Pharmaceutical composition for preventing or treating parkinson's disease, containing PDK inhibitor as active ingredient

Abstract

According to pharmaceutical composition for preventing or treating Parkinson's disease, containing a pyruvate dehydrogenase kinase (PDK) inhibitor as an active ingredient, a pharmaceutical preparation for preventing or treating Parkinson's disease, containing same, and a method for predicting a Parkinson's disease therapeutic effect of a candidate drug, of the present invention, it can be expected that a candidate drug treating Parkinson's disease and having an optimal therapeutic effect can be selected.

Description

Pharmaceutical composition for preventing or treating Parkinson's disease, comprising a PDK inhibitor as an active ingredient

[0001] An example of the present invention is:

[0002] The present invention relates to a pharmaceutical composition for preventing or treating Parkinson's disease, comprising a PDK (pyruvate dehydrogenase kinase) inhibitor as an active ingredient, a pharmaceutical preparation for preventing or treating Parkinson's disease, comprising the same, and a method for predicting the therapeutic effect of a candidate drug on Parkinson's disease.

[0003] Parkinson's disease (PD) is a disease that rarely appears before the age of 50 and its incidence increases with age. It is most prevalent between the ages of 85 and 89. Parkinson's disease is the second most common degenerative brain disease in the world, after Alzheimer's disease (AD), and is characterized by symptoms such as bradykinesia, tremors, and rigidity. The death of midbrain dopaminergic neurons (mDA neurons) located in the substantia nigra (SN) of the midbrain is the main cause of PD. Abnormal protein aggregates called Lewy bodies are commonly observed in the SN of Parkinson's patients. One of the main proteins that make up these Lewy bodies is α-synuclein (α-syn), and they appear in the form of abnormal aggregates. In a pathological environment, when alpha-synuclein (α-syn) forms a β-sheet structure, soluble monomers form oligomers, which gradually combine to form small protofibrils and eventually large, insoluble alpha-synuclein (α-syn) fibrils. Protein aggregates associated with degenerative brain diseases are removed through autophagy, and alpha-synuclein protein aggregates can also be removed by autophagy. When autophagy function is impaired, alpha-synuclein aggregates can occur, and these formed alpha-synuclein aggregates can also inhibit autophagy. Furthermore, alpha-synuclein protein aggregates can be removed by activating autophagy.

[0004] Phosphoinositide 3-kinase (PI3K) and pyruvate dehydrogenase kinase (PDK) are upstream regulators of the autophagy pathway and can influence autophagy activity. Activation of PI3K induces the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which activates protein kinase B (AKT). Activation of AKT inhibits autophagy by suppressing the ULK1 complex via the mTORC1 pathway.

[0005] PDK is a key regulator of the PI3K / AKT signaling pathway, interacting with PIP3 in the cell membrane to activate several downstream proteins, including AKT. PDK-mediated activation of AKT inhibits autophagy via the mTORC1 pathway. Inhibition of PDK reduces the activity of AKT and mTORC1, activating ULK1 and promoting autophagy.

[0006] The researchers began their study under the hypothesis that inhibiting PDK activity would induce autophagy activation, thereby removing or inhibiting the formation of abnormal alpha-synuclein aggregates, a hallmark of Parkinson's disease.

[0007] Based on this hypothesis, the inventors conducted a thorough study and found that:

[0008] A pharmaceutical composition for the prevention or treatment of Parkinson's disease, comprising a PDK (pyruvate dehydrogenase kinase) inhibitor as an active ingredient, a pharmaceutical preparation for the prevention or treatment of Parkinson's disease, comprising the same, and a method for predicting the therapeutic effect of a candidate drug on Parkinson's disease were designed.

[0009]

[0010] Accordingly, the present invention

[0011] The purpose is to provide a pharmaceutical composition for preventing or treating Parkinson's disease, which comprises a PDK (pyruvate dehydrogenase kinase) inhibitor as an active ingredient.

[0012]

[0013] In addition, the present invention

[0014] The purpose is to provide a pharmaceutical preparation for preventing or treating Parkinson's disease, comprising the above pharmaceutical composition.

[0015]

[0016] In addition, the present invention

[0017] The purpose of this invention is to provide a method for predicting the therapeutic effect of a candidate drug on Parkinson's disease using the following [Mathematical Formula 1].

[0018] [Mathematical Formula 1]

[0019]

[0020] Here,

[0021] A k agg is the area of ​​individual alpha-synuclein aggregates in the drug-treated group,

[0022] I k agg is the fluorescence intensity of individual alpha-synuclein aggregates in the drug-treated group,

[0023] C total is the total number of cells in the drug-treated group,

[0024] BA k agg is the area of ​​individual alpha-synuclein aggregates in the drug-untreated group,

[0025] BI k agg is the fluorescence intensity of individual alpha-synuclein aggregates in the drug-untreated group,

[0026] BC total refers to the total number of cells in the drug-untreated group.

[0027] In order to achieve the above purpose, the present invention

[0028] A pharmaceutical composition for preventing or treating Parkinson's disease is provided, comprising a PDK (pyruvate dehydrogenase kinase) inhibitor as an active ingredient.

[0029] In one embodiment of the present invention, the PDK inhibitor may be at least one selected from the group consisting of GSK2334470 (CAS no. 1227911-45-6), MP7 (CAS no. 1001409-50-2), and OSU03012 (CAS no. 742112-33-0).

[0030] In another embodiment of the present invention, the GSK2334470 (CAS no. 1227911-45-6) may be a compound of the following chemical formula 1.

[0031] [Chemical Formula 1]

[0032]

[0033]

[0034] In another embodiment of the present invention, the MP7 (CAS no. 1001409-50-2) may be a compound of the following chemical formula 2.

[0035] [Chemical Formula 2]

[0036]

[0037]

[0038] In another embodiment of the present invention, the OSU03012 (CAS no. 742112-33-0) may be a compound of the following chemical formula 3.

[0039] [Chemical Formula 3]

[0040]

[0041]

[0042] In another embodiment of the present invention, the PDK inhibitor may inhibit the aggregation of pathogenic alpha-synuclein.

[0043] In another embodiment of the present invention, the PDK inhibitor may inhibit the death of dopaminergic neuronal cells.

[0044]

[0045] In addition, the present invention

[0046] A pharmaceutical preparation for preventing or treating Parkinson's disease, comprising the above pharmaceutical composition, is provided.

[0047] In one embodiment of the present invention, the formulation may further comprise a pharmaceutically acceptable carrier, excipient or diluent.

[0048] In another embodiment of the present invention, the formulation may be in the form of an oral dosage form, an injection form, an infusion form, a spray form, or a liquid form.

[0049]

[0050] In addition, the present invention

[0051] A method for predicting the therapeutic effect of a candidate drug on Parkinson's disease is provided using the following [Mathematical Formula 1].

[0052] [Mathematical Formula 1]

[0053]

[0054] Here,

[0055] A k agg is the area of ​​individual alpha-synuclein aggregates in the drug-treated group,

[0056] I k agg is the fluorescence intensity of individual alpha-synuclein aggregates in the drug-treated group,

[0057] C total is the total number of cells in the drug-treated group,

[0058] BA k agg is the area of ​​individual alpha-synuclein aggregates in the drug-untreated group,

[0059] BI k aggis the fluorescence intensity of individual alpha-synuclein aggregates in the drug-untreated group,

[0060] BC total refers to the total number of cells in the drug-untreated group.

