Methods of treating and reducing the likelihood of developing epilepsy

Abstract

This disclosure relates to materials and method of using MEK inhibitors, such as the MAP2K inhibitor CI-1040 to treat a subject having an existing epileptic disorder or prevent epilepsy in a subject without an existing epileptic disorder following a brain injury alone or in combination with a second anti-epileptic drug such as an mTOR inhibitor.

Description

METHODS OF TREATING AND REDUCING THE LIKELIHOOD OF DEVELOPING EPILEPSYFIELD

[0001] This disclosure relates to materials and method of using MEK inhibitors, such as the MAP2K inhibitor CI-1040 to treat a subject having an existing epileptic disorder or prevent epilepsy in a subject without an existing epileptic disorder following a brain injury.BACKGROUND

[0002] Epilepsy is a common neurological disorder defined by spontaneous seizures and associated with extensive behavioral comorbidities. Interictal epileptiform discharges, or interictal spikes, are frequently observed in patients with epilepsy. Spikes are brief (< 200 ms) paroxysmal discharges that are generated over large brain regions and detected using EEG.1Over 90% of people with epilepsy have interictal spikes detected across repeated EEGs.23Following a brain injury, spikes may develop prior to seizures, suggesting that they could promote seizure development.45This argument is strengthened by the finding that removal of high-spiking areas correlates with improved surgical outcomes.6Not all spikes are associated with seizures; spikes have been recorded from patients with neurobehavioral disorders including anxiety, attention-deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), obsessive-compulsive disorder, depression, and schizoaffective disorder.7-12Between 5-60% of patients with ADHD and up to 70% of patients with ASD have spike activity on EEG, raising the possibility that spikes could contribute to both cognitive and behavioral disorders.12-15

[0003] Not surprisingly, patients with epilepsy experience cognitive and behavioral comorbidities that have been associated with interictal spikes: up to 50% of spikes may result in transitory cognitive impairment.16 17In adults with epilepsy, spikes impair memory maintenance and word retrieval, delay reaction times, disrupt executive functioning, and increase the risk of crashing a car in a virtual driving simulation.18-21Furthermore, neuronal synchrony induced by persistent spiking can disrupt cortical function in areas distant to the spike onset region, resulting in widespread cognitive deficits induced by localized spiking.2223Thus, spikes could serve as a biomarker for cognitive impairment in adults with epilepsy.

[0004] In animal models, spikes impair short-term memory, spatial memory, and object recognition.2425Spikes differentially affect behaviors depending on which brain regions they involve. We previously found that locomotor activity is strongly correlated with spike location.26Additionally, spikes during early life can have pronounced effects into adulthood; when induced in rat pups, spikes were associated with impaired spatial memoryperformance and reduced long-term potentiation in adult animals.27Early spikes also cause deficits in sociability and attention that persist into adulthood, suggesting interventions to reduce spikes during childhood could have long-lasting benefits.2829Currently, there are no therapeutics specifically designed to prevent or treat epileptic spiking.

[0005] Understanding the etiology of interictal spikes has proven challenging. In patients with epilepsy, spikes occur more frequently than seizures but often occur in the same brain regions, making it difficult to disentangle the pathophysiology of spikes from that of seizures. We curated an extensive collection of human cortical tissues, collected during resective epilepsy surgeries, where each sample is precisely mapped with long-term intracranial EEG in vivo recordings. Transcriptional comparisons of high- and low-spiking cortical regions show highly consistent changes including activation of the MAPK and CREB pathways in cortical layers l-lll.30-32MAPK signaling has an activity-dependent component, with calcium signaling initiating the MAPK cascade.33Increased neuronal activity near high-spiking areas causes persistent MAPK activation leading to increased CREB signaling, increased neuronal excitability, and strengthened synaptic connections between high-spiking neurons.34-37Within areas of increased MAPK activity, we also identified ‘microlesions’: areas with a marked reduction in neuronal (NeuN) staining and increased microglial activation centered in deeper cortical layers.3031Although nuclear NeuN staining was reduced, neuronal cell bodies were preserved within microlesion areas. Additionally, the presence of microlesions was significantly correlated with spike frequency in epileptic human neocortex.31SUMMARY

[0006] Disclosed herein is the identification of distinct molecular pathways upregulated in seizure areas and interictal spiking areas in the brains of subjects suffering from epileptic disorders. Interictal spiking was previously thought to be similar to seizures, and seizures and interictal spiking were thought to act through similar signaling pathways. However the instant disclosure provides the unexpected finding that the genes underlying seizure incidence are different from those that underly interictal spiking in human brain tissues and these same difference are seen in animal models. As reported herein, this disclosure enables the findings that the mTOR pathway is significantly upregulated in brain sections from patients exhibiting seizures, while the MAPK pathway is significantly upregulated in brain sections from patients exhibiting interictal spiking. Thus, the present disclosure contemplates therapies for reducing interictal spiking and combination therapies that reduce both the incidence of seizures and the incidence of interictal spiking in patients having an epileptic disorder or those having a likelihood of developing epilepsy following a brain injury. Since interictal spiking and seizures are known to profoundly affect cognition and behavior, and studies presented herein show that reducing spiking leads to improved behavior,methods to reduce interictal and spiking will also lead to improvements in cognition and behavior.

[0007] For example, effective anti-epileptic therapies based on the data disclosed herein, may target both mTOR and MAPK signaling. The data disclosed herein is the first report that a combination therapy comprising a mTOR inhibitor and a MEK inhibitor will effectively target these two distinct signaling pathways underlying seizures and interictal spiking, respectively. Such a combination of therapeutic agents is reasonably expected to reduce seizure incidence and interictal spiking in a subject suffering from an epileptic disorder. Furthermore, in subjects at risk of developing epilepsy, such as after a brain injury, the prophylactic combination of therapeutic agents disclosed herein is reasonably expected to prevent seizure onset and prevent interictal spiking. In both cases it is reasonably expected that these treatments will lead to improved cognition and behavior.

[0008] Interictal spikes are electroencephalographic discharges that occur at or near brain regions that produce epileptic seizures. While their role in generating seizures is not well understood, spikes have profound effects on cognition and behavior, depending on where and when they occur. It was previously demonstrated that spiking areas of human neocortex show sustained MAPK activation in superficial cortical layers l-lll and are associated with microlesions in deeper cortical areas characterized by reduced neuronal nuclear protein (NeuN) staining and increased microglial infiltration. Based on these findings, additional neuronal populations were investigated within microlesions, specifically inhibitory interneurons. Additionally, it was hypothesized that spiking would be sufficient to induce similar cytoarchitectonic changes within the rat cortex, and that inhibition of MAPK signaling, using a MAP2K inhibitor, would not only inhibit spike formation but also reduce these cytoarchitectonic changes and improve behavioral outcomes.

[0009] To test these hypotheses, tissue samples from 16 patients with intractable epilepsy who required cortical resections were analyzed. To further investigate the molecular underpinnings of spiking, a rat model of interictal spiking induced with tetanus toxin (TeNT) injection into layer II of the somatosensory cortex was optimized.3839This model reliably produces spontaneous epileptic spiking without seizures to permit analysis of spikes independent from seizures. It also replicates the activation of CREB and downstream transcriptional changes seen in human cortex.39It was previously shown that early inhibition of MAPK using a MAP2K (also known as MEK) inhibitor blocks CREB activation and significantly reduces interictal spiking in rats.39

[0010] Herein, the cytoarchitectonic changes in human epileptic spiking-associated microlesions were characterized, revealing a reduction of inhibitory interneurons that maylead to large-scale neuronal synchrony. These changes are reproducible in the TeNT rat model of interictal spiking. Use of the lipophilic MAP2K inhibitor, CI-1040, can block spike development, reduce epileptic spiking across time, and minimize spatial memory impairments. CI-1040 has already advanced to Phase II clinical trials as an anti-cancer agent, with few adverse effects reported, and could be readily repurposed to prevent the development of interictal spikes following brain injury.4041

[0011] Accordingly, in an aspect, the disclosure provides for a method of treating or preventing epilepsy in a subject following brain injury comprising administering to the subject an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof. In an embodiment, the brain injury results from trauma or stroke. In an embodiment, the CI-1040 is administered orally.

[0012] The disclosure provides for methods of treating a subject with an existing epileptic disorder or reducing the likelihood of developing epilepsy in a subject without an existing epileptic disorder following a brain injury, the method comprising administering to the subject an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof.

[0013] In addition, the disclosure provides for use of an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating a subject with an existing epileptic disorder or reducing the likelihood of developing epilepsy in a subject without an existing epileptic disorder following a brain injury.

[0014] The disclosure also provides for compositions for use in for treating a subject with an existing epileptic disorder or reducing the likelihood of developing epilepsy in a subject without an existing epileptic disorder following a brain injury, wherein the composition comprises an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof.

[0015] In another embodiment, the disclosure provides for methods of reducing the likelihood of developing epileptic seizures or reducing the number of epileptic seizures in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury, the method comprising administering to the subject an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof.

[0016] The disclosure also provides for use of an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof for the preparation of a medicament for reducing the development of epileptic seizures or reducing the number of epileptic seizures in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury.

[0017] The disclosure also provides for composition for use in reducing the likelihood of developing epileptic seizures or reducing the number of epileptic seizures in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury, wherein the composition comprises an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof.

[0018] In a further embodiment, the disclosure provides for methods of reducing the magnitude or number of interictal epileptic spikes in a subject with an existing epileptic disorder or in a subject without an existing epileptic disorder following a brain injury, the method comprising administering to the subject an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof.

[0019] In addition, the disclosure provides use of an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof for the preparation of a medicament for reducing the magnitude or number of interictal epileptic spikes in a subject with an existing epileptic disorder or in a subject without an existing epileptic disorder following a brain injury.

[0020] The disclosure also provides for compositions for use in reducing the magnitude or number of interictal epileptic spikes in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury, wherein the composition comprises an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof.

[0021] In another embodiment, the disclosure provides for methods of reducing the likelihood of developing cognitive decline in a subject with an existing epileptic disorder or in a subject without an existing epileptic disorder following a brain injury, the method comprising administering to the subject an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof.

[0022] The disclosure also provides for use of an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof for the preparation of a medicament for reducing the likelihood of developing cognitive decline in a subject with an existing epileptic disorder or in a subject without an existing epileptic disorder following a brain injury.

[0023] The disclosure also provides for compositions for use in reducing the likelihood of developing cognitive decline in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury, wherein the composition comprises an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof.

[0024] An epileptic disorder refers to a neurological disorder having recurrent seizures, which can be the result of one or more of the following: an inherited or non-inherited genetic defect, metabolic defect or dysfunction, structural or developmental abnormality in the brain,an autoimmunity disease, infection or brain injury. An epileptic seizure refers to a rhythmic burst of unprovoked epileptic discharges that may start focally and spread to other regions of the brain.

[0025] In any of the disclosed methods, uses or compositions for use, the subject is treated following a brain injury, wherein the brain injury results the brain injury results from trauma, stroke, infection, developmental abnormality, or one or more tumors.

[0026] In any of the disclosed methods, uses or compositions for use, an interictal epileptic spike refers to a localized paroxysmal discharge before or between seizures, which may be observed in patients with an epileptic disorder, and which may be used to identify regions of seizure onset.

[0027] In any of the disclosed methods, uses or compositions for use, CI-1040 or a pharmaceutically acceptable salt thereof, is administered orally or intravenously. In addition, any of the disclosed medicaments and compositions are formulated for oral or intravenous administration.

[0028] In some embodiments, any of the disclosed methods, uses or compositions for use, CI-1040 or a pharmaceutically acceptable salt thereof, is administered in combination with a second therapeutic agent, wherein the second therapeutic agent exhibits anti-epileptic properties. Anti-epileptic properties refer to therapeutic agents that are suitable for treatment or prevention of seizures or convulsions by controlling abnormal electrical activity in the brain. In particular, the second therapeutic agent can inhibit epileptogenesis, such as therapeutic agents that inhibit or slow the transformation of normal brain tissue to tissue that is capable of generating seizure activity.

[0029] In some embodiment, any of the disclosed methods, uses or compositions for use, CI-1040 or a pharmaceutically acceptable salt thereof, is administered in combination with a second anti-epileptic drug where that drug is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway. Exemplary inhibitors of the mTOR signaling pathway include rapamycin or an analogue thereof. Analogues of rapamycin include sirolimus, everolimus, temsirolimus and ridaforolimus. In some aspects, the CI-1040 or a pharmaceutically acceptable salt thereof and the second therapeutic agent are administered simultaneously, which includes administering the agents separately or in a single composition as an admixture.

[0030] The disclosure provides for compositions comprising i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits antiepileptic properties. In some compositions, the second therapeutic agent is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway. Exemplary inhibitors of themTOR signaling pathway include rapamycin or an analogue thereof. Analogues of rapamycin include sirolimus, everolimus, temsirolimus and ridaforolimus. In some compositions, the CI-1040 or a pharmaceutically acceptable salt thereof and the second therapeutic agent are administered simultaneously, which includes administering the agents separately or in a single composition as an admixture.

[0031] The disclosure also provides for methods of treating a subject with an existing epileptic disorder or reducing the likelihood of developing of epilepsy in a subject without an existing epileptic disorder following a brain injury, comprising administering a composition comprising i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits anti-epileptic properties.

[0032] The disclosure also provides for use of the composition comprising i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits antiepileptic properties for the preparation of a medicament for treating a subject with an existing epileptic disorder or reducing the likelihood of developing the development of epilepsy in a subject without an existing epileptic disorder following a brain injury.

[0033] In addition, the disclosure provides for a composition for use in treating a subject with an existing epileptic disorder or reducing the likelihood of developing epilepsy in a subject without an existing epileptic disorder following a brain injury, wherein the composition comprises i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits anti-epileptic properties.

[0034] The disclosure also provides for methods of reducing the likelihood of developing epileptic seizures or reducing the number of epileptic seizures in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury, comprising administering the composition comprising i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits anti-epileptic properties.

[0035] The disclosure also provides for use of the composition comprising i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits antiepileptic properties for the preparation of a medicament for reducing the likelihood of developing epileptic seizures or reducing the number of epileptic seizures in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury.

[0036] In addition, the disclosure provides for a composition for use in reducing the likelihood of developing epileptic seizures or reducing the number of epileptic seizures in a subject with an existing epileptic disorder or reducing the likelihood of developing epilepsy ina subject without an existing epileptic disorder following a brain injury, wherein the composition comprises i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits anti-epileptic properties.

[0037] The disclosure also provides for methods of reducing the likelihood of developing cognitive decline in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury, comprising administering the composition comprising i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits anti-epileptic properties.

[0038] The disclosure also provides for use of the composition comprising i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits antiepileptic properties for the preparation of a medicament for reducing the likelihood of developing cognitive decline in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury.

[0039] In addition, the disclosure provides for a composition for use in reducing the likelihood of developing cognitive decline in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury, wherein the composition comprises i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits anti-epileptic properties.

[0040] The disclosure also provides for methods of reducing the magnitude or number of interictal epileptic spikes in a subject with an existing epileptic disorder a subject with an existing epileptic disorder or in a subject without an existing epileptic disorder following a brain injury, comprising administering the composition comprising i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits antiepileptic properties.

