Methods for reducing neurodegeneration associated with neurodegenerative diseases

Abstract

The present disclosure relates to lemborexant, a dual orexin receptor antagonist, and compositions and methods for use in the treatment of Alzheimer's disease (AD), for example, in subjects having or at risk of developing AD. [Solution] A method for treating Alzheimer's disease (AD) in a subject having or at risk of developing AD, comprising administering to the subject a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, thereby treating AD.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 376,949, filed September 23, 2023, and U.S. Provisional Patent Application No. 63 / 382,278, filed November 3, 2023, the entire contents of which are incorporated herein by reference.

[0002] [Field of Disclosure]

[0002] Described herein are compositions and methods related to lemborexant, a dual orexin receptor antagonist, for use in the treatment of neurological disorders such as Alzheimer's disease (AD). [Background technology]

[0003] [background] Alzheimer's disease (AD) is a progressive neurodegenerative disease of unknown etiology and the most common form of dementia among the elderly. In 2006, there were 26.6 million cases of AD worldwide (range: 11.4 million to 59.4 million) (Brookmeyer, R., et al., Forecasting the global burden of Alzheimer's Disease. Alzheimer Dement. 2007;3:186-91), and it was reported that more than 5 million people in the United States suffered from AD (Alzheimer's Association, Alzheimer's Association report, 2010 Alzheimer's disease facts and figures. Alzheimer Dement. 2010;6:158-94). By 2050, the global prevalence of AD is projected to increase to 106.8 million (range: 47.22 million to 221.2 million), with an estimated prevalence of 11 to 16 million in the United States alone (Brookmeyer, supra, and 2010 Alzheimer's disease facts and figures, supra).

[0004]

[0004] The disease generally progresses slowly, accompanied by widespread cognitive decline, and in the final stages, subjects become bedridden. AD subjects typically survive only 3 to 10 years from the onset of symptoms, although extreme cases have been known to survive 2 to 20 years (Hebert, LE, et al., Alzheimer's disease in the US population: prevalence estimates using the 2000 census. Arch Neurol. 2003;60:1119-1122.). AD is the seventh leading cause of all deaths in the United States and the fifth leading cause of death among Americans over the age of 65, despite the fact that AD mortality rates are greatly underestimated because AD is rarely listed as the cause of death on death certificates.

[0005]

[0005] AD poses a significant economic burden throughout developed countries, significantly impacting healthcare systems and public resources as well as patients and their families. In the United States alone, total payments in 2010 were estimated at $172 billion, including $123 billion for Medicare and Medicaid.

[0006]

[0006] Histologically, this disease is characterized by senile plaques, which are found primarily in the association cortex, limbic system, and basal ganglia. The main component of these senile plaques is amyloid beta peptide (Aβ). Aβ exists in various conformational states: monomers, oligomers, protofibrils, and insoluble fibrils. The details of the mechanistic relationship between the development of Alzheimer's disease and Aβ production are unclear. However, some anti-Aβ antibodies are currently undergoing clinical investigation as potential therapeutic agents for Alzheimer's disease.

[0007] In addition to senile plaques, the disease is also characterized by tau aggregation and hyperphosphorylation, increased immune response, degenerating neurons, synaptic loss and eventual cognitive impairment, dementia and death.

[0008]

[0008] Insomnia has been implicated as a risk factor for AD. Historically, insomnia has been treated with a variety of medications, including doxepin, tricyclic antidepressants (TCAs), and dual orexin receptor antagonists (also known as DORAs), such as suvorexant or lemborexant. While the literature suggests an association between sleep disorders and the risk of AD, no direct association has been established. To illustrate the need for further elucidation of the association between insomnia and AD, the data disclosed herein suggest that at least two such sleep medications, namely doxepin and lemborexant, have different effects on AD pathology, despite both mediating sleep.

[0009]

[0009] Lemborexant is approved for the treatment of adult patients with insomnia, a condition characterized by difficulty falling asleep and / or staying asleep. Lemborexant and methods of use are disclosed, for example, in U.S. Patent Nos. 11,026,944 and 11,096,941, the contents of which are incorporated herein by reference.

[0010] Lemborexant has the following structure: [ka] and is also known as (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide or (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropane-1-carboxamide.

[0011]

[0011] Thus, there remains a need for improved treatments for AD, including early intervention before irreversible symptoms of this disease develop. The disclosure herein surprisingly demonstrates that lemborexant can be used for such treatments. Summary of the Invention

[0012] [overview]

[0012] One aspect of the present disclosure relates to a method for treating Alzheimer's disease (AD) in a subject having or at risk of developing AD, the method comprising administering a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof or a solvate thereof to the subject, thereby treating AD.

[0013] In some embodiments, treating AD comprises alleviating and / or slowing cognitive decline, hi some embodiments, treating AD comprises affecting (e.g., slowing, delaying, or reducing) changes in at least one marker of AD pathology.

[0014] In some embodiments, the marker is the level of tau phosphorylation, neurodegeneration, changes in microglial response, and / or the presence of Aβ plaques. In some embodiments, the marker is present in a brain region of the subject. In some embodiments, the brain region is the hippocampus, somatomotor cortex, somatosensory cortex, piriform cortex, and / or entorhinal cortex. In some embodiments, the marker is detected in a bodily fluid of the subject. In some embodiments, the bodily fluid is blood or cerebrospinal fluid (CSF).

[0015] In some embodiments, the subject does not exhibit signs of dementia and / or cognitive impairment. In some embodiments, the subject has mild cognitive impairment or mild dementia.

[0016] In some embodiments, the subject is amyloid positive. In some embodiments, the subject is at risk for further Aβ accumulation. In some embodiments, the subject is an ApoE4 carrier. In some embodiments, the subject has intermediate levels of amyloid PET (e.g., 20-40 centiloids). In some embodiments, the subject has high levels of amyloid PET (e.g., >40 centiloids).

[0017] In some embodiments, the subject has been diagnosed with AD based on brain imaging, cognitive function, and / or biomarker criteria. In some embodiments, the subject has early AD. In some embodiments, the subject has pre-AD.

[0018]

[0018] One aspect of the present disclosure relates to a method for reducing or maintaining tau (e.g., reducing or maintaining tau levels relative to levels before treatment begins, or slowing tau accumulation, tau phosphorylation and / or tau diffusion, or slowing the rate of any of these) in a subject having or at risk of developing AD, the method comprising administering a therapeutically effective amount of lemborexant to the subject, the therapeutically effective amount being sufficient to reduce or maintain tau in the subject.

[0019] In some embodiments, the subject is amyloid-negative. In some embodiments, tau levels are reduced or maintained relative to a reference. In some embodiments, the method comprises reducing and / or slowing tau accumulation and / or tau diffusion and / or slowing the rate thereof compared to the reference. In some embodiments, the reference is a baseline measurement from the subject before treatment. In some embodiments, the reference is a baseline measurement from a control subject. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0020] In some embodiments, the method comprises altering tau in a brain region of the subject. In some embodiments, the method comprises altering tau PET signal in a brain region of the subject. In some embodiments, the brain region is the hippocampus, entorhinal cortex, and / or piriform cortex. In some embodiments, the method comprises reducing tau in a bodily fluid of the subject. In some embodiments, the bodily fluid is blood or CSF.

[0021] In some embodiments, the tau is total tau. In some embodiments, the tau is insoluble tau. In some embodiments, the tau is aggregated tau. In some embodiments, the tau is phosphorylated tau (phospho-tau). In some embodiments, the phospho-tau is phosphorylated at one or more of T181, T217, S202, S205, or T231.

[0022] In some embodiments, the method comprises altering the ratio of phospho-tau to total tau. In some embodiments, the ratio of phospho-tau to total tau is decreased compared to the ratio of CSF phospho-tau to total tau in the subject prior to administration of lemborexant. In some embodiments, the ratio of phospho-tau to total tau is maintained within 10% of the ratio of phospho-tau to total tau in the subject prior to administration of lemborexant. In some embodiments, the method comprises increasing the rate of dephosphorylation of phospho-tau. In some embodiments, the method comprises decreasing the rate of tau phosphorylation. In some embodiments, the method comprises reducing or maintaining tau within 48 hours of administering the first dose of lemborexant. In some embodiments, the method comprises reducing phospho-tau in the hippocampus, entorhinal cortex, and / or piriform cortex.

[0023]

[0023] Another aspect of the present disclosure relates to a method for altering neurodegeneration (e.g., reducing and / or delaying and / or slowing the rate of neurodegeneration) in a subject having or at risk of developing AD, the method comprising administering to the subject a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof or a solvate thereof, which therapeutically effective amount is sufficient to alter neurodegeneration in the subject.

[0024] In some embodiments, the subject is amyloid-negative. In some embodiments, altering neurodegeneration comprises reducing and / or delaying and / or slowing the rate of neurodegeneration compared to a reference. In some embodiments, neurodegeneration is altered relative to a reference. In some embodiments, the reference is a baseline measurement from the subject before treatment. In some embodiments, the reference is a baseline measurement from a control subject. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0025] In some embodiments, the neurodegeneration is characterized by a decrease in at least one of cortical thickness and hippocampal volume. In some embodiments, altering neurodegeneration comprises maintaining or slowing the decline of cortical thickness and / or hippocampal volume. In some embodiments, the neurodegeneration is characterized by a decrease in at least one of pyramidal neurons in the cortex, pyramidal neurons in the hippocampus, or granule cells in the hippocampus. In some embodiments, altering neurodegeneration comprises maintaining or slowing the decline of pyramidal neurons and / or granule cells. In some embodiments, altering neurodegeneration comprises decreasing the rate of neurodegeneration. In some embodiments, altering neurodegeneration comprises altering neurofilament light chain (NfL) levels. In some embodiments, the method comprises altering NfL levels in the subject's blood and / or CSF.

[0026]

[0026] A further aspect of the present disclosure relates to a method for altering Aβ plaques (e.g., reducing or delaying the formation of Aβ plaques or slowing their growth rate) in a subject having or at risk of developing AD, the method comprising administering to the subject a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, which therapeutically effective amount is sufficient to alter Aβ plaques in the subject.

[0027] In some embodiments, Aβ plaques are altered relative to a reference. In some embodiments, altering Aβ plaques comprises reducing and / or delaying and / or slowing the rate of Aβ plaque formation compared to the reference. In some embodiments, the reference is a baseline measurement from a subject before treatment. In some embodiments, the reference is a baseline measurement from a control subject. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0028] In some embodiments, the Aβ plaques are fibrous plaques. In some embodiments, the Aβ plaques are all plaques (e.g., including diffuse plaques). In some embodiments, altering the Aβ plaques comprises reducing Aβ plaque growth. In some embodiments, the method comprises reducing Aβ plaque growth in the subject's hippocampus, the subject's somatomotor cortex, somatosensory cortex, and / or piriform cortex.

[0029] In some embodiments, altering Aβ plaques comprises altering an amyloid PET signal obtained from a brain region of the subject. In some embodiments, altering Aβ plaques corresponds to a decrease in the concentration of Aβ in the subject's CSF.

[0030]

[0030] In some embodiments, the Aβ is Aβ 38, Aβ 40 and / or Aβ 42. In some embodiments, Aβ plaques are altered within 48 hours of administration of the first dose of lemborexant.

[0031] In some embodiments, the subject does not exhibit signs of dementia and / or cognitive impairment. In some embodiments, the subject has mild cognitive impairment or mild dementia.

[0032] In some embodiments, the subject is at risk for further Aβ accumulation. In some embodiments, the subject is an ApoE4 carrier. In some embodiments, the subject has an intermediate level of amyloid PET (e.g., 20-40 centiloids). In some embodiments, the subject has a high level of amyloid PET (e.g., >40 centiloids).

[0033] In some embodiments, the subject has early stage AD. In some embodiments, the subject has pre-AD.

[0034]

[0034] One aspect of the present disclosure relates to a method for modulating microglial responses in a subject having Alzheimer's disease (AD) or at risk of developing AD, the method comprising administering to the subject a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof or a solvate thereof, wherein the therapeutically effective amount is sufficient to modulate microglial responses in the subject.

[0035] In some embodiments, modulating the microglial response comprises modulating the expression of at least one microglial marker. In some embodiments, the microglial marker is a general microglial marker. In some embodiments, the general microglial marker is Iba1, Clec7a, or CD68. In some embodiments, the microglial marker is a homeostatic microglial marker. In some embodiments, the homeostatic microglial marker is TMEM119 or P2RY12. In some embodiments, modulating the microglial response comprises modulating the activity of phagocytic microglia.

[0036] In some embodiments, the subject has mild cognitive impairment or mild dementia. In some embodiments, the subject does not show signs of dementia and / or cognitive impairment.

[0037] In some embodiments, the subject is amyloid-negative. In some embodiments, the subject has tau pathology. In some embodiments, the subject has neurodegeneration in a brain region. In some embodiments, the brain region is the hippocampus, entorhinal cortex, and / or piriform cortex. In some embodiments, the brain region is the CA1 region, CA2 region, CA3 region, or dentate gyrus of the hippocampus.

[0038] In some embodiments, modulating the microglial response comprises modulating a response in microglia associated with degenerating neurons. In some embodiments, modulating the microglial response comprises decreasing the expression of at least one general microglial marker. In some embodiments, the general microglial marker is Iba1, CD68, or Clec7a. In some embodiments, modulating the microglial response comprises increasing the expression of at least one homeostatic microglial marker. In some embodiments, the homeostatic microglial marker is TMEM119 or P2RY12.

[0039] In some embodiments, the subject has Aβ plaques. In some embodiments, the Aβ plaques are fibrillar Aβ plaques. In some embodiments, the subject is at risk for further Aβ accumulation. In some embodiments, the subject is an ApoE4 carrier. In some embodiments, the subject has intermediate levels of amyloid PET (e.g., 20-40 centiloids). In some embodiments, the subject has high levels of amyloid PET (e.g., >40 centiloids).

[0040] In some embodiments, the subject has early stage AD. In some embodiments, the subject has pre-AD.

[0041] In some embodiments, the Aβ plaques are present in the hippocampus, somatomotor cortex, somatosensory cortex, and / or piriform cortex.

[0042] In some embodiments, modulating the microglial response comprises modulating a response in microglia associated with Aβ plaques. In some embodiments, modulating the microglial response comprises increasing expression of a general microglial marker. In some embodiments, the general microglial marker is Iba1, Clec7a, or CD68. In some embodiments, modulating the microglial response comprises increasing engulfment of Aβ plaques by phagocytic microglia. In some embodiments, modulating the microglial response comprises decreasing expression of a homeostatic microglial marker. In some embodiments, the homeostatic microglial marker is TMEM119 or P2RY12.

[0043] In some embodiments of any of the methods disclosed herein, the therapeutically effective amount of lemborexant administered to a subject ranges from 5 mg to 50 mg per day. In some embodiments, the therapeutically effective amount of lemborexant administered to a subject ranges from 10 mg to 30 mg per day.

[0044]

[0044] In some embodiments, the therapeutically effective amount of lemborexant administered to a subject is selected from 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg and 30 mg per day.

[0045] In some embodiments, the therapeutically effective amount of lemborexant administered to a subject is 20-25 mg per day. In some embodiments, a single 25 mg dose of lemborexant is administered to a subject once per day.

[0046] In some embodiments, lemborexant is administered at a first dose for a first period of time, at a second dose for a second period of time, and optionally at a third dose for a third period of time. In some embodiments, each of the first period of time, the second period of time, and the third period of time is one week. In some embodiments, the first dose is lower than the second dose, and optionally, the second dose is lower than the third dose.

[0047] In some embodiments, the first dose is 5 mg of lemborexant once daily, the second dose is 10 mg of lemborexant once daily, and optionally, the third dose is 20-25 mg of lemborexant once daily. In some embodiments, the first dose is 5 mg or 7.5 mg of lemborexant once daily, the second dose is 10 mg, 12.5 mg, 15 mg, or 17.5 mg of lemborexant once daily, and the third dose is 20 mg, 22.5 mg, 25 mg, 27.5 mg, or 30 mg of lemborexant once daily.

[0048] In some embodiments, the first dose is higher than the second dose, and optionally, the second dose is higher than the third dose. In some embodiments, the first dose is 20-25 mg of lemborexant once daily, the second dose is 10 mg of lemborexant once daily, and optionally, the third dose is 5 mg of lemborexant once daily. In some embodiments, the first dose is 20 mg, 22.5 mg, 25 mg, 27.5 mg, or 30 mg of lemborexant once daily, the second dose is 10 mg, 12.5 mg, 15 mg, or 17.5 mg of lemborexant once daily, and optionally, the third dose is 5 mg or 7.5 mg of lemborexant once daily.

[0049] In some embodiments of any of the methods disclosed herein, lemborexant is administered to the subject for at least 6 months. In some embodiments, the method comprises administering lemborexant to the subject for at least 9 months, at least 12 months, or at least 15 months. In some embodiments, the method comprises administering lemborexant to the subject for at least 18 months. In some embodiments, the method comprises administering lemborexant to the subject for at least 24 months, 30 months, or 36 months.

[0050]

[0050] Another aspect of the present disclosure relates to a method for selecting a subject having Alzheimer's disease (AD) or at risk of developing AD for treatment with lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, the method comprising: (a) obtaining from the subject at least one measure of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial response, and biomarker expression; (b) comparing the measure from the subject with a measure from a reference; and (c) if the measure from the subject differs from the measure from the reference, selecting the subject for treatment with lemborexant.

[0051] In some embodiments, the subject has mild cognitive impairment or mild dementia. In some embodiments, the subject does not show signs of dementia and / or cognitive impairment.

[0052] In some embodiments, the subject is at risk for Aβ accumulation. In some embodiments, the subject is an ApoE4 carrier. In some embodiments, the subject has an intermediate level of amyloid PET (e.g., 20-40 centiloids). In some embodiments, the subject has a high level of amyloid PET (e.g., >40 centiloids).

[0053] In some embodiments, the subject has early stage AD. In some embodiments, the subject has pre-AD.

[0054] In some embodiments, the subject has been diagnosed with AD based on brain imaging, cognitive function, and / or biomarker criteria. In some embodiments, obtaining at least one measure comprises obtaining data from a brain scan of the subject and / or obtaining data from a biological sample from the subject. In some embodiments, the data from the brain scan indicates levels of tau phosphorylation, tau aggregation, Aβ plaque burden, and / or microglial response. In some embodiments, the biological sample is a bodily fluid. In some embodiments, the bodily fluid is cerebrospinal fluid (CSF), blood, or saliva.

[0055] In some embodiments, the reference is a control. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0056] In some embodiments, the control does not have AD. In some embodiments, the measured value from the subject is higher than the measured value from the control without AD. In some embodiments, the measured value from the subject is lower than the measured value from the control without AD.

[0057] In some embodiments, the control has AD. In some embodiments, the measurements from the subject are equal to or greater than the measurements from the control with AD. In some embodiments, the measurements from the subject are equal to or less than the measurements from the control with AD.

[0058] In some embodiments, the measure of tau phosphorylation comprises a measure of phosphorylation at one or more of T181, T217, S202, S205, or T231. In some embodiments, the measure of tau aggregation comprises a measure of insoluble tau aggregates (e.g., neurofibrillary tangles (NFTs)).

[0059] In some embodiments, the measure of neurodegeneration comprises a measure of cortical thickness and / or hippocampal volume or a measure of pyramidal or granular neuron loss.

[0060] In some embodiments, the measurement of Aβ plaque burden comprises a measurement of Aβ plaque volume and / or Aβ plaque volume growth, hi some embodiments, the measurement of Aβ plaque burden comprises a measurement of amyloid PET signal in a brain region of the subject or a measurement of Aβ in the CSF of the subject.

[0061] In some embodiments, the measure of microglial response is a change in expression of at least one microglial marker. In some embodiments, the microglial marker is Iba1, Clec7a, CD68, TMEM119, or P2RY12. In some embodiments, the measure of microglial response is a measure of microglial phagocytosis.

[0062]

[0062] A further aspect of the present disclosure relates to a method for monitoring the effectiveness of a treatment in a subject having Alzheimer's disease (AD) or at risk of developing AD, the method comprising: (a) obtaining from the subject a first measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression; (b) administering to the subject a dose of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof; (c) obtaining from the subject a second measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression; and (d) comparing the second measurement from the subject with the first measurement from the subject, wherein a difference between the first measurement and the second measurement indicates effective treatment with lemborexant.

[0063]

[0063] Another aspect of the present disclosure relates to a method of treating a subject having Alzheimer's disease (AD) or at risk of developing AD, the method comprising: (a) obtaining from the subject a first measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial response, and biomarker expression; (b) administering to the subject a first dose of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof; (c) obtaining from the subject a second measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression; (d) comparing the second measurement from the subject with the first measurement from the subject, and (e) administering a second dose of lemborexant if the first measurement differs from the second measurement.

[0064] In some embodiments, obtaining at least one measure comprises obtaining data from a brain scan of the subject and / or obtaining data from a biological sample from the subject. In some embodiments, the data from the brain scan indicates levels of tau phosphorylation, tau aggregation, Aβ plaque burden, and / or microglial response. In some embodiments, the biological sample is a bodily fluid. In some embodiments, the bodily fluid is cerebrospinal fluid (CSF), blood, or saliva.

[0065] In some embodiments, the first measurement from the subject is higher than the second measurement from the subject. In some embodiments, the first measurement from the subject is lower than the second measurement from the subject.

[0066] In some embodiments, the measure of tau phosphorylation comprises a measure of phosphorylation of one or more of T181, T217, S202, S205, or T231. In some embodiments, the measure of tau aggregation comprises a measure of insoluble tau aggregates (e.g., neurofibrillary tangles (NFTs)).

[0067] In some embodiments, the measure of neurodegeneration comprises a measure of cortical thickness and / or hippocampal volume or a measure of pyramidal or granular neuron loss.

[0068] In some embodiments, the measure of Aβ plaque burden comprises a measure of Aβ plaque volume and / or Aβ plaque volume growth, hi some embodiments, the measure of Aβ plaque burden comprises a measure of amyloid PET signal in a brain region of the subject or a measure of Aβ in the CSF of the subject.

[0069] In some embodiments, the measure of microglial response is the degree of expression of at least one microglial marker. In some embodiments, the microglial marker is Iba1, Clec71, P2RY12, or TMEM119. In some embodiments, the measure of microglial response is a measure of microglial phagocytosis. In some embodiments, the measure of biomarker expression is a measure of Ifnb1, MMP2, and / or Bace1 expression.

[0070] In some embodiments, the subject is amyloid-negative. In some embodiments, the subject has Aβ plaques.

[0071] In some embodiments, the subject has mild cognitive impairment and / or mild dementia. In some embodiments, the subject does not show signs of dementia and / or cognitive impairment.

[0072] In some embodiments, the subject is at risk for further Aβ accumulation. In some embodiments, the subject is an ApoE4 carrier. In some embodiments, the subject has an intermediate level of amyloid PET (e.g., 20-40 centiloids). In some embodiments, the subject has a high level of amyloid PET (e.g., >40 centiloids).

[0073] In some embodiments, the subject has early AD. In some embodiments, the subject has pre-AD. [Brief explanation of the drawings]

