Pharmaceutical composition for combined administration for preventing or treating vascular dementia, comprising rock inhibitor and PDE5 inhibitor as active ingredients

Abstract

The present invention relates to a pharmaceutical composition for combined administration for preventing or treating vascular dementia, comprising a ROCK inhibitor and a PDE5 inhibitor as active ingredients. As a result of performing various behavioral tests by administering the ROCK inhibitor and the PDE5 inhibitor in combination to a vascular dementia mouse model, the vascular dementia mouse model was found to restore learning, cognition, and memory abilities, have reduced motor activity and depression and anxiety behaviors, and exhibit a reduced expression level of pro-inflammatory cytokines, thereby demonstrating that the pharmaceutical composition for combined administration of the present invention has prophylactic or therapeutic effects on vascular dementia. Therefore, the pharmaceutical composition of the present invention can be advantageously used as a novel therapeutic agent for vascular dementia through the synergistic actions of the ROCK inhibitor and the PDE5 inhibitor, each targeting major pathological mechanisms of vascular dementia.

Description

Pharmaceutical composition for combination therapy for the prevention or treatment of vascular dementia comprising a ROCK inhibitor and a PDE5 inhibitor as active ingredients

[0001] The present application claims priority based on Korean Patent Applications No. 10-2025-0015621 and No. 10-2025-0200779, filed on February 7, 2025 and December 16, 2025, respectively, and all contents disclosed in the specifications and drawings of said applications are incorporated into the present application.

[0002] Vascular dementia (VD) is a neurodegenerative disease caused by damage to blood vessels in the brain, primarily triggered by stroke (ischemic and hemorrhagic stroke), vasculitis, and transient ischemic attacks (TIA). This vascular damage disrupts the supply of oxygen and nutrients to brain cells, leading to neuronal damage and consequently causing a decline in cognitive function. Pathologically, reduced cerebral blood flow, damage to the blood-brain barrier (BBB), inflammatory responses, oxidative stress, and neuronal apoptosis act as the main mechanisms. In particular, the loss of BBB integrity and impaired vascular function disrupt brain homeostasis, promoting neuroinflammation and neuronal damage, which accelerates the progression of the disease.

[0003] Vascular dementia causes various neurological abnormalities, including memory loss, cognitive decline, depression, and anxiety. These pathological changes impair connectivity between neurons and are closely associated with memory loss and symptoms of emotional instability. As blood flow to the brain decreases, the metabolic activity of brain cells declines, consequently affecting learning, judgment, and emotional regulation abilities. Vascular dementia is known as the second most common type of dementia after Alzheimer's disease, and its prevalence is continuously increasing with the advent of an aging society. Therefore, vascular dementia is recognized not merely as a disease of aging, but as a significant public health issue that increases socioeconomic burdens.

[0004] There are no FDA-approved treatments specifically for vascular dementia, and most patients currently use Alzheimer's disease medications informally to alleviate symptoms or prevent complications. However, these drugs have limitations in fundamentally improving vascular dementia, which is characterized by cognitive impairment caused by vascular lesions. Therefore, there is a great need for new treatments that target the pathophysiological mechanisms of vascular dementia by restoring cerebral vascular function or suppressing neuroinflammation, yet little is known about this.

[0005] The object of the present invention is to provide a pharmaceutical composition for co-administration for the prevention or treatment of vascular dementia comprising a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients.

[0006] Another objective of the present invention is to provide a kit for the prevention or treatment of vascular dementia comprising a pharmaceutical composition for concomitant administration for the prevention or treatment of vascular dementia.

[0007] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0008] The present invention provides a pharmaceutical composition for co-administration for the prevention or treatment of vascular dementia comprising a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients.

[0009] In one embodiment of the present invention, the ROCK inhibitor may be any one selected from the group consisting of Fasudil, Rifasudil, Netarsudil, Belumosudil, Y-27632, and H-1152, but is not limited thereto.

[0010] In one embodiment of the present invention, the phosphodiesterase 5 inhibitor may be any one selected from the group consisting of sildenafil, tadalafil, vardenafil, avanafil, and udenafil, but is not limited thereto.

[0011] In one embodiment of the present invention, the phosphodiesterase 5 inhibitor and the ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) may be administered in combination in a mass ratio of 1:0.1 to 5, but are not limited thereto.

[0012] In one embodiment of the present invention, the pharmaceutical composition may be characterized by satisfying one or more of the following features, but is not limited thereto.

[0013] (a) Enhances learning ability, memory, and cognitive function;

[0014] (b) Promotes physical activity;

[0015] (c) alleviating anxiety and avoidance behaviors; and

[0016] (d) Inhibits the expression of pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α).

[0017] In one embodiment of the present invention, the phosphodiesterase 5 inhibitor and the ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) may be administered simultaneously, sequentially, or individually, but are not limited thereto.

[0018] The present invention provides a kit for the prevention or treatment of vascular dementia comprising a pharmaceutical composition for concomitant administration for the prevention or treatment of vascular dementia.

[0019] In addition, the present invention provides a method for treating vascular dementia comprising the step of administering a pharmaceutical composition for co-administration, comprising a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients, to an individual in need of the same in a pharmaceutically effective amount.

[0020] In addition, the present invention provides a pharmaceutical composition for combination administration comprising a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients for the prevention or treatment of vascular dementia.

[0021] In addition, the present invention provides a use for preparing a pharmaceutical composition for co-administration containing a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients for the prevention or treatment of vascular dementia.

[0022] According to a pharmaceutical composition for combination administration for the prevention or treatment of vascular dementia comprising a ROCK inhibitor and a PDE5 inhibitor as active ingredients, when a ROCK inhibitor and a PDE5 inhibitor were administered in combination to a mouse model of vascular dementia and various behavioral tests were performed, it was confirmed that the learning, cognitive, and memory abilities of the mouse model of vascular dementia were restored, motor activity and depressive and anxious behaviors were alleviated, and the expression of pro-inflammatory cytokines was reduced, thereby proving that the pharmaceutical composition for combination administration of the present invention has a preventive or therapeutic effect on vascular dementia.