[0041] The disclosure also provides for use of the composition comprising i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits antiepileptic properties for the preparation of a medicament for reducing the magnitude or number of interictal epileptic spikes in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury.

[0042] In addition, the disclosure provides for a composition for use in reducing the magnitude or number of interictal epileptic spikes treating a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury, wherein the composition comprises i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits anti-epileptic properties.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figures 1A-1 B show that a seven-day oral dose of 250 mg / kg / day of Cl- 1040 significantly reduced cytoplasmic dpMAPK1 / 2 in the rat brain. Figure 1A provides rats that were assigned to one of three groups (CI-1040 treatment, vehicle only, or naive control). Rats in the drug-treated group received a 7-day dose of 250 mg / kg / day CI-1040 delivered orally in 2 g raw sugar cookie dough. Vehicle-only rats received 2 g of cookie dough for 7 days. Naive rats were maintained on a diet of ad libitium rat chow without supplementation. Rats were sacrificed 6 h after the final dose of drug or cookie dough and brains were collected and fractionated for western blotting. Each lane represents an individual rat. Figure 1 B provides quantification of protein levels of nuclear and cytoplasmic dpMAPK1 / 2, which were normalized by dividing the relative signal intensity of dpMAPK1 / 2 by p-actin for each lane. Fold change in dpMAPK1 / 2 protein levels for drug-treated and vehicle-only rats was calculated by dividing normalized dpMAPK1 / 2 values by the mean dpMAPK1 / 2 protein quantity for naive rats. Treatment with CI-1040 significantly reduced cytoplasmic levels of dpMAPK1 / 2 (t(4) = 4.56, P= 0.0052) and caused a substantial reduction in nuclear dpMAPK1 / 2 (t(4) = 1.98, P= 0.0595) in the rat brain. **P< 0.01 using an unpaired two-sided Student’s t-test. Error bars represent ± SEM.

[0044] Figures 2A-2F provide outlier analysis identifying six non-responders within the drug treatment groups. Figures 2A-2B show that there were no outliers within the vehicle- only sham or TeNT groups. Figure 2C provides ROUT analysis that identified one high- spiking outlier (animal 107) within the Sham + CI-1040 Early group. Figure 2D shows that two outliers (animals 78 and 106) were identified in the TeNT + CI-1040 Early group. Figure 2E shows that one outlier (animal 107) was identified in the Sham + CI-1040 Delayed group. Figure 2F provides that two outliers (animals 93 and 101) were identified in the TeNT + CI- 1040 Delayed group. Outliers were excluded from between-group analyses of drug or toxin effects but were included in behavioral studies which considered all animals together, regardless of group. Figure legends represent individual animal numbers.

[0045] Figures 3A-3D show that early treatment with CI-1040 reduces NeuN and increases microglial staining in sham animals. Figure 3A demonstrates that there was a significant effect of drug treatment on NeuN staining (F(3, 40) = 9.133, P< 0.0001) such that animals in the Sham + CI-1040 Early group had a significant reduction in NeuN staining in layers IV-VI and animals in the Sham + CI-1040 Delayed group had a significant reduction in NeuN staining in layers V-VI. Figure 3B provides parvalbumin staining, which was significantly reduced across all sham groups compared to naive animals (F(3, 40) = 6.239, P = 0.0014). Animals in the Sham no drug group had the largest reduction in parvalbumin staining in layers IV-V, while animals in the drug treatment groups had a smaller reduction inparvalbumin staining that was restricted to layer IV alone. Figure 3C provides that there were no significant differences between any of the sham groups and the naive animals in calbindin staining (F(3, 40) = 2.789, ns). Figure 3D shows that there was a significant group effect on Iba1 staining (F(3, 40) = 10.19, P< 0.0001), with early drug treatment causing a significant increase in microglia in layer IV and a trending increase in layers ll / lll (F= 0.069). There was also a trending increase in microglia in layer I for animals in the delayed drug treatment group (F= 0.083). Naive: n = 3; Sham: n = 3; Sham + CI-1040 (Days 0-6): n = 3; Sham + CI-1040 (Days 15-21): n = 3. Analysis performed using two-way ANOVA with T ukey-Kramer post-hoc tests. *F < 0.05, **F < 0.01 , ***F < 0.001 .

[0046] Figures 4A-4J demonstrate that high-spiking areas of human neocortex have fewer NeuN-positive neurons and inhibitory interneurons along with increased microglia. Figure 4A provides a schematic of long-term subdural electrodes used to identify areas that produce seizures and spikes before surgical resection. An example of interictal spike frequencies, for one patient, at individual electrode locations are shown as a three- dimensional heatmap. Relative spike frequencies for each region are represented as green and red for low- and high-spiking areas, respectively. The relationship between relative spike frequency and color scale is illustrated to the left of the brain schematic, with the maximum spike frequency reaching 486 spikes per 10 min recording segment for a single electrode. Figure 4B shows the low- and non-spiking areas used as internal controls, which have few areas of NeuN signal loss and microlesions compared to high-spiking cortical regions. Scale bar: 2 mm. Figure 4C demonstrates that microlesion areas have significantly reduced NeuN staining in cortical layers ll-VI (F(1 , 135) = 171.9, P< 0.0001 ). Control: n = 12; Microlesion: n = 17. Figure 4D shows that neurons with reduced NeuN staining (red arrowheads) are most frequently observed within layer V of microlesions. Figure 4E provides that PV interneurons are significantly reduced in layers ll-V of high-spiking cortex (F(1 , 130) = 278.8, P< 0.0001). Control: n = 12; Microlesion: n = 16. Figure 4F shows that PV reduction is most visually prominent in cortical layers Il-Ill of microlesions. Figure 4G demonstrates that CB staining is also reduced in layers ll-V of high-spiking cortex compared to control cortex. Control: n = 12; Microlesion: n = 14. Figure 4H shows that CB reduction is most prominent in layers Il-Ill of microlesions. Figure 41 demonstrates that high-spiking areas have increased Iba1 (microglial) staining in cortical layers l-IV (F(1 , 120) = 109.0, P< 0.0001 ). Control: n = 12; Microlesion: n = 14. Figure 4J provides that microlesions have larger microglia with more processes, most notably in cortical layers Il-Ill. Figures 4D, 4F, 4H, and 4J taken at 20X magnification. Scale bars represent 200 pm. *** P< 0.001 , **** P< 0.0001 using two-way ANOVA or mixed effects analysis with Sidak’s correction for multiple comparisons. Error bars: ± SEM.

[0047] Figures 5A-5I demonstrate that TeNT-induced spiking causes cytoarchitectonic changes in rat cortex that recapitulate those observed in high-spiking areas of human cortex. Figure 5A shows that spikes at the injection site (L2 electrode) have a unique short-duration and high-amplitude morphology. Scale bars: 0.5 sec (horizontal) x 1000 pV (vertical). Figure 5B provides that compared to sham animals, TeNT-injected rats had a significant increase in mean spikes / hour (across all EEG recording days) at the L2 electrode (F(5, 168) = 7.017, P < 0.0001 ). Subsequent histochemical analyses targeted this region. Sham: n = 6; TeNT : n = 7. Figure 5C provides 3-mm coronal sections of cortex, bisecting the L2 electrode site, that were collected for immunohistochemical staining. Brightfield images of the left hemisphere were divided into 3 cortical regions, which were extracted and subdivided into 10 horizontal regions of interest of equal size, corresponding to cortical layers l-VI . For each animal, and each cortical layer, one mean value was calculated across all three regions to provide a global measure of cell loss within that cortical layer. Figure 5D shows that TeNT rats have a loss of NeuN-positive neurons relative to surgically naive (control) rats (F(2, 35) = 9.351 , P = 0.0006). TeNT rats have a significant reduction in NeuN signal in layers IV-VI. Naive: n = 3; Sham: n = 3; TeNT: n = 4. Figure 5E demonstrates that compared to both sham and naive rats, TeNT rats had increased Iba1 (microglia) staining that was most pronounced in cortical layers Il-Ill but extended from layers l-IV (F(2, 35) = 21 .07, P< 0.0001). Naive: n = 3; Sham: n = 3; TeNT : n = 4. Figure 5F provides that TeNT rats had a loss of CB staining in layers ll / lll relative to naive rats (F(2, 35) = 3.764, P= 0.0331 ). Naive: n = 3; Sham: n = 3; TeNT: n = 4. Figure 5G shows that sham and TeNT animals had a loss of PV signal in cortical layers IV-V, with the greatest loss in layer IV (F(2, 35) = 14.69, P< 0.0001 ). Naive: n = 3; Sham: n = 3; TeNT: n = 4. Figure 5H demonstrates that NeuN loss is most pronounced in the neuronal nuclei within layer V (20X magnification). Black arrowheads: neurons with fully stained nuclei; red arrowheads: neurons with diminished nuclear NeuN staining. Figure 51 shows that microglia from TeNT animals had more processes and were more densely packed than those in naive animals (20X magnification). Scale bars represent 200 pm. * P< 0.05, ** P< 0.01 , *** P< 0.001 , **** P< 0.0001 using two-way ANOVAs with Tukey-Kramer post-hoc tests. Error bars: ± SEM.

[0048] Figures 6A-6C show that spiking does not affect somatostatin interneurons or astrocytes. Figure 6A provides a histological analysis that revealed no significant difference in somatostatin (SST) interneuron staining between control and microlesion areas of human cortex (F(1 , 125) = 0.658, ns). Control: n = 12; Microlesion: n = 15. Figure 6B provides that similar to the findings in human tissue, there are no significant differences in SST staining between surgically naive (control), Sham, and TeNT rats (F(2, 35) = 0.0299, ns). Naive: n = 3; Sham: n = 3; TeNT: n = 4. Figure 6C shows staining for GFAP (astrocytes) that revealeda significant group effect (F(2, 35) = 5.423, P= 0.0089); however, post hoc tests showed no significant differences between naive, Sham, and TeNT rats. Naive: n = 3; Sham: n = 3; TeNT: n = 4. Analysis performed using two-way ANOVA with Sidak’s or Tukey’s correction for multiple comparisons for human and rats, respectively. Error bars: ± SEM.

[0049] Figures 7A-7G demonstrate that both early and delayed treatment with CI-1040 significantly reduce interictal spikes. Figure 7A shows that both early and delayed CI-1040 drug treatments significantly reduced the mean spikes / hour (across all EEG recording days) at the L2 electrode (F(5, 168) = 7.017, P< 0.0001). TeNT: n = 7; TeNT + CI-1040 (Days 0- 6): n = 6; TeNT + CI-1040 (Days 15-21): n = 5. Figures 7B-7C demonstrate that low-level spiking was present across all groups in the anterior electrodes. Figure 7D shows that in TeNT-injected rats, spikes began to increase above sham levels at day 30, with maximum spiking occurring at day 49, followed by a steady decline in spiking that returned to sham levels by 4 months post-surgery (F(5, 28) = 6.164, P= 0.0006). Figure 7E demonstrates that low levels of spiking were also seen at the R2 electrode, contralateral to the TeNT injection site, across all groups. Figures 7F-7G show that the posterior electrodes showed almost no spikes in any group. L1 : left anterior electrode; L2: left middle electrode and saline / TeNT injection site; L3: left posterior electrode; R1 : right anterior electrode; R2: right middle electrode; R3: right posterior electrode. For Figures 7B-7G, Sham: n = 6; TeNT : n = 7; TeNT + CI-1040 (Days 0-6): n = 6; TeNT + CI-1040 (Days 15-21 ): n = 5. * P < 0.05, *** P < 0.001 , **** P< 0.0001 using two-way repeated measures ANOVAs or mixed effects analysis with Tukey-Kramer post-hoc tests. Error bars: ± SEM.

[0050] Figures 8A-8K provide that treatment with CI-1040 alters spike morphology and preserves cortical cytoarchitecture, which correlates with improved spatial memory. Figure 8A demonstrates that the mean percent area stained per cortical layer for each TeNT group was evaluated as a percent change relative to the naive group. White areas indicate no significant difference between TeNT and naive, blue areas indicate a significant reduction in TeNT animals, and red areas indicate a significant increase in TeNT animals. When compared to naive animals, all TeNT groups, including those with CI-1040 treatment, had a significant reduction in NeuN staining (F(3, 65) = 19.21 , P< 0.0001). Figure 8B shows that neither early nor delayed CI-1040 treatment mitigated parvalbumin interneuron loss in layer IV, and all groups had significantly less parvalbumin staining compared to naive animals (F(3, 65) = 11.13, P< 0.0001 ). Interestingly, both early and delayed drug treatments reduced parvalbumin staining in layers ll / lll, a finding not observed in non-drug treated animals. Figure 8C demonstrates that early drug treatment reduced CB interneuron loss in layers II- III by 46% and delayed drug treatment further restored CB interneurons to the level of naive animals (F(3, 65) = 3.088, P= 0.0332). Figure 8D provides that early drug treatmentnormalized microglia to naive levels in layers l-lll and reduced microglial staining in layer IV by 19%. Delayed drug treatment normalized microglia to the level of naive animals across all cortical layers (F(3, 65) = 9.447, P< 0.0001). Figure 8E shows that for each spike, we measured left and right half-wave spike amplitudes and durations, slopes, and total spike duration. Figure 8F demonstrates that among spikes which met the slope cutoff (> 71 pV / ms), those with maximum amplitude at least 1 o above the mean across all TeNT groups (> 1617 pV, red dashed line) were specific to TeNT animals. Figure 8G provides maximum amplitude distributions shown for all TeNT animals (regardless of treatment), demonstrating that both early and delayed MEK inhibition led to fewer spikes exceeding the maximum amplitude cutoff (red dashed line). Figure 8H-8I demonstrate that spike frequency on postoperative day 63 was significantly correlated with total distance traveled as well as off-target errors on the Barnes maze across all rats, regardless of treatment group. Figure 8J shows that among drug-sensitive spikes (slope > 71 pV / ms, amplitude > 1617 pV), there was a trending correlation between increased spiking and increased distance traveled on the final day of Barnes maze Trial 3. Figure 8K demonstrates that an increase in drug-sensitive spikes was significantly correlated with the number of off-target errors on the final day of Barnes maze Trial 3. n = 40 for each scatter plot. Sham: n = 6; Sham + CI-1040 (Days 0-6): n = 5; Sham + CI-1040 (Days 15-21 ): n = 5; TeNT: n = 7; TeNT + CI-1040 (Days 0-6): n = 6; TeNT + CI-1040 (Days 15-21): n = 5. Analysis performed using two-way ANOVA with Tukey- Kramer post-hoc tests. f < 0.1 , *P < 0.05, **P < 0.01 , ***P < 0.001 , ****P < 0.001 . For figures 8H-8K, significance was assessed as P< 0.05 using linear regression with Pearson’s correlation ( / ■).

