Pharmaceutical composition for extending healthspan and preventing or treating aging-related diseases, comprising phosphodiesterase 5 inhibitor

Abstract

The present invention relates to a pharmaceutical composition for extending healthspan and preventing or treating aging-related diseases, comprising a phosphodiesterase 5 (PDE5) inhibitor as an active ingredient. Specifically, the present invention relates to novel medical uses in which a PDE5 inhibitor, such as mirodenafil, induces AMP-activated protein kinase (AMPK) activation, improves the physical function of a subject through a dietary restriction (DR) mimetic mechanism, and provides effects of inhibiting TDP-43 protein aggregation and preserving intestinal barrier function.

Description

Pharmaceutical composition for extending healthy lifespan and preventing or treating aging-related diseases comprising a phosphodiesterase 5 inhibitor

[0001] The present invention relates to a pharmaceutical composition for extending healthspan and preventing or treating aging-related diseases, comprising a phosphodiesterase 5 (PDE5) inhibitor as an active ingredient. Specifically, the present invention relates to a novel medicinal use in which a PDE5 inhibitor, such as mirodenafil, induces AMP-activated protein kinase (AMPK) activation, improves the individual's physical function through a dietary restriction (DR) mimicking mechanism, and provides effects of inhibiting TDP-43 protein aggregation and preserving intestinal barrier function.

[0002]

[0003] Humanity is facing an unprecedented era of aging, and the importance of "healthspan"—living in a healthy, disease-free state beyond simply extending life expectancy—is being highlighted day by day. Aging is not merely a natural phenomenon that occurs with the passage of time, but a process accompanied by complex pathological changes, such as a decline in intracellular metabolic regulation, the breakdown of protein homeostasis, and the weakening of the barrier function of intestinal epithelial cells. The decline in physiological function associated with aging not only lowers an individual's quality of life but also imposes enormous social and economic burdens. Therefore, research aimed at extending healthspan by directly controlling the aging process is one of the core tasks of modern life science.

[0004] The most effective and reproducible method for extending lifespan known to date is 'Dietary Restriction (DR).' Dietary restriction has been reported to extend lifespan and slow age-related decline in a wide range of species, from invertebrates such as *C. elegans* to rodents and primates. The effects of such dietary restriction are known to manifest by regulating specific evolutionarily well-conserved signaling pathways, such as AMPK (AMP-activated protein kinase), insulin / IGF-1 signaling (IIS), and mTOR (mammalian target of rapamycin). In particular, AMPK serves as a key sensor for detecting the cell's energy state; it is activated under conditions of energy depletion (e.g., dietary restriction) and plays a crucial role in contributing to survival and lifespan extension by reorganizing cellular metabolic processes and maintaining homeostasis.

[0005] However, maintaining strict dietary restrictions for a long period is practically very difficult and has clear limitations for universal application to the human body, as it can cause side effects such as malnutrition or sarcopenia. For this reason, there is a very high demand for the development of 'dietary restriction mimetics (DR mimetics)' that can mimic the positive effects of dietary restriction without the side effects. An ideal dietary restriction mimetic should be able to control the aging process and extend healthy lifespan by pharmacologically activating key pro-longevity pathways, such as the AMPK pathway.

[0006] Meanwhile, phosphodiesterase-5 (PDE5) inhibitors are drugs that inhibit the PDE5 enzyme, which degrades type 2 cyclic guanosine monophosphate (cGMP). Mirodenafil, Sildenafil, and Tadalafil belong to this category and have been primarily used as treatments for erectile dysfunction or pulmonary arterial hypertension. Recent studies, including the applicant's prior registered patent (US 9,750,743), have reported that PDE5 inhibitors have a protective effect on neurons by inhibiting neuronal death that occurs after damage such as cerebral ischemia.

[0007] These prior technologies primarily focus on downstream mechanisms that enhance cell survival rates by inhibiting the 'execution phase,' the stage in which neurons die after damage. However, to more fundamentally control systemic functional decline associated with aging, as well as degenerative neurological diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), it is crucial to preemptively block the breakdown of proteostasis and the failure of intracellular energy metabolism regulation—steps preceding the onset of apoptosis signals. In particular, the abnormal aggregation of TDP-43 protein within neurons serves as the starting point for neurotoxicity, while weakened gut barrier function and the inactivation of the AMPK pathway due to aging are key factors that undermine systemic inflammation and neuronal defense mechanisms. However, these mechanisms have not been specifically elucidated.

[0008] The present invention aims to provide a new therapeutic application that goes a step further than existing cell death inhibition mechanisms by (1) fundamentally blocking the pathological aggregation of TDP-43 protein within neurons, and (2) reprogramming the cell's energy metabolism through AMPK pathway activation and strengthening the intestinal barrier function to control the upstream pathological mechanisms of neurodegeneration and aging.

[0009] The inventors focused on the novel potential of PDE5 inhibitors, which are proven safe drugs, as "metabolic reprogramming agents" that go beyond simple blood flow improvement or cell protection. By mimicking the in vivo Master Energy Sensor (AMPK) without reducing actual calorie intake, they can forcibly switch the biological system to an "energy conservation and repair mode." Furthermore, they identified that these inhibitors act through an upstream mechanism that inhibits the aggregation of the TDP-43 protein, which is the root cause of neurotoxicity. Through this novel pharmacological mechanism, the present invention aims to provide an innovative means to control aging and extend healthy lifespan without the difficulties of dietary restriction.

[0010]

[0011] The main problem that the present invention aims to solve is to provide an effective and safe pharmacological means for extending healthspan, the importance of which is increasing due to population aging.

[0012] More specifically, the present invention aims to solve the following multifaceted and specific problems arising during the aging process by overcoming the practical difficulties of dietary restriction, which is the most certain aging control method known to date, and by providing pharmacological means that mimic its core positive effects.

[0013] First, it is to provide a means to effectively improve and delay neuromuscular decline, a representative sign of aging, particularly sarcopenia characterized by a gradual decrease in muscle mass and strength, as well as physical decline and the resulting functional frailty.

[0014] Second, the invention provides a pharmaceutical composition capable of not only suppressing the decline in cognitive functions, such as memory and learning ability, which inevitably accompanies aging, but also fundamentally inhibiting neurodegeneration by directly blocking the abnormal aggregation (Proteinopathy) of the TDP-43 protein, which is a core pathology of degenerative brain diseases.

[0015] Third, it provides a method to prevent systemic inflammation and the spread of neuroinflammation through the Gut-Brain Axis by repairing damage to intestinal barrier integrity caused by the aging process, such as leaky gut, and reducing permeability.

[0016] Finally, beyond merely improving the aforementioned individual aging-related functional declines in a fragmentary manner, we aim to implement a true 'Dietary Restriction Mimetic' effect as a key higher-level molecular mechanism that induces all these improvement effects, which induces transcriptome reprogramming similar to a dietary restriction state through AMPK pathway activation without reducing actual food intake, thereby providing an advanced anti-aging technology concept that is differentiated from prior art.

[0017]

[0018] Meanwhile, the problems that this Institute seeks to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0019]

[0020] To solve the above problem, the present invention provides a pharmaceutical composition for extending healthy lifespan and preventing or treating aging-related diseases, comprising a phosphodiesterase-5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

[0021] The pharmaceutical composition according to the present invention extends the healthy lifespan of an individual by activating the intracellular AMP-activated protein kinase (AMPK) pathway, thereby inducing a transcriptome profile and metabolic reprogramming similar to that observed during Dietary Restriction (DR) without a reduction in actual calorie intake. In this case, the PDE5 inhibitor activates AMPK by regulating intracellular cGMP concentration, and this acts independently of the insulin / IGF-1 signaling deprivation mechanism (rIIS) or the mitochondrial deprivation mechanism (rMF).

[0022] In addition, the present composition prevents neuronal degeneration by directly inhibiting the abnormal aggregation of TAR DNA-binding protein 43 (TDP-43) in neurons during the aging process or by promoting the degradation of already formed aggregates, and can contribute to the prevention and treatment of TDP-43 proteinopathy-related diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

[0023] Furthermore, the present composition strengthens the tight junction structure between intestinal epithelial cells, thereby effectively improving the increase in intestinal permeability (intestinal leakage) associated with aging. Through this, it can block the systemic influx of intestinal toxins and inflammatory factors, and suppress aging-related systemic inflammation (inflammaging) and neuroinflammation mediated by the gut-brain axis.

[0024]

[0025] According to one embodiment,

[0026] A pharmaceutical composition for improving age-related metabolic decline or extending healthspan is provided, comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition is characterized by activating the intracellular AMP-activated protein kinase (AMPK) pathway to inhibit body fat accumulation without reduction in food intake and induce a transcriptome profile similar to diet restriction.

[0027] In the present invention, the extension of healthy lifespan may include one or more effects selected from the group consisting of delaying the decline in locomotion due to aging, maintaining associative memory, or preserving the tight junction structure of intestinal epithelial cells.

[0028] In the present invention, the individual is a human, and the AMPK pathway may be mediated through the activation of a complex agent comprising a human PRKAA1 or PRKAA2 subunit.

[0029]

[0030] According to another embodiment,

[0031] A pharmaceutical composition for the prevention or treatment of metabolic syndrome is provided, comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition is characterized by reprogramming lipid metabolism by activating the AMPK pathway independently of the insulin / IGF-1 signaling pathway (IIS).

[0032] In the present invention, the metabolic syndrome may include age-related obesity, insulin resistance, or visceral fat accumulation.

[0033]

[0034] According to another embodiment,

[0035] A pharmaceutical composition for the prevention or treatment of age-related intestinal barrier dysfunction or leaky gut syndrome is provided, comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

[0036] In the present invention, the composition can suppress systemic inflammation by blocking the systemic influx of endotoxins or inflammation-inducing factors derived from intestinal microorganisms by strengthening the tight junction structure between intestinal epithelial cells and reducing intestinal permeability.

[0037] In the present invention, the composition can alleviate neuroinflammation caused by increased intestinal permeability or inhibit blood-brain barrier (BBB) ​​damage by mediating the gut-brain axis.

[0038]

[0039] According to another embodiment,

[0040] A pharmaceutical composition for the prevention or treatment of diseases related to TDP-43 proteinopathy is provided, comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition is characterized by inhibiting abnormal aggregation of TDP-43 protein within nerve cells or maintaining proteostasis to reduce neurotoxicity.

[0041] In the present invention, the composition can block the formation of TDP-43 inclusion bodies in the cytoplasm of nerve cells or prevent the axon disconnection of motor neurons and the structural collapse of nerve cords.

[0042] In the present invention, the TDP-43 protein pathology-related disease may be selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), or limbic-dominant senile TDP-43 encephalopathy (LATE).

[0043]

[0044] According to another embodiment,

[0045] A pharmaceutical composition for the prevention or improvement of age-related physical decline or functional frailty is provided, comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

[0046] In the present invention, the improvement can be achieved by increasing the energy metabolic efficiency of muscle cells through AMPK pathway activation or by maintaining the structural integrity of the neuromuscular junction or nerve cord.

[0047]

[0048] According to another embodiment,

[0049] A pharmaceutical composition comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition can delay composite functional decline due to aging by inhibiting the aggregation of TDP-43 protein in neurons through the activation of the intracellular AMPK pathway and simultaneously preserving intestinal barrier function.

[0050]

[0051] In the pharmaceutical composition according to the above embodiment, the PDE5 inhibitor is Mirodenafil, Sildenafil, Tadalafil, Vardenafil, Udenafil, Avanafil, Lodenafil, NBB-116, Cimerafil, RT234, TPN-729, DDCI-01, TOP-N53, TR-422, Gisadenafil, PF-00489791, PF-03049423, SLx-2101, Youkenafil, Padanafil, NCX-1728, TOPV-122, Zaprinast, MSTM-102, TOPT-5, E4021 and It may be selected from the group consisting of tunodafil and their pharmaceutically acceptable salts.

[0052] In the present invention, the PDE5 inhibitor may be mirodenafil or sildenafil, or a pharmaceutically acceptable salt thereof.

[0053] In the present invention, the PDE5 inhibitor may be mirodenafil or a pharmaceutically acceptable salt thereof.

[0054] In the present invention, the composition is a formulation for oral administration, and the daily dosage of the active ingredient may be 1 mg to 100 mg.

[0055] In the present invention, the daily dosage of the active ingredient may be 1 mg to 20 mg.

[0056]

[0057] According to another embodiment,

[0058] A method for extending an individual's healthspan or delaying the decline in aging-related metabolic function, comprising the step of administering a therapeutically effective amount of a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof to an individual, wherein the administration activates the intracellular AMPK pathway to inhibit body fat accumulation without reduction in food intake and induce a transcriptome profile similar to diet restriction.

