KR101797010B1 - Composition for Improving Memory and Learning Abilities - Google Patents

Abstract

The present invention relates to a composition which induces nerve development in the adult hippocampus and is effective for improving cognitive ability including memory and learning ability, and more particularly, relates to a pharmaceutical composition for improving memory and learning ability using amodiaquine as an active ingredient.

Description

Composition for Improving Memory and Learning Abilities [

The present invention is to provide a composition effective in inducing neural development in the adult hippocampus to improve cognitive ability including memory and learning ability.

Neurogenesis is the process by which neurons are generated from neuronal precursor cells (NPCs). Adult mammalian brain cells are limited in number, and it has been postulated that no new neurons are generated after the developmental stage. However, many studies have shown that neurogenesis, ie neuronal generation and differentiation, develops during adulthood in the subventricular zone (SVZ) of the lateral ventricle and in the granulation zone (SGZ) of the hippocampal denatate gyrus . Neurogenesis in the adult hippocampus is associated with modulation of cognitive function. Evidence suggests that neonatal neurons induced by hippocampal NPC play a major role in long - term and short - term spatial memory as well as object recognition memory. Memory in psychology means that the change by learning is long lasting, and learning and memory are inseparable from each other, and the result of learning is measured through quantity and quality of memory. Therefore, the increase of nerve development by hippocampal stimulation by NPC is considered as a feasible strategy to increase cognitive function including learning and memory.

Studies on the improvement of cognitive function with the increase of the elderly population have been mainly approached in terms of treating diseases treating neurodegeneration caused by degenerative neurological diseases. Neurodegeneration is a progressive disease in which neurons in the central nervous system undergo degeneration due to the accumulation of abnormal protein aggregates and fall into dysfunction or cell death. Targeting to inhibit the formation of abnormal protein aggregates for the treatment of these diseases.

However, even in the case of the general public who do not have diseases such as neurodegeneration as well as the disease, in order to live in the modern society according to diversification of the social environment, prompt brain activities are required to accommodate many stimuli. Learning ability is directly related to intellectual activity ability and ultimately affects quality of life. In addition, modern medicine uses cholinester-raising inhibitor as a memory enhancer for learning retarded children whose intellectual ability related to memory is low due to inherent intellectual ability related to memory, but side effects such as dizziness, tremor, vomiting and liver toxicity are too severe, However, its application is limited. In addition, as the entrance examination competition becomes more intense, students who need long-term learning activities are more effective in improving memory and learning ability, as well as a drug that is used as a treatment for attention deficit hyperactivity disorder Good medicine ". Accordingly, there is a demand for a composition for improving memory and learning ability which can be used more safely.

Nurr1 (or Nr4a2) is a nuclear receptor that acts as an intracellular transcription factor and plays an important role in the development of NPCs. Previous studies involving the Nurr1 gene have shown that Nurr1 plays an important role in the proliferation and differentiation of NPCs in embryonic stem cells, embryonic olfactory neural stem cells and rat midbrain, striatum, cortex, external ganglion, SVZ and white matter in rats Show the charge. In vivo, Nurr1-engineered NPCs also showed increased neuronal differentiation in the striatum, while Nurr1 knockout mice showed decreased NPC neuronal differentiation in the bovine midbrain, including the black body and the ventral side. Previous studies have focused on the role of Nurr1 in promoting neurogenesis from dopaminergic precursors and the inhibition and treatment of Parkinsonism using it (Patent No. 10-2008-0106928). However, there has been no study on the expression of Nurr1 in the adult hippocampus as well as the influence of Nurr1 on the generation of neurons from the NPC of adult hippocampus.

Amodiaquine (AQ, amodiaquine) is an anti-malarial and anti-inflammatory drug that is characterized by low price, low toxicity, and rapid onset at around US $ 0.05 per wholesale price per dose in 2014. Nurr1 has been regarded as an orphan nuclear receptor, but very recent studies have reported that the anti-malaria agent amodiazin is an optional, potent agent that stimulates the transcriptional function of Nurr1 by direct interaction with the ligand binding domain of Nurr1 . However, as described above, the influence of Nurr1 on the generation of neurons from the NPC of adult hippocampus has not been studied, and the effect of amoediraine on neuronal development of the adult hippocampus is not known.

