WO2023068173A1

WO2023068173A1 - BIOMARKER FOR EVALUATING INTRACEREBRAL AMYLOID-β ACCUMULATION

Info

Publication number: WO2023068173A1
Application number: PCT/JP2022/038295
Authority: WO
Inventors: 義明佐藤; 大介伊東; 將三村; 鍾玉文; 肇田渕
Original assignee: エーザイ・アール・アンド・ディー・マネジメント株式会社; 慶應義塾
Priority date: 2021-10-18
Filing date: 2022-10-14
Publication date: 2023-04-27

Abstract

In view of the problems of the prior art, the present invention addresses the problem of providing a biomarker for evaluating intracerebral amyloid-β accumulation at low cost and with high diagnostic precision, and a method for evaluating intracerebral amyloid-β accumulation using said biomarker. This method for evaluating intracerebral amyloid-β accumulation includes: a step for measuring the amount of a phosphorylated tau (p-Tau) selected from the group consisting of p-Tau181, p-Tau217, p-Tau231, and a combination thereof, and the amount of neurofilament light chain in a blood sample obtained from a subject; and a step for comparing the measurement value obtained in the aforementioned step with a control value.

Description

A biomarker for assessing brain amyloid-β accumulation

The present disclosure relates to biomarkers for evaluating the accumulation of brain amyloid β.

Amyloid β is a type of protein produced in the brain, and the accumulation of amyloid β in the brain is believed to be closely related to the development of Alzheimer's disease (AD). Tau protein (also simply referred to as "tau") is also a type of protein produced in nerve cells such as the brain. Aberrant phosphorylation is believed to be involved in AD.

Accumulation of amyloid-β and abnormally phosphorylated tau (p-Tau) in the brain is clinically assessed by radiological studies such as positron emission tomography (PET) or by biomarkers of amyloid-β and tau in cerebrospinal fluid. be done. However, these detection methods are of limited utility as diagnostic tools due to their high invasiveness and high cost.

As a less invasive and low-cost method, the diagnosis of AD using biomarkers in blood or the evaluation of brain amyloid β accumulation has been proposed. For example, Non-Patent Document 1 discloses that the plasma p-tau-217/T217 ratio and p-tau-217 value distinguish between the amyloid β-negative group and the amyloid β-positive group, regardless of cognitive function. It is 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 AD screening, and that plasma p-Tau181 can be used to select individuals determined to be amyloid β-positive by PET. Non-Patent Document 4 discloses that plasma p-Tau181 can distinguish AD from young adults and elderly without cognitive impairment. Non-Patent Document 5 discloses that plasma p-Tau181 sorts 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.

Although these evaluation methods using phosphorylated tau in plasma are somewhat useful for screening Alzheimer's disease, the accuracy of evaluation of brain amyloid β accumulation is not necessarily high. Therefore, there is a great need for biomarkers to accurately assess brain amyloid-β accumulation.

On the other hand, there is also a report on prediction of progression to AD in patients with mild cognitive impairment (MCI) using a combined biomarker of plasma p-Tau181 and neurofilament light chain (NFL) (Non-Patent Document 7).

In view of the problems of the prior art, the present disclosure provides a low-cost, high-accuracy biomarker for evaluating brain amyloid β accumulation and a method for evaluating brain amyloid β accumulation using the biomarker. intended to provide

The present inventors have made intensive studies to solve the above problems, and as a result, the combination of phosphorylated tau 181 (p-Tau181) and neurofilament light chain (NFL) in plasma is useful for evaluating the accumulation of amyloid β in the brain. They are useful biomarkers, and it was found that the accumulation of brain amyloid β can be evaluated with high diagnostic accuracy by using these two biomarkers.

In one aspect, the disclosure provides a method for assessing brain amyloid-β accumulation, comprising phosphorylated Tau (p-Tau) 181, p-Tau217, p-Tau231 in a blood sample obtained from a subject. and a combination thereof, measuring the amount of p-Tau and the amount of neurofilament light chain (NFL), and comparing the measured value obtained in the above step with a control value. Provide a method that includes This method is a low-cost and minimally invasive method, and enables a method of evaluating brain amyloid-β accumulation with high diagnostic accuracy. A combination of predetermined p-Tau (p-Tau181, p-Tau217, p-Tau231) and NFL enables examination without failure while maintaining high specificity.

