Biomarker for evaluating intracerebral accumulation of amyloid β
Combining P-TAU and NFL in plasma allows for accurate, cost-effective assessment of brain amyloid β levels, addressing the limitations of invasive methods and improving diagnostic accuracy for Alzheimer's disease.
Patent Information
- Application Number
- JP2021170500
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2021-10-18
- Publication Date
- 2025-02-05
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Current methods for evaluating amyloid β accumulation in the brain, such as PET scans and cerebrospinal fluid analysis, are invasive and costly, limiting their applicability as diagnostic tools for Alzheimer's disease, and existing blood-based biomarkers do not provide high accuracy in assessing brain amyloid β accumulation.
Combining phosphorylated tau (P-TAU181, P-TAU217, P-TAU231) and neuropilament light chains (NFL) in plasma to measure and compare their levels with predetermined cut-off values for accurate diagnosis of amyloid β accumulation in the brain.
Provides a low-cost, non-invasive method with high diagnostic accuracy for amyloid β brain accumulation, enabling widespread use in initial diagnosis, health check-ups, and drug discovery.
Smart Images
Figure 2025016807000001_ABST
Abstract
Description
[Technical field]
[0001] The present disclosure relates to biomarkers for assessing brain amyloid-β accumulation. [Background technology]
[0002] Amyloid beta is a type of protein produced in the brain, and its accumulation in the brain is thought to be closely related to the onset of Alzheimer's disease (AD), etc. Tau protein (also simply called "tau") is also a type of protein produced in nerve cells in the brain, and while phosphorylation of tau is important for maintaining the cytoskeleton structure, abnormalities in this phosphorylation are thought to be involved in AD.
[0003] Accumulation of amyloid-β and abnormal phosphorylated tau (p-Tau) in the brain is clinically assessed by radiological examinations such as positron emission tomography (PET) or biomarkers of amyloid-β and tau in cerebrospinal fluid (CSF). However, these detection methods are highly invasive and expensive, limiting their use as diagnostic tools.
[0004] As a less invasive and less expensive method, the diagnosis of AD or the assessment of the accumulation of amyloid beta in the brain by a biomarker in blood has been proposed. For example, Non-Patent Document 1 discloses that the plasma p-tau-217 / T217 ratio and p-tau-217 value distinguish amyloid beta negative groups from amyloid beta positive groups regardless of cognitive function. Non-Patent Document 2 discloses that plasma P-Tau181 is a non-invasive diagnostic and prognostic biomarker for AD. Non-Patent Document 3 discloses that plasma p-Tau181 is useful for screening AD, and that plasma p-Tau181 can select individuals judged to be amyloid beta positive by PET. Non-Patent Document 4 discloses that plasma p-Tau181 can distinguish AD from young adults and elderly people without cognitive impairment. Non-Patent Document 5 discloses that plasma p-Tau181 can select AD from non-AD lesions with high accuracy. Furthermore, Non-Patent Document 6 discloses that plasma p-Tau217 can distinguish AD from other neurodegenerative diseases.
[0005] Although these methods of evaluating phosphorylated tau in plasma are useful to some extent for screening for Alzheimer's disease, the accuracy of evaluating the accumulation of amyloid beta in the brain is not necessarily high. Therefore, there is a great need for a biomarker that can accurately evaluate the accumulation of amyloid beta in the brain.
[0006] On the other hand, there has been a report on predicting progression of mild cognitive impairment (MCI) patients to AD using a combined biomarker of plasma p-Tau181 and neurofilament light chain (NFL) (Non-Patent Document 7). [Prior art documents] [Non-patent literature]
[0007] [Non-Patent Document 1] Barthelemy et al., “Blood plasma phosphorylated-tau isoforms track CNS change inAlzheimer's disease”, J Exp Med (2020) 217(11):e20200861
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[0008] In consideration of the problems of the conventional technology, the present disclosure aims to provide a biomarker for evaluating the accumulation of amyloid beta in the brain that is low cost and has high diagnostic accuracy, and a method for evaluating the accumulation of amyloid beta in the brain using the biomarker. [Means for solving the problem]
[0009] As a result of intensive research aimed at solving the above problems, the inventors have found that the combination of phosphorylated tau 181 (p-Tau181) and neurofilament light chain (NFL) in plasma is a useful biomarker for evaluating the accumulation of amyloid beta in the brain, and that the use of these two biomarkers makes it possible to evaluate the accumulation of amyloid beta in the brain with high diagnostic accuracy.
[0010] In one aspect, the present disclosure provides a method for evaluating the accumulation of amyloid beta in the brain, comprising the steps of measuring the amount of p-Tau selected from the group consisting of phosphorylated tau (p-Tau)181, p-Tau217, p-Tau231 and combinations thereof, and the amount of neurofilament light chain (NFL) in a blood sample obtained from a subject, and comparing the measured values obtained in the above steps with control values. This method is a low-cost and low-invasive method, and enables a method for evaluating the accumulation of amyloid beta in the brain with high diagnostic accuracy. The combination of a predetermined p-Tau (p-Tau181, p-Tau217, p-Tau231) and NFL enables a test without missing any cases while maintaining high specificity.
