Compositions and methods for preventing and treating neurodegenerative diseases

Abstract

The invention provides a composition containing a phosphodiesterase 5 inhibitor (PDE5 inhibitor) and an acetylcholin esterase inhibitor (AChEI) for preventing or treating neurodegenerative diseases and a use method of the composition. Wherein the PDE5 inhibitor is selected from the group consisting of mironafil, sildenafil, vardenafil, tadalafil, udenafil, dabigatafil, avanafil, and pharmaceutically acceptable salts, solvates, hydrates and mixtures thereof; and the AchEI is selected from the group consisting of donepezil, rivastigmine, galanthamine, poison hyacinth alkali, tacrine, metriofonate, phenylserine, tolserine, eosin, huperzine A and huperzine B, galangin, anacardol, donepezine-AP2238, donepezil-tacrine, tacrine-ferulic acid hybrids, tacrine-hydroxyquinoline, ladotegil, an indenyl derivative thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof. 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

[0001] Cross - Reference to Related Applications

[0002] This application claims the priority of U.S. Provisional Application No. 63 / 367,033, filed on June 24, 2022, the content of which is incorporated herein by reference. Technical Field

[0003] 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.

[0004] Sequence Listing

[0005] This application incorporates by reference in its entirety the Sequence Listing XML file named "04334900119_SequenceListing.xml (7KB)", which was created on June 23, 2023 and submitted electronically therewith. Background Art

[0006] In recent years, the number of patients suffering from degenerative neurological disorders has increased rapidly. When treating degenerative neurological disorders, the most important step is prevention. However, the cause of this disease is not yet clear, so treatment methods still need to be studied. The common pathological phenomenon of degenerative neurological disorders is the death of central nervous system cells. Different from cells in other organs, central nervous system cells are almost impossible to regenerate after cell death, resulting in permanent loss of function. Therefore, the treatment methods developed so far for such brain diseases mainly focus on the analysis of the death mechanism of nerve cells themselves and prevention based on this analysis.

[0007] Neurodegeneration, as a general term, involves the gradual 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), are posing a severe challenge to the aging population. Potential causes of neurodegeneration or neuronal cell death are oxidative stress, an increase in protein aggregates (such as α - or β - amyloid protein) in neurons, and chronic inflammation of the central nervous system (CNS).

[0008] For example, recent studies on Alzheimer's disease and Parkinson's disease have provided evidence that the inflammatory response in the brain is a major cause of neuronal death. In fact, it has been confirmed that the levels of inflammatory mediators and reactive oxygen species in the cerebrospinal fluid of patients with brain diseases are increased. In addition, a large number of activated microglial cells have been observed in the brain injury area, indicating that brain inflammation is a major cause of Parkinson's disease. Therefore, suppressing brain inflammation in glial cells has become a goal for the treatment of neurodegenerative disorders. However, the therapeutic agents developed so far can only effectively regulate the symptoms of the disease and cannot effectively treat the neurodegenerative disorder itself.

[0009] Therefore, there is a need to develop a preventive and therapeutic agent for neurodegenerative disorders based on a concept completely different from the traditional one.

[0010] For example, dementia is an acquired brain disease with a multi-faceted pathogenesis caused by a variety of genetic and environmental risk factors, and refers to a clinical condition that results in multiple cognitive deficits. The most representative disease leading to dementia is Alzheimer's disease, which mainly occurs in the elderly population and accounts for more than 60% of dementia cases.

[0011] Studies have shown that inflammation is associated with neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease. The traditional view is that due to the existence of the blood-brain barrier (BBB), the brain is an immune-privileged site. In contrast, recent studies have confirmed that the brain is fully capable of eliciting an immune response. Brain inflammation does not involve the peripheral immune system, nor antibodies or T cells. The immune response in the brain depends on the synthesis of inflammatory components by glial cells, especially resident phagocytes (microglial cells in the brain).

[0012] In the brain, glial cells play a key role in maintaining a homeostatic microenvironment that promotes neuronal survival. Microglial cells mediate the innate immune response to invading pathogens by secreting a variety of factors, including cytokines, chemokines, prostaglandins, reactive oxygen and nitrogen species, and growth factors. Therefore, the pro-inflammatory and anti-inflammatory responses must be strictly regulated to prevent the potentially harmful effects of oxidative stress caused by chronic inflammation on vulnerable neuronal populations.

