KR102687771B1 - Senomorphic combination therapy - Google Patents

Abstract

The present invention relates to a combination composition for senomorphics containing KU-60019 and Y-27632. More specifically, senescent cells with cell cycle arrest due to aging are combined with KU-60019 and Y-27632. By confirming the synergistic effect of restoring young cells to the level of young cells in various aspects by the combined treatment of -27632, the composition containing KU-60019 and Y-27632 was used as a combined pharmaceutical composition for senomorphics, a health functional food composition, and It can be used as a cosmetic composition.

Description

Combination composition for senomorphics {Senomorphic combination therapy}

The present invention relates to a combination composition for senomorphics comprising KU-60019 and Y-27632.

The more cells divide, the more they undergo aging. After a certain number of divisions, they reach Hayflick's limit, a state in which they can no longer divide, and cells that have reached this limit are called senescent cells. .

In the past, aging research was focused on identifying the causes of aging and methods for extending lifespan at the molecular level, but recently, research on methods and technology development on anti-aging for the prevention, recovery, and treatment of aging has been actively conducted. In particular, aging delay and aging research have been actively conducted. Research focused on developing therapeutic substances for related diseases is increasing.

Strategies for developing drugs to overcome aging can be largely divided into the development of drugs that remove only senescent cells (senolytics) and the development of drugs that improve the characteristics of senescent cells to those of young cells (senomorphics).

In aging individuals where tissue regeneration through stem cells is not actively progressing, there is a greater need for drugs that suppress the characteristics of senescent cells because there are concerns about the application of drugs that eliminate senescent cells.

Since around 2010, drugs that aim to suppress senescent cell traits, such as metformin, rapamycin, and NAD ⁺ booster, have been known. However, all reported drugs suppress the characteristics of aged cells by regulating metabolism or the activity of Sirt1, a DNA repair protein, and are therefore not very helpful in revitalizing the cell cycle mechanism of aged cells. However, no drug has yet been reported that restores the overall characteristics of senescent cells and induces them to actively divide like young cells.

Therefore, there is still a need to develop drugs (senomorphics) that improve the characteristics of senescent cells to those of young cells.

Republic of Korea Patent Publication No. 10-2020-0133511 (published on November 30, 2020)

The purpose of the present invention is to provide a combination composition for senomorphics that improves the characteristics of senescent cells to that of young cells.

The present invention provides a senomorphic composition comprising KU-60019 and Y-27632 that improves the characteristics of senescent cells to that of young cells.

Additionally, the present invention provides a pharmaceutical composition for treating or preventing diseases related to cellular aging, including KU-60019 and Y-27632.

Additionally, the present invention provides a pharmaceutical composition for treating or preventing diseases related to lipofuscin accumulation, including KU-60019 and Y-27632.

In addition, the present invention provides a health functional food composition for preventing or improving cellular aging-related diseases, comprising KU-60019 and Y-27632.

Additionally, the present invention provides a cosmetic composition for preventing or improving cellular aging-related diseases, comprising KU-60019 and Y-27632.

According to the present invention, by confirming the synergistic effect of recovering senescent cells whose cell cycle has been arrested due to aging to the level of young cells in various aspects by the combined treatment of KU-60019 and Y-27632, senomorphic It can be used as a combined pharmaceutical composition for senomorphics, a health functional food composition, and a cosmetic composition.

Figure 1 shows the CCK assay to determine the optimal cell division concentration of senescent cells according to the combined treatment of KU-60019 (KU) and Y-27632 (Y).
Figure 2 shows the degree of accelerated cell division of senescent cells according to the combined treatment of KU and Y, analyzed by HOECHST staining;
Figure 3 shows the effect of reducing the staining amount of the aging biomarker Senescence-associated-β-Galactosidase (SA-β-gal) due to the combined treatment of KU and Y using an optical microscope.
Figure 4 shows the induction of a decrease in the staining amount of the aging biomarker SA-β-gal due to the combined treatment of KU and Y, analyzed by an objective quantification method of SA-β-gal through FDG assay.
Figure 5 shows the induction of a decrease in the amount of lipofuscin in senescent cells due to the combined treatment of KU and Y through autofluorescence measurement of cells;
Figure 6 shows the induction of mitochondrial morphological changes due to the combined treatment of KU and Y through mitotracker staining;
Figure 7 shows the induction of intracellular oxygen radical reduction following the combined treatment of KU and Y through MitoSox assay.
Figure 8 shows the recovery of intracellular mitochondrial membrane potential following the combined treatment of KU and Y through TMRM assay.
Figure 9 shows the induction of increased mitochondrial biosynthesis due to the combined treatment of KU and Y through mitotimer assay.
Figure 10 shows the induction of a decrease in the degree of intracellular DNA damage due to the combined treatment of KU and Y through immunochemistry of γH2AX and 53BP1;
Figures 11 and 12 analyze the induction of activation of FOXM1 and E2F1 according to the combined treatment of KU and Y, respectively.
Figure 13 shows a schematic diagram of the mechanism of action for “sequential” activation of FOXM1 and E2F1 according to the combined treatment of Y and KU.

