Pharmaceutical composition for treating traumatic brain injury based on control of zinc toxicity and oxidative stress comprising PDE5 inhibitor as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating traumatic brain injury (TBI), comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

Description

Pharmaceutical composition for the treatment of traumatic brain injury based on the control of zinc toxicity and oxidative stress comprising a PDE5 inhibitor as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of traumatic brain injury (TBI) comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

[0002] More specifically, the present invention relates to a pharmaceutical composition for treating traumatic brain injury that effectively suppresses oxidative stress, neuronal degeneration, and neuroinflammation by suppressing the accumulation of free zinc, which is abnormally increased in brain tissue after traumatic brain injury, and by restoring glutathione (GSH) levels through the activation of the Nrf2 (Nuclear factor erythroid 2-related factor 2) / HO-1 (Heme oxygenase-1) signaling pathway.

[0003] In addition, the present invention relates to a treatment technology linked to companion diagnostics that improves therapeutic effects by measuring zinc concentration and oxidative stress-related indicators (e.g., GSH, 4-hydroxynonenal (4-HNE), Nrf2 activity levels, etc.) in brain tissue or biological samples to select a group of traumatic brain injury patients at high risk of secondary damage due to zinc toxicity, and administering the pharmaceutical composition to the selected patient group.

[0004] In addition, the present invention relates to a multi-mechanism-based brain function regeneration technology that promotes endogenous neurogenesis in the hippocampal dentate gyrus region after traumatic brain injury, and induces the maturation and functional differentiation of new cells so that new cells are effectively integrated into damaged neural circuits, thereby promoting long-term neuronal survival and recovery of motor and cognitive functions.

[0005]

[0006] In modern society, Traumatic Brain Injury (TBI) is a representative severe neurological disorder occurring across all age groups due to various causes, such as traffic accidents, falls, industrial accidents, and sports injuries, and is recognized globally as a serious public health and socioeconomic problem. TBI is not merely an acute injury; it causes long-term decline in motor, cognitive, and emotional functions, significantly lowering the individual patient's quality of life and imposing a massive medical and social burden on families and society as a whole. Consequently, the development of treatment strategies that go beyond simply ensuring survival immediately after injury to fundamentally restore damaged brain functions and enable patients to return to daily life has emerged as a core task in modern neuroscience and clinical medicine.

[0007] The pathological mechanism of traumatic brain injury (TBI) is divided into primary injury, which occurs immediately at the moment of impact, and secondary injury, which develops over the course of hours to days. Primary injury is an irreversible stage in which neuronal shearing, contusion, vascular rupture, and local hemorrhage occur due to mechanical forces caused by external impact; it is virtually impossible to directly repair this with current medical technology. On the other hand, secondary injury is a stage in which brain damage gradually spreads through biochemical and cellular chain reactions triggered by primary injury. Brain edema, breakdown of the blood-brain barrier (BBB), excitotoxicity, oxidative stress, and neuroinflammatory responses interact in a complex manner to determine the final neurological prognosis. Therefore, the key to modern TBI treatment lies not in reversing the primary injury that has already occurred, but in how effectively the pathological chain reactions of the secondary injury that unfolds thereafter can be blocked and controlled.

[0008] However, current clinical treatment for traumatic brain injury is limited primarily to supportive care aimed at controlling intracranial pressure (ICP), maintaining adequate cerebral blood flow, and preventing complications such as hypoxia and infection. Specifically, treatments include alleviating cerebral edema through the administration of osmotic diuretics (e.g., Mannitol), controlling excitotoxicity using anticonvulsants or sedatives, or surgical procedures such as decompression craniotomy when necessary. While these treatments may contribute to some extent in temporarily relieving physical pressure or symptoms, there are currently no approved pharmacological treatments that directly protect damaged neurons or induce the regeneration of destroyed neural circuits by blocking the key mechanisms of secondary injury.

[0009] Among the various factors contributing to the spread of such secondary damage, the abnormal accumulation of 'free zinc' has recently garnered attention as a key pathological mechanism, serving as an upstream regulatory factor that triggers oxidative stress and neuronal death in the early stages of injury. Although zinc is an essential trace element for normal synaptic transmission and neuroplasticity, when it is released in large quantities from presynaptic neurons following traumatic brain injury and accumulates in major brain regions, including the hippocampus, it disrupts mitochondrial electron transport systems and induces the excessive production of reactive oxygen species (ROS), thereby causing severe cytotoxicity. This zinc toxicity is reported to develop within hours of injury; if zinc accumulation is not effectively controlled within this critical window, glutathione (GSH), a key component of the endogenous antioxidant defense system, is rapidly depleted, leading to a chain reaction of irreversible neuronal death. Nevertheless, current standard treatments fail to provide the means to directly block or regulate this pathological trigger known as early zinc toxicity.

[0010] Meanwhile, phosphodiesterase-5 (PDE5) inhibitors have been used primarily for the treatment of erectile dysfunction or pulmonary arterial hypertension based on their vasodilating effects, and recently, studies on their potential for neuroprotection in central nervous system diseases are gradually being reported.

[0011] However, existing studies on traumatic brain injury involving PDE5 inhibitors (e.g., US 9,750,743 B2) have a fundamental limitation in that most focus on describing the effects of administration 24 to 72 hours after injury induction. According to the general pathological mechanism of traumatic brain injury, the translocation and accumulation of zinc released from synapses within neurons is observed during the acute phase spanning several hours to tens of hours (approximately 12 to 24 hours) after injury, and zinc kinetics during this period have been reported to be closely associated with neuronal death (Suh et al., 2000).

[0012] Therefore, administering the drug 24 hours after the zinc peak has swept through the brain tissue, as in the prior art, is a post-treatment measure that intervenes after mitochondrial breakdown and irreversible neuronal damage caused by zinc toxicity have been completed. In other words, while administration after 24 hours may partially delay the spread of secondary damage, there are clear limitations in fully preserving brain function by blocking zinc toxicity, which is the starting point of the pathology.

[0013] Accordingly, the inventors have determined that the only solution to prevent secondary damage at the source is to suppress the zinc accumulation curve itself by administering the drug within '24 hours' before the zinc concentration exceeds the irreversible threshold, within 12 hours during the rising phase before reaching the peak, and crucially within 3 hours (Golden Hour) when the initial surge occurs.

[0014] Furthermore, regarding endogenous neurogenesis, existing technologies (e.g., US 18 / 906,967 A) are limited to suggesting the possibility of a 'quantitative increase' in the number of neurons in chronic neurodegenerative diseases such as Alzheimer's disease. In the context of traumatic brain injury accompanied by rapid circuit disruption, intense oxidative stress, and an inflammatory environment, it is difficult to achieve functional recovery through simple cell proliferation alone. In other words, for new cells to differentiate into mature neurons and functionally integrate (wiring) into damaged existing neural circuits, a 'permissive microenvironment' in which the toxic environment is sufficiently suppressed must be established beforehand; however, a technical approach that organically links zinc toxicity control and neuroregeneration has not yet been established.

[0015] Accordingly, the inventors confirmed that the brain's endogenous regenerative capacity can be maximized by effectively controlling the accumulation of free zinc occurring in the early stages of traumatic brain injury through a PDE5 inhibitor, and by fundamentally suppressing oxidative stress and neuroinflammation through the restoration of the antioxidant defense system via the activation of the Nrf2 / HO-1 signaling pathway. Based on these findings, the present invention was completed to propose a novel therapeutic concept capable of controlling the pathological progression of traumatic brain injury stepwise, from the initial stage of injury to long-term functional recovery.

[0016]

[0017] The main problem that the present invention aims to solve is to provide an effective and safe pharmacological treatment method capable of fundamentally overcoming permanent neurological disabilities caused by traumatic brain injury (TBI) and substantially regenerating the cognitive and motor functions of the damaged brain.

[0018] Specifically, the present invention aims to overcome the fundamental limitations of existing conservative treatments, which are limited to symptom relief and survival maintenance, and to solve the following multifaceted and specific technical challenges by blocking the pathological biochemical chain reaction of secondary injury that unfolds after traumatic brain injury at a higher mechanism level.

[0019] First, it provides a therapeutic strategy that can fundamentally suppress the initial pathological chain reaction that serves as the starting point for mitochondrial dysfunction and neuronal death by preemptively blocking the abnormal accumulation of free zinc that occurs rapidly during the critical period when secondary damage progresses irreversibly after traumatic brain injury.

[0020] Second, the invention provides a pharmaceutical composition that restores glutathione (GSH) levels to normal levels, which are key components of the Nrf2 activation mechanism and endogenous antioxidant system disrupted by zinc toxicity, thereby rebuilding the self-defense capabilities of brain tissue and directly inhibiting neuronal degeneration and neuronal damage caused by excessive production of reactive oxygen species (ROS) and lipid peroxidation.

[0021] Third, the present invention provides a precision medicine platform linked with 'non-invasive diagnostic technologies,' such as 1H-MRS brain imaging or blood exosome analysis. This enables the administration of drugs to selected patients confirmed to have free zinc accumulation or GSH depletion, without the risk of open-skull tissue biopsies. This companion diagnostics approach offers high commercial value by not only minimizing side effects through reduced unnecessary drug administration but also maximizing the potential for clinical success by dramatically increasing the responder rate.

[0022] Finally, going beyond the control of the aforementioned individual pathological factors, we aim to realize an advanced brain function regeneration technology concept that goes beyond mere quantitative increase of newly generated neurons by creating a 'permissive microenvironment' capable of neuronal regeneration through a higher-level molecular mechanism that integrally induces these effects by suppressing the abnormal activation of toxic astrocytes. By maximizing the efficiency of qualitative differentiation into mature neurons, this technology enables functional integration into damaged neural circuits.

[0023]

[0024] 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.

[0025]

[0026] The present invention relates to a pharmaceutical composition for the prevention or treatment of traumatic brain injury (TBI) comprising a phosphodiesterase-5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

[0027] The composition according to the present invention effectively controls zinc toxicity and oxidative stress induced after traumatic brain injury by activating the Nrf2 / HO-1 signaling pathway through the regulation of cGMP signals within brain tissue, thereby inhibiting the resulting neuronal damage and death. Furthermore, by regulating the neuroinflammatory response to create a microenvironment favorable for neuroregeneration and promoting endogenous neurogenesis in the hippocampal dentate gyrus, it can induce the functional recovery of damaged neural circuits.

[0028] In addition, the present invention includes a companion diagnosis-linked treatment method for traumatic brain injury, which involves selecting patients at high risk of zinc toxicity or oxidative stress based on biological indicators or brain imaging information and administering a PDE5 inhibitor. Specific embodiments, active ingredients, timing and dosage of administration, and therapeutic effects of the present invention are described in more detail below.

[0029]

[0030] According to one embodiment,

[0031] A pharmaceutical composition for the prevention or treatment of traumatic brain injury (TBI) is provided, comprising mirodenafil or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition is characterized by controlling the accumulation of free zinc or oxidative stress in the brain tissue of a subject.

[0032] In the present invention, the mirodenafil or a pharmaceutically acceptable salt thereof may be mirodenafil 2HCl.

[0033] In the present invention, the composition may be administered to a selected patient group in which (a) the concentration of free zinc or oxidative stress indicators in biological samples separated from the subject are above or below a reference value; or (b) a patient group in which it is confirmed through brain imaging techniques that the accumulation of free zinc or the level of oxidative stress in the brain has changed significantly compared to a normal control group.

[0034] In the present invention, the oxidative stress indicator may be an increase in 4-HNE (4-hydroxynonenal) or a decrease in glutathione (GSH) levels.

[0035] In the present invention, the mirodenafil or a pharmaceutically acceptable salt thereof may be administered at a dose of 10 mg to 50 mg per day based on mirodenafil.

[0036]

[0037] According to another embodiment,

[0038] A pharmaceutical composition for the prevention or treatment of traumatic brain injury (TBI) is provided, comprising mirodenafil or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition is administered to a patient diagnosed with an increased concentration of free zinc in brain tissue compared to a normal control group or a decreased concentration of glutathione (GSH) compared to a normal control group.

[0039] In the present invention, the patient may be: (a) a patient in whom the accumulation of free zinc in the hippocampus region is confirmed through brain imaging; or (b) a patient in whom an increase in 4-HNE (4-hydroxynonenal) or a decrease in Nrf2 activity is confirmed as an indicator of oxidative stress in a biological sample.

[0040] In the present invention, the brain imaging includes magnetic resonance spectroscopy (1H-MRS) or positron emission tomography (PET), and the biological sample may include blood, plasma, serum, cerebrospinal fluid (CSF), or neural-derived exosomes in the blood.

[0041] In the present invention, the mirodenafil or a pharmaceutically acceptable salt thereof may be mirodenafil 2HCl.

[0042] In the present invention, the composition is administered at a dose of 10 mg to 100 mg per day, and the first administration may be initiated within 24 hours from immediately after the occurrence of traumatic brain injury until zinc accumulation in brain tissue reaches its peak.

[0043]

[0044] According to another embodiment,

[0045] A composition comprising mirodenafil or a pharmaceutically acceptable salt thereof for use in treating traumatic brain injury (TBI) is provided, wherein the composition is characterized by being administered within 24 hours immediately after the occurrence of traumatic brain injury to block mitochondrial damage caused by a zinc surge.

[0046] In the present invention, the composition can be administered repeatedly once a day for 7 days or more to inhibit the activation of microglia and astrocytes.

[0047]

[0048] According to another embodiment,

[0049] A pharmaceutical composition for the structural rewiring of damaged neural circuits and the recovery of cognitive function in patients with traumatic brain injury is provided, comprising mirodenafil or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition promotes the proliferation of endogenous neurons in the hippocampal dentate gyrus region and increases the efficiency of differentiation of the proliferated cells into mature neurons (NeuN-positive neurons).

