Compositions and methods for preventing and treating neurodegenerative diseases associated with diabetes

Abstract

The present invention provides a composition for preventing or treating neurodegenerative diseases, including antidiabetic therapeutic agents including phosphodiesterase 5 (PDE-5) inhibitors, sodium-glucose cotransporter 2 (SGLT2) inhibitors, and dipeptidyl peptidase 4 (PPD-4) inhibitors, and a method of using the same. The PDE5 inhibitor is selected from among sildenafil, tadalafil, vardenafil, tadalafil, udenafil, dasanafil, avanafil, pharmaceutically acceptable salts, solvates, hydrates, and mixtures thereof. The SGLT2 inhibitor is selected from among canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, tofogliflozin, remogliflozin etabonate, sergliflozin etabonate, pharmaceutically acceptable salts, solvates, hydrates, and mixtures thereof. The neurodegenerative disease is selected from the group consisting of 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 group of mixed etiologies thereof.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 367,278, 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 (PDE - 5) inhibitor, as well as antidiabetic therapeutic agents including a sodium - glucose cotransporter 2 (SGLT2) inhibitor and a dipeptidyl peptidase 4 (DPP - 4) inhibitor, for preventing or treating neurodegenerative diseases under diabetes, 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 "04334900120_SequenceListing.xml (7KB)", created on June 28, 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 explored. A common pathological phenomenon of neurodegenerative diseases is the death of central nervous system cells. Different from other organ cells, central nervous system cells can hardly 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, including neuron 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), 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 a major 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 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 has become a target for the treatment of neurodegenerative diseases. However, the therapeutic drugs developed so far have the effect of adjusting the symptoms of the disease, but have no effect on treating the neurodegenerative disease itself.

[0008] Therefore, there is a need to develop preventive and therapeutic drugs for neurodegenerative diseases based on a concept 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 related to 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 research has demonstrated 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, particularly 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 quiescent 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 shown 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, small amounts of Aβ are 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 signaling 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 in the treatment of dementia include acetylcholinesterase inhibitors (AchEI) and NMDA (N-methyl-D-aspartic acid) 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 symptom relief and delay, as well as improvement of cognitive function, and at present, 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] Furthermore, diabetics have a two-fold tendency to develop neurodegenerative diseases, including vascular dementia and Alzheimer's disease. Diabetes is known to cause atherosclerosis (accumulation of cholesterol in the blood vessel walls, narrowing the blood vessels), which can lead to cerebral infarction or cerebral hemorrhage. As a result, when brain tissue is damaged, brain function deteriorates, leading to dementia. In addition, insulin resistance and associated hyperinsulinemia in diabetics are also known to have a significant impact. Insulin plays an important role in signal transduction in the brain, regulates appetite and energy homeostasis, and is also involved in learning and memory. However, if there are problems with the function of insulin in the brain, Alzheimer's type dementia can occur. In the case of hyperinsulinemia, toxic proteins (amyloid beta protein) abnormally deposit in the brain. Furthermore, it is known that oxidative stress or inflammatory responses associated with diabetes affect the deposition of toxic proteins in the brain. Ultimately, diabetes causes various cerebrovascular diseases, and these cerebrovascular diseases can promote the progression of Alzheimer's disease.

[0017] Since the causes of neurodegenerative diseases are not fully understood, basic treatment remains a challenge. Commercially available drugs can only relieve symptoms in some diseases and cannot fundamentally change the progression of the disease. If severe side effects of these drugs appear after treatment, the improvement of symptoms in these patients is further limited. Therefore, the options for treating neurodegenerative diseases or disorders, including dementia, remain limited.

[0018] Nevertheless, the options for treating neurodegenerative diseases or disorders, including dementia, remain limited. SUMMARY OF THE INVENTION

[0019] The present invention provides a composition and method for treating neurodegenerative diseases under diabetic conditions by reducing neuroinflammation, particularly in the CNS system, and / or by reducing the expression of toxic proteins, such as beta amyloid (Ab).

