Assay for identifying disease

Abstract

Aspects of the disclosure provide assays for identifying diseases associated with orexin levels. The present teachings include methods for quantifying the concentration of orexin in a fluid sample, such as a cerebrospinal fluid sample, and methods for identifying and treating diseases based on the concentration of orexin, including but not limited to narcolepsy and Alzheimer's disease.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 655,404, filed June 3, 2024; U.S. Provisional Application No. 63 / 655,397, filed June 3, 2024; U.S. Provisional Application No. 63 / 612,777, filed December 20, 2023; and U.S. Provisional Application No. 63 / 612,784, filed December 20, 2023.

[0002] Statement regarding federally funded research or development This invention was completed with the support of the U.S. government under license number AG074151 granted by the National Institutes of Health. The U.S. government holds certain rights to this invention. Technical Field

[0003] This disclosure generally relates to assay methods for identifying and measuring diseases associated with altered orexin concentrations. Background Technology

[0004] Centrally induced narcolepsy includes type 1 narcolepsy, type 2 narcolepsy, and idiopathic narcolepsy, which can be diagnosed according to the International Classification of Sleep Disorders – Third Edition, Text Revised (ICSD-3-TR). Type 1 narcolepsy is characterized by one or both of the following: 1) cataplexy (as defined under the basic characteristics) and a) a multiple sleep latency test (MSLT) (a type of sleep study) showing a mean sleep latency of <8 minutes and two REM sleep onset periods or b) a sleep REM sleep onset period within 15 minutes of sleep onset on a nocturnal polysomnography, and / or 2) a low CSF orexin-A level <110 pg / ml or less than one-third of the mean CSF orexin-A value obtained in normal subjects using the same standardized assay. Type 2 narcolepsy is diagnosed using the MSLT with the same criteria as for type 1 narcolepsy. Idiopathic narcolepsy is diagnosed by a mean sleep latency of <8 minutes on the MSLT.

[0005] These disorders are associated with altered levels of orexin proteins in the cerebrospinal fluid (CSF). In particular, type 1 narcolepsy (NT1) is a sleep disorder characterized by a deficiency of orexin (hypothalamic hormone) and a loss of 85%-95% of orexinergic neurons in the hypothalamus. Figure 20The study described patients with NT1 who had a CSF orexin-A deficiency of ≤110 pg / ml as detected by radioimmunoassay (RIA). Despite a total loss of 33% of orexinergic neurons in patients with type 2 narcolepsy (NT2), NT2 patients had normal levels of CSF orexin-A. Previous work has shown that RIA measures <10% of intact orexin-A peptide, suggesting that RIA of CSF orexin-A primarily measures orexin-A-related metabolites.

[0006] Neurons that produce orexin are located only in the perifornium and the lateral and posterior hypothalamic regions, and project to brainstem nuclei, the amygdala, the hippocampus, and the cerebral cortex. Orexin-A is a 33-amino acid peptide with an N-terminal glutamine cyclized to pyroglutamate and two intrachain disulfide bonds. Orexin-B is a 28-amino acid peptide composed of two α-helices linked by short linkers. Figure 10 Both orexin A and B have their C-terminus after amidation. Human orexin-A and orexin-B share 46% homology (13 / 28 amino acids), and their C-terminal sides are highly conserved. Orexin binds to two G protein-coupled receptors, orexin receptor 1 (OXR1) and orexin receptor 2 (OXR2). The orexin system regulates wakefulness, eating behavior, energy homeostasis, and the reward system. Based on its role in the pathophysiology of narcolepsy, dual orexin receptor antagonists (DORAs) have been developed as a treatment for insomnia.

[0007] Most known information about orexin is based on studies measuring orexin-A. The role of orexin-B is less clear, but there is evidence that orexin-B differs from orexin-A. For example, orexin-B regulates behavior and other processes (such as thermoregulation), but is less involved in the control of energy metabolism or normal food intake. These differences in the regulatory functions of orexin-A and orexin-B may be due to differences in orexin receptor binding kinetics: orexin-A shows similar affinity for both OX1R and OX2R, while orexin-B shows a higher affinity for OX2R compared to OX1R.

[0008] The orexin system involves a precursor protein called prepro-orexin, which is proteolytically cleaved into orexin-A and orexin-B (orexin is also referred to as hypothalamic secretin in the literature). Radioimmunoassay (RIA) of orexin-A measures the N-terminus of the protein and is used to diagnose type 1 narcolepsy, but it cannot differentiate between other centrally originated forms of somnolence. Measurements of the C-terminus of orexin-A, as well as prepro-orexin and orexin-B, are insensitive and show no difference between different centrally originated forms of somnolence.

[0009] Alzheimer's disease (AD) is characterized by the deposition of Aβ in the brain as insoluble plaques (i.e., amyloid-positive), τ aggregation and hyperphosphorylation, neuronal degeneration, synaptic loss, and ultimately, cognitive impairment, dementia, and death. The soluble forms of Aβ and τ (proteins crucial for AD pathogenesis) vary in CSF during wakefulness: 1) CSF Aβ and τ concentrations increase during wakefulness and decrease during sleep in both mice and humans; 2) Overnight sleep deprivation increases CSF Aβ and τ levels by ≥30% via increased production / release; 3) τ hyperphosphorylation (p-τ) (an early step in τ-mediated neurodegeneration) is also affected by sleep deprivation, depending on the specific phosphorylation site. Furthermore, amyloid and τ symptoms are associated with sleep disruptions that reduce diurnal variations in CSF Aβ and τ. Based on these findings, sleep is a potential biomarker and / or modifiable risk factor for AD.

[0010] Considering the association between CSF Aβ and τ, sleep-wake activity, and AD symptoms, orexin-A was hypothesized to be a marker of AD. In cognitively normal healthy older adults, CSF orexin-A was positively correlated with CSF Aβ42, p-τ, and total τ concentrations. In contrast, orexin-B has previously been reported as undetectable in CSF samples. (Wenz, E. et al.) , European Journal of Neurology [European Journal of Neurology], (July 2022) Vol. 29, Supplement 1, p. 315. Abstract number: EPR-201.

[0011] Similar positive associations have been reported among CSF orexin-A, Aβ42, p-τ, and total τ in patients with Alzheimer's disease (AD). Furthermore, targeted proteomics studies using liquid chromatography / mass spectrometry (LC / MS) of markers of neurodegeneration have identified proorexin as one of four proteins specifically used to identify AD and Lewy body dementia. These studies suggest that CSF orexin has the potential to help stage AD (e.g., the number of years from asymptomatic to symptom onset and mild vs. moderate impact) and differentiate AD dementia from other neurodegenerative disorders that cause dementia and controls.

[0012] Therefore, improvements are still needed in the determination of orexin protein. Summary of the Invention

[0013] Several aspects of this disclosure provide improved assay methods for identifying diseases associated with orexin protein levels. This disclosure provides methods for quantifying concentrations in samples and their applications in relation to these diseases.

[0014] This instruction includes methods for quantifying orexin concentrations in fluid samples from patients. In one aspect, these methods may include obtaining a cerebrospinal fluid sample. In another aspect, the method may include mixing the sample with labeled anti-orexin antibody beads. In embodiments, the method further includes mixing with a solution containing NP40 (Sigma), guanidine (Sigma), and a mixture of protein inhibitors (Roche), and an internal orexin standard (13C-labeled orexin-A [Bachem], 13C15N-labeled orexin-B [Sigma], and 13C15N-labeled pro-orexin [Thermo Scientific]). In another aspect, these methods may include, for example, immunoprecipitation of the beads by three washes. In yet another embodiment, the method includes reduction, alkylation, trypsin digestion, and / or desalting (e.g., toptip desalting). On the other hand, these methods may include performing liquid chromatography / mass spectrometry (LC / MS) on the immunoprecipitated sample to quantify orexin concentration.

[0015] In some embodiments, the orexin analyte may be selected from OXA and OXB, these terms encompassing fragments or metabolites thereof, unless the context otherwise indicates. In some embodiments, the orexin analyte is a long OXA N-terminus, a long OXA C-terminus, a short OXA N-terminus, a complete long OXB N-terminus, a cleaved long OXB N-terminus, a complete short OXB N-terminus, a cleaved short OXB N-terminus, proorexin, or a combination thereof. In some embodiments, an antibody is used to bind to the target peptide sequence of the orexin analyte. In some embodiments, the target peptide sequence is one of the peptide sequences disclosed in Table 2 or a combination thereof.