Patent Document 10-2008-0106928

In accordance with the need for a composition for improving the cognitive function as described above, the present invention is not limited to cognitive dysfunction caused by neurodegeneration such as degenerative neuropathy, but is effective for improvement of memory capacity and learning ability of general adults, And to provide the above objects.

In order to achieve the above object, the present invention relates to a pharmaceutical composition for improving memory performance and learning ability using amodiquin as an active ingredient.

The present invention relates to a composition exhibiting an effect of enhancing memory and learning ability by increasing nerve development in the hippocampus of an adult. This is different from the cognitive dysfunction caused by neurodegeneration such as degenerative neurological diseases such as dementia, and its functional group is of course completely different. In the present invention, Nurr1 is expressed in adult hippocampal neuronal progenitor cells (NPC) through the following examples. Amodiaquine stimulates phosphorylation of ERK1 / 2 and AKT during ERK1 / 2 and AKT signaling to activate Nurr1 To promote nerve development in the hippocampus. This improves cognitive function, including memory and learning ability, by promoting neurogenesis in the normal hippocampus of a normal adult by an entirely different mechanism from that of eliminating or inhibiting the pathogenesis of the degenerative neurological disease resulting in cognitive dysfunction.

It is known that hippocampal neurogenesis is closely related to memory and learning ability enhancement. In addition to in vitro test results, amiodai queen is effective in improving memory and learning ability through Y-maze test and new object search test .

Amodiaquin is widely used as an anti-malarial drug and is being marketed as Flavoquine, Camoquin and Basoquin. However, the formulations are not limited thereto and may be prepared by mixing the active ingredient with a pharmaceutically acceptable carrier, a forming agent, a diluent or the like by a known method in the pharmaceutical field to prepare a powder, granule, tablet, And the like.

The dose of the active ingredient according to the present invention is appropriately selected according to the degree of absorption of the active ingredient in the body, the rate of water activation and the rate of excretion, the age, the sex and the condition, etc. Generally, To 100 mg per one or several divided doses.

The present invention also relates to a health functional food composition for improving memory performance and learning ability using amodiquinone as an active ingredient. Amodiaquine is added to the health functional food composition of the present invention so that it can be administered to an adult in an amount of 0.1 to 100 mg per kg of body weight per day per day to be administered to a health functional food composition such as tablets, capsules, pills, Can be used for manufacturing.

As described above, according to the composition of the present invention, toxicity is very weak and economical, and since it induces the nerve development of the adult hippocampus and has excellent effect on the control of the cognitive function, it is possible to improve the memory ability and learning ability of the examinee or the worker Can be usefully used as a composition.

In addition, the present invention can be usefully used to improve the cognitive function of a child with learning disabilities in place of a conventional therapeutic agent, which is required to be treated due to serious side effects but whose use is limited.

Figure 1 shows the results of RT-PCR, Western blot and immunocytochemical analysis showing the expression of Nurr1 mRNA or protein in adult hippocampal NPCs.
FIG. 2 is a graph showing the effect of amodiquine on cell proliferation of adult hippocampal NPCs.
FIG. 3 is a graph showing changes in the expression level during ERK1 / 2 and AKT signaling by treatment with amodimethine.
Figure 4 is a graph and image showing immunohistochemical analysis results for Ki67 in hippocampal DG.
FIG. 5 is a graph and image showing immunohistochemical analysis results for DCX in hippocampal DG.
6 is a graph showing the results of the Y-maze test.
Figure 7 is a graph showing the NORT results.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these embodiments are merely examples for explaining the content and scope of the technical idea of the present invention, and thus the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

[Example]

Example 1: Expression of Nurr1 in Adult Hippocampal Neuronal Progenitor Cells (NPCs)

Although expression of Nurr1 has been reported in several brain regions and NPCs in rodents, the expression of Nurr1 in adult hippocampal NPCs has never been confirmed. Thus, the level of mRNA and protein expression of Nurr1 in hippocampal NPCs of adult animals was measured by RT-PCR, Western blot and immunocytochemistry. The rodent brain was known to express large amounts of Nurr1, and the brain of rats was used as a positive control for Nurr1 detection.

