KR20220066602A - Pharmaceutical composition for preventing or treating neurodegenerative disease comprising carboplatin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases, particularly, FUS-related neurodegenerative diseases, comprising carboplatin as an active ingredient. The composition of the present invention inhibits FUS-induced apoptotic cell death and NMJ defects in neurons by increasing the expression of GSTO1 and GstO2, and thus can be used as a promising therapeutic agent for FUS-related neurodegenerative diseases.

Description

Pharmaceutical composition for preventing or treating neurodegenerative diseases comprising carboplatin {PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING NEURODEGENERATIVE DISEASE COMPRISING CARBOPLATIN}

The present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising kiboplatin as an active ingredient.

Protein aggregates are present in age-related neurodegenerative diseases (ND), such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD) and amyotrophic lateral sclerosis. (amyotrophic lateral sclerosis, ALS) is characteristically found.

Fused in Sarcoma (FUS) is an RNA/DNA binding protein, and it is known that the N-terminus of FUS is associated with transcriptional activity and the C-terminus is involved in protein and RNA binding. In addition, it has been confirmed that FUS has a recognition site for the transcription factors AP2, GCF, and Sp1, and it has been recently reported that several mutations of FUS were found in patients with Lou Gehrig's disease or frontotemporal dementia. In relation to these mutations, interestingly, it is reported that the FUS mutant protein is located in the stress granule to form an aggregate after it escapes from its original location in the nucleus and exits the nucleus.

Meanwhile, FUS mutations have been identified in most ALS patients. FUS ^P525L mutant shows cytoplasmic mislocalization and is associated with acute FUS-induced-ALS, FUS wild-type and its variant FUS ^P525L are known to exhibit the same phenotypes such as rough eyes and reduced motility in Drosophila.

However, studies on the mechanisms related to the aberrant location and aggregate accumulation of these FUS mutant proteins found in patients with ALS or frontotemporal dementia are scarcely known, and there is no treatment based thereon either.

Republic of Korea Patent Publication No. 10-2020-0053909

The inventors of the present invention found that carboplatin, which is mainly used as an anticancer agent, can be applied to the treatment of FUS-related neurodegenerative diseases by increasing the expression of GSTO1 (omega class glutathione transferase 1) and GstO2 (omega class glutathione transferase 2). The present invention was completed.

Accordingly, the present invention is to provide a pharmaceutical composition and functional health food for preventing or treating neurodegenerative diseases containing carboplatin as an active ingredient, and a method for preventing or treating neurodegenerative diseases using the same.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising carboplatin as an active ingredient.

In addition, the present invention provides a functional health food for preventing or improving neurodegenerative diseases comprising carboplatin as an active ingredient.

In addition, the present invention provides a method for preventing or treating a neurodegenerative disease comprising administering to an individual the pharmaceutical composition for the prevention or treatment of a neurodegenerative disease according to the present invention.

The composition of the present invention can be used as a promising therapeutic agent for FUS-related neurodegenerative diseases by increasing the expression of GSTO1 and GstO2 to suppress FUS-induced apoptotic cell death and NMJ defects in neurons. It may provide a promising treatment strategy for temporal lobe dementia.

1 shows the effect of improving the motor performance of FUS-induced larvae by carboplatin treatment.
2 shows the results of immunoblot analysis of brain extracts from FUS-expressing adult flies.
3 shows the ratio of insoluble/soluble FUS in FUS-expressing flies by carboplatin treatment.
4 shows the results of whole brain-immunostaining of FUS-expressing flies.
Figure 5a shows the expression of GstO2 protein in Drosophila exposed to hypoxia.
Figure 5b shows GstO2 expression levels in extracts from the heads of carboplatin-treated FUS-expressing flies.
Figure 5c shows that FUS-induced apoptotic cell death and NMJ defects are inhibited in fly neurons by carboplatin treatment.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of well-known techniques known to those skilled in the art may be omitted. In addition, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs.

Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

The present invention relates to a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases and a health functional food comprising carboplatin as an active ingredient.

The carboplatin (carboplatin) is a form of the anticancer drug cisplatin, and is mainly used in combination with other drugs for the treatment of advanced ovarian cancer and advanced metastatic non-small cell lung cancer.

