Method for treating epilepsy

Abstract

Provided herein are alpha? nicotinic receptor specific activators as anti-epileptogenic agents effective against epilepsy or epileptogenesis and associated comorbidities in a subject after a traumatic brain injury. Provided herein are methods for treating epilepsy in a subject, preventing seizures in a subject at risk for the same and preventing epileptogenesis in a traumatic brain injured subject and associated comorbidities. a7-nAChR activators are administered to the subject alone or in combination with established anti-epileptic or anti- epileptogenic drugs.

Description

[0001] METHOD FOR TREATING EPILEPSY

[0002] Cross-Reference to Related Applications

[0003] This international application claims benefit of priority under 35 C.F.R. §119(e) of provisional application U.S. Serial No. 63 / 562,305, filed March 7, 2024, the entirety of which is hereby incorporated by reference.

[0004] Field of the Invention

[0005] The present invention relates generally to the fields of neurology and pharmacotherapy. More specifically, the present invention relates to a method of treating, preventing or delaying progression of epilepsy and epileptic seizures with the use of alpha? nicotinic acetylcholine receptor (a7-nAChR) specific activators, such as, agonists and positive allosteric modulators (1 ).

[0006] Description of the Related Art

[0007] Epilepsy is a disorder characterized by recurrent spontaneous seizures (SRS) that are caused by abnormal electrical activity often involving the cortex and hippocampus in the central nervous system. In epilepsy, seizures can be severe, last for several minutes or hours, and may occur with high frequency even daily. Most seizures are convulsive, with loss of consciousness, and irregular and involuntary muscle twitching and muscle spasms. In severe cases, patients can experience status epilepticus when a single seizure is long-lasting which results in severe brain injuries and is potentially fatal. In general, life expectancy in subjects with epilepsy is reduced.

[0008] Anti-epileptic drugs are available for the treatment of epilepsy which target brain inhibitory GABA-A receptors or block voltage gated sodium channels to suppress neuronal activity. With long-term use, patients may develop tolerance to current anti-epileptic drugs making the medications ineffective. Furthermore, some patients do not respond to current medications. All currently approved anti-epileptic drugs have significant adverse effects, cause sedation and sensorimotor impairment and carry significant teratogenic risks to the fetus. A percentage of patients have treatment-resistant seizure disorders for which current anti-epileptic medications do not work. In the potentially fatal emergency situation of status epilepticus, patients need additional treatment options to prevent long-term harm and death. In addition, current anti-epileptic drugs have severe adverse effects including sedation and sensorimotor impairments. During pregnancy, current anti-epileptic drugs carry significant teratogenic risks for the unborn child which need to be balanced against the need to control seizures in the mother. Most cases of epilepsies are acquired following a trigger event such as a severe traumatic brain injury (TBI). After the initial brain insult, a latent period of variable length (months to years in humans) follows, during which the process of epileptogenesis takes place until spontaneous recurrent seizures (SRS) occur which define the neuropathological condition of epilepsy. During epileptogenesis, reorganization of excitatory and inhibitory neural connections takes place in brain networks including axonal sprouting and loss of inhibitory interneurons, which subsequently manifest in hyperexcitable networks and a propensity for spontaneous seizures. Neuroinflammation is causally involved in epileptogenesis and facilitates network restructuring. This latent period provides a window of opportunity to prevent, reduce or reverse the process of epileptogenesis, and reduce neuroinflammation following a traumatic brain injury. There are currently no pharmacological intervention therapies (2,3).

[0009] Thus, there is a need in the art for a pharmacological treatment option to prevent, reduce or delay the process of epileptogenesis in at-risks patients. The present invention fulfills this long-standing need in the art.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention shows that a7-nAChR agonists or positive allosteric modulators may be used in the treatment, prevention or delay of progression of epilepsy thereby acting like an anti-epileptic drug, and that a7-nAChR agonists or positive allosteric modulators may be used in a subject with a traumatic brain injury to prevent or delay processes of epileptogenesis leading to spontaneous recurrent seizures and to reduce comorbidities associated with a traumatic brain injury, thereby acting as an anti-epileptogenic drug.

[0012] The present invention is directed to a method for preventing an onset of or treating an epilepsy in a subject in need thereof. In the method an amount of a positive allosteric modulator pharmaceutically effective to act on an alpha7 nicotinic receptor is administered to the subject. The present invention is directed to a related invention further comprising administering at least one other drug effective to treat epilepsy in the subject. The present invention is directed to another related method further comprising repeating the administering step at least once.

[0013] The present invention also is directed to a method for preventing or delaying progression of an epilepsy in a subject in need thereof. In the method a pharmaceutically effective amount of an a7 nAChR agonist is administered to the subject. The present invention is directed to a related method further comprising repeating the administering step at least once. The present invention is directed further to a method for preventing a seizure in a subject at risk for having one. In this method, a pharmaceutically effective amount of at least one alpha? nicotinic receptor agonist is administered to the subject. The present invention is directed to a related invention further comprising administering at least one other drug to the subject effective to prevent a seizure in the subject. The present invention is directed to another related method further comprising repeating the administering step at least once.

[0014] The present invention is directed further still to a method for preventing an onset of post-traumatic epilepsy or at least one co-morbidity thereof in a subject with the traumatic brain injury. In this method, a pharmaceutically effective amount of at least one anti- epileptogenic drug that is a a7 nicotinic receptor agonist is administered to the subject. The present invention is directed to a related invention further comprising administering at least one other drug to the subject effective to prevent an onset of post-traumatic epilepsy to the subject. The present invention is directed to another related method further comprising repeating the administering step at least once.

