KR20200120057A - Pharmaceutical composition for treating ototoxicity hearing loss by additional effect of cisplatin comprising alpha-lipoic acid - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating ototoxic hearing loss or health functional food for alleviating ototoxic hearing loss by cisplatin containing alpha-lipoic acid (ALA) as an active component, wherein ALA reduces hearing loss during hearing tests in mice with ototoxic hearing loss caused by cisplatin, prevents damage to the auditory hair cells and spiral ganglion cells when the inner ear of the mouse is extracted and observed morphologically, alleviates dysfunction of the mitochondria by reducing a Bax gene or protein expression in auditory hair cells and increasing a Bcl-2 gene or protein expression, and thus reduces reactive oxygen species (ROS). Therefore, a composition of the present invention can be effectively used for treating ototoxicity.

Description

Pharmaceutical composition for treating ototoxicity hearing loss by cisplatin comprising alpha-lipoic acid as an active ingredient {Pharmaceutical composition for treating ototoxicity hearing loss by additional effect of cisplatin comprising alpha-lipoic acid}

The present invention relates to a pharmaceutical composition and a health functional food for treating ototoxic hearing loss by cisplatin containing alpha-lipoic acid as an active ingredient.

Since its anticancer effect was discovered in the 1960s, cis-diamminedichloroplatinum II has been widely used as an effective chemotherapy drug for solid tumors found in the ovaries, lungs, and bladder.

Cisplatin enters the cell by diffusion or transporter and is hydrated to lower the chloride concentration inside the cell, thereby releasing chloride ions. The hydrated form of cisplatin cross-links with the nucleotides of the nucleus and mitochondrial DNA to form adducts, thereby inhibiting the unlimited replication of cancer cells.

However, clinical treatment using cisplatin is limited in use because it causes nephrotoxicity, neurotoxicity, and ototoxicity as side effects of normal tissues. In particular, cisplatin-induced ototoxicity can cause irreversible sensorineural hearing loss, that is, hearing loss, and is a more serious problem, especially in the pediatric population.

Hearing loss can significantly impair a patient's quality of life. In particular, it can have a significant impact on social development, such as language acquisition and speaking of pediatric patients.

Although the underlying molecular mechanisms responsible for cisplatin ototoxicity have not been fully elucidated, previous studies have shown that cisplatin is the death receptor pathway, endoplasmic reticulum-stress pathway, and mitochondrial reactive oxygen species in normal cells. It has been shown to activate the (ROS) production pathway (mitochondrial reactive oxygen species (ROS)-generating pathway), which in turn leads to cell death.

Excessive ROS production is believed to be a major cause of cisplatin-induced ototoxic hearing loss, and it is also considered a direct attack of cisplatin on DNA.

In particular, the corti organs, spiral ganglions and lateral walls in the cochlea have been proposed as major targets of cisplatin for the production of strong ROS.

Alpha-lipoic acid (ALA) is Thioctic acid or 1,2-dithiolane-3-pentanoic acid; C ₈ H ₁₄ O ₂ S ₂ ), which is widely distributed in the cell membrane, cytoplasm and extracellular space, is soluble in water and lipids, and is an essential cofactor in mitochondrial dehydrogenase reactions.

The protective action of alpha-lipoic acid (ALA) was first reported by Rosenberg and Culik in 1959, and Rosenberg and Culik have a role in reducing the symptoms of scurvy caused by vitamin C and vitamin E deficiency. I found it.

It was later discovered that alpha-lipoic acid (ALA) has the ability to regenerate endogenous antioxidants in the body, including vitamin C and vitamin E and intracellular reduced glutathione (GSH).

Recently, alpha-lipoic acid (ALA) is receiving considerable attention as a universal antioxidant along with dihydrolipoic acid in a reduced form as a redox couple.

Meanwhile, cisplatin chemotherapy is essential for cancer patients, but previous studies have focused only on the protective pre-treatment effects of alpha-lipoic acid (ALA).

In addition, conventional studies have only demonstrated that major side effects caused by cisplatin, including nephrotoxicity and neurotoxicity, are effectively prevented by pre-administration of alpha-lipoic acid (ALA) in animal models. Until now, no treatment has been reported for the treatment of ototoxicity and hearing loss caused by cisplatin.

