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Epilepsia
2020 May 01;615:914-923. doi: 10.1111/epi.16497.
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Functional characterization of the antiepileptic drug candidate, padsevonil, on GABAA receptors.
Niespodziany I
,
Ghisdal P
,
Mullier B
,
Wood M
,
Provins L
,
Kaminski RM
,
Wolff C
.
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OBJECTIVE: The antiepileptic drug candidate, padsevonil, is the first in a novel class of drugs designed to interact with both presynaptic and postsynaptic therapeutic targets: synaptic vesicle 2 proteins and γ-aminobutyric acid type A receptors (GABAA Rs), respectively. Functional aspects of padsevonil at the postsynaptic target, GABAA Rs, were characterized in experiments reported here.
METHODS: The effect of padsevonil on GABA-mediated Cl- currents was determined by patch clamp on recombinant human GABAA Rs (α1β2γ2) stably expressed in a CHO-K1 cell line and on native GABAA Rs in cultured rat primary cortical neurons. Padsevonil selectivity for GABAA R subtypes was evaluated using a two-electrode voltage clamp on recombinant human GABAA Rs (α1-5/β2/γ2) in Xenopus oocytes.
RESULTS: In recombinant GABAA Rs, padsevonil did not evoke Cl- currents in the absence of the agonist GABA. However, when co-administered with GABA at effective concentration (EC)20 , padsevonil potentiated GABA responses by 167% (EC50 138 nmol/L) and demonstrated a relative efficacy of 41% compared with zolpidem, a reference benzodiazepine site agonist. Similarly, padsevonil demonstrated GABA-potentiating activity at native GABAA Rs (EC50 208 nmol/L) in cultured rat cortical neurons. Padsevonil also potentiated GABA (EC20 ) responses in GABAA Rs expressed in oocytes, with higher potency at α1- and α5-containing receptors (EC50 295 and 281 nmol/L) than at α2- and α3-containing receptors (EC50 1737 and 2089 nmol/L). Compared with chlordiazepoxide-a nonselective, full GABAA R agonist-the relative efficacy of padsevonil was 60% for α1β2γ2, 26% for α2β2γ2, 56% for α3β2γ2, and 41% for α5β2γ2; no activity was observed at benzodiazepine-insensitive α4β2γ2 receptors.
SIGNIFICANCE: Results of functional investigations on recombinant and native neuronal GABAA Rs show that padsevonil acts as a positive allosteric modulator of these receptors, with a partial agonist profile at the benzodiazepine site. These properties may confer better tolerability and lower potential for tolerance development compared with classic benzodiazepines currently used in the clinic.
Figure 1- Functional properties of padsevonil on recombinant γ‐aminobutyric acid type A (GABAA) receptors. A, Patch‐clamp recordings of human recombinant GABAA receptor (α1β2γ12) currents in CHO cells. Padsevonil (1 nmol/L–10 µmol/L) was tested in the presence of GABA at EC20 (5 µmol/L) (n = 7‐15 cells; ± SEM). Inset: Representative GABAA currents recorded from the same cell under control conditions (black), with padsevonil 10 µmol/L (red) and with 1 µmol/L zolpidem (gray). Horizontal bar represents 5 s and vertical bar 0.5 nA. B, Effects of padsevonil and GABA on GABAA receptor currents (n = 4‐7 cells; ± standard error of the mean [SEM]). C, Relative efficacy of padsevonil compared with zolpidem in potentiating GABA‐mediated (EC20) Cl− currents in human α1β2γ12 GABAA receptors (n = 7‐35 cells; ± SEM). Error bars are ± SEM
Figure 2- Padsevonil selectivity for γ‐aminobutyric acid type A (GABAA) receptor subtypes as determined by two‐electrode voltage clamp recordings (TEVC) on human recombinant GABAA receptors expressed in Xenopus oocytes. A, Dose–response of padsevonil (1 nmol/L–30 µmol/L). Data are shown as mean ± SEM (n = 6‐26 cells). B, Relative efficacy of padsevonil (10 µM) compared with chlordiazepoxide (CDP, 10 µmol/L) on GABAA receptor subtypes. Data are shown as mean ± SEM. SEM, standard error of mean
