WO2012067151A1 - Cartilage Acidic Protein 1蛋白質による脳梗塞の検査方法 - Google Patents
Cartilage Acidic Protein 1蛋白質による脳梗塞の検査方法 Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
Definitions
- the present invention relates to a method for examining cerebral infarction, a method for evaluating cerebral infarction treatment or prevention effect, a method for screening for cerebral infarction preventive or therapeutic agent, and cerebral infarction examination, cerebral infarction treatment effect evaluation or cerebral infarction prevention / treatment.
- the present invention relates to a drug candidate compound screening kit.
- Stroke is a general term for cerebrovascular disorders such as cerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage. Of these stroke types, cerebral infarction has been relatively increasing recently.
- Thrombolytic therapy, anticoagulant therapy, antiplatelet therapy, administration of cerebral protective drugs, etc. are used as drug therapy in the acute phase of cerebral infarction.
- Surgical surgery is performed for patients with severe or hemorrhagic cerebral infarction. Is done. Cerebral infarction symptoms are strongest in the acute phase and then gradually improve. This is because the necrotic brain tissue is swollen and the surrounding brain tissue is also compressed and damaged. As the swelling subsides, the surrounding tissues recover their function and the symptoms are fixed.
- free radicals released from cerebral ischemic sites have the function of necrosing surrounding tissues, continuous treatment is required to improve the functional prognosis even after the acute phase.
- the clinical types of cerebral infarction include lacunar infarction that develops when the intracerebral small artery is occluded, atherothrombotic cerebral infarction that develops when the intracerebral aorta is occluded by atheroma, and a thrombus in the heart becomes an obturator.
- Examples include cardiogenic cerebral embolism that develops by blocking an artery. Since appropriate treatment methods for the acute and chronic phases differ depending on these clinical types, a method for rapidly diagnosing the clinical type of patients with cerebral infarction has been demanded.
- As a method of classifying clinical types of cerebral infarction clinical symptom observation and echocardiography, MRI, MRA, cervical vascular echo, etc.
- Non-patent Document 1 a flowchart of clinical diagnosis of cerebral infarction based on TOAST classification (Non-patent Document 1), etc.
- molecular markers such as CRP, D-Dimer, RAGE, MMP-9, S100B, BNP within 24 hours of cerebral infarction
- BNP a specific cutoff value
- Non-patent Document 2 There was a need for molecular markers that could be diagnosed.
- the prognosis of cerebral infarction is to evaluate the degree of movement disorder in daily life by the modified Rankin Scale (Non-patent Document 3) or Barthel Index (Non-patent Document 4) between 3 months or 1 year after onset. It is judged by. Predicting the prognosis in advance is important for early determination of a treatment method for cerebral infarction. Although it has been disclosed that death can be predicted by setting a specific cutoff value with BNP in plasma measured at the time of admission (Non-patent Document 5), a molecular marker for predicting the degree of injury when alive is required. It was.
- the degree of cerebral infarction is assessed by the National Institute of Health Stroke Scale (NIHSS) between immediately after the onset of cerebral infarction and 3 months after the onset of cerebral infarction. It is judged by.
- NIHSS National Institute of Health Stroke Scale
- cerebral infarction worsens during hospitalization, it is possible to confirm whether it is progressive cerebral infarction by observing changes in NIHSS. Since progressive cerebral infarction often has a poor prognosis, it is important to predict treatment early and enhance treatment. However, a method for predicting progressive cerebral infarction has not been established, and a molecular marker to be predicted has been demanded.
- Patent Document 1 describes the use of Cartilage Acidic Protein 1 as a marker for cardiovascular disease, blood disease, neurological disease, respiratory disease, gastrointestinal disease and urological disease. However, there is no specific data showing that Cartilage Acidic Protein 1 can be used for diagnosis of these diseases, only showing that the expression level of Cartilage Acidic Protein 1 is high in ischemia / myocardial infarction model animals.
- the present invention has an object to provide a method for quickly and surely inspecting cerebral infarction using serum or plasma of patients with cerebral infarction and the like, and a method for evaluating cerebral infarction treatment effects. To do.
- Cartilage Acidic Protein 1 protein in the plasma of patients with normal or different concentrations in the plasma of patients with different disease types and prognoses.
- the present inventors have found that the amount of Cartilage Acidic Protein 1 protein is a useful index for examination of cerebral infarction, and have completed the present invention.
- the gist of the present invention is as follows. [1] It includes a step of measuring the amount of Cartilage Acidic Protein 1 protein in a blood sample collected from a test animal, and it is determined that the brain is infarcted when the amount of the protein is lower than that of a healthy subject A test method for diagnosing cerebral infarction in the animal. [2] The blood sample collected from the test animal is collected from the test animal at any time between the onset of cerebral infarction-like symptoms and the 20th day after the onset. [1] The examination method for diagnosis of cerebral infarction according to [1]. [3] A test method for diagnosing cerebral infarction in an animal, comprising measuring the amount of Cartilage Acidic Protein 1 protein in a blood sample collected from the test animal.
- [4] Disease type diagnosis diagnoses either lacunar infarction, atherothrombotic, cardiogenic cerebral embolism, aortic cerebral embolism, Branch atheromatous disease (BAD), arterial dissection, or unclassifiable The inspection method according to [3].
- a test method for predicting the prognosis of cerebral infarction in the animal comprising a step of measuring the amount of Cartilage Acidic Protein 1 protein in a blood sample collected from the test animal.
- a test method for predicting the progression of cerebral infarction in an animal comprising measuring the amount of Cartilage Acidic Protein 1 protein in a blood sample collected from the subject animal. [8] The prediction of the progression of cerebral infarction according to [7], wherein the blood sample collected from the test animal is collected from the test animal on the seventh day of the onset of cerebral infarction. Inspection method for. [9] Measuring the amount of Cartilage Acidic Protein 1 protein in a blood sample collected from an animal in need of prevention or treatment of cerebral infarction administered with a cerebral infarction preventive or therapeutic agent For evaluating the effect of preventing or treating cerebral infarction in Japan.
- a prophylactic or therapeutic agent for cerebral infarction comprising a step of contacting a subject with a candidate compound for a prophylactic or therapeutic agent for cerebral infarction and then measuring the amount of Cartilage Acidic Protein 1 protein in the subject Screening method.
- a kit for screening for cerebral infarction examination, cerebral infarction therapeutic effect, or screening for candidate compounds for preventing or treating cerebral infarction comprising a reagent capable of measuring the amount of Cartilage Acidic Protein 1 protein.
- Cartilage Acidic ⁇ Protein 1 protein whose plasma concentration in patients with cerebral infarction is different from that in plasma of healthy subjects or patients with different types of disease, prognosis, or progression is a molecular marker for cerebral infarction examination (This may be referred to herein as “CRTAC1 protein marker”).