[0023] Therefore, the pharmaceutical composition of the present invention can be usefully utilized as a novel treatment for vascular dementia through the synergistic action of each ROCK inhibitor and PDE5 inhibitor targeting the major pathological mechanisms of vascular dementia.

[0024] Figure 1a is a diagram showing the distance traveled to the exit (target zone) for each of the three groups of subjects, WT, VD, and ROCKi+PDE5i, in a Barnes maze experiment (based on Day 4, with the highest values ​​representing VD, WT, and ROCKi+PDE5i, respectively).

[0025] Figure 1b is a diagram showing the latency time to reach the exit (target zone) for each of the three groups of subjects, WT, VD, and ROCKi+PDE5i, in a Barnes maze experiment (based on Day 4, with the highest values ​​representing VD, WT, and ROCKi+PDE5i, respectively).

[0026] Figure 1c is a diagram showing the Time Spent in Target Quadrant (TSTQ) values ​​of three groups of objects, each with WT, VD, and ROCKi+PDE5i, in a Barnes maze experiment.

[0027] Figure 2 is a figure showing the degree of alternation (Alteration triplet) as a percentage (%) of each of the three groups of objects, WT, VD, and ROCKi+PDE5i, moving alternately across all three arms of the Y-maze in the Y-maze experiment.

[0028] Figure 3 is a diagram showing the total distance traveled in the open field for each of the three group objects, WT, VD, and ROCKi+PDE5i, in the open field test.

[0029] Figure 4 is a diagram showing the time in light of each of the three groups of objects, WT, VD, and ROCKi+PDE5i, in the light and dark box experiment.

[0030] Figure 5 is a diagram showing the entry latency of each of the five groups of subjects—WT, VD, Donepezil, Sildenafil, and ROCKi+PDE5i—in a passive avoidance experiment (based on Day 2, from highest to lowest, ROCKi+PDE5i, Donepezil, Sildenafil, WT, and VD, respectively).

[0031] Figure 6 shows the expression levels of IL-6, IL-1β, and TNF-α genes in each group after extracting mRNA from mouse brain tissues of five groups: WT, VD, Donepezil, Sildenafil, and ROCKi+PDE5i, followed by reverse transcription and real-time quantitative PCR (RT-qPCR).

[0032] Hypoxic brain damage caused by vascular stenosis or occlusion is a major mechanism of brain cell damage and a cause of vascular dementia (VD). While this occurs through various pathophysiological mechanisms, the core mechanism is mostly due to impaired blood flow.

[0033] The inventors of the present invention investigated whether the combined administration of a ROCKI inhibitor and a PDE5 inhibitor contributes to vasodilation and improves overall acute and chronic cerebral ischemia, thereby having a preventive or therapeutic effect on vascular dementia, in order to induce the activation of nitric oxide (NO), a major mechanism for the dilation and relaxation of cerebral vascular smooth muscle in an animal model of vascular dementia.

[0034] To this end, the inventors of the present invention evaluated whether the combined administration of a ROCK inhibitor and a PDE5 inhibitor could improve neurological abnormalities such as memory, depression, and anxiety in a mouse model of vascular dementia. As a result, they demonstrated that cognitive and motor impairments in a mouse model of vascular dementia treated with the combination of a ROCK inhibitor and a PDE5 inhibitor could be significantly improved, and confirmed that a neuroprotective effect in the vascular dementia model is exerted through the reduction of inflammatory cytokines (IL-6, IL-1β, TNF-α), thereby confirming that a pharmaceutical composition for the combined administration of a ROCK inhibitor and a PDE5 inhibitor can prevent or treat vascular dementia.

[0035] Accordingly, the present invention provides a pharmaceutical composition for co-administration for the prevention or treatment of vascular dementia comprising a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients.

[0036] In this specification, including all claims below, “vascular dementia (VD)” refers to a neurodegenerative disease caused by damage to blood vessels in the brain, primarily caused by cerebral blood flow impairment such as stroke (ischemic and hemorrhagic stroke), vasculitis, and transient ischemic attack. Vascular dementia occurs when nerve cells are exposed to a state of oxygen and nutrient deficiency due to reduced or blocked blood supply to the brain, resulting in damage or death, and consequently causes various cognitive and emotional abnormalities such as memory loss, impaired judgment, reduced attention, abnormal emotional regulation, depression, and anxiety.

[0037] The major pathological mechanisms of vascular dementia can be summarized as cerebral hypoperfusion, damage to the blood-brain barrier (BBB), endothelial dysfunction, oxidative stress, and neuroinflammation.

[0038] In all claims below, “ROCK (Rho-associated coiled-coil containing protein kinase)” is a protein belonging to the serine / threonine protein kinase group, ROCK1 and ROCK2 are involved in cytoskeletal reorganization, cell migration, inflammatory response, and vascular endothelial cell function, and the ROCK signaling pathway plays an important role in maintaining the structural and functional integrity of blood vessels and the blood-brain barrier (BBB). Excessive activation of ROCK signaling can cause vascular dysfunction and damage to the blood-brain barrier.

[0039] In the entirety of the following claims, “ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor)” may mean a substance that selectively or non-selectively inhibits the activation of ROCK1 and ROCK2.

[0040] Inhibition of ROCK primarily promotes the production of nitric oxide (NO) in endothelial cells, thereby exhibiting vasodilation and endothelial protective effects. Furthermore, blockade of the ROCK-MLC (Myosin Light Chain) pathway inhibits excessive cytoskeletal contraction and stress fiber formation, stabilizing inter-endothelial cell connections and relieving intercellular tension. In addition, ROCK inhibition significantly alleviates intravascular inflammatory responses by suppressing leukocyte adhesion and infiltration. These endothelial stabilization and anti-inflammatory effects enable the recovery of angiogenesis and enhance the survival and functional recovery capabilities of damaged endothelial cells, thereby restoring the structure and function of blood vessels.

