GABA a receptor modulator antiseizure treatments

Abstract

Described herein are methods of using 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d 3 )ethyl-2,2,2-d 3 ]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine or a salt or hydrate thereof, or compositions thereof, as an antiseizure or antiepileptic medicament.

Description

2700339.00162 GABAA RECEPTOR MODULATOR ANTISEIZURE TREATMENTS RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Patent Application No. 63 / 686,998, filed August 26, 2024, the entire content of which is incorporated herein by reference. BACKGROUND

[0002] Epilepsy is a disorder of the brain characterized by repeated seizures. A seizure is usually defined as a sudden alteration of behavior due to a temporary change in the electrical functioning of the brain. Normally, the brain continuously generates electrical impulses in an orderly pattern. These impulses travel along neurons—the network of nerve cells in the brain—and throughout the whole body via chemical messengers called neurotransmitters.

[0003] In epilepsy the brain’s electrical rhythms tend to become imbalanced, resulting in recurrent seizures. In patients with seizures, the normal electrical pattern is disrupted by sudden and synchronized bursts of electrical energy that may briefly affect their consciousness, movements, or sensations.

[0004] Epilepsy is usually diagnosed after a person has had at least two seizures that were not caused by some known medical condition, such as alcohol withdrawal or extremely low blood sugar. About 1% of adults are reported to have active epilepsy.

[0005] Accordingly, there remains a need for antiseizure medicaments and antiepileptics. SUMMARY

[0006] GABA receptors respond to the neurotransmitter gamma-aminobutyric acid (GABA), which is the major inhibitory compound of the vertebrate central nervous system. GABAA receptors occur in all organisms that have a nervous system.

[0007] It has been found that 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6- [(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine (Compound 1), an α2,3,5-selective GABAAreceptor modulator, is useful in disrupting alteration in the electrical functioning of the brain or nervous system and specifically as an antiseizure medicament or an antiepileptic medicament.

[0008] Thus, provided herein are methods of treating seizures in a subject in need thereof, comprising administration of Compound 1 or a pharmaceutically acceptable salt or hydrate thereof to the subject.2700339.00162

[0009] Also provided herein are methods of treating epilepsy in a subject in need thereof, comprising administration of Compound 1 or a pharmaceutically acceptable salt or hydrate thereof to the subject.

[0010] Compound 1 is shown below as a free base (wherein D is deuterium). D3C CD3N F3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 depicts Compound 1 in structural form as a hydrate.

[0012] FIG. 2 depicts SynchroPatch determination of the concentration-dependence of the positive modulation of recombinant GABAA receptors containing the α1, α2, α3, or α5 subunits stably expressed in heterologous cells. The graph plots fold-increase in chloride current with respect to GABA EC5–20values produced by Compound 1 at concentrations from 0.01 nM to 1 µM. Points indicate mean ± S.E.M.

[0013] FIG. 3A depicts a comparison of seizure severity scores of oral Compound 1 and diazepam in the rat amygdala kindling model. Compound 1 was tested in the amygdala kindling model at doses of 1, 6, 30, 100 mg / kg, p.o. Diazepam was tested at a dose of 16 mg / kg, p.o. Prior to drug treatment, all animals exhibited stage 5 seizures. Compound 1 treatment caused a dose-dependent reduction in the seizure severity score with maximal reduction comparable to that produced by diazepam. The number of animals in each group ranged from 12-16 except in the 100 mg / kg Compound 1 group, which consisted of 4 animals. Bars indicate mean ± S.E.M. *p<0.5, **p<0.01, ***p<0.001.

[0014] FIG.3B depicts a comparison of after-discharge duration (amygdala) of oral Compound 1 and diazepam in the rat amygdala kindling model. Compound 1 was tested in the amygdala kindling model at doses of 1, 6, 30, 100 mg / kg, p.o. Diazepam was tested at a dose of 16 mg / kg, p.o. Compound 1 treatment caused dose-dependent reductions in after-discharge2700339.00162 duration as assessed by the EEG recorded from depth electrodes in the amygdala. The number of animals in each group ranged from 12-16 except in the 100 mg / kg Compound 1 group, which consisted of 4 animals. Bars indicate mean ± S.E.M. *p<0.5, **p<0.01, ***p<0.001.

[0015] FIG. 3C depicts a comparison of after-discharge duration (cortex) of oral Compound 1 and diazepam in the rat amygdala kindling model. Compound 1 was tested in the amygdala kindling model at doses of 1, 6, 30, 100 mg / kg, p.o. Diazepam was tested at a dose of 16 mg / kg, p.o. Compound 1 treatment caused dose-dependent reductions in after-discharge duration as assessed by the EEG recorded from cortical surface recording. The number of animals in each group ranged from 12-16 except in the 100 mg / kg Compound 1 group, which consisted of 4 animals. Bars indicate mean ± S.E.M. *p<0.5, **p<0.01, ***p<0.001.

[0016] FIG.4A depicts a comparison of the effects of Compound 1 and diazepam administered by oral gavage on spikewave discharges in the GAERS genetic rat model of absence seizures. Specifically, a time course of the effect of the treatments on spikewave discharge (SWD) counts in successive 20 min. epochs beginning 10 min. after treatment administration. Each treatment group consisted of 4 to 10 rats. ***p<0.001, ****p<0.0001. Error bars show S.E.M.

[0017] FIG.4B depicts a comparison of the effects of Compound 1 and diazepam administered by oral gavage on spikewave discharges in the GAERS genetic rat model of absence seizures. Specifically, SWD rates during the entire post-treatment observation period from 10 min. to 130 min. after treatment administration expressed as a percent of the baseline rate for all groups. Each treatment group consisted of 4 to 10 rats. ***p<0.001, ****p<0.0001. Error bars show S.E.M.

[0018] FIG. 5 depicts the effect of Compound 1 on rat rotarod performance. Impact of Compound 1 (10, 30, 60, and 100 mg / kg, p.o.) on rotarod performance was tested in rats. During the training period, rats were placed on the rotarod (rotation speed accelerating from 4 rotations per minute (r / m) to 40 r / m within 5 min and trained twice a day for 3 days). Latency to fall off the rotarod was recorded. On the fourth day, rats were placed on the rotarod with the same setting of training, 30 min after drug dosing. Duration of time that the rat stayed on the rotarod was recorded. Data was analyzed using one-way ANOVA with Tukey post hoc test. Significance was considered at *p < 0.05. Pentobarbital was administered i.p. at 15 mg / kg as a positive control for this assay. n = 12 rats per treatment group.

[0019] FIG. 6 depicts a pharmacokinetic-pharmacodynamic analysis of the results in the mouse 6 Hz seizure test. The mean ± S.E.M Compound 1 plasma concentration values for the 3 doses of Compound 1 (30, 100, 300 mg / kg) at 0.25 hours post-dose are plotted against2700339.00162 the efficacy values representing the fraction of mice protected in groups subjected to 32 mA (circles) and 44 mA (squares) stimulation.

