Methods of reducing neurodegeneration associated with neurodegenerative disease

Abstract

The present disclosure relates to a dual orexin receptor antagonist, i.e., Labobolifera, as well as compositions and methods for use in the treatment of Alzheimer's disease (AD), e.g., in a subject suffering from or at risk of developing AD.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 376,949, filed on September 23, 2023, and U.S. Provisional Application No. 63 / 382,278, filed on November 3, 2023, the entire contents of which are hereby incorporated by reference in their entirety. Technical field

[0003] Compositions and methods involving the dual orexin receptor antagonist, lemborexant, are described herein for use in the treatment of neurological disorders such as Alzheimer's disease (AD). Background art

[0004] Alzheimer's disease (AD) is a progressive, neurodegenerative disorder of unknown etiology and is the most common form of dementia in the elderly. In 2006, there were 26.6 million cases of AD worldwide (range: 11.4 - 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 over 5 million people in the United States had AD (Alzheimer’s Association, Alzheimer’s Association report, 2010 Alzheimer’s disease facts and figures. Alzheimer Dement. 2010; 6:158 - 94). By 2050, it is predicted that the prevalence of AD will increase worldwide to 106.8 million (range: 47.2 - 221.2 million), and the prevalence in the United States alone is estimated to be 11 - 16 million. (Brookmeyer, supra, and 2010 Alzheimer's disease facts and figures, supra).

[0005] The disease typically involves a global decline in cognitive function, which progresses slowly and renders end-stage subjects bedridden. AD subjects typically survive only 3 to 10 years after symptom onset, although survival extremes of 2 years and 20 years are known. (Hebert, L.E., et al., Alzheimer disease in the U.S. population: prevalence estimates using the 2000 census. Arch Neurol. 2003;60:1119-1122.) Although the fact that death certificates rarely attribute the cause of death to AD results in a substantial underestimation of deaths due to AD, AD is still the seventh leading cause of all deaths in the United States and the fifth leading cause of death among Americans over 65 years of age.

[0006] AD represents a heavy economic burden in industrialized countries, with significant implications for the healthcare system and the treasury, as well as for subjects and their families. In the United States alone, the total cost in 2010 was estimated at $172 billion, including $123 billion for Medicare and Medicaid.

[0007] Histologically, the disease is characterized by neuritic plaques, which are mainly found in the association cortex, limbic system, and basal ganglia. The main component of these 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 onset of Alzheimer's disease and Aβ production are not yet clear. However, some anti-Aβ antibodies are currently being clinically investigated as potential therapeutic agents for Alzheimer's disease.

[0008] In addition to neuritic plaques, the disease is also characterized by tau aggregation and hyperphosphorylation, increased immune response, neuronal degeneration, synaptic loss, and ultimately cognitive dysfunction, dementia, and death.

[0009] Insomnia has been considered a risk factor for AD. Historically, insomnia has been treated with various medications, including doxepin, tricyclic antidepressants (TCA), and dual orexin receptor antagonists (also known as DORA) such as suvorexant or lemborexant. Although the literature has suggested a link between sleep disturbances and the risk of AD, a direct link has not been established. To illustrate the need for further clarification of the link between insomnia and AD, the data disclosed herein indicate that at least two such sleep medications, doxepin and lemborexant, have differential effects on AD pathology, although both mediate sleep.

[0010] Lemborexant has been approved for the treatment of adult patients with insomnia characterized by difficulty falling asleep and / or maintaining sleep. Lemborexant and its method 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.

[0011] Lemborexant has the following structure:

[0012]

[0013] 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.

[0014] Accordingly, there is still a need for improved AD treatment, including early intervention before the development of irreversible symptoms of the disease. The disclosure herein surprisingly demonstrates that lemborexant can be used for such treatment. SUMMARY OF THE INVENTION

[0015] One aspect of the present disclosure relates to a method for treating Alzheimer's disease (AD) in a subject suffering from 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, thereby treating AD.

[0016] In some embodiments, treating AD includes reducing and / or slowing cognitive decline. In some embodiments, treating AD includes affecting a change (e.g., slowing, delaying, or reducing) in at least one biomarker of AD pathology.

[0017] In some embodiments, the biomarker is the phosphorylation level of tau, neurodegeneration, changes in microglial response, and / or the presence of Aβ plaques. In some embodiments, the biomarker is present in a brain region of the subject. In some embodiments, the brain region is the hippocampus, somatic motor cortex, somatic sensory cortex, piriform cortex, and / or entorhinal cortex. In some embodiments, the biomarker is detected in a body fluid of the subject. In some embodiments, the body fluid is blood or cerebrospinal fluid (CSF).

[0018] In some embodiments, the subject does not show signs of dementia and / or cognitive impairment. In some embodiments, the subject has mild cognitive impairment or mild dementia.

[0019] In some embodiments, the subject is amyloid - positive. In some embodiments, the subject is at risk of further Aβ accumulation. In some embodiments, the subject is an ApoE4 carrier. In some embodiments, the subject has a moderate level of amyloid PET (e.g., 20 - 40 percentile units). In some embodiments, the subject has an elevated level of amyloid PET (e.g., >40 percentile units).

[0020] 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.

[0021] One aspect of the present disclosure relates to a method of reducing or maintaining tau in a subject having AD or at risk of developing AD (e.g., reducing or maintaining the tau level relative to the level before the start of treatment, or delaying tau accumulation, tau phosphorylation, and / or tau spreading, or slowing the rate of any of these), the method comprising administering to the subject a therapeutically effective amount of lemborexant, wherein the therapeutically effective amount is sufficient to reduce or maintain tau in the subject.

[0022] In some embodiments, the subject is amyloid - negative. In some embodiments, the tau level is reduced or maintained relative to a reference. In some embodiments, the method comprises reducing and / or delaying tau accumulation and / or tau spreading, and / or slowing its rate, compared 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.

[0023] In some embodiments, the method comprises altering tau in a brain region of the subject. In some embodiments, the method comprises altering the 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 body fluid of the subject. In some embodiments, the body fluid is blood or CSF.

[0024] 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 the phosphorylated form of tau (phosphorylated tau). In some embodiments, the phosphorylated tau is phosphorylated at one or more of T181, T217, S202, S205, or T231.

[0025] In some embodiments, the method includes altering the ratio of phosphorylated tau to total tau. In some embodiments, the ratio of phosphorylated tau to total tau is decreased compared to the ratio of CSF phosphorylated tau to total tau of the subject prior to administration of lemborexant. In some embodiments, the ratio of phosphorylated tau to total tau is maintained within 10% of the ratio of phosphorylated tau to total tau of the subject prior to administration of lemborexant. In some embodiments, the method includes increasing the rate of dephosphorylation of phosphorylated tau. In some embodiments, the method includes decreasing the rate of phosphorylation of tau. In some embodiments, the method includes reducing or maintaining tau within 48 hours of administration of a first dose of lemborexant. In some embodiments, the method includes reducing phosphorylated tau in the hippocampus, entorhinal cortex, and / or piriform cortex.

[0026] Another aspect of the present disclosure relates to a method of altering neurodegeneration (e.g., reducing and / or delaying neurodegeneration, and / or slowing its rate) in a subject having 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 alter neurodegeneration in the subject.

[0027] In some embodiments, the subject is amyloid negative. In some embodiments, altering neurodegeneration includes reducing and / or delaying neurodegeneration and / or slowing its rate compared to a reference. In some embodiments, the neurodegeneration is altered relative to a reference. In some embodiments, the reference is a baseline measurement from the subject prior to 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 neurodegeneration is characterized by loss of at least one of cortical thickness and hippocampal volume. In some embodiments, altering neurodegeneration includes maintaining cortical thickness and / or hippocampal volume or slowing the loss of cortical thickness and / or hippocampal volume. In some embodiments, the neurodegeneration is characterized by loss of 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 includes maintaining pyramidal neurons and / or granule cells or reducing the loss of pyramidal neurons and / or granule cells. In some embodiments, altering neurodegeneration includes decreasing the rate of neurodegeneration. In some embodiments, altering neurodegeneration includes altering neurofilament light chain (NfL) levels. In some embodiments, the method includes altering the NfL levels in the blood and / or CSF of the subject.

[0029] Another aspect of the present disclosure relates to a method of altering Aβ plaques (e.g., reducing or delaying the formation of Aβ plaques and / or slowing their growth rate) in a subject having 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 alter Aβ plaques in the subject.

[0030] In some embodiments, the Aβ plaques are altered relative to a reference. In some embodiments, altering Aβ plaques comprises reducing and / or delaying the formation of Aβ plaques and / or slowing their rate, compared to a reference. In some embodiments, the reference is a baseline measurement from the subject prior to 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.

[0031] In some embodiments, the Aβ plaques are fibrillar plaques. In some embodiments, the Aβ plaques are all plaques (e.g., including diffuse plaques). In some embodiments, altering Aβ plaques comprises reducing the growth of Aβ plaques. In some embodiments, the method comprises reducing the growth of Aβ plaques in the hippocampus of the subject, the somatic motor cortex of the subject, the somatosensory cortex, and / or the piriform cortex.

[0032] 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 CSF of the subject.

[0033] In some embodiments, the Aβ is Aβ38, Aβ40, and / or Aβ42. In some embodiments, Aβ plaques are altered within 48 hours of administering a first dose of lemborexant.

[0034] 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.

[0035] In some embodiments, the subject is at risk of further Aβ accumulation. In some embodiments, the subject is an ApoE4 carrier. In some embodiments, the subject has a moderate level of amyloid PET (e.g., 20 - 40 percentile units). In some embodiments, the subject has an elevated level of amyloid PET (e.g., >40 percentile units).

[0036] In some embodiments, the subject has early AD. In some embodiments, the subject has pre - AD.

[0037] One aspect of the present disclosure relates to a method of modulating the microglial response 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 the microglial response in the subject.

[0038] 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.

[0039] In some embodiments, the subject has mild cognitive impairment or mild dementia. In some embodiments, the subject does not exhibit signs of dementia and / or cognitive impairment.

[0040] 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 in the hippocampus.

[0041] In some embodiments, modulating the microglial response comprises modulating the response in microglia associated with degenerating neurons. In some embodiments, modulating the microglial response comprises reducing 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.

[0042] In some embodiments, the subject has Aβ plaques. In some embodiments, these Aβ plaques are fibrillar Aβ plaques. In some embodiments, the subject is at risk of further Aβ accumulation. In some embodiments, the subject is an ApoE4 carrier. In some embodiments, the subject has a moderate level of amyloid PET (e.g., 20 - 40 percentile units). In some embodiments, the subject has an elevated level of amyloid PET (e.g., >40 percentile units).

[0043] In some embodiments, the subject has early AD. In some embodiments, the subject has pre - AD.

[0044] In some embodiments, these Aβ plaques are present in the hippocampus, somatic motor cortex, somatic sensory cortex, and / or piriform cortex.

[0045] In some embodiments, modulating the microglial response includes modulating the response in microglia associated with Aβ plaques. In some embodiments, modulating the microglial response includes increasing the expression of general microglial markers. In some embodiments, the general microglial marker is 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 homeostatic microglial markers. In some embodiments, the homeostatic microglial marker is TMEM119 or P2RY12.

[0046] In some embodiments of any of the methods disclosed herein, the therapeutically effective amount of lemborexant administered to the subject is in the range of 5 mg to 50 mg per day. In some embodiments, the therapeutically effective amount of lemborexant administered to the subject is in the range of 10 mg to 30 mg per day.

[0047] In some embodiments, 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.

[0048] In some embodiments, the therapeutically effective amount of lemborexant administered to the subject is 20 - 25 mg per day. In some embodiments, a dose of 25 mg of lemborexant is administered to the subject once a day.

[0049] In some embodiments, lemborexant is administered in a first cycle at a first dose, in a second cycle at a second dose, and optionally in a third cycle at a third dose. In some embodiments, each of the first cycle, the second cycle, and the third cycle is 1 week. In some embodiments, the first dose is lower than the second dose, and optionally, the second dose is lower than the third dose.

[0050] 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.

[0051] 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.

[0052] In some embodiments of any of the methods disclosed herein, administering lemborexant to the subject for at least 6 months is included. In some embodiments, the method includes administering lemborexant to the subject for at least 9 months, at least 12 months, or at least 15 months. In some embodiments, the method includes administering lemborexant to the subject for at least 18 months. In some embodiments, the method includes administering lemborexant to the subject for at least 24 months, 30 months, or 36 months.

[0053] Another aspect of the present disclosure relates to a method of 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 a measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial response, and biomarker expression from the subject; (b) comparing the measurement from the subject with a measurement from a reference; and (c) selecting the subject for treatment with lemborexant if the measurement from the subject is different from the measurement from the reference.

[0054] In some embodiments, the subject has mild cognitive impairment or mild dementia. In some embodiments, the subject does not exhibit signs of dementia and / or cognitive impairment.

[0055] In some embodiments, the subject is at risk of Aβ accumulation. In some embodiments, the subject is an ApoE4 carrier. In some embodiments, the subject has a moderate level of amyloid PET (e.g., 20 - 40 percentile units). In some embodiments, the subject has an elevated level of amyloid PET (e.g., >40 percentile units).

[0056] In some embodiments, the subject has early AD. In some embodiments, the subject has pre - AD.

[0057] 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 measurement includes 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 the level of tau phosphorylation, tau aggregation, Aβ plaque burden, and / or microglial response. In some embodiments, the biological sample is a body fluid. In some embodiments, the body fluid is cerebrospinal fluid (CSF), blood, or saliva.

[0058] In some embodiments, the reference is a control. In some embodiments, the reference is a measurement from a control subject receiving a placebo.

[0059] In some embodiments, the control does not have AD. In some embodiments, the measurement from the subject is higher than the measurement from the control that does not have AD. In some embodiments, the measurement from the subject is lower than the measurement from the control that does not have AD.

[0060] In some embodiments, the control has AD. In some embodiments, the measurement value from the subject is similar to or higher than the measurement value from the control having AD. In some embodiments, the measurement value from the subject is similar to or lower than the measurement value from the control having AD.

[0061] In some embodiments, the measurement value of tau phosphorylation includes the measurement value of phosphorylation on one or more of T181, T217, S202, S205 or T231. In some embodiments, the measurement value of tau aggregation includes the measurement value of insoluble tau aggregates (e.g., neurofibrillary tangles (NFT)).

[0062] In some embodiments, the measurement value of neurodegeneration includes the measurement value of cortical thickness and / or hippocampal volume or the measurement value of pyramidal neuron or granule neuron loss.

[0063] In some embodiments, the measurement value of Aβ plaque burden includes the measurement value of Aβ plaque volume and / or the measurement value of Aβ plaque volume growth. In some embodiments, the measurement value of Aβ plaque burden includes the measurement value of amyloid PET signal in the brain region of the subject or the measurement value of Aβ in the CSF of the subject.

[0064] In some embodiments, the measurement value of microglial response is a change in the expression of at least one microglial marker. In some embodiments, the microglial marker is Iba1, Clec7a, CD68, TMEM119 or P2RY12. In some embodiments, the measurement value of microglial response is the measurement value of microglial phagocytosis.

[0065] Another aspect of the present disclosure relates to a method for monitoring the therapeutic efficacy of a subject having Alzheimer's disease (AD) or at risk of developing AD, the method comprising: (a) obtaining a first measurement value of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function and biomarker expression from the subject; (b) administering to the subject a dose of lemborexant, its pharmaceutically acceptable salt or its solvate; (c) obtaining a second measurement value of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function and biomarker expression from the subject; and (d) comparing the second measurement value from the subject with the first measurement value from the subject, wherein a difference between the first measurement value and the second measurement value indicates effective treatment with lemborexant.

[0066] 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 a first measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial response, and biomarker expression from the subject; (b) administering to the subject a first dose of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof; (c) obtaining a second measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial function, and biomarker expression from the subject; (d) comparing the second measurement from the subject with the first measurement from the subject, and (e) if the first measurement is different from the second measurement, administering a second dose of lemborexant.

[0067] In some embodiments, 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. 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 body fluid. In some embodiments, the body fluid is cerebrospinal fluid (CSF), blood, or saliva.

[0068] 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.

[0069] In some embodiments, the measurement of tau phosphorylation comprises the measurement of phosphorylation of one or more of T181, T217, S202, S205, or T231. In some embodiments, the measurement of tau aggregation comprises the measurement of insoluble tau aggregates (e.g., neurofibrillary tangles (NFTs)).

[0070] In some embodiments, the measurement of neurodegeneration comprises the measurement of cortical thickness and / or hippocampal volume or the measurement of pyramidal neuron or granule neuron loss.

[0071] In some embodiments, the measurement of Aβ plaque burden comprises the measurement of Aβ plaque volume and / or Aβ plaque volume growth. In some embodiments, the measurement of Aβ plaque burden comprises the measurement of amyloid PET signal in a brain region of the subject or the measurement of Aβ in the CSF of the subject.

[0072] In some embodiments, the measurement of microglial response is a measurement of the expression of at least one microglial marker. In some embodiments, the microglial marker is Iba1, Clec71, P2RY12, or TMEM 119. In some embodiments, the measurement of microglial response is a measurement of microglial phagocytosis. In some embodiments, the measurement of biomarker expression is a measurement of Ifnb1, MMP2, and / or Bace1 expression.

[0073] In some embodiments, the subject is amyloid negative. In some embodiments, the subject has Aβ plaques.

[0074] 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.

[0075] In some embodiments, the subject is at risk of further Aβ accumulation. In some embodiments, the subject is an ApoE4 carrier. In some embodiments, the subject has a moderate level of amyloid PET (e.g., 20 - 40 percentile units). In some embodiments, the subject has an elevated level of amyloid PET (e.g., >40 percentile units).

[0076] In some embodiments, the subject has early AD. In some embodiments, the subject has pre - AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 Shows the study protocol of a clinical trial. Figure 1 Depicts an overview of the study design with a habitual bedtime of 22:00.

[0078] Figure 2A -H depicts the data from the study of Example 2. Figure 2A Shows a schematic diagram of the study design. Electroencephalogram (EEG) analysis shows the percentage of time spent sleeping; Figure 2B Shows NREM sleep, Figure 2C Shows REM sleep, and Figure 2D Shows wakefulness (Veh or Lem - treated E4 (n = 10 mice / treatment group) and P301S / E4 mice (n = 8 mice / treatment group)). Two - way ANOVA, Tukey post - hoc comparison. Figure 2E Shows a representative spectrogram of EEG and electromyogram (EMG) analysis, illustrating NREM, REM, and wakefulness patterns in Veh - treated P301S / E4, and Figure 2FShows P301S / E4 mice treated with lemborexant. Figure 2G Shows the time-course analysis of the percentage of sleep observed over time in E4 mice (n = 10 mice / treatment group) from the start of vehicle or lemborexant treatment until 24 hours after oral gavage, while Figure 2H Shows the results for P301S / E4 mice (n = 8 mice / treatment group). White bars and black bars represent the light and dark phases, respectively. Data are represented as mean ± SEM; *p < 0.05, **p < 0.001, ***p < 0.0001.

[0079] Figure 3 depicts data from the study of Example 2. Figure 3A Shows representative images of phosphorylated tau stained with AT8 at serine 202 and threonine 205. The upper panel shows the hippocampus, and the lower panel shows the entorhinal cortex and piriform cortex. Scale bar - 500 μm. Figure 3B Shows representative images of tau stained with MC1. Scale bar - 500 μm. Figure 3C Shows the quantification of the percentage of the hippocampus covered by AT8 (nE4 and nP301S / E4 = 16 - 17 mice / treatment group), and Figure 3D Shows the entorhinal cortex / piriform cortex (nE4 = 16 - 18 mice / treatment group; nP301S / E4 = 15 - 19 mice / treatment group). Figure 3E Shows the percentage of the hippocampus stained with MC1 (nE4 = 18 mice / treatment group; nP301S / E4 = 16 - 17 mice / treatment group), and Figure 3F Shows the entorhinal cortex / piriform cortex (nE4 = 16 - 17 mice / treatment group; nP301S / E4 = 15 - 17 mice / treatment group). Figure 3G Shows representative images of cresyl violet-stained brains for volume analysis. Scale bar - 1 mm. Figure 3H Shows the quantification of the hippocampal volume, and Figure 3I Shows the piriform cortex volume. Figure 3J Shows the plasma neurofilament light chain (NfL) levels measured by SIMOA (nE4 and nP301S / E4 = 16 - 20 mice / treatment group). Data are represented as mean ± SEM; two-way ANOVA, Tukey's post hoc test; *p < 0.05, **p < 0.001, ***p < 0.0001.

[0080] Figure 4A -R depicts data from the study of Example 2. Figure 4A Shows representative images of microglia co-stained with IBA1 (green), CD68 (red), and DAPI (blue) in the CA3 region of the hippocampus. Figure 4BRepresentative images of microglia stained for TMEM119 (yellow) and DAPI (blue) in the CA3 region of the hippocampus are shown. Figure 4C Quantification of the percentage of CA3 covered by IBA1 is shown, and Figure 4D Quantification of the percentage of CA3 covered by CD68 is shown. Figure 4E Representative images of microglia stained for Clec7a (red) and DAPI (blue) are shown. Figure 4F Quantification of the percentage of CA3 covered by TMEM119 is shown, and Figure 4G Quantification of the percentage of CA3 covered by Clec7a is shown. Figure 4H Representative images of astrocytes co-stained for ApoE (green) and GFAP (red) are shown. Figure 4I Quantification of the percentage of ApoE co-localized with GFAP in CA3, or Figure 4J Quantification of the percentage of ApoE co-localized with IBA1 in CA3 is shown. Figure 4K Representative images of positive microglia co-stained for ApoE (green) and IBA1 (magenta) are shown. Figure 4L The percentage of the piriform / entorhinal cortex covered by Iba1 in lesinurad-treated mice compared to vehicle-treated control mice is shown. Figure 4M The percentage of the dentate gyrus covered by Iba1 in lesinurad-treated mice compared to vehicle-treated control mice is shown. Figure 4N The percentage of the piriform / entorhinal cortex covered by TMEM119 in lesinurad-treated mice compared to vehicle-treated control mice is shown. Figure 4O The percentage of the dentate gyrus covered by TMEM119 in lesinurad-treated mice compared to vehicle-treated control mice is shown. Figure 4P The percentage of the dentate gyrus covered by CD68 in lesinurad-treated mice compared to vehicle-treated control mice is shown. Figure 4Q The percentage of the piriform / entorhinal cortex covered by CD68 in lesinurad-treated mice compared to vehicle-treated control mice is shown. Figure 4R Representative images of the CA1 / CA2 region stained for GFAP are shown, Figure 4S A percentage map of CA1 / 2 covered by GFAP is shown, Figure 4T A percentage map of the dentate gyrus covered by GFAP is shown, and Figure 4UPercentage plot of GFAP coverage in the piriform / entorhinal cortex is shown. Scale bar - 50 μm. nE4 = 15 - 19 mice / treatment group; nP301S / E4 = 16 - 19 mice / treatment group. Data are represented as mean ± SEM; two-way ANOVA, Tukey's post hoc test; *p < 0.05, **p < 0.001, ***p < 0.0001.

[0081] Figure 5 depicts data from the study of Example 2. Figure 5A Volcano plot showing differentially regulated genes in P301S / E4 mice treated with vehicle compared to lemborexant. Cutoff for significance was set at two-fold Log fold change. nE4 and nP301S / E4 = 10 mice / treatment group. Figure 5B GO term analysis of genes significantly changed in P301S / E4 mice treated with vehicle compared to lemborexant is shown. Figure 5C Heatmap showing significance of all differentially expressed genes in P301S / E4 vehicle versus lemborexant reaching an adjusted p-value of < 0.05 at Log10. Figure 5D and 5E Representative images of VGLUT1 and PSD95 stained synapses in CA3 are shown. Scale bar - 50 μm. Figure 5F Quantification of the percentage of VGLUT1 puncta in CA3 is shown, Figure 5G showing results in the piriform cortex. Figure 5H Quantification of the percentage of PSD95 puncta in CA3 is shown, Figure 5I showing results in the piriform cortex. nE4 = 16 - 19 mice / treatment group; nP301S / E4 = 16 - 19 mice / treatment group. Data are represented as mean ± SEM; two-tailed unpaired T test; *p < 0.05, **p < 0.005, ***p < 0.0001.