[0016] In some embodiments, these methods can be used to diagnose a patient's sleep disorder based on orexin concentrations in a patient's fluid sample. In some embodiments, the diagnosed patient can be treated with appropriate treatment for the sleep disorder. In some embodiments, the treatment includes administration of an orexin receptor agonist, sodium oxybutyrate, amphetamine, modafinil, armodafinil, or a combination thereof. In some embodiments, the treatment includes administration of an orexin type 2 receptor agonist. In some embodiments, the orexin type 2 receptor agonist includes TAK-925 and / or TAK-861. See Fujimoto T et al. , Discovery of TAK-925 as a Potent, Selective, and Brain-Penetrant Orexin 2 Receptor Agonist [The discovery of TAK-925 as an effective, selective, and brain-osmotic orexin 2 receptor agonist] . ACS Med Chem Lett[ACS Medicinal Chemistry Letters] February 4, 2022; 13(3):457-462 and Mitsukawa K et al. , TAK-861, a potent, orally available orexin receptor 2-selective agonist, produces wakefulness in monkeys and improves narcolepsy-like phenotypes in mouse models [TAK-861 (a potent, orally available selective agonist of orexin receptor 2) induced wakefulness in monkeys and improved narcolepsy-like phenotypes in mouse models]. Sci Rep [Scientific Reports] Sep 6, 2024; 14(1):20838. In some embodiments, the treatment comprises administration of any agonist disclosed in WO2022 / 014680A1. For example, the agonist may be (2R)-2-cyclopropyl-2-{(1R,3S,5S)-3-[(3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl]-8-azabicyclo[3.2.1]oct-8-yl}acetamide, (R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]oct-8-yl)-3-methylbutyryl Amine, (R)-2-((lR,3S,5S)-3-((3S,4R)-1-(5-chloropyrimidin-2-yl)-3-ethoxypiperidin-4-yl)-8-azabicyclo[3.2.1]oct-8-yl)-2-cyclopropylacetamide or (R)-2-cyclopropyl-2-((lR,3S,5S)-3-((2S,4S)-l-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]oct-8-yl)acetamide.

[0017] In some embodiments, these methods can be used to diagnose or stage Alzheimer's disease in a patient based on orexin concentrations in a fluid sample (e.g., to determine the extent of disease progression). Previously, Alzheimer's disease staging was determined by evaluating changes associated with changes in brain architecture, cognitive abilities, memory, communication, and bodily control over one's own body. In some embodiments, these methods may include identifying Alzheimer's disease in a patient based on a combination of orexin concentrations in a fluid sample and one or more additional biomarkers, such as ratios of one or more of τ181, pτ181, τ217, pτ217, Aβ40, Aβ42, MTBR-τ243, or two or more thereof. (Horie K et al.) , CSF MTBR-tau243 is a specific biomarker of tau tangle pathology in Alzheimer's disease [CSF MTBR-τ243 is a specific biomarker for τ tangles pathology in Alzheimer's disease] Nat Med. [Nature Medicine] Aug 2023;29(8):1954-1963.

[0018] In some embodiments, these methods can be used to diagnose or stage a patient’s Parkinson’s disease, traumatic brain injury, Klein-Levin syndrome, somnolence attributable to medical disorders, somnolence attributable to drugs or substances, and somnolence associated with a patient’s mental disorders based on orexin concentrations in the patient’s fluid samples.

[0019] In some embodiments, these methods can be used to treat a patient’s insomnia based on the concentration of orexin in the patient’s fluid sample.

[0020] Other purposes and features will be clearly and partially indicated below. Attached Figure Description

[0021] Those skilled in the art will understand that the accompanying drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of this teaching in any way.

[0022] Figure 1 This study describes the differentiation of sleep disorders by orexin peptides in human cerebrospinal fluid. NT1: Narcolepsy type 1; NT2: Narcolepsy type 2; IH: Idiopathic hypersomnia; C: Control.

[0023] Figure 2 The ratio of the complete orexin-B N-terminal peptide to the cleaved orexin-B peptide was depicted as a way to differentiate patients with type 2 narcolepsy and idiopathic somnolence from controls.

[0024] Figure 3 The concentration of long orexin-AN terminus in the cerebrospinal fluid of patients with NT1, NT2, and IH was depicted compared with that of control patients who used Wako antiorexin-A antibody.

[0025] Figure 4 The concentration of the middle orexin-AC terminus in the cerebrospinal fluid of patients with NT1, NT2, and IH was depicted compared with that of control patients who used Wako Corporation's antiorexin-A antibody.

[0026] Figure 5 The concentrations of short orexin-AN termini in the cerebrospinal fluid of patients with NT1, NT2, and IH were depicted compared with control patients who used R&D Systems' antiorexin-A antibody.

[0027] Figure 6 The concentrations of intact long orexin-BN-termini in the cerebrospinal fluid of patients with NT1, NT2, and IH were depicted compared with control patients who used Wako Corporation's antiorexin-A antibody.

[0028] Figure 7The concentrations of cleaved long orexin-BN-termini in the cerebrospinal fluid of patients with NT1, NT2, and IH were depicted compared with those of control patients who used Wako antiorexin-A antibodies.

[0029] Figure 8 The concentrations of intact short orexin-BN-termini in the cerebrospinal fluid of patients with NT1, NT2, and IH were depicted compared with control patients using antiorexin-A antibodies from the R&D Systems Corporation.

[0030] Figure 9 The concentrations of cleaved short orexin-BN-termini in the cerebrospinal fluid of patients with NT1, NT2, and IH were depicted compared with control patients using antiorexin-A antibodies from the R&D Systems Corporation.

[0031] Figure 10 Potential binding epitopes for OXA, OXB, and proorexin were depicted.

[0032] Figure 11 The quantification of proorexogen in CSF was depicted using a Sigma antiorexogen antibody targeting the N-terminal portion of proorexogen.

[0033] Figure 12 The identification of NT1 by quantitative proorexin was described.

[0034] Figure 13 The study depicted a reduction in the amount of cleaved short OXB N-termini (precipitated by R&D Systems) compared to intact long OXB N-termini (precipitated by Wako Biotech antibody) in NT2 and IH patients.

[0035] Figure 14 The study depicted a reduction in the amount of cleaved short OXB N-terminus (precipitated by antibody from the R&D Systems company) compared to proorexin in NT2 and IH patients.

[0036] Figures 15a-15i depict the effects of sleep deprivation and the sleep medication sodium oxybutyrate on AD biomarkers. The red line represents sleep-deprived subjects, the green line represents sodium oxybutyrate subjects, and the blue line represents control subjects.

[0037] Figures 16a-16c depict the effects of orexin on amyloid symptoms in orexin-overexpressing mice (APP / PS1-21) and orexin knockout mice (APP / PS1-21 / OR- / -). Figure 17c The effects of the dual orexin receptor antagonist amolentide on amyloid deposition in the brain were described.

[0038] Figures 17a-17cThe effects of svoresen on the phosphorylation rates of CSF Aβ40, Aβ42 and p-τ-181 (pT181, T181) were described.

[0039] Figure 18a The workflow for first immunoprecipitation assay, second immunoprecipitation assay, and mass spectrometry assay for quantifying the concentrations of orexin-A metabolites and proorexin is described. Figure 18b The workflow for the first immunoprecipitation assay, second immunoprecipitation assay, and mass spectrometry assay for quantifying the concentration of orexin-B metabolites is described.

[0040] Figures 19a-19h The quantification of intact long OXA N-terminus, intact long OXA C-terminus, intact short OXA N-terminus, proorexinogen, intact long OXB N-terminus, cleaved long OXB N-terminus, intact short OXB N-terminus, and cleaved short OXB N-terminus using immunoprecipitation and mass spectrometry (IP / MS) or internal standard (IS) were described respectively.

[0041] Figure 20 The quantification of orexin-A in CSF samples from patients with narcolepsy type 1, narcolepsy type 2, and idiopathic somnolence was described using radioimmunoassay.

[0042] Figure 21 The concentrations of intact long OXA N-terminus, intact long OXA C-terminus, and intact short OXA N-terminus were depicted (as measured by IP / MS and in...). Figure 1 The concentrations of OXA (as described in the text) and OXA concentrations (as measured by radioimmunoassay and in...) Figure 20 The correlation described in the text.

[0043] Figure 22 a- Figure 22 e depicts the effect of overnight sleep deprivation on orexin peptide concentrations in the CSF. Four participants completed both the control group (normal sleep) and the sleep deprivation intervention group. All orexin peptides are shown as pg / mL concentrations. The overnight period during the intervention night was defined as hours 18–28 (01:00–11:00) to account for the transit time of CSF from the brain to the lumbar spinal canal (shaded area). Error bars indicate standard error. The vertical dashed line represents the intervention start time.

[0044] Figure 23 a- Figure 23e depicts the effect of overnight sleep deprivation on orexin peptides in the CSF, normalized to a percentage relative to baseline. Four participants completed both the control group (normal sleep) and the sleep deprivation intervention group. All orexin peptides were normalized to a percentage relative to baseline at hours 0–12 (07:00–19:00). The overnight period during the intervention night was defined as hours 18–28 (01:00–11:00) to account for the transit time of the CSF from the brain to the lumbar spinal canal (shaded area). Error bars indicate standard error. The vertical dashed line is the intervention start time. The horizontal dashed line is at 100% baseline.

