KR20220140960A - Composition for prevention and treating dementia with diabetes - Google Patents

Abstract

The present invention relates to a use as a therapeutic agent for Alzheimer's disease and dementia through removal of amyloid beta through concurrent use of mirodenafil, which is one of phosphodiesterase 5 inhibitors (PDE5 inhibitors), and empagliflozin, which is one of sodium-glucose cotransporter 2 inhibitors (SGLT 2 inhibitors). The present invention also relates to a use as a composition for preventing and treating dementia with diabetes.

Description

Composition for prevention and treatment of dementia with diabetes {Composition for prevention and treating dementia with diabetes}

The present invention is a phosphodiesterase 5 inhibitor (PDE5 inhibitor) mirodenafil (Mirodenafil) and sodium-glucose cotransporter 2 inhibitor (sodium-glucose cotransporter 2 inhibitor, SGLT2 inhibitor) one of It relates to a composition for preventing and treating dementia with diabetes, as well as use as a therapeutic agent for Alzheimer's disease and dementia through the removal of amyloid beta by the combination of empagliflozin.

A number of studies have demonstrated that diabetes is associated with dementia. Compared to non-diabetics, diabetic patients are twice as likely to develop vascular dementia and 1.6 times as likely to develop Alzheimer's disease. Diabetes is known to cause atherosclerotic lesions (the narrowing of blood vessels due to the accumulation of cholesterol on the walls of blood vessels), which can cause cerebral infarction or cerebral hemorrhage. In addition, it is known that the insulin resistance of diabetic patients and the resulting hyperinsulinemia have a great influence. Insulin also plays an important role in signal transduction in the brain, regulating appetite and energy homeostasis, and is also involved in learning and memory. However, problems with insulin action in the brain can lead to Alzheimer's dementia. In addition, when hyperinsulinemia occurs, a toxic protein (amyloid beta protein) is abnormally deposited in the brain. In addition, it is known that oxidative stress or inflammatory response related to diabetes affects the deposition of toxic proteins in the brain. In addition, diabetes causes various cerebrovascular diseases, and these cerebrovascular diseases also promote Alzheimer's disease.

Alzheimer's disease, which accounts for more than 50% of all dementia patients, is a representative senile degenerative brain disease that affects more than 36 million people worldwide. Aging is known as one of the common causes, and the prevalence of the elderly aged 65 to 85 is 0.6% to 8.4%, and about 5 million new patients are reported every year in the aging society, which is a significant burden in terms of social cost. Alzheimer's patients suffer from rapid memory loss, are accompanied by a decline in reason, and are characterized by a very slow onset and a gradual progression. - (2019): 1-8.)

The most characteristic pathological findings of this disease are the neurofibrillary tangle (NFT) composed of hyperphosphorylated tau protein inside the cell and the amyloid plaque existing outside the cell. The extracellular amyloid plate is mainly composed of a peptide called 'amyloid-β (Aβ)', which is a type of amyloid (starch-like) protein. Aβ is known to accumulate in the brain decades before the onset of Alzheimer's disease. The amyloid hypothesis is a theory that cells die due to neurotoxicity and chain reaction caused by this Aβ, which leads to a slow decline in the patient's cognitive function, leading to the progression of dementia (Hardy, John A., and Gerald A. Higgins. "Alzheimer's disease: the amyloid cascade hypothesis." Science 256.5054 (1992): 184-186.)

Amyloid precursor protein (hereinafter APP) is a protein that spans the cell membrane of nerve cells, and when some of these fragments are cut by a secretase, beta-amyloid (Aβ) is produced. Amyloid plaques (senile plaques) are formed between the nerve tissues due to insoluble beta-amyloid merging together.

Therefore, therapeutic agents are being developed that target each step of amyloid production, aggregation, and removal. Examples of such inhibitors include secretory enzyme inhibitors that block the production of beta-amyloid, aggregation inhibitors that prevent amyloid aggregation, and immunotherapeutic agents that immunologically removes beta-amyloid from the body through an antigen-antibody reaction. In addition, when several beta-amyloids aggregate to form oligomers, they adversely affect the mitochondrial energy production process, cause an inflammatory response, nerve cell loss, amyloid neurite formation, and tau hyperphosphorylation. These various pathways It can also be a target for drug development. (Kim, Seong Yoon. "Past and Future of Drug Treatments for Alzheimer's Disease." Journal of Korean Neuropsychiatric Association 57.1 (2018): 30-42.)

