WO2018078018A2

WO2018078018A2 - Detection of hypocretin-1 and -2

Info

Publication number: WO2018078018A2
Application number: PCT/EP2017/077449
Authority: WO
Inventors: Lars SCHACK KRUSE; Birgitte RAHBEK KORNUM; Poul Jørgen JENNUM; Helene Mathilde ÆGIDIUS
Original assignee: Glostrup Hospital; University Of Copenhagen
Priority date: 2016-10-27
Filing date: 2017-10-26
Publication date: 2018-05-03
Also published as: WO2018078018A3

Abstract

The present invention relates to methods for dissociating hypocretin-1 and/or hypocretin-2 from a carrier protein thereby allowing its detection in a body fluid, such as blood and blood components. Also provided are methods of diagnosing a sleep disorder or a neuropsychiatric disease or a disease of the nervous system or a traumatic brain injury or a stroke via detection of hypocretin-1 and/or hypocretin-2.

Description

Detection of Hypocretin-1 and -2 Technical field

The present invention relates to methods for dissociating hypocretin-1 and/or hypocretin-2 from a carrier protein thereby allowing its detection in a body fluid, such as blood and blood components. Also provided are methods of diagnosing a sleep disorder or a neuropsychiatric disease via detection of hypocretin-1 and/or hypocretin- 2. Background

It is well known that patients with the sleep disorder narcolepsy lack central hypocretin (hcrt), and that this deficiency is directly associated with their symptoms (Partinen et al. 2014). Consequently methods have been developed to determine the levels of hcrt in cerebrospinal fluid where reduced or undetectable concentrations are found in patients with narcolepsy when compared to age- and gender-matched normal controls.

Peripheral hcrt-1 levels have also been explored in patients with narcolepsy albeit with conflicting results (Dalai et al. 2001 ; Higuchi et al. 2002; Nishino et al. 2002). However, published individual plasma levels, including these studies, did not demonstrate to correspond to the real hcrt-1 levels.

Very large variations in peripheral hcrt-1 levels have been reported (1 -1000 pg/ml), often at levels below the RIA detection limit of the hcrt-1 (-40 pg/ml) and concerns have been raised about the specificity of the signal. Also the handling and method of collection is likely to affect hypocretin levels in blood (Dalai et al. 2001 ; Higuchi et al. 2002; Nishino et al. 2002).

Thus, results obtained in blood samples until now needs to be interpreted with caution and to date hcrt-1 is measured only in CSF to make a final diagnosis of narcolepsy type 1 .

Summary

The inventors have now surprisingly found that hypocretin peptides present in body fluids associate with one or more carrier proteins. This new finding allows for hypocretin-1 and/or hypocretin-2 levels to be measured also in non-CSF fluids with the previous step of dissociation of hypocretin-1 and/or hypocretin-2 from such carrier proteins. This in turn allows for sensitive and reliable detection of hypocretin also in peripheral body fluids such as blood and blood components, including serum and plasma and hence less invasive detection methods.

With the possibility of detecting hypocretin in peripheral body fluids a simpler test may be employed to reach more patients wherein it is advantageous to identify their hypocretin levels, such as in individuals having, suspected of having or at risk of having, a sleep disorder or a disease of the nervous system or a traumatic brain injury or a stroke or a neuropsychiatric disorder associated with changes in hypocretin levels.

It is an aspect to provide a method for dissociating hypocretin-1 and/or hypocretin-2 and/or a variant thereof from one or more carrier proteins in a sample, thereby allowing detection and/or quantification of said hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample.

Another aspect relates to a method for detection of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a sample, the method comprising one or more steps of a) providing a sample,

b) dissociating hypocretin-1 and/or hypocretin-2 and/or a variant thereof from one or more carrier proteins in said sample; and

c) detecting hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample. A further aspect relates to a method for diagnosis of a sleep disorder and/or of a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke in an individual, the method comprising:

a) providing a sample,

b) dissociating hypocretin-1 and/or hypocretin-2 and/or a variant thereof from one or more carrier proteins in said sample;

c) detecting hypocretin-1 and/or hypocretin-2 levels in said sample,

d) determining the level and/or concentration of said hypocretin-1 and/or hypocretin-2 and/or variant thereof in said sample. Description of Drawings

Figure 1 : Hcrt-1 peptide associates with albumin. A) Western blot using an antibody that detects hcrt-1 and cross-reacts with albumin. First two lanes depicts treatment of pooled CSF and hcrt-1 standard (contains BSA, thus labeled Hc-BSA) with 50 mM DTT-containing LDS reducing and denaturing sample buffer. For the last two lanes conventional Laemmli with beta-mercaptoethanol was used as sample buffer. A strong immunoreactivity is seen at the expected size of albumin, and signal from hcrt-1 only in the sample treated with LDS buffer indicating that dissociation from albumin is necessary. B) Western blot showing that the antibody recognizes albumin from different sources. BSA: bovine serum albumin. rHSA: recombinant human serum albumin produced in yeast cells and rice cells respectively. C) Human CSF was separated in two pools using a 10 kDa molecular weight cut off filter. After this the samples were fractionated using HPLC and hcrt-1 immunoreactivity in each fraction was measured by RIA. Dark gray indicates hcrt-1 still attached to carrier protein, light gray indicates hcrt-1 in solution. D) Synthetic Hcrt-1 and rHSA was incubated over night at 4C, immunoprecipitation of albumin was next performed, and finally hcrt-1 was detected by western blot (lane 3).

Figure 2: Separation of hcrt-1 from albumin allows for detection of hcrt-1 in human plasma and serum. A) Fractionation of human CSF followed by Western blotting. No hcrt-1 is detected in CSF by Western blotting, probably because of the low amounts present, but albumin is seen in fraction 26-29. rHSA: Recombinant human serum albumin used as size indicator and albumin control. B) Fractionation of hcrt-1 standard followed by Western blotting. Hcrt-1 peptide seen in fraction 20+21 . C) HPLC fractionation of 100 uL human CSF followed by detection of hcrt-1 in the individual fractions by RIA. D-E) HPLC fractionation of 100 uL human plasma and 10 uL serum followed by detection of hcrt-1 in the individual fractions by RIA. The plasma sample is from a healthy volunteer. The serum sample is a pool of human serum obtained from the Department of Clinical Biochemistry, Rigshospitalet - Glostrup. Only the fractions shown were included in the RIA.

Figure 3: Effect of pH-, heat- and o/n treatment of plasma on hcrt-1 concentration detectable with RIA. Each bar represents the mean of two measurements, duplicates of one sample, and the error bar shows the standard deviation. Samples of 250 ul of plasma were pH-, heat- and o/n (overnight) treated in various combinations. Samples were either subjected to o/n treatment at room temperature (RT), 37 or 45 degrees Celsius, or were immediately after thawing subjected to other treatment and was thus not left over night (-o/n). Some samples were heat treated for 10 min at 65°C (+65°C treatment) whilst other were not (-65°C treatment) and some were pH treated, pH 8, (+ buffer) whilst others were not (-buffer).

Figure 4: Effect of temperature and plasma volume on hcrt-1 concentration. Varying volumes of plasma was subjected to heat treatment at either 37°C or 65°C for 10 min with a buffer at pH 8.

Figure 5: Effect of pH on hcrt-1 concentration. 250 ul of plasma was subjected to heat treatment for 10 min at 65°C with a buffer with varying pH. Three biological replicates for each pH were measured. Each bar represents the mean of two measurements, duplicates of one sample, and the error bar shows the standard deviation (no error bar for one measurement).

Figure 6: Effect of heating temperature and pH on hcrt-1 concentration. Samples of 250 ul plasma were subjected to buffers of different pH (pH 8, 9 and 10) and heat treated for 10 min at either 50°C, 60°C, 65°C, 70 °C, 80 °C or 90 °C. Samples treated at 70 °C or above did not give any signal due to protein denaturation.

Figure 7: Effect of heating time on hcrt-1 concentration. Samples of 250 ul plasma were treated with a buffer at pH 9 and a temperature at 65°C for either 10, 20 or 30 minutes. Figure 8: Chymotrypsin digestion of plasma allows for detection of hcrt-1 in plasma. 250 μΙ_ plasma was subjected to either chymotrypsin digestion (+ treatment) or was non-treated (-treatment) and was subsequently analyzed using RIA to detect hcrt-1 . n=2 for each group. P-value = 0.0398 (^*). The dotted line signifies the detection limit of RIA.

Detailed description

The present disclosure relates to methods for dissociating hypocretin-1 and/or hypocretin-2 from one or more carrier proteins in a sample, thereby allowing detection of said hypocretin-1 and/or hypocretin-2 in said sample. Further, the present disclosure relates to methods for diagnosing a disease or disorder associated with hcrt-levels including a sleep disorder and/or a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke in an individual said method comprising determining the concentration of hypocretin-1 and/or hypocretin-2 in a sample taken from a body fluid of said individual.

Hypocretin

Hypocretin, also called orexin, is a neuropeptide that regulates arousal, wakefulness, and appetite. The most common form of narcolepsy, in which the sufferer briefly loses muscle tone (cataplexy), is caused by a lack of hypocretin in the brain due to destruction of the cells that produce it. There are approximately 70,000 hypocretin- producing neurons in the human brain that project from the lateral and posterior hypothalamus throughout the brain and spinal cord, including brain regions that modulate wakefulness. There are two types of hypocretin peptides: hypocretin-1 (hcrt-1 ; SEQ ID NO:1 ) and -2 (hcrt-2; SEQ ID NO:2) (aka. orexin-A and -B). They are excitatory neuropeptides produced by cleavage of a single 131 -amino acid long precursor protein (prepro- hypocretin; SEQ ID NO:3). Hypocretin-1 is 33 amino acid residues long and has two intrachain disulfide bonds (positions 34-66 of prepro-hypocretin); hypocretin-2 is a linear 28 amino acid residue peptide (positions 70-97 of prepro-hypocretin). Modified amino acid residues in hcrt occur.

The hypocretin peptides bind to the two G-protein coupled orexin receptors, HCRTR1 (OXi) and HCRTR2 (OX₂), with hypocretin-1 binding to both receptors with

approximately equal and high affinity while hypocretin-2 binds mainly to HCRTR2.

The biological functions of hypocretin are diverse, and include regulation of sleep- wakefulness, brown fat activation, lipid metabolism, appetite/food intake,

arousal/alertness and mood. A broader role in the homeostatic regulation of energy metabolism, autonomic function, hormonal balance and the regulation of body fluids, is also suggested.

Reference to hypocretin-1 (hcrt-1 , SEQ ID NO:1 ) and/or hypocretin-2 (hcrt-2, SEQ ID NO:2) and/or a variant thereof as used herein is meant to encompass native hcrt-1 peptide, native hcrt-2 peptide, any naturally occurring variant or fragment of hcrt-1 peptide, any naturally occurring variant or fragment of hcrt-2 peptide, as well as any other variants or fragments of hcrt-1 and hcrt-2.

In one embodiment a variant of hcrt-1 encompasses hcrt-1 modified at position 34 of prepro-hypocretin, corresponding to position 1 of hcrt-1 (SEQ ID NO:1 ), wherein glutamine (Glu, Q) is substituted with pyrrolidone carboxylic acid (Pyro-Glu).

In one embodiment a variant of hcrt-1 is

(Pyro-Glu)-PLPDCCRQKTCSCRLYELLHGAGNHAAGILTL (SEQ ID NO:7).

