KR20220153864A - Biomarker composition for early diagnosing of alzheimer's disease and use thereof - Google Patents

Abstract

The present application relates to a biomarker composition for diagnosis of Alzheimer's disease comprising N-methylalanine. A biomarker composition for diagnosing Alzheimer's disease according to one embodiment of the present application can diagnose Alzheimer's disease early by measuring and comparing the level of N-methylalanine in a subject. The composition can be applied to a kit for diagnosing Alzheimer's disease or to the prevention of Alzheimer's disease, or drug screening for treatment.

Description

Biomarker composition for early diagnosis of Alzheimer's disease and its use

The present application relates to a biomarker composition for early diagnosis of Alzheimer's disease and its use.

Currently, as the elderly population increases rapidly in Korea, brain diseases such as dementia and stroke, which are caused by the elderly, are increasing. Among them, Alzheimer's disease is the most common type of dementia, and it is a representative degenerative brain disease that makes daily life impossible by showing symptoms such as memory loss, cognitive ability loss, and motor function paralysis.

The pathogenesis and mechanism of Alzheimer's disease are not yet known precisely. The part that can be explained only by genotype is very limited, and the interaction between various risk factors and the development of complex diseases is also not known.

In Alzheimer's disease, appropriate emergency response at the onset is important for the prognosis of the disease, but early diagnosis is impossible with the currently used neuropsychological tests, blood tests, brain imaging tests, and genetic tests. The most commonly used magnetic resonance imaging (MRI) test is not only expensive, but can be confirmed only when brain atrophy has progressed considerably, and methods for detecting beta-amyloid protein and tau protein in cerebrospinal fluid or blood also have problems such as clinical accuracy. is being raised, and it is necessary to develop new early diagnostic markers.

Accordingly, the present inventors have made diligent efforts to develop a marker capable of diagnosing Alzheimer's disease at an early stage, and as a result, have developed a methylation metabolite biomarker capable of diagnosing Alzheimer's disease simply and quickly, thereby completing the present invention.

Korean Registered Patent Publication No. 10-2078500 Korean Registered Patent Publication No. 10-2164490

The present application is to provide a biomarker composition for diagnosing Alzheimer's disease containing N-methylalanine.

However, the problem to be solved by the present application is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

A first aspect of the present application provides a biomarker composition for diagnosing Alzheimer's disease comprising N-methylalanine.

The second aspect of the present application is,

(a) providing a biological sample of a subject;

(b) measuring the level of N-methylalanine in the biological sample; and

(c) providing a method for diagnosing Alzheimer's disease, comprising comparing the measured level of N-methylalanine with the measured value of a normal control sample.

A third aspect of the present application provides a kit for diagnosing Alzheimer's disease, including a quantification device for N-methylalanine.

The fourth aspect of the present application is,

(a) treating an individual suspected of having Alzheimer's disease with a candidate substance and measuring the level of N-methylalanine; and

(b) When the concentration of N-methylalanine is increased, a method for screening a drug for preventing or treating Alzheimer's disease is provided, comprising selecting the candidate substance as a drug for preventing or treating Alzheimer's disease.

The biomarker composition for diagnosing Alzheimer's disease according to one embodiment of the present application can be used to measure and compare the level of N-methylalanine in a subject, thereby diagnosing Alzheimer's disease at an early stage. It can be applied to drug screening for disease prevention or treatment.

1a to 1f show six brain regions (cerebellum, dorsal cortex, frontal lobe, hippocampus, brain stem, striatum) among 252 brain tissue-derived metabolites, in order to select age-specific disease-specific metabolites, normal group It is a diagram showing a volcano plot expressing metabolites with p-value and fold change (x-axis: fold change, y-axis: p-value) based on the Mann-Whitney test by comparing and childhood Alzheimer's disease group.
2a to 2f, in order to select disease-specific metabolites by six brain regions (cerebellum, dorsal cortex, frontal lobe, hippocampus, brain stem, striatum) among 252 brain tissue-derived metabolites, normal group It is a diagram showing a volcano plot expressing metabolites with p-value and fold change (x-axis: fold change, y-axis: p-value) based on the Mann-Whitney test by comparing the old Alzheimer's disease group.
3A and 3B are dot plots specifying N-methylalanine that was statistically significantly reduced in Alzheimer's disease compared to normal groups in all age groups and brain regions (hippocampus, frontal lobe, striatum, dorsal cortex, cerebellum, brain stem). .
4a and 4b are diagrams showing ROC curve graphs evaluating biomarker performance of N-methylalanine in all age groups and brain regions (hippocampus, frontal lobe, striatum, dorsal cortex, cerebellum, brain stem).
5A and 5B are ROC curves evaluating the performance of N-methylalanine as a biomarker in blood and dot plots showing a decrease in Alzheimer's disease compared to a normal group.

