Method for testing mild cognitive impairment, reagent for testing mild cognitive impairment, and method for screening candidate substance for therapeutic agent for mild cognitive impairment

Abstract

The present disclosure provides a test method capable of detecting MCI and a measurement reagent therefor. The method for testing MCI according to the present disclosure includes a measurement step in which the ability to eliminate hydroxyl radicals in a biological sample of a subject is measured.

Description

Technical Field

[0001] The present disclosure relates to a test method for mild cognitive impairment, a test reagent for mild cognitive impairment, and a method for screening candidate substances for treating mild cognitive impairment. Background Art

[0002] As society ages, the number of patients with cognitive impairment such as dementia and Alzheimer's disease or mild cognitive impairment, which is the pre-stage of the disease, is increasing. These cognitive impairments are diagnosed by doctors' interviews based on diagnostic criteria for each disease (Non-Patent Document 1).

[0003] Prior art literature

[0004] Non-patent literature

[0005] Non-patent literature 1: Japan Geriatrics Society, "Cognitive Function Evaluation Method and Dementia Diagnosis", [online], Japan Geriatrics Society, [Reiwa June 30, 2019], Internet<URL:https: / / www.jpn-geriat-soc.or.jp / tool / tool_02.html> Summary of the invention

[0006] Problems to be solved by the invention

[0007] Cognitive impairment can be prevented from developing by intervening in the early stages of cognitive impairment, namely, mild cognitive impairment (MCI). Therefore, a test method capable of detecting MCI is needed.

[0008] Therefore, an object of the present disclosure is to provide a test method capable of detecting MCI and a measuring reagent therefor.

[0009] Solution to the problem

[0010] In order to achieve the above-mentioned object, the test method for mild cognitive impairment (MCI) disclosed in the present invention (hereinafter also referred to as "test method") includes: a measuring step in which the ability to eliminate hydroxyl radicals in a biological sample of a subject is measured.

[0011] The test reagent for mild cognitive impairment of the present disclosure (hereinafter also referred to as “test reagent”) includes a measuring reagent for the ability to eliminate hydroxyl radicals.

[0012] The method for screening candidate substances for treating mild cognitive impairment disclosed herein (hereinafter referred to as "screening method") comprises: a selection step in which an activated substance that reduces the ability to eliminate hydroxyl radicals is selected from the test substances as a candidate substance for treating mild cognitive impairment.

[0013] The detection method disclosed herein is a method for detecting the scavenging ability of hydroxyl free radicals in a subject suspected of having mild cognitive impairment, and the detection method comprises:

[0014] A detection step in which the hydroxyl radical-eliminating ability in the biological sample of the subject is detected using a measuring reagent for the hydroxyl radical-eliminating ability.

[0015] Effects of the Invention

[0016] According to the present disclosure, whether a subject has MCI can be detected by using a biological sample from the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram showing a DMPO—OH spin adduct obtained by the ESR method. DETAILED DESCRIPTION

[0018] In the present specification, the "test for MCI" means, for example, detection of MCI, determination of MCI, determination of the preventive effect of MCI, determination of the therapeutic effect of MCI, determination of MCI patients for whom therapeutic drugs are effective, determination of therapeutic drugs that are effective for each MCI patient, a test method for diagnosing MCI, or a test for treating MCI, etc. The "determination of MCI" may be, for example, determination, test, detection or diagnosis of the presence or absence of MCI, determination, test, detection or diagnosis of the possibility (risk) of suffering from MCI, prediction of the prognosis after MCI treatment, or determination of the therapeutic effect of MCI therapeutic drugs, and may be replaced by any of them.

[0019] In the present specification, the term "suffering from" may mean, for example, a state of being suffering from the disease or the onset of the disease.

[0020] In this specification, "treatment" means therapeutic treatment and / or preventive treatment. In this specification, "treatment" refers to the treatment, cure, prevention, inhibition, relief, improvement of a disease, condition or disorder, or the cessation, inhibition, reduction or delay of the progression of a disease, condition or disorder. In this specification, "prevention" means the reduction of the possibility of the onset of a disease or condition, or the delay of the onset of a disease or condition. The "treatment" can be, for example, the treatment of a patient with a target disease, or the treatment of a model animal of a target disease.

[0021] In this specification, "mild cognitive impairment" means a disease classified as ICD code F06.7 (mild cognitive impairment) in the 11th edition of the International Classification of Diseases (ICD11), for example. The MCI can be evaluated using MoCA-J (Japanese version of MoCA), for example, and can be evaluated as MCI when the score is 25 points or less. In this specification, MCI can also be evaluated in combination with other evaluation methods, for example. As other evaluation methods, for example, Japanese version of Trail Making Test (TMT-J), Clinical Dementia Rating (CDR), Functional Assessment Staging (FAST), Modified Hasegawa Dementia Scale (Hasegawa's Dementia Scale-Revised, HDS-R), Mini-Mental State Examination (MMSE), DASC-21 (Dementia Assessment Sheet in Community-based Integrated Care System), etc. can be listed.