[0020] The composition includes a phosphodiesterase 5 (PDE-5) inhibitor and an antidiabetic therapeutic agent including a sodium-glucose cotransporter 2 (SGLT2) inhibitor or a dipeptidyl peptidase 4 (DPP-4) inhibitor,

[0021] The PDE-5 inhibitor is selected from among sildenafil, tadalafil, vardenafil, tadalifil, udenafil, dasanafil, avanafil, and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof,

[0022] The antidiabetic therapeutic agent is selected from among insulin, glucagon-like peptide 1 (GLP-1), and agents that (1) increase the amount of insulin secreted from the pancreas, (2) enhance the sensitivity of target organs to insulin, (3) reduce the rate of glucose absorption from the gastrointestinal tract, and (4) increase the loss of glucose via urination,

[0023] The SGLT2 inhibitor is selected from among canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, tofogliflozin, remogliflozin etabonate, sergliflozin etabonate, pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof,

[0024] The PPD-4 inhibitor is selected from, for example, but not limited to, Sitagliptin (trademark) (FDA approved in 2006, sold by Merck & Co. as Januvia), Vildagliptin (trademark) (EU approved in 2007, sold by Novartis as Galvus in the EU), Saxagliptin (trademark) (FDA approved in 2009, sold as Onglyza), Linagliptin (trademark) (FDA approved in 2011, sold by Eli Lilly and Company and Boehringer Ingelheim as Tradjenta), Gemigliptin (trademark) (approved in South Korea in 2012, sold by LG Life Sciences as Zemiglo), Anagliptin (trademark) (approved in Japan in 2012, sold by Sanwa Kagaku Kenkyusho Co., Ltd. and Kowa Company, Ltd.), Teneligliptin (trademark) (approved in Japan in 2012), Alogliptin (trademark) (FDA approved in 2013, sold by Takeda Pharmaceutical Company), Trelagliptin (trademark) (approved for use in Japan in 2015), Omarigliptin (trademark) (MK-3102) (approved in Japan in 2015, developed by Merck & Co., and through research, it was found that omarigliptin can be used as a once-weekly treatment and is generally well tolerated through the base test and extension test), Evogliptin (trademark) (approved for use in South Korea), Gosogliptin (trademark) (approved for use in Russia), Dutogliptin (trademark) (under development by Phenomix Corporation), Berberine (trademark) (an alkaloid found in plants of the Berberis genus), or mixtures thereof,

[0025] The composition inhibits inflammatory cytokines, such as IL1b, IL-6, or TNFa,

[0026] 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,

[0027] 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 group of mixed etiologies thereof.

Brief Description of the Drawings

[0028]

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DETAILED DESCRIPTION OF THE INVENTION

[0029] 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).

[0030] The composition includes anti-diabetic therapeutic agents including phosphodiesterase 5 (PDE-5) inhibitors and sodium-glucose co-transporter 2 (SGLT2) inhibitors.

[0031] The PDE-5 inhibitor is selected from the group consisting of milodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasanafil, avanafil, and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

[0032] The SGLT2 inhibitor is selected from canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, tofogliflozin, remogliflozin etabonate, sergliflozin etabonate, pharmaceutically acceptable salts, solvates, hydrates, and mixtures thereof.

[0033] The PPD-4 inhibitor is selected from, for example, but not limited to, Sitagliptin (trademark) (FDA approved in 2006, sold by Merck & Co. as Januvia), Vildagliptin (trademark) (EU approved in 2007, sold by Novartis as Galvus in the EU), Saxagliptin (trademark) (FDA approved in 2009, sold as Onglyza), Linagliptin (trademark) (FDA approved in 2011, sold by Eli Lilly and Company and Boehringer Ingelheim as Tradjenta), Gemigliptin (trademark) (approved in South Korea in 2012, sold by LG Life Sciences as Zemiglo), Anagliptin (trademark) (approved in Japan in 2012, sold by Sanwa Kagaku Kenkyusho Co., Ltd. and Kowa Company, Ltd.), Teneligliptin (trademark) (approved in Japan in 2012), Alogliptin (trademark) (FDA approved in 2013, sold by Takeda Pharmaceutical Company), Trelagliptin (trademark) (approved for use in Japan in 2015), Omarigliptin (trademark) (MK-3102) (approved in Japan in 2015, developed by Merck & Co., and through research, it was found that omarigliptin can be used as a once-weekly treatment and is generally well-tolerated throughout the base study and extension study), Evogliptin (trademark) (approved for use in South Korea), Gosogliptin (trademark) (approved for use in Russia), Dutogliptin (trademark) (under development by Phenomix Corporation), Berberine (trademark) (an alkaloid found in plants of the Berberis genus), or mixtures thereof.