[0045] Figure 24 a- Figure 24 e depicts the effect of overnight sleep deprivation on orexin peptides in the CSF, normalized as a percentage relative to the mean. Four participants completed both the control group (normal sleep) and the sleep deprivation intervention group. All orexin peptides were normalized as a percentage relative to the mean. The overnight period during the intervention night was defined as hours 18 to 28 (01:00–11:00) to account for the transit time of the CSF from the brain to the lumbar spinal canal (shaded area). Error bars indicate standard error. The vertical dashed line is the intervention start time. The horizontal dashed line is at 100% baseline. Detailed Implementation

[0046] This disclosure is based, at least in part, on the finding that the methods described herein can quantify orexin concentrations in fluid samples (such as CSF samples) to identify sleep disorders (including, but not limited to, narcolepsy type 1, narcolepsy type 2, and idiopathic hypersomnia) and Alzheimer's disease. In several aspects, these methods can be used to quantify orexin concentrations (including, but not limited to, proorexin (HCRT), orexin-A, orexin-B, and any combination or fragment or metabolite thereof). In some embodiments, orexin or a fragment or metabolite thereof is one of the peptides disclosed in Table 1 below: Table 1: Fragments of orexin A (Lang et al., J. Med Chem [Journal of Medicinal Chemistry] 2004).

[0047] One aspect of this disclosure provides mass spectrometry measurements of proorexin, orexin-A, and orexin-B peptides in human fluid samples (e.g., cerebrospinal fluid (CSF)). The disclosed data indicate that this assay can be used to distinguish samples from subjects with type 1 narcolepsy, type 2 narcolepsy, or idiopathic hypersomnia from samples from subjects without sleep disorders or from healthy groups (i.e., control samples) using all forms of orexin. These methods also enable the differentiation of type 2 narcolepsy and idiopathic hypersomnia from controls by measuring orexin-B peptides. Further analysis of the N-terminal peptide of orexin-B shows that the ratio of intact peptides to cleaved peptides also distinguishes type 2 narcolepsy and idiopathic hypersomnia from controls. By way of non-limiting example, Figure 1 and Figure 2 This study demonstrates the ability to differentiate centrally originating drowsiness based on the concentrations and ratios of proorexin, orexin A, and orexin B, where the concentrations are obtained using assays as disclosed herein. These methods can also be used to diagnose and differentiate other disorders characterized by low orexin concentrations in human fluid samples, such as Parkinson's disease, traumatic brain injury, Klein-Levin syndrome, drowsiness attributed to medical disorders, drowsiness attributed to drugs or substances, and drowsiness associated with a patient's mental disorder.

[0048] In several embodiments, this disclosure provides mass spectrometry measurements of proorexin, orexin-A, and orexin-B peptides in human fluid samples (e.g., cerebrospinal fluid (CSF)) for the diagnosis or staging of Alzheimer's disease (AD) in patients. In some embodiments, an increase in orexin peptides indicates that a subject has AD. In some embodiments, changes in orexin peptide levels between a first sample and a second sample can be used to determine that a patient has progressed to a more advanced stage of AD. In some embodiments, orexin measurements can be combined with one or more additional AD biomarkers, such as one or more of τ181, pτ181, τ217, pτ217, τ202, pτ202, Aβ38, Aβ40, Aβ42, MTBR-τ243, or ratios of two or more of these additional AD biomarkers.

[0049] In some embodiments, an increase in orexin peptides indicates that a subject with AD requires treatment (i.e., AD therapy). Such treatment may include therapy with anti-amyloid therapy (such as lencanezumab or donepezumab) or anti-tau therapy (such as E2814). See U.S. Patent No. 10,358,485, PCT Publication No. WO2023 / 079485 A1, and Roberts, M. et al. , Pre-clinical characterization of E2814, a high-affinity antibody targeting the microtubule-binding repeat domain of tau for passive immunotherapy in Alzheimer's disease[Preclinical characterization of E2814 (a high-affinity antibody targeting the microtubule-binding repeat domain of τ for passive immunotherapy in Alzheimer's disease)]. Acta Neuropath. Comm. [Journal of Neuropathology Communications] (2020) 8:13. Orexin peptide measurement can be used to indicate whether AD therapy should be started or stopped, or whether a subject should switch to a maintenance dose of AD therapy.

[0050] In several respects, the disclosed assay utilizes mass spectrometry to quantify orexin isolated from the sample, as described below. Unbound by theory, the disclosed mass spectrometry assay overcomes at least some limitations of existing orexin assays. Existing assays typically use immunoassays to quantify orexin in CSF, particularly CSF orexin-A. Other existing assays quantify orexin-B, rather than proorexin. Existing immunoassays are less sensitive than mass spectrometry assays and typically cannot characterize the whole protein. Furthermore, existing immunoassays cannot distinguish between orexin isolated from the sample. 13 For C6-leucine-labeled proteins, stable isotope labeling kinetics (SILK) can be achieved using MS. Furthermore, the mass spectrometry method offers higher sensitivity than existing immunoassays, providing a more precise measurement of orexin concentrations.

[0051] In several aspects, the disclosed assays include immunoprecipitation / mass spectrometry, which separates these proteins by contacting a CSF sample with various antibodies reactive to proorexin, orexin-A, and orexin-B. The assay further includes digestion of the separated orexin, followed by quantification using mass spectrometry.

[0052] In some embodiments, immunoprecipitation to separate proorexin, orexin-A, and orexin-B from a fluid sample (e.g., a CSF sample) comprises contacting the sample with a solid support functionalized with antibodies reactive to multiple portions of proorexin, orexin-A, and orexin-B. In some embodiments, the solid support comprises beads functionalized with antibodies reactive to multiple portions of proorexin, orexin-A, and orexin-B. Any suitable beads may be antibody-functionalized and are not limited to the immunoprecipitation portions of the disclosed assay, including but not limited to agarose beads.

[0053] In several respects, the antibodies used in the immunoprecipitation portion of the disclosed assay may include antibodies reactive to human proorexin, orexin-A, orexin-B. The antibody may be any suitable epitope specific to the N-terminus, C-terminus, intermediate domain, or full-length orexin. In several respects, the orexin protein or fragment targeted by the immunoprecipitation antibody may be selected for the disclosed assay based on one or more of at least several criteria relevant to accurate quantification using mass spectrometry. Non-limiting examples of criteria for the selected orexin protein fragment include peptide chemistry (ionization efficiency, e.g., lack of oxidation and alkylation sites), retention time, charge state, relative intensity, reproducible fragmentation pattern, and specificity to a particular orexin protein (orexin-A, orexin-B, etc.).

[0054] In several aspects, the immunoprecipitation portion of the disclosed assay can be performed in one or more stages. In some aspects, the stage may include a first immunoprecipitation step comprising contacting the sample with a first antibody to separate a first specific orexin, followed by a second immunoprecipitation step comprising contacting the sample with a second antibody to separate a second specific orexin. In some embodiments, the second antibody may be the same as the first antibody, or it may be different (e.g., it preferentially binds to different epitopes or different orexins). In other aspects, the stage may include fractionating the sample and digesting each fraction with different proteases to produce different aggregates of orexin fragments, followed by one or more immunoprecipitation steps, as described above.

[0055] By way of non-limiting example, the disclosed assay of the immunoprecipitation fraction can be performed in two separate stages using two portions of the sample. Each portion of the sample can be sequentially contacted with an antibody targeting a series of target peptides, as described above. By way of non-limiting example, the sample can be split into two portions and analyzed to quantify various forms of OXA and OXB, such as... Figures 3-9 As shown.

[0056] By way of non-limiting example, antibodies used in one or more immunoprecipitation steps may bind to one or more epitopes of orexin peptides.

[0057] In some embodiments, this disclosure provides the use of mass spectrometry. In some embodiments, a sample from a patient undergoes one or two immunoprecipitations as described, and the resulting immunoprecipitated sample is then analyzed by mass spectrometry. In some embodiments, the sample undergoes two immunoprecipitations, and the resulting immunoprecipitated sample is then analyzed by mass spectrometry. In some embodiments, mass spectrometry uses the quantification of target peptides to determine the type of orexin analyte in the immunoprecipitated sample. In some embodiments, mass spectrometry uses the quantification of target peptides to determine the amount of orexin analyte in the immunoprecipitated sample. In some embodiments, the quantified target peptides are those listed in Table 2 below.

[0058] Table 2: Target peptides quantified by mass spectrometry In some embodiments, a combination of selected antibodies can be used to detect and quantify orexin in fluid samples.

[0059] In some embodiments, antiorexin antibody beads are used for immunoprecipitation of orexin from a fluid sample. In some embodiments, the antiorexin antibody beads comprise an antibody that binds to orexin-A. In some embodiments, the antiorexin antibody beads are antiorexin-A antibody beads from R&D Systems, Inc. In some embodiments, the antiorexin antibody beads are antiorexin-A antibody beads from Wako Corporation. In some embodiments, the antiorexin antibody beads bind to orexin-B. In some embodiments, the antiorexin antibody beads are antiorexin-B antibody beads from R&D Systems, Inc. In some embodiments, the antiorexin antibody beads are antiorexin-B antibody beads from Wako Corporation. In some embodiments, the antiorexin antibody beads bind to pro-orexin. In some embodiments, the antiorexin antibody beads are antiorexin antibody beads from Sigma Corporation. In some embodiments, the antiorexin antibodies used in one or more immunoprecipitation steps are disclosed in Table 3.