[0022] In the present specification, the term "hydroxyl radical" refers to a radical corresponding to a hydroxyl group. The hydroxyl radical has particularly high reactivity among active oxygen species.

[0023] In this specification, "antioxidant" refers to a substance that captures active oxygen species. Examples of the active oxygen species include free radicals, singlet oxygen ( 1 O2), ozone (O3), hydrogen peroxide (H2O2) and other non-free radical clusters.

[0024] In this specification, "the ability to eliminate hydroxyl radicals" refers to the ability of a test substance to capture hydroxyl radicals.

[0025] <MCI markers>

[0026] On the one hand, the present disclosure discloses a marker as an indicator of mild cognitive impairment (MCI). The marker of MCI disclosed herein is the ability to eliminate hydroxyl radicals. The marker of mild cognitive impairment disclosed herein is characterized by the ability to eliminate hydroxyl radicals, and other structures and conditions are not particularly limited.

[0027] As a result of in-depth research by the inventors, it was found that the ability to eliminate hydroxyl radicals in a living body, especially the ability to eliminate hydroxyl radicals in saliva, is related to the onset of MCI, thereby establishing the present disclosure. According to the present disclosure, the possibility of a subject suffering from MCI (risk of suffering from MCI) can be tested by measuring the ability to eliminate hydroxyl radicals. In addition, in the present disclosure, since hydroxyl radicals become the target of MCI, candidate substances for the treatment of MCI can also be obtained by screening using the target. Therefore, the present disclosure is extremely useful in the clinical field and the field of biochemistry.

[0028] The ability to eliminate hydroxyl radicals can be used as a marker for MCI, and can be particularly suitable for use as a marker for MCI caused by Down syndrome and a marker for MCI caused by amyotrophic lateral sclerosis (ALS).

[0029] It is speculated that the ability to eliminate hydroxyl radicals in the organism is caused by the antioxidant contained in the organism. Therefore, in this specification, as a marker of MCI, the antioxidant can be used in addition to the ability to eliminate hydroxyl radicals, or the antioxidant can be used instead of the ability to eliminate hydroxyl radicals.

[0030] As described later, the MCI marker can also be used as a marker for diagnosing or detecting MCI, a marker for predicting the prognosis of MCI, or a marker for predicting or determining the therapeutic effect of MCI.

[0031] <MCI testing method>

[0032] On the other hand, the present specification discloses a method for testing the possibility of MCI. As described above, the MCI testing method disclosed herein includes: a measurement step, in which the ability to eliminate hydroxyl radicals in a biological sample of a subject is measured. The testing method disclosed herein is characterized in that the ability to eliminate hydroxyl radicals in a biological sample of the subject as an MCI marker is measured, and other steps and conditions are not particularly limited. For the testing method disclosed herein, the description of the marker of MCI disclosed herein can be cited.

[0033] According to the test method disclosed in the present invention, a test for the possibility of MCI can be implemented, for example. The "MCI test" means, for example, the detection of MCI, the determination of MCI, the screening of MCI, the determination of the preventive effect of MCI, the determination of the therapeutic effect of MCI, the determination of MCI patients for whom therapeutic drugs are effective, the determination of therapeutic drugs that are effective for each MCI patient, the inspection method for diagnosing MCI, or the inspection for the treatment of MCI. The "determination of MCI" can be, for example, the determination, test, detection or diagnosis of the presence or absence of MCI, the determination, test, detection or diagnosis of the possibility (risk) of suffering from MCI, the prediction of the prognosis after MCI treatment, or the determination of the therapeutic effect of MCI therapeutic drugs.

[0034] The subjects may include, for example, humans, non-human animals other than humans, and the like. As mentioned above, the non-human animals may include, for example, mammals such as mice, rats, dogs, monkeys, rabbits, sheep, horses, birds, fish, and the like.

[0035] There is no particular limitation on the type of the biological sample, and for example, body fluids, body fluid-derived cells, organs, tissues or cells separated from an organism can be listed. The body fluids can include, for example, blood, saliva, urine, lymph, synovial fluid of joints, bone marrow fluid, cerebrospinal fluid and other spinal fluids. As specific examples of the blood, for example, whole blood, serum, plasma and the like can be listed. The body fluid-derived cells can include, for example, blood-derived cells, specifically, blood cells such as red blood cells, white blood cells, and lymphocytes. In addition, based on the marker of the MCI, for example, the MCI of the subject can be tested by the ability to eliminate hydroxyl radicals in the saliva. Therefore, for example, since the burden on patients or doctors can be reduced, the biological sample is preferably saliva.