[0034] The composition inhibits inflammatory cytokines, such as IL1b, IL-6, or TNFa, and

[0035] the composition inhibits the growth and differentiation of nerve cells, as well as the degradation of learning and memory, induces a decrease in intracellular Aβ, thereby enhancing the protection of nerve cells and synaptic plasticity,

[0036] 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 group of their mixed etiologies.

[0037] In an embodiment of the present invention, there is provided a composition for preventing and treating dementia, which contains a phosphodiesterase 5 (PDE-5) inhibitor and an anti-diabetic therapeutic agent including a sodium-glucose cotransporter 2 (SGLT2) inhibitor or a PPD-4 inhibitor as an active ingredient.

[0038] In a specific embodiment of the present invention, the composition contains the phosphodiesterase 5 (PDE-5) inhibitor and an anti-diabetic therapeutic agent including a sodium-glucose cotransporter 2 (SGLT2) inhibitor or a PPD-4 inhibitor 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.

[0039] In another embodiment, the composition of the present invention is

[0040] A method for inhibiting Aβ oligomer / fibril formation by reducing Aβ aggregation,

[0041] A method for inhibiting β-amyloid formation processing through reducing BACE-1,

[0042] A method for reducing extracellular Aβ monomers, oligomers, and Aβ fibrils / plaques by increasing cerebral blood flow,

[0043] Method for inhibiting inhibition of neuronal cell death by activation of the NO / cGMP / PKG / CREB pathway, and promoting neurogenesis, synaptogenesis, and / or angiogenesis

[0044] Method for restoring synaptic plasticity by activation of Wnt signaling by inhibition of DKK-1, inhibiting APP production and reducing Aβ accumulation by inhibiting the positive feedback loop for Aβ production, and

[0045] Provided is a method for inhibiting the formation of Aβ fibrils / plaque by removing intracellular toxicity and soluble Aβ oligomers by activation of autophagy.

[0046] The phosphodiesterase 5 inhibitor of the present invention is at least one selected from the group consisting of sildenafil, tadalafil, vardenafil, tadalafil, udenafil, dasanafil, avanafil, and pharmaceutically acceptable salts, solvates, and hydrates thereof.

[0047] 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 salt is 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 compound of the present invention may be reacted with a base to form salts such as ammonium salts, alkali metal salts such as sodium or potassium salts, 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.

[0048] According to one embodiment of the present invention, examples of pharmaceutically acceptable salts can be milodenafil hydrochloride, sildenafil citrate, or vardenafil hydrochloride.

[0049] 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.

[0050] 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.

[0051] The SGLT2 inhibitor is selected from canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, tofogliflozin, remogliflozin etabonate, sergliflozin etabonate, pharmaceutically acceptable salts, solvates, hydrates, and mixtures thereof.

[0052] More preferably, the phosphodiesterase 5 inhibitor is selected from sildenafil, pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof, the anti-diabetic therapeutic agent is GLP-1, the SGLT2 inhibitor is selected from empagliflozin, pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof, and the PPD-4 inhibitor is selected from, but not limited to, Sitagliptin (trademark), Vildagliptin (trademark), Saxagliptin (trademark), Linagliptin (trademark), Gemigliptin (trademark), Anagliptin (trademark), Teneligliptin (trademark), Alogliptin (trademark), Trelagliptin (trademark), Omarigliptin (trademark), Evogliptin (trademark), Gosogliptin (trademark), Dutogliptin (trademark), Berberine (trademark), or mixtures thereof.

[0053] The pharmaceutical composition of the present invention may be administered orally or parenterally.

[0054] 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.