[0060] Table 3: Polyclonal and monoclonal orexin antibodies.

[0061] In some embodiments, a combination of selected antibodies can be used to detect and quantify orexin in fluid samples.

[0062] In some embodiments, a first antibody binding to orexin-A is mixed in a fluid sample, and then a second antibody binding to orexin-A is mixed in the fluid sample. In some embodiments, the first antibody binding to orexin-A is an anti-orexin-A antibody from He Guang Company, and the second antibody binding to orexin-A is an anti-orexin-A antibody from R&D Systems Company.

[0063] In some embodiments, a first antibody binding orexin-B is mixed in a fluid sample, and then a second antibody binding orexin-B is mixed in the fluid sample. In some embodiments, the first antibody binding orexin-B is an anti-orexin A antibody from He Guang Company, and the second antibody binding orexin-B is an anti-orexin B antibody from R&D Systems Company.

[0064] In some embodiments, a first antibody binding to proorexin is mixed in a fluid sample, and then a second antibody binding to proorexin is mixed in the fluid sample. In some embodiments, the first antibody binding to proorexin is an antiorexin A antibody from Harmony Optical Pharmaceuticals, and the second antibody binding to proorexin is an antiorexin antibody from Sigma-Aldrich.

[0065] By way of non-limiting example, the disclosed assay may include collecting a fluid sample and mixing it with labeled antiorexin antibody beads, τ-MM, and OXA-IS.

[0066] In some embodiments, the beads can then be immunoprecipitated using a washing step, a reduction step, an alkylation step, a trypsin digestion step, a desalting step (e.g., toptip desalting), or a combination thereof. In some embodiments, the resulting sample can then be subjected to liquid chromatography / mass spectrometry (LC / MS) to quantify orexin concentration.

[0067] In addition, the supernatant sample after immunoprecipitation can be analyzed to quantify the propeptide concentration. Labeled anti-OXA antibody beads, anti-propeptide beads, 4X concentration OXA-IS, and propeptide-IS can be mixed with the supernatant sample at room temperature for 2 hours. The sample can then be washed three times, followed by reduction, alkylation, trypsin digestion, and toptip desalting. The sample can then be subjected to LC / MS to quantify the orexin concentration.

[0068] In several embodiments, the methods disclosed herein can be used to diagnose sleep disorders associated with altered orexin levels (e.g., narcolepsy type 2 and idiopathic hypersomnia). Unbound by theory, this diagnosis can achieve higher accuracy than existing methods. Current methods for diagnosing these conditions are based on symptom and sleep research findings; this method would allow for diagnosis based on measurements of CSF orexin peptides.

[0069] In an exemplary embodiment, the mass spectrometry measurements can fully characterize the concentrations and kinetics of proorexitin, orexin-A, and orexin-B in the CSF of people with and without AD symptoms, and correlate them with sleep-wake activity.

[0070] In another instance, these methods can characterize orexin aberrations in human fluid samples (e.g., CSF samples) to diagnose or determine the stage of AD. For example, the mass spectrometry (MS) methods disclosed herein can be used to compare samples from subjects suspected of having AD with samples from subjects without Alzheimer's disease symptoms to quantify one or more of proorexin, orexin-A, orexin-B, and 13C6-leucine-labeled and unlabeled forms of these orexins. Differences between samples can be used to confirm that a subject suspected of having AD does indeed have AD or to determine the stage of AD (e.g., by comparing orexin levels with those of a subject at a known stage of AD).

[0071] In some embodiments, subjects identified as having AD or at risk of AD according to any of the MS methods described above may be treated with at least one sleep-inducing drug to enhance sleep. In some embodiments, the treatment reduces the orexin levels of the treated subject. In some embodiments, the reduction in the orexin levels of the treated subject is monitored, and treatment is continued if a change is observed. In some embodiments, the subject is treated with two sleep-inducing drugs. In some embodiments, the sleep-inducing drugs are leboresen, suvorexin, amolenide, daliresen, sodium oxybutyrate, or combinations thereof. In some embodiments, the sleep-inducing drugs are DORA, a GABA-A receptor agonist, an orexin type 2 receptor agonist, or combinations thereof. In some embodiments, administration of at least one sleep-inducing drug is continued for one month.

[0072] The definitions and methods described herein are provided to better define this disclosure and to guide those skilled in the art in practicing it. Unless otherwise stated, the terminology should be understood in accordance with its conventional usage by those skilled in the art.

[0073] In some embodiments, numerical values ​​for the amounts, properties (such as molecular weight, reaction conditions), etc., of the expressed components used to describe and claim certain embodiments of this disclosure should be understood to be modified by the term "about" in some cases. In some embodiments, the term "about" is used to indicate that the value includes the standard deviation of the average of the means or methods used to determine the value. In some embodiments, the numerical parameters set forth in the written description and appended claims are approximations that may vary depending on the desired properties sought to be obtained in a particular embodiment. In some embodiments, numerical parameters should be interpreted based on the number of significant digits reported and by applying common rounding techniques. Although the numerical ranges and parameters that set forth a broad range of some embodiments of this disclosure are approximations, the numerical values ​​set forth in specific instances are reported as precisely as possible where feasible. The numerical values ​​presented in some embodiments of this disclosure may contain some errors that are inevitably caused by the standard deviation found in their respective test measurements. The description of ranges of values ​​herein is intended only as a shorthand method of individually referring to each individual value falling within that range. Unless otherwise indicated herein, each individual value is incorporated into this specification as if it were described separately herein. The description of discrete values ​​should be understood to include the range between each value.

[0074] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing particular embodiments (especially in the context of certain claims below) may be interpreted to cover both the singular and the plural, unless otherwise specifically stated. In some embodiments, the term “or” as used herein (including the claims) is used to mean “and / or”, unless explicitly indicated to refer only to alternatives or that the alternatives are mutually exclusive.

[0075] The terms “comprise,” “have,” and “include” are open-ended connecting verbs. Any form or tense of one or more of these verbs (such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including”) is also open-ended. For example, any method that “comprises,” “has,” or “includes” one or more steps is not limited to having only those steps and may also cover other steps not listed. Similarly, any composition or apparatus that “comprises,” “has,” or “includes” one or more features is not limited to having only those features and may cover other features not listed.

[0076] All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise obviously contradicted by the context. The use of any and all instances or exemplary language (e.g., “such”) provided with respect to certain embodiments herein is intended only to better illustrate this disclosure and does not limit the scope of this disclosure as otherwise claimed. The language in this specification should not be construed as indicating that any unclaimed element is essential to the practice of this disclosure.

[0077] The grouping of alternative elements or embodiments disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of that group or other elements discovered herein. For convenience or patentability reasons, one or more members of a group may be included in or removed from the group. When any such inclusion or removal occurs, this specification is deemed to contain the modified group to satisfy the written description of all Markush groups used in the appended claims.

[0078] All publications, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes, as if each publication, patent, patent application or other reference were specifically and individually indicated as being incorporated herein by reference in its entirety for all purposes. References herein should not be construed as an admission that they are prior art to this disclosure.

[0079] Having described this disclosure in detail, it will be clear that modifications, variations, and equivalent embodiments are possible without departing from the scope of this disclosure as defined in the appended claims. Furthermore, it should be understood that all examples in this disclosure are provided as non-limiting examples.

[0080] Example The following non-limiting examples are provided to further illustrate this disclosure. It will be understood by those skilled in the art that the techniques disclosed in the following examples represent methods that the inventors have found to work well in the practice of this disclosure, and can therefore be considered examples constituting patterns of their practice. However, based on this disclosure, it will be understood by those skilled in the art that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of this disclosure and still obtaining similar or analogous results.

[0081] Example 1 Methods for detecting central nervous system-related drowsiness Mass spectrometry methods for measuring proorexin, orexin-A, and orexin-B peptides in human cerebrospinal fluid (CSF) have been developed. In short, orexin peptides are analyzed using a nanoAcquity ultra-high performance liquid chromatography system (Waters) connected to an Orbitrap Tribid Eclipse mass spectrometer (Thermo Scientific). Mass spectral transitions are extracted using Skyline (MacCoss Laboratory, University of Washington).

[0082] Orexin in CSF collected from patients diagnosed with NT1 (N = 15), NT2 (N = 15), IH (N = 15), and controls (N = 15) was measured using a sequential immunoprecipitation / mass spectrometry method. Patient characteristics are depicted in Table 4 below.

[0083] Table 4: Characteristics of NT1, NT2 and IH patients.