- CRTAC1 protein marker By using the CRTAC1 protein marker alone or in combination with other cerebral infarction molecular markers, it becomes possible to quickly and reliably diagnose cerebral infarction, or to classify the cerebral infarction and predict the prognosis.
- the first of the present invention is a method for examining cerebral infarction in an animal, comprising a step of measuring the amount of Cartilage Acidic Protein 1 protein in a blood sample collected from the subject animal.
- Cerebral infarction is a disease in which the cerebral artery is blocked by a thrombus or embolus and becomes ischemic, and the brain tissue is necrotized and damaged, and a lacunar infarction that develops due to blocking of a small intracerebral artery.
- Aortic cerebral embolism which is an embolism that develops when the obturator dissociates from the atherosclerotic lesion and occludes the intracerebral artery, and a plaque formed in the main artery obstructs the entrance of the penetrating branch “Branch atheromatous disease” (hereinafter sometimes referred to as “BAD”), an infarction of the penetrating branch region that occurs, arterial dissection where the intima of the cerebral blood vessel is damaged and blood enters the arterial wall and occludes the cerebral blood vessel Any of these are included.
- cerebral infarction includes any of acute phase, subacute phase, recovery phase (chronic phase) and the like.
- Cartilage Acidic Protein 1 protein (hereinafter sometimes referred to as “CRTAC1 protein”) means Cartilage Acidic Protein 1 protein, which is a target for measuring the content in a blood sample in the test method of the present invention. Any protein can be used as long as it is derived from a test animal. Specific examples of human-derived proteins include proteins represented by the specific amino acid sequences described below. Furthermore, fragments, derivatives, and mutants of proteins having the same functions as these are also included.
- the “test animal” may be any animal that may cause cerebral infarction, and specifically includes rodents such as humans, monkeys, and rats.
- the method for examining cerebral infarction of the present invention is particularly preferably performed in humans suspected of having cerebral infarction, humans after onset of cerebral infarction, or the like.
- the “blood sample” collected from the test animal is not particularly limited as long as it contains CRTAC1 protein and can measure the concentration thereof.
- EDTA plasma, citrate plasma, etc. any of plasma, serum, and whole blood may be used, but among these, EDTA plasma is preferably used because it can be easily collected, easily stored, and collected in a large amount.
- the timing for collecting the blood sample from the test animal may be any timing as long as the diagnosis of cerebral infarction is performed. Since cerebral infarction changes in symptoms and pathology from onset to onset, it will be described in detail below.
- Cartilage Acidic Protein 1 protein which is a target for measuring the content in a blood sample collected from a test animal, in the case of a human, from the amino acid sequence shown by Entry Name CRAC1_HUMAN of UniProtKB Is a protein.
- sequence information can be obtained by inputting the above Entry Name in a National Center for Biotechnology Information (NCBI) database.
- NCBI National Center for Biotechnology Information
- the amino acid sequence is human, but when the test animal is different, a homologous protein derived from the animal becomes the protein to be measured.
- the method for measuring the content of the protein in the blood sample is not particularly limited as long as the content of the protein can be measured.
- an existing immunoassay method using an antibody of the protein or a chromatographic method All components captured on a chromatographic carrier (eg, cation exchanger, anion exchanger, hydrophobic chromatographic carrier, metal ion, etc.) under certain conditions using a combination of holographic techniques and time-of-flight mass spectrometry (TOF-MS)
- TOF-MS time-of-flight mass spectrometry
- a method of measuring the mass of the solution at once, a two-dimensional gel electrophoresis method, or the like is used.
- the immunoassay a method of quantitative detection according to an enzyme immunoassay, a method of measurement by a fluorescence immunoassay, a chemiluminescence immunoassay, or the like is preferable.
- the enzyme immunoassay method is a detection method using an enzyme as a labeling substance among the labeled immunoassay methods.
- ELISA enzyme-linked immunosorbent assay
- the sandwich method is mentioned as one of the particularly preferable measurement modes in consideration of the convenience of operation, economic convenience, and general versatility in clinical examination.
- Antibodies and the like (including antibody fragments) used in the above measurement can be obtained by a known method using the target CRTAC1 protein as an antigen. However, it is not necessary to be produced using the target CRTAC1 protein as an antigen, and any protein may be used as long as it exhibits at least cross-reactivity with the protein and can measure its content. As such an immunoassay and an antibody used therefor, for example, Anti-human® CRTAC1 • Antibody (catalog No. AF5234) manufactured by R & D, etc. is preferably used.
- the blood sample used in the method of the present invention is preferably used immediately after being collected from the test animal for the above measurement, but a stored sample may be used.
- the method for storing the blood sample is not particularly limited as long as the amount of molecular marker for cerebral infarction examination does not change, but there are, for example, a low temperature condition such as 0 to 10 ° C. that does not freeze, a dark condition, and no vibration condition. preferable.
- a low temperature condition such as 0 to 10 ° C. that does not freeze, a dark condition, and no vibration condition.
- a method capable of avoiding marker molecule decomposition or oxidation reaction such as deep freezing is preferable.
- the content of the CRTAC1 protein marker in a blood sample collected from a test animal is measured.
- it is a composite index that combines the contents of specimens such as CRP and D-Dimer, which are existing cerebral infarction markers, and links the observation of clinical symptoms with the results of echocardiography, MRI, MRA, cervical vascular echo, etc. It is possible to inspect more accurately by using.
- the cerebral infarction test is performed using the content of the molecular marker for cerebral infarction test in the sample as an index
- the absolute value of the content of the molecular marker for cerebral infarction test in the sample is compared with that of a healthy person (healthy animal). It may be compared, or a method of inspecting by specifying an appropriate cutoff value may be used.
- the content in the blood sample of the cerebral infarction test marker of a healthy subject is obtained by collecting blood from a healthy subject who has been clinically confirmed in advance as not having cerebral infarction, and processing similar to that of blood collected from a test animal and It can be obtained by measuring and quantifying.
- the method for clinically confirming various cerebral infarctions is not particularly limited, but examples include methods for confirming from device diagnostic methods such as head X-ray CT, head MRI, MRA, cerebral angiography, cervical vascular echo, etc. It is done.
- the cut-off value is a value determined when determining a target disease group and a non-disease group (a group other than the target disease) by paying attention to a certain substance.
- determining the target disease and non-disease it is negative if it is below the cut-off value, positive if it is above the cut-off value, or positive if it is below the cut-off value, and if it is above the cut-off value
- the disease can be determined as negative (Kanai Masamitsu, Clinical Laboratory Proposal Kanehara Publishing Co., Ltd.).
- the index used for the purpose of evaluating the clinical usefulness of the cutoff value includes sensitivity and specificity.
- a certain population is determined using a cut-off value, and among the diseased patients, a (true positive) is positive in the determination, and b (false negative) is negative in the determination while being a disease patient.