[0055] The pharmaceutically acceptable carriers contained 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, in addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, and preservatives. Appropriate pharmaceutically acceptable carriers and drugs are detailed in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0056] The pharmaceutical composition of the present invention may be manufactured in unit dosage form by formulating it with pharmaceutically acceptable carriers and / or excipients, or may be prepared by incorporating it into a multi-dose container according to a method that can be easily carried out by those skilled in the technical field to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or may be in the form of an extract, powder, granule, tablet, film, or capsule, and may further contain a dispersing agent or a stabilizer.

[0057] In certain embodiments, the composition of the present invention has a synergistic effect on the inhibition of inflammatory factors and reduces neuroinflammation.

[0058] 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 (PDE-5 inhibitor) and an acetylcholinesterase inhibitor.

Examples

[0059] The following provides a more detailed description using the following embodiments. However, these embodiments are for illustrative purposes only and do not limit the scope of the present invention.

[0060] Experimental Example 1. Culture Method of IMG Cells

[0061] The IMG cells, a mouse microglia 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, Australia, HyClone, Logan, UT, USA) and 1% penicillin / streptomycin (P / S, HyClone). 2×10 5 cells were seeded in each 6-well plate and incubated in the above humidified CO2 incubator for 24 hours. Further, 100 ng / mL of LPS and drugs, namely, AR1001 and SGLT-2 inhibitor (empagliflozin), were administered individually or in combination at concentrations of 2, 10, and 20 μM.

[0062] Experimental Example 2. RNA Extraction and cDNA Synthesis

[0063] Cells were scraped using a cell scraper, and 2 mL of the culture medium was collected in 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. Furthermore, 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).

[0064] Experimental Example 3. Real-time RT-qPCR for Inflammatory Cytokines

[0065] 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 prior, 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.

[0066] [Table 1]

[0067] Experimental Example 4. Measurement Results of the Reduction Rate of Inflammatory Cytokines.

[0068] Figure 1 shows the synergistic effect on IL-1β in the combined treatment of milodenafil and empagliflozin of the present invention. Here, AR1001 refers to milodenafil.

[0069] In Figure 2, the reduction rate of IL-1β was 7.66% in the combined treatment with 2 μM of milodenafil and 2 μM of empagliflozin, 17.74% in the combination of 2 μM of milodenafil and 10 μM of empagliflozin, 31.62% in the combination of 2 μM of milodenafil and 20 μM of empagliflozin, 24.05% in the combination of 10 μM of milodenafil and 2 μM of empagliflozin, 34.67% in the combination of 10 μM of milodenafil and 10 μM of empagliflozin, 43.95% in the combination of 10 μM of milodenafil and 20 μM of empagliflozin, 54.66% in the combination of 20 μM of milodenafil and 2 μM of empagliflozin, and 61.99% in the combined treatment of 20 μM of milodenafil and 10 μM of empagliflozin. The reduction rates for various combinations were significantly higher than the sum of the individual reduction rates observed when treated with milodenafil or empagliflozin alone, and a synergistic effect exceeding the additive effect was confirmed.

[0070] Figure 3 shows the synergistic effect on TNF-α in the combined treatment of milodenafil and empagliflozin of the present invention. Here, AR1001 refers to milodenafil.

[0071] In Figure 4, the reduction rate of TNF-α was 12.02% for the combined treatment of 2 μM milodenafil and 2 μM empagliflozin, 19.41% for the combination of 2 μM milodenafil and 10 μM empagliflozin, 32.18% for the combination of 2 μM milodenafil and 20 μM empagliflozin, 34.75% for the combination of 10 μM milodenafil and 2 μM empagliflozin, 37.82% for the combination of 10 μM milodenafil and 10 μM empagliflozin, 48.65% for the combination of 10 μM milodenafil and 20 μM empagliflozin, 58.90% for the combination of 20 μM milodenafil and 2 μM empagliflozin, and 64.96% for the combined treatment of 20 μM milodenafil and 10 μM empagliflozin. The reduction rates for various combinations were significantly higher than the sum of the individual reduction rates observed when treated with milodenafil or empagliflozin alone, and a synergistic effect exceeding the additive effect was confirmed.

[0072] Experimental Example 5. Cell Culture

[0073] 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.

