KR101848231B1 - Compositions for preventing, improving or treating brain neurological disease comprising triiodo L thyronine, L thyroxine or its salt - Google Patents

Abstract

The present invention provides a composition for preventing, ameliorating or treating a cerebral neurological disease caused by dopamine deficiency comprising triiodothyronine and thyroxine or a salt thereof. According to the present invention, triiodothyronine, thyroxine, or a salt thereof remarkably enhances the expression and proliferation of dopamine neurons, thereby exhibiting an excellent effect for the prevention and treatment of brain nervous system diseases caused by dopamine deficiency such as Parkinson's disease.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing, ameliorating or treating a cerebral neurological disease, including, for example, thiazolidinediones, triiodothyronine, thyroxine or a salt thereof.

The present invention relates to a composition for preventing, ameliorating or treating a neurological disorder caused by dopamine deficiency comprising triiodothyronine (T ₃ ), thyroxine (T ₄ ) or a salt thereof.

Parkinson's disease is a chronic progressive degenerative disease of the cranial nervous system characterized by stable tremor, stiffness, gait and postural instability. Neuropathologic characteristics of dopaminergic neurons in the brain are gradually disappearing. Patients with Parkinson's disease are estimated to be approximately 1% of the population at approximately 60 years of age or older.

As drugs for the treatment of Parkinson's disease, dopamine receptor promoters and l-dopa preparations have been used. However, the dopamine receptor promoter has a low therapeutic efficiency, and the long-term effect of the L-dopa agent gradually deteriorates, resulting in side effects such as twisting of the body and abnormal movement in which the hands or feet move spontaneously. In addition to drug therapy, high frequency nerve stimulation such as radiofrequency ablation and deep brain stimulation is also performed, but it requires invasive surgery and is costly. In addition, there is an attempt to apply stem cell therapy, but the effect has not been verified as a very early stage, and the procedure is difficult. Accordingly, more effective drug development for treating Parkinson's disease is required.

Nurr1 (NR4A2) is a steroid receptor type transcription factor that is expressed in the midbrain and is a crucial factor in the development of neuronal dopamine neurons. Nurr1 is expressed not only in the midbrain, but also in the midbrain dopamine neurons of the adult brain. It has been reported that sustained expression of this transcription factor is crucial to the maintenance of the dopaminergic phenotype [3] and survival [4] [5]. In addition, decreased levels of Nurr1 or genetic deformation were observed in adult midbrain dopamine pathology [6] [7].

It has been reported that dopamine neurons are not generated in the midbrain of Nurr1-knockout mice, and that Nurr1 expression is decreased in elderly or Parkinson's disease autopsy. This means that Nurr1 plays a specific and essential role in the development of neuronal dopaminergic neurons, and thus a substance that regulates Nurr1 expression can be developed as a therapeutic agent for Parkinson's disease.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

One. Zetterstrom, RH et al., Science 276, 248-250, 1997 2. Saucedo-Cardenas, O. et al., Proc. Natl. Acad. Sci. USA 95, 4013-4018, 1998 3. Eells, JB, et al., Behav. Brain Res. 136, 267-275, 2002 4. Wallen, A. et al., Exp. Cell Res. 253, 737-746, 1999 5. Sousa, KM et al., Stem cells 25, 511-519, 2007 6. Jankovic, J. et al., Prog. Neurobiol. 77, 128-138, 2005 7. Chu, Y. et al., J. Comp. Neurol. 494, 495-514, 2006

The present inventors have made efforts to develop a drug for the treatment of neurological diseases caused by dopamine deficiency such as Parkinson's disease. As a result, it was confirmed that triiodothyronine and thyroxine, which are used as a therapeutic agent for thyroid disease, have a therapeutic effect on brain nervous system diseases caused by dopamine deficiency such as Parkinson's disease by promoting the expression and proliferation of dopaminergic neurons , Thereby completing the present invention.

It is therefore an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of neurological diseases caused by dopamine deficiency.

It is another object of the present invention to provide a health functional food composition for preventing or ameliorating diseases of the brain caused by dopamine deficiency.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cerebral neurological diseases caused by dopamine deficiency comprising triiodothyronine, thyroxine or a pharmaceutically acceptable salt thereof as an active ingredient.