The inventors of the present invention investigated the effect of alpha-lipoic acid (ALA) in a mouse model of hearing loss caused by cisplatin, and proapoptotic by alpha-lipoic acid (ALA) using auditory hair cells in vitro . It was confirmed that the expression of the protein (proapoptotic protein) Bax gene or protein decreased, and the expression of the antiapoptotic protein Bcl-2 gene or protein increased, thereby improving mitochondrial dysfunction and reducing hearing loss caused by ototoxicity. . In addition, it was confirmed that the antioxidant enzyme glutathione is directly involved in the oxidation-reduction reaction to lower free radicals (ROS). As well as the previously discovered effects of alpha-lipoic acid (ALA), as well as the therapeutic effect through post-treatment, the post-treatment effect of alpha-lipoic acid (ALA) is a therapeutic agent that treats hearing loss caused by cisplatin. It means the possibility of development. Through this, the inventors of the present invention have come to complete the present invention by revealing an antiapoptotic pathway activated by alpha-lipoic acid (ALA).

Korean Patent Registration No. 10-1891395 (registered on August 17, 2018)

Biologic activity of mitochondrial metabolites on aging and age-related hearing loss, The American Journal of Otology: March 2000-Volume 21, Issue 2, p 161-167

The present invention is to provide a pharmaceutical composition or health functional food for treating ototoxic hearing loss by cisplatin containing alpha-lipoic acid (ALA) as an active ingredient.

The present invention provides a pharmaceutical composition for treating ototoxic hearing loss by cisplatin containing alpha-lipoic acid (ALA) as an active ingredient.

In addition, it provides a health functional food for improving ototoxic hearing loss by cisplatin containing alpha-lipoic acid (ALA) as an active ingredient.

In the present invention, alpha-lipoic acid (ALA) significantly reduced hearing loss during hearing test in mice with ototoxic hearing loss caused by cisplatin, and auditory hair cells and spiral ganglion cells when morphological observation by extracting the inner ear of the mouse In the auditory hair cells, the expression of the Bax gene or protein was reduced, and the expression of the Bcl-2 gene or protein was increased to improve mitochondrial dysfunction, thereby reducing hearing loss due to ototoxicity, and the antioxidant enzyme glutathione. By confirming that it directly participates in the oxidation-reduction reaction and lowers the active oxygen (ROS), the alpha-lipoic acid of the present invention can be widely used as a therapeutic composition for ototoxic hearing loss or a health functional food for improvement that appears as a side effect of cisplatin.