[0074] [Figure 1] The study schema for the clinical trial is shown below. The study design, which assumes habitual bedtime at 10:00 PM, is outlined below. [Figure 2A] Figure 1 shows data from the study of Example 2. Figure 2 shows a schematic diagram of the study design. [Figure 2B] Data from the study in Example 2 are shown. Electroencephalography (EEG) analysis shows the percentage of time spent asleep. Non-REM sleep is shown in E4 (n=10 mice / treatment group) and P301S / E4 mice (n=8 mice / treatment group) treated with vehicle (Veh) or lemborexant (Lem). Two-way ANOVA with Tukey's post-hoc comparison. Data represent the mean ± SEM; *p<0.05, **p<0.001, ***p<0.0001. [Figure 2C] Data from the study in Example 2 are shown. Electroencephalography (EEG) analysis shows the percentage of time spent asleep. REM sleep is shown in E4 (n=10 mice / treatment group) and P301S / E4 mice (n=8 mice / treatment group) treated with vehicle (Veh) or lemborexant (Lem). Two-way ANOVA with Tukey's post-hoc comparison. Data represent the mean ± SEM; *p<0.05, **p<0.001, ***p<0.0001. [Figure 2D] Data from the study in Example 2 are shown. Electroencephalography (EEG) analysis shows the percentage of time spent asleep. Shown is wakefulness in E4 (n=10 mice / treatment group) and P301S / E4 mice (n=8 mice / treatment group) treated with vehicle (Veh) or lemborexant (Lem). Two-way ANOVA with Tukey's post-hoc comparison. Data represent the mean ± SEM; *p<0.05, **p<0.001, ***p<0.0001. [Figure 2E]1 shows data from the study in Example 2. Representative spectrograms of EEG and electromyography (EMG) analyses illustrating non-REM, REM, and wake patterns in vehicle-treated P301S / E4 are shown. [Figure 2F] 1 shows data from the study in Example 2. Lemborexant-treated P301S / E4 mice are shown. [Figure 2G] 1 shows data from the study in Example 2. A time course analysis of the percentage of sleep observed from the start of vehicle or lemborexant treatment through 24 hours post-gavage in E4 mice (n=10 mice / treatment group) is shown. Open and dark bars indicate light and dark periods, respectively. Data represent mean ± SEM; *p<0.05, **p<0.001, ***p<0.0001. [Figure 2H] Figure 1 shows data from the study in Example 2. Results are shown for P301S / E4 mice (n=8 mice / treatment group). White and dark bars indicate the light and dark periods, respectively. Data represent the mean ± SEM; *p<0.05, **p<0.001, ***p<0.0001. [Figure 3A] Figure 1 shows data from the study in Example 2. Representative images of AT8-stained phosphorylated tau at both serine 202 and threonine 205 are shown. The top panel shows the hippocampus, and the bottom panel shows the entorhinal and piriform cortices. Scale bar - 500 μm. [Figure 3B] Figure 1 shows data from the study in Example 2. Representative images of MC1 stained tau are shown. Scale bar - 500 μm. [Figure 3C] Figure 1 shows data from the study in Example 2. Quantification of the percentage of AT8-covered hippocampus (nE4 and nP301S / E4 = 16-17 mice per treatment group) is shown. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 3D]Data from the study in Example 2 are shown. The entorhinal cortex / piriform cortex (nE4 = 16-18 mice / treatment group; nP301S / E4 = 15-19 mice / treatment group) is shown. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001). [Figure 3E] Data from the study in Example 2 are shown. Percentages of MC1-stained hippocampi (nE4 = 18 mice / treatment group; nP301S / E4 = 16-17 mice / treatment group) are shown. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 3F] Data from the study in Example 2 are shown. Entorhinal cortex / piriform cortex (nE4 = 16-17 mice / treatment group; nP301S / E4 = 15-17 mice / treatment group) are shown. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001). [Figure 3G] Figure 1 shows data from the study in Example 2. Representative images of cresyl violet stained brains used for volumetric analysis are shown. Scale bar - 1 mm. [Figure 3H] Figure 1 shows data from the study in Example 2. Quantification of hippocampal volume is shown. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 3I] Figure 1 shows data from the study in Example 2. The volume of the piriform cortex is shown. Data represent the mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 3J] Data from the study in Example 2 are shown. Plasma neurofilament light chain (NfL) levels measured by SIMOA (nE4 and nP301S / E4 = 16-20 mice per treatment group) are shown. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4A]Figure 1 shows data from the study in Example 2. Representative images of IBA1 (green), CD68 (red), and DAPI (blue) co-stained microglia in the CA3 region of the hippocampus are shown. Scale bar - 50 μm. [Figure 4B] Figure 1 shows data from the study in Example 2. Representative images of TMEM119 (yellow) and DAPI (blue) stained microglia in the hippocampal CA3 region are shown. Scale bar - 50 μm. [Figure 4C] 1 shows data from the study of Example 2. Quantification of the percentage of IBA1 coverage is shown. nE4=15-19 mice / treatment group; nP301S / E4=16-19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4D] 1 shows data from the study in Example 2. Quantification of the percentage of CD68-coated CA3 is shown. nE4 = 15-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4E] Data from the study in Example 2 are shown. Representative images of Clec7a (red) and DAPI (blue) stained microglia are shown. Scale bar - 50 μm. [Figure 4F] 1 shows data from the study of Example 2. Quantification of percent TMEM119 coverage is shown. nE4 = 15-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4G] Data from the study in Example 2 are shown. Quantification of the percentage of Clec7a-covered CA3 is shown. nE4 = 15-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4H]Data from the study in Example 2 are shown. Representative images of ApoE (green) and GFAP (red) co-stained astrocytes are shown. Scale bar - 50 μm. [Figure 4I] 1 shows data from the study in Example 2. Quantification of the percentage of ApoE colocalized with GFAP is shown. nE4 = 15-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4J] Data from the study in Example 2 are shown. IBA1 in CA3 is shown. nE4 = 15-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4K] Data from the study in Example 2 are shown. Representative images of IBA1 (magenta) positive microglia co-stained with ApoE (green). Scale bar - 50 μm. [Figure 4L] 1 shows data from the study in Example 2. Percentage of Iba1-covered piriform / entorhinal cortex in lemborexant-treated mice compared to vehicle-treated control mice. nE4 = 15-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4M] 1 shows data from the study in Example 2. Percentage of Iba1-covered dentate gyrus in lemborexant-treated mice compared to vehicle-treated control mice. nE4 = 15-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4N]1 shows data from the study in Example 2. Percentage of TMEM119-covered piriform / entorhinal cortex in lemborexant-treated mice compared to vehicle-treated control mice. nE4 = 15-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4O] 1 shows data from the study in Example 2. Percentage of TMEM119-covered dentate gyrus in lemborexant-treated mice compared to vehicle-treated control mice. nE4 = 15-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4P] 1 shows data from the study in Example 2. Percentage of CD68-coated dentate gyrus in lemborexant-treated mice compared to vehicle-treated control mice is shown. nE4 = 15-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4Q] 1 shows data from the study in Example 2. Percentage of CD68-coated piriform / entorhinal cortex in lemborexant-treated mice compared to vehicle-treated control mice. nE4 = 15-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4R] 1 shows data from the study in Example 2. Representative images of the CA1 / CA2 region stained for GFAP are shown. [Figure 4S] Plots of the percentage of GFAP-covered CA1 / 2 are shown. nE4 = 15–19 mice / treatment group; nP301S / E4 = 16–19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4T] Plots of the dentate gyrus are shown. nE4 = 15–19 mice / treatment group; nP301S / E4 = 16–19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 4U] Plots of the piriform cortex / entorhinal cortex are shown. nE4 = 15–19 mice / treatment group; nP301S / E4 = 16–19 mice / treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 5A] Figure 1 shows data from the study in Example 2. Volcano plots comparing differentially regulated genes in vehicle- and lemborexant-treated P301S / E4 mice are shown. The cutoff value for significance is set at 2x LogFC. [Figure 5B] 1 shows data from the study in Example 2. GO term analysis of genes significantly altered in P301S / E4 mice treated with vehicle and lemborexant is shown. [Figure 5C] 1 shows data from the study in Example 2. A heatmap illustrating all genes differentially expressed in P301S / E4 vehicle and lemborexant that reached a significant Log10-adjusted p-value of <0.05 is shown. [Figure 5D] Data from the study in Example 2 are shown. Representative images of VGLUT1 and PSD95 stained synapses in CA3 are shown. Scale bar - 50 μm. [Figure 5E] Data from the study in Example 2 are shown. Representative images of VGLUT1 and PSD95 stained synapses in CA3 are shown. Scale bar - 50 μm. [Figure 5F] Data from the study in Example 2 are shown. Quantification of the percentage of VGLUT1 puncta in CA3 is shown. nE4 = 16-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; unpaired two-tailed t-test; *p<0.05, **p<0.005, ***p<0.0001. [Figure 5G] 1 shows data from the study in Example 2. Results are shown for the piriform cortex. nE4 = 16-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; unpaired two-tailed T-test; *p<0.05, **p<0.005, ***p<0.0001. [Figure 5H] Data from the study in Example 2 are shown. Quantification of the percentage of PSD95 puncta in CA3 is shown. nE4 = 16-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; unpaired two-tailed t-test; *p<0.05, **p<0.005, ***p<0.0001. [Figure 5I] 1 shows data from the study in Example 2. Results are shown for the piriform cortex. nE4 = 16-19 mice / treatment group; nP301S / E4 = 16-19 mice / treatment group. Data represent mean ± SEM; unpaired two-tailed T-test; *p<0.05, **p<0.005, ***p<0.0001. [Figure 6A] 1 shows data from the study in Example 2. Analysis of the time course of sleep percentage in E4 mice (nE4 = 7-9 mice / treatment group) is shown. White and dark bars indicate light and dark periods, respectively. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 6B] Data from the study in Example 2 are shown. Results are shown for P301S / E4 mice (nP301S / E4 = 8-10 mice per treatment group). White and dark bars indicate the light and dark periods, respectively. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 6C] Figure 1 shows data from the study in Example 2. The percentage of time spent sleeping during the light or dark phase in E4 and P301S / E4 mice (n=10 mice per genotype and treatment group) is shown. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 6D]Data from the study in Example 2 are shown. Sleep bout duration during the light or dark phase in E4 and P301S / E4 mice (n=10 mice per genotype and treatment group). s - seconds. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 6E] Data from the study in Example 2 are shown. The length of the wake period during the light or dark phase in E4 and P301S / E4 mice (n=10 mice per genotype and treatment group) is shown. s - seconds. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 7A] Data from the study in Example 2 are shown. Phosphorylated tau (pTau) was quantified in RAB fractions by ELISA (nE4 and nP301S / E4 = 18-20 mice per treatment group). Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001). [Figure 7B] Data from the study in Example 2 are shown. Phosphorylated tau (pTau) was quantified in RIPA fractions by ELISA (nE4 and nP301S / E4 = 18-20 mice per treatment group). Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001). [Figure 7C] Data from the study in Example 2 are shown. Phosphorylated tau (pTau) was quantified in the formic acid (FA) fraction by ELISA (nE4 and nP301S / E4 = 18-20 mice per treatment group). Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001). [Figure 7D] Data from the study in Example 2 are shown. Total tau (tTau) was quantified in the RAB fraction by ELISA (nE4 = 18-20 and nP301S / E4 = 18-20 mice per treatment group). Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001). [Figure 7E] Data from the study in Example 2 are shown. Total tau (tTau) was quantified in RIPA fractions by ELISA (nE4 = 18-20 and nP301S / E4 = 18-20 mice per treatment group). Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001). [Figure 7F] Data from the study in Example 2 are shown. Total tau (tTau) was quantified in the FA fraction by ELISA (nE4 = 18-20 and nP301S / E4 = 18-20 mice per treatment group). Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001). [Figure 7G] Data from the study in Example 2 are shown. The top panel illustrates a representative image of the hippocampus, including the granular layer, and the bottom panel shows the piriform cortex, including the pyramidal cell layer. Scale bar - 500 μm. [Figure 7H] Data from the study in Example 2 are shown. Volumetric analysis of cresyl violet-stained hippocampi is shown (nE4 and nP301S / E4 = 17-19 mice per treatment group). Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 7I] Data from the study in Example 2 are shown. Half of the brain with the ventricles removed is shown (nE4 and nP301S / E4 = 17-19 mice per treatment group). Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001). [Figure 7J] Data from the study in Example 2 are shown. The pyramidal cell layer of the piriform cortex is shown (nE4 and nP301S / E4 = 17-19 mice per treatment group). Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001). [Figure 7K]Data from the study in Example 2 are shown. The hippocampal granular layer is shown (nE4 and nP301S / E4 = 17-19 mice per treatment group). Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001). [Figure 8A] Data from the study in Example 2 are shown. Representative images of DAPI, IBA1, and P2RY12 co-stained microglia in CA3 are shown. Scale bar - 50 μm. [Figure 8B] 1 shows data from the study in Example 2. Quantified percentage of P2RY12-covered CA3 is shown. nE4 and nP301S / E4 = 16-19 mice per treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 8C] Figure 1 shows data from the study in Example 2. Results are shown for the dentate gyrus. nE4 and nP301S / E4 = 16-19 mice per treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. Percentage of P2RY12-covered dentate gyrus is shown. [Figure 8D] Data from the study in Example 2 are shown. Percentage of P2RY12-covered piriform cortex / entorhinal cortex is shown. nE4 and nP301S / E4 = 16-19 mice per treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey's post-hoc test; *p<0.05, **p<0.001, ***p<0.0001. [Figure 9A] 1 shows changes in sleep in APP / PS1dE9 mice administered doxepin or lemborexant from the study in Example 3. FIG. 1 is a schematic diagram of the experimental design. [Figure 9B] 1 shows changes in sleep in APP / PS1dE9 mice administered doxepin or lemborexant from the study of Example 3. 1 shows the effect on total sleep in mice treated with doxepin, lemborexant (10 mg or 30 mg), or vehicle. [Figure 9C]1 shows changes in sleep in APP / PS1dE9 mice administered doxepin or lemborexant from the study of Example 3. 1 shows the effects on light phase sleep in mice treated with doxepin, lemborexant (10 mg or 30 mg), or vehicle. [Figure 9D] 1 shows changes in sleep in APP / PS1dE9 mice administered doxepin or lemborexant from the study of Example 3. 1 shows the effects on dark-phase sleep in mice treated with doxepin, lemborexant (10 mg or 30 mg), or vehicle. [Figure 9E] 1 shows changes in sleep in APP / PS1dE9 mice administered doxepin or lemborexant from the study of Example 3. Percentage of sleep as a function of Zeitgeber time (ZT0 = lights on). [Figure 10A] 1 shows fibrillar amyloid plaque burden in APP / PS1dE9 mice administered doxepin or lemborexant from the study of Example 3. FIG. 1 is a schematic diagram of the timing of treatment of mice. [Figure 10B] 1 shows fibrillar amyloid plaque burden in APP / PS1dE9 mice treated with doxepin or lemborexant from the study in Example 3. Representative images of brain sections stained with X34 to label fibrillar amyloid plaques are shown. [Figure 10C]

[0023] Figure 1 shows fibrillar amyloid plaque burden in APP / PS1dE9 mice administered doxepin or lemborexant from the study in Example 3. Quantification of plaque burden (X34 stained area (%)) in different brain regions is shown. Error bars indicate mean ± SEM, and each dot represents a mouse. P values ​​from one-way ANOVA are shown. [Figure 11A] Figure 10A shows the total amyloid plaque burden in APP / PS1dE9 mice treated with doxepin or lemborexant, according to the study in Example 3. Representative brain sections stained for total amyloid plaque burden using the anti-Aβ antibody HJ3.4 are shown. [Figure 11B]Figure 10A shows the total amyloid plaque burden in APP / PS1dE9 mice treated with doxepin or lemborexant, as shown in Figure 10A. Quantification of plaque burden (HJ3.4 stained area (%)) in different brain regions. Error bars indicate mean ± SEM, and each dot represents a mouse. P values ​​from one-way ANOVA are shown. [Figure 12A] The study in Example 3 demonstrates APP processing / cleavage in APP / PS1dE9 mice treated with doxepin or lemborexant. Representative Western blot results for full-length APP and APP C-terminal fragments (CTF-α and -β) are shown. β-Tubulin is shown as a loading control. [Figure 12B]

[0023] Figure 1 shows APP processing / cleavage in APP / PS1dE9 mice treated with doxepin or lemborexant from the study in Example 3. Quantification of band intensity is shown. Error bars indicate mean ± SEM, and each dot represents a mouse. P values ​​from one-way ANOVA are shown. [Figure 13A] The study from Example 3 demonstrates clustering of microglia around plaques in APP / PS1dE9 mice treated with doxepin or lemborexant. Representative images of samples stained for plaques (X34) and microglia (Iba1) are shown. The volume of microglia around each plaque was calculated from a Z-stack of confocal images using Imaris software. [Figure 13B]

[0023] Figure 1 shows the clustering of microglia around plaques in APP / PS1dE9 mice treated with doxepin or lemborexant, according to the study in Example 3. Quantification of plaque volume (to demonstrate that similar sized plaques were quantified across conditions) and the volume of Iba1 around plaques are shown. Error bars represent the mean ± SEM, and each dot represents a mouse. P values ​​from one-way ANOVA are shown. [Figure 14A]Figure 1 shows the expression of periplaque microglial CD68 in APP / PS1dEP mice treated with doxepin or lemborexant from the study in Example 3. Representative images of samples stained for plaque (X34), microglia (Iba1), and phagosomes (CD68) are shown. [Figure 14B] Figure 1 shows the expression of periplaque microglial CD68 in APP / PS1dEP mice treated with doxepin or lemborexant from the study in Example 3. Iba1-colocalized CD68 was quantified around each plaque using Imaris software. Error bars indicate the mean ± SEM, and each dot represents an average of 8-10 plaques from one mouse. P values ​​from one-way ANOVA are shown. [Figure 14C]

[0023] Figure 1 shows the expression of peri-plaque microglial CD68 in APP / PS1dEP mice treated with doxepin or lemborexant from the study in Example 3. The volume of Iba1 (μm3) is shown. [Figure 14D] Figure 1 shows the expression of periplaque microglial CD68 in APP / PS1dEP mice treated with doxepin or lemborexant from the study in Example 3. Figure 2 shows the co-localization of Iba1 with CD68, measured as a percentage of Iba1. [Figure 14E] 1 shows the expression of periplaque microglial CD68 in APP / PS1dEP mice treated with doxepin or lemborexant from the study in Example 3. The volume of co-localized Iba1 and CD68 is shown. [Figure 15] The study in Example 3 demonstrates the effect of lemborexant treatment on gene expression. Following lemborexant treatment, the expression of transcripts encoding Ifnb1, Rab5a, and Mmp2 showed significant differences. Data are presented as fold changes (relative to the mean of VEH). Error bars represent the mean ± SEM, and each dot represents one mouse. P values ​​from one-way ANOVA are shown. [Figure 16A]

[0023] Figure 1 shows microglial phagocytosis of amyloid plaques in APP / PS1dEP mice treated with lemborexant, according to the study in Example 3.

[0024] Figure 1 shows a schematic diagram of the experimental design. [Figure 16B] 1 shows microglial phagocytosis of amyloid plaques in APP / PS1dEP mice treated with lemborexant, according to the study in Example 3. Flow cytometry gating strategy is shown. [Figure 16C] The study in Example 3 demonstrates microglial phagocytosis of amyloid plaques in APP / PS1dEP mice treated with lemborexant. The study demonstrates methoxy-X04 (MX04) positivity in a CD45 low CD11b + population isolated as potential microglia. [Figure 16D] The study from Example 3 shows microglial phagocytosis of amyloid plaques in APP / PS1dEP mice treated with lemborexant. Quantification of the percentage of MX04+ microglia by two-tailed T-test (p=0.0207). [Figure 17A] 1 shows the growth of amyloid plaques in mice with pre-existing plaques from the study of Example 3. FIG. 1 is a schematic diagram of the experimental design. [Figure 17B] The study from Example 3 shows the growth of amyloid plaques in mice with pre-existing plaques. Representative images of MX04, thiazine red, and overlay images are shown. P values ​​from one-way ANOVA are shown. [Figure 17C] The study in Example 3 demonstrates the development of amyloid plaques in mice with pre-existing plaques. Representative images of amyloid plaques labeled with X34, microglia labeled with IBA1, and microglial phagosomes labeled with CD68 are shown. [Figure 17D]The study from Example 3 shows amyloid plaque growth in mice with pre-existing plaques. The percentage increase in plaque volume in mice treated with VEH and lemborexant is shown. P values ​​are from a Mann-Whitney U test due to non-Gaussian distribution of the data. Graphs show mean ± SEM, with each dot representing one mouse. [Figure 17E] The study from Example 3 shows the growth of amyloid plaques in mice with existing plaques. Quantification of co-localized IBA1-CD68, shown as a percentage of total IBA1 (total microglia) area, is shown. P values ​​are from one-way ANOVA. The graph shows the mean ± SEM, and each dot represents one mouse. [Figure 17F] Figure 1 shows amyloid plaque growth in mice with pre-existing plaques from the study in Example 3. The percentage increase in plaque volume in mice treated with lemborexant, doxepin, or vehicle control is shown. Graph shows mean ± SEM, with each dot representing one mouse. [Figure 17G] The study from Example 3 shows the growth of amyloid plaques in mice with pre-existing plaques. Quantification of co-localized IBA1-CD68, shown as a percentage of the area of ​​total IBA1 (total microglia), is shown. The graph shows the mean ± SEM, and each dot represents one mouse. [Figure 18A] The study from Example 4 demonstrates the effect of lemborexant or doxepin on rhythmic activity patterns in arrhythmic Bmal1 KO mice. Representative actograms are shown. LD = 12h: 12h light / dark; DD = constant dark. [Figure 18B] The study from Example 4 demonstrates the effect of lemborexant or doxepin on rhythmic activity patterns in arrhythmic Bmal1 KO mice. Representative actograms are shown. LD = 12h: 12h light / dark; DD = constant dark. [Figure 18C]The study from Example 4 demonstrates the effect of lemborexant or doxepin on rhythmic activity patterns in arrhythmogenic Bmal1 KO mice. Quantification of circadian locomotor behavior in different parts of the experiment is shown (LD is indicated by the shaded area, DD+LEM is the area of ​​the shaded arrow, and DD is the remainder of the recording). Data analyzed by two-way ANOVA with Tukey's post-hoc test. DETAILED DESCRIPTION OF THE INVENTION

[0075] [Detailed explanation] [I. Definition]

[0092] The following are definitions of terms used in this application.

[0076]

[0093] As used herein, the singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0077]

[0094] As used herein, the phrase "and / or" means "either or both" of the elements so conjoined, i.e., elements that are present conjointly in some cases and separately in other cases. Thus, as a non-limiting example, "A and / or B," when used in combination with open-ended language such as "comprising," can, in some embodiments, refer to A only (optionally including elements other than B); in other embodiments, it can refer to B only (optionally including elements other than A); in still other embodiments, it can refer to both A and B (optionally including other elements), etc.

[0078]

[0095] As used herein, "at least one" means one or more of the elements in a list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements, nor does it exclude combinations of elements in the list of elements. This definition also allows that, optionally, elements other than those specifically identified in the list of elements to which the phrase "at least one" refers may be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B" or equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, optionally two or more, including A (optionally including elements other than B) but not including B; in another embodiment, to at least one, optionally two or more, including B (optionally including elements other than A) but not including A; in yet another embodiment, to at least one, optionally two or more, including A (optionally including other elements), and at least one, optionally two or more, including B (optionally including other elements), etc.

[0079]

[0096] When a numerical value is listed alone or as part of a numerical range, it is understood that the numerical value can vary above and below the stated value with a variance of 10% of the stated value.

[0080]

[0097] When a range of values ​​is recited herein, the range of values ​​is intended to encompass each value and subrange within that range. For example, "15 mg to 30 mg" includes, for example, 15.0 mg, 15.5 mg, 16.0 mg, 16.5 mg, 17.0 mg, 17.5 mg, 18.0 mg, 18.5 mg, 19.0 mg, 19.5 mg, 20.0 mg, 20.5 mg, 21.0 mg, 21.5 mg, 22.0 mg, 22.5 mg, 23.0 mg, 23.5 mg, 24.0 mg, 25.0 mg, 26.0 mg, 27.0 mg, 28.0 mg, 29.0 mg, 30.0 mg, 31.0 mg, 32.0 mg, 33.0 mg, 34.0 mg, 35.0 mg, 36.0 mg, 37.0 mg, 38.0 mg, 39.0 mg, 40.0 mg, 41.0 mg, 42.0 mg, 43.0 mg, 44.0 mg, 45.0 mg, 46.0 mg, 47.0 mg, 48.0 mg, 49.0 mg, 50.0 mg, 51.0 mg, 52.0 mg, 53.0 mg, 54.0 mg, 55.0 mg, 56.0 mg, 57.0 mg, 58.0 mg, 59.0 mg, 60.0 mg, 61.0 mg, 62.0 mg, 63.0 mg, 64.0 mg, 65.0 mg, 66.0 mg, 67.0 mg, 68.0 mg, 69.0 mg, 70.0 mg, 71.0 mg, 72 It is intended to encompass 4.5 mg, 25.0 mg, 25.5 mg, 26.0 mg, 26.5 mg, 27.0 mg, 27.5 mg, 28.0 mg, 28.5 mg, 29.0 mg, 29.5 mg, 30.0 mg, 15 mg to 15.5 mg, 15 mg to 16 mg, 15 mg to 17.5 mg, 17.5 mg to 21 mg, and 15 mg to 28 mg, etc.

[0081]

[0098] "Amyloid" refers to protein aggregates that form fibrillar morphologies. Amyloids are often formed from long, unbranched fibrils characterized by an extended beta-sheet secondary structure, approximately 7-13 nm wide and several micrometers long. Amyloids are typically found extracellularly and in vivo; in addition, fibrils bind the dye Congo red and exhibit green birefringence when viewed between crossed polarizers. Amyloid-forming proteins have been identified in association with serious diseases, including amyloid-beta peptide (Aβ), associated with Alzheimer's disease (AD), islet amyloid polypeptide (IAPP), associated with type 2 diabetes, and prion protein (PrP), associated with spongiform encephalopathies. As used herein, "amyloid," "amyloid-beta," "cerebral amyloid," and "amyloid-beta peptide (Aβ)" are used interchangeably.

[0082]

[0099] Amyloid β1-42 (Aβ42) refers to the amyloid β monomer consisting of amino acids 1-42 of the full-length protein (Table 5, SEQ ID NO: 13). Amyloid β1-40 (Aβ1-40) refers to the amyloid β monomer consisting of amino acids 1-40 of the full-length protein (Table 5, SEQ ID NO: 14).

[0083]

[0100] Amyloid levels from amyloid PET can be reported in "centiloid" units (CL) using the centiloid method (Klunk WE et al. The Centiloid Project: standardizing quantitative amyloid plaque estimation by PET. Alzheimer's Dement. 2015;11:1-15 e1-4). The centiloid method measures the tracer on a scale of 0 CL to 100 CL, with 0 considered the reference point and representing the average for young healthy controls, and 100 CL representing the average amyloid burden present in subjects with mild to moderate dementia due to AD (ibid.). As known to those skilled in the art, the centiloid threshold is variable and can be adjusted, for example, based on new or additional scientific information (see, e.g., http: / / www.gaain.org / centiloid-project). High levels of amyloid can be set relative to a baseline threshold for healthy controls, determined according to methods known to those skilled in the art.

[0084]

[0101] As used herein, whether a subject is "amyloid positive" or "amyloid negative" can be determined based on whether the subject has a positive amyloid load. In some embodiments, a subject is determined to be amyloid positive or amyloid negative as indicated by longitudinal positron emission tomography (PET) assessment of brain uptake of an amyloid imaging agent. In some embodiments, a subject is "amyloid negative" if their florbetapir amyloid PET SUVr negativity is less than 1.17. In some embodiments, a subject is determined to be amyloid positive or amyloid negative by CSF assessment of the presence of amyloid pathology using assessment of markers such as Aβ1-42 (e.g., soluble CSF biomarker analysis), alone or in combination with another method, such as PET measurement of brain amyloid. Methods for measuring Aβ38, Aβ40, and Aβ42 are known in the art, such as assays using LC-MS / MS. Methods may include the PrecivityAD™ assay (see, e.g., Kirmess et al., J. Clinica Chimica Acta 519:267-275 (2021)) and the Sysmex assay (https: / / www.eisai.com / news / 2019 / news201990.html) for measuring Aβ42 and Aβ40 in blood or plasma samples or CSF samples. In some embodiments, a qualitative visual reading of PET scans can be used to determine amyloid positivity and amyloid negativity by classifying subjects as having either "normal" or "abnormal" uptake based on PET image patterns. Readers are trained and certified to recognize brain PET images with abnormal or normal uptake patterns, or amyloid detection is performed by a semi-quantitative or quantitative approach. In some embodiments, a threshold is set to quantitatively determine whether Aβ brain burden from biomarkers (e.g., serum or CSF) and / or PET scans indicates that a subject is amyloid positive or amyloid negative. In some embodiments, the subject is determined to be amyloid positive or amyloid negative by MRI.In some embodiments, the whole brain or at least one region of the brain (e.g., cortical gray matter (i.e., cortex), lateral ventricles, frontal lobe, parietal lobe, temporal lobe, occipital lobe, cingulate cortex, amygdala, piriform cortex, entorhinal cortex, hippocampus, hippocampal CA3 (pyramidal neurons), and / or hippocampal dentate gyrus (granule cell neurons)) is analyzed by MRI.

[0085]

[0102] In some embodiments, a subject is determined to be amyloid positive or amyloid negative by retinal amyloid accumulation, hi some embodiments, a subject is determined to be amyloid positive or amyloid negative by behavioral / cognitive phenotype.

[0086]

[0103] The term "tau protein" or "tau" encompasses all tau isoforms, whether full-length, truncated, or post-translationally modified. In many animals, including but not limited to humans, non-human primates, rodents, fish, cows, frogs, goats, and chickens, tau is encoded by the gene MAPT. In humans, there are six isoforms of tau generated by alternative splicing of exons 2, 3, and 10 of MAPT. These isoforms range in length from 352 to 441 amino acids. Exons 2 and 3 each encode a 29-amino acid insert at the N-terminus (referred to as N), and full-length human tau isoforms can have both inserts (2N), one insert (1N), or no insert (0N). All full-length human tau isoforms also contain three repeats of the microtubule-binding domain (referred to as R). Inclusion of exon 10 at the C-terminus results in the inclusion of a fourth microtubule-binding domain encoded by exon 10. Thus, a full-length human tau isoform can consist of four repeats (4R) of the microtubule-binding domain (exon 10 included) or three repeats (3R) of the microtubule-binding domain (exon 10 not included). Human tau may or may not be post-translationally modified. For example, it is known in the art that tau can be phosphorylated, ubiquitinated, glycosylated, and glycosylated. Thus, the term "human tau" encompasses the (2N,3R), (2N,4R), (1N,3R), (1N,4R), (0N,3R), and (0N,4R) isoforms, isoforms that are N- and / or C-terminal truncated species, and all post-translationally modified isoforms. Alternative splicing of the gene encoding tau occurs in other animals as well. In animals where the gene has not been identified as MAPT, homologs can be identified by methods well known in the art.

[0087]

[0104] Phosphorylation of specific amino acids (i.e., "sites" or "residues") in tau results in phosphorylated tau (p-tau) isoforms. Phosphorylation can occur at different residues, such as T111, S113, T181, S199, S202, S208, T153, T175, T205, S214, T217, and T231.

[0088]

[0105] The term "p-tau" encompasses all phosphorylated tau (p-tau) isoforms, including, but not limited to, p-tau181, p-tau217, and p-tau231.

[0089]

[0106] Diseases associated with tau deposition in the brain are sometimes referred to as "tauopathies" or "tau pathology." A clinical sign of a tauopathy can be tau aggregates in the brain, including, but not limited to, neurofibrillary tangles. Other methods can be used to detect or measure tau phosphorylation at one or more amino acid residues, and optionally total tau, in a subject. For example, tau can be purified from blood or cerebrospinal fluid (CSF) obtained from a subject. CSF can be obtained by lumbar puncture.

[0090]

[0107] Methods for measuring tau phosphorylation include high-resolution mass spectrometry. Suitable types of mass spectrometers are known in the art. These include, but are not limited to, quadrupole mass spectrometers, time-of-flight mass spectrometers, ion trap mass spectrometers, and Orbitrap mass spectrometers, as well as hybrid mass spectrometers that combine different types of mass spectrometers into one architecture (e.g., Orbitrap Fusion™ Tribrid™ Mass Spectrometer from ThermoFisher Scientific). Other methods for measuring p-tau (phospho-tau) and t-tau (total tau) are known in the art, such as assays using liquid chromatography with tandem mass spectrometry (LC-MS-MS). Measurements of p-tau and t-tau can also be determined by positron emission tomography (PET) using radioactive tracers. The whole brain or at least one region of the brain (cortical gray matter (ie, cortex), frontal lobe, parietal lobe, temporal lobe, occipital lobe, cingulate cortex, amygdala, piriform cortex, entorhinal cortex, hippocampus) can be analyzed by PET.

[0091]

[0108] As used herein, "compared to placebo" refers to a comparison of a biomarker (e.g., p-tau, Aβ, etc.) between a subject receiving lemborexant and the same biomarker in another subject receiving a placebo (a substance with no therapeutic effect).

[0092]

[0109] As used herein, "compared to baseline" refers to a comparison of a biomarker (e.g., p-tau, Aβ) between a subject receiving lemborexant and the same biomarker in the same subject prior to treatment with lemborexant.

[0093]

[0110] As used herein, "maintenance" refers to a subject having or retaining the same level or approximately the same amount of a biomarker (e.g., p-tau, Aβ) in the subject's sample (e.g., CSF, blood) between two time points (one before administration of lemborexant and one after administration of lemborexant).

[0094]

[0111] As used herein, "MMSE" refers to the Mini-Mental State Examination, a cognitive performance instrument commonly used for screening purposes, but also often measured longitudinally in AD clinical trials, with a 30-point scale in which higher scores indicate less severe impairment and lower scores indicate more severe impairment. As used herein, seven items measuring orientation to time and place, memorization, recall, attention, language, and drawing were assessed. (Folstein, M.F. et al., "Mini-mental state. A practical method for grading the cognitive state of subjects for the clinician." J. Psychiatr. Res. 1975;12:189-98.)

[0095]

[0112] As used herein, "PSQI" refers to the Pittsburgh Sleep Quality Index, a self-assessment questionnaire assessing sleep quality and disturbances over a one-month time interval. Nineteen individual items generate seven "component" scores: subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleep medications, and daytime dysfunction. These seven component scores are summed to generate a global score ranging from 0 to 21, with lower scores indicating healthier sleep quality. Clinical and clinimetric characteristics of the PSQI were assessed over an 18-month period in "good" sleepers (healthy subjects, n = 52) and "poor" sleepers (depressed patients, n = 54; sleep-disordered patients, n = 62). (Buysse DJ et al., "The Pittsburgh Sleep Quality Index: a new instrument for psychiatric practice and research." Psychiatry Res. 1989;28(2):193-213.)

