A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat

Abstract

Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against Na(v)1.8 have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC(50) = 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Na(v)1.8 (IC(50) = 8 nM) and was >100-fold selective vs. human Na(v)1.2, Na(v)1.3, Na(v)1.5, and Na(v)1.7 (IC(50) values >or=1 microM). A-803467 (20 mg/kg, i.v.) blocked mechanically evoked firing of wide dynamic range neurons in the rat spinal dorsal horn. A-803467 also dose-dependently reduced mechanical allodynia in a variety of rat pain models including: spinal nerve ligation (ED(50) = 47 mg/kg, i.p.), sciatic nerve injury (ED(50) = 85 mg/kg, i.p.), capsaicin-induced secondary mechanical allodynia (ED(50) approximately 100 mg/kg, i.p.), and thermal hyperalgesia after intraplantar complete Freund's adjuvant injection (ED(50) = 41 mg/kg, i.p.). A-803467 was inactive against formalin-induced nociception and acute thermal and postoperative pain. These data demonstrate that acute and selective pharmacological blockade of Na(v)1.8 sodium channels in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

Fig. 1.

A-803467 is a selective blocker of Na_v1.8 channels. (A) Chemical structure of A-803467. (B) Block of recombinant and native Na_v1.8 currents by 100 nM A-803467. Current records were generated during voltage steps to 0 mV in control and after application of A-803467 (100 nM) for human Na_v1.8 or native DRG TTX-R currents. Holding potentials were set at −100 mV, and each test pulse was preceded by an 8-s prepulse to −40 mV, followed by 20 ms at −100 mV before a 20-ms depolarization to 0 mV. [Scale bars, 0.3 nA and 5 ms (Na_v1.8) and 1.5 nA and 5 ms (TTX-R).] (C) Representative concentration–response curves were generated after application of different concentrations of A-803467 and assessment of current block for human Na_v1.8 (IC₅₀ = 8 ± 1 nM), and TTX-R currents (IC₅₀ = 140 ± 14 nM) in rat DRG cells and hNa_v1.2, hNa_v1.3, hNa_v1.5, and hNa_v1.7. Data represent means ± SEM from at least three separate experiments. Percent inhibition was calculated as 1 − (test current/(control current + wash current/2)) and plotted vs. test concentration. Concentration-effect curves were fit by using the Hill equation to estimate IC₅₀ values and Hill coefficients. For hNa_v1.2, 1.3, 1.5, and 1.7, Hill coefficients were set to 0.7 as was found for Na_v1.8. Holding potentials for selectivity targets were −60 mV for hNa_v1.2, 1.3, and 1.7 and −90 mV for hNa_v1.5.

Fig. 2.

Voltage-dependent block and shifts in steady-state inactivation of Na_v1.8 currents by A-803467. Block of recombinant hNa_v1.8 currents by A-803467 was assessed after voltage protocols designed to vary the level of steady-state inactivation. Holding potential was −100 mV, and 500-ms prepulses were given to various potentials, followed by a 20-ms step to 0 mV. A 20-ms gap at −100 mV separated prepulse from test pulse. (A) Fractional current plotted against prepulse potential. Current values were normalized to the maximal current observed in control after prepulses to −100 mV. (B) Normalized fractional current vs. prepulse potential to illustrate shifts in V_1/2 values observed in the presence of drug. The current inset illustrates control, 1 μM A-803467, and recovery (e.g., wash) values. The smaller current record is in the presence of test compound. Symbols are the same as for A. Curve fits in both panels were derived by fitting Boltzmann equations to the data to obtain V_1/2 and slope factors (normalized current = [1 + exp[(V − V_1/2)/k]⁻¹ where V is the prepulse potential, V_1/2 is the holding potential that gave half-maximal inactivation, and k is the slope factor). [Scale bars, 0.5 nA and 5 msec (Na_v1.8).] Data represent means ± SEM from at least three separate experiments. (C and D) Effects of A-803467 on action potential firing in DRG neurons. Action potentials were recorded from small-diameter (18–25 μm) DRG neurons obtained from vehicle- or CFA-treated animals. (C) Action potentials were evoked in DRG neurons by injecting threshold current ranging from 100 to 300 pA from a resting state (resting membrane potential of ≈−53 mV, no current injection) or a depolarized state (−40 mV, by injecting 20- to 30-pA current). Evoked action potentials from resting state are not sensitive to 300 nM A-803467, which completely blocks evoked action potential from a depolarized state (representative of n = 4). A-803467 at 100 nM was ineffective in blocking evoked action potentials from both resting and depolarized states (data not shown). (D) Spontaneously firing neurons were obtained from small-diameter DRG neurons isolated 48 h after intraplantar CFA injection into the rat hindpaw. Approximately 10–12% of the neurons from CFA-treated animals fire spontaneously in our experiments. Representative responses (n = 4) are shown for control after application of 300 nM A-803467 and after wash out.

Fig. 3.

Administration of A-803467 (20 mg/kg, i.v.) significantly reduced von Frey hair-evoked (10 g) (A) and spontaneous spinal dorsal horn WDR neuronal (B) activity. Discharge activity was recorded from 13 WDR neurons located 756.3 ± 71.7 μm from the surface of the spinal cord in L5/L6 spinal nerve-injured rats. Before A-803467 injection, spontaneous WDR firing (first 5 min) was 2.8 ± 0.8 spikes per second, and the baseline evoked response (von Frey hair, 10 g) was 198 ± 32.9 spikes in 15 s. Administration of A-803467 (20 mg/kg, i.v.) reduced both evoked and spontaneous WDR neuron firing by 66.2% and 53.3%, respectively, from baseline levels. The effects of A-803467 were dose-dependent (data not shown). Data shown were collected 35 min after injection. n = 6–7 per group, ∗, P < 0.05 vs. baseline activity.

Fig. 4.

Antiallodynic effects of A-803467 in neuropathic pain. Two weeks after L5-L6 spinal nerve injury (SNL; A) or chronic constriction injury (CCI; B) A-803467 was injected 30 min (i.p.) before testing. A-803467 demonstrated significant antiallodynic effects in both neuropathic pain models [A: F (4, 65) = 21.94, P < 0.0001; B: F (4, 42) = 11.75, P < 0.0001). Circles represent paw withdrawal threshold ipsilateral to the injury; squares represent paw withdrawal threshold contralateral to the injury. Data represent mean ± SEM. ∗∗, P < 0.01 as compared with vehicle-treated animals. (n = 6–12 per group).

Fig. 5.

Antinociceptive effects of A-803467 in the capsaicin-induced secondary mechanical hypersensitivity and CFA-induced thermal hyperalgesia models. A-803467 was injected 30 min (i.p.) before testing in both assays. (A) A-803467 reversed the secondary mechanical hypersensitivity tested 3 h after intraplantar capsaicin injection [F (4, 43) = 133.9, P < 0.001)]. (B) A-803467 demonstrated significant antihyperalgesic effects when tested 2 days after CFA injection [F (7, 40) = 43.74, P < 0.0001]. Circles represent values ipsilateral to the injury; squares represent values contralateral to the injury. Data represent mean ± SEM. (n = 6–12 per group). ∗, P < 0.05, ∗∗, P < 0.01 as compared with vehicle-treated animals. ++, P < 0.01 as compared with ipsilateral paw.