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Article

KRM-II-81, a β3-Preferring GABAA Receptor Potentiator, Blocks Handling-Induced Seizures in Theiler’s Murine Encephalomyelitis Virus-Infected Mice

by
Dishary Sharmin
1,
Kamal P. Pandey
1,
Lalit K. Golani
1,2,
Sepideh Rezvanian
1,
Md Yeunus Mian
1,
Janet L. Fisher
3,
Arnold Lippa
4,
James M. Cook
1,4,
Daniel P. Radin
4,
Jodi L. Smith
5,
Jeffrey M. Witkin
1,4,5,
Hana Shafique
6 and
Rok Cerne
4,5,7,*
1
Department of Chemistry & Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA
2
Department of Chemistry and Chemical Biology, Neglected Diseases Laboratory Northeastern University, Boston, MA 02115, USA
3
Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, SC 29208, USA
4
RespireRx Pharmaceuticals Inc., Glen Rock, NJ 07452, USA
5
Laboratory of Antiepileptic Drug Discovery, Ascension St. Vincent, Indianapolis, IN 46260, USA
6
School of Medicine, Duke University, Durham, NC 27710, USA
7
Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia
*
Author to whom correspondence should be addressed.
Future Pharmacol. 2025, 5(2), 25; https://doi.org/10.3390/futurepharmacol5020025
Submission received: 14 March 2025 / Revised: 16 May 2025 / Accepted: 23 May 2025 / Published: 30 May 2025
Browse Figures
Versions Notes

Abstract

Background: The GABAA receptor (GABAAR) potentiator, KRM-II-81, is being developed as a novel antiseizure medication with reduced potential for sedation, tolerance development, and abuse liability. Although KRM-II-81 has been shown to provide antiseizure protection against a broad array of seizure induction paradigms, seizures induced by viral vectors have not been previously studied. GABAARs with specific α subunit compositions have been studied in relation to the reduced side-effect liability of KRM-II-81; however, the role of β subunit composition has yet to be determined. Methods: In the present study, KRM-II-81 was studied against handling-induced seizures in Theiler’s murine encephalomyelitis virus (TMEV)-infected mice. Results: An intracerebral infusion of TMEV on day 0 increased the cumulative seizure burden in mice when assessed for handling-induced seizures on days 3–7. KRM-II-81 (15 mg/kg, p.o., bid) nearly completely suppressed seizures in TMEV-infected mice over the course of daily treatments. The number of the most severe seizures (stage 5, tonic/clonic seizures) in the mice was suppressed to zero by KRM-II-81. Although the selectivity of KRM-II-81 for GABAAR α2/3 receptor subtypes might imbue KRM-II-81 with a reduced side-effect liability, other mechanisms are possible, and the potentiation of β1-containing GABAARs has been implicated in inducing sedation. The role of β subunit composition has yet to be determined for KRM-II-81. In electrophysiological studies with cells transfected with αxβ1γ2 or αxβ3γ2, KRM-II-81 preferentially potentiated GABA responses in cells containing β3 subunits in α2/3-containing GABAARs. Conclusions: The present findings confirm the robust antiseizure activity of KRM-II-81, now extended to a virus-induction model, and suggest a possible role of reduced β1-potentiation in the low side-effect profile of KRM-II-81.

