Compositions and methods for preventing and treating neurodegenerative disease with diabetes

Abstract

The invention provides a composition for preventing or treating neurodegenerative diseases and a use method thereof. The composition comprises a phosphodiesterase-5 (PDE-5) inhibitor and an anti-diabetes therapeutic agent comprising a sodium-glucose cotransporter 2 (SGLT2) inhibitor and a dipeptidyl peptidase-4 (PPD-4) inhibitor; wherein the PDE5 inhibitor is selected from the group consisting of mironafil, sildenafil, vardenafil, tadalafil, udenafil, dabigatafil, avanafil, pharmaceutically acceptable salts, solvates, hydrates, and mixtures thereof; and the SGLT2 inhibitor is selected from the group consisting of canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, ipagliflozin, luseogliflozin, tofogliflozin, repagliflozin etabate, sersegliflozin etabate, pharmaceutically acceptable salts, solvates, hydrates, and mixtures thereof; and 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), amyotrophic lateral sclerosis (ALS), frontotemporal dementia or a mixed cause thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 367,278, filed on June 29, 2022, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to a composition for preventing or treating a neurodegenerative disease under diabetes and a method of using the composition, wherein the composition contains a phosphodiesterase-5 (PDE-5) inhibitor and an anti-diabetic therapeutic agent including a sodium-glucose cotransporter 2 (SGLT2) inhibitor and a dipeptidyl peptidase-4 (PPD-4) inhibitor.

[0004] Sequence Listing

[0005] This application incorporates by reference in its entirety the sequence listing XML file entitled “04334900120_SequenceListing.xml (7KB)” created on June 28, 2023 and electronically submitted concurrently therewith. Background Art

[0006] In recent years, the number of patients suffering from degenerative neurological disorders has increased rapidly. In the treatment of degenerative neurological disorders, the most important step is prevention. However, the cause of the disease is not yet clearly understood, and therefore the treatment method remains to be studied. A common pathological phenomenon of degenerative neurological disorders 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 permanent functional loss. Therefore, the methods for treating this type of encephalopathy developed so far mainly focus on preventing nerve cell death.

[0007] Neurodegeneration as a general term involves the progressive loss of neuronal structure or function, including neuronal death 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) are posing a serious challenge to the aging population. The underlying causes of neurodegeneration or nerve cell death are oxidative stress, aggregation of toxic proteins such as beta-amyloid, and chronic inflammation of the central nervous system (CNS).

[0008] For example, recent studies on Alzheimer's disease and Parkinson's disease have shown that inflammatory reactions in the brain are 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. In addition, a large number of active microglia were observed in the brain injury area, indicating that brain inflammation is the main cause of Parkinson's disease. Inhibiting brain inflammation through glial cells has become a target for treating degenerative neurological disorders. However, the therapeutic agents developed so far are only effective in regulating the symptoms of the disease, but not in treating the degenerative neurological disorder itself.

[0009] Therefore, it is necessary to develop preventive and therapeutic agents for degenerative neurological disorders based on concepts that are completely different from conventional concepts. For example, dementia is an acquired brain disease with a multifaceted pathogenesis caused by various genetic and environmental risk factors, and refers to a clinical disease that causes a variety of cognitive deficits. The most common cause of dementia is AD, which occurs mainly in the elderly and accounts for more than 60% of dementia.

[0010] Studies have implicated inflammation in neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, etc. In contrast to the traditional belief that the brain is an immune privileged site due to the presence of the blood-brain barrier (BBB), recent studies have determined that the brain is fully capable of summoning an immune response. Inflammation of the brain does not involve the peripheral immune system, and does not involve antibodies or T cells. The immune response in the brain relies on glial cells, particularly resident phagocytes (which are microglia in the case of the brain) to synthesize inflammatory components.

[0011] In the brain, glial cells play a critical role in maintaining a homeostatic microenvironment that promotes neuronal survival. Microglia mediate innate immune responses to invading pathogens by secreting a large number of factors including cytokines, chemokines, prostaglandins, reactive oxygen and nitrogen species, and growth factors. Therefore, both pro- and anti-inflammatory responses must be tightly regulated to prevent the potential deleterious effects of chronic inflammation-induced oxidative stress on vulnerable neuronal populations.

[0012] In the normal adult brain, microglia are usually in a quiescent state. When activated, these cells are known to release various types of pro-inflammatory molecules, such as nitric oxide (NO) and cytokines, which lead to damage and cell death of surrounding neurons. For example, it has been found that activated microglia, accumulation of cytokines, and activation of the nuclear factor kappa B (NF-κB) pathway lead to the progression of neurodegenerative diseases.