The present inventors have made efforts to develop a drug for the treatment of neurological diseases caused by dopamine deficiency such as Parkinson's disease. As a result, it was confirmed that triiodothyronine and thyroxine, which are used as therapeutic agents for thyroid disease, promote the expression and proliferation of dopaminergic neurons and show therapeutic effects on brain nervous system diseases caused by dopamine deficiency such as Parkinson's disease.

As used herein, the term " prophylactic " means to inhibit or delay the progression of the neurological diseases caused by dopamine deficiency by the administration of the composition of the present invention, and the terms & It means that the symptoms caused by the neurological diseases caused by dopamine deficiency are improved or changed.

As used herein, the term " cranial nervous system disease due to dopamine deficiency " refers to a disease caused by a decrease in dopamine deficiency or dopamine levels in the central nervous system or a worsening of symptoms. A typical cause of dopamine deficiency and dopamine level decline is the loss of dopamine neurons.

As used herein, the term " dopamine neuron " or " dopamine neuron " refers to a neuron expressing Tyrosine Hydroxylase (TH).

According to one embodiment of the present invention, the neurological diseases caused by dopamine deficiency include Parkinson's disease, neurocognitive dysfunction, attention deficit hyperactivity disorder, restless legs syndrome and anxiety.

In one particular example, the neurological disorder due to dopamine deficiency is Parkinson ' s disease. Parkinson's disease is a typical cerebral neurological disease caused by a decrease in dopamine level and is characterized by stable tremor, stiffness, slowing of movement (slowing of movement) and postural instability. In addition, Parkinson's disease may be caused by other diseases such as autonomic nervous system symptoms, neuropsychiatric symptoms, cognitive dysfunctions, sleep disorders, pain, fatigue, smell disorders, gastrointestinal disorders, salivation, difficulty in swallowing, constipation, orthostatic hypotension, Of the patients. The composition of the present invention promotes the expression and proliferation of dopamine neurons and can be applied to the treatment of Parkinson's disease with various clinical symptoms as described above.

According to one embodiment of the present invention, the triiodothyronine, thyroxine, increases the dopamine level of the central nervous system.

According to one embodiment of the present invention, the triiodothyronine, thyroxine, promotes the proliferation of dopamine neurons or the differentiation of neural progenitor cells into dopamine neurons.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable salt of thiothiodin, thyroxine.

The term " pharmaceutically acceptable salt " as used herein means a formulation of a compound that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. The pharmaceutical salt may be prepared by dissolving the compound of the present invention in an organic solvent such as mineral acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid, sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, or p-toluenesulfonic acid, tartaric acid, formic acid, With an organic carboxylic acid such as acetic acid, acetic acid, benzenesulfonic acid, benzenesulfonic acid, benzenesulfonic acid, rosacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid and the like. The acid addition salt according to the present invention may be prepared by a conventional method, for example, by dissolving triiodothyronine or thyroxine in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, Followed by filtration and drying, or may be prepared by distillation of the solvent and excess acid under reduced pressure, followed by drying or crystallization in an organic solvent.

Further, the pharmaceutically acceptable salt of the present invention can be prepared by reacting triiodothyronine or thyroxine with a base to produce an alkali metal salt such as an ammonium salt, sodium or potassium salt, a salt such as an alkaline earth metal salt such as calcium or magnesium salt, For example, salts of organic bases such as triethylamine, triethylamine, triethylamine, triethylamine, chlorohexylamine, N-methyl-D-glucamine, tris (hydroxymethyl) methylamine and amino acid salts such as arginine and lysine.

According to one embodiment of the present invention, the pharmaceutically acceptable salts of the present invention are triiodothyronine sodium salt of the following formula (1) and thioxine sodium salt of the formula (2).

The scope of the present invention encompasses the triiodothyronine, thyroxine and pharmaceutically acceptable salts thereof as well as the solvates, hydrates and stereoisomers thereof which can be prepared therefrom. Also, derivatives of triiodothyronine and thyroxine are included in the scope of the present invention as long as they have an effect of promoting the expression or proliferation of dopamine neurons.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, .

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. Here, the formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, there is provided a health functional food composition for preventing or ameliorating a brain disease caused by dopamine deficiency comprising triiodothyronine, thyroxine or a salt thereof.

Since the health functional food composition of the present invention includes the components of the above-mentioned pharmaceutical composition, common description therebetween is omitted in order to avoid excessive complexity of the specification.