The carboplatin may increase the expression of omega class glutathione transferase 1 (GSTO1) or omega class glutathione transferase 2 (GstO2), thereby inhibiting FUS-related pathogenesis.

In the present invention, the neurodegenerative disease may be a FUS-related neurodegenerative disease, but is not limited thereto. The neurodegenerative diseases include stroke, stroke, memory loss, memory impairment, dementia, forgetfulness, Parkinson's disease, Alzheimer's disease, Pick's disease, Creutzfeld-Kacob's disease, Huntington's disease, amyotrophic lateral sclerosis (amyotrophic) It may include lateral sclerosis (ALS), frontotemporal dementia (FTD), Lou Gehrig's disease, and the like, and may include, for example, amyotrophic lateral sclerosis or frontotemporal dementia, but is not limited thereto.

In the present invention, "treatment" means, unless otherwise stated, a disease or condition to which the above term applies, or one or more symptoms of the disease or condition to reverse, alleviate, inhibit the progression, or prevent the disease or condition. and, as used herein, the term treatment refers to the act of treating when “treating” is defined as above. Accordingly, "treatment" or "therapeutic regimen" of a neurodegenerative disease in a mammal may include one or more of the following:

(1) inhibit the development of neurodegenerative diseases;

(2) prevent the spread of neurodegenerative diseases;

(3) alleviating neurodegenerative diseases;

(4) preventing the recurrence of neurodegenerative diseases; and

(5) alleviating the symptoms of neurodegenerative diseases (palliating)

The composition of the present invention comprising the carboplatin as an active ingredient may be used as a pharmaceutical composition or a food composition.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, a lubricant, A lubricant or flavoring agent may be used.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectables. For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with an orally, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or required, suitable binders, lubricants, disintegrants and color-developers may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, by using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by an appropriate method in the art, it can be preferably formulated according to each disease or component.

Carboplatin of the present invention may be included in an amount of 0.001 to 20% by weight based on the total weight of the composition.

The composition of the present invention may also be a food composition, and in addition to containing carboplatin as an active ingredient, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional food composition. have.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-mentioned flavoring agents can advantageously use natural flavoring agents (Taumatine), stevia extract (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements. There is this.

In addition, the food composition contains various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages.

The present invention also provides a health functional food for improving neurodegenerative diseases comprising carboplatin as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of improving neurodegenerative diseases.

In the present invention, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act, and provides nutrients for the structure and function of the human body. It refers to ingestion for the purpose of obtaining useful effects for health purposes, such as controlling or physiological effects.

The health functional food of the present invention may contain normal food additives, and unless otherwise specified, whether it is suitable as a food additive is related to the item according to the general rules and general test method of food additives approved by the Food and Drug Administration. It is judged according to the standards and standards.

The items listed in the "Food Additives Codex" include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodles-added alkalis, preservatives, and tar dye preparations.

For example, in the form of a tablet health functional food, a mixture of carboplatin, the active ingredient of the present invention, mixed with excipients, binders, disintegrants and other additives is granulated in a conventional manner, and then a lubricant is added and compressed. Or, the mixture may be directly compression molded. In addition, the health functional food in the form of tablets may contain a corrosive agent and the like, if necessary.

Among health functional foods in the form of capsules, hard capsules can be prepared by filling a conventional hard capsule with a mixture of carboplatin, the active ingredient of the present invention, mixed with additives such as excipients, and soft capsules include carboplatin as an excipient, etc. It can be prepared by filling the mixture mixed with the additives of the capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

A health functional food in the form of a ring can be prepared by molding a mixture of carboplatin, the active ingredient of the present invention, excipients, binders, disintegrants, etc., by a known method, and, if necessary, with sucrose or other coating agents. It can be peeled off, or the surface can be coated with a material such as starch or talc.

The health functional food in granular form can be prepared in granular form by a conventionally known method by mixing a mixture of carboplatin, the active ingredient of the present invention, excipients, binders, disintegrants, etc. and the like.

The health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.

The present invention also provides a method for preventing or treating neurodegenerative diseases, comprising administering to an individual carboplatin or a pharmaceutical composition containing carboplatin as an active ingredient.

The subject may be a mammal, for example, a human, but is not limited thereto.