[0050] In the present invention, the composition can restore spatial learning ability decline or long-term memory decline caused by traumatic brain injury and increase the proportion of BrdU and NeuN double-positive cells in brain tissue.

[0051] In the present invention, the composition can create a permissive microenvironment capable of nerve regeneration by being administered during the acute phase after the occurrence of traumatic brain injury and inhibiting zinc toxicity.

[0052]

[0053] According to another embodiment,

[0054] A pharmaceutical composition for the recovery of cognitive or motor function in patients with traumatic brain injury is provided, comprising mirodenafil or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition promotes endogenous neurogenesis in the hippocampal dentate gyrus region of the subject and induces the qualitative differentiation of newly formed cells into mature neurons to regenerate damaged neural circuits.

[0055] In the present invention, the composition may be administered within 24 hours after the occurrence of traumatic brain injury, or administration may begin at a time when 24 hours to 7 days have elapsed, and may be administered repeatedly for 1 week or more.

[0056] In the present invention, the recovery of cognitive function or motor function may be selected from one or more of the group consisting of (a) improvement of spatial learning ability and memory; (b) improvement of motor coordination ability and sense of balance; and (c) reduction of neurological severity indicators.

[0057] In the present invention, the promotion of endogenous neurogenesis can increase the proportion of BrdU and NeuN double-positive cells in brain tissue.

[0058]

[0059] According to another embodiment,

[0060] A pharmaceutical composition for inhibiting neuroinflammation caused by traumatic brain injury is provided, comprising mirodenafil or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition is characterized by inhibiting CD68 or Iba-1 expression in microglia within brain tissue, or GFAP or C3 expression in astrocytes.

[0061]

[0062] The pharmaceutical compositions according to the present invention may be administered in combination with one or more additional therapeutic agents selected from the group consisting of osmotic therapeutic agents, anti-inflammatory agents, neurotrophic factors, and stem cell therapeutic agents.

[0063]

[0064] According to another embodiment,

[0065] As a method for treating patients with traumatic brain injury (TBI),

[0066] (a) A step of determining the concentration of zinc or the level of oxidative stress indicators (GSH, 4-HNE, Nrf2) in the brain tissue of the patient;

[0067] (b) a step of selecting patients whose zinc concentration or oxidative stress indicators fall outside a preset reference range compared to a normal control group based on the above verification results; and

[0068] (c) a step of administering a therapeutically effective amount of mirodenafil or a pharmaceutically acceptable salt thereof to the selected patient.

[0069] A method for treating traumatic brain injury, including..., is provided.

[0070] In the present invention, the level verification of step (a) can be performed by analysis of a biological sample separated from a patient or by non-invasive measurement through brain imaging techniques.

[0071] In the present invention, the biological sample may be selected from the group consisting of cerebrospinal fluid, blood, plasma, and serum.

[0072] In the present invention, the brain imaging technique may include positron emission tomography (PET), magnetic resonance imaging (MRI), or fluorescence imaging targeting zinc or oxidative stress markers.

[0073]

[0074] According to another embodiment,

[0075] As a method to restore cognitive and motor functions in patients suffering from sequelae of traumatic brain injury,

[0076] (a) a step of screening patients after the acute phase has passed following the occurrence of traumatic brain injury; and

[0077] (b) a step of administering mirodenafil or a pharmaceutically acceptable salt thereof to the patient.

[0078] A method is provided that includes, wherein the administration promotes endogenous neurogenesis in the hippocampal dentate gyrus of the patient's brain to restore the function of damaged neural circuits.

[0079] In the present invention, the mirodenafil may be administered within 24 hours after the occurrence of traumatic brain injury, or administration may begin at a time when 24 hours to 7 days have elapsed, and may be administered repeatedly for more than 1 week.

[0080] In the present invention, the recovery of cognitive and motor functions may be one or more of (a) improvement of spatial learning ability and memory; (b) improvement of motor coordination ability and sense of balance; and (c) reduction of neurological severity.

[0081] In the present invention, the promotion of endogenous neurogenesis may entail an increase in the number of BrdU and DCX positive cells in brain tissue, or an increase in the proportion of BrdU and NeuN dual positive cells.

[0082]

[0083] According to another embodiment,

[0084] A method for treating a patient with traumatic brain injury (TBI) is provided, comprising: (a) a step of determining whether there is an excessive accumulation of zinc in brain tissue or the level of oxidative stress indicators (GSH, 4-HNE, Nrf2) by analyzing a biological sample isolated from the patient or taking non-invasive brain images; (b) a step of selecting patients in whom, as a result of the determination in step (a), the zinc concentration is above a reference value or the glutathione (GSH) concentration is below a reference value; and (c) a step of administering a therapeutically effective amount of mirodenafil or a pharmaceutically acceptable salt thereof to the selected patients.

[0085] In the present invention, the verification of step (a) can be performed by a non-invasive or minimally invasive method.

[0086] In the present invention, the administration of step (c) may be initiated within 24 hours after the occurrence of traumatic brain injury, before irreversible neuronal damage caused by zinc toxicity is completed.

[0087] In the present invention, the administration may be initiated within 12 hours after the occurrence of traumatic brain injury, which is the rising phase in which zinc concentration rapidly increases.

[0088]

[0089] A pharmaceutical composition comprising a PDE5 inhibitor or mirodenafil as an active ingredient according to the present invention provides the following significant and advanced effects.

[0090] First, the composition of the present invention can fundamentally suppress secondary damage caused by zinc toxicity occurring immediately after injury by directly controlling 'zinc accumulation,' which is a key pathology in the early stages of traumatic brain injury (TBI).

[0091] Second, the composition of the present invention can reactivate the Nrf2 / GSH pathway to restore the endogenous antioxidant defense system of brain tissue, thereby creating an environment in which the brain itself can resist oxidative stress and lipid peroxidation and delay neuronal degeneration.

[0092] Third, since the composition of the present invention can precisely evaluate a patient's pathological state based on specific biomarkers, treatment efficiency can be maximized by selecting a patient sub-population exhibiting specific pathological phenotypes, such as zinc overaccumulation or glutathione (GSH) depletion. In particular, the present invention provides a precision medicine platform linked with non-invasive diagnostic technologies, such as 1H-MRS brain imaging or blood exosome analysis, thereby possessing commercial utility in that it can select an optimal administration group without tissue biopsy.

[0093] Fourth, the composition of the present invention promotes endogenous neurogenesis and dramatically improves the efficiency of differentiation of newly formed cells into mature neurons by more than 2.5 times compared to the control group, thereby achieving the structural rewiring and functional integration of damaged neural circuits. This goes beyond simple anti-apoptosis and is the result of creating a 'permissive microenvironment' where stem cells can survive through the control of zinc toxicity, providing an unexpected neurogenesis effect that could not be predicted by existing technologies.

[0094] Fifth, since the composition of the present invention can utilize drugs such as mirodenafil, whose safety has already been verified, it can provide a safe treatment method that can be immediately applied to TBI patients requiring urgent treatment, while reducing the cost and time required for new drug development.

[0095] Sixth, the present invention has identified a significant difference (Singularity) in therapeutic effects depending on the timing of administration. By administering the drug during the "golden hour" corresponding to the acute phase of TBI—specifically within "24 hours," specifically within "12 hours" before zinc accumulation reaches its peak, and even more specifically within "3 hours" when a zinc surge occurs—it exerts the effect of "blocking the source of zinc toxicity," which cannot be achieved when administered after 24 hours. This is a decisive technical feature that not only dramatically improves neuronal survival rates compared to existing treatments but also preserves the brain microenvironment, which is an essential prerequisite for long-term cognitive function recovery.

[0096] In conclusion, the composition of the present invention can comprehensively regulate the entire pathological process from the early stages of TBI to the long-term recovery period, and through zinc control and qualitative nerve regeneration, it can achieve nerve function recovery and long-term recovery that were impossible with existing technologies.

[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 the experiment showing zinc toxicity induction and drug treatment in a hippocampal neuron culture model performed in Example 1. Figure 1B is a schematic diagram of the experiment showing zinc toxicity induction and drug treatment in a neuron-astrocytic co-culture model performed in Example 2. Figure 1C is a representative image showing the morphological changes of cells observed according to treatment with different concentrations of mirodenafil after zinc treatment in a hippocampal neuron culture model. Figure 1D is a graph analyzing cell viability according to treatment with different concentrations of mirodenafil after zinc treatment in a hippocampal neuron culture model. Figures 1E to 1H are fluorescence images and quantification graphs analyzing changes in intracellular glutathione (GSH), nNOS, and Nrf2 expression upon treatment with mirodenafil depending on the presence or absence of a zinc toxicity environment. Figures 1I to 1L are fluorescence images and quantitative graphs analyzing the number of NeuN+ neurons and changes in the expression of HO-1 and Nrf2 upon treatment with mirodenafil depending on the presence or absence of a zinc toxic environment.

[0101] Figure 2A is a representative image showing morphological changes in cells following treatment with different concentrations of mirodenafil after zinc treatment in a neuron-astrocytic co-culture model. Figure 2B is a graph analyzing cell viability following treatment with different concentrations of mirodenafil after zinc treatment in a neuron-astrocytic co-culture model. Figures 2C to 2F are fluorescence images and quantification graphs analyzing changes in intracellular expression of MAP2, GFAP, and S100B upon mirodenafil treatment depending on the presence or absence of a zinc toxic environment. Figures 2G to 2J are fluorescence images and quantification graphs analyzing changes in expression of glutathione (GSH) and GFAP and changes in the number of NeuN+ neurons upon mirodenafil treatment depending on the presence or absence of a zinc toxic environment.

[0102] Figures 3A to 3D are FJB stained images confirming neuronal degeneration in the hippocampus region 24 hours after treatment with different concentrations of mirodenafil (0.5, 1, 2 mg / kg) in a TBI-induced model. Figures 3E to 3H are graphs quantifying the stained images of Figures 3A to 3D by hippocampal region (CA1, CA3, GCL, and Hilus). Figures 3I to 3J are TSQ stained images confirming the accumulation of free zinc in the hippocampus region 3 hours after treatment with or without mirodenafil (2 mg / kg) in a TBI-induced model. Figures 3K to 3M are graphs quantifying the stained images of Figures 3I to 3J by hippocampal region (CA1, GCL, and Hilus).

[0103] Figure 4A is a fluorescence image analyzing changes in the expression of nNOS, Nrf2, and glutathione in brain tissue collected 24 hours after mirodenafil treatment depending on the presence or absence of a TBI-induced model. Figures 4B to 4D are graphs quantifying the stained image of Figure 4A by hippocampal region (CA1, CA3, GCL, and Hilus). Figure 4E is a fluorescence image of changes in the expression of 4-HNE, a lipid peroxidation indicator, in brain tissue collected 24 hours after mirodenafil treatment depending on the presence or absence of a TBI-induced model. Figure 4F is a quantification graph for the hippocampal region (CA1) based on the stained image of Figure 4E.

[0104] Figure 5A is a fluorescence image analyzing changes in the expression of Iba-1 and CD68 in brain tissue collected 24 hours after treatment with mirodenafil, depending on the presence or absence of a TBI-induced model. Figures 5B to 5C are quantitative graphs for the hippocampal region (CA1) for the stained image of Figure 5A. Figure 5D is a fluorescence image analyzing changes in the expression of GFAP and C3 in brain tissue collected 24 hours after treatment with mirodenafil, depending on the presence or absence of a TBI-induced model. Figures 5E to 5F are quantitative graphs for the hippocampal region (CA1) for the stained image of Figure 5D.

[0105] Figure 6A is a schematic diagram of an experiment performed in an animal model to evaluate the therapeutic effect of mirodenafil in a TBI model performed in Example 6. Figure 6B is a schematic diagram of an experiment performed in an animal model to evaluate the therapeutic effect of mirodenafil in a TBI model performed in Example 7. Figure 6C is a graph showing the degree of improvement (ΔmNSS) in sensory, motor, reflex, and balance functions in the mirodenafil treatment group. Figure 6D is a graph showing that neurological deficits (mNSS score) were significantly reduced in the mirodenafil treatment group (2 mg / kg) compared to the TBI-Vehicle group. Figure 6E is a representative NeuN staining image confirming surviving neurons in brain tissue 2 weeks after TBI induction. Figures 6F to 6H are the results of quantitative analysis of NeuN staining, and are graphs quantifying the mirodenafil treatment group (2 mg / kg) by hippocampal region (CA1, GCL, and Hilus) compared to the TBI-vehicle group.

[0106] Figure 7A shows images of representative tracks on days 1 and 6 of the Barnes Maze experiment. Figure 7B is a graph showing the quantitative analysis of escape latency in finding the target hole in the Barnes Maze experiment, demonstrating that the escape latency was significantly reduced in the mirodenafil treatment group (TBI-mirodenafil) compared to the vehicle group. Figure 7C shows images of representative swimming paths on days 1 and 5 of the Morris Water Maze experiment. Figures 7D and 7E are graphs showing escape latency and distance to target in the Morris Water Maze experiment, demonstrating that spatial learning ability was improved in the mirodenafil treatment group. Figures 7F and 7G are graphs showing the results of a probe test with the escape band removed, representing the number of target crossings and the time spent in the target quadrant, respectively. Figure 7H is a representative immunofluorescence staining image showing cell proliferation markers (BrdU, green), immature neuron markers (DCX, red), mature neuron markers (NeuN, white), and nuclear staining (DAPI, blue) performed in the hippocampus region. Figures 7I to 7K are graphs showing the quantitative analysis of the number of mature neurons (NeuN+), proliferated cells (BrdU+), and immature neurons (DCX+), respectively, and are quantified by hippocampal region (CA1, CA3, GCL, and Hilus). Figure 7L is a graph analyzing the proportion of cells exhibiting the phenotype of mature neurons (NeuN) among newly labeled BrdU cells, showing that the efficiency of neurogenesis and differentiation increased in the mirodenafil-treated group.