[0015] The present invention is directed further still to a method for preventing epileptogenesis and associated cognitive decline and memory loss in a subject with a traumatic brain injury. In the method a pharmaceutically effective amount of an anti- epileptogenic drug that is a a7 nicotinic receptor activator, is administered to the subject over a period of time following the traumatic brain injury. The present invention is directed to a related method further comprising re-administering the anti-epileptogenic drug at least once to the subject during the period of time.

[0016] Other and further aspects, features, benefits, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] So that the matter in which the above-recited features, advantages and objects of the invention, as well as others that will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof that are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

[0019] FIG. 1 is a schematic diagram of the homomeric pentamer that forms the alpha? nAChR cation channel and depicts binding sites for agonists, for example, acetylcholine, and for a positive allosteric modulator (PAM), for example, PNU-120596 (1-(5-chloro-2,4- dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea).

[0020] FIG. 2 illustrates proposed mechanisms of actions that are mediated by a7 nAChR activation resulting in control of seizures, reduced cell loss, reduced inflammatory responses and improve functional recovery after a traumatic brain injury (TBI). An a7-nAChR specific positive allosteric modulator, i.e. PNU-120596, enhances a7-nAChR activation, and thus, improves outcomes.

[0021] FIGS. 3A-3C show that a7 nAChR PAM, PNU-120596, reduces acute seizures. FIG. 3A provides a bar chart showing that the positive allosteric modulator, PNU-120596 (PNU), suppresses acute seizures induced with 6-Hz corneal stimulation (CS). FIG. 3B: shows a bar graph demonstrating that PNU-120596 dose-dependently suppressed CS-induced seizures with a low and a high dose of the drug. FIG. 3C: shows a line graph of the time course of an intraperitoneal (i.p.) injection of PNU-120596’s protection against acute psychomotor seizures. The results demonstrate anti-epileptic activity of PNU-120596.

[0022] FIGS. 4A-4B show long-term improved cognitive functions with PNU-120596 daily treatment for 21 days after a TBI. FIG. 4A shows a line graph of the improved spatial learning performance in the Morris Water Maze in TBI mice treated with PNU-120596 four months after the traumatic brain injury. FIG. 4B: shows a bar graph of % time spent exploring a novel object in the Novel Object Recognition Task performed at one to four months after TBI.

[0023] FIG 5 shows a bar graph of improved borrowing activity in a mouse traumatic brain injury cohort treated with PNU-120596 compared to the vehicle-treated traumatic brain injury cohort at four months after traumatic brain injury.

[0024] FIG. 6 shows a line graph of improved Body Weight in 120596-PNU-treated TBI mice compared to vehicle-treated traumatic brain injury mice four months after injury.

[0025] FIG. 7 shows a line graph of rapid recovery of neurological and motor functions with daily injections of 120596-PNU as indicated by improvement in the composite neuroscore.

[0026] FIGS. 8A-8B show Improved whole-body balance and motor coordination. FIG. 8A shows a line graph of the percentage of mice that completed the beam walk at day one, three and seven after traumatic brain injury. FIG. 8B shows a line graph demonstrating that over time the vehicle-treated traumatic brain injury cohort slowly improves and the PNU-TBI mice maintain their improved performance.

[0027] FIG. 9 shows a bar graph demonstrating performance in the Rotarod measuring the latency to fall off a rotating beam during a one-minute test. The performance continued to improve over time in traumatic brain injury mice receiving three weeks of PNU-120596 treatment after traumatic brain injury. FIGS. 10A-10D are bar graphs of reduced serum protein levels of markers of peripheral immune activation at day 7 post traumatic brain injury with 6 days of PNU-120596 treatment for interleukin-2 (IL-2) (FIG. 10A), tumor necrosis factor-alpha (TNF-a) (FIG.10B), granulocyte-macrophage colony-stimulating factor (GM-CSF) FIG. 10C, interferon-gamma (IFN-y) (FIG.10D).

[0028] FIGS. 11 A-11 B show bar graphs of home cage burrowing activity in mice at baseline (no injection), after i.p. injection of vehicle or PNU-120596 after two hours (FIG. 11A) and after 18 hours (FIG. 11 B) of burrowing, demonstrating that the drug is well tolerated.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] As used herein, the articles “a” and “an” when used in conjunction with the term “comprising” in the claims and / or the specification, may refer to “one”, but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”. Some embodiments of the invention may consist of or consist essentially of one or more elements, components, method steps, and / or methods of the invention.

[0031] As used herein, the term “or” in the claims refers to “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or”.

[0032] As used herein, the terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included.

[0033] As used herein, the terms “consists of” and “consisting of” are used in the exclusive, closed sense, meaning that additional elements may not be included.

[0034] As used herein, the term “includes” or “including” is used herein to mean “including, but not limited to”. The terms “includes”, “including” and “including but not limited to” are used interchangeably.

[0035] As used herein, the term “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., ± 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.

[0036] As used herein, the term “activator” refers to an a7 nAChR agonist or a a7 nAChR specific positive allosteric modulator (PAM). An a7 nAChR agonist is a compound that binds to a receptor comprised of a7 nAChR subunits in vivo and in vitro and is activating the receptor to perform a physiological function. An a7 nAChR positive allosteric modulator is a compound that binds to the a7 nAChR in vivo and in vitro and is potentiating the response to an agonist that activates the receptor to perform a physiological function.

[0037] In one embodiment of the present invention, there is provided a method for preventing an onset of or treating an epilepsy in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a positive allosteric modulator of an alpha? nicotinic receptor. Further to this embodiment the method comprises administering at least one other drug effective to treat epilepsy in the subject. Further to this embodiment, the method comprises repeating the administering step at least once.

