KR20230131810A - Composition for removing senescent cells comprising selenium-containing compound as active ingredient - Google Patents

Abstract

The composition for removing senescent cells according to the present invention contains a selenium compound or a pharmaceutically acceptable salt thereof as an active ingredient, thereby inducing the death of senescent cells and removing senescent cells, thereby preventing various aging-related diseases caused by senescent cells. It can be usefully applied to suppress and treat diseases and disorders. In addition, it can be usefully applied to medicines, cosmetics, health functional foods, and animal products in relation to age-related diseases and disorders caused by senescent cells.

Description

Composition for removing senescent cells containing a selenium compound as an active ingredient {COMPOSITION FOR REMOVING SENESCENT CELLS COMPRISING SELENIUM-CONTAINING COMPOUND AS ACTIVE INGREDIENT}

The present invention relates to a composition for removing senescent cells containing a selenium compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Aging refers to the process in which humans grow physically and cognitively as they grow older, leading to death. Recently, the World Health Organization (WHO) has assigned a disease code (MG2A) to old age itself, and various research and development and clinical trials to treat or delay aging are actively underway.

As we age, aging progresses in the human body due to internal factors such as decrease in telomeres and increase in reactive oxygen species, and as a result, various 'aging-related diseases & disabilities/disorders' occur. Various stress factors, including the administration of toxic substances such as anticancer drugs, metabolic disturbances such as obesity and diabetes, and tissue wounds and transplants, accelerate the occurrence of age-related diseases and disorders.

The fundamental cause of these age-related diseases and disorders is 'senescent cells'. According to recent research results, as we age, senescent cells accumulate in the human body due to various factors, and the accumulated senescent cells Secreted factors include senescence-promoting factors as well as senescence-associated pathogenic factors such as inflammation and fibrosis. The role of senescence-associated secretory phenotype (SASP) is (i) to gradually spread senescent cells by changing surrounding normal cells into senescent cells, and (ii) to change the structural and functional properties of all organs and tissues. (iii) cause various underlying pathological phenotypes such as chronic inflammation (immuno-aging) and fibro-aging due to aging, and (iv) eventually It is known to cause various age-related diseases and disorders throughout the body.

These senescent cells are removed by the body's immune cells, but as we age, the immune cells also age, so they cannot function properly and eventually senescent cells accumulate in the body. This cellular aging phenomenon occurs in all cells that make up the human body, and is observed not only in general somatic cells such as immune cells, but also in stem cells and various disease cells such as cancer. If aging progresses in stem cells, organs and tissues cannot be regenerated and maintained, and if aging progresses in diseased cells, the disease becomes worse.

Accordingly, just as cancer is treated by removing cancer cells, various research and development efforts are being conducted to treat age-related diseases and disorders by removing senescent cells (Robbins PD et al., Annuual Review of Pharmacological Toxicology 61, 779- 803, 2021). ABT263/737 and others have recently been reported as such innovative anti-aging treatments, but many side effects such as toxicity exist as problems.

Meanwhile, selenium is an essential mineral element that exists in trace amounts in the body and is a component of protein that acts as an antioxidant. It is known that synthetic compounds containing selenium, a natural element, have the activity of eliminating senescent cells, independently of the natural action of selenium, which protects cells and plays an important role in the survival and maintenance of cells. does not exist.

Robbins PD et al., Annual Review of Pharmacological Toxicology 61, 779-803, 2021.

Accordingly, the present inventors studied to develop a safe and effective drug that can remove senescent cells, and as a result confirmed that selenium compounds remove various types of senescent cells with high efficiency, thereby completing the present invention.

To achieve the above object, one aspect of the present invention provides a composition for removing senescent cells, comprising a selenium compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The composition for removing senescent cells according to the present invention significantly reduces the survival rate of not only naturally aged cells but also various senescent cells artificially induced by various factors over time, thereby preventing various age-related pathological diseases secreted from these senescent cells. It can fundamentally block and suppress the expression and secretion of human factors. Therefore, the composition for removing senescent cells according to the present invention can be usefully used for suppressing and treating age-related diseases and disorders. Furthermore, the composition for removing senescent cells according to the present invention can be usefully used in medicines, cosmetics, health functional foods, animal products, etc. in relation to aging-related diseases and disorders related to senescent cells.

Figure 1 shows the action of removing senescent cells using a selenium compound.
Figure 2 is a staining photograph of adipose tissue in mice in which aging was naturally induced, under conditions in which LX-112 was not treated and treated with LX-112.
Figure 3 is a staining photograph of adipose tissue in mice in which aging was induced by doxorubicin (DOX) treatment under conditions without and with LX-112 treatment.

Hereinafter, the present invention will be described in detail.

As used herein, the term “halogen” means F, Cl, Br or I unless otherwise specified.

As used herein, the term “alkyl,” unless otherwise specified, refers to a linear or branched, saturated hydrocarbon moiety. For example, “C _1-6 alkyl” means alkyl with a skeleton of 1 to 6 carbons. Specifically, C _1-6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, i-pentyl, t-pentyl, sec-pentyl, neopentyl. , may include hexyl.

As used herein, the term “haloalkyl” refers to alkyl substituted with one or more halogens. Specifically, haloalkyl may be an alkyl substituted with two or more halogens of the same type or with two or more types of halogens substituted.

As used herein, the term "alkoxy", unless otherwise specified, refers to a group having the formula -O-alkyl, wherein the alkyl group defined above is attached to the parent compound through an oxygen atom. The alkyl portion of the alkoxy group may have 1 to 20 carbon atoms (i.e., C ₁ -C ₂₀ alkoxy), 1 to 12 carbon atoms (i.e., C ₁ -C ₁₂ alkoxy), or 1 to 6 carbon atoms (i.e., C ₁ -C ₆ alkoxy). Examples of suitable alkoxy groups include methoxy (-O-CH ₃ or -OMe), ethoxy (-OCH ₂ CH ₃ or -OEt), t-butoxy (-OC(CH ₃ ) ₃ or -O-tBu). etc.

As used herein, the term “alkenyl” refers to a linear or branched hydrocarbon moiety containing one or more double bonds. For example, “C _2-6 alkenyl” refers to an alkenyl group with a skeleton of 2 to 6 carbon atoms. Specifically, C _2-6 alkenyl may include ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, etc. there is.

As used herein, the term “alkynyl” refers to a linear or branched hydrocarbon residue containing one or more triple bonds. For example, “C _2-6 alkynyl” refers to an alkenyl group with a skeleton of 2 to 6 carbons. Specifically, it may include ethynyl, n-propynyl, etc.

As used herein, the term “aryl” refers to an aromatic hydrocarbon radical derived by removing one hydrogen atom from six carbon atoms of the parent aromatic ring system. For example, an aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms. The aryl group may be phenyl.

As used herein, the term “heteroaryl” refers to an aromatic heterocyclyl having one or more heteroatoms in the ring. Non-limiting examples of heteroaryls include pyridinyl, pyrrolyl, oxazolyl, indolyl, isoindolyl, purinyl, furanyl, thienyl, benzofuranyl, benzothiophenyl, carbazolyl, imidazolyl, and thiazolyl. , isoxazolyl, pyrazolyl, isothiazolyl, quinolyl, isoquinolyl, pyridazyl, pyrimidyl, pyrazyl (which may have one or more substituents on the ring), etc.

As used herein, the term “cycloalkyl” refers to a saturated monocycle or poly-cycle containing only carbon atoms in the ring. For example, “3- to 9-membered cycloalkyl” means a cyclic hydrocarbon moiety containing 3 to 9 carbon atoms. Cycloalkyl may have 3 to 7 carbon atoms as a monocycle and 7 to 9 carbon atoms as a bicyclic cycloalkyl.

As used herein, the term “heterocycloalkyl” refers to a non-aromatic heterocyclyl having one or more heteroatoms in the ring. Heterocycloalkyl may have one or more carbon-carbon double bonds or carbon-heteroatom double bonds in the ring, to the extent that the ring is not aromatic due to the presence of the double bond. Non-limiting examples of heterocycloalkyls include azetidinyl, aziridinyl, pyrrolidinyl, piperidinyl, piperazinyl, homopiperazinyl, morpholino, thiomorpholino, tetrahydrofuranyl, tetrahydrothio. There are furanyl, tetrahydropyranyl, and pyranyl.

As used herein, the term “heteroatom” refers to an atom other than carbon (C), and may specifically be a selenium (Se), nitrogen (N), oxygen (O), or sulfur (S) atom.

One aspect of the present invention provides a composition for removing senescent cells, comprising a selenium compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

where R is a single bond, -(CH ₂ ) _n -, or -(CH ₂ ) _p -X-(CH ₂ ) _q -; n is an integer from 1 to 10; p and q are each independently an integer from 1 to 5; X is -S-, -Se- or phenylene; A is a group represented by Formula 2 below, or is phenyl, amine, or amide;

[Formula 2]

R ¹ , R ² , R ³ , R ⁴ , R ^{5 ,} R ⁶ , R ⁷ and R ⁸ are each independently selected from H, halogen, C _1-6 alkyl, haloC _1-6 alkyl, C _1-6 alkoxy, It is carboxy, amine, or nitro.

The amines and amides are each C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, 3-10 membered cycloalkyl, C _6-10 aryl, 3-10 membered heterocycloalkyl, or It may be substituted with 5-10 membered heteroaryl or may be unsubstituted.

In one embodiment, the selenium compound according to the present invention is a compound represented by the following formula (3).

[Formula 3]

where R is -(CH ₂ ) _n - or -(CH ₂ ) _p -X-(CH ₂ ) _q -; n is an integer from 1 to 10; p and q are each independently an integer from 1 to 5; X is -S-, -Se- or phenylene; R ¹ , R ² , R ³ , R ⁴ , R ^{5 ,} R ⁶ , R ⁷ and R ⁸ are each independently selected from H, halogen, C _1-6 alkyl, haloC _1-6 alkyl, C _1-6 alkoxy, It is carboxy, amine, or nitro.

In another embodiment, the selenium compound according to the present invention is a compound represented by the following formula (4).

[Formula 4]

where R is -(CH ₂ ) _n - or -(CH ₂ ) _p -X-(CH ₂ ) _q -; n is an integer from 1 to 10; p and q are each independently an integer from 1 to 5; X is -S-, -Se- or phenylene.

In another embodiment, the selenium compound according to the present invention is a compound represented by the following formula (5) or (6).

[Formula 5]

[Formula 6]

where n is an integer from 1 to 8; p and q are each independently an integer of 1 to 4; X is -S-, -Se- or phenylene.

In another embodiment, the selenium compound according to the present invention is a compound represented by any one of the following formulas 7 to 13.

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

The compound of Formula 7 is a type of selenium compound generally called ethaselen, and is referred to herein as LX-112.

The compound of Formula 8 is a type of selenium compound commonly called propylselen.

The compound of Formula 9 is a type of selenium compound generally called butaselen, and is referred to herein as LX-114.

The compound of Formula 10 is a type of selenium compound generally called butathiobiselen, and is referred to herein as LX-115.

The compound of Formula 11 is a type of selenium compound generally called butaselenobiselen, and is referred to herein as LX-116.

The compound of Formula 12 is a type of selenium compound generally called hexaselen, and is referred to herein as LX-117.

The compound of Formula 13 is a type of selenium compound generally called ebselen, PZ51, DR3305, and SPI-1005, and is referred to herein as LX-128.

In the present invention, the compound represented by Formula 1 and its pharmaceutically acceptable salt can selectively kill senescent cells.

The aging may be any one selected from the group consisting of natural aging over time, aging due to stress caused by anticancer drugs, and aging due to stress due to obesity. That is, the senescent cells may be related to any one selected from the group consisting of natural aging over time, aging due to stress caused by anticancer drugs, and aging due to stress due to obesity.

The senescent cells may be any one selected from the group consisting of somatic cells, stem cells, and cells abnormally changed due to disease. The senescent cells may be cells of organ tissue derived from liver, kidney, spleen, brain, lung, muscle, skin, fat, etc. The disease may be selected from the group consisting of inflammation, fibrosis, cancer, etc.

The senescent cells may exhibit any one or more of the following characteristics. (i) Cell division and growth are almost permanently halted. (ii) Compared to non-aging normal cells, the overall size of the cells is increased. In particular, the contents of lysosomes are increased and abnormal mitochondria are increased. (iii) Nuclear structural proteins such as Lamin B1 and chromatin-binding proteins such as HMGB1 are reduced. (iv) Expression of β-galactosidase (SA-β-gal) due to aging. (v) Lipofuscin is produced as a degenerative abnormal complex of proteins and lipids due to aging. (vi) CDK (cyclin-dependent kinase) inhibitory factors such as p16 and p21 are activated and expressed in large quantities. (vii) As a continuous stress response, signal transduction systems such as p53 are activated according to DDR (DNA damage response), and the expression of related target genes is regulated accordingly. In this way, in addition to cell division and growth cessation, which are common characteristics of senescent cells, senescence-associated markers may include SA-β-gal, lipofuscin, p16, p21, p53, etc. ( (see https://doi.org/10.1016/j.tcb.2018.02.001).

In one embodiment of the present invention, when the composition for removing senescent cells according to the present invention is treated with artificially induced senescence cells and naturally senescent cells through continuous subculture, senescent cells can be effectively removed. .

