Biomarker panel for assessing chronic stress states

Abstract

To provide a biomarker panel for objectively testing chronic stress levels in humans using saliva. [Solution] The present invention provides a biomarker panel containing at least three substances selected from the group consisting of metabolites listed in Table A as biomarkers, a method and kit for evaluating chronic stress states using the same, a method for screening and / or evaluating substances effective in improving chronic stress states, and an evaluation program.

Description

Technical Field

[0001] The present invention relates to a biomarker panel for evaluating a chronic stress state using saliva, a method and kit for evaluating a chronic stress state using the same, a method for screening and / or evaluating a substance effective for improving a chronic stress state, and an evaluation program.

Background Art

[0002] In order to lead a healthy daily life, it is necessary to maintain both a healthy body and a healthy mind. However, recently, due to factors such as increased stress, it has become a social problem such as a decline in mental health and an increase in the number of patients with depression. So far, exploration by metabolome analysis of biomarker candidates indicating the severity of depression (Patent Document 1), exploration of microRNAs that are biomarker candidates for depression (Patent Document 2), and exploration of candidates for blood biomarkers for the diagnosis of depression (Patent Document 3) have been conducted, and it has been disclosed that tryptophan or its metabolites are candidates as biomarkers for depression (Patent Document 4). However, all of these are disclosed regarding biomarkers in blood or cerebrospinal fluid.

[0003] Exploration of biomarkers by metabolome analysis using saliva samples has been reported to potentially lead to the diagnosis of oral diseases, various cancers, Alzheimer's disease, lifestyle-related diseases, etc. The present inventors have found that in saliva samples of depression model mice, specific metabolites and specific microRNAs are increased or decreased compared to saliva samples of control subjects, and have disclosed that these metabolites and microRNAs can be biomarkers for depression in saliva (Patent Document 5).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] Currently, subjective assessments of chronic stress states using questionnaires such as the Profile of Moods (POMS) are common, and objective methods for evaluating chronic stress states using biomarkers have not yet been established. Therefore, research is being conducted on biomarkers associated with chronic stress states, but most of these are detected in the blood or cerebrospinal fluid. To perform tests more easily, there is a need for the development of methods to detect biomarkers using saliva, which can be collected non-invasively. Patent document 5, mentioned above, discloses numerous metabolites and microRNAs that can serve as biomarkers for depression in saliva, but all of these were detected from the saliva of depression model mice. Therefore, there is a need for the development of methods to objectively test chronic stress states in humans using saliva, which can be collected non-invasively. [Means for solving the problem]

[0006] As a result of diligent research to solve the above problems, the inventors of the present invention discovered that in saliva samples from subjects with and without sleep problems, specific metabolites increased or decreased depending on differences in mood state as evaluated by POMS2 (Profile of Mood States 2nd Edition) and the OSA Sleep Questionnaire MA version (OSA-MA), and thus completed the present invention.

[0007] Therefore, the first embodiment of the present invention is the following biomarker panel for evaluating chronic stress states using saliva. [1] A biomarker panel for evaluating chronic stress status using saliva, A biomarker panel comprising at least three substances selected from the group consisting of metabolites listed in Table A below, as biomarkers. [2] The biomarker panel according to [1], comprising at least three substances selected as biomarkers from the group consisting of metabolites selected from one of the tables B to N described later. [3] The biomarker panel described in [2], wherein the chronic stress state is a total mood state (TMD) as defined in POMS2 (Profile of Mood States 2nd Edition), and the biomarkers include at least three substances selected from the group consisting of metabolites listed in Table B. [4] The biomarker panel according to [2], wherein the chronic stress state is anger / hostility (AH) as defined in POMS2, and the biomarkers include at least three substances selected from the group consisting of metabolites listed in Table C. [5] A chronic stress state is defined as confusion / embarrassment (CB) in POMS2, and includes at least three substances selected from the group consisting of metabolites listed in Table D as biomarkers [the biomarker panel described in 2]. [6] The biomarker panel according to [2], wherein the chronic stress state is depression (DD) as defined in POMS2, and the biomarkers include at least three substances selected from the group consisting of metabolites listed in Table E. [7] The biomarker panel according to [2], wherein the chronic stress state is fatigue-apathy (FI) as defined in POMS2, and the biomarkers include at least three substances selected from the group consisting of metabolites listed in Table F. [8] A chronic stress state is defined as tension / anxiety (TA) in POMS2, and includes at least three substances selected from the group consisting of metabolites listed in Table G as biomarkers [the biomarker panel described in 2]. [9] A biomarker panel according to [2], wherein a chronic stress state is defined as vitality / vitality (VA) in POMS2, and the biomarker includes at least three substances selected from the group consisting of metabolites listed in Table H.

[10] The biomarker panel according to [2], wherein the chronic stress state is friendly (F) as defined in POMS2, and comprises at least three substances selected from the group consisting of metabolites listed in Table I as biomarkers.

[11] A biomarker panel as described in [2], wherein a chronic stress state is morning sleepiness, which is factor 1 of the OSA-MA Sleep Questionnaire, and the biomarker includes at least three substances selected from the group consisting of metabolites listed in Table J.

[12] A biomarker panel according to [2], wherein a chronic stress state is sleep onset and sleep maintenance, which are the second factor of OSA-MA, and the biomarker includes at least three substances selected from the group consisting of metabolites listed in Table K.

[13] A biomarker panel according to [2] in which a chronic stress state is factor 3 of OSA-MA, and at least three substances selected from the group consisting of metabolites listed in Table L are used as biomarkers.

