A mitochondrial biosynthesis promoter containing 1,1-diethoxyethane as an effective ingredient and use thereof

Abstract

The present invention relates to a mitochondrial biosynthesis promoter containing 1,1-diethoxyethane (1,1-DEE) as an effective ingredient and use thereof. More specifically, the present invention relates to a mitochondrial biosynthesis promoter containing 1,1-diethoxyethane as an effective ingredient and use thereof, such as a composition for preventing, treating or ameliorating a disease in which mitochondrial biosynthesis is desired to be promoted, including a pharmaceutical composition, a cosmetic composition, a food composition and a feed composition.

Description

Technical Field

[0001] This invention relates to a mitochondrial biosynthesis promoter containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient and its uses. More specifically, this invention relates to a mitochondrial biosynthesis promoter containing 1,1-diethoxyethane as an active ingredient and its uses, such as compositions for the prevention, treatment, or improvement of diseases requiring the promotion of mitochondrial biosynthesis, including pharmaceutical compositions, cosmetic compositions, food compositions, and feed compositions. Background Technology

[0002] 5'-Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an enzyme in the evolutionarily conserved serine / threonine kinase series responsible for maintaining cellular energy homeostasis. This enzyme plays a crucial role in regulating energy balance [1,2]. AMPK consists of a catalytic α-subunit, a regulatory β-subunit, and a non-catalytic γ-subunit, each of which regulates a variety of physiological functions [3]. The α-subunit activates AMPK through phosphorylation of Thr172 residues, the β-subunit promotes the binding of AMPK to glycogen, and the γ-subunit binds to AMP and ADP, stimulating AMPK phosphorylation [4]. AMPK activation promotes catabolic metabolism, inhibits anabolic metabolism, generates ATP, and improves energy balance. Upstream kinases such as LKB1 (liver kinase B1), CAMKK2 (Ca2+ / calmodulin-dependent protein kinase kinase-2), and TAK (transforming growth factor-beta-activated kinase) play important roles in AMPK activation [5-7]. Drugs such as metformin, AICAR (5-aminoimidazole-4-carboxamide ribonucleotide), and resveratrol activate AMPK by regulating upstream kinases [8-10]. Energy states such as exercise and malnutrition can also activate AMPK. AMPK activation has potential implications for the prevention of obesity, cardiovascular disease (CVD), and cancer

[11] , especially playing an important preventive role in cardiovascular disease [17-22].

[0003] AMPK plays an important role in energy balance by regulating downstream effectors of catabolism processes such as fatty acid synthesis and glycolysis. Acetyl-CoA carboxylase (ACC) plays an important role in fatty acid biosynthesis. AMPK phosphorylation of Ser79 (ACC1) or Ser212 (ACC2) inhibits the enzyme activity of ACC and increases fatty acid oxidation [1]. In the heart, fatty acid oxidation is an important component of energy production. The AMPK-ACC signaling pathway regulates a variety of physiological processes such as platelet phospholipid and thrombus formation, cardiac hypertrophy and contractility, and ischemia regulation [24-26]. In addition, glycolysis is also regulated by PFKFB2 (phosphofructo-2-kinase / fructose-2,6-biphosphatase-2), which interferes with the formation and breakdown of fru-2,6-P2 (fructose-2,6-bisphosphate) and regulates the activity of PFK1 (phosphofructokinase-1). Phosphorylation of PFKFB2 at Ser466 and Ser483 promotes glycolysis, which is regulated by Akt (protein kinase B) and AMPK. Activation of PFKFB2 plays an important role in cardiac remodeling, inhibiting ferroptosis in I / R injury, and improving cardiac function under hypoxic conditions [27-29].

[0004] Mitochondrial biosynthesis is an important process for adapting to the energy needs of cells, and AMPK can promote this process. AMPK deficiency leads to mitochondrial dysfunction, and PGC-1α (peroxisome proliferator-activated receptor gammacoactivator 1-alpha), a major downstream regulator of AMPK, plays an important role in mitochondrial biosynthesis [2]. AMPK activates PGC-1α through transcription factor EB activation and direct binding, and phosphorylates it at Thr177 and Ser538 sites, promoting mitochondrial biosynthesis [32,33]. This process makes an important contribution to cardioprotection [34,35].

[0005] Slow down aging or extend lifespan Lifespan is finite; over time, organisms undergo the natural process of birth, growth, and aging, ultimately leading to death. Aging is one of the complex biological pathways that occur in all living organisms. To elucidate the causes and processes of aging, numerous studies have been conducted, primarily using two mechanisms to explain the phenomenon: the aging clock and cellular senescence. The aging clock theory explains aging through the loss of telomeres at the ends of DNA, arguing that the loss of telomeres (repetitive sequences located at the ends of chromosomes) leads to cellular and overall organismal aging. Cellular senescence posits that the accumulation of intracellular damage, such as oxidative damage and mitochondrial dysfunction, is the primary cause of aging. The mitochondrial damage theory emphasizes the importance of mitochondrial function in healthy cellular metabolism and bioenergy. Age-related declines in mitochondrial function include reduced ATP production, increased ROS production, decreased mitochondrial numbers, and changes in mitochondrial permeability.

[0006] As we age, the activity of pathways related to lipid metabolism is impaired. Specifically, reduced fat breakdown leads to decreased cellular uptake of low-density cholesterol (LDL), resulting in elevated LDL levels in the blood, which can trigger cardiovascular disease. Furthermore, aging causes a decline in cognitive and motor function, increasing the incidence of neurological disorders such as Alzheimer's and Parkinson's diseases, all of which reduce quality of life.

[0007] In addition to research focused on understanding the aging process, numerous studies have been conducted on slowing aging and extending lifespan. To date, the most successful method is dietary restriction. The effects of dietary restriction were initially reported in mice, and subsequently, longevity phenotypes resulting from dietary restriction have been observed in yeast, nematodes, fruit flies, and mice. However, as mentioned above, the beneficial effects of dietary restriction can only be observed when it is consistently maintained, making it difficult to apply to all living organisms.

[0008] To date, despite extensive research and efforts to slow down aging or extend lifespan, the mechanisms of aging have not been fully elucidated.

[0009] Cardiovascular disease Proper regulation of heart rate is essential for maintaining life. Conditions such as bradycardia and bradyrrhythmia, which cause abnormally low heart rates, can lead to serious health problems. A low heart rate can cause dizziness, fatigue, and fainting, and in severe cases, it can lead to heart failure or cardiac arrest. Therefore, there is a need for continuous development of therapeutic agents that can effectively increase heart rate.

[0010] To date, treatments that increase heart rate primarily work by stimulating the sympathetic nervous system. Representative drugs include beta-adrenergic agonists, antimuscarinic agents, and specific ion channel modulators.

[0011] Isoproterenol, a beta-adrenergic agonist, increases heart rate by non-selectively activating β1 and β2 receptors. However, isoproterenol affects not only heart rate but also myocardial contractility and blood pressure, and long-term use may lead to side effects such as hypertension, arrhythmia, and increased myocardial oxygen demand.

[0012] Antimuscarinic drugs (e.g., atropine) increase heart rate by inhibiting vagal nerve activity and reducing the influence of the parasympathetic nervous system. However, these drugs may be accompanied by side effects such as dry mouth, blurred vision, and urinary problems, and their heart rate regulation effect is relatively limited.

[0013] Some ion channel modulators work by increasing heart rate by regulating the flow of calcium or sodium ions within cardiomyocytes. However, these drugs directly affect cell membrane potential, thus requiring precise dosage control and potentially causing arrhythmias.

[0014] Therefore, there is a need for ongoing research into new substances that can rapidly and effectively increase heart rate while minimizing the side effects of existing drugs.

[0015] cancer Neuroblastoma is a malignant tumor originating from the neural crest, primarily expressing sympathetic ganglia and adrenal medulla. More than one-third of neuroblastomas are discovered in infancy (under 1 year old), with approximately 75% diagnosed before the age of 5. It is one of the most common and deadliest childhood cancers, characterized by its aggressiveness and poor prognosis during its progressive stage. Despite various efforts to treat neuroblastoma, including the development of multimodal therapy, immunotherapy, and clinical trials of novel targeted therapies, the prognosis for high-risk children remains poor, with a median five-year overall survival rate of approximately 50-70%. Recently, anticancer treatment has focused primarily on mechanisms that induce apoptosis. However, deficiencies in apoptosis regulation not only provide tumor cells with a growth advantage but also contribute significantly to treatment resistance. In fact, drug resistance remains one of the greatest threats to the treatment and control of neuroblastoma.

[0016] To date, research has primarily focused on the expression of multidrug resistance proteins and anti-apoptotic mechanisms in neuroblastoma cells, with relatively little attention paid to intracellular signaling pathways associated with treatment resistance. In fact, metabolic reprogramming, a key characteristic of cancer, is regulated through interactions between various signaling proteins and kinases. A deeper understanding of their synergistic effects could help identify promising therapeutic targets. In particular, AMP-activated protein kinase (AMPK) plays a central role in the regulation of metabolism and energy homeostasis in both normal and cancer cells, and it has attracted considerable attention as a key factor in this signaling axis. Numerous previous studies have demonstrated that the AMPK signaling pathway has a dual role in maintaining cancer cell homeostasis. Therefore, a strategy of modulating signaling systems, including AMPK, while employing treatments that specifically target tumor cells is considered a promising approach to inhibiting treatment resistance expression in cancer and improving patient prognosis.

[0017] Stroke Stroke is the third leading cause of death worldwide. Stroke survivors often experience motor impairments, cognitive deficits, and other challenges, severely hindering daily life and reducing quality of life. Furthermore, stroke is an independent risk factor for dementia of all causes. From 1990 to 2021, the global burden of stroke increased significantly, with stroke incidence increasing by 70%, associated mortality by 44%, and disability-adjusted life years lost by 32%. Despite medical advancements, the WSO / Lancet Neurology Commission on Stroke 2 (the second stroke commission jointly established by the World Stroke Organization and *The Lancet Neurology*) predicts that stroke mortality will increase by 50% between 2020 and 2050, placing a heavy burden on the world. Therefore, stroke remains a major global health problem in terms of mortality and disability rates, and the discovery of effective substances for stroke prevention, treatment interventions, and post-stroke rehabilitation is crucial.

[0018] dementia Dementia is a collective term for a range of neurodegenerative diseases that impair learning and memory abilities and disrupt daily life. The World Health Organization (WHO) reports that dementia is currently the seventh leading cause of death worldwide and a leading cause of disability and dependence among the elderly. With approximately 10 million new cases of dementia each year, it is considered a major global health problem affecting millions of lives and imposing a significant social and economic burden.

[0019] It is known that various risk factors, such as advanced age, hypertension, diabetes, obesity, smoking, and excessive alcohol consumption, are significantly associated with the onset of dementia. Furthermore, the impact of low to moderate alcohol consumption on dementia risk is currently unclear. Some studies suggest that a certain level of alcohol consumption may have a potential protective effect against dementia, but further, more precise research is needed.

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Cell Metab, 2005. 2(1): p. 21-3 [Non-patent document 7] Xie, M., et al., A pivotal role for endogenous TGF-beta-activated kinase-1 in the LKB1 / AMP-activated protein kinase energy-sensor pathway. Proc Natl Acad Sci USA, 2006. 103(46): p. 17378-83. [Non-Patent Literature 8] Shaw, RJ, et al., The kinase LKB1 mediates glucosehomeostasis in liver and therapeutic effects of metformin. Science, 2005. 310(5754): p. 1642-6. [Non-Patent Literature 9] Chan, AY, et al., Resveratrol inhibits cardiac hypertrophy via AMP-activated protein kinase and Akt. J Biol Chem, 2008. 283(35): p. 24194-201. [Non-patent document 10] Corton, JM, et al., 5-aminoimidazole-4-carboxamideribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells? Eur J Biochem, 1995. 229(2): p. 558-65. [Non-Patent Literature 11] Jeon, SM, Regulation and function of AMPK inphysiology and diseases. Exp Mol Med, 2016. 48(7): p. e245. [Reference 12] Marcq, P. and P. Schieberle, Characterization of theKey Aroma Compounds in a Commercial Fine and a Commercial Pedro XimenezSherry Wine by Application of the Sensomics Approach. J Agric Food Chem,2021. 69(17): p. 5125-5 [PubMed] Moreno, J., et al., Use of a flor velum yeast formodulating colour, ethanol and major aroma compound contents in red wine.Food Chem, 2016. 213: p. 90-97. [Published] Tan, C., et al., The Effects of Ultrasonic and GammaIrradiation on the Flavor of Potato Wines Investigated by Sensory Omics.Foods, 2023. 12(15). [CrossRef] [PubMed] 15] Dunkel, A., et al., Nature's chemical signatures inhuman olfaction: a foodborne perspective for future biotechnology. Engineering ChemInt Ed Engl, 2014. 53(28): p. 7124-4 [PubMed 16] Kawaguchi, D., H. Ogihara, and H. Kurokawa, Upgradingof Ethanol to 1,1-Diethoxyethane by Proton-Exchange Membrane Electrolysis.ChemSusChem, 2021. 14(20): p. 4431-4 [Non-Patent Document 17] Li, Y., et al., AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice. Cell Metab, 2011. 13(4): p. 376-388. [Non-Patent Document 18] Pulipaka, S., et al., Therapeutic efficacies of mitochondria-targeted esculetin and metformin in the improvement of age-associated atherosclerosis via regulating AMPK activation. Geroscience, 2024. 46(2): p. 2391-2408. [Non-Patent Document 19] Wang, Z., et al., Dexmedetomidine attenuates myocardial ischemia / reperfusion-induced ferroptosis via AMPK / GSK-3β / Nrf2 axis. Biomed Pharmacother, 2022. 154: p. 113572. [Non-Patent Document 20] Zhang, Y., et al., Melatonin attenuates myocardial ischemia-reperfusion injury via improving mitochondrial fusion / mitophagy and activating the AMPK-OPA1 signaling pathways. 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Hopkins, TA, JR Dyck, and GD Lopaschuk, AMP-activated protein kinase regulation of fatty acid oxidation in the ischemic heart. Biochem Soc Trans, 2003. 31(Pt 1): p. 207-1 [Reference 27] Harold, KM, et al., Loss of Cardiac PFKFB2 DrivesMetabolic, Functional, and Electrophysiological Remodeling in the Heart. J AmHeart Assoc, 2024. 13(7): p. e033676. [CrossRef] Fu, C., et al., PFKFB2 Inhibits Ferroptosis inMyocardial Ischemia / Reperfusion Injury Through Adenosine Monophosphate-Activated Protein Kinase Activation. J Cardiovasc Pharmacol, 2023. 82(2): p.128-137. [CrossRef] 29] Gao, J., et al., HIF-1 / AKT Signaling-Activated PFKFB2Alleviates Cardiac Dysfunction and Cardiomyocyte Apoptosis in Response toHypoxia. Int Heart J, 2021. 62(2): p. 350-358. [ PubMed ] Bergeron, R., et al., Chronic activation of AMP kinaseresults in NRF-1 activation and mitochondrial biogenesis. Am J PhysiolEndocrinol Metab. E1340-6. 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JPharmacol Sci, 2019. 139(4): p. 352-360. Summary of the Invention

[0021] The technical problem that the invention aims to solve Therefore, the inventors made efforts to study the physiological functions of 1,1-diethoxyethane (1,1-DEE). The results showed that 1,1-DEE can acutely inhibit mitochondrial oxidative phosphorylation (OXPHOS) (see Experiment 1) and long-term activate AMPK, increasing PGC-1α expression, thereby promoting mitochondrial biosynthesis (see Experiments 5 and 6). This confirms that 1,1-DEE can acutely inhibit mitochondrial respiration and also exert a long-term promoting effect on mitochondrial biosynthesis by activating AMPK, regulating mitochondrial function and energy metabolism. Based on this, the present invention was completed.

