Assay methods to identify a disease

Abstract

Among the various aspects of the present disclosure is the provision of assay methods to identify diseases associated with orexin levels. The present teachings include methods to quantify an orexin concentration in a fluid sample, such as a cerebrospinal fluid sample, and identifying and treating diseases, including but not limited to narcolepsy and Alzheimer's disease, from the orexin concentration.

Description

ASSAY METHODS TO IDENTIFY A DISEASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of 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.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under AG074151 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The present disclosure generally relates to assay methods to identify diseases associated with altered orexin concentrations.BACKGROUND

[0004] Hypersomnias of central origin include narcolepsy type 1, narcolepsy type 2, and idiopathic hypersomnia, which can be diagnosed according to the International Classification of Sleep Disorders - Third Edition, Text Revision (ICSD-3-TR). Narcolepsy type 1 is characterized by one or both of the following: 1) Cataplexy (as defined under Essential Features) and either a) a multiple sleep latency test (MSLT), a type of sleep study showing a mean sleep latency <8 minutes and two sleep onset REM periods, or b) a sleep onset REM period within 15 minutes of sleep onset on a nocturnal polysomnogram, and / or 2) a low CSF orexin-A level of <110 pg / ml on a radioimmunoassay (RIA) or less than one-third of mean CSF orexin-A values obtained innormal subjects with the same standardized assay. Narcolepsy type 2 is diagnosed with an MSLT using the same criteria as for narcolepsy type 1. Idiopathic hypersomnia is diagnosed with an MSLT showing a mean sleep latency of <8 minutes.

[0005] These disorders are associated with altered levels of orexin protein in the cerebrospinal fluid (CSF). In particular, narcolepsy type 1 (NT1) is a sleep disorder characterized by orexin (hypocretin) deficiency with 85-95% loss of orexinergic neurons in the hypothalamus. As depicted in FIG. 20, patients with NT1 have CSF orexin-A deficiency <110 pg / ml detected by radioimmunoassay (RIA). Although narcolepsy type 2 patients (NT2) have an overall loss of 33% of orexinergic neurons, NT2 patients have normal levels of CSF orexin-A. Previous work has shown that the RIA measures <10% of the intact orexin-A peptide, suggesting that the RIA for CSF orexin-A primarily measures orexin-A-related metabolites.

[0006] Neurons producing orexins are exclusively localized to the perifornical area and the lateral and posterior hypothalamic area and project to the brainstem nuclei, amygdala, hippocampus, and cerebral cortex. Orexin-A is a 33 amino acid residue peptide with an N- terminal glutamine cyclized to pyroglutamate and two intrachain disulfide bonds. Orexin-B is a 28 amino acid residue peptide consisting of two alpha-helices connected with a short linker (Figure 10). The C-termini of both orexins are amidated. Human orexin-A and orexin-B have 46% homology (13 / 28 amino acids) with their C-terminal side being well-conserved. Orexins bind to two G protein-coupled receptors, orexin receptor 1 (OXR1) and orexin receptor 2 (OXR2). The orexin system regulates sleep-wake activity, feeding behavior, energy homeostasis, and the reward system. Based on its role in narcolepsy pathophysiology, dual orexin receptor antagonists (DORAs) were developed as a treatment of insomnia.

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

[0008] The orexin system involves a precursor protein called prepro-orexin that is proteolytically cleaved into orexin-A and orexin-B (orexins are also called hypocretins in the literature). The available radioimmunoassay (RIA) for orexin-A measures the N-terminal end of the protein and is used to diagnose narcolepsy type 1, but is unable to differentiate other hypersomnias of central origin. Assays for the C-terminal end of orexin-A, and for prepro-orexin and orexin-B are not sensitive and have not shown differences between the different hypersomnias of central origin.

[0009] Alzheimer’s disease (AD) is characterized by the deposition of Ap in the brain as insoluble plaque (j.e., amyloid-positive), tau aggregation and hyperphosphorylation, neuronal degeneration, synaptic loss, and eventual cognitive dysfunction, dementia, and death. Soluble forms of Ap and tau, proteins critical to AD pathogenesis, change in CSF with sleep-wake activity: 1) CSF Ap and tau concentrations increase during wakefulness and decrease during sleep in both mice and humans; 2) overnight sleep deprivation increases CSF Ap and tau levels by >30% via increased production / release; 3) tau hyper-phosphorylation (p-tau), an early step in tau-mediated neurodegeneration, is also affected by sleep deprivation depending on the specificsite of phosphorylation. Further, amyloid and tau pathology are associated with sleep disruptions that decrease the diurnal oscillation of CSF Ap and tau. Based on these findings, sleep is a potential marker and / or modifiable risk factor for AD.

[0010] Given the associations between CSF Ap and tau, sleep-wake activity, and AD pathology, orexin-A is hypothesized to be a marker for AD. In cognitively normal healthy older adults, CSF orexin-A was positively associated with CSF Ap42, p-tau, and total tau concentrations. In contrast, it was also previously reported that orexin-B was not detected in CSF samples. Wenz, E. et al., European Journal of Neurology, (July 2022) Vol. 29, Supp. Supplement 1, pp. 315. Abstract Number: EPR-201.

[0011] Similar positive associations between CSF orexin-A, AP42, p-tau, and total tau have also been reported in patients with AD. Further, a targeted proteomics study of markers of neurodegeneration using liquid chromatography / mass spectrometry (LC / MS) found that prepro- orexin was one of four proteins that were specific for the identification of AD from Lewy Body Dementia. These studies show that CSF orexin has the potential to aid in staging AD (e.g., asymptomatic years to symptom onset vs. mildly vs. moderately affected) and to differentiate between AD dementia, other neurodegenerative disorders that cause dementia, and controls.

[0012] Thus, a need remains for improved assays to detect orexin proteins.SUMMARY OF THE INVENTION

[0013] Among the various aspects of the present disclosure is the provision of improved assay methods to identify diseases associated with orexin protein levels. The present disclosureprovides methods to quantify concentrations in samples and applications thereof related to the diseases.

[0014] The present teachings include methods to quantify an orexin concentration in a fluid sample from a patient. In one aspect, the methods can include acquiring a cerebrospinal fluid sample. In another aspect, the method can include mixing the sample with a labeled anti-orexin antibody bead. In an embodiment, the method further comprises mixing with solution containing NP40 (Sigma), guanidine (Sigma), and protein inhibitor cocktail (Roche), and orexin internal standards (13C isotope-labeled orexin-A [Bachem], 13C15N isotope-labeled orexin-B [Sigma] and 13C15N isotope-labeled prepro-orexin [Thermo Scientific]). In another aspect, the methods can include immunoprecipitating the beads, e.g., with three washes. In further embodiments, the method comprises reduction, alkylation, tryptic digest, and / or desalting (e.g., toptip desaltation). In yet another aspect, the methods can include performing liquid chromatography / mass spectrometry (LC / MS) on the immunoprecipitated sample to quantify orexin concentrations.