Hereinafter, the present invention will be described in more detail.

In order to solve the problem that most senomorphic drugs only focus on the presence or absence of drug efficacy in some aging traits and do not reveal detailed mechanisms through multifaceted investigation, raising concerns about overall anti-aging efficacy or side effects, the present inventor has developed a new As a result of diligent efforts to develop senomorphic drugs, the combined treatment of KU-60019 and Y-27632 resulted in the inactivated transcription factors FOXM1 and E2F1 being activated as cell cycle factors in senescent cells. The present invention was completed by discovering that reactivation promotes the transcription of most genes necessary for overcoming aging.

The present invention relates to a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof; and a senomorphic composition that improves the characteristics of senescent cells to those of young cells, comprising a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof:

[Formula 1]

[Formula 2]

The composition includes a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof; and a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof at a molar ratio of 1:(3-7), and more preferably at a molar ratio of 1:5, to It was confirmed that the synergistic effect of senomorphics, which improves the characteristics of young cells, was the best.

At this time, the compound represented by Formula 1 is KU-60019, an ATM (Ataxia telangiectasia mutated) kinase inhibitor, and the compound represented by Formula 2 is a rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor. It is Y-27632.

The pharmaceutically acceptable salt may be used in the form of either a pharmaceutically acceptable basic salt or an acidic salt. Basic salts can be used in the form of either organic base salts or inorganic base salts, such as sodium salts, potassium salts, calcium salts, lithium salts, magnesium salts, cesium salts, aminium salts, and ammonium salts. , triethylaminium salt, pyridinium salt, etc. can be used, but are not limited thereto.

Additionally, an acid addition salt formed by a free acid is useful as the acid salt. Inorganic and organic acids can be used as free acids. Hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, and phosphoric acid can be used as inorganic acids, and citric acid, acetic acid, maleic acid, fumaric acid, glucosan, and methanesulfonic acid can be used as organic acids. , benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glyconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid. etc. can be used. Preferably, hydrochloric acid can be used as the inorganic acid, and methanesulfonic acid can be used as the organic acid.

In addition, the composition may include not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.

The addition salt according to the present invention can be prepared by conventional methods, for example, by dissolving the compound of Formula 1 or Formula 2 in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, and adding an excess of organic base. It can be prepared by adding or adding an aqueous solution of an inorganic base and then precipitating or crystallizing it. Alternatively, an addition salt can be obtained by evaporating the solvent or excess base from this mixture and drying it, or it can be prepared by suction filtration of the precipitated salt.

The composition can improve the characteristics of senescent cells to that of young cells by reactivating cell cycle factors through sequential activation in which activation of FOXM1 is first induced and then activation of E2F1 is induced.

In addition, the composition reduces i) cell proliferation, ii) activity of β-galactosidase (senescence-associated β-galactosidase), iii) reduction of lipofuscin expression, iv) improvement of mitochondrial function, and v) generation of reactive oxygen species. The characteristics of senescent cells can be improved to those of young cells through one or more activities selected from the group consisting of reduction and vi) DNA damage repair.

In addition, the present invention relates to a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof; and a pharmaceutical composition for treating or preventing diseases related to cellular aging, comprising a compound represented by Formula 2 or a pharmaceutically acceptable salt thereof.

The cellular aging-related disease may be selected from the group consisting of skin wrinkles, wounds, sarcopenia, and progeria. The progeria may be selected from Wiener syndrome and Hutchinson-Gilford syndrome, but is not limited thereto.

In addition, the present invention relates to a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof; and a pharmaceutical composition for treating or preventing diseases related to lipofuscin accumulation, comprising a compound represented by Formula 2 or a pharmaceutically acceptable salt thereof.

