JP5704533B2 - Diagnostic method and diagnostic agent for Alzheimer's disease - Google Patents

Description

  The present invention relates to a diagnostic method and a diagnostic agent for Alzheimer's disease.

  Alzheimer's disease (hereinafter, also simply referred to as “AD”) is a disease that develops from the late 40s to the 50s in the elderly and in the late 60s in the elderly. When Alzheimer's disease develops, symptoms such as memory impairment, motivation disorder, judgment impairment, aphasia, apraxia, agnosia, personality disorder, and emotional disorder appear, and if it progresses, symptoms such as advanced intellectual disability appear. As pathological changes in the brain, senile plaques, neurofibrillary tangles, nerve cell loss, etc. are observed, and it is known that the lesions become more advanced as symptoms progress and cause significant brain atrophy. Senile plaque is the most characteristic brain lesion of AD, and its main component is β-amyloid protein (hereinafter also referred to as Aβ) such as β-amyloid 40 and β-amyloid 42 having a β sheet structure.

  The cause of AD is unknown, and the fundamental treatment and prevention methods have not yet been established. One of the biggest challenges in AD prevention and treatment drug design and testing is that a simple and reliable diagnostic method that can identify AD affected persons at an early stage has not been established. The social demand for early diagnosis of AD is high, and its rapid development is strongly desired.

  Currently, the diagnosis of AD relies heavily on neuropsychological evaluation and there are very few established objective indicators. Morphological evaluation by brain MRI (degree of atrophy), evaluation of cerebral blood flow by PET (positron emission tomography), or single photon emission tomography (SPECT) are used as supplementary methods. Since it is not an index that reflects the essence, there was a problem of lack of reliability.

  Β-amyloid protein and phosphorylated tau have been studied as candidates for AD biomarkers. The amount of β-amyloid protein and phosphorylated tau in cerebrospinal fluid are considered reliable indicators, and cerebrospinal fluid is collected and the concentration of β-amyloid protein in the cerebrospinal fluid is measured. Diagnosis is also performed. However, this method has a problem in that it is highly invasive because it collects cerebrospinal fluid, and it is difficult to apply in daily clinical practice because it places a heavy burden on the patient. In addition, there is a report of measuring β-amyloid protein in the blood in the examination of the AD diagnosis method, but it is said that a significant difference was not found between a healthy person and an AD patient (Non-Patent Document 1 and Non-Patent Document 1). Reference 2).

  Patent Document 1 discloses that after a human fasts for at least 4 hours, a sample of circulating fluid is separated from the human and the amyloid precursor protein (APP) 130 kDa form and / or 42 kDa form of the circulating fluid and / or a derivative of any form thereof. A method for testing human Alzheimer's disease comprising measuring a value relative to a normal control, wherein the relative increase in the 130 kDa form and / or its derivative and the relative decrease in the 42 kDa form and / or its derivative are A method that is an indicator of disease is disclosed. However, this method has room for improvement so that patients with AD can be more accurately determined at an early stage.

Recently, amyloid imaging using PET (positron emission tomography) has been performed in clinical diagnosis of AD. In this method, radioactive substances are injected into the body by intravenous injection, and then the brain is imaged by PET, so that amyloid deposition in the brain is detected. Therefore, this method is expected as a method for estimating the amount of β-amyloid protein in the brain in the living body. Has been. However, since amyloid imaging is still performed only in several facilities in Japan, there is a problem that it is expensive and difficult to use as a general diagnostic method.
Therefore, it is desired to develop an AD diagnostic method that is simple, minimally invasive, highly safe, and highly reliable using an index that reflects a disease state.

Japanese Patent No. 3277211

Mehta PD. Et al., “Plasma and cerebrospinal fluid levels of amyloid beta proteins 1-40 and 1-42 in Alzheimer disease.”, Arch Neurol 2000; 57: 100-105. Fukumoto H. et al., “Age but not diagnosis is the main predictor of plasma amyloid beta-protein levels.”, Arch Neurol 2003; 60: 958-964.

  The present invention is simple, low-cost, minimally invasive, highly safe for living bodies, and highly reliable for diagnosing the onset of Alzheimer's disease at an early stage. And a diagnostic agent for Alzheimer's disease.

  The present inventors have intensively studied to solve the above-mentioned problems. First, young (2 to 3 months) Alzheimer's disease (AD) model mice (APP) in which symptoms specific to Alzheimer's disease such as memory impairment do not appear. / PS1) and analyzing the difference between the blood β-amyloid 40 (Aβ40) value and the blood β-amyloid 42 (Aβ42) value between fasting blood sampling and occasional blood sampling (collected at an arbitrary timing), It was found that there was a statistically significant difference in Aβ40 and Aβ42 values on an empty stomach and at any time (FIGS. 1A and B). On the other hand, in wild-type (WT) mice (healthy mice), there was a slight difference in blood Aβ40 value between fasting blood sampling and occasional blood sampling, but there was no difference in blood Aβ42 value. (FIGS. 1A and B).