[0061]

[0062] In one embodiment of the present invention, it may be predicted that Parkinson's disease is treated by a candidate drug when the α-CAS value decreases compared to a group not treated with the drug.

[0063] In another embodiment of the present invention, the candidate drug may be a pyruvate dehydrogenase kinase (PDK) inhibitor.

[0064] In another embodiment of the present invention, the PDK inhibitor may be at least one selected from the group consisting of GSK2334470 (CAS no. 1227911-45-6), MP7 (CAS no. 1001409-50-2), and OSU03012 (CAS no. 742112-33-0).

[0065] In another embodiment of the present invention, the GSK2334470 (CAS no. 1227911-45-6) may be a compound of the following chemical formula 1.

[0066] [Chemical Formula 1]

[0067]

[0068]

[0069] In another embodiment of the present invention, the MP7 (CAS no. 1001409-50-2) may be a compound of the following chemical formula 2.

[0070] [Chemical Formula 2]

[0071]

[0072]

[0073] In another embodiment of the present invention, the OSU03012 (CAS no. 742112-33-0) may be a compound of the following chemical formula 3.

[0074] [Chemical Formula 3]

[0075]

[0076]

[0077] In addition, the present invention

[0078] A method for preventing or treating Parkinson's disease is provided, comprising administering a PDK inhibitor to a subject in need thereof.

[0079]

[0080] In addition, the present invention

[0081] Provided is a method for preventing or treating Parkinson's disease, comprising administering a PDK inhibitor to a subject in need thereof.

[0082]

[0083] By using a pharmaceutical composition for preventing or treating Parkinson's disease, which comprises the PDK (pyruvate dehydrogenase kinase) inhibitor of the present invention as an active ingredient, a pharmaceutical preparation for preventing or treating Parkinson's disease, which comprises the same, and a method for predicting the therapeutic effect of a candidate drug on Parkinson's disease, it is possible to expect the selection of a candidate drug having an optimal therapeutic effect and the treatment of Parkinson's disease.

[0084]

[0085] It should be understood that the effects of the present invention are not limited to the effects mentioned above, but include all effects that can be inferred from the detailed description of the present invention or the composition of the invention described in the claims.

[0086] Figure 1 is a diagram showing the molecular structures and CAS numbers of three PDK inhibitors (GSK2334470, MP7, OSU03012).

[0087] Figure 2 shows the results of quantitative analysis of the inhibitory effects of three PDK inhibitors on pathogenic α-syn aggregation. Figure 2A is a schematic diagram of the process by which pathogenic α-syn aggregation occurs in PD patients and the process by which pathogenic α-syn aggregation is induced by blue light in a light-induced α-syn aggregation promotion system, Figure 2B is a schematic diagram of a PD cell model-based drug activity screening system using a dopaminergic cell line (SH-SY5Y) in which the α-syn-Cry2PHR coding sequence is knocked in at the AAVS1 locus, Figure 2C is a formula for the comprehensive pathogenic α-syn aggregate formation index (α-synuclein comprehensive aggregation score, α-CAS), Figure 2D is a representative immunocytochemical staining image of pathogenic α-syn aggregates using the 5G4 antibody, and Figure 2E is a graph quantifying the degree of pathogenic α-syn aggregate formation by α-CAS.

[0088] Figure 3 shows the α-CAS quantification and IC of the inhibition of pathogenic α-syn aggregate formation by GSK2334470 treatment concentration. 50 Here are the analysis results. Figure 3A is a graph showing the degree of pathogenic α-syn aggregate formation quantified by α-CAS using immunocytochemistry using the 5G4 antibody, and Figure 3B is a graph showing the values ​​converted through min-max normalization.

[0089] Figure 4 shows the results of analyzing the pathogenic α-syn aggregation inhibitory effect and cell death inhibitory effect of GSK2334470 in midbrain dopaminergic neurons derived from PD patients. Figure 4A is a schematic diagram of a drug activity screening system based on a PD cell model that utilizes PD patient-derived hiPSCs that have an α-syn-mCherry-Cry2clust coding sequence inserted into the AAVS1 locus and differentiated into midbrain dopaminergic neurons (PD hiPSC-derived mDA neurons). Figure 4B is a representative image of simultaneous immunocytochemical staining of pathogenic α-syn aggregates using the 5G4 antibody and mDA neurons using the TH antibody. Figure 4C is a graph quantifying the degree of pathogenic α-syn aggregate formation in mDA neurons labeled with TH using α-CAS. Figure 4D is a representative image of mDA neuron immunocytochemical staining using the TH antibody. Figure 4E is a graph quantifying the survival rate of mDA neurons labeled with TH.

[0090] Figure 5 shows the results of analyzing the effects of oral administration of GSK2334470 on the inhibition of pathogenic α-syn aggregate lesions and protection of mDA neurons in a PD mouse model. Figure 5A is a schematic diagram summarizing the schedule for creating a PD mouse model by injecting AAV-α-syn or AAV-GFP into the SNpc region and α-syn microfibrils (αPFF) or PBS into the VTA region, and evaluating the therapeutic activity through oral administration of GSK2334470. Figure 5B is a representative image of simultaneous immunohistochemical staining of pathogenic α-syn aggregate lesions using pS129-α-syn antibody and mDA neurons using TH antibody in brain tissue sections, and Figure 5C is a quantitative graph for pS129-α-syn immunofluorescence signals.

[0091] Figure 6 shows the results of analyzing the effects of oral administration of GSK2334470 on alleviating bradykinesia and motor coordination abnormalities and protecting mDA neurons in a PD mouse model. Figures 6A and 6B are graphs showing the results of evaluating bradykinesia and motor coordination abnormalities using the pole test and the rotarod test, respectively. Figure 6C is a representative image of mDA neurons labeled with TH antibody in brain tissue sections confirmed by immunohistochemistry. Figure 6D is a quantitative graph showing the number of mDA neurons labeled with TH in the SNpc region.

[0092] Hereinafter, the present invention will be described in detail.

[0093]

[0094] In order to achieve the above purpose, the present invention

[0095] A pharmaceutical composition for preventing or treating Parkinson's disease is provided, comprising a PDK (pyruvate dehydrogenase kinase) inhibitor as an active ingredient.

[0096] As used herein, the term "PDK (pyruvate dehydrogenase kinase)" refers to an enzyme that plays an important role in regulating energy metabolism within cells, primarily by regulating the activity of the pyruvate dehydrogenase complex (PDC). PDK plays an important role in the process of converting pyruvate produced in glycolysis into acetyl-CoA, and this conversion process is the starting point of cellular respiration, which plays a key role in energy production in cells. PDK inhibits this conversion of pyruvate, allowing cells to regulate their metabolic processes, and is mainly activated in response to signals such as ATP concentration, hormonal signals, and glucose concentration. Excessive activation of PDK inhibits the oxidation of pyruvate within the mitochondria, and acts as an important mechanism for regulating energy metabolism even when the energy produced through glycolysis is insufficient.

