Pharmaceutical composition for treating Alzheimer's disease and application thereof

Abstract

The invention relates to a pharmaceutical composition for treating Alzheimer's disease and application thereof. The present invention provides a pharmaceutical composition for the treatment of Alzheimer's disease, comprising: a compound selected from the group consisting of: mironafil, sildenafil, vardenafil, tadalafil, udenafil, dabigatafil, and avanafil; and pharmaceutically acceptable salts, solvates and hydrates thereof. The invention also provides application of the pharmaceutical composition in preparation of medicines for treating Alzheimer's disease, and the pharmaceutical composition is used for reducing beta amyloid protein formation and treating diseases related to beta amyloid protein accumulation.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of March 24, 2020, application number 202080038090.7, and invention name “Compositions and methods for reducing the formation of β-amyloid protein and their compositions”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 822,975, filed on March 24, 2019, the contents of which are incorporated herein by reference. Technical Field

[0004] The present invention relates to methods for reducing beta-amyloid formation and for treating diseases associated with the accumulation of beta-amyloid. Background Art

[0005] Dementia refers to a clinical disease with multiple cognitive impairments (or MCD, multiple cognitive deficits), which is an acquired brain disease that exhibits a multifaceted pathogenesis caused by multiple genetic and environmental risk factors. The representative of the disease that causes dementia is Alzheimer's disease, which is mainly prevalent in the elderly and accounts for 60-70% of all dementias (Ann Neurol. 1993 May; 33 (5): 494-501.). With the rapid aging of the population recently, more progressive dementia in the elderly caused by Alzheimer's disease has increased, and the demand for the treatment of dementia has also increased. However, the difficulties in research and development have reduced the development process of new therapeutic agents and treatment methods for dementia under the additional low success rate. Based on the GBI research data in 2014, the success rate of the final approval of therapeutic agents for Alzheimer's disease is less than 1%, such as 72% in clinical phase II, 92% in clinical phase III, and 99.6% of failed NDA applications, and about 80% of the pipelines currently underway are only for "exploratory and / or preclinical".

[0006] The current general list of FDA-approved drugs includes AChE (acetylcholinesterase) inhibitors and NMDA (N-methyl-D-aspartate) receptor antagonists, and is also used in combination with antioxidants, NSAIDs (non-steroidal anti-inflammatory drugs), anti-inflammatory agents, cholesterol synthase inhibitor preparations or hormone preparations, etc. However, these drugs are only used to relieve symptoms and delay or improve cognitive impairment, but there is currently no fundamental treatment for dementia.

[0007] Representative AChE inhibitors include Donepezil (Aricept TM ), Galantamine (Reminyl TM)、Rivastigmine (ENA-713, Exelon TM ) and the like, and these drugs temporarily increase the concentration of the neurotransmitter acetylcholine to provide symptomatic treatment. In addition, these drugs are prescribed for patients with mild to moderate Alzheimer's disease, vascular dementia, Parkinson's disease dementia, and stroke or subcortical ischemic vascular disease (J Korean Med Assoc 2009; 52(4):417-425.).

[0008] Typical NMDA receptor antagonists include memantine (Ebixa TM ), which causes excitotoxicity to inhibit the glutamate transport system, which blocks synaptic plasticity to effectively reduce neuronal degeneration. Memantine has been shown to be effective for moderate or above dementia and Lewy body dementia, with relatively low side effects, but only shows limited activity in the earlier stages of the disease (Arch Neurol 2011 Aug; 68(8): 991-8). Therefore, compared with monotherapy, it is more often used with ChE inhibitors and is used for mild and severe dementia. However, so far, no clear evidence has been found that the combination of AChE inhibitors and memantine is more effective than AChE alone, and additional research is needed (Brain Neurorehabil 2015 Mar; 8(1): 19-23.).

[0009] However, although these drugs have long been used to treat dementia and Alzheimer's disease, there are no clear criteria for use, and furthermore, there are ongoing concerns about the use of these drugs as even at the maximum recommended human dose (MRHD), there is no improvement in the disease or continued disease progression leading to terminal disease in treated patients. However, until now, in the absence of any therapeutic agents with confirmed efficacy, there has been no alternative but to continue efforts to develop new treatments for dementia (Korean J Biol Psychiatry 2016 May; 23(2): 48-56.).

[0010] To date, the main targets of candidates developed for the treatment of dementia have included: (1) inhibitors of BACE-1 (β-secretase 1) or γ-secretase inhibitors to inhibit the production of Aβ (β-amyloid), (2) anti-Aβ monoclonal antibodies to remove Aβ (β-amyloid), (3) Tau aggregation inhibitors and TAOK inhibitors (an amino acid kinase) to inhibit Tau aggregation and phosphorylation, (4) AchE inhibitors and NMDA receptor antagonists to block AChE and NMDA receptors, etc. Recently, the pipeline targeting Aβ plaques and Tau proteins has increased, with approximately 70-80% of the candidates targeting the inhibition of Aβ production or the removal of Aβ. Additionally, approximately 30% of the pipeline consists of biopharmaceuticals with a predominately high ratio of monoclonal antibodies or peptides. However, due to the failure of clinical trials of candidates with high expectations recently, including anti-Aβ monoclonal antibodies targeting Aβ for treatment, such as Aducanumab (Biogen), Solanezumab (Eli Lilly), and Gantenerumab (Roche), the hypothesis of Aβ as a target for treating dementia has become uncertain. However, it is generally accepted that the key goal in developing a therapeutic agent for dementia is to inhibit Aβ formation and remove Aβ.

[0011] One of the problems in the development of Aβ-targeted drugs is that monoclonal antibodies with large molecular weights are difficult to penetrate cell membranes and thus can only effectively remove extracellular amyloid plaques and have only limited efficacy in removing intracellular Aβ oligomers. In addition, it has been reported that intracellular Aβ oligomers cause more cytotoxicity than extracellular Aβ oligomers and play a more important role in neuronal cell death (J Biol Chem. 2002 May 3; 277(18): 15666-70.). Therefore, small molecule drugs that can penetrate cell membranes better than macromolecules are developed, such as monoclonal antibodies for removing intracellular Aβ oligomers.

[0012] One of the viewpoints that has recently attracted attention in the field is that the guiding reason for the failure to develop therapeutic agents for dementia may be the conventional approach, the "one drug, one target" paradigm, because clinical impairment dementia is a disease with multifaceted pathogenesis. It is accepted that this traditional approach cannot achieve the successful development of therapeutic agents for dementia. Currently, there is increasing interest in developing new therapeutic agents based on the "one drug, multiple targets / mechanisms" paradigm to overcome the problems mentioned above. Approaches such as a compound with multi-target / multi-mechanism activity have become a new concept for new drug discovery and development (J Neural Transm (Vienna). 2013 Jun; 120 (6): 893-902., Future Med Chem. 2016 Apr; 8 (6): 697-711.). Therefore, in order to overcome the difficulties in developing therapeutic agents for dementia and increase the success rate of development, compared with the traditional "one drug, one target" paradigm, it is necessary to develop small molecule / multi-directional pharmacological drugs based on the "one drug, multiple targets / mechanisms" paradigm starting from candidate discovery and in the preclinical stage, especially considering the multifaceted pathogenesis of dementia (Expert Rev Clin Pharmacol 2013 Jan; 6(1):.10.1586 / ecp.12.74., Curr Pharm Des. 2016; 22(21):3171-81.). Summary of the invention

[0013] One embodiment of the present invention provides a method for inhibiting the formation of Aβ fibrils / plaques by removing toxic soluble Aβ oligomers in cells through administration of one of the following compounds: Mirodenafil, Sildenafil, Vardenafil, Tadalafil, Udenafil, Dasantafil and Avanafil; and pharmaceutically acceptable salts, solvates or hydrates thereof.

[0014] The method of the present invention provides, in one embodiment, the following: (1) inhibiting the formation of Aβ oligomers / fibrils by inhibiting Aβ aggregation; (2) inhibiting the amyloidogenic processing of β-protein by reducing BACE-1; (3) reducing the formation of extracellular Aβ monomers, oligomers, Aβ fibrils / plaques by increasing cerebral blood flow (vasodilation); (4) inhibiting neuronal cell death and accelerating neurogenesis, synaptogenesis and / or promoting angiogenesis by activating NO (nitric oxide) / cGMP (cyclic guanosine monophosphate) / PKG (protein kinase G) and CREB (cyclic AMP (adenosine monophosphate) response element binding protein) pathways; (5) inhibiting the formation of Aβ oligomers / fibrils by reducing BACE-1; (6) inhibiting the formation of Aβ oligomers / fibrils by reducing BACE-1; (7) inhibiting the formation of Aβ oligomers / fibrils / plaques by increasing cerebral blood flow (vasodilation); (8) inhibiting the formation of Aβ oligomers / fibrils / plaques by increasing cerebral blood flow (vasodilation); (9) inhibiting the formation of Aβ oligomers / fibrils / plaques by increasing cerebral blood flow (vasodilation); (10) inhibiting the formation of Aβ oligomers / fibrils by reducing BACE-1; (11) inhibiting the formation of Aβ oligomers / fibrils by reducing BACE-1; (12) inhibiting the formation of Aβ oligomers / fibrils by reducing BACE-1; (13) inhibiting the formation of Aβ oligomers / fibrils by reducing BACE-1; (14) inhibiting the formation of Aβ oligomers / fibrils by reducing BACE-1; (15) inhibiting the formation of Aβ oligomers / fibrils by reducing BACE-1; (16) inhibiting the formation of Aβ oligomers / fibrils WNT signaling pathway inhibitors 1) inhibit Wint signaling to restore synaptic plasticity (synaptic plasticity), and inhibit APP (amyloid precursor protein) production and reduce Aβ accumulation by blocking the positive feedback loop of Aβ production, and (6) inhibit the formation of Aβ fibrils / plaques by removing intracellular toxic and soluble Aβ oligomers through activating autophagy.

[0015] In another embodiment, the present invention provides a pharmaceutical composition comprising as an active ingredient a compound selected from the group consisting of milonafil, sildenafil, vardenafil, tadalafil, udenafil, daxantafil and avanafil; and pharmaceutically acceptable salts, solvates and hydrates thereof, which is used for (1) inhibiting Aβ oligomer / fibril formation by reducing Aβ aggregation, (2) inhibiting the processing of β-amyloidogenic protein by reducing BACE-1, (3) reducing extracellular Aβ monomers, oligomers and protofibrils by increasing cerebral blood flow, and (4) inhibiting the formation of Aβ oligomers and protofibrils by reducing BACE-1. The invention discloses a novel novel cytosolic Aβ inhibitory agent that inhibits the formation of Aβ fibrils / plaques, (4) inhibits neuronal cell death inhibition and promotes neurogenesis, synaptogenesis and / or angiogenesis by activating the NO / cGMP / PKG / CREB pathway, (5) restores synaptic plasticity by activating Wint signaling through the inhibition of DKK-1, and inhibits the production of APP and reduces Aβ accumulation by inhibiting the positive feedback loop of Aβ production, and (6) inhibits the formation of Aβ fibrils / plaques by removing intracellular toxic and soluble Aβ oligomers through the activation of autophagy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] I. Inhibition of Aβ oligomer / fibril formation by reducing Aβ aggregation

[0017] Figure 1 Results of a Thioflavin T assay showing dose-dependent inhibition of A[beta] fibril aggregation by the compositions of the invention are presented.

[0018] Figure 2Results of PICUP (photoinduced cross-linking of unmodified proteins) / SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis showing that the formation of Aβ oligomer aggregates has been reduced by treatment with the composition of the present invention are presented.

[0019] II. Suppression of β-amyloidogenic processing by BACE-1 reduction

[0020] Figure 3 The results of qRT-PCR (quantitative real-time polymerase chain reaction) showing that BACE-1 mRNA levels in retinoic acid-differentiated SH-SY5Y cells were increased by treatment with Aβ1-42 oligomers, but indicating that the increased BACE-1 mRNA levels were concentration-dependently reduced by treatment with the composition of the present invention.

[0021] III. Reducing extracellular Aβ monomers, oligomers and Aβ fibrils / plaques by increasing cerebral blood flow

[0022] Figure 4 Results are presented showing that intracellular calcium levels were quantified by treatment with hydrogen peroxide (H 2 O 2 ) treatment with pericytes, which was increased by treatment with the composition of the present invention, has been reduced.

[0023] IV. Inhibition of neuronal cell death by activating the NO / cGMP / PKG / CREB pathway

[0024] Figure 5 The results of cyclic GMP Complete ELISA showing that the amount of cGMP in retinoic acid-differentiated SH-SY5Y cells was decreased by the treatment of Aβ1-42 oligomers, but increased by the treatment with the composition of the present invention in a concentration-dependent manner are presented.

[0025] Figure 6 The results of Western blot analysis showing that the reduction in the ratio of apoptosis-related proteins (Capase-3 and PARP [poly(adenosine diphosphate-ribose) polymerase]): cleaved Capase-3 / Caspase-3 and cleaved PARP and cleaved PARP / PARP, which were increased in retinoic acid-differentiated SH-SY5Y cells, was reduced by treatment with the composition of the present invention are presented.

[0026] Figure 7 Results of the JC-1 mitochondrial membrane potential assay are presented showing that the concentration of mitochondrial membrane potential in retinoic acid-differentiated SH-SY5Y cells, which was decreased by treatment with Aβ1-42 oligomers, has been concentration-dependently increased by treatment with the composition of the present invention.

[0027] Figure 8 presents the results showing the following Results of S3 live cell analysis: The number of dead cells stained with Cytotox Red reagent in retinoic acid-differentiated SH-SY5Y cells, which was increased by treatment with Aβ1-42 oligomers, was decreased by treatment with the composition of the present invention.

[0028] V. Promotion of neurogenesis, synaptogenesis, and angiogenesis by activating the NO / cGMP / PKG / CREB pathway

[0029] Fig. 9 presents the results of Western blot analysis showing the following: The phosphorylation of Ser133 of CREB protein in retinoic acid-differentiated SH-SY5Y cells, which was decreased by treatment with Aβ1-42 oligomers, was increased in a concentration-dependent manner by treatment with the composition of the present invention.

[0030] Fig.10 presents the results of Western blot analysis showing the following: The protein levels of NGF (nerve growth factor) and BDNF (brain-derived neurotrophic factor) in retinoic acid-differentiated SH-SY5Y cells, which were decreased by treatment with Aβ1-42 oligomers, were increased in a concentration-dependent manner by treatment with the composition of the present invention.