[0038] In all embodiments, the positive allosteric modulator may be for an alpha? nicotinic receptor and may be an anti-seizure drug, an anti-epileptogenic drug, an anti-inflammatory drug, or a neuroprotective drug or a combination thereof. Particularly, the positive allosteric modulator is N-(5-Chloro-2,4-dimethoxyphenyl)-N’-(5-methyl-3-isoxazolyl)-urea (PNU- 120596), TQS, A-867744, AVL-3288, JNJ-1930942 or NS1738, or a combination thereof.

[0039] In all embodiments, the epilepsy may be selected from the group consisting of a traumatic brain injury-induced epilepsy, a genetic epilepsy, a chemical exposure-related epilepsy, a viral-induced epilepsy, a COVID 19 related epilepsy, an epilepsy induced by stroke, tumor or anoxia, an epilepsy related to drug withdrawal or alcohol use, and an acquired epilepsy. In one aspect, the epilepsy may be acquired from prolonged febrile seizures, status epilepticus, concussion, cardiac dysfunction, or hormonal impairment. In all embodiments, treating the epilepsy concomitantly delays progression thereof.

[0040] In another embodiment of the present invention, there is provided a method for preventing or delaying progression of an epilepsy in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of an a7 nAChR agonist. Further to this embodiment, the method comprises repeating the administering step at least once. In this embodiment delaying progression of the epilepsy concomitantly may treat the epilepsy.

[0041] In both embodiments the a7 nAChR agonist may be an anti-epileptic drug. Particularly, the anti-epileptic drug is N-(5-Chloro-2,4-dimethoxyphenyl)-N’-(5-methyl-3-isoxazolyl)-urea (PNU-120596), PNU-282987, FRM-17874, GTS-21 , ABT-126, TC-5619, AVL-3288, A- 867744, or JNJ1930942 or a combination thereof.

[0042] In both embodiments, the epilepsy may be selected from the group consisting of a traumatic brain injury-induced epilepsy, a genetic epilepsy, a chemical exposure-related epilepsy, a viral-induced epilepsy, a COVID 19 related epilepsy, an epilepsy induced by stroke, tumor or anoxia, an epilepsy related to drug withdrawal or alcohol use, and an acquired epilepsy. In an aspect thereof, the epilepsy may be acquired from prolonged febrile seizures, status epilepticus, concussion, cardiac dysfunction, or hormonal impairment. In yet another embodiment of the present invention, there is provided a method for preventing a seizure in a subject at risk for having one, comprising administering to the subject a pharmaceutically effective amount of at least one alpha? nicotinic receptor agonist. Further to this embodiment, the method comprises administering at least one other drug to the subject effective to prevent a seizure in the subject. In another further embodiment, the method comprises repeating the administering step at least once.

[0043] In all embodiments, the alpha? nicotinic receptor agonist may be an anti-epileptic drug. Particularly, the anti-epileptic drug is N-(5-Chloro-2,4-dimethoxyphenyl)-N’-(5-methyl-3- isoxazolyl)-urea (PNU-120596), TQS, A-867744, AVL-3288, JNJ-1930942, NS1738, PNU- 28987, FRM-17874, GTS-21 , ABT-126, TC-5619, AVL-3288, or A-867744, or a combination thereof. Also in all embodiments, the alpha? nicotinic receptor may be an alpha? nicotinic acetylcholine receptor.

[0044] In addition, in all embodiments, the subject may have a traumatic brain injury. In an aspect thereof the alpha? nicotinic receptor activator may be pharmaceutically effective for reducing in the subject with the traumatic brain injury an onset of post-traumatic epilepsy or at least one comorbidity associated therewith that is anxiety, depression, cognitive dysfunction, or motor dysfunctions. Furthermore, the seizure may be associated with an epilepsy selected from the group consisting of a genetic epilepsy, a chemical exposure-related epilepsy, a viral-induced epilepsy, a COVID 19 related epilepsy, an epilepsy induced by stroke, tumor or anoxia, an epilepsy related to drug withdrawal or alcohol use, and an acquired epilepsy.

[0045] In yet another embodiment of the present invention, there is provided a method for preventing an onset of post-traumatic epilepsy or at least one co-morbidity thereof in a subject with the traumatic brain injury, comprising administering to the subject a pharmaceutically effective amount of at least one anti-epileptogenic drug that is a a7 nicotinic receptor agonist. Further to this embodiment the method comprises administering at least one other drug effective to prevent an onset of post-traumatic epilepsy to the subject. In another further embodiment the method comprises re-administering the a7 nicotinic receptor agonist at least once to the subject.

[0046] In all embodiments, the a7 nicotinic receptor agonist may be a nicotinic receptor agonist may be PNU-282987, FRM-17874, GTS-21 , ABT107, ABT-126, TC-5619, PHA- 543613 or PHA-568487, or a combination thereof. In all embodiments, the comorbidity may be anxiety, depression, cognitive dysfunction, or motor dysfunction.

[0047] In yet another embodiment of the present invention, there is provided a method for preventing epileptogenesis and associated cognitive decline and memory loss in a subject with a traumatic brain injury, comprising administering to the subject over a period of time a pharmaceutically effective amount of an anti-epileptogenic drug that is an a7 nicotinic receptor activator following the traumatic brain injury. Further to this embodiment, the method comprises re-administering the anti-epileptogenic drug at least once to the subject during the period of time.