In one embodiment a variant of hcrt-1 encompasses hcrt-1 modified at position 66 of prepro-hypocretin, corresponding to position 33 of hcrt-1 (SEQ ID NO:1 ), wherein leucine (Leu, L) is substituted with leucine amide. In one embodiment a variant of hcrt-1 is

QPLPDCCRQKTCSCRLYELLHGAGNHAAGILTL-NH₂.

In one embodiment a variant of hcrt-2 encompasses hcrt-2 modified at position 97 of prepro-hypocretin, corresponding to position 28 of hcrt-2 (SEQ ID NO:2), wherein methionine (Met, M) is substituted with methionine amide.

In one embodiment a variant of hcrt-2 is

RSGPPGLQGRLQRLLQASGNHAAGILTM-NH₂. In one embodiment a variant of hcrt-1 and/or hcrt-2 encompasses hcrt-1 and/or hcrt-2 which have one or more amino acid substitutions or mutations compared to the native sequences, such as 1 , 2, 3, 4 or 5 amino acid substitutions compared to hcrt-1 (SEQ ID NO:1 ) and/or hcrt-2 (SEQ ID NO:2). In one embodiment said amino acid

substitutions comprise one or more conservative amino acid substitutions. In one embodiment said amino acid substitutions comprise one or more non-conservative amino acid substitutions.

In one embodiment said variant of hcrt-1 and/or hcrt-2 are naturally occurring variants. In one embodiment a variant of hcrt-1 and hcrt-2 is a fragment which is N-terminally truncated and/or C-terminally truncated.

In one embodiment a variant of hcrt-1 and hcrt-2 is a fragment which is N-terminally truncated, wherein said truncation comprises truncation of 1 -10 N-terminal amino acid residues, such as truncation of 1 -2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9 or 9-10 N-terminal amino acid residues.

In one embodiment a variant of hcrt-1 and hcrt-2 is a fragment which is C-terminally truncated, wherein said truncation comprises truncation of 1 -10 C-terminal amino acid residues, such as truncation of 1 -2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9 or 9-10 C-terminal amino acid residues.

In one embodiment a variant of hcrt-1 and hcrt-2 is a fragment which comprises at least 10 consecutive amino acids of SEQ ID NO:1 or SEQ ID NO:2, such as comprises at least 10-15, such as 15-20, such as 20-25 consecutive amino acids of SEQ ID NO:1 or SEQ ID NO:2.

In one embodiment a variant of hcrt-1 is a fragment derived from cleavage in the central cleavage site in the hcrt-1 protein. In one embodiment a variant of hcrt-1 is a fragment derived from cleavage in the central cleavage site in the hcrt-1 protein by chymotrypsin.

In one embodiment a variant of hcrt-1 is a fragment selected from the group consisting of:

QPLPDCCRQKTCSCRLY (SEQ ID NO:4),

QPLPDCCRQKTCSCRL (SEQ ID NO:5), and

QPLPDCCRQKTCSCR (SEQ ID NO:6). In one embodiment a variant of hcrt-1 and hcrt-2 as defined herein retains one or more biological functionalities of the native peptide. In one embodiment a variant of hcrt-1 bind to HCRTR1 (OX^ and HCRTR2 (OX₂). In one embodiment a variant of hcrt-2 bind to at least HCRTR2 (OX₂). The methods disclosed herein allows for detection of hcrt-1 , hcrt-2 and variants thereof in a multiplicity of body fluids.

Carrier protein

The present inventors have discovered that hypocretin-1 and hypocretin-2 are found in body fluids associated to carrier proteins. Therefore, when a sample of a body fluid is depleted of the protein part, it is likely that the sample is also depleted of hypocretin-1 and/or hypocretin-2 or variants thereof, unless the step of dissociating said hypocretin- 1 and/or hypocretin-2 or variants thereof from said carrier proteins is performed prior to removing proteins from the sample.

Carrier proteins are proteins that interact with other compounds, such as peptides or other proteins as well as non-peptidic compounds and ions. Carrier proteins thus help other compounds crossing barriers, for example membranes, and being transported within a tissue, an organ and/or an organism.

An example of carrier proteins is blood proteins, which help transporting various compounds from one site to another within the body of an animal, such as a human. Blood proteins, also termed plasma proteins or serum proteins, are proteins present in blood plasma. They serve many different functions, including transport of lipids, hormones, vitamins and minerals in the circulatory system and the regulation of acellular activity and functioning of the immune system. Other blood proteins act as enzymes, complement components, protease inhibitors or kinin precursors.

Serum albumin accounts for 50-60% of blood proteins, and is a major contributor to maintaining the osmotic pressure of plasma to assist in the transport of lipids and steroid hormones. Globulins make up 35-40% of blood proteins and transport ions, hormones, and lipids assisting in immune function. Fibrinogen comprises approx. 7% of blood proteins; conversion of fibrinogen to insoluble fibrin is essential for blood clotting. The remaining plasma proteins are regulatory proteins, such as enzymes, proenzymes, and hormones. In some embodiments of the present disclosure, hypocretin-1 and hypocretin-2, such as hypocretin-1 and hypocretin-2 in a sample, are associated to a carrier protein which has at least one binding domain for at least one hydrophobic compound. In some embodiments the carrier protein has at least one binding domain for hypocretin-1 and/or hypocretin-2 and/or a variant thereof.

In some embodiments the carrier protein is a blood carrier protein / blood plasma carrier protein.

Any blood carrier protein may be associated with hypocretin-1 and/or hypocretin-2 or a variant thereof. In some embodiments the carrier protein is selected from the group consisting of albumins, immunoglobulins (Igs), alpha- 1 -antitrypsin (A1 AT), fibrinogen, and haptoglobin (HG).

Albumins

In some embodiments of the present disclosure, hypocretin-1 and hypocretin-2, such as hypocretin-1 and hypocretin-2 in a sample, are associated to a carrier protein which is albumin. In some embodiments the carrier protein is albumin, such as an albumin selected from the group consisting of serum albumin, alpha-fetoprotein (alpha- fetoglobulin), vitamin D-binding protein and afamin.

The albumins are a family of globular proteins, the most common of which are the serum albumins. All the proteins of the albumin family are water-soluble, moderately soluble in concentrated salt solutions, and experience heat denaturation. Albumins are commonly found in blood plasma and differ from other blood proteins in that they are not glycosylated. A number of blood transport proteins are evolutionarily related, including serum albumin, alpha-fetoprotein (alpha-fetoglobulin), vitamin D-binding protein and afamin.

Serum albumin is the most abundant blood plasma protein and is produced in the liver and forms a large proportion of all plasma protein. The human version is human serum albumin, and it normally constitutes about 50-60% of human plasma protein. Serum albumins are important in regulating blood volume by maintaining the oncotic pressure (also known as colloid osmotic pressure) of the blood compartment. They also serve as carriers for molecules of low water solubility this way isolating their hydrophobic nature, including lipid-soluble hormones, bile salts, unconjugated bilirubin, free fatty acids (apoprotein), cations (such as Ca²⁺, Na⁺ and K⁺), ions (transferrin), and some drugs like warfarin, phenobutazone, clofibrate and phenytoin.

Albumins bind hydrophobic compounds and are abundant in blood; it was so discovered that hypocretin-1 and hypocretin-2 bind to albumins in blood.

In some embodiments the carrier protein is serum albumin.

Method for dissociation and detection of hcrt-1 and/or hcrt-2

Today only cerebrospinal fluid (CSF) is analyzed in order to measure hcrt-1 levels for clinical purposes. To obtain a CSF sample a lumbar puncture and extraction of the CSF is required. The procedure is time-consuming, painful, and may pose a danger for the patient. Moreover, as many patients in need of such a test are children, the laborious procedure may delay the execution of the test for years, leaving the patients without a diagnosis and thus without relevant clinical care and a treatment.

The present inventors have discovered that hypocretin-1 and hypocretin-2 are found peripherally in blood associated to one or more carrier proteins. They have so found a reliable method to detect and measure the presence of hypocretin-1 and hypocretin-2 in blood. The method comprises dissociating hypocretin-1 and/or hypocretin-2 from a carrier protein prior to its detection. The carrier protein can be any protein as defined herein; including albumin such as serum albumin.

Hypocretin-1 and hypocretin-2 in body fluids

In some embodiments of the present disclosure, the methods for dissociating and/or detecting hcrt-1 (SEQ ID NO:1 ) and/or hcrt-2 (SEQ ID NO:2) and/or a variant thereof is performed on a sample obtained from or obtainable from an individual.

In some embodiments of the present disclosure, the method for dissociating and/or detecting hcrt-1 and/or hcrt-2 and/or a variant thereof is performed on a body fluid sample, such as a sample selected from the group consisting of blood, whole blood, plasma, serum, urine, saliva, tears, cerebrospinal fluid and semen. In some embodiments of the present disclosure, the method for dissociating and/or detecting hcrt-1 and/or hcrt-2 and/or a variant thereof is performed on a sample selected from the group consisting of blood, whole blood, plasma, serum, urine, saliva, tears and semen.

In some embodiments of the present disclosure, the method for dissociating and/or detecting hcrt-1 and/or hcrt-2 and/or a variant thereof is performed on a sample selected from the group consisting of blood, whole blood, plasma and serum. These body fluids are easily collected and already routinely analyzed for other purposes than detection of hcrt-1 and/or hcrt-2, which makes their use advantageous over sampling and analysis of e.g. CSF.

In some embodiments of the present disclosure, the method for dissociating and/or detecting hcrt-1 and/or hcrt-2 and/or a variant thereof is performed on a sample, wherein the sample is plasma or serum.

In some embodiments of the present disclosure, the method for dissociating and/or detecting hcrt-1 and/or hcrt-2 and/or a variant thereof is performed on a sample, wherein the sample is plasma or serum which is not pre-depleted.

In some embodiments of the present disclosure, the method for dissociating and/or detecting hcrt-1 and/or hcrt-2 and/or a variant thereof is performed on a sample, wherein the sample is not cerebrospinal fluid (CSF). In some embodiments of the present disclosure, the method for dissociating and/or detecting hcrt-1 and/or hcrt-2 and/or a variant thereof is performed on a sample, wherein the sample is not pre-depleted plasma or pre-depleted serum.

The terms "pre-depleted plasma" and "pre-depleted serum" as used herein refer to a plasma sample or a serum sample from which some proteins, such as

immunoglobulins, albumin and other major protein constituents, have been removed by use of methods such as ultrafiltration, SCX (strong cation exchange) chromatography or immuno-affinity based resin. By such methods any peptides associated with said proteins are also removed. Dissociation of hcrt-1 and/or hcrt-2 from a carrier protein

The terms "dissociating" or "separating" as used herein in one embodiment refers to the act of breaking a covalent or any non-covalent bond between two molecules so that they are no longer bound to (or associated with) each other. The two molecules exist in the same sample prior to the dissociation and they may exist in the same sample also after the dissociation, though not bound to one another.

In a first aspect the present disclosure relates to a method for dissociating hypocretin-1 (hcrt-1 , SEQ ID NO:1 ) and/or hypocretin-2 (hcrt-2, SEQ ID NO:2), and/or a variant thereof, from one or more carrier proteins in a sample, thereby allowing detection and/or quantification of said hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample.

