KR20140042331A - Multiplex markers for diagnosing cognitive disorder and its use - Google Patents

Abstract

The present application discloses proteins that are differentially expressed in cognitive disorder diseases and uses thereof. Transthyretin, ApoE, alpha-synuclein, vitamin D binding protein, neurologranin, vimentin, stathmin, contactin, And a marker according to the present application selected from the group consisting of HDL-cholesterol, especially used in combination, can be usefully used as a target component in the diagnosis or treatment of cognitive disorder diseases.

Description

Multiple markers for diagnosing cognitive disorder and its use}

The present invention relates to the field of biomarkers for diagnosing cognitive disorders.

Alzheimers disease (AD), one of the cognitive disorders, is the most common form of dementia and is a representative neurodegenerative disease. It is estimated that more than 20% of elderly people over 80 years of age are affected by Alzheimer's disease, and the number is rapidly increasing in an aging society. Changes in the brain tissue of Alzheimer's disease (AD) patients are due to environmental and various etiological factors, and there is no clear diagnosis and treatment.

Currently, the most common AD diagnostic methods include imaging methods such as magnetic resonance imaging (MRI) and positron emission tomography (PET), and indirect methods such as mini mental state examination (MMSE) and questionnaire. Although it is used, the diagnosis through the MMSE is a result of the age, education, etc., the accuracy of the diagnosis is a problem.

Alzheimer's disease is characterized by senile plaque and microparticles in which β-amyloid (Aβ peptide), which is produced by sequentially cleaving amyloid precursor protein (APP) by β, γ-secretase, is deposited on brain tissue. Neurofibrillary tangles (NTFs) due to hyperphosphorylation of the canal associated protein Tau protein are the main pathological features. As a method of diagnosing AD, a method for identifying the above two lesions in brain tissue of AD patients is also used.However, there is no known method for diagnosing the disease or clarifying progress. to be. The search for biochemical markers for the diagnosis of AD is being conducted in various tissues, including body fluids such as blood and cerebrospinal fluid. Cerebrospinal fluid (CSF) is a site of direct contact with extracellular parts of the brain and reflects changes in the protein of the brain and decreases Aβ42 levels, total tau and autophosphorylation in CSF in patients with Alzheimer's disease, compared to the normal elderly. Tau increases have been reported.

However, considering that most AD patients are older than 65 years of age, there is a significant risk of lumbar puncture for cerebrospinal fluid. On the other hand, considering the relative ease of collection, the economics of the test, time saving, etc., there is a need for the development of new technologies including the discovery of new markers and therapeutic agents that can be detected in the blood.

Korean Patent No. 1003124 discloses a method for diagnosing Alzheimer's disease or a dementia-related neurological disease using gold nanoparticles, and a method for diagnosing Alzheimer's disease by binding beta amyloid to gold nanoparticles.

Korean Unexamined Patent Publication No. 2010-0049363 relates to a diagnosis apparatus and diagnostic method for Alzheimer's disease using a vitamin D binding protein, and discloses a diagnosis method and apparatus using an antibody binding to a vitamin D binding protein.

However, considering that it is a serious disease that is difficult to diagnose early, and if the disease progresses, it is difficult for normal life to be socially expensive. New markers include early detection and suppression, new markers for early treatment of high-risk groups, and discovery of therapeutics. There is a need for technology development.

The present application is to provide a diagnostic marker and its use.

The present application is a result of protein analysis that is differentially expressed in cognitive impairment patients and the control group was confirmed that the expression is significantly different in cognitive impairment patients.

In one embodiment, the present disclosure provides transthyretin, ApoE, alpha-cyuclein, vitamin D binding protein, neurologranin, vimentin, stathmin, contactin and a marker for diagnosing cognitive disorder disease, which is one or a combination of two or more selected from the group consisting of contactin and HDL-cholesterol. According to one embodiment the markers herein are used in combination of two or more.

In another embodiment, the present application also provides a method for diagnosing two or more cognitive impairment markers selected from transcyretin, ApoE, alpha-cynucleine, vitamin D binding protein, neurologrenin, bimentin, stesmine, contactin, or HDL-cholesterol. Provided are a kit for detecting a cognitive disorder disease, or a composition for diagnosing a cognitive disorder disease comprising the detection reagent.

In another embodiment the disclosure provides two or more recognitions selected from the group consisting of transcyretin, ApoE, alpha-cynucleine, vitamin D binding protein, neurologrenin, bimentin, stesmine, contactin, or HDL-cholesterol Detecting the presence or amount of a disorder diagnostic marker; And correlating the presence or amount of the detected marker with a diagnosis or prognosis of the test subject, detecting two or more of the markers from a sample of the test subject to provide information necessary for diagnosing or prognosis of the test subject. Provide a way to.

In another embodiment, the present invention provides a cognitive disorder comprising a modulator of one or more of transcyretin, ApoE, alpha-cynucleine, vitamin D binding protein, neurologrenin, bimentin, stesmine, contactin, or HDL-cholesterol It provides a composition for the prevention or treatment of.

