JP5455396B2 - Protein phosphatase-1 inhibitor - Google Patents

Description

The present invention relates to an inhibitor of protein phosphatase-1, an activator of Ca ²⁺ / calmodulin-dependent protein kinase II containing the inhibitor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor The present invention relates to a body expression enhancer and a synaptic transmission promoter.

  In recent years, dementia has become a major medical problem worldwide. Dementia is a disease with various symptoms centered on memory impairment and a decline in judgment, but the symptoms and their course differ depending on the disease that causes them. However, both cases are common in that the quality of life of the patient is significantly impaired. Dementia is also a serious social problem when considering the fact that it will cause a great deal of effort to caregivers including the patient's family. Since the increase in the elderly population due to prolonged lifespan is related to the increase in patients with dementia, it is predicted that the number of patients with dementia will increase in Japan in the future. Many people suffer from some cognitive impairment that is not classified as dementia.

Dementia associated with aging and neurodegenerative diseases such as Alzheimer's disease is mainly caused by cerebral nerve cell death (apoptosis) that causes brain atrophy, and in particular, the hippocampus atrophy. Neuronal apoptosis is largely induced by two pathways-oxidative stress and endoplasmic reticulum stress.
Examples of means for improving cognitive function include a method for suppressing apoptosis of the above-described brain neurons, a method for promoting synaptic transmission, and the like. Methods for promoting synaptic transmission include (1) stimulating neurotransmitter release from presynaptic terminals, (2) suppressing neurotransmitter degradation, and (3) suppressing neurotransmitter reabsorption. (4) Methods such as increasing the reactivity of neurotransmitter receptors expressed in post-synaptic cells are being studied.

On the other hand, in recent years, a mechanism for promoting hippocampal synaptic transmission has been elucidated. For example, activation of Ca ²⁺ / calmodulin-dependent protein kinase II (CaMKII) transports α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor to (synaptic) cell membranes. It has been reported that the expression level of AMPA receptor on the synapse is increased, the response is increased, and hippocampal synaptic transmission is promoted (Non-patent Document 1). Moreover, it is reported that CaMKII is activated by suppressing protein phosphatase-1 (PP-1), and that PP-1 is related to learning and memory (Non-patent Document 2).

  The present inventor has proposed 8- (2) as a compound having a long-term potentiating effect on synaptic transmission efficiency, which can delay metabolism in the body and can maintain a stable long-term potentiation (LTP) -like enhancement of synaptic transmission. -(2-pentyl-cyclopropylmethyl) -cyclopropyl) -octanoic acid (DCP-LA) has been found (Patent Document 1). LTP is thought to be involved in the development of various neurological and psychiatric disorders such as Alzheimer's disease, and thus substances that induce LTP expression are therapeutic or preventive agents for these neurological and psychiatric disorders including dementia. Have potential.

Several reports on DCP-LA have also been made. For example, DCP-LA selectively and directly activates PKC-ε (Non-patent Document 3), DCP-LA improves cognitive impairment in senescence-accelerated mice (Non-patent Document 4), DCP -LA increases the release of γ-aminobutyric acid from hippocampal neurons (Non-Patent Document 5), DCP-LA improves cognitive dysfunction in amyloid β peptide or scopolamine-treated rats (Non-Patent Document 6), It has been reported that DCP-LA promotes hippocampal synaptic transmission targeting α7 nicotinic acetylcholine receptor expressed in glutamatergic presynaptic cells (Non-patent Document 7). Furthermore, recently, it has been reported that DCP-LA has an action of suppressing neuronal cell death induced by oxidative stress (Patent Document 2).
However, the detailed mechanism of the synaptic transmission promoting action of DCP-LA has not yet been elucidated. Clarifying the mechanism and clarifying the action point of DCP-LA is the development of preventive / therapeutic drugs that have a different mechanism of action from existing drugs for various neurodegenerative diseases including Alzheimer's disease. Connected.

International Publication No. 02/50013 JP 2008-143819 A

Rongo, C., Bioessays, 2002, 24: 223-233 Genoux, D., Nature, 2002, 418: 970-975 Kanno T et al., J Lipid Res., 2006, 47 (6): 1146-56 Yaguchi T et al., Neuroreport, 2006, 23; 17 (1): 105-8. Kanno T et al., J Neurochem., 2005, 95 (3): 695-702. Nagata et al. T, Psychogeriatrics, 2005, 5: 122-126. Yamamoto et al., Neuroscience 2005, 130 (1): 207-213.

The present invention relates to an inhibitor of protein phosphatase-1, an activator of Ca ²⁺ / calmodulin-dependent protein kinase II containing the inhibitor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid An object is to provide a receptor expression enhancer and a synaptic transmission promoter.

As a result of intensive studies, the inventor activated Ca ²⁺ / calmodulin-dependent protein kinase II (hereinafter sometimes simply referred to as CaMKII) to a compound represented by the following formula (I) or a salt thereof. It was also found that the CaMKII activating action is derived from the protein phosphatase-1 (hereinafter sometimes simply referred to as PP-1) inhibitory action. Through activation of CaMKII, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (hereinafter sometimes simply referred to as AMPA) receptor is promoted to the cell membrane, and on the cell membrane The present invention was completed by confirming the promotion of hippocampal synaptic transmission accompanying the increase in the expression number of AMPA receptors. That is, the present invention is as follows.
[1] Formula (I)

[Where,
R is alkyl or alkenyl which may have one or more 1,2-cyclopropylene in the carbon chain and / or may have cyclopropyl at the chain end; Propylene may have a suitable substituent at the 3rd carbon atom,
X is a single bond or alkylene;
Subtracting the number of cyclopropane rings from the total number of carbons gives 10-25. ]
The protein phosphatase-1 (PP-1) inhibitor containing the carboxylic acid compound or its salt which has 1 or more cyclopropane rings represented by these.
[2] The above, wherein R is alkyl or alkenyl which may have one or more 1,2-cyclopropylene in the carbon chain and / or may have cyclopropyl at the chain end [ 1] The agent according to the above.
[3] R is

[Where,
R ′ is hydrogen or alkyl;
Y is a single bond or alkylene;
A is 1,2-cyclopropylene or vinylene;
m is an integer of 0 to 5,
However, when m is 2-5, A is the same or different. ]
The agent according to [2] above, wherein
[4] R is