In one embodiment of the present invention, the composition for removing senescent cells according to the present invention is applied to naturally aged mice or mice in which aging has been artificially induced due to stress such as anticancer drugs, fibrosis-inducing drugs, and obesity, or tumors. When treated with induced mice, senescent cells can be effectively removed from various organ tissues.

Therefore, the composition for removing senescent cells according to the present invention can be used to inhibit the expression and secretion of aging-related markers, aging-promoting factors, or aging-related secretory factors (SASP). Since these factors are expressed and secreted in senescent cells, the expression and secretion of factors may be fundamentally blocked as senescent cells are removed.

The aging-related marker is any one selected from the group including various factors including p16, p21, SA-β-gal, lipofuscin, etc. (https://doi.org/10.1016/j.tcb.2018.02.001) It can be one or more arguments.

The aging-related secreted factors (SASP) include various age-related pathological factors such as age-related inflammation and fibrosis, including IL1α, IL1β, IL6, IL10, MCP1, PAI1, VCAM1, MMP1, MMP2, MMP3, MMP12, MMP13, TGFβ, ACTA2, COL1A1, COL1A2, FN1, CXCL1, CXCL10, GM-CSF, GROα, GROβ, GROγ, IGFBP7, IL7, IL8, MIP1α, ENA78, GCP2, GITR, HGF, ICAM1, IGFBP2, IGFBP4, IGFBP5, IGFBP6, IL13, MCP4, MIF, MIP3α, MMP14, NAP2, PIGF, RANTES, sgp130, TIMP2, TRAILR3, Acrp30, Axl, bFGF, BLC, BTC, CTACK, EGFR, Fas, FGF7, GCSF, GDNF, HCC4, I309, IFNγ, IGFBP1, IGFBP3, IL11, IL15, IL2Rα, IL6R, ITAC, Leptin, LIF, MSPα, PAI2, PDGF-BB, SCF, SDF1, sTNF-RI, sTNF-RII, TIMP-1, tPA, uPA, uPAR, Aging-related inflammation and fibrosis factors such as VEGF, MCP3, IGF1, TGFβ3, MIP1, IL4, FGF7, PDGF-BB, IL16, BMP4, MDC, MCP4, IL10, TIMP1, ICAM1, Axl, CNTF, INFγ, EGF, BMP6, etc. It may be any one or more factors selected from the group including various pathological factors such as cancer (see http://www.saspatlas.com).

In one embodiment of the present invention, by using the composition for removing senescent cells according to the present invention, the expression and secretion of various age-related pathological factors (SASP) from senescent cells are eliminated in advance, thereby effectively preventing various age-related diseases and disorders. It can be suppressed and treated. In addition, by using the composition for removing senescent cells according to the present invention, it is possible to maintain resilience and resistance from various stresses, and to suppress, treat and improve various side effects and aftereffects caused by various stresses and diseases and disorders resulting therefrom. You can.

As such, the composition for removing senescent cells according to the present invention can be used to suppress and treat age-related diseases and disorders. That is, the present invention provides the use of the selenium compound represented by Formula 1 or a pharmaceutically acceptable salt thereof for suppressing and treating age-related diseases and disorders. In addition, the present invention provides the use of the selenium compound represented by Formula 1 or a pharmaceutically acceptable salt thereof for the production of medicines for suppressing and treating age-related diseases and disorders. Additionally, the present invention provides a method for suppressing and treating age-related diseases and disorders, comprising administering the selenium compound represented by Formula 1 or a pharmaceutically acceptable salt thereof to a subject in need. Additionally, the present invention provides a method for removing senescent cells, comprising treating the senescent cells with a selenium compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

The age-related diseases and disorders include those related to senescent cells, proliferative diseases and disorders, inflammatory or autoimmune diseases and disorders, neurological diseases and disorders, metabolic diseases and disorders, cardiovascular diseases and disorders, lung diseases and disorders, kidney diseases and disorders, Liver diseases and disorders, eye diseases and disorders, skin diseases and disorders, diseases and disorders related to regeneration and maintenance of stem cells or organ tissues, diseases and disorders related to foreign transplantation, fibrosis diseases and disorders, sclerosis diseases and disorders, β-galacto It may be any one or more selected from the group consisting of sidase-related diseases and disorders, other geriatric diseases and disorders, side effects and aftereffects due to stress, and age-related diseases and disorders resulting therefrom.

The above-mentioned aging-related diseases and disorders are related to aging cells, inflammation, fibrosis, cirrhosis, cancer, side effects and aftereffects due to anti-cancer drugs or stress caused by obesity, senescence, sarcopenia, lipoatrophy, liver and kidney damage, or skin aging and related related diseases. It may be a disease or disorder.

As an example, side effects and aftereffects caused by stress caused by anticancer drugs or obesity related to the senescent cells may include fatigue and weakness, weight loss, anxiety and depression, lethargy, or fatty liver.

The inflammation is senescence-related inflammation (inflamm-aging, immuno-senescence, senescence-associated inflammation) caused by senescent cells, and is observed in the elderly due to progressive chronic and degenerative inflammation or infections such as viruses, etc. The fibrosis and cirrhosis include diseases related to the liver, lungs, or kidneys, and the cancer refers to the aging process through prosenescence anti-cancer therapy. It is suppressed and treated by removing cancer cells and includes cancer, early cancer, prevention of cancer, recurrence, metastasis, resistance, malignancy or aftereffects, and the skin aging and related diseases and disorders include wrinkles, loss of elasticity, hair loss, and fur deterioration. , blemishes, birthmarks, spots, pigmentation, pigmentation, warts, hives, worsening of pores, or decreased wound healing.

In relation to the above frailty, frailty is, for example, a specific disease to which the R54 code was recently assigned, which includes age-related fatigue, weakness, asthenia, inactivity, and muscle weakness that occur with or without cognitive and memory impairment due to aging. Symptoms such as gait disturbance appear. Frailty can be analyzed by known symptom indicators (https://en.wikipedia.org/wiki/Frailty_syndrome).

Hereinafter, the various diseases and disorders known to be suppressed, improved, and treated by compositions for removing senescent cells are specifically indicated.

Proliferative diseases such as cancer, pre-cancerous/pre-malignant condition, cancer prevention, cancer relapse, cancer metastasis, cancer resistance, cancer malignancy, and various proliferative abnormalities. and disability; Arthritis, osteoarthritis, rheumatoid arthritis, degenerative articular cartilage, inflammatory bowel disease, ulcerative colitis, Crohn's disease, Mucositis, oral mucositis, chronic pancreatitis, tendinopathy, tendinosis, tendinitis, inflammation-aging, hyperamplification ( Inflammatory or autoimmune diseases and disorders, such as amplified hyperinflammation and COVID19 infection/complication; Memory loss, (mild) cognitive decline dysfunction, degenerative brain disease, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis ), amyotrophic lateral sclerosis, primary lateral sclerosis, motor neuron disease disorder, progressive supranuclear palsy, progressive bulbar paralysis, pseudobulbar paralysis, progressive muscular atrophy, lower motor neuropathy, spinal atrophy, spinal cord Spinal cord injury, chronic pain, post-polio syndrome, spastic paraplegia, anxiety, depression, neuropsychiatric dysfunction, (post-traumatic) stress disorder Neurological diseases and disorders such as traumatic stress disorder and sleep disorders; Obesity, diabetes, diabetic ulcer, diabetic cardiomyopathy, osteoporosis, intestinal microbiome disorder, lipodystrophy, Metabolic diseases and disorders such as various metabolic syndromes; Atherosclerosis, heart failure, congestive heart failure, myocardial infarction, cardiomyopathy, cardiomyocyte hypertrophy, cardiac dysfunction, heart Loss of cardiac stress tolerance, cardiac load resistance, atrial fibrillation abnormalities, angina pectoris, coronary artery aneurysm, aortic aneurysm, arrhythmia, carotid artery stenosis, dural arteriovenous fistulae ), coronary artery disease, peripheral vascular disease, renal artery stenosis, atherosclerosis, neointimal hyperplasia, hypertension, age-related vascular dysfunction, vascular calcification cardiovascular diseases and disorders such as hyporeactivity/calcification, blood coagulation disorders, brain aneurysm, stroke, thrombosis, endocarditis, hyperlipidemia, hypercholesterolemia, and mitral valve prolapse; Chronic obstructive pulmonary disease, chronic lung inflammation, pulmonary failure, decreased lung function, emphysema, bronchiectasis, hyperoxic lung injury, bronchopulmonary dysplasia , lung diseases and disorders such as respiratory distress syndrome, pulmonary hypertension, asthma, and viral pneumonia; Nephropathy, immunoglobulin A nephropathy, renal failure/dysfunction, glomerulosclerosis, chronic kidney disease, diabetic Kidney diseases and disorders such as chronic kidney disease and kidney stones; Cirrhosis, fatty liver, hepatitis, hepatic steatosis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, hepatopathy, cirrhosis, primary Liver diseases and disorders such as primary sclerosing cholangitis, primary biliary cirrhosis, and hepatic encephalopathy; Cataract, glaucoma, macular degeneration, macular edema, retinal degenerative disease, diabetic retinopathy, diabetic macular edema, presbyopia, Eye diseases and disorders such as dry eye disease, age-related vision loss, and hyperemia; Skin aging, wrinkles, loss of elasticity, epidermal atrophy, hair loss, allopecia, fur deterioration, melasma, blemishes, skin naevi, liver spot ), pigmentation, warts, urticaria, worsening pores, poor wound healing, psoriasis, herpes, pruritus, sensory disturbance, eczema, rash, dermatitis, eosinophilic dermatosis, immunobullous dermatosis, pustules, pemphigus , pemphigoid, lupus, blisters, keloids, dermal fibrohistiocytic proliferation, atopic dermatitis, cutaneous lymphoma, cutaneous lupus, systemic lupus erythematosus, erythroderma, lichen planus, lichenoid dermatosis, reactive Skin diseases and disorders such as neutrophilic dermatosis, strawberry nose, vitiligo, ichthyosis common, senescence in gingival tissues, dermatomyositis, actinic keratosis, and diseases related to photosensitivity and photoaging; Related to maintenance of regeneration of stem cells/bone marrow/organs/tissues, diseases and disorders, immunomodulatory diseases and disorders such as bone marrow hypoplasia, myelopathy, inflammation, sarcopenia, etc., and damage and decline in function of various organs/tissues. Diseases and Disorders; Foreign substances such as transplantation diseases and disorders of stem cells/bone marrow/organs/tissues/cells, such as graft-versus-host reaction or disease, corneal allograft rejection, etc. / Diseases and disorders related to external transplantation, such as transplant diseases and disorders of materials, immunomodulatory diseases and disorders such as inflammation, and damage and functional decline of various organs / tissues; Idiopathic pulmonary fibrosis, cystic fibrosis, pulmonary fibrosis, renal fibrosis, tubulointerstitial fibrosis, liver fibrosis ), cardiac fibrosis, pancreatic fibrosis, uterine fibrosis, oral submucous fibrosis, muscle fibrosis, glial fibrosis, retroperitoneal fibrosis, myelofibrosis, Fibrotic diseases and disorders such as mediastinal fibrosis, keloid, and fibrodysplasia ossificans progressiva; Sclerotic diseases and disorders such as scleroderma, systemic sclerosis, and multiple sclerosis; Progerias, Wiedmann-Lautenstrauch syndrome, Werner syndrome, Hutchinson-Gilford progeria syndrome, Rotmund-Thomson syndrome, Cockayne syndrome, Down-syndrome ), β-galactosidase-related diseases and disorders such as hypokeratosis, myasthenia congenita, aplastic anemia, and articular cartilage degeneration; Reduced lifespan/healthspan, frailty, inflammation-aging, sarcopenia, muscular dystrophy, muscle fibrosis, osteoporosis, muscle fatigue disorder , ataxia, bone disorder, chronic inflammation, delayed wound healing, delayed fracture healing, lipodystrophy, adipose atrophy, intervertebral disc degeneration), intervertebral disc herniation, kyphosis, fibrodysplasia ossificans progressiva, menopause, tremor, urinary incontinence, urinary dysfunction, splenomegaly, prostatic hypertrophy ), other geriatric diseases and disorders such as hearing loss/hearing loss, olfactory dysfunction, periodontal disease, etc.; Anticancer drugs such as radiation/chemistry/targeted/immunotherapy, drugs/substances with strong toxicity and side effects, physiological disturbances such as obesity/diabetes, diets such as high fat/high sugar/high salt, hemorrhagic shock, myocardial infarction, hypoxia, nutritional deficiency, war. Side effects and sequelae, including toxicity, caused by various acute and chronic stressors such as accidents, smoking, viral/bacterial infections, foreign transplants, inflammation, tissue wounds, and hyperthermia, as well as diseases and disorders resulting from them, such as fatigue and weakness ( fatigue, malaise, weight loss, reduced physical activity, anxiety, depression, lethargy, ataxia, cachexia, chronic inflammation, mucositis, thrombocytopenia ( thrombocytopenia, radiation ulcers, bone marrow dysfunction, heart functional disorder, immune disorder, hormonal system disorder, liver/nephrocomplication, stress neuropathy, liver/kidney /heart/gastrointestinal/ovaries/vascular system/skin toxicity, radiation ulcers, nausea, vomiting, diarrhea, peripheral neuropathy, syncope, anemia, hair loss, allopecia, pain, fluid retention, Rash, dermatitis, hyperpigmentation, anorexia, urticaria, lipodystrophy, adipose atrophy, panic disorder, cardiomyopathy, congestive heart failure, menopause, osteoporosis , infertility, preeclampsia, pre-eclampsia, cognitive decline dysfunction, cancer relapse, cancer metastasis, vision/hearing loss, decreased lung capacity, etc. These include, but are not limited to, various age-related diseases and disorders.

