Methods of treating generalized anxiety disorder

Abstract

Provided herein are methods of treating generalized anxiety disorder and anxiety, as well as improving psychic anxiety and somatic anxiety, in a subject in need thereof, comprising administering 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[1,2-b][1,2,4] triazin-7-yl)[l,l'-biphenyl]-2-carbonitrile, or a pharmaceutically acceptable salt thereof, such as a phosphate salt thereof, formulated in a pharmaceutical composition, to the subject.

Description

METHODS OF TREATING GENERALIZED ANXIETY DISORDERRELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Patent Application No. 63 / 559,133, filed February 28, 2024, and U.S. Provisional Patent Application No. 63 / 554,580, filed February 16, 2024, the entire content of each of which is incorporated herein by reference.BACKGROUND

[0002] Gamma-aminobutyric acid (GABA) is the major fast inhibitory neurotransmitter in humans fulfilling much of its role through activation of the GABAA receptor. GABAA receptors are ligand-gated chloride channels comprised of 5 protein subunits (ol-6, 01-3, yl-3, 5, £, 0, and n). GABAA receptors that contain al, a2, a3, or a5 subunits in combination with 0x subunits and a y2 subunit in a 2:2: 1 (a:0:y) ratio form an allosteric binding site separate from where GABA binds. This site, termed the benzodiazepine (BDZ) binding site, enables positive allosteric modulation of the ion channel. Nearly 90% of GABAA receptors in the central nervous system (CNS) contain a BDZ-binding site and those that do are classified into subtypes by their a subunit.

[0003] GABAA modulation is one pharmacologic approach for the treatment of anxiety (e.g., generalized anxiety disorder) or other disorders of the CNS, as demonstrated by broadspectrum GABAA agonists of the BDZ class (e.g., alprazolam, clonazepam, diazepam, lorazepam). However, long-term administration using this approach is generally limited due to significant side effects including sedation, ataxia, cognitive impairment, dependence, and withdrawal associated adverse events (AEs). Similarly, nonbenzodiazepines acting as GABAA receptor positive allosteric modulators of the benzodiazepine site may have limitations on their use as well. For example, zolpidem's effectiveness is nearly as much due to psychological effects as to the medication itself (Huedo-Medina TB, Kirsch I, Middlemass J, Klonizakis M, Siriwardena AN (December 2012). "Effectiveness of non-benzodiazepine hypnotics in treatment of adult insomnia: meta-analysis of data submitted to the Food and Drug Administration". BMJ. 345: e8343). Clinical development of alpidem, used to treat anxiety disorders, was discontinued due to liver toxicity. Clinical development of indiplon was terminated following FDA approvability conditioned on further safety studies. Clinical development of ocinaplon, an anxiolytic having low sedative or amnestic effects, was discontinued due to liver complications. Panadiplon, a high-affinity GABAA receptor partial agonist, showed a useful effects profile as a potent anxiolytic with little sedative effects, but panadiplon was discontinued from clinical development for use in humans after showing evidence of liver damage in both animals and human trials. Zaleplon has an ultrashortelimination half-life (Ihr), thereby limiting its utility at least due to dosing compliance challenges. Eszopiclone use may diminish the subject's ability to participate in activities requiring full alertness. Due to its propensity for dependence and attenuation, Zopiclone is recommended to be taken at the lowest effective dose for a short-term treatment duration (e.g., 2-3 weeks).

[0004] There remains a need for compounds other than broad-spectrum GABAA agonists for use in treating anxiety.SUMMARY

[0005] 2',6-difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,r- biphenyl]-2-carbonitrile does not potentiate GABAA receptor activity through the ol subtype. Provided herein are methods of treating anxiety (e.g., generalized anxiety disorder) comprising administering 2',6-difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2- b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile, or its pharmaceutically acceptable salt, to a subject in need thereof. Provided herein is also 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile, or its pharmaceutically acceptable salt, for use in a method of treating anxiety, as well as compositions for use of treating anxiety comprising 2',6-difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2- b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile, or its pharmaceutically acceptable salt (e.g., as a phosphate salt). In the following the description of the compositions for use and compounds for use are included in the description of the methods of treatment.DETAILED DESCRIPTION

[0006] 7-(l,l-Dimethylethyl)-6-[(l-ethyl-l / 7-l,2,4-triazol-5-yl)methoxy]-3-(2- fluorophenyl)-l,2,4-triazolo[4,3-b]pyridazine (CAS Registry Number #252977-51-8; shown below) is a mixed, subtype-selective ligand of the benzodiazepine site of al, a2, a3, and a5- containing GABAA receptors, where it acts as a partial agonist at benzodiazepine sites of the o2 and a3-containing subtypes, but as a silent antagonist at al and a5-containing subtypes. In human trials on healthy volunteers, the compound exhibited some similar attributes as lorazepam, but had much less side effect on cognition, memory, alertness or coordination. In Phase II trials, the compound was significantly superior to placebo without inducing sedation. However, the clinical development was halted due to preclinical toxicity in long term dosing studies.

