METHOD FOR TREATING SLEEP FRAGMENTATION DISORDERS USING DUAL ACTING H _I AND

5HT _2A INVERSE AGONIST/ANTAGONISTS

RELATED APPLICATION

[0001] This application claims priority to, and the benefits of U.S. Provisional Application No. 62/815,599, filed on March 8, 2019, the entire contents of which are incorporated by reference.

FIELD OF THE APPLICATION

[0002] The present disclosure relates to methods of alleviating a symptom of, treating, or preventing a sleep disorder (e.g., increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold) by administering a dual Hi receptor inverse agonist and 5HT2A receptor antagonist, or pharmaceutically acceptable salt thereof.

BACKGROUND

[0003] Breakthroughs in the field of sleep disorders research has brought about widespread scientific and popular appreciation for the health benefits of restful and restorative sleep. Sleep is now recognized along with diet and exercise as one of the three pillars of good health. The overall prevalence of current or previous sleep disorders in adults is estimated at 52.4% of the population. Almost two-thirds (64%) of the population report sleep difficulties at least a few times a week (National Sleep Foundation,“2012 Sleep in America” Poll). The International Classification of Sleep Disorders distinguishes over 80 different disorders and each can have profound health and economic implications. Whilst the daytime impairment caused by poor sleep has long been appreciated, poor sleep also has cascading negative impact upon alertness, cognition, learning and memory, vigilance, performance, and a broad range of co-morbid health conditions including acute and chrome pain and pain disorders, psychiatric conditions, neurodegenerative disease, developmental disorders, metabolic disease and diabetes, obesity, cardiovascular disease, immunological disorders, and many other medical conditions.

[0004] Sleep disorder patients are now readily segmented into a broader range of sleep disorder categories and conditions that are more amenable to new and better tailored therapies that hold promise for delivering better patient outcomes. Objective measures of sleep can play a vital role tow ^r ard understanding poor sleep and its amelioration. Despite sleeping 7-8 hours or more, patients, wherein the sleep is frequently interrupted or“fragmented” suffer all the consequences of sleep deprivation. Indeed, sleep consolidation is necessary for the restorative physiological benefits of sleep to he realized and comorbid conditions to be managed.

[0005 ] Pharmacological options are limited for the treatment of sleep fragmentation. In response to the lack of pharmacological options, there exists an unmet clinical need to develop new methods of treating sleep fragmentation, including pharmacological methods of treatment.

SUMMARY

[0006] In one aspect, the present disclosure provides compounds for alleviating a symptom of, treating, or preventing a sleep disorder.

[0007] In some embodiments, the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold. In some embodiments, the sleep disorder is increased disturbed sleep. In some embodiments, the sleep disorder is increased sleep fragmentation. In some embodiments, the sleep disorder is increased arousals. In some embodiments, the sleep disorder is decreased arousal threshold.

[0008] In one aspect, the present disclosure provides the identification of a pharmacological compound class having dual Hi receptor inverse agonist and 5HT2A receptor antagonist activity to alleviate the symptom of, treat, or prevent a sleep disorder. In some embodiments, the sleep disorder is characterized in whole or in part by sleep fragmentation. Compounds with dual Hi receptor inverse agonist and 5HT2A receptor antagonist pharmacology were studied for their effects on EEG sleep-wakefulness, locomotor activity, drink- and food-related activity, and body- temperature in rats using an enhanced and expanded version of SC()RE-2000 ^liy , a sleep- wake bioassay and analysis system known as“SCORE™”

[0009] In some embodiments, a compound of the present disclosure is a dual Hi receptor inverse agonist and SHTZA receptor antagonist. In some embodiments, the dual Hi receptor inverse agonist and SHTZA receptor antagonist is selected from: HY-10275, mirtazapine (HY10521), S- mirtazapine (HY 10378), quetiapine (HY10625), and amitriptyline, or a pharmaceutically acceptable salt thereof.

[0010] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering one or more compound of the present disclosure selected from: HY-10275, mirtazapine (HY10521), S-mirtazapine (HY10378), quetiapine (HY 10625), and amitriptyline, or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[001 1] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering HY-10275, or a pharmaceutically acceptable salt thereof

[0012] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering mirtazapine, or a pharmaceutically acceptable salt thereof

[0013] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering S-mirtazapine, or a pharmaceutically acceptable salt thereof

[0014] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering quetiapine, or a pharmaceutically acceptable salt thereof.

[0015] In some embodiments, the present discl osure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering amitriptyline, or a pharmaceutically acceptable salt thereof.

[0016] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with sleep apnea, restless legs syndrome, a high respiratory disturbance index (RDI), neurological disease, circadian rhythm disorder, pain, periodic leg movement disorder (PLMD), REM behavior disorder, elderly fragmented sleep, age-related sleep fragmentation, post-menopausal sleep disorder, substance abuse, substance abuse withdrawal, narcolepsy, mental disorder, or non-restorative sleep.

[0017] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with sleep apnea.

[0018] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousafs, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with restless legs syndrome.

[0019] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with a high respiratory disturbance index (RDI).

[0020] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with a neurological disease.

[0021] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with a circadian rhythm disorder.

[0022] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with pain.

[0023] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with periodic leg movement disorder (PLMD).

[0024] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with REM behavior disorder.

[0025] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousafs, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with elderly fragmented sleep.

[0026] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with age-related sleep fragmentation.

[0027] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with post-menopausal sleep disorder.

[0028] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with substance abuse.

[0029] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with substance abuse withdrawal.

[0030] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with narcolepsy.

[0031] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with a mental disorder.

[0032] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with non-restorative sleep. [0033] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousafs, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with snoring.

[0034] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with idiopathic hypersomnia.

[0035] In some embodiments, the methods of the present disclosure the subject is administered a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a compound of the present disclosure and an additional active agent. In some embodiments, the additional active agent is a sedative-hypnotic.

[0036] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder by administering a pharmaceutical composition comprising an Hi inverse agonist or antagonist and 5HT2A antagonist or inverse activity, or a pharmaceutically acceptable derivative thereof, in combination with an additional active agent.

[0037] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder by administering the compounds of the present disclosure and any additional active agents, if present, either hora somni, h.s. (at bedtime) or between about 0-4 hours before bedtime to a subject in need thereof.

[0038] Pharmaceutical therapies and methods of using such pharmaceutical therapies are provided herein for sleep fragmentation. Pharmaceutical therapies and methods of using such pharmaceutical therapies are provided for a sleep disorder in a subject with one or more co- morbid medical conditions. In some embodiments, these therapies and methods of using such therapies include pharmaceutical therapies to decrease disturbed sleep, decrease sleep fragmentation, decrease arousais, or increase arousal threshold. In some embodiments, a subject may have one or more of the following medical conditions: sleep apnea, restless legs syndrome, a high respiratory disturbance index (RDI), neurological disease, circadian rhythm disorder, pain, periodic leg movement disorder (PLMD), REM behavior disorder, elderly fragmented sleep, age-related sleep fragmentation, post-menopausal sleep disorder, substance abuse, substance abuse withdrawal, narcolepsy, mental disorder, or non-restorative sleep. [0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which tins disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0040] Other features and advantages of the disclosure will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

[0041] FIG. 1 depicts the number of wake bouts after treatment with HY-10275, wherein the thin line depicts HY-10275 administration at 3 mg/kg PO (CT-5, n=I2), and the thick line depicts methylcelluiose administration at 1 mL/kg PO (CT-5, n=12).

[0042] FIG. 2 depicts average aligned sleep bout each hour after treatment with HY-10275, wherein the thin line depicts HY-10275 administration at 3 mg/kg PO (CT-5, n=12), and the thick line depicts methylcelluiose administration at 1 mL/kg PO (CT-5, n=12).

[0043] FIG. 3 depicts the longest sleep bout each hour after treatment with HY-10275, wherein the thin line depicts HY-10275 administration at 10 mg/kg PO (n=17), and the thick line depicts methylcelluiose administration at 1 mL/kg PO (CT-18, n=17).

[0044] FIG. 4 depicts the normalized EEG delta power after treatment with HY-10275, wherein the thin line depicts HY-10275 administration at 10 mg/kg PO (n= ^:: l 7), and the thick line depicts methylcelluiose administration at 1 mL/kg PO (CT-18, n 1 7).

[0045 ] FIG 5 depicts longest wake bout each hour after treatment with HY-10275, wherein the thin line depicts HY-10275 administration at 10 mg/kg PO (n=T7), and the thick line depicts methylcelluiose administration at 1 mL/kg PO (CT-18, n=T7).

[ 0046] FIG 6 depicts the number of wake bouts each hour after treatment with mirtazapine, wherein the thin line depicts mirtazapine administration at 10 mg/kg PO (n=T9), and the thick line depicts methylcelluiose administration at 1 mL/kg PO (CT-18, n=T9). [0047] FIG. 7 depicts the average sleep bout each hour after treatment with mirtazapine, wherein the thin line depicts mirtazapine administration at 10 mg/kg PO (n=T9), and the thick line depicts methylcellulose administration at 1 mL/kg PO (CT-18, n 19).

[0048] FIG. 8 depicts the number of wake bouts each hour after treatment with S-mirtazapine, wherein the thin line depicts S-mirtazapine administration at 10 mg/kg PO (n=9), and the thick line depicts methylcellulose administration at 1 mL/kg PO (CT-18, n=10).

[0049] FIG. 9 depicts the average sleep bout each hour after treatment with S-mirtazapine, wherein the thin like depicts S-mirtazapine administration at 10 mg/kg PO (n=9), and the thick line depicts methylcellulose administration at 1 mL/kg PO (CT-18, n=10).

[0050] FIG. 10 depicts the number of wake bouts per hour after treatment with amitriptyline, wherein the thin line depicts amitriptyline administration at 10 mg/kg IP (CT-5, n=9), and the thick line depicts methylcellulose administration at 1 mL/kg IP (CT-5, n=10).

[0051] FIG. 11 depicts the average sleep bout each hour after treatment with amitriptyline, wherein the thin line depicts amitriptyline administration at 10 mg/kg IP (CT-5, n=9), and the thick line depicts methylcellulose administration at 1 mL/kg IP (CT-5, n=T0)

[0052] FIG. 12 depicts the number of wake bouts each hour after treatment with quetiapine, wherein the thin line depicts quetiapine administration at 3 mg/kg PO (n=7), and the thick line depicts methylcellulose administration at 1 mL/kg PO (CT-5, n=10).

[0053] FIG. 13 depicts the average sleep bout each hour after treatment with quetiapine, wherein the thin line depicts quetiapine administration at 30 mg/kg PO (n=7), and the thick line depicts methylcellulose administration at 1 mL/kg PO (CT-1 8, n=T0).

[0054] FIG. 14 depicts the number of wake bouts each hour after treatment with suvorexant, wherein the thin line depicts suvorexant administration at 30 mg/kg PO (CT-18, n=7), and the thick bne depicts IPGS (HY-16388) administration at 1 mL/kg PO (CT-18, n=9).

[0055] FIG. 15 depicts the average aligned sleep bout each hour after treatment with suvorexant, wherein the thin line depicts suvorexant administration at 30 mg/kg PO (CT-18, n ^:=: 7), and the thick line depicts TPGS (HY-16388) administration at 1 mL/kg PO (CT-18, n=9).

[0056] FIG. 16 depicts the normalized EEG delta power after treatment with zolpidem tartrate, wherein the thin line depicts zolpidem tartrate administration at 10 mg/kg PO (CT-18, EEG telemetry, n=8), and the thick line depicts methylcellulose administration at 1 mL/kg PO (CT-18, EEG telemetry, n=9). [ 0057] FIG. 17 depicts the number of wake bouts each hour after treatment with zolpidem tartrate, wherein the thin line depicts zolpidem tartrate administration at 10 mg/kg PO (CT-18, EEG telemetry, n=8), and the thick line depicts methylcellulose administration at 1 ml. kg PO (CT-18, EEG telemetry, n=9).

[0058] FIG. 18 depicts the average aligned sleep bout each hour after treatment with zolpidem tartrate, wherein the thin line depicts zolpidem tartrate administration at 10 mg/kg PO (CT-18, EEG telemetry, n=8), and the thick line depicts methylcellulose administration at 1 mL/kg PO

(CT-18, EEG telemetry, n=9).

[0059] FIG. 19 depicts the REM sleep after treatment with HY-10131, wherein the thin line depicts HY-10131 administration at 30 mg/kg PO (n=16), and the thick line depicts

methylcellulose administration at 1 mL/kg PO (CT-5, n=16).

[0060] FIG. 20 depicts the Locomotor Activity' Intensity (LMAi) after treatment with zolpidem, wherein the thin line depicts zolpidem administration at 30 mg/kg PO (CT-18, n=10), and the thick line depicts methylcellulose administration at 1 mL/kg PO (CT-18, n=10).

[0061] FIG. 21 depicts the number of wake bouts each hour after treatment with S-zopiclone, wherein the thin line depicts S-zopiclone administration at 10 mg/kg PO (CT-18, n=13), and the thick line depicts methylcellulose administration at 1 mL/kg PO (CT-18, n= 13).

[0001] FIG. 22 depicts the average sleep bout each hour after treatment with S-zopiclone, wherein (A) depicts S-zopiclone administration at 30 mg/kg PO (CT-18, n=T2), (B) depicts S- zopiclone administration at 10 mg/kg PO (CT-18, n=T0), (C) depicts S-zopiclone administration at 5 mg/kg PO (CT-18, n=T2), and (D) methylcellulose administration at 1 mL/kg PO (CT-18, n 12).