- the distribution of measured values between the target disease group and the non-disease group usually has some overlap. Therefore, sensitivity and specificity change by raising or lowering the cutoff value. Decreasing the cut-off value increases the sensitivity, but the specificity decreases, and increasing the cut-off value decreases the sensitivity, but increases the specificity. As a determination method, it is preferable that both sensitivity and specificity are higher. A determination method in which the values of sensitivity and specificity do not exceed 0.5 is not recognized as useful.
- the method for setting the cut-off value is to set 95% of the distribution of the non-disease group as one of the values at both ends from the center as the cut-off value.
- the distribution of the non-disease group shows a normal distribution
- a method of setting an average value +2 times standard deviation (SD) or an average value -2SD as a cut-off value is a normal distribution.
- the sensitivity and specificity vary depending on the cutoff value set as described above, so the sensitivity and specificity at a certain cutoff value are simply used. Rather than evaluating, it is desirable to evaluate with an index that maintains high sensitivity and specificity when the cut-off value is raised or lowered, for example, the AUC value of the ROC curve.
- the AUC value of the ROC curve becomes 1 when a cutoff value exists such that both sensitivity and specificity are 100%, and approaches 0.5 when the diagnostic performance is not good. Therefore, when the performance of a certain diagnostic method is judged by the AUC value of the ROC curve, it can be evaluated that the method is suitable as a diagnostic method if it is 0.7 or more. Since the CRTAC1 protein marker described in the present invention also has an ROC curve AUC value of 0.7 or more, it is used in the cerebral infarction examination method described later.
- CRTAC1 protein marker As a specific method of the cerebral infarction examination method using the CRTAC1 protein marker of the present invention, in order to distinguish healthy subjects from cerebral infarction patients. In other words, the amount of CRTAC1 protein in a blood sample collected from a test animal is measured, and if the CRTAC1 protein amount is lower than that of a healthy subject, it is determined that the subject has a cerebral infarction. And an inspection method.
- the timing of obtaining the specimen in this case is not particularly limited from immediately after the onset of cerebral infarction-like symptoms, but is preferred from the meaning of diagnosis from immediately after the onset to the 20th day after onset.
- a particularly preferable timing is between immediately after onset and 9 days after onset.
- the content of the CRTAC1 protein marker in the collected specimen may be measured, and this absolute value may be compared with that of a healthy person (healthy animal), or an appropriate cut-off value may be defined by the above method. An inspection method may be used.
- the absolute value of the content of the CRTAC1 protein marker in a blood sample collected from a subject suspected of having cerebral infarction is higher than that of a healthy person (healthy animal) or higher than the cutoff value, the subject The examiner can determine that the patient is a cerebral infarction patient.
- the content in the blood sample of the cerebral infarction test marker of a healthy subject is obtained by collecting blood from a healthy subject who has been clinically confirmed in advance as not having cerebral infarction, and processing similar to that of blood collected from a test animal and It can be obtained by measuring and quantifying.
- the method for clinically confirming various cerebral infarctions is not particularly limited, but examples include methods for confirming from device diagnostic methods such as head X-ray CT, head MRI, MRA, cerebral angiography, cervical vascular echo, etc. It is done.
- the disease type means a clinically distinct disease type known per se, and specifically includes “lacuna infarction” (sometimes referred to herein as “lacuna”), “atherothrombosis” Cerebral infarction “(sometimes referred to herein as” atherothrombotic “),” cardiogenic cerebral embolism “(sometimes referred to herein as” cardiogenic cerebral embolism ”) ), “Aortic cerebral embolism”, an embolism that develops when the obturator dissociates from the atherosclerotic lesion from the ascending aorta to the aortic arch and occludes the intracerebral artery, plaque formed in the main artery "Branch atheromatous disease” (hereinafter sometimes referred to as "BAD”), which is an infarction of the penetrating branch region caused by occlusion of the entrance of the penetrating branch, and blood in the arterial wall Does not fall under arterial dissection or any of them Disease type (hereinafter sometimes referred
- the cut-off value that can distinguish each disease type in the amount of CRTAC1 protein in the blood sample is determined as described above. And the amount of CRTAC1 protein in the sample is compared with the cut-off value to determine which disease type group it belongs to.
- the timing of obtaining the specimen when performing these measurements is not particularly limited immediately after the onset of cerebral infarction-like symptoms, but Table 1 shows preferred timings.
- the amount of CRTAC1 protein in a blood sample of a patient clinically determined as the above-mentioned distinct disease type is measured in advance, and the above method is used to distinguish between both disease type groups.
- (1-3) Test method for predicting prognosis of cerebral infarction using CRTAC1 protein marker As a specific method of the cerebral infarction test method using CRTAC1 protein marker of the present invention, the prognosis of cerebral infarction of a subject (animal) is determined. Examples include inspection methods for prediction. The prognosis of cerebral infarction is indicated by the degree of injury due to cerebral infarction of the patient after a certain period of time after the onset of cerebral infarction. The degree of failure can be assessed using an already established indicator known per se. Indicators include, for example, Japan's modified Rankin Scale (mRS) criteria (Yukito Shinohara et al. MRS Reliability Study Group, research on reliability of modified Rankin Scale-introduction of Japanese criteria and questionnaire, stroke 2007; 29: 6-13) etc. are used.
- mRS Japan's modified Rankin Scale
- the amount of CRTAC1 protein in a blood sample obtained from a subject about 2 weeks after onset, and the state of the subject 3 months after the onset is preferable.
- the amount of CRTAC1 protein in the blood sample at the time of measurement is measured in advance for each patient who has been clinically determined for the above mRS at the time of prediction. Then, a cutoff value for distinguishing both mRS groups is set by the above-described method. More specifically, for example, when distinguishing between patients with mRS 0-1 after 3 months and patients with 2-5, patients with mRS 0-5 3 months after the onset (for prediction purposes) 7 days after the onset of illness (at the time of measurement), the CRTAC1 protein level in the blood sample is measured, and the cut-off between the measured value of patients whose mRS is 0 to 1 and the measured value of patients 2 to 5 after 3 months Set the value. Thereafter, a sample at the measurement purpose is collected, the content of the CRTAC1 protein marker in the sample is measured, and the absolute value is compared with the cut-off value set by the above method. The state of the subject (animal) can be predicted.
- the prediction method of the present invention particularly preferably, about 2 weeks after onset, more preferably 3 months after onset by measuring the amount of CRTAC1 protein in a sample collected from a subject on the 9th day after onset.
- Examples include a method for predicting whether the mRS of the subject in the vicinity is 2 to 5 or less than 1 or the like.
- a preferred example is a method for predicting whether the mRS of the subject in the vicinity of 3 months after the onset is 4-5 or less than 3 by measuring the amount of CRTAC1 protein in a sample collected from It is done.