A group selected from
X is — (CH ₂ ) _n — wherein n, p, q and r are each an integer, the sum of p and n is 6 to 21, and the sum of q, r and n is 4 to 19. ],
The agent according to [3] above.
[5] R is

A group selected from
X is - _{(CH 2) n} - in which wherein, s, n are each integers, the sum is 3 to 18. ],
The agent according to [4] above.
[6] The agent according to [5] above, wherein s is 4 and n is 7.
[7] The agent according to [1] above, wherein the carboxylic acid compound or a salt thereof is 8- [2- (2-pentyl-cyclopropylmethyl) -cyclopropyl] -octanoic acid or a salt thereof.
[8] The agent according to any one of [1] to [7], which is a research reagent.
[9] An activator for Ca ²⁺ / calmodulin-dependent protein kinase II (CaMKII), comprising the agent according to any one of [1] to [8] above.
[10] An expression enhancer for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, comprising the agent according to [9] above.
[11] A synaptic transmission promoter comprising the agent according to [10] above.
[12] Formula (I)

[Where,
R is alkyl or alkenyl which may have one or more 1,2-cyclopropylene in the carbon chain and / or may have cyclopropyl at the chain end; Propylene may have a suitable substituent at the 3rd carbon atom,
X is a single bond or alkylene;
Subtracting the number of cyclopropane rings from the total number of carbons gives 10-25. ]
An activator of Ca ²⁺ / calmodulin-dependent protein kinase II, comprising a carboxylic acid compound having one or more cyclopropane rings or a salt thereof represented by:
[13] The agent according to [12] above, wherein the activation of Ca ²⁺ / calmodulin-dependent protein kinase II is due to inhibition of protein phosphatase-1.
[14] Formula (I)

[Where,
R is alkyl or alkenyl which may have one or more 1,2-cyclopropylene in the carbon chain and / or may have cyclopropyl at the chain end; Propylene may have a suitable substituent at the 3rd carbon atom,
X is a single bond or alkylene;
Subtracting the number of cyclopropane rings from the total number of carbons gives 10-25. ]
A carboxylic acid compound having one or more cyclopropane rings represented by
Expression enhancer of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor.
[15] The above, wherein the enhanced expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is due to activation of Ca ²⁺ / calmodulin-dependent protein kinase II [14] The agent according to [14].

  The agent of the present invention suppresses PP-1 and activates CaMKII, thereby promoting the transition of AMPA receptor to the cell membrane and promoting hippocampal synaptic transmission accompanying the increase in the number of AMPA receptor expression on the cell membrane. Can be made. That is, it leads to the development of a preventive / therapeutic agent for a disease (for example, neurodegenerative disease) in which it is beneficial to promote hippocampal synaptic transmission having an action mechanism different from that of an existing drug. Since it has a different mechanism of action from existing drugs, it is possible to avoid side effects that have been problematic in existing drugs. The agent of the present invention can also be used as a research reagent that can be a useful tool for the development of such preventive / therapeutic agents.

FIG. 1 was recorded from the CA1 region of a rat hippocampal slice before and after treatment with DCP-LA (100 nM) for 10 minutes in the presence and absence of GF109203X (100 nM) and / or KN-93 (3 μM). It is a graph which shows fEPSP. The upper part shows a potential chart. In the graph, each point represents an average value (± SEM) of EPSP (% reference slope) (n = 8). The vertical axis represents the elapsed time (minutes). fEPSP: excitatory postsynaptic field potential FIG. 2 measured CaMKII activity in cultured hippocampal neurons treated (or untreated) with a given concentration of DCP-LA in the presence or absence of KN-92 (3 μM) or KN-93 (3 μM). It is a figure which shows a result. The upper row shows the HPLC profile. In the HPLC profile, “S” and “P” indicate a non-phosphorylated substrate and a phosphorylated substrate, respectively. The vertical axis represents absorbance, and the horizontal axis represents retention time (minutes). The lower graph is a graph created using phosphorylated substrate peptide (pmol / min / μg cell protein) as an indicator of CaMKII activity. In the graph, each column represents the average value (± SEM) of CaMKII activity (n = 16). P value, unpaired t-test FIG. 3 is a diagram showing the results of measuring CaMKII activity and PP-1 activity in a cell-free system (A: CaMKII activity, B: PP-1 activity). The upper row of both A and B shows the HPLC profile. In the HPLC profile, “S” and “P” indicate a non-phosphorylated substrate and a phosphorylated substrate, respectively. The vertical axis represents absorbance, and the horizontal axis represents retention time (minutes). A (bottom): A graph prepared using phosphorylated substrate peptide (pmol / min / μg cell protein) as an indicator of CaMKII activity. In the graph, each column represents the mean value (± SEM) of CaMKII activity (n = 5). B (bottom): a graph prepared using a dephosphorylated substrate peptide (Δpmol / min) as an indicator of PP-1 activity. In the graph, each column represents the average value of PP-1 activity (± SEM) (n = 8). The amount of the dephosphorylated substrate peptide varies from (amount of phosphorylated substrate peptide in the presence of PP-1) to (in the absence of PP-1) in the presence and absence of microcystin (MC) or DCP-LA. This was determined by subtracting the amount of phosphorylated substrate peptide below. P value, unpaired t-test AMPA receptors composed of various combinations of GluR1, GluR2 and GluR3 subunits or mGluR1 (S831A) subunits were expressed in Xenopus oocytes. Each oocyte was bath-applied with kainic acid (50 μM) for 10 seconds at 10-minute intervals before and after 10 minutes of DCP-LA (100 nM) treatment in the presence and absence of KN-93 (3 μM). At a holding potential of −60 mV, the current was measured and recorded from 10 minutes before DCP-LA treatment to 70 minutes after treatment. FIG. 4 is a graph showing the results of current measurement in each oocyte. In addition, a chart of current measurement is shown. Application of kainic acid is indicated by a horizontal bar in the chart. In the graph, each point represents an average value (± SEM)% (n = 6-11) with respect to a reference amplitude (amplitude at −10 minutes). The vertical axis represents current amplitude (% reference value), and the horizontal axis represents elapsed time (minutes). A: Western blot with antibodies against GluR1 and GluR2 subunits of lysates from rat hippocampal slices immunoprecipitated with anti-GluR1 subunit antibody (GluR1 IP) or immunoprecipitated with anti-GluR2 subunit antibody (GluR2 IP) It is a figure which shows the result of having analyzed. B: It is a figure which shows the result of the Western blot analysis performed about the cytoplasm fraction (C) and the cell membrane fraction (M) isolate | separated from the lysate from a hippocampal slice using an anti-cadherin (cadherin) antibody and an anti- LDH antibody. . C: Hippocampal slices [GF109203X (GF) (100 nM), KN-93 (3 μM), H-89 (1 μM), PAO (3 μM) or BoTX-A (0.1 U) using antibodies against GluR1 and GluR2 subunits. / Ml) in the presence or absence of cytoplasmic fraction (C) and cell membrane fraction (M) isolated from lysates from DCP-LA at a given concentration or untreated] It is a figure which shows the result of a Western blot analysis. D: Graphed using C results. In the graph, each column represents the ratio of the signal intensity in the cell membrane fraction to the signal intensity in all cells as an average value (± SEM) for the signal intensity of the GluR1 or GluR2 subunit (n = 4). P value, one-way ANOVA