[0013] On the other hand, research reports on amyloid beta protein (Aβ), which is known to be a common cause of hereditary and sporadic Alzheimer's disease, report that even in normal people, Aβ is produced in small amounts in various parts of the body. In normal people, Aβ is rapidly degraded after production and does not accumulate in the body, but in the case of Alzheimer's patients, Aβ is abnormally produced in large quantities and accumulates in tissues without being degraded, resulting in the production of senile plaques or excessive accumulation in places such as the hippocampus or cerebral cortex, which play an important role in memory and learning. The accumulated Aβ triggers an inflammatory response in surrounding cells. As a result, nerve cells are damaged, and even the neural network that maintains the normal function of the brain is eventually damaged. In addition, the accumulated Aβ produces a large amount of reactive oxygen species, which activates the signal transduction system that kills nerve cells.

[0014] Aβ is a part of the amyloid precursor protein that is cleaved by β-secretase. Aβ comes in many forms, depending on the number of amino acids that make it up. In Alzheimer's patients, the proportion of Aβ consisting of 40 or 42 amino acids increases rapidly. There are many reports that Aβ induces neuronal cell death when treated with in vitro cultured nerve cells, and the mechanism of cell death is similar to the type of apoptosis observed in Alzheimer's patients. Damage to nerve cells by Aβ1-42 or Aβ1-43 proteins has been identified as one of the important causes of Alzheimer's disease, and Aβ25-35 is known to be an important toxic fragment of Aβ1-42 or 43 that can cause nerve cell damage.

[0015] The most common drugs currently approved by the FDA for the treatment of dementia include acetylcholinesterase inhibitors (AchEI) and NMDA (N-methyl-D-aspartate) receptor antagonists, and various other drugs such as antioxidants, nonsteroidal anti-inflammatory drugs (NSAIDs), anti-inflammatory drugs, statins and hormones are used in combination with them. However, these drugs are only used to alleviate and delay symptoms and improve cognitive function, and fundamental treatment of dementia is still needed.

[0016] Due to the reduction or loss of nerve cell function, degenerative neurological diseases including dementia show abnormalities in multiple functions, including all functions of the body that can be felt, such as the body's motor control function, cognitive function, perception function and sensory function, as well as autonomic nervous function, which regulates itself without the body's awareness.

[0017] In addition, the possibility of diabetic patients suffering from neurodegenerative diseases (including vascular dementia and Alzheimer's disease) is twice that of other patients. It is known that diabetes causes atherosclerotic lesions (causing blood vessels to narrow due to the accumulation of cholesterol in the blood vessel wall), which may lead to cerebral infarction or cerebral hemorrhage, and if the brain tissue is damaged, brain function will deteriorate, thereby leading to dementia. In addition, insulin resistance and the resulting hyperinsulinemia in known diabetic patients have a great impact. Insulin also plays an important role in signal transduction, regulating appetite and energy homeostasis in the brain, and participating in learning and memory. However, if there is a problem with the insulin function in the brain, Alzheimer's dementia may occur. In the case of hyperinsulinemia, toxic proteins (amyloid beta protein) are abnormally deposited in the brain. In addition, it is known that oxidative stress or inflammatory reactions associated with diabetes affect the deposition of toxic proteins in the brain. Finally, diabetes causes various cerebrovascular diseases, and these cerebrovascular diseases can promote the progress of Alzheimer's disease.

[0018] Because the causes of neurodegenerative diseases are not fully understood, fundamental treatments remain challenging. Commercially available drugs can only alleviate the symptoms of some diseases but cannot fundamentally change the progression of the disease. Severe side effects of drugs that occur after treatment further limit the improvement of symptoms in these patients. Therefore, options for treating neurodegenerative diseases or conditions, including dementia, remain limited.

[0019] However, options for treating neurodegenerative diseases or conditions, including dementia, remain limited. Summary of the invention

[0020] The present invention provides a composition and method for treating neurodegenerative diseases in diabetic state by reducing neuroinflammation, particularly in the CNS system and / or by reducing the expression of toxic proteins such as β-amyloid protein (Ab),

[0021] in,

[0022] The composition comprises a phosphodiesterase-5 (PDE-5) inhibitor and an anti-diabetic therapeutic agent including a sodium-glucose cotransporter 2 (SGLT2) inhibitor or a dipeptidyl peptidase-4 (PPD-4) inhibitor;

[0023] The PDE-5 inhibitor is selected from milonafil, sildenafil, vardenafil, tadalafil, udenafil, dasentafil, avanafil; and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof;