The composition for removing senescent cells of the present invention may also include a pharmaceutically acceptable salt of the selenium compound represented by Formula 1 above as an active ingredient. The pharmaceutically acceptable salts should have low toxicity to humans and should not have any negative effect on the biological activity and physicochemical properties of the parent compound. For example, the pharmaceutically acceptable salt may be an acid addition salt formed with a pharmaceutically acceptable free acid. The free acid may be an inorganic acid or an organic acid. In this case, the inorganic acid may be hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, hydrobromic acid, etc., and the organic acid may be acetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene sulfuric acid, etc. It may be fonic acid, fumaric acid, maleic acid, malonic acid, phthalic acid, succinic acid, lactic acid, citric acid, gluconic acid, tartaric acid, salicylic acid, malic acid, oxalic acid, benzoic acid, embonic acid, aspartic acid, glutamic acid, etc. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound of Formula 1 in an excess of acid aqueous solution and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone, or acetonitrile. It can be. Additionally, the pharmaceutically acceptable salt may be an alkali metal salt (sodium salt, etc.) or an alkaline earth metal salt (potassium salt, etc.). The alkali metal salt or alkaline earth metal salt can be obtained, for example, by dissolving the compound of Formula 1 in an excessive amount of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. .

The present invention provides a pharmaceutical composition for suppressing and treating age-related diseases and disorders, comprising the selenium compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, and silicic acid. Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline solution, PBS (phosphate buffered saline) ) or media, nanocapsules, nanospheres, nanogels, hydrogels, mesoporous silica, liposomes, exosomes, cyclodextrins, micelles (PLA/PLGA/PCL/PEG/SOL (Soluplus), etc.) It includes, but is not limited to, synthetically produced particles (particles, crystals, etc.) and polymers (polymeric complex, capsule, matrix, colloid, amphiphilic graft polymer, etc.), and in addition to the above ingredients, lubricants, wetting agents, sweeteners, and flavors. It may additionally contain agents, emulsifiers, suspending agents, preservatives, etc.

The pharmaceutical composition may further include pharmaceutically acceptable additives, and the additives may be, for example, pH adjusters, isotonic liquefaction agents, preservatives, and other excipients.

The dosage form of the pharmaceutical composition may vary depending on the method of use, but can be prepared as a tablet, granule, powder, syrup, solution, liquid extract, emulsion, suspension, steep, tablet, injection, capsule, pill, etc. In addition, the formulation of the pharmaceutical composition includes ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches that are formulations for topical or transdermal administration.

The pharmaceutical composition can be aseptically mixed with a pharmaceutically acceptable carrier and, if necessary, preservatives, buffers, etc. Ointments, pastes, creams and gel formulations according to the present invention additionally contain excipients such as animal and vegetable fat, oil, wax, paraffin, starch, toragacanto, cellulose derivatives, polyethylene glycol, silicone, bentonite, etc., silicic acid, talc, zinc oxide, etc. Or it may include a mixture thereof.

The term “inhibition” as used herein refers to any action that prevents, blocks, suppresses, or delays the progression of aging-related diseases and disorders by administering the composition according to the present invention, and refers to “treatment.” " means any action (alleviate, reverse or treat) in which the symptoms of aging-related diseases and disorders are improved, improved or beneficially changed by administration of the composition of the present invention.

The pharmaceutical composition of the present invention can be administered to a patient in a therapeutically effective amount or a pharmaceutically effective amount.

Here, “therapeutically effective amount” or “pharmaceutically effective amount” refers to the amount of a compound or composition effective in preventing or treating the target disease, which is sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment. This refers to an amount that does not cause side effects. Additionally, the “effective amount” refers to an amount sufficient to inhibit or reduce the activity of senescent cells in aging-related diseases, either in vitro or in vivo. The level of the effective amount is determined by factors including the patient's health status, type and severity of the disease, activity of the drug, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, combination or concurrent use of drugs, and It may be determined based on other factors well known in the medical field.

The pharmaceutical composition of the present invention may be administered by any suitable route, in a form of the pharmaceutical composition suitable for such route, and in a dosage effective for the intended treatment.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The composition for removing senescent cells according to the present invention may be used to remove senescent cells in vitro. Accordingly, the present invention provides a composition for removing senescent cells in vitro.

The composition for removing aging cells according to the present invention can be used as a cosmetic composition for skin regeneration. Accordingly, the present invention provides a cosmetic composition for skin regeneration.

In one embodiment of the present invention, when the composition for removing senescent cells according to the present invention is treated with naturally aged cells, the expression and secretion of skin aging-causing factors (SASP) can be effectively suppressed and reduced.

In one embodiment of the present invention, when the composition for removing senescent cells according to the present invention is treated with naturally aged mice, skin aging, etc. can be effectively improved.

Therefore, senescent cells secrete various factors (SASP) to gradually spread and accumulate senescent cells, causing various aging symptoms that occur in the skin (skin aging, wrinkles, loss of elasticity, hair loss, deterioration of fur, freckles, blemishes, birthmarks, birthmarks, etc.) Pigmentation, pigmentation, warts, moles, worsening pores, poor wound healing, psoriasis, herpes, pruritus, sensory disorders, decreased immunity, eczema, rash, dermatitis, eosinophilic dermatosis, immunobullous dermatosis, pus, pemphigus, pemphigoid. , lupus, blisters, fibrosis, keloids, dermal fibrous histiocytic proliferation, atopic dermatitis, cutaneous lymphoma, cutaneous lupus, diseases and disorders related to light sensation and photoaging, various skin diseases, etc.). Therefore, removing aging cells can suppress, improve, and regenerate skin aging.

As a specific example, the cosmetic composition of the present invention suppresses skin aging, wrinkles, loss of elasticity, hair loss, fur deterioration, freckles, blemishes, birthmarks, spots, discoloration, pigmentation, warts, hives, worsening of pores or decreased wound healing, etc. Can be improved or regenerated.

In order to use the composition for removing senescent cells according to the present invention as a cosmetic composition, it can be formulated into a formulation commonly prepared in the art, if necessary.

The cosmetic composition may be formulated, for example, as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing agent, oil, powder foundation, emulsion foundation, wax foundation, spray, etc. However, it is not limited to this. Specifically, it can be formulated as a softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder. In addition, when the formulation of the cosmetic composition is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide and mixtures thereof It may contain a carrier component selected from the group consisting of.

Additionally, when the formulation of the cosmetic composition is a solution or emulsion, it may contain a carrier component selected from the group consisting of solvents, solvating agents, emulsifying agents, and mixtures thereof. Examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, sorbitan fatty acid ester, and mixtures thereof. I can hear it.

In addition, when the formulation of the cosmetic composition is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and microcrystals It may contain a carrier component selected from the group consisting of cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and mixtures thereof. The carrier component may be included in an amount of about 1 to about 99.99% by weight, preferably about 80% by weight to about 90% by weight, based on the total weight of the cosmetic composition.

Additionally, the cosmetic composition may be in the form of an external skin preparation such as ointment, liquid, cream, spray, patch, etc.

The cosmetic composition can be applied directly to the skin or sprayed, depending on the formulation. At this time, the application amount and number of daily uses of the cosmetic composition can be appropriately set according to the user's age, gender, purpose, skin condition, etc.

The composition for removing senescent cells according to the present invention can be used as a health functional food. Accordingly, the present invention provides a health functional food composition for improving age-related diseases and disorders, comprising the selenium compound represented by Formula 1 above, or a pharmaceutically acceptable salt thereof, as an active ingredient.

Types of the health functional food may include various foods, beverages, gums, vitamin complexes, health supplements, etc.

In addition, the health functional food may be in the form of powder, granule, tablet, capsule, or beverage, and may additionally contain other natural or synthetic substances and additives for commercialization to improve and promote health functions.

Specifically, the liquid ingredients included in the health drink are not limited as long as they do not impede the effectiveness of the composition for removing aging cells, and like regular drinks, they may include various flavoring agents or natural carbohydrates as additional ingredients.

The natural carbohydrates include, for example, monosaccharides, disaccharides such as glucose and fructose; polysaccharides such as maltose and sucrose; Sugars such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol.

In addition, natural flavors (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavors (saccharin, aspartame, etc.) can be used as flavoring agents.

In addition, health functional foods include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), alginic acid and its salts, organic acids, protective colloidal thickeners, It may include pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

The composition for removing senescent cells according to the present invention can be used as a veterinary medicine, an external skin preparation for animals, or a health functional food for animals. Accordingly, the present invention provides a product composition for animals for improving aging-related diseases and disorders, comprising the selenium compound represented by Formula 1 above, or a pharmaceutically acceptable salt thereof, as an active ingredient. The animal product composition may be a veterinary drug, an external skin preparation for animals, or a health functional food for animals.

The veterinary medicine may be manufactured in an orally acceptable dosage form such as capsules, tablets, powders, solutions, syrups, etc., together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are not limited to this, but include starch, lactose, maltose, sucrose, fructose, dextrose, maltodextrin, sugar, citric acid, malic acid, other beverage flavorings, mannitol, sorbitol, xylitol, etc. It may include sugar alcohol, dicalcium phosphate, calcium sulfate, calcium carbonate, microcrystalline cellulose, etc. The external skin preparation for animals may be in a formulation such as ointment, liquid, cream, spray, patch, etc. The skin external preparation for animals may include applying directly to the skin or spraying, depending on the formulation. At this time, the application amount and number of daily uses of the external skin preparation for animals can be appropriately set according to the individual's age, gender, purpose, skin and fur condition, etc. The health functional food for animals may be an animal feed additive. The animal feed additives can be additionally added with additives permitted according to feed testing standards, and the feeding amount, feeding frequency, etc. can be adjusted according to the health status of the individual.

In this way, the composition for removing aging cells according to the present invention can be used in pharmaceuticals, cosmetics (for example, cosmetics for skin regeneration), health functional foods, or animal products.

[Example]

The above will be explained in more detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the examples is not limited to these only.

Example 1. Removal of senescent cells from senescent human cells

Example 1-1. Induction of cell aging

As we age, age-related diseases and disorders occur due to pathological factors (SASP) secreted by senescent cells that accumulate in the human body due to internal and external factors. To confirm that selenium compounds remove senescent cells, which play a fundamental role in this process, senescent cells were stained with SA-β-gal, an aging marker. The action of such a selenium compound is shown in Figure 1.

Specifically, foreskin cells derived from humans (source: Chungnam National University College of Medicine) were cultured in a designated medium (Iscove's Modified Dulbecco's Medium, source: ATCC; 10% FBS, source: GenDEPOT; 100 U/ml Penicillin & Streptomycin, source: Invitrogen). ), then treated with doxorubicin (DOX) dissolved in dimethyl sulfoxide (DMSO) at a concentration of 250 nM for 24 hours, and further cultured for 7 days to induce aging. The control group (-), which did not induce aging, was treated with DMSO only. To measure the proportion of aged cells, cells treated with doxorubicin and the control group were inoculated into a 24 well plate and stabilized for 24 hours, and then SA-β-gal, a marker related to aging, was tested using a kit (purchased by Cell Signaling Technology). was stained using , and the proportion of senescent cells stained with SA-β-gal was measured using an optical microscope and is shown in Table 1.

SA-β-gal
(%) - 9.0 (±5.0) DOX 90.0 (±7.0)

As a result, as shown in Table 1, the ratio of aging-related marker (SA-β-gal) in cells treated with doxorubicin (DOX) increased significantly compared to the control group (-), and the cells no longer grew. didn't From this, it was confirmed that aging was induced. Example 1-2. Removal of aged cells by LX-112

Cells treated with doxorubicin and the control group in the same manner as in Example 1-1 and cultured were treated with LX-112 (purchased by BOC) at concentrations of 0 μM, 1.1 μM, 3.3 μM, and 10 μM, respectively, and after 3 days, CellTiter- Cell viability and growth rate were measured using Glo Luminescent Cell Viability Assay (purchased by Promega) and are shown in Tables 2 and 3. At this time, N0 in Table 3 is calculated as 100% of the cell number before LX-112 treatment.

LX 0 μM 1.1 μM 3.3 μM 10 μM Survival (%) 100.2 (±4.0) 67.2 (±7.0) 27.0 (±4.0) 1.0 (±4.0)

LX N0 0 μM 1.1 μM 3.3 μM 10 μM Growth (%) 100.0 (±11.0) 253.0 (±4.0) 201.0 (±8.0) 161 (±7.0) 123.3 (±8.0)

As a result, as shown in Table 2, in cells where senescence was induced by treatment with doxorubicin, the higher the concentration of LX-112, the faster the cell survival rate decreased. Additionally, as shown in Table 3, there was no significant change in cell survival rate even when LX-112 was treated at various concentrations in cells in which senescence was not induced by treatment with the control group, and the cells grew normally. In addition to somatic cells, the death of senescent cells by LX-112 was confirmed to be about 50% in stem cells (umbilical cord-derived mesenchymal stem cells, purchased from ATCC) at a concentration of 10 μM. From this, it was confirmed that LX-112 can effectively remove senescent cells by inducing their death.

