KR20200025195A - Biomarker composition for diagnosing Alzheimer's dementia and use thereof - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing Alzheimer′s dementia and a use thereof. By a composition, a kit, and a method according to one embodiment, early diagnosis of Alzheimer′s dementia is possible with high accuracy using a blood sample. Accordingly, the present invention can be expected to be applied as a core technology in the field of Alzheimer′s dementia diagnosis.

Description

Biomarker composition for diagnosing Alzheimer's dementia and use thereof {@biomarker composition for diagnosing Alzheimer's dementia and use}

A biomarker composition for diagnosing Alzheimer's dementia and a use thereof.

As the aging society enters, various diseases of the elderly are emerging as big social problems. In particular, these diseases are causing a lot of attention due to the huge burden on medical expenses and the national economy, and active research is being conducted. Among them, Alzheimer's dementia (hereinafter referred to as 'AD') is accompanied by a variety of psychological and behavioral disorders with a lame progression of motor and cognitive decline. The damage is very serious, starting from the slightest symptoms in the early stages, and finally going to the point that voluntary personal life is impossible, causing huge economic burdens on the patient, family, and society, including medical expenses, and the loss of the economic population in the country. Alzheimer's dementia is a degenerative brain disease that emerges as a serious social problem, with 10% of people aged 65-74, 19% of people aged 75-84, and 47% of people over 85 years old, with an increasing incidence each year.

On the other hand, Alzheimer's dementia results from Alzheimer's disease and is one of the pathogenesis of Alzheimer's disease, the amyloid hypothesis, specifically, the beta-amyloid protein present in the tissues of patients and their accumulation. It is hypothesized that neural plaques induce continuous death of nerve cells and inhibit brain nerve transmission, causing cognitive impairment and causing disease. Thus, beta-amyloid protein, Aβ42, or acts as a major etiology of the disease of t-Tau, p-Tau181 Levels are used as a criterion to diagnose Alzheimer's dementia. In addition, the diagnosis of Alzheimer's dementia is performed using a neuropsychological test, MRI brain imaging test, genetic mutation test, and Aβ PET test.

 However, these conventional diagnostic methods require expensive examination costs or suffer severe pain in obtaining a sample, thus making it difficult to diagnose Alzheimer's dementia. In particular, it has been reported that the levels of Aβ42 or Aβ40 in cerebrospinal fluid and plasma increase in proportion to age in the animal model of Alzheimer's disease, but decrease as the cognitive impairment begins. Therefore, it is controversial to use the level of amyloid protein in cerebrospinal fluid and the like as a diagnostic index or prediction for onset of Alzheimer's dementia.

Under these technical backgrounds, various researches are being conducted to discover new biomarkers based on blood samples capable of early diagnosis of Alzheimer's dementia (Korea Patent Publication No. 10-2009-0009579).

One aspect is for diagnosing Alzheimer's dementia comprising any one protein from a contact system consisting of FXIIa (Activated factor XII), FXIa (Activated factor XI), FXa (Activated factor X), and Kallikrein. It is to provide a biomarker composition.

Another aspect is to provide a composition for diagnosing Alzheimer's dementia comprising an agent for measuring the activity level of the protein.

Another aspect is to provide a kit for diagnosing Alzheimer's dementia comprising an agent for measuring the activity level of the protein.

Another aspect is to establish an informational method for diagnosing Alzheimer's dementia, comprising measuring any one protein activity level from a contact system consisting of FXIIa, FXIa, FXa, and kallikrein from a subject's sample.

One aspect is a biomarker composition for diagnosing Alzheimer's dementia comprising any one plasma protein from a contact system composed of FXIIa (Activated factor XII), FXIa (Activated factor XI), FXa (Activated factor X), and Kallikrein. To provide.

As used herein, the term "marker" is a substance capable of diagnosing Alzheimer's dementia, and is a polypeptide, nucleic acid, lipid, glycolipid, glycoprotein, sugar (monosaccharide, which is closely related to the onset and / or progression of Alzheimer's dementia). Organic biomolecules such as disaccharides, oligosaccharides, and the like). For example, the marker can be FXIIa, FXIa, FXa, or kallikrein with increased activity compared to the normal control.

As used herein, the term "diagnosis" refers to confirming the presence or characteristics of a pathological state, and may include checking whether Alzheimer's dementia develops or whether Alzheimer's dementia progresses.

