Composition for preventing and treating neurodegenerative diseases

Abstract

The present invention provides a composition for preventing or treating neurodegenerative diseases, comprising a phosphodiesterase 5 inhibitor (PDE5 inhibitor) and an N-methyl-D-aspartic acid receptor (NMDA receptor) antagonist, wherein the PDE5 inhibitor is sildenafil, tadalafil, vardenafil, tadalafil, udenafil, dasantafil, avanafil, a pharmaceutically acceptable salt, solvate, hydrate, or a mixture thereof, and the NMDA receptor antagonist is selected from memantine, amantadine, ketamine, traxoprodil, lanicemine, lisinopril, petidine, levorphanol, methadone, dextropropoxyphene, tramadol, ketobemidone, dextromethorphan (DXM), phencyclidine (PCP), and methoxetamine (MXE), a pharmaceutically acceptable salt, solvate, hydrate, and mixtures thereof, and the neurodegenerative disease is dementia, Parkinson's disease (PD), dementia with Lewy bodies (DEB), Alzheimer's disease (AD), Huntington's disease (HD), multiple sclerosis (MS), vascular dementia (VaD), amyotrophic lateral sclerosis (AES), frontotemporal dementia, or a mixed etiology thereof.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 367,282, filed on June 29, 2022, the content of which is incorporated herein by reference.

[0002] The present invention relates to a composition comprising a phosphodiesterase 5 inhibitor (PDE5 inhibitor) and an N - methyl - D - aspartic acid receptor (NMDA receptor) antagonist for preventing or treating neurodegenerative diseases, and a method of using the same.

[0003] Sequence Listing

[0004] This application incorporates by reference in its entirety the XML file of the sequence listing entitled "04334900121_SequenceListing.xml (7KB)", created on June 23, 2023 and electronically submitted together with this specification.

Background Art

[0005] In recent years, the number of patients with neurodegenerative diseases has been increasing rapidly. In the treatment of neurodegenerative diseases, the most important step is prevention. However, since the cause of the disease has not yet been clearly understood, treatment methods still need to be studied. A common pathological phenomenon of neurodegenerative diseases is the death of central nervous system cells. Different from other organ cells, central nervous system cells are almost impossible to regenerate after cell death, leading to a permanent loss of function. Therefore, the treatment methods for such brain diseases developed so far mainly focus on preventing nerve cell death.

[0006] Neurodegeneration, in general, involves the progressive loss of the structure or function of neurons and includes the death of neurons in various regions of the brain. Neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and multiple sclerosis (MS), have emerged as a serious problem for aging. Potential causes of neurodegeneration or neuronal death are oxidative stress, aggregation of toxic proteins such as beta-amyloid, and chronic inflammation of the central nervous system (CNS).

[0007] For example, recent studies on Alzheimer's disease and Parkinson's disease have shown that the inflammatory response in the brain is the main cause of neuronal death. Increases in inflammatory mediators and reactive oxygen species have been confirmed in the cerebrospinal fluid of patients with brain diseases. Also, a large number of activated microglial cells have been observed in the damaged areas of the brain, indicating that brain inflammation is a major cause of Parkinson's disease. Suppression of brain inflammation by glial cells is a target for the treatment of neurodegenerative diseases. However, the therapeutic agents developed so far have the effect of adjusting the symptoms of the disease but do not have the effect of treating neurodegenerative diseases themselves.

[0008] Therefore, there is a need to develop preventive and therapeutic agents for neurodegenerative diseases based on concepts completely different from the conventional ones. For example, dementia is an acquired brain disease associated with a multifaceted etiology caused by various genetic and environmental risk factors and refers to clinical diseases that cause multiple cognitive impairments. The most common cause of dementia is AD, which mainly occurs in the elderly and accounts for more than 60% of dementia.

[0009] Research has shown that inflammation is involved in neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease. Contrary to the conventional view that the brain is an immune-privileged site due to the presence of the blood-brain barrier (BBB), recent studies have proven that the brain is capable of fully eliciting an immune response. Brain inflammation does not affect the peripheral immune system or antibodies or T cells. The immune response in the brain depends on the synthesis of inflammatory components by glial cells, especially resident phagocytes, microglia in the case of the brain.