1 is a hearing test result obtained by ALA pretreatment or ALA post-treatment and ABR measurement in cisplatin-injected mice (*p <0.05 vs cisplatin alone, control group; control group, cisplatin group; CP group, ALA pretreatment) Group; ALA pre-treatment group, ALA post-treatment group; ALA post-treatment group, ALA group; ALA group, ABR; auditory brainstem response).
FIG. 2 is a result of histological analysis of the inner ear of cisplatin-injected mice subjected to ALA pretreatment or ALA post treatment (n = 3), and FIG. 2A shows the basal turn of the inner ear of the control mouse. 2B is an observation of the stria vascularis [top] and the spiral ganglion [bottom] at high magnification (double arrow (↔) is the thickness of the vascular stria, and an asterisk (*) is a spiral ganglion It shows the degeneration of cells, and the scale bar is 50 μm), FIG. 2C is a result of quantitative comparison of the vascular striatal thickness (unit: μm), and FIG. 2D is the basal, middle, and apical This is the result of comparing the number of spiral ganglion cells between each group in turn (*p <0.05 vs cisplatin alone).
3 is a result of confirming the therapeutic effect before and after administration of ALA on the damage to auditory hair cells induced by cisplatin in cisplatin-injected mice.FIG. 3A shows internal hair cells to confirm the survival or degeneration of hair cells And Phalloidin staining (red) of the organ of Corti including external hair cells (white asterisk (*) indicates degenerative hair cells, scale bar is 50 μm), and Figure 3B shows the cochlea of each group. ) Is the result of histogram quantification of the number of internal and external hair cells in the basal, middle, and apical turns of (*p <0.05 vs cisplatin alone).
FIG. 4 is a result of confirming the effect of ALA treatment on cell viability in cisplatin-treated HEI-OC1 cells. FIG. 4A shows HEI-OC1 cells 0.5, 1, 2, 3 and 4 Incubation with mM ALA for 30 hours and cytotoxicity was measured by MTT assay.FIG. 4B shows HEI-OC1 cells treated with 30 μM cisplatin for 30 minutes and then treated with 0.5 to 4 mM ALA for 1 hour. This is the result of measuring cytotoxicity by MTT assay (*p <0.05 vs cisplatin alone).
5 is a result of confirming the effect of ALA on cell cycle arrest and apoptosis in cisplatin-treated HEI-OC1 cells, and FIG. 5A is a flow cytometry method (flow flow cytometry), and FIG. 5B is a result of comparing the sub-G0/G1 ratio between the CP-only treatment group and the ALA-only treatment group, and FIG. 5C is for detecting apoptotic cells. It is the result of TUNEL assay analysis (when observed with a fluorescence microscope, DNA-green, nuclear-blue, scale bar is 100 μm), Figure 5D shows caspase-3 expression in cells treated with cisplatin and ALA. It is a Western blot analysis result for confirming, and FIG. 5E is a result of measuring the caspase-3:β-actin ratio using densitometry (*p <0.05 vs cisplatin alone).
Figure 6 is a result showing the ROS scavenging ability of ALA in cisplatin-treated cells, Figure 6A is the result of measuring the intracellular ROS level (level) using a DCFH-DA probe (fluorescence intensity using a flow cytometer Detection), Figure 6B is a result of measuring the relative FACS fluorescence intensities (*p <0.05 vs cisplatin alone).
7 is a result of confirming the antiapoptotic effect of ALA on Bax-mediated apoptosis by cisplatin, and FIG. 7A is a loading control of β-actin and Bax Western Blot analysis result (n = 3 per lane), Figure 7B is the result of measuring the relative expression ratio of Bax and β-actin using densitometry, Figure 7C is a loading control (loading control) β Western blot analysis results of -actin and Bcl-2 (n = 3 per lane), Figure 7D is the result of measuring the relative expression ratio of Bcl-2 and β-actin using densitometry (* p <0.05 vs cisplatin alone).
8 is a result showing the expression levels of antioxidant enzymes GPx and GR in cells before or after ALA treatment, and FIG. 8A is a Western blot analysis result of β-actin and GPx, which are loading controls (n = 3 per lane), Figure 8B is a result of measuring the relative expression ratio of GR and β-actin using densitometry, Figure 8C is a Western blot analysis result of β-actin and GR as loading control And (n = 3 per lane), FIG. 8D is a result of measuring the relative expression ratios of GR and β-actin using densitometry, and FIG. 8E is a result showing the ratio of GSSG/GSH.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for treating ototoxic hearing loss by cisplatin containing alpha-lipoic acid (ALA) as an active ingredient.

The composition may inhibit the death of auditory hair cells.

In addition, the composition can reduce the expression of Bax gene or protein in auditory hair cells and increase the expression of Bcl-2 gene or protein, thereby reducing ototoxic hearing loss due to mitochondrial dysfunction.

The composition may increase the expression of the GPx gene or protein, and this may contribute to maintaining the reduced form of reduced glutathione (GSH) at a normal level by helping the GR role.

In one embodiment of the present invention, the pharmaceutical composition is any one selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or solutions according to a conventional method. Formulations of can be used.

In another embodiment of the present invention, the pharmaceutical composition is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, lubricant, flavoring agent, antioxidant, buffer solution, bacteriostatic agent, which are commonly used in the preparation of pharmaceutical compositions. It may further include one or more additives selected from the group consisting of diluents, dispersants, surfactants, binders, and lubricants.

Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules. And the like, and these solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used as the non-aqueous solvent and suspension. As a base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

According to an embodiment of the present invention, the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or It can be administered to the subject in a conventional manner via the intradermal route.

The dosage of the active ingredient according to the present invention may vary depending on the condition and weight of the subject, the type and extent of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be divided into several times, thereby not limiting the scope of the present invention.

In the present invention, the'subject' may be a mammal including a human, but is not limited to these examples.

In addition, the present invention provides a health functional food for improving ototoxic hearing loss by cisplatin containing alpha-lipoic acid (ALA) as an active ingredient.

The health functional foods include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, It may contain organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.

In addition, it may contain pulp for the manufacture of natural fruit juices, synthetic fruit juices and vegetable beverages. These components may be used independently or in combination. In addition, the health functional food composition is any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol and vitamin complex Can be

In addition, the above health functional food may additionally contain food additives, and whether it is suitable as a "food additive" is determined according to the general rules and general test methods of the Food Additive Code approved by the Food and Drug Administration, unless otherwise specified. It is judged according to the relevant standards and standards.