[0082] Figure 6 depicts data from the study of Example 2. Figure 6A Time course analysis of the percentage of sleep in E4 mice (n E4 = 7 - 9 mice / treatment group) is shown, and Figure 6B results in P301S / E4 mice (n P301S / E4 = 8 - 10 mice / treatment group) are shown. White and black bars represent light and dark phases, respectively. Figure 6C Percentage of time spent falling asleep during the light or dark phases in E4 and P301S / E4 mice (n = 10 mice / genotype and treatment group) is shown. Figure 6D Sleep episode length during the light or dark phases in E4 and P301S / E4 mice (n = 10 mice / genotype and treatment group) is shown.Figure 6E Shows the wake episode length. s - seconds. Data are presented as mean ± SEM; two - way ANOVA, Tukey's post - hoc test; *p < 0.05, **p < 0.001, ***p < 0.0001.

[0083] Figure 7 depicts data from the study of Example 2. Phosphorylated tau (pTau) in the RAB( Figure 7A ), RIPA( Figure 7B ), and formic acid (FA)( Figure 7C ) fractions was quantified by ELISA (nE4 and nP301S / E4 = 18 - 20 mice / treatment group). Total tau (tTau) in the RAB( Figure 7D ), RIPA( Figure 7E ), and FA( Figure 7F ) fractions was quantified by ELISA (nE4 = 18 - 20 and nP301S / E4 = 18 - 20 mice / treatment group). In Figure 7G , the upper panel shows a representative image of the hippocampus including the granular cell layer, and the lower panel shows the piriform cortex including the pyramidal cell layer. Figure 7H Shows the volumetric analysis of cresyl violet - stained hippocampus, Figure 7I shows the hemisphere minus the ventricles, Figure 7J shows the pyramidal cell layer of the piriform cortex, Figure 7K shows the granular cell layer of the hippocampus. (nE4 and nP301S / E4 = 17 - 19 mice / treatment group). Scale bar - 500 μm. Data are presented as mean ± SEM; two - way ANOVA, Tukey's post - hoc test; *p < 0.05, **p < 0.001, ***p < 0.0001.

[0084] Figure 8 depicts data from the study of Example 2. Figure 8A Shows representative images of microglia co - stained with DAPI, IBA1, and P2RY12 in CA3. Figure 8B Shows the percentage of CA3 covered by quantified P2RY12, Figure 8C shows the results in the dentate gyrus. nE4 and nP301S / E4 = 16 - 19 mice / treatment group. Scale bar - 50 μm. Data are presented as mean ± SEM; two - way ANOVA, Tukey's post - hoc test; *p < 0.05, **p < 0.001, ***p < 0.0001. Figure 8C Shows the percentage of the dentate gyrus covered by P2RY12. Figure 8D Shows the percentage of the piriform / entorhinal cortex covered by P2RY12.

[0085] Figure 9, from the study of Example 3, shows the sleep changes in APP / PS1dE9 mice administered with doxepin or lemborexant. Figure 9A is a schematic diagram of the experimental design. Figure 9B , Figure 9C and Figure 9D show the effects on total sleep, light-phase sleep, and dark-phase sleep in mice treated with doxepin, lemborexant (10 mg or 30 mg), or vehicle. Figure 9E shows the percentage of sleep as a function of Zeitgeber Time (ZT0 = lights on).

[0086] Figure 10, from the study of Example 3, shows the fibrillar amyloid plaque burden in APP / PS1dE9 mice administered with doxepin or lemborexant. Figure 10A is a schematic diagram of the mouse treatment time. Figure 10B shows representative images of brain sections stained with X34, which labels fibrillar amyloid plaques. Figure 10C shows the quantification of plaque burden (X34 staining area %) in different brain regions. Error bars indicate mean ± SEM, and each point represents one mouse. The P value from one-way ANOVA is shown.

[0087] Figure 11, from the study of Example 3, shows the total amyloid plaque burden in APP / PS1dE9 mice treated with doxepin or lemborexant as Figure 10A shown. Figure 11A shows representative brain sections stained for total amyloid plaque burden using the anti-Aβ antibody HJ3.4. Figure 11B is the quantification of plaque burden (HJ3.4 staining area %) in different brain regions. Error bars indicate mean ± SEM, and each point represents one mouse. The P value from one-way ANOVA is shown.

[0088] Figure 12, from the study of Example 3, shows APP processing / cleavage in APP / PS1dE9 mice treated with doxepin or lemborexant. Figure 12A shows representative immunoblot results for full-length APP and APP C-terminal fragments (CTF-α and -β). β-Tubulin is shown as a loading control. Figure 12B shows the quantification of band intensity. Error bars indicate mean ± SEM, and each point represents one mouse. The P value from one-way ANOVA is shown.

[0089] Figure 13, from the study of Example 3, shows perivascular microglial clustering in APP / PS1dE9 mice treated with doxepin or lemborexant. Figure 13ARepresentative images of samples stained for plaques (X34) and microglia (Iba1) are shown. The volume of microglia surrounding each plaque was calculated from the Z-stack of confocal images using Imaris software. Figure 13B Quantification of plaque volume (to indicate quantification of similarly sized plaques under different conditions) and perilesional Iba1 volume is shown. Error bars indicate mean ± SEM, and each point represents one mouse. P-values from one-way ANOVA are shown.

[0090] Figure 14, from the study of Example 3, shows perilesional microglial CD68 expression in APP / PS1dEP mice treated with doxepin or lemborexant. Figure 14A Representative images of samples stained for plaques (X34), microglia (Iba1), and phagosomes (CD68) are shown. Figure 14B CD68 co-localized with Iba1 quantified around each plaque using Imaris software is shown. Error bars represent mean ± SEM, and each point is the mean of 8 - 10 plaques from a single mouse. P-values from one-way ANOVA are shown. Figure 14C Shows the Iba1 volume (μm 3 ). Figure 14D Co-localization of Iba1 and CD68 measured as a percentage of Iba1 is shown. Figure 14E Volume of co-localized Iba1 and CD68 is shown.

[0091] Figure 15 , from the study of Example 3, shows the effect of lemborexant treatment on gene expression. After lemborexant treatment, the expression of transcripts encoding Ifnb1, Rab5a, and Mmp2 showed significant differences. Data are shown as fold change (relative to the mean of vehicle (VEH)). Error bars indicate mean ± SEM, and each point represents a single mouse. P-values from one-way ANOVA are shown.

[0092] Figure 16, from the study of Example 3, shows microglial amyloid plaque phagocytosis in APP / PS1dEP mice treated with lemborexant. Figure 16A A schematic diagram of the experimental design is shown. Figure 16B A flow cytometry gating strategy is shown. Figure 16C Positivity for methoxy-X04 (MX04) in the CD45-low, CD11b+ population as a potential microglial isolation is shown. Figure 16D Quantification of the percentage of MX04+ microglia by two-tailed T-test, p = 0.0207.

[0093] Figure 17, a study from Example 3, shows amyloid plaque growth in mice with pre-existing plaques. Figure 17A is a schematic of the experimental design. Figure 17B Shows representative images of MX04, thioflavin red, and the overlay image. P values are shown by one-way ANOVA. Figure 17C Shows representative images of amyloid plaques labeled with X34, microglia labeled with IBA1, and microglial phagosomes labeled with CD68. Figure 17D Shows the percentage of plaque volume growth in VEH- and lemborexant-treated mice. Due to the non-Gaussian distribution of the data, P values are from the Mann-Whitney U test. Figure 17E Shows the quantification of co-localized IBA1-CD68, shown as a percentage of the total IBA1 (total microglia) area. P values are from one-way ANOVA. Figure 17F Shows the percentage of plaque volume growth in mice treated with lemborexant, doxepin, or vehicle control. Figure 17G Shows the quantification of co-localized IBA1-CD68, shown as % of the total IBA1 (total microglia) area. All graphs show mean ± SEM, and each point is one mouse.

[0094] Figure 18, a study from Example 4, shows the effects of lemborexant or doxepin on the rhythmic activity patterns of arrhythmic Bmal1 KO mice. Figure 18A Shows representative activity plots. LD = 12 h:12 h light-dark; DD = constant dark. Figure 18B Shows the quantification of circadian locomotor behavior during different parts of the experiment (LD is indicated by the shaded area, DD + LEM is the area with the shaded arrow, and DD is the rest of the recording). Data were analyzed by two-way ANOVA and Tukey's post hoc test. DETAILED DESCRIPTION

[0095] I. Definitions

[0096] The following are definitions of terms used in this application.

[0097] Unless the context clearly indicates otherwise, the singular terms "a / an" and "the" as used herein include plural referents.

[0098] As used herein, the phrase "and / or" means "any one or both" of the elements so combined, i.e., the elements exist conjointly in some cases and separately in other cases. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising", "A and / or B" can in some embodiments refer only to A (optionally including elements other than B); in other embodiments, only to B (optionally including elements other than A); in still other embodiments, to both A and B (optionally including other elements); and so on.

[0099] As used herein, "at least one" means one or more 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, and does not exclude any combination of elements in the list. This definition also allows that other elements may optionally occur in addition to the specifically identified elements in the list referred to by the phrase "at least one", 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 in one embodiment refer to at least one, optionally including more than one A, without B (and optionally including elements other than B); in another embodiment, can refer to at least one, optionally including more than one B, without A (and optionally including elements other than A); in yet another embodiment, can refer to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0100] When numbers are recited individually or as part of a numerical range, it should be understood that the numerical values can vary above and below the stated value by up to 10% of the stated value.

[0101] When a range of values is listed herein, it is intended that each value and sub-range within that range be covered. For example, "15 mg to 30 mg" is intended to cover, 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, 24.5 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, 15 mg to 28 mg, etc.

[0102] "Amyloid" refers to protein aggregates that form a fibrillar morphology. Amyloids often consist of long, unbranched fibers characterized by an extended β-sheet secondary structure, which is approximately 7 - 13 nm wide and several micrometers long. Amyloids are typically extracellular and found in vivo; additionally, the fiber-binding dye Congo red, and then shows green birefringence when observed between crossed polarizers. Amyloid-forming proteins have been identified and associated with severe diseases, including amyloid-β 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-β", "cerebral amyloid", and "amyloid-β peptide (Aβ)" may be used interchangeably.

[0103] Amyloid-β 1-42 (Aβ42) refers to the amyloid-β monomer of amino acids 1 to 42 from the full-length protein (Table 5, SEQ ID NO: 13). Amyloid-β 1-40 (Aβ1-40) refers to the amyloid-β monomer of amino acids 1 to 40 from the full-length protein (Table 5, SEQ ID NO: 14).

[0104] Amyloid levels obtained from amyloid PET can be reported in "centiloid units" (CL) using the centiloid unit method. (Klunk WE et al. The Centiloid Project: standardizing quantitative amyloid plaque estimation by PET. [The Centiloid Project: standardizing quantitative amyloid plaque estimation by PET] Alzheimer’s Dement. [Alzheimer's Dementia] 2015; 11: 1–15e1–4). The centiloid unit method measures tracers in the range of 0 CL to 100 CL, where 0 is considered the anchor point and represents the mean of young healthy controls, and 100 CL represents the mean amyloid load present in subjects with mild to moderate severity dementia due to AD. (Ibid.). As is known to those of ordinary skill in the art, centiloid unit thresholds can vary, for example, they can be refined based on new or additional scientific information. (See, for example, http: / / www.gaain.org / centiloid-project). Elevated amyloid levels can be set relative to a baseline threshold in healthy controls determined according to methods known to those of ordinary skill in the art.

[0105] As used herein, whether a subject is "amyloid positive" or "amyloid negative" can be determined based on whether the subject has a positive amyloid burden. In some embodiments, a subject is determined to be amyloid positive or amyloid negative, as indicated by longitudinal positron emission tomography (PET) assessment of an amyloid imaging agent taken up by the brain. In some embodiments, a subject is "amyloid negative" if the florbetapir amyloid PET SUVr is less than 1.17. In some embodiments, the presence of amyloid pathology is determined by CSF assessment (e.g., soluble CSF biomarker analysis) of the amyloid using an assessment of a marker such as Aβ1-42, alone or in combination with another method (such as PET measurement of brain amyloid), to determine whether a subject is amyloid positive or negative. Methods for measuring Aβ38, Aβ40, and Aβ42 are known in the art, such as assays using LC MS / MS. Methods may include the PrecivityAD TM assay (see, e.g., Kirmess et al., J. Clinica Chimica Acta [Clinical Chemistry Acta] 519:267-275 (2021)) and the Sysmex assay (https: / / www.eisai.com / news / 2019 / news201990.html). In some embodiments, qualitative visual readings of PET scans can be used to determine amyloid positivity and negativity by classifying a subject as having "normal" or "abnormal" uptake based on the PET imaging pattern. Readers will have been trained and certified to identify brain PET images with abnormal or normal uptake patterns, or amyloid detection can be performed by semi-quantitative or quantitative methods. In some embodiments, thresholds will be set for quantitatively determining from biomarkers (e.g., serum or CSF) and / or PET scans whether the Aβ brain burden indicates that a subject is amyloid positive or negative. In some embodiments, a subject is determined to be amyloid positive or negative by MRI. In some embodiments, the entire 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, hippocampus CA3 (pyramidal neurons), and / or hippocampal dentate gyrus (granule cell neurons)) is analyzed by MRI.

[0106] In some embodiments, a subject is determined to be amyloid positive or negative by retinal amyloid accumulation. In some embodiments, a subject is determined to be amyloid positive or negative by behavioral / cognitive phenotypes.

[0107] 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, cattle, frogs, goats, and chickens, tau is encoded by the gene MAPT. In humans, there are six tau isoforms generated by alternative splicing of exons 2, 3, and 10 of MAPT. The lengths of these isoforms range from 352 to 441 amino acids. Exons 2 and 3 each encode a 29-amino acid insertion (termed N) at the N-terminus, and full-length human tau isoforms can have two insertions (2N), one insertion (1N), or no insertions (0N). All full-length human tau isoforms also have three repeats of the microtubule-binding domain (termed R). Inclusion of exon 10 at the C-terminus results in the inclusion of a fourth microtubule-binding domain encoded by exon 10. Thus, full-length human tau isoforms can consist of four repeats of the microtubule-binding domain (4R) (including exon 10) or three repeats of the microtubule-binding domain (3R) (excluding exon 10). Human tau can be or can not be post-translationally modified. For example, it is known in the art that tau can be phosphorylated, ubiquitinated, glycosylated, and glycated. Thus, the term "human tau" encompasses the (2N, 3R), (2N, 4R), (1N, 3R), (1N, 4R), (0N, 3R), and (0N, 4R) isoforms, isoforms as their N-terminal and / or C-terminal truncated species, and all post-translationally modified isoforms. Alternative splicing of the gene encoding tau similarly occurs in other animals. In animals in which the gene has not been identified as MAPT, homologs can be identified by methods well known in the art.

[0108] Phosphorylation of specific amino acids (i.e., "sites" or "residues") in tau gives rise to 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.

[0109] The term "p-tau" encompasses all phosphorylated tau (p-tau) isoforms, such as but not limited to p-tau181, p-tau217, and p-tau231.

[0110] Diseases associated with tau deposition in the brain can be referred to as "tauopathies" or "tau pathologies". Clinical signs of tauopathies can be aggregates of tau in the brain, including but not limited to neurofibrillary tangles. Other methods can be used to detect or measure tau phosphorylation and optionally total tau at one or more amino acid residues 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.

[0111] 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 traps, and Orbitrap mass spectrometers, as well as hybrid mass spectrometers that combine different types of mass analyzers into one architecture (e.g., the Orbitrap Fusion from ThermoFisher Scientific). TM Tribrid TM mass spectrometer). 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 a radioactive tracer. The entire brain or at least one region of the brain (cortical gray matter (i.e., cortex), frontal lobe, parietal lobe, temporal lobe, occipital lobe, cingulate cortex, amygdala, piriform cortex, entorhinal cortex, hippocampus) can be analyzed by PET.

[0112] As used herein, "relative to placebo" refers to a comparison of biomarkers (p-tau, Aβ, etc.) between the same biomarker in a subject administered lemborexant and another subject administered a placebo (a substance with no therapeutic effect).

[0113] As used herein, "relative to baseline" refers to a comparison of biomarkers (p-tau, Aβ, etc.) between the same biomarker in a subject administered lemborexant and the same subject before treatment with lemborexant.

[0114] As used herein, "maintain" means that a subject has or maintains the same level or approximately the same amount of a biomarker (p-tau, Aβ, etc.) in a subject sample (CSF, blood, etc.) between two time points (one time point before administration of lemborexant and another time point after administration of lemborexant).

[0115] As used herein, "MMSE" refers to the Mini-Mental State Examination, a cognitive tool commonly used for screening purposes and also typically longitudinally measured in AD clinical trials. It has a 30-point scale, where higher scores indicate a lower degree of impairment and lower scores indicate a higher degree of impairment. As used herein, seven items assessing time orientation, place orientation, registration, recall, attention, language, and drawing are evaluated. (Folstein, M.F. et al., "Mini-mental state. A practical method for grading the cognitive state of patients for the clinician." J. Psychiatr. Res. 1975; 12:189-98).

[0116] As used herein, "PSQI" refers to the Pittsburgh Sleep Quality Index, a self-rating questionnaire that assesses sleep quality and disturbances over a 1-month time interval. Nineteen individual items yield seven "component" scores: subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleep medications, and daytime dysfunction. The sum of the scores of these seven components results in a global score ranging from 0 to 21, where lower scores indicate a healthier sleep quality. The clinical and clinimetric properties of the PSQI were evaluated in "good" sleepers (healthy subjects, n = 52) and "poor" sleepers (depressed patients, n = 54; patients with sleep disorders, n = 62) over an 18-month period. (Buysse D.J. et al., "The Pittsburgh Sleep Quality Index: a new instrument for psychiatric practice and research." Psychiatry Res. 1989; 28(2):193-213).

[0117] As used herein, "STOP-Bang" refers to the Snoring, Tiredness, Observed apnea, high BP, BMI, Age, Neck circumference, and male sex (STOP-Bang) questionnaire. The questionnaire consists of eight dichotomous (yes / no) items related to the clinical features of sleep apnea. The total score ranges from 0 to 8. Patients can be classified for their obstructive sleep apnea (OSA) risk based on their respective scores. The STOP-Bang score ≥3 has sensitivities of 93% and 100% for detecting moderate to severe OSA (apnea-hypopnea index [AHI] >15) and severe OSA (AHI >30), respectively. (Chung F. et al., "STOP-Bang Questionnaire: A Practical Approach to Screen for Obstructive Sleep Apnea" Chest. 2016, 149(3):631-638).

[0118] As used herein, "PSG" refers to polysomnography, a standard diagnostic test for OSA. PSG provides an assessment of OSA in terms of the frequency of apnea and hypopnea per hour of sleep (apnea-hypopnea index or AHI). The severity classification of OSA is as follows: (a) none / minimal: AHI <5 per hour; (b) mild: AHI ≥5 but <15 per hour; (c) moderate: AHI ≥15 but <30 per hour; and (d) severe: AHI ≥30 per hour. (Alshaer H et al "Reproducibility and predictors of the apnea hypopnea index across multiple nights" Sleep Sci. 2018, 11(1):28-33).

[0119] Subjects with "preclinical AD" or "prodromal AD" as described herein are cognitively normal (e.g., unimpaired) individuals with moderate or elevated amyloid levels in the brain. The IWG criteria define at least two preclinical AD states in which the subject is cognitively unimpaired: pre-symptomatic AD and asymptomatic AD (or "asymptomatic, at risk"). Pre-symptomatic AD refers to a cognitively unimpaired subject with an autosomal dominant single gene mutation for AD (e.g., mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1), or presenilin 2 (PSEN2)). Pre-symptomatic subjects carrying an autosomal dominant single gene mutation are most likely to develop AD. Asymptomatic refers to a subject who has no clinical signs and symptoms of AD but has one or more biomarkers of AD pathology. The asymptomatic, at risk stage can be further classified. Deficits in episodic memory and executive function may occur later. Thus, subjects with prodromal AD can be identified through the asymptomatic stage of being cognitively unimpaired. Cognitively normal can include individuals with a CDR of 0, or individuals within the normal range of cognitive test scores (MMSE, International Shopping List Task, Logical Memory, etc.). Preclinical AD occurs before significant irreversible neurodegeneration and cognitive impairment, and is typically characterized by the presence of in vivo molecular biomarkers of AD and no clinical symptoms. Preclinical AD biomarkers that can indicate future development of Alzheimer's disease include, but are not limited to, moderate or elevated amyloid levels in the brain measured by amyloid or tau positron emission tomography (PET) (e.g., measured values on a scale of about 20 - 40, such as measured values of about 20 - 32). Additional biomarkers can be used alone or in combination, including one or more of the following: cerebrospinal fluid Aβ1-42 level and / or Aβ1-42 / 1-40 ratio, cerebrospinal fluid total tau level, cerebrospinal fluid neurogranin level, cerebrospinal fluid level of neurofilament light chain peptide (NfL), and biomarkers measured in serum or plasma (e.g., level of Aβ1-42, ratio of two forms of amyloid beta peptide (Aβ1-42 / 1-40 ratio, between about 0.092 - 0.094 or a ratio below about 0.092), plasma level 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 peptide (NfL)). In addition, certain risk factors contribute to the development of AD. For example, subjects carrying the apolipoprotein E (APOE) ε4 allele are also at greater risk of developing AD, and subjects with trisomy 21 (including the APP gene) are at greater risk of cerebral amyloidosis.Other risk factors associated with AD include, but are not limited to: a family history of having a first-degree relative with AD or dementia, being 65 years of age or older, being female, having had a traumatic brain injury or recovering from a traumatic brain injury, having other medical conditions (such as obesity, diabetes, heart and / or vascular diseases, cancer and / or immune system dysfunction, and / or sleep disorders such as insomnia or circadian rhythm sleep disorders), or having lifestyle risk factors (such as smoking, alcohol use, lack of exercise, lack of cognitive activity, and poor nutrition), as well as exposure to environmental risk factors (such as air pollution, metals (e.g., aluminum, copper, and zinc)).

[0120] As used herein, "early AD" or "early Alzheimer's disease" (EAD) is a continuum of AD severity from mild cognitive impairment to mild Alzheimer's dementia due to a moderate likelihood of AD. Subjects with early AD include subjects with mild Alzheimer's dementia as defined herein and subjects with mild cognitive impairment (MCI) due to a moderate likelihood of AD as defined herein. In some embodiments, subjects with early AD have an MMSE score of 22 to 30 and a Clinical Dementia Rating (CDR) total range of 0.5 to 1.0.

[0121] Other methods for detecting early AD disease can employ the following specified tests and assays, including the National Institute on Aging and Alzheimer's Association (NIA-AA) Core Clinical Criteria for probable Alzheimer's dementia among the following: McKhann, G.M. 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 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 AD have evidence of elevated amyloid in the brain or a positive amyloid burden. In some embodiments, the elevated amyloid in the brain or positive amyloid burden is indicated and / or confirmed by PET assessment. In some embodiments, the elevated amyloid in the brain or positive amyloid burden is indicated and / or confirmed by CSF assessment of markers such as Aβ1-42 (e.g., water-soluble CSF biomarker analysis). For example, subjects with AD can be selected according to the method in WO 2023 / 283650, the content of which is incorporated herein by reference. In some embodiments, the diagnostic threshold can be identified by amyloid PET either by visual reading (according to the label of the approved PET tracer) or by establishing a percentile unit threshold, above which subjects are considered to have elevated amyloid (e.g., varying between 15-40 percentile units).

[0122] In some embodiments, an elevated amyloid in the brain or a positive amyloid burden is indicated and / or confirmed by measuring the concentration of Aβ42 and the concentration of Aβ40 and calculating the ratio of Aβ42 to Aβ40 (Aβ42 / 40 ratio). In some embodiments, an elevated amyloid in the brain or a positive amyloid burden is indicated and / or confirmed by MRI or PET. In some embodiments, an elevated amyloid in the brain or a positive amyloid burden is indicated by retinal amyloid accumulation. 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 probable Alzheimer's dementia in the following document: McKhann, G.M. 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 higher at screening and at baseline.