In another aspect, the present invention provides a method of preparing a protein comprising the steps of providing at least one of a transcyretin, ApoE, alpha-cynucleine, vitamin D binding protein, neurologrenin, bimentin, stesmine, or contactin protein; Contacting the protein with a test substance; And comparing the expression of the protein in a control group not in contact with the protein in contact with the test substance.

The marker for diagnosing cognitive impairment according to the present invention may be usefully used for diagnosing cognitive impairment by reflecting physiological changes of various biochemical neurons occurring in patients with cognitive impairment. The marker according to the present invention can further improve the discriminative power of diagnosing cognitive impairment by a combination of multiple markers, and is useful for, for example, diagnosing Alzheimer's disease, determining the severity of disease, and treating the disease.

1 is a graphical representation of the effects of the markers identified herein (A) and the amount of expression increase and decrease in the sample in Table (B).
Figure 2 shows the results of the comparison of transcyretin concentrations in serum of Alzheimer's disease patients (test group) and normal control group by ELISA analysis.
Figure 3a shows the results of comparing the ApoE concentration in serum of patients with Alzheimer's disease (test group) and the normal control group by FACS analysis using microbeads with ApoE antibody.
3B is a graph showing the correlation between MMSE score and blood ApoE.
Figure 4 shows the result of comparing the alpha-cynuclein concentration in serum of patients with Alzheimer's disease (test group) and the normal control group by FACS analysis using microbeads with alpha-cynucleine antibody.
FIG. 5A shows the correlation between the serum BDBP concentrations of Alzheimer's disease patients (test group) and the normal control group by western blot (left) and the MMSE score (right).
5B shows the results of comparing the serum VDBP concentrations in patients with Alzheimer's disease (test group) and normal controls by FACS analysis using microbeads to which the VDBP antibody was attached.
6A and 6B show the results of Western blot comparison of neurologrenin concentration in serum of Alzheimer's disease patients (test group) and normal control group, and FACS analysis using microbeads to which neurologrenin antibody is attached. Indicates.
FIG. 7 shows the results of comparison of serum non-mentin concentrations in patients with Alzheimer's disease (test group) and normal controls by FACS analysis using microbeads with non-mentin antibodies.
FIG. 8 shows the results of comparing serum stesmin concentrations in patients with Alzheimer's disease (test group) and normal controls by FACS analysis using microbeads to which the stesmin antibody is attached.
9 shows the results of comparing serum concentrations of contactin in patients with Alzheimer's disease (test group) and normal controls by FACS analysis using microbeads to which contactin antibodies are attached.
10 shows the results of analyzing the HDL-cholesterol concentration in serum of Alzheimer's disease patients (test group) and normal control group.

In the present invention, platelets are isolated from blood of Alzheimer's (AD) patients and controls, and proteins are analyzed by various proteomics and bioinformatics methods to find AD diagnostic markers from proteins differentially expressed between the two groups. Based on the results.

Using bioinformatics technology, 49 proteins from Alzheimer's disease animal models were selected from known biochemical literature, and again, various factors such as primate expression, blood secretion and the presence of available antibodies. Nine candidates were selected based on their status: transthyretin, ApoE, alpha-synuclein, vitamin D-binding protein (VDBP), neurologrenin (neurogranin), vimentin (vimentin), stethmin (stathmin), contactin (contactin), HDL-cholesterol (High Density Lipoprotein-Cholesterol) was derived.

Thus in one aspect the present application relates to a marker for diagnosing cognitive impairment comprising one or a combination of two or more of the markers, or a composition comprising a marker.

"Cognitive impairment" in the context of the present invention refers to neurodegenerative diseases and includes, for example, Alzheimer's, Parkinson's dementia, Lewy body dementia or Huntington's disease dementia, or mild cognitive impairment, ie mild cognitive impairment, which is a stage before dementia. It is. The severity of these diseases may be classified by methods such as Mini mental state examination (MMSE) scores, and the markers of the present disclosure may be used for classification according to the progression of these diseases.

The marker of the present invention may be an indicator for the onset and progression of cognitive impairment, and may be used for onset, degree of disease progression, diagnosis or prognosis of disease.

According to an embodiment of the present invention, the marker protein of the present invention may be used in a method for providing information necessary for diagnosing cognitive disorders using a composition or kit for diagnosing cognitive disorders.

As used herein, the term "diagnosis" refers to determining the susceptibility of an object to a particular disease or condition, that is, a test subject, determining whether an object currently has a particular disease or condition, Determining the prognosis of a subject or diseased or diseased subject, or therametrics (eg, monitoring the subject's condition to provide information about treatment efficacy).

 As used herein, the term "diagnosis marker or diagnostic marker" is a substance capable of diagnosing cognitive impairment from normal, and an increase or increase in tissues or blood, such as a brain, from a subject having a cognitive impairment as compared to a normal control group. Proteins or nucleic acids exhibiting a declining aspect and the like. In the present invention, markers for diagnosing cognitive impairment are transthyretin, ApoE, alpha-synuclein, vitamin D-binding protein, neurologranin, non Two or more selected from the group consisting of vimentin, stathmin, contactin, HDL-cholesterol (High Density Lipoprotein-Cholesterol), the expression is increased or decreased in the appropriate tissue or blood, such as the brain. Among these markers, transcyretin, ApoE, and HDL-cholesterol have decreased expression in cognitive impairment patients compared to normal controls, and the rest increase expression.

