Biomarker composition for early diagnosing of alzheimer's disease and use thereof

Abstract

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

Description

Technology Field

[0001] The present invention relates to a biomarker composition for the early diagnosis of Alzheimer's disease and the use thereof. Background Technology

[0002] Currently, as the elderly population in Korea increases rapidly, brain diseases affecting the elderly, such as dementia and stroke, are on the rise. Among these, Alzheimer's disease is the most common type of dementia and is a representative degenerative brain disease that renders daily life impossible by exhibiting symptoms such as memory loss, cognitive impairment, and motor paralysis.

[0003] The exact causes and mechanisms of Alzheimer's disease are not yet known. The aspects that can be explained solely by genotype are very limited, and the interactions between the complex development of the disease and various risk factors have not yet been elucidated.

[0004] For Alzheimer's disease, appropriate emergency response in the early stages is crucial for the prognosis; however, early diagnosis is impossible with currently used neuropsychological tests, blood tests, brain imaging tests, and genetic tests. Magnetic resonance imaging (MRI), the most widely used method, is not only expensive but can only detect brain atrophy when it has already progressed significantly. Furthermore, methods for detecting beta-amyloid and tau proteins in cerebrospinal fluid or blood have raised issues regarding clinical accuracy, necessitating the development of new early diagnostic markers.

[0005] Accordingly, the inventors of the present invention have made diligent efforts to develop a marker capable of diagnosing Alzheimer's disease early, and have developed a methylation metabolite biomarker capable of diagnosing Alzheimer's disease simply and quickly, thereby completing the present invention. Prior art literature

[0006] Korean Registered Patent Publication No. 10-2078500 Korean Registered Patent Publication No. 10-2164490 The problem to be solved

[0007] The present invention aims to provide a biomarker composition for diagnosing Alzheimer's disease comprising N-methylalanine.

[0008] However, the problems that this invention seeks to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0009] The first aspect of the present invention provides a biomarker composition for diagnosing Alzheimer's disease comprising N-methylalanine.

[0010] The second aspect of the present invention is,

[0011] (a) A step of providing a biological sample of the subject;

[0012] (b) a step of measuring the level of N-methylalanine in the biological sample; and

[0013] (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.

[0014] A third aspect of the present invention provides a kit for diagnosing Alzheimer's disease comprising a quantitative device for N-methylalanine.

[0015] The fourth aspect of the present invention is,

[0016] (a) a step of treating a subject suspected of having Alzheimer's disease with a candidate substance and measuring the level of N-methylalanine; and

[0017] (b) A method for screening drugs for the prevention or treatment of Alzheimer's disease, comprising the step of selecting the candidate substance as a drug for the prevention or treatment of Alzheimer's disease when the concentration of the N-methylalanine increases. Effects of the invention

[0018] A biomarker composition for diagnosing Alzheimer's disease according to one embodiment of the present invention can diagnose Alzheimer's disease early by using it to measure and compare the level of N-methylalanine of a subject, and the composition can be applied to a kit for diagnosing Alzheimer's disease or to screening drugs for the prevention or treatment of Alzheimer's disease. Brief explanation of the drawing

[0019] Figures 1a to 1f are volcano plots representing metabolites based on the Mann-Whitney test p-value and fold change (x-axis: fold change, y-axis: p-value) to select age-specific metabolites by six brain regions (cerebellum, dorsal cortex, frontal lobe, hippocampus, brainstem, striatum) among 252 metabolites derived from brain tissue. Figures 2a to 2f are volcano plots representing metabolites based on the Mann-Whitney test p-value and fold change (x-axis: fold change, y-axis: p-value) to select age-specific metabolites by six brain regions (cerebellum, dorsal cortex, frontal lobe, hippocampus, brainstem, striatum) among 252 metabolites derived from brain tissue. Figures 3a and 3b are dot plots showing N-methylalanine that was statistically significantly reduced in Alzheimer's disease compared to the normal group across all age groups and brain regions (hippocampus, frontal lobe, striatum, dorsal cortex, cerebellum, brainstem). Figures 4a and 4b are ROC curve graphs evaluating the biomarker performance of N-methylalanine across all ages and brain regions (hippocampus, frontal lobe, striatum, dorsal cortex, cerebellum, brainstem). Figures 5a and 5b are drawings showing an ROC curve evaluating the performance of N-methylalanine in blood as a biomarker and a dot plot showing a decrease in Alzheimer's disease compared to the normal group. Specific details for implementing the invention