[0097] PDK is an enzyme involved in regulating cellular energy metabolism and is closely related to the PI3K (Phosphoinositide 3-kinase) / AKT signaling pathway. Activation of PI3K induces the production of PIP3 (Phosphatidylinositol 3,4,5-trisphosphate) in the cell membrane, which in turn activates AKT (Protein Kinase B). Activation of AKT inhibits the autophagy pathway through mTORC1 (mammalian target of rapamycin complex 1). PDK acts as a key regulator in this PI3K / AKT signaling pathway and can contribute to the inhibition of autophagy by activating downstream signaling molecules such as AKT depending on the cellular energy status. Furthermore, PDK regulates cellular metabolic processes by regulating the activity of the pyruvate dehydrogenase complex, which interacts with the mTORC1 pathway and plays a crucial role in determining the level of autophagy.

[0098] PDK is closely linked to the regulation of autophagy in neurodegenerative diseases such as Parkinson's disease. PDK activation inhibits autophagy by activating the AKT and mTORC1 pathways, which may contribute to the accumulation of abnormal protein aggregates such as alpha-synuclein. Conversely, PDK inhibition activates the unc-51-like autophagy activating kinase 1 (ULK1) complex, promoting autophagy and potentially clearing alpha-synuclein aggregates. Therefore, PDK inhibition is being studied as an important target for activating the autophagy pathway in diseases such as Parkinson's disease, with the potential for therapeutic effects.

[0099] As used herein, the term "PDK (pyruvate dehydrogenase kinase) inhibitor" refers to a drug that regulates cellular metabolism and energy production by inhibiting the activity of the PDK enzyme. PDK inhibits the activity of pyruvate dehydrogenase complex (PDC), a key step in the metabolic pathway that converts pyruvate to acetyl-CoA. When PDK is activated, PDC is inhibited, and pyruvate is not oxidized but instead converted to lactate. This process is particularly active in cancer cells and under hypoxic conditions, and it reorganizes cellular energy metabolism to aid survival. Therefore, the use of PDK inhibitors can convert pyruvate to acetyl-CoA, activating cellular respiration and suppressing lactate accumulation.

[0100] PDK inhibitors may play a key role in neurodegenerative diseases, particularly Parkinson's disease. Because PDK is linked to the PI3K / AKT / mTORC1 signaling pathway and contributes to the inhibition of autophagy, blocking PDK activity using PDK inhibitors promotes autophagy and increases the likelihood of removing abnormal protein aggregates such as α-synuclein. In particular, PDK inhibitors are being studied as potential treatments for Parkinson's disease, where they activate the autophagy pathway, inhibit the accumulation of abnormal protein aggregates, and contribute to reducing neuronal damage.

[0101] As used herein, the term "Parkinson's disease (PD)" refers to a degenerative disease affecting the central nervous system, characterized by the progressive loss of dopamine-producing neurons, particularly in the substantia nigra of the midbrain. This disease causes a decline in motor function, and the main symptoms include tremor, rigidity, bradykinesia, and postural instability. One of the pathological hallmarks of Parkinson's disease is the formation of protein aggregates called Lewy bodies, which are primarily composed of a protein called α-synuclein. Normally, α-synuclein exists in the form of monomers, but in Parkinson's disease, it aggregates into abnormal oligomers or fibrils and accumulates within nerve cells.

[0102] In Parkinson's disease, these protein aggregates are removed by intracellular clearance systems such as autophagy. However, impaired autophagy can exacerbate α-synuclein aggregation, potentially disrupting neuronal function. Signaling pathways such as the PI3K / AKT / mTORC1 pathway play a crucial role in regulating autophagy, and modulation of this pathway is being studied as a key factor in removing α-synuclein aggregates in Parkinson's disease. Furthermore, drugs such as PDK inhibitors have the potential to contribute to the treatment of Parkinson's disease by activating the autophagy pathway to reduce or eliminate α-synuclein aggregation.

[0103] The term "prevention" as used herein means any act of suppressing symptoms of Parkinson's disease or delaying the onset of symptoms in a subject by administering a pharmaceutical composition according to the present invention.

[0104] The term "treatment" as used herein means any action by which symptoms of Parkinson's disease in a subject are improved or beneficially changed by administration of a pharmaceutical composition according to the present invention.

[0105] As used herein, the term "administration" means providing a composition of the present invention to a subject by any suitable method.

[0106] As used herein, the term "subject" means a subject in need of treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, dog, cat, horse, or cow.

[0107] Additionally, the pharmaceutical composition may be provided as a pharmaceutical composition or pharmaceutical preparation containing the active ingredient alone or including one or more pharmaceutically acceptable carriers, excipients or diluents.

[0108] Specifically, the term "pharmaceutically acceptable carrier" as used herein is one commonly used in formulations, and may be a colloidal suspension, powder, saline solution, lipid, liposome, microspheres or nano-spheres. It also includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may further include other conventional additives such as antioxidants and buffers as needed. These may form a complex with or be associated with a carrier, and may be delivered in vivo using a carrier system known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids.

[0109] In addition, the term "pharmaceutically acceptable excipient" used herein means any substance that is used as a carrier or medium for delivery of the pharmaceutical composition according to the present invention, or added to the formulation to improve handling or storage properties, or to facilitate manufacturing the formulation in a dosage unit into an article such as a capsule or tablet suitable for oral administration. The excipient may include, but is not limited to, diluents, disintegrants, binders, adhesives, wetting agents, lubricants, glidants, fragrances, surfactants, and inclusion compounds.

[0110] When the above pharmaceutical composition is formulated, it can be prepared using excipients or diluents such as lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Regarding suitable pharmaceutically acceptable carriers and formulations, each ingredient can be preferably formulated using the methods disclosed in Remington's literature.

[0111] Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid preparations may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

[0112] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives.

[0113] Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin. When formulated as eye drops, known excipients or diluents may be used.

[0114] Additionally, the pharmaceutical composition may be administered alone or in combination with other therapeutic agents. Specifically, the pharmaceutical composition may be administered in combination with known compositions or other agents that have preventive or therapeutic effects for the disease, and may be administered simultaneously, separately, or sequentially, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum effect with the minimum amount possible without adverse effects, and this can be readily determined by those skilled in the art.

[0115] The above pharmaceutical composition can be administered orally or parenterally, and when administered parenterally, the method of injection can be selected from external application to the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intrathecal injection, percutaneous injection, intranasal injection, intraenteric injection, local injection, sublingual injection, rectal injection, or intrathoracic injection.

[0116] The above pharmaceutical composition is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on the type and severity of the patient's disease, the patient's age, sex, condition, and weight, the absorption rate of the active ingredient in the body, the activity, inactivation rate, and excretion rate of the drug, sensitivity to the drug, time of administration, route of administration, and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field. In addition, the dosage may vary depending on the patient's condition and weight, the extent of the disease, the drug form, route of administration, and time of administration, but can be appropriately selected by those skilled in the art.

[0117] Specifically, the pharmaceutical composition may be administered at a dose of 0.001 to 1000 mg / kg / day, more specifically 0.1 to 100 mg / kg / day. The administration may be administered once a day or divided into several doses. For example, the administration may be administered every other day or once a week. However, the dosage may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., and thus the dosage does not limit the scope of the present invention in any way.

[0118]

[0119] In one embodiment of the present invention, the PDK inhibitor may be at least one selected from the group consisting of GSK2334470 (CAS no. 1227911-45-6), MP7 (CAS no. 1001409-50-2), and OSU03012 (CAS no. 742112-33-0).

[0120] In another embodiment of the present invention, the GSK2334470 (CAS no. 1227911-45-6) may be a compound of the following chemical formula 1.