Although any one of the above markers may be used as a marker for diagnosing cognitive impairment, preferably, it is a complex marker including two or more of these markers. The markers may be used in a combination of two or more to improve diagnosis and / or prognosis of the cognitive impairment patient from the normal control group, and to discriminate the progress of the cognitive impairment. Among the markers according to the present application, a combination showing an optimal effect for this use can be selected and used by those skilled in the art.

Thus, in this respect, the present application also relates to the recognition of transcyretin, ApoE, alpha-cynucleine, VDBP, neurologrenin, bimentin, stesmine, contactin, and / or HDL-cholesterol (High Density Lipoprotein-Cholesterol). The present invention relates to a kit for diagnosing a disorder, wherein the kit includes a reagent for detecting the marker. The present application also relates to a composition for diagnosing cognitive disorders comprising the detection reagent.

As used herein, the term "biological sample or sample" includes, but is not limited to, any solid or liquid sample obtained from a human or mammal, such as tissue, urine, saliva, whole blood, platelets, plasma or serum samples from a particular organ. Do not. According to one embodiment herein, the marker protein is included in a tissue or blood sample.

The markers herein can be indicative of the onset and progression of the disease and can be used for the onset of the disease, the progression of the disease, the diagnosis or prognosis of the disease.

According to one embodiment of the present application, the marker of the present application may be used in a method for providing information necessary for diagnosing cognitive disorder using a composition or diagnostic kit for diagnosing cognitive disorder.

In one embodiment according to the present application, the sample in which the marker of the present application is used is for example one or more of platelets, whole blood, plasma or serum. In another embodiment, a sample used in the present invention may be a normal control group, a control group-based sample having a specific disease, as well as a sample of a subject to be diagnosed for comparative analysis.

Markers according to the present application are known and are as follows, for example: Transthyretin: Entrez Gene # 7276, ApoE: Entrez Gene # 348, Alpha-synuclein: Entrez Gene # 6622 , Vitamin D-binding protein: Entrez Gene # 2638, Neurologranin: Entrez Gene # 4900, Vimentin: Entrez Gene # 7431, stathmin: Entrez Gene # 3925, contactin: Entrez Gene # 1272), and functional equivalents thereof.

As used herein, the term "detection reagent" is a substance capable of detecting the marker of the present invention quantitatively or presence or pattern at a protein or nucleic acid such as mRNA level. Methods for analysis of the amount and presence expression pattern of a protein are well known and include, for example, Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Immunoprecipitation assays, Complement Fixation Assays, FACS, protein chips, and the like can be given as examples of the immunoassay method. Reagents for protein or nucleic acid level detection are well known, for example the former may be an antigen-antibody reaction, a substrate that specifically binds to the marker, a nucleic acid or peptide aptamer, a receptor that specifically interacts with the marker, Ligand or cofactor, or a mass spectrometer can be used. Reagents or substances that specifically interact or bind to the markers of the present invention may be used in conjunction with chip-based or nanoparticles.

In other words, the kit according to the present disclosure may include a reagent for detecting a marker at a protein or nucleic acid such as mRNA level. For example, reagents detectable at the protein level may include monoclonal antibodies, polyclonal antibodies, substrates, aptamers, receptors, ligands or cofactors, and the like. These reagents can be incorporated into nanoparticles or chips as needed. Proteins can also be detected using a mass spectrometer. The amount of detection and expression at the mRNA level can be detected in a manner using reverse transcriptase polymerase / polymerase chain reaction, RNase protection assay, or Northern blot and the like, and may include appropriate reagents necessary for this.

(RT-PCR) / polymerase chain reaction, competitive RT-PCR, real-time RT-PCR RNase protection assay, chip or Northern blot to detect the expression level, Methods can be used and these assays are well known and can also be performed using commercially available kits, and those skilled in the art will be able to select appropriate ones for the practice of the present application. For example, as a detection reagent in a method for measuring the presence and amount or pattern of the mRNA by RT-PCR, for example, a primer specific to the mRNA of the marker of the present invention. A primer refers to a nucleic acid sequence having a free 3 'hydroxyl group capable of complementarily binding with a template and allowing the reverse transcriptase or DNA polymerase to initiate replication of the template.

Detection herein includes quantitative and / or qualitative analysis, including detection of presence and absence and expression level detection, and such methods are well known in the art, and those skilled in the art will appreciate that methods suitable for the practice of the present application You will be able to choose.

According to an embodiment of the present disclosure, the detection reagent comprises an antibody, and the detection of the marker of the present disclosure is performed using an antibody molecule that specifically binds thereto.

Antibodies that may be used herein are polyclonal or monoclonal antibodies, preferably monoclonal antibodies. Antibodies can be produced using methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), the recombinant DNA method (US Patent No. 4,816,56) Or phage antibody library methods (Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 58, 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortal cell line with an antibody-producing lymphocyte, and the techniques required for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the past. The immunoassay format may include, but is not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay, capture-ELISA, inhibition or competitive assay, sandwich assay, flow cytometry, But are not limited to, fluorescent staining and immunoaffinity purification. Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984, which is incorporated herein by reference. By analyzing the final signal intensity by the above-described immunoassay process, that is, by performing a signal contrast with a normal sample, it is possible to diagnose whether the disease occurs.