[0022] Furthermore, it was confirmed that the 1,1-DEE according to the present invention not only induces mitochondrial biosynthesis through a physiological mechanism based on the AMPK (AMP-activated protein kinase) pathway, but also exhibits significant therapeutic effects in various disease models. Specifically, it was confirmed that 1,1-DEE (i) in the *C. elegans* model can prolong lifespan without hindering development; (ii) it helps prevent or improve cardiovascular disease by temporarily and drastically increasing heart rate. In addition, (iii) it was confirmed that it exerts anticancer effects by reducing cancer cell survival and inducing apoptosis; (iv) it was confirmed that in the ischemic stroke animal model (MCAO), it has effects such as reducing mortality, promoting recovery, improving cognitive function, and shrinking the brain injury site; (v) it was confirmed that after ischemic brain injury, it can not only restore cognitive function, but also improve spatial cognition and memory in normal mice.

[0023] Therefore, the object of the present invention is to provide a mitochondrial biosynthesis promoter containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

[0024] The present invention aims to provide a pharmaceutical composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for the prevention or treatment of diseases requiring the promotion of mitochondrial biosynthesis. Another object of the present invention is to provide a pharmaceutical composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for the prevention or treatment of age-related diseases, cardiovascular diseases, cancer, stroke, or dementia.

[0025] The present invention aims to provide a cosmetic composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for the prevention or improvement of diseases requiring the promotion of mitochondrial biosynthesis. Another object of the present invention is to provide a cosmetic composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for delaying aging or prolonging lifespan, and for the prevention or improvement of cardiovascular disease, cancer, stroke, dementia, cognitive decline, or memory loss.

[0026] The present invention aims to provide a food composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for the prevention or improvement of diseases requiring the promotion of mitochondrial biosynthesis. The present invention also aims to provide a food composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for delaying aging or prolonging lifespan, and for the prevention or improvement of cardiovascular disease, cancer, stroke, dementia, cognitive decline, or memory loss.

[0027] The present invention aims to provide a feed composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for the prevention or improvement of diseases requiring the promotion of mitochondrial biosynthesis. Another object of the present invention is to provide a feed composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for delaying aging or prolonging lifespan, and for the prevention or improvement of cardiovascular disease, cancer, stroke, dementia, cognitive decline, or memory loss.

[0028] However, the problems to be solved by the present invention are not limited to those mentioned above. Other problems not mentioned can be clearly understood by those skilled in the art from the following description.

[0029] [Solution to the Problem] This invention discloses a mitochondrial biosynthesis promoter containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient and its uses, such as compositions for the prevention, treatment or improvement of diseases requiring the promotion of mitochondrial biosynthesis, including pharmaceutical compositions, cosmetic compositions, food compositions and feed compositions.

[0030] Furthermore, the present invention discloses a composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for the prevention or treatment of age-related diseases and for delaying aging or prolonging lifespan.

[0031] Furthermore, this invention discloses a composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for the prevention, improvement or treatment of cardiovascular diseases.

[0032] Furthermore, the present invention discloses a composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for the prevention, improvement or treatment of cancer and cachexia arising from said cancer.

[0033] Furthermore, the present invention discloses a composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for the prevention, improvement or treatment of stroke.

[0034] Furthermore, the present invention discloses a composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for the prevention, improvement or treatment of dementia.

[0035] Furthermore, the present invention discloses a composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for preventing, improving or treating cognitive or memory decline.

[0036] In this invention, the molecular formula of 1,1-diethoxyethane (1,1-DEE) is C6H. 14 O2, represented by the following structural formula 1, is also known as acetaldehyde diethyl acetal or ethylidene diethyl ether.

[0037] [Structure 1] According to one embodiment, A mitochondrial biosynthesis promoter containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient is disclosed.

[0038] In this invention, the 1,1-diethoxyethane can increase the expression of PGC-1α (peroxisome proliferator-activated receptor gammacoactivator-1 alpha) and promote mitochondrial biosynthesis.

[0039] In this invention, the 1,1-diethoxyethane can enhance the transcriptional activity of Nrf1 and Nrf2, which in turn promotes mitochondrial biosynthesis by stimulating the expression of Tfam.

[0040] In this invention, the 1,1-diethoxyethane can also promote aerobic respiration.

[0041] According to another embodiment, A pharmaceutical composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient is disclosed for the prevention or treatment of diseases requiring the promotion of mitochondrial biosynthesis.

[0042] In this invention, the diseases requiring promotion of mitochondrial biosynthesis can include ischemic brain diseases, ischemic heart diseases, pulmonary embolism, ischemic-reperfusion injury, stroke, ischemic stroke, migraine, anemia, Parkinson's disease (PD), dementia, Alzheimer's disease (AD), Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis (ALS), Leigh syndrome, and mitochondrial myopathy. Myopathy, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), cirrhosis, Fanconi syndrome, atypical phenylketonuria, asthma, chronic obstructive pulmonary disease (COPD), diabetes, atherosclerosis, heart failure, hypertension, metabolic dysfunction, and metabolic dysfunction-associated steatotic liver disease.Choose one or more from the following groups: MASLD, insulin resistance, hypercholesterolemia, optic nerve diseases, Duchenne muscular dystrophy, myopathies, obesity, depression, osteoarthritis, cancer, cancer cachexia, inflammatory diseases, age-related diseases, cardiovascular diseases, and cognitive impairment or memory impairment.

[0043] In this invention, the pharmaceutical composition can be administered by any one or more methods selected from the group consisting of oral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, epithelial administration, local administration, intravaginal administration, pulmonary administration, rectal administration, sublingual administration, buccal administration, transdermal administration, ocular administration, inhalation, intracavernosal injection, intrathecal injection, epidural injection, and rectal administration.

[0044] According to another embodiment, A cosmetic composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient is disclosed for the prevention or improvement of diseases requiring the promotion of mitochondrial biosynthesis.

[0045] In this invention, the dosage form of the cosmetic composition can be any one or more selected from the group consisting of solutions, topical ointments, creams, foams, nourishing lotions, softening lotions, perfumes, facial masks, softening lotions, emulsions, makeup primers, serums, soaps, liquid cleansers, shower gels, sunscreens, sunscreen oils, suspensions, emulsions, pastes, gels, skin lotions, pressed powders, soaps, facial cleansers containing surfactants, oils, liquid foundations, emulsion foundations, wax powders, patches, and sprays.

[0046] According to another embodiment, A food composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient is disclosed for the prevention or improvement of diseases requiring the promotion of mitochondrial biosynthesis.

[0047] In this invention, the food may include meat, sausage, bread, chocolate, candy, snacks, pastries, pizza, instant noodles, other noodle products, chewing gum, dairy products including ice cream, various soups, beverages, tea, coffee drinks, energy drinks, alcoholic beverages, or vitamin complexes.

[0048] According to another embodiment, A feed composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient is disclosed for the prevention or improvement of diseases requiring the promotion of mitochondrial biosynthesis.

[0049] In this invention, the feed may include powdered feed, solid feed, wet pellet feed, dry pellet feed, EP (Extruder Pellet) feed or fresh feed.

[0050] Effect The 1,1-diethoxyethane (1,1-DEE) of this invention can rapidly and reversibly activate AMPK (AMP-activated protein kinase) by reducing ATP and generating ROS. Activated AMPK can induce ACC (acetyl-CoA carboxylase) phosphorylation, inhibit fatty acid synthesis, promote β-oxidation, and regulate glycolysis through phosphorylation of PFKFB2 (6-phosphofructo-2-kinase / fructose-2,6-bisphosphatase 2). Furthermore, AMPK activation can induce increased expression of PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha), enhancing mitochondrial biosynthesis and energy metabolism, and inducing the expression and structural formation of mitochondrial-related genes through the Nrf1 / Nrf2-Tfam axis.

[0051] Based on this physiological mechanism of inducing mitochondrial biosynthesis and enhancing metabolic function, 1,1-DEE has shown significant therapeutic effects in various disease models.

[0052] First, regarding its effects on delaying aging and extending lifespan, 1,1-DEE demonstrated a lifespan-extending effect in the *Caenorhabditis elegans* model without inhibiting development. These results suggest that 1,1-DEE can be used as a pharmaceutical composition, cosmetic composition, food composition, health functional food, or feed composition to effectively delay aging or extend lifespan.

[0053] Secondly, in terms of cardiovascular diseases, 1,1-DEE exhibits a transient and rapid increase in heart rate, and therefore can be used to effectively prevent, treat, or improve cardiovascular diseases. Furthermore, the compositions of the present invention are also applicable to functional foods or animal feed, for example, aiding in the health management of individuals requiring heart rate regulation or regulating the heart rate of livestock and pets. Therefore, the present invention provides a safe and effective method for increasing heart rate using 1,1-DEE, a promising technology that can be used for the prevention and treatment of cardiovascular diseases requiring heart rate regulation.

[0054] Third, regarding cancer and cachexia, 1,1-DEE exhibits anti-cancer effects by significantly reducing cancer cell survival and inducing apoptosis. In particular, it has been confirmed that it can provide a novel anti-cancer mechanism based on energy metabolism regulation through activation of the AMPK pathway, and in vivo experiments have also demonstrated therapeutic effects in inhibiting tumor growth, preventing cancer cachexia, and improving survival rates. Furthermore, unlike 1,2-DEE, 1,1-DEE exhibits selective effects and has been evaluated as an excellent anti-cancer candidate in terms of safety and efficacy. Therefore, the compositions according to the present invention can be effectively used as pharmaceutical compositions, food compositions, cosmetic compositions, or feed compositions for the prevention, treatment, or improvement of cancer and cachexia caused by it.

[0055] Fourth, regarding ischemic stroke, 1,1-DEE exhibited multiple therapeutic effects in the MCAO (mice middle cerebral artery occlusion) model, including reducing mortality, inhibiting weight loss and promoting recovery, improving spatial cognition and memory, and shrinking the site of brain damage. In particular, in the Morris water maze test, it confirmed a tendency to quickly find platforms in the early stages of learning, and MRI imaging analysis confirmed its brain-protective effect. These results suggest that 1,1-DEE may be an effective therapeutic substance for neuroprotection and cognitive function recovery in ischemic stroke.

[0056] Fifth, regarding cognitive function and memory decline, 1,1-DEE is effective not only in stroke models but also in normal mice, demonstrating an improvement in cognitive abilities. In stroke models, mice administered 1,1-DEE were able to find platforms more quickly, followed more direct paths, and covered significantly shorter distances. Even in normal mice, repeated administration showed a trend towards improved learning speed and memory, particularly in later tests where escape time was significantly shortened. These results indicate that 1,1-DEE can effectively prevent or improve cognitive function and memory decline.

[0057] Furthermore, the scope of the present invention is not limited by the effects described above. Attached Figure Description

[0058] Figure 1 The following images show the reversible inhibitory effect of acute exposure to 1,1-DEE on oxygen consumption and glycolysis in AC16 cells: (A) 2D structure of 1,1-DEE. (B) Viability of AC16 cells was assessed using EZ-Cytox reagent after culturing with a specified concentration of 1,1-DEE for 24 hours. (C) Mitochondrial respiration was assessed using the Agilent Seahorse XF96 Cell Mitochondrial Stress Test Kit after 30 minutes of acute exposure to 1,1-DEE, metformin, and 1,2-DEE. OCR was measured after sequentially adding different concentrations of 1,1-DEE, 1.5 µM oligomycin (Oligo), 1.5 µM FCCP, and 0.5 µM rotenone / antimycin (R / A). (D) Proton leakage, ATP production, maximum respiration, and residual gas volume were measured using the Seahorse XF96 Cell Mitochondrial Stress Test Kit. (E) Determine ECAR under the conditions described in (C).

[0059] Figure 2 The concentration- and time-dependent activation of AMP following acute exposure to 1,1-DEE was shown. Western blot analysis of AC16 cells was performed to assess AMPK phosphorylation under the following conditions: (A) co-cultured with different concentrations of 1,1-DEE for 10 min, (B) co-cultured with 15 mM 1,1-DEE for 30 min, (C) co-cultured with 15 mM 1,2-DEE for 30 min, and (D) co-cultured with 15 mM metformin for 60 min.

[0060] Figure 3 The effects of AMPK activation on fatty acid oxidation and glycolysis following acute exposure to 1,1-DEE were demonstrated: (A) AC16 cells were exposed to 15 mM 1,1-DEE for 30 min. Phosphorylation of AMPK, ACC, and PFKFB2 was assessed by Western blot analysis. (B) AC16 cells were exposed to 15 mM 1,1-DEE for 8 h. The mRNA expression levels of SREBP-1c and FASN were assessed using semi-quantitative PCR and qRT-PCR. (C) WT and AMPK DKO MEF cells were exposed to 15 mM 1,1-DEE for 30 min. (D) AC16 cells were pretreated with 10 µM compound C for 1 hour and then co-cultured with 15 mM 1,1-DEE for 120 min. Western blot analysis was performed to assess the phosphorylation of AMPK effectors, including ACC and PFKFB2.

[0061] Figure 4 This study showed that a portion of AMPK activation can be mediated by 1,1-DEE-induced ROS generation. AC16 cells were pretreated with 10 mM NAC for 2 h or with 10 µM ebselen for 1 h, followed by exposure to 15 mM 1,1-DEE for 30 min. ROS generation was observed using DCFH-DA immunofluorescence staining, and AMPK activation was assessed by Western blot analysis of phosphorylation of Thr172 remnants.

[0062] Figure 5 Long-term exposure to 1,1-DEE was shown to increase PGC-1α expression via AMPK activation. (A–C) AC16 cells were co-cultured with 15 mM 1,1-DEE for up to 8 hours: (A) AMPK phosphorylation was assessed by Western blot analysis. (B) PPARGC1A mRNA expression levels were analyzed by RT-PCR. (C) PGC-1α protein expression levels were confirmed by Western blot analysis. (D) MEF cells were co-cultured with 15 mM 1,1-DEE for up to 12 hours, and AMPK phosphorylation and PGC-1α expression were assessed by Western blot analysis.

[0063] Figure 6This study demonstrated that PGC-1α-mediated AMPK activation promotes mitochondrial biosynthesis. (A–B) AC16 cells were co-cultured with 15 mM 1,1-DEE for up to 8 hours: (A) mRNA expression levels of Nrf1, Nrf2, and Tfam were assessed by real-time PCR. (B) Protein expression levels of Nrf1, Nrf2, and Tfam were analyzed by Western blot. (C) After co-culturing WT and AMPKDKO MEF cells with 15 mM 1,1-DEE for up to 8 hours, protein expression levels of Nrf1, Nrf2, and Tfam were confirmed by Western blot. (D) AC16 cells were pretreated with compound C (10 µM) for 4 hours, then exposed to 15 mM 1,1-DEE for 24 hours, and Tfam protein expression levels were assessed by Western blot. (E) AC16 cells were pretreated with compound C (10 µM) for 4 hours, then exposed to 15 mM 1,1-DEE for 8 hours, and stained with MitoTracker Red CMXRos (200 nM) for 30 minutes. Mitochondrial biosynthesis was assessed by confocal microscopy at 40x magnification. (F) Flow cytometry analysis of AC16 cells was performed under the same conditions as in (E).

[0064] Figure 7 The effects of 1,1-DEE(A) according to the present invention on the development of Caenorhabditis elegans at concentrations of 100 μm and 1 mM are shown.