[0059] In the present invention, the method induces metabolic reprogramming to delay the decline in locomotion associated with aging or to maintain associative memory.

[0060]

[0061] According to another embodiment,

[0062] A method for preventing or treating age-related intestinal barrier dysfunction or leaky gut syndrome is provided, comprising the step of administering a therapeutically effective amount of a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof to an individual.

[0063] In the present invention, the administration can strengthen the tight junction structure of intestinal epithelial cells or reduce intestinal permeability.

[0064] In the present invention, the method can suppress systemic inflammation (inflammaging) or neuroinflammation by blocking the systemic influx of intestinal microorganism-derived toxins or inflammation-inducing factors.

[0065] In the present invention, the administration can suppress systemic inflammation (inflammaging) or neuroinflammation caused by increased intestinal permeability through the Gut-Brain Axis.

[0066]

[0067] According to another embodiment,

[0068] A method for the prevention or treatment of a disease associated with TDP-43 proteinopathy, comprising the step of administering a therapeutically effective amount of a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof to an individual, wherein the administration can reduce neurotoxicity by inhibiting abnormal aggregation of TDP-43 protein within neurons or maintaining proteostasis.

[0069] In the present invention, the TDP-43 protein pathology-related disease may be amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), or limbic-dominant senile TDP-43 encephalopathy (LATE).

[0070]

[0071] In the method according to the above embodiment, the PDE5 inhibitor is Mirodenafil, Sildenafil, Tadalafil, Vardenafil, Udenafil, Avanafil, Lodenafil, NBB-116, Simmerafil, RT234, TPN-729, DDCI-01, TOP-N53, TR-422, Gisadenafil, PF-00489791, PF-03049423, SLx-2101, Youkenafil, Padanafil, NCX-1728, TOPV-122, Zaprinast, MSTM-102, TOPT-5, E4021 and It may be selected from the group consisting of tunodafil and their pharmaceutically acceptable salts.

[0072] In the present invention, the PDE5 inhibitor may be mirodenafil or sildenafil, or a pharmaceutically acceptable salt thereof.

[0073] In the present invention, the PDE5 inhibitor may be mirodenafil or a pharmaceutically acceptable salt thereof.

[0074] In the present invention, the PDE5 inhibitor may be administered at a dose of 1 mg to 100 mg per day, or preferably at a low dose of 1 mg to 20 mg daily.

[0075]

[0076] According to another embodiment,

[0077] A phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof is provided for use in extending an individual’s healthspan or delaying the decline in age-related metabolic function, wherein the use is characterized by inducing a metabolic state similar to dietary restriction through intracellular AMPK pathway activation.

[0078]

[0079] According to another embodiment,

[0080] Phosphodiesterase 5 (PDE5) inhibitors or pharmaceutically acceptable salts thereof are provided for use in the prevention or treatment of age-related intestinal barrier dysfunction or leaky gut syndrome.

[0081] In the present invention, the use may be a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof, characterized by inhibiting systemic inflammation by blocking the systemic influx of intestinal toxins by strengthening the tight junction structure of intestinal epithelial cells and reducing intestinal permeability.

[0082]

[0083] According to another embodiment,

[0084] A phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof is provided for use in the prevention or treatment of a disease associated with TDP-43 proteinopathy, wherein the disease is characterized by abnormal aggregation of TDP-43 protein within neurons.

[0085] In the present invention, the phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof may be characterized in that the disease is amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), or limbic-dominant senile TDP-43 encephalopathy (LATE).

[0086]

[0087] In the phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof according to the above embodiment, the PDE5 inhibitor is mirodenafil or a pharmaceutically acceptable salt thereof, and the mirodenafil may be administered at a dose of 1 mg to 100 mg per day, or preferably at a low dose of 1 mg to 20 mg daily.

[0088]

[0089] A pharmaceutical composition comprising a phosphodiesterase-5 (PDE5) inhibitor or mirodenafil as an active ingredient according to the present invention provides the following significant effects.

[0090] First, unlike conventional PDE5 inhibitors which have been primarily used for hemodynamic effects (vasodilation and blood flow improvement), this invention has identified that the fundamental processes of aging can be regulated through dietary restriction mimicry and AMPK activation pathways, which are distinct from the insulin / IGF-1 signaling inhibition (rIIS) mechanism. In particular, it has for the first time identified a novel medicinal use in which mirodenafil plays a key role in extending an individual's healthy lifespan and delaying aging by activating AMPK through the regulation of cGMP concentration. In particular, this invention possesses technical novelty in that it identifies the AMPK pathway—an intracellular energy sensor—as the essential gateway for lifespan extension and anti-aging effects, rather than the cGMP-PKG (Protein Kinase G) pathway, which a person skilled in the art would expect as the mechanism of action of PDE5 inhibitors. As confirmed in Example 5, the fact that the life-extending effect of mirodenafil is completely lost in the absence of AMPK(aak-2) demonstrates that the drug acts through a novel metabolic regulatory mechanism independent of its existing simple blood flow improvement action.

[0091] Second, transcriptome analysis demonstrated that mirodenafil treatment induces gene expression patterns significantly similar to the Dietary Restriction (DR) model. This allows for the achievement of metabolic remodeling effects equivalent to dietary restriction without actually restricting calorie intake, thereby providing benefits such as inhibiting body fat accumulation and improving metabolic efficiency, as well as extending healthy lifespan.

[0092] Third, unlike conventional PDE5 inhibitor-related technologies (e.g., US 9,750,743) which focused on post-hoc / downstream mechanisms that inhibit the death of already damaged cells, the present invention is differentiated in that it controls upstream pathological mechanisms that serve as the starting point for aging and disease. The present invention provides a disease-modifying effect that controls the starting point of proteotoxicity by preventing aging and degeneration at a stage prior to cells turning on death signals through a fundamental preventive mechanism of inhibiting the pathological aggregation of TDP-43 protein, and defends against the progression of proteinopathy, such as ALS or FTD, at an early stage.

[0093] Fourth, by strengthening the tight junction structure of intestinal epithelial cells to suppress the increase in intestinal permeability associated with aging (leaky gut syndrome), it blocks the systemic influx of endotoxins and inflammatory factors derived from intestinal microorganisms and alleviates systemic inflammation (inflammaging). Accordingly, it can contribute to the prevention and improvement of leaky gut syndrome, inflammatory bowel disease, and irritable bowel syndrome.

[0094] Fifth, the present invention provides a significant effect of fundamentally inhibiting neurodegeneration by blocking the pathological link of the 'Gut-Brain Axis.' Unlike existing PDE5 inhibitor-related technologies (e.g., US 9,750,743), which were limited to a retrospective approach of inhibiting the death of already damaged brain neurons, the present invention blocks the influx of neurotoxicity at the source by inhibiting 'leaky gut,' an upstream cause of aging. Specifically, the present invention strengthens the structural integrity of intestinal epithelial cells, thereby preventing the bloodstream influx of intestinal toxins and inflammatory factors and alleviating systemic inflammation. This consequently inhibits damage to the blood-brain barrier (BBB) ​​and neuroinflammation, leading to a protective effect on motor neurons in a TDP-43 proteinopathy model. In other words, the present invention possesses clinical value not merely as a symptom reliever, but as a disease-modifying therapy that controls age-related neurodegeneration mediated by the Gut-Brain Axis. It can contribute to the prevention and improvement of neurological disorders related to the gut-brain axis, such as Alzheimer's disease, Parkinson's disease, dementia, mild cognitive impairment, and frontotemporal dementia. This represents a new paradigm that controls the causes of brain diseases in the gut rather than the brain.

[0095] Sixth, the present invention minimizes the costs and risks associated with the development of anti-aging agents requiring long-term use and provides high clinical and commercial value by repurposing a PDE5 inhibitor, which has already been used for decades for other indications and whose safety in the human body has been proven.

[0096] In conclusion, the composition of the present invention provides an innovative pharmacological solution capable of addressing the root causes of aging in a safe and effective manner, thereby achieving improvements in systemic physiological functions such as extending healthy lifespan, improving metabolic function, maintaining muscle and nerve function, protecting the intestinal barrier, and inhibiting TDP-43 proteinopathy.

[0097]

[0098] Meanwhile, the scope of the present invention is not limited by the effects described above.

[0099]

[0100] Figure 1A is a schematic diagram of an experiment showing the time of drug treatment initiation (late L4 to beginning of adult stage) to observe the effect of mirodenafil on the healthy lifespan of Caenorhabditis elegans. Figure 1B is a Kaplan-Meier survival curve showing the change in survival rate when Caenorhabditis elegans were treated with mirodenafil at different concentrations (0, 15, 30, 45, 60, 1000 μM). Figure 1C is a Kaplan-Meier survival curve showing the change in survival rate when Caenorhabditis elegans were treated with 60 μM of mirodenafil (Example 1).

[0101] Figure 2A is a graph showing the results of thrashing analysis in a liquid medium, representing the degree of maintenance of neuromuscular function in the mirodenafil-treated group and the control group. Figure 2B is a diagram showing the results of voluntary physical activity analysis (average movement speed and total distance traveled) measured using the WMicrotracker system in a solid medium. Figure 2C is a graph showing the food access rate over time for the mirodenafil-treated group and the control group, representing the results of evaluating food exploration performance (Example 2).

[0102] Figure 3 shows the results of the intestinal permeability (Smurf phenotype) evaluation for the mirodenafil-treated group and the control group in adult nematodes on day 5. Mirodenafil significantly reduces the incidence of intestinal leakage, demonstrating that it maintains the integrity of the intestinal epithelial cell structure (Example 3).

[0103] Figure 4 shows the results of a butanone-based associative learning memory essay. The mirodenafil-treated group maintained a significantly higher chemotactic index (CI) at 2 hours after learning compared to the control group, demonstrating that the memory retention ability of the higher nervous system was improved (Example 3).

[0104] Figure 5A shows the experimental design for collecting transcriptome samples by treating with 60 μM mirodenafil from the late L4 stage to the adult stage. Figure 5B shows the results of PCA analysis of the transcriptome profiles of the mirodenafil-treated group and the control group in adult nematodes on day 3. This demonstrates that the transcriptome signatures between the two groups are clearly distinguishable. Figure 5C shows the results of T-CLASS analysis, indicating that the transcriptome signature of the mirodenafil-treated group has high similarity to the diet restriction (DR) series longevity pathway and is distinguishable from the rIIS or rMF pathways (Example 4).

[0105] Figure 6A is a diagram specifically presenting the identity of the comparison dataset represented by each dot in the analysis of Figure 5C. Figure 6B shows the expression patterns of T-CLASS core genes as a heatmap, demonstrating that the mirodenafil treatment group is similar to the DR series model (Example 4).

[0106] Figure 7 shows survival curves comparing changes in lifespan upon treatment with 60 μM mirodenafil in wild-type (N2) and AMPK(aak-2) loss-of-function variant nematodes (Example 5).

[0107] Figure 8A shows the results of quantifying the degree of body fat accumulation in adult nematodes on day 5 after treatment with 60 μM mirodenafil using Oil Red O staining. Figure 8B shows that there was no change in feeding behavior by measuring the pharyngeal pumping rate of the mirodenafil-treated group and the control group under the same conditions (Example 6).

[0108] Figure 9 shows the pattern of TDP-43 aggregate formation and neuropathy after blue light stimulation on day 3 in adult KWK92 strains treated with 60 μM mirodenafil from late L4 (Example 7).

[0109] Figure 10 shows the results of comparing the degree of neuronal disconnection and motor function (thrashing) between the mirodenafil-treated group and the control group at the chronic pathological stage on day 6 of adulthood (Example 8).

[0110] Figure 11A is a survival curve showing the lifespan extension effect compared to the control group when treated with mirodenafil and sildenafil. Figure 11B is a graph comparing the effect of maintaining thrashing on day 5 of adulthood when treated with mirodenafil and sildenafil compared to the control group. Figure 11C is a graph comparing the effect of inhibiting intestinal permeability on day 5 of adulthood when treated with mirodenafil and sildenafil compared to the control group (Example 9).

[0111]

[0112] The inventors have confirmed through prior research that phosphodiesterase-5 (PDE5) inhibitors, specifically Mirodenafil, exhibit overall anti-aging effects, such as inducing transcriptome changes associated with aging and preserving neuronal function. This specification identifies the inevitable and specific mechanism of action through which such comprehensive anti-aging efficacy is manifested and includes examples demonstrating this.