[0036] For the elimination ability of hydroxyl radicals, for example, it can be implemented by measuring the elimination ability of hydroxyl radicals on the biological sample, or by measuring the elimination ability of hydroxyl radicals on the obtained antioxidant after extracting, crudely purifying or purifying the antioxidant from the biological sample. The measuring method of the elimination ability of hydroxyl radicals of antioxidants is not particularly limited, and a known method can be used. As a specific example, the measuring method of the elimination ability of hydroxyl radicals of the antioxidants can be listed as follows: for example, using divalent iron and H2O2 as generators of hydroxyl radicals and using electron spin resonance (Electron spin resonance, ESR) spin trapping method, 3-methylthiopropionaldehyde (methional) method, para-nitrosodimethylaniline (PNDMA) method, salicylate method, thiobarbituric acid (TBA) method, method using hydrogen atom transfer, etc., because it can suppress the influence of other active oxygen species such as hydrogen peroxide and singlet oxygen, it can specifically detect hydroxyl radicals, and the sensitivity is good, so the ESR spin trapping method is preferred. The measurement of the ability to eliminate hydroxyl radicals may be a qualitative measurement or a quantitative measurement. In the case where the measurement of the ability to eliminate hydroxyl radicals is a quantitative measurement, the ability to eliminate hydroxyl radicals may also be referred to as a measured value of the ability to eliminate hydroxyl radicals.

[0037] The test method disclosed in the present invention, for example, also includes a test step, in which the possibility of the subject suffering from MCI is tested by comparing the measured value of the ability to eliminate hydroxyl radicals in the biological sample of the subject (hereinafter also referred to as the "tested biological sample") with a threshold. The threshold is not particularly limited, for example, a threshold calculated from the measured value of the ability to eliminate hydroxyl radicals in the biological samples of MCI non-suffering patients, MCI patients, or from the MCI non-suffering patients and the MCI patients. In the case of prognostic evaluation, the threshold may also be, for example, the measured value of the ability to eliminate hydroxyl radicals before or after treatment (for example, just after treatment) of the same subject. In addition, in the present disclosure, "MCI non-suffering patients" may also be replaced by "healthy people". However, in the present disclosure, "MCI non-suffering patients" or "healthy people" refer to individuals who do not suffer from MCI, regardless of whether they suffer from diseases other than MCI. That is, in the present disclosure, "MCI non-suffering patients" or "healthy people" include individuals suffering from diseases other than MCI, and also include individuals who do not suffer from diseases other than MCI.

[0038] The threshold value can be obtained, for example, using a biological sample separated from a non-MCI patient and / or a MCI patient as described above (hereinafter also referred to as a "reference biological sample"). In addition, for the threshold value, for example, after separating the reference biological sample from a plurality of non-MCI patients and a plurality of MCI patients, the ability to eliminate hydroxyl radicals can be measured, and statistical calculations can be performed based on the obtained ability to eliminate hydroxyl radicals. In this case, as the threshold value, for example, clinical diagnostic values ​​such as diagnostic thresholds (cutoff values), treatment thresholds, and preventive medicine thresholds can be used. In addition, in the case of prognostic evaluation, for example, a reference biological sample separated from the same subject after treatment can also be used. The threshold value can be measured simultaneously with the subject's tested biological sample, or it can be measured in advance. In the latter case, for example, it is preferred because it is not necessary to obtain the threshold value each time the subject's tested biological sample is measured. Preferably, the subject's tested biological sample and the reference biological sample are collected, for example, under the same conditions, and the ability to eliminate hydroxyl radicals is measured under the same conditions.

[0039] In the test step, the evaluation method of the possibility of the subject suffering from MCI is not particularly limited and can be appropriately determined according to the type of the threshold. As a specific example, when the measured value of the ability to eliminate hydroxyl radicals in the tested biological sample of the subject is the same as the measured value of the ability to eliminate hydroxyl radicals in the baseline biological sample of the MCI non-suffering patient (when there is no significant difference), when it is significantly higher than the measured value of the ability to eliminate hydroxyl radicals in the baseline biological sample of the MCI non-suffering patient, when it is significantly higher than the measured value of the ability to eliminate hydroxyl radicals in the baseline biological sample of the MCI patient, and / or when it is higher than the threshold calculated from the measured values ​​of the ability to eliminate hydroxyl radicals in the biological samples of the MCI non-suffering patient and the MCI patient, the subject can be evaluated as having no possibility of suffering from MCI (also referred to as "risk" or "risk degree", the same below) or having a low possibility of suffering from MCI. In addition, the subject can be evaluated as having a possibility of suffering from MCI or having a high possibility (risk) of suffering from MCI when the measured value of the hydroxyl radical elimination ability in the tested biological sample of the subject is significantly lower than the measured value of the hydroxyl radical elimination ability in the baseline biological sample of the MCI non-suffering patient, when the measured value is the same as the measured value of the hydroxyl radical elimination ability in the baseline biological sample of the MCI patient (when there is no significant difference), when the measured value is significantly lower than the measured value of the hydroxyl radical elimination ability in the baseline biological sample of the MCI patient, and / or when the measured value is lower than the threshold calculated from the measured values ​​of the free radical scavenging ability in the biological samples of the MCI non-suffering patient and the MCI patient. In addition, in the testing step, the degree of MCI can be evaluated by comparing the measured value of the hydroxyl radical elimination ability in the tested biological sample of the subject with the measured value of the hydroxyl radical elimination ability in the baseline biological sample of MCI patients of each degree of MCI. Specifically, when the biological sample of the subject has a measured value of hydroxyl radical elimination ability that is the same as that of the baseline biological sample of MCI (without significant difference), the subject can be evaluated as having a possibility of having the said level of MCI or having a high possibility of having the said level of MCI.

