BAER‐101, a selective potentiator of α2‐ and α3‐containing GABAA receptors, fully suppresses spontaneous cortical spike‐wave discharges in Genetic Absence Epilepsy Rats from Strasbourg (GAERS)

BAER‐101 (formerly AZD7325) is a selective partial potentiator of α2/3‐containing γ‐amino‐butyric acid A receptors (GABAARs) and produces minimal sedation and dizziness. Antiseizure effects in models of Dravet and Fragile X Syndromes have been published. BAER‐101 has been administered to over 700 healthy human volunteers and patients where it was found to be safe and well tolerated. To test the extent of the antiseizure activity of BAER‐1010, we tested BAER‐101 in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model, a widely used and translationally relevant model. GAERS rats with recording electrodes bilaterally located over the frontal and parietal cortices were used. Electroencepholographic (EEG) signals in freely moving awake rats were analyzed for spike‐wave discharges (SWDs). BAER‐101 was administered orally at doses of 0.3–100 mg/kg and diazepam was used as a positive control using a cross‐over protocol with a wash‐out period between treatments. The number of SWDs was dose‐dependently reduced by BAER‐101 with 0.3 mg/kg being the minimally effective dose (MED). The duration of and total time in SWDs were also reduced by BAER‐101. Concentrations of drug in plasma achieved an MED of 10.1 nM, exceeding the Ki for α2 or α3, but 23 times lower than the Ki for α5‐GABAARs. No adverse events were observed up to a dose 300× MED. The data support the possibility of antiseizure efficacy without the side effects associated with other GABAAR subtypes. This is the first report of an α2/3‐selective GABA PAM suppressing seizures in the GAERS model. The data encourage proceeding to test BAER‐101 in patients with epilepsy.

BAER-101, formerly AZD7325 (Figure 1), is a positive allosteric modulator (PAM) of γ-amino-butyric acid A receptors (GABAARs).It is an N-substituted cinnoline structurally unrelated to the benzodiazepines.BAER-101 has been reported to be potent (~1 nM) and selective for α2 and α3 over other GABAAR subtypes, where it functions as a partial agonist (~15%-18% of maximal diazepam response at α2 and α3; ~8% at α5) and a neutral antagonist at α1 subtypes (Chen et al., 2014).This compound has been studied in healthy human volunteers and in patients with generalized anxiety disorder (GAD), where it displayed good tolerability and a relatively low level of sedation and dizziness, side-effects that are often seen with benzodiazepine GABAR PAMs like lorazepam (Chen et al., 2014;Gu et al., 2018;Zhou et al., 2012).The compound is now being developed as a possible treatment for epilepsy and specific anxiety disorders.
GABAAR PAMs are anticonvulsant and include the recent addition of ganaxolone into clinical practice for the treatment of seizures in CDKL5 deficiency disorder (Knight et al., 2022;Perucca et al., 2023).While ganaxolone is a neurosteroid which acts on both synaptic and extrasynaptic GABAARs, BAER-101 preferentially potentiates α2and α3-containing GABAARs and is expected to suppress overt convulsive and nonconvulsive seizures.Darigabat and ENX-101 are α2/3/5-preferening GABAAR PAMs being developed for epilepsy.KRM-II-81, like BAER-101, is an α2/3-selective GABAAR PAM, has robust effects in convulsive and electroencepholographic (EEG)-monitored seizure models and appears to be under development for epilepsy and pain (Witkin et al., 2022).
Preclinical studies have reported antiseizure effects of BAER-101.Hines and colleagues (2018) interrogated the role of collybistin association to the α2-subunit using strategies that included specific loss of function mutations.Gabra2-1 mice had enhanced susceptibility to seizures and early mortality.AZD7325 reduced the anxiety and heightened EEG δ power of surviving mice.In a mouse model of Dravet syndrome using Scn1a +/− mice, Nomura et al. (2019) showed that AZD7325 was protective against heat-induced convulsive seizures without notable sedation.Fragile X Mental Retardation Protein (FMRP) is functionally lost in fragile X syndrome.Schaefer et al. (2021) studied the effects of BAER 101 in FMRP-null mice.
BAER-101 reduced hyperexcitability in cortical circuits, partially corrected the alterations in cortical EEG power, reduced susceptibility to audiogenic seizures, and improved novel object recognition memory.
The rationale for the present study was to determine whether BAER-101 would reduce nonconvulsive seizures in an animal model of absence seizures where GABA has long been postulated to be a regulating mechanism (Cope et al., 2009).The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model presents a wellcharacterized system for evaluating absence seizures in rats using defined electrophysiological readouts (Depaulis et al., 2016;Vergnes et al., 1982).This breeding-derived rat strain exhibits spontaneous generalized cortical seizures in the form of spike-wave discharges (SWDs) during an absence seizure in which there is behavioral arrest (Figure 2).SWDs in the GAERS model (Wallengren et al., 2005).The data have predictive validity in patients (Depaulis et al., 2016;Klitgaard et al., 2002;van Luijtelaar et al., 2002).

