TW202425995A - Methods of reducing neurodegeneration associated with neurodegenerative diseases - Google Patents

Abstract

The disclosure relates to lemborexant, a dual orexin receptor antagonist, and compositions and methods for use in treatment of Alzheimer’s disease (AD), e.g., in a subject who has AD or who is at risk for developing AD.

Description

Methods of reducing neurodegeneration associated with neurodegenerative diseases

Described herein are compositions and methods involving the dual alpha-sensitizer receptor antagonist leborexant for use in treating neurological diseases such as Alzheimer's disease (AD).

Alzheimer's disease (AD) is a progressive, neurodegenerative disease of unknown etiology and the most common form of dementia among the elderly. In 2006, there were 26.6 million cases of AD worldwide (range: 11.4–59.4 million) (Brookmeyer, R. et al., Forecasting the global burden of Alzheimer's Disease. Alzheimer Dement. 2007; 3:186-91), and more than 5 million people in the United States were reported to have AD (Alzheimer's Association, Alzheimer's Association report, 2010 Alzheimer's disease facts and figures. Alzheimer Dement. 2010; 6:158-94). By 2050, the prevalence of AD is projected to grow to 106.8 million worldwide (range: 47.2–221.2 million), with an estimated prevalence of 11–16 million in the United States alone (Brookmeyer, supra, and 2010 Alzheimer's disease facts and figures, supra).

The disease usually involves an overall decline in cognitive function that slowly progresses and renders terminally ill subjects bedridden. AD subjects typically survive only 3 to 10 years after symptom onset, although survival extremes of 2 and 20 years are known. (Hebert, LE, et al., Alzheimer disease in the US population: prevalence estimates using the 2000 census. Arch Neurol. 2003;60:1119-1122.) Despite the fact that death certificates rarely attribute death to AD, deaths due to AD are greatly underestimated, AD is 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.

AD represents a heavy economic burden for industrialized countries, with significant consequences for health care systems and public coffers, as well as for patients and their families. In the United States alone, total costs were estimated at $172 billion in 2010, including $123 billion for Medicare and Medicaid benefits.

Histologically, the disease is characterized by neuroinflammatory plaques, which are mainly found in the synaptonemal cortex, limbic system, and basal ganglia. The main component of these plaques is the amyloid-like beta peptide (Aβ). Aβ exists in various conformational states: monomers, oligomers, protofibrils, and insoluble protofibrils. The details of the mechanistic relationship between the onset of Alzheimer's disease and the production of Aβ are still unclear. However, some anti-Aβ antibodies are currently being clinically studied as potential therapeutic agents for Alzheimer's disease.

In addition to neuroinflammatory plaques, the disease is characterized by tau aggregation and hyperphosphorylation, increased immune response, neuronal degeneration, synaptic loss, and ultimately cognitive impairment, dementia, and death.

Insomnia has been implicated as a risk factor for AD. Historically, insomnia has been treated with a variety of medications, including doxepin, tricyclic antidepressants (TCAs), and dual DORAs such as suvorexant or lemborexant. Although the literature has suggested a link between sleep disturbances and AD risk, a direct link has not been established. Indicating the need for further elucidation of the link between insomnia and AD, data disclosed herein suggest that at least two such sleep medications, doxepin and lemborexant, have differential effects on AD pathology, despite both mediating sleep.

Lebrexan has been approved for the treatment of adult patients with insomnia characterized by difficulty falling asleep and/or maintaining sleep. Lebrexan and methods of use thereof 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.

Labreson has the following structure: It is also called (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.

Therefore, there remains a need for improved AD treatments, including early intervention before irreversible symptoms of the disease develop. The disclosures herein provide striking evidence that Lebrexan may be useful for such treatments.

One aspect of the present disclosure relates to 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 leboraxant, a pharmaceutically acceptable salt thereof, or a solvate thereof, thereby treating AD.

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

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

In some embodiments, the subject shows no signs of dementia and/or cognitive impairment. In some embodiments, the subject has mild cognitive impairment or mild dementia.

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

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.

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

In some embodiments, the subject is amylin negative. In some embodiments, the tau level is reduced or maintained relative to a reference. In some embodiments, the method includes reducing and/or delaying tau aggregation and/or tau diffusion, 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.

In some embodiments, the method comprises altering tau in a brain region of the subject. In some embodiments, the method comprises altering tau PET information 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.

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 a phosphorylated form of tau (phosphorylated tau). In some embodiments, the phosphorylated tau is phosphorylated on one or more of T181, T217, S202, S205, or T231.

In some embodiments, the method includes changing the ratio of phosphorylated tau to total tau. In some embodiments, the ratio of phosphorylated tau to total tau is reduced compared to the ratio of phosphorylated tau to total tau in the CSF of the subject before the administration of leboraxan. 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 before the administration of leboraxan. In some embodiments, the method includes increasing the dephosphorylation rate of phosphorylated tau. In some embodiments, the method includes reducing the phosphorylation rate of tau. In some embodiments, the method includes reducing or maintaining tau within 48 hours of administering a first dose of leboraxan. In some embodiments, the method includes reducing phosphorylated tau in the hippocampus, entorhinal cortex, and/or piriform cortex.

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 lebrexan, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the therapeutically effective amount is sufficient to alter the neurodegeneration in the subject.

In some embodiments, the subject is amylin 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 of the subject before treatment. In some embodiments, the reference is a baseline measurement of a control subject. In some embodiments, the reference is a measurement of a control subject administered a placebo.

In some embodiments, the neurodegeneration is characterized by a 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 reducing the loss of cortical thickness and/or hippocampal volume. In some embodiments, the neurodegeneration is characterized by a loss of at least one of pyramidal neurons in the cortex, pyramidal neurons in the hippocampus, or granule cells in the hippocampus. 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 reducing the rate of neurodegeneration. In some embodiments, altering neurodegeneration comprises altering neurofilament light chain (NfL) levels. In some embodiments, the method comprises altering the NfL level in the blood and/or CSF of the subject.

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 leboraxant, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the therapeutically effective amount is sufficient to alter Aβ plaques in the subject.

In some embodiments, the Aβ plaques are altered relative to a reference. In some embodiments, altering Aβ plaques includes reducing and/or delaying the formation of Aβ plaques, and/or slowing its rate, as 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.

In some embodiments, the Aβ plaques are primitive 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 subject's hippocampus, the subject's somatomotor cortex, somatosensory cortex, and/or piriform cortex.

In some embodiments, altering Aβ plaques comprises altering amyloid protein PET signals obtained from a brain region of the subject. In some embodiments, altering Aβ plaques corresponds to a decrease in Aβ concentration in the CSF of the subject.

In some embodiments, the Aβ is Aβ38, Aβ40 and/or Aβ42. In some embodiments, Aβ plaques are altered within 48 hours of administration of the first dose of leboraxan.

In some embodiments, the subject shows no signs of dementia and/or cognitive impairment. In some embodiments, the subject has mild cognitive impairment or mild dementia.

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

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

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

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

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.

In some embodiments, the subject is amylin 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.

In some embodiments, modulating the small neuroglia response comprises modulating a response in small neuroglia associated with degenerating neurons. In some embodiments, modulating the small neuroglia response comprises reducing the expression of at least one general small neuroglia marker. In some embodiments, the general small neuroglia marker is Iba1, CD68, or Clec7a. In some embodiments, modulating the small neuroglia response comprises increasing the expression of at least one homeostatic small neuroglia marker. In some embodiments, the homeostatic small neuroglia marker is TMEM119 or P2RY12.

In some embodiments, the subject has Aβ plaques. In some embodiments, the Aβ plaques are fibrillar Aβ plaques. In some embodiments, the subject is at risk for further Aβ accumulation. In some embodiments, the subject is an ApoE4 carrier. In some embodiments, the subject has moderate levels of amyloid protein PET (e.g., 20-40 percentile units). In some embodiments, the subject has elevated levels of amyloid protein PET (e.g., >40 percentile units).

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

In some embodiments, the Aβ plaques are present in the hippocampus, motor cortex, somatosensory cortex, and/or piriform cortex.

In some embodiments, modulating the microglial response comprises modulating the response in microglial cells associated with Aβ plaques. In some embodiments, modulating the microglial response comprises 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 comprises increasing the phagocytosis of Aβ plaques by phagocytic microglial cells. In some embodiments, modulating the microglial response comprises decreasing the expression of homeostatic microglial markers. In some embodiments, the homeostatic microglial cell marker is TMEM119 or P2RY12.

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

In some embodiments, the therapeutically effective amount of leboraxant 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.

In some embodiments, the therapeutically effective amount of lebrexan administered to the subject is 20-25 mg per day. In some embodiments, a dose of 25 mg of lebrexan is administered to the subject once daily.

In some embodiments, Lebrexan is administered at a first dose for a first cycle, at a second dose for a second cycle, and optionally at a third dose for a third cycle. 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.

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

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 lebrexan once a day, the second dose is 10 mg of lebrexan once a day, and optionally, the third dose is 5 mg of lebrexan once a day. In some embodiments, the first dose is 20 mg, 22.5 mg, 25 mg, 27.5 mg, or 30 mg of lebrexan once a day, the second dose is 10 mg, 12.5 mg, 15 mg, or 17.5 mg of lebrexan once a day, and optionally, the third dose is 5 mg or 7.5 mg of lebrexan once a day.

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

Another aspect of the disclosure relates to a method of selecting a subject having Alzheimer's disease (AD) or at risk of developing AD for treatment with lebrexan, 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 cell response, and biomarker expression from the subject; (b) comparing the measurement from the subject to a measurement from a reference; and (c) selecting the subject for treatment with lebrexan if the measurement from the subject is different from the measurement from the reference.

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.

In some embodiments, the subject is at risk for Aβ accumulation. In some embodiments, the subject is an ApoE4 carrier. In some embodiments, the subject has moderate levels of amyloid protein PET (e.g., 20-40 percentile units). In some embodiments, the subject has elevated levels of amyloid protein PET (e.g., >40 percentile units).

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

In some embodiments, the subject has been diagnosed with AD based on brain imaging, cognitive function, and/or biomarker criteria. 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 cell response. In some embodiments, the biological sample is a body fluid. In some embodiments, the body fluid is cerebrospinal fluid (CSF), blood, or saliva.

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

In some embodiments, the control does not have AD. In some embodiments, the measured value from the subject is higher than the measured value from the control that does not have AD. In some embodiments, the measured value from the subject is lower than the measured value from the control that does not have AD.

In some embodiments, the control has AD. In some embodiments, the measurement from the subject is similar to or higher than the measurement from the control with AD. In some embodiments, the measurement from the subject is similar to or lower than the measurement from the control with AD.

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

In some embodiments, measures of neurodegeneration include measures of cortical thickness and/or hippocampal volume or measures of pyramidal or granule neuron loss.

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

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

Another aspect of the present disclosure relates to a method for monitoring the efficacy of a treatment in a subject having Alzheimer's disease (AD) or at risk for developing AD, the method comprising: (a) obtaining a first measurement 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 leboraxan, 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; and (d) The second measurement from the subject is compared to the first measurement from the subject, wherein a difference between the first measurement and the second measurement indicates effective treatment with leborax.

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

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 cell response. In some embodiments, the biological sample is a body fluid. In some embodiments, the body fluid is cerebrospinal fluid (CSF), blood, or saliva.

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.

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

In some embodiments, measures of neurodegeneration include measures of cortical thickness and/or hippocampal volume or measures of pyramidal or granule neuron loss.

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

In some embodiments, the measure of small neuroglia response is a measure of expression of at least one small neuroglia marker. In some embodiments, the small neuroglia marker is Iba1, Clec71, P2RY12, or TMEM 119. In some embodiments, the measure of small neuroglia response is a measure of phagocytosis of small neuroglia. In some embodiments, the measure of biomarker expression is a measure of Ifnb1, MMP2, and/or Bace1 expression.

In some embodiments, the subject is amylin negative. In some embodiments, the subject has Aβ plaques.

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.

In some embodiments, the subject is at risk for further Aβ accumulation. In some embodiments, wherein the subject is an ApoE4 carrier. In some embodiments, wherein the subject has moderate levels of amyloid protein PET (e.g., 20-40 percentile units). In some embodiments, wherein the subject has elevated levels of amyloid protein PET (e.g., >40 percentile units).

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

Cross-references to related applications

This application claims priority to 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 incorporated herein by reference in their entirety. I. Definitions

The following are definitions of terms used in this application.

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

As used herein, the phrase "and/or" means "any one or both" of the elements so joined together, i.e., the elements are present in conjunction in some cases and separately in other cases. Thus, as a non-limiting example, when used in conjunction with open language such as "comprising," "A and/or B" may refer to just A in some embodiments (optionally including elements other than B); to just B (optionally including elements other than A) in other embodiments; to both A and B (optionally including other elements) in still other embodiments; and so forth.

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 of elements. This definition also allows that other elements may appear as necessary in addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers, 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") may refer in one embodiment to at least one, optionally including more than one A, without B (and optionally including elements other than B); in another embodiment, it may refer to at least one, optionally including more than one B, without A (and optionally including elements other than A); in yet another embodiment, it may 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.

When a number is recited either alone or as part of a range of values, it is understood that the number can vary above and below the stated value by 10%.

When a range of values is listed herein, it is intended that all values and sub-ranges are included within the range. .0 mg, 27.5 mg, 28.0 mg, 28.5 mg, 29.0 mg, 29.5 mg, 30.0 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.

"Amyloid" refers to protein aggregates that form a protofibrillar morphology. Amyloids are often formed by long, unbranched fibrils characterized by an extended β-folded secondary structure with a width of approximately 7-13 nm and a length of several micrometers. Amyloids are usually found outside of cells and in vivo; in addition, the fibers bind the dye Congo red and then display green birefringence when viewed between crossed polarizers. Amyloid-forming proteins have been identified and associated with serious diseases, including amyloid beta peptide (Aβ) associated with Alzheimer's disease (AD), islet amyloid polypeptide (IAPP) associated with type 2 diabetes, and proteoglycans (PrP) associated with cavernous encephalopathy. As used herein, "amyloid", "amyloid beta", "brain amyloid" and "amyloid beta peptide (Aβ)" are used interchangeably.

Amylin β 1-42 (Aβ42) refers to an amylin β monomer from amino acids 1 to 42 of the full-length protein (Table 5, SEQ ID NO: 13). Amylin β 1-40 (Aβ1-40) refers to an amylin β monomer from amino acids 1 to 40 of the full-length protein (Table 5, SEQ ID NO: 14).

Amyloid levels from an amyloid PET can be reported in “centigrade units” (CL) using the centigrade method (Klunk WE et al. The Centiloid Project: standardizing quantitative amyloid plaque estimation by PET. Alzheimer’s Dement. 2015;11:1–15 e1–4). The centigrade method measures the tracer in a 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 average amyloid load present in subjects with mild to moderately severe dementia due to AD. (Ibid.) As is known to those skilled in the art, centiloid thresholds may vary, for example, may be modified based on new or additional scientific information. (See, for example, http://www.gaain.org/centiloid-project.) Elevated amylin levels may be set relative to baseline thresholds in healthy controls determined according to methods known to those skilled in the art.

As used herein, whether a subject is "amyloid positive" or "amyloid negative" can be determined based on whether the subject has a positive amyloid load. In some embodiments, a subject is determined to be amyloid positive or amyloid negative as indicated by longitudinal positron emission tomography (PET) assessment of amyloid imaging agents captured into the brain. In some embodiments, a subject is "amyloid negative" if the flubiprofen amyloid PET SUVr negativity is less than 1.17. In some embodiments, a subject is determined to be amyloid positive or amyloid negative by CSF assessment for the presence of amyloid pathology using markers such as Aβ1-42 (e.g., soluble CSF biomarker analysis), alone or in combination with another method such as PET measurement of brain amyloid. Methods for measuring Aβ38, Aβ40, and Aβ42 are known in the art, such as assays using LC MS/MS. Methods may include the PrecivityAD ^™ assay (see, e.g., Kirmess et al., J. Clinica Chimica Acta 519: 267-275 (2021)) and the Sysmex assay (https://www.eisai.com/news/2019/news201990.html) for measuring Aβ42 and Aβ40 in a blood or plasma sample or a CSF sample. In some embodiments, a qualitative visual readout of a PET scan can be used to determine amylin positivity and amylin negativity by classifying a subject as having "normal" or "abnormal" uptake based on the PET image pattern. Readers will have been trained and tested to identify brain PET images with abnormal or normal uptake patterns, or to perform amyloid detection by semi-quantitative or quantitative methods. In some embodiments, thresholds will be set for quantitative determination of Aβ brain burden from biomarkers (e.g., serum or CSF) and/or PET scans to indicate whether the subject is positive or negative for amyloid. In some embodiments, a subject is determined to be positive for amyloid or negative for amyloid 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 lobes, parietal lobes, temporal lobes, occipital lobes, cingulate cortex, amygdala, piriform cortex, entorhinal cortex, hippocampus, hippocampal CA3 (pyramidal neurons), and/or hippocampal dentate gyrus (granule cell neurons)) is analyzed by MRI.

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

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 produced 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 insert at the N-terminus (referred to as N), and full-length human tau isoforms can have two inserts (2N), one insert (1N), or no insert (0N). All full-length human tau isoforms also have three repeats of the microtubule binding domain (referred to as R). The inclusion of exon 10 at the C-terminus results in the inclusion of a fourth microtubule binding domain encoded by exon 10. Thus, a full-length human tau isoform may consist of four repeats of a microtubule binding domain (4R) (including exon 10) or three repeats of a microtubule binding domain (3R) (excluding exon 10). Human tau may or may not be post-translationally modified. For example, it is known in the art that tau can be phosphorylated, ubiquitinated, glycosylated, and glycated. Thus, the term "human tau" encompasses (2N, 3R), (2N, 4R), (1N, 3R), (1N, 4R), (0N, 3R), and (0N, 4R) isoforms, as well as N-terminal and/or C-terminal truncated isoforms thereof and all post-translationally modified isoforms. Alternative splicing of the gene encoding tau occurs similarly in other animals. In animals where the gene has not been identified as MAPT, homologs can be identified by methods well known in the art.

Phosphorylation of specific amino acids (i.e., "sites" or "residues") in tau produces 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.

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

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

Methods for measuring tau phosphorylation include high resolution mass spectrometry. Suitable types of mass spectrometers are known in the art. These include, but are not limited to, quadrupole mass spectrometers, time-of-flight mass spectrometers, ion trap and orbitrap mass spectrometers, and hybrid mass spectrometers that combine different types of mass analyzers into one architecture (e.g., the Orbitrap Fusion™ Tribrid™ mass spectrometer from ThermoFisher Scientific). Other methods for measuring p-tau (phospho-tau) and t-tau (total tau) are known in the art, such as assays using liquid chromatography coupled to 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. PET can be used to analyze 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).

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

As used herein, "relative to baseline" refers to a comparison of a biomarker (p-tau, Aβ, etc.) between a subject administered lebrexan and the same biomarker in the same subject prior to treatment with lebrexan.

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

As used herein, "MMSE" refers to the Mini-Mental State Examination, a cognitive tool commonly used for screening purposes and also commonly measured longitudinally in AD clinical trials, with a 30-point scale in which higher scores indicate lower levels of impairment and lower scores indicate higher levels of impairment. As used herein, the assessment measures seven items of time orientation and location orientation, recording, memory, attention, language, and drawing. (Folstein, MF, 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).

As used herein, "PSQI" refers to the Pittsburgh Sleep Quality Index, a self-report questionnaire that assesses sleep quality and disturbances at 1-month time intervals. Nineteen individual items generate seven "component" scores: subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbance, use of sleep medication, and daytime dysfunction. The sum of the scores for these seven components yields a global score ranging from 0 to 21, with lower scores indicating healthier sleep quality. The clinical and clinicometric properties of the PSQI were assessed in "good" sleepers (healthy subjects, n = 52) and "poor" sleepers (depressed patients, n = 54; patients with sleep disturbances, n = 62) over an 18-month period. (Buysse DJ et al. “The Pittsburgh Sleep Quality Index: a new instrument for psychiatric practice and research.” Psychiatry Res. 1989; 28(2):193-213).

As used herein, "STOP-Bang" refers to the Snoring, Fatigue, Observed Apnea, High BP, BMI, Age, Neck Circumference, and Male Gender (STOP-Bang) Questionnaire. The questionnaire consists of eight dichotomous (yes/no) items related to clinical features of sleep apnea. The total score ranges from 0 to 8. Patients can be categorized for their risk of sleep apnea (OSA) based on their individual scores. A STOP-Bang score ≥ 3 has a sensitivity 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).

As used herein, "PSG" refers to multi-channel sleep recording, a standard diagnostic test for OSA. PSG provides an assessment of OSA based on the frequency of apneas and hypopneas per hour of sleep (apnea-hypopnea index or AHI). The severity of OSA is classified 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).