[0113] Specifically, the 'anti-aging' and 'extension of healthy lifespan' effects described in this specification are the result of inevitable metabolic reprogramming that occurs as the AMPK (AMP-activated protein kinase) pathway, an intracellular energy sensor, is activated upon administration of mirodenafil, as demonstrated through specific examples (Figs. 5 to 8). This means that the composition of the present invention induces the same metabolic benefits as dietary restriction at the molecular level without actual reduction in calorie intake.

[0114] In addition, it was confirmed that the comprehensive 'neuroprotective effect' identified in prior research is achieved by a mechanism that goes beyond simple inhibition of cell death and fundamentally blocks the pathological aggregation of TDP-43 protein, a major causative agent of neurodegeneration (Figs. 9 and 10). Furthermore, the present invention was completed by identifying that these systemic and neurological improvement effects are closely associated with the recovery of gut barrier function, which weakens with aging.

[0115]

[0116] The present invention will be described in detail below. However, this is presented as an example of the invention and does not limit the scope of the invention, and it is obvious to those skilled in the art that various modifications to the embodiments are possible within the scope of the invention. Throughout this specification, unless otherwise specifically stated, "includes" or "contains" refers to the inclusion of any component (or constituent) without any particular limitation and should not be interpreted as excluding the addition of other components (or constituents).

[0117] Definition of Terms

[0118] As used herein, "Healthspan" refers to the period during which an individual lives in good health without the debilitation associated with aging or serious physical or mental impairments. This is a concept that emphasizes qualitative aspects rather than "lifespan," which is merely the period of survival.

[0119] As used herein, "Pharmaceuticalally acceptable" means a compound, substance, composition and / or formulation that can be administered to an individual without undesirable biological or other effects.

[0120] As used herein, "pharmaceuticalally effective amount" or "therapeutically effective amount" means an amount of an active ingredient sufficient to produce the intended pharmacological, therapeutic, or prophylactic effect when administered to an individual.

[0121] As used herein, the term "treatment" (Treating or Treatment) includes reversing, alleviating, inhibiting, or preventing the progression of a disease, disorder, or condition. This may include palliating the symptoms of a disease.

[0122] As used in this specification, "Subject" means a mammal, preferably a human, but is not limited thereto.

[0123] As used in this specification, "Dietary Restriction Mimetic" means a substance that provides metabolic and physiological benefits similar to those of dietary restriction (DR) at a molecular level without actually reducing calorie intake.

[0124] As used in this specification, “TDP-43 proteinopathy” refers to a group of diseases caused by the abnormal aggregation or displacement of TAR DNA-binding protein 43 (TDP-43), including amyotrophic lateral sclerosis (ALS), frontolateral dementia (FTD), and limbic-dominant senile TDP-43 encephalopathy (LATE).

[0125] As used in this specification, "PDE5 inhibitor" means any compound that inhibits the activity of cGMP-specific phosphodiesterase type 5 enzyme (PDE5) to reduce the degradation of cGMP.

[0126]

[0127] 1. Composition for extending healthy lifespan or delaying aging

[0128] The present invention relates to a composition comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition is characterized by reorganizing cell metabolism into a dietary restriction-like state through the activation of the AMP-activated protein kinase (AMPK) pathway, thereby extending the lifespan of an individual and maintaining or restoring physiological and functional homeostasis that decline with aging, thereby enhancing the healthspan or delaying the progression of aging.

[0129] The composition of the present invention enhances intracellular cGMP signaling through PDE5 inhibition and induces AMPK activation as a downstream signaling pathway, thereby inducing transcriptome reprogramming and metabolic remodeling similar to dietary restriction without accompanying a reduction in actual calorie intake.

[0130] The nematode *C. elegans*, used as the primary efficacy model in the present invention, provides high validity in demonstrating the effects of the present invention and exploring its mechanisms. Key mechanisms of aging and longevity regulation, such as the AMPK signaling pathway and the insulin / IGF-1 signaling pathway (IIS), are evolutionarily highly conserved in mammals, including humans. Furthermore, pathological mechanisms of specific neurodegenerative diseases, such as the aggregation of TDP-43 protein, are also effectively reproduced in the aforementioned model animals. Therefore, efficacy data from the *C. elegans* model serve as strong scientific grounds for reasonably predicting the effects in mammals that possess the same conserved pathways. Consequently, there is a reasonable expectation of success that the composition of the present invention, which targets the aforementioned conserved pathways, will exhibit the same anti-aging effects in mammals, particularly humans.

[0131] The PDE5 inhibitors available for use in the present invention are Mirodenafil (AR1001), Sildenafil, Tadalafil, Vardenafil, Udenafil, Avanafil, Lodenafil, NBB-116, Cimerafil, RT234, TPN-729, DDCI-01, TOP-N53, TR-422, Gisadenafil, PF-00489791, PF-03049423, SLx-2101, Youkenafil, Padanafil, NCX-1728, TOPV-122, Zaprinast, MSTM-102, TOPT-5, E4021, and It may be selected from the group consisting of tunodafil and pharmaceutically acceptable salts thereof, but is not limited thereto.

[0132] In the embodiments of the present invention, among the above PDE5 inhibitors, mirodenafil was selected as the representative compound, and the composition of the present invention was intensively verified to determine whether it exhibits effects of promoting healthy lifespan and delaying aging, and its mechanism of action. The above mirodenafil (development code name: AR1001) is a pyrrolopyrimidinone class compound represented by [Chemical Formula 1].

[0133] [Chemical Formula 1]

[0134]

[0135] IUPAC name: 5-Ethyl-3,5-dihydro-2-[5-([4-(2-hydroxyethyl)-1-piperazinyl]sulfonyl)-2-propoxyphenyl]-7-propyl-4H-pyrrolo[3,2-d]pyrimidin-4-one

[0136] Free Base: C 26 H 37 N5O5S / Molecular weight approx. 531.67 g / mol

[0137] Dihydrochloride (2HCl): C 26 H 39 Cl2N5O5S / Molecular weight approx. 604.6 g / mol

[0138] In the present invention, mirodenafil may be used in the form of a free base or a pharmaceutically acceptable salt, and in the examples, mirodenafil dihydrochloride (AR1001·2HCl) was used.

[0139] The present invention is the first to identify, through examples, that the known cGMP signaling pathway following PDE5 inhibition operates in functional linkage with AMPK activation in a physiological context related to aging and healthy lifespan. In particular, prior art has never suggested or indicated that PDE5 inhibitors can induce a diet-restriction-like metabolic state or express effects of promoting healthy lifespan and delaying aging through the AMPK pathway.

[0140] The present invention is not limited to specific PDE5 inhibitors but can be applied to the entire drug class of PDE5 inhibitors. Despite some differences in chemical structure, the PDE5 inhibitors described in the claims of the present invention share a clear and identical mechanism of action, which is the 'selective inhibition of the PDE5 enzyme.' Since PDE5 inhibition leads to the common result of increased intracellular cGMP concentration and subsequent AMPK activation, the effect demonstrated by the representative compound mirodenafil can be understood as a class effect that can be reasonably expected from other PDE5 inhibitors with the same mechanism of action.

[0141] In fact, as confirmed in Example 9 of the present invention (Fig. 11), sildenafil, a homologous drug with a chemical structure different from mirodenafil, also exhibited equivalent levels of lifespan extension, preservation of motility, and intestinal barrier strengthening effects in a Caenorhabditis elegans model. This is a decisive basis supporting the fact that the technical concept of the present invention is not limited to mirodenafil but can be commonly applied to all PDE5 inhibitors and their pharmaceutically acceptable salts described herein. Therefore, the composition of the present invention should be interpreted as a broad anti-aging solution encompassing the entire library of PDE5 inhibitors, including mirodenafil, sildenafil, tadalafil, etc.

[0142] Furthermore, mirodenafil according to a preferred embodiment of the present invention shares a drug class effect of metabolic reprogramming through PDE5 inhibition, while having a unique pharmacological profile that distinguishes it from other PDE5 inhibitors. Specifically, mirodenafil possesses high selectivity for PDE5 as well as an additional mechanism of action that modulates glucocorticoid receptors (GR), thereby providing neuroprotective and anti-inflammatory synergistic effects beyond simple PDE5 inhibition.

[0143] The fact that the mechanism of action identified in this invention can be effectively applied to humans is strongly supported by the evolutionary conservation of the target protein. The AMPK pathway, which is the core mechanism of this invention, is an evolutionarily highly conserved metabolic regulatory system. Specifically, the nematode AAK-2 protein is a functional and structural ortholog to the human AMPKα subunit (PRKAA1 / 2). In the kinase domain responsible for enzyme activity, the two proteins share more than 80% of amino acid sequence homology, and the phosphorylation sites essential for enzyme activation (Thr243 in nematodes, Thr172 in humans) are perfectly conserved. Therefore, the result of 'loss of mirodenafil effect upon aak-2 deficiency' confirmed in Example 5 (Fig. 7) suggests that when this drug is administered to humans, it will exert the same metabolic reprogramming and anti-aging effects through the activation of human PRKAA1 / 2 (AMPK). Furthermore, the fact that sildenafil, whose safety in the human body has already been proven, showed the same effect in this model (Example 9) further increases the clinical applicability of the present invention.

[0144] Accordingly, the present invention provides a novel technical concept that the signaling axis leading from PDE5 to AMPK is functionally involved in regulating aging and promoting healthy lifespan.

[0145]

[0146] (1) Effect of maintaining systemic metabolic homeostasis and inhibiting body fat accumulation

[0147] The composition according to the present invention can normalize whole-body energy metabolism disrupted by aging or metabolic abnormalities, and can maintain or improve whole-body metabolic homeostasis by restoring the balance between energy consumption and storage in the body, and can suppress body fat accumulation without changes in actual food intake.

[0148] According to the example, it was confirmed that in the experimental group administered the composition according to the present invention, body fat accumulation was significantly reduced and metabolism-related indicators were improved without any change in food intake (Example 6).

[0149] Accordingly, the composition according to the present invention can be used for the prevention, treatment, or improvement of obesity, abdominal obesity, dyslipidemia, hypertriglyceridemia, hypercholesterolemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic syndrome, type 2 diabetes, insulin resistance, hyperinsulinemia, etc.

[0150]

[0151] (2) Dietary restriction-like metabolic remodeling and anti-aging effects

[0152] The composition according to the present invention can alleviate age-related metabolic decline and systemic decline and extend lifespan by activating the intracellular AMPK pathway to induce transcriptome-level metabolic remodeling similar to dietary restriction.

[0153] According to the example, transcriptome analysis results confirmed that the composition of the present invention alleviates aging-related metabolic decline by reorganizing the entire metabolic network in the body, similar to the pattern observed under dietary restriction conditions, rather than regulating a single gene (Example 4).

[0154] Accordingly, the composition according to the present invention can be used for the prevention, treatment, or improvement of aging, progeroid syndrome, senescence, age-associated metabolic decline, metabolic syndrome, etc.

[0155]

[0156] (3) Effects on maintaining motor and neuromuscular function

[0157] The composition according to the present invention can preserve the activity of an individual by maintaining or improving motor skills, muscle function, and neuromuscular junction integrity that deteriorate with aging.

[0158] According to the examples, it was confirmed that the distance traveled, exercise endurance, and foraging behavior were significantly improved in the treatment group of the composition of the present invention (Example 2), which suggests a preventive effect against sarcopenia, muscle weakness, muscle atrophy, age-related decline in motor function, and frailty syndrome.

[0159] Accordingly, the composition according to the present invention can be used for the prevention, treatment, or improvement of sarcopenia, age-related muscle weakness, muscle atrophy, age-related motor dysfunction, frailty syndrome, etc.

[0160]

[0161] (4) Effects of protecting the intestinal barrier and maintaining the gut-brain axis

[0162] The composition according to the present invention can protect the intestinal barrier function that is damaged due to aging, strengthen the tight junction structure, and alleviate systemic inflammation and maintain nerve function by inhibiting the systemic influx of intestinal-derived inflammatory factors.

[0163] According to the example, it was confirmed that intestinal permeability-related indicators were improved in the treatment group of the composition of the present invention, and memory retention ability and neural function indicators were significantly improved (Example 3).

[0164] Accordingly, the composition according to the present invention can be used for the prevention, treatment, or improvement of Leaky Gut Syndrome, Inflammatory Bowel Disease, Irritable Bowel Syndrome, Dementia, Alzheimer's Disease, Parkinson's Disease, Mild Cognitive Impairment, Frontotemporal Dementia (FTD), Age-related Cognitive Decline, Neuropathy, etc.

[0165]

[0166] (5) Metabolic regulation and aging delay effects dependent on AMPK activation

[0167] The composition of the present invention can inhibit the progression of aging and the decline in metabolic function by enhancing intracellular cGMP signaling through PDE5 inhibition and activating the AMPK pathway based on this.