Items listed in the "Food Additives Code", for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as reduced pigment, licorice extract, crystalline cellulose, high cooling pigment, guar gum, L -Mixed preparations, such as a sodium glutamate preparation, a noodle-added alkali agent, a preservative preparation, and a tar color preparation, etc. are mentioned.

At this time, the content of the pravastatin and antiplatelet agents added to the food in the process of manufacturing the health functional food can be appropriately added or subtracted as needed, and preferably added to include 1 to 90 parts by weight to 100 parts by weight of the food. I can.

In addition, the present invention inhibits Bax protein expression, increases Bcl-2 protein expression, or GPx protein expression in vitro , which contains alpha-lipoic acid as an active ingredient. It provides a composition for a reagent that increases.

In addition, animals other than humans, characterized by treating a composition containing alpha-lipoic acid as an active ingredient, inhibiting Bax protein expression, or increasing Bcl-2 protein expression, Methods of increasing GPx protein expression are provided. That is, the alpha-lipoic acid according to the present invention may inhibit only Bax protein expression, may increase only Bcl-2 protein expression, or may increase only GPx protein expression.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experiment method>

1) mouse preparation

Female and male mice (8 weeks old) were purchased from Hyochang Science (Daegu, Korea). All animal procedures were performed in accordance with the Institutional Animal Care guidelines issued by the Committee of Animal Research at Kyungpook National University.

2) In vivo experimental design

Mice were divided into control group, cisplatin group (CP group), ALA pre-treatment group, ALA post-treatment group, and ALA group (a total of 5 groups). ).

The control group was treated with 0.9% saline for only 3 days.

In the ALA pre-treatment group, mice were injected with ALA for 2 days and then cisplatin was administered.

In the ALA post-treatment group, mice were injected with cisplatin and then ALA was injected for the next 2 days.

The ALA group injected only ALA into mice for 3 days.

In the cisplatin group (CP group), only cisplatin was injected into mice.

Specifically, 20 m/kg cisplatin (Sigma, St. Louis, MO, USA) and/or 100 mg/kg ALA (Thioctacid inj®, Bukwang Pharmaceutical Co., Ltd., Seoul, Korea) were administered intraperitoneally. .

In addition, in the case of the cisplatin-administered group, the ABR threshold was measured before injection and 4.5 days after cisplatin injection in order to evaluate the change in hearing ability.

3) Auditory brainstem response (ABR)

To evaluate auditory function, ABR measurements were performed using an ABR meter (System 3, Tucker Davis Technology, Inc., Alachua, FL, USA). All tests were done in a soundproof room. Prior to the ABR measurement, alfaxalone (40 mg/kg) was injected intramuscularly to anesthetize the mouse, and the mouse was placed on a heating pad to maintain the body temperature at 37°C. Mouse body temperature was measured and monitored with a rectal thermometer. In order to record ABR, the subcutaneous scanning electrode was inserted into the crown (+charge), the mastoid process (-e), and the hind limb (ground). Acoustic stimuli are tone-burst stimulus with 1-ms rise/fall time and 5-ms plateau at frequencies of 8, 16, and 32 kHz, or temporary click stimuli. And applied in a monaural way through a speaker.

Stimulus signals were generated using SigGenRP and an RP2.1 real-time processor and then transmitted through attenuators (PA5, TDT), speaker drivers (ED1, TDT) and electrostatic speakers (EC1, TDT). Stimulation was generated by repeating 500 times at a 5-dB decrement from the 90-dB sound pressure level to the acoustic threshold.

4) Paraffin sectioning and histological analysis

Mice were perfused with 1X phosphate buffered saline (PBS), and inner ears were separated. In the case of paraffin sectioning, the inner ear was fixed in PBS containing 4% paraformaldehyde (PFA) for 24 hours at 4°C, and then 10% ethylenediaminetetraacetic acid was added at 4°C for 24 hours. It was decalcified with PBS. The inner ears were dehydrated with a graded ethanol series, xylene was permeated, and embedded in paraffin at room temperature.