  As used herein, alkyl in R is linear or branched, and the alkyl includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, Examples include heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, eicosanyl, docosanyl and the like. The alkyl may have 1,2-cyclopropylene in the carbon chain and / or cyclopropyl at the chain end. When 1,2-cyclopropylene is present in the carbon chain when alkyl is n-pentyl, for example, 1,2-cyclopropylene is present between the 2- and 3-position carbon atoms of n-pentyl. In the case it means 2- (2-propylcyclopropan-1-yl) ethyl. Two or more 1,2-cyclopropylenes may be present. When alkyl is n-pentyl and cyclopropylene is present at the chain end, 5-cyclopropylpentan-1-yl is substituted when cyclopropyl is present at the terminal carbon atom (5th carbon atom) of n-pentyl. means. The alkyl in the present invention is alkyl having 1,2-cyclopropylene in the carbon chain and / or cyclopropyl at the chain end (for example, 2- (2- (3-cyclopropylpropyl) -cyclopropane-1- Yl) ethyl). Examples of alkyl at R having 1,2-cyclopropylene in the carbon chain and cyclopropyl at the chain end are preferably octyl, (2-pentylcyclopropan-1-yl) methyl, (2- ( (2-Ethyl-cyclopropan-1-yl) methyl) cyclopropan-1-yl) methyl, (2-((2-((2-pentyl-cyclopropan-1-yl) methyl) cyclopropane-1- Yl) methyl) -cyclopropan-1-yl) methyl, (2-((2-pentyl-cyclopropan-1-yl) methyl) cyclopropan-1-yl) methyl, octyl, (2-pentyl) Cyclopropan-1-yl) methyl is particularly preferred.

  The alkenyl in R is linear or branched, and the alkenyl includes ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, sec-butenyl, tert-butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, Dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, eicosenyl, dococenyl, butadienyl, pentadienyl, hexadienyl, heptadienyl, octadienyl, nonadienyl, decadienyl, undecadienyl, tridecadienyl, tridecadienyl, dodecacadenyl Dienyl, heptadecadienyl, octadecadienyl, eicosadienyl, docosadienyl, Xatrienyl, heptatrienyl, octatrienyl, nonatrienyl, decatrienyl, undecatrienyl, dodecatrienyl, tridecatrienyl, tetradecatrienyl, pentadecatrienyl, hexadecatrienyl, heptadecatrienyl, octadecatrienyl, eico Satrienyl, docosatrienyl, octatetraenyl, nonatetraenyl, decatetraenyl, undecatetraenyl, dodecatetraenyl, tridecatetraenyl, tetradecatetraenyl, pentadecatetraenyl, hexadecatetraenyl, heptadecatetraenyl, octa Decatetraenyl, eicosatetraenyl, docosatetraenyl and the like. The alkenyl may have 1,2-cyclopropylene in the carbon chain and / or cyclopropyl at the chain end. When 1,2-cyclopropylene is present in the carbon chain when alkenyl is 1-pentenyl, for example, when 1,2-cyclopropylene is present between the 2- and 3-position carbon atoms of 1-pentenyl. Means 2- (2-propylcyclopropan-1-yl) ethenyl. Two or more 1,2-cyclopropylenes may be present. When alkenyl is 1-pentenyl, when cyclopropyl is present at the chain end, when cyclopropyl is present at the terminal carbon atom (5-position carbon atom) of 1-pentenyl, it means 5-cyclopropyl-1-pentenyl. To do. The alkenyl in the present invention is an alkenyl having 1,2-cyclopropylene in the carbon chain and cyclopropyl at the chain end (for example, 2- (2- (3-cyclopropylpropyl) cyclopropan-1-yl) ethenyl). Is included. Alkenyl at R having 1,2-cyclopropylene in the carbon chain and / or cyclopropyl at the chain end is preferably octenyl.

  1,2-Cyclopropylene in R may have a suitable substituent such as alkyl at the 3-position carbon atom. Examples of alkyl at the 3-position carbon atom of 1,2-cyclopropylene in R are preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, tert- Examples include pentyl and hexyl.

  The alkylene in X is linear or branched, and as the alkylene, methylene, ethylene, propylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene, decamethylene, undecamethylene, dodecamethylene , Tridecamethylene, tetradecamethylene, pentadecamethylene, hexadecamethylene, heptadecamethylene, octadecamethylene, nonadecamethylene, eicosamethylene and the like. Heptamethylene, trimethylene and tetramethylene are preferred.

  In the compounds of formula (I) according to the invention, the number of cyclopropane rings minus the total number of carbons can be 10-25, preferably 18-22. p, q, r, m, n, and s are each an integer (including 0).

  For example, the compound of formula (I) is 8- (2- (2-pentyl-cyclopropylmethyl) -cyclopropyl) -octanoic acid (wherein DCP-LA as required) having the following structural formula: Abbreviated).

  Alternatively, examples of the compound of the formula (I) include 8- (2- (2- (Z) -octenyl) cyclopropyl) octanoic acid, 8-((2-octyl) cyclopropyl) octanoic acid, 8- ( 2-((2-((2-ethyl-cyclopropan-1-yl) methyl) cyclopropan-1-yl) methyl) cyclopropyl) octanoic acid, 8- (2-((2-((2- ( (2-Pentyl-cyclopropan-1-yl) methyl) cyclopropan-1-yl) methyl) cyclopropan-1-yl) methyl) cyclopropyl) butanoic acid, 8- (2-((2-((2 -Pentyl-cyclopropan-1-yl) methyl) cyclopropan-1-yl) methyl) cyclopropyl) pentanoic acid.