[0031] Fig.11 presents the results of NGF immunocytochemistry showing the following: The protein level of NGF (nerve growth factor) in retinoic acid-differentiated SH-SY5Y cells, which was decreased by treatment with Aβ1-42 oligomers, was increased by treatment with the composition of the present invention.

[0032] VI. Restoration of synaptic plasticity by activating Wnt signaling via DKK-1 inhibition

[0033] Fig.12 presents the results of qRT-PCR showing the following: The levels of DKK-1 mRNA in retinoic acid-differentiated SH-SY5Y cells and differentiated HT-22 mouse hippocampal neuronal cells, which were increased by treatment with Aβ1-42 oligomers, were decreased by treatment with the composition of the present invention.

[0034] Fig.13 presents the results of qRT-PCR showing the following: The protein levels of DKK-1 in retinoic acid-differentiated SH-SY5Y cells and differentiated HT-22 mouse hippocampal neuronal cells, which were increased by treatment with Aβ1-42 oligomers, were decreased by treatment with the composition of the present invention.

[0035] Fig.14Presented are the results of qRT-PCR showing that the level of Wnt3a mRNA in retinoic acid differentiated SH-SY5Y cells and differentiated HT-22 mouse hippocampal neuronal cells, first treated with Aβ1-42 oligomers, was increased by treatment with the composition of the present invention compared to the control group.

[0036] Fig.15 Results of Western blot analysis showing that decreased Wnt1 protein levels in the hippocampus of an Alzheimer's disease animal model (5XFAD transgenic mice) have been increased by treatment with the composition of the present invention are presented.

[0037] Fig.16 The results of a Wnt / β-catenin TF Array showing that the activities of Wnt / β-catenin-related factors, VAX2, c-Myc, NR5A2, Mitf, TCF / LEF, NFAT, CEBP, GLI-1, GBX2, and AP-1, in retinoic acid-differentiated SH-SY5Y cells and differentiated HT-22 mouse hippocampal neuronal cells, first treated with Aβ1-42 oligomers, were increased more than two-fold by treatment with the composition of the invention compared to the control group.

[0038] Fig.17 Results of a TOPFLASH reporter gene assay (Wnt / β-catenin signaling) are presented showing that Wnt / β-catenin activity in retinoic acid differentiated SH-SY5Y cells and differentiated HT-22 mouse hippocampal neuronal cells is reduced by treatment with Aβ1-42 oligomers and concentration-dependently increased by treatment with the composition of the invention.

[0039] Fig.18 Results of Western blot analysis showing that the Ser9 phosphorylation level of GSK3β protein in retinoic acid-differentiated SH-SY5Y cells, which was decreased by the treatment of Aβ1-42 oligomers, had been increased by the treatment with the composition of the present invention are presented.

[0040] Fig.19 Results of a phospho-GSK-3b (Ser9) sandwich ELISA showing that the Ser9 phosphorylation level of the GSK3β protein in retinoic acid differentiated SH-SY5Y cells, which was decreased by the treatment of Aβ1-42 oligomers, was increased by the treatment with the composition of the present invention are presented.

[0041] Fig. 20Results of Western blot analysis showing that the increased Ser9 phosphorylation level of Tau protein in the hippocampus of an Alzheimer's disease animal model (NSE-hAPP-C105) has been reduced by treatment with the composition of the present invention are presented.

[0042] VII. Reduction of APP formation and Aβ accumulation by suppressing the positive feedback loop of Aβ production via inhibition of DKK-1

[0043] Fig.21 Results of Western blot analysis showing that APP and Aβ1-42 protein levels in retinoic acid differentiated SH-SY5Y cells, which were increased by treatment with Aβ1-42 oligomers, were reduced by treatment with the composition of the invention are presented.

[0044] Fig. 22 The results of Western blot analysis showing the following: APP and Aβ1-42 protein levels in retinoic acid-differentiated SH-SY5Y cells, expressed by H 2 O 2 has been reduced by treatment with the composition of the present invention.

[0045] VIII. Inhibition of Aβ fibril / plaque formation by removing intracellular toxic and soluble Aβ oligomers via activation of autophagy

[0046] Fig.23 Results of Western blot analysis showing that increased Aβ protein levels in the hippocampus of an Alzheimer's disease animal model (NSE-hAPP-C105) have been reduced by treatment with the composition of the present invention are presented.

[0047] Fig.24 The results of Thioflavin S staining / GFAP (glial fibrillary acid protein) immunohistochemistry showing that the number of Aβ plaques in the hippocampus of an Alzheimer's disease animal model (NSE-hAPP-C105) has been reduced by treatment with the composition of the present invention. The number of Aβ plaques was counted by counting the number of Aβ plaques (green) stained by Thioflavin S and the number of co-localized points (yellow) of astrocytes (red) stained by GFAP.

[0048] Fig.25 Presented are the results of Western blot analysis showing that the Thr172 phosphorylation level of the AMPK catalytic subunit α in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells, which was reduced by the treatment of Aβ1-42 oligomers, had been concentration-dependently increased by the treatment with the composition of the present invention.

[0049] Fig.26The results of Western blot analysis showing the following are presented: the level of LC3B protein (an autophagy marker) in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells, which is reduced by the treatment of Aβ1-42 oligomers, has increased, while inducing autophagic flux (converted by LC3B-1 to LC3B-II) to increase the LC3B-II / I ratio, and also increasing the expression of ubiquitin-binding protein p62 (SQSTM, Sequestosome 1) (autophagosome cargo protein), which is reduced by the treatment of Aβ1-42 oligomers. In addition, 3-MA (3-methyladenine) (autophagy / PI3K inhibitor) is used in combination with the composition of the present invention at the same time, and the level of autophagic flux (LC3B-II / I ratio) increased by the composition of the present invention is reduced, and the expression of p62 reduced by the composition of the present invention has increased. In addition, when 3-MA is used together with the composition of the present invention, the levels of APP and Aβ1-42 proteins reduced by the composition of the present invention are increased, and the results of Western blot analysis are in Fig.26 Shown in.

[0050] Fig. 27 The results of Western blot analysis showing that the levels of ATG7 and ATG5 / 12 proteins (i.e., autophagy markers) in the hippocampus of an Alzheimer's disease animal model (5XFAD transgenic mice) were increased, and the level of ATG5 / 12 protein in the cortex was increased by treatment with the composition of the present invention are presented.

[0051] IX. Effects of improving cognitive ability and behavioral learning ability

[0052] Fig.28 Results of the Morris water maze test are presented showing that in evaluating the improvement of cognitive function levels in an Alzheimer's dementia animal model (NSE-hAPP-C105), the group treated with the composition of the present invention obtained reduced latency to the target and distance to the target, as well as increased time in the target quadrant and number of platform crossings compared to the transgenic mouse control.

[0053] Fig.29 Results of a passive avoidance test are presented showing that in evaluating the improvement of cognitive ability and behavioral learning ability in an Alzheimer's dementia animal model (NSE-hAPP-C105), the group treated with the composition of the present invention achieved reduced latency time compared to the transgenic mouse control. DETAILED DESCRIPTION

[0054] One embodiment of the present invention provides a method for the following purposes by administering one of the compounds selected from milonafil, sildenafil, vardenafil, tadalafil, udenafil, daxantfil and avanafil; and pharmaceutically acceptable salts, solvates or hydrates thereof: (1) inhibiting the formation of Aβ oligomers / fibrils by inhibiting Aβ aggregation, (2) inhibiting the processing of β-amyloidogenic protein by reducing BACE-1, (3) reducing the formation of extracellular Aβ monomers, oligomers, Aβ fibrils / plaques by increasing cerebral blood flow (vasodilation), (4) activating NO (nitric oxide) / cGMP (cyclic guanosine monophosphate) / P KG (protein kinase G), CREB (cyclic AMP (adenosine monophosphate) response element binding protein) pathway to inhibit neuronal cell death and accelerate neurogenesis, synaptogenesis and / or promote angiogenesis, (5) restore synaptic plasticity (synaptic plasticity) by activating Wint signaling through the inhibition of DKK-1 (Dickkopf WNT signaling pathway inhibitor 1), and inhibit the production of APP (amyloid precursor protein) and reduce Aβ accumulation by suppressing the positive feedback loop of Aβ production, and (6) inhibit the formation of Aβ fibrils / plaques by removing intracellular toxic and soluble Aβ oligomers through the activation of autophagy.

[0055] In another embodiment, the present invention provides a pharmaceutical composition comprising as an active ingredient a compound selected from the group consisting of milonafil, sildenafil, vardenafil, tadalafil, udenafil, daxantafil and avanafil; and pharmaceutically acceptable salts, solvates and hydrates thereof, which is used for (1) inhibiting Aβ oligomer / fibril formation by reducing Aβ aggregation, (2) inhibiting the processing of β-amyloidogenic protein by reducing BACE-1, (3) reducing extracellular Aβ monomers, oligomers and protofibrils by increasing cerebral blood flow, and (4) inhibiting the formation of Aβ oligomers and protofibrils by reducing BACE-1. The invention discloses a novel novel cytosolic Aβ inhibitory agent that inhibits the formation of Aβ fibrils / plaques, (4) inhibits neuronal cell death inhibition and promotes neurogenesis, synaptogenesis and / or angiogenesis by activating the NO / cGMP / PKG / CREB pathway, (5) restores synaptic plasticity by activating Wint signaling through the inhibition of DKK-1, and inhibits the production of APP and reduces Aβ accumulation by inhibiting the positive feedback loop of Aβ production, and (6) inhibits the formation of Aβ fibrils / plaques by removing intracellular toxic and soluble Aβ oligomers through the activation of autophagy.

[0056] The present invention has multiple mechanisms that are different from other Alzheimer's therapeutic agents previously or currently developed. The present invention provides significantly improved efficacy in treating Alzheimer's disease compared to current drugs on the market by adopting a "one drug, multiple targets / mechanisms" strategy that exceeds conventional discovery strategies based on the "one drug, one target" paradigm to provide (1) inhibition of Aβ oligomer / fibril formation by reducing Aβ aggregation, (2) inhibition of β-amyloidogenic processing by reducing BACE-1, (3) reduction of extracellular Aβ monomers, oligomers and Aβ fibrils / plaques by increasing cerebral blood flow, (4) (5) by activating the NO / cGMP / PKG / CREB pathway to inhibit neuronal cell death and promote neurogenesis, synaptogenesis and / or angiogenesis, (6) by activating Wint signaling through the inhibition of DKK-1 to restore synaptic plasticity (synaptic plasticity), and by inhibiting the positive feedback loop of Aβ production to inhibit APP production and reduce Aβ accumulation, and (7) by activating autophagy to remove intracellular toxic and soluble Aβ oligomers to inhibit the formation of Aβ fibrils / plaques.

[0057] Treatment mechanism of Alzheimer's disease

[0058] 1. Inhibition of Aβ oligomer / fibril formation by inhibiting Aβ aggregation

[0059] The common pathological feature of sporadic and familial Alzheimer's dementia is the accumulation of a peptide called Aβ to form extracellular senile plaques (J Alzheimers Dis 2018; 64 (s1): S567-S610.). Although Aβ is also produced in normal people, it is degraded quickly enough and does not accumulate in the body. On the other hand, in the case of Alzheimer's patients, Aβ is not only formed in large quantities, but also does not degrade and accumulate in tissues (Alzheimers Res Ther 2013 Nov 29; 5 (6): 60.). These abnormal Aβ accumulations induce the formation of extracellular senile plaques to interfere with signal transmission between neurons, and also accumulate in the hippocampus, which plays an important role in memory and learning ability and cortex, to cause cell inflammation and neuronal damage, and ultimately damage the neural network required for normal function (EBioMedicine 2016 Apr; 6: 42-49.). In addition, accumulated Aβ monomers aggregate to form toxic Aβ oligomers to increase the production of ROS (reactive oxygen species) and RNS (reactive nitrogen species) to activate signaling networks associated with neuronal cell death to increase cell apoptosis (Mt Sinai J Med. 2010 Jan-Feb; 77(1):43-9.).

[0060] The composition of the present invention inhibits the aggregation of Aβ fibrils ( Figure 1) to inhibit Aβ oligomer / fibril formation ( Figure 2 ).

[0061] Therefore, the composition of the present invention inhibits Aβ oligomer aggregation to inhibit Aβ oligomer / fibril formation.

[0062] 2. Inhibition of β-amyloidogenic processing by decreasing BACE-1

[0063] Aβ is produced by APP because various secretases cut APP to generate Aβ with various numbers of amino acids. Among them, in the case of Alzheimer's patients, the Aβ ratio with 42 or 43 amino acids formed by BACE-1 increases rapidly. At present, it is known that the damage to nerve cells by Aβ1-42 or Aβ1-43 is one of the important reasons for the progression of Alzheimer's disease, and it is known that Aβ25-35 is a toxic fragment of Aβ1-42 or Aβ1-43 that causes neuronal cell damage (J AminoAcids 2011; 2011: 198430., PLoS One 2013; 8 (1): e53117, J Alzheimers Dis 2018; 62 (3): 1345-1367). In particular, it has been recently reported that sildenafil (PDE-5 inhibitor) suppresses the expression of BACE-1 and cathepsin B to inhibit the β-amyloidogenic processing of APP to reduce the production of Aβ1-42 or Aβ1-43, and it is expected that PDE-5 inhibitors can suppress the expression of BACE-1 to reduce the formation of Aβ1-42 or Aβ1-43 that causes neuronal cell damage, so as to have a therapeutic effect on Alzheimer's disease (J Gerontol a Biol Sci Med Sci 2015 Jun; 70(6): 675-85., J Urol. 2016 Apr; 195(4Pt 1): 1171.).

[0064] The composition of the present invention inhibits the expression of BACE-1, which plays a key role in the processing of β-amyloid production ( Figure 3 ).

[0065] Therefore, the composition of the present invention inhibits the processing of β-amyloidogenic protein by APP to reduce the formation of Aβ1-42 or Aβ1-43 that causes neuronal cell damage.

[0066] 3. Reduce Aβ monomers, oligomers and Aβ fibrils / plaques by increasing cerebral blood flow through other cells

[0067] One of the first steps in the onset of dementia is the damage of cells constituting cerebral blood vessels (including endothelial cells, pericytes, and vascular smooth muscle cells) by various genetic and environmental factors, followed by the destruction of the blood-brain barrier, allowing neurotoxic proteins such as Aβ proteins or cells to invade brain tissue to cause brain tissue swelling or various degenerative lesions. In the brain, it penetrates into the tissue, causing brain tissue inflammation and various degenerative lesions, which is known there (Nat Rev Neurol 2018 Mar; 14(3.): 133-150.).

