captured under 63x magnification. D.W., distilled water. n = 6 per group; data expressed as mean ± SEM.
Figure 4.
Figure 4.
R-47 induces CPP in mice with paclitaxel-induced neuropathy. A) Timeline depiction of the CPP test. B) The administration of R-47 at a dose of 10 mg/kg p.o. induces CPP in paclitaxel-treated mice, but not in vehicle-treated mice. *P < 0.05 vs D.W.-Paclitaxel. BL, Baseline (pre-conditioning day); Test represents post-conditioning drug-free preference; D.W., Distilled Water; Veh, vehicle; Pac, paclitaxel. n = 15 per group; data expressed as mean ± SEM.
Figure 5.
Figure 5.
Effects of R-47 on Nicotine Reward in the Conditioned Place Paradigm. Mice were conditioned with either subcutaneous (s.c.) saline or nicotine (0.5mg/kg) for 3 days. Distilled water or R-47 (1, and 10 mg/kg; p.o) pretreatment were given 120-min prior to nicotine-conditioning. Nicotine itself induces a robust place preference but R-47 pretreatment doesn’t block the effect of nicotine. * Denotes p<0.05 from vehicle control. Each point represents the mean ± SEM of n=10–12 mice per group.
Figure 6.
Figure 6.
R-47 Fails to Precipitate Nicotine Physical and Affective Signs. Male mice were chronically infused with saline or nicotine (24 mg/kg/day) for 14 days. On day 15, mice received subcutaneous (s.c.) injection of saline, R-47 (10 mg/kg, p.o., 120 min pretreatment) or mecamylamine (2 mg/kg; s.c., 10 min pretreatment) prior to behavioral assessment of A) somatic signs, B) hyperalgesia (hot plate latency), C) time spent in the light side (Light-Dark Box test), D) # of entries (Light-Dark Box test). Mecamylamine-Nicotine group shows withdrawal symptoms (i.e. increased # of somatic signs), but Mecamylamine-Nicotine group shows a decrease in the hot plate latency and time in the light chamber of the Light-Dark Box tests. * Denotes p< 0.05 vs. Saline minipump group. Each point represents the mean ± S.E.M. of n=8 mice per group. MP: minipump; Veh: vehicle; Mec: mecamylamine.
Figure 7.
Figure 7.
R-47 does not stimulate NSCLC cell proliferation alone or interfere with paclitaxel-induced growth inhibition of NSCLC cells. The “start” time point represents the initial number of cells after seeding. A 24-hour R-47 pretreatment period occurred from Start to Day 0 for the R-47 and Pac + R-47 conditions, then all subsequent treatments lasted 24 hours; no drugs were present after Day 1. The number of viable cells was determined via trypan blue exclusion. Insets display the Pac and Pac + R-47 data on a smaller scale. ***P < 0.001, ****P < 0.0001 vs control. Data are expressed as the mean ± SEM of three independent experiments. Pac, paclitaxel.
Figure 8.
Figure 8.
R-47 does not interfere with paclitaxel-induced apoptosis of NSCLC cells. Cells were treated with R-47, paclitaxel, or the combination for 48 hours. Quantification of apoptotic cells was determined by the Annexin V/PI assay. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs control; n.s., not significant (P > 0.05). Data are expressed as the mean ± SEM of three independent experiments. Pac, paclitaxel.
Figure 9.
Figure 9.
R-47 does not enhance A549 NSCLC tumor growth alone or in combination with paclitaxel in NSG mice. Mice were subcutaneously injected with 1.5 × 106 cells in each flank. Once tumors became palpable, mice were given R-47 (10 mg/kg, p.o.) twice daily for 3 days starting on Day 1, then once daily in combination with paclitaxel (10 mg/kg, i.p.) for 4 days starting on Day 4. A) The left and right flank tumor volumes (l × w × h) were determined with calipers and values were averaged for each mouse. Data are expressed as mean + SEM; n = 8–9 mice per group. *P < 0.05, ***P < 0.001 vs Veh-Water. B) Mice were euthanized on day 17, after which tumors were extracted and weighed; tumor weights were averaged for each mouse. *P < 0.05, **P < 0.01 vs Veh-Water. Data are expressed as mean + SEM; n = 8–9 mice per group. Veh, vehicle; Pac, paclitaxel.
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