[0096]

[0113] As used herein, "STOP-Bang" refers to the STOP-Bang questionnaire, which assesses snoring, fatigue, apnea indications, hypertension, BMI, age, neck circumference, and men. The questionnaire consists of eight dichotomous (yes / no) items related to clinical features of sleep apnea syndrome. The total score ranges from 0 to 8. Patients can be classified as to their risk of obstructive sleep apnea (OSA) based on their respective scores. The sensitivity of a STOP-Bang score of ≥ 3 for detecting moderate to severe OSA (apnea-hypopnea index [AHI] > 15) and severe OSA (AHI > 30) is 93% and 100%, respectively. (Chung F. et al., "STOP-Bang Questionnaire: A Practical Approach to Screen for Obstructive Sleep Apnea," Chest. 2016, 149(3):631-638).

[0097]

[0114] As used herein, "PSG" refers to polysomnography, a standard diagnostic test for OSA. PSG provides an assessment of OSA as the frequency of apneas and hypopneas per hour of sleep (the apnea-hypopnea index or AHI). The severity of OSA is classified as follows: (a) none / minimal: AHI<5 / hour; (b) mild: AHI≥5 but <15 / hour; (c) moderate: AHI≥15 but <30 / hour; and (d) severe: AHI≥30 / hour (Alshaer H et al. "Reproducibility and predictors of the apnea-hypopnea index across multiple nights" Sleep Sci. 2018, 11(1):28-33).

[0098]

[0115] A subject with "preclinical AD" or "pre-AD" as described herein is a cognitively normal (e.g., unimpaired) individual with intermediate or high levels of amyloid in the brain. At least two clinical pre-AD states in which a subject does not have cognitive impairment are defined by the IWG criteria: presymptomatic AD and asymptomatic AD (or "asymptomatic at-risk"). Presymptomatic AD refers to a subject with an autosomal dominant single-gene mutation for AD, such as a mutation in amyloid precursor protein (APP), presenilin 1 (PSEN1), or presenilin 2 (PSEN2), and who does not have cognitive impairment. Asymptomatic subjects with autosomal dominant single-gene mutations who do not show symptoms are most likely to develop AD. Asymptomatic refers to a subject who does not have clinical signs and symptoms of AD, but who has one or more biomarkers of AD pathology. The asymptomatic at-risk stage can be further classified. Impairments in episodic memory and executive function may appear later. Thus, a pre-AD subject can be identified by the asymptomatic stage, which lacks cognitive impairment. Cognitively normal individuals may include individuals with a CDR of 0 or cognitive test scores within the normal range (e.g., MMSE, International Shopping List Task, Logical Memory, etc.). Preclinical AD occurs before significant, irreversible neurodegeneration and cognitive impairment and is typically characterized by the appearance of in vivo molecular biomarkers of AD and the absence of clinical symptoms. Preclinical AD biomarkers that may indicate future development of Alzheimer's disease include, but are not limited to, intermediate or elevated levels of amyloid in the brain (e.g., about 20-40 centimeters, e.g., about 20-32 centimeters) as measured by amyloid or tau positron emission tomography (PET).Additional biomarkers include the level of Aβ1-42 and / or the Aβ1-42 / 1-40 ratio in cerebrospinal fluid, the level of total tau in cerebrospinal fluid, the level of neurogranin in cerebrospinal fluid, the level of neurofilament light chain peptide (NfL) in cerebrospinal fluid, and biomarkers measured in serum or plasma (e.g., the level of Aβ1-42, the ratio of two forms of amyloid-b peptide (e.g., about 0.092-0.094 or about 0.0 These include one or more of the following: Aβ1-42 / 1-40 ratio less than 92; plasma total tau (T-tau) levels; phosphorylated tau (P-tau) isoform levels (including tau phosphorylated at 181 (P-tau181), 217 (P-tau217), and 231 (P-tau231)); glial fibrillary acidic protein (GFAP); and neurofilament light chain (NfL), which may be used alone or in combination. Additionally, certain risk factors contribute to the development of AD. For example, subjects with the ε4 allele of apolipoprotein E (APOE) are also at increased risk of developing AD, and subjects with trisomy 21 involving the APP gene are at increased risk of cerebral amyloidosis. Other risk factors associated with AD include, but are not limited to, having a family history of first-degree relatives with AD or dementia, being 65 years of age or older, being female, having or having recovered from a traumatic brain injury, suffering from other conditions such as obesity, diabetes, heart disease and / or vascular disease, cancer, and / or immune system dysfunction, and / or a sleep disorder such as insomnia or a circadian rhythm sleep disorder, or having lifestyle risk factors such as smoking, alcohol consumption, physical inactivity, lack of cognitive activity, and malnutrition, and being exposed to environmental risk factors such as air pollution, metals (e.g., aluminum, copper, zinc, etc.).

[0099]

[0116] As used herein, "early AD" or "early Alzheimer's disease" (EAD) is a continuum of AD severity ranging from mild cognitive impairment due to AD - moderate likelihood to mild Alzheimer's dementia. Subjects with early AD include subjects with mild Alzheimer's dementia as defined herein and subjects with mild cognitive impairment (MCI) - moderate likelihood due to AD as defined herein. In some embodiments, subjects with early AD have an MMSE score of 22-30 and a global range of Clinical Dementia Rating (CDR) of 0.5-1.0.

[0100]

[0117] Other methods for detecting early AD disease can use the tests and assays specified below. Such tests and assays include the National Institute of Aging-Alzheimer's Association (NIA-AA) Core Clinical Criteria for Probable Alzheimer's Dementia in McKhann, GM et al., "The Diagnosis of Dementia Due to Alzheimer's Disease: Recommendations from the National Institute on Aging-Alzheimer's Association Workgroups on Diagnostic Guidelines for Alzheimer's Disease." Alzheimer Dement. 2011;7:263-9. Other methods include the CDR-SB, ADCOMS Composite Clinical Score, Mini-Mental State Examination, ADAS-Cog, ADAS MCI-ADL, modified iADRS, Wechsler Memory Scale-IV Logical Memory (Subscale) I (WMS-IV LMI), and Wechsler Memory Scale-IV Logical Memory (Subscale) II (WMS-IV LMII). In some embodiments, subjects with early-stage AD have evidence of high amyloid or positive amyloid burden in the brain. In some embodiments, high amyloid or positive amyloid burden in the brain is indicated and / or confirmed by PET assessment. In some embodiments, high amyloid or positive amyloid burden in the brain is indicated and / or confirmed by CSF assessment of markers such as Aβ1-42 (e.g., soluble CSF biomarker analysis). For example, subjects with AD can be selected according to the methods of WO 2023 / 283650, the contents of which are incorporated herein by reference.In some embodiments, a diagnostic threshold can be identified by amyloid PET either visually (by labeling of an approved PET tracer) or by establishing a centiloid threshold (e.g., varying between 15 and 40 centiloids) at which a subject is considered to have high amyloid.

[0101]

[0118] In some embodiments, high amyloid or positive amyloid burden in the brain is indicated and / or confirmed by measuring Aβ42 and Aβ40 concentrations and calculating the ratio of Aβ42 to Aβ40 (Aβ42 / 40 ratio). In some embodiments, high amyloid or positive amyloid burden in the brain is indicated and / or confirmed by MRI or PET. In some embodiments, high amyloid or positive amyloid burden in the brain is indicated by amyloid accumulation in the retina. In some embodiments, more than one assessment method is used. As used herein, a subject with "mild Alzheimer's dementia" is a subject who meets the NIA-AA core clinical criteria for possible Alzheimer's dementia in McKhann, GM et al., "The diagnosis of dementia due to Alzheimer's disease: Recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease." Alzheimer Dement. 2011;7:263-9. Also included herein are subjects with a CDR score of 0.5 to 1.0 and a Memory Box score of 0.5 or greater at screening and baseline.

[0102]

[0119] In some embodiments, the subject has "high amyloid" or "intermediate amyloid." Those skilled in the art will understand that amyloid levels by amyloid PET can be reported in "centiloid" units (CL) using the centiloid method (Klunk WE et al. The Centiloid Project: standardizing quantitative amyloid plaque estimation by PET. Alzheimer's Dement. 2015;11:1-15 el-4). The centiloid method measures tracer on a scale of 0 CL to 100 CL, where 0 is considered the reference point and represents the average in young, healthy controls, and 100 CL represents the average amyloid burden present in subjects with mild to moderate dementia due to AD (ibid.). As known to those skilled in the art, the centiloid threshold can vary and can be refined, for example, based on new or additional scientific information (see, e.g., http: / / www.gaain.org / centiloid-project.). High levels of amyloid can be set relative to a baseline threshold for healthy controls determined according to methods known to POSA. For example, a centimeter value of 32.5 can be used as the threshold for "high amyloid," and "intermediate amyloid" levels refer to Aβ amyloid PET in the range of 20-32.5 CL (e.g., 30 CL). In another example, a centimeter value of 40 can be used as the threshold for "high amyloid," and "intermediate amyloid" levels refer to Aβ amyloid PET in the range of 20-40 CL.

[0103]

[0120] As used herein, a subject with "moderate likelihood of MCI due to Alzheimer's disease" is a subject who has been confirmed as such according to the NIA-AA core clinical criteria for mild cognitive impairment due to Alzheimer's disease - moderate likelihood. For example, AD subjects who have symptoms but are not dementia due to evidence of brain amyloid pathology are less heterogeneous in cognitive decline as measured by the ADCOMS Composite Clinical Score defined herein, and are more similar to subjects with mild Alzheimer's dementia. Also included herein are subjects with a CDR score of 0.5 and a Memory Box score of 0.5 or higher at screening and baseline. Additionally, herein, subjects who report a history of subjective memory decline that has begun gradually and progresses slowly, as corroborated by informants, starting one year before screening.

[0104]

[0121] As used herein, "ADAS-cog" refers to the Alzheimer's Disease Assessment Scale-Cognitive. The ADAS-cog is a cognitive scale widely used in Alzheimer's disease testing, with structured scales assessing memory (word recall, delayed word recall, and word recognition), reasoning (following commands), language (reading aloud, comprehension), orientation, conceptual practice (writing letters on an envelope), and constructive practice (copying geometric designs) (Rosen, W. Get et al., "A new rating scale for Alzheimer's disease." Am. J. Psychiatry 1984;141:1356-64). Assessments of spoken language, verbal comprehension, word-finding difficulties, ability to remember test instructions, mazes, and crossing out target digits from a random number table were also obtained. The modified version used herein is scored from 0 to 90, with a score of 0 indicating no impairment and a score of 90 indicating maximal impairment.

[0105]

[0122] As used herein, "CDR-SB" refers to the Clinical Dementia Rating Scale (Sum of Boxes). The CDR is a clinical scale that represents five levels of impairment in performance in each of six functional categories, including memory, orientation, judgment and problem-solving, community problems, home and hobbies, and personal care (Berg, L. et al., "Mild senile dementia of the Alzheimer type 2: Longitudinal assessment," Ann. Neurol. 1988;23:477-84). The ratings of the degree of impairment obtained in each of the six categories of function are combined into a single global assessment (ranging from 0 to 3) of the Dementia CDR score. The Sum of Boxes score provides an additional measure of change; each category has a maximum possible score of 3, and the total score is the sum of the category scores, resulting in a possible total score of 0 to 18, with higher scores indicating more severe impairment. The global score can be used as a clinical measure of dementia severity.

[0106]

[0123] As used herein, "ADCOMS" refers to the Alzheimer's Disease Composite Score, a composite clinical score based on analysis of four ADAS-Cog items (delayed word retrieval, orientation, word recognition, and word-finding difficulties), two MMSE items (orientation to time and drawing), and all six CDR-SB items (personal care, community problems, home and hobbies, memory, orientation, and judgment and problem solving), as described in the Examples and Wang, J. et al., "ADCOMS: a composite clinical outcome for prodromal Alzheimer's disease trials," J. Neurol. Neurosurg. Psychiatry. 2016;87:993-999. ADCOMS was developed to be particularly sensitive to disease progression in the early stages of AD, i.e., prodromal and mild AD.

[0107]

[0124] As used herein, "ApoE4-positive" subject and "ApoE4 carrier" refer to a subject who has the ε4 variant of the apolipoprotein E gene. The ε4 variant is one of several major alleles of the apolipoprotein E gene. This gene is generally involved in fat metabolism. Apolipoprotein E ε4 carriers have been found to exhibit significantly higher amyloid burden compared to non-carriers (Drzezga, A. et al., "Effect of APOE genotype on amyloid plaque load and gray matter volume in Alzheimer's disease." Neurology. 2009;72:1487-94.). In some embodiments, the subject is a heterozygous carrier of an allele of the apolipoprotein E ε4 gene. In some embodiments, the subject is a homozygous carrier of an allele of the apolipoprotein E ε4 gene.

[0108]

[0125] As used herein, the term "clinical deterioration" refers to a worsening of one or more clinical symptoms of AD. Methods for measuring clinical deterioration can use the tests and assays specified herein. In some embodiments, clinical deterioration is determined by worsening of ADCOMS. In some embodiments, clinical deterioration is determined by worsening of MMSE. In some embodiments, clinical deterioration is determined by worsening of ADAS-Cog. In some embodiments, clinical deterioration is determined by worsening of FAQ. In some embodiments, clinical deterioration is determined by worsening of CDR-SB. In some embodiments, clinical deterioration is determined by worsening of Wechsler Memory Scale-IV Logical Memory (subscale) I and / or (subscale) II. In some embodiments, clinical deterioration is determined by worsening CDR score. In some embodiments, clinical deterioration refers to a worsening of one or more biomarkers of AD or brain measurements (e.g., by PET or MRI), such as brain atrophy and / or amyloid accumulation.

[0109]

[0126] As used herein, the term "treat" (also "treating" or "treatment") refers to any administration or application of a therapeutic agent to a subject having a disease or disorder, including inhibiting the disease, slowing the progression of the disease, delaying progression, arresting the onset, reversing the progression of the disease (e.g., reversing Aβ fiber accumulation), preventing the onset of the disease or at least one symptom of the disease or preventing further progression of the disease, alleviating or ameliorating one or more symptoms or underlying conditions of the disease, curing the disease, improving one or more clinical indicators, or preventing the recurrence of one or more symptoms of the disease. In some embodiments, treating can include maintaining the severity of at least one symptom of the disease (i.e., preventing worsening), for example, when the condition is expected to progress and / or worsen in the absence of the administration or application of the therapeutic agent to the subject. In some embodiments, maintaining symptoms can refer to no change (e.g., no significant change, such as no statistically significant change) in symptoms following administration or application of a therapeutic agent to a subject, compared to a control (e.g., a subject not receiving treatment or administered a placebo) in which a change (e.g., a significant change, such as a statistically significant change) in symptoms occurs. Complete treatment is not necessary. In some embodiments, treating AD in a subject includes administering, e.g., intravenously injecting, e.g., a dual orexin receptor antagonist, e.g., lemborexant, to a subject at risk of developing AD but who does not yet show evidence of dementia.

[0110]

[0127] As used herein, the term "preventing" is encompassed by the term "treating," unless the context dictates otherwise, and refers to obtaining a beneficial or desired preventative effect. For a preventative effect, the composition can be administered to a subject who is at risk of developing Alzheimer's disease (e.g., based on biomarkers and / or family history), even if a clinical diagnosis of Alzheimer's disease has not been made, a subject who has one or more preclinical symptoms of Alzheimer's disease but no clinical symptoms, and / or a subject who reports one or more physiological symptoms of Alzheimer's disease. As used herein, "prevention" can further include a prophylactic effect, i.e., eradication or amelioration of one or more of the underlying symptoms being treated or physiological symptoms associated therewith. Prevention also encompasses arresting or slowing the further progression of one or more symptoms of the disease.

[0111]

[0128] As used herein, "control" (or "control sample") refers to a biological sample obtained from a subject with a known AD condition, distinct from the sample being evaluated. In some embodiments, the control sample is obtained from a subject who has not been diagnosed with Alzheimer's disease, e.g., according to one or more of the definitions above. For example, the control sample can be obtained from a subject who does not have clinical symptoms of AD (e.g., cognitive impairment and / or dementia) and / or does not have any markers of AD pathology (e.g., PET scan or CSF analysis of biomarkers such as amyloid or tau). In some embodiments, the control sample can be obtained from a healthy subject. In some embodiments, the control can be obtained from a subject with a comorbidity not associated with AD. In some embodiments, the control can be obtained from a subject who has been diagnosed along the spectrum of AD disease, including, for example, preclinical AD or mild cognitive impairment. In some embodiments, the control sample can be a baseline sample taken from the subject prior to the initiation of any treatment. In some embodiments, the control sample can be a sample taken from a control subject administered a placebo.

[0112]

[0129] As used herein, the term "therapeutically effective amount" refers to an amount of a compound or pharmaceutical composition sufficient to produce a desired therapeutic effect, e.g., reverse, prevent, delay, or slow cognitive decline and / or reverse, prevent, delay, or slow the rate of change in one or more biomarkers of AD. Those skilled in the art will understand that the therapeutically effective amount of lemborexant administered to a subject will depend on several factors, including pharmacodynamic properties, route of administration, frequency of treatment, and the health, age, and weight of the subject being treated, and will be able to determine the appropriate amount for each subject using the information disclosed herein.

[0113] [II. Method]

[0130] Disclosed herein are methods for reducing the amount of p-tau in a subject, comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof. Disclosed herein are methods for reducing neurodegeneration in a subject, comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof. Disclosed herein are methods for reducing amyloid beta in a subject, comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof.

[0114]

[0131] In one aspect of the present disclosure, lemborexant affects at least one marker of AD pathology. Without being bound by theory, in some embodiments, the surprising effects of lemborexant on AD and AD pathology may be related to lemborexant's role in regulating sleep and treating insomnia, among other potential effects on AD pathology. In particular, lemborexant provides benefits not seen with other sleep aids, such as doxepin, another drug approved for the treatment of insomnia. For example, as discussed in the Examples, administration of lemborexant and doxepin to an animal model of AD showed distinct effects on Aβ plaque development, activation of phagocytic microglia, and expression of biomarkers involved in membrane receptor trafficking and inflammatory activity. In one animal model, lemborexant reduced total amyloid plaque burden (including both diffuse and fibrous plaques), whereas doxepin reduced only total amyloid burden, but not fibrous plaque burden (Example 3, Section B). Lemborexant, but not doxepin, also increased the activation of phagocytic microglia surrounding Aβ plaques (Example 3, Section E). Finally, lemborexant, but not doxepin, significantly upregulated the expression of the inflammatory mediator IFN-β Ifnb1, the lysosomal protein Rab5a, and the Aβ-degrading enzyme Mmp2 (Example 3, Section F). Without being bound by theory, these data may indicate that lemborexant and doxepin act by different mechanisms, suggesting that the drug's effects on sleep may, in some conditions, differ from their effects on AD pathology.

[0115]

[0132] In various embodiments, methods are disclosed herein for reducing or maintaining the level of p-tau and / or t-tau in a subject, comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof. Methods for reducing or maintaining the level of p-tau in a subject, increasing tau dephosphorylation, decreasing the ratio of p-tau to tau, and / or decreasing the rate of tau phosphorylation in a subject are also disclosed, comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof. In some embodiments, the level of p-tau and / or t-tau in the CSF of a subject is reduced or maintained after administration of a therapeutically effective amount of lemborexant compared to the level of p-tau and / or t-tau in the CSF of the subject prior to such administration. In some embodiments, the level of p-tau and / or t-tau in the CSF of a subject is reduced or maintained after administration of a therapeutically effective amount of lemborexant compared to the level of p-tau and / or t-tau in the CSF of the subject after administration of a placebo.

[0116]

[0133] Also disclosed herein are methods for reducing neurodegeneration in a subject, comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof. Also disclosed herein are methods for reducing or maintaining amyloid beta in a subject, comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof.

[0117]

[0134] In some embodiments, a subject in need of the methods of the invention shows evidence of at least one disease selected from Alzheimer's disease, pre-Alzheimer's disease, early Alzheimer's disease, mild cognitive impairment, cerebral amyloid angiopathy, frontotemporal dementia, Lewy body disease, dementia with Lewy bodies, Parkinson's disease, vascular dementia, limbic-predominant age-related TDP-43 encephalopathy, frontotemporal lobar degeneration, corticobasal degeneration, Pick's disease, multiple system atrophy, and progressive supranuclear palsy.

[0118] [III. Subjects in need of treatment]

[0135] In some embodiments, a subject in need of one or more of the disclosed methods shows evidence of at least one disease selected from Alzheimer's disease, pre-Alzheimer's disease, early Alzheimer's disease, mild cognitive impairment, cerebral amyloid angiopathy, frontotemporal dementia, Lewy body disease, dementia with Lewy bodies, Parkinson's disease, vascular dementia, limbic-predominant age-related TDP-43 encephalopathy, frontotemporal lobar degeneration, corticobasal degeneration, Pick's disease, multiple system atrophy, and progressive supranuclear palsy. In some embodiments, a subject in need thereof shows evidence of at least one disease selected from Alzheimer's disease, pre-Alzheimer's disease, and early Alzheimer's disease. In some embodiments, a subject in need thereof shows evidence of mild cognitive impairment. In some embodiments, the subject in need thereof shows evidence of cerebral amyloid angiopathy, frontotemporal dementia, Lewy body disease, dementia with Lewy bodies, vascular dementia, limbic-predominant age-related TDP-43 encephalopathy, frontotemporal lobar degeneration, corticobasal degeneration, hi some embodiments, the subject in need thereof shows evidence of at least one disease selected from Parkinson's disease, Pick's disease, multiple system atrophy, and progressive supranuclear palsy.

[0119]

[0136] One aspect of the present disclosure relates to a method of treating Alzheimer's disease (AD) in a subject having or at risk of developing AD, the method comprising administering a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof to the subject, thereby treating AD. In some embodiments, the subject is in need of treatment (e.g., has AD, pre-AD, or is otherwise at risk of developing AD).

[0120]

[0137] In some embodiments, treating AD refers to one or more of inhibiting progression, slowing progression, slowing the rate of progression, retarding progression, preventing the onset, and reversing progression of AD, AD pathology, symptoms of AD, and / or underlying symptoms of AD. In some embodiments, treating refers to preventing the onset or preventing the onset of AD, AD pathology, symptoms of AD, and / or underlying symptoms of AD. In some embodiments, treating refers to alleviating or ameliorating one or more symptoms or underlying symptoms of AD (e.g., ameliorating Aβ fibril accumulation) and / or improving one or more clinical indicators of AD (e.g., cognitive function, brain amyloid or tau levels, and / or biomarker expression). In some embodiments, treating refers to preventing the onset or recurrence of one or more symptoms of AD.

[0121]

[0138] In some embodiments, treating AD includes reducing, preventing, or slowing cognitive decline.

[0122]

[0139] In some embodiments, the subject in need of a treatment described herein is a subject with AD, e.g., a subject diagnosed with AD. Diagnosis may be based on cognitive assessment. Diagnosis may be based on measurements of AD pathology obtained by brain imaging (e.g., amyloid PET or tau PET) and / or expression of biomarkers in the subject. In some embodiments, the biomarkers include brain amyloid levels, brain tau levels, cerebrospinal fluid Aβ1-42 levels, cerebrospinal fluid total tau levels, cerebrospinal fluid neurogranin levels, and cerebrospinal fluid neurofilament light chain (NfL) levels. In some embodiments, the subject with AD exhibits cognitive impairment and AD pathology. For example, the subject with AD may have a t-tau level greater than 400 ng / L, an Aβ1-42 level less than 550 ng / L, and / or an Aβ1-42 / Aβ1-40 ratio less than 0.065.

[0123]

[0140] In some embodiments, the subject in need of treatment has early Alzheimer's disease (also referred to as "early AD" or "EAD"). In some embodiments, the subject with early AD has symptoms on the AD severity continuum, ranging from mild cognitive impairment due to AD - moderate likelihood to mild Alzheimer's dementia. In some embodiments, the subject with early AD has mild Alzheimer's dementia as defined herein and / or mild cognitive impairment due to AD (MCI) - moderate likelihood as defined herein. In some embodiments, the subject has an MMSE score of 22-30 and a Clinical Dementia Rating (CDR) global range of 0.5-1.0. In some embodiments, the subject with early AD has evidence of high amyloid or positive amyloid burden in the brain. In some embodiments, high amyloid or positive amyloid burden in the brain is demonstrated and / or confirmed by PET assessment. In some embodiments, high amyloid or positive amyloid burden in the brain is indicated and / or confirmed by CSF assessment of markers such as Aβ1-42 (e.g., soluble CSF biomarker analysis). In some embodiments, high amyloid or positive amyloid burden in the brain is indicated and / or confirmed by measuring the ratio of Aβ42 to Aβ40 (Aβ42 / 40 ratio). In some embodiments, high amyloid or positive amyloid burden in the brain is indicated and / or confirmed by MRI. In some embodiments, high amyloid or positive amyloid burden in the brain is indicated by retinal amyloid accumulation. In some embodiments, multiple assessment methods are used.

[0124]

[0141] In some embodiments, a subject has preclinical Alzheimer's disease (also referred to as "pre-AD"). Subjects with pre-AD can be cognitively normal and have intermediate or high levels of amyloid in the brain. In some embodiments, subjects with pre-AD can be identified by an asymptomatic stage with or without memory symptoms and impairments in new episodic memory and executive function. In some embodiments, subjects with pre-AD have a CDR 0 and / or scores within the normal range on cognitive tests (e.g., MMSE, International Shopping List Task, Logical Memory, etc.). In some embodiments, the subject is also diagnosed with other biomarkers indicative of future development of AD, such as intermediate or high levels of amyloid in the brain by amyloid or tau positron emission tomography (PET) (e.g., a centiloid measurement of about 20-40, e.g., a measurement of about 20-32), levels of Aβ1-42 in the cerebrospinal fluid and / or the Aβ1-42 / 1-40 ratio, levels of total tau in the cerebrospinal fluid, levels of neurogranin in the cerebrospinal fluid, levels of neurofilament light peptide (NfL) in the cerebrospinal fluid, and blood biomarkers measured in serum or plasma (e.g., levels of Aβ1-42, the ratio of two forms of amyloid-β peptide (Aβ1-42 / 1-40). The subjects may have one or more of the following: a 0.092-0.094 ratio (e.g., a ratio of about 0.092-0.094 or less than about 0.092), plasma levels of plasma total tau (T-tau), levels of phosphorylated tau (P-tau) isoforms (including tau phosphorylated at 181 (P-tau181), 217 (P-tau217), and 231 (P-tau231)), glial fibrillary acidic protein (GFAP), and neurofilament light chain (NfL). In some embodiments, subjects with pre-AD may have intermediate amyloid (e.g., about 20-40 centiloid). In some embodiments, subjects with pre-AD may have high amyloid (e.g., >40 centiloid).

[0125]

[0142] In some embodiments, a subject may be at risk for developing AD. The subject may have one or more risk factors for developing AD, such as having a familial AD gene (e.g., the apolipoprotein E ε4 allele, also known as "APOE4" or "ApoE4"), a family history of a first-degree relative with AD or dementia, being 65 years of age or older, being female, having or recovering from a traumatic brain injury, or suffering from other conditions such as obesity, diabetes, heart disease, and / or vascular disease. In some embodiments, a subject with pre-AD may be at risk for developing AD. This risk of developing AD may be significantly higher than the risk of developing AD in control subjects and / or may be at risk for developing AD earlier than expected in control subjects. For example, a subject with pre-AD who is cognitively normal but has intermediate amyloid PET levels (approximately 20-40 centimeters) may be at risk for further Aβ accumulation and early spread of tau pathology over a four-year period compared to control subjects. Subjects with pre-AD and who are cognitively normal but have high levels of amyloid PET (>40 centiloid) will be at increased risk of cognitive decline over 4 years compared to control subjects.

[0126]

[0143] In some embodiments, treating AD includes affecting (eg, slowing, delaying, or reducing) changes in at least one marker of AD pathology.

[0127]

[0144] In some embodiments, the marker of AD pathology is the level of tau phosphorylation, neurodegeneration, changes in microglial response, and / or the presence of Aβ plaques. The marker of AD pathology may be present in a brain region of the subject, such as the hippocampus, somatomotor cortex, somatosensory cortex, piriform cortex, and / or entorhinal cortex. In some embodiments, the marker of AD pathology is detected in a brain scan. For example, tau phosphorylation can be detected by tau PET; Aβ can be detected by amyloid PET. In some embodiments, the marker of AD pathology is detected in a subject's body fluids, such as blood (e.g., plasma) or CSF. For example, various species of phosphorylated tau and Aβ can be detected in the subject's plasma or CSF.

[0128]

[0145] In some embodiments, the subject does not exhibit signs of dementia and / or cognitive impairment, hi some embodiments, the subject has mild cognitive impairment or mild dementia.

[0129]

[0146] In some embodiments, the subject is amyloid positive. The subject may be at risk for further Aβ accumulation and / or spreading tau pathology. The subject may be at risk for cognitive decline. In some embodiments, the subject may have intermediate levels of amyloid PET (e.g., about 20-40 centiloids). In some embodiments, the subject may have high levels of amyloid PET (e.g., >40 centiloids). In some embodiments, the subject may have the APOE4 gene. In some embodiments, the subject may have one or more risk factors for developing AD, such as having a family history of AD or dementia in a first-degree relative, being 65 years of age or older, being female, having or recovering from a traumatic brain injury, and suffering from other conditions, such as obesity, diabetes, heart disease and / or vascular disease.