As used herein, a subject with "preclinical AD" or "pre-AD" is a cognitively normal (e.g., unimpaired) individual 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: presymptomatic AD and asymptomatic AD (or "asymptomatic, at risk"). Presymptomatic AD refers to a cognitively unimpaired subject with an autosomal dominant single gene mutation for AD. A presymptomatic subject will most likely develop AD as a result of the autosomal dominant single gene mutation. 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 categorized. Deficits in episodic memory and executive function may appear later. Thus, subjects with pre-AD can be identified by an asymptomatic stage in which cognition is not impaired. Cognitively normal can include individuals with CDR 0, or individuals within the normal range on 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 the absence of clinical symptoms. Preclinical AD biomarkers that may indicate the future development of Alzheimer's disease include, but are not limited to, moderate or elevated levels of amyloid in the brain as measured by amyloid or tau positron emission tomography (PET) (e.g., a percentile measurement of about 20-40, e.g., a measurement of about 20-32). Additional biomarkers may be used alone or in combination, including one or more of the following: cerebrospinal fluid Aβ1-42 levels and/or Aβ1-42/1-40 ratio, cerebrospinal fluid total tau levels, cerebrospinal fluid neurogranin levels, cerebrospinal fluid neurofilament light chain peptide (NfL) levels, and biomarkers measured in serum or plasma (e.g., Aβ1-42 levels, the ratio of two forms of amyloid b peptide (Aβ1-42/1-40 ratio)). ratio between about 0.092-0.094 or a ratio below about 0.092), plasma levels of 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), fibroblastic acidic protein (GFAP), and neurofilament light chain peptide (NfL).

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

Other methods for detecting early AD disease may employ tests and assays as specified below, including the National Institute on Aging and Alzheimer’s Association (NIA-AA) core clinical guidelines for possible Alzheimer’s dementia as described in 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, Weys Memory Scale-IV Logical Memory (Subscale) I (WMS-IV LMI), and Weys Memory Scale-IV Logical Memory (Subscale) II (WMS-IV LMII). In some embodiments, the subject with early AD has evidence of elevated amyloids or positive amyloid load in the brain. In some embodiments, elevated amyloids or positive amyloid load in the brain is indicated and/or confirmed by PET assessment. In some embodiments, elevated amyloids or positive amyloid load in the brain is indicated and/or confirmed by CSF assessment of markers such as Aβ1-42 (e.g., water-soluble CSF biomarker analysis). For example, a subject with AD can be selected according to the methods in WO 2023/283650, the contents of which are incorporated herein by reference. In some embodiments, diagnostic thresholds can be identified by the amyloid PET by visual reading (according to the label of the approved PET tracer) or by establishing a percentile unit threshold, above which a subject is considered to have elevated amyloids (e.g., varying between 15-40 percentile units).

In some embodiments, elevated amyloids or positive amyloid load in the brain 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, elevated amyloids or positive amyloid load in the brain is indicated and/or confirmed by MRI or PET. In some embodiments, elevated amyloids or positive amyloid load in the brain 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 possible Alzheimer's dementia as described in 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 were subjects with a CDR score of 0.5 to 1.0 and a Memory Box score of 0.5 or higher at Screening and Baseline.

In some embodiments, the subject has "elevated amyloid" or "moderate amyloid." Those skilled in the art will recognize that amyloid levels from an amyloid PET can be reported in "centigrade" (CL) using the centigrade method. (Klunk WE et al. The Centiloid Project: standardizing quantitative amyloid plaque estimation by PET. Alzheimer's Dement. 2015; 11 : 1-15 el-4). The centiloid method measures tracers in a 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 average amylin load present in subjects with mild to moderately severe dementia due to AD. (Ibid.) As known to those skilled in the art, the centiloid thresholds can vary, for example, can be modified based on new or additional scientific information. (See, for example, http://www.gaain.org/centiloid-project.) Elevated amylin levels can be set relative to baseline thresholds in healthy controls determined according to methods known to POSA. For example, a percentile value of 32.5 may be used as a threshold for "elevated amyloids," and a "moderate amyloid" level refers to an Aβ amyloid PET in the range of 20-32.5 CL (e.g., 30 CL). In another example, a percentile value of 40 may be used as a threshold for "elevated amyloids," and a "moderate amyloid" level refers to an Aβ amyloid PET in the range of 20-40 CL.

As used herein, a subject with "MCI due to moderate likelihood of AD" is a subject identified as such according to the NIA-AA core clinical criteria for mild cognitive impairment due to moderate likelihood of Alzheimer's disease. For example, AD subjects with symptomatic but non-demented AD with brain amyloid pathology make them less heterogeneous and more similar to subjects with mild Alzheimer's dementia in terms of cognitive and functional decline as measured by the ADCOMS Composite Clinical Score defined herein. Also included are subjects with a CDR score of 0.5 and a memory frame area score of 0.5 or higher at screening and baseline. In addition, subjects who reported a history of subjective memory decline with gradual onset and slow progression within the last year before screening, as confirmed by an informant, were included.

As used herein, "ADAS-cog" refers to the Alzheimer's Disease Assessment Scale-Cognitive. ADAS-cog is a cognitive scale commonly used in Alzheimer's trials that has structured scales that assess memory (verbal recall, delayed verbal recall, and word recognition), reasoning (following commands), language (naming, comprehension), orientation, conceptual practice (placing a letter in an envelope), and construction practice (copying a geometric design). (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 spoken language, language comprehension, difficulty with calling words, ability to remember test instructions, mazes, and number crossing. The modified form used herein is scored on a scale of 0 to 90, with 0 indicating no impairment and 90 indicating the highest degree of impairment.

As used herein, "CDR-SB" refers to the clinical dementia rating—sum of boxes. The CDR is a clinical rating scale that describes 5 levels of impairment in the performance of 6 functional categories including memory, orientation, judgment and problem solving, group affairs, family 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 level of impairment obtained for each of the 6 functional categories is combined into 1 total level of the dementia CDR score (ranging from 0 to 3). The sum of the box scores provides an additional measure of variation, with each category having a maximum possible score of 3 points and the total score being the sum of the category scores, resulting in a total possible score of 0 to 18, with higher scores indicating a greater degree of impairment. The total score can be used as a clinical measure of the severity of dementia.

As used herein, "ADCOMS" refers to the Alzheimer's Disease Composite Score, a composite clinical score based on four ADAS-Cog items (delayed verbal recall, orientation, word recognition, and word difficulty), two MMSE items (temporal orientation and drawing), and all six CDR-SB items (personal care, group affairs, family and hobbies, memory, orientation, and judgment and problem solving), as in the examples and Wang, J. et al., "ADCOMS: a composite clinical outcome for prodromal Alzheimer's disease trials." J. Neurol. Neurosurg. Psychiatry. 2016; ADCOMS was developed to be particularly sensitive to disease progression in the early stages of AD, i.e., prodromal and mild AD.

As used herein, "ApoE4 positive" subjects and "ApoE4 carriers" refer to subjects who have the ε4 variant of the apoE4 gene. The ε4 variant is one of several major alleles of the apoE4 gene. This gene is generally responsible for fat metabolism. It has been found that carriers of apoE4 ε4 show significantly higher amyloid retention when compared to non-carriers. (Drzezga, A. et al., "Effect of APOE genotype on amyloid plaque load and gray matter volume in Alzheimer disease." Neurology. [Neurology] 2009; 72:1487-94). In some embodiments, the subject is a heterozygous carrier of the apolipoprotein E ε4 allele. In some embodiments, the subject is a homozygous carrier of the apolipoprotein E ε4 allele.

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 a worsening of ADCOMS. In some embodiments, clinical decline is determined by a worsening of MMSE. In some embodiments, clinical decline is determined by a worsening of ADAS-Cog. In some embodiments, clinical decline is determined by a worsening of FAQ. In some embodiments, clinical decline is determined by a worsening of CDR-SB. In some embodiments, clinical decline is determined by a worsening of 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 worsening of one or more biomarkers of AD or brain measurements (e.g., by PET or MRI) such as brain atrophy and/or amyloid protein accumulation.

As used herein, the term "treat" (also "treating" or "treatment") refers to any administration or application of a therapeutic agent to a subject suffering from a disease or disorder, and includes inhibiting the disease, slowing the progression of the disease, delaying the progression, arresting its development, reversing the progression of the disease (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 of the disease or one or more potential conditions, curing the disease, improving one or more clinical indicators, or preventing the recurrence of one or more symptoms of the disease. In some embodiments, treatment may include maintaining the severity of at least one symptom of the disease (i.e., preventing worsening), for example, when the symptom would be expected to progress and/or worsen without the administration or application of the therapeutic agent to the subject. In some embodiments, maintaining symptoms may mean that there is no change (e.g., no significant change, such as no statistically significant change) in the symptom after the administration or application of the therapeutic agent to the subject, compared to a control (e.g., a subject not receiving treatment or receiving a placebo), for which there is a change (e.g., a significant change, such as a statistically significant change) in the symptom. Complete treatment is not required. In some embodiments, treatment of AD in a subject comprises administering (e.g., intravenous infusion) a dual alpha-deoxyglucose receptor antagonist (e.g., lebrexant) to a subject who is, for example, at risk for developing AD but has not yet shown signs of dementia.

As used herein, unless the context indicates otherwise, the term "prevention," which is encompassed within the term "treatment," refers to obtaining a beneficial or desired preventive benefit. For preventive benefit, the composition may be administered to a subject at risk for 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 physiological symptoms of Alzheimer's disease, even though a clinical diagnosis of Alzheimer's disease has not yet been made. As used herein, "prevention" may further include a therapeutic benefit, which means eradication or amelioration of the underlying condition being treated or one or more physiological symptoms associated therewith. Prevention also covers stopping or slowing the further progression of one or more symptoms of a disease.

As used herein, "control" (also "control sample") refers to a biological sample obtained from a subject that is different from those samples being evaluated and has a known AD status. In some embodiments, the control sample is obtained from a subject who has not been diagnosed with Alzheimer's disease, for example, according to one or more of the above definitions. For example, the control sample can be obtained from a subject who has no clinical symptoms of AD (e.g., cognitive impairment and/or dementia) and/or no markers of AD pathology (e.g., PET scans or CSF analysis of biomarkers such as amyloid proteins or tau). In some embodiments, the control sample can be obtained from a healthy subject. In some embodiments, the control can be obtained from a subject with a comorbidity not related to AD. In some embodiments, controls can be obtained from subjects diagnosed along the AD disease spectrum, including, for example, preclinical AD or mild cognitive impairment. In some embodiments, control samples can be baseline samples collected from subjects before starting any treatment. In some embodiments, control samples can be samples collected from control subjects administered a placebo.

As used herein, the term "therapeutically effective amount" refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired therapeutic effect, such as reversing, preventing, delaying or slowing cognitive decline and/or reversing, preventing, delaying or slowing the rate of change of one or more biomarkers of AD. Those skilled in the art will understand that the therapeutically effective amount of leboraxant administered to a subject can 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. II. Methods

Disclosed herein are methods of reducing the amount of p-tau in a subject, the method comprising administering a therapeutically effective amount of lebrexan to a subject in need thereof. Also disclosed herein are methods of reducing neurodegeneration in a subject, the method comprising administering a therapeutically effective amount of lebrexan to a subject in need thereof. Also disclosed herein are methods of reducing amyloid beta in a subject, the method comprising administering a therapeutically effective amount of lebrexan to a subject in need thereof.

In one aspect of the present disclosure, lebrexan affects at least one marker of AD pathology. Without being bound by theory, in some embodiments, the surprising effects of lebrexan on AD and AD pathology may be related to the role of lebrexan in regulating sleep and treating insomnia, in addition to other potential effects on AD pathology. Notably, lebrexan provides benefits that other sleep agents such as doxepin (another drug approved for the treatment of insomnia) do not have. For example, as discussed in the examples, when each was administered to an animal model of AD, lebrexan and doxepin showed differential effects on the development of Aβ plaques, activation of phagocytic microglia, and expression of biomarkers involved in membrane receptor trafficking and inflammatory activity. In an animal model, leborexant reduced total amyloid plaque burden (including diffuse and protofibrillary plaques), whereas doxepin reduced total amyloid burden but not protofibrillary plaque burden (Example 3, Part B). Leborexant also increased the activation of phagocytic microglia surrounding Aβ plaques, whereas doxepin did not (Example 3, Part E). Finally, leborexant significantly upregulated the expression of Ifnb1 , the inflammatory mediator IFN-β, the lysosomal protein Rab5a , and the Aβ-degrading enzyme Mmp2 , whereas doxepin did not (Example 3, Part F). Without being bound by theory, these data may indicate that leborabine and doxepin act via different mechanisms and suggest that the effects of the drugs on sleep may differ in some conditions from their effects on AD pathology.

In various embodiments, disclosed herein are methods of reducing or maintaining the amount of p-tau and/or t-tau in a subject, the methods comprising administering a therapeutically effective amount of lebrexan to a subject in need thereof. Also disclosed are methods of 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 a therapeutically effective amount of lebrexan to a subject in need thereof. In some embodiments, the amount of p-tau and/or t-tau in the CSF of a subject after administration of a therapeutically effective amount of lebrexan is reduced or maintained compared to the amount of p-tau and/or t-tau in the CSF of the subject before such administration. In some embodiments, the amount of p-tau and/or t-tau in the CSF of a subject after administration of a therapeutically effective amount of Lebron is reduced or maintained as compared to the amount of p-tau and/or t-tau in the CSF of the subject after administration of a placebo.

Also disclosed herein is a method of reducing neurodegeneration in a subject, the method comprising administering a therapeutically effective amount of lebrexan to a subject in need thereof. Also disclosed herein is a method of reducing or maintaining amyloid beta in a subject, the method comprising administering a therapeutically effective amount of lebrexan to a subject in need thereof.

In some embodiments, the subject in need of the methods of the present invention has shown 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 vascular disease, frontotemporal dementia, dementia with Lewy bodies, Lewy body dementia, Parkinson's disease, vascular dementia, marginal dominant age-related TDP-43 encephalopathy, frontotemporal degeneration, cortical basal ganglia degeneration, Pick's disease, multiple system atrophy, and progressive supranuclear neuropathy. III. Subjects in need of treatment

In some embodiments, a subject in need of one or more of the disclosed methods has shown 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 vascular disease, frontotemporal dementia, dementia with Lewy bodies, dementia with Lewy bodies, Parkinson's disease, vascular dementia, marginal dominant age-related TDP-43 encephalopathy, frontotemporal degeneration, corticobasal degeneration, Pick's disease, multiple system atrophy, and progressive supranuclear neuropathy. In some embodiments, a subject in need has shown signs of at least one disease selected from the group consisting of Alzheimer's disease, pre-Alzheimer's disease, and early Alzheimer's disease. In some embodiments, the subject in need has shown signs of mild cognitive impairment. In some embodiments, the subject in need has shown signs of cerebral vascular disease, frontotemporal dementia, dementia with Lewy bodies, dementia with Lewy bodies, vascular dementia, marginal dominant age-related TDP-43 encephalopathy, frontotemporal degeneration, corticobasal degeneration. In some embodiments, the subject in need has shown signs of at least one disease selected from Parkinson's disease, Pick's disease, multiple system atrophy and progressive supranuclear neuropathy.

One aspect of the present disclosure relates to 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 lebrexan, a pharmaceutically acceptable salt thereof, or a solvate thereof, thereby treating AD. In some embodiments, the subject is in need of treatment (e.g., has AD, pre-AD, or is at risk of developing AD).

In some embodiments, treating AD refers to inhibiting AD, AD pathology, AD symptoms, and/or potential conditions of AD, slowing its progression, slowing its rate of progression, delaying its progression, arresting its development, and reversing its progression. In some embodiments, treating refers to preventing the onset of AD, AD pathology, AD symptoms, and/or potential conditions of AD or preventing their development. In some embodiments, treating refers to alleviating or ameliorating one or more symptoms or potential conditions of AD (e.g., improving the accumulation of Aβ fibrils) and/or improving one or more clinical measures of AD (e.g., cognitive function, brain amyloid or tau levels and/or biomarker expression). In some embodiments, treating refers to preventing the occurrence or recurrence of one or more symptoms of AD.

In some embodiments, treating AD comprises reducing, halting and/or slowing cognitive decline.

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 assessment. The diagnosis can be based on measurements of AD pathology and/or the expression of biomarkers in the subject obtained by brain imaging (e.g., amyloid protein PET or tau PET). In some embodiments, biomarkers include brain amyloid protein levels, brain tau levels, cerebrospinal fluid levels of Aβ1-42, cerebrospinal fluid levels of total tau, cerebrospinal fluid levels of neurogranin, and cerebrospinal fluid levels of neurofilament light chain (NfL). In some embodiments, subjects with AD show cognitive impairment and AD pathology. For example, a subject with AD may have a t-tau level greater than 400 ng/L, an Aβ1-42 level less than 550 ng/L, and/or an Aβ1-42/Aβ1-40 ratio less than 0.065.

In some embodiments, the subject in need of treatment has early Alzheimer's disease (also referred to as "early AD" or "EAD"). In some embodiments, the subject with early AD has symptoms ranging from mild cognitive impairment with moderate likelihood of AD to mild Alzheimer's dementia. In some embodiments, the subject with early AD has mild Alzheimer's dementia as defined herein and/or mild cognitive impairment (MCI) with moderate likelihood of AD as defined herein. In some embodiments, the subject has an MMSE score of 22 to 30 and a total range of the Clinical Dementia Rating Scale (CDR) of 0.5 to 1.0. In some embodiments, the subject with early AD has evidence of elevated amyloids or positive amyloid load in the brain. In some embodiments, an increase in amyloid protein or positive amyloid protein load in the brain is indicated and/or confirmed by PET assessment. In some embodiments, an increase in amyloid protein or positive amyloid protein load in the brain is indicated and/or confirmed by CSF assessment of markers such as Aβ1-42 (e.g., water-soluble CSF biomarker analysis). In some embodiments, an increase in amyloid protein or positive amyloid protein load in the brain is indicated and/or confirmed by measuring the ratio of Aβ42 to Aβ40 (Aβ42/40 ratio). In some embodiments, an increase in amyloid protein or positive amyloid protein load in the brain is indicated and/or confirmed by MRI assessment. In some embodiments, elevated amyloids or positive amyloid load in the brain is indicated by retinal amyloid accumulation. In some embodiments, more than one assessment method is used.

In some embodiments, the subject has preclinical Alzheimer's disease (also referred to as "pre-AD"). Subjects with pre-AD may be cognitively normal, with moderate or elevated levels of amylin in the brain. In some embodiments, subjects with pre-AD can be identified by an asymptomatic phase with or without memory complaints and the presence of deficits in episodic memory and executive function. In some embodiments, subjects with pre-AD have CDR 0 and/or scores 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 suggestive of future development of AD, such as one or more of: moderate or elevated levels of brain amyloid (e.g., a percentile measurement of about 20-40, such as a measurement of about 20-32) as determined by amyloid or tau positron emission tomography (PET), cerebrospinal fluid Aβ1-42 levels, and/or Aβ 1-42/1-40 ratio, cerebrospinal fluid total tau levels, cerebrospinal fluid neurogranin levels, cerebrospinal fluid neurofilament light chain peptide (NfL) levels, and blood biomarkers as measured in serum or plasma (e.g., levels of Aβ1-42, the ratio of the two forms of amyloid beta peptide (Aβ1-42/1-40 ratio, e.g., between about 0.092-0.094 or less In some embodiments, subjects with pre-AD may have moderate amylin (e.g., about 20-40 percentile units). In some embodiments, subjects with pre-AD may have elevated amylin (e.g., > 40 percentile units).

In some embodiments, a subject may be at risk for developing AD. A subject may have one or more risk factors for developing AD, such as carrying a familial AD gene (e.g., apolipoprotein E ε4 allele, also known 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 or recovering from a traumatic brain injury, having other conditions such as obesity, diabetes, heart and/or vascular disease, etc. In some embodiments, a subject with pre-AD is at risk for developing AD. The risk of developing AD may be greater than the risk of developing AD in control subjects and/or the risk of developing AD more quickly than estimated for control subjects. For example, subjects with pre-AD and normal cognition but with intermediate amyloid PET levels (approximately 20-40 percentiles) may be at risk for further Aβ accumulation and early spread of tau pathology within 4 years compared to control subjects. Subjects with pre-AD and normal cognition but with elevated amyloid PET levels (> 40 percentiles) may be at high risk for cognitive decline within 4 years compared to control subjects.