[14] The biomarker panel according to [2], wherein the chronic stress state is fatigue recovery, which is the fourth factor of OSA-MA, and the biomarkers include at least three substances selected from the group consisting of metabolites listed in Table M.

[15] The biomarker panel according to [2], wherein a chronic stress state is sleep duration, which is the fifth factor of the OSA-MA, and the biomarker includes at least three substances selected from the group consisting of metabolites listed in Table N.

[16] A biomarker panel according to any one of [1] to

[15] , wherein at least one of the biomarkers is a nucleic acid-related metabolite.

[17] A biomarker panel according to any one of the items [1] to

[15] , comprising at least four substances as biomarkers.

[0008] A second embodiment of the present invention is a method for evaluating chronic stress states using a biomarker panel.

[18] A method for evaluating a chronic stress state using a biomarker panel described in any one of items [1] to

[15] , comprising the steps of: quantifying a biomarker contained in the biomarker panel from a saliva sample derived from a subject; and comparing a reference value based on the amount of biomarker present in a control saliva sample with the quantitative value of the biomarker in the saliva sample derived from the subject, wherein the chronic stress state is evaluated using the significant difference between the reference value and the quantitative value of the biomarker as an indicator.

[19] The method according to

[18] , wherein the control saliva sample is a saliva sample from a person who is judged not to be in a chronic stress state by evaluation using POMS2 and / or OSA-MA.

[0009] A third embodiment of the present invention is a kit for evaluating chronic stress states using a biomarker panel.

[20] A kit for evaluating chronic stress status using a biomarker panel described in any one of items [1] to

[15] , the kit comprising reagents for quantifying biomarkers contained in the biomarker panel from a saliva sample derived from a subject.

[0010] A fourth embodiment of the present invention is a method for screening and / or evaluating substances effective in improving chronic stress conditions using a biomarker panel.

[21] A method for screening and / or evaluating substances effective in improving chronic stress conditions using a biomarker panel described in any one of items [1] to

[15] , comprising the steps of: quantifying the biomarkers contained in the biomarker panel in a saliva sample from a subject before administration of a candidate substance and obtaining quantitative value 1; quantifying the biomarkers contained in the biomarker panel in a saliva sample from a subject after administration of a candidate substance and obtaining quantitative value 2; and comparing quantitative value 1 and quantitative value 2, wherein the significant difference between quantitative value 1 and quantitative value 2 is used as an indicator to screen and / or evaluate substances effective in improving chronic stress conditions.

[22] The method according to

[21] , wherein the subjects are those who have been judged to be in a state of chronic stress by evaluation using POMS2 and / or OSA-MA.

[23] The method according to

[21] , wherein the substance is used to treat a chronic stress condition.

[0011] A fifth embodiment of the present invention is a program for evaluating chronic stress states using a biomarker panel.

[24] A program for assessing chronic stress conditions, A program to instruct the computer to perform the following steps (1) and (2): (1) A procedure for calculating whether there is a significant difference between the measurement data from the subject and the reference value, based on the measurement data including the quantitative value of a biomarker included in any one of the biomarker panels described in item [1] to

[15] in the saliva sample from the subject, and the reference value based on the amount of biomarker present in the control saliva sample. (2) A procedure for displaying evaluation results using the statistical significance as an indicator when there is a statistically significant difference between the calculated measurement data and the reference value. [Effects of the Invention]

[0012] By using the biomarker panel of the present invention, it becomes possible to objectively evaluate a chronic stress state using saliva that can be non-invasively collected. Furthermore, by using the biomarker panel of the present invention, it also becomes possible to screen and / or evaluate substances effective for improving a chronic stress state.

Mode for Carrying Out the Invention

[0013] 1. Biomarker Panel for Evaluating Chronic Stress State A first embodiment of the present invention is a biomarker panel for evaluating a chronic stress state using saliva, the biomarker panel containing at least three substances selected from the group consisting of metabolites described in Table A below as biomarkers.

[0014]

Table 1-1

Table 1-2

Table 1-3

Table l-4

Table 1-5

Table 1-6

[0015] In all the tables described in the present application, two or more substances described in one row are substances detected as the same substance. Also, amino acids are shown in three-letter notation.

[0016] A "chronic stress state" refers to a stress state that persists over a long period of time. Unlike temporary stress (acute stress), it refers to a state of being subjected to stress repeatedly experienced in daily life or stress caused by long-term problems. While there are no particular limitations on the criteria or methods for determining a chronic stress state in this invention, chronic stress can be assessed using scales that evaluate mood states and depressive symptoms, such as POMS2 (Profile of Mood States 2), BDI (Beck Depression Inventory), and CES-D (Center for Epidemiologic Studies Depression Scale). Furthermore, since stress significantly affects sleep, the presence and degree of a chronic stress state can also be assessed based on evaluation results from scales such as PSQI (Pittsburgh Sleep Quality Index) and OSA-MA (OSA Sleep Questionnaire MA version) to evaluate sleep quality and patterns.

[0017] In this invention, a "biomarker" is a substance that serves as an indicator of the presence of a specific disease or disorder, and its concentration can usually represent the state or progression of the disease or disorder. In this invention, the substance used as a biomarker is a substance that correlates with the presence and degree of chronic stress.

[0018] In this invention, "biomarker panel" refers to a collection of multiple biomarkers (panelized) to improve evaluation accuracy compared to evaluation using a single biomarker. The biomarker panel of this invention is a panel containing at least three, preferably at least four, and more preferably at least five substances selected from the group consisting of metabolites listed in Table A above. There is no upper limit to the number of substances included in the biomarker panel, and it may include all the substances listed in the table. It is believed that the accuracy of the evaluation results will improve by testing a larger number of biomarkers.