[0024] The antidiabetic therapeutic agent is selected from insulin, glucagon-like peptide-1 (GLP-1), an agent that (1) increases the amount of insulin secreted by the pancreas, (2) increases the sensitivity of target organs to insulin, (3) reduces the rate of glucose absorption from the gastrointestinal tract, and (4) increases the loss of glucose through urination;

[0025] The SGLT2 inhibitor is selected from canagliflozin, dapagliflozin, empagliflozin, erpagliflozin, ipagliptin, rupagliflozin, togliflozin, repagliflozin etabonate, sergliflozin etabonate, or a pharmaceutically acceptable salt, solvate, hydrate or mixture thereof;

[0026] The PPD-4 inhibitor is selected from, for example, but not limited to, Sitagliptin TM (FDA approved in 2006, marketed by Merck & Co. as Januvia), Vildagliptin TM (Approved by the EU in 2007 and marketed by Novartis as Galvus in the EU), Saxagliptin TM (FDA approved in 2009, marketed as Onglyza), Linagliptin TM (FDA approved in 2011, marketed as Tradjenta by Eli Lilly and Company and Boehringer Ingelheim), Gemigliptin TM (approved in South Korea in 2012, marketed by LG Life Sciences as Zemiglo), Anagliptin TM (approved in Japan in 2012, marketed by Sanwa Kagaku Kenkyusho Co., Ltd. and Kowa Company, Ltd.), Teneligliptin TM (approved in Japan in 2012), Alogliptin TM (FDA approved in 2013, marketed by Takeda Pharmaceutical Company), Trelagliptin TM (Approved for use in Japan in 2015), Omarigliptin TM (MK-3102) (approved in Japan in 2015, developed by Merck & Co.; studies have shown that omaglitin can be used as a once-weekly treatment and was generally well tolerated throughout the basic and extension studies), Evogliptin TM (approved for use in South Korea), Gosogliptin TM(approved for use in Russia), Dutogliptin TM (Developed by Phenomix Corporation), Berberine TM (alkaloids found in plants of the genus Berberis) or mixtures thereof;

[0027] The composition inhibits pro-inflammatory cytokines such as IL1b, IL-6 or TNFa;

[0028] The composition inhibits the growth and differentiation of nerve cells and degrades learning and memory to induce a reduction in intracellular Aβ, thereby increasing protection of nerve cells and synaptic plasticity; and

[0029] 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), amyotrophic lateral sclerosis (ALS), frontotemporal dementia or mixed causes thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 and Figure 2 Compositions containing milonafil and empagliflozin were shown to provide a synergistic effect on IL1β inhibition, for example, at a ratio of milonafil:empagliflozin = 10:1, 5:1, 2:1, 1:1, 1:2, or 1:5.

[0031] Figure 3 and Figure 4 Compositions containing milonafil and empagliflozin were shown to provide a synergistic effect on TNFα inhibition, for example, at a ratio of milonafil:empagliflozin = 10:1, 5:1, 2:1, 1:1, 1:2 or 1:5.

[0032] Figures 5 to 8 It was shown that at a milonafil:empagliflozin ratio of, for example, 50:1, 10:1, 1:1, 1:5 or 1:10 wt / wt%, milonafil and empagliflozin had a synergistic effect in reducing Aβ42 accumulation. DETAILED DESCRIPTION

[0033] The present invention provides a composition and method for treating neurodegenerative diseases by reducing neuroinflammation, particularly in the CNS system and / or by reducing the expression of toxic proteins such as β-amyloid protein (Ab),

[0034] in,

[0035] The composition comprises a phosphodiesterase-5 (PDE-5) inhibitor and an anti-diabetic therapeutic agent including a sodium-glucose cotransporter 2 (SGLT2) inhibitor;

[0036] The PDE-5 inhibitor is selected from milonafil, sildenafil, vardenafil, tadalafil, udenafil, dasentafil, avanafil; and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof;

[0037] The SGLT2 inhibitor is selected from canagliflozin, dapagliflozin, empagliflozin, erpagliflozin, ipagliptin, rupagliflozin, togliflozin, repagliflozin etabonate, sergliflozin etabonate, and pharmaceutically acceptable salts, solvates, hydrates and mixtures thereof;