Accordingly, the present disclosure provides, for example, [1] to [26] below.
[1]
A method for evaluating the accumulation of brain amyloid β, comprising:
The amount of p-Tau selected from the group consisting of phosphorylated Tau (p-Tau) 181, p-Tau217, p-Tau231 and combinations thereof, and neurofilament light chain (NFL ) and
and comparing the measured value obtained in the above step with a control value.
[2]
The method of [1], wherein the control value is a predetermined cutoff value.
[3]
The cut-off values are the measured values of the p-Tau and the NFL in a blood sample obtained from a control subject, and the p-Tau and the p-Tau in a blood sample obtained from a subject with amyloid β accumulation in the brain. The method of [2], wherein the method is determined by comparing the measured values of NFL above.
[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 of aiding the diagnosis of Alzheimer's disease (AD), mild cognitive impairment in the setting of AD or preclinical AD, comprising:
The amount of p-Tau selected from the group consisting of phosphorylated Tau (p-Tau) 181, p-Tau217, p-Tau231 and combinations thereof, and neurofilament light chain (NFL ) and
and comparing the measured value obtained in the above step with a control value to evaluate the accumulation of brain amyloid β.
[11]
The method of [10], wherein the control value is a predetermined cutoff value.
[12]
The cut-off values are the measured values of the p-Tau and the NFL in a blood sample obtained from a control subject, and the p-Tau and the p-Tau in a blood sample obtained from a subject with amyloid β accumulation in the brain. The method of [11], determined by comparing each of the NFL measurements.
[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 one of [10] to [13], wherein the p-Tau is a combination of p-Tau181, p-Tau217 and p-Tau231.
[18]
The method of any one of [10]-[17], wherein the blood sample is whole blood, serum, or plasma.
[19]
A method for obtaining an index for assisting diagnosis of Alzheimer's disease (AD), mild cognitive impairment in the background of 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;
and comparing the measured value obtained in the above step with a control value to obtain information on the accumulation of brain amyloid β.
[20]
A kit for evaluating the accumulation of brain amyloid β, 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 specific to neurofilament light chain (NFL) and an antibody that binds to.
[21]
A kit for evaluating the accumulation of brain amyloid β, comprising:
phosphorylated tau (p-Tau) 181, p-Tau217, p-Tau231, and a neurofilament light chain (NFL) comprising an antibody that specifically binds to p-Tau selected from the group consisting of combinations thereof; A kit for use with a kit containing an antibody that specifically binds to.
[22]
A kit for evaluating the accumulation of brain amyloid β, comprising:
p-Tau comprising an antibody that specifically binds to a neurofilament light chain (NFL) and is selected from the group consisting of phosphorylated tau (p-Tau) 181, p-Tau217, p-Tau231 and combinations thereof A kit for use with a kit containing an antibody that specifically binds to.
[23]
The kit according to any one of [20] to [22], wherein the p-Tau is p-Tau181.
[24]
The kit according to any one of [20] to [22], wherein the p-Tau is p-Tau217.
[25]
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.

The present disclosure also provides, for example, [27] to [45] below.
[27]
A method of treating Alzheimer's disease (AD), mild cognitive impairment in the setting of AD or preclinical AD, comprising:
The amount of p-Tau selected from the group consisting of phosphorylated Tau (p-Tau) 181, p-Tau217, p-Tau231 and combinations thereof, and neurofilament light chain (NFL ) and
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;
administering a therapeutically effective amount of a therapeutic agent for Alzheimer's disease, mild cognitive impairment in the setting of AD, or preclinical AD to a subject assessed for accumulation of brain amyloid beta.
[28]
The method of [27], wherein said control value is a predetermined cutoff value.
[29]
The cut-off values are the measured values of the p-Tau and the NFL in a blood sample obtained from a control subject, and the p-Tau and the p-Tau in a blood sample obtained from a subject with amyloid β accumulation in the brain. The method of [28], determined by comparing each of the above NFL measurements.
[30]
The method of [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 of any of [27]-[30], wherein the p-Tau is a combination of p-Tau181, p-Tau217 and p-Tau231.
[35]
The method of any of [27]-[34], wherein the blood sample is whole blood, serum, or plasma.
[36]
The subject evaluated for accumulation of brain amyloid β is diagnosed with Alzheimer's disease, mild cognitive impairment in the background of AD, or preclinical AD before administration of the therapeutic agent, [27]- The method according to any one of [35].
[37]
A method of diagnosing Alzheimer's disease (AD), mild cognitive impairment in the setting of AD or preclinical AD, comprising:
The amount of p-Tau selected from the group consisting of phosphorylated Tau (p-Tau) 181, p-Tau217, p-Tau231 and combinations thereof, and neurofilament light chain (NFL ) and
and comparing the measured value obtained in the above step with a control value to evaluate the accumulation of brain amyloid β.
[38]
The method of [37], wherein said control value is a predetermined cutoff value.
[39]
The cut-off values are the measured values of the p-Tau and the NFL in a blood sample obtained from a control subject, and the p-Tau and the p-Tau in a blood sample obtained from a subject with amyloid β accumulation in the brain. The method of [38], determined by comparing each of the above NFL measurements.
[40]
The method of [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 one of [37] to [40], wherein the p-Tau is p-Tau181.
[42]
The method according to any one 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 of any of [37]-[40], wherein said p-Tau is a combination of p-Tau181, p-Tau217 and p-Tau231.
[45]
The method of any of [37]-[44], wherein the blood sample is whole blood, serum, or plasma.