[0020] Embodiments of the present invention are described below with reference to the attached drawings to enable those skilled in the art to easily implement the invention. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein. Furthermore, in order to clearly explain the present invention in the drawings, parts unrelated to the explanation have been omitted, and similar parts throughout the specification are denoted by similar reference numerals.

[0021] Throughout this specification, when a component is described as being located “on” another component, this includes not only cases where a component is in contact with another component, but also cases where another component exists between the two components.

[0022] Throughout this specification, when a part is described as “comprising” a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components. Throughout this specification, terms of degree such as “about,” “substantially,” etc., are used to mean at or near the stated value when inherent manufacturing and material tolerances are presented in the said meaning, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosure in which precise or absolute values ​​are mentioned to aid in understanding this specification. Throughout this specification, terms of degree such as “step” or “step of” do not mean “step for”.

[0023] Throughout this specification, the term “combination(s) of these” included in the Markush-form expression means one or more mixtures or combinations selected from the group consisting of the components described in the Markush-form expression, and means including one or more selected from the group consisting of said components.

[0024] Throughout this specification, the description “A and / or B” means “A or B, or A and B”.

[0026] Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the attached drawings. However, the present invention may not be limited to these embodiments and examples and drawings.

[0028] The first aspect of the present invention provides a biomarker composition for diagnosing Alzheimer's disease comprising N-methylalanine.

[0029] The term "methylation" as used throughout this specification refers to a reaction in which a hydrogen atom bonded to a carbon, nitrogen, oxygen, sulfur atom, etc., of an organic compound is replaced with a methyl group (-CH3). In addition to providing carbon to organic compounds in various biosynthetic processes such as the synthesis of amino acids and nucleotides, it is involved in various functions such as gene regulation through the methylation of DNA / RNA and the methylation of proteins such as histones.

[0030] As used throughout this specification, the term "alanine" refers to an organic compound with the chemical formula C3H7NO2, which is an amino acid. It exists in two structural isomer forms: L-alanine and D-alanine.

[0031] As used throughout this specification, the term "metabolite" refers to a general term for substances produced as a result of metabolic processes within a living organism, and refers to a quantifiable low-molecular-weight molecule that best represents the organism's phenotype.

[0032] As used throughout this specification, the term "Alzheimer's disease" refers to the most common degenerative neurological disease causing dementia, characterized by a gradual onset and progressive deterioration of cognitive functions, including memory. Alzheimer's disease according to this invention includes, but is not necessarily limited to, sporadic Alzheimer's disease or familial Alzheimer's disease.

[0033] In one embodiment of the present invention, the diagnosis may include determining the susceptibility of an individual to a specific disease or condition, determining whether an individual currently has a specific disease or condition, determining the prognosis of an individual with a specific disease or condition, or therametrics (e.g., monitoring the condition of an individual to provide information on therapeutic efficacy).

[0035] The second aspect of the present invention is,

[0036] (a) A step of providing a biological sample of the subject;

[0037] (b) a step of measuring the level of N-methylalanine in the biological sample; and

[0038] (c) A method for diagnosing Alzheimer's disease is provided, comprising the step of comparing the measured level of N-methylalanine with the measured value of a normal control sample. Content overlapping with the first aspect also applies to the diagnostic method of the second aspect.

[0039] The term "subject" as used throughout this specification refers to a subject diagnosed with Alzheimer's disease, and preferably may be a human.

[0040] The above biological sample may be isolated from the subject for which the risk of Alzheimer's disease is to be assessed, 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 blood, but not limited thereto.

[0042] The third aspect of the present invention provides a kit for diagnosing Alzheimer's disease comprising a quantitative device for N-methylalanine. Contents overlapping with the first and second aspects also apply to the kit of the third aspect.