[0038] The PPD-4 inhibitor is selected from, for example, but not limited to, Sitagliptin TM (FDA approved in 2006, marketed by Merck & Co. as Januvia), Vildagliptin TM (Approved by the EU in 2007 and marketed by Novartis as Galvus in the EU), Saxagliptin TM (FDA approved in 2009, marketed as Onglyza), Linagliptin TM (FDA approved in 2011, marketed as Tradjenta by Eli Lilly and Company and Boehringer Ingelheim), Gemigliptin TM (approved in South Korea in 2012, marketed by LG Life Sciences as Zemiglo), Anagliptin TM (approved in Japan in 2012, marketed by Sanwa Kagaku Kenkyusho Co., Ltd. and Kowa Company, Ltd.), Teneligliptin TM (approved in Japan in 2012), Alogliptin TM (FDA approved in 2013, marketed by Takeda Pharmaceutical Company), Trelagliptin TM (Approved for use in Japan in 2015), Omarigliptin TM (MK-3102) (approved in Japan in 2015, developed by Merck & Co.; studies have shown that omaglitin can be used as a once-weekly treatment and was generally well tolerated throughout the basic and extension studies), Evogliptin TM (approved for use in South Korea), Gosogliptin TM (approved for use in Russia), DutogliptinTM (Developed by Phenomix Corporation), Berberine TM (alkaloids found in plants of the genus Berberis) or mixtures thereof;

[0039] The composition inhibits pro-inflammatory cytokines such as IL1b, IL-6 or TNFa;

[0040] The composition inhibits the growth and differentiation of nerve cells and degrades learning and memory to induce a reduction in intracellular Aβ, thereby increasing protection of nerve cells and synaptic plasticity; and

[0041] 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), amyotrophic lateral sclerosis (ALS), frontotemporal dementia or mixed causes thereof.

[0042] In one embodiment of the present invention, a composition for preventing and treating dementia is provided, which comprises a phosphodiesterase-5 (PDE-5) inhibitor and an anti-diabetic therapeutic agent including a sodium-glucose co-transporter 2 (SGLT2) inhibitor or a PPD-4 inhibitor as active ingredients.

[0043] In a certain embodiment of the invention, the composition comprises a weight % of a phosphodiesterase-5 (PDE-5) inhibitor to an anti-diabetic therapeutic agent including a sodium-glucose co-transporter 2 (SGLT2) inhibitor or a PPD-4 inhibitor 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.

[0044] In another embodiment, the composition of the invention provides a method for:

[0045] Inhibit Aβ oligomer / fibril formation by reducing Aβ aggregation;

[0046] Inhibits β-amyloidogenic processing by reducing BACE-1;

[0047] Reduce extracellular Aβ monomers, oligomers, and Aβ fibrils / plaques by increasing cerebral blood flow;

[0048] Inhibits neuronal cell death and promotes neurogenesis, synaptogenesis and / or angiogenesis by activating the NO / cGMP / PKG / CREB pathway.

[0049] Restoring synaptic plasticity by activating Wint signaling via inhibition of DKK-1 (synaptic plasticity), and inhibiting APP production and reducing Aβ accumulation by curbing the positive feedback loop of Aβ production, and

[0050] Inhibits the formation of Aβ fibrils / plaques by removing intracellular toxic and soluble Aβ oligomers via activation of autophagy.

[0051] The phosphodiesterase 5 inhibitor of the present invention is at least one selected from the group consisting of milonafil, sildenafil, vardenafil, tadalafil, udenafil, dasenstafil, avanafil; and pharmaceutically acceptable salts, solvates and hydrates thereof.

[0052] Pharmaceutically acceptable salt refers to the preparation of a compound, which does not cause severe stimulation to the organism to which the compound is applied and does not impair the biological activity and properties of the compound. Pharmaceutically acceptable salts can be prepared by conventional methods well known in the art using pharmaceutically acceptable and substantially nontoxic organic and inorganic acids. Acids include inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid and phosphoric acid; and organic acids such as sulfonic acid (such as methanesulfonic acid, ethanesulfonic acid and p-toluenesulfonic acid), tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutane 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 a base to form an ammonium salt; an alkali metal salt, such as a sodium salt or a potassium salt; a salt such as an alkaline earth metal salt, such as a calcium salt or a magnesium salt; a salt of an organic base, such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine; and an amino acid salt, such as arginine and lysine.

[0053] According to one embodiment of the present invention, examples of the pharmaceutically acceptable salt may be milonafil hydrochloride, sildenafil citrate or vardenafil hydrochloride.

[0054] 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.

[0055] Solvate refers to a compound of the 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.

[0056] The SGLT2 inhibitor is selected from canagliflozin, dapagliflozin, empagliflozin, erpagliflozin, ispagliflozin, rupagliflozin, topagliflozin, repagliflozin etabonate, sergliflozin etabonate, and pharmaceutically acceptable salts, solvates, hydrates and mixtures thereof.