[0013] In the normal adult brain, microglial cells are usually in a resting state. When these cells are activated, they are known to release various types of pro-inflammatory molecules, such as nitric oxide (NO) and cytokines, which can cause damage and cell death of surrounding neurons. For example, it has been found that the activation of microglial cells, the accumulation of cytokines, and the activation of the nuclear factor κB (NF-κB) pathway can lead to the progression of neurodegenerative diseases.

[0014] On the other hand, research on beta-amyloid (Aβ), which is known to be a common cause of both hereditary and sporadic Alzheimer's disease, reports that even in normal individuals, small amounts of Aβ are produced in various parts of the body. In normal people, the Aβ produced in the body is rapidly degraded and does not accumulate in the body. However, in patients with Alzheimer's disease, Aβ is produced in abnormally large amounts and accumulates in tissues without being degraded, leading to the formation of senile plaques or excessive accumulation in parts such as the hippocampus or cerebral cortex, which play important roles in memory and learning. The accumulated Aβ triggers an inflammatory response in surrounding cells. As a result, nerve cells are damaged, and eventually, the neural network that maintains the normal functions of the brain is damaged. In addition, the accumulated Aβ also generates a large amount of reactive oxygen species, thereby activating the signal transduction system that kills nerve cells.

[0015] Aβ is a part of the amyloid precursor protein that is cleaved by beta-secretase. Aβ has various forms depending on the number of amino acids that make it up. In the case of patients with Alzheimer's disease, the proportion of Aβ composed of 40 or 42 amino acids increases rapidly. There are many reports that Aβ induces neuronal cell death when treated with nerve cells cultured in vitro, and the mechanism of cell death is similar to the type of apoptosis seen in patients with Alzheimer's disease. The damage of Aβ1-42 or Aβ1-43 proteins to nerve cells has been identified as one of the important causes leading to Alzheimer-type diseases, and Aβ25-35 is an important toxic fragment known to cause nerve cell damage in Aβ1-42 or 43.

[0016] The most common drugs currently approved by the FDA for the treatment of dementia include AChEIs and NMDA (N-methyl-D-aspartic acid) receptor antagonists. In addition, various other drugs are used in combination with them, such as antioxidants, non-steroidal anti-inflammatory drugs (NSAIDs), anti-inflammatory agents, statins, and hormones. However, these drugs are only used to relieve, delay symptoms, and improve cognitive function, and currently, dementia still requires fundamental treatment.

[0017] Representative AChEIs include donepezil, rivastigmine, and galantamine. These drugs play a symptomatic treatment role by temporarily increasing the concentration of the neurotransmitter acetylcholine. In addition, these drugs are also prescribed to patients with mild to moderate Alzheimer's disease, vascular dementia, Parkinson's disease dementia, and stroke or patients with subcortical ischemic vascular diseases.

[0018] Due to the decline or loss of nerve cell function, neurodegenerative diseases including dementia cause abnormalities in various functions of the human body, including all perceivable body functions such as the body's motor control function, cognitive function, perceptual function, and sensory function, as well as the autonomic nerve function that regulates itself without the body's awareness.

[0019] Since the etiology of neurodegenerative diseases including dementia has not been fully understood, radical treatment remains challenging. Commercially available drugs can only relieve the symptoms of some diseases, but cannot fundamentally change the progression of the diseases, and drug tolerance and serious side effects will occur after treatment, which further limits the improvement of symptoms in these patients.

[0020] However, the options for treating neurodegenerative diseases or conditions including dementia are still limited. Summary of the Invention

[0021] The present invention provides a composition and method for treating neurodegenerative diseases by reducing neuroinflammation especially in the CNS system and / or by reducing the expression of toxic proteins (such as β-amyloid (Aβ)),

[0022] wherein,

[0023] the composition comprises a PDE-5 inhibitor and an acetylcholinesterase inhibitor (AChEI),

[0024] the PDE-5 inhibitor is selected from milronafide, sildenafil, vardenafil, tadalafil, udenafil, dasenafil, avanafil; and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof;

[0025] the AchEI is selected from donepezil, galantamine, rivastigmine or mixtures thereof;

[0026] the composition inhibits pro-inflammatory cytokines, such as IL1β, IL-6 or TNFα;

[0027] the composition inhibits the growth and differentiation of nerve cells and degenerates learning and memory ability, induces the reduction of intracellular Aβ, thereby increasing the protective effect of nerve cells and the plasticity of synapses; and

[0028] the neurodegenerative diseases are 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 their mixed etiologies. Brief Description of the Drawings

[0029] Figure 1 and Figure 2 show that the composition containing milronafide and donepezil has a synergistic effect on IL1β inhibition, for example, at a ratio of milronafide:donepezil of 5:1, 2:1, 1:1, 1:2, 1:5 or 1:10.