[0121] [Chemical Formula 1]

[0122]

[0123]

[0124] In another embodiment of the present invention, the MP7 (CAS no. 1001409-50-2) may be a compound of the following chemical formula 2.

[0125] [Chemical Formula 2]

[0126]

[0127]

[0128] In another embodiment of the present invention, the OSU03012 (CAS no. 742112-33-0) may be a compound of the following chemical formula 3.

[0129] [Chemical Formula 3]

[0130]

[0131]

[0132] In another embodiment of the present invention, the PDK inhibitor may inhibit the aggregation of pathogenic alpha-synuclein.

[0133] As used herein, the term "pathogenic alpha-synuclein (α-syn)" refers to a small protein found primarily in neurons in the brain, known to play a crucial role in signal transmission between nerve cells. Alpha-synuclein is involved in regulating the formation and release of synaptic vesicles, and under normal conditions exists as a soluble monomer. However, under pathological conditions, alpha-synuclein transforms into abnormal aggregates, which are associated with various neurodegenerative diseases. In particular, abnormal accumulation of alpha-synuclein is observed in diseases such as Parkinson's disease, Lewy body dementia, and multiple system atrophy.

[0134] In Parkinson's disease, alpha-synuclein, a major component of Lewy bodies, forms abnormal aggregates in dopaminergic neurons in the substantia nigra of the midbrain. These aggregates can cause cytotoxicity within neurons, disrupting their function and ultimately leading to cell death. Cellular clearance systems, such as autophagy, are involved in this aggregate formation process, and if autophagy malfunctions, alpha-synuclein accumulation becomes more severe. Approaches that activate the autophagy pathway or degrade alpha-synuclein aggregates are emerging as important areas of research for the treatment of Parkinson's disease.

[0135] In another embodiment of the present invention, the PDK inhibitor may inhibit the death of dopaminergic neuronal cells.

[0136] As used herein, the term "dopaminergic neuron" refers to a nerve cell that produces and secretes the neurotransmitter dopamine, which plays a crucial role in the central nervous system. Dopaminergic neurons are primarily located in the substantia nigra and ventral tegmental area of ​​the midbrain, and these cells are involved in regulating various brain functions, including motor control, emotion, the reward system, and motivation. Damage or death of dopaminergic neurons can lead to various neurological problems due to insufficient dopamine production and transmission.

[0137] Parkinson's disease (PD) is primarily caused by the loss of dopaminergic neurons, which rapidly die, particularly in the substantia nigra. This leads to decreased dopamine levels, which in turn causes motor dysfunction, leading to symptoms such as tremors, rigidity, and bradykinesia. Dopaminergic neuron death is closely linked to pathological changes such as the formation of alpha-synuclein aggregates, and methods to inhibit these aggregates or protect neurons are a key part of Parkinson's treatment research.

[0138]

[0139] In addition, the present invention

[0140] A pharmaceutical preparation for preventing or treating Parkinson's disease, comprising the above pharmaceutical composition, is provided.

[0141] The above terms “Parkinson’s disease”, “prevention”, “treatment”, etc. may fall within the scope mentioned above.

[0142] In one embodiment of the present invention, the formulation may further comprise a pharmaceutically acceptable carrier, excipient or diluent.

[0143] The above terms “carrier”, “excipient”, “diluent”, etc. may be within the scope described above.

[0144] In another embodiment of the present invention, the formulation may be in the form of an oral dosage form, an injection form, an infusion form, a spray form, or a liquid form.

[0145] The term "injection" as used herein refers to a solution, suspension, emulsion, or solid sterile preparation that is administered directly to body tissues or organs, such as subcutaneously, intramuscularly, or intravascularly, or that is dissolved or suspended in a solvent when used.

[0146] The term "injection" as used herein refers to a drug that is inserted into the body through the urethra, anus, vagina, etc. and is dissolved by body temperature or secretions to produce an effect.

[0147] The term "spray" as used herein refers to a medicine that is sprayed like a mist using a device.

[0148] The term "liquid formulation" as used herein refers to a pharmaceutical form that provides a drug in liquid form, and a dosage form that contains an active ingredient in a liquid form.

[0149] As used herein, the term "patch" refers to a formulation that adheres to the skin and provides sustained therapeutic effects. Patches have a lower risk of adverse effects, such as gastrointestinal or liver damage, due to oral medications. They can be administered to patients who have difficulty taking oral medications. Furthermore, they are absorbed directly into the bloodstream through the skin without undergoing liver metabolism, offering the advantage of delivering the same therapeutic effects without side effects, even at lower doses, compared to oral medications.

[0150]

[0151] In addition, the present invention

[0152] A method for predicting the therapeutic effect of a candidate drug on Parkinson's disease is provided using the following [Mathematical Formula 1].

[0153] [Mathematical Formula 1]

[0154]

[0155] Here,

[0156] A k agg is the area of ​​individual alpha-synuclein aggregates in the drug-treated group,

[0157] I k agg is the fluorescence intensity of individual alpha-synuclein aggregates in the drug-treated group,

[0158] C total is the total number of cells in the drug-treated group,

[0159] BA k agg is the area of ​​individual alpha-synuclein aggregates in the drug-untreated group,

[0160] BI k agg is the fluorescence intensity of individual alpha-synuclein aggregates in the drug-untreated group,

[0161] BC total refers to the total number of cells in the drug-untreated group.

[0162]

[0163] In one embodiment of the present invention, it may be predicted that Parkinson's disease is treated by a candidate drug when the α-CAS value decreases compared to a group not treated with the drug.

[0164] In another embodiment of the present invention, the candidate drug may be a pyruvate dehydrogenase kinase (PDK) inhibitor.

[0165] In another embodiment of the present invention, the PDK inhibitor may be at least one selected from the group consisting of GSK2334470 (CAS no. 1227911-45-6), MP7 (CAS no. 1001409-50-2), and OSU03012 (CAS no. 742112-33-0).

[0166] In another embodiment of the present invention, the GSK2334470 (CAS no. 1227911-45-6) may be a compound of the following chemical formula 1.

[0167] [Chemical Formula 1]

[0168]

[0169]

[0170] In another embodiment of the present invention, the MP7 (CAS no. 1001409-50-2) may be a compound of the following chemical formula 2.

[0171] [Chemical Formula 2]

[0172]

[0173]

[0174] In another embodiment of the present invention, the OSU03012 (CAS no. 742112-33-0) may be a compound of the following chemical formula 3.

[0175] [Chemical Formula 3]

[0176]

[0177]

[0178] To facilitate understanding of the present invention, the following examples will be described in more detail. However, these examples are intended only to exemplify the content of the present invention and are not intended to limit the scope of the present invention. These examples are provided to more fully explain the present invention to those with average knowledge in the technical field to which the invention pertains.

[0179]

[0180] [Example]

[0181] Example 1. Experimental materials and methods

[0182] 1-1. Cell culture and differentiation of midbrain dopaminergic neurons

[0183] SH-SY5Y cell lines with α-syn-Cry2PHR or Cry2PHR coding sequence knocked in at the AAVS1 locus were cultured in Dulbecco's Modified Eagle Medium / F12 (DMEM / F-12) medium supplemented with 15% heat-inactivated FBS and 2 mM L-alanyl-L-glutamine dipeptide. Human induced pluripotent stem cells (SNCAtriplication PD hiPSCs (ND27760-8; 46XX) derived from a Parkinson's disease patient with SNCA gene triplication with α-syn-mCherry-Cry2clust or mCherry-Cry2clust coding sequence knocked in at the AAVS1 locus. 20) were cultured with mitotically inactivated mouse embryonic fibroblasts (MEFs) in DMEM / F-12 medium supplemented with 20% knockout serum replacement (KSR), 0.1 mM MEM-NEAA, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM β-mercaptoethanol, and 10 ng / mL FGF2 to maintain colony morphology. All cells were cultured in a humidified incubator maintained at 37°C and 5% CO2, and the presence of mycoplasma infection was periodically checked.