[0007] The compound 2',6-difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2- b][l / 2,4]triazin-7-yl)-[l,r-biphenyl]-2-carbonitrile does not potentiate GABAA receptor activity through the ol subtype. Instead, the compound is an o2,3,5-subtype-selective modulator of the GABAA receptor. Put another way, the compound 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile has partial positive modulation at a2, a3, and a5 receptor subtypes but is an antagonist at the al subtype. Without being theory-bound, this is an attractive and desirable profile for a GABA- modulator as it could have less propensity for adverse effects compared to non-selective BDZs since side effects of non-selective GABAA agonists may result from al subunit activity.

[0008] Thus, in some embodiments, provided herein are methods of treating anxiety in a subject in need thereof, comprising administering 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile, or a pharmaceutically acceptable salt thereof, to the subject. For brevity, 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile may be referred to herein as Compound 1.

[0009] Thus, in some embodiments, provided herein are methods of treating anxiety in a subject in need thereof, comprising administering 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile as a phosphate salt to the subject. For brevity, 2',6-difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin- 7-yl)-[l,l'-biphenyl]-2-carbonitrile phosphate may be referred to herein as Compound 2.Definitions

[0010] Certain terms, whether used alone or as part of a phrase or another term, are defined below.

[0011] The articles "a" and "an" refer to one or to more than one of the grammatical object of the article.

[0012] Numerical values relating to measurements are subject to measurement errors that place limits on their accuracy. For this reason, the term "about" may explicitly or implicitly modify all numerical values provided herein, unless otherwise indicated. The term "about" generally indicates a possible variation of no more than 10%, 5%, or 1% of a numerical value. In some embodiments, the last decimal place of a numerical value provided herein indicates its degree of accuracy. In some embodiments, where no other error margins are given, the maximum margin is ascertained by applying the rounding-off convention to the last decimal place or last significant digit when a decimal is not present in the given numerical value.

[0013] The term "amelioration" means a lessening of severity of at least one indicator 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.

[0014] 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.

[0015] The term "anxiety" includes, but is not limited to generalized anxiety disorder, psychic anxiety, panic disorder, social anxiety disorder, phobia-related disorders, agoraphobia, separation anxiety, somatic anxiety, and childhood anxiety disorder.

[0016] 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 may be performed in any suitable order, and may include a combination of one or more embodiments herein, unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language ("for example," "such as," etc.) provided herein is intended merely to better illuminate the described subject matter and does not pose a 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.

[0017] Each group member of a grouping of alternative elements or embodiments of this disclosure 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.

[0018] Reference made to a patent or printed publication document throughout this specification incorporate herein by reference the document's entire content.

[0019] Embodiments of this disclosure are illustrative. Accordingly, the present disclosure is not limited to that precisely as shown and described.Methods

[0020] In some embodiments, provided herein are methods of treating anxiety in a subject in need thereof, comprising administration of a pharmaceutical composition comprising 2’,6- difluoro-5’-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,T-biphenyl]-2- carbonitrile, or a pharmaceutically acceptable salt thereof, (e.g., Compound 1 or Compound 2) once per day for at least 7 consecutive days to the subject.

[0021] In some embodiments, provided herein are methods of treating anxiety in a subject in need thereof, comprising administration of a pharmaceutical composition comprising 2’,6- difluoro-5’-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,T-biphenyl]-2- carbonitrile phosphate once per day for at least 7 consecutive days to the subject.

[0022] In some embodiments, provided herein are methods of treating anxiety in a subject in need thereof, comprising administration of a pharmaceutical composition comprising 2',6- difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2- carbonitrile once per day for at least 7 consecutive days to the subject.

[0023] In some embodiments, provided herein are methods of improving psychic anxiety in a subject in need thereof, comprising administration of a pharmaceutical composition once per day for at least 14 consecutive days to the subject, wherein the pharmaceutical composition comprises about 2.5 mg of 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile, or a pharmaceutically acceptable salt thereof (e.g., about 2.0 mg of Compound 1 or about 2.5 mg of Compound 2), improving psychic anxiety is determined by a statistically significant mean change from a baseline of the subject on the Hamilton Anxiety Rating Scale (HAM-A) subscale for psychic anxiety, the baseline of the subject on the HAM-A subscale for psychic anxiety is determined by assessing the subject on the HAM-A subscale for psychic anxiety on the first day of administration of the pharmaceutical composition but prior to administration of the pharmaceutical composition.