[0015] In some embodiments, the orexin analyte can be selected from OXA and OXB, which terms encompass a fragment or metabolite thereof unless context indicates otherwise. In some embodiments, the orexin analyte is long OXA N-terminus, long OXA C-terminus, short OXA N-terminus, long OXB N-terminus intact, long OXB N-terminus cleaved, short OXB N-terminus intact, Short OXB N-terminus cleaved, prepro-orexin, or a combination thereof. In some embodiments, an antibody is used to bind to a target peptide sequence of an 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, the methods can be used to diagnose a sleep disorder in a patient based on orexin concentrations in the fluid sample of the patient. In some embodiments, a diagnosed patient may be treated with a suitable treatment for the sleep disorder. In some embodiments, the treatment comprises administering an orexin receptor agonist, sodium oxybate, an amphetamine, modafinil, armodafinil, or a combination thereof. In some embodiments, the treatment comprises administering an orexin type 2 receptor agonist. In some embodiments, the orexin type 2 receptor agonist comprises TAK-925 and / or TAK-861. See Fujimoto T, el al., Discovery of TAK-925 as a Poteat, Selective, and Brain-Penetrant Orexin 2 Receptor Agonist. ACS Med Chem Lett. 2022 Feb 4;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. Sci Rep. 2024 Sep 6; 14(l):20838. In some embodiments, the treatment comprises administering any agonist disclosed in W02022 / 014680A1. For example, the agonist may be one or more of (2R)-2-cyclopropyl-2- {(lR,3S,5S)-3-[(3S,4R)-l-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl]-8- azabicyclo[3.2.1]octan-8-yl}acetamide, (R)-2-((lR,3S,5S)-3-((3S,4R)-l-(5-fluoropyrimidin-2- yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-3-methylbutaneamide, (R)-2- ((lR,3S,5S)-3-((3S,4R)-l-(5-chloropyrimidin-2-yl)-3-ethoxypiperidin-4-yl)-8- azabicyclo[3.2.1]octan-8-yl)-2-cyclopropyl acetamide, 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]octan-8- yljacetamide.

[0017] In some embodiments, the methods can be used to diagnose or stage (e.g., determine the extent of disease progression) Alzheimer’s disease in a patient based on orexin concentrations inthe fluid sample of the patient. Preivously, Alzheimer’s disease staging was determined by evaluating the changes associated with changes in brain architecture, cognitive abilities, memory, communication, physical control of one’s own body. In some embodiments, the methods can include identifying Alzheimer’s disease in a patient based on a combination of orexin concentrations and one or more additional biomarkers in the fluid sample of the patient, e.g., one or more of Taul81, pTau!81, Tau217, pTau217, AP40, AP42, MTBR-tau243, or a ratio of two or more thereof. Horie K, et al., CSF MTBR-tan243 is a specific biomarker of tan tangle pathology in Alzheimer's disease. Nat Med. 2023 Aug;29(8): 1954-1963.

[0018] In some embodiments, the methods can be used to diagnose or stage Parkinson’s Disease, traumatic brain injury, Kleine-Levin syndrome, hypersomnia due to a medical disorder, hypersomnia due to a medication or substance, and hypersomnia associated with a psychiatric disorder in a patient, in a patient based on orexin concentrations in the fluid sample of the patient.

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

[0020] Other objects and features will be in part apparent and in part pointed out hereinafter.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0022] FIG. 1 depicts 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] FIG. 2 depicts how the ratio of orexin-B N-terminus intact peptide and cleaved orexin-B peptide can be used to differentiate between patients with narcolepsy type 2 and idiopathic hypersomnia from controls.

[0024] FIG. 3 depicts the concentration of long orexin-A N-terminus in cerebrospinal fluid from patients with NT1, NT2, and IH compared to control patients using a Wako anti-orexin-A antibody.

[0025] FIG. 4 depicts the concentration of middle orexin-A C-terminus in cerebrospinal fluid from patients with NT1, NT2, and IH compared to control patients using a Wako anti-orexin-A antibody.

[0026] FIG. 5 depicts the concentration of short orexin-A N-terminus in cerebrospinal fluid from patients with NT1, NT2, and IH compared to control patients using an R&D Systems anti-orexin- A antibody.

[0027] FIG. 6 depicts the concentration of long orexin-B N-terminus intact in cerebrospinal fluid from patients with NT1, NT2, and IH compared to control patients using a Wako anti-orexin-A antibody.

[0028] FIG. 7 depicts the concentration of long orexin-B N-terminus cleaved in cerebrospinal fluid from patients with NT1, NT2, and IH compared to control patients using a Wako anti- orexin-A antibody.

[0029] FIG. 8 depicts the concentration of short orexin-B N-terminus intact in cerebrospinal fluid from patients with NT1, NT2, and IH compared to control patients using an R&D Systems anti- orexin-A antibody.

[0030] FIG. 9 depicts the concentration of short orexin-B N-terminus cleaved in cerebrospinal fluid from patients with NT1, NT2, and IH compared to control patients using an R&D Systems anti-orexin-A antibody.

[0031] FIG. 10 depicts potential binding epitopes of OXA, OXB, and prepro-orexin.

[0032] FIG. 11 depicts the quantification of prepro-orexin in CSF using Sigma anti-prepro- orexin antibody targeting the N-terminal portion of prepro-orexin.

[0033] FIG. 12 depicts the discrimination of NT1 by quantification of prepro-orexin.

[0034] FIG. 13 depicts the reduced amount of short OXB N-terminus cleaved (precipitated by R&D Systems antibody) compared to long OXB N-terminus intact (precipitated by Wako antibody) in NT2 and IH patients.

[0035] FIG. 14 depicts the reduced amount of short OXB N-terminus cleaved (precipitated by R&D Systems antibody) compared to prepro-orexin in NT2 and IH patients.

[0036] FIGs. 15a-l 5i depict the effect of sleep deprivation and the sleep drug sodium oxybate on AD biomarkers. Red lines denote sleep-deprived subjects, green lines denote sodium oxybate subjects, and blue lines denote control subjects.

[0037] FIGs. 16a- 16c depict the effect of orexin on amyloid pathology in mice overexpressing orexin (APP / PS1-21) and mice with orexin knocked out (APP / PS1-21 / OR- / -). FIG. 17c depicts the effect of a dual orexin receptor antagonist, almorexant, on amyloid deposition in the brain.

[0038] FIGs. 17a- 17c depict the effect of suvorexant on CSF Ap40, Ap42, and p-tau-181 phosphorylation rate (pT181.T181), respectively.

[0039] FIG. 18a depicts the workflow of the first immunoprecipitation assay, second immunoprecipitation assay, and mass spectrometry assay used to quantify concentrations of orexin-A metabolites and prepro-orexin. FIG. 18b depicts the workflow of the first immunoprecipitation assay, second immunoprecipitation assay, and mass spectrometry assay used to quantify concentrations of orexin-B metabolites.

[0040] FIGs. 19a-h depict the quantification of long OXA N-terminus intact, long OXA C- terminus intact, short OXA N-terminus intact, Prepro-orexin, long OXB N-terminus intact, long OXB N-terminus cleaved, short OXB N-terminus intact, and short OXB N-terminus cleaved, respectively, using either the immunoprecipitation and mass spectrometry assay (IP / MS) or internal standards (IS).

[0041] FIG. 20 depicts the quantification of orexin-A in CSF samples from patients with narcolepsy type 1, narcolepsy type 2, and idiopathic hypersomnia, using radioimmunoassay.

[0042] FIG. 21 depicts the correlation of concentration of long OXA N-terminus intact, long OXA C-terminus intact, and short OXA N-terminus intact as measured by IP / MS and depicted in FIG. 1, with the concentration of OXA as measured by radioimmunoassay and depicted in FIG. 20.