[0062] FIG. 23 depicts the Locomotor Activity Intensity (LMAi) after treatment with S- zopiclone, wherein (A) depicts S-zopiclone administration at 30 mg/kg PO (CT-18, n=T2), (B) depicts S-zopiclone administration at 10 g/kg PO (CT-18, n=T0), (C) depicts S-zopiclone administration at 5 mg/kg PO (CT-18, n=T2), and (D) methy lcellulose administration at 1 mL/kg PO (CT-18, n 12).

[0063] FIG 24 depicts the integrated electromyograph (EMG) activity after treatment with S- zopiclone, wherein the thin line depicts S-zopiclone administration at 10 mg/kg PO (CT-18, n=T3), and the thick line depicts methylcellulose administration at 1 mL/kg PO (CT-18, n ^:=: 12).

[0064] FIG. 25 depicts the percent nonREM (NREM) sleep after treatment with S-zopiclone, wherein (A) depicts S-zopiclone administration at 30 mg/kg PC) (CT-18, n=T2), (B) depicts S- zopiclone administration at 10 mg/kg PO (CT-18, n=T0), (C) depicts S-zopiclone administration at 5 mg/kg PO (CT-18, n=T2), and (D) methylcellulose administration at 1 niL/'kg PO (CT-18, n 12).

DETAILED DESCRIPTION

Definitions

[0065] As used herein,“uninterrupted bouts of sleep” (average sleep bout duration) refers to the average duration of all bouts of uninterrupted sleep that occurred each hour, measured in minutes.“Interruption” of sleep refers to 2 or more consecutive 10 second epochs of

wakefulness.“Interruption” of wake refers to 2 or more consecutive 10 second epochs of sleep. The value for the length of a bout that extends into the subsequent hour is assigned to the hour in which it began. Analogous quantification was carried out for bouts of wakefulness. Sleep bout length may reflect the human tendency to awaken periodically through the night (such awakenings are normally not recalled), which in turn may be an important factor in determining the restorative value of sleep in a subject (e.g., humans). Pre-clinical measures of sleep bout duration are also strong predictors of soporific efficacy in a subject (e.g., humans).

[0066] As used herein,“NREM” and“nonREM” refers to non-rapid eye movement sleep stages.

[0067] As used herein,“reduced number of arousals” refers to the reduced number of wake bouts per hour.

[0068] As used herein,“REM” refers to the rapid eye movement sleep stage.

[0069] As used herein,“SEM” refers to standard error of the mean.

[0070] As used herein,“CT” refers to circadian time.

[0071] As used herein“sleep consolidation” refers to the measurement of the average sleep bout duration per hour.

[0072] As used herein,“sleep continuity" refers to the measurement of sleep-bout length.

[0073] As used herein, the“depth” of sleep is characterized by EEG slow wave activity, which may subserve sleep continuity or sleep consolidation, which is one of several determinants of sleep quality.

[0074] As used herein,“SWA” refers to slow wave activity, which may be exemplified as EEG delta po wer by use of Fourier analysis. [0075] As used herein,“LMA intensity” (LMAi) refers to locomotor activity (LMA) counts per minute of EEG-defined wakefulness. This variate allows an assessment of LMA that is independent of the amount of time awake, which may be used to quantify the specificity of a wake- or sleep-promoting effect.

[0076] As used herein,“number of wake bouts” refers to the number of uninterrupted bouts of wakefulness that occurred each hour, measured in minutes. Number of wake bouts are of interest because it may closely reflect the number of arousais from sleep that occur at the time of measurement. Interpreted together, average sleep bout duration and number of wake bouts provide a highly reliable assessment of a drug or novel molecular entity (NME) effect on sleep fragmentation. Drugs that improve sleep fragmentation have been shown to improve the restorative benefits of sleep.

[0077] As used herein, the term“co-morbid” refers to a disease or disorder which is concurrent with a sleep disorder, but may not be the cause, in whole or m part, of the sleep disorder.

[0078] As used herein, the term“pharmaceutically acceptable” refers to compounds, anions, cations, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0079] It is to be understood that the present disclosure also provides pharmaceutical compositions comprising any compound described herein in combination with at least one pharmaceutically acceptable excipient or carrier.

[0080] As used herein, the term“pharmaceutical composition” is a formulation comprising a compound of the present disclosure in a form suitable for administration to a subject. In some embodiments, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler or a vial. The quantity of active ingredient (e.g., a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the subject. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. Dosage forms for the topical or transdermal administration of a compound of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants in some embodiments, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

[0081] As used herein, the term“preventing,”“prevent,” or“protecting against” describes reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder.

[0082] As used herein, the term“rat” and“laboratory rat” are used interchangeably.

[0083] It is to be appreciated that references to“treating” or“treatment” include the alleviation of established symptoms of a condition.“Treating” or“treatment” of a state, disorder or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclimcal symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclimcal symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclimcal symptoms.

[0084] As used herein, the term“subject” is interchangeable with the term“subject in need thereof’, both of which refer to a subject having a disease or having an increased risk of developing the disease. A“subject” includes a mammal. The mammal can be, for example, a human or appropriate non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig. The subject can also be a bird or fowl. In some embodiments, the mammal is a rat. In some embodiments, the mammal is a human. In some embodiments, a subject in need has been previously diagnosed or identified as having a disease or disorder disclosed herein. A subject in need thereof can also be one who is suffering from a disease or disorder disclosed herein. Alternatively, a subject in need thereof can be one who has an increased risk of developing such disease or disorder relative to the population at large (i.e., a subject who is predisposed to developing such disorder relative to the population at large). A subject in need thereof can have a refractory or resistant disease or disorder disclosed herein (i.e., a disease or disorder disclosed herein that does not respond or has not yet responded to treatment). The subject may he resistant at start of treatment or may become resistant during treatment. In some embodiments, the subject in need thereof received and failed all known effective therapies for a disease or disorder disclosed herein. In some embodiments, the subject m need thereof received at least one prior therapy.

[0085] Unless explicitly indicated otherwise, the terms“approximately” and“about” are synonymous. In some embodiments,“approximately” and“about” refer to the recited value, amount, dose or duration ± 20%, ± 15%, ± 10%, ± 8%, ± 6%, ± 5%, ± 4%, ± 2%, ± 1%, or ± 0.5%. In another embodiment,“approximately” and“about” refer to the listed amount, value, dose, or duration ± 10%, ± 8%, ± 6%, ± 5%, ± 4%, or ± 2%. In some embodiments,

“approximately” and“about” refer to the listed amount, value, dose, or duration ± 5%. In some embodiments,“approximately” and“about” refer to the listed amount, value, dose, or duration ± 2%. In some embodiments,“approximately” and“about” refer to the listed amount, value, dose, or duration ± 1%.

[0086] In some embodiments, a“compound” is the same as an“active ingredient”, an“

H1/5HT2A molecule”, an“Hi inverse agonist”, an“HI/SHTIA antagonist”, a“dual acting Hi inverse agonist”, a“5HT2A antagonist molecule (HI/5HT2A) “a dual Hi receptor inverse agonist and 5-HT2A receptor antagonist”, and a“compound of the present disclosure”.

[0087] In some embodiments,“HY-10275” may also be referred to as 3-(4- Dibenzo[b,f][l,4]oxazepin-l 1 -yl-piperazin-l-yl)-2,2-dimethyl-propionic acid; di-hydrogen

chloride salt pharmaceutically acceptable salt thereof. [0088] In some embodiments,“mirtazapine” may also be referred to as HY10521, 2-methyl-

l,2,3,4,10,14b-hexahydrobenzo[c]pyrazino[l,2-a]pyrido[3,2 -f]azepine

or a pharmaceutically acceptable salt thereof.

[0089] In some embodiments,“S-mirtazapine” may also be referred to as HY10378, (S)-2- methyl- 1 ,2,3,4, 10, 14b-hexahydrobenzo[c]pyrazino[l ,2-a]pyrido[3,2-f]azepine (i.e. ,

pharmaceutically acceptable salt thereof.

[0090] In some embodiments,“quetiapme” may also be referred to as HY10625, 2-(2-(4- (dibenzo[b,f|[l,4]thiazepm-l l-yl)piperazin-l-yl)ethoxy)ethan-l -ol (i.e.,

pharmaceutically acceptable salt thereof.

[0091] In some embodiments,“amitriptyline” is 3-(10,11 -dihydro- 5H-dibenzo[a,d][7]annulen-5-

ylidene)-N,N-dimethylpropan-l -amine pharmaceutically acceptable salt thereof. [ 0092] In some embodiments, zolpidem may also be referred to as Ambien®, HY-10131 , N,N- dimethyl-2-[6-methyl-2-(4-methylphenyl)imidazo[l ,2-a]pyridin-3-yl]acetamide (i.e.,

pharmaceutically acceptable salt thereof.

[0093] In some embodiments, zolpidem is a pharmaceutically acceptable salt.

[0094] In some embodiments, zolpidem is zolpidem tartrate.

[0095] In some embodiments, S-zopiclone may also be referred to as Lunesta®, eszopiclone,

[(7S)-6-(5-chloropyridin-2-yl)-5-oxo-7//-pyrrolo[3,4-b]py razin-7-yl] 4-methylpiperazine-l -

carboxylate pharmaceutically acceptable salt thereof.

[0096] In some embodiments, suvorexant may also be referred to as Be!somra®, [(7i?)-4-(5- chloro-l,3-benzoxazol-2-yl)-7-methyl-l,4-diazepan-l-yl]-[5-m ethyl-2-(triazol-2-

yl)pheny Ijmethanone pharmaceutically acceptable salt thereof.

Methods of Use

[0097] Sleep fragmentation, a condition m humans characterized by poor sleep consolidation, frequent brief arousals or microarousals (defined by the American Academy of Sleep Medicine as episodes of cortical EEG activation lasting at least 2 seconds and up to 16 seconds in duration and interrupting sleep), and frequent transitions to lighter stages of sleep, results in significant daytime impairment that may include impaired attention and concentration, excessive sleepiness, impaired judgement, impaired memory and learning, increased risk of accidents, and secondary morbidity and mortality when sleep fragmentation is a concomitant of pain, sleep disordered breathing, and other disease states. Patients suffering from sleep fragmentation are often unaware of the hundreds of brief arousals that may occur during the night, and primarily complain of severe daytime impairment. Sleep fragmentation patients often complain that their sleep is not beneficial, refreshing, or restorative.

[0098] The diagnosis of sleep fragmentation and its methods of treatment must not be equated with insomnia, which is a separate and distinct medical diagnosis. Insomnia is typically characterized by patient awareness and dissatisfaction with their sleep. Most insomnia patients have a hyperarousal disorder that makes it difficult to fall asleep and/or difficult to stay asleep but enable them to function well during the daytime. Insomnia is diagnosed by measuring the latency to persistent sleep, i.e., LPS (LPS of >30 minutes satisfies the definition of sleep-onset insomnia) and/or measuring the amount of time awake after sleep onset, i.e., WASO (WASO of >50 minutes satisfies the definition of sleep-maintenance insomnia). It is common for insomnia patients, and particularly the elderly, to awaken m the middle of the night and be unable to return to sleep. Unlike patients suffering from sleep fragmentation, insomnia patients are almost always highly aware of their inability to fall asleep or stay asleep at night and complain about their nighttime experience, and typically do not complain about daytime impairment.

[0099] An extensive body of research heretofore undisclosed has identified that molecules in a pharmacological class best described as having dual Hi receptor inverse agonist and 5HT2A receptor antagonist activity improve sleep fragmentation as evidenced by reduced number of arousals (measured preclinieally as reduced number of wake bouts per hour) and increased sleep consolidation (measured preclinieally by average sleep bout duration per hour).

[0100] Molecules with the requisite Hi inverse agonist and SH ^' UA antagonist receptor pharmacology may be studied for their effects on EEC sleep- wakefulness, locomotor activity, drink- and food-related activity and body temperature in rats using an improved and expanded version of SCORE-2000 ^® , a sophisticated sleep- wake bioassay and analysis system which shall henceforth be referred to as“SCORE™”. The technology is well suited for sleep-wake efficacy and physiological and behavioral side effect assessment. Associated with this technology is an extensive pharmacological database with standardized sleep-wake, physiological, and behavioral data for over 500 distinct molecules. The standardized nature of the experimental designs, data quality control, and data analysis methods enable direct comparisons between molecules.

[0101] The utility of dual acting Hi inverse agonist and 5HT2A antagonist molecules to alleviate a symptom of, treat, or prevent a sleep disorder may be assessed by evaluating the sleep architecture and sleep quality endpoints of compounds with affinity and inverse

agonist/antagonist properties for these receptors.

Rat and Human Sleep

[0102] The present disclosure provides a pre-clinical evaluation using rats. Without washing to be bound by theory, rat sleep and human sleep have all of the necessary fundamental similarities to permit the rat to be used as a preciinical model. As such, compounds that are soporific m a human may have soporific effects in a rat, and compounds that are soporific in a rat may have soporific effects in a human. Both rat and human exhibit robust circadian modulation of sleep tendency and sleep architecture.

[0103] The“homeostatic” control of sleep shares similarity across mammalian species, including humans, in that loss of sleep increases a homeostatic drive for sleep evidenced by a reduction in latency to sleep onset, increase in the depth of sleep that can be reflected by the amount of low- frequency“delta” EEG (“EEG slow waves”) during nonREM, an increase in sleep consolidation as measured by sleep bout duration, or an increase in total sleep time. Sleep deprivation in a subject may cause the subject to fall asleep faster, sleep deeper, sleep more efficiently (e.g., more consolidated bouts of sleep), or sleep more (e.g., an increase of sleep time) until the homeostatic drive for sleep becomes adequately discharged through the sleeping process.