- a subject (animal) diagnosed with cerebral infarction by the method of the present invention has a very good prognostic course and therapeutic effect by receiving a treatment suitable for each disease.
- (1-4) Examination Method for Predicting Progression of Cerebral Infarction Using CRTAC1 Protein Marker As a specific method of the cerebral infarction examination method using CRTAC1 protein marker of the present invention, progression of cerebral infarction in a subject (animal) Examples include inspection methods for predicting sex. Progression of cerebral infarction is indicated by a change in the severity of the patient due to cerebral infarction within a certain period of time after the onset of cerebral infarction. Severity can be assessed using known indicators already known per se. As an index, for example, National Institute of Health Stroke Scale (NIHSS) (Brott T. et al., Measurement of acute cerebral infarction: A clinical examination scale. Stroke, 20, 864 (1989)) is used.
- NIHSS National Institute of Health Stroke Scale
- the amount of CRTAC1 protein in the blood sample obtained from the subject within about one week immediately after the onset is preferable.
- the amount of CRTAC1 protein in the blood sample at the time of measurement is measured in advance for each patient who has been clinically determined for the NIHSS at the time of the prediction purpose, and the change in NIHSS that is desired to be distinguished at the time of the prediction purpose.
- the cutoff value for distinguishing between progressive and non-progressive is set by the above-described method.
- NIHSS immediately after the onset of cerebral infarction
- NIHSS determined within 2 weeks (at the time of prediction) increases by 1 or more, it is progressive, if it does not change or decreases by 1 or more
- It is defined as non-progressive and the amount of CRTAC1 protein in blood samples from immediately after onset to the seventh day of onset (during measurement) is measured, and patients with advanced or non-progressive NIHSS within 2 weeks Set the cutoff value between the measured values.
- a sample at the measurement purpose is collected, the content of the CRTAC1 protein marker in the sample is measured, and the absolute value is compared with the cut-off value set by the above method.
- the state of the subject (animal) can be predicted.
- Examples include a method of predicting whether the examiner's NIHSS will increase or decrease by 1 or more or decrease by 1 or more immediately after the onset of cerebral infarction.
- Kit for measuring cerebral infarction The present invention further provides a kit for use in the above-described cerebral infarction examination, or evaluation of the prevention or treatment effect of cerebral infarction described below, or a screening method for a cerebral infarction prevention or treatment drug.
- the kit is also included.
- the contents of the kit are configured by a combination of equipment or reagents.
- the configuration or form of the kit is not limited as long as it includes a substance that is essentially the same as each component described below, or a substance that is essentially the same as a part thereof. Even if they are different, they are included in the kit of the present invention.
- the reagent include an anti-CRTAC1 protein marker antibody when the CRTAC1 protein marker is measured by an immunoassay.
- a biological sample diluent an antibody-immobilized solid phase, a reaction buffer, a washing solution, a labeled secondary antibody or an antibody fragment thereof, a labeled detection reagent, a standard substance, and the like are also included as necessary.
- the diluted solution of the biological sample include an aqueous solution containing a protein such as BSA or casein in a surfactant or a buffer.
- the antibody-immobilized solid phase a material in which various polymer materials are shaped to suit the application and anti-molecular marker antibodies or their antibody fragments are solid-phased is used. Shapes are tubes, beads, plates, fine particles such as latex, sticks, etc., and materials are polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, polyethylene terephthalate, etc. Polymer materials, glass, ceramics and metals. Examples of the method for immobilizing an antibody include known methods such as a physical method and a chemical method, or a combination method thereof. For example, a 96-well polystyrene immunoassay microterplate prepared by subjecting an antibody or antibody fragment to a hydrophobic solid phase can be used.
- reaction buffer Any reaction buffer may be used as long as it provides a solvent environment for the binding reaction between the antibody on the antibody-immobilized solid phase and the antigen in the biological sample.
- surfactants buffers, BSA, Reaction buffers containing proteins such as casein, preservatives, stabilizers, reaction accelerators and the like can be mentioned.
- the labeled secondary antibody or antibody fragment thereof is labeled with a labeling enzyme such as horseradish peroxidase (HRP), bovine small intestine alkaline phosphatase, ⁇ -galactosidase, or buffering agent. , BSA, casein and other proteins, preservatives and the like are used.
- HRP horseradish peroxidase
- BSA bovine small intestine alkaline phosphatase
- ⁇ -galactosidase or buffering agent.
- BSA casein and other proteins, preservatives and the like are used.
- a substrate for absorption measurement such as tetramethylbenzidine or orthophenylenediamine, or a fluorescence such as hydroxyphenylpropionic acid or hydroxyphenylacetic acid.
- a substrate for absorption measurement such as tetramethylbenzidine or orthophenylenediamine
- a fluorescence such as hydroxyphenylpropionic acid or hydroxyphenylacetic acid.
- the substrate or the luminescent substrate such as luminol is alkaline phosphatase
- examples include an absorbance measurement substrate such as 4-nitrophenyl phosphate, and a fluorescent substrate such as 4-methylumbelliferyl phosphate.
- the second aspect of the present invention is blood collected from an animal in need of prevention or treatment of cerebral infarction administered with a cerebral infarction preventive or therapeutic agent.
- a method for evaluating the effect of preventing or treating cerebral infarction in an animal comprising a step of measuring the amount of CRTAC1 protein marker in a sample.
- an animal that requires prevention or treatment of cerebral infarction is a human who exhibits symptoms of cerebral infarction or an animal described in (1) (these may be simply referred to as “subjects”), Examples include patients diagnosed with cerebral infarction by the method (1) above.
- the cerebral infarction preventive or therapeutic agent may be any as long as it is used as a cerebral infarction preventive or therapeutic agent.
- thrombolytic agents such as urokinase and tissue plasminogen activator, antiparin such as heparin, etc.
- anticoagulants such as coagulants, cyclooxygenase inhibitors, phosphodiesterase inhibitors, thromboxen A2 (TXA2) synthesis inhibitors, and brain protective agents such as free radical scavengers.
- the amount of CRTAC1 protein marker in a blood sample collected from an animal in need of prevention or treatment of cerebral infarction administered with a cerebral infarction preventive or therapeutic agent is increased or decreased compared to the amount before administration, or When approaching the amount in a control animal that has not developed cerebral infarction, it can be determined that there was a preventive or therapeutic effect. Since the cerebral infarction preventive or therapeutic agent to be treated differs depending on the disease type, it is possible to perform appropriate treatment for the patient if the disease type is diagnosed with the CRTAC1 protein marker.
- a lacunar infarction if a lacunar infarction is diagnosed, it can be treated with a thromboxen A2 (TXA2) synthesis inhibitor and a cyclooxygenase inhibitor can prevent recurrence of cerebral infarction.