[0065] Figure 8 The effect of 1,1-DEE(A) according to the present invention on the development of Caenorhabditis elegans at a concentration of 20 mM was demonstrated.

[0066] Figure 9 The effects of isoform 1,2-DEE of the 1,1-DEE(A) according to the present invention on the development of Caenorhabditis elegans at concentrations of 100 μm and 1 mM are shown.

[0067] Figure 10 The effects of 1,1-DEE(A) according to the present invention on the lifespan extension of Caenorhabditis elegans at concentrations of 100 μm and 1 mM were demonstrated.

[0068] Figure 11 The effects of isoform 1,2-DEE of the 1,1-DEE(A) according to the present invention on the lifespan extension of Caenorhabditis elegans at concentrations of 100 μm and 1 mM are shown.

[0069] Figure 12The changes in EHT beat rate (BPM: beats per minute) over time before and after treatment with different concentrations of 1,1-DEE and 1,2-DEE are shown.

[0070] Figure 13 The changes in EHT beat rate (BPM: beats per minute) over time before and after treatment with different concentrations of 1,1-DEE and 1,2-DEE are shown. (Top: 1,1-DEE and 1,2-DEE concentrations are 26 mM) Figure 14 The changes in the twist force of EHT over time before and after treatment with different concentrations of 1,1-DEE and 1,2-DEE are shown.

[0071] Figure 15 The changes in EHT twist force over time before and after treatment with different concentrations of 1,1-DEE and 1,2-DEE are shown. (Top: 1,1-DEE and 1,2-DEE concentrations are 26 mM) Figure 16 Representative images of EHT pulsation changes after processing with 1,1-DEE and 1,2-DEE (50 mM) are shown.

[0072] Figure 17 The changes in EHT pulsation velocity and twist force were shown from the moment of treatment to 10 minutes after treatment with 1,1-DEE at a concentration of 25 mM.

[0073] Figure 18 The changes in cell viability and morphology in SHSY-5Y cells with varying concentrations and treatment times of 1,1-diethoxyethane (1,1-DEE) and 1,2-1,1-diethoxyethane (1,2-DEE) are shown. (A) Cell viability, as determined by MTT assay, is shown after 24 hours of treatment with 1,1-DEE or 1,2-DEE. Each experiment was repeated three times. (B) Morphological changes in SHSY-5Y cells over time (6, 12, 24 hours) are shown under a 20x magnification backlight microscope after treatment with 1,1-DEE or 1,2-DEE at various concentrations (1, 5, 10, 20 mM).

[0074] Figure 19 The results show the effects of flow cytometry analysis on the apoptosis-inducing effect of 1,1-DEE in SHSY-5Y cells. After 24 hours of treatment, the apoptosis rate increased significantly with increasing 1,1-DEE concentration. The results are expressed as mean ± standard deviation (SD) based on three independent experiments.

[0075] Figure 20 The changes in 1,1-DEE-induced AMPK activity over time were shown in SHSY-5Y cells. Cells were treated with 1,1-DEE for 0–120 min, and the expression of total AMPK and phosphorylated AMPK protein was determined by Western blot analysis.

[0076] Figure 21 This study demonstrates tumor progression and the inhibitory effect of 1,1-DEE on tumors in a xenograft mouse model using SHSY-5Y cells. (A) shows changes in tumor volume in the 1,1-DEE treatment group and the control group, and (B) shows the results obtained by assessing the effect on the overall condition of mice through changes in body weight.

[0077] Figure 22 The study shows survival rates and weight changes following prophylactic administration of 1,1-DEE in a xenograft mouse model. (A) shows the difference in survival rates between the treatment and control groups, and (B) shows weight changes from the start of administration to the end of the survival period.

[0078] Figure 23 This study demonstrated the protective effect of 1,1-DEE against mortality in ischemic stroke. Following middle cerebral artery occlusion (MCAO), the mortality rate was 30% in the control group and 0% in the 1,1-DEE-treated group. Data are expressed as mean ± standard error (SE). A p < 0.05, relative risk (RR) = 0.3684, and a 95% confidence interval (CI) of 0.1915–0.9505 were considered statistically significant.

[0079] Figure 24 This study demonstrates the beneficial effect of 1,1-DEE on weight change after stroke. Weight was measured every 6 days from baseline to the end of the study in both the experimental and control groups. (A): Compared to baseline, the control group experienced a mean weight loss of 3.77%, while the treatment group experienced a mean weight gain of 5.46% (t-test, p < 0.001). (B): During the study, the control group experienced a continuous decrease in weight, while the treatment group experienced a steady increase in weight. At the end of the study, the mean weight of the treatment group was significantly higher than that of the control group (27.68 g vs. 26.29 g, t-test, p < 0.05). All data are expressed as mean ± standard error.

[0080] Figure 25The spatial cognition and memory-enhancing effects of 1,1-DEE were demonstrated. (A): The cumulative percentage of mice finding the hidden platform was plotted as a time-to-event graph and analyzed using an accelerated time to failure (AFT) survival analysis model. At time 20 seconds, the success rate in the treatment group was significantly higher than that in the control group (33% vs. 0%, p < 0.05). However, no statistical significance was observed at time points 40 and 60 seconds. (B): Representative swimming path analysis showed that mice treated with 1,1-DEE swam to the platform along a direct and efficient path, while control mice swam to the platform along irregular and disoriented paths. (C): Comparison of swimming distances among mice that successfully found the platform showed that the path to the platform was significantly shorter in the treatment group than in the control group (146.3 cm vs. 820.8 cm, t-test, p < 0.01). All data are presented as mean ± standard error.

[0081] Figure 26 The study demonstrated that 1,1-DEE can alleviate ischemic stroke and improve motor function after stroke. (A): Representative MRI results from each experimental group are presented, showing that the damage caused by middle cerebral artery occlusion (MCAO) was relatively small in mice administered 1,1-DEE. (B): Maximum swimming speeds measured in the Morris Water Maze (MWM) test were compared. The maximum swimming speeds recorded in the treatment group were significantly higher than those in the control group (33.83 cm / s vs. 27.77 cm / s, t-test, p < 0.05). All data are expressed as mean ± standard error (SE).

[0082] Figure 27The results show that 1,1-DEE can improve spatial cognition and memory in mice after stroke. (A): Time-to-event curves represent the cumulative percentage of mice that found the hidden platform, analyzed based on an accelerated failure time (AFT) survival analysis model. Overall, the success rate of reaching the platform was higher in the 1,1-DEE-treated group than in the control group, especially at the initial 20-second time point, where the success rate in the treated group was 33%, significantly higher than that in the control group (0%) (p<0.05). On the other hand, there was no statistically significant difference between the two groups at the 40-second and 60-second time points. (B): Mice treated with 1,1-DEE showed a direct swimming path toward the platform, while mice in the control group showed an irregular random path. (C): Comparison of the swimming path length of mice that successfully found the platform showed that the mean length in the 1,1-DEE-treated group was 146.3 cm, significantly shorter than that in the control group (mean 820.8 cm) (p < 0.01, t-test). All data are expressed as mean ± standard error (SE).

[0083] Figure 28 The results showed that 1,1-DEE can enhance learning and memory in normal mice. ([A–D]: Time-to-event curves representing the cumulative rate of reaching the platform over time show the results of the pre-test (A), the test after the first round of dosing (B), the test after the second round of dosing (C), and the test after the third round of dosing (D). Although there were no statistically significant differences between the 1,1-DEE treatment group and the control group in each stage (p>0.05), the 1,1-DEE treatment group generally showed a faster trend of reaching the platform after repeated dosing. (E): Comparing the mean escape time of each group during the pre-test and the subsequent three rounds of testing showed that both groups showed similar initial execution ability. However, the escape time of the 1,1-DEE treatment group gradually decreased with the increase of the number of dosings, while no significant change was observed in the control group. However, there were no statistically significant differences between the groups (p>0.05). (F) In the control group, the time-to-event curves between the pre-test and the subsequent three rounds of testing showed consistent execution without significant changes (p>0.05). (G) Conversely, a significant improvement in performance was observed during testing in the time-event curves of the 1,1-DEE treatment group. In particular, the escape time was significantly reduced in the second round of testing compared to the pre-test (p < 0.01), and a significant improvement was also confirmed in the third round of testing (p < 0.05).

[0084] Figure 29This study demonstrates the effect of AMPK activation through long-term 1,1-DEE treatment on improving mitochondrial respiration and glycolysis. (A–O) AC16 cells were treated for 8 hours with various concentrations of 1,1-DEE, 1,2-DEE, and metformin. Mitochondrial respiration was assessed using the Agilent Seahorse XF96 Cell Mito Stress Test Kit. Mitochondrial parameters were assessed for each treatment group (1,1-DEE, 1,2-DEE, metformin), including oxygen consumption rate (OCR, A, E, I), basal respiration (B, F, J), ATP-linked production (C, G, K), and maximal respiration (D, H, L). Glycolysis was assessed after treatment with 1,1-DEE (M), 1,2-DEE (N), and metformin (O). p–t, AC16 cells were treated under three conditions. The conditions were 1,1-DEE (15 mM) and compound C (10 μM, 4 h pretreatment) with 1,1-DEE (15 mM) and metformin (15 mM) for 8 h. Parameters measured included mitochondrial OCR (P), basal respiration (Q), ATP-associated production (R), maximal respiration (S), and ECAR (T). Data are expressed as mean ± SEM of three independent experiments (1,1-DEE) and 2–3 independent experiments (1,2-DEE, metformin). Multiple t-detection was used to calculate p-values ​​(nsp>0.05, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001).

[0085] Figure 30The results of 1,1-DEE treatment in C57BL / 6J mice are shown as an assessment of improved mitochondrial function. Eight-week-old C57BL / 6J mice (n=3) were administered 1,1-DEE for up to 8 weeks. RNA-seq analysis was performed using mouse livers. (A–G) Preranked analysis using GSEA was used to assess the enriched genomic processes (GOBPs) in the 1,1-DEE-treated groups. The assessed processes were “GOBP NADH dehydrogenase complex assembly” (A), “GOBP oxidative phosphorylation” (B), “GOBP mitochondrial respiratory chain complex assembly” (C), “GOBP aerobic respiration” (D), “GOBP aerobic electron transport chain” (E), “GOBP ATP biosynthesis” (F), and “GOBP ATP synthesis coupled with electron transport” (G), which were not enriched in the untreated groups. (H) Heatmap shows the major genetic differences in expression between the untreated and 1,1-DEE-treated groups.

[0086] Figure 31 Survival curves of rats after injection with 1,1-DEE (300 μl) according to the invention or normal saline (NS, 300 μl) are shown.

[0087] Figure 32 These are photographs used to confirm the health indicators (hair condition, weight, activity, etc.) of rats injected with 1,1-DEE (300 μl) or normal saline (NS, 300 μl) according to the present invention. (A) Rats administered 1,1-DEE (left: 688 g, right: 796 g) (B) Rats administered normal saline (NS) (left: 620 g, right: 781 g) Figure 33 The results show a comparison of cell migration status at 0 HR (after template removal) and 12 HR after treatment with different concentrations (1 mM, 10 mM, 50 mM) of 1,1-DEE according to the present invention.

[0088] Figure 34 The migration (μm) of the treatment groups with various concentrations (1 mM, 10 mM, 50 mM) of 1,1-DEE according to the present invention is shown. 2 Quantitative results for migration percentage (%) and cell island expansion (fold change).

[0089] Figure 35The results of TMRE metabolic dye staining (A) for treatment groups with various concentrations (1 mM, 10 mM, 50 mM) of 1,1-DEE according to the present invention, and the results of CTCF (Corrected Total Cell Fluorescence) quantitative analysis based on the images (B) are shown.

[0090] Figure 36 The results of SA-β-gal staining for EA.hy926 endothelial cells to assess cellular senescence are shown. Cells were pretreated with 1,1-DEE for 8 hours and then exposed to oxidized low-density lipoprotein (ox-LDL) for 48 hours. Panel (A) shows representative microscopic images obtained at 200× magnification, and panel (B) shows the quantitative percentage of SA-β-gal positive cells. Data are expressed as mean ± standard deviation (mean ± SD) from at least three independent experiments. Statistical significance is indicated by *P < 0.05 and **P < 0.01. Detailed Implementation

[0091] The following provides a detailed description of the mitochondrial biosynthesis promoter containing 1,1-diethoxyethane as an active ingredient and its uses in specific embodiments of the present invention. However, this is merely an example of the present invention and is not intended to limit the scope of the invention. Various modifications can be made to the embodiments within the scope of the present invention, which will be apparent to those skilled in the art. In this specification, unless otherwise expressly stated, "comprising" or "containing" should be understood as meaning the non-limiting inclusion of a certain constituent element (or component) and should not be construed as excluding other constituent elements (or components).

[0092] As used in this specification, the term "treatment" refers to any form of treatment or prevention that provides an individual with a disease or at risk of developing the disease with effects such as improving the individual's condition, slowing the progression of the disease, delaying the onset of symptoms, or alleviating the progression of symptoms. Therefore, the term "treatment" includes preventative treatment for individuals to prevent the onset of symptoms. Furthermore, "treatment" and "prevention" do not imply a cure or complete elimination of symptoms.

[0093] The term “improvement” as used in this instruction manual may refer to at least a reduction in parameters related to the relief or treatment of the condition, such as the degree of symptom reduction, and all behaviors.

[0094] As used in this specification, the term "object" refers to animals including cows, monkeys, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs. For example, the object can be a mammal, particularly a human.

[0095] In this specification, the terms “mitochondrial biosynthesis” and “mitochondrial biogenesis” are used interchangeably. Although “mitochondrial biogenesis” is the preferred term for the formation of new mitochondria in the technical sense, in the context of this specification, both terms are intended to encompass the induction of mitochondrial formation and its functional enhancement.

[0096] 1. Mitochondrial biosynthesis promoters This invention The aim is to provide a mitochondrial biosynthesis promoter containing 1,1-diethoxyethane (1,1-DEE) as the active ingredient.

[0097] Regarding the mitochondrial biosynthesis promoter in this invention, the 1,1-diethoxyethane can increase the expression of PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha), thereby promoting mitochondrial biosynthesis.

[0098] According to the mitochondrial biosynthesis promoter of the present invention, the 1,1-diethoxyethane can increase the transcriptional activity of Nrf1 and Nrf2, which promotes mitochondrial biosynthesis by stimulating the expression of Tfam.

[0099] According to the mitochondrial biosynthesis promoter of the present invention, the 1,1-diethoxyethane can promote aerobic respiration.

[0100] According to the mitochondrial biosynthesis promoter of the present invention, the mitochondrial biosynthesis promoter can be used to prevent, treat or improve ischemic brain diseases, ischemic heart diseases, pulmonary embolism, ischemic-reperfusion injury, stroke, ischemic stroke, migraine, anemia, Parkinson's disease (PD), dementia, Alzheimer's disease (AD), Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis (ALS), Leigh syndrome, mitochondrial myopathy, and mitochondrial encephalopathy. Encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), cirrhosis, Fanconi syndrome, atypical phenylketonuria, asthma, chronic obstructive pulmonary disease (COPD), diabetes, atherosclerosis, heart failure, hypertension, metabolic dysfunction, and metabolic dysfunction-associated steatotic liver disease.Choose one or more from the following groups: MASLD, insulin resistance, hypercholesterolemia, optic nerve diseases, Duchenne muscular dystrophy, myopathies, obesity, depression, osteoarthritis, cancer, cancer cachexia, inflammatory diseases, age-related diseases, cardiovascular diseases, and cognitive impairment or memory impairment.