In one embodiment the present disclosure relates to a method for dissociating hypocretin-1 (hcrt-1 , SEQ ID NO:1 ) and/or hypocretin-2 (hcrt-2, SEQ ID NO:2), and/or a variant thereof, from one or more carrier proteins in a sample, thereby allowing detection and/or quantification of said hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample,

wherein said carrier protein is albumin, and wherein said sample is a body fluid.

wherein said carrier protein is serum albumin, and wherein said sample is a body fluid selected from the group consisting of blood, whole blood, plasma, serum, urine, saliva, tears and semen. In one embodiment said sample, such as said body fluid sample, such as said body fluid sample selected from the group consisting of blood, whole blood, plasma, serum, urine, saliva, tears and semen, is obtained from or obtainable from an individual. In one embodiment said body fluid is selected from the group consisting of blood, whole blood, plasma and serum. In one embodiment said body fluid is selected from the group consisting plasma and serum. In one embodiment the method is for dissociating hypocretin-1 (hcrt-1 , SEQ ID NO:1 ) and/or a variant thereof, from one or more carrier proteins in a sample, thereby allowing detection and/or quantification of said hypocretin-1 and/or a variant thereof in said sample. In some embodiments the method for dissociating hypocretin-1 and/or hypocretin-2 and/or a variant thereof from one or more carrier proteins in a sample, thereby allowing detection and/or quantification of said hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample is an in vitro method. Any method that provides dissociation of hypocretin-1 or hypocretin-2 from another protein may be used for the purpose of dissociating hypocretin-1 and/or hypocretin-2 from a carrier protein.

In some embodiments of the present disclosure, the dissociation step comprises breaking at least one covalent or non-covalent bond between hypocretin-1 or hypocretin-2 and the carrier protein in said sample. Proteins and peptides can interact with and associate to other proteins and/or peptides via a variety of bonds. The interactions or bonds can be of covalent or non-covalent nature. Examples of non- covalent interactions that can occur between hypocretin-1 , hypocretin-2 or a variant thereof and another protein or peptide, such as a carrier protein are dipole-dipole interactions (between permanent and/or induced dipoles), hydrogen bonds, ionic bonds, hydrophobic interactions. Examples of covalent bonds are disulfide bonds and peptide bonds. In some embodiments the dissociation step comprises one or more steps of heating the sample.

In some embodiments the dissociation step comprises one or more steps of increasing (or adjusting) the pH of the sample. In some embodiments the dissociation step comprises one or more steps of heating the sample and increasing (or adjusting) the pH of the sample.

In some embodiments the dissociation step comprises one or more steps of heating the sample to a temperature of 50 to 75°C; such as 50 to 55°C, such as 50 to 60°C, such as 60 to 61 °C, such as 61 to 62°C, such as 62 to 63°C, such as 63 to 64°C, such as 64 to 65°C, such as 65 to 66°C, such as 66 to 67°C, such as 67 to 68°C, such as 68 to 69°C, such as 69 to 70°C, such as 70 to 75°C. In some embodiments the

dissociation step comprises one or more steps of heating the sample to a temperature of 60 to 70°C.

In some embodiments the dissociation step comprises one or more steps of heating the sample to a temperature of above 50°C, such as above 55°C, such as 60°C or above 60°C.

In some embodiments the dissociation step comprises one or more steps of heating the sample to a temperature of below 75°C, such as 70°C or below 70°C.

In some embodiments the dissociation step comprises one or more steps of heating the sample to a temperature of approx. 65°C.

In some embodiments the dissociation step comprises one or more steps of heating the sample, wherein said sample is not heated over-night / is heated less than overnight.

In some embodiments the dissociation step comprises one or more steps of heating the sample for a period of 1 minute to 5 hours; such as 1 minute to 5 minutes, such as 5 minutes to 10 minutes, such as 15 minutes to 20 minutes, such as 20 minutes to 25 minutes, such as 25 minutes to 30 minutes, such as 30 minutes to 40 minutes, such as 40 minutes to 50 minutes, such as 50 minutes to 60 minutes, such as 1 hour to 1 ½ hours, such as 1 ½ to 2 hours, such as 2 to 2½ hours, such as 2½ to 3 hours, such as 3 to 3½ hours, such as 3½ to 4 hours, such as 4 to 4½ hours, such as 4½ to 5 hours. In some embodiments the dissociation step comprises one or more steps of heating the sample for a period of 5 minutes to 60 minutes, such as 5 minutes to 45 minutes, such as 10 to 40 minutes, such as 10 to 30 minutes. In some embodiments the dissociation step comprises one or more steps of heating the sample for a period of approx. 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 45 minutes, 50 minutes or 60 minutes. In some embodiments the dissociation step comprises one or more steps of heating the sample for a period of approx. 30 minutes.

In some embodiments the dissociation step comprises one or more steps of increasing or adjusting the pH of the sample.

In some embodiments the dissociation step comprises one or more steps of increasing or adjusting the pH of the sample to a pH of above 7.

In some embodiments the dissociation step comprises one or more steps of increasing or adjusting the pH of the sample to a pH of 7 to 1 1 , such as a pH of 8 to 10; such as a pH of 7 to 8, a pH of 8 to 9, a pH of 9 to 10 or a pH of 10 to 1 1 .

In some embodiments the dissociation step comprises one or more steps of increasing or adjusting the pH of the sample to a pH of 8, a pH of 9 or a pH of 10. In some embodiments the dissociation step comprises one or more steps of increasing or adjusting the pH of the sample to a pH of approx. 9.

In some embodiments the dissociation step comprises one or more steps of

i) heating the sample to a temperature of 50 to 75°C for a period of 5 minutes to 5 hours; and

ii) adjusting the pH of the sample to a pH of 7 to 1 1 .

In some embodiments the dissociation step comprises one or more steps of

i) heating the sample to a temperature of 60 to 70°C, or approx. 65°C, for a period of 5 minutes to 60 minutes; and

ii) adjusting the pH of the sample to a pH of 8 to 10. In some embodiments the dissociation step does not comprise the use of a solvent. In some embodiments the dissociation step does not comprise the use of a filter. In some embodiments the dissociation step does not comprise the use of a solvent and a filter. In one embodiment the present disclosure relates to a method for dissociating hypocretin-1 (hcrt-1 , SEQ ID NO:1 ) and/or hypocretin-2 (hcrt-2, SEQ ID NO:2), and/or a variant thereof, from one or more carrier proteins in a sample, thereby allowing detection and/or quantification of said hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample,

wherein said carrier protein is an albumin, and wherein said sample is a body fluid, and wherein the dissociation step comprises one or more steps of

ii) adjusting the pH of the sample to a pH of 7 to 1 1 .

wherein said carrier protein is serum albumin, and wherein said sample is a body fluid selected from the group consisting of blood, whole blood, plasma, serum, urine, saliva, tears and semen, and

wherein the dissociation step comprises one or more steps of

ii) adjusting the pH of the sample to a pH of 8 to 10.

In some embodiments, the dissociation step is a chemical or an enzymatic reaction.

In some embodiments, the dissociation step is a chromatography separation.

In some embodiments, the dissociation step is chromatography separation on a high- performance liquid chromatography (HPLC) column. The chromatography column is preferably resistant to raw serum/plasma degeneration as well as able to resolve the sample enough for hypocretin-1 and hypocretin-2 detection.

For example, a C18 reverse column having the same features of Waters: XSelect Peptide CSH C18 Column, 13θΑ, 3.5 μηι, 4.6 mm X 150 mm, 1/pkg or μBondapak C18 Column, 125A, 10 μηι, 3.9 mm X 300 mm, 1 /pkg can be used.

A suitable protocol for chromatographic separation using any one of the columns mentioned above is a protocol comprising plasma or serum fractionation using a gradient, such as a hydrophobic to hydrophilic gradient. For example an acetonitrile gradient, such as a 10-60% acetonitrile gradient over a certain period of time, for example at least 20 minutes, or at least 30 minutes, or at least 40 minutes or at least 50 minutes. In some embodiments, the dissociation step comprises use of a protease. Proteases are enzymes that hydrolyze peptide bonds between amino acids. Many different proteases exist, each having different preferences for the target amino acid residue. The function of proteases is that of degrading peptides and proteins. In some embodiments, the dissociation step comprises use of a protease and said protease preferably digests said carrier protein and/or does not digest said hypocretin- 1 and/or hypocretin-2 and/or a variant thereof.

In some embodiments, said protease is a chymotrypsin-like serine protease.

In some embodiments, said protease is chymotrypsin (EC 3.4.21 .1 ).

Chymotrypsin preferentially cleaves peptide amide bonds where the carboxyl side of the amide bond (the P1 position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). Chymotrypsin also hydrolyzes other amide bonds in peptides at slower rates, particularly those containing leucine and methionine at the P1 position.

In some embodiments, the dissociation step does not comprise use of a protease. In one embodiment the method for dissociating hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) and/or a variant thereof, from one or more carrier proteins in a sample results in the partial or complete dissociation of said hypocretin from said carrier proteins. The dissociation should be sufficient to allow for the reliable, sensitive and/or specific detection of said hypocretin in a sample, such as a peripheral body fluid sample, such as a blood sample as defined herein.

Detection of hcrt- 1 and/or hcrt-2

In a further aspect the present disclosure relates to a method for detection of hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) and/or a variant thereof in a sample, the method comprising one or more steps of

a) providing a sample,

c) detecting hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said

sample.

In one embodiment the method is for detection of hypocretin-1 (SEQ ID NO:1 ) and/or a variant thereof in a sample, the method comprising one or more steps of

a) providing a sample,

b) dissociating hypocretin-1 and/or a variant thereof from one or more carrier proteins in said sample; and

c) detecting hypocretin-1 and/or a variant thereof in said sample. The dissociation step in some embodiments is performed as outlined herein above.

These include one or more steps of heating the sample and/or increasing the pH of the sample; a chemical or an enzymatic reaction; a chromatography separation and use of a protease. In one embodiment the sample is a body fluid, such as a body fluid selected from the group consisting of blood, whole blood, plasma, serum, urine, saliva, tears and semen.

In one embodiment said sample, such as said body fluid sample, such as said body fluid sample selected from the group consisting of blood, whole blood, plasma, serum, urine, saliva, tears and semen, is obtained from or obtainable from an individual. Once the sample has undergone a dissociation step, said hypocretin-1 and/or hypocretin-2 as well as any variant thereof can be detected. Any method suitable for detection of proteins or peptides can be used to detect said hypocretin-1 and/or hypocretin-2.

In some embodiments, the method for detection of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a sample is an in vitro method. In some embodiments, the detecting step comprises an assay selected from the group consisting of an immunoassay, a protein assay and a mass spectrometry assay.

In some embodiments, the detecting step comprises an immunoassay selected from the group consisting of RIA (radioimmunoassay), MIA (magnetic immunoassay), EIA (enzyme immunoassay), FIA (fluorescent immunoassay) and ELISA (enzyme-linked immunosorbent assay). Protocols describing how to perform these methods are readily available in the literature. Antibodies specific for hcrt-1 and/or hcrt-2 as well as for their variants are commercially available. Non-limiting examples of methods based on immunodetection are immunoassays selected from the group consisting of: affinity chromatography, Immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), ELISA-derived assays such as immune-PCR in which the detecting antibody is labeled with a DNA-label, immunofluorescent assay, Western blotting, and the like. These methods are well known by a person skilled in the art of detecting and analyzing proteins.

In some embodiments, the detecting step comprises a protein assay selected from the group consisting of centrifugation, electrophoresis, chromatography and western blotting.