[0084] Compared with NT2, IH, and the control, NT1 showed a deficiency in ubiquitin, with lower levels of proorexin, orexin-A metabolites, and orexin-B metabolites (all p < 0.05 after Tukey correction for multiple comparisons). Orexin-B metabolites distinguished NT2 and IH from NT1 and the control (all p < 0.05 after Tukey correction for multiple comparisons). Furthermore, compared with IH and the control, NT2 showed a relatively lower ratio of the shortest form of orexin-B to the longer orexin-B metabolites (all p < 0.05 after Tukey correction for multiple comparisons).

[0085] This study tested whether a novel mass spectrometry assay of orexin in different forms of CSF could differentiate between centrally originating narcolepsy. As expected, all forms of orexin were reduced in CSF collected from NT1 patients compared to all other groups. Short metabolites of orexin-B were also found to be reduced in participants with NT2 and IH. Without being bound by theory, aberrant orexin transmission may also be involved in the pathophysiology of type 2 narcolepsy and IH, with varying effects on orexin peptide types.

[0086] Figure 1 The results show that this assay can be used to differentiate between type 1 narcolepsy and type 2 narcolepsy, idiopathic hypersomnia, and controls using all forms of orexin. It can also differentiate between type 2 narcolepsy and idiopathic hypersomnia and controls by measuring orexin-B peptide. Figure 2 The ratio of intact orexin-B peptide to cleaved orexin-B peptide also distinguished type 2 narcolepsy and idiopathic somnolence from controls.

[0087] The orexin system involves a precursor protein called proorexin, which is proteasically cleaved into orexin-A and orexin-B (orexin is also referred to as hypothalamic secretin in the literature). Radioimmunoassay (RIA) for orexin-A measures the N-terminus of the protein and is used to diagnose type 1 narcolepsy, but... Figure 20 The drowsiness shown cannot be distinguished from other central nervous system sources. Furthermore, Figure 21 The results will show that, as measured by immunoprecipitation and mass spectrometry, and in... Figure 1 The concentrations of orexin-A metabolites described herein are similar to those measured by RIA and in Figure 20 The correlation between the concentration of orexin-A described in the study demonstrates that the RIA primarily measures erroneous orexin-A metabolites, and that the RIA measures orexin-A near the N-terminus.

[0088] The assays for the C-terminus of orexin-A, as well as for proorexin and orexin-B, were insensitive and showed no difference between different sources of somnolence from the central nervous system.

[0089] This disclosure will allow for the diagnosis of narcolepsy type 2 and idiopathic hypersomnia with greater accuracy than existing methods.

[0090] Example 2 Quantitative determination of orexin-A by immunoprecipitation-mass spectrometry The immunoprecipitation assay for detecting and quantifying orexin-A is disclosed below. Figure 18a .

[0091] To prepare the OXA-IS (internal standard) (“OXA-1-IP”) for analysis, 10 µL of 10 µg / mL OXA-IS was mixed with 990 µL of 1% HSA to obtain 100 ng / mL OXA-IS in 1% HSA. Two tubes were prepared, totaling 2 mL of this solution. Next, in 15 mL Falcon tubes, 2 x 980 µL of 100 ng / mL OXA-IS was mixed with 8 x 980 µL (7840 µL) of 1% HSA (totaling approximately 9.8 mL of 20 ng / mL OXA-IS). Then, 10 µL of 20 ng / mL OXA-IS (200 pg) was aliquoted into 1.5 mL Eppendorf Protein-Lobind tubes and stored at -80°C until use.

[0092] To prepare OXA-IS and propeptide-IS (“OXA-propeptide-2-IP”) for analysis, 10 µL of 10 µg / mL OXA-IS was mixed with 990 µL of 1% HSA to obtain 100 ng / mL OXA-IS in 1% HSA. Three tubes were prepared, totaling 3 mL of this solution. Next, in 15 mL Falcon tubes, 3 x 980 µL of 100 ng / mL OXA-IS was mixed with 3 x 980 µL of 100 ng / mL propeptide-IS (totaling approximately 5.88 mL of 50 ng / mL OXA-IS and 50 ng / mL propeptide-IS). Then, aliquot 10 µL of the mixture of “50 ng / mL OXA-IS and propeptide-IS” (500 pg each of OXA-IS and propeptide-IS) into 1.5 mL Eppendorf Protein-Lobind tubes and store at -80°C until use.

[0093] On Day 1 (the first immunoprecipitation of OXA (“IP”), 220 µL of CSF was pipetted into a 1.5 mL Eppendorf Protein LoBind tube containing 10 µL of 20 ng / mL “OXA-IP”. Then, 12.5 µL of τ premix (containing NP40, guanidine, and PI in PBS) was added. Next, 20 µL of 5x diluted anti-OXA monoclonal antibody and photopolymer beads (50% slurry) were added to this mixture. The resulting mixture was incubated at 4°C for more than 16 hours.

[0094] On day 2 (after transfer of IP supernatant, washing, reduction / alkylation, and digestion), the mixture was centrifuged at 3901 rcf for 5 minutes using a basket rotor to precipitate the beads. The collected 200 µL (fixed volume) of supernatant was transferred to a 1.5 mL Eppendorf Protein LoBind tube containing 10 µL of “OXA-propeptide-second-IP”. The tube was stored at -80°C until immunoprecipitation began.

[0095] For washing, set the Eppendorf ThermoMixer C to 65°C and close the cap to allow incubation of the sample during DTT reduction. After transfer following immunoprecipitation, aspirate the residual supernatant. (1) Add 1 mL of 25 mM TEABC. (2) Invert the sample 30 times to wash the beads. (3) Centrifuge the sample at 390 rpm for 5 minutes using a basket spinner to precipitate the beads. (4) Aspirate the supernatant. Repeat steps (1)-(4) above for a total of 3 washes.

[0096] For reduction / alkylation, add 20 µL of 5 mM DTT in 25 mM TEABC to the sample, followed by vortexing and rapid shake centrifugation. Then incubate the sample at 65 °C and 1000 rpm for 30 min (+ / - min) using an Eppendorf ThermoMixer C. Next, add 20 µL of 10 mM IAA in 25 mM TEABC to the sample, followed by vortexing. Then incubate the sample in the dark at room temperature for 30 min (+ / - 1 min), followed by rapid shake centrifugation.

[0097] For trypsin digestion, add 40 µL of 10 µg / mL trypsin in 25 mM TEABC to the sample, followed by vortexing and rapid centrifugation. Then incubate the sample at 37°C for 16–20 hours.

[0098] On day 3 (solid-phase extraction via Oasis HLB Plated), 100 µL of 25 mMTEABC was added to the sample, followed by vortexing. The sample was then centrifuged at 390 rcf for 5 min using a basket rotor to precipitate beads. The Oasis HLB μElution plate was pretreated with 200 µL MeOH, 200 µL 60% acetonitrile and 0.1% formic acid (FA), and 200 µL 0% acetonitrile and 0.1% FA (x2). After pretreatment of the Oasis HLB μElution plate, the entire supernatant was added to the plate. The plate was then washed twice with 200 µL of 0% acetonitrile and 0.1% FA. Elution was performed by adding 100 µL of 60% acetonitrile and 0.1% FA. The collected plate was rapidly rotated using a swing centrifuge. The sample was then transferred from the collected plate to a new 1.5 mL Eppendorf Protein LoBind tube. The samples were then frozen on dry ice and Speed-Vac at 4 °C to remove the solvent. Optionally, these dried samples could be stored at -80 °C. When ready for immediate LC / MS analysis, the samples were resuspended in 27.5 µL of 3% acetonitrile and 3% FA under vortex. The samples were centrifuged at 21130 rcf for 10 min at 4 °C using an angle rotor. 25 µL of the supernatant was then transferred to an MS vial for LC / MS analysis using the Oribtrap Ellipse.

[0099] Example 3 Quantitative determination of orexin-A by immunoprecipitation-mass spectrometry The immunoprecipitation assay for detecting and quantifying orexin-A is disclosed below. Figure 18a .

[0100] To prepare OXA-IS and propeptide-IS (“OXA-propeptide-2-IP”) for analysis, 10 µL of 10 µg / mL OXA-IS was mixed with 990 µL of 1% HSA to obtain 100 ng / mL OXA-IS in 1% HSA. Three tubes (100 ng / mL OXA-IS) were prepared, totaling 3 mL of this solution. Then, 10 µg / mL propeptide-IS was mixed with 990 µL of 1% HSA to obtain 100 ng / mL propeptide-IS in 1% HSA. In 15 mL Falcon tubes, 3 x 980 µL of 100 ng / mL OXA-IS was mixed with 3 x 980 µL of 100 ng / mL propeptide-IS (totaling approximately 5.88 mL of 50 ng / mL OXA-IS and 50 ng / mL propeptide-IS). Next, aliquot 10 µL of the mixture of “50 ng / mL OXA-IS and propeptide-IS” (500 pg each of OXA-IS and propeptide-IS) into 1.5 mL Eppendorf Protein-Lobind tubes and store at -80°C until use.