[0011] Therefore, the present disclosure provides, for example, the following [1] to
[26] . [1] A method for evaluating brain amyloid beta accumulation, comprising: Measuring the amount of p-Tau selected from the group consisting of phosphorylated tau (p-Tau) 181, p-Tau 217, p-Tau 231 and combinations thereof, and the amount of neurofilament light chain (NFL) in a blood sample obtained from the subject; comparing the measured value obtained in the above step with a control value; A method comprising: [2] The method according to [1], wherein the control value is a predetermined cutoff value. [3] The method according to [2], wherein the cut-off value is determined by comparing the measured values of p-Tau and NFL in a blood sample obtained from a control subject with the measured values of p-Tau and NFL in a blood sample obtained from a subject having accumulation of amyloid beta in the brain. [4] The method according to [2] or [3], wherein the cutoff value of the p-Tau and / or the cutoff value of the NFL is one or more. [5] The method according to any one of [1] to [4], wherein the p-Tau is p-Tau181. [6] The method according to any one of [1] to [4], wherein the p-Tau is p-Tau217. [7] The method according to any one of [1] to [4], wherein the p-Tau is p-Tau231. [8] The method according to any one of [1] to [4], wherein the p-Tau is a combination of p-Tau181, p-Tau217 and p-Tau231. [9] The method according to any one of [1] to [8], wherein the blood sample is whole blood, serum, or plasma.
[10] A method for assisting in the diagnosis of Alzheimer's disease (AD), mild cognitive impairment due to AD, or preclinical AD, comprising: Measuring the amount of p-Tau selected from the group consisting of phosphorylated tau (p-Tau) 181, p-Tau 217, p-Tau 231 and combinations thereof, and the amount of neurofilament light chain (NFL) in a blood sample obtained from the subject; A step of comparing the measured value obtained in the above step with a control value to evaluate the accumulation of amyloid β in the brain. A method comprising:
[11] The method according to
[10] , wherein the control value is a predetermined cutoff value.
[12] The method according to
[11] , wherein the cut-off value is determined by comparing the measured values of p-Tau and NFL in a blood sample obtained from a control subject with the measured values of p-Tau and NFL in a blood sample obtained from a subject having accumulation of amyloid beta in the brain.
[13] The method according to
[11] or
[12] , wherein the cutoff value of the p-Tau and / or the cutoff value of the NFL is one or more.
[14] The method according to any one of
[10] to
[13] , wherein the p-Tau is p-Tau181.
[15] The method according to any one of
[10] to
[13] , wherein the p-Tau is p-Tau217.
[16] The method according to any one of
[10] to
[13] , wherein the p-Tau is p-Tau231.
[17] The method according to any of
[10] to
[13] , wherein the p-Tau is a combination of p-Tau181, p-Tau217 and p-Tau231.
[18] The method according to any one of
[10] to
[17] , wherein the blood sample is whole blood, serum, or plasma.
[19] A method for obtaining an index for assisting in the diagnosis of Alzheimer's disease (AD), mild cognitive impairment due to AD, or preclinical AD, comprising: measuring the amount of p-Tau selected from the group consisting of p-Tau181, p-Tau217, p-Tau231 and combinations thereof, and the amount of NFL in a blood sample obtained from the subject; A step of comparing the measured value obtained in the above step with a control value to obtain information on the accumulation of amyloid β in the brain. A method comprising:
[20] A kit for assessing accumulation of amyloid beta in the brain, comprising an antibody that specifically binds to p-Tau selected from the group consisting of phosphorylated tau (p-Tau) 181, p-Tau217, p-Tau231, and combinations thereof, and an antibody that specifically binds to neurofilament light chain (NFL). [twenty one] A kit for assessing accumulation of amyloid beta in the brain, comprising an antibody that specifically binds to p-Tau selected from the group consisting of phosphorylated tau (p-Tau) 181, p-Tau217, p-Tau231, and combinations thereof, and used in conjunction with a kit comprising an antibody that specifically binds to neurofilament light chain (NFL). [twenty two] A kit for assessing accumulation of amyloid beta in the brain, comprising an antibody that specifically binds to neurofilament light chain (NFL) and is used in conjunction with a kit comprising an antibody that specifically binds to phosphorylated tau (p-Tau) 181, p-Tau 217, p-Tau 231, and combinations thereof. [twenty three] The kit according to any one of
[20] to
[22] , wherein the p-Tau is p-Tau181. [twenty four] The kit according to any one of
[20] to
[22] , wherein the p-Tau is p-Tau217. [twenty five] The kit according to any one of
[20] to
[22] , wherein the p-Tau is p-Tau231.
[26] The kit according to any one of
[20] to
[22] , wherein the p-Tau is a combination of p-Tau181, p-Tau217 and p-Tau231.
[0012] The present disclosure also provides, for example, the following
[27] to
[45] .
[27] A method for treating Alzheimer's disease (AD), mild cognitive impairment due to AD, or preclinical AD, comprising: Measuring the amount of p-Tau selected from the group consisting of phosphorylated tau (p-Tau) 181, p-Tau 217, p-Tau 231 and combinations thereof, and the amount of neurofilament light chain (NFL) in a blood sample obtained from the subject; A step of comparing the measured value obtained in the above step with a control value to evaluate the accumulation of amyloid beta in the brain; Administering a therapeutically effective amount of a therapeutic agent for Alzheimer's disease, mild cognitive impairment due to AD, or preclinical AD to a subject evaluated to have accumulated amyloid beta in the brain; A method comprising:
[28] The method according to
[27] , wherein the control value is a predetermined cutoff value.
[29] The method according to
[28] , wherein the cut-off value is determined by comparing the measured values of p-Tau and NFL in a blood sample obtained from a control subject with the measured values of p-Tau and NFL in a blood sample obtained from a subject having accumulation of amyloid beta in the brain, respectively.