The level or concentration of hcrt-1 and/or hcrt-2 or variants thereof may be determined through direct specific binding or by indirect competitive binding to a ligand. In a particular embodiment of the disclosure, the determination of the concentration of hcrt- 1 and/or hcrt-2 or variants thereof is performed via a RIA wherein hcrt-1 and/or hcrt-2 or variants thereof from the test sample and an iodine 125-labelled hypocretin molecule compete for binding to an anti-hcrt antibody.

In a particular embodiment, a method according to the invention comprises the determination of the concentration of hcrt-1 and/or hcrt-2 or variants thereof by performing a direct binding assay such as Surface Plasmon Resonance (SPR).

In some embodiments, the detecting step comprises a mass spectrometry assay, such as a quantitative mass spectrometry assay.

Methods for the specific detection of a protein based on mass spectrometry include, but are not limited to, Selected Reaction Monitoring (SRM) and Multiple Reaction Monitoring (MRM). Methods based on flow cytometry include, but are not limited to, a multiplex assay such as Luminex®XMAP, combining flow cytometry with microspheres and lasers. In some embodiments, the detecting step comprises a mass spectrometry assay selected from SRM and MRM.

Methods for diagnosis

The methods of dissociating hcrt-1 (SEQ ID NO:1 ) and/or hcrt-2 (SEQ ID NO:2), and/or a variant thereof, from a carrier protein and so determining its concentration in a sample of body fluid as disclosed herein can be used for diagnosis of a disease or disorder associated with abnormal levels of said hcrt-1 and/or hcrt-2.

A disease or disorder associated with abnormal levels of hcrt-1 and/or hcrt-2 in one embodiment is selected from the group consisting of a sleep disorder and/or of a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke.

The term "abnormal" as used herein refers to levels or concentrations of hcrt-1 and/or hcrt-2 that differ from the levels or concentrations found in healthy human individuals, such as human individuals who do not present with a sleep disorder and/or a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke. Thus, an aspect of the present disclosure relates to a method for diagnosis of a sleep disorder and/or of a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke in an individual, the method comprising: a) providing a sample,

b) dissociating hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) and/or a variant thereof from one or more carrier proteins in said sample;

c) detecting hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample, d) determining the level and/or concentration of said hypocretin-1 and/or hypocretin-2 and/or variant thereof in said sample.

Also disclosed is a method for diagnosis of a sleep disorder in an individual, the method comprising:

a) providing a sample,

c) detecting hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample, d) determining the level and/or concentration of said hypocretin-1 and/or hypocretin-2 and/or variant thereof in said sample. Also disclosed is a method for diagnosis of a neuropsychiatric disease in an individual, the method comprising:

a) providing a sample,

In one embodiment said sample is obtained from said individual or obtainable from said individual. In one embodiment said sample is a body fluid sample obtained from or obtainable from said individual.

The dissociation step in some embodiments is performed as outlined herein above. These include the one or more steps of heating the sample and/or increasing the pH of the sample; a chemical or an enzymatic reaction; a chromatography separation and/or use of a protease.

The sample in some embodiments is as outlined herein above and includes body fluids, such as a body fluid selected from the group consisting of blood, whole blood, plasma, serum, urine, saliva, tears and semen.

The detection step in some embodiments is performed as outlined herein above. These include one or more assays selected from the group consisting of an immunoassay, a protein assay and a mass spectrometry assay.

In one embodiment said variant of hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) is a naturally occurring variant or fragment of hypocretin-1 and/or a naturally occurring variant or fragment of hypocretin-2.

In one embodiment said variant of hypocretin-1 and/or hypocretin-2 is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 as well as C-terminally amidated versions of any one of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

In one embodiment said sleep disorder and/or neuropsychiatric disease is associated with abnormal levels of hcrt-1 and/or hcrt-2. In one embodiment said sleep disorder and/or neuropsychiatric disease and/or disease of the nervous system and/or traumatic brain injury and/or stroke is associated with abnormal levels of at least hcrt-1 .

Reference to a method for diagnosis of a sleep disorder and/or of a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke, or to a method for diagnosis of narcolepsy, as used herein, is meant also to encompass methods of identifying individuals who has, who are at risk of having and/or who are at risk of developing said disorders.

In one embodiment the method for diagnosis of a sleep disorder and/or of a

neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke comprises:

a) providing a sample, b) dissociating hypocretin-1 (SEQ ID NO:1 ) and/or a variant thereof from one or more carrier proteins in said sample;

c) detecting said hypocretin-1 and/or a variant thereof in said sample,

d) determining the level and/or concentration of said hypocretin-1 and/or variant thereof in said sample.

Thus, an aspect of the present disclosure relates to a method for diagnosis of a sleep disorder and/or of a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke in an individual, the method comprising: a) providing a sample,

c) detecting hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample, d) determining the level and/or concentration of said hypocretin-1 and/or hypocretin-2 and/or variant thereof in said sample;

wherein said sample of step a) is a sample obtained from or obtainable from an individual; such as a body fluid sample; such as a sample selected from the group consisting of blood, whole blood, plasma, serum, urine, saliva, tears, cerebrospinal fluid and semen; such as plasma or serum;

wherein said carrier protein of step b) is albumin such as serum albumin; and wherein said dissociation step comprises breaking at least one covalent or non-covalent bond between hypocretin-1 or hypocretin-2 and the carrier protein in said sample;

and wherein said detecting step c) optionally comprises an assay selected from the group consisting of an immunoassay, a protein assay, a binding assay and a mass spectrometry assay.

In some embodiments the dissociation step b) comprises one or more steps of heating the sample and/or increasing/adjusting the pH of the sample; such as heating the sample to a temperature of 50 to 75°C, such as 60 to 70°C for a period of from 5 minutes to 60 minutes and/or adjusting the pH of the sample to a pH of 7 to 1 1 such as pH 8 to 10, as is outlined herein in detail. In some embodiments the dissociation step b) comprises a chemical reaction, an enzymatic reaction, a chromatography separation or use of a protease, as is outlined herein in detail.

In one embodiment, the sleep disorder is a hypersomnia. In one embodiment the hypersomnia is central hypersomnia, including idiopathic hypersomnia; recurrent hypersomnia such as Klein-Levin syndrome; or narcolepsy.

In some embodiments the sleep disorder is narcolepsy. In some embodiments the sleep disorder is narcolepsy including narcolepsy with cataplexy (narcolepsy type 1 ; narcolepsy-cataplexy syndrome; NC; NRCLP1 ; narcolepsy with low hypocretin) and narcolepsy without cataplexy (narcolepsy type 2; NwC; narcolepsy with normal hypocretin).

In one embodiment the sleep disorder is narcolepsy with cataplexy (narcolepsy type 1 ; narcolepsy-cataplexy syndrome; NRCLP1 ; NC; narcolepsy with low hypocretin).

In some embodiments the sleep disorder is insomnia, including but not limited to transient insomnia, acute insomnia and chronic insomnia. In some embodiments the neuropsychiatric disease is selected from the group consisting of schizophrenia, depression, seasonal affective disorder, attention deficit hyperactivity disorder (ADHD), bipolar disorder and hallucinations.

In some embodiments the disease of the nervous system is selected from the group consisting of demyelinating diseases of the central nervous system, inflammatory polyneuropathies and neurodegenerative disorders.

In some embodiments, the demyelinating disease of the central nervous system is multiple sclerosis.

In some embodiments, the inflammatory polyneuropathy is acute inflammatory demyelination polyneuropathy or chronic inflammatory demyelinating polyneuropathy.

In some embodiments, the neurodegenerative disorder is Parkinson's disease, Parkinsonism or dementia. The so determined level and/or concentration of hypocretin-1 and/or hypocretin-2 in some embodiments is compared to a cut-off value, which is a reference concentration range corresponding to what is normally measured in individuals who are not affected by any sleep disorder or neuropsychiatric disease or disease of the nervous system or traumatic brain injury or stroke.

In some embodiments the method comprises the further step of e) comparing said level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or the variant thereof with the level in healthy human individuals, such as human individuals not presenting with any sleep disorder or neuropsychiatric disease or disease of the nervous system or traumatic brain injury or stroke.

Thus, in some embodiments the method for diagnosis of a sleep disorder and/or of a neuropsychiatric disease and/or of a disease of the nervous system and/or of a traumatic brain injury and/or of a stroke comprises the further step of e) comparing said level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or the variant thereof with a cut-off value,

wherein said cut-off value is determined from the concentration range of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in healthy human individuals, such as human individuals who do not present with a sleep disorder and/or a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke,

wherein a level and/or concentration that is lower or greater than the cut-off value indicates the presence or absence of said sleep disorder and/or neuropsychiatric disease and/or disease of the nervous system and/or traumatic brain injury and/or stroke.

By "lower" it is intended that the measured value in the sample is smaller than the average value; smaller than the 25% quantile value; or smaller than the lowest value of the concentration range of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in healthy human individuals, such as human individuals who do not present with a sleep disorder and/or a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke. By "greater" it is intended that the measured value is larger than the average value; larger than the 75% quantile value; or larger than the highest value of the concentration range of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in healthy human individuals, such as human individuals who do not present with a sleep disorder and/or a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke.

In one embodiment a healthy human individual as used herein are age- and gender- matched normal controls. In one embodiment a healthy human individual are human individuals who do not present with a sleep disorder and/or a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke.

In one embodiment a healthy human individual are human individuals who do not present with narcolepsy, such as narcolepsy type 1 .

Thus, in some embodiments the method for diagnosis of a sleep disorder and/or of a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke comprises the further step of classifying the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof as:

a) Low: when the level and/or concentration is lower than the cut-off value

b) Normal: when the level and/or concentration is equal (or comparable) to the cut-off value; and

c) High: when the level and/or concentration is higher than the cut-off value. In some embodiments the cut-off value is a range or an interval and therefore the method for diagnosis of a sleep disorder and/or of a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke comprises the further step of classifying the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof as:

a) Low: when the level and/or concentration is lower than the lower limit of the cut-off value

b) Normal: when the level and/or concentration is comprised (or within) the cut-off value; and

c) High: when the level and/or concentration is higher than the upper limit cut-off value. In some embodiments said cut-off value is a concentration range (or a concentration interval), such as the concentration range of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in healthy human individuals, such as human individuals who do not present with a sleep disorder and/or a neuropsychiatric disease and/or disease of the nervous system and/or traumatic brain injury and/or stroke.

Several sleep disorders and neuropsychiatric diseases are associated with abnormal levels of hypocretin-1 and/or hypocretin-2 and can therefore be diagnosed using the method disclosed herein.

Moreover, several diseases of the nervous system, for example demyelinating diseases of the central nervous system such as multiple sclerosis, inflammatory polyneuropathies such as acute inflammatory demyelination polyneuropathy and chronic inflammatory demyelinating polyneuropathy, and neurodegenerative disorders such as Parkinson's disease, Parkinsonism and dementia are also associated with abnormal levels of hypocretin-1 and/or hypocretin-2 and can therefore be diagnosed using the method disclosed herein.

Traumatic brain injuries and stroke may also be associated with changes in the levels of hypocretin-1 and/or hypocretin-2, which become abnormal in an individual affected by a traumatic brain injury or stroke, and can therefore be diagnosed using the method disclosed herein.

In one embodiment of the disclosure, the method is used for diagnosis of narcolepsy, such as narcolepsy with cataplexy (narcolepsy type 1 ; narcolepsy-cataplexy syndrome; NC; NRCLP1 ; narcolepsy with low hypocretin).