[0168] According to the example, under conditions where AMPK function was impaired, the lifespan extension effect of the composition was lost, but under normal conditions, significant lifespan extension and metabolic improvement were confirmed (Example 5).

[0169] Accordingly, the composition of the present invention can be used for the prevention, treatment, or improvement of age-related metabolic decline, breakdown of energy homeostasis, insulin resistance, hyperinsulinemia, type 2 diabetes, metabolic syndrome, hyperglycemia, etc.

[0170]

[0171] (6) Inhibition of TDP-43 protein pathology and improvement of progressive neurodegenerative diseases

[0172] The composition of the present invention inhibits the pathological aggregation of TDP-43 protein or increases its solubility, thereby inhibiting nerve damage and functional decline and fundamentally delaying the course of progressive diseases.

[0173] The nematode *C. elegans*, used as the primary efficacy model in this invention, provides high validity in demonstrating the effects of the invention and exploring its mechanisms. Pathological mechanisms of specific neurodegenerative diseases, such as the aggregation of TDP-43 protein, are evolutionarily highly conserved in mammals, including humans. Therefore, efficacy data from the *C. elegans* model serve as strong scientific grounds for reasonably predicting the effects in mammals that possess the same conserved pathways.

[0174] According to the example, it was confirmed that the decline in neurological function over time was suppressed and motor ability and survival indicators were improved in a TDP-43 protein pathology model (Example 7).

[0175] Accordingly, the composition of the present invention can be used for the prevention, treatment, or delay of progression of amyotrophic lateral sclerosis (ALS), frontolateral dementia (FTD), limbic-predominant age-related TDP-43 encephalopathy (LATE), and other TDP-43-related neurodegenerative diseases.

[0176]

[0177] (7) Effects of maintaining overall function and extending healthy lifespan

[0178] The composition according to the present invention can extend healthy lifespan by not only extending lifespan but also suppressing age-related decline in systemic functions in various ways, such as maintaining exercise function, improving metabolic homeostasis, and neuroprotection.

[0179] In the examples, life extension, maintenance of motor function, improvement of metabolism, and neuroprotection were comprehensively confirmed (Examples 1–7). This demonstrates an unpredictable synergistic effect that maximizes overall healthy lifespan through interaction, going beyond the simple sum of each mechanism, thereby supporting the remarkable inventiveness of the present invention.

[0180] Accordingly, the composition of the present invention can be used to prevent, treat, or improve frailty, age-related functional decline syndrome, healthspan reduction, and quality of life deterioration in the elderly.

[0181]

[0182] 2. Uses of compositions for extending healthy lifespan or delaying aging

[0183] A composition comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient according to the present invention can be implemented as a pharmaceutical composition, food composition, feed composition, or cosmetic composition for extending healthy lifespan or delaying aging.

[0184]

[0185] (1) Pharmaceutical composition

[0186] The above pharmaceutical composition may be administered orally, intravenously, subcutaneously, intramuscularly, intraperitoneally, epithelially, topically, vaginally, pulmonaryly, rectally, sublingually, buccally, transdermally, ocularly, inhaled, intracavernously, intrathecally, epidurally, and rectally. When administered orally, for example, the above pharmaceutical composition may be formulated as a tablet, or the active agent may be coated or protected from degradation in the stomach. Additionally, the above composition may be administered by any device capable of delivering 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.

[0187] The above pharmaceutical composition may be carried on a carrier, and the carrier may include one or more selected from virus particles, vesicles, nanoparticles, microparticles, liposomes, transposons, micelles, antibodies, and exosomes, but is not limited thereto.

[0188] The appropriate dosage of the above pharmaceutical composition varies depending on factors such as the formulation method, mode of administration, patient's age, weight, sex, pathological condition, diet, time of administration, route of administration, elimination rate, and response sensitivity, and a moderately skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. For example, the above pharmaceutical composition may be administered as a single or multiple doses, or divided into 1 to 4 doses per day. For example, mirodenafil is a drug that has already been marketed as a treatment for erectile dysfunction (generally administered orally at a dose of 50 mg or 100 mg once daily) and is currently undergoing Phase 3 clinical trials as a treatment for Alzheimer's disease. Sildenafil, a drug in the same class, is used at doses of 25 mg, 50 mg, and 100 mg, and Tadalafil at doses of 5 mg, 10 mg, and 20 mg. Considering these existing clinical applications, for the purpose of the present invention to improve age-related physiological decline, the active ingredient may comprise a range of about 1 mg to 500 mg per day for adults, preferably 1 mg to 100 mg; depending on the degree of disease progression or the purpose of administration, it may be administered as a therapeutic dose of about 10 mg to about 50 mg, particularly about 30 mg, or subdivided into low-dose ranges such as about 1 mg to about 25 mg, about 1 mg to about 20 mg, about 2 mg to about 25 mg, about 5 mg to about 25 mg, about 5 mg to about 20 mg, or about 5 mg to about 15 mg for minimizing side effects and long-term maintenance therapy; specifically, the said daily dose may be among about 1 mg, 1.5 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, or 50 mg It can be set to any one capacity.The dosage may be administered once a day, or divided into several doses per day (e.g., 2 to 3 times) as needed. In particular, for the purpose of the invention of anti-aging and extending healthy lifespan, it is desirable to adopt a daily or chronic administration method to ensure stable reprogramming of cell metabolism and maintenance of long-term effects.

[0189] The above pharmaceutical composition may be prepared in a unit dose form or contained in a multi-dose container by formulation using pharmaceutically acceptable carriers and / or excipients according to methods that can be easily practiced by a person skilled in the art to which the invention pertains. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer. Furthermore, the above pharmaceutical composition may be administered in the form of a suppository, spray, ointment, cream, gel, inhalant, or skin patch. Additionally, the above pharmaceutical composition may be prepared for administration to mammals, more preferably for administration to humans.

[0190] The above pharmaceutically acceptable carrier may be solid or liquid and may be one or more selected from excipients, antioxidants, buffers, bacteriostatic agents, dispersants, adsorbents, surfactants, binders, preservatives, disintegrants, sweeteners, flavorings, lubricants, release regulators, wetting agents, stabilizers, suspending agents, and lubricants. Additionally, the pharmaceutically acceptable carrier may be selected from saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof.

[0191]

[0192] (2) Food composition

[0193] As used in this specification, the term “food” refers to a natural product or processed product containing one or more nutrients, preferably one that has undergone some degree of processing to become ready for direct consumption, and in a conventional sense may include food, food additives, functional foods, and beverages.

[0194] As used in this specification, the terms “functional food” or “health functional food” refer to a group of foods to which added value has been added to the food by using physical, biochemical, or biotechnological methods to act or manifest the function of the food for a specific purpose, or to foods designed and processed to sufficiently express in vivo regulatory functions regarding the regulation of biological defense rhythms, disease prevention, and recovery, which are inherent in the food composition; specifically, they may be health functional foods. The functional food may include food science-acceptable food additives and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods. The types of health functional foods may include, but are not limited to, powder, granules, tablets, capsules, or beverage forms.

[0195] The above food composition may be used to improve fatigue or enhance exercise performance. For example, the above food composition may be used to prevent or improve physical fatigue, muscle fatigue, muscle pain, decreased muscle function, muscle diseases caused by muscle wasting or muscle degeneration, muscle wasting, decreased explosiveness, or decreased endurance, but is not limited thereto.

[0196] The above food is characterized by being meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, coffee beverages, stamina drinks, alcoholic beverages, or vitamin complexes.

[0197] The above food composition 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, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, or vegetable beverages. These ingredients may be used independently or in combination.

[0198] The term “functional food or health functional food” refers to a group of foods to which added value has been imparted by using physical, biochemical, or biotechnological methods to make the functions of the food act or manifest for a specific purpose, or to a food processed by designing it to sufficiently express in the body the in vivo regulatory functions regarding the regulation of biological defense rhythms, disease prevention, and recovery possessed by the food composition; specifically, it may be a health functional food. The above functional food may include food science-acceptable food additives and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.

[0199]

[0200] (3) Feed composition

[0201] The above feed contains nutrients such as energy, protein, lipids, vitamins, and minerals required by animals, and may be plant-based feed such as grains, root vegetables, food processing by-products, algae, fibers, oils and fats, starches, meal, and grain by-products, or animal-based feed such as proteins, inorganic substances, oils and fats, minerals, oils and fats, and single-cell proteins, but is not limited thereto.

[0202] The above feed may be powder feed, solid feed, moist pellet feed, dry pellet feed, EP (Extruder Pellet) feed, raw feed, etc., but is not limited thereto.

[0203] The above feed composition may include binders, emulsifiers, preservatives, etc. added to prevent quality degradation, and the above feed composition may include feed additives. Amino acid preparations, vitamin preparations, enzyme preparations, flavoring agents, non-protein nitrogen compounds, silicate preparations, buffering agents, extractants, oligosaccharides, etc. added to the feed to increase utility may be included. In addition, feed mixing agents, etc. may be additionally included, but are not limited thereto.

[0204]

[0205] (4) Cosmetic composition

[0206] The above cosmetic composition may additionally include a dermatologically acceptable carrier. The dermatologically acceptable carrier may include, but is not limited to, purified water, oil, wax, fatty acid, fatty acid alcohol, fatty acid ester, surfactant, hygroscopic agent, thickener, antioxidant, viscosity stabilizer, chelating agent, buffer, preservative, lower alcohol, etc., and its type and concentration may vary and may include parts that a person skilled in the art can modify within the scope of the present invention.

[0207] In addition to the active ingredients of the present invention, the above cosmetic composition may include functional substances as needed, such as whitening agents, moisturizers, anti-inflammatory agents, antibacterial agents, antifungal agents, vitamins, sunscreens, antibiotics, anti-acne agents, perfumes, and dyes, and these may be included in the cosmetic composition according to the present invention in amounts commonly used in the field of cosmetics. To enhance the functional effects, the cosmetic composition of the present invention may additionally contain one or more moisturizing active ingredients exhibiting the same or similar functions.

[0208] The above cosmetic composition may be prepared in the form of a general emulsion formulation and a solubilizing formulation. Cosmetics in the form of an emulsion include nourishing lotions, creams, and essences, while cosmetics in the form of a solubilizing formulation include softening lotions. In addition to the active ingredient of the present invention, the above cosmetic composition may also be prepared in the form of an adjuvant for topical or systemic application commonly used in the art by containing a dermatologically acceptable medium or base. Suitable cosmetic formulations may be provided, for example, in the form of a solution, gel, solid or paste anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranulocyte, or an ionic (liposome) or non-ionic vesicular dispersant, or in the form of a cream, skin toner, lotion, powder, ointment, spray, or conceal stick. Additionally, it may be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

[0209] The above cosmetic composition may be formulated into one or more selected from the group consisting of solutions, topical ointments, creams, foams, nourishing lotions, softening lotions, perfumes, packs, softening waters, emulsions, makeup bases, essences, soaps, liquid cleansers, bath additives, sunscreen creams, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing products, oils, powder foundations, emulsion foundations, wax foundations, patches, and sprays.

[0210]

[0211] Various embodiments are presented below to aid in understanding the invention. The following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of protection of the invention to the following embodiments.

[0212]

[0213] <Example>

[0214] Example 1. Confirmation of life extension effect by Mirodenafil

[0215] 1) Materials and Methods

[0216] (1) Test organisms and rearing conditions

[0217] In the present invention, the wild-type (N2) strain of *Caenorhabditis elegans*, a standard model animal for aging and lifespan research, was used as the test organism. The nematode was reared using Nematode Growth Medium (NGM) under constant temperature conditions of 20°C. The NGM medium was composed to contain 51.3 mM NaCl, 2.5% (w / v) peptone, 2% (w / v) agar, 10 mg / L cholesterol, 1 mM CaCl₂, 1 mM MgSO₄, and 25 mM KH₂PO₄.

[0218] (2) Synchronization

[0219] To ensure the reliability of the experimental results, a synchronization process was performed to secure individuals at the same developmental stage. Adult nematodes were treated with a bleaching solution (1% NaOCl, 0.5 M NaOH) for 3 minutes to isolate internal eggs. Subsequently, embryos were collected by repeatedly washing with M9 buffer (composition: 42 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.5 mM NaCl, 1 mM MgSO₄). The collected embryos were inoculated into NGM medium at 20°C for approximately 12 hours to incubate L1 stage larvae, thereby aligning the developmental stages between the experimental groups.

[0220] (3) Preparation of feed (dead OP50)

[0221] To prevent the concentration or activity of the active ingredient mirodenafil from being disturbed by the metabolic activity of Escherichia coli (E. coli OP50), inactivated bacteria were used as a food source. For this purpose, kanamycin was added to an OP50 culture medium incubated overnight in LB medium at a final concentration of 50 μg / mL, and metabolism was inhibited at 36°C for 1.5 hours. Subsequently, the bacteria were completely killed by heat treatment at 95°C for 1 hour.