  It should be noted that the compounds of formula (I) may contain one or more stereoisomers (eg optical isomers, geometric isomers) resulting from asymmetric carbon atoms or double bonds. All such isomers and mixtures thereof are included within the scope of the present invention.

  The compound of the formula (I) can be produced, for example, by the method shown in WO02 / 50013. The compound of formula (I) may also be extracted from natural products.

  The salt of the compound of the formula (I) is not particularly limited, but is preferably a pharmaceutically or food acceptable salt, for example, an inorganic base (eg, an alkali metal such as sodium or potassium; an alkaline earth metal such as calcium or magnesium; Aluminum, ammonium), organic bases (eg, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N-dibenzylethylenediamine), inorganic acids (eg, hydrochloric acid, hydrobromic acid) , Nitric acid, sulfuric acid, phosphoric acid), organic acids (eg, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid), basic amino acids (eg, Arginine, lysine, ornithine), or acidic amino acid (e.g., aspartic acid, salts and the like and glutamic acid).

  As used herein, a subject can be a mammal. Examples of such mammals include primates (eg, humans, monkeys, chimpanzees), rodents (eg, mice, rats, guinea pigs), pets (eg, dogs, cats, rabbits), working animals or livestock. (Eg, cows, horses, pigs, sheep, goats), and humans are preferred.

The compounds of formula (I) and salts thereof have a PP-1 inhibitory action and activate CaMKII by this action.
PP-1 (protein phosphatase-1) is one of the enzymes that dephosphorylate phosphorylated proteins, and is classified as serine / threonine phosphatase. PP-1 exhibits a wide range of tissue distribution and intracellular distribution, and mainly controls a wide range of cell functions such as sugar metabolism, cell movement, cell cycle, DNA replication, transcription, translation, and cell adhesion. In recent years, attention has been paid to the involvement of protein phosphorylation in neural activity, and it is considered to play an important role in higher brain function represented by plasticity of synaptic transmission and memory / learning together with CaMKII described later. CaMKII (Ca ²⁺ / calmodulin-dependent protein kinase II) is abundant in neurons and phosphorylates neurotransmitter synthase, synaptic vesicle binding protein, ion channel, neurotransmitter receptor, etc. It is thought to play an important role in the higher brain functions represented by plasticity of synaptic transmission and memory / learning by regulating these functions. In a normal state of neural activity, the active state of CaMKII is maintained in an equilibrium state by a balance of intracellular Ca ²⁺ / calmodulin, CaMKII activity, and phosphatase (including PP-1) activity.

Further, activation of CaMKII promotes the transport (exocytosis) of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor. The AMPA receptor is a receptor that plays the most central role among glutamate receptors that control excitatory synaptic transmission in the central nervous system, and includes GluR1, GluR2, GluR3, and GluR4 subunits, and is a tetramer. It is believed that a functional channel is formed by forming Promotion of AMPA receptor transport increases AMPA receptor expression on the cell membrane, and enhanced receptor response promotes hippocampal synaptic transmission.
Therefore, the agent of the present invention (PP-1 inhibitor, CaMKII activator) having a PP-1 inhibitory action and capable of activating CaMKII by the action or a salt thereof is an AMPA receptor. It is useful as an expression enhancer on the cell membrane of the body, and further as a synaptic transmission promoter.

  As described above, since the agent of the present invention can promote synaptic transmission, it is useful as an agent for preventing or treating various neurodegenerative diseases, particularly diseases or conditions associated with cognitive impairment. The disease or condition associated with cognitive impairment specifically includes dementia (eg, senile dementia, Alzheimer's dementia, cerebrovascular dementia, post-traumatic dementia, dementia caused by brain tumor, chronic dura mater Dementia caused by hematoma, dementia caused by normal pressure cerebral edema, dementia caused by various diseases such as postmeningitis dementia and Parkinsonian dementia, non-demented cognitive impairment (eg, mild cognition) Disorders (MCI)), learning or memory disorders (eg, learning and memory disorders associated with brain development disorders) and the like. Furthermore, the agent of the present invention can be used for improving learning and memory (eg, short-term memory, long-term memory).

  In addition, since the agent of the present invention can drive the cascade of “PP-1 inhibitory action → CaMKII activation action → AMPA receptor expression enhancing action → hippocampal synaptic transmission promoting action”, various neurodegenerative diseases, particularly It is also useful as a research reagent for developing a prophylactic or therapeutic agent for a disease or condition associated with cognitive impairment.

  The agent of the present invention may be useful for prevention or treatment of various neurodegenerative diseases, for example, when used as a medicine. The agent of the present invention can also be used as a food, but in that case, a subject who has a high risk of developing a neurodegenerative disease or a subject who is eager to not develop the disease should eat regularly. Thus, the risk of developing the disease in such a subject can be reduced.

  As used herein, “prevention” of a neurodegenerative disease prevents the manifestation of the symptom in a subject who does not exhibit symptoms of the neurodegenerative disease (cognitive impairment, learning / memory impairment, etc.). "Treatment" means that a subject who exhibits symptoms of a neurodegenerative disease (cognitive impairment, learning / memory impairment, etc.) alleviates the symptoms or prevents or delays the deterioration of the symptoms Means that.

  The agent of the present invention may contain any carrier, for example, a pharmaceutically or food acceptable carrier, in addition to the compound of formula (I) or a salt thereof. Examples of such carriers include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate and other excipients, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin Binders such as gum arabic, polyethylene glycol, sucrose, starch, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol-starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate, aerosil , Lubricants such as talc and sodium lauryl sulfate, fragrances such as citric acid, menthol, glycyllysine / ammonium salt, glycine and orange powder, sodium benzoate Preservatives such as sodium bisulfite, methylparaben, propylparaben, stabilizers such as citric acid, sodium citrate, acetic acid, suspension agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, dispersants such as surfactants, water, Examples include, but are not limited to, saline, diluents such as orange juice, cocoa butter, polyethylene glycol, base waxes such as white kerosene.