[0041] In the entire specification including the following claims, “phosphodiesterase 5 inhibitor (PDE5 inhibitor)” may refer to a substance that activates the cGMP signaling pathway by inhibiting the activity of phosphodiesterase type 5 (PDE5) which degrades intracellular cGMP (cyclic guanosine monophosphate), and is primarily used in the treatment of cardiovascular disease and pulmonary arterial hypertension by inducing vasodilation.

[0042] PDE5 inhibitors promote vasodilation and vasodilation by enhancing nitric oxide (NO) signaling through the induction of increased activity of Protein Kinase G (PKG) within vascular smooth muscle. This leads to increased and improved blood flow in microvessels and macrovessels, resulting in improved oxygen supply and metabolic environments in local tissues. Furthermore, they enhance the functional stability of endothelial cells to reduce oxidative stress and apoptosis, protect the vascular barrier to maintain the structural integrity of the blood-brain barrier (BBB), reduce the expression of inflammatory cytokines, and exhibit anti-inflammatory and anti-fibrotic effects.

[0043] Including all claims below, the strategy of co-administering a ROCK inhibitor and a PDE5 inhibitor may be characterized by simultaneously targeting the complex pathological mechanisms of vascular dementia. Through this, the present invention may be an effective approach capable of overcoming the limitations of existing single-drug treatments. That is, including all claims below, the present invention can expect a synergistic effect that enhances neuroprotective effects by inducing vasodilation and neovascularization through the PDE5 inhibitor to improve cerebral blood flow, inhibiting vascular smooth muscle contraction through the ROCK inhibitor, and promoting vascular improvement and endothelial cell survival. That is, including all claims below, the complementary mechanisms of action of these two drugs improve major pathological characteristics of VD, such as reduced cerebral blood flow, BBB damage, and inflammatory responses, in various ways, and consequently inhibit the progression of neuronal damage and contribute to the recovery of cognitive function.

[0044] Accordingly, as per the entire claim below, the composition of the present invention and / or the method of combination using the same may be characterized by promoting angiogenesis and restoring the integrity of the blood-brain barrier (BBB) ​​to improve cerebral blood flow, thereby restoring cognitive function. Furthermore, as a result of co-administering a ROCK inhibitor and a PDE5 inhibitor, the expression levels of the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α were significantly reduced, confirming that the co-administration exerted neuroprotective effects, such as inhibiting neuroinflammation, by suppressing the inflammatory response.

[0045] Accordingly, as per the entire claim below, combination therapy of ROCK inhibitors and PDE5 inhibitors is a therapeutic strategy capable of simultaneously achieving improved vascular function and neuroprotection, and may present potential therapeutic possibilities not only for vascular dementia but also for various neurodegenerative diseases or ischemic cerebral diseases.

[0046] In this specification, including all claims below, “concomitant administration” is a concept encompassing all forms of administration in which a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor are administered simultaneously or sequentially with a time interval, and refers to a case where the drugs are present during overlapping periods of pharmacological action in vivo, thereby exhibiting mutually complementary or additive / synergistic pharmacological effects. Concomitant administration includes not only cases where the drugs are mixed and administered within the same formulation, but also cases where the pharmacokinetic windows of action overlap and a complex pharmacological effect is manifested, even if the drugs are administered separately in different formulations or via different routes of administration (e.g., oral, intravenous, intraperitoneal, subcutaneous, etc.). Furthermore, even if the two drugs are not administered completely simultaneously, cases where the second drug is administered within a certain time after the first drug, so that the two drugs reach pharmacologically active concentrations simultaneously in vivo, also constitute concomitant administration.

[0047] Accordingly, in this specification, including the entire claim below, concomitant administration is a concept that includes all methods of administration such as (i) simultaneous administration, (ii) sequential or staggered administration, and (iii) administration with adjusted doses and intervals according to clinical or preclinical purposes, and encompasses all cases where two drugs jointly exhibit physiological or pharmacological effects through said methods.

[0048] Including all claims below, the ROCK inhibitor may be any one selected from the group consisting of Fasudil, Rifasudil, Netarsudil, Belumosudil, Y-27632, and H-1152, but is not limited thereto.

[0049] In the present invention, “Fasudil” is a small molecule compound that selectively inhibits the active site of ROCK, and exhibits a vasodilating effect by blocking the RhoA-ROCK pathway that causes vascular smooth muscle contraction, and has been reported to improve endothelial function, increase cerebral blood flow, and have anti-inflammatory effects.

[0050] In the present invention, “Ripasudil” is a ROCK inhibitor having high selectivity for ROCK2 and is used clinically primarily as a glaucoma treatment for the purpose of reducing intraocular pressure. Rifasudil has been reported to increase aqueous humor outflow by inhibiting cytoskeletal rearrangement and promoting intercellular relaxation.

[0051] In the present invention, “Netarsudil” is a dual-function drug that exhibits both ROCK inhibitory action and norepinephrine transporter (NET) inhibitory activity, and is a ROCK inhibitor approved by the U.S. FDA as an intraocular pressure-lowering treatment (Rhopressa®).

[0052] In the present invention, “Belumosudil” is an oral drug having selective inhibitory activity against ROCK2 and has been approved by the U.S. FDA as a treatment for chronic graft-versus-host disease.

[0053] In the present invention, “Y-27632” is a representative small molecule pyridine compound that acts on both ROCK1 and ROCK2. It possesses properties that potently control downstream responses of the ROCK signaling axis, such as reducing cytoskeletal contraction and increasing cell adhesion and survival. Furthermore, it is widely utilized to increase the initial survival rate in stem cell and various organoid cultures by inhibiting cellular stress and apoptosis induced during the process of single-cell formation.

[0054] In this invention, “H-1152” is a ROCK inhibitor with high selectivity and potent ROCK inhibitory activity, and has been reported to improve vascular function and exhibit tissue protective effects in various preclinical models.

[0055] Including all claims below, the ROCK inhibitor may preferably be Y-27632, but is not limited thereto.

