The riluzole derivative 2-amino-6-trifluoromethylthio-benzothiazole (SKA-19), a mixed KCa2 activator and NaV blocker, is a potent novel anticonvulsant

Abstract

Inhibitors of voltage-gated sodium channels (Na(v)) have been used as anticonvulsants since the 1940s, while potassium channel activators have only been investigated more recently. We here describe the discovery of 2-amino-6-trifluoromethylthio-benzothiazole (SKA-19), a thioanalog of riluzole, as a potent, novel anticonvulsant, which combines the two mechanisms. SKA-19 is a use-dependent NaV channel blocker and an activator of small-conductance Ca(2+)-activated K(+) channels. SKA-19 reduces action potential firing and increases medium afterhyperpolarization in CA1 pyramidal neurons in hippocampal slices. SKA-19 is orally bioavailable and shows activity in a broad range of rodent seizure models. SKA-19 protects against maximal electroshock-induced seizures in both rats (ED50 1.6 mg/kg i.p.; 2.3 mg/kg p.o.) and mice (ED50 4.3 mg/kg p.o.), and is also effective in the 6-Hz model in mice (ED50 12.2 mg/kg), Frings audiogenic seizure-susceptible mice (ED50 2.2 mg/kg), and the hippocampal kindled rat model of complex partial seizures (ED50 5.5 mg/kg). Toxicity tests for abnormal neurological status revealed a therapeutic index (TD50/ED50) of 6-9 following intraperitoneal and of 33 following oral administration. SKA-19 further reduced acute pain in the formalin pain model and raised allodynic threshold in a sciatic nerve ligation model. The anticonvulsant profile of SKA-19 is comparable to riluzole, which similarly affects Na(V) and KCa2 channels, except that SKA-19 has a ~4-fold greater duration of action owing to more prolonged brain levels. Based on these findings we propose that compounds combining KCa2 channel-activating and Na(v) channel-blocking activity exert broad-spectrum anticonvulsant and analgesic effects.

Fig. 1

Chemical structures of riluzole and 2-amino-6-trifluoromethylthio-benzothiazole (SKA-19)

Fig. 2

2-Amino-6-trifluoromethylthio-benzothiazole (SKA-19) protects mice and rats in the maximal electroshock (MES)-induced seizure model. (a) Dose–response curves for seizure protection in the MES test (□) and neurological impairment (■) following intraperitoneal (i.p.) administration in mice (n = 8 per dose, 2-h time point). ED₅₀ 4.8 mg/kg [95 % confidence interval (CI) 4.05–5.37], TD₅₀ 29.8 mg/kg (95 % CI 25.35–35.98); protective index (PI) 6.2. (b) Dose–response curves for i.p. administration in rats (n = 8 per dose, 1-h time point). ED₅₀ 1.6 mg/kg (95 % CI 1.24–1.94), TD₅₀ of 14.33 mg/kg (95 % CI 10.72–17.9); PI 8.9. (c) Dose–response curves for oral administration in rats (n = 8 per dose, 4-h time point). ED₅₀ 2.33 mg/kg (95 % CI 1.3–3.39), TD₅₀ 77.38 mg/kg (95 % CI 62.55–91.01); PI 33.2

Fig. 3

2-Amino-6-trifluoromethylthio-benzothiazole (SKA-19) shows efficacy in multiple seizure models. (a) 6-Hz seizure test in mice: ED₅₀ 12.19 mg/kg [95 % confidence interval (CI) 8.2–17.45, n = 8 per dose]. See Fig. 2 for mouse TD₅₀ 2 h after intraperitoneal (i.p.) application. (b) Hippocampal kindled rats: ED₅₀ 5.47 mg/kg (95 % CI 2.92–8.92, n = 8 per dose). (c) Frings audiogenic seizure (AGS)-susceptible mouse model: ED₅₀ 2.15 mg/kg (95 % CI 1.52–2.65, n = 8 per dose). In all cases, testing was performed 2 h after i.p. application of SKA-19

Fig. 4

Pharmacokinetics of 2-amino-6-trifluoromethylthio-benzothiazole (SKA-19). (a) Total SKA-19 plasma concentrations (mean ± SD) following intravenous administration of 10 mg/kg in CremophorEL (Sigma-Aldrich, St. Louis, MO, USA)/phosphate-buffered saline to male Sprague–Dawley rats (n = 3). The inset shows the same data on a log scale. The data were best fitted as biexponential decay and with a quick distribution into tissue followed by elimination (t_1/2 = 2.16 ± 0.023 h). (b) SKA-19 plasma concentrations following oral gavage application at 10 mg/kg in solution (n = 3) or as a methylcellulose suspension (n = 3). (c) Plasma concentrations following intraperitoneal (i.p.) application at 10 and 30 mg/kg (n = 3). (d) Tissue concentrations 2 h after i.p. administration of SKA-19 and riluzole at 10 mg/kg (n = 3)