Also, using an old (18-19 months) AD model mouse (APP / PS1) showing symptoms peculiar to AD such as memory impairment, the blood Aβ40 value and blood in fasting blood sampling and occasional blood sampling as described above When the difference in the middle Aβ42 value was analyzed, it was found that there was a statistically significant difference in both mice (FIGS. 2A and B). On the other hand, in old wild-type (WT) mice (healthy mice), there was no difference in either the blood Aβ40 value or the blood Aβ42 value between fasting blood and occasional blood collection (FIGS. 2A and B).
These findings were considered to suggest that it is necessary to take into account the blood glucose state at the time of blood collection when the blood Aβ value is used as a diagnostic index for Alzheimer's disease.

  Therefore, based on the above findings, the AD model mouse (APP / PS1) was administered with an aqueous glucose solution to give a glucose load, and the blood Aβ40 value and Aβ42 value in the glucose load state were analyzed. In comparison, it was found that both the Aβ40 value and the Aβ42 value in blood were significantly increased (FIGS. 5A to 5E and 6A to 6E). On the other hand, when a similar test was performed on wild-type (WT) mice (healthy mice), there was almost no difference in blood Aβ40 value and Aβ42 value even in the glucose load state compared to fasting (FIG. 5A). ~ E and Figures 6A-E). In addition, the change of the blood glucose level and insulin level when glucose load was performed did not have a difference between AD model mice and healthy mice (FIGS. 3A to 4D and 4A to 4D).

Thus, the amount of blood Aβ increase (degree of increase) during glucose loading was clearly greater in AD model mice than in healthy mice. From this finding, the present inventors have found that the difference in blood Aβ value between the fasting state and the glucose load state (the amount of increase in blood Aβ due to glucose load) is useful as a diagnostic indicator of Alzheimer's disease.
Furthermore, even in the same AD model mouse, when the young AD model mouse and the old AD model mouse were compared, the increase in blood Aβ due to glucose load was larger in the old AD model mouse. For this reason, it came to the mind that the amount of increase in blood Aβ due to glucose load is an index that reflects the degree of progression of the disease state (FIGS. 5A to 5E and 6A to 6E).

AD model mice (APP / PS1) are considered to reflect the pathology of human Alzheimer's disease (McGowan E. et al, Amyloid phenotype characterization of transgenic mice overexpressing both mutant amyloid precursor protein and mutant presenilin 1 transgenes Neurobiol Dis. 1999 Aug; 6 (4): 231-44.). Since the young model mouse (APP / PS1) used by the present inventors was young, it was an individual in which AD-specific symptoms such as memory impairment did not appear, so the human early stage (symptoms did not appear) It was thought to reflect the pathology of AD-affected individuals. In addition, it was considered that the aged model mice reflect the pathology of human AD sufferers who have AD-specific symptoms such as memory impairment.
Also, for example, Alzheimer's disease vaccine that was effective in AD model mice was also effective in human AD patients in clinical practice (Schenk D. et al., Immunization with amyloid-beta attenuates Alzheimer-disease-like Pathology in the PDAPP mouse.Nature. 1999 Jul 8; 400 (6740) 173-7. and Nicoll JA. et al, Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report.Nat Med. 2003 Apr; 9 (4): 448-52.), The above results in AD model mice were considered to be applicable to humans.

Thus, no studies have been reported so far that have found an index that reflects the pathology of Alzheimer's disease from the relationship between brain and systemic glucose metabolism. In the pathological analysis of the disease model animal, the present invention is the first to find a pathological index that links Alzheimer's disease, sugar metabolism, and cognitive dysfunction, and conceived of applying it to diagnosis. The present inventors further conceived that the technique used for the diagnosis of diabetes can be applied to the diagnosis of Alzheimer's disease. This is because of safety, low invasiveness, and low cost. It can be a very good diagnostic technique.
The present inventors have further studied based on the above findings, and have completed the present invention.

That is, the present invention relates to the following (1) to (12).
(1) A method for diagnosing Alzheimer's disease, comprising a step of measuring a β-amyloid protein value in a blood sample collected from a mammal subjected to an energy load.
(2) Further, the β-amyloid protein value in the blood sample collected from the mammal subjected to energy load is compared with the β-amyloid protein value in the blood sample collected from the mammal on an empty stomach. The diagnostic method according to (1), further comprising the step of obtaining an increase in blood β-amyloid protein due to energy load.
(3) The method further comprises a step of comparing the amount of increase in blood β-amyloid protein due to energy load in the mammal with the amount of increase in blood β-amyloid protein due to energy load in a healthy mammal. The diagnostic method according to.
(4) The diagnostic method according to any one of (1) to (3), wherein the energy load is a sugar load.
(5) The diagnostic method according to any one of (1) to (4), wherein the energy load is a glucose load.
(6) The diagnostic method according to any one of (1) to (5), wherein the β-amyloid protein is β-amyloid 40 and / or β-amyloid 42.
(7) β-amyloid protein value in a blood sample collected from a mammal at any time point between 15 minutes and 8 hours after the energy load, and a blood sample collected from the mammal on an empty stomach The diagnostic method according to (2), wherein the β-amyloid protein value in the medium is compared.
(8) The diagnostic method according to any one of (1) to (7), wherein the blood sample is plasma.
(9) The diagnostic method according to any one of (1) to (8), wherein the blood sample is obtained by diluting a plasma component separated from blood by 4 to 200 times.
(10) Use of β-amyloid protein value in a blood sample collected from a mammal subjected to an energy load to determine Alzheimer's disease.
(11) Use of a sample of blood collected from a mammal subjected to an energy load for producing a sample for measuring β-amyloid protein value in the determination of Alzheimer's disease.
(12) A diagnostic agent for Alzheimer's disease comprising glucose.