[0010] In the brain, glial cells play an important role in maintaining the homeostatic microenvironment that promotes the survival of neurons. Microglia mediate the innate immune response against invading pathogens by secreting a diverse array of factors, including cytokines, chemokines, prostaglandins, reactive oxygen and nitrogen species, and growth factors. Therefore, inflammatory and anti-inflammatory responses must be tightly regulated to prevent the potential adverse effects of long-term inflammation-induced oxidative stress on vulnerable neuronal populations.

[0011] In the normal adult brain, microglial cells are usually in a resting state. When activated, these cells are known to release various types of inflammatory molecules, such as nitric oxide (NO) and cytokines, which can cause damage and cell death to surrounding neurons. For example, activated microglia, cytokine accumulation, and activation of the nuclear factor kappa B (NF-κB) pathway have been found to contribute to the progression of neurodegenerative diseases.

[0012] On the other hand, in studies on amyloid-beta protein (Aβ), which is known as a common cause of both familial and sporadic Alzheimer's disease, it has been reported that even in normal individuals, a small amount of Aβ is produced in various parts of the body. In normal individuals, Aβ is rapidly degraded after production and does not accumulate in the body. However, in patients with Alzheimer's disease, Aβ is abnormally overproduced, not degraded, and accumulates in tissues, resulting in the formation of senile plaques or excessive accumulation in areas 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 even the neural network for maintaining normal brain function is damaged. Furthermore, the accumulated Aβ produces a large amount of reactive oxygen species and activates the signal transduction system that kills nerve cells.

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

[0014] The most common drugs currently approved by the FDA and used for the treatment of dementia include acetylcholinesterase inhibitors (AchEI) and NMDA receptor antagonists, and various other drugs, such as antioxidants, non-steroidal anti-inflammatory drugs (NSAID), anti-inflammatory agents, statins, and hormones, are used in combination with them. However, these drugs are only used for the relief and delay of symptoms and the improvement of cognitive function, and currently, a fundamental treatment for dementia is still needed.

[0015] Neurodegenerative diseases, including dementia, show abnormalities in a variety of functions, including all functions of the body that can be felt, due to a decrease or loss of neuronal function, such as the body's motor control function, cognitive function, perceptual function, and sensory function, as well as the autonomic nervous function that is automatically controlled in a state where the human body is not conscious.

[0016] Since the causes of neurodegenerative diseases are not fully understood, basic treatment is still an issue. Commercially available drugs can only relieve symptoms in some diseases and cannot fundamentally change the progression of the diseases. If serious side effects of the drugs appear after treatment, the improvement of the symptoms of these patients is further limited. Therefore, the options for treating neurodegenerative diseases or disorders, including dementia, are still limited.

SUMMARY OF THE INVENTION

[0017] The present invention provides compositions and methods for treating neurodegenerative diseases by reducing neuroinflammation, particularly in the CNS system, and / or by reducing the expression of toxic proteins, such as beta-amyloid (Ab).

[0018] The composition includes a PDE-5 inhibitor and an NMDA receptor antagonist.

[0019] The PDE-5 inhibitor is selected from among sildenafil, tadalafil, vardenafil, tadalifil, udenafil, dasanafil, avanafil, and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

[0020] The NMDA receptor antagonist is selected from among memantine, amantadine, ketamine, traxoprodil, lanicemine, lisuridem, perzinfotel, levorphanol, methadone, dextropropoxyphene, tramadol, ketobemidone, dextromethorphan (DXM), phencyclidine (PCP), and methoxetamine (MXE), pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

[0021] The composition inhibits inflammatory cytokines, such as IL1b, IL-6, or TNFa.

[0022] The composition inhibits the growth and differentiation of neurons, as well as the degradation of learning and memory, induces a decrease in intracellular Aβ, thereby enhancing the protection of neurons and synaptic plasticity.

[0023] The neurodegenerative disease is selected from among dementia, Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease (AD), Huntington's disease (HD), multiple sclerosis (MS), vascular dementia (VaD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, or mixed etiologies thereof.

Brief Description of the Drawings

[0024]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Modes for Carrying Out the Invention

[0025] The present invention provides compositions and methods for treating neurodegenerative diseases by reducing neuroinflammation, particularly in the CNS system, and / or by reducing the expression of toxic proteins, such as beta amyloid (Ab).