[0130]

[0147] In some embodiments, the subject has been diagnosed with AD based on brain imaging, cognitive function, and / or biomarker criteria. In some embodiments, the subject has early AD. In some embodiments, the subject has pre-AD.

[0131]

[0148] In some embodiments, treatment can slow cognitive decline and / or reduce the rate of change in biomarkers of AD.

[0132] [IV. Tau and Aβ]

[0149] The ratio of the concentration of p-tau (also referred to herein as "phospho-tau" or "phosphorylated tau") to the concentration of t-tau (also referred to herein as "total tau") in a subject's CSF (herein the "CSF p-tau / t-tau ratio") can be used to assess the degree of tau phosphorylation. The concentrations of p-tau and t-tau in a subject's CSF are measured by liquid chromatography with tandem mass spectrometry (LC MS / MS).

[0133]

[0150] In some embodiments, the ratio of CSF p-tau / t-tau in subjects administered a therapeutically effective amount of lemborexant is reduced compared to the ratio of CSF p-tau / t-tau in subjects administered a placebo. In some embodiments, the ratio of CSF p-tau / t-tau in subjects administered a therapeutically effective amount of lemborexant is maintained within 10% (i.e., + / -) of the ratio of CSF p-tau / t-tau in subjects administered a placebo. In some embodiments, the ratio of CSF p-tau / t-tau in subjects administered a therapeutically effective amount of lemborexant is within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the ratio of CSF p-tau / t-tau in subjects administered a placebo.

[0134]

[0151] In some embodiments, the ratio of CSF p-tau / t-tau in a subject administered a therapeutically effective amount of lemborexant is reduced compared to the ratio of CSF p-tau / t-tau in the subject before administration of lemborexant.

[0135]

[0152] In some embodiments, the ratio of CSF p-tau / t-tau in a subject administered a therapeutically effective amount of lemborexant is maintained within 10% (i.e., + / -) of the ratio of CSF p-tau / t-tau in the subject prior to administration of lemborexant. In some embodiments, the ratio of CSF p-tau / t-tau in a subject administered a therapeutically effective amount of lemborexant is within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the ratio of CSF p-tau / t-tau in the subject prior to administration of lemborexant.

[0136]

[0153] In some embodiments, the concentration of amyloid beta (Aβ) in the CSF is lower than the concentration of Aβ in the CSF of a subject administered a placebo. In some embodiments, the concentration of Aβ in the CSF is lower than the concentration of Aβ in the CSF of the subject prior to administration of lemborexant. In some embodiments, the concentration of Aβ38, Aβ40, and / or Aβ42 in the CSF is reduced. In some embodiments, the concentration of Aβ in the CSF is maintained compared to the concentration of Aβ in the CSF of the subject prior to administration of lemborexant. In some embodiments, the concentration of Aβ38, Aβ40, and / or Aβ42 in the CSF is maintained compared to the concentration of Aβ in the CSF of the subject prior to administration of lemborexant. In some embodiments, the amyloid PET signal in the brain of a subject administered a therapeutically effective amount of lemborexant is lower than the amyloid PET signal in the brain of a subject administered a placebo. In some embodiments, the amyloid PET signal in the brain of a subject administered a therapeutically effective amount of lemborexant is lower than or maintained below the amyloid PET signal in the brain of the subject prior to administration of lemborexant. In some embodiments, the increase in Aβ levels in the CSF of subjects administered lemborexant is less than the increase in Aβ levels in the CSF of subjects administered a placebo.

[0137]

[0154] In some embodiments, subjects administered lemborexant have CSF Aβ concentrations that are at least 5% lower compared to subjects administered a placebo, hi some embodiments, subjects administered lemborexant have CSF Aβ concentrations that are at least 10%, at least 15%, at least 20%, or at least 25% lower compared to subjects administered a placebo.

[0138]

[0155] In some embodiments, the concentration of Aβ in CSF is measured using liquid chromatography with tandem mass spectrometry (LC MS / MS). In some embodiments, the concentration of Aβ38, Aβ40, and / or Aβ42 in CSF is measured using LC MS / MS. Methods for measuring Aβ38, Aβ40, and / or Aβ42 are known in the art, such as assays using LC MS / MS. Methods can include the PrecivityAD™ assay (see, e.g., Kirmess et al., J. Clinica Chimica Acta 519:267-275 (2021)) and the Sysmex assay (https: / / www.eisai.com / news / 2019 / news201990.html) for measuring Aβ42 and Aβ40 in blood or plasma samples or CSF samples. In some embodiments, the concentration of Aβ in CSF is measured using ELISA. In some embodiments, the concentrations of Aβ38, Aβ40, and / or Aβ42 in CSF are measured using ELISA. Methods for measuring Aβ are known in the art. See Englund, H. et al., J. Neurochem. 103:334-45 (2007). In some embodiments, the reduction or maintenance of Aβ38, Aβ40, and / or Aβ42 concentrations is compared to the subject prior to administration of lemborexant. In some embodiments, administration of a therapeutically effective amount of a composition comprising lemborexant to a subject reduces the concentration of Aβ38, Aβ40, and / or Aβ42 in cerebrospinal fluid by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, or at least 13% relative to baseline.

[0139]

[0156] In some embodiments, p-tau and t-tau are reduced. In some embodiments, p-tau, t-tau, and / or aggregated tau are reduced. In some embodiments, the reduction in tau phosphorylation occurs in the whole brain or in regions of the brain, such as the frontal lobe, parietal lobe, temporal lobe, occipital lobe, cingulate cortex, amygdala, hippocampus, entorhinal cortex, and / or piriform cortex. In some embodiments, tau PET signal in the brain is reduced compared to placebo. In some embodiments, tau PET signal in the brain is reduced or maintained compared to baseline.

[0140]

[0157] In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces the level of p-tau in the CSF of the subject compared to the level of p-tau in the CSF of a subject administered a placebo. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces the level of p-tau in the CSF of the subject compared to the level of p-tau in the CSF of the subject prior to administration of lemborexant. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject maintains the level of p-tau in the CSF of the subject compared to the level of p-tau in the CSF of a subject administered a placebo. In some embodiments, the level of p-tau in the CSF of the subject prior to administration of lemborexant is maintained by administration of a therapeutically effective amount of lemborexant to the subject.

[0141]

[0158] In some embodiments, the concentration of p-tau in the CSF is measured using liquid chromatography with tandem mass spectrometry (LC MS / MS).

[0142]

[0159] In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject maintains the level of p-tau in the subject's CSF compared to the level of p-tau in the CSF of a subject administered a placebo. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces the level of p-tau in the subject's CSF by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, or at least 13% compared to the level of p-tau in the CSF of a subject administered a placebo. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject results in a reduction in the amount of p-tau in the subject's CSF by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, or at least 13% compared to the amount of p-tau in the subject's CSF before administration of lemborexant.

[0143]

[0160] In some embodiments, the increase in CSF p-tau levels in subjects administered lemborexant is less than the increase in CSF p-tau levels in subjects administered a placebo.

[0144]

[0161] In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject maintains or reduces the level of p-tau in the subject's CSF compared to the level of p-tau in the subject's CSF at baseline for 18 months following administration of lemborexant. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces the level of p-tau in the subject's CSF by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, or at least 13% compared to the level of p-tau in the subject's CSF at baseline.

[0145]

[0162] In some embodiments, the reduction in the amount of p-tau or t-tau in the subject's CSF is due to a decrease or maintenance of the amount of p-tau or t-tau in the subject, comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof. In some embodiments, the reduction in the amount of p-tau in the subject's CSF is due to an increase in the dephosphorylation of p-tau. In some embodiments, the reduction in the amount of p-tau in the subject's CSF is due to a decrease in the amount of tau. In some embodiments, the reduction in the amount of p-tau in the subject's CSF is due to a decrease in the ratio of p-tau / t-tau.

[0146]

[0163] In some embodiments, administration of a therapeutically effective amount of lemborexant disclosed herein to a subject reduces or maintains the level of p-tau in the subject's CSF compared to the level of p-tau in the CSF of a subject administered a placebo. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces the level of p-tau in the cerebrospinal fluid by at least about 5 pg / mL, at least about 10 pg / mL, at least about 15 pg / mL, at least about 20 pg / mL, at least about 25 pg / mL, at least about 30 pg / mL, at least about 35 pg / mL, or at least about 40 pg / mL compared to placebo. In some embodiments, administration of a composition comprising a therapeutically effective amount of lemborexant disclosed herein to a subject reduces the level of p-tau in the cerebrospinal fluid by at least about 40 pg / mL compared to placebo.

[0147]

[0164] In some embodiments, administration of a therapeutically effective amount of lemborexant disclosed herein to a subject reduces or maintains the level of p-tau in the subject's CSF compared to the level of p-tau in the subject's CSF prior to administration of lemborexant. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces the level of p-tau in cerebrospinal fluid by at least about 5 pg / mL, at least about 10 pg / mL, at least about 15 pg / mL, at least about 20 pg / mL, at least about 25 pg / mL, at least about 30 pg / mL, at least about 35 pg / mL, or at least about 40 pg / mL compared to baseline. In some embodiments, administration of a composition comprising a therapeutically effective amount of lemborexant disclosed herein to a subject reduces the level of p-tau in cerebrospinal fluid by at least about 40 pg / mL compared to baseline.

[0148]

[0165] In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces the level of p-tau in cerebrospinal fluid by at least about 5 pg / mL, at least about 10 pg / mL, at least about 15 pg / mL, at least about 20 pg / mL, at least about 25 pg / mL, at least about 30 pg / mL, at least about 35 pg / mL, or at least about 40 pg / mL relative to baseline for 18 months following administration of a composition comprising a therapeutically effective amount of lemborexant. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces the level of p-tau in cerebrospinal fluid by at least 40 pg / mL relative to baseline for at least 18 months following administration of a composition comprising a therapeutically effective amount of lemborexant.

[0149]

[0166] In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces the level of p-tau in cerebrospinal fluid compared to placebo by at least about 5 pg / mL, at least about 10 pg / mL, at least about 15 pg / mL, at least about 20 pg / mL, at least about 25 pg / mL, at least about 30 pg / mL, at least about 35 pg / mL, or at least about 40 pg / mL for 18 months following administration of a composition comprising a therapeutically effective amount of lemborexant. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces the level of p-tau in cerebrospinal fluid compared to placebo by at least 40 pg / mL for 18 months following administration of a composition comprising at least a therapeutically effective amount of lemborexant.

[0150]

[0167] In some embodiments, the amount of p-tau is reduced relative to the subject's baseline within 48 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of p-tau is reduced relative to the subject's baseline within 24 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of p-tau is reduced relative to the subject's baseline within 12 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of p-tau is reduced relative to the subject's baseline within 6 hours of administering a first dose of lemborexant to the subject.

[0151]

[0168] In some embodiments, the amount of t-tau is reduced relative to the subject's baseline within 48 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of t-tau is reduced relative to the subject's baseline within 24 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of t-tau is reduced relative to the subject's baseline within 12 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of t-tau is reduced relative to the subject's baseline within 6 hours of administering a first dose of lemborexant to the subject.

[0152]

[0169] In some embodiments, the amount of Aβ is reduced relative to the subject's baseline within 48 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of Aβ is reduced relative to the subject's baseline within 24 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of Aβ is reduced relative to the subject's baseline within 12 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of Aβ is reduced relative to the subject's baseline within 6 hours of administering a first dose of lemborexant to the subject.

[0153]

[0170] In some embodiments, the amount of p-tau in the subject's CSF is reduced compared to a subject administered a placebo within 48 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of tau phosphorylation in the subject's CSF is reduced compared to a subject administered a placebo within 24 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of tau phosphorylation in the subject's CSF is reduced compared to a subject administered a placebo within 12 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of tau phosphorylation in the subject's CSF is reduced compared to a subject administered a placebo within 6 hours of administering a first dose of lemborexant to the subject.

[0154] [A. Altering tau]

[0171] A further aspect of the present disclosure relates to a method of altering tau (e.g., reducing, slowing, and / or slowing the rate of tau accumulation, tau phosphorylation, and / or tau diffusion) in a subject having or at risk of developing AD, the method comprising administering to the subject a therapeutically effective amount of lemborexant, the therapeutically effective amount sufficient to alter tau in the subject. In some embodiments, altering tau comprises reducing, slowing, or slowing the rate of any of tau accumulation, tau phosphorylation, or tau diffusion.

[0155]

[0172] In some embodiments, altering tau is, for example, slowing, slowing the rate of progression, delaying progression, preventing the onset, or reversing the progression of tau pathology (e.g., tau accumulation, tau phosphorylation, and / or tau diffusion) in a brain region. The brain region can be the cortex or hippocampus. The brain region can be the CA1, CA2, CA3 regions of the hippocampus and / or the dentate gyrus. The brain region can be the entorhinal cortex and / or the piriform cortex. In some embodiments, altering tau is preventing the onset or preventing the onset of tau pathology. Altering tau can reduce, delay, or slow the rate of onset and / or progression of symptoms of tau pathology. In some embodiments, altering tau is alleviating or ameliorating one or more symptoms of tau pathology and / or improving one or more clinical indicators of tau pathology (e.g., cognitive function, brain amyloid or tau levels and / or biomarker expression). In some embodiments, altering tau prevents the occurrence or recurrence of one or more symptoms of tau pathology.

[0156]

[0173] In some embodiments, the subject is amyloid-negative. The subject may have mild cognitive impairment or mild dementia. In some embodiments, the subject does not show signs of dementia and / or cognitive impairment.

[0157]

[0174] In some embodiments, tau is altered relative to a reference. Thus, the methods described herein can include reducing and / or slowing tau accumulation, tau phosphorylation, and / or tau diffusion, and / or slowing the rate of any of these, compared to the reference. In some embodiments, the reference is a baseline measurement from a subject before treatment. In some embodiments, the reference is a baseline measurement from a control subject. The reference can be measurements obtained from multiple control subjects and used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0158]

[0175] In some embodiments, the methods herein involve altering tau (e.g., reducing, preventing, or slowing the growth of tau) in a brain region of a subject. Altering tau in a brain region can include altering tau PET signal in the brain region. In some embodiments, the brain region is the hippocampus, entorhinal cortex, and / or piriform cortex.

[0159]

[0176] In some embodiments, altering tau includes altering tau in a bodily fluid of the subject. For example, tau levels in the brain of a subject can be detected in the bodily fluid of the subject. In some embodiments, the bodily fluid is blood (e.g., plasma) or CSF.

[0160]

[0177] In some embodiments, one or more forms of tau can be altered. In some embodiments, the tau is total tau. In some embodiments, the tau is aggregated tau. In some embodiments, the tau is phosphorylated tau (phospho-tau). Phospho-tau can be tau that is phosphorylated at one or more of T181, T217, S202, S205, or T231.

[0161]

[0178] In some embodiments, altering tau comprises altering the ratio of phospho-tau to total tau. In some embodiments, the ratio of phosphorylated tau to total tau is altered such that the ratio is maintained within 10% of the ratio of phosphorylated tau to total tau in the subject prior to administration of lemborexant. In some embodiments, the rate of dephosphorylation of phospho-tau is increased. In some embodiments, the rate of tau phosphorylation is decreased. In some embodiments, altering tau comprises altering tau within 48 hours of administering the first dose of lemborexant. For example, tau can be reduced within 48 hours of administering the first dose of lemborexant. In some embodiments, reducing tau comprises altering phospho-tau in the hippocampus, entorhinal cortex, and / or piriform cortex.

[0162] [B. Maintaining Tau]

[0179] A further aspect of the present disclosure relates to a method for maintaining tau (e.g., tau accumulation, tau phosphorylation, and / or tau proliferation) in a subject having or at risk of developing AD, the method comprising administering to the subject a therapeutically effective amount of lemborexant, the therapeutically effective amount sufficient to maintain tau in the subject.

[0163]

[0180] In some embodiments, maintaining tau refers to maintaining tau accumulation, tau phosphorylation, and / or tau diffusion when tau is expected to progress and / or worsen in the absence of the administration or application of the therapeutic agent to the subject. In some embodiments, maintaining tau can refer to no change (e.g., no significant change, such as no statistically significant change) in tau (e.g., tau accumulation, tau phosphorylation, and / or tau diffusion) after administration or application of the therapeutic agent to a subject compared to a control in which administration or application of the therapeutic agent results in a change (e.g., a significant change, such as a statistically significant change) in tau.

[0164]

[0181] In some embodiments, maintaining tau includes, for example, maintaining tau pathology (e.g., tau accumulation, tau phosphorylation, and / or tau diffusion) in a brain region. The brain region can be the cortex or hippocampus. The brain region can be the CA1 region, CA2 region, CA3 region, and / or dentate gyrus of the hippocampus. The brain region can be the entorhinal cortex and / or piriform cortex. In some embodiments, maintaining tau is preventing the onset or occurrence of tau pathology, e.g., because tau pathology is maintained unchanged. Maintaining tau can reduce, delay, or slow the rate of onset and / or progression of symptoms of tau pathology, e.g., because tau pathology is maintained unchanged. In some embodiments, maintaining tau can result in alleviation or amelioration of one or more symptoms of tau pathology and / or improvement of one or more clinical indicators of tau pathology (e.g., cognitive function, brain amyloid or tau levels and / or biomarker expression). In some embodiments, maintaining tau may result in the prevention of the onset or recurrence of one or more symptoms of tau pathology.

[0165]

[0182] In some embodiments, the subject is amyloid-negative. The subject may have mild cognitive impairment or mild dementia. In some embodiments, the subject does not show signs of dementia and / or cognitive impairment.

[0166]

[0183] In some embodiments, the subject exhibits symptoms of cognitive impairment. In some embodiments, the subject is amyloid-positive. In some embodiments, the subject has a diagnosis of AD, e.g., early stage AD.

[0167]

[0184] In some embodiments, tau is maintained relative to a reference. Thus, the methods described herein can include maintaining tau accumulation, tau phosphorylation, and / or tau diffusion (or the rate of any of these) compared to a reference. In some embodiments, the reference is a baseline measurement from a subject prior to treatment. In some embodiments, the reference is a baseline measurement from a control subject. The reference can be measurements obtained from multiple control subjects and can be used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0168]

[0185] In some embodiments, maintaining tau comprises maintaining tau in a brain region of the subject. Maintaining tau in a brain region can include altering, reducing, or maintaining tau PET signal in the brain region. In some embodiments, the brain region is the hippocampus, entorhinal cortex, and / or piriform cortex.

[0169]

[0186] In some embodiments, maintaining tau includes maintaining tau in a bodily fluid of the subject. The level of tau in the brain of the subject can be related to the level in the subject's bodily fluid. In some embodiments, the bodily fluid is blood (e.g., plasma) or CSF.

[0170]

[0187] In some embodiments, one or more forms of tau can be maintained. In some embodiments, the tau is total tau. In some embodiments, the tau is aggregated tau. In some embodiments, the tau is phosphorylated tau (phospho-tau). Phospho-tau can be tau phosphorylated at one or more of T181, T217, S202, S205, or T231.

[0171]

[0188] In some embodiments, maintaining tau comprises maintaining the ratio of phospho-tau to total tau. In some embodiments, the ratio of phospho-tau to total tau is maintained within 10% of the ratio of phospho-tau to total tau in the subject prior to administration of lemborexant. In some embodiments, maintaining tau comprises maintaining tau within 48 hours of administration of the first dose of lemborexant. In some embodiments, phospho-tau is maintained in the hippocampus, entorhinal cortex, and / or piriform cortex.

[0172] [V. Microglial Response]

[0189] In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject increases the number of activated microglial cells compared to placebo. In some embodiments, the increase in the number of activated microglial cells is measured by PET. In some embodiments, the activated microglial cells are microglial phagocytes.

[0173]

[0190] In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject increases the number of activated microglial cells compared to baseline. In some embodiments, the increase in the number of activated microglial cells is measured by PET. In some embodiments, the activated microglial cells are microglial phagocytes.

[0174]

[0191] Methods for measuring microglia are known in the art, such as PET. In some embodiments, the whole brain or at least one region of the brain (e.g., cortical gray matter (i.e., cortex), lateral ventricles, frontal lobe, parietal lobe, temporal lobe, occipital lobe, cingulate cortex, amygdala, piriform cortex, entorhinal cortex, hippocampus, hippocampal CA3 (pyramidal neurons), and / or hippocampal dentate gyrus (granule cell neurons)) is analyzed by PET.

[0175] [A. Modulating microglial responses]

[0192] One aspect of the present disclosure relates to a method of modulating microglial responses in a subject having or at risk of developing Alzheimer's disease (AD), the method comprising administering to the subject a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, sufficient to modulate microglial responses in the subject.

[0176]

[0193] In some embodiments, modulating refers to increasing or decreasing the microglial response and / or increasing or slowing the rate of the microglial response. In some embodiments, the number of microglia does not change, but the microglial response (e.g., activation, reactivation, reactivity, differentiation, etc.) is modulated. Modulation may vary by brain region (e.g., microglial activation or other response may occur differently in different brain regions).

[0177]

[0194] In some embodiments, modulation of microglial response is measured in a subject and compared to the microglial response in a reference. In some embodiments, the reference is a baseline measurement from the subject before treatment. In some embodiments, the reference is a baseline measurement from a control subject. The reference can be measurements obtained from multiple control subjects and used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0178]

[0195] In some embodiments, modulating the microglial response includes modulating the expression of at least one microglial marker. The microglial marker can be a general microglial marker. The general microglial marker can be a general marker of microglia in a particular condition or disease state (e.g., a general marker of activated microglia, reactive microglia, and / or microglia in a disease state). For example, the general microglial marker can be Iba1, Clec7a, or CD68. The microglial marker can be a homeostatic microglial marker. For example, the homeostatic microglial marker is TMEM119 or P2RY12.

[0179]

[0196] In some embodiments, modulating a microglial response comprises modulating the activity of phagocytic microglia.

[0180]

[0197] In some embodiments, the subject has mild cognitive impairment or mild dementia. In some embodiments, the subject does not show signs of dementia and / or cognitive impairment.

[0181]

[0198] In some embodiments, the subject is amyloid-negative. The subject may have tau pathology. The subject may have neurodegeneration in a brain region, such as the hippocampus, entorhinal cortex, and / or piriform cortex. In some embodiments, the brain region is the CA1, CA2, or CA3 region of the hippocampus, or the dentate gyrus.

[0182]

[0199] In some embodiments, in a subject with neurodegeneration, modulating the microglial response comprises modulating the response of microglia associated with degenerating neurons. Microglia associated with neurodegenerating neurons may be located adjacent to the neurons, for example, when observed in a scan or sample obtained from the subject. In some embodiments, microglia associated with degenerating neurons may phagocytose the degenerating neurons and / or debris resulting therefrom. Thus, in some embodiments, administering a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof to such a subject may comprise decreasing the expression of at least one general microglial marker. The general microglial marker may be Iba1, CD68, or Clec7a. In some embodiments, modulating the microglial response comprises increasing the expression of at least one homeostatic microglial marker. The homeostatic microglial marker may be TMEM119 or P2RY12.

[0183]

[0200] In some embodiments, the subject is amyloid-positive, e.g., the subject has Aβ plaques. The Aβ plaques can be fibrillar Aβ plaques. In some embodiments, the Aβ plaques are present in the hippocampus, somatomotor cortex, somatosensory cortex, and / or piriform cortex of the subject.

[0184]

[0201] In some embodiments, the subject does not exhibit signs of dementia and / or cognitive impairment, hi some embodiments, the subject has mild cognitive impairment or mild dementia.

[0185]

[0202] In some embodiments, the subject is at risk for further Aβ accumulation. The subject may be at risk for the spread of tau pathology. The subject may be at risk for cognitive decline. In some embodiments, the subject may have intermediate levels of amyloid PET (e.g., about 20-40 centiloids). In some embodiments, the subject may have high levels of amyloid PET (e.g., >40 centiloids). In some embodiments, the subject may be an ApoE4 carrier. In some embodiments, the subject may have one or more risk factors for developing AD, such as having a family history of AD or dementia in a first-degree relative, being 65 years of age or older, being female, having or recovering from a traumatic brain injury, and suffering from other conditions, such as obesity, diabetes, heart disease and / or vascular disease.

[0186]

[0203] In some embodiments, the subject has early stage AD, hi some embodiments, the subject has pre-AD.

[0187]

[0204] In some embodiments, in a subject who is amyloid-positive, modulating the microglial response includes modulating a response in microglia associated with Aβ plaques. Microglia associated with Aβ plaques may be located adjacent to Aβ plaques, for example, as observed in a scan or sample obtained from the subject. In some embodiments, microglia associated with Aβ plaques may phagocytose the Aβ plaques. Thus, in some embodiments, administering a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof to such a subject may include increasing the expression of a general microglial marker. The general microglial marker may be Iba1, Clec7a, or CD68. In some embodiments, modulating the microglial response includes increasing the phagocytosis of Aβ plaques by phagocytic microglia. In some embodiments, modulating the microglial response includes decreasing the expression of a homeostatic microglial marker. The homeostatic microglial marker may be TMEM119 or P2RY12.

[0188] [VI. Amyloid Plaques]

[0205] In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces or maintains amyloid plaques compared to placebo. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces or maintains fibrillar amyloid plaques compared to placebo. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces or maintains amyloid plaques compared to baseline. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject reduces or maintains fibrillar amyloid plaques compared to baseline. In some embodiments, the amyloid plaques are fibrillar amyloid plaques.

[0189] [A. Alters Aβ plaques]

[0206] Another aspect of the present disclosure relates to a method for altering Aβ plaques (e.g., reducing or delaying the formation of, or slowing the rate of growth of, Aβ plaques) in a subject having or at risk of developing AD, the method comprising administering to the subject a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, sufficient to alter Aβ plaques in the subject.

[0190]

[0207] In some embodiments, altering Aβ plaques includes slowing, slowing the rate of progression, retarding, preventing the onset, and reversing the progression of Aβ plaque formation and / or Aβ plaque growth. In some embodiments, altering Aβ plaques includes preventing the onset or occurrence of Aβ plaque pathology (e.g., any pathology resulting from or consistent with Aβ plaque formation and / or Aβ plaque growth). Altering Aβ plaques can reduce, retard, or slow the rate of onset and / or progression of symptoms of this pathology. In some embodiments, altering Aβ plaques includes alleviating or ameliorating one or more symptoms of Aβ plaque pathology and / or improving one or more clinical indicators of Aβ plaque pathology (e.g., cognitive function, brain amyloid or tau levels, and / or biomarker expression). In some embodiments, altering Aβ plaques includes preventing the onset or recurrence of one or more symptoms of Aβ plaque formation and / or Aβ plaque growth.

[0191]

[0208] In some embodiments, Aβ plaques are altered relative to a reference. Thus, altering Aβ plaques can include reducing and / or delaying and / or slowing the rate of Aβ plaque formation compared to the reference. In some embodiments, the reference is a baseline measurement from a subject prior to treatment. In some embodiments, the reference is a baseline measurement from a control subject. The reference can be measurements obtained from multiple control subjects and used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0192]

[0209] In some embodiments, the Aβ plaques are fibrous plaques. In some embodiments, the Aβ plaques are total plaques, which can include non-fibrous (e.g., diffuse) plaques.

[0193]

[0210] In some embodiments, altering Aβ plaques comprises reducing the growth or growth rate of Aβ plaques. The reduction in Aβ plaque growth can occur in the subject's hippocampus, the subject's somatomotor cortex, somatosensory cortex, and / or piriform cortex. In some embodiments, altering Aβ plaques comprises altering an amyloid PET signal obtained from a brain region of the subject. In some embodiments, altering Aβ plaques corresponds to a reduction in the concentration of Aβ in the subject's CSF. The Aβ can be Aβ38, Aβ40, and / or Aβ42.

[0194]

[0211] In some embodiments, altering Aβ comprises altering Aβ plaques within 48 hours of administering the first dose of lemborexant.

[0195]

[0212] In some embodiments, the subject does not exhibit signs of dementia and / or cognitive impairment, hi some embodiments, the subject has mild cognitive impairment or mild dementia.

[0196]

[0213] In some embodiments, the subject is at risk for further Aβ accumulation. The subject may also be at risk for the spread of tau pathology. The subject may be at risk for cognitive decline. In some embodiments, the subject may have intermediate levels of amyloid PET (e.g., about 20-40 centiloids). In some embodiments, the subject may have high levels of amyloid PET (e.g., >40 centiloids). In some embodiments, the subject may be an ApoE4 carrier. In some embodiments, the subject may have one or more risk factors for developing AD, such as having a family history of AD or dementia in a first-degree relative, being 65 years of age or older, being female, having or recovering from a traumatic brain injury, and suffering from other conditions, such as obesity, diabetes, heart disease, and / or vascular disease.

[0197]

[0214] In some embodiments, the subject has early stage AD, hi some embodiments, the subject has pre-AD.