The method of evaluating the accumulation of amyloid β in the brain of the present disclosure not only has high diagnostic accuracy, but also is low cost, so the range of use can be greatly expanded, and large-scale utilization such as initial diagnosis and health checkup is possible. Be expected. Therefore, in particular, as a primary screening for amyloid β test, a high true positive rate and diagnostic accuracy can be achieved, and it can be a promising evaluation method for drug discovery based on amyloid β accumulation in the brain.

According to the kit of the present disclosure, accumulation of brain amyloid β can be evaluated with high diagnostic accuracy and at low cost.

FIG. 3 is a diagram showing the concentration of p-Tau181 in the blood of 60 cases in the negative group and 34 cases in the positive group in an amyloid β PET test. FIG. 2 is a diagram showing the concentrations of p-Tau181 and NFL in the blood of 60 cases in the negative group and 34 cases in the positive group in an amyloid β PET test.

[Method for evaluating the accumulation of amyloid β in the brain]
In one embodiment, the method of assessing the accumulation of brain amyloid-β comprises phosphorylated Tau181 (p-Tau181), phosphorylated Tau217 (p-Tau217), phosphorylated Tau231 ( measuring the amount of p-Tau selected from the group consisting of p-Tau231) and combinations thereof, and the amount of neurofilament light chain (NFL), and comparing the measured value obtained in the above step with a control value. and the step of

Here, a subject means mammals including, but not limited to, humans, chimpanzees, orangutans, gorillas, baboons, monkeys, mice, rats, pigs, horses, dogs, and cows. The subject is human in one embodiment.

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. A blood sample may be whole blood, serum, or plasma, and may be a blood sample obtained from a human subject.

Amyloid β (also referred to as “Aβ”) is a type of protein made in the brain known to those skilled in the art. Assessing the accumulation of cerebral amyloid β means determining whether or not a greater accumulation of cerebral amyloid β is observed in comparison with control subjects. Accumulation of cerebral amyloid β means that amyloid β is not excreted in the brain and accumulates in the brain. A control subject means a brain amyloid β-negative subject without accumulation of brain amyloid β. Accumulation of brain amyloid-β is conventionally assessed clinically by radiological studies such as positron emission tomography (PET) or by biomarkers of amyloid-β and phosphorylated tau in cerebrospinal fluid. For example, in an amyloid β PET examination using the PET probe ¹⁸ F-Florbetaben, if the accumulation of brain amyloid β is found to be greater than the diagnostic criteria according to the visual judgment criteria of the ¹⁸ F-Florbetaben manufacturer, brain amyloid β If the test is positive and the accumulation of brain amyloid β is less than the diagnostic criteria, it is determined to be negative for brain amyloid β.

Accumulation of amyloid β in the brain is considered to be one of the main pathologies mainly seen in Alzheimer's disease (AD) and mild cognitive impairment (MCI due to AD) in the background of AD. There are many patients who do not have amyloid-β accumulation in the brain, and the gap between clinical diagnosis and pathological diagnosis is an issue. Also, corticobasal ganglia syndrome (CBS), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), Parkinson's disease or Parkinson's syndrome (PD), usually not associated with accumulation of amyloid β in the brain ), or geriatric psychiatric disorders, which may also be associated with accumulation of brain amyloid-β in clinically diagnosed patients. Furthermore, although cognitive function is normal, accumulation of cerebral amyloid β may occur, and such a preclinical stage of Alzheimer's disease is called preclinical AD. If the evaluation method of the present embodiment is positive, accumulation of brain amyloid β is considered to be suspected regardless of the clinically diagnosed disease name. Therefore, the evaluation method of this embodiment is useful as a simple primary screening method for the accumulation of brain amyloid β. 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.

The 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 , in one embodiment p-Tau181. These p-Tau are biomarkers for evaluating the accumulation of brain amyloid β, and can improve diagnostic accuracy when used in combination with neurofilament light chain (NFL). Tau is a protein known to those skilled in the art that is expressed in neurons and glial cells of the central and peripheral nervous systems. Phosphorylation of tau takes place in the brain, and some abnormal phosphorylation is known. Among them, p-Tau181 is phosphorylated Tau threonine 181, p-Tau217 is Tau threonine 217 phosphorylated, and p-Tau231 is Tau threonine 231 phosphorylated. is.

When using p-Tau in combination as a measurement target, the amount of any two of p-Tau181, p-Tau217, and p-Tau231 may be measured, and all three p-Tau may be measured. Two p-Tau combinations include p-Tau181 and p-Tau217, p-Tau181 and p-Tau231, and p-Tau217 and p-Tau231.

The amount of p-Tau, ie, p-Tau181, p-Tau217, and p-Tau231, in a blood sample can be measured according to known protein quantification methods. For example, absorbance measurement and immunological measurement methods using antigen-antibody reaction such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), immunofluorescence method, immunoprecipitation method, and , liquid chromatography/mass spectrometry (LC/MS). In addition, it can be easily quantified using a commercially available measurement kit such as SIMOA (registered trademark) p-Tau181 Advantage V2 Kit (manufactured by Quanterix). Here, the amount of p-Tau is a measured value (absolute value).