[0030] Figure 3 and Figure 4 ​​shows that the composition containing milonafide and donepezil has a synergistic effect on TNFα inhibition, for example, at milonafide:donepezil ratios of 10:1, 5:1, 2:1, 1:1, 1:2 or 1:5.

[0031] Figure 5 and Figure 6 shows that the composition containing milonafide and galantamine has a synergistic effect on IL1β inhibition, for example, at milonafide:galantamine ratios of 10:1, 5:1, 2:1, 1:1, 1:2, 1:5 or 1:10.

[0032] Figure 7 and Figure 8 shows that the composition containing milonafide and galantamine has a synergistic effect on IL1β inhibition, for example, at milonafide:galantamine ratios of 10:1, 5:1, 2:1, 1:1, 1:2, 1:5 or 1:10.

[0033] Figure 9 and Figure 10 shows that the composition containing milonafide and rivastigmine has a synergistic effect on IL1β inhibition, for example, at milonafide:rivastigmine ratios of 5:1, 2:1, 1:1, 1:2 or 1:5.

[0034] Figure 11 and Figure 12 shows that the composition containing milonafide and rivastigmine has a synergistic effect on TNFα inhibition, for example, at milonafide:rivastigmine ratios of 5:1, 2:1, 1:1, 1:2, 1:5 or 1:10.

[0035] Figures 13 - 18 AR1001 (milonafide) and all three AChEIs have a synergistic effect on reducing Aβ42 accumulation at AR1001:AChEI ratios of, for example, 5:1, 1:1 or 1:5.

[0036] Figure 13 and Figure 14 shows the results of the treatment with the combination of milonafide and donepezil according to an embodiment of the present invention, resulting in a reduction of intracellular Aβ.

[0037] Figure 15 and Figure 16 shows the synergistic effect of the composition of the present invention containing milonafide and galantamine on reducing intracellular Aβ.

[0038] Figure 17 and Figure 18 shows the synergistic effect of the composition of the present invention containing milonafide and rivastigmine on reducing intracellular Aβ.​​​​​​​ Detailed implementation mode

[0039] The present invention provides a composition and method for treating neurodegenerative diseases by reducing neuroinflammation, especially in the CNS system, and / or by reducing the expression of toxic proteins (such as beta-amyloid (Aβ)).

[0040] Wherein,

[0041] The composition comprises a PDE-5 inhibitor and an acetylcholinesterase inhibitor (AChEI).

[0042] The PDE-5 inhibitor is selected from milronafil, sildenafil, vardenafil, tadalafil, udenafil, dasenafil, avanafil; and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof;

[0043] The AChEI is selected from donepezil, galantamine, rivastigmine or mixtures thereof;

[0044] The composition inhibits pro-inflammatory cytokines, such as IL1β, IL-6 or TNFα;

[0045] The composition inhibits the growth and differentiation of nerve cells, degenerates learning and memory abilities, induces a reduction in intracellular Aβ, thereby increasing the protective effect on nerve cells and the plasticity of synapses; and

[0046] The neurodegenerative diseases are 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 their mixed etiologies.

[0047] An embodiment of the present invention provides a composition for preventing and treating dementia, which comprises a phosphodiesterase 5 inhibitor and an acetylcholinesterase inhibitor as active ingredients.

[0048] In a certain embodiment of the present invention, the weight percentage of the PDE-5 inhibitor to the AChEI in the composition is 1:0.1 to 1:10 or 50:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5 or 1:10.