A further aspect of the present disclosure relates to a method for diagnosis of narcolepsy, the method comprising:

a) providing a sample,

c) detecting said hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample, and d) determining the level and/or concentration of said hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample.

In some embodiments the method comprises the further step of e) comparing said level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or the variant thereof with the level in healthy human individuals, such as human individuals not presenting with narcolepsy; or e) comparing said level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or the variant thereof with a cut-off value. In one embodiment of the disclosure, the method is used for diagnosis of narcolepsy, and

a) an individual has, or is likely to have, narcolepsy if the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof is lower than the level in healthy human individuals, such as human individuals who do not present with narcolepsy, and

b) an individual does not have, or is not likely to have, narcolepsy if the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or variant thereof is similar to the level in healthy human individuals, such as human individuals who do not present with narcolepsy.

In one embodiment the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a subject having, or likely to have, narcolepsy is lower than the level in healthy human individuals. In one embodiment the level and/or

concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof from a subject having narcolepsy is between 0 pg/ml and 200 pg/ml, such as between 0 pg/ml and 180 pg/ml, such as between 0 pg/ml and 160 pg/ml, such as between 0 pg/ml and 140 pg/ml, such as between 0 pg/ml and 120 pg/ml, such as between 0 pg/ml and 100 pg/ml, such as between 0 pg/ml and 80 pg/ml, such as between 0 pg/ml and 60 pg/ml, such as 0 pg/ml and 40 pg/ml, such as between 0 pg/ml and 20 pg/ml.

In one embodiment the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a subject having, or likely to have, narcolepsy is between 0 pg/ml and 10 pg/ml, such as between 10 pg/ml and 20 pg/ml, such as between 20 pg/ml and 40 pg/ml, such as between 40 pg/ml and 60 pg/ml, such as between 60 pg/ml and 80 pg/ml, such as between 80 pg/ml and 100 pg/ml, such as between 100 pg/ml and 125 pg/ml, such as between 125 pg/ml and 150 pg/ml, such as between 150 pg/ml and 200 pg/ml.

In one embodiment of the disclosure, the method is used for diagnosis of narcolepsy and:

a) an individual has, or is likely to have, narcolepsy if the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof is about 0 pg/ml, such as 0 to 50 pg/ml, such as 0 to 100 pg/ml, such as 0 to 1 10 pg/ml;

b) an individual does not have, or is not likely to have, narcolepsy if the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or variant thereof is 200 pg/ml or more, such as 220 pg/ml or more, such as 400 pg/ml or more.

a) an individual has, or is likely to have, narcolepsy if the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof is between 50% and 100% lower than the level and/or concentration in healthy human individuals,

b) an individual does not have, or is not likely to have, narcolepsy if the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or variant thereof is equal to or not more than 50% lower than the level and/or concentration in healthy human individuals.

In one embodiment the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a subject having narcolepsy is lower than the level in healthy human individuals when said level is at least 50% lower than the level in healthy human individuals, such as between 55% and 100% lower, such as between 60% and 100% lower, such as between 65% and 100% lower, such as between 70% and 100% lower, such as between 75% and 100% lower, such as between 80% and 100% lower, such as between 85% and 100% lower, such as between 90% and 100% lower, such as between 95% and 100% lower than the level in healthy human individuals.

In one embodiment the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a subject having narcolepsy is lower than the level in healthy human individuals when said level is 55% to 60% lower, such as 60% to 65% lower, such as 65% to 70% lower, such as 70% and 75% lower, such as 75% to 80% lower, such as 80% to 85% lower, such as 85% to 90% lower, such as 90% to 95% lower, such as 95% to 100% lower than the level in healthy human individuals.

In some embodiments, the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a subject having, or likely to have, narcolepsy is about 0 pg/ml.

In some embodiments, the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a subject having, or likely to have, narcolepsy is about 50 pg/ml.

In some embodiments, the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a subject having, or likely to have, narcolepsy is about 100 pg/ml.

In some embodiments, the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a subject having, or likely to have, narcolepsy is about 150 pg/ml. In some embodiments, the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a healthy human individual is about 300 pg/ml.

In some embodiments, the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a healthy human individual is about 400 pg/ml.

In some embodiments, the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in a healthy human individual is about 500 pg/ml.

Several sleep disorders, neuropsychiatric diseases, diseases of the nervous system, traumatic brain injuries as well as stroke that can be diagnosed with the method disclosed herein can be treated by administering a drug to a subject in need thereof. The terms "treatment" and "treating" as used herein refer to the management and care of a patient for the purpose of combating a condition, disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the active compound for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, disease or disorder; curing or eliminating the condition, disease or disorder; and/or preventing the condition, disease or disorder. The subject to be treated is preferably a mammalian, in particular a human being. The subjects to be treated can be of various ages.

Thus, in some embodiments of the present disclosure, the method for diagnosis of a sleep disorder and/or of a neuropsychiatric disease and/or disease of the nervous system and/or traumatic brain injury and/or stroke further comprises the step of treating said sleep disorder and/or neuropsychiatric disease and/or disease of the nervous system and/or traumatic brain injury and/or stroke.

In one embodiment the method for diagnosis of narcolepsy further comprises the step of treating said narcolepsy.

In some embodiments, the treatment comprises administration of one or more CNS stimulants, non-stimulants such as a norepinephrine reuptake inhibitor (NRI) or antidepressants including SSRI and tricyclic antidepressants. In some embodiments the treatment comprises administration of one or more medicaments selected from the group consisting of modafinil, amphetamine, dextroamphetamine, armodafinil, gamma-hydroxybutyrate (GHB), methylphenidate, sodium oxybate, methylphenidate, desvenlafaxine, ephedrine, atomoxetine, venlafaxine, fluoxetine, clomipramine, imipramine, protriptyline, pitolisant and JZP-1 10.

Sleep disorders and neuropsychiatric diseases

The present disclosure in one embodiment relates to methods for diagnosis of a sleep disorder and/or a neuropsychiatric disease in an individual comprising determining the level and/or concentration of hypocretin-1 and/or hypocretin-2 and/or the variant thereof in a sample taken from a body fluid of said individual.

Sleep disorders

The methods disclosed herein can be used to diagnose a sleep disorder. Various types of sleep disorders exist, for example hypersomnia, such as central hypersomnia, including idiopathic hypersomnia, recurrent hypersomnia such as Klein-Levin syndrome and narcolepsy; other sleep disorders are insomnia, including transient insomnia, acute insomnia and chronic insomnia.

Individuals affected by narcolepsy often present symptoms such as excessive daytime sleepiness (EDS), sleep fragmentation, cataplexy (in narcolepsy with cataplexy), abnormal REM sleep, nocturnal sleep disruption, paralysis during sleep onset or during awakening; and/or hypnagogic hallucinations. Similar symptoms are shown also by individuals affected by Narcolepsy Due to Medical Condition (NDMC), a group of disorders also known as secondary or symptomatic narcolepsy. Cataplexy is a sudden loss of muscle tone triggered by emotions, which is the most valuable clinical feature used to diagnose narcolepsy. Therefore, sleep disorders are neurologically disabling. Human narcolepsy is primarily a sporadically occurring disorder but familial clustering has been observed. Examples of medical conditions commonly causing narcolepsy with cataplexy are: tumors, sarcoidosis, arteriovenous malformations affecting the hypothalamus, multiple sclerosis plaques impairing the hypothalamus, paraneoplastic syndrome antt-Ma2 antibodies, Neimann-Pick type C disease or Coffin-Lowry syndrome. Examples of medical conditions commonly causing narcolepsy without cataplexy are: head trauma, myotonic dystrophy, Prader-Willi syndrome, Parkinson's disease or multisystem atrophy.

The characteristics of excessive daytime sleepiness (EDS) include e.g. repeated episodes of naps or lapses into sleep of short duration, usually less than an hour. The patient e.g. sleeps for 10 to 20 minutes and awakens refreshed but begins to feel sleepy again and the pattern repeats itself. In patients afflicted with EDS sleep usually occurs in situations in which tiredness is common, such as traveling in transport, attending a monotonous meeting that requires no active participation, or listening to a play, concert, movie or lecture, but there may also be sudden and irresistible sleep attacks in situations where sleep normally never occurs, including during an examination, at interactive business talks, while eating, walking or driving and when actively conversing. Usually sleep attacks occur on a background of drowsiness that is a common daily feature. Cataplexy is characterized by sudden loss of muscle tone. The duration of cataplexy is usually short, ranging from a few seconds to several minutes and recovery is immediate and complete. The loss of muscle tone varies in severity and ranges from a mild sensation of weakness with head droop, facial sagging, jaw drop, slurred speech and buckling of the knees to complete postural collapse, with a fall to the ground.

Cataplexy is usually precipitated by emotion that usually has a pleasant or exciting component, such as laughter, elation, pride, anger or surprise. Cataplexy may be associated with narcolepsy. Cataplexy may be associated with specific lesions located primarily in the lateral and posterior hypothalamus, as e.g. tumors (astrocytoma, glioblastoma, glioma, craniopharyngioma and subependynoma) and arterio-venous malformations. Conditions in which cataplexy can be seen include ischemic events, multiple sclerosis, head injury, paraneoplastic syndromes, and infections, such as encephalitis. Cataplexy may occur transiently or permanently due to lesions of the hypothalamus that were caused by surgery, especially in difficult tumor resections. In infancy, cataplexy can be seen in association with other neurological syndromes such as Niemann-Pick type C disease.

A measurement of hypocretin-1 and/or hypocretin-2 values is used for diagnosing narcolepsy and other sleep disorder (Mignot et al. 2003; Einhaus 1999; Malik et al. 2001 ; Marcus et al. 2002; Vankova et al. 2003). However, only CSF samples are used for the analysis at the moment and therefore the number of tested individuals is limited.

In some embodiments the present disclosure relates to a method for diagnosis of a sleep disorder, said sleep disorder being a hypersomnia, such as central hypersomnia, including idiopathic hypersomnia, recurrent hypersomnia such as Klein-Levin syndrome and narcolepsy.

In some embodiments of the present disclosure, the sleep disorder is narcolepsy including with cataplexy (narcolepsy type 1 ; narcolepsy-cataplexy syndrome; NRCLP1 ; narcolepsy with low hypocretin) and narcolepsy without cataplexy (narcolepsy type 2; narcolepsy with normal hypocretin).

Narcolepsy 1 is a disease is caused by mutations affecting the hypocretin gene.

Human narcolepsy is associated with a deficient hypocretin system. Hypocretins are absent and/or greatly diminished in the brain and cerebrospinal fluid (CSF) of most narcoleptic patients. Thanks to the methods of the present disclosure, it will be possible to detect the absence or the scarce presence of hypocretin without the need of sampling CFS, but by analyzing samples of body fluids that can easily be collected, such as blood, whole blood, plasma, serum, urine, saliva, tears, and semen.

In some embodiments the present disclosure relates to a method for diagnosis of a sleep disorder, said sleep disorder being insomnia, including transient insomnia, acute insomnia and chronic insomnia. Neuropsychiatnc diseases

Neuropsychiatric diseases also known as mental and behavioral disorders, are psychological syndromes or patterns associated with distress (e.g. via a painful symptom), disability (impairment in one or more important areas of functioning), increased risk of death, or causes a significant loss of autonomy. Examples of neuropsychiatric diseases or mental and behavioral disorders which are affected by abnormal values of hypocretin-1 and/or hypocretin-2 are schizophrenia, depression, seasonal affective disorder, attention deficit hyperactivity disorder (ADHD), bipolar disorder and hallucinations. In some embodiments the present disclosure relates to a method for diagnosis of a neuropsychiatric disease, said neuropsychiatric disease being selected from the group consisting schizophrenia, depression, seasonal affective disorder, attention deficit hyperactivity disorder (ADHD), bipolar disorder and hallucinations. Schizophrenia is a mental disorder characterized by abnormal social behavior and failure to understand what is real. Common symptoms include false beliefs, unclear or confused thinking, hearing voices, reduced social engagement and emotional expression, and a lack of motivation. People with schizophrenia often have additional mental health problems such as anxiety disorders, major depressive illness, or substance use disorder. The causes of schizophrenia include environmental and genetic factors.