[0101] On day 1 (OXA second immunoprecipitation, transfer of supernatant after IP, washing, reduction / alkylation, and digestion), thaw 200 µL of supernatant and 10 µL of “OXA-propeptide-second-IP” from a 1.5 mL Eppendorf Protein LoBind tube. Centrifuge the sample to combine the sample volumes to the bottom of the tube. Add 40 µL of a 1:1 mixture (50% slurry) of anti-OXA monoclonal R&D System beads and anti-pro-OX promonoclonal Sigma beads to the sample. Rotate the resulting mixture at room temperature for 150 min (+ / - 30 min).

[0102] After transferring the IP supernatant, centrifuge the sample at 390 rcf for 5 minutes using a basket rotor to precipitate the beads. Transfer the supernatant to a 1.5 mL Eppendorf Protein LoBind tube without disturbing the beads. Store the tube at -80°C.

[0103] For washing, set the Eppendorf ThermoMixer C to 65°C and close the cap to allow incubation of the sample during DTT reduction. After transfer following immunoprecipitation, aspirate the residual supernatant. (1) Add 1 mL of 25 mM TEABC. (2) Invert the sample 30 times to wash the beads. (3) Centrifuge the sample at 390 rpm for 5 minutes using a basket spinner to precipitate the beads. (4) Aspirate the supernatant. Repeat steps (1)-(4) above for a total of 3 washes.

[0104] For reduction / alkylation, add 20 µL of 5 mM DTT in 25 mM TEABC to the sample, followed by vortexing and rapid shake centrifugation. Then incubate the sample at 65 °C and 1000 rpm for 30 min (+ / - min) using an Eppendorf ThermoMixer C. Next, add 20 µL of 10 mM IAA in 25 mM TEABC to the sample, followed by vortexing. Then incubate the sample in the dark at room temperature for 30 min (+ / - 1 min), followed by rapid shake centrifugation.

[0105] For trypsin digestion, add 40 µL of 10 µg / mL trypsin in 25 mM TEABC to the sample, followed by vortexing and rapid centrifugation. Then incubate the sample at 37°C for 16–20 hours.

[0106] On day 2 (solid-phase extraction via Oasis HLB Plated), 100 µL of 25 mMTEABC was added to the sample, followed by vortexing. The sample was then centrifuged at 390 rcf for 5 min using a basket rotor to precipitate the beads. The Oasis HLB μElution plate was pretreated with 200 µL MeOH, 200 µL 60% acetonitrile and 0.1% formic acid (FA), and 200 µL 0% acetonitrile and 0.1% FA (x2). After pretreatment of the Oasis HLB μElution plate, the entire supernatant was added to the plate. The plate was then washed twice with 200 µL of 0% acetonitrile and 0.1% FA. Elution was performed by adding 100 µL of 60% acetonitrile and 0.1% FA. The plate was collected by rapid rotation using a swing centrifuge. The sample was then transferred from the collected plate to a new 1.5 mL Eppendorf Protein LoBind tube. The samples were then frozen on dry ice and Speed-Vac at 4 °C to remove the solvent. Optionally, these dried samples could be stored at -80 °C. When ready for immediate LC / MS analysis, the samples were resuspended in 27.5 µL of 3% acetonitrile and 3% FA under vortex. The samples were centrifuged at 21130 rcf for 10 min at 4 °C using an angle rotor. 25 µL of the supernatant was then transferred to an MS vial for LC / MS analysis using the Oribtrap Ellipse.

[0107] Example 4 Quantitative determination of orexin-B by immunoprecipitation-mass spectrometry The immunoprecipitation assay for detecting and quantifying orexin-B is disclosed below. Figure 18b .

[0108] To prepare the OXB-IS (internal standard) (“OXB-1-IP”) for the first immunoprecipitation assay, 10 µL of 10 µg / mL OXB-IS was mixed with 990 µL of 1% HSA to obtain 100 ng / mL OXB-IS in 1% HSA. Two tubes were prepared, totaling 2 mL of this solution. Next, in 15 mL Falcon tubes, 2 x 980 µL of 100 ng / mL OXB-IS was mixed with 8 x 980 µL (7840 µL) of 1% HSA (totaling approximately 9.8 mL of 20 ng / mL OXB-IS). Then, 10 µL of 20 ng / mL OXB-IS (200 pg) was aliquoted into 1.5 mL Eppendorf Protein-Lobind tubes and stored at -80°C until use.

[0109] To prepare OXB-IS (“OXB-2-IP”) for the second immunoprecipitation assay, 10 µL of 10 µg / mL OXB-IS was mixed with 990 µL of 1% HSA to obtain 100 ng / mL OXB-IS in 1% HSA. Three tubes were prepared, totaling 3 mL of this solution. Next, in 15 mL Falcon tubes, 3 x 980 µL of 100 ng / mL OXB-IS was mixed with 3 x 980 µL of 1% HSA (totaling approximately 5.88 mL of 50 ng / mL OXB-IS). Then, 10 µL of the 50 ng / mL OXB-IS was aliquoted into 1.5 mL Eppendorf Protein-Lobind tubes and stored at -80°C until use.

[0110] On day 1 (the first immunoprecipitation of OXB (“IP”), 200 µL of CSF was pipetted into a 1.5 mL Eppendorf Protein LoBind tube containing 10 µL of 20 ng / mL “OXB-IP”. Then, 12.5 µL of τ premix (containing NP40, guanidine, and PI in PBS) was added. Next, 20 µL of 5x diluted anti-OXA monoclonal antibody and photopolymer beads (50% slurry) were added to this mixture. The resulting mixture was incubated at 4°C for more than 16 hours.

[0111] On day 2 (after transfer of IP supernatant, washing, and digestion), the mixture was centrifuged at 390 rpm for 5 minutes using a basket rotor to precipitate the beads. The collected 200 µL (fixed volume) of supernatant was transferred to a 1.5 mL Eppendorf Protein LoBind tube containing 10 µL of 50 ng / mL “OXB-2-IP”. The tube was stored at -80°C until immunoprecipitation began.

[0112] For washing, after transfer following immunoprecipitation, aspirate the residual supernatant. (1) Add 1 mL of 25 mM TEABC. (2) Then invert the sample 30 times to wash the beads. (3) Then centrifuge the sample at 390 rpm for 5 minutes using a basket rotor to precipitate the beads. (4) Then aspirate the supernatant. Repeat steps (1)-(4) above for a total of 3 washes.

[0113] For trypsin digestion, add 40 µL of 10 µg / mL trypsin in 25 mM TEABC to the sample, followed by vortexing and rapid centrifugation. Then incubate the sample at 37°C for 16–20 hours.

[0114] On day 3 (solid-phase extraction via Oasis HLB Plated), 100 µL of 25 mMTEABC was added to the sample, followed by vortexing. The sample was then centrifuged at 390 rcf for 5 min using a basket rotor to precipitate beads. The Oasis HLB μElution plate was pretreated with 200 µL MeOH, 200 µL 60% acetonitrile and 0.1% formic acid (FA), and 200 µL 0% acetonitrile and 0.1% FA (x2). After pretreatment of the Oasis HLB μElution plate, the entire supernatant was added to the plate. The plate was then washed twice with 200 µL of 0% acetonitrile and 0.1% FA. Elution was performed by adding 100 µL of 60% acetonitrile and 0.1% FA. The collected plate was rapidly rotated using a swing centrifuge. The sample was then transferred from the collected plate to a new 1.5 mL Eppendorf Protein LoBind tube. The samples were then frozen on dry ice and Speed-Vac at 4 °C to remove the solvent. Optionally, these dried samples could be stored at -80 °C. When ready for immediate LC / MS analysis, the samples were resuspended in 27.5 µL of 2% acetonitrile and 2% FA under vortex. The samples were centrifuged at 21130 rcf for 10 min at 4 °C using an angle rotor. 25 µL of the supernatant was then transferred to an MS vial for LC / MS analysis using the Oribtrap Ellipse.

[0115] Example 5 Quantitative determination of orexin-B by immunoprecipitation-mass spectrometry The immunoprecipitation assay for detecting and quantifying orexin-B is disclosed below. Figure 18b .

[0116] To prepare OXB-IS and propeptide-IS (“OXB-2-IP”) for analysis, 10 µL of 10 µg / mL OXB-IS was mixed with 990 µL of 1% HSA to obtain 100 ng / mL OXB-IS in 1% HSA. Three tubes (100 ng / mL OXB-IS) were prepared, totaling 3 mL of this solution. In 15 mL Falcon tubes, 3 x 980 µL of 100 ng / mL OXB-IS was mixed with 3 x 980 µL of 1% HSA (approximately 5.88 mL of 50 ng / mL OXB-IS in total). Next, 10 µL of 50 ng / mL OXB-IS was aliquoted into 1.5 mL Eppendorf Protein-Lobind tubes and stored at -80°C until use.

[0117] On day 1 (OXB second immunoprecipitation, transfer of supernatant after IP, washing and digestion), thaw 200 µL of supernatant and 10 µL of “OXB-2-IP” from a 1.5 mL Eppendorf Protein LoBind tube. Centrifuge the sample to combine the sample volumes to the bottom of the tube. Add 20 µL of anti-OXB monoclonal R&D system beads (50% slurry) to the sample. Rotate the resulting mixture at room temperature for 150 min (+ / - 30 min).