According to another embodiment of the invention the detection reagent is a reagent for RNA analysis, the detection of the marker of the present application is carried out at the mRNA level. Detection of mRNA is usually performed through Northern blot or reverse transcription PCR (polymerase chain reaction). In the latter case, it is possible to detect a specific gene in a specimen by isolating RNA of the specimen, specifically mRNA, synthesizing cDNA therefrom, and then using a specific primer or a combination of a primer and a probe to detect the presence / Or the amount of expression can be determined. Such a method is described in, for example, Han, H., Bearss, DJ; Browne, LW; Calaluce, R .; Nagle, RB; Von Hoff, DD. Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray. Cancer Res 2002 , 62, (10), 2890-6).

According to another embodiment of the present application, mass spectrometry can be used to detect the marker and the protein can be separated from the sample and then analyzed, for example, in the manner described in the examples herein, J Protein Res 2010, 9, (2), and (3), the present inventors have found that, for example, 689-99 / Anderson, L .; Hunter, CL, Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol Cell Proteomics 2006, 5, (4), 573-88.

Reagents capable of detecting such markers of the present invention can be present separately in divided containers, and in this sense, the present invention also relates to a device / apparatus that includes the marker detection reagent of the present invention in a divided manner.

In another aspect, the present disclosure provides a method of detecting a marker or a protein, comprising detecting a presence or an amount of a marker according to the present application; And correlating the presence or amount of the detected marker with a diagnosis or prognosis of the test subject, wherein the marker is detected from a sample of the test subject to provide information necessary for diagnosing or prognosis of the test subject. It is about. The detection of the presence / absence or amount of expression of a marker in the methods herein can be determined at protein and / or nucleic acid, for example mRNA expression levels, as mentioned above.

The method of the present invention may further use non-protein clinical information of the patient, that is, non-marker clinical information, in addition to the marker analysis result, to provide information regarding the diagnosis or prognosis of the cognitive disorder. Such nonprotein clinical information may include, for example, the age, sex, weight, diet, body mass, underlying disease, magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), or mini-mental status of a patient. examination or positron emission tomography (PET) Including but not limited to one or more of the following.

The method includes correlating a detection result of a marker with a diagnosis or prognosis of a cognitive impairment, and according to one embodiment, the correlating step includes determining the amount or presence of a protein of each marker in the normal control group. This is compared with the detection result, for example, the increase and decrease, and then diagnosed based on this. For example, when one or more of the markers according to the present application are significantly increased or decreased in comparison with the value of the control group, information for diagnosing the disease as occurring in the subject may be provided.

According to an embodiment of the present disclosure, the step of associating a sample of the subject with a normal control group, sets a threshold value for diagnosing the onset of each marker, and then detects the detection result of the subject with the threshold value. Can be compared.

The marker according to the present invention is a differential increase or decrease in expression in cognitive disorders compared to normal tissues, and agents that control the expression of such substances can be usefully used as candidates for treatment.

Furthermore, the present application is based on protein analysis that is differentially expressed in cognitive disorders, and in one embodiment, transcyretin, ApoE, alpha-cynuclein, vitamin D binding protein, neurologrenin, bimentin, stesmin, contactin, Or a method for screening a substance for treating cognitive impairment targeting at least one of HDL-cholesterol. The substances selected by the method are particularly useful as cognitive impairment inhibitors / therapies.

The method includes providing a cell expressing the protein; Contacting the protein with a test substance; And comparing the increase and decrease in the expression and / or activity of the protein in a control group not in contact with the protein in contact with the test substance.

The test substance used in the method of the present invention is a substance which is expected to modulate the expression and / or activity of the protein. For example, for the purpose of screening a drug, the compound may have a low molecular weight therapeutic effect. For example, compounds of about 1000 Da in weight such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may form part of a compound library, and the number of compounds constituting the library may vary from tens to millions. Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and small molecule compounds based on templates such as benzodiazepines, hydantoin, biaryls, carbocycles and polycycle compounds (such as naphthalene, phenoty) Azine, acridine, steroids, and the like), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but these are merely illustrative. It is not limited to this.

Proteins that can be used as targets in the screening methods herein are as described above, and depending on the specific method, those from a variety of hosts can be used in the method, including both full length or truncated forms. In addition, even the same host, for example, a human, may have sequence variation depending on a specific individual, region, environment, and the like. Of course, this sequence may be modified (deletion, substitution, addition) Can be used in the present invention. In one embodiment, it is human-derived, and such is the same as mentioned above.

Cells expressing such proteins can be used in the methods herein, or animal models of cognitive impairment diseases, such as those used in the Examples herein, can be used, including such cells.

Cell and animal models expressing the protein can be prepared using methods known in the art. In one embodiment, the DNA or RNA sequence encoding the protein used in the screening is expressed in an appropriate host cell by using recombinant DNA technology. For example, a plasmid containing the corresponding gene encoding the protein is used in a stable or transient manner after delivery to eukaryotic cells such as insect cells and mammalian cells. The cells are contacted with a test substance under conditions that enable the growth of cells to be maintained, and the expression of the markers of the present application is examined at the RNA and protein levels. The detection method at the RNA and protein levels of the marker is as mentioned above.