The health functional food composition of the present invention may contain, as an active ingredient, triiodothyronine, thyroxine or a salt thereof, as well as a component that is ordinarily added in food production, for example, a protein, a carbohydrate, a fat, Flavoring agents, flavoring agents and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings.

In addition to the above, the health functional food composition of the present invention can be used as a nutritional supplement, vitamins, minerals (electrolytes), a dietary ingredient, a flavoring agent such as a synthetic flavoring agent and a natural flavoring agent, a coloring agent and a thickening agent And carbonates used in alginic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonated drinks and the like.

Considering the easy accessibility to food, the health functional food composition of the present invention is very useful for prevention and improvement of brain nervous system diseases due to dopamine deficiency.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a composition for preventing, ameliorating or treating a cerebral neurological disease caused by dopamine deficiency comprising triiodothyronine, thyroxine or a salt thereof.

(Ii) According to the present invention, triiodothyronine, thyroxine or a salt thereof remarkably enhances the expression and proliferation of dopamine neurons, thereby exhibiting an excellent effect for the prevention and treatment of cerebral neurological diseases caused by dopamine deficiency such as Parkinson's disease.

FIG. 1 shows that expression of tyrosine hydroxylase (TH), a marker of dopamine neuron, was increased in a concentration-dependent manner when Nurr1 gene was expressed in rat neural stem cells and fetal bovine serum (FBS) Show.
FIG. 2 shows that when the Nurr1 gene was expressed in rat neural stem cells and treated with 0.01% FBS, the increase in TH expression was markedly increased as the differentiation date was lengthened as compared with the control.
Fig. 3 shows that when the Nurr1 gene was expressed in rat neural stem cells and treated with 0.01% FBS, the mRNA and protein of TH and Nurr1 increased depending on the differentiation date.
FIG. 4 shows a comparison of the expression of the Nurr1 gene in rat neural stem cells and the treatment with a mixture of 18 candidate substances (Table 1) contained in the FBS component, similar to the treatment with 0.01% FBS in a 0.1% And the expression of TH was increased.
FIG. 5 shows the results of measuring the TH promoter activity after expressing the Nurr1 gene in rat neural stem cells and treating each of the 18 candidate substances, one by one, in order to discover the component exhibiting the most effective effect in the mixture of Table 1.
FIG. 6 shows the expression of TH when the Nurr1 gene is expressed in rat neural stem cells and the 18 candidate substances are treated separately, one by one.
FIG. 7 shows that the Nurr1 gene was expressed in rat neural stem cells 4, 5, and 16, which were judged to be effective among them, and the TH promoter activity was measured after removing Nurr1 gene from 18 candidate substances, Show the results.
FIG. 8 shows changes in the expression of TH when the Nurr1 gene is expressed in rat neural stem cells and Candidates 4, 5 and 16 are removed from 18 candidate substances, one by one, or in various combinations and treated.
Figure 9 and expressing Nurr1 gene in rat neural stem cells, tri iodine tea Ronin (T ₃₎ the case of processing by each concentration, concentration-dependent increase in the expression of TH significantly and, 6 pg / ㎖ than T ₃ is 0.01 during processing % FBS or a 0.1% mixture (chemical mixture).
FIG. 10 shows that the TH promoter activity was increased in a concentration-dependent manner when the Nurr1 gene was expressed in rat neural stem cells and T ₃ was treated at different concentrations.
FIG. 11 shows the results of differentiation of rat fetal mesencephalic neuronal progenitor cells and the treatment of T ₃ and thyroxine (T ₄ ), respectively, as in the case of artificially expressing Nurr1 in rat cerebral cortical neuronal progenitor cells And that the expression of TH was increased in a concentration-dependent manner at the time of administration of T ₃ and T ₄ .
FIG. 12 shows that expression of Nurr1 gene in human neuroblastoma cell line and treatment of T ₃ and T ₄ , respectively, increased expression of TH and TH promoter activity.
FIG. 13 also shows that the TH promoter activity was also increased when T ₃ and T ₄ were respectively treated with human neural stem cell line expressing Nurr 1 itself.
FIG. 14 shows that TH expression was increased when T ₃ and T ₄ were treated respectively with cells differentiated directly into neural progenitor cells of a human embryonic stem cell line.
After 15 is induced dopaminergic neuron damage treating the neurotoxic substance such as and expressing Nurr1 gene in stem cells neural rats, 6-hydroxydopamine (6-OHDA ) and free radicals (H ₂ O ₂₎ sinkin T ₃ and T ₄ , respectively, show that TH expression is restored.
FIG. 16 shows that treatment of 6-OHDA with rat prenatal cerebral neuronal progenitor cells induced dopaminergic neuronal damage and then treated with T ₃ and T ₄ , respectively, to restore TH expression.
FIG. 17 shows that treatment of T ₃ and T ₄ after treatment with 6-OHDA to induce dopaminergic neuronal cell damage in cells in which human embryonic stem cell lines were directly differentiated into neural progenitor cells restored TH expression.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  One : Rat  Effects of fetal dopamine neurons on the expression and differentiation Effect of FBS  Confirm