Paraffin-embedded inner ears were successively sectioned into 6 μm-thick pieces using a microtome (Leica RM2235, Leica Microsystems, Germany), and a Superfrost Plus® microscope slide ( Fisher Scientific, Pittsburgh, PA, USA). All slides were kept at 4°C until use. A slide with an inner ear encapsulated with paraffin was incubated at 65° C. for 1 hour, deparaffinized with xylene, and then rehydrated using a graded ethanol series. Nuclei and cytoplasm were stained with hematoxylin and eosin reagents (Sigma).

To determine the density of spiral ganglion cells, in each turn (basal, middle and apical) of the cochlea, 1 mm ² area of the paraffin section. The percentage of cells was calculated by calculating the average number of inner spiral ganglion cells.

5) Phalloidin staining

The cochlea was fixed with PBS containing 4% PFA for 20 minutes at room temperature, washed with 1X PBS, and incubated with 0.1% Triton X-100 for 15 minutes at room temperature. The organ of Corti was dissected from the cochlea, stained with PBS containing Alexa Fluor 555 phalloidin (Invitrogen-Molecular Probes, Eugene, OR, 1:1000) for 2 hours in the dark, and washed 3 times with PBS. Using a confocal microscope (LSM 700, Zeiss, Oberkochen, Germany) to observe the corti organs with hole mounts, normal hair cells within the 200 μm area of each turn of the cochlea. ) Counted.

6) Cell culture and viability assay

HEI-OC1 auditory hair cells were treated with antibiotic-free 10% fetal bovine serum (Hyclone, Logan, UT, USA) in Dulbecco's modified Eagle's medium (Hyclone, Logan, UT, USA) and 50 units/mL IFN-γ (PeproTech). , EC, London, UK) for 16 hours under acceptable conditions (33° C., 10% CO ₂ ).

In all experiments in vitro, each group of cells (7 × 10 ³ cells/well of 96-well plate) was pre-treated or post-treated with 1 mM ALA before and after 30 μM cisplatin treatment for 30 hours.

Cell viability is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide( MTT; Sigma, St. Louis, MO, USA)]. After the cells were treated with 0.5, 1, 2, 3, 4 mM ALA and/or 30 μM cisplatin for 30 hours, 0.5 mg/mL of MTT solution was added to the cell culture medium, followed by 10% CO at 33°C. with ₂ and cultured for 2 hours. The optical density of each well was measured at 550 nm using a microplate reader.

7) Measurement of intracellular ROS production

HEI-OC1 cells were incubated for 16 hours and then treated with 30 μM cisplatin for 24 hours with or without ALA pre- and post-treatment. The intracellular ROS level was measured using a fluorescent dye DCFH-DA (Invitrogen-Molecular Probes, Eugene, OR, USA). The cells were washed twice with 1 μM PBS and incubated for 5 minutes at 33° C. and 10% CO ₂ with 10 μM DCF-DA. Then, flow analysis of cells cultured in DCFH-DA was performed using a BD FACS Aria III flow cytometer (BD Biosciences, San Diego, CA, USA) (10,000 events per sample).

8) Cell cycle analysis

HEI-OC1 cells were trypsinized, counted, centrifuged, treated with 30 μM cisplatin, and fixed in 70% ethanol for 30 hours. HEI-OC1 cells were washed twice with 1X PBS and centrifuged. The centrifuged pellet was resuspended in a solution of RNase A (0.2 mg/mL) and propidium iodide (PI, 10 μg/mL) and incubated at 4° C. for 30 minutes. Cell cycle distribution was measured using a BD FACS Aria III flow cytometer (BD Biosciences, San Diego, CA, USA).

9) Detection of DNA fragmentation

Apatosis was analyzed using an in situ cell death detection kit (Roche Biochemicals, Mannheim, Germany) based on the terminal TUNEL technique. HEI-OC1 cells were cultured on a 24-well culture plate and treated with cisplatin for 30 hours. Then, the cells of each experimental group were fixed in 4% PFA at room temperature for 15 minutes, washed, and permeabilized at 4° C. for 5 minutes with water containing 0.1% Triton X-100 and 0.1% sodium citrate. 50 μL of the TUNEL reaction mixture was added to the sample and incubated at 37° C. for 60 minutes. All samples were washed 3 times with 1X PBS for 5 minutes each. Then, the nuclei were stained with 4',6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole) for 5 minutes. Specimens were visualized using a fluorescence microscopy (Carl Zeiss, Oberkochen, Germany).

