Compositions and Methods for the Prevention and Treatment of Neurodegenerative Diseases

Abstract

The present invention provides a composition for preventing or treating neurodegenerative diseases containing a phosphodiesterase 5 inhibitor (PDE5 inhibitor) and an acetylcholinesterase inhibitor (AChEI), and a method of using the same, wherein the PDE5 inhibitor is selected from among sildenafil, tadalafil, vardenafil, tadalafil, udenafil, dasanafil, avanafil, their pharmaceutically acceptable salts, solvates, hydrates, and mixtures, the AChEI is selected from among donepezil, rivastigmine, galantamine, physostigmine, tacrine, metrifonate, phenserine, tolserine, eceroline, huperzine A and B, galangin, cardanol, donepezil-AP2238, donepezil-tacrine, tacrine-ferulic acid hybrid, tacrine-hydroxyquinoline, ladostigil, indenyl derivatives, their pharmaceutically acceptable salts, solvates, hydrates, and mixtures, and the neurodegenerative disease is dementia, Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), or multiple sclerosis (MS).

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 367,033, filed on June 24, 2022, the content of which is incorporated herein by reference.

[0002] The present invention relates to a composition containing a phosphodiesterase 5 inhibitor (PDE5 inhibitor) and an acetylcholinesterase inhibitor (AChEI) for preventing or treating neurodegenerative diseases, and a method of using the same.

[0003] Sequence Listing This application incorporates by reference in its entirety the XML file of the sequence listing entitled "04334900119_SequenceListing.xml (7KB)", created on June 23, 2023, and electronically filed together with this specification.

Background Art

[0004] In recent years, the number of patients with degenerative neuropathy has been increasing rapidly. In the treatment of degenerative neuropathy, the most important stage is prevention. However, the cause of this disease has not yet been clearly understood, and therefore, research on treatment methods is still needed. A common pathological phenomenon of degenerative neuropathy is the death of central nervous system cells. Unlike cells of other organs, central nervous system cells are almost impossible to regenerate after cell death, resulting in a permanent loss of function. Therefore, the treatment methods for such brain diseases developed so far have mainly focused on the analysis of the mechanism of nerve cell death itself and the prevention of death based on such analysis.

[0005] Neurodegeneration, in general terms, involves the progressive loss of the structure or function of neurons, including the death of neurons in various regions of the brain. Neurodegenerative diseases, including dementia, Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and multiple sclerosis (MS), have emerged as a serious problem for the aging population. Potential causes of neurodegeneration or neuronal cell death are oxidative stress, increased aggregates of proteins such as alpha-amyloid or beta-amyloid in neurons, and chronic inflammation in the central nervous system (CNS).

[0006] For example, recent studies on Alzheimer's disease and Parkinson's disease have shown that the inflammatory response in the brain is a major cause of neuronal death. In fact, increased inflammatory mediators and reactive oxygen species have been confirmed in the cerebrospinal fluid of patients with brain diseases. Also, in the areas of brain damage, many active microglial cells have been observed, indicating that brain inflammation is a major cause of Parkinson's disease. Therefore, inhibiting brain inflammation by glial cells has become a goal for treating neurodegenerative disorders. However, the therapeutic drugs developed so far are only effective in controlling the symptoms of the disease and not in treating neurodegeneration itself.

[0007] Therefore, it is necessary to develop preventive and therapeutic drugs for neurodegenerative disorders based on a concept completely different from the conventional ones.

[0008] For example, dementia is an acquired brain disease with a multifaceted etiology caused by various genetic and environmental risk factors, which refers to a clinical disease that causes multiple cognitive impairments. The most typical disease causing dementia is Alzheimer's disease, which mainly occurs in the elderly and accounts for more than 60% of dementia cases.

[0009] Multiple studies have suggested the involvement of inflammation in neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease. Contrary to the traditional view that the brain is an immunologically privileged site due to the presence of the blood-brain barrier (BBB), recent studies have demonstrated that the brain is fully equipped with the ability to initiate an immune response. The peripheral immune system is not involved in inflammation within the brain, nor are antibodies or T cells. The immune response within the brain depends particularly on the synthesis of inflammatory components by glial cells, which are resident phagocytes (microglia in the case of the brain).

[0010] Within the brain, glial cells play an important role in maintaining a homeostatic microenvironment that promotes the survival of neurons. Microglia mediate the innate immune response to invading pathogens by secreting a diverse range of factors including cytokines, chemokines, prostaglandins, reactive oxygen and nitrogen species, and growth factors. Therefore, it is necessary to tightly regulate inflammatory and anti-inflammatory responses to prevent the potential harmful effects of long-term inflammatory oxidative stress on vulnerable neuronal populations.