  The agent of the present invention can also contain two or more compounds of the formula (I) or salts thereof as active ingredients, or can be combined in use, or such compounds or salts thereof and other neurodegenerative diseases. It can be a combination drug in combination with one or more other components useful for the prevention or treatment of the disease. Such a combination agent can prevent or treat neurodegenerative diseases additively or synergistically.

  Examples of components useful for the prevention or treatment of other neurodegenerative diseases include, for example, polyphenol, coenzyme Q1, β-sitosterol, isoflavone, mevinic acid, vitamin C, vitamin E, flavonoids, terpenes, folic acid, vitamin B6, vitamin B12, sesquipene lactone, urokinase, nattokinase, dilinoleoylphosphatidylethanolamine, propyl sulfide, apple pectin, acetic acid, EPA, DHA, and the like can be mentioned.

  When the agent of the present invention is used as a food, examples of the food include general foods (eg, bread, dairy products (eg, milk, yogurt), confectionery, candy, rice cake, chocolate, cake, pudding, jelly, refreshing Drinking water, noodles), health foods, dietary supplements, and foods for specified health use and nutritional function foods stipulated in the Health Functional Food System of the Ministry of Health, Labor and Welfare. The food may be in any form such as a liquid (water-soluble or insoluble), a solid such as a powder, a granule, a tablet or a capsule, or a semi-solid such as a jelly. The agent of the present invention can be used by dissolving in water or a predetermined aqueous solution. In such a case, the agent of the present invention may contain a solubilizer (eg, linoleic acid) and a stabilizer.

  When the agent of the present invention is used as a food, the intake amount is the form used (eg, liquid, solid, semi-solid), the concentration of the compound of formula (I) or a salt thereof, and prevention of neurodegenerative diseases. Or, depending on the presence / absence, type and amount of components useful for treatment, it cannot be generally stated, but usually the unit intake of the compound of formula (I) or a salt thereof (intake or intake per day) About 5 mg to about 500 mg can be mentioned.

  When the agent of the present invention is used as a food, the food may contain the compound of formula (I) or a salt thereof in a unit intake or divided amount thereof. The food product comprising a unit intake of a compound of formula (I) or a salt thereof may be a single liquid, solid or semi-solid containing a unit intake of a compound of formula (I) or a salt thereof. Multiple (for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10) foods containing the compound of the formula (I) or a salt thereof in divided amounts of unit intake may be taken once or per day. ) Can be solids (eg, tablets, capsules) or semi-solids recommended for consumption.

  When the agent of the present invention is used as a food, the unit intake or divided amount of the compound of formula (I) or a salt thereof is individually packaged or filled, or the compound of formula (I) or a salt thereof A large number of unit intakes or divided amounts thereof may be comprehensively packaged or filled. As the food in which the unit intake of the compound of the formula (I) or a salt thereof or a divided amount thereof is individually packaged or filled, for example, the unit intake of the compound of the formula (I) or a salt thereof or the divided amount thereof, Examples include ordinary packages (for example, PTP (press through packing) sheets, paper containers, film (eg, plastic film) containers, glass containers, plastic containers) individually packed or filled. Such individually packaged or filled foods are further combined and packaged or filled together in one container (eg, paper container, film (eg, plastic film) container, glass container, plastic container). It may be. As a food product in which a large number of unit intakes of the compound of the formula (I) or a salt thereof or a divided amount thereof are comprehensively packaged or filled, for example, a single container ( For example, what was packaged or filled in the paper container, the film (for example, plastic film) container, the glass container, the plastic container) is mentioned. The food of the present invention can also be used for a long time (for example, 3 days or more, preferably 7 days, 10 days, 14 days, 21 days or more, or 1 Months, 2 months, 3 months or more).

  When the agent of the present invention is used as a medicine, a medicine suitable for oral administration includes a solution obtained by dissolving an effective amount of a substance in a diluent such as water or physiological saline, and an effective amount of the substance as a solid or a granule. Capsules, sachets or tablets, suspensions in which an effective amount of a substance is suspended in an appropriate dispersion medium, and solutions in which an effective amount of the substance is dissolved are dispersed in an appropriate dispersion medium and emulsified. Emulsions, powders, granules, etc.

  Suitable pharmaceuticals for parenteral administration (eg, intravenous injection, subcutaneous injection, intramuscular injection, local injection, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants. Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. Also, aqueous and non-aqueous sterile suspensions can be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The drug can be enclosed in a container such as an ampoule or a vial by unit dose or multiple doses. In addition, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.

  When the agent of the present invention is used as a medicine, the dose is determined depending on the activity and type of the active ingredient, the mode of administration (eg, oral or parenteral), the severity of the disease, the animal species to be administered, the type of the subject to be administered. Although it varies depending on drug acceptability, body weight, age, etc., it cannot be generally stated, but usually an effective amount of the compound of formula (I) or a salt thereof per day for an adult can be about 5 mg to about 500 mg.

  The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

(Materials and methods)
1. Animals All animal handling is approved by the Hyogo College of Medicine Animal Experimentation Committee and complies with NIH (National Institutes of Health) guidelines for the management and use of laboratory animals.

2. Measurement of excitatory postsynaptic potential (fEPSP) Hippocampal slices (400 μm) were prepared from male Wistar rats (6 weeks old). Hippocampal sections were treated with DCP-LA (100 nM) for 10 minutes in the presence and absence of GF109203X (100 nM) and / or KN-93 (3 μM). Before and after 10 minutes of DCP-LA treatment, CA1 was electrically stimulated (0.03 Hz, 0.1 milliseconds) on the side branch of the shuffler in standard artificial cerebrospinal fluid (hereinafter abbreviated as ACSF). The fEPSP from the area was recorded. ACSF was oxygen-treated at 34 ° C., 95% O ₂ and 5% CO ₂ . The composition of ACSF is as follows:
ACSF : 117 mM NaCl, 3.6 mM KCl, 1.2 mM NaH ₂ PO ₄ , 1.2 mM MgCl ₂ , 2.5 mM CaCl ₂ , 25 mM NaHCO ₃ and 11.5 mM glucose