[0068] In addition, it has been reported that with the progression of cognitive decline caused by Alzheimer's dementia, the blood-brain barrier collapses, the cerebral blood flow in the brain area responsible for memory decreases, and the area continues to expand (Alzheimers Dement 2017 May; 13 (5): 531-540.). In addition, recent studies have shown that in the brain tissue of patients with Alzheimer's dementia, ROS increased by Aβ increases the calcium concentration in pericytes to cause vascular pericyte contraction, and thus reduces the amount of cerebral blood flow (Science. 2019 Jul 19; 365 (6450)). Therefore, appropriate cerebral blood flow should be maintained, not only to provide a large amount of oxygen and nutrition to the brain, but also to effectively remove neurotoxins, including carbon dioxide and Aβ brain, or protein wastes naturally generated by metabolism (Nat Rev Neurosci 2017 Jul; 18 (7): 419-434).

[0069] PDE5 inhibitors are used to treat pulmonary hypertension or erectile dysfunction because they can inhibit the activation of PDE5 to prevent the conversion of cGMP to 5'-GMP, and ultimately increase the concentration of cGMP in pericytes to induce vascular dilation (Int JImpot Res. 2004 Jun; 16 Suppl 1: S4-7.). In particular, in recent studies, PDE5 inhibitors have shown Alzheimer's patients (J Cereb Blood Flow Metab 2018 Feb; 38 (2): 189-203.).

[0070] The compositions of the present invention reduce the intracellular calcium concentration in pericytes, which is 2 O 2 treatment, indicating that the composition can inhibit the decrease in cerebral blood flow by inhibiting the contraction of pericytes caused by increased calcium concentration ( Figure 4 ).

[0071] Therefore, the composition of the present invention seems to be expected to have an inhibitory effect on the same neurotoxicity of the protein that shrinks vascular pericytes and accumulates by suppressing cerebral blood flow via increasing brain tissue Aβ.

[0072] 4. Inhibit neuronal cell death by activating the NO / cGMP / PKG / CREB pathway and promote neurogenesis, synaptogenesis, and angiogenesis

[0073] Aβ monomers are transfected into cells through endocytosis aggregates to form toxic soluble Aβ oligomers. These aggregates can not only induce cell dysfunction associated with neuronal cell death, but also increase the hydrogen phosphorylation of Tau protein (microtubule-associated protein) to promote the formation of NFT (neurofibrillary tangles). As a result, the increased ROS in the process cause mitochondrial damage and activate Caspase-3 to cause neuronal cell death. (Nat Rev Neurosci. 2007 Jul; 8 (7): 499-509., Neuron. 2008 Nov 26; 60 (4): 534-42., Nat Rev Neurosci. 2011 Feb; 12 (2): 65-72.).

[0074] PDE5 (phosphodiesterase 5) is expressed in the cerebral cortex and hippocampus, and plays an important role in cognitive function in brain tissue (J Comp Neurol 2003 Dec 22; 467 (4): 566-80.), and in particular, it has been reported that PDE5 expression in the cerebral cortex of brain tissue of patients with Alzheimer's disease is increased compared with normal people (Neuropathol Appl Neurobiol. 2015 Jun; 41 (4): 471-82.). In addition, it has been reported that in the mouse hippocampus, synaptic plasticity important for brain cognitive function reduced by Aβ is restored by the action of NO / cGMP / CREB pathway (J Neurosci 2005 Jul 20; 25 (29): 6887-97.).

[0075] It is reported that PDE5 inhibitors can also inhibit neuronal cell apoptosis, and this is because PED5 inhibitors can inhibit PED5 activity to prevent the conversion of cGMP to 5'-GMP. As a result, cGMP accumulates in the brain to activate PKG (Mol Neurobiol. 2010 Jun; 41 (2-3): 129-37., ACS Chem Neurosci. 2012 Nov 21; 3 (11): 832-44., Exp Neurol. 2014 Nov; 261: 267-77.). Activated PKG inhibits Caspase-3 activated by toxic soluble Aβ oligomers to inhibit neuronal cell apoptosis. (Neurobiol Aging 2014 Mar; 35 (3): 520-31, Neuroscience 2016 Jul 22; 328: 69-79, Front Pharmacol. 2017 Mar 8; 8: 106.). In addition, phosphorylation of Ser133 of CREB protein (which is a gene transcription factor) activates PKG, which will increase the expression of factors related to neurogenesis, synaptogenesis, and angiogenesis (Behav Brain Res 2013 Aug 1; 250: 230-7, DNA Cell Biol. 2018 Nov; 37(11): 861-865.).

[0076] The composition of the present invention inhibits the activation of PDE5 to prevent the conversion of cGMP to 5'-GMP, thereby increasing the amount of cGMP in cells ( Figure 5 ), inhibiting the activity of Caspase-3 ( Figure 6 ), restore mitochondrial membrane potential ( Figure 7 ) to reduce Aβ-induced neuronal death ( Figure 8 ).

[0077] The composition of the present invention increases the expression of factors related to neurogenesis, synaptogenesis and angiogenesis by increasing the phosphorylation of Ser133 in the CREB protein ( Fig. 9 ).

[0078] The composition of the present invention increases the expression of NGF and BDNF which are mainly involved in regulating the growth, maintenance, proliferation and survival of neurons ( Fig.10 and 11 ).

[0079] Thus, the present invention provides effects for stimulating neuronal apoptosis, neurogenesis, synaptogenesis and angiogenesis in the brain of dementia patients by activating the NO / cGMP / PKG / CREB pathway.

[0080] 5. Restoration of synaptic plasticity by activating Wnt signaling via inhibition of DKK-1, and reduction of APP production and inhibition of Aβ accumulation by inhibiting the positive feedback loop

[0081] The Wnt / β-catenin pathway is a crucial signaling pathway that controls many cellular processes including cell survival. In particular, the Wnt / β-catenin pathway in the brain is not only important for neuronal survival and neuronal signal generation, but also plays a vital role in controlling synaptic plasticity, blood-brain barrier integrity and controlling its function (Biomed Res Int 2014; 2014: 301575.). Currently, it is known that the expression of the Wnt antagonist DKK-1 increases in brain tissue of Alzheimer's patients to inhibit the Wnt / β-catenin pathway and increase synaptic plasticity (J Neurosci 2004 Jun 30; 24 (26): 6021-7., Front Cell Neurosci 2013 Nov 5; 7: 162). It is reported that Aβ accumulated in hippocampal neurons increases the expression of DKK-1 to reduce synaptic plasticity (J Neurosci 2012 Mar 7; 32 (10): 3492-8.). In addition, it is reported that DKK-1 acts as a positive feedback loop on Aβ production, and this gives an expectation that, instead of conventionally targeting Aβ, it may be possible to slow down the progression of Alzheimer's disease by reducing Aβ production by inhibiting DKK-1 (DKK-1 is a crucial controller of the positive feedback loop of Aβ production) to reduce synaptic loss (J Mol Cell Biol. 2014 Feb; 6(1): 75-80., Neuron. 2014 Oct 1; 84(1): 63-77., Cell Death Dis. 2014 Nov 27; 5: e1544., Curr Biol. 2016 Oct 10; 26(19): 2551-2561., Front Neurosci. 2016 Oct 19; 10: 459., Transl Psychiatry. 2018 Sep 20; 8(1): 179.).

[0082] On the other hand, in several degenerative brain diseases including frontotemporal dementia, progressive supranuclear palsy and Alzheimer's disease, NFTs formed by aggregation of hyperphosphorylated Tau proteins are the main cause of the disease, and these diseases are collectively referred to as 'Tauopathy' (Front Mol Neurosci. 2011 Oct 5; 4: 24., Front Neurosci. 2019 Dec 13; 13: 1274.). It is known that the current Tauopathy is caused by the inhibition of the Wnt / β-catenin pathway by various pathogenic mechanisms in the brain (Mol Brain. 2019 Dec 4; 12 (1): 104.), especially by the accumulation of Aβ in the brain, the Wnt / β-catenin pathway is inhibited to reduce Ser9 phosphorylation in GSK3β protein and increase Tyr216 phosphorylation to activate GSK3β and enhance the phosphorylation of Tau protein in mouse hippocampal neurons. As a result, NFT formation is enhanced to reduce neuronal survival, neuronal generation, synaptic plasticity, blood-brain barrier integrity and functionality, and enhance the progression of Tauopathy (Neurosci Res. 1998 Aug; 31(4): 317-23., Annu Rev Pathol. 2019 Jan 24; 14: 239-261.).

[0083] In summary, the expression of DKK-1 increased by the accumulation of Aβ in the brain acts as a Wnt antagonist to suppress the Wbt / β-catenin pathway, and activates GSK3β to increase the formation of NFTs, thereby reducing synaptic plasticity, and also acts as a positive feedback loop for Aβ production to enhance the production and accumulation of Aβ to promote the progression of degenerative brain diseases. Therefore, therapies targeting the inhibition of DKK-1 expression are expected to inhibit degenerative brain diseases including Alzheimer's disease by restoring synaptic plasticity through activating Wnt signaling and reducing the formation and accumulation of Aβ through suppressing the positive feedback loop of Aβ production.

[0084] The compositions of the invention reduce the expression of DKK-1, a Wnt antagonist and regulator of the positive feedback loop of Aβ production ( Fig.12 , 13 ).

[0085] The composition of the present invention increases Wnt3a ( Fig.14 ) and Wnt1( Fig.15 ) expression, the activity of Wnt / β-catenin-related transcription factors (VAX2, c-Myc, NR5A2, Mitf, TCF / LEF, NFAT, CEBP, GLI-1, GBX2, AP-1) ( Fig.16 ), and increased the activity of Wnt / β-catenin ( Fig.17 ).

[0086] The composition of the present invention increases the Ser9 phosphorylation of the GSK3β protein to inhibit activity (GSK3β activity) ( Fig.18 , 19 ).

[0087] The composition of the present invention reduces the Ser199 / 202 phosphorylation of Tau protein increased in the Alzheimer's dementia animal model (NSE-hAPP-C105) Fig. 20 ).

[0088] The compositions of the present invention inhibit the Aβ production positive feedback loop and result in lowering of APP and Aβ protein levels ( Fig.21 , 22 ).

[0089] Therefore, the composition of the present invention is expected to restore synaptic plasticity by activating Wnt signaling from DKK-1 inhibition and reduce the formation of APP and the accumulation of Aβ by suppressing the positive feedback loop of Aβ production.

[0090] 6. Activate autophagy to inhibit the formation of toxic soluble Aβ oligomers and Aβ plaques

[0091] Although the brain consumes 20% or more of the total oxygen consumed by the human body, the defense mechanism to remove the reactive oxygen species (ROS) inevitably generated in cells is weak, and therefore the resistance to oxidative stress is weak. ROS accumulated in cells by oxidative stress induce oxidation of proteins in the endoplasmic reticulum to inhibit protein folding and accumulate misfolded proteins, and ultimately endoplasmic reticulum stress (J Neurochem. 2006 Jun; 97 (6): 1634-58., Antioxid Redox Signal. 2007 Dec; 9 (12): 2277-93.). As a result, intracellular aβ accumulation caused by endoplasmic reticulum stress further increases endoplasmic reticulum stress as well as endosomal and lysosomal leakage and mitochondrial dysfunction to enhance neuronal apoptosis (J Neurosci Res. 2011 Jul; 89 (7): 1031-42.). In addition, recent genome-wide association studies of patients with Alzheimer's dementia have revealed gene mutations associated with Aβ release or removal (SORL1, BIN1, CD2AP, PICALM), which confirms that intracellular accumulation of Aβ is the main cause of the progression of Alzheimer's dementia (Trends Neurosci. 2017 Oct; 40(10): 592-602., Nat Rev Mol Cell Biol. 2018 Dec; 19(12): 755-773.).

[0092] Recently, autophagy has attracted attention as the main cause of neurodegenerative diseases. Autophagy is one of the main cellular mechanisms, and it is known that autophagy plays a role in decomposing long-lived proteins and organelles (Nat Med. 2013 Aug; 19 (8): 983-97.). Autophagy dysfunction causes the accumulation of misfolded protein aggregates in cells, thereby inducing various diseases, and is especially closely related to the onset of four major neurodegenerative diseases, i.e., Alzheimer's disease, Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis (Mol Cells. 2015 May; 38 (5): 381-9., Nat Rev Drug Discov. 2018 Sep; 17 (9): 660-688.). In particular, the two main factors of the onset of Alzheimer's disease, i.e., the accumulation of Aβ and Tau proteins, are induced by dysfunctional autophagy in the brain of Alzheimer's patients (J Syst Integr Neurosci. 2017; 3 (4): 1-6.). As a result, Aβ and Tau proteins accumulate in the brain to form senile plaques, causing necrosis of neuronal cells responsible for cognitive ability in the hippocampus and cortex, and ultimately leading to decreased cognitive function in Alzheimer's patients (J Cell Biol. 2005 Oct10; 171(1): 87-98., Front Aging Neurosci. 2018 Jan 30; 10: 04.).

[0093] AMPK (adenosine monophosphate-activated protein kinase) is responsible for controlling several biological functions, including insulin sensitivity, cell survival, proliferation and apoptosis, among which, in particular, it maintains cellular homeostasis by activating autophagy via inhibition of mTOR (mammalian target of rapamycin) protein (Nat Rev Mol Cell Biol. 2018 Feb; 19(2): 121-135., Mol Cell. 2017 Jun 15; 66(6): 789-800.). Currently, Thr172 phosphorylation by AMPK catalytic subunit a is the main starting point of the signal transduction pathway that activates autophagy (Exp Mol Med. 2016 Apr 1; ​​48: e224., Nat Rev Mol Cell Biol. 2018 Feb; 19(2): 121-135., Nat Rev Drug Discov. 2019 Jul; 18(7): 527-551.), and recently, in degenerative brain diseases, it has been reported that neuronal degeneration is inhibited by removing misfolded protein aggregates associated with degenerative brain diseases through autophagy activation from activated AMPK, including Aβ and Tau, which are the main onset factors of Alzheimer's disease (Front Neurosci. 2018 May 22; 12: 255., Nat Rev Drug Discov. 2018 Sep; 17(9): 660-688. J Alzheimers Dis. 2019; 68(1): 33-38). Considering that 30-40% of Alzheimer's disease patients suffer from mixed pathology with other pathological symptoms, removing intracellular accumulated misfolded protein aggregates (including Aβ and Tar aggregates) by activating autophagy from activated AMPK will be a new target for the future development of therapies for degenerative brain diseases including Alzheimer's disease (Front Neurosci. 2018 May 22; 12: 255., Nat Rev Drug Discov. 2018 Sep; 17(9): 660-688. J Alzheimers Dis. 2019; 68(1): 33-38.).