[0056] Including all claims below, the phosphodiesterase 5 inhibitor may be any one selected from the group consisting of sildenafil, tadalafil, vardenafil, avanafil, and udenafil, but is not limited thereto.

[0057] In the present invention, “Tadalafil” is a PDE5 inhibitor characterized by a long half-life and high PDE5 selectivity, and provides a sustained vasodilating effect by inhibiting cGMP degradation.

[0058] In the present invention, “Vardenafil” is an inhibitor having high affinity and selectivity for PDE5, and exhibits a potent increase in cGMP and vasodilation effect even at low doses.

[0059] In the present invention, “Avanafil” is a second-generation PDE5 inhibitor characterized by rapid drug absorption and high selectivity, which induces vascular smooth muscle relaxation by increasing cGMP concentration within a short time after administration.

[0060] In the present invention, “Udenafil” is a PDE5 inhibitor having an intermediate half-life and excellent safety, and exhibits a sustained vasodilating effect through the inhibition of cGMP degradation. Udenafil has been reported to have vascular smooth muscle relaxation and anti-inflammatory effects.

[0061] Including all claims below, the phosphodiesterase 5 inhibitor may preferably be sildenafil, but is not limited thereto.

[0062] Sildenafil is a selective phosphodiesterase type 5 (PDE5) inhibitor represented by Chemical Formula 1 below, which is a drug that induces the sustainment and amplification of cGMP signaling by inhibiting the degradation of cGMP within cells. Sildenafil exhibits a potent vasodilating effect by activating PKG in vascular smooth muscle cells and inhibiting calcium-dependent contractile signals; consequently, it provides physiological and pharmacological benefits such as improved blood flow in microvessels and peripheral vessels, enhanced oxygen supply, and stabilization of endothelial function. Furthermore, sildenafil exhibits endothelial protective effects through increased activity of endothelial nitric oxide synthase (eNOS), reduced oxidative stress, and inhibition of inflammatory mediating signals, and contributes to maintaining the structural and functional integrity of the vascular barrier and blood-brain barrier (BBB) ​​through the stabilization of tight junction proteins. Therefore, when sildenafil is used in combination with ROCK inhibitors, mechanisms such as vasodilation, endothelial protection, and anti-inflammatory effects can act in a complementary and synergistic manner. The figure below shows the structural formula of sildenafil, illustrating Chemical Formula 1.

[0063]

[0064] [Chemical Formula 1] Sildenafil (C 22 H 30 N6O4S)

[0065]

[0066] Including all claims below, the phosphodiesterase 5 inhibitor and the ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) may be administered in combination in a mass ratio of 1:0.1 to 5, but are not limited thereto.

[0067] The content of the phosphodiesterase 5 inhibitor or ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the degree of progression of symptoms, the condition of the patient, etc. For example, it may be 0.0001 to 99.9% by weight or 0.001 to 50% by weight based on the total weight of the composition, but is not limited thereto. The above content ratio is a value based on the dry weight after removing the solvent.

[0068] In the present specification, including all claims below, Sildenafil 20 mg / kg (saline dissolved) and ROCK inhibitor 10 mg / kg (DMSO dissolved) were administered in combination, and Sildenafil : ROCK inhibitor was administered in three mass ratios of 20 mg / kg : 10 mg / kg, 20 mg / kg : 20 mg / kg, and 20 mg / kg : 30 mg / kg, and behavioral analysis was performed, and as a result, there was no difference in behavioral indicators between the three mass ratios and no side effects were observed.

[0069] Accordingly, in the entirety of the following claims, a phosphodiesterase 5 inhibitor and a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) may be administered in combination in a mass ratio of 1:0.5 to 1.5 or in combination in a mass ratio of 1:0.5.

[0070] In the design of combination therapies in the preclinical stage, it is common for the optimal dose of a drug to be adjusted within a range of ±2 times due to differences in metabolic, absorption, distribution, and excretion processes among individuals. In particular, in mg / kg-based small animal models, even for the same drug, the actual in vivo exposure can vary significantly depending on the individual's body weight and metabolic variation, so a certain range of dose adjustment is naturally assumed. Considering these conventional dose adjustment principles and pharmacokinetic variability in the industry, a person skilled in the art can predict that pharmacological efficacy is essentially maintained even if the combination ratio of the two drugs is expanded from at least half (0.25) to at least twice (3) based on the center value (1:0.5 to 1.5) of the example.

[0071] Accordingly, in all claims below, the phosphodiesterase 5 inhibitor and the ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) of the present invention may be administered in combination in a mass ratio of 1:0.25 to 3 based on embodiments of the present invention.

[0072] The mass ratio of the co-administration of the PDE5 inhibitor and the ROCK inhibitor of the present invention is 1 : 0.1 to 5, 1 : 0.1 to 4. 1 : 0.1 to 3, 1 : 0.1 to 2.5, 1 : 0.1 to 2, 1 : 0.1 to 1.5, 1 : 0.1 to 1, 1 : 0.1 to 0.5, 1 : 0.3 to 5, 1 : 0.3 to 4. 1 : 0.3 to 3, 1 : 0.3 to 2.5, 1 : 0.3 to 2, 1 : 0.1 to 1.5, 1 : 0.3 to 1, 1 : 0.3 to 0.5, 1 : 0.5 to 5, 1 : 0.5 to 4. 1 : 0.5 to 3, 1 : 0.5 to It may be 2.5, 1 : 0.5 to 2, 1 : 0.5 to 1.5, 1 : 0.5 to 1, 1 : 0.5, but is not limited thereto.

[0073] Including all claims below, the pharmaceutical composition may be characterized by satisfying one or more of the following features, but is not limited thereto.

[0074] (a) Enhances learning ability, memory, and cognitive function;

[0075] (b) Promotes physical activity;

[0076] (c) alleviating anxiety and avoidance behaviors; and

[0077] (d) Inhibits the expression of pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α).

[0078] The pharmaceutical composition for concomitant administration according to the present invention has been confirmed to significantly improve learning ability, memory, and cognitive function through complex pharmacological mechanisms such as improved blood flow, restoration of endothelial cell function, and reduction of neuroinflammation. In this specification, including all claims below, the memory may be a concept that includes short-term memory, long-term memory, working memory, and avoidance memory.