[0049] In another embodiment, the composition of the present invention provides a synergistic effect in the following aspects:

[0050] (1) Inhibiting the formation of Aβ oligomers / fibrils by reducing Aβ aggregation;

[0051] (2) Inhibiting beta-amyloidogenic processing by reducing BACE-1;

[0052] (3) Reducing extracellular Aβ monomers, oligomers, and Aβ protofibrils / plaques by increasing cerebral blood flow;

[0053] (4) Suppressing neuronal cell death, inhibiting and promoting neurogenesis, synaptogenesis, and / or angiogenesis by activating the NO / cGMP / PKG / CREB pathway,

[0054] (5) Activating Wnt signaling by inhibiting DKK-1 to restore synaptic plasticity, and inhibiting the production of APP and reducing the accumulation of Aβ by suppressing the positive feedback loop of Aβ production, and

[0055] (6) Inhibiting the formation of Aβ protofibrils / plaques by activating autophagy to remove intracellular toxic and soluble Aβ oligomers.

[0056] The phosphodiesterase 5 inhibitor of the present invention is at least one selected from the group consisting of milronafide, sildenafil, vardenafil, tadalafil, udenafil, dasanafil, avanafil; and pharmaceutically acceptable salts, solvates, and hydrates thereof.

[0057] A pharmaceutically acceptable salt refers to a compound formulation 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 known in the art using pharmaceutically acceptable and substantially non-toxic organic and inorganic acids. The 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, p-toluenesulfonic acid, etc., 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 react with bases to form ammonium salts; 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.

[0058] According to one embodiment of the present invention, examples of pharmaceutically acceptable salts can be milronafide hydrochloride, sildenafil citrate, or vardenafil hydrochloride.

[0059] A hydrate refers to the compound of the present invention, or its salt, that contains stoichiometric or non-stoichiometric water bound by non-covalent intermolecular forces.

[0060] A solvate refers to a compound of the present invention, or a salt thereof, that contains a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Thus, preferred solvents are volatile, non-toxic, and / or suitable for administration to humans.

[0061] The acetylcholinesterase inhibitors of the present invention are at least one selected from the group consisting of donepezil, rivastigmine, galantamine; and pharmaceutically acceptable salts, solvates, and hydrates thereof.

[0062] More preferably, the phosphodiesterase 5 inhibitor is selected from milronafide, its pharmaceutically acceptable salts, solvates, hydrates or mixtures, and the acetylcholinesterase inhibitor is selected from donepezil, galantamine, rivastigmine, their pharmaceutically acceptable salts, solvates, hydrates or mixtures.

[0063] The pharmaceutical composition of the present invention can be administered orally or parenterally.

[0064] According to an embodiment of the present invention, the pharmaceutical composition of the present invention is administered to a subject orally or parenterally to a site other than the head. In other words, even if the composition of the present invention is not directly administered to the brain tissue, the body tissue around the brain tissue (such as the scalp) and its adjacent parts, the desired effects of the present invention can be exerted. In a specific example, the parenteral administration is subcutaneous administration, intravenous administration, intraperitoneal injection, transdermal administration or intramuscular administration. In another specific example, it is subcutaneous administration, intravenous administration or intramuscular administration.

[0065] The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention is a carrier commonly used in formulations, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, 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 Edition, 1995).

[0066] The pharmaceutical composition of the present invention can be formulated using pharmaceutically acceptable carriers and / or excipients to be prepared in unit dosage form, or can be prepared by internalization in a multi-dose container by a method that can be easily implemented by those skilled in the art to which the present invention pertains. In this case, the formulation can be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or can be in the form of an extract, powder, granule, tablet, film or capsule, and can also contain a dispersant or stabilizer.

[0067] In one embodiment, the composition of the present invention provides a synergistic effect on inhibiting pro-inflammatory factors, thereby reducing neuroinflammation.

[0068] In another embodiment, the composition of the present invention reduces amyloid-β protein by jointly using phosphodiesterase 5 (PDE5 inhibitor) and acetylcholinesterase inhibitor, thereby providing a synergistic effect on reducing the accumulation of Aβ42 to prevent and / or treat dementia.

[0069] Examples

[0070] The following will be described in more detail using the following embodiments. However, these embodiments are only for illustrating the present invention, and the scope of the present invention is not limited by these embodiments.

[0071] Experimental Example 1. Culture method of IMG cells

[0072] The IMG cells used in the experiment are a mouse microglial cell line, which is cultured in a CO2 incubator (311-TIF, ThermoFisher Scientific Forma, MA, USA) under the conditions of 37 °C and 5% CO2 using DMEM complete medium (HyClone), and the medium contains 10% fetal bovine serum (FBS; Australian Orgin, HyClone, Logan, UT, USA) and 1% penicillin / streptomycin (P / S; HyClone).