[0043] In one embodiment of the present invention, the quantitative device may be a chromatograph and a mass spectrometer.

[0044] As used throughout this specification, the term "chromatograph" refers to an apparatus for performing chromatography.

[0045] In one embodiment of the present invention, the chromatograph may be one or more selected from the group consisting of a gas chromatograph, an ion chromatograph, and a liquid chromatograph, and preferably may be a gas chromatograph, but is not limited thereto.

[0046] The term "chromatography" as used throughout this specification refers to an analytical method that combines the principles of an immune response based on antigen-antibody reactions, the binding principles of ligands and receptors, and the binding principles of nucleic acids and complementary nucleic acids with the principles of chromatography, in which a sample and reagent move along a medium by means of a mobile phase.

[0047] As used throughout this specification, the term "mass spectrometer" refers to a device that quantitatively analyzes molecules or atoms of different masses by applying a magnetic field and an electric field to an anode ray to bend its path.

[0048] In one embodiment of the present invention, the Alzheimer's disease diagnostic kit may additionally include a biomarker composition of the present invention in addition to a quantitative device.

[0050] The fourth aspect of the present invention is,

[0051] (a) a step of treating a subject suspected of having Alzheimer's disease with a candidate substance and measuring the level of N-methylalanine; and

[0052] (b) A screening method for a drug for the prevention or treatment of Alzheimer's disease is provided, comprising the step of selecting the candidate substance as a drug for the prevention or treatment of Alzheimer's disease when the level of the N-methylalanine increases. Contents overlapping with aspects 1 through 3 also apply to the screening method of aspect 4.

[0053] As used throughout this specification, the term "screening" refers to a process of measuring one or more characteristics of one or more biomolecules. For example, a typical screening process involves measuring one or more characteristics of one or more members of one or more libraries. Screening may be performed computationally using computational models of biomolecules and virtual environments of biomolecules.

[0054] As used throughout this specification, the term "drug" refers to a substance administered to a living organism by means such as ingestion, topical application, or direct injection to treat or alleviate diseases, injuries, or other bodily abnormalities. Nutritional supplements for nutrient replenishment, analgesics or anesthetics for pain relief, and food may also be included in the category of drugs.

[0055] In one embodiment of the present invention, the screening method may include the steps of: treating an individual suspected of having Alzheimer's disease with a candidate drug substance to confirm its preventive or therapeutic effect against Alzheimer's disease; measuring the level of N-methylalanine in a biological sample isolated from the individual treated with the candidate substance; and selecting the candidate substance as a drug for the prevention or treatment of Alzheimer's disease if the measured level of N-methylalanine has increased compared to before treatment with the candidate substance.

[0057] The present invention will be explained in more detail below through embodiments thereof; however, the following embodiments are merely illustrative to aid in understanding the present invention, and the content of the present invention is not limited to the following embodiments.

[0059] [Example]

[0060] Example 1: Preparation of brain tissue and blood samples according to the onset time of Alzheimer's disease

[0061] B6; 129-Psen1, in which all core pathological mechanisms of Alzheimer's disease were induced tm1Mpm Tg(APPSwe, tauP301L) 1Lfa / J rats (3xAD-Tg) were used as the disease group animal model. 3xAD-Tg, which had induced genetic mutations in APP Swedish, MAPT P301L, and PSEN1 M146V, was purchased from Jackson Laboratory. C57BL / 6 rats were used as the normal control rats.

[0062] For both the disease group and the normal group mice, the hippocampus, frontal cortex, dorsal cortex, striatum, cerebellum, and brainstem of the brain were isolated at 5 and 12 months of age. In addition, blood samples were obtained from the mice by collecting blood using blood collection tubes.

[0064] Example 2: Extraction of metabolites derived from brain tissue and blood

[0065] For metabolite extraction, brain tissue samples were placed in a round-bottom 2.0 mL e-tube, freeze-dried, and then disrupted using a Mixer Mill MM400 (Retsch GmbH & Co., Germany) with a 5-mm steel ball.