[30] The method according to
[28] or
[29] , wherein the cutoff value of the p-Tau and / or the cutoff value of the NFL is one or more.
[31] The method according to any one of
[27] to
[30] , wherein the p-Tau is p-Tau181.
[32] The method according to any one of
[27] to
[30] , wherein the p-Tau is p-Tau217.
[33] The method according to any one of
[27] to
[30] , wherein the p-Tau is p-Tau231.
[34] The method according to any of
[27] to
[30] , wherein the p-Tau is a combination of p-Tau181, p-Tau217 and p-Tau231.
[35] The method according to any one of
[27] to
[34] , wherein the blood sample is whole blood, serum, or plasma.
[36] The method according to any of
[27] to
[35] , wherein the subject assessed to have accumulated amyloid beta in the brain is diagnosed with Alzheimer's disease, mild cognitive impairment due to AD, or preclinical AD before administration of the therapeutic agent.
[37] A method for diagnosing Alzheimer's disease (AD), mild cognitive impairment due to AD, or preclinical AD, comprising: Measuring the amount of p-Tau selected from the group consisting of phosphorylated tau (p-Tau) 181, p-Tau 217, p-Tau 231 and combinations thereof, and the amount of neurofilament light chain (NFL) in a blood sample obtained from the subject; A step of comparing the measured value obtained in the above step with a control value to evaluate the accumulation of amyloid β in the brain. A method comprising:
[38] The method according to
[37] , wherein the control value is a predetermined cutoff value.
[39] The method according to
[38] , wherein the cut-off value is determined by comparing the measured values of p-Tau and NFL in a blood sample obtained from a control subject with the measured values of p-Tau and NFL in a blood sample obtained from a subject having accumulation of amyloid beta in the brain, respectively.
[40] The method according to
[38] or
[39] , wherein the cutoff value of the p-Tau and / or the cutoff value of the NFL is one or more.
[41] The method according to any of
[37] to
[40] , wherein the p-Tau is p-Tau181.
[42] The method according to any of
[37] to
[40] , wherein the p-Tau is p-Tau217.
[43] The method according to any one of
[37] to
[40] , wherein the p-Tau is p-Tau231.
[44] The method according to any of
[37] to
[40] , wherein the p-Tau is a combination of p-Tau181, p-Tau217 and p-Tau231.
[45] The method according to any one of
[37] to
[44] , wherein the blood sample is whole blood, serum, or plasma. Effect of the Invention
[0013] The method for evaluating the accumulation of amyloid beta in the brain according to the present disclosure not only has high diagnostic accuracy but also is low cost, so that the scope of use can be significantly expanded and it is expected to be used on a large scale in early diagnosis, health checkups, etc. Therefore, in particular, as a primary screening for amyloid beta testing, it can achieve a high true positive rate and diagnostic accuracy, and it can be an evaluation method that is expected to be used in drug discovery based on the accumulation of amyloid beta in the brain.
[0014] The kit of the present disclosure makes it possible to evaluate the accumulation of amyloid β in the brain with high diagnostic accuracy and at low cost. [Brief description of the drawings]
[0015] [Figure 1] FIG. 1 shows the concentration of p-Tau181 in the blood of 60 negative cases and 34 positive cases in an amyloid β PET test. [Diagram 2] FIG. 1 shows the concentrations of p-Tau181 and NFL in the blood of 60 negative cases and 34 positive cases in an amyloid β PET test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0016] [Method for assessing accumulation of amyloid beta in the brain] In one embodiment, the method for assessing accumulation of amyloid beta in the brain includes measuring the amount of p-Tau selected from the group consisting of phosphorylated tau 181 (p-Tau181), phosphorylated tau 217 (p-Tau217), phosphorylated tau 231 (p-Tau231) and combinations thereof, and the amount of neurofilament light chain (NFL) in a blood sample obtained from a subject, and comparing the measured values obtained in the above steps with control values.
[0017] As used herein, subject refers to a mammal, including but not limited to a human, chimpanzee, orangutan, gorilla, baboon, monkey, mouse, rat, pig, horse, dog, and cow. In one embodiment, the subject is a human.
[0018] The blood sample may contain the biomarkers to be measured, p-Tau and NFL, selected from the group consisting of p-Tau181, p-Tau217, p-Tau231 and combinations thereof. The blood sample may be whole blood, serum or plasma, and may be a blood sample obtained from a human subject.
[0019] Amyloid beta (also referred to as "Aβ") is a type of protein produced in the brain that is known to those skilled in the art. Evaluating the accumulation of amyloid beta in the brain refers to determining whether or not a greater amount of amyloid beta accumulation is observed in the brain compared to a control subject. Accumulation of amyloid beta in the brain means that amyloid beta is not excreted from the brain and accumulates in the brain. In addition, a control subject means a subject that is amyloid beta-negative in the brain, without accumulation of amyloid beta in the brain. In clinical practice, accumulation of amyloid beta in the brain is conventionally evaluated by radiological tests such as positron emission tomography (PET), or biomarkers of amyloid beta and phosphorylated tau in cerebrospinal fluid. For example, a PET probe 18 In amyloid β PET testing using F-Florbetaben, 18 According to the visual assessment criteria established by the manufacturer of F-Florbetaben, if the accumulation of amyloid beta in the brain is greater than the diagnostic criteria, the patient is judged to be amyloid beta positive in the brain, and if the accumulation of amyloid beta in the brain is less than the diagnostic criteria, the patient is judged to be amyloid beta negative in the brain.