[0079] Including all claims below, the pharmaceutical composition according to the present invention has been confirmed to increase overall motor activity through effects such as cerebral blood flow redistribution and neuroinflammation reduction.

[0080] Including the full claims below, it has been confirmed that the composition of the present invention is characterized by alleviating anxiety-like behavior and avoidance behavior through the reduction of neuroinflammation and the stabilization of synaptic function.

[0081] In all claims below, “Interleukin-6 (IL-6)” refers to a representative inflammatory cytokine that increases rapidly in acute inflammatory and tissue damage responses, and is closely associated with neuronal damage and increased BBB permeability when overexpressed.

[0082] In all claims below, “Interleukin-1 beta (IL-1β)” refers to an inflammatory cytokine that promotes microglia activation and amplifies neuroinflammation, and is closely associated with memory impairment and synaptic damage.

[0083] Including all claims below, “Tumor Necrosis Factor-alpha (TNF-α)” is a potent inflammatory cytokine that is closely associated with nervous system dysfunction by inducing apoptosis signals, increasing oxidative stress, and causing damage to the blood-vascular barrier.

[0084] Including all claims below, the pharmaceutical composition for co-administration according to the present invention may be characterized by significantly reducing the expression of the cytokines, thereby inducing effects of alleviating cerebrovascular inflammation, restoring BBB structure, protecting neurons, and improving cognitive function.

[0085] Including all claims below, the phosphodiesterase 5 inhibitor and the ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) may be administered simultaneously, sequentially, or separately, but are not limited thereto.

[0086] Even when administered individually or sequentially, the order of administration is not restricted, and the order of administration or administration regimen can be appropriately adjusted depending on the type of inhibitor, the patient's condition, etc.

[0087] The present invention provides a kit for the prevention or treatment of vascular dementia comprising a pharmaceutical composition for concomitant administration for the prevention or treatment of vascular dementia.

[0088] In the present invention, the term "Kit" refers to a combination of substances or devices, etc., for preventing or treating vascular dementia using a pharmaceutical composition according to the present invention, and there are no limitations on the specific form. The kit according to the present invention may include not only the compound according to the present invention for preventing and / or treating diseases related to vascular dementia, but also one or more other constituent compositions, solutions, or devices suitable for the prevention, improvement, or treatment of the disease.

[0089] In the present invention, the kit may include, in addition to the above-mentioned formulation, other components, compositions, solutions, devices, etc., that are typically required for the measurement or detection methods thereof. Specific examples may include, but are not limited to, components necessary for sample collection tools for individuals, blood storage, management, etc. In this case, each component may be applied one or more times without limitation on the number of times, and there is no restriction on the order in which each substance is applied, and the application of each substance may proceed simultaneously or sequentially.

[0090] In the present invention, the kit may include a container; instructions; etc. The container may serve to package the formulation and may also serve to store and secure it. The material of the container may take the form, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., and these may be formed partially or wholly from plastic, glass, paper, foil, wax, etc. The container may be equipped with a cap that is initially part of the container or can be attached to the container by mechanical, adhesive, or other means and is fully or partially detachable, and may also be equipped with a stopper that allows access to the contents by a needle. The kit may include an outer package, and the outer package may include instructions regarding the use of the components.

[0091] In the present invention, the kit may further include instructions, but is not limited thereto. Additionally, in the present invention, the instructions may describe a method for providing information necessary for the prevention or treatment of vascular dementia according to the present invention, but is not limited thereto.

[0092] The pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration are expected, for example, oral administration, subcutaneous injection, intraperitoneal administration, intramuscular injection, intrathecal (intradural) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spray through the mouth or nose, skin administration, transdermal administration, etc.

[0093] The pharmaceutical composition according to the present invention may further include a suitable carrier, excipient, and diluent commonly used in the manufacture of pharmaceutical compositions. The excipient may be one or more selected from the group consisting of, for example, diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

[0094] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric granules, liquids, eye drops, oxylic agents, emulsions, suspensions, ethanol tablets, troches, fragrances, limonene adzes, tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, warning agents, lotions, pastes, sprays, inhalants, patches, sterile injectable solutions, or aerosols, according to conventional methods, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, or cataplasms.

[0095] Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0096] When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0097] Excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, refined lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, primogel, etc., as additives to tablets, powders, granules, capsules, pills, and lozenges according to the present invention; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium casein, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch paste, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. may be used as binders, and hydroxypropylmethylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silica, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, hard anhydrous silica, etc. disintegrants;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium pods, kaolin, petroleum jelly, sodium stearate, cocoa paste, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silica, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and hard anhydrous silica may be used.

[0098] As additives to the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearic acid sucroses, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, water ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. may be used.

[0099] In the syrup preparation according to the present invention, a solution of white sugar, other sugars or sweeteners, etc. may be used, and if necessary, flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, viscosity enhancers, etc. may be used.

[0100] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0101] In the suspension agent according to the present invention, suspending agents such as acacia, tragacanthus, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910 may be used, and surfactants, preservatives, stabilizers, coloring agents, and fragrances may be used as needed.

[0102] The injectable preparation according to the present invention comprises solvents such as distilled water for injection, 0.9% sodium chloride injection solution, Ringer's injection solution, dextrose injection solution, dextrose + sodium chloride injection solution, PEG, lactated Ringer's injection solution, ethanol, propylene glycol, non-volatile oils—sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nijungtinamide, hexamine, and dimethylacetamide; and buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums. It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and sodium bisulfite acetone; non-inflammatory agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0103] The suppository according to the present invention comprises cocoa dough, lanolin, Witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, Tween or Spandex, Imhausen, monollene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydrokote SP, S-70-XXA, S-70-XX75 (S-70-XX95), and Hydrokote. Bases such as 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Masa-MF, Masupol, Masupol-15, Neosupostal-N, Paramount-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), and Tegestor triglyceride base (TG-95, MA, 57) may be used.