[0026] The composition contains a PDE-5 inhibitor and an NMDA receptor antagonist,

[0027] The PDE-5 inhibitor is selected from among sildenafil, tadalafil, vardenafil, tadalafil, udenafil, dasantafil, avanafil, and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof,

[0028] The NMDA receptor antagonist is memantine, amantadine, ketamine, traxoprodil, lanicemine, lisrengemuzad, petidine, levorphanol, methadone, dextropropoxyphene, tramadol, ketobemidone, dextromethorphan (DXM), phencyclidine (PCP), and methoxetamine (MXE), pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof,

[0029] The composition inhibits inflammatory cytokines, such as IL1b, IL-6, or TNFa,

[0030] The composition inhibits the growth and differentiation of nerve cells, as well as the degeneration of learning and memory, induces a decrease in intracellular Aβ, thereby enhancing the protection of nerve cells and synaptic plasticity,

[0031] The neurodegenerative disease is selected from among dementia, Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease (AD), Huntington's disease (HD), multiple sclerosis (MS), vascular dementia (VaD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, or a mixed etiology thereof.

[0032] In an embodiment of the present invention, there is provided a composition for preventing and treating dementia, containing a phosphodiesterase 5 inhibitor and an NMDA receptor antagonist as active ingredients.

[0033] In certain embodiments of the present invention, the composition comprises a PDE-5 inhibitor and an NMDA receptor antagonist in a weight ratio 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.

[0034] In another embodiment, the composition of the present invention

[0035] inhibits Aβ oligomer / fibril formation by reducing Aβ aggregation,

[0036] inhibits β-amyloid formation processing by reducing BACE-1,

[0037] reduces extracellular Aβ monomers, oligomers, and Aβ fibrils / plaques by increasing cerebral blood flow,

[0038] inhibits the inhibition of neuronal cell death by activating the NO / cGMP / PKG / CREB pathway, and promotes neurogenesis, synaptogenesis, and / or angiogenesis,

[0039] restores synaptic plasticity by activating the Wnt signaling pathway through inhibition of DKK-1, and inhibits APP production and reduces Aβ accumulation by suppressing the positive feedback loop for Aβ production, and

[0040] provides a synergistic effect on inhibiting the formation of Aβ fibrils / plaques by removing intracellular toxicity and soluble Aβ oligomers by activating autophagy.

[0041] The phosphodiesterase 5 inhibitor of the present invention is at least one selected from the group consisting of sildenafil, tadalafil, vardenafil, tadalafil, udenafil, dasantafil, avanafil, and pharmaceutically acceptable salts, solvates, or hydrates thereof.

[0042] The pharmaceutically acceptable salt refers to a pharmaceutical formulation of the 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. The 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 such acids include inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, and p-toluenesulfonic acid, 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. Further, the compounds of the present invention may be reacted with a base to form ammonium salts, alkali metal salts such as sodium or potassium salts, salts such as alkaline earth metal salts such as calcium or magnesium salts, organic bases such as dicyclohexylamine, N-methyl-D-glucamine, salts of tris(hydroxymethyl)methylamine, and amino acid salts such as arginine and lysine.

[0043] According to one embodiment of the present invention, examples of pharmaceutically acceptable salts can be milodenafil hydrochloride, sildenafil citrate, or vardenafil hydrochloride.

[0044] The hydrate refers to the compound of the present invention or a salt thereof containing a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0045] The solvate refers to the 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 therefor are those that are volatile, non-toxic, and / or suitable for administration to humans.

[0046] The NMDA receptor antagonist is memantine, amantadine, ketamine, traxoprodil, lanisemin, lisreneremdaz, petidine, levorphanol, methadone, dextropropoxyphene, tramadol, ketobemidone, dextromethorphan (DXM), phencyclidine (PCP), and methoxetamine (MXE), a pharmaceutically acceptable salt, solvate, hydrate, or a mixture thereof.

[0047] More preferably, the phosphodiesterase 5 inhibitor is selected from the group consisting of sildenafil, a pharmaceutically acceptable salt, solvate, hydrate, or a mixture thereof, and the NMDA receptor antagonist is memantine, a pharmaceutically acceptable salt, solvate, hydrate, or a mixture thereof.

[0048] The pharmaceutical composition of the present invention may be administered orally or parenterally.