[0011] In the brains of healthy adults, microglial cells are normally in a resting state. These cells are known to release various types of inflammatory molecules such as nitric oxide (NO) and cytokines that cause damage and cell death to surrounding neurons when activated. For example, activated microglia, the accumulation of cytokines, and the activation of the nuclear factor kappa B (NF-kappa B) pathway have been found to contribute to the progression of neurodegenerative diseases.

[0012] On the one hand, in research on amyloid-beta protein (Aβ), which is known to be a common cause of hereditary and sporadic Alzheimer's disease, it has been reported that small amounts of Aβ are produced in various parts of the body even in healthy individuals. In healthy individuals, Aβ is rapidly degraded after being produced and does not accumulate in the body. However, in the case of Alzheimer's disease patients, Aβ is produced abnormally in large amounts, accumulates in tissues without being degraded, and as a result, the formation of senile plaques or excessive accumulation occurs in places such as the hippocampus or cerebral cortex, which play important roles in memory and learning. The accumulated Aβ causes an inflammatory response in surrounding cells. As a result, nerve cells are damaged, and ultimately, even the neural network for maintaining the normal function of the brain is damaged. Furthermore, the accumulated Aβ generates a large amount of reactive oxygen species that activate the signal transduction system for killing nerve cells.

[0013] Aβ is a part of the amyloid precursor protein that is cleaved by β-secretase. Depending on the number of amino acids that make up Aβ, there are various forms of Aβ. In the case of Alzheimer's disease patients, the proportion of Aβ composed of 40 or 42 amino acids rapidly increases. There are many reports that treating neurons cultured in vitro with Aβ induces neuronal cell death and that the mechanism of cell death is often similar to the type of apoptosis seen in Alzheimer's disease patients. The damage to nerve cells by Aβ1-42 or Aβ1-43 proteins has been identified as one of the important causes of Alzheimer's-type diseases, and Aβ25-35 is known to be an important toxic fragment of Aβ1-42 or 43 that causes nerve cell damage.

[0014] The most common drugs currently approved by the FDA and used in the treatment of dementia include AChEIs and NMDA (N-methyl-D-aspartic acid) receptor antagonists, and various other drugs such as antioxidants, non-steroidal anti-inflammatory drugs (NSAIDs), anti-inflammatory agents, statins, and hormones are used in combination with them. However, these drugs are only used to relieve and delay symptoms and improve cognitive function, and currently, a fundamental treatment for dementia is still needed.

[0015] Typical AChEIs include donepezil, rivastigmine, and galantamine, and these drugs show a therapeutic effect on symptoms by temporarily increasing the concentration of the neurotransmitter acetylcholine. Furthermore, these drugs are prescribed to patients with mild to moderate Alzheimer's disease, vascular dementia, Parkinson's disease dementia, and stroke or associated subcortical ischemic vascular disease.

[0016] Due to the decline or loss of nerve cell function, neurodegenerative diseases including dementia show abnormalities in a variety of functions, including all perceptible physical functions of the human body such as motor control function, cognitive function, perceptual function, and sensory function, as well as the autonomic nerve function that is self-regulated in a state not recognized by the human body.

Summary of the Invention

Problems to be Solved by the Invention

[0017] Since the causes of neurodegenerative diseases including dementia are not fully understood, fundamental treatment remains a difficult problem. Commercially available drugs can relieve the symptoms of some diseases, but cannot fundamentally change the progression of the diseases, and drug tolerance and severe side effects occur after treatment, further limiting the improvement of the symptoms of these patients.

[0018] Under such circumstances, the options for treating neurodegenerative diseases or disorders including dementia are still limited.

Means for Solving the Problems

[0019] The present invention provides compositions and methods for treating neurodegenerative diseases by reducing neuroinflammation in the CNS system and / or reducing the expression of toxic proteins such as beta-amyloid (Ab). Here, The composition includes a PDE-5 inhibitor and an acetylcholinesterase inhibitor (AChEI). The PDE-5 inhibitor is selected from among sildenafil, tadalafil, vardenafil, tadalifil, udenafil, dasanafil, avanafil, and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. The AChEI is selected from among donepezil, galantamine, rivastigmine, or mixtures thereof. The composition inhibits inflammatory cytokines such as IL1b, IL-6, or TNFa. The composition inhibits the growth and differentiation of nerve cells, as well as the deterioration of learning and memory, induces a decrease in intracellular Aβ, thereby increasing the protection of nerve cells and synaptic plasticity. The neurodegenerative disease is selected from the group consisting of dementia, Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease (AD), Huntington's disease (HD), multiple sclerosis (MS), vascular dementia (VaD), or a group of mixed etiologies thereof. Brief Description of the Drawings