  According to the present invention, Alzheimer's disease or its possibility can be easily diagnosed, examined or determined easily, at low cost, with minimal invasiveness, and at an early stage. This makes it possible to develop new therapeutic agents and preventive agents for Alzheimer's disease.

FIGS. 1A and B are diagrams showing the results of measurement of β-amyloid protein values (FIG. 1A: Aβ40, FIG. 1B: Aβ42) in fasting blood and blood samples collected from young wild-type mice and Alzheimer's disease model mice. It is. FIGS. 2A and 2B are graphs showing the results of measuring β-amyloid protein values (FIG. 2A: Aβ40, FIG. 2B: Aβ42) in fasting and occasional blood plasma of old wild-type mice and Alzheimer's disease model mice. is there. FIGS. 3A to 3D show time-dependent changes in blood glucose level and plasma insulin concentration after glucose load in young wild-type mice and Alzheimer's disease model mice (FIG. 3A: blood glucose level, FIG. 3C: plasma insulin concentration), blood glucose level and It is a figure (FIG. 3B: blood glucose level, FIG. 3D: insulin level) which shows AUC of an insulin level. 4A to 4D show changes in blood glucose level and plasma insulin concentration with time after glucose loading in old wild-type mice and Alzheimer's disease model mice (FIG. 4A: blood glucose level, FIG. 4C: plasma insulin concentration), and blood glucose level and insulin. It is a figure which shows AUC (FIG. 4B: blood glucose level, FIG. 4D: insulin level) of a value. FIGS. 5A to 5E are diagrams showing time-dependent changes in β-amyloid 40 value in plasma (FIG. 5A to D) and AUC of changes in β-amyloid 40 value after glucose loading in wild-type mice and Alzheimer's disease model mice (FIG. 5). 5E). FIGS. 6A to 6E show time-dependent changes in β-amyloid 42 value in plasma after glucose loading (FIG. 6A to D) and AUC of β-amyloid 42 value change (FIG. 6E) in wild type mice and Alzheimer's disease model mice. FIG.

The method for diagnosing Alzheimer's disease of the present invention includes a step of measuring β-amyloid protein level in a blood sample collected from a mammal subjected to an energy load.
Hereinafter, the β amyloid protein value in a blood sample collected from a mammal is also simply referred to as “blood Aβ value”. The blood Aβ value is usually the Aβ concentration or the amount of Aβ in a blood sample.

  In the present invention, “energy loaded” means that a nutrient that becomes energy in the body is ingested, or that the nutrient is orally or parenterally administered. A mammal subjected to an energy load is usually a mammal whose blood glucose level, blood insulin level, etc. are increased compared to fasting. Examples of mammals include humans, monkeys, cows, sheep, goats, horses, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like.

  In this specification, “fasting” is usually after a fast (drinking is allowed) of about 12 hours or more. For example, in the case of humans, they are usually fasted (drinkable) for at least about 12 hours or more, preferably about 16 hours or more. The upper limit of fasting time is usually about 18 hours.

  The method of applying energy load to the mammal is not particularly limited, and it is sufficient to cause the mammal to ingest foods and drinks containing nutrients, or to administer drugs or the like containing nutrients to mammals orally or parenterally. In the present invention, it is preferable to apply an energy load by ingesting nutrients to mammals, orally administering them, or intravenously injecting them.

As a nutrient administered to a mammal in order to apply an energy load, at least one selected from the group consisting of sugar, protein and lipid is preferable, and a nutrient that increases blood glucose level is more preferable. Particularly preferred is sugar.
Examples of the sugar include monosaccharides such as glucose, xylose, arabinose, galactose, fructose or mannose; disaccharides such as cellobiose, sucrose, lactose and maltose; polysaccharides such as dextrin and soluble starch. Among these, monosaccharides are preferable, and glucose is more preferable. That is, in the present invention, the energy load is preferably a sugar load, and more preferably a glucose load.