[0040] In the test step, when evaluating the state of the prognosis, for example, the evaluation can be performed in the same manner as described above, or the measured value of the ability to eliminate hydroxyl radicals in the baseline biological sample after treatment of the same subject can be used as a threshold for evaluation. As a specific example, when the measured value of the ability to eliminate hydroxyl radicals in the subject's tested biological sample is the same as the threshold (when there is no significant difference), and / or when it is significantly lower than the threshold, the subject can be evaluated as having a possibility of recurrence or deterioration after the treatment, or a high possibility of recurrence or deterioration. In addition, when the measured value of the ability to eliminate hydroxyl radicals in the subject's tested biological sample is significantly higher than the threshold, the subject can be evaluated as having no possibility of recurrence or deterioration after the treatment, or a low possibility of recurrence or deterioration.

[0041] In the test step, for example, biological samples of the same subject may be collected over time, and the measured values ​​of the ability to eliminate hydroxyl radicals in the biological samples may be compared. Thus, in the test step, for example, if the measured value increases over time, it can be determined that the possibility of suffering from the disease is decreasing or cured, and if the measured value decreases over time, it can be determined that the possibility of suffering from the disease is increasing.

[0042] The test method disclosed herein may, for example, also treat the subject based on the result of the test step. Specifically, the test method disclosed herein may, for example, also include an administration step, in which a therapeutic drug for MCI is administered to the subject who obtains a test result of suffering from MCI in the test step. The conditions such as the administration method, administration period, administration amount, and administration interval of the therapeutic drug for MCI may be appropriately set according to the type of the therapeutic drug for MCI. The therapeutic drug for MCI may also be a candidate therapeutic drug substance obtained by the screening method described later.

[0043] In the test method disclosed herein, although the ability to eliminate the hydroxyl radical as an indicator (marker) of the MCI is used, in the test method disclosed herein, the antioxidant may be used in addition to the ability to eliminate the hydroxyl radical, or the antioxidant may be used to replace the ability to eliminate the hydroxyl radical. In this case, in the test method disclosed herein, the "ability to eliminate the hydroxyl radical" may be interpreted as "antioxidant", and the "measured value of the ability to eliminate the hydroxyl radical" may be interpreted as "the amount of the antioxidant", and the description thereof may be cited.

[0044] The subject is not particularly limited, and may be, for example, an MCI patient, an individual whose MCI is unknown, or an individual suspected of having MCI.

[0045] As described above, the test method disclosed herein can be used, for example, as a method for diagnosing or detecting MCI, a method for predicting the prognosis of MCI, or a method for predicting or determining the effect of MCI treatment. Therefore, the test method disclosed herein can also be used as a method for selecting patients (responders) who respond to MCI therapeutic drugs, and as an auxiliary diagnosis method for adjusting the dosage of MCI therapeutic drugs.

[0046] <Test Reagent>

[0047] On the other hand, the present specification discloses a test reagent capable of testing the possibility of MCI. As described above, the test reagent of the present disclosure includes a measuring reagent for the ability to eliminate hydroxyl radicals. The test reagent of the present disclosure is characterized in that it includes a measuring reagent for the ability to eliminate hydroxyl radicals, and other structures and conditions are not particularly limited. According to the test reagent of the present disclosure, the test method of the present disclosure can be easily implemented. For the test reagent of the present disclosure, the description of the markers and test methods of MCI of the present disclosure can be cited.

[0048] The measuring reagent of the elimination ability of the hydroxyl radical can be appropriately determined according to the measuring method of the elimination ability of the hydroxyl radical, for example. As a specific example, when the elimination ability of the hydroxyl radical is measured by the ESR method, the measuring reagent of the elimination ability of the hydroxyl radical can list a generator of hydroxyl radicals and a detector of hydroxyl radicals. The generator of the hydroxyl radical can list, for example, a combination of divalent iron and H2O2, etc. Examples of the hydroxyl radical detector include 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), and 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline-N-oxide (CYPMPO).

