Composition and method for reducing amyloid beta formation and composition thereof

Abstract

To provide a pharmaceutical composition for treating Alzheimer's disease, a method for reducing amyloid beta formation, and a method for treating a disease associated with the accumulation of amyloid beta.SOLUTION: The present invention provides a pharmaceutical composition comprising a compound selected from the group consisting of mirodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasantafil, and avanafil, and a pharmaceutically acceptable salt, solvate, and hydrate thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority from U.S. Provisional Application No. 62 / 822,975, filed on March 24, 2019, the content of which is incorporated herein by reference.

[0002] The present invention relates to methods for reducing amyloid - beta formation and for treating diseases associated with the accumulation of amyloid - beta.

Background Art

[0003] Dementia refers to a clinical disease that suffers from multiple cognitive impairments (or MCD, Multiple Cognitive Deficits) as an acquired brain disease showing a multifaceted etiology caused by various genetic and environmental risk factors. The representative disease causing dementia is Alzheimer's disease, which mainly prevails among the elderly and accounts for 60 - 70% of all dementia cases (see Non - Patent Document 1). In recent years, with the rapid aging of the population, the more progressive dementia of the elderly due to Alzheimer's type dementia has increased, and the need for the treatment of dementia has also increased. However, the further difficulties in research and development with a low success rate have delayed the development process of new therapeutic drugs and treatment methods for dementia. Based on the data of GBI Research in 2014, the success rate of the final approval of Alzheimer's disease therapeutic drugs is less than 1%, 72% in Phase II clinical trials, 92% in Phase III clinical trials, 99.6% of NDA applications fail, and about 80% of the current ongoing pipeline is only in the "exploratory and / or pre - clinical stage".

[0004] The current conventional list of FDA-approved drugs includes AChE (acetylcholinesterase) inhibitors and NMDA (N-methyl-D-aspartic acid) receptor antagonists, which are used in combination with antioxidants, NSAIDs (non-steroidal anti-inflammatory drugs), anti-inflammatory agents, statin preparations, or hormone preparations. However, these drugs are only used to relieve symptoms and delay and improve cognitive impairment, and there is currently no fundamental treatment for dementia.

[0005] Typical AChE inhibitors include donepezil (Aricept (trademark)), galantamine (Reminyl (trademark)), rivastigmine (ENA-713, Exelon (trademark)), etc. These drugs temporarily increase the concentration of the neurotransmitter acetylcholine and provide symptomatic treatment. In addition, these drugs are prescribed to patients with mild to moderate Alzheimer's disease, vascular dementia, dementia due to Parkinson's disease, and stroke or subcortical ischemic vascular disease (see Non-Patent Document 2).

[0006] Typical NMDA receptor antagonists include memantine (Ebixa (trademark)), which inhibits the glutamate transport system that causes excitotoxicity, blocks synaptic plasticity, and effectively reduces neuronal degeneration. Memantine has been confirmed to be effective for moderate to severe dementia and Lewy body dementia with relatively low side effects, but it showed only limited activity in earlier stages of the disease (see Non-Patent Document 3). Therefore, it has been used for mild and severe dementia in combination with ChE inhibitors rather than as monotherapy. However, no clear evidence has been found so far that the combination of AChE inhibitor and memantine is more effective than AChE alone, and additional research is needed (see Non-Patent Document 4).

[0007] However, although these drugs have been used for a long time in the treatment of dementia and Alzheimer's disease, there are no clear criteria for their use. Moreover, even at the maximum recommended human dose (MRHD), only either improvement was not seen to lead patients to the end stage of treated patients or the disease progression continued, and there has been a continuous concern about the use of these drugs. However, there are no therapeutics whose efficacy has been confirmed to date, and there is no option but to continue the efforts for new treatment development for dementia (see Non-Patent Document 5).

[0008] The main targets for candidates for the treatment of dementia developed so far include the following: (1) inhibitors of BACE-1 (β-secretase 1) or γ-secretase inhibitors for inhibiting the production of Aβ (amyloid β), (2) anti-Aβ monoclonal antibodies for removing Aβ (amyloid β), (3) tau aggregation inhibitors and inhibitors of (one amino acid kinase) TAOK inhibitors for inhibiting tau aggregation and phosphorylation, (4) AChE inhibitors and NMDA receptor antagonists for blocking AchE and NMDA receptors, etc. Recently, the pipelines targeting Aβ plaques and tau proteins have been increasing, and among them, about 70 - 80% of the candidates target the suppression of Aβ production or Aβ removal. In addition, about 30% of the pipelines are biopharmaceuticals with a predominately high ratio of monoclonal antibodies or peptides. However, the clinical trials of promising candidates, including anti-Aβ monoclonal antibodies targeting Aβ such as Aducanumab (Biogen), Solanezumab (Eli Lilly), and Gantenerumab (Roche), have recently failed, making the assumption of Aβ as a target for the treatment of dementia uncertain. Nevertheless, it is generally accepted that the main targets for the development of therapeutics for dementia are the suppression of Aβ formation and its removal.

[0009] One of the challenges in the development of Aβ-targeted drugs is that monoclonal antibodies with large molecular weights have great difficulty penetrating cell membranes. Therefore, it may be effective only by removing extracellular amyloid plaques, and its effectiveness for removing intracellular Aβ oligomers is limited. Furthermore, intracellular Aβ oligomers have been reported to cause more cytotoxicity than extracellular Aβ oligomers and play a more important role in neuronal death (see Non-Patent Document 6). Therefore, the development of small molecule drugs that can penetrate cell membranes rather than macromolecules such as monoclonal antibodies for removing intracellular Aβ oligomers.

[0010] One of the recent views attracting attention in this field is that the reason for the failure in the development direction of dementia therapeutic drugs may be the "one drug, one target" paradigm, which is a conventional approach, because dementia, a clinical disorder, is a disease with a multifaceted etiology. It is recognized that this conventional approach cannot achieve the success of developing dementia therapeutic drugs. Currently, in order to overcome the above problems, the interest in the development of new therapeutic drugs based on the "one drug, multiple targets / mechanisms" paradigm is increasing. Approaches such as using one compound with multiple target / multiple mechanism activities represent a new concept in the discovery and development of new drugs (see Non-Patent Documents 7 and 8). Therefore, in order to overcome the difficulties in the development of dementia therapeutic drugs and increase the success rate of development, it is necessary to develop small molecule / combination pharmacological drugs based on the "one drug, multiple targets / mechanisms" paradigm rather than the conventional "one drug, one target" paradigm, especially considering the multifaceted etiology of dementia, from the initial stage of candidate discovery and in the preclinical stage (see Non-Patent Documents 9 and 10).

Prior Art Documents

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

[0012] One embodiment of the present invention provides a method for inhibiting the formation of Aβ fibrils / plaques by removing intracellular toxic soluble Aβ oligomers by administering one of a compound selected from milodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasantafil, and avanafil, and a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0013] Another embodiment of the present invention is for (1) inhibition of Aβ oligomer / fibril formation by suppression of Aβ aggregation, (2) inhibition of β-amyloid formation processing by reduction of BACE-1, (3) reduction of extracellular Aβ monomer, oligomer, Aβ fibril / plaque formation by increased cerebral blood flow (vasodilation), (4) inhibition of neuronal cell death and promotion of neurogenesis, synaptogenesis and / or angiogenesis by activation of the NO (nitric oxide) / cGMP (cyclic guanosine monophosphate) / PKG (protein kinase G), CREB (cyclic AMP (adenosine monophosphate) response element binding protein) pathway, (5) restoration of synaptic plasticity (synaptic plasticity) by activation of Wint signaling by inhibition of DKK-1 (Dickkopf WNT signaling pathway inhibitor 1), and reduction of Aβ accumulation by inhibition of APP (amyloid precursor protein) production and suppression of the positive feedback loop for Aβ production, and (6) inhibition of Aβ fibril / plaque formation by removal of intracellular toxicity and soluble Aβ oligomers by activation of autophagy.

[0014] In another embodiment, the present invention provides a pharmaceutical composition for (1) inhibition of Aβ oligomer / fibril formation by reduction of Aβ aggregation, (2) inhibition of BACE-1 with reduced β-amyloid formation processing, (3) reduction of extracellular Aβ monomer, oligomer and Aβ fibril / plaque by increased cerebral blood flow, (4) inhibition of neuronal cell death and promotion of neurogenesis, synaptogenesis and / or angiogenesis by activation of the NO / cGMP / PKG / CREB pathway, (5) restoration of synaptic plasticity (synaptic plasticity) by activation of Wint signaling by inhibition of DKK-1, and reduction of Aβ accumulation by inhibition of APP production and suppression of the positive feedback loop for Aβ production, and (6) inhibition of Aβ fibril / plaque formation by removal of intracellular toxicity and soluble Aβ oligomers by activation of autophagy, comprising one compound selected from milodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasanafil, and avanafil, and pharmaceutically acceptable salts, solvates, and hydrates thereof.

Brief Description of the Drawings

[0015]

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Mode for Carrying Out the Invention

[0016] One embodiment of the present invention is (1) inhibition of the formation of Aβ oligomers / fibrils by suppression of Aβ aggregation, (2) inhibition of β-amyloid formation processing by reduction of BACE-1, (3) reduction of the formation of extracellular Aβ monomers, oligomers, Aβ fibrils / plaque by increased cerebral blood flow (vasodilation), (4) inhibition of neuronal cell death and promotion of neurogenesis, synaptogenesis and / or angiogenesis by activation of the NO (nitric oxide) / cGMP (cyclic guanosine monophosphate) / PKG (protein kinase G), CREB (cyclic AMP (adenosine monophosphate) response element binding protein) pathway, (5) recovery of synaptic plasticity by activation of the Wint signaling pathway by inhibition of DKK-1 (Dickkopf WNT signaling pathway inhibitor 1), and reduction of Aβ accumulation by inhibition of the production of APP (amyloid precursor protein) and suppression of the positive feedback loop for Aβ production, and (6) inhibition of the formation of Aβ fibrils / plaque by activation of autophagy and removal of intracellular toxicity and soluble Aβ oligomers by administration of one of the compounds selected from milodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasanafil, and / or avanafil, and pharmaceutically acceptable salts, solvates, and hydrates thereof. A method is provided for this.

[0017] In another embodiment, the present invention provides a pharmaceutical composition for (1) inhibiting Aβ oligomer / fibril formation by reducing Aβ aggregation, (2) inhibiting BACE-1 which reduces β-amyloid forming processing, (3) reducing extracellular Aβ monomer, oligomer and Aβ fibril / plaque by increasing cerebral blood flow, (4) suppressing the inhibition of neuronal cell death and promoting neurogenesis, synaptogenesis and / or angiogenesis by activating the NO / cGMP / PKG / CREB pathway, (5) restoring synaptic plasticity (synaptic plasticity) by activating the Wnt signaling by inhibiting DKK-1, and reducing Aβ accumulation by inhibiting the production of APP and suppressing the positive feedback loop for Aβ production, and (6) inhibiting the formation of Aβ fibril / plaque by removing intracellular toxicity and soluble Aβ oligomers by activating autophagy, comprising one compound selected from milodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasanafil, and avanafil, and a pharmaceutically acceptable salt, solvate, and hydrate thereof.

[0018] The present invention has a plurality of mechanisms that distinguish it from other Alzheimer's therapeutics developed previously or currently under development. The present invention provides for (1) inhibition of Aβ oligomer / fibril formation by reduction of Aβ aggregation, (2) inhibition of BACE-1 which reduces β-amyloid forming processing, (3) reduction of extracellular Aβ monomer, oligomer and Aβ fibril / plaque by increase in cerebral blood flow, (4) suppression of neuronal cell death, and promotion of neurogenesis, synaptogenesis and / or angiogenesis by activation of the NO / cGMP / PKG / CREB pathway, (5) restoration of synaptic plasticity (synaptic plasticity) by activation of the Wnt signaling by inhibition of DKK-1, and reduction of Aβ accumulation by inhibition of APP production and suppression of the positive feedback loop for Aβ production, and (6) inhibition of Aβ fibril / plaque formation by removal of intracellular toxicity and soluble Aβ oligomers by activation of autophagy. By adopting a "one drug, multiple targets / mechanisms" strategy that is superior to the conventional discovery strategies based on the "one drug, one target" paradigm, it provides significantly improved efficacy for the treatment of Alzheimer's disease compared to current drugs on the market.