Major depressive disorder (MDD), also known as simply depression, is a mental disorder characterized by at least two weeks of low mood that is present across most situations. It is often accompanied by low self-esteem, loss of interest in normally enjoyable activities, low energy, and pain without a clear cause. People may also occasionally have false beliefs or see or hear things that others cannot. Major depressive disorder can negatively affects a person's family, work or school life, sleeping or eating habits, and general health (American Psychiatric Association, 2013, pp. 160-168). An atypical depression may be, but is not limited to, a subtype of dysthymia and major depression characterized by mood reactivity and by reversed vegetative symptoms, namely over-eating and over-sleeping as described by The Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR) and The

International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10).

Bipolar disorder, formerly manic depression, is a mental disorder with periods of depression and periods of elevated mood. The elevated mood is significant and is known as mania or hypomania, depending on its severity, or whether symptoms of psychosis are present. During mania an individual behaves or feels abnormally energetic, happy or irritable. Individuals often make poorly thought out decisions with little regard to the consequences. The need for sleep is usually reduced during manic phases. Other mental health issues such as anxiety disorders and substance use disorder are commonly associated. The causes are not clearly understood, but both environmental and genetic factors play a role (Anderson et al. 2012).

Seasonal affective disorder (SAD), also known as winter depression, winter blues, summer depression or seasonal depression, is a mood disorder in which, people who have normal mental health throughout most of the year, experience depressive symptoms at the same time each year, most commonly in the winter. Symptoms of

SAD may consist of difficulty waking up in the morning, nausea, tendency to oversleep and over eat, especially a craving for carbohydrates, which leads to weight gain. Other symptoms include a lack of energy, difficulty concentrating on or completing tasks, withdrawal from friends, family, and social activities, and decreased sex drive. People who experience spring and summer depression show symptoms of classic depression including insomnia, anxiety, irritability, decreased appetite, weight gain or loss, social withdrawal, and decreased sex drive. As many as 20% of the people affected by seasonal affective disorder may also have a bipolar disorder (American Psychiatric Association, 2013, pp. 123-154). Attention deficit hyperactivity disorder (ADHD) is a mental disorder of the

neurodevelopmental type. It is characterized by problems paying attention, excessive activity, or difficulty controlling behavior which is not appropriate for a person's age. Primary sleep disorders may affect attention and behavior and the symptoms of ADHD may affect sleep. Sleep disorders and ADHD commonly co-exist. They can also occur as a side effect of medications used to treat ADHD. In children with ADHD, insomnia is the most common sleep disorder with behavioral therapy the preferred treatment. Problems with sleep initiation are common among individuals with ADHD but often they will be deep sleepers and have significant difficulty getting up in the morning.

References

American Psychiatric Association (2013), Diagnostic and Statistical Manual of Mental Disorders (DSM-5®) (5th ed.), Arlington: American Psychiatric Publishing, pp. 160-168 American Psychiatric Association (2013), Diagnostic and Statistical Manual of Mental Disorders (5th ed.), Arlington: American Psychiatric Publishing, pp. 123-154

Anderson IM, Haddad PM, Scott J (2012), Bipolar disorder. British Medical Journal Dalai MA, Schuld A, Haack M, Uhr M, Geisler P, Eisensehr I, Noachtar S, Pollmacher T (2001 ) Normal plasma levels of orexin A (hypocretin-1 ) in narcoleptic patients.

Neurology 56:1749-51

Einhaus SI, S R., Craniopharyngioma, in Principles and Practice of Pediatric

Neurosurgery, P.I. Albright AL, Adelson PD, Editor. 1999, Thieme: New York. p. 545- 562

Higuchi S, Usui a, Murasaki M, et al (2002) Plasma orexin-A is lower in patients with narcolepsy. Neurosci Lett 318:61 ^

Malik, S., Boeve BF, Krahn LE, Silber MH (2001 ) Narcolepsy associated with other central nervous system disorders; Neurology 57(3): p. 539-41

Marcus, C.L., Trescher WH, Halbower AC, Lutz J. (2002) Secondary narcolepsy in children with brain tumors; Sleep 25(4): p. 435-9

Mignot, E., W. Chen, and J. Black (2003) On the value of measuring CSF hypocretin-1 in diagnosing narcolepsy. Sleep 26(6): p. 646-9

Nishino S, Mignot E (2002) Plasma orexin-A is lower in patients with narcolepsy. Sleep Med 3:377-8

Partinen M, Kornum BR, Plazzi G, Jennum P, Julkunen I, Vaarala O (2014) Narcolepsy as an autoimmune disease: the role of H1 N1 infection and vaccination. Lancet Neurol 13:600-613 Vankova, J., Stepanova I, Jech R, Elleder M, Ling L, Mignot E, Nishino S, Nevsimalova S. (2003) Sleep disturbances and hypocretin deficiency in Niemann-Pick disease type C. Sleep 26(4): p. 427-30

Sequences

hcrt-1 (Orexin-A) sp|043612|34-66 (SEQ ID NO:1 ):

QPLPDCCRQKTCSCRLYELLHGAGNHAAGILTL Modified residue position 34: Pyrrolidone carboxylic acid (Pyroglutamic acid, Pyro-Glu) Modified residue position 66: Leucine amide (Leu-NH₂)

Disulphide bonds: between amino acid residues no. 39 and 45 and between amino acid residues no. 40 and 47. (Pyro-Glu)PLPDCCRQKTCSCRLYELLHGAGNHAAGILTL (SEQ ID NO:7)

PLPDCCRQKTCSCRLYELLHGAGNHAAGILTL-NH₂

(Pyro-Glu)PLPDCCRQKTCSCRLYELLHGAGNHAAGILTL- NH₂ (SEQ ID NO:7) hcrt-2 (Orexin-B) sp|O43612|70-97 (SEQ ID NO:2):

RSGPPGLQGRLQRLLQASGNHAAGILTM

Modified residue position 97: Methionine amide

RSGPPGLQGRLQRLLQASGNHAAGILTM-NH2

Prepro-hypocretin (UniProtKB - 043612 (OREX HUMAN) - SEQ ID NO:3):

MNLPSTKVSW AAVTLLLLLL LLPPALLSSG AAAQPLPDCC RQKTCSCRLY ELLHGAGNHA AGILTLGKRR SGPPGLQGRL QRLLQASGNH AAGILTMGRR AGAEPAPRPC LGRRCSAPAA ASVAPGGQSG I

Cleaved hcrt-1 / fragments of hcrt-1 :

QPLPDCCRQKTCSCRLY (SEQ ID NO:4)

QPLPDCCRQKTCSCRL (SEQ ID NO:5)

QPLPDCCRQKTCSCR (SEQ ID NO:6) Examples

Example 1. Hcrt-1 binds to albumin

Western blot analysis indicates that hcrt-1 binds to albumin and that some of the hcrt-1 is retained in the heavy fraction when running human CSF over a 10 kDa molecular weight filter (Figure 1 A-C). This indicates that hcrt-1 is retained in the heavy fraction because of binding to carrier proteins such as albumin, as also shown by co- immunoprecipitation experiments (Figure 1 D). Example 2. Method for dissociation of hypocretin-1 from albumin and

measurement of its concentration

Plasma or serum was freshly collected. For serum, blood was collected in a test tube, where it coagulates within 30 min at room temperature. Afterwards, the test tube was centrifuged at 2000 x g. The serum (top layer) was transferred to a new test tube (low- bind Eppendorf/Sarstedt, 1 .5 ml microtube) without blood contamination and then frozen (-20 eller -80) or analyzed directly.

10-100 μΙ serum was transferred by an autosampler to 0.3 ml test tubes. The HPLC (Shimadzu) had the following setup:

a. Mobile phase A: MilliQ H₂0 + 5% acetonitrile + 0,1 % trifluoroacetic acid b. Mobile phase B: Acetonitrile + 0,1 % trifluoroacetic acid

c. Column: 3,9x300 mm, 125A, μBondaPack C18 reverse phase column from Waters (#WAT027324)

d. Program:

i. Gradient 0-40 min, 1 ml/min, fra 10 til 60% mobile phase B

40- 41 min, 1 ml/min: 60 til 100% mobile phase B

41 - 46 min, 2ml/min: 100% mobile phase B

iv. 46-47 min, 1 ml/min: 100% til 10% mobile phase B (if more samples were to be run - or changed to isocratic flow 100% mobile phase B, 0,05 ml/min)

v. 47-67 min, 1 ml/min: 10% mobile phase B

e. Fractions collection (fractions 0-30) took place between 2 and 33 min (1

ml/fraction) in 3,5 ml test tubes The fractions were concentrated using N₂ gas at 37 ^<C for ca. 3 hours or until drying. Afterwards the fractions were covered with parafilm and stored at -20 'C until analysis the day after. RIA was performed according the producer's protocol and an internal standard was included in each run (K3, CSF clarified and aliquoted by Biolab, Rigshospitalet - Glostrup)

Once the sample had been collected, the timer of the gamma counter (PerkinElmer, Wizard2) was set on 1 min measurement - Standard A. The measurement was carried on until 10.000 counts were obtained and the result converted in counts per seconds (for example if Std A in average gives 333 cpm, one should count for 30 min to reach 10.000 counts, which corresponds to 1800 seconds). The concentration of the unknown samples was calculated from previously obtained standard curve, done in GraphPad Prism and based on a non-linear regression analysis (variable slope, four parameters), from which the concentration of the unknown samples was extrapolated. When using this HPLC protocol on CSF, plasma or serum followed by detection of hcrt- 1 by the conventional hcrt-1 RIA, a peak signal from hcrt-1 was detected in fractions 20-21 (Figure 2B). Albumin was retained longer in the column and eluted in fraction 26- 28 (Figure 2B). Results from plasma and serum also revealed a specific hcrt-1 signal and surprisingly high levels of hcrt-1 (Figure 2C-E). No hcrt-1 was detected by fractionation of human CSF followed by Western blotting, but albumin was found in fraction 26-29 (Figure 2A).

Example 3. pH- and heat-treatment of plasma

The aim is to alter the structure of albumin so the interaction between hcrt-1 , hcrt-2 and albumin is disrupted and enables detection of hcrt-1 and -2.

Background

Human serum albumin (HSA) is the most abundant protein in the blood plasma constituting about 50-60% of all human plasma proteins, and is present in the blood at around 35-50 mg ml-1 . The physiological roles are many; HSA is involved in regulating colloid osmotic blood pressure and aid in transport, distribution and metabolism of multiple endo- and exogenous ligands. HSA binds at multiple sites a chemically diverse range of ligands comprising cations, fatty acids, hormones and pharmaceuticals, providing a depot of ligands available in concentrations well beyond their solubility in blood plasma. HSA binds with the highest affinity to molecules of low water solubility such as fatty acids, isolating their hydrophobic nature and thus allows for transport of these molecules in blood circulation which are otherwise insoluble.

