Stereospecific antiseizure activity in mouse and rat epilepsy models by a pyridinium inhibitor of TNFα/NFκB signaling

Abstract

Epilepsy affects over 50 million people worldwide and increases the risk of death. An intrinsic state of central inflammation, mainly driven by TNFα/NFκB signaling, may contribute to the refractory nature of some epilepsies. We have therefore hypothesized that inhibitors of this signaling pathway might be therapeutic. To test this hypothesis, we have measured the antiseizure properties of the enantiomeric compounds MRS-2481 and MRS-2485 in rodent seizure model systems. In the 6 Hz (44 mA) induced seizure test in mice, the (S) species, MRS-2485, was found to have higher protective potency and lower toxicity than the (R) species MRS-2481. However, neither of these enantiomers were protective in the MES-induced seizure test. MRS-2485 was also found to be protective in the corneal kindled mouse test. Finally, MRS-2485 reduced the post-kainate rat hippocampal slice electrical burst rate and duration. We conclude that MRS-2485, the (S)-enantiomer, is a potent inhibitor of seizure activity in mouse and rat models of epilepsy.

1. Introduction

Epilepsy affects over 50 million people worldwide and increases the risk of death [1]. The problem is that although there are at least 20 approved drugs for epilepsy, 20–30% of cases remain refractory to treatment [2]. However, it has recently been proposed that an intrinsic state of central inflammation may contribute to the refractory nature of some epilepsies Some intractable epilepsies may either be caused by, be enhanced by, or be accompanied by chemokines such as IL-8 which are found at epileptic foci within the brain [3–6]. IL-8 is a product of TNFα/NFκB signaling. Other cytokines and chemokines regulated by this pathway have been found to be elevated in some epilepsy patients, including the T-cell chemokines TARC and ICAM5, which we recently reported to be plasma biomarkers for temporal lobe epilepsy [7]. Other proinflammatory signaling pathways, including COX2 and the inflammasome, have been intensively studied as possible targets for candidate antiseizure medications [8,9]. Our approach here has therefore been to focus exclusive attention on a medicinal chemistry approach to inhibitors of TNFα/NFκB signaling, and to test candidate leads for anti-epilepsy activity using the NIH’s ASP/ETSP program.

Using IL-8 as a biomarker for this pathway, we identified the amphiphilic pyridinium salts as one such class of small molecules [10]. Medicinal chemistry to optimize anti-inflammatory properties allowed us to further identify a highly active set of enantiomers MRS-2481 and MRS-2485 [10]. These compounds were identical except at the optically active alpha carbon (see Fig. 1 and Supplemental Figs. 1–3). Importantly, we showed that MRS-2481 is more potent than MRS-2485 as an inhibitor of TNFα/NFκB signaling [10]. However, we were unable to identify the specific site of action. Furthermore, it has been reported that MRS-2481 and MRS-2485 were equally potent inhibitors of calcium conductance and neuronal cell death induced by beta-amyloid, a peptide associated with Alzheimer’s disease [11]. Thus, the stereoselectivity observed in inhibition of TNFα/NFκB signaling was not discernible when these amphiphilic pyridinium compounds were tested as beta-amyloid channel blockers. Nonetheless, in the general case where there is stereoselectivity of one enantiomer over the opposite antipode, as in the case of MRS-2481 for TNFα/NFκB signaling, it has frequently been found that specific interactions with a target protein or other biopolymer are responsible for the differential activity [12].

Fig. 1. Structures and optical properties of MRS-2481 and MRS-2485.

(a) MRS-2481. (b) MRS-2485.

Based on consideration of their inhibitory properties on TNFα/NFκB signaling, we initially hypothesized that these enantiomeric compounds might also have stereospecific antiseizure properties. Based on preliminary data, MRS-2485 rather than MRS-2481 was found to have the most seizure protection and the least toxicity when compared in mice with the 6 Hz seizure test. By contrast there was no protection in the MES-induced seizure test. We therefore asked whether MRS-2485 could be quantitated for antiseizure potency and toxicity (1) in a more complete mouse 6 Hz test; (2) in the corneal kindled mouse test; and (3) by reduction of post-kainate hippocampal slice electrical burst rate and duration in rats. We found positive responses in all three tests and concluded that MRS-2485 could now be considered for further testing as a candidate antiseizure medication.