[0049] As described above, the test reagent of the present disclosure can easily implement the test method of the present disclosure. Therefore, the test reagent of the present disclosure is preferably used in the test method of the present disclosure.

[0050] The present disclosure may also be, for example, an application of a reagent for measuring the ability of eliminating hydroxyl radicals to test the possibility of MCI.

[0051] The reagents may be mixed or part or all of them may be separately configured. In the latter case, the test reagent of the present disclosure may also be referred to as a test kit.

[0052] The test reagent disclosed in the present invention may also include other structures, such as pre-treatment reagents for biological samples, instructions for use, and the like.

[0053] The test reagent disclosed in the present invention may also include a measuring reagent for an antioxidant to replace the measuring reagent for the ability to eliminate the hydroxyl radical. The measuring reagent for the antioxidant can be appropriately determined, for example, according to the type of the antioxidant. In the case where the antioxidant is a protein, the test reagent can be, for example, a measuring reagent for the antioxidant protein, or a measuring reagent for the mRNA encoding the antioxidant protein. As a measuring reagent for the antioxidant protein, for example, antibodies against antioxidants can be used. In the case where the antioxidant is a nucleic acid, for the measuring reagent for the mRNA encoding the antioxidant protein, for example, reverse transcriptase, deoxynucleotide triphosphate (dNTP), polymerase, primers, etc. can be listed. For the measuring reagent of the amount of the antioxidant, for example, in the oxygen radical absorbance capacity (ORAC) method, high-purity Trolox standards as standard substances can be listed.

[0054] <MCI diagnosis method and diagnostic reagent>

[0055] On the other hand, the present specification discloses a method for diagnosing the possibility of MCI and a diagnostic reagent. The diagnostic method of MCI disclosed herein includes a step of measuring the ability to eliminate hydroxyl radicals in a biological sample of a subject. In addition, the diagnostic reagent of MCI disclosed herein includes a measuring reagent for the ability to eliminate hydroxyl radicals. In addition, for the present disclosure, the description of the test method and test reagent disclosed herein can be cited.

[0056] <Screening method>

[0057] On the other hand, the present specification discloses a method for screening candidate therapeutic drug substances for MCI. As described above, the screening method for candidate therapeutic drug substances for MCI disclosed in the present invention includes a selection step, in which an active substance that improves the ability to eliminate hydroxyl radicals is selected from the test substance as a candidate therapeutic drug substance for MCI. The present disclosure is characterized in that the target of the candidate therapeutic drug substance for MCI is the ability to eliminate hydroxyl radicals, and other steps and conditions are not particularly limited. For the screening method disclosed in the present invention, the description of the markers, test methods and test reagents for MCI disclosed in the present invention can be cited.

[0058] The test substance may include, for example, low molecular weight compounds, peptides, proteins, nucleic acids, etc. For the low molecular weight compounds, for example, a library of known low molecular weight compounds may be included. The peptide may be, for example, a linear, branched or cyclic peptide, and each amino acid constituting the peptide may be a natural amino acid, a modified amino acid, an artificial amino acid or a combination thereof. The protein may be any one of a natural protein or an artificial protein. For the protein, for example, an antibody, a growth factor, a proliferation factor, or variants thereof may be included.

[0059] The selection step includes, for example, a measurement step, in which the test substance is made to coexist in a hydroxyl radical generating system to measure the ability to eliminate hydroxyl radicals; and a selection step, in which, when the ability to eliminate hydroxyl radicals obtained in the measurement step is higher than that of a control coexistence system in which the test substance does not coexist, the test substance is selected as the candidate therapeutic drug. In the measurement step, for example, the description of the method for measuring the ability to eliminate hydroxyl radicals can be cited for the measurement of the ability to eliminate hydroxyl radicals. The hydroxyl radical generating system includes, for example, cells derived from MCI patients.

[0060] <Detection method>

[0061] On the other hand, the present specification discloses a method for detecting the ability to eliminate hydroxyl radicals that can be used to detect the possibility of MCI in a subject suspected of MCI. The detection method disclosed herein is a method for detecting the ability to eliminate hydroxyl radicals in a subject suspected of MCI, comprising a detection step, in which a measuring reagent for the ability to eliminate hydroxyl radicals is used to detect the ability to eliminate hydroxyl radicals in a biological sample of the subject. The detection method disclosed herein is characterized in detecting the ability to eliminate hydroxyl radicals in the biological sample of the subject, and other steps and conditions are not particularly limited. For the detection method disclosed herein, the description of the markers, test methods and test reagents for MCI can be cited.