[0066] 30 μl of blood samples were transferred to 1.5 mL e-tubes. 1,400 μl of an extraction solvent, a 9:5 mixture of methanol and water, was added to the powdered brain tissue samples and blood samples. After a stabilization time of 15 minutes on ice, metabolites were extracted by sonication for 15 minutes while maintaining the temperature at 4°C. Subsequently, after centrifugation at 14,000 g for 5 minutes at 4°C, 700 μl of the supernatant was transferred to new 1.5 mL e-tubes for GC-TOF MS analysis and 550 μl for LC-Orbitrap MS analysis. The supernatant samples were concentrated and dried using a high-speed vacuum concentrator (SCANVAC, Korea) and stored at -80°C until mass spectrometry.

[0068] Example 3: Analysis of brain tissue and blood metabolite profiles using multi-platform mass spectrometry

[0069] Qualitative and quantitative analyses of various primary and secondary metabolites and energy metabolites derived from brain tissue and blood were performed using non-target metabolite profiling techniques.

[0070] For the analysis of basal metabolites, 5 μl of 40 mg / ml methoxyamine hydrochloride dissolved in pyridine was added to the dried extract for GC-TOF MS analysis, and methoxylation was performed by incubating at 30°C for 90 minutes. Subsequently, for trimethylsilylation, 45 μl of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA +1% TMCS; Thermo, USA) was added, followed by 2 μl of a mixture of 13 fatty acid methyl esters (C8, C9, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28, and C30) as a retention time index marker, and incubation was performed at 37°C for 60 minutes.

[0071] Derivatized samples were analyzed using an Agilent 7890B gas chromatograph (Agilent Technologies) and a mounted Leco Pegasus HT time-of-flight mass spectrometer (LECO, St. Joseph, MI, USA). All analytical procedures were performed in a random order to minimize instrument system error. 0.5 μl of derivatized metabolite samples were 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 and held at 330°C for 5 minutes. Mass spectra were obtained at a scan rate of 20 spectral / s in the range of 85–500 m / z.

[0072] Data acquisition and preprocessing were performed using ChromaTOF software, and the resulting generic text (.txt) and generic netCDF files were subjected to peak alignment and identification based on the BinBase algorithm.

[0073] For the analysis of secondary metabolites and energy metabolites, 50 μl of 70% ACN was added to the dried extracts for LC-Orbitrap MS analysis, sonicated for 10 minutes, and then filtered using a spin column before being transferred to vials. Chromatographic separation was performed on an Ultimate-3000 UPLC system (Thermo Fisher Scientific, USA) using a 150 × 2.1 mm UPLC BEH 1.7 μm HILIC column (Waters, USA) equipped with a 5.0 mm × 2.1 mm UPLC BEH 1.7 μm HILIC VanGuard pre-column (Waters, USA). 19 mM ammonium acetate and 68 mM ammonium hydroxide 5% acetonitrile were used as mobile phase solvent A, and 100% acetonitrile was used as mobile phase solvent B. The flow rate was 0.3 mL / min, and mobile phase B was maintained at 95%, then reduced to 85% over 10 minutes, then reduced to 10% over 10 minutes and to 95% over 5 minutes, and then maintained at 95% for 5 minutes.

[0074] Mass spectrometry analysis was performed using the Q-Exactive plus instrument (Thermo Fisher Scientific, MA, USA) in positive ionization mode. Data acquisition and preprocessing were performed using Xcalibur software (Thermo Fisher Scientific, San José, CA, USA), and subsequent data processing was performed using Compound Discoverer 2.0 software (Thermo Fisher Scientific, San José, CA, USA).

[0076] Example 4: Elucidation of disease-specific metabolic regulatory mechanisms by brain region and exploration of representative molecular markers

[0077] To elucidate the pathological metabolic abnormality mechanisms of juvenile dementia, metabolite mobility in six brain regions (cerebellum, dorsal cortex, frontal lobe, hippocampus, brainstem, and striatum) was compared and analyzed between a disease group and a normal group (C57BL / 6) of 5-month-old transgenic mice (3xTg-AD model). The Mann-Whitney (MW) test, a representative nonparametric univariate analysis method, was applied to normal genotype and Alzheimer's disease (AD) genotype mouse samples, and metabolites that changed significantly depending on the presence or absence of the disease were selected.