[0020] Accumulation of amyloid beta in the brain is considered to be one of the main pathologies mainly seen in Alzheimer's disease (AD) and mild cognitive impairment due to AD (MCI due to AD), but there are many cases in which patients diagnosed with AD by clinical diagnosis do not have accumulation of amyloid beta in the brain, and the discrepancy between clinical diagnosis and pathological diagnosis is an issue. In addition, even patients who have been clinically diagnosed with corticobasal syndrome (CBS), frontotemporal dementia (FTLD), progressive supranuclear palsy (PSP), Parkinson's disease or Parkinson's syndrome (PD), or geriatric psychiatric disorders, which usually do not have accumulation of amyloid beta in the brain, may have accumulation of amyloid beta in the brain. Furthermore, cognitive function may be normal but accumulation of amyloid beta in the brain may occur, and such a preclinical stage of Alzheimer's disease is called preclinical AD. If the evaluation method of this embodiment is judged to be positive, it is considered that there is a suspicion of accumulation of amyloid beta in the brain regardless of the disease name by clinical diagnosis. Therefore, the evaluation method of this embodiment is useful as a simple primary screening method for accumulation of amyloid beta in the brain. The clinical diagnostic criteria for these neurological diseases are known to those skilled in the art, and include, for example, the diagnostic criteria described in the Examples.
[0021] Phosphorylated Tau (p-Tau) is selected from the group consisting of phosphorylated Tau 181 (p-Tau181), phosphorylated Tau 217 (p-Tau217), phosphorylated Tau 231 (p-Tau231), and combinations thereof, and in one embodiment is p-Tau181. These p-Taus are biomarkers for evaluating the accumulation of amyloid beta in the brain, and can improve diagnostic accuracy when used in combination with neurofilament light chain (NFL). Tau (Tau) is a protein known to those skilled in the art that is expressed in neurons and glial cells in the central and peripheral nervous systems. Tau phosphorylation occurs in the brain, and some abnormal phosphorylation is known. Among them, p-Tau181 is Tau phosphorylated at threonine 181, p-Tau217 is Tau phosphorylated at threonine 217, and p-Tau231 is Tau phosphorylated at threonine 231.
[0022] The amount of p-Tau in a blood sample, i.e., p-Tau181, p-Tau217, and p-Tau231, can be measured according to known protein quantification methods. For example, absorbance measurement, immunoassays utilizing antigen-antibody reactions such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), immunofluorescence, and immunoprecipitation, and liquid chromatography / mass spectrometry (LC / MS) can be included. In addition, it can be easily quantified by a commercially available measurement kit, for example, SIMOA® p-Tau181 Advantage V2 Kit (Quanterix). Here, the amount of p-Tau is a measured value (absolute value).
[0023] Neurofilament light chain (NFL) is known to be a biomarker of axonal injury, but is not known to be used to evaluate the accumulation of amyloid beta in the brain. Neurofilament (NF) is mainly composed of three types of subunits: NFL (low or light; light chain), NFM (medium; medium chain), and NFH (heavy or high; heavy chain), of which NFL is the light chain.
[0024] The amount of NFL in a blood sample can be measured according to known protein quantification methods, similar to p-Tau. Examples include absorbance measurement, immunological measurement methods using antigen-antibody reactions such as ELISA, EIA, RIA, immunofluorescence, and immunoprecipitation, and LC / MS. In addition, it can be easily quantified using a commercially available measurement kit, such as SIMOA (registered trademark) NFL Advantage (SR-X) Kit (Quanterix). Here, the amount of NFL is a measured value (absolute value).
[0025] In this specification, "control value" refers to both the control value of p-Tau and the control value of NFL, unless otherwise specified. The control value is a value for predicting the accumulation of amyloid beta in the brain, and can be a predetermined cut-off value. In one embodiment, the predetermined cut-off value is a cut-off value determined by the same assay as that used in the measurement step for measuring the amount of p-Tau and NFL.
[0026] The "cut-off value" in this specification refers to both the cut-off value of p-Tau and the cut-off value of NFL, unless otherwise specified. The cut-off value may be determined by comparing the measurement values of p-Tau and NFL in blood samples obtained from a control subject with the measurement values of p-Tau181 and NFL in blood samples obtained from a subject with brain amyloid-β accumulation, respectively. The subject with brain amyloid-β accumulation is a subject that is brain amyloid-β positive by conventional diagnostic methods, such as amyloid-β PET, and the control subject is a subject that is brain amyloid-β negative.
[0027] The cut-off value may be determined by the desired sensitivity of diagnosis. The sensitivity of diagnosis may be arbitrarily set depending on the purpose of evaluation, for example, 70%, 80%, 90%, 95% or 100%. Since the evaluation method of the present disclosure is expected to be a pre-diagnosis of amyloid β PET examination, which is a definitive diagnosis of brain amyloid β accumulation, it is considered that a preliminary test using blood before amyloid β PET is clinically required to have as few false negatives as possible, in order to provide appropriate treatment to as many subjects as possible. Therefore, in one embodiment, the sensitivity may be set to 100% (i.e., sensitivity without false negatives).
[0028] The measured values of p-Tau and NFL in blood samples obtained from control subjects are compared with the measured values of p-Tau and NFL in blood samples obtained from subjects with brain amyloid β accumulation, and cut-off values can be determined according to a predetermined sensitivity.The cut-off value of p-Tau and the cut-off value of NFL can be one, two or more, for example, two, three or four.