The diseases related to the accumulation of lipofuscin include neuronal ceroid lipofuscinoses (NCL), age-related macular degeneration, neurofibrillary tangles, and brown atrophy of the heart. of the heart), Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), acromegaly, denervation atrophy, lipid myopathy ) and chronic obstructive pulmonary disease (COPD).

In one embodiment of the present invention, the pharmaceutical composition is any one selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops, or solutions according to conventional methods. The formulation can be used.

In another embodiment of the present invention, the pharmaceutical composition includes suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, and diluents commonly used in the preparation of pharmaceutical compositions. , it may further include one or more additives selected from the group consisting of dispersants, surfactants, binders, and lubricants.

Specifically, carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, and microcrystalline. Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agents, etc., and such solid preparations can be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

According to one embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. It can be administered to the subject.

The preferred dosage of the composition may vary depending on the subject's condition and weight, type and degree of disease, drug form, administration route and period, and may be appropriately selected by a person skilled in the art. According to one embodiment of the present invention, but is not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be divided into several administrations, and the scope of the present invention is not limited thereby.

In the present invention, the 'subject' may be a mammal, including humans, but is not limited to these examples.

In addition, the present invention relates to a compound represented by Formula 1; and a health functional food composition for preventing or improving cellular aging-related diseases, comprising a compound represented by Formula 2.

The term "health functional food" refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with the Health Functional Food Act, and "functional" refers to food that is related to the structure and function of the human body. It means ingestion for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects.

The health functional food composition may contain common food additives, and its suitability as a “food additive” is determined in accordance with the general provisions and general test methods of the food additive code approved by the Ministry of Food and Drug Safety, unless otherwise specified. The decision is made based on the specifications and standards for the item.

Items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as subchromic pigment, licorice extract, crystalline cellulose, high-liquid pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations.

The effective dose of the compound contained in the health functional food can be used in accordance with the effective dose of the therapeutic agent, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range, Since the active ingredient poses no safety issues, it is certain that it can be used in amounts exceeding the above range.

There are no particular restrictions on the types of health functional foods, and examples include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, and tea. , drinks, alcoholic beverages, and vitamin complexes.

In addition, the present invention relates to a compound represented by Formula 1; and a cosmetic composition for preventing or improving cellular aging-related diseases, comprising a compound represented by Formula 2.

The cosmetic composition may include, in addition to the compound represented by Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof, conventional auxiliaries such as stabilizers, solubilizers, vitamins, pigments, and fragrances, and a carrier.

The cosmetic composition can be prepared in any formulation commonly prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion foundation, It may be formulated as a wax foundation or spray, but is not limited thereto. More specifically, it can be manufactured in the form of sun cream, softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, pack, spray, or powder.

When the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicon, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. .

If the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as the carrier ingredient. In particular, in the case of a spray, chlorofluorohydrocarbon, propane/butane may be used as the carrier ingredient. Alternatively, it may include a propellant such as dimethyl ether.

When the formulation is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -Butyl glycol oil, fatty esters of glycerol, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and microcrystalline cellulose. , aluminum metahydroxide, bentonite, agar or tracant, etc. can be used.

In the present invention, the term “prevention” refers to any action that suppresses or delays disease by administering the composition according to the present invention. In the present invention, the term “treatment” refers to any action that improves or beneficially changes the symptoms of a disease by administering the composition according to the present invention. In the present invention, “improvement” means any action that improves the bad condition of a disease by administering or ingesting the composition of the present invention to an individual.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Example> Examination of the effect of restoring aging characteristics of human dermal fibroblasts according to the combined treatment of KU-60019 and Y-27632

1. Experimental method

1) Cell culture

Human dermal fibroblasts used in the experiment were samples collected from newborn skin (PCS-201-010; ATCC), and the cells were grown in Dulbecco's modified Eagle's medium [25 mM glucose, 10% FBS (S001-01; Welgene), 100 units/ml penicillin, 10 μg/ml streptomycin, and 25 ng/ml amphotericin B (LS203-01; Welgene)] at 37°C and 5% CO ₂ conditions.

Subculture was carried out whenever the cell confluency (cell distribution rate on the bottom of the cell culture tank) did not exceed 80%, and passage was continued by cultivating a quarter of cells from the 1st to 45th generation collected from newborn skin. . From the 45th to the 47th, it was 1/3, and after the 48th, it was 1/2.