Example 2. Removal of senescent cells by LX-112 from senescent human cancer cells and tumors

Example 2-1. Induction of senescence in cancer cells

SAOS-2 (purchased by ATCC), a human-derived osteosarcoma cancer cell line, was cultured in the designated medium (DMEM, purchased by GenDEPOT; 10% FBS, purchased by GenDEPOT; 100 U/ml Penicillin & Streptomycin, purchased by Invitrogen). Then, senescence was induced in the same manner as in Example 1-1, and then the ratio of cellular senescence-related marker (SA-β-gal) was measured, and the results are shown in Table 4.

SA-β-gal
(%) - 5.0 (±6.0) DOX 66.0 (±5.0)

As a result, as shown in Table 4, the ratio of aging-related marker (SA-β-gal) in cells treated with doxorubicin (DOX) increased significantly compared to the control group (-), and the cells no longer grew. didn't From this, it was confirmed that aging was induced. Example 2-2. LX-112 eliminates aged cancer cells

The standard of care for various cancers, including osteosarcoma, is repeated chemotherapy. Accordingly, aging was induced in SAOS-2 in the same manner as in Example 2-1, and then (i) treated again with doxorubicin (DOX → DOX) or (ii) treated with LX-112 (5 μM) composition (DOX →LX-112). Then, after 3 days (3d) and 7 days (7d), cell viability was measured using CellTiter-Glo Luminescent Cell Viability Assay (purchased by Promega), and the results are shown in Tables 5 and 6.

- DOX→DOX 3d 7d Survival (%) 100.0 (±6.0) 72.0 (±7.0) 55.0 (±5.0)

- DOX→LX 3d 7d Survival (%) 100 (±3.0) 35.0 (±4.0) 14.0 (±5.0)

As a result, as shown in Tables 5 and 6, the combined and sequential treatment of doxorubicin and LX-112 was more effective than in the case of repeated treatment with doxorubicin (DOX → DOX) in SAOS-2, which lacks the intracellular anticancer genes Rb and p53. In one case (DOX→LX-112), it more effectively reduced the survival rate of cancer cells. From this, it was confirmed that the combined and sequential treatment with an anticancer drug that induces aging and LX-112, a composition for removing senescent cells, exhibited a more effective anticancer effect than in the case of repeated treatment with anticancer drugs through chemotherapy. Example 2-3. Suppression of aged tumors by LX-112 in mice

After 7-week-old male BALB/c nude mice (purchased by KRIBB Laboratory Animal Resource Center) were adapted to the experimental environment for one week, the mice were randomized into an experimental group and a control group. SAOS-2 (10 ⁶ cells), a human osteosarcoma cancer cell line cultured in the same manner as in Example 2-1, was mixed with PBS (purchased by Welgene) and then mixed with 2% isoflurane (purchased by Sigma). It was administered subcutaneously to anesthetized mice. When the size of the tumor reached about 50 mm ² , doxorubicin was mixed at a concentration of 10 mg/kg in a 1:1 solution of distilled water and PBS (pH 7.2) and administered intraperitoneally to induce tumor aging. After 10 days, LX-112 mixed with 0.5% CMC-Na solution (purchased by Sigma) at a concentration of 100 mg/kg was orally administered daily for 21 days, and the size of the tumor was measured. The control group was repeatedly administered a solution mixed with doxorubicin, and then the size of the tumor was measured. Table 7 shows the comparison results.

Tumor Diameter (mm) DOX→DOX 15.4 (±1.2) DOX→LX 6.2 (±4.8)

As a result, as shown in Table 7, when tumors that induced aging in mice were treated with LX-112 (DOX → LX), the size of the tumor increased further than when treated with doxorubicin repeatedly (DOX → DOX). effectively suppressed and reduced. From this, it was confirmed that treatment with LX-112, a composition for inducing senescence and removing senescent cells in the formed tumor, exhibits a more effective anticancer effect than when repeatedly treating anticancer drugs with chemotherapy in mice.

Example 3. Removal of senescent cells by LX-112 from senescent human cells

Example 3-1. Inducing natural aging of cells

Foreskin cells derived from humans were subcultured in designated media (Iscove's Modified Dulbecco's Medium, ATCC; 10% FBS, GenDEPOT; 100 U/ml Penicillin & Streptomycin, Invitrogen) until no longer growing. REP; replicative senescence) induced natural aging. As a control group (-), foreskin cells before natural aging were used. Afterwards, the ratio of cell aging-related marker (SA-β-gal) was measured in the same manner as in Example 1-1, and the results are shown in Table 8.

SA-β-gal
(%) - 7.2 (±2.0) REP 93.8 (±5.0)

As a result, as shown in Table 8, the ratio of aging-related marker (SA-β-gal) significantly increased in cells that were continuously subcultured (REP) until they no longer grew. From this, it was confirmed that aging was induced. Example 3-2. Removal of aged cells by LX-112

Natural aging of human-derived foreskin cells was induced in the same manner as in Example 3-1, then treated with LX-112 at concentrations of 0 μM, 1.1 μM, 3.3 μM, and 10 μM, and after 3 days, CellTiter-Glo Luminescent Cell Viability Using Assay (purchased by Promega), the cell survival rate was measured in Table 9, and the cell growth rate for cells in which senescence was not induced was measured and shown in Table 10. At this time, in Table 10, N0 is calculated as 100% of the cell number before LX-112 treatment.

LX 0 μM 1.1 μM 3.3 μM 10 μM Survival (%) 100.5 (±4.0) 59.0 (±6.0) 29.0 (±5.0) 1.0 (±9.0)

LX N0 0 μM 1.1 μM 3.3 μM 10 μM Growth (%) 100.0
(±6.0) 262.0
(±13.0) 206.0
(±15.0) 151.0
(±12.0) 122.7
(±16.0)

As a result, as shown in Tables 9 and 10, the higher the concentration of LX-112 in cells in which senescence was induced, the cell survival rate decreased rapidly, and in cells in which senescence was not induced, treatment with LX-112 at various concentrations There was no change in cell survival rate, and cells grew normally. From this, it was confirmed that LX-112 effectively removes senescent cells by inducing their death.

Example 4. Reduction of the amount of aging-related markers expressed in senescent cells by LX-112

In the same manner as Example 3, natural aging of human foreskin cells was induced and treated with LX-112 at a concentration of 3 μM, and then the aging-related markers p16 and p21 were analyzed by qRT-PCR (quantitative real-time PCR). The amount of mRNA was measured. As a control group, foreskin cells before senescence induction were used. Specifically, cells were collected and treated with TRIzol Reagent (purchased by Thermo Fisher Scientific) to extract total RNA. The concentration of extracted total RNA was quantified using Nanodrop (purchased by Micro Digital). Genomic DNA was removed from the extracted total RNA using SuperScript IV VILO Master Mix with ezDNase Enzyme (purchased by Thermo Fisher Scientific), and cDNA was prepared. Using the synthesized cDNA, using the housekeeping gene GAPDH as a standard, qRT-PCR for p16 and p21 was performed using SFCgreen I qPCR Master Mix (purchased by SFC Probe), and CFX Connect Real-Time PCR Detection Analysis was performed using System (purchased by Bio Rad). Table 11 shows the results showing the mRNA amounts of aging-related markers p16 and p21 before and after inducing aging, and Table 12 shows the mRNA amounts of p16 and p21, which are aging-related markers, under conditions in which aging was induced and LX-112 was not treated and treated. Table 12 shows the mRNA amounts of p16 and p21 after senescence was induced, each calculated as 100%, and compared with the mRNA amounts of p16 and p21 after treatment with LX-112. The primers used were synthesized through bionics, and the sequences are shown in Table 13 below.

mRNA (Fold) p16 p21 - 1.0 (±0.1) 1.0 (±0.3) REP 5.0 (±0.5) 4.0 (±0.2)

mRNA (%) p16 p21 - 100.0 (±4.0) 100.0 (±6.0) LX 43.0 (±4.0) 39.0 (±7.0)

Gene Primer 1 sequence number Primer 2 sequence number GAPDH GGAAGGGCTCATGACCACAG One ACAGTCTTTCTGGGTGGCAGTG 2 p16 TGAGCTTTGGTTCTGCCATT 3 AGCTGTCGACTTCATGACAAG 4 p21 GAGACTAAGGCAGAAGATGTAGAG 5 GCAGACCAGCATGACAGAT 6

As a result, as shown in Table 11, when senescence was induced in cells, the mRNA amounts of p16 and p21 rapidly increased. Additionally, as shown in Table 12, when senescence-induced foreskin cells were treated with LX-112, the mRNA amounts of p16 and p21 decreased. From this, it was confirmed that LX-112 effectively reduces the amount of p16 and p21, aging-related markers that increase with cellular aging.

Example 5. Reduction of the amount of age-related inflammation and fibrosis secretion factors (SASP) expressed and secreted in senescent cells by LX-112

In the same manner as in Example 3, natural aging of human foreskin cells was induced and treated with LX-112 at a concentration of 3 μM, and then an immunoassay (ProcartaPlex Immunoassay, purchased from Thermo Fisher Scientific) was performed using the obtained culture medium. The expression levels of aging-related inflammation and fibrosis secretion factors (SASP), IL1α, IL1β, IL6, IL10, MCP1, PAI1, VCAM1, MMP1, MMP2, MMP3, MMP12, and TGFβ, were measured. Table 14 shows the age-related inflammation and fibrosis secretion factor (SASP) before (-) and after (REP) inducing senescence, and Table 15 shows the age-related inflammation and fibrosis secretion factor (SASP) in the conditions without and with LX-112 treatment after inducing senescence. ) This is the result showing the amount. Table 15 calculates the amount of each factor after inducing aging as 100% and compares it with the amount of each factor after treatment with LX-112.

Protein (Fold) IL1α IL1β IL6 IL10 MCP1 PAI1 - 1.0
(±0.3) 1.0
(±0.2) 1.0
(±0.1) 1.0
(±0.1) 1.0
(±0.2) 1.0
(±0.1) REP 14.0
(±6.0) 11.0
(±3.0) 64.0
(±6.0) 16.0
(±4.0) 25.0
(±5.0) 69.0
(±9.0) Protein (Fold) VCAM1 MMP1 MMP2 MMP3 MMP12 TGFβ - 1.0
(±0.2) 1.0
(±0.1) 1.0
(±0.1) 1.0
(±0.2) 1.0
(±0.2) 1.0
(±0.1) REP 32.0
(±4.0) 3.6
(±1.0) 18.0
(±8.0) 48.2
(±8.0) 13.0
(±4.0) 151.0
(±9.0)

Protein (%) IL1α IL1β IL6 IL10 MCP1 PAI1 - 100.0
(±4.0) 100.0
(±3.0) 100.0
(±6.0) 100.0
(±4.0) 100.0
(±3.0) 100.0
(±7.0) LX 57.0
(±5.0) 46.0
(±6.0) 41.0
(±4.0) 57.0
(±7.0) 38.0
(±8.0) 41.0
(±4.0) Protein (%) VCAM1 MMP1 MMP2 MMP3 MMP12 TGFβ - 100.0
(±5.0) 100.0
(±2.0) 100.0
(±8.0) 100.0
(±4.0) 100.0
(±3.0) 100.0
(±4.0) LX 45.0
(±5.0) 43.0
(±3.0) 39.0
(±9.0) 38.0
(±8.0) 41.0
(±6.0) 49.0
(±9.0)

As a result, as shown in Table 14, when senescence was induced in cells, the expression levels of IL1α, IL1β, IL6, IL10, MCP1, PAI1, VCAM1, MMP1, MMP2, MMP3, MMP12, and TGFβ rapidly increased. Additionally, as shown in Table 15, when senescence-induced foreskin cells were treated with LX-112, the amount of each factor decreased. From this, it was confirmed that LX-112 effectively reduces the amount of various age-related inflammation and fibrosis secretion factors (SASP) that increase with cell aging.

Example 6. Induction of fibrosis by senescence-related secreted factor (SASP) and inhibition of fibrosis by LX-112 in mice

Example 6-1. Induction of fibrosis in normal cells by aging-related secretion factor (SASP) expressed and secreted by senescent cells.