[0019] Therapeutic Mechanisms for Alzheimer's Disease 1. Inhibition of Aβ oligomer / fibril formation by inhibition of Aβ aggregation The common pathological features in sporadic and familial Alzheimer's dementia are peptides known as Aβ accumulation that forms extracellular senile plaques (J Alzheimers Dis 2018;64(s1):S567-S610.). Aβ is produced even in normal people, but it is degraded quickly enough not to accumulate in the body. On the other hand, in the case of Alzheimer's disease patients, Aβ is not only formed in large amounts but also not degraded in tissues and accumulates (Alzheimers Res Ther 2013 Nov 29;5(6):60.). These abnormal Aβ accumulations prevent signal transmission between nerve cells, form extracellular senile plaques, induce accumulations in the hippocampus and cortex, which play important roles in memory and learning abilities, cause cell inflammation and nerve cell damage, and ultimately damage the nerve cell network necessary for normal functions (EBioMedicine 2016 Apr;6:42-49.). In addition, the accumulated Aβ monomers aggregate to form toxic Aβ oligomers, increase the production of ROS (reactive oxygen species) and RNS (reactive nitrogen species), and activate the signal network related to nerve cell death that increases apoptosis (Mt Sinai J Med. 2010 Jan-Feb;77(1):43-9.).

[0020] The composition of the present invention inhibited Aβ fibril aggregation (Figure 1) and (Figure 2) in order to inhibit Aβ oligomer / fibril formation.

[0021] Therefore, the composition of the present invention inhibits Aβ oligomer aggregation oligomers and inhibits Aβ oligomer / fibril formation.

[0022] 2. Suppression of β-amyloid formation processing by reducing BACE-1 Aβ is produced from APP as various secretase-cleaved APPs to generate Aβ with various numbers of amino acids. Among these, in the case of Alzheimer's patients, the ratio of Aβ to 42 or 43 amino acids formed by BACE-1 increases rapidly. Currently, it is known that neuronal damage by Aβ1-42 or Aβ1-43 is one of the important causes in the progression of Alzheimer's disease, and Aβ25-35 is known to be a toxic fragment of Aβ1-42 or Aβ1-43 that causes neuronal damage (J Amino Acids 2011;2011:198430., PLoS One 2013;8(1):e53117, J Alzheimers Dis 2018;62(3):1345-1367). In particular, recently, sildenafil, a PDE-5 inhibitor, has been reported to suppress BACE-1 and cathepsin B expression, inhibit the β-amyloid forming processing of APP, and reduce the production of Aβ1-42 or Aβ1-43. It was predicted that PDE-5 inhibitors would have a therapeutic effect on Alzheimer's disease by suppressing the expression of BACE-1 and reducing the formation of Aβ1-42 or Aβ1-43 that causes neuronal damage (J Gerontol a Biol Sci Med Sci 2015 Jun;70(6):675-85., J Urol.2016 Apr;195(4 Pt 1):1171.).

[0023] The composition of the present invention suppressed the expression of BACE-1, which plays an important role in the β-amyloid forming process (Figure 3).

[0024] Therefore, the composition of the present invention inhibits the β-amyloid forming process by APP and reduces the formation of Aβ1-42 or Aβ1-43 that causes damage to neurons.

[0025] 3. Reduction of cerebral blood flow by Aβ monomers, oligomers and Aβ fibrils / plaque increased by other cells One of the first steps in the onset of dementia is the damage of cells that make up the blood vessels in the brain, including endothelial cells, pericytes, and vascular smooth muscle cells, due to various genetic and environmental factors, followed by disruption in the blood-brain barrier that enables neurotoxic proteins such as Aβ protein or cells to penetrate into the brain tissue and cause brain tissue expansion or various degenerative changes. In the brain, it is known that there is brain tissue inflammation and organization that infiltrates various degenerative changes (Nat Rev Neurol 2018 Mar;14(3):133-150.).

[0026] In addition, as the blood-brain barrier breaks down with the progression of cognitive function decline caused by Alzheimer's disease, a decrease in cerebral blood flow in the brain regions responsible for memory and a continuous expansion of those regions have been reported (Alzheimers Dement 2017 May;13(5):531-540.). Furthermore, recent research has shown that in the brain tissue of Alzheimer's disease patients, an increase in ROS due to Aβ causes contraction of perivascular cells, and thus increases the calcium concentration in pericytes to reduce cerebral blood flow (Science.2019 Jul 19;365(6450)). Therefore, appropriate cerebral blood flow needs to be maintained not only to provide a large amount of oxygen and nutrients to the brain, but also to effectively remove neurotoxins including carbon dioxide and Aβ brain, or protein waste products naturally generated through metabolism (Nat Rev Neurosci 2017 Jul;18(7):419-434).

[0027] PDE5 inhibitors are used in the treatment of pulmonary hypertension or erectile dysfunction because they can inhibit the activation of PDE5 to increase the concentration of cGMP in pericytes, which prevents the conversion of cGMP to 5'-GMP and ultimately induces vasodilation (Int J Impot Res.2004 Jun;16 Suppl 1:S4-7.). In particular, in recent research, PDE5 inhibitors have been shown in Alzheimer's patients (J Cereb Blood Flow Metab 2018 Feb;38(2):189-203.).

[0028] The composition of the present invention reduces the intracellular calcium concentration in pericytes increased by H2O2 treatment, and inhibits the contraction of pericytes caused by the increase in calcium concentration, indicating that the composition can inhibit the reduction of cerebral blood flow (Figure 4).

[0029] Therefore, the composition of the present invention inhibits the contraction of pericytes in order to increase cerebral blood flow and reduce the accumulation of neurotoxins such as Aβ in brain tissue.

[0030] 4. Inhibition of neuronal death and promotion of neurogenesis, synaptogenesis, and angiogenesis by activation of the NO / cGMP / PKG / CREB pathway Aβ monomers were transfected into cells by endocytic aggregates to form toxic soluble Aβ oligomers. The aggregates not only induce disorders of cell functions related to neuronal death, but also increase the hyperphosphorylation of tau protein, a microtubule-associated protein, to promote the formation of NFT (Neurofibrillary Tangle). As a result, the increase in ROS in this process causes mitochondrial damage and activates caspase-3, leading to neuronal 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.).

[0031] PDE5 (phosphodiesterase 5) is expressed in the cerebral cortex and hippocampus, which play important roles in cognitive function among brain tissues (see J Comp Neurol 2003 Dec 22;467(4):566-80). In particular, it has been reported that PDE5 expression increases in the cerebral cortex of the brain tissues of Alzheimer's disease patients compared to normal individuals (Neuropathol Appl Neurobiol.2015 Jun;41(4):471-82.). Furthermore, in the hippocampus of mice, it has been reported that synaptic plasticity, which is important for brain cognitive function reduced by Aβ, is restored by the action of the NO / cGMP / CREB pathway (J Neurosci 2005 Jul 20;25(29):6887-97.).

[0032] PDE5 inhibitors can also inhibit neuronal apoptosis, which is reported to be because PDE5 inhibitors can inhibit PDE5 activity and prevent the conversion of cGMP to 5'-GMP. As a result, cGMP accumulates in the brain and activates 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 suppresses caspase-3 activated by toxic soluble Aβ oligomers and inhibits neuronal 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 the CREB protein, a gene transcription factor, activates PKG, which increases the expression of factors related to neurogenesis, synaptogenesis, and angiogenesis (Behav Brain Res 2013 August 1;250:230-7,DNA Cell Biol.2018 Nov;37(11):861-865.).

[0033] The composition of the present invention inhibits the activation of PDE5, increases the amount of cGMP in cells, prevents the conversion of cGMP to 5'-GMP (Figure 5), suppresses the activity of caspase-3 (Figure 6), restores the mitochondrial membrane potential (Figure 7), and reduces Aβ-induced neuronal cell death (Figure 8).

[0034] The composition of the present invention increased the expression of factors related to neurogenesis, synaptogenesis, and angiogenesis by increasing the phosphorylation of Ser133 in the CREB protein (Figure 9).

[0035] The composition of the present invention increased the expression of NGF and BDNF, which are mainly involved in the regulation of the growth, maintenance, proliferation, and survival of neurons (Figures 10 and 11).

[0036] Therefore, the present invention provides the effect of stimulating neuronal apoptosis, neurogenesis, synaptogenesis, and angiogenesis in the brain of dementia patients by activating the NO / cGMP / PKG / CREB pathway.

[0037] 5. Restoration of synaptic plasticity by activation of Wnt signaling through inhibition of DKK-1, and inhibition of Aβ accumulation by reduction of APP production and suppression of the positive feedback loop The Wnt / β-catenin pathway is an essential 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 signaling but also plays an indispensable role in synaptic plasticity, blood-brain barrier integrity, and the control of their functions (Biomed Res Int 2014;2014:301575.). At present, it is known that the expression of the Wnt antagonist DKK-1 increases in the brain tissue of Alzheimer's patients, inhibits the Wnt / β-catenin pathway, and increases synaptic plasticity (J Neurosci 2004 Jun 30;24(26):6021-7., Front Cell Neurosci 2013 Nov 5;7:162). It has been reported that the expression of DKK-1 is increased by Aβ accumulated in hippocampal neurons, leading to a decrease in synaptic plasticity (J Neurosci 2012 Mar 7;32(10):3492-8.). Furthermore, DKK-1 has been reported to play a role in a positive feedback loop for Aβ production, suggesting that it may be possible to delay the progression of Alzheimer's disease by inhibiting DKK-1, an essential regulator of the positive feedback loop for Aβ production, to reduce Aβ production and synaptic loss, instead of targeting Aβ as in the past (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.).

[0038] On the one hand, in some neurodegenerative diseases including frontotemporal dementia, progressive supranuclear palsy, and Alzheimer's disease, neurofibrillary tangles (NFTs) formed by the aggregation of tau protein due to hyperphosphorylation are the main cause of this disease, and these disorders are collectively called "tauopathies" (Front Mol Neurosci. 2011 Oct 5;4:24., Front Neurosci. 2019 Dec 13;13:1274.). Current tauopathies are known to be caused because the Wnt / β-catenin pathway is inhibited by various pathogenic mechanisms in the brain (Mol Brain. 2019 Dec 4;12(1):104.), and in particular, the inhibition of the Wnt / β-catenin pathway by the accumulation of Aβ in the brain reduces Ser9 phosphorylation in the GSK3β protein and increases Tyr 216 phosphorylation, activates GSK3β, and enhances the phosphorylation of tau protein in mouse hippocampal neurons. As a result, the formation of NFTs reduces neuronal survival, neurogenesis, synaptic plasticity, blood-brain barrier integrity and functionality, and enhances the progression of tauopathy (Neurosci Res. 1998 Aug;31(4):317-23., Annu Rev Pathol. 2019 Jan 24;14:239-261.).

[0039] In summary, the increased expression of DKK-1 due to the accumulation of Aβ in the brain functions as a Wnt antagonist to inhibit the Wnt / β-catenin pathway and activate GSK3β, increasing the formation of NFTs and reducing synaptic plasticity, and also functions as a positive feedback loop for the production of Aβ to enhance the production and accumulation of Aβ and promote the progression of neurodegenerative disorders. Therefore, therapeutic agents targeting the inhibition of DKK-1 expression are expected to inhibit neurodegenerative diseases including Alzheimer's disease by restoring synaptic plasticity through the activation of Wnt signaling and reducing the formation and accumulation of Aβ by inhibiting the positive feedback loop for the production of Aβ.

