Use of c-FOS inhibitor for treatment of parkinson's disease with depressive / anxiety disorders

Abstract

The present invention found that neural activity plays an important role in the interaction between non-motor symptoms such as depression / anxiety and neuropathies in Parkinson's disease, and particularly, demonstrates that T-5224, which is a c-FOS inhibitor, targets both non-motor symptoms and neuropathies, thereby effectively alleviating both of them. When used as a therapeutic agent in patients with early Parkinson's disease, a c-FOS inhibitor exhibits excellent therapeutic effects by targeting both non-motor symptoms and neuropathy, and thus has an advantage in that the progression of diseases in Parkinson's disease patients can be delayed and the quality of life of Parkinson's disease patients can be improved.

Description

Use of C-FOS inhibitors for the treatment of Parkinson's disease with depressive / anxiety disorders

[0001] The present invention relates to the use of a c-Fos inhibitor for treating Parkinson's disease, and more particularly, to the use of a c-Fos inhibitor for treating Parkinson's disease, including the effect of treating depression / anxiety, a non-motor disorder that appears in the early stages of Parkinson's disease.

[0002] Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons, primarily in the substantia nigra, and the accumulation of protein aggregates known as Lewy bodies (LNs) and Lewy bodies (LBs). Clinically, PD presents with resting tremor, bradykinesia, and gait disturbances. In addition to motor symptoms, non-motor symptoms include anosmia, constipation, autonomic instability, rapid eye movement sleep behavior disorder, apathy, anhedonia, depression, anxiety, and impulse control disorders. Non-motor symptoms can have a more pronounced impact on quality of life than motor symptoms. Among non-motor symptoms, anxiety and depression often present as early premotor signs, sometimes preceding the onset of motor symptoms by up to 20 years (1, 2). Up to 35% of patients with PD experience depressive symptoms, and the prevalence of major depressive disorder is estimated to be approximately 17% (3). Anxiety and depression are important independent variables predicting health-related quality of life decline in Parkinson's disease. Depression in Parkinson's disease has been reported to accelerate cognitive and motor decline and increase mortality compared to non-depressed Parkinson's disease patients (4, 5). Furthermore, Parkinson's disease patients with comorbid depression and anxiety are known to have more severe symptoms, a worse response to antidepressant treatment, and higher levels of functional disability (6). Despite these observations, the underlying mechanisms and pathophysiology of depression in Parkinson's disease remain poorly understood, and a definitive causal relationship between depression and the progression of Parkinson's disease has not yet been established. Current evidence suggests that Lewy pathology initially manifests in the olfactory bulb and dorsal motor nucleus of the vagus nerve and progressively spreads to additional regions of the nervous system, ultimately affecting cortical areas as the disease progresses (7).Substantial in vitro and in vivo experimental evidence has identified prion-like spread of α-synuclein (α-syn) as a novel mechanism underlying the progression of Parkinson's disease (PD), significantly advancing our understanding of the disease's pathophysiology. Notably, this pathological process appears to occur prior to the onset of Parkinson's disease motor symptoms (8, 9).

[0003] Accordingly, the inventors of the present invention have completed the present invention as a result of their efforts to develop a novel treatment agent capable of simultaneously treating depression / anxiety behavior as a novel treatment agent for Parkinson's disease.

[0004] 1. Rodriguez-Violante M, de Sarachaga AJ, Cervantes-Arriaga A, Millan-Cepeda R, Leal-Ortega R, Estrada-Bellmann I, et al. Self-Perceived Pre-Motor Symptoms Load in Patients with Parkinson's Disease: A Retrospective Study. J Parkinsons Dis. 2016;6(1):183-90.

[0005] 2. Weintraub D, Mamikonyan E. The Neuropsychiatry of Parkinson Disease: A Perfect Storm. Am J Geriatr Psychiatry. 2019;27(9):998-1018.

[0006] 3. Reijnders JS, Ehrt U, Weber WE, Aarsland D, Leentjens AF. A systematic review of prevalence studies of depression in Parkinson's disease. Move Disord. 2008;23(2):183-9; quiz 313.

[0007] 4. Dobkin RD, Allen LA, Menza M. Cognitive-behavioral therapy for depression in Parkinson's disease: a pilot study. Mov Disord. 2007;22(7):946-52.

[0008] 5. Hughes TA, Ross HF, Mindham RH, Spokes EG. Mortality in Parkinson's disease and its association with dementia and depression. Acta Neurol Scand. 2004;110(2):118-23.

[0009] 6. Dissanayaka NN, Sellbach A, Silburn PA, O'Sullivan JD, Marsh R, Mellick GD. Factors associated with depression in Parkinson's disease. J Affect Disord. 2011;132(1-2):82-8.

[0010] 7. Braak H, Del Tredici K, Rub U, de Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiology of aging. 2003;24(2):197-211.

[0011] 8. Choi YR, Kim JB, Kang SJ, Noh HR, Jou I, Joe EH, et al. The dual role of c-src in cell-to-cell transmission of alpha-synuclein. EMBO Rep. 2020;21(7):e48950.

[0012] 9. Choi YR, Park SJ, Park SM. Molecular events underlying the cell-to-cell transmission of alpha-synuclein. FEBS J. 2021;288(23):6593-602.

[0013]

[0014] The purpose of the present invention is to provide a novel therapeutic agent that exhibits excellent therapeutic effects in the early stages of Parkinson's disease accompanied by non-motor disorders.

[0015] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease, which comprises a c-Fos inhibitor as an active ingredient.

[0016] In the present invention, the c-Fos inhibitor may be characterized as being T-5224 or a pharmaceutically acceptable salt thereof.

[0017] In the present invention, the composition may be characterized by restoring non-motor impairment in the early stages of Parkinson's disease.

[0018] In the present invention, the non-motor disorder may be characterized as a depressive / anxiety disorder.

[0019] In the present invention, the composition may be characterized in that it is administered to a patient in whom α-synuclein diffusion is observed and / or a pSer129 α-synuclein positive patient.

[0020] In the present invention, the composition may be characterized in that it is administered to a patient in whom astrocytosis and / or microgliosis is observed.

[0021] In the present invention, the astrocytosis may be characterized by an increase in the expression of GFAP compared to the normal control group, and the microgliosis may be characterized by an increase in the expression of Iba-1 compared to the normal control group.

[0022] In the present invention, the patient has tyrosine hydroxylation (TH) in the substantia nigra. It may be characterized by a decrease in positive neurons.

[0023] The present invention also provides a method for providing information for determining a treatment for Parkinson's disease, the method comprising:

[0024] (a) A step of identifying one or more of the following neuropathological signs in the brain tissue of a patient suspected of having Parkinson's disease:

[0025] (i) α-syn diffusion;

[0026] (ii) Confirmation of astrocytosis and / or microgliosis;

[0027] (iii) identification of pSer129 α-synuclein positive neuronal cell bodies; and

[0028] (iv) Tyrosine hydroxylation (TH) in the substantia nigra (SN) Identification of positive neuronal cell bodies;

[0029] (b) a step of determining administration of a c-Fos inhibitor as a treatment for Parkinson's disease when any one or more of the above neuropathological signs is confirmed; provides an information providing method including;

[0030] In the present invention, the c-Fos inhibitor may be characterized as being T-5224 or a pharmaceutically acceptable salt thereof.