1. Introduction

5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole (KRM-II-81) is a GABAA receptor (GABAAR) potentiator with antiseizure, antianxiety, and antinociceptive pharmacological effects that is approaching clinical development [1] The potential therapeutic value gleaned from preclinical studies shows the antiseizure efficacy of KRM-II-81 often exceeds that of standard of care medications (see [1,2] for context and discussion). Although there is an extensive database describing the antiseizure efficacy of KRM-II-81, the compound has not been studied in any models of virus-induced seizures. Furthermore, although molecular pharmacology and molecular modeling have described the rationale for the reduced sedative liability of KRM-II-81, the potential for a role for specific β-configurations of GABAARs has not been explored.
In patients, epilepsy secondary to CNS viral infection is often intractable, and surgery to alleviate focal generators is hampered by the diffuse CNS damage from viral mediators (see [3] for a review). Theiler’s murine encephalomyelitis virus (TMEV)-induced seizures have been described as a method for the development of epilepsy by viral induction [4,5,6,7,8]. Viral infection in this model results in hippocampal sclerosis-associated mesial temporal lobe epilepsy [5]. Inflammatory mechanisms, specifically interleukin-6 production, have been implicated in seizure development [5,9,10]. Thus, this model system enables scrutiny of novel potential treatment agents under these viral-induction methods [4,7,11]. In addition, data from multiple standard antiseizure medications are available for comparative purposes. Valproate and levetiracetam were active, whereas carbamazepine, clonazepam, and NBQX were either not active or exacerbated, rather than protected, against seizures induced by TMEV [4,11,12]. Thus, there is at least some evidence to suggest that the TMEV model represents some degree of pharmacoresistance and provides a novel platform for screening new potential antiseizure medications [4,7,11].
KRM-II-81 has a relatively low liability for producing sedative and motor-impairing effects [1,13]. Some data have suggested that its broad efficacy in epilepsy and pain models, among others, is at least in part due to its relatively mild sedative liability, enabling dosing to efficacious plasma levels [1]. Although its actions on a select population of GABAARs, such as α2/3-containing GABAARs, might underlie its relatively modest sedation [1], there are potentially other molecular substrates that could be responsible, an examination of which is one of the exploratory aims of the present study.
GABAARs are notable for their structural diversity, with specific subunit combinations likely contributing to their functional activity [14]. Our previous studies have demonstrated that KRM-II-81 selectively modulates receptors containing α2 and α3 subunits [15,16]. It has long been hypothesized from abundant data sources that preferential potentiation of α2/3-containing GABAARs will be anxiolytic, antinociceptive, anti-seizure, and importantly, will have these beneficial therapeutic effects at doses that are below those that produce sedation and motor impairment [2,17]. Indeed, a host of GABAAR potentiators, such as KRM-II-81, are less sedating than clinically approved GABAAR potentiators, such as diazepam, that also potentiate other populations of GABAARs (see [1] for overview and discussion). However, that selective potentiation of α2/3-containing (vs. α1) GABAARs is necessary and sufficient for more modest sedative effects remains uncertain (see [1,2,18] for data and discussion).
The alpha subunits comprising GABAARs have been studied mostly for their influence on GABAAR pharmacology [2,17]. That the β subunit composition could be a determinant of reduced levels of sedation was raised by Gee and colleagues. Their work identified compounds that produced little or no potentiation of β1-containing GABAARs, which exhibited anxiolytic-like [19,20] and antinociceptive [21] effects in rodent models while remaining non-sedating. They suggested from their findings that the potentiation of β1-associated GABAARs is linked to sedation. In the present report, we describe the results of electrophysiological studies to determine the functional selectivity of KRM-II-81 in αxβ1γ2- vs. αxβ3γ2-configured GABAARs. The finding that KRM-II-81 selectively potentiated β3- over β1-containing GABAARs suggests the possibility that the reduced activation of αxβ1γ2 receptors contributes to the lower sedation liability of KRM-II-81 compared to non-selective potentiators.
The results of experiments reported here have extended the diversity of epilepsy models for which KRM-II-81 is active and connect these findings to its predicted efficacy in temporal lobe seizures. The present findings also suggest another potential molecular substrate underlying the novel pharmacological properties of KRM-II-81 in reduced potentiation of β1-containing GABAARs.

2. Materials and Methods

Compounds. KRM-II-81 was synthesized by us as described [15,22]. Other compounds were obtained from Sigma-Aldrich (St. Louis, MO, USA).
KRM-II-81 was finely suspended in 0.5% methylcellulose, which was used as the vehicle in the vehicle-treatment group in these experiments. Compounds were administered to the mice in a volume of 10 mL/kg.