  Although it does not specifically limit as food and drink containing a nutrient, For example, if it is sugar, sugar itself or the aqueous solution containing sugar is preferable. Examples of preparations for oral administration containing nutrients include solid preparations such as tablets, powders, granules and capsules; and liquid preparations such as emulsions, syrups and suspensions. In the case of parenteral administration, for example, nutrients can be administered in dosage forms such as injections, drops, sprays, sprays, suppositories and the like.

  When a glucose load is applied, it is preferable to administer glucose or an aqueous solution containing glucose by an administration route selected from the group consisting of oral and intravenous. Preferably, it is administered orally. For the glucose load, a commercially available glucose aqueous solution can be used, and a commercially available glucose solution used in a glucose tolerance test used for diagnosis of diabetes, for example, a 75 g glucose solution (for example, manufactured by Ajinomoto Pharma Co., Ltd.) is preferably used. Can be used.

The amount of glucose to be administered for applying a glucose load may be appropriately selected according to the administration route, the body weight of the mammal, and the like, and in the case of oral administration, usually about 500 to 5000 mg of glucose per kg body weight, preferably Is preferably administered at about 1000 to 3000 mg. It is also preferable to orally administer this amount of glucose at a time.
For example, in the case of humans, it is preferable to administer about 50 to 200 g of glucose at a time, or more preferably about 75 to 100 g of glucose at a time. For example, a glucose load can be easily applied by orally administering a glucose solution such as the 75 g glucose solution described above at a time.

  In the case of intravenous administration, the glucose load can be applied, for example, by intravenous injection or intravenous infusion of about 5-70% aqueous glucose solution, preferably about 30-50% aqueous glucose solution. The dosage of the aqueous glucose solution is usually about 500 to 5000 mg, preferably about 1000 to 3000 mg of glucose per kg of body weight. For humans, the glucose aqueous solution is usually administered intravenously over about 5 to 10 minutes, usually about 50 to 500 mL, preferably about 50 to 200 mL, more preferably about 50 to 100 mL.

  The mammal to which the energy load is applied in the present invention is preferably an animal in which the blood glucose level is increased compared to the fasting state due to the energy load.

  The blood sample only needs to contain a component of mammalian blood subjected to an energy load, and as the blood component, for example, blood, plasma or serum is preferable. More preferably, it is plasma. The blood sample can be prepared by a known method from a mammal, for example, from blood collected by intravenous blood collection. Preferably, a blood sample is prepared using blood collected by heparin blood collection. Plasma is prepared by separating plasma components from collected blood using a known method, for example, a β amyloid ELISA kit manufactured by Wako Pure Chemical Industries, according to the method described in the package insert attached to the kit. be able to.

In the present invention, the blood sample is preferably a blood plasma component diluted from about 4 to 200 times, more preferably about 4 to 100 times diluted, more preferably about 10 to 20 times. More preferably diluted to 1. The method for diluting the plasma component and the diluted solution may be appropriately selected according to the method for measuring the β amyloid protein value, and are not particularly limited. The dilution of the plasma component is preferably performed using, for example, a specimen dilution solution attached to the β amyloid ELISA kit sold by Wako Pure Chemical Industries.
The blood sample in the present invention may be further treated with a surfactant / denaturant, etc., depending on the Aβ measurement method described below.

  A sample of blood collected from a mammal subjected to an energy load is suitable as a sample for β-amyloid protein measurement used for determination, examination or diagnosis of Alzheimer's disease, for example, used as it is as a sample for β-amyloid protein measurement However, as long as the effects of the present invention are exhibited, other components may be appropriately added according to the method for measuring the β-amyloid protein value. A composition containing such a blood sample is also included in the blood sample of the present invention.

  Examples of β-amyloid protein (Aβ) include β-amyloid 40 (Aβ40), β-amyloid 42 (Aβ42), oligomer Aβ, and the like. Among them, in the present invention, β-amyloid 40 and / or β -Preferably the value of amyloid 42 is measured.

  The method for measuring the Aβ value in the blood sample is not particularly limited, and can be measured according to a known method. For example, the Aβ value can be easily measured by ELISA (Enzyme-Linked ImmunoSorbent Assay), Western blotting, MS or the like. When measuring by ELISA, use a commercially available β amyloid ELISA kit (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) and easily measure the Aβ value according to the method described in the instructions attached to the kit. Can do. The measurement of Aβ value by Western blotting is, for example, Huang SM et al., Neprilysin-sensitive synapse-associated amyloid-beta peptide oligomers impair neuronal plasticity and cognitive function. J Biol Chem. 2006 Jun 30; 281 (26): 17941 This can be done according to the method described in -51. The measurement of Aβ value by MS is, for example, Wang R et al., The profile of soluble amyloid beta protein in cultured cell media.Detection and quantification of amyloid beta protein and variants by immunoprecipitation-mass spectrometry. J Biol Chem. 1996 Dec 13 ; 271 (50): 31894-902.