[0031] In the present invention, the patient suspected of having Parkinson's disease may be characterized by exhibiting non-motor disorder symptoms in the early stages of Parkinson's disease.

[0032] In the present invention, the patient suspected of having Parkinson's disease may be characterized as not yet having developed motor disorder due to Parkinson's disease or being in the early stage of developing motor disorder.

[0033] The present invention also provides a method for preventing or treating Parkinson's disease, comprising administering a c-Fos inhibitor to a subject in need thereof.

[0034] In another aspect, the present invention provides the use of a c-Fos inhibitor for the prevention or treatment of Parkinson's disease.

[0035] In another aspect, the present invention provides a c-Fos inhibitor for use in the prevention or treatment of Parkinson's disease.

[0036] In another aspect, the present invention provides the use of a c-fos inhibitor for the manufacture of a drug for the prevention or treatment of Parkinson's disease.

[0037] The present invention has clarified that neural activity plays an important role in the interaction between non-motor symptoms such as depression / anxiety and neuropathology in Parkinson's disease, and in particular, has proven that T-5224, a c-Fos inhibitor, can effectively improve both non-motor symptoms and neuropathology by targeting them. Therefore, when a c-Fos inhibitor is used as a treatment for patients with early-stage Parkinson's disease, it can exhibit excellent therapeutic effects by targeting both non-motor symptoms and neuropathology, and thus has the advantage of slowing the progression of the disease in Parkinson's disease patients and improving the quality of life of Parkinson's disease patients.

[0038] Figure 1 shows that injection of α-synuclein preformed fibers (α-syn PFFs) exacerbates anxiety and depressive-like behaviors in A53T Tg mice when exposed to chronic restraint stress (CRS).

[0039] Figure 1a is a schematic diagram of the experimental procedure.

[0040] Figure 1b shows the results of monitoring body weight changes over time during the 4-week CRS.

[0041] Figure 1c shows the results of evaluating motor activity by measuring the total distance traveled and the time in the center area of ​​the open field in the open field test.

[0042] Figure 1d shows the results of the elevated plus maze test, which assesses anxiety-like behavior in the elevated plus maze by measuring the time and frequency in the closed arm (CA) and open arm (OA), and representative trajectories showing movement in the elevated plus maze.

[0043] Figure 1e shows an assessment of depressive-like behavior by measuring immobility time and high activity time in the forced swim test.

[0044] Figure 1f shows an evaluation of motor function by measuring the latency to fall from the bar in a pole climbing test.

[0045] Figure 1g shows the results of evaluating motor function based on hindlimb stride length differences through footprint analysis. This represents hindlimb stride length (mm) recorded from footprint analysis, and the dotted line indicates the walking direction.

[0046] For Figures 1a to 1d, n=16-21 per group, and for Figures 1e to 1g, n=8-11 per group. *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA and Tukey's multiple comparison test.

[0047] Figure 2 shows that CRS exacerbates α-syn proliferation induced by PFF injection.

[0048] Figure 2a (top) shows representative pS129 α-syn pathology images in the Cg, PrL, M, Ins, BLA, Ect, and Ent of PFF+NCRS or PFF+CRS brains. Black arrows indicate pSer129 α-syn-positive inclusion bodies. The scale bar represents 50 μm. Figure 2a (bottom) shows the percentage of area occupied by α-syn pathology in the symmetrical region at day 31 after PFF+NCRS (n = 12) and PFF+CRS (n = 11) treatment in A53Tg mice. *** p < 0.001, ** p < 0.01, * p < 0.05, unpaired t-test and two-tailed comparison test.

[0049] Figure 2b shows a bar graph summarizing the quantitative results of IBA-1 immunohistochemistry in Cg, PrL, M, Ins, CPu, BLA, Ect, Ent, and SN, and presented in PBS+NCRS (n = 5), PBS+CRS (n = 5), PFF+NCRS (n = 4), and PFF+CRS (n = 5) for microgliosis. *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA followed by Tukey's multiple comparison test. Scale bars represent 50 μm.

[0050] Figure 2c shows a bar graph summarizing the quantitative results of GFAP immunohistochemistry in Cg, PrL, M, Ins, CPu, BLA, Ect, Ent, and SN, and astrocytosis in PBS+NCRS (n = 4), PBS+CRS (n = 4), PFF+NCRS (n = 5), and PFF+CRS (n = 6). *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA followed by Tukey's multiple comparison test. Scale bars represent 50 μm.

[0051] Figure 2d shows immunohistochemical analysis of TH in the SN and stereological counting analysis of TH-positive neurons in the PBS+NCRS (n = 14), PBS+CRS (n = 12), PFF+NCRS (n = 11), or PFF+CRS (n = 10) groups. Scale bar represents 200 μm.

[0052] Figure 2e shows immunohistochemistry of TH in CPu and striatal TH density analysis in the PBS+NCRS (n = 5), PBS+CRS (n = 4), PFF+NCRS (n = 5), or PFF+CRS (n = 5) groups. *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA and Tukey's multiple comparison test.

[0053] Figure 3 shows that CRS induces increased α-syn proliferation associated with c-Fos induction.

[0054] Figure 3a shows images of c-Fos in the Cg, M, and BLA regions of PBS+NCRS or PBS+CRS, PFF+NCRS, or PFF+CRS brains.

[0055] Figure 3b shows the percentage evaluation of c-Fos area in the PBS+NCRS or PBS+CRS, PFF+NCRS or PFF+CRS brain regions, indicating the percentage of area occupied by c-Fos in the symmetrical region. Quantitative results are summarized in a bar graph for PBS+NCRS (n = 3), PBS+CRS (n = 5), PFF+NCRS (n = 5), and PFF+CRS (n = 7). *** p < 0.001, ** p < 0.01, * p < 0.05, unpaired t-test and two-tailed comparison test.

[0056] Figure 3c shows representative images of c-fos and pSer129 α-syn immunostaining. Scale bar, 50 μm.

[0057] Figure 3d is a correlation curve showing the relationship between the change in PFF / PBS c-Fos intensity and the percentage of area occupied by pSer129 α-syn. * p < 0.05.

[0058] Figure 4 shows that c-Fos inhibitors can alleviate non-motor symptom-related behavioral deficits and α-syn proliferation induced by PFF injection or CRS exposure.

[0059] Figure 4a is a schematic diagram of the experimental procedure for the effect of T-5224 (c-Fos / AP-1 inhibitor, Med Chem Express).

[0060] Figure 4b evaluates general locomotor activity across all groups in the open-field test, observing distance traveled and time spent in the central area of ​​the open-field arena. Two-way ANOVA with Tukey's multiple comparisons, n=3-16 per group.