In one embodiment according to the invention, the cell expressing the protein is for example a mammalian cell expressing all or part of the protein (transient or Stable transduction or endogenous expression), such as but not limited to the brain Neuroblast-like subclone of SK-N-SH (SH-SY5Y), Hippocampal neuronal cell line (HT22), bEnd.3 (Immortalized mouse brain endothelial cell), U373 (Astrocytoma), rat or mouse primary culture , CHO (Chinese hamster ovary cell), or HEK (Human embryonic kidney 293) can be used. After culturing these cells in a cell culture plate, the test substance was added thereto, and then, after a certain time, a Western blot in which the total protein was extracted from the cells was performed to compare with the control (when the test substance was not treated), Changes in expression can be selected as candidates for the treatment of cognitive impairment.

The amount of protein used in the present method, the type of cells and the amount and type of test substance vary depending on the specific test method used and the type of test substance, and those skilled in the art will be able to select an appropriate amount. As a result of the experiment, a substance which causes a decrease in protein expression or activity in the presence of the test substance as compared with the control group not in contact with the test substance is selected as a substance inhibiting the interaction. Less than about 99%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60% A reduction, about 55% reduction, about 50% or less reduction, about 45% or less reduction, about 40% or less reduction, about 30% or less reduction, or about 20% or less reduction, but the range is not excluded.

In addition, as a method of screening such a therapeutic agent, a method of fixing the marker of the present application to an affinity column and contacting with a sample to purify [Pandya et al, Virus Res 87: 135-143, 2002], a two-hybrid method Methods [Fields, S and Song, O., Nature 340: 245-246, 1989], Western Blot ("Molecular Cloning-A Laboratory Manual" Cold Spring Habor Laboratory, NY, Maniatis, T. at al. (1982) section 18. 30-18.74], a number of known methods can be used, including high-volume screening methods [Aviezer et al, J Biomol Screen 6: 171-7, 2001], and those skilled in the art can select appropriate methods according to specific embodiments. Samples containing test substances for use in screening include but are not limited to tissue extracts, expression products of gene libraries, synthetic compounds, synthetic peptides, natural compounds, and the like.

The term "treatment" as used herein is a concept involving inhibiting, eliminating, alleviating, ameliorating, and / or preventing a disease or condition or condition due to a disease.

The present application relates to the regulation of transthyretin, ApoE, alpha-cynucleine, vitamin D binding protein, neurologrenin, bimentin, stesmin, contactin, or HDL-cholesterol, and various known substances having this function. Included herein. Modulator herein refers to a substance that binds to or interacts with the substance to alter at least one biological characteristic and / or activity thereof. In one embodiment according to the invention a modulator is a substance that increases the expression (transcription and translation) and / or activity of the protein. Inhibitors herein are substances that can bind or interact with nucleic acids, including the proteins or mRNAs thereof, to inhibit / modulate the expression (transcription and translation) and / or activity of these proteins. Such materials include, for example, low molecular weight compounds, proteins including antibodies and polypeptides, and nucleic acid molecules including RNA and DNA, or any combination thereof. Herein, expression refers to transcription of a gene and expression of a transcript into a protein. The term " activity " means a biological action that allows an expressed protein to function in cells.

In one embodiment of the invention, the inhibitor or modulator is selected from the group consisting of antisense oligonucleotide, siRNA (small interfering RNA), shRNA (small hairpin RNA) that can specifically control the transcription of the coding gene and / ) Or miRNA (microRNA). siRNA, shRNA and miRNA can be transcribed through RNA interference, for example, short-length interfering RNA (siRNA) binds specifically to a transcript to form an RNA Induced Silencing Complex (RISC). The RNA cannot be expressed as a protein in the cell (silencing). By inhibiting such protein expression, the expression of other proteins affected by the protein may be increased. For example, an RNA that binds to an endogenous interfering RNA that inhibits the expression of a particular protein can upregulate expression of the protein. Such siRNA, shRNA or miRNA have a sequence that is highly complementary to its target sequence. A highly complementary sequence means at least about 70% complementarity, at least about 80% complementarity, at least about 90% complementarity, or about 100% complementarity with at least 15 consecutive bases in length of the target gene. Antisense oligonucleotides, siRNAs, shRNAs and / or miRNAs that target markers of various origins can be used as long as they bind to the target nucleic acid and function as silencing, including biological equivalents, derivatives and analogs thereof. will be.

Antisense oligonucleotides are as known in the art and are, for example, short synthetic nucleic acids that bind to any coding sequence of a target protein, thereby inhibiting / reducing the expression of the target protein. The antisense RNA may have an appropriate length depending on the target gene and the delivery method, for example, about 6, 8 or 10 to 40, 60 or 100 nucleotides.