When the population doubling time (DT) exceeded 14 days and the SA-β-gal ratio exceeded 90%, the cells were considered sufficiently aged and the experiment was conducted. On the other hand, only cells with a population doubling time (DT) of 1 day or less were considered young cells and the experiment was performed. The cells passed the MycoAlert Mycoplasma Detection Kit (LT07-318; Lonza) contamination test before the experiment.

2) Cell proliferation experiment

Cells to be used in the experiment were transplanted into a well plate 3 days before the experiment. After treatment with drugs KU-60019 (hereinafter referred to as KU) and Y-27632 (hereinafter referred to as Y), the amount of cells was quantified by treatment with D-Plus™ CCK reagent (Dongin) for 4 hours. A plate reader (TECAN Infinite M200 PRO) was used in the 450nm wavelength range. In the case of Hoechst 33342, cells treated with KU and Y drugs were stained with 10 μg/ml Hoechst 33342 for 30 minutes, washed twice with phosphate-buffered saline (PBS), and ex/em: plate reader at 355/460nm wavelength range. The amount of cells was quantified using (TECAN Infinite M200 PRO).

3) SA-β-gal staining experiment

SA-β-gal staining was performed using fluorescein di-β-d-galactopyranoside (FDG; Thermo Fisher Scientific) or X-gal-based Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, #9860). X-gal was processed according to the kit manufacturer's protocol, and in the case of FDG, the cells were treated so that the final concentration of FDG was 100 μM in the complete solution of the kit excluding X-gal. Cells were transplanted 3 days before the experiment, and for imaging, the area where the cells were distributed was randomly photographed using an Olympus CKX41 microscope for the The degree of staining was quantified using a reader (TECAN Infinite M200 PRO). Next, the amount of cells was calculated using Hoechst 33342, and the degree of FDG staining per unit cell was calculated.

4) Lipofuscin quantification experiment

The amount of lipofuscin per cell was quantified using an LSR Fortessa (Beckton Dickson) flow cytometer. By applying the fact that lipofuscin is autofluorescent, the values were measured using a 530/30 nm bandpass filter (FITC channel) in the 488 nm wavelength range without staining.

5) Immunochemical experiment

Cells were transplanted into a 4-well chamber cell culture slide (SPL) 3 days before the experiment. After Y and KU treatment, cells were fixed with 4% paraformaldehyde for 10 minutes. The primary antibody was diluted in gelatin dilution buffer (GDB) and then applied onto the cells and reacted at 4°C overnight. After two rinses with PBS, the secondary antibody was diluted 1:1,000 in GDB and reacted at room temperature for 1 hour. DAPI staining was performed using mounting solution with DAPI (Vector Laboratories). Finally, imaging was performed with a ZEISS Axiocam microscope. The antibodies used in the above experiment are as follows.

rabbit anti-γH2AX (9218S; 1:500 dilution; Cell Signaling Technology), mouse anti-53BP1 (05-726; 1:500 dilution; MilliporeSigma)

6) Measurement of mitochondrial function and status

To measure the shape or total amount of mitochondria, cells were reacted with 30 nM MitoTracker Deep Red (M22426; Thermo Fisher) for 30 minutes at 37°C under 5% CO ₂ conditions. All relevant experiments were measured using an LSR Fortessa (Beckton Dickson) flow cytometer, and to quantify the amount of ROS generated, 5 μM MitoSOX (M36008; Thermo Fisher) (excitation wavelength: 561 nm, 615/24 nm bandpass filter, PE-Texas Red channel), 37℃, 5% CO ₂ 30 minutes reaction; To quantify mitochondrial membrane potential, react with tetramethylrhodamine, methyl ester (TMRM; I34361; Thermo Fisher) (excitation wavelength: 561 nm, 615/24 nm bandpass filter, PE-Texas Red channel) at 37°C, 5% CO ₂ for 30 minutes. proceeded.

For mitochondrial biogenesis experiments, pLenti6.3/V5-DEST plasmid containing the MitoTimer reporter (#50547l, Addgene) was transduced into 45s cells, and then green fluorescence (excitation wavelength of 488 nm with a) was measured using an LSR Fortessa flow cytometer. The ratio of 530/30 nm bandpass filter, FITC channel)/red fluorescence (excitation wavelength of 561 nm with a 583/22 nm bandpass filter, PE channel) was obtained and quantified.