- TXA2 thromboxen A2
- a cyclooxygenase inhibitor can prevent recurrence of cerebral infarction.
- the prognosis is improved by using several kinds of therapeutic agents such as free radical scavengers from the early stage of treatment, or by enhancing the content of rehabilitation. It is possible to perform such treatment.
- an anticoagulant such as argatroban or an antiplatelet agent such as a phosphodiesterase inhibitor is used in combination with a free radical scavenger from the early stage of treatment.
- a free radical scavenger By enhancing drug treatment, it becomes possible to prevent the progression of symptoms by preventing or suppressing the degree of progression.
- CRTAC1 in a blood sample of a subject is contacted with a subject and a candidate compound for a prophylactic or therapeutic agent for cerebral infarction.
- a screening method for a preventive or therapeutic agent for cerebral infarction comprising a step of measuring the amount of a protein marker.
- a non-human animal individual, tissue, cell or the like in which the content of the CRTAC1 protein marker exhibits the same abnormality as in the cerebral infarction state is used.
- animals having cerebral infarction include non-human cerebral infarction animals in which a cerebral infarction state has been formed by a surgical technique or the like (Yuji Kuge, Kazuo Minematsu et al. Nylon Monofilament for Intraluminal Middle Cerebral Artery Occlusionin Rats. Stroke , 26, 1655 (1995)).
- Yuji Kuge Kazuo Minematsu et al. Nylon Monofilament for Intraluminal Middle Cerebral Artery Occlusionin Rats. Stroke , 26, 1655 (1995)
- CRTAC1 protein marker content similar to that in the normal state (normal) as a control Based on the CRTAC1 protein marker, if a pathological condition similar to a certain disease type in humans can be constructed in a non-human animal with cerebral infarction, an experimental system for developing a therapeutic agent in a patient with cerebral infarction of a certain disease type will be established be able to. In addition, by monitoring the prognosis of a non-human animal individual with the marker, it is possible to develop a therapeutic agent effective for the prognosis of human cerebral infarction.
- candidate compounds for preventing or treating cerebral infarction include peptides, proteins, non-peptidic compounds, low-molecular synthetic compounds, and the like. These compounds may be novel compounds or known compounds. Moreover, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like containing these compounds may be used. The dose, administration method, treatment time and the like may be appropriately selected according to the subject to be used.
- the method for measuring the content of the CRTAC1 protein marker in the subject after contacting with the candidate compound can be performed by a method suitable for each subject.
- the subject when it is a cell, it may be prepared as a cell extract according to a known method and used as a sample, or the cell may be immobilized on a culture plate or a slide glass and used as a sample. Also good.
- detection can be performed by ELISA or the like. It is preferable that these samples have the same number of cells used for extraction, or the amount of purified RNA or the amount of extracted protein so that the numerical values obtained as a result of detection can be compared and analyzed accurately. If the amount of CRTAC1 protein marker in the subject contacted with the candidate compound is increased or decreased compared to the amount before contact, or approaches the amount in the control subject, the candidate compound is It can be determined to have a prophylactic or therapeutic effect.
- Example 1 Evaluation of Test Method for Discriminating Healthy Persons from Cerebral Infarction Patients Using Cartilage Acidic Protein 1 Protein Marker Immediately after the onset of cerebral infarction (described as “DAY0” in the table), 7 days after onset (in the table) Blood was collected from patients with cerebral infarction at the time (described as “DAY7”) and healthy individuals (number of persons described in Table 2), and EDTA plasma was obtained.
- the AUC value of the ROC curve was calculated according to the method described in Fawcett, T. (2004) ROC Graphs: NotesNoteand Practical Considerations for researchers. The results are shown in Table 2. It is shown that a value exceeding 0.7 at each time of blood collection is useful as a test method for discriminating between a cerebral infarction patient and a healthy person.
- blood can be obtained from cerebral infarction patients immediately after onset and on the 7th day, and the test for distinguishing between healthy individuals and patients with cerebral infarction can be performed by comparing the concentration of CRTAC1 protein in healthy subjects and plasma. all right.
- Example 2 Evaluation of a test method for discriminating cerebral infarction type using CRTAC1 protein marker Patients with a confirmed diagnosis for each type of cerebral infarction, specifically, “lacuna infarction” (in the table (Referred to as “lacuna”), “atherothrombotic cerebral infarction” (referred to as “atherothrombotic” in the table), “cardiogenic cerebral embolism” (referred to as “cardiogenic cerebral embolism” in the table) ), “Aortic cerebral embolism” (referred to as “aortic cerebral embolism” in the table), “Branch atheromatous disease” (referred to as “BAD” in the table), “arterial dissection” (in the table) , Referred to as “arterial dissection”, or patients with a disease type that does not fall into any of these (referred to as “non-classifiable type” in the table), immediately after the onset of cerebral infarction (described as “DAY0” in the table) ), 7 days after onset (in
- the concentration of CRTAC1 protein in the plasma was measured in the same manner as in the method of Example 1, and the AUC value of the ROC curve was calculated from the measured value in the same manner as in Example 1. It is shown that a value exceeding 0.7 at each time of blood collection is useful as a test method for determining the disease type of a cerebral infarction patient.
- Example 3 Evaluation of a test method for predicting the prognosis of cerebral infarction using CRTAC1 protein marker
- the prognosis of cerebral infarction is indicated by the degree of injury due to cerebral infarction of the patient after a certain period of time after the onset of cerebral infarction. It is.
- Japanese version modified Rankin Scale (mRS) criteria Yukito Shinohara et al. MRS Reliability Study Group, reliability of modified Rankin Scale-Japanese version criteria and questionnaire
- Blood was collected 7 days after the onset of cerebral infarction (indicated as “DAY7” in the table) for patients classified as 0 to 5 using the introduction of Stroke 2007; 29: 6-13) (each in Table 4) EDTA plasma was obtained.
- the concentration of CRTAC1 protein in the plasma was measured in the same manner as in the method of Example 1, and the AUC value of the ROC curve was calculated from the measured value in the same manner as in Example 1.
- the results are shown in Table 4. It is shown that a value exceeding 0.7 at each time of blood collection is useful as a test method for determining the prognosis of a cerebral infarction patient.
- the degree of injury (prognosis) 3 months after the onset of cerebral infarction was determined by comparing the CRTAC1 protein concentration in the plasma on the 7th day after the onset of patients ranked 3 months later. 3 months later, rank 2-5 patients, rank 0-1 patients, rank 3-5 patients, rank 0-2 patients, rank 4-5 patients, rank 0-3 patients It turned out that the test
- Example 4 Evaluation of Test Method for Predicting Progression of Cerebral Infarction Using CRTAC1 Protein Marker 3 days after onset, 7 onset of NIHSS immediately after the onset of cerebral infarction (described as “DAY0” in the table) One day after the onset and 14 days after the onset, NIHSS increased by 1 or more points as progressive, and NIHSS did not change or decreased by 1 or more points as non-progressive. To eliminate bias between severity and disease type subgroups, patients with NIHSS> 15 immediately after onset were excluded. The concentration of CRTAC1 protein in the plasma was measured in the same manner as in the method of Example 1, and the AUC value of the ROC curve was calculated from the measured value in the same manner as in Example 1.