[0020]

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Mode for Carrying Out the Invention

[0021] The present invention provides a composition and a method for treating neurodegenerative diseases by reducing neuroinflammation in the CNS system in particular, and / or by reducing the expression of toxic proteins such as beta amyloid (Ab). Here, The composition includes a PDE-5 inhibitor and an acetylcholinesterase inhibitor (AChEI). The PDE-5 inhibitor is selected from milodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasanafil, avanafil, and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. The AChEI is selected from donepezil, galantamine, rivastigmine, or mixtures thereof. The composition inhibits inflammatory cytokines, for example, IL1b, IL-6, or TNFa. The composition inhibits the growth and differentiation of nerve cells, as well as the deterioration of learning and memory, thereby inducing a decrease in intracellular Aβ, thereby increasing the protection of nerve cells and synaptic plasticity. The neurodegenerative disease is selected from dementia, Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease (AD), Huntington's disease (HD), multiple sclerosis (MS), vascular dementia (VaD), or a mixed etiology thereof.

[0022] In one embodiment of the present invention, the present invention provides a composition for preventing and treating dementia, comprising a phosphodiesterase 5 inhibitor and an acetylcholinesterase inhibitor as active ingredients.

[0023] In a particular embodiment of the present invention, the composition comprises a weight ratio of PDE-5 inhibitor to AChEI of 1:0.1 to 1:10, or 50:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, or 1:10.

[0024] In another embodiment, the composition of the present invention provides a synergistic effect on the following: (1) Inhibition of Aβ oligomer / fibril formation by reduction of Aβ aggregation; (2) Inhibition of β-amyloid formation processing by reduction of BACE-1; (3) Reduction of extracellular Aβ monomers, oligomers, and Aβ fibrils / plaques by increasing cerebral blood flow; (4) Suppression of neuronal cell death by activation of the NO / cGMP / PKG / CREB pathway, and promotion of neurogenesis, synaptogenesis, and / or angiogenesis; (5) Restoration of synaptic plasticity (synaptic plasticity) by activation of the Wint signaling pathway by inhibition of DKK-1, and inhibition of APP production and reduction of Aβ accumulation by suppression of the positive feedback loop of Aβ production, and (6) Inhibition of Aβ fibril / plaque formation by removing intracellular toxicity and soluble Aβ oligomers by activation of autophagy.

[0025] The phosphodiesterase 5 inhibitor of the present invention is at least one selected from the group consisting of milodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasanafil, avanafil, and pharmaceutically acceptable salts, solvates, and hydrates thereof.

[0026] A pharmaceutically acceptable salt refers to a formulation of a compound that does not cause severe irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. Pharmaceutically acceptable salts are prepared by conventional methods well known in the art using pharmaceutically acceptable, substantially non-toxic organic and inorganic acids. Examples of acids include inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, and phosphoric acid, and sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, and p-toluenesulfonic acid, and organic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutyric acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, and salicylic acid. In addition, the compounds of the present invention can be reacted with bases to form ammonium salts; salts such as alkali metal salts such as sodium salts or potassium salts; salts such as alkaline earth metal salts such as calcium salts or magnesium salts; salts of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, and salts of amino acids such as arginine and lysine.

[0027] According to one embodiment of the present invention, examples of pharmaceutically acceptable salts can be milodenafil hydrochloride, sildenafil citrate, or vardenafil hydrochloride.

[0028] A hydrate refers to a compound of the present invention or a salt thereof that contains a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0029] A solvate refers to a compound of the present invention or a salt thereof containing a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents for this purpose are solvents that are volatile, non-toxic, and / or suitable for administration to humans.

[0030] The acetylcholinesterase inhibitor of the present invention is at least one selected from the group consisting of donepezil, rivastigmine, galantamine, and pharmaceutically acceptable salts, solvates, and hydrates thereof.

[0031] More preferably, the phosphodiesterase 5 inhibitor is selected from the group consisting of sildenafil, pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof, and the acetylcholinesterase inhibitor is selected from the group consisting of donepezil, galantamine, rivastigmine, pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

[0032] The pharmaceutical composition of the present invention can be administered orally or parenterally.