[0019] The substances listed in Table A above are metabolites found through metabolome analysis of human saliva. The inventors selected metabolites that showed a statistically significant difference (p<0.05) when comparing the differences between two groups—a group of the top 10 individuals and a group of the bottom 10 individuals—for mood states scored using the POMS2 questionnaire, namely, Total Mood Disturbance (TMD), Tension-Anxiety (TA), Depression-Dejection (DD), Anger-Hostility (AH), Vigor-Activity (VA), Fatigue-Inertia (FI), Confusion-Bewilderment (CB), and Friendliness (F), using a t-test. Furthermore, for each of the 435 substances listed above, a correlation analysis was performed using Spearman's rank correlation to examine the concentrations of the aforementioned 435 substances for each of the five items scored by OSA-MA: morning sleepiness, falling asleep and maintaining sleep, dreaming, fatigue recovery, and sleep duration. Metabolites showing a significant correlation (p<0.05) were selected. Table A summarizes the substances that showed a correlation with the PSQI score and those that showed a correlation with the OSA-MA score.

[0020] Of the substances listed in Table A above, those that showed a correlation with the PSQI score (a significant difference between high-scoring and low-scoring individuals) are shown in Tables B to I below, and those that showed a correlation with the OSA-MA score are shown in Tables J to N below. The biomarker panel of the present invention may include at least three substances selected from the group consisting of metabolites listed in one of the tables B to N below as biomarkers.

[0021] The metabolites listed in Table B below have been shown to be associated with total mood state (TMD) as defined in POMS2. Therefore, a biomarker panel containing at least three substances selected from the group of metabolites listed in Table B may be useful for evaluating total mood state (TMD).

[0022] [Table 2]

[0023] The metabolites listed in Table C below are those that have been shown to be associated with anger / hostility (AH) as defined in POMS2. Therefore, a biomarker panel containing at least three substances selected from the group consisting of the metabolites listed in Table C may be useful for evaluating anger / hostility (AH).

[0024] [Table 3]

[0025] The metabolites listed in Table D below are those that have been shown to be associated with confusion / embarrassment (CB) as defined in POMS2. Therefore, a biomarker panel containing at least three substances selected from the group consisting of the metabolites listed in Table D may be useful for evaluating confusion / embarrassment (CB).

[0026] [Table 4-1] [Table 4-2]

[0027] The metabolites listed in Table E below have been shown to be associated with depression (DD) as defined in POMS2. Therefore, a biomarker panel containing at least three substances selected from the group consisting of the metabolites listed in Table E may be useful for evaluating depression (DD).

[0028] [Table 5]

[0029] The metabolites listed in Table F below are those that have been shown to be associated with fatigue-apathy (FI) as defined in POMS2. Therefore, a biomarker panel containing at least three substances selected from the group consisting of the metabolites listed in Table F may be useful for evaluating fatigue-apathy (FI).

[0030] [Table 6]

[0031] The metabolites listed in Table G below are those that have been shown to be associated with tension and anxiety (TA) as defined in POMS2. Therefore, a biomarker panel containing at least three substances selected from the group consisting of the metabolites listed in Table G may be useful for evaluating tension and anxiety (TA).

[0032] [Table 7]

[0033] The metabolites listed in Table H below are those that have been shown to be associated with vitality / vitality (VA) as defined in POMS2. Therefore, a biomarker panel containing at least three substances selected from the group consisting of the metabolites listed in Table H may be useful for evaluating vitality / vitality (VA).

[0034] [Table 8]

[0035] The metabolites listed in Table I below are those that have been shown to be associated with friendliness (F) as defined in POMS2. Therefore, a biomarker panel containing at least three substances selected from the group consisting of the metabolites listed in Table I may be useful for evaluating friendliness (F).

[0036] [Table 9-1] [Table 9-2]

[0037] The metabolites listed in Table J below have been shown to correlate with morning sleepiness, which is the first factor of OSA-MA. Therefore, a biomarker panel containing at least three substances selected from the group of metabolites listed in Table J may be useful for evaluating morning sleepiness.

[0038] [Table 10]

[0039] The metabolites listed in Table K below have shown correlations with sleep onset and sleep maintenance, which are the second factors of the OSA-MA. Therefore, a biomarker panel containing at least three substances selected from the group consisting of the metabolites listed in Table K may be useful for evaluating sleep onset and sleep maintenance.

[0040] [Table 11]

[0041] The metabolites listed in Table L below have shown correlation with Yumemi, the third factor of OSA-MA. Therefore, a biomarker panel containing at least three substances selected from the group of metabolites listed in Table L may be useful for evaluating Yumemi.

[0042] [Table 12]

[0043] The metabolites listed in Table M below are those that have shown a correlation with fatigue recovery, which is the fourth factor of OSA-MA. Therefore, a biomarker panel containing at least three substances selected from the group consisting of the metabolites listed in Table M may be useful for evaluating fatigue recovery.

[0044] [Table 13]

[0045] The metabolites listed in Table N below have been shown to correlate with sleep duration, which is the fifth factor of the OSA-MA. Therefore, a biomarker panel containing at least three substances selected from the group consisting of the metabolites listed in Table N may be useful for evaluating sleep duration.

[0046] [Table 14]

[0047] When the biomarker panel of the present invention includes substances selected from the group consisting of metabolites listed in one of the tables B to N above as biomarkers, it includes at least three, preferably at least four, and more preferably at least five substances as biomarkers. There is no upper limit to the number of substances included in the biomarker panel, and it may include all the substances listed in the table. It is believed that the accuracy of the evaluation results will improve by testing a larger number of biomarkers.