Culture medium (REP) of human foreskin cells in which natural aging was induced was obtained in the same manner as in Example 5, and the foreskin cells that were not induced to age and were growing normally were treated for 1 day. Next, the mRNA amounts of age-related fibrosis factors ACTA2, COL1A1, COL1A2, FN1, and SDF1 were measured by qRT-PCR in the same manner as in Example 4, and are shown in Table 16. The control group (-) used foreskin cells that were not treated with the culture medium of senescent cells. The primers used were synthesized through bionics, and the sequences are shown in Table 17 below.

mRNA (Fold) ACTA2 COL1A1 COL1A2 FN1 SDF1 - 1.0 (±0.3) 1.0 (±0.2) 1.0 (±0.1) 1.0 (±0.2) 1.0 (±0.2) REP 3.3 (±0.5) 4.5 (±0.6) 3.9 (±1.0) 4.1 (±0.4) 0.2 (±0.4)

Gene Primer 1 sequence number Primer 2 sequence number ACTA2 GTGAAGAAGAGAGACAGCACTG 7 CCCATTCCCACCATCACC 8 COL1A1 AAGGGACACAGAGGTTTCAGTGG 9 CAGCACCAGTAGCACCATCATTTC 10 COL1A2 CTTGCAGTAACCTTATGCCTAGCA 11 CCCATCTAACCTCTCTACCCAGTCT 12 FN1 TGTCAGTCAAAGCAAGCCCG 13 TTAGGACGCTCATAAGTGTCACCC 14 SDF1 TGCCAGAGCCAACGTCAAG 39 CAGCCGGGGCTACAATCTGAA 40

As a result, as shown in Table 16, it was confirmed that the factors regulated by aging-related secretion factor (SASP) include ACTA2, COL1A1, COL1A2, FN1, and SDF1, which are regulators of fibrosis and pigmentation. . From this, it was confirmed that the amount of factors that induce fibrosis increases with aging. It was confirmed that the amount of SDF1, which suppresses pigmentation, decreases with aging. Example 6-2. Inhibition of fibrosis by LX-112 in mice

After 8-week-old male C57BL6J mice (purchased by KRIBB Laboratory Animal Resource Center) were adapted to the experimental environment for 1 week, the mice were randomized into an experimental group and a control group. To induce peritoneal fibrosis, 0.1% chlorhexidine gluconate (CHG, purchased by Sigma) was dissolved in 15% ethanol phosphate buffer solution and administered intraperitoneally at a concentration of 10 ml/kg every 2 days for 20 days. From day 9, LX-112 was mixed with 0.5% CMC-Na solution (purchased by Sigma) and administered intraperitoneally at a concentration of 70 mg/kg every day. The control group (-) was administered only the solution in the same volume. On the 20th day, paraffin sections of peritoneal tissue prepared by fixation in 4% paraformaldehyde solution were stained with H/E (Hematoxylin/Eosin) and trichrome, and the peritoneal mesothelial cell layer was examined under a light microscope. The thickness was measured. Table 18 shows the results quantitatively showing the thickness of the peritoneal mesothelial cell layer in mice with peritoneal fibrosis induced by chlorhexidine gluconate (CHG) in conditions without and with LX-112 treatment.

Submesothelial Thickness
(μm) - - 10.0 (±1.1) CHG - 69.1 (±6.4) LX 43.1 (±10.4)

As a result, as shown in Table 18, the thickness of the peritoneum was increased by chlorhexidine gluconate, and LX-112 suppressed and reduced the increased thickness. From this, it was confirmed that LX-112 effectively inhibits and treats fibrosis.

Example 7. Elimination of senescent cells and related anti-aging activity of LX-112 in naturally induced aging mice

Example 7-1. Establishment of a naturally induced aging mouse model

After 22-month-old female C57BL6J mice (purchased by KRIBB Laboratory Animal Resource Center) were adapted to the experimental environment for one week, the aged mice were randomized into an experimental group and a control group. 8-week-old mice that were not aged were used as controls. LX-112 was mixed with 0.5% CMC-Na solution (purchased by Sigma) and administered orally at a concentration of 70 mg/kg daily for 21 days. The control group was administered only the solution in the same volume. After completion of drug administration, the method described in each example below was performed.

Example 7-2. Decreased amount of aging-related marker (p16) in LX-112

The mouse was sacrificed, RNA was extracted from the organ tissue of each region, and qRT-PCR was performed using the synthesized cDNA. Specifically, in order to maintain the quality of RNA during the RNA extraction process, the tissue from each collected area was immediately cut into 30 mg to 50 mg pieces, placed in RNA stabilization solution (purchased by Thermo Fisher Scientific), and absorbed into the tissue for 1 day. Afterwards, it was stored at -80°C until use. The collected tissue was placed together with the lysate in a tube containing beads for tissue homogenization (purchased by MP Bio) and homogenized using tissue homogenization equipment (purchased by MP Bio). Total RNA was extracted from the homogenized tissues. The extraction method used an RNA extraction kit (PureLink RNA Mini Kit, purchased from Thermo Fisher Scientific) for soft tissues including liver, kidney, lung, and brain, and for muscle. , hard tissue including skin, fat, etc. used TRIzol Reagent (purchased by Thermo Fisher Scientific). The concentration of extracted total RNA was quantified using Nanodrop (purchased by Micro Digital) or SpectraMax iD3 Multi-Mode Microplate Reader (purchased by Molecular Device). Genomic DNA was removed from the extracted total RNA using a cDNA synthesis kit (purchased by Thermo Fisher Scientific), and cDNA was prepared. Using the synthesized cDNA, qRT-PCR was performed for each gene using the housekeeping gene β-actin as a standard, using Taqman Fast Abvanced Master Mix (purchased by Applied Biosystems), SFCgreen I qPCR Master Mix (purchased by SFC Probe), or AmfiSure. It was performed using qGreen qPCR Master Mix (purchased by GenDEPOT) and analyzed using the CFX Connect Real-Time PCR Detection System (purchased by Bio Rad). Table 19 shows the results showing the mRNA amount of p16, an aging marker, before (Young) and after (Old) inducing aging, and Table 20 shows the mRNA amount of p16, an aging marker, under conditions in which aging was induced and LX-112 was not treated and treated. am. Table 20 calculates the amount of p16 mRNA after inducing senescence as 100% and compares it with the amount of p16 mRNA after treatment with LX-112. The primers used were synthesized through bionics, except that p16 and IL6 used TaqMan primers (purchased by Thermo Fisher Scientific). The sequences of the primers used are shown in Table 21 below.

p16 mRNA
(Fold) Liver Kidney Lung Brain eWAT Muscle Skin Young 1.0
(±0.7) 1.0
(±0.6) 1.0
(±0.1) 1.0
(±0.3) 1.0
(±0.4) 1.0
(±0.5) 1.0
(±0.0) Old 53.9
(±10.8) 18.6
(±2.4) 3.3
(±1.0) 9.0
(±3.2) 3.5
(±1.1) 11.0
(±9.8) 7.3
(±2.5)

p16 mRNA
(%) Liver Kidney Lung Brain eWAT Muscle Skin - 100.0
(±4.8) 100.0
(±3.8) 100.0
(±5.7) 100.0
(±3.8) 100.0
(±9.7) 100.0
(±8.4) 100.0
(±1.5) LX 62.0
(±3.5) 52.0
(±7.9) 69.0
(±1.0) 89.0
(±5.0) 79.0
(±2.0) 76.0
(±3.1) 74.0
(±6.7)

Gene Primer 1 sequence number Primer 2 sequence number β-actin CATCCGTAAAGACCTCTATGCCAAC 15 ATGGAGCCACCGATCCACA 16 p21 GCAGATCCACAGCGATATCCA 17 AACAGGTCGGACATCACCAG 18 IL1α TCCATAACCCATGATCTGGAA 19 TTGGTTGAGGGAATCATTCAT 20 IL1β GTATGGGCTTGGACTGTTTC 21 GCTGTCTGCTCATTCACG 22 CXCL1 ACCGAAGTCATAGCCACACTC 23 CTCCGTTACTTGGGGACACC 24 CXCL10 GCTGCCGTCATTTTCTGC 25 TCTCACTGGCCCCGTCATC 26 MMP3 GTTGGAGAACATGGAGACTTTGT 27 CAAGTTCATGAGCAGCAACCA 28 MMP12 TGCACTCTGCTGAAAGGAGTCT 29 GTCATTGGAATTCTGTCCTTTCCA 30 MMP13 AAGGGGATAACAGCCACTACAA 31 ACCAACATAAAAATTAAGCCAAATG 32 MCP1 GCATCTGCCCTAAGGTCTTCA 33 GTGGAAAGGTAGTGGATGCATT 34 COL1A1 CAGTCGATTCACCTACAGCAC 35 TGGAGGGAGTTTACACG 36 Agtr1a AAGGGCCATTTGCTTTTCT 37 AACTCACAGCAACCCTCCAA 38

As a result, as shown in Table 19, when a mouse ages, liver, kidney, lung, brain, epididymal fat (eWAT), muscle, skin, etc. The amount of p16 mRNA rapidly increased in various organ tissues. Additionally, as shown in Table 20, when mice with induced aging were treated with LX-112, the amount of p16 mRNA decreased in various organ tissues. From this, it was confirmed that LX-112 effectively reduces the amount of p16, an aging marker, that increases in various organ tissues of mice by removing senescent cells that increase as mice age. Example 7-3. Decreased amount of aging-related marker (SA-β-gal) in LX-112

The mouse was sacrificed, tissue sections were prepared from the organ tissues of each region, and stained for an aging marker (SA-β-gal) in the same manner as in Example 1-1. Specifically, fat was removed from the tissues of each area collected from mice and fixed in 4% formalin at room temperature for more than 24 hours. For cryopreservation, the cells were placed in 30% sucrose for more than 8 hours and then rapidly frozen in cryoprotectant OCT medium for cryosectioning. The rapidly frozen tissue was commissioned to Histoa, a company specializing in histopathology services, and frozen sections were produced. The prepared frozen tissue sections were cut to a thickness of 8 μm, sectioned on a slide cover, dried at room temperature for 30 minutes, and then stored at -80°C until use. After staining with an aging marker (SA-β-gal), the images were taken through an optical microscope, and the area of the stained area was calculated using the Image J program and quantified by normalizing it based on the total area. Table 22 shows staining of SA-β-gal, an aging marker, before (Young) and after (Old) inducing aging, and Table 23 shows staining of SA-β-gal, an aging marker, under conditions in which aging was induced and LX-112 was not treated and treated. This is the result showing the area. Figure 2 is a photo result of staining of adipose tissue.

SA-β-gal (Fold) Liver Kidney Lung Skin eWAT Young 1.0 (±0.5) 1.0 (±0.6) 1.0 (±0.2) 1.0 (±0.3) 1.0 (±1.0) Old 4.5 (±0.2) 3.0 (±0.4) 1.1 (±0.5) 2.8 (±0.3) 45.9 (±9.3)

SA-β-gal
(%) Liver Kidney Lung Skin eWAT - 100.0 (±9.0) 100.0 (±8.0) 100.0 (±6.0) 100.0 (±9.0) 100.0 (±7.0) LX 58.0 (±7.0) 55.0 (±17.0) 67.0 (±6.0) 68.0 (±9.0) 38.0 (±7.0)

As a result, as shown in Table 22, when mice age, SA-β-gal is stored in various organ tissues such as liver, kidney, lung, skin, and epididymal fat (eWAT). The staining area increased. In addition, as shown in Table 23 and FIG. 2, when mice with induced aging were treated with LX-112, the staining area of SA-β-gal decreased in various organ tissues. From this, it was confirmed that LX-112 effectively reduces the amount of SA-β-gal, an aging marker, that increases in various organ tissues of mice by removing senescent cells that increase as mice age. Example 7-4. LX-112 reduces the amount of aging-related degenerative marker (lipofuscin)

The mouse was sacrificed and tissue sections were prepared from muscle tissue in the same manner as in Example 7-3, and the degenerative abnormal complex of proteins and lipids produced in the lysosomes of cells with aging, which are age-related degenerative markers, were extracted. The amount of lipofuscin was stained and photographed using Sudan Black B (purchased by Sigma), and then the area of the stained area was calculated using the Image J program and quantified by normalizing it based on the total area. Table 24 shows the results showing the staining area of lipofuscin, an age-related degenerative marker, before and after inducing aging, and Table 25 shows the staining area of lipofuscin, an age-related degenerative marker, under conditions in which aging was induced and LX-112 was not treated and treated.

Muscle Lipofuscin
(Fold) Young 1.0 (±0.2) Old 1.5 (±0.4)

Muscle Lipofuscin
(%) - 100.0 (±8.0) LX 71.0 (±6.0)

As a result, as shown in Table 24, as mice aged, the lipofuscin staining area increased in muscle tissue. Additionally, as shown in Table 25, when senescence-induced mice were treated with LX-112, the lipofuscin staining area decreased in various tissues. From this, it was confirmed that LX-112 effectively reduces the amount of lipofuscin, an age-related degenerative marker, that increases in muscle tissue of mice by removing senescent cells that increase as mice age. Example 7-5. LX-112 reduces the amount of age-related inflammatory secretory factor (SASP) (IL6)

Serum collected from mice was stored at -80°C until use. The amounts of IL6 and MCP1, which are age-related inflammatory secretory factors (SASPs), were measured in serum using an enzyme-linked immunosorbent assay (ELISA Kit, purchased from R&D Systems). Table 26 shows the results showing the amounts of IL6 and MCP1, which are age-related inflammatory secretory factors (SASP), before and after inducing aging, and Table 27 shows the amounts of IL6 and MCP1, which are age-related inflammatory secretory factors (SASP), under conditions in which aging was induced and LX-112 was not treated and treated. .

