Inhibition of α9α10 nicotinic acetylcholine receptors prevents chemotherapy-induced neuropathic pain

Abstract

Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α10 nicotinic acetylcholine receptors (nAChRs) have been proposed as an important nonopioid mechanism based on studies demonstrating prevention of neuropathology after trauma-induced nerve injury. However, the key α9α10 ligands characterized to date are at least two orders of magnitude less potent on human vs. rodent nAChRs, limiting their translational application. Furthermore, an alternative proposal that these ligands achieve their beneficial effects by acting as agonists of GABA_B receptors has caused confusion over whether blockade of α9α10 nAChRs is the fundamental underlying mechanism. To address these issues definitively, we developed RgIA4, a peptide that exhibits high potency for both human and rodent α9α10 nAChRs, and was at least 1,000-fold more selective for α9α10 nAChRs vs. all other molecular targets tested, including opioid and GABA_B receptors. A daily s.c. dose of RgIA4 prevented chemotherapy-induced neuropathic pain in rats. In wild-type mice, oxaliplatin treatment produced cold allodynia that could be prevented by RgIA4. Additionally, in α9 KO mice, chemotherapy-induced development of cold allodynia was attenuated and the milder, temporary cold allodynia was not relieved by RgIA4. These findings establish blockade of α9-containing nAChRs as the basis for the efficacy of RgIA4, and that α9-containing nAChRs are a critical target for prevention of chronic cancer chemotherapy-induced neuropathic pain.

Keywords: alpha9; chemotherapy; nicotinic; pain.

Fig. 1.

Peptide sequence of α-conotoxins Vc1.1 and RgIA, as well as RgIA point mutation analogs. (A) Amino acid sequences of α-conotoxin Vc1.1 from Conus victoriae and α-conotoxin RgIA from C. regius (shell shown) indicating disulfide bridges between the first and third cysteines and between the second and fourth cysteines. (B) Single substitutions (red) were made for several RgIA amino acids (bold). In general, conservative mutations (similar hydrophobicity, size, or charge) were chosen. We also used monoido-Tyr based on our prior experience that substituting this residue for Tyr in the second disulfide loop increased the potency of α-conotoxin MI (57). (C) Effect (100 nM peptide) on blocking ACh-induced current on human α9α10 nAChR currents expressed in X. laevis oocytes. Error bars represent the mean ± SEM from three to seven separate oocytes. Red bars indicate a significant difference from RgIA (P < 0.05).

Fig. 2.

Concentration response of RgIA analogs on human and Rat α9α10 nAChRs. (A) Amino acid sequences. Concentration responses of RgIA (B) and analogs (C) on human α9α10 nAChRs. Concentration responses of RgIA (D) and analogs (E) on rat α9α10 nAChRs. Oocytes expressing α9α10 nAChRs were voltage-clamped at −70 mV and subjected to a 1-s pulse of 10 μM ACh every minute as described in Materials and Methods. The IC₅₀s and Hill slopes are depicted in Table 1. Data points are the mean ± SEM from three to six separate oocytes.

Fig. S1.

Blockade by RgIA and analogs on human nAChR subtypes. (A) Blockade of human nAChRs by RgIA and analogs. Oocytes expressing the indicated human nAChR subtype were voltage-clamped at −70 mV and subjected to several 1-s pulses of ACh, followed by an additional pulse of ACh with the indicated RgIA compound (10 μM). Data are shown as a percentage of 100 μM ACh-induced current (I_ACh) in the presence of RgIA compound compared with the control response in the absence of compound. Data points are the mean from three to five oocytes. (B) Concentration response of RgIA4 and RgIA5 on human α9α10 and α7 nAChRs. Xenopus oocytes expressing nAChRs were subjected to two-electrode voltage clamp as described in Materials and Methods, and the IC₅₀ values for blockade of I_ACh were determined. The IC₅₀ values for RgIA4 and RgIA5 on α9α10 were 1.5 ± 0.5 nM and 0.44 ± 0.09 nM, respectively. Hill slopes were 1.14. ± 0.2 and 0.86 ± 0.1, respectively. The IC₅₀ values for RgIA4 and RgIA5 on α7 were 1.8 ± 0.7 μM and 0.33 ± 0.09 μM, respectively. Hill slopes were 0.97 ± 0.13 and 1.3 ± 0.4, respectively. Error bars denote the mean ± SEM from three to four oocytes for each experimental determination.