10) Protein preparation

HEI-OC1 cells were washed with ice-cold 1X PBS, and in 100 μL of RIPA buffer (Elpis Biotech, Daejeon, Korea) containing a cocktail of protease inhibitor (Calbiochem Protease Inhibitor Cocktail Set 1, La Jolla, CA, USA). Suspended. This suspension was transferred to a pre-cooled 1.5 mL tube, incubated on ice for 15 minutes, centrifuged at 13,000 rpm at 4° C. for 30 minutes, and vortexed for 1 minute. The supernatant was taken and placed in a new tube on ice and the pellet was discarded.

11) Western blot analysis

The HEI-OC1 cell extract was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated protein was electrically transferred to a nitrocellulose membrane. After blocking the membrane for 1 hour with a solution of 20 mM Tris-HCl (pH 7.6), 137 mM NaCl and 0.01% Tween-20 (TBS-T) containing 5% skim milk, the primary antibody (1: 100-1: 2000 dilution) was probed at room temperature. Next, the membrane (membrane) was washed three times for 5 minutes each with TBS-T, and a secondary antibody (1: 2000 dilution) was incubated. After undergoing a series of washing procedures, measurements were made using an improved chemiluminescent detection system.

Rabbit anti-Bax and anti-caspase-3 antibodies are Cell Signaling (Cell Signaling, Danvers, MA, USA), rabbit anti-Bcl-2, anti-GR, and anti-GPx antibodies are Abcam (Abcam, Cambridge, MA, UK). And Goat anti-rabbit-lgG-HRP was used as a secondary antibody (Cell Signaling, Danvers, MA, USA).

12) Measuring glutathione levels

Cellular GSH levels were measured using a GSH colorimetric detection kit (BioVision Inc., Milpitas, CA, USA) of HEI-OC1 cell lysates exposed to cisplatin for 24 hours. Briefly, cells were scraped and suspended in 60 μL of ice-cold glutathione buffer, and each sample was mixed with 20 μL of perchloric acid. Samples were kept on ice for 5 minutes and centrifuged at 13,000 rpm for 2 minutes at 4°C. The supernatant was collected and 20 μL of cold 6 N KOH solution was added to 40 μL of the resulting sample. These samples were kept on ice for 5 minutes and centrifuged in a cold place. Next, 10 μL of the supernatant (neutralized supernatant) was transferred to a 96-well plate. To detect GSH, 80 μL assay buffer was added to each well. 70 μL of a total GSH sample was added to 10 μL reducing agent mix and incubated for 10 minutes. GSSG samples were added to 60 μL assay buffer and 10 μL GSH quencher for 10 minutes, then 10 μL reducing agent was added, and GSSG samples were incubated for 10 minutes. Finally, 10 μL of σ-phthalaldehyde (o-phthalaldehyde) was added to all samples and incubated at room temperature for 40 minutes.

13) Statistical analyses

Data were analyzed using the SPSS statistical software package (version 23; SPSS, Chicago, IL, USA). Continuous variables were expressed as mean±standard deviation. One-way ANOVA (one-way analysis of variance) was used to analyze 5 or 6 groups of variables. Two groups were analyzed using Student's t test. P values less than 0.05 (P<0.05) were considered statistically significant.

<Example 1> Confirmation of the effect of ALA to protect hearing and inhibit sensory cell death by cisplatin-induced ototoxic

1) Confirmation through measurement of auditory brainstem response

The effect of ALA on cisplatin-induced ototoxic hearing loss was investigated through a mouse model. In the case of the control group and the ALA group, the change in the ABR threshold was less than 5 dB over the entire frequency range including the click sound, indicating that ALA treatment did not affect hearing ability in normal mice. I could see that. In addition, the cisplatin group (CP group) showed a significant change in the ABR threshold, confirming that cisplatin treatment caused severe hearing loss (FIG. 1).

The ALA pre-treatment group demonstrated that hearing was almost completely protected (5-10 dB change in ABR threshold), demonstrating that ALA can protect against cisplatin-induced hearing loss. . The most interesting result was the alleviative effect of ALA, as can be seen by the ALA post-treatment group. A small difference in ABR threshold change of 5-10 dB was observed between ALA pre-treatment group and ALA post-treatment group, but in ALA post-treatment group. Most of the hearing loss was alleviated after cisplatin administration. These results suggest that ALA is effective as a therapeutic drug.

2) Confirmation through morphological observation of stria vascularis and spiral ganglion

Histological analysis using hematoxylin and eosin staining was performed to determine whether changes in listening ability were related to morphological abnormalities. Since the stria vascularis and the spiral ganglion are the main targets of ototoxicity due to cisplatin, the study focused on these two areas (Fig. 2A).