[0073] 2x10 5 cells are seeded in each well of a 6-well plate and incubated for 24 hours under the above conditions.

[0074] After incubation for 24 hours, they are treated or combinedly treated with 100 ng / ml LPS and drugs AR1001 and AchEI (donepezil, rivastigmine, galantamine) at concentrations of 2 μM, 10 μM, and 20 μM respectively.

[0075] Experimental Example 2. RNA extraction and cDNA synthesis

[0076] Scrape the cells in the hole with a scraper, put 2 mL of culture medium into a 15 mL conical tube, centrifuge at 3,000 RPM for 5 minutes, discard the culture medium other than the precipitate, and treat each cell with 1 mL of Trizol.

[0077] Transfer to a 1.5 mL e-tube, add 0.2 mL of chloroform, vortex for 1 minute, and store at room temperature for 2 minutes.

[0078] After centrifuging at 12,000 g and 4 °C for 10 minutes, separate 500 μL of the supernatant, add 500 μL of isopropanol to the separated supernatant, let stand at room temperature for 10 minutes, and then centrifuge at 12,000 g and 4 °C for 10 minutes.

[0079] Discard the supernatant and wash the precipitate twice with 75% EtOH.

[0080] Discard all EtOH, dry the RNA precipitate and dissolve it in 10 μL of DEPC-treated water.

[0081] According to PrimeScript TM II First Strand cDNA Synthesis Kit (Takara) for cDNA synthesis.

[0082] Experimental Example 3. Real-time RT-qPCR of Pro-inflammatory Cytokines

[0083] Real-time RT-qPCR was performed using Quant Studio 5 (Applied biosystems). The primer sequence of the IL1β forward primer is 5’-AGCTTCAGGCAGGCAGTATC-3’ (SEQ ID NO:1); the IL1β reverse primer is 5’-AAGGTCCACGGGAAAGAC AC-3’ (SEQ ID NO:2); the TNFα forward primer is 5’-AAATGGCCTCCCTCTCATCAG-3’ (SEQ ID NO:3); the TNFα reverse primer is 5’-GTCACTC GAATTTTGAGAAGATGATC-3’ (SEQID NO:4); the β-actin forward primer is 5’-CGTGCGTGACATCAAAGAGAA-3’ (SEQ ID NO:5); the β-actin reverse primer is 5’–TGGATGCCACAGGATTCCAT-3’ (SEQ ID NO:6). SYBR Green PCR Master Mix (ThermoFisher) was used for the polymerase.

[0084] Experimental Example 4. Results of Measuring the Reduction Rate of Pro-inflammatory Cytokines.

[0085] Figure 1 The results of an experiment for examining whether milonafide and donepezil in combination in the present invention have a synergistic effect on IL-1β are shown.

[0086] Figure 1 In Figure 2 , when treated with a combination of 2 μM milonafide and 2 μM donepezil, the reduction rate of IL-1β was 11.89%; when treated with a combination of 2 μM milonafide and 10 μM donepezil, the reduction rate of IL-1β was 26.53%; when treated with a combination of 2 μM milonafide and 20 μM donepezil, the reduction rate of IL-1β was 54.49%; and when treated with a combination of 10 μM milonafide and 2 μM donepezil, the reduction rate of IL-1β was 27.44%; and when treated with a combination of 10 μM milonafide and 10 μM donepezil, the reduction rate of IL-1β was 41.36%; and when treated with a combination of 10 μM milonafide and 20 μM donepezil, the reduction rate of IL-1β was 76.51%; and when treated with a combination of 20 μM milonafide and 2 μM donepezil, the reduction rate of IL-1β was 50.28%; and when treated with a combination of 20 μM milonafide and 10 μM donepezil, the reduction rate of IL-1β was 78.92%; which was significantly higher than the sum of the increase rates A and B when milonafide or donepezil was treated alone, demonstrating that the confirmable effect exceeded the additive effect.

[0087] Figure 3 The results of an experiment for examining whether milonafide and donepezil in combination in the present invention have a synergistic effect on TNF-α are shown.