[0020] FIG. 7 depicts plasma concentrations of Compound 1 following oral administration in rats at doses of 0.075, 0.75, 1, 2, 6, 30 mg / kg. Closed symbols are taken from the amygdala kindling experiment and were collected 2 h after dosing (3 rats per dose). Open symbols are from the GAERS experiment and were taken 0.5 h after the end of the last EEG recording of the study, about 2.5 h after dosing (3 rats per dose, except the 0.75 mg dose which was 2 rats). Symbols and error bars indicate mean ± S.E.M. Where error bars are not shown they are smaller than the size of the symbols. DETAILED DESCRIPTION

[0021] Provided herein are methods of treating seizure or epilepsy in a subject in need thereof, comprising administration of Compound 1 or a pharmaceutically acceptable salt thereof to the subject. Definitions

[0022] Certain terms, whether used alone or as part of a phrase or another term, are defined below.

[0023] The articles “a” and “an” refer to one or to more than one of the grammatical object of the article.

[0024] Numerical values relating to measurements are subject to measurement errors that place limits on their accuracy. For this reason, all numerical values provided herein, unless otherwise indicated, are to be understood as being modified by the term “about.”

[0025] The term “amelioration” means a lessening of severity of at least one indicator or symptom of a condition or disease, such as a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.

[0026] The terms “composition” and “pharmaceutical composition” refer to a mixture of at least one compound described herein with a carrier or a pharmaceutically acceptable carrier, respectively. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a composition exist including, but not limited to, intravenous, oral, nasal, rectal, intravaginal, aerosol, parenteral, buccal, sublingual, ophthalmic, pulmonary, transdermal, and topical administration.2700339.00162

[0027] The terms “effective amount” and “therapeutically effective amount” refer to an amount of therapeutic compound, such as a compound described herein, administered to a subject, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect.

[0028] The term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid filler, solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent, or encapsulating material, involved in carrying or transporting at least one compound described herein within or to the patient such that the compound may perform its intended function. A given carrier must be “acceptable” in the sense of being compatible with the other ingredients of a particular formulation, including the compounds described herein, and not injurious to the patient. Other ingredients that may be included in the pharmaceutical compositions described herein are known in the art and described, for example, in “Remington’s Pharmaceutical Sciences” (Genaro (Ed.), Mack Publishing Co., 1985), the entire content of which is incorporated herein by reference.

[0029] The term “pharmaceutically acceptable salt” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Lists of salts are found in “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” (P. Henrich Stahl & Camille G. Wermuth (Eds.), VHCA & Wiley- VCH, 2002), the entire content of which is incorporated herein by reference.

[0030] The terms “substituted”, or “substitution” refers to replacement of hydrogen attached to another group with an atom or group of atoms as the replacement substituent, wherein each substituent is independently selected.

[0031] The terms “treatment” or “treating” refer to the application of one or more specific procedures used for the amelioration of a disease. A “prophylactic” treatment, refers to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset.

[0032] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the described subject matter and does not pose a2700339.00162 limitation on the scope of the subject matter otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to practicing the described subject matter.

[0033] Groupings of alternative elements or embodiments of this disclosure are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. Furthermore, a recited member of a group may be included in, or excluded from, another recited group for reasons of convenience or patentability.

[0034] References have been made to patents and printed publications throughout this specification, each of which are individually incorporated herein by reference in their entirety.

[0035] It is to be understood that the embodiments of this disclosure are illustrative. Accordingly, the present disclosure is not limited to that precisely as shown and described. Compounds

[0036] 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4- triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine (Compound 1) is a GABAA receptor modulator that can act at the benzodiazepine site of the GABAAreceptor as a selective allosteric modulator of the α2, α3, and α5 subtypes.

[0037] Compound 1 and its salts as referred to herein are synthesized using any suitable procedures starting from compounds that are available from commercial sources, or are prepared using procedures described herein. General methods for the preparation of a compound as described herein are modified by the use of appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein.

[0038] In some embodiments, Compound 1 may be prepared using the methods described in U.S. Patent Nos. 8,003,646, 8,399,467, or 8,921,366, the entire content of each of which are incorporated by reference. The preparation of a compound corresponding to a non-deuterated form of Compound 1 is described in the Journal of Medicinal Chemistry, 48 (23): 7089–92 (Carling et al., “7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2- fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine: a functionally selective gamma-aminobutyric acid(A) (GABA(A)) alpha2 / alpha3-subtype selective agonist that exhibits potent anxiolytic activity but is not sedating in animal models”). Thus, Compound 1 may be prepared in a similar manner as Carling et al. by substitution with the appropriate corresponding deuterated reagents.2700339.00162

[0039] Preparation of a salt of Compound 1 may occur by, for example, contacting Compound 1 with an acid in a solvent solution, and isolating the salt of Compound 1 by removing the solvent. In order to prepare a particular salt, such as a hemi-, mono-, or di-salt, the corresponding molar ratio of Compound 1 and acid is provided in the solvent solution.

[0040] Thus, in some embodiments of the methods herein, Compound 1 is used in the form of a fumarate (e.g., mono- or di-fumarate salt) salt of 3-(2,5-difluorophenyl)-7-[1,1- di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4- triazolo[4,3-b]pyridazine.

[0041] In some embodiments of the methods herein, Compound 1 is used in the form of a hemi-fumarate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1- methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

[0042] In some embodiments of the methods herein, Compound 1 is used in the form of the formula: D3C CD3N X.

[0043] In some embodiments of the methods herein, Compound 1 is used in the form of a sulfate salt (e.g., mono- or di-sulfate salt) of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl- d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine.

[0044] In some embodiments of the methods herein, Compound 1 is used in the form of a hydrochloride salt (e.g., mono- or di-hydrochloride salt) of 3-(2,5-difluorophenyl)-7-[1,1- di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4- triazolo[4,3-b]pyridazine.

[0045] In some embodiments of the methods herein, Compound 1 is used in the form of a phosphate salt (e.g., mono- or di-phosphate salt) of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl- d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine.2700339.00162

[0046] In some embodiments of the methods herein, Compound 1 is used in the form of a tosylate salt (e.g., mono- or di-tosylate salt) of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl- d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine.

[0047] In some embodiments of the methods herein, Compound 1 is used in the form of a malonate salt (e.g., mono- or di-malonate salt) of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl- d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine.

[0048] In some embodiments of the methods herein, Compound 1 is used in the form of a maleate salt (e.g., mono- or di-maleate salt) of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl- d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine.

[0049] In some embodiments of the methods herein, Compound 1 or its salt is provided as an anhydrate, hemihydrate, monohydrate, or dihydrate. In some embodiments, Compound 1 or its salt is a solvate. In some embodiments, Compound 1 or its salt is in a solid form. Compositions

[0050] In some embodiments of the methods herein, Compound 1 or its salt is administered in the form of a composition comprising one or more of Compound 1 or its various salt forms described herein. In some embodiments, the compositions are pharmaceutical compositions. The compositions may further comprise a pharmaceutically acceptable carrier.

[0051] In some embodiments, the composition is a pharmaceutical composition consisting essentially of Compound 1 or a salt or hydrate thereof.

[0052] In some embodiments, the composition is a pharmaceutical composition comprising Compound 1 or a salt or hydrate thereof.

[0053] In some embodiments, the compositions described herein comprise a first pharmaceutical active, which is Compound 1 or a salt thereof, and a second pharmaceutical active, which may be a compound useful in treating a disease or disorder of the central nervous system. Methods

[0054] Provided herein are methods of treating seizure in a subject in need thereof, comprising administration of Compound 1 or a pharmaceutically acceptable salt thereof to the subject. For example, disclosed methods can comprise treatment of focal seizures affecting a2700339.00162 part of the body, secondary seizures resulting from focal seizures, generalized seizures affecting the entire body, “absence” seizures comprising brief staring spells, or convulsions.

