The Positive Allosteric Modulator of α 2/3-Containing GABAA Receptors, KRM-II-81, Is Active in Pharmaco-Resistant Models of Epilepsy and Reduces Hyperexcitability after Traumatic Brain Injury

Abstract

The imidizodiazepine, 5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole (KRM-II-81), is selective for α2/3-containing GABA_A receptors. KRM-II-81 dampens seizure activity in rodent models with enhanced efficacy and reduced motor-impairment compared with diazepam. In the present study, KRM-II-81 was studied in assays designed to detect antiepileptics with improved chances of impacting pharmaco-resistant epilepsies. The potential for reducing neural hyperactivity weeks after traumatic brain injury was also studied. KRM-II-81 suppressed convulsions in corneal-kindled mice. Mice with kainate-induced mesial temporal lobe seizures exhibited spontaneous recurrent hippocampal paroxysmal discharges that were significantly reduced by KRM-II-81 (15 mg/kg, orally). KRM-II-81 also decreased convulsions in rats undergoing amygdala kindling in the presence of lamotrigine (lamotrigine-insensitive model) (ED₅₀ = 19 mg/kg, i.p.). KRM-II-81 reduced focal and generalized seizures in a kainate-induced chronic epilepsy model in rats (20 mg/kg, i.p., three times per day). In mice with damage to the left cerebral cortex by controlled-cortical impact, enduring neuronal hyperactivity was dampened by KRM-II-81 (10 mg/kg, i.p.) as observed through in vivo two-photon imaging of layer II/III pyramidal neurons in GCaMP6-expressing transgenic mice. No notable side effects emerged up to doses of 300 mg/kg KRM-II-81. Molecular modeling studies were conducted: docking in the binding site of the α1β3γ2L GABA_A receptor showed that replacing the C8 chlorine atom of alprazolam with the acetylene of KRM-II-81 led to loss of the key interaction with α1His102, providing a structural rationale for its low affinity for α1-containing GABA_A receptors compared with benzodiazepines such as alprazolam. Overall, these findings predict that KRM-II-81 has improved therapeutic potential for epilepsy and post-traumatic epilepsy. SIGNIFICANCE STATEMENT: We describe the effects of a relatively new orally bioavailable small molecule in rodent models of pharmaco-resistant epilepsy and traumatic brain injury. KRM-II-81 is more potent and generally more efficacious than standard-of-care antiepileptics. In silico docking experiments begin to describe the structural basis for the relative lack of motor impairment induced by KRM-II-81. KRM-II-81 has unique structural and anticonvulsant effects, predicting its potential as an improved antiepileptic drug and novel therapy for post-traumatic epilepsy.

Fig. 1.

Effects of KRM-II-81 in corneal-kindled mice (left) and the mesial temporal lobe epilepsy model in mice (right). (Left panel) KRM-II-81 was given orally, 2 hours prior to testing in corneal-kindled mice. KRM-II-81 dose dependently increased the percentage of mice that were protected against convulsions (inset) and decreased seizure severity in the mice. Each point or bar represents the mean ± S.E.M. of eight mice. Seizure prevalence (inset) was assessed with Fisher’s exact probability test with *P < 0.05 with vehicle-treated mice. Seizure severity was evaluated with ANOVA followed by Dunnett’s post-hoc test with ***P < 0.001 compared with vehicle control mice. (Right panel) Mice with kainate-induced mesial temporal lobe seizures exhibited spontaneous recurrent hippocampal paroxysmal discharges. KRM-II-81 (15 mg/kg, orally, 2 hours prior) was tested in four mice (t₃ = 8.6, P < 0.01 as indicated by **).

Fig. 2.

Daily seizure events for each of 12 rats undergoing kainate-induced chronic epilepsy. Data are shown for baseline, vehicle, and drug-treatment conditions. Drug treatment was with KRM-II-81(20 mg/kg, i.p., three times per day).

Fig. 3.

KRM-II-81 inhibited cortical neuronal hyperactivity in a CCI model of post-traumatic epilepsy in vivo. (A) In vivo two-photon imaging of cortical neurons in layer II/III showed great increases in integrated fluorescence (left, P < 0.001, Student’s t test) and fraction of active neurons (right, P < 0.01) in mice with CCI injury for 3 months (n = 3 in both groups). (B) Representative images of maximal projections of cortical layer II/III GCaMP6-expressing neurons at baseline and 0.5, 1, 2, and 4 hours after injection of KRM-II-81 in vivo (top row). ΔF/F traces of calcium transients of neurons corresponding to the color-circled neurons are shown on the bottom. Note the dramatic decrease in calcium activity 0.5 hours after injection of KRM-II-81. (C) There were dramatic decreases in integrated fluorescence and fraction of active cells 0.5 hours after drug injection, suggesting a decrease in cortical excitatory activity. One-way ANOVA was followed by Bonferroni test. *P < 0.05; **P < 0.01, ***P < 0.001. n = 3 mice.

Fig. 4.

Panel A. Chemical structures of DZP and QH-II-66. The DZP and pendent aromatic rings of DZP are numbered. The benzene (A) and pendent phenyl (B) rings of DZP are labeled. Panel B. Chemical structures of ALP and KRM-II-81. The DZP, pendent aromatic, and triazole rings of ALP are numbered. The benzene (A), pendent phenyl (B), and triazole (C) rings of ALP are labeled.

Fig. 5.

(A) Bound DZP (orange) and docked QH-II-66 (pink) overlay; α1 (yellow) and γ2 (turquoise) subunits of α1β3γ2L GABA_A receptor 6HUP. (B) Ligand-protein interactions; bound DZP (orange) in complex with α1 (yellow) and γ2 (turquoise) subunits of α1β3γ2L GABA_A receptor 6HUP. (C) Ligand-protein interactions; docked QH-II-66 (pink) in complex with α1 (yellow) and γ2 (turquoise) subunits of α1β3γ2L GABA_A receptor 6HUP. Dashed lines indicate π–π interactions and hydrogen bonds; hydrogen bond (green), hydrophobic interaction (magenta).

Fig. 6.

Bound ALP (gray) and docked KRM-II-81 (sienna) overlay; α1 (yellow) and γ2 (turquoise) subunits of α1β3γ2L GABA_A receptor 6HUO.

Fig. 7.

(A) Ligand-protein interactions; bound ALP (gray) in complex with α1 (yellow) and γ2 (turquoise) subunits of α1β3γ2L GABA_A receptor 6HUO. (B) Showing ligand-protein interactions; docked KRM-II-81 (sienna) in complex with α1 (yellow) and γ2 (turquoise) subunits of α1β3γ2L GABA_A receptor 6HUO. Dashed lines indicate π–π interactions, hydrogen bonds and halogen bond; hydrogen bond (green), hydrophobic interaction (magenta), and halogen bond (black).