[0101] In the mitochondrial biosynthesis promoter according to the present invention, the mitochondrial biosynthesis promoter can be used to manufacture compositions for preventing or treating aging-related diseases, as well as compositions for delaying aging or prolonging lifespan.

[0102] In the mitochondrial biosynthesis promoter according to the present invention, the mitochondrial biosynthesis promoter can be used to manufacture compositions for the prevention, improvement or treatment of cardiovascular diseases.

[0103] In the mitochondrial biosynthesis promoter according to the present invention, the mitochondrial biosynthesis promoter can be used to manufacture compositions for the prevention, improvement or treatment of cancer and the cachexia it causes.

[0104] In the mitochondrial biosynthesis promoter according to the present invention, the mitochondrial biosynthesis promoter can be used to manufacture compositions for the prevention, improvement or treatment of stroke.

[0105] In the mitochondrial biosynthesis promoter according to the present invention, the mitochondrial biosynthesis promoter can be used to manufacture compositions for the prevention, improvement or treatment of dementia.

[0106] In the mitochondrial biosynthesis promoter according to the present invention, the mitochondrial biosynthesis promoter can be used to manufacture compositions for preventing, improving or treating cognitive or memory decline.

[0107] In the mitochondrial biosynthesis promoter according to the present invention, the mitochondrial biosynthesis promoter can be used to manufacture compositions that enhance immunity, improve physical strength, increase physical endurance, increase energy levels, enhance vitality, enhance physical recovery ability, or support sustained physical activity.

[0108] In the mitochondrial biosynthesis promoter according to the present invention, the mitochondrial biosynthesis promoter can also be used as a (stem) cell differentiation and maturation promoter.

[0109] In the mitochondrial biosynthesis promoter according to the present invention, the concentration of 1,1-diethoxyethane can be 1 mM to 25 mM.

[0110] 2. Uses of mitochondrial biosynthesis promoters The present invention aims to provide a composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for the prevention, treatment or improvement of diseases requiring the promotion of mitochondrial biosynthesis, including pharmaceutical compositions, cosmetic compositions, food compositions and feed compositions.

[0111] 2.1. Diseases requiring promotion of mitochondrial biosynthesis According to one embodiment, The purpose of this invention is to provide a composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient for the prevention, improvement or treatment of diseases requiring the promotion of mitochondrial biosynthesis.

[0112] According to the composition of the present invention, the diseases requiring promotion of mitochondrial biosynthesis can be from ischemic brain diseases, ischemic heart diseases, pulmonary embolism, ischemic-reperfusion injury, stroke, ischemic stroke, migraine, anemia, Parkinson's disease (PD), dementia, Alzheimer's disease (AD), Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis (ALS), Leigh syndrome, and mitochondrial myopathy. Myopathy, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), cirrhosis, Fanconi syndrome, atypical phenylketonuria, asthma, chronic obstructive pulmonary disease (COPD), diabetes, atherosclerosis, heart failure, hypertension, metabolic dysfunction, and metabolic dysfunction-associated steatotic liver disease.Choose one or more from the following groups: MASLD, insulin resistance, hypercholesterolemia, optic nerve diseases, Duchenne muscular dystrophy, myopathies, obesity, depression, osteoarthritis, cancer, cancer cachexia, inflammatory diseases, age-related diseases, cardiovascular diseases, and cognitive impairment or memory impairment.

[0113] (1) Delay aging or prolong lifespan As used in this specification, "aging" refers to the various physiological changes that occur over time after birth, including decreased cell differentiation and proliferation, functional decline, decreased homeostasis of organs and organ systems, and increased susceptibility to external stress and disease. The term "aging-related diseases" refers to diseases that occur more frequently with aging. In this invention, delaying individual aging means reducing the rate of aging over time compared to a control group, i.e., individuals who have not been treated or administered the composition according to one embodiment, thus slowing down the progression of aging.

[0114] As used in this specification, the term "extended lifespan" refers to a longer lifespan of an individual compared to natural conditions. For example, "extended lifespan" can refer to extending the lifespan of an individual determined by innate or acquired factors such as genetic factors. Specifically, "extended lifespan" can include appropriately controlling lifespan control factors determined by an individual's genetic factors, such as the expression level of relevant genes and the production level of relevant proteins, thereby extending individual lifespan; and appropriately controlling lifespan control factors determined by acquired factors such as environmental factors, thereby extending individual lifespan. For example, extended lifespan can refer to extending the natural death period of an organism. In this invention, extended individual lifespan means surviving longer than a control group, i.e., individuals not treated or administered the composition according to one embodiment.

[0115] According to an exemplary embodiment, A composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient is disclosed for the prevention or treatment of age-related diseases and for delaying aging or prolonging lifespan.

[0116] In this invention, the age-related diseases can be one or more diseases selected from the group consisting of Alzheimer's disease, diabetes, Parkinson's disease, Huntington's disease, degenerative joint disease, stroke, angina pectoris, osteoporosis, myocardial infarction, muscular dystrophy, amyotrophic lateral sclerosis, sarcopenia, cirrhosis, chronic kidney disease, macular degeneration, heart failure, geriatric depression, degenerative intervertebral disc disease, spinal stenosis, metabolic syndrome, and decreased lung function.

[0117] In this invention, the age-related disease can be a cognitive impairment disease caused by age, and the cognitive function can specifically be one or more selected from the group consisting of perception, memory, attention, speech comprehension, speech generation, reading comprehension, creation of imagery, learning, and reasoning.

[0118] In this invention, when the disease requiring promotion of mitochondrial biosynthesis is an aging-related disease, the 1,1-diethoxyethane can be added at a concentration of 100 μM or higher to less than 20 mM. Preferably, the 1,1-diethoxyethane can be added at a concentration of 1 mM or higher to less than 10 mM. When the concentration of 1,1-diethoxyethane is below 100 μM, it may not have an effective effect, while a concentration above 20 mM may inhibit development.

[0119] In one embodiment, the 1,1-diethoxyethane (1,1-DEE) according to the invention exhibited a lifespan-extending effect in a Caenorhabditis elegans model without inhibiting development. Therefore, the 1,1-diethoxyethane (1,1-DEE) according to the invention holds promise for effective application in pharmaceutical compositions, cosmetic compositions, food compositions, health functional foods, or feed compositions for delaying aging or extending lifespan.

[0120] (2) Cardiovascular diseases According to an exemplary embodiment, A composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient is disclosed for the prevention, improvement or treatment of cardiovascular diseases.

[0121] In this invention, the pharmaceutical composition can reduce the twist force of myocardial cells.

[0122] In this invention, the pharmaceutical composition can temporarily and rapidly increase heart rate. For example, after administration, the pharmaceutical composition can rapidly increase heart rate within 10 to 60 minutes.

[0123] In this invention, the cardiovascular disease can be one or more diseases selected from the group consisting of hypertension, arrhythmia, heart failure, heart attack, myocardial infarction, atherosclerosis, angina, stroke, cerebral hemorrhage, aneurysm, coronary artery disease, aortic disease, cerebrovascular disease, ischemic disease, pulmonary embolism, peripheral vascular disease, and cardiovascular diseases requiring heart rate promotion. In the pharmaceutical composition according to this invention, the cardiovascular disease requiring heart rate promotion can be one or more diseases selected from the group consisting of bradycardia, bradyrrhythmia, bradycardic angina, heart failure, neurogenic syncope, hypotensive shock, and hypothyroidism.

[0124] In this invention, the bradycardia can be one or more diseases selected from the group consisting of acute bradycardia, vagal bradycardia, beta-blocker overdose-induced bradycardia, bradycardia during anesthesia induction, bradycardia after cardiac arrest, and bradycardia due to neurogenic shock, but is not limited thereto.

[0125] In this invention, the bradycardia may include, but is not limited to, sick sinus syndrome (SSS) or atrioventricular block (AV block).

[0126] In this invention, the hypotensive shock may include cardiogenic shock or septic shock, but is not limited to these.

[0127] In one embodiment, 1,1-diethoxyethane (1,1-DEE) according to the invention can temporarily and dramatically increase heart rate, and is therefore effective for the prevention, treatment, or improvement of cardiovascular diseases. The compositions of the invention can be used not only for pharmaceutical purposes but also as functional foods and animal feed, thus providing a variety of application possibilities. For example, when used as functional foods, it can aid in personal health management requiring heart rate regulation; when used as animal feed, it can help regulate the heart rate of livestock or pets. Therefore, the present invention provides a novel treatment method that safely and effectively increases heart rate using 1,1-DEE, and proposes an innovative technology that helps prevent and treat cardiovascular diseases requiring heart rate promotion.

[0128] (3) Cancer According to an exemplary embodiment, A composition containing 1,1-diethoxyethane as an active ingredient for the prevention, improvement or treatment of cancer is disclosed.

[0129] In this invention, the cancers mentioned can include pancreatic cancer, lung cancer, mesothelioma, stomach cancer, esophageal cancer, liver cancer, iliary tract cancer, bladder cancer, head and neck cancer, oral cancer, nasopharyngeal cancer, adult brain cancer, colon cancer, rectal cancer, colorectal cancer, prostate cancer, ovarian cancer, cervical cancer, uterine cancer, testicular cancer, lymphoma, leukemia, skin cancer, breast cancer, kidney cancer, neuroblastoma, and Merkel cell carcinoma. The disease is selected from the group consisting of cellcarcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma.

[0130] In this invention, the 1,1-diethoxyethane can activate adenosine monophosphate activated protein kinase (AMPK).

[0131] In this invention, the 1,1-diethoxyethane can induce apoptosis.

[0132] In this invention, the 1,1-diethoxyethane can prevent or treat cachexia caused by the cancer.

[0133] In one embodiment, the 1,1-diethoxyethane (1,1-DEE) according to the present invention can significantly reduce cancer cell survival and induce apoptosis, thereby exhibiting an anti-cancer effect, and can provide a novel anti-cancer mechanism by regulating energy metabolism through activation of the AMPK pathway. Furthermore, tumor growth inhibition effects have been confirmed through in vivo experiments, and it can also provide additional therapeutic effects such as prevention of cancer cachexia and improvement of survival. Moreover, unlike 1,2-DEE, 1,1-DEE exhibits selective effects, and therefore can be used as an anti-cancer candidate with excellent safety and efficacy. Therefore, compositions containing 1,1-diethoxyethane according to the present invention can be effectively used as pharmaceutical compositions, food compositions, cosmetic compositions, or feed compositions for the prevention, treatment, or improvement of cancer and its cachexia.

[0134] (4) Stroke In this invention, "stroke" refers to sudden localized neurological deficits caused by abnormal cerebral blood flow. Such strokes may include cerebral infarction (due to thrombosis or other obstruction of cerebral blood vessels leading to brain cell death) or cerebral hemorrhage (due to rupture of cerebral blood vessels). Specific symptoms of stroke include sudden headache and vomiting, possible hemiplegia or localized paralysis, localized sensory numbness and loss, and possible speech disorders (aphasia or speech impairment), facial nerve disorders, and motor incoordination. In this invention, stroke may also refer to apoplexy.

[0135] According to an exemplary embodiment, A composition containing 1,1-diethoxyethane as an active ingredient is disclosed for the prevention, improvement or treatment of stroke.

[0136] In this invention, the stroke can be an ischemic stroke or a hemorrhagic stroke.

[0137] In this invention, the composition can promote the recovery of motor function impaired by stroke or reduce the infarct area.

[0138] In one embodiment, 1,1-diethoxyethane (1,1-DEE) according to the present invention exhibits therapeutic effects in ischemic stroke (mouse MCAO model) such as improving survival rate by reducing mortality, improving overall condition by restoring weight, improving spatial cognition and memory, and protecting nerves by reducing brain damage, thereby indicating that the substance may be an effective therapeutic substance for neuroprotection and cognitive recovery in ischemic stroke.

[0139] (5) Dementia According to an exemplary embodiment, A composition containing 1,1-diethoxyethane as an active ingredient for the prevention, improvement or treatment of dementia is disclosed.

[0140] In this invention, the dementia can be vascular dementia, Alzheimer's disease, dementia with Lewy bodies, or frontotemporal dementia (FTD).

[0141] In one embodiment, 1,1-diethoxyethane (1,1-DEE) according to the present invention exhibited effects such as increasing the success rate of reaching the platform, enhancing cognitive function in the early stages of learning, ensuring an effective swimming path, and shortening swimming distance in an ischemic stroke (MCAO-induced mouse model). In particular, it induced improved cognitive function regardless of motor ability, and combined with results obtained by confirming the location of brain injury via MRI, this suggests that 1,1-DEE may be an effective therapeutic substance for cognitive function recovery and neuroprotection after ischemic brain injury.

[0142] (6) Cognitive ability or memory According to an exemplary embodiment, A composition containing 1,1-diethoxyethane as an active ingredient is disclosed for the prevention, improvement or treatment of cognitive or memory decline, or for the enhancement of cognitive or memory abilities.

[0143] In one embodiment, and in another embodiment, repeated administration of 1,1-diethoxyethane (1,1-DEE) according to the invention to a normal mouse model induced an increase in the success rate of reaching the platform and a reduction in the average latency, as measured by the Morris Water Maze test, demonstrating an effect on improving spatial cognition and memory. In particular, although there was no difference in performance between the two groups initially, a significant improvement in performance was observed after repeated administration, indicating that 1,1-DEE is a substance that can improve cognitive function even in normal individuals.

[0144] 2.2. Examples of the Composition (1) Pharmaceutical composition According to the pharmaceutical composition of the present invention, the pharmaceutical composition can be administered orally, intravenously, subcutaneously, intramuscularly, intraperitoneally, epithelially, topically, vaginally, pulmonaryly, rectally, sublingually, buccally, transdermally, ocularly, by inhalation, intracavernosal injection, intrathecal injection, epidural injection, and rectal administration. For oral administration, for example, the pharmaceutical composition can be formulated as an uncoated tablet, or coated with an active agent or otherwise protected from degradation in the stomach. Furthermore, the composition can be administered via any device capable of transporting the active substance to target cells. The route of administration may vary depending on the general condition and age of the patient, the nature of the treatment conditions, and the selected active ingredient.

[0145] According to the pharmaceutical composition of the present invention, the pharmaceutical composition may also be carried on a delivery carrier, which may include, but is not limited to, any one or more selected from viral particles, vesicles, nanoparticles, microparticles, liposomes, transposons, micelles, antibodies and exosomes.

[0146] In the pharmaceutical composition according to the present invention, the appropriate dosage of the pharmaceutical composition varies depending on factors such as the method of formulation, route of administration, patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate, and responsiveness. A skilled physician can easily determine and prescribe a dosage that effectively achieves the desired therapeutic or preventative purpose. For example, the pharmaceutical composition can be administered in single or multiple doses, divided into 1 to 4 daily doses. For example, the dosage of the pharmaceutical composition may include 0.01 mg / kg to 100 mg / kg per adult, preferably 0.02 mg / kg to 90 mg / kg, more preferably 0.03 mg / kg to 80 mg / kg.

[0147] According to the pharmaceutical composition of the present invention, the pharmaceutical composition can be formulated using pharmaceutically acceptable carriers and / or excipients in a manner readily practiced by those skilled in the art, to prepare a single-dose dosage form or dispense it into a multi-dose container. In this case, the dosage form can be a solution, suspension, or emulsion in an oily or aqueous medium, or an extract, powder, granule, tablet, or capsule, and may additionally include dispersants or stabilizers. Furthermore, the pharmaceutical composition can be administered in the form of suppositories, sprays, ointments, creams, gels, inhalers, or skin patches. Moreover, the pharmaceutical composition can be formulated for administration to mammals, more preferably for administration to humans.