[0061] Figure 4c shows the recovery of PFF-induced behavioral changes in the elevated plus maze test by T-5224 administration. Two-way ANOVA and Tukey's multiple comparisons, n=3-16 per group.

[0062] Figure 4d shows that immobility time was reduced by T-5224 administration in the forced swim test. Two-way ANOVA and Tukey multiple comparisons, n=3-16 per group.

[0063] Figure 4e shows that the reduction in descent time was effectively restored by T-5224 administration in a pole test. Two-way ANOVA and Tukey multiple comparisons, n=3-16 per group.

[0064] Figure 4f shows that T-5224 administration reversed the effects of α-syn PFF or CRS on α-syn pathology. *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA followed by Tukey's multiple comparison test.

[0065]

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0067] As used herein, the terms "about" or "approximately" mean within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, and will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation, depending on the practice of the art. Alternatively, "about" may mean within 20% or less, 10% or less, 5% or less, or 1% or less of a given value or range. Alternatively, particularly in relation to biological systems or processes, the terms may mean within a factor of 5 or a factor of 2 of a given value. Where a particular value is described herein and in the claims, the term "about" should be assumed to mean within an acceptable error range for the particular value, unless otherwise stated.

[0068]

[0069] In this invention, we have demonstrated that depressive / anxious behaviors interact with α-syn diffusion to exacerbate symptoms and accelerate the progression of Parkinson's disease. This interaction is mediated by the induction of c-Fos, a neuronal activity marker. Furthermore, we have demonstrated that administration of T-5224, a c-Fos inhibitor, can exhibit remarkable therapeutic effects on Parkinson's disease accompanied by depressive / anxious behaviors.

[0070] Accordingly, the present invention relates, in one aspect, to a composition comprising a c-Fos inhibitor as an active ingredient, specifically, to a pharmaceutical composition for preventing or treating Parkinson's disease.

[0071] In the present invention, the c-Fos inhibitor may be characterized as being T-5224 or a pharmaceutically acceptable salt thereof, but is not limited thereto.

[0072] In the present invention, T-5224 is a transcription factor c-Fos / activator protein (AP)-1 inhibitor with anti-inflammatory effects, and is known as a substance that specifically inhibits the DNA binding activity of c-Fos / c-Jun without affecting other transcription factors. T-5224 has the following chemical structure.

[0073]

[0074] T-5224 chemical structure

[0075] CAS No.: 530141-72-1

[0076] The compound may contain multiple asymmetric centers and may exist in the form of optically pure enantiomers, mixtures of enantiomers, such as, for example, racemates, optically pure diastereomers, mixtures of diastereomers, diastereomeric racemates or mixtures of diastereomeric racemates. According to the Cahn-Ingold-Prelog convention, the asymmetric carbon atoms may be in the "R" or "S" configuration.

[0077] In the present invention, the composition may be characterized by restoring non-motor disorders in the early stages of Parkinson's disease, and the non-motor disorders may be characterized by being depressive / anxiety disorders.

[0078] In one embodiment, the composition may be characterized in that it is administered to a patient in whom α-synuclein diffusion is observed and / or a pSer129 α-synuclein positive patient.

[0079] In the present invention, α-synuclein diffusion in a Parkinson's disease patient can be confirmed by pathological changes such as abnormal folding of α-synuclein protein, accumulation in the form of aggregates or fibrils, or formation of Lewy bodies and Lewy neurites within nerve cells, or by confirming a diffusion pattern in which α-synuclein is released from a nerve cell and spreads to an adjacent nerve cell, or by using a biomarker such as a change in the concentration of α-synuclein in cerebrospinal fluid (CSF) and blood, but is not limited thereto, and can be determined by a diagnostician or clinician diagnosing a Parkinson's disease patient according to conventional diagnostic criteria.

[0080] Methods for measuring α-synuclein diffusion in patients with Parkinson's disease include, but are not limited to, measuring the total amount of α-synuclein protein or the concentration of a specific form (e.g., phosphorylated α-synuclein) through cerebrospinal fluid (CSF) or blood tests, detecting it using an enzyme-linked immunosorbent assay (ELISA), analyzing biopsied brain tissue to confirm the presence of α-synuclein, and identifying α-synuclein within Lewy bodies and Lewy neurites.

[0081] In the present invention, a patient in whom α-synuclein diffusion is observed may mean a patient in whom significant concentration or morphological changes are observed compared to a normal control group, and the change may mean a patient in whom a change of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% is observed when quantified.

[0082] Meanwhile, characteristic protein aggregates called Lewy bodies and Lewy neurites are formed within the nerve cells of patients with Parkinson's disease, and these aggregates mainly contain abnormally modified α-synuclein, especially pSer129 α-synuclein phosphorylated at the Ser129 position (phosphorylated α-synuclein at serine 129). To detect Ser129 α-synuclein in brain tissue samples of patients suspected of having Parkinson's disease, a specific antibody can be used, or the stained tissue can be observed under a microscope to confirm the presence of pSer129 α-synuclein in Lewy bodies and Lewy neurites, thereby selecting patients who are positive for pSer129 α-synuclein. Additionally, patients positive for pSer129 α-synuclein can be screened through cerebrospinal fluid (CSF) or blood tests, or by using an enzyme-linked immunosorbent assay (ELISA).

[0083] In another embodiment, the composition may be administered to a patient in whom astrocytosis and / or microgliosis is observed in biopsied brain tissue. The astrocytosis may be characterized by increased expression of GFAP compared to a normal control, and the microgliosis may be characterized by increased expression of Iba-1 compared to a normal control.

[0084] In the present invention, an increase in the expression of GFAP and / or Iba-1 compared to a normal control group may mean that, when the change is quantified, the expression is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0085] The above expression can be measured by a variety of methods that measure protein levels, including but not limited to single molecule detection (e.g., SIMOA® assay), Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC) mass spectrometry, microcytometry, microscopy, fluorescence-activated cell sorting (FACS), flow cytometry, and various assays based on properties of the protein, including but not limited to enzymatic activity or interactions with other protein partners.

[0086] In the present invention, the patient may be characterized in that the drug is administered to a patient with a decrease in tyrosine hydroxylation (TH) positive neurons in the substantia nigra.

[0087] In Parkinson's disease, dopaminergic neurons are gradually damaged and die, and this mainly occurs in the substantia nigra region. Tyrosine hydroxylation (TH) staining is performed by collecting brain biopsy tissue from a Parkinson's disease patient, staining it, and analyzing the number and distribution of tyrosine hydroxylation (TH) neurons to evaluate the degree of dopaminergic neuron death, thereby selecting patients suitable for administration of the pharmaceutical composition of the present invention.

[0088]

[0089] The present invention, from another aspect, relates to a method for providing information for determining a treatment for Parkinson's disease.