[0057] More preferably, the phosphodiesterase 5 inhibitor is selected from milonafil, a pharmaceutically acceptable salt, solvate, hydrate or mixture thereof, and the antidiabetic therapeutic agent is GLP-1, the SGLT2 inhibitor is selected from empagliflozin, a pharmaceutically acceptable salt, solvate, hydrate or mixture thereof, and the PPD-4 inhibitor is selected from but not limited to SitagliptinTM, VildagliptinTM, SaxagliptinTM, LinagliptinTM, GemigliptinTM, AnagliptinTM, TeneligliptinTM, AlogliptinTM, TrelagliptinTM, OmarigliptinTM, EvogliptinTM, GosogliptinTM, DutogliptinTM, BerberineTM or a mixture thereof.

[0058] The pharmaceutical composition of the present invention can be administered orally or parenterally.

[0059] According to one embodiment of the present invention, the pharmaceutical composition of the present invention is orally administered to a subject or non-orally administered to a site other than the head. In other words, the composition of the present invention can also show the expected effect of the present invention even when it is not directly administered to brain tissue, body tissue surrounding the brain tissue (e.g., scalp), and sites adjacent thereto. In a specific example, non-oral administration is subcutaneous administration, intravenous administration, intraperitoneal injection, transdermal administration, or intramuscular administration, and in another specific example, non-oral administration is subcutaneous administration, intravenous administration, or intramuscular administration.

[0060] The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is a carrier that is generally used for preparation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum, magnesium stearate and mineral oil, but is not limited thereto. The pharmaceutical composition of the present invention may also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents and preservatives in addition to the above-mentioned components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th edition, 1995).

[0061] The pharmaceutical composition of the present invention can be prepared in unit dosage form by using pharmaceutically acceptable carriers and / or excipients, or can be prepared by internalization in a multi-dose container according to methods that can be easily performed by those skilled in the art to which the present invention belongs. In this case, the preparation can be in the form of a solution, suspension or emulsion in an oil 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.

[0062] In a certain embodiment, the compositions of the invention provide a synergistic effect of inhibition of pro-inflammatory factors to provide a reduction in neuroinflammation.

[0063] In another embodiment, the composition of the present invention provides a synergistic effect on reducing Aβ42 accumulation by reducing amyloid β using a combination of phosphodiesterase-5 (PDE-5 inhibitor) and an acetylcholinesterase inhibitor to prevent and / or treat dementia.

[0064] Example

[0065] Hereinafter, a more detailed description will be given 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.

[0066] Experimental Example 1. IMG cell culture method

[0067] The mouse microglial cell line IMG cells used in the experiment were cultured in DMEM complete medium (HyClone) containing 10% fetal bovine serum (FBS; Australian Orgin, HyClone, Logan, UT, USA) and 1% penicillin / streptomycin (P / S; HyClone) at 37°C and 5% CO 2 Humidified CO 2 The cells were cultured and maintained in an incubator (311-TIF, Thermo Fisher Scientific Forma, MA, USA). 2 × 10 5 cells, and the cells were placed in a humidified CO 2 The cells were incubated in an incubator for 24 hours. In addition, 100 ng / mL of LPS and the drugs AR1001 and SGLT-2 inhibitor (empagliflozin) were treated at concentrations of 2, 10, and 20 μM, either alone or in combination.

[0068] Experimental Example 2. RNA extraction and cDNA synthesis

[0069] Use a cell scraper to scrape the cells and collect 2 mL of the culture solution in a 15 mL conical tube. Centrifuge it at 3,000 RPM for 5 minutes and discard the supernatant (culture solution). Resuspend the precipitate in 1 mL of Trizol and transfer it to a 1.5 mL microcentrifuge tube. In addition, add 0.2 mL of chloroform and vortex it for 1 minute. Let the microcentrifuge tube stand at room temperature for 2 minutes. After centrifugation at 12,000 × g for 10 minutes at 4°C, separate the supernatant (approximately 500 μL) in a fresh microcentrifuge tube. Add an equal volume of isopropanol to the supernatant and mix the solution thoroughly. Place it in a microcentrifuge tube rack and let it stand at room temperature for 10 minutes, and then centrifuge it at 12,000 × g for 10 minutes at 4°C. Discard the supernatant and wash the precipitate twice with 75% ethanol. Dry the RNA precipitate and dissolve it in 10 μL of DEPC-treated water. After quantifying the RNA, follow PrimeScript TM It was converted into cDNA using the protocol of II First Strand cDNA Synthesis Kit (Takara).