[0107]

[0108] 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).

[0109] Definition of Terms

[0110] 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.

[0111] 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.

[0112] 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.

[0113] As used in this specification, "Subject" means a mammal, preferably a human, but is not limited thereto.

[0114] As used herein, "Functional Recovery" does not merely refer to behavioral improvements, such as a reduction in the mNSS score, but is interpreted as a concept that essentially encompasses the processes of "long-term survival of neurons," "structural integration" of newly generated neurons, and "neural circuit rewiring" as biological mechanisms enabling such improvements within damaged brain tissue. In other words, since the functional recovery mentioned in this invention is an outcome impossible to achieve without the physical reconstruction of neural networks, the description of functional recovery includes the description of neural circuit reconstruction.

[0115] The term "neuroprotection" as used in this specification includes primary defense against apoptosis immediately after traumatic brain injury, and further encompasses the process of maintaining a "permissive microenvironment" in which the brain's inherent endogenous neurogenesis ability can be expressed by removing inhibitory factors such as zinc toxicity. Therefore, the "protective" effect mentioned in this invention inevitably contains the effect of promoting "regeneration" and "differentiation."

[0116] As used herein, “phosphodiesterase 5 (PDE5) inhibitor” refers to any compound and any pharmaceutically acceptable salt thereof that selectively inhibits the activity of PDE5, an enzyme that degrades intracellular cGMP, thereby increasing the concentration of cGMP. The PDE5 inhibitor in the present invention is defined as a multi-mechanistic active ingredient that not only improves blood flow but also rebuilds the antioxidant defense system and controls zinc toxicity by activating the downstream signaling pathway, the Nrf2 / HO-1 pathway.

[0117] As used in this specification, “Traumatic Brain Injury (TBI)” refers to structural and functional damage to the brain caused by the transmission of mechanical energy, such as physical impact, acceleration, or deceleration. This encompasses both primary injury, which occurs immediately at the time of the accident, and secondary injury, which is a biochemical destruction process that continues for several hours to several days thereafter. The present invention is particularly intended to regenerate brain function by controlling the initial pathological mechanism that triggers secondary injury.

[0118] As used herein, "zinc toxicity" or "zinc accumulation" refers to cytotoxicity caused by the excessive release of zinc stored in vesicles of presynaptic neurons into the synaptic cleft immediately after TBI, leading to an excessive influx into postsynaptic neurons. This is a leading pathological factor of secondary damage that results in mitochondrial breakdown and depletion of the antioxidant system (GSH).

[0119] As used in this specification, "oxidative stress" refers to a state in which the balance between the generation of reactive oxygen species (ROS) in the body and the antioxidant defense system that removes them is disrupted. In this invention, it specifically refers to a neurodegenerative state accompanied by the depletion of glutathione (GSH) and the accumulation of lipid peroxides (4-HNE).

[0120] As used herein, the "permissive microenvironment" refers to a biological foundation optimized to suppress toxic substances (such as zinc) and inflammatory factors (such as Type A1 astrocytes) occurring after brain injury, thereby allowing newly formed neurons to grow normally without dying and to be wired with existing neural circuits. This concept is contrasted with the "hostile microenvironment," where regeneration is inhibited.

[0121] As used herein, "Quality Differentiation Efficiency" refers to the proportion of newly generated cells (BrdU+) that go beyond mere survival to develop into mature neurons capable of performing actual functions by acquiring an indicator (NeuN+) representing the characteristics of a mature neuron. The present invention is characterized by significantly increasing the proportion of dual-positive (BrdU+ / NeuN+) cells, which has not been achieved by existing technologies.

[0122] As used herein, "Functional Integration" refers to a step of substantially restoring cognitive and motor functions by having qualitatively differentiated new neurons structurally connect within a destroyed neural network and exhibit synaptic activity.

[0123] As used herein, "Companion Diagnostics" refers to diagnostic tests performed in conjunction with drug administration to identify patient groups with high therapeutic responsiveness or low risk of side effects to a specific drug therapy, and the present invention includes a precision medical approach through the measurement of zinc concentration and oxidative stress indicators.

[0124] As used in this specification, the term "biological sample" includes, but is not limited to, tissues, cells, whole blood, serum, plasma, and cerebrospinal fluid (CSF) separated from an individual, as well as saliva, urine, or exhaled breath condensate. In particular, the biological sample in the present invention is interpreted to encompass all body fluids that can indirectly reflect the pathological state of brain tissue at the periphery, including "neural-derived exosomes" or "extracellular vesicles" circulating in the blood.

[0125]

[0126] 1. Composition for treating traumatic brain injury

[0127] 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 effectively inhibits acute neuronal damage, oxidative stress, neuroinflammation, neural network breakdown, and the resulting decline in motor and cognitive functions caused by traumatic brain injury (TBI), and further induces functional recovery and neuroregeneration of damaged brain tissue.

[0128] The composition of the present invention enhances intracellular cyclic guanosine monophosphate (cGMP) signaling through PDE5 inhibition, and thereby maintains nitric oxide synthase (nNOS) activity, activates the antioxidant transcription factor Nrf2, restores glutathione (GSH) homeostasis, and induces antioxidant enzyme expression as downstream signaling pathways, thereby [reducing] the zinc (Zn] that occurs in the early stages of traumatic brain injury. 2+ It can fundamentally normalize toxic and extreme oxidative stress environments. In particular, the inventors confirmed through prior research that the effects of 'inhibiting zinc accumulation' and 'reducing oxidative stress' are not merely passive protection preventing the death of existing cells, but are indispensable prerequisites for the functional recovery of brain tissue. High concentrations of free zinc and reactive oxygen species generated after traumatic brain injury create an 'adversarial environment' that inhibits the proliferation and differentiation of endogenous neurons.

[0129] Therefore, the action of the composition according to the present invention in controlling zinc toxicity and restoring glutathione levels is a direct cause of reactivating the endogenous neurogenesis mechanism that had been suppressed. That is, the neurogenesis and neural network reconstruction effects identified in the present invention (Fig. 7) are not separate new effects, but rather continuous biological phenomena inevitably induced by the core mechanism of the present invention, which is the control of zinc toxicity.

[0130] 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, TOPN-53, 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.

[0131] In the embodiments of the present invention, among the above PDE5 inhibitors, mirodenafil was selected as the representative compound, and the neuroprotective effect, long-term neuronal survival, neuroregeneration, and functional recovery effect and the mechanism of action in a traumatic brain injury model were intensively verified. The above mirodenafil (development code name: AR1001) is a pyrrolopyrimidinone class compound represented by [Chemical Formula 1].

[0132] [Chemical Formula 1]

[0133]

[0134] 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

[0135] Free Base: C 26 H 37 N5O5S / Molecular weight approx. 531.67 g / mol

[0136] Dihydrochloride (2HCl): C 26 H 39 Cl2N5O5S / Molecular weight approx. 604.6 g / mol

[0137] 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.

[0138] The primary mechanism of action of mirodenafil is the reversible and selective inhibition of the PDE5 enzyme, which leads to an increase in intracellular cGMP concentration. The present invention is the first to identify that the PDE5-cGMP signaling axis integrally regulates antioxidant defense, neuronal survival, and neuroregeneration in the pathogenesis of traumatic brain injury.

[0139]

[0140] (1) Acute neuroprotective effect through inhibition of zinc toxicity and oxidative stress

[0141] The composition of the present invention can suppress neurotoxicity caused by excessively released zinc ions in the early stages of traumatic brain injury and effectively block intracellular glutathione (GSH) depletion and reactive oxygen species (ROS) accumulation.

[0142] In one embodiment, the composition is administered during the acute phase within 72 hours, preferably within 48 hours, and more preferably within 24 hours after the onset of TBI, and in particular, may be first administered within 12 hours before full-scale accumulation of free zinc in brain tissue, or within 6 hours, more preferably within 3 hours, before the initiation of an irreversible neuronal death cascade. According to the examples, in a zinc-treated neuronal death model, the composition of the present invention was confirmed to substantially normalize the oxidative stress environment by increasing neuronal survival rates by 20% or more, 30% or more, or 50% or more compared to the control group, significantly maintaining nNOS expression, and restoring intracellular GSH levels by 10% or more, 20% or more, or 30% or more (Example 1).

[0143] Accordingly, the composition according to the present invention can be usefully used for acute neuronal protection in acute neurological injuries such as traumatic brain injury, cerebral contusion, concussion, ischemia-reperfusion injury, and hypoxic brain injury.

[0144]

[0145] (2) Activation effect of Nrf2-HO-1 antioxidant defense axis

[0146] The composition according to the present invention can induce enhancement of cGMP signaling through PDE5 inhibition, and thereby promote nuclear translocation and activation of the antioxidant transcription factor Nrf2, which can induce increased expression of HO-1 and antioxidant enzymes.

[0147] In one embodiment, the composition is characterized by activating the Nrf2 signaling pathway when administered during the acute phase following traumatic brain injury to increase HO-1 expression levels by 10%, 20%, or 30% or more compared to the control group, and in conjunction with this, restoring endogenous glutathione (GSH) levels by 10%, 20%, or 30% or more. According to an example, the composition of the present invention restored Nrf2 activation that had been suppressed under zinc toxicity conditions and significantly increased downstream antioxidant enzyme expression, thereby significantly reducing oxidative damage to neurons (Example 1).

[0148] Therefore, the composition according to the present invention can be used not as a simple ROS scavenger, but as a mechanism-based therapeutic agent that fundamentally reactivates the endogenous antioxidant defense system.

[0149]

[0150] (3) Effect of restoring motor and balance function after traumatic brain injury

[0151] The composition according to the present invention can induce functional recovery of neural circuits damaged by traumatic brain injury, thereby substantially improving motor skills and sense of balance.

[0152] According to the example, in an animal model of severe TBI, the group administered mirodenafil within 24 hours of injury showed an improvement in mNSS scores of 10% or more or 20% or more compared to the control group, and a performance time in the beam balance test improved by 10% or more or 20% or more, and these motor function recovery effects were maintained continuously for more than 1 week after injury (Example 6).

[0153] Therefore, the composition according to the present invention can be usefully used to improve post-traumatic movement disorders, gait disorders, balance disorders, and neurological sequelae.

[0154]

[0155] (4) Long-term neuronal survival and brain tissue preservation effects

[0156] The composition according to the present invention is not limited to the protective effect on acute neuronal cells, but can maintain neuronal survival over a long period of more than 2 weeks after injury.

[0157] According to the example, at 14 days after TBI, it was confirmed that the number of NeuN-positive neurons in the hippocampal CA1, dentate gyrus (GCL), and Hilus regions increased by more than 10%, more than 20%, or more than 30% compared to the control group (Example 6).

[0158] Accordingly, the composition according to the present invention can substantially inhibit delayed neuronal loss caused by traumatic brain injury and provide a brain tissue preservation effect.

[0159]

[0160] (5) Inducing effect of endogenous neural stem cell proliferation and neural regeneration

[0161] The composition according to the present invention can promote the proliferation and differentiation into neurons of endogenous neural stem cells that are inhibited after traumatic brain injury.

[0162] In one embodiment, the composition is BrdU in the hippocampal dentate gyrus region + or DCX + It increases the number of newly generated cells by more than 10%, more than 20%, or more than 50% compared to the control group, and in particular BrdU + / NeuN + Improves the double positive cell ratio by approximately 2.5 times (11% → 28%) (Example 7)

[0163] Therefore, the composition according to the present invention can go beyond merely inducing cell division and induce 'qualitative differentiation' into functional mature neurons.

[0164] Furthermore, the inventors confirmed that free zinc, which increases rapidly after traumatic brain injury, acts as a potent 'regeneration inhibitor' that suppresses the proliferation and differentiation of endogenous neural stem cells. Therefore, the composition of the present invention blocking zinc toxicity in the early stages of injury is an essential prerequisite for establishing a foundation for the subsequent normal differentiation of neural stem cells.

[0165] In other words, the groundbreaking neurogenesis and cognitive function recovery effects confirmed in Fig. 7 are not separate, independent phenomena, but are inevitable biological results induced by the 'early zinc surge control' which is a feature of the present invention. Through this, the present invention integrally achieves two heterogeneous effects, 'toxicity control (early)' and 'tissue regeneration (late)', through a single drug mechanism.

[0166]

[0167] (6) Effects on the recovery of cognitive function and learning and memory abilities

[0168] The composition of the present invention can significantly improve learning and memory abilities impaired by traumatic brain injury.

[0169] According to the example, the results of the behavioral evaluation confirmed that the spatial learning performance indicator of the group treated with the composition of the present invention improved by 15% or more or 30% or more compared to the control group, and the long-term memory evaluation indicator also improved by 15% or more or 30% or more (Example 7).

[0170] Accordingly, the composition according to the present invention can be used for the prevention or treatment of post-traumatic cognitive impairment, memory loss, concentration disorders, and post-traumatic dementia.

[0171]

[0172] (7) Potential for functional brain repair and regenerative medical applications

[0173] The composition according to the present invention can reduce the accumulation of free zinc by 10%, 20%, 30%, or 50% or more in a traumatic brain injury environment, significantly suppress oxidative stress and inflammatory responses, and reduce the expression of toxic astrocytes (C3, GFAP) and microglia activation markers (Iba-1, CD68) by 10%, 20%, 30%, or 50% or more, thereby actively establishing a permissive microenvironment in which newly generated neurons can survive and mature. Through this, beyond simple neuronal protection, it enables the functional restoration of damaged neural circuits and long-term recovery of brain function.

[0174] Therefore, the composition according to the present invention can present a regenerative medicine treatment paradigm that goes beyond the treatment of traumatic brain injury.