[0118] After transferring the IP supernatant, centrifuge the sample at 390 rcf for 5 minutes using a basket rotor to precipitate the beads. Transfer the supernatant to a 1.5 mL Eppendorf Protein LoBind tube without disturbing the beads. Store the tube at -80°C.

[0119] For washing, after transfer following immunoprecipitation, aspirate the residual supernatant. (1) Add 1 mL of 25 mM TEABC. (2) Then invert the sample 30 times to wash the beads. (3) Then centrifuge the sample at 390 rpm for 5 minutes using a basket rotor to precipitate the beads. (4) Then aspirate the supernatant. Repeat steps (1)-(4) above for a total of 3 washes.

[0120] For trypsin digestion, add 40 µL of 10 µg / mL trypsin in 25 mM TEABC to the sample, followed by vortexing and rapid centrifugation. Then incubate the sample at 37°C for 16–20 hours.

[0121] On day 2 (solid-phase extraction via Oasis HLB Plated), 100 µL of 25 mMTEABC was added to the sample, followed by vortexing. The sample was then centrifuged at 390 rcf for 5 min using a basket rotor to precipitate the beads. The Oasis HLB μElution plate was pretreated with 200 µL MeOH, 200 µL 60% acetonitrile and 0.1% formic acid (FA), and 200 µL 0% acetonitrile and 0.1% FA (x2). After pretreatment of the Oasis HLB μElution plate, the entire supernatant was added to the plate. The plate was then washed twice with 200 µL of 0% acetonitrile and 0.1% FA. Elution was performed by adding 100 µL of 60% acetonitrile and 0.1% FA. The plate was collected by rapid rotation using a swing centrifuge. The sample was then transferred from the collected plate to a new 1.5 mL Eppendorf Protein LoBind tube. The samples were then frozen on dry ice and Speed-Vac at 4 °C to remove the solvent. Optionally, these dried samples could be stored at -80 °C. When ready for immediate LC / MS analysis, the samples were resuspended in 27.5 µL of 2% acetonitrile and 2% FA under vortex. The samples were centrifuged at 21130 rcf for 10 min at 4 °C using an angle rotor. 25 µL of the supernatant was then transferred to an MS vial for LC / MS analysis using the Oribtrap Ellipse.

[0122] Example 6 Detection and quantification of orexin-A Cerebrospinal fluid samples were obtained from patients with type 1 narcolepsy (n = 15), type 2 narcolepsy (n = 15), idiopathic hypersomnia (n = 15), and healthy patients without sleep disorders (n = 15). Example 2 and... Figure 18a The immunoprecipitation-mass spectrometry method disclosed in the report is used to detect and quantify the N-terminus and C-terminus of long OXA molecules. The concentrations of the N-terminus and C-terminus of long OXA molecules in each patient cohort are disclosed in [the report / document / etc.]. Figure 3 and Figure 4 middle.

[0123] By using Example 3 and Figure 18a The second immunoprecipitation-mass spectrometry assay disclosed in the report is used to detect and quantify short OXAN-terminals. The concentration of short OXAN-terminals is disclosed in [the report / document / etc.]. Figure 5 middle.

[0124] Example 7 Detection and quantification of orexin-B Cerebrospinal fluid samples were obtained from patients with type 1 narcolepsy (n = 15), type 2 narcolepsy (n = 15), idiopathic hypersomnia (n = 15), and healthy patients without sleep disorders (n = 15). Example 4 and... Figure 18b The immunoprecipitation-mass spectrometry assay disclosed herein is used to detect and quantify intact and cleaved long OXB N-termini. The concentrations of intact and cleaved long OXB N-termini for each patient cohort are disclosed in [the data / details]. Figure 6 and Figure 7 middle.

[0125] By using Example 5 and Figure 18b The second immunoprecipitation-mass spectrometry assay disclosed herein is used to detect and quantify intact and cleaved short OXB N-termini. The concentrations of intact and cleaved short OXB N-termini are disclosed in [the following text is missing from the original extract]. Figure 8 and Figure 9 middle.

[0126] Example 8 Detection and quantification of proorexin Using Sigma's anti-proorgasmogen antibody and Example 3 and Figure 18a The study disclosed the determination of proorexin in cerebrospinal fluid samples. Figure 11 This demonstrates successful quantification of the N-terminus of proorexogen, while the C-terminus of proorexogen cannot be adequately quantified.

[0127] In addition, cerebrospinal fluid samples were obtained from patients with type 1 narcolepsy (n = 15), type 2 narcolepsy (n = 15), idiopathic hypersomnia (n = 15), and healthy patients without sleep disorders (n = 15). (Usage Example 3 and...) Figure 18a The method of immunoprecipitation coupled with mass spectrometry for detecting and quantifying proorexin was disclosed in [the document / document / etc.]. The concentration of proorexin was disclosed in [the document / document / etc.]. Figure 12 middle.

[0128] Example 9 Comparison of cut short OXB N-terminus, intact long OXB N-terminus, and proorexin. The concentrations of the cleaved short OXB N-terminus, the intact long OXB N-terminus, and proorexogen obtained in Examples 7 and 8 above were compared (the sequences of the cleaved short OXB N-terminus, the intact long OXB N-terminus, and proorexogen are shown in Table 2). A comparison of the concentrations of the cleaved short OXB N-terminus with the intact long OXB N-terminus is disclosed in [the table below]. Figure 13 The comparison of the concentrations of the N-terminus of cleaved short OXB with those of proorexin is disclosed in [the journal / publication]. Figure 14 middle.

[0129] Example 10 The effect of sleep deprivation on Alzheimer's disease biomarkers Subjects were assigned to different sleep protocols, and samples were taken to measure T181, S202, and T217. Their concentrations were normalized relative to each subject's baseline (mean over hours 07:00–19:00) prior to intervention in each group. Four subjects completed the control, sleep deprivation, and drug intervention protocols. Four subjects completed both protocols. Compared to the control and drug (sodium oxybutyrate) groups, the mean overnight concentrations of Aβ38, Aβ40, and Aβ42 were approximately 30% higher than baseline in sleep deprivation participants (Figures 15a–15c). Compared to the control and drug groups, the mean overnight concentrations of unphosphorylated T181, S202, and T217 were 30%–50% higher than baseline in sleep deprivation participants (Figures 15d–15f). The mean overnight variation of pT181 was similar to that of unphosphorylated T181. In contrast, phosphorylated S202 (pS202) showed no difference between the sleep deprivation group and the control group, or between the drug group and the control group. For phosphate-τT217 (pT217), there was a significant increase of 60%-80% in the sleep deprivation group compared with the control, while there was no change in the drug group.

[0130] To measure the phosphorylation rate at each site, the ratio of phosphate-τ to unphosphorylated forms was compared, and it was shown that each phosphate-τ species responded differently to sleep deprivation (Fig. 15g–Fig. 15i). Under sleep deprivation, the mean overnight phosphorylation ratio of pT217 / T217 increased by 15%–20% relative to baseline, while pT181 / T181 showed no difference between groups, and pS202 / S202 decreased more relative to baseline during sleep deprivation compared to controls. These findings demonstrate the importance of the p-τ / τ ratio and avoid the confounding effect of increased p-τ concentrations due solely to increased τ concentrations without altering the relative phosphorylation rate (e.g., pT181 increases due to increased T181, compared to the increase in pT217 exceeding the change in T217). Since participants were amyloid-negative based on their CSFAβ42 / 40 ratio, future research is urgently needed to determine how CSF p-τ is affected by sleep at different stages of AD symptoms (e.g., amyloid-negative vs. amyloid-positive).

[0131] Example 11 Effects of orexin on amyloid symptoms This study investigated transgenic mice with orexin gene knockout or overexpression of amyloid precursor protein (APP). Knockout of orexin resulted in a significant reduction in amyloid symptom in the brain, while overexpression in the hippocampus did not alter amyloid deposition (Fig. 16a-16b).

[0132] Treatment with DORA (amorente) decreased soluble Aβ concentrations, while intraventricular administration of orexin increased them. Furthermore, prolonged treatment with amorente for 8 weeks reduced amyloid deposition (Figure 16c). In humans, patients with narcolepsy (i.e., those with orexin deficiency) showed reduced CSF Aβ, τ, p-τ, and amyloid deposition on amyloid PET compared to age- and sex-matched controls. Moreover, CSF orexin levels were higher in early AD and associated with sleep disturbances. These findings strongly suggest that blocking orexin can modulate AD symptoms in the brain.