3. Cell culture The hippocampus was removed from the brain of Wister rat fetuses (gestational age, 18 days). Dissociated hippocampal cells were seeded in a 96-well plate and cultured in a culture solution at 37 ° C. in a humid environment of 5% CO ₂ and 95% air. The composition of the culture solution is as follows:
Culture medium : Neurobasal supplemented with B27 (1:50), 2.5 mM glutamine, 50 μM glutamate, penicillin (final concentration 100 U / ml) and streptomycin (final concentration, 0.1 mg / ml)

4). In situ CaMKII assay CaMKII activity in cultured rat hippocampal neurons was measured according to previous reports (Kanno T. et al, J. Lipid Res. 47: 1146-1156, 2006). Cultured neurons were treated with DCP-LA in extracellular fluid for 10 minutes at 37 ° C. in the presence or absence of KN-92 or KN-93. The cells were then washed with 100 μl Ca ²⁺ -free phosphate buffered saline (PBS) and 50 μl extracellular fluid (50 μg / ml digitonin, 25 mM glycerol 2-phosphate, 200 μM ATP and 100 μM for 15 minutes at 30 ° C. Autocamtide-2 (Calbiochem, San Diego, USA); CaMKII synthetic substrate peptide). The supernatant was collected and boiled at 100 ° C. for 5 minutes to stop the reaction. A part (20 μl) of the solution was subjected to reverse phase high performance liquid chromatography (HPLC) (LC-10ATvp, Shimadzu Co., Kyoto, Japan). The peak of the substrate peptide and the peak of the newly produced product were detected at an absorbance of 214 nm (SPD-10Avp UV-VIS detector, Shimadzu Co., Kyoto, Japan). The molecular weight of each peak was measured from the standard spectra of bradykinin (MW 1060.2) and neurotensin (MW 1672.9). According to the analysis with MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry; Voyager ST-DER, PE Biosystems Inc., Foster City, USA), the peak of the substrate and the peak of the new product are respectively The molecular weight was 1508 and the molecular weight was 1588. Since the molecular weight of 1588 matches the molecular weight of the unphosphorylated substrate protein (MW1508) plus the molecular weight of HPO ₃ (MW80), the newly produced product peak was phosphorylated. It turns out that it corresponds to. The areas of unphosphorylated CaMKII substrate peptide and phosphorylated CaMKII substrate peptide were measured (the total area corresponds to the concentration of CaMKII substrate peptide used here). The amount of phosphorylated substrate peptide (pmol / min / μg cell protein) was calculated and used as an indicator of CaMKII activity.
The composition of the extracellular fluid is as follows.
Extracellular fluid : 137 mM NaCl, 5.4 mM KCl, 10 mM MgCl ₂ , 5 mM ethylene glycol-bis (2-aminoethyl ether) -N, N, N ', N'-tetraacetic acid (EGTA), 0.3 mM Na ₂ HPO ₄ , 0.4 mM K ₂ HPO ₄ and 20 mM 4- (2-hydroxyethyl) -1-piperazine-ethanesulfonic acid, pH 7.2

5. Cell-free assay for measurement of CaMKII activity and PP-1 activity To measure CaMKII activity in a cell-free system, in the reaction medium (25 μl, pH 8.0), in the presence and absence of DCP-LA, Synthetic CaMKII substrate peptide (10 μM) was reacted with CaMKII (1.1 × 10 ⁶ U) (Calbiochem, San Diego, USA) at 35 ° C. for 10 minutes.
To measure PP-1 activity in a cell-free system, 10 minutes at 35 ° C. in the same reaction medium used in the CaMKII assay in the presence and absence of microcystin (Sigma) or DCP-LA The synthetic CaMKII substrate peptide (10 μM) was reacted with CaMKII (1.1 × 10 ⁶ U) (Calbiochem, San Diego, USA) and PP-1 (0.1 U) (Sigma, St. Louis, MO, USA). . Both reactions were terminated by treatment at 100 ° C. for 5 minutes. A part (10 μl) of each solution was injected into a column (250 mm × 4.6 mm) (COSMOSIL 5C ₁₈ -AR-II, Nacalai Tesque, Kyoto, Japan) and subjected to a reverse phase HPLC system (LC-10ATvp, Shimadzu) . Unphosphorylated and phosphorylated peptides were detected at an absorbance of 214 nm (SPD-10Avp UV-Vis detector, Shimadzu). The amount of phosphorylated substrate peptide (pmol / min) was used as an indicator of CaMKII activity. The amount of dephosphorylated substrate peptide (Δpmol / min) was used as an indicator of PP-1 activity. The amount of the dephosphorylated substrate peptide varies from (amount of phosphorylated substrate peptide in the presence of PP-1) to (in the absence of PP-1) in the presence and absence of microcystin or DCP-LA. This was determined by subtracting the amount of phosphorylated substrate peptide).
The composition of the reaction medium is as follows.
Reaction medium : 40 mM 4- (2-hydroxyethyl) -1-piperazine-ethanesulfonic acid (HEPES), 5 mM Mg-acetate, 0.4 mM CaCl ₂ , 0.1 mM ATP, 0.1 mM EGTA, 1 μM calmodulin (Calbiochem, San Diego, USA)

6). In vitro transcription and translation mRNAs encoding GluR1, GluR2 and GluR3 subunits were synthesized by in vitro transcription. Ser ⁸³¹ was replaced with Ala, and a mutant GluR1 subunit lacking the CaMKII phosphorylation site was prepared [mGluR1 (S831A)]. Xenopus oocytes isolated manually from the ovary were injected with mRNA with various combinations of GluR1, GluR2 and GluR3 subunits or mGluR1 (S831A) subunits and incubated at 18 ° C.