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

[0044] FIGs. 23a-e depict the effect of overnight sleep deprivation on orexin peptides in CSF normaliezed to percent baseline. Four participants completed both the control normal sleep and the sleep-deprived intervention groups. All orexin peptides were normalized to percent of the hours 0-12 (07:00-19:00) baseline. The overnight period during the intervention night was defined as hours 18 to 28 (01 :00-11 :00) to account for transit time of CSF from the brain to lumbar catheter (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] FIGs. 24a-e depict the effect of overnight sleep deprivation on orexin peptides in CSF normalized to percent of the mean. Four participants completed both the control normal sleep and the sleep-deprived intervention groups. All orexin peptides were normalized to percent of the mean. The overnight period during the intervention night was defined as hours 18 to 28 (01 :00-l 1 :00) to account for transit time of CSF from the brain to lumbar catheter (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 DESCRIPTION

[0046] The present disclosure is based, at least in part, on the discovery that the methods described herein can quantify orexin concentrations in a fluid sample, such as a CSF sample, to identify a sleep disorder including, but not limited to, narcolepsy type 1, narcolepsy type 2, andidiopathic hypersomnia, as well as Alzheimer’s disease. In various aspects, the methods can be used to quantify orexin concentrations including, but not limited to, prepro-orexin (HCRT), orexin-A, orexin-B, and any combination or fragment or metabolite thereof. In some embodiments, the orexin or fragment or metabolite thereof is one of the peptides disclosed in Table 1 below:Table 1 : Orexin A fragments (Lang et al., J. Med Chem 2004).

[0047] One aspect of the present disclosure provides a mass spectrometry assay to measure prepro-orexin, orexin-A, and orexin-B peptides in a human fluid sample, e.g., a cerebrospinal fluid (CSF). The disclosed data shows that this assay may be used to differentiate samples from a subject with narcolepsy type 1, narcolepsy type 2, idiopathic hypersomnia, and a sample from a subject without the sleep disorder or an average in a healthy population (i.e., a control sample) using all forms of orexin. The methods are also able to differentiate narcolepsy type 2 and idiopathic hypersomnia from controls by measuring orexin-B peptides. Further analysis of the orexin-B N-terminus peptides shows that the ratio of the intact vs. the cleaved peptide alsodifferentiates narcolepsy type 2 and idiopathic hypersomnia from controls. By way of nonlimiting example, FIGS. 1 and 2 demonstrate the ability to differentiate between hypersomnias of central origin based on concentrations of prepro-orexin, orexin A, orexin B, and ratios thereof, wherein the concentrations are obtained using the assay method as disclosed herein. The methods can also be used to diagnose and distinguish between other disorders characterized by low orexin concentrations in a human fluid sample, such as Parkinson’s disease, traumatic brain injury, Kleine-Levin Syndrome, hypersomnia due to a medical disorder, hypersomnia due to a medication or substance, and hypersomnia associated with a psychiatric disorder in a patient.

[0048] In various embodiments, the present disclosure provides a mass spectrometry assay to measure prepro-orexin, orexin-A, and orexin-B peptides in a human fluid sample, e.g., a cerebrospinal fluid (CSF) to diagnose or stage Alzheimer’s disease (AD) in a patient. In some embodiments, an increase in an orexin peptide indicates a subject with AD. In some embodiments, a change in the level of an orexin peptide level between a first and second sample can be used to determine that a patient has progressed to a more advanced stage of AD. In some embodiments, an orexin measurement may be combined with one or more additional AD biomarkers, e.g., one or more of Taul81, pTaul81, Tau217, pTau217, Tau202, pTau202, Ap38, A04O, Ap42, MTBR-tau243 or a ratio of two or more thereof.

[0049] In some embodiments, an increase in orexin peptide indicates a subject with AD who is need of treatment, i.e., is in need of an AD therapy. Such treatment may include treatment with an anti-amyloid therapy, such as lecanemab or donanemb, or an anti-tau therapy, such as E2814. See U.S. Patent No. 10,358,485, PCT Publ. No. WO2023 / 079485 Al and Roberts, M. etal., Pre- clinical characterization of E2814, a high-affinity antibody targeting the microtubule-bindingrepeat domain of tau for passive immunotherapy in Alzheimer ’s disease. Acta Neuropath. Comm. (2020) 8:13. An orexin peptide measurement may be used to indicate treatment with an AD therapy should be initiated or discontinued, or that a subject should switched to a maintenance therapy dose of an AD therapy.

[0050] In various aspects, the disclosed assay makes use of mass spectrometry quantification of orexins isolated from samples as described below. Without being bound by theory, the disclosed mass spectrometry assay may overcome at least some of the limitations of existing orexin assay methods. Existing assays typically quantify orexins in the CSF, in particular CSF orexin-A, using an immunoassay. Other existing assays quantify orexin-B, but not prepro-orexin protein. Existing immunoassay methods are less sensitive than mass spectrometry assay methods and are typically unable to characterize the entire protein. In addition, existing immunoassays are unable to differentiate proteins labeled with13C6-leucine such as is done with MS for stable isotope labeling kinetics (SILK). Further, mass spectrometry assay methods are more sensitive than existing immunoassays, providing for more precise measurements of orexin concentrations.

[0051] In various aspects, the disclosed assay comprises an immunoprecipitation / mass spectrometry assay that isolates prepro-orexin, orexin-A, and orexin-B by contacting the CSF sample with various antibodies reactive to these proteins. The assay further includes digesting the isolated orexins followed by quantification using mass spectrometry.

[0052] In some embodiments, immunoprecipitation to isolate the prepro-orexin, 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 various portions of prepro-orexin, orexin-A, and orexin-B. In some embodiments, the solid support comprises a bead functionalized withantibodies reactive to various portions of prepro-orexin, orexin-A, and orexin-B. Any suitable beads may be functionalized with antibodies and used in the immunoprecipitation portion of the disclosed assay without limitation including, but not limited to, sepharose beads.

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

[0054] In various aspects, the immunoprecipitation portion of the disclosed assay may be accomplished in one or more stages. In some aspects, a stage may include a first immunoprecipitation step comprising contacting the sample with a first antibody to isolate a first specific orexin, followed by a second immunoprecipitation step comprising contacting the sample with a second antibody to isolate 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 a different epitope or different orexin). In other aspects, a stage may include splitting the sample into portions and digesting each portion with a different proteinase to produce differentassemblages of orexin fragments, followed by one or more immunoprecipitation steps as described above.

[0055] By way of non-limiting example, the immunoprecipitation portion of the disclosed assay may be conducted in two separate stages with two portions of the sample. Each portion of the sample may be sequentially contacted with antibodies targeting a series of targeted peptides as described above. By way of non-limiting example, the sample may be divided into two portions and analyzed to quantify various forms of OXA and OXB as illustrated in Figs. 3-9.

[0056] By way of non-limiting example, antibodies used in the immunoprecipitation step(s) may bind one or more epitopes of orexin peptides.

[0057] In some embodiments, the present disclosure provides for the use of mass spectrometry. In some embodiments, a sample from a patient undergoes one or two immunoprecipitations as described, then the resulting immunoprecipitation sample is analyzed by mass spectrometry. In some embodiments, the sample undergoes two immunoprecipitations, then the resulting immunoprecipitation sample is analyzed by mass spectrometry. In some embodiments, mass spectrometry uses quantification of target peptides to determine the type of orexin analyte in the immunoprecipitation sample. In some embodiments, mass spectrometry uses quantification of target peptides to determine the amount of orexin analyte in the immunoprecipitation sample. In some embodiments, the quantification target peptide is one listed in Table 2 below.Table 2: Target Peptides Quantified by Mass Spectrometry

[0058] In some embodiments, selecting a combination of antibodies is useful for detecting and quantifying orexin in a fluid sample.

[0059] In some embodiments, anti-orexin antibody beads are used to immunoprecipitate the orexin from the fluid sample. In some embodiments, the anti-orexin antibody bead comprises an antibody that binds to orexin-A. In some embodiments, the anti-orexin antibody bead is an R&D Systems anti-orexin-A antibody bead. In some embodiments, the anti-orexin antibody bead is a Wako anti-orexin-A antibody bead. In some embodiments, the anti-orexin antibody bead binds to orexin-B. In some embodiments, the anti-orexin antibody bead is an R&D Systems anti- orexin-B antibody bead. In some embodiments, the anti-orexin antibody bead is a Wako anti- orexin-B antibody bead. In some embodiments, the anti-orexin antibody bead binds to prepro- orexin. In some embodiments, the anti-orexin antibody bead is a Sigma anti-orexin antibodybead. In some embodiments, the anti-orexin antibody used in the immunoprecipitation step(s) is disclosed in Table 3.Table 3: Polyclonal and Monoclonal Orexin Antibodies.

