Evaluation of anticonvulsant actions of dibromophenyl enaminones using in vitro and in vivo seizure models

Abstract

Epilepsy and other seizure disorders are not adequately managed with currently available drugs. We recently synthesized a series of dibromophenyl enaminones and demonstrated that AK6 and E249 were equipotent to previous analogs but more efficacious in suppressing neuronal excitation. Here we examined the actions of these lead compounds on in vitro and in vivo seizure models. In vitro seizures were induced in the hippocampal slice chemically (zero Mg2+ buffer and picrotoxin) and electrically using patterned high frequency stimulation (HFS) of afferents. In vivo seizures were induced in rats using the 6 Hz and the maximal electroshock models. AK6 (10 µM) and E249 (10 µM) depressed the amplitude of population spikes recorded in area CA1 of the hippocampus by -50.5±4.3% and -40.1±3.1% respectively, with partial recovery after washout. In the zero Mg2+ model, AK6 (10 µM) depressed multiple population spiking (mPS) by -59.3±6.9% and spontaneous bursts (SBs) by -65.9±7.2% and in the picrotoxin-model by -43.3±7.2% and -50.0±8.3%, respectively. Likewise, E249 (10 µM) depressed the zero-Mg2+-induced mPS by -48.8±9.5% and SBs by -55.8±15.5%, and in the picrotoxin model by -37.1±5.5% and -56.5±11.4%, respectively. They both suppressed post-HFS induced afterdischarges and SBs. AK6 and E249 dose-dependently protected rats in maximal electroshock and 6 Hz models of in vivo seizures after 30 min pretreatment. Their level of protection in both models was similar to that obtained with phenytoin Finally, while AK6 had no effect on locomotion in rats, phenytoin significantly decreased locomotion. AK6 and E249, suppressed in vitro and in vivo seizures to a similar extent. Their in vivo activities are comparable with but not superior to phenytoin. The most efficacious, AK6 produced no locomotor suppression while phenytoin did. Thus, AK6 and E249 may be excellent candidates for further investigation as potential agents for the treatment of epilepsy syndromes with possibly less CNS side effects.

Figure 1. Summarized synthetic scheme for synthesis of AK6 (compound 25) and E249 (compound 21).

Figure 2. AK6 and E249 irreversibly depress population spikes (PS) recorded in the CA1 area of the rat hippocampal slice.

A: Sample PS traces in control, after 6 minutes bath perfusion with 10 µM AK6 and following 15 min washout of the compound. B: Average time-effect plot of the effect of 10 µM AK6 on PS amplitude. C: Average time-effect plot of the effect of 10 µM E249 on PS amplitude.

Figure 3. AK6 and E249 suppress zero Mg²⁺-induced epileptiform activity in the rat hippocampus.

A: Sample traces of a single PS and multiple PS (mPS) induced by bath perfusion with aCSF in which magnesium was removed and effect of AK6 on mPS. B: Sample voltage traces (recorded in gap-free mode) showing that AK6 and E249 (only AK6 is shown) also suppress spontaneous bursts (SBs) frequency induced by the zero Mg²⁺ buffer. C1&2: Bar graphs summarizing the effects of AK6 on the number of spikes and SB frequency, respectively. D1&2: Bar graphs summarizing the effects of E249 on the number of spikes and SB frequency, respectively. In this and all other figures, * indicates statistical significance at p<0.05 compared to control.

Figure 4. AK6 and E249 suppress picrotoxin induced epileptiform activity in the rat hippocampus.

A: Sample traces of a single PS and multiple PS (mPS) induced by bath perfusion with aCSF containing 100 µM picrotoxin and the effect of 10 µM AK6 on the resulting mPS. B: Sample voltage traces (recorded in gap-free mode) showing that AK6 and E249 (only AK6 is shown) also suppress SBs (arrow heads) frequency induced by picrotoxin. C1&2: Bar graphs summarizing the effects of AK6 on the number of spikes and SB frequency, respectively. D1&2: Bar graphs summarizing the effects of E249 on the number of spikes and SB frequency, respectively.

Figure 5. AK6 and E249 depress high frequency afferent stimulation (HFS)-induced epileptiform activity in the rat hippocampus.

A: Sample traces of a single PS and multiple PS (mPS) induced by a patterned HFS (see methods). B: Sample voltage traces (recorded in gap-free mode) showing that AK6 suppresses afterdischarge (downward arrowheads) immediately following HFS. C: Bar graphs summarizing the effect of AK6 on the AD frequency. D: Sample voltage traces (recorded in gap-free mode) showing that E249 suppresses SBs, referred to as stimulus train-induced bursts (STIBs, upwards arrowheads). E: Bar graphs summarizing the effect of E249 on STIBs.

Figure 6. AK6 and E249 suppress maximal electroshock (MES) and 6 Hz seizures in a dose-dependent manner in male rats.

A: Bar graphs summarizing the AK6 dose-dependent inhibition of seizures induced by the 6 Hz (A1, n = 20/5/5/8: control to highest dose of drug) and MES (A2, n = 18/5/5/8: control to highest dose of drug) models of seizures. B: Bar graphs summarizing the E249 dose-dependent inhibition of seizures induced by the 6 Hz (B1, n = 20/10/5/10: control to highest dose of drug) and MES (B2, n = 18/10/5/10: control to highest dose of drug) models of seizures. Note that the control bars and the 20 mg/kg bars for AK6 and E249 in this figure and those in figure 7 are the same.

Figure 7. AK6 and E249 suppression of MES- and 6 Hz-induced seizures are comparable to those of phenytoin.

Bar graphs summarizing the effect of AK6 (20 mg/kg), E249 (20 mg/kg) and phenytoin (40 mg/kg) on the 6 Hz (A, n = 5–20) and MES (B, n = 5–18) models of seizures.

Figure 8. AK6 has no effect on locomotor activity in male rats.

A: A time to effect plot of the horizontal motor activity of rats in control (saline injection), after AK6 (20 mg/kg) and after phenytoin (40 mg/kg) all given IP. B: Bar graph summarizing total horizontal locomotor activity (2 hours) of all rats in A.