Fig. S2.

Lack of functional effect of RgIA4 on GABA_B receptors. (A) Concentration response of GABA (control) and RgIA4 on GABA_B1a/B2 receptors using a cAMP-based fluorescence detection assay as described in Materials and Methods. RgIA4 activity on GABA_B1a/B2 receptors was calculated as a percentage of maximal control 30 μM GABA. (B) Concentration response of 3-aminopropyl-methylphosphinic acid (3-APMPA) (control) and RgIA4 on GABA_B1a/B2 receptors assessed by bioimpedance measurements using a cellular dielectric spectroscopy assay as described in Materials and Methods. RgIA4 activity on GABA_B1a/B2 receptors was calculated as a percentage of maximal control 3 μM 3-APMPA. (C) RgIA4 failed to inhibit Ca_V currents in isolated rat DRG neurons. The effect of either 1 μM RgIA4 or 30 μM baclofen is shown as a percentage of control Ca_V current. Baclofen inhibits Ca_V current by 28 ± 8% (*P < 0.05, n = 6) via GABA_B receptor activation. However, RgIA4 failed to affect Ca_V current significantly (4 ± 10%; P = 0.32, n = 8). Of the six neurons exposed to both RgIA4 and baclofen, there was no correlation between the effect of RgIA4 vs. baclofen on the Ca_V current. Calculation of the Pearson correlation yielded R = 0.45 (not significant).

Fig. 3.

RgIA4 prevents chemotherapy-induced neuropathic pain in rats. Chemotherapy-induced peripheral neuropathy in rats was induced by repeated i.p. injection of oxaliplatin as described in Materials and Methods. Control animals were treated with vehicle. RgIA4 was dissolved in saline and injected s.c. daily. Withdrawal latency (A) and paw pressure (B) were used as a measure of cold allodynia and mechanical hyperalgesia, respectively. The cold allodynia test was performed on days 8, 15, and 22 at 24 h after RgIA4 administration (A), and the mechanical hyperalgesia test was performed at 1-wk time points 30 min (B) and 24 h (C) after RgIA4 administration. Values are expressed as the mean ± SEM from eight rats for each experimental determination. ***P < 0.001 significantly different from vehicle.

Fig. 4.

RgIA4 prevents chemotherapy-induced neuropathic pain in wild-type but not α9 KO mice. Chemotherapy-induced peripheral neuropathy in mice was induced by repeated i.p. injection of oxaliplatin. Control animals were treated with vehicle. RgIA4 was dissolved in saline and injected s.c. daily. Mice were assessed in a cold-plate chamber in which the temperature was decreased from room temperature at rate of 10 °C per minute. Time to response is indicated on the y axis. (A) Mice were injected i.p. 5 d each week with vehicle or oxaliplatin, and injected s.c. with RgIA4 (40 μg/kg) or vehicle 5 d each week. Cold-plate testing was performed 30 min after and 24 h after the last injection on indicated days. (B) In a separate experiment, mice were injected as described in A but assessed at 72 h after the last injection. Values are expressed as the mean ± SEM (n= 7–9) for each experimental determination. *P < 0.05, **P < 0.01, ***P < 0.001 significantly different from vehicle/vehicle control. ^oP < 0.05, ^ooP < 0.01, ^ooo P < 0.001 significantly different compared with oxaliplatin/vehicle-treated mice.

Fig. 5.

RgIA4 and α9 KO prevent chronic cold allodynia. Repeated oxaliplatin injection induces progressive cold allodynia in wild-type mice. Cold allodynia is not detected 72 h after oxaliplatin administration either in wild-type mice that received RgIA4 or in α9 KO mice (additional details are provided in Fig. 4). Data shown are 72 h after the last injection. The control was i.p. saline administered five times per week + s.c. saline administered five times per week (vehicle/vehicle) at the indicated time points; i.p. oxaliplatin was administered five times per week; and s.c. saline or RgIA4 was administered five times per week. *P < 0.05, **P < 0.01 significantly different from vehicle/vehicle controls at indicated time points for respective genotypes.