In the stria vascularis, no significant histological differences were observed, including the thickness between the control group and all other groups (FIGS. 2B (top) and FIG. 2C). The most severe damage caused by cisplatin was found in the spiral ganglion, and the number of cells was 70% lower in the basin turn of the cisplatin group (CP group) than in the control group. In contrast, in the ALA pre-treatment group and the ALA post-treatment group, the number of spiral ganglion cells was similar to that of the control group (FIGS. 2B (bottom) and FIG. 2D).

These results suggest that the loss of helical ganglion cells can induce hearing loss directly from cisplatin, which is in good agreement with the ABR threshold result.

3) Confirmation through observation of histological changes in hair cells

To determine whether hair cells are affected by cisplatin-induced ototoxic hearing loss, corti organs were stained with whole mount phalloidin. As a result, remarkable changes in internal hair cells and external hair cells due to the administration of cisplatin could be confirmed (FIG. 3).

The area of the basal turn showed more severe damage than the area of the middle and apical turns, and the outer hair cells were inner hair cells. It was more sensitive to cisplatin (Fig. 3A). However, it was confirmed that severe damage was significantly protected or alleviated by pretreatment or post-treatment with ALA (Fig. 3B).

<Example 2> Confirmation of cytotoxicity and cell viability of ALA

To investigate whether ALA affects the cytotoxicity induced by cisplatin, we used HEI-OC1 (House Ear Institute-Organ of Corti 1) cells to investigate the intracellular mechanisms regulated by ALA. First, the cytotoxicity of ALA was tested by measuring the viability of cells not treated with ALA and cells treated with ALA.

It could be observed that there was no cytotoxicity in ALA at a concentration of 0.5 to 4 mM (Fig. 4A). However, when only cisplatin was treated (cisplatin group (CP group)), the cell viability was reduced to 40%. However, with ALA pretreatment or post treatment, it could be confirmed that the cell viability was significantly increased (Fig. 4B). When the cells were pretreated with 2 mM ALA, the highest cell viability, 87%, was confirmed. In addition, when ALA was post-treated, cell damage caused by cisplatin was significantly alleviated, and a cell viability of 78% was confirmed in 1 mM ALA. In consideration of these results, 1 mM ALA was used in the next experiment.

<Example 3> Confirmation of the effect of ALA on the cell cycle and apoptosis

To demonstrate the protective role of ALA on cisplatin apoptosis, the cell cycle and apoptosis signaling pathway were analyzed and investigated.

Since conventional studies have only suggested that cross-linking of cisplatin and DNA causes cell cycle arrest, in the present invention, the sub-G0/G1 fraciton ratio was measured through flow cytometry.

It was confirmed that the cisplatin group (CP group) had a larger cell population at the sub-G0/G1 stage than the control group (17% vs. 4%). However, in the ALA pre-treatment group and ALA post-treatment group, only 6-7% of the cell population was in the sub-G0/G1 stage (FIGS. 5A and 5B ). These results suggest that ALA can protect cells and mitigate cisplatin-induced cell cycle arrest.

The cisplatin group (CP group) showed a high TUNEL signal that was not seen in the control group when analyzed by the TUNEL assay. In contrast, only the minimum TUNEL signal was detected in the ALA pre-treatment group and the ALA post-treatment group, and the signal intensity was significantly lower than that of the cisplatin group (CP group) (Fig. 5C).

In addition, it was observed through Western blot analysis that ALA has the ability to reduce apoptosis signals through caspase-3 expression analysis. It was confirmed that caspase-3 expression was high in the cisplatin-treated cells, but the cisplatin-treated control group did not express enough to be conspicuous. In contrast, it was observed that caspase-3 expression was suppressed in the ALA pre-treatment group and ALA post-treatment group (FIGS. 5D and 5E).

These results show that not only the pretreatment with ALA can protect cells against cisplatin cytotoxicity, but the post-treatment with ALA is the caspase-3-dependent apoptosis signaling pathway induced by cisplatin. ), which means that cell death can be alleviated.

<Example 4> Confirmation of the effect of reducing ROS in mitochondria and increase or decrease of related proteins

One of the main causes of cisplatin-induced apoptosis is excessive accumulation of ROS in cells, and ALA is well known as an effective antioxidant. Accordingly, DCFHDA (2′, 7′-dichlorofluorescein diacetate) analysis was performed to confirm whether ALA reduces intracellular ROS produced by cisplatin in HEI-OC1 cells.