Hereinafter, embodiments of the present application will be described in detail so that those skilled in the art can easily practice with reference to the accompanying drawings. However, the present disclosure may be implemented in many different forms and is not limited to the embodiments described herein. And in order to clearly describe the present application in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are attached to similar parts throughout the specification.

Throughout the present specification, when a member is said to be located “on” another member, this includes not only a case where a member is in contact with another member, but also a case where another member exists between the two members.

Throughout the specification, when a part "includes" a certain component, it means that it may further include other components without excluding other components unless otherwise stated. As used throughout this specification, the terms "about", "substantially", and the like, are used at or approximating that number when manufacturing and material tolerances inherent in the stated meaning are given, and do not convey the understanding of this application. Accurate or absolute figures are used to help prevent exploitation by unscrupulous infringers of the disclosed disclosure. The term "step of (doing)" or "step of" as used throughout the present specification does not mean "step for".

Throughout this specification, the term “combination(s) of these” included in the expression of the Markush form means a mixture or combination of one or more selected from the group consisting of the components described in the expression of the Markush form, It means including one or more selected from the group consisting of the above components.

Throughout this specification, reference to “A and/or B” means “A or B, or A and B”.

Hereinafter, embodiments and embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. However, the disclosure may not be limited to these embodiments and examples and drawings.

A first aspect of the present application provides a biomarker composition for diagnosing Alzheimer's disease comprising N-methylalanine.

The term "methylation" used throughout the present specification refers to a reaction in which hydrogen atoms bonded to carbon, nitrogen, oxygen, and sulfur atoms of organic compounds are replaced with methyl groups (-CH ₃ ). In addition to providing carbon to organic compounds in various biosynthetic processes such as amino acid synthesis and nucleotide synthesis, it is involved in various functions such as gene regulation through methylation of DNA/RNA and methylation of proteins such as histones.

The term "alanine" used throughout the present specification is an organic compound having a chemical formula of C ₃ H ₇ NO ₂ and is one of amino acids. It exists in two structural isomeric forms, L-alanine and D-alanine.

The term "metabolite" used throughout the present specification is a general term for substances produced as a result of metabolic processes in a living body, and refers to small molecules that can be quantified to best represent the phenotype of a living body.

As used throughout the specification, the term "Alzheimer's disease" is the most common degenerative neurological disease that causes dementia, and refers to a disease in which cognitive function, including memory, gradually develops and gradually deteriorates.

In one embodiment of the present application, the diagnosis is to determine a subject's susceptibility to a specific disease or disorder, to determine whether an individual currently has a specific disease or disorder, to determine a specific disease or disorder determining the prognosis of an individual suffering from the disease, or therametrics (eg, monitoring a subject's condition to provide information about the efficacy of a treatment).

The second aspect of the present application is,

(a) providing a biological sample of a subject;

(b) measuring the level of N-methylalanine in the biological sample; and

(c) providing a method for diagnosing Alzheimer's disease, comprising comparing the measured level of N-methylalanine with the measured value of a normal control sample. Content overlapping with the first aspect is also applied to the diagnosis method of the second aspect.

The term "subject" used throughout the present specification refers to a subject diagnosed with Alzheimer's disease, and may preferably be a human.

The biological sample may be isolated from the subject for which the risk of Alzheimer's disease is to be evaluated, and may be one or more selected from the group consisting of tissue, cell, blood, plasma, peritoneal fluid, synovial fluid, saliva, urine, and feces, preferably. It may be blood, but is not limited thereto.

A third aspect of the present application provides a kit for diagnosing Alzheimer's disease, including a quantification device for N-methylalanine. Contents overlapping with the first and second aspects are also applied to the kit of the third aspect.