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

In some embodiments, markers of AD pathology are phosphorylation levels of tau, neurodegeneration, changes in microglial cell responses, and/or the presence of Aβ plaques. Markers of AD pathology can be present in brain regions of the subject, such as the hippocampus, motor cortex, somatosensory cortex, piriform cortex, and/or entorhinal cortex. In some embodiments, markers of AD pathology are detected in brain scans. For example, tau phosphorylation can be detected by tau PET; Aβ can be detected by amyloid protein PET. In some embodiments, markers of AD pathology are detected in body fluids of the subject, 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.

In some embodiments, the subject shows no signs of dementia and/or cognitive impairment. In some embodiments, the subject has mild cognitive impairment or mild dementia.

In some embodiments, the subject is amyloid positive. The subject may be at risk for further Aβ accumulation and/or tau pathological 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 having a family history of a first-degree relative with AD or dementia, being 65 years of age or older, being female, having or recovering from a traumatic brain injury, and having other conditions such as obesity, diabetes, heart disease, and/or vascular disease.

In some embodiments, the subject has been diagnosed as having 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.

In some embodiments, treatment can slow cognitive decline and/or reduce the rate of change of AD biomarkers. IV. Tau and Aβ

The ratio of the concentration of p-tau (also referred to herein as "phosphorylated tau" or "phosphorylated tau") to the concentration of t-tau (also referred to herein as "total tau") in the CSF of a subject (hereinafter "CSF p-tau/t-tau ratio") can be used to assess the amount of tau phosphorylation. The concentrations of p-tau and t-tau in the CSF of a subject are measured using liquid chromatography-tandem mass spectrometry (LC MS/MS).

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

In some embodiments, the ratio of CSF p-tau/t-tau in a subject administered a therapeutically effective amount of lebrexan is reduced compared to the ratio of CSF p-tau/t-tau in the subject prior to administration of lebrexan.

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

In some embodiments, the concentration of amyloid beta (Aβ) in the CSF is lower than the Aβ concentration in the CSF of a subject administered a placebo. In some embodiments, the Aβ concentration in the CSF is lower than the Aβ concentration in the CSF of a subject prior to administration of leboraxan. In some embodiments, the concentration of Aβ38, Aβ40, and/or Aβ42 in the CSF is reduced. In some embodiments, the Aβ concentration in the CSF is maintained compared to the Aβ concentration in the CSF of a subject prior to administration of leboraxan. In some embodiments, the concentration of Aβ38, Aβ40, and/or Aβ42 in the CSF is maintained compared to the Aβ concentration in the CSF of a subject prior to administration of leboraxan. In some embodiments, the amyloid PET message in the brain of a subject administered a therapeutically effective amount of lebrexan is lower than the amyloid PET message in the brain of a subject administered a placebo. In some embodiments, the amyloid PET message in the brain of a subject administered a therapeutically effective amount of lebrexan is lower or maintained compared to the amyloid PET message in the brain of the subject prior to administration of lebrexan. In some embodiments, the increase in Aβ concentration in the CSF of a subject administered lebrexan is less than the increase in Aβ concentration in the CSF of a subject administered a placebo.

In some embodiments, the Aβ concentration in the CSF of subjects administered lebrexan is at least 5% lower than that of subjects administered a placebo. In some embodiments, the Aβ concentration in the CSF of subjects administered lebrexan is at least 10%, at least 15%, at least 20%, or at least 25% lower than that of subjects administered a placebo.

In some embodiments, the Aβ concentration in CSF is measured using liquid chromatography-tandem mass spectrometry (LC MS/MS). In some embodiments, the concentration of Aβ38, Aβ40, and/or Aβ42 in CSF is measured using LC MS/MS. Methods for measuring Aβ38, Aβ40, and Aβ42 are known in the art, such as assays using LC MS/MS. Methods may include the PrecivityAD ^™ assay for measuring Aβ42 and Aβ40 in blood or plasma samples or CSF samples (see, e.g., Kirmess et al., J. Clinica Chimica Acta 519: 267-275 (2021)) and the Sysmex assay (https://www.eisai.com/news/2019/news201990.html). In some embodiments, the concentration of Aβ in CSF is measured using ELISA. In some embodiments, the concentration of Aβ38, Aβ40 and/or Aβ42 in CSF is 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 before administration of leborexant. In some embodiments, administration of a composition comprising a therapeutically effective amount of leboraxant to a subject results in a decrease in cerebrospinal fluid level concentrations relative to baseline of Aβ38, Aβ40, and/or Aβ42 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, p-tau and t-tau are reduced. In some embodiments, p-tau, t-tau and/or aggregated tau are reduced. In some embodiments, the reduction in tau phosphorylation occurs 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 message in the brain is reduced compared to placebo. In some embodiments, the tau PET message in the brain is reduced or maintained compared to baseline.

In some embodiments, administering a therapeutically effective amount of lebrexan to a subject results in a decrease in the p-tau concentration in the subject's CSF compared to the p-tau concentration in the CSF of a subject administered a placebo. In some embodiments, administering a therapeutically effective amount of lebrexan to a subject results in a decrease in the p-tau concentration in the subject's CSF compared to the p-tau concentration in the subject's CSF prior to administration of lebrexan. In some embodiments, administering a therapeutically effective amount of lebrexan to a subject results in a maintenance of the amount of p-tau in the subject's CSF compared to the p-tau concentration in the CSF of a subject administered a placebo. In some embodiments, administering a therapeutically effective amount of lebrexan to a subject causes the amount of p-tau in the subject's CSF to be maintained prior to administration of lebrexan.

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

In some embodiments, administering a therapeutically effective amount of lebrexan to a subject causes the amount of p-tau in the subject's CSF to be maintained as compared to the p-tau concentration in the CSF of a subject administered a placebo. In some embodiments, administering a therapeutically effective amount of lebrexan to a subject causes the amount of p-tau in the subject's CSF to be 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% as compared to the amount of p-tau in the CSF of a subject administered a placebo. In some embodiments, administering a therapeutically effective amount of lebrexan to a subject causes a decrease in the amount of p-tau in the subject's CSF by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, or at least 13% compared to the amount of p-tau in the subject's CSF prior to administration of lebrexan.

In some embodiments, the increase in the amount of p-tau in the CSF of a subject administered lebrexan is less than the increase in the amount of p-tau in the CSF of a subject administered a placebo.

In some embodiments, administering a therapeutically effective amount of lebrexan to a subject results in a decrease in p-tau concentration in the subject's CSF up to 18 months after administration of lebrexan, compared to p-tau concentration in the subject's CSF at baseline. In some embodiments, administering a therapeutically effective amount of lebrexan to a subject results in a decrease in p-tau concentration in the subject's CSF by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, or at least 13%, compared to p-tau concentration in the subject's CSF at baseline.

In some embodiments, the reduction in the amount of p-tau or t-tau in the CSF of a subject is due to a reduction or maintenance of the amount of p-tau or t-tau in the subject, comprising administering a therapeutically effective amount of leboraxant to a subject in need thereof. In some embodiments, the reduction in the amount of p-tau in the CSF of a subject is due to an increase in p-tau dephosphorylation. In some embodiments, the reduction in the amount of p-tau in the CSF of a subject is due to a reduction in the amount of tau. In some embodiments, the reduction in the amount of p-tau in the CSF of a subject is due to a reduction in the ratio of p-tau/t-tau.

In some embodiments, administration of a disclosed therapeutically effective amount of lebrexan to a subject results in a decrease or maintenance of p-tau concentration in the subject's CSF as compared to the p-tau concentration in the CSF of a subject administered a placebo. In some embodiments, administration of a therapeutically effective amount of lebrexan to a subject results in a decrease in the amount of p-tau in the CSF 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. In some embodiments, administration of a composition disclosed herein comprising a therapeutically effective amount of leboraxant to a subject results in a decrease in cerebrospinal fluid levels of p-tau by at least about 40 pg/mL relative to placebo.

In some embodiments, administration of a disclosed therapeutically effective amount of lebrexan to a subject results in a decrease or maintenance of p-tau concentration in the subject's CSF as compared to the p-tau concentration in the subject's CSF prior to administration of lebrexan. In some embodiments, administration of a therapeutically effective amount of lebrexan to a subject results in a decrease in the amount of p-tau in the cerebrospinal fluid relative to baseline 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. In some embodiments, administration of a composition disclosed herein comprising a therapeutically effective amount of lebrexan to a subject results in a decrease in the amount of p-tau in the cerebrospinal fluid relative to baseline by at least about 40 pg/mL.

In some embodiments, administration of a therapeutically effective amount of lebrexan to a subject results in a decrease in the amount of p-tau in the cerebrospinal fluid by at least about 5 pg/mL, at least about 10 pg/mL, at least about 15 pg/mL, at least about 20 pg/mL, at least about 25 pg/mL, at least about 30 pg/mL, at least about 35 pg/mL, or at least about 40 pg/mL relative to baseline, up to 18 months after administration of the composition comprising the therapeutically effective amount of lebrexan. In some embodiments, administration of a composition comprising a therapeutically effective amount of lebrexan to a subject results in a decrease in the amount of p-tau in the cerebrospinal fluid by at least 40 pg/mL relative to baseline, up to 18 months after administration of the composition comprising the therapeutically effective amount of at least lebrexan.

In some embodiments, administration of a therapeutically effective amount of lebrexan to a subject results in a decrease in the amount of p-tau in the cerebrospinal fluid by at least about 5 pg/mL, at least about 10 pg/mL, at least about 15 pg/mL, at least about 20 pg/mL, at least about 25 pg/mL, at least about 30 pg/mL, at least about 35 pg/mL, or at least about 40 pg/mL, relative to placebo, up to 18 months after administration of the composition comprising the therapeutically effective amount of lebrexan. In some embodiments, administration of a composition comprising a therapeutically effective amount of lebrexan to a subject results in a decrease in the amount of p-tau in the cerebrospinal fluid by at least 40 pg/mL, relative to placebo, up to 18 months after administration of the composition comprising the therapeutically effective amount of at least lebrexan.

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

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

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

In some embodiments, the amount of p-tau in the CSF of a subject is reduced within 48 hours of administering the first dose of lebrexan to the subject, relative to subjects administered a placebo. In some embodiments, the amount of tau phosphorylation in the CSF of a subject is reduced within 24 hours of administering the first dose of lebrexan to the subject, relative to subjects administered a placebo. In some embodiments, the amount of tau phosphorylation in the CSF of a subject is reduced within 12 hours of administering the first dose of lebrexan to the subject, relative to subjects administered a placebo. In some embodiments, the amount of tau phosphorylation in the CSF of a subject is reduced within 6 hours of administering the first dose of lebrexan to the subject, relative to subjects administered a placebo. A. Alteration of tau

Another aspect of the disclosure relates to a method of altering tau (e.g., reducing or delaying, and/or slowing the rate of, tau accumulation, tau phosphorylation, and/or tau diffusion) in a subject having AD or at risk of developing AD, the method comprising administering to the subject a therapeutically effective amount of lebrexan, wherein the therapeutically effective amount is sufficient to alter tau in the subject. In some embodiments, altering tau comprises reducing or delaying, or slowing the rate of, tau accumulation, tau phosphorylation, or tau diffusion.

In some embodiments, altering tau is slowing the progression, slowing the rate of progression, delaying the progression, arresting the progression, and reversing the progression of tau pathology (e.g., tau aggregation, tau phosphorylation, and/or tau diffusion), for example, in a brain region. The brain region may be the cortex or the hippocampus. The brain region may be the CA1 region, the CA2 region, the CA3 region, and/or the dentate gyrus of the hippocampus. The brain region may be the entorhinal cortex and/or the piriform cortex. In some embodiments, altering tau is preventing the onset of tau pathology or preventing the progression of tau pathology. Altering tau may reduce, delay, or slow the onset of symptoms of tau pathology and/or the rate of progression. In some embodiments, altering tau is to alleviate or ameliorate one or more symptoms of tau pathology and/or improve one or more clinical measures of tau pathology (e.g., cognitive function, brain amyloid or tau levels and/or biomarker expression). In some embodiments, altering tau is to prevent the occurrence or recurrence of one or more symptoms of tau pathology.

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

In some embodiments, tau is altered relative to a reference. Thus, the methods described herein may include reducing and/or delaying tau accumulation, tau phosphorylation, and/or tau diffusion, and/or slowing the rate of any of the above, 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. The reference may 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.

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

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

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

In some embodiments, altering tau comprises 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 in the subject prior to administration of leboraxan. 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 comprises altering tau within 48 hours of administering a first dose of leboraxan. For example, tau may be reduced within 48 hours of administering a first dose of leboraxan. In some embodiments, reducing tau comprises altering phosphorylated tau in the hippocampus, entorhinal cortex, and/or piriform cortex. B. Maintaining tau

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

In some embodiments, maintaining tau refers to maintaining tau accumulation, tau phosphorylation, and/or tau diffusion when tau would be expected to progress and/or worsen without the administration or application of the therapeutic agent to the subject. In some embodiments, maintaining tau may refer to the absence of a change (e.g., no significant change, such as no statistically significant change) in tau (e.g., tau accumulation, tau phosphorylation, and/or tau diffusion) after administration or application of the therapeutic agent to the subject, as compared to a control in which administration or application of the therapeutic agent results in a change (e.g., a significant change, such as a statistically significant change) in tau.

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

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

In some embodiments, the subject shows signs of cognitive impairment. In some embodiments, the subject is positive for amyloid protein. In some embodiments, the subject is diagnosed with AD, such as early AD.

In some embodiments, tau is maintained relative to a reference. Thus, the methods described herein may include maintaining tau accumulation, tau phosphorylation, and/or tau diffusion (or the rate of any of these) compared to a reference. In some embodiments, the reference is a baseline measurement from the subject before treatment. In some embodiments, the reference is a baseline measurement from a control subject. A reference may be a measurement obtained from more than one control subject and may be used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

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

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

In some embodiments, one or more forms of tau may be maintained. In some embodiments, the tau is total tau. In some embodiments, the tau is aggregated tau. In some embodiments, the tau is a phosphorylated form of tau (phosphorylated tau). Phosphorylated tau may be tau phosphorylated on one or more of T181, T217, S202, S205, or T231.

In some embodiments, maintaining tau comprises maintaining a 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 leboraxan. In some embodiments, maintaining tau comprises maintaining tau within 48 hours of administration of the first dose of leboraxan. In some embodiments, phosphorylated tau is maintained in the hippocampus, entorhinal cortex, and/or piriform cortex. V. Microglial Cell Responses

In some embodiments, administration of a therapeutically effective amount of leboraxan to a subject results in 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.

In some embodiments, administration of a therapeutically effective amount of leboraxan to a subject results in 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.

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. A. Modulation of Microglia Response

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

In some embodiments, modulation refers to increasing or decreasing a microglia response, and/or refers to increasing or decreasing the rate of microglia response. In some embodiments, the number of microglia is not changed, but the microglia response (e.g., activation, reactivation, differentiation, etc.) is modulated. Modulation can vary by brain region (e.g., microglia activation or other responses can occur differently in different brain regions).

In some embodiments, modulation of the microglial cell response is measured in a subject and compared to the microglial cell 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. A reference can be a measurement obtained from more than one control subject that is used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

In some embodiments, regulating the microglia response comprises regulating the expression of at least one microglia marker. The microglia marker may be a general microglia marker. For example, the general microglia marker may be Iba1, Clec7a, or CD68. The microglia marker may be a homeostatic microglia marker. For example, the homeostatic microglia marker is TMEM119 or P2RY12.

In some embodiments, modulating the microglial response comprises modulating the activity of phagocytic microglial cells.

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.

In some embodiments, the subject is amylin negative. The subject may have tau pathology. The subject may 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.

In some embodiments, for those subjects with neurodegeneration, modulating the microglial response includes modulating the response in microglial cells associated with degenerating neurons. For example, microglial cells associated with neurodegenerative neurons can be in close proximity to neurons when observed in a scan or sample obtained from the subject. In some embodiments, microglial cells associated with degenerating neurons can engulf degenerating neurons and/or fragments thereof. Thus, in some embodiments, administering a therapeutically effective amount of leboraxant, a pharmaceutically acceptable salt thereof, or a solvate thereof to the subjects can include reducing the expression of at least one general microglial marker. The general microglia marker may be Iba1, CD68, or Clec7a. In some embodiments, modulating the microglia response comprises increasing the expression of at least one homeostatic microglia marker. The homeostatic microglia marker may be TMEM119 or P2RY12.

In some embodiments, the subject is amyloid positive, for example, the subject has Aβ plaques. The Aβ plaques may be fibrillar Aβ plaques. In some embodiments, the Aβ plaques are present in the hippocampus, motor cortex, somatosensory cortex, and/or piriform cortex of the subject.

In some embodiments, the subject shows no signs of dementia and/or cognitive impairment. In some embodiments, the subject has mild cognitive impairment or mild dementia.

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

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

In some embodiments, in those subjects who are amyloid positive, modulating the microglia response includes modulating the response in microglia associated with Aβ plaques. For example, microglia associated with Aβ plaques can be in close proximity to Aβ plaques when observed in a scan or sample obtained from the subject. In some embodiments, microglia associated with Aβ plaques can phagocytose Aβ plaques. Thus, in some embodiments, administering a therapeutically effective amount of leboraxant, a pharmaceutically acceptable salt thereof, or a solvate thereof to the subjects can include increasing the expression of general microglia markers. The general microglia marker may be Iba1, Clec7a, or CD68. In some embodiments, modulating microglia response comprises increasing phagocytic microglia phagocytosis of Aβ plaques. In some embodiments, modulating the microglia response comprises reducing the expression of a homeostatic microglia marker. The homeostatic microglia marker may be TMEM119 or P2RY12. VI. Amylin Plaques

In some embodiments, administration of a therapeutically effective amount of leboraxan to a subject results in a reduction or maintenance of amyloid plaques compared to a placebo. In some embodiments, administration of a therapeutically effective amount of leboraxan to a subject results in a reduction or maintenance of profibrillar amyloid plaques compared to a placebo. In some embodiments, administration of a therapeutically effective amount of leboraxan to a subject results in a reduction or maintenance of profibrillar amyloid plaques compared to a baseline. In some embodiments, administration of a therapeutically effective amount of leboraxan to a subject results in a reduction or maintenance of profibrillar amyloid plaques compared to a baseline. In some embodiments, the amyloid plaques are protofibrillar amyloid plaques. A. Alteration of Aβ Plaques

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 leboraxant, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the therapeutically effective amount is sufficient to alter Aβ plaques in the subject.

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, preventing 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 occurring concurrently with Aβ plaque formation and/or Aβ plaque growth) or preventing the development of Aβ plaque pathology. Modifying Aβ plaques can reduce, delay or slow the onset and/or rate of progression of symptoms of the pathology. In some embodiments, modifying Aβ plaques includes relieving or ameliorating one or more symptoms of Aβ plaque pathology and/or improving one or more clinical measures of Aβ plaque pathology (e.g., cognitive function, brain amyloid or tau levels and/or biomarker expression). In some embodiments, modifying Aβ plaques includes preventing the occurrence or recurrence of Aβ plaque formation and/or one or more symptoms of Aβ plaque growth.

In some embodiments, the Aβ plaques are altered relative to a reference. Thus, altering Aβ plaques may include reducing and/or delaying Aβ plaque formation, and/or slowing its 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. A reference may be a measurement obtained from more than one control subject that is used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

In some embodiments, the Aβ plaques are protofibrillar plaques. In some embodiments, the Aβ plaques are total plaques and may include non-protofibrillar (e.g., diffuse) plaques.

In some embodiments, altering Aβ plaques comprises reducing the growth or growth rate of Aβ plaques. The reduction in Aβ plaque growth may be in the subject's hippocampus, the subject's motor cortex, somatosensory cortex, and/or piriform cortex. In some embodiments, altering Aβ plaques comprises altering amyloid protein PET information obtained from a brain region of the subject. In some embodiments, altering Aβ plaques corresponds to a reduction in Aβ concentration in the subject's CSF. The Aβ may be Aβ38, Aβ40, and/or Aβ42.

In some embodiments, altering Aβ comprises altering Aβ plaques within 48 hours of administering the first dose of leboraxant.

In some embodiments, the subject shows no signs of dementia and/or cognitive impairment. In some embodiments, the subject has mild cognitive impairment or mild dementia.