7.2 Electrode Voltage Clamp Measurement Two to three days after injecting the mRNA, the oocytes were transferred to a measurement chamber and continuously perfused at 22 ° C. with a standard amphibian Ringer's solution. Kainic acid (50 μM) was bath-applyed into the oocytes at 10 minute intervals before and after 10 minutes of DCP-LA treatment. All cell membrane currents were measured and recorded with a GeneClamp-500 amplifier (Axon Instruments, Inc., Foster City, Calif., USA) and computer analyzed using pClamp software (version 6.0.3, Axon Instruments, Inc.).
The composition of a standard amphibian Ringer's solution is as follows.
Amphibian Ringer's solution ; 115 mM NaCl, 2 mM KCl, 1.8 mM CaCl ₂ and 5 mM HEPES, pH 7.0

8). Analysis of AMPA receptor transport Rat hippocampal slices (male, Wistar, 6w) (400 μm) were incubated in standard ACSF for 20 minutes at 34 ° C. in the presence and absence of DCP-LA. The incubation is carried out in the presence and absence of GF109203X (100 nM), KN-93 (3 μM) and H-89 (1 μM), or phenylarsine oxide (PAO) (3 μM) 20 minutes before DCP-LA treatment. Or in the presence or absence of botulinum toxin A (BoTX-A) (0.1 U / ml) 12 hours before DCP-LA treatment.
The sections were then homogenized by sonication in chilled mitochondrial buffer containing 1% protease inhibitor cocktail, followed by centrifugation (3,000 rpm, 5 min, 4 ° C.). The supernatant was centrifuged (11,000 rpm, 15 min, 4 ° C.), and the collected supernatant was further ultracentrifuged (100,000 rpm, 60 min, 4 ° C.) to separate it into a cytoplasmic fraction and a membrane fraction.
The protein concentration of each fraction was measured using a BCA protein assay kit (Pierce, Rockford, IL, USA). The protein of the cell membrane fraction was resuspended in mitochondrial buffer containing 1% sodium dodecyl sulfate (SDS). The proteins of each fraction were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. After blocking with TTBS (150 mM NaCl, 0.1% Tween20 and 20 mM Tris, pH 7.5) containing 5% bovine serum albumin (BSA), the transferred membrane was treated with an antibody against GluR1 subunit (Upstate Biotechnology, Lake Placid, NY). , USA), antibodies to GluR2 subunit (Chemicon, Temecula, CA, USA), antibodies to cadherin (Sigma, St Louis, MO, USA) and antibodies to lactate dehydrogenase (LDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by reaction with goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP). Immunoreactivity was detected with ECL kit (GE Healthcare, Piscataway, NJ, USA) and visualized using the chemiluminescence detection system (FUJIFILM, Tokyo, Japan). The signal density was measured with Image Gauge software (FUJIFILM). The composition of the mitochondrial buffer is as follows.
Mitochondrial buffer ; 210 mM mannitol, 70 mM sucrose and 1 mM EDTA, 10 mM HEPES, pH 7.5

9. Immunoprecipitation and Western blot analysis Rat hippocampal slices (male Wistar, 6w) (400 μm) were lysed with lysis solution. After the obtained lysate was centrifuged, the extract (500 μg in terms of protein) was incubated overnight at 4 ° C. in the presence of anti-GluR1 subunit antibody (Upstate Biotechnology) or anti-GluR2 subunit antibody (Chemicon). 20 μl of protein G agarose (GE healthcare) was then added to the extract and incubated at 4 ° C. overnight. The pellet was washed twice with PBS and dissolved with 40 μl of SDS sample buffer (0.2 mM Tris-HCl, 0.04% SDS, 20% glycerol, pH 6.8). After boiling for 5 minutes, the proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The transcribed membrane was blocked with 5% BSA, reacted with an antibody against GluR1 subunit (Upstate Biotechnology), an antibody against GluR2 subunit (Chemicon) and an antibody against GluR3 subunit (Chemicon), and then HRP-conjugated goat anti-goat Reaction was performed with IgG antibody or goat anti-mouse IgG antibody. Immunoreactivity was detected with ECL kit (GE Healthcare).
The composition of the dissolving solution is as follows.
Dissolution solution : 0.1% Tween20, 0.1% SDS, 20 mM Tris-HCl, 150 mM NaCl and 1% protease inhibitor cocktail

10. Statistical analysis Statistical analysis was performed using unpaired t-test, Fisher's Protected Least Significant Difference (PLSD) test and one-way analysis of variance (ANOVA).

Example 1: Role of CaMKII in hippocampal synaptic transmission promoting action of DCP-LA DCP-LA (100 nM) has been reported to induce a transient surge in the slope of fEPSP recorded from the CA1 region of rat hippocampal slices. (Yaguchi, T. et al., Mol. Brain Res. 133: 320-324, 2005). Following a sustained increase of about 200%, a maximum of about 900% of the reference level is reached (FIG. 1). The transient increase in fEPSP slope by DCP-LA was only partially inhibited by the PKC inhibitor GF109203X (100 nM), but the sustained increase was inhibited by this inhibitor (FIG. 1). KN-93 (3 μM), an inhibitor of activated CaMKII, suppresses a transient increase in fEPSP slope due to DCP-LA to about 200% of the reference level, and further adds fEPSP by adding GF109203X (100 nM). The increase in slope was completely inhibited (FIG. 1).
From these results, it was shown that the hippocampal synaptic transmission promoting action induced by DCP-LA is mediated by both CaMKII and PKC, and CaMKII contributes greatly.

Example 2: Effect of DCP-LA on CaMKII Since it was revealed in Example 1 that CaMKII greatly contributed to the action of DCP-LA to promote hippocampal synaptic transmission, next, DCP-LA was CaMKII itself. It was investigated whether it was activated.
CaMKII activity was measured using reverse phase HPLC. In the experimental system using cultured rat hippocampal neurons, the reference value for CaMKII activity was 7.2 ± 0.3 pmol / min / μg protein (FIG. 2). DCP-LA enhanced CaMKII activity in a concentration (0.01-1 μM) dependent manner. The promoting effect of DCP-LA on CaMKII activity was markedly inhibited by KN-93 (3 μM), an inhibitor of active CaMKII, but was inhibited by KN-92 (3 μM), an inhibitor of inactive CaMKII. Not (Figure 2). From this result, it was shown that DCP-LA can activate CaMKII.
Next, it was examined whether DCP-LA promotes CaMKII activity in a cell-free system. Surprisingly, no enhancement of CaMKII activity by DCP-LA (100 μM) was observed in the cell-free system (FIG. 3A). From this result, it was considered that DCP-LA does not directly activate CaMKII, but activates CaMKII by acting on factors present in cells.
CaMKII is activated through its autophosphorylation and inactivated by PP-1 catalyzed dephosphorylation. Therefore, the effect of DCP-LA on PP-1 was examined.
In a cell-free PP-1 assay, the inhibitor of PP-1 microcystin (0.1 and 1 μM) significantly reduced PP-1 activity (FIG. 3B). DCP-LA also decreased PP-1 activity significantly depending on the concentration (1-100 μM), although its effect was weaker than microcystin (FIG. 3B).
These results suggest a mechanism of action that DCP-LA activates CaMKII by inhibiting PP-1.