[0049] According to an 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 in the present invention even when not directly administered to brain tissue, body tissue surrounding the brain tissue (e.g., scalp), and adjacent sites thereof. In one specific example, the parenteral administration is subcutaneous administration, intravenous administration, intraperitoneal injection, transdermal administration, or intramuscular administration, and in another specific example, it is subcutaneous administration, intravenous administration, or intramuscular administration.

[0050] The pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulation, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, and preservatives in addition to the above components. Appropriate pharmaceutically acceptable carriers and drugs are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0051] The pharmaceutical composition of the present invention may be manufactured in unit dosage form by formulating it using pharmaceutically acceptable carriers and / or excipients, or may be prepared by incorporating it into a multiple-dose container according to a method that can be easily performed by those skilled in the technical field to which the present invention belongs. In this case, the preparation 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.

[0052] In certain embodiments, the composition of the present invention has a synergistic effect on the inhibition of inflammatory factors and reduces neuroinflammation.

[0053] In another embodiment, the composition of the present invention has a synergistic effect on the reduction of Aβ42 accumulation and prevents and / or treats dementia through the reduction of amyloid beta by the combined use of a phosphodiesterase 5 (PDE5 inhibitor) and an NMDA receptor antagonist.

Example

[0054] The following provides a more detailed description using the embodiments below. However, these embodiments are for illustrative purposes only and do not limit the scope of the present invention.

[0055] Experimental Example 1. Culture Method of IMG Cells

[0056] The IMG cells, a mouse microglial cell line used in the experiment, were cultured and maintained at 37 °C and 5% CO2 in a humidified CO2 incubator (311-TIF, Thermo Fisher Scientific Forma, MA, USA) in DMEM complete medium (HyClone) containing 10% fetal bovine serum (FBS, from Australia, HyClone, Logan, UT, USA) and 1% penicillin / streptomycin (P / S, HyClone). A total of 2×10 5 cells were seeded in each well of a 6-well plate and incubated in the above humidified CO2 incubator for 24 hours. Furthermore, 100 μM of glutamate and drugs, namely, AR1001 and an NMDA receptor antagonist (memantine), were administered individually or in combination at concentrations of 2, 10, and 20 μM.

[0057] Experimental Example 2. RNA Extraction and cDNA Synthesis

[0058] Cells were scraped using a cell scraper, and 2 mL of the culture medium was collected into a 15 mL conical tube. It was centrifuged at 3,000 RPM for 5 minutes, and the supernatant (culture medium) was discarded. The pellet was resuspended in 1 mL of TRIzol and transferred to a 1.5 mL microtube. Further, 0.2 mL of chloroform was added, and it was vortexed for 1 minute. This microtube was placed on the bench at room temperature for 2 minutes. After centrifugation at 12,000×g for 10 minutes at 4°C, the supernatant (about 500 μL) was separated into a new microtube. An equal volume of isopropanol was added to the supernatant, and the solution was mixed well. This was placed on the bench of the microtube at room temperature for 10 minutes and then centrifuged at 12,000×g for 10 minutes at 4°C. The supernatant was discarded, and the pellet was washed twice with 75% ethanol. This RNA pellet was dried and dissolved in 10 μL of DEPC-treated water. After quantifying this RNA, it was converted to cDNA according to the protocol of the PrimeScript™ II 1st strand cDNA Synthesis Kit (Takara).

[0059] Experimental Example 3. Real-time RT-qPCR of Inflammatory Cytokines

[0060] This sample cDNA was amplified using gene-specific primers and SYBR Green PCR Master Mix (ThermoFisher) on a Quant Studio 5 thermal cycler model (Applied biosystems). The amplification conditions were as follows: polymerase activation at 50°C for 2 minutes, pre-denaturation at 95°C for 10 minutes, a total of 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The specific primer sequences are described in Table 1.

[0061]

Table 1

[0062] Experimental Example 4. Measurement of Inflammatory Cytokine Levels.

[0063] Figure 1 shows the synergistic effect on IL-1β in the combined treatment of milodenafil and memantine of the present invention. Here, AR1001 refers to milodenafil.