[0088] In Figure 3 , AR1001 refers to milonafide, see Figure 4 , when treated with a combination of 2 μM milonafide and 2 μM donepezil, the reduction rate of TNF-α was 11.68%; when treated with a combination of 2 μM milonafide and 10 μM donepezil, the reduction rate of TNF-α was 38.46%; and when treated with a combination of 10 μM milonafide and 2 μM donepezil, the reduction rate of TNF-α was 28.59%; and when treated with a combination of 10 μM milonafide and 10 μM donepezil, the reduction rate of TNF-α was 57.62%; and when treated with a combination of 10 μM milonafide and 20 μM donepezil, the reduction rate of TNF-α was 83.61%; and when treated with a combination of 20 μM milonafide and 2 μM donepezil, the reduction rate of TNF-α was 59.85%; and when treated with a combination of 20 μM milonafide and 10 μM donepezil, the reduction rate of TNF-α was 83.08%; which was significantly higher than the sum of the increase rates A and B when milonafide or donepezil was treated alone, demonstrating that the confirmable effect exceeded the additive effect.

[0089] Figure 5The results of an experiment for examining whether milonafide and galantamine used in combination in the present invention have a synergistic effect on IL-1β are shown.

[0090] Figure 5 In [reference], AR1001 refers to milonafide, see Figure 6 , when treated with a combination of 2 μM milonafide and 2 μM galantamine, the reduction rate of IL-1β was 9.52%; when treated with a combination of 2 μM milonafide and 10 μM galantamine, the reduction rate of IL-1β was 26.28%; when treated with a combination of 2 μM milonafide and 20 μM galantamine, the reduction rate of IL-1β was 38.57%; and when treated with a combination of 10 μM milonafide and 2 μM galantamine, the reduction rate of IL-1β was 27.34%; and when treated with a combination of 10 μM milonafide and 10 μM galantamine, the reduction rate of IL-1β was 40.29%; and when treated with a combination of 10 μM milonafide and 20 μM galantamine, the reduction rate of IL-1β was 59.91%; and when treated with a combination of 20 μM milonafide and 2 μM galantamine, the reduction rate of IL-1β was 56.17%; and when treated with a combination of 20 μM milonafide and 10 μM galantamine, the reduction rate of IL-1β was 71.15%; and when treated with a combination of 20 μM milonafide and 20 μM [galantamine], the reduction rate of IL-1β was 83.74%, which was significantly higher than the sum of the increase rates A and B when milonafide or galantamine was treated alone, demonstrating that the confirmable effect exceeded the additive effect.

[0091] Figure 7 The results of an experiment for examining whether milonafide and galantamine used in combination in the present invention have a synergistic effect on TNF-α are shown.

[0092] Figure 7 In [reference], AR1001 refers to milonafide, see Figure 8 , when treated with a combination of 2 μM milonafide and 2 μM galantamine, the reduction rate of TNF-α was 12.01%; when treated with a combination of 2 μM milonafide and 10 μM galantamine, the reduction rate of TNF-α was 25.22%; and when treated with a combination of 10 μM milonafide and 2 μM galantamine, the reduction rate of TNF-α was 38.08%; and when treated with a combination of 10 μM milonafide and 10 μM galantamine, the reduction rate of TNF-α was 47.14%; and when treated with a combination of 10 μM milonafide and 20 μM galantamine, the reduction rate of TNF-α was 76.08%; and when treated with a combination of 20 μM milonafide and 10 μM galantamine, the reduction rate of TNF-α was 72.41%; which was significantly higher than the sum of the increase rates A and B when milonafide or galantamine was treated alone, demonstrating that the confirmable effect exceeded the additive effect.

[0093] Figure 9Shows the results of an experiment for examining whether there is a synergistic effect of the combined use of milonafide and rivastigmine in the present invention on IL-1β.

[0094] Figure 9 In it, AR1001 refers to milonafide, see Figure 10 , when treated with the combination of 2 μM milonafide and 2 μM rivastigmine, the reduction rate of IL-1β is 8.17%; when treated with the combination of 2 μM milonafide and 10 μM rivastigmine, the reduction rate of IL-1β is 23.70%; and when treated with the combination of 10 μM milonafide and 2 μM rivastigmine, the reduction rate of IL-1β is 25.29%; and when treated with the combination of 10 μM milonafide and 10 μM rivastigmine, the reduction rate of IL-1β is 39.91%; and when treated with the combination of 10 μM milonafide and 20 μM rivastigmine, the reduction rate of IL-1β is 53.79%; and when treated with the combination of 20 μM milonafide and 10 μM rivastigmine, the reduction rate of IL-1β is 67.53%; which is significantly higher than the sum of the increase rates A and B when milonafide or rivastigmine is treated alone, which proves that the confirmable effect exceeds the additive effect.