For this purpose, hippocampal NPC (rAHP) of adult rat isolated from the brain of adult Fisher 344 rats was purchased from Chemicon (Catalog No. SCR022, Billerica, MA, USA). The NPC was supplemented with Dulbecco's modified Eagles's medium (DMEM) / F12 medium (Gibco / Invitrogen) supplemented with B27 supplement, L-glutamine, 1 x penicillin solution, fungizone, streptomycin and basic FGF (bFGF, 20 ng / , Carlsbad, Calif.). Tissue culture glass or plastic products used to culture hippocampal NPCs were coated with laminin (5 μg / ml) and poly-L-ornithine (10 μg / ml). Cells were cultured in a humidified incubator at 37 ° C, 5% CO ₂ , and subcultured every 3 to 4 days.

The expression of Nurr1 m-RNA or protein was confirmed by RT-PCR, Western blotting and immunocytochemical methods, and the results are shown in Fig. Specific experimental methods are as follows.

1) RT-PCR

Total RNA from cortical neurons was extracted using Qiagen RNeasy Mini Kit (Qiagen Inc.) according to the manufacturer's manual and reverse transcribed with Superscript II reverse transcriptase (Life Technologies, Inc.). In order to confirm the contamination of the genomic DNA, all reverse transcription reagents except the reverse transcriptase were mixed and cultured as a negative control. In order to confirm the expression of Nurr1 in the hippocampus NPC of adult mouse, the reverse transcribed cDNA was amplified with GeneAmp PCR System StepOnePlus (Applied Biosystems, Helios, Singapore) using primers of SEQ ID NO: 1 and SEQ ID NO: 2 specific to Nurr1. Amplification conditions were as follows: 95 캜, 5 min; 95 C, 30 sec, 38 cycles; 60 캜, 1 min; 72 ° C, 1 minute.

SEQ ID NO: 1: 5'-GAC ACT TCA CAA CTT CCA CCA GAA CT-3 '

SEQ ID NO: 2: 5'-ACT GCG ATG CGT GGC CGA TCT GC-3 '

2) Western blot

Cells were incubated with 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 140 mM NaCl, 1% (w / v) Nonidet P-40, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF and 10 μg / ml aprotinin Lt; / RTI > buffer. Cell lysates were separated by 12% SDS-PAGE and electrophoresed on PVDF (polyvinylidene difluoride) membrane (Bio-Rad). The PVDF membrane was immersed in blocking buffer (1 × Tris-buffered saline, 1% BSA, 1% nonfat dry milk) for 1 hour and then incubated with primary antibody against Nurr1 (Santa Cruz Biotechnology; 1: 500) at 4 ° C . Blots were developed with anti-rabbit IgG-conjugated peroxidase and chemiluminescent detection system (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Bands were visualized with a ChemicDoc XRS system (Bio-Rad) and quantified using Quantity One imaging software (Bio-Rad).

3) Immunocytochemistry

Adult rat hippocampal NPCs were fixed with 4% paraformaldehyde (Sigma) in PBS for 10 minutes at room temperature. After blocking with 3% normal goat serum (Vector Laboratories, Burlingame, CA, USA) and 1% BSA (Sigma), slides were incubated overnight at 4 ° C with primary antibody against Nurr1 (Santa Cruz Biotechnology, Respectively. After washing, the slide was incubated with Cy3-goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA, USA, 1: 400) for 4 hours at room temperature. Cells were cross-stained with 4-6-diamidino-2-phenylindole (DAPI) and images were observed with a confocal microscope (Carl Zeiss LSM 700 Meta, Oberkochen, Germany).

1 is an image showing the expression level of Nurr1 mRNA by RT-PCR, and B and C are images showing Western blot and immunocytochemical results showing the expression level of Nurr1 protein, respectively. From FIG. 1, it was confirmed that Nurr1 mRNA and Nurr1 protein were expressed at a level similar to that in rat brain in not only rat brain but also hippocampal NPC of adult rat.