[0070] Experimental Example 3. Real-time RT-qPCR of pro-inflammatory cytokines

[0071] Sample cDNA was amplified using gene specific primers and SYBR green PCR master mix (ThermoFisher) in a QuantStudio 5 thermal cycler (Applied biosystems). Amplification conditions were as follows - enzyme activation at 50°C for 2 minutes, pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds and extension at 72°C for a total of 40 cycles for 30 seconds. Specific primer sequences are mentioned in Table-1.

[0072] Table-1: Primer sequences used for real-time RT-PCR

[0073]

[0074] Experimental Example 4. Results of measuring the reduction rate of pro-inflammatory cytokines.

[0075] Figure 1 The results show the synergistic effect of the combination treatment of milonafil and empagliflozin on IL-1β in the present invention. Here, AR1001 refers to milonafil.

[0076] exist Figure 2For the combination of 2μM milopfil and 2μM empagliflozin, the IL-1β reduction rate was 7.66%; for the combination of 2μM milopfil and 10μM empagliflozin, the reduction rate was 17.74%; for the combination of 2μM milopfil and 20μM empagliflozin, the reduction rate was 31.62%; for the combination of 10μM milopfil and 2μM empagliflozin, the reduction rate was 24.05%; for the combination of 10μM milopfil and 10μM empagliflozin, the reduction rate was 34.67%; for the combination of 10μM milopfil and 20μM empagliflozin, the reduction rate was 43.95%; for the combination of 20μM milopfil and 2μM empagliflozin, the reduction rate was 54.66%; and for the combination treatment of 20μM milopfil and 10μM empagliflozin, the IL-1β reduction rate was 61.99%. The reduction rates of the different combinations were significantly higher than the sum of the individual reduction rates observed with either mironafil or empagliflozin alone, confirming a synergistic effect that outweighs an additive effect.

[0077] Figure 3 The results show that the combination of milonafil and empagliflozin in the present invention has a synergistic effect on TNF-α. Here, AR1001 refers to milonafil.

[0078] exist Figure 4 In the present study, the TNF-α reduction rate for the combination treatment of 2 μM milonafil and 2 μM empagliflozin was 12.02%; for the combination of 2 μM milonafil and 10 μM empagliflozin, the reduction rate was 19.41%; for the combination of 2 μM milonafil and 20 μM empagliflozin, the reduction rate was 32.18%; for the combination of 10 μM milonafil and 2 μM empagliflozin, the reduction rate was 34.75%; for the combination of 10 μM milonafil and 10 μM empagliflozin, the reduction rate was 37.82%; for the combination of 10 μM milonafil and 20 μM empagliflozin, the reduction rate was 48.65%; for the combination of 20 μM milonafil and 2 μM empagliflozin, the reduction rate was 58.90%; and for the combination treatment of 20 μM milonafil and 10 μM empagliflozin, the reduction rate was 64.96%. The reduction rates of the different combinations were significantly higher than the sum of the individual reduction rates observed with either mironafil or empagliflozin alone, confirming a synergistic effect that outweighs an additive effect.

[0079] Experimental Example 5. Cell culture

[0080] The SH-SY5Y human neuroblastoma cell line used in the experiment was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and was cultured at 37°C and 5% CO2. CO2The cells were cultured in an incubator (311-TIF, ThermoFisher Scientific Forma, MA, USA) 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).

[0081] Experimental Example 6. Neuronal differentiation of SH-SY5Y cells using all-trans retinoic acid (RA)

[0082] To examine the changes in the amount of amyloid β, 2 × 10 5 For cell fixation and stabilization, complete DMEM / F12 medium (HyClone) containing 10% FBS (HyClone) and 1% P / S (HyClone) was used to culture at 37°C and 5% CO 2 Under the conditions of CO 2 The cells were cultured in an incubator (Thermo Fisher Scientific Forma) for 24 hours.

[0083] 24 hours after cell distribution, the cell culture medium was removed for neuronal 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).

[0084] On the third day of differentiation, the medium was replaced with new DMEM / F12 differentiation medium. On the sixth day of differentiation, the medium for the untreated control group was replaced with new DMEM / F12 differentiation medium, and the sample treatment groups were replaced by adding new DMEM / F12 differentiation medium under different conditions.