[0060] In some embodiments, selecting a combination of antibodies is useful for detecting and quantifying orexin in a fluid sample.

[0061] In some embodiments, a first antibody that binds to orexin-A is mixed in a fluid sample, and then a second antibody that binds to orexin-A is mixed in the fluid sample. In some embodiments, the first antibody that binds to orexin-A is a Wako anti-orexin A antibody, and the second antibody that binds to orexin-A is an R&D Systems anti-orexin A antibody.

[0062] In some embodiments, a first antibody that binds to orexin-B is mixed in a fluid sample, and then a second antibody that binds to orexin-B is mixed in the fluid sample. In some embodiments, the first antibody that binds to orexin-B is a Wako anti-orexin A antibody, and the second antibody that binds to orexin-B is an R&D Systems anti-orexin B antibody.

[0063] In some embodiments, the first antibody that binds to prepro-orexin is mixed in a fluid sample, and then a second antibody that binds to prepro-orexin is mixed in the fluid sample. In some embodiments, the first antibody that binds to prepro-orexin is a Wako anti-orexin A antibody, and the second antibody that binds to prepro-orexin is a Sigma anti-prepro-orexin antibody.

[0064] By way of non-limiting example, the disclosed assay may include collecting a fluid sample and mixing it with a labeled anti-orexin antibody bead, Tau-MM, and OXA-IS.

[0065] In some embodiments, the beads can then be immunoprecipitated with a wash step, a reduction step, an alkylation step, a tryptic digestion step, a desalting step (e.g., toptip desaltation), or a combination thereof. In some embodiments, liquid chromatography / mass spectrometry (LC / MS) can then be performed on the resulting sample to quantify orexin concentrations.

[0066] Further, the supernatant sample after immunoprecipitation can be analyzed to quantify propeptide concentrations. A tagged anti-OXA antibody bead, anti-propeptide bead, 4X concentration OXA-IS, and Propeptide-IS can be mixed with the supernatant sample at room temperature for 2 hours, and then the sample can be washed three times, followed by reduction, alkylation, trypic digestion, and toptip desaltation. LC / MS can then be performed on the sample to quantify orexin concentrations.

[0067] In various embodiments, the methods disclosed herein may be used to diagnose sleep disorders associated with altered orexin levels, e.g., narcolepsy type 2 and idiopathic hypersomnia. Without being bound by theory, such diagnosis may have greater accuracy than provided by existing methods. Current methods to diagnose these conditions are based onsymptoms and findings from a sleep study; this method will allow for these conditions to be diagnosed based on the measurement of CSF orexin peptides.

[0068] In an exemplary embodiment, the described mass spectrometry assay can fully characterize prepro-orexin, orexin- A, and orexin-B concentrations and kinetics in human CSF with and without AD pathology and correlate them with sleep-wake activity.

[0069] In another example, the methods can characterize orexin isoforms in a human fluid sample (e.g., a CSF sample) to diagnose or determine the stage of AD. For instance, a sample from a subject suspected of having AD may be compared to a sample from a subject without Alzheimer’s disease pathology using the mass spectrometry (MS) methods disclosed herein to quantify one or more of prepro-orexin, orexin- A, orexin-B, and 13C6-leucine labeled and unlabeled versions of those orexins. Differences between the samples may be used to confirm that the subject suspected of having AD does have AD or to determine the stage of AD (e.g., by comparing to orexin levels in subjects at known AD stages).

[0070] In some embodiments, a subject identified with AD or at risk of AD according to any of the MS methods above may be treated to enhance sleep with at least one sleep-inducing medication. In some embodiments, treatment reduces orexin levels in a treated subject. In some embodiments, the treated subject is monitored for reduced orexin levels, and treatment is continued if a change is observed. In some embodiments, the subject is treated with two sleepinducing medications. In some embodiments, the sleep-inducing drug is lemborexant, suvorexant, almorexant, dardiorexant, sodium oxybate, or a combination thereof. In some embodiments, the sleep-inducing medication is a DORA, a GABA-A receptor agonist, an orexintype 2 receptor agonist, or a combination thereof. In some embodiments, the at least one sleepinducing medication is administered for one month.

[0071] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0072] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within therange. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0073] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0074] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0075] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to the practice of the present disclosure.

[0076] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0077] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.EXAMPLES

[0078] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in theexamples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1 Method to Detect Hypersomnias of Central Origin

[0079] We have developed a mass spectrometry assay to measure prepro-orexin, orexin-A, and orexin-B peptides in human cerebrospinal fluid (CSF). Briefly, orexin peptides were analyzed by a nanoAcquity ultra-performance liquid chromatography system (Waters) coupled to Orbitrap Tribid Eclipse mass spectrometer (Thermo Scientific). Mass spectrometry transitions were extracted using Skyline (MacCoss lab, University of Washington).

[0080] Using a sequential immunoprecipitation / mass spectrometry method, orexins were measured in CSF collected from patients diagnosed with NT1 (N=15), NT2 (N=15), IH (N=15), and controls (N=15). Patient characteristics are depicted below in Table 4.Table 4: NT1, NT2, and IH Patient Characteristics.

[0081] NT1 showed pan-orexin deficiency with lower levels of prepro-orexin, orexin-A metabolites, and orexin-B metabolites compared to NT2, IH, and controls (all p<0.05 after Tukey correction for multiple comparisons). Metabolites of orexin-B differentiated both NT2 and IH from NT1 and from controls (all p<0.05 after Tukey correction for multiple comparisons). Further, the ratio of the shortest form of orexin-B / longer orexin-B metabolites was relatively decreased in NT2 compared to IH and controls (all p<0.05 after Tukey correction for multiple comparisons).

[0082] This study tested if a novel mass spectrometry assay for different forms of CSF orexin differentiated among the hypersomnias of central origin. As expected, all forms of orexin were decreased in CSF collected from NT1 patients compared to all other groups. We also found that short metabolites of orexin-B are reduced in participants with NT2 and IH. Without being bound by theory, abnormal orexin transmission may also be involved in the pathophysiology of narcolepsy type 2 and IH, with differential impact on orexin peptide species.

[0083] FIG. 1 shows that this assay may be used to differentiate narcolepsy type 1 from narcolepsy type 2, idiopathic hypersomnia, and controls using all forms of orexin. We are also able to differentiate narcolepsy type 2 and idiopathic hypersomnia from controls by measuring orexin-B peptides. FIG. 2 shows that the ratio of the intact vs. the cleaved peptide of orexin-B also differentiates narcolepsy type 2 and idiopathic hypersomnia from controls.

[0084] The orexin system involves a precursor protein called prepro-orexin that is proteolytically cleaved into orexin-A and orexin-B (orexins are also called hypocretins in the literature). The radioimmunoassay (RIA) for orexin-A measures the N-terminal end of the protein and is used todiagnose narcolepsy type 1, but, as shown in FIG. 20, is unable to differentiate other hypersomnias of central origin. Furthermore, FIG. 21 shows that correlating the concentration of orexin-A metabolites as measured by the immunoprecipitation and mass spectrometry assays and depicted in FIG. 1 with the concentration of orexin-A as measured by RIA and depicted in FIG. 20 demonstrates that RIA primarily measures unauthentic orexin-A metabolites, and that RIA measures orexin-A near the N-terminus.