Neurofilament light chain (NFL) is known to be a biomarker of nerve axonal damage, and is not known to be used to assess brain amyloid β accumulation. Neurofilament (NF) is mainly composed of three types of subunits: NFL (low or light; light chain), NFM (medium; medium chain), NFH (heavy or high; heavy chain). light chain.

The amount of NFL in a blood sample can be measured according to known protein quantification methods, similar to p-Tau. Examples thereof include absorbance measurement, immunological measurement methods using antigen-antibody reactions such as ELISA, EIA, RIA, immunofluorescence, 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 (manufactured by Quanterix). Here, the amount of NFL is a measured value (absolute value).

"Control value" herein refers to both the p-Tau control value and the NFL control value unless otherwise specified. The control value is a value for predicting accumulation of brain amyloid β, and can be a predetermined cutoff value. In one embodiment, the predetermined cutoff value is a cutoff value determined by the same assay used in the measuring step to measure the amount of p-Tau and NFL. Also, in another embodiment, the predetermined cutoff value is a cutoff value determined by an assay different from the measuring assay used in the measuring step of measuring the amounts of p-Tau and NFL.

Unless otherwise specified, the "cutoff value" in this specification refers to both the p-Tau cutoff value and the NFL cutoff value. The cut-off values were obtained by combining the measured values of p-Tau and NFL in blood samples obtained from control subjects and the measured values of p-Tau181 and NFL in blood samples obtained from subjects with brain amyloid β accumulation. It may be determined by comparing each. Subjects with brain amyloid-β accumulation are brain amyloid-β-positive subjects by conventional diagnostic methods, eg, amyloid-β PET, and control subjects are brain amyloid-β-negative subjects.

The cut-off value may be determined by the desired diagnostic sensitivity. The diagnostic sensitivity can be arbitrarily set depending on the purpose of evaluation, and can be set to 70%, 80%, 90%, 95% or 100%, for example. Since the evaluation method of the present disclosure is expected as a pre-diagnosis of amyloid β PET examination, which is a definitive diagnosis of cerebral amyloid β accumulation, blood before amyloid β PET was used for the purpose of appropriate treatment of more subjects. Preliminary tests are considered clinically necessary with as few false negatives as possible. Therefore, in one embodiment, the sensitivity may be 100% (ie sensitivity with no false negatives).

A predetermined A cut-off value can be determined according to the sensitivity. Also, the p-Tau cutoff value and the NFL cutoff value may each be one, or two or more, for example, two, three, or four.

Specifically, the cut-off value is determined, for example, as shown in FIG. 2, p-Tau (p-Tau181 in FIG. 2) and Measurements of NFL and p-Tau and NFL in blood samples obtained from subjects with brain amyloid-β accumulation (cerebral amyloid-β positive subjects) are graphed to obtain the desired sensitivity (e.g., 100%), cut-off values (2 for p-Tau181 and 1 for NFL in FIG. 2) can be derived. The measured values of p-Tau and NFL depend on the measurement method, so if the cutoff value is determined in advance for one measurement method (assay), it is not necessary to measure and determine each time. .

Also, in one embodiment, when determining the cutoff value, the population of control subjects and subjects with brain amyloid β accumulation is a population having a statistically significant number. For example, each number may be 10 or more, and as a further example, 30 or more, 50 or more, or even 100 or more.

In the step of evaluating the accumulation of cerebral amyloid β by comparing the measured value obtained in the above measurement step with the control value, the combination of p-Tau and NFL is evaluated. When there is one p-Tau (eg, p-Tau is p-Tau181) and the cutoff values of p-Tau and NFL are one, for example, the cutoff value is p - If the Tau181 cutoff value is 1 and the NFL cutoff value is 1, the p-Tau181 measured value is p-Tau181 cutoff value 1 or more, and the NFL measured value is NFL cutoff value 1 or more In some cases, the subject may be determined as positive for brain amyloid β, and the others may be determined as negative for brain amyloid β.

Among the cutoff values of p-Tau181 and NFL, if one cutoff value is 1 and the other cutoff value is 2 or more, for example, the cutoff value is the NFL cutoff value 1 and p-Tau181 cutoff value 1 and p-Tau181 cutoff value 2 (where p-Tau181 cutoff value 1 < p-Tau181 cutoff value 2), the measured value of p-Tau181 is p-Tau181 If the cutoff value is 2 or more, and the measured value of NFL is less than the NFL cutoff value 1, and the measured value of p-Tau181 is p-Tau181 cutoff value 1 or more p-Tau181 cutoff value If it is less than 2, it may be determined as brain amyloid β-positive, and otherwise, it may be determined as brain amyloid β-negative. If each cutoff value is 2 or more, it is assumed. Furthermore, when using two or more p-Tau in combination, set the cutoff value of each p-Tau according to the above, in combination with the cutoff value of NFL, or the total amount of each p-Tau , and combined with the NFL cutoff value for determination.