[0048] In both embodiments the anti-epileptogenic drug may be at least one activator selected from the group consisting of PNU-120596, TQS, A-867744, AVL-3288, NS1738, PNU- 282987, FRM-17874, GS021 , ABT-126, TC-5619, AVL-3288, A-86774, and JNJ1930942. In both embodiments, the anti-epileptogenic drug may be pharmaceutically effective to modulate an activity of an alpha7 nicotinic acetylcholine receptor. In addition, the step of administering the anti-epileptogenic drug may further reduce at least one comorbidity associated with the traumatic brain injury in the subject. Particularly, the comorbidity may be anxiety, depression, cognitive dysfunction, or motor dysfunction. In one aspect of both embodiments the anti- epileptogenic drug may be a neuroprotective drug effective to prevent tissue and neuronal loss in the subject. In another aspect the anti-epileptogenic drug may be effective to prevent or reduce inflammation in the brain and in the periphery thereof.

[0049] The present invention relates to pharmacological uses of alpha7 nicotinic acetylcholine receptor (a7-nAChR) specific activators, i.e. agonists and positive allosteric modulators for the treatment of epilepsy from any cause, and of epileptogenic processes following a traumatic brain injury (TBI) to prevent, delay or reduce the development of spontaneous recurrent seizures and associated comorbidities following TBI. Table 1 lists specific alpha7 nAChR activators, positive allosteric modulator (PAM) and agonists.

[0050] TABLE 1

[0051] Thus, provided herein are methods for treating, reducing or preventing seizures or epileptogenesis in a subject, for example, but not limited to, a subject with or without an epilepsy or associated condition such as a subject with a traumatic brain injury and / or associated comorbidities. Examples of epilepsy are traumatic brain injury-induced epilepsy and related comorbidities, a genetic epilepsy, a chemical exposure-related epilepsy, a viral- induced epilepsy, a COVID 19 related epilepsy, an epilepsy induced by stroke, tumor or anoxia, an epilepsy related to drug withdrawal or alcohol use, and an acquired form of epilepsy such as prolonged febrile seizures, status epilepticus, concussion, cardiac dysfunction and hormonal impairment.

[0052] These conditions may be treated, prevented or attenuated via administration one or more times of at least one pharmaceutically or therapeutically effective nicotinic receptor activator agent, i.e. an agonist or positive allosteric modifiers, or anti-epileptic drugs or agents that modify or effect an activity of, target or activate an alpha? nAChR which is the alpha? nicotinic acetylcholine receptor. Examples of these modifiers, activators and anti- epileptogenic drugs may be, but are not limited to, PNU-120596, TQS, A-867744, AVL-3288, NS1738, PNU-282987, FRM-17874, GTS-21 , ABT-126, TC-5619, AVL-3288, A-867744, or JNJ1930942 or pharmaceutical compositions thereof suitable for oral, liquid, or injectable administration. These drugs may be used alone or in combination with other antiepileptic drugs.

[0053] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

[0054] EXAMPLE 1

[0055] Materials and methods g7 nAChR

[0056] The o7 nAChR is a pentameric homomer composed of five alpha7 protein subunits that form a cation channel (FIG. 1 ). The o7 nAChR has binding sites for agonists, i.e. the endogenous neurotransmitter acetylcholine, and for allosteric modulators, i.e. PNU-120596. Binding of an agonist to the receptor activates the o7 nAChR, and this activation is enhanced by a positive allosteric modulator which binds to a different site of the receptor.

[0057] Activation of o7 nAChRs (i) increases activity of inhibitory interneurons and inhibitory networks, thereby controlling excitatory synaptic activity (4), (ii) increases actions of the antiinflammatory cholinergic pathway, thereby regulating the peripheral immune response (5), (iii) suppress neuroinflammatory activity, thereby reducing astrogliosis and microgliosis in the brain (6), and (iv) activates neuroprotective pathways (7), thereby reducing neuronal loss in epilepsy disorders and after a traumatic brain injury. As such, o7 nAChR activators act as anti-epileptic, anti-epileptogenic, anti-inflammatory and neuroprotective agents (FIG. 2).

[0058] Assessment of the efficacy of PNU-120596 as an antiepileptic drug that reduces acute seizures

[0059] The g7-selective PAM, PNU-120596 (4), robustly enhances the effects of the endogenous agonist acetylcholine on o7 nAChR activation and augments functional responses. Anti-epileptic efficacy of PNU-120596 is tested using the acute seizure model of 6-Hz cornea electrical stimulation (CS) causing intensity-dependent acute psychomotor seizures, a test often applied for screening of new anti-epileptic drugs (AEDs) (8). In accordance with increased activation of inhibitory circuits, PNU-120596 significantly increased individual seizure thresholds in 10 out of 12 mice by an average increase of 33.6% (FIG. 3A). Using an individualized electrical stimulus intensity that caused seizures in 100% of vehicle- treated mice (10 out of 10), an acute injection of 0.01 or 3 mg / kg PNU120596 protected against seizures in ~50 and -80% of mice, respectively, demonstrating the dose dependent increase in drug efficacy (FIG. 3B). The time course of antiseizure effects of PNU-120596 in 6-Hz CS-induced focal seizures revealed the lasting anti-seizure effects following one intraperitoneal (i.p.) injection of 3 mg / kg (FIG. 3C). Thus, the a7-selective PAM, PNU-120596 demonstrated anti-seizure efficacy in an acute seizure model.

[0060] EXAMPLE 2

[0061] Mouse model of cortical contusion injury of traumatic brain injury to assess outcomes after a traumatic brain injury

[0062] A validated mouse model of traumatic brain injury is used with a 2mm cortical contusion injury (CCI), which has shown consistent and longitudinal incidence of spontaneous recurring seizures (SRS), an essential feature for testing novel therapeutics for PTE prevention (9,10).