Example 3: Effect of DCP-LA on AMPA receptor responsiveness CaMKII is known to enhance AMPA receptor responsiveness. Thus, the effect of DCP-LA on AMPA receptor response in oocytes expressing GluR1, GluR2 and GluR3 subunits in various combinations was examined.
In the two-electrode voltage clamp measurement, a potential of −60 mV is temporarily obtained by adding kainate, which is an AMPA receptor agonist (FIGS. 4A to I). DCP-LA (100 nM) is a step-up and sustained current potentiating action on AMPA receptors composed of GluR1, GluR3, GluR1 / GluR2, GluR1 / GluR3 and GluR1 / GluR2 / GluR3 subunits. Induced. After treatment with DCP-LA for 10 minutes, at the time when 70 minutes passed, the reference levels reached 193 ± 19%, 157 ± 10%, 136 ± 8%, 134 ± 6%, and 141 ± 7%, respectively. Both were markedly inhibited by KN-93 (3 μM), an inhibitor of active CaMKII (P <0.001; compared to the effect of DCP-LA in the absence of KN-93; Fisher's PLSD test (FIGS. 4A, C, D, F, G). In contrast, for AMPA receptors composed of GluR2 and GluR2 / GluR3 subunits, DCP-LA (100 nM) showed little or no potentiation (FIG. 4B, E).
These results suggest that the GluR1 subunit is required for the enhancement of AMPA receptor responsiveness induced by DCP-LA, and that the enhancement is CaMKII-dependent.
The GluR1 subunit contains a Ser ⁸³¹ CaMKII phosphorylation site. In order to determine whether the enhancing effect of DCP-LA on AMPA receptor responsiveness is due to phosphorylation of GluR1 subunit by CaMKII, a mutant GluR1 AMPA receptor lacking the CaMKII phosphorylation site [MGluR1 (S831A) receptor] was expressed in oocytes. DCP-LA (100 nM) also enhanced the current induced by kainic acid against the mGluR1 (S831A) receptor (FIG. 4I). This result suggests that DCP-LA enhances AMPA receptor responsiveness by an action mechanism independent of phosphorylation of GluR1 subunit by CaMKII.

Example 4: Effect of DCP-LA on AMPA receptor exocytosis Based on the above results, CaMKII activated by DCP-LA has an effect of enhancing AMPA receptor responsiveness induced by DCLP-LA. It was thought that it contributed to the increase in the expression on the cell membrane of the AMPA receptor which bears. Therefore, the effect of DCP-LA on the expression of AMPA receptor on the cell membrane was examined.
Lysates obtained from rat hippocampal slices were immunoprecipitated with anti-GluR1 subunit antibody or anti-GluR2 subunit antibody, and Western blot analysis was performed using immunoprecipitates (GluR1 IP, GluR2 IP). In any of the immunoprecipitates, a signal that was immunoresponsive to the anti-GluR1 subunit antibody and the anti-GluR2 subunit antibody was detected, but no signal was observed for the anti-GluR3 subunit antibody ( FIG. 5A). This result suggests that the GluR1 and GluR2 subunits form AMPA receptors expressed in the rat hippocampus. Therefore, the present inventors focused on the GluR1 and GluR2 subunits in order to investigate the effect of DCP-LA on AMPA receptor transport.
Lysates prepared from rat hippocampal slices were separated into two fractions, a pellet and a supernatant, by ultracentrifugation. Immunoreactive signals against an antibody against the cytoplasmic marker LDH (anti-LDH antibody) and an antibody against the cell membrane marker cadherin (anti-cadherin antibody) were detected in the supernatant and the pellet, respectively (FIG. 5B). This result suggests that the supernatant fraction and the pellet fraction are composed of a cytoplasmic component and a fine membrane component, respectively. In lysates obtained from hippocampal slices not treated with DCP-LA, comparable immune response signals to GluR1 and GluR2 subunits were observed in both the cytoplasmic and cell membrane fractions (FIG. 5C). , D). Lysates obtained from DCP-LA treated hippocampal slices enhanced the signal in the cell membrane fraction in a bell-shaped manner (1 nM-1 μM) dependent manner (FIGS. 5C, D). This indicates that DCP-LA increases the expression of GluR1 and GluR2 subunits on the cell membrane.
The effect of DCP-LA was inhibited by KN-93 (3 μM), an inhibitor of active CaMKII, but GF109203X (100 nM) and H-89 (1 μM), inhibitors of protein kinase A (PKA). Was not inhibited (FIGS. 5C and D). This indicates that DCP-LA increases the expression of the GluR1 and GluR2 subunits on the plasma membrane by inhibiting endocytosis from the plasma membrane to the cytoplasm via the CaMKII pathway. ing.
Furthermore, the increased expression on the cell membrane of GluR1 and GluR2 subunits induced by DCP-LA (100 nM) was inhibited by BoTX-A (0.1 U / ml), an inhibitor of exocytosis, and endocytosis. PAO (3 μM), which is an inhibitor of NO, did not show any effect (FIGS. 5C and D).
From the above results, DCP-LA stimulates exocytosis of GluR1 and GluR2 subunits by activating CaMKII, thereby increasing the expression of AMPA receptor on the cell membrane. Became clear.

  By inhibiting PP-1 and activating CaMKII, the transition of AMPA receptor to the cell membrane can be promoted, and hippocampal synaptic transmission accompanying the increase in the number of AMPA receptor expression on the cell membrane can be promoted. This agent leads to the development of a preventive / therapeutic agent for diseases (for example, neurodegenerative diseases) that have a mechanism of action different from that of existing drugs and that are beneficial to promote hippocampal synaptic transmission. Furthermore, the agent of the present invention can be provided as a research reagent that can be a useful tool for developing a preventive / therapeutic agent for such diseases.

Claims (4)

A protein phosphatase-1 (PP-1) inhibitor comprising 8- [2- (2-pentyl-cyclopropylmethyl) -cyclopropyl] -octanoic acid or a salt thereof. The agent according to claim 1, which is a research reagent. An activator of Ca ²⁺ / calmodulin-dependent protein kinase II (CaMKII), comprising the agent according to claim 1 or 2 . An expression enhancer for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, comprising the agent according to claim 3 .

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