[0024] In some embodiments, provided herein are methods of improving somatic anxiety in a subject in need thereof, comprising administration of a pharmaceutical composition once per day for at least 14 consecutive days to the subject, wherein the pharmaceutical composition comprises about 2.5 mg of 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile phosphate, improving somatic anxiety is determined by a statistically significant mean change from a baseline of the subject on the Hamilton Anxiety Rating Scale (HAM-A) subscale for somatic anxiety, the baseline of the subject on the HAM-A subscale for somatic anxiety is determined by assessing the subject on the HAM-A subscale for somatic anxiety on the first day of administration of the pharmaceutical composition prior to administration of the pharmaceutical composition.

[0025] In some embodiments, provided herein are methods of improving somatic anxiety in a subject in need thereof, comprising administration of a pharmaceutical composition once per day for at least 14 consecutive days to the subject, wherein the pharmaceutical composition comprises about 2.0 mg of 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile, improving somatic anxiety is determined by a statistically significant mean change from a baseline of the subject on the Hamilton Anxiety Rating Scale (HAM-A) subscale for somatic anxiety, the baseline of the subject on the HAM-A subscale for somatic anxiety is determined by assessing the subject on the HAM-A subscale for somatic anxiety on the first day of administration of the pharmaceutical composition prior to administration of the pharmaceutical composition.

[0026] In some embodiments, provided herein are methods of treating generalized anxiety disorder in a subject in need thereof, comprising administration of a pharmaceutical composition comprising 2',6-difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2- ^][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile phosphate once per day for at least 14 consecutive days to the subject, wherein the subject is a human.

[0027] In some embodiments, provided herein are methods of treating generalized anxiety disorder in a subject in need thereof, comprising administration of a pharmaceutical composition comprising 2',6-difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2- b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile once per day for at least 14 consecutive days to the subject, wherein the subject is a human.

[0028] In some embodiments of the methods herein, the 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile or 2',6- difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2- carbonitrile phosphate is the sole active pharmaceutical ingredient in the pharmaceutical composition. In some embodiments of the methods herein, the 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile or 2',6- difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2- carbonitrile phosphate is the sole active pharmaceutical ingredient administered to the subject in the method (e.g., another prescribed active pharmaceutical ingredient is not needed to impart the desired therapeutic effect), is the sole active pharmaceutical ingredient administered to the subject during the method (the subject is not being administered anyother prescribed active pharmaceutical ingredient), or both. In some embodiments, the pharmaceutical composition comprises about 1.9 to about 6.3 mg of 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile or 2',6- difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2- carbonitrile phosphate. In some embodiments, the pharmaceutical composition comprises about 2.5 mg of 2',6-difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7- yl)-[l,l'-biphenyl]-2-carbonitrile phosphate. In some embodiments, the pharmaceutical composition comprises about 2.0 mg of 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile.

[0029] In some embodiments of the methods herein, the 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile or 2',6- difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2- carbonitrile phosphate is administered in an amount to achieve an AUCtau plasma concentration (e.g., a steady state plasma concentration) of about 500 to about 2,000 ng-h / mL, e.g., about 700 to about 1,000 ng-h / mL, e.g., about 820 ng-h / mL, at about day 14 of administration of the pharmaceutical composition. In some embodiments, the plasma concentration is a steady state plasma concentration. In some embodiments, the daily dosage administered to the subject achieves a plasma steady state concentration of 2',6-difluoro-5'- (3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l, 2, 4]triazin-7-yl)-[ 1,1 '-biphenyl ]-2-carbonitrile having an about 10-20% differential in peak to trough GABAA receptor occupancy of the 2',6- difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2- carbonitrile. In some embodiments, the peak receptor occupancy is about 80-90%. In some embodiments, the trough receptor occupancy is about 70-80%. In some embodiments, a daily dosage of about 2.5 mg Compound 2 (or about 2.0 mg Compound 1) achieves an a2 or o5, or both, receptor subunit functional activity at plasma steady state of Compound 2 (or Compound 1) of about 30-40%, and an a3 receptor subunit functional activity at plasma steady state of Compound 2 (or Compound 1) of about 45-55%.

[0030] In some embodiments of the methods herein, the 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile or 2',6- difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2- carbonitrile phosphate is administered in an amount to achieve a score of 1 or 2 on a clinical global impression - improvement scale (CGI-I) assessment of generalized anxiety disorder of the subject.