2.1. TMEV Model

These experiments were expertly conducted at the NIH Epilepsy Therapy Screening Program contract site within the Department of Pharmacology and Toxicology at the University of Utah, Salt Lake City (see Acknowledgments).
Animals. Male C57BL/6J mice (5–6 weeks) obtained from The Jackson Laboratories (Bar Harbor, ME, USA) were used. The mice had free access to food (TEKLAD GLOBAL 2920X-011620M) and water. Housing and experiments were conducted under guidelines from the National Research Council and the Institutional Animal Care and Use Committee at the University of Utah.
TMEV model. Mice were briefly anesthetized with isoflurane (1.5–3% isoflurane). Forty mice began the study. They were given 20 μL of the Daniels strain of TMEV (3 × 105) plaque-forming units intra-cortically to a depth of 2 mm in the temporal region of the right hemisphere (posterior and medial to the right eye). The viral titer was from the same batch for the experiment. No sham injections were utilized as titer-induced seizures have been previously documented by this method [4,7,11], and in the current study, all mice were treated with TMEV. The TMEV-treated mice were then compared based upon dosing group—those receiving KRM-II-81 were compared vs. those receiving vehicle treatments. The inoculation occurred on a Friday (day 0), so that day 3–7 monitoring was conducted on Monday–Friday of the following week.
Mice were divided into two groups: one group received KRM-II-81 (15 mg/kg, PO, bid), and the other group received the drug vehicle (PO, bid) and served as the control group. The 15 mg/kg dose was selected based upon prior data on the potency of KRM-II-81 in other seizure models and tolerability under repeat dosing [1]. The number of mice/group was based upon prior studies with the TMEV model that established the ability to detect seizure suppression [4,7,11].
Beginning on the day of viral inoculation (day 0), either vehicle (control group) or KRM-II-81 was administered (experimental group) by oral gavage, bid (~0900 and 1500 h). Vehicle or KRM-II-81 was given each subsequent day for the next 7 days. If a mouse had body weight loss of 20% or more, it was eliminated from the study.
Handling for seizure induction was required to pick up the mouse and dose it. Handlers were highly trained and utilized, as best as possible, in handling and dosing methods that were consistent from occasion to occasion. Seizures were monitored immediately post gavage (bid) as well as 2 h post gavage on days 3–7 by the methods described [4]. Trained observers were blinded to groups. The 2 h time period for observation was chosen based upon prior pharmacokinetic and anti-seizure data of KRM-II-81 [1,23,24].
Data collection and analysis. The following data were collected: the presence or absence of seizures and the severity of each seizure measured by a modified Racine scoring (scores 1–5) [25]. Body weights were also measured daily.
The cumulative seizure burden was calculated using the post-dosing observations designated AM and PM across all observation days 3 to 7. Seizure burden is the total seizure score for each mouse during AM and PM (e.g., 0 and 5 = 5 for that day). Thus, seizure burden utilized all seizure scores. These values for each mouse are cumulated over days. These data provide a key analysis combining both frequency and severity of seizures [4]. The mean ± SEM cumulative score of the mice over these experimental days was evaluated by ANOVA to compare the vehicle-treated group with the KRM-II-81-treated group. Post-hoc comparisons of results from all days were evaluated by Šidák’s multiple comparison test to control for multiple statistical testing under one alpha level with greater power than the Bonferroni correction.
A mean seizure severity score was obtained by averaging all scores, including zeros for each treatment period and condition separately. Vehicle vs. KRM-II-81 values were assessed by ANOVA. These values relative to the number of observations were compared, vehicle to KRM-II-81, by Fisher’s Exact Probability test, a conservative statistical method for assessing differences in quantal data.
The total number of seizures, as well as the number of stage 5 or stage 4 seizures, were also counted for each experimental day, 3 to 7, post inoculation. These seizure stages are the most severe and their potential reduction by KRM-II-81 was used to provide an index of the efficacy of the compound against severe seizure endpoints. These values relative to the number of observations were compared, vehicle to KRM-II-81, by Fisher’s Exact Probability test.
Body weights of mice for both the vehicle and KRM-II-81 groups were averaged for each observation day (3 to 7) and compared by ANOVA. All statistical comparisons were considered significant by the a priori setting of p < 0.05 by two-tailed tests (GraphPad Prizm, ver. 9.4.1., San Diego, CA, USA).

2.2. Subunit Composition

Experiments to define the selectivity of KRM-II-81 for specific GABAAR subunit composition were conducted in collaboration with the Department of Pharmacology, Physiology & Neuroscience in the School of Medicine at the University of South Carolina in Columbia, SC, USA.
Transfected mammalian cells. Full-length cDNAs encoding mammalian GABAARs in pCMVNeo expression vectors were transfected (2 mg of each subunit) into the human embryonic kidney cell line HEK-293T (GenHunter, Nashville, TN, USA) using calcium phosphate precipitation. The clones for the GABAAR subunits were generously provided by Dr. Robert Macdonald (Vanderbilt University, Nashville TN, USA) and Dr. David Weiss (University of Texas Health Sci. Center, San Antonio, TX, USA). 1 µg of the plasmid pHookTM-1 (Invitrogen, Carlsbad, CA, USA) containing cDNA encoding the surface antibody sFv was also transfected into the cells for selection of the transfected cells. After incubation for 4–6 h at 3% CO2, the cells were treated with a glycerol solution (15%) in BBS buffer (50 mM BES(N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), 280 mM NaCl, 1.5 mM Na2HPO4) for 30 s. Cells were maintained in a media of Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 mg/mL streptomycin. After 44–52 h, the selection procedure for pHook expression was performed. The cells were passaged by a 5 min incubation using a 0.05% trypsin/0.02% EDTA solution in phosphate buffered saline (10 mM Na2HPO4, 150 mM NaCl, @ pH 7.3) that was mixed with magnetic beads coated with antigen (3–5 µL) for the pHook antibody (~6 × 105 beads). Beads and bead-coated cells were isolated using a magnetic stand after a 30–60 min incubation that permitted the beads to bind to positively transfected cells. The selected cells were then resuspended in DMEM and then plated onto glass coverslips, treated with poly L-lysine and coated with collagen. These were used for electrical recordings 18–28 h later.
Recording of GABA currents. Whole-cell GABA currents were recorded in voltage-clamp mode with an Axon 200B patch clamp amplifier (Molecular Devices, San Jose, CA, USA). The external solutions used for electrical recordings were as follows in mM: 142 NaCl, 8.1 KCl, 6 MgCl2, 1 CaCl2, and 10 HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) with pH = 7.4 and osmolarity adjusted to 295–305 mOsm. The internal solutions of recording electrodes used were as follows: 153 KCl, 1 MgCl2, 5 K-EGTA (ethylene glycol-bis (β-aminoethyl ether N,N,N′N′-tetraacetate)), 2 MgATP and 10 HEPES with pH = 7.4 and osmolarity adjusted to 295–305 mOsm. GABA was diluted into an external solution from freshly made or frozen stocks in water. KRM-II-81 was diluted from stocks in DMSO (final DMSO concentration < 0.01%). Patch pipettes were pulled from borosilicate glass with an internal filament (World Precision Instruments, Sarasota, FL, USA) on a two-stage puller (Narishige Interntional, Amityville, NY, USA) to a resistance of 5–10 MΩ. For whole-cell recordings, the application of GABA was performed with a stepper solution exchanger with a complete exchange time of <50 ms (open tip, SF-77B, Warner Instruments, Hamden, CT, USA).
Data analysis. Whole-cell currents were digitized at 10 kHz and analyzed with the pClamp suite of programs (MDC, Sunnyvale, CA, USA). Data are reported as mean ± SEM for a minimum of four replicates. Two-way ANOVA was followed by Bonferroni multiple comparisons tests with an a priori p level of 0.05.