2. Materials and methods

2.1. Animals and test substances

The animal testing described here were all performed by the Epilepsy Therapy Screening Program (ETSP), National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), Bethesda, MD, 20892 (ETSP)/NINDS/NIH [13]. The ETSP testing sites at the University of Utah and University of Washington are blinded to the source and structure of all participant compounds that are submitted for testing. Male albino CF-I were obtained from Charles River (18–30 g; Charles River, Kingston, NY. Five-to six-week-old male C57BL/6 mice (15–20 g) were obtained from Charles River, Raleigh, NC, U.S.A.). Rats were obtained from the same source. All animals were allowed free access to food (ENVIGO 2920X for all Charles River-derived animals) and water except when they were removed from their cages for the experimental procedure. All animals were housed, fed, and handled in a manner consistent with the recommendations in the National Research Council publication, “Guide for the Care and Use of Laboratory Animals” [14]. No insecticides capable of altering hepatic drug metabolism enzymes were used in the animal facilities. Animals were used once. All animals were euthanized in accordance with the Institute of Laboratory Resources policies on the humane care of laboratory animals. All protocols involving the use of animals have been approved by the Institutional Animal Care and Use Committee at the University of Utah.

2.2. Synthesis and characterization of MRS-2481 and MRS-2485

MRS-2481 (R)-1-(8-((2-phenylpropanoyl)oxy)octyl)pyridin-1-ium bromide) and MRS-2485 (S)-1-(8-((2-phenylpropanoyl)oxy)octyl)pyridin-1-ium bromide) were synthesized as described in Ref. [10]. One gram of each compound was obtained by scaling up the method of [10] by Natlab, Inc. (Research Triangle, NC). As further described, samples of each compound synthesized by Natlab Inc. were determined to be pure by analytical HPLC (Supplemental Fig. 1), and characterized by nuclear magnetic resonance (NMR) (Supplemental Fig. 2) and mass spectrometry (C₂₂H₃₀N0₂+, exact mass: 340.2274) for both compounds (Supplemental Fig. 3). Samples were also solubilized in MeOD-D₄ at 10 mg/mL, temperature = 21.7 ° C, and analyzed for optical rotation on a P-2000 Digital Polarimeter (Jasco, Inc., Easton, MD, USA). Under these conditions, the specific rotation of MRS-2481 was –15.41 ± 0.05 deg.dm⁻¹ g⁻¹cm³, and the specific rotation of MRS-2485 was +14.26 ± 0.01 deg.dm⁻¹ g⁻¹ cm³.

2.3. Compound Preparation for administration

The test substances were prepared by weighing out 255 mg of each compound. 8.5 mL of methylcellulose (MC) was added, and a mortar and pestle (20 min) were used to bring it into solution. After that the solution was sonicated using a Sona bath at room temperature for 20 min. Each compound was administered intraperitoneally (i.p.) to a mouse in a volume of 0.01 mL/g body weight.

2.4. Prescreen testing paradign

Each acute seizure model included N - 4 animals/dose/time point. These animals were cumulatively evaluated for rotarod impairment, giving a total group size for the rotarod component of N = 8/dose/time point. Animals were tested for each acute seizure test at two time points and three doses given by i.p. administration in a volume of 0.01 mL/g body weight in mice. Tests of impairment were based on performance on the rotarod [15]. Any adverse effects, including deaths, were noted. An observer blinded to treatment confirmed behavioral observations.

2.5. 6 Hz, 44 mA Mouse seizure model

A drop of 0.5% tetracaine hydrochloride in 0.9% saline (anesthetic/electrolyte) solution was applied to the eyes of each animal prior to placement of the corneal electrodes. The electrical stimulus was delivered, for a 3 s duration, via corneal electrodes by an apparatus similar to that originally described by Ref. [16]. Typically, 6 Hz seizures were characterized by an initial momentary stun followed immediately by jaw clonus, forelimb clonus, twitching of the vibrissae, and Straub tail lasting for at least 1 s. Mice not displaying seizures were considered protected.

2.6. Maximal Electroshock test

For the Maximal Electroshock (MES) tests, a drop of 0.5% tetracaine hydrochloride in 0.9% saline (anesthetic/electrolyte) solution was applied to the eyes of each animal prior to placement of the corneal electrodes. The electrical stimulus in the MES test was 50 mA, 60 Hz, 0.2 s for mice, and was delivered via corneal electrodes by an apparatus similar to that originally described by Woodbury and Davenport [16]. Mice not displaying hindlimb tonic extension were considered protected.