[0104] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the extract. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used.

[0105] Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0106] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined according to factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0107] The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all the above-mentioned factors, and this can be easily determined by a person skilled in the art to which the present invention belongs.

[0108] The pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration are expected, for example, oral administration, subcutaneous injection, intraperitoneal administration, intramuscular injection, intrathecal (intradural) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spray through the mouth or nose, skin administration, transdermal administration, etc.

[0109] The dosage of the pharmaceutical composition of the present invention is determined according to the type of active ingredient drug, along with various relevant factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and the severity of the disease. Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, gender, and weight, and may be increased or decreased depending on the route of administration, the severity of the disease, gender, weight, age, etc.

[0110] In the present invention, the term “individual” refers to a subject requiring treatment for a disease and can be used interchangeably with a patient. More specifically, the term “individual” refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0111] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0112] In the present invention, “prevention” refers to any act of suppressing or delaying the onset of a target disease, “treatment” refers to any act of improving or beneficially altering the target disease and associated metabolic abnormality symptoms through the administration of a pharmaceutical composition according to the present invention, and “improvement” refers to any act of reducing parameters related to the target disease, such as the severity of symptoms, through the administration of a composition according to the present invention.

[0113] Including all claims below, the pharmaceutical composition of the present invention may be administered to a group of patients with vascular dementia who do not show a therapeutic effect when any one selected from the group consisting of ROCK inhibitors, Donepezil ((±)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]methyl]-1H-inden-1-one hydrochloride), phosphodiesterase 5 inhibitors, and sildenafil is administered alone.

[0114] Including all claims below, the pharmaceutical composition of the present invention may target a group of patients with vascular dementia who do not show a therapeutic effect when a ROCK inhibitor is administered alone.

[0115] Including all claims below, the pharmaceutical composition of the present invention may be administered to a group of patients with vascular dementia who did not show a therapeutic effect when Donepezil ((±)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]methyl]-1H-inden-1-one hydrochloride), an adjuvant drug for improving symptoms of vascular dementia, was administered alone.

[0116] Including all claims below, the pharmaceutical composition of the present invention may target a group of patients with vascular dementia who do not show a therapeutic effect when a phosphodiesterase 5 inhibitor is administered alone.

[0117] Specifically, as per the entire claim below, the pharmaceutical composition of the present invention may target a group of patients with vascular dementia who do not show a therapeutic effect when sildenafil is administered alone.

[0118] The present invention provides a pharmaceutical composition for combination therapy to improve vascular dementia, comprising a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients.

[0119] In addition, the present invention provides a method for preventing or treating vascular dementia, comprising the step of administering a pharmaceutical composition for co-administration containing a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients to an individual in need of the same in a pharmaceutically effective amount.

[0120] In addition, the present invention provides a method for preventing, improving, or treating vascular dementia, comprising the step of administering a pharmaceutical composition for co-administration containing a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients to an individual in need of the same in a pharmaceutically effective amount.

[0121] In addition, the present invention provides a pharmaceutical composition for combination administration comprising a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients for the prevention or treatment of vascular dementia.

[0122] In addition, the present invention provides a pharmaceutical composition for combination administration comprising a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients for the prevention, improvement, or treatment of vascular dementia.

[0123] In addition, the present invention provides a use for preparing a pharmaceutical composition for co-administration containing a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients for the prevention or treatment of vascular dementia.

[0124] In addition, the present invention provides a use for preparing a pharmaceutical composition for combination administration comprising a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients for the prevention, improvement, or treatment of vascular dementia.

[0125] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0126]

[0127] [Experimental Method]

[0128]

[0129] 1. Behavioral Test using a Vascular Dementia Mouse Model

[0130] All behavioral analyses were performed at the Experimental Animal Center of the Biomedical Industry Research Institute at the Catholic University of Korea in Seoul, South Korea. To minimize variability, experiments were conducted using domestically manufactured (custom chamber) equipment, and approximately 60 dB of white noise was applied during the experiments. All behavioral experiments, except for the passive avoidance test, were performed using Smart v3.0 (Panlab, Barcelona, ​​Spain), while the passive avoidance test utilized a shut-avoidance program (Panlab, Barcelona, ​​Spain). Each experiment was repeated three times, and significant differences were confirmed using an unpaired t-test.

[0131]

[0132] 1.1 Behavioral Analysis Test to Confirm the Effects of ROCKi + PDE5i Combination Therapy 1

[0133] In Behavioral Analysis Test 1, the experiment was conducted with three groups: the WT (Wild Type) group as the normal control group, the VD (vascular dementia) group as the negative control group, and the ROCKi + PDE5i combination therapy group as the experimental group.

[0134]

[0135] 1.1.1 Assessment of Spatial Learning and Cognitive Function Using Barnes Mazes

[0136] To evaluate the learning ability, memory, and cognitive flexibility of rats, a Barnes maze experiment was performed using a maze device consisting of a circular black platform with a diameter of 92 cm, manufactured in Korea, with 18 holes arranged around it.

[0137] During the training phase, each rat was allowed to freely explore the maze for 3 minutes, find the exit, and enter it. Subsequently, during the experiment phase, the escape box was removed, and the time spent in the zone where the escape box was originally located was measured. Each rat was placed in the center of the device, and the time spent in the target quadrant, target hole, and error zone was recorded. This experiment was repeated for a total of 4 days, and the lighting intensity within the maze was maintained at 390 lux.

[0138]

[0139] 1.1.2 Assessment of Short-term Memory and Motor Activity Using a Y-Maze

[0140] To evaluate the short-term memory and motor activity of each rat, an 8-minute experiment was conducted using a domestically produced Y-maze. The maze consisted of three zones—A, B, and C—and a central zone, and each rat was placed in zone A at the start of the experiment. During the experiment, the total number of arm entries and the correct alternation ratio were measured, and the lighting intensity within the maze was maintained at 390 lux.

