Silexan and pregabalin show similar inhibitory effects on VOCCs in primary hippocampal neurons.

<p>Concentration-dependent effect of Silexan (A, 0.01–10 µM) and pregabalin (3–30 µM) after 10 min preincubation on KCl (60 mM) induced Ca²⁺-influx in primary hippocampal neurons. (C) shows representative traces of intracellular Ca²⁺ concentration in primary hippocampal neurons stimulated with KCl (60 mM) preincubated with Silexan (1 µg/ml), pregabalin (10 µM) compared to ctl. (D)–(F) show representative fields of fura-2 loaded hippocampal neurons preincubated with Silexan 1 µg/ml (D), pregabalin 10 µM (E) or control (F) under basal conditions. (G)–(I) show the same samples after stimulation with KCl (60 mM). All data presented are means ± SEM (n = 10–14, paired t-test).</p

Silexan does neither share the binding site of pregabalin, nor inhibits VOCCs via G_i-coupled receptors.

<p>(A) Displacement studies conducted with [³H]-gabapentin in partially purified synaptic membranes. Synaptic membranes were incubated with [³H]-gabapentin in presence of pregabalin (A, 0.001–100 µM) or Silexan (B, 0.1–100 µM; n = 3). (C) Primary hippocampal neurons were incubated in the presence or absence of PTX (200 ng/ml) for 18 h. Afterwards cells were treated with Silexan (1 µg/ml) or AEA (1 µM) for 10 min and then stimulated with KCl (60 mM; n = 11–12). (D) Additive inhibitory effects of P/Q-type and N-type channel blockers on KCl-induced Ca²⁺-influx in murine synaptosomes. Synaptosomes were preincubated with ω-agatoxin IVA (100 nM), ω-conotoxin GVIA (30 nM) or a combination of both inhibitors for 10 min. Afterwards, they were stimulated with KCl (80 mM). Silexan (1 µg/ml) causes no significant additive effects when combined with the N-type VOCCs inhibitor ω-conotoxin GVIA (30 nM, E), or the P/Q-type inhibitor ω-agatoxin IVA (100 nM, F; n = 9–12). All data presented are mean values ± SEM (paired t-test).</p

Silexan reduces anxiety-related behaviour in the elevated plus maze and increases pentobarbital-induced sleeping time.

<p>(A–C) Mice were treated orally with diazepam (2.5 mg/kg BW), pregabalin (100 mg/kg BW) or Silexan (1–30 mg/kg BW) for three days. Diazepam, pregabalin and Silexan increased the time spent in the open arm (A, B) and entry number (C). (D) Mice were treated orally with pregabalin (100 mg/kg BW) or Silexan (1–30 mg/kg BW) for nine days. Sleeping time was determined after i.p. application of pentobarbital (45 mg/kg BW). All data presented are mean values ± SEM using 8 mice per treatment group (unpaired t-test).</p

Silexan dose-dependently inhibits VOCCs in murine synaptosomes.

<p>Dose-dependent effects of pregabalin (A), Silexan (B) and linalool (D) on KCl-induced Ca²⁺-influx. Murine synaptosomes were preincubated (10 min) with different concentrations of pregabalin (A, 0.03–100 µM), Silexan (B, 0.01–10 µg/ml) or linalool (D, 0.01–1 µM) and afterwards stimulated with KCl (80 mM). (C) Representative Ca²⁺-transients in murine synaptosomes after KCl-induced activation of VOCCs in the presence and absence of pregabalin (3 µM) and Silexan (1 µg/ml). (E) Effect of the preincubation (10 min) with linalool (1 µM) and linalyl acetate (1 µM) on KCl-induced Ca²⁺-influx in murine synaptosomes. (F) Several other monterpenes have no effect on KCl-induced Ca²⁺-influx in murine synaptosomes under similar conditions. All data presented are mean values ± SEM (n = 10–12), paired t-test.</p