[0031] In some embodiments of the methods herein, the 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile or 2',6- difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2- carbonitrile phosphate is administered in an amount to achieve a >50% reduction from a baseline of the subject on the Hamilton Anxiety Rating Scale (HAM-A) 14 days after the first administration of the pharmaceutical composition, wherein the baseline of the subject on the HAM-A is determined by assessing the subject on the HAM-A on the first day of administration of the pharmaceutical composition but prior to administration of the pharmaceutical composition.

[0032] In some embodiments of the methods herein, the subject is clinically depressed, clinically an insomniac, or both.

[0033] In some embodiments of the methods herein, the pharmaceutical composition is in the form of a single unit, an oral dosage form, administered more than 2 hours subsequent to oral food intake (e.g., not within 2 hours, whether before or after, of oral food intake; e.g., on an empty stomach), or a combination thereof.

[0034] In some embodiments of the methods herein, the methods include an efficacy or improvement metric, or a combination thereof, described herein.

[0035] The following examples further illustrate embodiments of the present disclosure. However, they are in no way a limitation of the teachings or disclosure as described herein.EXAMPLESExample 1: Animal Toxicology Study.

[0036] Three-month toxicology studies in dog and rats are completed and safety margins are calculated based on a no-observed adverse-effect level (NOAEL) of 4.8 mg / kg / day of compound 1 in dog and 3.2 mg / kg / day of compound 1 in rat based on toxicokinetic data from the exposures observed. Calculated Safety margins, based on human plasma steady state studies, at a dose of 2.5 mg (corresponds to 2 mg of 2',6-difluoro-5'-(3-(2-hydroxypropan- 2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile as the free base / neutral form) are shown in Table 1. Taken together, these data indicate adequate safety margins for Compound 2 to support dose selection of 2.5 mg Compound 2 for further study.Table 1. Safety Margins Based on Plasma Exposure Levels in the Dog 3-month Toxicology Report and the Rat 3-month Toxicology Report.1 Compound 2 AUC of 17,000-29,000 ng-h / mL in males and females, respectively, represents the exposure in dogs at the NOAEL of 4.8 mg / kg / day of the free base / neutral form.2 Compound 2 AUC of 78,400-81,900 ng-h / mL in males and females, respectively, represents the exposure in rats at the NOAEL of 3.2 mg / kg / day free base / neutral form.3 Compound 2 geometric mean AUCtau of 819 ng-h / mL at Compound 2, 2.5 mg QD, at plasma steady state (Day 12).Abbreviations: AUC = area under the concentration curve; NOAEL = no-observed adverse- effect level; QD = once dailyExample 2: Treatment of Patients with Generalized Anxiety Disorder.

[0037] The efficacy and safety of using 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile phosphate (Compound 2), as compared to placebo, for treatment of generalized anxiety disorder in patients is demonstrated using the below dosage and regimen. Assessments include, but are not limited to those shown in Table 3 below.

[0038] A randomized, double-blind, placebo-controlled, monotherapy treatment study is performed. Each patient in the study is randomized to a pre-defined placebo lead-in period and follow-out period such that patients randomized to Compound 2 will receive a total of 2 weeks of placebo at the start and / or at the end of the 9-week Treatment Period, and 7 weeks of Compound 2, including a 4-week fixed dose period of 2.5 mg and a 3-week taper. Patients randomized to placebo will receive placebo for 9 weeks and will be sequence-matched to patients randomized to Compound 2. Patients will receive: Compound 2 Group: 4 weeks of a fixed dose of Compound 2 (2.5 mg), 3 weeks of a Compound 2 taper (1.875 mg for 1 week,1.25 mg for 1 week, and 0.625 mg for 1 week), and 2 weeks of placebo (before and / or after Compound 2)— all doses will be administered as 4 capsules per day (one size 3 capsule and three size 4 capsules) to maintain blinding; and Placebo Group: a total of 9 weeks of daily placebo capsules matched to the 4 capsules per day administered to patients in the Compound 2 group (one size 3 capsule and three size 4 capsules) to maintain blinding. Patients will receive one Size 3 capsule (2.5 mg Compound 2 or matching placebo) and three Size 4 capsules (0.625 mg Compound 2 or matching placebo) every day for the duration of the 9- week treatment period. The 4-week treatment and the 3-week taper will therefore be "hidden" in the 9-week period of study drug dosing. Details of study drugs are shown in Table 2 below.

[0039] Blood is collected for measurement of plasma concentrations of Compound 2 (e.g., as the free base / neutral form) and its metabolites (as applicable), which may be isolated from a sample in the form of a salt, on Day 1, Day 21, Day 49, and Day 77.Table 2. Study DrugsTable 3.1 Change from baseline to the last day of the 4-week randomized study treatment period (Compound 2 or placebo), not including the placebo lead-in, taper, or placebo follow-out period, will be assessed.2 Measured at the last day of the 4-week randomized study treatment period (Compound 2 or placebo), not including the placebo lead-in, taper, or placebo follow-out period.3 The adverse event will be calculated as treatment-emergent adverse events.