[0184] To differentiate PD hiPSCs into midbrain dopaminergic neurons (mDA neurons), some PD hiPSCs maintained with MEFs were converted to a feeder-free state and subcultured. The coating material used was iMatrix-511i Silk (Matrixome), and the medium used was StemFit Basic04 Complete Type (Ajinomoto). PD hiPSCs that had been passaged 2-5 times under feeder-free conditions were dissociated into single cells using Accutase, and then differentiated into mDA neurons using a differentiation protocol developed based on the CHIR-boosting technique published by Lorenz Studer's group in the Cell Stem Cell journal in 2021 (Kim, TWet et al. Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use. Cell Stem Cell 28, 343-355 e345 (2021)).

[0185]

[0186] 1-2. Blue light irradiation

[0187] Blue light was irradiated within the cell culture incubator using a custom-made TouchBright W-Series (Live Cell Instrument) light irradiation device. Light irradiation intensity, cycle, and duration were controlled via the provided software, and accurate light intensity was measured at least once a month using LaserCheck (Coherent) equipment to maintain consistent light irradiation intensity.

[0188]

[0189] 1-3. Immunocytochemistry (ICC)

[0190] Cells to be immunofluorescently stained were fixed with 2% paraformaldehyde (PFA) for 5 minutes in a humidified incubator maintained at 37°C and 5% CO2, and then additionally fixed with 4% PFA for 10 minutes in a humidified incubator maintained at 37°C and 5% CO2. They were then washed twice with phosphate-buffered saline (PBS). They were washed once for 15 minutes with immunocytochemical solution (ICC solution: PBS supplemented with 0.5% bovine serum albumin (BSA) and 0.1% Triton X-100). Primary antibodies were diluted in the ICC solution, and incubation was performed at 4°C for 1 hour for SH-SY5Y cells and overnight for mDA neurons. Afterwards, one wash was performed for 15 minutes with ICC solution at room temperature. Afterwards, the secondary antibody was diluted in ICC solution and incubated for 1 hour for SH-SY5Y cells and 2 hours for mDA neurons at room temperature. Then, 1 μg / mL DAPI (4′,6-diamidino-2-phenylindole) was added during the wash with ICC solution, and cell nuclei were simultaneously stained for 5 minutes at room temperature. After two additional washes with ICC solution, the buffer was completely replaced with PBS.

[0191]

[0192] 1-4. Objective numerical analysis through automated image acquisition and image analysis for quantification of pathogenic α-syn aggregates.

[0193] Fluorescent images were acquired using a fully automated fluorescence microscope, Lionheart FX (BioTek). The auto laser focus function was used to automatically obtain clear cell images, and images were automatically captured using the same detailed parameter settings to ensure objectively quantifiable images. When SH-SY5Y cells were stained, the parameters were set and captured for each channel as follows: DAPI channel (illumination = 5, integration time = 5, gain = 15) for obtaining images stained with DAPI, and Cy5 channel (illumination = 3, integration time = 120, gain = 24) for obtaining images stained with 5G4 antibody. A total of nine areas were captured in a checkerboard pattern per well, with the interval between images (403 × 297 μm) set to 200 μm, centered on the center of the well, and these images are shown at the bottom of Fig. 2B. When mDA neurons were stained, the parameters for each channel were set and captured as follows: DAPI channel (illumination = 5, integration time = 5, gain = 15) to obtain images stained with DAPI, GFP channel (illumination = 5, integration time = 50, gain = 24) to obtain images stained with TH antibody, and Cy5 channel (illumination = 5, integration time = 70, gain = 24) to obtain images stained with 5G4 antibody.

[0194] The acquired images were automatically analyzed using Gen5 software (BioTek). DAPI was used through the 'Cellular Analysis' function within the software. + Cell nucleus and 5G4 + The selection criteria for aggregates were established and applied, and based on this, DAPI+ Cell count, 5G4 + Aggregate area, 5G4 + Objective numerical values such as aggregate fluorescence intensity could be extracted. The parameters applied to each screening criterion are as follows: DAPI + Nucleus screening criterion (threshold = 1500, advanced detection options_background flattening_rolling ball diameter = 10, advanced detection options_image smoothing strength = 1, advanced detection options_evaluate background on = 5, object selection_Min. object size = 10 μm, object selection_Max. object size = 20 μm, subpopulation_integral <7200000>0, subpopulation_mean < 30000, subpopulation_peak < 65000, subpopulation_integral > 9000000), 5G4[[ID=⑧]] +Aggregate selection criteria (threshold = 3000, advanced detection options_background flattening_rolling ball diameter = 1, advanced detection options_image smoothing strength = 0, advanced detection options_evaluate background on = 5, object selection_Min. object size = 0.2 μm, object selection_Max. object size = 6 μm, subpopulation_integral <1500000>0, subpopulation_circularity > 0.1, subpopulation_peak < 64000, subpopulation_mean < 15100).

[0195]

[0196] 1-5. Comprehensive α-synuclein aggregation index (α-CAS) formula

[0197]

[0198] A comprehensive etiological α-synuclein aggregation index (α-synuclein comprehensive aggregation score, α-CAS) formula that can be used as a drug efficacy search criterion for Parkinson's disease treatment agents was developed.

[0199] That is, after treating with the drug, pathogenic α-synuclein aggregation is induced with blue light, and the cells are immunofluorescently stained with the 5G4 antibody. Then, the area (A k agg ) of each individual alpha-synuclein aggregate and the fluorescence intensity (I k agg ) of the corresponding alpha-synuclein aggregate are multiplied and calculated for all aggregates, and the sum is obtained. This is divided by the total number of cells (C total) was calculated. This value was normalized to the value calculated in the positive control group (untreated group) that was exposed only to blue light without drug treatment. BA k agg is the area of ​​individual alpha-synuclein aggregates in the positive control group (untreated group), BI k agg is the fluorescence intensity of individual alpha-synuclein aggregates in the positive control group (untreated group), BC total refers to the total number of cells in the positive control group (untreated group).

[0200]

[0201] 1-6. Creation of a Parkinson's disease mouse model and drug administration

[0202] Male C57BL / 6N mice (3 months old) were purchased from Orient Bio Animal Co. (Seongnam, Korea). AAV-GFP or AAV-α-syn was injected into the substantia nigra pars compacta (SNpc) (coordinates are bregma, L: 1.3, AP: -3.4, DV: -4.3 mm), and PBS or α-syn PFF was injected into the ventral tegmental area (VTA) (coordinates are bregma, L: 0.5, AP: -3.4, DV: -4.3). To evaluate the effect of GSK2334470, 1 mg / kg / day was administered orally for 3 weeks, starting 1 week after stereotaxic injection.