[0175]

[0176] 2. Uses of composition for treating traumatic brain injury

[0177] A composition comprising a phosphodiesterase 5 (PDE5) inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient according to the present invention may be implemented as a pharmaceutical composition, food composition, feed composition, or cosmetic composition for the prevention, treatment, or improvement of traumatic brain injury (TBI).

[0178] The composition according to the present invention can be used to suppress acute neuronal damage, zinc toxicity, oxidative stress, neuroinflammation, and the resulting decline in motor and cognitive functions caused by traumatic brain injury, and further to promote functional recovery of damaged brain tissue and neuroregeneration. In particular, the composition of the present invention is applicable at all pathological stages, from immediately after the occurrence of traumatic brain injury to the acute, subacute, and recovery phases, and the therapeutic effect can be maximized by administering it before the onset of or during the progression of the secondary injury chain reaction following traumatic brain injury.

[0179] In addition, the composition of the present invention may also be applied to the prevention, treatment, or improvement of acute or subacute neurological injury diseases such as cerebral contusion, concussion, diffuse axonal injury, ischemia-reperfusion injury, and hypoxic brain injury, which share pathophysiological mechanisms similar to traumatic brain injury.

[0180]

[0181] (1) Pharmaceutical composition

[0182] The above pharmaceutical composition may be administered by means of oral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, epithelial administration, local administration, vaginal administration, pulmonary administration, rectal administration, sublingual administration, buccal administration, transdermal administration, ocular administration, inhalation, intracavernous injection, intrathecal injection, epidural injection, and rectal administration. The route of administration may be appropriately selected according to the condition of the patient, the severity of traumatic brain injury, the stage of the pathology, the age and general condition of the patient, and the characteristics of the selected active ingredient. In the case of oral administration, the above pharmaceutical composition may be provided in the form of a tablet, or may be formulated to coat the active ingredient or protect it from degradation in the stomach. Additionally, the composition may be administered through any delivery device including a targeted delivery system so that the active ingredient can be efficiently delivered to brain tissue or the site of injury.

[0183] 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.

[0184] The appropriate dosage of the above pharmaceutical composition may vary depending on factors such as the formulation method, method of administration, patient's age, weight, gender, pathological condition, severity and stage of progression of traumatic brain injury, timing of administration, route of administration, excretion rate, and drug responsiveness, and a person skilled in the art can easily determine a dosage effective for treatment or prevention. For example, PDE5 inhibitors are a group of drugs with a history of clinical use for indications such as erectile dysfunction; considering this existing clinical dosage range, the active ingredient of the present invention may be administered in a range of about 1 mg to 500 mg per day, preferably about 1 mg to 100 mg, for the prevention or treatment of traumatic brain injury in adults. In one embodiment, for acute patients in whom elevated zinc concentration, GSH depletion, or worsening of oxidative stress indicators are observed, the daily dose of a PDE5 inhibitor or mirodenafil may be administered as an initial loading dose of about 1 mg to 200 mg, preferably 10 mg to 100 mg, more preferably 30 mg to 50 mg. Alternatively, for the minimization of side effects and long-term maintenance therapy, it may be administered in subdivided 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, and specifically, the daily dose may be set to any one of the doses of 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.

[0185] The above daily dosage may be administered once a day or divided into several doses per day (e.g., 2 to 3 times) as needed, and it may be preferable to administer it repeatedly for a short period or for a certain period to suppress secondary damage and restore neurological function after traumatic brain injury.

[0186] The above pharmaceutical composition may be prepared in a unit dose or multi-dose form using pharmaceutically acceptable carriers and / or excipients, and the formulation may include, but is not limited to, a solution, suspension, emulsion, extract, powder, granule, tablet, capsule, suppository, spray, ointment, cream, gel, inhalant, or skin patch. The above composition may be prepared for administration to mammals, preferably humans.

[0187] 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.

[0188] 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.

[0189]

[0190] (2) Food composition

[0191] 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.

[0192] 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.

[0193] The above food composition may be used to assist in alleviating oxidative stress in nerve tissue, maintaining an anti-inflammatory state, protecting neurological function, and improving general fatigue during the recovery or subacute phase following traumatic brain injury. For example, the above food composition may help alleviate or improve central nervous system fatigue, cognitive fatigue, general weakness, delayed recovery, or stress response that may accompany traumatic brain injury, but is not limited thereto.

[0194] 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.

[0195] 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.

[0196] 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.

[0197]

[0198] (3) Feed composition

[0199] 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.

[0200] 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.

[0201] The above feed composition may be used to assist in neuroprotection, alleviation of oxidative stress, inhibition of inflammatory responses, and maintenance of recovery phase functions in experimental animals or companion animals that have suffered traumatic brain injury or equivalent central nervous system injury.

[0202] 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.

[0203]

[0204] (4) Cosmetic composition

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209]

[0210] 3. Precision Medical Treatment Methods for Traumatic Brain Injury

[0211] The treatment method according to the present invention essentially includes a step of diagnosing the pathological condition of the patient prior to administration. Patients with traumatic brain injury exhibit significant heterogeneity in zinc accumulation and oxidative stress susceptibility depending on the mechanism of injury and individual genetic and metabolic characteristics.

[0212] Experimental results of the present invention (Figs. 1 and 4) confirmed that the neuroprotective effect of mirodenafil depends directly on the recovery of glutathione (GSH) levels through the activation of the Nrf2 / HO-1 pathway. Therefore, rather than a patient group with GSH levels within the normal range or minimal zinc toxicity, the therapeutic benefit is maximized when administered to selected patients: (i) a 'zinc overload' patient group in which the concentration of free zinc in brain tissue or cerebrospinal fluid has increased by more than 20% compared to the normal control group, or (ii) an 'antioxidant deficient' patient group in which the GSH concentration has decreased by more than 20% compared to the normal control group. This patient selection process overcomes the limitations of therapeutic efficiency inherent in existing comprehensive TBI treatments and completes a technical configuration that provides intensive treatment to patients with high drug response.

[0213] A precision medical treatment method for traumatic brain injury according to one embodiment is,

[0214] A step of measuring one or more pathological markers from a biological sample separated from a subject or performing a non-invasive imaging technique;

[0215] A step of selecting a patient group for treatment based on the above pathological indicators; and

[0216] The method is characterized by including the step of administering a composition containing a phosphodiesterase 5 (PDE5) inhibitor as an active ingredient to a selected patient group.

[0217] In one embodiment, the pathological indicator may be one or more selected from the group consisting of (a) free zinc concentration; (b) glutathione (GSH) concentration; (c) 4-hydroxynonenal (4-HNE) concentration; (d) Nrf2 activity level; (e) expression level of microglia activation markers (Iba-1, CD68, etc.); and (f) expression level of astrocyte activation markers (C3, GFAP, etc.).

[0218] In the present invention, it is preferable that the measurement of the pathological indicators be performed using a non-invasive or minimally invasive method for the safety of the patient and for rapid diagnosis. Since tissue biopsy can pose additional risks to patients with brain damage, the present invention specifically measures the indicators and selects patients through the following non-invasive technical means.

[0219] (1) Direct measurement through neuroimaging: By quantifying the concentration of glutathione (GSH) in the hippocampus or damaged area in real time using magnetic resonance spectroscopy (1H-MRS) without physically collecting brain tissue, the presence of antioxidant deficiency is diagnosed non-invasively. Positron emission tomography (PET) is used to visualize the distribution of activated microglia and the level of neuroinflammation using TSPO ligands, or to image areas of excessive accumulation of free zinc using zinc-specific radiotracers.

[0220] (2) Indirect measurement via liquid biopsy: Through analysis of blood or cerebrospinal fluid, the concentrations of circulating 4-HNE (lipid peroxide), oxidized proteins (protein carbonyls), or zinc present in the patient's plasma, serum, or cerebrospinal fluid are precisely measured using HPLC, ICP-MS, or ELISA (Enzyme-linked immunosorbent assay). Through neural-derived exosome analysis, neuronal or astrocyte-derived exosomes are isolated from peripheral blood, and by analyzing the levels of Nrf2 and HO-1 proteins or mRNA expression contained within the exosomes, they are used as non-invasive markers representing the oxidative stress state within brain tissue.

[0221] The introduction of such diagnostic technology accurately identifies the patient's pathological condition without the need for a brain tissue biopsy, thereby providing crucial information for selecting the patient group (Responder) for whom administration of the composition according to the present invention is most urgent and effective.

[0222] In one embodiment, the patient group to be treated may be a patient group satisfying one or more of the following cases compared to a normal control group: a refractory zinc concentration increased by 10% or more, a GSH concentration decreased by 10% or more, a 4-HNE concentration increased by 10% or more, or the expression level of a glial cell activation marker increased by 10% or more.

[0223] Preferably, a patient group in which the concentration of free zinc increases by 20% or more, the concentration of GSH decreases by 20% or more, the concentration of 4-HNE increases by 20% or more, or the expression of glial cell activation markers increases by 20% or more can be identified as a treatment target group.

[0224] More preferably, a group of patients in whom the measured free zinc concentration exceeds the normal range by 10% to 50%, 20% to 50%, or 30% to 50% or more, or in whom the GSH concentration is depleted by 10%, 20%, or 30% or more compared to the reference value, can be determined as a high-risk secondary injury group due to traumatic brain injury.

[0225]

[0226] 4. Stepwise control method for the secondary injury chain reaction following traumatic brain injury

[0227] The PDE5 inhibitor-based composition according to the present invention is characterized by a core technical feature of intervening before the accumulation of free zinc reaches its peak in the pathological mechanism of traumatic brain injury (TBI), thereby blocking the spread of secondary damage at the source.

[0228] In one embodiment, the composition according to the present invention is administered within 24 hours immediately after the occurrence of traumatic brain injury. As mentioned above in the background art (Suh et al., 2000), the migration and accumulation of synaptic zinc within neurons proceeds to an acute phase over approximately 12 to 24 hours after injury and reaches a peak.

[0229] Unlike the prior art (US 9,750,743 B2), which is limited to 'post-treatment' that repairs damage already incurred by administering a drug 24 hours after the zinc peak has passed, the present invention administers a drug within 24 hours before the zinc concentration exceeds the irreversible neuronal death threshold. By doing so, the slope of the zinc accumulation curve is suppressed and the peak itself is prevented, thereby preventing fatal intracellular organelle damage such as mitochondrial breakdown.

[0230] Preferably, the composition is administered within 12 hours (0 to 12 hours), which is the rising phase, during the period when zinc accumulation accelerates and surges toward a peak. Administration during this period has a decisive effect in maximizing the survival rate of nerve cells by suppressing the reaching of the peak of the zinc surge.

[0231] More preferably, the composition is administered within 3 hours (0 to 3 hours) immediately after injury, which is the 'Golden Hour' when initial zinc release begins. Early administration at this point can block the onset of zinc accumulation at the source, thereby most effectively blocking the pathological chain reaction that continues for up to 24 hours thereafter.

[0232] In the present invention, the selection of the timing of administration is not merely a matter of convenience but a key technical component that determines treatment efficiency. The inventors have identified that the success or failure of treating traumatic brain injury depends on how effectively the zinc surge, which occurs during the 'golden hour'—that is, within 3 hours immediately after the injury—is blocked.

[0233] When the composition according to the present invention is administered within '24 hours (especially within 3 hours)', it provides the following qualitatively distinct therapeutic mechanism compared to administration after '24 hours' recommended by the prior art. It preemptively protects neurons by activating the PDE5-cGMP pathway at the pre-mitochondrial collapse stage, before zinc is excessively introduced into the neurons and causes the mitochondrial membrane potential to collapse. Furthermore, preserving the endogenous defense system by preventing depletion itself (effect of administration within 3 hours) is significantly superior in terms of neuronal survival rate compared to restoring already depleted GSH afterward (effect of administration after 24 hours).

[0234] Accordingly, the present invention achieves an unpredictable and significant increase in therapeutic efficiency by selecting a specific time point (0 to 24 hours, preferably 0 to 3 hours) outside the administration range (24 to 72 hours) of the prior art.

[0235] In one embodiment, the present invention acts during the acute phase to inhibit the accumulation of free zinc released from presynaptic neurons in the hippocampus by 10%, 20%, 30%, or 50% or more compared to the control group.

[0236] In one embodiment, the acute phase control can improve the indicators of neuronal degeneration and death by 10%, 15%, 20%, 25%, 30%, or 50% or more compared to the control group by maintaining intracellular cGMP concentration to block the breakdown of mitochondrial membrane potential.

[0237] In addition, it can prevent rapid glutathione (GSH) depletion immediately after injury and protect the endogenous antioxidant defense system by restoring GSH levels by 10%, 20%, 30%, or more than 50% compared to the control group.

[0238] In one embodiment, the present invention acts during the subacute phase between 24 hours and 7 days after injury to block the spread of neuroinflammation by inhibiting the abnormal activation of microglia (Iba-1, CD68) and astrocytes (C3, GFAP) by 10%, 15%, 20%, 25%, 30%, or 50% or more compared to the control group.

[0239] In addition, it can delay the breakdown of tight junctions of the blood-brain barrier (BBB) ​​and reduce the cerebral edema index by 10%, 15%, 20%, 25%, 30%, or more than 50% compared to the control group.

[0240] In one embodiment, the present invention acts during the recovery period after 7 days following injury to promote the proliferation and differentiation of endogenous neural stem cells under a permissive neural microenvironment established in the preceding step.

[0241] Specifically, functional neural circuit rewiring can be achieved by improving the differentiation efficiency of nascent cells (BrdU+, DCX+) into mature neurons (NeuN+) by 10%, 15%, 20%, 25%, 30%, or 50% or more compared to the control group.

[0242] In addition, behavioral cognitive function or motor function indicators can be restored by 10%, 15%, 20%, 25%, 30%, or 50% or more compared to the control group.