[0062] The subject suspected of having MCI may be, for example, an individual who the subject himself / herself has a subjective suspicion of having MCI, or an individual who is determined to be suspected of having MCI or has a possibility of having MCI as a result of a medical examination by a doctor or the like. For the individual who the subject himself / herself has a subjective suspicion of having MCI, for example, an individual with certain subjective symptoms, an individual who wishes to undergo a preventive examination, etc. may be listed.

[0063] [Example]

[0064] The following are examples of the present disclosure. However, the present disclosure is not limited to the following examples. Unless otherwise specified, commercially available reagents are used based on their specifications.

[0065] (1) Subject

[0066] It was confirmed that the ability to eliminate hydroxyl radicals in saliva from MCI patients is reduced.

[0067] A cognitive function test (Japanese version of MoCA, MoCA-J) was performed on 27 subjects who obtained written consent to detect cognitive functions (visual space, executive function, naming, memory, attention, repetition, language recall, abstract concept, delayed regeneration, and sense of direction). Subjects with a MoCA-J result of 25 points or less were considered MCI patients (MCI group, n=24, average cognitive function was 17.25), and subjects with a score of 26 or more were considered MCI non-patients (MCI non-patient group, n=3, average cognitive function was 27). In the selection of subjects, individuals who took adrenocortical hormones or immunosuppressants for a long time, individuals who had a history of taking antibiotics in the past 3 months, and individuals who had received antifungal treatment in the past 6 weeks were excluded.

[0068] (2) Saliva collection

[0069] The collection of saliva is performed as follows using a commercially available saliva collection tube (Salikids (registered trademark), manufactured by Sarstedt Co., Ltd.). First, each subject is asked to hold the cotton roll attached to the Salikids (registered trademark) in the oral cavity for 5 minutes to allow the cotton roll to fully absorb the subject's saliva. Then, the cotton roll containing saliva is inserted into the hanging insert of the Sarikids (registered trademark), the lid is closed, and centrifugation is performed at 3000×g for 3 minutes. After separation, the supernatant portion in the tube is used as a saliva sample of each subject and stored at -40°C. In addition, when collecting saliva, each subject is prohibited from eating, taking oral medications, brushing teeth, and professional oral cleaning (Professional Mechanical Tooth Cleaning, PMTC) from 1 hour before collection.

[0070] (3) Measurement of saliva's ability to eliminate hydroxyl radicals

[0071] The ability of each saliva sample to eliminate hydroxyl radicals was confirmed by the electron spin resonance (ESR) spin trapping method using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent. The generation system of hydroxyl radicals is a divalent iron and H2O2 generation system. Specifically, 20 μl of 20 mmol / l H2O2 was added to 180 μl of 10 mmol / l phosphate buffered saline (pH 7.2) containing 20 μl of 50 mmol / l DMPO and 20 μmol / l of divalent iron to generate hydroxyl radicals. Then, the mixture (200 μl) was transferred to a flat cell, and the DMPO-OH spin adduct was measured using the X-band ESR spin trapping method to confirm the generation of hydroxyl radicals. The measurement conditions of ESR are described below.

[0072] As shown in the following formula (A), the generated hydroxyl radical reacts with DMPO added to the reaction system to generate a stable free radical nitroxide (DMPO-OOH) that can be detected by ESR. When a saliva sample is added to the reaction system, the signal intensity obtained by ESR changes. Figure 1 The DMPO-OH spin adduct obtained by the ESR method is shown.

[0073] [Chemical formula A]

[0074]

[0075] For the measurement of ESR, an electron spin resonance device (JES-RE3X, X-band spectrometer, manufactured by JEOL) is connected to a WIN-RAD ESR data analyzer (Radical Research, Tokyo, Japan), and the measurement conditions are as follows. The hyperfine coupling constant is calculated using the resonant frequency measured by a microwave frequency counter and the resonant electric field measured by an electric field measuring device ES-FC5 (manufactured by JEOL). The detected spin adducts are quantitatively measured according to the ESR spectrum of manganese oxide standard material. The signal intensity of the actual measurement is represented by the relative height of the signal intensity of the ESR spectrum normalized to manganese oxide standard material. In addition, for the elimination ability of hydroxyl radicals, the saliva sample is 4 to 6 samples collected from each subject respectively, and the elimination ability of the hydroxyl radicals of multiple samples is measured, and the average value is calculated, and the average value is used as the elimination ability of the hydroxyl radicals of each subject.

[0076] (ESR measurement conditions)

[0077] Device:

[0078] Electron spin resonance device (JES-RE3X, X-band spectrometer, manufactured by JEOL Ltd.)