Alzheimer's dementia is a pathological phenomenon distinguished from normal aging, and Alzheimer's disease caused by accumulation of beta-amyloid is a major etiology. Currently, a diagnostic technique targeting beta-amyloid protein is commonly used, but development of a new diagnostic technique is needed due to severe pain in obtaining a cerebrospinal fluid sample, difficulty in early diagnosis of Alzheimer's dementia, and the like.

According to one embodiment, FXIIa, FXIa, FXa, or kallikrein with increased activity in plasma is a normal (ND) control, a prognostic stage of Alzheimer's AD and / or Alzheimer's dementia (AD). ) Can be used as a useful indicator or criterion for identifying groups. Thus, FXIIa, FXIa, FXa, or kallikrein with increased activity can be used as a biomarker for diagnosing Alzheimer's dementia.

Another aspect provides a composition for diagnosing Alzheimer's dementia, comprising an agent for measuring any one plasma protein activity level selected from a contact system consisting of FXIIa, FXIa, FXa, and kallikrein.

In the composition for diagnosing Alzheimer's dementia, the same terms or elements mentioned above are the same as those mentioned above.

The measurement of the activity level may be to evaluate the concentration or activity ability (eg, proteolytic activity) of the activated protein, etc., which may be appropriately quantified and evaluated according to conventional methods in the art. As an agent for measuring the activity level, a substrate peptide or an antibody that specifically binds to the activated protein may be applied.

For example, the substrate peptide is substrate S-2302 [HD-Pro-Phe-Arg- p NA] of FXIIa, substrate S-2366 [pyroGlu-Pro-Arg- p NA] of FXIa, substrate HD of kallikrein. -Val-Leu-Arg-AFC, or substrate S-2765 [ZD-Arg-Gly-Arg- p NA] of FXa can be applied. In addition, since the production of an antibody that specifically binds to the protein having a known amino acid sequence corresponds to the obvious matter in the art, antibodies specific for the activating protein may also be applied. Such antibodies may include polyclonal antibodies and recombinant antibodies, and in addition to the full form having the full length of two heavy and two light chains, Fab, F (ab '), which is a functional fragment that retains at least antigen binding function, F (ab ') 2, Fv, and the like.

According to one embodiment, increased activity of FXIIa, FXIa, FXa, or kallikrein in blood samples has been found to be associated with the onset or progression of Alzheimer's dementia. In particular, these activity changes were able to accurately predict even early stages of Alzheimer's dementia, that is, prodromal Alzheimer's dementia. Thus, agents for measuring the activity of FXIIa, FXIa, FXa, or kallikrein can be used to early diagnose Alzheimer's dementia.

In addition, the composition is beta amyloid _1-42 (Aβ _1-42 ), beta amyloid _1-40 (Aβ _1-40 ), t-Tau, p-Tau181 And biomarkers or diagnostic compositions known in the art for diagnosing Alzheimer's dementia, such as agents for measuring any level or concentration selected from the group consisting of a combination thereof. According to one embodiment, in combination with the biomarker or diagnostic composition can improve the reliability or accuracy of the prediction of the onset and progression of Alzheimer's dementia, for example, an agent for measuring the activity level of FXIIa And agents for measuring the levels of beta amyloid _1-42 .

Another aspect provides a kit for diagnosing Alzheimer's dementia, comprising the diagnostic composition.

In the kit for diagnosing Alzheimer's dementia, the same terms or elements mentioned above are the same as those mentioned above.

The kit is, for example, a secondary material labeled with a substrate, a suitable buffer, a chromophore or a fluorophore for the detection of FXIIa, FXIa, FXa, or a substrate peptide that reacts with kallikrein or an antibody that specifically binds. Or chromogenic substrates. The substrate may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, or a glass slide glass. Peroxidase, or alkaline phosphatase may be used as the chromophore. As the fluorescent material, AFC, FITC, or RITC may be used. As the chromogenic substrate, ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), TMB (tetramethyl benzidine) and the like can be used.

In addition, the kit may be, for example, a microarray capable of measuring the mRNA expression level of FXIIa, FXIa, FXa, or kallikrein protein or gene encoding the same. The microarray can be easily prepared by those skilled in the art using the FXIIa, FXIa, FXa, or kallikrein gene according to methods known in the art. The microarray may be one in which cDNA of a sequence corresponding to mRNA or fragment thereof of a gene encoding FXIIa, FXIa, FXa, or kallikrein protein is attached to a substrate as a probe.