[0104] Uninterrupted, well consolidated sleep can determine sleep quality in both a rat and a human. Without wishing to be bound by theory, no matter how much a subject sleeps or what frequency of EEG dominates during sleep, the beneficial work of the sleeping process requires that sleep is not fragmented (interrupted) by frequent arousals.

[0105] Without wishing to be bound by theory, compounds of the present disclosure which affect REM sleep by decreasing the latency to sleep onset, increasing sleep time, increasing the depth and/or consolidation of sleep, or reduce arousals, or a combination of the aforementioned effects in a subject, have the same effects in a different subject. The compounds of the present disclosure, which affect REM sleep by decreasing the latency to sleep onset effects in a subject, may have the same effects on a subject of a different species. The compounds of the present disclosure, which affect ITEM sleep by decreasing the latency to sleep onset effects in rats, may have the same effects on a subject of different species. The compounds of the present disclosure, which affect REM sleep by decreasing the latency to sleep onset effects m rats, may have the same effects on a human.

[0106] Without washing to be bound by theory, compounds of the present disclosure which affect REM sleep by increasing sleep time in a subject, have the same effects on a different subject. The compounds of the present disclosure which affect REM sleep by increasing sleep time in a subject, may have the same effects on a subject of a different species. The compounds of the present disclosure which affect REM sleep by increasing sleep time in rats, may have the same effects on a subject of a different species. The compounds of the present disclosure which affect REM sleep by increasing sleep time in rats, may have the same effects on a human.

[0107] Without wishing to be bound by theory, compounds of the present disclosure which affect REM sleep by increasing the depth and/or consolidation of sleep in a subject, have the same effects in a different subject. Compounds of the present disclosure which affect REM sleep by increasing the depth and/or consolidation of sleep in a subject, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect REM sleep by increasing the depth and/or consolidation of sleep in rats, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect REM sleep by increasing the depth and/or consolidation of sleep in rats, may have the same effects in a human.

[0108] Without wishing to be bound by theory, compounds of the present disclosure which affect REM sleep by increasing the depth and consolidation of sleep in a subject, have the same effects in a different subject. Compounds of the present disclosure which affect REM sleep by increasing the depth and consolidation of sleep in a subject, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect REM sleep by increasing the depth and consolidation of sleep in rats, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect REM sleep by increasing the depth and consolidation of sleep in rats, may have the same effects in a human.

[0109] Without wishing to be bound by theory, compounds of the present disclosure which affect REM sleep by increasing the depth or consolidation of sleep in a subject, have the same effects in a different subject. Compounds of the present disclosure winch affect REM sleep by increasing the depth or consolidation of sleep in a subject, may have the same effects in a subject of a different species. Compounds of the present disclosure winch affect REM sleep by increasing the depth or consolidation of sleep in rats, may have the same effects in a subject of a different species. Compounds of the present disclosure winch affect REM sleep by increasing the depth or consolidation of sleep in rats, may have the same effects in a human.

[0110] Without wishing to be bound by theory, compounds of the present disclosure which affect REM sleep by increasing the depth of sleep in a subject, have the same effects in a different subject. Compounds of the present disclosure which affect REM sleep by increasing the depth of sleep in a subject, may have the same effects in a subject of a different species.

Compounds of the present disclosure which affect REM sleep by increasing the depth of sleep in rats, may have the same effects m a subject of a different species. Compounds of the present disclosure which affect REM sleep by increasing the depth of sleep in rats, may have the same effects in a human.

[011 1] Without wishing to be bound by theory', compounds of the present disclosure which affect REM sleep by increasing the consolidation of sleep in a subject, have the same effects in a different subject. Compounds of the present disclosure which affect REM sleep by increasing the consolidation of sleep in a subject, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect REM sleep by increasing the consolidation of sleep in rats, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect REM sleep by increasing the consolidation of sleep in rats, may- have the same effects in a human.

[01 12] Without wishing to be bound by theory, compounds of the present disclosure which affect REM sleep by reducing arousals in a subject, have the same effects in a different subject. Compounds of the present disclosure which affect REM sleep by reducing arousals in a subject, have the same effects in a subject of a different species. Compounds of the present disclosure which affect REM sleep by reducing arousals in rats, have the same effects in a subject of a different species. Compounds of the present disclosure which affect REM sleep by reducing arousals in rats, have the same effects in a human.

[0113] Without wishing to be bound by theory, compounds of the present disclosure which affect NREM sleep by decreasing the latency to sleep onset, increasing sleep time, increasing the depth and/or consolidation of sleep, or reduce arousals, or a combination of the aforementioned effects in a subject, have the same effects in a different subject. The compounds of the present disclosure, which affect NREM sleep by decreasing the latency to sleep onset effects in a subject, may have the same effects on a subject of a different species. The compounds of the present disclosure, which affect NREM sleep by decreasing the latency to sleep onset effects m rats, may have the same effects on a subject of different species. The compounds of the present disclosure, which affect NREM sleep by decreasing the latency to sleep onset effects in rats, may have the same effects on a human.

[0114] Without wishing to be bound by theory, compounds of the present disclosure which affect NREM sleep by increasing sleep time in a subject, have the same effects on a different subject. The compounds of the present disclosure which affect NREM sleep by increasing sleep time in a subject, may have the same effects on a subject of a different species. The compounds of the present disclosure which affect NREM sleep by increasing sleep time in rats, may have the same effects on a subject of a different species. The compounds of the present disclosure which affect NREM sleep by increasing sleep time in rats, may have the same effects on a human.

[0115] Without wishing to be bound by theory, compounds of the present disclosure which affect NREM sleep by increasing the depth and/or consolidation of sleep in a subject, have the same effects in a different subject. Compounds of the present disclosure which affect NREM sleep by increasing the depth and/or consolidation of sleep in a subject, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect NREM sleep by increasing the depth and/or consolidation of sleep in rats, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect NREM sleep by increasing the depth and/or consolidation of sleep in rats, may have the same effects in a human.

[0116] Without wishing to be bound by theory, compounds of the present disclosure which affect NREM sleep by increasing the depth and consolidation of sleep in a subject, have the same effects in a different subject. Compounds of the present disclosure which affect NREM sleep by increasing the depth and consolidation of sleep in a subject, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect NREM sleep by- increasing the depth and consolidation of sleep in rats, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect NREM sleep by increasing the depth and consolidation of sleep in rats, may have the same effects in a human. [0117] Without wishing to he bound by theory, compounds of the present disclosure winch affect NREM sleep by increasing the depth or consolidation of sleep in a subject, have the same effects in a different subject. Compounds of the present disclosure which affect NREM sleep by increasing the depth or consolidation of sleep in a subject, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect NREM sleep by increasing the depth or consolidation of sleep in rats, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect NREM sleep by increasing the depth or consolidation of sleep in rats, may have the same effects in a human.

[0118] Without wishing to be bound by theoiy, compounds of the present disclosure which affect NREM sleep by increasing the depth of sleep m a subject, have the same effects in a different subject. Compounds of the present disclosure which affect NREM sleep by increasing the depth of sleep in a subject, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect NREM sleep by increasing the depth of sleep in rats, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect NREM sleep by increasing the depth of sleep in rats, may have the same effects in a human.

[0119] Without wishing to be bound by theory , compounds of the present disclosure which affect NREM sleep by increasing the consolidation of sleep in a subject, have the same effects in a different subject. Compounds of the present disclosure which affect NREM sleep by increasing the consolidation of sleep in a subject, may have the same effects in a subject of a different species. Compounds of the present disclosure which affect NREM sleep by increasing the consolidation of sleep in rats, may have the same effects in a subject of a different species.

Compounds of the present disclosure which affect NREM sleep by increasing the consolidation of sleep in rats, may have the same effects in a human.

[0120] Without wishing to be bound by theory, compounds of the present disclosure which affect NREM sleep by reducing arousals in a subject, have the same effects in a different subject. Compounds of the present disclosure which affect NREM sleep by reducing arousals in a subject, have the same effects in a subject of a different species. Compounds of the present disclosure which affect NREM sleep by reducing arousals in rats, have the same effects in a subject of a different species. Compounds of the present disclosure which affect NREM sleep by- reducing arousals in rats, have the same effects m a human. [0121] Without wishing to he bound by theory, sleep continuity can be measured as the duration of NREM“bouts” or the duration of REM bouts, or the duration NREM+REM“bouts”, wherein an arousal or bout of wakefulness interrupts the NREM-REM cycle.

[0122 ] in some embodiments, sleep bout can he comprised of NREM, REM, or NREM+REM.

[0123] In some embodiments, sleep bout can be comprised of NREM. In some embodiments, sleep bout can be comprised of REM. In some embodiments, sleep bout can be comprised of NREM+REM.

[0124] In some embodiments, NREM and REM sleep alternate in what may be called the NREM-REM cycle. In some embodiments, NREM precedes REM.

[0125] In some embodiments, the proportion of time spent in NREM versus REM is the same for different subjects. In some embodiments, the proportion of time spent in NREM versus REM is the same for different subjects of different species. In some embodiments, the proportion of time spent in NREM versus REM is the same for a rat and a subject of a different species. In some embodiments, the proportion of time spent in NREM versus REM is the same for a rat and a human.

[0126] In some embodiments, the proportion of time spent in NREM versus REM is about 5:1.

In some embodiments, the proportion of time spent in NREM versus REM is about 4: 1. In some embodiments, the proportion of time spent in NREM versus REM is about 3: 1. In some embodiments, the proportion of time spent in NREM versus REM is about 2: 1.

[0127] In some embodiments, the proportion of time spent in NREM versus REM is from about 100: 1 to about 1 : 1. In some embodiments, the proportion of time spent in NREM versus REM is from about 90: 1 to about 1 : 1. In some embodiments, the proportion of time spent in NREM versus REM is from about 80: 1 to about 1 : 1. In some embodiments, the proportion of time spent in NREM versus REM is from about 70: 1 to about 1 : 1. In some embodiments, the proportion of time spent in NREM versus REM is from about 60: 1 to about 1 : 1. In some embodiments, the proportion of time spent in NREM versus REM is from about 50: 1 to about 1 : 1. In some embodiments, the proportion of time spent in NREM versus REM is from about 40: 1 to about 1 : 1. In some embodiments, the proportion of time spent in NREM versus REM is from about 30: 1 to about 1 : 1. In some embodiments, the proportion of time spent in NREM versus REM is from about 20: 1 to about 1 : 1. In some embodiments, the proportion of time spent in NREM versus REM is from about 10: 1 to about 1 : 1. In some embodiments, the proportion of time spent in NREM versus REM is from about 5: 1 to about 1 : 1. In some embodiments, the species is a mouse. In some embodiments, the species is a hoofed animal. In some embodiments, the hoofed animal is a horse or cow. In some embodiments, the species is not a laboratory rat. In some embodiments, the species is not a human.

[0128] In some embodiments, hypnotics reduce REM sleep to some degree, and several classes of sleep disorder medicines can strongly suppress REM sleep. Without wishing to be bound by theory, REM sleep suppression is relevant to learning, memory, and/or psychiatric health.

[0129] Without wishing to be bound by theory, the relative effect of some classes of medicines for sleep disorders, neuropsychiatric disorders, and cardiovascular disease that either inhibit or stimulate REM sleep translates from a subject to a different subject. The relative effect of some classes of medicines for sleep disorders, neuropsychiatric disorders, and cardiovascular disease that either inhibit or stimulate REM sleep translates from a subject to a subject of a different species. The relative effect of some classes of medicines for sleep disorders, neuropsychiatric disorders, and cardiovascular disease that either inhibit or stimulate ITEM sleep translates from a rat to a subject of a different species. The relative effect of some classes of medicines for sleep disorders that either inhibit or stimulate REM sleep translate from a rat to a human.

[0130] In some embodiments, a class of medicine which may inhibit or stimulate REM or NREM sleep is selected from a sleep therapeutic, a neuropsychiatric antidepressant, and a cardiovascular medicine.

[0131] In some embodiments, a sleep therapeutic is a sedative hypnotic GABAA positive allosteric modulators that binds to the GABAA benzodiazepine receptor. In some embodiments, a neuropsychiatric antidepressant is a selective serotonin reuptake inhibitor (SSRI) or an atypical antipsychotics. In some embodiments, a cardiovascular medicine is a therapeutic that may bind to an alpha-adrenergic receptor.

[0132] There are two differences which may be present between rat and human sleep. First, rats are night-active, whereas humans are day-active. This difference may have no importance per se for testing drug effects on sleep and wakefulness. The timing of the dose relative to the normal sleep period can be relied upon when evaluating drug efficacy on certain sleep related variables (e.g. inhibition of REM sleep) when comparing rat and human sleep. The difference between a rat and a human is sleep-bout length, also referred to as“sleep continuity.” Further, humans may consolidate sleep into a single period per day, interrupted normally only by very short (e.g., less than 2 hours, less than 1 hour, less than 45 minutes, less than 30 minutes, less than 25 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, or less than 1 minute) bouts of wakefulness. The abnormal conditions may result in human sleep becoming fragmented, diminishing the restorative benefits of sleep. Rats may have shorter bouts of sleep that occur throughout the 24-hour day (e.g., on average, every 20 minutes, a rat completes a sleep-wake cycle). During darkness (when the rat may be most active), sleep typically occupies about 1/3 of each 20-minute cycle, and REM sleep is rare. During the day (lights-on), the rat typically sleeps about 2/3 of each 20-minute cycle. The polyphasic nature of sleep and shorter spontaneous sleep bout durations in the rat, enable highly sensitive assessments of drug effects such as those that increase sleep consolidation (sleep bout duration), decrease the number of arousals (number of wake bouts), and a variety of secondary but desirable measures of sleep quality, for example such as EEG slow wave activity' in nonREM sleep, and measures of wake maintenance as measured by wake bout duration. Sleep bout-length may also be a sensitive measure of physiological sleepiness and is a pre-clinica! predictor of soporific efficacy in humans.