[0085] Assays for the C-terminal end of orexin-A, and for prepro-orexin and orexin-B are not sensitive and have not shown differences between the different hypersomnias of central origin.

[0086] The present disclosure will allow for narcolepsy type 2 and idiopathic hypersomnia to be diagnosed with greater accuracy than existing methods.Example 2 Immunoprecipitation assay with Mass Spectrometry to Quantify Long Orexin-A

[0087] An immunoprecipitation assay to detect and quantify long orexin-A is disclosed as follows and FIG. 18a.

[0088] To prepare OXA-IS (internal standard) for analysis (“OXA-lst-IP), 10 pL of 10 pg / mL OXA-IS was mixed with 990 pL of 1% HSA to obtain 100 ng / mL OXA-IS in 1% HSA. Two tubes were made, totaling 2 mL of this solution. Next, in a 15-mL Falcon tube, 2 x 980 pL of 100 ng / mL OXA-IS was mixed with 8 x 980 pL (7840 pL) of 1% HSA (total approximately 9.8 mL of 20 ng / mL OXA-IS). Then, 10 pL of 20 ng / mL OXA-IS (200 pg) was aliquoted to a 1.5 mL Eppendorf Protein-Lobind tube and stored at -80°C until use.

[0089] To prepare OXA-IS and propeptide-IS for analysis (“OXA-Propeptide 2nd-IP”), 10 pL of10 pg / mL OXA-IS was mixed with 990 pL of 1% HSA to obtain 100 ng / mL OXA-IS in 1%HSA. Three tubes were made, totaling 3 mL of this solution. Next, in a 15-mL Falcon tube, 3 x 980 pL of 100 ng / mL OXA-IS was mixed with 3 x 980 pL of 100 ng / mL propeptide-IS (total approximately 5.88 mL of 50 ng / mL OXA-IS and 50 ng / mL propeptide-IS). Then, 10 pL of mix-“50 ng / mL OXA-IS and propeptide-IS” (500 pg each of OXA-IS and propeptide-IS) was aliquoted to a 1.5 mL Eppendorf Protein-Lobind tube and stored at -80°C until use.

[0090] On day 1 (1stimmunoprecipitation (“IP”) of OXA), 220 pL of CSF was pipetted into an1.5 mL Eppendorf Protein LoBind tube containing 10 pL of “OXA-lst-IP” at 20 ng / mL. Then,12.5 pL Tau master mix (contains NP40, guanidine, and PI in PBS) was added. Then, 20 pL of 5x Diluted Anti-OXA Monoclonal Wako Beads (50% slurry) were added to this mixture. The resulting mixture was rotated for more than 16 hours at 4°C.

[0091] On day 2 (transfer post-IP supernatant, wash, reduction / alkylation, and digestion), the mixtures were centrifuged at 3901 ref for 5 minutes using a swinging bucket rotor to pellet the beads. The collected 200 pL (fixed volume) of supernatant was transferred into 1.5 mL Eppendorf Protein LoBind tubes containing 10 pL of “OXA-Propeptide 2nd-IP.” The tubes were stored at -80°C until the immunoprecipitation was initiated.

[0092] For the wash, the Eppendorf ThermoMixer C was set to 65°C and the cover closed so that the samples coule be incubated during reduction with DTT. The residual supernatant was aspirated once the post-immunoprecipiation transfer was completed. (1) 1 mL of 25 mM TEABC was added. (2) The samples were then inverted 30 times to wash the beads. (3) The samples were then centrifuged at 3901 ref for 5 minutes using a swinging bucket rotor to pellet the beads. (4) The supernatant was then aspirated. Steps (l)-(4) described above were repeated for a total of 3 washes.

[0093] For the reduction / alkylation, 20 pL of 5 mM DTT in 25 mM TEABC was added to the samples with subsequent vortexing and quick swing centrifugation. The samples were then incubated at 65°C for 30 min (+ / - min) at 1000 rpm using an Eppendorf ThermoMixer C. Then, 20 pL of 10 mM IAA in 25 mM TEABC was added to the samples with subsequent vortexing. The samples were then incubated at room temperature for 30 min (+ / - 1 min) in the dark and then subjected to quick swing centrifugation.

[0094] For trypsin digestion, 40 pL of 10 pg / mL trypsin in 25 mM TEABC was added to the samples with subsequent vortexing and quick swing centrifugation. The samples were then incubated for 16-20 hours at 37°C.

[0095] On day 3 (solid phase extraction by Oasis HLB Plated), 100 pL of 25 mM TEABC was added to the samples with subsequent vortexing. The samples were then centrifuged at 3901 ref for 5 minutes using a swinging bucket rotor to pellet the beads. The Oasis HLB pElution plate was conditioned with 200 pL MeOH, 200 pL 60% acetonitrile and 0.1% formic acid (FA), and 200 pL 0% acetonitrile and 0.1% FA (x2). Once the Oasis HLB pElution plate was conditioned, the entire supernatant was added to the plate. The plate was then washed twice with 200 pL of 0% acetonitrile and 0.1% FA. Elution was performed by adding 100 pL of 60% acetonitrile and 0.1% FA. A quick spin was performed on the collection plate using a swing centrifuge. Then, the samples were transferred from the collection plate to new 1.5 mL Eppendorf Protein LoBind tubes. The samples were then frozen on dry ice and Speed-Vac’d at 4°C to remove solvent. Optionally, these dried samples could be stored at -80°C. When ready for immediate LC / MS analysis, the samples were resuspended with 27.5 pL of 3% acetonitrile and 3% FA with vortexing. The samples were centrifuged at 21130 ref for 10 minutes at 4°C using an angledrotor. 25 L of supernatant was then transferred into a MS vial with subsequent LC / MS analysis using an Oribtrap Ellipse.Example 3 Immunoprecipitation assay with Mass Spectrometry to Quantify Short Orexin-A

[0096] An immunoprecipitation assay to detect and quantify short orexin-A is disclosed as follows and FIG. 18a.

[0097] To prepare OXA-IS and propeptide-IS for analysis (“OXA-Propeptide 2nd-IP”), 10 pL of 10 pg / mL OXA-IS was mixed with 990 pL of 1% HSA to obtain 100 ng / mL OXA-IS in 1% HSA. Three tubes (100 ng.mL OXA-IS) were made, totaling 3 mL of this solution. Then, 10 pg / mL of propeptide-IS was mixed with 990 pL of 1% HAS to obtain 100 ng / mL propeptide-IS in 1% HSA. In 15 mL Falcon tubes, 3 x 980 pL of 100 ng / mL OXA-IS was mixed with 3 x 980 pL of 100 ng / mL propeptide-IS (total approximately 5.88 mL of 50 ng / mL OXA-IS and 50 ng / mL propeptide-IS). Next, 10 pL of mix-“50 ng / mL OXA-IS and propeptide-IS” (500 pg each of OXA-IS and propeptide-IS) was aliquoted to a 1.5 mL Eppendorf Protein-Lobind tube and stored at -80°C until use.

[0098] On day 1 (2ndimmunoprecipitation of OXA, transfer post-IP supernatant, wash, reduction / alkylation, and digestion), 200 pL of supernatant and 10 pL of “OXA-Propeptide 2nd- IP” was thawed from 1.5 mL Eppendorf Protein LoBind tubes. The samples were centrifuged to consolidate the sample volume to the bottom of the tuves. 40 pL of 1 : 1 mixture of anti-OXA monoclonal R&D beads to anti-PreProOX monoclonal Sigma beads (50% slurry) was added to the samples. The resulting mixture was rotated at room temperature for 150 minutes (+ / - 30 minutes).