[0095] Figure 11 Shows the results of an experiment for examining whether there is a synergistic effect of the combined use of milonafide and rivastigmine in the present invention on TNF-α.

[0096] In Figure 12 , AR1001 refers to milonafide, see Figure 8 , when treated with the combination of 2 μM milonafide and 2 μM rivastigmine, the reduction rate of TNF-α is 11.93%; when treated with the combination of 2 μM milonafide and 10 μM rivastigmine, the reduction rate of TNF-α is 21.14%; when treated with the combination of 2 μM milonafide and 20 μM rivastigmine, the reduction rate of TNF-α is 34.44%; and when treated with the combination of 10 μM milonafide and 2 μM rivastigmine, the reduction rate of TNF-α is 30.21%; and when treated with the combination of 10 μM milonafide and 10 μM rivastigmine, the reduction rate of TNF-α is 44.63%; and when treated with the combination of 10 μM milonafide and 20 μM rivastigmine, the reduction rate of TNF-α is 55.00%; and when treated with the combination of 20 μM milonafide and 10 μM rivastigmine, the reduction rate of TNF-α is 65.93%; which is significantly higher than the sum of the increase rates A and B when milonafide or rivastigmine is treated alone, which proves that the confirmable effect exceeds the additive effect.

[0097] Experimental Example 5. Cell culture

[0098] The SH-SY5Y human neuroblastoma cell line used in the experiment was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). It was cultured in a CO2 incubator (311-TIF, Thermo Fisher Scientific Forma, MA, USA) at 37 °C and 5% CO2 using DMEM / F12 complete medium (HyClone), which contained 10% fetal bovine serum (FBS; Australian Orgin, HyClone, Logan, UT, USA) and 1% penicillin / streptomycin (P / S; HyClone).

[0099] Experimental Example 6. Neuronal-like differentiation of SH-SY5Y cells using all-trans retinoic acid (RA)

[0100] 2×104 [sic] cells / well were seeded into 96-well plates to evaluate cytotoxicity, and 2×105 [sic] cells were seeded into T-25 flasks to examine neuronal cell death, neuronal inflammatory responses, changes in protein expression related to neurotransmitters and synaptic plasticity, and acetylcholinesterase (AChE) activity. For cell fixation and stabilization, the cells were cultured in DMEM / F12 complete medium (HyClone) containing 10% FBS (HyClone) and 1% P / S (HyClone) in a CO2 incubator (Thermo Fisher Scientific Forma) at 37 °C and 5% CO2 for 24 hours. Twenty-four hours after cell seeding, 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 medium of the sample treatment groups was replaced by adding fresh DMEM / F12 differentiation medium under various conditions.

[0101] Experimental Example 7. Formation and treatment of amyloid-β protein (Aβ) 1-42

[0102] To form Aβ1-42 oligomers, human Aβ1-42 (Abcam, Cambridge, MA, USA) was added to complete DMEM / F12 medium (HyClone) containing 1% FBS (HyClone) and 1% P / S (HyClone) to a concentration of 10 μM, and incubated in a CO2 incubator (Thermo Fisher Scientific Forma) at 37 °C and 5% CO2 for three hours to form Aβ1-42 oligomers.

[0103] To examine the changes in Aβ1-42, the existing cell culture medium was removed from SH-SY5Y neuron-like cells differentiated from RA and replaced with complete DMEM / F12 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 cell damage caused by Aβ1-42 oligomers.

[0104] After 72 hours, the medium was removed, and the cells were treated with complete DMEM / F12 medium (HyClone) alone or in combination with milonafide, 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 conducted.

[0105] Experimental Example 8. Measurement results of human Aβ42 ELISA (enzyme-linked immunosorbent assay)

[0106] To measure the amount of Aβ42 (pg / mL) in cells, the cells were harvested and treated with cell lysis buffer. Then, centrifuged at 14,000 rpm and 4 °C for 10 minutes, after which the supernatant was transferred and the protein was recovered. Using Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific) to quantify the amount of protein. Subsequently, the amount of Aβ42 in cells was measured using a human Aβ42 ELISA kit (Invitrogen).