[0063] This mouse model was used for evaluation of treatment outcomes of the a7 nAChR PAM, PNU-120596, as an example of an a7 nAChR activator acting as a disease modifier. The cortical contusion injury model causes cortical and hippocampal tissue damage (primary injury) and delayed secondary changes which follow in the days, weeks and months after the injury. These changes include but are not limited to neuroinflammation with microgliosis and astrogliosis, loss of cortical and hippocampal inhibitory neurons, restructuring of excitatory brain pathways, cognitive deficiencies and loss of muscle strength and coordination. Eventually, these changes initiate epileptogenic processes that result in epileptic discharges and SRS. This cortical contusion injury mouse model has been described using histological, molecular, physiological, electrophysiological, immunological and behavioral methods which allow the assessment of long-term and short term out comes of pharmacological intervention therapies including but not limited to lesion size, neuroinflammation, hyperexcitability, muscle strength, balance and motor coordination, seizure onset and severity, and cognitive and emotional behavioral deficiencies that follow in the days, weeks and months after the injury (10). Building on this knowledge, PNU-120596 was tested as an example of an a7 nAChR selective activator.

[0064] For testing of the drug in the PTE model, mice are anesthetized, and a computer assisted impact injury is induced with a depth of 1 - 2mm. This model simulates the traumatic brain injury experienced during blunthead injuries on the battlefield, in vehicular accidents or falls. Drug treatment with intraperitoneal (i.p.) injections, is started 3 hours post traumatic brain injury and then once daily for 21 days. Mice are randomly assigned to three different treatment groups (sham: surgery but no cortical contusion injury and no i.p. injections; PNU: surgery, cortical contusion injury at a depth of 2 mm and daily injections with PNU-120596 (1 or 3mg / kg) dissolved in a vehicle solvent solution; TBI: surgery, cortical contusion injury at a depth of 2 mm and daily injections of vehicle solvent solution), n = 8 to 10 adult male mice. Testing of long-term outcomes is done three to four months post traumatic brain injury. PNU-120596 improved long-term outcomes of impaired cognitive functions that are associated with TBI.

[0065] Spatial memory is assessed in the Morris Water Maze (MWM) four months post injury (FIG. 4A). The Sham cohort rapidly learned the location of an escape platform outperforming the traumatic brain injury groups, TBI and PNU, on day one of the four daily learning trials. The CCI caused significantly impaired spatial learning indicated by the long latency of the traumatic brain injury cohort (TBI) to locate an escape platform hidden in the water tank, and the improvement during the 4 days of training was minimal. In contrast, the traumatic brain injury cohort receiving PNU-120596 (PNU) rapidly learned to locate the escape platform by day 2 of training and performed similar to Sham control thereafter. Thus, PNU-120596 rescued the impairment in spatial learning caused by traumatic brain injury in mice.

[0066] The Novel Object Recognition Task (NORT) is a widely used behavioral test in rodents to assess recognition memory. It takes advantage of a rodent's natural tendency to explore new objects more than familiar ones, and unimpaired mice easily differentiate between two objects. The test allows for repeated testing to evaluate improvement over time (FIG: 4B). The Sham control cohort spent about 80% of test time exploring a novel unfamiliar object with little change between tests at one, two, three, or four months post traumatic brain injury. The traumatic brain injury mice spent less than 50% of test time exploring a new object with little change over time which is close to chance. This indifference in time spent with a novel object suggests recognition memory deficits caused by the CCI. Three weeks of PNU-120596 treatment significantly improved recognition memory at one, two , three and four months but there is no improvement in object memory over time.

[0067] PNU-120596 improved long-term outcome measures of well-being and neurological dysfunctions after TBI

[0068] A) Burrowing activity (FIG. 5): Burrowing as a natural activity in rodents, and mice are self-motivated to burrow (11 ). Testing home cage burrowing activity is a highly sensitive method to assess general well-being, motor and neurological impairments. Reduced burrowing activity is commonly viewed as an indicator of reduced overall well-being, motor dysfunction and / or cognitive impairment. Borrowing activity was used to assess long-term outcome of a traumatic brain injury following daily injections of vehicle (TBI) or PNU-120596 (PNU) administered for 21 days starting at day 0 post TBI. Sham control mice removed all pellets (50 g) from a tube placed in their home cage during the two hours of burrowing time. Four months after the CCI, the traumatic brain injury mice showed almost no burrowing activity. In contrast, PNU-treated traumatic brain injury mice showed greatly improved burrowing activity (FIG. 5). This indicates that the treatment with the a7 nAChR activator rescues neurological and motor dysfunctions associated with traumatic brain injury.

[0069] B) Improved recovery of body weight (FIG. 6): After a severe CCI, mice lose up to 15 percent of their body weight in the month following the injury. Thereafter, the traumatic brain injury mice gradually recover but do not reach control levels four months post CCI. In traumatic brain injury mice treated with PNU-120596, recovery of body weight is significantly improved.

[0070] C) Improved recovery of neurological functions after traumatic brain injury (FIG. 7): The composite neuroscore is a behavioral assessment tool used to evaluate neurological and motor function in animal models following a neurological insult. It combines multiple motor and sensory tests into a single score to provide a comprehensive and quantitative measure of functional recovery. In here, seven tests are used, each scored from 0 to 4, for a maximum composite neuroscore of 28 points (fully functional, achieved before CCI), and a minimum score of 0 points (complete impairment). TBI cohorts (TBI and PNU) were treated with vehicle or PNU-120596 from day 0 to 6 post-CCL Following the CCI, performance was assessed at day 1 , 3 and 7. Scores fell for all groups including sham controls (due to sham surgery). The decline was most drastic for the TBI group at day 1 , whereas TBI-cohorts treated with PNU- 120596 which started 3 hours after CCI, exhibited a less severe drop in neuroscores. All groups showed recovery during the first week post CCI, however, traumatic brain injury mice treated with PNU-120596 showed better performance scores at all test points, and those treated with the lower dose (1 mg / kg) had full recovery.