[0040] The composition of the present invention reduced the expression of DKK-1, which is a Wnt antagonist and a regulator of the positive feedback loop of Aβ production (Figures 12 and 13).

[0041] The composition of the present invention increased the expression of Wnt3a (Figure 14) and Wnt1 (Figure 15), the activities of Wnt / β-catenin-related transcription factors (VAX2, c-Myc, NR5A2, Mitf, TCF / LEF, NFAT, CEBP, GLI-1, GBX2, AP-1) (Figure 16), and increased the activity of Wnt / β-catenin (Figure 17).

[0042] The composition of the present invention increased the phosphorylation of Ser9 of the GSK3β protein, thereby inhibiting the activity of GSK3β (Figures 18 and 19).

[0043] The composition of the present invention reduced the phosphorylation of Ser199 / 202 of tau protein that increases in an Alzheimer's disease animal model (NSE-hAPP-C105) (Figure 20).

[0044] The composition of the present invention inhibited the Aβ production positive feedback loop, and as a result, reduced the APP and Aβ protein levels (Figures 21 and 22).

[0045] Therefore, it is expected that the composition of the present invention restores synaptic plasticity by activating Wnt signaling from DKK-1 inhibition and reduces the formation of APP and the accumulation of Aβ by suppressing the positive feedback loop for Aβ production.

[0046] 6. Activation of autophagy to inhibit the formation of toxic soluble Aβ oligomers and Aβ plaques The brain consumes more than 20% of all the oxygen consumed by the human body. However, because the defense mechanism for removing reactive oxygen species (ROS) that are inevitably generated within cells is weak, it is vulnerable to oxidative stress. ROS accumulated within cells due to oxidative stress induces the oxidation of proteins in the endoplasmic reticulum, inhibits protein folding, accumulates misfolded proteins, and ultimately accumulates 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 endosome and lysosome leakage and mitochondrial dysfunction, enhancing neuronal apoptosis (J Neurosci Res. 2011 Jul;89(7):1031-42.). Furthermore, recent genome-wide association studies of Alzheimer's dementia patients have shown mutations in genes involved in the release or removal of Aβ (SORL1, BIN1, CD2AP, PICALM), confirming 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.).

[0047] Recently, autophagy has attracted attention as a major cause of neurodegenerative diseases. Autophagy is one of the major cellular mechanisms and is known to function to degrade long-lived proteins and organelles (Nat Med. 2013 August; 19(8): 983-97). Dysfunction of autophagy causes intracellular accumulation of misfolded protein aggregates, inducing various diseases, and is particularly closely related to the onset of four major neurodegenerative diseases, namely 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, dysfunctional autophagy in the brains of Alzheimer's disease patients induces aggregation of Aβ and tau proteins, the two major pathogenic factors of Alzheimer's disease (J Syst Integr Neurosci. 2017; 3(4): 1-6). As a result, Aβ and tau proteins accumulated in the brain form senile plaques, causing necrosis or neurons in the hippocampus and cortex responsible for cognitive ability, and ultimately leading to a reduction in cognitive function in Alzheimer's disease patients (J Cell Biol. 2005 Oct 10; 171(1): 87-98., Front Aging Neurosci. 2018 Jan 30; 10:04).

[0048] AMPK (adenosine monophosphate-activated protein kinase) is responsible for the regulation of several biological functions including insulin sensitivity, cell survival, proliferation, and apoptosis. Among these, in particular, it controls autophagy by inhibiting the mTOR (mammalian target of rapamycin) protein in order to maintain cellular homeostasis (Nat Rev Mol Cell Biol. 2018 Feb;19(2):121-135), Mol Cell. 2017 Jun 15;66(6):789-800.). Currently, Thr172 phosphorylation by the AMPK catalytic subunit a is the main starting point of the signaling pathway for autophagy activation (Exp Mol Med. 2016 April 1;48:e224., Nat Rev Mol Cell Biol. 2018 Feb;19(2):121-135., Nat Rev Drug Discov. 2019 Jul;18(7):527-551.). Recently, in neurodegenerative diseases, it has been reported that neuronal degeneration is inhibited by removing misfolded protein aggregates related to neurodegenerative diseases including Aβ and tau, which are the main pathogenic factors of Alzheimer's disease, by autophagy activation from activated AMPK (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 have a mixed pathology with other pathological symptoms, removing misfolded protein aggregates including Aβ and Tar aggregates accumulated intracellularly by autophagy activation from activated AMPK will be a new target in the future development of therapeutic drugs for neurodegenerative 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.).

[0049] The composition of the present invention reduced the Aβ protein level in the hippocampus of an Alzheimer's disease animal model (NSE-hAPP-C105) (Figure 23).

[0050] The composition of the present invention reduced the number of Aβ plaques in the hippocampus and cortex of an Alzheimer's disease animal model (5XFAD transgenic mice) (Figure 24).

[0051] The composition of the present invention increased the AMPK catalytic subunit α of Thr172 phosphorylation (Figure 25).

[0052] The composition of the present invention induced the autophagic flux of the autophagy marker LC3B (conversion from LC3B-I to LC3B-II), increased the LC3B-II / I ratio, and reduced the autophagosome cargo protein, ubiquitin-binding protein p62 (SQSTM, sequestosome 1) (Figure 26).

[0053] The composition of the present invention enhanced the expression of the autophagy markers, ATG7 and ATG5 / 12, and the expression of ATG5 / 12 in the cortex of the hippocampus of an Alzheimer's disease animal model (5XFAD transgenic mice) (Figure 26).

[0054] Therefore, the composition of the present invention increased the LC3BII / I ratio of the autophagy marker, reduced the ubiquitin-binding protein p62 of the autophagosome cargo protein, increased the expression of ATG7 and ATG5 / 12, and activated the autophagy cascade.

[0055] 7. Alzheimer's disease animal model (NSE-hAPP-C105) for improving cognitive and behavioral learning skills To test the effect of the composition of the present invention on the gradual memory decline and behavioral disorders that appear in Alzheimer's disease, a neurodegenerative disease caused by the formation and accumulation of Aβ42, the composition of the present invention was administered intraperitoneally to 13-month-old C57BL / 6-Tg(NSE-hAPP-C105)Kor transgenic mice at a dose of 4 mg / kg once a day for 4 weeks before testing cognitive ability and behavioral learning skills by passive avoidance test and Morris water maze test to analyze changes in cognitive ability and behavioral learning skills.

[0056] It was confirmed that the cognitive ability and behavioral learning skills in the group treated with the composition of the present invention were improved compared to the transgenic cow control group in the Alzheimer's disease animal model (NSE-hAPP-C105) (Figs. 28, 29).

Example

[0057] Example 1. PDE5 (IC 50 , inhibition concentration 50) test for the selectivity of the composition of the present invention Example 1-1. Test method for PDE5 selectivity To test the selective inhibitory activity (IC 50 , inhibition concentration 50) of the composition of the present invention against PDE5 (phosphodiesterase 5), this test was conducted by MDS Pharma Services, an analytical institution CRO, and Scottish Biomedical. The results were measured using a PDE SPA analysis kit (Amersham Pharmacia Biotech). MDS Pharma Services analyzed PDE1 (bovine heart), PDE2, 3, 5 (human platelets), PDE4 (human U937 cells), and PDE6 (bovine retinal rods), and Scottish Biomedical analyzed PDE5, 7-11 (recombinant human enzymes). The test samples (100 μL each) were added to PDE family proteins (10 μL) in Tris-HCl buffer (15 mM, pH 7.5), 3It was added to a mixture of [H]-cGMP (5 Ci / mL), bovine serum albumin (0.5 mg / mL), and MgCl2 (5 mM). The reaction was initiated by the addition of the PDE family protein. Each sample was incubated in a water bath at 30 °C for 30 minutes, and then the reaction was terminated by the addition of SPA beads (PerkinElmer) (50 μL). The test tubes were allowed to stand for 20 minutes and then measured with a liquid scintillation counter (Tri-carb 1500, Packard). To determine the degree of inhibition of the PDE family of protein activity, the compositions of the present invention and the test samples were dissolved in DMSO (dimethyl sulfoxide), and then the solutions were diluted with distilled water to a final DMSO concentration of at least 0.2% (v / v). All inhibition tests were performed with the hydrolysis rate of cGMP (cyclic guanosine monophosphate) not exceeding 15%. The amount of GMP formed increased dose-dependently and proportionally depending on time and the PDE5 family protein.

[0058] Example 1-2. Results of the PDE5 selectivity test for the composition of the present invention 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 was 0.338 nM (MDS Pharma Services), which was 30 - 376,471 times more selective compared to the remaining 10 PDE family proteins (Table 1).

[0059]

Table 1

[0060] Example 2. Test on the inhibition of Aβ aggregation by the composition of the present invention- Example 2-1. Test method for Aβ aggregation inhibitory (Aβ Aggregation Inhibition) Example 2-1-1. Thioflavin T assay One of the main biomarkers of Alzheimer's, the causative Aβ1-42 peptide, when incubated at 37°C for 3 days, generates Aβ fibrils and oligomers. To test the activity of the composition of the present invention for inhibiting Aβ fibril and oligomer formation, the mixture was incubated at 37°C for 3 days, and then Aβ1-42 (50 μM) was treated with the composition of the present invention at various concentrations (5, 50, 500 μM) before measuring the amount of Aβ1-42 fibrils and oligomers. Using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, USA), the fluorescence intensity from thioflavin T at an excitation of 450 nm / emission of 485 was measured. The thioflavin T assay measures the fluorescence response from β-sheets formed as peptide aggregates and showed higher values because more aggregates were formed. The thioflavin T assay cannot distinguish between Aβ oligomers and fibrils, but it has the advantage that it can quantitatively indicate the total amount of aggregated protein.

[0061] Example 2-1-2. PICUP / SDS-PAGE analysis The production of Aβ oligomers was observed by the PICUP (photoinduced cross-linking of unmodified proteins) assay method. The PICUP process was used to immobilize the generated Aβ oligomers and fibrils / plaques, which were then separated by size using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Silver staining was used to observe individual Aβ1-42 peptides.

[0062] Example 2-2. Results of the inhibition of Aβ aggregation by the composition of the present invention The results of inhibiting Aβ-42 aggregation showed that the composition of the present invention inhibited the inhibitory effect on Aβ1-42 aggregation formation, which was discussed dose-dependently based on the thioflavin T assay, compared to the untreated control group (Figure 1), and the present invention inhibited Aβ1-42 oligomer and fibril / plaque formation by PICUP / SDS-PAGE analysis compared to the untreated control group (Figure 2). Considering the results, it is expected that the composition of the present invention inhibits Aβ oligomer and fibril / plaque formation.

[0063] Example 3. Change in BACE-1 expression in retinoic acid-differentiated SH-SY5Y cells by the composition of the present invention Example 3-1. Method for culturing SH-SY5Y nerve cells Example 3-1-1. Subculture of cells A mixture of DMEM / F12 complete medium, DPBS, and trypsin-EDTA was preheated in a 37°C constant temperature water bath for 30 minutes. The cell culture flask was taken out of the incubator, all the medium was removed, and it was rinsed once with DPBS (T25: 5 ml, T75: 10 ml, and T175: 20 ml). After discarding all the DPBS, trypsin-EDTA (T25: 2 mL, T75: 5 mL, and T175: 10 mL) was added, and the mixture was left in an incubator with 5% CO2 at 37°C for 4 minutes. In a 50 ml conical tube, the cells were mixed with fresh complete medium (T25 ml, T75: 10 ml, and T175: 20 mL) and centrifuged at 1500 rpm for 4 minutes. All the supernatant was removed, 1 mL of fresh DMEM / F12 complete medium was added, and the cells were resuspended either by tapping or pipetting. The cells and the medium were added to the cell culture flask and adjusted to T75: 20 ml and T175: 40 ml before incubating the mixture in an incubator at 37°C under 5% CO2.