[0048] It is preferable that at least one of the biomarkers included in the biomarker panel of the present invention is a nucleic acid-related metabolite. Metabolites detected in saliva include nucleic acid-related metabolites such as guanosine, cytidine, adenine, thymine, 2'-deoxyadenosine, 5'-deoxyadenosine, XMP, dTMP, and dCMP (see Table A in the Examples for other nucleic acid-related metabolites). Therefore, by constructing a biomarker panel that includes these nucleic acid-related metabolites as biomarkers, it is considered possible to evaluate chronic stress states by measuring nucleic acid-related metabolites in saliva.

[0049] 2. Methods for evaluating chronic stress states A second embodiment of the present invention is a method for evaluating a chronic stress state using the biomarker panel of the present invention. This method includes the steps of quantifying the biomarkers contained in the biomarker panel from a saliva sample derived from a subject, and comparing a reference value based on the amount of biomarker present in a control saliva sample with the quantified value of the biomarker in the saliva sample derived from the subject, and evaluating the chronic stress state using the significant difference between the reference value and the quantified value of the biomarker as an indicator.

[0050] The subjects of the method for evaluating chronic stress states of the present invention are not particularly limited, as long as they are subjects capable of developing chronic stress states. While the subjects are typically humans, they may be any mammal, such as pets like dogs and cats, livestock animals like cows, horses, and pigs, or laboratory animals like mice and rats.

[0051] In the method for evaluating chronic stress states according to the present invention, first, biomarkers included in the biomarker panel of the present invention are quantified from a saliva sample derived from the subject. The biomarker panel used is not particularly limited, as long as it includes biomarkers selected from any of the tables A to N above. For example, when evaluating chronic stress states as an alternative to the POMS2 questionnaire, biomarkers selected from tables B to I above that correspond to the Total Mood State (TMD), Tension-Anxiety (TA), Depression-Depression (DD), Anger-Hostility (AH), Vitality-Vitality (VA), Fatigue-Apathy (FI), Confusion-Bewilderment (CB), and Friendliness (F), which can be scored using the POMS2 questionnaire, can be used. Also, when evaluating chronic stress states as an alternative to the OSA-MA, biomarkers selected from tables J to N above that correspond to morning sleepiness, sleep onset and maintenance, dreaming, fatigue recovery, and sleep duration, which can be scored using the OSA-MA, can be used.

[0052] Saliva samples can be collected non-invasively from the subject, which is advantageous because it reduces the physical and / or mental burden on the subject. Saliva samples can be collected at any time of day. If the amount of a biomarker fluctuates throughout the day, collecting saliva samples at specific times can lead to a more accurate diagnosis.

[0053] The method of saliva collection is not particularly limited and can be carried out by methods known to those skilled in the art. Commercially available saliva collection kits can also be used.

[0054] Saliva samples may be frozen and stored after being collected from the subjects, or they may be prepared as needed. Pretreatment may be performed as desired, for example, by adjusting the concentration of an arbitrary internal standard substance in the saliva samples to be constant across all samples. Such adjustment may be performed by ultrafiltration or other methods.

[0055] The biomarkers included in the biomarker panel of the present invention are metabolites present in human saliva and are all substances disclosed in databases such as PubChem and HMDB (The Human Metabolome Database). The method for quantifying each biomarker is not particularly limited as long as it can quantify the biomarker, but can be performed, for example, by metabolome analysis. Metabolome analysis can be performed by CE-FTMS (capillary electrophoresis-Fourier transform mass spectrometry).

[0056] Furthermore, if the biomarker is a nucleic acid-related metabolite, it can be quantified using methods such as liquid chromatography-mass spectrometry (LC-MS) or high-performance liquid chromatography (HPLC).

[0057] Next, a reference value based on the amount of biomarker present in the control saliva sample is compared with the quantitative value of the biomarker in the saliva sample derived from the subject, and the significant difference between the reference value and the quantitative value of the biomarker is used as an indicator to evaluate the state of chronic stress. Here, the "control saliva sample" is a saliva sample collected from a healthy mammal of the same species as the subject, that is, a mammal that does not have a state of chronic stress. In particular, when the subject is human, it is preferable that the control saliva sample is a saliva sample derived from a person who has been judged not to be in a state of chronic stress by evaluation using POMS2 and / or OSA-MA.

[0058] The amount of biomarkers present in a control saliva sample can be quantified in the same way as the biomarkers present in the subject's saliva sample. This quantification can be performed when quantifying the biomarkers present in the subject's saliva sample to determine the reference value, or the reference value can be set in advance by quantifying the biomarkers present in the control saliva sample.

[0059] The assessment of chronic stress status is performed using the significant difference between the reference value and the quantitative value of biomarkers in the subject's saliva sample as an indicator. The significance test can be performed using methods known to those skilled in the art, such as Welch's t-test or Student's t-test. Significant significance is not particularly limited, but for example, a p-value of less than 0.05, less than 0.01, or less than 0.001 may be considered significant.

[0060] The presence or severity of chronic stress is determined by whether the amount of a biomarker in a saliva sample collected from the subject is significantly increased or significantly decreased compared to the reference value. Here, whether an increase or decrease relative to the reference value is considered to indicate chronic stress depends on the type of biomarker. In either case, if there is a significant difference between the reference value and the quantitative value of the subject, the subject can be evaluated as having and / or having a severe chronic stress state. Alternatively, by setting a reference value in advance, a diagnosis of chronic stress can be made if the amount of a biomarker in a saliva sample collected from the subject exceeds or falls below that reference value.