[0094] The composition of the present invention reduces the Aβ protein level in the hippocampus of the Alzheimer's dementia animal model (NSE-hAPP-C105) Fig.23 ).

[0095] The composition of the present invention reduces the number of Aβ plaques in the hippocampus and cortex of an Alzheimer's disease animal model (5XFAD transgenic mice) Fig.24 ).

[0096] The composition of the present invention increases the phosphorylation of Thr172 of AMPK catalytic subunit α ( Fig.25 ).

[0097] The composition of the present invention induces the autophagic flux of the autophagic marker LC3B (conversion of LC3B-I to LC3B-II) to increase the LC3B-II / I ratio and reduce the autophagosome cargo protein ubiquitin-binding protein p62 (SQSTM, Sequestosome 1) ( Fig.26 ).

[0098] The composition of the present invention enhances the expression of autophagy markers ATG7 and ATG5 / 12 in the cortex of the hippocampus of an Alzheimer's disease animal model (5XFAD transgenic mice) and the expression of ATG5 / 12 ( Fig.26 ).

[0099] Therefore, the composition of the present invention increases the autophagy marker LC3BII / I ratio, decreases the autophagosome cargo protein ubiquitin-binding protein p62, and increases the expression of ATG7 and ATG5 / 12 to activate the autophagy cascade.

[0100] 7. Alzheimer's dementia animal model for improving cognitive and behavioral learning skills (NSE-hAPP-C105)

[0101] In order to test the effect of the composition of the present invention on the gradual memory loss and behavioral disorders that occur in Alzheimer's disease, a neurodegenerative disease caused by the formation and accumulation of Aβ42, 13-month-old C57BL / 6-Tg(NSE-hAPP-C105)Kor transgenic mice were intraperitoneally injected with the composition of the present invention (at 4 mg / kg, once a day for 4 weeks), and then the cognitive ability and behavioral learning skills were tested by passive avoidance test and Morris water maze test to analyze any changes in cognitive ability and behavioral learning skills.

[0102] It was confirmed that in an Alzheimer's dementia animal model (NSE-hAPP-C105), cognitive ability and behavioral learning skills were improved in the group treated with the composition of the present invention compared with the transgenic mouse control group ( Fig.28 , 29 ).

[0103] Example

[0104] Example 1. Selective test of the composition of the present invention on PDE5 (IC 50 , inhibitory concentration 50)

[0105] Example 1-1. Test method for PDE5 selectivity

[0106] In order to test the selective inhibitory activity (IC 50 , inhibition concentration 50), the test was performed by MDS Pharma Services, analysis agency CRO and Scottish Biomedical. The results were measured using the PDE SPA assay kit (Amersham Pharmacia Biotech). MDS Pharma Services analyzed PDE1 (bovine heart); PDE 2, 3, 5 (human platelets); PDE4 (human U937 cells); and PDE6 (bovine rod cells) and Scottish Biomedical analyzed PDE 5, 7-11 (recombinant human enzyme). Test samples (100 μL each) were added to PDE family proteins (10 μl), [ 3 H]-cGMP (5 Ci / mL), bovine serum albumin (0.5 mg / mL), and MgCl 2 (5mM) in a mixture of Tris-HCl buffer (15mM, pH 7.5). The reaction started with the addition of PDE family proteins. Each sample was stored in a 30°C water bath for 30 minutes, and then SPA beads (PerkinElmer) (50μL) were added to terminate the reaction. The test tubes were allowed to stand for 20 minutes and then measured by a liquid scintillation counter (Tri-carb 1500, Packard). In order to determine the degree of inhibition of PDE family protein activity, the composition of the present invention and the test sample were dissolved in DMSO (dimethyl sulfoxide), and the solution was then diluted with distilled water to a final DMSO concentration of at least 0.2% (v / v). All inhibition tests were performed under conditions where the cGMP (cyclic guanosine monophosphate) hydrolysis rate did not exceed 15%. The amount of GMP formed depends on time and PDE5 family proteins, increasing in a dose-dependent and proportional manner.

[0107] Example 1-2. Results of PDE5 selectivity testing of compositions of the present invention

[0108] Based on the inhibitory activity of the composition of the present invention against 11 PDE families (MDS Pharma Services and Scottish Biomedical), the IC of the composition of the present invention against PDE5 50 The concentration of 5-nitro-1-deoxy-1-nitropropene was 0.338 nM (MDS Pharma Services) and the selectivity was 30-376,471-fold compared to the remaining 10 PDE family proteins (Table 1).

[0109] Table 1. Selectivity results for the PDE family of the present invention

[0110]

[0111] a MDS Pharma Services; PDE1 (bovine heart); PDE 2, 3, 5 (human platelets); PDE4 (human U937 cells); PDE6 (bovine rod photoreceptor cells).

[0112] b Scottish Biomedical; PDE 5, 7-11 (recombinant human enzyme); NI = no inhibition observed.

[0113] Example 2. Test of inhibition of Aβ aggregation by the composition of the present invention

[0114] Example 2-1. Test method for inhibition of Aβ aggregation (Aβ aggregation inhibition)

[0115] Example 2-1-1. Thioflavin T determination

[0116] If Aβ1-42 peptide (one of the main biomarkers and causes of Alzheimer's disease) is incubated at 37°C for 3 days, Aβ fibrils and oligomers are generated. In order to test the activity of the composition of the present invention for inhibiting the formation of Aβ fibrils and oligomers, Aβ1-42 (50 μM) was treated with various concentrations (5, 50, 500 μM) of the composition of the present invention, and the mixture was then incubated at 37°C for 3 days, and the amount of Aβ1-42 fibrils and oligomers was subsequently measured. Using Varioskan LUX Multimode Microplate Reader (Multimode Microplate Reader) (Thermo Fisher Scientific, USA), the fluorescence intensity of Thioflavin T at excitation 450nm / emission 485 was measured. The Thioflavin T assay measures the fluorescence response from the β-sheet formed as a peptide aggregate and shows higher values ​​because more aggregates are formed. Although the Thioflavin T assay cannot distinguish between Aβ oligomers and fibrils, the advantage is that it can quantitatively show the total amount of aggregated protein.

[0117] Example 2-1-2. PICUP / SDS-PAGE analysis

[0118] The generation of Aβ oligomers was observed by PICUP (photoinduced cross-linking of unmodified proteins) analysis. Using the PICUP method, the generated Aβ oligomers and fibrils / plaques were fixed and then separated by xize using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The separated Aβ1-42 peptides were observed using silver staining.

[0119] Example 2-2. Results of inhibition of Aβ aggregation by the composition of the present invention

[0120] The results of Aβ-42 aggregation inhibition showed that, based on the Thioflavin T assay, the composition of the present invention inhibited Aβ1-42 aggregation formation compared with the untreated control group, and the inhibitory effect was dose-dependent ( Figure 1 ), and also by PICUP / SDS-PAGE analysis, compared with the untreated control group, the present invention inhibited Aβ1-42 oligomer and fibril / plaque formation ( Figure 2 ). Considering this result, the composition of the present invention is expected to inhibit Aβ oligomer and fibril / plaque formation.

[0121] Example 3. Changes in BACE-1 expression in retinoic acid-differentiated SH-SY5Y cells by the composition of the present invention

[0122] Example 3-1. Cell culture method for SH-SY5Y neurons

[0123] Example 3-1-1. Cell subculture

[0124] Preheat the mixture of DMEM / F12 complete medium, DPBS and trypsin-EDTA in a constant water bath at 37°C for 30 minutes. Take out the cell culture flask from the incubator to remove all the medium and rinse once with DPBS (T25: 5ml, T75: 10ml and T175: 20ml). After discarding all the DPBS, add trypsin-EDTA (T25: 2mL, T75: 5mL and T175: 10mL), and place the mixture at 37°C with 5% CO 2 Incubator for 4 minutes. In a 50 ml conical tube, mix new complete medium (T25 ml, T75: 10 ml and T175: 20 ml) and cells and centrifuge at 1500 rpm for 4 minutes. Remove all supernatant and add 1 ml of fresh DMEM / F12 complete medium to resuspend cells by tapping or pipetting. Add cells and medium to cell culture flasks to make T75: 20 ml and T175: 40 ml, and then incubate the mixture at 37 ° C in 5% CO 2 Incubate in an incubator.

[0125] Example 3-1-2. SH-SY5Y neuroblastoma differentiation

[0126] Preheat a mixture of DMEM / F12 complete medium (Hyclone), DPBS (Hyclone) and trypsin-EDTA (Hyclone) in a 37°C constant water bath for 30 minutes. Take out the cell culture flask from the incubator to remove all the medium and rinse once with DPBS (T75: 10 ml and T175: 20 ml). After discarding all the DPBS, add trypsin-EDTA (T75: 5 mL and T175: 10 mL), and place the mixture in a 37°C incubator with 5% CO. 2 Incubate in 400 mL of incubator for 4 minutes. In a 50 mL conical tube, mix new complete medium (10 mL for T75 and 20 mL for T175) and cells and centrifuge at 1500 rpm for 4 minutes. Remove all supernatant and add 1 mL of fresh DMEM / F12 complete medium to resuspend cells by tapping or pipetting. Add a certain amount of DMEM / F12 complete medium and transfer 4 × 10 4 cells / cm 2 Transfer to a 6-well plate coated with type I collagen or a 60 mm, 100 mm cell culture dish coated with type I collagen (Corning). After 24 hours, replace the medium with DMEM / F12 differentiation medium [+1% FBS+1% penicillin / streptomycin (Hyclone)+10 μM retinoic acid (Sigma-Aldrich)]. On day 5, replace the medium again with new DMEM / F12 differentiation medium. On day 8, replace the medium again with new DMEM / F12 differentiation medium. On day 10, complete the differentiation.

[0127] Example 3-2. Aβ and preparation and treatment methods of the present invention

[0128] DMEM / F12 (+1% FBS+1% penicillin / streptomycin) was prepared to have 1 μM human Aβ1-42 (Abcam). The Aβ solution was allowed to stand at 37°C for 3 hours to form Aβ oligomers. The original cell culture medium was discarded, and the newly prepared Aβ1-42 oligomer (1 μM) solution was treated alone or with the composition of the present invention (maximum concentration of 40 μM), and then incubated at 37°C under 5% CO 2 The cells were incubated in an incubator at 4 °C for 72 hours. After 72 hours, the culture medium was removed, and the cells were washed once with PBS and collected before continuing the following experiment.

[0129] Example 3-3. qRT-PCR method

[0130] Using Easy-Blue TM Total RNA was prepared using a total RNA extraction kit (Intron Biotechnology) and analyzed using PrimeScriptTM II 1st strand cDNA synthesis kit (TAKARA), reverse transcribe 1 μg RNA. For BACE-1 and β-actin PCR, provide cDNA as template, PCR premix (TAKARA) and the next primer: human BACE-1 (forward 5′-CTGGTATACACCCATCCGGC-3′, reverse 5′-CTTGGGCAAACGAAGGTTGG-3′); human β-actin (forward 5′-CCAGGTCATCACCATTGG-3′, reverse 5′-CAGAGTACTTGCGCTCAG-3′). PCR cycling conditions are as follows: denaturation (95°C for 45 seconds); annealing, human BACE-1 (60°C for 45 seconds), β-actin (56°C for 45 seconds); extension (72°C for 45 seconds); 40 cycles. QuantStudio TM qRT-PCR was performed using DNA Sequencing (Thermo Scientific).

[0131] Example 3-4. Results of altering BACE-1 expression in retinoic acid-differentiated SH-SY5Y cells by the composition of the present invention

[0132] The present invention dose-dependently reduces the expression of BACE-1 mRNA, which is increased by treating retinoic acid-differentiated SH-SY5Y cells with Aβ1-42 ( Figure 3 ). Considering these results, the present invention is expected to suppress the formation of Aβ1-42 or Aβ1-43 causing neuronal cell damage by suppressing the expression of BACE-1 which plays a major role in the β-amyloidogenic processing of APP.

[0133] Example 4. Changes in calcium concentration in pericytes by the composition of the present invention

[0134] Example 4-1. Method for culturing pericytes

[0135] Example 4-1-1. Cell subculture

[0136] Using Attachment Factor TM (Cell Systems, #4Z0-210), extracellular matrix (ECM) coated several T-75cm 2 Cell culture flask (SPL, #70075) for 1 minute, followed by 15 mL of serum and CultureBoost TM Complete classical medium (Cell Systems, #4Z0-500) was added to the flask and then incubated at 37°C in a CO 2The incubated primary human brain pericytes (Cell Systems, #ACBRI 498P) were washed twice with PBS buffer, followed by the addition of 3 mL of 0.25% trypsin (Hyclone, #SH30042.02) and incubated at 37°C in a CO incubator. 2 The mixture was allowed to react in an incubator for 2 minutes. 7 mL of serum and CultureBoost TM Complete classical medium was added and cell clusters were separated by repeated pipetting and the supernatant was removed by centrifugation (1,500 rpm, 4 minutes). Cell clusters were placed in 1 mL of complete classical medium with serum and CultureBoost TM The mixture was homogenized in complete classical medium and then plated in a stable T-75cm 2 Cell culture flasks at optimal CO 2 Incubate cells at 4 °C.

[0137] Example 4-2. For use with H 2 O 2 Preparation and treatment methods of , and measurement of changes in intracellular calcium concentration after treatment

[0138] Use Attachment Factor TM Coat a flat-bottom 96-well plate (SPL, #30096) and place the plate with serum and CultureBoost TM Complete classical culture medium (100 μL / well) was added to each well, and the plate was then incubated at 37°C in a CO 2 Primary human brain pericytes treated with 0.25% Tyrypsin were added to a flask containing serum and CultureBoost TM Attachment of complete classical culture medium to a Facotr-coated 96-well plate and incubate the mixture at 37 °C in CO. 2 Incubate in an incubator for 16 hours (5 × 10 3 The culture medium of the cells was removed and the cells were washed with HEPES-buffered saline (132 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl 2 , 1.5 mM CaCl 2 , 11.5 mM glucose, 11.5 mM HEPES, 1.2 mM NaH 2 PO 4 ) and transferred to a 96-well plate containing pericytes, where the ratio of intracellular Ca 2+The dye (Fura-2) (Molecular Probes, #F1201) was diluted to 1 μM and then incubated at room temperature for 30 minutes. The supernatant was removed from the 96-well plate, and the cells were washed three times with HEPES-buffered saline and H 2 O 2 The cells are treated with a diluted composition of the present invention and then the fluorescence is measured to measure changes in intracellular calcium concentration.