[0064] Example 3-1-2. Differentiation of SH-SY5Y neuroblastoma A mixture of DMEM / F12 complete medium (Hyclone), DPBS (Hyclone), and trypsin-EDTA (Hyclone) was preheated in a 37°C constant temperature water bath for 30 minutes. The cell culture flask was taken out of the incubator, all the medium was removed, and it was rinsed once with DPBS (T75: 10 ml and T175: 20 ml). After discarding all the DPBS, trypsin-EDTA (T75: 5 mL and T175: 10 mL) was added, and the mixture was left in an incubator with 5% CO2 at 37°C for 4 minutes. In a 50 ml conical tube, the new complete medium (T75: 10 ml and T175: 20 ml) and the cells were mixed and centrifuged at 1500 rpm for 4 minutes. All the supernatant was removed, 1 mL of fresh DMEM / F12 complete medium was added, and the cells were resuspended either by tapping or pipetting. A certain amount of DMEM / F12 complete medium was added, and 4×10 4 cells / cm 2 was transferred to a collagen type I-coated 6-well plate or a collagen type I-coated 60 mm or 100 mm cell culture dish (Corning). After 24 hours, the medium was replaced with DMEM / F12 differentiation medium [+1% FBS +1% penicillin / streptomycin (Hyclone) +10 μM retinoic acid (Sigma-Aldrich)]. On the 5th day, the medium was replaced again with fresh DMEM / F12 differentiation medium. On the 8th day, the medium was replaced again with fresh DMEM / F12 differentiation medium. On the 10th day, the differentiation was completed.

[0065] Example 3-2. Aβ of the present invention and preparation and treatment methods DMEM / F12 (+1% FBS +1% penicillin / streptomycin) was prepared to have 1 μM of human Aβ1-42 (Abcam). The Aβ solution was left standing 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 40 μM), and then incubated in an incubator at 37°C for 72 hours under 5% CO2. After 72 hours, the medium was removed, the cells were washed once with PBS, collected, and then the following experiments were continued.

[0066] Example 3-3. qRT-PCR method Total RNA was prepared using the Easy-Blue™ Total RNA Extraction Kit (Intron Biotechnology) and reverse-transcribed from 1 μg of RNA using the PrimeScript™ II First Strand cDNA Synthesis Kit (TAKARA). The cDNA was provided as a template for EmeraldAmp® PCR Master Mix (TAKARA) with the following primers for BACE-1 and β-actin PCR: human BACE-1 (forward 5’-CTGGTATACACCCATCCGGC-3’, reverse 5’-CTTGGGCAAACGAAGGTTGG-3’); human β-actin (forward 5’-CCAGGTCATCACCATTGG-3’, reverse 5’-CAGAGTACTTGCGCTCAG-3’). The conditions for the PCR cycle were as follows: denaturation (45 seconds at 95°C); annealing, human BACE-1 (45 seconds at 60°C), β-actin (45 seconds at 56°C); extension (45 seconds at 72°C); 40 cycles. qRT-PCR was performed using QuantStudio™ 5 (Thermo Scientific).

[0067] Example 3-4. Results of changes in BACE-1 expression by the composition of the present invention in retinoic acid-differentiated SH-SY5Y cells The present invention dose-dependently reduced the increased expression of BACE-1 mRNA by treating retinoic acid-differentiated SH-SY5Y cells with Aβ1-42 (Figure 3). Considering the results, the present invention is expected to suppress the formation of Aβ1-42 or Aβ1-43 that causes nerve cell damage by suppressing the expression of BACE-1, which plays a major role in the β-amyloid forming process of APP.

[0068] Example 4. Changes in calcium concentration in pericytes by the composition of the present invention Example 4-1. Method for culturing pericytes Example 4-1-1. Subculture of cells Several T-75 cm 2A cell culture flask (SPL, #70075) was coated with Attachment Factor (trademark) (Cell Systems, #4Z0-210), which is an extracellular matrix (ECM), for 1 minute. Subsequently, 15 mL of complete classical medium containing serum and CultureBoost (trademark) (Cell Systems, #4Z0-500) was added to the flask, and then the mixture was stabilized in an incubator at 37 °C under CO2. The incubated normal human brain pericytes (Cell Systems, #ACBRI498P) were washed twice with PBS buffer, and then 3 mL of 0.25% trypsin (Hyclone, #SH30042.02) was added. The mixture was reacted in a CO2 incubator at 37 °C for 2 minutes. 7 mL of complete classical medium containing serum and CultureBoost (trademark) was added, and cell clusters were separated by repeated pipetting. The supernatant was removed by centrifugation (1,500 rpm, 4 minutes). After homogenizing the cell pellet with 1 mL of complete classical medium containing serum and CultureBoost (trademark), the cells were stabilized in a T-75 cm 2 The cells were incubated in an optimal CO2 environment in the cell culture flask.

[0069] Example 4-2. Method of preparation and treatment with H2O2, and measurement of changes in intracellular calcium concentration after treatment A flat-bottom 96-well plate (SPL, #30096) was coated with Attachment Factor (trademark), and complete classical medium (100 μL / well) containing serum and CultureBoost (trademark) was added to each well. Subsequently, the plate was stabilized in a CO2 incubator at 37 °C for 1 hour. Normal human brain pericytes treated with 0.25% trypsin were added to an Attachment Facotr-Coated-96-Well Plate containing complete classical medium with serum and CultureBoost (trademark), and the mixture was incubated in a CO2 incubator at 37 °C for 16 hours (5×10 3(Cell / Well). The culture medium of the cultured cells was removed, and the cells were washed once with HEPES-buffered saline (132 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.5 mM CaCl2, 11.5 mM glucose, 11.5 mM HEPES, 1.2 mM NaH2PO4) and transferred to 96-well containing pericytes, where the ratio measurement intracellular Ca 2+ The dye, Fura-2 (Molecular Probes, #F1201), was diluted to 1 μM with HEPES-buffered saline and then incubated at room temperature for 30 minutes. The supernatant was removed from the 96-well plate, the cells were washed three times with HEPES-buffered saline, and after treatment with the composition of the present invention diluted with HEPES-buffered saline and H2O2, fluorescence was measured to measure changes in intracellular calcium concentration.

[0070] Example 4-4. Results of changes in calcium concentration in pericytes of retinoic acid-differentiated SH-SY5Y cells by the composition of the present invention The composition of the present invention reduced the intracellular calcium concentration in perivascular cells (pericytes) increased by treatment with H2O2 (Figure 4). As a result, it is expected that the composition of the present invention can inhibit the reduction of cerebral blood flow caused by the contraction of pericytes caused by an increase in intracellular calcium concentration in pericytes due to excessive ROS production.

[0071] Example 5. Changes in intracellular cGMP in retinoic acid-differentiated SH-SY5Y cells by the composition of the present invention Example 5-1. Method for cell culture of SH-SY5Y neurons Example 5-1-1. Subculture of cells Using the method described in Example 3-1-1, the cells were subcultured.

[0072] Example 5-1-2. Differentiation of SH-SY5Y neuroblastoma Using the method described in Example 3-1-2, SH-SY5Y neuronal cell differentiation was performed.

[0073] Example 5-2. Aβ and the preparation and treatment methods of the present invention Using the method described in Example 3-2, preparation and treatment were carried out.

[0074] Example 5-3. Method for Measuring Intracellular cGMP The experiment was carried out according to the manual of the cyclic GMP complete ELISA kit (Abcam, #ab133052, USA). After removing the cell culture medium and treating the cells with 0.1 mL of 0.1 M HCl, the cells were left standing at room temperature for 10 minutes. The cells were collected by centrifugation at 600 g, and only the supernatant was collected. When 0.1 M HCl was contained in the sample, 50 μl of neutralization reagent was added to each well. According to the manual, 100 μl of each agent 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 the cyclic GMP complete alkaline phosphatase conjugate was also added to each well. After adding 50 μl of the cyclic GMP complete antibody, the reaction was carried out at room temperature for 2 hours. After removing all the reaction solutions, the wells were washed 3 times with 400 μL of washing buffer. After completely removing the washing buffer, 5 μl of the cyclic GMP complete alkaline phosphatase conjugate was added. After adding 200 μl of the pNpp substrate solution to each well, the reaction was carried out at room temperature for 1 hour. 50 μl of the stop solution was added, and the absorbance value was immediately measured using a plate reader at 405 nm. The results were calculated using the kit manual.

[0075] Example 5-4 Results of Changes in Intracellular cGMP in Retinoic Acid-Differentiated SH-SY5Y Cells According to the Present Invention The composition of the present invention concentration-dependently increased the concentration of cGMP reduced by treating retinoic acid-differentiated SH-SY5Y cells with Aβ1-42 oligomers (Figure 5). Considering this result, the present invention is expected to selectively inhibit the activity of PDE5 (Table 1), inhibit the conversion of cGMP to 5'-GMP, increase intracellular cGMP, inhibit the activity of caspase-3 (Figure 6), and restore the mitochondrial membrane potential that may inhibit neuronal cell death (Figure 8). In addition, the increased intracellular cGMP according to the present invention increases the activity of CREB (Figure 9) that promotes the expression of neurotrophins including NGF and BDNF to improve cognitive function (Figures 10 and 11).

[0076] Example 6. Changes in the expression of apoptosis-regulating related proteins in retinoic acid-differentiated SH-SY5Y cells according to the present invention Example 6-1. Method for culturing SH-SY5Y neurons Example 6-1-1. Subculture of cells Using the method described in Example 3-1-1, subculture of cells was performed.

[0077] Example 6-1-2. Differentiation of SH-SY5Y neuroblastoma Using the method described in Example 3-1-2, SH-SY5Y neurons were differentiated.

[0078] Example 6-2. Method for the preparation and treatment of Aβ according to the present invention 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 newly prepared Aβ1-42 oligomer solution (10 μM) was incubated at 37 °C for 72 hours in a 5% CO2 incubator with or without the composition of the present invention (maximum concentration 40 μM). The medium was removed after 72 hours, and the cells were washed once with PBS and collected. Further experiments were performed according to the following procedure.

[0079] Example 6-5. Western blot analysis method To extract proteins, cells were treated with RIPA lysis buffer (Bio-Rad), homogenized, and centrifuged (14,000 rpm, 10 minutes, 4°C) to obtain the supernatant. A 5% stacking gel (DW, 30% acrylamide:bisacrylamide, 1M Tris pH6.8, 10% SDS, TEMED, 10% ammonium persulfate) and a 12% separating gel (DW, 30% acrylamide:bisacrylamide for SDS-PAGE, 1.5M Tris pH8.8, 10% SDS, TEMED, 10% ammonium persulfate) were used. The supernatant obtained by centrifugation was mixed with 4× 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 a well-prepared stacking gel in a Mini-Protein II Dual-Slab Apparatus (Bio-Rad) and electrophoresed at 150 volts until all had settled to the bottom. A polyvinylidene difluoride (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 electricity was passed at 200 mA for 60 minutes to deposit the membrane. After deposition, the membrane was blocked with 5% skim milk (TBS-T: 10 mM Tris-Base pH8.0, 150 mM NaCl, 0.1% Tween-20) on a platform shaker for 60 minutes. The primary antibodies used in the current experiment are provided in Table 2. Each primary antibody was diluted in 5% skim milk at the optimal concentration and placed on a platform shaker at 4°C for 16 hours. The primary antibodies were recovered and washed 3 times with TBS-T, 10 minutes each time, and the secondary antibodies (goat anti-rabbit IgG H&L (HRP), Abcam; goat anti-mouse IgG H&L (HRP), Abcam) were diluted in 5% skim milk at a ratio of 1:10,000, followed by placement on a platform shaker at room temperature for 60 minutes and washing 3 times with the TBS-T solution, 10 minutes each time.Finally, the membrane was added to SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific™) to properly develop the color before scanning the membrane using an image analysis system, and the amount of protein was calculated using Image J Software (NIH, USA).