  In the present invention, a blood sample collected from a mammal after an increase in blood glucose level reaches a peak due to energy load (intake or administration of nutrients such as sugar) and before the blood glucose level decreases to a fasting blood glucose level It is preferable to measure the Aβ value in the medium. For example, an energy-loaded mammal in the present invention (or an energy-loaded state) is usually about 15 minutes to 8 hours after the energy load (at the time of energy load), preferably about 40 hours. Between minutes and 8 hours, more preferably after about 1 hour and after 8 hours, more preferably between about 1 hour and after 6 hours, particularly preferably after about 1 hour and 2 hours. Mammals until after hours. In the present invention, it is preferable that the mammal is not ingested with nutrients until all of the blood samples are collected from the mammal after the mammal has been subjected to an energy load. Specifically, after applying the energy load, until the collection of the blood sample is completed, the mammal is not allowed to ingest foods and drinks containing nutrients, and a drug containing the nutrients is orally administered to the mammal. And preferably not administered parenterally.

  In the present invention, one or more time points from the energy load (at the time of energy load) to usually after about 15 minutes to 8 hours, preferably from about 40 minutes to 8 hours, more preferably Is one or more time points between about 1 hour and 8 hours later, more preferably about 1 time or more time points between about 1 hour and 6 hours later, particularly preferably after about 1 hour Aβ levels are measured in samples of blood taken from mammals at one or more time points up to 2 hours later. When blood is collected at two or more different time points after a certain time from the energy load, it is preferable to measure the Aβ value separately for each blood sample obtained at each time point. That is, for blood samples collected at X, Y and Z minutes (X <Y <Z) after the energy load, the blood Aβ value after X minutes from the energy load It is preferable to measure the blood Aβ value after Y minutes and the blood Aβ value after Z minutes, respectively.

In the present invention, the change in blood Aβ value (0) at the time of energy load (for example, at the time of glucose administration) in a graph in which the vertical axis represents blood Aβ value change (ΔAβ) and the horizontal axis represents time (minutes). It is also preferable to obtain an area surrounded by a straight line parallel to the passing time axis and a curve of ΔAβ transition (hereinafter also referred to as “AUC of blood Aβ value change” (Area Under the blood concentration time Curve)). Alzheimer's disease can also be determined using the AUC of middle Aβ value change as an index. For example, as will be described later, if the AUC of the change in blood Aβ value of the mammal at a certain time after the energy load is larger than the AUC of change in blood Aβ value of the healthy animal, the possibility of Alzheimer's disease is high. Can be determined.
It is preferable to evaluate the AUC of the change in blood Aβ value based on the result of normal energy load up to about 120 minutes, preferably up to about 480 minutes.

In the present invention, (1) β-amyloid protein value (blood Aβ value) or (2) AUC of blood Aβ value change in a blood sample collected from a mammal subjected to energy load is expressed as Alzheimer's. It is preferably used as an index for disease determination.
When (1) is used for the determination of Alzheimer's disease, β-amyloid protein value in a blood sample collected from a mammal subjected to energy load, and β in a blood sample collected from the mammal on an empty stomach -It is preferable to compare the amyloid protein value and determine the amount of increase in blood β-amyloid protein due to energy load, and use this amount as an index for determining Alzheimer's disease. By comparing the energy load state and fasting blood Aβ value in the same individual, Alzheimer's disease can be more reliably determined. In this case, it is determined that the possibility of Alzheimer's disease is high when the amount of increase (increase) in the blood Aβ value due to energy load is significantly greater than when fasting.

That is, the diagnostic method of the present invention further includes a β-amyloid protein value in a blood sample collected from a mammal subjected to an energy load, and a β-amyloid protein value in a blood sample collected from the mammal on an empty stomach. It is preferable to include the process of calculating | requiring the increase amount of the beta-amyloid protein in the blood by energy load.
The above “increase amount of blood Aβ due to energy load” is calculated by the following formula, the fasting blood Aβ value (blood Aβ (0)), and the energy load state (X minutes from energy load ( Or the difference from the blood Aβ value (blood Aβ (X)) after (after X hours). The fasting blood Aβ value (blood Aβ (0)) is preferably the blood Aβ value immediately before the energy load.
(Increased amount of blood Aβ due to load of energy) (ΔAβ (X)) = blood Aβ (X) −blood Aβ (0)

  The amount of increase in blood Aβ due to the energy load is, for example, any of the period from about 15 minutes to 8 hours, preferably from about 40 minutes to 8 hours after the energy load (when loading the energy). More preferably, any time point between about 1 hour and 8 hours later, more preferably any time point between about 1 hour and 6 hours later, particularly preferably about 1 hour. It is preferably the difference between the blood Aβ value at any point in time after and after 2 hours and the fasting blood Aβ value. In addition, it is also preferable to determine the amount of increase in blood Aβ due to the energy load at two or more different points in time.