[0222] (4) Drug and treatment conditions

[0223] Mirodenafil dihydrochloride (mirodenafil 2HCl) was used as the active ingredient. To evaluate only the anti-aging effect while excluding the influence of developmental stages, drug treatment was initiated from the late L4 stage, when nematode development was nearly complete, to the adult-onset stage (Fig. 1A). Specifically, 40 mL of E. coli (OP50) culture was centrifuged to obtain a pellet, which was then resuspended in 2 mL of a solution containing mirodenafil at each concentration. 200 μL of the bacteria-drug mixed suspension was plated onto the surface of an NGM plate, dried overnight at room temperature, and administered. The test concentrations of mirodenafil were set to 0, 15, 30, 45, 60, and 1000 μM. In addition, to prevent population ingress due to offspring generation, the DNA synthesis inhibitor FUdR (5-fluoro-2'-deoxyuridine) was added to the medium to achieve a final concentration of 40 μM, and the medium was dried overnight to ensure complete absorption.

[0224] (5) Life measurement

[0225] L4 end-stage individuals were transferred to NGM plates coated with inactivating OP50 containing various concentrations of mirodenafil, and their survival was monitored periodically until death. Death was determined when there was no reaction when the head or tail of the nematode was lightly stimulated with a platinum wire. Individuals that migrated to the plate wall and disappeared or died abnormally during the experiment were censored for statistical analysis.

[0226] (6) Statistical analysis

[0227] The collected life expectancy data were plotted using Kaplan-Meier survival curves. Statistical significance between the control and experimental groups was tested using the log-rank test, and OASIS2 software was used for the analysis. Other data were analyzed using GraphPad Prism 9, and Student's t-tests or ANOVA were performed for comparisons between groups. For all statistical analyses, the significance level was set at p < 0.05, and significance notations were defined as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0228] 2) Result

[0229] As a result of administering mirodenafil to adult Caenorhabditis elegans, it was confirmed that the survival period was statistically significantly extended compared to the control group (Fig. 1B, Table 1). Mirodenafil showed a consistent lifespan extension effect across the entire range of test concentrations from 15 μM to 1000 μM, which indicates excellent reproducibility of the effect with changes in concentration. In particular, analysis of Kaplan-Meier survival curves calculated for each concentration group showed that the survival curves of the mirodenafil-treated groups generally shifted to the right compared to the control group, and the log-rank test results showed a statistically significant difference (p < 0.05). This data-based proof demonstrates that the overall survival distribution of individuals was significantly improved by the administration of mirodenafil.

[0230] The life-prolonging effect of mirodenafil was most pronounced at a concentration of 60 μM, at which the median lifespan increased by approximately 28.8% compared to the control group (p < 1×10⁻⁶). 16 (Fig. 1C). This significant increase in median lifespan implies that survival characteristics of the entire population have been structurally improved, going beyond a simple increase in average lifespan. Meanwhile, no acute toxicity, abnormal mortality patterns, or instability of the survival curve were observed in individuals throughout the experiment, including high-concentration conditions, suggesting that mirodenafil exhibits a stable lifespan-extending effect over a wide concentration range.

[0231] From the above results, it was experimentally confirmed that mirodenafil is an active anti-aging ingredient capable of effectively delaying the progression of aging, as it statistically significantly extends the survival period of Caenorhabditis elegans even when administered after the adult stage.

[0232] Concentrations (μM)Mean LifespanS.Ep-Value017.120.47ND1520.320.926.00e-043021.680.632.30e-084520.610.374.40e-086022.630.45< 1.0e-10100021.40.568.70e-08

[0233]

[0234] Example 2. Effect of Mirodenafil on Preserving Motor Function and Foraging Ability

[0235] In this invention, to determine whether mirodenafil substantially improves the functional health status (healthspan) of individuals that deteriorates during the aging process, beyond merely extending lifespan, multifaceted motility and behavioral analyses were performed on Caenorhabditis elegans.

[0236] 1) Materials and Methods

[0237] (1) Analysis of liquid medium motility (Thrashing)

[0238] To quantitatively evaluate changes in neuromuscular function associated with aging, thrashing in a liquid environment was measured. Droplets were formed by dropping 200 μL of M9 buffer solution onto a 60 mm unseeded NGM plate, and adult individuals on days 4 to 6 were transferred into the droplets. The spontaneous movement of the nematodes within the liquid was recorded for a certain period using a microscope camera, and motility was automatically analyzed by calculating the number of body bends per second using the WormTracker plugin of ImageJ software.

[0239] (2) Solid medium motility (WMicrotracker) analysis

[0240] To evaluate voluntary physical activity in a more natural habitat, the WMicrotracker SMART system (Phylumtech) was used. After transferring 5-day-old adult individuals to 35 mm unseeded NGM plates, it was confirmed that the M9 buffer solution on the plate surface had completely dried. Subsequently, the average movement speed (speed, mm / s) and total distance traveled (distance, mm / part) of the individuals moving voluntarily over a certain period were precisely measured using an infrared sensor-based automatic tracking system.

[0241] (3) Food exploration analysis

[0242] A foraging study was conducted to evaluate the integrated preservation of sensory and motor functions. A pellet obtained by centrifuging an E. coli (OP50) culture was resuspended at a high concentration in M9 buffer solution; 100 μL of this solution was dispensed onto one end of a 90 mm NGM plate and allowed to dry completely to form a food layer. 80 to 120 nematodes were dispensed onto the opposite end of the plate, and the remaining buffer solution was removed using a Kimwipe to ensure it did not hinder movement. Subsequently, the number of individuals reaching the food layer was recorded at 5-minute intervals for one hour. To reflect the characteristics of time-series data, statistical analysis was performed using a Generalized Linear Mixed Model (GLMM).

[0243] 2) Result

[0244] It was confirmed that the mirodenafil administration group maintained significantly superior overall motor function and sensory-based behavioral abilities compared to the control group, even under conditions of progressive aging (Fig. 2). This suggests that mirodenafil not only extends survival time but also substantially improves the functional health status of individuals by effectively suppressing the functional decline associated with aging. The results for each analysis item are as follows.

[0245] (1) Inhibition of neuromuscular function decline and maintenance of motor ability

[0246] Analysis of thrashing in liquid revealed that in the control group, the number of body bends tended to gradually decrease with the progression of aging after day 5 of adulthood. In contrast, the mirodenafil administration group maintained a higher frequency of thrashing compared to the control group even at the relatively advanced stage of day 6 of adulthood, and this difference was statistically significant (Fig. 2A, Table 2).

[0247] Pulsating movement is known to be an indicator that comprehensively reflects the contraction and relaxation capabilities of body wall muscles and the function of the motor nervous system that regulates them. Therefore, these results provide data-based support that mirodenafil effectively alleviates the decline in neuromuscular function associated with aging and stably maintains the functional linkage between muscles and nerves over a long period. In particular, the significant difference observed on days 5 and 6 in adults implies that the function-preserving effect of mirodenafil is not limited to the early stages of aging but persists into later stages.

[0248] DayVehicleMirodenafilp-ValueMeanSEMMeanSEM426.93.85283.420.127526.144.229.22.30.002622.924.625.435.40.011

[0249] (2) Effects of maintaining voluntary physical activity and delaying frailty

[0250] Automated tracking analysis using the WMicrotracker system under solid medium conditions confirmed that the mirodenafil administration group maintained an overall higher level of spontaneous physical activity compared to the control group (Fig. 2B, Table 3). At the 5th day of adulthood, the mirodenafil administration group showed a statistically significant increase compared to the control group in both average movement speed and total movement distance.

[0251] The voluntary movement speed and distance of nematodes are known as representative behavioral indicators reflecting the decline in neuromuscular function and general vitality associated with aging. In this regard, the results of this study suggest that mirodenafil delays the decline in physical activity caused by aging and effectively maintains the general functional vitality of the individual.

[0252] CountyAverage_Speed ​​[mm / s]Travelled_Distance [mm / part]MeanSEMp-valueMeanSEMp-ValueVehicle0.020.0033ND9.211.83N.D.Mirodenafil0.050.00830.02921.584.770.026

[0253] (3) Preservation of sensorimotor integration ability and survival instinct

[0254] The food exploration assay is a representative behavioral analysis experiment that evaluates sensory-motor integration, a high-order coordination ability in which the sensory nervous system detects chemical signals and the motor nervous system operates organically to move to a target point after recognizing them. This experiment comprehensively reflects not only simple locomotion ability but also the ability to perform behaviors based on environmental stimulus perception, decision-making, directional movement, and survival instinct.

[0255] As a result of the experiment, the mirodenafil administration group reached the food layer at a significantly faster rate compared to the control group, and the cumulative proportion of individuals reaching the food also remained significantly high over time (Fig. 2C, Table 4). In particular, GLMM analysis reflecting time-series repeated measurements data showed that the mirodenafil administration group consistently maintained a statistically significant superior search success rate compared to the control group after 90 minutes of observation (p=0.00746).

[0256] This means that mirodenafil does not merely increase muscle contractility or movement speed, but also preserves chemosensory perception, neural signal processing efficiency, and nervous system responsiveness—abilities that tend to decline with aging. In other words, by maintaining the functional integrity of the entire behavioral execution circuit extending from sensory nerves to the central nervous system and motor nerves, mirodenafil provides a neuroprotective effect that enables the individual to actively respond to changes in the external environment and perform behaviors advantageous for survival.

[0257] These results strongly support the fact that mirodenafil contributes substantially not only to lifespan extension but also to the qualitative improvement of healthspan, where the behavioral adaptive capacity and survival instincts of aging individuals are maintained.

[0258] Time (min)VehicleWorms on the lawn (%)Mirodenafil Worms on the lawn (%)51.2891.407106.4918.9971516.02420.142022.66630.0242530.28639.5093039.86547.6893547.079 54.4034050.00357.9814552.38160.0355056.84162.9175559.62165.7726061.33766.3699066.07674.835

[0259] In conclusion, the comprehensive results of Example 2 clearly demonstrate that mirodenafil not only extends life support but also significantly improves physical activity and sensorimotor integration abilities, which decline rapidly with aging. This effect implies that mirodenafil induces a state in which physical vitality and behavioral performance are maintained even in old age by preserving the functional integrity of the entire neuromuscular system over the long term. Therefore, it strongly suggests that mirodenafil acts as an active 'healthy lifespan enhancer' that fundamentally alleviates age-related functional decline and substantially improves the individual's quality of life.

[0260]

[0261] Example 3. Effects of Mirodenafil on Intestinal Barrier (Leakage) Inhibition and Memory Retention Improvement

[0262] In order to determine the effects of mirodenafil on physical barrier function and higher-order cognitive function in an individual, a leaky gut assessment and an associative learning and memory retention essay were performed.

[0263] 1) Materials and Methods

[0264] (1) Evaluation of intestinal permeability (Leaky gut, Smurf phenotype)

[0265] A 'Smurf assay' was performed to measure changes in the integrity of intestinal epithelial cells due to aging. Day 5 adult specimens were placed in wells containing a non-absorbent blue dye (FD&C Blue No. 1) mixed with an E. coli (OP50) pellet suspension and exposed for 2 hours. Afterward, the specimens were transferred to unseeded NGM plates, dried for 30 minutes, washed with M9 buffer to remove residual dye from the body surface, and observed under a microscope. Species in which the dye was confined only to the inside of the intestinal tract were classified as 'Normal,' while those in which the dye leaked into the body cavity outside the intestinal tract, causing the entire body to turn blue, were classified as 'Leaky Gut' (or 'Smurf Phenotype'), and the proportions of each group were calculated.

[0266] (2) Assessment of associative learning and memory retention (Butanone-based conditioning)

[0267] To evaluate the function of the higher-order nervous system, associative learning and memory essays using butanone were performed.

[0268] Conditioning: Nematodes were washed with M9 buffer to remove residual food and kept starved for 1 hour. Afterward, they were transferred to a plate coated with food (OP50), and 2 μL of a 10% butanone dilution was dropped onto the inside of the plate lid and sealed. This was maintained for 1 hour to induce associative learning between the food and the butanone scent in the nematodes.

[0269] Chemotaxis assay: Chemotaxis was measured on a 90 mm test plate immediately after learning (0 hr) and 2 hours later (2 hr). 4 μL of 10% butanone was dropped onto one end of the plate and 4 μL of 99% ethanol (control) was dropped onto the opposite end, and 1 μL of the anesthetic NaN3 was added to both ends to fix the reached specimens.