The evaluation (judgment) process may be performed by humans according to the above criteria, or may be performed automatically using a computer system. When using a computer system, control values (one or more cutoff values for each biomarker, respectively) may be pre-entered into the system or may be entered at the same time as the measurements. After the measurement and cutoff values are entered, the system compares the measurement and cutoff values and outputs "+ (positive)" or "- (negative)" according to the algorithm.

By using the combination of p-Tau and NFL, the evaluation method of the present embodiment has 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. According to this, the evaluation method of the present embodiment can provide true positive results and high diagnostic accuracy, and thus can be used for primary screening.

The evaluation method of the present disclosure can be clinically used as a preliminary examination (primary screening). Subjects determined to be intracerebral amyloid β-positive by this evaluation method are preferably subjected to further detailed examination/diagnosis and appropriate treatment.

[Method for Assisting Diagnosis of Alzheimer's Disease (AD), Mild Cognitive Impairment in the Background of AD, or Preclinical AD]
In one embodiment, a method to aid in diagnosing Alzheimer's disease (AD), mild cognitive impairment in the setting of AD, or preclinical AD comprises p-Tau181, p-Tau217, p- A step of measuring the amount of p-Tau selected from the group consisting of Tau231 and combinations thereof and the amount of NFL, and comparing the measured value obtained in the above step with a control value to determine the accumulation of brain amyloid β and evaluating.

If the accumulation of amyloid β in the brain is determined (evaluated) as positive, it is highly likely that you have Alzheimer's disease (AD), mild cognitive impairment with AD (MCI due to AD), or preclinical AD. Accordingly, the methods of the present disclosure are useful for primary screening for AD or MCI due to AD or preclinical AD.

In one embodiment, a method of obtaining an index to aid in the diagnosis of Alzheimer's disease (AD), mild cognitive impairment in the background of AD, or preclinical AD comprises p-Tau181, p - measuring the amount of p-Tau selected from the group consisting of Tau217, p-Tau231 and combinations thereof, and the amount of NFL; and obtaining information on the accumulation of internal amyloid β. In one embodiment, the indicator is information of amyloid-β accumulation in the subject's brain.

[Kit for evaluating the accumulation of amyloid β in the brain]
In one embodiment, the kit for evaluating accumulation of brain amyloid β comprises p-Tau selected from the group consisting of phosphorylated tau (p-Tau) 181, p-Tau217, p-Tau231, and combinations thereof. Antibodies that specifically bind and antibodies that specifically bind to a neurofilament light chain (NFL).

In another embodiment, the kit for assessing brain amyloid-β accumulation comprises p-Tau selected from the group consisting of phosphorylated tau (p-Tau) 181, p-Tau217, p-Tau231 and combinations thereof. and includes an antibody that specifically binds to a neurofilament light chain (NFL).

A kit for assessing brain amyloid-β accumulation of a further embodiment comprises an antibody that specifically binds to a neurofilament light chain (NFL) and phosphorylated tau (p-Tau) 181, p-Tau 217 , p-Tau231 and combinations thereof.

In one embodiment, p-Tau is p-Tau181.

The above antibody may be any antibody or antigen-binding fragment thereof that specifically binds to p-Tau or NFL, and may be a polyclonal antibody or a monoclonal antibody. Also, in one embodiment, the antibody is a detectably labeled antibody, and labeling can be accomplished by methods known to those skilled in the art, depending on the method of measuring antigen-antibody binding.

The term "specifically binds" is a term well known to those skilled in the art, and methods for determining specific binding of an antibody or antigen-binding fragment thereof to an antigen or epitope are also well known. . In one embodiment, "specifically binds" means, for example, that the anti-p-Tau181 antibody or antigen-binding fragment thereof binds to other target molecules with greater binding affinity, avidity, and more rapidly. and/or for a longer duration to bind to p-Tau181 by an immunological response. In another embodiment, "specifically binds" means, for example, that the anti-p-Tau181 antibody or antigen-binding fragment thereof binds to p-Tau181 by at least about 10 ^-7 M, or at least about 10 ^-8 M, or by an antibody or antigen-binding fragment thereof with a KD of at least about 10 ⁻⁹ M, or at least 10 ⁻¹⁰ M or less. In yet another embodiment, "specifically binds" means that, for example, an anti-p-Tau181 antibody or an antigen-binding fragment thereof binds to p-Tau181 through an immunological reaction, but other p-Tau (eg, p-Tau217, p-Tau231).