The diagnostic method of the present invention preferably further comprises a step of comparing the increase in blood Aβ due to energy load in the mammal and the increase in blood Aβ due to energy load in a healthy mammal. A method for applying an energy load to a healthy mammal, a method for preparing a blood sample from the healthy mammal, and the like are the same as those described above.
As described above, the amount of increase in blood Aβ due to the energy load (the degree of increase in blood Aβ) is larger in mammals suffering from Alzheimer's disease than in normal mammals, and therefore is constant from the energy load calculated by the above formula. Alzheimer's disease can be determined by comparing the increase in blood Aβ (ΔAβ (X)) after time (X minutes) between the test mammal and a healthy animal. For example, when the amount of increase in blood Aβ due to an algae load in the mammal (ΔAβ (X)) is larger than the amount of increase in blood Aβ due to an algae load in healthy animals (ΔAβ (X)) (for example, Usually, about 1.5 times or more, preferably about 3 times or more, more preferably about 4 times or more, and further preferably about 5 times or more), it is determined that the possibility of Alzheimer's disease is high.

  For comparison between the increase in blood Aβ due to energy load in the mammal (test mammal) and the increase in blood Aβ due to energy load in a healthy mammal, the blood Aβ value when energy load is applied AUC of change can also be used, and when the AUC of blood Aβ change in the mammal when an energy load is applied is greater than the AUC of blood Aβ change in a healthy mammal (eg, usually about 1.. 5 times or more, preferably about 3 times or more, more preferably about 4 times or more, still more preferably about 5 times or more), and it can be determined that the possibility of Alzheimer's disease is high. It is preferable to evaluate the AUC of change in blood Aβ value based on the result of normal energy load up to about 120 minutes, preferably up to about 480 minutes.

  A diagnostic agent for Alzheimer's disease containing glucose is suitably used when an energy load is applied to a mammal in the diagnosis, test or determination of the Alzheimer's disease. Such diagnostic agents are also encompassed by the present invention. The content of glucose is not particularly limited as long as the effects of the present invention are exhibited. For example, a diagnostic agent containing glucose and administered once by an administration route selected from the group consisting of oral and intravenous and about 15 minutes to 8 hours before blood collection is suitable. Such a diagnostic agent is preferably administered to a mammal fasted for about 12 to 16 hours at a time by an administration route selected from the group consisting of oral and intravenous. The diagnostic agent preferably contains about 50 to 200 g of glucose, more preferably about 50 to 100 g, and even more preferably about 75 to 100 g.

  A diagnostic agent containing glucose is suitably used when an energy load is applied to a mammal in the above diagnosis, examination or determination. A diagnostic agent administered to a mammal containing glucose and fasted for about 12 to 16 hours once by an administration route selected from the group consisting of oral and intravenous, about 15 minutes to 8 hours before blood collection is also described above. It is preferably used when an energy load is applied to a mammal in diagnosis, examination or determination. Such a diagnostic agent for Alzheimer's disease is also one aspect of the present invention. In the case of oral administration, the diagnostic agent is an aqueous solution containing about 50 to 200 g (more preferably about 50 to 100 g) of glucose and having a glucose concentration of about 0.2 to 0.5 g / mL. In the case of intravenous administration, an aqueous solution containing about 50 to 200 g (more preferably about 50 to 100 g) and having a glucose concentration of about 0.2 to 0.5 g / mL is preferable. Such a diagnostic agent is preferably administered to a mammal fasted for about 12 to 16 hours, once orally or intravenously, and about 40 minutes to 8 hours before blood collection.

  A diagnostic kit for Alzheimer's disease containing the above diagnostic agent is also a preferred embodiment of the present invention. Kits include devices for intravenous injection, blood collection devices for collecting blood; antibodies, reagents, etc. for measuring β-amyloid protein; other buffers, instructions, surfactants, denaturing agents, etc. May be included.

  According to the present invention, Alzheimer's disease can be easily diagnosed, tested or determined with high reliability, and early diagnosis of Alzheimer's disease becomes possible. In addition, the progression of the pathology of Alzheimer's disease can be diagnosed, examined or determined.

  EXAMPLES Next, although an Example demonstrates this invention concretely, this invention is not limited to these.

1. Animals used PSAPP transgenic Alzheimer's disease model mice (male, young: 2-3 months old, old: 18-19 months, each n = 5-7, hereinafter referred to as Tg) and healthy wild type mice (male, young) Age: 2-3 months old, old age: 18-19 months old, each n = 5-7, hereinafter referred to as WT), blood Aβ value (plasma Aβ concentration), blood glucose level, blood insulin level, etc. Was measured. Tg and WT were purchased from The Jackson Laboratory.

2. Blood collection and sample preparation method and measurement method Blood collection was performed from the tail vein of awake mice. After heparin blood collection, plasma was obtained by centrifugation at 4 ° C. and 1200 g for 20 minutes.
The blood glucose measurement was performed using a human blood glucose measurement instrument (Glutest Ace, manufactured by Sanwa Chemical Laboratory Co., Ltd.).
Plasma insulin concentration was measured by ELISA using an ultrasensitive mouse insulin measurement kit (manufactured by Morinaga Bioscience Institute).
The plasma Aβ concentration was measured by ELISA using Human / Rat βAmyloid (40) and (42) ELISA Kit wako (manufactured by Wako Pure Chemical Industries, Ltd.).