In addition, the kit may be a kit including essential elements necessary for performing RT-PCR, for example, when measuring the expression level of mRNA of FXIIa, FXIa, FXa, or kallikrein gene. The RT-PCR kits include enzymes such as test tubes or other suitable containers, reaction buffers, deoxyribonucleotides (dNTPs), Taq-polymerases and reverse transcriptases, in addition to the respective primers specific for the mRNA of the marker gene. Inhibitors, DEPC-water, or sterile water. It may also include primer pairs specific for the genes used as quantitative controls.

Another aspect provides an informational method for diagnosing Alzheimer's dementia, comprising measuring a protein activity level selected from a contact system consisting of FXIIa, FXIa, FXa, and kallikrein from a subject's sample. .

In the information providing method for diagnosing Alzheimer's dementia, the same terms or elements mentioned above are the same as those mentioned above.

As used herein, the term "individual" refers to an individual who wants to confirm the onset or progression of Alzheimer's dementia or to predict the risk of developing it. The subject is not limited if the animal that can develop Alzheimer's dementia, but may specifically be a mammal, for example, human (Homo sapiens).

As used herein, the term "sample" may include tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, samples such as urine, and the like, which are separated from the subject's body, for example, plasma days Can be.

As a method for measuring the activity level, absorbance measurement, western blotting, magnetic bead-antibody immunoprecipitation, ELISA, mass spectrometry, radioimmunoassay, immunoprecipitation method and the like can be used.

The method may comprise comparing the measured activity level with that of a normal control sample. If the activity level in the sample is higher than that of the normal control sample, the subject may determine that Alzheimer's dementia is more likely to develop or progress. The change in activity level is that the activity level of the individual is similar or 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50% compared to the normal control or negative control. Can include increasing over 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% and 1000% . According to an embodiment, when the measured FXIIa concentration in the sample has 26 to 26.5 U / ml or more or 27 to 27.5 U / ml or more, the sample may be Alzheimer's dementia or Alzheimer's dementia, respectively, in a prognostic stage. It can be judged that it is derived from the patient.

In addition, the method comprises a group consisting of beta amyloid _1-42 (Aβ _1-42 ), beta amyloid _1-40 (Aβ _1-40 ), t-Tau, p-Tau181 and combinations thereof from a cerebral spinal fluid sample of an individual. It may further comprise the step of measuring any one level selected from. This step may improve the reliability or accuracy of predicting the onset and progression of Alzheimer's dementia.

In one embodiment, the method comprises measuring the activity level of FXIIa (Activated factor XII) from a subject's sample, and measuring the level of beta amyloid _1-42 (Aβ _1-42 ) from the brain spinal fluid sample of the subject; And calculating, from the measured levels, ratio values of beta amyloid _1-42 (Aβ _1-42 ) levels / active levels of FXIIa (Activated factor XII).

If the ratio value derived from the sample is lower than the level of the normal control sample, the subject may determine that Alzheimer's dementia is more likely to develop or progress. The change in the ratio value indicates that the percentage value of the individual is comparable to the normal control or negative control, or 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%. It may include decreasing by more than 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% and 1000%. . According to one embodiment, when the value of Aβ _1-42 / FXIIa measured in the sample has a 33 to 34 pg / ml or less and 27 to 28 pg / ml or less, the sample is Alzheimer's dementia of the pre-symptom stage, respectively Or from Alzheimer's dementia patients.

According to the composition and method according to one aspect, it is possible to predict the onset or progression of Alzheimer's dementia early by measuring the activity of a specific plasma protein in the blood sample, thereby contributing to improving the efficacy of the conventional treatment or treatment method.

The compositions and methods according to one aspect exhibit high reliability or accuracy in predicting the onset and progression of Alzheimer's dementia, which can be further enhanced through combination with existing biomarkers.