[0061] The biomarker panel used in the method for evaluating chronic stress states according to the present invention contains at least three substances as biomarkers. Therefore, by combining the measurement results of at least three biomarkers, a more accurate evaluation of chronic stress states can be performed compared to an evaluation method that measures only one biomarker. Furthermore, a risk score can be calculated based on the measurement results of at least three biomarkers included in the biomarker panel of the present invention, and the evaluation can be performed using that risk score.

[0062] 3. Kits for assessing chronic stress states A third embodiment of the present invention is a kit for evaluating chronic stress states using the biomarker panel of the present invention. Specifically, the kit includes reagents for quantifying biomarkers contained in the biomarker panel from a saliva sample derived from a test subject.

[0063] As described above in relation to the biomarker panel of the present invention, the biomarkers included in the biomarker panel of the present invention are at least three substances selected from the group consisting of metabolites listed in Table A above, which are metabolites present in human saliva. All of these substances are disclosed in databases such as PubChem and HMDB (The Human Metabolome Database), and information on their quantification methods is also known in the industry. Therefore, the reagents included in the kit of the present invention are not particularly limited as long as they are reagents that can be used to quantify any of the substances listed in Table A above.

[0064] 4. Methods for screening and / or evaluating substances effective in improving chronic stress conditions. A fourth embodiment of the present invention is a method for screening and / or evaluating substances effective in improving chronic stress conditions using the biomarker panel of the present invention. This method includes the steps of: obtaining quantitative value 1 by quantifying the biomarkers contained in the biomarker panel of the present invention in a saliva sample from a subject before administration of a candidate substance; obtaining quantitative value 2 by quantifying the biomarkers contained in the biomarker panel in a saliva sample from a subject after administration of the candidate substance; and comparing quantitative value 1 and quantitative value 2. The method screens and / or evaluates substances effective in improving chronic stress conditions using the significant difference between quantitative value 1 and quantitative value 2 as an indicator.

[0065] There are no particular limitations on the candidate substances to be screened and / or evaluated by the method of the present invention, but it is preferable that they be substances whose safety to the organism and / or human body of the subject being tested has been confirmed.

[0066] The subjects used in the method of the present invention are not particularly limited, as long as they are subjects capable of developing a chronic stress state. The subjects may be any mammal, such as pets like dogs and cats, livestock animals like cattle, horses, and pigs, laboratory animals like mice and rats, or humans. In particular, by using animal models of diseases associated with chronic stress states, such as depression models, or by using individuals judged to be in a chronic stress state based on evaluations using POMS2 and / or OSA-MA, it is possible to screen and / or evaluate substances that are effective in improving chronic stress states.

[0067] The biomarker panel used is not particularly limited, as long as it includes biomarkers selected from any of the tables A to N above. For example, when screening and / or evaluating substances effective in improving chronic stress states scored by the POMS2 questionnaire, biomarkers selected from tables B to I above that correspond to the Total Mood State Score (TMD), Tension-Anxiety (TA), Depression-Depression (DD), Anger-Hostility (AH), Vitality-Energy (VA), Fatigue-Apathy (FI), Confusion-Bewilderment (CB), and Friendliness (F), which can be scored by the POMS2 questionnaire, can be used. Also, when screening and / or evaluating substances effective in improving chronic stress states scored by the OSA-MA, biomarkers selected from tables J to N above that correspond to morning sleepiness, sleep onset and maintenance, dreaming, fatigue recovery, and sleep duration, which can be scored by the OSA-MA, can be used.

[0068] There are no particular limitations on the method of administering the candidate substance to the test subject, but administration methods such as oral, intravenous, or topical application can be used. If the test subject is an animal, the test substance can also be added to the feed or water and administered to the animal.

[0069] The method for collecting saliva from subjects and the method for quantifying biomarkers are as described in "2. Method for evaluating chronic stress states" above.

[0070] Furthermore, in the method of the present invention, a "saliva sample derived from the subject before administration of the candidate substance" is required to obtain quantitative value 1, and a "saliva sample derived from the subject after administration of the candidate substance" is required to obtain quantitative value 2. These two saliva samples may be collected from a single individual or from separate individuals.

[0071] An example of a specific procedure for carrying out the method of the present invention is described below. Saliva samples are collected from subjects before and after administration of the candidate substance. The quantitative value of the biomarker in the saliva sample before administration is designated as quantitative value 1, and the quantitative value of the biomarker in the saliva sample after administration is designated as quantitative value 2. Quantitative value 1 and quantitative value 2 are then compared. If a significant difference is found between quantitative value 1 before administration of the candidate substance and quantitative value 2 after administration of the candidate substance, the candidate substance can be screened and / or evaluated as a substance effective in improving chronic stress conditions.

[0072] Furthermore, multiple subjects are divided into two groups: one group receives no candidate substance, and the other group receives the candidate substance. Saliva samples are collected from each of the two groups, and the biomarkers included in the biomarker panel are quantified from the collected saliva samples. The biomarker quantification value for the group that did not receive the candidate substance is designated as Quantitative Value 1, and the biomarker quantification value for the group that received the candidate substance is designated as Quantitative Value 2. Next, Quantitative Value 1 and Quantitative Value 2 are compared. If, as a result of the comparison, there is a significant difference between Quantitative Value 1 for the group that did not receive the candidate substance and Quantitative Value 2 for the group that received the candidate substance, the candidate substance can be screened and / or evaluated as a substance effective in improving chronic stress conditions.