[0148] In the pharmaceutical compositions according to the present invention, the pharmaceutically acceptable carrier may be a solid or a liquid, and may be selected from one or more of excipients, antioxidants, buffer solutions, antibacterial agents, dispersants, adsorbents, surfactants, binders, preservatives, disintegrants, sweeteners, flavorings, glidants, controlled-release agents, wetting agents, stabilizers, suspending agents, and lubricants. Furthermore, pharmaceutically acceptable carriers may be selected from saline, sterile water, Ringer's solution, buffered saline, glucose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof.

[0149] In one embodiment, suitable fillers may be sugars (e.g., glucose, sucrose, maltose, and lactose), starches (e.g., corn starch), sugar alcohols (e.g., mannitol, sorbitol, maltitol, erythritol, and xylitol), starch hydrolysates (e.g., dextrin and maltodextrin), cellulose or cellulose derivatives (e.g., microcrystalline cellulose), or mixtures thereof, but are not limited thereto.

[0150] In one embodiment, a suitable binder may be povidone, copovidone, methylcellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, gelatin, gums, sucrose, starch, or mixtures thereof, but is not limited thereto.

[0151] In one embodiment, a suitable preservative may be benzoic acid, sodium benzoate, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol, gallate, hydroxybenzoate, EDTA, or mixtures thereof, but is not limited thereto.

[0152] In one embodiment, a suitable disintegrant may be sodium starch glycolate, croscarmellose, croscarmellose cellulose, starch, microcrystalline cellulose, or mixtures thereof, but is not limited thereto.

[0153] In one embodiment, suitable sweeteners may be sucralose, saccharin, sodium saccharin or potassium saccharin or calcium saccharin, acesulfame potassium or sodium cyclohexylsulfamate, mannitol, fructose, sucrose, maltose or mixtures thereof, but are not limited thereto.

[0154] In one embodiment, a suitable glidant may be silica, colloidal silica, talc, etc., but is not limited thereto.

[0155] In one embodiment, a suitable lubricant may be a long-chain fatty acid and its salts, such as magnesium stearate and stearic acid, talc, glyceryl ester wax or mixtures thereof, but is not limited thereto.

[0156] (2) Cosmetic Composition According to the cosmetic composition of the present invention, the cosmetic composition can be used for skin regeneration, improvement of wrinkles, or skin moisturization.

[0157] According to the cosmetic composition of the present invention, the cosmetic composition may further include a dermatologically acceptable carrier. The dermatologically acceptable carrier may include, but is not limited to, purified water, oil, wax, fatty acids, fatty alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, preservatives, lower alcohols, etc., and their types and concentrations vary, including those that can be modified within the scope of the present invention by those skilled in the art.

[0158] In addition to the active ingredients of the present invention, the cosmetic composition of the present invention may also contain functional substances as needed, such as whitening agents, moisturizers, anti-inflammatory agents, antibacterial agents, antifungal agents, vitamins, ultraviolet blocking agents, antibiotics, anti-acne agents, fragrances, dyes, etc. The content of these substances can be included in the cosmetic composition of the present invention according to commonly used amounts in the cosmetic field. To enhance its functional effects, the cosmetic composition of the present invention may also contain one or more additional moisturizing active ingredients with the same or similar functions.

[0159] According to the cosmetic compositions of the present invention, the cosmetic compositions can be formulated into common emulsifier and solubilizer types. Emulsifier-type cosmetics include nourishing toners, creams, and serums, while solubilizer-type cosmetics include softening toners. In addition to the active ingredients of the present invention, the cosmetic compositions may also contain dermatologically acceptable mediators or bases, prepared in an adjuvant form commonly used in this field for topical or systemic application. Suitable cosmetic dosage forms may include, for example, solutions, gels, solid or paste-like anhydrous products, emulsions obtained by dispersing an oil phase into an aqueous phase, suspensions, microemulsions, microcapsules, microspheres or ionic (liposomes), nonionic microcapsule dispersants, as well as creams, lotions, emulsions, powders, ointments, sprays, or concealer sticks. Furthermore, they can also be prepared in the form of foam or aerosol compositions containing more compressed propellant.

[0160] According to the cosmetic composition of the present invention, the dosage form of the cosmetic composition may be any one or more selected from the group consisting of solutions, topical ointments, creams, foams, nourishing lotions, softening lotions, perfumes, face masks, softening lotions, emulsions, primers, serums, soaps, liquid cleansers, shower gels, sunscreens, sun oils, suspensions, emulsions, pastes, gels, skin lotions, pressed powders, soaps, facial cleansers containing surfactants, oils, liquid foundations, emulsion foundations, wax powders, patches, and sprays.

[0161] (3) Food composition The term "food" as used in this instruction manual refers to natural or processed products containing one or more nutrients, preferably processed to a certain degree to reach a state that is ready for direct consumption. In a conventional sense, it can include food, food additives, functional foods, and beverages.

[0162] The terms "functional food" or "health functional food" as used in this specification refer to food products that are given added value through physical, biochemical, or bioengineering methods to play a role and demonstrate function for a specific purpose. Alternatively, they refer to foods that, through design and processing, allow the biological defense rhythm regulation, disease prevention, and recovery-related regulatory functions of the food combination to be fully expressed in the body. Specifically, they can be health functional foods. These functional foods may include food additives that are acceptable in food science, as well as appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods. The types of health supplements are not limited to these and may include powders, granules, tablets, capsules, or beverage forms.

[0163] According to the food composition of the present invention, the food composition can be used to improve fatigue or enhance motor performance. For example, the food composition can be used to prevent or improve symptoms such as physical fatigue, muscle fatigue, muscle pain, decreased muscle function, muscle loss or degeneration leading to muscle diseases, muscle atrophy, low explosive power or low endurance, etc., but is not limited thereto.

[0164] According to the food composition of the present invention, the food is characterized in that it is meat, sausage, bread, chocolate, candy, snacks, pastries, pizza, instant noodles, other noodle products, chewing gum, dairy products including ice cream, various soups, beverages, tea, coffee drinks, energy drinks, alcoholic beverages, or vitamin complexes.

[0165] The food composition according to the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Furthermore, the composition of the present invention may contain fruit pulp used in the manufacture of natural fruit juices, fruit juice beverages, or vegetable beverages. These ingredients may be used alone or in combination.

[0166] In the food composition of the present invention, the "functional food" or "health functional food" in the food composition refers to a food that is given added value through physical, biochemical, or bioengineering methods, and plays a role and embodies a function for a specific purpose; or it refers to a food that, through design and processing, allows the in vivo regulatory functions related to biological defense rhythm regulation, disease prevention, and recovery possessed by the food composition to be fully expressed in the organism. Specifically, it can be a health functional food. The functional food may include food additives that are acceptable in food science, and may also include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.

[0167] (4) Feed composition According to the feed composition of the present invention, the feed contains nutrients such as energy, protein, lipids, vitamins, and minerals required by animals. It can be plant-based feed such as grains, roots and fruits, food processing by-products, algae, fiber, oils, starches, gourds, and grain by-products, or animal-based feed such as protein, inorganic substances, oils, minerals, and single-cell proteins, but is not limited thereto.

[0168] According to the feed composition of the present invention, the feed may be powdered feed, solid feed, wet pellet feed, dry pellet feed, EP (extruder pellet) feed, fresh feed, etc., but is not limited thereto.

[0169] According to the feed composition of the present invention, the feed composition may contain binders, emulsifiers, preservatives, etc., added to prevent quality degradation, and the feed composition may contain feed additives. To enhance efficacy, amino acid preparations, vitamin preparations, enzymes, flavoring agents, non-protein nitrogen compounds, silicate preparations, buffers, extractants, oligosaccharides, etc., may be added to the feed. Furthermore, feed mixing agents may be included, but the invention is not limited thereto.

[0170] 3. Treatment methods for diseases requiring mitochondrial biosynthesis promoters This invention The object of the present invention is to provide a method for the prevention or treatment of diseases requiring the promotion of mitochondrial biosynthesis, including the step of administering a pharmaceutical composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient to an individual. According to an exemplary embodiment, the object of the present invention is to provide a method for the prevention or treatment of age-related diseases, cardiovascular diseases, cancer, stroke, dementia, or cognitive or memory decline, including the step of administering a pharmaceutical composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient to an individual.

[0171] According to the method of the present invention, the individuals may include, but are not limited to, humans, cattle, monkeys, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs.

[0172] According to the method of the present invention, the route of administration, dosage, and frequency of administration of the pharmaceutical composition can be adjusted according to the patient's condition and the presence or absence of side effects, and the optimal route of administration, dosage, and frequency of administration can be selected by a person skilled in the art within an appropriate range. In this invention, the preferred dosage of the exosomes or pharmaceutical composition can be controlled within the range of 0.001 mg / kg to 100 mg / kg per day for adults, depending on the patient's condition, weight, sex, age, severity of illness, and route of administration. It can be administered once daily or in multiple divided doses. This dosage should not be construed as limiting the scope of the invention in any way.

[0173] The following embodiments are provided to aid in understanding the present invention. These embodiments are merely for illustrative purposes and the scope of protection of the invention is not limited to these embodiments.

[0174] <Materials and Methods> 1. Materials 1,1-DEE (A902), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (#35845), N-acetyl-L-cysteine ​​(A9165), and hydrogen peroxide (H2O2, #88579) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1,2-DEE was purchased from Tokyo Chemical Industry (Tokyo, Japan). The EZ-Cytox assay kit (EZ-3000) was purchased from DoGenBio (Geumcheon, Seoul, Republic of Korea). MitoTracker® Red CMXRos (#9082) and protease / phosphatase inhibitor cocktail (100×, #5872) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

[0175] The following antibodies were used: AMPK (#2532, 1:1000), phosphorylated AMPK (Thr172) (#2535, 1:1000), ACC (#3676, 1:1000), phosphorylated ACC (Ser79) (#11818, 1:1000), PFKFB2 (#13029, 1:1000), phosphorylated PFKFB2 (Ser483) (#13064, 1:1000), PGC-1α (#2178, 1:1000), Nrf1 (nuclear respiratory factor 1) (#46743, 1:1000), Nrf2 (#12721, 1:1000), and β-actin (#4970, 1:1000) antibody (CST). In addition, PGC-1α (#PA5-72948, 1:1000) and mitochondrial transcription factor A (Tfam) (#MA5-16148, 1:1000) antibodies were purchased from Invitrogen. GAPDH (LF-PA0212, 1:1000) and anti-rabbit IgG horseradish peroxidase-binding (LF-SA8002, 1:5000) antibodies were purchased from AbFrontier (Daejeon, Korea).

[0176] 2. Cell Culture and Processing AC16 cells were maintained in Dulbecco modified Eagle medium / nutrient mixture F-12 (DMEM / F12, LM002-05) supplemented with FBS (fetal bovine serum, S001-01) and 1× penicillin / streptomycin (P / S, LS 202-02) (Welgene, Gyeongsan, Korea). Cells were enzymatically induced with 1× trypsin-EDTA (#25300-062) at 70% density and then passaged. For specific experiments, cells were seeded in 6-well plates and allowed to attach overnight. 1,1-DEE was diluted in serum-free medium before co-culturing with the cells.

[0177] Wild-type (WT) and AMPKα double knockout (DKO) mouse embryonic fibroblast (MEF) cell lines were provided by Gachon University School of Medicine and cultured in DMEM (LM 001-05) supplemented with 10% (v / v) FBS and 1× P / S (Welgene).

[0178] 3. Viability Measurement According to the manufacturer's protocol, the cytotoxicity of 1,1-DEE was assessed using the EZ-Cytox assay. Cells were seeded in 96-well plates (17,000 cells / 10 µL / well) and incubated overnight at 37°C, 5% CO2. The next day, cells were co-cultured with 1,1-DEE for 24 hours. After 24 hours of culture, cells were treated with EZ-Cytox reagent (1:10 v / v) and cultured for 30 minutes. The signal was detected at 450 nm using a microplate reader. Cell viability was calculated using the following mathematical formula 1, and the results were analyzed using GraphPad Prism 8.0.1 (San Diego, CA, USA).

[0179] [Mathematical Expression 1] In the mathematical formula 1, "blank" represents the absorbance of wells containing cell-free culture medium and EZ-Cytox, "control" represents the absorbance of wells containing cells and EZ-Cytox but not treated with 1,1-DEE, and "exp" represents the absorbance of wells containing cells, 1,1-DEE, and EZ-Cytox.

[0180] 4. Assessment of mitochondrial oxygen consumption (OCR) and extracellular acidification rate (ECAR) According to the manufacturer's protocol, mitochondrial OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) were assessed using the Agilent Seahorse Mito Stress Test Kit (#103015-100, Seahorse Bioscience, Houston, TX, USA). Prior to the assay, AC16 cells were cultured at 1.8 × 10⁻⁶ cells / cells. 4 Cells / well were seeded at a density of 96-well Seahorse cell culture microplates and allowed to adhere overnight. The sensor cartridge was hydrated in 200 µL of Seahorse XF calibrator (#100840-000) and incubated overnight in a CO2-free 37°C incubator. Following the modified protocol, cells were washed on the day of assay and replaced with 180 µL / well of Seahorse XF DMEM (#103575-100). Using a Seahorse Xfe96 analyzer (Agilent, California, USA), 1,1-DEE, oligomycin (1.5 µM), carbonyl cyanide-4 (trifluoromethoxy)phenylhydrazone (FCCP) (1.5 µM), and rotenone / antimycin (0.5 µM) were injected sequentially into the injection port to assess OCR and ECAR. Results were analyzed using GraphPad Prism 8.0.1.

[0181] 5. Immunofluorescence staining and flow cytometry analysis Cells were seeded in 6-well plates and incubated overnight at 37°C and 5% CO2. Cells were then exposed to 1,1-DEE. For immunofluorescence staining, cells were washed with PBS after specific treatment and cultured in 10 µM DCFH-DA for 30 min at 37°C and 5% CO2 for ROS detection, or cultured in 200 nM MitoTracker® Red CMXRos for mitochondrial labeling. Cells were washed twice with PBS after culture. Images were captured using a fluorescence microscope. For flow cytometry analysis, cells were co-cultured with DCFH-DA or MitoTracker, treated with trypsin, collected, and fixed in 70% ethanol at 4°C for 30 min. DCFH-DA signals were detected using FACS Canto II at excitation and emission wavelengths of 485 nm and 535 nm, respectively, and MitoTracker Red signals were detected at excitation and emission wavelengths of 579 nm and 599 nm, respectively.

[0182] 6. Western blot analysis Cells were washed twice with Dulbecco phosphate-buffered saline (DPBS, LM 001-01, Welgene) and then lysed using lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.1% NP-40, and 1× protease / phosphatase inhibitor. The lysates were placed on ice and vortexed every 5 minutes for 30 minutes. Cell debris was removed by centrifugation at 13,000 rpm for 10 minutes. The supernatant was then collected and protein concentrations were determined using a BCA protein assay kit (Pierce, USA). The lysates were mixed with sample buffer containing 60 mM Tris (pH 6.8), 25% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.5% bromophenol, and 2-mercaptoethanol, and then heated at 95°C for 5 minutes. The samples were then subjected to SDS-PAGE, electrophoresis, and transferred to an Immobilon PVDF membrane. The membrane was added to TBST containing 3% BSA, blocked at room temperature for 1 hour, and then immunoblotted overnight at 4°C with primary antibody. The next day, the membrane was washed three times with TBST and then incubated for another hour with equivalent secondary antibody. Finally, protein expression was detected using Immobilon chemiluminescent HRP matrix (#P90720, Merck Millipore) and Fusion Solo Vilber Lourmat (Vilber GmbH, Eberhardzell, Germany). Protein quantification was performed using ImageJ software (ImageJ 1.5i, National Institutes of Health, Bethesda, MD, USA).