Fig. 5

Comparison of seizure protection time course between riluzole and 2-amino-6-trifluoromethylthio-benzothiazole (SKA-19). (a) Time course of seizure protection in the mouse maximal electroshock (MES) test following intraperitoneal (i.p.) administration of riluzole as a solution (n = 8 per time point). (b) Dose–response curves for seizure protection in MES test (□) and neurological impairment (■) following i.p. administration of increasing riluzole doses in mice (n = 8 per dose, 10-min time point). ED₅₀ 5.37 mg/kg [95 % confidence interval (CI) 5.17–5.57], TD₅₀ 15.77 mg/kg (95 % CI 11.94–20.83); protective index (PI) 2.9. (c) Time course of seizure protection in the mouse MES test following i.p. administration of SKA-19 as a solution (n = 6–8 per time point). (d) Dose–response curves for seizure protection in MES test (□) and neurological impairment (■) following i.p. administration of increasing SKA-19 doses in mice (n = 6–8 per dose, 10-min time point). ED₅₀ 4.93 mg/kg (95 % CI 4.15–5.86), TD₅₀ 16.08 mg/kg (95 % CI 13.06–19.02); PI 3.2

Fig. 6

2-Amino-6-trifluoromethylthio-benzothiazole (SKA-19) is effective in pain models. (a) SKA-19 (5 mg/kg administered 2 h prior to formalin injection) significantly decreased the time mice spent licking the affected hindpaw in a 2-min period recorded at 5-min intervals in the formalin pain test (n = 8 per group). (b) Threshold for foot withdrawal in response to a series of calibrated Von Frey fibers in rats 7 days (n = 8) after recovery from nerve ligation surgery following administration of SKA-19 (5 m/kg)

Fig. 7

Increasing concentrations of 2-amino-6-trifluoromethylthio-benzothiazole (SKA-19) inhibit (a) spontaneous, and (b) 4-aminopyridine (4-AP)- and (c) picrotoxin-induced Ca²⁺ oscillations in cultured 14 days in vitro (DIV) hippocampal neurons. (a) SKA-19 concentrations of 1 μM or less have no effect on spontaneous Ca²⁺ oscillations. The arrow indicates addition of SKA-19. (b) 4-AP (1 μM, arrow indicates addition) produces an immediate but transient elevation in neuronal intracellular Ca²⁺ followed by increased Ca²⁺ oscillation frequency with lower amplitude. The initial rise and the oscillations are inhibited by SKA-19. SKA-19 was added 10 min before 4-AP. (c) Picrotoxin (PTX; 10 μM, arrow indicates addition) induces higher amplitude Ca²⁺ oscillations, which are inhibited by SKA-19 (added 10 min before PTX). VEH = vehicle

Fig. 8

2-Amino-6-trifluoromethylthio-benzothiazole (SKA-19) reduces action potential (AP) firing of hippocampal pyramidal neurons by activating KCa2 channels and inhibiting voltage-gated Na⁺ channels (Na_V) channels. (a) Overlay of representative AP traces recorded before (black) and after (red) perfusion of 1 μM SKA-19 (left) or 25 μM SKA-19 (right). Cells were held at –65 mV and a train of APs evoked by a 1-s 150-pA current injection. (b) Overlay of AP traces recorded in the absence and presence of 10 μM SKA-19 (left), and plot of the number of APs elicited in the presence (red) and absence of SKA-19 in response to stimulating current injections of increasing amplitude (right). Data points represent means and SDs for 3 independent neurons. (c) SKA-19 enhances the medium afterhyperpolarization (AHP). SKA-19 at a concentration of 10 μM increases the amplitude of the current underlying the medium AHP. Shown on a compressed (left) and expanded time scale (middle). Bar graph of normalized mI_AHP current amplitude (right). Shown are means and SDs for 3 neurons. (d) SKA-19 activates rKCa2.2 and hKCa2.3 stably expressed in human embryonic kidney-293 cells. (e) Concentration–response curve for the activation of KCa2.1, KCa2.2, and KCa2.3 recorded in the presence of 250 nM of free intracellular Ca²⁺. See Table 1 for EC₅₀ values. (f) SKA-19 is a state- and use-dependent inhibitor of Nav1.2 currents in N1E-115 neuroblastoma cells. Sample Na_V1.2 current traces blocked by 1 μM and 10 μM SKA-19 (left). State-dependence (middle): IC₅₀ at –70 mV holding potential is 0.86 ± 0.70 μM; IC₅₀ at –90 mV holding potential is 7.90 ± 0.01 μM; IC₅₀ at –120 mV holding potential is 9.50 ± 1.72 μM. Use-dependence (right): IC₅₀ at 0.1 Hz is 7.90 ± 0.01 μM; IC₅₀ at 20 Hz is 0.52 ± 0.23 μM. Data points represent means plus SDs extracted from recordings from at least 3 independent cells