[0123] In some embodiments, a subject has "elevated amyloid" or "moderate amyloid". Those of ordinary skill in the art will recognize that amyloid levels from amyloid PET can be reported in "centiloid units" (CL) using a centiloid unit 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 unit method measures tracers in the range of 0 CL to 100 CL, where 0 is considered the anchor and represents the mean of young healthy controls, and 100 CL represents the mean amyloid burden present in subjects with mild to moderate severity dementia due to AD. (Ibid.) As is known to those of ordinary skill in the art, centiloid unit thresholds can vary, for example, can be refined based on new or additional scientific information. (See, e.g., http: / / www.gaain.org / centiloid-project.) An elevated amyloid level can be set relative to a baseline threshold in healthy controls determined according to methods known in the art. For example, a centiloid unit value of 32.5 can be used as the threshold for "elevated amyloid", and a "moderate amyloid" level refers to Aβ amyloid PET in the range of 20 - 32.5 CL (e.g., 30 CL). In another example, a centiloid unit value of 40 can be used as the threshold for "elevated amyloid", and a "moderate amyloid" level refers to Aβ amyloid PET in the range of 20 - 40 CL.

[0124] As used herein, a subject with "MCI due to moderate likelihood of AD" is a subject so identified according to the NIA-AA core clinical criteria for mild cognitive impairment due to moderate likelihood of Alzheimer's disease. For example, as measured by the ADCOMS Composite Clinical Score as defined herein, symptomatic but non-demented AD subjects with cerebral amyloid pathology have less heterogeneity with mild Alzheimer's dementia subjects and are more similar in terms of cognitive and functional decline. Also included are subjects with a CDR score of 0.5 and a memory box score of 0.5 or higher at screening and at baseline. In addition, subjects with a history of subjective memory decline and gradual onset and slow progression reported by an informant within the most recent 1 year prior to screening are also included herein.

[0125] As used herein, "ADAS-cog" refers to the Alzheimer's Disease Assessment Scale-Cognitive. The ADAS-cog is a widely used cognitive scale in Alzheimer's disease trials and has structured scales that assess memory (word recall, delayed word recall, and word recognition), reasoning (following commands), language (naming, comprehension), orientation, ideational praxis (putting letters in envelopes), and constructional praxis (copying geometric designs). (Rosen, W.G. et al., "A new rating scale for Alzheimer's disease." Am. J. Psychiatry 1984; 141:1356-64). Ratings are also obtained for speech, language comprehension, word-finding difficulty, ability to remember test instructions, mazes, and digit cancellation. The modified form used herein is scored on a scale of 0 to 90, where 0 indicates no impairment and 90 indicates the highest degree of impairment.

[0126] As used herein, "CDR-SB" refers to the clinical dementia rating - sum of boxes. The CDR is a clinical scale that describes five levels of impairment in the performance aspects of six functional categories including memory, orientation, judgment and problem solving, community affairs, 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 for each of the six functional categories are combined into one overall rating for the dementia CDR score (range 0 to 3). The sum of the box scores provides an additional measure of change, where each category has a maximum possible score of 3 points and the total score is the sum of the scores for each category, giving a total possible score of 0 to 18, with higher scores indicating a higher degree of impairment. The total score can be used as a clinical measure of the severity of dementia.

[0127] As used herein, "ADCOMS" refers to the Alzheimer's Disease Composite Score, a composite clinical score based on four ADAS-Cog items (delayed word recall, orientation, word recognition, and word finding difficulty), two MMSE items (time orientation and drawing), and all six CDR-SB items (personal care, community affairs, home and hobbies, memory, orientation, and judgment and problem solving), as discussed in the Examples and in 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 during the early, i.e., prodromal and mild, stages of AD.

[0128] As used herein, "ApoE4 positive" subjects and "ApoE4 carriers" refer to subjects having the ε4 variant of the apolipoprotein gene. The ε4 variant is one of several major alleles of the apolipoprotein gene. This gene is generally responsible for lipid metabolism. It has been found that carriers of apolipoprotein ε4 show significantly higher amyloid retention rates compared to non-carriers. (Drzezga, A. et al., "Effect of APOE genotype on amyloid plaque load and gray matter volume in Alzheimer disease.", Neurology 2009; 72: 1487-94). In some embodiments, the subject is a heterozygous carrier of the apolipoprotein E ε4 gene allele. In some embodiments, the subject is a homozygous carrier of the apolipoprotein E ε4 gene allele.

[0129] As used herein, the term "clinical decline" refers to the worsening of one or more clinical symptoms of AD. Methods for measuring clinical decline can employ the tests and assays specified herein. In some embodiments, clinical decline is determined by worsening of ADCOMS. In some embodiments, clinical decline is determined by worsening of MMSE. In some embodiments, clinical decline is determined by worsening of ADAS-Cog. In some embodiments, clinical decline is determined by worsening of FAQ. In some embodiments, clinical decline is determined by worsening of CDR-SB. In some embodiments, clinical decline is determined by worsening of the Wechsler Memory Scale-IV Logical Memory (subscale) I and / or (subscale) II. In some embodiments, clinical decline is determined by worsening of the CDR score. In some embodiments, clinical decline refers to the worsening of one or more biomarkers of AD or brain measurements such as brain atrophy and / or amyloid accumulation (e.g., by PET or MRI).

[0130] As used herein, the term "treat" (and also "treating" or "treatment") refers to any administration or application of a therapeutic agent to a subject having a disease or disorder, and includes inhibiting the disease, slowing disease progression, delaying progression, halting its development, reversing disease progression (e.g., reversing the accumulation of Aβ fibrils), preventing the onset of the disease or at least one symptom of the disease, or preventing further development of the disease, alleviating or improving one or more symptoms or one or more underlying conditions of the disease, curing the disease, improving one or more clinical metrics, or preventing the recurrence of one or more symptoms of the disease. In some embodiments, treatment can include maintaining the severity of at least one symptom of the disease (i.e., preventing worsening), e.g., when symptoms are expected to progress and / or worsen in the absence of administration or application of a therapeutic agent to the subject. In some embodiments, maintaining a symptom can refer to no change (e.g., no significant change, such as no statistically significant change) in the symptom after administration or application of a therapeutic agent to the subject as compared to a control (e.g., a subject not receiving treatment or receiving a placebo), for which the symptom changes (e.g., significantly, such as statistically significantly). Complete treatment is not required. In some embodiments, treatment of AD in a subject includes administering (e.g., by intravenous infusion) a dual orexin receptor antagonist (e.g., lemborexant) to a subject, such as one at risk of developing AD but not yet showing signs of dementia.

[0131] As used herein, unless the context indicates otherwise, the term "prevention" subsumed within the term "treatment" refers to obtaining a beneficial or desired prophylactic benefit. For prophylactic benefits, a composition may be administered to a subject at risk of developing Alzheimer's disease (e.g., based on biomarkers and / or family history); to a subject having one or more preclinical symptoms but not clinical symptoms of Alzheimer's disease; and / or to a subject reporting one or more physical symptoms of Alzheimer's disease, although a clinical diagnosis of having Alzheimer's disease has not been made. As used herein, "prevention" may further include a therapeutic benefit, which means eradicating or ameliorating a latent condition being treated or one or more physical symptoms associated therewith. Prevention also encompasses preventing or slowing further progression of one or more symptoms of a disease.

[0132] As used herein, "control" (and also "control sample") refers to a biological sample obtained from a subject that is different from those being evaluated and has a known AD status. In some embodiments, a control sample is obtained from, for example, a subject not diagnosed with Alzheimer's disease according to one or more of the definitions above. For example, a control sample may be obtained from a subject without clinical symptoms of AD (e.g., cognitive impairment and / or dementia) and / or without any markers of AD pathology (e.g., a PET scan or CSF analysis of biomarkers such as amyloid or tau). In some embodiments, a control sample may be obtained from a healthy subject. In some embodiments, a control may be obtained from a subject having a comorbidity unrelated to AD. In some embodiments, a control may be obtained from a subject diagnosed along the AD disease spectrum, which includes, for example, preclinical AD or mild cognitive impairment. In some embodiments, a control sample may be a baseline sample collected from a subject prior to the initiation of any treatment. In some embodiments, a control sample may be a sample collected from a control subject administered a placebo.

[0133] As used herein, the term "therapeutically effective amount" refers to the amount of a compound or pharmaceutical composition sufficient to produce a desired therapeutic effect, e.g., reversing, arresting, delaying or slowing cognitive decline and / or reversing, arresting, delaying or slowing the rate of change of one or more biomarkers of AD. One of ordinary skill in the art will understand that the therapeutically effective amount of lemborexant administered to a subject may depend on a variety of factors, including pharmacodynamic characteristics, route of administration, frequency of treatment, and the health, age and weight of the subject to be treated, and with the information disclosed herein, will be able to determine the appropriate amount for each subject.

[0134] II. Methods

[0135] The present disclosure provides methods for reducing the amount of p-tau in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of lemborexant. The present disclosure also provides methods for reducing neurodegeneration in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of lemborexant. The present disclosure also provides methods for reducing amyloid-β in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of lemborexant.

[0136] In one aspect of the present disclosure, lemborexant affects at least one biomarker of AD pathology. Without being bound by theory, in some embodiments, in addition to other potential effects on AD pathology, the surprising effects of lemborexant on AD and AD pathology may be related to the role of lemborexant in regulating sleep and treating insomnia. Notably, lemborexant provides benefits not shared by other sleep agents such as doxepin (another drug approved for treating insomnia). For example, as discussed in the examples, when administered to an AD animal model, lemborexant and doxepin showed differential 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 the total amyloid plaque burden (including diffuse and fibrillar plaques), while doxepin only reduced the total amyloid burden without reducing the fibrillar plaque burden (Example 3, Part B). Lemborexant also increased the activation of phagocytic microglia surrounding Aβ plaques, while doxepin did not (Example 3, Part E). Finally, lemborexant significantly upregulated the expression of Ifnb1, the inflammatory mediator IFN-β, the lysosomal protein Rab5a, and the Aβ-degrading enzyme Mmp2, while doxepin did not (Example 3, Part F). Without being bound by theory, these data may indicate that lemborexant and doxepin act through different mechanisms and suggest that the effects of a drug on sleep may, in some conditions, be different from its effects on AD pathology.

[0137] In various embodiments, methods are disclosed herein for reducing or maintaining the amount of p-tau and / or t-tau in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of lemborexant. Also disclosed are methods for reducing or maintaining the amount of p-tau in a subject, increasing the dephosphorylation of tau in a subject, reducing the ratio of p-tau to tau and / or reducing the rate of tau phosphorylation, the methods comprising administering to a subject in need thereof a therapeutically effective amount of lemborexant. In some embodiments, the amount of p-tau and / or t-tau in the CSF of a subject after administering a therapeutically effective amount of lemborexant is reduced or maintained compared to the amount of p-tau and / or t-tau in the CSF of such subject prior to such administration. In some embodiments, the amount of p-tau and / or t-tau in the CSF of a subject after administering a therapeutically effective amount of lemborexant is reduced or maintained compared to the amount of p-tau and / or t-tau in the CSF of a subject after administering a placebo.

[0138] Also disclosed herein are methods for reducing neurodegeneration in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of lemborexant. Also disclosed are methods for reducing or maintaining amyloid-β in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of lemborexant.

[0139] In some embodiments, a subject in need of the methods of the invention has exhibited signs of at least one disease selected from the group consisting of Alzheimer's disease, pre-Alzheimer's disease, early Alzheimer's disease, mild cognitive impairment, cerebral amyloid angiopathy, frontotemporal dementia, dementia with Lewy bodies, Lewy body dementia, 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.

[0140] III. Subjects in Need of Treatment

[0141] In some embodiments, a subject in need of one or more of the disclosed methods has exhibited signs of at least one disease selected from the following: Alzheimer's disease, pre - Alzheimer's disease, early Alzheimer's disease, mild cognitive impairment, cerebral amyloid angiopathy, frontotemporal dementia, Lewy body dementia, 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 has exhibited signs 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 has exhibited signs of mild cognitive impairment. In some embodiments, a subject in need has exhibited signs of cerebral amyloid angiopathy, frontotemporal dementia, Lewy body dementia, dementia with Lewy bodies, vascular dementia, limbic predominant age - related TDP - 43 encephalopathy, frontotemporal lobar degeneration, corticobasal degeneration. In some embodiments, a subject in need has exhibited signs of at least one disease selected from Parkinson's disease, Pick's disease, multiple system atrophy, and progressive supranuclear palsy.

[0142] One aspect of the present disclosure relates to a method for treating Alzheimer's disease (AD) in a subject suffering from 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, thereby treating AD. In some embodiments, the subject is in need of treatment (e.g., suffers from AD, pre - AD, or is at risk of developing AD).

[0143] In some embodiments, treating AD refers to inhibiting AD, AD pathology, AD symptoms, and / or underlying conditions of AD, slowing its progression, slowing its rate of progression, delaying its progression, preventing its development, and reversing its progression, one or more of these. In some embodiments, treatment refers to preventing the onset or development of AD, AD pathology, AD symptoms, and / or underlying conditions of AD. In some embodiments, treatment refers to alleviating or improving one or more symptoms or underlying conditions of AD (e.g., improving the accumulation of Aβ fibrils) and / or improving one or more clinical metrics of AD (e.g., cognitive function, cerebral amyloid or tau levels, and / or biomarker expression). In some embodiments, treatment refers to preventing the occurrence or recurrence of one or more symptoms of AD.

[0144] In some embodiments, treating AD includes reducing, halting, and / or slowing cognitive decline.

[0145] In some embodiments, the subject in need of the treatment described herein is a subject with AD, such as a subject who has been diagnosed with AD. The diagnosis can be based on cognitive evaluation. The diagnosis can be based on measurements of AD pathology obtained by brain imaging (e.g., amyloid PET or tau PET) and / or the expression of biomarkers in the subject. In some embodiments, the biomarkers include brain amyloid levels, brain tau levels, cerebrospinal fluid (CSF) levels of Aβ1-42, CSF levels of total tau, CSF levels of neurogranin, and CSF levels of neurofilament light chain (NfL). In some embodiments, a subject with AD shows cognitive impairment and AD pathology. For example, a subject with AD may have a t-tau level higher than 400 ng / L, an Aβ1-42 level lower than 550 ng / L, and / or an Aβ1-42 / Aβ1-40 ratio lower than 0.065.

[0146] 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, a subject with early AD has a continuum of AD severity symptoms from mild cognitive impairment due to moderate likelihood of AD to mild Alzheimer's dementia. In some embodiments, a subject with early AD has mild Alzheimer's dementia as defined herein and / or mild cognitive impairment (MCI) due to moderate likelihood of AD as defined herein. In some embodiments, the subject has an MMSE score of 22 to 30 and a Clinical Dementia Rating (CDR) total score range of 0.5 to 1.0. In some embodiments, a subject with early AD has evidence of elevated amyloid in the brain or a positive amyloid burden. In some embodiments, elevated amyloid in the brain or a positive amyloid burden is indicated and / or confirmed by PET assessment. In some embodiments, elevated amyloid in the brain or a positive amyloid burden is indicated and / or confirmed by CSF assessment of a marker such as Aβ1-42 (e.g., soluble CSF biomarker assay). In some embodiments, elevated amyloid in the brain or a positive amyloid burden is indicated and / or confirmed by measuring the ratio of Aβ42 to Aβ40 (Aβ42 / 40 ratio). In some embodiments, elevated amyloid in the brain or a positive amyloid burden is indicated and / or confirmed by MRI assessment. In some embodiments, elevated amyloid in the brain or a positive amyloid burden is indicated by retinal amyloid accumulation. In some embodiments, more than one assessment method is used.

[0147] In some embodiments, the subject has preclinical Alzheimer's disease (also referred to as "pre-AD"). A subject with pre-AD can be cognitively normal and have moderate or elevated levels of amyloid in the brain. In some embodiments, a subject with pre-AD can be identified by an asymptomatic stage with or without memory complaints and emerging episodic memory and executive function deficits. In some embodiments, a subject with pre-AD has a CDR of 0 and / or a score within the normal range on cognitive test scores (e.g., MMSE, International Shopping List Task, Logical Memory, etc.). In some embodiments, the subject has other biomarkers indicative of future development of AD, such as one or more of the following: moderate or elevated levels of brain amyloid determined by amyloid or tau positron emission tomography (PET) (e.g., a measurement value on a scale of about 20 - 40, such as a measurement value of about 20 - 32), cerebrospinal fluid Aβ1-42 level and / or Aβ1-42 / 1-40 ratio, cerebrospinal fluid total tau level, cerebrospinal fluid neurogranin level, cerebrospinal fluid level of neurofilament light chain peptide (NfL), and blood biomarkers measured in serum or plasma (e.g., level of Aβ1-42, ratio of two forms of amyloid beta peptide (Aβ1-42 / 1-40 ratio, such as a ratio between about 0.092 - 0.094 or a ratio below about 0.092), plasma level 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 peptide (NfL)). In some embodiments, a subject with pre-AD can have moderate amyloid (e.g., about 20 - 40 scale units). In some embodiments, a subject with pre-AD can have elevated amyloid (e.g., >40 scale units).

[0148] In some embodiments, a subject may be at risk of developing AD. The subject may have one or more risk factors for developing AD, such as carrying a familial AD gene (e.g., the apolipoprotein E ε4 allele, also referred to as “APOE4” or “ApoE4”), having a family history of a first-degree relative with AD or dementia, being 65 years of age or older, being female, having had a traumatic brain injury or recovering from a traumatic brain injury, having other medical conditions such as obesity, diabetes, heart and / or vascular diseases, etc. In some embodiments, a subject with pre-AD is at risk of developing AD. The risk of developing AD may be greater than the risk of developing AD in a control subject and / or the risk of developing AD may be greater than in a control subject at an estimated faster time. For example, compared to a control subject, a subject with pre-AD who is cognitively normal but has moderate amyloid PET levels (about 20 - 40 percentile units) may be at risk of further Aβ accumulation and early spread of tau pathology within 4 years. Compared to a control subject, a subject with pre-AD who is cognitively normal but has elevated amyloid PET levels (>40 percentile units) may be at high risk of cognitive decline within 4 years.

[0149] In some embodiments, treating AD includes affecting a change (e.g., slowing, delaying, or reducing) in at least one marker of AD pathology.

[0150] In some embodiments, a marker of AD pathology is the phosphorylation level of tau, neurodegeneration, changes in microglial response, and / or the presence of Aβ plaques. Markers of AD pathology may be present in brain regions of the subject, such as the hippocampus, primary motor cortex, primary somatosensory cortex, piriform cortex, and / or entorhinal cortex. In some embodiments, markers of AD pathology are 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, markers of AD pathology are detected in a subject's body fluid such as blood (e.g., plasma) or CSF. For example, various phosphorylated tau and Aβ can be detected in plasma or CSF from the subject.

[0151] In some embodiments, the subject does not show signs of dementia and / or cognitive impairment. In some embodiments, the subject has mild cognitive impairment or mild dementia.

[0152] In some embodiments, the subject is amyloid positive. The subject may be at risk for further Aβ accumulation and / or tau pathology spread. The subject may be at risk for cognitive decline. In some embodiments, the subject may have a moderate level of amyloid PET (e.g., about 20 - 40 percentile units). In some embodiments, the subject may have an elevated level of amyloid PET (e.g., >40 percentile units). In some embodiments, the subject may carry the APOE4 gene. In some embodiments, the subject may have one or more risk factors for developing AD, such as a family history of having a first-degree relative with AD or dementia, being 65 years of age or older, being female, having had a traumatic brain injury or recovering from a traumatic brain injury, and having other medical conditions such as obesity, diabetes, heart disease, and / or vascular disease.

[0153] In some embodiments, based on brain imaging, cognitive function, and / or biomarker criteria, it has been diagnosed whether the subject has AD. In some embodiments, the subject has early AD. In some embodiments, the subject has pre-AD.

[0154] In some embodiments, the treatment can slow cognitive decline and / or reduce the rate of change of AD biomarkers.

[0155] IV. Tau and Aβ

[0156] The ratio of the concentration of p-tau (also referred to herein as "phosphorylated tau" or "phospho-tau") to the concentration of t-tau (also referred to herein as "total tau") in the subject's CSF (the "CSF p-tau / t-tau ratio" herein) can be used to evaluate the amount of phosphorylation of tau. The concentrations of p-tau and t-tau in the subject's CSF are measured using liquid chromatography - tandem mass spectrometry (LC MS / MS).

[0157] In some embodiments, the CSF p-tau / t-tau ratio of a subject administered a therapeutically effective amount of lemborexant is reduced compared to the CSF p-tau / t-tau ratio of a subject administered a placebo. In some embodiments, the CSF p-tau / t-tau ratio of a subject administered a therapeutically effective amount of lemborexant is maintained within 10% (i.e., + / -) of the CSF p-tau / t-tau ratio of a subject administered a placebo. In some embodiments, the CSF p-tau / t-tau ratio of a subject administered a therapeutically effective amount of lemborexant is within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the CSF p-tau / t-tau ratio of a subject administered a placebo.

[0158] In some embodiments, the ratio of CSF p-tau / t-tau in a subject administered a therapeutically effective amount of lemborexant is decreased compared to the ratio of CSF p-tau / t-tau in the subject before administration of lemborexant.

[0159] In some embodiments, the ratio of CSF p-tau / t-tau in a subject administered a therapeutically effective amount of lemborexant is maintained within (i.e., + / -) 10% of the ratio of CSF p-tau / t-tau in the subject before 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 before administration of lemborexant.

[0160] In some embodiments, the concentration of amyloid beta (Aβ) in CSF is lower than the Aβ concentration in the CSF of subjects administered a placebo. In some embodiments, the Aβ concentration in CSF is lower than the Aβ concentration in the CSF of the subject before administration of lemborexant. In some embodiments, the concentration of Aβ38, Aβ40, and / or Aβ42 in CSF is decreased. In some embodiments, the Aβ concentration in CSF is maintained compared to the Aβ concentration in the CSF of the subject before administration of lemborexant. In some embodiments, the concentration of Aβ38, Aβ40, and / or Aβ42 in CSF is maintained compared to the Aβ concentration in the CSF of the subject before 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 or maintained compared to the amyloid PET signal in the brain of the subject before administration of lemborexant. In some embodiments, the increase in the Aβ concentration in the CSF of a subject administered lemborexant is less than the increase in the Aβ concentration in the CSF of a subject administered a placebo.

[0161] In some embodiments, the Aβ concentration in the CSF of a subject administered lemborexant is at least 5% lower than the Aβ concentration in the CSF of a subject administered a placebo. In some embodiments, the Aβ concentration in the CSF of a subject administered lemborexant is at least 10%, at least 15%, at least 20%, or at least 25% lower than the Aβ concentration in the CSF of a subject administered a placebo.

[0162] In some embodiments, the concentration of Aβ in CSF is measured using liquid chromatography-tandem mass spectrometry (LC MS / MS). In some embodiments, the concentrations of Aβ38, Aβ40, and / or Aβ42 in CSF are measured using LC MS / MS. Methods for measuring Aβ38, Aβ40, and Aβ42 are known in the art, such as assays using LC MS / MS. The methods may include PrecivityAD assays for measuring Aβ42 and Aβ40 in blood or plasma samples or CSF samples (see, for example, Kirmess et al., J. Clinica Chimica Acta 519:267-275 (2021)) and Sysmex assays (https: / / www.eisai.com / news / 2019 / news201990.html). 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 the concentration of Aβ38, Aβ40, and / or Aβ42 is compared to the subject prior to administration of lemborexant. In some embodiments, administering to a subject a composition comprising a therapeutically effective amount of lemborexant such that the cerebrospinal fluid level concentration of Aβ38, Aβ40, and / or Aβ42 is reduced 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 the baseline. TM assays (see, for example, Kirmess et al., J. Clinica Chimica Acta 519:267-275 (2021)) and Sysmex assays (https: / / www.eisai.com / news / 2019 / news201990.html). 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 the concentration of Aβ38, Aβ40, and / or Aβ42 is compared to the subject prior to administration of lemborexant. In some embodiments, administering to a subject a composition comprising a therapeutically effective amount of lemborexant such that the cerebrospinal fluid level concentration of Aβ38, Aβ40, and / or Aβ42 is reduced 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 the baseline.

[0163] 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 of tau phosphorylation occurs throughout the 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, the tau PET signal in the brain is reduced compared to a placebo. In some embodiments, the tau PET signal in the brain is reduced or maintained compared to the baseline.