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

In some embodiments, the subject has early AD. In some embodiments, the subject has pre-AD. VII. Neurodegeneration

Also disclosed herein is a method of reducing neurodegeneration in a subject, the method comprising administering a therapeutically effective amount of leboraxan to a subject in need thereof. In some embodiments, the subject in need thereof 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 vascular disease, frontotemporal dementia, dementia with Lewy bodies, dementia with Lewy bodies, Parkinson's disease, vascular dementia, marginal dominant age-related TDP-43 encephalopathy, frontotemporal degeneration, cortical basal ganglia degeneration, Pick's disease, multiple system atrophy, and progressive supranuclear neuropathy. In some embodiments, the subject in need has shown signs of at least one disease selected from Alzheimer's disease, pre-Alzheimer's disease, and early Alzheimer's disease. In some embodiments, the subject in need has shown signs of mild cognitive impairment. In some embodiments, the subject in need has shown signs of cerebral vascular lesions, frontotemporal dementia, Lewy body dementia, Lewy body dementia, vascular dementia, marginal dominant age-related TDP-43 encephalopathy, frontotemporal degeneration, corticobasal degeneration. In some embodiments, the subject in need has shown signs of at least one disease selected from Parkinson's disease, Pick's disease, multiple system atrophy, and progressive supranuclear neuropathy.

In some embodiments, the reduction in neurodegeneration is observed by maintaining cortical thickness or slowing the reduction in cortical thickness relative to subjects administered a placebo. In some embodiments, the reduction in neurodegeneration is observed by maintaining cortical thickness or slowing the reduction in cortical thickness relative to the subject's baseline. In some embodiments, the reduction in neurodegeneration is observed by maintaining hippocampal size or slowing the reduction in hippocampal size. In some embodiments, the reduction in neurodegeneration is observed by maintaining or reducing the loss of pyramidal neurons relative to the subject's baseline or relative to subjects administered a placebo. In some embodiments, a reduction in neurodegeneration is observed by maintaining or reducing the loss of granulocytes relative to the subject's baseline or relative to subjects administered a placebo.

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 the CA1 have been observed in AD, and volumetric studies indicate that CA1 atrophy measurements can improve the diagnostic accuracy of the MCI stage. Newer MRI techniques, such as quantitative susceptibility mapping (QSM) or T2* transverse relaxation time, have shown that iron levels and their accumulation rate are heterogeneous in the human brain and are associated with cognitive impairment and reduced 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 measured indirectly with functional magnetic resonance imaging (fMRI). The whole brain or at least one region of the brain (e.g., cortical gray matter (i.e., cortex), lateral ventricles, frontal lobe, parietal lobe, temporal lobe, occipital lobe, cingulate cortex, amygdala, piriform cortex, entorhinal cortex, hippocampus, hippocampal CA3 (pyramidal neurons), and/or hippocampal dentate gyrus (granule cell neurons)) can be analyzed by MRI. In some embodiments, neurodegeneration is reduced within 48 hours of administering the first dose of lebrexan to the subject relative to the subject's baseline. In some embodiments, the amount of p-tau is reduced within 24 hours of administering the first dose of lebrexan to the subject relative to the subject's baseline. In some embodiments, the neurodegeneration is reduced within 12 hours of administering the first dose of lebrexan to the subject, relative to the subject's baseline. In some embodiments, the neurodegeneration is reduced within 6 hours of administering the first dose of lebrexan to the subject, relative to the subject's baseline.

In some embodiments, the neurodegeneration is reduced or maintained for at least 30 days after administration of the first dose of Lebrexan. In some embodiments, the amount of p-tau in the CSF of the subject is reduced or maintained for at least 30 days after administration of the first dose of Lebrexan.

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 administration of the first dose of lebrexan. In some embodiments, the amount of p-tau in the CSF of the subject is reduced for at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 days after administration of the first dose of lebrexan.

In some embodiments, the neurodegeneration is reduced or maintained within 30 days of administration of the first dose of lebrexan. In some embodiments, the amount of p-tau in the CSF of the subject is reduced or maintained for at least 30 days after administration of the first dose of lebrexan. In some embodiments, the neurodegeneration is reduced or maintained within 60 days of administration of the first dose of lebrexan.

In some embodiments, the neurodegeneration is reduced or maintained within 45 days of administering the first dose of lebrexan. In some embodiments, the amount of p-tau in the CSF of the subject is reduced or maintained for at least 45 days after administering the first dose of lebrexan.

In some embodiments, the neurodegeneration is reduced or maintained within 60 days of administration of the first dose of lebrexan. In some embodiments, the amount of p-tau in the CSF of the subject is reduced or maintained for at least 60 days after administration of the first dose of lebrexan.

In some embodiments, the neurodegeneration is reduced or maintained for at least 120 days after administration of the first dose of Lebrexan. In some embodiments, the amount of p-tau in the CSF of the subject is reduced or maintained for at least 120 days after administration of the first dose of Lebrexan.

In some embodiments, the neurodegeneration is reduced or maintained for at least 180 days after administration of the first dose of Lebrexan. In some embodiments, the amount of p-tau in the CSF of the subject is reduced or maintained for at least 180 days after administration of the first dose of Lebrexan.

In some embodiments, the effect of leborexant on neurodegeneration or p-tau is evident after a period of treatment, such as after 3 months, after 6 months, or after 9 months. In some embodiments, neurodegeneration begins to decrease after at least 6 months of treatment. In some embodiments, neurodegeneration decreases after at least 3 months of treatment, such as after 6 months or 9 months of treatment. In some embodiments, the amount of p-tau in the CSF of a 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 a 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, the neurodegeneration is reduced or maintained for at least 6 months after administration of the first dose of Lebrexan. In some embodiments, the amount of p-tau in the CSF of the subject is reduced or maintained for at least 6 months after administration of the first dose of Lebrexan.

In some embodiments, neurodegeneration is reduced for at least 1 year after administration of the first dose of Lebroxan. In some embodiments, the amount of p-tau in the CSF of a subject is reduced for at least 1 year after administration of the first dose of Lebroxan. In some embodiments, the amount of t-tau in the CSF of a subject is reduced for at least 1 year after administration of the first dose of Lebroxan. In some embodiments, the amount of Aβ in the CSF of a subject is reduced for at least 1 year after administration of the first dose of Lebroxan. In some embodiments, the amount of pro-fibrillary plaques is reduced for at least 1 year after administration of the first dose of Lebroxan. In some embodiments, the amount of activated microglia increases for at least 1 year after administration of the first dose of leborexant. A. Modification of Neurodegeneration

Thus, 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 leboraxant, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the therapeutically effective amount is sufficient to alter the neurodegeneration in the subject.

In some embodiments, altering neurodegeneration includes slowing the progression of neurodegeneration, slowing the rate of progression of neurodegeneration, delaying the progression of neurodegeneration, preventing the development of neurodegeneration, and reversing the progression of neurodegeneration. In some embodiments, altering neurodegeneration includes preventing the onset of a pathology caused by or occurring simultaneously with neurodegeneration or preventing its development). Altering neurodegeneration can reduce, delay, or slow the onset of symptoms of the pathology and/or the rate of progression. In some embodiments, altering neurodegeneration includes alleviating or ameliorating one or more symptoms of neurodegeneration and/or improving one or more clinical measures of a pathology caused by or occurring simultaneously with neurodegeneration (e.g., cognitive function, brain amyloid or tau levels, and/or biomarker expression). In some embodiments, altering neurodegeneration comprises preventing the occurrence or recurrence of one or more symptoms of neurodegeneration.

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

In some embodiments, the neurodegeneration is altered relative to a reference. Thus, altering neurodegeneration may include reducing and/or delaying neurodegeneration, 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. A reference may 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.

In some embodiments, the neurodegeneration is characterized by at least one of a loss of cortical thickness or a decrease in hippocampal volume. In some embodiments, altering neurodegeneration comprises maintaining cortical thickness in a subject or slowing a decrease in cortical thickness. In some embodiments, the neurodegeneration is characterized by at least one of a loss of pyramidal neurons in the cortex or a loss of pyramidal or granule neurons in the hippocampus. In some embodiments, altering neurodegeneration comprises maintaining hippocampal volume in a subject or slowing a decrease in hippocampal volume. In some embodiments, altering neurodegeneration comprises maintaining pyramidal neurons or granule neurons or reducing a loss of pyramidal neurons or granule neurons. In some embodiments, altering neurodegeneration comprises reducing the rate of neurodegeneration. In some embodiments, altering neurodegeneration comprises altering neurofilament light chain (NfL) levels. NfL levels in the blood and/or CSF of a subject may be altered. VIII. Selection of Subjects for Treatment

Another aspect of the disclosure relates to a method of selecting a subject having Alzheimer's disease (AD) or at risk of developing AD for treatment with lebrexan, 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 cell response, and biomarker expression from the subject; (b) comparing the measurement from the subject to a measurement from a reference; and (c) selecting the subject for treatment with lebrexan if the measurement from the subject is different from the measurement from the reference.

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.

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

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

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 may indicate levels of tau phosphorylation, tau aggregation, Aβ plaque burden, and/or microglial cell response.

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

In some embodiments, the reference is a control. In some embodiments, the reference is a baseline measurement from a control subject. A reference can be a measurement obtained from more than one control subject that is used as a standard or threshold measurement. In some embodiments, the reference is a measurement from a control subject administered a placebo.

In some embodiments, the control does not have AD. The measured value from the subject may be higher than the measured value from the control that does not have AD. The measured value from the subject may be lower than the measured value from the control that does not have AD.

In some embodiments, the control has AD. The measurement from the subject is similar to or higher than the measurement from the control having AD. The measurement from the subject may be similar to or lower than the measurement from the control having AD.

In some embodiments, the measurement of tau phosphorylation comprises a measurement of phosphorylation at one or more of T181, T217, S202, or S205.

In some embodiments, a measure of tau aggregation comprises a measure of insoluble tau aggregates (e.g., neurofibrillary tangles (NFTs)).

In some embodiments, measures of neurodegeneration include measures of cortical thickness and/or hippocampal volume or measures of pyramidal or granule neuron loss.

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

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

In some embodiments, the measure of small neuroglia response is a change in the expression of at least one small neuroglia marker. The small neuroglia marker can be Iba1, Clec7a, CD68, TMEM119, or P2RY12. In some embodiments, the measure of small neuroglia response is a measure of small neuroglia phagocytosis. IX. Therapeutic Efficacy

Disclosed herein are methods of treating a subject with leboraxant, a pharmaceutically acceptable salt thereof, or a solvate thereof, and methods for monitoring the efficacy of treatment in a subject.

One aspect of the present disclosure relates to a method for monitoring the efficacy of a treatment in a subject having Alzheimer's disease (AD) or at risk for developing AD, the method comprising: (a) obtaining a first measurement 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 leboraxant, 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; and (d) The second measurement from the subject is compared to the first measurement from the subject, wherein a difference between the first measurement and the second measurement indicates effective treatment with leborax.

Another aspect of the present disclosure relates to a method of treating a subject having Alzheimer's disease (AD) or at risk for developing AD, the method comprising: (a) obtaining a first measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial cell response, and biomarker expression from the subject; (b) administering to the subject a first dose of leboraxan, a pharmaceutically acceptable salt thereof, or a solvent complex thereof; (c) obtaining a second measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial cell function, and biomarker expression from the subject; (d) administering to the subject a first dose of leboraxan, a pharmaceutically acceptable salt thereof, or a solvent complex thereof; comparing the second measurement from the subject to the first measurement, and (d) administering a second dose of leborax if the first measurement differs from the second measurement in the compared measurements. The first measurement and the second measurement can differ in that the second measurement is higher than the first measurement. Alternatively, the second measurement can be lower than the first measurement. For example, if the measurement is of small neuroglia response, in some embodiments, the first measurement of small neuroglia response can be higher than the second measurement of small neuroglia response.

Yet another aspect of the present disclosure relates to a method for monitoring the efficacy of a treatment in a subject having Alzheimer's disease (AD) or at risk for developing AD, the method comprising: (a) obtaining a first measurement 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 leboraxan, 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; and (d) A second measurement from the subject is compared to a first measurement from the subject to obtain a comparison measurement, wherein a difference in the comparison measurement between the first measurement and the second measurement or a difference between the comparison measurement and the reference measurement indicates effective treatment with leborax.

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

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 cell response. In some embodiments, the biological sample is a body fluid. In some embodiments, the body fluid is cerebrospinal fluid (CSF), blood, or saliva.

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.

In some embodiments, for example, in those methods including comparison measurements and reference measurements, the reference measurement is obtained from at least one control. In some embodiments, the reference measurement is a comparison of a first measurement from the control and a second measurement from the control. In some embodiments, the comparison measurement is higher than the reference measurement. In some embodiments, the comparison measurement is lower than the reference measurement. For example, a comparison measurement comparing a first measurement from a subject and a second measurement from a subject can indicate that no change has occurred. At the same time, the reference measurement can indicate that a change has occurred in the control. Therefore, the difference between the comparison measurement and the reference measurement can indicate whether the treatment is effective and/or whether a second dose of leborax should be administered.

In some embodiments, the reference measurement is a measurement from a control. A reference measurement can be obtained from more than one control subject, which is used as a standard or threshold measurement. In some embodiments, the reference measurement is a measurement from a control subject administered a placebo.

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

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

In some embodiments, the measurement of tau phosphorylation comprises a measurement of phosphorylation of one or more of T181, T217, S202, S205, or T231.

In some embodiments, a measure of tau aggregation comprises a measure of insoluble tau aggregates (e.g., neurofibrillary tangles (NFTs)).

In some embodiments, measures of neurodegeneration include measures of cortical thickness and/or hippocampal volume or measures of pyramidal or granule neuron loss.

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

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

In some embodiments, the measure of small neuroglia response is a measure of the expression of at least one small neuroglia marker. The small neuroglia marker can be Iba1, Clec71, P2RY12, or TMEM 119. In some embodiments, the measure of small neuroglia response is a measure of small neuroglia phagocytosis.

In some embodiments, the measure of biomarker expression is a measure of Ifnb1, MMP2 and/or Bace1 expression.

In some embodiments, the subject is negative for amylin.

In some embodiments, the subject has Aβ plaques.

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.

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

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

As disclosed herein, a dose of lebrexan may refer to a therapeutically effective amount of lebrexan, a pharmaceutically acceptable salt thereof, or a solvate thereof. In some embodiments, the methods disclosed herein comprise orally administering 5 mg to 20 mg of lebrexan to a subject once daily. In some embodiments, a dose of 20 mg of lebrexan is administered to the subject once daily. In some embodiments, a dose of 25 mg of lebrexan is administered to the subject once daily. In some embodiments, a dose of 25 mg of lebrexan is administered to the subject once daily for at least 2 days. In some embodiments, a dose of 25 mg of lebrexan is administered to the subject once daily. In some embodiments, a dose of 25 mg of Reboraxant is administered to the subject once daily for at least 5 days. In some embodiments, a dose of 25 mg of Reboraxant is administered to the subject once daily. In some embodiments, a dose of 25 mg of Reboraxant is administered to the subject once daily for at least one week. In some embodiments, a dose of 25 mg of Reboraxant is administered to the subject once daily. In some embodiments, a dose of 25 mg of Reboraxant is administered to the subject once daily for at least one month.

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

The dosage forms of the present disclosure contain a therapeutically effective amount of leboraxant for treatment when administered according to the teachings of the present disclosure. The unit dose of the effective amount in the dosage form is 0.5 mg to 100 mg, 2 mg to 75 mg, 2 mg to 70 mg, 2 mg to 65 mg, 2 mg to 60 mg, 2 mg to 55 mg, 2 mg to 50 mg, 2 mg to 45 mg, 2 mg to 40 mg, 2 mg to 35 mg, 2 mg to 30 mg, 2 mg to 25 mg, 2 mg to 20 mg, 2 mg to 15 mg, 2 mg to 15 mg, 2 mg, 2.5 mg, 4 mg, 5 mg, 8 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg. The unit dose is not limited by the type of dosage form or the number of unit 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.

Thus, a therapeutically effective amount of leboraxant, a pharmaceutically acceptable salt thereof, or a solvent thereof as administered herein may include an amount falling within a range, for example, within the range of 5 mg to 50 mg per day.

In some embodiments, the therapeutically effective amount of lebrexan administered to the subject is in the range of 5 mg to 50 mg per day. For example, the therapeutically effective amount of lebrexan administered to the subject can be in the range of 10 mg to 30 mg per day. In some embodiments, the therapeutically effective amount of lebrexan 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.

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

In some embodiments, a dose of 20 mg of lebrexan is administered to the subject once daily.

In some embodiments, Lebrexan 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. Each of the first cycle, the second cycle, and the third cycle can be 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. For example, in some embodiments, the first dose is 5 mg of lebrexan once a day, the second dose is 10 mg of lebrexan once a day, and optionally, the third dose is 20-25 mg of lebrexan once a day. In some embodiments, the first dose is 5 mg or 7.5 mg of lebrexan once a day, the second dose is 10 mg, 12.5 mg, 15 mg, or 17.5 mg of lebrexan once a day, and the third dose is 20 mg, 22.5 mg, 25 mg, 27.5 mg, or 30 mg of lebrexan once a day.

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 lebrexan once a day, the second dose is 10 mg of lebrexan once a day, and optionally, the third dose is 5 mg of lebrexan once a day. In some embodiments, the first dose is 20 mg, 22.5 mg, 25 mg, 27.5 mg, or 30 mg of lebrexan once a day, the second dose is 10 mg, 12.5 mg, 15 mg, or 17.5 mg of lebrexan once a day, and optionally, the third dose is 5 mg or 7.5 mg of lebrexan once a day.

In some embodiments, lebrexan may be administered to a subject over a period of time. In some embodiments, the methods described herein comprise administering lebrexan to a subject for at least 6 months. In some embodiments, the methods described herein comprise administering lebrexan to a subject for at least 9 months, at least 12 months, or at least 15 months. In some embodiments, the methods described herein comprise administering lebrexan to a subject for at least 18 months. In some embodiments, the methods described herein comprise administering lebrexan to a subject for at least 24 months, 30 months, or 36 months. In some embodiments, lebrexan may be administered for the remainder of the subject's life. XI. PHARMACEUTICAL COMPOSITIONS

In some embodiments, the dosage forms of the present disclosure may constitute one or more pharmaceutical compositions comprising leboraxant together with a pharmaceutically acceptable excipient.

As used herein, the term "composition" used herein includes a product containing a specific ingredient in a specific amount and any product resulting directly or indirectly from the combination of a plurality of specific ingredients in a specific amount. 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.

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 non-toxic to the subject.

The solid dosage forms of the present disclosure include capsules, granules, tablets, pellets, granules, powders, suspensions and tablets.

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 the Japanese Pharmacopoeia, 16th edition; and the pharmaceutical dosage forms of Chapter 1151 of the U.S. Pharmacopoeia-NF.

In some embodiments, the pharmaceutical composition comprises leboraxant. 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.

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 may be used include parenteral, oral, inhalation spray, topical, rectal, nasal, buccal, vaginal, and implanted depot administration. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intracisternal, intrathecal, intrahepatic, 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 composition disclosed herein may be, for example, an aqueous or oily suspension. Such suspensions can be prepared according to techniques known in the art using suitable dispersants or wetting agents and suspending agents known in the art. Sterile injectable preparations can also be sterile injectable solutions or suspensions in nontoxic parenterally acceptable diluents or solvents, for example, as solutions 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. In addition, sterile, nonvolatile oils can be used as solvents and/or suspending media.

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 to prepare injectables, as can natural pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated forms. Such oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants commonly used in the preparation of pharmaceutically acceptable dosage forms (including emulsions and suspensions). For the purpose of preparation, other commonly used surfactants can also be used, such as Tweens, Spans and other emulsifiers or bioavailability enhancers, which are commonly used to make pharmaceutically acceptable solids, liquids and/or other dosage forms.