[0051] Figures 9A-9D how that CI-1040 increases calbindin staining and reduces microglial activation in TeNT animals. Figure 9A demonstrates that there was a significant reduction in NeuN staining in layers IV-VI across all TeNT groups, regardless of drug treatment (F(3, 65) = 19.21 , P< 0.0001). Figure 9B shows that both early and delayed CI- 1040 treatment increased parvalbumin interneuron loss in layer IV, relative to untreated TeNT animals (F(3, 65) = 11.13, P< 0.0001). Early drug treatment also resulted in a significant reduction in parvalbumin interneuron loss in layer V, compared to naive animals. Interestingly, both early and delayed drug treatments reduced parvalbumin staining in layers ll / lll, a finding not observed in the TeNT no drug group. Figure 9C demonstrates that there was a significant group effect of CI-1040 treatment on calbindin staining (F(3, 65) = 3.088, P = 0.0332). TeNT animals in the drug treatment groups had similar levels of calbindin staining in layers Il-Ill compared to naive animals. Furthermore, delayed drug treatment significantly increased the level of calbindin staining compared to untreated TeNT animals. Figure 9D provides that while untreated TeNT animals had a significant increase in Iba1 staining inlayers ll-IV compared to naive animals (F(3, 65) = 9.447, P< 0.0001), animals treated with CI-1040 had no significant differences in Iba1 staining compared to the naive group. Naive: n = 3; TeNT: n = 4; TeNT + CI-1040 (Days 0-6): n = 5; TeNT + CI-1040 (Days 15-21 ): n = 5. Analysis performed using two-way ANOVA with Tukey-Kramer post-hoc tests. *P< 0.05, **P < 0.01 , ***P < 0.001 , **** P < 0.0001 .

[0052] Figures 10A-10D provide an exemplary cluster analysis of interictal spikes based on morphologic parameters. We performed a cluster analysis on all spikes observed at the toxin injection site (electrode L2) on post-operative day 49 for Sham, TeNT, and TeNT + CI- 1040 groups. Each spike is visualized as a single datapoint on the scatterplot showing the relationship of the Right (second-half) Amplitude (y-axis) to the Right (second-half) Duration (x-axis). Amplitude and duration values are displayed as z-scores. Different colors indicate distinct clusters of spikes determined independently for each experimental group via k- means clustering. Separation among clusters is also indicated by the convex polygons drawn around adjacent sets of points. Figure 10A provides that spikes in Sham animals fell into three distinct clusters with a broad spread of Right Amplitude and Right Duration, representing a unique distribution of spike morphologies compared to those observed in TeNT animals with or without drug treatment. Figures 10B-10D provide that spikes in TeNT animals were distributed into two distinct clusters and were more concentrated in the upper right quadrant of the scatterplots, corresponding to higher values both Right Amplitude and Right Duration (Right Slope).

[0053] Figures 11A-11 D provide an overview of the experimental design. Figure 11A shows subdural electrodes that are used to identify human cortical areas that produce seizures and epileptic spiking ahead of surgical resection for patients with intractable seizures. Figure 11 B demonstrates that seizing and spiking areas are electrically defined, with non- and / or low-spiking areas used as internal controls. Figure 11C shows how cortical layers are carefully dissected and alternate strips used for histology and omics. Figure 11 D provides a schematic for how results from genomics are analyzed, validated and tested for therapeutic intervention.

[0054] Figures 12A-12B demonstrate that human brain seizure genes are different from spiking genes. Figure 12A provides a heatmap showing the seizure genes and spiking genes in function of their differential expression. Figure 12B provides a Venn diagram representation of the seizure genes and spiking genes indicate a minor overlap between the two transcriptomes.

[0055] Figures 13A-13B show that human seizure and spiking have distinct pathway signatures. Figure 13A presents major differentially expressed gene pathways associatedwith spiking and seizures as -log (p value). Figure 13B provides a schematic of MAPK / ERK pathway activation associated with spiking, and Figure 13C provides a schematic of mTOR activation as seen in seizures.

[0056] Figures 14A-14L provide a comparison of seizure onset zones and controls, showing sustained mTOR activation in specific cortical layers of human brain cortex. Figure 14A shows western blotting of cortical brain lysates from control / spiking only and seizure areas from 11 patients using mTOR specific antibodies. Figure 14B-14C provide quantitative analysis of the western blotting in Figure 14A. Figure 14D-14F shows western blotting of cortical brain lysates from control / spiking only and seizure areas from 11 patients using p70S6kinase specific antibodies, and corresponding quantitative analysis. Figure 14G and Figure 141 provides immunostaining for mTOR and phosphor-mTOR in cortex layers, with quantification of Figure 141 provided in Figure 14J. Figure 14H provides immunostaining for pCREB in cortex layers that is known to be induced by MAPK. Figure 14K provides immunostaining for phosphor-p70S6kinase, which is quantified in Figure 14L.

[0057] Figures 15A-15F show that a high dose tetanus toxin-induced seizure model in rat cortex recapitulates that seizures are associated with increased mTOR activation in rat cortex, similar to epithelial human cortex. Figure 15A provides a schematic of tetanus toxin administration into the left somatosensory cortex (LS) of a rat. Figure 15B shows representative spike and seizure tracings recorded. Figure 15C demonstrates that phosphor-mTOR staining in seizure, spiking, and control tissues. Figure 15D provides quantification of the Figure 15C phosphor-mTOR staining. Figure 15E provides phosphor- CREB staining in seizure, spiking, and control tissues. Figure 15F shows quantification of the Figure 15E phospho-CREB staining.

[0058] Figures 16A-16D demonstrate that seizure and spiking genes overlap with OMIM genes of genetic epilepsy and neurocognitive developmental disorders. Figure 16A provides a Venn diagram showing the overlap between spiking and seizure genes with OMIM genes. Figure 16B lists ion channel and excitability genes that are upregulated or downregulated in the seizure and spiking gene dataset. Genes in italic are known to be transcriptionally regulated by the mTOR pathway. Two such genes are CACNA1C, as shown in Figure 16C, and KCNQ2, as shown in Figure 16D. Insets show higher magnification (63X) of layer V / VI of seizure zone cortex.DETAILED DESCRIPTION

[0059] Disclosed herein are materials and methods for the treatment of epilepsy or the prevention of epilepsy, wherein the materials comprise a MEK inhibitor or a pharmaceutically acceptable salt thereof, and wherein the methods comprise administration of a MEK inhibitoror pharmaceutically acceptable salt thereof to a subject in need thereof, wherein the subject suffers from epilepsy or the subject has suffered a brain injury. Further contemplated are combination therapies, wherein a MEK inhibitor of the disclosure is administered in combination with one or more additional therapeutics. In some embodiments, the one or more additional therapeutics is an mTOR inhibitor.

[0060] The disclosure is based, in part, on new data presented herein showing that while CI-1040 treatment can reduce the development of interictal spiking, there is the added, unexpected finding that a different pathway, namely mTOR, will need to be disrupted to reduce the development of seizures. Thus, this is the first report to suggest that treating seizures and interictal spiking requires targeting at least two different underlying molecular pathways. The disclosure further provides that mTOR signaling is active in seizure areas of an epileptic brain, but not in areas experiencing interictal spiking. This is the first report evaluating active molecular pathways in particular brain regions of human epileptic patients and validated in an animal model, and the data provided herein supports the benefit of treating patients with epileptic disorders using both MEK inhibitors and mTOR inhibitors to target areas of the brain exhibiting interictal spiking and areas of the brain experiencing seizures simultaneously. The present disclosure is the first report of such a combination therapy strategy for treating epileptic disorders as well as the embodiment for the use of CI- 1040 as the MEK inhibitor.

[0061] “Modulating” or “modulate” refers to the treating, prevention, suppression, enhancement or induction of a function, condition or disorder.

[0062] “Treating” or “treatment” includes the treatment of a disease or disorder described herein, in a subject, preferably a human, and includes: i.) inhibiting a disease or disorder, i.e., arresting its development; ii.) relieving a disease or disorder, i.e., causing regression of the disorder or its symptoms; iii.) slowing progression of the disorder; and / or iv.) inhibiting, relieving, or slowing the onset or progression of one or more symptoms of the disease or disorder.

[0063] “Subject” refers to a warm-blooded animal such as a mammal, preferably a human, or a human child, which is afflicted with, or has the potential to be afflicted with one or more diseases and disorders described herein.

[0064] “Effective amount” refers to the amount of a disclosed therapeutic agent provided herein that is sufficient to effect beneficial or desired biological, emotional, medical, or clinical response of a cell, tissue, system, animal, or human. An effective amount can be administered in one or more administrations, applications, or dosages. The term can alsoinclude within its scope amounts effective to enhance or restore to substantially normal physiological function.

[0065] “Therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific therapeutic agent employed; the duration of the treatment; drugs used in combination or coincidental with the specific therapeutic agent employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition. In other aspects, the desired response is to reduce the likelihood of developing the disease or condition, or to reduce the likelihood of developing the symptoms of the disease or condition. In certain embodiments, wherein a subject has an existing epileptic disorder, a therapeutically effective amount of a therapeutic agent reduces the number and / or frequency of seizures in the subject. In some embodiments, a therapeutically effective amount prevents or reduces the likelihood of developing seizures after administration of the disclosed treatment. In other aspects, a therapeutically effective amount of a therapeutic agent reduces the magnitude, number or frequency of interictal epileptic spikes in the subject or prevents or reduces the likelihood of developing additional interictal spikes or developing interictal spikes in additional unaffected regions of the brain. In another aspect, a therapeutically effective amount of a therapeutic agent reduces, slows or prevents cognitive decline in the subject.

[0066] For example, it is well within the skill of the art to start doses of a therapeutic agent at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications.It is generally preferred that a maximum dose of the therapeutic agent (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0067] A response to a therapeutically effective dose of a disclosed therapeutic agent and / or composition, for example, can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. In some embodiments, a decrease of disease symptoms is a reduction in the frequency and / or magnitude of seizures experienced by a subject with an existing epileptic disorder, or a reduction in structural and functional brain abnormalities as measured by electroencephalography (EEG), magnetic resonance imaging (MRI), computed tomography (CT) scans, positron emission tomography (PET) scans, or single photon emission computed tomography (SPECT) scans. In other embodiments, a decrease of disease symptoms is a reduction or elimination of interictal spiking, as measured for example by EEG or functional MRI.

[0068] In still other embodiments, a decrease of disease symptoms is a reduction in cognitive decline, as measured by a neuropsychological assessment. For example, a neuropsychological assessment can be conducted by analyzing general intellect, attention and speed of processing, memory, language, spatial functions, executive functions, sensory and motor functions. The neuropsychological assessment may be measured using standardized tests including the Mini-Mental State Examination (MMSE), the Montreal Cognitive Assessment (MoCA), Mini-Cog, and the Saint Louis University Mental Status (SLUMS) exam. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.

[0069] The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed therapeutic agent and / or composition, by changing the disclosed therapeutic agent and / or composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0070] Also included are “prophylactic” treatments, which can be directed to reducing the rate of progression of the disease or disorder being treated, reducing the chance of developing symptoms of the disorder, delaying the onset of the disease or condition, preventing the onset of symptoms of the disease or condition, and / or reducing the severity ofits onset. “Treatment” or “prophylaxis” does not necessarily indicate complete eradication, cure, or prevention of the disease or disorder, or associated symptoms thereof.

[0071] “Prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition or reduces the likelihood of developing a disease or condition. In some embodiments, a prophylactically effective amount is an amount that prevents or reduces the likelihood of a subject from developing an epileptic disorder, that prevents a subject from having a seizure, that prevents a subject from developing interictal spiking, or a combination thereof. In various embodiments, a subject administered a prophylactically effective amount experiences interictal spiking, but does not develop an epileptic disorder.

[0072] “Prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. For example, the disclosed methods prevent or reduce the chance of developing an epileptic condition, epileptic seizures or interictal spiking in an affected or unaffected region of the brain. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0073] “Pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

[0074] “Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient or carrier with which a therapeutic agent of the disclosure is administered. The terms “effective amount” or “pharmaceutically effective amount” refer to a nontoxic but sufficient amount of the agent to provide the desired biological result. That result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate “effective” amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

[0075] “Pharmaceutically acceptable carriers” for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990). For example, sterile saline and phosphate-buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid can be added as preservatives. Id. at 1449. In addition, antioxidants and suspending agents can be used. Id.

[0076] “Pharmaceutically acceptable prodrug” or “prodrug” represents those prodrugs of the therapeutic agents of the present disclosure which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use. Prodrugs of the present disclosure can be rapidly transformed in vivo to a parent therapeutic agent having a structure of a disclosed therapeutic agent, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).

[0077] “Contacting” refers to bringing a disclosed therapeutic agent in proximity to a cell, a target protein, or other biological entity together in such a manner that the disclosed therapeutic agent can affect the activity of the a cell, target protein, or other biological entity, either directly; i.e., by interacting with the cell, target protein, or other biological entity itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the cell, target protein, or other biological entity itself is dependent.Epilepsy

[0078] Epilepsy is a disabling neurological disorder of recurrent seizures affecting up to 1% of the population70. An epileptic disorder refers to a neurological disorder having recurrent seizures, which can be the result of one or more of the following: an inherited or non-inherited genetic defect, metabolic defect or dysfunction, structural or developmental abnormality in the brain, an autoimmunity disease, infection or brain injury. A seizure refers to a rhythmic burst of epileptic discharge that may start focally and spread to other regions of the brain.

[0079] While single gene defects in ion channels or neurotransmitter receptors are associated with some inherited forms of epilepsy, these mutations cannot account for the majority of patients with epilepsy. In most patients with partial epilepsy71, seizures start in focal brain regions in response to a wide variety of brain insults often with no clear histopathological abnormalities72. Regardless of the original brain insult, neocortical epileptic foci show a remarkably similar electrophysiological pattern of localized, abnormal electrical discharges that can become rhythmic and spread to widespread brain regions to produce clinical seizures. Between seizures, and far more frequent than seizures, these focal brain regions generate localized “interictal” discharges (referred to herein as “interictal spikes”) that can be used to help identify regions of seizure onset73.

[0080] One goal in the treatment of patients with intractable epilepsy is to reduce or eliminate clinical seizures, which have overt behavioral signs and are often lead to major disability and death. Intractable epilepsy, also known as refractory or drug-resistant epilepsy, is a type of epilepsy that cannot be adequately controlled with anti-epileptic therapeutic agents. While an electrographic seizure is a rhythmic burst of epileptic discharges that can start focally and spread to large regions of the brain, interictal spikes generally occur singly or in small groups, remain localized to specific brain regions, and are more frequent than seizures. Interictal spikes are paroxysmal discharges commonly observed in patients with epilepsy which represent an abnormally synchronized population of hyperexcitable neurons firing as an aggregate.Interictal Spikes

[0081] Interictal epileptic spike refers to a localized paroxysmal discharge which may be observed in patients with an epileptic disorder, and which may be used to identify regions of seizure onset. Interictal spikes are known to develop long before actual seizures, and thus may represent an important driving force in epileptogenesis. Interictal spikes are routinely detected on surface EEG and their presence is used to identify epileptic brain regions. Determining their location, patterns of propagation, latency, and frequency can be helpful clinically to identify ictal-onset zones and thereby improve post-surgical outcomes74. In vitro, interictal spiking and seizure activity can arise from the same neural networks75and, in hippocampal slice cultures, these neuronal networks become fully active when there is sufficient neurotransmitter release to initiate action potentials in a critical number of neurons76. It has been hypothesized that positive feedback from synapses of neurons lead to the propagation of excitation throughout the neural network77, which in turn becomes an important driving force for the generation of seizures.