3. Results

3.1. TMEV Model

Viral injection on day 0 induced an escalating increase in handling-induced seizure burden as assessed on experimental days 3 through 7. Figure 1 shows the mean cumulative seizure burden of mice as observed in the post-dosing periods. At 15 mg/kg (p.o., bid), KRM-II-81 significantly decreased seizure burden over days 3 to 7 (F4,185 = 2.92, p < 0.05). KRM-II-81 reduced the seizure burden compared to mice treated with drug vehicle (F1,185 = 47.9, p < 0.0001) where there was also a significant interaction of experimental day × treatment (F4,185 = 2.81, p < 0.05).
The body weights of the mice in both groups were comparable over days 3 to 7 post-viral inoculation. Day 3 weights ± SEM were 18.1 ± 0.47 g and 18.1 ± 0.57 g for the vehicle and KRM-II-81 treatment groups, respectively. On day 7, the mean body weights of the mice were 17.9 ± 0.62 g and 17.9 ± 0.49 g for the vehicle and KRM-II-81-treated mice, respectively. Comparing day 3 to day 7 body weights separately for the vehicle and KRM-II-81 groups did not reveal statistical differences (t38 = 0.26, p = 0.80 and t36 = 0.26, p = 0.79, for Vehicle and KRM-II-81 groups, respectively). The behaviors of the mice were visually observed during all observation periods, and no unusual behavioral changes were reported.
Statistical comparisons of effects of seizure severity relative to their respective vehicle control values are shown in Table 1 for the 2 h observation periods after dosing each day bid. KRM-II-81 significantly suppressed seizure severity.
The total number of seizures for vehicle- and KRM-II-81-treated groups is shown in Table 2 over successive days post-viral inoculation at 2 h post dosing for the two dosing periods per day. KRM-II-81 produced large and statistically significant decreases in the total number of seizures compared to the vehicle-treated mice on days 3, 4, 5, and 6 post-viral inoculation. The number of seizures on day 7 was low in comparison to the other days in both the vehicle and drug-treatment groups, and statistically significant differences were not observed.
Stage 5 seizures were evaluated separately to determine whether KRM-II-81 decreased these most serious tonic/clonic convulsions (Table 3). As with total seizures, Stage 5 seizures decreased in the presence of oral KRM-II-81 (to zero for Stage 5 seizures), an effect statistically differentiated from vehicle-treated mice on days 3, 4, 5, and 6 post-viral inoculation. Stage 4 seizures also showed a reduction in the mice treated with KRM-II-81, again demonstrating the efficacy of KRM-II-81 against these most severe seizure endpoints. Vehicle-treated mice exhibited 10 Stage 4 seizures, whereas KRM-II-81-treated mice only had one.

3.2. Subunit Composition

The effects of KRM-II-81 strongly depended on the subunit composition of recombinant receptors. The four benzodiazepine-sensitive α subunits (α1,2,3,5) were co-expressed with either β1/γ2L, or with β3/γ2L subunits in HEK cells, and 1 µM KRM-II-81 was co-applied with an EC3–5 concentration of GABA established separately for each isoform. In agreement with previously reported studies, KRM potentiated GABA current responses at all recombinant receptors (Figure 2). The potentiation significantly depended on the type of alpha subunit (F = 6.5, p < 0.01), with a larger potentiation of α2 and α3 subunit-containing GABAARs than α1 subunit-containing GABAARs. In addition to dependence on the alpha subunit, the effect of KRM-II-81 depended on the expressed beta subunit (F = 6.1, p < 0.05), with more robust potentiation of GABA currents by KRM-II-81 in β3- than of β1-containing GABAARs.
The potentiation of GABA currents by KRM-II-81 was most pronounced with receptors containing a combination of β3/α2 and β3/α3 subunits (Figure 2). In contrast, the statistical interaction term for alpha subunit (1, 2, 3, and 5) × beta subunit (1 and 3) lacked statistical significance (F = 2.0, p = 1.0). When the GABAARs expressed α3 subunits, the co-expression of β3 subunit led to significantly larger potentiation of GABA currents by KRM-II-81 than with co-expression of β1 (p < 0.05). KRM-II-81 is only active in the presence of GABA when tested in concentrations up to 300 μM.