[0080]

Table 2

[0081] Example 6-6 Changes in the Expression of Apoptosis-Regulating Related Proteins in Retinoic Acid-Differentiated SH-SY5Y Cells According to the Present Invention The present invention suppressed the formation of cleaved caspase-3 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 was reduced, and thus the amount of formed cleaved PARP was reduced (Figure 6). Considering these results, the present invention suppresses apoptosis caused by Aβ1-42 oligomers, reduces neuronal cell death, and increases the survival of nerve cells.

[0082] Example 7. Changes in Mitochondrial Membrane Potential in Retinoic Acid-Differentiated SH-SY5Y Cells According to the Present Invention Example 7-1. Method for Culturing SH-SY5Y Neurons Example 7-1-1. Subculture of Cells Subculture of cells was performed using the method described in Example 3-1-1.

[0083] Example 7-1-2. Differentiation of SH-SY5Y Neuroblastoma SH-SY5Y neurons were differentiated using the method described in Example 3-1-2.

[0084] Example 7-2. Method for Preparation and Treatment of Aβ According to the Present Invention Preparation and treatment were performed using the method described in Example 3-2.

[0085] Example 7-3. Method for Measuring Mitochondrial Membrane Potential After removing the cell culture medium, the cells were washed once with CPBS. The JC-1 solution of JC-1 - Mitochondrial Membrane Potential Assay Kit (Abcam, USA) was mixed with serum-free medium, and the cells were treated with the mixture. The cells were left in an incubator at 37 °C for 15 minutes. The medium was removed, the cells were washed once with DPBS, and fresh medium was added. Fluorescence of JC-1 monomer at an excitation of 475 nm / emission of 530 nm and that of JC-1 dimer at an excitation of 535 nm / emission of 590 nm were measured using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, USA).

[0086] Example 7-4. Changes in Mitochondrial Membrane Potential in Retinoic Acid-Differentiated SH-SY5Y Cells The present invention dose-dependently increased the mitochondrial membrane potential reduced by the treatment of retinoic acid-differentiated SH-SY5Y cells with Aβ1-42 oligomers (Figure 7). Considering the results, the present invention reduced mitochondrial damage caused by excessive ROS formed by Aβ1-42 oligomers and increased neuronal cell viability.

[0087] Example 8. Live Cell Analysis by the Effect of Suppressing Neuronal Cell Death in Retinoic Acid-Differentiated SH-SY5Y Cells According to the Present Invention Example 8-1. Method for Cell Culture of SH-SY5Y Neurons Example 8-1-1. Subculture of Cells Using the method described in Example 3-1-1, subculture of cells was performed.

[0088] Example 8-1-2. Differentiation of SH-SY5Y Neuroblastoma Using the method described in Example 3-1-2, SH-SY5Y neuroblastoma cells were differentiated.

[0089] Example 8-2. Preparation and Treatment of Aβ According to the Present Invention Using the method described in Example 3-2, preparation and treatment were carried out.

[0090] Example 8-3. IncuCyte S3 Live Cell Analysis (Satorius, USA) A cytotoxic red reagent (Essen Bioscience Cat #4632) was added at a concentration of 1:1,000 (v / v), and the cells were incubated at 37 °C in 5% CO2 for 24 hours. To analyze cytotoxicity, the final magnification of the microscope was adjusted to x200, and pictures were taken every 1.5 hours. Using the software provided by IncuCyte, images were generated to be at 1.5 fps, and for the analysis of cytotoxicity, the total area of the red objects read in the images was compared. The experiment was repeated 3 times, and Student's T-test was used for statistical analysis. All significance tests were carried out at the P < 0.05 level.

[0091] Example 8-4. Results of live cell analysis on the inhibitory effect of neuronal cell death in retinoic acid-differentiated SH-SY5Y cells according to the present invention The present invention reduced apoptotic cells stained by a cytotoxic red reagent increased in retinoic acid-differentiated SH-SY5Y cells by treatment with Aβ1-42 oligomers (Figure 8). Considering this result, the present invention can increase neuronal cell survival rate by suppressing neuronal cell death induced by Aβ1-42 oligomers.

[0092] Example 9. Changes in CREB expression in retinoic acid-differentiated SH-SY5Y cells according to the present invention Example 9-1. Method for cell culture of SH-SY5Y neurons Example 9-1-1. Subculture of cells Using the method described in Example 3-1-1, subculture of cells was carried out.

[0093] Example 9-1-2. Differentiation of SH-SY5Y neuroblastoma Using the method described in Example 3-1-2, SY5Y neurons were differentiated.

[0094] Example 9-2. Aβ and Preparation and Treatment Methods of the Present Invention Using the method described in Example 3-2, preparation and treatment were carried out.

[0095] Example 9-4. Western Blot Analysis Method To extract proteins, cells were treated with RIPA lysis buffer (Biorad), homogenized, and centrifuged (14,000 rpm, 10 minutes, 4 °C) to obtain the supernatant. A 5% stacking gel (DW, 30% acrylamide:bisacrylamide, 1 M Tris pH6.8, 10% SDS, TEMED, 10% ammonium persulfate) and a 12% separating gel (DW, 30% acrylamide:bisacrylamide, 1.5 M Tris pH8.8, 10% SDS, TEMED, 10% ammonium persulfate) were used for SDS-PAGE. The supernatant obtained by centrifugation and 4× Laemmli buffer (Bio-Rad) were mixed 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 and rotated. Protein size markers (Bio-Rad) and each sample were injected into the stacking gel wells provided in the Mini-Protein II Dual-Slab Apparatus (Bio-Rad) and electrophoresed at 150 volts until all had settled to the bottom. The polyvinylidene difluoride (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 the Mini Trans-Bolt Cell (Bio-Rad), and electricity was passed through at 200 mA for 60 minutes to deposit the membrane. After deposition, the membrane was blocked with a 3% BSA (bovine serum albumin) solution (TBS-T: 10 mM Tris-Base pH8.0, 150 mM NaCl, 0.1% Tween-20) on a platform shaker for 60 minutes. The primary antibodies used in the current experiment are provided in Table 3. Each primary antibody was diluted in a 5% non-fat milk solution at the optimal concentration and placed on a platform shaker at 4 °C for 16 hours. The primary antibodies were recovered and washed 3 times with TBS-T for 10 minutes each, and the secondary antibodies (goat anti-rabbit IgG H&L (HRP), Abcam; goat anti-mouse IgG H&L (HRP), Abcam) were diluted at a ratio of 1:10,000 with 5% non-fat milk, then placed on a platform shaker at room temperature for 60 minutes and washed 3 times with the TBS-T solution for 10 minutes each.Finally, the membrane was added to SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific™), and the color was appropriately developed before scanning the membrane using an image analysis system, and the amount of protein was calculated using Image J Software (NIH, USA).

[0096]

Table 3

[0097] Example 9-5. Changes in CREB expression in retinoic acid-differentiated SH-SY5Y cells according to the present invention The present invention increased the Ser133 phosphorylation of CREB protein, which was decreased in retinoic acid-differentiated SH-SY5Y cells by treatment with Aβ1-42 oligomers, in a concentration-dependent manner (Figure 9). Considering this result, the present invention is expected to increase the Ser133 phosphorylation in CREB protein reduced by Aβ1-42 oligomers, restore the activity of CREB protein, a cell transcription factor, and induce the expression of factors related to neurogenesis, synaptogenesis, and angiogenesis to improve cognitive function.

[0098] Example 10. NGF and BDNF in expression changes in retinoic acid-differentiated SH-SY5Y cells according to the present invention Example 10-1. Method for culturing SH-SY5Y nerve cells Example 10-1-1. Subculture of cells Using the method described in Example 3-1-1, subculture of cells was performed.

[0099] Example 10-1-2. Differentiation of SH-SY5Y neuroblastoma Using the method described in Example 3-1-2, SH-SY5Y neuroblastoma was differentiated.

[0100] Example 10-2. Preparation and treatment method of Aβ and the present invention The preparation and treatment were carried out using the method described in Example 3-2.

[0101] Example 10-3. Western blot analysis Western blot analysis was performed using the method described in Example 6-5, and the primary antibodies and the conditions used in this experiment are provided in Table 4.

[0102] [Table 4]

[0103] Example 10-4. NGF immunocytochemistry (ICC) SH-SY5Y was incubated in Corning BioCoat Collagen I culture slides (Corning, #354630), and 4.8×10 4 cells / well were treated with Aβ1-42 oligomers (72 hours) and the present invention (24 hours) for 7 days, and an NGF immunostaining experiment was performed. The cells were fixed with 4% paraformaldehyde (pH 7.4) for 10 minutes and washed three times with ice-cold PBS buffer. After permeabilizing 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 with a blocking solution (10% normal goat serum in PBS) for 30 minutes. The primary antibody used in the current experiment is the rabbit anti-NGF antibody (Abcam, #ab52918) in Table 4. The cells were incubated in the 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 three times with PBS buffer. The cells were treated with VectorShield mounting medium, and fluorescence images were obtained using a super-resolution confocal laser microscope (Carl Zeiss, #LSM800).

[0104] Example 10-5. NGF and BDNF expression changes in retinoic acid-differentiated SH-SY5Y cells according to the present invention When examining the effect of increasing the expression of neurotrophin according to the present invention, Western blot analysis showed that the present invention increased the expression of NGF and BDNF, which decreased in retinoic acid-differentiated SH-SY5Y cells by treatment with Aβ1-42 oligomers, in a concentration-increasing dependent manner (Figure 10). Immunocytochemistry also showed that the present invention increased the expression of NGF in retinoic acid-differentiated SH-SY5Y cells by treatment with Aβ1-42 oligomers (Figure 11). Based on these results, the present invention is expected to increase the expression of neurotrophins including NGF and BDNF, which play an important role in inducing the survival, development and function of nerve cells in order to stimulate the survival and differentiation of nerve cells to improve cognitive function.

[0105] Example 11. Influence on the expression of DKK-1 in retinoic acid-differentiated SH-SY5Y cells and differentiated HT-22 mouse hippocampal neurons according to the present invention Example 11-1. Cell culture method for SH-SY5Y neurons Example 11-1-1. Subculture of cells Using the method described in Example 3-1-1, subculture of cells was performed.

[0106] Example 11-1-2. Differentiation of SH-SY5Y neuroblastoma Using the method described in Example 3-1-2, SH-SY5Y cells were differentiated.

[0107] Example 11-2. Cell culture method for HT-22 mouse hippocampal neuron cell line Example 11-2-1. Subculture of cells DMEM / high glucose medium (10% FBS, 1% penicillin-streptomycin), DPBS, and 0.25% trypsin-EDTA were preheated in a 37°C constant temperature bath for 30 minutes. The cell culture flask was taken out of the incubator, the existing medium was discarded, and it was washed once with DPBS (T25: 5 ml, T75: 10 ml, and T175: 20 ml). All the DPBS was discarded, 0.25% trypsin-EDTA (T25: 2 ml, T75: 5 ml, and T175: 10 mL) was added, and the mixture was left in the incubator at 37°C under 5% CO2 for 2 minutes. Fresh complete medium (T25: 4 ml, T75: 10 ml, and T175: 20 mL), the cells were mixed in a 50 mL conical tube and centrifuged at 1500 rpm for 4 minutes. After removing all the supernatant, fresh DMEM / high glucose was added, and the cells were resuspended either by tapping or pipetting. The cells and the medium were added to a cell culture flask (T75~15 ml and T175~40 ml) and incubated at 37°C under 5% CO2.