Timing of treatment

[0133] Empirical optimization can be performed by assessing sleep-related compounds by administering such compounds at two circadian times of day, CT-18 and CT-5, wherein CT-0 is defined as lights-on. CT-18 is the mid-point of the activity phase of the rat’s circadian cycle, 6 hours after lights-off, and may be sensitive to soporific drug effects on sleep bout length, although such effects can be observed at both CT-18 and CT-5. CT-5 begins several hours of peak abundance of REM sleep and thus is a sensitive time to reveal drug-related inhibition of REM sleep. Both CT-18 and CT-5 are suitable times of the day for the assessment of drug effects on sleep fragmentation as measured by arousals (number of wake bouts), sleep consolidation (sleep bout duration), as well as assessments of maintenance of wakefulness (wake bout length) and drug-related side effects.

[0134] Preclmical effects observed at either CT-5 (treatments administered at a time of day- corresponding to 5 hours after lights-on) and/or CT-18 (treatments administered at a time of day corresponding to 18 hours after lights-on or 6 hours after light-off when animals are housed in a 24 hour light dark cycle consisting of 12-hours of light and 12 hours of dark) are considered sufficient for purposes of identifying molecules that can reduce sleep fragmentation.

Biological Assay

[0135] The subject is surgically prepared for EEG and EMG recording and administered an analgesic with an antibiotic, followed by therapeutic delivery via intraperitoneal or oral administration. The sleep and wakefulness is determined using SCORE™.

[0136] Statistically significant differences between drug and vehicle were screened using a post- hoc Student’s T-test applied to hourly binned data and adjusted for repeated measures.

[0137] The compounds of the present disclosure exhibit improved sleep fragmentation, as assessed by evaluating the sleep architecture and sleep quality endpoints of several neurosteroid molecules with established affinity and functional activity at this target.

[0138] in some embodiments, sleep fragmentation is improved by (i) reducing the number of arousals (as measured by the number of wake bouts per hour), or (ii) increasing sleep consolidation (as measured by average sleep bout duration per hour). In some embodiments, sleep fragmentation is improved by (i) reducing the number of arousals (as measured by the number of wake bouts per hour), and (ii) increasing sleep consolidation (as measured by average sleep bout duration per hour). In some embodiments, sleep fragmentation is improved by reducing the number of arousals (as measured by the number of wake bouts per hour. In some embodiments, sleep fragmentation is improved by increasing sleep consolidation (as measured by average sleep bout duration per hour).

Methods of Use

[0139] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder.

[0140] In some embodiments, the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold.

[0141] In some embodiments, the sleep disorder is increased disturbed sleep. In some embodiments, the sleep disorder is increased sleep fragmentation. In some embodiments, the sleep disorder is increased arousals. In some embodiments, the sleep disorder is decreased arousal threshold. [0142] The present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering one or more compounds of the present disclosure or pharmaceutically acceptable salt thereof to a subject in need thereof

[0143] in some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering a dual acting H inverse agonist and SHTYi antagonist molecules (HI/5HT2A), or pharmaceutically acceptable salts thereof, to a subject m need thereof.

[0144] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering one or more compound selected from: HY- 10275, mirtazapine, amitriptyline, and quetiapine, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0145] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of or treating a sleep disorder by administering one or more compound selected from: HY-10275, mirtazapine, amitriptyline, and quetiapine, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0146] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of a sleep disorder by administering one or more compound selected from: HY-10275, mirtazapine, S -mirtazapine, amitriptyline, and quetiapine, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0147] In some embodiments, the present disclosure provides one or more compound selected from: HY-10275, mirtazapine, S-mirtazapine, amitriptyline, and quetiapine, or a

pharmaceutically acceptable salt thereof, for use in alleviating a symptom of, treating, or preventing a sleep disorder.

[0148] In some embodiments, the present disclosure provides one or more compound selected from: HY-10275, mirtazapine, S-mirtazapine, amitriptyline, and quetiapine, or a

pharmaceutically acceptable salt thereof, for use in alleviating a symptom of or treating a sleep disorder.

[0149] In some embodiments, the present disclosure provides one or more compound selected from: HY-10275, mirtazapine, S-mirtazapine, amitriptyline, and quetiapine, or a

pharmaceutically acceptable salt thereof, for use in alleviating a symptom of treating a sleep disorder. [0150] In some embodiments, the present disclosure provides use of one or more compound selected from: HY-10275, nurtazapine, S-mirtazapine, amitriptyline, and quetiapine, or a pharmaceutically acceptable salt thereof, for alleviating a symptom of, treating, or preventing a sleep disorder.

[0151] In some embodiments, the present disclosure provides use of one or more compound selected from: HY-10275, mirtazapine, S-mirtazapine, amitriptyline, and quetiapine, or a pharmaceutically acceptable salt thereof, for alleviating a symptom of or treating a sleep disorder.

[0152] In some embodiments, the present disclosure provides use of one or more compound selected from: HY-10275, mirtazapine, S-mirtazapine, amitriptyline, and quetiapine, or a pharmaceutically acceptable salt thereof, for alleviating a symptom of a sleep disorder.

[0153] In some embodiments, the present disclosure provides use of one or more compound selected from: HY-10275, mirtazapine, S-mirtazapine, amitriptyline, and quetiapine, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament, for alleviating a symptom of, treating, or preventing a sleep disorder.

[0154] In some embodiments, the present disclosure provides use of one or more compound selected from: HY-10275, mirtazapine, S-mirtazapine, amitriptyline, and quetiapine, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament, for alleviating a symptom of or treating a sleep disorder.

[0155] In some embodiments, the present disclosure provides use of a one or more compound selected from: HY-10275, mirtazapine, S-mirtazapine, amitriptyline, and quetiapine, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament, for alleviating a symptom of a sleep disorder.

[0156] In some embodiments, the compound is HY-10275, mirtazapine (HY10521), S- mirtazapine (HY 10378), quetiapine (HY10625), or amitriptyline, or a combination thereof, or a pharmaceutically acceptable salt thereof.

[0157] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering HY-10275, or a pharmaceutically acceptable salt thereof, to a subject in need thereof. [0158] In some embodiments, the present disclosure provides HY-10275, or a pharmaceutically acceptable salt thereof, for use in alleviating a symptom of, treating, or preventing a sleep disorder.

[0159] in some embodiments, the present disclosure provides use of HY-10275, or a

pharmaceutically acceptable salt thereof, for alleviating a symptom of, treating, or preventing a sleep disorder.

[0160] In some embodiments, the present disclosure provides use of HY-10275, or a

pharmaceutically acceptable salt thereof, in the manufacture of a medicament, for alleviating a symptom of, treating, or preventing a sleep disorder.

[0161] In some embodiments, the compound is HY-10275, or a pharmaceutically acceptable salt thereof.

[0162] In some embodiments, HY-10275 is administered at a dose between about 0.010 mg/kg and about 50 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.05 mg/kg and about 50 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.1 mg/kg and about 50 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.1 mg/kg and about 45 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.1 mg/kg and about 40 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0. 1 mg/kg and about 35 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.1 mg/kg and about 30 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.1 mg/kg and about 25 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.1 rng/kg and about 20 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.1 mg/kg and about 15 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.5 mg/kg and about 50 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.5 mg/kg and about 45 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.5 mg/kg and about 40 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.5 mg/kg and about 35 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.5 mg/kg and about 30 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.5 mg/kg and about 25 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.5 mg/kg and about 20 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 0.5 mg/kg and about 15 mg/kg. In some embodiments, HY- 10275 is administered at a dose between about 1 mg/kg and about 30 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 1 mg/kg and about 25 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 1 mg/kg and about 20 mg/kg. In some embodiments, HY- 10275 is administered at a dose between about 1 mg/kg and about 15 mg/kg. In some

embodiments, HY-10275 is administered at a dose between about 3 mg/kg and about 30 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 3 mg/kg and about 25 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 3 mg/kg and about 20 mg/kg. In some embodiments, HY-10275 is administered at a dose between about 3 mg/kg and about 15 mg/kg.

[0163] In some embodiments, HY-10275 is administered at a dose of about 0.010 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 0.015 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 0.020 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 0.025 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 0.05 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 0.1 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 0.2 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 0.3 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 0.4 mg/kg. In some

embodiments, HY-10275 is administered at a dose of about 0.5 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 0.6 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 0 7 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 0.8 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 0.9 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 1 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 3 mg/kg. In some embodiments, HY- 10275 is administered at a dose of about 5 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 10 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 15 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 20 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 25 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 30 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 35 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 40 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 45 mg/kg. In some embodiments, HY-10275 is administered at a dose of about 50 mg/kg.

[0164] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering mirtazapine, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0165] In some embodiments, the present disclosure provides mirtazapine, or a pharmaceutically acceptable salt thereof for use in alleviating a symptom of treating, or preventing a sleep disorder.

[0166] In some embodiments, the present disclosure provides use of mirtazapine, or a pharmaceutically acceptable salt thereof for alleviating a symptom of, treating, or preventing a sleep disorder.

[0167] In some embodiments, the present disclosure provides use of mirtazapine, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament, for alleviating a symptom of, treating, or preventing a sleep disorder.

[0168] In some embodiments, the compound is mirtazapine, or a pharmaceutically acceptable salt thereof.

[0169] In some embodiments, mirtazapine is administered at a dose between about 0.010 mg/kg and about 50 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 0.05 mg/kg and about 50 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 0.1 mg/kg and about 50 mg/kg. In some embodiments, mirtazapine is

administered at a dose between about 0.5 mg/kg and about 50 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 1 mg/kg and about 50 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 1 mg/kg and about 45 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 1 mg/kg and about 40 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 1 mg/kg and about 35 g/kg. In some embodiments, mirtazapine is administered at a dose between about 1 mg/kg and about 30 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 1 mg/kg and about 25 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 1 mg/kg and about 20 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 1 mg/kg and about 15 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 5 mg/kg and about 30 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 5 mg/kg and about 25 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 5 mg/kg and about 20 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 5 mg/kg and about 15 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 10 mg/kg and about 30 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 10 mg/kg and about 25 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 10 mg/kg and about 20 mg/kg. In some embodiments, mirtazapine is administered at a dose between about 10 mg/kg and about 15 mg/kg.

[0170] In some embodiments, mirtazapine is administered at a dose of about 0.010 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 0.015 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 0.020 mg/kg. In some

embodiments, mirtazapine is administered at a dose of about 0.025 mg/kg. In some

embodiments, mirtazapine is administered at a dose of about 0.05 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 0.1 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 0.2 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 0.3 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 0.4 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 0.5 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 1 mg/kg. In some

embodiments, mirtazapine is administered at a dose of about 1.5 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 2 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 2 5 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 3 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 3.5 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 4 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 4.5 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 5 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 10 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 15 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 20 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 25 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 30 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 35 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 40 mg/kg. In some embodiments. mirtazapine is administered at a dose of about 45 mg/kg. In some embodiments, mirtazapine is administered at a dose of about 50 mg/kg.

[0171] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering S-mirtazapme, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0172] In some embodiments, the present disclosure provides S-mirtazapine, or a

pharmaceutically acceptable salt thereof, for use in alleviating a symptom of, treating, or preventing a sleep disorder.

[0173] In some embodiments, the present disclosure provides use of S-mirtazapme, or a pharmaceutically acceptable salt thereof, for alleviating a symptom of, treating, or preventing a sleep disorder.

[0174] In some embodiments, the present disclosure provides use of S-mirtazapme, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament, for alleviating a symptom of, treating, or preventing a sleep disorder.

[0175] In some embodiments, the compound is S-mirtazapine, or a pharmaceutically acceptable salt thereof.

[0176] In some embodiments, S-mirtazapine is administered at a dose between about 0.010 mg/kg and about 50 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 0.05 mg/kg and about 50 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 0.1 mg/kg and about 50 mg/kg. In some embodiments, S- mirtazapine is administered at a dose between about 0 5 mg/kg and about 50 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 1 mg/kg and about 50 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 1 mg/kg and about 45 g/kg. In some embodiments, S-mirtazapine is administered at a dose between about 1 mg/kg and about 40 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 1 mg/kg and about 35 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 1 mg/kg and about 30 mg/kg. In some embodiments, S- mirtazapme is administered at a dose between about 1 mg/kg and about 25 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 1 mg/kg and about 20 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 1 mg/kg and about 15 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 5 mg/kg and about 30 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 5 mg/kg and about 25 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 5 mg/kg and about 20 mg/kg. In some embodiments, S- mirtazapine is administered at a dose between about 5 mg/kg and about 15 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 10 mg/kg and about 30 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 10 mg/kg and about 25 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 10 mg/kg and about 20 mg/kg. In some embodiments, S-mirtazapine is administered at a dose between about 10 mg/kg and about 15 mg/kg.