[0079] Measurement conditions:

[0080] Microwave output: 8.00mW

[0081] Scan time: 1 minute

[0082] Scan width: 334.8±5mT

[0083] Magnetic field modulation: 100kHz 0.079mT

[0084] Gain: ×400

[0085] Scan time: 1 minute

[0086] Time constant: 0.03 seconds

[0087] These results are shown in the following Table 1. As shown in the following Table 1, it was found that the saliva of the MCI group had a significantly lower hydroxyl radical scavenging ability than the saliva of the MCI non-suffering group.

[0088] [Table 1]

[0089]

[0090] From the above, it is known that since the ability to eliminate hydroxyl radicals in the saliva of MCI patients is reduced, the ability to eliminate hydroxyl radicals functions as a marker for MCI.

[0091] The present disclosure is described above with reference to the embodiments and examples, but the present disclosure is not limited to the above embodiments and examples. The composition and details of the present disclosure can be changed in various ways that can be understood by those skilled in the art within the scope of the present disclosure. In addition, each embodiment can be appropriately combined with other embodiments.

[0092] <Notes>

[0093] A part or all of the above-mentioned embodiments and examples may be described as the following supplementary notes, but are not limited to the following.

[0094] (Note 1)

[0095] A method for testing mild cognitive impairment (MCI) includes a measuring step in which the ability to eliminate hydroxyl free radicals in a biological sample of a subject is measured.

[0096] (Note 2)

[0097] According to the testing method described in Supplementary Note 1, the measured value of the ability to eliminate hydroxyl radicals is measured in the measuring step.

[0098] (Note 3)

[0099] The test method according to Supplementary Note 2, wherein the test method comprises:

[0100] a testing step, in which the possibility of the subject suffering from MCI is tested by comparing the measured value of the hydroxyl radical elimination ability in the biological sample of the subject with a threshold value,

[0101] The threshold value is a threshold value calculated from a measured value of the ability to eliminate hydroxyl radicals in a biological sample of a non-MCI patient or from a measured value of the ability to eliminate hydroxyl radicals in a biological sample of an MCI patient.

[0102] (Note 4)

[0103] According to the testing method described in Note 3, wherein in the testing step, if the measured value of the ability to eliminate hydroxyl radicals in the biological sample of the subject is lower than the measured value of the ability to eliminate hydroxyl radicals in the biological sample of the MCI non-patient, if it is the same as the measured value of the ability to eliminate hydroxyl radicals in the biological sample of the MCI patient, or if it is lower than the measured value of the ability to eliminate hydroxyl radicals in the biological sample of the MCI patient, the subject is evaluated as having the possibility of suffering from MCI.

[0104] (Note 5)

[0105] The testing method according to any one of Notes 1 to 4, wherein the biological sample is saliva.

[0106] (Note 6)

[0107] A test method according to any one of Notes 1-5, wherein the test for MCI is detection of MCI, determination of MCI, determination of preventive effect of MCI, determination of therapeutic effect of MCI, screening of MCI, determination of MCI patients who are responsive to therapeutic drugs, determination of therapeutic drugs that are effective for individual MCI patients, an examination method for diagnosing MCI, or an examination for the treatment of MCI.

[0108] (Note 7)

[0109] The test method according to any one of Supplements 1 to 6, wherein the test method includes an administration step in which an MCI therapeutic drug is administered to a subject who has received a test result indicating that he or she suffers from MCI.

[0110] <MCI test reagent>

[0111] (Note 8)

[0112] A test reagent for mild cognitive impairment, comprising a reagent for measuring the ability to eliminate hydroxyl radicals.

[0113] (Note 9)

[0114] The test reagent according to Supplementary Note 8, wherein the test reagent comprises a generator of hydroxyl radicals and a detector of hydroxyl radicals.

[0115] (Note 10)

[0116] The test reagent according to Note 8 or 9, wherein the test reagent is used in the test method according to any one of Notes 1-7.

[0117] <Method for Screening Therapeutic Drugs>

[0118] (Note 11)

[0119] A method for screening candidate substances for treating mild cognitive impairment comprises a selection step in which an activated substance that improves the ability to eliminate hydroxyl radicals is selected from test substances as a candidate substance for treating mild cognitive impairment.

[0120] (Note 12)

[0121] The screening method according to Supplementary Note 11, wherein the screening method comprises:

[0122] a measuring step, in which the test substance is allowed to coexist in a hydroxyl radical generating system to measure the hydroxyl radical elimination ability; and

[0123] A selection step, in which, when the hydroxyl radical elimination ability obtained in the measurement step is higher than that of a control coexistence system in which the test substance does not coexist, the test substance is selected as the therapeutic drug candidate substance.

[0124] (Note 13)

[0125] The screening method according to Supplement 11 or 12, wherein the test substance is at least one selected from the group consisting of low molecular weight compounds, peptides, proteins and nucleic acids.