Effects of compounds in the GAERS
In the present study, BAER-101 was given orally to GAERS animals in doses of 3-100 mg/kg.When all of these doses were found to suppress SWDs, an overlapping dose range of 0.1-3 mg/kg was subsequently explored.BAER-101 suppressed SWDs in the GAERS model with a minimal effective dose of 0.3 mg/kg that resulted in plasma levels that were greater than the K i for α2 or α3 but less than the K i for α5 GABAARs.When taken together with the antiseizure effects reported in the literature, these data combined with their human translational power, encourage investigation in patients.The GABA PAM, Darigabat, suppressed SWDs in the GAERS model (Duveau et al., 2019) and also significantly suppressed seizures in patients with photosensitive epilepsy (Gurrell et al., 2019).
The data with BAER-101 are the first to support the anti-absence efficacy of a GABA PAM selective for the α2 and α3 subunitcontaining GABAAR constructs.

| Animals and conditions
A total of 14 experimentally naïve adult male GAERS were obtained from Dr. Antoine Depaulis (INSERM, Grenoble Institute of Neurosciences) and delivered to the laboratory at the age of ~3 months.The rats were first housed in groups in cages on wood litter with free access to food and water until surgery.Animal housing was maintained under artificial lighting between 7:30 a.m. and 7:30 p.m. at a controlled ambient temperature (22 ± 2°C) and relative humidity.

| Compounds
BAER-101 was provided by Avenue Therapeutics.A vial containing the compound (BAER-101 batch C423/4) was shipped at RT. Upon delivery, the compound was stored at RT, protected from light.Test compound BAER-101 was formulated at the appropriate concentrations for a dose volume of 5 mL/kg.Diazepam (Hoffmann-La Roche) was formulated for a dose volume of 10 mL/kg.BAER-101 was formulated at 0.02-20 mg/mL, for a dosing volume of 5 mL/kg, in a 20% hydroxypropyl beta cyclodextrin (HPbCD) in water.The vehicle was prepared in advance and kept at 4°C for a maximum of 4 weeks.HPbCD (batch A0434679, ref. 297561000, Fischer Scientific) was weighed and transferred in 50% of the final required volume of water for injection (Cooper).The mixture was stirred until complete dissolution, then the volume was adjusted to the final required volume.The first solution of BAER-101 was prepared at 20 mg/mL (phase 1) or 0.6 mg/kg (phase 2).BAER-101 material was weighed in a glass vial.Then the appropriate volume of vehicle was added little by little while stirring.The mixture was vortexed, then sonicated for 15 min.The resulting formulation was a clear solution.Lower concentrations for each phase were then prepared using serial dilutions from the initial concentrated solution described above.An appropriate volume of BAER-101 solution was measured, and then the appropriate volume of the vehicle was added.The mixture was vortexed.All formulations were then aliquoted in appropriate volumes for one EEG recording.
Aliquots were protected from light and stored at 4°C for a maximum duration of 10 days.One formulation was used for two EEG recordings.When formulations were stored at 4°C before use, they were left at room temperature for 10-15 min before use.Then they were placed in an ultrasound bath for 15 min.All formulations were still clear solutions.