[0040] Efficacy endpoints / metrics of the methods herein can include one or more of the following:• Mean change from baseline (to the last day of the 4-week randomized study treatment period, i.e., Compound 2 or placebo, not including the placebo lead-in, taper, or placebo follow-out period) on the HAM-A total score;• Mean value of the CGI-I;• Percentage of responders (defined as patients with scores of 1 or 2) on the CGI-I;• Mean change from baseline in the CGI-S;• Percentage of responders (defined as patients who experience >50% reduction in the HAM-A total score from baseline);• Percentage of remitters (defined as patients who experience HAM-A total score <7 at endpoint);• Mean change from baseline in the HAM-A subscale for psychic anxiety;• Mean change from baseline in the HAM-A subscale for somatic anxiety;• Mean change from baseline and time-course of response of treatment in the GAD-7 total score;• Mean change from baseline and time-course of response in the HAM-D 6 total score;• Mean change from baseline and time-course of response in the Functioning Survey;• Mean change from baseline and time-course of response in the PSWQ-10 total score;• Mean change from baseline and time-course of response in the ISI total score; or• Mean change from baseline and time-course of response in the Treatment Satisfaction Question.

[0041] Additionally, the methods herein can include one or more of the following improvement metrics:• Improvements in insomnia in patients with generalized anxiety disorder, defined as clinically significant insomnia with an Insomnia Severity Index (ISI) score >14 at baseline, and measured by change from baseline ISI score;• Improvements in depression in patients with generalized anxiety disorder, defined as co- morbid symptoms of depression with a 6-Item Hamilton Depression Rating Scale (HAM-D 6) score >4 at baseline, and measured by change from baseline in HAM-D 6 score;• Improvements in anxiety as measured by change from baseline on the HAM-A total score in certain subgroups of patients, such as:• Patients with a history of prior childhood trauma as measured by the Childhood Trauma Questionnaire Short Form (CTQ-SF) :• With dimensional physical abuse scores >8;• With dimensional physical neglect scores >8;• With dimensional emotional abuse scores >9;• With dimensional emotional neglect scores >10; or• With dimensional sexual abuse scores >6;• Patients with high levels (above the median score) of psychic anxiety at baseline;• Patients with high levels (above the median score) of somatic anxiety at baseline;• Patients with clinically significant insomnia with an Insomnia Severity Index (ISI) score >14 at baseline; or• Patients with co-morbid symptoms of depression with a 6-Item Hamilton Depression Rating Scale (HAM-D 6) score >4 at baseline.

[0042] Hamilton Anxiety Rating Scale (HAM-A): The HAM-A scale assesses the severity of different anxiety-related symptoms (Hamilton MA. The assessment of anxiety states by rating. British journal of medical psychology. 1959; Hamilton M. Standardised assessment and recording of depressive symptoms. Psychiat Neurol Neurochir. 1969, 72:201) As the original HAM-A is semi-structured and lacks instructions for administration and clear anchor points for the assignment of severity ratings, a structured interview guide version (SIGH-A) will be used in the current study (Shear MK, Vander Bilt J, Rucci P,et al. Reliability and validity of a structured interview guide for the Hamilton Anxiety Rating Scale (SIGH-A). Depressionand anxiety. 2001; 13(4): 166-78). The SIGH-A has been shown to have high inter-rater and test-retest reliability and highly correlates with a self- report measure of overall anxiety (Shear et al.). The HAM-A will be administered at Screening, Day 1 (pre-randomization), Day 7, Day 14, Day 21, Day 28, Day 35, Day 42, Day 49, Day 56, and Day 63. Patients will be eligible for inclusion in the study if, at Screening and Day 1, the HAM-A score is >22 and the patient has at least moderately severe core symptoms of anxious mood and tension as measured by HAM-A Items 1 and 2, respectively, with scores each >2.

[0043] Ecological Momentary Assessment (EMA): EMA contains frequently administered questions targeted towards collecting real-time information and observations that take place in a patient's daily environment. In this study, an EMA will be delivered on a smartphone twice per day (morning and evening) from the first day of the Screening Period to Day 63. During the Screening Period, scores in EMA clinical measures (i.e., GAD-7 and HAM-D 6) and general EMA completion frequency will be used to determine adherence eligibility, per independent adjudication. At each twice-daily EMA there will be a Functioning Survey, that examines social context, location, and activities, followed by 2 clinical rating scales, GAD-7 and HAM-D 6. Once per week a Treatment Satisfaction question will be included in the EMA.

