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. 2018 Nov 21;38(47):10180-10201.
doi: 10.1523/JNEUROSCI.1049-18.2018. Epub 2018 Oct 9.

Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors

Shannon D Shields  1 , Lunbin Deng  2 , Rebecca M Reese  2 , Michelle Dourado  2 , Janet Tao  3 , Oded Foreman  3 , Jae H Chang  4 , David H Hackos  1
Affiliations

Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors

Shannon D Shields et al. J Neurosci. .

Abstract

Strong human genetic evidence points to an essential contribution of the voltage-gated sodium channel Nav1.7 to pain sensation: loss of Nav1.7 function leads to congenital insensitivity to pain, whereas gain-of-function mutations in the SCN9A gene that encodes Nav1.7 cause painful neuropathies, such as inherited erythromelalgia, a syndrome characterized by episodic spontaneous pain. Selective Nav1.7 channel blockers thus hold promise as potential painkillers with improved safety and reduced unwanted side effects compared with existing therapeutics. To determine the maximum effect of a theoretically perfectly selective Nav1.7 inhibitor, we generated a tamoxifen-inducible KO mouse model enabling genetic deletion of Nav1.7 from adult mice. Electrophysiological recordings of sensory neurons from these mice following tamoxifen injection demonstrated the loss of Nav1.7 channel current and the resulting decrease in neuronal excitability of small-diameter neurons. We found that behavioral responses to most, but surprisingly not all, modalities of noxious stimulus are abolished following adult deletion of Nav1.7, pointing toward indications where Nav1.7 blockade should be efficacious. Furthermore, we demonstrate that isoform-selective acylsulfonamide Nav1.7 inhibitors show robust analgesic and antinociceptive activity acutely after a single dose in mouse pain models shown to be Nav1.7-dependent. All experiments were done with both male and female mice. Collectively, these data expand the depth of knowledge surrounding Nav1.7 biology as it relates to pain, and provide preclinical proof of efficacy that lays a clear path toward translation for the therapeutic use of Nav1.7-selective inhibitors in humans.SIGNIFICANCE STATEMENT Loss-of-function mutations in the sodium channel Nav1.7 cause congenital insensitivity to pain in humans, making Nav1.7 a top target for novel pain drugs. Targeting Nav1.7 selectively has been challenging, however, in part due to uncertainties in which rodent pain models are dependent on Nav1.7. We have developed and characterized an adult-onset Nav1.7 KO mouse model that allows us to determine the expected effects of a theoretically perfect Nav1.7 blocker. Importantly, many commonly used pain models, such as mechanical allodynia after nerve injury, appear to not be dependent on Nav1.7 in the adult. By defining which models are Nav1.7 dependent, we demonstrate that selective Nav1.7 inhibitors can approximate the effects of genetic loss of function, which previously has not been directly established.

Keywords: Nav1.7; acylsulfonamide; congenital insensitivity to pain; drug development; pain; tamoxifen.