The retrovirus used in the present invention was prepared as follows. The 292GPG cells used for the retrovirus production were maintained in DMEM, 10% FBS, 2 mM L-Glutamine, 1% penicillin-streptomycin in a medium supplemented with 1 μg / ml doxycycline, 2 μg / ml puromycin and 0.3 mg / ml G418. The day before transfection, 1.8 x ¹⁰⁶ / well of 293GPG cells were placed on a ^6- well plate. 293GPG cells were cultured in DMEM medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and 2 mM L-glutamine (Sigma) supplemented at 5% CO _{2 and} 37 ° C until the next day. Two tubes were prepared, and 250 μl of Opti-MEM (Gibco) was added with 4 μg of retroviral vector plasmid, and the other one was vortexed with 250 μl of Opti-MEM and 10 μl of Lipofectamine 2000 ™ (Invitrogen) I have. Five minutes later, the two tubes were slowly mixed using a pipet. After incubation for 20 minutes at room temperature, the medium of 293 GPG cells laid on the previous day was removed, washed once with Opti-MEM medium, and then added to 293 GPG cells. After incubation for 4 hours at 37 ° C in 5% CO _2, the medium was removed and replaced with 1.2 ml of DMEM, 10% FBS, 2 mM L-Glutamine and 1% penicillin-streptomycin medium. The next day, the culture medium was replaced with 1.2 ml of DMEM, 10% FBS, 2 mM L-Glutamine and 1% penicillin-streptomycin medium, and polybrene (hexadimethrine bromide: Sigma H9268) was obtained at 24 hour intervals until the 293GPG cells were killed. 2 ㎍ / ㎖, stored at 70 ℃, and dissolved before retroviral transduction.

Cerebral cortical neural stem cells were isolated from rat embryos (14.5 days of age) and infected with retroviruses expressing the Nurr1 gene in the presence of bFGF. bFGF was induced to induce the differentiation of neural stem cells. FBS (fetal bovine serum) was treated at different concentrations.

Immunofluorescence staining was performed using an antibody (Sigma-Aldrich or Pel-Freez) that responds to tyrosine hydroxylase (TH), a marker of dopamine neuron, resulting in increased expression of TH from 0.001% And increased in a concentration-dependent manner up to 0.1% (Fig. 1). The immunofluorescence salt method was carried out as follows.

The cultured cells were fixed with 4% paraformaldehyde for 20 minutes. Then, the cells were washed three times with 0.1% BSA / PBS solution for 5 minutes each. 0.1% BSA / PBS solution and 10% normal goat serum and 0.03% Triton X-100 (Sigma) were mixed and blocked for 1 hour before the primary antibody reaction. The primary antibody was diluted in a mixture of 0.1% BSA / PBS solution and 10% normal goat serum, and incubated at 4 ° C until the next day. The primary antibody was washed three times with 0.1% BSA / PBS solution for 5 minutes on the next day. The biotin-conjugated secondary antibody was diluted in 0.1% BSA / PBS solution, incubated at room temperature for 30 minutes, and washed three times with 0.1% BSA / PBS for 5 minutes. (DTAF, 1: 1000 or Rhodamine, 1: 400) secondary antibody (Jackson Immuno Research Laboratories, West Grove, Pa.) Was diluted in 0.1% BSA / Washed three times for 5 minutes with BSA / PBS solution and once with 3 times DW. After staining the cells, VECTASHIELD with DAPI (Vector Laboratories, Burlingame, Calif.) Mounting solution was dropped on the slides and a coverslip with attached cells was attached.