[0044] Functioning Survey: The Functioning Survey (Strassnig MT, Miller ML, Moore R, Depp CA, Pinkham AE, Harvey PD. Evidence for avolition in bipolar disorder? A 30-day ecological momentary assessment comparison of daily activities in bipolar disorder and schizophrenia. Psychiatry research. 2021 Jun l;300: 113924) captures social context and activities with a time frame of "now" for location / social context, and "the past hour" for the activity items. The first 2 survey items in each EMA capture location (home vs away) and social context (alone versus with someone). Based on the answer provided to location and social context, additional customized surveys for home-alone, home-with someone, and away are delivered excluding assessments of irrelevant activities (e.g., number of in-person social interactions in the past hour while home alone). In addition, the Functioning Survey completed in the evening will capture whether the patient has used caffeine- and / or nicotine-containing products during the day.

[0045] Generalized Anxiety Disorder-7 (GAD-7): The GAD-7 (Spitzer RL, Kroenke K, Williams JB, Lowe B. A brief measure for assessing generalized anxiety disorder: the GAD-7. Archives of internal medicine. 2006 May 22; 166(10) : 1092-7) will be presented as an item- by-item survey on the EMA device screen, with all 7 items presented in the same order at each assessment. It measures different facets of anxiety, including nervousness, worrying, worrying about multiple things, inability to relax, restlessness, irritability, catastrophizingthoughts. Scores for each item range from 0-3, yielding a potential total score of 21. Patients will receive prompts after 30 seconds of inactivity. All item scores will be collected by the application and the total scores will be calculated automatically in the database. The GAD-7 survey will launch immediately after the Functioning Survey during each EMA survey sequence. In line with the general principles of this survey, the morning survey will ask the question in terms of how the patient is feeling "now" and the evening survey will ask about "today".

[0046] 6-item Hamilton Depression Rating Scale (HAM-D 6): The HAM-D 6 (O'Sullivan RL, Fava M, Agustin C, Baer L, Rosenbaum JF. Sensitivity of the six-item Hamilton depression rating scale. Acta Psychiatrica Scandinavica. 1997 May; 95(5) : 379-84) includes 6 core symptoms of depression: depressed mood, guilt, loss of interest in work and activities, anxiety (psychic), somatic symptoms (general), and psychomotor retardation. These items are extracted from the longer HAM-D 17. All items are presented one at a time, in a fixed order, on the EMA device screen. Patients will receive prompts after 30 seconds of inactivity. Items 1 through 5 of the scale are scored 0 to 4 and item 6 is scored 0 to 2 (in line with the original HAM-D 17 scoring). Thus, total scores for the HAM-D 6 can range from 0 to 22. All item scores will be collected by the application and the total scores will be calculated automatically in the database. The HAM-D 6 assessment will occur immediately after the GAD-7 assessment and before the Treatment Satisfaction question (when delivered). In line with the general principles of this survey, the morning survey will answer the question in terms of how the patient is feeling "now" and the evening survey will ask about "today".

[0047] Treatment Satisfaction Question: A Treatment Satisfaction question will be included in the evening survey at a pseudo-randomized weekly interval aiming to capture satisfaction (from not at all to extremely), in each of nine (9) 1-week treatment periods.

[0048] Clinical Global Impression: CGI is a commonly used, brief, investigator-rated assessment (Guy W, editor. ECDEU Assessment Manual for Psychopharmacology. Rockville, MD, U.S. Department of Health Education, and Welfare. 1976). In this study, CGI for improvement (CGI-I) and severity (CGI-S) will be assessed by the Investigator or qualified designee following an interview with the patient or study partner to query the status of the patient with regards to, for example, anxiety symptoms, AEs, or other symptoms. Both assessments are rated on a 7-point scale where raters will be asked about improvement from baseline (where 1 indicates "very much improved" to 7 "very much worse"), as well as severity of illness (where 1 corresponds to "normal, not at all ill", and 7 is "among the most extremely ill patients"). The CGI-S will be administered at Screening, Day 1 (pre-randomization), Day7, Day 14, Day 21, Day 28, Day 35, Day 42, Day 49, Day 56, and Day 63. The CGI-I will be administered on Day 7, Day 14, Day 21, Day 28, Day 35, Day 42, Day 49, Day 56, and Day 63. Both assessments will be administered by the Investigator or designee.