[0073] In particular, even when the subjects have a chronic stress state, it is possible to quantify biomarkers from saliva samples of healthy individuals who do not have a chronic stress state (for example, a group consisting of individuals judged not to be in a chronic stress state based on evaluation using POMS2 and / or OSA-MA) to obtain quantitative value 3, and then compare quantitative value 2 (the value after administration of the candidate substance) with quantitative value 3 to confirm that there is no significant difference. If there is no significant difference between quantitative value 2 and quantitative value 3, it can be confirmed that the candidate substance improved the chronic stress state to a healthy state.

[0074] Furthermore, while quantitative value 3 can be measured at the same time as quantitative values ​​1 and 2, it is also possible to quantify the biomarkers present in saliva samples from healthy control groups beforehand and set reference values.

[0075] A significant difference between quantitative value 1 and quantitative value 2, or between quantitative value 2 and quantitative value 3, can be determined by statistical tests known to those skilled in the art, such as Welch's t-test or Student's t-test. A significant difference is not particularly limited, but for example, a p-value less than 0.05, less than 0.01, or less than 0.001 may be considered significant.

[0076] Substances screened and / or evaluated as "substances effective in improving chronic stress states" by the method of the present invention may be used as active ingredients in pharmaceuticals, supplements, and health foods (including Foods for Specified Health Uses and Foods with Function Claims) for improving chronic stress states, or as feed additives. In particular, substances in which the test subjects were sleep disorder model mice or subjects judged to be in a chronic stress state by POMS2, and a significant difference was observed between quantitative value 1 and quantitative value 2, may be candidate substances for improving chronic stress states or for treating diseases known to be associated with chronic stress states, such as depression, anxiety disorders, bipolar disorder, sleep disorders, and chronic fatigue syndrome.

[0077] 5. Programs for assessing chronic stress states A fifth embodiment of the present invention is a program for evaluating a chronic stress state, which is a program for causing a computer to perform the following steps (1) and (2). (1) A procedure for calculating whether there is a significant difference between the measurement data from the subject and the reference value, based on measurement data including quantitative values ​​of biomarkers included in the biomarker panel of the present invention in a saliva sample from the subject, and a reference value based on the amount of biomarkers present in a control saliva sample. (2) A procedure for displaying an evaluation result using the significant difference as an indicator when there is a significant difference between the calculated measurement data and the reference value.

[0078] The method for calculating whether there is a significant difference between the measurement data including the quantitative value of the biomarker, the reference value based on the amount of biomarker present in the control saliva sample, and the measurement data from the subject and the reference value in procedure (1) above, as well as the method for obtaining the evaluation result using the significant difference as an indicator in procedure (2), are as described in "2. Method for evaluating chronic stress state" above.

[0079] The program of the present invention may further include a step for inputting measurement data.

[0080] The program of the present invention can be implemented on a computer by methods known to those skilled in the art. For example, it can be implemented on a computer via a computer-readable recording medium on which the program of the present invention is stored.

[0081] The program of the present invention can be used in the above-mentioned "2. Method for evaluating chronic stress states" and "4. Method for screening and / or evaluating substances effective in improving chronic stress states." [Examples]

[0082] The present invention will be described below using examples, but the present invention is not limited to these examples.

[0083] The following trials are registered in the clinical trial registry system. Registration body: UMIN-CTR Registration number: UMIN000053021

[0084] Participants in the study were selected based on the following criteria. i. Japanese ii. Male iii. Middle-aged and older adults (45 years old and over, under 60 years old) iv. Healthy individuals v. Persons working consecutive shifts (5 consecutive days) with a full two-day weekend system. vi. Of the persons described in i. to v. above, those who meet either condition a. or b. below 365 people each, 730 people in total a. Those who have trouble with the quality of their sleep b. Those who do not have problems with sleep quality vii. Of those listed in i. to vi. above, those who underwent the screening in Pittsburgh during the screening period. Based on the results of the Japanese version of the Sleep Quality Index (PSQI), either a. or b. below 50 people each who meet the conditions, for a total of 100 people. a. Individuals with a PSQI score (PSQIG) of 2 or less (good group) b. Individuals with a PSQI score (PSQIG) of 6 or higher (bad group)

[0085] Furthermore, individuals who met the following exclusion criteria were excluded from participating in the study. i. Individuals currently receiving treatment for or with a history of malignant tumors, heart failure, or myocardial infarction. ii. Individuals with implanted pacemakers or cardioverter-defibrillators. iii. Individuals currently receiving treatment for any of the following chronic diseases arrhythmia, liver damage, chronic kidney disease, cerebrovascular disease, rheumatic disease, diabetes, Dyslipidemia, hypertension, and other chronic diseases iv. Persons who are taking Foods for Specified Health Uses or Foods with Function Claims v. Persons taking pharmaceuticals (including herbal medicines) or supplements. vi. Individuals with allergies (to medications) vii. Pregnant, breastfeeding, or intending to become pregnant during the examination period. viii. Individuals who participated in other clinical trials within the 28 days prior to the date of obtaining consent. or those who plan to participate during the exam period ix. Any other person whom the principal investigator deems unsuitable to be included in this study.

[0086] Each participant was asked to complete the OSA-MA (OSA Sleep Questionnaire MA version) and their sleep reflection upon waking was evaluated. The survey conditions and content are as follows: Implementation details: Each participant will visit the hospital on their first day off after a series of consecutive workdays. Patients were asked to complete the OSA-MA upon waking up on the day of their visit. Survey items: Morning drowsiness, falling asleep and maintaining sleep, dreaming, fatigue recovery, sleep duration, and individual questions. Study period: This observation period Evaluation details: Measured values Evaluation method: Answers to each question item will be scored to measure morning drowsiness, sleep onset and maintenance, and dreaming. We calculated scores for fatigue recovery and sleep duration.