[0177] In some embodiments, S-mirtazapine is administered at a dose of about 0.010 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 0.015 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 0.020 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 0.025 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 0.05 mg/kg. In some

embodiments, S-mirtazapine is administered at a dose of about 0.1 mg/kg. In some

embodiments, S-mirtazapine is administered at a dose of about 0.2 mg/kg. In some

embodiments, S-mirtazapine is administered at a dose of about 0.3 mg/kg. In some

embodiments, S-mirtazapine is administered at a dose of about 0.4 mg/kg. In some

embodiments, S-mirtazapine is administered at a dose of about 0.5 mg/kg. In some

embodiments, S-mirtazapine is administered at a dose of about 1 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 1.5 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 2 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 2.5 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 3 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 3.5 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 4 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 4.5 mg/kg. In some

embodiments, S-mirtazapine is administered at a dose of about 5 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 10 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 1 5 mg/kg. In some embodiments, S-mirtazapine is

administered at a dose of about 20 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 25 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 30 mg/kg. In some embodiments, S-mirtazapme is administered at a dose of about 35 mg/kg. In some embodiments, S-mirtazapme is administered at a dose of about 40 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 45 mg/kg. In some embodiments, S-mirtazapine is administered at a dose of about 50 mg/kg.

[0178] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering amitriptyline, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0179] In some embodiments, the present disclosure provides amitriptyline, or a

pharmaceutically acceptable salt thereof, for use in alleviating a symptom of, treating, or preventing a sleep disorder.

[0180] In some embodiments, the present disclosure provides use of amitriptyline, or a pharmaceutically acceptable salt thereof, for alleviating a symptom of treating, or preventing a sleep disorder.

[0181] In some embodiments, the present disclosure provides use of amitriptyline, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament, for alleviating a symptom of, treating, or preventing a sleep disorder.

[0182] In some embodiments, the compound is amitriptyline, or a pharmaceutically acceptable salt thereof.

[0183] In some embodiments, amitriptyline is administered at a dose between about 0.010 mg/kg and about 50 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 0.05 mg/kg and about 50 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 0.1 mg/kg and about 50 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 0.5 mg/kg and about 50 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 1 mg/kg and about 50 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 1 mg/kg and about 45 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 1 mg/kg and about 40 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 1 mg/kg and about 35 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 1 mg/kg and about 30 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 1 mg/kg and about 25 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 1 mg/kg and about 20 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 1 mg/kg and about 15 mg/kg. in some embodiments, amitriptyline is administered at a dose between about 5 mg/kg and about 30 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 5 mg/kg and about 25 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 5 mg/kg and about 20 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 5 mg/kg and about 15 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 10 mg/kg and about 30 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 10 mg/kg and about 25 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 10 mg/kg and about 20 mg/kg. In some embodiments, amitriptyline is administered at a dose between about 10 mg/kg and about 15 mg/kg.

[0184] In some embodiments, amitriptyline is administered at a dose of about 0.010 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 0.015 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 0.020 mg/kg. In some

embodiments, amitriptyline is administered at a dose of about 0.025 mg/kg. In some

embodiments, amitriptyline is administered at a dose of about 0.05 mg/kg. In some

embodiments, amitriptyline is administered at a dose of about 0.1 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 0 2 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 0.3 mg/kg. In some embodiments, amitriptyline is

administered at a dose of about 0.4 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 0.5 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 1 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 1.5 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 2 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 2.5 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 3 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 3.5 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 4 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 4.5 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 5 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 10 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 15 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 20 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 25 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 30 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 35 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 40 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 45 mg/kg. In some embodiments, amitriptyline is administered at a dose of about 50 mg/kg.

[0185] In some embodiments, the present disclosure is directed to a method of alleviating a symptom of, treating, or preventing a sleep disorder by administering quetiapine, or a

pharmaceutically acceptable salt thereof, to a subject m need thereof.

[0186] In some embodiments, the present disclosure provides quetiapine, or a pharmaceutically acceptable salt thereof, for use in alleviating a symptom of, treating, or preventing a sleep disorder.

[0187] In some embodiments, the present disclosure provides use of quetiapine, or a

pharmaceutically acceptable salt thereof, for alleviating a symptom of, treating, or preventing a sleep disorder.

[0188] In some embodiments, the present disclosure provides use of quetiapine, or a

pharmaceutically acceptable salt thereof, for the manufacture of a medicament, for alleviating a symptom of treating, or preventing a sleep disorder.

[0189] In some embodiments, the compound is quetiapine, or a pharmaceutically acceptable salt thereof

[0190] In some embodiments, quetiapine is administered at a dose between about 0.010 mg/kg and about 50 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.05 mg/kg and about 50 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.1 mg/kg and about 50 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.5 mg/kg and about 50 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.1 mg/kg and about 45 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.1 mg/kg and about 40 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.1 mg/kg and about 35 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.1 mg/kg and about 30 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.1 mg/kg and about 25 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.1 mg/kg and about 20 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.1 mg/kg and about 15 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.5 mg/kg and about 50 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.5 mg/kg and about 45 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.5 mg/kg and about 40 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.5 mg/kg and about 35 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.5 mg/kg and about 30 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.5 mg/kg and about 25 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.5 mg/kg and about 20 mg/kg. In some embodiments, quetiapine is administered at a dose between about 0.5 mg/kg and about 15 mg/kg. In some embodiments, quetiapine is administered at a dose between about 1 mg/kg and about 30 mg/kg. In some embodiments, quetiapine is administered at a dose between about 1 mg/kg and about 25 mg/kg. In some embodiments, quetiapine is administered at a dose between about 1 mg/kg and about 20 mg/kg. In some embodiments, quetiapine is administered at a dose between about I mg/kg and about 15 mg/kg. In some embodiments, quetiapine is administered at a dose between about 3 mg/kg and about 30 mg/kg. In some embodiments, quetiapine is administered at a dose between about 3 mg/kg and about 25 mg/kg. In some embodiments, quetiapine is administered at a dose between about 3 mg/kg and about 20 mg/kg. In some embodiments, quetiapine is administered at a dose between about 3 mg/kg and about 1 5 mg/kg.

[0191 ] In some embodiments, quetiapine is adm imstered at a dose of about 0.010 mg/kg. In some embodiments, quetiapine is administered at a dose of about 0.015 mg/kg. In some embodiments, quetiapine is administered at a dose of about 0.020 mg/kg. In some embodiments, quetiapine is administered at a dose of about 0.025 mg/kg. In some embodiments, quetiapine is administered at a dose of about 0.05 mg/kg. In some embodiments, quetiapine is administered at a dose of about 0.1 mg/kg. In some embodiments, quetiapine is administered at a dose of about 0.2 mg/kg. In some embodiments, quetiapine is administered at a dose of about 0.3 mg kg. In some embodiments, quetiapine is administered at a dose of about 0.4 mg/kg. In some

embodiments, quetiapine is administered at a dose of about 0.5 mg/kg. In some embodiments, quetiapine is administered at a dose of about 0.6 mg/kg. In some embodiments, quetiapine is administered at a dose of about 0.7 mg/kg. In some embodiments, quetiapine is administered at a dose of about 0.8 mg/kg. In some embodiments, quetiapine is administered at a dose of about 0.9 mg/kg. In some embodiments, quetiapine is administered at a dose of about 1 mg/kg. In some embodiments, quetiapine is administered at a dose of about 3 mg/kg. In some embodiments, quetiapine is administered at a dose of about 5 mg/kg. In some embodiments, quetiapine is administered at a dose of about 10 mg/kg. In some embodiments, quetiapine is administered at a dose of about 15 mg/kg. In some embodiments, quetiapine is administered at a dose of about 20 mg/kg. In some embodiments, quetiapine is administered at a dose of about 25 mg/kg. In some embodiments, quetiapine is administered at a dose of about 30 mg/kg. In some embodiments, quetiapine is administered at a dose of about 35 mg/kg. In some embodiments, quetiapine is administered at a dose of about 40 nig/kg. In some embodiments, quetiapine is administered at a dose of about 45 mg/kg. In some embodiments, quetiapine is administered at a dose of about 50 mg/kg.

[0192] In some embodiments, the compound is administered orally. In some embodiments, the compound is administered parenterally. In some embodiments, the compound is administered transdermally. In some embodiments, the compound is administered subcutaneously. In some embodiments, the compound is administered intravenously.

[0193] In some embodiments, a compound of the present disclosure is not suvorexant, zolpidem, S-zopiclone, or a pharmaceutically acceptable salt thereof.

[0194] In some embodiments, a compound of the present disclosure is not suvorexant or a pharmaceutically acceptable salt thereof.

[0195] In some embodiments, a compound of the present disclosure is not zolpidem or a pharmaceutically acceptable salt thereof.

[0196] In some embodiments, a compound of the present disclosure is not S-zopiclone or a pharmaceutically acceptable salt thereof.

[0197] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with sleep apnea, restless legs syndrome, a high respiratory disturbance index (RDI), neurological disease, circadian rhythm disorder, pain, periodic leg movement disorder f PLMD), REM behavior disorder, elderly fragmented sleep, age-related sleep fragmentation, post-menopausal sleep disorder, substance abuse, substance abuse withdrawal, narcolepsy, mental disorder, or non-restorative sleep. [0198] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by sleep apnea, restless legs syndrome, a high respiratory disturbance index (RDI), neurological disease, circadian rhythm disorder, pain, periodic leg movement disorder (PLMD), REM behavior disorder, elderly fragmented sleep, age-related sleep fragmentation, post-menopausal sleep disorder, substance abuse, substance abuse withdrawal, narcolepsy, mental disorder, or non-restorative sleep.

[0199] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with sleep apnea, restless legs syndrome, high respiratory disturbance index (RDI), neurological disease, circadian rhythm disorder, pain, periodic leg movement disorder (PLMD), REM behavior disorder, elderly fragmented sleep, age-related sleep fragmentation, post-menopausal sleep disorder, substance abuse, substance abuse withdrawal, narcolepsy, mental disorder, or non-restorative sleep.

[0200] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with sleep apnea.

[0201] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by sleep apnea.

[0202] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with sleep apnea.

[0203] In some embodiments, sleep apnea is obstructive sleep apnea. [0204] In some embodiments, sleep apnea is obstructive sleep apnea due to a high respiratory disturbance index (RDI) associated with an elevated respiratory event related arousal (RERA) with or without a concomitant apnea.

[0205] In some embodiments, sleep apnea is obstructive sleep apnea due to a high respiratory disturbance index (RDI) associated with an elevated respiratory event related arousal (RERA) with a concomitant apnea.

[0206] In some embodiments, sleep apnea is obstructive sleep apnea due to a high respiratory disturbance index (RDI) associated with an elevated respiratory event related arousal (RERA) with or without a concomitant hypopnea.

[0207] In some embodiments, sleep apnea is obstructive sleep apnea due to a high respiratory disturbance index (RDI) associated with an elevated respiratory event related arousal (RERA) with a concomitant hypopnea.

[0208] In some embodiments, sleep apnea is obstructive sleep apnea due to a high respiratory disturbance index (RDI) associated with an elevated respiratory' event related arousal (RERA) with or without concomitant acute hemoglobin desaturation.

[0209] In some embodiments, sleep apnea is obstructive sleep apnea due to a high respiratory disturbance index (RDI) associated with an elevated respiratory event related arousal (RERA) with concomitant acute hemoglobin desaturation.

[0210] In some embodiments, sleep apnea is central sleep apnea.

[021 1 ] In some embodiments, sleep apnea is low-arousal threshold sleep apnea.

[0212] In some embodiments, sleep apnea is hypopnea.

[0213] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with restless legs syndrome.

[0214] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by restless legs syndrome.

[0215] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousafs, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with restless legs syndrome.

[0216] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with a high respiratory disturbance index

(RDI).

[0217] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by a high respiratory disturbance index (RDI).

[0218] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with a high respiratory' disturbance index (RDI).

[0219] In some embodiments, the RDI is associated with an elevated respiratory' event related arousal (RERA) with or without a concomitant apnea.

[0220] In some embodiments, the RDI is associated with an elevated respiratory' event related arousal (RERA) with a concomitant apnea.

[0221 ] In some embodiments, the RDI is associated with an elevated respiratory event related arousal (RERA) with or without a concomitant hypopnea.

[0222] In some embodiments, the RDI is associated with an elevated respiratory event related arousal (RERA) with a concomitant hypopnea.

[0223] In some embodiments, the RDI is associated with an elevated respiratory event related arousal (RERA) with or without concomitant acute hemoglobin desaturation.

[0224] In some embodiments, the RDI is associated with an elevated respiratory event related arousal (RERA) with concomitant acute hemoglobin desaturation.

[0225] In some embodiments, the RDI is associated with an elevated respiratory event related arousal (RERA) with or without concomitant hemoglobin desaturation.

[0226] In some embodiments, the RDI is associated with an elevated respiratory event related arousal (RERA) with concomitant hemoglobin desaturation. [0227] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousafs, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with a neurological disease.

[0228] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by a neurological disease.

[0229] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with a neurological disease.

[0230] In some embodiments, the neurological disease is a neurodegenerative disease.

[0231] In some embodiments, the neurodegenerative disease is Lewy body disease (i.e., Lewy body dementia). In some embodiments, the Lewy body disease is diffuse.

[0232] In some embodiments, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). In some embodiments, the neurodegenerative disease is Huntington’s disease. In some embodiments, the neurodegenerative disease is Parkinson’s disease. In some embodiments, the neurodegenerative disease is Alzheimer’s disease. In some embodiments, the neurodegenerative disease is a synucleinopathy.

[0233] In some embodiments, a synucleinopathy is Alzheimer’s disease, Parkinson’s disease, or Lewy body dementia. In some embodiments, a synucleinopathy is Alzheimer’s disease. In some embodiments, a synucleinopathy is Parkinson’s disease. In some embodiments, a synucleinopathy is dementia with Lewy bodies. In some embodiments, a synucleinopathy is multiple system atrophy.