The cells were treated with 0.01% FBS and subjected to TH immunofluorescence staining for the differentiation date of dopaminergic neurons. As a result of the differentiation date, the increase of TH expression was superior to that of the control (Fig. 2). The expression of mRNA and protein associated with dopaminergic neuron expression was observed. As a result, TH and Nurr1 mRNA were increased according to the differentiation date, and TH and Nurr1 protein were increased by Western blot (Fig. 3).

mRNA measurement was carried out as follows. To synthesize the cDNA, the cultured cells were removed and RNA was extracted using Trizol (TRI Reagent, Molecular Research Center, Cincinnati, Ohio). The reverse transcription reaction was performed using a Superscript kit (Invitrogen) in a total volume of 20 μl. 1 μl of superscript II, 4 μl of 5x superscript solution, 1 μl of 10 mM dNTP mixture, 1 μl of random primer, 0.5 μl of RNase inhibitor, 2 μl of 0.1 mM DTT, RNA (5 μg) CDNA was synthesized by reacting at 42 DEG C for 45 minutes, 70 DEG C for 15 minutes, and 4 DEG C for 15 minutes. Quantitative RT-PCR was performed on the synthesized cDNA. 3 μl of a 10 × reaction solution, 3 μl of a 2.5 mM dNTP mixture, 0.5 μl of cDNA, 0.5 μl of DNA polymerase (Cosmo genetech), 0.15 μl of a primer mixture Lt; / RTI > 22 μl was placed in a 0.5 ml tube and reacted.

 Western blotting was carried out as follows. The cultured neural progenitor cells were washed twice with PBS, and lysis solution (0.5% Triton X, 20 mM Tris 7.4, 10 mM EDTA) containing protease inhibitor (phenylmethanesulfonyl fluoride, PMSF) Min. Protein lysates were collected using a scraper. Protein samples were quantitated with protein assay solution (Bid-rad, CA) and 4x sampling solution was added and boiled for 10 minutes. And then electrophoresed on an SDS-PAGE gel. After electrophoresis, the cells were transferred to a PVDF membrane pre-activated with methanol at 30 V for 1 hour using a transfer solution (25 mM Tris base, 0.2 M glycin, 20% methanol, pH 8.5). After blocking with the blocking solution (5% skim milk) for 1 hour, the primary antibody was diluted 1: 1000 in the washing solution and reacted overnight at 4 ° C. Then, the cells were washed three times with washing solution (1 × TBS, 0.1% Tween 20) every 5 minutes, and the biotin-conjugated secondary antibody was diluted with 1: 2000 ratio and reacted at 4 ° C. for 1 hour. Then, the rinsing solution was washed three times at intervals of 5 minutes, diluted with 1: 3000 horseradish peroxidase (HRP), reacted for 15 minutes, and washed three times with washing solution every 5 minutes. The chemiluminescence (ECL) solution (Perkinelmer, MA) was treated with chemiDoc to visualize protein expression.

Example  2 : FBS  On the expression of dopamine neurons

Cerebral cortical neural stem cells were isolated from the rat embryo (14.5 days of age) and infected with retrovirus expressing the Nurr1 gene in the presence of bFGF as in Example 1. bFGF was induced to induce the differentiation of neural stem cells. From this time, the mixture of FBS and Table 1 was treated by concentration. The concentrations in Table 1 were considered as 100% FBS concentrations. Immunofluorescence staining was performed as in Example 1 using antibodies reactive with tyrosine hydroxylase (TH), a marker of dopamine neuronal cells.

As a result, as shown in Fig. 4, the expression of TH was increased similarly to the case of treatment with 0.01% FBS at 0.1% mixture treatment (Fig. 4).