[0141]

[0142] 1.1.3 Assessment of Motor Activity and Anxiety Behavior Using Open Field Experiments

[0143] An open field experiment was conducted using a domestically manufactured device to evaluate the motor activity and anxiety behavior of rats. Each rat was placed in the center of a square open field measuring 45 × 45 × 40 cm and allowed to move freely for 10 minutes. Anxiety levels and exploratory behavior were evaluated by analyzing the distance traveled by the rats during the experiment. The lighting intensity in the central and surrounding areas was maintained at 390 lux.

[0144]

[0145] 1.1.4 Assessment of Anxiety and Avoidance Behaviors Using the Light-Dark Box Experiment

[0146] A light-dark box experiment was conducted to evaluate the anxiety levels and avoidance behaviors of rats. This experiment utilized the rats' innate avoidance of bright spaces and preference for dark spaces, characterized by the fact that the time spent in the dark zone increased as the level of anxiety increased.

[0147] The device used in the experiment was manufactured in Korea and was divided into two sections: a light compartment and a dark compartment, connected by a small opening. Each rat was placed in the light compartment and allowed to move freely between the two compartments for 10 minutes, during which time the rat spent time in the light compartment (Time in light, sec) was measured.

[0148]

[0149] 1.2 Behavioral Analysis Test to Confirm the Effects of ROCKi + PDE5i Combination Therapy 2

[0150] In Behavioral Analysis Test 2, the experiment was conducted with five groups: the WT (Wild Type) group as the normal control group, the VD (Vascular Dementia) group as the negative control group, the Donepezil monotherapy group and the Sildenafil monotherapy group as the positive control group, and the ROCKi + PDE5i combination therapy group as the experimental group.

[0151]

[0152] 1.2.1 Evaluation of Long-Term Memory Using Passive Avoidance Experiments

[0153] To evaluate long-term memory based on fear-based responses in rats, passive avoidance experiments were conducted using the Harvard Apparatus (USA) equipment.

[0154] Each rat was first placed in the light compartment and allowed to freely explore the main box for one minute, and then moved to the dark compartment, which the rat instinctively preferred, when the door was opened. Two seconds after the rat had completely entered the dark compartment, the door was closed and a weak electric stimulus was applied for five seconds.

[0155] This experiment was repeated for 4 days using the same procedure, and the degree of learning and memory retention was evaluated by measuring the entry latency—the time it takes for a rat to enter a dark area—in each experiment. The lighting intensity inside the main box was maintained at 390 lux.

[0156]

[0157] 2. Reverse-Transcription Quantitative Real-Time PCR

[0158] After the experiment was completed, the left cerebral hemisphere (approx. 200 mg) of each individual was collected in a frozen state and homogenized. Subsequently, total RNA was extracted from the brain tissue using the easy-BLUE™ Total RNA Extraction Kit (iNtRON, Daejeon, South Korea) according to the manufacturer's protocol. The concentration and purity of the isolated RNA were measured using a NanoDrop spectrophotometer (NanoDrop Technologies, LLC; Wilmington, DE, USA).

[0159] cDNA was synthesized by performing reverse transcription using All-in-One 5×First Strand cDNA Master Mix (CellScript, Madison, WI, USA) with 1 μg of total RNA. Subsequently, quantitative real-time PCR (qRT-PCR) was performed using TOPreal™ qPCR 2×PreMIX (Enzynomics, Daejeon, South Korea) with the synthesized cDNA as a template, following the manufacturer's instructions.

[0160] Gene expression analysis was performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), and the mRNA expression levels of the following genes were measured: IL-6, IL-1β, TNF-α, α-SMA, Vcam1, Zo-1, Zo-2, Cdh5, Vegf, Kdr, Ang2, Eng, Nrp1, Nr2a, Nr2b, Creb1, Bdnf, and Map2.

[0161]

[0162] 3. Dosage of concomitantly administered drugs

[0163] Prior to the experiment on the combination therapy of Sildenafil and the ROCK inhibitor Y-27632, the optimal dosage concentrations of each drug were determined. Eight-week-old male Sprague-Dawley (SD) rats were used as the animal model, and all drugs were administered by intraperitoneal injection.

[0164] For Sildenafil, the general recommended dose based on the literature (20 mg / kg, saline solution) was applied. Meanwhile, for the ROCK inhibitor Y-27632, behavioral analysis was performed at three concentrations: 10 mg / kg, 20 mg / kg, and 30 mg / kg (DMSO solution). The results showed no differences in behavioral indicators among the three concentrations, and no adverse effects were observed. Accordingly, considering efficacy and safety, the minimum effective concentration of 10 mg / kg was determined as the final administration concentration.

[0165] Finally, in this example, Sildenafil 20 mg / kg (saline dissolved) and Y-27632 10 mg / kg (DMSO dissolved) were used as co-administration conditions.

[0166]

[0167] [Example]

[0168]

[0169]

[0170] Example 1. Effect of combined administration of ROCKi and PDE5i on the recovery of learning, cognitive, and memory functions in mice with vascular dementia

[0171] Barnes maze experiments were conducted to evaluate learning ability and memory in a mouse model of vascular dementia (VD) and to verify the combined effects of ROCK inhibitors (ROCKi) and PDE5 inhibitors (PDE5i). The experiment consisted of three groups administered WT, VD, and ROCKi+PDE5i, and the distance traveled and the time taken for individuals in each group to reach the food location (target area) were compared.

[0172] As a result, the WT group had a long travel distance to the food on the first day, but it gradually shortened through repeated training. On the other hand, the VD group showed a decrease in learning ability as the travel distance actually increased over time. In contrast, an improvement effect was confirmed in the ROCKi+PDE5i combination administration group, with a significant decrease in travel distance (Fig. 1a).

[0173] In addition, an analysis of the time to reach the target point (latency time) over time for each group showed that while the time to reach the target point for the VD group gradually increased over time, the ROCKi+PDE5i combination therapy group showed a tendency to be continuously shortened (Fig. 1b).