In one embodiment of the present application, the quantitative device may be a chromatograph and a mass spectrometer.

The term "chromatograph" used throughout the present specification refers to a device for chromatography.

In one embodiment of the present application, the chromatograph may be at least one selected from the group consisting of a gas chromatograph, an ion chromatograph, and a liquid chromatograph, preferably a gas chromatograph, but is not limited thereto.

The term "chromatography" used throughout the present specification refers to the principle of immune response based on antigen-antibody reaction, the principle of binding a ligand to a receptor, the principle of binding of a nucleic acid to a complementary nucleic acid, etc. It refers to an analytical method that combines the principle of chromatography to move along.

The term "mass spectrometer" used throughout the specification refers to a device that quantitatively analyzes molecules or atoms having different masses by applying a magnetic field and an electric field to an anode wire to bend the path.

In one embodiment of the present application, the kit for diagnosing Alzheimer's disease may further include the biomarker composition of the present application in addition to the quantitative device.

The fourth aspect of the present application is,

(a) treating an individual suspected of having Alzheimer's disease with a candidate substance and measuring the level of N-methylalanine; and

(b) When the level of N-methylalanine is increased, a method for screening for a drug for preventing or treating Alzheimer's disease is provided, comprising the step of selecting the candidate substance as a drug for preventing or treating Alzheimer's disease. Content overlapping with the first to third aspects is also applied to the screening method of the fourth aspect.

As used throughout this specification, the term "screening" refers to the process of measuring one or more properties of one or more biomolecules. For example, a typical screening process involves measuring one or more properties of one or more members of one or more libraries. Screening can be performed computationally using computational models of biomolecules and virtual environments of biomolecules.

The term "medicine" used throughout the present specification refers to a substance administered to an organism by eating, applying, or directly injecting to treat or alleviate disease, injury, or other bodily abnormality. Nutrients for replenishment of nutrients, pain relievers or anesthetics to reduce pain, food, etc. may also be included in the category of drugs.

In one embodiment of the present application, the screening method comprises the steps of treating an individual suspected of having Alzheimer's disease with a drug candidate whose preventive or therapeutic effect on Alzheimer's disease is to be confirmed, and a biological sample isolated from the individual treated with the candidate substance. Measuring the level of N- methylalanine in; and selecting the candidate substance as a drug for preventing or treating Alzheimer's disease when the measured level of N-methylalanine is increased compared to before treatment with the candidate substance.

Hereinafter, the present invention will be described in more detail through examples of the present application, but the following examples are only exemplified to aid understanding of the present application, and the content of the present application is not limited to the following examples.

[Example]

Example 1: Preparation of brain tissue and blood samples according to the onset of Alzheimer's disease

B6, in which all the key pathological mechanisms of Alzheimer's disease are induced; 129-Psen1 ^tm1Mpm Tg (APPSwe, tauP301L) 1Lfa/J rats (3xAD-Tg) were used as disease group animal models. 3xAD-Tg was derived from APP Swedish, MAPT P301L and PSEN1 M146V genetically modified, and was purchased from Jackson Laboratory. C57BL/6 was used as a normal control rat.

In both the diseased group and the normal group, rats were examined at 5 months and 12 months after birth, in which the hippocampus, frontal cortex, dorsal cortex, striatum, cerebellum, and brainstem of the brain were examined. Separated. In addition, 'blood' samples were obtained by 'blood collection' from the 'above' rats using a 'blood collection' tube.

Example 2: Extraction of metabolites derived from brain tissue and blood

For metabolite extraction, the brain tissue samples were put into a round-bottom 2.0mL e-tube, frozen and dried, and then 5-mm steel balls were put into it and disrupted using a Mixer Mill MM400 (Retsch GmbH & Co., Germany). .

Blood samples were transferred to 1.5 mL e-tubes by 30 μl each. 1400 μl of “extraction solvent” mixed with “9:5” of “methanol” and “water” was added to the “brain” tissue sample and “blood” sample in the form of powder, and after “stabilization” time in “ice” for 15 minutes, “sonication” was performed while maintaining 4℃ for 5 minutes. Thus, metabolites were extracted. After centrifugation at 14,000g at 4℃ for 5 minutes, the supernatant was transferred to a new 1.5mL e-tube, 700 μl for GC-TOF MS analysis and 550 μl for LC-Orbitrap MS analysis. The supernatant sample was "concentrated" and dried using a "high-speed" vacuum concentrator (SCANVAC, Korea) and stored at -80°C until "mass" analysis.