The treatment of Alzheimer's disease over the past 20 years has mainly been to relieve symptoms and delay disease progression. -aspartate receptor antagonist) has been used. After tacrine was first developed as an acetylcholinesterase inhibitor, drugs that reduced liver toxicity (donepezil, rivastigmine, galantamine) were developed and used one after another, and memantine was approved as an NMDA receptor antagonist in 2003. Acetylcholinesterase inhibitors are used in mild cases and memantine as monotherapy or in combination with acetylcholinesterase inhibitors in moderate to severe cases (Nygaard, Haakon B. "Current and emerging therapies for Alzheimer's disease"). 'disease." Clinical therapeutics 35.10 (2013): 1480-1489.)

However, although these drugs temporarily relieve some symptoms, the disease-modifying effect is not proven, so it is necessary to develop a therapeutic agent that fundamentally inhibits or prevents the progression of the disease.

On the other hand, degenerative neurological diseases, including dementia, are caused by the decrease or loss of nerve cell function, resulting in motor control function, cognitive function, perceptive function, and sensory function. In addition to all the functions of the body that we can feel, it shows abnormalities in a wide variety of functions, including the autonomic nervous system that is self-regulating in a state that we do not perceive.

Until now, the cause of dementia is unknown, so fundamental treatment is impossible, and the five commercially available drugs can only relieve symptoms in some diseases. Post-resistance and serious drug side effects have emerged, further limiting the improvement of symptoms in dementia patients.

Republic of Korea Patent Registration No. 10-0358083 Republic of Korea Patent Registration No. 10-2000382 Republic of Korea Patent Registration No. 10-2020-0010224 Republic of Korea Patent No. 10-1605576

The present invention has been devised to solve the above problems, and an object of the present invention is to inhibit the growth and differentiation of nerve cells and to inhibit the growth and differentiation of neurons and to inhibit the learning and memory deterioration of the toxic protein Aβ with mirodenafil and empagliflo To provide a composition capable of treating dementia by inducing a decrease in intracellular Aβ by reducing the expression of toxic protein by concurrent use of gin, thereby increasing the protection of neurons and synaptic plasticity.

In addition, an object of the present invention is to prevent and treat dementia with diabetes by using empagliflozin, which is used as a therapeutic agent for diabetes, together with mirodenafil with respect to amyloid beta acting as a direct cause of neuronal cell death. To provide a composition for

The present invention relates to a phosphodiesterase 5 inhibitor; And SGLT 2 inhibitor (sodium-glucose cotransporter 2 inhibitor); relates to a composition for preventing and treating dementia with diabetes containing as an active ingredient.

The phosphodiesterase 5 inhibitor of the present invention includes mirodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasantafil, avanafil; and at least one selected from the group consisting of pharmaceutically acceptable salts, solvates and hydrates thereof.

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

According to one embodiment of the present invention, the pharmaceutically acceptable salt may be mirodenafil hydrochloride, sildenafil citrate, or vardenafil hydrochloride.

The hydrate is a compound of the present invention containing a stoichiometric or non-sto ichiometric amount of water bound by a non-covalent intermolecular force. or salts thereof.

The solvate means a compound of the present invention or a salt thereof containing a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents therefor are those that are volatile, non-toxic and/or suitable for administration to humans.

SGLT 2 inhibitor (sodium-glucose cotransporter 2 inhibitor) canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, ipragl Reflozin (ipragliflozin), luceogliflozin (lus eogliflozin), tofogliflozin (Tofogliflozin), remogliflozin etabonate (remogliflozin etabonate), sergliflozin etabonate (sergliflozin etabonate) ); and at least one selected from the group consisting of pharmaceutically acceptable salts, solvates and hydrates thereof.

More preferably, the phospho diesterase 5 inhibitor of the present invention is mirodenafil, and the SGLT 2 inhibitor is empagliflozin and pharmaceutically acceptable salts and solvents thereof. It is characterized in that at least one selected from the group consisting of cargo and luggage.

Here, the mirodenafil and empagliflozin may be co-administered in a concentration ratio of 1:0.1 to 10.

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

According to one embodiment of the present invention, the pharmaceutical composition of the present invention is administered orally to a subject or non-orally administered to a site other than the head. That is, the composition of the present invention may exhibit the intended effect in the present invention even when it is not directly administered to the brain tissue, the body tissue surrounding the brain tissue (eg, the scalp), and a region adjacent thereto. In one specific example, the non-oral administration is subcutaneous administration, intravenous administration, intraperitoneal injection, transdermal administration or intramuscular administration, and in another specific example, subcutaneous administration, intravenous administration or intramuscular administration.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet, film or capsule, and may additionally include a dispersant or stabilizer.