| Electrode implantation
Electrodes for SWD recording were implanted in all 14 rats under general anesthesia (isoflurane; 2% in oxygen) using stereotaxic methods.Animals first received an injection of the analgesic drug buprenorphine (0.05 mg/kg, SC), about 2 h before induction of anesthesia.After anesthesia induction, rats were placed in the stereotaxic frame.The body temperature was monitored and maintained constant during the surgery.An ophthalmic gel was placed on the eyes to avoid the drying of the cornea.The depth of anesthesia was controlled before starting the surgery and the breathing rate and the cardiac rhythm were visually controlled during the surgery.The incision zone was first cleaned, and betadine was applied.The skin was incised, and the skull cleaned.Five monopolar electrodes (ref: E363/ 96/1.6/SP,Plastics One) were positioned over the frontal and parietal cortices, on both sides of the brain.The approximate coordinates for the frontal cortex were AP + 2 mm, ML ± 3 mm (from bregma as reference).The approximate coordinates for the parietal cortex were AP − 7 mm, ML ± 3 mm.The rats were equipped with a female connector fixed on the skull to allow chronic EEG recordings.A fifth electrode was placed on the cerebellum as reference.
After surgery, animals were maintained in individual cages.
Within 8 h from surgery, animals received a second injection of buprenorphine (0.05 mg/kg, SC).Animals were left in their home cage for at least 1 week of recovery.
Two weeks after electrode implantation, a 1-h preliminary EEG was recorded on all animals in control conditions.EEG signals were analyzed to count SWDs.From the 14 implanted GAERS, all 14 animals were found valid for EEG experiments, according to our quality criteria of signal-to-noise ratio and number of SWDs per hour: they all presented a satisfactory signal-to-noise ratio and a sufficient number of SWDs (above 20 per hour).A total of 12 animals were randomly selected for the pharmacological experiments.The two remaining animals were kept in a reserve pool.

| Phase 1
The initial study plan with BAER-101 included the following six conditions: Vehicle (20% HPbCD in water) PO, diazepam at 2 mg/kg IP, BAER-101 at 100 mg/kg PO, BAER-101 at 30 mg/kg PO, BAER-101 at 10 mg/kg PO, and BAER-101 at 3 mg/kg PO.Compound conditions were administered in a cross-over protocol, following the rules: each animal received each condition in a random order and in separate recording sessions, two animals received each condition in each recording session, and a minimal wash-out period of 7 days was allowed between each administration.
An intermediate check on study results after the second EEG of this cross-over protocol indicated that compound doses all produced substantial effects on EEG.The cross-over was interrupted, and doses were changed in phase 2 to a lower overlapping dose set.

| Phase 2
Four EEG recordings were completed with the following set of compound conditions: Vehicle (20% HPbCD in water) PO, diazepam 2 mg/kg IP, and BAER-101 (0.1, 0.3, 1, and 3 mg/kg, PO).Compound conditions were still administered in a cross-over protocol, following the rules: each animal received four different conditions from the six indicated above, in a random order and in separate recording sessions, animals having received the vehicle, diazepam or BAER-101 at 3 mg/kg during phase 1 were not ascribed again to these conditions, and a minimal wash-out period of 7 days was allowed between each administration.