[0243]

[0244] 5. Precision medical administration method for traumatic brain injury based on pathological markers

[0245] A precision medical administration method for traumatic brain injury based on pathological indicators according to one embodiment of the present invention is characterized by including the step of adjusting the dosage or administration cycle of a composition containing a PDE5 inhibitor according to changes in the patient's pathological indicators.

[0246] In one embodiment, when the free zinc concentration increases by 10%, 15%, 20%, 25%, 30%, or 50% or more compared to the reference value, or when the oxidative stress indicator worsens, the daily dose of mirodenafil may be set in the range of 1 mg to 200 mg.

[0247] Preferably, it may be administered as an initial (loading) dose of 10 mg to 100 mg, more preferably 30 mg to 50 mg, and more preferably 30 mg per day. Alternatively, for the minimization of side effects and long-term maintenance therapy, it may be administered in a subdivided low-dose range 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, and specifically, the daily dose may be set to any one of 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.

[0248] In one embodiment, by monitoring biomarkers during administration, if the GSH level recovers by 10%, 15%, 20%, 25%, 30%, or 50% or more compared to baseline, it can be determined as a treatment responder and switched to a maintenance dose.

[0249] In addition, the administration period or administration cycle can be adjusted in stages based on whether the rate of improvement in behavioral motor function or cognitive function indicators improves by 10%, 15%, 20%, 25%, 30%, or 50% or more compared to the control group.

[0250]

[0251] 6. Method of combination administration for the treatment of traumatic brain injury

[0252] In one embodiment of the present invention, the PDE5 inhibitor-based composition may be administered in combination with a standard treatment or regenerative adjuvant therapy for traumatic brain injury to exhibit a synergistic effect.

[0253] In the case of patients with traumatic brain injury accompanied by acute intracranial pressure elevation, the above composition can be administered in combination with mannitol or hypertonic saline, which are osmotic treatment agents, to simultaneously suppress cerebral edema and block free zinc toxicity.

[0254] In another embodiment, the composition may be used in combination with stem cell therapy or administration of neurotrophic factors, thereby further improving the endogenous neurogenesis efficiency by 10%, 15%, 20%, 25%, 30%, or 50% or more compared to the control group.

[0255] In addition, the above composition is provided in parallel with cognitive and motor rehabilitation protocols, thereby maximizing the efficiency of functional integration of drug-matured new neurons into existing neural circuits through rehabilitation stimulation.

[0256]

[0257] 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.

[0258]

[0259] <Example>

[0260] Example 1. Confirmation of zinc toxicity inhibition and antioxidant mechanism in a neuronal cell culture model

[0261] 1) Materials and Methods

[0262] (1) Isolation and culture of hippocampal neurons

[0263] Hippocampus tissue was isolated from Sprague-Dawley rat fetuses at embrryonic day 19 (E19). The isolated hippocampus tissue was enzymatically treated to dissociate it into single cells and seeded into culture dishes coated with poly-L-lysine. Neurobasal medium supplemented with B27 was used as the culture medium, and the cells were cultured in an incubator at 37°C with 5% CO₂ for 7 days (in vitro day 7, DIV 7) to induce neuronal maturation before being used in the experiment.

[0264] (2) Zinc toxicity induction and drug treatment

[0265] To simulate the acute zinc toxicity environment that occurs during traumatic brain injury, hippocampal neurons on day 7 of culture were treated with zinc chloride (ZnCl₂, Sigma-Aldrich) at a concentration of 300 μM. Three hours after zinc treatment, mirodenafil, the experimental substance of the present invention, was added to the culture medium to final concentrations of 5 μM, 10 μM, and 15 μM, respectively. After drug treatment, the cells were cultured for an additional 21 hours under the same culture conditions.

[0266] (3) Cell viability assay

[0267] To quantitatively evaluate the neuroprotective effect of mirodenafil, the WST-based EZ-CytoX assay was performed. At the time when the reaction was completed 21 hours after drug treatment, EZ-CytoX solution (DoGenBio Co.) was added to each culture well to equal 1 / 10 of the medium volume and reacted at 37°C. Subsequently, the absorbance at a wavelength of 450 nm was measured using a microplate reader (SpectraMax), and the cell viability (%) of the mirodenafil-treated group compared to the zinc-alone treated group was calculated.

[0268] (4) Immunocytochemistry

[0269] Immunocytochemical staining was performed to confirm the antioxidant effect and glutathione (GSH) recovery as the mechanism of action of mirodenafil. Cultured neurons were fixed with 4% paraformaldehyde (PFA), non-specific binding was blocked, and the following primary antibodies were incubated overnight at 4°C.

[0270] Antioxidant Indicators: Anti-Nrf2 (Cell Signaling), Anti-HO-1 (Abcam), Anti-nNOS (Invitrogen)

[0271] Glutathione Indicator: Anti-GS-NEM (Millipore, N-ethylmaleimide adduct form)

[0272] Neuronal marker: Anti-NeuN (Millipore)

[0273] Afterwards, the fluorescently labeled secondary antibody was reacted at room temperature for 2 hours, and the fluorescence intensity of each indicator was analyzed using a confocal microscope (Olympus or Zeiss).

[0274] (5) Statistical analysis

[0275] All experimental results were expressed as mean ± standard error (SEM). Depending on the data distribution characteristics, one-way ANOVA or the Kruskal-Wallis test was performed to test for significance between groups, and the Bonferroni test was applied as a post-hoc test. For all statistical analyses, the significance level was set at p < 0.05, and significance was indicated as follows:

[0276] *p < 0.05; **p < 0.01; ***p < 0.001.

[0277] 2) Result

[0278] (1) Concentration-dependent neuroprotective effect of mirodenafil (Cell viability analysis)

[0279] To evaluate the toxicity of the high-concentration zinc environment formed during traumatic brain injury (TBI) on neurons and the protective effect of mirodenafil against it, cell viability was analyzed after treating hippocampal neurons with 300 μM zinc chloride (ZnCl₂). EZ-CytoX analysis revealed that the cell viability of the zinc-alone treatment group (ZnCl₂-Vehicle) was significantly reduced to approximately 50% compared to the normal control group (Control-Vehicle), confirming that severe neuronal death was induced.

[0280] In contrast, a concentration-dependent recovery of cell viability was observed in the group treated with mirodenafil. Specifically, a trend of increasing viability was observed starting from the 5 μM mirodenafil treatment group, and statistically significant improvement in viability (p < 0.001) was confirmed in the 10 μM and 15 μM treatment groups compared to the zinc-alone treatment group (see Figs. 1C, 1D and Table 1). These results suggest that mirodenafil possesses a potent and remarkable efficacy that goes beyond simple inhibition of apoptosis to directly protect neurons in a specific neurotoxic environment induced by zinc overload.

[0281] GroupZnCl₂(-)ZnCl₂(+)MeanSEMp-ValueMeanSEMp-ValueMirodenafil0 μM1003.848053.4362.486# ZnCl₂(-)p < 0.001Mirodenafil5 μM100.1075.1970.98762.0682.038p < 0.001Mirodenafil10 μM103.4475.0740.59481.6232.697p < 0.001Mirodenafil15 μM103.5207.3710.67784.2441.338p < 0.001

[0282] (2) Activation of endogenous antioxidant defense mechanism (Nrf2 / HO-1)

[0283] To determine whether the neuroprotective effect of mirodenafil is due to the activation of intracellular defense mechanisms rather than a simple physical blocking effect, changes in the expression of Nrf2 (Nuclear factor erythroid 2-related factor 2) and HO-1 (Heme oxygenase-1), key antioxidant-related markers, were analyzed using immunocytochemical staining.

[0284] Confocal microscopy analysis revealed that in zinc-toxicity-induced neurons (ZnCl₂-Vehicle), the expression of nuclear Nrf2 was reduced, and consequently, the expression of the downstream antioxidant enzyme HO-1 was also suppressed. In contrast, in the mirodenafil-treated group, the fluorescence intensity of nuclear Nrf2 increased significantly compared to the zinc-alone treated group (p < 0.05), and accordingly, the expression of HO-1, known as a potent antioxidant enzyme, also increased statistically significantly (p < 0.001) (see Figs. 1I, 1K, 1L and Table 2).

[0285] These results clearly demonstrate the specific mechanism of action by which mirodenafil protects neurons by promoting the nuclear activation of transcription factor Nrf2 under conditions of oxidative stress and effectively activating the endogenous antioxidant defense system, including its downstream antioxidant enzyme HO-1.

[0286] Marker groupZnCl₂(-)ZnCl₂(+)MeanSEMp-ValueMeanSEMp-ValueNrf2Vehicle10.06100.4460.039# ZnCl₂(-)p < 0.001Mirodenafil1.0190.0490.8080.7110.033p < 0.033HO-1Vehicle10.03600.5440.021# ZnCl₂(-)p < 0.001Mirodenafil1.0540.0270.2420.7300.028p < 0.001

[0287] (3) Inhibition of glutathione (GSH) depletion and restoration of NO signaling

[0288] We analyzed changes in the expression of neuronal nitric oxide synthase (nNOS), an enzyme involved in glutathione (GSH) depletion and nitric oxide (NO) signaling, which are known as major causes of oxidative stress induced by zinc toxicity. To this end, immunocytochemical staining using GS-NEM antibodies was performed.

[0289] Analysis results showed that while intracellular GSH levels decreased sharply in zinc-treated neurons, GSH levels depleted due to zinc toxicity in the mirodenafil-treated group were significantly restored to normal control levels (p < 0.001). These results suggest that mirodenafil of the present invention effectively maintains intracellular antioxidant capacity by promoting the synthesis of GSH, a key antioxidant factor responding to oxidative stress, or by inhibiting its consumption.

[0290] Meanwhile, the expression of nNOS, an enzyme essential for maintaining normal neuronal function, was also significantly reduced by zinc toxicity, but it was confirmed that nNOS expression was preserved at a statistically significant level when treated with mirodenafil (p < 0.001) (see Figs. 1E, 1F, 1G and Table 3).

[0291] Marker groupZnCl₂(-)ZnCl₂(+)MeanSEMp-ValueMeanSEMp-ValueGSHVehicle10.03600.5440.021# ZnCl₂(-)p < 0.001Mirodenafil1.0540.0270.2420.7300.028p < 0.001nNOSVehicle10.05700.2480.017# ZnCl₂(-)p < 0.001Mirodenafil1.0970.0550.2270.4910.043p < 0.001

[0292] From the results of Example 1 above, it was demonstrated that mirodenafil significantly inhibits neuronal death by activating the Nrf2 / HO-1 signaling pathway and effectively restoring depleted glutathione (GSH) under a zinc toxic environment formed during the early stages of traumatic brain injury induction. In particular, this neuroprotective effect was confirmed to be due to the fundamental enhancement of intracellular antioxidant defense mechanisms, unlike existing drugs that merely non-specifically inhibit cell death.

[0293] Furthermore, the mirodenafil of the present invention suggests the possibility of selectively exhibiting excellent therapeutic effects in patient groups with elevated zinc levels or reduced antioxidant capacity, particularly GSH levels, which strongly supports the fact that the drug of the present invention can be effectively applied to precision medicine-based treatment strategies utilizing zinc overload or antioxidant indicators as biomarkers.

[0294]

[0295] Example 2. Protective effect in a neuronal-astrocytic co-culture model

[0296] 1) Materials and Methods

[0297] (1) Establishment of a neuron-astrocyte co-culture model (Primary neuron-astrocyte co-culture)

[0298] To simulate the interaction environment between neurons and astrocytes within actual brain tissue, a neuron-astrocytic co-culture system was established. First, cerebral cortex tissue was excised from Sprague-Dawley rats at embrryonic day 19 (E19), and astrocytes were isolated. The isolated astrocytes were sufficiently matured by culturing them for approximately 4 weeks in T-75 flasks coated with poly-L-lysine using glial medium supplemented with horse serum and glucose. After transplanting the matured astrocytes into a 24-well plate, neurons isolated from the hippocampus of the same E19 rats were seeded onto the astrocytes. Subsequently, an in vitro co-culture model in which neurons and astrocytes coexist was established by co-culturing for 10 days at 37°C and 5% CO₂ using Neural Maintenance Medium (Gibco).

[0299] (2) Zinc toxicity induction and drug treatment

[0300] To create a toxic environment similar to that of traumatic brain injury in co-cultured cells, zinc chloride (ZnCl₂, Sigma-Aldrich) at a concentration of 300 μM was pre-treated for 3 hours. Subsequently, mirodenafil of the present invention was added to the culture medium (post-treatment) to final concentrations of 5 μM, 10 μM, and 15 μM, respectively. Drug treatment was continued for a total of 21 hours, and cell viability and biomarker analysis were performed after the treatment was completed.

[0301] (3) Cell viability assay

[0302] EZ-CytoX analysis was performed to evaluate the cytoprotective effect in a co-culture environment. After adding EZ-CytoX solution (DoGenBio Co.) to a culture medium containing cells that had completed drug response and allowing the reaction to occur, the absorbance at a wavelength of 450 nm was measured using a microplate reader. Through this, the total cell viability (%) of the mirodenafil-treated group was calculated compared to the zinc-alone-treated group.

[0303] (4) Immunocytochemistry

[0304] Immunocytochemical staining using specific antibodies was performed to confirm the structural integrity of neurons, the activity status of astrocytes, and intracellular glutathione (GSH) levels. The following primary antibodies were reacted with cells fixed in 4% paraformaldehyde (PFA) at 4°C.