[0071] D) Improved whole-body balance and motor coordination (FIGS. 8-9): Body balance and motor coordination are assessed using the beam walk and the rotarod task, which determine motor function, coordination, balance, and endurance. In the beam walk, mice are tested on their ability to complete crossing a narrow beam to a safe platform. In the rotarod, mice are test for their ability to stay on a rotating rod and the time to fall off is recorded.

[0072] Beam walk performance was assessed at day 1 , 3 and 7 post CCI. All groups experienced a decline in performance at day 1 post traumatic brain injury, which for mice in the sham group reflects the impact of the sham surgery and anesthesia on their ability to cross the beam. The 1stday decline was most dramatic for the TBI mice which showed significant impairment even on day 7 (FIG. 8A). PNU-treated TBI mice performed similar to Sham mice at the 1stday, and were indistinguishable from Sham at day 7 post traumatic brain injury. Thirty days after CCI, traumatic brain injury mice without PNU treatment (TBI) had not yet fully recovered whereas the PNU cohort maintained their improved performance (FIG. 8B).

[0073] Performance on the rotarod was assessed at day 10, 30 and 60 post-CCI (FIG. 9). Sham mice were able to stay on the rotating beam for the 60 seconds of testing at all test days. Mice in the traumatic brain injury cohort (TBI) performed poorly and were unable to stay on the rotating rod for one minute, but slowly improved over time. Mice in the TBI-PNU cohort had better performance at day 10 and 30 post CCI, and fully had recovered by day 60 post traumatic brain injury. Given that PNU-120596 treatment was given for the first 21 days post traumatic brain injury, the treatment benefits the long-term recovery even when the drugs is no longer administered.

[0074] Effect of PNU-120586 to reduce immune system activation:

[0075] Activation of the a7 nAChR on immune cells results in reduced peripheral inflammation which can significantly improve outcomes following traumatic brain injury by limiting secondary injury processes and promoting neurological recovery. TBI initiates a cascade of inflammatory responses, both in the brain (neuroinflammation) and throughout the body (peripheral inflammation). Controlling peripheral inflammation is crucial because it can amplify neuroinflammation and worsen brain damage. The effects of PNU-120596 on blood serum markers were evaluated 7 days post traumatic brain injury. Blood serum levels of seven cytokines were significantly increased compared to Sham (IL-2, TNF-a, MIP-1 a (CCL3), IL- 12, GM-CSF, IFN-y, and IL-13). Of those, all but MIP-1 a exhibited lower serum levels in TBI- PNU compared to TBI-vehicle treated group, and three cytokines were no longer significantly different from Sham control (IFN-y, GM-CSF, IL-12). Examples are shown in FIGS. 10A-10D. The results underscore that the augmented activation of a7 nAChRs by the PAM PNU- 120596, suppresses peripheral immune responses.

[0076] Protective effect of PNU-120596 therapy on epileDtogenesis in mouse PTE model

[0077] In the established CCI model, the occurrence of epileptogenesis with robust SRS is observed in 85% of severe traumatic brain injury mice (9). The model also exhibits progressive neuronal damage, neurodegeneration, and neuroinflammation in multiple brain regions. The onset of SRS is correlated with interneuron loss following traumatic brain injury, and contralateral hippocampal sclerosis is observed, indicating a potential network basis for epileptogenesis.

[0078] Control, traumatic brain injury (TBI) and TBI-PNU groups are subjected to continuous 24 / 7 video with EEG monitoring for a duration of 4 months. The primary outcome measures include: (a) Latency to SRS, (b) Percentage of animals exhibiting SRS, (c) Frequency of SRS occurrences, (d) Severity or duration of SRS episodes, (e) Cumulative seizure counts. The collected data are analyzed to assess the progression of epileptogenesis, the presence of an epileptic state, and the severity of seizures within various subgroups. Epileptiform discharges and high frequency oscillations (HFOs) are examined and analyzed in all experimental groups. The Racine scale is utilized for evaluation of observed seizures exhibited by the TBI animals in + / - PNU-treated groups. This scale provides a standardized measure to assess the intensity and progression of seizures. Lastly, the survival rate over a 120-day period is recorded, and Kaplan-Meier curves are employed to analyze and compare the survival rates between the two groups.

[0079] Effect of PNU-120596 to prevent hyperexcitabilitv.

[0080] Control, traumatic brain injury (TBI) and TBI-PNU mice are used to determine seizure thresholds in the 6-Hz CS test which is an indication of hyperexcitability of neuronal networks. A lower seizure threshold indicates an increase in seizure susceptibility and vice versa.

[0081] Effect of chronic PNU-120596 treatment to reduce TBI-related long-term neuro-inflammation and neurodeqeneration

[0082] Control, traumatic brain injury (TBI) and TBI-PNU groups will undergo neuropathological examination using immunohistochemistry with target specific antibodies to quantify neurodegeneration in NeuN-positive (NeuN+) neurons, parvalbumin-positive (PV+) interneurons, and assess neuroinflammation using GFAP and IBA1 immunostaining in multiple brain regions. Neuronal loss will be assessed by quantifying analysis using a comprehensive stereology method (12). This analysis will help determine the extent of neuronal loss in TBI nice with and without PNU-treatment. Quantifying the loss of PV(+) interneurons will provide insights on neuroprotectant potential of PNU-120596 on the interneuron population. Neuroinflammation will also be assessed using immunostaining for GFAP+ and IBA1 +. GFAP immunostaining will allow to evaluate astrogliosis, an increase in the number and activation of astrocytes, which is a hallmark of neuroinflammation (13,14). IBA1 immunostaining will enable us to assess microgliosis, which is an increase in the number and activation of microglia, another key component of inflammatory response.