| EEG recordings
The day of the recording session, the 12 selected animals were placed in a recording chamber and connected to their recording cable.An habituation period (~30 min) was allowed before the EEG recording session.EEG recordings were collected on freely moving animals for 40 min preadministration (baseline period) and 90 min postadministration using SystemPlus Evolution (Micromed).The EEG signal was band-pass filtered between 0.8 Hz and 1 kHz and digitized at 512 Hz (SDLTM128 Channels; Micromed).Animals were maintained in quiet wakefulness state during the recording session.A quiet wakefulness is required to record SWD activity, as sleep will disrupt the epileptic activity in the GAERS model.An experimenter was monitoring the animals and was maintaining wakefulness by gently stimulating the animals when required.Whenever an animal was showing some signs of sleep, as in showing a curled-up posture with closed eyes and some delta waves on EEG signals, the experimenter softly prodded the animal or introduced a novel object in the rat environment as described (Duveau et al., 2019).

| Data analysis
EEG recordings were subsequently analyzed offline and quantified blindly by an expert on SynapCell's proprietary platform to identify SWDs (Figure 2).SWDs were analyzed during a 40 min baseline period (immediately before compound administration), and for a period of 80 min between 10 and 90 min after compound administration.The first 10 min immediately after administration were not analyzed since administration markedly disturbs the occurrence of SWDs.
Data are expressed as the mean ± SEM.For each animal and administration, data were computed for number, average duration, and cumulated duration of SWD per 20 min periods.Posttreatment data were also pooled over the 80 min duration of the posttreatment period and normalized to % of baseline values, providing a single data point measuring the percentage of inhibition for each animal and each dose.ED50 was calculated from these data, using a nonlinear regression in GraphPad Prism v9.5 (GraphPad).

| Statistical analyses
Statistical analyses were performed using GraphPad Prism v9.5.The significance level was set at p < .05 a priori.Absolute data measured per 20 min periods were analyzed using a two-way analysis of variance (ANOVA) with the compound condition and the time from administration as factors.As the crossover design was not fully completed, not all animals received all doses of BAER-101.Consequently, measures were considered repeated only within the time factor.When significant, ANOVA was followed by paired comparisons using Dunnett's test, with comparisons versus the second baseline period, versus vehicle, and versus diazepam.
Pooled data over the whole posttreatment period were analyzed using a one-way ANOVA, with the compound condition as factor.
When significant, the ANOVA was followed by paired comparisons using a Dunnett test versus the vehicle condition.

| Plasma concentrations of BAER-101
Drug concentrations were determined for the purpose of correlating with pharmacodynamic effects.
For the purpose of determining plasma drug concentrations, blood was sampled from each animal.Directly after each EEG recording, blood was collected via tail sampling on each animal from the crossover for analysis and plasma collection.A sample was collected at the end of each EEG from each phase.All animals included in EEG recording were sampled.
Plasma samples were collected from each animal at the end of each recording session (i.e., six recordings in total) about 2 h after compound administration by tail vein.Blood was collected directly into a Sarstedt Minivette point of care testing system.Blood samples were then transferred into a 0.2 mL polymerase chain reaction tube.All tubes were kept on ice for <30 min before centrifugation.The centrifugation was conducted at 3000g, for 10 min, at 4°C.The resultant plasma, at least 25 µL, was collected and transferred into a labeled tube and stored at −80°C.

| GAERS model
Although different doses of BAER-101 were evaluated in two separate phases, results from both phases were combined to provide a better overview of the effects of BAER-101 on SWDs.
As a result of the dose change between phase 1 (3-100 mg/kg) and phase 2 (0.1-3 mg/kg), the number of data points for each condition was not equal (Table 1).

| Drug vehicle
Per os (PO) administration of the vehicle composed of 20% HPbCD in water had no significant effect on the occurrence of SWDs (Figure 3).The number and the duration of SWDs remained unchanged after administration, as compared to baseline.For example, the number of SWDs during the 80 min posttreatment period remained stable at 101.8 ± 7.5% of the baseline value.
T A B L E 1 Number of rats tested for suppression of SWDs by compound administration across the study.