[0064] In Figure 2, the reduction rate of IL-1β was 6.11% in the combined treatment of 2 μM milodenafil and 2 μM memantine, 14.70% in the combination of 2 μM milodenafil and 10 μM memantine, 30.48% in the combination of 2 μM milodenafil and 20 μM memantine, 16.99% in the combination of 10 μM milodenafil and 2 μM memantine, 27.47% in the combination of 10 μM milodenafil and 10 μM memantine, 40.56% in the combination of 10 μM milodenafil and 20 μM memantine, 28.35% in the combination of 20 μM milodenafil and 2 μM memantine, 39.11% in the reduction rate of IL-1β in the combination of 20 μM milodenafil and 10 μM memantine, and 45.10% in the reduction rate of IL-1β in the combined treatment of 20 μM milodenafil and 20 μM memantine. The reduction rates for various combinations were significantly higher than the sum of the individual reduction rates observed when treated with milodenafil or memantine alone, and a synergistic effect exceeding the additive effect was confirmed.

[0065] Figure 3 shows the synergistic effect on TNF-α in the combined treatment of milodenafil and memantine of the present invention. Here, AR1001 refers to milodenafil.

[0066] In Figure 4, the reduction rate of TNF-α was 16.38% in the combined treatment with 2 μM of milodenafil and 2 μM of memantine, 24.26% in the combination of 2 μM of milodenafil and 10 μM of memantine, 28.51% in the combination of 2 μM of milodenafil and 20 μM of memantine, 21.22% in the combination of 10 μM of milodenafil and 2 μM of memantine, 29.86% in the combination of 10 μM of milodenafil and 10 μM of memantine, 32.53% in the combination of 10 μM of milodenafil and 20 μM of memantine, 34.58% in the combination of 20 μM of milodenafil and 2 μM of memantine, 39.26% in the combination of 20 μM of milodenafil and 10 μM of memantine, and 41.34% in the combined treatment with 20 μM of milodenafil and 20 μM of memantine. The reduction rates by various combinations were significantly higher than the sum of the individual reduction rates observed when treated with milodenafil or memantine alone, and a synergistic effect exceeding the additive effect was confirmed.

[0067] Experimental Example 5. Cell Culture

[0068] The SH-SY5Y human neuroblastoma cell line used in this 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) using DMEM / F12 complete medium (HyClone) containing 10% fetal bovine serum (FBS, Australia, HyClone, Logan, UT, USA) and 1% penicillin / streptomycin (P / S, HyClone) under the conditions of 37 °C and 5% CO2.

[0069] Experimental Example 6. Neuronal Differentiation of SH-SY5Y Cells Using All-Trans Retinoic Acid (RA)

[0070] To evaluate cytotoxicity, 2×10 4Cells / wells were dispensed into a 96-well plate. To confirm changes in protein expression related to neuronal cell death, neuroinflammatory responses, neurotransmitters, and synaptic plasticity, as well as the activity of acetylcholinesterase (AChE), 2×10 5 cells were dispensed into a T-25 flask. For cell fixation and stabilization, DMEM / F12 complete medium (HyClone) containing 10% FBS (HyClone) and 1% P / S (HyClone) was used and cultured in a CO2 incubator (Thermo Fisher Scientific Forma) at 37 °C and 5% CO2 for 24 hours. Twenty-four hours after dispensing the cells, the cell 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 treatment group was replaced by adding fresh DMEM / F12 differentiation medium under various conditions.

[0071] Experimental Example 7. Formation and Treatment of Amyloid-β (Aβ) 1-42

[0072] 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 left standing in a CO2 incubator (Thermo Fisher Scientific Forma) at 37 °C and 5% CO2 for 3 hours to form Aβ1-42 oligomers.

[0073] To confirm the changes in BDNF, the cell culture medium present in RA-differentiated SH-SY5Y neuron-like cells was removed and replaced with DMEM / F12 complete medium (HyClone) containing Aβ1-42 oligomers (1 μM), and cultured in a CO2 incubator (Thermo Fisher Scientific Forma) for 72 hours under the conditions of 37 °C and 5% CO2 to induce Aβ1-42 oligomer-induced cell damage.

[0074] After 72 hours, the medium was removed, and then the cells were treated with DMEM / F12 complete medium (HyClone) containing Aβ1-42 oligomers (1 μM or 10 μM) alone or in combination with milodenafil and memantine, and cultured in a CO2 incubator (Thermo Fisher Scientific Forma) for 24 hours under the conditions of 37 °C and 5% CO2 before the experiment was conducted.