4. Discussion

The results of the present study demonstrated that the GABAAR potentiator KRM-II-81 is active against handling-induced seizures in the TMEV mouse model, a model that represents some degree of pharmaco resistance and provides a novel platform for screening new potential antiseizure medications [4,7,11]. The suppression, in some cases complete, of seizures in this model by KRM-II-81 documents the high efficacy of this compound as an antiseizure agent. The ability of KRM-II-81 to block the most severe tonic/clonic seizures induced by TMEV infection attests to the robust antiseizure activity of this compound. This is the first study of viral-induced seizures with KRM-II-81. Further work will be required to establish whether KRM-II-81 can also prevent other impairments in biological function that are induced by viral infection, including behavioral alterations. Barker-Haliski and colleagues reported that minocycline was able to improve behavioral deficits induced by viral exposure, whereas, in their study, valproate did not [26].
When administering KRM-II-81 orally at 15 mg/kg, bid, in virus-infected mice, there were only two adverse events: one mouse died and another exhibited weight loss of greater than 20%. Despite the loss of weight by one mouse, the overall group showed no significant weight changes with KRM-II-81 treatment. This observation stands in contrast to other medications shown to dampen viral-induced seizures in this model—minocycline was effective in reducing seizures in TMEV mice, but did so while producing marked loss of body weight [26]. In contrast, valproate decreased seizure burden without decreasing the body weights of the mice. Doses of KRM-II-81 higher than 15 mg/kg are well tolerated in other seizure models [1]. Since 15 mg/kg markedly suppressed seizures in TMEV-infected mice in the present study, the minimum effective dose of KRM-II-81 is ≤15 mg/kg, and the exact value could be determined in future studies.
Subchronic dosing with KRM-II-81 has not shown tolerance to its anticonvulsant effects over 5 days [1,22] or to its antinociceptive effects over 22 days of dosing [27]. In the present study, KRM-II-81 was dosed for 8 days, bid, starting on day 0, the day of viral infection. The total number of seizures, total Stage 5 seizures, seizure burden, and seizure severity were all suppressed across the observation periods, showing another example of the lack of tolerance development with this compound. Direct comparisons with other anticonvulsants will be needed to translate these findings across available medications. Nonetheless, it is well known that tolerance development is a major impediment to antiseizure efficacy, and that KRM-II-81 has not exhibited tolerance in preclinical studies [1]; however, clinical testing of this compound has not yet commenced.
Seizures arising from viral infections in humans are often pharmacoresistant, and guidance on effective antiseizure medications has not been established [3,28]. Table 4 summarizes the data on anti-seizure protection in the TMEV model. While valproate was active in reducing seizure severity, another Na+ channel blocker, carbamazepine, increased, rather than decreased, seizure frequency [4]. The AMPA receptor antagonist, NBQX, is a potent anticonvulsant across multiple animal models, and perpampanel is an AMPA receptor antagonist used as an antiseizure agent in patients (Fycompa®). Despite the broad anticonvulsant activity of NBQX, it increased rather than suppressed TMEV-induced seizures [12]. The recent identification of cannabidiol [29] and soticlestat as active compounds in this model (Table 4) is encouraging for patients.
The chronic epilepsy engendered by TMEV can also be generated by application of kainate into the hippocampus or amygdala. As with the TMEV model, the kainate models of mesial temporal lobe epilepsy (mTLE) exhibit chronic epilepsy that is characteristic of the temporal lobe epilepsies seen in patients [30,31]. Other in vivo models of mTLE have also been developed, including those that use oral kainate [1] or pilocarpine [31,32]. An intra-hippocampal kainate model has also been described using rats [33] and Rhesus monkeys [34]. In these kainate models, few compounds suppress both seizure frequency or seizure burden as well as seizure severity. KRM-II-81 is effective against both aspects of kainate-induced seizures in rats as well as in the intra-hippocampal mTLE seizures model in mice at a single dose of 15 mg/kg, p.o. [1]. Further, some compounds only suppress mTLE seizures at doses that produce untoward effects (e.g., valproate, carbamazepine, lamotrigine [35]. KRM-II-81 in both a mouse and a rat model showed suppression of seizures in the absence of untoward effects [1]. As with the rodent mTLE modeling, patients with mTLE are often pharmacoresistant to standard anti-seizure medications [36]. mTLE patients often have depression and anxiety as comorbid symptoms, which are associated with more difficult seizure control issues [37].