[0090] In the present invention, the method comprises:

[0091] (a) A step of identifying one or more of the following signs in the brain tissue of a patient suspected of having Parkinson's disease:

[0092] (i) α-synuclein diffusion;

[0093] (ii) Confirmation of astrocytosis and / or microgliosis;

[0094] (iii) identification of pSer129 α-synuclein positive neuronal cell bodies; and

[0095] (iv) Identification of tyrosine hydroxylation (TH)-positive neuronal cell bodies in the substantia nigra (SN);

[0096] (b) a step of determining administration of a c-Fos inhibitor as a treatment for Parkinson's disease when one or more of the above symptoms is confirmed;

[0097] In the present invention, the c-Fos inhibitor may be characterized as being T-5224 or a pharmaceutically acceptable salt thereof.

[0098] In the present invention, the patient suspected of having Parkinson's disease may be characterized by exhibiting non-motor disorder symptoms in the early stages of Parkinson's disease.

[0099] In the present invention, the patient suspected of having Parkinson's disease may be characterized as not yet having developed motor disorder due to Parkinson's disease or being in the early stage of developing motor disorder.

[0100]

[0101] In the present invention, a "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the free base or free acid, but is not biologically undesirable or otherwise undesirable. The salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, especially hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and N-acetylcysteine. The salts can also be prepared by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, and polyimine resins.

[0102] In the present invention, “administration” of a compound means providing the compound or a prodrug of the compound to a subject in need of treatment.

[0103] The pharmaceutical composition of the present invention may be administered to a patient singly or co-administered. Co-administration refers to the simultaneous or sequential administration of the compounds, either individually or in combination (with one or more compounds or agents). Accordingly, the preparation may also be combined with other active substances, if desired.

[0104] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0105] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method.

[0106] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0107] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives.

[0108] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0109] The pharmaceutical composition may be a sterile injectable preparation as a sterile aqueous or oily suspension for injection. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Acceptable vehicles and solvents include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, a sterile fixed oil may be conventionally used as a solvent or suspending medium. For this purpose, any fixed oil with minimal irritation, including synthetic mono- or diglycerides, may be used. Fatty acids such as oleic acid and its glyceride derivatives are useful in injectable preparations, as are pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), especially their polyoxyethylated forms.

[0110] The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration. These compositions can be prepared by mixing the compound of the present invention with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0111] Parenteral administration of the pharmaceutical composition of the present invention is particularly useful when the desired treatment involves a site or organ easily accessible by topical application. When applied topically to the skin, the pharmaceutical composition should be formulated as a suitable ointment containing the active ingredient suspended or dissolved in a carrier. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying waxes, and water. In another embodiment, the pharmaceutical composition of the present invention may be formulated as a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. The pharmaceutical composition of the present invention may also be applied topically to the lower intestinal tract by rectal suppository or as a suitable enema. Topically applied transdermal patches are also encompassed by the present invention.

[0112] The pharmaceutical composition of the present invention can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0113] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in the pharmaceutical composition of the present invention will vary depending on the identity, size, and / or disorder of the subject being treated and the route by which the composition is administered. For example, the content of the active ingredient included in the pharmaceutical composition of the present invention is not particularly limited thereto, but may be included in an amount of 0.0001 to 100 wt%, for example, 0.001 to 50 wt%, and more preferably 0.01 to 10 wt%, based on the total weight of the final composition.

[0114] In the present invention, pharmaceutically acceptable excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like suitable for a particular dosage form purpose. Remington, The Science and Practice of Pharmacy, 21st Edition, AR Gennaro, (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients used in the formulation of pharmaceutical compositions and known techniques for their preparation. Any conventional carrier medium is contemplated to be within the scope of the present invention, except that it is not compatible with the substance or its derivatives by providing any undesired biological effect or otherwise interacting in a deleterious manner with any other ingredients of the pharmaceutical composition. A pharmaceutically acceptable excipient is at least 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0115] The above excipients are approved for human and veterinary use. In some embodiments, the excipients are approved by the U.S. Food and Drug Administration. In some embodiments, the excipients are pharmaceutical grade. In some embodiments, the excipients meet the standards of the United States Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopeia, and / or International Pharmacopeia (EP).

[0116] Pharmaceutically acceptable excipients used in the preparation of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils.

[0117] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and combinations thereof.

[0118] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (Starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Vegum), sodium lauryl sulfate, quaternary ammonium compounds, and combinations thereof.

[0119] Exemplary surface active agents and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, Chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and veegum [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, Hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [Miruz 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Breeze 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laureate, sodium lauryl sulfate, Pluronic F68,including but not limited to poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0120] Exemplary binders include, but are not limited to, starches (e.g., corn starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extracts of Irish moss, panwar gum, shattie gum, mucilage of Isaphol fuskus, carboxymethyl cellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (veegum), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohols; and combinations thereof.

[0121] Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol. Exemplary acid preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxymethylene mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Fenonipp, methylparaben, Germoll 115, Germaben II, Neolon, Carton, and Euxyl. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0122] Exemplary buffers include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium globionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, Pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof, including but not limited to.

[0123] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0124] Exemplary oils include almond, apricot kernel, avocado, babassu palm, bergamot, black currant seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, flax seed, geraniol, pumpkin, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litchia cubeba, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange-colored orange raffi, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquamana, savory, sea buckthorn, Examples of oils include, but are not limited to, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

[0125] These excipients may optionally be included in the formulations of the present invention. Excipients such as cocoa butter and suppository wax, colorants, coating agents, sweeteners, flavoring agents, and perfumes may be present in the compositions at the discretion of the formulator.

[0126] The term "prevention" of the present invention includes preventing or delaying the appearance of clinical or subclinical symptoms of a condition, disorder or condition in the future in a mammal, particularly an individual that may be suffering from or susceptible to the condition, disorder or condition, but has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder or condition.

[0127] The term "treatment" of the present invention refers to:

[0128] (1) inhibiting a condition, disorder or pathology (e.g., inhibiting, reducing or delaying the onset of a disease, or in the case of maintenance treatment, the recurrence of at least one of its clinical or subclinical symptoms); and / or

[0129] (2) alleviating a condition, disorder or condition (i.e., causing regression of the condition, disorder or condition, or at least one of its clinical or subclinical symptoms).

[0130] The benefits to the patient receiving treatment will be statistically significant, or at least perceptible to the patient or physician. However, those skilled in the art will understand that when a drug is administered to a patient to treat a disease, the results may not always be effective.

[0131] While the pharmaceutical compositions provided in the present invention are principally intended for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to all types of animals. That is, the pharmaceutical compositions according to the present invention can also be administered to other mammals, such as animals requiring veterinary treatment, such as livestock (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). A skilled veterinary pharmacologist, with a thorough understanding of the modifications of pharmaceutical compositions for administration to various animals, can design and / or implement such modifications simply through routine experimentation, if necessary.

[0132] The pharmaceutical compositions described herein may be prepared by any method known in the art of pharmacology or described herein. Typically, such methods for preparing tablets involve the steps of bringing the active ingredient into association with excipients and / or one or more other auxiliary ingredients, followed by, if necessary or desired, shaping and / or packaging the product into desired single- or multi-dose units.