[0085] Experimental Example 7. Formation and treatment of amyloid β (Aβ) 1-42

[0086] 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 incubated at 37°C and 5% CO 2 Under the conditions of CO 2The cells were placed in an incubator (Thermo Fisher Scientific Forma) for three hours to allow the Aβ1-42 oligomers to form.

[0087] After 72 hours, the culture medium was removed, and the cells were then treated with DME M / F12 complete medium (HyClone) containing Aβ1-42 oligomers (10 μM) alone or in combination with milonafil and empagliflozin and incubated at 37°C and 5% CO 2 Under the conditions of CO 2 The cells were cultured in an incubator (Thermo Fisher Scientific Forma) for 24 hours before the experiment.

[0088] Experimental Example 8. Measurement of amyloid β (Aβ) by human ELISA (enzyme-linked immunosorbent assay) kit 42

[0089] To measure the amount of Aβ42 (pg / mL), 50 μL of cell culture medium was placed in a 96-well plate [sic: plate]. In addition, 50 μL of Hu Aβ42 detection antibody solution was added to each well and incubated on an orbital shaker 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 a dark room for 30 minutes. Finally, 100 μL of stop solution was added thereto, and the absorbance at 450 nm was measured within 2 hours.

[0090] Implementation options 1 to 3.

[0091] Below Figure 6 In the embodiment 1, the combination treatment of 1 μM milonafil and 1 μM empagliflozin, the combination treatment of 1 μM milonafil and 10 μM empagliflozin, and the combination treatment of 1 μM milonafil and 10 μM empagliflozin, Figure 8 The combination of 5 μM mironafil and 0.5 μM empagliflozin was used for treatment.

[0092] Comparative Examples 1 to 6

[0093] exist Figure 6 Among them, Comparative Example 1 was treated with 1 μM milonafil alone, and Comparative Examples 2 and 3 were treated with 1 μM and 10 μM empagliflozin alone, respectively.

[0094] exist Figure 8 In the present invention, Comparative Example 4 was treated with 5 μM milonafil alone, and Comparative Example 5 was treated with 0.5 μM empagliflozin alone.

[0095] In addition, Comparative Example 5 is Figure 6 In the combination treatment of 1 μM milonafil and 20 μM empagliflozin, and Comparative Example 6 is Figure 8 The combination of 5 μM milonafil and 0.05 μM empagliflozin was used for treatment.

[0096] In summary, the 26.0% Aβ reduction rate of the combination treatment of 1 μM milonafil and 1 μM empagliflozin in Implementation Option 1; the 31.5% Aβ reduction rate of the combination treatment of 1 μM milonafil and 10 μM empagliflozin in Implementation Option 2; and the 39.3% Aβ reduction rate of the combination treatment of 5 μM milonafil and 0.5 μM empagliflozin in Example 3 showed statistically significant differences compared to the sum of the reduction rates A and B of the treatment with milonafil or empagliflozin alone, which confirms that effects exceeding the additive effect can be recognized.

[0097] In other words, the Aβ reduction rates of the 1 μM and 5 μM mildenafil treatments of Comparative Examples 1 and 4 are referred to as A1 and A2, respectively, and the Aβ reduction rates of the 1 μM, 10 μM and 0.5 μM empagliflozin treatments of Comparative Examples 2, 3 and 5 are referred to as B1, B2 and B3, respectively. It can be seen that the Aβ reduction rate of 26.0% in Implementation Option 1 is significantly higher than 'A1+B1=8.6%', the Aβ reduction rate of 31.5% in Implementation Option 2 is significantly higher than 'A1+B2=21.1%', and the Aβ reduction rate of 39.3% in Implementation Option 3 is significantly higher than 'A2+B3=21.3%'.

[0098] In addition, Figure 6 Comparative Example 5 of the combined treatment of 1 μM milonafil and 20 μM empagliflozin and Figure 8 In the case of Comparative Example 6 in which 5 μM milonafil and 0.05 μM empagliflozin were treated in combination, it can be seen that an antagonistic effect much weaker than the sum of the effects of each drug was exhibited.

[0099] It can be seen from these experimental results that even if milonafil and empagliflozin are treated in combination, it has a synergistic effect or synergistic effect on the removal of amyloid β (Aβ) at a concentration ratio of milonafil to empagliflozin of 1:0.1 to 10, but the sum of the effects decreases at a concentration ratio of 1:0.01 (which exceeds the lower limit of the range) and 1:20 (which exceeds the upper limit).

[0100] Therefore, preferably, the concentration ratio of milonafil and empagliflozin in the combination treatment is 1:0.1 to 10.

[0101] The present invention described above is illustrative only, and those skilled in the art to which the present invention belongs will understand that various modifications and other equivalent embodiments are possible. Therefore, it should 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 concept 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.