[0198] [VII. Neurodegeneration]

[0215] Also disclosed herein are methods of reducing neurodegeneration in a subject, the methods comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof. In some embodiments, the subject in need thereof shows evidence of at least one disease selected from Alzheimer's disease, pre-Alzheimer's disease, early Alzheimer's disease, mild cognitive impairment, cerebral amyloid angiopathy, frontotemporal dementia, Lewy body disease, dementia with Lewy bodies, Parkinson's disease, vascular dementia, limbic-predominant age-related TDP-43 encephalopathy, frontotemporal lobar degeneration, corticobasal degeneration, Pick's disease, multiple system atrophy, and progressive supranuclear palsy. In some embodiments, the subject in need thereof shows evidence of at least one disease selected from Alzheimer's disease, pre-Alzheimer's disease, and early Alzheimer's disease. In some embodiments, the subject in need thereof shows evidence of mild cognitive impairment. In some embodiments, the subject in need thereof shows evidence of cerebral amyloid angiopathy, frontotemporal dementia, Lewy body disease, dementia with Lewy bodies, vascular dementia, limbic-predominant age-related TDP-43 encephalopathy, frontotemporal lobar degeneration, corticobasal degeneration, hi some embodiments, the subject in need thereof shows evidence of at least one disease selected from Parkinson's disease, Pick's disease, multiple system atrophy, and progressive supranuclear palsy.

[0199]

[0216] In some embodiments, the reduction in neurodegeneration is observed by a maintenance of, or a reduced loss of, cortical thickness relative to subjects administered a placebo. In some embodiments, the reduction in neurodegeneration is observed by a maintenance of, or a reduced loss of, cortical thickness relative to the subject's baseline. In some embodiments, the reduction in neurodegeneration is observed by a maintenance of, or a reduced reduction in hippocampal size. In some embodiments, the reduction in neurodegeneration is observed by a maintenance of, or a reduced loss of, pyramidal neuron cells relative to the subject's baseline or subjects administered a placebo. In some embodiments, the reduction in neurodegeneration is observed by a maintenance of, or a reduced loss of, granule neuron cells relative to the subject's baseline or subjects administered a placebo.

[0200]

[0217] Measuring the thickness of brain regions such as the cortex or the size of the hippocampus can be achieved using magnetic resonance imaging (MRI). High-spatial resolution sMRI now allows for volumetric measurements of subregions of the hippocampus. Early changes in CA1 have been observed in AD, and volumetric studies have shown that measuring atrophy of CA1 may improve diagnostic accuracy at the MCI stage. Quantitative susceptibility mapping (QSM) or T2 *Novel MRI techniques, such as transverse relaxation time, have shown that iron levels and their accumulation rates are heterogeneous in the human brain and correlate with cognitive impairment and motor decline. Neuronal dysfunction and changes in the connectivity of different brain networks are believed to occur early in the course of neurodegenerative diseases and can be indirectly measured with functional magnetic resonance imaging (fMRI). The whole brain or at least one region of the brain (e.g., cortical gray matter (i.e., cortex), lateral ventricles, frontal lobe, parietal lobe, temporal lobe, occipital lobe, cingulate cortex, amygdala, piriform cortex, entorhinal cortex, hippocampus, hippocampal CA3 (pyramidal neurons), and / or hippocampal dentate gyrus (granule cell neurons)) can be analyzed by MRI. In some embodiments, neurodegeneration is reduced relative to the subject's baseline within 48 hours of administering a first dose of lemborexant to the subject. In some embodiments, the amount of p-tau is reduced relative to the subject's baseline within 24 hours of administering a first dose of lemborexant to the subject. In some embodiments, neurodegeneration is reduced relative to the subject's baseline within 12 hours of administering a first dose of lemborexant to the subject, hi some embodiments, neurodegeneration is reduced relative to the subject's baseline within 6 hours of administering a first dose of lemborexant to the subject.

[0201]

[0218] In some embodiments, neurodegeneration is reduced or maintained for at least 30 days following administration of the first dose of lemborexant, hi some embodiments, the amount of p-tau in the subject's CSF is reduced or maintained for at least 30 days following administration of the first dose of lemborexant.

[0202]

[0219] In some embodiments, neurodegeneration is reduced within 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, or 55 days after administration of the first dose of lemborexant. In some embodiments, the amount of p-tau in the subject's CSF is reduced at least 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, or 55 days after administration of the first dose of lemborexant.

[0203]

[0220] In some embodiments, neurodegeneration is reduced or maintained within 30 days of administering the first dose of lemborexant. In some embodiments, the amount of p-tau in the subject's CSF is reduced or maintained for at least 30 days after administering the first dose of lemborexant. In some embodiments, neurodegeneration is reduced or maintained within 60 days of administering the first dose of lemborexant.

[0204]

[0221] In some embodiments, neurodegeneration is reduced or maintained for at least 45 days after administration of the first dose of lemborexant, hi some embodiments, the amount of p-tau in the subject's CSF is reduced or maintained for at least 45 days after administration of the first dose of lemborexant.

[0205]

[0222] In some embodiments, neurodegeneration is reduced or maintained within 60 days of administration of the first dose of lemborexant, hi some embodiments, the amount of p-tau in the subject's CSF is reduced or maintained for at least 60 days after administration of the first dose of lemborexant.

[0206]

[0223] In some embodiments, neurodegeneration is reduced or maintained for at least 120 days following administration of the first dose of lemborexant, hi some embodiments, the amount of p-tau in the subject's CSF is reduced or maintained for at least 120 days following administration of the first dose of lemborexant.

[0207]

[0224] In some embodiments, neurodegeneration is reduced or maintained for at least 180 days following administration of the first dose of lemborexant, hi some embodiments, the amount of p-tau in the subject's CSF is reduced or maintained for at least 180 days following administration of the first dose of lemborexant.

[0208]

[0225] In some embodiments, the effect of lemborexant on neurodegeneration or p-tau becomes apparent after a period of treatment, e.g., after 3 months, 6 months, or 9 months. In some embodiments, neurodegeneration begins to decrease after at least 6 months of treatment. In some embodiments, neurodegeneration decreases after at least 3 months of treatment, e.g., after 6 months or 9 months of treatment. In some embodiments, the amount of p-tau in the subject's CSF is reduced or maintained after at least a 3-month period. In some embodiments, the amount of p-tau in the subject's CSF is reduced or maintained after at least a 6-month period of treatment or after at least a 9-month period of treatment. In some embodiments, neurodegeneration is reduced or maintained for at least 6 months after administration of the first dose of lemborexant. In some embodiments, the amount of p-tau in the subject's CSF is reduced or maintained for at least 6 months after administration of the first dose of lemborexant.

[0209]

[0226] In some embodiments, neurodegeneration is reduced for at least one year after administration of the first dose of lemborexant. In some embodiments, the amount of p-tau in the subject's CSF is reduced for at least one year after administration of the first dose of lemborexant. In some embodiments, the amount of t-tau in the subject's CSF is reduced for at least one year after administration of the first dose of lemborexant. In some embodiments, the amount of Aβ in the subject's CSF is reduced for at least one year after administration of the first dose of lemborexant. In some embodiments, the amount of fibrous plaques is reduced for at least one year after administration of the first dose of lemborexant. In some embodiments, the amount of activated microglial cells is increased for at least one year after administration of the first dose of lemborexant.

[0210] [A. Alters neurodegeneration]

[0227] Accordingly, one aspect of the present disclosure relates to a method for altering neurodegeneration (e.g., reducing or delaying neurodegeneration or slowing its rate of growth) in a subject having or at risk of developing AD, the method comprising administering to the subject a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, sufficient to alter neurodegeneration in the subject.

[0211]

[0228] In some embodiments, altering neurodegeneration includes slowing the progression, slowing the rate of progression, retarding progression, preventing the onset, and reversing the progression of neurodegeneration. In some embodiments, altering neurodegeneration includes preventing the onset or occurrence of pathology resulting from or consistent with neurodegeneration. Altering neurodegeneration can reduce, retard, or slow the rate of onset and / or progression of symptoms of this pathology. In some embodiments, altering neurodegeneration includes alleviating or ameliorating one or more symptoms of neurodegeneration and / or improving one or more clinical indicators of pathology resulting from or consistent with neurodegeneration (e.g., cognitive function, brain amyloid or tau levels and / or biomarker expression). In some embodiments, altering neurodegeneration includes preventing the onset or recurrence of one or more symptoms of neurodegeneration.

[0212]

[0229] In some embodiments, the subject is amyloid-negative. The subject may have mild cognitive impairment or mild dementia. In some embodiments, the subject does not show signs of dementia and / or cognitive impairment.

[0213]

[0230] In some embodiments, neurodegeneration is altered relative to a reference. Thus, altering neurodegeneration can include reducing and / or delaying neurodegeneration and / or slowing its rate compared to the reference. In some embodiments, the reference is a baseline measurement from a subject before treatment. In some embodiments, the reference is a baseline measurement from a control subject. The reference can be measurements obtained from multiple control subjects and used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0214]

[0231] In some embodiments, the neurodegeneration is characterized by at least one of a decrease in cortical thickness or a decrease in hippocampal volume. In some embodiments, altering neurodegeneration comprises maintaining or slowing the decrease in cortical thickness in the subject. In some embodiments, the neurodegeneration is characterized by at least one of a decrease in pyramidal neurons in the cortex or a decrease in pyramidal or granule neurons in the hippocampus. In some embodiments, altering neurodegeneration comprises maintaining or slowing the decrease in hippocampal volume in the subject. In some embodiments, altering neurodegeneration comprises maintaining or reducing the decrease in pyramidal or granule neurons. In some embodiments, altering neurodegeneration comprises reducing the rate of neurodegeneration. In some embodiments, altering neurodegeneration comprises altering neurofilament light chain (NfL) levels. The level of NfL can be altered in the blood and / or CSF of the subject.

[0215] VIII. Selecting Subjects for Treatment

[0232] A further aspect of the present disclosure relates to a method of selecting a subject having or at risk of developing Alzheimer's disease (AD) for treatment with lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, the method comprising: (a) obtaining from the subject a measure of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial response, and biomarker expression; (b) comparing the measure from the subject with a measure from a reference; and (c) selecting the subject for treatment with lemborexant if the measure from the subject differs from the measure from the reference.

[0216]

[0233] In some embodiments, the subject has mild cognitive impairment or mild dementia. In some embodiments, the subject does not show signs of dementia and / or cognitive impairment.

[0217]

[0234] In some embodiments, the subject is at risk for further Aβ accumulation. The subject may be at risk for the spread of tau pathology. The subject may be at risk for cognitive decline. In some embodiments, the subject may have intermediate levels of amyloid PET (e.g., about 20-40 centiloids). In some embodiments, the subject may have high levels of amyloid PET (e.g., >40 centiloids). In some embodiments, the subject may be an ApoE4 carrier. In some embodiments, the subject may have one or more risk factors for developing AD, such as having a family history of AD or dementia in a first-degree relative, being 65 years of age or older, being female, having or recovering from a traumatic brain injury, and suffering from other conditions, such as obesity, diabetes, heart disease and / or vascular disease.

[0218]

[0235] In some embodiments, the subject has early stage AD. In some embodiments, the subject has pre-AD. In some embodiments, the subject has been diagnosed with AD based on brain imaging, cognitive function, and / or biomarker criteria.

[0219]

[0236] In some embodiments, obtaining at least one measure comprises obtaining data from a brain scan of the subject and / or obtaining data from a biological sample from the subject, in some embodiments, the brain scan data may be indicative of levels of tau phosphorylation, tau aggregation, Aβ plaque burden, and / or microglial response.

[0220]

[0237] In some embodiments, the biological sample is a bodily fluid, hi some embodiments, the bodily fluid is cerebrospinal fluid (CSF), blood, or saliva.

[0221]

[0238] In some embodiments, the reference is a control. In some embodiments, the reference is a baseline measurement from a control subject. The reference can be measurements obtained from multiple control subjects and used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0222]

[0239] In some embodiments, the control does not have AD. Measurements from the subject will be higher than measurements from controls without AD. Measurements from the subject will be lower than measurements from controls with AD.

[0223]

[0240] In some embodiments, the control has AD. The measured value from the subject is equal to or greater than the measured value from the control with AD. The measured value from the subject can be equal to or less than the measured value from the control with AD.

[0224]

[0241] In some embodiments, the measure of tau phosphorylation comprises a measure of phosphorylation at one or more of T181, T217, S202, or S205.

[0225]

[0242] In some embodiments, the measure of tau aggregation comprises a measure of insoluble tau aggregates (eg, neurofibrillary tangles (NFTs)).

[0226]

[0243] In some embodiments, measurements of neurodegeneration include measurements of cortical thickness and / or hippocampal volume, or measurements of pyramidal or granular neuron loss.

[0227]

[0244] In some embodiments, the measure of Aβ plaque burden comprises a measure of Aβ plaque volume and / or Aβ plaque volume growth.

[0228]

[0245] In some embodiments, the measure of Aβ plaque burden comprises a measure of amyloid PET signal in a brain region of the subject or a measure of Aβ in the CSF of the subject.

[0229]

[0246] In some embodiments, the measure of microglial response is a change in expression of at least one microglial marker. The microglial marker can be Iba1, Clec7a, CD68, TMEM119, or P2RY12. In some embodiments, the measure of microglial response is a measure of microglial phagocytosis.

[0230] [IX. Therapeutic Effects]

[0247] Disclosed herein are methods of treating a subject with lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, and methods of monitoring the effectiveness of treatment in a subject.

[0231]

[0248] One aspect of the present disclosure relates to a method for monitoring the effectiveness of a treatment in a subject having Alzheimer's disease (AD) or at risk of developing AD, the method comprising: (a) obtaining from the subject a first measure of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression; (b) administering to the subject a dose of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof; (c) obtaining from the subject a second measure of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression; and (d) comparing the second measure from the subject with the first measure from the subject, wherein a difference between the first measure and the second measure indicates effective treatment with lemborexant.

[0232]

[0249] Another aspect of the present disclosure relates to a method of treating a subject having or at risk of developing Alzheimer's disease (AD), the method comprising: (a) obtaining from the subject a first measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial response, and biomarker expression; (b) administering to the subject a first dose of lemborexant, a pharmaceutically acceptable salt, or a solvate thereof; (c) obtaining from the subject a second measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression; (d) comparing the second measurement from the subject to the first measurement; and (d) administering a second dose of lemborexant if the first measurement differs from the second measurement in the comparison measurements. The first and second measurements may differ in that the second measurement is higher than the first measurement. Instead, the second measurement will be lower than the first measurement. For example, if the measurement is a microglial response, in some embodiments, the first measurement of the microglial response will be higher than the second measurement of the microglial response.

[0233]

[0250] Yet another aspect of the present disclosure relates to a method for monitoring the effectiveness of a treatment in a subject having Alzheimer's disease (AD) or at risk of developing AD, the method comprising: (a) obtaining from the subject a first measure of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression; (b) administering to the subject a dose of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof; (c) obtaining from the subject a second measure of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression; and (d) comparing the second measure from the subject with the first measure from the subject to obtain a comparative measure, wherein a difference in the comparative measure between the first measure and the second measure or a difference between the comparative measure and a reference measure indicates effective treatment with lemborexant.

[0234]

[0251] Another aspect of the present disclosure relates to a method of treating a subject having or at risk of developing Alzheimer's disease (AD), the method comprising: (a) obtaining from the subject a first measure of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial response, and biomarker expression; (b) administering to the subject a first dose of lemborexant, a pharmaceutically acceptable salt, or a solvate thereof; (c) obtaining from the subject a second measure of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression; (d) comparing the second measure from the subject with the first measure from the subject to obtain a comparative measure; and (e) administering the second dose of lemborexant if the comparative measure differs from the second measure in the comparative measure or if the comparative measure differs from a reference measure.

[0235]

[0252] In some embodiments, obtaining at least one measure comprises obtaining data from a brain scan of the subject and / or obtaining data from a biological sample from the subject. In some embodiments, the brain scan data indicates levels of tau phosphorylation, tau aggregation, Aβ plaque burden, and / or microglial response. In some embodiments, the biological sample is a bodily fluid. In some embodiments, the bodily fluid is cerebrospinal fluid (CSF), blood, or saliva.

[0236]

[0253] In some embodiments, the first measurement from the subject is higher than the second measurement from the subject, hi some embodiments, the first measurement from the subject is lower than the second measurement from the subject.

[0237]

[0254] In some embodiments, for example, in those methods including a comparative measurement and a reference measurement, the reference measurement is obtained from at least one control. In some embodiments, the reference measurement is a comparison of a first measurement from the control with a second measurement from the control. In some embodiments, the comparative measurement is higher than the reference measurement. In some embodiments, the comparative measurement is lower than the reference measurement. For example, a comparative measurement comparing a first measurement from a subject with a second measurement from the subject may indicate that no change has occurred, while the reference measurement may indicate a change that has occurred in the control. Thus, the difference between the comparative measurement and the reference measurement may indicate whether treatment has been effective and / or whether a second dose of lemborexant needs to be administered.

[0238]

[0255] In some embodiments, the reference measurement is a measurement from a control. Reference measurements can be obtained from multiple control subjects and are used as standard or threshold measurements. In some embodiments, the reference measurement is a measurement from a control subject administered a placebo.

[0239]

[0256] In some embodiments, the control does not have AD. In some embodiments, the comparative measurement is higher than the reference measurement. In some embodiments, the comparative measurement is lower than the reference measurement.

[0240]

[0257] In some embodiments, the control has AD, e.g., untreated AD. In some embodiments, the comparative measurement is higher than the reference measurement. In some embodiments, the comparative measurement is lower than the reference measurement.

[0241]

[0258] In some embodiments, the measure of tau phosphorylation comprises a measure of phosphorylation of one or more of T181, T217, S202, S205, or T231.

[0242]

[0259] In some embodiments, the measure of tau aggregation comprises a measure of insoluble tau aggregates (eg, neurofibrillary tangles (NFTs)).

[0243]

[0260] In some embodiments, measurements of neurodegeneration include measurements of cortical thickness and / or hippocampal volume, or measurements of pyramidal or granular neuron loss.

[0244]

[0261] In some embodiments, the measure of Aβ plaque burden comprises a measure of Aβ plaque volume and / or Aβ plaque volume growth.

[0245]

[0262] In some embodiments, the measure of Aβ plaque burden comprises a measure of amyloid PET signal in a brain region of the subject or a measure of Aβ in the CSF of the subject.

[0246]

[0263] In some embodiments, the measure of microglial response is the degree of expression of at least one microglial marker. The microglial marker can be Iba1, Clec71, P2RY12, or TMEM119. In some embodiments, the measure of microglial response is a measure of microglial phagocytosis.

[0247]

[0264] In some embodiments, the measure of biomarker expression is a measure of Ifnb1, MMP2 and / or Base1 expression.

[0248]

[0265] In some embodiments, the subject is amyloid-negative.

[0249]

[0266] In some embodiments, the subject has Aβ plaques.

[0250]

[0267] In some embodiments, the subject has mild cognitive impairment or mild dementia. In some embodiments, the subject does not show signs of dementia and / or cognitive impairment.

[0251]

[0268] In some embodiments, the subject is at risk for further Aβ accumulation. The subject may be at risk for the spread of tau pathology. The subject may be at risk for cognitive decline. In some embodiments, the subject may have intermediate levels of amyloid PET (e.g., about 20-40 centiloids). In some embodiments, the subject may have high levels of amyloid PET (e.g., >40 centiloids). In some embodiments, the subject may be an ApoE4 carrier. In some embodiments, the subject may have one or more risk factors for developing AD, such as having a family history of AD or dementia in a first-degree relative, being 65 years of age or older, being female, having or recovering from a traumatic brain injury, and suffering from other conditions, such as obesity, diabetes, heart disease and / or vascular disease.

[0252]

[0269] In some embodiments, the subject has early stage AD. In some embodiments, the subject has pre-AD. In some embodiments, the subject has been diagnosed with AD based on brain imaging, cognitive function, and / or biomarker criteria.

[0253] [X.Dose]

[0270] As disclosed herein, a dose of lemborexant may refer to a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt, or a solvate thereof. In some embodiments, the methods disclosed herein comprise orally administering to a subject a dose of 5 mg to 20 mg of lemborexant once per day. In some embodiments, a 20 mg dose of lemborexant is administered to a subject once per day. In some embodiments, a 25 mg dose of lemborexant is administered to a subject once per day. In some embodiments, a 25 mg dose of lemborexant is administered to a subject once per day for at least two days. In some embodiments, a 25 mg dose of lemborexant is administered to a subject once per day. In some embodiments, a 25 mg dose of lemborexant is administered to a subject once per day for at least five days. In some embodiments, a 25 mg dose of lemborexant is administered to a subject once per day. In some embodiments, a 25 mg dose of lemborexant is administered to a subject once per day for at least one week. In some embodiments, a 25 mg dose of lemborexant is administered to a subject once per day. In some embodiments, a 25 mg dose of lemborexant is administered to a subject once per day for at least one month.

[0254]

[0271] In some embodiments, the methods disclosed herein comprise orally administering to a subject a dosage form comprising lemborexant. In some embodiments, the methods disclosed herein comprise orally administering to a subject a dosage form comprising 10 mg of lemborexant. In some embodiments, the methods disclosed herein comprise orally administering to a subject a dosage form comprising 15 mg of lemborexant. In some embodiments, the methods disclosed herein comprise orally administering to a subject a dosage form comprising 20 mg of lemborexant. In some embodiments, the methods disclosed herein comprise orally administering to a subject a dosage form comprising 25 mg of lemborexant. In some embodiments, the methods disclosed herein comprise orally administering to a subject a dosage form comprising 30 mg of lemborexant. In some embodiments, the methods disclosed herein comprise orally administering to a subject a dosage form comprising 35 mg of lemborexant. In some embodiments, the methods disclosed herein comprise orally administering to a subject a dosage form comprising 40 mg of lemborexant. In some embodiments, the methods disclosed herein comprise orally administering to a subject a dosage form comprising 45 mg of lemborexant. In some embodiments, the methods disclosed herein comprise orally administering to a subject a dosage form comprising 50 mg of lemborexant.

[0255]

[0272] The dosage forms of the present disclosure comprise a therapeutically effective amount of lemborexant when administered for treatment in accordance with the instructions of the present disclosure. The effective unit dose in the dosage form is 0.5 mg to 100 mg, 2 mg to 75 mg, 2 mg to 70 mg, 2 mg to 65 mg, 2 mg to 60 mg, 2 mg to 55 mg, 2 mg to 50 mg, 2 mg to 45 mg, 2 mg to 40 mg, 2 mg to 35 mg, 2 mg to 30 mg, 2 mg to 25 mg, 2 mg to 20 mg, 2 mg to 15 mg, 2 mg to 15 mg, 2 mg, 2.5 mg, 4 mg, 5 mg, 8 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg. The unit dose is not limited by the type of dosage form or the number of dosage forms for a single administration. In some embodiments, the unit dose can be 2.5 mg. In some embodiments, the unit dose can be 5 mg. In some embodiments, the unit dose may be 10 mg. In some embodiments, the unit dose may be 7.5 mg. In some embodiments, the unit dose may be 12.5 mg. In some embodiments, the unit dose may be 15 mg. In some embodiments, the unit dose may be 18 mg. In some embodiments, the unit dose may be 20 mg. In some embodiments, the unit dose may be 22 mg. In some embodiments, the unit dose may be 25 mg. In some embodiments, the unit dose may be 30 mg. In some embodiments, the unit dose may be 32 mg. In some embodiments, the unit dose may be 35 mg. In some embodiments, the unit dose may be 40 mg. In some embodiments, the unit dose may be 50 mg.

[0256]

[0273] Thus, the therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof administered herein can include a dose that falls within a range, for example, 5 mg to 50 mg per day.

[0257]

[0274] In some embodiments, the therapeutically effective amount of lemborexant administered to a subject ranges from 5 mg to 50 mg per day. For example, the therapeutically effective amount of lemborexant administered to a subject can range from 10 mg to 30 mg per day. In some embodiments, the therapeutically effective amount of lemborexant administered to a subject is selected from 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, and 30 mg per day.

[0258]

[0275] In some embodiments, the therapeutically effective amount of lemborexant administered to a subject is 20-25 mg per day.

[0259]

[0276] In some embodiments, a 20 mg dose of lemborexant is administered to a subject once per day.

[0260]

[0277] In some embodiments, lemborexant is administered at a first dose for a first period of time, at a second dose for a second period of time, and optionally at a third dose for a third period of time, wherein the first period of time, the second period of time, and the third period of time may each be one week.

[0261]

[0278] In some embodiments, the first dose is lower than the second dose, and optionally, the second dose is lower than the third dose. For example, in some embodiments, the first dose is 5 mg of lemborexant once daily, the second dose is 10 mg of lemborexant once daily, and optionally, the third dose is 20-25 mg of lemborexant once daily. In some embodiments, the first dose is 5 mg or 7.5 mg of lemborexant once daily, the second dose is 10 mg, 12.5 mg, 15 mg, or 17.5 mg of lemborexant once daily, and the third dose is 20 mg, 22.5 mg, 25 mg, 27.5 mg, or 30 mg of lemborexant once daily.

[0262]

[0279] In some embodiments, the first dose is higher than the second dose, and optionally, the second dose is higher than the third dose. For example, in some embodiments, the first dose is 20-25 mg of lemborexant once daily, the second dose is 10 mg of lemborexant once daily, and optionally, the third dose is 5 mg of lemborexant once daily. In some embodiments, the first dose is 20 mg, 22.5 mg, 25 mg, 27.5 mg, or 30 mg of lemborexant once daily, the second dose is 10 mg, 12.5 mg, 15 mg, or 17.5 mg of lemborexant once daily, and optionally, the third dose is 5 mg or 7.5 mg of lemborexant once daily.

[0263]

[0280] In some embodiments, lemborexant can be administered to a subject over a period of time. In some embodiments, the methods described herein comprise administering lemborexant to a subject for at least 6 months. In some embodiments, the methods described herein comprise administering lemborexant to a subject for at least 9 months, at least 12 months, or at least 15 months. In some embodiments, the methods described herein comprise administering lemborexant to a subject for at least 18 months. In some embodiments, the methods described herein comprise administering lemborexant to a subject for at least 24 months, 30 months, or 36 months. In some embodiments, lemborexant can be administered to a subject for life.

[0264] XI. Pharmaceutical Compositions

[0281] In some embodiments, dosage forms of the present disclosure may comprise one or more pharmaceutical compositions containing lemborexant together with pharmaceutically acceptable excipients.

[0265]

[0282] As used herein, the term "composition" includes a product containing specific amounts of specific ingredients and any product resulting directly or indirectly from the combination of specific amounts of specific ingredients. Such terms, in reference to pharmaceutical compositions, are intended to include products containing active ingredients and inactive ingredients that constitute a carrier, and include any product resulting directly or indirectly from the combination, complexation, or aggregation of any two or more ingredients, or the dissociation of one or more ingredients, or any other type of reaction or interaction. Thus, pharmaceutical compositions of the present disclosure include any composition prepared by mixing a compound of the present disclosure with a pharmaceutically acceptable carrier.

[0266]

[0283] As used herein, the term "pharmaceutically acceptable" means the carrier, diluent, excipient or vehicle is compatible with the other ingredients of the formulation and non-toxic to the subject.

[0267]

[0284] Solid dosage forms of the present disclosure include capsules, granules, lozenges, pellets, pills, powders, suspensions, and tablets.

[0268]

[0285] The pharmaceutical compositions of the present disclosure can be prepared using standard techniques and manufacturing processes generally known in the art (see, for example, monographs: Japanese Pharmacopoeia, 16th Edition; and Pharmaceutical Dosage Forms of US Pharmacopoeia-NF, Chapter 1151).

[0269]

[0286] In some embodiments, the pharmaceutical composition comprises lemborexant. In some embodiments, the pharmaceutical composition further comprises at least one additional ingredient selected from a pharmaceutically acceptable carrier, a pharmaceutically acceptable vehicle, and a pharmaceutically acceptable excipient.

[0270]

[0287] In some embodiments, the at least one additional component in the pharmaceutical composition is selected depending on the intended route of administration of the pharmaceutical composition. Non-limiting examples of suitable routes of administration for which the pharmaceutical composition can be used include parenteral administration, oral administration, inhalation spray administration, topical administration, rectal administration, nasal administration, buccal administration, intravaginal administration, and administration via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intracisternal, intraspinal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In some embodiments, the mode of administration is selected from intravenous, oral, subcutaneous, and intramuscular administration. Sterile injectable forms of the compositions of the present disclosure may be, for example, aqueous or oily suspensions. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents known in the art. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, solutions in 1,3-butanediol. Non-limiting examples of vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils can be used as solvents and / or suspending media.

[0271]

[0288] For this purpose, any sterile fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful for preparing injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or oil suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers, or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, and / or other dosage forms, can also be used for formulation purposes.

[0272]

[0289] For oral administration, lemborexant can be provided in any acceptable oral dosage form, including, but not limited to, suspensions, capsules, tablets, orally disintegrating tablets, sprinkles, and other oral formulations that are easy to swallow. In some embodiments, lemborexant is provided in the form of a tablet or capsule. In some embodiments, lemborexant is provided in the form of a crushable tablet. For tablets for oral use, commonly used carriers include lactose and cornstarch. Lubricants, such as magnesium stearate, can also be added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and / or suspending agents. Specific sweeteners, flavorings, or coloring agents can also be added, if desired.

[0273]

[0290] In order that the disclosure described herein may be more fully understood, the following examples are set forth, it being understood that these examples are for illustrative purposes only and should not be construed as limiting the disclosure in any way.