[0029] Specifically, the cutoff value can be determined by graphing the measured values of p-Tau (p-Tau181 in FIG. 2) and NFL in blood samples obtained from control subjects (subjects negative for amyloid beta in the brain) and the measured values of p-Tau and NFL in blood samples obtained from subjects with amyloid beta accumulation in the brain (subjects positive for amyloid beta in the brain), as shown in FIG. 2, and deriving cutoff values (two for p-Tau181 and one for NFL in FIG. 2) according to the desired sensitivity (for example, 100%). Note that the measured values of p-Tau and NFL depend on the measurement method, so if the cutoff values are determined in advance for one measurement method (assay), there is no need to measure and determine them every time.
[0030] In one embodiment, when determining the cutoff value, the populations of the control subjects and the subjects with cerebral amyloid β accumulation are populations having statistically significant numbers. For example, each may be 10 or more, and as a further example, may be 30 or more, 50 or more, or even 100 or more.
[0031] In the step of evaluating the accumulation of amyloid-β in the brain by comparing the measured value obtained in the above measurement step with a control value, it is evaluated by the combination of the above p-Tau and NFL. When there is one p-Tau (for example, p-Tau is p-Tau181) and there is one cut-off value for each of p-Tau and NFL, for example, when the cut-off values are p-Tau181 cut-off value 1 and NFL cut-off value 1, if the measured value of p-Tau181 is greater than or equal to p-Tau181 cut-off value 1 and the measured value of NFL is greater than or equal to NFL cut-off value 1, it may be determined as positive for amyloid-β in the brain, and otherwise it may be determined as negative for amyloid-β in the brain.
[0032] When one of the cut-off values of p-Tau181 and NFL is one and the other cut-off value is two or more, for example, when the cut-off values are NFL cut-off value 1 and p-Tau181 cut-off value 1 and p-Tau181 cut-off value 2 (where p-Tau181 cut-off value 1 < p-Tau181 cut-off value 2), if the measured value of p-Tau181 is greater than or equal to p-Tau181 cut-off value 2, and when it is less than NFL cut-off value 1 and the measured value of p-Tau181 is greater than or equal to p-Tau181 cut-off value 1 and less than p-Tau181 cut-off value 2, it may be determined as positive for amyloid-β in the brain, and otherwise it may be determined as negative for amyloid-β in the brain. When each cut-off value is two or more, it can be analogized. Further, when there are two or more p-Taus, in accordance with the above, the cut-off value of each p-Tau can be set and combined with the cut-off value of NFL, or the cut-off value can be set as the total amount of each p-Tau and combined with the cut-off value of NFL for determination.
[0033] The evaluation (judgment) step may be performed by a human being according to the above judgment criteria, or may be performed automatically using a computer system. When a computer system is used, control values (one or more cutoff values for each biomarker) may be input to the system in advance, or may be input simultaneously with the measured values. After the measured values and cutoff values are input, the system compares the measured values with the cutoff values and outputs "+ (positive)" or "- (negative)" according to an algorithm.
[0034] The evaluation method of this embodiment uses a combination of p-Tau and NFL, and has a higher diagnostic accuracy (specificity) than conventional evaluation methods, particularly evaluation methods using only p-Tau. The specificity of this evaluation method may be, for example, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more at 100% sensitivity. Thus, the evaluation method of this embodiment can provide high diagnostic accuracy while realizing true positives, and can therefore be used as a primary screening.
[0035] The evaluation method of the present disclosure can be used as a preliminary test (primary screening) in clinical practice. For subjects determined to be positive for intracerebral amyloid β by the present evaluation method, it is preferable to conduct further detailed examinations and diagnosis and provide appropriate treatment.
[0036] [Method for assisting in the diagnosis of Alzheimer's disease (AD), mild cognitive impairment due to AD, or preclinical AD] In one embodiment, a method for assisting in the diagnosis of Alzheimer's disease (AD), mild cognitive impairment in the context of AD, or preclinical AD comprises measuring the amount of p-Tau selected from the group consisting of p-Tau181, p-Tau217, p-Tau231, and combinations thereof, and the amount of NFL in a blood sample obtained from a subject, and comparing the measured values obtained in the above steps with control values to assess accumulation of amyloid beta in the brain.
[0037] When the accumulation of amyloid beta in the brain is judged (evaluated) to be positive, it is considered that there is a high possibility of Alzheimer's disease (AD), mild cognitive impairment due to AD (MCI due to AD), or preclinical AD. Therefore, the above-mentioned method of the present disclosure is useful for primary screening of AD, MCI due to AD, or preclinical AD.
[0038] In one embodiment, a method for obtaining an index for assisting in the diagnosis of Alzheimer's disease (AD), mild cognitive impairment due to AD, or preclinical AD includes the steps of measuring the amount of p-Tau selected from the group consisting of p-Tau181, p-Tau217, p-Tau231, and combinations thereof, and the amount of NFL in a blood sample obtained from a subject, and comparing the measured value obtained in the above step with a control value to obtain information on the accumulation of amyloid beta in the brain. In one embodiment, the index is information on the accumulation of amyloid beta in the subject's brain.
[0039] [Kit for assessing the accumulation of amyloid beta in the brain] In one embodiment, a kit for assessing accumulation of amyloid beta in the brain comprises an antibody that specifically binds to phosphorylated tau (p-Tau) 181, p-Tau 217, p-Tau 231, and combinations thereof, and an antibody that specifically binds to neurofilament light chain (NFL).