| Diazepam
Diazepam significantly reduced the number of SWDs immediately after administration (Figure 3).From an average frequency of SWDs at 25.6 ± 1.1 per 20 min during baseline, the epileptic activity was reduced to 1.2 ± 1.1 SWDs during the first 20 min period after treatment (10-30 min after administration).This reduction remained stable for the whole posttreatment period, up to 90 min after administration.The average effect on SWDs over the 80 min after treatment was 93.1 ± 6.5% inhibition from baseline.
The average duration of remaining SWDs was not accurately measured, as only two animals were still showing continuous epileptic events after treatment.In the other 10 animals, SWDs were totally inhibited for at least 70 min after administration.These effects of diazepam at 2 mg/kg IP are standard in the GAERS model and match reference data from SynapCell (Duveau et al., 2019).
Diazepam was chosen over other GABAergic antiseizure medications as the positive control since it is a nonselective GABAAR potentiator with which to compare effects of the alpha-selective BAER-101.

| BAER-101
BAER-101 was evaluated at doses ranging from 3 to 100 mg/kg PO during phase 1.After evaluation of results from the first two recordings, a near complete inhibition of SWD was seen with all doses.
Doses were then reassessed and reduced down to doses ranging from 0.1 to 30 mg/kg PO in four additional recordings (Figure 3).
No gross (visually observed) behavioral modifications or adverse effects were reported after the administration of BAER-101 at any dose or time including sedation up to the dose of 100 mg/kg.
When considering the number of SWD per 20 min periods after administration, BAER-101 induced a significant reduction of events at all doses above 1 mg/kg PO, as compared to both the baseline and the vehicle (F 40,305 = 9.56, p < .0001).The effect was immediate, as it was already significant during the first measurement time-point, 10-90 min after administration.The effect remained stable over the posttreatment period, as it was still significant 70-90 min after administration.Thus, T max occurred during the 10-30 min observation period after oral dosing.This maximal effect for each dose was maintained across the full observation period.
When posttreatment data were pooled over 80 min and expressed as a percentage of baseline (Figure 4), the SWD inhibition was significant for all doses at 0.3 mg/kg and above, as compared to the vehicle (F 8,61 = 33.2,p < .0001).The ED 50 was estimated at 0.51 mg/kg (95% confidence interval [CI]: 0.47-0.56).
The average duration of remaining SWDs could only be measured with doses of BAER-101 up to 3 mg/kg.Above that dose, only a few animals have been tested, and almost no SWD remained, preventing accurate measure of SWD duration.
At 1 and 3 mg/kg, BAER-101 induced a significant reduction of the average duration of SWDs, as compared to the vehicle (Figure 5) (F 20,167 = 3.35, p < .0001).For example, SWD lasted on average for 15.9 ± 0.8 s during the 80 min after vehicle administration, and they were reduced to 8.1 ± 0.8 s after treatment with BAER-101 at 3 mg/kg (Figure 6).The effect on SWD duration was less stable than the reduction of the number of SWDs: at the end of recordings, 70-90 min after administrations, the average duration of SWDs in all BAER-101 conditions were returned to values that were not significantly different than in the vehicle condition (Figure 5).
For diazepam, the average duration of SWDs could not be accurately measured, as only two animals were still showing continuous epileptic events after treatment.In the other 10 animals, SWDs were totally inhibited for at least 70 min after administration.p < .01;### p < .001;#### p < .0001as compared to vehicle (n = 4-12 as per Table 1).
Thus, the lack of statistical significance in Figure 5 is due to this variability across animals for this measure.
The total time spent in SWD is a combined parameter, affected by both the number of remaining SWDs and their duration.As both measures were significantly reduced by BAER-101, the total time in SWD was significantly reduced by BAER-101 (F 40,305 = 9.46, p < .0001).BAER-101 significantly reduction the total time as compared to the vehicle at all doses above 1 mg/kg (Figure 7).The reduction was immediately obtained, 10-30 min after administration, with all doses.The reduction was still significant at the end of recordings, 70-90 min after administration.
Pooled data over the 80 min posttreatment period showed a dose-dependent effect, with a significant reduction as compared to vehicle at all doses above 0.3 mg/kg (Figure 8) (F 8,61 = 50.5,p < .0001).At doses above 1 mg/kg, the effect of BAER-101 was not significantly different from the effect of diazepam at 2 mg/kg IP.