2.7. Corneal kindled mouse test

The test was conducted as previously described [17,18]. Briefly, 5–6 week-old male C57BL/6 mice were kindled electrically by a 1.5 mA, 60 Hz, 3 s stimulation via corneal electrodes to a criterion of 5 consecutive Stage 5 seizures (facial clonus and head nodding progressing to forelimb clonus, and finally rearing and falling accompanied by a generalized clonic seizure. Testing of compounds began at least 7 days after the last stimulation. At least 3 doses, sufficient to produce between 0% and 100% protection were evaluated in groups of 8 fully kindled mice.)

2.8. Post-Kainate hippocampal slice in rat

A modified low-dose kainic acid administration protocol was used to induce status epilepticus in adult male Sprague–Dawley rats weighing 150–200 g, as previously described [19–21]. Rats were anesthetized using sodium pentobarbital (50 mg/kg, IP) and the brains are quickly removed and placed in ice cold, oxygenated 95% 02–5% C02 sucrose artificial cerebrospinal fluid (ACSF) solution containing (in mM): sucrose (200), KCI (3.0), NaH₂PO₄ (1.4), MgSO₄ (2.0), NaHCO₃ (26.0), glucose (10.0), and CaCl₂ (2.0). The brains were sliced in 350 pm thick horizontal sections in a ventral to dorsal orientation and incubated at room temperature for 1–2 h prior to experiments in oxygenated. artificial cerebrospinal fluid (ACSF) containing NaCl (126 mM) instead of sucrose, and 10 μM glycine (pH = 7.33–7.38; osmolarity = 290–310 mOsm). Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded from eight brain slices simultaneously. MRS-2485 was tested at a minimum of 4 concentrations, and concentration-response curves were fit to data, thus allowing the half maximal effective concentration (EC₅₀) to be calculated.

2.9. Behavioral assessment by rotarod assay

Motor impairment was identified in mice by the rotarod test [15]. A mouse was placed on a 1-inch knurled rod that rotated at a speed of 6 r.p.m. The mouse was considered impaired if it fell off this rotating rod three times during a 1-min period.

2.10. Absorption, distribution, metabolism, Excretion and Toxicity (ADMET)

Predicted Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) were measured by two methods. Method #1 was the pkCSM method of predicting small molecule pharmacokinetic and toxicity properties using graph-based signatures [22]. Method #2 was the method used by StarDrop Software (v7.2) [23].

3. Results

3.1. MRS-2485 passed the prescreen paradigm for antiseizure potency and toxicity

Table 1 shows the efficacy of MRS-2481 and MRS-2485 to protect treated mice from 6 Hz induced seizures and relative inefficacy against MES induced seizures following administration of 30, 100 and 300 mg/kg of both compounds. The data shown are from one experiment that is representative of four studies testing MRS-2481 and three studies testing MRS-2485. At 30 mg/kg, MRS-2481 protected only 2 of 4 tested mice from seizure activity at 30 min, and none of 4 mice 2 h after dosing. In the MES-induced seizure test, none of 4 mice were protected. Importantly, no toxicity was observed at this dose. By contrast, at the same 30 mg/kg dose for MRS-2485, all four of the four tested mice were protected from the 6 Hz stimulus at 30 min. Furthermore, no toxicity was detected. However, as for the mouse MES-induced seizure test, neither MRS-2481 nor MRS-2485 protected the mice from seizures.

Table 1.

Influence of MRS-2481 and MRS-2485 on 6 Hz seizure protection and MES-Induced seizure protection.

DRUG	DOSE, mg/kg, I.P.	6 Hz 44 mA seizure, (protected/total animals)	Observed Toxicity (Motor Impairment)	MES-induced seizure, (protected/total)	Observed Toxicity (Motor Impairment) at 2 h
MRS-2481	30	2/4 at 30 min 0/4 at 2 h	none	0/4	None
	100	2/4 at 30 min 0/4 at 2 h	none	1/4	“observed”
	300		Immediate death		Immediate death
MRS-2485	30	4/4 at 30 min.	none	0/4	none
	100	4/4 at 2 h	Rotarod grasp	0/4	“observed”
	300	Not measured	Immediate death	Not measured	Immediate death

Upon escalating the doses to 100 mg/kg in the 6 Hz test, all 4 mice treated with MRS-2485 were protected at the 2-h time point. However, at this dose toxicity was manifest by trouble grasping the rotarod. By contrast, MRS-2481 was found to have similar modest protective capabilities to those observed at the lower 30 mg/kg dose. Protective activity by 100 mg/kg MRS-2481 was noted for one mouse in the MES-induced seizure test, but toxicity was also observed at that dose. Finally, when the mice were treated with either MRS-2481 or MRS-2485 at the 300 mg/kg dose, immediate death was recorded. These experiments were repeated twice with similar results.