[0164] In some embodiments, administering a therapeutically effective amount of lemborexant to a subject causes a decrease in the concentration of p-tau in the subject's CSF as compared to the concentration of p-tau in the CSF of a subject administered a placebo. In some embodiments, administering a therapeutically effective amount of lemborexant to a subject causes a decrease in the concentration of p-tau in the subject's CSF as compared to the concentration of p-tau in the subject's CSF prior to administering lemborexant. In some embodiments, administering a therapeutically effective amount of lemborexant to a subject causes the amount of p-tau in the subject's CSF to be maintained as compared to the concentration of p-tau in the CSF of a subject administered a placebo. In some embodiments, administering a therapeutically effective amount of lemborexant to a subject causes the amount of p-tau in the subject's CSF prior to administering lemborexant to be maintained.

[0165] In some embodiments, the concentration of p-tau in the CSF is measured using liquid chromatography-tandem mass spectrometry (LC MS / MS).

[0166] In some embodiments, administering a therapeutically effective amount of lemborexant to a subject causes the amount of p-tau in the subject's CSF to be maintained as compared to the concentration of p-tau in the CSF of a subject administered a placebo. In some embodiments, as compared to the amount of p-tau in the CSF of a subject administered a placebo, administering a therapeutically effective amount of lemborexant to a subject causes the amount of p-tau in the subject's CSF to decrease 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%. In some embodiments, as compared to the amount of p-tau in the subject's CSF prior to administering lemborexant, administering a therapeutically effective amount of lemborexant to a subject causes the amount of p-tau in the subject's CSF to decrease 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%.

[0167] In some embodiments, the increase in the amount of p-tau in the CSF of a subject administered lemborexant is lower than the increase in the amount of p-tau in the CSF of a subject administered a placebo.

[0168] In some embodiments, administering a therapeutically effective amount of lemborexant to a subject causes the p-tau concentration in the subject's CSF to be maintained or decreased until 18 months after administration of lemborexant, as compared to the p-tau concentration in the subject's CSF at baseline. In some embodiments, administering a therapeutically effective amount of lemborexant to a subject causes the p-tau concentration in the subject's CSF to be decreased 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% as compared to the p-tau concentration in the subject's CSF at baseline.

[0169] In some embodiments, the decrease in the amount of p-tau or t-tau in the subject's CSF is due to a decrease or maintenance in the amount of p-tau or t-tau in the subject, and includes administering a therapeutically effective amount of lemborexant to a subject in need thereof. In some embodiments, the decrease in the amount of p-tau in the subject's CSF is due to an increase in p-tau dephosphorylation. In some embodiments, the decrease 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 decrease in the amount of p-tau in the subject's CSF is due to a decrease in the p-tau / t-tau ratio.

[0170] In some embodiments, administering the disclosed therapeutically effective amount of lemborexant to a subject causes the p-tau concentration in the subject's CSF to be decreased or maintained as compared to the p-tau concentration in the CSF of subjects administered placebo. In some embodiments, administering a therapeutically effective amount of lemborexant to a subject causes the cerebrospinal fluid amount of p-tau to be decreased 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 placebo. In some embodiments, administering a composition comprising a therapeutically effective amount of lemborexant disclosed herein to a subject causes the cerebrospinal fluid amount of p-tau to be decreased by at least about 40 pg / mL relative to placebo.

[0171] In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject causes a decrease or maintenance of the p-tau concentration in the subject's CSF compared to the p-tau concentration in the subject's CSF before administration of lemborexant. In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject causes a decrease in the amount of CSF p-tau of 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. In some embodiments, administration of a composition comprising a therapeutically effective amount of lemborexant disclosed herein causes a decrease in the amount of CSF p-tau of at least about 40 pg / mL relative to baseline.

[0172] In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject causes a decrease in the amount of CSF p-tau of 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, up to 18 months after administration of the composition comprising a therapeutically effective amount of lemborexant. In some embodiments, administration of a composition comprising a therapeutically effective amount of lemborexant to a subject causes a decrease in the amount of CSF p-tau of at least 40 pg / mL relative to baseline, up to 18 months after administration of the composition comprising a therapeutically effective amount of at least lemborexant.

[0173] In some embodiments, administration of a therapeutically effective amount of lemborexant to a subject causes a decrease in the amount of CSF p-tau of 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 placebo, up to 18 months after administration of the composition comprising a therapeutically effective amount of lemborexant. In some embodiments, administration of a composition comprising a therapeutically effective amount of lemborexant to a subject causes a decrease in the amount of CSF p-tau of at least 40 pg / mL relative to placebo, up to 18 months after administration of the composition comprising a therapeutically effective amount of at least lemborexant.

[0174] In some embodiments, the amount of p-tau is reduced within 48 hours after administering the first dose of lemborexant to the subject, relative to the subject's baseline. In some embodiments, the amount of p-tau is reduced within 24 hours after administering the first dose of lemborexant to the subject, relative to the subject's baseline. In some embodiments, the amount of p-tau is reduced within 12 hours after administering the first dose of lemborexant to the subject, relative to the subject's baseline. In some embodiments, the amount of p-tau is reduced within 6 hours after administering the first dose of lemborexant to the subject, relative to the subject's baseline.

[0175] In some embodiments, the amount of t-tau is reduced within 48 hours after administering the first dose of lemborexant to the subject, relative to the subject's baseline. In some embodiments, the amount of t-tau is reduced within 24 hours after administering the first dose of lemborexant to the subject, relative to the subject's baseline. In some embodiments, the amount of t-tau is reduced within 12 hours after administering the first dose of lemborexant to the subject, relative to the subject's baseline. In some embodiments, the amount of t-tau is reduced within 6 hours after administering the first dose of lemborexant to the subject, relative to the subject's baseline.

[0176] In some embodiments, the amount of Aβ is reduced within 48 hours after administering the first dose of lemborexant to the subject, relative to the subject's baseline. In some embodiments, the amount of Aβ is reduced within 24 hours after administering the first dose of lemborexant to the subject, relative to the subject's baseline. In some embodiments, the amount of Aβ is reduced within 12 hours after administering the first dose of lemborexant to the subject, relative to the subject's baseline. In some embodiments, the amount of Aβ is reduced within 6 hours after administering the first dose of lemborexant to the subject, relative to the subject's baseline.

[0177] In some embodiments, the amount of p-tau in the subject's CSF is reduced within 48 hours after administering the first dose of lemborexant to the subject, relative to subjects administered a placebo. In some embodiments, the amount of tau phosphorylation in the subject's CSF is reduced within 24 hours after administering the first dose of lemborexant to the subject, relative to subjects administered a placebo. In some embodiments, the amount of tau phosphorylation in the subject's CSF is reduced within 12 hours after administering the first dose of lemborexant to the subject, relative to subjects administered a placebo. In some embodiments, the amount of tau phosphorylation in the subject's CSF is reduced within 6 hours after administering the first dose of lemborexant to the subject, relative to subjects administered a placebo.

[0178] A. Altering tau

[0179] Another aspect of the present disclosure relates to methods of altering tau (e.g., reducing or delaying tau aggregation, tau phosphorylation, and / or tau spreading, and / or slowing the rate thereof) in a subject having AD or at risk of developing AD, the method comprising administering to the subject a therapeutically effective amount of lemborexant, wherein the therapeutically effective amount is sufficient to alter tau in the subject. In some embodiments, altering tau comprises reducing or delaying tau aggregation, tau phosphorylation, or tau spreading, or slowing the rate of any of these.

[0180] In some embodiments, altering tau is slowing the progression of tau pathology (e.g., tau aggregation, tau phosphorylation, and / or tau spreading) in a brain region, such as, slowing the rate of its progression, delaying its progression, preventing its development, and reversing its progression. The brain region can be the cortex or the 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 the piriform cortex. In some embodiments, altering tau is preventing the onset or preventing the development of tau pathology. Altering tau can reduce, delay, or slow the onset and / or the rate of progression of symptoms of tau pathology. In some embodiments, altering tau is alleviating or improving one or more symptoms of tau pathology and / or improving one or more clinical metrics of tau pathology (e.g., cognitive function, brain amyloid, or tau levels, and / or biomarker expression). In some embodiments, altering tau is preventing the occurrence or recurrence of one or more symptoms of tau pathology.

[0181] In some embodiments, the subject is amyloid negative. The subject can have mild cognitive impairment or mild dementia. In some embodiments, the subject does not show signs of dementia and / or cognitive impairment.

[0182] In some embodiments, tau is altered relative to a reference. Thus, compared to the reference, the methods described herein can include reducing and / or delaying tau aggregation, tau phosphorylation, and / or tau spreading, and / or slowing the rate of any of these. In some embodiments, the reference is a baseline measurement from the subject prior to treatment. In some embodiments, the reference is a baseline measurement from a control subject. The reference can be a measurement obtained from more than one control subject, which is used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0183] In some embodiments, the methods herein include altering tau in a brain region of the subject (e.g., reducing, preventing, or slowing the growth of tau). Altering tau in a brain region can include altering the tau PET signal in the brain region. In some embodiments, the brain region is the hippocampus, the entorhinal cortex, and / or the piriform cortex.

[0184] In some embodiments, altering tau includes altering tau in a subject's body fluid. For example, the level of tau in the subject's brain can be detected in the body fluid of the subject. In some embodiments, the body fluid is blood (e.g., plasma) or CSF.

[0185] In some embodiments, one or more forms of tau can be altered. In some embodiments, tau is total tau. In some embodiments, tau is aggregated tau. In some embodiments, the tau is a phosphorylated form of tau (phosphorylated tau). Phosphorylated tau can be tau phosphorylated at one or more of T181, T217, S202, S205, or T231.

[0186] In some embodiments, altering tau includes altering the ratio of phosphorylated 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 of the subject before the administration of lemborexant. In some embodiments, the rate of dephosphorylation of phosphorylated tau is increased. In some embodiments, the rate of phosphorylation of tau is decreased. In some embodiments, altering tau includes altering tau within 48 hours of administering a first dose of lemborexant. For example, tau can be decreased within 48 hours of administering a first dose of lemborexant. In some embodiments, decreasing tau includes altering phosphorylated tau in the hippocampus, entorhinal cortex, and / or piriform cortex.

[0187] B. Maintaining tau

[0188] Another aspect of the present disclosure relates to a method of maintaining tau (e.g., tau aggregation, tau phosphorylation, and / or tau spreading) in a subject having AD or at risk of developing AD, the method comprising administering to the subject a therapeutically effective amount of lemborexant, wherein the therapeutically effective amount is sufficient to maintain tau in the subject.

[0189] In some embodiments, maintaining tau means maintaining tau aggregation, tau phosphorylation, and / or tau spreading when tau is expected to progress and / or deteriorate without administering or applying a therapeutic agent to the subject. In some embodiments, maintaining tau can mean that there is no change (e.g., no significant change, such as no statistically significant change) in tau (e.g., tau aggregation, tau phosphorylation, and / or tau spreading) after administering or applying a therapeutic agent to the subject compared to a control in which administering or applying a therapeutic agent would result in a change in tau (e.g., a significant change, such as a statistically significant change).

[0190] In some embodiments, maintaining tau includes maintaining tau pathology (e.g., tau aggregation, tau phosphorylation, and / or tau spreading) in, for example, a brain region. The brain region can be the cortex or the 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 the piriform cortex. In some embodiments, maintaining tau is preventing the onset or preventing the development of tau pathology, e.g., because the tau pathology remains unchanged. Maintaining tau can reduce, delay, or slow the onset and / or rate of progression of symptoms of tau pathology, e.g., because the tau pathology remains unchanged. In some embodiments, maintaining tau can cause remission or improvement of one or more symptoms of tau pathology and / or improvement of one or more clinical metrics of tau pathology (e.g., cognitive function, brain amyloid, or tau levels and / or biomarker expression). In some embodiments, maintaining tau can cause prevention of the occurrence or recurrence of one or more symptoms of tau pathology.

[0191] In some embodiments, the subject is amyloid negative. The subject can have mild cognitive impairment or mild dementia. In some embodiments, the subject does not show signs of dementia and / or cognitive impairment.

[0192] In some embodiments, the subject shows signs of cognitive impairment. In some embodiments, the subject is amyloid positive. In some embodiments, the subject is diagnosed with AD, e.g., early AD.

[0193] In some embodiments, tau is maintained relative to a reference. Thus, the methods described herein can include maintaining tau aggregation, tau phosphorylation, and / or tau spreading (or the rate of any of these) relative to a reference. In some embodiments, the reference is a baseline measurement from the subject prior to treatment. In some embodiments, the reference is a baseline measurement from a control subject. The reference can be a measurement obtained from more than one control subject 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.

[0194] In some embodiments, maintaining tau includes maintaining tau in a brain region of a subject. Maintaining tau in the brain region can include altering, reducing, or maintaining the tau PET signal in the brain region. In some embodiments, the brain region is the hippocampus, the entorhinal cortex, and / or the piriform cortex.

[0195] In some embodiments, maintaining tau includes maintaining tau in a body fluid of a subject. The level of tau in the subject's brain can be correlated with the level in the subject's body fluid. In some embodiments, the body fluid is blood (e.g., plasma) or CSF.

[0196] 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 the phosphorylated form of tau (phosphorylated tau). The phosphorylated tau can be tau phosphorylated at one or more of T181, T217, S202, S205, or T231.

[0197] In some embodiments, maintaining tau includes maintaining the ratio of phosphorylated tau to total tau. In some embodiments, the ratio of phosphorylated tau to total tau is maintained within 10% of the ratio of phosphorylated tau to total tau in the subject prior to administration of lemborexant. In some embodiments, maintaining tau includes maintaining tau within 48 hours of administration of the first dose of lemborexant. In some embodiments, phosphorylated tau is maintained in the hippocampus, entorhinal cortex, and / or piriform cortex.

[0198] V. Microglial Response

[0199] In some embodiments, administering a therapeutically effective amount of lemborexant to a subject causes an increase in the number of activated microglia compared to a placebo. In some embodiments, the increase in the number of activated microglia is measured by PET. In some embodiments, the activated microglia are phagocytic microglia.

[0200] In some embodiments, administering a therapeutically effective amount of lemborexant to a subject causes an increase in the number of activated microglia compared to baseline. In some embodiments, the increase in the number of activated microglia is measured by PET. In some embodiments, the activated microglia are phagocytic microglia.

[0201] 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 ventricle, 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.

[0202] A. Modulating Microglial Response

[0203] One aspect of the present disclosure relates to a method of modulating the microglial response of 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 the microglial response of the subject.

[0204] In some embodiments, modulation refers to increasing or decreasing the microglial response, and / or refers to increasing or decreasing the rate of the microglial response. In some embodiments, the number of microglia remains unchanged, but the response of microglia is modulated (e.g., activation, reactivation, reactivity, differentiation, etc.). Modulation can vary according to brain region (e.g., microglial activation or other responses can occur differently in different brain regions).

[0205] In some embodiments, the modulation of the 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 prior to treatment. In some embodiments, the reference is a baseline measurement from a control subject. The reference can be a measurement obtained from more than one control subject, which is used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0206] 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 context or disease environment (e.g., a general marker of activated microglia, reactive microglia, and / or microglia in a disease environment). 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.

[0207] In some embodiments, modulating the microglial response includes modulating the activity of phagocytic microglia.

[0208] 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.

[0209] In some embodiments, the subject is amyloid-negative. The subject may have tau pathology. The subject can have neurodegeneration in brain regions such as 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 in the hippocampus.

[0210] In some embodiments, for those subjects with neurodegeneration, modulating the microglial response includes modulating the response in microglia associated with degenerating neurons. For example, when observed in a scan or in a sample obtained from a subject, microglia associated with neurodegenerating neurons can be very close to the neurons. In some embodiments, microglia associated with degenerating neurons can phagocytose degenerating neurons and / or their debris. Thus, in some embodiments, administering a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof to these subjects can include reducing the expression of at least one general microglial marker. The general microglial marker can be Iba1, CD68, or Clec7a. In some embodiments, modulating the microglial response includes increasing the expression of at least one homeostatic microglial marker. The homeostatic microglial marker can be TMEM119 or P2RY12.

[0211] In some embodiments, the subject is amyloid positive, e.g., the subject has Aβ plaques. These Aβ plaques can be fibrillar Aβ plaques. In some embodiments, these Aβ plaques are present in the hippocampus, somatic motor cortex, somatic sensory cortex, and / or piriform cortex of the subject.

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

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

[0214] In some embodiments, the subject has early AD. In some embodiments, the subject has preclinical AD.

[0215] In some embodiments, in those subjects who are amyloid positive, modulating the microglial response includes modulating the response in microglia associated with Aβ plaques. For example, when observed in scans or samples obtained from a subject, microglia associated with Aβ plaques can be very close to the Aβ plaques. In some embodiments, microglia associated with Aβ plaques can phagocytose Aβ plaques. Thus, in some embodiments, administering a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof to these subjects can include increasing the expression of general microglial markers. The general microglial markers can 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 homeostatic microglial markers. The homeostatic microglial markers can be TMEM119 or P2RY12.

[0216] VI. Amyloid Plaques

[0217] In some embodiments, compared to placebo, administering a therapeutically effective amount of lemborexant to a subject results in a reduction or maintenance of amyloid plaques. In some embodiments, compared to placebo, administering a therapeutically effective amount of lemborexant to a subject results in a reduction or maintenance of fibrillar amyloid plaques. In some embodiments, compared to baseline, administering a therapeutically effective amount of lemborexant to a subject results in a reduction or maintenance of amyloid plaques. In some embodiments, compared to baseline, administering a therapeutically effective amount of lemborexant to a subject results in a reduction or maintenance of fibrillar amyloid plaques. In some embodiments, the amyloid plaques are fibrillar amyloid plaques.

[0218] A. Altering Aβ Plaques

[0219] Another aspect of the present disclosure relates to a method of altering Aβ plaques (e.g., reducing or delaying the formation of Aβ plaques, and / or slowing their growth rate) in a subject having 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 alter the Aβ plaques in the subject.

[0220] In some embodiments, altering Aβ plaques includes slowing the progression of Aβ plaque formation and / or Aβ plaque growth, slowing the rate of progression of Aβ plaque formation and / or Aβ plaque growth, delaying the progression of Aβ plaque formation and / or Aβ plaque growth, arresting the development of Aβ plaque formation and / or Aβ plaque growth, and reversing the progression of Aβ plaque formation and / or Aβ plaque growth. In some embodiments, altering Aβ plaques includes preventing the onset of Aβ plaque pathology (e.g., any pathology caused by or concurrent with Aβ plaque formation and / or Aβ plaque growth) or preventing the development of Aβ plaque pathology. Altering Aβ plaques can reduce, delay, or slow the onset and / or rate of progression of the symptoms of the pathology. In some embodiments, altering Aβ plaques includes alleviating or improving one or more symptoms of Aβ plaque pathology and / or improving one or more clinical metrics of Aβ plaque pathology (e.g., cognitive function, cerebral amyloid or tau levels, and / or biomarker expression). In some embodiments, altering Aβ plaques includes preventing the occurrence or recurrence of one or more symptoms of Aβ plaque formation and / or Aβ plaque growth.

[0221] In some embodiments, these Aβ plaques are altered relative to a reference. Thus, altering Aβ plaques can include reducing and / or delaying Aβ plaque formation, and / or slowing its rate, compared to the reference. In some embodiments, the reference is a baseline measurement from the subject prior to treatment. In some embodiments, the reference is a baseline measurement from a control subject. The reference can be a measurement obtained from more than one control subject and used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0222] In some embodiments, these Aβ plaques are fibrillar plaques. In some embodiments, the Aβ plaques are total plaques and can include non-fibrillar (e.g., diffuse) plaques.

[0223] In some embodiments, altering Aβ plaques includes reducing the growth or growth rate of Aβ plaques. The reduction in Aβ plaque growth can be in the hippocampus of the subject, the primary motor cortex of the subject, the somatosensory cortex, and / or the piriform cortex. In some embodiments, altering Aβ plaques includes altering the 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 CSF of the subject. The Aβ can be Aβ38, Aβ40, and / or Aβ42.

[0224] In some embodiments, altering Aβ includes altering Aβ plaques within 48 hours of administering a first dose of suvorexant.

[0225] In some embodiments, the subject does not show signs of dementia and / or cognitive impairment. In some embodiments, the subject has mild cognitive impairment or mild dementia.

[0226] In some embodiments, the subject is at risk of further Aβ accumulation. The subject may also be at risk of tau pathology spreading. The subject may be at risk of cognitive decline. In some embodiments, the subject may have a moderate level of amyloid PET (e.g., about 20 - 40 percentile units). In some embodiments, the subject may have an elevated level of amyloid PET (e.g., >40 percentile units). 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 a family history of having a first-degree relative with AD or dementia, being 65 years of age or older, being female, having had a traumatic brain injury or recovering from a traumatic brain injury, and having other medical conditions such as obesity, diabetes, heart disease, and / or vascular disease.

[0227] In some embodiments, the subject has early AD. In some embodiments, the subject has preclinical AD.

[0228] VII. Neurodegeneration

[0229] Also disclosed herein is a method of reducing neurodegeneration in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of lemborexant. In some embodiments, the subject in need thereof has shown signs of at least one disease selected from the group consisting of Alzheimer's disease, preclinical Alzheimer's disease, early Alzheimer's disease, mild cognitive impairment, cerebral amyloid angiopathy, frontotemporal dementia, Lewy body dementia, 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 has shown signs of at least one disease selected from Alzheimer's disease, preclinical Alzheimer's disease, and early Alzheimer's disease. In some embodiments, the subject in need thereof has shown signs of mild cognitive impairment. In some embodiments, the subject in need thereof has shown signs of cerebral amyloid angiopathy, frontotemporal dementia, Lewy body dementia, dementia with Lewy bodies, vascular dementia, limbic-predominant age-related TDP-43 encephalopathy, frontotemporal lobar degeneration, corticobasal degeneration. In some embodiments, the subject in need thereof has shown signs of at least one disease selected from Parkinson's disease, Pick's disease, multiple system atrophy, and progressive supranuclear palsy.

[0230] In some embodiments, a reduction in neurodegeneration is observed by maintaining cortical thickness or slowing the reduction of cortical thickness relative to subjects administered a placebo. In some embodiments, a reduction in neurodegeneration is observed by maintaining cortical thickness or slowing the reduction of cortical thickness relative to the baseline of the subject. In some embodiments, a reduction in neurodegeneration is observed by maintaining hippocampal size or slowing the reduction of hippocampal size. In some embodiments, a reduction in neurodegeneration is observed by maintaining or reducing the loss of pyramidal neuron cells relative to the baseline of the subject or relative to subjects administered a placebo. In some embodiments, a reduction in neurodegeneration is observed by maintaining or reducing the loss of granule neuron cells relative to the baseline of the subject or relative to subjects administered a placebo.

[0231] Methods for measuring the thickness of brain parts such as the cortex or measuring the size of the hippocampus can be achieved using magnetic resonance imaging (MRI). High-spatial-resolution sMRI now allows volumetric determination of hippocampal subregions. Early changes in CA1 have been observed in AD, and volumetric studies indicate that CA1 atrophy measurements can improve the diagnostic accuracy at the MCI stage. Novel MRI techniques, such as quantitative susceptibility mapping (QSM) or T2* transverse relaxation time, have shown that iron levels and their accumulation rates are heterogeneous in the human brain and are associated with cognitive impairment and slowing of motor performance. Neuronal dysfunction and altered connectivity of different brain networks are thought to occur early in the course of neurodegenerative diseases and can be indirectly measured using functional magnetic resonance imaging (fMRI). The entire brain or at least one region of the brain (e.g., cortical gray matter (i.e., the 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 within 48 hours of administering a first dose of lemborexant to the subject relative to the baseline of the subject. In some embodiments, the amount of p-tau is reduced within 24 hours of administering a first dose of lemborexant to the subject relative to the baseline of the subject. In some embodiments, neurodegeneration is reduced within 12 hours of administering a first dose of lemborexant to the subject relative to the baseline of the subject. In some embodiments, neurodegeneration is reduced within 6 hours of administering a first dose of lemborexant to the subject relative to the baseline of the subject.

[0232] In some embodiments, neurodegeneration is reduced or maintained for at least 30 days after administering a first dose of lemborexant. In some embodiments, the amount of p-tau in the CSF of the subject is reduced or maintained for at least 30 days after administering a first dose of lemborexant.

[0233] In some embodiments, neurodegeneration is reduced within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 days of administering the first dose of lemborexant. In some embodiments, the amount of p-tau in the subject's CSF is reduced for at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 days after administering the first dose of lemborexant.