[0055] Provided herein are methods of treating epilepsy in a subject in need thereof, comprising administration of Compound 1 or a pharmaceutically acceptable salt thereof to the subject.

[0056] Actual dosage levels of the active ingredients (e.g., the pharmaceutical active compound, e.g., Compound 1 or a salt thereof), the compositions, or the pharmaceutical compositions provided herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0057] In particular, the selected dosage level will depend upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health, or prior medical history of the patient being treated.

[0058] Routes of administration include, without limitation, oral, nasal, rectal, intravaginal, aerosol, parenteral, buccal, sublingual, ophthalmic, pulmonary, or topical administration. In some embodiments, the oral or nasal route of administration is an oral inhalational or nasal inhalational route of administration. The compounds for use as described herein may be formulated for administration by any suitable route to achieve the particular method being applied.

[0059] Accordingly, administration of a compound, a composition, or a combination disclosed herein includes a variety of enteral or parenteral approaches selected from, without limitation: oral administration in any acceptable form, such as, e.g., tablet, liquid (e.g., liquid suspension of particles), capsule, powder, or the like; topical or transdermal administration in any acceptable form, such as, e.g., drops, spray, creams, gels ointments, or patches; buccal, nasal, sublingual, ophthalmic, pulmonary, and / or inhalation administration in any acceptable form; rectal administration in any acceptable form; vaginal administration in any acceptable form; peri- and intra-tissue administration in any acceptable form, such as, e.g., intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, or intraarticular injection; intravascular administration in any acceptable form, such as, e.g., catheter instillation; and by placement device, such as, e.g., an implant, a stent, a patch, a pellet, a catheter, an osmotic pump, a suppository, a bioerodible delivery system, a non-bioerodible delivery system or another implanted extended or slow release system.2700339.00162

[0060] Local administration results in significantly more delivery of a compound, a composition, or a combination to a specific location as compared to the entire body of the mammal, whereas systemic administration results in delivery of a compound, a composition, or a combination to essentially the entire body of the individual. Routes of administration suitable for treating a central nervous system related disease or disorder as disclosed herein also include both central and peripheral administration. Central administration results in delivery of a compound, a composition, or a combination to essentially the central nervous system of the individual and includes, e.g., nasal administration, intrathecal administration, epidural administration as well as a cranial injection or implant. In some embodiments, central administration is used to administer the compound, composition, or combinations described herein.

[0061] Central administration by the nasal route, which targets drug absorption through the vascular plexus of the nasal cavity, is distinct from administration by nasal inhalation, which delivers drug through the pulmonary system. Whereas the latter typically uses liquid or dry powder aerosols with mean particle sizes less than about 10 microns, central administration may be accomplished using mean particle sizes of about 10 microns or larger. Mists and aerosols can be generated using nebulizers, dry powder inhalers, pressurized aerosols, and atomization pumps. It is also feasible to use nose drops (e.g., a suspension of particles in a liquid) for central administration by the nasal route.

[0062] Peripheral administration results in delivery of a compound, a composition, or a combination to essentially any area of an individual outside of the central nervous system and encompasses any route of administration other than direct administration to the spine or brain. Kits

[0063] In some embodiments, provided herein are packaged Compound 1 or its salt or hydrate, packaged compositions, or packaged pharmaceutical compositions, comprising a container holding a therapeutically effective amount of Compound 1 or its salt or hydrate, or a composition herein, and instructions for using the same in accordance with one or more of the methods provided herein.

[0064] Compound 1 or its salt or hydrate and associated materials can be finished as a commercial product by the usual steps performed in the present field, for example by appropriate sterilization and packaging steps. For example, the material can be treated by UV / vis irradiation (200–500 nm), for example using photo-initiators with different absorption wavelengths (e.g., Irgacure 184, 2959), preferably water-soluble initiators (e.g., Irgacure2700339.00162 2959). Such irradiation is usually performed for an irradiation time of 1-60 min, but longer irradiation times may be applied, depending on the specific method. The material according to the present disclosure can be finally sterile-wrapped so as to retain sterility until use and packaged (e.g. by the addition of specific product information leaflets) into suitable containers (boxes, etc.).

[0065] According to further embodiments, Compound 1 or its salt or hydrate can also be provided in kit form combined with other components necessary for administration of the material to the patient. For example, disclosed kits, such as for use in the treatment of seizure or epilepsy, can further comprise, for example, administration materials.

[0066] The kits may be designed in various forms based on the specific deficiencies they are designed to treat.

[0067] The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings or disclosure as described herein. EXAMPLES Example 1

[0068] The purpose of this study was to characterize the α-subunit selectivity of Compound 1 and evaluate its antiseizure potential in preclinical seizure and epilepsy models.

[0069] Methods: Compound 1 and diazepam potentiation of GABA chloride current responses in cells expressing recombinant GABAA receptors were evaluated using an automated patch clamp assay. Antiseizure effects of Compound 1 were examined in the mouse 6 Hz test at 32 mA and 44 mA, the rat amygdala kindling model, and genetic absence epilepsy rats from Strasbourg (GAERS). Diazepam belongs to a group of medicines called benzodiazepines. It is a positive allosteric modulator (PAM) of GABAA receptors and lacks α-subunit selectivity. It is used to treat anxiety, muscle spasms, and seizures or fits.

[0070] Results: Compound 1 displayed partial PAM activity with respect to diazepam at GABAAreceptors containing α2, α3, or α5 subunits but did not enhance GABA responses of GABAAreceptors containing α1 subunits. Compound 1 (30, 100, and 300 mg / kg, i.p.) and diazepam (1 mg / kg) protected most animals in the 6 Hz model at 32 mA but was less effective at 44 mA. In the rat amygdala kindling model, Compound 1 (1–100 mg / kg, p.o.) reduced behavioral seizure severity and after-discharge duration in a dose-dependent manner. Compound 1 (0.075–100 mg / kg, p.o.) caused dose-dependent, persistent (>130 min.) inhibition of spontaneous spike-wave discharges in GAERs rats whereas diazepam (3 mg / kg, p.o.) transiently inhibited discharges.2700339.00162

[0071] Significance: Compound 1 is an α2,α3,α5-selective GABAA receptor PAM that has low potency and partial efficacy. The drug is highly effective in seizure and epilepsy rodent models. Compound 1 is most potent in the GAERS model of absence epilepsy. At higher doses it is active in the 6 Hz test and amygdala kindling model. These results demonstrate that a partial, subtype-selective GABAA receptor PAM, Compound 1 or its salt, can have activity in translationally validated preclinical epilepsy screening models, and Compound 1 or its salt may be used as a treatment for seizure, and focal or generalized epilepsies.

[0072] Compound 1 is a α2,α3,α5-selective GABAA receptor partial positive allosteric modulator.

[0073] Compound 1 showed dose-related antiseizure effects in clinically-validated rodent seizure models.

[0074] Compound 1 was most potent at suppressing spike-wave discharges in GAERS, a model of generalized absence epilepsy.