As used herein, the term “therapeutically effective amount” refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human as conceived by a researcher, veterinarian, physician or other clinician, which an amount that induces amelioration of the symptoms of the disease or disorder being treated. It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration for the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other components contained in the composition, the type of formulation, and the age, weight, and general health of the patient It can be adjusted according to various factors including state, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. In the treatment method of the present invention, in the case of adults, it is preferable to administer the serrated mother extract of the present invention once or several times a day, in a dose of 0.01 mg/kg to 100 mg/kg.

In the treatment method of the present invention, the composition comprising carboplatin of the present invention as an active ingredient is administered via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. It can be administered in a conventional manner.

Hereinafter, the present invention will be described in more detail by way of Examples. The following examples are merely for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.

<Example>

Example 1

transgenic flies

The UAS - FUS line was donated by the University of Pennsylvania. The pan-neuron driver elav ^3A - Gal4 and eye-specific driver GMR-Gal4 lines were obtained from the Bloomington Drosophila Stock Center. Depending on the genetic background, W ¹¹¹⁸ flies were used as controls.

Drug Supply Analysis

The thawed standard fly diet was mixed with various concentrations of carboplatin (LKT Laboratories). An equal volume of distilled water was used as an excipient control for carboplatin. Parent flies were bred on a diet supplemented with carboplatin. Tertiary-young larvae were selected and used in the experiment.

motor activity

For larval crawling analysis, tertiary-young larvae were washed with PBS to remove residual food medium. Larvae were dried on clean clean filter paper and placed on 2% grape juice-agar Petri plates. Larvae of each gene line were allowed to crawl for 90 seconds. To quantify the crawling behavior of the larvae, a line was drawn by tracking the larva and the distance was measured using ImageJ software. Results from at least 10 larvae were averaged for each transgenic line.

Immunoblot analysis

For Western blot analysis, ten fly heads were homogenized and prepared in LDS sample buffer (Invitrogen) to prepare protein extracts. The total protein extract was separated using a 4-12% gradient SDS-PAGE gel and transferred to a PVDF membrane (Millipore). The membrane was blocked with TBS (Tris-buffered saline) containing 4% nonfat dry milk or 4% bovine serum albumin (BSA) for 1 hour and incubated with primary antibody at 4°C for 12 hours. The primary antibodies used were: rabbit anti-FUS (1:1000; Bethyl Laboratories); rabbit anti- Drosophila GstO2 (1:1000); and rabbit anti-β-actin (1:4000; Cell Signaling). Analytes were washed with TBA containing 0.1% Tween-20 (TBST) and incubated with secondary antibody. Primary antibodies were detected with the following horseradish peroxidase (HRP)-conjugated secondary antibodies: goat anti-rabbit IgG HRP conjugate and goat anti-mouse IgG HRP conjugate (1:2000; Millipore). It was detected using the ECL-Plus kit (Amersham).

Protein solubility assay

With minor modifications to the known protocol, total proteins were fractionated according to their solubility. The heads of 12 flies were homogenized in SDS-free lysis buffer (50 mm Tris-HCl, 150 mm NaCl, 5 mM EDTA, 0.1% NP-40, and 10% glycerol, pH 7.5). The homogenized sample was centrifuged at 100,000× g for 30 minutes at 4° C. and the supernatant was collected as a soluble fraction. The remaining pellet was further extracted by treatment with 50 μl of 2× SDS loading buffer with 2% SDS, sonicated, and heated at 95° C. for 10 minutes. The supernatant was then collected as an insoluble fraction.

Whole-brain immunostaining

The brains of tertiary-young larvae were fixed with 4% formaldehyde in fixation buffer (100 mm PIPES, 1 mm EGTA, 1% Triton X-100, and 2 mm MgSO ₄ , pH6.9) for 25 min and 10 mg/ml Blocked with wash buffer containing BSA (50 mm Tris-HCl, 150 mm NaCl, 0.1% Triton X-100, and 0.5 mg/ml BSA, pH 6.8) at 25° C. for 1 hour. The brains were then incubated with primary antibody diluted in blocking buffer at 4°C for 12 h. The antibodies used were as follows: anti-cleaved caspase-3 (1:500; Cell Signaling). Samples were then incubated with h Alexa 488-conjugated secondary antibody (1:200; Invitrogen), and DAPI (1:500; Sigma-Aldrich). The brain was washed 3 times for 10 minutes each with washing buffer, and added to SlowFade ^TM Gold antifade reagent (Invitrogen). All images were acquired with a Carl Zeiss confocal microscope (LSM710).