[0139] Example 4-4. Results of changes in calcium concentration in pericytes of SH-SY5Y cells differentiated with retinoic acid using the composition of the present invention

[0140] The composition of the present invention reduces the intracellular calcium concentration in vascular pericytes (pericytes), which is 2 O 2 The treatment increases ( Figure 4 ). Based on this result, it is expected that the composition of the present invention can inhibit the decrease in cerebral blood flow caused by pericyte contraction, which in turn is caused by increasing the calcium concentration in pericytes through the excessive production of ROS.

[0141] Example 5. Changes in intracellular cGMP in retinoic acid-differentiated SH-SY5Y cells by the composition of the present invention

[0142] Example 5-1. Cell culture method of SH-SY5Y neuronal cells

[0143] Example 5-1-1. Cell subculture

[0144] The cells were subcultured using the method described in Example 3-1-1.

[0145] Example 5-1-2. SH-SY5Y neuroblastoma differentiation

[0146] Using the method described in Example 3-1-2, SH-SY5Y neuronal differentiation was performed.

[0147] Example 5-2. Aβ and preparation and treatment methods of the present invention

[0148] The preparation and processing were carried out using the methods described in Example 3-2.

[0149] Example 5-3. Intracellular cGMP measurement method

[0150] The experiment was performed according to the instruction manual of the cyclic GMP Complete ELISA kit (Abcam, #ab133052, USA). The cell culture medium was removed and the cells were treated with 0.1 mL of 0.1 M HCl and then left to stand at room temperature for 10 minutes. The cells were recovered by 600 g centrifugation and only the supernatant was collected. Wherein 0.1 M HCl was included in the sample, and 50 μl of neutralizing reagent was added to each well. According to the instruction manual, 100 μl per dose, and standard solutions (0, 0.8, 4, 20, 100, 500 pmol / ml) were added to each well of a 96-well plate, and 50 μl of cyclic GMP complete alkaline phosphatase conjugate was also added to each well. After adding 50 μl of cyclic GMP complete antibody, the reaction was carried out at room temperature for 2 hours. Once all the reaction solutions were removed, the wells were washed three times with 400 μl of washing buffer. After completely removing the washing buffer, 5 μl of cyclic GMP complete alkaline phosphatase conjugate was added. After adding 200 μl of pNpp substrate solution to each well, the reaction was carried out at room temperature for 1 hour. 50 μl of stop solution was added and the absorbance was immediately measured at 405 nm using a plate reader. The results were calculated using the instruction manual in the kit.

[0151] Example 5-4 Results of changes in intracellular cGMP in retinoic acid-differentiated SH-SY5Y cells by the present invention

[0152] The composition of the present invention concentration-dependently increased the cGMP concentration ( Figure 5 Considering this result, it is expected that the present invention selectively inhibits the activity of PDE5 (Table 1) to inhibit the conversion of cGMP to 5'-GMP and increase intracellular cGMP, which inhibits the activity of Caspase-3 ( Figure 6 ) and restore mitochondrial membrane potential to inhibit neuronal cell death ( Figure 8 In addition, the intracellular cGMP increased by the present invention increases the activity of CREB ( Fig. 9 ) to promote the expression of neurotrophic factors including NGF and BDNF ( Fig.10 , 11 ) to improve cognitive function.

[0153] Example 6. Changes in the expression of proteins related to apoptosis regulation in retinoic acid-differentiated SH-SY5Y cells by the present invention

[0154] Example 6-1. Cell culture method of SH-SY5Y neuronal cells

[0155] Example 6-1-1. Cell subculture

[0156] The cell subculture was performed using the method described in Example 3-1-1.

[0157] Example 6-1-2. SSH-SY5Y neuroblastoma differentiation

[0158] Using the method described in Example 3-1-2, SH-SY5Y neuronal cells were differentiated.

[0159] Example 6-2. Method for preparing Aβ and method for treating by the present invention

[0160] Human Aβ1-42 (Abcam) was mixed with DMEM / F12 (+1% FBS+1% penicillin / streptomycin) to a concentration of 10 μM. The Aβ solution was allowed to stand at 37°C for 3 hours to form Aβ oligomers. The culture medium was removed, and the freshly prepared Aβ1-42 oligomer solution (10 μM) was incubated with or without the composition of the present invention (maximum concentration 40 μM) at 37°C in 5% CO 2 The cells were incubated in an incubator for 72 hours. After 72 hours, the culture medium was removed and the cells were washed once with PBS to collect the cells. Additional experiments were performed by the following procedure.

[0161] Example 6-5. Western blot analysis method

[0162] To extract proteins, cells were treated with RIPA lysis buffer (Bio-Rad), homogenized, and centrifuged (14,000 rpm, 10 minutes, 4°C) to obtain supernatants. 5% stacking gel (DW, 30% acrylamide: bisacrylamide, 1M Tris pH 6.8, 10% SDS, TEMED, 10% ammonium persulfate) and 12% separation gel (DW, 30% acrylamide: bisacrylamide, 1.5M Tris pH 8.8, 10% SDS, TEMED, 10% ammonium persulfate) were used for SDS-PAGE. The supernatant obtained by centrifugation was mixed with 4X Laemmli buffer (Bio-Rad) at a ratio of 3: 1 and boiled at 95°C for 10 minutes to denature the proteins. The mixture was cooled in an ice bath for 10 minutes and spun down. Protein size markers (Bio-Rad) and each sample were added to the wells of the stacking gel prepared in a Mini-Protein II Dual-Slab apparatus (Bio-Rad) and electrophoresed at 150 volts until all precipitated to the bottom. The polyvinylidene fluoride (PVDF) membrane was wetted with methanol for activation and washed with transfer buffer (190 mM glycine, 50 mM Tris-base, 0.05% SDS, 20% methanol). Whatman 3M paper wetted with transfer buffer was stacked in a Mini Trans-Bolt cell (Bio-Rad) and energized at 200 mA for 60 minutes to deposit the membrane. After deposition, the membrane was blocked with a 5% skim milk solution (in TBS-T: 10 mM Tris-base pH 8.0, 150 mM NaCl, 0.1% Tween-20) on a platform shaker for 60 minutes. The primary antibodies used in the current experiments are provided in Table 2. The primary antibodies were each diluted at the optimal concentration in 5% skim milk and placed on a platform shaker at 4°C for 16 hours. The primary antibodies were recovered and washed three times with TBS-T for 10 minutes each. The secondary antibodies (goat anti-rabbit IgG H&L (HRP), Abcam; goat anti-mouse IgG H&L (HRP), Abcam) were diluted with 5% skim milk at a ratio of 1:10,000, then placed on a platform shaker at room temperature for 60 minutes and washed three times with TBS-T solution for 10 minutes. Finally, the membrane was added to SuperSignal TM West Femto Highest Sensitivity Substrate (SuperSignal TM West Femto MaximumSensitivity Substrate)(Thermo Scientific TM) and the color was correctly expressed (colro) before scanning the membrane using an image analysis system, and the amount of protein was calculated using Image J software (NIH, USA).

[0163] Table 2. List of primary antibodies

[0164]

[0165] Example 6-6 Changes in the expression of proteins related to apoptosis regulation in retinoic acid-differentiated SH-SY5Y cells by the present invention

[0166] The present invention inhibits the formation of cleaved Capase-3 which is increased by treating retinoic acid-differentiated SH-SY5Y cells with Aβ1-42 oligomers. As a result, the amount of RARP cleaved by cleaved Caspase-3 is reduced, and thus the amount of cleaved PARP formed is reduced ( Figure 6 ). In view of these results, the present invention suppresses apoptosis caused by Aβ1-42 oligomers and reduces neuronal cell death to increase neuronal cell survival.

[0167] Example 7. Changes in mitochondrial membrane potential in retinoic acid-differentiated SH-SY5Y cells by the present invention

[0168] Example 7-1. Method for culturing SH-SY5Y neurons

[0169] Example 7-1-1. Cell subculture

[0170] The cell subculture was performed using the method described in Example 3-1-1.

[0171] Example 7-1-2. SH-SY5Y neuroblastoma differentiation

[0172] Using the method described in Example 3-1-2, SH-SY5Y neuronal cells were differentiated.

[0173] Example 7-2. Method for preparing Aβ and method for treating with the present invention

[0174] The preparation and processing were carried out using the methods described in Example 3-2.

[0175] Example 7-3. Method for measuring mitochondrial membrane potential

[0176] After removing the cell culture medium, the cells were washed once with CPBS. The JC-1 solution of the JC-1-mitochondrial membrane potential assay kit (Abcam, USA) was mixed with a culture medium without FBS, and the cells were treated with the mixture. The cells were placed in a 5% incubator for 15 minutes at 37°C. The culture medium was removed and the cells were washed once with DPBS and clean culture medium was added. Using Varioskan LUX multifunctional microplate reader (Thermo Fisher Scientific, USA), the fluorescence of JC-1 monomer at excitation 475nm / emission 530nm and JC-1 dimer at excitation 535nm / emission 590nm was measured.

[0177] Example 7-4. Changes in mitochondrial membrane potential in retinoic acid-differentiated SH-SY5Y cells

[0178] The present invention dose-dependently increases the mitochondrial membrane potential ( Figure 7 ). In view of these results, the present invention reduces mitochondrial damage caused by excessive ROS formed by Aβ1-42 oligomers to increase neuronal cell survival.

[0179] Example 8. Live cell analysis of the effects of the present invention on suppressing neuronal cell death in SH-SY5Y cells differentiated by retinoic acid

[0180] Example 8-1. Cell culture method of SH-SY5Y neurons

[0181] Example 8-1-1. Cell subculture

[0182] The cell subculture was performed using the method described in Example 3-1-1.

[0183] Example 8-1-2. SH-SY5Y neuroblastoma differentiation

[0184] SH-SY5Y neuroblastoma cells were differentiated using the method described in Example 3-1-2.

[0185] Example 8-2. Preparation of Aβ and treatment with the present invention

[0186] The preparation and processing were carried out using the methods described in Example 3-2.

[0187] Example 8-3. IncuCyte S3 live cell analysis (Satorius, USA)

[0188] Cytotox Red reagent (Essen Bioscience catalog number 4632) was added at a concentration of 1:1,000 (v / v) and the cells were incubated at 5% CO. 2 Incubate at 37 ° C for 24 hours. In order to analyze cytotoxicity, the final ratio of the microscope was adjusted to x200, and a photo was taken every 1.5 hours. Using the software provided by IncuCyte, images were produced at 1.5fps, and in order to analyze cytotoxicity, the total red object area readings in the image were compared. The experiment was repeated three times, and Student's T-Test was used for statistical analysis. All significance tests were performed at P < 0.05 level.

[0189] Example 8-4. Results of live cell analysis of the effects of the present invention on suppressing neuronal cell death in SH-SY5Y cells differentiated by retinoic acid

[0190] The present invention reduces the number of apoptotic cells ( Figure 8 ). In view of this result, the present invention can increase neuronal cell survival by suppressing neuronal cell death induced by Aβ1-42 oligomers.

[0191] Example 9. Changes in CREB expression in retinoic acid-differentiated SH-SY5Y cells by the present invention

[0192] Example 9-1. Cell culture method of SH-SY5Y neurons

[0193] Example 9-1-1. Cell subculture

[0194] The cell subculture was performed using the method described in Example 3-1-1.

[0195] Example 9-1-2. SH-SY5Y neuroblastoma differentiation

[0196] Using the method described in Example 3-1-2, SY5Y neuronal cells were differentiated.

[0197] Example 9-2. Aβ and the preparation and treatment methods of the present invention

[0198] The preparation and processing were carried out using the methods described in Example 3-2.

[0199] Example 9-4. Western blot analysis method

[0200] To extract protein, cells were treated with RIPA lysis buffer (Biorad), homogenized, and centrifuged (14,000 rpm, 10 minutes, 4°C) to obtain supernatant. 5% stacking gel (DW, 30% acrylamide: bisacrylamide, 1M Tris pH 6.8, 10% SDS, TEMED, 10% ammonium persulfate) and 12% separation gel (DW, 30% acrylamide: bisacrylamide, 1.5M Tris pH 8.8, 10% SDS, TEMED, 10% ammonium persulfate) were used for SDS-PAGE. The supernatant obtained by centrifugation was mixed with 4X Laemmli buffer (Bio-Rad) at a ratio of 3: 1 and boiled at 95°C for 10 minutes to denature the protein. The mixture was cooled in an ice bath and spun down. Protein size markers (Bio-Rad) and each sample were injected into the stacking gel wells equipped in the Mini-Protein II dual plate apparatus (Bio-Rad) and electrophoresed at 150 volts until all settled at the bottom. The polyvinylidene fluoride (PVDF) membrane was wetted with methanol for activation and washed with transfer buffer (190 mM glycine, 50 mM Tris-base, 0.05% SDS, 20% methanol). Whatman 3M paper wetted with transfer buffer was stacked in a Mini Trans-Bolt tank (Bio-Rad) and energized at 200 mA for 60 minutes to deposit the membrane. After deposition, the membrane was blocked with 3% BSA (bovine serum albumin) solution (in TBS-T: 10 mM Tris-base pH 8.0, 150 mM NaCl, 0.1% Tween-20) on a platform shaker for 60 minutes. The primary antibodies used in the current experiments are provided in Table 3. The primary antibodies were each diluted at their respective optimal concentrations in a 5% skim milk solution and placed on a platform shaker at 4°C for 16 hours. The primary antibodies were recovered and washed three times with TBS-T for 10 minutes each. The secondary antibodies (goat anti-rabbit IgG H&L (HRP), Abcam; goat anti-mouse IgG H&L (HRP), Abcam) were diluted with 5% skim milk at a ratio of 1:10,000, then placed on a platform shaker at room temperature for 60 minutes and washed three times with TBS-T solution for 10 minutes. Finally, the membrane was added to SuperSignal TM West Femto Highest Sensitivity Substrate (Thermo Scientific TM ) and the colors were correctly expressed before scanning the membrane using an image analysis system, and the amount of protein was calculated using Image J software (NIH, USA).