| Drug concentrations
Concentrations of BAER-101 in plasma of the rats were consistent across each EEG and blood sampling period and showed dosedependence (Figure 9).F I G U R E 8 Number of spike-wave discharges (SWDs) over the 80 min recording period after administration, expressed as a % of the baseline values, for all conditions.### p < .001;#### p < .0001,as compared to vehicle.
The minimally effective dose (MED) for SWD suppression was 0.3 mg/kg which generated plasma levels in rats of 3.58 ng/ mL = 10.1 nM.

| DISCUSSION
We have demonstrated in the present study that BAER-101 suppresses SWDs in the GAERS model of absence seizures.BAER-101 is the most potent compound yet reported in this model (see Depaulis et al., 2016 andDuveau et al., 2019  Plasma drug exposures were dose-dependent.The MED for SWD suppression was 0.3 mg/kg which generated plasma levels in rats of 3.58 ng/mL = 10.1 nM.Since the K i of BAER-101 at α2 and α3-associated GABAARs of BAER-101 is reported to be about 1 nM (Chen et al., 2014) and the K i at α5 is 230 nM, plasma drug concentrations at the MED were about 10× the K i for α2 and α3 but <23× the K i at α5-containing GABAARs.BAER-101 is reported to be a neutral antagonist at α1-subtype GABAARs (Chen et al., 2014).
In healthy human volunteers, patients with GAD, and Fragile X patients, BAER-101 demonstrated the low sedative effects reported in rats here and elsewhere (Hines et al., 2018;Nomura et al., 2019;Schaefer et al., 2021).
There are other molecules that have been designed to selectively potentiate α2 and α3 while avoiding activation of α1 with the goal of minimizing sedation and dizziness (Cerne et al., 2022;Skolnick, 2012;Witkin et al., 2022).Two such compounds are currently being developed for epilepsy, darigabat and ENV-101.
Darigabat (PF-06372865), an α2/3/5-preferring GABA PAM was reported to partially suppress SWDs in the GAERS model at an MED of 1 mg/kg (Duveau et al., 2019), and was also shown to demonstrate antiseizure effects in humans in a photosensitive model at a single dose of 17.5 mg (Gurrell et al., 2019).However, along with data from other epilepsy models (Nomura et al., 2019;Schaefer et al., 2021), leads us to believe that BAER-101 will have antiseizure effects in patients.Darigabat suppressed SWDs in GAERS rats and also completely suppressed generalized seizures in a photosensitive model at a single dose of 17.5 mg (Gurrell et al., 2019).
The data in photosensitive epileptic patients appears to be predictive of antiseizure properties in a broad population of patients (Yuen & Sims, 2014), suggesting broad antiseizure efficacy of BAER-101 in epileptic patients.
The tolerability and low sedation observed in humans with AZD7325 (Chen et al., 2014), along with the lack of sedation up to a dose of 100 mg/kg in the current study, suggest that BAER-101 will be less sedating at therapeutic doses than currently used GABAAR PAMS.The molecular basis for a nonsedating GABAAR PAM is not known (Cerne et al., 2022;Duke et al., 2018;Skolnick, 2012).BAER-101 (AZD7325) was discovered along with other GABAAR PAMs aiming to minimize sedation with the strategy of lowering efficacy at α1-containing GABAARs (Cerne et al., 2022;Skolnick, 2012) as in the work of Merck and Company-where α1 activity was reduced to zero or near zero levels in their overlapping chemical series (Atack, 2011).