[0174] In addition, when comparing the Time Spent in Target Quadrant (TSTQ) values, the VD group showed significantly lower values ​​compared to the WT group, but the ROCKi+PDE5i combination therapy group showed a pattern of significant recovery in TSTQ values, confirming the spatial memory improvement effect of combination therapy (Fig. 1c).

[0175]

[0176] In addition, a Y-maze experiment was performed to evaluate working memory. This Y-maze experiment consisted of three groups: WT, VD, and ROCKi+PDE5i. Mice in each group freely explored the three arms within the maze; among them, the ROCKi+PDE5i administration group exhibited a correct alternation pattern in which all three arms crossed and moved alternately, demonstrating improved working memory function compared to the VD group (Fig. 2).

[0177] These results suggest that the combination of ROCKi and PDE5i can improve both short-term working memory and avoidance memory abilities in a vascular dementia model.

[0178]

[0179] Example 2. Effects of ROCKi+PDE5i combination therapy on improving depression and anxiety avoidance

[0180] The overall motor activity and anxiety behavior of vascular dementia (VD) mice were evaluated through an open field test to verify the effects of combination therapy with ROCK inhibitors (ROCKi) and PDE5 inhibitors (PDE5i). When the total movement distance was compared among three groups—WT, VD, and ROCKi+PDE5i—the VD group showed a relatively short movement distance, indicating reduced activity; however, the ROCKi+PDE5i combination therapy group showed a higher level of movement distance than the WT group, demonstrating a significant recovery in motor activity (Fig. 3).

[0181] In addition, the anxiety and avoidance behaviors of each group were evaluated through a light and dark box experiment. The VD group showed high levels of anxiety as they spent a long time in the dark compartment, whereas the ROCKi+PDE5i combination therapy group showed a pattern of reduced anxiety behavior as their time in light increased (Fig. 4).

[0182]

[0183] Example 3. Effect of ROCKi+PDE5i combination therapy on improving avoidance memory ability

[0184] Finally, a passive avoidance experiment was conducted on all groups to evaluate fear-based avoidance learning. As a result, improvement in avoidance response (latency time) was observed in all groups except the VD group. However, only the ROCKi+PDE5i combination therapy group showed consistent and stable avoidance behavior starting from day 3 of the experiment (Fig. 5).

[0185] These results suggest that the combination of ROCKi and PDE5i can improve avoidance memory ability in a vascular dementia model through synergistic effects.

[0186]

[0187] Example 4. Neuroprotective effects of ROCKi+PDE5i combination therapy through improvement of vascular endothelial function and modulation of inflammatory response

[0188] To evaluate the effect of co-administration of ROCK inhibitors (ROCKi) and PDE5 inhibitors (PDE5i) on the recovery from vascular dementia (VD), mRNA was extracted from mouse brain tissues of each group, and gene expression levels were analyzed through reverse transcription and real-time quantitative PCR (RT-qPCR).

[0189] As a result, in the group administered ROCKi and PDE5i in combination, the expression levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α were significantly reduced compared to the VD model (Fig. 6). These results suggest that the two inhibitors may have a synergistic effect to improve vascular endothelial cell function and regulate inflammatory responses in the brain and exhibit neuroprotective effects by suppressing neuroinflammation.

[0190]

[0191] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0192] According to a pharmaceutical composition for combination administration for the prevention or treatment of vascular dementia comprising a ROCK inhibitor and a PDE5 inhibitor as active ingredients, when a ROCK inhibitor and a PDE5 inhibitor were administered in combination to a mouse model of vascular dementia and various behavioral tests were performed, it was confirmed that the learning, cognitive, and memory abilities of the mouse model of vascular dementia were restored, motor activity and depressive and anxious behaviors were alleviated, and the expression of pro-inflammatory cytokines was reduced, thereby proving that the pharmaceutical composition for combination administration of the present invention has a preventive or therapeutic effect on vascular dementia.

[0193] Therefore, the pharmaceutical composition of the present invention can be usefully utilized as a novel treatment for vascular dementia through the synergistic action of each ROCK inhibitor and PDE5 inhibitor targeting the major pathological mechanisms of vascular dementia, and thus its industrial applicability is recognized.

Claims

1. A pharmaceutical composition for co-administration for the prevention or treatment of vascular dementia comprising a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients.

2. In Paragraph 1, A pharmaceutical composition in which the above-mentioned ROCK inhibitor is any one selected from the group consisting of Fasudil, Rifasudil, Netarsudil, Belumosudil, Y-27632, and H-1152.

3. In Paragraph 1, A pharmaceutical composition in which the above phosphodiesterase 5 inhibitor is any one selected from the group consisting of sildenafil, tadalafil, vardenafil, avanafil, and udenafil.

4. In Paragraph 1, A pharmaceutical composition characterized by the above phosphodiesterase 5 inhibitor and ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) being administered in combination in a mass ratio of 1:0.1 to 5.

5. In Paragraph 1, The above pharmaceutical composition is characterized by satisfying one or more of the following features: (a) Enhances learning ability, memory, and cognitive function; (b) Promotes physical activity; (c) alleviating anxiety and avoidance behaviors; and (d) Inhibits the expression of pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α).

6. In Paragraph 1, A pharmaceutical composition characterized in that the above phosphodiesterase 5 inhibitor and ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) are administered simultaneously, sequentially, or individually.

7. A kit for the prevention or treatment of vascular dementia comprising a pharmaceutical composition for concomitant administration for the prevention or treatment of vascular dementia according to any one of claims 1 to 6.

8. A method for treating vascular dementia comprising the step of administering a pharmaceutical composition for co-administration, comprising a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients, to an individual in need of the same in a pharmaceutically effective amount.

9. Use of a pharmaceutical composition for combination therapy containing a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients for the prevention or treatment of vascular dementia.

10. Use for manufacturing a pharmaceutical composition for combination administration comprising a ROCK inhibitor (Rho-associated coiled-coil containing protein kinase inhibitor) and a phosphodiesterase 5 inhibitor as active ingredients for the prevention or treatment of vascular dementia.

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