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

In order to more fully understand the disclosure described herein, the following examples are described. It should be understood that the examples are for illustrative purposes only and should not be construed as limiting the disclosure in any way. Non-limiting embodiments of the disclosure: 1. A method for reducing or maintaining the amount of p-tau or t-tau in a subject, the method comprising administering a therapeutically effective amount of leborax to a subject in need thereof. 2. The method as described in embodiment 1, wherein the amount of p-tau or t-tau in the subject is reduced or maintained relative to the subject's baseline. 3. The method as described in 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. 4. The method of any one of embodiments 1-3, wherein the ratio of p-tau to tau in the subject is lower after administration of a therapeutically effective amount of lebrexan, relative to the subject's baseline or relative to a placebo. 5. The method of any one of embodiments 1-4, wherein the rate of dephosphorylation of tau in the subject is higher after administration of a therapeutically effective amount of lebrexan, relative to the subject's baseline or relative to a placebo. 6. The method of any one of embodiments 1-5, wherein the rate of tau phosphorylation in the subject is lower after administration of a therapeutically effective amount of lebrexan, relative to the subject's baseline or relative to a placebo. 7. The method of any one of embodiments 1-6, wherein the ratio of p-tau/t-tau in the subject's CSF is reduced compared to the ratio of p-tau/t-tau in the subject's CSF prior to administration of lebrexan. 8. The method of embodiment 7, wherein the ratio of p-tau/t-tau in the subject's CSF is maintained within 10% of the ratio of p-tau/t-tau in the subject prior to administration of lebrexan. 9. The method of any one of embodiments 1-8, wherein the Aβ concentration in the subject's CSF is less than or equal to the Aβ concentration in the subject's CSF prior to administration of lebrexan. 10. The method of embodiment 9, wherein the amyloid PET message in the brain of the subject administered a therapeutically effective amount of leboraxan is lower or the same as the amyloid PET message in the brain of the subject prior to administration of leboraxan. 11. The method of any of embodiments 1-10, wherein the tau PET message in the brain of the subject is reduced relative to baseline. 12. The method of any of embodiments 1-11, wherein the tau is p-tau, t-tau, or aggregated tau. 13. The method of any of embodiments 1-12, wherein p-tau and t-tau are reduced. 14. The method of any of embodiments 1-13, wherein the reduction in tau phosphorylation occurs in the hippocampus, entorhinal cortex, and/or piriform cortex. 15. The method of any one of embodiments 1-14, wherein the Aβ in the CSF of the subject is Aβ38, Aβ40 and/or Aβ42. 16. The method of any one of embodiments 1-15, wherein the amount of p-tau is reduced within 48 hours of administering a first dose of lebrexan to the subject relative to the subject's baseline. 17. A method of reducing neurodegeneration in a subject, the method comprising administering a therapeutically effective amount of lebrexan to a subject in need thereof. 18. The method of embodiment 17, wherein the reduction in neurodegeneration is observed by maintaining cortical thickness or reducing the reduction in cortical thickness relative to the subject's baseline or relative to a subject administered a placebo. 19. The method of embodiment 17 or embodiment 18, wherein the reduction in neurodegeneration is observed by maintaining hippocampal size or slowing the reduction in hippocampal size relative to the subject's baseline or relative to subjects administered a placebo. 20. The method of any of embodiments 17-19, wherein the reduction in neurodegeneration is observed by maintaining or reducing the loss of pyramidal neurons relative to the subject's baseline or relative to subjects administered a placebo. 21. The method of any of embodiments 17-20, wherein the reduction in neurodegeneration is observed by maintaining or reducing the loss of granule neurons relative to the subject's baseline or relative to subjects administered a placebo. 22. The method of any one of embodiments 1-21, wherein the therapeutically effective amount of lebrexan administered to the subject is in the range of 10 mg to 50 mg per day. 23. The method of embodiment 22, wherein the therapeutically effective amount of lebrexan administered to the subject is in the range of 15 mg to 30 mg per day. 24. The method of embodiment 22, wherein the therapeutically effective amount of lebrexan administered to the subject is 25 mg per day. 25. The method of any one of embodiments 1-24, wherein a dose of 25 mg of lebrexan is administered to the subject once daily. 26. The method of embodiment 25, wherein a dose of 25 mg of lebrexan is administered to the subject once daily for at least two days. 27. The method of any one of embodiments 1-26, wherein a dose of 2.5 mg, 5 mg, 10 mg, 15 mg, or 20 mg of lebrexan is administered to the subject once daily at least two days after a dose of 25 mg of lebrexan is administered once daily. 28. A method of reducing Aβ concentration in a subject, the method comprising administering a therapeutically effective amount of lebrexan to a subject in need thereof. 29. The method of any one of embodiments 1-28, wherein the Aβ concentration in the subject's CSF is lower than the Aβ concentration in the subject's CSF prior to administration of lebrexan. 30. The method of any one of embodiments 1-29, wherein the amyloid protein PET message in the subject's brain of the subject administered lebrexan is lower than the amyloid protein PET message in the subject's brain before administration of lebrexan. 31. The method of embodiment 30, wherein the Aβ in the subject's CSF is Aβ38, Aβ40 and/or Aβ42. 32. The method of any one of embodiments 1-29, wherein the concentration of Aβ is reduced within 48 hours of administration of lebrexan. 33. A method for increasing the number of activated microglia in a subject, the method comprising administering a therapeutically effective amount of lebrexan to a subject in need thereof. 34. The method of embodiment 33, wherein the number of activated microglia is increased relative to baseline. 35. The method of any of embodiments 33 or 34, wherein the activated microglia are phagocytic microglia. 36. The method of any of embodiments 33-35, wherein the number of activated microglia is measured by PET or CSF biomarkers of microglia activation. 37. The method of any of embodiments 33-36, wherein the whole brain or at least one region of the brain is analyzed by PET. 38. The method of embodiment 37, wherein at least one region of the brain analyzed by PET is selected from the group consisting of 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). Examples Example 1 : Clinical study protocol a. Trial 1 : Acute effects of leboraxan on CSF β - amyloid protein and Tau

The acute effects of leborax will be studied in cognitively normal, amyloid-positive participants.

Inclusion criteria will be: o Age 60-80 years o Any gender o Any race/ethnicity o Mini-Mental State Examination score (MMSE) ≥ 27 o Positive plasma Aβ test (i.e., amyloid positive) o Pittsburgh Sleep Quality Index > 5

Exclusion criteria will be: o Cognitive impairment as determined by history of MMSE < 27 o Inability to speak or understand English o Any sleep disorder other than insomnia ▪   No history of moderate to severe sleep-disordered breathing with STOP-Bang score > 5 ▪   History or reported symptoms suggestive of restless legs syndrome, narcolepsy, or other sleep disorders ▪   No more than mild sleep apnea on PSG (AHI < 16) o Sleep schedule outside of bedtime range 22:00-midnight o Contraindications to lumbar catheter (anticoagulants; bleeding disorders; allergy to lidocaine or antiseptics; previous CNS or low back surgery) o Cardiovascular disease requiring medication, excluding controlled hypertension (PI judgment) o Stroke o Liver or kidney damage o Lung disease (PI judgment) o Type 1 diabetes o HIV or AIDS o Neurological or psychiatric disorder requiring medication (PI judgment) o Suicidal ideation o Alcohol or tobacco use (PI judgment) o Use of sedative medication (PI judgment) o Inability to get out of bed independently o Participants with abnormal physical examinations that, in the opinion of the investigator, should be excluded. o Current pregnancy o Body mass index > 35 o History of migraine (PI judgment) o History of substance abuse in the past 6 months o History or presence of any clinically significant medical condition, behavioral or psychiatric disorder (including suicidal ideation) that could affect subject safety or interfere with study assessments or PI participant judgment, or surgical history that would not make the subject a good candidate based on medical history or patient report. o Urinary or fecal incontinence o Concurrent enrollment in another trial of an investigational drug or device

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

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

At approximately 20:00, a lumbar catheter and two IV catheters will be placed in each participant to collect 6 ml of CSF every 2 hours for 48 hours. The lumbar catheter ports will be placed on the outside of the gown sleeve for easy access to minimize disturbance during fluid collection. The sampling start time will begin approximately 1 hour before the typical bedtime defined by each participant's sleep diary to allow for ^13C ₆ -leucine infusion and frequent blood sampling before bedtime. 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) . [ Table 1 ] Timeline of catheter placement and removal, bedtime, and sampling Hours 0 1 2 4 6 8 10 12 14 16 18 20 twenty two twenty four event C 、X B X X X X X X X X X X X X Hours 25 26 28 30 32 34 36 38 40 42 44 46 48 49 event B X X X X X X X X X X X X. R B C : catheter placement; B : bedtime; X : CSF/blood sampling; R : catheter removal

Approximately 1 hour before habitual bedtime (t = 0) on day 1, all participants will begin an infusion of 800 mg labeled ¹³ C ₆ -leucine to label the protein in vivo during intracellular translation to monitor Aβ kinematics.

Participants will be allowed to sleep as they will be able to turn off the lights immediately at their habitual bedtime (bedtime is at hour 1, approximately 22:00-00:00). A dim red light (safety light) will be used when collecting CSF and blood in the dark. Participants will sleep until the last awakening in the morning.

During Day 2, all participants will be kept awake in a well-lit room, monitored regularly, and no naps will be allowed. At approximately 22:00 (hour 25) on Day 2, all participants will follow the same sleep routine as they did the night before, except that there will be no infusion of labeled ^13C ₆ -leucine and the time of "hour 26" will depend on the participant's regular bedtime. Participants will receive the same placebo or Lebrexan dose as the night before. The study will end at approximately 20:00 (hour 48) on Day 3, when the IV catheter and lumbar catheter will be removed. All participants will then sleep overnight and will be monitored for at least 8 hours after catheter removal before being discharged.

CSF tau, phosphorylated tau, and Aβ kinetics will be quantified by mass spectrometry. For each participant's baseline, AD biomarkers (Aβ38, Aβ40, Aβ42, T181, S202, T217, pT181, pS202, pT217) will be normalized to the mean of the first 6 hours (t = 0-6, 20:00-02:00) before intervention changes are observed in CSF. 0 hour concentrations 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). The changes relative to these baselines were then plotted over time by treatment arm to examine whether the changes in concentrations or ratios were linear.

For statistical analysis, a (linear mixed-effects) LME model with random intercepts and slopes will be used if the linearity assumption is valid, otherwise a mixed model with 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. Normality assumptions will be checked using residual plots, and appropriate transformations (e.g., logarithmic) will be considered. An unstructured covariance matrix will be used, and if there are convergence issues, various other covariance matrix structures will be compared (e.g., compound symmetry, first-order autoregression), and the best-fitting structure will be selected for the final analysis based on the Akaike information criterion (AIC). b. Trial 2 : Treatment of subjects with preclinical or early Alzheimer's disease

The trial will evaluate the efficacy of leborabine in preventing or delaying Aβ accumulation, downstream spread of tau pathology, and cognitive decline in a range of preclinical and early AD.

Aβ aggregation often precedes the clinical stage of Alzheimer's disease (AD) by more than a decade and is thought to play a key role in accelerating the spread of tauopathy and neurodegeneration during the preclinical phase of the disease. Multiple neuroimaging and biomarker observational studies have demonstrated that Aβ aggregation is associated with an increased risk of cognitive decline in clinically normal elderly individuals.

The trial will utilize NAV4694 (flutafuranol) amyloid PET imaging to assess eligibility and longitudinal outcome of fibrillar amyloid pathology, and MK6240 tau PET tracer to assess neurofibrillary tangles and longitudinal spread of tau neurite pathology. Clinical outcomes include the Preclinical Alzheimer's Disease Cognitive Composite 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.

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. i. Group 1 : Patients with preclinical Alzheimer's disease - moderate levels of amyloid

The trial aims to determine whether leborabine treatment leads to primary prevention or delay of AD by preventing or slowing early Aβ accumulation in the brain. The trial will enroll cognitively normal individuals with intermediate levels of amyloid proteins (approximately 20-40 percentiles) on screening PET imaging who are considered to be in the earliest preclinical stages of AD and at risk for further Aβ accumulation and early spread of tau pathology within four years.

Patient inclusion criteria. Patients will be selected for intact cognition, as determined by an education-adjusted Mini-Mental State Examination (MMSE) score ≥ 27, with a global clinical dementia rating (CDR) of 0. Patients will have intermediate levels of amyloid on PET imaging (20-40 percentile).

Lebrexan Administration. Participants will be administered Lebrexan or matching placebo monthly during in-person study visits where treatment adherence and adverse events will be assessed. Lebrexan is FDA-approved for the treatment of insomnia at doses of 5-10 mg. Lebrexan doses may be increased to 20 mg following interim analyses of safety and efficacy. At the start of the study, participants will take Lebrexan 5 mg once daily for 1 week, then increase to Lebrexan 10 mg once daily; there will be a matching placebo tablet for both Lebrexan 5 mg and Lebrexan 10 mg. At the end of the study, participants will take Lebrexan 5 mg once daily for 1 week before discontinuing treatment.

Medication Titration. Because participants will be lebrexan naive, lebrexan 5 mg once daily will be offered for 1 week, followed by titration to lebrexan 10 mg once daily; there will be matching placebo tablets for lebrexan 5 mg and lebrexan 10 mg. At the end of the study, participants will titrate down to lebrexan 5 mg once daily within 1 week before discontinuation.

If the leboraxan dose is increased to 20 mg after the interim analysis, the same titration-up and titration-down schedule will be followed, except there will be an additional week of taking leboraxan 10 mg: Titration-up: leboraxan 5 mg-1 week o             leboraxan 10 mg-1 week o             leboraxan 20 mg-1 week Titration-down: leboraxan 10 mg-1 week o             leboraxan 5 mg-1 week o             Stop

Study outcomes. Anticipate measures of primary, secondary, and exploratory outcomes.

Primary Outcome: The primary outcome measure of the trial will be amyloid PET SUVr at 6 months, measured and compared to placebo. Biomarker outcomes will include tau PET, measurements of Aβ, phosphorylated tau, and basal fibrils in CSF and plasma.

Secondary outcomes: tau PET will be measured.

Exploratory Outcomes: In CSF, the biomarkers Aβ, tau, phosphorylated tau, neurogranin (NG), 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 testing of the Preclinical Alzheimer's Disease Cognitive Composite 5 (PACC5) scale and the Cognitive Function Index (CFI) to obtain cognition. ii. Preclinical Alzheimer's Disease - Elevated levels of amyloid

The trial aims to determine whether leborabine treatment leads to primary prevention or delay of AD by preventing early Aβ accumulation in the brain. The trial will enroll cognitively normal individuals with elevated levels of amyloid protein (approximately >40 percentiles) on screening PET imaging who are at high risk for cognitive decline over a four-year period.

Patient inclusion criteria. Patients will be selected for intact cognition, as determined by an education-adjusted Mini-Mental State Examination (MMSE) score ≥ 27, with a global clinical dementia rating (CDR) of 0. Patients will have elevated amyloid PET levels > 40.

Lebrexan Administration. Participants will be administered Lebrexan or matching placebo monthly during in-person study visits where treatment adherence and adverse events will be assessed. Lebrexan is FDA-approved for the treatment of insomnia at doses of 5-10 mg. Lebrexan doses may be increased to 20 mg following interim analyses of safety and efficacy. At the start of the study, participants will take Lebrexan 5 mg once daily for 1 week, then increase to Lebrexan 10 mg once daily; there will be a matching placebo tablet for both Lebrexan 5 mg and Lebrexan 10 mg. At the end of the study, participants will take Lebrexan 5 mg once daily for 1 week before discontinuing treatment.

Medication Titration. Because participants will be lebrexan naive, lebrexan 5 mg once daily will be offered for 1 week, followed by titration to lebrexan 10 mg once daily; there will be matching placebo tablets for lebrexan 5 mg and lebrexan 10 mg. At the end of the study, participants will titrate down to lebrexan 5 mg once daily within 1 week before discontinuation.

If the leboraxan dose is increased to 20 mg after the interim analysis, the same titration-up and titration-down schedule will be followed, except there will be an additional week of taking leboraxan 10 mg: Titration-up: leboraxan 5 mg-1 week o             leboraxan 10 mg-1 week o             leboraxan 20 mg-1 week Titration-down: leboraxan 10 mg-1 week o             leboraxan 5 mg-1 week o             Stop

Study outcomes. Anticipate measures of primary, secondary, and exploratory outcomes.

Primary Outcome. To test the effect of leborabine on cognitive decline in patients with elevated amyloid proteins, the primary outcome measure of the trial will be the Preclinical AD Cognitive Composite 5 (PACC5) at 6 months. Biomarker outcomes will include tau PET, and measures of Aβ, phosphorylated tau, and basal fibrils in CSF and plasma.

Secondary outcomes. Cognitive Function Index (CFI) will be measured. Amylin PET and tau PET will be measured.

Exploratory endpoints. Clinical measures will be ADCS ADL-Prevention, Computerized Cognitive Composite, ISLT, Follow-up, CDR-SB, and time to CDR 0.5. vMRI and rs-MRI will be determined. In CSF, biomarkers Aβ, tau, phospho-tau, neurogranin (NG), neurofilament light chain (NfL) will be measured. In plasma, NfL, phospho-tau 181, and phospho-tau 217 will be measured. c. Trial 3 : Treatment of subjects with preclinical or early Alzheimer's disease

This trial will evaluate the efficacy of lebrexan in preventing or delaying Aβ accumulation, downstream spread of tau pathology, and cognitive decline in a range of preclinical and early AD. This trial will follow the protocol of Trial 2 except that different doses will be used. 7.5 mg of lebrexan will be used instead of 5 mg of lebrexan. 15 mg of lebrexan will be used instead of 10 mg of lebrexan. 25 mg of lebrexan will be used or 30 mg of lebrexan will be used instead of 20 mg of lebrexan. Example 2 : Mouse Study of Tau -Mediated Neurodegeneration

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

Changes in sleep-wake behavior

Changes in sleep-wake behavior were confirmed by electroencephalography (EEG) analysis. E4 and P301S/E4 mice treated with Lebrexan showed an increase of approximately 25% in the time spent in NREM sleep and a decrease of approximately 20% in the time spent awake (Fig. 2b-f). No changes in REM sleep time were observed, consistent with previous findings.

A reduction in tau-dependence of NREM sleep was observed in P301S/E4 mice compared to E4 mice (Fig. 2b), indicating that pathological tau is linked to sleep. The effects of leborexant on sleep (Fig. 2g and 2h) and sleep-related motor activity (Fig. 6), measured by EEG and Piezosleep pad, persisted for approximately five hours after treatment, with no additional phase delay or changes in sleep-wake activity during the day or night.

When the drug is administered, Lebrexan induces sleep during the active phase of the dark phase. Figure 2I shows that after administration of Lebrexan in E4 and P301S/E4 mice, the percentage of sleep during the dark phase increased. Figure 2J shows that after administration of Lebrexan in E4 and P301S/E4 mice, the length of sleep episodes (in seconds) during the dark phase increased. Figure 2K shows that after administration of Lebrexan in E4 and P301S/E4 mice, the length of wake episodes (in seconds) decreased.

Implications for tau pathology and neurodegeneration

To determine whether leborexant could affect tau pathology and neurodegeneration, the hippocampus and entorhinal and piriform cortices, regions that show significant tau-mediated degeneration, were studied. A significant reduction of approximately 20% in AT8+ and MC1+ tau staining control was observed in leborexant-treated P301S/E4 mice compared to vehicle-treated mice (Figure 3a-f). In addition, leborexant-treated P301S/E4 mice had significantly reduced insoluble phosphorylated and total tau levels (Figure 7a-f).

We found that brain atrophy was strongly reduced in P301S/E4 mice treated with leborexant compared with controls (Fig. 3g-i and Fig. 7h,i). More specifically, atrophy of the hippocampus and piriform/entorhinal cortex was significantly improved by approximately 50%, accompanied by reduced enlargement of the lateral ventricles. Granulocyte and pyramidal neuronal layers were significantly thicker in P301S/E4 mice treated with leborexant (Fig. 7g to 7k), as confirmed by reduced plasma neurofilament light chains levels (Fig. 3j), demonstrating robust improvement in neuronal damage and degeneration.

Microglial cell responsiveness

To investigate whether the significant reduction in tau-mediated neurodegeneration in leborexant-treated mice was linked to reduced microglial reactivity, different markers of reactive microglial cells were quantified in the spectrum of disease-associated or stable populations in the hippocampus and piriform cortex. Significant changes were observed (Figure 4), mainly in the hippocampus and most notably in the CA3 region (Figure 4).

A significant decrease in ionized calcium binding adapter 1 (IBA1) was observed, indicating an overall decrease in the reactive microglial population in leborabine-treated P301S/E4 mice compared with vehicle-treated P301S/E4 mice (Fig. 4A, C, L, and M).

Disease-associated microglial cell markers, such as Clec7a (Fig. 3E, G) and CD68 (Fig. 4A, D, P, Q), a marker of phagolysosomal activity, were significantly increased in P301S/E4 mice relative to E4 mice, but leborexant strongly reduced these markers compared with vehicle-treated P301S/E4.

A significant increase in the TMEM119 marker of homeostatic microglia was observed in P301S/E4 mice treated with Lebrexan (Fig. 4B, F, N, and O), whereas no changes were observed in P2RY12 (Fig. 8).

These results suggest that reduced reactive microglia are involved in the role of leborexant in modulating tau-mediated neurodegeneration. Furthermore, both astrocyte and microglia APOE colocalization was significantly reduced in leborexant-treated P301S/E4 mice compared with controls (Fig. 4H-K), with the latter being more frequent only during periods of elevated inflammatory and injurious pathology. Leborexant treatment reduced GFAP+ astrocyte reactivity in the hippocampus of P301S/E4 mice (Fig. 4R). No changes in microglia reactivity were found in E4 knock-in mice in the absence of tau pathology.