[0033] According to one embodiment of the present invention, the pharmaceutical composition of the present invention is administered orally to a subject or parenterally to a site other than the head. In other words, the composition of the present invention can exhibit the intended effects of the present invention even when not directly administered to brain tissue, body tissue surrounding the brain tissue (e.g., scalp), and adjacent sites. In a specific example, parenteral administration is subcutaneous administration, intravenous administration, intraperitoneal injection, transdermal administration, or intramuscular administration, and in another specific example, this is subcutaneous administration, intravenous administration, or intramuscular administration.

[0034] The pharmaceutically acceptable carriers contained in the pharmaceutical composition of the present invention are those commonly used in formulations, and these include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, and preservatives. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington’s Pharmaceutical Sciences (19th ed., 1995).

[0035] The pharmaceutical composition of the present invention may be prepared in unit dosage form by formulating with pharmaceutically acceptable carriers and / or excipients, or may be prepared by internalizing in multiple-dose containers according to methods readily practicable by those skilled in the technical field to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or may be in the form of an extract, powder, granule, tablet, film, or capsule, and may further contain a dispersing agent or a stabilizer.

[0036] In certain embodiments, the composition of the present invention provides a synergistic effect against the inhibition of inflammatory factors, resulting in the reduction of neuroinflammation.

[0037] In another embodiment, the composition of the present invention provides a synergistic effect against the reduction of Aβ42 accumulation for preventing and / or treating dementia through the reduction of amyloid beta by the combined use of a phosphodiesterase 5 (PDE5 inhibitor) and an acetylcholinesterase inhibitor.

Example

[0038] Hereinafter, a more detailed description will be given using the following embodiments. However, these embodiments are merely for exemplifying the present invention, and the scope of the present invention is not limited by these embodiments.

[0039] Example 1. Culture method of IMG cells IMG cells, a mouse microglia cell line used in the experiment, were cultured in a CO2 incubator (311-TIF, Thermo Fisher Scientific Forma, MA, USA) under the conditions of 37 °C and 5% CO2 using DMEM complete medium (HyClone) containing 10% fetal bovine serum (FBS; Australian Orgin, HyClone, Logan, UT, USA) and 1% penicillin / streptomycin (P / S; HyClone).

[0040] 2×10 5 cells were seeded into each well of a 6-well plate and incubated for 24 hours under the above conditions.

[0041] After 24 hours of incubation, 100 ng / ml of LPS, as well as drugs, AR1001, and AChEIs (donepezil, rivastigmine, galantamine), were treated individually or in combination at concentrations of 2, 10, and 20 μM.

[0042] Example 2. RNA extraction and cDNA synthesis The cells in the well were scraped off using a scraper, 2 mL of the culture solution was placed in a 15 mL conical tube, centrifuged at 3,000 RPM for 5 minutes, the culture solution excluding the pellet was discarded, and each cell was treated with 1 mL of Trizol.

[0043] Transferred to a 1.5 mL e-tube, 0.2 mL of chloroform was added, vortexed for 1 minute, and stored at room temperature for 2 minutes.

[0044] After centrifugation at 12,000 g for 10 minutes at 4°C, 500 μL of the supernatant was separated, 500 μL of isopropanol was added to the separated supernatant, and it was left at room temperature for 10 minutes. Then, it was centrifuged at 12,000 g for 10 minutes at 4°C.

[0045] The supernatant was discarded, and the pellet was washed twice with 75% EtOH.

[0046] All EtOH was discarded, the RNA pellet was dried, and it was dissolved in 10 μL of DEPC-treated water.

[0047] cDNA synthesis was performed according to the PrimeScript™ II 1st strand cDNA Synthesis Kit (Takara).

[0048] Example 3. Real-time RT-qPCR of inflammatory cytokines Real-time RT-qPCR was performed using Quant Studio 5 (Applied biosystems). The primer sequence of the IL1 beta forward primer was 5’-AGCTTCAGGCAGGCAGTATC-3’ (SEQ ID NO: 1). The IL1 beta reverse primer was 5’-AAGGTCCACGGGAAAGACAC-3’ (SEQ ID NO: 2). The TNF alpha forward primer was 5’-AAATGGCCTCCCTCTCATCAG-3’ (SEQ ID NO: 3). The TNF alpha reverse primer was 5’-GTCACTCGAATTTTGAGAAGATGATC-3’ (SEQ ID NO: 4). The beta-actin forward primer was 5’-CGTGCGTGACATCAAAGAGAA-3’ (SEQ ID NO: 5). The beta-actin reverse primer was 5’-TGGATGCCACAGGATTCCAT-3’ (SEQ ID NO: 6). SYBR Green PCR Master Mix (ThermoFisher) was used for the polymerase.