[0083] Effect of PNU-120596 on TBI-induced aberrant neuroqenesis and mossy fiber sprouting in the hippocampus.

[0084] Hippocampal neurogenesis is assessed by conducting stereological analysis of doublecortin immuno-positive (DCX+) neurons specifically in the subgranular zone and granule cell layer regions of the hippocampus. This approach provides accurate measurements of the number and distribution of DCX+ neurons allowing one to estimate neurogenesis in the hippocampus regions (12). The Timm staining enables visualization and quantification of the extent of mossy fiber sprouting using Timm densitometry for quantification Effect of PNU-120596 on TBI-related chronic anxiety and depression behavior

[0085] To evaluate anxiety and emotional behavior in the animals, two behavioral tests are utilized: the open-field test and the elevated plus-maze (16). The open-field test assesses exploratory behavior and anxiety levels by measuring the time spent in the center versus the periphery of an open arena. The elevated plus-maze evaluates anxiety-related behavior by measuring the time spent in the open arms versus the closed arms of a plus-shaped maze (17). To assess behavioral depression, the social isolation test is conducted (16). This test involves subjecting the mice to periods of social isolation and then assessing their behavioral responses to determine signs of depressive-like behavior.

[0086] EXAMPLE 3

[0087] PNU-120596 does not cause sickness behavior, sedation, motor impairment

[0088] An aspect of treatment of an epilepsy or of a traumatic brain injury, should be that the medicament has minimal adverse effects and does not cause sickness behavior, cognitive impairment, sedation, reduced motor coordination, or depression. Home cage burrowing activity is a highly sensitive method to assess these aspects. The higher dose of 3 mg / kg was tested in adult male mice without injury or prior drug treatment (naive mice) for effects on burrowing activity 2 hours and 18 hours post injection. The drug effects were compared to baseline burrowing activity (no injection), vehicle (solvent) and PNU-120596 (3 mg / kg) injection (FIGS. 11A-11 B). The alpha? nAChR PAM had no effect on burrowing 2 hours after an i.p. injection, indicating a lack of adverse effects immediately after treatment or after 18 hours, indicating no longer lasting adverse effects.

[0089] The following references are cited herein.

[0090] 1. Papke and Horenstein, Pharmacol Rev. 73(3):1118-1149, 2021 Jul.

[0091] 2. Lbscher W. Neuropharmacology, 167:107605, 2020 May 1.

[0092] 3. Klein et al., Nat Rev Drug Discov.23(9):682-708, 2024 Sep.

[0093] 4. Hurst et al., J Neurosci. 25(17):4396-405, 2005 Apr 27.

[0094] 5. Wang et al., Nature. 421 :384-388, 2003

[0095] 6. Kalkman & Feuerbach, Cell Mol Life Sci., 73(13):2511-30, 2016 Jul.

[0096] 7. Kume et al. Nicotinic Acetylcholine Receptor Signaling: Roles in Neuroprotection. Singapore: Springer, Chapter 4, 2018.

[0097] 8. Golub et al. Exp Neurol. 360:114294, 2023 Feb.

[0098] 9. Golub & Reddy, Exp Neurol. 348:113946, 2022 Feb.

[0099] 10. Golub & Reddy, Pharmacol Rev. 2022 Apr;74(2):387-438, 2022. 11. Deacon RM. Burrowing in rodents: a sensitive method for detecting behavioral dysfunction. Nat Protoc. 2006;1 (1 ):118-21.

[0100] 12. Reddy et al. Curr Protoc. 2024 Dec;4(12):e70053.

[0101] 13. Kuruba et al. Biochim Biophys Acta Mol Basis Dis. 2018 Sep; 1864(9 Pt B):2845- 2858.

[0102] 14. Wu et al. J Pharmacol Exp Ther. 2018 Nov;367(2):302-321 , 2018.

[0103] 15. Rao et al. J Neurosci Res. 2006;83(6):1088-105, 2006.

[0104] 16. Reddy et al. Curr Protoc. 2024 0ct;4(10):e70019, 2024.

[0105] 17. Pellow & File, Pharmacol Biochem Behav. 1986 Mar;24(3):525-9, 1986.

Claims

WHAT IS CLAIMED IS:1 . A method for preventing an onset of or treating an epilepsy in a subject in need thereof, comprising: administering to the subject an amount of a positive allosteric modulator pharmaceutically effective to act on an alpha? nicotinic receptor.

2. The method of claim 1 , further comprising administering at least one other drug effective to treat epilepsy in the subject.

3. The method of claim 1 , further comprising repeating the administering step at least once.

4. The method of claim 1 , wherein the positive allosteric modulator for an alpha? nicotinic receptor is an anti-seizure drug, an anti-epileptogenic drug, an anti-inflammatory drug, or a neuroprotective drug or a combination thereof.

5. The method of claim 4, wherein the positive allosteric modulator is N-(5-Chloro- 2,4-dimethoxyphenyl)-N’-(5-methyl-3-isoxazolyl)-urea (PNU-120596), TQS, A-867744, AVL- 3288, JNJ-1930942 or NS1738, or a combination thereof.

6. The method of claim 1 , wherein the epilepsy is selected from the group consisting of a traumatic brain injury-induced epilepsy, a genetic epilepsy, a chemical exposure-related epilepsy, a viral-induced epilepsy, a COVID 19 related epilepsy, an epilepsy induced by stroke, tumor or anoxia, an epilepsy related to drug withdrawal or alcohol use, and an acquired epilepsy.