[0040] In another embodiment, a kit for assessing accumulation of amyloid beta in the brain comprises an antibody that specifically binds to p-Tau selected from the group consisting of phosphorylated tau (p-Tau)181, p-Tau217, p-Tau231, and combinations thereof, and is used in conjunction with a kit comprising an antibody that specifically binds to neurofilament light chain (NFL).
[0041] In a further embodiment, a kit for assessing brain amyloid beta accumulation comprises an antibody that specifically binds to neurofilament light chain (NFL) and is used in conjunction with a kit comprising an antibody that specifically binds to phosphorylated tau (p-Tau)181, p-Tau217, p-Tau231, and combinations thereof.
[0042] In one embodiment, the p-Tau is p-Tau181. The antibody may be any antibody or antigen-binding fragment thereof that specifically binds to p-Tau or NFL, and may be a polyclonal or monoclonal antibody. In one embodiment, the antibody is a detectably labeled antibody, and the labeling may be performed by methods known to those skilled in the art depending on the method for measuring antigen-antibody binding.
[0043] The above kit is used to measure the amount of p-Tau and NFL, and the measured value is compared with the control value to evaluate the accumulation of amyloid beta in the brain. The method for measuring the amount of p-Tau and NFL is not particularly limited, and examples thereof include ELISA, EIA, RIA, immunofluorescence, immunoprecipitation, radioimmunoassay, chemiluminescence immunoassay, electrochemiluminescence immunoassay, latex turbidimetric immunoassay, latex photometric immunoassay, and immunochromatography assay. In addition to the antibody, the kit may include any reagent or instrument that can be used to measure the amount of p-Tau and NFL.
[0044] [Methods for diagnosing and treating Alzheimer's disease (AD), mild cognitive impairment due to AD, or preclinical AD] In one embodiment, a method for treating Alzheimer's disease (AD), mild cognitive impairment due to AD, or preclinical AD includes the steps of measuring the amount of p-Tau selected from the group consisting of phosphorylated tau (p-Tau)181, p-Tau217, p-Tau231, and combinations thereof, and the amount of neurofilament light chain (NFL) in a blood sample obtained from a subject, comparing the measured value obtained in the above step with a control value to evaluate the accumulation of amyloid beta in the brain, and administering a therapeutically effective amount of a therapeutic agent for Alzheimer's disease, mild cognitive impairment due to AD, or preclinical AD to the subject evaluated to have accumulated amyloid beta in the brain.
[0045] The therapeutic agent for Alzheimer's disease, mild cognitive impairment due to AD, or preclinical AD is not particularly limited, and examples thereof include anti-Aβ antibodies, agents that inhibit Aβ production or accumulation, anti-tau antibodies, and the like.
[0046] The subject evaluated as having the accumulation of amyloid β in the brain is diagnosed with Alzheimer's disease, mild cognitive impairment due to AD, or preclinical AD before administering the therapeutic agent. Here, the diagnosis is a diagnosis including a step of evaluating the accumulation of amyloid β in the brain according to the present disclosure in one embodiment, a diagnosis including a radiological examination such as positron emission tomography (PET) in another embodiment, and a diagnosis including a radiological examination such as positron emission tomography (PET) in addition to the step of evaluating the accumulation of amyloid β in the brain according to the present disclosure in a further embodiment.
[0047] In one embodiment, a method for diagnosing Alzheimer's disease, mild cognitive impairment due to AD, or preclinical AD includes measuring the amount of p-Tau selected from the group consisting of p-Tau181, p-Tau217, p-Tau231, and combinations thereof, and the amount of NFL in a blood sample obtained from a subject, and comparing the measured values obtained in the above steps with control values to evaluate accumulation of amyloid beta in the brain.
[0048] The present disclosure will be described in detail below by way of examples, but the present disclosure is not limited to these examples in any way. EXAMPLES
[0049] <Clinical diagnosis> A total of 94 subjects (51 men, 43 women, age: 69.7±9.0 years) were recruited for this study and underwent brain MRI and various neuropsychological tests (Mini-Mental-State Examination (MMSE: Non-Patent Document 8), Wechsler Memory Scale-Revised Logical Memory I&II Story A (WMS-R Logical Memory I&II; Non-Patent Document 9), and Alzheimer's Disease Assessment Scale- Cognitive subscale (ADAS-COG; Non-Patent Document 10), verbal fluency (Non-Patent Document 11), Trail Making Test A&B, and Japanese Adult Reading Test (JART; Non-Patent Document 12)). Clinical diagnoses were made according to the current consensus diagnostic criteria (Table 1). [Table 1]
[0050] As a result, the breakdown of diseases of the 94 subjects was as follows: healthy (37 subjects), mild cognitive impairment (MCI, 14 subjects), Alzheimer's disease (AD, 19 subjects), corticobasal syndrome (CBS, 4 subjects), frontotemporal dementia (FTLD, 10 subjects), progressive supranuclear palsy (PSP, 4 subjects), Parkinson's disease or parkinsonian syndrome (PD, 2 subjects), and geriatric psychiatric disorders (4 subjects).
[0051] Next, to check for the presence of amyloid beta accumulation in the brain, a positron emission tomography (PET) probe, 18 After administering 300MBq of F-florbetaben intravenously to the subjects, they were imaged using an amyloid beta PET scanner (True Point Biograph 40 / 64 (Siemens Asahi Meditec Co., Ltd.)). 18The study was conducted according to the visual assessment criteria set by the manufacturer of F-Florbetaben, and a total of 94 subjects were divided into a group of 60 subjects with negative brain amyloid beta accumulation (hereinafter simply referred to as the "negative group") and a group of 34 subjects with positive brain amyloid beta accumulation (hereinafter simply referred to as the "positive group").