In another embodiment herein, the modulators of transthyretin, ApoE, alpha-cynucleine, vitamin D binding protein, neurologrenin, bimentin, stesmine, contactin, or HDL-cholesterol specifically recognize this It can be an antibody. As used herein, the term antibody is intended to include intact antibodies, antigen-binding fragments of antibody molecules and antibodies or fragments functionally equivalent to these. In addition, the antibody of the present invention may be of the IgG, IgM, IgD, IgE, IgA or IgY type, and may be of the IgGl, IgG2, IgG3, IgG4, IgA1 or IgA2 class or subclass thereof. Antibodies of the invention include monoclonal, polyclonal, chimeric, short chain, heterologous, humanized and humanized antibodies and active fragments thereof.

In other embodiments herein, modulators of transthyretin, ApoE, alpha-cynucleine, vitamin D binding protein, neurologrenin, bimentin, stesmine, contactin, or HDL-cholesterol are active and / or functioning thereof A polypeptide that can modulate, interfere with, interfere with and / or inhibit mechanisms. The term polypeptide, as used herein, refers to a polymer of amino acids, either natural or synthetic, and does not refer to a particular length as long as it has this action, and includes peptides, oligopeptides. Such polypeptides can function as competitive inhibitors that act competitively with and interfere with normal proteins. Polypeptides include those that are naturally or artificially modified, such as glycosylation, acetylation, phosphorylation, and the like.

The therapeutic agent herein may further contain at least one active ingredient exhibiting the same or similar function in addition to the above-mentioned modulators or a compound which maintains / increases the solubility and / or absorption of the active ingredient. In addition, the therapeutic agents of the present application may be used alone or in combination with methods using surgery, drug treatment and biological response modifiers for the treatment or prevention of cognitive impairment diseases.

In addition to the above-mentioned effective ingredients, the therapeutic agent of the present invention may further comprise at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and one or more of these components. Other conventional additives such as buffers, bacteriostats and the like may be added. In addition, it can be formulated into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants, Specific antibody or other ligand can be used in combination with the carrier. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

The method of administering the therapeutic agent of the present application is not particularly limited thereto, and known administration methods may be applied, and parenteral administration (for example, intravenous, subcutaneous, intraperitoneal, or topical) or oral, depending on the desired method. In the case of parenteral administration, it can be administered through a patch-type, nasal / respirator attached to the skin, and administration by intravenous injection is preferable to obtain a rapid therapeutic effect. The dosage may vary widely depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, and severity of the disease. Parenteral administration may be preferred for protein preparations including siRNAs, miRNAs, antisense oligonucleotides, shRNA preparations and polypeptides targeting the markers identified herein, but do not exclude other routes and means. For typical drugs, dosage units include, for example, about 0.01 mg to 100 mg but do not exclude the below and above ranges. The daily dose may be about 1 μg to 10 g, and may be administered once to several times a day.

Hereinafter, the present invention will be described in more detail by way of examples. It should be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the subject matter of the present invention .

Example  1: Experiment and Method

Patient group and control group

In 2008 and 2010, blood collected from Alzheimer's disease (MCI) patients and mild cognitive impairment patients (MCI) and healthy people from Samsung Medical Center, Korea were used. Patients diagnosed with Alzheimer's disease may be diagnosed with medical examinations, neurological examinations, magnetic resonance imaging (MRI), or positron emission tomography (PET). and Stroke / Alzheimer's disease and Related Disorders Association (NINCDS-ADRDA).

Blood Collection and Serum Separation

The blood is collected by patient's blood with standard blood collection techniques, transferred to a polypropylene vacuum blood collection tube (SST advance, BD, UK) and centrifuged to remove fibrinogen and cellular components. Obtained and used in the following experiment.

In the blood Biomarker  Protein analysis

For analysis of each biomarker present in the blood, FACS (Fluorescence activated cell sorting, FACS Calibur ™, BD Bioscience), ELISA, or Western blot methods were used as conventional unless otherwise noted. In the case of FACS, microbeads, which are known to be more sensitive than conventional protein measurement methods, were used to accurately detect and quantify each marker known to be present in the blood. In addition, in order to overcome the experimental constraints that can be used for the commercialized antibody-adhered microbeads, which are not commercially available, a method of attaching an antibody directly to the microbead was performed. Biomarker candidates were selected directly by Bioinformatics and attached to the beads to perform all processes of detection in the blood. Antibody attachment of beads is described, for example, in Choi et al. See, 2008 Journal of Clinical Neurology 4: 84-88, Chen R et al., 1999, Clin Chem 45: 1693-1694, wherein antigen-antibody binding determines the amount or presence of proteins present in a particular sample. Enzyme-linked immunosorbent assay (ELISA), a common method of detection, is described by Han SH, et al., J Alzheimers Dis. 2011; 25 (1): 77-84, and Western blot may be referred to those disclosed in Korean Patent 815504.

The MMSE assay was performed using Folstein, MF, Folstein, SE, and McHugh, PR (1975) Journal of Psychiatric Research 12, 189-198, and the correlation between the concentration of each marker in the blood and the MMSE score.

Statistical analysis for the comparison of the two groups between the control group and the patient group (test group) was performed using the Student t-test and p <0.05 was considered a significant result. Disease prediction rates were analyzed using receiver-operator curve (ROC) analysis.