[0133] The pharmaceutical compositions of the present invention may be manufactured, packaged, and / or sold unpackaged as single unit doses and / or multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient to be administered to a subject and / or a convenient fraction of such a dosage, such as, for example, one-half or one-third of the dosage.

[0134] In the present invention, "administration" means introducing a given substance into a patient by any suitable method, and the pharmaceutical composition may be administered via any common route as long as the drug can reach the target tissue. Routes of administration may include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0135] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount.

[0136] The term "pharmaceutically effective amount" herein may mean a "therapeutically effective amount," which refers to an amount of a compound or composition (e.g., a compound or composition of the present invention) sufficient to achieve a beneficial or desired result. A pharmaceutically effective amount may be administered in one or more dosages, applications, or doses, and is not intended to be limited to a particular formulation or route of administration.

[0137] Effective dosage levels may be determined based on factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, and sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the excretion rate, the duration of treatment, drugs used in combination or concurrently with the composition of the present invention used, and other factors well known in the medical field.

[0138] The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. It can also be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer the amount that achieves maximum efficacy with the minimum amount possible without causing side effects.

[0139] The dosage of the pharmaceutical composition of the present invention can be determined by a person skilled in the art in consideration of the purpose of use, the degree of toxicity of the disease, the patient's age, weight, sex, medical history, or the type of substance used as an active ingredient.

[0140] In addition, the pharmaceutical composition comprising a c-Fos inhibitor as an active ingredient in the present invention may be characterized in that, when administered to a patient with early-stage Parkinson's disease who has not developed motor disorder symptoms but has neuropathological abnormalities with non-motor disorder symptoms, (i) improves or recovers non-motor disorders, (ii) improves or recovers neuropathological abnormalities, and / or (iii) prevents or delays the onset of motor disorders.

[0141] In another aspect, the present invention relates to a method for preventing or treating Parkinson's disease, comprising administering a c-Fos inhibitor to a subject in need thereof.

[0142] In another aspect, the present invention relates to the use of a c-Fos inhibitor for the prevention or treatment of Parkinson's disease.

[0143] In another aspect, the present invention relates to a c-Fos inhibitor for use in the prevention or treatment of Parkinson's disease.

[0144] In another aspect, the present invention relates to the use of a c-fos inhibitor for the manufacture of a drug for the prevention or treatment of Parkinson's disease.

[0145] In the present invention, the detailed description of the treatment method, use, and use for manufacturing the drug is the same as the detailed description of the pharmaceutical composition, unless mutually contradictory, and the overlapping description is omitted.

[0146] The term "subject" of the present invention means any animal, including humans, that has already developed or is likely to develop Parkinson's disease, and by administering the composition of the present invention to the subject, the disease can be effectively prevented and / or treated.

[0147] In another aspect, the present invention relates to a food composition comprising a c-Fos inhibitor.

[0148] In a preferred embodiment, the food may be a health functional food for preventing or improving Parkinson's disease.

[0149] The term “improvement” in the present invention means any action that at least reduces a parameter related to the condition being treated, for example, the severity of a symptom.

[0150] When the food composition of the present invention is used as a food additive, the composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. Generally, when manufacturing a food or beverage, the composition of the present invention may be added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for health purposes or health control purposes, it may be added in an amount of 5% by weight or less, and since there are no safety issues, the active ingredient may be added in an amount exceeding the above range.

[0151] The food composition of the present invention can be manufactured in all forms, such as food additives, nutritional supplements, and health functional foods.

[0152] For example, as a health functional food, the composition of the present invention can be manufactured and consumed in the form of tea, juice, and drinks, or can be granulated, encapsulated, and powdered and consumed. In addition, the composition of the present invention can be manufactured by adding it to health drinks (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and processed foods thereof (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.).

[0153] When a c-fos inhibitor is incorporated into a health drink, it may contain various flavorings or natural carbohydrates as additional ingredients, similar to conventional beverages. The natural carbohydrates described above may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, or synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates may be appropriately determined by those skilled in the art.

[0154] In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination. The proportions of these additives may also be appropriately selected by those skilled in the art.

[0155] The preventive, therapeutic, and improving effects of the c-fos inhibitor in the compositions of various embodiments according to the present invention can be equally applied to Parkinson's disease caused by various causes, and in particular, it is preferable to apply it to Parkinson's disease accompanied by depressive / anxiety disorder.

[0156] In the present invention, "individual", "patient" and "subject" are used interchangeably and include any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, including humans.

[0157]

[0158] Example

[0159] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0160]

[0161] Example 1. Experimental method

[0162]

[0163] 1-1. Experimental animals

[0164] Experiments were conducted using M83 mice (B6; C3-Tg (Prnp-SNCA*A53T)83Vle / J, The Jackson Laboratory) overexpressing A53T mutant human α-synuclein under the control of the mouse prion protein promoter (Prnp) and C57BL / 6 mice (Orient Bio). M83 mice were obtained by mating hemizygous M83 mice produced by crossing homozygous male M83 mice with female C57BL / 6 mice. All animal experimental procedures were performed in accordance with the guidelines of the Ajou University College of Medicine Ethics Committee (IACUC No. 2022-0014).

[0165]

[0166] 1-2. Stereotactic surgery and drug treatment

[0167] Purification of recombinant α-synuclein and production of α-synuclein preformed fibers (PFFs) were performed according to (8, 9).

[0168] Two- to three-month-old hemizygous M83 mice were anesthetized by intraperitoneal injection with 2% 2,2,2-tribromoethanol in saline (250 mg / kg, Sigma) and secured in a stereotaxic apparatus (David Kopf Instruments, USA). 10 μg of sterile PBS or recombinant human WT α-synuclein fibrils (5 mg / ml) were injected at a rate of 400 nL using a single needle (33 gauge) with a 10 μl syringe (Hamilton, NV), and the needle was maintained at the injection site for 10 min. The coordinates used for unilateral injection were anterior-posterior +1.0 mm, medial-lateral +1.8 mm, and dorso-ventral -3.2 mm, targeting the dorsal striatum. Each injected mouse was administered T-5224 (a c-Fos / AP-1 inhibitor, Med Chem Express), 3-{5-[4-(Cyclopentyloxy)-2-hydroxybenzoyl]-2-[(3-hydroxy-1,2-benzisoxazol-6-yl)methoxy]phenyl}propionic acid) 3 days after injection. T-5224 was dissolved in 10% DMSO and 90% polyvinylpyrrolidone (PVP) solution or corn oil, and T-5224 (30 mg per kg) or the solvent was administered orally (po) for 4 weeks, followed by behavioral analysis and immunohistochemical analysis. 2-Methyl-6-(phenylethanyl)-pyridine (MPEP hydrochloride, Caymon Chemical) was dissolved in 10% Tween 80 (vol / vol) and 90% sterile saline, and MPEP (3 mg per kg) or the solvent was administered intraperitoneally (ip) for 4 weeks, followed by immunohistochemical analysis.