Antibodies that specifically bind to p-Tau selected from the group consisting of p-Tau181, p-Tau217, p-Tau231 and combinations thereof are anti-p-Tau181 antibody, anti-p-Tau217 antibody or anti-p-Tau231 It may be any one of the antibodies or an antigen-binding fragment thereof, and any two of the anti-p-Tau181 antibody, anti-p-Tau217 antibody and anti-p-Tau231 antibody (anti-p-Tau181 antibody and anti- p-Tau217 antibody, anti-p-Tau181 antibody and anti-p-Tau231 antibody and p-Tau217 and p-Tau231) or an antigen-binding fragment thereof, anti-p-Tau181 antibody, anti-p-Tau217 antibody and anti- It may be three antibodies of p-Tau231 antibody or antigen binding fragments thereof.

Using the above kit, the amount of p-Tau and NFL is measured, and the amount of amyloid β in the brain is evaluated by comparing the measured value with the control value. Methods for measuring the amounts of p-Tau and NFL are not particularly limited, but examples include ELISA, EIA, RIA, immunofluorescence, immunoprecipitation, radioimmunoassay, chemiluminescence immunoassay, electrochemiluminescence immunoassay, Methods include latex turbidimetric immunoassays, latex photometric immunoassays, immunochromatographic assays, and the like. The kit may contain, in addition to the antibody, any reagents, instruments, or instructions for using the kit that can be used in measuring the amount of p-Tau and NFL. For example, it may contain p-Tau or NFL, which can be used as controls when preparing a standard curve.

[Method for diagnosis and treatment of Alzheimer's disease (AD), mild cognitive impairment in the background of AD or preclinical AD]
In one embodiment, the method of treating Alzheimer's disease (AD), mild cognitive impairment in the setting of AD, or preclinical AD comprises in a blood sample obtained from the subject phosphorylated Tau (p-Tau) 181, p-Tau217, measuring the amount of p-Tau selected from the group consisting of p-Tau231 and combinations thereof and the amount of neurofilament light chain (NFL), and comparing the measured value obtained in the above step with a control value. and administering a therapeutically effective amount of Alzheimer's disease, AD-induced mild cognitive impairment or preclinical and administering an AD therapeutic agent.

The therapeutic agent for Alzheimer's disease, mild cognitive impairment on the background of AD, or preclinical AD is not particularly limited, but includes, for example, anti-Aβ antibodies, agents that inhibit Aβ production or accumulation, anti-tau antibodies, and the like.

The subject evaluated to have accumulated amyloid β in the brain is diagnosed with Alzheimer's disease, mild cognitive impairment in the background of AD, or preclinical AD before administration of the therapeutic agent. Here, the diagnosis is, in one embodiment, a diagnosis comprising the step of evaluating the accumulation of brain amyloid β of the present disclosure, and in another embodiment, a diagnosis comprising a radiological examination such as positron emission tomography (PET) and, in a further embodiment, a diagnosis comprising a radiological examination, such as positron emission tomography (PET), in addition to the step of assessing brain amyloid-β accumulation of the present disclosure.

In one embodiment, the method of diagnosing Alzheimer's disease, mild cognitive impairment in the setting of AD, or preclinical AD comprises: a step of measuring the amount of p-Tau and the amount of NFL selected from the group consisting of; comparing the measured value obtained in the above step with a control value to evaluate the accumulation of brain amyloid β; include.

The present disclosure will be described in detail below with examples, but the present disclosure is not limited to these.

<Clinical diagnosis>
A total of 94 subjects recruited in this study (male: 51, female: 43, age: 69.7 ± 9.0 years old) brain MRI examination and various neuropsychological examinations (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; Patent Document 10), Verbal Fluency (Non-Patent Document 11), Trail Making Test A & B, Japanese Adult Reading Test (JART; Non-Patent Document 12)) were performed and clinically evaluated according to the current consensus diagnostic criteria (Table 1). A diagnosis was made.

As a result, the breakdown of the diseases of the 94 subjects was healthy subjects (37 people), mild cognitive impairment (MCI, 14 people), Alzheimer's disease (AD, 19 people), corticobasal ganglia syndrome (CBS, 4 people). , frontotemporal dementia (FTD, 10), progressive supranuclear palsy (PSP, 4), Parkinson's disease or syndrome (PD, 2), and senile psychosis (4). rice field.

Next, in order to examine the presence or absence of amyloid β accumulation in the brain, ¹⁸ F-Florbetaben, which is a positron emission tomography (PET) probe, was intravenously administered to the subject in an amount equivalent to 300 MBq, and then amyloid Images were taken using a βPET inspection device (True Point Biograph 40/64 (Siemens Asahi Meditec Co., Ltd.)). Determination of brain amyloid β accumulation was performed according to visual criteria by the manufacturer of ¹⁸ F-Florbetaben. The subjects were divided into 34 patients positive for internal amyloid β accumulation (hereinafter simply referred to as "positive group").

<Measurement of biomarkers>
Plasma specimens were then collected from 94 subjects. That is, without breakfast, blood was collected from each subject in the morning and immediately ice-cooled. Subsequently, plasma components were centrifuged at 1200 g×10 minutes and 2800 g×10 minutes within 2 hours after blood collection, and stored in an ultra-low temperature freezer at −80° C. until analysis.