[0075] Experimental Example 8. Measurement of Amyloid β (Aβ) 42 by Human ELISA (Enzyme-Linked Immunosorbent Assay) Kit

[0076] To measure the amount of Aβ42 (pg / mL), 50 μL of cell culture medium was placed in a 96-well plate. Furthermore, 50 μL of Hu Aβ42 detection antibody solution was added to each well, placed on an orbital shaker, and incubated at room temperature for 3 hours. The solution was discarded, washed with 1× wash buffer, 100 μL of anti-rabbit IgG HRP antibody was added thereto, and incubated at room temperature for 30 minutes. The solution was completely discarded again, washed with 1× wash buffer, 100 μL of stabilized chromogen was added thereto, and incubated at room temperature in the dark for 30 minutes. Finally, 100 μL of stop solution was added, and the absorbance at 450 nm was measured within 2 hours.

[0077] Examples 5 to 8.

[0078] In FIG. 6, Embodiment 1 is a combined treatment with 0.1 μM of milodenafil and 0.1 μM of memantine, Embodiment 2 is a combined treatment with 0.5 μM of milodenafil and 0.1 μM of memantine, and Embodiment 3 is a combined treatment with 0.5 μM of milodenafil and 0.5 μM of memantine. For reference, AR1001 in FIG. 6 refers to milodenafil.

[0079] Comparative Examples 1 to 4.

[0080] In FIG. 6, Comparative Examples 1 and 2 are treatments with 0.1 μM and 0.5 μM of milodenafil alone, and Comparative Examples 3 and 4 are treatments with 0.1 μM and 0.5 μM of memantine alone.

[0081] These results show that the Aβ reduction rate of 15.97% in the combined treatment of 0.1 μM of milodenafil and 0.5 μM of memantine in Embodiment 1, the Aβ reduction rate of 13.15% in the combined treatment of 0.5 μM of milodenafil and 0.1 μM of memantine in Embodiment 2, and the Aβ reduction rate of 23.18% in the combined treatment of 0.5 μM of milodenafil and 0.5 μM of memantine in Embodiment 3 were significantly higher than the sum of the reduction rates of A and B in the treatment with milodenafil or memantine alone, and it was confirmed that an effect exceeding the additive effect could be recognized.

[0082] In other words, referring to the Aβ reduction rates of Comparative Examples 1 and 2 regarding the treatments with 0.1 μM and 0.5 μM of milodenafil alone as A1 and A2 respectively, and referring to the Aβ reduction rates of Comparative Examples 3 and 4 regarding the treatments with 0.1 μM and 0.5 μM of memantine alone as B1 and B2 respectively, it can be seen that the Aβ reduction rate of 15.97% in Embodiment 1 is significantly higher than "A1 + B2 = -0.18%", the Aβ reduction rate of 15.97% in Embodiment 2 is significantly higher than "A2 + B1 = -1.63%", and the Aβ reduction rate of 15.97% in Embodiment 3 is significantly higher than "A2 + B2 = -1.72%".

[0083] The above-described invention of the present application is merely an example, and it will be understood by those skilled in the art to which the present invention pertains that various modifications and other equivalent embodiments are possible therefrom. Therefore, it will be understood that the present invention is not limited to the forms mentioned in the above detailed description. Therefore, the true scope of the technical protection of the present invention should be determined by the technical idea of the appended claims. Furthermore, the present invention is understood to cover all modifications, equivalents, and alternatives within the spirit and scope of the present invention as defined by the appended claims.

[0084] Sequence Listing

[0085] [Table 2]

Claims

1. At least one phosphodiesterase 5 inhibitor, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and At least one N-methyl-D-aspartate receptor (NMDA receptor) antagonist, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. A pharmaceutical composition containing the following:

2. The pharmaceutical composition according to claim 1, wherein the phosphodiesterase 5 inhibitor comprises mirodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasantafil, avanafil, or derivatives thereof or mixtures thereof.

3. The pharmaceutical composition according to claim 1, wherein the phosphodiesterase 5 inhibitor is mirodenafil.

4. The pharmaceutical composition according to claim 1, wherein the NMDA receptor antagonist comprises memantine, amantadine, ketamine, traxoprodil, ranisemin, risrenemdaz, pethidine, levorphanol, methadone, dextropropoxifen, tramadol, ketobemidone, dextromethorphan (DXM), phencyclidine (PCP), or methoxetamine (MXE), or a mixture thereof.