HSA is a 66.5 kDa protein which comprises three homologous domains (I, II and III) that assemble to form a heart-shaped molecule at physiological pH. Each domain is a product of two subdomains with similar structural motifs. HSA has several ligand binding sites and show great conformational adaptability that enables binding of several ligands to HSA simultaneously. Furthermore, HSA undergo pH- and allosteric effector dependent reversible conformational isomerizations.

Under pH 2.7 HSA shows an expanded (E) form where HSA becomes fully uncoiled within the limits of its disulphide bonds. Between pH 2.7 and 4.3 HSA shows a fast form (F) characterized by a dramatic increase in viscosity, low solubility and a significant loss in the alfa-helical content. Between pH 4.3 and 8, and in the absence of allosteric effectors, HSA displays the neutral form (N) which is characterized by a heart-shaped structure. Between pH 4.3 and 8.0 in the presence of allosteric effectors (e.g., drugs, long-chain FAs or ions) and at pH greater than 8 in the absence of ligands, HSA changes conformation to the basic (B) form with the loss of a-helix and rigidity, as well as an increased affinity for some ligands. E.g. the presence of Ca²⁺ can elicit the N->B transition at pH >8 brining the structural change into a pH range encountered in blood plasma suggesting that the N->B transition plays a role in ligand transport. The aged (A) form occurs with time at pH greater than 8. Thus, the pH- and allosteric dependent conformational changes of HSA facilitate binding and release of a variety of molecules/ligands during transportation in the circulatory system allowing for delivery to target tissues.

The structure of a protein is referred to as the primary, secondary, tertiary and quaternary structure. The three latter structures relate to the three-dimensional structure of a protein. The three-dimensional structure is held together by a number of different bonds and interactions, such as hydrogen bonds, ionic bonds, hydrophobic interactions and disulphide bridges. They all help to achieve maximal stability of the protein. The weak nature of the interactions controlling the three-dimensional structure of proteins makes proteins sensitive to external stress factors such as heat, pH, hydrophobic surfaces, high shear and presence of metal ions. The native state of a protein, in which it is in its most stable natural conformation in situ, can be disrupted by such factors and thus facilitate the loss of secondary, tertiary and quaternary structure, a process called denaturation. Denaturation of a proteins results in an unfolding or misfolding of the protein, often with formation of aggregates of denatured polypeptide chains, and severity-dependent loss of biological activity.

Heat denaturation of proteins occurs due to the disruption of hydrogen bonds and non- polar hydrophobic interactions that normally confer structure rigidity. An increase in the kinetic energy is observed with heat exposure and facilitates molecular vibrations so strong and rapid that the bonds are disrupted.

The melting point (TM) of a protein is defined as the temperature at which the protein denatures also explained as where the unfolding transition occurs. Melting points differ significantly from protein to protein due to the unique primary sequences of amino acids they harbour. Solution conditions can also have great impact on the stability of the protein structure and hence influence the melting temperature. Factors such as pH, ionic strength, salt concentration, and post-translational modifications, e.g.

glycosylation have all shown to have a marked influence on TM.

The degree of heat denaturation of a protein is dependent on the time in which the protein is exposed to the heat. A change in protein structure as observed with denaturation is a gradual transition and different types of aggregates predominate at different heating times. Whether the structural changes observed are reversible or irreversible is dependent on heating time and temperature. Heat denaturation of albumin has been studied extensively for decades. The most famous is the boiled egg studies where the albumins of the egg white denature and form a solid white mass. The TM range reported for HSA at pH 7.4 is 59-64°C.

However, it has been reported that TM changes with pH e.g. at pH 9.9 TM is 65.6°C and at pH 5.4 TM is 65.4°C. The secondary structure content shifts with increasing temperature. Heat treatment decreases a-helix structure, increases albumin aggregate formation and β structure content in a concentration and temperature dependent manner. Furthermore, changes in the secondary structure of HSA have also been observed with varying pH and heating time. In accordance to the reported TM values, at temperatures up to 60°C almost no denaturation of HSA is observed. However, at 65°C observable changes in structure can be observed and at 80°C it has been reported that the degree of denaturation reaches 50%. Denaturation is reversible at lower temperatures, but show structural irreversibility at higher temperatures. It has been shown that HSA heated to 80°C will be subject to an irreversible denaturation of about 40%. It has been suggested that HSA do not refold to its native structure after heating beyond the temperature of unfolding. Studies on bovine serum albumin (BSA) structure have also shown that pH affects the structure of BSA during heat

denaturation and interestingly that different buffer systems at same pH also influence BSA structure and display different denaturation patterns. Furthermore, the strength of the buffer also influenced the structural changes accompanied by heat denaturation. These data suggest that pH, heating time and temperature, and buffer type and strength all influence albumin denaturation.

In this study, we have pH- and heat-treated plasma to alter the structure of HSA to disrupt the interaction between HSA and hcrt-1 which allows for detection of hcrt-1 in blood.

Procedure

Depicted in the scheme below is the stepwise procedure with the aim of dissociating hcrt-1 from albumin. The variables of the dissociation method are pH of buffer, heating time and temperature, ± solvent, ± filter and ± evaporation.

Sample Plasma and Serum

type Volumes 250, 500, 750 and 1000 ul

Step 1 Buffer addition

pH 2, 4, 8, 9 and 10

Step 2 Heating

Time: 10, 20, 30 min, or overnight (o/n)

Temperature: 37, 50, 60, 65, 70, 80, 90 °C

Step 3 ±Solvent

Type: DMSO (1 -5%), MeCN(35%), MeCN:MeOH (2:1 ) Step 4 ±Filter

10-30 kDa filter

Step 5 ±Evaporation

N2 evaporation, 37°C for 2-3 hours

Step 6 Detection

e.g. RIA analysis

Example of a protocol for a dissociation method

The combination of steps that gives the highest yield is:

Plasma was thawed on wet ice and 250 ul of the plasma was mixed with 250 ul of water and 250 ul of buffer at pH 9. The sample was mixed briefly by vortexing and was allowed to mix for 15 min before heat treatment. The sample was heated for 30 min at 65°C and N2 evaporated for 2-3 hours at 37°C. The sample was then analysed by RIA and the concentration of hcrt-1 was determined.

Results

Effect ofpH-, heat- and o/n treatment of plasma on hcrt-1 concentration detectable with RIA

Samples of 250 ul of plasma (EDTA) were pH-, heat- and o/n (overnight) treated in various combinations. Samples were either subjected to o/n treatment at room temperature (RT), 37 or 45 degrees Celsius, or were immediately after thawing subjected to other treatment and was thus not left over night (-o/n). Some samples were heat treated for 10 min at 65°C (+65°C treatment) whilst other were not (-65°C treatment) and some were pH treated, pH 8, (+ buffer) whilst others were not (-buffer). The results are shown in Figure 3. This experiment shows that both pH treatment and heat treatment (65°C at 10 min) influence the detection of hcrt-1 levels above the detection limit of the RIA (10 pg/ml). This suggests that both heat and pH affect the hcrt-1 / albumin interaction and that both are required to sufficiently unmask hcrt-1 in the plasma. This experiment also shows that o/n treatment of plasma decrease the hcrt-1 concentration suggesting a degradation of hcrt-1 with time. The sample that was not subjected to o/n treatment shows the highest hcrt-1 concentration. No difference is observed between o/n at 37 and 45 degrees and little difference in Hcrt-1 concentration is observed between 37°C, 45°C and RT.

Effect of temperature and plasma volume on hcrt- 1 concentration

Varying volumes of plasma was subjected to heat treatment at either 37°C or 65°C for

10 min with a buffer at pH 8. Results are shown in Figure 4. This experiment shows that 500 ul of plasma heated at 65°C for 10 min gives the highest hcrt-1 yield. Furthermore, heating the sample at 37°C degrees for 10 min does not seem to have any effect on the detectable levels of hcrt-1 which is under the detection limit for all plasma volumes. This suggests that heating temperature has an effect on the yield and that 65°C is better than 37°C to liberate hcrt- 1 from albumin.

Effect of pH on hcrt- 1 concentration

250 ul of plasma was subjected to heat treatment for 10 min at 65°C with a buffer with varying pH.

The results are shown in Figure 5. This experiment shows that plasma treated for 10 min at 65°C with a buffer at pH 8, 9 or 10 enables us to detect hcrt-1 levels above that of the detection limit of the RIA. At pH 9 we see the highest average concentration (B) suggesting that treatment with a buffer at this pH gives the highest yield. In was not possible to detect any hcrt-1 in any of the 6 samples treated with pH 2 or 4.

Effect of heating temperature and pH on hcrt- 1 concentration

Samples of 250 ul plasma were subjected to buffers of different pH (pH 8, 9 and 10) and heat treated for 10 min at either 50°C, 60°C, 65°C, 70°C, 80°C, or 90°C. The results are shown in Figure 6. This experiment shows that 65°C gives the highest hcrt-1 yield for both pH 9 and 10 whereas the samples treated with 60°C and 50°C show hcrt-1 concentrations under the detection limit of the RIA. At pH 8, heat treatment at 65°C gives a signal under the detection limit and at 60°C a high signal can be observed. However the standard deviation of the two measurements is large and errors in the measurements cannot be excluded. When plasma was heat treated with temperatures from 70 and up, visible protein denaturation occurred hindering further treatment of the sample. Effect of heating time on hcrt- 1 concentration

Samples of 250 ul plasma were treated with a buffer at pH 9 and a temperature at 65°C for either 10, 20 or 30 minutes.

The results are shown in Figure 7. This experiment shows that heating for 30 minutes gives the highest hcrt-1 yield. 10 min gives second most and 20 min the least. All time points provide detectable results.

Example 4. Chymotrypsin-treatment of plasma

Five reactions of each setup with and without chymotrypsin as shown in the table below were mixed and left at 37^<Ό o/n. After approximately 19 hours each sample were briefly spun down and heated for 10 min at 65 °C. The five reactions of each setup were then mixed into two tubes. The two new tubes either containing 250 ul of digested or undigested plasma were then N2 evaporated for 2-3 hours at 37 ^<Ό and then analyzed using RIA. Data are shown in Figure 8.

Table: Chymotrypsin digestion setup

5x Digestion setup of plasma (+ treatment) 5x Control (- treatment)

50 μΙ_ plasma

50 μΙ_ 5x digestion buffer (Tris:HCI pH 8, 10

50 μΙ_ 5x digestion buffer (Tris:HCI pH mM CaCI2)

8, 10 mM CaCI2)

25 μΙ_ chymotrypsin (10 mg/mL) [0.25 mg]

250 μΙ_ MiliQ

Claims

A method for dissociating hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) and/or a variant thereof from one or more carrier proteins in a sample, thereby allowing detection and/or quantification of said hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample.

A method for detection of hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) and/or a variant thereof in a sample, the method comprising one or more steps of

a) providing a sample,

c) detecting hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample.

The method of claim 2, further comprising a step of determining the level and/or concentration of said hypocretin-1 and/or said hypocretin-2 and/or a variant thereof in said sample.

The method of any one of the preceding claims, wherein the method is an in vitro method.

The method of any one of the preceding claims, wherein the sample is obtained from or obtainable from an individual such as a human being.

The method of any one of the preceding claims, wherein the sample is a body fluid.

The method of any one of the preceding claims, wherein the sample is a body fluid selected from the group consisting of blood, whole blood, plasma, serum, urine, saliva, tears, cerebrospinal fluid and semen.