[0075] Compound 1 was also effective in the 6 Hz test and the amygdala kindling model, indicating possible utility in treating focal onset seizures. INTRODUCTION

[0076] GABAAreceptors, members of the pentameric ligand-gated ion channel superfamily, are chloride channels that serve as the main mediators of inhibitory neurotransmission in the brain. The 5 protein subunits that compose GABAA receptors commonly include 2 α-subunits, 2 β-subunits and a γ2-subunit, that can exist in long (γ2L) and short (γ2S) variants. Genetic or acquired defects in GABA mediated neurotransmission are believed to underlie some forms of epilepsy. Benzodiazepines in clinical use today, such as 1,4-benzodiazepines including diazepam, lorazepam, midazolam and clonazepam, and the 1,5-benzodiazepine clobazam, act as PAMs on GABAA receptors containing α1, α2, α3, or α5 subunits and can be characterized as “non-selective.” Such non-selective GABAAPAMS have broad-spectrum antiseizure activity but, except for clobazam, are not widely used in the chronic treatment of epilepsy because they induce dose-limiting side effects, including sedation and ataxia, and are subject to tolerance issues. Use of selective α2,3,5 PAMs for the treatment of epilepsy, may provide improved treatment options, at least, by reducing sedation due to lack of activity on receptors that contain α1 subunits.

[0077] L-838,417, a non-benzodiazepine, is an α2,3,5 selective PAM that confers partial positive modulation at α2-, α3-, and α5-containing GABAA receptors but is an antagonist at α1-subunit containing receptors. Benzodiazepines are considered “full” PAMs when the degree2700339.00162 of positive modulation they confer is equal to the most efficacious agents, with diazepam often considered as a prototype full PAM. Due to its poor pharmacokinetic profile in preclinical models, L-838,417, a non-deuterated version of Compound 1, was not advanced into clinical trials, and therefore its side effect profile and liability for tolerance in humans is unknown.

[0078] Herein it is demonstrated that Compound 1 is a GABAA receptor PAM with selectivity for α2, α3, and α5 using patch clamp recording that possesses antiseizure activity in several animal models that are known to be sensitive to GABAAreceptor PAMs. MATERIALS AND METHODS Automated patch clamp assay

[0079] Human GABAA receptor subunit combinations α1β3γ2L, α2β2γ2L, α2β3γ2L, α3β3γ2L and α5β3γ2L stably expressed in Chinese hamster ovary (CHO) cells were cultured using standard techniques. GABAAreceptor chloride currents were assessed using the SynchroPatch automated platform at room temperature. To test for PAM activity, GABA was applied at its EC20 concentration and then GABA is applied again in the presence of the test compound, either Compound 1 hydrate (FIG. 1) or diazepam, both in 0.2% DMSO. Compound 1 was tested at 10 concentrations (0.01 nM, 0.1 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 10 μM) in duplicates. Diazepam was tested at five concentrations (0.01, 0.1, 1, 10, 100 μM) in duplicates. The fold over GABA alone was determined with the formula (Icomp / IGABA) − 1, where Icomp is the current amplitude in the presence of the test compound and IGABA is the current amplitude in the presence of GABA EC20alone. The compound EC50is determined from a plot of the fold value versus concentration. The % activation relative to agonist (GABA EC20) was determined with the formula [(Icomp / IGABA) − 1] ×100, where Icomp is the current amplitude in the presence of the compound and IGABA is the current amplitude in the presence of GABA alone. Emax is the largest % activation value obtained with any compound concentration. 6 Hz seizure test All mouse experimentation was approved by the University of Washington Institutional Animal Care and Use Committee (protocol 4387-01). Adult male CF-1 mice (20– 35 g) were obtained from Envigo (Haslett, MI). Compound 1 was formulated in 0.5% methylcellulose 4000 (Sigma Aldrich M0512) and administered by the intraperitoneal (i.p.) route 0.25 h prior to challenge with a 6 Hz (hertz) 32 mA (milliampere) or 44 mA equivalent current for 3 s (second) delivered through corneal electrodes to elicit a psychomotor seizure characterized by unilateral or bilateral forelimb clonus, vibrissae twitching, and Straub tail. For the 6 Hz test, the i.p. route of administration was chosen deliberately to ensure sufficient exposure for efficacy in the2700339.00162 mouse 6 Hz model due to prior pharmacokinetic studies with the parent compound demonstrating that i.p. administration in mice gives superior and desired exposure profile as compared to oral administration. The experimenter was blinded to the treatment condition. Mice not displaying all these behaviors within the immediate (5–10 s) period after stimulation were considered “protected.” Ten mice were tested in each treatment group. Diazepam was dosed at 1 mg / kg, i.p., as supplied from Hospira.Rotarod test of minimal motor impairment

[0080] Rotarod test of minimal motor impairment (MMI) in mice was established by mouse performance on a fixed-speed rotarod, which was assessed at the University of Washington in all mice immediately prior to the 6 Hz seizure stimulation for each current intensity. When a normal mouse is placed on a rod that rotates at a speed of 6 rpm, the animal can maintain its equilibrium for long periods of time. If a mouse falls off the rotarod three times during the 60 s trial period, it is considered “impaired.” Each time a mouse falls off the rotarod in the 60 s trial period, it is immediately returned to the rotarod until the 60 s trial period has elapsed. Evaluation on the rotarod is a binary outcome measure (Yes / No fall) and scoring is conducted by an experimenter who is blinded to treatment condition. The number of mice that exhibit minimal motor impairment on the rotarod assay is reported, with a total of n = 10 mice per treatment group from each current intensity in the 6 Hz study (Table 2).

[0081] Performance of rats in the rotarod assay was examined 30 min after oral administration of Compound 1. During the training session, rats were placed on the rotarod, with the rotation speed accelerating from 4 r / m (rotations / minute) to 40 r / m within 5 min. Rats were trained twice a day for 3 days. The latency to fall off the rotarod in the last trial was recorded and considered as baseline. On the fourth day, during the testing phase, rats were randomly assigned to test groups and 30 min after drug administration, placed on the rotarod with the same settings as that of the training. The duration of time that each rat remained on the rotarod was recorded. Dosing and rotarod measurement were conducted by two scientists separately. Pentobarbital was used as a positive control for assay sensitivity. Data were analyzed using one-way analyses of variance (ANOVAs) with post hoc analysis using Tukey's test and significance considered at p < 0.05. Amygdala kindling model

[0082] Male Wistar rats (Janvier Labs) were anesthetized with isoflurane and injected with carprofen (5 mg / kg, s.c.). Screw electrodes were placed over the fronto-parietal cortex, a depth electrode in the amygdala, a screw electrode was placed over the right occipital cortex to serve as ground; and an anchor screw was placed over the left occipital cortex. The animals were allowed at least 10 days to recover from surgery. Rats were stimulated via the amygdala electrode twice daily, 5 days per week until they displayed 4 consecutive seizures with scores2700339.00162 of 4 or 5 on the Racine scale. Each rat received the 4 treatments according to an incomplete Latin Square design, by investigators blinded to the test substance. Testing of each treatment required 2 days, with a washout time of two days between treatments. On Day 1 (baseline session) the animal received vehicle (0.5% methylcellulose) orally and was stimulated 60 or 120 min. later. On Day 2 (test session) approximately 24 h after the baseline session the test substance was administered orally, and the animal was stimulated 60 or 120 min. later. After each stimulation, the seizure behavior was scored using the 6-point (0–5) Racine scale. In addition, cortical and amygdala EEG recordings were collected from the amygdala and cortex electrodes and were analyzed off-line to determine the after-discharge duration. Mean seizure severity scores in the baseline and test sessions were compared with the Wilcoxon test; mean after-discharge durations were compared using the paired t-test. GAERS model