[0305] Neuronal structural marker: Anti-MAP2 (Microtubule-associated protein 2) - Confirmation of neurites and structures

[0306] Astrocyte Markers: Anti-GFAP (Glial fibrillary acidic protein), Anti-S100B (S100 calcium-binding protein B) - Evaluation of astrocyte damage and responsiveness

[0307] Antioxidant Indicator: Anti-GS-NEM (Millipore) - Verification of intracellular glutathione (GSH) levels

[0308] Cell identification markers: Anti-NeuN (neuronal nucleus), Anti-GFAP (astrocytocyte)

[0309] Afterward, a fluorescently labeled secondary antibody was reacted, and the degree of damage and GS-NEM fluorescence intensity for each cell type were quantitatively analyzed using a confocal microscope.

[0310] (5) Data Analysis

[0311] The area (Area %) of MAP2, GFAP, and S100B positive regions and the fluorescence intensity of GS-NEM were measured using image analysis software (ImageJ, etc.). Statistical significance between groups was verified through one-way ANOVA and Bonferroni post-hoc tests, with the significance level set at p < 0.05.

[0312] 2) Result

[0313] (1) Concentration-dependent cell viability in co-culture environment

[0314] The protective effect of mirodenafil against zinc toxicity was evaluated in a neuronal-astrocytic co-culture model, an environment similar to actual brain tissue. EZ-CytoX analysis showed that treatment with zinc at a concentration of 300 μM significantly reduced the viability of co-cultured cells. In contrast, in the experimental group treated with mirodenafil, cell viability was significantly improved in a concentration-dependent manner within the concentration ranges of 5 μM, 10 μM, and 15 μM.

[0315] In particular, in the mirodenafil 10 μM and 15 μM treatment groups, cell viability was restored with very high statistical significance at the p < 0.001 level compared to the zinc-alone treatment group (see Figures 2A, 2B and Table 4). These results clearly demonstrate that mirodenafil exhibits excellent cytoprotective efficacy not only under single neuronal culture conditions but also in complex brain cell environments where interactions with astrocytes are present.

[0316] GroupZnCl₂(-)ZnCl₂(+)MeanSEMp-ValueMeanSEMp-ValueMirodenafil0 μM1003.119054.2943.431# ZnCl₂(-)p < 0.001Mirodenafil5 μM101.3521.4670.70064.9994.2960.065Mirodenafil10 μM106.5991.0600.06679.1021.916p < 0.001Mirodenafil15 μM106.1121.5550.79882.9572.366p < 0.001

[0317] (2) Maintaining neuronal structure and inhibiting astrocyte damage (Structural integrity)

[0318] To more specifically confirm the protective effects of each cell type in a co-culture environment, immunofluorescence staining was performed using the neuronal cell marker MAP2 and the astrocyte markers GFAP and S100B.

[0319] Analysis results showed that in co-cultured cells induced with zinc toxicity, neurite discontinuity and cell body atrophy were clearly observed in neurons (MAP2). In contrast, in the mirodenafil 10 μM treatment group, the neurite network remained generally intact, and the MAP2 positive area increased statistically significantly compared to the zinc-alone treatment group (p = 0.037).

[0320] Meanwhile, in the case of astrocytes (GFAP, S100B), zinc treatment induced rapid structural damage along with changes in excessive reactivity. In contrast, in co-cultured cells treated with mirodenafil, such astrocyte damage was effectively suppressed, and the structural morphology of GFAP and S100B-positive cells was maintained at a level similar to that of normal controls (p < 0.001 and p = 0.0428, respectively) (see Figs. 2C, 2D, 2E, 2F and Table 5).

[0321] Marker groupZnCl₂(-)ZnCl₂(+)MeanSEMp-ValueMeanSEMp-ValueMAP2Vehicle10.07000.4350.052# ZnCl₂(-)p < 0.001Mirodenafil1.0420.0710.6780.6000.0560.037S100BVehicle10.06900.4090.036# ZnCl₂(-)p < 0.001Mirodenafil0.9990.0650.9920.5690.0670.043GFAPVehicle10.06000.2020.023# ZnCl₂(-)p < 0.001Mirodenafil1.0080.0480.9140.35540.036p < 0.001

[0322] (3) Restoration of the intercellular glutathione (GSH) antioxidant network

[0323] Astrocytes play a key role in maintaining the antioxidant defense system by synthesizing glutathione (GSH) within brain tissue and supplying it to neurons. To investigate the effect of the mirodenafil of the present invention on this antioxidant interaction between neurons and astrocytes, intracellular GSH levels were evaluated through immunocytochemical analysis using GS-NEM antibodies.

[0324] Analysis results showed that GSH levels were rapidly depleted in both neurons (NeuN-positive cells) and astrocytes (GFAP-positive cells) under a zinc toxic environment. In contrast, in co-cultured cells treated with 10 μM mirodenafil, the GSH fluorescence intensity in both neurons and astrocytes increased statistically significantly (p < 0.001).

[0325] In addition, the degree of recovery of GSH levels showed a strong positive correlation with the actual number of surviving neurons (number of NeuN-positive cells) and the degree of preservation of astrocytes (GFAP-positive area) (see Figs. 2G, 2H, 2I, 2J and Table 6). These results clearly demonstrate that mirodenafil effectively alleviates oxidative stress throughout the brain microenvironment by restoring the antioxidant network between neurons and astrocytes beyond the single-cell level.

[0326] Marker groupZnCl₂(-)ZnCl₂(+)MeanSEMp-ValueMeanSEMp-ValueGSHVehicle10.04600.2280.020# ZnCl₂(-)p < 0.001Mirodenafil1.0090.0480.8910.4410.020p < 0.001GFAPVehicle10.06000.2020.023# ZnCl₂(-)p < 0.001Mirodenafil1.0080.0480.9140.3550.0360.006NeuNVehicle10.03700.3980.028# ZnCl₂(-)p < 0.001Mirodenafil0.9930.0350.8990.4850.0310.044

[0327] From the results of Example 2 above, it was confirmed that mirodenafil not only merely inhibits neuronal death but also effectively protects astrocytes, which play a key role in maintaining homeostasis in brain tissue, and significantly enhances the antioxidant defense capacity of the entire brain tissue by normalizing the glutathione (GSH) supply and circulation system between astrocytes and neurons.

[0328] These results clearly support the fact that the pharmaceutical composition of the present invention possesses a differentiated mechanism of action capable of fundamentally regulating the complex brain injury environment accompanied by oxidative stress and neuroinflammation, going beyond protective effects at the individual cell level.

[0329]

[0330] Example 3. Inhibition of zinc accumulation and prevention of neuronal death in an animal model of traumatic brain injury (TBI).

[0331] 1) Materials and Methods

[0332] (1) Induction in experimental animals and traumatic brain injury (TBI) models

[0333] 8-week-old male Sprague-Dawley rats (body weight 300-350 g) were anesthetized with 1.5% isoflurane and fixed to a stereotactic fixation device. A 3 mm diameter craniotomy was performed in the right cerebral cortex (3.0 mm posterior and 2.8 mm lateral relative to Lambda), and severe traumatic brain injury was induced using an electromagnetically controlled shock device (Impact One™ Stereotaxic Impactor) under conditions of a shock depth of 3.0 mm and a speed of 5.0 m / s.

[0334] (2) Drug administration

[0335] Immediately after traumatic brain injury (TBI), Mirodenafil was administered subcutaneously (sc) at doses of 0.5 mg / kg, 1 mg / kg, and 2 mg / kg, respectively. The same volume of physiological saline was administered to the control group (vehicle). In this experiment, brain tissue was analyzed at a set time point after a single administration to evaluate the acute effects of the drug.

[0336] (3) Analysis of zinc accumulation in brain tissue (TSQ staining)

[0337] To evaluate the excessive release and accumulation of zinc in the brain following TBI, experimental animals were sacrificed 3 hours after injury induction. The excised brains were frozen and prepared into 10 μm thick unfixed sections. For zinc-specific fluorescence staining, the sections were incubated in a 4.5 μM TSQ (N-(6-methoxy-8-quinolyl)-para-toluene sulfonamide) solution in pH 10.5 buffered conditions for 60 seconds, followed by washing with physiological saline. Subsequently, the degree of zinc accumulation in the CA1 region of the hippocampus and the Granule Cell Layer (GCL) and Hilus region within the Dentate Gyrus (DG) was imaged and quantified using a fluorescence microscope at a wavelength of 360 nm ultraviolet (UV).

[0338] (4) Neuronal cell death analysis (Fluoro-Jade B staining)

[0339] To evaluate neuronal degeneration, brain tissue was excised 24 hours after TBI induction and fixed with 4% paraformaldehyde (PFA). Subsequently, 30 μm thick brain sections were pretreated with 0.06% potassium permanganate solution and stained in 0.001% Fluoro-Jade B (FJB) solution for 30 minutes. The number of FJB-positive neurons (degenerated neurons) was counted in the CA1, CA3, dentate gyrus (GCL), and Hilus regions of the hippocampus using a fluorescence microscope under blue excitation conditions of 450–490 nm wavelength.

[0340] (5) Statistical analysis

[0341] Comparisons between groups were performed using the Kruskal-Wallis test and the Mann-Whitney U test, and a p-value < 0.05 was determined to be statistically significant.

[0342] 2) Result

[0343] (1) Inhibitory effect of zinc accumulation in the hippocampus region during early TBI (Zinc accumulation)

[0344] Traumatic brain injury is known to induce the release of excessive free zinc within neurons, leading to secondary neurotoxicity. To determine whether the administration of mirodenafil according to the present invention can control this initial zinc toxicity, TSQ staining was performed 3 hours after injury induction.

[0345] As a result, in the TBI-induced vehicle administration group, strong zinc fluorescence signals were observed throughout the CA1 region of the hippocampus, the granular cell layer (GCL) of the dentate gyrus, and the Hilus region, indicating an excessive accumulation of zinc at the site of injury. On the other hand, in the mirodenafil 2 mg / kg administration group, it was confirmed that the zinc fluorescence intensity was significantly reduced, even visually, in all regions of the CA1, GCL, and Hilus.

[0346] Quantitative analysis showed that the mirodenafil 2 mg / kg administration group exhibited a statistically significant inhibitory effect on zinc accumulation in the CA1 region compared to the vehicle group (p = 0.023), and also showed significantly lower zinc accumulation in the GCL and Hilus regions within the dentate gyrus with p = 0.037 and p = 0.007, respectively (see Fig. 3I-3M and Table 7). These results clearly demonstrate that mirodenafil effectively inhibits the excessive intracellular zinc accumulation that occurs immediately after traumatic brain injury, thereby creating a neuroprotective microenvironment from the early stages of injury.

[0347] Domain MeanSEMp-ValueCA1Vehicle30.6332.3790Mirodenafil202.9220.023GCLVehicle50.8335.61420Mirodenafil33.4673.7750.037HilusVehicle27.0331.8930Mirodenafil17.0332.0570.007

[0348] The results of this embodiment demonstrate the superiority of the present invention in conjunction with the known pathological mechanism of TBI (Suh et al., 2000). According to the literature, zinc accumulation after TBI peaks over 12 to 24 hours and leads to neuronal death. As confirmed in Figures 3I to 3M, the administration of mirodenafil significantly inhibited zinc concentration in the hippocampus at the 3-hour mark, which is the early stage of zinc accumulation.

[0349] This suggests that mirodenafil prevented the formation of the 'Zinc Peak' expected to be reached at the 12–24 hour mark by blocking the 'starting point' of zinc accumulation. As a result, as shown in Figures 3A–3H (analysis at the 24-hour mark), while extensive neuronal degeneration occurred in the vehicle group while undergoing the zinc peak, this degeneration was significantly suppressed in the mirodenafil administration group.

[0350] If mirodenafil is administered after 24 hours have elapsed as taught in the prior art (US 9,750,743 B2), the zinc accumulation observed in Fig. 3 has already triggered oxidative damage (ROS generation) and mitochondrial death signals within the neuronal cells, so it is difficult to expect a potent neuroprotective effect (Figs. 3A to 3H) as confirmed in this embodiment. In other words, the 'administration within 3 hours' configuration of the present invention is not merely a temporal change of the prior art, but is a physical and biological essential requirement to achieve a new mechanism of blocking zinc toxicity.

[0351] In conclusion, unlike prior art which administers after 24 hours have passed since the zinc peak, the present invention has been confirmed to exhibit an excellent neuroprotective effect through a 'preventive treatment mechanism' that intervenes in the acute phase (0 to 24 hours) during which zinc accumulates, particularly in the early stages, thereby fundamentally blocking the formation of zinc toxicity.

[0352] (2) Inhibition of neuronal degeneration and death of hippocampal neurons

[0353] To determine whether inhibition of zinc accumulation leads to actual neuronal survival, Fluoro-Jade B (FJB) staining was performed 24 hours after inducing traumatic brain injury, and the number of degenerating neurons was analyzed.

[0354] Analysis results showed that in the vehicle administration group, numerous FJB-positive cells (green fluorescence) were observed throughout the CA1, CA3, dentate gyrus (GCL), and Hilus regions of the hippocampus, indicating that extensive neuronal degeneration and death had occurred. In contrast, in the mirodenafil-administered experimental group, a dose-dependent decrease in the number of FJB-positive cells was observed in the dose ranges of 0.5 mg / kg, 1 mg / kg, and 2 mg / kg (see Figures 3A-3H and Table 8).

[0355] In particular, the mirodenafil 2 mg / kg administration group statistically significantly reduced the number of degenerated neurons in the CA1 region (p = 0.0252), dentate gyrus (GCL, p = 0.0195), and hilus (p = 0.0066) regions compared to the vehicle group. These results clearly demonstrate that mirodenafil effectively prevents subsequent neuronal degeneration and death by inhibiting zinc accumulation in the early stages following traumatic brain injury.