For quantification of biomarkers, p-Tau181 and NFL were obtained using SIMOA® p-Tau181 Advantage V2 Kit (manufactured by Quanterix) or SIMOA® NFL Advantage (SR-X) Kit (manufactured by Quanterix), respectively. was used, and according to the attached protocol, an ultra-sensitive auto ELISA (SIMOA (registered trademark) HD-1 or SR-X) was used as a measuring instrument, and N = 2 samples were used. The measured value of N=2 obtained for each sample was averaged for each sample and used as a measured value, and after correction for the dilution rate (4 times), was used as a quantitative value for each sample. Each measured value was expressed as an absolute value (pg/ml).

<Setting biomarker evaluation criteria>
Diagnosis of the presence or absence of cerebral amyloid β accumulation using blood will be used for pre-diagnosis of amyloid β PET examination, which is a definitive diagnosis of cerebral amyloid β accumulation, if drug development for cerebral amyloid β accumulation becomes common in the future. is expected. For the purpose of appropriate treatment for a larger number of subjects, it is clinically necessary to have as few false negatives as possible in the preliminary blood test before the amyloid β PET test. At present, there are no clinically available test methods with high accuracy and sensitivity, so it is difficult to set criteria (diagnostic criteria) for clinical sensitivity. However, if as many patients with amyloid-β accumulation in the brain as possible are correctly identified as positive, an amyloid-β PET test that provides a definitive diagnosis will be performed, and an environment in which access to appropriate drugs will be established, the benefit to patients will be maximized. Therefore, we decided to evaluate various biomarkers by establishing criteria that can correctly determine positive patients with amyloid β accumulation in the brain.

(Diagnosis by p-Tau)
As a result of the amyloid β PET test, the concentrations of p-Tau181 in the blood of 60 cases of the negative group and 34 cases of the positive group were graphically shown in FIG. As shown in FIG. 1, p-Tau181 in blood was confirmed to be increased in the amyloid β PET test-positive group, reproducing past reports. Considering the sensitivity of 100%, which is the target sensitivity of diagnosis, the cut-off value used in p-Tau diagnosis is around 1.45 pg/ml, and the specificity at this time was found to be 47%. bottom.

(Combination of p-Tau and NFL)
As a result of the amyloid β PET test, the concentrations of p-Tau181 and NFL in the blood of 60 cases of the negative group and 34 cases of the positive group were graphically shown in FIG. As a result, p-Tau181 in blood was 0 to less than 1.45 pg/mL, 1.45 to less than 3.5 pg/mL, 3 groups of 3.5 pg/mL or more, and NFL in blood was 0 to 30 pg/mL. When divided into two groups of less than and 30 pg/mL or more, when p-Tau181 is less than 1.45 to 3.5 pg/mL and NFL is 30 pg/mL or more, many subjects have negative amyloid β PET test results, It was newly found that when p-Tau181 was less than 1.45-3.5 pg/mL and NFL was less than 0-30 pg/ml, many subjects had positive amyloid β PET test results.

Based on this, it was determined that more accurate diagnosis would be possible by performing diagnosis based on the matrix shown in Table 2 as a new diagnostic criterion.

Next, when the diagnostic sensitivity was set at 100% (that is, sensitivity without false negatives), the diagnostic accuracy of the combination of p-Tau181 and NFL was compared with that of p-Tau181 alone. As a result, the specificity of diagnosis by the combination of p-Tau181 and NFL obtained when using the above cutoff value is 70%, and the diagnostic accuracy is higher than 47% of the specificity of diagnosis with p-Tau181 alone. increased by 23%. Therefore, when the cut-off value for diagnosing without false negatives is set by the combination marker of p-Tau181 and NFL, the accuracy of diagnosis is greatly increased compared to the case of diagnosis with p-Tau181 alone. was done.

<Differential diagnosis by combination of p-Tau and NFL>
It is a very important index for administering drugs that target amyloid β accumulation in the brain to evaluate whether or not patients who have been clinically diagnosed as having some type of neurodegenerative disease have amyloid β accumulation in their brains. becomes. Therefore, among the 94 subjects who entered this time, 57 subjects who were diagnosed as having some kind of neurodegenerative disease in the clinical diagnosis before PET also used the combination of p-Tau and NFL in the present disclosure, In comparison with the evaluation of p-Tau181 alone, it was examined whether the accumulation of amyloid β in the brain can be predicted with high accuracy. The breakdown of the clinical diagnosis of 57 subjects (male: 29, female: 28, age: 69.7 ± 9.0 years old) is MCI (14), AD (19), CBS (4 people), FTD (10 people), PD (2 people), PSP (4 people) and senile mental illness (4 people). There were 26 cases in the group.