[0083] Fourteen adult male rats (age 4–8 months) of the Genetic Absence Epilepsy Rat from Strasbourg (GAERS) strain were obtained from Dr. Antoine Depaulis (INSERM, Grenoble Institute of Neurosciences, Grenoble, France) under exclusive license to SynapCell. EEG recordings were obtained from right and left, frontal and parietal cortices of freely moving animals using SystemPlus Evolution software (Micromed). One week after electrode implantation, the EEG was recorded for 1 h and 12 rats that demonstrated ≥20 spike-wave discharges (SWDs) during the recording were selected for the study. A crossover design was used in which each animal received treatments in a randomized fashion including vehicle (0.5% methylcellulose), diazepam (3 mg / kg), and doses of Compound 1 from 0.075 mg / kg to 100 mg / kg, on different days by oral gavage. SWDs were counted during a 20 min. baseline period (from 25 to 5 min. before treatment administration), and for successive 20 min. epochs up to 120 min. starting 10 min. after administration. The initial 10 min. was ignored as dosing disturbs the occurrence of SWDs. When required, animals were returned to a state of quiet wakefulness by gentle stimulation. SWD counts per epoch were expressed as mean ± S.E.M. Statistical analysis was performed by ANOVA using a mixed model with repeated measures, followed by paired comparisons (Dunnett) vs. vehicle (Graphpad Prism v9.1). ED50 values were calculated using the dose-response curve fitting of Prism v9.1, with a variable slope model and an R-squared method. Plasma Compound 1 Collection and Assay

[0084] Following completion of in-life testing, blood was collected from a subset of animals (n = 3 / dose group) post testing in the 6 Hz, amygdala kindled rats, and GAERS model (n = 2– 3 / dose group) to determine plasma Compound 1 concentrations. Whole blood samples were2700339.00162 collected 0.25 h after dosing in mice in the 6 Hz study and 2–2.5 h after dosing in the amygdala kindled rats and GAERS experiments. Blood samples were either from the trunk following decapitation or through retroorbital sinus puncture, in accordance with institutionally approved methods, and isolated plasma was frozen and stored at −80°C (with anticoagulant K2 EDTA (dipotassium ethylenediamine tetraacetic acid) until assayed by LC / MS / MS (liquid chromatography tandem mass spectrometry) using an internal standard (IS). After addition of the IS, plasma samples were extracted by protein precipitation using acetonitrile. Reversed-phase HPLC (high performance liquid chromatography) separation was achieved with a Phenomenex, Synergi Max-RP column (50 × 2.0 mm, 4 micron). MS / MS (tandem mass spectrometry) detection was conducted in Thermo-Ion Spray (TIS) positive mode. Quantitation was based on linear regression of analyte / IS area ratio vs. a weighting factor. The dynamic range of detection for both the mouse and rat methods was 5.00–5000 ng / mL for Compound 1. RESULTS Compound 1 is a selective GABAAreceptor α2,3,5 PAM To demonstrate the positive modulatory activity and selectivity of Compound 1 on GABAA receptors with different α-subunits, single cell electrophysiological studies using an automated patch clamp system applied to cells that expressed recombinant GABAA receptors was conducted. Compound 1 (FIG. 1) potently enhanced currents generated by GABA in receptors containing α2, α3, and α5 subunits but caused negligible potentiation of α1 subunit– containing receptors (FIG. 2). In contrast, diazepam was more efficacious on α2, α3, and α5 subunit–containing receptors and was an effective PAM of α1 subunit– containing receptors. Despite its greater functional activity (Emax values), diazepam had weaker potency (i.e., exhibited larger EC50 values) than Compound 1 on receptors with α2, α3, and α5 subunits (Table 1). Thus, on α2, α3, and α5 subunit–containing receptors, Compound 1 displayed potent but partial PAM activity relative to diazepam. Evaluation of Compound 1 in the mouse 6 Hz seizure test and rotarod test

[0085] Compound 1 was evaluated in the mouse 6 Hz model at simulation intensities of 32 mA and 44 mA, with diazepam, which is known to be effective in this test, as a positive control. As shown in Table 2, all vehicle pretreated animals at either stimulation intensity exhibited seizures. Diazepam pretreatment protected all mice stimulated with the lower intensity current from seizures but only partially protected the group stimulated with the higher intensity current. The reduced protection conferred by diazepam with higher intensity current is consistent with the results of a prior study in the same mouse strain. Similarly,2700339.00162 Compound 1 protected most animals simulated at 32 mA but fewer animals at 44 mA. It is noteworthy that Compound 1 at 30–100 mg / kg is equally or more effective than diazepam at 1 mg / kg. Importantly, none of the mice receiving Compound 1 exhibited impairment in the rotarod test at any of the doses tested (Table 2). Antiseizure effects of Compound 1 in the amygdala kindling model

[0086] Compound 1 was evaluated for its ability to inhibit behavioral seizures and EEG after- discharge in the rat amygdala kindling model. Fully kindled rats were administered either vehicle, Compound 1 at 4 escalating test doses (1, 6, 30, and 100 mg / kg), or diazepam (16 mg / kg) as a positive control. With vehicle treatment (baseline), stage 5 seizures were observed in all animals (FIG. 3A). Diazepam (16 mg / kg), administered orally 60 min. prior to the electrical stimulation, substantially reduced the mean severity seizure score (−3.7, p<0.001, FIG. 3A), and mean after-discharge duration in the amygdala (−69%, p ˂ 0.001), FIG. 3B) and the cortex (−75%, p ˂ 0.001, FIG. 3C). Compound 1, administered 2 hours prior to electrical stimulation, also significantly reduced the mean seizure severity score and the mean after-discharge duration in both the amygdala and the cortex. At doses of 1, 6, and 30 mg / kg, Compound 1 reduced the mean seizure severity score by −1.8 (p <0.01), −3.2 (p<0.001) and −3.8 (p<0.01), respectively (FIG. 4A). Compared to baseline, Compound 1 at 1, 6, and 30 mg / kg also reduced mean after-discharge duration in the amygdala by −26% (p<0.01), −46% (p<0.01), and −64% (p <0.001), respectively (FIG. 4B); and the after- discharge duration in the cortex (−30%, p ˂ 0.01; −53%, p ˂ 0.01 and −66%, p ˂ 0.001) (FIG. 4C). The 100 mg / kg Compound 1 group showed the greatest reductions in seizure severity score (−4.0) and after-discharge duration in the cortex (−76%, p ˂ 0.05) and in the amygdala (−78%) but because there were fewer animals in the group it was not possible to demonstrate statistical significance with respect to baseline for two of the measures. Overall, Compound 1 produced a dose-dependent inhibition of the behavioral seizure response and the EEG after-discharge in the amygdala and the cortex over the dose-range of 1 mg / kg to 100 mg / kg. The maximal effect obtained with Compound 1 was comparable to that produced by diazepam at 16 mg / kg. Plasma exposures 2 hours after dosing in a separate set of rats treated with 1, 6, and 30 mg / kg oral doses of Compound 1 were 191 ± 46.6 ng / ml, 487 ± 91.3 ng / ml, and 1102 ± 115 ng / ml (mean ± S.D, 3 rats per dose). Antiseizure effects of Compound 1 in the genetic absence epilepsy rat from Strasbourg (GAERS)