Comparison of the in vitro pharmacology of BAER-101 to that of a few of the Merck compounds is shown in Table 2. BAER is the only compound shown in this table with selectivity over α5.All three compounds produced some dizziness and sedation in humans with MRK-409 exhibiting more than the others (Atack, 2011;Atack et al., 2011;Cerne et al., 2022;Chen et al., 2014).Some of these papers drew the conclusion that α1 was a primary contributor to sedation.However, since direct comparisons of these compounds have not been made in humans at comparable receptor occupancies (e.g., TPA023B was not evaluated to full receptor occupancy -Atack et al., 2011), it is not possible to unambiguously interpret the relationship of these data to sedation.The low oral doses of BAER-101 that fully suppressed SWDs in GAERS rats highlights another feature that could allow for antiseizure efficacy without sedation.
To determine whether BAER-101 incurs less of a risk of tolerance development than with other antiseizure medications like diazepam (Löscher & Schmidt, 2006) will require clinical data.A lack of tolerance over the short term to an α2/3-selective GABAAR potentiator, KRM-II-81, has been reported (Witkin et al., 2018(Witkin et al., , 2020) ) and over a longer period of 22 days in a neuropathic pain model using mice (Biggerstaff et al., 2020).Data from point mutation experiments have also suggested that potentiation of α2-containing GABAARs might be the means of avoiding tolerance (Ralvenius et al., 2015).
Based on prior patient safety and tolerability data, coupled with the high potency and full efficacy in the GAERS model, BAER-101 is considered to be a drug suitable for advancing into human clinical trials for epilepsy.
model allow researchers to determine if their compounds are effective in suppressing these generalized electrographic seizures.The model has additional utility in predicting seizure exacerbating effects of compounds.The latter is important because some antiseizure medications are contraindicated in patients with absence seizures-carbamazepine is one of these antiseizure medications, and also produces increases in F I G U R E 1 Structure of BAER 101.BAER-101 (AZD7325 or AZ12466186) is a nonbenzodiazepine potentiator of α2 and α3-containing γ-amino-butyric acid A receptors with molecular formula C 19 H 19 FN 4 O 2 and molecular weight 354.38.F I G U R E 2 A representative EEG tracing showing two well-defined spike-wave discharges (SWDs) that occur spontaneously in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model during the generalized absence seizure period in these rats.Different SWDs adopt similar patterns but can have variable durations.
At the time of experiments (compound testing), animals were 4-5 months old.The study was performed following the local Standard Operating Procedures of SynapCell.All experiments were conducted at SynapCell's facility.They were approved by the ethical committee of the Grenoble Institute of Neurosciences, University Grenoble Alpes, and performed in accordance with the European Committee Council directive of September 22, 2010 (2010/63/EU).All efforts were made to minimize animal suffering and reduce the number of animals used.
2.7.3 | Bioanalytical data statistics Concentrations were evaluated for each experimental phase and at each dose level.The mean ± SEM concentration of BAER-101 at each dose was determined and assessed for dose-dependence by ANOVA and Dunnett's test using the concentration of BAER-101 at 0.1 mg/kg as the control.The relationship of suppression of SWD EEG signals to plasma concentrations of BAER-101 were made using Pearson's linear regression model.

F
I G U R E 3 Number of spike-wave discharges (SWDs) per 20 min in the vehicle condition (dark blue), diazepam at 2 mg/kg intraperitoneal (IP) (light blue), and BAER-101 at 0.1-100 mg/kg PO (other colors).The gray arrow indicates the time of compound administration (admin.).# p < .05;## The dose of orally administered BAER-101 was proportionally related to the concentrations of BAER-101 achieved in rat plasma (Figure 10, left panel)-(F 6,17 ) = 732.5,p < .0001.The concentration of BAER-101 in plasma was significantly associated with suppression of the number of SWD (Figure 10, right panel).