[0234] 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.

[0235] In some embodiments, neurodegeneration is reduced or maintained within 45 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 45 days after administering the first dose of lemborexant.

[0236] In some embodiments, neurodegeneration is reduced or maintained within 60 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 60 days after administering the first dose of lemborexant.

[0237] In some embodiments, neurodegeneration is reduced or maintained for at least 120 days after 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 120 days after administering the first dose of lemborexant.

[0238] In some embodiments, neurodegeneration is reduced or maintained for at least 180 days after 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 180 days after administering the first dose of lemborexant.

[0239] In some embodiments, after a period of treatment, such as after 3 months, 6 months, or 9 months, the effect of lemborexant on neurodegeneration or p-tau is significant. 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, such as after 6 months or 9 months of treatment. In some embodiments, the amount of p-tau in the CSF of the subject decreases or is maintained after a period of at least 3 months. In some embodiments, the amount of p-tau in the CSF of the subject decreases or is maintained after a period of at least 6 months after treatment or after a period of at least 9 months of treatment. In some embodiments, neurodegeneration decreases or is maintained for at least 6 months after administration of the first dose of lemborexant. In some embodiments, the amount of p-tau in the CSF of the subject decreases or is maintained for at least 6 months after administration of the first dose of lemborexant.

[0240] In some embodiments, neurodegeneration decreases for up to at least 1 year after administration of the first dose of lemborexant. In some embodiments, the amount of p-tau in the CSF of the subject decreases for up to at least 1 year after administration of the first dose of lemborexant. In some embodiments, the amount of t-tau in the CSF of the subject decreases for up to at least 1 year after administration of the first dose of lemborexant. In some embodiments, the amount of Aβ in the CSF of the subject decreases for up to at least 1 year after administration of the first dose of lemborexant. In some embodiments, the amount of fibrillar plaques decreases for up to at least 1 year after administration of the first dose of lemborexant. In some embodiments, the amount of activated microglia increases for up to at least 1 year after administration of the first dose of lemborexant.

[0241] A. Altering Neurodegeneration

[0242] Accordingly, one aspect of the present disclosure relates to a method of altering neurodegeneration (e.g., reducing or delaying neurodegeneration, or slowing its growth rate) in a subject having 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 alter the neurodegeneration of the subject.

[0243] In some embodiments, altering neurodegeneration includes slowing the progression of neurodegeneration, reducing the rate of progression of neurodegeneration, delaying the progression of neurodegeneration, halting the development of neurodegeneration, and reversing the progression of neurodegeneration. In some embodiments, altering neurodegeneration includes preventing the onset or development of a pathology caused by or concurrent with neurodegeneration. Altering neurodegeneration can reduce, delay, or slow the onset and / or rate of progression of symptoms of the pathology. In some embodiments, altering neurodegeneration includes alleviating or improving one or more symptoms of neurodegeneration and / or improving one or more clinical metrics of a pathology caused by or concurrent with neurodegeneration (such as cognitive function, cerebral amyloid or tau levels, and / or biomarker expression). In some embodiments, altering neurodegeneration includes preventing the occurrence or recurrence of one or more symptoms of neurodegeneration.

[0244] 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 exhibit signs of dementia and / or cognitive impairment.

[0245] In some embodiments, the neurodegeneration is altered relative to a reference. Thus, compared to the reference, altering neurodegeneration can include reducing and / or delaying neurodegeneration and / or slowing its rate. In some embodiments, the reference is a baseline measurement from the subject prior to treatment. In some embodiments, the reference is a baseline measurement from a control subject. The reference can be a measurement obtained from more than one control subject and used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0246] In some embodiments, neurodegeneration is characterized by at least one of loss of cortical thickness or reduction in hippocampal volume. In some embodiments, altering neurodegeneration includes maintaining cortical thickness in the subject or slowing the reduction of cortical thickness. In some embodiments, neurodegeneration is characterized by at least one of loss of pyramidal neurons in the cortex or loss of pyramidal or granule neurons in the hippocampus. In some embodiments, altering neurodegeneration includes maintaining hippocampal volume in the subject or slowing the reduction of hippocampal volume. In some embodiments, altering neurodegeneration includes maintaining pyramidal neurons or granule neurons or reducing the loss of pyramidal neurons or granule neurons. In some embodiments, altering neurodegeneration includes reducing the rate of neurodegeneration. In some embodiments, altering neurodegeneration includes altering neurofilament light chain (NfL) levels. The NfL levels in the blood and / or CSF of the subject can be altered.

[0247] VIII. Subjects for Selective Treatment

[0248] Another aspect of the present disclosure relates to a method of 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 a measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial response, and biomarker expression from the subject; (b) comparing the measurement from the subject with a measurement from a reference; and (c) selecting the subject for treatment with lemborexant if the measurement from the subject is different from the measurement from the reference.

[0249] In some embodiments, the subject has mild cognitive impairment or mild dementia. In some embodiments, the subject does not exhibit signs of dementia and / or cognitive impairment.

[0250] In some embodiments, the subject is at risk of further Aβ accumulation. The subject may be at risk of tau pathology spread. The subject may be at risk of cognitive decline. In some embodiments, the subject may have a moderate level of amyloid PET (e.g., about 20 - 40 percentile units). In some embodiments, the subject may have an elevated level of amyloid PET (e.g., > 40 percentile units). 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 a family history of having a first-degree relative with AD or dementia, being 65 years of age or older, being female, having had a traumatic brain injury or recovering from a traumatic brain injury, and having other medical conditions such as obesity, diabetes, heart disease, and / or vascular disease.

[0251] In some embodiments, the subject has early AD. In some embodiments, the subject has preclinical AD. In some embodiments, the subject has been diagnosed with AD based on brain imaging, cognitive function, and / or biomarker criteria.

[0252] In some embodiments, obtaining at least one measurement includes 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 may indicate levels of tau phosphorylation, tau aggregation, Aβ plaque burden, and / or microglial response.

[0253] In some embodiments, the biological sample is a body fluid. In some embodiments, the body fluid is cerebrospinal fluid (CSF), blood, or saliva.

[0254] 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 a measurement obtained from more than one control subject and used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

[0255] In some embodiments, the control does not have AD. The measurement from the subject can be higher than the measurement from the control without AD. The measurement from the subject can be lower than the measurement from the control without AD.

[0256] In some embodiments, the control has AD. The measurement from the subject is similar to or higher than the measurement from the control with AD. The measurement from the subject can be similar to or lower than the measurement from the control with AD.

[0257] In some embodiments, the measurement of tau phosphorylation includes the measurement of phosphorylation at one or more of T181, T217, S202, or S205.

[0258] In some embodiments, the measurement of tau aggregation includes the measurement of insoluble tau aggregates (e.g., neurofibrillary tangles (NFT)).

[0259] In some embodiments, the measurement of neurodegeneration includes the measurement of cortical thickness and / or hippocampal volume or the measurement of pyramidal neuron or granule neuron loss.

[0260] In some embodiments, the measurement of Aβ plaque burden includes the measurement of Aβ plaque volume and / or the measurement of Aβ plaque volume growth.

[0261] In some embodiments, the measurement of Aβ plaque burden includes the measurement of amyloid PET signal in the brain region of the subject or the measurement of Aβ in the CSF of the subject.

[0262] In some embodiments, the measurement of microglial response is a change in the expression of at least one microglial marker. The microglial marker can be Iba1, Clec7a, CD68, TMEM119, or P2RY12. In some embodiments, the measurement of microglial response is a measurement of microglial phagocytosis.

[0263] IX. Therapeutic Efficacy

[0264] Disclosed herein are methods of treating a subject with lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, and methods for monitoring the therapeutic efficacy of a subject.

[0265] One aspect of the present disclosure relates to a method of monitoring the therapeutic efficacy of a subject having Alzheimer's disease (AD) or at risk of developing AD, the method comprising: (a) obtaining a first measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque load, microglial function, and biomarker expression from the subject; (b) administering to the subject a dose of suvorexant, a pharmaceutically acceptable salt thereof, or a solvate thereof; (c) obtaining a second measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque load, microglial function, and biomarker expression from the subject; 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 suvorexant.

[0266] 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 a first measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque load, microglial response, and biomarker expression from the subject; (b) administering to the subject a first dose of suvorexant, a pharmaceutically acceptable salt thereof, or a solvate thereof; (c) obtaining a second measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque load, microglial function, and biomarker expression from the subject; (d) comparing the second measurement from the subject with the first measurement, and (d) if the first measurement is different from the second measurement in the comparison of the measurements, administering a second dose of suvorexant. The first measurement and the second measurement may be different because the second measurement is higher than the first measurement. Alternatively, the second measurement may be lower than the first measurement. For example, if the measurement is microglial response, in some embodiments, the first measurement of microglial response may be higher than the second measurement of microglial response.

[0267] Another aspect of the present disclosure relates to a method for monitoring the therapeutic efficacy in a subject having Alzheimer's disease (AD) or at risk of developing AD, the method comprising: (a) obtaining a first measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque load, microglial function, and biomarker expression from the subject; (b) administering to the subject a dose of suvorexant, a pharmaceutically acceptable salt thereof, or a solvate thereof; (c) obtaining a second measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque load, microglial function, and biomarker expression from the subject; and (d) comparing the second measurement from the subject with the first measurement from the subject to obtain a comparison measurement, wherein a difference between the first measurement and the second measurement in the comparison measurement, or a difference between the comparison measurement and a reference measurement, indicates effective treatment with suvorexant.

[0268] Another aspect of the present disclosure relates to a method for treating a subject having Alzheimer's disease (AD) or at risk of developing AD, the method comprising: (a) obtaining a first measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque load, microglial response, and biomarker expression from the subject; (b) administering to the subject a first dose of suvorexant, a pharmaceutically acceptable salt thereof, or a solvate thereof; (c) obtaining a second measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque load, microglial function, and biomarker expression from the subject; (d) comparing the second measurement from the subject with the first measurement from the subject to obtain a comparison measurement; and (e) administering a second dose of suvorexant if the first measurement and the second measurement are different in the comparison measurement, or if the comparison measurement is different from a reference measurement.

[0269] In some embodiments, 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. In some embodiments, the data from the brain scan indicates the levels of tau phosphorylation, tau aggregation, Aβ plaque load, and / or microglial response. In some embodiments, the biological sample is a body fluid. In some embodiments, the body fluid is cerebrospinal fluid (CSF), blood, or saliva.

[0270] 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.

[0271] In some embodiments, for example, in those methods that include comparing a measurement value and a reference measurement value, the reference measurement value is obtained from at least one control. In some embodiments, the reference measurement value is a comparison of a first measurement value from a control and a second measurement value from the control. In some embodiments, the comparison measurement value is higher than the reference measurement value. In some embodiments, the comparison measurement value is lower than the reference measurement value. For example, a comparison measurement value that compares a first measurement value from a subject and a second measurement value from the subject may indicate that no change has occurred. Meanwhile, the reference measurement value may indicate that a change has occurred in the control. Thus, the difference between the comparison measurement value and the reference measurement value may indicate whether the treatment is effective and / or whether a second dose of lemborexant should be administered.

[0272] In some embodiments, the reference measurement value is a measurement value from a control. The reference measurement value can be obtained from more than one control subject and is used as a standard or threshold measurement value. In some embodiments, the reference measurement value is a measurement value from a control subject administered a placebo.

[0273] In some embodiments, the control does not have AD. In some embodiments, the comparison measurement value is higher than the reference measurement value. In some embodiments, the comparison measurement value is lower than the reference measurement value.

[0274] In some embodiments, the control has AD, such as untreated AD. In some embodiments, the comparison measurement value is higher than the reference measurement value. In some embodiments, the comparison measurement value is lower than the reference measurement value.

[0275] In some embodiments, the measurement value of tau phosphorylation includes the measurement value of phosphorylation of one or more of T181, T217, S202, S205, or T231.

[0276] In some embodiments, the measurement value of tau aggregation includes the measurement value of insoluble tau aggregates (e.g., neurofibrillary tangles (NFT)).

[0277] In some embodiments, the measurement value of neurodegeneration includes the measurement value of cortical thickness and / or hippocampal volume or the measurement value of pyramidal neuron or granule neuron loss.

[0278] In some embodiments, the measurement value of Aβ plaque burden includes the measurement value of Aβ plaque volume and / or the measurement value of Aβ plaque volume growth.

[0279] In some embodiments, the measurement value of Aβ plaque burden includes the measurement value of amyloid PET signal in the brain region of the subject or the measurement value of Aβ in the CSF of the subject.

[0280] In some embodiments, the measurement of the microglial response is a measurement of the expression of at least one microglial marker. The microglial marker can be Iba1, Clec71, P2RY12, or TMEM 119. In some embodiments, the measurement of the microglial response is a measurement of the phagocytosis of microglia.

[0281] In some embodiments, the measurement of biomarker expression is a measurement of the expression of Ifnb1, MMP2, and / or Bace1.

[0282] In some embodiments, the subject is amyloid negative.

[0283] In some embodiments, the subject has Aβ plaques.

[0284] 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.

[0285] In some embodiments, the subject is at risk of further Aβ accumulation. The subject may be at risk of tau pathology spread. The subject may be at risk of cognitive decline. In some embodiments, the subject may have a moderate level of amyloid PET (e.g., about 20 - 40 percentile units). In some embodiments, the subject may have an elevated level of amyloid PET (e.g., >40 percentile units). 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 a family history of having a first-degree relative with AD or dementia, being 65 years of age or older, being female, having had a traumatic brain injury or recovering from a traumatic brain injury, and having other medical conditions such as obesity, diabetes, heart disease, and / or vascular disease.

[0286] In some embodiments, the subject has early AD. In some embodiments, the subject has preclinical AD. In some embodiments, the subject has been diagnosed with AD based on brain imaging, cognitive function, and / or biomarker criteria.

[0287] X. Dose

[0288] As disclosed herein, the dose of lemborexant can refer to a therapeutically effective amount of lemborexant, a pharmaceutically acceptable salt thereof, or a solvate thereof. In some embodiments, the methods disclosed herein include orally administering to a subject from 5 mg to 20 mg of lemborexant once daily. In some embodiments, a dose of 20 mg of lemborexant is administered to the subject once daily. In some embodiments, a dose of 25 mg of lemborexant is administered to the subject once daily. In some embodiments, a dose of 25 mg of lemborexant is administered to the subject once daily for at least 2 days. In some embodiments, a dose of 25 mg of lemborexant is administered to the subject once daily. In some embodiments, a dose of 25 mg of lemborexant is administered to the subject once daily for at least 5 days. In some embodiments, a dose of 25 mg of lemborexant is administered to the subject once daily. In some embodiments, a dose of 25 mg of lemborexant is administered to the subject once daily for at least one week. In some embodiments, a dose of 25 mg of lemborexant is administered to the subject once daily. In some embodiments, a dose of 25 mg of lemborexant is administered to the subject once daily for at least one month.

[0289] In some embodiments, the methods disclosed herein include orally administering to a subject a dosage form comprising lemborexant. In some embodiments, the methods disclosed herein include orally administering to a subject a dosage form comprising 10 mg of lemborexant. In some embodiments, the methods disclosed herein include orally administering to a subject a dosage form comprising 15 mg of lemborexant. In some embodiments, the methods disclosed herein include orally administering to a subject a dosage form comprising 20 mg of lemborexant. In some embodiments, the methods disclosed herein include orally administering to a subject a dosage form comprising 25 mg of lemborexant. In some embodiments, the methods disclosed herein include orally administering to a subject a dosage form comprising 30 mg of lemborexant. In some embodiments, the methods disclosed herein include orally administering to a subject a dosage form comprising 35 mg of lemborexant. In some embodiments, the methods disclosed herein include orally administering to a subject a dosage form comprising 40 mg of lemborexant. In some embodiments, the methods disclosed herein include orally administering to a subject a dosage form comprising 45 mg of lemborexant. In some embodiments, the methods disclosed herein include orally administering to a subject a dosage form comprising 50 mg of lemborexant.

[0290] The dosage forms disclosed herein contain a therapeutically effective amount of lemborexant for treatment when administered in accordance with the teachings disclosed herein. The unit dose of the effective amount in the dosage form is from 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 single-dose dosage forms. In some embodiments, the unit dose may be 2.5 mg. In some embodiments, the unit dose may 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.

[0291] Thus, the therapeutically effective amount of lemborexant, its pharmaceutically acceptable salts, or its solvates as administered herein may include doses falling within a range, such as within the range of 5 mg to 50 mg per day.

[0292] In some embodiments, the therapeutically effective amount of lemborexant administered to the subject is within the range of 5 mg to 50 mg per day. For example, the therapeutically effective amount of lemborexant administered to the subject may be within the range of 10 mg to 30 mg per day. In some embodiments, 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.

[0293] In some embodiments, the therapeutically effective amount of lemborexant administered to the subject is 20 - 25 mg per day.

[0294] In some embodiments, a dose of 20 mg of lemborexant is administered to the subject once a day.

[0295] In some embodiments, lemborexant is administered in a first cycle at a first dose, in a second cycle at a second dose, and optionally in a third cycle at a third dose. Each of the first cycle, the second cycle, and the third cycle can be 1 week.

[0296] 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.

[0297] 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.

[0298] In some embodiments, lemborexant can be administered to a subject over a period of time. In some embodiments, the methods described herein include administering lemborexant to a subject for at least 6 months. In some embodiments, the methods described herein include 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 include administering lemborexant to a subject for at least 18 months. In some embodiments, the methods described herein include administering lemborexant to a subject for at least 24 months, 30 months, or 36 months. In some embodiments, lemborexant can be administered for the remainder of the subject's life.

[0299] XI. Pharmaceutical Compositions

[0300] In some embodiments, the dosage forms disclosed herein can constitute one or more pharmaceutical compositions that contain lemborexant together with a pharmaceutically acceptable excipient.

[0301] As used herein, the term "composition" used herein includes a product containing specific ingredients in specific amounts and any product resulting directly or indirectly from the combination of multiple specific ingredients in specific amounts. Such terms related to pharmaceutical compositions are intended to include products containing active ingredients and inert ingredients constituting carriers, and include every product resulting directly or indirectly from the combination, complexation or aggregation of any two or more ingredients, or the dissociation, other types of reactions or interactions of one or more ingredients. Therefore, the pharmaceutical composition of the present disclosure includes every composition prepared by mixing the compound of the present disclosure with a pharmaceutically acceptable carrier.

[0302] As used herein, the term "pharmaceutically acceptable" means that the carrier, diluent, excipient, or vehicle is compatible with the other ingredients of the formulation and is not toxic to the subject.

[0303] Solid dosage forms of the present disclosure include capsules, granules, lozenges, pellets, pills, powders, suspensions, and tablets.

[0304] The pharmaceutical compositions disclosed herein can be prepared using standard techniques and manufacturing methods generally known in the art. See, for example, the monographs of Japanese Pharmacopoeia [Japanese Pharmacopoeia], 16th edition; and US Pharmacopoeia-NF [United States Pharmacopoeia-National Formulary], Chapter 1151 of pharmaceutical dosage forms.

[0305] In some embodiments, the pharmaceutical composition comprises lebrexan. In some embodiments, the pharmaceutical composition further comprises at least one additional component selected from a pharmaceutically acceptable carrier, a pharmaceutically acceptable vehicle, and a pharmaceutically acceptable excipient.

[0306] In some embodiments, the at least one additional component in the pharmaceutical composition is selected according to the intended route of administration of the pharmaceutical composition. Non-limiting examples of suitable routes of administration of the pharmaceutical composition include parenteral, oral, inhaled spray, topical, rectal, nasal, buccal, vaginal, and implant depot administration. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intracisternal, intrathecal, intraliver, intralesional, and intracranial injection or infusion techniques. In some embodiments, the mode of administration is selected from intravenous, oral, subcutaneous, and intramuscular administration. The sterile injectable form of the compositions of the present disclosure can be, for example, an aqueous or oily suspension. 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. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Non-limiting examples of vehicles and solvents that can be employed include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, a sterile, non-volatile oil can be used as a solvent and / or suspending medium.

[0307] For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives can be used in the preparation of injectables, as can natural pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxylated forms. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersing agents, such as carboxymethyl cellulose or similar dispersing agents commonly used in formulating pharmaceutically acceptable dosage forms, including emulsions and suspensions. For formulating purposes, other commonly used surfactants, such as Tweens, Spans, and other emulsifying agents or bioavailability enhancers, which are commonly used in manufacturing pharmaceutically acceptable solid, liquid, and / or other dosage forms, can also be used.

[0308] For oral administration, lemborexant can be provided in acceptable oral dosage forms, including but not limited to suspensions, capsules, tablets, orally disintegrating tablets, sprays, and other orally administrable formulations that are easy to swallow. In some embodiments, lemborexant is provided in the form of tablets or capsules. In some embodiments, lemborexant is provided in the form of compressible tablets. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants, such as magnesium stearate, can also be added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When an aqueous suspension for oral use is required, the active ingredient is combined with an emulsifying and / or suspending agent. If desired, certain sweetening, flavoring, or coloring agents can also be added.

[0309] To more fully understand the disclosures described herein, the following examples are presented. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the disclosure in any way.

[0310] Non-limiting embodiments of the disclosure:

[0311] 1. A method of reducing or maintaining the amount of p-tau or t-tau in a subject, the method comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof.

[0312] 2. The method according to embodiment 1, wherein the amount of p-tau or t-tau in the subject is reduced or maintained relative to the baseline of the subject.

[0313] 3. The method according to embodiment 1 or embodiment 2, wherein the amount of p-tau or t-tau in the subject is reduced or maintained relative to a placebo.

[0314] 4. The method according to any one of embodiments 1-3, wherein the ratio of p-tau to tau in the subject is lower relative to the baseline of the subject or relative to a placebo after administering a therapeutically effective amount of lemborexant.

[0315] 5. The method according to any one of embodiments 1-4, wherein the rate of dephosphorylation of tau in the subject is higher relative to the baseline of the subject or relative to a placebo after administering a therapeutically effective amount of lemborexant.

[0316] 6. The method according to any one of embodiments 1-5, wherein the rate of phosphorylation of tau in the subject is lower relative to the baseline of the subject or relative to a placebo after administering a therapeutically effective amount of lemborexant.

[0317] 7. The method according to any one of embodiments 1-6, wherein the ratio of p-tau / t-tau in the CSF of the subject is reduced compared to the ratio of CSF p-tau / t-tau of the subject before administering lemborexant.

[0318] 8. The method according to embodiment 7, wherein the ratio of p-tau / t-tau in the CSF of the subject is maintained within 10% of the ratio of p-tau / t-tau of the subject before administering lemborexant.

[0319] 9. The method according to any one of embodiments 1-8, wherein the Aβ concentration in the CSF of the subject is lower than or equal to the Aβ concentration in the CSF of the subject before administering lemborexant.

[0320] 10. The method according to embodiment 9, wherein the amyloid PET signal in the brain of a subject administered a therapeutically effective amount of lemborexant is lower or the same as the amyloid PET signal in the brain of the subject before administration of lemborexant.

[0321] 11. The method according to any one of embodiments 1-10, wherein the tau PET signal in the brain of the subject is reduced relative to the baseline.

[0322] 12. The method according to any one of embodiments 1-11, wherein the tau is p-tau, t-tau or aggregated tau.

[0323] 13. The method according to any one of embodiments 1-12, wherein p-tau and t-tau are reduced.

[0324] 14. The method according to any one of embodiments 1-13, wherein the reduction in tau phosphorylation occurs in the hippocampus, entorhinal cortex and / or piriform cortex.

[0325] 15. The method according to any one of embodiments 1-14, wherein the Aβ in the CSF of the subject is Aβ38, Aβ40 and / or Aβ42.

[0326] 16. The method according to any one of embodiments 1-15, wherein the amount of p-tau is reduced within 48 hours of administering the first dose of lemborexant to the subject, relative to the baseline of the subject.

[0327] 17. A method of reducing neurodegeneration in a subject, the method comprising administering a therapeutically effective amount of lemborexant to a subject in need thereof.

[0328] 18. The method according to embodiment 17, wherein a reduction in neurodegeneration is observed by maintaining the cortical thickness or slowing the reduction in cortical thickness relative to the baseline of the subject or relative to a subject administered a placebo.