7) Western blot technique

Cells were dissolved in 2ㅧLaemmli sample buffer (#161-0737, Bio-Rad) containing 5% β-mercaptoethanol (Sigma) and then boiled at 95°C for 5 minutes. The dissolved proteins were size-sequentially electrophoresed through SDS-PAGE and transferred from the SDS-PAGE gel to polyvinylidene difluoride (PVDF) membranes (Millipore). To prevent antibodies from sticking anywhere, the PVDF membrane was previously blocked with tris-buffered saline + 0.05% Tween 20 dissolved in 5% bovine serum albumin (BSA; Sigma), and then treated with primary antibodies. Next, the membrane was reacted with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour. To detect the proteins, they were made to emit light with an enhanced chemiluminescence solution (P90720, Millipore) and then visualized with an ImageQuant LAS-4000 digital imaging system (GE Healthcare). Quantification of imaging results was performed with ImageQuant TL software 8.1.

2. Experiment results

Senescent cells that have reached the Hayflick limit through subculture no longer undergo cell division. However, as shown in Figures 1 and 2, it was confirmed that cell division of senescent cells resumed when treated with Y and KU. In particular, when Y 2.5 μM and KU 0.5 μM were treated simultaneously, a synergistic effect on cell division induction was demonstrated. It has been done.

Unlike young cells, senescent cells are stained blue using senescence-associated-β-galactosidase staining, which is one of the biomarkers. Figures 3 and 4 confirm that when senescent cells were simultaneously treated with Y and KU, the number of unstained cells decreased compared to the total number of cells.

Senescent cells produce lipofuscin, a type of metabolic waste produced when digesting or processing various metabolites in intracellular lysosomes. Lipofuscin, a yellow-brown pigment granule, is not produced in young cells that actively process metabolic waste, but when produced in old cells, it causes various degenerative diseases. As shown in Figure 5, it was confirmed that lipofuscin was greatly reduced when Y and KU were treated simultaneously.

Mitochondria of aging cells have an elongated and stretched shape compared to mitochondria of young cells. As shown in Figure 6, when Y and KU were treated together, it was confirmed that the mitochondrial shape of aged cells existed in fragments similar to that of young cells.

Mitochondria of aged cells have higher ROS production, lower mitochondrial membrane potential, and lower mitochondrial turnover than mitochondria of young cells. However, it was confirmed that the patterns of these various aged mitochondria changed to resemble those of young cells when treated with Y and KU, as shown in Figures 7 to 9.

Aged cells do not have the ability to repair DNA damage, so they have a lot of broken DNA. As shown in Figure 10, it was confirmed that abnormal DNA was significantly recovered when Y and KU were treated simultaneously.

Summarizing the above results, it was confirmed that the recovery action of aged cells was greatly improved by the combined treatment of Y and KU.

Meanwhile, by revealing how cell cycle factors are affected by the combined treatment of Y and KU, the synergistic effect of the recovery of senescent cells due to the combined treatment of Y and KU was identified, Figures 11 and 12 As shown, following the combined treatment of Y and KU, the inactivated transcription factors FOXM1 and E2F1 in senescent cells are reactivated by cell cycle factors, promoting the transcription of most genes necessary to overcome aging. It was judged that In particular, the “sequential” activation of FOXM1 in the early stage and E2F1 in the latter stage according to the combined treatment of Y and KU was judged to be very important for the recovery function of senescent cells.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof; and
An in vitro method of improving the characteristics of senescent cells to that of young cells by treating senescent cells with a composition containing a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof at a molar ratio of 1:5. ) Senomorphic composition:
[Formula 1]

[Formula 2]
delete The method of claim 1, wherein the composition improves the characteristics of senescent cells to those of young cells by reactivating cell cycle factors through sequential activation in which activation of FOXM1 is first induced and then activation of E2F1 is induced. A composition made of. The method of claim 1, wherein the composition i) reduces cell proliferation, ii) reduces the activity of β-galactosidase (senescence-associated β-galactosidase), iii) reduces the expression level of lipofuscin, iv) improves mitochondrial function, and v) A composition characterized in that it improves the characteristics of senescent cells to those of young cells through one or more activities selected from the group consisting of reducing active oxygen production and vi) DNA damage repair. delete delete delete A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof; and
Pharmaceutical composition for treating or preventing neuronal ceroid lipofuscinoses (NCL), comprising a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof at a molar ratio of 1:5:
[Formula 1]

[Formula 2]
delete A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof; and
Sequential treatment of FOXM1 and E2F1, comprising treating senescent cells in vitro with a composition containing a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof at a molar ratio of 1:5: How to activate:
[Formula 1]

[Formula 2]
delete