Fold IL6 MCP1 Young 1.0 (±0.3) 1.0 (±0.2) Old 4.2 (±0.3) 2.8 (±1.1)

% IL6 MCP1 - 100.0 (±29.0) 100.0 (±27.1) LX 63.0 (±12.8) 58.0 (±9.8)

As a result, as shown in Table 26, when mice aged, the amounts of IL6 and MCP1 increased in the serum. Additionally, as shown in Table 27, when senescence-induced mice were treated with LX-112, the amounts of IL6 and MCP1 in the serum decreased. From this, it was confirmed that LX-112 effectively reduces the amount of IL6 and MCP1, which are age-related inflammatory secretory factors (SASP), that increase in the serum of mice by removing senescent cells that increase as mice age. Example 7-6. LX-112 reduces the amount of age-related inflammation and fibrosis secretion factors (SASP)

Using cDNA synthesized in the same manner as in Example 7-2, qRT-PCR was performed and analyzed for MMP3, IL1β, MMP12, IL6, and MMP13, which are age-related inflammation and fibrosis secretion factors (SASP). Table 28 shows MMP3, IL1β, MMP12, age-related inflammation and fibrosis secretion factor (SASP), before and after inducing senescence, and Table 29 shows MMP3, IL1β, MMP12, MMP3, IL1β, and This result shows the mRNA amount of IL6 and MMP13. Table 29 calculates the mRNA amount of each factor after senescence was induced as 100% and compared it with the mRNA amount of each factor after treatment with LX-112.

mRNA (Fold) Liver Kidney Lung Muscle Skin MMP3 IL1β MMP12 IL6 MMP3 MMP12 MMP13 Young 1.0
(±0.2) 1.0
(±0.4) 1.0
(±0.5) 1.0
(±0.1) 1.0
(±0.3) 1.0
(±0.4) 1.0
(±0.1) Old 2.2
(±0.2) 2.0
(±0.2) 3.6
(±1.9) 2.9
(±1.3) 1.5
(±0.2) 10.0
(±6.3) 1.3
(±0.6)

mRNA
(%) Liver Kidney Lung Muscle Skin MMP3 IL1β MMP12 IL6 MMP3 MMP12 MMP13 - 100.0
(±5.1) 100.0
(±9.9) 100.0
(±15.0) 100.0
(±12.0) 100.0
(±38.0) 100.0
(±8.4) 100.0
(±49.0) LX 75.0
(±13.0) 67.0
(±3.9) 81.0
(±13.0) 65.0
(±21.0) 92.0
(±6.0) 78.0
(±14.0) 95.0
(±2.0)

As a result, as shown in Table 28, when mice age, MMP3, IL1β, MMP12, The amount of mRNA such as IL6 and MMP13 increased. Additionally, as shown in Table 29, when senescence-induced mice were treated with LX-112, the mRNA amount of each age-related inflammation and fibrosis secretion factor (SASP) was decreased in various organ tissues. From this, LX-112 eliminates senescent cells that increase as the mouse ages, thereby increasing age-related inflammation and fibrosis secretion factors (SASP) such as MMP3, IL1β, MMP12, IL6, and MMP13 in various organ tissues of the mouse. It was confirmed that the amount was effectively reduced. Example 7-7. LX-112 improves aging-related frailty (activity, endurance, muscle strength and grip strength, sarcopenia, lipoatrophy, etc.)

To analyze aging-related senescence in mice, activity evaluation (open field test), endurance evaluation (wire hanging test), and muscle strength and grip strength test (grip strength test) were performed. Specifically, in the open field test, the mouse was placed in a white square box and the trajectory it moved for a certain period of time was measured. The time spent in the center was quantified by normalizing it based on the total distance moved. In the wire hanging test, the time the mouse could endure hanging on a wire was measured. In the muscle strength and grip test (grip strength test), the mouse was held by its tail and made to hold a grip strength measuring device (purchased by Jeongdo B&P) with its front paws, then pulled slightly until it missed, and the force applied to the measuring device just before missing was measured. Table 30 shows the quantitative data on activity evaluation, endurance evaluation, muscle strength, and grip strength evaluation, which are criteria for determining senescence, before and after inducing aging, and under conditions in which aging was induced and LX-112 was not treated and LX-112 was treated. This is the result expressed as . Table 31 calculates the amount of each evaluation after aging was induced as 100% and compared it with the amount of each evaluation after treatment with LX-112.

Open Field Test
Time in Center (%) Wire Hanging Test
Time (Sec) Grip Sterenght Test
Strength (N) Young 13.1 (±3.93) 64.0 (±10.0) 1.0 (±0.05) Old 5.2 (±1.51) 45.0 (±7.0) 0.8 (±0.12)

Open Field Test
Time in Center (%) Wire Hanging Test
Time (Sec) Grip Sterenght Test
Strength (N) Muscle Mass (g) - 100.0 (±18.0) 100.0 (±15.0) 100.0 (±14.0) 0.5 (±0.0) LX 121.0 (±9.0) 119.0 (±10.0) 132.0 (±11.0) 0.7 (±0.1)

As a result, as shown in Table 30, the activity, endurance, muscle strength, and grip strength of the mouse decreased as aging was induced. Additionally, as shown in Table 31, treatment of mice with induced aging with LX-112 increased activity, endurance, muscle strength, and grip strength. In addition, in muscle tissue, the aging-related markers p16 (Tables 19 and 20) and SA-β-gal (Tables 22 and 23), the aging-related degenerative marker lipofuscin (Tables 24 and 25), and the aging-related inflammatory secretion factor. (SASP), IL6 (Tables 26, 27 and 29), etc. were also decreased by treatment with LX-112. From this, it was confirmed that LX-112 improves aging in mice by removing senescent cells that increase as mice age. Consistent with this improvement effect, it was confirmed from the results in Table 31 that LX-112 increased the weight of muscle tissue and overall fat tissue of the quadriceps femoris in aged mice. Examples 7-8. LX-112 improves kidney damage with aging

The amounts of urea and creatinine, markers of kidney damage, in serum collected from mice were measured by spectrophotometry using a kit (purchased by BioAssay Systems and Sigma). Additionally, the amount of Agtr1a mRNA, a kidney damage marker, was measured by qRT-PCR as described in Example 7-2. Table 32 shows the quantitative results of urea, creatinine, and Agtr1a mRNA, which are kidney damage markers, under conditions that induced aging and were not treated with LX-112 and treated with LX-112, respectively. The amount of each factor after inducing aging was calculated as 100% and compared with the amount of each factor after treatment with LX-112.

Urea (%) Creatine (%) Agtr1a mRNA (%) - 100.0 (±4.0) 100.0 (±11.0) 100.0 (±9.0) LX 69.0 (±6.0) 87.0 (±7.8) 64.0 (±7.0)

As a result, as shown in Table 32, when mice with induced aging were treated with LX-112, the amounts of urea, creatinine, and Agtr1a mRNA decreased. From this, it was confirmed that LX-112 improves kidney damage and dysfunction in mice by eliminating senescent cells that increase as mice age. Example 7-9. Improvement of skin aging caused by aging with LX-112

Frozen sections of skin tissue were stained with H/E (Hematoxylin/Eosin), and the thickness of the dermis was observed under an optical microscope. Additionally, the mRNA amount of collagen (COL1A1) was measured by qRT-PCR as described in Example 7-2. Table 33 quantitatively shows the degree of skin aging according to the thickness of the skin (dermal) and the amount of collagen (COL1A1) mRNA in mice in which aging was naturally induced, under conditions without and with LX-112 treatment.

Dermal Thickness (μm) Collagen mRNA (Fold) - 221.0 (±22.1) 1.0 (±0.2) LX 265.0 (±17.2) 2.3 (±0.4)

As a result, as shown in Table 33, when mice with induced aging were treated with LX-112, skin thickness and collagen amount increased. In addition, in skin tissue, aging-related markers p16 (Tables 19 and 20) and SA-β-gal (Tables 22 and 23), age-related inflammation and fibrosis secretion factor (SASP) and collagen decomposing enzymes MMP1, MMP12, MMP13 (Table 29) was also decreased by treatment with LX-112. From this, it was confirmed that LX-112 improves skin aging in mice by removing senescent cells that increase as the mouse ages.

Example 8. Removal of senescent cells and related anti-aging activity of LX-112 in aging mice induced by anticancer drugs

Example 8-1. Establishment of a mouse model of aging induced by anticancer drugs

After adapting 8-week-old male C57BL6J mice (purchased by KRIBB Laboratory Animal Resource Center) to the experimental environment for 1 week, doxorubicin, an anticancer drug, was administered intraperitoneally at a concentration of 10 mg/kg, and mice that were induced to age for more than 10 days were assigned to the experimental group. and randomized to control group. LX-112 was mixed with 0.5% CMC-Na solution (purchased by Sigma) and administered orally at a concentration of 70 mg/kg daily for 21 days. The control group was administered only the solution in the same volume. After completion of drug administration, the method described in each example below was performed.

Example 8-2. Decreased amount of aging-related marker (p16) in LX-112

Mice were sacrificed in the same manner as in Example 7-2, RNA was extracted from organ tissues of each region, and qRT-PCR was performed and analyzed using the synthesized cDNA. Table 34 shows the aging-related markers p16 and p21 before and after inducing aging by treatment with anticancer drugs, and Table 35 shows aging-related markers p16 and p21 under conditions in which aging was induced by treatment with anticancer drugs and without and with LX-112 treatment. This is a result showing the amount of mRNA. Table 35 shows the mRNA amounts of p16 and p21 after senescence was induced by treatment with anticancer drugs, calculated as 100%, and compared with the mRNA amounts of p16 and p21 after treatment with LX-112.

Liver Kidney eWAT p16 mRNA
(Fold) - 1.0 (±0.3) 1.0 (±0.3) 1.0 (±0.3) DOX 2.6 (±0.8) 1.3 (±0.3) 3.2 (±1.2) p21 mRNA
(Fold) - 1.0 (±0.6) 1.0 (±0.4) 1.0 (±.0.2) DOX 3.8 (±2.3) 2.6 (±2.1) 3.9 (±3.0)

Liver Kidney eWAT p16 mRNA
(%) - 100.0 (±24.0) 100.0 (±9.89) 100.0 (±9.8) LX 70.0 (±8.3) 75.0 (±14.81) 72.0 (±18.75) p21 mRNA
(%) - 100.0 (±18.58) 100.0 (±25.8) 100.0 (±18.51) LX 78 (±11.5) 83.8 (±9.5) 68.4 (±15.1)

As a result, as shown in Table 34, when mice were aged by treatment with anticancer drugs, the mRNA amounts of p16 and p21 increased in various organ tissues such as liver, kidney, and epididymal fat (eWAT). Additionally, as shown in Table 35, when mice whose aging was induced by treatment with anticancer drugs were treated with LX-112, the mRNA amounts of p16 and p21 were decreased in various organ tissues. From this, it was confirmed that LX-112 effectively reduces the amount of aging-related markers p16 and p21 that increase in various organ tissues of mice by removing senescent cells that increase in mice due to treatment with anticancer drugs. Example 8-3. Reduction of the amount of the aging-related marker (SA-β-gal) in LX-112. The mice described in Example 7-3 were sacrificed and tissue sections were prepared from the organ tissues of each region to remove the aging-related marker, SA-β-gal. β-gal was stained. Table 36 shows SA-β, an aging marker, before and after inducing aging by treatment with anticancer drugs, and Table 37 shows SA-β, an aging marker, under conditions in which aging was induced by treatment with anticancer drugs and without and with LX-112 treatment. This is the result showing the staining area of -gal. Figure 3 is a photo result of staining of adipose tissue.

SA-β-gal (Fold) Liver Kidney Lung eWAT - 1.0 (±0.6) 1.0 (±0.9) 1.0 (±0.2) 1.0 (±0.1) DOX 3.7 (±0.9) 4.1 (±1.5) 1.3 (±0.4) 4.2 (±0.9)

SA-β-gal (%) Liver Kidney Lung eWAT - 100.0 (±7.2) 100.0 (±10.2) 100.0 (±6.2) 100.0 (±8.4) LX 67.4 (±9.9) 69.2 (±10.2) 64.2 (±9.2) 51.4 (±13.4)

As a result, as shown in Table 36, when mice are aged by treatment with anticancer drugs, SA-β-gal is lost in various organ tissues such as liver, kidney, lung, and epididymal fat (eWAT). The staining area increased. Additionally, as shown in Table 37 and Figure 3, when mice whose aging was induced by treatment with anticancer drugs were treated with LX-112, the staining area of SA-β-gal decreased in various organ tissues. From this, it can be seen that LX-112 effectively reduces the amount of SA-β-gal, an aging marker, that increases in various organ tissues of mice by removing senescent cells that increase with the aging of mice by treatment with anticancer drugs. Confirmed. Example 8-4. LX-112 reduces the amount of aging-related degenerative marker (lipofuscin)

Mice were sacrificed in the same manner as in Example 7-4, and tissue sections were prepared from muscle tissue and stained with lipofuscin, an age-related degenerative marker. Table 38 shows lipofuscin, an age-related degenerative marker, before and after inducing aging by treatment with anticancer drugs, and Table 39 shows lipofuscin, an age-related degenerative marker, under conditions in which aging was induced by treatment with anticancer drugs and without and with LX-112 treatment. This is the result showing the staining area.

Muscle Lipofuscin
(Fold) - 1.0 (±0.2) DOX 2.2 (±0.5)

Muscle Lipofuscin
(%) - 100.0 (±19.3) LX 77.1 (±8.0)

As a result, as shown in Table 38, when mice aged by treatment with anticancer drugs, the area of lipofuscin staining in muscle tissue increased. Additionally, as shown in Table 39, when mice whose aging was induced by treatment with anticancer drugs were treated with LX-112, the area of lipofuscin staining in muscle tissue decreased. From this, it was confirmed that LX-112 effectively reduces the amount of lipofuscin, an age-related degenerative marker, that increases in muscle tissue of mice by removing senescent cells that increase as mice age through treatment with anticancer drugs. Example 8-5. LX-112 reduces the amount of age-related inflammatory secretory factor (SASP) (IL6)

The amount of IL6, an age-related inflammatory secretory factor (SASP), was measured in serum collected from mice in the same manner as in Example 7-5 using an enzyme-linked immunosorbent assay (ELISA Kit, purchased from R&D Systems). Table 40 shows the aging-related inflammatory secretion factor (SASP) before and after inducing aging by treatment with anticancer drugs, and Table 41 shows aging-related inflammation secretion factor (SASP) before and after inducing aging by treatment with anticancer drugs and under conditions without and with LX-112 treatment. This is a result showing the amount of IL6.