[0329] 19. The method according to embodiment 17 or embodiment 18, wherein a reduction in neurodegeneration is observed by maintaining the hippocampal size or slowing the decrease in hippocampal size relative to the baseline of the subject or relative to a subject administered a placebo.

[0330] 20. The method according to any one of embodiments 17-19, wherein a reduction in neurodegeneration is observed by maintaining or reducing the loss of pyramidal neuron cells relative to the baseline of the subject or relative to a subject administered a placebo.

[0331] 21. The method according to any one of embodiments 17-20, wherein a reduction in neurodegeneration is observed by maintaining or reducing the loss of granule neurons relative to the baseline of the subject or relative to a subject administered a placebo.

[0332] 22. The method according to any one of embodiments 1-21, wherein the therapeutically effective amount of suvorexant administered to the subject is in the range of 10 mg to 50 mg per day.

[0333] 23. The method according to embodiment 22, wherein the therapeutically effective amount of suvorexant administered to the subject is in the range of 15 mg to 30 mg per day.

[0334] 24. The method according to embodiment 22, wherein the therapeutically effective amount of suvorexant administered to the subject is 25 mg per day.

[0335] 25. The method according to any one of embodiments 1-24, wherein a dose of 25 mg of suvorexant is administered to the subject once a day.

[0336] 26. The method according to embodiment 25, wherein a dose of 25 mg of suvorexant is administered to the subject once a day for at least two days.

[0337] 27. The method according to any one of embodiments 1-26, wherein after administering a dose of 25 mg of suvorexant to the subject once a day for at least two days, a dose of 2.5 mg, 5 mg, 10 mg, 15 mg, or 20 mg of suvorexant is administered to the subject once a day.

[0338] 28. A method of reducing the Aβ concentration in a subject, the method comprising administering a therapeutically effective amount of suvorexant to a subject in need thereof.

[0339] 29. The method according to any one of embodiments 1-28, wherein the Aβ concentration in the CSF of the subject is lower than the Aβ concentration in the CSF of the subject before administering suvorexant.

[0340] 30. The method according to any one of embodiments 1-29, wherein the amyloid PET signal in the brain of the subject administered suvorexant is lower than the amyloid PET signal in the brain of the subject before administering suvorexant.

[0341] 31. The method according to embodiment 30, wherein the Aβ in the CSF of the subject is Aβ38, Aβ40, and / or Aβ42.

[0342] 32. The method according to any one of embodiments 1-29, wherein the concentration of Aβ is reduced within 48 hours of administering suvorexant.

[0343] 33. A method of increasing the number of activated microglia in a subject, the method comprising administering a therapeutically effective amount of suvorexant to a subject in need thereof.

[0344] 34. The method according to embodiment 33, wherein the number of activated microglia is increased relative to a baseline.

[0345] 35. The method according to any one of embodiments 33 or 34, wherein the activated microglia are phagocytic microglia.

[0346] 36. The method according to any one of embodiments 33-35, wherein the number of activated microglia is measured by a PET or CSF biomarker of microglial activation.

[0347] 37. The method according to any one of embodiments 33-36, wherein the whole brain or at least one region of the brain is analyzed by PET.

[0348] 38. The method according to embodiment 37, wherein at least one region of the brain analyzed by PET is selected from the cortical gray matter, lateral ventricle, 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).

[0349] Examples

[0350] Example 1: Clinical study protocol

[0351] a. Trial 1: Acute effects of suvorexant on CSF β-amyloid and Tau

[0352] The acute effects of suvorexant will be studied in cognitively normal amyloid-positive participants.

[0353] The inclusion criteria will be:

[0354] ○ Aged 60-80 years

[0355] ○ Any gender

[0356] ○ Any race / ethnicity

[0357] ○ Mini-Mental State Examination score (MMSE) ≥ 27

[0358] ○ Plasma Aβ test positive (i.e., amyloid-positive)

[0359] ○ Pittsburgh Sleep Quality Index > 5

[0360] The exclusion criteria will be:

[0361] ○Cognitive impairment determined by a history of MMSE < 27

[0362] ○Unable to speak or understand English

[0363] ○Any sleep disorder other than insomnia

[0364] ■No history of moderate to severe sleep disordered breathing and STOP - Bang score > 5

[0365] ■History or reported symptoms suggestive of restless legs syndrome, narcolepsy, or other sleep disorders

[0366] ■No more than mild sleep apnea on PSG (AHI < 16)

[0367] ○Sleep schedule outside the bedtime range of 22:00 - midnight

[0368] ○Contraindications to lumbar catheter (anticoagulants; bleeding disorders; allergy to lidocaine or disinfectant; previous central nervous system or lower back surgery)

[0369] ○Cardiovascular diseases requiring medical treatment, except for controlled hypertension (PI judgment)

[0370] ○Stroke

[0371] ○Liver or kidney impairment

[0372] ○Lung diseases (PI judgment)

[0373] ○Type 1 diabetes

[0374] ○HIV or AIDS

[0375] ○Neurological or psychiatric disorders requiring medical treatment (PI judgment)

[0376] ○Suicidal ideation

[0377] ○Alcohol use or smoking (PI judgment)

[0378] ○Use of sedative medications (PI judgment)

[0379] ○Unable to get out of bed independently

[0380] ○In the opinion of the investigator, participants with abnormal physical examinations should be excluded.

[0381] ○Current pregnancy

[0382] ○Body mass index > 35

[0383] ○History of migraine (PI judgment)

[0384] ○History of substance abuse in the past 6 months

[0385] ○ History or presence of any clinically significant medical condition, behavior, or mental disorder (including suicidal ideation) that may affect the safety of the subject or interfere with the study assessment or judgment of PI participants, or a surgical history based on medical records or patient reports that is not a good candidate.

[0386] ○ Urinary or fecal incontinence

[0387] ○ Simultaneous enrollment in another trial of the study drug or device

[0388] Twelve (or more) participants will be randomly assigned to receive placebo (N = 4 or more) or lemborexant 25 mg (N = 8 or more).

[0389] Procedure: Randomized subjects will be admitted in the early afternoon (Night 1). All participants will have their sleep monitored with an unattended full assembly PSG (TrackIt TM ; Lifelines, Troy, IL) that will allow sleep staging according to the gold standard of the American Academy of Sleep Medicine criteria and has been used to monitor sleep for 36 - 48 hours in similar studies.

[0390] At approximately 20:00, a lumbar catheter and two IV catheters will be placed in each participant, and 6 ml of CSF will be collected 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 perturbation during fluid collection. The sampling start time will be approximately 1 hour before the typical bedtime defined by each participant's sleep log to allow for 13 C 6 - leucine infusion and frequent blood sampling. Six milliliters of blood will be 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).

[0391] Table 1. Timeline of catheter placement and removal, bedtime, and sampling

[0392]

[0393] C: Catheter placement; B: Bedtime; X: CSF / Blood sampling; R: Catheter removal

[0394] Approximately 1 hour before their habitual bedtime (t = 0) on Day 1, all participants will begin an infusion of 800 mg of labeled 13 C 6 -leucine to in vivo label proteins during intracellular translation to monitor Aβ kinetics.

[0395] Participants will be allowed to sleep as they will be able to turn off the lights immediately at their habitual bedtime (bedtime is between hours 1, approximately 22:00 - 00:00). Dim red light (safety light) will be used when collecting CSF and blood in the dark. Participants will sleep until they wake up in the morning.

[0396] During Day 2, all participants will be kept awake and regularly monitored in a well - lit room and napping will not be permitted. At approximately 22:00 (hour 25) on Day 2, except that no infusion of labeled 13 C 6 -leucine and the time of "hour 26" will depend on the participants' regular bedtime, all participants will follow the same sleep routine as they did the previous night. Participants will receive the same placebo or dose of lemborexant as the previous night. The study will end at approximately 20:00 (hour 48) on Day 3, at which time the IV catheter and lumbar catheter will be removed. Then all participants will sleep overnight and will be monitored for at least 8 hours after catheter removal and then discharged from the study.

[0397] CSF tau, phosphorylated tau, and Aβ kinetics will be quantified by mass spectrometry. For the determination of the baseline for each participant, AD biomarkers (Aβ38, Aβ40, Aβ42, T181, S202, T217, pT181, pS202, pT217) will be normalized to the mean over the first 6 hours (t = 0 - 6, 20:00 - 02:00) before any intervention - induced changes are observed in the CSF. The 0 - hour concentration will also be normalized. Phosphorylated tau ratios (pT181 / T181, pS202 / S202, pT217 / T217, p - tau / t - tau) will be normalized to the first time point (0 hour). Then the trajectories of changes over time relative to these baselines will be plotted by treatment - arm group to examine whether the changes in concentration or ratio are linear.

[0398] For statistical analysis, if the linear hypothesis is valid, a (linear mixed effects) LME model with random intercepts and slopes will be used for analysis; otherwise, a mixed model for repeated measures (MMRM) will be used. Fixed effects in the model will include treatment group, time, and their interaction. Baseline will be included as a covariate. The normality assumption will be checked using residual plots, and appropriate transformations (e.g., logarithm) will be considered. An unstructured covariance matrix will be used, and if there are convergence problems, various other covariance matrix structures (e.g., compound symmetry, first-order autoregressive) will be compared, and the best-fitting structure will be selected based on the Akaike information criterion (AIC) for the final analysis.

[0399] b. Trial 2: Treatment of Subjects with Preclinical or Early Alzheimer's Disease

[0400] This trial will evaluate the efficacy of lemborexant in preventing or delaying Aβ accumulation, downstream tau pathology spread, and cognitive decline in a series of preclinical and early AD.

[0401] Amyloid-β (Aβ) accumulation often begins more than a decade 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 during the preclinical stage of the disease. Multiple neuroimaging and biomarker observational studies have demonstrated that Aβ accumulation is associated with an increased risk of cognitive decline in clinically normal elderly individuals.

[0402] This trial will use NAV4694 (flutafuranol) amyloid PET imaging to assess eligibility and longitudinal outcomes of fibrillar amyloid pathology, and the MK6240 tau PET tracer to evaluate the longitudinal spread of neurofibrillary tangles and tau neurite pathology. Clinical outcomes include the Preclinical Alzheimer's Cognitive Composite Scale 5 (PACC-5) consisting 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), participant and study partner reports of cognitive function.

[0403] In addition, scans of the patient's cortex and hippocampus will be performed to assess the extent of neurodegeneration. CSF and blood will be collected to measure AD biomarkers such as Aβ, tau, p-tau, and NfL.

[0404] i. Group 1: Patients with Preclinical Alzheimer's Disease - Moderate Level of Amyloid

[0405] The purpose of this trial is to determine whether lemborexant treatment results in primary prevention or delay of AD by preventing or slowing early Aβ accumulation in the brain. This trial will enroll cognitively normal individuals with moderate levels of amyloid on screening PET imaging (approximately 20 - 40 percentile units), who are considered to be in the earliest preclinical stage of AD and at risk of further Aβ accumulation and early spread of tau pathology within four years.

[0406] Patient inclusion criteria. Patients will be selected for intact cognition, determined by an education-adjusted Mini-Mental State Examination (MMSE) score ≥ 27 and a global Clinical Dementia Rating (CDR) of 0. Patients will have moderate levels of amyloid (20 - 40 percentile units) on PET imaging.

[0407] Lemborexant administration. Lemborexant or a matching placebo will be administered to participants monthly during in-person study visits where treatment compliance and adverse events will be evaluated. Lemborexant is FDA-approved for the treatment of insomnia at a dose of 5 - 10 mg. After an interim analysis of safety and efficacy, the lemborexant dose can be increased to 20 mg. At the start of the study, participants will take 5 mg of lemborexant once daily for 1 week and then increase to 10 mg of lemborexant once daily; there will be matching placebo tablets for both 5 mg and 10 mg of lemborexant. At the end of the study, participants will take 5 mg of lemborexant once daily for 1 week before drug discontinuation.

[0408] Drug titration. Since participants will be naive to lemborexant, 5 mg of lemborexant will be provided once daily for 1 week, followed by titration to 10 mg of lemborexant once daily; there will be matching placebo tablets for both 5 mg and 10 mg of lemborexant. At the end of the study, participants will be titrated down to 5 mg of lemborexant once daily for 1 week before drug discontinuation.

[0409] If the lemborexant dose is increased to 20 mg after the interim analysis, the same up-titration and down-titration protocol will be followed, except that there will be an additional week of taking 10 mg of lemborexant:

[0410] Up-titration: 5 mg of lemborexant - 1 week

[0411] ○ 10 mg of lemborexant - 1 week

[0412] ○ 20 mg of lemborexant - 1 week

[0413] Down-titration: 10 mg of lemborexant - 1 week

[0414] ○ 5 mg of lemborexant - 1 week

[0415] ○ Stop

[0416] Study outcomes. Anticipate measurements of primary, secondary, and exploratory outcomes.

[0417] Primary outcome: The primary outcome measure of the trial is amyloid PET SUVr at 6 months, measured and compared to placebo. Biomarker outcomes will include measurements of tau PET, Aβ, phosphorylated tau, and oligomers in CSF and plasma.

[0418] Secondary outcome: Tau PET will be measured.

[0419] Exploratory outcomes: In CSF, biomarkers Aβ, tau, phosphorylated tau, neurogranin (NG), and neurofilament light chain (NfL) will be measured. In plasma, NfL, phosphorylated tau 181, and phosphorylated tau 217 will be measured. Clinical outcomes measuring cognition will include tests of the Preclinical Alzheimer's Cognitive Composite 5 (PACC5) scale and the Cognitive Function Index (CFI).

[0420] ii. Preclinical Alzheimer's disease - elevated levels of amyloid

[0421] The aim of the trial is to determine whether lemborexant treatment results in primary prevention or delay of AD by preventing early Aβ accumulation in the brain. The trial will recruit cognitively normal individuals with elevated levels of amyloid (approx. >40 percentile units) on screening PET imaging, who are at high risk of cognitive decline within four years.

[0422] Patient inclusion criteria. Patients will be selected for intact cognition, determined by an education-adjusted Mini-Mental State Examination (MMSE) score ≥27 and a global Clinical Dementia Rating (CDR) of 0. Patients will have elevated amyloid PET levels >40.

[0423] Lemborexant administration. Lemborexant or a matching placebo will be administered monthly to participants during in-person study visits where treatment compliance and adverse events will be evaluated. Lemborexant is FDA-approved for the treatment of insomnia at a dose of 5 - 10 mg. After an interim analysis of safety and efficacy, the lemborexant dose can be increased to 20 mg. At the start of the study, participants will take lemborexant 5 mg once daily for 1 week and then increase 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 take lemborexant 5 mg once daily for 1 week before drug withdrawal.

[0424] Drug titration. Since the participants will be naive to lemborexant, a once-daily dose of 5 mg of lemborexant will be provided for 1 week, followed by titration to a once-daily dose of 10 mg of lemborexant; matching placebo tablets will be available for both 5 mg and 10 mg of lemborexant. At the end of the study, the participants will be titrated down to a once-daily dose of 5 mg of lemborexant during the 1-week period prior to drug discontinuation.

[0425] If the lemborexant dose is increased to 20 mg after the interim analysis, the same up-titration and down-titration regimens will be followed, except that there will be an additional week of taking 10 mg of lemborexant:

[0426] Up-titration: 5 mg of lemborexant - 1 week

[0427] ○ 10 mg of lemborexant - 1 week

[0428] ○ 20 mg of lemborexant - 1 week

[0429] Down-titration: 10 mg of lemborexant - 1 week

[0430] ○ 5 mg of lemborexant - 1 week

[0431] ○ Stop

[0432] Study outcomes. Measurements of primary, secondary, and exploratory outcomes are foreseen.

[0433] Primary outcome. To test the effect of lemborexant on cognitive decline in patients with elevated amyloid, the primary outcome measure of the trial is the Preclinical Alzheimer's Cognitive Composite 5 (PACC5) at 6 months. Biomarker outcomes will include measurements of tauPET, Aβ, phosphorylated tau, and fibrils in CSF and plasma.

[0434] Secondary outcomes. The Cognitive Function Index (CFI) will be measured. Amyloid PET and tau PET will be measured.

[0435] Exploratory outcomes. Clinical measurements will be the ADCS ADL-Prevention, Computerized Cognitive Composite, ISLT, Trail Making, CDR-SB, and time to reach CDR 0.5. vMRI and rs-MRI will be determined. In CSF, the biomarkers Aβ, tau, phosphorylated tau, neurogranin (NG), and neurofilament light chain (NfL) will be measured. In plasma, NfL, phosphorylated tau 181, and phosphorylated tau 217 will be measured.

[0436] c. Trial 3: Treatment of Subjects with Preclinical or Early Alzheimer's Disease

[0437] This trial will evaluate the efficacy of lemborexant in preventing or delaying Aβ accumulation, downstream tau pathology spread, and cognitive decline in a series of preclinical and early AD settings. This trial will follow the protocol of Trial 2, with the difference being that different doses will be used. Lemborexant at 7.5 mg will be used instead of 5 mg of lemborexant. Lemborexant at 15 mg will be used instead of 10 mg of lemborexant. Lemborexant at 25 mg or 30 mg will be used instead of 20 mg of lemborexant.

[0438] Example 2: Mouse study of tau-mediated neurodegeneration

[0439] When tau-mediated neuroinflammation was observed without significant neuronal loss, P301S / E4 and E4 knock-in non-tau-depositing mice were orally gavaged daily with 30 mg / kg lemborexant or vehicle starting at 7.5 months of age until 9.5 months of age (Figure 2a).

[0440] A. Changes in sleep-wake behavior

[0441] Changes in sleep-wake behavior were confirmed by electroencephalogram (EEG) analysis. E4 and P301S / E4 mice treated with lemborexant showed an approximately 25% increase in the time spent in NREM sleep and an approximately 20% decrease in the time spent awake ( Figures 2B - 2F ). No changes in REM sleep time were observed, consistent with previous findings.

[0442] Compared to E4 mice, a tau-dependent decrease in NREM sleep was observed in P301S / E4 mice ( Figure 2B ), indicating a link between pathological tau and sleep. The effects of lemborexant on sleep ( Figure 2G and Figure 2H ) and sleep-related motor activity (Figure 6) measured by EEG and Piezosleep pad persisted for approximately five hours after treatment, without additional phase delay or changes in circadian sleep-wake activity.

[0443] B. Effects on tau pathology and neurodegeneration

[0444] To determine whether lemborexant could affect tau pathology and neurodegeneration, the hippocampus, entorhinal cortex, and piriform cortex, which are regions showing significant tau-mediated degeneration, were studied. A significant approximately 20% decrease in AT8+ and MC1+ tau staining was observed in lemborexant-treated P301S / E4 mice compared to vehicle-treated mice ( Figures 3A - 3F ). Additionally, lemborexant-treated P301S / E4 mice had significantly reduced insoluble phosphorylated and total tau levels ( Figures 7A - 7F ).

[0445] It was found that compared with the control, the degree of brain atrophy in P301S / E4 mice treated with lemborexant was strongly attenuated ( Figures 3G - 3I and Figure 7H –7K). More specifically, atrophy of the hippocampus and piriform / entorhinal cortex was significantly improved by approximately 50%, accompanied by a reduction in the enlargement of the lateral ventricles. In P301S / E4 mice treated with lemborexant, the granular cell and pyramidal cell neuronal layers were significantly thicker ( Figure 7G – Figure 7K ), which was confirmed by reduced plasma neurofilament light chain levels ( Figure 3J ), demonstrating a robust improvement in neuronal injury and degeneration.

[0446] C. Microglial reactivity

[0447] To investigate whether the significant reduction in tau-mediated neurodegeneration in lemborexant-treated mice was associated with reduced microglial reactivity, different markers of reactive microglia were quantified in the spectra of disease-related or homeostatic populations in the hippocampus and piriform cortex. Significant changes were observed (Figure 4), mainly in the hippocampus and most prominently in the CA3 region (Figure 4).

[0448] A significant reduction in ionized calcium-binding adaptor molecule 1 (IBA1) was observed, indicating an overall reduction in the reactive microglial population in P301S / E4 mice treated with lemborexant compared to vehicle-treated P301S / E4 mice ( Figure 4A 、 Figure 4C 、 Figure 4L and Figure 4M ).

[0449] Disease-related microglial markers such as Clec7a ( Figure 4E and Figure 4G ) and CD68 ( Figure 4A 、 Figure 4D 、 Figure 4P 、and Figure 4Q ), a marker of phagolysosomal activity, were significantly increased in P301S / E4 mice compared to E4 mice, but lemborexant strongly reduced these markers compared to vehicle-treated P301S / E4.

[0450] A significant increase in the TMEM119 marker of homeostatic microglia was observed in P301S / E4 mice treated with lemborexant ( Figure 4B 、 Figure 4F 、 Figure 4N and Figure 4O ), while no change was observed in P2RY12 (Figure 8).

[0451] These results suggest that reducing reactive microglia is involved in the regulation of lemborexant-mediated tau neurodegeneration. Moreover, compared with controls, there was a significant decrease in the co-localization of APOE in astrocytes and microglia in lemborexant-treated P301S / E4 mice ( Figures 4H - 4K ), which is more common only during elevated inflammatory and pathological conditions. Lemborexant treatment reduced GFAP+ astrocyte reactivity in the hippocampus of P301S / E4 mice ( Figures 4R - 4T ). In the absence of tau pathology, no changes in microglial reactivity were found in E4 knock-in mice.

[0452] D. RNA sequencing

[0453] To gain insight into the mechanisms underlying the observed changes in microglial reactivity and tau-mediated neurodegeneration induced by lemborexant-induced NREM sleep, RNA sequencing was performed on bulk hippocampal tissue. Gene expression changes were found and were involved in multiple functional modules, including hormone and GPCR ligand binding, glial cell differentiation, synaptic regulation, particularly excitatory synapses, and the response to DNA damage (Figure 5). In addition to normal aging, defective DNA repair has been associated with age-related neurodegenerative diseases such as AD. Hyperphosphorylated and aggregated tau can impede DNA repair by interacting with DNA repair proteins.

[0454] Based on these data, sleep, which is characterized by an overall reduction in neuronal metabolism and likely synaptic activity, may play a key role in neuronal genome maintenance and DNA repair functions. However, 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, Foxp2 are genes that regulate sleep. More particularly, thyrotropin-releasing hormone (Trh) and its receptor (Trhr2) regulate behavioral arousal, in part, through orexin. TRH application converts GABAergic neurons from the burst-firing mode typically associated with synchronous cortical activity that occurs during NREM sleep to the tonic, single-spike mode of action potential generation associated with desynchronized cortical activity that occurs during wakefulness and REM sleep. Downregulation of Trh and Trhr2 indicates that DORA-induced NREM sleep further interacts with the humoral regulation of sleep-wake behavior to promote sleep, particularly in the presence of tau, as these effects are absent in non-tau E4 mice. To support these findings, we observed changes in the expression of Slc22a6, Pla2g2f, Ptgdr, which regulate sleep-wake through potent endogenous hypnotics such as prostaglandins. In addition to catalyzing the biosynthesis of prostaglandins, phospholipase A2 plays a major role in cell growth differentiation and inflammation and is associated with metabolic changes in patients with obstructive sleep apnea. Similarly, genes involved in glial cell differentiation (including Tmem119, Tmem114, Cd68, Aif1, Cd300a, Pea15a, and H2-Q1 expressed by microglia) are differentially regulated in P301S / E4 mice treated with suvorexant ( Figure 5C ), indicating that promoting suvorexant-mediated NREM sleep or inhibiting orexin signaling affects important immune functions such as T cell antigen expression, response to injury, and regulation of apoptosis, all of which can directly alter tau-mediated neurodegeneration. Further supporting this principle is the transcriptional and immunohistochemical reduction of presynaptic vesicular glutamate transporter (VGLUT1, Slc17a7) and postsynaptic density markers (PSD95, Shank1, Shank2) ( Figures 5C - 5I ). Transcriptional changes in synaptic receptor activity and tissues such as Otof, Nrxn3, Mrgprf, Mapk13, and Fmod ( Figure 5C ) confirm that promoting NREM sleep or inhibiting orexin receptor signaling reduces tau-mediated neurodegeneration and associated synaptic loss.

[0455] E. Methods and Analysis

[0456] Mouse : All animal procedures and protocols were approved by the Animal Studies Committee of Washington University School of Medicine. PS19 tau transgenic mice carrying 1N4R tau and overexpressing the human P301S tau mutation were used 24. These mice were backcrossed to C57BL / 6 for more than ten generations. Human apoE4 knock-in mice were generated as described in Mol. Neurodegener. [Molecular Neurodegeneration] 14, (2019) and hybridized with P301S mice for several generations to produce 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 and under the same 12-hour light / dark cycle, ambient room temperature, and with free access to food and water.