Example 2: Blood Transcyretin  Concentration comparison

Serum prepared in Example 1 was added to the ELISA kit (Standard Diagnostic, INC, Korea, reference: Korean Patent No. 815504 and Han SH, et al., J Alzheimers Dis. 2011; 25 (1): 77-84) of The expression level was examined by ELISA analysis. The results are described in FIG. As shown in FIG. 1, the concentration of transthyretin in AD patients has decreased, indicating that it is useful as a diagnostic marker or as a therapeutic agent.

The characteristics of the control group (normal person) and the test group (Alzheimer's disease patients, mild cognitive impairment patients) are summarized in Table 1 below.

[Table 1]

Example  3: blood ApoE  Concentration comparison

The blood ApoE concentrations of 44 patients and 45 normal patients of Example 1 were reacted with a detection antibody and a fluorescence-attached secondary antibody (Cat # 3712-3-1000, 3712-6-1000 monoclonal IgG1, Mabtech). Quantitative analysis was performed. For FACS analysis, ApoE was isolated from blood using functional beads to which the antibody was attached, and analyzed by FACS.

The characteristics of the control group (normal person) and the test group (Alzheimer's disease patients, mild cognitive impairment patients) are summarized in Table 2 below.

[Table 2]

The results are described in FIGS. 3A and 3B and show significant decreases in blood ApoE concentrations in Alzheimer's and mild cognitive impairment patients, which is useful as diagnostic markers or therapeutic targets. In addition, as shown in FIG. 3B, the decrease in blood ApoE concentrations in Alzheimer's and mild cognitive impairment patients was correlated with the Mini mental state examination (MMSE) score suggesting the severity of the disease. It can also be used as.

Example  4: Alpha in the blood Cynuclein  Concentration comparison

Blood alpha-cynuclein in blood (n = 23) of Alzheimer's and mild cognitive impairment patients of Example 1 The concentration was detected by reacting the detection antibody with the fluorescent antibody attached to the secondary antibody (Cat # 250917, 250918, Abbiotech) and then quantitative analysis using FACS. For FACS analysis, alpha-cynuclein in blood was separated using functional beads to which the antibody was attached, and analyzed by FACS.

The results are shown in FIG. 4, which showed a significant increase in blood alpha-cynuclein concentrations in Alzheimer's and mild cognitive impairment patients, indicating that they are useful as diagnostic markers or as therapeutic agents.

Example  5: blood VDBP  Concentration comparison

In order to observe the expression level of VDBP in the serum of Alzheimer's and mild cognitive impairment patients of Example 1, Western blot was performed using antibodies (anti-human DBP, Santacruz) against the serum of 21 AD patients and 21 control groups. Was carried out. In addition, beads were attached to antibodies to VDBP (Cat # LS-C42213, Lifespan Bioscience, anti-human DBP, SD) and reacted with serum and analyzed using FACS.

The results are described in FIGS. 5A and 5B. As shown in the results, significantly increased VDBP was observed in the serum of Alzheimer's disease and mild cognitive impairment. In addition, in order to confirm the correlation between the MMSE score and the DBP, the correlation between the MMSE score and the DBP concentration of 37 patients who performed the MMSE test among the AD patients and the control group was analyzed. As shown in FIG. 5B, the MMSE score was The lower the level, the greater the correlation between the amount of VDBP in serum (r = -0.4432, p <0.05). This indicates that the present markers are useful as diagnostic markers or as therapeutic agents.

Example  6: blood Neurologrenin  Concentration comparison

Western blot was performed using the antibody (a44, anti-humnan neurogranin, SD) for the serum of AD patients and the control group of Example 1 and the antibody against Ng (Cat # LS-C16823, Lifespan bioscience, a44, anti). -Human neurogranin (SD) was also reacted with serum using beads attached and analyzed using FACS.

The results are shown in Figures 6a and 6b, the results of the experiment was able to observe a significantly increased neurologrenin in the serum of both Alzheimer's disease and mild cognitive impairment. This indicates that the present markers are useful as diagnostic markers or as therapeutic agents.

Example  7: Serum Vimentin  Concentration comparison

The serum of the AD patient and the control group of Example 1 was reacted with the serum using a bead attached with antibodies to bimentin (Cat # ab 61780, ab 8978, abcam) to the serum of the AD patient and the control group. The analysis was carried out using FACS.

The results are shown in FIG. 7, and all of the results of the experiment showed a markedly increased nonmentin in the serum of Alzheimer's disease and mild cognitive impairment. This indicates that the present markers are useful as diagnostic markers or as therapeutic agents.

Example  8: blood Stethmin  Concentration comparison

For the sera of the AD patients and the control group of Example 1, using beads attached to the antibody (Cat # ab 47464, abcam, Cat # H00003925-M01A, Abnova) to stesmin with respect to the serum of the AD patients and the control group After reacting with serum was analyzed using FACS.

The results are shown in Figure 8, the results of the experiment was able to observe a markedly increased stethmin in the serum of both Alzheimer's disease and mild cognitive impairment. This indicates that the present markers are useful as diagnostic markers or as therapeutic agents.

Example  9: blood Contactin  Concentration comparison

The serum of the AD patient and the control group of Example 1 was reacted with the serum using beads attached to the contactin antibody (sc-50429, 73858, Santa cruz biotech) to the serum of the AD patient and the control group. The analysis was carried out using FACS.