[0169]

[0170] 1-3. Immunohistochemical analysis

[0171] Mouse brains were perfused with PBS, dissected, fixed in PBS containing 4% paraformaldehyde, stored overnight, incubated in PBS solution containing 30% sucrose overnight, and cut into 35 μm-thick coronal sections using a cryotome (Model CM-3050; Leica Biosystems). The coronal brain sections (≥ 3 slices / mouse) were permeabilized by immersion in a PBS solution containing 5% bovine serum albumin and 1% Triton X-100 for 60 min, and then incubated for 8-12 h at 4°C with primary antibodies anti-c-Fos (Cell signaling Technology #2250; 1:500), anti-pSer129 α-syn (Abcam #ab51253; or Wako #015-25191; 1:500), anti-calbindin (Swant, #CB300; 1:1000), and anti-CamKII (Abcam #ab22609; 1:500) diluted in blocking solution for fluorescent immunostaining. Sections were washed three times with PBS and incubated with Alexa Fluor 488- (Invitrogen, #A21202, #21206) or 568- (Invitrogen, #A10037, #10042)-conjugated secondary antibodies for 1 hour at room temperature. The sections, washed three times with PBS, were mounted on silane-coated slide glass (#5116-20F; Muto Glass Ind., Ltd., Japan) using Vectashield mounting medium (H-1200; Vector Laboratories). The section suspensions were washed three times in PBS for 10 minutes, treated with 3% H2O2 (Sigma, St. Louis, MO) dissolved in PBS for 5 minutes to inactivate endogenous peroxidase, and then blocked with 1% BSA / 0.2% Triton X-100 solution dissolved in PBS for 1 hour.The primary antibody anti-Iba-1 (Wako, 019-19741; 1:1000), anti-GFAP (Neuromics, #RA22101; 1:5000), or anti-Tyrosine hydroxylase (Abcam, #ab13772; 1:1000) was added to the sections and incubated overnight at 4°C. After incubation with biotin-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA) for 1 h at room temperature, the sections were washed with PBS and incubated with ABC reagent (Vector Laboratories, Burlingame, CA) for 1 h. The sections were stained with 3',3'-diaminobenzidine (DAB) (Sigma, St. Louis, MO) at room temperature and mounted on slides. The sections were dehydrated with ethanol and xylene, mounted with permount solution, and coverslipped.

[0172]

[0173] 1-4. Confocal Microscopy Images and Analysis

[0174] Fluorescent immunostained sections were visualized on a Carl Zeiss (Jena, Germany) laser scanning confocal (LSM800) microscope and a Leica TCS SP8 microscope. Counts, density, and intensity were assessed and quantified using MetaMorph (Molecular Devices). To measure GFAP intensity, 5–6 fields were measured for each region (3 sections per mouse). Background intensity was measured in an area adjacent to the measurement area, and after subtracting the background intensity, the ratio of intensities was converted to a percentage using ImageJ software. Tyrosine hydroxylase (TH) was measured in the substantia nigra (SN) in six coronal sections approximately 3.1–3.6 mm posterior to the bregma in both cerebral hemispheres of the mice to quantify dopamine neurons. + Neurons were counted.

[0175]

[0176] 1-5. Quantitative Pathology

[0177] Slides were digitally scanned using a slide scanner (Axio Scan.Z1; Carl Zeiss, Germany) at 20x magnification. All image settings were maintained constant during image acquisition. Z-stack images obtained from the slide scanner were converted to maximum projection, and the acquired images were further processed using the ZEN software installed on the Axio Scan.Z1. Digitized slides were used for quantitative pathology. For quantification of α-synuclein pathology, reference coronal section images relative to bregma were selected: 2.10 mm, 0.98 mm, −1.58 mm, and −3.08 mm. Reference images were imported into MetaMorph (Molecular Devices) software to allow annotation and quantification of the percentage area occupied by pSer129 α-synuclein pathology. Standardized annotations were created to allow independent quantification of 50 brain regions corresponding to the mouse brain atlas map of Paxinos and Franklin.

[0178]

[0179] 1-6. Chronic Restraint Stress (CRS)

[0180] Mice were randomly assigned to non-restrained control (NCRS) and restrained (CRS) groups. Chronic restraint stress (CRS) experiments were performed for 28 days, 2 h daily (15:00-17:00) in an acrylic cylindrical, large, flat-bottomed enclosure (Jeungdo Bio & Plant Co., Seoul, Korea). The restraint device had multiple slots to restrain mice according to their size and suppressed physical limb movement without pain. After restraint, the mice were immediately released into their original cages. Non-restrained mice remained in their original cages without the CRS procedure, and CRS mice had no access to food and water during the CRS exposure period. Mice were weighed weekly before the experiment.

[0181]

[0182] 1-7. Mouse Behavior Analysis

[0183] All behavioral experiments were performed between 9:00 AM and 6:00 PM under normal room lighting. All mice were fed ad libitum and housed on a 12-hour light / dark cycle. Animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Ajou University College of Medicine (IACUC No. 2022-0014).

[0184]

[0185] 1-7-1. Open field test (OFT)

[0186] Mice were placed in the testing room and allowed to become acclimated to the environment. Mice were placed in a white acrylic 40 × 40 × 35 cm open-field box and monitored for 10 min under 3–50 lux conditions. Distance traveled and time spent in the center zone were recorded, and each recorded video was analyzed using SMART 3.0 software (Panlab Harvard Apparatus., Barcelona, ​​Spain).

[0187]

[0188] 1-7-2. Sucrose Preference Test (SPT)

[0189] A sugar preference test was performed at the end of the CRS procedure and was performed using a dual-bottle choice paradigm. Briefly, mice were acclimated to sugar by being provided with two bottles of 1% sucrose solution (wt / vol) for 24 h. After this, one bottle of the 1% sucrose solution (wt / vol) was replaced with plain water for 24 h. After 2 days of acclimation, mice were water-deprived for 18 h and were provided with two pre-weighed bottles. One bottle contained 200 ml of 2% sucrose and the other contained plain water. After 12 h (i.e., overnight), fluid consumption was measured by the change in bottle weight. Sugar preference was expressed as the ratio of sugar consumption to total fluid consumption.

[0190]

[0191] 1-7-3. Elevated Plus-Maze Test (EPT)

[0192] The elevated maze consisted of two open arms, two closed arms (30 × 6 cm, ~20 lux), and a central area, which was elevated 50 cm above the floor. Mice were initially positioned in the central area facing the closed arms and were allowed to freely move throughout the maze for 5 min. The number of maze entries into the open and closed arms and the time spent in each arm were measured using SMART 3.0 software (Panlab Harvard Apparatus, Barcelona, ​​Spain).