[0203]

[0204] 1-7. Mouse motor ability measurement method

[0205] Pole test: Mice were allowed to acclimate to the pole (exercise test post) for at least 20 minutes (a mandatory acclimation phase was necessary because mice tend to become fearful and refuse to descend when first placed on the pole). The pole was made of a 10 mm diameter, 58 cm high metal pole covered with gauze bandage. Acclimated mice were placed on top of the pole. The total time taken to reach the lower end from the upper end of the pole was recorded. Each mouse was evaluated for six sessions, and the total time and average were recorded.

[0206] Rota-rod test: The Rota-rod device was set to free-run for approximately 30 seconds (to stabilize the posture of the mouse being evaluated). After the free-run, the RPM was gradually increased (40 rpm / 300 s) and recording was performed. When the mouse descended, the corresponding time and RPM were recorded. After two pre-sessions, six sessions for the main experiment were conducted.

[0207]

[0208] 1-8. Perfusion and brain tissue immunostaining

[0209] Animals were anesthetized and secured to a mouse surgical platform for open heart surgery. They were perfused with 1X PBS and 4% PFF (w / v). Mouse brains were extracted, postfixed in 4% PFF (w / v), and cryopreserved in 30% sucrose (w / v). For immunohistochemistry, the fixed brains were sectioned coronally at 35 μm thickness. Sections including the substantia nigra were used for the primary analysis. To analyze dopaminergic neurons, brain sections were incubated with tyrosine hydroxylase (TH) or pS129-α-synuclein antibodies (1:1000 dilution). For subsequent visualization, the brain sections were sequentially incubated with biotinylated goat anti-rabbit IgG or anti-mouse IgG and streptavidin-conjugated horseradish peroxidase (HRP). The sections were then exposed to 3,3-diaminobenzidine to determine the degree of TH staining in each brain section. After staining, the number of dopaminergic neurons was counted using an Optical Fractionator probe of Stereo Investigator equipment for the TH-stained samples. For fluorescence imaging analysis, after labeling with the primary antibody (TH, pS129-a-syn), the secondary antibody conjugated to a fluorescent dye was used to stain, and then magnified images were obtained to determine the degree of progression of pathogenic α-syn aggregation lesions in the brain region where dopaminergic neurons are distributed.

[0210]

[0211] Example 2. Experimental Results

[0212] 2-1. Evaluation and quantitative analysis of the alpha-synuclein aggregation inhibition effects of three PDK inhibitors.

[0213] The aim was to evaluate the alpha-synuclein aggregation inhibition effect of three well-known PDK inhibitors (GSK2334470 (CAS no. 1227911-45-6), MP7 (CAS no. 1001409-50-2), and OSU03012 (CAS no. 742112-33-0)), and the molecular structures and CAS numbers of these PDK inhibitors are shown in Figure 1.

[0214] Because alpha-synuclein protein aggregation in Parkinson's disease patients progresses over decades, a system that accelerates this process is necessary to evaluate the aggregation-inhibiting effects of drugs. Using a recently reported light-induced alpha-synuclein aggregation-promoting system, aggregation of pathogenic alpha-synuclein can be induced within hours by irradiating with blue light, as shown in Figure 2A (Kim, M.S. et al. Advanced human iPSC-based preclinical model for Parkinson's disease with optogenetic alpha-synuclein aggregation. Cell Stem Cell 30, 973-986 e911 (2023)).

[0215] The inhibitory effect of PDK inhibitors on alpha-synuclein aggregation was evaluated using a dopaminergic cell line (SH-SY5Y) in which the α-syn-Cry2PHR coding sequence was inserted (knocked-in) into the AAVS1 locus, and a schematic diagram is shown in Fig. 2B.

[0216] To objectively quantify the formation of pathogenic alpha-synuclein aggregates stained with the 5G4 antibody, images taken by an automated microscope were analyzed to extract the area and fluorescence intensity of the aggregates, and a formula was developed to calculate the comprehensive pathogenic alpha-synuclein aggregate formation index (α-synuclein comprehensive aggregation score, α-CAS) based on the extracted values, which is shown in Fig. 2C.

[0217] Based on this, the inhibitory effect of PDK inhibitors on alpha-synuclein aggregation was quantitatively evaluated. Dopaminergic cell lines with α-syn-Cry2PHR or Cry2PHR coding sequence inserted into AAVS1 locus were pretreated with vehicle or PDK inhibitor at a concentration of 1 μM for 24 hours, and then irradiated with 26 μW / mm for 1.5 hours. 2 Cells were cultured in a dark environment or exposed to blue light (470 nm) of the highest intensity (0.5 Hz, 1 s pulse) (vehicle group, n = 40; PDK inhibitor pretreatment group, GSK2334470 and MP7, n = 7, OSU03012, n = 8). Quantitative data are expressed as mean ± standard deviation and were analyzed using one-way ANOVA, followed by Sidak's post hoc test (****P < 0.0001. ns indicates no significance. Scale bar length in the image is 20 μm).

[0218] As a result, as shown in Fig. 2D, in the first row (AAVS1::Cry2PHRSH-SY5Y cells), only the nuclei of cells were stained, but in the second row (AAVS1::α-syn-Cry2PHRSH-SY5Y cells), it was confirmed that pathogenic α-syn aggregates were stained together in red when irradiated with blue light, and in the third row (AAVS1::α-syn-Cry2PHRSH-SY5Y cells), it was confirmed that the aggregation of pathogenic α-syn stained in red was inhibited when treated with GSK2334470, MP7, and OSU03012.

[0219] In addition, as shown in the graph of the quantitative analysis results in Figure 2E, the control group, AAVS1::Cry2PHRSH-SY5Y cells, showed an α-CAS value of 0.00, confirming that α-syn aggregation did not occur. On the other hand, in the experimental group AAVS1::α-syn-Cry2PHRSH-SY5Y cells, the group that was irradiated with blue light and administered vehicle showed an α-CAS value of 1.00 (0% aggregation inhibition effect), whereas the group administered GSK2334470 showed an α-CAS value of 0.27 (73% aggregation inhibition effect), the group administered MP7 showed an α-CAS value of 0.60 (40% aggregation inhibition effect), and the group administered OSU03012 showed an α-CAS value of 0.67 (33% aggregation inhibition effect), confirming that all three PDK inhibitors showed statistically significant inhibitory effects on pathogenic alpha-synuclein aggregation, and among them, the alpha-synuclein aggregation inhibition effect of GSK2334470 was confirmed to be the strongest.

[0220]

[0221] 2-2. Concentration-dependent inhibitory effect of GSK2334470 on pathogenic alpha-synuclein aggregation and IC 50 analyze

[0222] For GSK2334470, which showed the most potent inhibitory effect on pathogenic alpha-synuclein aggregation, we aimed to evaluate the aggregation inhibition effect according to treatment concentration.

[0223] Dopaminergic cell lines harboring the α-syn-Cry2PHR coding sequence inserted into the AAVS1 locus were pretreated with the indicated concentrations of GSK2334470 for 24 h, followed by irradiation with 26 μW / mm for 1.5 h. 2 Blue light (470 nm) of the century was applied (0.5 Hz, 1 s pulse) (n = 4 for 10 nM, 16 for 100 nM, 8 for 200, 300, 510 nM, 24 for 1, 10 μM). Quantitative data are expressed as mean ± standard deviation.

[0224] As a result, as shown in Figure 3A, the inhibitory effect of GSK2334470 on pathogenic alpha-synuclein aggregation was concentration dependent, and as shown in Figure 3B, the IC 50 The value was confirmed to be 802.9 nM.