Example 3: Brain tissue and blood metabolome profile analysis through multi-platform mass spectrometry

By applying the non-targeted “metabolite” profiling technique, “brain” tissue, “blood” derived “various” work, secondary “metabolites and “energy” metabolites “qualitative,” “quantitative” analyzes were performed.

For the basic metabolite analysis, 5μl of 40mg/ml methoxyamine hydrochloride dissolved in pyridine was added to the dried extract for GC-TOF MS analysis, followed by incubation at 30°C for 90 minutes to achieve methoxylation. Then, for trimethylsilylation, 45 μl N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA +1% TMCS; Thermo, USA) was added, and 13 fatty acid methyl esters were mixed (C8, C9, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28 and C30) were added as a retention time index marker, and then incubated at 37°C for 60 minutes.

Derivatized samples were analyzed using an Agilent 7890B gas chromatograph (Agilent Technologies) equipped with a Leco Pegasus HT time-of-flight mass spectrometer (LECO, St. Joseph, MI, USA). All “analysis” processes were performed in a “random” order to “minimize” “devices” and “systems” errors. 0.5 μl of the derivatized metabolite sample was separated on an RTX-5Sil MS column (Restek, Gellefonte, PA, USA), and the GC oven temperature was increased from 50 °C to 330 °C at a rate of 20 °C / min. It was held for 5 minutes. Mass spectra were obtained at a scan rate of 20 spectra/s at 85-500 m/z.

ChromaTOF software was used for data acquisition and preprocessing, and peak alignment and identification were performed based on the BinBase algorithm for the generic text (.txt) file and generic netCDF file obtained here.

For the analysis of secondary metabolites and energy metabolites, 70% ACN was added to the dried extract for LC-Orbitrap MS analysis, 50 μl was added, sonicated for 10 minutes, and filtered using a spin column before transfer to a vial. ． Chromatographic separation was performed on a 150 × 2.1 mm UPLC BEH 1.7-μm HILIC column equipped with a 5.0 mm × 2.1 mm UPLC BEH 1.7 μm HILIC VanGuard pre-column (Waters, USA) on an Ultimate-3000 UPLC system (Thermo Fisher Scientific, USA). (Waters, USA). 19mM ammonium acetate and 68mM ammonium hydroxide = 5% acetonitrile were used as the mobile phase "solvent" A, and "100%" acetonitrile was used as the "mobile phase" solvent B. The flow rate was carried out at 0.3 mL/min, and the mobile phase B was maintained at 95%, then dropped to 85% for 10 minutes, then dropped to 10% for 10 minutes and 95% for 5 minutes, and then dropped to 95% for 5 minutes. It was maintained at 95%.

The mass analysis system was analyzed in positive ionization mode using a Q-Exactive plus instrument (Thermo Fisher Scientific, MA, USA). Data acquisition and preprocessing were performed using Xcalibur software (Thermo Fisher Scientific, San Josι, CA, USA), and then data processing was based on Compound Discoverer 2.0 software (Thermo Fisher Scientific, San Josι, CA, USA).

Example 4: Identification of metabolic regulation mechanisms by disease-specific brain regions and exploration of representative molecular indicators

To identify the mechanism of childhood, onset, dementia, pathological, metabolic, and abnormalities, the 6 types of “brain” regions (cerebellum, dorsal cortex, frontal lobe, hippocampus, brain stem, The “metabolite” fluidity of the striatum) was “comparative” and analyzed. The Mann-Whitney (MW) test, a representative “non-parametric” univariate analysis method, was applied to “normal genotype” and “Alzheimer’s disease (AD) genotype” mouse samples, and “metabolites” that changed “significantly” according to “presence” of “disease” were selected.

The “fold change (FC),” which is a ratio that indicates how much “increases and decreases” in the normal group, the childhood, and the Alzheimer’s disease group, and the “volcano plot” that shows the p-value obtained through the MW test on the x and y axes, respectively, are “significantly” between the groups. Metabolites that showed a large “difference” were screened, and as a result, it was confirmed that “only” N-methylalanine “significantly” decreased “more than 2 times” in all “brain” areas (see FIGS. 1a to 1f and 2a to 2f).