The results are shown in FIG. 9, and the results of the above experiments showed that significantly increased contactin in the serum of Alzheimer's disease and mild cognitive impairment. This indicates that the present markers are useful as diagnostic markers or as therapeutic agents.

Example  10: blood HDL Cholesterol Concentration Ratio

The blood HDL-cholesterol of 212 Alzheimer's and mild cognitive impairment patients and 80 normal subjects of Example 1 was measured using the HDL-Cholesterol Assay kit (Cell biolabs) according to the manufacturer's method.

The results are shown in FIG. 10, and the concentration of HDL-cholesterol was significantly increased in Alzheimer's disease and mild cognitive impairment patients. This indicates that the present markers are useful as diagnostic markers or as therapeutic agents.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (22)

Transthyretin, ApoE, alpha-synuclein, vitamin D binding protein, neurologranin, vimentin, stathmin, contactin, And one or two or more combinations selected from the group consisting of HDL-cholesterol.
The cognitive disorder marker of claim 1, wherein the cognitive disorder is Alzheimer's disease, Parkinson's disease dementia, Lewy body dementia, Huntington's disease dementia, or mild cognitive disorder.
The marker for diagnosing cognitive disorder disease according to claim 1, wherein the marker is used for diagnosing severity of cognitive disorder.
The marker of claim 1, wherein the marker is detected at the protein or nucleic acid level or at the protein and nucleic acid level.
Specimens comprising a detection reagent for a diagnostic marker for diagnosing two or more cognitive disorders selected from transcyretin, ApoE, alpha-cynucleine, vitamin D binding protein, neurologrenin, bimentin, stesmine, contactin, or HDL-cholesterol , Kit for diagnosing cognitive disorders.
According to claim 5, The sample is tissue, urine, saliva, whole blood, plasma, platelets or serum samples, Cognitive disorder disease diagnosis kit.
The kit of claim 5, wherein the detection reagent is a reagent capable of detecting the marker at the protein or nucleic acid level.
The cognitive disorder of claim 7, wherein the reagent detectable at the protein level is a monoclonal antibody, a polyclonal antibody, a substrate, an aptamer, a receptor, a ligand or a cofactor, or a reagent for detecting a mass spectrometer. Diagnostic kit.
The kit for diagnosing cognitive disorders according to claim 7, wherein the reagent that can be detected at the nucleic acid level is a reagent used in reverse transcriptase chain reaction, RNase protection assay, or Northern blot.
The kit of claim 4, wherein the cognitive disorder is Alzheimer's disease, Parkinson's disease dementia, Lewy body dementia, Huntington's disease dementia, or mild cognitive impairment.
The presence of two or more cognitive impairment diagnostic markers selected from the group consisting of transthyretin, ApoE, alpha-cynucleine, vitamin D binding protein, neurologrenin, bimentin, stesmine, contactin, or HDL-cholesterol, or Detecting the amount; And
Correlating the presence or amount of the detected marker with a diagnosis or prognosis of a test subject's cognitive impairment,
A method of detecting two or more such markers from a subject's sample to provide information necessary for diagnosing or prognostic cognitive impairment.
The method of claim 11, wherein the presence or amount of the marker is determined at the protein or mRNA expression level.
The method of claim 11, wherein said associating further uses non-marker clinical information of said test subject.
The method of claim 13, wherein the non-marker clinical information of the test subject is age, sex, weight, diet, body mass, underlying disease, magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), or Mini-mental status examination (MMSE) or positron emission tomography (PET) Which is one or more of the methods.
12. The method of claim 11, wherein the associating step compares the amount or presence of the determined markers with the detection results for each of the markers determined in the normal control group.
The method of claim 11, wherein the cognitive disorder is Alzheimer's disease, Parkinson's disease dementia, Lewy body dementia, Huntington's disease dementia, or mild cognitive impairment.
Composition for the prevention or treatment of cognitive disorders comprising modulators of one or more of transcyretin, ApoE, alpha-cynucleine, vitamin D binding protein, neurologrenin, bimentin, stesmine, contactin, or HDL-cholesterol .
18. The composition of claim 17, wherein the modulator comprises a low molecular compound, an antibody, an antisense oligonucleotide, siRNA, shRNA, miRNA or polypeptide.
Providing at least one of transthyretin, ApoE, alpha-cynucleine, vitamin D binding protein, neurologrenin, bimentin, stesmine, or contactin protein;
Contacting the protein with a test substance; And
Comparing the expression of the protein in a control group that is not in contact with the protein in contact with the test substance, a method for screening a substance for preventing or treating a cognitive disorder disease.
20. The method of claim 19, wherein the protein is provided in the form of a cell or animal model expressing it.
21. The method of claim 20, wherein the cell is SH-SY5Y (Neuroblast-like subclone of SK-N-SH), HT22 (Hippocampal neuronal cell line), bEnd.3 (Immortalized mouse brain endothelial cell), U373 (Astrocytoma), rat Or mouse primary culture, Chinese hamster ovary cell (CHO), or human embryonic kidney 293 (HEK).
The method of claim 18, wherein the cognitive disorder is Alzheimer's disease, Parkinson's disease dementia, Lewy body dementia, Huntington's disease dementia, or mild cognitive impairment.

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