[0183] 7. RNA extraction, semi-quantitative PCR-grade quantitative real-time PCR (qRT-PCR) Total RNA was extracted using Trizol reagent (#155960626, Ambion, Carlsbad, California, USA). RNA concentration was determined using a NanoDrop spectrophotometer; an A260 / A280 ratio of 1.8–2.0 indicated purity. Improm-II was used according to the manufacturer's protocol. TM Complementary DNA (cDNA) was synthesized using reverse transcriptase (A3802, Promega, Madison, USA). The cDNA product was then applied to semi-quantitative PCR using AccuPower® PCR Premix (Bioneer, Daejeon, Korea). Additionally, TOPreal was used...TM qRT-PCR was performed using a SYBR Green qPCR Premix (#RT500S, Enzynomics, Daejeon, Korea). Primers were purchased from Bioneer and are listed in Table 1 below.

[0184] Table 1 8. Statistical Analysis Statistical analysis was performed using GraphPad Prism 8.0.1. Data are expressed as mean ± standard error (SEM). For statistical significance, a two-way ANOVA was used, and p ≤ 0.05 was considered statistically significant.

[0185] <Results> 1. Does 1,1-DEE temporarily inhibit OCR and ECAR in AC16 cells?

[0186] EZ-Cytox analysis was performed to evaluate 1,1-DEE ( Figure 1 A) Effect on cell viability: Determining the concentration used in the experiment. Cells were treated with different concentrations of 1,1-DEE for 24 hours, followed by the addition of EZ-Cytox reagent. Cell viability was assessed, and the results showed that no cytotoxicity was observed at concentrations below 25 mM. Figure 1 B). Treatment with the same concentration of metformin also showed safe results, therefore 25 Mm was selected as the maximum treatment concentration in subsequent experiments.

[0187] Mitochondrial respiration was assessed using the Agilent Seahorse XF96 Cell Mito Stress Test Kit. Cells were acutely injected with different concentrations of 1,1-DEE over 30 minutes, followed by sequential injection of oligomycin, FCCP, and rotenone / antimycin as mitochondrial modulators. OCR and ECAR were monitored. Results showed a reversible response: a sharp decrease in basal OCR followed by rapid recovery after 1,1-DEE injection. OCR decreased at concentrations of 10–20 mM, but did not fully recover after 30 minutes within this concentration range. However, no significant change in OCR was observed after 1,2-DEE injection, while treatment with 5–20 mM metformin showed a gradual decreasing trend in OCR after 30 minutes. Figure 1 C). Furthermore, mitochondrial ATP-coupled respiration, maximal respiration, and residual volume were assessed by injection of oligomycin, FCCP, and rotenone / antimycin. Acute injection of 10-20 mM 1,1-DEE reduced ATP-coupled respiration, maximal respiration, and residual volume, but proton leakage did not change significantly. Figure 1 D).

[0188] ECAR is measured by the amount of pyruvate released per minute after conversion to lactate, reflecting the rate of cellular glycolysis. Upon acute injection of 1,1-DEE, ECAR, like basal OCR, reversibly decreases. Figure 1 However, when treated with 1,2-DEE and metformin, ECAR did not change significantly. Figure 1 E).

[0189] In summary, these results indicate that 1,1-DEE can rapidly modulate mitochondrial and glycolytic functions, inducing a reversible reduction in mitochondrial oxidative phosphorylation (OXPHOS) and cellular glycolysis.

[0190] 2. Does 1,1-DEE activate AMPK? When oxidative phosphorylation (OXPHOS) and glycolysis are reduced due to mitochondrial toxins, eukaryotes possess a highly conserved homeostatic system to cope with the decrease in ATP levels. A key component of this system is AMPK (AMP-activated protein kinase), a major regulator of cellular energy. AMPK is fully activated within minutes of OXPHOS inhibition, enhancing catabolism and reducing anabolism.

[0191] To confirm whether 1,1-DEE affects AMPK activation by inhibiting phosphorylation of AMPK Ser172 residues by OXPHOS, AC16 cells were treated with different concentrations of 1,1-DEE for 10 minutes, and phosphorylated AMPK expression was analyzed. The results showed that 1,1-DEE treatment increased phosphorylated AMPK expression after 10 minutes. Figure 2 A).

[0192] Furthermore, to confirm whether 1,1-DEE transiently induces AMPK activation, cells were exposed to 1,1-DEE at specific time intervals. Phosphorylated AMPK expression transiently increased at 5 and 10 minutes, then decreased, thus confirming that acute exposure to 1,1-DEE can reversibly activate AMPK. Figure 2 B). However, exposure to 1,2-DEE showed no difference in AMPK activation within 30 minutes (B). Figure 2 C), when treated with metformin, AMPK phosphorylation gradually increased after 60 minutes. Figure 2 D).

[0193] 3. To elucidate the mechanism by which AMPK activation induced by 1,1-DEE regulates fatty acid oxidant glycolysis. AMPK regulates fatty acid metabolism through phosphorylation of ACC (acetyl-CoA carboxylase). When AMPK phosphorylates ACC, ACC activation is inhibited, thereby suppressing synthesis. The inhibition of ACC activation by AMPK reduces the production of malonyl-CoA, the matrix of fatty acid synthase (FAS). Malonyl-CoA inhibits fatty acid β-oxidation in mitochondria, enabling AMPK to regulate fatty acid oxidation.

[0194] After treating AC16 cells with different concentrations of 1,1-DEE for 30 minutes, the phosphorylation levels of AMPK's downstream target proteins ACC and PFKFB2 were analyzed. AMPK activated by 1,1-DEE phosphorylates the Ser79 residue of ACC, inhibiting ACC activity, reducing fatty acid synthesis, and thus promoting fatty acid β-oxidation in mitochondria. Figure 3 A). Furthermore, it was confirmed that 1,1-DEE treatment reduced the expression of sterol regulatory element-binding protein-1c (SREBP-1c) and FASN genes, and inhibited ACC activity. Figure 3 B). AMPK is known to also regulate glycolysis by enhancing PFKFB2 activation, and increased phosphorylation of Ser466 and Ser483 residues of PFKFB2 has been observed after exposure to 1-DEE, thereby enhancing PFKFB2 activation and glycolysis. Figure 3 A).

[0195] To confirm whether ACC and PFKFB2 phosphorylation is regulated by AMPK, wild-type (WT) and AMPK double knockout (DKO) MEF cells were treated with 1,1-DEE for 30 minutes. The results showed that, similar to those in AC16 cells, AMPK and ACC phosphorylation was enhanced when WT MEF cells were treated with 1,1-DEE, while this enhanced phosphorylation was not observed in AMPK DKO MEF cells. Figure 3 C). Furthermore, pretreatment with the AMPK inhibitor compound C reduced phosphorylation of the Ser483 residue of PFKFB2, confirming that PFKFB2 activation depends on 1,1-DEE-induced AMPK activation. Figure 3 D).

[0196] 4. Confirm whether 1,1-DEE-induced AMPK activation is mediated by ROS. AMPK activation is primarily promoted by changes in the ADP / ATP and AMP / ATP ratios. However, some studies have reported that AMPK can be activated by ROS, especially H2O2. Therefore, experiments were conducted to confirm whether AMPK is activated by ROS generation.

[0197] After treatment with 1,1-DEE, ROS levels were measured using DCFH-DA and analyzed by live-cell fluorescence staining. Results showed that, compared to the control group, intracellular ROS levels increased after 10 minutes of 1,1-DEE treatment. Figure 4 A). After pretreatment with the ROS scavenger NAC (N-acetylcysteine), the generation of RPS caused by 1,1-DEE was reduced. Figure 4 A).

[0198] Cells were then pretreated with NAC and ebselen, and exposed to 1,1-DEE for 30 minutes. AMPK phosphorylation levels were then analyzed by Western blotting. The results showed that AMPK phosphorylation decreased with the use of a ROS scavenger, indicating that the ROS scavenger inhibited AMPK activation. Figure 4 B).

[0199] 5. Confirm whether long-term treatment with 1,1-DEE increases PGC-1α expression through AMPK activation. This study evaluated the level of AMPK activation after prolonged exposure to 1,1-DEE. After treating AC16 cells with 15 mM 1,1-DEE for 8 hours, AMPK phosphorylation was enhanced at 4 and 8 hours, confirming AMPK activation. Figure 5 A).

[0200] Among AMPK targets, PGC-1α is a crucial factor regulating most genes related to mitochondrial metabolism and is considered a core regulator of mitochondrial biosynthesis. After treating AC16 cells with 1,1-DEE for 8 hours, the mRNA expression of the gene encoding PPARG1Ca for PGC-1α increased. Figure 5 B), Western blot analysis also confirmed that 1,1-DEE-induced AMPK activation increased PGC-1α expression ( Figure 5 C).

[0201] To confirm whether the increase in PGC-1α expression was regulated by AMPK activation, wild-type (WT) and AMPK dual knockout (DKO) MEF cells were treated with 15 mM 1,1-DEE for up to 12 hours. Results showed that in WT MEF cells, AMPK phosphorylation induced by 1,1-DEE was associated with increased PGC-1α expression starting at 2 hours, while no difference in PGC-1α expression was observed in AMPK DKO MEF cells. Figure 5 D).

[0202] 6. Investigating the effects of AMPK on PGC-1 α Mechanisms regulating mitochondrial biosynthesis PGC1-α is a key transcription factor regulating mitochondrial biosynthesis, increasing mitochondrial mass through transcriptional machinery. PGC-1α binds to Nrf1 and Nrf2, enhancing the transcriptional levels of mitochondrial genes associated with the electron transport chain (ETC) complex, while simultaneously promoting the expression of Tfam, responsible for mitochondrial transcription and genome replication. This facilitates the formation of new mitochondria.

[0203] AC16 cells were treated with 15 mM 1,1-DEE for up to 8 hours to assess whether PGC-1α induces mitochondrial biosynthesis via the Nrf1 / Nrf2–Tfam axis. Semi-quantitative PCR and qRT-PCR results showed that Nrf1 and Nrf2 mRNA expression increased after 8 hours of treatment, while Tfam expression significantly increased from 4 hours onwards. Figure 6 A). Western blot analysis also showed a significant increase in the protein expression of Nrf2 and Tfam, while Nrf1 did not ( Figure 6 B).

[0204] To confirm whether AMPK regulates increased expression of the Nrf1 / Nrf2–Tfam axis, wild-type (WT) and AMPK dual knockout (DKO) MEF cells were treated with 15 mM 1,1-DEE for up to 8 hours. Results showed increased protein expression of Nrf1 and Nrf2 in WT MEF cells, but no difference was observed in AMPK DKO cells. Figure 6 C).

[0205] Pretreatment of AC16 cells with the AMPK inhibitor compound C inhibited Tfam protein expression exposed to 1,1-DEE, confirming that mitochondrial biosynthesis via the Nrf1 / Nrf2 / Tfam axis depends on 1,1-DEE-induced AMPK activation. Figure 6 D).

[0206] In flow cytometry analysis using MitoTracker Red, AC16 cells exposed to 1,1-DEE showed increased fluorescence signal and increased mitochondrial activity; pretreatment with compound C resulted in decreased signal. Figure 6 F). Confocal microscopy revealed mitochondrial morphology fragmentation (green arrow) after treatment with 1,1-DEE; this fragmentation was reduced after pretreatment with compound C. No such change was observed with treatment with 1,2-DEE. Figure 6 E).

[0207] 7. Regarding the improvement of mitochondrial respiration and glycolysis by AMPK activation induced by long-term 1,1-DEE treatment. Evaluate After confirming that AMPK activation promotes mitochondrial fatty acid oxidation and biosynthesis signals, mitochondrial respiration capacity was assessed using the Seahorse XF96 Mito Stress Test Kit after 8 hours of 1,1-DEE treatment. Treatment with different concentrations of 1,1-DEE gradually increased mitochondrial OCR (Oxygen Response Rate) over time. Figure 29 A), which is reflected in the treatment group by mitochondrial basal respiration ( Figure 29 B) ATP-related generation ( Figure 29 C) Maximum respiratory volume of the machine ( Figure 29 D) has increased.

[0208] Furthermore, during this treatment, glycolysis was enhanced, and the ECAR value gradually increased, confirming the presence of cellular glycogen. The fermentation process is activated ( Figure 29 M This result indicates that long-term 1,1-DEE treatment promotes mitochondrial oxygen production for ATP generation. OXPHOS and glycolysis processes are impaired. Conversely, 1,2-DEE treatment did not alter mitochondrial OCR and ECAR (oxophosphorylation). Figure 29 E, 29F, 29G, 29H, 29N ）。

[0209] During metformin treatment, the OCR value gradually decreased ( Figure 29 I 、 Figure 29 J 、 Figure 29 K 、 Figure 29 L However, the ECAR value increased. This indicates that cells generate ATP through glycolysis after inhibiting mitochondrial respiration. Figure 29 O Cells were pretreated with Compound C. At that time, compared with the 1,1-DEE treatment group, mitochondrial OCR was reduced, and basal respiration ( Figure 29 P ), ATP-related generation ( Figure 29 Q ) and maximum respiratory volume ( Figure 29 R 、 29S As ) decreases, ECAR is also suppressed. Figure 29 T ）。

[0210] In summary, it can be seen that 1,1-DEE improves cellular energy, specifically by increasing mitochondrial respiration and cellular glycolysis. The strength is mediated through AMPK activation.

[0211] In addition, RNA-seq analysis was performed on the livers of male C67BL / 6 mice after 8 weeks of treatment with 1,1-DEE. GSEA pre- Ranking analysis showed that 1,1-DEE treatment induced the enrichment expression of multiple mitochondrial-associated biological processes (GOBP) (Figure 1). 30A~G). Heatmap analysis of major genes in the "GOBP NADH dehydrogenase complex assembly" showed that, with Compared to the untreated group, in the 1,1-DEE treated group, the NADH: ubiquinone oxidoreductase series genes (ndufv1, ... Moderately increased expression of ndufs2, etc., indicates enhanced mitochondrial oxidative capacity and respiration. Figure 30 H).

[0212] <Experimental Example> Experimental Example 1. Evaluation of the effects of 1,1-DEE on lifespan extension and inhibition of cellular senescence. 1-1. Developmental Experiment of Caenorhabditis elegans In this experiment, the wild-type *C. elegans* Bristol N2 strain was used. Bleached eggs were placed in an S-basal buffer with 1,1-DEE or 1,2-DEE added and cultured in liquid at 20°C until the L1 larval stage. During culture, bleached eggs were placed in 50 ml test tubes and rotated at low speed on a vortex mixer to ensure uniform mixing. Once the worms reached the L1 larval stage, they were separated from the S-basal buffer by centrifugation and released into liquid nematodegrowth media (NGM). At this point, *E. coli* OP50, used as food for the worms, was added to the liquid NGM. The worms were then aliquoted into 96-well plates. Each well contained liquid NGM, OP50, approximately 20 worms, 1,1-DEE or 1,2-DEE (0, 100 μM, 1 mM, 20 mM), and amptorelicin B (0.1 μg / ml), for a total volume of 200 μl. Place 96 wellplates on a shaker and gently agitate to mix, preventing drug precipitation, and incubate in liquid at 20°C. After 2-3 days of incubation, observe the developmental status of *C. elegans* under a microscope to evaluate the effects of 1,1-DEE and 1,2-DEE.