[0234] In some embodiments, the neurological disease is a neurodevelopmental disease. In some embodiments, the neurodevelopmental disease is autism. In some embodiments, the neurological disease is a muscular dystonia. In some embodiments, the dystonia is neuromuscular dystonia. In some embodiments, the neuromuscular dystonia is spasmodic torticollis.

[0235] In some embodiments, the neurological disease is multiple sclerosis (MS). [0236] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousafs, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with a circadian rhythm disorder.

[0237] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by a circadian rhythm disorder.

[0238] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with a circadian rhythm disorder.

[0239] In some embodiments, the circadian rhythm disorder is advanced sleep- wake phase disorder. In some embodiments, the circadian rhythm disorder is irregular sleep-wake rhythm disorder. In some embodiments, the circadian rhythm disorder is jet lag. In some embodiments, the circadian rhythm disorder is shift work sleep disorder. In some embodiments, the circadian rhythm disorder is delayed sleep phase syndrome. In some embodiments, the circadian rhythm disorder is non-24 hour rhythm disorder.

[0240] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with pain.

[0241] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by pain.

[0242] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with pain. [0243] In some embodiments, the pain is selected from an inflammatory pain, a nociceptive pain, a neuropathic pain, a mixed nociceptive and neuropathic pain, a post-operative pain, a post herpetic pain, a traumatic pain, a phantom-limb pam, a fibromyalgia, a back pain, a cancer pain, and an osteoarthritic pam.

[0244] In some embodiments, the pam is an inflammatory pain. In some embodiments, the inflammatory pain is arthritis. In some embodiments, the arthritis is rheumatoid arthritis. In some embodiments, the arthritis is osteoarthritis.

[0245] In some embodiments, the pain is a nociceptive pam. In some embodiments, the nociceptive pain is acute. In some embodiments, the nociceptive pain is chronic. In some embodiments, the nociceptive pam is caused by a cancer therapy. In some embodiments, the nociceptive pain is caused by a surgery.

[0246] In some embodiments, the pain is a neuropathic pain. In some embodiments, the neuropathic pam is chronic. In some embodiments, the neuropathic pain is acute. In some embodiments, the neuropathic pain is back pain. In some embodiments, the neuropathic pain is caused by a spinal cord injury . In some embodiments, the neuropathic pain is caused by multiple sclerosis. In some embodiments, the neuropathic pam is caused by a stroke. In some

embodiments, the neuropathic pain is caused by diabetes. In some embodiments, the neuropathic pain is caused by a metabolic condition.

[0247] In some embodiments, the pam is a mixed nociceptive and neuropathic pain

[0248] In some embodiments, the pain is a post-operative pam.

[0249] In some embodiments, the pain is a post-herpetic pain

[0250] In some embodiments, the pam is a traumatic pain. In some embodiments, traumatic pain is caused by causalgia.

[0251] In some embodiments, the pam is a phantom-limb pain.

[0252] In some embodiments, the pain is a fibromyalgia.

[0253] In some embodiments, the pam is a back pam. In some embodiments, the pam is a low back pam.

[0254] In some embodiments, the pam is a cancer pain. In some embodiments, the cancer pam is cancer. In some embodiments, the cancer pain is caused by a tumor. In some embodiments, the cancer pain is caused by a cancer treatment. In some embodiments, the cancer pain is caused by chemotherapy. In some embodiments, the cancer pain is radiation therapy. In some embodiments, the cancer pain is caused by surgery.

[0255] In some embodiments, the pam is an osteoarthritic pain.

[0256] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with periodic leg movement disorder

(PLMD).

[0257] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by periodic leg movement disorder (PLMD).

[0258] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with periodic leg movement disorder (PLMD).

[0259] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with REM behavior disorder

[0260] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by REM behavior disorder.

[0261] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with REM behavior disorder.

[0262] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousafs, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with elderly fragmented sleep.

[0263] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by elderly fragmented sleep.

[0264] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with elderly fragmented sleep.

[0265] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with age-related sleep fragmentation.

[0266] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by age-related sleep fragmentation.

[0267] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with age-related sleep fragmentation

[0268] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with post-menopausal sleep disorder.

[0269] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by post-menopausal sleep disorder. [0270] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousafs, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with post-menopausal sleep disorder.

[0271] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with substance abuse.

[0272] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by substance abuse.

[0273] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with substance abuse.

[0274] In some embodiments, the substance abuse is opioid abuse or alcoholism. In some embodiments, the substance abuse is opioid abuse. In some embodiments, the substance abuse is alcoholism.

[0275] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with substance abuse withdrawal.

[0276] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by substance abuse withdrawal.

[0277] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with substance abuse withdrawal. [0278] In some embodiments, the substance abuse withdrawal is opioid withdrawal or alcohol withdrawal. In some embodiments, the substance abuse withdrawal is opioid withdrawal. In some embodiments, the substance abuse is alcohol withdrawal.

[0279] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with narcolepsy.

[0280] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by narcolepsy.

[0281] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with narcolepsy.

[0282] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with a mental disorder.

[0283] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by a mental disorder

[0284] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with a mental disorder.

[0285] In some embodiments, the mental disorder is depression, major depressive disorder, post- traumatic stress disorder, anxiety disorder, bipolar disorder, or schizophrenia.

[0286] In some embodiments, the mental disorder is depression. In some embodiments, the mental disorder is major depressive disorder. In some embodiments, the mental disorder is post- traumatic stress disorder. In some embodiments, the mental disorder is anxiety disorder. In some embodiments, the mental disorder is bipolar disorder. In some embodiments, the mental disorder is schizophrenia.

[0287] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with non-restorative sleep.

[0288] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by non-restorative sleep.

[0289] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with non-restorative sleep.

[0290] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with snoring.

[0291 ] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by snoring.

[0292] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with snoring.

[0293] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousals, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by or co-morbid with idiopathic hypersomnia. [0294] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousafs, or decreased arousal threshold in a subject, wherein the sleep disorder is caused by idiopathic hypersomnia.

[0295] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder wherein the sleep disorder is increased disturbed sleep, increased sleep fragmentation, increased arousais, or decreased arousal threshold in a subject, wherein the sleep disorder is co-morbid with idiopathic hypersomnia.

[0296] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder by administering to a subject in need thereof a pharmaceutical composition comprising an Hi inverse agonist or antagonist and 5HT'2A antagonist or inverse activity and an additional active agent. In some embodiments, the additional active agent is a sedative-hypnotic.

[0297] In some embodiments, the sedative-hypnotic is selected from: zo!pidem, suvorexant, butabarbital, quazepam, triazolam, tasimelteon, eszopiclone, temazepam, ramelteon, secobarbital, doxepin, estazolam, or flurazepam.

[0298] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder by administering a pharmaceutical composition comprising an Hi inverse agonist or antagonist and 5HT2A antagonist or inverse activity, or a pharmaceutically acceptable derivative thereof, either alone, or in combination with a single additional active agent.

[0299] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder by administering a pharmaceutical composition comprising an Hi inverse agonist or antagonist and 5HT2A antagonist or inverse activity, or a pharmaceutically acceptable derivative thereof, in combination with a single additional active agent.

[0300] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder by administering a pharmaceutical composition comprising an Hi inverse agonist or antagonist and 5HT2A antagonist or inverse activity, or a pharmaceutically acceptable derivative thereof, either alone, or in combination with one or more additional active agent. [0301] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder by administering a pharmaceutical composition comprising an Hi inverse agonist or antagonist and 5HT2A antagonist or inverse activity, or a pharmaceutically acceptable derivative thereof, in combination with one or more additional active agent.

[0302] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder by administering one or more compound of the present disclosure either hora somni, h.s. (at bedtime) or between 0-4 hours before bedtime.

[0303] In some embodiments, the present disclosure is directed to alleviating a symptom of, treating, or preventing a sleep disorder by administering one or more compound of the present disclosure and an additional active agents, if present, either hora somni, h.s. (at bedtime) or between 0-4 hours before bedtime.

[0304] In some embodiments, the number of arousals is decreased by up to about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% reduction in the number of wake bouts per hour post-treatment relative to vehicle treatment.

[0305] In some embodiments, HY-10275 alleviates a symptom of, treats, or prevents a sleep disorder.

[0306] In some embodiments, HY-10275 reduces sleep fragmentation . In some embodiments, HY-10275 decreases the number of arousals (e.g., about up to 50% reduction in the number of wake bouts per hour post-treatment relative to vehicle treatment) and increases sleep consolidation (e.g., about 10-fold in average sleep bout duration per hour post-treatment relative to vehicle treatment).

[0307] In some embodiments, HY-10275 reduces wake bout length.

[0308] In some embodiments, mirtazapine alleviates a symptom of, treats, or prevents a sleep disorder.

[0309] In some embodiments, mirtazapine reduces sleep fragmentation . In some embodiments, mirtazapine decreases the number of arousals (e.g., about up to 50% reduction in the number of wake bouts per hour post-treatment relative to vehicle treatment) and increases sleep consolidation (e.g., about 10-fold in average sleep bout duration per hour post-treatment relative to vehicle treatment).

[0310] In some embodiments, mirtazapine reduces wake bout length.

[0311] In some embodiments, S-mirtazapine alleviates a symptom of, treats, or prevents a sleep disorder.

[0312] In some embodiments, S-mirtazapine reduces sleep fragmentation . In some

embodiments, S-mirtazapine decreases the number of arousals (e.g., about up to 50% reduction m the number of wake bouts per hour post-treatment relative to vehicle treatment) and increases sleep consolidation (e.g., about 10-fold m average sleep bout duration per hour post-treatment relative to vehicle treatment).

[0313] In some embodiments, S-mirtazapine reduces wake bout length.

[0314] In some embodiments, quetiapine alleviates a symptom of) treats, or prevents a sleep disorder.

[0315] In some embodiments, quetiapine reduces sleep fragmentation . In some embodiments, quetiapine decreases the number of arousals (e.g., about up to 50% reduction in the number of wake bouts per hour post-treatment relative to vehicle treatment) and increases sleep

consolidation (e.g., about 10-fold in average sleep bout duration per hour post-treatment relative to vehicle treatment).

[0316] In some embodiments, quetiapine reduces wake bout length.

[0317] In some embodiments, amitriptyline alleviates a symptom of, treats, or prevents a sleep disorder.

[0318] In some embodiments, amitriptyline reduces sleep fragmentation . In some embodiments, amitriptyline decreases the number of arousals (e.g., about up to 50% reduction in the number of wake bouts per hour post-treatment relative to vehicle treatment) and increases sleep

consolidation (e.g., about 10-fold in average sleep bout duration per hour post-treatment relative to vehicle treatment).

[0319] In some embodiments, amitriptyline reduces wake bout length.

[0320] In some embodiments, one or more compound of the present disclosure produces improvements in sleep fragmentation in comparison to compounds prescribed comprising the pharmacological standard of care for insomnia. In some embodiments, the standard of care compounds for insomnia are, for example, but not limited to, the orexin antagonists (e.g., suvorexant (BELSOMRA®)) and non-benzodiazepine, benzodiazepine receptor dependent GABAA allosteric modulators (e.g., zolpidem (AMBIEN®) and eszopiclone (LUNESTA®)), which do not adequately improve the requisite preclmical measures of sleep fragmentation.

[0321 ] in some embodiments, orexin OX1/OX2 antagonist suvorexant fails to improve sleep fragmentation at 3 mg/kg, 10 mg/kg, and 30 mg/kg. In some embodiments, suvorexant does not reduce the number of arousals as measured by the number of wake bouts per hour, but instead, increases the number of arousals. In some embodiments, suvorexant produces small

improvements in sleep consolidation at the highest dose (30 mg/kg) as measured by average sleep bout duration per hour post-treatment.

[0322] In some embodiments, zolpidem, at doses sufficient to produce increases m soporific efficacy as measured by EEG slow wave activity (EEG delta power in nonREM sleep), fails to reduce the number of arousals as measured by the number of wake bouts per hour and fails to produce improvement in sleep consolidation as measured by average sleep bout duration per hour. Zolpidem can produce sedative effects (up to and including non-responsiveness of the subject); however, increasing the dose of zolpidem may be complicated by a short kinetic half- life and can be contraindicated due to significant unwanted side effects including, but not limited to, REM sleep inhibition, severe motor coordination impairment, memory impairment, rebound insomnia, and other unwanted adverse effects, or a combination thereof.

[0323] In some embodiments, S-zopiclone (marketed as LUNESTA®) fails to reduce the number of arousals as measured by the number of wake bouts per hour and produces only small improvements in sleep consolidation as measured by average sleep bout duration per hour. In some embodiments, the dose of S-zopiclone tested (i.e., from about 5 mg/kg to about 30 mg/kg) does not increase sleep consolidation above the levels exhibited during the baseline rest phase (lights-on; circadian time 0:00-12:00 prior to treatment). While the immediate sedative hypnotic effects of S-zopiclone can increase with dose, driving additional soporific efficacy with high doses is contraindicated due to dose-dependent unwanted side effects. In some embodiments, S- zopiclone decreases Locomotor Activity Intensity (LMAi). In some embodiments, S-zopiclone dose-dependently inhibits REM sleep, which may be important for memory and learning. In some embodiments, standard of care insomnia drugs (e.g., zolpidem and S-zopiclone) can also reduce muscle tone, which is not only important for ambulation (e.g., getting up from sleep to use the bathroom), but also may have unwanted consequences for subjects diagnosed with or at risk for obstructive sleep apnea. In some embodiments, relaxation of upper airway muscles is undesirable as it could worsen obstructive sleep apnea and its comorbid health risks. Direct electromyographic (EMG) assessment of muscle tone may reveal unwanted myorelaxation (decrease in skeletal muscle electrical activity) after treatment with S-zopie!one. The improvement in nonREM sleep time induced by S-zopiclone can be followed by an undesirable dose-dependent rebound interference in sleep that would not enable improvement in sleep fragmentation.