These data indicate firstly that at the 30 mg/kg dose, 6 Hz protection by MRS-2485 was general for all the tested mice, while protection by MRS-2481 was only partial. Secondly, at the 100 mg/kg dose some toxicity could be detected with both compounds; yet again, MRS-2485 protected all the tested mice, even at 2 h. Finally, neither of these compounds could protect in the MES-induced seizure test. We have therefore interpreted these comparative data to indicate that MRS-2485 is likely to be superior to MRS-2481 as a candidate antiseizure drug.

3.2. MRS-2485 stratifies efficacy from toxicity in the 6 Hz seizure protection test

Table 2A shows a titration of MRS-2485 between 17 and 100 mg/kg for the 6 Hz seizure protection test, with 8 mice/concentration. After 1 h at 100 mg/kg, 7 of 8 mice were protected without toxicity. However, after 24 h, toxicity began to be observed at > 75 mg/kg. Nonetheless, as the titration extended to higher doses, protection was still observed in spite of toxicity dominating the higher concentration range. Table 2B shows that from these data the 50% Effective Dose (ED₅₀) could be calculated to be 39.44 mg/kg; Confidence Interval = [23.42–60.71]. The 50% Toxicity Dose (TD₅₀) could also be calculated from these data to be 135.87 mg/kg; Confidence Interval = [124.76–148.31]. The non-overlap of the Confidence Intervals indicates that protection and toxicity can be clearly stratified. Consistently, the slope differences for both the ED₅₀ and the TD₅₀ titration curves are different, suggesting that the protection and toxicity mechanisms are different.

Table 2.

Calculation of ED₅₀ and TD₅₀ for the 6 Hz test of MRS-2485.

A. Dose Protection and Toxicity Relationships in Mice for MRS-2485
TEST	TIME, hrs	Dose, mg/kg	Observed Toxicity (Motor Impairment)	Deaths	Fraction of Mice Protected
6 Hz 44 mA	1.0	17	none	0	1/8
6 Hz 44 mA	1.0	30	none	0	3/8
6 Hz 44 mA	1.0	60	none	0	6/8
6 Hz 44 mA	1.0	100	none	0	7/8
6 Hz 44 mA	24.0	75	observed	0	0/8
6 Hz 44 mA	24.0	110	unable to grasp rod	0	1/8
6 Hz 44 mA	24.0	130	unable to grasp rod	0	2/8
6 Hz 44 mA	24.0	140	observed	2	3/8
6 Hz 44 mA	24.0	150	observed	7	8/8

B, Calculation of ED-50 and TD-50 for MRS-2485
Metric	Test, 6 Hz 44 mA (Protection, ED50)	Test, 6 Hz 44 mA(Toxicity/Motor Impairment, TD50)
Time	1 Hour	24 h
ED50 or TD50	39.44 mg/kg	135.87 mg/kg
Confidence Interval	[23.42–60.71]	[124.76–148.31]
Slope	3.05 ± 0.95 (SE)	18.08 ± 6.32 (SE)

Since many approved anti-epilepsy drugs (AEDs) have been characterized by the same ASP/ETSP laboratory at the University of Utah, it was possible to compare MRS-2485 with the ability of at least eight different AEDs to protect in the 6 Hz test. Fig. 2 shows the relative potencies of the eight AEDs as a function of drug concentration. Here we have superimposed data for MRS-2485 (coded red) on this collection of approved AEDs, which includes Levetiracetam (Keppra ^R; coded green). Interestingly, both MRS-2485 and Levetiracetam not only titrate very closely, but are also inactive in the MES test [24]. Based on these data it appears that further tests for compound-specific protection in the absence of toxicity should focus on MRS-2485, with an emphasis on the drug concentration range within the ED₅₀ Confidence Interval.

Fig. 2. Fraction of animals protected from the 6 Hz seizure test by MRS-2485 (Red), compared to levetiracetam (Keppra®) (Green) and other anti-epilepsy drugs.