The present invention relates to a dementia inhibitory effect through amyloid beta reduction by using a phosphodiesterase 5 inhibitor and a SGLT 2 inhibitor in combination. It can be widely used for prophylactic and therapeutic pharmaceutical uses as therapeutic agents.

1 and 2 are experimental results for the reduction of intracellular Aβ according to the combination treatment of mirodenafil and memantine according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[Experimental Example 1] Cell culture

The SH-SY5Y human neuroblastoma cell line used in the experiment was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and was supplemented with 10% fetal bovine serum (FBS; Australian Orgin, HyClone, Logan, UT, USA) and 1% Using DMEM/F12 Complete Medium (HyClone) containing penicillin/streptomycin (P/S;HyClone) at 37°C and 5% CO ₂ condition, CO ₂ incubator (311-TIF, Thermo Fisher Scientific Forma, MA, USA) cultured in

[Experimental Example 2] Neuron-like differentiation of SH-SY5Y cells using All-trans-retinoic acid (RA)

To check the amount of change in amyloid beta, 2×10 ⁵ cells were seeded in a T-25 flask. For cell fixation and stabilization, use DMEM/F12 Complete Medium (HyClone) containing 10% FBS (HyClone) and 1% P/S (HyClone) under _{a CO 2 incubator} (Thermo Fisher Scientific Forma) for 24 hours.

24 hours after cell dispensing, the cell culture medium was removed for neuron-like differentiation, and 1% FBS (HyClone), 1% P/S (HyClone), 10 μM all-trans-retinoic acid (RA; Sigma-Aldrich, St. Louis, MO, USA) was replaced with DMEM/F12 differentiation medium.

On the third day of differentiation, the medium was replaced with new DMEM/F12 differentiation medium. On the 6th day of differentiation, the untreated control group was replaced with new DMEM/F12 differentiation medium, and the sample treated group was replaced by adding new DMEM/F12 differentiation medium under various conditions.

[Experimental Example 3] Amyloid β(Aβ)1-42 Formation and Treatment

To form Aβ1-42 oligomer, human Aβ1-42 (Abcam, Cambridge, MA, USA) was added to DMEM/F12 Complete Medium (HyClone) containing 1% FBS (HyClone) and 1% P/S (HyClone). It was added so as to become 10 μM, and left in a CO ₂ incubator (Thermo Fisher Scientific Forma) for 3 hours under 37° C., 5% CO ₂ conditions to form Aβ1-42 oligomers.

After 72 hours, after removing the culture medium, DME M/F12 Complete Medium (HyClone) containing 10 μM of Aβ1-42 oligomer was treated alone or in combination with mirodenafil and empagliflozin to CO ₂ under the condition of 5% CO ₂ at 37 ° C. After incubation for 24 hours in an incubator (Thermo Fisher Scientific Forma), subsequent experiments were performed.

[Experimental Example 4] Amyloid beta 42 Human ELISA (enzyme-linked immunosor bent assay) measurement result

To measure the amount (pg/mL) of amyloid beta 42, put Aβ1-42, 50 μL of cell culture media in a 96 well pltate, add 50 μL of Hu Aβ42 Detection Antibody Solution to each well, and react at room temperature for 3 hours while shaking. The solution was all discarded, washed with 1X Wash Buffer, and 100 μL of Anti-Rabbit IgG HRP was added, followed by reaction at room temperature for 30 minutes. The solution was again discarded, washed with 1X Wash Buffer, and 100 μL of Stabilized Chromogen was added, followed by reaction at room temperature in the dark for 30 minutes. Then, after adding 100 μL of Stop Solution, the absorbance value was measured at 450 nm within 2 hours, and the results are shown in Tables 1 and 2 and FIGS. 1 and 2 below.

[Examples 1-3]

In Table 1 below, Example 1 is a combination treatment of mirodenafil 1 μM and empagliflozin 1 μM, Example 2 is a combination treatment of mirodenafil 1 μM and empagliflozin 10 μM, and Example 3 is In Table 2, 5 μM of mirodenafil and 0.5 μM of memantine were treated in combination.

[Comparative Examples 1 to 6]

In Table 1 below, Comparative Example 1 was treated with 1 μM of mirodenafil alone, and Comparative Examples 2 and 3 were treated with 1 μM and 10 μM of empagliflozin alone, respectively.

In Table 2 below, Comparative Example 4 was treated with 5 μM of mirodenafil alone, and Comparative Example 5 was treated with 0.5 μM of empagliflozin alone.

In Comparative Example 5, 1 μM of mirodenafil and 20 μM of empagliflozin were treated in combination in Table 1, and Comparative Example 6 was treated with 5 μM of mirodenafil and 0.05 μM of empagliflozin in Table 2.