[0270] Calculation of Index: After allowing individuals to move freely at room temperature for 30 minutes, the number of individuals in each zone was counted, and the Chemotaxis Index (CI) was calculated as follows: CI = (#butanone - #ethanol) / (Total)

[0271] 2) Result

[0272] Experimental results confirmed that mirodenafil effectively inhibits the breakdown of tissue barrier function associated with aging, while significantly preserving the ability to retain learned memories.

[0273] (1) Preservation of intestinal barrier function and prevention of systemic inflammation

[0274] As a result of the intestinal permeability evaluation (Smurf assay), the control group (Vehicle) exhibited a smudge phenotype in which dye leaked out of the intestinal tract in more than 30% of individuals on day 5 of adulthood, indicating a rapid decline in the integrity of intestinal epithelial cells due to aging. In contrast, in the mirodenafil administration group, the proportion of individuals exhibiting smudge phenotype decreased statistically significantly compared to the control group, and the proportion of normal individuals with the dye confined to the inside of the intestinal tract increased significantly (Fig. 3, Table 5).

[0275] The intestinal barrier acts as a primary line of defense, blocking the entry of external antigens, microbial toxins, and endotoxins (such as LPS) into the body. Increased intestinal permeability associated with aging is known to be a major cause of "inflammaging," which continuously triggers systemic inflammatory responses; these inflammatory signals can lead to neuroinflammation and cognitive decline via the Gut-Brain axis. Therefore, the results of this study suggest that mirodenafil provides a mechanistic basis for suppressing systemic inflammation and blocking the cascading progression of nervous system decline by effectively preserving the structural and functional integrity of the intestinal epithelium.

[0276] CountyNormal(N)leaky gut(N)Total(N)leaky gut ratio(%)p-ValueVehicle23211234432.56N.D.Mirodenafil2494329214.730.026

[0277] (2) Maintenance of cognitive function and protection of higher nervous system

[0278] In the butanone-based associative learning and memory retention essay, both the control group and the mirodenafil group showed a high chemotaxis index (CI) immediately after learning (0 hr) (control group 0.68 ± 0.06, mirodenafil group 0.66 ± 0.05), and no significant difference was observed in the initial learning ability itself. This means that mirodenafil does not affect the learning induction phase.

[0279] However, when evaluating memory retention ability at 2 hours after learning (2 hr), the chemotactic index in the control group decreased sharply to 0.39 ± 0.08, whereas the mirodenafil administration group maintained a relatively high index of 0.58 ± 0.08, confirming that the learned memory was statistically significantly preserved (Fig. 4, Table 6).

[0280] In particular, the significant suppression of 'memory decay,' which occurs over time following memory formation, suggests that mirodenafil protects the functional integrity of higher-order nervous systems related to synaptic function and neural plasticity, going beyond mere improvements in sensory responses or motor function. This result supports the potential to mitigate the decline in memory retention frequently observed during the aging process and to ensure the long-term continuity of cognitive function.

[0281] In conclusion, the results of Example 3 clearly demonstrate that mirodenafil simultaneously exerts an inhibitory effect on systemic inflammation through the protection of intestinal integrity and an effect on improving memory retention through the preservation of higher-order nervous system function. This dual action suggests that age-related cognitive decline and systemic functional decline can be comprehensively alleviated by inhibiting the functional breakdown of the gut-brain axis. Therefore, mirodenafil fully supports the potential as an effective composition for substantially improving an individual's healthspan and quality of life.

[0282] CI0 hr (p-Value = 0.562)2 hr (p-Value = 0.010)VehicleMirodenafilVehicleMirodenafil 1st Experiment 0.817520.857140.577980.77778 2nd Experiment 0.846850.704920.227270.56522 3rd Experiment 0.800000.666670.323530.33333 4th Experiment 0.478260.553190.200000.51163 5th Experiment 0.565220.526320.312500.45161 6th Experiment 0.585940.639640.669900.85294

[0283]

[0284] Example 4. Demonstration of Transcriptome Changes and Dietary Restriction (DR)-like Pathway Activation Induced by Mirodenafil

[0285] In this embodiment, an in-depth analysis of transcriptome data was performed to clarify the specific molecular orientation of the overall anti-aging effect of mirodenafil confirmed through prior research.

[0286] 1) Materials and Methods

[0287] (1) Test organism and drug treatment (for transcriptome sample preparation)

[0288] Wild-type Caenorhabditis elegans (N2) were cultured in standard NGM medium at 20°C. To exclude the developmental effects of the active ingredient, treatment with mirodenafil (60 μM) was initiated from late L4 to adult-onset (Fig. 5A). The control group was set as a vehicle treatment group without the same dose of the active ingredient. Inactivated OP50 bacteria were provided as a feed to minimize drug disturbance caused by bacterial metabolism, and FUdR 40 μM was included to prevent transcriptome interference due to reproduction.

[0289] (2) Time of sample collection

[0290] Samples for transcriptome analysis were collected on day 3 of adults. This is a point in time before distinct external changes of bodily aging appear, intended to precisely capture the early metabolic reprogramming process induced by mirodenafil (Fig. 5A).

[0291] (3) RNA extraction and quality check

[0292] Total RNA was extracted from collected nematode samples using TRIzol reagent (Invitrogen) and purified through chloroform extraction and isopropan precipitation. The integrity and concentration of the extracted RNA were strictly verified using the Agilent Bioanalyzer system.

[0293] (4) mRNA library construction and sequencing

[0294] 200 ng of total RNA per sample was used as the starting material for library construction. mRNA with a Poly(A) tail was selectively concentrated using the Poly(A) mRNA Capture Module (ABclonal), and a library was constructed using the mRNA-seq Lib Prep Module for Illumina (ABclonal). The completed library was sequenced on the Illumina platform, and the generated raw data was registered in the NCBI GEO database (Access No.: GSE308543).

[0295] (5) Transcriptome data processing and analysis pipeline

[0296] The quality of the sequencing data was evaluated using FastQC, and adapters and low-quality reads were removed using Cutadapt. The purified reads were aligned to the reference genome (WBcel235) using STAR software. Raw read counts and TPM (Transcripts Per Kilobase Million) values ​​were calculated using RSEM, and normalization and Variance-Stabilizing Transformation (VST) were performed using the DESeq2 package in the R (v4.3.2) environment. Subsequently, Principal Component Analysis (PCA) was performed to confirm the separation of overall gene expression patterns between groups.

[0297] (6) T-CLASS Analysis and Drawing Configuration

[0298] T-CLASS (Transcriptome-based Classification of Aging and Senescence Signatures) analysis was performed to compare the transcriptome signature induced by mirodenafil with existing longevity-inducing pathways. An optimal gene set was selected using ANOVA and the elbow method. The ComBat algorithm was used to correct for batch effects, and similarity to known longevity pathways, such as dietary restriction (DR), insulin / IGF-1 signaling impairment (rIIS), and mitochondrial dysfunction (rMF), was calculated based on Spearman correlation. The dot identity of the comparison dataset serving as the basis for the analysis was specified, and the expression patterns of the corresponding genes were visualized as a heatmap to elucidate the mechanistic locus of mirodenafil.

[0299] 2) Result

[0300] (1) Clear distinction of PCA-based transcriptome profiles

[0301] PCA analysis of VST-normalized transcriptome data revealed that the mirodenafil-treated group and the control group formed clearly separated clusters in the principal component space (Fig. 5B). In particular, despite the relatively early stage of adulthood (day 3), the two groups were distinctly separated along the PC1 and PC2 axes, which implies that mirodenafil actively induces transcriptomic reprogramming from the early stages of aging, rather than a compensatory response to end-stage aging.

[0302] These results provide decisive evidence at the transcriptome level supporting that mirodenafil acts not merely as a survival-extending factor, but as a regulatory factor that fundamentally alters the individual's metabolic state and physiological homeostasis.

[0303] (2) Confirmation of Dietary Restriction (DR) Mimicking Mechanism through T-CLASS Analysis

[0304] To determine the specific direction of the aforementioned transcriptome changes, a comparative analysis (T-CLASS) was performed with a pre-established database of longevity-inducing pathways. As a result, it was confirmed that the gene expression pattern induced by mirodenafil treatment corresponded highly to the Dietary Restriction (DR) pathway rather than the insulin signaling impairment (rIIS) or mitochondrial function impairment (rMF) pathways (Fig. 5C). This data specifically supports the fact that the composition according to the present invention mimics the effects of dietary restriction at the molecular level without physical dietary control. In other words, this analysis proved that the changes in metabolism-related genes observed in prior studies were not random fluctuations, but rather part of 'Metabolic Reprogramming' that switches the body's energy metabolism to an efficient longevity mode.

[0305] Dimensional contribution analysis showed that Dimension 1 and Dimension 2 accounted for 32% and 20% of the explanatory power, respectively, indicating that the transcriptome changes induced by mirodenafil form statistically significant independent axes. In particular, the fact that it stably clustered at a position most closely aligned with the DR family, going beyond merely partially activating one of the existing longevity mechanisms, clearly demonstrates that mirodenafil selectively activates pathways with 'dietary restriction mimetic (DR mimetic)' characteristics.

[0306] (3) Specifying the dot identity of the comparison dataset and heatmap-based verification

[0307] To clarify the meaning of each dot shown in Fig. 5C, Fig. 6A specifically presents the identity of the longevity path dataset used for comparison.

[0308] rIIS lineage: daf-2(e1370), daf-2(e1368) variants and daf-2 RNAi

[0309] DR lineages: eat-2(ad1116), eat-2(ad465) variants and direct dietary restriction model

[0310] rMF family: isp-1, clk-1, nuo-6 variants and cco-1 RNAi

[0311] As a result of correlation analysis with the above dataset, the mirodenafil transcriptome signature was consistently located within the DR family cluster in repeated analyses and showed a pattern clearly distinguishable from the rIIS or rMF families. Furthermore, when the expression patterns of the key genes used in the T-CLASS analysis were visualized as a heatmap, the gene expression pattern of the mirodenafil treatment group showed a high level of agreement with the DR model (Fig. 6B).

[0312] This result simultaneously demonstrates, both visually and quantitatively, that mirodenafil reproduces the systemic metabolic adaptation program observed in DR conditions at the gene expression level, rather than inhibiting a single signaling axis or inducing a specific stress response.

[0313] In conclusion, the transcriptome analysis results of the present embodiment clearly demonstrate that mirodenafil exerts anti-aging effects by inducing a metabolic state similar to 'dietary restriction (DR)' through gene expression redesign throughout the body, rather than being limited to specific genes or restricted signaling pathways. This transcriptome-level evidence supports the fact that mirodenafil is an active ingredient that activates a unique DR-like pathway distinct from existing longevity-inducing mechanisms. This result demonstrates a high level of selectivity unpredictable from existing technologies, suggesting that it can selectively induce only the target efficacy without unnecessarily affecting other pathways, and strongly suggests its potential as a new molecular strategy for enhancing healthy lifespan.

[0314]

[0315] Example 5. Confirmation of the lifespan extension mechanism of mirodenafil dependent on energy sensor AMPK (AAK-2) activation

[0316] To confirm the molecular pathways through which the lifespan-extending and diet-restriction-mimicking effects of mirodenafil identified in the previous examples are mediated, verification experiments were conducted using a model of AMPK (nematode aak-2), a key factor in regulating longevity. This is a specific example demonstrating that the metabolic regulatory mechanism suggested in previous studies is essentially dependent on the activation of AMPK, an actual in vivo energy sensor.

[0317] 1) Materials and Methods

[0318] (1) Selection of test organisms and variants

[0319] For the experiment, wild-type (N2) strains of Caenorhabditis elegans were used along with a loss-of-function mutant strain aak-2 (ok524), which is deficient in the aak-2 gene corresponding to the catalytic subunit of AMPK. The nematode AAK-2 protein is known to have a high degree of structural and functional homology with the mammalian AMPKα subunit, and is used as a standard model for energy sensing and longevity regulation research.

[0320] (2) Experimental conditions and measurements

[0321] All experimental conditions were set identically to those in Example 1. Specifically, to exclude developmental effects in both wild-type and aak-2(lf) variant nematodes, treatment with mirodenafil (60 μM) was initiated from the late L4 stage. The control group was treated with vehicles under the same conditions. Killed OP50s were provided as feed, and to prevent confusion due to reproduction, lifespan was measured at 20°C on NGM plates containing FUdR at a final concentration of 40 μM. The survival curves of each group were plotted using the Kaplan-Meier method, and statistical significance was verified via the log-rank test.