[0052] <Biomarker measurement> Next, plasma samples were collected from 94 subjects. Blood was drawn from each subject in the morning without breakfast and immediately cooled on ice. The plasma components were then centrifuged at 1200g x 10 minutes and 2800g x 10 minutes within 2 hours of blood collection, and stored in a -80°C ultra-low temperature freezer until analysis.
[0053] Regarding the quantification of biomarkers, p-Tau181 and NFL were measured using SIMOA® p-Tau181 Advantage V2 Kit (Quanterix) or SIMOA® NFL Advantage (SR-X) Kit (Quanterix), respectively, according to the attached protocol, using an ultrasensitive auto ELISA (SIMOA® HD-1 or SR-X) as a measuring device, and N=2 for each specimen. The measured values of N=2 for each sample obtained were averaged for each sample to obtain the measured value, and after correction for the dilution factor (4 times), the quantitative value for each sample was obtained. The measured values were each shown as absolute values (pg / ml).
[0054] <Establishing evaluation criteria for biomarkers> When drug development for brain amyloid-β accumulation becomes commonplace in the future, blood-based diagnosis of brain amyloid-β accumulation is expected to be used for prediagnosis of amyloid-β PET testing, which is a definitive diagnosis of brain amyloid-β accumulation. In order to provide appropriate treatment to as many subjects as possible, preliminary blood tests before amyloid-β PET testing require clinically low false negatives. At present, there are no clinically available tests with high accuracy and sensitivity, making it difficult to set clinical sensitivity criteria (diagnostic standards). However, since maximizing the benefits to patients would be achieved by correctly determining as many patients with brain amyloid-β accumulation as possible as positive, and by providing an environment in which they can undergo amyloid-β PET testing, which is a definitive diagnosis, and access appropriate drugs, we decided to set criteria that can correctly determine patients with brain amyloid-β accumulation as positive and evaluate various biomarkers.
[0055] (Diagnosis by pTau) The concentration of p-Tau181 in the blood of 60 negative cases and 34 positive cases based on the results of the amyloid-β PET test is shown in Figure 1. As shown in Figure 1, an increase in p-Tau181 in the blood was confirmed in the amyloid-β PET test positive group, reproducing previous reports. Considering a sensitivity of 100%, which is the target sensitivity for diagnosis, the cutoff value used in pTau diagnosis is around 1.45 pg / ml, and the specificity at this time was found to be 47%.
[0056] (pTau and NFL combination) As a result of the amyloid-β PET examination, the concentrations of p-Tau181 and NFL in the blood of 60 subjects in the negative group and 34 subjects in the positive group were graphed and shown in Fig. 2. As a result, when the blood p-Tau181 was divided into three groups of less than 0 to 1.45 pg / mL, less than 1.45 to 3.5 pg / mL, and 3.5 pg / mL or more, and the blood NFL was divided into two groups of less than 0 to 30 pg / ml and 30 pg / mL or more, it was newly found that when p-Tau181 was less than 1.45 to 3.5 pg / mL and NFL was 30 pg / mL or more, there were many subjects with negative amyloid-β PET examination results, and when p-Tau181 was less than 1.45 to 3.5 pg / mL and NFL was less than 0 to 30 pg / ml, there were many subjects with positive amyloid-β PET examination results.
[0057] From this, it was judged that more accurate diagnosis was possible by performing diagnosis based on the matrix shown in Table 2 as a new diagnostic criterion.
Table 2
[0058] Next, when the diagnostic sensitivity was set to 100% (i.e., the sensitivity with no false negatives), the diagnostic accuracy by the combination of p-Tau181 and NFL was compared with the case of diagnosis with p-Tau181 alone. As a result, the specificity of the diagnosis by the combination of p181Tau and NFL obtained when using the above cut-off value was 70%, and the diagnostic accuracy increased by 23% compared to the specificity of 47% in the case of diagnosis with p-Tau181 alone. Therefore, when the cut-off value for diagnosing without false negatives was set with the combination marker of p-Tau181 and NFL, it was shown that the diagnostic accuracy increased significantly compared to the case of diagnosis with p-Tau181 alone.
[0059] <Differential diagnosis by the combination of pTau and NFL> For patients who have been diagnosed with some kind of neurodegenerative disease in clinical practice, evaluating whether they have amyloid beta accumulation in the brain is a very important indicator when administering a drug that targets amyloid beta accumulation in the brain. Therefore, among the 94 subjects enrolled this time, 57 subjects who were diagnosed with some kind of neurodegenerative disease in clinical diagnosis before PET were examined whether the combination of pTau, NF, and L disclosed herein can accurately predict amyloid beta accumulation in the brain, compared with the evaluation of p-Tau181 alone. The clinical diagnoses of the 57 subjects (29 men, 28 women, age: 69.7±9.0 years) were as follows: MCI (14), AD (19), CBS (4), FTLD (10), PD (2), PSP (4), and geriatric psychiatric disorders (4). The amyloid beta PET test results were negative in 31 cases and positive in 26 cases.