Figure 1 shows a standard curve for measuring the contact system activity in the blood, the results of measuring the activity of (A) FXIIa, (B) FXIa, (C) FXa, and (D) Kallikrein.
2 shows the correlation between the onset and / or progression of Alzheimer's dementia (normal, prodromal AD, AD) and contact system activity, (A) FXIIa, (B) FXIa, (C) FXa, and (D) Kallikrein activity was measured.
Figure 3 is a correlation analysis between FXIIa and Alzheimer's dementia biomarkers, the results confirmed the correlation between (A) Aβ _1-42 , (B) t-Tau and (C) p-Tau181.
4 shows the possibility of using FXIIa as a novel marker for evaluating the onset and / or progression of Alzheimer's dementia. (A) The distinction between normal group (ND) and Alzheimer's dementia group (prodromal AD) ROC curve for, (B) ROC curve for the distinction between normal group (ND) and Alzheimer's dementia group (AD).
5 shows the possibility of using Aβ _1-42 / FXIIa as a novel marker for evaluating the onset and / or progression of Alzheimer's dementia, (A) the normal group (ND) and the Alzheimer's dementia group of prognostic symptoms ( ROC curves for the distinction between prodromal AD) and (B) ROC curves for the distinction between normal (ND) and Alzheimer's dementia (AD).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Construction of Contact Meter Activation Measurement System

First, to measure the activity of FXIIa, FXIa, FXa, or Kallikrein in plasma, each standard was diluted in PBS at the following concentrations (FXIIa: 0, 0.5, 1, 1.5, 2, 2.5, 4). , 10 U / ml, FXIa: 0.02, 0.05, 0.1, 0.2, 0.4, 1 U / ml, FXa: 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1 U / ml, Kallikrein: 0, 0.02, 0.04, 0.08, 0.1, 0.2, 0.4, 0.8, 1, 2, 4 U / ml). Subsequently, 90 μl of diluted standard and 10 μl of 4 mM various synthetic peptide substrates (substrate S-2302 [FX-Pro-Phe-Arg- p NA] of FXIIa, substrate S-2366 [pyroGlu-Pro- of FXIa) Arg- p NA], kallikrein substrate HD-Val-Leu-Arg-AFC, FXa substrate S-2765 [ZD-Arg-Gly-Arg- p NA]) were mixed and reacted at 37 ° C. for 60 minutes. , Absorbance A ₄₀₅ was measured using a 96-well plate leader (Molecular Devices, San Jose, Calif., USA). On the other hand, the substrate of FXIIa, FXIa, or FXa was obtained from Chromogenix (Milan, Italy), the substrate of kallikrein from Merck (Darmstadt, Germany), and the experimental standard curve was prepared using a sigmoidal 4-parameter curve. .

As a result, as shown in A to D of FIG. 1, in the standard curve for each of FXIIa, FXIa, FXa, or Kallikrein, the R-square values were 0.9694, 0.9916, 0.998, 0.9871, respectively, and all the values were 0.96 or more. . From these experimental results, it can be seen that the measurement system according to the present example has high accuracy and reproducibility with respect to FXIIa, FXIa, FXa or Kallikrein activity.

Example 2. Analysis of FXIIa, FXIa, FXa and Kallikrein Activity in Plasma

90 μl of 10% plasma diluted in PBS and 10 μl of 4 mM various synthetic peptide substrates (substrate S-2302 of FXIIa, substrate S-2366 of FXIa, substrate of kallikrein HD-Val-Leu-Arg-AFC, FXa Substrate S-2765) was mixed and reacted at 37 ° C. for 60 minutes, and then their activity was measured in a 96-well plate leader (Molecular Devices, San Jose, CA, USA). The plasma was obtained from a normal control group (Normal), a prognostic stage of Alzheimer's dementia (Prodromal AD), Alzheimer's dementia (AD). The activity measurement was repeated three times each, after which the absorbance average value was converted to units (U) and multiplied by the dilution factor (10-fold) to calculate the activity of each sample .

As a result, as shown in A to D of Figure 2, the activity of FXIIa increased to 23.9, 28.0, and 30.6 U / ml as the disease progressed to normal, prodromal AD, and AD, FXIa, FXa, or Kallikrein The activity of showed similar tendency as above. The results indicate that the onset and / or progression of Alzheimer's dementia correlates closely with the activity of FXIIa, FXIa, FXa, or Kallikrein.

Example 3 Correlation Analysis between CSF-derived Biomarkers and FXIIa

Correlation analysis was performed between conventional cerebrospinal fluid-derived biomarkers (Aβ _1-42 , t-Tau and p-Tau181) and contact system-derived biomarkers (FXIIa) according to this embodiment. In this experiment, SPSS 24.0 version was used to perform Pearson correlation coefficient analysis.