[0074] Experimental Example 6. Neuronal Differentiation of SH-SY5Y Cells Using All-Trans Retinoic Acid (RA)

[0075] To confirm the change amount of amyloid beta, 2×10 5Cells 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.

[0076] 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).

[0077] 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.

[0078] Experimental Example 7. Formation and Treatment of Amyloid-β (Aβ) 1-42

[0079] 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.

[0080] After 72 hours, the medium was removed, and then the cells were treated with milodenafil and empagliflozin alone or in combination in DMEM / F12 complete medium (HyClone) containing 10 μM of Aβ1-42 oligomers, and cultured in a CO2 incubator (Thermo Fisher Scientific Forma) for 24 hours under the conditions of 37 °C and 5% CO2, and then the experiment was conducted.

[0081] Experimental Example 8. Measurement of Amyloid-β (Aβ) 42 by Human ELISA (Enzyme-Linked Immunosorbent Assay) Kit

[0082] To measure the amount of Aβ42 (pg / mL), 50 μL of cell culture medium was placed in a 96-well plate [as it is in the original text (sic): plate]. Further, 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.

[0083] Embodiments 1 to 3.

[0084] In Figure 6 below, Embodiment 1 is the combined treatment of 1 μM of milodenafil and 1 μM of empagliflozin, Embodiment 2 is the combined treatment of 1 μM of milodenafil and 10 μM of empagliflozin, and Embodiment 3 is the combined treatment of 5 μM of milodenafil and 0.5 μM of empagliflozin in Figure 8.

[0085] Comparative Examples 1 to 6.

[0086] In Figure 6, Comparative Example 1 is the treatment with 1 μM of milodenafil alone, and Comparative Examples 2 and 3 are the treatments with 1 μM and 10 μM of empagliflozin alone, respectively.

[0087] In FIG. 8, Comparative Example 4 is the treatment with 5 μM of sildenafil alone, and Comparative Example 5 is the treatment with 0.5 μM of empagliflozin alone.

[0088] Furthermore, Comparative Example 5 is the combined treatment with 1 μM of sildenafil and 20 μM of empagliflozin in FIG. 6, and Comparative Example 6 is the combined treatment with 5 μM of sildenafil and 0.05 μM of empagliflozin in FIG. 8.

[0089] As a conclusion, the Aβ reduction rate of 26.0% in the combined treatment with 1 μM of sildenafil and 1 μM of empagliflozin in Embodiment 1, the Aβ reduction rate of 31.5% in the combined treatment with 1 μM of sildenafil and 10 μM of empagliflozin in Embodiment 2, and the Aβ reduction rate of 39.3% in the combined treatment with 5 μM of sildenafil and 0.5 μM of empagliflozin in Embodiment 3 showed statistically significant differences compared with the sum of the reduction rates of A and B in the treatment with sildenafil or empagliflozin alone. Therefore, it was confirmed that an effect exceeding the additive effect could be recognized.

[0090] In other words, referring to the Aβ reduction rates of Comparative Examples 1 and 4 regarding the treatments with 1 μM and 5 μM of sildenafil alone as A1 and A2, respectively, and referring to the Aβ reduction rates of Comparative Examples 2, 3, and 5 regarding the treatments with 1 μM, 10 μM, and 0.5 μM of empagliflozin alone as B1, B2, and B3, respectively, it can be seen that the Aβ reduction rate of 26.0% in Embodiment 1 is significantly higher than "A1 + B1 = 8.6%", the Aβ reduction rate of 31.5% in Embodiment 2 is significantly higher than "A1 + B2 = 21.1%", and the Aβ reduction rate of 39.3% in Embodiment 3 is significantly higher than "A2 + B3 = 21.3%".

[0091] Furthermore, in Comparative Example 5 regarding the combined treatment of 1 μM of milodenafil and 20 μM of empagliflozin in FIG. 6, and in the case of Comparative Example 6 regarding the combined treatment of 5 μM of milodenafil and 0.05 μM of empagliflozin in FIG. 8, it was found that an antagonistic effect was shown, which was rather weaker than the sum of the effects of each drug.