F
I G U R E 4 Number of spike-wave discharge (SWD) over the 80 min after administration, expressed as % of the baseline values, for all conditions.## p < .01;#### p < .0001,as compared to vehicle.F I G U R E 5 Average duration of remaining spike-wave discharges (SWDs) per 20 min in the vehicle condition (dark blue), diazepam at 2 mg/kg IP (light blue), and BAER-101 at 0.1-1 mg/kg PO (shades of green), and 3 mg/kg BAER-101 represented in yellow.BAER-101 at doses above 3 mg/kg was not considered in the analysis, as very few SWDs durations could be measured.The gray arrow indicates the administration time (admin.).# p < .05;### p < .001;#### p < .0001as compared to vehicle (n = 4-12).FI G U R E 6 Average duration of spike-wave discharges (SWDs) remaining after administration, over the 80 min after administration, expressed as % of the baseline values.Vehicle, diazepam and BAER-101 doses from 0.1 to 3 mg/kg were considered.Doses of BAER-101 above 3 mg/kg were not considered, as very few SWDs could be measured.### p < .001;#### p < .0001,as compared to vehicle.F I G U R E 7 Total time spent in spike-wave discharges (SWDs) per 20 min, in the vehicle condition (dark blue), diazepam at 2 mg/kg IP (light blue), and BAER-101 at 0.1 to 100 mg/kg PO (other colors).The gray arrow indicates the time of administration (admin.).## p < .01;### p < .001;#### p < .0001as compared to vehicle (n = 4-12).
for reference compounds) with an MED of 0.3 mg/kg, PO.The data are the first to show that a GABA PAM that is selective for the α2 and α 3-subtype GABAARs is active in this model.The pharmacology of BAER-101, which lacks activity at the α1-subtype of GABAAR, might be the reason for the low occurrence of dizziness and somnolence observed in both animal and human studies with the compound.That potentiation of α2 and α3-containting GABAARs is sufficient for antiabsence effects in the GAERS model is supported by the drug's on-target engagement with these GABAAR subtypes.
the α5 GABAAR is thought to play a key role in synaptic plasticity, cognition, and memory, suggesting that engagement of α5 risks anticognitive effects.BAER-101 is the first clinical candidate which is selective for only α2, 3 and not for α1 or α5, a pharmacology consistent with antiseizure activity while avoiding adverse side effects common to the GABAA PAM class.Absence epilepsy is not completely controlled by antiseizure drugs and has a high rate of drug resistance(Jiang et al., 2023).Absence seizures have been characterized as being dependent on extrasynaptic GABA.Antiseizure targets were hypothesized to be thalamic extrasynaptic or GABA transport mechanisms arising from data from a series of elegant studies(Cope et al., 2009).However, the present findings along with the data presented byDuveau et al. (2019) indicate that targeting a selective subset of synaptic GABAARs is sufficient to suppress absence seizures.The GAERS model has pharmacological predictive validity(Depaulis et al., 2016) and the efficacy of BAER-101 in generalized seizures of the GAERS model as monitored by EEG in awake rats, F I G U R E 9 Concentrations of BAER-101 across each of the six EEG and blood sampling periods of the study.F I G U R E 10 Concentrations of BAER-101 from rat plasma as a function of dose are shown in the left panel.The relationship of BAER-101 concentration in plasma to suppression of spike-wave discharge (SWD) in the GAERS model is presented on the right.The bars are means ± SD.The points are means.The line in the right panel was fitted by the regression equation shown.
Abbreviations: GABAAR, γ-amino-butyric acid A receptor; PAM, positive allosteric modulator.a Data for BAER-101 (AZD7325) are fromChen et al. (2014) and fromAtack (2011) for the other compounds.pKi are in nM and Pot refers to the relative efficacy of the compounds at each of the alpha subtypes where 0 refers to the absence of relative potentiation of GABA currents.