[0082] Since normal neuronal activity is a critical force that shapes nervous system development and plasticity78, it is likely that ongoing ictal and interictal epileptic activity influence the functional and structural changes that could lead to and maintain hyperexcitability and hyperconnectivity. Consistently, genes encoding neurotransmitter receptors, ion channels, transcription factors and neurotrophic factors have been found to be differentially expressed in various animal models of epilepsy and in human epileptic brain tissues79.

[0083] Without wishing to be bound to any particular theory, it is hypothesized that local regions of neocortex that display spontaneous ictal and interictal activities are maintained in an epileptic state through the local expression of specific genes and proteins. Comparing these abnormal regions in human cortex to nearby less abnormal regions provides a methodof identifying molecular pathways that maintain the epileptic state. Disclosed herein is the identification of a number of pathway signatures human epileptic activity that appear to be independent of the “cause” of epilepsy, and provide options for determining the functional and spatial relationship of these activity-dependent genes within “normal” appearing 6- layered neocortex and in adjacent cortical regions80.

[0084] Determining the functional and spatial relationship of these activity-dependent genes within the neocortex and adjacent cortical regions is a systems biology approach, wherein data from multiple experimental sources is obtained, integrated, and analyzed81. The approach disclosed herein condenses and focuses diverse and highly variant types of data, yielding insights that would not be apparent from singular data sources. Through this unbiased, big data mining approach, highly consistent patterns of differentially expressed genes, proteins, and metabolites, together with their associated signaling pathway can be discovered from carefully localized human neocortical tissues from patients undergoing epilepsy surgery. Such an experimental approach generates large, unbiased data sets that can readily organized into a multivariate interactome, that also includes clinical data such as medications and seizure characteristics. Using a variety of correlation analyses, statistically significant clusters containing a number of these variables can be visualized, and the resultant interactome is a discovery machine that can be used through reverse-translation back into animal and in vitro models to validate novel biomarkers as well as therapeutic targets that can then be forward translated back to patients with epilepsy.

[0085] Disclosed herein are genes which are differentially induced at human epileptic foci in proportion to the amount of interictal spikes, rather than the frequency of seizures, suggesting that interictal activity may be an important driving force for activity-dependent gene expression and plasticity at epileptic foci82.Subjects

[0086] A subject in need of the materials and methods of the disclosure is a subject hat would benefit from the treatment or prevention of epilepsy. In various embodiments, the subject is a subject with an existing epileptic disorder. As used herein, the term “epileptic disorder” refers to a medical condition characterized by episodes of uncontrolled electrical activity in the brain, thus producing symptoms that include two or more seizures.

[0087] In some embodiments, the subject is a subject without an existing epileptic disorder, but is experiencing interictal spiking. In various embodiments, the subject is a subject with an existing epileptic disorder, and is experiencing interictal spiking.

[0088] Other factors can cause epilepsy such as brain or head trauma, brain tumors, arteriovenous malformations and cavernous malformations, developmental abnormalities,stroke, metabolic disorders, exposure to toxins or substances, infections such as meningitis, HIV, viral encephalitis and some parasitic infections can cause epilepsy. In some embodiments, the subject is a subject without an existing epileptic disorder and is not experiencing interictal spiking, but has experienced a brain injury.

[0089] In some embodiments, a subject with an existing epileptic disorder has genetic epilepsy, wherein the subject comprises one or more mutations in one or more genes associated with epilepsy.

[0090] In various embodiments, a subject with an existing epileptic disorder is diagnosed by neuroimaging, including magnetic resonance imaging (MRI), computed tomography (CT) scans, and positron emission tomography (PET) scans. In some embodiments, a subject with an existing epileptic disorder is diagnosed with electroencephalogram (EEG), wherein optionally the EEG is video EEG to monitor the patient’s behavior during a seizure and correlate it with the electrical activity in the brain. In various embodiments, a subject with an existing epileptic disorder is diagnosed with neuropsychological testing, wherein the testing assesses cognitive function and memory, which can be affected by epilepsy.MEK Inhibitors

[0091] The term “MEK” refers to a MAP kinase kinase / ERK kinase (MEK), which is part of the Raf / MEK / ERK kinase or RAS / MAPK signal transmission pathway. MEK phosphorylates and activates mitogen-activated protein kinase (MAPK) (see Figure 13B). MEK proteins are coded by seven different genes, among which MEK1 and MEK2 are of greatest significance. A MEK inhibitor is understood herein to refer to an inhibitor of MEK, that is, any therapeutic agent that downregulates, inhibits, reduces or ceases MEK activity, levels and / or function. MEK inhibitors for use in the present disclosure preferably inhibit MEK1 / 2 of a subject. The MEK inhibitor for use in the present disclosure may be a dual inhibitor - in that case, the MEK inhibitor may not only inhibit a MEK, preferably MEK1 / 2, but also inhibits its upstream kinase (i.e. MAPKKK). MEK1 / 2 is the MAPKK in the Ras / Raf pathway, whereby Ras / Raf acts as MAPKKK and ERK1 / 2 acts as MAPK. An example of such a dual inhibitor for use in the present disclosure is PLX-4032. The term “MEK inhibitor” as used herein is understood to encompass pharmaceutically acceptable salts thereof. The term “MEK inhibitor” as used herein may refer to one MEK inhibitor or a combination of two or more MEK inhibitors.

[0092] The MEK inhibitor used in the materials and methods disclosed herein may be a MEK1 / 2 inhibitor or a pharmaceutically acceptable salt thereof. The MEK inhibitor may be an allosteric inhibitor. The MEK inhibitor may be a selective allosteric inhibitor of MEK1 and MEK2 (MEK1 / 2). The MEK inhibitor may be CI-1040 (PD 184352, CAS No.: 212631-79-3) or a pharmaceutically acceptable salt thereof. In other embodiments, the MEK inhibitor maybe REC-4881 (TAK-733 or REC-2029 or REC-4881 ) or a pharmaceutically acceptable salt thereof. TAK-733 is an example of a selective, allosteric MEK1 / 2 inhibitor. The MEK inhibitor may be selected from the group: Binimetinib (MEK162, ARRY-438162, ARRY-162), Cobimetinib (GDC-0973, XL-518, RG7421 ), Selumetinib (AZD6244, ARRY-142,886), Trametinib (GSK1120212, JTP-74057), Mirdametinib (PD0325901 ), RO5126766 (CH5126766), RO4987655 (CH4987655), Refametinib (RDEA119, BAY 869766) and Pimasertib (MSC1936369, AS703026), or a pharmaceutically acceptable salt thereof. The MEK inhibitor may be selected from the group: PD98059, PD184352 (2-(2-chloro-4-iodo- phenylamino)- N-cyclopropylmethoxy-3,4-difluoro-benzamide), AZD8330, RDEA-1 19 (BAY- 869766), AS703026 and PLX-4032 (Zelboraf ® (Vemurafenib)), or a pharmaceutically acceptable salt thereof. The MEK inhibitor may be selected from the group: CI-1040, REC- 4881 , Binimetinib, Cobimetinib, Trametinib and Selumetinib, or a pharmaceutically acceptable salt thereof. The MEK inhibitor may be Binimetinib. The MEK inhibitor PD98059 inhibits the activation of MEK by the kinase Raf. The MEK inhibitor R05126766 is a protein kinase inhibitor specific for the Raf and MEK mitogen-activated protein kinases (MAPKs) with potential anti-neoplastic activity. Raf / MEK dual kinase inhibitor R05126766 specifically inhibits the kinase activities of Raf and MEK, resulting in the inhibition of target gene transcription that promotes malignant transformation of cells. The MEK inhibitor AS703026 is a highly selective and potent allosteric inhibitor of MEK1 / 2.

[0093] CI-1040 (PD 184352, CAS No.: 212631 -79-3) refers an orally active, highly specific, small-molecule inhibitor of MEK of formula:

[0094] "Pharmaceutically acceptable salts" refers to salts or zwitterionic forms of the CI- 1040 (“compound”). The salts can be prepared by reacting the compound with an acid having a suitable cation. The pharmaceutically acceptable salts of the compound can be acid addition salts formed with pharmaceutically acceptable acids. Examples of acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, tartaric, and citric. Nonlimiting examples of salts of compounds of the disclosure include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide,sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzene sulphonate, and p-toluenesulfonate salts. In addition, available amino groups present in the compounds of the disclosure can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference to compounds of the present disclosure appearing herein is intended to include the present compounds as well as pharmaceutically acceptable salts thereof. “MEK inhibitor” includes all inhibitors of MEK1 / 2 known to the skilled in the art. Exemplary MEK inhibitors include is PD0325901 (CAS Number: 391210-10-9), CI-1040 (CAS Number: 212631-79-3), Refametinib, Selumetinib, Trametinib, Cobimetinib, GDC-0623 (RG 7421) and TAK-733 (CAS Number: 1035555-63-5).

[0095] “MAPK inhibitor” or “ERK1 / 2 inhibitor” include all inhibitors of ERK1 / 2 known to the skilled in the art. For example, the ERK1 / 2 inhibitor include Ulixertinib, SCH772984 (CAS number: 942183-80-4), Ravoxertinib (GDC-0994) and LY3214996 (CAS Number: 1951483- 29-6).

[0096] Treatment (i.e. the MEK inhibitor or a pharmaceutically acceptable salt thereof) may be combined with one or more additional therapeutic agent. As such, the method may further comprise a step of administering one or more additional therapeutic agents to the subject. The MEK inhibitor or a pharmaceutically acceptable salt thereof and the one or more additional therapeutic agents may be administered simultaneously, sequentially or separately. The MEK inhibitor or a pharmaceutically acceptable salt thereof and the additional therapeutic agent may be administered in combination. The MEK inhibitor or a pharmaceutically acceptable salt thereof may be administered contemporaneously, previously or subsequently to the one or more additional therapeutic agents. The additional therapeutic agent may be a mTOR inhibitor.Anti Epileptic or Anti Epileptogenic (second therapeutic agent)

[0097] The disclosure provides for therapeutic agents that inhibit epileptogenesis, such as therapeutic agents that inhibit or slow the transformation of normal brain tissue to tissue thatis capable of generating spontaneous seizure activity. For example, the second therapeutic agent may inhibit the activation of genes associated with epileptogenesis, such as mTOR.

[0098] Mammalian target of rapamycin (mTOR) is a highly conserved Serine / Threonine (Ser / Thr) protein kinase, which belongs to the phosphatidylinositol-3-kinase-related (PIKK) protein family. In mammals, mTOR integrates growth factor, amino acid, nutrient and energy sensing signals, and thus plays a major role in key cellular processes, such as cellular growth, proliferation, cytoskeletal organization, transcription, protein synthesis and ribosomal biogenesis. Aberrant mTOR regulation has been implicated in several disease processes, including cancer, diabetes, ocular diseases and neurodegenerative disorders.

[0099] As used herein, an “mTOR inhibitor” refers to any agent that inhibits signaling of mTOR. The disclosure provides for compositions comprising MEK inhibitor in comprising a mTOR inhibitor and in particular using a combination of MEK inhibitor and mTOR inhibitor to treat epilepsy in a subject in need. Exemplary mTOR inhibitors include, rapamycin, temsirolimus, everolimus, PI-103, CC-223, INK128, AZD8055, KU 0063794, Voxtalisib (XL765, SAR245409), Ridaforolimus (Deforolimus, MK-8669), NVP-BEZ235, CZ415, Torkinib (PP242), Torin 1 , Omipalisib (GSK2126458, GSK458), OSI-027, PF-04691502, Apitolisib (GDC-0980, RG7422), WYE-354, Vistusertib (AZD2014), Torin 2, Tacrolimus (FK506), GSK1059615, Gedatolisib (PF-05212384, PKI-587), WYE-125132 (WYE-132), BGT226 (NVP-BGT226), Palomid 529 (P529), PP121 , WYE-687, CH5132799, WAY-600, ETP-46464, GDC-0349, XL388, Zotarolimus (ABT-578), and Chrysophanic Acid. In some embodiments, the mTOR inhibitor is rapamycin or analogues of rapamycin including sirolimus, everolimus, temsirolimus or ridaforolimus.

[0100] Other mTOR inhibitors that can be used in conjunction include neutralizing antibodies against mTOR, therapeutic agents inhibiting the activity of mTORs, therapeutic agents inhibiting the transcription of a gene encoding an mTOR (e.g., antisense nucleic acids, siRNAs, ribozymes), peptides, or combinations thereof.Combination Therapy

[0101] Prior to the instant disclosure, it was not known that distinct molecular pathways were upregulated in human seizure and interictal spiking brain regions. Disclosed herein is the unexpected finding that mTOR signaling is only upregulated in seizure-occurring areas of the brain of a subject suffering from an epileptic disorder, while MEK signaling is only upregulated in areas of an epileptic brain exhibiting interictal spiking. Success of a therapeutic regimen designed to treat epilepsy in a subject that almost always exhibits both seizures and interictal spiking depends on the ability of the therapeutic agents to target both mTOR and MEK signaling, underlying seizures and interictal signaling, respectively. Theinstant disclosure provides the first report of this differential pathway expression in epileptic human and rodent brains, and the disclosure contemplates a combination therapy comprising a MEK inhibitor and a mTOR inhibitor for the treatment of epilepsy.

[0102] The terms “co-administering” and “combination therapy” mean that a MEK inhibitor, such as CI-1040, and a second therapeutic agent that exhibits anti-epileptic properties are administered in a manner that permits both to exert therapeutic effects during an overlapping period of time or independently at different periods of time. In any combination therapy disclosed herein, the MEK inhibitor and the second therapeutic agent that exhibits antiepileptic properties may be administered in the same pharmaceutical composition (e.g., an admixture) or in separate compositions, via the same or different routes of administration. As disclosed herein, anti-epileptic properties describe the ability of a therapeutic agent to control abnormal electrical activity in the brain.

[0103] A MEK inhibitor, such as CI-1040, and a second therapeutic agent that exhibits anti-epileptic properties may be co-administered concurrently, i.e., simultaneously, or at different times, as long as both exert therapeutic effects during an overlapping period of time. For example, the MEK inhibitor, such as CI-1040, and the second therapeutic agent that exhibits anti-epileptic properties may both be administered to a subject within a time period of about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, about 5 hours, or longer. If the MEK inhibitor, such as CI-1040, and the second therapeutic agent that exhibits anti-epileptic properties are not co-administered concurrently, either the MEK inhibitor, such as CI-1040, or the second therapeutic agent that exhibits anti-epileptic properties may be administered first. As long as the subsequent therapeutic agent is administered while a therapeutic effect of the first administered agent is present, the MEK inhibitor, such as CI-1040, and the second therapeutic agent that exhibits anti-epileptic properties are considered to be co-administered and used in combination therapy in accordance with the teachings of the disclosure.