Table 4. Effects of standard and novel anti-seizure compounds in the TMEV mouse model studied.
Table 4. Effects of standard and novel anti-seizure compounds in the TMEV mouse model studied.
CompoundMechanism(s)TMEV Model
KRM-II-81GABAA α2/3Active (Present manuscript)
DarigabatGABAA α2/3------------
DiazepamGABAA------------
PhenobarbitalGABAAActive [7]
TiagabineGABA------------
VigabatrinGABA------------
ClonazepamGABANot Active [7]
PhenytoinNa+ ChannelsActive [7]
LamotrigineNa+ ChannelsNot Active [7]
ValproateNa+ ChannelsActive [4,7]
TopiramateNa+ Channels------------
CarbamazepineNa+ ChannelsIncrease [4] a
GabapentinCa2+ Channels------------
EthosuxamideCa2+ ChannelsNot Active [7]
LevetiracetamSV2AActive [7,11]
Increase [38]
NBQXAMPAIncrease [12] a
PerampanelAMPA------------
CannabidiolCannabinoidActive [29]
CeftriaxoneCephalosporin antibioticNot Active [39]
VU0360172mGlu5 receptor potentiatorActive [40]
CCPAAdenosine 1 receptor agonist------------
MinocyclineTetracycline antibioticActive [41]
Not Active [7]
Soticlestat (TAK-935)Cholesterol 24-hydroxylase inhibitorActive [42]
DexamethasoneCorticosteroidActive [7]
PrednisoneCorticosteroidActive [7]
IbuprofenNSAIDNot Active [7]
DiclofenacNSAIDNot Active [7]
CelocoxibCox-2 inhibitorActive [7]
a Exacerbation rather than protection. CCPA: 2-chloro-N6-cyclopentyladenosine; NSAID: non-steroidal anti-inflammatory drug. Active indicates that the compound decreased seizures. Not active indicates that the compound did not decrease seizures. Increase indicates that the compound exacerbated seizures. Not reported indicates that the specific measure was not reported in the paper. --------- indicates there are no literature data.
The literature describing the mechanisms associated with the generally low sedative liability of potentiators of α2/3-containing GABAARs typically have focused discussion on their lack of α1-potentiating properties [2,17,18]. However, the necessary and sufficient molecular pharmacology of non-sedating GABAAR potentiators has not been solved (e.g., compounds with prominent potentiation of α1-containing GABAARs have been identified that also have non-sedating profiles in humans, such as ocinaplon) [1,2,18]. Gee and colleagues suggested that specific sets of compounds can produce anxiolytic-like effects in rodents without sedation by virtue of their low propensity for potentiating GABAARs containing β1 subunits [19]. This work was confirmed by Yoshimura et al. [20] and expanded into the pain domain as non-sedating analgesics [21]. The relevance of the β2/3-prefering potentiators for epilepsy has also been discussed. The suppression of the rate of hippocampal kindling in mice has been reported with such compounds [43]. Furthermore, the compounds with low β-1 potentiation did not demonstrate memory impairment or tolerance development, as seen in preclinical studies with KRM-II-81 [1]. The findings, in the present study, showing that KRM-II-81 has a reduced propensity for activating these β1-assocatiated GABAARs that might be related to sedation is a novel and potentially telling addition to the molecular contributions to sedation and possibly other known side-effects of non-selective GABAAR potentiators. Nonetheless, whether the reduced potentiation of β1-containing GABAARs is relevant to this action will require further scrutiny. The β2/3-preferening compounds described by Gee et al. [19] and Yoshimura et al. [20] are extrasynaptically acting compounds and are not α-subtype preferring. In contrast, KRM-II-81 is a synaptic potentiator with α2/3-selectivity. The definitive contribution of these pharmacological differences to the benign side-effect profile of these compounds will require further experimental scrutiny.