[0107] Figure 14 The results of an experiment for examining whether the combined use of milonafide and donepezil in the present invention has a synergistic effect on Aβ reduction are shown.

[0108] Figure 14 In this, AR1001 refers to milonafide, see Figure 14, when treated with a combination of 0.1 μM milonafil and 0.1 μM donepezil, the Aβ reduction rate was 5.49%; when treated with a combination of 0.1 μM milonafil and 0.5 μM donepezil, the Aβ reduction rate was 8.35%; when treated with a combination of 0.5 μM milonafil and 0.1 μM donepezil, the Aβ reduction rate was 11.65%; and when treated with a combination of 0.5 μM milonafil and 0.5 μM donepezil, the Aβ reduction rate was 18.27%, which was significantly higher than the sum of the increase rates A and B when milonafil or donepezil was treated alone, demonstrating that the confirmable effect exceeded the additive effect.

[0109] Figure 16 The results of an experiment for examining whether there is a synergistic effect on Aβ reduction when milonafil and galantamine are used in combination in the present invention are shown.

[0110] See Figure 16 , when treated with a combination of 0.1 μM milonafil and 0.1 μM galantamine, the Aβ reduction rate was 4.32%; when treated with a combination of 0.1 μM milonafil and 0.5 μM galantamine, the Aβ reduction rate was 19.08%; when treated with a combination of 0.5 μM milonafil and 0.1 μM galantamine, the Aβ reduction rate was 11.15%; and when treated with a combination of 0.5 μM milonafil and 0.5 μM galantamine, the Aβ reduction rate was 20.43%, which was significantly higher than the sum of the increase rates A and B when milonafil or galantamine was treated alone, demonstrating that the confirmable effect exceeded the additive effect.

[0111] Figure 18 The results of an experiment for examining whether there is a synergistic effect on Aβ reduction when milonafil and rivastigmine are used in combination in the present invention are shown.

[0112] See Figure 18 , when treated with a combination of 0.1 μM milonafil and 0.1 μM rivastigmine, the Aβ reduction rate was 8.43%; when treated with a combination of 0.1 μM milonafil and 0.5 μM rivastigmine, the Aβ reduction rate was 14.88%; when treated with a combination of 0.5 μM milonafil and 0.1 μM rivastigmine, the Aβ reduction rate was 8.94%; and when treated with a combination of 0.5 μM milonafil and 0.5 μM rivastigmine, the Aβ reduction rate was 17.91%, which was significantly higher than the sum of the increase rates A and B when milonafil or rivastigmine was treated alone, demonstrating that the confirmable effect exceeded the additive effect.

[0113] The present invention as described above is merely illustrative, and those skilled in the art to which the present invention pertains will understand that various modifications and other equivalent embodiments can be made therefrom. Therefore, it will be understood that the present invention is not limited to the form mentioned in the above detailed description. Thus, the true technical protection scope of the present invention should be determined by the technical idea of the appended claims. In addition, it should be understood that the present invention covers all modifications, equivalents, and substitutions within the spirit and scope of the present invention defined by the appended claims.

[0114] Sequence Listing

[0115]

Claims

1. A composition comprising: a phosphodiesterase 5 inhibitor; and an acetylcholinesterase inhibitor as active ingredients.

2. The composition according to claim 1, wherein the phosphodiesterase 5 inhibitor is selected from the group consisting of milronafide, sildenafil, vardenafil, tadalafil, udenafil, dasenafil, avanafil, 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, beneserine, toserine, eserine, huperzine A and huperzine B, galangin, cardanol, donepezil-AP2238, donepezil-tacrine, tacrine-ferulic acid hybrid, tacrine-hydroxyquinoline, ladostigil, its indenyl derivatives, pharmaceutically acceptable salts, solvates, hydrates and mixtures thereof.

4. The composition for preventing and treating dementia according to claim 1, wherein the phosphodiesterase 5 inhibitor is selected from the group consisting of milronafide, 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.

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

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 β-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 their mixed etiologies.

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 production and 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 inhibiting 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 activating Wnt signaling to restore synaptic plasticity 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 the production of APP and reducing Aβ accumulation by inhibiting 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 activating autophagy to remove toxic and soluble Aβ oligomers within cells, comprising: Administering an effective amount of a pharmaceutical composition comprising the composition according to claim 1.

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