[0049] Example 4. Measurement results of the reduction rate of inflammatory cytokines. Figure 1 shows the results of an experiment to confirm whether the combined use of milodenafil and donepezil in the present invention has a synergistic effect on IL-1β.

[0050] In Figure 1, AR1001 refers to milodenafil. Referring to Figure 2, the reduction rate of IL-1β in the combined treatment of 2 μM milodenafil and 2 μM donepezil is 11.89%; the reduction rate of IL-1β in the combined treatment of 2 μM milodenafil and 10 μM donepezil is 26.53%; the reduction rate of IL-1β in the combined treatment of 2 μM milodenafil and 20 μM donepezil is 54.49%; and the reduction rate of IL-1β in the combined treatment of 10 μM milodenafil and 2 μM donepezil is 27.44%; and the reduction rate of IL-1β in the combined treatment of 10 μM milodenafil and 10 μM donepezil is 41.36%; and the reduction rate of IL-1β in the combined treatment of 10 μM milodenafil and 20 μM donepezil is 76.51%; and the reduction rate of IL-1β in the combined treatment of 20 μM milodenafil and 2 μM donepezil is 50.28%; and the reduction rate of IL-1β in the combined treatment of 20 μM milodenafil and 10 μM donepezil is 78.92% are significantly higher than the sum of the increase rates A and B when treated with milodenafil or donepezil alone, which proves that an effect exceeding the additive effect can be observed.

[0051] Figure 3 shows the results of an experiment to confirm whether the combined use of milodenafil and donepezil in the present invention has a synergistic effect on TNF-α.

[0052] In Figure 3, AR1001 refers to milodenafil. Referring to Figure 4, the reduction rate of TNF-α in the combined treatment of 2 μM milodenafil and 2 μM donepezil was 11.68%; the reduction rate of TNF-α in the combined treatment of 2 μM milodenafil and 10 μM donepezil was 38.46%; and the reduction rate of TNF-α in the combined treatment of 10 μM milodenafil and 2 μM donepezil was 28.59%; and the reduction rate of TNF-α in the combined treatment of 10 μM milodenafil and 10 μM donepezil was 57.62%; and the reduction rate of TNF-α in the combined treatment of 10 μM milodenafil and 20 μM donepezil was 83.61%; and the reduction rate of TNF-α in the combined treatment of 20 μM milodenafil and 2 μM donepezil was 59.85%; and the reduction rate of TNF-α in the combined treatment of 20 μM milodenafil and 10 μM donepezil was 83.08% were significantly higher than the sum of the increase rates A and B when treated with milodenafil or donepezil alone, which confirmed that an effect exceeding the additive effect could be observed.

[0053] Figure 5 shows the results of an experiment to confirm whether the combination of milodenafil and galantamine in the present invention has a synergistic effect on IL-1β.

[0054] In Figure 5, AR1001 refers to milodenafil. Referring to Figure 6, the reduction rate of IL-1β in the combined treatment of 2 μM milodenafil and 2 μM galantamine is 9.52%; the reduction rate of IL-1β in the combined treatment of 2 μM milodenafil and 10 μM galantamine is 26.28%; the reduction rate of IL-1β in the combined treatment of 2 μM milodenafil and 20 μM galantamine is 38.57%; and the reduction rate of IL-1β in the combined treatment of 10 μM milodenafil and 2 μM galantamine is 27.34%; and the reduction rate of IL-1β in the combined treatment of 10 μM milodenafil and 10 μM galantamine is 40.29%; and the reduction rate of IL-1β in the combined treatment of 10 μM milodenafil and 20 μM galantamine is 59.91%; and the reduction rate of IL-1β in the combined treatment of 20 μM milodenafil and 2 μM galantamine is 56.17%; and the reduction rate of IL-1β in the combined treatment of 20 μM milodenafil and 10 μM galantamine is 71.15%; and the reduction rate of IL-1β in the combined treatment of 20 μM milodenafil and 20 μM galantamine is 83.74% are significantly higher than the sum of the increase rates A and B when treated with milodenafil or galantamine alone, which proves that an effect exceeding the additive effect can be observed.

[0055] Figure 7 shows the results of an experiment to confirm whether the combination of milodenafil and galantamine in the present invention has a synergistic effect on TNF-α.