[0201] Table 3. List of primary antibodies

[0202]

[0203] Example 9-5. Changes in CREB expression in retinoic acid-differentiated SH-SY5Y cells by the present invention

[0204] The present invention concentration-dependently increased the Ser133 phosphorylation of CREB protein that was reduced by treatment with Aβ1-42 oligomers in retinoic acid-differentiated SH-SY5Y cells ( Fig. 9 ). Considering this result, the present invention contemplates increasing Ser133 phosphorylation in CREB protein reduced by Aβ1-42 oligomers to restore the activity of CREB protein activity, a cellular transcription factor, to induce the expression of factors associated with neurogenesis, synaptogenesis, and angiogenesis to improve cognitive function.

[0205] Example 10. Changes in NGF and BDNF expression in retinoic acid-differentiated SH-SY5Y cells by the present invention

[0206] Example 10-1. Cell culture method of SH-SY5Y neurons

[0207] Example 10-1-1. Cell subculture

[0208] The cell subculture was performed using the method described in Example 3-1-1.

[0209] Example 10-1-2. SH-SY5Y neuroblastoma differentiation

[0210] SH-SY5Y neuroblastoma cells were differentiated using the method described in Example 3-1-2.

[0211] Example 10-2. Aβ and the preparation and treatment methods of the present invention

[0212] The preparation and processing were carried out using the methods described in Example 3-2.

[0213] Example 10-3. Western blot analysis method

[0214] Using the method described in Example 6-5, Western blot analysis has been performed, and the primary antibodies and conditions used in the current experiments are provided in Table 4.

[0215] Table 4. List of primary antibodies

[0216]

[0217] Example 10-4. NGF immunocytochemistry (ICC)

[0218] SH-SY5Y were incubated in Corning BioCoat collagen I culture slides (Corning, #354630) at 4.8×10 4 Cells / well were treated with Aβ1-42 oligomers (72 hours) and the present invention (24 hours) for 7 days, and NGF immunostaining experiments were performed. The cells were fixed in 4% paraformaldehyde (pH 7.4) for 10 minutes and washed three times with ice-cold PBS buffer. After permeabilization of the cells with 0.1% Triton X-100 in PBS, the cells were washed three times with ice-cold PBS buffer. The fixed cells were blocked for 30 minutes using a blocking solution (10% normal goat serum in PBS). The primary antibody used in the current experiment was the rabbit anti-NGF antibody in Table 4 (Abcam, #ab52918). The cells were incubated in rabbit anti-NGF antibody (X300 dilution) solution at 4°C for 16 hours, washed three times with PBS buffer, incubated with Alexa 488 anti-rabbit IgG secondary antibody (X500 dilution) at room temperature for 1 hour, washed once with PBS, reacted with DAPI staining solution (Abcam, #ab228549) for nuclear staining for 1 minute, and washed 3 times with PBS buffer. Cells were treated with Vector Shield mounting medium and fluorescent images were obtained using a super-resolution confocal laser microscope (Carl Zeiss, #LSM800).

[0219] Example 10-5. Changes in NGF and BDNF expression in retinoic acid-differentiated SH-SY5Y cells by the present invention

[0220] After reviewing the effect of increasing the expression of neurotrophic factors by the present invention, Western blot analysis showed that the present invention concentration-dependently increased the expression of NGF and BDNF decreased by treatment with Aβ1-42 oligomers in retinoic acid-differentiated SH-SY5Y cells ( Fig.10 ), and immunocytochemistry also showed that the present invention increased the expression of NGF in retinoic acid-differentiated SH-SY5Y cells by treating with Aβ1-42 oligomers ( Fig.11 ). Based on these results, the present invention is expected to increase the expression of neurotrophic factors including NGF and BDNF, which play an important role in inducing neuronal survival, development and function, to stimulate the survival and differentiation of neuronal cells to improve cognitive function.

[0221] Example 11. Effect of the present invention on the expression of DKK-1 in retinoic acid-differentiated SH-SY5Y cells and differentiated HT-22 mouse hippocampal neuronal cells

[0222] Example 11-1. Cell culture method of SH-SY5Y neurons

[0223] Example 11-1-1. Cell subculture

[0224] The cell subculture was performed using the method described in Example 3-1-1.

[0225] Example 11-1-2. SH-SY5Y neuroblastoma differentiation

[0226] SH-SY5Y cells were differentiated using the method described in Example 3-1-2.

[0227] Example 11-2. Cell culture method of HT-22 mouse hippocampal neuron cell line

[0228] Example 11-2-1. Cell subculture

[0229] Preheat DMEM / high glucose medium (10% FBS, 1% penicillin-streptomycin), DPBS and 0.25% trypsin-EDTA in a constant temperature bath at 37°C for 30 minutes. Remove the cell culture flask from the incubator, discard the existing medium and wash once with DPBS (T25: 5ml, T75: 10ml and T175: 20ml). Discard all DPBS, add 0.25% trypsin-EDTA (T25: 2ml, T75: 5ml and T175: 10ml), and place the mixture at 37°C under 5% CO 2 50ml conical tube and centrifuge at 1500rpm for 4 minutes. After removing all supernatant, add fresh DMEM / high glucose and resuspend the cells by tapping or pipetting. Add cells and culture medium to cell culture flasks (T75-15ml and T175-40ml) and incubate at 37°C in 5% CO. 2 Incubate under.

[0230] Example 11-2-2. Neuronal differentiation of HT-22 cells

[0231] Preheat DMEM / high glucose medium (10% FBS, 1% penicillin-streptomycin), DPBS and 0.25% trypsin-EDTA in a constant temperature bath at 37°C for 30 minutes. Remove the cell culture flask from the incubator, discard the existing medium and wash once with DPBS (T25: 5ml, T75: 10ml and T175: 20ml). Discard all DPBS, add 0.25% trypsin-EDTA (T25: 2ml, T75: 5ml and T175: 10ml), and place the mixture at 37°C under 5% CO 2400 μl, 100 μl, and 200 μl of new complete medium (T25: 4 ml, T75: 10 ml, and T175: 20 ml) and cells were mixed in a 50 ml conical tube and centrifuged at 1500 rpm for 4 minutes. After removing all supernatant, 1 ml of fresh DMEM / high glucose was added and the cells were resuspended by tapping or pipetting. Further, the optimal amount of DMEM / high glucose medium was added and 2 × 10 5 cells / cm 2 Add to a 6-well plate coated with type I collagen. After 24 hours, the medium was replaced with differentiation medium [Neurobasal plus medium (Gibco) + B-27 supplement (Gibco) + 1% penicillin-streptomycin]. After 24 hours, the medium was replaced with new differentiation medium and treated with Aβ1-42 oligomers and the present invention for 6 hours.

[0232] Example 11-3. Preparation and treatment of Aβ and the present invention

[0233] The preparation and processing were carried out using the methods described in Example 3-2.

[0234] Example 11-4. qRT-PCR method

[0235] Using Easy-Blue TM Total RNA was prepared using a total RNA extraction kit (Intron Biotechnology) and analyzed using PrimeScript TM II 1st strand cDNA synthesis kit (TAKARA), reverse transcribe 1 μg RNA. For human DKK and β-actin PCR, provide cDNA as template, use PCR master mix (TAKARA) and the next primer: human DKK1 (forward 5′-ATTCCAACGCTATCAAGAACC-3′, reverse 5′-CCAAGGTGCTATGATCATTACC-3′); human β-actin (forward 5′-CCAGGTCATCACCATTGG-3′, reverse 5′-CAGAGTACTTGCGCTCAG-3′); mouse DKK1 (forward 5'-TCTGCTAGGAGCCAGTGCC-3', reverse 5'-GATGGTGATCTTTCTGTATCC-3'); mouse β-actin (forward 5'-CTGTCCCTGTATGCCTCTG-3', reverse 5'-ATGTCACGCACGATTTCC-3'). PCR cycling conditions were as follows: denaturation, 95°C for 45 seconds; annealing, 56°C for 45 seconds; extension, 72°C for 45 seconds; 40 cycles. QuantStudio TMqRT-PCR was performed using DNA Sequencing (Thermo Scientific).

[0236] Example 11-5. Western blot analysis method

[0237] Western blot analysis was performed using the method described in Example 6-5, and the primary antibodies and conditions used in the current experiments are provided in Table 5.

[0238] Table 5. List of primary antibodies

[0239]

[0240] Example 11-6. Changes in DKK-1 expression in retinoic acid-differentiated SH-SY5Y cells and differentiated HT-22 mouse hippocampal neuronal cells by the present invention

[0241] The present invention reduces the expression of DKK-1 in mRNA ( Fig.12 ) and at the protein level ( Fig.13 ) in the expression. Considering this result, it is expected that the present invention reduces the DKK-1 expression increased by Aβ1-42 oligomers and restores synaptic plasticity by activating Wnt signaling ( Figure 14-20 ) and by inhibiting the positive feedback loop of Aβ production to reduce APP formation and Aβ accumulation ( Fig.21 , 22 ).

[0242] Example 12. Changes in Wnt gene expression in differentiated HT-22 mouse hippocampal neuronal cells by the present invention

[0243] Example 12-1. Cell culture method of HT-22 mouse hippocampal neuron cell line

[0244] Example 12-1-1. Cell subculture

[0245] The cell subculture was performed using the method described in Example 11-2-1.

[0246] Example 12-1-2. Neuronal differentiation of HT-22 cells

[0247] The cell subculture was performed using the method described in Example 11-2-2.

[0248] Example 12-3. Aβ and the preparation and treatment methods of the present invention

[0249] A 10 μM solution of human Aβ1-42 (Abcam) in DMEM / F12 (+1% FBS+1% penicillin / streptomycin) was prepared. The Aβ solution was placed at 37°C for 3 hours to form Aβ oligomers. The existing culture medium was discarded, and the cells were treated with a freshly prepared Aβ1-42 oligomer (10 μM) solution with or without the present invention (maximum concentration 5 μM) and incubated at 37°C under 5% CO 2 Incubate for 72 hours at 4 °C. After 72 hours, the culture medium was removed and the cells were washed once with PBS and recovered. Additional experiments were performed using the following procedure.

[0250] Example 12-3. qRT-PCR method

[0251] Using Easy-Blue TM Total RNA was prepared using a total RNA extraction kit (Intron Biotechnology) and PrimeScript TM II 1st strand cDNA synthesis kit (TAKARA), reverse transcribe 1 μg RNA. For Wnt and β-actin PCR, provide cDNA as template, use PCR master mix (TAKARA) and the following primers: mouse Wnt1 (forward 5′-CTCTTTGGCCGAGAGTTCGTGG-3′, reverse 5′-CCTCGGTTGCCGTAAAGGACGC-3′); mouse Wnt3a (forward 5′-CTCGCATGGCATAGATGGGTGC-3′, reverse 5′-GCAGGTGTGCACGTCATAGAC-3′); mouse Wnt5a (forward 5′-CATGGAGTGTCTGGCTCCTG-3′, reverse 5′-GTCCATCCCCTCTGAGGTCTTG-3′); mouse Wnt7a (forward 5′-CGGGAGATCAAGCAGAATGC-3′, reverse 5′-GCCTAGCTCTCGGAACTGTGGC-3′); mouse β-actin (forward 5′-CTGTCCCTGTATGCCTCTG-3′, reverse 5′-ATGTCACGCACGATTTCC-3′). PCR cycling conditions were as follows: denaturation (95°C for 45 seconds); annealing, Wnt 1, Wnt3a, Wnt 5a (60°C for 45 seconds); Wnt7a (57°C for 45 seconds); β-actin (56°C for 45 seconds); extension (72°C for 45 seconds); 40 cycles. TM qRT-PCR was performed using DNA sequencing (DNA PCR) using the R&D software (ThermoScientific).

[0252] Example 12-4. Changes in Wnt gene expression in differentiated HT-22 mouse hippocampal neurons by the present invention

[0253] Compared with the control group, the present invention increased the expression of Wnt3a gene in differentiated HT-22 mouse hippocampal neurons treated with Aβ1-42 oligomers ( Fig.14 ). Considering this result, it is expected that the present invention increases the expression of Wnt3a, which is known to increase synaptic activity important for cognitive function and activate the canonical Wnt signaling to restore synaptic plasticity.

[0254] Example 13. Changes in Wnt expression in an animal model of Alzheimer's disease (5XFAD transgenic mice) by the present invention

[0255] Example 13-1 Alzheimer's disease animal model (5XFAD transgenic mice)

[0256] The internationally widely used transgenic mouse model of Alzheimer's disease 5XFAD (C57BL / 6x SJL) was used. The characteristics of this model are that the progression of Alzheimer's disease is significantly faster than that of other mouse models genetically transformed via 5 different genes (APP KM670 / 671NL (Swedish), APP I716V (Florida), APP V717I (London), PSEN1 M146L (A>C), PSEN1 L286V). In other words, it is expected that any composition effective in treating in this model will be effective in other APP models with relatively slow progression. Brain tissues in the cortical and hippocampal regions related to cognitive function were used for analysis.

[0257] Example 13-2. Dosage of the present invention

[0258] Mice at a prime age with progressive Alzheimer's disease pathology and deteriorating cognitive function (6 months old, male) were administered daily via intraperitoneal injection at 5 mg / kg / day or 10 mg / kg / day for 4 weeks.

[0259] Example 13-3. Preparation of tissues

[0260] The present invention was administered daily via intraperitoneal injection for 4 weeks. The animals were anesthetized in the animal room using CO 2 gas, the brains were excised to measure the amount of protein expression, and the hippocampus and cerebral cortex were separated and stored in a -80 °C DeepFreezer until the protein expression was analyzed.

[0261] Example 13-5. Western blot analysis method

[0262] Using the method described in Example 6-5, Western blot analysis was performed, and the primary antibodies and conditions used for the current experiment are provided in Table 6.

[0263] Table 6. List of primary antibodies

[0264]

[0265] Example 13-6. Changes in Wnt1 expression by the present invention in an animal model of Alzheimer's disease (5XFAD transgenic mice)

[0266] The present invention increased the decreased Wnt1 expression in the hippocampus of an animal model of Alzheimer's disease (5XFAD transgenic mice) ( Fig.15 ). Considering this result, it is expected that the present invention increases the expression of Wnt1, providing an important function for synaptogenesis between neurons to restore synaptic plasticity.