[0356] CountyCA1GCLHilusMeanSEMp-ValueMeanSEMp-ValueMeanSEMp-ValueMirodenafil0 μM174.06735.33701133.766114.603046.1335.2760Mirodenafil(0.5mg / kg)128.215.245070.282950.158.7380.20443.42.4300.656Mirodenafil( 1mg / kg)103.73314.1890.121860.03340.2940.07428.12.0040.023Mirodenafil(2mg / kg)55.913.1000.025697.36794.4650.02021.9333.3310.007

[0357] The results of Example 3 above demonstrate a clear causal relationship in that mirodenafil effectively blocks the excessive accumulation of zinc in the hippocampal region during the acute phase within about 3 hours after injury in a traumatic brain injury (TBI) model, and as a result, significantly inhibits neuronal degeneration and death observed 24 hours after injury.

[0358] These results suggest that the neuroprotective and restorative effects of mirodenafil are not merely secondary phenomena, but are directly attributed to the control of specific mechanisms known as 'inhibition of zinc accumulation' and 'prevention of GSH depletion.' In other words, mirodenafil specifically corrects pathological conditions where zinc surge and subsequent rapid oxidative damage are the primary causes, rather than the general pathology of TBI.

[0359] Therefore, when applying the drug to clinical settings, selecting patients based on zinc accumulation or GSH depletion is a critical factor in predicting and ensuring the therapeutic efficacy of the drug of the present invention. This demonstrates a selective therapeutic effect on a specific patient sub-population that was not predicted by the prior art.

[0360]

[0361] Example 4. Activation of antioxidant defense mechanisms and inhibition of reactive oxygen species (ROS) by Mirodenafil in a traumatic brain injury (TBI) model

[0362] 1) Materials and Methods

[0363] (1) Animal model and drug administration

[0364] Traumatic brain injury (TBI) was induced in 8-week-old male Sprague-Dawley rats in the same manner as in Example 3 above. Immediately after the injury, mirodenafil was administered subcutaneously (sc) at a dose of 2 mg / kg, and the same volume of physiological saline was administered to the control group (Vehicle).

[0365] (2) Brain tissue preparation (24-hour mark)

[0366] To analyze changes in antioxidant defense mechanisms, experimental animals were sacrificed 24 hours after TBI induction and brain tissue was extracted. The extracted brains were fixed with 4% paraformaldehyde (PFA) and prepared into 30 μm thick frozen sections for use as samples for immunohistochemistry.

[0367] (3) Analysis of oxidative stress and defense mechanisms (Immunofluorescence)

[0368] To evaluate the degree of oxidative damage and changes in the expression of antioxidant defense factors within brain tissue, immunofluorescence staining was performed using the following specific antibodies. The stained tissues were imaged using a confocal microscope, and the fluorescence intensity in the hippocampus region was quantitatively analyzed.

[0369] Oxidative Stress (ROS) Indicator: Anti-4-HNE (4-hydroxynonenal), lipid peroxidation products generated by reactive oxygen species

[0370] Nitric Oxide Synthase: Anti-nNOS (neuronal Nitric Oxide Synthase), an enzyme involved in regulating cerebral blood flow and maintaining neurological function

[0371] Antioxidant Transcription Factor: Anti-Nrf2 (Nuclear factor erythroid 2-related factor 2), a key transcription factor regulating antioxidant enzyme expression

[0372] Glutathione (GSH): Anti-GS-NEM (N-ethylmaleimide adduct), an antibody used to measure the levels of glutathione, a major intracellular antioxidant.

[0373] (4) Statistical analysis

[0374] Comparisons between groups were analyzed using the Kruskal-Wallis test, and a p-value of less than 0.05 was considered to indicate a statistically significant difference.

[0375] 2) Result

[0376] (1) Recovery of the endogenous antioxidant defense system (Nrf2 / GSH / nNOS)

[0377] Traumatic brain injury is known to induce rapid oxidative stress within brain tissue, significantly impairing the endogenous antioxidant defense system. In this experiment, the TBI-Vehicle group showed significantly reduced expression of nNOS, Nrf2, and GSH compared to the Sham control group.

[0378] In contrast, in the mirodenafil (2 mg / kg) administration group, the expression of these antioxidant defense factors was significantly restored compared to the vehicle group. Specifically, the expression of nNOS, which was reduced due to TBI, was significantly increased by mirodenafil administration (p = 0.0136), suggesting that mirodenafil contributes to maintaining cerebral blood flow and protecting neurofunction by promoting normal nitric oxide (NO) production. Furthermore, the expression of Nrf2, a key regulator of the antioxidant response, also increased statistically significantly in the mirodenafil administration group compared to the vehicle group (p = 0.004). Following this Nrf2 activation, intracellular glutathione (GSH) levels (GS-NEM fluorescence intensity), which had been depleted due to TBI, were significantly restored by mirodenafil administration (p = 0.0118).

[0379] MilitaryGSHnNOSNRF-2MeanSEMp-ValueMeanSEMp-ValueMeanSEMp-ValueSham-Vehicle10.038010.028010.0 470Sham-Mirodenafil1.0790.0200.1681.0020.0310.9701.0170.0590.832TBI-Vehicle0.4210.024# Sham-vehp < 0.0010.7180.041# Sham-vehp < 0.0010.2990.017#Sham-vehp < 0.001TBI-Mirodenafil24.1331.2470.0120.8970.0400.0140.5700.0520.004

[0380] (2) Inhibition of lipid peroxidation caused by reactive oxygen species (ROS)

[0381] To determine whether the recovery of the above-mentioned endogenous antioxidant defense system leads to a reduction in actual oxidative damage, the expression of 4-hydroxynonenal (4-HNE), a lipid peroxidation product generated by reactive oxygen species (ROS), was analyzed. As a result, a strong 4-HNE red fluorescence signal was observed throughout the hippocampus region in the TBI-Vehicle group, confirming that severe oxidative damage caused by traumatic brain injury was induced.

[0382] In contrast, in the mirodenafil (2 mg / kg) administration group, the fluorescence intensity of 4-HNE was significantly reduced compared to the vehicle group (p < 0.001), and showed a recovery pattern similar to that of the sham control group. These results demonstrate that mirodenafil effectively inhibits lipid peroxidation induced by reactive oxygen species, thereby substantially reducing oxidative cell damage occurring after traumatic brain injury.

[0383] Gun4HNEMeanSEMp-ValueSham-Vehicle10.0420Sham-Mirodenafil0.9000.1140.480TBI-Vehicle3.9190.341# Sham-vehp < 0.001TBI-Mirodenafil1.6090.1700.001

[0384] This embodiment demonstrates a specific pharmacological mechanism by which mirodenafil blocks the vicious cycle of oxidative stress induced after traumatic brain injury (TBI). Specifically, mirodenafil restores the expression of glutathione (GSH) depleted by TBI and reduced nNOS, and significantly enhances the endogenous antioxidant defense capacity of the brain tissue itself by activating the antioxidant transcription factor Nrf2. As a result, it was confirmed to effectively inhibit secondary neuronal damage induced by reactive oxygen species (ROS), particularly lipid peroxidation.

[0385] These results strongly support the fact that the pharmaceutical composition of the present invention is not merely a simple symptom reliever, but a precision medicine-based therapeutic agent that can be selectively applied to patient groups whose antioxidant systems (GSH / Nrf2) have collapsed due to oxidative stress.

[0386]

[0387] Example 5. Effect of alleviating neuroinflammation by inhibiting microglia and astrocyte activity after traumatic brain injury

[0388] One of the major causes of secondary injury occurring after traumatic brain injury (TBI) is a neuroinflammatory response induced by overactive glial cells. In this example, we investigated whether the administration of mirodenafil can suppress the response of microglia and astrocytes that are pathologically activated after traumatic brain injury.

[0389] 1) Materials and Methods

[0390] (1) Animal model and drug treatment

[0391] Severe traumatic brain injury was induced in 8-week-old Sprague-Dawley rats (SD Rat) in the same manner as in Example 3 above. Immediately after the injury, mirodenafil was administered subcutaneously (sc) at a dose of 2 mg / kg, and the same volume of physiological saline was administered to the control group (Vehicle).

[0392] (2) Analysis of neuroinflammation markers (Immunofluorescence)

[0393] To evaluate the extent of neuroinflammation progression after traumatic brain injury, animals were sacrificed 24 hours after TBI induction, brain tissue was excised, and frozen sections were prepared. Immunofluorescence staining using the following glial cell-specific antibodies was performed on the prepared sections.

[0394] Microglia Markers: Anti-Iba-1 (Ionized calcium-binding adapter molecule 1): A universal marker for confirming the morphological activation of microglia; Anti-CD68 (Cluster of Differentiation 68): A functional marker reflecting the phagocytosis and lysosomal activity of activated microglia.

[0395] Astrocyte Markers: Anti-GFAP (Glial Fibrillary Acidic Protein): An indicator of reactive astrogliosis, whose expression increases upon injury / Anti-C3 (Complement Component 3): An inflammatory marker distinguishing neurotoxic A1 astrocytes.

[0396] (3) Data Analysis

[0397] Images of the hippocampus CA1 region were acquired using a confocal microscope, and the fluorescence intensity and activation score of each marker were quantified. Statistical significance between groups was analyzed using the Kruskal-Wallis test.

[0398] 2) Result

[0399] (1) Activation of microglia and inhibition of phagocytosis

[0400] In the TBI-Vehicle group, Iba-1 positive microglia in the hippocampal CA1 region exhibited a typical amoeboid morphology characterized by enlarged cell bodies and shortened processes, and the expression of CD68, an indicator of phagocytic activity, also increased rapidly. This indicates that microglia were pathologically overactivated due to traumatic brain injury.

[0401] In contrast, in the mirodenafil (2 mg / kg) administration group, microglia maintained a relatively ramified morphology, and quantitative analysis showed that the expression level of Iba-1 was statistically significantly reduced compared to the vehicle group (p = 0.0033). In addition, the CD68 activation score was also significantly reduced compared to the vehicle group (p = 0.0011). These results suggest that mirodenafil blocks excessive inflammatory responses in neural tissue by effectively inhibiting the excessive activation and phagocytosis of microglia, which are immune cells in the brain.

[0402] CountyIba-1CD68MarkerMeanSEMp-ValueMeanSEMp-ValueShamVehicle1.8330.40801.4430.0570Mirodenafil20.1670.6491.4000.0670.647TBIVehicle6.90.506# Sham-vehp < 0.0017.60.441# Sham-vehp < 0.001Mirodenafil3.7330.3240.0033.70.3920.001

[0403] (2) Inhibition of reactivity and neurotoxic transformation of astrocytes

[0404] Analysis of changes in the reactivity of astrocytes revealed that in the TBI-Vehicle group, GFAP expression in the hippocampal CA1 region was significantly increased, leading to hypertrophy of the astrocytes. In particular, the expression of complement component 3 (Complement protein C3), an inflammatory marker that induces neurotoxicity, was strongly increased. This indicates that astrocytes are pathologically overactivated by traumatic brain injury, and transformation into neurotoxic type A1 reactive astrocytes is induced.

[0405] On the other hand, in the mirodenafil (2 mg / kg) administration group, the fluorescence intensity of GFAP was statistically significantly reduced compared to the vehicle group (p = 0.0022), and the expression of C3 was also significantly inhibited (p = 0.0002). Since C3 is known as a key factor that activates the complement system to induce synaptic damage and neuronal death, these results demonstrate that mirodenafil effectively blocks the pathological phenotypic change in which astrocytes lose their neuroprotective A2 state and switch to the neurotoxic A1 state.

[0406] Marker group GFAPC3MeanSEMp-ValueMeanSEMp-ValueShamVehicle10.091010.1350Mirodenafil0.8680.0140.2820.9130.0810.617TBIVehicle5.7691.029# Sham-vehp < 0.0018.3260.394# Sham-vehp < 0.001Mirodenafil2.9250.0550.0023.9510.225p < 0.001

[0407] The results of Example 5 clearly demonstrate that mirodenafil possesses a powerful anti-neuroinflammatory effect that regulates the altered brain immune environment following traumatic brain injury, going beyond a simple neuroprotective effect. Specifically, mirodenafil reduces the expression of CD68 in microglia to alleviate secondary neurological damage caused by excessive phagocytosis, and effectively blocks the formation of a neurotoxic inflammatory environment by inhibiting the expression of complement protein C3 in astrocytes.

[0408] Such mechanistic data provides strong grounds for the pharmaceutical composition of the present invention to be patented for a new medical use as a “composition for the treatment of brain diseases accompanied by neuroinflammation,” supporting the fact that this is an independent and differentiated inventive effect clearly distinguishable from the simple ‘inhibition of apoptosis’ effect of existing technology.

[0409]

[0410] Example 6. Long-term neuronal survival and motor function recovery effects after traumatic brain injury (Long-term outcome)

[0411] In this embodiment, we evaluated whether the administration of mirodenafil not only had a neuroprotective effect during the acute phase after traumatic brain injury but also demonstrated sustained therapeutic efficacy in neuronal survival and motor function recovery during the long-term phase of 1 to 2 weeks after injury.

[0412] 1) Materials and Methods

[0413] (1) Animal model and drug administration schedule

[0414] Severe traumatic brain injury (TBI) was induced in 8-week-old SD rats in the same manner as in [Example 3]. Mirodenafil was administered as a single subcutaneous injection (sc) immediately after injury at doses of 0.5, 1.0, and 2 mg / kg, and then repeated once daily for one week. The same volume of physiological saline was administered to the control group (Vehicle).

[0415] (2) Motor function assessment (mNSS test)

[0416] To quantitatively evaluate neurological deficits and the degree of recovery, the modified Neurological Severity Score (mNSS) was measured daily from day 1 to day 7 after TBI induction. The mNSS is an indicator that comprehensively evaluates motor, sensory, reflex, and balance functions; a higher score (maximum 18 points) indicates more severe neurological damage. In addition, the beam balance test was performed to precisely evaluate the sense of balance.