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Figures

Figure 1.
Figure 1.
Expression distribution of scn9a mRNA and characterization of Nav1.7 deletion in cKO mice. A, Time course of loss of scn9a mRNA from DRG of Nav1.7 cKO mice (red circles). Signal is undetectable at all post-tamoxifen time points. Black squares represent control littermates. n = 6 mice/genotype. B, Tissue distribution of scn9a mRNA in Nav1.7 cKO mice and control mice. Relative expression of Nav1.7 cKO and control was normalized to the level observed in WT trigeminal ganglia. Tissues are as indicated. OB, Olfactory bulb; OE, olfactory epithelium. C, Example Western blot of Nav1.7 protein from DRG lysates 3 d and 1, 2, 4, or 6 weeks following tamoxifen injection. Tubulin immunostaining is shown as a loading control. D, Time course of loss of Nav1.7 protein from DRG of Nav1.7 cKO mice (red) as measured by Western blot. Decay constant (τ) = 0.98 ± 0.19 weeks. No detectable Nav1.7 protein loss was observed in DRGs from control mice (black). E–G, Expression of other Nav channel mRNAs in DRGs (E), TGs (F), and SCGs (G) following tamoxifen injection (after 8 weeks) from Nav1.7 cKO mice (gray) or control mice (black). No significant differences in Nav channel mRNA levels were observed (except for scn9a). H, Representative images of scn9a ISH in DRG from a Nav1.7 cKO mouse and a control mouse. scn9a mRNA was detected in 838 of 933 (90%) DRG neurons pooled from 3 control mice, representing all soma cross-sectional areas. Black bars represent scn9a-positive. White bars represent scn9a-negative. scn9a mRNA was detected in 46 of 770 (6%) DRG neurons pooled from 3 Nav1.7 cKO mice. Black bars represent scn9a-positive. White bars represent scn9a-negative. Gray bars represent nuclear aggregates. Error bars indicate SEM.
Figure 2.
Figure 2.
Voltage-gated sodium currents from DRG neurons derived from Nav1.7 cKO mice or control littermates. A, TTX-resistant and TTX-sensitive voltage-gated sodium currents were measured from small-, medium-, or large-diameter DRG neurons acutely isolated from either control (cre−) mice or Nav1.7 cKO (cre+) mice. Mice were dissected 7–9 weeks following tamoxifen dosing to ensure complete decay of Nav1.7 protein. Whole-cell voltage clamp was done using a holding voltage of −80 mV, then hyperpolarizing to −120 mV for 20 ms and depolarizing to 0 mV for 20 ms with a stimulus frequency of 1 Hz; 500 nm TTX was used to isolate the TTX-resistant component of the sodium current. B, TTX-sensitive current density measured from small-, medium-, and large-diameter DRG neurons obtained from control (cre−) or Nav1.7 cKO (cre+) mice. TTX-sensitive currents were found to be significantly decreased in small- and medium-diameter Nav1.7 cKO DRG neurons (but not large-diameter) relative to control DRG neurons. C, TTX-sensitive current density versus membrane capacitance as measured on a single-cell basis. D, TTX-resistant current density measured from small-, medium-, and large-diameter DRG neurons obtained from control (cre−) or Nav1.7 cKO (cre+) mice. No significant difference in TTX-resistant currents between DRG neurons from control versus Nav1.7 cKO mice was observed. Most large-diameter neurons did not have detectable TTX-resistant currents. E, TTX-resistant current density versus membrane capacitance as measured on a single-cell basis. ***p < 0.001. Error bars indicate SEM.
Figure 3.
Figure 3.
Electrophysiological properties of DRG neurons derived from WT and Nav1.7 cKO mice without adjusting resting membrane potential. A, Current-clamp recordings of small-diameter DRG neurons acutely isolated from either control (cre−) mice or Nav1.7 cKO (cre+) mice. Mice were dissected 7–9 weeks following tamoxifen dosing to ensure complete decay of Nav1.7 protein. Cells were stimulated with short 10 ms depolarizing current pulses of increasing amplitude until the cell fired an AP (left) or with long 500 ms depolarizing current pulses to examine AP firing frequency (right) with and without 500 nm TTX. Large-diameter DRG neurons were also examined (far right). B, Resting membrane potential did not differ between genotypes and did not significantly change after adding 500 nm TTX. C, AP threshold voltage was measured before and after adding 500 nm TTX. AP threshold voltages did not differ between genotypes, and TTX had no significant effect. D, Current threshold for firing an AP was measured before and after adding 500 nm TTX. Current thresholds did not differ between genotypes, and TTX had no significant effect. E, Number of APs per 500 ms was measured before and after adding 500 nm TTX. No genotype difference was observed between control and Nav1.7 cKO neurons, and TTX had no significant effect. Error bars indicate SEM.