  About the measured value, the presence or absence of a statistically significant difference was calculated | required by performing t test. A case where P <0.05 was regarded as a statistically significant difference.

3. AUC Calculation Method Glucose AUC is a straight line parallel to the time axis passing through the blood glucose level at the time of glucose load (dose) in the graph with the vertical axis representing blood glucose level (mg / dL) and the horizontal axis representing time (minutes). And an area surrounded by a curve of blood glucose level transition.
Insulin AUC is a line parallel to the time axis passing through the plasma insulin concentration at the time of glucose load (during administration) in a graph with the vertical axis representing plasma insulin concentration (ng / mL) and the horizontal axis representing time (minutes), It was defined as the area surrounded by a curve with plasma insulin concentration transition.
Glucose AUC and insulin AUC were evaluated by results from glucose load up to 480 minutes.

  ΔAβ AUC (change in blood Aβ concentration) is a line parallel to the time axis passing through ΔAβ at the time of glucose load (administration) in a graph with ΔAβ on the vertical axis and time (minutes) on the horizontal axis, and ΔAβ It was defined as the area surrounded by a curve with transition. The AUC of ΔAβ was evaluated based on the results from glucose load up to 480 minutes.

Test example 1
Measurement of difference in blood Aβ value between fasting and occasional First, the presence or absence of the difference in blood Aβ value (plasma Aβ concentration) between fasting and occasional was evaluated in Tg and WT respectively. Fasting blood collection was performed after 16 hours of fasting. Both fasting and occasional blood collection were performed at approximately the same time in the morning (light period). There were no differences between Tg and WT for young and old mice in terms of body weight, blood glucose levels (fasting and ad hoc), and plasma insulin levels (fasting and ad hoc) (Table 1).

  Table 1 shows metabolic parameters of PSAPP transgenic mice (Tg (“TG” in Table 1))) and wild-type mice (WT) used in the test. Metabolic parameters are body weight, blood glucose level, and plasma insulin concentration. In Table 1, the numerical values are mean ± SE (standard error). “NS” means that there is no significant difference.

  As can be seen from Table 1, there was no significant difference in metabolic parameters between PSAPP mice (Tg) and wild-type mice (WT) in both young and old mice.

  FIG. 1A and FIG. 1B show the results of measuring β-amyloid protein levels in fasting plasma and blood samples collected from young wild-type mice and Alzheimer's disease model mice (FIG. 1A: Aβ40, FIG. 1B: Aβ42). . “TG” in FIGS. 1 to 6 means an Alzheimer's disease model mouse (Tg). FIG. 2A and FIG. 2B show the results of measuring β-amyloid protein levels in fasting plasma and blood samples collected from old wild-type mice and Alzheimer's disease model mice (FIG. 2A: Aβ40, FIG. 2B: Aβ42).

In young mice, the plasma Aβ40 concentration was significantly (** P <0.01) higher than that of WT in both fasting and as needed (FIG. 1A). In addition, when comparing fasting and as needed, there was a significant difference (## P <0.01) in plasma Aβ40 concentration in both Tg and WT (FIG. 1A). The plasma Aβ42 concentration was also significantly higher (** P <0.01) than the WT Tg, but only Tg showed a significant difference (#P <0.05) between fasting and ad libitum (FIG. 1B). The plasma Aβ40 concentration (FIG. 2A) and Aβ42 concentration (FIG. 2B) of old mice were also Tg significantly higher than WT (** P <0.01), but only fasting and occasional values only at Tg. A significant difference (#P <0.05) was observed.
From this result, it was found that the plasma Aβ concentration was changed between fasting and at any time particularly in Alzheimer's disease mice.

Test example 2
Next, the change in plasma Aβ concentration by the glucose tolerance test was evaluated. Glucose loading was performed by intraperitoneal administration of 2 g / kg body weight glucose using a 20% glucose solution after fasting for 16 hours. Blood was collected 15, 30, 60, 120, 240, and 480 minutes after glucose loading. There was no difference in changes in blood glucose level and plasma insulin concentration after glucose loading in young mice (FIG. 3) and old mice (FIG. 4). 3A shows the change in blood glucose level over time from energy load (0 minute), FIG. 3B shows the glucose AUC, FIG. 3C shows the change over time in plasma insulin concentration from the energy load (0 minute), and FIG. AUC is shown respectively (all are young mice). 4A shows the change in blood glucose level over time from energy load (0 minute), FIG. 4B shows the glucose AUC, FIG. 4C shows the change over time in plasma insulin concentration from the energy load (0 minute), and FIG. AUC is shown respectively. In the figure, NS means that there is no significant difference.
From this, it was confirmed that the same glucose load was applied to both Tg and WT mice.