RNA sequencing

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

Based on these data, sleep, in which there is an overall decrease in neuronal metabolism and possibly synaptic activity, may play a key role in neuronal genome maintenance and DNA repair function. However, further research is needed to understand the connection between sleep, DNA damage and neurodegeneration. Interestingly, genes such as Adra2b, Trh, Trhr2, Mpzl2, Slc22a6, Pla2g2f, Ptgdr, Foxp2 are genes that regulate sleep. More specifically, thyrotropin-releasing hormone (Trh) and its receptor (Trhr2) regulate behavioral arousal in part through wakefulness. TRH application switches GABAergic neurons from a burst-firing mode normally associated with synchronized cortical activity occurring during NREM sleep to a tonic, single-spike mode of action potential generation associated with desynchronized cortical activity occurring during wakefulness and REM sleep. Downregulation of Trh and Trhr2 suggests that DORA-induced NREM sleep further interacts with 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. In support of these findings, we observed changes in the expression of Slc22a6, Pla2g2f, and Ptgdr, which modulate sleep-wakefulness by potent endogenous hypnotic agents such as prostaglandins. In addition to catalyzing the biosynthesis of prostaglandins, phospholipase A2 plays a major role in cell growth, differentiation, and inflammation and has been associated with metabolic changes in patients with sleep apnea. Similarly, genes involved in glial differentiation, including microglial-expressed Tmem119, Tmem114, Cd68, Aif1, Cd300a, Pea15a, and H2-Q1, were differentially regulated in P301S/E4 mice treated with leborexant (Figure 5C), suggesting that promoting leborexant-mediated NREM sleep or inhibiting leborexant signaling affects important immune functions, such as T cell antigen presentation, 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 glutamine transporters (VGLUT1, Slc17a7) and postsynaptic density markers (PSD95, Shank1, Shank2) (Figure 5C-5I). Transcriptional changes in the activity and organization of synaptic receptors such as Otof, Nrxn3, Mrgprf, Mapk13, and Fmod (Figure 5C) demonstrate that promoting NREM sleep or inhibiting ghrelin receptor signaling reduces tau-mediated neurodegeneration and associated synaptic loss.

Methods and analysis

Mice: All animal procedures and protocols were approved by the Animal Research Committee of the 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 with C57BL/6 for more than ten generations. Human apoE4 knock-in mice were generated as described in Mol. Neurodegener. 14 , (2019) and outcrossed with P301S mice for several generations to generate experimental P301S/E4 mice. Littermates of the same sex were randomly assigned to experimental groups. Only male animals were used and sacrificed at 9.5 months of age. All mice were housed under specific pathogen-free conditions with the same 12-h light/dark cycle, ambient room temperature, and ad libitum access to food and water.

Treatment: Mice were housed daily with a single 30 mg/kg dose of leboraxan or 0.5% methylcellulose vehicle at ZT13 (1 hour after the onset of darkness) starting at 7.5 months of age until euthanasia at 9.5 months of age.

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

Sleep-wake state measurements and analysis: Sleep-wake behavior of mice was monitored using electroencephalography (EEG) and independently using the PiezoSleep mouse behavior tracking system (SignalSolutions).

For EEG experiments, animals were anesthetized with isoflurane (0.5%-3%). Any signs of pain were assessed by toe pinching before the incision was made. Screw electrodes were then surgically implanted in the skull of mice for EEG and stainless steel wire electrodes were implanted in the dorsal cervical muscles for electromyography (EMG). After a midline vertical incision to expose the skull, tweezers and 3% hydrogen peroxide were used to remove any connective tissue and dry the skull for electrode placement. 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 the screw was fixed in the skull. Using the same technique as the reference electrode, two bilateral active recording electrodes were placed on the parietal cortex (−2.5 mm posterior, ±1.5 lateral; bregma) and the foot screws were fixed to the cerebellum (−6.2 mm posterior, ±0.5 lateral; bregma). The exposed skull, screws, and all metal 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 remainder of the incision was closed using tissue glue (Vetbond, 3M). After the procedure, mice were placed in a warm chamber to fully recover from anesthesia and housed individually in monitoring cages with fresh bedding, water, food, and Carprofen (oral; ¼ of a 5 g tablet; ad libitum) supplement. After three days of recovery from surgery, mice were habituated to the recording cages for two weeks, followed by two consecutive days of undisturbed EEG/EMG recordings in freely moving mice. Bilateral cortical EEG information was acquired by P511K A.C. Preamplifiers (Grass-Telefactor Instruments, Warwick, RI USA), digitized with a BIOPAC MP150, digitally recorded at a sampling rate of 250 Hz using BIOPAC's AcqKnowlege software, and converted to (.edf) format for analysis. EEG was processed in MATLAB (MathWorks) with a 1–30 Hz bandpass filter to remove DC offset and high-frequency noise. EEG/EMG recordings were manually scored for wakefulness, NREM, and REM sleep in 10-second epochs to create calibration files containing a hybrid-z-score variable specific to the subject being recorded. The calibration file was fed into AccuSleep, an automated sleep scoring program based on machine learning in MATLAB, to complete the rest of the scoring.

The PiezoSleep mouse behavioral tracking system (Signal Solutions, LLC, Lexington, KY, USA) was used. The non-invasive method consists of a thin dielectric piezoelectric inductor pad that generates a voltage signal in response to changes in real-time fluctuations in pressure on its surface. Mice were individually housed with the piezoelectric pad placed underneath fresh bedding with ad libitum access to fresh water and food and recorded undisturbed over six days. Data were acquired using SleepStats software (Signal Solutions, LLC, Lexington, KY, USA).

Volumetric analysis: Volumetric analysis of the hippocampus, entorhinal/piriform cortex, and ventricles was performed by stereologic evaluation of 180 μm sections starting from bregma -1.3 mm to bregma -3.1 mm (16-18 sections per mouse, depending on the severity of brain atrophy). 30 μm microtome-cut sections mounted on slides were briefly immersed in distilled water and then incubated in pre-warmed 0.1% cresol violet at 37°C for six minutes. After this, the tissues were rinsed in distilled water and sequentially transferred to 70%, 95%, and 100% ethanol for two minutes each. Slides were then washed in xylene and coverslipped with cytoseal60 mounting medium (Thermo Fisher Scientific). Slides were scanned at 20X magnification using a Hamamatsu Nanozoomer microscope. The hippocampus, EC/PC, and ventricle were tracked using NDP.view 2. Volume was calculated using the formula volume = (sum of areas) * 0.3 mm.

Neuronal layer thickness measurements: The dentate granule and entorhinal cone cell layers were measured in three sections by drawing tick marks through the cell layers using NDP.view 2 and calculating the mean for each mouse.

Immunohistochemical analysis: Free-floating sections were briefly washed with TRIS-buffered saline (TBS-Tx) containing 1% TritonX100, and then quenched with 0.3% hydrogen peroxide for 20 minutes at room temperature to quench endogenous peroxidase. After a brief wash, sections were blocked with 5% goat serum for 30 minutes at room temperature, and then incubated with primary antibodies 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 in HRP-conjugated secondary antibodies for one hour at room temperature. AT8- and MC1-stained tissues were then developed with 3,3'-diaminobenzidine (DAB, Sigma) for 10 and 14 minutes, respectively. Tissue sections were mounted on slides and dehydrated using a series of increasing ethanol concentrations before a final immersion in xylene and coverslipping using Cytoseal mounting medium. Slides were scanned at 20X magnification using a Hamamatsu Nanozoomer microscope.

Immunofluorescence staining: Free-floating sections were briefly washed with PBS and blocked in 5% donkey serum for 1 hour at room temperature. Unless otherwise stated, primary antibodies were diluted in blocking buffer and incubated overnight at 4°C with gentle agitation. Primary antibodies were used as follows: IBA1 (1:500; 019-19741, Fujifilm or NB100-1028, Novus Biologicals), CD68 (1:00; FA-11, BioRad), P2RY12 (1:100 at room temperature, HPA013796, Sigma-Aldrich), TMEM119 (1:500; E3E1O, Cell Signaling Technology), Clec7a (1:50 at room temperature; mabg-mdect, InvivoGen), GFAP (1:2000; 2E1.E9 Alexa Flour 488-conjugated, BioLegend), APOE (1:300; D7I9N, Cell Signaling Technologies), PSD-95 (1:200, 51-6900, Thermo Fisher Scientific), VGLUT1 (1:200, AB5905, Merck Millipore). The next day, sections were washed and incubated with secondary antibodies diluted in blocking buffer, followed by 4′,6-diamidino-2-phenylindole (DAPI, 5 μg/mL), and then mounted on slides (Prolong™ Gold Antifade Reagent, Thermo Fisher Scientific).

Confocal imaging and analysis: Images were acquired using a Leica Stellaris 5 confocal microscope and Leica Application Suite X software (4.2.1.23810). For the acquisition of each immunostaining, the laser and detector settings remained unchanged. For all analyses, at least two images per brain region and slide were acquired 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 synaptic imaging, super-resolution confocal images based on self-adjusted deconvolution were generated using a Leica Stellaris 8 Lightning with 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 projection). Microglia were then segmented using an automatic thresholding method in Fiji and presented as % area covered by the selected stain in the hippocampus or entorhinal/piriform cortex.

Protein extraction: Frozen mouse hippocampal tissue was weighed and homogenized with 200 μl RAB buffer pH 7.0 (100 mM MES, 1 mM EGTA, 0.5 mM MgSO4, 750 mM NaCl, 20 mM NaF, 1 mM Na3VO4) supplemented with 1x protease inhibitor (cOmplete™, Roche) and 1x phosphatase inhibitor (PhosSTOP, Roche) in a bullet mixer homogenizer (Next Advance) using a molten bead tube. The homogenate was centrifuged at 5000 × g for five minutes at 4°C to precipitate RAB insoluble material, and the supernatant was ultracentrifuged at 50'000 × g for 20 minutes in an Optima MAX-XP ultracentrifuge (Beckman Coulter) using an MLA-130 rotor to obtain a RAB extract. Proteins were extracted from the remaining cell pellet using 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 solubilized with ice-cold 70% formic acid (FA) and sonicated at 30% amplitude in short pulses for one minute at room temperature using an ultrasonic oscillator (model FB120, Fisher Scientific), followed by a final ultracentrifugation at 50'000 × g for 20 minutes at 4°C. The protein concentration of the RIPA fraction was measured using the BCA assay (Pierce). All samples were aliquoted and frozen at -80°C until use.

Tau ELISA: Human tau and pTau in RAB, RIPA, and 70% FA fractions were measured using sandwich ELISA and normalized to tissue weight as described28. 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. 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.

NFL concentration: Plasma NFL concentration was measured using the NF-Light Simoa Advantage Kit and the Quanterix. The measurements were performed according to the manufacturer's instructions.

RNA extraction: Frozen hippocampal tissue was weighed and homogenized in chloroform containing TRIzol™ in an RNase-free bead tube (REDE, Next Progress). Samples were centrifuged at 12'000xg for 15 min at 4°C, and the aqueous supernatant was transferred for RNA isolation using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. RNA quality was controlled using a Bioanalyzer and then next-generation sequencing was performed by Clontech SMARTer.

RNA sequencing and analysis: Samples were prepared, indexed, pooled, and sequenced on an Illumina NovaSeq 6000 according to the library kit manufacturer's protocol. Base calls and demultiplexing were performed with Illumina's bcl2fastq software and a custom python demultiplexer with a maximum of one mismatch in the indexed reads. RNA-seq reads were then aligned to the Ensembl release 76 master element using STAR version 2.7.9a (Doblin et al.). Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 2.0.3. Isoform representation of known Ensembl transcripts was quantified using Salmon version 1.5.2. Sequencing performance was assessed for 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. All gene counts were then imported into the R/Bioconductor package EdgeR, and TMM normalized size factors were calculated to adjust for differences in library size across samples. Ribosomal genes and genes not represented in samples with the minimum group size minus one count per million were excluded from further analysis. TMM size factors and count matrices were then imported into the R/Bioconductor package Limma. The weighted likelihood based on the observed mean-variant relationship for each gene and sample was then calculated for all samples using the voomWithQualityWeights function and fit using the Limma generalized linear model with additional unknown potential effects identified by substitution variable analysis (SVA). The performance of all genes was assessed using a plot of the standard deviation of the residuals for each gene versus their mean log counts, where the trend lines of the residuals fit very well. A differential performance analysis was then performed to analyze differences between conditions, and results were filtered only for those genes with a Benjamini-Hochberg pseudodiscovery rate-adjusted p-value less than or equal to 0.05.

For each control extracted with Limma, global perturbations in known gene ontology (GO) terms, MSigDb, and KEGG pathways were detected using the R/Bioconductor package GAGE9 to test the performance change of the reported log2 fold change reported by Limma in each term relative to the background log2 fold change 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 with a Benjamini-Hochberg pseudo-discovery rate-adjusted p-value less than or equal to 0.05. The perturbed KEGG pathways were presented as annotated KEGG plots using the R/Bioconductor package Pathview, where the observed log2 fold changes of genes within the term were significantly perturbed relative to background in a single direction or compared to other genes within a given term in any direction with a p-value less than or equal to 0.05.

To find the most critical genes, Limma voomWithQualityWeights transformed log2 counts per million expression data were then analyzed by weighted gene correlation network analysis using the R/Bioconductor package WGCNA. In other words, all genes were correlated with each other by Pearson correlation and clustered into unsigned modules by expression similarity using thresholds empirically determined from the data. Eigengenes were then created for each de novo cluster and their expression profiles were then correlated with all coefficients of the model matrix. Because the gene clusters were created by expression profiles rather than known functional similarities, the clustering modules were given random colored names, with gray being the only module with any pre-existing definitions containing genes that did not cluster well with other genes. The de novo clustered genes were then tested for functional enrichment of known GO terms using a hypergeometric test available in the R/Bioconductor package clusterProfiler. Valid terms with a Benjamini-Hochberg adjusted p-value less than 0.05 were then compressed by similarity into a clusterProfiler class network graph to display 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 whether these features were also found to be significantly differentially represented.

Statistical analysis: All statistical analyses were performed using Graphpad Prism 8.0. Unless otherwise stated, data are presented as mean ± SEM. Data were examined for normality using the Shapiro-Wilk method, D'Agostino and Pearson normality tests, and KS normality tests. Unless otherwise stated, statistical significance between groups with normally distributed data was calculated by unpaired T-test or two-way analysis of variance followed by Tukey post hoc test for group-by-group comparisons. P less than 0.05 was considered significant: *p < 0.05, **p < 0.01, and ***p < 0.001. Example 3 : Effects of leptin and doxorubicin in the Aβ load model A. Sleep - wake behavior

Sleep changes were assessed in APPswe/PS1deltaE9 (also referred to as "PSAPP") mice after administration of doxepin or lebrexan. In other words, 4-5 month old PSAPP mice (mixed sex) were individually housed and cages were placed on the Information Solutions piezoelectric sleep monitoring base (Adapt-A-Base). Sleep-wake behavior was monitored for 7 days while mice were treated daily at ZT 0 (lights on) with vehicle, doxepin 35 mg/kg, or lebrexan (10 or 30 mg/kg) by oral gavage.

FIG9A shows a schematic diagram of the experimental design and a graph of the effects of doxepin and levofloxacin on total sleep ( FIG9B ), light sleep ( FIG9C ), and dark sleep ( FIG9D ). P values from one-way ANOVA are shown. Each dot represents one mouse. FIG9E shows the percentage of sleep at time points during the day after drug injection. P values are from two-way repeated measures ANOVA.

The data indicate that leborexant and doxepin each increase total sleep time in PSAPP mice, and the effects are similar after treatment with each drug. Notably, DOX-induced sleep occurs throughout the day, whereas LEM-induced sleep is restricted to the light period (the natural resting period for mice). B. Amyloid plaque deposition

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

Figure 10A shows a schematic diagram of the treatment time of PSAPP mice. Females develop plaques faster/younger, so males and females are staggered to allow the combination of data. Figure 10B shows representative images of brain sections stained with X34, which mark protofilamentous amyloid plaques. Figure 10C shows the quantification of plaque load (X34 staining area%) in different brain regions (hippocampus, motor cortex, somatosensory cortex and piriform cortex). Error bars indicate mean ± SEM, and each point is a mouse. P values from single-factor variance analysis are shown. The data indicate that long-term administration of leborexant reduces protofilamentous amyloid plaque load in PSAPP mice, while leborexant does not. A dose-response effect was observed for levofloxacin.

Figure 11 shows that long-term leborax reduces total amyloid plaque load in PSAPP mice more effectively than doxepin. Figure 11A shows brain sections stained for total amyloid plaque load using anti-Aβ antibody HJ3.4. Figure 11B shows quantification of plaque load in different brain regions (hippocampus, motor cortex, somatosensory cortex, and piriform cortex) (measured as the % area showing HJ3.4 staining). Error bars indicate mean ± SEM, and each point is one mouse. P values from single-factor ANOVA are shown.

Overall, the data showed that leboraxan reduced total amyloid plaque burden (including diffuse and fibrillary plaques) in PSAPP mice. Doxorubicin also significantly reduced total amyloid burden, but not fibrillary plaque burden, and did not do so to the same extent as leboraxan (30 mg/kg dose), which showed a significant effect only in the piriform cortex.

The differential response of levofloxacin compared with doxepin suggests that the effect on plaque development can be separated from the similar sleep effects induced by both drugs. C. Amyloid protein processing

To determine whether leborexant and doxepin affect amyloid protein processing, cortical lysates were obtained from PSAPP mice treated with either drug, and the levels of full-length APP and APP C-terminal fragment (CTF) were measured by Western blotting. 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.

The results showed that neither leborexant nor doxepin altered APP processing/cleavage in PSAPP mice. D. Size of amyloid plaques and number of small neuroglia surrounding the plaques

The number of microglia around amyloid plaques in PSAPP mice was determined after administration of leboraxan or doxepin. 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 Z-stacks of confocal images using Imaris software. Figure 13A shows representative images of brain sections obtained from PSAPP mice after administration of leboraxan or doxepin. Figure 13B shows quantification of plaque volume (indicating that plaques of similar size were quantified under different conditions) and peri-plaque Iba1 volume. Error bars indicate mean ± SEM, each point represents one mouse. P values from one-way ANOVA are shown.

The results showed that the number of small neuroglia cells around plaques remained unchanged after the administration of leborexant or doxepin. E. Phagocytic subtypes of small neuroglia cells around plaques of profibrillary amyloid protein

To determine the subtype of microglia around protofibrillar amyloid plaques, CD68 expression in microglia of PSAPP mice was determined after administration of leborexant or doxorubicin. Brain sections from PSAPP mice were stained with markers of protofibrillar plaques (X34), microglia (Iba1), and phagosomes (CD68). Figure 14A shows representative images of brain sections. Figure 14B shows the quantification of CD68 colocalized with Iba1 around each plaque obtained using Imaris software. Error bars represent mean ± SEM, and each point is the average of 8-10 plaques from a single mouse. P values from single-factor ANOVA are shown.

The results show that administration of leborabine but not doxorubicin increased the phagocytic subtype of microglia surrounding promyelin plaques. The total number of microglia counts was unchanged in terms of area density, and the total number of microglia colocalized to the plaques was unchanged. Figure 14C shows Iba1+ volume, Figure 14D shows colocalized Iba1+ and CD68+ (as a percentage of Iba1+ staining), and Figure 14E shows colocalized Iba1+ and CD68+. Increased CD68 in microglia surrounding plaques indicates increased phagocytic activation. F. Gene expression

To identify changes in gene expression following administration of leborabine or doxorubicin to PSAPP mice, qPCR arrays were performed on cortical tissue from mice. FIG. 15 is a quantitative plot showing three transcripts that showed significant differences in expression. Ifnb1 encodes the inflammatory mediator IFN-β, which is implicated in microglial cell 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 (mean relative to vehicle (VEH)). Error bars indicate mean ± SEM, and each point is a single mouse. P values from single-factor ANOVA are shown.