[0104] In combination therapy, the MEK inhibitor, such as CI-1040, and the a second therapeutic agent that exhibits anti-epileptic properties are administered in a manner that permits both to exert physiological effects during an overlapping period of time. In one aspect, the therapeutic agents are administered within 30 minutes of each other, for example, in a window of about 5 minutes to 15 minutes, e.g., 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, or 15 minutes, with either the MEK inhibitor, such as CI-1040, and the second therapeutic agent that exhibits anti-epileptic properties administered first. In various aspects, the MEKinhibitor, such as CI-1040, and the second therapeutic agent that exhibits anti-epileptic properties is administered one, two, or three times per day. Optionally, the MEK inhibitor, such as CI-1040, and the second therapeutic agent that exhibits anti-epileptic properties are administered orally, for example, both agents are administered orally, or the MEK inhibitor is administered orally and the second agent is administered intravenously. Alternatively, the second agent is administered orally and the MEK inhibitor is administered intravenously.

[0105] In still further embodiments, the combination therapy may further comprise an additional therapeutic agent, wherein an additional therapeutic agent is any therapeutic agent that exhibits an activity that benefits a subject having an epileptic disorder or a subject without an existing epileptic disorder following brain injury. For example, an additional therapeutic agent can exhibit anti-epileptic properties, can be suitable for treating concomitant diseases or disorders presenting in the subject, or can be useful for ameliorating side effects associated with administration of the combination of a MEK inhibitor and a mTOR inhibitor.

[0106] In some exemplary embodiments, a therapeutically effective amount of the combination therapies disclosed herein is an amount sufficient to reduce the frequency of seizures experienced by the subject. In various embodiments, a therapeutically effective amount of the combination therapies disclosed herein is an amount sufficient to reduce the number and / or frequency of interictal epileptic spiking, the magnitude of interictal epileptic spiking, or a combination thereof. In further embodiments, a therapeutically effective amount of the combination therapies disclosed herein is an amount sufficient to prevent the development of interictal epileptic spiking and / or seizures in a subject that has suffered a brain injury. In various embodiments, a therapeutically effective amount of the combination therapies disclosed herein is an amount sufficient to reduce or prevent cognitive decline in a subject with an existing epileptic disorder or in a subject without an existing epileptic disorder following a brain injury.

[0107] In some embodiments, the single therapeutic agent or combination therapy disclosed herein serves to treat an existing epileptic disorder or prevent the development of an epileptic disorder. Treatment efficacy is measured by a reduction in the frequency of seizures experienced by a subject suffering from an epileptic disorder, or a reduction in structural and functional brain abnormalities as measured by electroencephalography (EEG), magnetic resonance imaging (MRI), computed tomography (CT) scans, positron emission tomography (PET) scans, or single photon emission computed tomography (SPECT) scans. Treatment efficacy is further measured by a reduction in the number and / or frequency, or magnitude, of interictal spiking as measured by EEG or functional MRI (fMRI). Treatment efficacy is still further measured by a reduction or prevention of cognitive decline.Pharmaceutical Compositions

[0108] In certain aspects, this disclosure provides for a pharmaceutical composition comprising an amount of a MEK inhibitor, such as CI-1040, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, excipients, or carriers. In addition, this disclosure provides for a pharmaceutical composition comprising an amount of a mTOR inhibitor, such as rapamycin or analogues thereof such as sirolimus, everolimus, temsirolimus or ridaforolimus, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, excipients, or carriers. The disclosure also provides for pharmaceutical compositions comprising an amount of a MEK inhibitor, such as the CI-1040 or a pharmaceutically acceptable salt thereof, in combination with a second therapeutic agent, wherein the second therapeutic agent exhibits anti-epileptic properties, such as a mTOR inhibitor such as rapamycin or analogues thereof such as sirolimus, everolimus, temsirolimus or ridaforolimus or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, excipients, or carriers.

[0109] Suitable excipients for non-liquid formulations are also known to those of skill in the art. A thorough discussion of pharmaceutically acceptable excipients and salts is available in Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990).

[0110] Additionally, auxiliary substances, such as wetting or emulsifying agents, biological buffering substances, surfactants, and the like, can be present in such vehicles. A biological buffer can be any solution which is pharmacologically acceptable and which provides the formulation with the desired pH, i.e., a pH in the physiologically acceptable range. Examples of buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank’s buffered saline, and the like.

[0111] Depending on the intended mode of administration, the pharmaceutical compositions can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, can include other therapeutic agents, adjuvants, diluents, buffers, and the like.

[0112] In general, the composition of the disclosure will be administered in a therapeutically effective amount by any of the accepted modes of administration. Suitabledosage ranges depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the therapeutic agent used, the route and form of administration, the indication towards which the administration is directed, and the preferences and experience of the medical practitioner involved. One of ordinary skill in the art of treating such diseases will be able, without undue experimentation and in reliance upon personal knowledge and the disclosure of this application, to ascertain a therapeutically effective amount of the compositions of the disclosure for a given epileptic disorder or condition.

[0113] Thus, the composition of the disclosure can be administered as pharmaceutical formulations including those suitable for oral (including buccal and sub-lingual), rectal, nasal, topical, pulmonary, vaginal or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.

[0114] For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, and the like, an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitanmonolaurate, triethanolamine sodium acetate, triethanolamineoleate, and the like. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington’s Pharmaceutical Sciences, referenced above.

[0115] Yet another aspect is the use of permeation enhancer excipients including polymers such as: polycations (chitosan and its quaternary ammonium derivatives, poly-L- arginine, aminated gelatin); polyanions (N-carboxymethyl chitosan, poly-acrylic acid); and, thiolated polymers (carboxymethyl cellulose-cysteine, polycarbophil-cysteine, chitosanthiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugates).

[0116] For oral administration, the composition will generally take the form of a tablet, capsule, softgel capsule, or can be an aqueous or nonaqueous solution, suspension or syrup. Tablets and capsules are preferred oral administration forms. Tablets and capsules for oral use can include one or more commonly used carriers such as lactose and cornstarch. Lubricating agents, such as magnesium stearate, are also typically added. Typically, the compositions of the disclosure can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl callulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

[0117] When liquid suspensions are used, the active agent can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like and with emulsifying and suspending agents. If desired, flavoring, coloring and / or sweetening agents can be added as well. Other optional components for incorporation into an oral formulation herein include, but are not limited to, preservatives, suspending agents, thickening agents, and the like.

[0118] Parenteral formulations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solubilization or suspension in liquid prior to injection, or as emulsions. Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents and suspending agents. The sterile injectable formulation can also be a sterile injectable solution or a suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media. In addition, parenteral administration can involve the use of a slow release or sustained release system such that a constant level of dosage is maintained.

[0119] Parenteral administration includes intraarticular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Administration via certain parenteral routes can involve introducing the formulations of the disclosure into thebody of a patient through a needle or a catheter, propelled by a sterile syringe or some other mechanical device such as a continuous infusion system. A formulation provided by the disclosure can be administered using a syringe, injector, pump, or any other device recognized in the art for parenteral administration.

[0120] Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents and suspending agents. The sterile injectable formulation can also be a sterile injectable solution or a suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media. In addition, parenteral administration can involve the use of a slow release or sustained release system such that a constant level of dosage is maintained.

[0121] Preparations according to the disclosure for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of nonaqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms can also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They can be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.

[0122] Sterile injectable solutions are prepared by incorporating one or more of the compounds of the disclosure in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-f iltered solution thereof. Thus, for example, a parenteral composition suitable for administration by injection is prepared by stirring 1 .5% by weight of active ingredient in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.

[0123] Alternatively, the pharmaceutical compositions of the disclosure can be administered in the form of suppositories for rectal administration. These can be prepared bymixing the agent with a suitable nonirritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

[0124] Preferred formulations for topical drug delivery are ointments and creams. Ointments are semisolid preparations which are typically based on petrolatum or other petroleum derivatives. Creams containing the selected active agent, are, as known in the art, viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also sometimes called the “internal” phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. The specific ointment or cream base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum drug delivery. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing.

[0125] Formulations for buccal administration include tablets, lozenges, gels and the like. Alternatively, buccal administration can be effected using a transmucosal delivery system as known to those skilled in the art. The compounds of the disclosure can also be delivered through the skin or mucosal tissue using conventional transdermal drug delivery systems, i.e., transdermal “patches” wherein the agent is typically contained within a laminated structure that serves as a drug delivery device to be affixed to the body surface. In such a structure, the drug composition is typically contained in a layer, or “reservoir,” underlying an upper backing layer. The laminated device can contain a single reservoir, or it can contain multiple reservoirs. In one aspect, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, can be either a polymeric matrix as described above, or it can be a liquid or gel reservoir, or can take some other form. The backing layer in these laminates, which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility. The material selected for the backing layer should be substantially impermeable to the active agent and any other materials that are present.

[0126] The pharmaceutical compositions of the disclosure can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, propellants such as fluorocarbons or nitrogen, and / or other conventional solubilizing or dispersing agents.

[0127] The compositions of the disclosure can be formulated for aerosol administration, particularly to the respiratory tract and including intranasal administration. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and / or other conventional solubilizing or dispersing agents. The compound will generally have a small particle size for example of the order of 5 microns or less. Such a particle size can be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The aerosol can conveniently also contain a surfactant such as lecithin. The dose of drug can be controlled by a metered valve. Alternatively, the active ingredients can be provided in a form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). The powder carrier will form a gel in the nasal cavity. The powder composition can be presented in unit dose form for example in capsules or cartridges of e.g., gelatin or blister packs from which the powder can be administered by means of an inhaler.

[0128] A pharmaceutically or therapeutically effective amount of a compound of the disclosure will be delivered to the subject. The precise effective amount will vary from subject to subject and will depend upon the species, age, the subject’s size and health, the nature and extent of the condition being treated, recommendations of the treating physician, and the therapeutics or combination of therapeutics selected for administration. Thus, the effective amount for a given situation can be determined by routine experimentation. The subject can be administered as many doses as is required to reduce and / or alleviate the signs, symptoms, or causes of the disorder in question, or bring about any other desired alteration of a biological system. When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient.

[0129] The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the activecomponent. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0130] The disclosed pharmaceutical compositions comprising the disclosed therapeutic agents are further contemplated for use in a method of treating a subject in need thereof, wherein the method comprises administering to the subject an effective amount of CI-1040 or a pharmaceutically acceptable salt thereof. In some embodiments, the disclosed method is a method of treating a subject with an existing epileptic disorder or reduces the likelihood of developing the development of epilepsy in a subject without an existing epileptic disorder following a brain injury. In various embodiments, the disclosed method is a method of reducing the likelihood of developing epileptic seizures or reducing the number of epileptic seizures in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury. In some embodiments, the disclosed method is a method of reducing the magnitude or number of interictal spikes in a subject with an existing epileptic disorder or in a subject without an existing epileptic disorder following a brain injury. In various embodiments, the disclosed method is a method of reducing or preventing cognitive decline in a subject with an existing epileptic disorder or in a subject without an existing epileptic disorder following a brain injury.

[0131] The disclosure further provides pharmaceutical compositions for use in treating or preventing epileptic disorders, wherein the pharmaceutical composition comprises an effective amount of CI-1040 or a pharmaceutically acceptable salt thereof. In some embodiments, the disclosure provides pharmaceutical compositions for use in treating a subject with an existing epileptic disorder or preventing the development of epilepsy in a subject without an existing epileptic disorder following a brain injury. In various embodiments, the disclosure provides pharmaceutical compositions for use in preventing epileptic seizures or reducing the number of epileptic seizures in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury. In some embodiments, the disclosure provides pharmaceutical compositions for use in reducing the magnitude or number of interictal spikes in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury. In various embodiments, the disclosure provides pharmaceutical compositions for use in reducing or preventing cognitive decline in a subject with an existing epileptic disorder or a subject without an existing epileptic disorder following a brain injury.Kits

[0132] The disclosed compounds and / or pharmaceutical compositions comprising the disclosed therapeutic agents can conveniently be presented as a kit, whereby two or more components, which may be active or inactive ingredients, carriers, diluents, and the like, are provided with instructions for preparation of the actual dosage form by the patient or person administering the drug to the patient. Such kits may be provided with all necessary materials and ingredients contained therein, or they may contain instructions for using or making materials or components that must be obtained independently by the patient or person administering the drug to the patient. In further aspects, a kit can include optional components that aid in the administration of the unit dose to patients, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, a kit can contain instructions for preparation and administration of the compositions. The kit can be manufactured as a single use unit dose for one patient, multiple uses for a particular patient (at a constant dose or in which the individual compounds may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple patients (“bulk packaging”). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

[0133] In a further aspect, the disclosed kits can be packaged in a daily dosing regimen (e.g., packaged on cards, packaged with dosing cards, packaged on blisters or blow-molded plastics, etc.). Such packaging promotes products and increases patient compliance with drug regimens. Such packaging can also reduce patient confusion. The present disclosure also features such kits further containing instructions for use.

[0134] In a further aspect, the present disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the disclosure. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0135] In various aspects, the disclosed kits can also comprise compounds and / or products co-packaged, co-formulated, and / or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and / or product and another component for delivery to a patient.

[0136] It is contemplated that the disclosed kits can be used in connection with the disclosed methods of making, the disclosed methods of using or treating, and / or the disclosed compositions.EXAMPLES

[0137] The foregoing may be better understood by reference to the following, which are illustrative of certain embodiments of the disclosure, and are not intended to limit its scope.Example 1 Materials and Methods

[0138] Human Tissue Collection. Human neocortical tissue samples were obtained from 16 patients who underwent surgery for drug-resistant epilepsy. Patients were enrolled following informed consent as approved by an Institutional Review Board at Wayne State University (IRB #2015-0457). Only brain tissues that would otherwise be discarded were used, and no additional tissue was removed beyond what was indicated for seizure treatment. Tissues were subsequently transferred to the University of Illinois at Chicago NeuroRepository. Patients underwent a two-stage surgery with long-term subdural electrocorticography (ECoG) and at least 3 days of continuous in vivo monitoring to identify regions of seizure onset and interictal spiking, which were then removed during the second stage of the procedure.31’4243Tissue was divided into 1 cm3samples corresponding to a precise brain location overlying a single ECoG electrode. Quantitative measures of electrical activity were calculated as the number of spikes in 10 minutes averaged from three independent ten-minute recordings using an automatic algorithm described previously.31’4445Tissue samples were stained with Luxol fast blue and hematoxylin and eosin and reviewed by a neuropathologist to confirm that they were devoid of histopathological abnormalities or surgical hemorrhage.

[0139] Tissue Processing. For immunohistochemistry, tissue samples corresponding to precise electrode locations were fixed with 4% paraformaldehyde, cryoprotected in 30% sucrose, and embedded in OCT (TissueTek) before mounting as described previously.3020 pm sections were prepared and stored frozen until use. Immunohistochemistry was performed using VECTASTAIN® Elite® ABC Universal PLUS Kit (#PK-8200, Vector Labs). Tissue samples were probed with the following antibodies: NeuN (1 :4000, #MAB337, Millipore), lba-1 (1 :1000, #019-019741 , Wako), calbindin (1 :800, #131767, Cell Signaling Technologies), somatostatin (1 :50, #ab11 1912, Abeam), and parvalbumin (1 :4000, #ab1 1427, Abeam).