As a result, as shown in A to C of Figure 3, FXIIa was found to have a significant correlation with cerebrospinal fluid biomarkers of Alzheimer's dementia [Aβ _1-42 (r = -0.463, P <0.01) , t-Tau (r = 0.509, P <0.001), p-Tau181 (r = 0.452; P <0.001)].

Example 4 ROC Curve Curve Analysis for Alzheimer's Dementia Diagnosis

The ROC curve curves of FXIIa related to the diagnosis of Alzheimer's dementia and Alzheimer's dementia at the stage of prognostic symptoms (SPSS 24.0 version) were analyzed to identify the respective AUC, sensitivity and specificity. In addition, as well as the use of FXIIa alone, in combination with the conventional cerebrospinal fluid-derived biomarker Aβ _1-42 , that is, ROC curve curves for the ratio of Aβ _1-42 / FXIIa were also analyzed in the same manner.

As a result, as shown in Table 1 and FIG. 4, the cut-off values of FXIIa for distinguishing the prodromal AD group from the AD group from the normal group were 26.3 U / ml and 27.2 U / ml, respectively. At this time, its sensitivity and specificity were 73.9% and 74.0% for the prodromal AD group and 78.6% and 78.0% for the AD group, showing high confidence in predicting the onset and progression of Alzheimer's dementia. In addition, as shown in Table 1 and FIG. 5, cut-off values of Aβ _1-42 / FXIIa for distinguishing the prodromal AD and AD groups from the normal group were 33.8 pg / ml and 27.4 pg / ml. In particular, its sensitivity and specificity were 87.0% and 86.0% for the prodromal AD group, 100% and 100% for the AD group, and the reliability was remarkably improved.

TABLE 1

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (14)

A biomarker composition for diagnosing Alzheimer's dementia, comprising any one protein selected from a contact system composed of FXIIa (Activated factor XII), FXIa (Activated factor XI), FXa (Activated factor X), and Kallikrein.
Alzheimer's, comprising an agent for measuring any one protein activity level selected from a contact system consisting of FXIIa (Activated factor XII), FXIa (Activated factor XI), FXa (Activated factor X), and Kallikrein Composition for diagnosing sexual dementia.
The diagnostic composition of claim 2, wherein the agent comprises a substrate peptide that reacts with the protein or an antibody that specifically binds to the protein.
The method according to claim 2, any one selected from the group consisting of beta amyloid _1-42 (Aβ _1-42 ), beta amyloid _1-40 (Aβ _1-40 ), t-Tau, p-Tau 181 and combinations thereof Further comprising a formulation for measuring the level of, diagnostic composition.
The composition of claim 4, wherein the composition is an agent for measuring the activity level of FXIIa; And a formulation for measuring the level of beta amyloid _1-42 .
The diagnostic composition of claim 2, wherein the Alzheimer's dementia comprises Alzheimer's dementia in a prognostic stage.
A kit for diagnosing Alzheimer's dementia, comprising the composition of any one of claims 2 to 6.
Measuring a protein activity level of any one selected from a contact system consisting of an activated factor XII (FXIIa), an activated factor XI (FXIa), an activated factor X (Fxa), and a kallikrein from a subject's sample Informational method for diagnosing Alzheimer's dementia.
The method of claim 8, comprising determining that Alzheimer's dementia is when the measured activity level is higher than that of a normal control sample.
The method of claim 8, wherein the sample is blood or plasma.
The method according to claim 8, wherein the Alzheimer's dementia comprises Alzheimer's dementia in a prognostic stage.
The group of claim 8, wherein the group consists of beta amyloid _1-42 (Aβ _1-42 ), beta amyloid _1-40 (Aβ _1-40 ), t-Tau, p-Tau181, and combinations thereof And measuring the level of any one selected from.
The method of claim 12, further comprising: (a) measuring the activity level of FXIIa (Activated Factor XII) from a subject's sample, and measuring the level of beta amyloid _1-42 (Aβ _1-42 ) from the cerebrospinal fluid sample of the subject; And
(b) calculating, from the measured levels, ratio values of beta amyloid _1-42 (Aβ _1-42 ) levels / active levels of FXIIa (Activated factor XII).
The method of claim 13, comprising determining that Alzheimer's dementia is when the calculated ratio value is lower than that of a normal control sample.

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