[0225]

[0226] 2-3. Inhibitory Effect of GSK2334470 on Alpha-Synuclein Aggregation and Apoptosis in Midbrain Dopaminergic Neurons Derived from Induced Pluripotent Stem Cells of Parkinson's Disease Patients

[0227] GSK2334470, which was confirmed to have an inhibitory effect on pathogenic alpha-synuclein aggregation in a dopaminergic cell line (SH-SY5Y) into which a light-induced alpha-synuclein aggregation promoting system was introduced, was also verified to have an inhibitory effect on pathogenic alpha-synuclein aggregation lesions and selective cell death in mesencephalic dopaminergic neurons (mDA neurons) derived from Parkinson's disease patient-induced pluripotent stem cells (PD hiPSCs), and this is shown in Fig. 4.

[0228] Using Parkinson's disease patient induced pluripotent stem cell-derived midbrain dopaminergic neurons (PD hiPSC-derived mDA neurons) in which the α-syn-mCherry-Cry2clust coding sequence was knocked-in at the AAVS1 locus, the inhibitory effect of the PDK inhibitor GSK2334470 on alpha-synuclein aggregation and dopaminergic neuron-specific cell death was evaluated, and a schematic diagram is shown in Fig. 4A.

[0229] PD hiPSC-derived mDA neurons, in which the α-syn-mCherry-Cry2clust or mCherry-Cry2clust coding sequence was inserted into the AAVS1 locus, were treated with vehicle or 100 nM GSK2334470 three times at 48-h intervals for 7 days, and simultaneously exposed to 14 μW / mm 2The cells were cultured in a dark environment or exposed to blue light (470 nm) (0.17 Hz, 0.5 s pulse) (AAVS1::mCherry-Cry2clustPD hiPSC-derived mDA neuron group, n = 25 in dark condition, n = 21 in blue light irradiation condition; AAVS1::α-syn-mCherry-Cry2clustPD hiPSC-derived mDA neuron group, n = 32 in dark condition, n = 26 in vehicle-treated group under blue light irradiation condition, n = 9 in GSK2334470-treated group under blue light irradiation condition). Quantitative data were expressed as mean ± standard deviation and analyzed using one-way ANOVA, followed by Sidak's post hoc test (**P < 0.01. ****P < 0.0001. ns indicates no significance. Scale bar in the image is 20 μm).

[0230] As a result, as shown in Fig. 4B, it was confirmed that the formation of pathogenic alpha-synuclein aggregates in TH-stained midbrain dopaminergic neurons was inhibited by treatment with the PDK inhibitor GSK2334470. In addition, as shown graphically in Fig. 4C as a result of quantitative analysis, the control AAVS1::mCherry-Cry2clustPD hiPSC-derived mDA neurons showed an α-CAS value of 0.00, confirming that α-syn aggregation did not occur. On the other hand, in the experimental group, AAVS1::mCherry-Cry2clustPD hiPSC-derived mDA neurons, the group irradiated with blue light and administered vehicle showed an α-CAS value of 1.00 (0% aggregation inhibition effect), whereas the group administered GSK2334470 showed an α-CAS value of 0.17 (83% aggregation inhibition effect), confirming that the PDK inhibitor GSK2334470 showed a statistically significant effect of inhibiting pathogenic alpha-synuclein aggregation.

[0231] These results suggest that GSK2334470 also exhibits inhibitory effects on pathogenic alpha-synuclein aggregation in Parkinson's disease patient-derived induced pluripotent stem cell-derived midbrain dopaminergic neurons.

[0232] In addition, as shown in Fig. 4D, we confirmed that the specific cell death of TH-stained midbrain dopaminergic neurons due to blue light-induced pathogenic alpha-synuclein aggregation was inhibited by treatment with the PDK inhibitor GSK2334470. In addition, as shown graphically in Fig. 4E as a result of quantitative analysis, when the ratio of TH-stained midbrain dopaminergic neurons in the control group AAVS1::mCherry-Cry2clustPD hiPSC-derived mDA neurons cultured under dark conditions was set to 100%, we confirmed that the ratio showed a statistically insignificant difference in the control group due to blue light irradiation. On the other hand, in the experimental group AAVS1::mCherry-Cry2clustPD hiPSC-derived mDA neurons, the group that was irradiated with blue light and administered vehicle showed a 53% proportion of midbrain dopaminergic neurons stained with TH, whereas the group that was administered GSK2334470 showed a 77% proportion, confirming that the PDK inhibitor GSK2334470 showed a statistically significant effect in inhibiting the death of midbrain dopaminergic neurons stained with TH.

[0233] These results suggest that GSK2334470 also inhibits specific cell death of Parkinson's disease patient-derived induced pluripotent stem cells-derived midbrain dopaminergic neurons caused by blue light-induced pathological alpha-synuclein aggregation.

[0234]

[0235] 2-4. Verification of the inhibitory effect of GSK2334470 on pathogenic alpha-synuclein aggregation and therapeutic effects in an animal model of Parkinson's disease.

[0236] We aimed to verify the therapeutic effects of GSK2334470, which has been shown to inhibit alpha-synuclein aggregation in a Parkinson's disease cell model, in an in vivo animal model of Parkinson's disease. To this end, we established an α-syn-PFF (pre-formed fibril) / AAV-α-syn complex injection model, known to induce accelerated alpha-synuclein aggregation, dopaminergic neuronal cell death, and motor dysfunction. The therapeutic effects of oral administration of GSK2334470 were evaluated, and the overall schematic diagram is shown in Figure 5A.

[0237] After oral administration of vehicle or GSK2334470 (1 mg / kg / day) to the PBS / AAV-GFP injection control group and the αPFF / AAV-α-syn complex injection group for 3 weeks, simultaneous immunohistochemical staining of brain sections using pS129-α-syn antibody for pathogenic α-syn aggregation lesions and TH antibody for mDA neurons was performed, and pS129-α-syn immunofluorescence signals were quantified (vehicle group, n = 8; GSK2334470 group, PBS / AAV-GFP injection control, n = 7, αPFF / AAV-α-syn complex injection group, n = 8). Quantitative data are expressed as mean ± standard deviation and were analyzed using two-way ANOVA, followed by Sidak's post hoc test (****P< 0.0001. Scale bar length in the image is 20 μm).

[0238] When α-syn-PFF / AAV-α-syn complex was injected into the midbrain substantia nigra region of 3-month-old mice and treated with vehicle, as shown in Fig. 5B, it was confirmed that pathogenic α-synuclein aggregate lesions were significantly induced compared to the control group after 1 month, as shown by an increase in the red pS129-α-syn immunofluorescence signal compared to the control group (first column) (second column of the first row).

[0239] On the other hand, in the experimental group (fourth column) that was administered GSK2334470 orally one week after the stereotactic injection of α-syn-PFF / AAV-α-syn, the pS129-α-syn immunofluorescence signal was confirmed to be inhibited by 50.74% compared to the animal model of Parkinson's disease that was administered the vehicle (second column), as shown in Figs. 5B and 5C. This result suggests that the pathogenic α-synuclein aggregation lesions were significantly suppressed by the administration of GSK2334470.

[0240]

[0241] These results suggest that GSK2334470 has significant activity in inhibiting pathogenic alpha-synuclein aggregate lesions in an animal model of Parkinson's disease.