[0108] Example 11-2-2. Neuronal Differentiation of HT-22 Cells DMEM / high glucose medium (10% FBS, 1% penicillin-streptomycin), DPBS, and 0.25% trypsin-EDTA were preheated in a 37°C constant temperature bath for 30 minutes. The cell culture flask was taken out of the incubator, the existing medium was discarded, and it was washed once with DPBS (T25: 5 ml, T75: 10 ml, and T175: 20 ml). All the DPBS was discarded, 0.25% trypsin-EDTA (T25: 2 ml, T75: 5 ml, and T175: 10 mL) was added, and the mixture was left in the incubator at 37°C under 5% CO2 for 2 minutes. Fresh complete medium (T25: 4 ml, T75: 10 ml, and T175: 20 mL), the cells were mixed in a 50 mL conical tube and centrifuged at 1500 rpm for 4 minutes. After removing all the supernatant, 1 mL of fresh DMEM / high glucose was added, and the cells were resuspended either by tapping or pipetting. An optimal amount of DMEM / high glucose medium was further added, 2×10 5Cells / cm2 were added to a collagen type I-coated 6-well plate. After 24 hours, the medium was replaced with a differentiation medium [Neurobasal Plus medium (Gibco) + B-27 supplement (Gibco) + 1% penicillin-streptomycin]. After 24 hours, the medium was replaced with fresh differentiation medium, and Aβ1-42 oligomers and the present invention were treated for 6 hours.

[0109] Example 11-3. Preparation and treatment of Aβ and the present invention Using the method described in Example 3-2, preparation and treatment were performed.

[0110] Example 11-4: qRT-PCR method Total RNA was prepared using the Easy-Blue™ Total RNA Extraction Kit (Intron Biotechnology), and 1 μg of RNA was reverse-transcribed using the PrimeScript™ II First Strand cDNA Synthesis Kit (TAKARA). The cDNA was provided as a template using EmeraldAmp® PCR Master Mix (TAKARA) and the following primers for human DKK and β-actin PCR: 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’). The conditions for the PCR cycle were as follows: denaturation, 45 seconds at 95°C; annealing, 45 seconds at 56°C; extension, 45 seconds at 72°C; 40 cycles. qRT-PCR was performed using QuantStudio™ 5 (Thermo Scientific).

[0111] Example 11-5. Western blot analysis method Using the method described in Example 6-5, Western blot analysis was performed, and the primary antibody and the conditions used in this experiment are provided in Table 5.

[0112]

Table 5

[0113] Example 11-6. Changes in DKK-1 expression in retinoic acid-differentiated SH-SY5Y cells and differentiated HT-22 mouse hippocampal neurons according to the present invention The present invention decreased the expression of DKK-1 at the mRNA (Figure 12) and protein levels (Figure 13) in retinoic acid-differentiated SH-SY5Y cells and differentiated HT-22 mouse hippocampal neurons increased by treatment with Aβ1-42 oligomers. Considering the results, the present invention is expected to reduce the increased DKK-1 expression by Aβ1-42 oligomers, restore synaptic plasticity by activating Wnt signaling (Figures 14-20), and reduce APP formation and Aβ accumulation by suppressing the positive feedback loop of Aβ production (Figures 21, 22).

[0114] Example 12. Changes in Wnt gene expression in differentiated HT-22 mouse hippocampal neurons according to the present invention Example 12-1. Cell culture method for HT-22 mouse hippocampal neuron cell line Example 12-1-1. Cell passage culture Using the method described in Example 11-2-1, cell passage culture was performed.

[0115] Example 12-1-2. Neuronal differentiation of HT-22 cells Using the method described in Example 11-2-2, cell passage culture was performed.

[0116] Example 12-3. Preparation and treatment methods of Aβ and the present invention A 10 μM human Aβ1-42 (Abcam) solution was prepared in DMEM / F12 (+1% FBS +1% penicillin / streptomycin). The Aβ solution was left at 37 °C for 3 hours to form Aβ oligomers. The existing medium was discarded, and the cells were treated with freshly prepared Aβ1-42 oligomer (10 μM) solution (maximum concentration 5 μM) regardless of the presence or absence of the present invention and incubated at 37 °C for 72 hours under 5% CO2. After 72 hours, the medium was removed, and the cells were washed once with PBS and collected. Further experiments were performed using the following procedure.

[0117] Example 12-3: qRT-PCR method Total RNA was prepared using the Easy-Blue (trademark) Total RNA Extraction Kit (Intron Biotechnology), and 1 μg of RNA was reverse-transcribed using the PrimeScript (trademark) II First Strand cDNA Synthesis Kit (TAKARA). The cDNA was provided as a template for Wnt and β-actin PCR using EmeraldAmp (registered trademark) 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'). The conditions for the PCR cycle were as follows. Denaturation (45 seconds at 95°C); annealing, Wnt1, Wnt3a, Wnt5a (45 seconds at 60°C); Wnt7a (45 seconds at 57°C); β-actin (45 seconds at 56°C); extension (45 seconds at 72°C); 40 cycles. qRT-PCR was performed using QuantStudio (trademark) 5 (Thermo Scientific).

[0118] Example 12-4. Changes in Wnt gene expression in differentiated HT-22 mouse hippocampal neurons according to the present invention The present invention increased the expression of the Wnt3a gene in differentiated HT-22 mouse hippocampal neurons upon treatment with Aβ1-42 oligomers as compared to the control group (Figure 14). Considering the results, it is expected that the present invention increases the expression of Wnt3a, which is known to increase synaptic activity important for cognitive function, and activates the classical Wnt signaling to restore synaptic plasticity.

[0119] Example 13. Changes in Wnt expression in an Alzheimer's disease animal model (5XFAD transgenic mouse) according to the present invention Example 13-1 Alzheimer's disease animal model (5XFAD transgenic mouse) The internationally widely used transgenic mouse model of Alzheimer's, 5XFAD (C57BL / 6xSJL), was used. The characteristic of this model is that the progression of Alzheimer's disease is significantly faster than other mouse models through five different gene conversions (APP KM670 / 671NL (Sweden), APP I716V (Florida), APP V717I (London), PSEN1 M146L (A>C), PSEN1 L286V). In other words, any composition therapeutically effective in this model is expected to be effective in other APP models with relatively slow progression. Analyses were performed using brain tissues in the cortical and hippocampal regions related to cognitive function.

[0120] Example 13-2. Dosage of the present invention Mice at an active age (6 months, male) with the progression of the pathological condition of Alzheimer's disease and deterioration of cognitive function were administered daily by intraperitoneal injection at 5 mg / kg / day or 10 mg / kg / day for 4 weeks.

[0121] Example 13-3. Preparation of tissues The present invention was administered daily by intraperitoneal injection for 4 weeks. The animals were anesthetized in the animal room using CO2 gas, the brains were excised to measure the protein expression level, the hippocampus and cerebral cortex were separated, and stored in an ultra-low temperature freezer at -80°C until analysis of protein expression.

[0122] Example 13-5. Western blot analysis method Using the method described in Example 6-5, Western blot analysis was performed, and the primary antibodies and the conditions used in this experiment are shown in Table 6.

[0123]

Table 6

[0124] Example 13-6. Changes in Wnt1 expression in an Alzheimer's disease animal model (5XFAD transgenic mouse) according to the present invention The present invention increased the reduced Wnt1 expression in the hippocampus of an Alzheimer's disease animal model (5XFAD transgenic mouse) (Figure 15). Considering the results, the present invention is expected to increase the expression of Wnt1, which provides an important function for synaptogenesis between nerve cells and restores synaptic plasticity.

[0125] Example 14. Confirmation of activation of factors related to Wnt / β-catenin in differentiated HT-22 mouse hippocampal neurons according to the present invention using a Wnt / β-catenin TF activation profiling plate array Example 14-1. Cell culture method for HT-22 mouse hippocampal neuron cell line Example 14-1-1. Subculture of cells Using the method described in Example 11-2-1, subculture of cells was performed.

[0126] Example 14-1-2. Neuronal differentiation of HT-22 cells Using the method described in Example 11-2-2, subculture of cells was performed.

[0127] Example 14-2. Preparation and treatment method of Aβ according to the present invention A 10 μM solution of human Aβ1-42 (Abcam) in DMEM / F12 (+1% FBS +1% penicillin / streptomycin) was prepared. The Aβ solution was left at 37 °C for 3 hours to form Aβ oligomers. The existing medium was discarded, and the cells were treated with freshly prepared Aβ1-42 oligomer (1 μM) solution (maximum concentration 5 μM) regardless of the presence or absence of the present invention and incubated at 37 °C under 5% CO2 for 6 hours. After 6 hours, the medium was removed, and the cells were washed once with PBS and collected. Further experiments were performed using the following procedure.

[0128] Example 14-3. Wnt / β-catenin TF activation profiling plate array method Nuclear extracts were extracted from the cells, and a Wnt / β-catenin TF activation profiling plate array (Signosis, #FA-1007) was performed according to the procedure provided by the vendor. The relative luminescence was measured using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, USA).

[0129] Example 14-4. Activation of Wnt / β-catenin-related transcription-related factors measured by transcription factor array The present invention increased the activities of Wnt / β-catenin-related transcription factors including VAX2, c-Myc, NR5A2, Mitf, TCF / LEF, NFAT, CEBP, GLI-1, GBX2, and AP-1 in differentiated HT-22 mouse hippocampal neurons treated three or more times with Aβ1-42 oligomers (Figure 16). Considering the results, it is expected that the present invention increases the activities of Wnt / β-catenin-related transcription factors to stimulate Wnt signaling activation and restore synaptic plasticity.

[0130] Example 15. Measurement of changes in Wnt / β-catenin activity in differentiated HT-22 mouse hippocampal neurons according to the present invention using the TOPFLASH reporter gene assay Example 15-1. Cell culture method for HT-22 mouse hippocampal neuron cell line Example 15-1-1. Subculture of cells Cell passage culture was performed using the method described in Example 11-2-1.

[0131] Example 15-1-2. Neuronal Differentiation of HT-22 Cells Cell passage culture was performed using the method described in Example 11-2-2.

[0132] Example 15-2-3. Transfection of TOPFLASH Reporter Plasmid DNA In a 6-well plate, Fugene HD transfection reagent (Promega, #E2311) and TOPFLASH reporter plasmid DNA (pcDNA-β-galactosidase, TOPFLASH) were added to HT-22 cells differentiated in 2×10 5 cells / well, and the cells were incubated at 37 °C under 5% CO2 for 24 hours.

[0133] Example 15-2-3. Preparation and Method for Treatment of Aβ and the Present Invention A 1 μM solution of human Aβ1-42 (Abcam) in DMEM / F12 (+1% FBS +1% penicillin / streptomycin) was prepared. The Aβ1-42 solution was left at 37 °C for 3 hours to form Aβ oligomers. After 24 hours of transfection, the existing cell medium was discarded, and the cells were treated with freshly prepared Aβ1-42 oligomer (1 μM) solution regardless of the presence or absence of the present invention (maximum concentration 5 μM), and incubated at 37 °C under 5% CO2 for 6 hours. After 6 hours, the medium was removed, the cells were washed once with PBS, and collected. Further experiments were performed according to the following procedure.

[0134] Example 15-3. TOPFLASH Reporter Gene Assay Method The HT-22 cells were washed with PBS buffer, and cell lysates 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-Tablet) (100 μl / well), and the proteins were quantified. A part of the cell lysates was used to perform a β-galactosidase assay, and the light absorption was measured at 420 nm. The remaining part of the cell lysates was used to perform a luciferase assay to measure the luminescence. The obtained luminescence values were normalized with the protein concentration using the β-galactosidase activity (absorbance at 420 nm) to obtain the relative luciferase activity values.