The NINDS-ETSP laboratory performing the tests of the 6 Hz model at the University of Utah were responsible for all above data, which included levetiracetam (LEV). Thus, MRS-2485 and levetiracetam have ED₅₀ values in the range of 30 mg/kg. CODE: CZP (carbamazepine); TGB (tiagabine); LEV (levetiracetam); PB (phenobarbital); FBM (felbamate); VPA (valproic acid); ESM (ethosuximide); TMO (trimethadione). (Source: Barton ME et al., Epilepsy Res 47(2001)217–227 [29]). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3.3. MRS-2485 is protective in the corneal kindled mouse test

Anti-epilepsy drug protection in the corneal kindled mouse test has a very close quantitative relationship with protection in the 6 Hz test when comparing more than nine different anti-epilepsy drugs [17] (viz., Rowley and White, 2010; r² = 0.9519). We therefore tested MRS-2485 for ability to protect in the corneal kindled mouse test. Table 3A shows titration data between 10 and 75 mg/kg for MRS-2485. Protection increased with dose from 0 of 8 mice to 8 of 8 mice, with the average seizure score reducing from 5.0 to 0.125. Finally, toxicity was absent from the entire titration series. Table 3B shows that from these data, the ED₅₀ for protection can be calculated to be 33.74 mg/kg; Confidence Interval [22.6–45.46], with a slope of 3.89 ± 0.959. Importantly, these data correspond closely to values of the ED₅₀ for the 6 Hz protection test (viz., 39.44 mg/kg; see Table 2B) and with the slope of the 6 Hz data from which the ED₅₀ was calculated (3.05 ± 0.95; see Table 2B). Thus, the mechanisms underlying protection by MRS-2485 appear to share at least this similarity with some other anti-epilepsy drugs.

Table 3.

Calculation of ED₅₀ for corneal-kindled mouse test of MRS-2485.

A. Corneal Kindled Mouse (CKM) Test for MRS-2485
Dose, mg/kg	Time, hours	Protection (N/T)	Average Seizure Score	Observed Toxicity (motor impairment, N/T), 3–4 days
10	1.0	0/8	5.0	0/8
20	1.0	2/8	4.5	0/8
40	1.0	5/8	3.0	0/8
65	1.0	6/8	2.0	0/8
75	1.0	8/8	0.125	0/8

Metric	Test, CKM Protection	Observed Toxicity (motor impairment, N/T)
B. Measurement of ED50 for Protection of Corneal Kindled Mouse (CKM) by MRS-2485
Time	1 h	3–4 days
ED50 or TD50	33.74 mg kg	none
Confidence Interval	[22.6–45.46]	none
Slope	3.89 ± 0.959	none

Cv.

3.4. MRS-2485 is protective in the rat post-kainate hippocampal slice

Chronic kainate administration to rats can result in a status epilepticus state that appears to resemble temporal lobe epilepsy. Horizontal brain slices containing the medial entorhinal cortex exhibit recurrent epileptiform discharges (REDs) that are pharmacoresistant to many traditional antiseizure drugs [21]. Table 4 shows that 100 mM MRS-2485 reduces the burst rate by 73 ± 9% (SEM, N = 5) of control, and reduces the bust duration by 53 ± 7% (SEM, N = 5) of control. We conclude that MRS-2485 is protective in the rat post-kainate hippocampal slice test.

Table 4.

Inhibition of the post-kainate hippocampal slice electrical burst rate and duration in the rat for MRS-2485.

Reduction of Post-Kainate Hippocampal Slice Electrical Burst Rate and Duration in Rat by MRS-2485
Metric	Medial entorhinal cortex hippocampal slices from kainic acid-treated rat
Compound Concentration	100 μM
Number of Slices	5
% Control Burst Rate or Burst Duration	100
% Control Burst Rate	73 ± 9 (SEM)
% Control Burst Duration	53 ± 7 (SEM)

3.5. Predicted Absorption, distribution, metabolism, Excretion and Toxicity (ADMET)

Supplemental Table S1 shows results from two different methods of predicting physicochemical and ADMET properties for MRS-2485. The pkCSM method identifies 27 features. The StarDrop (v.7.2) method adds 11 features. These predicted features are calculated from comparisons of the structure of MRS-2485 with data from hundreds of experimentally measured drugs. Multiple examples of measurement and interpretation for the pkCSM method are shown in Supplemental Table S2. These datasets are both supportive of prediction that MRS-2485 may penetrate into the brain. For example, Supplemental Table S1 shows that StarDrop (v.7.2) predicts the measurement of Blood Brain Barrier (BBB) permeability by MRS2485 to be 0.8384. The maximum measure of BBB permeability by this method is 1, and numbers close to 1 are said to represent likely BBB permeability. The value 0.8384 could be considered close to I. Supplemental Table S1 also shows that the pkCSM method predicts a measurement for the log of BBB permeability to be 0.65. Based the use of 320 compounds whose logBB has been measured experimentally, a value of logBB >0.3 is considered to indicate that MRS-2485 can readily cross the blood brain barrier (see Supplemental Table S2, by Drs. Pires and Asher, co-authors of [22]). We conclude that although not measured directly, this predicted property is consistent with the anti-seizure activity of MRS-2485.