8. The method of any one of the preceding claims, wherein the sample is plasma or serum.

9. The method of any one of the preceding claims, wherein the sample is not cerebrospinal fluid (CSF).

10. The method of any one of the preceding claims, wherein the sample is not pre- depleted plasma or pre-depleted serum.

1 1 . The method of any one of the preceding claims, wherein the dissociation step comprises breaking at least one covalent or non-covalent bond between hypocretin-1 or hypocretin-2 and said one or more carrier proteins in said sample.

12. The method of any one of the preceding claims, wherein the dissociation step comprises one or more steps of heating the sample and/or increasing or adjusting the pH of the sample.

13. The method of any one of the preceding claims, wherein the dissociation step comprises one or more steps of heating the sample to a temperature of 50 to 75°C; such as 50 to 55°C, such as 50 to 60°C, such as 60 to 61 °C, such as 61 to 62°C, such as 62 to 63°C, such as 63 to 64°C, such as 64 to 65°C, such as 65 to 66°C, such as 66 to 67°C, such as 67 to 68°C, such as 68 to 69°C, such as 69 to 70°C, such as 70 to 75°C.

14. The method of any one of the preceding claims, wherein the dissociation step comprises one or more steps of heating the sample for a period of 1 minute to 5 hours; such as 1 minute to 5 minutes, such as 5 minutes to 10 minutes, such as 15 minutes to 20 minutes, such as 20 minutes to 25 minutes, such as 25 minutes to 30 minutes, such as 30 minutes to 40 minutes, such as 40 minutes to 50 minutes, such as 50 minutes to 60 minutes, such as 1 hour to 1 ½ hours, such as 1 ½ to 2 hours, such as 2 to 2½ hours, such as 2½ to 3 hours, such as

3 to 3½ hours, such as 3½ to 4 hours, such as 4 to 4½ hours, such as 4½ to 5 hours.

15. The method of any one of the preceding claims, wherein the dissociation step comprises one or more steps of increasing or adjusting the pH of the sample to a pH of 7 to 1 1 , such as a pH of 8 to 10; such as a pH of 7 to 8, a pH of 8 to 9, a pH of 9 to 10 or a pH of 10 to 1 1 .

16. The method of any one of the preceding claims, wherein the dissociation step comprises one or more steps of

a) heating the sample to a temperature of 50 to 75°C for a period of 5

minutes to 5 hours; and

b) adjusting the pH of the sample to a pH of 7 to 1 1 .

17. The method of any one of the preceding claims, wherein the dissociation step comprises one or more steps of

a) heating the sample to a temperature of 60 to 70°C, or approx. 65°C, for a period of 5 minutes to 60 minutes; and

b) adjusting the pH of the sample to a pH of 8 to 10.

18. The method of any one of the preceding claims, wherein the dissociation step is a chemical or an enzymatic reaction.

19. The method of any one of the preceding claims, wherein the dissociation step is a chromatography separation.

20. The method of any one of the preceding claims, wherein the dissociation step is chromatography separation on a high-performance liquid chromatography (HPLC) column.

21 . The method of any one of the preceding claims, wherein the dissociation step comprises use of a protease.

22. The method of any one of the preceding claims, wherein said protease

preferably digests said one or more carrier proteins and/or does not digest said hypocretin-1 and/or hypocretin-2 and/or a variant thereof.

23. The method of any one of the preceding claims, wherein said protease is a chymotrypsin-like serine protease, such as chymotrypsin.

24. The method of any one of the preceding claims, wherein said carrier protein has at least one binding domain for at least one hydrophobic compound, such as has at least one binding domain for hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) and/or a variant thereof.

25. The method of any one of the preceding claims, wherein said carrier protein is a blood carrier protein, such as a blood plasma carrier protein.

26. The method of any one of the preceding claims, wherein said carrier protein selected from a group consisting of albumins, immunoglobulins (Ig), alpha-1 antitrypsin (A1 AT), fibrinogen, and haptoglobin (HG).

27. The method of any one of the preceding claims, wherein said carrier protein is albumin, such as an albumin selected from the group consisting of serum albumin, alpha-fetoprotein (alpha-fetoglobulin), vitamin D-binding protein and afamin.

28. The method of any one of the preceding claims, wherein said carrier protein serum albumin.

29. The method of any one of the preceding claims, wherein the detecting step comprises an assay selected from the group consisting of an immunoassay, protein assay and a mass spectrometry assay.

30. The method of any one of the preceding claims, wherein said immunoassay selected from the group consisting of RIA, MIA, EIA, FIA and ELISA.

31 . The method of any one of the preceding claims, wherein said protein assay selected from the group consisting of centrifugation, electrophoresis, chromatography and western blotting.

32. The method of any one of the preceding claims, wherein the detecting step comprises quantitative mass spectrometry assay.

33. The method of any one of the preceding claims, wherein said variant of hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) comprises any naturally occurring variant or fragment of hypocretin-1 and/or any naturally occurring variant or fragment of hypocretin-2.

34. The method of any one of the preceding claims, wherein said variant of

hypocretin-1 and/or hypocretin-2 is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 as well as C-terminally amidated versions of any one of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

35. A method for diagnosis of a disease or disorder associated with abnormal levels of hcrt-1 and/or hcrt-2, such as a sleep disorder and/or of a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke in an individual, the method comprising:

a) providing a sample,

c) detecting hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample,

d) determining the level and/or concentration of said hypocretin-1 and/or hypocretin-2 and/or variant thereof in said sample.

36. The method of claim 35 wherein said sample is a sample such as a body fluid sample obtained from or obtainable from said individual.

37. The method of any one of claims 35-36 comprising the further step of

comparing said level and/or concentration of said hypocretin-1 and/or hypocretin-2 and/or a variant thereof with a cut-off value,

wherein said cut-off value is determined from the concentration range of hypocretin-1 and/or hypocretin-2 and/or a variant thereof in healthy human individuals, such as human individuals who do not present with a sleep disorder and/or a neuropsychiatric disease and/or a disease of the nervous system and/or a traumatic brain injury and/or a stroke, and wherein a level and/or concentration that is lower or greater than the cut-off value indicates the presence or absence of said sleep disorder and/or neuropsychiatric disease and/or disease of the nervous system and/or traumatic brain injury and/or stroke.

38. The method of any one of claims 35-37, comprising the further step of

classifying the level and/or concentration of said hypocretin-1 and/or hypocretin- 2 and/or a variant thereof in said sample as:

a) Low: When the level and/or concentration is lower than the cut-off value b) Normal: When the level and/or concentration is equal to the cut-off

value; and

c) High: When the level and/or concentration is higher than the cut-off value.

39. The method of any one of claims 35-38, wherein said cut-off value is a

concentration range, such as the concentration range of said hypocretin-1 and/or hypocretin-2 and/or a variant thereof in healthy human individuals, such as human individuals who do not present with a sleep disorder and/or a neuropsychiatric disease and/or disease of the nervous system and/or traumatic brain injury and/or stroke.

40. The method of any one of claims 35-39, said method further comprising the step of treating said sleep disorder and/or said neuropsychiatric disease and/or said disease of the nervous system and/or said traumatic brain injury and/or stroke.

41 . The method of any one of claims 35-40, wherein the sleep disorder is a

hypersomnia, such as central hypersomnia, including idiopathic hypersomnia, recurrent hypersomnia such as Klein-Levin syndrome and narcolepsy.

42. The method of any one of claims 35-41 , wherein the sleep disorder is

narcolepsy, including narcolepsy with cataplexy (narcolepsy type 1 ; narcolepsy- cataplexy syndrome; NC; NRCLP1 ; narcolepsy with low hypocretin) and narcolepsy without cataplexy (narcolepsy type 2; NwC; narcolepsy with normal hypocretin).

43. The method of any one of claims 35-42, wherein the sleep disorder is insomnia, including transient insomnia, acute insomnia and chronic insomnia.

44. The method of any one of claims 35-43, wherein the neuropsychiatric disease is selected from the group consisting of schizophrenia, depression, seasonal affective disorder, attention deficit hyperactivity disorder (ADHD), bipolar disorder and hallucinations.

45. The method of any one of claims 35-44, wherein the disease of the nervous system is selected from the group consisting of demyelinating diseases of the central nervous system, inflammatory polyneuropathies and neurodegenerative disorders.

46. A method for diagnosis of narcolepsy in an individual, the method comprising: a) providing a sample, such as a sample obtained from or obtainable from said individual, such as a body fluid sample obtained from or obtainable from said individual,

c) detecting said hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample, and

47. The method of claim 46, wherein:

a) an individual has, or is likely to have, narcolepsy if the level and/or

concentration of hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) and/or a variant thereof is lower than the level in healthy human individuals, such as human individuals who do not present with narcolepsy, and

b) an individual does not have, or is not likely to have, narcolepsy if the level and/or concentration of hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) and/or variant thereof is comparable to the level in healthy human individuals, such as human individuals who do not present with narcolepsy.

48. The method of any one of claims 46-47, wherein:

a) an individual has, or is likely to have, narcolepsy if the level and/or

concentration of hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) and/or a variant thereof is about 0 pg/ml, such as 0 to 50 pg/ml, such as 0 to 100 pg/ml, such as 0 to 1 10 pg/ml;

b) an individual does not have, or is not likely to have, narcolepsy if the level and/or concentration of hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) and/or variant thereof is 200 pg/ml or more, such as 220 pg/ml or more, such as 400 pg/ml or more.

49. The method of any one of claims 46-48, wherein:

a) an individual has, or is likely to have, narcolepsy if the level and/or

concentration of hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) and/or a variant thereof is 50% to 100% lower than the level in healthy human individuals, such as human individuals who do not present with narcolepsy, and

b) an individual does not have, or is not likely to have, narcolepsy if the level and/or concentration of hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) and/or variant thereof is equal to the level, or not more than 50% lower than the level in healthy human individuals such as human individuals who do not present with narcolepsy.

50. The method of any one of claims 46-49, said method further comprising the step of treating said narcolepsy.

51 . The method of claim 50, wherein the treatment comprises administration of one or more CNS stimulants, non-stimulants such as a norepinephrine reuptake inhibitor (NRI), or antidepressants including SSRI and tricyclic antidepressants.

52. The method of any one of claims 50-51 , wherein the treatment comprises

administration of one or more medicaments selected from the group consisting of modafinil, amphetamine, dextroamphetamine, armodafinil, gamma- hydroxybutyrate (GHB), methylphenidate, sodium oxybate, methylphenidate, desvenlafaxine, ephedrine, atomoxetine, venlafaxine, fluoxetine, clomipramine, imipramine, protriptyline, pitolisant and JZP-1 10.

The method of any of claims 35-45 or 46-52, wherein

a) said sample is as specified in any one of claims 5-10, and/or

b) said step b) of dissociating hypocretin-1 (SEQ ID NO:1 ) and/or

hypocretin-2 (SEQ ID NO:2) and/or a variant thereof from one or more carrier proteins in said sample is as specified in any one of claims 1 1 - 28, and/or

c) said step c) of detecting said hypocretin-1 and/or hypocretin-2 and/or a variant thereof in said sample is as specified in any one of claims 29-32, and/or

d) said variant of hypocretin-1 and/or hypocretin-2 is a naturally occurring variant or fragment of hypocretin-1 and/or a naturally occurring variant or fragment of hypocretin-2; such as a variant of hypocretin-1 (SEQ ID NO:1 ) and/or hypocretin-2 (SEQ ID NO:2) selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.