5. Conclusions

KRM-II-81 blocks temporal lobe seizures induced by viral infection in mice, and the literature data show that it is one of the few compounds that suppresses mesial temporal lobe seizure burden and severity in kainate-induction models. KRM-II-81 preferentially potentiates β3- vs. β1-containing GABAA receptors in addition to being a selective potentiator of α2/3-containing GABAA receptors. The later findings suggest additional molecular pharmacology underlying the non-sedating- and non-tolerance-producing preclinical profile of this novel antiseizure compound.

Author Contributions

Conceptualization, J.M.W., D.P.R., H.S., R.C., J.M.C., D.S. and J.L.F.; methodology, J.M.W. and R.C.; validation, J.M.W., H.S., J.L.F. and R.C.; formal analysis, J.L.F., J.M.W. and R.C.; investigation, J.L.F.; resources, D.S., K.P.P., L.K.G., S.R., M.Y.M., J.L.F., A.L., J.L.S., J.M.W. and J.M.C.; data curation, J.L.F., H.S., R.C. and J.M.W.; writing—original draft preparation, all authors.; writing—review and editing, all authors; visualization, J.M.W.; supervision, J.M.C. and J.L.F.; project administration, J.M.W. and J.M.C.; funding acquisition, J.M.W., J.M.C. and A.L. All authors have read and agreed to the published version of the manuscript.

Funding

We thank the following granting agencies for support: DA-043204, DA054177, AA029023, and NS-076517 and the National Science Foundation, Division of Chemistry [Grant CHE-1625735]. We also acknowledge the UW-Milwaukee Shimadzu Laboratory for Advanced and Applied Analytical Chemistry and support from the Milwaukee Institute of Drug Discovery and the University of Wisconsin-Milwaukee Research Foundation.

Institutional Review Board Statement

All experiments conformed to the standards of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Utah’s Institutional Animal Care and Use Committee (IACUC), 10 September 2001. NIH Contract No: HHSN271201600048C, Salt Lake City; PI: Karen S. Wilcox.

Informed Consent Statement

No human data are presented.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

We are grateful for the generous support of the Lucas family and from the Henry and Nelly Pence Foundation Trust for their support of this research. Their dedication to the relief of suffering in epileptic patients has enabled advances in new antiseizure medications. We are indebted to the NIH Epilepsy Therapy Screening Program in Bethesda Maryland for their acceptance of KRM-II-81 into their program. In particular, we thank the thoughtful dedication, guidance, and oversight of Shalini Sharma on this collaborative project. This work would also not have been possible without the excellence of the screening site for this program in the Department of Pharmacology and Toxicology at the University of Utah, Salt Lake City. The direction of this group by Karen S. Wilcox and its management by Cameron S. Metcalf have enabled this and other novel screening models for the advancement of anticonvulsant drug candidates.

Conflicts of Interest

A.L. is the CEO of RespireRx Pharmaceuticals; J.M.W., J.M.C., D.P.R., and R.C. are unpaid employees of RespireRx Pharmaceuticals. RespireRx Pharmaceuticals has a license agreement with the University of Wisconsin-Milwaukee to develop KRM-II-81. J.M.C. is a patent holder for the invention of KRM-II-81.