EXAMPLES

[0324] The following examples illustrate the disclosure. These examples are not intended to limit the scope of the present disclosure, but rather to provide guidance to the skilled artisan to prepare and use the methods of the present disclosure. While particular embodiments of the present disclosure are described, the skilled artisan will appreciate that various changes and modifications can be made without departing from the spirit and scope of the disclosure.

Abbreviations

[0325] The following abbreviations are used in the examples, which follow. This list is not meant to be an all-inclusive list of abbreviations used in the application as additional standard abbreviations, which are readily understood by those skilled in the art, can also be used in the examples.

C Celsius

EEG Electro- encephalograph

EMG electromyograph

g grams

hr hour(s)

Hz hertz

kg kilograms

mL milliliters

Example 1. Experimental details and methods for determining sleep continuity, wakefulness, number of arousals, locomotor activity, and soporific efficacy

Animal preparation rats

[0326] Adult, male Wistar rats (approximately 270 g at time of surgery, Charles River

Laboratories) were anesthetized (2% isoflourane in 95/5 oxygen) and surgically prepared with a cranial implant that permitted chrome electro-encephalogram (EEG) and electromyogram (EMG) recording. Body temperature and locomotor activity were monitored via a miniature transmitter (Minimitter Series 4000 E-Mitter, Bend, OR) surgically placed m the abdomen during the same anesthetic event the cranial portion was implanted. The cranial implant consisted of stainless steel screws (2 frontal [+3.9 AP from bregma, ±2.0 ML] and 2 occipital [-6.4 AP, ±5.5 ML]) for EEG recording. Two Teflon-coated stainless steel wires were positioned under the nuchal trapezoid muscles for EMG recording. All leads were soldered to a miniature connector (Microtech, Boothwyn, PA) and gas sterilized with ethylene oxide prior to surgery. The implant assembly was affixed to the skull by the combination of the EEG recording screws,

cyanoacrylate applied between the hermetically sealed implant connector and skull, and dental acrylic. An analgesic (buprinorphine 0.3 mg/kg IP) was administered pre-operatively and daily SC for 2 days post-surgery. An antibiotic was administered before surgery' (chloramphenicol 40 mg/kg IM) and for 7-10 days after surgery' (Clavamox b.i.d). At least three weeks were allowed for surgical recovery' prior to any data collection.

Recording environment

[0327] Rats were housed individually within specially modified Nalgene ^® microisolator cages equipped with an ultra-low-torque slip-ring commutator and a custom polycarbonate filter-top riser. These cages were located within separate, ventilated compartments of a stainless steel sleep-wake recording chamber. Food and water were available ad libitum and the ambient temperature was 23±1° C. A 24-hr light-dark cycle (LD 12: 12) was maintained throughout the study using fluorescent light. Light intensity averaged 35-40 lux at mid-level inside the cage. Relative humidity averaged 50% approximately. Animals were undisturbed for two days before and after each treatment.

Automated data collection

[0328] Sleep and wakefulness were determined using SCORE™— a microcomputer-based sleep- wake and physiological monitoring system. Validation of the SCORE™ sleep stage identification algorithm in rodents and utility in pre-cimical drug evaluation have been previously described (Van Gelder et al. 1991 ; Edgar et ai. Psychopharmacology 1991 , 105, 374; J. Pharmacology & Experimental Therapeutics 1997, 283, 757; Seidel et al J Pharmacology & Experimental Therapeutics 275, 263; J. Pharmacology & Experimental Therapeutics, 285,

1073). For the studies described herein, the system monitored amplified EEG (xl 0,000, bandpass 1-30 Hz; initial digitization rate 400 Hz [Grass Corp., Quincy, MA]), integrated EMG (bandpass 10-100 Hz, RMS integration), and telemetered body temperature and non-specific locomotor activity (LMA), and drink- and food-related activity, from up to 150 rodents simultaneously. Arousal states were classified on-line as NREM sleep, REM sleep, wake, or theta-dominated wake every 10 seconds using EEG period and amplitude feature extraction and ranked membership algorithms. Individually taught EEG-arousal-state templates and EMG criteria differentiated states of arousal. LMA and drink-related activity were automatically recorded as counts per minute, and body temperature was recorded each minute. LMA was detected in both horizontal and vertical planes by a customized telemetry receiver (ER4000, Minimitter, Bend, OR) beneath the cage. Drink-related activity and food-related activity were detected by beam break sensors closely situated around recessed access portals to the lixit and the food bin, respectively. The beam break area for the food bin was, however, relatively large, and these data have not been validated as an endpoint for food consumption per se. Telemetry measures (LMA and body temperature) were not part of the SCORE arousal-state determination algorithm; thus, sleep-scoring and telemetry' data were concurrent but independent measures. In addition to frequent on-line inspection of the EEG and EMG signals, quality control of the data was assured by expert analysts with a minimum of 4 years of experience using a proprietary suite of programs ( SCOREVIEW™, Hypnion, Inc., Lexington, MA) that allowed data quality of all variables to be flexibly scrutinized at the level of (i) individual visual examination of raw EEG and EMG signals, (U) individual hourly mean timeseries, and (Hi) group mean timeseries, using a combination of graphical and statistical assessments. An integrated relational database was updated with data quality control decisions for each individual treatment, and this database controlled all subsequent use of these data. Complete, digitized raw EEG and integrated EMG data have been written to CD-R and are permanently archived for all treatments.

Treatments and drug preparation

[0329] Drug dose, route of administration, and timing of administration are described for each compound and variable within the data exemplifications. Where applicable, methyleeliulose vehicle was prepared as a sterile 0.25% solution of methyleeliulose ( 15 centipoise, Sigma, St. Louis, MO., USA). D-a-Tocopheryl polyethylene glycol 1000 succinate (TPGS, HY-16388)

20% solution was used to solubilize the compound suvorexant (HY-16387). [0330] Drugs were weighed using a Metier AX205 analytical balance (d=0.01 mg). Compound was mixed with vehicle using a sterile 2 mL ground glass pestle and mortar until completely dissolved, and then transferred to a sterile Vacutainer (red top) tube. Solutions were agitated immediately before being drawn into a syringe. Oral gavage administration (PO) was typically in a volume of 1 ml. kg. To administer the treatment, each rat was removed from its cage for about 60-90 seconds to be weighed and treated (the home cage is the recording cage m SCORE™ systems). Note that this procedure caused no prior sleep loss, unlike cases in which the animal must first be acclimated to a special recording chamber. Rats in this experiment lived

permanently in their“home cage” within the recording chamber. Prior sleep loss— for instance, the“acclimation” commonly used by other investigators— significantly influences the measurement of sleep-w¾kefuiness responses to drugs (Edgar et al. Psychopharmacology 1991, 105, 374; Meltzer & Serpa, Drug Dev Res , 1988, 14, 151).

Study design

[0331] The standard recording duration for SCORE data was not less than 30 hours before and after treatment. The 30 hours pre-treatment baseline recording was itself preceded by at least 24 hours in which the animal was undisturbed in the home/recording cage. Rats were randomly assigned to treatments in parallel groups. Some rats received more than one active treatment, in which cases at least 7 days“washout” elapsed between each treatment.

Statistical Ana lysis

[0332] Statistically significant differences between drug and vehicle were screened using a post- hoc Student’s T-test applied to hourly binned data and adjusted for repeated measures.

Figure formats and graphics conventions

[0333] Two types of time-series plots are depicted: Post-treatment detail plots the first five hours post-treatment. Variables were computed in 5-minute bins, aligned to the minute of treatment. The first time bin, labeled 0, represents the first 5 minutes post treatment. Pre- and post-treatment time series plot ±30 hours before and after CT-18 treatments (or 29 hr before and 31 hr after CT- 5 treatments). Treatment occurred at the beginning of the hour marked by an arrow. Variables w¾re computed in hourly bins.

[0334] The following graphics convention have been used: All data are plotted as group mean ± SEM. The thick gray 3me encompasses the vehicle treatment mean ± SEM Along the x-axis, time of treatment is marked by a triangle unless noted otherwise. Along the x-axis, light/dark bars indicate lights on/off.

Example 2. HY-10275 effect on number of arousals, sleep continuity, depth of sleep, and maintenance of wakefulness

HY-10275 reduces number of arousals

[0335] The Hi inverse agonist and SHITA antagonist HY-10275 administered to male Wistar rats at CT-5 (5 hours after light-on; time of treatment indicated by the triangle on the abscissa), reduced the number of arousals (indicated by the arrow), as measured by the number of wake bouts. Number of wake bouts are plotted as the population (N=12) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant difference from methylcellulose vehicle control indicated by asterisk. 24 hour light-dark cycle (LD 12: 12) indicated on the abscissa (FIG. I). HY-10275 produced a statistically significant reduction m arousals, evidenced by 50% reductions in the number of wake bouts per hour post-treatment.

HY-10275 increases sleep continuity/consolidation at CT5

[0336] The Hi inverse agonist and 51 P ^' .Ά antagonist HY-10275 administered to male Wistar rats at CT-5 (5 hours after light-on; time of treatment indicated by the triangle on the abscissa), increased sleep consolidation (indicated by the arrow), as measured by the average sleep bout duration per hour. Average sleep bout duration was calculated as the mean duration of all sleep bouts initiated in a given hour for an individual animal, plotted as the population (N ^: =12) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from methylcellulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 2). HY-10275 produced a robust and statistically significant increase in sleep consolidation as exemplified by a 2-fold increase in sleep bout- length per hour as compared to vehicle controls.

HY-10275 increases sleep continuity/consolidation at CT-18

[0337] The Hi inverse agonist and 5HT?.A antagonist HY-10275 administered to Wistar rats at CT-18 (6 hours after lights-off; time of treatment indicated by the triangle on the abscissa), increased sleep consolidation (indicated by the arrow), as measured by the longest sleep bout duration observed per hour. Longest sleep bout duration was calculated as the population mean of the longest sleep bout duration initiated in a given hour for an individual animal, plotted as the population (N=T7) hourly mean ± SEM 30 hours before (baseline) and after treatment.

Statistically significant differences from methyicellulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 3). HY-10275 produced a statistically significant increase in sleep consolidation as exemplified by a 2-fold increase in the longest sleep bout-length per hour as compared to vehicle controls.

HY-10275 increases depth of sleep as measured by EEG slow wave activity

[0338] The Hi inverse agonist and SHITA antagonist HY-10275 administered to male Wistar rats at CT-18 (6 hours after lights-off; time of treatment indicated by the triangle on the abscissa) increased EEG slow wave activity in nonREM sleep (indicated by the arrow), as measured by the normalized (percent change from baseline) EEG delta power during nonREM sleep per hour. Normalized EEG delta pow¾r (power in the EEG at frequencies of 0.5-4. OHz, computed using Fourier analysis) is ploted m this example as the population (N=17) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from

methyicellulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12:12) indicated on the abscissa (FIG. 4).

HY-10275 reduces maintenance of wakefulness

[0339] The Hi inverse agonist and 5HT2A antagonist HY-10275 administered to male Wistar rats at CT-18 (6 hours after lights-off; time of treatment indicated by the triangle on the abscissa), decreased wake bout length (a measure of maintenance of wakefulness, and indirect measure of latency to return to sleep; indicated by the arrow), as measured by the longest wake bout duration per hour. Longest wake bout duration was calculated as the mean duration of the single longest wake bouts initiated in a given hour for an individual animal, plotted as the population (N=T7) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from methyicellulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 5).

Example 3. Mirtazapine effects on number of arousals and sleep continuity

Mirtazapine reduces number of arousals

[0340] The Hi inverse agonist and 5HT?.A antagonist racemic mirtazapine (HY-10521) administered to Wistar rats at CT-18 (6 hours after light-off; time of treatment indicated by the triangle on the abscissa), reduced the number of arousals (indicated by the arrow), as measured by the number of wake bouts. Number of wake bouts are plotted as population (N=19) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from methylce!lulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 6). Mirtazapine significantly reduced the number of arousals during the initial 6 hours post-treatment (arrow).

Mirtazapine increases sleep continuity/consolidation

[0341] The Hi inverse agonist and SHITA antagonist racemic mirtazapine (HY- 10521) administered to Wistar rats at CT-18 (6 hours after light-off; time of treatment indicated by the triangle on the abscissa), increased sleep consolidation (indicated by the arrow), as measured by the average sleep bout duration per hour. Average sleep bout duration was calculated as the mean duration of all sleep bouts initiated in a given hour for an individual animal, plotted as the population (N=T9) hourly mean ± SEM 30 hours before (baseline) and after treatment.

Statistically significant differences from mefhylce!!ulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 7). Mirtazapine increased sleep consolidation as evidenced by a statistically significant approximately 2-fold increase in the average sleep bout duration relative to vehicle.

Example 4. S-mirtazapine effects on number of arousals and sleep continuity

S-mirtazapine reduces number of arousals

[0342] The Hi inverse agonist and 5HT2A antagonist S-mirtazapine (HY-10378; the single S- enantiomer of mirtazapine) administered to Wistar rats at CT-18 (6 hours after light-off; time of treatment indicated by the triangle on the abscissa), reduced the number of arousals (indicated by the arrow), as measured by the number of wake bouts. Number of wake bouts plotted as the population (N=9) hourly mean ± SEM 30 hours before (baseline) and after treatment.