[0213] The results showed that treatment with 1,1-DEE at concentrations of 100 μM and 1 mM did not affect the development of *C. elegans*, but treatment with a concentration of 20 mM inhibited its development. Figure 7 and 8 Furthermore, regarding 1,2-DEE, it was confirmed that neither 100 μM nor 1 mM concentrations caused any problems with the development of *C. elegans*. Figure 9 Based on this, concentrations of 100 M and 1 mM, which would not cause developmental problems, were selected for the Caenorhabditis elegans lifespan experiment.

[0214] 1-2. Lifespan Experiment of Caenorhabditis elegans The 96-well plate setup for the lifespan experiments was the same as for the developmental experiments. However, when the worms reached the L4 stage, 5-fluoro-2'-deoxyuridine (FUDR) was added to prevent egg hatching. Each well had a total volume of 200 μl and consisted of liquid NGM, OP50, approximately 20 worms, 1,1-DEE or 1,2-DEE (0, 100 μM, 1 mM, 20 mM), ampoulecin B (0.1 μg / ml), and FUDR (200 μM). The 96-well plates were gently shaken on a shaker to prevent drug precipitation and cultured in liquid at 20°C. The survival rate of *C. elegans* was measured every 2–3 days; worms not active in the liquid medium were considered dead. All experiments were performed independently by two researchers. Lifespan differences were calculated based on the area under the curve (AUC). The experiments were conducted identically to real-world experiments.

[0215] The results showed that when 1,1-DEE was treated at a concentration of 1 mM, experimenters 1 and 2 observed lifespan extensions of 10% and 13%, respectively. Figure 10 Furthermore, it was confirmed that treatment with 1,1-DEE at a concentration of 100 μM had no significant effect on lifetime. On the other hand, for 1,2-DEE, treatment at a concentration of 1 mM resulted in a -5% and -15% reduction in lifetime observed by Experimenter 1 and Experimenter Observer, respectively, and it was confirmed that treatment with 1,2-DEE at a concentration of 100 μM had no significant effect on lifetime. Figure 11 ).

[0216] This demonstrates that the 1,1-DEE of the present invention, at a concentration of 1 mM, can extend the lifespan of *C. elegans* without affecting its development.

[0217] 1-3. Rat lifespan experiment To confirm the life-extending effect of 1,1-DEE in this invention in a mammalian model, a lifespan experiment was conducted using 48-week-old rats. Twenty rats were randomly divided into two groups (n=10). The control group received a single tail vein injection of physiological saline (300 μl), while the experimental group received a single injection of 300 μL of 1,1-DEE solution at a dose of 300 mg / kg via the same route. All injections were performed at 48 weeks of age under isoflurane anesthesia. For the subsequent 20 weeks, the rats were housed under identical environmental conditions, with two rats from each group in each cage, and consistent feeding and lighting conditions were maintained.

[0218] The results showed that the survival rate of the control group gradually decreased from week 62, with one death at week 62, one death at week 63, one death at week 64, one death at week 65, two deaths at week 66, and two deaths at week 67. In contrast, the 1,1-DEE-treated group only experienced one death at week 67, demonstrating a significant improvement in survival rate. Kaplan-Meier survival curve analysis showed that the survival rate of the 1,1-DEE-treated group was significantly higher than that of the control group (p<0.01, **). Figure 31 Based on the end of week 68, only 2 out of 10 animals in the control group survived, while 9 out of 10 animals in the 1,1-DEE-treated group survived.

[0219] In addition, general health indicators such as hair condition, respiratory rate, and spontaneous activity were assessed. Compared with the control group, the 1,1-DEE-treated group had more lustrous and healthier hair, and their spontaneous activity also showed a significant trend of improvement. Figure 32 These results indicate that 1,1-DEE not only helps extend lifespan but also helps improve health-span.

[0220] 1-4. Endothelial cell senescence experiment To evaluate the anti-aging activity of 1,1-diethoxyethane (1,1-DEE), a cellular senescence model was established using human vascular endothelial EA.hy926 cells. Cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum and antibiotics in a 37°C, humidity-controlled 5% CO2 incubator. Prior to treatment, cells were incubated overnight in serum-free DMEM. Subsequently, cells were pretreated with different concentrations of 1,1-DEE (0, 0.01, 0.1, and 1 mM) for 8 hours, followed by exposure to oxidized low-density lipoprotein (ox-LDL, 100 µg / mL) for 48 hours to induce senescence.

[0221] Senescence-associated β-galactosidase (SA-β-gal) staining was performed using a commercial kit according to the manufacturer's instructions. After fixation, cells were incubated in staining solution at 37°C for 18–24 hours. Stained cells were observed under an optical microscope at 200× magnification, and the proportion of SA-β-gal-positive cells was quantified based on at least three independent experiments.

[0222] Exposure to ox-LDL significantly increased cellular senescence, resulting in a three-fold increase in the proportion of SA-β-gal positive cells compared to the untreated control group (41.3% vs. 14%). Pretreatment with 1 mM 1,1-DEE significantly inhibited ox-LDL-induced senescence, reducing the proportion of positive cells to 23.0% (p < 0.01), a decrease of 44.3%. Lower concentrations (0.01 and 0.1 mM) did not show significant effects. The calculated effect size (Cohen's d = 4.1) indicated that 1 mM 1,1-DEE had a strong biological effect. Figure 36 ).

[0223] These results confirm that 1,1-DEE can effectively inhibit endothelial cell senescence induced by dyslipidemia stress. Combined with the lifespan extension observed in *C. elegans* and rats, these findings suggest that 1,1-DEE exerts both lifespan extension and health-span improvement effects by slowing down cellular senescence.

[0224] Experimental Example 2. Confirming the effect of 1,1-DEE on promoting heart rate. 2-1. Formation of Engineered Heart Tissue (EHT) Cardiomyocytes derived from adult induced stem cells (iPSC-CM) and primary cardiac fibroblasts were mixed at a ratio of 9:1 with 12.5 mg / mL fibrin gel and then loaded into a mold made of 2% agar. A silica gel column was suspended with its tip in contact with the cell and hydrogel mixture, and the mixture was then incubated at 37°C for 30 minutes to harden. After removing the hardened hydrogel from the mold, it was cultured in vitamin A RPMI 1640 cell culture medium for 8 days. The medium was changed every two days, and 20 μg / mL LAPortin was added to the cell culture medium from day 1 to day 7 to prevent fibrin degradation.

[0225] 2-2. Measurement of pulsations treated with 1,1-DEE and 1,2-DEE On day 8 of culture, fine culture medium (RPMI 1640 - vitamin A) was prepared, with 1,1-DEE and 1,2-DEE diluted to final concentrations of 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, and 75 mM, respectively, to replace the old cell culture medium. Before changing the cell culture medium, and 10 minutes, 1 hour, 6 hours, 12 hours, and 24 hours later, the movement of the rod was recorded for 15 seconds using a 4x objective lens. Using self-developed MATLAB code, the rod movement was numerically quantified from the recorded video, and the `findpeaks` function in R was used to analyze the rod's movement distance and the number of movements within 15 seconds. Based on this, the twist force and BPMBPM (Beat per Minute) generated by EHT were calculated.

[0226] Figure 12 and Figure 13 The changes in EHT beat rate (BPM: beats per minute) over time before and after treatment with different concentrations of 1,1-DEE and 1,2-DEE are shown.

[0227] Figure 14 and Figure 15 The changes in the twist force of EHT over time before and after treatment with different concentrations of 1,1-DEE and 1,2-DEE are shown.

[0228] Figure 16 Representative images of EHT pulsation changes resulting from 1,1-DEE and 1,2-DEE treatment (50 mM) are shown.

[0229] Following treatment with 1,1-DEE and 1,2-DEE, in the 1,1-DEE-treated group, the pulsation velocity of EHT decreased proportionally with increasing 1,1-DEE concentration, while the twist force decreased. This change persisted for 1 hour after compound treatment, after which there was no significant difference compared to the 1,2-DEE-treated group. Furthermore, a decreasing trend in pulsation amplitude was observed with increasing pulsation velocity, which was attributed to the decrease in twist force (see [link to article]). Figure 12 Up to 16).

[0230] A comprehensive analysis of the results from high- and low-concentration experiments showed that BPM increased with increasing 1,1-DEE concentration, while twist force decreased. Notably, the effect on BPM recovered approximately 6 hours after treatment, while the effect on twist force persisted for 24 hours.

[0231] also, Figure 17The changes in EHT pulsation velocity and twist force are shown from the immediate post-treatment period to 10 minutes after treatment with 1,1-DEE at a concentration of 25 mM. Figure 17 As shown, BPM began to increase after 1 minute of treatment, and the effect of increasing heart rate became more pronounced over time. On the other hand, twist force gradually decreased over the same time interval, falling to less than 10% of the pre-treatment level at 10 minutes. The results indicate that 1,1-DEE treatment rapidly increases heart rate while inhibiting contractility, suggesting that 1,1-DEE may be a promising composition for promoting acute cardiac pulsation.

[0232] Therefore, the results of this experiment confirm that 1,1-DEE has a sharp increase in EHT heart rate from the time of treatment until 10 minutes to 1 hour, after which this effect disappears. This demonstrates that 1,1-DEE can be used as a drug to temporarily increase heart rate.

[0233] Experimental Example 3. Confirming the effect of 1,1-DEE on cachexia 3-1. Experimental Methods (1) Cell viability detection Cells were packed at 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 µl in 96-well plates and placed in RPMI containing 10% FBS and 1% penicillin and streptomycin. The following day, over 24 hours, different concentrations of 1,1-DEE and 1,2-DEE containing serial dilutions were added to each well (total 120 µl). Next, 10 µl of MTT (DoGenBio Co., Ltd.) was added to each well, and the plates were incubated at 37°C for 2 hours. Cell viability was analyzed by measuring absorbance at 450 nm using a microplate spectrophotometer (Epoch, Biotek, USA).

[0234] (2) Cell culture SHSY-5Y (Human Neuroblastoma) cells were purchased from the Korean Cell Bank (Daehak-ro, Jongno-gu, Seoul, South Korea) and cultured in RPMI 1640 growth medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified incubator containing 5% CO2.

[0235] (3) Western Blot To cultivate SHSY-5Y cells (5 × 10⁻⁶) from 60 × 15 mm cell culture dishes (SPL Life Sciences, Gyeonggi-do, Republic of Korea) 5 Proteins were extracted using a solution of iNtRON Biotechnology (Gyeonggi-do, South Korea) at a concentration of 150 μL / mL. PVDF membranes and Western chemiluminescent HRP matrix were purchased from Millipore Corporation (Billerica, MA, USA).

[0236] After isolating 30 μg of total protein, separation was performed using 10% sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS-PAGE). The proteins were then transferred to a PVDF membrane for further analysis. The membrane was blocked for 1–2 hours with 0.1% Tween-20 added to TBST containing 5% skim milk, followed by overnight incubation at 4°C with primary antibody (1:1000) in TBST. The membrane was then washed three times with TBST for 10 minutes each time, and immunoreactive proteins were detected by chemiluminescence using horseradish peroxidase-conjugated secondary antibody (1:2000).

[0237] Western blot assays used multiple antibodies from Cell Signaling Technology (Danvers, MA, USA), including anti-phosphorylated AMPK (Cell Signaling, #2535S), anti-AMPK (Cell Signaling, #2532S), and anti-β-actin (Cell Signaling, #5125S) monoclonal antibodies. To assess total protein levels, the blot membrane was washed for 30 minutes at 56°C with Restore™ Western Blot Stripping buffer (Thermo Fisher Scientific, Meridian Rd., Rockford, IL, USA).

[0238] (4) Apoptosis detected by Annexin V / FITC According to the manufacturer's protocol, the likelihood of apoptosis in SHSY-5Y cells was assessed using the Annexin V fluorescence kit (BD Pharmingen, San Diego, USA). Cells were cultured at 2.0 × 10⁶ cells per well. 5 Cells were aliquoted into 6-well plates containing 10% FBS and cultured in RPMI medium for 24 hours. Subsequently, the cells were treated with the desired concentration of 1,1-DEE and cultured at 37°C for 24 hours.

[0239] After treatment, cells were harvested and precipitated, then resuspended in 400 µL of binding buffer and stained with 5 µL of FITC-Annexin-V and 10 µL of PI provided in the kit. Following staining, flow cytometry analysis was performed using BD FACS Calibur (BD Biosciences, CA, USA), and plotting and data analysis were performed using FlowJo™ software (BD Biosciences, CA, USA).

[0240] (5) Animal management and administration Four-week-old male NSG mice were purchased from the National Center for Immunotherapy Innovation at Chonnam National University (CNU) in South Korea and housed at the Laboratory Animal Research Center of CNU. Mice were housed in an environment with a temperature of 23°C and humidity of 60%, maintaining a 16-hour day / 8-hour night cycle. Food and water were provided freely before the experimental procedures began. Five animals were housed in each cage, and their weight was recorded three times per week. All experimental procedures involving live animals followed the institutional guidelines of Chonnam National University (CNUIACUC-H-2024-42).

[0241] SHSY-5Y cells cultured in RPMI medium containing 10% FBS were subjected to 10 7 Cells were suspended in 100 μL phosphate-buffered saline (PBS) and administered subcutaneously to the right rib area of ​​each mouse. Mice were divided into a control group and a treatment group. The treatment group received three intraperitoneal (IP) injections of 1,1-DEE (110 mg / kg) (alternating daily) prior to tumor induction for prophylaxis. The treatment group received alternating injections from day 1 to day 105, while the control group received PBS injections. Animals were used for survival studies the day after administration.

[0242] 3-2. Results (1) Evaluation of the cytotoxicity of 1,1-DEE and 1,2-DEE (negative control group) on SHSY-5Y cell line The viability of SHSY-5Y cells treated with 1,1-DEE was determined using the MTT assay, and the results are as follows: Figure 18 As shown in the figure. The results showed that 1,1-DEE at concentrations higher than 10 mM reduced the survival rate of SHSY-5Y cells. Figure 18 A). Conversely, no significant effect was observed in the 1,2-DEE treatment group ( Figure 18 B).

[0243] In addition, to assess changes in cell morphology, the effects of time- and dose-dependent treatment with 1,1-DEE on the morphology of SHSY-5Y cells were observed using an inverted optical microscope. The results showed that 1,1-DEE induced significant morphological changes in a concentration- and time-dependent manner, reducing cell viability, while this phenomenon was not observed in the isoform 1,2-DEE treatment group. Figure 18 C).

[0244] (2) Assessment of 1,1-DEE-induced apoptosis To confirm whether 1,1-DEE induces apoptosis, SHSY-5Y cells were treated with 1,1-DEE for 24 hours, followed by Annexin V and PI staining. The results showed that 1,1-DEE increased the proportion of apoptotic cells corresponding to the Q2 region in a concentration-dependent manner. Figure 19 ).

[0245] (3) Evaluation of AMPK activation induced by 1,1-DEE To confirm the effect of 1,1-DEE on AMPK activation, experiments were conducted using the SHSY-5Y cell line. The results showed that 1,1-DEE activated AMPK in a time-dependent manner, with significant activation observed between 30 minutes and 2 hours. Furthermore, it was confirmed that 1,1-DEE at a concentration of 2 mM exhibited a particularly pronounced concentration-dependent induction of AMPK activation. Figure 20 ).