[0056] In Figure 7, AR1001 refers to milodenafil. Referring to Figure 8, the reduction rate of TNF-α in the combined treatment of 2 μM milodenafil and 2 μM galantamine is 12.01%; the reduction rate of TNF-α in the combined treatment of 2 μM milodenafil and 10 μM galantamine is 25.22%; and the reduction rate of TNF-α in the combined treatment of 10 μM milodenafil and 2 μM galantamine is 38.08%; and the reduction rate of TNF-α in the combined treatment of 10 μM milodenafil and 10 μM galantamine is 47.14%; and the reduction rate of TNF-α in the combined treatment of 10 μM milodenafil and 20 μM galantamine is 76.08%; and the reduction rate of TNF-α in the combined treatment of 20 μM milodenafil and 10 μM galantamine is 72.41% are significantly higher than the sum of the increase rates A and B when treated with milodenafil or galantamine alone, which proves that an effect exceeding the additive effect can be observed.

[0057] Figure 9 shows the results of an experiment to confirm whether the combination of milodenafil and rivastigmine in the present invention has a synergistic effect on IL-1β.

[0058] In Figure 9, AR1001 refers to milodenafil. Referring to Figure 10, the reduction rate of IL-1β in the combined treatment of 2 μM milodenafil and 2 μM rivastigmine is 8.17%; the reduction rate of IL-1β in the combined treatment of 2 μM milodenafil and 10 μM rivastigmine is 23.70%; and the reduction rate of IL-1β in the combined treatment of 10 μM milodenafil and 2 μM rivastigmine is 25.29%; and the reduction rate of IL-1β in the combined treatment of 10 μM milodenafil and 10 μM rivastigmine is 39.91%; and the reduction rate of IL-1β in the combined treatment of 10 μM milodenafil and 20 μM rivastigmine is 53.79%; and the reduction rate of IL-1β in the combined treatment of 20 μM milodenafil and 10 μM rivastigmine is 67.53% are significantly higher than the sum of the increase rates A and B when treated with milodenafil or rivastigmine alone, which proves that an effect exceeding the additive effect can be observed.

[0059] Figure 11 shows the results of an experiment to confirm whether the combined use of milodenafil and rivastigmine in the present invention has a synergistic effect on TNF-α.

[0060] In Figure 12, AR1001 refers to milodenafil. Referring to Figure 8, the reduction rate of TNF-α in the combined treatment of 2 μM milodenafil and 2 μM rivastigmine was 11.93%; the reduction rate of TNF-α in the combined treatment of 2 μM milodenafil and 10 μM rivastigmine was 21.14%; the reduction rate of TNF-α in the combined treatment of 2 μM milodenafil and 20 μM rivastigmine was 34.44%; and the reduction rate of TNF-α in the combined treatment of 10 μM milodenafil and 2 μM rivastigmine was 30.21%; and the reduction rate of TNF-α in the combined treatment of 10 μM milodenafil and 10 μM rivastigmine was 44.63%; and the reduction rate of TNF-α in the combined treatment of 10 μM milodenafil and 20 μM rivastigmine was 55.00%; and the reduction rate of TNF-α in the combined treatment of 20 μM milodenafil and 10 μM rivastigmine was 65.93% were significantly higher than the sum of the increase rates A and B when treated with milodenafil or rivastigmine alone, which confirmed that an effect exceeding the additive effect could be observed.

[0061] Example 5. Cell culture The SH-SY5Y human neuroblastoma cell line used in the experiment was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in a CO2 incubator (311-TIF, Thermo Fisher Scientific Forma, MA, USA) under the conditions of 37 °C and 5% CO2 using DMEM / F12 complete medium (HyClone) containing 10% fetal bovine serum (FBS; Australian Orgin, HyClone, Logan, UT, USA) and 1% penicillin / streptomycin (P / S; HyClone).

[0062] Example 6. Neuronal-like Differentiation of SH-SY5Y Cells Using All-Trans Retinoic Acid (RA) 2×10 4 Cells / well were dispensed into 96-well plates to evaluate cytotoxicity, and 2×10 5 Cells were dispensed into T-25 flasks to confirm changes in protein expression related to neuronal cell death, neuronal inflammatory responses, neurotransmitters, and synaptic plasticity, as well as the activity of acetylcholinesterase (AChE). For cell attachment and stabilization, DMEM / F12 complete medium (HyClone) containing 10% FBS (HyClone) and 1% P / S (HyClone) was used, and the cells were cultured in a CO2 incubator (Thermo Fisher Scientific Forma) at 37 °C and 5% CO2 for 24 hours. Twenty-four hours after cell dispensing, the cell culture medium was removed for neuronal-like differentiation and replaced with DMEM / F12 differentiation medium containing 1% FBS (HyClone), 1% P / S (HyClone), and 10 μM all-trans retinoic acid (RA; Sigma-Aldrich, St. Louis, MO, USA). On the third day of differentiation, the medium was replaced with fresh DMEM / F12 differentiation medium. On the sixth day of differentiation, the medium of the untreated control group was replaced with fresh DMEM / F12 differentiation medium, and the sample-treated groups were replaced by adding fresh DMEM / F12 differentiation medium under various conditions.