IL6
(Fold) - 1.0 (±1.1) DOX 11.7 (±0.5)

IL6
(%) - 100.0 (±38.4) LX 69.8 (±12.2)

As a result, as shown in Table 40, when mice aged by treatment with anticancer drugs, the amount of IL6 in the serum increased. Additionally, as shown in Table 41, when mice whose aging was induced by treatment with anticancer drugs were treated with LX-112, the amount of IL6 in the serum decreased. From this, it was confirmed that LX-112 effectively reduces the amount of IL6, an age-related inflammatory secretory factor (SASP), that increases in the serum by removing senescent cells that increase with aging in mice by treatment with anticancer drugs. Example 8-6. LX-112 reduces the amount of age-related inflammation and fibrosis secretion factors (SASP)

Using cDNA synthesized in the same manner as in Example 7-6, qRT-PCR was performed and analyzed for age-related inflammation and fibrosis secretion factors (SASP) such as IL6, CXCL1, CXCL10, IL1β, etc. Table 42 shows aging-related inflammation and fibrosis secretion factors before and after inducing aging by treatment with anticancer drugs, and Table 43 shows aging-related inflammation and fibrosis secretion factors under conditions in which aging was induced by treatment with anticancer drugs and without and with LX-112 treatment. This result shows the mRNA amount of IL6, CXCL1, CXCL10, and IL1β (SASP). Table 43 calculates the mRNA amount of each factor after aging was induced by treatment with an anticancer drug as 100% and compared it with the mRNA amount of each factor after treatment with LX-112.

mRNA (Fold) IL6 CXCL1 CXCL10 IL1β Liver - 1.0 (±0.5) 1.0 (±0.6) 1.0 (±0.3) 1.0 (±0.5) DOX 1.8 (±0.3) 3.0 (±0.9) 2.6 (±0.9) 1.6 (±0.3) eWAT - 1.0 (±0.5) 1.0 (±0.2) 1.0 (±0.4) 1.0 (±0.5) DOX 3.2 (±3.3) 1.3 (±0.4) 3.0 (±1.2) 1.6 (±2.2)

mRNA (%) IL6 CXCL1 CXCL10 IL1β Liver - 100.0 (±35.4) 100.0 (±38.5) 100.0 (±13.6) 100.0 (±28.4) LX 42.0 (±23.5) 68.0 (±21.1) 84.0 (±11.5) 42.0 (±35.2) eWAT - 100.0 (±38.4) 100.0 (±8.5) 100.0 (±38.7) 100.0 (±78.7) LX 48.6 (±12.5) 94.8 (±1.2) 74.0 (±11.0) 38.0 (±37.0)

As a result, as shown in Table 42, when mice aged by treatment with anticancer drugs, the mRNA amounts of IL6, CXCL1, CXCL10, and IL1β increased in organ tissues such as liver and epididymal fat (eWAT). In addition, as shown in Table 43, when mice whose aging was induced by treatment with anticancer drugs were treated with LX-112, the mRNA amount of each age-related inflammation and fibrosis secretion factor (SASP) was decreased in various organ tissues. From this, LX-112 removes senescent cells that increase as mice age due to treatment with anticancer drugs, thereby increasing age-related inflammation and fibrosis secretion factors (SASP), such as IL6, CXCL1, and CXCL1, that increase in various organ tissues of mice. , it was confirmed that the amount of IL1β was effectively reduced. Example 8-7. Improved fatigue, weakness, and weight loss due to anticancer drug LX-112

Activity evaluation (open field test), endurance evaluation (wire hanging test), muscle strength and grip strength evaluation (grip strength) were performed in the same manner as in Example 7-7 to analyze fatigue and weakness that occur as a side effect due to treatment with anticancer drugs in mice. test) was performed. Table 44 shows before and after inducing aging by treatment with anticancer drugs, and Table 45 shows activity evaluation, which is a criterion for determining senescence, under conditions in which aging was induced by treatment with anticancer drugs and without and with LX-112 treatment, This is a quantitative result of the endurance evaluation, muscle strength, and grip strength evaluation. Table 46 shows body weight results before and after inducing aging by treatment with anticancer drugs, and under conditions in which aging was induced by treatment with anticancer drugs and treated with LX-112. In Table 45, the amount of each evaluation after aging was induced by treatment with anticancer drugs was calculated as 100% and compared with the amount of each evaluation after treatment with LX-112.

Open Field Test
Time in Center (%) Wire Hanging Test
Time (Sec) Grip Sterenght Test
Strength (N) - 11.9 (±4.2) 69.0 (±7.0) 1.3 (±0.1) DOX 4.9 (±2.2) 32.0 (±11.0) 0.7 (±0.1)

Open Field Test
Time in Center (%) Wire Hanging Test
Time (Sec) Grip Sterenght Test
Strength (N) - 100.0 (±22.0) 100.0 (±25.0) 100.0 (±29.0) LX 129.0 (±15.0) 127.0 (±15.0) 122.0 (±18.0)

Mean Body Weight (g) - DOX DOX+LX 26.5 (±5.7) 21.3 (±2.6) 22.8 (±3.1)

As a result, as shown in Tables 44 to 46, when mice whose aging was induced by treatment with anticancer drugs were treated with LX-112, activity, endurance, muscle strength, and grip strength increased, and weight loss was improved. In addition, lipofuscin (Tables 38 and 39), an age-related degenerative marker in muscle tissue, was also reduced by treatment with LX-112. From this, it was confirmed that LX-112 improves fatigue and weakness in mice that occur as a side effect of anticancer drug treatment by removing senescent cells that increase as the mouse ages. Consistent with this improvement effect, it was confirmed from the results in Table 46 that LX-112 increased the body weight of aged mice by treatment with anticancer drugs. Example 8-8. Improvement of liver damage caused by anticancer drug LX-112

The amount of AST (aspartate aminotransferase) and ALT (alanine transaminase) released into the blood upon liver damage was measured in serum collected from mice in the same manner as in Example 7-5 using a kit (purchased by Sigma). It was measured by spectrophotometry according to the method described in the manual. Table 47 shows AST and ALT, liver damage markers, before and after inducing aging by treatment with anticancer drugs, and Table 48 shows liver damage markers, AST and ALT, under conditions in which aging was induced by treatment with anticancer drugs and without and with LX-112 treatment. This is the result showing the amount of. In Table 48, the amount of each factor after aging was induced by treatment with anticancer drugs was calculated as 100% and compared with the amount of each factor after treatment with LX-112.

Fold AST ALT - 1.0 (±0.1) 1.0 (±0.1) DOX 1.3 (±0.0) 1.4 (±0.5)

% AST ALT - 100.0 (±14.4) 100.0 (±9.2) LX 78.0 (±17.3) 72.2 (±14.1)

As a result, as shown in Table 47, when mice aged by treatment with anticancer drugs, the amounts of AST and ALT in the serum increased. Additionally, as shown in Table 48, when mice whose aging was induced by treatment with anticancer drugs were treated with LX-112, the amounts of AST and ALT in the serum decreased. From this, it was confirmed that LX-112 improves liver damage and dysfunction in mice by eliminating senescent cells that increase as mice age by treatment with anticancer drugs. Example 8-9. Improvement of kidney damage caused by anticancer drug LX-112

The amounts of urea and creatinine, which are markers of kidney damage, were measured in serum collected from mice in the same manner as in Examples 7-8 using spectrophotometry. Table 49 shows the quantitative results of urea, creatinine, and Agtr1a mRNA, which are kidney damage markers, under conditions in which aging was induced by treatment with anticancer drugs and LX-112 was not treated and treated with LX-112. The amount of each factor after aging was induced by treatment with anticancer drugs was calculated as 100% and compared with the amount of each factor after treatment with LX-112.

% Urea Creatinine Agtr1a mRNA - 100.0 (±0.2) 100.0 (±5.0) 100.0 (±6.0) LX 91.2 (±1.2) 89.0 (±4.0) 75.0 (±9.0)

As a result, as shown in Table 49, when mice whose aging was induced by treatment with anticancer drugs were treated with LX-112, the amounts of urea, creatinine, and Agtr1a mRNA decreased. From this, it was confirmed that LX-112 improves kidney damage and dysfunction in mice by eliminating senescent cells that increase as mice age through treatment with anticancer drugs.

Example 9. Removal of senescent cells and related anti-aging activity of LX-112 in aging mice induced by obesity

Example 9-1. Establishment of a mouse model of obesity-induced aging

15-week-old male C57BL6J mice (purchased by Jackson Laboratories), whose obesity was induced by a high-fat diet, were adapted to the experimental environment for one week, and then the mice were randomized into an experimental group and a control group. LX-112 was mixed with 0.5% CMC-Na solution (purchased by Sigma) and administered orally at a concentration of 70 mg/kg daily for 21 days. The control group was administered only the solution in the same volume. After completion of drug administration, the method described in each example below was performed. Mice were sacrificed and the weight of adipose tissue in each area was measured. Table 50 shows body weight, each adipose tissue, epididymal fat (eWAT), inguinal fat (iWAT), subcutaneous fat (asWAT), and mesentery, before and after inducing obesity in mice induced with high-fat diet (DIO). This is a quantitative result of the weight of fat (mWAT) and brown fat between the shoulder blades (iBAT).

Body Weight (g) eWAT (mg) iWAT (mg) asWAT (mg) mWAT (mg) iBAT (mg) - 20.7
(±0.1) 169.1
(±70.1) 188.4
(±2.1) 205.6
(±88.4) 96.6
(±19.1) 100.5
(±35.9) DIO 41.5
(±3.8) 2437.4
(±161.4) 1471.9
(±162.8) 1269.6
(±24.7) 1018.3
(±481.5) 344.7
(±140.7)

As a result, as shown in Table 50, the weight of adipose tissue in various areas increased along with body weight. From this, it was confirmed that obesity was induced. Example 9-2. Decreased amount of aging-related marker (p16) in LX-112

Mice were sacrificed in the same manner as in Example 7-2, RNA was extracted from organ tissues of each region, and qRT-PCR (quantitative real-time PCR) was performed and analyzed using the synthesized cDNA. Table 51 shows the mRNA amounts of p16 and p21, aging-related markers, before and after inducing aging by obesity, and Table 52 shows the mRNA amounts of p16 and p21, aging-related markers, under conditions in which aging was induced by obesity and without and with LX-112 treatment. This is the result shown. Table 52 shows the mRNA amounts of p16 and p21 after aging induced by obesity were calculated as 100% and compared with the mRNA amounts of p16 and p21 after treatment with LX-112.

Fold Liver Muscle Lung Skin eWAT iWAT asWAT iBAT p16
mRNA - 1.0
(±0.1) 1.0
(±0.6) 1.0
(±0.3) 1.0
(±0.3) 1.0
(±0.4) 1.0
(±0.0) 1.0
(±0.2) 1.0
(±1.0) DIO 1.2
(±0.5) 2.2
(±0.2) 1.1
(±0.2) 1.3
(±0.5) 3.3
(±2.2) 1.2
(±0.1) 1.2
(±0.2) 2.1
(±1.0) p21 mRNA - 1.0
(±0.7) 1.0
(±0.1) 1.0
(±0.2) 1.0
(±0.1) 1.0
(±0.4) 1.0
(±0.5) 1.0
(±0.8) 1.0
(±0.1) DIO 20.4
(±1.9) 1.2
(±0.1) 1.1
(±0.2) 1.2
(±0.0) 1.7
(±0.3) 2.9
(±0.2) 1.2
(±0.3) 1.4
(±1.1)

% Liver Muscle Lung Skin eWAT iWAT asWAT iBAT p16
mRNA - 100.0
(±3.4) 100.0
(±4.7) 100.0
(±5.5) 100.0
(±1.6) 100
(±3.0) 100.0
(±3.4) 100.0
(±1.5) 100.0
(±1.3) LX 71.3
(±4.5) 77.8
(±7.9) 81.0
(±3.0) 79.1
(±3.0) 61.2
(±4.3) 79.1
(±7.0) 84.1
(±3.3) 89.2
(±2.6) p21 mRNA - 100.0
(±7.9) 100.0
(±9.4) 100.0
(±9.6) 100.0
(±9.4) 100.0
(±8.2) 100.0
(±4.5) 100.0
(±6.2) 100.0
(±4.3) LX 62.9
(±9.7) 73.2
(±7.2) 79.3
(±8.4) 78.1
(±9.2) 67.2
(±6.8) 71.2
(±3.3) 82.1
(±7.7) 70.0
(±6.1)

As a result, as shown in Table 51, when mice age due to aging, liver, muscle, lung, skin, epididymal fat (eWAT), inguinal fat (iWAT), and subcutaneous fat The mRNA amounts of p16 and p21 increased in various organ tissues, such as fat (asWAT) and interscapular brown fat (iBAT). Additionally, as shown in Table 52, when LX-112 was treated with mice in which aging was induced, the mRNA amounts of p16 and p21 were decreased in various organ tissues. From this, it was confirmed that LX-112 effectively reduces the amount of aging-related markers p16 and p21 that increase in various organ tissues of mice by removing senescent cells that increase in mice due to aging due to obesity. Example 9-3. LX-112 reduces the amount of age-related inflammatory secretory factor (SASP) (IL6)

The amount of IL6 and activin A, which are age-related inflammatory secretion factors (SASP), were measured in serum collected from mice in the same manner as in Example 7-5 using an enzyme-linked immunosorbent assay (ELISA Kit, place of purchase - R&D Systems). Table 53 is before and after inducing aging by obesity, and Table 54 is IL6, an age-related inflammatory secretory factor (SASP), and fluid levels under conditions in which aging was induced by obesity and without and with LX-112 treatment. This is a result showing the amount of thivin A.