Mixture component density One Cortisol 0.5 ng / ml 2 Growth hormone 39 ng / ml 3 Parathyroid hormone (PTH) 1.72 ng / ml 4 Triiodo- L- thyronine (T ₃ ) 1.2 ng / ml 5 Thyroxine (L -Thyroxine, T ₄₎ 0.12 ng / ml 6 Follicle-stimulating hormone (FSH) 0.095 ng / ml 7 Luteinizing hormone (LH) 0.008 ng / ml 8 Prostaglandin E1 (Prostaglandin E1) 6 ng / ml 9 Prostaglandin E2 (Prostaglandin E2) 6 ng / ml 10 Prostaglandin F1a (Prostaglandin F1a) 12.3 ng / ml 11 Retinoic acid 9-cis 90 ng / ml 12 Retinoic acid all-trans 90 ng / ml 13 cholesterol 310000 ng / ml 14 Lactate-dehydrogenase (LDH) 864 mU / ml 15 Aspartate-aminotransferase (aspartate-aminotransferase) 130 mU / ml 16 Alkaline phosphatase (Alkaline phosphatase) 255 mU / ml 17 Thyroid-stimulating hormone (TSH) 1.22 ng / ml 18 Prolactin (LTH) 0.176 ng / ml

from Lindl, T. (2002): " Zell- und Gewebekultur ". 5th ed. Spektrum Akademischer Verlag, Heidelberg

Example  3: Identification of the active substances in the mixture

In order to identify the most effective components of the mixture, the Nurr1 gene was expressed in rat neural stem cells and one candidate substance was treated in each of 18 candidate substances. TH promoter activity measurement (TH promoter assay) was performed as follows. After transfection of 6.0TH-GL3B plasmid and pRSV-R-Luc plasmid with lipofectamine2000, the cells were washed with cold PBS, 100ul of lysis buffer was added, and the mixture was stirred for 10 minutes. The cell lysate was transferred to a tube and centrifuged, and the supernatant was transferred to a white 96-well plate in 20 ul increments. The expression level of the promoter was measured by a luminometer using a firefly luciferase reagent, and the value obtained by reacting with the renilla luciferase reagent was used as a correction value. As a result, the candidate C4 [triiodo- L -thyronine T ₃ )], No. 5 [C5, L -thyroxine (T ₄ )], and No. 16 (C16, alkaline phosphatase).

Expression of TH was confirmed by immunofluorescence staining (Fig. 6). Immunofluorescent staining was performed as follows. The cultured cells were fixed with 4% paraformaldehyde for 20 minutes. Then, the cells were washed three times with 0.1% BSA / PBS solution for 5 minutes each. 0.1% BSA / PBS solution and 10% normal goat serum and 0.03% Triton X-100 (Sigma) were mixed and blocked for 1 hour before the primary antibody reaction. The primary antibody was diluted in a mixture of 0.1% BSA / PBS solution and 10% normal goat serum, and incubated at 4 ° C until the next day. The primary antibody was washed three times with 0.1% BSA / PBS solution for 5 minutes on the next day. The biotin-conjugated secondary antibody was diluted in 0.1% BSA / PBS solution, incubated at room temperature for 30 minutes, and washed three times with 0.1% BSA / PBS for 5 minutes. (DTAF, 1: 1000 or Rhodamine, 1: 400) secondary antibody (Jackson Immuno Research Laboratories, West Grove, Pa.) Was diluted in 0.1% BSA / Washed three times for 5 minutes with BSA / PBS solution and once with 3 times DW. After staining the cells, VECTASHIELD with DAPI (Vector Laboratories, Burlingame, Calif.) Mounting solution was dropped on the slides and a coverslip with attached cells was attached.

In order to confirm whether candidate substances thought to be effective for dopaminergic neuron activity are indeed important factors in FBS treatment efficacy, 4, 5, 16 were removed from 18 candidates, respectively, TH promoter activity. As a result, the promoter activity was greatly reduced in the experimental group in which Candidates 4 and 5 were removed together (FIG. 7), and fluorescence immunohistochemistry showed that TH expression was also greatly reduced (FIG. 8). These results show that T₃, T₄Is the main factor of the FBS treatment effect.

Example  4: T ₃ By concentration  Effect of treatment on the expression and differentiation of dopamine neuron

If one were expressing Nurr1 gene in rat neural stem cells, treatment at different concentrations diluted stairs to T ₃ in two steps from 200 pg / ㎖, was dose-dependently increase the expression of TH significantly, 6 pg / ㎖ than T ₃ processed with (Fig. 9) than in the case of 0.01% FBS or 0.1% mixture treatment. In addition, the promoter activity was also increased in a concentration-dependent manner in the TH promoter activity measurement (FIG. 10).