In conclusion, Aβ reduction rate of 26.0% of Example 1 in which mirodenafil 1 μM and empagliflozin 1 μM were combined; Aβ reduction rate of 31.5% of Example 2 in which 1 μM of mirodenafil and 10 μM of empagliflozin were combined; The Aβ reduction rate of 39.3% of Example 3, in which mirodenafil 5 μM and empagliflozin 0.5 μM were combined, was a statistically significant difference compared to the sum of reduction rates A and B when mirodenafil and empagliflozin alone were treated. It could be confirmed that the effect more than the shopping mall was recognized.

That is, the Aβ reduction rates of Comparative Examples 1 and 4 in which mirodenafil 1 μM and 5 μM were treated alone were A1 and A2, respectively, and Comparative Examples 2, 3, and 5 in which empagloflozin 1 μM and 10 μM and 0.5 μM were treated alone. When the Aβ reduction rates of B1, B2, and B3 are respectively, the Aβ reduction rate of 26.0% of Example 1 is significantly higher than 'A1+B1 = 8.6%', and the Aβ reduction rate of Example 2 31.5% is 'A1+B2 = 21.1' %', and it can be seen that the Aβ reduction rate of 39.3% of Example 3 is significantly higher than that of 'A2+B3 = 21.3%'.

On the other hand, in Table 1, in Comparative Example 5, in which 1 μM of mirodenafil and 20 μM of empagliflozin were combined, and in Table 2, in Comparative Example 6, in which 5 μM of mirodenafil and 0.05 μM of empagliflozin were combined. Rather than the sum of the effects of each drug, it was confirmed that the weakening sangje effect appeared.

According to these experimental results, even if mirodenafil and empagliflozin were treated in combination, the concentration ratio of mirodenafil and empagliflozin was 1:0.1 to 10, synergistic or synergistic for the removal of amyloid beta (Aβ). ), but it was confirmed that the sum of the effects decreased at a concentration ratio of 1:0.01 outside the lower limit of this range and 1:20 outside the upper limit.

Therefore, it is preferable that the concentration ratio during the combined treatment of mirodenafil and empagliflozin is 1:0.1-10.

The present invention described above is merely exemplary, and those of ordinary skill in the art to which the present invention pertains will appreciate that various modifications and equivalent other embodiments are possible therefrom. Therefore, it will be well understood that the present invention is not limited to the forms recited in the above detailed description. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims. It is also to be understood that the present invention includes all modifications, equivalents and substitutions falling within the spirit and scope of the invention as defined by the appended claims.

Claims (6)

phosphodiesterase 5 inhibitors; and
SGLT 2 inhibitor (sodium-glucose cotransporter 2 inhibitor);
A composition for preventing or treating dementia, characterized in that it contains as an active ingredient.
According to claim 1,
The phosphodiesterase 5 inhibitor (phosphodiesterase 5 inhibitor),
mirodenafil, sildenafil, vardenafil, tadalafil, udenafil, dasantafil, avanafil; And a composition for preventing or treating dementia, characterized in that at least one selected from the group consisting of pharmaceutically acceptable salts, solvates and hydrates thereof.
According to claim 1,
The SGLT 2 inhibitor (sodium-glucose cotransporter 2 inhibitor) is canagliflozin (canagliflozin), dapagliflozin (dapagliflozin), empagliflozin (empagliflozin), ertugliflozin (ertugliflozin), ipragliflozin, lus eogliflozin, tofogliflozin, remogliflozin etabonate, sergliflozin etabonate ( sergliflozin etabonate) and pharmaceutically acceptable salts, solvates and hydrates thereof, a composition for preventing or treating dementia, characterized in that at least one selected from the group consisting of.
According to claim 1,
The phosphodiesterase 5 inhibitor is at least one selected from the group consisting of mirodenafil and pharmaceutically acceptable salts, solvates and hydrates thereof;
The SGLT 2 inhibitor (sodium-glucose cotransporter 2 inhibitor) is at least one selected from the group consisting of empagliflozin and its pharmaceutically acceptable salts, solvates and hydrates Prevention of dementia, characterized in that or a therapeutic composition.
5. The method of claim 4,
The phosphodiesterase 5 inhibitor and the SGLT2 inhibitor are co-administered in a concentration ratio of 1: 0.1 to 10 to have a synergistic effect on the removal of amyloid beta (Aβ). A composition for preventing or treating dementia.
According to claim 1,
The dementia is Alzheimer's dementia or a composition for preventing or treating dementia, characterized in that it includes dementia with diabetes.

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