[0087] Compound 1 and diazepam were assessed for their ability to reduce the occurrence of spontaneous SWDs in the GAERS rat. Compound 1 was evaluated at 6 oral doses of 0.075,2700339.00162 0.75, 2, 6, 30, 100 mg / kg and diazepam was evaluated at a single 3 mg / kg oral dose. As shown in FIG. 4A, following treatment with Compound 1 at a dose of 0.075 mg / kg there was a numerical decrease in the mean SWD counts in each epoch compared to the counts in the vehicle group but the reductions were not statistically significant. However, as seen in FIG. 4B, the overall SWD rate during the post-treatment observation period (10 min. to 130 min.) was significantly reduced even at this low dose. At doses of 0.75, 2, 6, 30 and 100 mg / kg, Compound 1 significantly reduced the SWD counts per epoch and the overall SWD rate when compared to the vehicle group values. The reduction was observed even in the first epoch after dosing (10–30 min). For each of these doses there was a monotonic reduction in mean SWD count in successive epochs with complete suppression of the discharges occurring more rapidly at the higher doses. Once suppressed there was no recovery of activity during the observation period. A dose-response curve constructed from the overall SWD rate data in FIG. 4B provided a calculated ED50value for Compound 1 of 0.10 mg / kg (95% CI 0.05–0.21).

[0088] Diazepam at 3 mg / kg caused a rapid and almost complete elimination of SWDs in the first epoch (10–30 min.) but, in contrast to Compound 1 where the suppression was persistent, the mean number of discharges progressively increased during the remainder of the observation period. This indicates an initial high efficacy followed by a “wearing off” for diazepam that was surprisingly not observed for Compound 1. Compound 1 did not affect rat rotarod performance

[0089] Compound 1 was administered orally to rats at four dose levels, 10, 30, 60, 100 mg / kg, prior to testing performance in a rotarod assay (FIG.5). Duration of time that the rat remained on an accelerating rotarod was recorded. Compound 1- treated animals, at all doses tested, remained on the rotarod for a similar amount of time as compared to vehicle-treated animals. These data indicate that Compound 1 did not cause motor impairments at anti-seizure doses including the highest tested dose of 100 mg / kg. Compound 1 pharmacokinetic-pharmacodynamic relationships

[0090] Blood was collected from selected mice in the 6 Hz test experiments and rats in the amygdala kindling and GAERS experiments after the completion of testing to allow assessment of the plasma levels associated with antiseizure efficacy. FIG. 6 plots the extent of protection in the 6 Hz test at the two stimulation intensities with the plasma levels of Compound 1 achieved in animals receiving Compound 1 at doses of 30, 100, and 300 mg / kg. At the lower stimulation intensity, nearly all animals were protected so no concentration- dependence was discernable. However, at the higher stimulation intensity only a portion of the animals were protected and there is a clear concentration-dependence to the extent of2700339.00162 protection. With the 32 mA stimulation intensity, it is apparent that robust seizure protection is associated with plasma concentration of 100 ng / ml to 1000 ng / ml.

[0091] FIG. 7 plots the plasma concentration values obtained in rats in the amygdala kindling and GAERS experiments. Efficacy in the amygdala kindling model was associated with plasma Compound 1 concentrations in the same range as those that were effective in the 6 Hz test with 32 mA stimulation. In contrast, efficacy in GAERS rats occurs with much lower plasma exposures (<200 ng / mL), suggesting, without being bound to theory, that substantially less receptor occupancy is required to treat absence seizures. DISCUSSION

[0092] In the present study, the antiseizure activity of Compound 1 was characterized in a battery of well-established rodent seizure and epilepsy models. The results indicate that Compound 1 has robust antiseizure activity when administered parenterally and orally. The rodent models selected for the present study represent some of the most frequently used for discovery of antiseizure medication, and for which a high degree of predictive validity exists. The 6 Hz test is a well-characterized mouse model of focal-onset seizures that demonstrates not only pharmacoresistance at high stimulation intensities but also provides a valuable degree of differentiation between antiseizure medication standards of care. The amygdala kindled rat represents a validated rat model of secondarily generalized focal onset seizures that can identify clinically beneficial therapies (i.e., levetiracetam) and is also sensitive to benzodiazepines. Finally, the GAERS model is highly reflective of SWDs associated with absence epilepsy, and informative for identifying compounds that may reduce or aggravate SWDs. Therefore, these models were selected to characterize and differentiate the preclinical profile of Compound 1 and provide insight for predicting clinical potential. At the highest dose tested (100mg / kg), Compound 1 did not cause motor impairment as assessed with the rotarod assays, in both mice and rats (Figure 5, Table 2). Based on patch clamp recordings, in cells expressing recombinant GABAA receptors with various α subunits, Compound 1 displayed selective GABAA receptor PAM activity, with partial functional activity at GABAA receptor subtypes containing α2, α3, or α5 subunits but devoid of activity at receptors with the α1 subunit. The pharmacological profile of Compound 1 differs substantially from that of diazepam, which is a fully active PAM at α1-containing GABAA receptors. The in vitro potencies of Compound 1 at the various α subunit–containing receptors is 10-to 20-fold greater than that of diazepam but the functional activity is substantially less, with diazepam maximally potentiating GABAA receptor chloride currents as much as 5-fold greater than Compound 1.2700339.00162