Example 2: Confirmation of proliferative stimulation of adult rat hippocampal NPC by amodimyquin

In order to evaluate the effect of Nurr1 on the proliferation of adult hippocampal NPC, Amodiaquin, a Nurr1 agonist, was treated in hippocampal NPC of adult rats and the changes of cell proliferation were confirmed.

(CCK-8) method (Enzo life science Icn.Lausen, Switzerland) was used to evaluate the effect of amodiazin (Sigma-Aldrich, St. Louis, USA) on cell proliferation. The proliferation index of each group was determined according to the manual. Briefly, growth-indexed cells were seeded in 24-well plates at a concentration of 3 × 10 ⁵ cells / well and treated with amodiquine at 0, 10, 100, or 1,000 nm to obtain F12 / And cultured in DMEM (500 μL). Subsequently, 20 μL of CCK-8 solution was added to each well containing 200 μL of medium, incubated at 37 ° C. for 2 hours, and absorbance of each group at 450 nm was measured with an absorbance microplate reader (Molecular Devices LLC, CA, USA) Were used as an indicator of cell proliferation.

FIG. 2 is a graph showing the results. It shows that the treatment of amoediraine significantly increased the proliferation of hippocampal NPCs in adult rats. The degree of cell proliferation increased significantly with the passage of time after treatment, but did not show any dependence on the amount of treatment. This is the first result that demonstrates that Amodiaquin stimulates proliferation of adult hippocampal NPCs.

To confirm the mechanism of NPC proliferative stimulation by Nurr1 activation, the signaling process activated during NPC proliferation by Nurr1 agonist was confirmed. For this, expression of ERK1 / 2 and AKT was measured by Western blotting and the results are shown in Fig. FIG. 3 shows that phosphorylation of ERK1 / 2 and AKT is activated in adult hippocampal NPC by treatment with amodimethine, indicating that the proliferation of adult hippocampal NPC by amodimethyne is increased by ERK1 / 2 and AKT signaling It suggests.

Example 3: Animal experiment

1) Assessment of the effect of amoediraine on adult hippocampal nerve development

Immunohistochemical analysis of Ki67, a marker of adult neurogenesis, and DCX (doublecortin), a marker of adult neural differentiation in mouse hippocampus was performed through animal experiments.

Male C57BL / 6 mice at 8 weeks of age were purchased from Koatech (Pyeongtaek, South Korea) and purified for 1 week before the experiment. All animals were raised according to the guidelines for the protection and use of laboratory animals. The experimental procedure was approved and verified by the protocol of the Experimental Animal Management Committee of. Amiodiquin diluted in 0.9% saline was intraperitoneally injected at a concentration of 20 mg / kg for 12 days twice a day for 12 hours. Two weeks after the injection of Amodiaquin, the mice were sacrificed and the brain was harvested. Six sections from both hemispheres per animal were analyzed by blind analysis.

Brain sections were washed with PBS and treated with 1% hydrogen peroxide for 15 minutes. The sections were incubated overnight at 4 ° C with mouse anti-Ki67 antibody (1: 500, abcam, Cambridge, England) or goat anti-doublecortin (DCX) antibody (1: 1000, Santa Cruz Biotechnology, Texas, USA). Subsequently, the sections were incubated with biotinylated horse anti-mouse IgG or biotinylated horse anti-goat IgG (1: 200, VECTOR, Burlingame, USA) and avidin-biotin-peroxidase complex solution and SIGMA FASTTM 3.3'-Diaminobenzidine (Sigma-Aldrich, St. Louis, USA). Images were processed with Image-Pro Plus 6.0 program (Media Cybernetics, Bethesda, USA) to quantify Ki67 and DCX positive cells in dentate gyrus (DG) of the hippocampus.

FIG. 4 is a graph and an image showing immunohistochemical analysis of Ki67 in hippocampal DG, showing that Ki67-positive cells were significantly increased in the amodiazene-treated group compared with the control group treated with saline. This suggests that the activation of Nurr1 by amodiquine increases the proliferation of adult rat hippocampal NPCs.