Figure 4.
Figure 4.
Electrophysiological properties of DRG neurons derived from WT and Nav1.7 cKO mice. A, Current-clamp recordings of small-diameter DRG neurons acutely isolated from either control (cre.neg) mice or Nav1.7 cKO (cre.pos) mice. Mice were dissected 7–9 weeks following tamoxifen dosing to ensure complete decay of Nav1.7 protein. Current was injected to maintain a resting membrane potential of −70 mV in these recordings. Cells were stimulated with depolarizing current pulses of increasing amplitude until the cell fired an AP. The overall shape of the AP did not differ between genotypes. B, AP threshold voltage was measured before and after adding 500 nm TTX. Neurons from Nav1.7 cKO mice have significantly more depolarized AP threshold voltages relative to neurons from control mice. Furthermore, TTX significantly shifted the AP threshold voltage to more depolarized voltages in control neurons but not Nav1.7 cKO neurons. C, Current threshold for firing an AP was measured before and after adding 500 nm TTX. Neurons from Nav1.7 cKO mice required significantly larger current pulses to fire an AP relative to neurons from control mice. Furthermore, TTX significantly increased the amount of current needed to fire an AP in control neurons but not Nav1.7 cKO neurons. D, Resting membrane potential, set to −70 mV before the addition of TTX, did not significantly change after adding 500 nm TTX. E, AP overshoot amplitude was measured before and after adding 500 nm TTX. No difference was observed between control and Nav1.7 cKO neurons; however, addition of TTX significantly decreased AP overshoot amplitude in control neurons but not Nav1.7 cKO neurons. F, The 10%–90% peak rise rate was measured before and after adding 500 nm TTX. No difference was observed between control and Nav1.7 cKO neurons; however, addition of TTX significantly decreased upstroke slope in control neurons but not Nav1.7 cKO neurons. G, AP half-width was measured before and after adding 500 nm TTX. No difference was observed between control and Nav1.7 cKO neurons; however, addition of TTX significantly increased AP half-width in control neurons but not Nav1.7 cKO neurons. Significance (paired two-tailed t test): *p < 0.05; ***p < 0.001; ###p < 0.001. Error bars indicate SEM.
Figure 5.
Figure 5.
General neurological function and olfaction are intact in Nav1.7 cKO mice. A, Nav1.7 cKO mice perform similarly to control littermates in several tests of general neurological function. B, Mice of both genotypes displayed a normal motor and postural phenotype in the tail suspension test. C, Negative geotaxis, a measure of vestibulomotor reflexes, was normal in Nav1.7 cKO mice. D, Latency to fall from a rotating beam was unaltered by deletion of Nav1.7. E, In the wire hang test of motor coordination, Nav1.7 cKO and control littermates performed similarly. F, In the novel odor recognition test, mice are presented with three olfactory cues. Habituation to repeated presentation of the same odor, as well as increased approaches to a novel odor, are prominent features of this test that indicate correct olfactory function. Performance on this test was identical between genotypes, reflecting intact olfaction in Nav1.7 cKO mice. Error bars indicate SEM.
Figure 6.
Figure 6.
Nav1.7 cKO mice are insensitive to most modalities of acute noxious stimulus. A, Hot plate test, 55°C. Control and Nav1.7 cKO mice have similar withdrawal latency before tamoxifen administration; after tamoxifen-mediated deletion, cKO mice show prolonged latency to respond. Cutoff = 30 s. B, Tail immersion test, 55°C. Cutoff = 5 s. C, Hindpaw radiant heat (Hargreaves) test. Cutoff = 30 s. D, Randall–Selitto test (pressure applied to the tail). Cutoff = 750 g. E, Intraplantar capsaicin (1 μg in 10 μl) induced nocifensive behavior measured in the first 5 min after injection in control, but not Nav1.7 cKO, mice. F, Intraperitoneal injection of dilute