[0093] There has been considerable interest in the potential of subtype-selective GABAA receptor PAMs, as well as partial agonists, for the treatment of seizures and epilepsy. High- affinity ligands of the benzodiazepine binding site with partial efficacy, such as the beta- carboline derivative abecarnil (ZK 112119), considered to be “anxioselective” due to anxiolytic-like effects, also showed anticonvulsant effects in number of chemically and electrically induced seizure models, in both rodents and primates. It has long been recognized that GABAA receptors containing the α1-subunit contribute to the antiseizure actions of nonselective GABAA receptor PAMs. However, α2 subunit– containing receptors have also been demonstrated to play a role. More recently, compounds with α2, α3 subunit selectivity, such as the imidazopyridazine derivative darigabat, have been reported to exhibit antiseizure effects in animal models such as the pentylenetetrazol seizure test and amygdala kindled rats. The α2, α3-selective PAM BAER-101 (AZD7325), an N-substituted cinnoline, has been reported to have activity in a mouse model of Dravet syndrome and in GAERS. A large body of data is also available for the imidazodiazepine KRM-II- 81, which is an α2 ,α3-selective PAM. Surprisingly, KRM-II-81 demonstrated superior efficacy to diazepam in some models. Relative to diazepam, darigabat exhibits greater potency at α2 and α3 subunits, whereas KRM-II-81 has lower potency on these receptor configurations. It is important to note, however, that KRM-II-81 has comparable functional activity to diazepam on GABAA receptors containing these subunits, whereas darigabat has lower functional activity than diazepam. In contrast, Compound 1 shows lower functional activity but greater potency compared to diazepam, and displays robust antiseizure activity. Therefore, partial PAM activity is sufficient to confer antiseizure efficacy animal seizure and epilepsy models relevant to absence seizures and focal-onset seizures. BAER-101 (AZD7325) has also been reported to be a partial α2, α3 PAM. BAER-101 has recently been reported to have efficacy in GAERS, consistent with the idea that partial PAM activity is sufficient to confer antiseizure efficacy, at least for SWD seizures. A liability of GABAA recepto PAMs for the acute treatment of seizures and the chronic treatment of epilepsy is the development of refractoriness or tolerance. There is evidence that partial GABAA receptor PAMs may have reduced liability for tolerance. It will be of interest to determine if Compound 1 has reduced tolerance compared with PAMs that act in a benzodiazepine-like fashion on GABAA receptors and reduced tolerance in relation to subtype- selective compounds such as darigabat and KRM-II-81. Despite its partial PAM activity at the GABAA receptor and lack of activity at α1 subunit–containing receptors, Compound 1 demonstrated robust antiseizure activity in the various models tested. Compound 1 was effective in the mouse 6 Hz model with stimulation intensity of 32 mA, and higher doses showed antiseizure efficacy with the higher 44 mA stimulation intensity. Notably, no signs of2700339.00162 motor impairment in the rotarod were observed at any of the doses tested in mice. Diazepam (1 mg / kg, i.p.) protected all animals tested with the stimulation intensity of 32 mA, but only 3 of 10 animals at 44 mA. Most antiseizure drugs that are active in the 6 Hz test exhibit greater potency with 32 mA stimulation than with 44 mA stimulation. Benzodiazepines, including diazepam and clonazepam, exhibit this property. Thus, the selectivity of Compound 1 does not cause it to act differently from established drugs in the 6 Hz test. In kindled rats, Compound 1 produced dose-dependent (1–100 mg / kg, oral) inhibition of behavioral seizures as assessed with the Racine scale. There was a corresponding attenuation of the after- discharge duration. Benzodiazepines are well recognized to inhibit behavioral seizures and the after-discharge duration in amygdala kindled rats. We confirmed this effect with diazepam at an oral dose of 16 mg / kg. The magnitude of the antiseizure action of Compound 1 in amygdala kindled rats was similar to that obtained with diazepam. Given that higher doses of Compound 1 reduced seizure severity to under stage 2 but did not fully block it, it appears that Compound 1, like diazepam, does not completely block focal seizures but may substantially reduce secondary or generalized seizures. Darigabat has been reported to have antiseizure effects in the kindled rats, but insufficient data were provided to allow a direct comparison.

[0094] Compound 1 effectively reduced SWDs in GAERS, which is a model of absence epilepsy. Seizures in the GAERS model are highly sensitive to diazepam, and also to the α2, α3 selective compounds BAER-101 and darigabat. Although diazepam cannot be used in the prolonged treatment of absence seizures in humans due to the rapid development of tolerance, the benzodiazepine clonazepam is approved by the U.S. Food and Drug Administration (FDA) for that indication but is used only rarely because of side effects, such as drowsiness and the liability for tolerance. Compound 1 was effective in GAERS at non-sedating doses that were associated with plasma exposures substantially lower than those that were required in the other seizure paradigms. The remarkable efficacy of these GABAA receptor PAMs in GAERS is not fully understood. Thalamocortical mechanisms are well recognized to play a role in absence seizure in GAERS and other absence epilepsy models. Dysfunction of GABAergic neurons in the nucleus reticularis thalami (nRT) that project to thalamic nuclei, may lead to SWDs, including in GAERS. More specifically, it has been demonstrated that cortico-thalamic and nRT neurons are hyperexcitable in the GAERS. GABAA receptors containing α3 subunits are highly enriched in the nRT. GABAA receptors containing α3 subunits are therefore positioned to suppress the pathological hyperexcitability of these neurons, which could be an important factor in the therapeutic activity of α3-targeting PAMs.

[0095] Thus, although the α2-driven activity of α2,α3,α5-selective PAMs such as Compound 1 may exceed α3-driven activity in treating focal-onset seizures, it can be speculated that α32700339.00162 PAM activity may be of particular significance in the anti-absence seizure activity in GAERS. Support for this concept comes from studies in mice showing that a point mutation in α3 renders the receptors benzodiazepine insensitive and benzodiazepines fail to suppress absence seizures in these animals. Consistent with the role of α3-subunit containing GABAA receptors in regulating absence seizures is the observation that certain individuals with rare loss-of- function variants in the GABRA3 gene encoding the α3 subunit exhibit absence seizures. Loss-of- function gene variants in humans GABRA2 and GABRA5, encoding α2 and α5 subunits, respectively, have been associated with non-absence seizures. CONCLUSION

[0096] The in vitro pharmacological studies presented here show that Compound 1 is a partial GABAA receptor PAM active at receptors containing α2, α3, and α5 subunits but devoid of activity at receptors with the α1 subunit. Compound 1 displayed efficacy in multiple rodent seizure and epilepsy models, and was particularly efficacious in the GAERS rat, a validated model of absence epilepsy, the most common idiopathic generalized epilepsy. These results support the use of Compound 1 as an antiseizure treatment in patients with focal and generalized epilepsies. Table 1. SynchroPatch determination of the potency and efficacy of Compound 1 and diazepam for positive modulation of recombinant GABAA receptor subtypes stably expressed in heterologous cells. Subunit Compound 1 Diazepam d

[0097] Emax(%) values are the maximum percent increase in chloride current with respect to GABA EC5–20values. EC5–20-% of maximal effective concentration, EC50-half maximal effective concentration, Emax -maximal effective concentration, SEM-standard error of mean. Table 2. Comparison of Compound 1 and diazepam in the mouse 6 Hz seizure test.2700339.00162 Treatment Dose (mg / kg, Stimulation Intensity Rotarod Impairment i.p.)ac rea men was es e n separa e groups o mce w s mu a on n ens es of 32 and 44 mA. Seizure protection column entries indicate the number of mice protected (out of a total of 10 mice tested) under the different treatment conditions: vehicle, diazepam (1 mg / kg), or Compound 11 (30, 100, or 300 mg / kg). Minimal motor impairment in mice was established by performance on a fixed-speed rotarod immediately prior to in-life seizure testing. Each mouse was considered impaired if it fell off the rotarod 3 times during a 1-min period. i.p.-intraperitoneal, mA-milliampere, min-minute, mg / kg-milligram per kilogram. Example 2

[0099] A therapeutically-effective amount of Compound 1 is administered orally to a patient with epilepsy who is experiencing focal seizures. Following administration, the frequency of the seizures is decreased by 50%. Example 3

[0100] A therapeutically-effective amount of Compound 1 is administered parenterally to a patient with epilepsy who is experiencing focal seizures. Following administration, the frequency of the seizures is decreased by 50%. Example 4

[0101] A therapeutically-effective amount of a pharmaceutically-acceptable salt of Compound 1 is administered orally to a patient with epilepsy who is experiencing focal seizures. Following administration, the frequency of the seizures is decreased by 50%. Example 5

[0102] A therapeutically-effective amount of a pharmaceutically-acceptable salt of Compound 1 is administered parenterally to a patient with epilepsy who is experiencing focal seizures. Following administration, the frequency of the seizures is decreased by 50%.2700339.00162 Example 6

[0103] A therapeutically-effective amount of Compound 1 is administered orally to a patient with epilepsy who is experiencing secondary seizures. Following administration, the frequency of the seizures is decreased by 50%. Example 7

