GalR2-positive allosteric modulator exhibits anticonvulsant effects in animal models

Abstract

Galanin receptors type 1 (GalR1) and/or type 2 (GalR2) represent unique pharmacological targets for treatment of seizures and epilepsy. Previous studies have shown that the endogenous peptide ligand galanin exerts powerful anticonvulsant effect through activation of these two G protein-coupled receptors, which are highly expressed in the temporal lobe of rodent brain. Here we report the characterization of a putative GalR2-positive allosteric modulator CYM2503. CYM2503 potentiated the galanin-stimulated IP1 accumulation in HEK293 cells stably expressing GalR2 receptor, whereas it exhibited no detectable affinity for the (125)I galanin-binding site of GalR2 receptor, an effect consistent with that of a positive allosteric modulator. In the rat Li-pilocarpine status epilepticus model, CYM2503, injected intraperitoneally, increased the latency to first electrographic seizure and the latency to first stage 3 behavioral seizure, decreased the latency to the establishment of status epilepticus, and dramatically decreased the mortality. In a Li-pilocarpine seizure model in mice, CYM2503 increased the latency to first electrographic seizure and decreased the total time in seizure. CYM2503 also attenuated electroshock-induced seizures in mice. Thus, CYM2503 provides a starting point for the development of anticonvulsant therapy using the galanin R2 receptor as target.

Fig. 1.

Structure of CYM2503.

Fig. 2.

CYM2503 mediated potentiation of the agonist-stimulated GalR2 activation and the lack of affinity of CYM2503 for GalR2 orthosteric site in HEK293-GalR2 cells. IP1 accumulation was used as an index of GalR2 activation. (A) CYM2503 potentiated the effect of 100 nM galanin. (B) CYM2503 shifted the galanin dose–response curve to the left. (C) Iodine-125 porcine galanin, at 0.15 nM, was not displaced by CYM2503 at up to 100 μM concentration. For comparison, unlabeled rat galanin displaced ¹²⁵I porcine galanin with an IC₅₀ of 10.1 nM in the same experiment. (D) Lack of effect of CYM2503 on IP1 accumulation. CYM2503 at concentrations up to 100 μM did not change baseline IP1 level. As a comparison, rat galanin stimulated IP1 production in the same experiment. All data shown were from one experiment performed in triplicate and are typical of two to six independent experiments that yielded similar results.

Fig. 3.

CYM2503 exhibited no affinity for GalR1 and it did not modulate GalR1 signaling. Agonist-induced inhibition of forskolin-stimulated cAMP production in CHO-GalR1 cells was used as an index of GalR1 activation. Forskolin was used at a concentration of 4 μM. (A) Iodine-125 porcine galanin, at 0.1 nM, was not displaced by CYM2503 at up to 100 μM concentration. For comparison, unlabeled rat galanin displaced ¹²⁵I porcine galanin with an IC₅₀ of 5.4 nM in the same experiment. (B) Rat galanin inhibited forskolin-stimulated cAMP accumulation with an IC₅₀ of 7.8 pM in CHO-GalR1 cells, whereas it had no effect in the parent CHO cell line that does not express GalR1. (C) Lack of effect of CYM2503 on cAMP accumulation. CYM2503 at concentrations up to 100 μM had no effect on forskolin-stimulated cAMP production. (D) CYM2503 did not shift the galanin dose–response curve. The data shown here were from one experiment performed in triplicate and are typical of three or four independent experiments that yielded similar results.

Fig. 4.

Anticonvulsant effect of CYM2503 in the Li-pilocarpine model in mice. (A) Experimental paradigm. (B) Examples of electrographic seizure activity and spikes induced by Li-pilocarpine. (C) Statistic analysis of EEG. Data are mean ± SEM. (*P < 0.05, ANOVA followed by Dunnett multiple comparison with respective controls, n = 6–10.) LEV, levetiracetam 50 mg/kg i.p.

Fig. 5.

CYM2503 protected mice in the electroshock induced seizure model. A 15-min pretreatment with 60 mg/kg i.p. (A) decreased the percent of mice that developed hindlimb extension and decreased the mortality. The number of animals in each group is shown above the bar graph. (B) Latency to hindlimb extension was also increased. Data are mean ± SEM. (*P < 0.05, one-way ANOVA followed by Dunnett multiple comparison with vehicle, n = 9–11.)

Fig. 6.

Anticonvulsant effect of CYM2503 in the Li-pilocarpine model in rats. (A) Latency to first EEG seizure and latency to first stage 3 or higher behavioral seizure. (B) Latency to the establishment of SE. Data are mean ± SEM. (*P < 0.05 and **P < 0.01, ANOVA followed by Dunnett multiple comparison vs. respective controls, n = 4–5.) LEV, levetiracetam 50 mg/kg i.p. CYM2503 dose was 60 mg/kg i.p.