acetic acid (0.9% in saline, 10 ml/kg) induced abdominal constrictions in control, but not Nav1.7 cKO, mice. G–I, Formalin test (2% formalin i.pl.). G, Time course of flinching, licking, and biting behaviors scored in 5 min bins for 1 h. H, Nav1.7 cKO mice display approximately half as much formalin-induced behavior as control mice in Phase 1 (0–10 min) and an equivalent amount in Phase 2 (10–60 min). I, Latency to begin displaying nocifensive behavior after formalin injection is increased in Nav1.7 cKO mice. J, Scratch bouts in response to intradermal cheek injection of the nonhistaminergic pruritogen chloroquine were markedly suppressed after adult-onset deletion of Nav1.7 compared with control conditions. K, Scratch bouts in response to intradermal cheek injection of histamine were significantly reduced in Nav1.7 cKO mice compared with control littermates. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analysis described in Table 1. Error bars indicate SEM.
Figure 7.
Figure 7.
Nav1.7 cKO mice are sensitive to some noxious stimuli and time course of behavioral effects. A, von Frey test of mechanical threshold. Tamoxifen treatment did not change the response of Nav1.7 cKO mice to von Frey filaments. Cutoff = 7.5 g. B, Intensity-response relationship to electrical stimuli delivered through a floor grid did not differ between genotypes. C, Nav1.7 cKO mice learn an active avoidance paradigm where electrical stimuli are an effective aversive teaching signal in a manner indistinguishable from control mice. n = 24 control, 18 cKO mice. D–F, Time courses to develop insensitivity to the hot plate test (D), the tail immersion test (E), and the Hargreaves test (F) when measured at weekly intervals following tamoxifen dosing. Statistical analysis described in Table 1. Error bars indicate SEM.
Figure 8.
Figure 8.
Evidence for reduced activation of spinal nociceptive pathways in Nav1.7 cKO mice. Noxious heat (A), strong mechanical pressure (B), or intraplantar formalin injection (C) applied to the hindpaw induces an increase in Fos-immunoreactive profiles in the ipsilateral laminae I-II in control, but not Nav1.7 cKO, mice. D, Compared with unstimulated mice, electrical shock delivered through a floor grid induced a significant increase in Fos-positive profiles in laminae I-II of both control and Nav1.7 cKO mice. n = 4 mice/group. Black bars represent control littermates. White bars represent Nav1.7 cKO mice. Statistical analysis described in Table 1. Error bars indicate SEM.
Figure 9.
Figure 9.
Mechanical allodynia is surprisingly preserved in several models of chronic pain. A, Mice developed cooling-evoked allodynia in the SNI model of neuropathic pain as measured by responses to acetone evaporation. After 2 weeks, tamoxifen was administered (arrow) to induce deletion of Nav1.7. Nav1.7 cKO mice (open symbols) showed a gradual reversal of cooling-evoked allodynia. B, Established neuropathic mechanical allodynia was not reversed by Nav1.7 deletion. C, When Nav1.7 was deleted before SNI (arrow), cKO mice were protected from developing cooling-evoked allodynia. D, Mechanical allodynia developed mostly normally (though to a slightly reduced extent) when SNI took place after Nav1.7 deletion. E, Representative sections of spinal cord dorsal horn from control and Nav1.7 cKO mice at 3 d after SNI. Microglia, stained for Iba-1, are activated ipsilateral to the nerve injury in both genotypes. F, Quantification of Iba-1 staining intensity in mice that have undergone SNI. Increase in pixel density ipsilateral to the injury is similar in control and Nav1.7 cKO mice. G, Heat hypersensitivity in the CFA model is absent in Nav1.7 cKO mice. H, Mechanical hypersensitivity in the CFA model develops to a mostly similar degree in both genotypes. I, Hindpaw edema after CFA injection is similar in both genotypes. J, Fos-like immunoreactivity in the spinal cord dorsal horn following CFA inflammation and exposure to a normally non-noxious mechanical stimulus (forced walking). Ipsilateral to an inflammatory insult, the number of Fos-positive profiles is increased specifically in laminae I-II upon mechanical stimulation in both control and Nav1.7 cKO mice, serving as an additional indication of intact mechanical allodynia in the absence of Nav1.7. K, In the incision model of postoperative pain, heat hypersensitivity is absent in Nav1.7 cKO mice. L, Incision-induced mechanical hypersensitivity in Nav1.7 cKO mice is comparable with control mice. M, Unlike control littermates, Nav1.7 cKO mice do not develop mechanical allodynia secondary to an extraterritorial capsaicin injection. Statistical analysis described in Table 1. Error bars indicate SEM.