[0060] The results of the evaluation of the above 57 subjects using the above cutoff values are shown in Table 3. As a result of differential diagnosis using p-Tau181 alone, 9 subjects were negative (9 in the negative group, 0 in the positive group) and 48 subjects were positive (22 in the negative group, 26 in the positive group), with diagnostic sensitivity and specificity of 100% and 29%, respectively. On the other hand, as a result of differential diagnosis using the combination of p-Tau181 and NFL, 20 subjects were negative (20 in the negative group, 0 in the positive group) and 37 subjects were positive (11 in the negative group, 26 in the positive group), with diagnostic sensitivity and specificity of 100% and 65%, respectively. That is, the diagnostic accuracy of the diagnosis using the combination of p-Tau181 and NFL was 36% higher than that of the diagnosis using p-Tau181 alone. This demonstrated that when the sensitivity was set at 100%, the combination of p-Tau181 and NFL increased the accuracy of differential diagnosis compared to p-Tau181 alone, demonstrating the usefulness of the combination marker. [Table 3]
[0061] In addition, 37 healthy subjects (33 cases in the negative group and 4 cases in the positive group) were evaluated in the same manner as above, and the results are shown in Table 4. As a result of differential diagnosis using p-Tau181 alone, 21 subjects were negative (21 in the negative group and 0 in the positive group) and 16 subjects were positive (12 in the negative group and 4 in the positive group), with diagnostic sensitivity and specificity of 100% and 64%, respectively. On the other hand, as a result of differential diagnosis using the combination of p-Tau181 and NFL, 25 subjects were negative (25 in the negative group and 0 in the positive group) and 12 subjects were positive (8 in the negative group and 4 in the positive group), with diagnostic sensitivity and specificity of 100% and 76%, respectively. That is, the diagnosis using the combination of p-Tau181 and NFL had a diagnostic accuracy 12% higher than that using p-Tau181 alone. [Table 4] [Industrial Applicability]
[0062] Since amyloid-β PET testing is expensive and facilities that can perform the testing are limited, and since the measurement of amyloid-β and tau biomarkers in cerebrospinal fluid is an invasive method, the present disclosure can provide a method for evaluating the accumulation of amyloid-β in the brain that is useful as a primary screening to replace these methods and has high diagnostic accuracy. Currently, development of drugs targeting the accumulation of amyloid-β in the brain is progressing, and since this disclosure can be an important indicator for providing such drugs to more patients who want treatment, it is expected that this disclosure will lead to a reduction in national medical expenses and an improvement in access to medicines.
Claims
1. A method for evaluating brain amyloid beta accumulation, comprising: measuring the amount of phosphorylated Tau (p-Tau) selected from the group consisting of p-Tau 181, p-Tau 217, p-Tau 231 and combinations thereof, and the amount of neurofilament light chain (NFL) in a blood sample obtained from the subject; comparing the measured value obtained in the step with a control value; A method comprising:
2. The method of claim 1 , wherein the control value is a predetermined cutoff value.
3. 3. The method of claim 2, wherein the cut-off value is determined by comparing the p-Tau and NFL measurements in blood samples obtained from control subjects with the p-Tau and NFL measurements in blood samples obtained from subjects with brain amyloid-β accumulation, respectively.
4. The method according to claim 2 or 3, wherein the cut-off value of the p-Tau and / or the cut-off value of the NFL is one or more.
5. The method of any one of claims 1 to 4, wherein the p-Tau is p-Tau181.
6. The method according to any one of claims 1 to 5, wherein the blood sample is whole blood, serum or plasma.
7. A method for assisting in the diagnosis of Alzheimer's disease (AD), mild cognitive impairment in the setting of AD, or preclinical AD, comprising: measuring the amount of phosphorylated Tau (p-Tau) selected from the group consisting of p-Tau 181, p-Tau 217, p-Tau 231 and combinations thereof, and the amount of neurofilament light chain (NFL) in a blood sample obtained from the subject; comparing the measured value obtained in the above step with a control value to evaluate the accumulation of amyloid β in the brain; A method comprising:
8. The method of claim 7 , wherein the control value is a predetermined cutoff value.
9. 9. The method of claim 8, wherein the cut-off value is determined by comparing the p-Tau and NFL measurements in blood samples obtained from control subjects with the p-Tau and NFL measurements in blood samples obtained from subjects with brain amyloid-β accumulation, respectively.
10. The method according to claim 8 or 9, wherein the cut-off value of the p-Tau and / or the cut-off value of the NFL is one or more.
11. The method of any one of claims 7 to 10, wherein the p-Tau is p-Tau181.
12. The method according to any one of claims 7 to 11, wherein the blood sample is whole blood, serum or plasma.
13. A kit for assessing accumulation of amyloid beta in the brain, comprising an antibody that specifically binds to p-Tau selected from the group consisting of phosphorylated tau (p-Tau) 181, p-Tau 217, p-Tau 231, and combinations thereof, and an antibody that specifically binds to neurofilament light chain (NFL).
14. A kit for assessing accumulation of amyloid beta in the brain, comprising an antibody that specifically binds to p-Tau selected from the group consisting of phosphorylated tau (p-Tau) 181, p-Tau 217, p-Tau 231 and combinations thereof, and used in conjunction with a kit comprising an antibody that specifically binds to neurofilament light chain (NFL).
15. A kit for assessing accumulation of amyloid beta in the brain, comprising an antibody that specifically binds to neurofilament light chain (NFL), and used in conjunction with a kit comprising an antibody that specifically binds to phosphorylated tau (p-Tau) 181, p-Tau 217, p-Tau 231, and combinations thereof.
16. The kit according to any one of claims 13 to 15, wherein the p-Tau is p-Tau181.