[0135] Results of Wnt / β-catenin activity according to the present invention in differentiated HT-22 mouse hippocampal neurons measured by the TOPFLASH reporter gene assay The present invention increased the Wnt / β-catenin activity in differentiated HT-22 mouse hippocampal neurons, which was reduced by treatment with Aβ1-42 oligomers, in a concentration-dependent manner (Figure 17). Considering the results, it is expected that the present invention promotes the activation of Wnt signaling to restore synaptic plasticity by increasing Wnt expression (Figures 14 and 15) and the activity of Wnt / β-catenin-related transcription factors (Figure 16).

[0136] Example 16. Changes in GSK3β expression in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells and differentiated HT-22 mouse hippocampal neurons according to the present invention Example 16-1. Cell culture method for SH-SY5Y neurons Example 16-1-1. Cell subculture Cell subculture was performed using the method described in Example 3-1-1.

[0137] Example 16-1-2. Differentiation of SH-SY5Y neuroblastoma SH-SY5Y cells were differentiated using the method described in Example 3-1-2.

[0138] Example 16-2. Method for Cell Culture of HT-22 Mouse Hippocampal Neuron Cell Line Example 16-2-1. Subculture of Cells Using the method described in 11-2-1, subculture of cells was performed.

[0139] Example 16-2-2. Neural Cell Differentiation of HT-22 Cells Using the method described in 11-2-2, subculture of cells was performed.

[0140] Example 16-3. Preparation of Aβ and Method for Treatment According to the Present Invention Using the method described in Example 3-2, preparation and treatment were performed.

[0141] Example 16-4. Western Blot Analysis Method Using the method described in Example 9-4, Western blot analysis was performed, and the primary antibodies and conditions are provided in Table 7.

[0142]

Table 7

[0143] Example 16-5. PathScan (registered trademark) Phospho-GSK-3b (Ser9) Sandwich ELISA HT-22 cells (1.5×10 5Cells / well) were incubated in a 6-well plate (SPL, #30006). The medium was changed to Neurobal Plus Medium containing B-27 supplement. After 24 hours, the 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. Cell lysis buffer (Cell Signaling, #9803) was added to each well (100 μl / well). The cells were placed on ice for 5 minutes, and the cell lysates were scraped and transferred to 1.5 mL. The cell lysates were homogenized using sonication with a Bioruptor device. The homogenized cell lysates were centrifuged (14,000 rpm, 10 minutes), and the supernatant was analyzed to quantify the amount of protein using the Pierce (trademark) BCA Protein Assay Kit. GSK-3β mouse mAb-coated microwells included in the PathScan Phospho-GSK-3β (Ser9) Sandwith ELISA Kit (Cell Signaling, #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 an ELISA experiment was performed using Phospho-GSK-3β (Ser9) rabbit detection mAb and anti-rabbit IgG, HRP-conjugated antibody (formulated ELISA). The TMB substrate solution was used as the ELISA substrate, and the absorption at 450 nm was measured to determine the relative amount of Phospho-GSK-3β (Ser9).

[0144] Example 16-6. Changes in GSK3β expression according to the present invention in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells and differentiated HT-22 mouse hippocampal neurons When examining the effect of the present invention on the phosphorylation of GSK3β protein, as shown by Western blot analysis, the present invention increases the Ser9 phosphorylation of the protein in retinoic acid-differentiated SH-SY5Y cells reduced by Aβ1-42 oligomers (Figure 18), and Phospho-GSK-3b(Ser9) Sandwich ELISA indicates that the present invention increased the Ser9 phosphorylation of the protein in differentiated HT-22 mouse hippocampal neurons reduced by Aβ1-42 oligomers (Figure 19). Considering the results, it is expected that the present invention increases the Ser9 phosphorylation of the protein that inactivates GSK3 and reduces the phosphorylation of tau protein to reduce tau pathology.

[0145] Example 17. Change in Tau Expression According to the Present Invention in an Alzheimer's Disease Animal Model (NSE-Happ-C105) Example 17-1. Experimental Animals The animals for the experiment were C57BL / 6-Tg(NSE-hAPP-C105)Kor transgenic mice (13 months old), and Alzheimer's dementia was induced by overexpressing a mutant APP gene that expresses only the APP C-terminal 105 amino acids in the brain tissue. The animals were housed under controlled conditions of temperature 20±2°C, humidity 50%, lights on from 08:00 to 20:00, and lights off from 20:00 to 08:00. The groups were identified as non-control / treatment groups [non-tg control, NTC (n = 6), Alzheimer's disease group [tg-control TC (n = 6)], Alzheimer's disease group treated with the present invention [tg-the present invention, TM (n = 6)], and food and water were supplied ad libitum during the experiment.

[0146] Example 17-2. Administration Method of the Present Invention After a single-dose based test on an Alzheimer's disease model including a mouse memory test, a behavioral test using a maze in a water motor function test, and a passive avoidance test, the daily human dose of the present invention was administered once a day at 4 mg / kg for 4 weeks by intraperitoneal injection to 13-month-old Alzheimer's disease model mice based on Reagan-Shaw et al. (2008). The same amount of physiological saline was administered to the control group (NTC) and the Alzheimer's disease group (TC).

[0147] Example 17-3. Tissue Preparation The present invention was administered daily for 4 weeks by intraperitoneal injection. The animals were anesthetized in the animal room using CO2 gas, the brains were excised to measure the protein expression level, the hippocampus and cerebral cortex were separated, and stored in an ultra-low temperature freezer at -80 °C until the analysis of protein expression.

[0148] Example 17-4. Western Blot Analysis Method Using the method described in Example 9-4, Western blot analysis was performed, and the primary antibodies and conditions are provided in Table 8.

[0149]

Table 8

[0150] Example 17-5. Tau Expression Changes According to the Present Invention in an Alzheimer's Dementia Animal Model (NSE-Happ-C105) The present invention reduced the Ser199 / 202 phosphorylation in tau protein increased in the hippocampus of an Alzheimer's dementia animal model (NSE-hAPP-C105) (Figure 20). Considering this result, it is expected that the present invention reduces the phosphorylation of Ser199 / 202 in tau protein, suppresses the aggregation in tau protein, reduces the formation of NFT, and reduces tau pathology.

[0151] Example 18. Changes in APP Formation and Aβ Accumulation According to the Present Invention in Retinoic Acid-Differentiated SH-SY5Y Human Neuroblastoma Cells Example 18-1. Cell culture method of SH-SY5Y neurons Example 18-1-1. Cell subculture Cell subculture was performed using the method described in Example 5-1-1.

[0152] Example 18-1-2. Differentiation of SH-SY5Y neuroblastoma SH-SY5Y neurons were differentiated using the method described in Example 5-1-2.

[0153] Example 18-2. Method for the preparation and treatment of Aβ according to the present invention Example 18-2-1. Method for the preparation and treatment of Aβ according to the present invention Preparation and treatment were carried out using the method described in Example 5-2.

[0154] Example 18-2-2. Preparation of H2O2 and method for treatment according to the present invention DMEM / F12 containing 100 μM H2O2 (+1% FBS +1% penicillin / streptomycin) was added to differentiated SH-SY5Y cells every 24 hours, refreshed 3 times, and the present invention was added at various concentrations (10, 20, 40 μM) for 24 hours.

[0155] Example 18-3. Western blot analysis method Western blot analysis was performed using the method described in Example 6-5, and the primary antibodies and conditions are provided in Table 9.

[0156]

Table 9

[0157] Example 18-4. Changes in APP formation and Aβ accumulation according to the present invention in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells The present invention reduced APP formation and Aβ1-42 accumulation increased by treatment with Aβ1-42 oligomers (Figure 21) and H2O2 (Figure 22) in retinoic acid-differentiated SH-SY5Y cells. Considering the results, it is expected that the present invention reduces APP formation and Aβ accumulation by suppressing the positive feedback loop of Aβ production.

[0158] Example 19. Change in Aβ expression according to the present invention in an Alzheimer's disease animal model (NSE-Happ-C105) Example 19-1. Experimental animals The same type of animal model described in Example 17-1 was used.

[0159] Example 19-2. Administration method of the present invention The present invention was administered using the same method described in Example 17-2.

[0160] Example 19-3. Preparation of tissues Tissue samples were prepared and stored using the method described in Example 17-3.

[0161] Example 19-4. Western blot analysis Western blot analysis was performed using the method described in Example 6-5, and the primary antibodies and conditions are provided in Table 10.

[0162] [Table 10]

[0163] Example 19-5. Results of changes in Aβ expression in an Alzheimer's disease animal model (NSE-hAPP-C105) The present invention effectively reduced the expression of Aβ in the hippocampus in an Alzheimer's disease animal model (NSE-hAPP-C105) (Figure 23). Based on this result, it is expected that the present invention effectively suppresses the formation and accumulation of Aβ.

[0164] Example 20. Aβ plaque changes according to the present invention in an Alzheimer's disease animal model (5XFAD transgenic mouse) Example 20-1. Alzheimer's disease animal model (5XFAD transgenic mouse) The animal model described in Example 13-1 was used.

[0165] Example 20-2. Administration of the composition of the present invention The composition of the present invention was administered using the method described in Example 13-2.

[0166] Example 20-3. Preparation and fixation of tissue samples The present invention was administered daily by intraperitoneal injection for 4 weeks. After the animals were anesthetized in the animal room using CO2 gas, the chest was opened and 50 mM PBS (phosphate buffered saline) was administered to the left ventricle for 3 minutes, and then a fixing solution in which 4% PFA (paraformaldehyde) was dissolved in 0.1 M phosphate buffer for 10 minutes was perfused. After reflux and fixation, the brain was excised, fixed by adding to a 4% PFA fixing solution at 4°C for 12 hours, and then the tissue was precipitated in a 30% sucrose solution for 5 days, and sliced into 40-μm thickness by continuous tubular intercept using a cryostat (Leica) for storage.

[0167] Example 20-4. Aβ plaque staining method using thioflavin S stain G and GFAP (glial fibrillary acidic protein) immunohistochemistry (IHC) method After fixing mouse brain tissue in 4% paraformaldehyde (pH 7.4) solution for 24 hours, the tissue was dehydrated using 30% sucrose, and frozen sections were prepared. The primary antibodies and conditions are provided in Table 11. After performing antigen retrieval using 1% SDS on frozen sections of the train tissue, the samples were reacted with glial fibrillary acidic 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 the GFAP antibody / thioflavin S-treated frozen brain tissue sections, and Hoechst 33342 (Sigma-Aldrich) was used for nuclear staining.

[0168]

Table 11

[0169] Example 20-5. Changes in Aβ plaques according to the present invention in an Alzheimer's disease animal model (5XFAD transgenic mouse) 7260 The present invention reduces the number of Aβ plaques in the hippocampus and cerebral cortex of an Alzheimer's disease animal model (5XFAD transgenic mouse) (Figure 24), which was determined by counting the number of Aβ plaques (green) stained with thioflavin S and the co-localization spots (yellow) of astrocytes (red) stained with GFAP. Considering this result, the present invention is expected to suppress Aβ production and Aβ accumulation and reduce extracellular Aβ plaque formation.

[0170] Example 21. Changes in AMPK expression according to the present invention in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells Example 21-1. Method for culturing SH-SY5Y nerve cells Example 21-1-1. Subculture of cells Using the method described in Example 3-1-1, subculture of cells was performed.

[0171] Example 21-1-2. Differentiation of SH-SY5Y Neuroblastoma Using the method described in Example 3-1-2, SH-SY5Y nerve cells were differentiated.

[0172] Example 21-2. Method for Preparation and Treatment of Aβ According to the Present Invention A 1 μM human Aβ1-42 (Abcam) solution in DMEM / F12 (+1% FBS +1% penicillin / streptomycin) was prepared. The Aβ solution was left at 37 °C for 3 hours to form Aβ oligomers. The existing medium was discarded, and the cells were treated with freshly prepared Aβ1-42 oligomer (1 μM) solution (maximum concentration 10 μM) regardless of the presence or absence of the present invention and incubated at 37 °C under 5% CO2 for 72 hours. After 72 hours, the medium was removed, the cells were washed once with PBS, and collected. Further experiments were performed using the following procedure.