In order to select the old age, onset, pathological, state, and specific metabolite, 'metabolite' and 'fluidity' were compared by 'brain' area in the 'disease group' and 'normal group' in the same way as 'after birth', '12 months old' rats, '5 months after birth', and 'rats'. Through this, it was confirmed that N-methylalanine, which was selected as a “disease specific” metabolite in the “childhood” onset disease group, “significantly” decreased in “all” brain “regions” in the “elderly” onset disease group (see FIGS. 3a and 3b).

Example 5: Alzheimer's disease-specific brain tissue biomarker discovery and verification

In order to verify the possibility of clinical application of N-methylalanine as a biomarker, a “representative” molecular “indicator” for selected “diseases,” a Receiver Operating Characteristic (ROC) analysis was performed for all “brain” areas and for each age, and a high level of AUC value was confirmed.

As the AUC value is closer to 1, it is possible to accurately predict the presence or absence of disease with high sensitivity and specificity. It was verified that it is a “biomarker” with “excellent” verification power with “AUC” 1, “specificity” 100, and “sensitivity” 100 in all “brain” areas of childhood, “old age,” onset, and disease. All of the AUC curves have a significance level of 0.0001 or less (see Figs. 4a and 4b).

Example 6: Verification of predictive power of blood-derived N-methylalanine as a non-invasive disease biomarker

N-Methylalanine, which is a “brain”, tissue, origin, biomarker, material, was analyzed “qualitatively and quantitatively” through “blood”, internal, and metabolite profiling, and “connected” “biological” “indicator” was discovered.

The 　abundance of N-methylalanine in 　in the normal group and in the diseased group by age was expressed as a dot plot, and as a result, it was confirmed that it decreased significantly in 　in the 　brain 　in both 　young, old age,onset, and diseased groups. (See Figures 5a and 5b). In addition, by applying the ROC analysis, the AUC value was 0.867 (specificity 66.67, sensitivity 100, p = 0.0013) in 　 childhood disease, 1 (specificity 100, sensitivity 100, p < 0.0001) in the elderly disease. It was confirmed that N-methylalanine is a “biomarker” with “very” high “predictive power” not only in brain tissue but also in “non-invasive” biological fluids.

Overall, it was confirmed that the biomarker composition containing N-methylalanine of the present application has very high predictive power for Alzheimer's disease, and thus can contribute to early diagnosis of Alzheimer's disease.

The above description of the present application is for illustrative purposes, and those skilled in the art will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present application. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present application is indicated by the following claims rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts thereof should be construed as being included in the scope of the present application.

Claims (9)

A biomarker composition for diagnosing Alzheimer's disease comprising N-methylalanine.
The biomarker composition for diagnosing Alzheimer's disease according to claim 1, wherein the Alzheimer's disease is sporadic Alzheimer's disease or familial Alzheimer's disease.
(a) providing a biological sample of a subject;
(b) measuring the level of N-methylalanine in the biological sample; and
(c) a method for diagnosing Alzheimer's disease comprising the step of comparing the measured level of N-methylalanine with the measured value of a normal control sample.
The method for diagnosing Alzheimer's disease according to claim 3, wherein the Alzheimer's disease is sporadic Alzheimer's disease or familial Alzheimer's disease.
A kit for diagnosing Alzheimer's disease, comprising a quantification device for N-methylalanine.
The kit for diagnosing Alzheimer's disease according to claim 5, wherein the quantitative device is a chromatograph and a mass spectrometer.
The kit for diagnosing Alzheimer's disease according to claim 5, wherein the Alzheimer's disease is sporadic Alzheimer's disease or familial Alzheimer's disease.
(a) treating an individual suspected of having Alzheimer's disease with a candidate substance and measuring the level of N-methylalanine; and
(b) when the level of N-methylalanine is increased, a method for screening a drug for preventing or treating Alzheimer's disease, comprising selecting the candidate substance as a drug for preventing or treating Alzheimer's disease.
The method of screening for drugs for preventing or treating Alzheimer's disease according to claim 8, wherein the Alzheimer's disease is sporadic Alzheimer's disease or familial Alzheimer's disease.

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