The results showed that after administration of leborexant, genes related to Aβ degradation were upregulated in PSAPP mice. There was no significant change in gene expression in the doxorubicin-treated group. G. Number of microglia that actively phagocytose amyloid plaques

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

Results showed that LEM treatment acutely increased microglial phagocytosis of amyloid plaques in vivo. No MX04+ cells were observed in wild-type mice that lack amyloid plaques. In contrast, 1.8% of microglial cells in vehicle-treated PSAPP mice took up MX04, while 3.75% of microglial cells took up MX04 in the LEM-treated group. H. Amyloid Plaque Deposition in Elderly Subjects

The effect of administering Lebrexan was evaluated in aged PSAPP mice. A schematic diagram of the experimental design is shown in FIG17A . In other words, 9-month-old PSAPP mice with amyloid plaques were treated daily with vehicle (Veh) or Lebrexan (LEM, 30 mg/kg) for 30 days. On the first day of treatment, existing plaques were marked in vivo by intraperitoneal (i.p.) injection of methoxy-X04 (MX04). After 30 days of treatment, mice were sacrificed and all plaques were labeled with thiocyanate red, which is similar to X34 and binds to protofibrillar amyloid. Plaque growth during the 30-day treatment period was calculated by comparing MX04 volume to thiazolidine red volume on a plaque-by-plaque basis. Figure 17B shows representative images of MX04, thiazolidine red, and overlay images. Figure 17D shows a graph in which at least 10 plaques were analyzed per mouse, and the mean value for each mouse is shown on the graph with each circle. The LEM-treated group showed a trend of reduced plaque growth during the 30-day treatment period. Due to the non-Gaussian distribution of the data, the P value is from the Mann-Whitney U test. Figure 17C shows representative images of amyloid plaques labeled with X34, microglia labeled with IBA1, and microglia phagosomes labeled with CD68. The volume of IBA1-CD68 colocalization within a 20 μm sphere of each amyloid plaque was calculated to determine peri-plaque microglia CD68 expression. Figure 17E shows quantification of colocalized IBA1-CD68, shown as a percentage of total IBA1 (total microglia) area. Each circle represents the mean of a single mouse, and 10 plaques were quantified per mouse. In Figures 17D and 17E, comparisons between VEH and LEM treatment groups were analyzed by two-tailed t-tests.

The effect of doxepin administration was also evaluated in aged PSAPP mice. Neither lebrexan nor doxepin administration resulted in significant changes in total plaque number in aged mice. Similar no significant changes were observed in plaque volume, IBA1+ cells, IBA1 volume, and colocalization of IBA1-CD68.

Finally, the volume of dystrophic neurites surrounding amyloid plaques was quantified by measuring the increase in BACE1 at presynaptic terminals. Neither lebrexan nor doxepin administration resulted in significant changes in the volume of dystrophic neurites.

The effects of multiplex administration on plaque growth and phagocytic microglia surrounding amyloid plaques and data for administration of leboraxan are shown in Figures 17F and 17G. In Figures 17F and 17G, comparisons of VEH, DOX, and leboraxan treatment groups were analyzed by one-way ANOVA.

Overall, the results showed that leboraxan and doxepin each showed a trend to slow the growth of amyloid plaques in aged mice with pre-existing plaques. Each drug increased phagocytic microglia surrounding amyloid plaques in aged mice. Leboraxan, but not doxepin, also showed this effect on phagocytic microglia in young mice. The effects of leboraxan and doxepin on clearing established plaques in aged mice were less than their effects on preventing plaque accumulation in young mice. I. Methods a. Animals

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

APP/PS1 is a bi-transgenic mouse expressing chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and mutant human presenilin 1 (PS1-dE9), both proteins targeting CNS neurons. b. Methods for measuring sleep - wake behavior

5-month-old APPswe/PS1deltaE9 (PSAPP) mice were orally administered VEH, LEM (10 mg/kg/day or 30 mg/kg/day), or DOX (35 mg/kg/day) daily at ZT0 for 6 days. Sleep and wakefulness were determined using a non-invasive piezoelectric system, Adapt-A-Base (Information Solutions). Mice were individually housed in cages on a piezoelectric inductor base in a soundproof, light-proof cabinet (Circadian cabinet, ClockLab) and recorded without disturbance for 7 days. At the start of the recording, each cage was equipped with 160 g corncob bedding, 220 g food pellets, and 380 mL water, so that each cage had the same weight except for minor variability between mouse weights. Sleep-wake states were analyzed by SleepStats software (Information Solutions, Inc.). Sleep episode length was scored using 30-second epochs according to the manufacturer's default settings in the current version of SleepStats software. c. Methods for evaluating the effects of long-term administration of leborabine or doxepin on amyloid plaque deposition

Young PSAPP mice (3-month-old females and 3.5-month-old males) were orally (po) administered VEH, LEM (10 mg/kg/day or 30 mg/kg/day), or DOX (35 mg/kg/day) daily at ZT0 for 6 weeks. Female and male mice were subsequently perfused at 4.5 and 5 months, respectively. Brains were extracted and processed for IHC/IF, total transcriptomics , or proteomic analysis. d. Methods to evaluate the effects of leborax and doxepin on microglial Aβ phagocytic activity

5-month-old PSAPP mice were given VEH or LEM (30 mg/kg/day) daily at ZT0 for 7 days. On day 7, mice were injected intraperitoneally with methoxy-X-04 (MX-04, 10 mg/kg) to label plaques in vivo. Mice were perfused 3 hours after MX04 injection, and brains were processed for flow cytometry assays to estimate MX-04-positive microglia. e. Methods to evaluate the effects of LEM on amyloid plaque deposition in aged PSAPP mice

9-month-old PSAPP mice were injected intraperitoneally (ip) with methoxy-X-04 (MX-04, 10 mg/kg) to mark plaques in vivo, and VEH or LEM (30 mg/kg/day) were administered orally daily at ZT0 for 4 weeks. At 10 months, mice were perfused, and brains were extracted and processed for IHC/IF analysis. Fixed brain sections were stained with thiophene red and compared with MX-04 staining to estimate plaque growth. f. Drugs

Mice were orally administered vehicle (VEH), doxorubicin (DOX, Cayman Chemical #15888, dissolved in PBS), or leborexant (LEM, suspended in 0.5% methylcellulose) at ZT0 (lights on). VEH mice were given a similar volume of vehicle daily. The tip of a 22-gauge oral feeding needle was dipped into 100% sucrose solution immediately before feeding. Methoxy-X-04 (Tocris #4920, 10 mg/kg) was injected intraperitoneally for time-stamping experiments and analysis of microglial phagocytic activity. g. IHC/IF :

All mice were perfused between ZT5 and ZT7 (1100 and 1300 h). Mice were deeply anesthetized with pentobarbital (150 mg/kg) intraperitoneally and then perfused transcardially with ice-cold Dulbecco's modified PBS (DPBS) containing 3 g/l heparin. The brain was carefully extracted and the left hemisphere was postfixed in 4% paraformaldehyde for 48 h (4°C) and then cryoprotected in 30% sucrose in PBS (4°C) for 24 h. The brain was then sectioned into 40 μm serial coronal sections on a cryo-sliding microtome (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, quickly frozen, and kept at -80°C until analysis.

Protofibrillar Aβ was stained with X-34 dye (SML-1954, 1:5,000; MilliporeSigma) or thiazolidine red. For X-34 staining, free-floating sections were washed three times in PBS for 5 minutes each and then permeabilized in 0.25% Triton X-100 PBS (PBS-X) for 30 minutes. Tissue sections were then incubated in X34-0.1M NaOH for 20 minutes, washed in X34 buffer (40% EtOH in PBS), and then washed 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), sections were washed three times in TBS and then incubated in 0.3% hydrogen peroxide for 10 minutes. Sections were washed three more times in TBS and then blocked in 3% milk diluted in TBS + 0.25% Triton X-100 for 30 minutes. Sections were incubated overnight at 4°C in biotinylated HJ3.4 in TBS + 0.25% Triton X-100 + 1% milk. The next day, sections were washed and then developed using ABC Elite (Vector PK-6100) for 60 minutes. The sections were then incubated in 3,3-diaminobenzidine (DAB, Sigma-Aldrich) as a chromogen and 0.05% hydrogen peroxide as a substrate and dehydrated before coverslipping using Cytoseal 60 (8310; Thermo Fisher Scientific).

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

Epifluorescence imaging: All fluorescence imaging was performed on a Keyence BZ-X810 microscope. Typically, laser intensity and exposure time were selected for each queue of specimens after the tissue was examined so that appropriate parameters were chosen and then kept constant for all slides during that imaging session. These values varied with the antibody, but all sections in a given queue 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.

Confocal imaging and analysis: Patches located in the cortical gray matter and completely contained within the thickness of the slice were selected for imaging. 16-bit image stacks were acquired in sequential mode with 0.26 µm z-steps using a Zeiss LSM-980 Airyscan 2 confocal microscope and ZEN software (v 3.7, blue version). Uniform pinhole, laser power, and PMT detector gain settings were used in all experiments. For all analyses, at least 2 images were taken per brain region and slide using a 40× oil immersion objective at a minimum resolution of 1,024 × 1,024. Confocal images of IBA1 and CD68 volumes around X34+ plaques were quantified on a semiautomated 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 colocalized with the surfaces of the various immunostains. IBA1+ and CD68+ surface areas were then colocalized within a 20 μm extended shell around the plaques. To quantify the number of plaque-associated IBA1+ microglia, a threshold was applied on all images to assign a plaque to each cell body or spot. The X34 surface was expanded to 20 μm, and the spots within the X34+ expanded surface were counted. Any spot that is completely within the extended surface or partially touching the extended surface is included in the analysis. i. Western Blot

Tissue samples were homogenized by sonication on 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 on an iBright CL1500 imaging system (Thermo) using Lumigen TMA-6 chemiluminescent reagent. Band intensities were quantified using Fiji software (NIH) and normalized to a β-tubulin loading control. j. Statistical data

Statistical tests were performed and graphs were drawn using GraphPad Prizm software, version 9.0.1. Power analysis was used to determine the variability of outcome measures and to estimate the number of observations required to test the null hypothesis. All graphs show the mean (with bars), the individual data points within the bars, and error bars depicting the SEM. For Experiments 1 and 2, a one-way analysis of variance 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 data sets with a single dependent variable and 2 groups to determine whether the variance was significantly different. If not significantly different, a two-tailed unpaired t test was performed. If the variance was different, the nonparametric Mann-Whitney U test was performed. Outliers were identified and excluded using the Grubbs test. All P values are indicated in the figures. Example 4 : Effects on rhythmic activity patterns

The effects of leboraxan administration on rhythmic activity patterns were evaluated in arrhythmic Bmal1 knockout (KO) mice in order to assess the restorative effects of leboraxan in mice lacking a functional clock. Bmal1 KO mice lack a functional clock and therefore lose their 24-hour rhythm in continuous darkness (DD conditions). The arrhythmic behavior of Bmal1 KO mice is masked in a normal 12-hour light:12-hour dark cycle (L:D conditions).

Control (Cre-) or Bmal1 KO (CAG-CreERT2; Bmal1 (f/f)) mice were treated with tamoxifen to delete Bmal1. One month later, infrared activity recordings were recorded under 12h:12h L:D conditions (yellow areas) or continuous darkness (DD conditions). Mice were administered leborexant (LEM) at a dose of 30 mg/kg by oral gavage at 6 am (previous ZT 0) for 9 days (indicated by red bars and red arrows). Activity recordings were collected for another 2 weeks after drug administration was stopped. Figure 18A shows representative activity graphs of control and Bmal1 knockout mice. Figure 18B is a quantification of the endpoints of the activity graph during different parts of the experiment (in Figure 18A, LD indicates during the yellow region, DD+LEM is the region with red bars, and DD is the rest of the recording). Data were analyzed by two-way ANOVA with Tukey's post hoc test.

Results showed that Bmal1 knockout mice showed greater daytime activity than wild-type mice under all conditions (LD, DD+LEM, and DD). Bmal1 KO mice were able to maintain locomotor activity similar to that of LD under DD conditions (CT0 was similar to ZT0 under DD conditions) when they were given leborax every day at CT0. Relative amplitude, which indicates the ratio of the average activity in the most active 10 hours to the average activity in the least active 5 hours, was lower in Bmal1 knockout mice under all conditions. In general, higher relative amplitudes are associated with stable rhythms. Diurnal stability (IS), which measures the synchronization between daily 24-hour rhythms, was lower in Bmal1 knockout mice under all conditions. High IS indicates good synchronization of rhythms. Identical form and range

Those skilled in the art will recognize, or be able to determine using only routine experimentation, many equivalents to the specific embodiments provided herein. The scope of the present disclosure is not intended to be limited to the above embodiments, but is as described in the appended claims.

When a range is given, the endpoints are included. In addition, it should be understood that unless otherwise indicated or otherwise obvious from the context and the understanding of those skilled in the art, unless the context clearly indicates otherwise, the values expressed as ranges can assume any specific value or sub-range within the range specified in the various embodiments of the present disclosure, to one tenth of the unit of the lower limit of the range.

In addition, it is 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. Because such embodiments are deemed to be known to those skilled in the art, they may be excluded even if the exclusion is not expressly stated herein. Any particular embodiment of the composition of the present disclosure (e.g., any composition, treatment or active ingredient; any method of production; 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.

It is to be understood that the words which have been used are words of description rather than limitation and may be varied within the purview of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

Although the present disclosure has been described in considerable detail with a certain particularity with respect to several described embodiments, it is not intended that the present disclosure should be limited to any such details or embodiments or any particular embodiments, but rather it is to be interpreted with reference to the appended claims so as to provide the broadest possible interpretation of such claims in light of the art to effectively encompass the intended scope of the present disclosure.

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

[ Figure 1 ] shows the study design of the clinical trial. Figure 1 depicts the overview of the study design with a habitual bedtime of 22:00.

[ Figure 2A-H ] depicts data from the study of Example 2. Figure 2A shows a schematic diagram of the study design. Electroencephalographic (EEG) analysis of the percentage of time spent in NREM sleep in Figure 2B, REM sleep in Figure 2C, and wakefulness in E4 (n = 10 mice/treatment group) and P301S/E4 mice (n = 8 mice/treatment group) treated with vehicle (Veh) or Lebrexan (Lem). Two-way analysis of variance, Tukey post hoc comparisons. Figure 2E shows representative spectra of EEG and electromyography (EMG) analysis, illustrating NREM, REM, and wakefulness patterns in vehicle-treated P301S/E4, and Figure 2F shows Lebrexan-treated P301S/E4 mice. Figure 2G shows a time course analysis of the percentage of sleep observed over time from the start of vehicle or Lebrexan treatment until 24 hours after oral gavage in E4 mice (n = 10 mice/treatment group), and Figure 2H shows P301S/E4 mice (n = 8 mice/treatment group). White and black bars represent light and dark periods, respectively. Data represent mean ± SEM; *p < 0.05, **p < 0.001, ***p < 0.0001.

[ Figure 3 ] depicts data from the study of Example 2. Figure 3A shows representative images of AT8-stained phosphorylated tau at both 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 MC1-stained tau. Scale bar -500 μm. Figure 3C shows quantification of the percentage of 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). Fig. 3E shows the percentage of MC1-stained hippocampus (nE4 = 18 mice/treatment group; nP301S/E4 = 16-17 mice/treatment group), and Fig. 3F shows the entorhinal cortex/piriform cortex (nE4 = 16-17 mice/treatment group; nP301S/E4 = 15-17 mice/treatment group). Fig. 3G shows representative images of cresol violet-stained brains for volume analysis. Scale bar -1 mm. Fig. 3H shows quantification of hippocampal volume, and Fig. 3I shows piriform cortex volume. Fig. 3J shows plasma neurofilament light chain (NfL) levels measured by SIMOA (nE4 and nP301S/E4 = 16-20 mice/treatment group). Data are mean ± SEM; two-way analysis of variance, Tukey post hoc test; *p ＜ 0.05, **p ＜ 0.001, ***p ＜ 0.0001.

[ Figure 4A-R ], from the study of Example 2, depicts (a) representative images of microglia co-stained with IBA1 (green), CD68 (red), and DAPI (blue) in the CA3 region of the hippocampus. (b) representative images of microglia stained with TMEM119 (yellow) and DAPI (blue) in the CA3 region of the hippocampus. (c) Quantification of the percentage of CA3 covered by IBA1 and (d) quantification of the percentage of CA3 covered by CD68. (e) representative images of microglia stained with Clec7a (red) and DAPI (blue). (f) Quantification of the percentage of CA3 covered by TMEM119 and (g) Clec7a. (h) representative images of astrocytes co-stained with ApoE (green) and GFAP (red). (i) Quantification of the percentage of ApoE co-localized with GFAP in CA3 or (j) Quantification of the percentage of ApoE co-localized with IBA1 in CA3. (k) Representative images of positive microglia co-stained with ApoE (green) and IBA1 (magenta). Figure 4L shows the percentage of Iba1-covered piriform/entorhinal cortex in leborexant-treated mice compared to vehicle-treated control mice. Figure 4M shows the percentage of Iba1-covered dentate gyrus in leborexant-treated mice compared to vehicle-treated control mice. Figure 4N shows the percentage of TMEM119-covered piriform/entorhinal cortex in leborexant-treated mice compared to vehicle-treated control mice. FIG. 4O shows the percentage of TMEM119 coverage of the dentate gyrus in Lebrexan-treated mice compared to vehicle-treated control mice. FIG. 4P shows the percentage of CD68 coverage of the dentate gyrus in Lebrexan-treated mice compared to vehicle-treated control mice. FIG. 4Q shows the percentage of CD68 coverage of the piriform/entorhinal cortex in Lebrexan-treated mice compared to vehicle-treated control mice. FIG. 4R shows representative images of the CA1/CA2 region stained for GFAP, and a graph of the percentage of GFAP coverage of the CA1/2, dentate gyrus, and piriform/entorhinal cortex. Scale bar - 50 μm. nE4 = 15-19 mice/treatment group; nP301S/E4 = 16-19 mice/treatment group. Data represent mean ± SEM; two-way ANOVA, Tukey post hoc test; *p ＜ 0.05, **p ＜ 0.001, ***p ＜ 0.0001.

[ Figure 5 ] depicts data from the study of Example 2. Figure 5A shows a volcano plot comparing differentially regulated genes in P301S/E4 mice treated with vehicle versus lebrexan. The cutoff for significance was set at two times the Log fold change. nE4 and nP301S/E4 = 10 mice/treatment group. Figure 5B shows a GO term analysis of genes significantly changed in P301S/E4 mice treated with vehicle versus lebrexan. Figure 5C shows a heat map illustrating all differentially expressed genes in P301S/E4 vehicle versus lebrexan that achieved significance with a Log10 adjusted p value < 0.05. Figure 5D shows representative images of VGLUT1 (green) and PSD95-stained (magenta) synapses in CA3. Scale bar -50 μm. Figure 5F shows quantification of the percentage of VGLUT1 puncta in CA3 and piriform cortex (Figure 5G). Figure 5H shows quantification of the percentage of PSD95 puncta in CA3 and piriform cortex (Figure 5I). nE4 = 16-19 mice/treatment group; nP301S/E4 = 16-19 mice/treatment group. Data represent mean ± SEM; two-tailed unpaired T test; *p < 0.05, **p < 0.005, ***p < 0.0001.

[ Figure 6 ] depicts data from the study of Example 2. Figure 6A shows a time course analysis of the percentage of sleep in E4 mice (n ^E4 = 7-9 mice/treatment group), and Figure 6B shows data in P301S/E4 mice (n ^P301S/E4 = 8-10 mice/treatment group). White bars and black bars represent the light period and dark period, respectively. Figure 6C shows the percentage of time spent asleep during the light period or dark period for E4 and P301S/E4 mice (n = 10 mice/genotype and treatment group). Figure 6D shows the length of sleep episodes during the light period or dark period for E4 and P301S/E4 mice (n = 10 mice/genotype and treatment group), and Figure 6E shows the length of wake episodes. s-seconds. Data are mean ± SEM; two-way analysis of variance, Tukey post hoc test; *p ＜ 0.05, **p ＜ 0.001, ***p ＜ 0.0001.

[ Figure 7 ] depicts data from the study of Example 2. Phosphorylated tau (pTau) in RAB (Figure 7A), RIPA (Figure 7B), and formic acid (FA) (Figure 7C) fractions were quantified by ELISA (nE4 and nP301S/E4 = 18-20 mice/treatment group). Total tau (tTau) in RAB (Figure 7D), RIPA (Figure 7E), and FA (Figure 7F) fractions were 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 volume analysis of the hippocampus stained with cresyl violet, Figure 7I shows hemibrains minus ventricles, Figure 7J shows the pyramidal cell layer of the piriform cortex, and Figure 7K shows the granulocyte layer of the hippocampus. (nE4 and nP301S/E4 = 17-19 mice/treatment group). Scale bar -500 μm. Data represent mean ± SEM; two-way analysis of variance, Tukey post hoc test; *p < 0.05, **p < 0.001, ***p < 0.0001.

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

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

[ Figure 10 ], from the study of Example 3, shows the protofilamentous amyloid plaque load in APP/PS1dE9 mice administered doxepin or levofloxacin. Figure 10A is a schematic diagram of the treatment time of mice. Figure 10B shows representative images of brain sections stained with X34, which marks protofilamentous amyloid plaques. Figure 10C shows the quantification of plaque load (% of X34 stained area) in different brain regions. Error bars indicate mean ± SEM, and each point is one mouse. P values from one-way analysis of variance are shown.

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

[ Figure 12 ], from the study of Example 3, shows APP processing/cleavage in APP/PS1dE9 mice treated with doxepin or levofloxacin. Figure 12A shows representative Western blot 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 quantification of band intensity. Error bars indicate mean ± SEM, each point is one mouse. P values from one-way ANOVA are shown.