[0087] Furthermore, each participant was asked to complete the Japanese version of POMS2 (Profile of Mood States 2nd Edition) to evaluate their mood state at the time. The survey conditions and content are as follows: Implementation details: Each participant will visit the clinic on the morning of their first day off after a series of working days, as described below. After collecting saliva, the Japanese version of POMS2 was used to assess the mood state at the time. I received feedback. Survey items: Total Mood State (TMD) score, Tension-Anxiety (TA), Depression - Depression (DD), Anger - Hostility (AH), Vigor - Energy (VA), Fatigue-Apathy (FI), Confusion-Confusion (CB), Friendliness (F), each questionnaire item Study period: This observation period Evaluation details: Measured values Evaluation method: Answers to each question item will be scored, and TA, DD, AH, VA, FI, CB will be assigned. Calculate the F and TMD scores.

[0088] Furthermore, saliva tests were conducted for each subject. Specifically, saliva was collected from subjects who visited the clinic on the morning of their first day off after a series of consecutive workdays. Saliva was collected using a saliva collection tube (LSI Medience, Japan) according to the prescribed procedure. The collected saliva samples were stored in a -80°C freezer until shipment. The analysis was outsourced to Human Metabolome Technologies, Inc., where metabolome analysis (ωScan 100 samples + 353 substances quantified) was performed using CE-FTMS (capillary electrophoresis-Fourier transform mass spectrometry). When sending the saliva samples to Human Metabolome Technologies, Inc., they were packaged with dry ice and shipped frozen.

[0089] Metabolome analysis detected 683 substances in saliva. Of these, 435 substances were selected as the metabolites for analysis, as they were detected in more than 50% of subjects in either the good group (PSQI score of 2 or less, N=50) or the bad group (PSQI score of 6 or more, N=50). For these 435 substances, the relative area was calculated according to the following formula. Relative area value = Relative area value of candidate compound sheet × Sample amount (mg)

[0090] Data Analysis 1 We analyzed the relationship between human salivary metabolome data and mood state assessment results using POMS2. Specifically, for each mood state scored on the POMS2 questionnaire—namely, Total Mood State (TMD), Tension-Anxiety (TA), Depression-Depression (DD), Anger-Hostility (AH), Vitality-Vitality (VA), Fatigue-Apathy (FI), Confusion-Bewilderment (CB), and Friendliness (F)—participants were divided into two groups: the top 10 and the bottom 10. Using the relative area values ​​of the 435 substances mentioned above, we compared the differences between the two groups for each mood state using t-tests, and calculated the magnification change and p-value. Metabolites showing a significant difference (p<0.05) were selected and summarized in Tables B-1 to I-1 below. In each table, two or more substances listed on the same line are considered to be the same substance.

[0091] [Table 15]

[0092] [Table 16]

[0093] [Table 17-1] [Table 17-2]

[0094] [Table 18]

[0095] [Table 19]

[0096] [Table 20]

[0097] [Table 21]

[0098] [Table 22-1] [Table 22-2]

[0099] By measuring the concentrations of each metabolite listed in Tables B-1 to I-1 above in saliva, it is possible to evaluate each mood state that can be assessed using POMS2. Therefore, the metabolites listed in Tables B-1 to I-1 above are biomarkers that can be used to evaluate various mood states, and a biomarker panel containing three or more of these is considered effective for evaluating mood states using saliva.

[0100] Data Analysis 2 We analyzed the correlation between human salivary metabolome data and the evaluation results of the OSA-MA. Specifically, we performed a correlation analysis using Spearman's rank correlation between the five items scored by the OSA-MA (morning sleepiness, sleep onset and maintenance, dreaming, fatigue recovery, and sleep duration) and the concentrations of the 435 substances mentioned above. Spearman's rank correlation was performed without substituting missing values ​​(missing values ​​were treated as they were), and the Spearman's rank correlation coefficient and p-value were calculated. Metabolites showing significant correlation (p<0.05) were selected and summarized in Tables J-1 to N-1 below. In each table, two or more substances listed on the same line are considered to be the same substance.

[0101] [Table 23-1] [Table 23-2]

[0102] [Table 24]

[0103] [Table 25]

[0104] [Table 26]

[0105] [Table 27]

[0106] The metabolites listed in Tables J-1 to N-1 above can be evaluated using OSA-MA by measuring their respective concentrations in saliva, thereby enabling assessment of five items: morning sleepiness, sleep onset and maintenance, dreaming, fatigue recovery, and sleep duration. Therefore, the metabolites listed in Tables J-1 to N-1 above are biomarkers that can be used to evaluate various mood states, and a biomarker panel containing three or more of these metabolites can objectively demonstrate each item of OSA-MA.

[0107] summary Based on the data analysis 1 and data analysis 2 described above, the substances considered effective as biomarkers (i.e., the materials listed in Tables B-1 to N-1) were compiled to obtain Table A below.

[0108] [Table 28-1] [Table 28-2] [Table 28-3] [Table 28-4] [Table 28-5] [Table 28-6]

[0109] The substances listed in Table A above are all effective biomarkers for evaluating chronic stress states using saliva. Compared to measuring a single biomarker, measuring multiple effective biomarkers can improve the accuracy of the assessment. Therefore, a biomarker panel containing at least three substances selected from this group of substances is considered effective for objectively examining chronic stress states using saliva.