[0063] Example 7. Formation and Treatment of Amyloid-β (Aβ) 1-42 To form Aβ1-42 oligomers, human Aβ1-42 (Abcam, Cambridge, MA, USA) was added to DMEM / F12 complete medium (HyClone) containing 1% FBS (HyClone) and 1% P / S (HyClone) to a concentration of 10 μM, and the mixture was left in a CO2 incubator (Thermo Fisher Scientific Forma) at 37 °C and 5% CO2 for 3 hours to form Aβ1-42 oligomers.

[0064] To confirm the change of Aβ1-42, the existing cell culture medium was removed from RA-differentiated SH-SY5Y neuron-like cells, and replaced with DMEM / F12 complete medium (HyClone) containing Aβ1-42 oligomers (10 μM), and cultured in a CO2 incubator (Thermo Fisher Scientific Forma) at 37 °C and 5% CO2 for 72 hours to induce Aβ1-42 oligomer-induced cell damage.

[0065] After 72 hours, the culture medium was removed, and then DMEM / F12 complete medium (HyClone) was treated alone or in combination with milodifenil, donepezil, galantamine, or rivastigmine, and cultured in a CO2 incubator (Thermo Fisher Scientific Forma) at 37 °C and 5% CO2 for 24 hours, and then the experiment was carried out.

[0066] Example 8. ELISA (Enzyme-Linked Immunosorbent Assay) Measurement Results of Human Aβ42 To measure the amount of Aβ42 (pg / mL) in cells, the cells were collected and treated with cell lysis buffer. Then, after centrifugation at 4 °C and 14,000 rpm for 10 minutes, the supernatant was transferred to recover the protein. The amount of protein was quantified using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Subsequently, the amount of Aβ42 in the cells was measured using a human Aβ42 ELISA kit (Invitrogen).

[0067] Figure 14 shows the results of an experiment to confirm whether the combined use of milodifenil and donepezil in the present invention has a synergistic effect on the reduction of Aβ.

[0068] In FIG. 14, AR1001 refers to milodenafil. Referring to FIG. 14, the reduction rate of Aβ in the combined treatment of 0.1 μM milodenafil and 0.1 μM donepezil is 5.49%; the reduction rate of Aβ in the combined treatment of 0.1 μM milodenafil and 0.5 μM donepezil is 8.35%; the reduction rate of Aβ in the combined treatment of 0.5 μM milodenafil and 0.1 μM donepezil is 11.65%; and the reduction rate of Aβ in the combined treatment of 0.5 μM milodenafil and 0.5 μM donepezil is 18.27% are significantly higher than the sum of the increase rates A and B when treated with milodenafil or donepezil alone, which proves that an effect exceeding the additive effect can be recognized.

[0069] FIG. 16 shows the results of an experiment to confirm whether the combined use of milodenafil and galantamine in the present invention has a synergistic effect on the reduction of Aβ.

[0070] Referring to FIG. 16, the reduction rate of Aβ in the combined treatment of 0.1 μM milodenafil and 0.1 μM galantamine is 4.32%; the reduction rate of Aβ in the combined treatment of 0.1 μM milodenafil and 0.5 μM galantamine is 19.08%; the reduction rate of Aβ in the combined treatment of 0.5 μM milodenafil and 0.1 μM galantamine is 11.15%; and the reduction rate of Aβ in the combined treatment of 0.5 μM milodenafil and 0.5 μM galantamine is 20.43% are significantly higher than the sum of the increase rates A and B when treated with milodenafil or galantamine alone, which proves that an effect exceeding the additive effect can be recognized.

[0071] FIG. 18 shows the results of an experiment to confirm whether the combined use of milodenafil and rivastigmine in the present invention has a synergistic effect on the reduction of Aβ.

[0072] Referring to Fig. 18, the reduction rate of Aβ in the combined treatment with 0.1 μM of milodenafil and 0.1 μM of rivastigmine was 8.43%; the reduction rate of Aβ in the combined treatment with 0.1 μM of milodenafil and 0.5 μM of rivastigmine was 14.88%; the reduction rate of Aβ in the combined treatment with 0.5 μM of milodenafil and 0.1 μM of rivastigmine was 8.94%; and the reduction rate of Aβ in the combined treatment with 0.5 μM of milodenafil and 0.5 μM of rivastigmine was 17.91%, which was significantly higher than the sum of the increase rates A and B in the case of treatment with milodenafil or rivastigmine alone, thereby proving that an effect exceeding the additive effect can be observed.