Figure 10.
Figure 10.
Selectivity profiles of GX-585 and GX-201. A, Chemical structures of GX-201 and GX-585. B, Example trace showing a voltage-clamp recording of human Nav1.7 where each point represents the peak current amplitude during a 20 ms pulse to 0 mV. The blocking effect of 100 nm GX-201 as well as the very slow off-rate are shown. Right, Linear relationship between K_obs and GX-201 concentration, which was used to calculate the on-rate (K_on). C, Example trace showing a measurement of the IC50 of GX-201 on human Nav1.5. D, IC50 curves showing the selectivity pattern of GX-201 against human Nav1.1–1.8 channels. Red dashed trace represents the expected IC50 curve for GX-201 on hNav1.7 based on the Kd measured using kinetics. E, Selectivity pattern for GX-585 against human Nav1.1–1.8 channels. F, IC50 values of GX-201 on WT hNav1.7 and S4 voltage-sensor arginine mutants. G, IC50 values of GX-201 on hNav1.7, hNav1.1, and hNav1.7 mutants showing the residues driving selectivity. H, Table showing holding voltages used for each channel subtype, equilibrium IC50 values, kinetics parameters, and calculated Kd values. mTTX_S and mTTX_S(KO) refer to TTX-sensitive channels recorded in WT and Nav1.7 cKO mouse DRG neurons, respectively. mTTX_R and mTTX_R(1.9) refer to mouse TTX-resistant channels recorded in WT and Nav1.8 KO DRG neurons, respectively. In the absence of Nav1.8, the TTX-resistant current is carried entirely by Nav1.9 channels. Error bars indicate SEM.
Figure 11.
Figure 11.
Nav1.7-selective inhibitors have antinociceptive and analgesic properties in WT mice. A, B, Response latency on the 55°C hot plate test was prolonged after oral administration of GX-201 (A) or GX-585 (B), but not vehicle. C, D, Latency to withdraw the tail from a 55°C water bath was significantly longer after dosing of GX-201 (C) or GX-585 (D) compared with vehicle. E, Escape threshold in the Randall–Selitto assay was elevated in mice administered GX-585 compared with vehicle-treated controls. F, von Frey threshold was not significantly different between mice treated with GX-201 or vehicle, as expected because Nav1.7 cKO mice also do not differ from WT littermates in this assay. A, C, F, GX-201 (30 mg/kg, p.o., average total plasma concentration 10.5 ± 0.7 μm). B, D, E, GX-585 (60 mg/kg, p.o., average total plasma concentration 49.3 ± 5.4 μm). G, H, Baseline withdrawal latency from a radiant heat stimulus was recorded; then an intraplantar injection of CFA was performed. Withdrawal latencies were recorded again 24 h later (post-CFA, predose). GX-201 (30 mg/kg, p.o., average total plasma concentration 16.1 ± 1.5 μm; G) or GX-585 (60 mg/kg, p.o., average total plasma concentration 67.9 ± 15.6 μm; H) were administered, and testing was performed again 1 h later (post-CFA, postdose). Both Nav1.7 inhibitors reversed established hypersensitivity back to near pre-CFA baseline levels. I, C57BL/6 mice (n = 12/group) were administered the indicated dose of GX-585, and precapsaicin baseline von Frey thresholds were measured at the plantar footpad 1 h later. Capsaicin was injected extraterritorially at the ankle, and plantar von Frey thresholds were measured again after 30 min. GX-585 provided a dose-dependent protection from developing mechanical allodynia in this model. Average total plasma concentrations were 15.7 ± 3.1, 31.1 ± 5.3, and 60.8 ± 8.0 μm for 10, 30, and 60 mg/kg doses, respectively. J, C57BL/6 mice were dosed with vehicle (0.5% methylcellulose/0.2% Tween 80) or 60 mg/kg GX-585 1 h before testing. Vehicle-treated mice (black bars) demonstrated the expected pattern of habituation/dishabituation to the presentation of novel/familiar odor stimuli. In contrast, GX-585-treated mice were relatively indifferent to odor presentation. This may indicate perturbed olfaction after systemic administration of the drug. *p < 0.05, **p < 0.01. Statistical analysis described in Table 1. Error bars indicate SEM.
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