[0110] Furthermore, the substances listed in italics and underline in Table A above are all nucleic acid-related metabolites. Therefore, by constructing a biomarker panel that includes these nucleic acid-related metabolites as biomarkers, it is thought that it will be possible to evaluate chronic stress states by measuring nucleic acid-related metabolites in saliva. [Industrial applicability]

[0111] This invention provides a biomarker panel for objectively examining chronic stress states in humans using saliva. By using the biomarker panel of this invention, it becomes possible to create a method and kit for evaluating chronic stress states, a method for screening and / or evaluating substances effective in improving chronic stress states, and an evaluation program.

Claims

1. A biomarker panel for evaluating chronic stress states using saliva, A biomarker panel comprising at least three substances selected from the group consisting of metabolites listed in Table A below, as biomarkers. Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6

2. The biomarker panel according to claim 1, comprising at least three substances selected as biomarkers from the group consisting of metabolites selected from one of the tables B to N below. Table 2 Table 3 Table 4-1 Table 4-2 Table 5 Table 6 Table 7 Table 8 Table 9-1 Table 9-2 Table 10 Table 11 Table 12 Table 13 Table 14

3. The biomarker panel according to claim 2, wherein the chronic stress state is a total mood state (TMD) as defined in POMS2 (Profile of Mood States 2nd Edition), and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table B.

4. The biomarker panel according to claim 2, wherein the chronic stress state is anger / hostility (AH) as defined in POMS2, and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table C.

5. The biomarker panel according to claim 2, wherein the chronic stress state is confusion / embarrassment (CB) as defined in POMS2, and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table D.

6. The biomarker panel according to claim 2, wherein the chronic stress state is depression (DD) as defined in POMS2, and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table E.

7. The biomarker panel according to claim 2, wherein the chronic stress state is fatigue / apathy (FI) as defined in POMS2, and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table F.

8. The biomarker panel according to claim 2, wherein the chronic stress state is tension / anxiety (TA) as defined in POMS2, and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table G.

9. The biomarker panel according to claim 2, wherein the chronic stress state is vitality / vitality (VA) as defined in POMS2, and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table H.

10. The biomarker panel according to claim 2, wherein the chronic stress state is friendly (F) as defined in POMS2, and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table I.

11. The biomarker panel according to claim 2, wherein the chronic stress state is morning sleepiness, which is the first factor of the OSA Sleep Questionnaire MA version (OSA-MA), and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table J.

12. The biomarker panel according to claim 2, wherein the chronic stress state is sleep onset and sleep maintenance, which are the second factor of OSA-MA, and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table K.

13. The biomarker panel according to claim 2, wherein the chronic stress state is the third factor of OSA-MA, and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table L.

14. The biomarker panel according to claim 2, wherein the chronic stress state is fatigue recovery, which is the fourth factor of OSA-MA, and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table M.

15. The biomarker panel according to claim 2, wherein the chronic stress state is sleep duration, which is the fifth factor of OSA-MA, and the biomarker panel includes at least three substances selected from the group consisting of metabolites listed in Table N.

16. The biomarker panel according to any one of claims 1 to 15, wherein at least one of the biomarkers is a nucleic acid-related metabolite.

17. A biomarker panel according to any one of claims 1 to 15, comprising at least four substances as biomarkers.

18. A method for evaluating a chronic stress state using a biomarker panel according to any one of claims 1 to 15, A step of quantifying the biomarkers included in the biomarker panel from a saliva sample derived from the subject, A step of comparing a reference value based on the amount of the biomarker present in a control saliva sample with a quantitative value of the biomarker in a saliva sample derived from the subject being tested. A method for evaluating a chronic stress state, including the above-mentioned reference value and using the significant difference between the quantitative value of the biomarker as an indicator.

19. The method according to claim 18, wherein the control saliva sample is a saliva sample derived from a person who is judged not to be in a chronic stress state by evaluation using POMS2 and / or OSA-MA.

20. A kit for evaluating a chronic stress state using a biomarker panel according to any one of claims 1 to 15, Reagents for quantifying biomarkers included in the biomarker panel from saliva samples derived from the test subject A kit that includes this.

21. A method for screening and / or evaluating substances effective in improving chronic stress conditions using a biomarker panel according to any one of claims 1 to 15, A step of quantifying the biomarkers contained in the biomarker panel in a saliva sample derived from a subject before administration of the candidate substance, and obtaining a quantitative value 1, The process involves quantifying the biomarkers contained in the biomarker panel in a saliva sample derived from a subject after administration of a candidate substance, and obtaining a quantitative value 2. A step of comparing the quantitative value 1 with the quantitative value 2. A method for screening and / or evaluating substances that are effective in improving chronic stress conditions, including the above, using the significant difference between quantitative value 1 and quantitative value 2 as an indicator.

22. The method according to claim 21, wherein the subject being tested is determined to be in a state of chronic stress based on evaluation using POMS2 and / or OSA-MA.

23. The method according to claim 21, wherein the substance is a therapeutic agent for a chronic stress state.

24. This is a program for evaluating chronic stress states. A program to instruct the computer to perform the following steps (1) and (2): (1) A procedure for calculating whether there is a significant difference between the measurement data from the subject and the reference value, based on measurement data including quantitative values ​​of biomarkers contained in the biomarker panel described in any one of claims 1 to 15 in a saliva sample derived from the subject, and a reference value based on the amount of the biomarker present in a control saliva sample. (2) A procedure for displaying an evaluation result using the significant difference as an indicator when there is a significant difference between the measurement data and the reference value as a result of the calculation described above.

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