Statistically significant differences from methyicellulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 8). S- mirtazapine significantly reduced the number of arousals during the initial 6 hours post-treatment (arrow).

S-mirtazapine increases sleep continuity/consolidation

[0343] The Hi inverse agonist and 5HT2A antagonist S-mirtazapine (HY-10378; the single S- enantiomer of mirtazapine) administered to Wistar rats at CT-18 (6 hours after light-off; time of treatment indicated by the triangle on the abscissa), increased sleep consolidation (indicated by the arrow), as measured by the average sleep bout duration per hour. Average sleep bout duration was calculated as the mean duration of all sleep bouts initiated in each hour for an individual animal, plotted as the population (N=9) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from methylcelluiose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 9). S- mirtazapine increased sleep continuity/consolidation as evidenced by a statistically significant 2- fold increase in average sleep bout duration (arrow).

Example 5. Amitriptyline effects on number of arousals and sleep continuity

Amitriptyline reduces number of arousals

[0344] The Hi inverse agonist and 51 GG.Ά antagonist tricyclic antidepressant amitriptyline administered to Wistar rats at CT-5 (5 hours after light-on; time of treatment indicated by the triangle on the abscissa), can reduce the number of arousals (indicated by the arrow), as measured by the number of wake bouts. Number of wake bouts ploted as population (N=9) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from methylcelluiose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 10). Amitriptyline significantly reduced the number of arousals during the initial 6 hours post- treatment (arrow).

Amitriptyline increases sleep conti nuiiy /consolidation

[0345] The Hi inverse agonist and 5HT2A antagonist tricyclic antidepressant amitriptyline administered to male Wistar rats at CT-5 (5 hours after light-on; time of treatment indicated by the triangle on the abscissa), increased sleep consolidation (indicated by the arrow), as measured by the average sleep bout duration per hour. Average sleep bout duration was calculated as the mean duration of all sleep bouts initiated in each hour for an individual animal, plotted as the population (N=9) hourly mean ± SEM 30 hours before (baseline) and after treatment.

Statistically significant differences from methylcelluiose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 1 1 ).

Amitriptyline increased sleep consolidation/continuity as evidenced by a statistically significant approximately 2-fold increase in average sleep bout lengths (arrow). Example 6. Quetiapine effects on number of arousals and sleep continuity

Quetiapine reduces number of arousals

[0346] The HI inverse agonist and 5HT2a antagonist antipsychotic/antidepressant quetiapine (HY-! 0625) administered to male Wistar rats at CT-5 (5 hours after light-on; time of treatment indicated by the triangle on the abscissa), modestly reduces the number of arousals (indicated by the arrow), as measured by the number of wake bouts. Number of wake bouts plotted as the population (N=7) hourly mean ± SEM 30 hours before (baseline) and after treatment.

Statistically significant differences from methyicellulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 12). Quetiapine produced a statistically significant reduction in arousals post-treatment (arrow).

Quetiapine increases sleep continuity/consolidation

[0347] The Hi inverse agonist and 5HT?A antagonist antidepressant/antipsychotic quetiapine (HY-10625) administered to Wistar rats at CT-18 (6 hours after light-off; time of treatment indicated by the triangle on the abscissa), increased sleep consolidation (indicated by the arrow), as measured by the average sleep bout duration per hour. Average sleep bout duration was calculated as the mean duration of all sleep bouts initiated in each hour for an individual animal, plotted as the population (N=7) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from methyicellulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 13).

Quetiapine (HY-10625) increased sleep continuity/ consolidation as evidenced by a statistically significant >50% increase in average sleep bout lengths relative to vehicle control treatment (arrow).

Example 7. Suvorexant effects on number of arousals and sleep continuity

Suvorexant fails to reduce the number of arousals

[0348] The orexin OX1/QX2 receptor antagonist standard of care sedative hypnotic for insomnia suvorexant (HY-16387) administered to male Wistar rats at CT-18 (6 hours after light-off; time of treatment indicated by the triangle on the abscissa), failed to reduce the number of arousals (indicated by the arrow), as measured by the number of wake bouts per hour. Number of wake bouts plotted as the population (N=7) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from vehicle control (TPGS) are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 14).

Weak effect of suvorexant on sleep continuity/ consolidation

[0349] The orexin OX 1/0X2 receptor antagonist standard of care sedative hypnotic for insomnia suvorexant (HY-16387) administered to Wistar rats at CT-5 (5 hours after light-on; time of treatment indicated by the triangle on the abscissa), produced a weak increase in sleep consolidation (statistical trend 0.05<P< 1). as measured by the average sleep bout duration per hour (weak effect observed in the first two hours post-treatment; indicated by the arrow).

Average sleep bout duration was calculated as the mean duration of all sleep bouts initiated in a given hour for an individual animal, plotted as the population (N=7) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from

methylcellu!ose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 15).

Example 8, Zolpidem effects on EEG slow wave activity, number of arousals, sleep

continuity, MEM sleep, and locomotor activity

Zolpidem soporific efficacy as measured by EEG slow wave activity.

[0350] The standard of care treatment for insomnia, zo!pidem, administered to Wistar rats at CT- 18 (6 hours after iights-off; time of treatment indicated by the triangle on the abscissa) increased EEG slow wave activity in nonREM sleep (indicated by the arrow), as measured by the normalized (percent change from baseline) EEG delta power during nonREM sleep per hour. Normalized EEG delta power (power in the EEG at frequencies of 0.5-4.0Hz, computed using Fourier analysis) is plotted in this example as the population (N=8) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from methylceilulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 16).

Soporific doses of zolpidem fail to reduce the number of arousals

[0351] The insomnia standard of care, zolpidem, administered at a dose that produces robust efficacy as measured by EEG slow wave activity (FIG.16) failed to reduce the number of arousals as measured by the number of wake bouts per hour (indicated by the arrow). Zolpidem is a GAB AA positive allosteric modulator acting at the benzodiazepine binding site on GABAA. in this example, zolpidem (HY-10131) was administered to Wistar rats at CT-18 (6 hours after light-off; time of treatment indicated by the triangle on the abscissa). These are the same animals and the same drug treatment above. Number of wake bouts were plotted as the population (N=8) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from vehicle control (methylcellulose) are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 17). Despite the robust increase m EEG delta power during nonREM sleep (FIG. 16), zolpidem failed to reduce arousals as measured by the number of wake bouts per hour (FIG. 17).

Modest effect of zolpidem on sleep continuity/ consolidation

[0352] At a dose sufficient to produce robust soporific efficacy as measured by EEG slow wave activity (FIG. 16), the benzodiazepine receptor mediated GAB AA positive allosteric modulator, zolpidem (HY-I0131), administered to male Wistar rats at CT-18 (6 hours after light-off; time of treatment indicated by the triangle on the abscissa), produced only a small increase in sleep consolidation (statistical trend 0 05<P< 1), as measured by the average sleep bout duration per hour (weak effect observed in the first two hours post-treatment; indicated by the arrow).

Average sleep bout duration was calculated as the mean duration of all sleep bouts initiated in each hour for an individual animal, plotted as the population (N=8) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from methylcellulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 18).

Zolpidem inhibits REM sleep

[0353] Increasing the dose of zolpidem in an attempt to further reduce arousals or increase sleep continuity /'consolidation produces limited benefits, and triggers robust unwanted adverse events. In this Example, the GABAA positive allosteric modulator, zolpidem (HY-10131) administered to male Wistar rats at CT-5 (5 hours after light-on; tune of treatment indicated by the triangle on the abscissa), inhibited REM sleep (indicated by the arrow), as measured by the percent RE sleep that occurred per hour. Data are plotted as the population (N=16) hourly mean ± SEM 30 hours before (baseline) and after treatment. Statistically significant differences from

methylcellulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG 19).

Zolpidem interferes with locomotor activity [0354] The GABAA positive allosteric modulator sedative hypnotic drug for insomnia, zolpidem (HY-10131), administered to male Wistar rats at CT-18 (6 hours after light-off; time of treatment indicated by the triangle on the abscissa), inhibited Locomotor Activity Intensity (LMAi), as measured by the counts of locomotor activity per minute of wakefulness (indicated by the arrow), averaged hourly for each animal. LMAi reveals changes m motor activity (movements) that are disproportionate from that normally observed during wakefulness. Hypoactivity during w¾kefulness, as shown here, is indicative of motor coordination impairment and/or myorelaxant properties of zolpidem and related benzodiazepine receptor ligand sedative hypnotics. The magnitude of undesirable motor impairment (reduction in LMAi) increases with dose and is evident 4-5 hours post-treatment in this zolpidem 30 mg/kg example (arrow). Data are plotted as the population (N=10) hourly mean ± SEM 30 hours before (baseline) and after treatment.

Statistically significant differences from methylcellulose vehicle control animals are indicated by asterisks. 24 hour light-dark cycle (LD 12:12) is indicated on the abscissa (FIG. 20).

Example 9, S-zopiclone effects on number of arousals, sleep continuity, locomotor activity, muscle tone, and rebound sleep disturbance

S-zopiclone failed to reduce the number of arousals

[0355] The insomnia standard of care drug S-zopiclone, administered to male Wistar rats at CT- 18 (6 hours after light-off) failed to reduce the number of arousals (indicated by the arrow), as measured by the number of wake bouts per hour post-treatment. Number of wake bouts are plotted as the population (N=T3) hourly mean ± SEM 30 hours before (baseline) and after treatment (red triangle on the abscissa). Statistically significant differences from vehicle control (methylcellulose) are indicated by asterisks 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 21).

Effect of S-zopiclone on sleep continuity/ consolidation

[0356] Three doses of S-zopiclone (5 mg/kg, 10 mg/kg, and 30 mg/'kg) administered to Wistar rats at CT-18 (6 hours after light-off; time of treatment indicated by the triangle on the abscissa), are illustrated in this Example. All doses, including the highest dose of 30 mg/'kg produced only small increase in sleep consolidation. Only the highest dose (30 mg/kg S-zopiclone) produced a statistically significant increase in average sleep bout duration in the first treatment hour post treatment (indicated by the arrow). S-zopiclone effects on sleep consolidation were not dose proportional. Data are plotted as the population (N=T 0-12) mean ± SEM each hour for 30 hours before (baseline) and after treatment. Statistically significant differences from methylcelluiose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 22).

S-zopiclone dose-dependently interferes with locomotor activity

[0357] The insomnia standard of care sedative hypnotic S-zopiclone, administered to male Wistar rats at CT-18 (6 hours after light-off; time of treatment indicated by the triangle on the abscissa), dose-dependently inhibited Locomotor Activity Intensity (LMAi) as measured by the counts of locomotor activity per minute of wakefulness, averaged hourly for each animal. LMAi reveals whether changes m motor activity (movements) are disproportionate from that normally observed during wakefulness. A disproportionate reduction in locomotor activity during wakefulness, as shown here, is indicative of motor impairment that may be due in part to the skeletal muscle myorelaxant properties of S-zopiclone (see also FIG. 24) and functionally like other benzodiazepine receptor GABAA allosteric modulators (e.g., zolpidem, Example 20). The magnitude of undesirable motor impairment (reduction in LMAi) increases with dose, and is evident 1-6 hours post-treatment in the S-zopiclone 10 mg/kg and 30 mg/kg example. Data are plotted as the population (N=10-12) mean ± SEM on an hourly basis for 30 hours before (baseline) and after treatment. Statistically significant differences from methylcelluiose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 23).

S-zopiclone decreases muscle tone as measured by electromyogram (EMG)

[0358] The insomnia standard of care sedative hypnotic s-zopiclone, administered to Wistar rats at CT-18 (6 hours after light-off), decreased EMG activity (indicated by the arrow) as measured by root-mean-square analyses of EMG power in 5 minute intervals (treatment occurred at time zero). Data are plotted as population (N=T 3) mean ± SEM. Statistically significant differences from methylcelluiose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 24).

S-zopiclone soporific effects are followed by rebound sleep disturbance

[0359] The insomnia standard of care sedative hypnotic s-zopiclone, administered to male Wistar rats at CT-18 (6 hours after light-off; time of treatment indicated by the triangle on the abscissa), initially increased the amount of nonREM (NREM) sleep per hour post-treatment, but these initial soporific effects were follo wed by an interval of sleep interference (6-18 hours post treatment) characterized by a reduction in sleep time (indicated by the arrow _'" ). This“rebound” sleep disturbance corresponded with other markers of sleep disturbance such as concomitant reductions in sleep bout durations during the next circadian sleep phase (circadian time 0: GO- 12: 00; also see FIG. 22). Data are plotted as the population mean ± SEM on an hourly basis for 30 hours before (baseline) and after treatment. Statistically significant differences from methyleeliulose vehicle control are indicated by asterisks. 24 hour light-dark cycle (LD 12: 12) is indicated on the abscissa (FIG. 25). Latent sleep disturbance is an undesirable feature of drugs intended to treat sleep fragmentation. Thus, the insomnia drug S-zopiclone is not an appropriate medicine to treat sleep fragmentation.

EQUIVALENTS

[0360] The details of one or more embodiments of the disclosure are set forth in the

accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. to which this disclosure belongs. All patents and publications cited in this specification are incorporated by reference.

[0361 ] The foregoing description has been presented only for the purposes of illustration and is not intended to limit the disclosure to the precise form disclosed, but by the claims appended hereto.