  FIG. 5A (young age) and FIG. 5C (old age) show the time course of plasma Aβ40 concentration after glucose loading in wild type mice and Alzheimer's disease model mice, respectively. FIG. 5B (young age) and FIG. 5D (old age) show the amount of change in plasma Aβ40 concentration (ΔAβ (40)) after glucose loading, respectively. The AUC for ΔAβ (40) is shown in FIG. 5E. In the figure, white circles (◯) indicate Tg, and black circles (●) indicate WT.

  In young Tg mice, the plasma Aβ40 concentration increased from 30 minutes after glucose loading, reached a peak after 60 minutes, and then gradually decreased (FIG. 5A). A similar trend was seen in young WT, but the degree was minor (FIG. 5A). When the plasma Aβ40 value was evaluated as a concentration change amount (ΔAβ) from the 0 minute value, the change amount (increase amount) tended to be larger in Tg than in WT (FIGS. 5B and 5E). At 480 minutes later, there was no difference between the two.

Similarly, an increase in plasma Aβ40 concentration after glucose loading was observed in old Tg mice (FIG. 5C), but unlike young Tg mice, at least 480 minutes after peaking at 60 to 120 minutes after glucose loading. Until then, the price remained high.
The amount of change in plasma Aβ40 concentration from the 0 minute value (ΔAβ) was significantly larger in Tg than WT (** P <0.01, FIGS. 5D and E), and this difference is apparent even at 480 minutes after glucose loading Met. Comparing the AUC of ΔAβ (40) (FIG. 5E), the amount of change (increase) in the old Tg mice was significantly larger than that in the old WT mice (** P <0.01). It was significantly larger than the amount of change in mice (** P <0.01).

From this result, the change in blood Aβ40 value in the glucose tolerance test shows significantly different kinetics between Tg and WT and reflects the progression of Tg pathology (difference between young Tg and old Tg). I understood. A similar trend was observed for plasma Aβ42 concentrations (FIGS. 6A-E).
The time course of plasma Aβ42 concentration after glucose loading is shown in FIG. 6A (young age) and FIG. 6C (old age), respectively. Plasma Aβ42 concentration change (ΔAβ (42)) after glucose load is shown in FIG. 6B (young age) and FIG. 6D (old age), respectively. The AUC for ΔAβ (42) is shown in FIG. 6E. In the figure, white circles (◯) indicate Tg, and black circles (●) indicate WT.
Particularly, the amount of change in blood Aβ42 concentration (ΔAβ (42) AUC) in the old age Tg was significantly (** P <0.01) larger than that in the old age WT (FIG. 6E).

Test example 3
Patients with Alzheimer's disease are subjected to energy load or glucose load (sugar is administered orally or intravenously), and blood is collected before and after glucose load at 15, 30, 60, and 120 minutes. The amount of medium Aβ is measured. Alzheimer's disease is diagnosed using the amount of blood Aβ before glucose load and the amount of blood Aβ after glucose load.

  The present invention is useful in the medical and research fields because it is useful in the diagnosis of Alzheimer's disease, the development of therapeutic and preventive drugs, and the like.

Claims (12)

  A measurement method for determining Alzheimer's disease, comprising a step of measuring a β-amyloid protein value in a blood sample collected from a mammal subjected to an energy load.   Further, the β-amyloid protein value in the blood sample collected from the mammal subjected to the energy load is compared with the β-amyloid protein value in the blood sample collected from the mammal on an empty stomach, and the energy load is compared. The measurement method according to claim 1, comprising a step of determining the amount of increase in blood β-amyloid protein due to the blood.   The measurement according to claim 2, further comprising the step of comparing the amount of increase in blood β-amyloid protein due to energy load in the mammal with the amount of increase in blood β-amyloid protein due to energy load in a healthy mammal. Method.   The measurement method according to claim 1, wherein the energy load is a sugar load.   The measuring method according to claim 1, wherein the energy load is a glucose load.   The measuring method according to claim 1, wherein the β-amyloid protein is β-amyloid 40 and / or β-amyloid 42.   Β-amyloid protein value in a sample of blood collected from a mammal at any time between 15 minutes and 8 hours after the energy load, and β in a sample of blood collected from the mammal on an empty stomach The method according to claim 2, wherein the amyloid protein value is compared.   The measurement method according to claim 1, wherein the blood sample is plasma.   The measurement method according to claim 1, wherein the blood sample is obtained by diluting a plasma component separated from blood by 4 to 200 times.   Use of β-amyloid protein value in a sample of blood collected from an energy loaded mammal for Alzheimer's disease determination.   Use of a sample of blood collected from an energy-loaded mammal for producing a sample for measuring β-amyloid protein level in the determination of Alzheimer's disease. A diagnostic agent for Alzheimer's disease comprising glucose and administered once by an administration route selected from the group consisting of oral and intravenous, 15 minutes to 8 hours before blood collection, wherein A diagnostic agent for Alzheimer's disease for measuring β-amyloid protein in a sample of blood .

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