The cisplatin group (CP group) showed about 1.6 times the fluorescence intensity compared to the control group (control group), which indicates that the increase in ROS was caused by cisplatin. In contrast, the ALA pre-treatment group maintained a similar fluorescent intensity as the control group. In addition, the ALA post-treatment group showed significantly lower fluorescence intensity compared to the cisplatin group (CP group) (FIGS. 6A and 6B). These results indicate that ALA not only reduces overexpressed ROS in cells after cisplatin treatment, but also prevents cisplatin-induced ROS production before treatment.

Bax present in the cytoplasm moves to the mitochondrial intermembrane space according to oxidative stress and releases cytochrome c, thereby inducing apoptosis. On the other hand, Bcl-2 inhibits Bax activity, inhibits apoptosis and promotes cell survival. Thus, increased ROS accumulation by cisplatin induces Bax to cause mitochondrial dysfunction that induces cell death, and ALA plays a central role in protecting mitochondria from oxidative stress induced by cisplatin.

ROS scavenging of ALA has been shown to eventually inhibit apoptosis through the regulation of Bax, a proapoptotic protein, and Bcl-2, an antiapoptotic protein. In the cisplatin group (CP group), the Bax protein expression level was 1.6 times higher than that of the control group, but the Bax protein expression was significantly attenuated in the ALA-treated cell group (FIGS. 7A and 7B). Conversely, cisplatin treatment down-regulated Bcl-2 protein expression and ALA-treated cells restored Bcl-2 protein expression (FIGS. 7C and 7D ).

What is important here is that although the cells have already been damaged by cisplatin (in the case of the ALA post-treatment group), ALA successfully inhibits Bax protein expression, reducing apoptosis. That is, this means that ALA is effective as a therapeutic agent after the onset of ototoxic hearing loss caused by cisplatin.

<Example 5> Confirmation of ALA involved in the oxidation-reduction reaction of glutathione

It is known that ALA is involved in the oxidation and reduction of glutathione, a representative antioxidant. GSH, a reduced form of glutathione, is oxidized to glutathione disulfide (GSSG) by glutathione peroxidase (GPx) to reduce intracellular ROS, and GSSG reduces GSH by glutathione reductase (GR). Let it. Since the glutathione redox cycle is known to play a key role in reducing the oxidized form of GSSG to GSH, the expression levels of GPx and GR were confirmed by Western blot.

Surprisingly, GPx expression increased by 4-5 times in all groups treated with ALA, suggesting that GPx expression induced by ALA can actively eliminate ROS (Figs. 8A and 8B). Interestingly, the increase in GR expression was not significant compared to GPx expression (FIGS. 8C and 8D ). This indicates that increased GSSG cannot be reduced to GSH due to insufficient expression of GR.

However, although GR expression did not increase significantly, the GSSG/GSH ratio of the ALA pre-treatment group and ALA post-treatment group was similar to that of the control group (Fig. 8E). ). These results imply that ALA plays a role of GR to reduce GSSG to GSH, thereby continuously reducing ROS.

From the above results, it was confirmed that alpha-lipoic acid is effective in improving ototoxic hearing loss caused by cisplatin. Therefore, alpha-lipoic acid according to the present invention can be used as a pharmaceutical composition for treating ototoxic hearing loss caused by cisplatin or as a health functional food for improvement.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (5)

A pharmaceutical composition for the treatment of ototoxic hearing loss by cisplatin containing alpha-lipoic acid as an active ingredient.
The method of claim 1,
The composition is a pharmaceutical composition for the treatment of ototoxic hearing loss by cisplatin, characterized in that inhibiting the death of auditory hair cells.
The method of claim 1,
The composition is a pharmaceutical composition for the treatment of ototoxic hearing loss by cisplatin, characterized in that the Bax gene or protein expression is reduced and the Bcl-2 gene or protein expression is increased in auditory hair cells.
The method of claim 1,
The composition is a pharmaceutical composition for the treatment of ototoxic hearing loss by cisplatin, characterized in that increasing the expression of the GPx gene or protein in the auditory hair cells.
A health functional food for improving ototoxic hearing loss by cisplatin containing alpha-lipoic acid as an active ingredient.

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