[0274] Non-limiting embodiments of the present disclosure: 1. A method for reducing or maintaining the amount of p-tau or t-tau in a subject, comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof. 2. The method of embodiment 1, wherein the amount of p-tau or t-tau in the subject is reduced or maintained relative to the subject's baseline. 3. The method of embodiment 1 or 2, wherein the amount of p-tau or t-tau in the subject is reduced or maintained compared to placebo. 4. The method of any one of embodiments 1-3, wherein the ratio of p-tau to tau in the subject is lowered relative to the subject's baseline or relative to placebo following administration of a therapeutically effective amount of lemborexant. 5. The method of any one of embodiments 1-4, wherein the rate of tau dephosphorylation in the subject is increased following administration of a therapeutically effective amount of lemborexant relative to the subject's baseline or relative to placebo. 6. The method of any one of embodiments 1-5, wherein the rate of tau phosphorylation in the subject is reduced following administration of a therapeutically effective amount of lemborexant relative to the subject's baseline or relative to placebo. 7. The method of any one of embodiments 1-6, wherein the ratio of p-tau / t-tau in the subject's CSF is reduced compared to the CSF p-tau / t-tau ratio before administration of lemborexant. 8. The method of embodiment 7, wherein the ratio of p-tau / t-tau in the subject's CSF is maintained within 10% of the subject's ratio of p-tau / t-tau before administration of lemborexant. 9. The method of any one of embodiments 1-8, wherein the concentration of Aβ in the subject's CSF is equal to or less than the concentration of Aβ in the subject's CSF prior to administration of lemborexant. 10. The method of embodiment 9, wherein the amyloid PET signal in the brain of the subject administered a therapeutically effective amount of lemborexant is equal to or less than the amyloid PET signal in the brain of the subject prior to administration of lemborexant. 11. The method of any one of embodiments 1-10, wherein the tau PET signal in the subject's brain is decreased relative to baseline. 12. The method of any one of embodiments 1 to 11, wherein tau is p-tau, t-tau or aggregated tau. 13. The method of any one of embodiments 1 to 12, wherein p-tau and t-tau are reduced. 14. The method of any one of embodiments 1-13, wherein the reduction in tau phosphorylation occurs in the hippocampus, entorhinal cortex and / or piriform cortex. 15. The method of any one of embodiments 1-14, wherein the Aβ in the subject's CSF is Aβ38, Aβ40 and / or Aβ42. 16. The method of any one of embodiments 1-15, wherein the amount of p-tau is reduced relative to the subject's baseline within 48 hours of administering to the subject a first dose of lemborexant. 17. A method for reducing neurodegeneration in a subject, comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof. 18. The method of embodiment 17, wherein the reduction in neurodegeneration is observed by maintenance of or a reduction in the loss of cortical thickness relative to the subject's baseline or to subjects administered a placebo. 19. The method of embodiment 17 or 18, wherein the reduction in neurodegeneration is observed by a maintenance of, or a reduction in the decrease in, hippocampal size relative to the subject's baseline or to subjects administered a placebo. 20. The method of any one of embodiments 17-19, wherein the reduction in neurodegeneration is observed by preservation of or a reduction in the loss of pyramidal neuron cells relative to the subject's baseline or to subjects administered a placebo. 21. The method of any one of embodiments 17-20, wherein the reduction in neurodegeneration is observed by preservation of or a reduction in the loss of granule neuron cells relative to the subject's baseline or to subjects administered a placebo. 22. The method of any one of embodiments 1-21, wherein the therapeutically effective amount of lemborexant administered to the subject ranges from 10 mg to 50 mg per day. 23. The method of embodiment 22, wherein the therapeutically effective amount of lemborexant administered to the subject ranges from 15 mg to 30 mg per day. 24. The method of embodiment 22, wherein the therapeutically effective amount of lemborexant administered to the subject is 25 mg per day. The method of any one of embodiments 1-24, wherein one dose of 25.25 mg of lemborexant is administered to the subject once per day. The method of embodiment 25, wherein one dose of 26.25 mg of lemborexant is administered to the subject once per day for at least two days. 27. The method of any one of embodiments 1-26, wherein a 2.5 mg, 5 mg, 10 mg, 15 mg, or 20 mg dose of lemborexant is administered to the subject once per day following administration of a 25 mg dose of lemborexant once per day for at least two days. 28. A method for reducing the concentration of Aβ in a subject, comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof. 29. The method of any one of embodiments 1-28, wherein the concentration of Aβ in the subject's CSF is lower than the concentration of Aβ in the subject's CSF before administration of lemborexant. 30. The method of any one of embodiments 1-29, wherein the amyloid PET signal in the brain of the subject administered lemborexant is lower than the amyloid PET signal in the brain of the subject prior to administration of lemborexant. 31. The method of embodiment 30, wherein the Aβ in the subject's CSF is Aβ38, Aβ40 and / or Aβ42. 32. The method of any one of embodiments 1-29, wherein the concentration of Aβ is reduced within 48 hours of administration of lemborexant. 33. A method for increasing the number of activated microglial cells in a subject, comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof. 34. The method of embodiment 33, wherein the number of activated microglial cells is increased relative to baseline. 35. The method of any one of embodiments 33 or 34, wherein the activated microglial cells are microglial phagocytes. 36. The method of any one of embodiments 33 to 35, wherein the number of activated microglial cells is measured by PET or CSF biomarkers of microglial activation. 37. The method according to any one of embodiments 33 to 36, wherein the whole brain or at least one region of the brain is analyzed by PET. 38. The method of embodiment 37, wherein at least one region of the brain analyzed by PET is selected from cortical gray matter, lateral ventricles, frontal lobe, parietal lobe, temporal lobe, occipital lobe, cingulate cortex, amygdala, piriform cortex, entorhinal cortex, hippocampus, hippocampal CA3 (pyramidal neurons), and hippocampal dentate gyrus (granule cell neurons). [Example]

[0275] Example 1 Clinical Study Protocol [a. Study 1: Acute effects of lemborexant on CSF amyloid beta and tau]

[0291] The acute effects of lemborexant will be investigated in cognitively normal amyloid-positive participants.

[0276]

[0292] The selection criteria are as follows: Age 60-80 · Gender-neutral Any race / ethnicity Mini-Mental Status Examination score (MMSE) ≥ 27 Positive plasma Aβ test (i.e., amyloid positive) Pittsburgh Sleep Quality Index >5

[0277]

[0293] The exclusion criteria were as follows: Cognitive impairment <27 as determined by MMSE history · Can't speak or understand English Sleep disorders other than insomnia - No history of moderate to severe sleep-disordered breathing and a STOP-Bang score >5 - History or reported symptoms suggestive of restless legs syndrome, narcolepsy, or other sleep disorders - Mild sleep apnea (AHI<16) on PSG - Sleep schedule outside of 10:00 PM to midnight Contraindications for lumbar catheterization (anticoagulants; bleeding disorders; allergies to lidocaine or disinfectants; previous central nervous system or lumbar surgery) Cardiovascular disease requiring medication other than controlled hypertension (PI determination) Stroke Liver or kidney damage ·Lung disease (PI determination) ·Type 1 diabetes HIV or AIDS Neurological or psychiatric disorders requiring medication (PI determination) Suicidal thoughts Alcohol or tobacco use (PI determination) Use of sedatives (PI determination) Can't get out of bed by myself In the opinion of the investigator, participants should be excluded due to abnormalities on physical examination. ·Pregnant Body Mass Index > 35 History of migraine (PI determination) - Substance abuse history in the past 6 months · History or presence of clinically significant medical condition, behavioral or psychiatric disorder (including suicidal ideation) or surgical history based on medical records or patient reports that may affect the subject's safety or interfere with the study evaluation or judgment of the PI participant are not appropriate candidates. Urinary or fecal incontinence Concurrent enrollment of an investigational drug or device in another trial

[0278]

[0294] Twelve (or more) participants will be randomly assigned to receive placebo (N=4 or more) or lemborexant 25 mg (N=8 or more).

[0279]

[0295] Procedure: Randomized participants will be admitted to the hospital in the early afternoon (Night 1). All participants will have their sleep monitored with an unattended full-montage PSG (TrackIt™; Lifelines, Troy, IL). This full-montage PSG allows for sleep stage classification according to the gold-standard American Academy of Sleep Medicine criteria and has already been used in similar studies to monitor 36–48 h of sleep.

[0280]

[0296] At approximately 8:00 PM, a lumbar catheter and two IVs will be placed in each participant to collect 6 ml of CSF every 2 hours for 48 hours. The lumbar catheter port will be placed on the outside of the gown sleeve for easy access to minimize disturbance during fluid collection. The start time of sampling will be: 13To allow for frequent blood sampling before bedtime, the C6-leucine infusion and sleep monitoring began approximately one hour before typical bedtime, as defined by each participant's sleep log. Six milliliters of blood were collected at the following time points: 0 (baseline), 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, and 48 hours (Table 1).

[0281] [Table 1]

[0282]

[0297] Approximately 1 hour before habitual bedtime on day 1 (t = 0), all participants received 800 mg of labeled Aβ during intracellular translation to monitor Aβ dynamics. 13 Infusion of C6-leucine is initiated to label proteins in vivo.

[0283]

[0298] Participants will be asked to sleep immediately at their usual bedtime (bedtime is time 1, approximately 22:00–00:00) with the lights off. A dim red light (safety light) will be used when collecting CSF and blood samples in the dark. Participants will sleep until their final awakening in the morning.

[0284]

[0299] On day 2, all participants were kept awake in a well-lit room, monitored regularly, and not allowed to nap. At approximately 22:00 (hour 25) on day 2, all participants followed the same sleep routine as the previous night, but were not allowed to nap. 13 No C6-leucine infusion will be administered, and the time of "Time 26" will be determined by the participant's usual bedtime. Participants will receive the same dose of placebo or lemborexant as the previous night. The study will end at approximately 8:00 PM on Day 3 (Time 48), at which point the IV and lumbar catheters will be removed. All participants will then sleep overnight, be monitored for at least 8 hours after catheter removal, and then be discharged.

[0285]

[0300] CSF tau, phosphorylated tau, and Aβ dynamics are quantified by mass spectrometry. To determine each participant's baseline, AD biomarkers (Aβ38, Aβ40, Aβ42, T181, S202, T217, pT181, pS202, pT217) are normalized to the average of the first 6 hours (t = 0-6, 20:00-02:00) before intervention-induced changes are seen in the CSF. Concentrations at time 0 are also normalized. Phosphorylated tau ratios (T181 / T181, pS202 / S202, pT217 / T217, p-tau / t-tau) are normalized to the first time point (time 0). Trajectories of these changes from baseline over time are plotted by treatment group to determine whether changes in concentrations or ratios are linear.

[0286]

[0301] For statistical analysis, if the assumption of linearity is valid, a (linear mixed effects) LME model with a random intercept and slope is used for the analysis; otherwise, a mixed model for repeated measures (MMRM) is used. Fixed effects in the model include treatment group, time, and their interaction. The baseline is included as a covariate. The assumption of normality is examined using residual plots and appropriate transformations (e.g., log) are considered. If an unstructured covariance matrix is ​​used and convergence is an issue, various other covariance matrix structures (e.g., compound symmetry, first-order autoregressive) are compared and the best structure is selected for the final analysis based on the Akaike Information Criterion (AIC).

[0287] b. Study 2: Treatment of Subjects with Preclinical or Early Alzheimer's Disease

[0302] The study will evaluate the efficacy of lemborexant in preventing or slowing Aβ accumulation, downstream spread of tau pathology, and cognitive decline across the preclinical and early AD continuum.

[0288]

[0303] Amyloid-β (Aβ) accumulation often begins more than 10 years before the clinical stage of Alzheimer's disease (AD) and is thought to play a key role in accelerating the spread of tauopathy and neurodegeneration in the preclinical stages of AD. Multiple neuroimaging and biomarker observational studies have shown that Aβ accumulation is associated with an increased risk of cognitive decline in clinically normal older adults.

[0289]

[0304] The study will utilize NAV4694 (flutafuranol) amyloid PET imaging to assess fibrillar amyloid pathology for eligibility and long-term outcomes, and MK6240 tau PET tracer to assess the spread of neurofibrillary tangles and tau neurite pathology over time. Clinical outcomes will include the Preclinical Alzheimer Cognitive Composite-5 (PACC-5), which is comprised of the Free and Cued Selective Reminding Test, Paragraph Recall IIa, Digit-Symbol, MMSE, and Semantic Category Fluency, as well as the Cognitive Function Index (CFI), a participant- and study partner-report of cognitive function.

[0290]

[0305] Additionally, patients will undergo cortical and hippocampal scans to assess the extent of neurodegeneration, and CSF and blood samples will be collected to measure AD biomarkers, including Aβ, tau, p-tau, and NfL.

[0291] [i. Group 1: Preclinical Alzheimer's disease - patients with intermediate levels of amyloid]

[0306] The purpose of this study is to determine whether lemborexant treatment provides primary prevention or delay of AD by preventing or slowing early Aβ accumulation in the brain. The study will enroll cognitively normal individuals with intermediate levels of amyloid on screening PET imaging (approximately 20-40 centimeters), considered to be in the earliest preclinical stage of AD, at risk for further Aβ accumulation and early spread of tau pathology over a 4-year period.

[0292]

[0307] Patient Inclusion Criteria: Select patients with an overall Clinical Dementia Rating (CDR) of 0 and cognitively intact cognitive function as determined by an education-adjusted Mini-Mental State Examination (MMSE) score of ≥ 27. Patients will have intermediate levels of amyloid (20-40 centimeters) on PET imaging.

[0293]

[0308] Lemborexant Administration. Participants will receive lemborexant or a matching placebo during monthly in-person study visits where treatment adherence and adverse events will be assessed. Lemborexant is FDA-approved for the treatment of insomnia at doses of 5 to 10 mg. Following an interim analysis of safety and efficacy, the lemborexant dose may be increased to 20 mg. At the start of the study, participants will take lemborexant 5 mg once daily for one week, then increase the dose to lemborexant 10 mg once daily; both lemborexant 5 mg and lemborexant 10 mg will have matching placebo tablets. At the end of the study, participants will take lemborexant 5 mg once daily for one week until discontinuation.

[0294]

[0309] Titration Dosing. Participants will be lemborexant-naive and will receive lemborexant 5 mg once daily for 1 week, then increased to lemborexant 10 mg once daily; there will be matching placebo tablets for both lemborexant 5 mg and lemborexant 10 mg. At the end of the study, participants will receive a reduction in lemborexant to 5 mg once daily for 1 week until discontinuation of the drug.

[0295]

[0310] If the lemborexant dose is increased to 20 mg after the interim analysis, the same titration and tapering schedule will be followed, except for an additional week of lemborexant 10 mg: Titration: lemborexant 5 mg - 1 week Lemborexant 10mg - 1 week Lemborexant 20mg - 1 week Tapering: lemborexant 10 mg - 1 week Lemborexant 5mg - 1 week ·cancel.

[0296]

[0311] Study outcomes. Estimates of primary, secondary, and exploratory outcome measures.

[0297]

[0312] Primary Outcome: The primary outcome measure of the study is amyloid PETSUVr measured at 6 months compared with placebo. Biomarker outcomes include tau PET, CSF and plasma measurements of Aβ, phosphorylated tau, and protofibrils.

[0298]

[0313] Secondary outcomes: measuring tau PET.

[0299]

[0314] Exploratory outcomes: Biomarkers Aβ, tau, phosphorylated tau, neurogranin (NG), and neurofilament light chain (NfL) will be measured in CSF. In plasma, NfL, phosphorylated tau 181, and phosphorylated tau 217 will be measured. Clinical outcomes to measure cognition include the Preclinical Alzheimer's Disease Cognitive Composite 5 (PACC5) scale for cognition and testing to obtain the Cognitive Functional Index (CFI).

[0300] [ii. Preclinical Alzheimer's Disease – High Levels of Amyloid]

[0315] The purpose of this study is to determine whether lemborexant treatment provides primary prevention or delay of AD by preventing early Aβ accumulation in the brain. The study will enroll cognitively normal individuals with elevated amyloid levels (approximately >40 cTc) on screening PET imaging, who are at high risk for cognitive decline over four years.

[0301]

[0316] Patient Inclusion Criteria: Select patients with a global Clinical Dementia Rating (CDR) of 0 and cognitively intact as determined by an education-adjusted Mini-Mental State Examination (MMSE) score of ≥ 27. Patients will have elevated levels of amyloid PET > 40.

[0302]

[0317] Lemborexant Administration. Participants will receive lemborexant or a matching placebo during monthly in-person study visits where treatment adherence and adverse events will be assessed. Lemborexant is FDA-approved for the treatment of insomnia at doses of 5 to 10 mg. Following an interim analysis of safety and efficacy, the lemborexant dose may be increased to 20 mg. At the start of the study, participants will take lemborexant 5 mg once daily for one week, then increase the dose to lemborexant 10 mg once daily; both lemborexant 5 mg and lemborexant 10 mg will have matching placebo tablets. At the end of the study, participants will take lemborexant 5 mg once daily for one week until discontinuation.

[0303]

[0318] Titration Dosing. Participants will be lemborexant-naive and will receive lemborexant 5 mg once daily for 1 week, after which lemborexant will be increased to 10 mg once daily; there will be matching placebo tablets for both lemborexant 5 mg and lemborexant 10 mg. At the end of the study, participants will receive a reduction in lemborexant to 5 mg once daily for 1 week until discontinuation of the drug.

[0304]

[0319] If the lemborexant dose is increased to 20 mg after the interim analysis, the same titration and tapering schedule will be followed, except for an additional week of lemborexant 10 mg: Titration: lemborexant 5 mg - 1 week Lemborexant 10mg - 1 week Lemborexant 20mg - 1 week Tapering: lemborexant 10 mg - 1 week Lemborexant 5mg - 1 week ·cancel.

[0305]

[0320] Study outcomes. Estimates of primary, secondary, and exploratory outcome measures.

[0306]

[0321] Primary Outcome: To examine the effect of lemborexant on cognitive decline in patients with elevated amyloidosis, the primary outcome measure of this study is the Preclinical AD Cognitive Composite 5 (PACC5) at 6 months. Biomarker outcomes include tau PET, and measurements of Aβ, phosphorylated tau, and protofibrils in CSF and plasma.

[0307]

[0322] Secondary outcomes: Cognitive Function Index (CFI), amyloid PET and tau PET will be measured.

[0308]

[0323] Exploratory outcomes. Clinical measures include ADCS ADL prevention, Computerized Cognitive Composite, ISLT, Trails, CDR-SB, and time to CDR0.5. vMRI and rs-MRI will be determined. Biomarkers Aβ, tau, phospho-tau, neurogranin (NG), and neurofilament light chain (NfL) will be measured in CSF. Plasma NfL, phospho-tau 181, and phospho-tau 217 will be measured.

[0309] c. Study 3: Treatment of Subjects with Preclinical or Early Alzheimer's Disease

[0324] This study will evaluate the efficacy of lemborexant in preventing or slowing Aβ accumulation, downstream spread of tau pathology, and cognitive decline across the preclinical and early AD continuum. This study will follow the protocol of Study 2, except different doses will be used: 7.5 mg of lemborexant will be used instead of 5 mg of lemborexant; 15 mg of lemborexant will be used instead of 10 mg of lemborexant; and 25 mg or 30 mg of lemborexant will be used instead of 20 mg of lemborexant.

[0310] Example 2: Mouse studies of tau-mediated neurodegeneration

[0325] P301S / E4 and E4 knock-in tau-free mice were orally gavaged with 30 mg / kg lemborexant or vehicle daily from 7.5 months to 9.5 months of age, when tau-mediated neuroinflammation without obvious neuronal loss was observed (Figure 2a).

[0311] [A. Changes in sleep-wake behavior]

[0326] Changes in sleep-wake behavior were verified by electroencephalography (EEG). E4 and P301S / E4 mice treated with lemborexant showed an approximately 25% increase in non-REM sleep time and an approximately 20% decrease in wake time (Figures 2B-2F). Consistent with previous findings, no changes in REM sleep time were observed.

[0312]

[0327] A tau-dependent reduction in non-REM sleep was observed in P301S / E4 mice compared with E4 mice (Figure 2B), indicating that pathological tau is associated with sleep. The effects of lemborexant on sleep (Figures 2G and 2H) and sleep-related locomotor activity (Figure 6), as measured by EEG and piezoelectric sleep pad, persisted for approximately 5 hours after treatment, without any additional phase delay or changes in circadian sleep-wake activity.

[0313]

[0328] B. Effects on Tau Pathology and Neurodegeneration

[0314]

[0329] To determine whether lemborexant could affect tau pathology and neurodegeneration, we examined the hippocampus, entorhinal cortex, and piriform cortex, regions that exhibit significant tau-mediated degeneration. In P301S / E4 mice treated with lemborexant, a significant reduction of approximately 20% in AT8+ and MC1+ tau staining was observed compared to vehicle-treated controls (Figures 3A-F). Additionally, P301S / E4 mice treated with lemborexant had significantly reduced levels of insoluble phosphorylated tau and total tau (Figures 7A-F).

[0315]

[0330] We found that lemborexant treatment significantly reduced the extent of brain atrophy in P301S / E4 mice compared with controls (Figures 3G-3I and 7H-7K). More specifically, atrophy of the hippocampus and piriform / entorhinal cortex was significantly reduced by approximately 50%, and lateral ventricle enlargement was reduced. In P301S / E4 mice treated with lemborexant, the neuronal layers of granule cells and pyramidal cells were significantly thickened (Figures 7G-7K), which was confirmed by reduced plasma neurofilament light chain levels (Figure 3J), demonstrating robust improvement in neuronal damage and degeneration.

[0316] [C. Microglial Responsiveness]

[0331] To investigate whether the marked reduction in tau-mediated neurodegeneration in mice treated with lemborexant is associated with reduced microglial responsiveness, we quantified different markers of reactive microglia across the spectrum of disease-associated or homeostatic populations in both the hippocampus and piriform cortex. Substantial changes were observed primarily in the hippocampus, most notably in the CA3 region (Figure 4).

[0317]

[0332] A significant decrease in ionized calcium-binding adaptor molecule 1 (IBA1) was observed, indicating an overall reduction in the reactive microglial population in lemborexant-treated P301S / E4 mice compared to vehicle-treated P301S / E4 mice (Figures 4A, 4C, 4L, and 4M).

[0318]

[0333] Disease-associated microglial markers such as Clec7a (Figures 4E and 4G), a marker of phagolysosomal activity, and CD68 (Figures 4A, 4D, 4P, and 4Q) were significantly increased in P301S / E4 mice relative to E4 mice, with lemborexant potently reducing these markers relative to vehicle-treated P301S / E4 mice.

[0319]

[0334] In P301S / E4 mice treated with lemborexant, a significant increase in the homeostatic microglial marker TMEM119 was observed (Figures 4B, 4F, 4N, and 4O), while no changes were observed in P2RY12 (Figure 8).

[0320]

[0335] These results suggest that a reduction in reactive microglia is involved in the effects of lemborexant in modulating tau-mediated neurodegeneration. Furthermore, APOE colocalization in astroglia and microglia was significantly reduced in lemborexant-treated P301S / E4 mice compared with controls (Figures 4H-K), the latter being more commonly observed only in highly inflammatory and damaging conditions. Lemborexant treatment reduced GFAP+ astroglial reactivity in the hippocampus of P301S / E4 mice (Figures 4R-T). No changes in microglial reactivity were observed in E4 knock-in mice, which lack tau pathology.

[0321] [D.RNA Sequencing]

[0336] To elucidate the mechanisms behind the changes in microglial responsiveness and tau-mediated neurodegeneration observed with lemborexant-induced non-REM sleep, we performed RNA sequencing in bulk hippocampal tissue. Changes in gene expression were found, implicating multiple functional modules, including hormone and GPCR ligand binding, glial differentiation, synaptic regulation, particularly excitatory synapses, and response to DNA damage (Figure 5). In addition to normal aging, defective DNA repair is also associated with age-related neurodegenerative diseases, such as AD. Hyperphosphorylated and aggregated tau can interfere with DNA repair by interacting with DNA repair proteins.

[0322]

[0337] Based on these data, sleep, which results in a global decrease in neuronal metabolism and, in some cases, decreased synaptic activity, may play an important role in maintaining the neuronal genome and DNA repair function. Nevertheless, further research is needed to understand the link between sleep, DNA damage, and neurodegeneration. Interestingly, genes such as Adra2b, Trh, Trhr2, Mpzl2, Slc22a6, Pla2g2f, Ptgdr, and Foxp2 regulate sleep. More specifically, thyrotropin-releasing hormone (Trh) and its receptor (Trhr2) regulate behavioral wakefulness, in part through orexin. Application of TRH converts GABAergic neurons from the burst-firing mode normally associated with synchronous cortical activity occurring during non-REM sleep to the tonic single-spike mode of action potential generation associated with asynchronous cortical activity occurring during wakefulness and REM sleep. The downregulation of Trh and Trhr2 suggests that DORA-induced non-REM sleep further interacts with humoral regulation of sleep-wake behavior to promote sleep, particularly in the presence of tau (since these effects were absent in non-tau E4 mice). To support these findings, we observed changes in the expression of Slc22a6, Pla2g2f, and Ptgdr, which regulate sleep-wake behavior by potent endogenous hypnotics such as prostaglandins. In addition to catalyzing the biosynthesis of prostaglandins, phospholipase A2 plays a major role in cell proliferation, differentiation, and inflammation, and is associated with metabolic alterations in patients with obstructive sleep apnea. In contrast, genes involved in glial differentiation, including microglia expressing Tmem119, Tmem114, Cd68, Aif1, Cd300a, Pea15a, and H2-Q1, were differentially regulated in lemborexant-treated P301S / E4 mice (Figure 5C), suggesting that lemborexant-mediated promotion of non-REM sleep or inhibition of orexin signaling may affect important immune functions such as T cell antigen presentation, response to injury, and regulation of apoptosis, all of which may directly modify tau-mediated neurodegeneration.Further supporting this principle is the reduction of presynaptic vesicular glutamate transporters (VGLUT1, Slc17a7) and postsynaptic density markers (PSD95, Shank1, Shank2) both transcriptionally and immunohistochemically (Figures 5C-5I). Transcriptional changes in synaptic receptor activity and synaptic organization, particularly Otof, Nrxn3, Mrgprf, Mapk13, and Fmod (Figure 5C), support that promoting NREM sleep or inhibiting orexin receptor signaling reduces tau-mediated neurodegeneration and associated synapse loss.

[0323] E. Methods and Analysis

[0338] Mice: All animal procedures and protocols were approved by the Animal Studies Committee at Washington University School of Medicine. PS19 tau transgenic mice, which harbor 1N4R tau and overexpress the human P301S tau mutation, were used. These mice have been backcrossed to C57BL / 6 mice for over 10 generations. Human apoE4 knock-in mice were generated as described in Mol. Neurodegener. 14, (2019), and P301S mice were crossed over several generations to generate experimental P301S / E4 mice. Same-sex littermates were randomly assigned to experimental groups. Only male animals were used and sacrificed at 9.5 months of age. All mice were housed under specific pathogen-free conditions with the same 12-hour light-dark cycle, ambient room temperature, and free access to food and water.

[0324]

[0339] Treatment: Mice were administered a single 30 mg / kg dose of lemborexant or 0.5% methylcellulose vehicle by oral gavage daily at ZT13, 1 hour after the onset of the dark phase, until euthanasia at 7.5 months of age and 9.5 months of age.

[0325]

[0340] Tissue collection: All mice were perfused between ZT3 and ZT7, a time when mice are sleep-deprived, to avoid circadian influences on transcriptional changes in microglial gene expression. Before transcardial perfusion, mice were anesthetized with pentobarbital (50 mg / kg, intraperitoneally). Blood was collected from the heart and centrifuged at 5,000 × g for 5 minutes at 4°C to obtain plasma. Mice were perfused transcardially with ice-cold phosphate-buffered saline containing 0.3% heparin. One hemibrain was dissected, flash-frozen, and stored at -80°C for biochemical analysis. The other hemibrain was immersion-fixed in 4% paraformaldehyde for 24 hours, subsequently cryoprotected in 30% sucrose for 48 hours, and frozen at -80°C until tissue samples were sectioned for immunohistochemical analysis.

[0326]

[0341] Measurement and analysis of sleep-wake states: Sleep-wake behavior of mice was monitored using electroencephalography (EEG) and independently using the PiezoSleep mouse behavior tracking system (SignalSolutions).