[0267] Example 14. Confirmation of the activation of factors related to Wnt / β-catenin-related factors in differentiated HT-22 mouse hippocampal neuronal cells by the present invention using a Wnt / β-catenin TF Activation Profiling Plate Array

[0268] Example 14-1. Method for culturing cells of the HT-22 mouse hippocampal neuronal cell line

[0269] Example 14-1-1. Subculture of cells

[0270] Using the method described in Example 11-2-1, subculture of cells was performed.

[0271] Example 14-1-2. Neuronal differentiation of HT-22 cells

[0272] Using the method described in Example 11-2-2, subculture of cells was performed.

[0273] Example 14-2. Method for preparing Aβ and method of treating with the present invention

[0274] Prepare a 10 μM solution of human Aβ1-42 (Abcam) in DMEM / F12 (+1% FBS + 1% penicillin / streptomycin). Place the Aβ solution at 37 °C for 3 hours to form Aβ oligomers. Discard the existing medium, and treat the cells with the newly prepared Aβ1-42 oligomer (1 μM) solution, with or without the present invention (maximum concentration 5 μM), and incubate at 37 °C in 5% CO 2 2. Incubate for 6 hours. After 6 hours, remove the medium, wash the cells once with PBS and harvest. Perform additional experiments using the following procedure.

[0275] Example 14-3. Wnt / β-catenin TF activation assay plate array method

[0276] Nuclear extracts were obtained from cells and subjected to Wnt / β-catenin TF activation assay plate array (Signosis, #FA-1007) according to the protocol provided by the supplier. Relative light units were measured using a Varioskan LUX multi-function microplate reader (Thermo Fisher Scientific, USA).

[0277] Example 14-4. Measurement of activation of Wnt / β-catenin-related transcription factors by transcription factor array

[0278] The present invention increases the activity of Wnt / β-catenin-related transcription factors including VAX2, c-Myc, NR5A2, Mitf, TCF / LEF, NFAT, CEBP, GLI-1, GBX2 and AP-1 ( Fig.16 ). Considering this result, it is expected that the present invention increases the activity of Wnt / β-catenin related transcription factors to stimulate Wnt signaling activation to restore synaptic plasticity.

[0279] Example 15. Measurement of changes in Wnt / β-catenin activity in differentiated HT-22 mouse hippocampal neuronal cells by the present invention using the TOPFLASH reporter gene assay

[0280] Example 15-1. Cell culture method of HT-22 mouse hippocampal neuron cell line

[0281] Example 15-1-1. Cell subculture

[0282] The cell subculture was performed using the method described in Example 11-2-1.

[0283] Example 15-1-2. Neuronal differentiation of HT-22 cells

[0284] The cell subculture was performed using the method described in Example 11-2-2.

[0285] Example 15-2-3. Transfection of TOPFLASH reporter plasmid DNA

[0286] In a 6-well plate, Fugene HD transfection reagent (Promega, #E2311) and TOPFLASH reporter plasmid DNA (pcDNA-β-galactosidase, TOPFLASH) were added to 2×10 5cells / well and incubate the cells at 37°C in 5% CO 2 Incubate for 24 hours.

[0287] Example 15-2-3. Methods for preparing and treating Aβ and the present invention

[0288] A 1 μM solution of human Aβ1-42 (Abcam) in DMEM / F12 (+1% FBS+1% penicillin / streptomycin) was prepared. The solution of Aβ1-42 was placed at 37°C for 3 hours to form Aβ oligomers. After 24 hours of transfection, the existing cell culture medium was discarded, and the cells were treated with a freshly prepared Aβ1-42 oligomer (1 μM) solution, with or without the present invention (maximum concentration 5 μM), and incubated at 37°C under 5% CO 2 After 6 hours, the culture medium was removed and the cells were washed once with PBS and recovered. Additional experiments were performed by the following procedure.

[0289] Example 15-3. TOPFLASH reporter gene assay method

[0290] HT-22 cells were washed twice with PBS buffer, and cell lysates (100 μl / well) were prepared with lysis buffer (0.1% Triton X-100, 200 mM Tris-Cl (pH 8.0), Complete Mini protease inhibitor cocktail (Roche) and Pierce phosphatase inhibitor mini tablets) to quantify protein. Using a portion of the cell lysate, β-galactosidase was measured and light absorption was measured at 420 nm. Using the remaining cell lysate, luciferase was measured to measure luminescence. The luminescence values ​​obtained were normalized to protein concentration using β-galactosidase activity (absorbance at 420 nm) to obtain relative luciferase activity values.

[0291] Example 15-4. Results of measuring Wnt / β-catenin activity in differentiated HT-22 mouse hippocampal neuronal cells by the present invention by TOPFLASH reporter gene assay

[0292] The present invention concentration-dependently increases the Wnt / β-catenin activity in differentiated HT-22 mouse hippocampal neuronal cells that was reduced by treatment with Aβ1-42 oligomers ( Fig.17 ). Considering this result, it is expected that the present invention increases Wnt expression ( Fig.14 and 15 ) and the activity of Wnt / β-catenin-related transcription factors ( Fig.16 ), to promote the activity of Wnt signaling to restore synaptic plasticity.

[0293] Example 16. Changes in GSK3β expression in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells and differentiated HT-22 mouse hippocampal neuronal cells by the present invention

[0294] Example 16-1. Cell culture method of SH-SY5Y neurons

[0295] Example 16-1-1. Cell subculture

[0296] The cell subculture was performed using the method described in Example 3-1-1.

[0297] Example 16-1-2. SH-SY5Y neuroblastoma differentiation

[0298] SH-SY5Y cells were differentiated using the method described in Example 3-1-2.

[0299] Example 16-2. Cell culture method of HT-22 mouse hippocampal neuron cell line

[0300] Example 16-2-1. Cell subculture

[0301] Cell subculture was performed using the method described in 11-2-1.

[0302] Example 16-2-2. Neuronal differentiation of HT-22 cells

[0303] Cell subculture was performed using the method described in 11-2-2.

[0304] Example 16-3. Method for preparing Aβ and method for treating with the present invention

[0305] The preparation and processing were carried out using the methods described in Example 3-2.

[0306] Example 16-4. Western blot analysis method

[0307] Western blot analysis was performed using the method described in Example 9-4, and the primary antibodies and their conditions are provided in Table 7.

[0308] Table 7. List of primary antibodies

[0309]

[0310] Example 16-5. Phospho-GSK-3b (Ser9) Sandwich ELISA

[0311] HT-22 cells (1.5×10 5Cells / well) were incubated in 6-well plates (SPL, #30006). The culture medium was replaced with Neurobal plus culture medium with B-27 supplement, and 24 hours later, cells were treated with Aβ1-42 oligomers (1 μM) and the present invention (1, 2, 5 μM) for 6 hours. The cell culture medium was removed and the cells were washed three times with PBS buffer, and cell lysis buffer (Cell Signaling, #9803) was added to each well (100 μl / well). The cells were placed on top of ice for 5 minutes, and the cell lysate was discarded to change to 1.5 ml, and the cell lysate was homogenized using ultrasound using a Bioruptor device. The homogenized cell lysate was centrifuged (14,000 rpm, 10 minutes) and the Pierce TM The supernatant was analyzed by BCA protein assay kit to quantify the amount of protein. The microwells coated with GSK-3β mouse mAb included in the PathScan phosphorylated-GSK-3β (Ser9) sandwich ELISA kit (CellSignaling, #7311C) were stabilized at room temperature for 30 minutes, and the same amount of protein (30 μg / well) diluted with ELISA sample diluent was added to each microwell, and the mixture was reacted at 4 ° C for 16 hours. Each microwell was washed four times with ELISA wash buffer, and ELISA experiments were performed using phosphorylated-GSK-3β (Ser9) rabbit detection mAb and anti-rabbit IgG (HRP-linked antibody) (ELISA formulated). TMB substrate solution was used as an ELISA substrate to measure the absorption at 450nm to determine the relative amount of phosphorylated-GSK-3β (Ser9).

[0312] Example 16-6. Changes in GSK3β expression in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells and differentiated HT-22 mouse hippocampal neuronal cells by the present invention

[0313] Reviewing the effects of the present invention on the phosphorylation of GSK3β protein, as shown by Western blot analysis, the present invention increased Ser9 phosphorylation of the protein decreased by Aβ1-42 oligomers in retinoic acid-differentiated SH-SY5Y cells ( Fig.18 ), and phosphorylated-GSK-3b (Ser9) sandwich ELISA showed that the present invention increased Ser9 phosphorylation of proteins decreased by Aβ1-42 oligomers in differentiated HT-22 mouse hippocampal neuronal cells ( Fig.19 ). Considering these results, it is expected that the present invention increases Ser9 phosphorylation of proteins to inactivate GSK3 to reduce phosphorylation of Tau proteins to reduce Tau pathology.

[0314] Example 17. Changes in Tau expression in an Alzheimer's dementia animal model (NSE-Happ-C105) by the present invention

[0315] Example 17-1. Experimental Animals

[0316] The animals used in the experiment were C57BL / 6-Tg (NSE-hAPP-C105) Kor transgenic mice (13 months) in which Alzheimer's dementia was induced by overexpressing a mutant APP gene expressing only the 105 amino acids at the C-terminus of APP in brain tissue. The animals were housed in conditions such as temperature of 20±2°C, humidity of 50%, lighting of 08:00-20:00, and lights out of 20:00-08:00. The groups were determined as control / untreated groups [non-tg control, NTC (n=6), Alzheimer's disease groups [tg-control, TC (n=6)], Alzheimer's disease groups treated with the present invention [tg-present invention, TM (n=6)], and food and water were supplied without restriction during the experiment.

[0317] Example 17-2. Administration method of the present invention

[0318] After a basic test of the Alzheimer's disease model including mouse memory test, water maze behavior test, motor function test and passive avoidance test, the human daily dose of the present invention, based on Reagan-Shaw, et al. (2008), was administered to 13-month-old Alzheimer's disease model mice once a day by intraperitoneal injection at 4 mg / kg for 4 weeks. The control group (NTC) and the Alzheimer's disease group (TC) were administered with the same amount of normal saline.

[0319] Example 17-3. Tissue preparation

[0320] The present invention is administered daily by intraperitoneal injection for 4 weeks using CO 2 The animals were anesthetized with gas in an animal room, the brains were removed to measure the amount of protein expression, and the hippocampus and cerebral cortex were separated and stored in a -80°C deep freezer until the protein expression was analyzed.

[0321] Example 17-4. Western blot analysis method

[0322] Western blot analysis was performed using the method described in Example 9-4, and the primary antibodies and their conditions are provided in Table 8.

[0323] Table 8. List of primary antibodies

[0324]

[0325] Example 17-5. Changes in Tau expression in an Alzheimer's dementia animal model (NSE-Happ-C105) by the present invention

[0326] The present invention reduces the Ser199 / 202 phosphorylation in the Tau protein increased in the hippocampus of the Alzheimer's dementia animal model (NSE-hAPP-C105) Fig. 20 ). Considering this result, it is expected that the present invention reduces Ser199 / 202 phosphorylation in Tau protein to suppress the aggregation of Tau protein, thereby reducing the formation of NFTs and reducing Tau pathology.

[0327] Example 18. Changes in APP formation and Aβ accumulation in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells by the present invention

[0328] Example 18-1. Cell culture method of SH-SY5Y neurons

[0329] Example 18-1-1. Cell subculture

[0330] The cell subculture was performed using the method described in Example 5-1-1.

[0331] Example 18-1-2. SH-SY5Y neuroblastoma differentiation

[0332] Using the method described in Example 5-1-2, SH-SY5Y neuronal cells were differentiated.

[0333] Example 18-2. Method for preparing Aβ and method for treating with the present invention

[0334] Example 18-2-1. Method for preparing Aβ and method for treating with the present invention

[0335] The preparation and processing were carried out using the methods described in Example 5-2.

[0336] Example 18-2-2. For the preparation of H 2 O 2 Method and method of treating with the present invention

[0337] Every 24 hours, 100 μM H 2 O 2 DMEM / F12 (+1% FBS+1% penicillin / streptomycin) was added to the differentiated SH-SY5Y cells and renewed three times, and the present invention was added at various concentrations (10, 20, 40 μM) for 24 hours.

[0338] Example 18-3. Western blot analysis method

[0339] Western blot analysis was performed using the method described in Example 6-5, and the primary antibodies and their conditions are provided in Table 9.

[0340] Table 9. List of primary antibodies

[0341]

[0342] Example 18-4. Changes in APP formation and Aβ accumulation in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells by the present invention

[0343] In retinoic acid-differentiated SH-SY5Y cells, the present invention reduces the expression of Aβ1-42 oligomers ( Fig.21 ) and H 2 O 2 ( Fig. 22 Considering this result, it is expected that the present invention can reduce APP formation and Aβ accumulation by inhibiting the positive feedback loop of Aβ production.

[0344] Example 19. Changes in Aβ expression in an Alzheimer's dementia animal model (NSE-Happ-C105) by the present invention

[0345] Example 19-1. Experimental Animals

[0346] The same type of animal model described in Example 17-1 was used.

[0347] Example 19-2. Administration method of the present invention

[0348] The present invention was administered using the same method described in Example 17-2.

[0349] Example 19-3. Preparation of tissue

[0350] Tissue samples were prepared and stored using the methods described in Example 17-3.

[0351] Example 19-4. Western blot analysis method

[0352] Western blot analysis was performed using the method described in Example 6-5, and the primary antibodies and their conditions are provided in Table 10.

[0353] Table 10. List of primary antibodies

[0354]

[0355] Example 19-5. Results of changes in Aβ expression in an Alzheimer's dementia animal model (NSE-hAPP-C105)

[0356] The present invention effectively reduces the expression of Aβ in the hippocampus of an Alzheimer's dementia animal model (NSE-hAPP-C105). Fig.23 ). Based on this result, it is expected that the present invention effectively suppresses the formation and accumulation of Aβ.

[0357] Example 20. Changes in Aβ plaques in an Alzheimer's disease animal model (5XFAD transgenic mice) by the present invention

[0358] Example 20-1 Alzheimer's disease animal model (5XFAD transgenic mice)

[0359] The animal model described in Example 13-1 was used.

[0360] Example 20-2. Administration of the composition of the present invention

[0361] The composition of the present invention was administered using the method described in Example 13-2.