[0322] 2) Result

[0323] (1) Confirmation of loss of efficacy of mirodenafil in case of AMPK deficiency

[0324] Experimental results clearly demonstrated that the lifespan extension effect caused by the administration of mirodenafil was reproduced in wild-type (N2) nematodes, but the lifespan extension effect caused by mirodenafil was completely lost in AMPK loss-of-function mutants (aak-2 null) (Fig. 7, Table 7). This proves that the anti-aging efficacy exhibited by mirodenafil of the present invention is achieved through a systematic metabolic regulatory mechanism mediated by the activation of the AMPK pathway, going beyond simple antioxidant action. In particular, since nematode AAK-2 is a highly conserved ortholog functionally and structurally similar to the human AMPKα subunit (Apfeld et al., 2004; Lee et al., 2008), these results strongly suggest that mirodenafil will act through the same AMPK pathway when administered to humans.

[0325] These results clearly demonstrate that the life-extending effect of mirodenafil is not due to simple non-specific physiological activity or stress response, but is a mechanism-dependent effect that is expressed only when normal function of the AMPK (AAK-2) pathway is present. In other words, since the anti-aging efficacy of mirodenafil is not expressed under conditions where AMPK activity is blocked, it implies that AMPK functions as an essential mediator of mirodenafil's action.

[0326] CountyMeanLifespanS.Ep-Valuewild type23.320.17NDwild type (+Mirodenafil)24.890.13< 1.0e-10aak-2(If)17.770.26< 1.0e-10aak-2(lf)(+Mirodenafil)17.660.26< 1.0e-10

[0327] These results clearly demonstrate that the anti-aging effect of mirodenafil is not due to a simple non-specific physiological response, but is expressed on the premise of the normal function of the AMPK pathway, a key regulator of bioenergetic metabolism. In other words, mirodenafil can be interpreted as exhibiting an anti-aging effect by activating a series of molecular reactions involving the AMPK pathway, mediated by an increase in intracellular cGMP signaling following PDE5 inhibition. This mechanism of action based on the 'PDE5-AMPK axis' supports the unique technical principles of the present invention, which are distinguished from previously known anti-aging or life extension approaches.

[0328] To support the human applicability of the mechanism of action identified in this invention, the conservation between the key factor of Caenorhabditis elegans and its human homolog was analyzed. The nematode's AAK-2 protein is an enzyme corresponding to the α subunit of AMP-activated protein kinase (AMPK), and is known to be a highly conserved functional and structural ortholog of the human AMPKα subunit (PRKAA1 / 2). Specifically, in the kinase domain responsible for enzyme activity, nematode AAK-2 shares **high amino acid identity** of approximately 71% with the human AMPKα subunit (particularly AMPKα1) (approximately 80% for AAK-1), and has been reported to have a conserved threonine residue in which the phosphorylation site essential for enzyme activation (Nematode Thr243, Human AMPKα Thr172) is located at the corresponding position (Apfeld et al., Genes Dev., 2004; Lee et al., J. Biol. Chem., 2008). In fact, it has been experimentally proven that the functional homology between nematode AAK-2 Thr243 and human AMPKα Thr172 is demonstrated by the fact that anti-phospho-AMPK (Thr172) antibodies specifically recognize Thr243 phosphorylation in wild-type AAK-2, and that the signal is lost in a mutant (aak-2 T243A) with this site substituted (Carman et al., microPublication Biology, 2022).

[0329] Therefore, the data on the 'loss of lifespan extension effect upon aak-2 deficiency' confirmed in this example (Fig. 7) demonstrates that this is an essential mechanism by which mirodenafil exerts metabolic regulation and anti-aging effects through the same pathway (AMPK activation) even when administered to humans. Previous studies have also reported that AAK-2, a catalytic subunit of AMPK in C. elegans, acts as a central factor regulating lifespan and healthspan, and that upon aak-2 deficiency, the lifespan extension effect is lost or reduced in various lifespan extension models (e.g., certain forms of dietary restriction, increased stress resistance, etc.). Notably, genetic analysis results have shown that the antidiabetic drug metformin improves health lifespan indicators in nematodes, such as reducing fat accumulation, maintaining mobility, and extending lifespan, and that these effects depend on the AMPKα ortholog AAK-2 and its upstream factor LKB1 (PAR-4) (Onken and Driscoll, PLoS One, 2010). This mechanism is consistent with the metabolic improvement and anti-aging effects observed in mammals through the metformin-AMPK axis (Moreno-Arriola et al., PLoS One, 2016). As such, the fact that the AMPK-aak-2 axis identified in the nematode model is well conserved in terms of both structure and function with the AMPK-PRKAA1 / 2 axis in mammals and humans, and that cases of mechanism congruence between nematodes and mammals have already been reported with metformin, strongly supports the result that mirodenafil of the present invention exhibits an AAK-2-dependent lifespan extension effect in C. elegans as a valid and significant indicator for predicting AMPK-mediated metabolic regulation and anti-aging efficacy in humans.

[0330]

[0331] Example 6. Effect of inducing metabolic reprogramming and inhibiting body fat accumulation without change in dietary intake

[0332] In order to determine whether the life extension and health life enhancement effects of mirodenafil are due to a reduction in actual food intake or are induced by metabolic reprogramming at the molecular level, the degree of lipid accumulation in the body and eating behavior were each analyzed in detail.

[0333] 1) Materials and Methods

[0334] (1) Quantification of body fat accumulation (Oil Red O staining)

[0335] To visualize and quantify the energy storage status of the organisms, Oil Red O (ORO) staining, which selectively stains neutral lipids, was used. Immediately before use, the 60% ORO stain solution was filtered through a 0.22 μm syringe filter to remove insoluble particles. Adult nematodes were washed with 1× PBST and fixed in 60% isopropanol for 3 minutes. Afterward, they were incubated in the ORO stain solution for 2 hours, excess dye was removed, and images were taken using an optical microscope.

[0336] Staining intensity was quantified by extracting the red channel of the image using the OpenCV library and summing the pixel intensities relative to the total area of ​​the specimen, and this was calculated as the relative neutral lipid accumulation amount compared to the control group.

[0337] (2) Measurement of feeding activity (Pharyngeal pumping rate)

[0338] To evaluate whether mirodenafil treatment affects the actual food intake of nematodes, pharyngeal pumping rate, a direct indicator of feeding behavior, was measured. For adult nematodes on day 5, the number of posterior pharyngeal movements occurring per minute on a culture plate under minimized external stimulation was directly counted.

[0339] 2) Result

[0340] Experimental results confirmed that mirodenafil significantly improves energy storage and metabolic efficiency in the body without causing significant changes in the individual's feeding behavior.

[0341] (1) Significant reduction in body fat without change in food intake

[0342] Oil Red O staining and quantitative analysis performed on adult nematodes on day 5 showed that the mirodenafil (60 μM) administration group had a statistically significant decrease in the accumulation of neutral lipids in the body compared to the control group (Fig. 8A, Table 8). This pattern of reduced lipid accumulation is consistent with typical physiological characteristics generally observed under dietary restriction (DR) conditions.

[0343] This suggests that mirodenafil inhibits the storage of excess energy in the body without physical nutritional intake restrictions and remodels the metabolic state in a direction that enhances lipid breakdown and energy utilization efficiency.

[0344] MilitaryMeanSEMp-ValueVehicle control10.00014N.D.Mirodenafil0.770.040.001

[0345] (2) Verification as a True DR Mimetic

[0346] Importantly, it was clearly confirmed that the aforementioned body fat reduction effect was not the result of appetite suppression or feeding inhibition due to drug toxicity. When pharyngeal pumping speed, a direct indicator of feeding behavior, was measured, no statistically significant difference was observed between the mirodenafil administration group and the control group (Fig. 8B, Table 9).

[0347] MilitaryMeanSEMp-ValueVehicle148.1813.889NDMirodenafil166.923.0160.474

[0348] Generally, many compounds that exhibit life-extending effects often inhibit feeding behavior or reduce an individual's ability to consume, resulting in a state of 'forced fasting.' However, mirodenafil of the present invention is interpreted to induce diet restriction-like metabolic effects, such as promoting fat burning and optimizing energy efficiency, through molecular signaling including the activation of the AMPK pathway, even under conditions where actual food intake is maintained normally.

[0349] In conclusion, the data from Example 6 support the fact that mirodenafil functions as a 'True DR Mimetic' that effectively replicates the metabolic benefits of dietary restriction (DR), known as a key mechanism of anti-aging, without impairing the individual's feeding behavior or nutrient supply. This clearly suggests that mirodenafil possesses distinct clinical value and technological advancement, capable of providing potent anti-aging and healthy lifespan enhancement effects without the risk of nutritional imbalance or weight loss that can be problematic in the elderly.

[0350]

[0351] Example 7. Specification of protein toxicity control and neuroprotective mechanisms in a TDP-43 proteinpathy model

[0352] In order to evaluate whether the effects of preserving motor function and improving cognitive function confirmed in the preceding Examples 2 and 3 are valid in the specific pathological environment of neurodegenerative diseases, experiments were conducted using a TDP-43 protein pathology model, a major causative agent of neurodegeneration. This example specifically demonstrates that the neuroprotective effect of mirodenafil goes beyond simple function maintenance and includes a proteotoxicity defense mechanism that inhibits the aggregation of toxic proteins within neurons.

[0353] 1) Materials and Methods

[0354] (1) Experimental model and treatment conditions

[0355] We used the KWK92 strain, an ALS nematode model in which human TDP-43 protein aggregation is induced by blue light stimulation and GFP is expressed in GABAergic motor neurons. To evaluate the prophylactic effect of mirodenafil, 60 μM of mirodenafil was administered starting from the late L4 stage, while the control group was treated with the same amount of vehicle. All groups were continuously irradiated with blue light to induce TDP-43 proteinopathy.

[0356] (2) Evaluation method by stage of pathological progression (Adult, Day 3)

[0357] At 72 hours of blue light exposure, the number of TDP-43 inclusions formed in processes within 20 μm of the neuronal cell body, focusing on DD2, VD3, and VD4 neurons, was quantified. In addition, the occurrence of neural gaps was observed (Fig. 9).

[0358] 2) Result

[0359] Experimental results confirmed that mirodenafil exhibits more pronounced and significant neuroprotective and functional improvement effects as TDP-43 proteinopathy progresses and moves toward the chronic stage where the pathology intensifies.

[0360] (1) Neuropathological features in the early pathological stage (3rd day of adulthood)

[0361] Analysis on day 3 of adulthood revealed that the formation of TDP-43 aggregates and the onset of neurodegeneration had occurred in all experimental groups stimulated by blue light. At this point, the mirodenafil administration group did not show a statistically significant difference in the number of TDP-43 aggregates or the rate of severe neuronal disconnection compared to the control group (Fig. 9).

[0362] This suggests that in the early stages of pathology, molecular-level metabolic reprogramming induced by mirodenafil is in progress, a stage prior to when anatomically clear improvements in neural structure have not yet become visible.

[0363]

[0364] Example 8. Verification of efficacy in preserving neuronal integrity and disease-modifying in the chronic pathological stage

[0365] To confirm whether the initial aggregation inhibition mechanism identified in Example 7 leads to the preservation of long-term neural structures, the effect of preventing neuronal discontinuity and motor function decline in the aging stage (adult day 6) with chronic pathology was analyzed. This is an example demonstrating that mirodenafil has disease-modifying efficacy that fundamentally delays the progression of the disease, rather than providing temporary symptom relief.

[0366] 1) Materials and Methods

[0367] (1) Chronic pathology evaluation (adult on day 6)

[0368] The degree of degeneration of the ventral nerve cords was observed at 144 hours of blue light exposure and classified into three stages: “Normal (no interruption),” “Mild (1 interruption),” and “Severe (2 or more interruptions).” In addition, to evaluate functional changes due to motor neuron damage, the number of thrashing movements in a liquid medium was measured (Fig. 10).

[0369] 2) Result

[0370] (1) Significant neuroprotective effect in the chronic pathological stage

[0371] The analysis results for adults on day 6 with chronic pathology showed a clear difference from the initial pathological stage. In the control group (Vehicle), the proportion of individuals in the “Severe” grade showing severe neuronal disconnection was overwhelmingly high, whereas in the mirodenafil administration group, the proportion of individuals showing severe neurodegeneration was significantly reduced by more than 50% compared to the control group (Fig. 10, Table 10). This suggests that in order to achieve the ‘motor function preservation’ effect confirmed in previous studies of mirodenafil, it is essential to involve a mechanism that fundamentally blocks the structural breakdown of neurons.

[0372] Furthermore, the proportion of subjects maintaining a normal ventral nerve cord structure significantly increased in the mirodenafil administration group, confirming that mirodenafil effectively protects the integrity of neural structures even under continuous TDP-43 proteotoxicity stress. This supports the fact that the functional preservation effect identified in the previous thrashing assessment was achieved through the actual structural maintenance of the neural network.