[0417] (3) Long-term neuronal survival analysis (NeuN staining)

[0418] To confirm the correlation between motor function recovery and neuronal preservation, brain tissue was extracted 2 weeks (14 days) after the completion of the behavioral experiment and TBI induction. Immunofluorescence staining was performed using NeuN antibodies, which are neuronal nucleus-specific markers, and the number of surviving neurons was counted in the CA1, granule cell layer (GCL), and Hilus regions of the hippocampus.

[0419] (4) Statistical analysis

[0420] Behavioral experimental data (mNSS) were analyzed using two-way ANOVA, and histological quantitative data were analyzed using the Kruskal-Wallis test.

[0421] 2) Result

[0422] (1) Significant recovery of neurological motor function

[0423] The TBI-Vehicle group maintained high mNSS scores even 7 days after injury, indicating persistent sensory and motor dysfunction. In contrast, the mirodenafil (2 mg / kg) group showed a gradual recovery trend starting from day 1 of administration, and showed statistically significantly lower mNSS scores compared to the vehicle group, particularly from day 3 to day 7 after injury (p < 0.05).

[0424] In addition, analysis of the change in Δscore showed that the mirodenafil group demonstrated superior recovery ability compared to the vehicle group across sensory, motor, reflex, and balance functions (Figs. 6C, 6D). This suggests that mirodenafil alleviates damage to motor neural circuits caused by traumatic brain injury or promotes functional reorganization.

[0425] (2) Increased long-term neuronal survival within the hippocampus

[0426] To determine whether the recovery of motor function is associated with the structural preservation of actual brain tissue, NeuN staining was performed 2 weeks after injury. As a result, extensive neuronal loss was observed across the hippocampal CA1, dentate gyrus (GCL), and hilus regions in the vehicle group.

[0427] In contrast, in the mirodenafil (2 mg / kg) administration group, the density of NeuN-positive neurons in the aforementioned hippocampal regions remained visibly significantly high (Fig. 6E). Quantitative analysis showed that the mirodenafil administration group exhibited statistically significantly increased neuronal viability in all regions—CA1 (p = 0.0464), GCL (p = 0.05), and Hilus (p = 0.0248)—compared to the vehicle group (Figs. 6F–6H, Table 13).

[0428] CountyCA1GCLHilusMeanSEMp-ValueMeanSEMp-ValueMeanSEMp-ValueSham-Vehicle452.20819.75301184.87523.3390115. 754.4770Sham-Mirodenafil475.37517.1020.4101185.2549.4980.995119.8752.6260.464TBI-Vehicle84.5667.967# Sham-vehp < 0.001438.73343.362# Sham-vehp < 0.00129.10.777# Sham-vehp < 0.001TBI-Mirodenafil107.3334.7800.046697.366666794.4650.05046.46775.0150.025

[0429] Example 6 demonstrates that the therapeutic effect of mirodenafil is not limited to short-term neuronal protection after traumatic brain injury, but exhibits a long-term protective effect (Long-term neuronal survival) that stably maintains neuronal survival rates up to 2 weeks after injury, and that this structural preservation is directly linked to functional recovery of motor ability and balance function.

[0430]

[0431] Example 7. Effects of promoting hippocampal neurogenesis and improving cognitive function (learning and memory) after traumatic brain injury

[0432] This embodiment is not intended to present a new invention, but rather to conduct a confirmatory study to verify the biological mechanism by which the long-term recovery of motor function and the significant improvement in mNSS scores (Fig. 6) of the mirodenafil administration group confirmed in Example 6 are attributed.

[0433] As disclosed in Example 3, mirodenafil blocks zinc toxicity (Fig. 3) and restores glutathione (Fig. 4), thereby improving the brain microenvironment. In Example 7, this improvement of the microenvironment led to actual proliferation (BrdU) and functional maturation (NeuN / DCX) of endogenous neurons, and was histologically visualized to support the previously observed 'functional recovery'.

[0434] 1) Materials and Methods

[0435] (1) Animal model and drug administration

[0436] Severe traumatic brain injury (TBI) was induced in 8-week-old Sprague-Dawley rats in the same manner as in Example 3 above. Mirodenafil was administered subcutaneously (sc) once daily for one week starting immediately after injury at a dose of 2 mg / kg.

[0437] (2) Cognitive Function Tests

[0438] After the drug administration was terminated, behavioral experiments measuring spatial learning and memory were performed to evaluate whether hippocampal function had recovered.

[0439] Barnes Maze: Performed from day 1 to day 6 after TBI, and spatial learning ability was evaluated by measuring the time taken to find an escape hole among multiple holes located at the edge of a circular platform (Escape latency).

[0440] Morris Water Maze (MWM): Performed from day 7 to day 12 after TBI. For the first 5 days, training to find hidden escape platforms was conducted to measure latency and distance traveled to the platforms, and on day 6, a probe trial was performed with the platforms removed to evaluate long-term memory by measuring the time spent in the quadrant where the platforms were located and the number of crossings passed through that location.

[0441] (3) Neurogenesis analysis (BrdU labeling and immunohistochemistry)

[0442] To track newly generated neurons, BrdU (5-bromo-2′50 mg / kg), which is inserted into DNA during cell division, was administered intraperitoneally twice daily for 4 days starting immediately after TBI. After 4 weeks of TBI, the animals were sacrificed and their brains were extracted, after which immunofluorescence staining was performed in the hippocampal dentate gyrus (DG) region using the following markers.

[0443] Anti-BrdU: Confirmation of proliferating cells

[0444] Anti-DCX (Doublecortin): Identification of migrating immature neurons

[0445] Anti-NeuN: Identification of mature neurons

[0446] BrdU / NeuN Double Staining: Analysis of the differentiation rate of newly generated cells into neurons

[0447] (4) Statistical analysis

[0448] Behavioral experiment data were analyzed using two-way ANOVA, and histological data were analyzed using the Kruskal-Wallis test.

[0449] 2) Result

[0450] (1) Significant recovery of spatial learning and memory abilities

[0451] As a result of the cognitive function assessment, the TBI-Vehicle group exhibited significant learning impairment, failing to significantly shorten escape time despite repeated training. In contrast, the mirodenafil (2 mg / kg) administration group showed statistically significantly shorter escape times compared to the Vehicle group starting from the third day of training.

[0452] Barnes Miro: The mirodenafil group showed significantly reduced escape time compared to the vehicle group from day 3 to day 6 of training (p < 0.05) (Fig. 7A, B).

[0453] Morris Underwater Maze: During the training period (1–5 days), the mirodenafil group showed a significant decrease in time to reach the escape zone and distance traveled compared to the vehicle group (p < 0.05) (Figs. 7C–E). In addition, in the probe test, the mirodenafil group showed a significant increase in time in target quadrant (p = 0.03) and target crossing (p = 0.01) compared to the vehicle group (Figs. 7F, G). This indicates that mirodenafil effectively restores hippocampus-dependent spatial learning ability and long-term memory.

[0454] (2) Promotion of neurogenesis in the hippocampal dentate gyrus

[0455] To confirm the histological mechanism of cognitive function recovery, neurogenesis was analyzed in the hippocampal dentate gyrus (DG). The results showed that while the TBI-vehicle group exhibited a significant decrease in overall neurogenesis ability, the mirodenafil administration group showed a significant increase in all stages of neurogenesis.

[0456] Cell proliferation and increase in immature neurons: The mirodenafil administration group showed a significant increase in the number of BrdU-positive cells (p = 0.005) and DCX-positive immature neurons (p = 0.001) compared to the vehicle group (Fig. 7H, J, K).

[0457] Promotion of differentiation into mature neurons: Analysis of the proportion of BrdU-positive cells that differentiated into NeuN-positive mature neurons showed that the vehicle group was only about 11%, while the mirodenafil administration group was about 28%, showing a differentiation rate about 2.5 times higher (Fig. 7L).

[0458] GunNeuN+ cellsBrdU+ cellsDCX+ cellsMeanSEMp-ValueMeanSEMp-ValueMeanSEMp-ValueSham-Vehicle1205.43335.700052.0674.722047.51.40 40Sham-Mirodenafil1192.16755.6130.84768.5675.3750.050557.8674.3700.076TBI-Vehicle591.33315.372# Sham-vehp < 0.001113.3333.609# Sham-vehp < 0.001101.64.505# Sham-vehp < 0.001TBI-Mirodenafil741.143.8240.023205.511.0210.005146.9334.015p < 0.001

[0459] The results of Example 7 above clearly demonstrate that mirodenafil exerts a potent neurogenesis effect that significantly promotes the proliferation and differentiation of endogenous stem cells in damaged brain tissue following traumatic brain injury (TBI). Through this neurogenesis-promoting effect, it was confirmed that cognitive functions, particularly learning and memory abilities, which can remain as permanent disabilities after TBI, were statistically significantly improved. In particular, this neuroregenerative effect mediated by the activation of the cGMP signaling pathway serves as a decisive basis supporting the inventiveness of the present invention for functional restoration and regenerative medicine therapeutic applications, going beyond the conventional concept of a simple neuroprotectant.

[0460]

[0461] Meanwhile, the neurogenesis-promoting effect of the PDE5 inhibitor according to the present invention is technically clearly distinct from the simple neuroprotective effect of existing technologies reported in chronic degenerative neurological diseases such as Alzheimer's disease. The present invention has an essential difference in that it proactively normalizes the extreme oxidative stress environment, characterized by acute zinc toxicity and glutathione depletion specific to TBI, thereby actively establishing a permissive microenvironment in which newly generated neurons can survive and functionally settle.

[0462] Furthermore, the present invention possesses the ability to induce qualitative differentiation, which goes beyond merely increasing cell division (BrdU-positive cells) and significantly improves the rate at which proliferated cells differentiate into mature neurons (NeuN-positive cells) that perform actual neural functions by approximately 2.5 times (increasing from 11% to 28%) compared to the control group. This effect corresponds to a significant effect not achieved by existing technologies and provides a regenerative medical treatment method that substantially regenerates the function of brain tissue physically lost due to traumatic brain injury.

[0463]

[0464] Specific parts of the present invention have been described in detail above. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

A pharmaceutical composition for the prevention or treatment of traumatic brain injury (TBI), comprising mirodenafil or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition is administered to a patient diagnosed with an increased concentration of free zinc in brain tissue compared to a normal control group or a decreased concentration of glutathione (GSH) compared to a normal control group. A pharmaceutical composition according to claim 1, characterized in that the patient is: (a) a patient in whom urea zinc accumulation in the hippocampus region is confirmed through brain imaging; or (b) a patient in whom an increase in 4-HNE (4-hydroxynonenal) or a decrease in Nrf2 activity is confirmed as an indicator of oxidative stress in a biological sample. A pharmaceutical composition according to claim 2, wherein the brain imaging comprises magnetic resonance spectroscopy (1H-MRS) or positron emission tomography (PET), and the biological sample comprises blood, plasma, serum, cerebrospinal fluid (CSF), or neural-derived exosomes in the blood. A pharmaceutical composition according to claim 1, characterized in that the mirodenafil or a pharmaceutically acceptable salt thereof is mirodenafil 2HCl. A pharmaceutical composition according to claim 1, wherein the composition is administered at a dose of 10 mg to 100 mg per day, and the first administration is initiated within 24 hours from immediately after the occurrence of traumatic brain injury until zinc accumulation in brain tissue reaches its peak. A method for treating a patient with traumatic brain injury (TBI), comprising: (a) a step of determining whether there is an excessive accumulation of zinc in brain tissue or the level of oxidative stress indicators (GSH, 4-HNE, Nrf2) by analyzing a biological sample isolated from the patient or taking non-invasive brain images; (b) a step of selecting patients in whom, as a result of the determination in step (a), the zinc concentration is above a reference value or the glutathione (GSH) concentration is below a reference value; and (c) a step of administering a therapeutically effective amount of mirodenafil or a pharmaceutically acceptable salt thereof to the selected patients. A method according to claim 6, characterized in that the verification of step (a) is performed by a non-invasive or minimally invasive method. A method according to claim 6, characterized in that the administration of step (c) is initiated within 24 hours after the occurrence of traumatic brain injury, before irreversible neuronal damage caused by zinc toxicity is completed. A method according to claim 8, characterized in that the administration is initiated within 12 hours after the occurrence of traumatic brain injury, during the rising phase in which zinc concentration rapidly increases. A composition comprising mirodenafil or a pharmaceutically acceptable salt thereof for use in the treatment of traumatic brain injury (TBI), wherein the composition is characterized by being administered within 24 hours immediately following the occurrence of traumatic brain injury to block mitochondrial damage caused by a zinc surge. A composition according to claim 10, characterized in that the above composition is administered repeatedly once a day for 7 days or more to inhibit the activation of microglia and astrocytes. A pharmaceutical composition for the structural rewiring of damaged neural circuits and the recovery of cognitive function in patients with traumatic brain injury, comprising mirodenafil or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition promotes the proliferation of endogenous neurons in the hippocampal dentate gyrus region and increases the efficiency of differentiation of the proliferated cells into mature neurons (NeuN-positive neurons). A pharmaceutical composition according to claim 12, characterized in that the composition restores spatial learning ability decline or long-term memory decline caused by traumatic brain injury and increases the ratio of BrdU and NeuN double-positive cells in brain tissue. A pharmaceutical composition according to claim 12, characterized in that the composition is administered during the acute phase after the occurrence of traumatic brain injury to suppress zinc toxicity, thereby creating a permissive microenvironment capable of nerve regeneration. A pharmaceutical composition according to any one of claims 1, 6, 10 and 12, characterized in that the composition is administered in combination with one or more additional therapeutic agents selected from the group consisting of osmotic therapeutic agents, anti-inflammatory agents, neurotrophic factors and stem cell therapeutic agents.

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