[0242]

[0243] 2-5. Evaluation of the motor impairment and dopaminergic neuron death inhibition effects of GSK2334470 in an animal model of Parkinson's disease.

[0244] Next, we aimed to evaluate whether pathological features such as bradykinesia, motor coordination abnormalities, and dopaminergic neuronal death induced in an animal model of Parkinson's disease could be improved by administration of GSK2334470. To this end, bradykinesia and motor coordination abnormalities were evaluated using the pole test and rota-rod test in the αPFF / AAV-α-syn complex injection group and the PBS / AAV-GFP injection control group, which were orally administered GSK2334470 (1 mg / kg / day) or vehicle for 3 weeks, respectively (vehicle group, n = 10; GSK2334470 group, PBS / AAV-GFP injection control group, n = 7, αPFF / AAV-α-syn complex injection group, n = 8). Quantitative data were expressed as mean ± standard deviation and analyzed using two-way ANOVA, followed by Sidak's post hoc test (**P<0.01. ****P<0.0001. ns indicates no significance).

[0245] As a result, as shown in Fig. 6A and Fig. 6B, the α-syn-PFF / AAV-α-syn complex injection group showed an increase in the completion time in the pole test and a significant decrease in the performance duration in the rota-rod test compared to the PBS / AAV-GFP injection control group, confirming that stereotaxic injection of α-syn-PFF / AAV-α-syn into the midbrain substantia nigra induced bradykinesia and motor coordination abnormalities. On the other hand, in the oral administration group of GSK2334470, the therapeutic effect on motor coordination abnormalities was not observed in the Parkinson's disease animal model, whereas the motor disorder manifested as bradykinesia was confirmed to be recovered almost to the level of the control group. In the case of the PBS / AAV-GFP injection control group, it was confirmed that there was no change in motor ability due to GSK2334470 administration compared to the vehicle treatment group.

[0246]

[0247] We aimed to perform a quantitative analysis of dopaminergic neurons distributed in the substantia nigra of the midbrain, which plays an important role in motor coordination. To this end, we labeled dopaminergic neurons in the substantia nigra of the midbrain using immunohistochemical staining for tyrosine hydroxylase (TH), a dopaminergic neuron marker.

[0248] After oral administration of vehicle or GSK2334470 (1 mg / kg / day) for 3 weeks to the PBS / AAV-GFP control group and the αPFF / AAV-α-syn complex injection group, immunohistochemical staining and quantification of mDA neurons labeled with TH antibody in brain tissue sections were performed. Quantitative data are expressed as mean ± standard deviation and were analyzed using two-way ANOVA, followed by Sidak's post hoc test (**P<0.01. ****P<0.0001. ns indicates no significance. Scale bar in the image is 200 μm).

[0249] As a result, as shown in Fig. 6C, stereotaxic injection of α-syn-PFF / AAV-α-syn into the midbrain substantia nigra induced significant dopaminergic neuronal death (second column of the first row) compared to the control group (first column of the first row) after 1 month, and this progressive dopaminergic neuronal death was significantly inhibited by administration of GSK2334470, as confirmed through stereological counting analysis (second column of the second row).

[0250] In addition, as shown in Fig. 6D, stereotaxic injection of α-syn-PFF / AAV-α-syn into the midbrain substantia nigra region induced significant dopaminergic neuronal cell death (second bar graph) compared to the control group (first bar graph) after 1 month, and it was confirmed that this progressive dopaminergic neuronal cell death was significantly inhibited by administration of GSK2334470 (fourth bar graph).

[0251] These results suggest that bradykinesia and dopaminergic neuronal cell death, which are major Parkinson's disease clinical lesions induced by stereotactic midbrain injection of α-syn-PFF / AAV-α-syn, can be effectively controlled by GSK2334470.

[0252]

[0253] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A pharmaceutical composition for preventing or treating Parkinson's disease, comprising a PDK (pyruvate dehydrogenase kinase) inhibitor as an active ingredient.

2. In claim 1, A pharmaceutical composition, characterized in that the PDK inhibitor is at least one selected from the group consisting of GSK2334470 (CAS no. 1227911-45-6), MP7 (CAS no. 1001409-50-2), and OSU03012 (CAS no. 742112-33-0).

3. In claim 2, A pharmaceutical composition characterized in that the above GSK2334470 (CAS no. 1227911-45-6) is a compound of the following chemical formula 1. [Chemical Formula 1] 4. In claim 2, A pharmaceutical composition characterized in that the above MP7 (CAS no. 1001409-50-2) is a compound of the following chemical formula 2. [Chemical Formula 2] 5. In claim 2, A pharmaceutical composition characterized in that the above OSU03012 (CAS no. 742112-33-0) is a compound of the following chemical formula 3. [Chemical Formula 3] 6. In claim 1, A pharmaceutical composition characterized in that the PDK inhibitor inhibits the aggregation of pathogenic alpha-synuclein.

7. In claim 1, A pharmaceutical composition characterized in that the above PDK inhibitor inhibits the death of dopaminergic neuron cells.

8. A pharmaceutical preparation for preventing or treating Parkinson's disease, comprising the pharmaceutical composition of any one of claims 1 to 7.

9. In claim 8, A pharmaceutical preparation, characterized in that the above preparation further comprises a pharmaceutically acceptable carrier, excipient or diluent.

10. In claim 8, A pharmaceutical preparation characterized in that the above preparation is in the form of an oral preparation, injection preparation, infusion preparation, spray preparation or liquid preparation.

11. A method for predicting the therapeutic effect of a candidate drug on Parkinson's disease using [Mathematical Formula 1]. [Mathematical Formula 1] Here, A k agg is the area of ​​individual alpha-synuclein aggregates in the drug-treated group, I k agg is the fluorescence intensity of individual alpha-synuclein aggregates in the drug-treated group, C total is the total number of cells in the drug-treated group, BA k agg is the area of ​​individual alpha-synuclein aggregates in the drug-untreated group, BI k agg is the fluorescence intensity of individual alpha-synuclein aggregates in the drug-untreated group, BC total refers to the total number of cells in the drug-untreated group.

12. In claim 11, A method for predicting a therapeutic effect, characterized in that it is predicted that Parkinson's disease is cured by a candidate drug when the above α-CAS value decreases compared to the untreated group.

13. In claim 11, A method for predicting therapeutic effect, characterized in that the above candidate drug is a PDK inhibitor.

14. In claim 13, A method for predicting therapeutic effect, characterized in that the PDK inhibitor is at least one selected from the group consisting of GSK2334470 (CAS no. 1227911-45-6), MP7 (CAS no. 1001409-50-2), and OSU03012 (CAS no. 742112-33-0).

15. In claim 14, A method for predicting therapeutic effect, characterized in that the above GSK2334470 (CAS no. 1227911-45-6) is a compound of the following chemical formula 1. [Chemical Formula 1] 16. In claim 14, A method for predicting therapeutic effect, characterized in that the above MP7 (CAS no. 1001409-50-2) is a compound of the following chemical formula 2. [Chemical Formula 2] 17. In claim 14, A method for predicting therapeutic effect, characterized in that the above OSU03012 (CAS no. 742112-33-0) is a compound of the following chemical formula 3. [Chemical Formula 3] 18. A method for preventing or treating Parkinson's disease, comprising administering a PDK inhibitor to a subject in need thereof.

19. A use for preventing or treating Parkinson's disease, comprising administering a PDK inhibitor to a subject in need thereof.

Images