[0373] CountyNormal(%)Mild(%)Severe(%)p-ValueVehicle20.430.6148.97N.D.Mirodenafil40.6749.1510.160.0367

[0374] (2) Recovery and preservation of motor function in pathology models

[0375] The aforementioned neuroprotective effect was also confirmed by improvements in functional indicators. Unlike the control group, in which motility was severely impaired due to chronic TDP-43 aggregation, the mirodenafil administration group showed a significant increase in the number of thrashings measured in liquid medium compared to the control group.

[0376] This result demonstrates that mirodenafil can maintain functional connectivity between the neuromuscular system in the long term by maintaining proteostasis, going beyond the downstream mechanism of merely inhibiting neuronal apoptosis.

[0377] MilitaryMeanSEMp-ValueVehicle20.311.55NDMirodenafil25.541.840.034

[0378] In conclusion, the comprehensive data of Example 7 clearly demonstrates that mirodenafil exerts potent neuroprotective and functional recovery effects in the chronic stage, where the pathology intensifies as the disease progresses, based on metabolic regulatory effects that accumulate from the early stages of pathology. In particular, the comparative results of day 3 and day 6 in adults support the fact that the composition of the present invention possesses technical value as a disease-modifying treatment capable of fundamentally delaying the course of progressive neurodegenerative diseases, rather than merely providing temporary symptom relief.

[0379]

[0380] Example 9. Verification of Human Applicability and Drug Class Effect

[0381] To demonstrate that the anti-aging and health-life-promoting effects of mirodenafil identified in the preceding examples are not inherent characteristics of the specific compound but rather a 'class effect' based on the pharmacological mechanism of PDE5 enzyme inhibition, the inventors conducted a comparative experiment using sildenafil, a drug of the same class already proven for its safety and efficacy in humans and used worldwide, as a positive control.

[0382] 1) Materials and Methods

[0383] (1) Drug treatment and experimental conditions

[0384] Experiments were conducted on wild-type (N2) Caenorhabditis elegans. To ensure a fair comparison of drug effects, mirodenafil and sildenafil (Sigma-Aldrich) were treated at the same concentration of 60 μM. A vehicle without drugs was used as a control. To exclude effects during developmental stages, individuals were transferred to a medium containing drugs and continuously exposed from the late L4 larval stage to the beginning of the adult stage (adult-onset treatment), and killed E. coli OP50 was provided as food to prevent metabolic disturbance.

[0385] (2) Analysis of lifespan and healthy lifespan indicators

[0386] The following three major indicators were compared and analyzed for each group.

[0387] Lifespan: A Kaplan-Meier survival curve was constructed by measuring the survival period of an individual until death.

[0388] Locomotion: To evaluate the maintenance of neuromuscular function, Day 5 adults were transferred to a liquid medium (M9 buffer) and the thrashing rate was measured.

[0389] Intestinal Barrier Integrity: To evaluate intestinal permeability, a Smurf assay using a blue dye was performed on 5-day-old adult individuals, and the proportion of individuals in which the dye leaked into the body cavity was quantified.

[0390] 2) Result

[0391] (1) Confirmation of equivalence between drugs in life-extending effects

[0392] As a result of measuring lifespan, both the mirodenafil (60 μM) treatment group and the sildenafil (60 μM) treatment group showed a statistically significant lifespan extension effect compared to the control group (Vehicle) (Fig. 11A). This suggests that PDE5 inhibition is a common key mechanism that induces lifespan extension.

[0393] (2) Proof of commonality between exercise function and intestinal barrier strengthening effects

[0394] As a result of evaluating functional decline due to aging, the two drugs showed equivalent improvement effects in health lifespan indicators as well.

[0395] Motility: In aging individuals on day 5 of adulthood, the control group showed a rapid decline in motility, whereas both mirodenafil and sildenafil-treated groups maintained significantly higher motility (thrashing) compared to the control group (Fig. 11B).

[0396] Intestinal barrier: As a result of evaluating intestinal permeability, the incidence of smurf phenotype was significantly lower in both drug-treated groups compared to the control group. This demonstrates that mirodenafil and sildenafil have a common effect of inhibiting age-related barrier breakdown by strengthening the structural integrity of intestinal epithelial cells (Fig. 11C).

[0397]

[0398] In conclusion, the above results clearly demonstrate that the anti-aging, motor function preservation, and intestinal barrier strengthening effects exhibited by the mirodenafil of the present invention are effects commonly observed in PDE5 inhibitor class drugs (e.g., sildenafil). This supports the idea that the technical concept of the present invention is not limited to mirodenafil but can be extended to compositions containing PDE5 inhibitors as active ingredients, and strongly supports human applicability through mechanistic identity with drugs already in use in the human body.

Claims

1. A pharmaceutical composition for improving age-related metabolic decline or extending healthspan, comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition is characterized by activating the intracellular AMP-activated protein kinase (AMPK) pathway to inhibit body fat accumulation without reduction in food intake and induce a transcriptome profile similar to diet restriction.

2. A pharmaceutical composition according to claim 1, characterized in that the extension of healthy lifespan comprises one or more effects selected from the group consisting of delaying the decline in locomotion associated with aging, maintaining associative memory, or preserving the tight junction structure of intestinal epithelial cells.

3. A pharmaceutical composition according to claim 1, wherein the individual is a human, and the AMPK pathway is mediated through the activation of a complex agent comprising a human PRKAA1 or PRKAA2 subunit.

4. A pharmaceutical composition for the prevention or treatment of metabolic syndrome comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition is characterized by reprogramming lipid metabolism by activating the AMPK pathway independently of the insulin / IGF-1 signaling pathway (IIS).

5. A pharmaceutical composition according to claim 4, characterized in that the metabolic syndrome includes age-related obesity, insulin resistance, or visceral fat accumulation.

6. A pharmaceutical composition for the prevention or treatment of age-related intestinal barrier dysfunction or leaky gut syndrome, comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

7. A pharmaceutical composition according to claim 6, characterized in that the composition inhibits systemic inflammation by blocking the systemic influx of endotoxins or inflammation-inducing factors derived from intestinal microorganisms by strengthening the tight junction structure between intestinal epithelial cells to reduce intestinal permeability.

8. A pharmaceutical composition according to claim 6, characterized in that the composition alleviates neuroinflammation caused by increased intestinal permeability or inhibits blood-brain barrier (BBB) ​​damage through the Gut-Brain Axis.

9. A pharmaceutical composition for the prevention or treatment of diseases related to TDP-43 proteinopathy, comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition is characterized by inhibiting the abnormal aggregation of TDP-43 protein within nerve cells or maintaining proteostasis to reduce neurotoxicity.

10. A pharmaceutical composition according to claim 9, characterized in that the composition blocks the formation of TDP-43 inclusion bodies in the cytoplasm of nerve cells or prevents the axon severance of motor neurons and the structural collapse of nerve cords.

11. A pharmaceutical composition according to claim 9, characterized in that the TDP-43 proteinopathy-related disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), or limbic-dominant senile TDP-43 encephalopathy (LATE).

12. A pharmaceutical composition for the prevention or improvement of age-related physical decline or functional frailty, comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

13. A pharmaceutical composition according to claim 12, characterized in that the improvement is achieved by increasing the energy metabolic efficiency of muscle cells through AMPK pathway activation or by maintaining the structural integrity of the neuromuscular junction or nerve cord.

14. A pharmaceutical composition comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition is characterized by delaying composite functional decline associated with aging by inhibiting the aggregation of TDP-43 protein in neurons through the activation of the intracellular AMPK pathway and simultaneously preserving intestinal barrier function.

15. In any one of claims 1, 4, 6, 9, 12, or 14, the PDE5 inhibitor is Mirodenafil, Sildenafil, Tadalafil, Vardenafil, Udenafil, Avanafil, Lodenafil, NBB-116, Simmerafil, RT234, TPN-729, DDCI-01, TOP-N53, TR-422, Gisadenafil, PF-00489791, PF-03049423, SLx-2101, Youkenafil, Padanafil, NCX-1728, TOPV-122, A pharmaceutical composition characterized by being selected from the group consisting of Zaprinast, MSTM-102, TOPT-5, E4021, and Tunodafil and their pharmaceutically acceptable salts.

16. A pharmaceutical composition according to claim 15, wherein the PDE5 inhibitor is mirodenafil or sildenafil, or a pharmaceutically acceptable salt thereof.

17. A pharmaceutical composition according to claim 16, characterized in that the PDE5 inhibitor is mirodenafil or a pharmaceutically acceptable salt thereof.

18. A pharmaceutical composition according to any one of claims 1 to 17, wherein the composition is a formulation for oral administration and the daily dose of the active ingredient is 1 mg to 100 mg.

19. A pharmaceutical composition according to claim 18, characterized in that the daily dosage of the active ingredient is 1 mg to 20 mg.

20. A method for extending an individual's healthspan or delaying the decline in aging-related metabolic function, comprising the step of administering a therapeutically effective amount of a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof to an individual, wherein the administration activates the intracellular AMPK pathway to inhibit body fat accumulation without reduction in food intake and induce a Dietary Restriction-like transcriptome profile.

21. A method according to claim 20, characterized in that the method induces metabolic reprogramming to delay the decline in locomotion associated with aging or to maintain associative memory.

22. A method for preventing or treating age-related intestinal barrier dysfunction or leaky gut syndrome, comprising the step of administering a therapeutically effective amount of a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof to an individual.

23. A method according to claim 22, characterized in that the administration strengthens the tight junction structure of intestinal epithelial cells or reduces intestinal permeability.

24. A method according to claim 22, characterized in that it blocks the systemic influx of intestinal microorganism-derived toxins or inflammation-inducing factors to suppress systemic inflammation (inflammaging) or neuroinflammation.

25. A method according to claim 22, characterized in that the administration suppresses systemic inflammation (inflammaging) or neuroinflammation caused by increased intestinal permeability via the gut-brain axis.

26. A method for preventing or treating a disease associated with TDP-43 proteinopathy, comprising the step of administering a therapeutically effective amount of a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof to an individual, wherein the administration is characterized by inhibiting abnormal aggregation of TDP-43 protein within nerve cells or maintaining proteostasis to reduce neurotoxicity.

27. In paragraph 26, the above TDP-43 proteinopathy-related disease is amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), or limbic-dominant senile TDP-43 encephalopathy (LATE).

28. In any one of paragraphs 20, 22, or 26, the PDE5 inhibitor is Mirodenafil, Sildenafil, Tadalafil, Vardenafil, Udenafil, Avanafil, Lodenafil, NBB-116, Simmerafil, RT234, TPN-729, DDCI-01, TOP-N53, TR-422, Gisadenafil, PF-00489791, PF-03049423, SLx-2101, Youkenafil, Padanafil, NCX-1728, TOPV-122, Zaprinast, MSTM-102, A method characterized by being selected from the group consisting of TOPT-5, E4021 and Tunodafil and their pharmaceutically acceptable salts.

29. A method according to claim 28, wherein the PDE5 inhibitor is mirodenafil or sildenafil, or a pharmaceutically acceptable salt thereof.

30. A method according to claim 29, characterized in that the PDE5 inhibitor is mirodenafil or a pharmaceutically acceptable salt thereof.

31. A method according to any one of claims 20 to 30, wherein the PDE5 inhibitor is administered at a dose of 1 mg to 100 mg per day, or preferably at a low dose of 1 mg to 20 mg daily.

32. A phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof for use in extending an individual’s healthspan or delaying the decline of age-related metabolic functions, wherein the use is characterized by inducing a metabolic state similar to dietary restriction through intracellular AMPK pathway activation.

33. Phosphodiesterase 5 (PDE5) inhibitors or pharmaceutically acceptable salts thereof for use in the prevention or treatment of age-related intestinal barrier dysfunction or leaky gut syndrome.

34. In paragraph 33, the above use is a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof, characterized by inhibiting systemic inflammation by blocking the systemic influx of intestinal toxins by reinforcing the tight junction structure of intestinal epithelial cells to reduce intestinal permeability.

35. A phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of a disease associated with TDP-43 proteinopathy, wherein the disease is characterized by abnormal aggregation of TDP-43 protein within neurons.

36. A phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof, characterized in that the disease in paragraph 35 is amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), or limbic-dominant senile TDP-43 encephalopathy (LATE).

37. A phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof, wherein, in any one of claims 32 to 36, the PDE5 inhibitor is mirodenafil or a pharmaceutically acceptable salt thereof, and the mirodenafil is administered at a dose of 1 mg to 100 mg per day, or preferably at a low dose of 1 mg to 20 mg daily.

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