- diagnosis of cerebral infarction diagnosis of cerebral infarction, prognosis prediction test and evaluation of treatment / prevention effect can be performed, which is useful in the medical and diagnostic fields.
- a therapeutic / prophylactic agent for cerebral infarction can be screened, which is also useful in the pharmaceutical field.
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Abstract
Description
脳梗塞の臨床病型の分類方法としては、臨床症候の観察と心エコー、MRI、MRA、頚部血管エコー等を施行してTOAST分類に準拠した脳梗塞臨床診断のフローチャート(非特許文献1)等により分類する方法や、分子マーカーを用いた方法、具体的には、CRP、D-Dimer、RAGE、MMP-9、S100B、BNP等の分子マーカーを脳梗塞発症24時間以内に同時測定した結果、BNPとD-Dimerで、特定のカットオフ値を設定すると、心原性脳塞栓症を他病型と分類できること(非特許文献2)などが開示されているが、さらに迅速で確実な病型診断が可能となる分子マーカーが求められていた。
脳梗塞の予後は、発症後3ヶ月後ないしは1年後までの間にmodified Rankin Scale(非特許文献3)、またはBarthel Index(非特許文献4)により日常生活における動作の障害度を評価することで判断されている。予後を事前に予測することは、脳梗塞の治療方法を早期に決める上で重要である。入院時に測定した血漿中のBNPで特定のカットオフ値を定めることにより死亡が予測できること(非特許文献5)が開示されているが、生存した場合の障害の程度を予測する分子マーカーが求められていた。
脳梗塞の症状の程度(重症度)は、脳梗塞発症直後から発症後3ヶ月後までの間にNational Institute of Health Stroke Scale(NIHSS)により意識障害、運動障害や高次機能障害などを評価することで判断されている。脳梗塞は少なからず入院期間中に症状が悪化するが、NIHSSの変化を観察することにより進行性脳梗塞かどうかを確認できる。進行性脳梗塞は予後不良となることが多いことから、早期に予測して治療を充実させることが重要である。しかしながら、進行性脳梗塞を予測する方法は確立されておらず、予測する分子マーカーが求められていた。
特許文献1には、Cartilage Acidic Protein 1を循環器疾患、血液疾患、神経疾患、呼吸器疾患、消化器疾患および泌尿器疾患のマーカーとして使用することが記載されている。しかしながら、虚血・心筋梗塞モデル動物でCartilage Acidic Protein 1の発現量が多いことを示すのみで、Cartilage Acidic Protein 1がこれらの疾患の診断に使用できることを示す具体的なデータはない。
[1]被検動物より採取された血液試料中のCartilage Acidic Protein 1蛋白質の量を測定する工程を含み、該蛋白質量が健常者と比較して低下していた場合に脳梗塞であると判断することを特徴とする、該動物における脳梗塞の診断のための検査方法。
[2]被検動物より採取された血液試料が、脳梗塞様症状の発症直後から発症後20日目の間のいずれかの時期において被検動物より採取されたものであることを特徴とする[1]に記載の脳梗塞の診断のための検査方法。
[3]被検動物より採取された血液試料中のCartilage Acidic Protein 1蛋白質の量を測定する工程を含む、該動物における脳梗塞の病型診断のための検査方法。
[4]病型診断が、ラクナ梗塞、アテローム血栓性、心原性脳塞栓症、大動脈原性脳塞栓症、Branch atheromatous disease(BAD)、動脈解離、分類不能のいずれかを診断するものである[3]に記載の検査方法。
[5]被検動物より採取された血液試料中のCartilage Acidic Protein 1蛋白質の量を測定する工程を含む、該動物における脳梗塞の予後の予測のための検査方法。
[6]被検動物より採取された血液試料が、脳梗塞発症7日目に被検動物より採取されたものであることを特徴とする[5]に記載の脳梗塞の予後の予測のための検査方法。
[7]被検動物より採取された血液試料中のCartilage Acidic Protein 1蛋白質の量を測定する工程を含む、該動物における脳梗塞の進行性の予測のための検査方法。
[8]被検動物より採取された血液試料が、脳梗塞発症7日目に被検動物より採取されたものであることを特徴とする[7]に記載の脳梗塞の進行性の予測のための検査方法。
[9]脳梗塞の予防薬もしくは治療薬が投与された脳梗塞の予防もしくは治療を必要とする動物から採取された血液試料中のCartilage Acidic Protein 1蛋白質の量を測定する工程を含む、該動物における脳梗塞の予防もしくは治療効果の評価方法。
[10]被検体と脳梗塞の予防薬または治療薬の候補化合物を接触させた後、該被検体中のCartilage Acidic Protein 1蛋白質の量を測定する工程を含む、脳梗塞の予防薬または治療薬のスクリーニング方法。
[11]Cartilage Acidic Protein 1蛋白質の量を測定し得る試薬を含んでなる、脳梗塞検査用、脳梗塞治療効果評価用、または脳梗塞予防・治療薬候補化合物のスクリーニング用キット。
(1)脳梗塞検査方法
本発明の第1は、被検動物より採取された血液試料中の、Cartilage Acidic Protein 1蛋白質量を測定する工程を含む、該動物における脳梗塞の検査方法である。
カットオフ値の臨床的有用性を評価する目的で用いられる指標としては、感度と特異度が挙げられる。ある母集団をカットオフ値を用いて判定し、疾病患者のうち、判定で陽性とされたものをa(真陽性)、疾病患者でありながら判定で陰性とされたものをb(偽陰性)、疾病患者でないにも関わらず判定で陽性とされたものをc(偽陽性)、疾病患者でなく判定で陰性とされたものをd(真陰性)と表したときに、a/(a+b)で表される値を感度(真陽性率)、d/(c+d)で表される値を特異度(真陰性率)として表すことができる。