[0457] Treatment : Starting at 7.5 months of age, mice were gavaged daily at ZT13 (one hour after the onset of darkness) with a single 30 mg / kg dose of lemborexant or 0.5% methylcellulose vehicle until euthanasia at 9.5 months of age.

[0458] Tissue collection: All mice were perfused between ZT3 and ZT7 during the time window when the mice were sleep-deprived to avoid the influence of the circadian rhythm on the transcriptional fluctuations of microglial gene expression. Before transcardial perfusion, mice were anesthetized with pentobarbital (50 mg / kg, intraperitoneal). Blood was collected from the heart before transcardial perfusion and centrifuged at 5000 x g for 5 minutes at 4 °C to obtain plasma. Mice were transcardially perfused with ice-cold phosphate-buffered saline containing 0.3% heparin. One hemisphere of the brain was dissected, snap-frozen, and stored at -80 °C for biochemical analysis. The other hemisphere of the brain was immersion-fixed in 4% paraformaldehyde for 24 hours, then cryoprotected in 30% sucrose for 48 hours and frozen at -80 °C until tissue samples were sectioned for immunohistochemical analysis.

[0459] Measurement and analysis of sleep - wake states: Electroencephalography (EEG) was used and the PiezoSleep mouse behavior tracking system (Signal Solutions) was independently used to monitor the sleep-wake behavior of the mice.

[0460] For EEG experiments, animals were anesthetized with isoflurane (0.5%-3%). Before making an incision, any signs of pain were evaluated by toe pinch. Then, a screw electrode was surgically implanted in the mouse skull for EEG and a stainless-steel wire electrode was implanted in the nuchal muscles for electromyography (EMG). After a midline vertical incision was made to expose the skull, any connective tissue was removed using forceps and 3% hydrogen peroxide and the skull was dried to place the electrodes. A drill hole for the frontal reference electrode was made using a microdrill with a 0.9 mm tip (anterior +0.5 mm, lateral ±0.5 mm; bregma) and a screw was fixed in the skull. Using the same technique as for the reference electrode, two bilateral active recording electrodes were placed on the parietal cortex (posterior -2.5 mm, lateral ±1.5; bregma) and ground screws were fixed on the cerebellum (posterior -6.2 mm, lateral ±0.5; bregma). The exposed skull, screws, and all wires were covered in a layer of dental cement (SNAP, Parkell), and the needle was fixed to the head for subsequent recording. The skin was sutured around the exposed dental cement / needle and the remaining part of the incision was closed using tissue glue (Vetbond, 3M). After this procedure, the mice were placed in a warming chamber to fully recover from anesthesia and were housed individually in a monitored cage with fresh bedding, water, food, and a carprofen (oral; 1 / 4 of a 5 g tablet; ad libitum) supplement. Three days after surgical recovery, the mice were habituated in the recording cage for two weeks, followed by two days of continuous, undisturbed EEG / EMG recording in freely moving mice. Bilateral cortical EEG signals were acquired via a P511K A.C. preamplifier (Grass-Telefactor Instruments, Warwick, RI USA), digitized with a BIOPAC MP150, digitally recorded at a sampling rate of 250 Hz using BIOPAC's AcqKnowledge software, and converted to the (.edf) format for analysis. The EEG was processed in MATLAB (MathWorks) with a band-pass filter of 1-30 Hz to remove DC offset and high-frequency noise. EEG / EMG recordings were manually scored for wakefulness, NREM, and REM sleep in 10-second epochs to create a calibration file containing subject-specific, mixed-z-score variables. The calibration file was input into the machine learning-based automated sleep scoring program AccuSleep in MATLAB to complete the remaining part of the scoring.

[0461] Use the PiezoSleep mouse behavior tracking system (Signal Solutions, LLC, Lexington, KY, USA). The non-invasive method includes a thin dielectric piezoelectric sensor pad that generates a voltage signal in response to changes in real-time fluctuations of pressure on its surface. Mice were housed individually, with the piezoelectric pad placed under fresh bedding, and had free access to fresh water and food, and were recorded undisturbed for six days. Data was acquired using SleepStats software (Signal Solutions, LLC, Lexington, KY, USA).

[0462] Volume analysis: Volume analysis of the hippocampus, entorhinal cortex / piriform cortex, and ventricles was performed via stereology by evaluating sections (16 - 18 sections per mouse depending on the severity of brain atrophy) at 180 μm intervals from -1.3 mm to -3.1 mm from bregma. Sections cut with a 30 μm microtome and fixed on slides were briefly immersed in distilled water and then incubated in pre-warmed 0.1% cresyl violet at 37°C for six minutes. After that, the tissue was rinsed in distilled water and transferred successively to 70%, 95%, and 100% ethanol for two minutes each. The slides were then cleaned in xylene and finally coverslipped with cytoseal60 mounting medium (Thermo Fisher Scientific). The slides were scanned at 20X magnification using a Hamamatsu Nanozoomer microscope. The hippocampus, EC / PC, and ventricles were traced using NDP.view 2. The volume calculation formula is volume = (sum of areas) * 0.3 mm.

[0463] Measurement of neuronal layer thickness: The dentate granule and entorhinal pyramidal cell layers were measured in three sections by using NDP.view 2 to draw scale lines across the cell layers and calculating the average for each mouse.

[0464] Immunohistochemical analysis:Free-floating sections were briefly washed with Tris-buffered saline containing 1% Triton X-100 (TBS-Tx), and then endogenous peroxidase was quenched with 0.3% hydrogen peroxide for 20 minutes at room temperature. After a brief wash, the sections were blocked with 5% goat serum for 30 minutes at room temperature, and then incubated overnight at 4 °C in biotinylated AT8 (phospho-tau Ser202, Thr205; 1:500, MN1020B, Thermo Fisher Scientific) or MC1 (1:500, kindly provided by Dr. Peter Davies). The next day, MC1-stained sections were briefly washed and incubated for one hour at room temperature in an HRP-conjugated secondary antibody. Then, AT8- and MC1-stained tissues were developed with 3,3'-diaminobenzidine (DAB, Sigma) for 10 minutes and 14 minutes, respectively. The tissue sections were mounted on slides and dehydrated using a series of increasing concentrations of ethanol, then finally immersed in xylene and coverslipped using Cytoseal mounting medium. The slides were scanned at 20X magnification using a Nanozoomer microscope from Hamamatsu.

[0465] Immunofluorescence staining:Wash the free-floating sections briefly with PBS and block them for 1 hour in 5% donkey serum at room temperature. Unless otherwise specified, dilute the primary antibodies in blocking buffer and incubate them overnight with gentle agitation at 4°C. Use the primary antibodies as follows: IBA1 (1:500; 019-19741, Fujifilm or NB100-1028, Novus Biologicals), CD68 (1:100; 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 Technology), PSD-95 (1:200, 51-6900, Thermo Fisher Scientific), VGLUT1 (1:200, AB5905, Merck Millipore). The next day, wash the sections and incubate them with secondary antibodies diluted in blocking buffer, followed by incubation with 4′,6-diamidino-2-phenylindole (DAPI, 5 μg / mL), and then mount the sections onto slides (Prolong TM Gold Antifade Reagent, Thermo Fisher Scientific).

[0466] 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 of each brain region and slide were taken at 1024×1024 pixel resolution with a 15-μm z-step thickness 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. For synapse imaging, Leica Stellaris 8 Lightning was used to generate super-resolution confocal images based on adaptive deconvolution using a 63x oil immersion objective. Image analysis was performed using Fiji (ImageJ). For quantitative feasibility, all layers from a single image stack were projected onto a single slice (Stack\Z Project). Subsequently, microglia were segmented using an automatic thresholding method in Fiji and presented as the % area covered by the selected stain in the hippocampus or entorhinal cortex / piriform cortex.

[0467] Protein extraction: The frozen mouse hippocampal tissue was weighed and homogenized in a bullet blender homogenizer (Next Advance) using bead tubes with 1x protease inhibitor cocktail (cOmplete TM, homogenized in 200 μl of RAB buffer pH 7.0 (100 mM MES, 1 mM EGTA, 0.5 mM MgSO4, 750 mM NaCl, 20 mM NaF, 1 mM Na3VO4) containing Roche's protease inhibitor cocktail and 1x phosphatase inhibitor (PhosSTOP, Roche). The homogenate was centrifuged at 5000×g for five minutes at 4 °C to pellet RAB-insoluble material, and the supernatant was ultracentrifuged at 50,000×g for 20 minutes using an MLA-130 rotor in an Optima MAX-XP ultracentrifuge (Beckman Coulter) to obtain the RAB extract. From the remaining cell pellet, proteins were extracted with RIPA buffer pH 8.0 (150 mM NaCl, 50 mM TRIS, 0.5% deoxycholic acid, 1% Triton-X 100, 0.1% sodium deoxycholate, 5 mM EDTA, 20 mM NaF, 1 mM Na3VO4) supplemented with protease and phosphatase inhibitors. After clearing RIPA-insoluble material at 5000×g for five minutes at 4 °C, the supernatant was ultracentrifuged again 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 for one minute at 30% amplitude in short pulses at room temperature using a sonicator (model FB120, Fisher Scientific), followed by a final ultracentrifugation at 50,000×g for 20 minutes at 4 °C. Protein concentrations of the RIPA fractions were measured using a BCA assay (Pierce). All samples were aliquoted and frozen at -80 °C until use.

[0468] Tau ELISA : Human tau and pTau in the RAB, RIPA, and 70% FA fractions were measured using sandwich ELISA and normalized to tissue weight as described28. The coating antibodies for total human tau and pTau were TAU-5 (mouse monoclonal antibody, 20 μg / ml) and HJ14.5 (mouse monoclonal antibody, 20 μg / ml), respectively. The capture antibodies for total human tau and pTau were HT7-biotinylated antibody (MN1000B, Thermo Fisher Scientific) and AT8-biotinylated antibody (MN1020B, Thermo Fisher Scientific), respectively.

[0469] NFL concentration: Plasma NFL concentration was measured using the NF-Light Simoa Advantage Kit with Quanterix. Measurements were performed according to the manufacturer's instructions.

[0470] RNA extraction:The frozen hippocampal tissue was weighed and homogenized in chloroform containing TRIzol in RNase-free bead tubes (REDE, Next Advance). TM The samples were centrifuged at 12,000 x g for 15 minutes 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, and then next-generation sequencing was performed by Clontech SMARTer.

[0471] RNA sequencing and analysis: Samples were prepared according to the library kit manufacturer's protocol, indexed, pooled, and sequenced on an Illumina NovaSeq 6000. Base calling and demultiplexing were performed using Illumina's bcl2fastq software and a custom Python demultiplexing program, with a maximum of one mismatch in the index reads. The 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 obtained from the number of uniquely mapped 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 evaluated against the total number of aligned reads, the total number of uniquely aligned reads, and the features detected. Ribosome scores, known junction saturation, and read distribution on known gene models were quantified using RSeQC version 4.0. Then all gene counts were input into the R / Bioconductor package EdgeR, and TMM-normalized size factors were calculated to adjust for differences in the sample library sizes. Ribosomal genes and genes not expressed in samples with a minimum size minus one count per million were excluded from further analysis. Then the TMM size factors and count matrix were input into the R / Bioconductor package Limma. Then the weighted likelihood based on the observed mean-variance relationship for each gene and sample was calculated for all samples using the voomWithQualityWeights function and fitted using the Limma generalized linear model, which has additional unknown latent effects determined by surrogate variable analysis (SVA). The performance of all genes was evaluated using a plot of the residual standard deviation for each gene against their average log counts, where the trend line of the residuals was very well-fitted. Then differential expression analysis was performed to analyze the differences between conditions, and the results were filtered only for those genes with Benjamini-Hochberg false discovery rate-adjusted p-values less than or equal to 0.05.

[0472] For each control extracted by Limma, the R / Bioconductor package GAGE9 was used to detect global perturbations in known gene ontology (GO) terms, MSigDb, and KEGG pathways to test for expression changes in the reported log2 fold changes reported by Limma in each term relative to the background log2 fold changes of all genes found outside the corresponding term. The R / Bioconductor package heatmap3 was used to display heatmaps between sample groups for each GO or MSigDb term where the Benjamini-Hochberg false discovery rate adjusted p-value was less than or equal to 0.05. The perturbed KEGG pathways were presented as annotated KEGG graphs using the R / Bioconductor package Pathview where the log2 fold changes of genes within the observed term were significantly perturbed in a single direction relative to the background or in any direction compared to other genes within a given term with a p-value less than or equal to 0.05.

[0473] To find the most critical genes, the R / Bioconductor package WGCNA was then used to analyze the Limma voomWithQualityWeights transformed log2 counts per million expression data by weighted gene co-expression network analysis. Briefly, all genes were correlated to each other by Pearson correlation and clustered into unsigned modules by expression similarity using a power threshold empirically determined from the data. Eigengenes were then created for each de novo cluster and their expression profiles were then correlated to all coefficients of the model matrix. Since these gene clusters were created by expression profiles rather than known functional similarities, these clustered modules were given random color names where grey is the only module with any pre-existing definition containing genes that did not cluster well with other genes. The functional enrichment of known GO terms for these de novo clustered genes was then tested using the hypergeometric test available in the R / Bioconductor package clusterProfiler. The significant terms with Benjamini-Hochberg adjusted p-values less than 0.05 were then collapsed by similarity into a clusterProfiler category network graph to show the most significant terms for each module of hub genes, thereby interpolating the functions of each significant module. The information of all clustered genes for each module was then combined with their respective statistical significance results from Limma to determine if these features were also found to be significantly differentially expressed.

[0474] Statistical analysis:All statistical analyses were performed using Graphpad Prism 8.0. Data are presented as mean ± SEM unless otherwise stated. The normality of the data was checked using the Shapiro-Wilk method, D’Agostino and Pearson normality tests, and KS normality test. Unless otherwise stated, statistical significance between groups with normally distributed data was calculated by unpaired T-test or two-way ANOVA followed by Tukey's post hoc test for pairwise comparisons. P < 0.05 was considered significant: *p < 0.05, **p < 0.01, and ***p < 0.001.

[0475] Example 3: Effects of Lemborexant and Doxepin in an Aβ Load Model

[0476] A. Sleep-Wake Behavior

[0477] Sleep changes were evaluated in APPswe / PS1deltaE9 (also known as “PSAPP”) mice after administration of doxepin or lemborexant. Briefly, 4- to 5-month-old PSAPP mice (mixed sex) were housed individually and their cages were placed on a piezoelectric sleep monitoring base (Adapt-A-Base) from Signal Solutions. Sleep-wake behavior was monitored for 7 days while mice were treated orally by gavage with vehicle, doxepin 35 mg / kg, or lemborexant (10 or 30 mg / kg) daily at ZT 0 (lights on).

[0478] Figure 9A A schematic of the experimental design and graphs showing the effects of doxepin and lemborexant on total sleep ( Figure 9B ), light-phase sleep ( Figure 9C ), and dark-phase sleep ( Figure 9D ) are shown. P values from one-way ANOVA are shown. Each point represents one mouse. Figure 9E Sleep percentages at time points during the day after drug injection are shown. P values are from two-way repeated measures ANOVA.

[0479] Data indicate that both lemborexant and doxepin increased total sleep time in PSAPP mice, and the effects were similar after treatment with each drug. Notably, DOX-induced sleep was spread throughout the day, while LEM-induced sleep was restricted to the light phase (the natural rest period for mice).

[0480] B. Amyloid Plaque Deposition

[0481] PSAPP mice were chronically administered lemborexant or doxepin to determine the long-term effects of each drug on amyloid plaque deposition. PSAPP mice were treated orally by gavage with the drugs 6 days per week at ZT0 (lights on) for 1.5 months.

[0482] Figure 10A Shows a schematic of the treatment times in PSAPP mice. Females develop plaques faster / younger, so males and females were staggered to allow for combination of the data. Figure 10B Shows a representative image of a brain section stained with X34, which labels fibrillar amyloid plaques. Figure 10C Shows quantification of plaque burden (X34 staining area %) in different brain regions (hippocampus, somatomotor cortex, somatosensory cortex, and piriform cortex). Error bars indicate mean ± SEM, and each point is one mouse. P values from one-way ANOVA are shown. Data indicate that long-term administration of lemborexant reduced fibrillar amyloid plaque burden in PSAPP mice, whereas doxepin did not. A dose response for the effect of lemborexant was observed.

[0483] Figure 11 shows that long-term lemborexant is more effective than doxepin in reducing total amyloid plaque burden in PSAPP mice. Figure 11A Shows a brain section 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 mean ± SEM, and each point is one mouse. P values from one-way ANOVA are shown.

[0484] Overall, the data show that lemborexant reduces total amyloid plaque burden (including diffuse and fibrillar plaques) in PSAPP mice. Doxepin also significantly reduces total amyloid burden but not fibrillar plaque burden and does not reduce it to the same extent as lemborexant (30 mg / kg dose), as only lemborexant (30 mg / kg dose) shows a significant effect in the piriform cortex.

[0485] The different responses of lemborexant compared to doxepin suggest that the effects on plaque development can be separated from the similar sleep effects induced by these two drugs.

[0486] C. Amyloid Processing

[0487] To determine whether lemborexant and doxepin affect amyloid processing, cortical lysates were obtained from PSAPP mice treated with either drug, and the levels of full-length APP and APP C-terminal fragments (CTFs) were measured by Western blot. Figure 12A Shows a representative Western blot with β-tubulin as a loading control. Figure 12B Shows quantification of band intensity. Error bars indicate mean ± SEM, and each point is one mouse. P values from one-way ANOVA are shown.

[0488] The results showed that neither lemborexant nor doxepin altered APP processing / cleavage in PSAPP mice.

[0489] D. Size of amyloid plaques and number of microglia around the plaques

[0490] After administration of lemborexant or doxepin, the number of microglia around amyloid plaques in PSAPP mice was determined. Brain sections from PSAPP mice were stained for plaques (using X34) and microglia (using Iba1). Plaques of approximately the same size in all mice were selected. The volume of microglia around each plaque was calculated from the Z-stack of confocal images using Imaris software. Figure 13A Representative images of brain sections obtained from PSAPP mice after administration of lemborexant or doxepin are shown. Figure 13B Quantification of plaque volume (indicating quantification of similarly sized plaques under different conditions) and perilesional Iba1 volume is shown. Error bars indicate mean ± SEM, and each point represents one mouse. P-values from one-way ANOVA are shown.

[0491] The results showed that the number of microglia around the plaques remained unchanged after administration of lemborexant or doxepin.

[0492] E. Phagocytic subtypes of microglia around fibrillar amyloid plaques

[0493] To determine the subtypes of microglia around fibrillar amyloid plaques, CD68 expression in microglia of PSAPP mice was determined after administration of lemborexant or doxepin. Brain sections from PSAPP mice were stained with markers for fibrillar plaques (X34), microglia (Iba1), and phagosomes (CD68). Figure 14A Representative images of brain sections are shown. Figure 14B Quantification of CD68 co-localized with Iba1 around each plaque obtained using Imaris software is shown. Error bars represent mean ± SEM, and each point is the mean of 8 - 10 plaques from a single mouse. P-values from one-way ANOVA are shown.

[0494] The results showed that administration of lemborexant, but not doxepin, increased the phagocytic subtypes of microglia around fibrillar amyloid plaques. The total microglia count remained unchanged in terms of area density, and the total number of microglia co-localized to the plaques remained unchanged. Figure 14C The Iba1+ volume is shown, Figure 14D The co-localized Iba1+ and CD68+ (as a percentage of Iba1+ staining) is shown, Figure 14ECo-localized Iba1+ and CD68+ are shown. Increased CD68 in perivascular microglia indicates increased phagocytic activation.

[0495] F. Gene expression

[0496] To identify changes in gene expression following administration of lemborexant or doxepin to PSAPP mice, qPCR arrays were performed on cortical tissue from the mice. Figure 15 Is a quantitative plot of three transcripts showing 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 metalloproteinase that has been shown to degrade Aβ. Data are shown as fold change (relative to the mean of vehicle (VEH)). Error bars indicate mean ± SEM, and each point is a single mouse. P values from one-way ANOVA are shown.

[0497] Results showed that genes associated with Aβ degradation were upregulated in PSAPP mice following administration of lemborexant. Changes in gene expression in the doxepin treatment group were not significant.

[0498] G. Number of microglia actively phagocytosing amyloid plaques

[0499] The effect of lemborexant on microglial amyloid plaque phagocytosis was evaluated in PSAPP mice. Figure 16A A schematic of the experimental design is shown (Lau et al., STAR Protoc. 2021). Briefly, 5-month-old PSAPP mice were treated daily with vehicle (Veh) or lemborexant (LEM) 30 mg / kg by oral gavage for 7 days. On day 7 after treatment, amyloid plaques were labeled in vivo via intraperitoneal (i.p.) injection of methoxy-X04 (MX04). Three hours later, the mice were sacrificed and microglia were isolated from the brain and subjected to flow cytometry. Figure 16B A flow cytometry gating strategy for isolating possible microglia is shown. After gating on side and forward scatter to identify live single cells, the CD45-low, CD11b+ population was isolated as possible microglia. Figure 16C Analysis of methoxy-X04 positivity in this cell population is shown. Figure 16D Quantification of the percentage of MX04+ microglia, indicating microglia that had phagocytosed the labeled amyloid, is shown. By two-tailed T test, P = 0.0207.

[0500] Results showed that LEM treatment acutely increased microglial amyloid plaque phagocytosis in vivo. MX04+ cells were not observed in wild-type mice lacking amyloid plaques. In contrast, 1.8% of microglia took up MX04 in vehicle-treated PSAPP mice, while 3.75% of microglia took up MX04 in the LEM-treated group.

[0501] H. Amyloid Plaque Deposition in Aged Subjects

[0502] The effect of administering Lemborexant was evaluated in aged PSAPP mice. Figure 17A A schematic of the experimental design is shown. Briefly, 9-month-old PSAPP mice with 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 via intraperitoneal (i.p.) injection of Methoxy-X04 (MX04). After 30 days of treatment, the mice were sacrificed and all plaques were labeled with Thioflavin Red, which is similar to X34 and binds to fibrillar amyloid. Plaque growth during the 30-day treatment was calculated by comparing the volume of MX04 with the volume of Thioflavin Red on a per-plaque basis. Figure 17B Representative images of MX04, Thioflavin Red, and overlay images are shown. Figure 17D A graph is shown in which at least 10 plaques were analyzed per mouse, and the mean for each mouse is shown as a circle on the graph. The LEM-treated group showed a trend towards reduced plaque growth during the 30-day treatment. Due to the non-Gaussian distribution of the data, the P value was from a Mann-Whitney U test. Figure 17C Representative images of amyloid plaques labeled with X34, microglia labeled with IBA1, and microglial phagosomes labeled with CD68 are shown. The co-localization volume of IBA1-CD68 within a 20-μm sphere of each amyloid plaque was calculated to determine perilesional microglial CD68 expression. Figure 17E Quantification of co-localized IBA1-CD68 is shown as a percentage of the total IBA1 (total microglia) area. Each circle represents the mean for a single mouse, and 10 plaques were quantified per mouse. In Figure 17D and Figure 17E comparisons between the VEH and LEM treatment groups were analyzed by two-tailed t test.

[0503] The effect of administering Doxepin was also evaluated in aged PSAPP mice. Neither Lemborexant nor Doxepin administration resulted in a significant change in the total number of plaques in aged mice. Similar non-significant changes were observed in plaque volume, IBA1+ cells, IBA1 volume, and co-localization of IBA1-CD68.

[0504] Finally, the volume of dystrophic neurites around amyloid plaques was quantified by measuring increased BACE1 at the presynaptic terminal. Administration of either lemborexant or doxepin did not result in a significant change in the volume of dystrophic neurites.

[0505] Figure 17F and Figure 17G show the effects of doxepin administration on plaque growth and phagocytic microglia around amyloid plaques and data for lemborexant administration. In Figure 17F and Figure 17G , comparisons of the VEH, DOX, and lemborexant treatment groups were analyzed by one-way ANOVA.