[ Figure 13 ], from the study of Example 3, shows clusters of peri-plaque microglia in APP/PS1dE9 mice treated with doxorubicin or levofloxacin. Figure 13A shows representative images of samples stained for plaques (X34) and microglia (Iba1). The volume of microglia surrounding each plaque was calculated from Z-stacks of confocal images using Imaris software. Figure 13B shows quantification of plaque volume (to indicate quantification of plaques of similar size under different conditions) and peri-plaque Iba1 volume. Error bars indicate mean ± SEM, and each point is one mouse. P values from one-way ANOVA are shown.

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

[ Figure 15 ], from the study of Example 3, shows the effect of leborexant treatment on gene expression. The expression of transcripts encoding Ifnb1 , Rab5a , and Mmp2 showed significant differences after leborexant treatment. 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.

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

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

[ Figure 18 ], from the study of Example 4, shows the effects of leborax or doxepin on the rhythmic activity patterns of arrhythmic Bmal1 KO mice. Figure 18A shows representative activity graphs. LD = 12 h:12 h light-dark; DD = continuous darkness. Figure 18B shows quantification of diurnal rhythmic motor behavior during different parts of the experiment (LD indicates during the yellow area, DD+LEM is the area with red bars, and DD is the rest of the recording). Data were analyzed by two-way ANOVA and Tukey post hoc test.

Claims (186)

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 lebrexan, a pharmaceutically acceptable salt thereof, or a solvate thereof, thereby treating AD. The method of claim 1, wherein treating AD comprises reducing and/or slowing cognitive decline. The method of claim 1, wherein treating AD comprises affecting a change (e.g., slowing, delaying, or reducing) in at least one marker of AD pathology. The method of claim 3, wherein the marker is tau phosphorylation level, neurodegeneration, changes in microglial cell response and/or the presence of Aβ plaques. The method of claim 4, wherein the marker is present in a brain region of the subject. The method of claim 5, wherein the brain region is the hippocampus, motor cortex, somatosensory cortex, piriform cortex and/or entorhinal cortex. The method of claim 4, wherein the marker is detected in a body fluid of the subject. The method of claim 7, wherein the body fluid is blood or cerebrospinal fluid (CSF). The method of claim 1, wherein the subject does not show signs of dementia and/or cognitive impairment. The method of claim 1, wherein the subject has mild cognitive impairment or mild dementia. The method of claim 1, wherein the subject is positive for amylin. The method of claim 11, wherein the subject is at risk for further Aβ accumulation. The method of claim 12, wherein the subject is an ApoE4 carrier. The method of claim 12, wherein the subject has an intermediate level of amylin PET (e.g., 20-40 percentiles). The method of claim 12, wherein the subject has elevated levels of amylin PET (e.g., >40 percentile units). The method of claim 1, wherein the subject has been diagnosed with AD based on brain imaging, cognitive function and/or biomarker criteria. The method of claim 16, wherein the subject suffers from early-stage AD. The method of claim 16, wherein the subject suffers from pre-AD. A method of reducing or maintaining tau in a subject having AD or at risk for developing AD (e.g., reducing or maintaining tau, or delaying tau accumulation, tau phosphorylation and/or tau diffusion, or slowing the rate of any of these), the method comprising administering to the subject a therapeutically effective amount of leboraxant, wherein the therapeutically effective amount is sufficient for tau in the subject. The method of claim 19, wherein the subject is negative for amylin. The method of claim 19, wherein tau levels are reduced or maintained relative to a reference. The method of claim 19, wherein the method comprises reducing and/or delaying tau accumulation and/or tau diffusion, and/or slowing the rate thereof, as compared to a reference. The method of claim 22, wherein the reference is a baseline measurement of the subject before treatment. The method of claim 22, wherein the reference is a baseline measurement from a control subject. The method of claim 22, wherein the reference is a measurement from a control subject administered a placebo. The method of claim 19, wherein the method comprises altering tau in a brain region of the subject. The method of claim 19, wherein the method comprises altering tau PET signal in a brain region of the subject. The method of claim 27, wherein the brain region is the hippocampus, entorhinal cortex and/or piriform cortex. The method of claim 19, wherein the method comprises reducing tau in a bodily fluid of the subject. The method of claim 29, wherein the body fluid is blood or CSF. The method of claim 19, wherein the tau is total tau. The method of claim 19, wherein the tau is insoluble tau. The method of claim 19, wherein the tau is aggregated tau. The method of claim 19, wherein the tau is a phosphorylated form of tau (phosphorylated tau). The method of claim 34, wherein the phosphorylated tau is phosphorylated at one or more of T181, T217, S202, S205 or T231. The method of claim 34, wherein the method comprises altering the ratio of phosphorylated tau to total tau. The method of claim 36, wherein the ratio of phosphorylated tau to total tau is reduced compared to the ratio of phosphorylated tau to total tau in the subject's CSF prior to administration of leborax. The method of 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 leborabine. The method of claim 34, comprising increasing the rate of dephosphorylation of phosphorylated tau. The method of claim 34, comprising reducing the phosphorylation rate of tau. The method of claim 19, comprising reducing or maintaining tau within 48 hours of administering the first dose of lebrexan. The method of claim 34, wherein the method comprises reducing phosphorylated tau in the hippocampus, entorhinal cortex, and/or piriform cortex. 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 leboraxant, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the therapeutically effective amount is sufficient to alter the neurodegeneration in the subject. The method of claim 43, wherein the subject is negative for amylin. The method of claim 43, wherein altering neurodegeneration comprises reducing and/or delaying neurodegeneration, and/or slowing its rate, compared to a reference. The method of claim 43, wherein the neural degeneration is changed relative to a reference. The method of claim 46, wherein the reference is a baseline measurement of the subject before treatment. The method of claim 46, wherein the reference is a baseline measurement from a control subject. The method of claim 46, wherein the reference is a measurement from a control subject administered a placebo. The method of claim 43, wherein the neurodegeneration is characterized by a loss of at least one of cortical thickness and hippocampal volume. The method of claim 50, wherein altering neurodegeneration comprises maintaining cortical thickness and/or hippocampal volume or reducing loss of cortical thickness and/or hippocampal volume. 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. The method of claim 52, wherein altering neurodegeneration comprises maintaining pyramidal neurons and/or granule cells or reducing the loss of pyramidal neurons and/or granule cells. The method of claim 43, wherein altering neurodegeneration comprises reducing the rate of neurodegeneration. The method of claim 43, wherein altering neurodegeneration comprises altering neurofilament light chain (NfL) levels. The method of claim 55, comprising altering the level of NfL in the blood and/or CSF of the subject. 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 leboraxant, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the therapeutically effective amount is sufficient to alter Aβ plaques in the subject. The method of claim 57, wherein the Aβ plaques are altered relative to a reference. The method of claim 58, wherein altering Aβ plaques comprises reducing and/or delaying the formation of Aβ plaques, and/or slowing the rate thereof, compared to a reference. The method of claim 58, wherein the reference is a baseline measurement of the subject before treatment. The method of claim 58, wherein the reference is a baseline measurement from a control subject. The method of claim 58, wherein the reference is a measurement from a control subject administered a placebo. The method of claim 57, wherein the Aβ plaques are fibrillary plaques. The method of claim 57, wherein the Aβ plaques are all plaques. The method of claim 57, wherein altering Aβ plaques comprises reducing the growth of Aβ plaques. The method of claim 65, comprising reducing the growth of Aβ plaques in the subject's hippocampus, the subject's motor cortex, the somatosensory cortex, and/or the piriform cortex. The method of claim 57, wherein altering Aβ plaques comprises altering amyloid protein PET signals obtained from a brain region of the subject. The method of claim 57, wherein the altered Aβ plaques correspond to a decrease in Aβ concentration in the subject's CSF. The method of claim 68, wherein the Aβ is Aβ38, Aβ40 and/or Aβ42. The method of claim 57, comprising altering Aβ plaques within 48 hours of administering the first dose of lebrexan. The method of claim 57, wherein the subject does not show signs of dementia and/or cognitive impairment. The method of claim 57, wherein the subject has mild cognitive impairment or mild dementia. The method of claim 57, wherein the subject is at risk for further Aβ accumulation. The method of claim 73, wherein the subject is an ApoE4 carrier. The method of claim 73, wherein the subject has an intermediate level of amylin PET (e.g., 20-40 percentile). The method of claim 73, wherein the subject has elevated levels of amylin PET (e.g., >40 percentile units). The method of claim 57, wherein the subject suffers from early-stage AD. The method of claim 57, wherein the subject suffers from pre-AD. A method for modulating microglial cell responses in a subject having Alzheimer's disease (AD) or at risk for developing AD, the method comprising administering to the subject a therapeutically effective amount of leboraxant, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the therapeutically effective amount is sufficient to modulate the microglial cell responses in the subject. The method of claim 79, wherein modulating the microglia response comprises modulating the expression of at least one microglia marker. The method of claim 80, wherein the microglia marker is a general microglia marker. The method of claim 81, wherein the common microglia marker is Iba1, Clec7a or CD68. The method of claim 80, wherein the microglial cell marker is a stable microglial cell marker. The method of claim 83, wherein the homeostatic microglia marker is TMEM119 or P2RY12. The method of claim 79, wherein modulating the microglia response comprises modulating the activity of phagocytic microglia. The method of claim 79, wherein the subject has mild cognitive impairment or mild dementia. The method of claim 79, wherein the subject does not show signs of dementia and/or cognitive impairment. The method of claim 79, wherein the subject is negative for amylin. The method of claim 79, wherein the subject has tau pathology. The method of claim 79, wherein the subject has neurodegeneration in a brain region. The method of claim 90, wherein the brain region is the hippocampus, entorhinal cortex and/or piriform cortex. The method of claim 90, wherein the brain region is the CA1 region, CA2 region, CA3 region or dentate gyrus in the hippocampus. The method of claim 88, wherein modulating the microglia response comprises modulating the response in microglia associated with degenerating neurons. The method of claim 93, wherein modulating the microglia response comprises reducing the expression of at least one general microglia marker. The method of claim 94, wherein the common microglia marker is Iba1, CD68 or Clec7a. The method of claim 93, wherein modulating the microglia response comprises increasing the expression of at least one homeostatic microglia marker. The method of claim 96, wherein the homeostatic microglia marker is TMEM119 or P2RY12. The method of claim 79, wherein the subject has Aβ plaques. The method of claim 98, wherein the Aβ plaques are fibrillar Aβ plaques. The method of claim 98, wherein the subject is at risk for further Aβ accumulation. The method of claim 100, wherein the subject is an ApoE4 carrier. The method of claim 100, wherein the subject has an intermediate level of amylin PET (e.g., 20-40 percentile). The method of claim 100, wherein the subject has elevated levels of amylin PET (e.g., >40 percentile units). The method of claim 98, wherein the subject suffers from early-stage AD. The method of claim 98, wherein the subject suffers from pre-AD. The method of claim 98, wherein the Aβ plaques are present in the hippocampus, motor cortex, somatosensory cortex and/or piriform cortex. The method of claim 98, wherein modulating the microglia response comprises modulating the response in microglia associated with Aβ plaques. The method of claim 107, wherein modulating the microglia response comprises increasing the expression of a general microglia marker. The method of claim 108, wherein the common microglia marker is Iba1, Clec7a or CD68. The method of claim 107, wherein regulating microglial response comprises increasing phagocytosis of Aβ plaques by phagocytic microglial cells. The method of claim 107, wherein modulating the microglial cell response comprises reducing the expression of a homeostatic microglial cell marker. The method of claim 111, wherein the homeostatic microglia marker is TMEM119 or P2RY12. The method of any one of claims 1 to 112, wherein the therapeutically effective amount of lebrexan administered to the subject is in the range of 5 mg to 50 mg per day. The method of claim 113, wherein the therapeutically effective amount of leboraxant administered to the subject is in the range of 10 mg to 30 mg per day. The method of claim 113, wherein the therapeutically effective amount of leboraxant 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. The method of claim 113, wherein the therapeutically effective amount of leboraxant administered to the subject is 20-25 mg per day. The method of any one of claims 1 to 112, wherein a dose of 25 mg of lebrexan is administered to the subject once daily. The method of claim 113, wherein Lebrexant 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. The method of claim 118, wherein each of the first cycle, the second cycle, and the third cycle is 1 cycle. The method of claim 118, wherein the first dose is less than the second dose, and optionally, the second dose is less than the third dose. The method of claim 120, wherein the first dose is 5 mg of lebrexan once daily, the second dose is 10 mg of lebrexan once daily, and optionally, the third dose is 20-25 mg of lebrexan once daily. The method of claim 120, wherein the first dose is 5 mg or 7.5 mg of lebrexan once daily, the second dose is 10 mg, 12.5 mg, 15 mg, or 17.5 mg of lebrexan once daily, and the third dose is 20 mg, 22.5 mg, 25 mg, 27.5 mg, or 30 mg of lebrexan once daily. The method of claim 118, wherein the first dose is greater than the second dose, and optionally, the second dose is greater than the third dose. The method of claim 123, wherein the first dose is 20-25 mg of lebrexan once daily, the second dose is 10 mg of lebrexan once daily, and optionally, the third dose is 5 mg of lebrexan once daily. The method of claim 123, wherein the first dose is 20 mg, 22.5 mg, 25 mg, 27.5 mg or 30 mg of lebrexan once daily, the second dose is 10 mg, 12.5 mg, 15 mg or 17.5 mg of lebrexan once daily, and optionally, the third dose is 5 mg or 7.5 mg of lebrexan once daily. The method of any one of claims 1 to 125, comprising administering lebrexan to the subject for at least 6 months. The method of claim 126, comprising administering lebrexan to the subject for at least 9 months, at least 12 months, or at least 15 months. The method of claim 126, comprising administering Lebrexan to the subject for at least 18 months. The method of any of claims 126 to 128, comprising administering lebrexan to the subject for at least 24 months, 30 months, or 36 months. A method of selecting a subject having Alzheimer's disease (AD) or at risk of developing AD for treatment with leboraxant, 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 cell response, and biomarker expression from the subject; (b) comparing the measurement from the subject to a measurement from a reference; and (c) selecting the subject for treatment with leboraxant if the measurement from the subject is different from the measurement from the reference. The method of claim 130, wherein the subject has mild cognitive impairment or mild dementia. The method of claim 130, wherein the subject does not show signs of dementia and/or cognitive impairment. The method of claim 130, wherein the subject is at risk for Aβ accumulation. The method of claim 133, wherein the subject is an ApoE4 carrier. The method of claim 133, wherein the subject has an intermediate level of amylin PET (e.g., 20-40 percentiles). The method of claim 133, wherein the subject has elevated levels of amylin PET (e.g., >40 percentile units). The method of claim 130, wherein the subject suffers from early-stage AD. The method of claim 130, wherein the subject suffers from pre-AD. The method of claim 130, wherein the subject has been diagnosed with AD based on brain imaging, cognitive function and/or biomarker criteria. The method of claim 130, wherein obtaining at least one measurement comprises obtaining data from a brain scan of the subject and/or obtaining data from a biological sample from the subject. The method of claim 140, wherein the data from the brain scan indicates levels of tau phosphorylation, tau aggregation, Aβ plaque burden, and/or microglial cell response. The method of claim 140, wherein the biological sample is a body fluid. The method of claim 140, wherein the body fluid is cerebrospinal fluid (CSF), blood, or saliva. The method of claim 130, wherein the reference is a comparison. The method of claim 130, wherein the reference is a measurement from a control subject administered a placebo. The method of claim 144, wherein the control does not have AD. The method of claim 146, wherein the measurement from the subject is higher than the measurement from the control who does not have AD. The method of claim 146, wherein the measurement from the subject is lower than the measurement from the control who does not have AD. The method of claim 144, wherein the control suffers from AD. The method of claim 149, wherein the measurement from the subject is similar to or higher than the measurement from the control having AD. The method of claim 149, wherein the measurement from the subject is similar to or lower than the measurement from the control having AD. The method of claim 130, wherein the measurement of tau phosphorylation comprises a measurement of phosphorylation at one or more of T181, T217, S202, S205, or T231. The method of claim 130, wherein the measure of tau aggregation comprises a measure of insoluble tau aggregates (e.g., neurofibrillary tangles (NFTs)). The method of claim 130, wherein the measure of neurodegeneration comprises a measure of cortical thickness and/or hippocampal volume or a measure of pyramidal or granule neuron loss. The method of claim 130, wherein the measure of Aβ plaque burden comprises a measure of Aβ plaque volume and/or Aβ plaque volume growth. The method of claim 130, wherein the measure of Aβ plaque burden comprises a measure of amyloid PET signal in a brain region of the subject or a measure of Aβ in the CSF of the subject. The method of claim 130, wherein the measure of microglial response is a change in the expression of at least one microglial marker. The method of claim 157, wherein the microglia marker is Iba1, Clec7a, CD68, TMEM119 or P2RY12. The method of claim 157, wherein the measure of microglial cell response is a measure of microglial cell phagocytosis. A method for monitoring the efficacy of a treatment in a subject having Alzheimer's disease (AD) or at risk for developing AD, the method comprising: (a) obtaining a first measurement 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 leboraxan, a pharmaceutically acceptable salt thereof, or a solvent complex 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; and (d) Comparing the second measurement from the subject to the first measurement from the subject; wherein a difference between the first measurement and the second measurement indicates effective treatment with leborax. A method for treating a subject having Alzheimer's disease (AD) or at risk for developing AD, the method comprising: (a) obtaining a first measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial cell response, and biomarker expression from the subject; (b) administering a first dose of leboraxan, a pharmaceutically acceptable salt thereof, or a solvent complex thereof to the subject; (c) obtaining a second measurement of at least one of tau phosphorylation, tau aggregation, neurodegeneration, Aβ plaque burden, microglial cell function, and biomarker expression from the subject; (d) comparing the second measurement from the subject to the first measurement from the subject; and (e) administering a second dose of leborax if the first measurement is different from the second measurement. The method of claim 160 or claim 161, wherein obtaining at least one measurement comprises obtaining data from a brain scan of the subject and/or obtaining data from a biological sample from the subject. The method of claim 162, wherein the data from the brain scan indicates levels of tau phosphorylation, tau aggregation, Aβ plaque burden, and/or microglial cell response. The method of claim 162, wherein the biological sample is a body fluid. The method of claim 164, wherein the body fluid is cerebrospinal fluid (CSF), blood, or saliva. The method of claim 160 or claim 161, wherein the first measurement value from the subject is higher than the second measurement value from the subject. The method of claim 160 or claim 161, wherein the first measurement value from the subject is lower than the second measurement value from the subject. The method of claim 160 or claim 161, wherein the measurement of tau phosphorylation comprises a measurement of phosphorylation of one or more of T181, T217, S202, S205, or T231. The method of claim 160 or claim 161, wherein the measure of tau aggregation comprises a measure of insoluble tau aggregates (e.g., neurofibrillary tangles (NFTs)). The method of claim 160 or claim 161, wherein the measure of neurodegeneration comprises a measure of cortical thickness and/or hippocampal volume or a measure of pyramidal neuron or granule neuron loss. The method of claim 160 or claim 161, wherein the measure of Aβ plaque burden comprises a measure of Aβ plaque volume and/or Aβ plaque volume growth. The method of claim 160 or claim 161, wherein the measure of Aβ plaque burden comprises a measure of amyloid PET signal in a brain region of the subject or a measure of Aβ in the CSF of the subject. The method of claim 160 or claim 161, wherein the measure of microglial response is a measure of expression of at least one microglial marker. The method of claim 173, wherein the microglia marker is Iba1, Clec71, P2RY12 or TMEM119. The method of claim 173, wherein the measure of microglial cell response is a measure of microglial cell phagocytosis. The method of claim 160 or claim 161, wherein the measure of biomarker expression is a measure of Ifnb1, MMP2 and/or Bace1 expression. The method of claim 160 or claim 161, wherein the subject is negative for amylin. As claimed in claim 160 or the method of claim 160, wherein the subject has Aβ plaques. The method of claim 160 or claim 161, wherein the subject has mild cognitive impairment and/or mild dementia. The method of claim 160 or claim 161, wherein the subject does not show signs of dementia and/or cognitive impairment. The method of claim 160 or claim 161, wherein the subject is at risk for further Aβ accumulation. The method of claim 181, wherein the subject is an ApoE4 carrier. The method of claim 181, wherein the subject has an intermediate level of amylin PET (e.g., 20-40 percentiles). The method of claim 181, wherein the subject has elevated levels of amylin PET (e.g., >40 percentile units). The method of claim 160 or claim 161, wherein the subject suffers from early AD. The method of claim 160 or claim 161, wherein the subject suffers from pre-AD.