本発明のCRTAC1蛋白質マーカーを用いた脳梗塞検査方法の具体的方法として、健常者と脳梗塞患者を区別するための検査方法、すなわち、被検動物より採取された血液試料中のCRTAC1蛋白質量を測定し、CRTAC1蛋白質量が健常者と比較して低下していた場合に脳梗塞であると判断することを特徴とする検査方法が挙げられる。この場合の検体の取得のタイミングは、脳梗塞様症状の発症直後から特に制限はないが、発症直後から発症後20日目までが診断を行う意味からも好ましい。特に好ましいタイミングとしては、発症直後~発症後9日目の間である。採取された検体中の上記CRTAC1蛋白質マーカーの含有量を測定して、この絶対値を健常者(健常動物)のそれと比較してもよいし、上記の方法で適当なカットオフ値を規定して検査する方法でもよい。脳梗塞が疑われる被検者から採取された血液試料中のCRTAC1蛋白質マーカーの含有量の絶対値が、健常者(健常動物)のそれより高い場合や、カットオフ値より高い場合に、該被検者は脳梗塞患者であると判定することができる。健常者の上記脳梗塞検査用マーカーの血液試料中の含有量は、予め脳梗塞でないことを臨床的に確認された健常者から血液を採取し、被検動物から採取した血液と同様の処理及び測定を行って定量することにより得ることができる。臨床的に各種脳梗塞を確認する方法は特に制限がないが、例えば頭部X線CT、頭部MRI、MRA、脳血管造影、頚部血管エコー等の機器診断方法等から確認する方法等が挙げられる。また、既存の脳梗塞マーカーであるCRPやD-Dimer等の検体中含有量とを組み合わせることや、臨床症候の観察と心エコー、MRI、MRA、頚部血管エコー等の結果とを関連付けた複合指標を用いることで、より的確に検査することが可能である。
本発明のCRTAC1蛋白質マーカーを用いた脳梗塞検査方法の具体的方法として、脳梗塞の病型を区別するための検査方法が挙げられる。病型とは、それ自体既知の臨床的に区別されている病型を意味し、具体的には、「ラクナ梗塞」(本明細書中では「ラクナ」と称することがある)、「アテローム血栓性脳梗塞」(本明細書中では、「アテローム血栓性」と称することがある)、「心原性脳塞栓症」(本明細書中では、「心原性脳塞栓」と称することがある)、上行大動脈から大動脈弓部にできた粥状硬化巣から栓子が解離して脳内動脈を閉塞して発症する塞栓症である「大動脈原性脳塞栓症」、主幹動脈にできたプラークが穿通枝の入口部を閉塞することによって生じる穿通枝領域の梗塞である「Branch atheromatous disease」(以下、「BAD」と称することがある)、脳血管の内膜が損傷し血液が動脈壁内に侵入して脳血管を閉塞する動脈解離あるいはそれらのいずれにも該当しない病型(以下、これを「分類不能型」と称することがある)が挙げられる。
本発明のCRTAC1蛋白質マーカーを用いた脳梗塞検査方法の具体的方法として、被検者(動物)の脳梗塞の予後を予測するための検査方法が挙げられる。脳梗塞の予後は、脳梗塞発症後、ある一定期間の後に、その患者の脳梗塞による障害の程度にて示される。障害の程度は、それ自体公知の既に確定している指標を用いて評価することができる。指標としては、例えば、日本版modified Rankin Scale(mRS)判定基準(篠原幸人らmRS信頼性研究グループ、modified Rankin Scaleの信頼性に関する研究-日本語版判定基準書および問診表の紹介、脳卒中 2007;29:6-13)等が用いられる。
本発明のCRTAC1蛋白質マーカーを用いた脳梗塞検査方法の具体的方法として、被検者(動物)の脳梗塞の進行性を予測するための検査方法が挙げられる。脳梗塞の進行性は、脳梗塞発症後、ある一定期間以内に、その患者の脳梗塞による重症度の変化にて示される。重症度は、それ自体公知の既に確定している指標を用いて評価することができる。指標としては、例えば、National Institute of Health Stroke Scale(NIHSS)(Brott T. et al., Measurement of acute cerebral infarction: A clinical examination scale. Stroke, 20, 864 (1989))等が用いられる。
本発明は、さらに上記脳梗塞検査に用いるためのキット、あるいは下述する脳梗塞の予防もしくは治療効果の評価、あるいは脳梗塞予防もしくは治療薬のスクリーニング方法を行うためのキットも含まれる。キットの内容は、機器または試薬の組み合わせにより構成されるが、以下に述べる各構成要素と本質的に同一、またはその一部と本質的に同一な物質が含まれていれば、構成または形態が異なっていても、本発明のキットに包含される。試薬としては、例えば、免疫測定法によりCRTAC1蛋白質マーカーを測定する場合には、抗CRTAC1蛋白質マーカー抗体を含む。また、必要に応じ、生体試料の希釈液、抗体固定化固相、反応緩衝液、洗浄液、標識された二次抗体またはその抗体断片、標識体の検出用試薬、標準物質なども含まれる。生体試料の希釈液としては、界面活性剤、緩衝剤などにBSAやカゼインなどの蛋白質を含む水溶液などが挙げられる。
本発明の第2の態様は、脳梗塞の予防薬もしくは治療薬が投与された脳梗塞の予防もしくは治療を必要とする動物から採取された血液試料中のCRTAC1蛋白質マーカーの量を測定する工程を含む、該動物における脳梗塞の予防もしくは治療効果の評価方法である。
脳梗塞の予防薬もしくは治療薬が投与された脳梗塞の予防もしくは治療を必要とする動物から採取された血液試料におけるCRTAC1蛋白質マーカー量が、投与前の量に比べて増加又は減少するか、あるいは、脳梗塞を発症していない対照の動物における量に近づいた場合、予防または治療効果があったと判定することができる。
病型により治療する脳梗塞予防もしくは治療薬が異なるため、CRTAC1蛋白質マーカーで病型診断を行えば患者に対して適切な治療を施すことが可能となる。例えば、ラクナ梗塞と診断されればスロンボキセンA2(TXA2)合成阻害薬で治療を行い、サイクロオキシゲナーゼ阻害薬で脳梗塞の再発を予防できる。また、該マーカーで予後不良となる可能性が予測できた場合には、フリーラジカルスカベンジャー等の数種の治療薬を治療早期から併用したり、リハビリテーションの内容を充実させることにより、予後が好転するような治療を施すことが可能となる。さらに、該マーカーで進行性となる可能性が予測できた場合には、治療早期からフリーラジカルスカベンジャーにアルガトロバン等の抗凝固剤やフォスフォリジエステラーゼ阻害薬等の抗血小板薬を併用するなどして薬物治療を充実させることにより、進行性を予防あるいは進行の程度を抑えて症状の重症化を防ぐことが可能となる。
本発明の第3の態様は、被検体と脳梗塞の予防薬もしくは治療薬の候補化合物を接触させた後、該被検体の血液試料中のCRTAC1蛋白質マーカー量を測定する工程を含む、脳梗塞の予防もしくは治療薬のスクリーニング方法である。
CRTAC1蛋白質マーカーをもとに、脳梗塞状態の非ヒト動物個体でヒトにおけるある病型に類似した病態を構築できれば、ある病型の脳梗塞患者における治療薬を開発するための実験系を確立することができる。また、非ヒト動物個体の予後を該マーカーでモニタリングすることにより、ヒトの脳梗塞の予後に有効な治療薬を開発することが可能となる。