[0193]

[0194] 1-7-4. Forced swim test (FST)

[0195] A 5-L glass beaker was filled with 3-3.5 L of tap water (25°C), and the mice were exposed to water for 15 min on day 1 and for 5 min on the following day. Inactive floating behavior and active swimming behavior were quantified using SMART 3.0 software (Panlab Harvard Apparatus., Barcelona, ​​Spain).

[0196]

[0197] 1-7-5. Pole-climbing test (PCT)

[0198] The pole climbing test consisted of a 50 cm high, 1 cm diameter wooden bar, wrapped with a bandage to prevent slipping. The bar was placed in the home cage, with a mat on the floor to allow the mice to descend without injury. Mice were placed head-up on the top of the bar (3 inches from the top of the bar) and trained to descend the bar. Each mouse was tested after three training sessions, and the time taken to descend the bar was recorded. The maximum time to stop the test was set at 120 seconds. Results were expressed as turns and total time (seconds).

[0199]

[0200] 1-7-6. Rotarod Test

[0201] To assess motor coordination and balance, each mouse was placed on a lane of a rotarod apparatus (Bioseb, USA), with an empty lane between each mouse. The rotarod was programmed to accelerate from 4 to 400 rpm over 300 s. To test the latency to fall from the rotarod, three tests were performed, spaced at least 30 minutes apart.

[0202]

[0203] 1-7-7. Footprint Test

[0204] To obtain footprints, the forelimbs and hindlimbs of mice were dipped in non-toxic paint, and the mice were placed on a track in a closed box. The track's floor was covered with white paper, and the footprints were recorded. The average distance between the forelimbs and hindlimbs (step length, SL) was analyzed. Data from mice that did not cross the track smoothly were excluded from the analysis.

[0205]

[0206] 1-8. Quantification and Statistical Analysis

[0207] All statistical analyses were performed using GraphPad Prism 8 software (San Diego, CA, USA). Statistical comparisons between groups were performed as follows: Normally distributed data were compared using Student's t-test for the area percentage occupied by pS129 α-synuclein between the CRS and NCRS conditions. Regular two-way ANOVA was used to compare the CRS / NCRS and PFF / PBS conditions. Tukey's post hoc test was used to correct for multiple comparisons. Pearson coefficients were calculated for correlation studies. The results of significance tests were as follows: ***p < 0.001; **p < 0.01; *p < 0.05. Data are presented as mean ± SEM.

[0208]

[0209] Example 2. Acceleration of depressive / anxious behaviors following injection of preformed α-synuclein fibrils.

[0210]

[0211] Chronic Restraint Stress (CRS) is a commonly used analytical technique to study depressive-like behaviors in rodents. To explore the bidirectional relationship between depressive- / anxiety-like behaviors and α-syn proliferation, we induced depressive- / anxiety-like behaviors using the CRS model and induced α-syn proliferation using the preformed α-syn fibril (PFF) injection model (Fig. 1a). After 3 weeks, no significant differences in body weight were observed between the chronic restraint stress (CRS) and non-chronic restraint stress (NCRS) groups. However, A53T TG mice injected with PFF and exposed to CRS showed slower weight gain compared to mice treated with PBS but not exposed to CRS. This was observed at the end of the 4-week experiment (Fig. 1b).

[0212] General activity levels and exploratory behavior were assessed using the Open Field Test (OFT). Total distance traveled was not significantly altered by PFF injection or CRS treatment, confirming that neither intervention exerted sedative or excitatory effects. Although not statistically significant, there was a trend toward a decrease in the time spent in the central region of the OFT in PFF-injected mice treated with CRS (Fig. 1c).

[0213] Anxiety-like phenotypes were further assessed using the elevated plus-maze test (EPM) under CRS treatment and PFF injection conditions, and significant changes included decreased time spent in the open arms and increased time spent in the closed arms, both of which were accelerated by PFF injection. The CRS group exhibited a more pronounced depressive / anxious phenotype compared to the NCRS group. Although A53T TG mice alone did not significantly affect the depressive / anxious phenotype, PFF / CRS-treated A53T TG mice showed increased anxiety-related behaviors in the EPM, including the number of open-arm entries and the time spent in the closed arms, compared to PBS / CRS-treated A53T mice (Fig. 1d).

[0214] Depressive-like behaviors were assessed using the forced swim test (FST). CRS significantly prolonged inactivity time and decreased activity time. In particular, PFF / CRS-treated A53T mice exhibited significantly longer inactivity time compared to PBS / CRS-treated mice, suggesting that PFF injection exacerbates depressive symptoms (Fig. 1e).

[0215] Meanwhile, in CRS-treated PFF-injected mice, the time spent in the light compartment in the LDT tended to increase and sugar consumption tended to decrease in the Sucrose Preference Test (SPT) (data not shown).

[0216] Motor coordination was assessed using the pole-climbing test (PCT) and the rotarod test. A53T mice injected with PFF and exposed to CRS showed slower descending times in the pole-climbing test compared to mice not exposed to CRS (Fig. 1f). However, the rotarod test was not affected by PFF injection or CRS treatment (data not shown).

[0217] To further assess motor function, footprint analysis was performed on the last day of CRS treatment. Differences in hindlimb stride length were observed after 4 weeks of CRS (Fig. 1g). However, no significant differences were observed between the PBS and PFF groups exposed to CRS. Forelimb strength was similar between the CRS and NCRS groups and was not affected by PFF injections (data not shown).

[0218]

[0219] In conclusion, PFF amplified depressive / anxiety-like behaviors in the EPM and FST, suggesting a link between α-syn proliferation and the enhancement of depressive / anxiety-like behaviors.

[0220]

[0221] Example 3. Acceleration of α-syn pathology induced by chronic restraint stress following injection of preformed α-synuclein fibrils.

[0222]

[0223] We aimed to explore the combined effect of CRS and PFF on the proliferation of pathological α-syn. Using immunohistochemical analysis of pSer129 α-syn, a marker of Lewy neurites (LNs) and Lewy bodies (LBs), we observed significant pSer129 α-syn-positive inclusions throughout the brain of A53T TG mice one month after injection. In particular, A53T TG mice injected with PFF and exposed to CRS had more widespread inclusions, particularly in the cingulate cortex (Cg), anterior cingulate cortex (PrL), motor cortex (M1), insular cortex (Ins), basolateral limbic nucleus (BLA), lateral entorhinal cortex (Ect), and medial entorhinal cortex (Ent) regions (Fig. 2a). These results indicate that PFF injection into the striatum induces marked α-syn pathology, and that this pathology is further exacerbated by CRS in specific brain regions.

[0224] CRS is known to induce microgliosis and astrogliosis in the brain. Therefore, to analyze changes in microglia and astrocytes, we stained for the microglial marker Iba-1 and the astroglial reactivity marker GFAP to analyze micro / astrogliosis. As a result, we confirmed that CRS significantly activated microglia and was associated with α-syn proliferation in PFF-injected mice (Fig. 2b). Similarly, GFAP expression was significantly increased by PFF injection, and CRS exposure further exacerbated astrogliosis and α-syn proliferation in these mice (Fig. 2c).