[0506] Overall, the results showed that lemborexant and doxepin each showed a tendency to slow amyloid plaque growth in aged mice with pre-existing plaques. Each drug increased phagocytic microglia around amyloid plaques in aged mice. Lemborexant, but not doxepin, also showed this effect on phagocytic microglia in young mice. The effects of lemborexant and doxepin on clearing established plaques in aged mice were less than their effects on preventing plaque accumulation in young mice.

[0507] I. Methods

[0508] J. Animals

[0509] Male and female APPswe / PS1deltaE9 (PSAPP) mice were used for all experiments.

[0510] APP / PS1 is a double transgenic mouse expressing the chimeric mouse / human amyloid precursor protein (Mo / HuAPP695swe) and mutant human presenilin 1 (PS1-dE9), both proteins being targeted to CNS neurons.

[0511] K. Methods for Measuring Sleep-Wake Behavior

[0512] VEH, LEM (10 mg / kg / day or 30 mg / kg / day), or DOX (35 mg / kg / day) was orally administered to 5-month-old APPswe / PS1deltaE9 (PSAPP) mice at ZT0 daily for 6 days. The sleep and wake states were determined using a non-invasive piezoelectric system, Adapt-A-Base (Signal Solutions). Mice were individually housed in cages placed on piezoelectric sensor bases within a soundproof and light-tight cabinet (Circadian cabinet, ClockLab) and were recorded without perturbation for 7 days. At the start of the recording, each cage was equipped with 160 g of corncob bedding, 220 g of food pellets, and 380 mL of water such that each cage had the same weight except for the minor variability in mouse body weight. The sleep-wake states were analyzed using SleepStats software (Signal Solutions). Sleep bout lengths were scored using 30-second epochs according to the manufacturer's default settings in the current version of SleepStats software.

[0513] L. Method for evaluating the effect of long-term administration of lemborexant or doxepin on amyloid plaque deposition

[0514] VEH, LEM (10 mg / kg / day or 30 mg / kg / day), or DOX (35 mg / kg / day) was orally (p.o.) administered to young PSAPP mice (3-month-old females and 3.5-month-old males) at ZT0 daily for 6 weeks. Subsequently, female and male mice were perfused at 4.5 months and 5 months, respectively. Brains were extracted and processed for IHC / IF, transcriptomics, or proteomics analysis.

[0515] M. Method for evaluating the effect of lemborexant and doxepin on microglial Aβ phagocytic activity

[0516] VEH or LEM (30 mg / kg / day) was administered to 5-month-old PSAPP mice at ZT0 daily for 7 days. On day 7, mice were intraperitoneally injected with methoxy-X-04 (MX-04, 10 mg / kg) to label the plaques in vivo. Mice were perfused 3 hours after MX04 injection, and the brains were processed for flow cytometry to estimate MX-04-positive microglia.

[0517] N. Method for evaluating the effect of LEM on amyloid plaque deposition in aged PSAPP mice

[0518] 9-month-old PSAPP mice were injected intraperitoneally (i.p.) with methoxy-X-04 (MX-04, 10 mg / kg) to label plaques in vivo, and VEH or LEM (30 mg / kg / day) was administered orally at ZT0 daily for 4 weeks. At 10 months, the mice were perfused, the brains were extracted and processed for IHC / IF analysis. Fixed brain sections were stained with thioflavin S and compared with MX-04 staining to estimate plaque growth.

[0519] O. Drugs

[0520] Mice were orally administered vehicle (VEH), doxepin (DOX, Cayman Chemical #15888, dissolved in PBS), or suvorexant (LEM, suspended in 0.5% methylcellulose) at ZT0 (lights on). VEH mice were given a similar volume of vehicle daily. Immediately before gavage, the tip of a 22-gauge orogastric feeding needle was dipped into 100% sucrose solution. Intraperitoneal injection of methoxy-X-04 (Tocris #4920, 10 mg / kg) was performed for the timestamp experiment and analysis of microglial phagocytic activity.

[0521] P. IHC / IF:

[0522] All mice were perfused between ZT5 and ZT7 (1100 and 1300 h). Mice were deeply anesthetized intraperitoneally with pentobarbital (150 mg / kg) and then perfused transcardially with ice-cold Dulbecco's modified PBS (DPBS) containing 3 g / l heparin. The brains were carefully extracted and the left hemisphere was post-fixed in 4% paraformaldehyde for 48 h (4°C) and then cryoprotected in a PBS solution of 30% sucrose (4°C) for 24 h. The brains were then sectioned into 40-μm serial coronal sections on a cryostat (SM1020R; Leica) and stored in cryoprotectant solution (30% ethylene glycol, 15% sucrose, 15% phosphate buffer in ddH20). For biochemical analysis, the right hemisphere was dissected to isolate the cortex and hippocampus, snap-frozen, and kept at -80°C until analysis.

[0523] Stain the fibrillar Aβ with X-34 dye (SML-1954, 1:5,000; MilliporeSigma) or thiazine red. For X-34 staining, wash the free-floating sections three times for 5 minutes each in PBS, then permeabilize in 0.25% Triton X-100 PBS (PBS-X) for 30 minutes. Then incubate the tissue sections in X34-0.1M NaOH for 20 minutes, wash in X34 buffer (PBS solution with 40% EtOH), and then wash twice in PBS. For staining of total Aβ (HJ3.4 biotinylated, anti-Aβ1-13, mouse monoclonal antibody, 1:1,000, 2.81 μg / mL; generated in-house), wash the sections three times in TBS and then incubate in 0.3% hydrogen peroxide for 10 minutes. Wash the sections three more times in TBS and then block in 3% milk diluted in TBS+0.25% Triton X-100 for 30 minutes. Incubate the sections overnight at 4°C in biotinylated HJ3.4 in TBS+0.25% Triton X-100+1% milk. The next day, wash the sections and then develop for 60 minutes using ABC Elite (Vector PK-6100). Then incubate the sections in 3,3-diaminobenzidine (DAB, Sigma-Aldrich) as the chromogen and 0.05% hydrogen peroxide as the substrate and dehydrate before covering with a coverslip using Cytoseal 60 (8310; Thermo Fisher Scientific).

[0524] For immunofluorescence (IF) staining with IBA1 (goat, Abcam ab5076, 1:500) or CD68 (rat, BioRad MCA1957, 1:500), wash the sections three times in TBS, block in TBSX (TBS+0.4% Triton X-100) containing 3% donkey serum for 60 minutes, and incubate overnight at 4°C in the primary antibody diluted in TBSX containing 1% donkey serum. Then incubate the sections at room temperature in PBSX (or TBSX) with a 1:1000 donkey fluorescent secondary antibody for 1 hour. Mount the sections, seal in Fluoromount-G (0100-01; SouthernBiotech), and store at 4°C in the dark until imaging.

[0525] Q. Imaging

[0526] Epifluorescence imaging: All fluorescence imaging was performed on a Keyence BZ-X810 microscope. Typically, after examining the tissue, the laser intensity and exposure time were selected for the samples of each cohort to select appropriate parameters, which could then remain constant for all slides during that imaging session. These values varied with the antibody, but all sections within a given cohort were imaged at the same magnification under the same conditions. Images were processed using the BZ-X800 analyzer program (Keyence Corp.) and quantified using Fiji version 2.1.0 (NIH). All areas were quantified in 2-3 sections per mouse.

[0527] Confocal imaging and analysis: Plaques located in the cortical gray matter and completely contained within the section 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 (v 3.7, blue edition). Uniform pinhole, laser power, and PMT detector gain settings were used in all experiments. For all analyses, at least 2 images were taken of each brain region and slide at a minimum resolution of 1,024 × 1,024 using a 40× oil immersion objective. Confocal images of the IBA1 and CD68 volumes around X34+ plaques were quantified on a semi-automated platform using MATLAB and Imaris 10.0.1 software (Bitplane). To create a surface for each stain based on a threshold applied to all images, the X34+ surface was expanded by 20 μm and co-localized with the surfaces of the various immunostains. The IBA1+ and CD68+ surface areas were then co-localized 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 spots to each cell body or puncta. The X34 surface was expanded to 20 μm, and the spots within the expanded X34+ surface were counted. Any spots that were completely within the extended surface or partially in contact with the extended surface were included in the analysis.

[0528] R. Western Blot

[0529] Tissue samples were homogenized by sonication in ice in radioimmunoprecipitation (RIPA) buffer (Pierce, Thermo Scientific) containing complete protease inhibitor and PhosSTOP phosphatase inhibitor (Roche). PAGE and western blot analysis were performed using Invitrogen Novex gels and reagents. Bands were visualized using Lumigen TMA-6 chemiluminescent reagent on an iBright CL1500 imaging system (Thermo). Band intensities were quantified using Fiji software (NIH) and normalized relative to the β-tubulin loading control.

[0530] S. Statistics

[0531] Statistical tests were performed and graphs were plotted using GraphPad Prism software version 9.0.1. Power analysis was used to determine the variability of the outcome measures and to estimate the number of observations required to test the null hypothesis. All graphs show the mean (using bar graphs), individual data points within the bars, and error bars depicting the SEM. For Experiments 1 and 2, one-way ANOVA was performed. If the main effect was significant, the following post hoc multiple comparison tests were performed: Tukey (for equal group sizes) or Tukey-Kramer (for unequal group sizes). For Experiments 3 and 4, an F-test was first performed on the dataset with a single dependent variable and 2 groups to determine if the variances were significantly different. If not significantly different, a two-tailed unpaired t-test was performed. If the variances were different, a non-parametric Mann-Whitney U test was performed. Grubbs test was used to identify and exclude outliers. All P values are indicated in the graphs.

[0532] Example 4: Effect on the rhythmic activity pattern

[0533] The effect of administering suvorexant on the rhythmic activity pattern was evaluated in arrhythmic Bmal1 knockout (KO) mice in order to assess the restorative effect of suvorexant on mice lacking a functional clock. Bmal1 KO mice lack a functional clock and thus lose their 24-hour rhythm in constant darkness (DD condition). The arrhythmic behavior of Bmal1 KO mice is masked in the normal 12-hour light:12-hour dark cycle (L:D condition).

[0534] Control (Cre-) or Bmal1 KO (CAG-CreERT2; Bmal1(f / f)) mice were treated with tamoxifen to ablate Bmal1. One month later, infrared activity recordings were made under 12h:12h L:D conditions (yellow area) or constant darkness (DD condition). At 6 am (previous ZT 0), a dose of 30 mg / kg of lemborexant (LEM) was administered to the mice via oral gavage for 9 days (indicated by red bars and red arrows). Activity recordings were collected for an additional 2 weeks after drug administration was stopped. Figure 18A Representative activity plots of control and Bmal1 knockout mice are shown. Figure 18B Is the quantification of the endpoints of the activity plots during different parts of the experiment (in Figure 18A , LD indicates during the yellow area, DD+LEM is the area with the red bar, and DD is the rest of the recording). Data were analyzed by two-way ANOVA and Tukey's post hoc test.

[0535] The results showed that under all conditions (LD, DD+LEM, and DD), Bmal1 knockout mice showed greater daytime activity than wild-type mice. Bmal1 KO mice, when they were given lemborexant daily at CT0, were able to maintain locomotor activity similar to LD under DD conditions (CT0 is similar to ZT0 under DD conditions). Under all conditions, the relative amplitude was lower in Bmal1 knockout mice, and the relative amplitude indicates the ratio of the average activity during the 10 most active hours to the average activity during the 5 least active hours. Generally, a higher relative amplitude is associated with a stable rhythm. Under all conditions, the interday stability (IS) that measures the synchronization between daily 24-hour rhythms was lower in Bmal1 knockout mice. A high IS indicates good synchronization of the rhythm.

[0536] Equivalent forms and ranges

[0537] Those skilled in the art will recognize, or be able to determine using only routine experimentation, many equivalent forms of specific embodiments of the particular embodiments provided herein. The scope of this disclosure is not intended to be limited to the above specific embodiments, but rather as set forth in the appended claims.

[0538] When ranges are given, endpoints are included. In addition, it should be understood that unless otherwise indicated or otherwise obvious from the context and the understanding of one of ordinary skill in the art, values expressed as ranges may be assumed to be any specific value or subrange within the stated range in different embodiments of this disclosure, to one-tenth of the unit of the lower limit of the range.

[0539] 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. Since such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if such exclusion is not expressly set forth herein. Any specific embodiment of the compositions of the present disclosure (e.g., any composition, therapeutic or active ingredient; any production method; any method of use; etc.) may be excluded from any one or more of the claims for any reason, whether or not it involves the existence of prior art.

[0540] It should be understood that the words that have been used are descriptive words rather than restrictive words and may vary within the scope of the appended claims without departing from the true scope and spirit of the present disclosure in its broader aspects.

[0541] Although the present disclosure has been described with a certain degree of particularity with respect to several described embodiments, it is not intended that the present disclosure be limited to any such details or embodiments or any particular embodiment, but rather it is to be construed with reference to the appended claims so as to provide the broadest possible interpretation of such claims in light of the art to effectively cover the intended scope of the present disclosure.

[0542] 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) shall control. Additionally, the section headings, materials, methods, and examples are illustrative only and not intended to be restrictive.

Claims

1. A method for treating Alzheimer's disease (AD) in a subject having 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, thereby treating AD.

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

3. The method according to claim 1, wherein treating AD comprises affecting a change (e.g., slowing, delaying, or reducing) in at least one biomarker of AD pathology.

4. The method according to claim 3, wherein the biomarker is the phosphorylation level of tau, neurodegeneration, a change in microglial response, and / or the presence of Aβ plaques.

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

6. The method according to claim 5, wherein the brain region is the hippocampus, somatic motor cortex, somatic sensory cortex, piriform cortex, and / or entorhinal cortex.

7. The method according to claim 4, wherein the biomarker is detected in a body fluid of the subject.

8. The method according to claim 7, wherein the body fluid is blood or cerebrospinal fluid (CSF).

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

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

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

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

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

14. The method according to claim 12, wherein the subject has a moderate level of amyloid PET (e.g., 20 - 40 percentile units).

15. The method according to claim 12, wherein the subject has an elevated level of amyloid PET (e.g., > 40 percentile units).

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

17. The method according to claim 16, wherein the subject has early AD.

18. The method according to claim 16, wherein the subject has pre - AD.

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

20. The method according to claim 19, wherein the subject is amyloid negative.

21. The method according to claim 19, wherein the tau level is reduced or maintained relative to a reference.

22. The method according to claim 19, wherein the method comprises reducing and / or delaying tau accumulation and / or tau spreading, and / or slowing its rate, as compared to a reference.

23. The method according to claim 22, wherein the reference is a baseline measurement from the subject prior to treatment.

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

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

26. The method according to claim 19, wherein the method comprises altering tau in a brain region of the subject.

27. The method according to claim 19, wherein the method comprises altering the tau PET signal in a brain region of the subject.

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

29. The method according to claim 19, wherein the method comprises reducing tau in a body fluid of the subject.

30. The method according to claim 29, wherein the body fluid is blood or CSF.

31. The method according to claim 19, wherein the tau is total tau.

32. The method according to claim 19, wherein the tau is insoluble tau.

33. The method according to claim 19, wherein the tau is aggregated tau.

34. The method according to claim 19, wherein the tau is a phosphorylated form of tau (phosphorylated tau).

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

36. The method according to claim 34, wherein the method comprises altering the ratio of phosphorylated tau to total tau.

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

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

39. The method according to claim 34, the method comprises increasing the rate of dephosphorylation of phosphorylated tau.

40. The method according to claim 34, the method comprises reducing the rate of phosphorylation of tau.

41. The method according to claim 19, the method comprises reducing or maintaining tau within 48 hours of administering a first dose of lemborexant.

42. The method according to claim 34, wherein the method comprises reducing phosphorylated tau in the hippocampus, entorhinal cortex, and / or piriform cortex.

43. A method of altering neurodegeneration (e.g., reducing and / or delaying neurodegeneration, and / or slowing its rate) in a subject having 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 alter neurodegeneration in the subject.

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

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

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

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

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

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

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

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

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

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

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

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

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

57. A method of altering Aβ plaques (e.g., reducing or delaying Aβ plaque formation, and / or slowing their growth rate) in a subject having 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 alter Aβ plaques in the subject.

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

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

60. The method according to claim 58, wherein the reference is a baseline measurement from the subject prior to treatment.

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

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

63. The method according to claim 57, wherein the Aβ plaque is a fibrillar plaque.

64. The method according to claim 57, wherein the Aβ plaque is all plaques.

65. The method according to claim 57, wherein altering the Aβ plaque comprises reducing the growth of the Aβ plaque.

66. The method according to claim 65, the method comprising reducing the growth of Aβ plaques in the hippocampus of the subject, the primary motor cortex of the subject, the somatosensory cortex, and / or the piriform cortex.

67. The method according to claim 57, wherein altering the Aβ plaque comprises altering the amyloid PET signal obtained from a brain region of the subject.

68. The method according to claim 57, wherein altering the Aβ plaque corresponds to a decrease in the concentration of Aβ in the CSF of the subject.

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

70. The method according to claim 57, the method comprising altering the Aβ plaque within 48 hours of administering a first dose of suvorexant.

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

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

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

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

75. The method according to claim 73, wherein the subject has a moderate level of amyloid PET (e.g., 20 - 40 percentile units).

76. The method according to claim 73, wherein the subject has an elevated level of amyloid PET (e.g., >40 percentile units).

77. The method according to claim 57, wherein the subject has early AD.

78. The method according to claim 57, wherein the subject has pre - AD.

79. A method of modulating the microglial response 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 suvorexant, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the therapeutically effective amount is sufficient to modulate the microglial response of the subject.

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

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

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

83. The method according to claim 80, wherein the microglial marker is a homeostatic microglial marker.

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

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

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

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

88. The method according to claim 79, wherein the subject is amyloid negative.

89. The method according to claim 79, wherein the subject has tau pathology.

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

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

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

93. The method according to claim 88, wherein modulating the microglial response comprises modulating the response in microglia associated with degenerating neurons.

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

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

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

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

98. The method according to claim 79, wherein the subject has Aβ plaques.

99. The method according to claim 98, wherein the Aβ plaques are fibrillar Aβ plaques.

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

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

102. The method according to claim 100, wherein the subject has a moderate level of amyloid PET (e.g., 20 - 40 percentile units).

103. The method according to claim 100, wherein the subject has an elevated level of amyloid PET (e.g., >40 percentile units).

104. The method according to claim 98, wherein the subject has early AD.

105. The method according to claim 98, wherein the subject has pre-AD.

106. The method according to claim 98, wherein the Aβ plaques are present in the hippocampus, somatic motor cortex, somatic sensory cortex, and / or piriform cortex.

107. The method according to claim 98, wherein modulating the microglial response comprises modulating the response in microglia associated with Aβ plaques.

108. The method according to claim 107, wherein modulating the microglial response comprises increasing the expression of general microglial markers.

109. The method according to claim 108, wherein the general microglial markers are Iba1, Clec7a, or CD68.

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

111. The method according to claim 107, wherein modulating the microglial response comprises reducing the expression of homeostatic microglial markers.

112. The method according to claim 111, wherein the homeostatic microglial markers are TMEM119 or P2RY12.

113. The method according to any one of claims 1-112, wherein the therapeutically effective amount of suvorexant administered to the subject is in the range of 5 mg to 50 mg per day.

114. The method according to claim 113, wherein the therapeutically effective amount of suvorexant administered to the subject is in the range of 10 mg to 30 mg per day.

115. The method according to claim 113, wherein the therapeutically effective amount of suvorexant 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. The method according to claim 113, wherein the therapeutically effective amount of suvorexant administered to the subject is 20-25 mg per day.

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

118. The method according to claim 113, wherein suvorexant is administered in a first dose for a first cycle, in a second dose for a second cycle, and optionally in a third dose for a third cycle.

119. The method according to claim 118, wherein each of the first cycle, the second cycle, and the third cycle is 1 week.

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

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

122. The method according to claim 120, wherein 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.

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

124. The method according to claim 123, wherein 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.

125. The method according to claim 123, wherein 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.

126. The method according to any one of claims 1 - 125, the method comprising administering lemborexant to the subject for at least 6 months.

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

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

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

130. A method of 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 a measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque load, microglial response and biomarker expression from the subject; (b) comparing the measurement from the subject with a measurement from a reference; and (c) selecting the subject for treatment with lemborexant if the measurement from the subject is different from the measurement from the reference.

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

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

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

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

135. The method according to claim 133, wherein the subject has a moderate level of amyloid PET (e.g., 20 - 40 units on a percentile scale).

136. The method according to claim 133, wherein the subject has an elevated level of amyloid PET (e.g., > 40 units on a percentile scale).

137. The method according to claim 130, wherein the subject has early AD.

138. The method according to claim 130, wherein the subject has pre - AD.

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

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

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

142. The method according to claim 140, wherein the biological sample is a body fluid.

143. The method according to claim 140, wherein the body fluid is cerebrospinal fluid (CSF), blood, or saliva.

144. The method according to claim 130, wherein the reference is a control.

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

146. The method according to claim 144, wherein the control does not have AD.

147. The method according to claim 146, wherein the measurement from the subject is higher than the measurement from the control that does not have AD.

148. The method according to claim 146, wherein the measurement from the subject is lower than the measurement from the control that does not have AD.

149. The method according to claim 144, wherein the control has AD.

150. The method according to claim 149, wherein the measurement from the subject is similar to or higher than the measurement from the control that has AD.

151. The method according to claim 149, wherein the measurement from the subject is similar to or lower than the measurement from the control that has AD.

152. The method according to claim 130, wherein the measurement of tau phosphorylation includes the measurement of phosphorylation at one or more of T181, T217, S202, S205, or T231.

153. The method according to claim 130, wherein the measurement of tau aggregation includes the measurement of insoluble tau aggregates (e.g., neurofibrillary tangles (NFT)).

154. The method according to claim 130, wherein the measurement of neurodegeneration includes the measurement of cortical thickness and / or hippocampal volume or the measurement of pyramidal neuron or granule neuron loss.

155. The method according to claim 130, wherein the measured value of Aβ plaque burden comprises a measured value of Aβ plaque volume and / or a measured value of Aβ plaque volume growth.

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

157. The method according to claim 130, wherein the measured value of microglial response is a change in the expression of at least one microglial marker.

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

159. The method according to claim 157, wherein the measured value of microglial response is a measured value of microglial phagocytosis.

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

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

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

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

164. The method according to claim 162, wherein the biological sample is a body fluid.

165. The method according to claim 164, wherein the body fluid is cerebrospinal fluid (CSF), blood, or saliva.

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

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

168. The method according to claim 160 or claim 161, wherein the measurement value of tau phosphorylation includes the measurement value of phosphorylation of one or more of T181, T217, S202, S205, or T231.

169. The method according to claim 160 or claim 161, wherein the measurement value of tau aggregation includes the measurement value of insoluble tau aggregates (e.g., neurofibrillary tangles (NFT)).

170. The method according to claim 160 or claim 161, wherein the measurement value of neurodegeneration includes the measurement value of cortical thickness and / or hippocampal volume or the measurement value of pyramidal neuron or granule neuron loss.

171. The method according to claim 160 or claim 161, wherein the measurement value of Aβ plaque load includes the measurement value of Aβ plaque volume and / or the measurement value of Aβ plaque volume growth.

172. The method according to claim 160 or claim 161, wherein the measurement value of Aβ plaque load includes the measurement value of amyloid PET signal in the brain region of the subject or the measurement value of Aβ in the CSF of the subject.

173. The method according to claim 160 or claim 161, wherein the measurement value of microglial response is the measurement value of the expression of at least one microglial marker.

174. The method according to claim 173, wherein the microglial marker is Iba1, Clec71, P2RY12, or TMEM 119.

175. The method according to claim 173, wherein the measurement value of microglial response is the measurement value of microglial phagocytosis.

176. The method according to claim 160 or claim 161, wherein the measurement value of biomarker expression is the measurement value of Ifnb1, MMP2, and / or Bace1 expression.

177. The method according to claim 160 or claim 161, wherein the subject is amyloid negative.

178. The method according to claim 160 or claim 160, wherein the subject has Aβ plaques.

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

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

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

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

183. The method according to claim 181, wherein the subject has a moderate level of amyloid PET (e.g., 20 - 40 percentile units).

184. The method according to claim 181, wherein the subject has an elevated level of amyloid PET (e.g., >40 percentile units).

185. The method according to claim 160 or claim 161, wherein the subject has early AD.

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

Images