5. The pharmaceutical composition according to claim 1 for a method of preventing and / or treating dementia.

6. A pharmaceutical composition according to claim 1 for a method of preventing or treating neuroinflammation.

7. A pharmaceutical composition according to claim 1 for a method of preventing and / or inhibiting the formation and / or accumulation of beta-amyloid.

8. A pharmaceutical composition according to claim 1 for a method of preventing or treating neurodegenerative diseases.

9. The pharmaceutical composition according to claim 8, wherein the neurodegenerative disease is dementia, Parkinson's disease (PD), Lewy body dementia (DLB), Alzheimer's disease (AD), Huntington's disease (HD), multiple sclerosis (MS), vascular dementia (VaD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, or a mixture thereof.

10. The pharmaceutical composition according to claim 1 for a method of inhibiting Aβ oligomer / fibrillation formation by reducing Aβ aggregation.

11. The pharmaceutical composition according to claim 1 for a method of inhibiting β-amyloid formation processing by reducing BACE-1.

12. The pharmaceutical composition according to claim 1 for a method of reducing extracellular Aβ monomers, oligomers, and Aβ fibrils / macula by increasing cerebral blood flow.

13. The pharmaceutical composition according to claim 1 for a method of inhibiting neuronal cell death, promoting neurogenesis, synapse formation, and / or angiogenesis by activation of the NO / cGMP / PKG / CREB pathway.

14. The pharmaceutical composition according to claim 1 for a method of restoring synaptic plasticity by inhibition of DKK-1 and / or activation of Wint signaling.

15. The pharmaceutical composition according to claim 1 for a method of inhibiting APP production and / or reducing Aβ accumulation by suppressing a positive feedback loop for Aβ production.

16. The pharmaceutical composition according to claim 1 for a method of inhibiting intracellular toxicity by activation of autophagy and the formation of Aβ fibrils / plaques by removal of soluble Aβ oligomers.

17. Myrodenafil, or a pharmaceutically acceptable salt, solvate, hydrate, or derivative thereof, and Memantine, or its pharmaceutically acceptable salts, solvates, hydrates, or derivatives. A pharmaceutical composition containing the following:

18. The pharmaceutical composition according to claim 17 for a method of preventing and / or treating dementia.

19. The pharmaceutical composition according to claim 17 for a method of preventing or treating neuroinflammation.

20. The pharmaceutical composition according to claim 17 for a method of preventing and / or inhibiting the formation and / or accumulation of beta-amyloid.

21. The pharmaceutical composition according to claim 17 for a method of preventing or treating a neurodegenerative disease.

22. The pharmaceutical composition according to claim 21, wherein the neurodegenerative disease is dementia, Parkinson's disease (PD), Lewy body dementia (DLB), Alzheimer's disease (AD), Huntington's disease (HD), multiple sclerosis (MS), vascular dementia (VaD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, or a combination thereof.

23. The pharmaceutical composition according to claim 17 for a method of inhibiting Aβ oligomer / fibrillation formation by reducing Aβ aggregation.

24. The pharmaceutical composition according to claim 17 for a method of inhibiting β-amyloid formation processing by reducing BACE-1.

25. The pharmaceutical composition according to claim 17 for a method of reducing extracellular Aβ monomers, oligomers, and Aβ fibrils / macula by increasing cerebral blood flow.

26. The pharmaceutical composition according to claim 17 for a method of inhibiting neuronal cell death, promoting neurogenesis, synapse formation, and / or angiogenesis by activation of the NO / cGMP / PKG / CREB pathway.

27. ​​The pharmaceutical composition according to claim 17 for a method of restoring synaptic plasticity by inhibiting DKK-1 and / or activating Wint signaling.

28. The pharmaceutical composition according to claim 17 for a method of inhibiting APP production and / or reducing Aβ accumulation by suppressing a positive feedback loop for Aβ production.

29. The pharmaceutical composition according to claim 17 for a method of inhibiting intracellular toxicity by activation of autophagy and the formation of Aβ fibrils / plaques by removal of soluble Aβ oligomers.

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