その投与量、投与方法、処理時間等は、用いる被検体に従って適宜選択すればよい。
候補化合物と接触させた被検体におけるCRTAC1蛋白質マーカーの量が、接触前の量に比べて増加、又は減少するか、あるいは、対照の被検体における量に近づいた場合、該候補化合物は脳梗塞の予防または治療効果を有すると判定することができる。
脳梗塞発症直後(表中「DAY0」と記載されている)、発症7日後(表中「DAY7」と記載されている)時点の脳梗塞患者と健常者(それぞれ表2中に記載の人数)から採血を行い、EDTA血漿を取得した。R&D社のAnti-human CRTAC1 Antibody(カタログNo.AF5234)、R&D社のBiotinylated Anti-Human CRTAC1 Antibody(カタログNo.BAF5234)、及びR&D社のStreptavidin-HRP (カタログNo.DY998)を用いた免疫測定法により、EDTA血漿中のCRTAC1蛋白質濃度を測定した。具体的には脳梗塞患者由来の血漿(数十人分を混合)中のCRTAC1蛋白質濃度を任意に100U/mLと定め、これを用いて検量線を作成し、この検量線から血漿中のCRTAC1蛋白質の相対濃度(U/mL)を算出した。
脳梗塞の各病型に診断が確定している患者、具体的には、「ラクナ梗塞」(表中では「ラクナ」と称する)、「アテローム血栓性脳梗塞」(表中では、「アテローム血栓性」と称する)、「心原性脳塞栓症」(表中では、「心原性脳塞栓」と称する)、「大動脈原性脳塞栓症」(表中では、「大動脈原性脳塞栓」と称する)、「Branch atheromatous disease」(表中では「BAD」と称する)、「動脈解離」(表中では、「動脈解離」と称する)、あるいはそれらのいずれにも該当しない病型(表中では「分類不能型」と称する)の患者について、脳梗塞発症直後(表中「DAY0」と記載されている)、発症7日後(表中「DAY7」と記載されている)、時点で採血を行い(それぞれ表3中に記載の人数)からそれぞれEDTA血漿を取得した。
該血漿中のCRTAC1蛋白質の濃度を実施例1の方法と同様に測定し、該測定値から実施例1と同様にROC 曲線の AUC 値を算出した。
それぞれの採血時点において、0.7を上回ったものが脳梗塞患者の病型を判別するための検査方法として有用であることが示される。
脳梗塞の予後は、脳梗塞発症後、ある一定期間の後に、その患者の脳梗塞による障害の程度にて示される。脳梗塞発症3ヶ月後の障害の程度を、日本版modified Rankin Scale(mRS)判定基準(篠原幸人らmRS信頼性研究グループ、modified Rankin Scaleの信頼性に関する研究-日本語版判定基準書および問診表の紹介、脳卒中 2007;29:6-13)を用いて0~5に分類した患者について、脳梗塞発症7日後(表中「DAY7」と記載されている)に採血を行い(それぞれ表4中に記載の人数)EDTA血漿を取得した。
該血漿中のCRTAC1蛋白質の濃度を実施例1の方法と同様に測定し、該測定値から実施例1と同様にROC 曲線の AUC 値を算出した。その結果を表4に示す。
それぞれの採血時点において、0.7を上回ったものが脳梗塞患者の予後を判別するための検査方法として有用であることが示される。
脳梗塞発症直後(表中「DAY0」と記載されている)のNIHSSに対して,発症3日後,発症7日後,発症14日後のいずれかのポイントでNIHSSが1点以上増加した場合を進行性,NIHSSが変化しなかった場合及び1点以上減少した場合を非進行性と定義した。重症度および病型のサブグループ間の偏りをなくすため、発症直後のNIHSSが15以上の患者は除外した。該血漿中のCRTAC1蛋白質の濃度を実施例1の方法と同様に測定し、該測定値から実施例1と同様にROC 曲線の AUC 値を算出した。その結果を表5に示す。
発症7日後の採血時点において、0.7を上回ったものが進行性患者と非進行性患者の判別するための検査方法として有用であることが示される。つまり、発症7日目の脳梗塞患者から血液を取得して、血漿中のCRTAC1蛋白質の濃度を比較することにより進行性患者と非進行性患者を判別するための検査が行えることがわかった。
Claims (11)
- 被検動物より採取された血液試料中のCartilage Acidic Protein 1蛋白質の量を測定する工程を含み、該蛋白質量が健常者と比較して低下していた場合に脳梗塞であると判断することを特徴とする、該動物における脳梗塞の診断のための検査方法。
- 被検動物より採取された血液試料が、脳梗塞様症状の発症直後から発症後20日目の間のいずれかの時期において被検動物より採取されたものであることを特徴とする請求項1に記載の脳梗塞の診断のための検査方法。
- 被検動物より採取された血液試料中のCartilage Acidic Protein 1蛋白質の量を測定する工程を含む、該動物における脳梗塞の病型診断のための検査方法。
- 病型診断が、ラクナ梗塞、アテローム血栓性、心原性脳塞栓症、大動脈原性脳塞栓症、Branch atheromatous disease、動脈解離、分類不能のいずれかを診断するものである請求項3に記載の検査方法。
- 被検動物より採取された血液試料中のCartilage Acidic Protein 1蛋白質の量を測定する工程を含む、該動物における脳梗塞の予後の予測のための検査方法。
- 被検動物より採取された血液試料が、脳梗塞発症7日目に被検動物より採取されたものであることを特徴とする請求項5に記載の脳梗塞の予後の予測のための検査方法。
- 被検動物より採取された血液試料中のCartilage Acidic Protein 1蛋白質の量を測定する工程を含む、該動物における脳梗塞の進行性の予測のための検査方法。
- 被検動物より採取された血液試料が、脳梗塞発症7日目に被検動物より採取されたものであることを特徴とする請求項7に記載の脳梗塞の進行性の予測のための検査方法。
- 脳梗塞の予防薬もしくは治療薬が投与された脳梗塞の予防もしくは治療を必要とする動物から採取された血液試料中のCartilage Acidic Protein 1蛋白質の量を測定する工程を含む、該動物における脳梗塞の予防もしくは治療効果の評価方法。
- 被検体と脳梗塞の予防薬または治療薬の候補化合物を接触させた後、該被検体中のCartilage Acidic Protein 1蛋白質の量を測定する工程を含む、脳梗塞の予防薬または治療薬のスクリーニング方法。
- Cartilage Acidic Protein 1蛋白質の量を測定し得る試薬を含んでなる、脳梗塞検査用、脳梗塞治療効果評価用、または脳梗塞予防・治療薬候補化合物のスクリーニング用キット。
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