References

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Futurepharmacol 05 00025 g001
Figure 1. Effects of KRM-II-81 (p.o., bid) on the cumulative handling-induced seizure burden measured in the AM and PM post-dosing periods in mice inoculated with TMEV on day 0. Each data point represents the cumulative mean ± SEM for N = 20 (vehicle) or N = 18–20 (KRM-II-81) mice due to attrition in the initial N = 20 due to criteria weight loss (one mouse) or death (one mouse) (see Results). The seizure values were obtained from the post-dosing observation periods in the AM and PM. * p < 0.05 compared to day 3 values; ** p < 0.01; *** p < 0.0001 for KRM-II-81 treatment compared to respective data from vehicle-treated mice on the same day by ANOVA followed by Šidák’s multiple comparison test.
Figure 1. Effects of KRM-II-81 (p.o., bid) on the cumulative handling-induced seizure burden measured in the AM and PM post-dosing periods in mice inoculated with TMEV on day 0. Each data point represents the cumulative mean ± SEM for N = 20 (vehicle) or N = 18–20 (KRM-II-81) mice due to attrition in the initial N = 20 due to criteria weight loss (one mouse) or death (one mouse) (see Results). The seizure values were obtained from the post-dosing observation periods in the AM and PM. * p < 0.05 compared to day 3 values; ** p < 0.01; *** p < 0.0001 for KRM-II-81 treatment compared to respective data from vehicle-treated mice on the same day by ANOVA followed by Šidák’s multiple comparison test.
Futurepharmacol 05 00025 g001
Futurepharmacol 05 00025 g002
Figure 2. Effect of the GABAAR subunit subtype on modulation by KRM-II-81. GABA (EC3–5) was co-applied for 5 s with 1 µM KRM-II-81. The effect of GABA alone for each alpha subtype did not exceed EC5 levels, and potentiation by KRM-II-81 is expressed relative to the effects of GABA alone (100%). Recombinant receptors were expressed with different α subunits as indicated (X-axis) and either β1 (white) or β3 (gray) subunits. All recombinant receptors contained the γ2L subunit. Each bar represents the mean ± SEM of 3–6 replicates. The dashed line denotes the response to GABA alone (100%). * p < 0.05 by Bonferroni multiple-comparisons test.
Figure 2. Effect of the GABAAR subunit subtype on modulation by KRM-II-81. GABA (EC3–5) was co-applied for 5 s with 1 µM KRM-II-81. The effect of GABA alone for each alpha subtype did not exceed EC5 levels, and potentiation by KRM-II-81 is expressed relative to the effects of GABA alone (100%). Recombinant receptors were expressed with different α subunits as indicated (X-axis) and either β1 (white) or β3 (gray) subunits. All recombinant receptors contained the γ2L subunit. Each bar represents the mean ± SEM of 3–6 replicates. The dashed line denotes the response to GABA alone (100%). * p < 0.05 by Bonferroni multiple-comparisons test.
Futurepharmacol 05 00025 g002
Table 1. Mean seizure severity during injection periods and in the AM or PM 2 h after injection observation sessions.
Table 1. Mean seizure severity during injection periods and in the AM or PM 2 h after injection observation sessions.
VehicleKRM-II-81ANOVA
1.39 ± 0.390.03 ± 0.01F1,369 = 114, p < 0.0001
Values shown are means ± SEM for observations of all mice on days 3–7 post-viral inoculation. ANOVA values are for KRM-II-81 data compared to its respective vehicle control.
Table 2. Total number of seizures observed in vehicle-treated and in KRM-II-81-treated groups over successive days post-viral inoculation (dpi).
Table 2. Total number of seizures observed in vehicle-treated and in KRM-II-81-treated groups over successive days post-viral inoculation (dpi).
Treatment3 dpi4 dpi5 dpi6 dpi7 dpi
Vehicle13/4015/4015/4015/403/40
KRM-II-811/40 ***0/39 ***0/36 ***0/36 ***3/36
Values are the number of mice exhibiting a convulsion/number of seizure observation periods (number of mice × 2 observation periods). The reduction in the number of seizure observation periods from 40 was due to one death and one mouse euthanization, as noted in Table 2. *** p < 0.001 by Fisher’s Exact Probability test.
Table 3. Total number of Stage 5 seizures observed in vehicle-treated and in KRM-II-81-treated groups over successive days post-viral inoculation (dpi).
Table 3. Total number of Stage 5 seizures observed in vehicle-treated and in KRM-II-81-treated groups over successive days post-viral inoculation (dpi).
Treatment3 dpi4 dpi5 dpi6 dpi7 dpi
Vehicle3/409/4015/4014/402/40
KRM-II-810/400/39 **0/36 ***0/36 ***0/36
Values are the number of mice exhibiting a convulsion/number of seizure observation periods (number of mice × 2 observation periods per day). The reduction in the number of seizure observation periods from 40 was due to one death and one mouse euthanization as noted in Table 2. ** p < 0.01; *** p < 0.001 by Fisher’s Exact Probability test.
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Sharmin, D.; Pandey, K.P.; Golani, L.K.; Rezvanian, S.; Mian, M.Y.; Fisher, J.L.; Lippa, A.; Cook, J.M.; Radin, D.P.; Smith, J.L.; et al. KRM-II-81, a β3-Preferring GABAA Receptor Potentiator, Blocks Handling-Induced Seizures in Theiler’s Murine Encephalomyelitis Virus-Infected Mice. Future Pharmacol. 2025, 5, 25. https://doi.org/10.3390/futurepharmacol5020025

AMA Style

Sharmin D, Pandey KP, Golani LK, Rezvanian S, Mian MY, Fisher JL, Lippa A, Cook JM, Radin DP, Smith JL, et al. KRM-II-81, a β3-Preferring GABAA Receptor Potentiator, Blocks Handling-Induced Seizures in Theiler’s Murine Encephalomyelitis Virus-Infected Mice. Future Pharmacology. 2025; 5(2):25. https://doi.org/10.3390/futurepharmacol5020025

Chicago/Turabian Style

Sharmin, Dishary, Kamal P. Pandey, Lalit K. Golani, Sepideh Rezvanian, Md Yeunus Mian, Janet L. Fisher, Arnold Lippa, James M. Cook, Daniel P. Radin, Jodi L. Smith, and et al. 2025. "KRM-II-81, a β3-Preferring GABAA Receptor Potentiator, Blocks Handling-Induced Seizures in Theiler’s Murine Encephalomyelitis Virus-Infected Mice" Future Pharmacology 5, no. 2: 25. https://doi.org/10.3390/futurepharmacol5020025

APA Style

Sharmin, D., Pandey, K. P., Golani, L. K., Rezvanian, S., Mian, M. Y., Fisher, J. L., Lippa, A., Cook, J. M., Radin, D. P., Smith, J. L., Witkin, J. M., Shafique, H., & Cerne, R. (2025). KRM-II-81, a β3-Preferring GABAA Receptor Potentiator, Blocks Handling-Induced Seizures in Theiler’s Murine Encephalomyelitis Virus-Infected Mice. Future Pharmacology, 5(2), 25. https://doi.org/10.3390/futurepharmacol5020025

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