[0099] For transfer of the post- IP supernatant, the samples were centrifuged at 3901 ref for 5 minutes using a swinging bucket rotor to pellet the beads. The supernatant was transferred into 1.5 mL Eppendorf Protein LoBind tubes without disturbing the beads. The tubes were stored at -80°C.

[0100] For the wash, the Eppendorf ThermoMixer C was set to 65°C and the cover closed so that the samples coule be incubated during reduction with DTT. The residual supernatant was aspirated once the post-immunoprecipiation transfer was completed. (1) 1 mL of 25 mM TEABC was added. (2) The samples were then inverted 30 times to wash the beads. (3) The samples were then centrifuged at 3901 ref for 5 minutes using a swinging bucket rotor to pellet the beads. (4) The supernatant was then aspirated. Steps (l)-(4) described above were repeated for a total of 3 washes.

[0101] For the reduction / alkylation, 20 pL of 5 mM DTT in 25 mM TEABC was added to the samples with subsequent vortexing and quick swing centrifugation. The samples were then incubated at 65°C for 30 min (+ / - min) at 1000 rpm using an Eppendorf ThermoMixer C. Then, 20 pL of 10 mM IAA in 25 mM TEABC was added to the samples with subsequent vortexing. The samples were then incubated at room temperature for 30 min (+ / - 1 min) in the dark and then subjected to quick swing centrifugation.

[0102] For trypsin digestion, 40 pL of 10 pg / mL trypsin in 25 mM TEABC was added to the samples with subsequent vortexing and quick swing centrifugation. The samples were then incubated for 16-20 hours at 37°C.

[0103] On day 2 (solid phase extraction by Oasis HLB Plated), 100 pL of 25 mM TEABC was added to the samples with subsequent vortexing. The samples were then centrifuged at 3901ref for 5 minutes using a swinging bucket rotor to pellet the beads. The Oasis HLB pElution plate was conditioned with 200 pL MeOH, 200 pL 60% acetonitrile and 0.1% formic acid (FA), and 200 pL 0% acetonitrile and 0.1% FA (x2). Once the Oasis HLB pElution plate was conditioned, the entire supernatant was added to the plate. The plate was then washed twice with 200 pL of 0% acetonitrile and 0.1% FA. Elution was performed by adding 100 pL of 60% acetonitrile and 0.1% FA. A quick spin was performed on the collection plate using a swing centrifuge. Then, the samples were transferred from the collection plate to new 1.5 mb Eppendorf Protein LoBind tubes. The samples were then frozen on dry ice and Speed-Vac’d at 4°C to remove solvent. Optionally, these dried samples could be stored at -80°C. When ready for immediate LC / MS analysis, the samples were resuspended with 27.5 pL of 3% acetonitrile and 3% FA with vortexing. The samples were centrifuged at 21130 ref for 10 minutes at 4°C using an angled rotor. 25 pL of supernatant was then transferred into a MS vial with subsequent LC / MS analysis using an Oribtrap Ellipse.Example 4 Immunoprecipitation assay with Mass Spectrometry to Quantify Long Orexin-B

[0104] An immunoprecipitation assay to detect and quantify long orexin-B is disclosed as follows and FIG. 18b.

[0105] To prepare OXB-IS (internal standard) for the first immunoprecipitation analysis (“OXB-lst-IP), 10 pL of 10 pg / mL OXB-IS was mixed with 990 pL of 1% HSA to obtain 100 ng / mL OXB-IS in 1% HSA. Two tubes were made, totaling 2 mL of this solution. Next, in a 15-mL Falcon tube, 2 x 980 pL of 100 ng / mL OXB-IS was mixed with 8 x 980 pL (7840 pL) of 1% HSA (total approximately 9.8 mL of 20 ng / mL OXB-IS). Then, 10 pL of 20 ng / mL OXB-IS (200 pg) was aliquoted to a 1.5 mL Eppendorf Protein-Lobind tube and stored at -80°C until use.

[0106] To prepare OXB-IS for the second immunoprecipitation analysis (“OXB-2nd-IP”), 10 pL of 10 pg / mL OXB-IS was mixed with 990 pL of 1% HSA to obtain 100 ng / mL OXB-IS in 1% HSA. Three tubes were made, totaling 3 mL of this solution. Next, in a 15-mL Falcon tube, 3 x 980 pL of 100 ng / mL OXB-IS was mixed with 3 x 980 pL of 1% HAS (total approximately 5.88 mL of 50 ng / mL OXB-IS). Then, 10 pL of 50 ng / mL OXB-IS was aliquoted to a 1.5 mL Eppendorf Protein-Lobind tube and stored at -80°C until use.

[0107] On day 1 (1stimmunoprecipitation (“IP”) of OXB), 200 pL of CSF was pipetted into an 1.5 mL Eppendorf Protein LoBind tube containing 10 pL of “OXB-lst-IP” at 20 ng / mL. Then, 12.5 pL Tau master mix (contains NP40, guanidine, and PI in PBS) was added. Then, 20 pL of 5x Diluted Anti-OXA Monoclonal Wako Beads (50% slurry) were added to this mixture. The resulting mixture was rotated for more than 16 hours at 4°C.

[0108] On day 2 (transfer post-IP supernatant, wash, and digestion), the mixtures were centrifuged at 3901 ref for 5 minutes using a swinging bucket rotor to pellet the beads. The collected 200 pL (fixed volume) of supernatant was transferred into 1.5 mL Eppendorf Protein LoBind tubes containing 10 pL of “OXB-2nd-IP” at 50 ng / mL. The tubes were stored at -80°C until the immunoprecipitation was initiated.

[0109] For the wash, the residual supernatant was aspirated once the post- immunoprecipiation transfer was completed. (1) 1 mL of 25 mM TEABC was added. (2) The samples were then inverted 30 times to wash the beads. (3) The samples were then centrifugedat 3901 ref for 5 minutes using a swinging bucket rotor to pellet the beads. (4) The supernatant was then aspirated. Steps (l)-(4) described above were repeated for a total of 3 washes.

[0110] For trypsin digestion, 40 pL of 10 pg / mL trypsin in 25 mM TEABC was added to the samples with subsequent vortexing and quick swing centrifugation. The samples were then incubated for 16-20 hours at 37°C.

[0111] On day 3 (solid phase extraction by Oasis HLB Plated), 100 pL of 25 mM TEABC was added to the samples with subsequent vortexing. The samples were then centrifuged at 3901 ref for 5 minutes using a swinging bucket rotor to pellet the beads. The Oasis HLB pElution plate was conditioned with 200 pL MeOH, 200 pL 60% acetonitrile and 0.1% formic acid (FA), and 200 pL 0% acetonitrile and 0.1% FA (x2). Once the Oasis HLB pElution plate was conditioned, the entire supernatant was added to the plate. The plate was then washed twice with 200 pL of 0% acetonitrile and 0.1% FA. Elution was performed by adding 100 pL of 60% acetonitrile and 0.1% FA. A quick spin was performed on the collection plate using a swing centrifuge. Then, the samples were transferred from the collection plate to new 1.5 mL Eppendorf Protein LoBind tubes. The samples were then frozen on dry ice and Speed-Vac’d at 4°C to remove solvent. Optionally, these dried samples could be stored at -80°C. When ready for immediate LC / MS analysis, the samples were resuspended with 27.5 pL of 2% acetonitrile and 2% FA with vortexing. The samples were centrifuged at 21130 ref for 10 minutes at 4°C using an angled rotor. 25 pL of supernatant was then transferred into a MS vial with subsequent LC / MS analysis using an Oribtrap Ellipse.Example 5Immunoprecipitation assay with Mass Spectrometry to Quantify Short Orexin-B

[0112] An immunoprecipitation assay to detect and quantify short orexin-B is disclosed as follows and FIG. 18b.