[0225] In summary, PFF / CRS-treated A53T TG mice exhibit accelerated α-syn pathology and an accompanying neuroinflammatory phenotype.

[0226] In the present invention, when evaluating the degeneration of dopaminergic neurons, CRS or PFF injection alone did not significantly change the tyrosine hydroxylase (TH) intensity of the striatum or the number of TH-positive cells in the substantia nigra. Nevertheless, CRS / PFF-treated A53T TG mice showed decreased TH intensity in the striatum and a decreased number of TH-positive cells in the substantia nigra, suggesting that CRS may contribute to the progressive nigrostriatal dopaminergic neuron degeneration by enhancing the initial seeding and pathological spreading of α-syn (Figs. 2d-2f). In addition, Fluoro-Jade C (FJC), a marker of neurodegeneration, increased in the substantia nigra of PFF-injected mice, and the number of these cells was even higher in CRS / PFF-treated mice (data not shown). These results suggest that enhanced initial formation and rapid pathological proliferation of α-syn induced by CRS may lead to progressive nigrostriatal dopaminergic neuron degeneration.

[0227]

[0228] Example 4. Correlation between c-Fos induction and increased α-syn diffusion induced by CRS.

[0229]

[0230] To investigate whether altered neural activity in various brain regions of PFF / CRS-treated A53T TG mice may underlie the development of α-syn pathology and behavioral deficits, we assessed c-Fos expression across experimental groups.

[0231] CRS was found to induce c-Fos expression in various brain regions, including the cingulate cortex, motor / sensory cortex, amygdala, hypothalamus, insular cortex, and substantia nigra (Fig. 3a, 3b). Notably, c-Fos induction was more pronounced in the PFF / CRS group, suggesting a potential association between depressive / anxiety-like behavior and α-syn diffusion. Interestingly, PFF injection alone induced c-Fos expression in various brain regions, including the cingulate cortex, motor / sensory cortex, entorhinal cortex, amygdala, and lateral / periorbital / medial cortices. Some neuronal cell bodies showing pSer129 α-synuclein positivity coexisted with c-Fos induction (Fig. 3c). Moreover, a positive correlation was observed between the degree of pSer129 α-syn detection and the degree of c-Fos induction (Fig. 3d). These results suggest that CRS and PFF injections can synergistically increase neural activity in various brain regions.

[0232]

[0233] Example 5. Improvement of depressive behavior by α-syn diffusion through c-Fos inhibition.

[0234]

[0235] To determine the role of c-Fos induction in the interaction between depression and α-synuclein proliferation, we administered T-5224, a c-Fos inhibitor that targets transcription regulated by AP-1 (Fig. 4a).

[0236] After T-5224 administration, the OFT assessed general activity levels and exploratory behaviors, revealing no significant differences between CRS and NCRS (Fig. 4b). However, in the EPM, T-5224 successfully reversed the depressive / anxiety-like behavior observed in PFF / CRS-treated mice (Fig. 4c). Similarly, in the FST, T-5224 reversed the increased inactivity duration and decreased hyperactivity duration observed in PFF / CRS treatment (Fig. 4d). In the pole test, T-5224 effectively reversed the slightly slower fall time observed in A53T TG mice exposed to CRS. These data indicate that c-Fos inhibitors are effective in alleviating depressive behaviors exacerbated by α-syn diffusion (Fig. 4e). Furthermore, IHC analysis demonstrated that T-5224 ameliorated the increased α-syn diffusion induced by CRS. Interestingly, α-synuclein diffusion induced by PFF injection was also reduced by c-Fos inhibition (Fig. 4f). This suggests that T-5224 effectively mitigates α-synuclein pathology induced under both CRS / PFF and PFF alone conditions. These findings support the role of c-Fos induction in the complex interplay between depression and α-synuclein diffusion.

[0237]

[0238] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0239]

[0240] [National Research and Development Project Supporting This Invention]

[0241] [Project ID] 1711191592

[0242] [Assignment Number] 2019R1A5A2026045

[0243] [Ministry Name] Ministry of Science and ICT

[0244] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0245] [Research Project Name] Group Research Support - Leading Research Center Support Project (MRC)

[0246] [Research Project Name] Brain Disease Convergence Research Center

[0247] [Name of Project Performing Organization] Ajou University

[0248] Research Period: June 1, 2019 - February 28, 2026

Claims

1. A pharmaceutical composition for preventing or treating Parkinson's disease, comprising a c-Fos inhibitor as an active ingredient.

2. A pharmaceutical composition for preventing or treating Parkinson's disease, characterized in that the c-Fos inhibitor in claim 1 is T-5224 or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition for preventing or treating Parkinson's disease, characterized in that the composition restores non-motor impairment in the early stage of Parkinson's disease, in accordance with paragraph 1.

4. A pharmaceutical composition for preventing or treating Parkinson's disease, characterized in that the non-motor disorder in the third paragraph is a depressive / anxiety disorder.

5. A pharmaceutical composition for preventing or treating Parkinson's disease, characterized in that the composition is administered to a patient in whom α-synuclein diffusion is observed and / or a pSer129 α-synuclein positive patient in paragraph 1.

6. A pharmaceutical composition for preventing or treating Parkinson's disease, characterized in that the composition is administered to a patient in whom astrocytosis and / or microgliosis is observed in the first paragraph.

7. A pharmaceutical composition for preventing or treating Parkinson's disease, wherein in paragraph 6, the astrocytosis is characterized by an increase in the expression of GFAP compared to the normal control group, and the microgliosis is characterized by an increase in the expression of Iba-1 compared to the normal control group.

8. A pharmaceutical composition for preventing or treating Parkinson's disease, characterized in that the composition is administered to a patient characterized by a decrease in tyrosine hydroxylation (TH) positive neurons in the substantia nigra, in accordance with paragraph 1.

9. A method for providing information for determining a treatment for Parkinson's disease, the method comprising: (a) A step of identifying one or more of the following neuropathological signs in the brain tissue of a patient suspected of having Parkinson's disease: (i) α-syn diffusion; (ii) Confirmation of astrocytosis and / or microgliosis; (iii) identification of pSer129 α-synuclein positive neuronal cell bodies; and (iv) identification of tyrosine hydroxylation (TH)-positive neuronal cell bodies in the substantia nigra (SN); and (b) A method for providing information, comprising a step of determining administration of a c-Fos inhibitor as a treatment for Parkinson's disease when any one or more of the above neuropathological signs are confirmed.

10. A method for providing information, characterized in that the c-Fos inhibitor in claim 9 is T-5224 or a pharmaceutically acceptable salt thereof.

11. A method for providing information, wherein the patient suspected of having Parkinson's disease in paragraph 9 exhibits non-motor disorder symptoms in the early stages of Parkinson's disease.

12. A method for providing information, characterized in that, in paragraph 9, the patient suspected of having Parkinson's disease has not yet developed motor disorder due to Parkinson's disease or is in the early stage of onset of motor disorder.

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