7. The method of claim 6, wherein the epilepsy is acquired from prolonged febrile seizures, status epilepticus, concussion, cardiac dysfunction, or hormonal impairment.

8. The method of claim 1 , wherein treating the epilepsy concomitantly delays progression thereof.

9. A method for preventing or delaying progression of an epilepsy in a subject in need thereof, comprising:administering to the subject a pharmaceutically effective amount of an a7 nAChR agonist.

10. The method of claim 9, further comprising repeating the administering step at least once.11 . The method of claim 9, wherein the a7 nAChR agonist is an anti-epileptic drug.

12. The method of claim 11 , wherein the anti-epileptic drug is (PNU-120596), PNU- 282987, FRM-17874, GTS-21 , ABT-126, TC-5619, AVL-3288, A-867744, or JNJ1930942 or a combination thereof.

13. The method of claim 9, wherein the epilepsy is selected from the group consisting of a traumatic brain injury-induced epilepsy, a genetic epilepsy, a chemical exposure-related epilepsy, a viral-induced epilepsy, a COVID 19 related epilepsy, an epilepsy induced by stroke, tumor or anoxia, an epilepsy related to drug withdrawal or alcohol use, and an acquired epilepsy.

14. The method of claim 13, wherein the epilepsy is acquired from prolonged febrile seizures, status epilepticus, concussion, cardiac dysfunction, or hormonal impairment.

15. The method of claim 9, wherein delaying progression of the epilepsy concomitantly treats the epilepsy.

16. A method for preventing a seizure in a subject at risk for having one, comprising: administering to the subject a pharmaceutically effective amount of at least one alpha7 nicotinic receptor agonist.

17. The method of claim 16, further comprising administering at least one other drug to the subject effective to prevent a seizure in the subject.

18. The method of claim 16, further comprising repeating the administering step at least once.

19. The method of claim 16, wherein the alpha? nicotinic receptor agonist is an anti-epileptic drug.

20. The method of claim 19, wherein the anti-epileptic drug is N-(5-Chloro-2,4- dimethoxyphenyl)-N’-(5-methyl-3-isoxazolyl)-urea (PNU-120596), TQS, A-867744, AVL- 3288, JNJ-1930942, NS1738, PNU-28987, FRM-17874, GTS-21 , ABT-126, TC-5619, AVL- 3288, or A-867744, or a combination thereof.

21. The method of claim 16, wherein the alpha? nicotinic receptor is an alpha? nicotinic acetylcholine receptor.

22. The method of claim 16, wherein the subject has a traumatic brain injury.

23. The method of claim 16, wherein the alpha? nicotinic receptor activator is pharmaceutically effective for reducing in the subject with the traumatic brain injury an onset of post-traumatic epilepsy or at least one comorbidity associated therewith that is anxiety, depression, cognitive dysfunction, or motor dysfunctions.

24. The method of claim 16, wherein the seizure is associated with an epilepsy selected from the group consisting of a genetic epilepsy, a chemical exposure-related epilepsy, a viral-induced epilepsy, a COVID 19 related epilepsy, an epilepsy induced by stroke, tumor or anoxia, an epilepsy related to drug withdrawal or alcohol use, and an acquired epilepsy.

25. A method for preventing an onset of post-traumatic epilepsy or at least one comorbidity thereof in a subject with the traumatic brain injury, comprising: administering to the subject a pharmaceutically effective amount of at least one anti- epileptogenic drug that is an a7 nicotinic receptor agonist.

26. The method of claim 25, further comprising administering at least one other drug effective to prevent an onset of post-traumatic epilepsy to the subject.

27. The method of claim 25, further comprising re-administering the a7 nicotinic receptor agonist at least once to the subject.

28. The method of claim 27, wherein the a7 nicotinic receptor agonist is a nicotinic receptor agonist may be PNU-282987, FRM-17874, GTS-21 , ABT107, ABT-126, TC-5619, PHA-543613 or PHA-568487, or a combination thereof.

29. The method of claim 25, wherein the comorbidity is anxiety, depression, cognitive dysfunction or motor dysfunction.

30. A method for preventing epileptogenesis and associated cognitive decline and memory loss in a subject with a traumatic brain injury, comprising: administering to the subject over a period of time a pharmaceutically effective amount of an anti-epileptogenic drug that is an a7 nicotinic receptor activator, following the traumatic brain injury.

31. The method of claim 30, further comprising re-administering the anti- epileptogenic drug at least once to the subject during the period of time.

32. The method of claim 30, wherein the anti-epileptogenic drug is at least one activator selected from the group consisting of PNU-120596, TQS, A-867744, AVL-3288, NS1738, PNU0282987, FRM-17874, GS021 , ABT-126, TC-5619, AVL-3288, A-86774, and JNJ 1930942.

33. The method of claim 30, wherein the anti-epileptogenic drug is pharmaceutically effective to modulate an activity of an alpha7 nicotinic acetylcholine receptor.

34. The method of claim 30, wherein the step of administering the anti- epileptogenic drug further reduces at least one comorbidity associated with the traumatic brain injury in the subject.

35. The method of claim 34, wherein the comorbidity is anxiety, depression, cognitive dysfunction or motor dysfunction.

36. The method of claim 30, wherein the anti-epileptogenic drug is a neuroprotective drug effective to prevent tissue and neuronal loss in the subject.

37. The method of claim 30, wherein the anti-epileptogenic drug is effective to prevent or reduce inflammation in the brain and in the periphery thereof.

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