[0092] From these experimental results, even when the combined treatment of milodenafil and empagliflozin was carried out, there was a synergistic effect or synergistic action on the removal of amyloid beta (Aβ) at a concentration ratio of milodenafil to empagliflozin of 1:0.1 to 10. However, it was found that the total effect decreased at concentration ratios outside this range, specifically 1:0.01 below the lower limit value and 1:20 above the upper limit value.

[0093] Therefore, the concentration ratio of the combined treatment of milodenafil and empagliflozin is preferably 1:0.1 to 10.

[0094] The above invention is merely illustrative, 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.

[0095] Sequence Listing

[0096]

Table 2

Claims

1. At least one phosphodiesterase 5 inhibitor, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and At least one antidiabetic drug, 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 a pharmaceutically acceptable salt, solvate, hydrate, or mixture thereof.

3. The pharmaceutical composition according to claim 1, wherein the antidiabetic drug is a drug that (1) increases the amount of insulin secreted from the pancreas, (2) increases the sensitivity of a target organ to insulin, (3) decreases the rate of glucose absorption from the gastrointestinal tract, or (4) increases glucose loss through urination, or a combination thereof.

4. The pharmaceutical composition according to claim 1, wherein the antidiabetic drug comprises 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 pharmaceutical composition according to claim 4, wherein the SGLT2 inhibitor comprises canagliflozin, dapagliflozin, empagliflozin, erzgliflozin, ipragliflozin, luseogliflozin, tofogliflozin, remogliflozin etahoneate, or cergliflozin etahoneate, or a mixture thereof.

6. The pharmaceutical composition according to claim 4, wherein the DPP-4 inhibitor comprises Sitagliptin (trademark), Vildagliptin (trademark), Saxagliptin (trademark), Linagliptin (trademark), Gemigliptin (trademark), Anagliptin (trademark), Teneligliptin (trademark), Alogliptin (trademark), Trelagliptin (trademark), Omarigliptin (trademark), Evogliptin (trademark), Gosogliptin (trademark), Dutogliptin (trademark), or Berberine (trademark), or a mixture thereof.

7. A pharmaceutical composition according to claim 1, The phosphodiesterase 5 inhibitor comprises mirodenafil, and The aforementioned antidiabetic drug includes N,N-dimethylbiguanide, a DPP-4 inhibitor, or an SGLT2 inhibitor. The aforementioned pharmaceutical composition.

8. The pharmaceutical composition according to claim 1 for a method of preventing or treating neuroinflammation.

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

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

11. The pharmaceutical composition according to claim 10, 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), or frontotemporal dementia, or a combination thereof.

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

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

14. 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.

15. 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.

16. 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.

17. 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.

18. 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.

19. Myrodenafil, or a pharmaceutically acceptable salt, solvate, hydrate or derivative thereof, and Empagliflozin, or any pharmaceutically acceptable salt, solvate, hydrate, or derivative thereof. A pharmaceutical composition containing the following:

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

21. The pharmaceutical composition according to claim 19 for a method of preventing and / or inhibiting the formation and / or accumulation of beta-amyloid.

22. The pharmaceutical composition according to claim 19 for a method of preventing or treating a neurodegenerative disease.

23. The pharmaceutical composition according to claim 22, 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), or frontotemporal dementia, or a mixture thereof.

24. The pharmaceutical composition according to claim 19 for a method of inhibiting Aβ oligomer / fibrillation formation by reducing Aβ aggregation.

25. The pharmaceutical composition according to claim 19 for a method of inhibiting β-amyloid formation processing by reducing BACE-1.

26. The pharmaceutical composition according to claim 19 for a method of reducing extracellular Aβ monomers, oligomers, and Aβ fibrils / macula by increasing cerebral blood flow.

27. ​​The pharmaceutical composition according to claim 19 for a method of inhibiting neuronal cell death, promoting neurogenesis, synapse formation, and / or angiogenesis by activation of the NO / cGMP / PKG / CREB pathway.

28. The pharmaceutical composition according to claim 19 for a method of restoring synaptic plasticity by inhibition of DKK-1 and / or activation of Wint signaling.

29. The pharmaceutical composition according to claim 19 for a method of inhibiting APP production and / or reducing Aβ accumulation by suppressing a positive feedback loop for Aβ production.

30. The pharmaceutical composition according to claim 19 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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