Table 3 shows the results of evaluating the above 57 subjects using the above cutoff values. As a result of the differential diagnosis of 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), and the sensitivity and specificity of diagnosis were 100% and 29%, respectively. On the other hand, as a result of differential diagnosis by 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 (negative 11 in the group and 26 in the positive group), and the sensitivity and specificity of diagnosis were 100% and 65%, respectively. That is, diagnosis by the combination of p-Tau181 and NFL increased the diagnostic accuracy by 36% over diagnosis by p-Tau181 alone. From this, when the sensitivity is set to 100%, the diagnosis by the combination of p-Tau181 and NFL is compared with the diagnosis by p-Tau181 alone, It is shown that the accuracy of differential diagnosis increases, and the combination marker The usefulness of

Furthermore, 37 healthy subjects (33 cases in the negative group and 4 cases in the positive group) among the subjects were also evaluated in the same manner as above, and the results are shown in Table 4. As a result of the differential diagnosis of p-Tau181 alone, 21 subjects were negative (21 in the negative group, 0 in the positive group), and 16 subjects were positive (12 in the negative group , 4 in the positive group), and the sensitivity and specificity of diagnosis were 100% and 64%, respectively. On the other hand, as a result of the differential diagnosis by the combination of p-Tau181 and NFL, 25 subjects were negative (25 in the negative group, 0 in the positive group), and 12 subjects were positive (negative 8 in the group and 4 in the positive group), and the sensitivity and specificity of diagnosis were 100% and 76%, respectively. That is, diagnosis by the combination of p-Tau181 and NFL increased the diagnostic accuracy by 12% over diagnosis by p-Tau181 alone.

Due to the high cost and limited availability of Aβ PET tests, as well as the invasive method of measuring Aβ and tau biomarkers in cerebrospinal fluid, the present disclosure is a primary alternative to these methods. It is possible to provide a method for evaluating brain amyloid β accumulation that is useful for screening and has high diagnostic accuracy. Currently, the development of drugs that target the accumulation of amyloid β in the brain as a therapeutic target is progressing, and the present disclosure can be an important index for providing such drugs to patients who desire more treatment. It is expected that this will lead to a reduction in national medical expenses and an improvement in access to medicines.

Claims

A method for evaluating the accumulation of brain amyloid β, comprising:
The amount of p-Tau selected from the group consisting of phosphorylated Tau (p-Tau) 181, p-Tau217, p-Tau231 and combinations thereof, and neurofilament light chain (NFL ) and
and comparing the measured value obtained in said step with a control value.
The method according to claim 1, wherein said control value is a predetermined cutoff value.
The cutoff values are the measured values of the p-Tau and the NFL in a blood sample obtained from a control subject, and the p-Tau and the NFL in a blood sample obtained from a subject with accumulation of brain amyloid β. 3. The method of claim 2, determined by comparing each of the NFL measurements.
The method according to claim 2 or 3, wherein the cutoff value of the p-Tau and/or the cutoff value of the NFL is one or more than two.
The method according to any one of claims 1 to 4, wherein the p-Tau is p-Tau181.
The method according to any one of claims 1 to 5, wherein the blood sample is whole blood, serum, or plasma.
A method of aiding the diagnosis of Alzheimer's disease (AD), mild cognitive impairment in the setting of AD or preclinical AD, comprising:
The amount of p-Tau selected from the group consisting of phosphorylated Tau (p-Tau) 181, p-Tau217, p-Tau231 and combinations thereof, and neurofilament light chain (NFL ) and
and comparing the measured value obtained in said step with a control value to assess the accumulation of brain amyloid-β.
The method according to claim 7, wherein said control value is a predetermined cutoff value.
The cutoff values are the measured values of the p-Tau and the NFL in a blood sample obtained from a control subject, and the p-Tau and the NFL in a blood sample obtained from a subject with accumulation of brain amyloid β. 9. The method of claim 8, determined by comparing each of said NFL measurements.
The method according to claim 8 or 9, wherein the cutoff value of the p-Tau and/or the cutoff value of the NFL is one or more than two.
The method according to any one of claims 7 to 10, wherein the p-Tau is p-Tau181.
The method according to any one of claims 7 to 11, wherein the blood sample is whole blood, serum or plasma.
A kit for evaluating the accumulation of brain amyloid β, 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 specific to neurofilament light chain (NFL) and an antibody that binds to.
A kit for evaluating the accumulation of brain amyloid β, comprising:
phosphorylated tau (p-Tau) 181, p-Tau217, p-Tau231, and a neurofilament light chain (NFL) comprising an antibody that specifically binds to p-Tau selected from the group consisting of combinations thereof; A kit for use with a kit containing an antibody that specifically binds to.
A kit for evaluating the accumulation of brain amyloid β, comprising:
p-Tau comprising an antibody that specifically binds to a neurofilament light chain (NFL) and is selected from the group consisting of phosphorylated tau (p-Tau) 181, p-Tau217, p-Tau231 and combinations thereof A kit for use with a kit containing an antibody that specifically binds to.
The kit according to any one of claims 13 to 15, wherein the p-Tau is p-Tau181.