Figure 3- Effects of padsevonil, zolpidem, and the reference compound Ro 15‐4513 on potentiation of the γ‐aminobutyric acid (GABA; control) response on α4β2γ2 receptors expressed in Xenopus oocytes. Currents were recorded using two‐electrode voltage clamp in the presence of GABA at EC20. Error bars are mean ± SEM. SEM, standard error of mean
Figure 4- Effects of padsevonil on native γ‐aminobutyric acid type A (GABAA) receptors in cultured neurons. A, Dose–response of padsevonil on native GABAA receptor currents in rat primary cortical neurons (days in vitro [DIV] 10‐14). Data have been normalized to the basal evoked current with GABA at EC10 (2 µmol/L) (n = 8 neurons; mean ± SEM). Inset: Representative traces of GABAA receptor currents evoked under control conditions (in black), with padsevonil 10 µmol/L (in red) and with zolpidem 1 µmol/L (in gray) in the same cortical neuron (DIV 11). Horizontal bar represents 5 s and vertical bar 1 nA. B, quantitative polymerase chain reaction analysis of GABAA receptor subunits in rat primary cortical neurons (average of three different cultures; DIV 10‐14). Data have been normalized to the maximal levels (mean ± standard deviation)
Figure 1. Functional properties of padsevonil on recombinant γ‐aminobutyric acid type A (GABAA) receptors. A, Patch‐clamp recordings of human recombinant GABAA receptor (α1β2γ12) currents in CHO cells. Padsevonil (1 nmol/L–10 µmol/L) was tested in the presence of GABA at EC20 (5 µmol/L) (n = 7‐15 cells; ± SEM). Inset: Representative GABAA currents recorded from the same cell under control conditions (black), with padsevonil 10 µmol/L (red) and with 1 µmol/L zolpidem (gray). Horizontal bar represents 5 s and vertical bar 0.5 nA. B, Effects of padsevonil and GABA on GABAA receptor currents (n = 4‐7 cells; ± standard error of the mean [SEM]). C, Relative efficacy of padsevonil compared with zolpidem in potentiating GABA‐mediated (EC20) Cl− currents in human α1β2γ12 GABAA receptors (n = 7‐35 cells; ± SEM). Error bars are ± SEM
Figure 2. Padsevonil selectivity for γ‐aminobutyric acid type A (GABAA) receptor subtypes as determined by two‐electrode voltage clamp recordings (TEVC) on human recombinant GABAA receptors expressed in Xenopus oocytes. A, Dose–response of padsevonil (1 nmol/L–30 µmol/L). Data are shown as mean ± SEM (n = 6‐26 cells). B, Relative efficacy of padsevonil (10 µM) compared with chlordiazepoxide (CDP, 10 µmol/L) on GABAA receptor subtypes. Data are shown as mean ± SEM. SEM, standard error of mean
Figure 3. Effects of padsevonil, zolpidem, and the reference compound Ro 15‐4513 on potentiation of the γ‐aminobutyric acid (GABA; control) response on α4β2γ2 receptors expressed in Xenopus oocytes. Currents were recorded using two‐electrode voltage clamp in the presence of GABA at EC20. Error bars are mean ± SEM. SEM, standard error of mean
Figure 4. Effects of padsevonil on native γ‐aminobutyric acid type A (GABAA) receptors in cultured neurons. A, Dose–response of padsevonil on native GABAA receptor currents in rat primary cortical neurons (days in vitro [DIV] 10‐14). Data have been normalized to the basal evoked current with GABA at EC10 (2 µmol/L) (n = 8 neurons; mean ± SEM). Inset: Representative traces of GABAA receptor currents evoked under control conditions (in black), with padsevonil 10 µmol/L (in red) and with zolpidem 1 µmol/L (in gray) in the same cortical neuron (DIV 11). Horizontal bar represents 5 s and vertical bar 1 nA. B, quantitative polymerase chain reaction analysis of GABAA receptor subunits in rat primary cortical neurons (average of three different cultures; DIV 10‐14). Data have been normalized to the maximal levels (mean ± standard deviation)
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