[0049] Penn State Worry Questionnaire-10 (PSWQ-10): The Penn State Worry Questionnaire-10 (PSWQ-10) is an abridged version of the Penn State Worry Questionnaire (PSWQ; Meyer TJ, Miller ML, Metzger RL, Borkovec TD. Development and validation of the penn state worry questionnaire. Behaviour research and therapy. 1990 Jan 1, 28(6):487-95; Brown TA, Antony MM, Barlow DH. Psychometric properties of the Penn State Worry Questionnaire in a clinical anxiety disorders sample. Behaviour research and therapy. 1992 Jan 1, 30(l):33-7) with excellent internal consistency, convergent and discriminant validity, and criterion group validity. It measures state pathological worry, a core symptom of GAD. Patients are presented with 10 statements and asked to rate each of them (from 0 / never to 6 / almost always) according to how often each applied to them (Yao B, Sripada RK, Klumpp H, Abelson JL, Muzik M, Zhao Z, Rosenblum K, Briggs H, Kaston M, Warren R. Penn State Worry Questionnaire-10: A new tool for measurement-based care. Psychiatry Research. 2016 May 30, 239:62-7). The PSWQ-10 will be performed by the patient in the presence of the Investigator or designee on as described in the Schedule of Evaluations.

[0050] Insomnia Severity Index (ISI): The ISI is a 7-item self-report questionnaire assessing the subjective symptoms regarding the nature, severity, and impact of insomnia. The dimensions evaluated are severity of sleep onset, sleep maintenance, and early morning awakening problems, sleep dissatisfaction, interference of sleep difficulties with daytime functioning, noticeability of sleep problems by others, and distress caused by the sleep difficulties. A 5-point Likert scale is used to rate each item (i.e., 0 = no problem; 4 = very severe problem), yielding a total score ranging from 0 to 28. Higher scores indicate more severe insomnia (Bastien CH, Vallieres A, Morin CM. Validation of the Insomnia Severity Index as an outcome measure for insomnia research. Sleep medicine. 2001 Jul 1, 2(4):297-307). The ISI will be performed by the patient in the presence of the Investigator or designee as described in the Schedule of Evaluations.

[0051] 2.5 mg Compound 2 administered once daily in subjects in need thereof is found to treat generalized anxiety disorder, which may, in some embodiments, be determined by one or more efficacy endpoints described herein.Example 3: Treatment of Patients with Generalized Anxiety Disorder.

[0052] The efficacy and safety of using 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile (Compound 1), ascompared to placebo, for treatment of generalized anxiety disorder in patients may be demonstrated using a 2.0 mg dosage and regimen as in Example 2. A summary of the study protocol is provided in Table 4.Table 4.

[0053] 2.0 mg Compound 1 administered once daily in subjects in need thereof is found to treat generalized anxiety disorder, which may, in some embodiments, be determined by one or more efficacy endpoints described herein.

Claims

CLAIMSWhat is claimed is:

1. A method of treating generalized anxiety disorder in a subject in need thereof, comprising administration of a pharmaceutical composition comprising 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile or a pharmaceutically acceptable salt thereof, once per day for at least 14 consecutive days to the subject, wherein the subject is a human.

2. The method of claim 1, wherein the pharmaceutical composition comprises 2',6- difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2- carbonitrile.

3. The method of claim 1, wherein the pharmaceutical composition comprises 2',6- difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2- carbonitrile phosphate.

4. The method of claim 3, wherein the 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile phosphate is administered not in combination with another active pharmaceutical ingredient at the same time in the same pharmaceutical composition.

5. The method of claim 3 or 4, wherein the pharmaceutical composition comprises about 1.9 to about 6.3 mg of 2',6-difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2- b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile phosphate.

6. The method of claim 3 or 4, wherein the pharmaceutical composition comprises about 2.5 mg of 2',6-difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'- biphenyl]-2-carbonitrile phosphate.

7. The method of claim 1-4, wherein the 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile, or pharmaceuticallyacceptable salt thereof, is administered in an amount to achieve an AUCtau plasma concentration of about 500 to about 2,000 ng-h / mL at about day 14 of administration of the pharmaceutical composition.

8. The method of claim 1-4, wherein the 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,r-biphenyl]-2-carbonitrile, or pharmaceutically acceptable salt thereof, is administered in an amount to achieve an AUCtau plasma concentration of about 700 to about 1,000 ng-h / mL at about day 14 of administration of the pharmaceutical composition.

9. The method of claim 1-4, wherein the 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile, or pharmaceutically acceptable salt thereof, is administered in an amount to achieve a score of 1 or 2 on a clinical global impression - improvement scale (CGI-I) assessment of generalized anxiety disorder of the subject.

10. The method of claim 1-4, wherein the 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile, or pharmaceutically acceptable salt thereof, is administered in an amount to achieve a >50% reduction from a baseline of the subject on the Hamilton Anxiety Rating Scale (HAM-A) 14 days after the first administration of the pharmaceutical composition, wherein the baseline of the subject on the HAM-A is determined by assessing the subject on the HAM-A on the first day of administration of the pharmaceutical composition but prior to administration of the pharmaceutical composition.