Hypoxia exposure

The flies were placed in a closed chamber at 25° C. supplied with 5% oxygen/95% nitrogen.

immunohistochemistry

To analyze the neuromuscular junction (NMJ) of Drosophila, tertiary-young larvae were excised in PBS and fixed in 4% formaldehyde in PBS for 15 minutes. Samples were washed 3 times with PBS containing 0.1% Triton X-100 for 10 minutes and then blocked with 5% BSA in PBST. Then, the sample was incubated with a primary antibody at 4° C. for 12 hours. As a primary antigen, FITC-conjugated anti-HRP (Jackson Immuno Research Laboratories) was used at 1:150. Larvae preparations were fixed in SlowFade ^™ Gold antifade reagent (Invitrogen). All images were acquired from a Leica TCS SP5 AOBS confocal microscope.

<Result>

Recently, several research results have been published that inactivation of GSTO1 can increase the sensitivity to various diseases such as neurodegenerative diseases. Based on the discovery that Drosophila GstO2 downregulates disease progression in extracts from the head of a FUS-induced model, the present invention focused on activating or inducing GSTO1 as a neuroprotective effector. In addition, several factors that can increase GSTO1-specific expression have recently been discovered. Transient hypoxia is a symptom that upregulates GSTO-1 expression to increase mitochondrial ROS levels and prolong lifespan in C. elegans . Exposure of breast cancer cells to carboplatin induces hypoxia-inducible factor-dependent expression of GSTO1. Hypoxia exposure did not alter GstO2 protein expression in Drosophila (Fig. 5a). Therefore, to confirm that pharmacological restoration of GstO2 expression could solve the FUS-induced toxicity problem observed in the nervous system of FUS-expressing flies, FUS-expressing flies were treated with carboplatin. Drosophila larvae expressing FUS were continuously fed a carboplatin-supplemented diet during larval stage. Carboplatin treatment (2 mg/ml) significantly increased the GstO2 expression level in extracts from the heads of FUS-expressing flies ( FIG. 5b ). It was found that the dyskinesias of FUS-induced larvae were significantly suppressed by carboplatin treatment in a dose-dependent manner ( FIG. 1 ). Immunoblot analysis of brain extracts from FUS-expressing adult flies indicated that carboplatin treatment did not affect FUS levels ( FIG. 2 ). Then, the effect of carboplatin on FUS solubility was confirmed. When soluble and insoluble FUS were quantified, it was confirmed that the ratio of insoluble/soluble FUS in FUS-expressing flies treated with carboplatin was significantly reduced by 50% or more compared to untreated flies ( FIG. 3 ). In addition, it was confirmed that carboplatin treatment inhibited FUS-induced apoptotic cell death and NMJ defects in fly neurons ( FIGS. 4 and 5c ). Taken together, these results suggest that reduced GSTO1/GstO2 activity influences FUS-related pathogenesis and that modulation of GSTO1/GstO2 activity may be a promising therapeutic strategy for FUS-related neurodegenerative diseases such as ALS and FTD. .

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (6)

A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising carboplatin as an active ingredient. The method of claim 1,
The neurodegenerative disease is a FUS-related neurodegenerative disease, a pharmaceutical composition for the prevention or treatment of a neurodegenerative disease. The method of claim 1,
The degenerative neurological disease is stroke, stroke, memory loss, memory impairment, dementia, forgetfulness, Parkinson's disease, Alzheimer's disease, Pick's disease, Creutzfeld-Kacob disease, Huntington's disease, amyotrophic lateral sclerosis (amyotrophic) A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases selected from the group consisting of lateral sclerosis; ALS), frontotemporal dementia (FTD), and Lou Gehrig's disease. The method of claim 1,
The carboplatin is a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases that increases the expression of GSTO1 (omega class glutathione transferase 1) or GstO2 (omega class glutathione transferase 2). A health functional food for the prevention or improvement of neurodegenerative diseases containing carboplatin as an active ingredient. [Claim 5] A method for preventing or treating neurodegenerative diseases comprising administering the composition of any one of claims 1 to 4 to a subject other than a human.

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