[0073] The above-described invention of the present invention is merely illustrative, and those skilled in the art to which the present invention pertains will understand that various modifications and other equivalent embodiments are possible therefrom. Therefore, it should be understood that the present invention is not limited to the forms mentioned in the forms for carrying out the above invention. Therefore, the true scope of the technical protection of the present invention should be determined by the technical idea of the appended claims. Furthermore, it should be understood that the present invention includes all modifications, equivalents, and substitutions within the spirit and scope of the present invention defined by the appended claims.

[0074]

Table 1

Claims

1. A phosphodiesterase 5 inhibitor; and an acetylcholinesterase inhibitor, a composition comprising the same as an active ingredient.

2. The composition according to claim 1, wherein the phosphodiesterase 5 inhibitor is selected from the group consisting of sildenafil, tadalafil, vardenafil, tadalafill, udenafil, dasanafil, avanafil, and pharmaceutically acceptable salts, solvates, hydrates, and mixtures thereof.

3. The composition according to claim 1, wherein the acetylcholinesterase inhibitor is selected from the group consisting of donepezil, rivastigmine, galantamine, physostigmine, tacrine, metrifonate, phenserine, tolserine, eceroline, fuperidine A and B, galangin, cardanol, donepezil-AP2238, donepezil-tacrine, tacrine-ferulic acid hybrid, tacrine-hydroxyquinoline, ladostigil, indenyl derivatives, pharmaceutically acceptable salts, solvates, hydrates, and mixtures thereof.

4. The phosphodiesterase 5 inhibitor is selected from the group consisting of sildenafil, pharmaceutically acceptable salts, solvates, hydrates, and mixtures thereof; and the acetylcholinesterase inhibitor is at least one selected from the group consisting of donepezil, galantamine, rivastigmine, pharmaceutically acceptable salts, solvates, hydrates, and mixtures thereof, a composition for preventing and treating dementia according to claim 1.

5. The composition according to claim 1, wherein the phosphodiesterase 5 inhibitor is sildenafil.

6. A method for preventing or treating neuroinflammation, comprising administering an effective amount of a pharmaceutical composition comprising the composition according to claim 1.

7. A method for preventing or inhibiting the formation and / or accumulation of beta amyloid, comprising administering an effective amount of a pharmaceutical composition comprising the composition according to claim 1.

8. A method for preventing or treating neurodegenerative diseases, comprising administering an effective amount of a pharmaceutical composition comprising the composition according to claim 1.

9. The method according to claim 8, wherein the neurodegenerative disease is selected from the group consisting of dementia, Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease (AD), Huntington's disease (HD), multiple sclerosis (MS), vascular dementia (VaD), and mixed etiologies thereof. ​ ​ ​

10. A method for inhibiting Aβ oligomer / fibril formation by reducing Aβ aggregation, comprising administering an effective amount of a pharmaceutical composition comprising the composition according to claim 1.

11. A method for inhibiting β-amyloid formation processing by reducing BACE-1, comprising administering an effective amount of a pharmaceutical composition comprising the composition according to claim 1.

12. A method for reducing extracellular Aβ monomers, oligomers, and Aβ fibrils / plaques by increasing cerebral blood flow, comprising administering an effective amount of a pharmaceutical composition comprising the composition according to claim 1.

13. A method for suppressing neuronal cell death, promoting neurogenesis, synaptogenesis, and / or angiogenesis by activating the NO / cGMP / PKG / CREB pathway, comprising administering an effective amount of a pharmaceutical composition comprising the composition according to claim 1.

14. A method for restoring synaptic plasticity by activating Wnt signaling by inhibiting DKK-1, comprising administering an effective amount of a pharmaceutical composition comprising the composition according to claim 1.

15. A method for inhibiting APP production and reducing Aβ accumulation by suppressing the positive feedback loop of Aβ production, comprising administering an effective amount of a pharmaceutical composition comprising the composition according to claim 1.

16. A method for inhibiting Aβ fibril / plaque formation by removing intracellular toxicity and soluble Aβ oligomers by activating autophagy, comprising administering an effective amount of a pharmaceutical composition comprising the composition according to claim 1. ​ ​ ​ ​ ​ ​ ​

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