[0140] Image analysis of human histological sections. Slides were imaged using an Aperio AT2 microscope system (Leica). Images were exported in a TIF format forsubsequent analysis. For each sample section, 6 regions of interest (ROIs) spanning cortical layers l-VI, with a width of 1500 ± 100 pixels were selected. Each ROI was subdivided into 10 equally sized horizontal layers representing the cortical layers. To quantify any changes in the microlesions as compared to control areas, each individual layer was analyzed using Imaged (Analyze Particles function).46Percent area stained (signal area divided by total area) was used to compare staining intensity. Data were exported into GraphPad Prism (Version 9) for graphing and statistical analyses.

[0141] Animals. Two-month-old male Sprague-Dawley rats were acquired from Envigo. Animals were housed in individual cages (to reduce the risk of premature headcap detachment) with ad libitum access to food and water throughout the study. Rats were entrained on a 12 h light-dark cycle with lights-on beginning at sunrise. Rats were sacrificed at 8 months of age. Additionally, three surgery-naive male Sprague-Dawley rats were included to control for effects of epidural electrode placement. Rats were monitored daily by UIC veterinary care staff. All experimental protocols were approved by the UIC Animal Care Committee (ACC Protocol #21-081) and adhered to institutional guidelines.

[0142] TeNT injection and intracranial electrode implantation surgery. Rats were anesthetized with 5% isoflurane in 100% O2 (induction) followed by 2.5% isoflurane in 100% O2 (maintenance) at a rate of 2 L / min. The scalp was shaved and cleaned with iodine and isopropanol. A subcutaneous injection of 1% lidocaine (0.2 mL) and 0.125% bupivacaine (0.3 mL) was delivered under the scalp 10 minutes before the first incision. Rats were placed in a Kopf stereotaxic frame to stabilize the skull. A rectal temperature probe and infrared heating pad were used to monitor and maintain the animal’s temperature at 37°C throughout surgery. An incision was made from the midline of the skull above the nasal sinus to the posterior occiput (~3 cm in length). The scalp was retracted and periosteum was removed. The area was cleaned with 10% H2O2 and dried with sterile gauze. An electrocautery tool was used to achieve hemostasis along the skull surface. Seven holes were drilled into the skull surface using a 1 / 16-inch drill bit (3 left and 3 right electrode holes: AP +4 mm, ML 3.5 mm; AP -1 mm, ML 3.5 mm; AP -6 mm, ML 3.5 mm; 1 reference electrode hole above the nasal sinus: AP +10.5 mm, ML 0.5 mm left), exposing the surface of the dura. A 1 pL Hamilton syringe attached to a micromanipulator was used to inject rats in the left somatosensory cortex at the L2 electrode hole (AP -1 mm, ML 3.5 mm left, DV -1 .5 mm). Rats were randomly assigned to receive either TeNT or sham injections: TeNT-injected rats received 80 ng TeNT in 1 pL sterile PBS, sham-injected rats received 1 pL sterile PBS. The injection was delivered over 4 min (0.25 pL / min) and the needle was left in place for 10 min to ensure localized toxin delivery. After removing the needle, 7 customized epidural screw electrodes (3.2 mm wide, 7 mm long; P1 Technologies) were screwed into the skull to adepth of 1 mm and fixed to two 6-channel plastic electrode pedestals (#MS363, P1 Technologies). A thin layer of Vetbond tissue adhesive (3M) was applied to the surface of the skull to aid in headcap retention. Dental cement was applied around the electrode screws and pedestals to create a headcap apparatus. The scalp incision was closed with a metal wound clip and antibiotic ointment was applied to the scalp margins. Rats were placed in a warming chamber set to 37°C and allowed to recover from anesthesia for 1 h. Liquid acetaminophen (32 mg / mL) was added to the rats’ drinking water at a final concentration of 4 mg / mL to provide post-operative analgesia for 5 days following surgery.

[0143] MAP2K inhibitor dosing and delivery. Rats are fitted with headcaps to enable EEG acquisition for the duration of the study. The rate of premature headcap detachment (a humane endpoint) is approximately 20%, and excessive manipulation of the skull can increase headcap loss. To avoid drug delivery methods that require restraint, a voluntary oral delivery method was optimized.47To acclimate rats to the oral drug delivery method, 2 g raw sugar cookie dough (Pillsbury) was provided to all rats at 0800 h each day for five consecutive days prior to surgery. Rats were randomly divided into three drug treatment groups: control, early drug dose, and delayed drug dose [Sham: n = 6; Sham + CI-1040 Early: n = 6; Sham + CI-1040 Delayed: n = 6; TeNT : n = 7; TeNT + CI-1040 Early: n = 8;TeNT + CI-1040 Delayed: n = 8.]. Rats in the drug treatment groups received 250 mg / kg / day of the non-competitive MAP2K1 / 2 inhibitor, CI-1040 (MedChemExpress), delivered in 2 g raw cookie dough. This dose effectively reduced MAPK activation as shown in Figure 1 . The early drug group received drug beginning on the day of surgery (first dose administered 2 h prior to surgery) and at 0800 h each day thereafter through post-operative day 6; these rats also received 2 g cookie dough without drug on post-operative days 15-21 . Rats in the delayed drug group received 2 g cookie dough without drug on the day of surgery through post-operative day 6, and cookie dough with drug from post-operative days 15-21 . The control group received 2 g raw cookie dough on the day of surgery through post-operative day 6 and again on post-operative days 15-21 . All rats were monitored to ensure complete consumption.

[0144] EEG and video recordings. EEG data was acquired at a sampling rate of 1000 Hz using Stellate HARMONIE software (Version 6.0, Stellate Systems Inc.) and a time- locked video recording system. Continuous video-EEG monitoring was carried out for up to 24 h per recording day. For EEG acquisition, rats were removed from their home cages and placed in individual acrylic recording cages with an EEG tether attached to the cage lid. Following attachment to the EEG tethers, animals were able to move freely throughout their recording cages. Rats received ad libitum access to food and water during recordings. EEG signal quality was assessed at the beginning of each recording by examining electrodeimpedance: electrodes with impedance exceeding 50 kQ were excluded from subsequent analyses. EEG recordings occurred at the following intervals: every 5 days (beginning on post-operative day 5 through post-operative day 35), every 7 days (post-operative days 35- 84), and monthly (post-operative day 84 through the 6-month study endpoint). Stellate EEG files were converted into European Data Format (EDF) files for subsequent visualization (EDFbrowser, Version 1.76; Free Software Foundation) and data analysis. EDF files were filtered using a 1-35 Hz bandpass filter and a fourth-order Butterworth filter. All figures containing EEG data were generated using a referential montage in EDFbrowser. EDF files were manually reviewed for recording artifacts before implementing our automated spike detection algorithm.

[0145] Barnes maze. To assess spatial memory function, Barnes maze testing was carried out using a custom-made 20-hole maze adapted from Rosenfeld et al.48Testing was conducted over three 5-day trials: Trial 1 (pre-surgery), Trial 2 (post-operative days 15-19), Trial 3 (post-operative days 56-60). On day 1 of each trial, rats were acclimated to the Barnes maze layout and directed into the escape hole prior to testing. From days 2-5 rats were allowed to explore the maze without being directed into the escape hole. Testing ended after the rat entered the escape hole or after 5 min elapsed. The test was recorded as a failure if the rat: (1) did not enter the escape hole within the 5 min testing period; or (2) jumped or fell from the maze and was not captured and returned to the center of the maze within 10 s. Between trials, the location of the escape hole was moved relative to a set of fixed visual cues within the testing room. Barnes maze testing was conducted between 0700-1100 daily, and rats were assigned to a random testing order each day. Each test was video recorded and performance metrics were assessed with EthoVision XT software (Version 16; Noldus).

[0146] Tissue collection. In preparation for perfusion, rats received an overdose of isoflurane anesthesia. Rats were perfused with 500 mL of heparinized PBS and subsequently decapitated. Brains were removed and divided into six coronal sections of either 2- or 3-mm thickness using a brain matrix. Sections were cut so that each pair of electrodes was bisected along the coronal plane. The posterior region was designated for histology (3-mm thick section). Sections designated for histology were post-fixed for 48 h in 4% PFA in PBS and cryoprotected in 30% sucrose in TBS. Tissue was flash-frozen in OCT blocks, cryosectioned into 20-pm thick sections, and stored at -20°C prior to immunohistochemical staining.

[0147] Immunohistochemistry. Tissue sections were washed in TBS and placed in blocking solution (0.05% Triton X-100, 5% goat or horse serum in TBS) for 1 h. Endogenous peroxidases were quenched with two 30 min incubations in a peroxide-sodium azide solution(0.1% sodium azide, 0.3% H2O2 in TBS). Tissue was placed in primary antibody (lba-1 : 1 :750, #019-019741 , Wako; calbindin: 1 :150, #131767, Cell Signaling Technologies; somatostatin: 1 :50, #ab111912, Abeam; parvalbumin: 1 : 4000, #ab11427, Abeam; NeuN: 1 :4000, #MAB337, Millipore; GFAP: 1 :800, #MAB360, Millipore) overnight at 4°C. Immunohistochemistry was performed using VECTASTAIN Elite ABC-HRP Kit (1 :250, goat anti-rabbit (#PK-6101 ) or horse anti-mouse (#PK-6102)). Images were acquired with an Aperio AT2 microscope (Leica) and exported in a .tif format for subsequent analysis in Imaged.

[0148] Image analysis of rat histological sections. A minimum of three animals from each treatment group and three naive animals (no surgical or drug exposure) were included in the histological analysis (Sham: n = 3; Sham + CI-1040 Early: n = 3; Sham + CI-1040 Delayed: n = 3; TeNT : n = 4; TeNT + CI-1040 Early: n = 5; TeNT + CI-1040 Delayed: n = 5; Naive: n = 3). Brightfield images were converted to grayscale and inverted in Adobe Photoshop. For each histological section, three ROIs in the left cortex were identified using Imaged software. ROIs extended from the cortical surface to the corpus callosum and were oriented so that each ROI was perpendicular to the cortical surface. The height of each ROI varied based on the cortical thickness and the width was maintained at 1500 ± 100 pixels. ROI thresholding was performed by a blinded observer using Imaged. ROIs were then subdivided into ten equally sized horizontal layers, based on the initial image height, with each subdivision representing approximately the same cortical depth across all ROIs. The Analyze Particles function in Imaged (with a particle size threshold of 100-2000 pixels2) was used to quantify the signal for each ROI. Percent area stained (signal area divided by total area) was used to compare signal intensity across regions. For each animal, we calculated the mean percent area stained within each cortical layer across all three ROIs.

[0149] Statistical analysis. An a priori power analysis using existing spike data for sham and TeNT-injected rats was conducted.39To achieve a power > 0.80 with an alpha value of 0.05 and an estimated Coehn’s do 1 .36, a minimum of four animals per experimental group was required. Additionally, a 20% rate of premature headcap loss was estimated and therefore two additional animals were included per group. Rats were assigned to groups using a random sequence generator. Investigators were blinded to experimental groups during data analysis and automated algorithms were used to mitigate bias when quantifying spikes. For histological analysis of human cortex, two-way ANOVA or mixed effects analysis with Sidak’s multiple comparisons test was used to compare control versus microlesion areas. For histological analysis of rat cortex, two-way ANOVAs with Tukey-Kramer multiple comparisons tests were used to identify significant differences between surgical (Sham vs TeNT) groups or within surgical groups and between drug treatment groups. Whencomparing individual values for mean spikes / hour, robust regression and outlier removal (ROUT) analysis (0 = 1%) was used to identify high-spiking outliers within each group (Figure 2). Prior to outlier removal, there were no significant differences between in spiking between the TeNT only and TeNT + drug treatment groups (TeNT vs. TeNT + CI-1040 Early, P= 0.99; TeNT vs. TeNT + CI-1040 Delayed, P= 0.97) or the TeNT Early vs. Delayed groups (P = 0.82). These results were driven by several animals with extremely high spike frequencies (ROUT identified outliers) likely reflecting a Type II error. Outliers were excluded from subsequent drug efficacy analyses but included in grouped behavioral analyses to elucidate relationships between excessive spiking and behavior. To analyze the relationship between Barnes maze outcomes and spiking, a linear regression analysis was performed and Pearson’s correlation coefficient was calculated. Statistical analysis was performed using R and GraphPad Prism.49A P-value cutoff of < 0.05 was considered statistically significant. All data are presented as mean ± SEM. Figures were constructed using GraphPad Prism, EDFbrowser, Adobe Illustrator and the ggplot2 package in R.

[0150] CI-1040 dose selection. A 7-day dosing study to ensure that CI-1040 could cross the blood-brain barrier and effectively inhibit MAPK activity when delivered orally was conducted (Figure 1 ). Rats were divided into three groups: 7-days CI-1040 (n = 3), 7-days cookie dough (n = 3), or naive (n = 3) (Figure 1 A). Rats in the CI-1040 group received a dose of 250 mg / kg / day of CI-1040 in 2 g raw sugar cookie dough at 0800 h each day for seven consecutive days. Rats in the cookie dough group received 2 g raw cookie dough for seven consecutive days. Rats in the naive group did not receive drug or cookie dough. Six hours after the final drug dose (or at 1400 h on day 7 for rats that did not receive drug) all rats were sacrificed via cardiac perfusion with heparinized PBS. Rats were decapitated and brains were collected. Each brain was bisected along the midline into left and right hemispheres. The left hemisphere was flash-frozen for subsequent homogenization and western blotting.

[0151] Tissue homogenization and western blotting. Fresh frozen brain tissue was added to a buffer solution (320 mM sucrose, 10 mM Tris-HCI, 1.7 mM sodium pyrophosphate, 10 mM NaF, 1 mM PMSF, 2 mM NasVC , Complete Mini Protease Inhibitor Cocktail (Roche, #11836153001 ; Basel, Switzerland)) and homogenized on ice using an immersion disperser (Kinematica AG; Malters, Switzerland). Tissue lysate was centrifuged at 2300 rpm for 10 min at 4°C. The pellet was collected and resuspended in sucrose-free buffer solution as the nuclear fraction. The supernatant was centrifuged again at 16,000 rpm for 1 h at 4°C. The supernatant was collected as the cytosolic fraction. Fractionated tissue lysate was stored at -80°C prior to western blotting.