In FIG. 5 showing the results of immunohistochemistry for DCX in hippocampal DG, DCX-positive cells were significantly increased in the amodiazene-treated group compared to the control group treated with saline, And the NPC differentiation also increased.

Taken together, these results confirm that the administration of amoediraine increases neurogenesis in the adult hippocampus.

2) Y-Maze Test (Y-Maze Test)

Improvement in cognitive memory of mice treated with amodiquine by the method described in 1) was measured by the Y-maze test after two weeks of Amodiaquin injection. As a control group, saline was injected instead of amodiaquine.

The Y-maze equipment consists of three branches (30 cm long, 8 cm wide and 15 cm high) arranged at an angle of 120 ° to the central space (8 × 8 cm). Each mouse was placed on one branch and allowed to move freely for 5 minutes to evaluate the spontaneous rate of change. The spontaneous rate of change is defined as the number of times that the system goes into three different branches without repeats (ie, ABC, BCA, etc., not ABA).

Voluntary change rate (%) = [successive entries / (total arm entries - 2) × 100]

FIG. 6 is a graph showing the results of the Y-maze test, showing that the spontaneous rate of change was significantly increased, although there was no significant difference in the number of times to enter the whole branch. This means that amodiakune improves cognitive memory without affecting motor function.

3) Novel object recognition test (NORT)

The cognitive memory improvement of the mice treated with amodiquine by the method described in 1) was measured by NORT in an open field box (45 x 45 x 45 cm) two weeks after Amodiaquin injection. As a control group, saline was injected instead of amodiaquine.

Prior to the test, the mice were allowed to acclimate for 5 minutes in a test box with no items for 3 consecutive days. After acclimation, the mice were placed in a test box and two identical objects were searched for 3 minutes (familiarization session). The objects used in this study were wooden blocks with different shapes of the same size. Familiarization session After 24 hours, one familiarized object and one new object were searched for 3 minutes (test session). All sessions were recorded on a video tracking system (EthoVision XT 10.0, Noldus Information Technology, Wageningen, Netherlands) and analyzed for each session the time it took for the mouse to navigate and recognize each item. The recognition time of an object means the time to face, smell, bite, or stay within 2cm. The test result (identification index) is expressed as a ratio of recognition time for new objects, and is calculated by the following equation.

Identification index = t _novel / (t _novel + t _familiar ) × 100

FIG. 7 shows the result of NORT, where A is the identification index of the familiar session, B is the identification index of the test session, and C is the result of detection of the trace in the test session by Ethovision software. The identification index of the familiarization session is expressed as the time to recognize two identical objects in the whole time.

As shown in FIG. 7, there was no significant difference between the two groups in the familiarization session, but the identification index increased significantly in the test session.

Overall, these results show that Amodiaquin enhances cognitive function in adult rats by increasing hippocampal neurogenesis.

<110> KONYANG UNIVERSITY INDUSTRIAL COOPERATION GROUP <120> Composition for Improving Memory and Learning Abilities <130> P0716-376 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gacacttcac aacttccacc agaact 26 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 actgcgatgc gtggccgatc tgc 23

Claims (5)

A pharmaceutical composition for enhancing memory and learning ability of a normal person, not a cognitive dysfunction caused by a degenerative neurological disease comprising Amodiaquin as an active ingredient.
The method according to claim 1,
Wherein the composition enhances nerve development of the hippocampus, and is not a cognitive dysfunction caused by a neurodegenerative disease but enhances memory ability and learning ability of a normal person.
3. The method of claim 2,
Wherein said neurogenesis is caused by activation of Nurr1 in a hippocampal neuronal precursor cell (NPC), which is not a cognitive dysfunction caused by degenerative neurological disease, but which enhances memory performance and learning ability of a normal person.
A health functional food composition for enhancing memory and learning ability of a normal person, not a cognitive dysfunction due to degenerative neurological disease comprising Amodiaquin as an active ingredient.
5. The method of claim 4,
Wherein the composition enhances nerve development of the hippocampus, and is a cognitive dysfunction caused by a degenerative neurological disease.

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