[0246] (4) Evaluation of the tumor growth inhibition effect of 1,1-DEE To confirm whether 1,1-DEE inhibits tumor growth in vivo, a subcutaneous tumor model was established using 4-week-old NSG mice. In the experimental group, 1,1-DEE was administered intraperitoneally three times, followed by stable subcutaneous injection of SHSY-5Y cells. Tumor size was measured three weeks after inoculation, and a specific concentration of 1,1-DEE was administered every two days, with tumor size being monitored. The results showed that 1,1-DEE significantly inhibited tumor growth in vivo. Figure 21Tumor growth curves for each group in (A) showed that, during the period of 21 to 33 days from tumor palpation, the tumor volume in the 1,1-DEE-treated group was significantly reduced. Additionally, Figure 21 (B) shows the changes in mouse body weight measured during the experiment and assesses the in vivo antitumor effect by measuring survival rate and tumor size.

[0247] (5) Evaluation of the effects of 1,1-DEE on increasing survival rate and preventing cancer cachexia To confirm whether 1,1-DEE plays a preventative role before cancer induction, 1,1-DEE was administered in a metronome pattern at three 1-day intervals before tumor cell injection, and continued thereafter. Figure 22 (A) shows the Kaplan-Meier survival curves, confirming that the survival rate of the 1,1-DEE treatment group was significantly higher than that of the PBS treatment group.

[0248] in addition, Figure 22 (B) shows the weight changes in mice during their survival, which can be used to indirectly assess the occurrence of cancer cachexia. The results showed that the 1,1-DEE treatment group exhibited a trend of inhibiting weight loss, confirming that it may have a preventive effect against cancer cachexia.

[0249] Experimental Example 4. Confirming the efficacy of 1,1-DEE in ischemic stroke. 4-1. Experimental Methods (1) Animals All animal experiments were conducted in accordance with the Chonnam National University Guidelines for the Management and Use of Laboratory Animals. Mice were housed under a 12-hour circadian rhythm with free access to food and water (CNUHIACUC-25015). Eight-week-old male C57BL / 6 (Black 6) inbred mice weighing 27–28 g were used.

[0250] (2) Drugs and administration The test drug was diluted with physiological saline and then administered via a single intravenous injection at a dose of 20 mg / kg through the tail vein.

[0251] (3) Inducing ischemic stroke For the ischemic stroke model, induction was performed via middle cerebral artery occlusion (MCAO). Anesthesia was induced by inhalation of isoflurane mixed with oxygen (Hana Pharm, Seoul, Republic of Korea). After a midline incision in the neck, the left common carotid artery, external carotid artery, and internal carotid artery were exposed. A monofilament approximately 10 mm in diameter was inserted through the external carotid artery to occlude the origin of the middle cerebral artery. The occlusion was maintained for 45 minutes, after which the monofilament was removed to allow reperfusion. The surgical site was sutured, and the mice were monitored during recovery.

[0252] (4) Brain MRI Mice were kept in a supine position, and brain images were acquired using MRI equipment.

[0253] (5) Morris Water Maze (MWM) A water tank containing opaque white water, 100 cm in diameter, 50 cm high, and 30 cm deep, with a water temperature of 24 ± 1°C, was used in an environment where external visual stimuli were blocked. The tank was divided into four zones clockwise, with a hidden platform set up in Zone 1. Mice underwent two days of free exploration training to acclimatize to the environment. During training, each attempt lasted 60 seconds. If the platform was not found, the mice were guided to it and kept there for 10 seconds. Even if the platform was successfully found, the mice were kept there for 10 seconds. All experiments started from Zone 3. During the testing period, exploration time, path, speed, and success rate were recorded and analyzed using the EthoVision XT 14 program.

[0254] 4-2. Results (1) Evaluate the effect of 1,1-DEE on mortality after ischemic stroke. Following MCAO, the control group (n=10) and the treatment group (n=12) were compared. Body weight was measured the day before MCAO, and the treatment group received 1,1-DEE. MCAO was performed under blinded conditions. MRI was performed 36 hours after MCAO, and the microwave-wise mass (MWM) test was performed 48 hours later. Three days later, the mice were sacrificed after their final body weight was measured. Results showed a 30% mortality rate in the control group, while all mice in the 1,1-DEE-treated group survived. Statistical analysis (Chi-square test, p < 0.05) showed a relative risk (RR) of 0.3684 (95% CI: 0.1915–0.9505), indicating a 63.16% reduction in mortality risk in the treatment group. Figure 23 ).

[0255] (2) Confirm the effect of 1,1-DEE on weight loss induced by stroke. Weight loss after stroke is a common phenomenon caused by systemic inflammation and neurological damage, and may lead to sarcopenia and cachexia. Weight loss is closely related to functional recovery. This study also assessed changes in body weight. During the 6-day experimental period, the control group experienced a mean weight loss of 3.77%, while the treatment group gained 5.46% (t-test, p < 0.001). Figure 24 A). It was also confirmed that the final body weight of the treatment group (27.68 g) was significantly higher than that of the control group (26.29 g) (t-test, p < 0.05). Figure 24 B).

[0256] (3) Confirm the effect of 1,1-DEE on enhancing spatial memory and cognitive function. MWM (Milestone Memory Model) is a widely used method for assessing spatial learning and memory in rodents. This study used time-event survival curves integrating arrival time and success rate for analysis. Analysis using the AFT (accelerated failure time) model showed that the success rate in the treatment group was 50% in the last 60 seconds, while it was only 25% in the control group (p > 0.05). At the 20th second, 33% of the treatment group had a success rate, while the control group had zero success, a significant difference (p < 0.05). Figure 25 A). The swimming speeds of the two groups were similar (t-test, p>0.05), indicating that swimming ability did not affect the results.

[0257] Furthermore, the swimming path analysis results showed that the treatment group used a direct path to the target point, while the control group used a random path. Figure 25 B). Comparison of path lengths in successful mice also showed that the path length in the treatment group was significantly shorter than that in the control group (146.3 cm vs. 820.8 cm, t-test, p < 0.01). Figure 25 C). This indicates that 1,1-DEE has a positive effect on the acquisition and maintenance of spatial memory.

[0258] These results confirm that 1,1-DEE treatment can enhance spatial cognition or memory recall, especially in the early stages of topic learning.

[0259] (4) Confirm the effect of 1,1-DEE in relieving cerebral ischemia-reperfusion injury and improving motor function after stroke. Both experimental groups underwent brain magnetic resonance imaging (MRI) within 36 hours of middle cerebral artery occlusion (MCAO). Representative images showed that the infarct area observed in mice administered 1,1-DEE was smaller than that in the control group, suggesting that 1,1-DEE has a mitigating effect on stroke-induced brain injury. Figure 26 A).

[0260] In addition to cognitive impairment, ischemic stroke can also cause motor dysfunction. Although the water maze (MWM) test is not the best method for assessing motor dysfunction, we hypothesized that MCAO-induced stroke might reduce swimming ability in mice. Therefore, the maximum swimming speeds observed in each test were determined. Interestingly, the maximum swimming speeds of mice administered 1,1-DEE were significantly higher than those of the control group (33.83 cm / s vs. 27.77 cm / s, t-test, p < 0.05). This suggests that 1,1-DEE may promote the recovery of motor function after ischemic stroke. Figure 26 B).

[0261] Experiment Example 5. Confirming the effects of 1,1-DEE on cognitive function. 5-1. Experimental Methods The experimental animals, drugs, administration methods, and the Morris water maze test were consistent with those in Example 4.

[0262] 5-2. Results (1) Confirm the effect of 1,1-DEE on improving cognitive function in a mouse model induced by ischemic stroke. Vascular dementia is the second most common type of dementia after Alzheimer's disease, and stroke is one of the major risk factors for dementia. In this embodiment, to confirm the impact of stroke on cognitive function, an ischemic stroke mouse model induced by middle cerebral artery occlusion (MCAO) was used. Experiments were conducted to confirm the effect of 1,1-DEE in this model. Figure 27 Male C57BL / 6 mice were trained in the Morris Water Maze (MWM) experiment and then randomly assigned to either a control group (n=10) or a 1,1-DEE treatment group (n=12). In the treatment group, 1,1-DEE was administered intravenously one day prior to MCAO surgery. MCAO surgery was performed blinded, and the location of the cerebral infarction was confirmed by MRI 36 hours post-surgery. Figure 26 A). The MWM test was performed 48 hours post-surgery, and mice were sacrificed 3 days after the test. The MWM test was evaluated based on latency time and the success rate of reaching the platform, and statistical analysis was performed based on accelerated failure time (AFT).

[0263] The results showed that the rate of reaching the plateau within 60 seconds was higher in the 1,1-DEE treatment group than in the control group, but this was not statistically significant (p>0.05). However, at the 20-second time point, the success rate in the 1,1-DEE treatment group was 33%, while no individuals in the control group achieved success. Figure 27 A). This difference was statistically significant (p < 0.05). Furthermore, there was no significant difference in swimming speed between the two groups (p > 0.05, data not shown), confirming that the difference in cognitive function was not caused by differences in motor ability. Analysis of representative swimming paths for each group showed that the 1,1-DEE treatment group used a strategy of directly approaching the platform, while the control group used a randomized path (…). Figure 27 B). The swimming distances of the mice in the control group were compared. The results showed that the average swimming distance was 820.8 cm, while the average swimming distance of the mice in the 1,1-DEE treatment group was 146.3 cm, which was statistically significantly shorter (p<0.01). Figure 27 C).

[0264] The above results indicate that 1,1-DEE has a positive effect on the recovery of cognitive function after ischemic brain injury.

[0265] (2) Confirm the cognitive function enhancement effect of 1,1-DEE in normal mouse models. This study used normal male C57BL / 6 mice and employed the Morris Water Maze (MWM) test to evaluate the cognitive function-enhancing effect of 1,1-DEE. First, basic executive function was confirmed through a pre-test, and mice were then randomly assigned to a control group (n=12) and a 1,1-DEE treatment group (n=12). The control group received intravenous PBS, while the treatment group received intravenous 1,1-DEE at a dose of 20 mg / kg. Administration was performed on days 1, 5, and 9, with the MWM test conducted two days after each administration, on days 3, 7, and 11. Figure 28 ).

[0266] Preliminary assessment results showed no statistically significant difference in executive function between the two groups. Figure 28 A). After the first dose, 16.7% of the control group reached the plateau within 20 seconds, while 41.7% of the 1,1-DEE-treated group did. After the second dose ( Figure 28 B), the difference was even more pronounced: in the control group, 5 out of 9 (55.6%) successfully reached the platform within 20 seconds, while in the treatment group, 9 out of 10 (90%) successfully reached the platform. Figure 28 C). A similar trend was observed after the third dose. Figure 28D). Although there was no statistically significant difference in cumulative success rate at each time point (p>0.05), the treatment group consistently performed better. The mean latency time (time to reach the platform) was 40.07 seconds for the control group and 39.52 seconds for the treatment group in the pre-test; 38.04 seconds and 33.93 seconds in the first round of testing; 32.00 seconds and 21.14 seconds in the second round of testing; and 41.48 seconds and 29.78 seconds in the third round of testing. Figure 28 E). Although there were no statistically significant differences at any time point (p > 0.05), the results of AFT analysis during repeated testing showed that the latency time in the treatment group was significantly shorter than that in the pre-test (Round 2: p < 0.01, Round 3: p < 0.05). Figure 28 G). These results indicate that 1,1-DEE also improves spatial cognition and memory in mice in a normal state.

[0267] Experimental Example 6. Confirming the effect of 1,1-DEE on cancer. 6-1. Experimental Methods We assessed changes in cell motility and mitochondrial function in MCF7 breast cancer cells treated with 1,1-DEE using MCF7 cells. Cells were constructed into 2D Icelandic morphologies on collagen-coated polyacrylamide gels using a PDMS template. 1.5 × 10⁶ cells were then... 6 Cell suspensions at a concentration of cells / mL were aliquoted into templates at 200 µL and attached at 37°C with 5% CO2 for 3 hours. After attachment, the PDMS templates were removed, washed with PBS, and culture medium containing 1 mM, 10 mM, and 50 mM 1,1-DEE was added. Cell motility was quantified using time-lapse imaging and MATLAB analysis. Mitochondrial function was assessed by TMRE fluorescence staining.

[0268] 6-2. Results The results showed that, in the cell motility analysis, no motility was observed in the 50 mM treatment group due to cell apoptosis. Figure 33 In the quantitative analysis of motility, no significant motility was detected in the 50 mM treatment group. Figure 34 TMRE fluorescence signal analysis showed that mitochondrial function was decreased and functional damage was observed in the 50 mM treatment group. Figure 35 Therefore, it is evident that AMPK activation is closely related to the metabolic regulation and cellular function of cancer cells, and 1,1-DEE can exert anti-cancer effects within an appropriate concentration range.

[0269] Supervising Authority / Funding Agency: Chonnam Technopark Research Project: Support Project for Intellectual Property Capacity Building in Hwasun Vaccine Industrial Park Project Name: IP-R&D Undertaking organization: LuxAnima Ltd. The foregoing has described specific parts of the invention in detail. It will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the invention is not thereby limited. Therefore, the actual scope of the invention should be considered to be defined by the appended claims and their equivalents.

Claims

1. A mitochondrial biosynthesis promoter containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

2. The mitochondrial biosynthesis promoter according to claim 1, characterized in that, The 1,1-diethoxyethane promotes mitochondrial biosynthesis by increasing the expression of PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha).

3. The mitochondrial biosynthesis promoter according to claim 1, characterized in that, The 1,1-diethoxyethane increases the transcriptional activity of Nrf1 and Nrf2, which in turn promotes mitochondrial biosynthesis by stimulating Tfam expression.

4. The mitochondrial biosynthesis promoter according to claim 1, characterized in that, The 1,1-diethoxyethane can promote aerobic respiration.

5. A pharmaceutical composition containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient, for the prevention or treatment of diseases requiring the promotion of mitochondrial biosynthesis, characterized in that, The diseases requiring the promotion of mitochondrial biosynthesis include ischemic brain diseases, ischemic heart diseases, pulmonary embolism, ischemic-reperfusion injury, stroke, ischemic stroke, migraine, anemia, Parkinson's disease (PD), dementia, Alzheimer's disease (AD), Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis (ALS), Leigh syndrome, mitochondrial myopathy, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like events. Acidosis and Stroke-like Episodes (MELAS), Cirrhosis, Fanconi Syndrome, Atypical Phenylketonuria, Asthma, Chronic Obstructive Pulmonary Disease (COPD), Diabetes, Atherosclerosis, Heart Failure, Hypertension, Metabolic Dysfunction, and Metabolic Dysfunction-Associated Steatotic Liver Disease.Choose from the following groups: MASLD, insulin resistance, hypercholesterolemia, optic nerve diseases, Duchenne muscular dystrophy, myopathies, obesity, depression, osteoarthritis, cancer, cancer cachexia, inflammatory diseases, age-related diseases, cardiovascular diseases, and cognitive impairment or memory impairment.

6. A pharmaceutical composition for the prevention or treatment of age-related diseases and for delaying aging or prolonging life, comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

7. A pharmaceutical composition for the prevention or treatment of cardiovascular diseases, comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

8. A pharmaceutical composition for the prevention or treatment of cancer, comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

9. A pharmaceutical composition for the prevention or treatment of stroke, comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

10. A pharmaceutical composition for the prevention or treatment of cognitive or memory decline and impairment, comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

11. A pharmaceutical composition for the prevention or treatment of dementia, comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

12. A cosmetic composition for the prevention or improvement of diseases requiring the promotion of mitochondrial biosynthesis, comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

13. A food composition for the prevention or improvement of diseases requiring the promotion of mitochondrial biosynthesis, comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

14. A feed composition for the prevention or improvement of diseases requiring the promotion of mitochondrial biosynthesis, comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

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