11. The method of one of claims 1-10, wherein the subject in need is clinically depressed.

12. The method of one of claims 1-11, wherein the subject in need is clinically an insomniac.

13. The method of one of claims 1-12, wherein the 2',6-difluoro-5'-(3-(2-hydroxypropan- 2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile, or a pharmaceutically acceptable salt thereof, is administered not in combination with another active pharmaceutical ingredient at the same time in the same pharmaceutical composition.

14. The method of one of claims 1-12, wherein the 2',6-difluoro-5'-(3-(2-hydroxypropan- 2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile phosphate is administered not in combination with another active pharmaceutical ingredient at the same time in the same pharmaceutical composition.

15. The method of one of claims 1-14, wherein the pharmaceutical composition is in the form of a single unit.

16. The method of one of claims 1-15, wherein the administration is oral administration.

17. The method of claim 16, wherein the oral administration is more than 2 hours subsequent to oral food intake.

18. A method of improving psychic anxiety in a subject in need thereof, comprising administration of a pharmaceutical composition once per day for at least 14 consecutive days to the subject, wherein the pharmaceutical composition comprises about 2.0 mg of 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile, or a pharmaceutically acceptable salt thereof, improving psychic anxiety is determined by a statistically significant mean change from a baseline of the subject on the Hamilton Anxiety Rating Scale (HAM-A) subscale for psychic anxiety, the baseline of the subject on the HAM-A subscale for psychic anxiety is determined by assessing the subject on the HAM-A subscale for psychic anxiety on the first day of administration of the pharmaceutical composition but prior to administration of the pharmaceutical composition.

19. A method of improving psychic anxiety in a subject in need thereof, comprising administration of a pharmaceutical composition once per day for at least 14 consecutive days to the subject, wherein the pharmaceutical composition comprises about 2.5 mg of 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,r-biphenyl]-2-carbonitrile phosphate, improving psychic anxiety is determined by a statistically significant mean change from a baseline of the subject on the Hamilton Anxiety Rating Scale (HAM-A) subscale for psychic anxiety, the baseline of the subject on the HAM-A subscale for psychic anxiety is determined by assessing the subject on the HAM-A subscale for psychic anxiety on the first day of administration of the pharmaceutical composition but prior to administration of the pharmaceutical composition.

20. A method of improving somatic anxiety in a subject in need thereof, comprising administration of a pharmaceutical composition once per day for at least 14 consecutive days to the subject, wherein the pharmaceutical composition comprises about 2.0 mg of 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile, or a pharmaceutically acceptable salt thereof, improving somatic anxiety is determined by a statistically significant mean change from a baseline of the subject on the Hamilton Anxiety Rating Scale (HAM-A) subscale for somatic anxiety, the baseline of the subject on the HAM-A subscale for somatic anxiety is determined by assessing the subject on the HAM-A subscale for somatic anxiety on the first day of administration of the pharmaceutical composition prior to administration of the pharmaceutical composition.

21. A method of improving somatic anxiety in a subject in need thereof, comprising administration of a pharmaceutical composition once per day for at least 14 consecutive days to the subject, whereinthe pharmaceutical composition comprises about 2.5 mg of 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile phosphate, improving somatic anxiety is determined by a statistically significant mean change from a baseline of the subject on the Hamilton Anxiety Rating Scale (HAM-A) subscale for somatic anxiety, the baseline of the subject on the HAM-A subscale for somatic anxiety is determined by assessing the subject on the HAM-A subscale for somatic anxiety on the first day of administration of the pharmaceutical composition prior to administration of the pharmaceutical composition.

22. A method of treating an anxiety disorder in a subject in need thereof, comprising administration of a pharmaceutical composition comprising 2',6-difluoro-5'-(3-(2- hydroxypropan-2-yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,r-biphenyl]-2-carbonitrile or a pharmaceutically acceptable salt thereof, once per day for at least 7 consecutive days to the subject, wherein the subject is a human.

23. The method of claim 22, wherein the 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile is a phosphate salt.

24. The method of claim 23, wherein the 2',6-difluoro-5'-(3-(2-hydroxypropan-2- yl)imidazo[l,2-b][l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile phosphate is administered not in combination with another active pharmaceutical ingredient at the same time in the same pharmaceutical composition.

25. The method of claim 23 or 24, wherein the pharmaceutical composition comprises about 1.9 to about 6.3 mg of 2',6-difluoro-5'-(3-(2-hydroxypropan-2-yl)imidazo[l,2- £>][ l,2,4]triazin-7-yl)-[l,l'-biphenyl]-2-carbonitrile phosphate.

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