[0133] Example 12 Use orexin antagonists to reduce Aβ levels and τ phosphorylation In an ongoing study, the effects of sovoraxone (the first-in-class dual orexin receptor antagonist (DORA)) on CSF Aβ levels and p-τ-181 phosphorylation were measured. Cognitively normal adults aged 45–65 years were randomized to receive sovoraxone 10 mg, sovoraxone 20 mg, or placebo. CSF samples were collected every 2 hours via an indwelling lumbar catheter for 36 hours. At 9 pm, participants received a blinded slice, and the lights were off. Upon waking on day 2 (approximately 6–7 am), participants remained awake until approximately 9 pm, at which point they received their second blinded slice (no change in the intervention group). The study ended at 36 hours (day 3, approximately 8 am). After normalization relative to t = 0, compared to placebo, CSF Aβ40, Aβ42, and pT181 / T181 ratio (a measure of phosphorylation rate) were significantly reduced in the sovoraxone 20 mg group. Figures 17a-17c ).

Claims

1. A method for quantitatively determining the concentration of at least one orexin selected from orexin-A, orexin-B, proorexin, and any combination thereof in a fluid sample, preferably a cerebrospinal fluid sample, the method comprising: a. Obtain the fluid sample, b. Mix the fluid sample with at least one labeled anti-orexin antibody bead to immunoprecipitate the at least one orexin from the fluid sample to form at least one isolated orexin sample containing at least one of orexin-A, orexin-B and proorexin. as well as c. Perform liquid chromatography / mass spectrometry (LC / MS) on each of the at least one isolated orexin sample to quantify the concentration of the at least one orexin.

2. The method of claim 1, wherein the cerebrospinal fluid sample is mixed with orexin-A-binding anti-orexin antibody beads.

3. The method of claim 2, wherein the antiorexin antibody beads are antiorexin-A antibody beads from the R&D Systems Company.

4. The method of claim 2, wherein the antiorexin antibody beads are antiorexin-A antibody beads from Wako Corporation.

5. The method of claim 1, wherein the cerebrospinal fluid sample is mixed with orexin-binding anti-orexin antibody beads.

6. The method of claim 5, wherein the antiorexin antibody beads are antiorexin-B antibody beads from R&D Systems.

7. The method of claim 5, wherein the antiorexin antibody beads are antiorexin-B antibody beads from Wako Corporation.

8. The method of claim 1, wherein the cerebrospinal fluid sample is mixed with antiorexin beads that bind to proorexin.

9. The method of claim 8, wherein the antiorexin antibody beads are Sigma antiorexin antibody beads.

10. The method of any one of claims 1-9, the method further comprising a second mixing step, wherein the separated orexin sample is mixed with a second anti-orexin antibody bead, and then the separated orexin sample is subjected to LC / MS.

11. The method of claim 10, wherein the cerebrospinal fluid sample is mixed with a first anti-orexin antibody bead that binds orexin-A, and then mixed with a second anti-orexin antibody bead that binds orexin-A.

12. The method of claim 10, wherein the cerebrospinal fluid sample is mixed with a first anti-orexin antibody bead that binds orexin-A, and then mixed with a second anti-orexin antibody bead that binds pre-orexinogen.

13. The method of claim 10, wherein the cerebrospinal fluid sample is mixed with a first anti-orexin antibody bead that binds orexin-B, and then mixed with a second anti-orexin antibody bead that binds orexin-B.

14. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing the long-anoretin-AN-terminal quantitative target peptide.

15. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing the long orexin-AC terminus to quantify the target peptide.

16. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing the short-anoretin-AN-terminal quantitative target peptide.

17. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing the intact long orexin-BN-terminus quantitative target peptide.

18. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing the quantitative target peptide at the cleaved long orexin-BN terminus.

19. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing the intact short orexin-BN-terminus to quantify the target peptide.

20. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing the cleaved short orexin-BN-terminus quantitative target peptide.

21. The method of any one of claims 10-20, the method further comprising a washing step between the first mixing step and the second mixing step.

22. The method of any one of claims 10-21, further comprising a washing step between the second mixing step and the LC / MS step.

23. The method of any one of claims 1-22, wherein the method further comprises a reduction step.

24. The method of any one of claims 1-23, wherein the method further comprises an alkylation step.

25. The method of any one of claims 1-24, wherein the method further comprises a trypsin digestion step.

26. A method for diagnosing a sleep disorder in a patient selected from narcolepsy type 1, narcolepsy type 2, and idiopathic somnolence, the method comprising measuring the concentration of at least one orexin selected from orexin-A, orexin-B, proorexin, and any combination thereof in a fluid sample from the patient, wherein the concentration of the at least one orexin is obtained using the method as described in any one of the preceding claims, and wherein a decrease in the level of the at least one orexin protein relative to the level in a control sample indicates that the patient has the sleep disorder.

27. The method of claim 26, wherein the fluid sample is a cerebrospinal fluid sample.

28. The method of claim 26 or 27, wherein the sleep disorder is narcolepsy type 2.

29. The method of claim 26 or 27, wherein the sleep disorder is idiopathic hypersomnia.

30. The method of claim 26 or 27, wherein the concentration of orexin-A is used to identify the patient's type 1 narcolepsy.

31. The method of claim 26 or 27, wherein the concentration of orexin-B is used to identify the patient's type 2 narcolepsy.

32. The method of claim 26 or 27, wherein the concentration of orexin-B is used to identify idiopathic somnolence in the patient.

33. A method for treating a patient with a sleep disorder selected from narcolepsy type 1, narcolepsy type 2, and idiopathic hypersomnia, the method comprising administering a treatment for the sleep disorder to a patient diagnosed with the sleep disorder, wherein the patient has been diagnosed by means of measuring the concentration of at least one orexin selected from orexin-A, orexin-B, proorexin, and any combination thereof in a fluid sample from the patient, wherein the concentration of the at least one orexin is obtained using the method as described in any one of the preceding claims, and wherein the level of the at least one orexin protein is increased relative to the level in a control sample taken from the patient prior to the administration of the treatment.

34. The method of claim 33, wherein the fluid sample is a cerebrospinal fluid sample.

35. The method of claim 33 or 34, wherein the sleep disorder is narcolepsy type 2.

36. The method of claim 33 or 34, wherein the sleep disorder is idiopathic hypersomnia.

37. The method of any one of claims 33-36, wherein the treatment comprises administering an orexin receptor agonist, sodium hydroxybutyrate, amphetamine, modafinil, armodafinil, or a combination thereof.

38. The method of claim 37, wherein the orexin receptor agonist is an orexin type 2 receptor agonist.

39. The method of claim 38, wherein the orexin type 2 receptor agonist is TAK-925 or TAK-861.

40. A method for differentiating between type 1 narcolepsy, type 2 narcolepsy, and idiopathic hypersomnia in a patient presenting with a sleep disorder, the method comprising measuring the concentration of at least one orexin selected from orexin-A, orexin-B, proorexin, and any combination thereof in a fluid sample from the patient, wherein the concentration of the at least one orexin is obtained using the method of any one of claims 1-25, and wherein a change in the level of the at least one orexin protein relative to the level in a control sample indicates whether the patient has type 1 narcolepsy, type 2 narcolepsy, or idiopathic hypersomnia.

41. The method of claim 40, wherein a decrease in the patient’s orexin-A level relative to the level in the control sample indicates that the patient has type 1 narcolepsy.

42. The method of claim 40, wherein a decrease in the patient’s orexin-B level relative to the level in the control sample indicates that the patient has type 2 narcolepsy or idiopathic somnolence.

43. A method for diagnosing Alzheimer's disease (AD) in a patient, the method comprising measuring the concentration of at least one orexin selected from orexin-A, orexin-B, proorexin, and any combination thereof in a fluid sample from the patient, wherein the concentration of the at least one orexin is obtained using the method as described in any one of the preceding claims, and wherein an increase in the level of the at least one orexin protein relative to the level in a control sample indicates that the patient has AD.

44. A method for staging Alzheimer's disease (AD) in a patient, the method comprising measuring the concentration of at least one orexin selected from orexin-A, orexin-B, proorexin, and any combination thereof in a fluid sample from the patient, wherein the concentration of the at least one orexin is obtained using the method as described in any of the preceding claims, and comparing the orexin level with the level in a sample from a subject having a known AD stage, thereby staging the AD in the patient.

45. The method of claim 44, further comprising measuring the concentration of at least one orexin selected from orexin-A, orexin-B, proorexin, and any combination thereof in a second fluid sample from the patient, wherein the concentration of the at least one orexin is obtained using the method as described in any of the preceding claims, wherein an increase in orexin levels between the two samples indicates that the subject has progressed to a more advanced stage of AD.

46. ​​The method of any one of claims 43-45, the method further comprising measuring the ratio of one or more, or two or more, of one or more of the following AD biomarkers in a sample from the patient: τ181, pτ181, τ217, pτ217, τ202, pτ202, Aβ38, Aβ40, Aβ42, MTBR-τ243.

47. The method of any one of claims 43-46, wherein the sample is a CSF sample.

48. A method of treating AD, the method comprising administering AD treatment to a subject diagnosed with AD according to the method of any of the preceding claims.

49. The method of claim 48, wherein the treatment comprises administering suvorexin, amolenide, daliresen, sodium hydroxybutyrate, leboresen, or a combination thereof.

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