[0102] Sequence Listing

[0103]

Claims

1. A composition comprising: Phosphodiesterase 5 inhibitors; and Antidiabetic therapeutic agent as active ingredient.

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

3. The composition of claim 1, wherein the antidiabetic therapeutic agent is an agent that (1) increases the amount of insulin secreted by the pancreas, (2) increases the sensitivity of target organs to insulin, (3) reduces the rate of glucose absorption from the gastrointestinal tract, or (4) increases glucose loss through urination, or a mixture thereof.

4. The composition of claim 1, wherein the antidiabetic therapeutic agent is insulin or a derivative thereof, glucagon-like peptide-1 (GLP-1) or a derivative thereof, a sodium-glucose cotransporter 2 (SGLT2) inhibitor or a derivative thereof, a dipeptidyl peptidase-4 (PPD-4) inhibitor or a derivative thereof, or a mixture thereof.

5. The composition of claim 4, wherein the SGLT2 inhibitor is selected from the group consisting of canagliflozin, dapagliflozin, empagliflozin, erpagliflozin, ispagliflozin, rupagliflozin, topagliflozin, repagliflozin ectoate, sergliflozin ectoate, and pharmaceutically acceptable salts, solvates, hydrates and mixtures thereof.

6. The composition of claim 4, wherein the DPP-4 inhibitor is selected from Sitagliptin TM , Vildagliptin TM , Saxagliptin TM , Linagliptin TM , Gemigliptin TM 、Anagliptin TM , Teneligliptin TM 、Alogliptin TM , Trelagliptin TM , Omarigliptin TM , Evogliptin TM , Gosogliptin TM , Dutogliptin TM , Berberine TM or a mixture thereof.

7. The composition for preventing and treating dementia according to claim 1, in The phosphodiesterase 5 inhibitor is selected from the group consisting of milonafil, a pharmaceutically acceptable salt, solvate, hydrate and mixture thereof; The antidiabetic therapeutic agent is N,N-dimethylbiguanide, a DPP-4 inhibitor or a SGLT2 inhibitor.

8. The composition of claim 1, wherein the phosphodiesterase 5 inhibitor is milonafil.

9. A method for preventing or treating neuroinflammation in a diabetic patient, the method comprising: An effective amount of a pharmaceutical composition comprising the composition of claim 1 is administered to a subject in need thereof.

10. A method for preventing or inhibiting the formation and / or accumulation of β-amyloid protein, the method comprising: An effective amount of a pharmaceutical composition comprising the composition of claim 1 is administered to a subject in need thereof.

11. A method for preventing or treating a neurodegenerative disease, the method comprising: An effective amount of a pharmaceutical composition comprising the composition of claim 1 is administered to a subject in need thereof.

12. The method of claim 8, wherein the neurodegenerative disease is 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.

13. A method for inhibiting Aβ oligomer / fibril formation by reducing Aβ aggregation, the method comprising: An effective amount of a pharmaceutical composition comprising the composition of claim 1 is administered to a subject in need thereof.

14. A method for inhibiting the generative processing of beta-amyloid by reducing BACE-1, the method comprising: An effective amount of a pharmaceutical composition comprising the composition of claim 1 is administered to a subject in need thereof.

15. A method for reducing extracellular Aβ monomers, oligomers and Aβ fibrils / plaques by increasing cerebral blood flow, the method comprising: An effective amount of a pharmaceutical composition comprising the composition of claim 1 is administered to a subject in need thereof.

16. A method for suppressing neuronal cell death and promoting neurogenesis, synaptogenesis and / or angiogenesis by activating the NO / cGMP / PKG / CREB pathway, the method comprising: An effective amount of a pharmaceutical composition comprising the composition of claim 1 is administered to a subject in need thereof.

17. A method for restoring synaptic plasticity by activating Wint signaling via inhibition of DKK-1, the method comprising: An effective amount of a pharmaceutical composition comprising the composition of claim 1 is administered to a subject in need thereof.

18. A method for inhibiting the production of APP and reducing Aβ accumulation by suppressing the positive feedback loop of Aβ production, the method comprising: An effective amount of a pharmaceutical composition comprising the composition of claim 1 is administered to a subject in need thereof.

19. A method for inhibiting the formation of Aβ fibrils / plaques by removing intracellular toxic and soluble Aβ oligomers via activation of autophagy, the method comprising: An effective amount of a pharmaceutical composition comprising the composition of claim 1 is administered to a subject in need thereof.

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