[0113] To prepare OXB-IS and propeptide-IS for analysis (“OXB-2nd-IP”), 10 pL of 10 pg / mL OXB-IS was mixed with 990 pL of 1% HSA to obtain 100 ng / mL OXB-IS in 1% HSA. Three tubes (100 ng.mL OXA-IS) were made, totaling 3 mL of this solution. In 15 mL Falcon tubes, 3 x 980 pL of 100 ng / mL OXB-IS was mixed with 3 x 980 pL 1% HAS (total approximately 5.88 mL of 50 ng / mL OXB-IS). Next, 10 pL of 50 ng / mL OXB-IS was aliquoted to a 1.5 mL Eppendorf Protein-Lobind tube and stored at -80°C until use.

[0114] On day 1 (2ndimmunoprecipitation of OXB, transfer post-IP supernatant, wash, and digestion), 200 pL of supernatant and 10 pL of “OXB-2nd-IP” was thawed from 1.5 mL Eppendorf Protein LoBind tubes. The samples were centrifuged to consolidate the sample volume to the bottom of the tuves. 20 pL of anti-OXB monoclonal R&D beads (50% slurry) was added to the samples. The resulting mixture was rotated at room temperature for 150 minutes (+ / - 30 minutes).

[0115] For transfer of the post-IP supernatant, the samples were centrifuged at 3901 ref for 5 minutes using a swinging bucket rotor to pellet the beads. The supernatant was transferred into 1.5 mL Eppendorf Protein LoBind tubes without disturbing the beads. The tubes were stored at -80°C.

[0116] For the wash, the residual supernatant was aspirated once the post- immunoprecipiation transfer was completed. (1) 1 mL of 25 mM TEABC was added. (2) Thesamples were then inverted 30 times to wash the beads. (3) The samples were then centrifuged at 3901 ref for 5 minutes using a swinging bucket rotor to pellet the beads. (4) The supernatant was then aspirated. Steps (l)-(4) described above were repeated for a total of 3 washes.

[0117] For trypsin digestion, 40 pL of 10 pg / mL trypsin in 25 mM TEABC was added to the samples with subsequent vortexing and quick swing centrifugation. The samples were then incubated for 16-20 hours at 37°C.

[0118] On day 2 (solid phase extraction by Oasis HLB Plated), 100 pL of 25 mM TEABC was added to the samples with subsequent vortexing. The samples were then centrifuged at 3901 ref for 5 minutes using a swinging bucket rotor to pellet the beads. The Oasis HLB pElution plate was conditioned with 200 pL MeOH, 200 pL 60% acetonitrile and 0.1% formic acid (FA), and 200 pL 0% acetonitrile and 0.1% FA (x2). Once the Oasis HLB pElution plate was conditioned, the entire supernatant was added to the plate. The plate was then washed twice with 200 pL of 0% acetonitrile and 0.1% FA. Elution was performed by adding 100 pL of 60% acetonitrile and 0.1% FA. A quick spin was performed on the collection plate using a swing centrifuge. Then, the samples were transferred from the collection plate to new 1.5 mb Eppendorf Protein LoBind tubes. The samples were then frozen on dry ice and Speed-Vac’d at 4°C to remove solvent. Optionally, these dried samples could be stored at -80°C. When ready for immediate LC / MS analysis, the samples were resuspended with 27.5 pL of 2% acetonitrile and 2% FA with vortexing. The samples were centrifuged at 21130 ref for 10 minutes at 4°C using an angled rotor. 25 pL of supernatant was then transferred into a MS vial with subsequent LC / MS analysis using an Oribtrap Ellipse.Example 6Detection and Quantification of Orexin-A

[0119] Cerebrospinal fluid samples were obtained from patients with narcolepsy type 1 (n=15), narcolepsy type 2 (n=15), idiopathic hypersomnia (n=15), and healthy patients not presenting with a sleep disorder (n=15). Long OXA N-terminus and long OXA C-terminus were detected and quantified first using the immunoprecipitation assay with mass spectrometry disclosed in Example 2 and FIG. 18a. The concentration of long OXA N-terminus and long OXA C-terminus for each patient cohort is disclosed in FIG. 3 and FIG. 4, respectively.

[0120] Short OXA N-terminus was detected and quantified by use of a second immunoprecipitation assay with mass spectrometry disclosed in Example 3 and FIG. 18a. The concentration of short OXA N-terminus is disclosed in FIG. 5.Example 7 Detection and Quantification of Orexin-B

[0121] Cerebrospinal fluid samples were obtained from patients with narcolepsy type 1 (n=15), narcolepsy type 2 (n=15), idiopathic hypersomnia (n=15), and healthy patients not presenting with a sleep disorder (n=l 5). Long OXB N-terminus intact and long OXB N-terminus cleaved were detected and quantified first using the immunoprecipitation assay with mass spectrometry disclosed in Example 4 and FIG. 18b. The concentration of long OXB N-terminus intact and long OXB N-terminus cleaved for each patient cohort is disclosed in FIG. 6 and FIG. 7, respectively.

[0122] Short OXB N-terminus intact and short OXB N-terminus cleaved were detected and quantified by use of a second immunoprecipitation assay with mass spectrometry disclosedin Example 5 and FIG. 18b. The concentration of short OXB N-terminus intact and short OXB N-terminus cleaved are disclosed in FIG. 8 and FIG. 9, respectively.Example 8 Detection and Quantification of Prepro-Orexin

[0123] Prepro-orexin was detected in cerebrospinal fluid samples using a Sigma anti- prepro-orexin antibody and the assay disclosed in Example 3 and FIG. 18a. Fig. 11 shows successful quantification of the N-terminus of prepro-orexin, while the C-terminus of prepro- orexin was not able to be sufficiently quantified.

[0124] Additionally, cerebrospinal fluid samples were obtained from patients with narcolepsy type 1 (n=15), narcolepsy type 2 (n=15), idiopathic hypersomnia (n=15), and healthy patients not presenting with a sleep disorder (n=15). Prepro-orexin was detected and quantified using the immunoprecipitation assay with mass spectrometry disclosed in Example 3 and FIG. 18a. The concentration of prepro-orexin is disclosed in FIG. 12.Example 9Comparison of Short OXB N-Terminus Cleaved, Long OXB N-Terminus Intact, and Prepro-orexin

[0125] The concentration of short OXB N-terminus cleaved, long OXB n-terminus intact, and prepro-orexin obtained in Examples 7 and 8 above were compared (sequences for short OXB N-terminus cleaved, long OXB N-terminus intact, and prepro-orexin are in Table 2). The comparison of the concentration of short OXB N-terminus cleaved to long OXB N-terminusintact is disclosed in FIG. 13. The comparison of the concentration of short OXB N-terminus cleaved to prepro-orexin is disclosed in FIG. 14.Example 10 Effect of Sleep Deprivation on Alzheimer’s Disease Biomarkers

[0126] Subjects were assigned to different sleep regimens and samples were taken to measure T181, S202, and T217, and their concentrations were normalized to a baseline for each subject before each group’s intervention (average of hours 07:00-19:00). Four subjects completed the control, sleep-deprived, and drug intervention drugs. Four subjects completed two of the groups. Mean overnight concentrations of AP38, AP40, and AP42 increased approximately 30% above baseline levels in sleep-deprived participants compared to control and drug (sodium oxybate) groups (FIGS. 15a-c). Mean overnight concentrations for unphosphorylated T181, S202, and T217 increased 30-50% above baseline levels in sleep-deprived participants compared to control and drug groups (FIGS. 15d-f). Mean overnight changes in pT181 were similar to unphosphorylated T181. In contrast, phosphorylated S202 (pS202) showed no differences between the sleep-deprived and control groups or the drug and control groups. For phospho-tau T217 (pT217), there was a large increase of 60-80% in the sleep-deprived group compared to the control with no change in the drug group.