[0362] Example 20-3. Preparation and fixation of tissue samples

[0363] The invention was administered daily by intraperitoneal injection for 4 weeks and CO 2 The animals were anesthetized with gas in an animal room, after which the chest was opened to administer 50 mM PBS (phosphate buffered saline) to the left ventricle for 3 minutes and perfused with a fixative solution in which 4% PFA (paraformaldehyde) was dissolved in 0.1 M phosphate buffer for 10 minutes. After perfusion and fixation, the brain was removed and added to a 4% PFA fixative solution at 4°C for 12 hours of fixation, followed by precipitation of the tissue in a 30% sucrose solution for 5 days and cutting through the

[0364] Serial tubular sections at 40 μm thickness were cut using a cryostat (Leica) for storage.

[0365] Example 20-4. Aβ plaque staining method using Thioflavin S staining G and GFAP (glial fibrillary acid protein) immunohistochemistry (IHC) method

[0366] Mouse brain tissue was fixed in 4% paraformaldehyde (pH 7.4) solution for 24 hours, and then dehydrated with 30% sucrose and frozen sections were prepared. The primary antibodies and conditions are provided in Table 11. After antigen retrieval of frozen sections of brain tissue using 1% SDS, the samples were reacted with glial fibrillary acid protein (GFAP) antibody at 4°C for 16 hours. The samples were treated with 500 μM Thioflavin S in 50% ethanol for 7 minutes for Thioflavin S staining. Alexa Fluor 594 goat anti-rabbit (IgG) secondary antibody was reacted with GFAP antibody / Thioflavin S-treated brain tissue cryosections, and Hoechst 33342 (Sigma-Aldrich) was used for nuclear staining.

[0367] Table 11. List of primary antibodies

[0368]

[0369] Example 20-5. Changes in Aβ plaques in the Alzheimer's disease animal model (5XFAD transgenic mice) 7260 by the present invention

[0370] The present invention reduces the number of Aβ plaques in the hippocampus and cerebral cortex of an Alzheimer's disease animal model (5XFAD transgenic mice) Fig.24 ), which was determined by counting the number of Aβ plaques (green) stained by Thioflavin S and the number of co-localized points (yellow) of astrocytes (red) stained with GFAP. In view of this result, it is expected that the present invention inhibits Aβ production and Aβ accumulation to reduce extracellular Aβ plaque formation.

[0371] Example 21. Changes in AMPK expression in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells by the present invention

[0372] Example 21-1. Cell culture method of SH-SY5Y neurons

[0373] Example 21-1-1. Cell subculture

[0374] The cell subculture was performed using the method described in Example 3-1-1.

[0375] Example 21-1-2. SH-SY5Y neuroblastoma differentiation

[0376] Using the method described in Example 3-1-2, SH-SY5Y neuronal cells were differentiated.

[0377] Example 21-2. Method for preparing Aβ and method for treating with the present invention

[0378] A 1 μM solution of human Aβ1-42 (Abcam) in DMEM / F12 (+1% FBS+1% penicillin / streptomycin) was prepared. The Aβ solution was placed at 37°C for 3 hours to form Aβ oligomers. The existing culture medium was discarded, and the cells were treated with a freshly prepared Aβ1-42 oligomer (1 μM) solution with or without the present invention (maximum concentration 10 μM) and incubated at 37°C under 5% CO 2Incubate for 72 hours at 4 °C. After 72 hours, the culture medium was removed and the cells were washed once with PBS and recovered. Additional experiments were performed using the following procedure.

[0379] Example 21-3. Western blot analysis method

[0380] Western blot analysis was performed using the method described in Example 9-4, and the primary antibodies and their conditions are provided in Table 12.

[0381] Table 12. List of primary antibodies

[0382]

[0383] Example 21-4. Changes in AMPK expression in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells by the present invention

[0384] The present invention concentration-dependently increases the phosphorylation of Thr172 of AMPK catalytic subunit α, which is decreased by Aβ1-42 oligomer treatment in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells ( Fig.25 ). Considering this result, the present invention is expected to increase the phosphorylation of Thr172 in the AMPK catalytic subunit α decreased by Aβ to restore AMPK activity to activate autophagy to remove misfolded protein aggregates associated with degenerative brain diseases and inhibit neuronal degeneration.

[0385] Example 22. Changes in the expression of autophagy markers including LC3B and P62 by the present invention in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells

[0386] Example 22-1. Cell culture method of SH-SY5Y neurons

[0387] Example 22-1-1. Cell subculture

[0388] The cell subculture was performed using the method described in Example 2-1-1.

[0389] Example 22-1-2. SH-SY5Y neuroblastoma differentiation

[0390] Using the method described in Example 2-1-2, SH-SY5Y neuronal cells were differentiated.

[0391] Example 22-2. Method for preparing Aβ and method for treating with the present invention

[0392] A 1 μM solution of human Aβ1-42 (Abcam) in DMEM / F12 (+1% FBS+1% penicillin / streptomycin) was prepared. The Aβ solution was placed at 37°C for 3 hours to form Aβ oligomers. The existing culture medium was discarded, and the cells were treated with a freshly prepared Aβ1-42 oligomer (1 μM) solution, with or without the present invention (maximum concentration 40 μM) and 3-MA (3-methyladenine, Sigma-Aldrich) (5 mM), autophagy / PI3K (phosphoinositide 3-kinase) inhibitor, and incubated at 37°C in 5% CO 2 Incubate for 72 hours at 4 °C. After 72 hours, the culture medium was removed and the cells were washed once with PBS and recovered. Additional experiments were performed using the following procedure.

[0393] Example 22-3. Western blot analysis method

[0394] Western blot analysis was performed using the method described in Example 6-5, and the primary antibodies and their conditions are provided in Table 13.

[0395] Table 13. List of primary antibodies

[0396]

[0397] Example 22-4. Changes in the expression of autophagy markers including LC3B and P62 caused by the present invention in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells

[0398] The present invention not only increases the expression of the autophagy marker LC3B reduced by Aβ1-42 oligomers in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells, but also induces autophagic flux (LC3B-I to LC3B-II conversion) to simultaneously increase the LC3B-II / I ratio, and reduces the expression of ubiquitin-binding protein p62 (SQSTM, Sequestosome 1) (autophagosome cargo protein), which is increased by Aβ1-42 oligomers. In addition, using 3-MA (autophagy / PI3K inhibitor) together with the present invention reduces the autophagic flux (LC3B-II / I ratio) increased by the present invention, and increases the expression of p62 reduced by the present invention. In addition, when cells are treated with the present invention and 3-MA together, APP formation and Aβ1-42 accumulation increase ( Fig.26 ). Considering this result, it is expected that the present invention suppresses APP formation and Aβ1-42 accumulation by activating autophagy.

[0399] Example 23. Changes in the expression of autophagy markers including ATG7 and ATG5 / 12 by the present invention in an Alzheimer's disease animal model (5XFAD transgenic mice)

[0400] Example 23-1 Alzheimer's disease animal model (5XFAD transgenic mice)

[0401] The animal model described in Example 13-1 was used.

[0402] Example 23-2. Administration of the composition of the present invention

[0403] The composition was administered using the same method described in Example 13-2.

[0404] Example 23-3. Preparation of tissue samples

[0405] Tissue samples were prepared and stored using the methods described in Example 13-3.

[0406] Example 23-4. Western blot analysis method

[0407] Western blot analysis was performed using the method described in Example 6-5, and the primary antibodies and their conditions are provided in Table 14.

[0408] Table 14. List of primary antibodies

[0409]

[0410] Example 23-5. Changes in the expression of autophagy markers including ATG7 and ATG5 / 12 by the present invention in an Alzheimer's disease animal model (5XFAD transgenic mice)

[0411] The present invention increases the expression of autophagy markers including ATG7 and ATG5 / 12 in the hippocampus of an Alzheimer's disease animal model (5XFAD transgenic mice), and increases the expression of ATG5 / 12 in the cerebral cortex ( Fig. 27 Considering this result, it is expected that the present invention increases autophagic flux (LC3BII / I ratio), autophagic markers, and decreases p62 ( Fig.26 ), and increase the expression of ATG7 and ATG5 / 12 to activate the autophagy cascade.

[0412] Example 24. Effect of the present invention on improving cognitive and behavioral learning abilities in an Alzheimer's dementia animal model (NSE-hAPP-C105)

[0413] Example 23-1. Experimental Animals

[0414] The animal model described in Example 17-1 was used.

[0415] Example 23-2. Administration method of the present invention

[0416] The composition was administered using the same method described in Example 17-2.

[0417] Example 23-3. Testing methods for cognitive ability and behavioral learning ability

[0418] Example 23-3-1. Morris water maze test

[0419] Before using the water maze test to measure changes in cognitive ability, the present invention was administered by intraperitoneal injection at 4 mg / kg, once a day, for 4 weeks. The test was carried out in a circular water bath (1 m in diameter × 40 cm in height) at 22-25 ° C of water, wherein the target (target: 12 cm in diameter) was set about 3 cm lower than the water surface. Dry milk powder was added to the water to make the target invisible, and a SMART 3.0protram (Panlab) was installed on the ceiling above the water bath to monitor the time (latency to the target) of the test animals reaching the target before and after 4 weeks of the test (latency to the target), the swimming distance (distance to the target) and the swimming pattern (swimming pattern). The test subjects were trained in the first 5 days, twice a day, starting from the starting point and ending at the target. Mice that did not find the target twice were made to identify the location of the target. Each test was performed at an interval of 5 minutes, and the target was removed on the 6th day to perform a test lasting 1 minute from the starting point, and the results were used as experimental data.

[0420] Example 23-3-2. Passive avoidance test

[0421] The present invention was administered by intraperitoneal injection with a 4mg / kg dosage, once a day, for 4 weeks, and then a passive avoidance test was performed to evaluate memory. The passive avoidance test includes a front room (18×18×25cm) for a white bright room, a back room (18×18×25cm) for a black dark room, and a sturdy metal stainless steel is installed on the floor of the dark room, and a hole with a 4cm diameter is provided on the wall of the front room and the back room, which is opened and closed with a gate type. Each test animal is isolated in each cage for 1 minute, moved to the front room for 10 seconds to adjust, and the gate type door is opened simultaneously to allow the test animal to move freely between the rooms. Once all four feet of the test animal enter the back room, the door is closed rapidly, and the time (initial latency time) of the test animal moving from the front room to the back room is recorded, and energization (0.5mA) lasts for 2 seconds. After 5 seconds, the test animal is moved to its living cage. After 72 hours, the same test was performed to measure the latency to enter darkness, moving from the front chamber to the back chamber until a maximum of 300 seconds.

[0422] Example 23-3-3. Data processing method

[0423] The collected data were processed using the SPSS 20.0 statistical program to calculate statistical errors (mean ± SD), and the variables between species and groups were verified by one-way analysis of variance (One-Way ANOVA). If there were significant differences between the groups, the Bonferroni method was used for post-validation. For this purpose, the hypothesis acceptance was set to α = 0.05.

[0424] Example 23-4. Improvement of cognitive and behavioral learning skills in an Alzheimer's dementia animal model (NSE-hAPP-C105) by the present invention

[0425] Example 23-4-1. Results of the present invention in improving cognitive function in an Alzheimer's dementia animal model (NSE-hAPP-C105): Water maze test (Morris water maze test)

[0426] C57BL / 6-Tg(NSE-hAPP-C105)Kor transgenic mice were subjected to the Morris water maze test to measure the effects of the present invention on cognitive function after 4 weeks of treatment. First, the analysis of the time to reach the target showed that the TC group was statistically increased compared with the NTC group (P=0.001), and the TM group was statistically decreased compared with the TC group (P=0.001). Second, the swimming distance to reach the target was analyzed, and the TC group was statistically increased compared with the NTC group (P=0.001), and the TM group was statistically decreased compared with the TC group (P=0.001). Third, the analysis of the swimming time from the target in the quadrant showed that the TC group was statistically decreased compared with the NTC group (P=0.001), and the TM group was statistically increased compared with the TC group (P=0.001). Fourth, analysis of the time to pass the target showed that the TC group was statistically lower than the NTC group (P = 0.001), and the TM group was statistically higher than the TC group (P = 0.037) ( Fig.28 ).

[0427] Example 23-4-2. Results of the present invention on improvement of action learning and cognitive ability in an Alzheimer's dementia animal model (NSE-hAPP-C105): Passive avoidance test

[0428] The passive avoidance test was performed using C57BL / 6-Tg(NSE-hAPP-C105)Kor transgenic mice to evaluate the effects of the present invention on motor learning and cognitive abilities for 4 weeks ( Figure 6 The results showed that the TC group was statistically lower than the NTC group (P = 0.001), and the TM group was statistically higher than the TC group (P = 0.004) ( Fig.29 ).

Claims

1. A pharmaceutical composition for treating Alzheimer's disease, include: A compound selected from the group consisting of milonafil, sildenafil, vardenafil, tadalafil, udenafil, dasenstafil and avanafil; and pharmaceutically acceptable salts, solvates and hydrates thereof.

2. The pharmaceutical composition according to claim 1, in, The compounds increase blood flow in the brain through vasodilation to inhibit the formation and accumulation of extracellular A[beta] monomers, oligomers and / or A[beta] fibrils / plaques.

3. The pharmaceutical composition according to claim 1, in, The compounds activate the NO / cGMP / PKG / CREB pathway to reduce neuronal cell death and promote neurogenesis, synaptogenesis, and angiogenesis.

4. The pharmaceutical composition according to claim 1, in, The compound inhibits DKK-1 (Dickkopf WNT signaling pathway inhibitor 1) to activate Wnt signaling to restore synaptic plasticity and inhibits the positive feedback loop of Aβ production to reduce the formation of APP (amyloid precursor protein) and the accumulation of Aβ.

5. The pharmaceutical composition according to claim 1, in, The compounds activate autophagy to remove toxic soluble Aβ oligomers within cells to suppress the formation and accumulation of Aβ fibrils / plaques.

6. The pharmaceutical composition according to claim 1, in, The compounds improve behavioral and cognitive functions.

7. Use of a pharmaceutical composition in the preparation of a medicament for treating Alzheimer's disease, The pharmaceutical composition comprises a compound selected from the group consisting of milonafil, sildenafil, vardenafil, tadalafil, udenafil, dasenstafil and avanafil; and pharmaceutically acceptable salts, solvates and hydrates thereof.

8. The use according to claim 7, in, The compounds inhibit the formation of Aβ aggregation by reducing the formation of Aβ oligomers or fibrils.

9. The use according to claim 7, in, The compounds inhibit the processing of beta-amyloidogenic protein through BACE-1 reduction.

10. The use according to claim 7, in, The compounds reduce the extracellular formation and accumulation of amyloid-β monomers, oligomers and / or amyloid-β fibrils and plaques through vasodilation.

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