[0327]

[0342] For EEG experiments, animals were anesthetized with isofluorane (0.5–3%). Pain signs were assessed by toe pinch before incision. Mice were then surgically implanted with screw electrodes for EEG in the skull and stainless steel wire electrodes for electromyography (EMG) in the neck muscles. After a vertical incision was made in the midline to expose the skull, forceps and 3% hydrogen peroxide were used to remove any connective tissue and dry the skull for electrode placement. A 0.9 mm microdrill was used to drill a burr hole for the frontal reference electrode (bregma +0.5 mm, lateral ±0.5 mm; bregma), and a screw was fixed into the skull. Using the same technique as for the reference electrode, two bilateral active recording electrodes were placed in the parietal cortex (posterior -2.5 mm, lateral ±1.5 mm; bregma), and a ground screw was fixed into the cerebellum (posterior -6.2 mm, lateral ±0.5 mm; bregma). The exposed skull, screws, and all wires were covered with a layer of dental cement (SNAP, Parkell), and pin headers were attached to the head for later recording. The skin was sutured around the exposed dental cement / pin headers, and the remainder of the incision was closed using tissue adhesive (Vetbond, 3M). After the procedure, mice were placed in a warm chamber to fully recover from anesthesia and housed individually in monitoring cages with fresh bedding, water, food, and Carprofen (oral; 1 / 4 tab of a 5g tab; ad libitum) supplements. After a 3-day recovery period, mice were habituated to the recording cages for 2 weeks, after which undisturbed EEG / EMG recordings were performed on freely moving mice over two consecutive days. Bilateral cortical EEG signals were acquired using a P511K AC Preamplifier (Grass-Telefactor Instruments, Warwick, RI, USA), digitized using a BIOPAC MP150, and digitally recorded at a sampling rate of 250 Hz using BIOPAC's AcqKnowledge software. The EEG was processed in MATLAB (MathWorks) with a 1-30 Hz bandpass filter to remove DC offset and high-frequency noise. EEG / EMG recordings were manually scored for 10-s epochs of wakefulness, non-REM sleep, and REM sleep, and a calibration file containing a subject-specific mixed z-scoring variable was created.The calibration file was imported into AccuSleep, a machine learning-based automated sleep scoring program in MATLAB, and the remaining scoring was completed.

[0328]

[0343] We used the PiezoSleep mouse behavior tracking system (Signal Solutions, LLC, Lexington, KY, USA). This noninvasive method involves a thin dielectric piezo sensor pad that generates a voltage signal in response to real-time changes in pressure on its surface. Mice were individually housed with piezo pads under fresh bedding and free access to fresh water and food. Data were recorded undisturbed over a 6-day period. SleepStats software (Signal Solutions, LLC, Lexington, KY, USA) was used to acquire data.

[0329]

[0344] Volumetric analysis: Volumetric analysis of the hippocampus, entorhinal / piriform cortex, and ventricles was performed using stereological methods by evaluating 180-µm-spaced sections (16–18 sections per mouse, depending on the severity of brain atrophy) from bregma -1.3 mm to bregma -3.1 mm. Slide-mounted 30-µm microtome sections were briefly immersed in distilled water and then incubated in preheated 0.1% cresyl violet at 37°C for 6 minutes. Following this, the tissue was rinsed in distilled water and transferred sequentially to 70%, 95%, and 100% ethanol for 2 minutes each. Slides were then washed in xylene before finally being coverslipped with cytoseal 60 mounting medium (Thermo Fisher Scientific). Slides were scanned at 20x magnification using a Hamamatsu Nanozoomer microscope. The hippocampus, EC / PC, and ventricles were traced using NDP.view2. The formula for calculating the volume was volume = (total area) × 0.3 mm.

[0330]

[0345] Measurement of neuronal layer thickness: The dentate granule and entorhinal pyramidal cell layers were measured in three sections by drawing a scale line across the cell layer using NDP.view2 and calculating the average value for each mouse.

[0331]

[0346] Immunohistochemistry: Free-floating sections were briefly washed in TRIS-buffered saline containing 1% Triton X100 (TBS-Tx), followed by quenching endogenous peroxidase with 0.3% hydrogen peroxide for 20 minutes at room temperature. After a brief wash, sections were blocked with 5% goat serum for 30 minutes at room temperature, followed by overnight incubation with biotinylated AT8 (Phospho-Tau Ser202, Thr205; 1:500, MN1020B, Thermo Fisher Scientific) or MC1 (1:500, kindly provided by Dr. Peter Davies) primary antibodies at 4°C. The following day, MC1-stained sections were briefly washed and incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Both AT8- and MC1-stained tissues were then developed with 3,3'-diaminobenzidine (DAB, Sigma) for 10 and 14 minutes, respectively. Tissue sections were mounted on slides, dehydrated through an increasingly concentrated series of ethanol, and finally immersed in xylene and coverslipped using Cytoseal mounting medium. Slides were scanned at 20x magnification using a Hamamatsu Nanozoomer microscope.

[0332]

[0347] Immunofluorescence staining: Free-floating sections were washed briefly with PBS and blocked in 5% donkey serum for 1 hour at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C with gentle agitation unless otherwise indicated. The primary antibodies used were IBA1 (1:500; 019-19741, Fujifilm or NB100-1028, Novus Biologicals), CD68 (1:00; FA-11, BioRad), P2RY12 (1:100 at room temperature; HPA013796, Sigma-Aldrich), TMEM119 (1:500 E3E1O, Cell Signaling Technology), Clec7a (1:50 at room temperature; mabg-mdect, InvivoGen), GFAP (1:2000; 2E1.E9 Alexa Flour 488-conjugated, BioLegend), APOE (1:300; D7I9N, Cell Signaling), and PSD-95 (1:200; 51-6900, Thermo Fisher Scientific). The following day, sections were washed and incubated with secondary antibodies diluted in blocking buffer, followed by 4',6-diamidin-2-phenylindole (DAPI, 5 μg / mL) if necessary, before mounting on slides (Prolong™ Gold Antifade Reagent, Thermo Fisher Scientific).

[0333]

[0348] Confocal imaging and analysis: Images were acquired using a Leica Stellaris 5 confocal microscope and Leica Application Suite X software (4.2.1.23810). Laser and detector settings were kept constant for each immunostaining acquisition. For all analyses, at least two images per brain region and slide were acquired using 20x (Apo CS 10x / 0.40 dry), 40x (Apo CS 40.0x 1.25), and 63x (Apo CS 63.0x 1.4 Oil) differential interference contrast objectives, respectively, at a resolution of 1024 x 1024 pixels with a z-step thickness of 15 µm. For synaptic imaging, a Leica Stellaris 8 Lightning microscope was used to generate adaptive deconvolution-based super-resolution confocal images using a 63x oil objective. Image analysis was performed using Fiji (ImageJ). For feasibility of quantification, all layers of one image stack were projected onto one slice (stack\Z projection). Microglia were then segmented using an automatic thresholding method in Fiji and presented as the area (%) covered by the selected stain in the hippocampus or entorhinal / piriform cortex.

[0334]

[0349] Protein extraction: Frozen mouse hippocampal tissue was weighed and homogenized in a Bullet Blender homogenizer (Next Advance) using a bead tube containing 200 μl of RAB buffer pH 7.0 (100 mM MES, 1 mM EGTA, 0.5 mM MgSO, 750 mM NaCl, 20 mM NaF, 1 mM NaVO) supplemented with 1× protease inhibitor (cOmplete™, Roche) and 1× phosphatase inhibitor (PhosSTOP, Roche). The homogenate was centrifuged at 5,000 × g for 5 min at 4 °C to pellet insoluble RAB material, and the supernatant was ultracentrifuged at 50,000 × g for 20 min in an MLA-130 rotor of an Optima MAX-XP ultracentrifuge (Beckman Coulter) to obtain the RAB extract. Proteins were extracted from the remaining cell pellet with RIPA buffer pH 8.0 (150 mM NaCl, 50 mM TRIS, 0.5% deoxycholate, 1% Triton-X 100, 0.1% sodium deoxycholate, 5 mM EDTA, 20 mM NaF, 1 mM Na3VO4) supplemented with protease and phosphatase inhibitors. After clarification of the RIPA-insoluble material at 5,000 × g for 5 minutes at 4 °C, the supernatant was again ultracentrifuged at 50,000 × g for 30 minutes to obtain the RIPA-soluble protein fraction. The RIPA-insoluble pellet was dissolved in ice-cold 70% formic acid (FA) and sonicated at room temperature for 1 minute with a short pulse at 30% amplitude using a Fisher Scientific ultrasonicator (Model FB120), followed by a final ultracentrifugation at 50,000 × g for 20 minutes at 4 °C. Protein concentrations of RIPA fractions were measured using the BCA assay (Pierce). All samples were aliquoted and frozen at -80°C until use.

[0335]

[0350] Tau ELISA: Human tau and p-tau were measured in the RAB, RIPA, and 70% FA fractions using sandwich ELISA and normalized to tissue weight as described. The coating antibodies for total human tau and p-tau were TAU-5 (mouse monoclonal, 20 μg / ml) and HJ14.5 (mouse monoclonal, 20 μg / ml), respectively. The capture antibodies for total human tau and p-tau were HT7-biotinylated (MN1000B, ThermoFisher Scientific) and AT8-biotinylated (MN1020B, ThermoFisher Scientific), respectively.

[0336]

[0351] NFL concentrations: Plasma NFL concentrations were measured using the Quanterix NF-Light Simoa Assay Advantage kit according to the manufacturer's instructions.

[0337]

[0352] RNA extraction: Frozen hippocampal tissue was weighed and homogenized in chloroform containing TRIzol™ in an RNAase-free bead tube (REDE, Next Advance). Samples were centrifuged at 12,000 × g for 15 min at 4°C, and the aqueous supernatant was transferred for RNA isolation using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNA quality was controlled using a Bioanalyzer prior to Next Generation Sequencing using a Clontech SMARTer.

[0338]

[0353] RNA Sequencing and Analysis: Samples were prepared, indexed, pooled, and sequenced on an Illumina NovaSeq 6000 according to the library kit manufacturer's protocol. Base calling and demultiplexing were performed using Illumina's bcl2fastq software and a custom Python demultiplexing program, with a maximum of one mismatch in indexed reads. RNA-seq reads were then aligned to the Ensembl release 76 primary assembly using STAR version 2.7.9a (Doblin et al.). Gene counts were derived from the number of uniquely aligned, unambiguous reads using Subread:featureCount version 2.0.3. Isoform expression of known Ensembl transcripts was quantified using Salmon version 1.5.2. Sequencing performance was assessed by the total number of aligned reads, the total number of uniquely aligned reads, and the number of detected features. Ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified using RSeQC version 4.0. All gene counts were then imported into the R / Bioconductor package EdgeR, and a TMM normalized size coefficient was calculated to adjust samples for differences in library size. Ribosomal genes and genes not expressed at the minimum group size minus one sample exceeding 1 CPM (counts per million) were excluded from further analysis. The TMM size coefficient and count matrix were then imported into the R / Bioconductor package Limma. Weighted likelihoods based on the observed mean-variance relationships of all genes and samples were then calculated for all samples using the voomWithQualityWeights function and fitted using a Limma generalized linear model with additional unknown latent effects determined by surrogate variable analysis (SVA). Performance of all genes was assessed by plotting the residual standard deviation of all genes against the mean logarithm, with a robustly fitted line of the residuals.Differential expression analysis was then performed to analyze differences between conditions, and the results were filtered for only genes with a Benjamini-Hochberg false discovery rate adjusted p-value of 0.05 or less.

[0339]

[0354] For each imaging agent extracted by Limma, we used the R / Bioconductor package GAGE9 to detect global perturbations of known Gene Ontology (GO) terms, MSigDb, and KEGG pathways. We examined the log2 fold change reported by Limma for each term and the background log2 fold change of all genes found outside the respective term. We used the R / Bioconductor package heatmap3 to display heatmaps between groups of samples for Go or MSigDb terms with Benjamini-Hochberg false discovery rate-adjusted p-values ​​of 0.05 or less. Perturbed KEGG pathways in which the observed log2 fold change of genes within a term was significantly perturbed in one direction relative to background or in any direction relative to other genes within a given term with p-values ​​of 0.05 or less were rendered as annotated KEGG graphs using the R / Bioconductor package Pathview.

[0340]

[0355] To identify the most significant genes, Limma voomWithQualityWeights-transformed log2 CPM expression data were analyzed by weighted gene correlation network analysis using the R / Bioconductor package WGCNA. Briefly, all genes were correlated with each other by Pearson correlation and clustered into unsigned modules by expression similarity using an output threshold empirically determined from the data. An eigengene was then created for each de novo cluster to correlate its expression profile with all coefficients in the model matrix. Because these clusters of genes were created by expression profile rather than known functional similarity, the clustered modules were given random color names, with gray being the only module with a pre-existing definition of containing genes that do not cluster well with other genes. These de novo clustered genes were then tested for functional enrichment of known GO terms using the hypergeometric test available in the R / Bioconductor package clusterProfiler. Significant terms with a Benjamini-Hochberg adjusted p-value less than 0.05 were then collapsed by similarity into a clusterProfiler categorical network plot to display the most significant terms for each module of hub genes and appended features for each significant module. The information for all clustered genes in each module was then combined with the respective statistical significance results from Limma to determine whether those features were also found to be significantly differentially expressed.

[0341]

[0356] Statistical analysis: All statistical analyses were performed using Graphpad Prism 8.0. Data are expressed as mean ± SEM unless otherwise specified. Data were checked for normality using the Shapiro-Wilk method, D'Agostino and Pearson normality test, and KS normality test. Statistical significance between groups with normally distributed data was calculated by unpaired T-test or two-way analysis of variance, followed by Tukey's post-hoc test for group-by-group comparisons unless otherwise specified. P less than 0.05 was considered significant: * p<0.05, ** p<0.01 and *** p<0.001.

[0342] Example 3: Effects of lemborexant and doxepin in an Aβ burden model [A. Sleep-wake behavior]

[0357] Sleep changes were assessed in APPswe / PS1deltaE9 (also referred to as "PSAPP") mice following administration of doxepin or lemborexant. Briefly, 4- to 5-month-old PSAPP mice (mixed sex) were housed individually and placed in a SignalSolutions piezoelectric sleep monitoring base (Adapt-A-Base). Sleep-wake behavior was monitored for 7 days, and mice were administered vehicle, 35 mg / kg doxepin, or lemborexant (10 or 30 mg / kg) by oral gavage daily at ZT0 (lights on).

[0343]

[0358] Figure 9A shows a schematic of the experimental design and graphs of the effects of doxepin and lemborexant on total sleep (Figure 9B), light phase sleep (Figure 9C), and dark phase sleep (Figure 9D). P values ​​from a one-way ANOVA are shown. Each dot represents one mouse. Figure 9E shows the percentage of sleep at one day post-drug injection. P values ​​are obtained from a two-way repeated measures ANOVA.

[0344]

[0359] Data show that lemborexant and doxepin each increase total sleep time in PSAPP mice, with effects comparable after treatment with each drug. Notably, DOX-induced sleep is throughout the day, whereas LEM-induced sleep is restricted to the light phase (the mouse's natural resting period).

[0345] [B. Amyloid plaque deposition]

[0360] To determine the long-term effects of either lemborexant or doxepin on amyloid plaque deposition, PSAPP mice were administered either drug chronically by oral gavage 6 days a week for 1.5 months at ZT0 (lights on).

[0346]

[0361] Figure 10A shows a schematic diagram of treatment timing for PSAPP mice. Because females develop plaques earlier / at a younger age, males and females were alternated to allow for data combination. Figure 10B shows representative images of brain sections stained with X34, which labels fibrillar amyloid plaques. Figure 10C shows quantification of plaque burden (% X34 stained area) in different brain regions (hippocampus, somatomotor cortex, somatosensory cortex, and piriform cortex). Error bars indicate mean ± SEM, and each dot represents a mouse. P values ​​from one-way ANOVA are shown. The data indicate that chronic administration of lemborexant, but not doxepin, reduced fibrillar amyloid plaque burden in PSAPP mice. A dose-response response was observed for lemborexant.

[0347]

[0362] Figure 11 shows that long-term lemborexant reduces total amyloid plaque burden more effectively than doxepin in PSAPP mice. Figure 11A shows brain sections stained for total amyloid plaque burden using the anti-Aβ antibody HJ3.4. Figure 11B shows quantification of plaque burden (measured as the area (%) showing HJ3.4 staining) in different brain regions (hippocampus, somatomotor cortex, somatosensory cortex, and piriform cortex). Error bars indicate the mean ± SEM, and each dot represents a mouse. P values ​​from one-way ANOVA are shown.

[0348]

[0363] Taken together, these data demonstrate that lemborexant reduces total amyloid plaque burden (including both diffuse and fibrous plaques) in PSAPP mice. Doxepin also significantly reduces total amyloid burden, but not fibrous plaque burden, to a lesser extent than lemborexant (at a dose of 30 mg / kg), as only lemborexant (at a dose of 30 mg / kg) showed significant effects in the piriform cortex.

[0349]

[0364] The differential response of lemborexant compared with doxepin suggests that the effect on plaque development is separable from the similar hypnotic effects induced by these two drugs.

[0350] [C. Amyloid Processing]

[0365] To determine whether lemborexant and doxepin affect amyloid processing, cortical lysates were obtained from PSAPP mice treated with either drug, and levels of full-length APP and APP C-terminal fragments (CTFs) were measured by Western blot. Figure 12A shows a representative Western blot using β-tubulin as a loading control. Figure 12B shows quantification of band intensity. Error bars indicate mean ± SEM, and each dot represents a mouse. P values ​​from one-way ANOVA are shown.

[0351]

[0366] The results show that neither lemborexant nor doxepin alters APP processing / cleavage in PSAPP mice.

[0352] [D. Amyloid plaque size and number of microglia surrounding the plaque]

[0367] The number of microglia surrounding amyloid plaques was determined in PSAPP mice after administration of either lemborexant or doxepin. Brain sections from PSAPP mice were stained for plaques (using X34) and microglia (using Iba1). Plaques of approximately the same size were selected in all mice. Microglial volume around each plaque was calculated from Z-stacks of confocal images using Imaris software. Figure 13A shows representative images of brain sections obtained from PSAPP mice after administration of either lemborexant or doxepin. Figure 13B shows quantification of plaque volume (demonstrating that plaques of similar size were quantified across conditions) and the volume of Iba1 around plaques. Error bars represent the mean ± SEM, and each dot represents a mouse. P values ​​from one-way ANOVA are shown.

[0353]

[0368] The results show that the number of microglia around plaques does not change after administration of lemborexant or doxepin.

[0354] [E. Phagocytic subtypes of microglia surrounding fibrillar amyloid plaques]

[0369] To determine the subtype of microglia surrounding fibrillar amyloid plaques, we measured CD68 expression in microglia from PSAPP mice after administration of either lemborexant or doxepin. Brain sections from PSAPP mice were stained with markers for fibrous plaques (X34), microglia (Iba1), and phagosomes (CD68). Figure 14A shows a representative image of a brain section. Figure 14B shows quantification of Iba1-colocalized CD68 around each plaque, obtained using Imaris software. Error bars indicate the mean ± SEM, and each dot represents the average of 8–10 plaques from one mouse. P values ​​from one-way ANOVA are shown.

[0355]

[0370] The results demonstrate that administration of lemborexant, but not doxepin, increases the phagocytic subtype of microglia surrounding fibrillar amyloid plaques. The total number of microglia, in terms of area density, remains unchanged, and the total number of microglia co-localized with plaques remains unchanged. Figure 14C shows the volume of Iba1+, Figure 14D shows co-localized Iba1+ and CD68+ (as a percentage of Iba1+ staining), and Figure 14E shows co-localized Iba1+ and CD68+. Increased CD68 in microglia surrounding plaques indicates increased phagocytic activation.

[0356] [F. Gene Expression]

[0371] To determine changes in gene expression after administration of lemborexant or doxepin to PSAPP mice, qPCR arrays were performed on mouse cortical tissue. Figure 15 shows quantification plots of three transcripts that showed significant differences in expression. Ifnb1 encodes the inflammatory mediator IFN-β, which is involved in microglial regulation in AD. Rab5a encodes a lysosomal protein, and Mmp-2 encodes a metalloprotease that has been shown to degrade Aβ. Data are presented as fold changes (relative to the mean of VEH). Error bars indicate the mean ± SEM, and each dot represents one mouse. P values ​​from one-way ANOVA are shown.

[0357]

[0372] The results showed that genes related to Aβ degradation were upregulated in PSAPP mice after lemborexant treatment, but the changes in gene expression were not significant in the doxepin-treated group.

[0358] [G. Number of microglia actively phagocytosing amyloid plaques]

[0373] The effect of lemborexant on microglial amyloid plaque phagocytosis was evaluated in PSAPP mice. Figure 16A shows a schematic diagram of the experimental design (Lau et al., STAR Protoc. 2021). Briefly, 5-month-old PSAPP mice were treated daily by oral gavage with 30 mg / kg vehicle (Veh) or lemborexant (LEM) for 7 days. After treatment on day 7, amyloid plaques were labeled in vivo by intraperitoneal (ip) injection of methoxy-X04 (MX04). Three hours later, mice were sacrificed, and microglia were isolated from the brain and analyzed by flow cytometry. Figure 16B shows the flow cytometry gating strategy for isolating potential microglia. Following side-scatter and forward-scatter gating to identify viable single cells, the CD45 low CD11b+ population was isolated as potential microglia. Figure 16C shows the analysis of this cell population for methoxy-MX04 positivity. Figure 16D shows the quantification of the percentage of MX04+ microglia, indicating microglia with phagocytosed labeled amyloid. P=0.0207 by two-tailed T-test.

[0359]

[0374] The results show that LEM treatment dramatically increases microglial amyloid plaque phagocytosis in vivo. MX04 cells were not observed in wild-type mice, which lack amyloid plaques. In contrast, 1.8% of microglia in vehicle-treated PSAPP mice internalized MX04, compared with 3.75% of microglia in the LEM-treated group.

[0360] H. Amyloid Plaque Deposition in Aging Subjects

[0375] The effects of lemborexant administration were evaluated in aged PSAPP mice. Figure 17A shows a schematic diagram of the experimental design. Briefly, 9-month-old PSAPP mice bearing amyloid plaques were treated daily with vehicle (Veh) or lemborexant (LEM, 30 mg / kg) for 30 days. On day 1 of treatment, existing plaques were labeled in vivo by intraperitoneal (ip) injection of methoxy X04 (MX04). After 30 days of treatment, mice were sacrificed, and all plaques were labeled with thiazine red, which binds to fibrillar amyloid, similar to X34. Plaque growth during the 30-day treatment period was calculated by comparing the volume of MX04 and thiazine red per plaque. Figure 17B shows representative images of MX04, thiazine red, and overlay images. Figure 17D shows a graph in which at least 10 plaques were analyzed per mouse, with each circle representing the average per mouse. The LEM-treated group showed a trend toward reduced plaque growth over the 30-day treatment period. P values ​​are from a Mann-Whitney U test due to the non-Gaussian distribution of the data. Figure 17C shows representative images of X34-labeled amyloid plaques, IBA1-labeled microglia, and CD68-labeled microglial phagosomes. The volume of IBA1-CD68 colocalization was calculated within a 20-µm sphere from each amyloid plaque to determine periplaque microglial CD68 expression. Figure 17E shows quantification of colocalized IBA1-CD68 as a percentage of total IBA1 (total microglia) area. Each circle represents the average for one mouse; 10 plaques were quantified per mouse. In Figures 17D and 17E, comparisons between VEH-treated and LEM-treated groups were analyzed by two-tailed t-test.

[0361]

[0376] The effects of doxepin treatment were also evaluated in aged PSAPP mice. Neither lemborexant nor doxepin treatment significantly altered the total number of plaques in aged mice. Similarly, no significant changes were observed in plaque volume, IBA1+ cells, IBA1 volume, or IBA1-CD68 colocalization.

[0362]

[0377] Finally, we quantified the volume of dystrophic neurites surrounding amyloid plaques by measuring the elevation of BACE1 in presynaptic terminals.Neither lemborexant nor doxepin treatment significantly altered the volume of dystrophic neurites.

[0363]

[0378] The effects of doxepin administration on plaque growth and phagocytic microglia surrounding amyloid plaques are shown in Figures 17F and 17G, along with the data for lemborexant administration. In Figures 17F and 17G, comparisons between VEH, DOX, and lemborexant treatment groups were analyzed by one-way analysis of variance.

[0364]

[0379] In summary, lemborexant and doxepin each showed a tendency to slow amyloid plaque growth in aged mice with pre-existing plaques. Each drug increased phagocytic microglia surrounding amyloid plaques in aged mice. Lemborexant, but not doxepin, also exhibited this effect on phagocytic microglia in young mice. Both lemborexant and doxepin were less effective at removing existing plaques in aged mice than at preventing plaque buildup in young mice.

[0365] [I. Method] [J.Animals]

[0380] Male and female APPswe / PS1deltaE9 (PSAPP) mice were used in all experiments.

[0366]

[0381] APP / PS1 is a double transgenic mouse model expressing chimeric mouse / human amyloid precursor protein (Mo / HuAPP695swe) and mutant human presenilin 1 (PS1-dE9), both of which are induced in central nervous system neurons.

[0367] [K. Methods for measuring sleep-wake behavior]

[0382] Five-month-old APPswe / PS1deltaE9 (PSAPP) mice were orally administered VEH, LEM (10 mg / kg / day or 30 mg / kg / day), or DOX (35 mg / kg / day) daily for 6 days at ZT0. Sleep and wakefulness were determined using a noninvasive piezoelectric system, Adapt-A-Base (Signal Solutions). Mice were individually housed in cages placed on piezoelectric sensor bases in a sound- and light-proof cabinet (Circadian Cabinets, ClockLab) and recorded without disturbance for 7 days. At the start of recording, each cage contained 160 g of corncob bedding, 220 g of food pellets, and 380 mL of water, resulting in identical cage weights except for slight variations in mouse body weight. Sleep-wake states were analyzed using SleepStats software (Signal Solutions). Epochs of 30 seconds were used to score sleep bout lengths according to the manufacturer's default settings in the current version of SleepStats software.

[0368] [L. Method for assessing the effect of long-term administration of lemborexant or doxepin on amyloid plaque deposition]

[0383] Young PSAPP mice (3-month-old females and 3.5-month-old males) were orally administered VEH, LEM (10 mg / kg / day or 30 mg / kg / day), or DOX (35 mg / kg / day) daily for 6 weeks at ZT0. Subsequently, female and male mice were perfused at 4.5 and 5 months, respectively. Brains were removed and processed for IHC / IF, transcriptomics, or proteomics analysis.

[0369] [M. Methods for assessing the effects of lemborexant and doxepin on microglial Aβ phagocytic activity]

[0384] Five-month-old PSAPP mice were administered VEH or LEM (30 mg / kg / day) daily for 7 days at ZT0. On day 7, mice were intraperitoneally injected with methoxy X-04 (MX-04, 10 mg / kg) to label plaques in vivo. Three hours after MX-04 injection, mice were perfused, and brains were processed for flow cytometry assays to estimate MX-04-positive microglial cells.

[0370] [N. Methods for assessing the effect of LEM on amyloid plaque deposition in aged PSAPP mice]

[0385] Nine-month-old PSAPP mice were intraperitoneally (ip) injected with methoxy X-04 (MX-04, 10 mg / kg) to label plaques in vivo and orally administered VEH or LEM (30 mg / kg / day) daily for 4 weeks at ZT0. Mice were perfused at 10 months of age, and brains were removed and processed for IHC / IF analysis. Fixed brain sections were stained with thiazine red and compared with MX-04 staining to estimate plaque development.

[0371] [O. Drugs]

[0386] Mice were orally administered either vehicle (VEH), doxepin (DOX, Cayman Chemical #15888, solubilized in PBS), or lemborexant (LEM, suspended in 0.5% methylcellulose) at ZT0 (lights on). VEH mice received a similar volume of vehicle daily. The tip of a 22-gauge oral gavage needle was dipped in 100% sucrose solution immediately prior to oral gavage. MethoxyX-04 (Tocris #4920, 10 mg / kg) was injected intraperitoneally for timestamp experiments and analysis of microglial phagocytic activity.

[0372] [P.IHC / IF:]

[0387] All mice were perfused from ZT5 to ZT7 (1100 to 1300 hours). Mice were deeply anesthetized intraperitoneally with pentobarbital (150 mg / kg) and then transcardially perfused with ice-cold Dulbecco's modified PBS (DPBS) containing 3 g / L heparin. Brains were carefully removed, and the left hemisphere was postfixed in 4% paraformaldehyde for 48 hours (4°C), followed by cryoprotection in 30% sucrose in PBS (4°C) for 24 hours. Brains were then serially sectioned at 40 μm coronally using a freezing slide microtome (SM1020R; Leica) and stored in cryoprotectant solution (30% ethylene glycol, 15% sucrose, 15% phosphate buffer in ddH2O). For biochemical analysis, the right hemisphere was cut to separate the cortex and hippocampus, which were then snap-frozen and kept at -80°C until analysis.

[0373]

[0388] Fibrillar Aβ was stained with X-34 dye (SML-1954, 1:5,000; MilliporeSigma) or thiazine red. For X-34 staining, free-floating sections were washed three times with PBS for 5 min each and then permeabilized with 0.25% Triton X-100 in PBS (PBS-X) for 30 min. The tissue sections were then incubated with X34-0.1 M NaOH for 20 min, washed with X34 buffer (40% EtOH in PBS), and then washed twice with PBS. To stain total Aβ (HJ3.4 biotinylated, anti-Aβ1-13, mouse monoclonal, 1:1,000, 2.81 μg / mL; in-house), sections were washed three times with TBS and incubated with 0.3% hydrogen peroxide for 10 min. Sections were washed again with TBS × 3 and blocked for 30 minutes with 3% milk diluted in TBS + 0.25% Triton X-100. Sections were incubated overnight at 4°C with biotinylated HJ3.4 in TBS + 0.25% Triton X-100 + 1% milk. The next day, sections were washed and then developed for 60 minutes using ABC Elite (Vector PK-6100). Sections were then incubated with 3,3-diaminobenzidine (DAB, Sigma-Aldrich) as the chromogen and 0.05% hydrogen peroxide as the substrate, dehydrated, and coverslipped using Cytoseal 60 (8310; Thermo Fisher Scientific).