[0127] To measure the phosphorylation rate of each site, the ratio of phospho-tau was compared to the unphosphorylated form and showed that each phospho-tau species responded to sleep deprivation differently (FIGS. 15g-i). The mean overnight phosphorylation ratio of pT217 / T217 increased 15-20% above baseline with sleep deprivation while pT 181 / T181 did not differ between groups and pS202 / S202 decreased from baseline more during sleep deprivationcompared to controls. These findings demonstrate the importance of the p-tau / tau ratio and avoid the confounding effect of p-tau concentration increasing solely due to increasing tau concentration without altering the relative phosphorylation rate (for example, pT 181 is increasing because T181 is increasing vs. pT217 is increasing in excess of the change in T217). Since participants were amyloid-negative based on their CSF AP42 / 40 ratios, future investigations are critically needed to determine how CSF p-tau is affected by sleep at different stages of AD pathology (e g., amyloid-negative vs. amyloid-positive).Example 11 Effect of Orexin on Amyloid Pathology

[0128] Amyloid precursor protein (APP) transgenic mice were studied with either the orexin gene knocked out or over-expressed. Knocking out orexin led to a marked decrease in amyloid pathology in the brain while over-expression in the hippocampus did not alter amyloid deposition (FIGs. 16a-b).

[0129] Treatment with a DORA (almorexant) decreased soluble Ap concentrations while intra-cerebroventricular administration of orexin increased them. Further, prolonged treatment with almorexant for 8 weeks decreased amyloid deposition (FIG 16c). In humans, patients with narcolepsy (i.e., with orexin deficiency) have reduced CSF Ap, tau, p-tau, and amyloid deposition on amyloid PET compared to age- and sex-matched controls. Further, CSF orexin levels are higher in early AD and are associated with sleep disturbance. These findings strongly suggest that blocking orexin may modulate AD pathology in the brain.Example 12Reduction of Ap Levels and Tan Phosphorylation with Orexin Antagonist

[0130] In an ongoing study, the effect of suvorexant, a first-in-class dual orexin receptor antagonist (DORA), on CSF A levels and p-tau-181 phosphorylation was measured. 45-65 year old cognitively normal adults were randomized to receive suvorexant 10 mg, suvorexant 20 mg, or placebo. CSF was sampled every 2 hours for 36 hours via an indwelling lumbar catheter. At 9 pm, participants received a blinded tablet, and the lights were turned off. Upon waking on Day 2 (~6-7 am), participants remained awake until ~9 pm when they received their second blinded tablet (no change in the intervention group). The study ended at hour 36 (Day 3, ~8 am). After normalizing to t=0, CSF Ap40, A 42, and pTl 81 / T 181 ratio (a measure of the phosphorylation rate) decreased significantly in the suvorexant 20 mg group compared to placebo (FIGS. 17a-c).

Claims

WHAT IS CLAIMED IS:

1. A method to quantify a concentration of at least one orexin selected from orexin-A, orexin-B, prepro-orexin, and any combination thereof in a fluid sample, preferably a cerebrospinal fluid sample, the method comprising: a. acquiring the fluid sample, b. mixing 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 comprising at least one of orexin-A, orexin-B, and prepro-orexin; and c. performing liquid chromatography / mass spectrometry (LC / MS) on each of the at least one isolated orexin samples to quantify the concentration of the at least one orexin.

2. The method of claim 1, wherein the cerebrospinal fluid sample is mixed with an anti-orexin antibody bead that binds to orexin-A.

3. The method of claim 2, wherein the anti-orexin antibody bead is an R&D Systems anti-orexin-A antibody bead.

4. The method of claim 2, wherein the anti-orexin antibody bead is a Wako anti- orexin-A antibody bead.

5. The method of claim 1, wherein the cerebrospinal fluid sample is mixed with an anti-orexin antibody bead that binds to orexin-B.

6. The method of claim 5, wherein the anti-orexin antibody bead is an R&D Systems anti-orexin-B antibody bead.

7. The method of claim 5, wherein the anti-orexin antibody bead is a Wako anti- orexin-B antibody bead.

8. The method of claim 1, wherein the cerebrospinal fluid sample is mixed with an anti-orexin bead that binds to prepro-orexin.

9. The method of claim 8, wherein the anti-orexin antibody bead is a Sigma anti- orexin antibody bead.

10. The method of any one of claims 1-9, further comprising a second mixing step wherein the isolated orexin sample is mixed with a second anti-orexin antibody bead before performing LC / MS on the isolated orexin sample.

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

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

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

14. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing a long orexin-A N-terminus quantification target peptide.

15. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing a long orexin-A C-terminus quantification target peptide.

16. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing a short orexin-A N-terminus quantification target peptide.

17. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing a long orexin-B N-terminus intact quantification target peptide18. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing a long orexin-B N-terminus cleaved quantification target peptide.

19. The method of any one of claims 1-13, wherein the LC / MS step comprises analyzing a short orexin-B N-terminus intact quantification target peptide.

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

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

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

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

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

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

26. A method of diagnosing a sleep disorder selected from narcolepsy type 1, narcolepsy type 2, and idiopathic hypersomnia in a patient, the method comprising measuring the concentration of at least one orexin selected from orexin-A, orexin-B, prepro-orexin, and any combination thereof in a fluid sample from a patient, wherein the concentration of the at least one orexin is obtained using the method of 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 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 a concentration of orexin-A is used to identify narcolepsy type 1 in a patient.

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

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

33. A method of treating a sleep disorder selected from narcolepsy type 1, narcolepsy type 2, and idiopathic hypersomnia in a patient, the method comprising administering a treatment for a sleep disorder to a patient diagnosed as having the sleep disorder, wherein the patient has been diagnosed by a method comprising measuring the concentration of at least one orexin selected from orexin-A, orexin-B, prepro-orexin, and any combination thereof in a fluid sample from a patient, wherein the concentration of the at least one orexin is obtained using the method of 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 before administering 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 oxybate, an 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 of distinguishing between narcolepsy type 1, narcolepsy type 2, and idiopathic hypersomnia in a patient presenting with a sleep disorder, the method comprising measuring the concentration of at least one orexin selected from long orexin-A, orexin-B, prepro-orexin, and any combination thereof in a fluid sample from a 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 narcolepsy type 1, narcolepsy type 2, or idiopathic hypersomnia.

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

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

43. A method of 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, prepro-orexin, and any combination thereof in a fluid sample from a patient, wherein the concentration of the at least one orexin is obtained using the method of 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 the patient has AD.

44. A method of 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, prepro-orexin, and any combination thereof in a fluid sample from a patient, wherein the concentration of the at least one orexin is obtained using the method of any one of the preceding claims, and comparing the level of orexin to the level in a sample from a subject with 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, prepro-orexin, and any combination thereof in a second fluid sample from a patient, wherein the concentration of the at least one orexin is obtained using the method of any one of the preceding claims, wherein an increase in orexin level between the two samples indicates the subject has progressed to a more advanced stage of AD.

46. The method of any one of claims 43-45, further comprising measuring one or more additional AD biomarkers in a sample from the patient, e.g., one or more of Taul81, pTaul81, Tau217, pTau217, Tau202, pTau202, Ap38, A04O, A 42, MTBR-tau243 or a ratio of two or more thereof.

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

48. A method of treating AD, comprising administering an AD treatment to a subject diagnosed with having AD according to the method of any prior claim.

49. The method of claim 48, wherein the treatment comprises administering suvorexant, almorexant, dardiorexant, sodium oxybate, lemborexant, or a combination thereof.

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