[0173] Example 21-3. Western Blot Analysis Using the method described in Example 9-4, Western blot analysis was performed, and the primary antibodies and their conditions are provided in Table 12.

[0174]

Table 12

[0175] Example 21-4. Changes in AMPK Expression According to the Present Invention in Retinoic Acid-Differentiated SH-SY5Y Human Neuroblastoma Cells The present invention reduced the concentration-dependent increase in phosphorylation of Thr172 of the AMPK catalytic subunit α in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells by Aβ1-42 oligomer treatment (Figure 25). Considering this result, the present invention is expected to increase the phosphorylation of Thr172 in the AMPK catalytic subunit α reduced by Aβ, restore AMPK activity, activate autophagy, remove misfolded protein aggregates related to degenerative brain diseases, and inhibit nerve cell degeneration.

[0176] Example 22. Changes in the expression of autophagy markers including LC3B and P62 according to the present invention in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells Example 22-1. Method for culturing SH-SY5Y nerve cells Example 22-1-1. Subculture of cells Using the method described in Example 2-1-1, subculture of cells was performed.

[0177] Example 22-1-2. Differentiation of SH-SY5Y neuroblastoma Using the method described in Example 2-1-2, SH-SY5Y nerve cells were differentiated.

[0178] Example 22-2. Method for preparation and treatment of Aβ according to the present invention A 1 μM human Aβ1-42 (Abcam) solution in DMEM / F12 (+1% FBS + 1% penicillin / streptomycin) was prepared. The Aβ solution was left at 37°C for 3 hours to form Aβ oligomers. The existing medium was discarded, and the cells were treated with 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), an autophagy / PI3K (phosphoinositide 3-kinase) inhibitor, and incubated at 37°C under 5% CO2 for 72 hours. After 72 hours, the medium was removed, the cells were washed once with PBS, and collected. Further experiments were performed using the following procedure.

[0179] Example 22-3. Western blot analysis method Using the method described in Example 6-5, Western blot analysis was performed, and the primary antibodies and conditions are provided in Table 13.

[0180]

Table 13

[0181] Example 22-4. Changes in the expression of autophagy markers including LC3B and P62 according to the present invention in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells The present invention not only increases the expression of LC3B, an autophagy marker reduced by Aβ1-42 oligomers in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells, and simultaneously induces autophagic flux (conversion from LC3B-I to LC3B-II) to increase the LC3B-II / I ratio, but also reduces the expression of ubiquitin-binding protein p62 (SQSTM, sequestosome 1), an autophagosome cargo protein increased by Aβ1-42 oligomers. In addition, 3-MA (an autophagy / PI3K inhibitor) was used together with the present invention to reduce the autophagic flux (LC3B-II / I ratio) increased by the present invention and increase the expression of p62 reduced by the present invention. Furthermore, when the cells were treated with the present invention and 3-MA together, APP formation and Aβ1-42 accumulation increased (Figure 26). Considering this result, it is expected that the present invention suppresses APP formation and Aβ1-42 accumulation by activating autophagy.

[0182] Example 23. Changes in the expression of autophagy markers including ATG7 and ATG5 / 12 according to the present invention in an Alzheimer's disease animal model (5XFAD transgenic mice) Example 23-1. Alzheimer's disease animal model (5XFAD transgenic mice) The animal model described in Example 13-1 was used.

[0183] Example 23-2. Administration of the composition of the present invention The composition was administered using the same method described in Example 13-2.

[0184] Example 23-3. Preparation of tissue samples Tissue samples were prepared and stored using the method described in Example 13-3.

[0185] Example 23-4. Western blot analysis method Using the method described in Example 6-5, Western blot analysis was performed, and the primary antibodies and their conditions are provided in Table 14.

[0186] [Table 14]

[0187] Example 23-5. Changes in the expression of autophagy markers including ATG7 and ATG5 / 12 according to the present invention in an Alzheimer's disease animal model (5XFAD transgenic mice) The present invention increased the expression of autophagy markers including ATG7 and ATG5 / 12 in the hippocampus of an Alzheimer's disease animal model (5XFAD transgenic mice) and increased the expression of ATG5 / 12 in the cerebral cortex (Figure 27). Considering the results, the present invention is expected to increase autophagic flux (LC3BII / I ratio), increase autophagy markers, reduce p62 (Figure 26), and activate the autophagy cascade by increasing the expression of ATG7 and ATG5 / 12.

[0188] Example 24. Effect of improving cognitive and behavioral learning abilities in an Alzheimer's disease animal model (NSE-hAPP-C105) for the present invention Example 23-1. Experimental animals The animal model described in Example 17-1 was used.

[0189] Example 23-2. Administration method of the present invention The composition was administered using the same method described in Example 17-2.

[0190] Example 23-3. Test method for cognitive ability and behavioral learning ability Example 23-3-1. Morris water maze test The present invention was administered once a day at 4 mg / kg by intraperitoneal injection for 4 weeks before measuring changes in cognitive ability using the water maze test. The test was conducted in water in a circular water tank (diameter 1 m × height 40 cm) at 22 - 25°C, and the target (target: diameter 12 cm) was set approximately 3 cm below the water surface. Dried milk powder was added to the water to make the target invisible, and SMART 3.0 protram (Panlab) was installed on the ceiling above the water tank. Before and 4 weeks after the experiment, the target arrival time (latency to the target), swimming distance (distance to the target), and swimming pattern (swimming pattern) of the test animals were monitored. The test subjects were trained twice a day for the first 5 days to start from the starting point and end at the target. Mice that did not find the target at both time points were made to recognize the position of the target. Each test was conducted every 5 minutes, and the target was removed on the 6th day and a 1-minute test was conducted, starting from the starting point, and the results were used as experimental data.

[0191] Example 23 - 3 - 2. Passive Avoidance Test The present invention was administered once a day at a dose of 4 mg / kg by intraperitoneal injection for 4 weeks before conducting the passive avoidance test to evaluate memory. The passive avoidance test consisted of a front room in a white, bright room (18×18×25 cm), and the back room was a black, dark room (18×18×25 cm). A strong metal stainless steel was installed on the floor of the dark room in the front and back rooms, and there were holes with a diameter of 4 cm that opened and closed in a guillotine type on the walls of the front and back rooms. Each experimental animal was isolated in each cage for 1 minute and adjusted while the guillotine-type door was opened so that the test animal could move freely between the rooms, and then moved to the front room in 10 seconds. When all four feet of the test animal were in the back room, the door was quickly closed, and the time (initial latency) for the test animal to move from the front room to the back room was recorded, and it was electrified for 2 seconds (0.5 mA). After 5 seconds, the test sample animal was moved to the housing cage. 72 hours later, the same test was conducted to measure the latency until entering the dark, which is the time to move from the front room to the back room up to a maximum of 300 seconds.

[0192] Example 23-3-3. Data Processing Method The collected data was a process of calculating statistical errors (Mean±SD) using the SPSS 20.0 statistical program, and the verification of variables between species and groups was performed by one-way analysis of variance (one-way ANOVA). When there was a significant difference between groups, a post hoc test was performed using the Bonferroni method. Therefore, the significance level was set at α = 0.05.

[0193] Example 23-4. Improvement of Cognitive and Behavioral Learning Skills According to the Present Invention in an Alzheimer's Disease Animal Model (NSE-hAPP-C105) Example 23-4-1. Results of Improvement of Cognitive Function According to the Present Invention in an Alzheimer's Disease Animal Model (NSE-hAPP-C105): Morris Water Maze Test The Morris water maze test was performed on C57BL / 6-Tg(NSE-hAPP-C105)Kor transgenic mice to measure the effect of the present invention on cognitive function after 4 weeks of treatment. First, analysis of the time to reach the target showed that it was statistically increased in the TC group compared to the NTC group (P = 0.001) and statistically decreased in the TM group compared to the TC group (P = 0.001). Second, analysis of the swimming distance to reach the target showed that it was statistically increased in the TC group compared to the NTC group (P = 0.001) and statistically decreased in the TM group compared to the TC group (P = 0.001). Third, analysis of the swimming time from the target within the quadrant showed that it was statistically decreased in the TC group compared to the NTC group (P = 0.001) and statistically increased in the TM group compared to the TC group (P = 0.001). Fourth, analysis of the number of times passing through the target showed that it was statistically decreased in the TC group compared to the NTC group (P = 0.001) and statistically increased in the TM group compared to the TC group (P = 0.037) (Figure 28).

[0194] Example 23-4-2. Results of Improvement of Active Learning and Cognitive Ability According to the Present Invention in an Alzheimer's Disease Animal Model (NSE-hAPP-C105): Passive Avoidance Test The passive avoidance test was performed using C57BL / 6-Tg(NSE-hAPP-C105)Kor transgenic mice to evaluate the effect of the 4-week treatment according to the present invention on active learning and cognitive ability (Figs. 6A and 6B). The results are shown to be statistically decreased in the TC group (P = 0.001) compared to the NTC group and statistically increased in the TM group (P = 0.004) compared to the TC group (Fig. 29).

Claims

1. A pharmaceutical composition for treating Alzheimer's disease, comprising a compound selected from the group consisting of milodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasanafil, and avanafil, and pharmaceutically acceptable salts, solvates, and hydrates thereof.

2. The pharmaceutical composition according to claim 1, wherein the compound increases blood flow in the brain by vasodilation to inhibit the formation and accumulation of extracellular Aβ monomers, oligomers, and / or Aβ fibrils / plaque.

3. The pharmaceutical composition according to claim 1, wherein the compound activates 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, wherein the compound inhibits DKK-1 (Dickkopf WNT signaling pathway inhibitor 1) to activate Wnt signaling to restore synaptic plasticity and inhibit 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, wherein the compound activates autophagy to remove intracellular toxic soluble Aβ oligomers and inhibit the formation and accumulation of Aβ fibrils / plaque.

6. The pharmaceutical composition according to claim 1, wherein the compound improves behavioral and cognitive functions.

7. A method for treating Alzheimer's disease, comprising administering a pharmaceutical composition comprising a compound selected from the group consisting of milodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasanafil, and avanafil, and pharmaceutically acceptable salts, solvates, and hydrates thereof.

8. The method according to claim 7, wherein the compound inhibits the formation of Aβ aggregation inhibition by reducing the formation of Aβ oligomers or fibrils.

9. The method according to claim 7, wherein the compound inhibits β-amyloid formation processing by reducing BACE-1.

10. The method according to claim 7, wherein the compound reduces the extracellular formation and accumulation of amyloid beta monomers, oligomers, and / or amyloid beta fibrils and plaques by vasodilation.

11. The method according to claim 7, wherein the compound reduces neuronal cell death and enhances neurogenesis, synaptogenesis, or angiogenesis by activating the NO / cGMP / PKG / CREB pathway.

12. The method according to claim 7, wherein the compound restores synaptic plasticity by activating Wnt signaling through DKK-1 inhibition.

13. The method according to claim 7, wherein the compound inhibits DKK-1 by inhibiting the positive feedback loop of Aβ production to inhibit the formation of APP and Aβ accumulation.

14. The method according to claim 7, wherein the compound activates autophagy to remove intracellular toxic soluble Aβ oligomers and inhibits the formation and accumulation of Aβ fibrils / plaque.

15. The method according to claim 7, wherein the compound improves behavioral and cognitive functions.

16. The method according to claim 7, wherein the compound activates autophagy to remove intracellular toxic soluble Aβ oligomers and inhibits the formation and accumulation of Aβ fibrils / plaque.

17. The method according to claim 7, wherein the compound improves behavioral and cognitive functions.

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