[0374]

[0389] For immunofluorescence staining (IF) with IBA1 (goat, Abcam ab5076, 1:500) or CD68 (rat, BioRad MCA1957, 1:500), sections were washed with TBS x 3, blocked with 3% donkey serum in TBSX (TBS + 0.4% Triton X-100) for 60 min, and incubated overnight at 4°C with primary antibodies diluted in 1% donkey serum in TBSX. Sections were then incubated for 1 hour at room temperature with donkey fluorescent secondary antibodies at 1:1000 in PBSX (or TBSX). Sections were mounted and sealed with Fluoromount-G (0100-01; SouthernBiotech) and stored in the dark at 4°C until imaging.

[0375] [Q. Imaging]

[0390] Epifluorescence imaging: All fluorescence imaging was performed on a Keyence BZ-X810 microscope. In general, laser intensity and exposure time were selected for each cohort of tissue samples to select appropriate parameters that could be maintained constant across all slides during an imaging session. Although these values ​​varied depending on the antibody, all sections of a given cohort were imaged under the same conditions and at the same magnification. Images were processed with the BZ-X800 Analyzer program (Keyence Corp.) and quantified with Fiji version 2.1.0 (NIH). All regions were quantified in 2–3 sections per mouse.

[0376]

[0391] Confocal imaging and analysis: Plaques located in the cortical gray matter and completely contained within the slice thickness were selected for imaging. 16-bit image stacks were acquired in sequential mode with a 0.26 μm z-step using a Zeiss LSM-980 Airyscan 2 confocal microscope and ZEN software (v3.7, blue edition). Uniform pinhole, laser power, and PMT detector gain settings were used in all experiments. For all analyses, at least two images were taken per brain region and slide at a resolution of 1,024 × 1,024 or higher using a 40× oil immersion objective. Quantification of confocal images for the volume of IBA1 and CD68 around X34+ plaques was performed on a semi-automated platform using MATLAB and Imaris 10.0.1 software (Bitplane). To form a surface for each staining based on a threshold applied to all images, the X34+ surface was extended by 20 μm and colocalized with the various immunostained surfaces. The surface areas of IBA1+ and CD68+ were then colocalized within a 20 μm extended shell around the plaque. To quantify the number of plaque-associated IBA1+ microglia, a threshold was applied to all images to assign a spot to each cell body or puncta. The X34 surface was extended by 20 μm, and spots were counted within the X34+ extended surface. Spots completely contained within or partially contacting the extended surface were included in the analysis.

[0377] [R. Western blotting]

[0392] Tissue samples were homogenized by sonication on ice in radioimmunoprecipitation (RIPA) buffer (Pierce, Thermo Scientific) containing complete protease inhibitors and PhosSTOP phosphatase inhibitor (Roche). PAGE and Western blotting were performed using Invitrogen Novex gels and reagents. Bands were visualized with an iBright CL1500 imaging system (Thermo) using Lumigen TMA-6 chemiluminescence reagent. Band intensity was quantified using Fiji software (NIH) and normalized to a β-tubulin loading control.

[0378] [S.Statistics]

[0393] Statistical tests were performed and graphs plotted using GraphPad Prizm software, version 9.0.1. Power analysis was used to determine the variability of outcome measures and estimate the number of observations required to test the null hypothesis. All graphical representations show means with bar graphs, individual data points within the bar graphs, and error bars representing the SEM. In Experiments 1 and 2, one-way ANOVAs were performed. If main effects were significant, the following post-hoc multiple comparison tests were performed: Tukey (if group sizes were equal) or Tukey-Kramer (if group sizes were unequal). In Experiments 3 and 4, to determine whether variances were significantly different, an F-test was first performed for data sets with one dependent variable and two groups. If not, an unpaired two-tailed t-test was performed. If variances differed, a nonparametric Mann-Whitney U test was performed. Outliers were identified and excluded using a Grubbs' test. All P values ​​are noted in the figures.

[0379] Example 4: Effects on rhythmic activity patterns

[0394] The effects of lemborexant administration on rhythmic activity patterns were evaluated in arrhythmogenic Bmal1 knockout (KO) mice to assess the restorative effects of lemborexant on mice lacking a functional clock. Bmal1 KO mice lack a functional clock and consequently lose their 24-hour rhythm in constant darkness (DD condition). The arrhythmic effects of Bmal1 KO mice are masked under a normal 12-hour light:dark cycle (L:D condition).

[0380]

[0395] Control (Cre-) or Bmal1 KO (CAG-CreERT2; Bmal1(f / f)) mice were treated with tamoxifen to delete Bmal1. One month later, infrared actigraphy was recorded under 12:12 h L:D conditions (yellow area) or constant darkness (DD condition). Lemborexant (LEM) was administered by oral gavage at a dose of 30 mg / kg at 6:00 AM (traditionally ZT0) for 9 days (red bar and red arrow). Actigraphy was collected for an additional 2 weeks after cessation of treatment. Figures 18A and 18B show representative actograms from control and Bmal1 KO mice. Figure 18C shows quantification of actigraphy endpoints during different parts of the experiment (in Figures 18A and 18B, LD indicates the yellow area, DD+LEM is the area with the red bar, and DD is the remainder of the recording). Data were analyzed by two-way analysis of variance with Tukey's post-hoc test.

[0381]

[0396] The results show that Bmal1 knockout mice exhibited higher daytime activity than wild-type mice under all conditions (LD, DD+LEM, and DD). Daily administration of lemborexant at CT0 allowed Bmal1 KO mice to maintain locomotor activity under DD conditions similar to LD (CT0 is equivalent to ZT0 under DD conditions). Relative amplitude, which represents the ratio of the average activity during the most active 10 hours to the average activity during the least active 5 hours, was lower in Bmal1 knockout mice under all conditions. Generally, higher relative amplitude correlates with more stable rhythms. Interdaily stability (IS), which measures synchronization between daily 24-hour rhythms, was lower in Bmal1 knockout mice under all conditions. Higher IS indicates better synchronization of rhythms.

[0382] [Equivalents and Scope]

[0397] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments given the detailed description provided herein. The scope of the present disclosure is not limited to the above detailed description, but rather is as defined by the appended claims.

[0383]

[0398] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, it is to be understood that values ​​expressed as ranges can assume any particular value or subrange within the ranges set forth in different embodiments of this disclosure to the tenth of the unit of the lower limit of that range, unless otherwise apparent from the context.

[0384]

[0399] In addition, it should be understood that any particular embodiment of the present disclosure that falls within the prior art may be expressly excluded from any one or more of the claims. Such embodiments may be excluded even if the exclusion is not explicitly set forth herein, because they are deemed to be known to those of ordinary skill in the art. Any particular embodiment of the compositions of the present disclosure (e.g., any composition, therapeutic or active ingredient; any method of manufacture; any method of use, etc.) may be excluded from any one or more claims for any reason, whether related to the existence of prior art or not.

[0385]

[0400] It is to be understood that the terms used are terms of description rather than of limitation and may be changed within the purview of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

[0386]

[0401] While the present disclosure has been described in considerable detail and with certain particularity with respect to several described embodiments, it is not intended to be limited to any such details or embodiments or to any particular embodiment, but rather should be construed with reference to the appended claims in a manner that provides the broadest possible interpretation of such claims in light of the prior art and thus effectively encompasses the intended scope of the present disclosure.

[0387]

[0402] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the section headings, materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

1. A method for treating Alzheimer's disease (AD) in a subject having or at risk of developing AD, comprising administering to the subject a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, thereby treating AD.

2. 10. The method of claim 1, wherein treating AD comprises reducing and / or slowing cognitive decline.

3. 10. The method of claim 1, wherein treating AD comprises affecting (e.g., slowing, delaying, or reducing) changes in at least one marker of AD pathology.

4. 4. The method of claim 3, wherein the marker is the level of tau phosphorylation, neurodegeneration, changes in microglial response and / or the presence of Aβ plaques.

5. The method of claim 4 , wherein the marker is present in a brain region of the subject.

6. 6. The method of claim 5, wherein the brain region is the hippocampus, somatomotor cortex, somatosensory cortex, piriform cortex and / or entorhinal cortex.

7. The method of claim 4 , wherein the marker is detected in a bodily fluid of the subject.

8. 8. The method of claim 7, wherein the bodily fluid is blood or cerebrospinal fluid (CSF).

9. 10. The method of claim 1, wherein the subject does not exhibit signs of dementia and / or cognitive impairment.

10. 10. The method of claim 1, wherein the subject has mild cognitive impairment or mild dementia.

11. The method of claim 1 , wherein the subject is amyloid positive.

12. 12. The method of claim 11, wherein the subject is at risk for further Aβ accumulation.

13. The method of claim 12, wherein the subject is an ApoE4 carrier.

14. 13. The method of claim 12, wherein the subject has an intermediate level of amyloid PET (e.g., 20-40 centiloids).

15. 13. The method of claim 12, wherein the subject has a high level of amyloid PET (e.g., >40 centiloid).

16. 10. The method of claim 1, wherein the subject has been diagnosed with AD based on brain imaging, cognitive function and / or biomarker criteria.

17. 17. The method of claim 16, wherein the subject has early stage AD.

18. 17. The method of claim 16, wherein the subject has pre-AD.

19. A method for reducing or maintaining tau (e.g., reducing or maintaining tau, or slowing tau accumulation, tau phosphorylation and / or tau diffusion, or slowing the rate of any of these) in a subject having or at risk of developing AD, comprising administering a therapeutically effective amount of lemborexant to the subject, wherein the therapeutically effective amount is sufficient to reduce tau in the subject.

20. 20. The method of claim 19, wherein the subject is amyloid-negative.

21. 20. The method of claim 19, wherein tau levels are reduced or maintained relative to a reference.

22. 20. The method of claim 19, comprising reducing and / or delaying and / or slowing the rate of tau accumulation and / or tau spreading compared to a reference.

23. 23. The method of claim 22, wherein the reference is a baseline measurement from the subject before treatment.

24. 23. The method of claim 22, wherein the reference is a baseline measurement from a control subject.

25. 23. The method of claim 22, wherein the reference is a measurement from a control subject administered a placebo.

26. 20. The method of claim 19, comprising altering tau in a brain region of the subject.

27. 20. The method of claim 19, comprising altering tau PET signal in a brain region of the subject.

28. 28. The method of claim 27, wherein the brain region is the hippocampus, the entorhinal cortex, and / or the piriform cortex.

29. 20. The method of claim 19, comprising reducing tau in a bodily fluid of the subject.

30. 30. The method of claim 29, wherein the bodily fluid is blood or CSF.

31. 20. The method of claim 19, wherein the tau is total tau.

32. 20. The method of claim 19, wherein the tau is insoluble tau.

33. 20. The method of claim 19, wherein the tau is aggregated tau.

34. 20. The method of claim 19, wherein the tau is phosphorylated tau (phospho-tau).

35. 35. The method of claim 34, wherein the phospho-tau is phosphorylated at one or more of T181, T217, S202, S205, or T231.

36. 35. The method of claim 34, comprising altering the ratio of phospho-tau to total tau.

37. 37. The method of claim 36, wherein the ratio of phospho-tau to total tau is reduced compared to the ratio of CSF phospho-tau to total tau in the subject prior to administration of lemborexant.

38. 37. The method of claim 36, wherein the ratio of phospho-tau to total tau is maintained within 10% of the ratio of phospho-tau to total tau in the subject prior to the administration of lemborexant.

39. 35. The method of claim 34, comprising increasing the rate of dephosphorylation of phospho-tau.

40. 35. The method of claim 34, comprising decreasing the rate of tau phosphorylation.

41. 20. The method of claim 19, comprising reducing or maintaining tau within 48 hours of administration of the first dose of lemborexant.

42. 35. The method of claim 34, comprising reducing phospho-tau in the hippocampus, entorhinal cortex and / or piriform cortex.

43. A method for altering neurodegeneration (e.g., reducing and / or delaying and / or slowing the rate of neurodegeneration) in a subject having or at risk of developing AD, comprising administering to the subject a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, sufficient to alter neurodegeneration in the subject.

44. 44. The method of claim 43, wherein the subject is amyloid negative.

45. 44. The method of claim 43, wherein altering neurodegeneration comprises reducing and / or delaying and / or slowing the rate of neurodegeneration compared to a reference.

46. 44. The method of claim 43, wherein the neurodegeneration is altered relative to a reference.

47. 47. The method of claim 46, wherein the reference is a baseline measurement from the subject before treatment.

48. 47. The method of claim 46, wherein the reference is a baseline measurement from a control subject.

49. 47. The method of claim 46, wherein the reference is a measurement from a control subject administered a placebo.

50. 44. The method of claim 43, wherein the neurodegeneration is characterized by a decrease in at least one of cortical thickness and hippocampal volume.

51. 51. The method of claim 50, wherein altering neurodegeneration comprises maintaining or slowing the decline of cortical thickness and / or hippocampal volume.

52. 44. The method of claim 43, wherein the neurodegeneration is characterized by a loss of at least one of pyramidal neurons in the cortex, pyramidal neurons in the hippocampus, or granule cells in the hippocampus.

53. 53. The method of claim 52, wherein altering neurodegeneration comprises preserving or reducing the loss of pyramidal neurons and / or granule cells.

54. 44. The method of claim 43, wherein altering neurodegeneration comprises reducing the rate of neurodegeneration.

55. 44. The method of claim 43, wherein altering neurodegeneration comprises altering neurofilament light chain (NfL) levels.

56. 56. The method of claim 55, comprising altering the NfL level in the blood and / or CSF of the subject.

57. A method for altering Aβ plaques (e.g., reducing or delaying the formation of Aβ plaques or slowing their growth rate) in a subject having or at risk of developing AD, comprising administering to the subject a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, sufficient to alter Aβ plaques in the subject.

58. 58. The method of claim 57, wherein the Aβ plaques are altered relative to a reference.

59. 59. The method of claim 58, wherein altering Aβ plaques comprises reducing and / or delaying the formation of Aβ plaques and / or slowing the rate thereof compared to a reference.

60. 59. The method of claim 58, wherein the reference is a baseline measurement from the subject before treatment.

61. 59. The method of claim 58, wherein the reference is a baseline measurement from a control subject.

62. 59. The method of claim 58, wherein the reference is a measurement from a control subject administered a placebo.

63. 58. The method of claim 57, wherein the Aβ plaque is a fibrous plaque.

64. 58. The method of claim 57, wherein the Aβ plaques are all plaques.

65. 58. The method of claim 57, wherein altering Aβ plaques comprises reducing Aβ plaque growth.

66. 66. The method of claim 65, comprising reducing Aβ plaque growth in the subject's hippocampus, the subject's somatomotor cortex, somatosensory cortex, and / or piriform cortex.

67. 58. The method of claim 57, wherein altering Aβ plaques comprises altering an amyloid PET signal obtained from a brain region of the subject.

68. 58. The method of claim 57, wherein altering Aβ plaques corresponds to a decrease in the concentration of Aβ in the subject's CSF.

69. 69. The method of claim 68, wherein the Aβ is Aβ38, Aβ40 and / or Aβ42.

70. 58. The method of claim 57, comprising altering Aβ plaques within 48 hours of administering the first dose of lemborexant.

71. 58. The method of claim 57, wherein the subject does not exhibit signs of dementia and / or cognitive impairment.

72. 58. The method of claim 57, wherein the subject has mild cognitive impairment or mild dementia.

73. 58. The method of claim 57, wherein the subject is at risk for further Aβ accumulation.

74. 74. The method of claim 73, wherein the subject is an ApoE4 carrier.

75. 74. The method of claim 73, wherein the subject has an intermediate level of amyloid PET (e.g., 20-40 centiloids).

76. 74. The method of claim 73, wherein the subject has a high level of amyloid PET (e.g., >40 centiloid).

77. 58. The method of claim 57, wherein the subject has early stage AD.

78. 58. The method of claim 57, wherein the subject has pre-AD.

79. 1. A method of modulating microglial responses in a subject having or at risk of developing Alzheimer's disease (AD), comprising administering to the subject a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the therapeutically effective amount is sufficient to modulate the microglial response in the subject.

80. 80. The method of claim 79, wherein modulating the microglial response comprises modulating expression of at least one microglial marker.

81. 81. The method of claim 80, wherein the microglial marker is a general microglial marker.

82. 82. The method of claim 81, wherein the general microglial marker is Iba1, Clec7a or CD68.

83. 81. The method of claim 80, wherein the microglial marker is a homeostatic microglial marker.

84. 84. The method of claim 83, wherein the homeostatic microglial marker is TMEM119 or P2RY12.

85. 80. The method of claim 79, wherein modulating the microglial response comprises modulating the activity of phagocytic microglia.

86. 80. The method of claim 79, wherein the subject has mild cognitive impairment or mild dementia.

87. 80. The method of claim 79, wherein the subject does not exhibit signs of dementia and / or cognitive impairment.

88. 80. The method of claim 79, wherein the subject is amyloid-negative.

89. 80. The method of claim 79, wherein the subject has tau pathology.

90. 80. The method of claim 79, wherein the subject has neurodegeneration in a brain region.

91. 91. The method of claim 90, wherein the brain region is the hippocampus, entorhinal cortex, and / or piriform cortex.

92. 91. The method of claim 90, wherein the brain region is the CA1 region, CA2 region, CA3 region or dentate gyrus in the hippocampus.

93. 89. The method of claim 88, wherein modulating the microglial response comprises modulating a response in microglia associated with degenerating neurons.

94. 94. The method of claim 93, wherein modulating the microglial response comprises decreasing the expression of at least one general microglial marker.

95. 95. The method of claim 94, wherein the general microglial marker is Iba1, CD68 or Clec7a.

96. 94. The method of claim 93, wherein modulating the microglial response comprises increasing expression of at least one homeostatic microglial marker.

97. 97. The method of claim 96, wherein the homeostatic microglial marker is TMEM119 or P2RY12.

98. 80. The method of claim 79, wherein the subject has Aβ plaques.

99. 99. The method of claim 98, wherein the Aβ plaque is a fibrillar Aβ plaque.

100. 99. The method of claim 98, wherein the subject is at risk for further Aβ accumulation.

101. The method of claim 100, wherein the subject is an ApoE4 carrier.

102. 101. The method of claim 100, wherein the subject has an intermediate level of amyloid PET (e.g., 20-40 centiloids).

103. 101. The method of claim 100, wherein the subject has a high level of amyloid PET (e.g., >40 centiloid).

104. 99. The method of claim 98, wherein the subject has early stage AD.

105. 99. The method of claim 98, wherein the subject has pre-AD.

106. 99. The method of claim 98, wherein the Aβ plaques are present in the hippocampus, somatomotor cortex, somatosensory cortex, and / or piriform cortex.

107. 99. The method of claim 98, wherein modulating the microglial response comprises modulating a response in microglia associated with Aβ plaques.

108. 108. The method of claim 107, wherein modulating the microglial response comprises increasing expression of a general microglial marker.

109. 109. The method of claim 108, wherein the general microglial marker is Iba1, Clec7a or CD68.

110. 108. The method of claim 107, wherein modulating the microglial response comprises increasing phagocytosis of Aβ plaques by phagocytic microglia.

111. 108. The method of claim 107, wherein modulating the microglial response comprises reducing expression of a homeostatic microglial marker.

112. 112. The method of claim 111, wherein the homeostatic microglial marker is TMEM119 or P2RY12.

113. 113. The method of any one of claims 1 to 112, wherein the therapeutically effective amount of lemborexant administered to the subject ranges from 5 mg to 50 mg per day.

114. 114. The method of claim 113, wherein the therapeutically effective amount of lemborexant administered to the subject ranges from 10 mg to 30 mg per day.

115. 114. The method of claim 113, wherein the therapeutically effective amount of lemborexant administered to the subject is selected from 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, and 30 mg per day.

116. 114. The method of claim 113, wherein the therapeutically effective amount of lemborexant administered to the subject is 20-25 mg per day.

117. 113. The method of any one of claims 1-112, wherein a single dose of 25 mg of lemborexant is administered to the subject once per day.

118. 114. The method of claim 113, wherein lemborexant is administered at a first dose for a first period of time, at a second dose for a second period of time, and optionally at a third dose for a third period of time.

119. 119. The method of claim 118, wherein each of the first period, the second period, and the third period is one week.

120. 119. The method of claim 118, wherein the first dose is lower than the second dose, and optionally, the second dose is lower than the third dose.

121. 121. The method of claim 120, wherein the first dose is 5 mg of lemborexant once per day, the second dose is 10 mg of lemborexant once per day, and optionally, the third dose is 20-25 mg of lemborexant once per day.

122. 121. The method of claim 120, wherein the first dose is 5 mg or 7.5 mg of lemborexant once per day, the second dose is 10 mg, 12.5 mg, 15 mg, or 17.5 mg of lemborexant once per day, and the third dose is 20 mg, 22.5 mg, 25 mg, 27.5 mg, or 30 mg of lemborexant once per day.

123. 119. The method of claim 118, wherein the first dose is higher than the second dose, and optionally the second dose is higher than the third dose.

124. 124. The method of claim 123, wherein the first dose is 20-25 mg of lemborexant once per day, the second dose is 10 mg of lemborexant once per day, and optionally, the third dose is 5 mg of lemborexant once per day.

125. 124. The method of claim 123, wherein the first dose is 20 mg, 22.5 mg, 25 mg, 27.5 mg, or 30 mg of lemborexant once per day, the second dose is 10 mg, 12.5 mg, 15 mg, or 17.5 mg of lemborexant once per day, and optionally, the third dose is 5 mg or 7.5 mg of lemborexant once per day.

126. 126. The method of any one of claims 1-125, comprising administering lemborexant to the subject for at least six months.

127. 127. The method of claim 126, comprising administering lemborexant to the subject for at least 9 months, at least 12 months, or at least 15 months.

128. 127. The method of claim 126, comprising administering lemborexant to the subject for at least 18 months.

129. 129. The method of any one of claims 126-128, comprising administering lemborexant to the subject for at least 24 months, 30 months, or 36 months.

130. 1. A method of selecting a subject having or at risk of developing Alzheimer's disease (AD) for treatment with lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, comprising: (a) obtaining from the subject a measure of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial response, and biomarker expression; (b) comparing the measurements from the subject with measurements from a reference; and (c) selecting the subject for treatment with lemborexant if the measured value from the subject differs from the measured value from the reference. A method comprising:

131. 131. The method of claim 130, wherein the subject has mild cognitive impairment or mild dementia.

132. 131. The method of claim 130, wherein the subject does not exhibit signs of dementia and / or cognitive impairment.

133. 131. The method of claim 130, wherein the subject is at risk for Aβ accumulation.

134. The method of claim 133, wherein the subject is an ApoE4 carrier.

135. 134. The method of claim 133, wherein the subject has an intermediate level of amyloid PET (e.g., 20-40 centiloids).

136. 134. The method of claim 133, wherein the subject has a high level of amyloid PET (e.g., >40 centiloid).

137. 131. The method of claim 130, wherein the subject has early stage AD.

138. 131. The method of claim 130, wherein the subject has pre-AD.

139. 131. The method of claim 130, wherein the subject has been diagnosed with AD based on brain imaging, cognitive function and / or biomarker criteria.

140. 131. The method of claim 130, wherein obtaining at least one measurement comprises obtaining data from a brain scan of the subject and / or obtaining data from a biological sample from the subject.

141. 141. The method of claim 140, wherein the data from the brain scan indicates levels of tau phosphorylation, tau aggregation, Aβ plaque burden, and / or microglial response.

142. 141. The method of claim 140, wherein the biological sample is a bodily fluid.

143. 141. The method of claim 140, wherein the bodily fluid is cerebrospinal fluid (CSF), blood or saliva.

144. 131. The method of claim 130, wherein the reference is a control.

145. 131. The method of claim 130, wherein the reference is a measurement from a control subject administered a placebo.

146. 145. The method of claim 144, wherein the control does not have AD.

147. 147. The method of claim 146, wherein the measured value from the subject is higher than the measured value from the control without AD.

148. 147. The method of claim 146, wherein the measured value from the subject is lower than the measured value from the control without AD.

149. 145. The method of claim 144, wherein the subject has AD.

150. 150. The method of claim 149, wherein the measurement from the subject is equal to or greater than the measurement from the control with AD.

151. 150. The method of claim 149, wherein the measurement from the subject is equal to or less than the measurement from the control with AD.

152. 131. The method of claim 130, wherein the measure of tau phosphorylation comprises a measure of phosphorylation at one or more of T181, T217, S202, S205 or T231.

153. 131. The method of claim 130, wherein the measure of tau aggregation comprises a measure of insoluble tau aggregates (e.g., neurofibrillary tangles (NFTs)).

154. 131. The method of claim 130, wherein the measure of neurodegeneration comprises a measure of cortical thickness and / or hippocampal volume or a measure of pyramidal or granular neuron loss.

155. 131. The method of claim 130, wherein the measure of Aβ plaque burden comprises a measure of Aβ plaque volume and / or Aβ plaque volume growth.

156. 131. The method of claim 130, wherein the measure of Aβ plaque burden comprises a measure of amyloid PET signal in a brain region of the subject or a measure of Aβ in the CSF of the subject.

157. 131. The method of claim 130, wherein the measure of microglial response is a change in expression of at least one microglial marker.

158. 158. The method of claim 157, wherein the microglial marker is Iba1, Clec7a, CD68, TMEM119 or P2RY12.

159. 158. The method of claim 157, wherein said measure of microglial response is a measure of phagocytosis by microglia.

160. 1. A method for monitoring the effectiveness of a treatment in a subject having or at risk of developing Alzheimer's disease (AD), comprising: (a) obtaining from the subject a first measure of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression; (b) administering to the subject a dose of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof; (c) obtaining from the subject a second measure of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression; and (d) comparing the second measurement from the subject with the first measurement from the subject. wherein a difference between the first measurement and the second measurement indicates effective treatment with lemborexant.

161. 1. A method of treating a subject having or at risk of developing Alzheimer's disease (AD), comprising: (a) obtaining from the subject a first measure of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial response, and biomarker expression; (b) administering to the subject a first dose of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof; (c) obtaining from the subject a second measure of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression; (d) comparing the second measurement from the subject with the first measurement from the subject; and (e) administering a second dose of lemborexant if the first measured value is different from the second measured value. A method comprising:

162. 162. The method of claim 160 or 161, wherein obtaining at least one measurement comprises obtaining data from a brain scan of the subject and / or obtaining data from a biological sample from the subject.

163. 163. The method of claim 162, wherein the data from the brain scan indicates levels of tau phosphorylation, tau aggregation, Aβ plaque burden, and / or microglial response.

164. 163. The method of claim 162, wherein the biological sample is a bodily fluid.

165. 165. The method of claim 164, wherein the bodily fluid is cerebrospinal fluid (CSF), blood, or saliva.

166. 162. The method of claim 160 or 161, wherein the first measurement value from the subject is higher than the second measurement value from the subject.

167. 162. The method of claim 160 or 161, wherein the first measurement value from the subject is lower than the second measurement value from the subject.

168. 162. The method of claim 160 or 161, wherein the measure of tau phosphorylation comprises a measure of phosphorylation of one or more of T181, T217, S202, S205 or T231.

169. 162. The method of claim 160 or 161, wherein the measure of tau aggregation comprises a measure of insoluble tau aggregates (e.g., neurofibrillary tangles (NFTs)).

170. 162. The method of claim 160 or 161, wherein the measure of neurodegeneration comprises a measure of cortical thickness and / or hippocampal volume or a measure of pyramidal or granular neuron loss.

171. 162. The method of claim 160 or 161, wherein the measure of Aβ plaque burden comprises a measure of Aβ plaque volume and / or Aβ plaque volume growth.

172. 162. The method of claim 160 or 161, wherein the measure of Aβ plaque burden comprises a measure of amyloid PET signal in a brain region of the subject or a measure of Aβ in the CSF of the subject.

173. 162. The method of claim 160 or 161, wherein the measure of microglial response is the degree of expression of at least one microglial marker.

174. 174. The method of claim 173, wherein the microglial marker is Iba1, Clec71, P2RY12 or TMEM119.

175. 174. The method of claim 173, wherein said measure of microglial response is a measure of phagocytosis by microglia.

176. 162. The method of claim 160 or 161, wherein the measure of biomarker expression is a measure of Ifnb1, MMP2 and / or Base1 expression.

177. 162. The method of claim 160 or 161, wherein the subject is amyloid negative.

178. 161. The method of claim 160 or 160, wherein the subject has Aβ plaques.

179. 162. The method of claim 160 or 161, wherein the subject has mild cognitive impairment and / or mild dementia.

180. 162. The method of claim 160 or 161, wherein the subject does not exhibit signs of dementia and / or cognitive impairment.

181. 162. The method of claim 160 or 161, wherein the subject is at risk for further Aβ accumulation.

182. The method of claim 181, wherein the subject is an ApoE4 carrier.

183. 182. The method of claim 181, wherein the subject has an intermediate level of amyloid PET (e.g., 20-40 centiloids).

184. The method of claim 181, wherein the subject has a high level of amyloid PET (e.g., >40 centiloids).

185. 162. The method of claim 160 or 161, wherein the subject has early stage AD.

186. 162. The method of claim 160 or 161, wherein the subject has pre-AD.

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