4. Discussion

We report here that the small molecule MRS-2485 is protective against seizure activity in three models of epilepsy in mice and rats. In the study of protection in the 6 Hz test in mice, MRS-2485 was found to have an ED₅₀ of 39 mg/kg and a TD₅₀ of 136 mg/kg. The confidence intervals for these metrics did not overlap, indicating a clear separation of efficacy from toxicity. In addition, when plotted in a collective 6 Hz dose-response panel with eight other AEDs, MRS-2485 was found to be slightly less potent than the AED levetiracetam (Keppra ^R), but slightly more potent than the AED Felbamate (Felbatol ^R). Furthermore, virtually equivalent protection by MRS-2485, and relative lack of overlapping toxicity, was also found when tested in the Corneal Kindled Mouse Test. For example, the ED₅₀ values were 39.44 mg/kg for the 6 Hz test, and 33.74 mg/kg for the corneal kindled mouse test. The TD₅₀ for the 6 Hz test was 135.87 mg/kg, while no toxicity was noted below the top of the testing concentration range of 75 mg/kg for the corneal kindled mouse test. Finally, MRS-2485 was found to be protective in the rat post-kainate hippocampal slice test. Thus MRS-2485 passed all three tests for identification of a potential anti-epilepsy drug as defined by the Epilepsy Therapy Screening Program (ETSP)/NINDS/NIH (www.ninds.nih.gov/Current-Research/Focus-Research/Focus-Epilepsy/ETSP).

The decision to test MRS-2485 and its optical isomer MRS-2481 was based on our earlier discovery that MRS-2481 was a potent stereoselective inhibitor of TNFα/NFκB signaling, and also a suppressor of cytokine and chemokine hypersecretion from cystic fibrosis lung epithelial cells [10]. Building evidence from the literature had pointed to inflammation, especially localized to microglia and astrocytes, as having a central role to play in some epilepsies [2,25–28]. In addition, some intractable epilepsies might be either caused by, enhanced by, or accompanied by accumulation of TNFα/NFκB-driven pro-inflammatory mediators at epileptic foci within the brain. Such mediators included Interleukin-8 (IL-8) [3–6], CCL2 (MCP-1) [26,27], brain-derived neurotropic factor (BNDF) [28] and others. Yet, we were aware that inhibitors of TNFα/NFκB signaling still remained an under-pursued pro-inflammatory therapeutic target for epilepsy [29,30]. We were therefore surprised to learn through these experiments that the stereoselectivity for seizure protection by these amphiphilic pyrdinium salts in all testing paradigms displayed the opposite stereoselectivity to that of TNFα/NFκB inhibition. Thus, for antiseizure activity, the (S)-stereoisomer MRS-2485, the less potent in vitro TNFα/NFκB inhibitor, was more effective than the more potent in vitro (R)-stereoisomer MRS-2481. Nonetheless, MRS-2485 is still an inhibitor of TNFα/NFkB signaling, just far less potent than MRS2481.

The comparison of MRS-2485 with properties of approved anti-epilepsy drugs suggests that this compound may deserve further consideration as a candidate drug (See Fig. 2 and [31]). MRS-2485 is also protective against 6 Hz-induced seizures, but not MES-induced seizures, similar to the widely used AED levetiracetam (Keppra^R) [24]. In addition, the ED₅₀ of MRS-2485, and slope of the dose-response curves in both the 6 Hz test and the Corneal Kindled mouse test, are similar to currently used anti-epilepsy drugs [17]. Finally, the systematic selectivity of one optically active isomer, MRS-2485, over the other, MRS-2481, suggests that there may be specific sites of interaction, yet to be discovered, that may be responsible for seizure protection, both in vivo and in vitro. We therefore predict that MRS-2485, or a relevant derivative, may prove therapeutically useful when tested on subjects with epileptic seizures.