Fold IL6 Activin A - 1.0 (±0.6) 1.0 (±0.5) DIO 2.0 (±0.5) 2.9 (±1.2)

% IL6 Activin A - 100.0 (±9.2) 100.0 (±13.2) LX 51.8 (±14.2) 64.5 (±12.3)

As a result, as shown in Table 53, when mice aged due to obesity, the amounts of IL6 and activin A in the serum increased. Additionally, as shown in Table 54, when mice with aging induced by obesity were treated with LX-112, the amounts of IL6 and activin A in the serum decreased. From this, it was found that LX-112 effectively reduces the amount of IL6 and activin A, which are age-related inflammatory secretion factors (SASP), that increase in the serum of mice by removing senescent cells that increase as mice age due to obesity. Confirmed

Example 9-4. LX-112 improves anxiety, depression, and lethargy caused by obesity

It has been revealed that anxiety and depression caused by obesity have no causal relationship with increased body weight and are caused by the accumulation of senescent cells. Accordingly, an open field test was performed to analyze lethargy due to anxiety and depression caused by obesity in mice in the same manner as in Example 7-7. Table 55 is before and after inducing aging by obesity, and Table 56 is activity evaluation, which is a criterion for judging lethargy due to anxiety and depression, under conditions in which aging was induced by obesity and without and with LX-112 treatment. This is a quantitative result. Table 56 calculates the amount of activity evaluation after aging induced by obesity as 100% and compares it with the amount of activity evaluation after treatment with LX-112.

Open Field Test Time in Center (%) - 7.3 (±0.4) DIO 2.8 (±0.0)

Open Field Test Time in Center (%) - 100.0 (±21.0) LX 137.0 (±15.0)

As a result, as shown in Table 55, activity decreased in mice whose aging was induced by obesity. Additionally, as shown in Table 56, when mice with aging induced by obesity were treated with LX-112, activity increased. From this, it was confirmed that LX-112 improves anxiety, depression, and lethargy in mice caused by obesity by eliminating senescent cells that increase as mice age. Example 9-5. Improvement of fatty liver due to obesity with LX-112

Mice were sacrificed in the same manner as in Example 7-4, sections were prepared from liver tissue, and fat accumulation was stained with Oil Red O. Table 57 shows the staining area of Oil Red O, which is a criterion for determining fatty liver, before and after inducing aging by obesity, and Table 58 shows the staining area of Oil Red O, a criterion for determining fatty liver, under conditions in which aging was induced by obesity and without treatment with LX-112. This is the result shown. Table 58 shows the staining area of Oil Red O after aging induced by obesity was calculated as 100% and compared with the staining area of Oil Red O after treatment with LX-112.

Oil Red O
(Fold) - 5.8 (±1.4) DIO 47.7 (±4.2)

Oil Red O
(%) - 100.0 (±11.3) LX 74.0 (±7.3)

As a result, as shown in Table 57, when mice aged due to obesity, the area of Oil Red O staining increased. Additionally, as shown in Table 58, when mice with aging induced by obesity were treated with LX-112, the area of Oil Red O staining decreased. From this, it was confirmed that LX-112 reduces the amount of fat accumulated in the liver tissue of mice by removing senescent cells that increase as mice age due to obesity. Example 10. Obtaining a variant selenium compound of LX-112 that has senescent cell removal and related anti-aging activity

Example 10-1. Removal of senescent cells in LX-114, LX-115, LX-116, LX-117, LX-128

LX-114 (purchased by REONSIMI), LX-115 (purchased by REONSIMI), LX-116 (purchased by REONSIMI), and LX-117 (purchased by REONSIMI) were added to the cells treated and cultured with doxorubicin and the control group in the same manner as in Example 1. REONSIMI), LX-128 (purchased by Abcam), etc. were treated at concentrations of 0 μM, 1.1 μM, 3.3 μM, and 10 μM and cultured for 3 days. Next, cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay (purchased by Promega). Tables 59 and 60 show cells after treating human-derived foreskin cells with doxorubicin (DOX) or the control group and then treating each composition such as LX-114, LX-115, LX-116, LX-117, and LX-128. This is the result showing survival rate and growth rate.

Survival (%) 0 μM 1.1 μM 3.3 μM 10 μM LX-114 100.8 (±5.1) 69.1 (±2.3) 27.4 (±5.1) 6.2 (±3.4) LX-115 100.1 (±8.1) 68.9 (±3.9) 24.2 (±6.7) 3.1 (±4.9) LX-116 100.2 (±6.9) 51.5 (±6.7) 21.9 (±5.9) 3.1 (±7.8) LX-117 100.4 (±8.3) 59.4 (±2.1) 25.6 (±6.6) 3.9 (±2.9) LX-128 100.0 (±3.0) 96.0 (±5.4) 89.0 (±4.2) 58.0 (±6.1)

Growth (%) N0 0 μM 1.1 μM 3.3 μM 10 μM LX-114 100.3
(±3.9) 260.4
(±14.4) 204.7
(±9.3) 163.7
(±14.2) 121.7
(±21.1) LX-115 100.7
(±1.9) 249.7
(±11.1) 195.3
(±7.1) 174.5
(±16.9) 116.0
(±19.4) LX-116 100.6
(±4.1) 261.9
(±19.3) 199.1
(±9.3) 163.2
(±19.4) 126.9
(±17.8) LX-117 100.2
(±5.8) 257.7
(±14.9) 197.1
(±8.4) 165.4
(±21.0) 126.8
(±15.8) LX-128 100.0
(±24) 301.0
(±8.9) 298.4
(±11.2) 290.6
(±20.1) 259.4
(±11.7)

As a result, as shown in Table 59, the concentration of compositions such as LX-114, LX-115, LX-116, LX-117, and LX-128 containing selenium was high in cells in which senescence was induced by treatment with doxorubicin. The cell survival rate decreased. In addition, as shown in Table 60, there was no significant change in cell survival rate even when all compositions were treated at various concentrations in cells in which senescence was not induced by treatment with the control group, and the cells were growing normally. From this, it was confirmed that the LX-112 variant composition containing selenium can effectively remove senescent cells by inducing their death. In addition, the generation of reactive oxygen species (ROS) in senescent cells was analyzed. Each composition, including LX-112, LX-114, LX-115, LX-116, and LX-117, was treated at a concentration of 30 μM and cultured cells were treated with DCFDA/H2DCFDA (2',7'-Dichlorofluorescein diacetate, purchased from Abcam). ) was treated with 20 μM and reacted at 37°C for 45 minutes. Fluorescently stained cells were analyzed using a plate reader (SpectraMax ID3, Ex/Em=485 nm/535 nm, purchased from Molecular Device). As a result, when each composition (LX-112, LX-114, LX-115, LX-116, LX-117) induced the death of senescent cells, reactive oxygen species increased by 1.8 to 2.1 times compared to before treatment with the compound. Sikkim was confirmed. From this, it was confirmed that the LX-112 variant composition containing selenium can effectively remove senescent cells by inducing the death of senescent cells by generating reactive oxygen species. Example 10-2. Decreased amounts of aging-related marker (p16) and age-related inflammation and fibrosis secretion factor (SASP) in LX-114, LX-115, LX-116, and LX-117.

In the same manner as Example 7-1, aged mice were mixed with LX-114, LX-115, LX-116, and LX-117 in 0.5% CMC-Na solution (purchased by Sigma) and administered 70 mg/kg daily for 21 days. It was administered orally at any concentration. The control group was administered only the solution in the same volume. After completion of drug administration, the method described in each example below was performed. Mice were sacrificed in the same manner as in Example 7-2, and qRT-PCR was performed and analyzed using cDNA synthesized by extracting RNA from liver tissue. Table 61 shows the aging-related marker p16 and age-related inflammation and fibrosis secretion factors ( This is the result showing the mRNA amount of MMP3 (SASP). Table 61 shows the mRNA amounts of p16 and MMP3 after natural aging was induced, calculated as 100%, and compared with the mRNA amounts of p16 and MMP3 after treatment with LX-114, LX-115, LX-116, and LX-117. .

LX-114 LX-115 LX-116 LX-117 p16 mRNA (%) 76.9 (±3.6) 75.2 (±5.5) 63.0 (±7.0) 71.3 (±4.0) MMP3 mRNA (%) 84.3 (±6.9) 81.2 (±6.3) 71.3 (±6.7) 69.3 (±7.2)

As a result, as shown in Example 7-2 and Table 19 or Example 7-6 and Table 28, when mice aged due to natural aging, the mRNA amounts of p16 and MMP3 increased in liver tissue, but this Treatment with LX-114, LX-115, LX-116, and LX-117 decreased the mRNA amounts of p16 and MMP3 (Table 61). From this, LX-114, LX-115, LX-116, and LX-117 remove senescent cells that increase in mice due to natural aging, thereby suppressing aging-related marker p16 and age-related inflammation and fibrosis secretion factors ( It was confirmed that the amount of MMP3 (SASP) was effectively reduced.

<110> Lifeceutix Co., Ltd. <120> COMPOSITION FOR REMOVING SENESCENT CELLS COMPRISING SELENIUM-CONTAINING COMPOUND AS ACTIVE INGREDIENT <130> FPD/202309-0014 <150> KR 10-2021-0085169 <151> 2021-06-29 <160>40 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_GAPDH <400> 1 ggaagggctc atgaccacag 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_GAPDH <400> 2 acagtcttct gggtggcagt g 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_p16 <400> 3 tgagctttgg ttctgccatt 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_p16 <400> 4 agctgtcgac ttcatgacaa g 21 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_p21 <400> 5 gagactaagg cagaagatgt agag 24 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_p21 <400> 6 gcagaccagc atgacagat 19 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_ACTA2 <400> 7 gtgaagaaga ggacagcact g 21 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_ACTA2 <400> 8 cccattccca ccatcacc 18 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_COL1A1 <400> 9 aaggggacaca gaggtttcag tgg 23 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_COL1A1 <400> 10 cagcaccagt agcaccatca tttc 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_COL1A2 <400> 11 cttgcagtaa ccttatgcct agca 24 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_COL1A2 <400> 12 cccatctaac ctctctaccc agtct 25 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_FN1 <400> 13 tgtcagtcaa agcaagcccg 20 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_FN1 <400> 14 ttaggacgct cataagtgtc accc 24 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_beta-actin <400> 15 catccgtaaa gaccctctatg ccaac 25 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_beta-actin <400> 16 atggagccac cgatccaca 19 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_p21 <400> 17 gcagatccac agcgatatcc a 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_p21 <400> 18 aacaggtcgg acatcaccag 20 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_IL1-alpha <400> 19 tccataaccc atgatctgga a 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_IL1-alpha <400> 20 ttggttgagg gaatcattca t 21 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_IL1-beta <400> 21 gtatgggctg gactgtttc 19 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_IL1-beta <400> 22 gctgtctgct cattcacg 18 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_CXCL1 <400> 23 accgaagtca tagccacact c 21 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_CXCL1 <400> 24 ctccgttact tggggacacc 20 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_CXCL10 <400> 25 gctgccgtca ttttctgc 18 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_CXCL10 <400> 26 tctcactggc ccgtcatc 18 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_MMP3 <400> 27 gttggagaac atggagactt tgt 23 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_MMP3 <400> 28 caagttcatg agcagcaacc a 21 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_MMP12 <400> 29 tgcactctgc tgaaaggagt ct 22 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_MMP12 <400>30 gtcattggaa ttctgtcctt tcca 24 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_MMP13 <400> 31 aaggggataa cagccactac aa 22 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_MMP13 <400> 32 accaacataa aaattaagcc aaatg 25 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_MCP1 <400> 33 gcatctgccc taaggtcttc a 21 <210> 34 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_MCP1 <400> 34 gtggaaaagg tagtggatgc att 23 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_COL1A1 <400> 35 cagtcgattc acctacagca c 21 <210> 36 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_COL1A1 <400> 36 tggagggagtttacacg 17 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_Agtr1a <400> 37 aagggccatt ttgcttttct 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_Agtr1a <400> 38 aactcacagc aaccctccaa 20 <210> 39 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 1_SDF1 <400> 39 tgccagagcc aacgtcaag 19 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer 2_SDF1 <400> 40 cagccgggct acaatctgaa 20

Claims (1)

A composition for removing foreskin cells that have stopped cell division due to doxorubicin treatment in vitro, comprising a compound represented by the following formula (7) as an active ingredient:
[Formula 7]