[0152] Samples were prepared for western blotting by adding 20 pg of nuclear or cytoplasmic protein to Laemmli sample buffer (2x, BioRad, #1610737) and boiling at 95°C for 5 min. Prepared samples were loaded into a 10% TGX electrophoresis gel (BioRad, #456-1036) and run at 150 V for 1 h in Tris-Glycine-SDS buffer. Transfer components and PVDF membranes were equilibrated in a 20% methanol Tris-glycine buffer. Proteins were transferred at 100 V for 1 h and 15 min at 4°C. Membranes were removed from the transfer apparatus and placed in blocking solution (5% milk, 1% BSA, 0.1% TBST) for 1 h at room temperature, followed by overnight incubation at 4°C in primary antibody (Phospho-p44 / 42 MAPK1 / 2 1 :1500; Cell Signaling Technologies, #4370S) diluted in blocking solution.Membranes were washed in TBST and incubated with secondary antibody (HRP-linked goat anti-rabbit 1 :4000; BioRad, #170-6515) for 1 h at room temperature. ECL substrate (Pierce, #32106) was applied to the membranes for 1 min and membranes were placed into an autoradiography cassette. Membranes were exposed to film for 5 min (cytoplasmic fraction) or 10 min (nuclear fraction) and developed in an X-Omat film processer (Kodak; Rochester, NY). Membranes were stripped for 7 min with Restore PLUS Stripping Buffer (ThermoFisher, #46428) and re-blocked for 1 h at room temperature, followed by overnight incubation at 4°C in primary antibody (P-actin 1 :3000; Cell Signaling Technologies, #3700S) diluted in blocking solution. Membranes were washed in TBST and incubated with secondary antibody (HRP- linked goat anti-mouse 1 :4000; BioRad, #170-6516) for 1 h at room temperature. ECL substrate was applied to the membranes for 1 min and membranes were placed into an autoradiography cassette. Membranes were exposed to film for 5 min (cytoplasmic fraction) or 10 min (nuclear fraction) and developed in an X-Omat film processer. Films were digitally scanned and band intensities were quantified using Imaged. Unpaired two-sided Student’s t- tests were used to compare normalized protein levels of diphosphorylated MAPK1 / 2 (dpMAPK1 / 2) in the cytoplasmic and nuclear fractions of rat brain homogenate (Figure 1 B).

[0153] Algorithm-based interictal spike detection. To analyze EEG data for the presence of interictal spikes, we implemented a custom spike-detection algorithm developed using MATLAB software (R2021 b; MathWorks). The spike algorithm was adapted from previously published versions to accommodate a 1000 Hz sampling rate and optimize calculation of spike morphologic parameters.2850Briefly, EDF files were imported into MATLAB using the edfread function. EDF files were filtered with a 1-35 Hz fourth-order Butterworth bandpass filter. Each electrode was analyzed for the presence of spikes; spikes were defined as high-amplitude events with negative polarity, shorter than 200 ms in duration, with a maximum voltage greater than one standard deviation above the median background signal. Both spikes (< 70 ms) and sharp waves (70-200 ms) were included in the overall spike count. Spikes were then divided into two half waves, for which amplitude,duration, and slope were calculated. Estimation of these metrics depends on accurate identification of the start and end points (edges) of each spike. These can be identified visually as the locations of the first trough points (local minima) that occur just before (start) and after (end) the point of maximum voltage. After identifying the spike location (point of maximum voltage), edge detection was performed in three essential steps. First, the raw signals were filtered using a fourth-order Butterworth highpass filter with 7 Hz cutoff frequency to eliminate the confounding effect of low-frequency background or prominent slow-waves. Next, the filtered signals were smoothed using a 12-point moving average window. Finally, the raw, filtered, and smoothed signals were inverted, and potential edge points were identified using the findpeaks function. True edges were designated as those which were detected consistently across the raw, filtered, and smoothed signals. Moreover, the maximum amplitude and total duration for each spike was determined. For each recording day, spike counts were normalized using the total recording length (mean spikes / hour). In addition to the spikes automatically detected by our algorithm, we also manually reviewed early EEG files (prior to post-operative day 30) to look for smaller spikes that the algorithm may have missed; however, for the purposes of this study we focused only on spikes that were detected by the algorithm to ensure an unbiased assessment of drug effects.

[0154] Exploratory data analysis of spike morphology. Exploratory data analysis was used to classify spikes based on individual morphology. Cluster analysis and plotting was performed in R using the tidyverse, factoextra, and gridExtra packages. We developed an R script which takes a dataframe with all spike morphology data as input and, leveraging a user-defined set of morphologic parameters, automatically implements k-means clustering and outputs plots like those shown in Figure 3. Morphologic data are analyzed and clustered independently for each experimental group. Briefly, the data are first scaled (i.e. converted to z-scores) to ensure that parameters with different ranges of values can be fairly compared. Next, outliers were removed via the built-in outlier detection in the R boxplot() function to avoid distorting the cluster analysis. Silhouette analysis [1 ,2] was then performed to determine the optimal number clusters, after which k-means clustering was performed using the the kmeans() function (stats package). Finally, the fviz_nbclust() function (factoextra package) was used to generate the annotated plots from the k-means clustering results. Various combinations of the fundamental spike morphologic parameters (i.e. Left Amplitude, Left Duration, Right Amplitude, Right Duration) were computed by the spike detection algorithm. For simplicity, and since it provided the most clear and informative results, provided is the iteration where we included only Right Amplitude and Right Duration (Figure 3).

[0155] A population of spikes with a steep second-half slope that were seen across all animals injected with TeNT were identified but these were infrequent in sham animals. A specific cutoff value of 71 pV / ms (3.2o cutoff) was obtained by: (1) computing an individual cutoff for both sham and TeNT animals (p + 3.2o) of the second-half slope of all spikes at the L2 electrode across treatment groups; and (2) taking the average of these two cutoffs. Using this second-half slope cutoff, we were able to separate spikes observed in TeNT- injected animals from those of sham-injected animals (number of spikes above the 3.2o cutoff: TeNT: 1576 spikes, 1.73 % of total spikes; TeNT + CI-1040 Early: 671 spikes, 1.18 % of total spikes; TeNT + CI-1040 Delayed: 549 spikes, 1.36 % of total spikes; Sham: 138 spikes, 0.284 % of total spikes; Sham + CI-1040 Early: 39 spikes, 0.19 % of total spikes; Sham + CI-1040 Delayed:163 spikes, 0.537 % of total spikes). These TeNT-specific spikes were predominantly observed at the L2 injection site, with peak spiking occurring on day 70, nearly one month after the overall spike peak at day 49. Spikes above the slope cutoff were both shorter in duration and higher in amplitude than spikes below the cutoff.

[0156] Within this group of TeNT-specific spikes, a subpopulation of spikes that responded to CI-1040 treatment were identified. These drug-sensitive spikes had a maximum amplitude 1 o above the mean value for all TeNT spikes exceeding the slope threshold (amplitude cutoff = (((mean max. amplitude + 1 o of TeNT spikes > 71 pV / ms on L2) + (mean max. amplitude + 1 o TeNT Early spikes > 71 pV / ms on L2) + (mean max. amplitude + 1 o TeNT Delayed spikes > 71 pV / ms on L2)) / 3) = 1617 pV). CI-1040 treatment reduced spikes above the amplitude threshold in both early and delayed drug treatment groups, narrowing the distribution of spike amplitudes below the cutoff value (number of spikes above the 1 o cutoff: TeNT: 516 spikes, 35% of all spikes > 71 pV / ms on L2; TeNT + CI-1040 Early: 47 spikes, 7.4% of all spikes > 71 pV / ms on L2; TeNT + CI-1040 Delayed: 51 spikes, 9.7% all spikes > 71 pV / ms on L2).Example 2 Microlesions in Spiking Human Neocortex have Reduced Inhibitory Interneurons and Increased Microglia

[0157] . Twenty-nine samples, devoid of other pathologies, from high-spiking, low-spiking, and non-spiking cortical brain regions from 16 different patients were selected as shown in Figure 4A. Each sample was precisely mapped to long-term intracranial electrical brain recordings as described.43High-spiking samples had significantly more microlesions with reduced NeuN staining compared to low- / non-spiking control samples (Figure 4B-4D). This corresponds with previously published findings showing that microlesions occurred more frequently in high-spiking areas, and that the loss of nuclear NeuN within microlesions is not associated with a reduction in the number of neurons.31

[0158] Cortical layer-specific quantitative analysis of NeuN staining showed a significant reduction in layers ll-VI in high-spiking samples compared to control brain regions, with the greatest decrease observed in layer V (Figure 4C-4D). These same layers showed a significant reduction in inhibitory interneurons. Parvalbumin (PV) interneurons were decreased in layers I l-V of high-spiking human cortex as compared to low- / non-spiking cortex, with layer IV showing the largest reduction in PV staining (Figure 4E-4F). Calbindin (CB) interneurons were also reduced in microlesion areas compared to low / non-spiking regions, with the greatest loss occurring in layers Il-Ill (Figure 4G-4H). Microlesions also had increased microglia across cortical layers l-IV (Figure 4I-4J). These results suggest that cortical epileptic spiking could result from a reduction in inhibitory interneurons in deeper cortical layers leading to increased neuronal synchrony, excitability, and connectivity in superficial layers l-lll.30Example 3 Epileptic Spiking in Rodents Replicates Cytoarchitectonic Changes seen in Human Spiking Neocortex

[0159] TeNT was injected into the left somatosensory cortex, corresponding to the L2 electrode site (Figure 5A), resulting in focal epileptic spikes not seen in sham (saline- injected) animals (Figure 5B). Video and EEG data were manually reviewed for all animals to confirm that spikes occurred in the absence of seizures. High-spiking cortical regions were subsequently probed for neuronal and microglial markers (Figure 5C). Neuronal NeuN staining was significantly reduced within the left cortex of the TeNT animals compared to surgically naive controls, with the greatest reduction in layers V-VI. (Figure 5D). There were no significant differences in the number of neuronal cell bodies within layer V.

[0160] Paralleling the findings in spiking human cortex, TeNT-injected spiking animals also had increased microglial staining across layers l-IV of the left cortex relative to both sham and naive animals, with the greatest increase in layers ll-IV (Figure 5E). In these same spiking regions, TeNT animals also had a loss of inhibitory interneurons relative to naive and sham animals. For CB interneurons, TeNT animals had significant reduction in staining in layers ll / lll (Figure 5F). Interestingly, for PV interneurons, both sham and TeNT animals had a loss of staining in cortical layers IV-V (Figure 5G), suggesting that these neurons may be sensitive to even low levels of spiking activity. Closer histological analysis of NeuN staining in naive and TeNT animals showed that cytoplasmic NeuN signal persisted within neurons, but nuclear NeuN was almost completely lost (Figure 5H). A similar comparison of Iba1 histology showed that TeNT animals had an increase in the density of microglia and the number of microglial processes (Figure 5I). It was previously shown that there were no significant differences in gene expression or histology for astroglial markers between highand low-spiking areas of human tissue.31Similarly, there were no significant differences in GFAP staining in rat tissue (Figure 6). Somatostatin (SST) staining was also unaffected by spiking in both human and rat tissue (Figure 6).Example 4 Early and Delayed Treatment with CI-1040 Reduces Interictal Spiking, Interneuron Loss and Microglial Activation

[0161] Given that MAPK activation occurs in spiking human and rat cortex3039and that MAP2K inhibition can significantly prevent the development of epileptic spiking when delivered acutely39, the effect of MAP2K inhibition was examined at both early (0-7 days after TeNT injection) and delayed timepoints (15-21 days after TeNT injection) to assess the effects of MAP2K inhibition after spikes have already developed. To confirm drug-target engagement, a 7-day oral treatment with 250 mg / kg / day of the MAP2K1 / 2 inhibitor, CI-1040, led to a significant reduction in cytoplasmic diphosphorylated MAPK1 / 2 (also known as diphosphorylated ERK1 / 2) in the rat brain (P = 0.0052) (Figure 1) and a decrease in nuclear diphosphorylated MAPK1 / 2 (P = 0.0595). This demonstrates the ability of CI-1040 to cross the blood-brain barrier and inhibit MAPK activity within the brain. As discussed in the Statistical Analysis section, ROUT analysis identified six high-spiking outliers (Figure 2) that were excluded from between-group analyses of drug or toxin effects. Final sample sizes for between group analyses of drug effects were as follows: Sham, n = 6; Sham + CI-1040 Early, n = 5; Sham + CI-1040 Delayed, n = 5; TeNT, n = 7; TeNT + CI-1040 Early, n = 6; TeNT + CI-1040 Delayed, n = 5.

[0162] TeNT-injected animals had the highest spike frequency at the L2 electrode, corresponding to the toxin injection site (Figure 7A). Placement of electrodes in the sham- injected animals produced minimal spiking that was evenly distributed across the L1 , L2, R1 , and R2 electrodes (Figure 7B-7E). In both groups, spikes were rarely observed on the posterior L3 and R3 electrodes (Figure 7F-7G). Repeated-measures two-way ANOVA of mean spikes / hour by electrode showed a significant reduction in spikes in the TeNT + CI- 1040 early (P= 0.0001) and delayed groups (P< 0.0001) relative to the untreated TeNT group at the L2 electrode, suggesting that MAP2K inhibition can reduce spiking both acutely (concurrent with TeNT injection) and after spikes have started to develop (Figure 7A).

[0163] Examination of mean spikes / hour on each recording day revealed three phases of spike development in TeNT animals: an acute phase of spike development (month 1), a subacute phase of rapidly increasing spike frequency at the L2 injection site (months 2-3), and a delayed phase where spike frequency returned to sham levels (months 4-6). A mixed- effects analysis of mean spikes / hour per recording day on the L2 electrode showed asignificant increase in spike frequency within the TeNT group compared to the Sham group at post-operative days 42 (P= 0.0392), 49 (P= 0.0181), 56 (P= 0.0291), and 63 (P = 0.00132). There was also a significant increase in spike frequency in the TeNT + CI-1040 Early group compared to the Sham group at day 63 (P= 0.0331). Both early and delayed CI- 1040 treatment blunted the peak number of mean spikes / hour from post-operative days 42- 63 (Figure 7D).

[0164] CI-1040 treatment did not rescue NeuN or PV staining in TeNT animals (Figure 8A-8B; Figure 9); however, both early and delayed CI-1040 treatment significantly reduced the loss of CB staining in layers Il-Ill of the left cortex of TeNT animals, the region where CB staining was lowest within untreated TeNT animals (Figure 8C). Delayed drug treatment in TeNT animals significantly reduced Iba1 staining relative to untreated animals, and early drug treatment caused a trending reduction in Iba1 staining in cortical layer IV (P= 0.0775) (Figure 8D). In contrast, early drug-treated sham animals had significantly less NeuN staining compared to naive animals and significantly more Iba1 staining compared to nondrug sham and naive animals (Figure 3).Example 5 Spikes in TeNT-injected Rats are Morphologically Distinct and Highly Sensitive to CI- 1040 treatment

[0165] . Spikes were subclassified based on their morphology. As shown in Figure 8E, each waveform was approximated as a triangle from which the amplitude, duration, and slope in both the first half (left) and second half (right) could be computed. A population of spikes on the L2 electrode that distinguished TeNT-injected animals from sham animals was identified. Specifically, spikes with a second-half slope of at least 71 pV / ms (slope cutoff) were observed in all animals injected with TeNT but present in only half of the sham animals. These spikes accounted for a larger fraction of the total spike count in TeNT animals compared to shams (1 .36% vs 0.284%). Similar results were observed when comparing TeNT and sham animals treated with CI-1040. TeNT animals in both early and delayed drug treatment groups showed greater percentages of spikes above the slope cutoff than the corresponding sham groups (Early: 1.18% (TeNT) vs. 0.19% (Sham); Delayed: 1.36% (TeNT) vs 0.537% (Sham)). These TeNT-specific spikes were predominantly observed at the L2 injection site, with peak spiking occurring on day 70, nearly one month after the overall spike peak at day 49. Spikes above the slope cutoff had both shorter duration and higher amplitude than spikes below the cutoff.