[0104] A therapeutically-effective amount of Compound 1 is administered parenterally to a patient with epilepsy who is experiencing secondary seizures. Following administration, the frequency of the seizures is decreased by 50%. Example 8

[0105] A therapeutically-effective amount of a pharmaceutically-acceptable salt of Compound 1 is administered orally to a patient with epilepsy who is experiencing secondary seizures. Following administration, the frequency of the seizures is decreased by 50%. Example 9

[0106] A therapeutically-effective amount of a pharmaceutically-acceptable salt of Compound 1 is administered parenterally to a patient with epilepsy who is experiencing secondary seizures. Following administration, the frequency of the seizures is decreased by 50%. Example 10

[0107] A therapeutically-effective amount of Compound 1 is administered orally to a patient with epilepsy who is experiencing generalized seizures. Following administration, the frequency of the seizures is decreased by 50%. Example 11

[0108] A therapeutically-effective amount of Compound 1 is administered parenterally to a patient with epilepsy who is experiencing generalized seizures. Following administration, the frequency of the seizures is decreased by 50%. Example 12

[0109] A therapeutically-effective amount of a pharmaceutically-acceptable salt of Compound 1 is administered orally to a patient with epilepsy who is experiencing generalized seizures. Following administration, the frequency of the seizures is decreased by 50%.2700339.00162 Example 13

[0110] A therapeutically-effective amount of a pharmaceutically-acceptable salt of Compound 1 is administered parenterally to a patient with epilepsy who is experiencing generalized seizures. Following administration, the frequency of the seizures is decreased by 50%. Embodiments

[0111] Embodiment 1- A method of treating seizure in a subject in need thereof, comprising administration of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6- [(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine or a pharmaceutically acceptable salt thereof to the subject.

[0112] Embodiment 2- A method of treating epilepsy in a subject in need thereof, comprising administration of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6- [(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine or a pharmaceutically acceptable salt thereof to the subject.

[0113] Embodiment 3- The method of embodiment 1 or 2, wherein the 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine or a pharmaceutically acceptable salt thereof is a fumarate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl- 1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

[0114] Embodiment 4- The method of embodiment 1 or 2, wherein the 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine or a pharmaceutically acceptable salt thereof is a sulfate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H- 1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

[0115] Embodiment 5- The method of embodiment 1 or 2, wherein the 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine or a pharmaceutically acceptable salt thereof is a hydrochloride salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1- methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

[0116] Embodiment 6- The method of embodiment 1 or 2, wherein the 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine or a pharmaceutically acceptable salt thereof is2700339.00162 a phosphate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl- 1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

[0117] Embodiment 7- The method of embodiment 1 or 2, wherein the 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine or a pharmaceutically acceptable salt thereof is a tosylate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl- 1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

[0118] Embodiment 8- The method of embodiment 1 or 2, wherein the 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine or a pharmaceutically acceptable salt thereof is a malonate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl- 1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

[0119] Embodiment 9- The method of embodiment 1 or 2, wherein the 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine or a pharmaceutically acceptable salt thereof is a maleate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl- 1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

[0120] Embodiment 10- A compound, selected from: a monohydrate of 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine; a. a fumarate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]- 6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine; b. a sulfate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6- [(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine; c. a hydrochloride salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2- d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine; d. a phosphate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2- d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine; e. a tosylate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6- [(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine;2700339.00162 f. a malonate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]- 6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine; or g. a maleate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6- [(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

[0121] Embodiment 11- A composition, comprising the compound of claim 10.

[0122] Embodiment 12- The composition of embodiment 11, which is a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier.

[0123] Embodiment 13- A method of treating seizure in a subject in need thereof, comprising administration of the composition of embodiment 11 or 12, or a monohydrate of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine, to the subject.

[0124] Embodiment 14- A method of treating epilepsy in a subject in need thereof, comprising administration of the composition of embodiment 11 or 12, or a monohydrate of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine, to the subject.

[0125] Embodiment 15- The method of embodiments 1, 3-9 or 13, wherein said seizure comprises a focal seizure.

[0126] Embodiment 15- The method of embodiments 1, 3-9 or 13, wherein said seizure comprises a secondary seizure.

[0127] Embodiment 15- The method of embodiments 1, 3-9 or 13, wherein said seizure comprises a generalized seizure.

Claims

2700339.00162 CLAIMS We claim:

1. A method of treating seizure in a subject in need thereof, comprising administration of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol- 5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine or a pharmaceutically acceptable salt thereof to the subject.

2. A method of treating epilepsy in a subject in need thereof, comprising administration of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol- 5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine or a pharmaceutically acceptable salt thereof to the subject.

3. The method of claim 1 or 2, wherein the 3-(2,5-difluorophenyl)-7-[1,1-di(methyl- d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine or a pharmaceutically acceptable salt thereof is a fumarate salt of 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

4. The method of claim 1 or 2, wherein the 3-(2,5-difluorophenyl)-7-[1,1-di(methyl- d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine or a pharmaceutically acceptable salt thereof is a sulfate salt of 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

5. The method of claim 1 or 2, wherein the 3-(2,5-difluorophenyl)-7-[1,1-di(methyl- d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine or a pharmaceutically acceptable salt thereof is a hydrochloride salt of 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.2700339.00162 6. The method of claim 1 or 2, wherein the 3-(2,5-difluorophenyl)-7-[1,1-di(methyl- d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine or a pharmaceutically acceptable salt thereof is a phosphate salt of 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

7. The method of claim 1 or 2, wherein the 3-(2,5-difluorophenyl)-7-[1,1-di(methyl- d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine or a pharmaceutically acceptable salt thereof is a tosylate salt of 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

8. The method of claim 1 or 2, wherein the 3-(2,5-difluorophenyl)-7-[1,1-di(methyl- d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine or a pharmaceutically acceptable salt thereof is a malonate salt of 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

9. The method of claim 1 or 2, wherein the 3-(2,5-difluorophenyl)-7-[1,1-di(methyl- d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3- b]pyridazine or a pharmaceutically acceptable salt thereof is a maleate salt of 3-(2,5- difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5- yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

10. A compound, selected from: a monohydrate of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl- 1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine; a fumarate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl- 1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine; a sulfate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H- 1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine;2700339.00162 a hydrochloride salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1- methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine; a phosphate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl- 1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine; a tosylate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl- 1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine; a malonate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl- 1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine; or a maleate salt of 3-(2,5-difluorophenyl)-7-[1,1-di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl- 1H-1,2,4-triazol-5-yl)methoxy]-1,2,4-triazolo[4,3-b]pyridazine.

11. A composition, comprising the compound of claim 10.

12. The composition of claim 11, which is a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier.

13. A method of treating seizure in a subject in need thereof, comprising administration of the composition of claim 11 or 12, or a monohydrate of 3-(2,5-difluorophenyl)-7-[1,1- di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4- triazolo[4,3-b]pyridazine, to the subject.

14. A method of treating epilepsy in a subject in need thereof, comprising administration of the composition of claim 11 or 12, or a monohydrate of 3-(2,5-difluorophenyl)-7-[1,1- di(methyl-d3)ethyl-2,2,2-d3]-6-[(1-methyl-1H-1,2,4-triazol-5-yl)methoxy]-1,2,4- triazolo[4,3-b]pyridazine, to the subject.

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