[0078] Metabolites showing a statistically significant difference between the two groups were selected using a volcano plot in which the fold change (FC), representing the increase or decrease in the juvenile Alzheimer's disease group compared to the normal group, and the p-value obtained from the MW test were plotted on the x and y axes, respectively. As a result, it was confirmed that N-methylalanine was the only one that decreased significantly by more than twofold in all brain regions (see Figs. 1a to 1f, 2a to 2f).

[0079] To identify metabolites specific to pathological conditions onset in old age, metabolite mobility by brain region was compared and analyzed in 12-month-old mice, similar to 5-month-old mice. Through this, it was confirmed that N-methylalanine, identified as a disease-specific metabolite in the childhood-onset disease group, was significantly reduced in all brain regions in the old-age-onset disease group (see Figures 3a and 3b).

[0081] Example 5: Discovery and Validation of Alzheimer's Disease-Specific Brain Tissue Biomarkers

[0082] To verify the clinical applicability of N-methylalanine, a selected representative molecular marker of the disease, as a biomarker, Receiver Operating Characteristic (ROC) analysis was performed across all brain regions and age groups, and high AUC values ​​were confirmed.

[0083] The closer the AUC value is to 1, the more accurately the presence or absence of disease can be predicted with high sensitivity and specificity. In this invention, it was verified that N-methylalanine is a biomarker with excellent efficacy, exhibiting an AUC of 1, specificity of 100, and sensitivity of 100 in all brain regions of diseases onset in childhood and old age, with the exception of the cerebellum (AUC: 0.972, specificity: 100, sensitivity: 83.33) on the senile side. All AUC curves have a significance level p of 0.0001 or less (see Figures 4a and 4b).

[0085] Example 6: Verification of the predictive power of blood-derived N-methylalanine as a non-invasive disease biomarker

[0086] We discovered linked biomarkers by qualitatively and quantitatively analyzing N-methylalanine, a previously selected brain tissue-derived biomarker, through blood metabolite profiling.

[0087] The age-specific abundance of N-methylalanine in the normal and disease groups in the blood was plotted, and the results confirmed that, similar to brain tissue, levels were significantly reduced compared to the normal group in both childhood and elderly disease groups (see Figures 5a and 5b). Additionally, ROC analysis was applied to confirm that the AUC value was 0.867 (specificity 66.67, sensitivity 100, p=0.0013) for childhood-onset disease and 1 (specificity 100, sensitivity 100, p<0.0001) for elderly-onset disease, confirming that N-methylalanine is a biomarker with very high predictive power not only in brain tissue but also in non-invasive bodily fluids.

[0089] Overall, it was confirmed that the biomarker composition containing N-methylalanine of the present invention possesses very high predictive power for Alzheimer's disease and can contribute to the early diagnosis of Alzheimer's disease.

[0091] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical concept or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0092] The scope of the present invention is defined by the claims set forth below rather than by the detailed description above, and all modifications or variations derived from the meaning and scope of the claims and the concept of equivalents thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 Biomarker composition for diagnosing Alzheimer's disease containing N-methylalanine. Claim 2 delete Claim 3 A method for diagnosing Alzheimer's disease comprising: (a) providing a biological sample of a subject; (b) measuring the level of N-methylalanine in the biological sample; and (c) comparing the measured level of N-methylalanine with a measurement of a normal control sample. Claim 4 delete Claim 5 A kit for diagnosing Alzheimer's disease comprising a quantitative device for N-methylalanine. Claim 6 In claim 5, the above quantitative device is a chromatograph and a mass spectrometer, an Alzheimer's disease diagnostic kit. Claim 7 delete Claim 8 A method for screening drugs for the prevention or treatment of Alzheimer's disease, comprising: (a) treating a subject suspected of having Alzheimer's disease with a candidate substance and measuring the level of N-methylalanine; and (b) selecting the candidate substance as a drug for the prevention or treatment of Alzheimer's disease when the level of N-methylalanine increases. Claim 9 delete

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