[0126] <Method for detecting the ability to eliminate hydroxyl radicals>

[0127] (Note 14)

[0128] A detection method for detecting the ability to eliminate hydroxyl radicals in a subject suspected of having mild cognitive impairment, the detection method comprising:

[0129] A detection step in which the hydroxyl radical-eliminating ability in the biological sample of the subject is detected using a measuring reagent for the hydroxyl radical-eliminating ability.

[0130] (Note 15)

[0131] The detection method according to Note 14, wherein the biological sample is saliva.

[0132] <Application>

[0133] (Note 16)

[0134] Application of a reagent measuring the ability to eliminate hydroxyl radicals for testing the possibility of mild cognitive impairment.

[0135] Industrial availability

[0136] As described above, according to the present disclosure, the possibility of a subject suffering from MCI (risk of suffering from MCI) can be tested by measuring the ability to eliminate hydroxyl radicals. In addition, in the present disclosure, since the ability to eliminate hydroxyl radicals becomes a target for MCI, candidate substances for treating MCI can also be obtained by screening using the target. Therefore, the present disclosure is extremely useful in the clinical field and the biochemical field.

[0137] This application claims the benefit of priority based on Japanese patent application No. 2022-106269, filed on June 30, 2022, the disclosure of which is incorporated herein in its entirety.

Claims

1. A method for testing mild cognitive impairment (MCI), the method comprising: A measuring step in which the hydroxyl radical elimination ability in the biological sample of the subject is measured.

2. The testing method according to claim 1, wherein a measured value of the ability to eliminate hydroxyl radicals is measured in the measuring step.

3. The testing method according to claim 2, wherein the testing method comprises: a testing step, in which the possibility of the subject suffering from MCI is tested by comparing the measured value of the hydroxyl radical elimination ability in the biological sample of the subject with a threshold value, The threshold value is a threshold value calculated from a measured value of the ability to eliminate hydroxyl radicals in a biological sample of a non-MCI patient or from a measured value of the ability to eliminate hydroxyl radicals in a biological sample of an MCI patient.

4. The testing method according to claim 3, wherein in the testing step, if the measured value of the ability to eliminate hydroxyl radicals in the biological sample of the subject is lower than the measured value of the ability to eliminate hydroxyl radicals in the biological sample of the MCI non-patient, is the same as the measured value of the ability to eliminate hydroxyl radicals in the biological sample of the MCI patient, or is lower than the measured value of the ability to eliminate hydroxyl radicals in the biological sample of the MCI patient, the subject is evaluated as having the possibility of suffering from MCI.

5. The testing method according to any one of claims 1 to 4, wherein the biological sample is saliva.

6. The test method according to any one of claims 1 to 5, wherein the test for MCI is detection of MCI, determination of MCI, determination of preventive effect of MCI, determination of therapeutic effect of MCI, screening of MCI, determination of MCI patients for whom therapeutic drugs are effective, determination of therapeutic drugs that are effective for individual MCI patients, an examination method for diagnosing MCI, or an examination for the treatment of MCI.

7. The test method according to any one of claims 1 to 6, wherein the test method comprises an administration step in which an MCI therapeutic drug is administered to the subject who has obtained a test result indicating that he or she suffers from MCI.

8. A test reagent for mild cognitive impairment, comprising a measuring reagent for the ability to eliminate hydroxyl radicals. 9 . The test reagent according to claim 8 , wherein the test reagent comprises a generator of hydroxyl radicals and a detector of hydroxyl radicals.

10. The test reagent for mild cognitive impairment according to claim 8 or 9, which is used in the test method according to any one of claims 1 to 7.

11. A method for screening candidate substances for treating mild cognitive impairment, comprising: A selection step of selecting an activating substance that improves the ability to eliminate hydroxyl radicals from the test substances as a candidate substance for the therapeutic drug for mild cognitive impairment.

12. The screening method according to claim 11, wherein the screening method comprises: a measuring step, in which the test substance is allowed to coexist in a hydroxyl radical generating system to measure the hydroxyl radical elimination ability; and A selection step, in which, when the hydroxyl radical elimination ability obtained in the measurement step is higher than that of a control coexistence system in which the test substance does not coexist, the test substance is selected as the therapeutic drug candidate substance. 13 . The screening method according to claim 11 or 12 , wherein the test substance is at least one selected from the group consisting of low molecular weight compounds, peptides, proteins, and nucleic acids.

14. A detection method for detecting the ability of a subject suspected of having mild cognitive impairment to eliminate hydroxyl radicals, the detection method comprising: A detection step is performed in which the hydroxyl radical-eliminating ability in the biological sample of the subject is detected using a measuring reagent for the hydroxyl radical-eliminating ability. The detection method according to claim 14 , wherein the biological sample is saliva.

16. Use of a reagent for measuring the ability of eliminating hydroxyl radicals for testing the possibility of mild cognitive impairment.

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