[0166] Within this group of TeNT-specific spikes, a subpopulation of spikes that responded to CI-1040 treatment was observed (Figure 8F). These drug-sensitive spikes hada maximum amplitude of at least 1617 pV (amplitude cutoff), which was one standard deviation above the mean value for all TeNT spikes (across all treatment groups) that exceeded the slope cutoff (Figure 10). CI-1040 treatment reduced spikes above the amplitude cutoff in both early and delayed drug treatment groups, which showed narrower distributions of spike amplitudes (Figure 8G).Example 6Drug-sensitive Spikes are Correlated with Impaired Spatial Memory Performance.

[0167] To understand the relationship between increased spiking and behavioral performance, the mean spikes / hour on post-operative day 63 were correlated, the EEG recording day closest to the final day of Barnes maze trial 3, with Barnes maze performance outcomes across all groups. Here, high-spiking outliers were included to further elucidate the effects of increased spiking on behavior. There was a significant correlation between increased spiking and increased distance traveled across all animals on the final day of Barnes maze trial 3 (Figure 8H). Similarly, increased spiking was correlated with an increased number of off-target errors, defined as exploration of any maze hole other than the escape hole (Figure 8I). It was then determined whether drug-sensitive spikes (second-half slope > 71 pV / ms, maximum amplitude > 1617 pV) also correlated with Barnes maze performance. The relationship between increased drug-sensitive spiking and increased distance traveled was not significant (Figure 8J), but there was a significant correlation between drug-sensitive spikes and off-target errors: more spikes correlated with poorer performance (Figure 8K). There were no significant differences in Barnes-maze performance across time between surgical groups or drug treatment groups.Example 7 Spatially Distinct Electrical, Transcriptional, and Molecular Networks Define the Human Epileptic Brain

[0168] Patients who fail to respond to seizure medications greatly benefit from surgical removal of epileptic brain regions. These patients undergo long-term intracranial recordings to identify regions of seizure onset and frequent spiking between seizures. The relationship between brain regions generating ‘interictal’ spikes and seizures is not known, but spikes occur near the site of seizure onset and are associated with significant brain dysfunction. Comparing electrically-defined seizure and spiking brain regions from 24 epilepsy patients by transcriptomic analyses revealed distinct but highly consistent transcriptional profiles implicating unique signaling pathways in seizure versus spiking human cortex. Only a small subset of genes were common to both seizures and spiking. While the mammalian target of rapamycin - mTOR pathway was implicated in seizure brain regions and localized to layersll / lll and V / VI cortical neurons, MAPK and CREB activation were implicated in spiking brain regions and expressed primarily in superficial neuronal layers. Mechanistically, an animal model that generates either seizure or spiking brain regions showed parallel patterns of mTOR and CREB activation in the same neuronal layers. Seizure and spiking brain regions differentially expressed a large proportion of genes with known mutations in genetic epileptic and neurocognitive disorders. Two of these genes, CACNA2 and KCNQ2, colocalized within mTOR activated neurons in seizures-producing brain regions. Taken together, these findings demonstrate highly unique electrical, transcriptional, and molecular differences in brain regions that produced seizures and spiking within human epileptic networks implicating distinct and combined therapeutic strategies.

[0169] An integrated analysis was carried out to identify cortical areas that produce seizure or epileptic spiking ahead of surgical resection of these brain regions as a means to surgically treat patients with intractable seizures. A schematic of the integrated analysis is provided in Figure 11 . Seizing and spiking areas were electrically defined together with non- and / or low-spiking areas that were part of the planned resection and used as internal controls. All of the cortical layers were carefully dissected away from white matter, and alternative strips from each block of tissue corresponding to a different brain region (seizure, spiking, control) were used for histology and omics. Results from the genomic analysis were analyzed, validated, and tested for therapeutic intervention.

[0170] From the genomic analysis, it was determined that seizure genes were different than spiking genes. The heatmap generated from the genomic analysis provides differentially expressed genes in various subjects, wherein seizure genes and spiking genes were shown in function of their differential expression (Figure 12A). The upper quadrant corresponds to spiking genes, the lower quadrant corresponds to seizure genes. Each row corresponds to the expression of a gene, and each column corresponds to a sample. The columns were grouped into four quadrants. The first two quadrants were from the 20 patients analyzed by microarrays; the first quadrant was from brain parts that are non-spiking, while the samples provided in the second quadrant were from brain parts with high spiking brain activity. Quadrants three and four were from 14 patients analyzed by RNAseq, and correspond to samples from the brain regions without and with seizures, respectively. Further provided is a Venn diagram representing the seizure genes and the spiking genes, indicating a minor overlap between the two transcriptomes (Figure 12B). The unexpected finding was that a majority of identified seizure genes and spiking genes were entirely different, with a small overlap of only 526 genes.

[0171] Pathway analysis of differentially expressed gene pathways associated with spiking and seizures (as indicated by -log (p value)) revealed that seizure and spikingpathway signatures were distinct. The identified pathways for spiking include transcriptional activity of the SMAD2 / SMAD3:SMAD4 heterotrimer, NGF-stimulated transcription, the protein ubiquitination pathway, ATF4 activating genes in response to endoplasmic reticulum stress, ABRA signaling pathway, TNF signaling, ERK / MAPK signaling, neuroinflammation signaling pathway, EIF2AK1 (HRI) response to heme deficiency, and TEC kinase signaling. Pathway analysis further revealed that seizure pathway signatures include eukaryotic translation, selenoamino acid metabolism, EIF2AK4 (GCN2) response to amino acid deficiency, ROBO receptor signaling, EIF2 signaling, mTOR signaling, Netrin signaling, GP6 signaling, ROBO SLIT signaling pathway, and regulation of elF4 and p70S6K signaling (Figure 13A). In conclusion, the most significant pathway activations were MAPK / ERK pathway associated with a spiking (Figure 13B), and mTOR activation associated with a seizures (Figure 13C).Example 8 Sustained mTOR Activation was Observed in Specific Cortical Layers in Seizure Onset Zones

[0172] Western blotting was conducted on samples of cortical brain lysates from control (spiking only) and seizure areas from 11 patients using mTOR specific antibodies (Figure 14A). Quantitative analyses showed specific increases in phospho-mTOR levels in seizure tissues over control tissues, while the total mTOR levels did not change (Figures 14B-14C). A similar increase in the levels of activated p70S6kinase downstream of mTOR was also detected via western blotting (Figures 14D-14F).

[0173] Immunostaining for phospho-mTOR confirmed increased activation, especially in layers ll / lll and V / VI of the cortex, as shown in Figure 14G-14I, and the data in Figure 141 was quantified and presented in Figure 14J. Immunostaining confirmed that phospho-CREB was activated in cortical areas with high spiking, but not seizure areas (Figure 14H). A similar layer-specific increase in phospho-P70S6kinase is shown by immunostaining in Figure 14K, and that data was quantified in Figure 14L. Thus, this data demonstrated that sustained mTOR activation was observed in specific cortical layers that correspond to seizure onset zones.

[0174] Analysis of the tetanus toxin (TeNT)-induced seizure rat model recapitulates that animals with seizures show sustained mTOR activation. 1 pL volume of tetanus toxin (80 or 250 ng / pL) or its vehicle (sterile phosphate-buffered saline, PBS) was administered into layer V of the left somatosensory cortex (LS) using a Hamilton syringe. The injection was performed 1 .5 mm below the dura at the following doses: 80 ng / pL and 250 ng / pL As shown in Figure 15, both doses of TeNT injected resulted in interictal spiking, but seizures wereonly observed in mice injected with 250 ng / pL, and the vehicle receiving group served as the control. Electroencephalogram (EEG) recordings were conducted and recorded starting 2 days after the intracortical injection of tetanus toxin and continued for up to 6 months. Representative spike and seizure tracings recordings are provided in Figure 15B.Increased phosphor-mTOR staining was seen in tissues that produced seizures, but not spiking or control tissue as shown in Figure 15C, and this data is quantified in Figure 14D. No changes in total mTOR levels were observed. In rats with spiking, but no seizures, there was increased pCREB staining in the superficial layers as compared to the seizure and control tissue (Figures 15E-15F). This analysis demonstrated that this high dose tetanus toxin-induced seizure model recapitulates that seizures are associated with increase mTOR activation while spiking is associated with increased MAPK and CREB activation in rat cortex in the same cortical layers similar to epileptic human cortex.

[0175] This analysis determined that seizure and spiking genes overlap with OMIM genes associated with genetic epilepsy and neurocognitive developmental disorders. In Figure 16A, a Venn diagram is provided, showing the overlap between spiking and seizure genes with OMIM genes known to be involved in epilepsy and neurocognitive disorders. Figure 16B provides a list of ion channel and excitability genes that were upregulated or downregulated in the seizure and spiking gene dataset. Genes in italics are known to be transcriptionally regulated by the mTOR pathway. Two such genes, CACNA1C (Figure 16C) and KCNQ2 (Figure 16D), showed increased co-localization with phosphor-mTOR staining in seizure areas of human epileptic cortex, as compared to control areas. 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Claims

What is claimed is:1 . A method of treating a subject with an existing epileptic disorder or reducing the likelihood of developing epilepsy in a subject without an existing epileptic disorder following a brain injury, the method comprising administering to the subject an effective amount of CI- 1040, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1 wherein administration of an effective amount of CI-1040 reduces the magnitude and / or number of interictal epileptic spikes in the subject.

3. The method of claim 1 wherein administration of an effective amount of CI-1040 reduces in cognitive decline in the subject.

4. The method of any one of claims 1-3, wherein the brain injury results from trauma, stroke, infection, developmental abnormality, or a tumor.

5. The method of any one of claims 1 -4, wherein CI-1040 or a pharmaceutically acceptable salt thereof is administered orally or intravenously.

6. The method of any one of claims 1 -4, wherein CI-1040 or a pharmaceutically acceptable salt thereof is administered orally.

7. The method of any one of claims 1 -6, wherein the CI-1040 or a pharmaceutically acceptable salt thereof, is administered in combination with a second therapeutic agent, wherein the second therapeutic agent exhibits anti-epileptic properties.

8. The method of claim 7, wherein the second therapeutic agent is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway.

9. The method of claim 8, wherein the inhibitor of the mTOR signaling pathway is rapamycin or an analogue thereof.

10. The method of claim 8, wherein the inhibitor of mTOR signaling pathway is sirolimus, everolimus, temsirolimus or ridaforolimus.11 . The method of any one of claims 7-10, wherein CI-1040 or a pharmaceutically acceptable salt thereof and the second therapeutic agent are administered simultaneously.

12. The method of any one of claims 7-11 , wherein CI-1040 or a pharmaceutically acceptable salt thereof and the second agent are administered separately.

13. A composition comprising i) CI-1040 or a pharmaceutically acceptable salt thereof and ii) a second therapeutic agent that exhibits anti-epileptic properties.

14. The composition of claim 13 wherein the second therapeutic agent is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway.

15. The composition of claim 14 wherein the inhibitor of the mTOR signaling pathway is rapamycin or an analogue thereof.

16. The method of claim 14, wherein the inhibitor of mTOR signaling pathway is sirolimus, everolimus, temsirolimus or ridaforolimus.

17. The composition of any one of claims 13-16, wherein the CI-1040 or a pharmaceutically acceptable salt thereof and the second therapeutic agent are administered simultaneously.

18. The composition of any one of claims 13-17, wherein the CI-1040 or a pharmaceutically acceptable salt thereof and the second therapeutic agent are administered separately.

19. The composition of any one of claims 13-18 wherein the CI-1040 or a pharmaceutically acceptable salt thereof and the second therapeutic agent are administered as an admixture.

20. A method of treating a subject with an existing epileptic disorder or reducing the likelihood of developing epilepsy in a subject without an existing epileptic disorder following a brain injury, comprising administering the composition of any one of claims 13-19.21 . Use of a composition of any one of claims 13-19 for the preparation of a medicament for treating a subject with an existing epileptic disorder or reducing the likelihood of developing the epilepsy in a subject without an existing epileptic disorder following a brain injury.

23. A composition for use in treating a subject with an existing epileptic disorder or reducing the likelihood of developing epilepsy in a subject without an existing epileptic disorder following a brain injury, wherein the composition comprises the composition of any one of claims 13-19.

24. Use of an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating a subject with an existing epileptic disorderor reducing the likelihood of developing of epilepsy in a subject without an existing epileptic disorder following a brain injury.

25. The use of claim 24 wherein administration of an effective amount of CI-1040 reduces the magnitude and / or number of interictal epileptic spikes in the subject.

26. The use of claim 24 wherein administration of an effective amount of CI-1040 reduces cognitive decline in the subject.

27. The use of any one of claims 24-26, wherein the brain injury results from trauma, stroke, infection, development abnormality or a tumor.

28. The use of any one of claims 24-27, wherein CI-1040 or a pharmaceutically acceptable salt thereof is formulated for oral or intravenous administration.

29. The use of any one of claims 24-27, wherein CI-1040 or a pharmaceutically acceptable salt thereof is formulated for oral or intravenous administration.

30. The use of any one of claims 24-29, wherein medicament is administered in combination with a second therapeutic agent, wherein the second therapeutic agent exhibits anti-epileptic properties.31 . The use of claim 30, wherein the second therapeutic agent is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway.

32. The use of claim 31 , wherein the inhibitor of the mTOR signaling pathway is rapamycin an analogue thereof.

33. The use of claim 31 wherein the inhibitor of the mTOR signaling pathway is sirolimus, everolimus, temsirolimus or ridaforolimus.

34. The use of any one of claims 30-33, wherein the medicament and the second therapeutic agent are formulated for simultaneous administration.

35. The use of any one of claims 30-33, wherein medicament and the second agent are formulated for separate administration.

36. A composition for use in for treating a subject with an existing epileptic disorder or reducing the likelihood of developing epilepsy in a subject without an existing epileptic disorder following a brain injury, wherein the composition comprises an effective amount of CI-1040, or a pharmaceutically acceptable salt thereof.

37. The composition of claim 36 wherein administration of an effective amount of CI- 1040 reduces the magnitude and / or number of interictal epileptic spikes in the subject.

38. The composition of claim 36 wherein administration of an effective amount of CI- 1040 reduces in cognitive decline in the subject.

39. The composition of any one of claims 36-38, wherein the brain injury results from trauma, stroke, infection, developmental abnormality, or tumors.

40. The composition of any one of claims 36-39, wherein CI-1040 or a pharmaceutically acceptable salt thereof is formulated for oral or intravenous administration.41 . The composition of any one of claims 36-39, wherein CI-1040 or a pharmaceutically acceptable salt thereof is formulated for oral or intravenous administration.

42. The composition of any one of claims 36-41 , wherein medicament is administered in combination with a second therapeutic agent, wherein the second therapeutic agent exhibits anti-epileptic properties.

43. The composition of claim 42, wherein the second therapeutic agent is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway.

44. The composition of claim 43, wherein the inhibitor of the mTOR signaling pathway is rapamycin or an analogue thereof.

45. The composition of claim 43, wherein the inhibitor of mTOR signaling pathway is sirolimus, everolimus, temsirolimus or ridaforolimus.

46. The composition of any one of claims 42-45, wherein the medicament and the second therapeutic agent formulated for simultaneous administration.

47. The composition of any one of claims 42-45, wherein medicament and the second agent are formulated for separate administration.

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