Example 5: Effects on the expression and differentiation of midbrain dopaminergic neurons promote T _3, Check the effect of T ₄

While the differentiation of the rat embryo mesencephalic neural progenitor cells were treated with T ₃ and T _4, respectively. Neural progenitor cell culture was performed as follows. Female Sprague Dawley (SD) rat Embryos were extracted from the uterus at 14 days of gestation and the midbrain part of the embryo was isolated. Neuronal progenitor cells were prepared from single cells. Cells with 0.2 x 10 ⁶ / well in advance PLO [poly-L-ornithine ( 15 mg / ㎖, sigma, St Louis, MO)] / FN [fibronectin (1 mg / ㎖, sigma)] a 24well plate coated with I laid it down. The next day 37 ℃, 5% CO ₂ under incubate conditions 20 ng / ㎖ of basic fibroblast growth factor (bFGF; R & D Systems) are added to the serum-free media using the (. N2, Johe et al 1996 ) 1 ilgan cultured After the next day, N2 + 0.2 mM ascorbicacid (Sigma) was added for differentiation and the medium was changed every 2 days.

T After a process at the time of the neural stem cells differentiated to ₃ and T _4, as is the case in which artificially expressing Nurr1 in rat fetal cerebral cortex neural progenitor cells, while differentiation of midbrain neuronal precursor cells of the rat fetus to T ₃ and T ₄ (Fig. 11), the expression of TH that occurred in midbrain dopamine neurons increased in a concentration-dependent manner. These results demonstrate that in vivo treatment of T ₃ and T ₄ in vivo can improve the expression of dopamine neurons in vivo, suggesting that T ₃ and T ₄ may be induced by dopamine deficiency such as Parkinson's disease And can be used as a remedy for the diseases caused by the disease.

Example  6: T in human cell line ₃ And T ₄ Dopaminergic neuron expression and enhancement

Expressing Nurr1 gene in human neural stem cell line BE (2) C cells were, T ₃ and T differentiation by treating the ₄ each were subjected to immunofluorescence staining with TH promoter activity measurements, T _3, T ₄ are both dopamine neurons It was shown to be effective in increasing cell expression (Fig. 12). In addition, SH-SY5Y cells, which are human neuroblastoma cell lines expressing Nurr1 alone, also showed increased TH promoter activity in both T ₃ and T ₄ (FIG. 13). In addition, H9 neural precursor cells in which human embryonic stem cell line H9 was directly differentiated into neural stem cells were treated with T ₃ and T ₄ , respectively, and differentiated into neurons, which showed the same effect (FIG. 14). These results indicate that T ₃ and T ₄ are potentially effective when administered as a drug to humans.

Example  7: T for neurotoxic substances ₃ And T ₄ Effect of dopamine neuron repair

In order to be used as a therapeutic agent, it is necessary to confirm the recovery effect of damage when dopaminergic neurons are damaged. Therefore, 6-hydroxydopamine (6-OHDA (20uM, MP biomedical) and reactive oxygen species (H ₂ O ₂ ) (100uM, sigma) inducing apoptosis in neurons, respectively. The dopaminergic neurons were treated by treatment with T ₃ and T ₄ for one week, respectively. As a result, it was confirmed that dopamine neurons were significantly increased compared to the untreated group (FIG. 15). In addition, 6-OHDA (10 uM) was treated with both T ₃ and T ₄ for 2 weeks, and dopaminergic neurons were similarly recovered (Fig. 16 and 17). These results show that T ₃ and T ₄ actually have a restorative effect on neurotoxic substances, indicating that they can be used as a treatment for Parkinson's disease.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

A pharmaceutical composition for preventing or treating Parkinson's disease comprising triiodothyronine, thyroxine or a pharmaceutically acceptable salt thereof as an active ingredient.
delete 2. The composition of claim 1, wherein the pharmaceutically acceptable salt is a sodium triiodothyronine salt or a thixocin sodium salt.
2. The composition of claim 1, wherein the triiodothyronine or thyroxine increases dopamine levels in the central nervous system.
2. The composition of claim 1, wherein the triiodothyronine or thyroxine promotes the proliferation of dopamine neurons or the differentiation of neural progenitor cells into dopamine neurons.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition has a formulation selected from the group consisting of powders, granules, tablets, capsules, suspensions, syrups, aerosols, coated tablets, pills, gels, juices, Composition.
A dietary supplement composition for preventing or ameliorating Parkinson's disease comprising triiodothyronine, thyroxine or a salt thereof.

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