WO2024225709A1 - Composition for treating ototoxic hearing loss caused by aminoglycoside antibiotics - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating hearing loss, the composition containing berberine chloride as an active ingredient. The pharmaceutical composition was found to effectively inhibit hair cell apoptosis caused by reactive oxygen species and exhibit an excellent protective effect on hair cells, and thus can be effectively used as a drug or health supplement for preventing or treating hearing loss.

Description

Composition for treating ototoxic hearing loss caused by aminoglycoside antibiotics

The present invention provides a composition for treating ototoxic hearing loss caused by an aminoglycoside antibiotic.

Hearing loss (deafness) is a disease that affects 15 to 20% of the entire population. The number of people with hearing loss is increasing due to environmental pollution and aging in modern society. Since hearing impairment once occurs is permanent, it is very important to prevent it before it occurs. Hearing loss is mostly caused by environmental factors such as sudden, drug-induced (anticancer drugs, antibiotics, etc.), noise-induced, traumatic, geriatric, and congenital, as well as genetic factors, and is mainly caused by damage and death of auditory cells. Most research on auditory cell damage has been on noise-induced hearing loss, while research on hearing loss caused by ototoxic drug damage mechanisms is insufficient.

Meanwhile, aminoglycosides, a pharmaceutical category of antibiotics, are known to be used in the treatment of gram-negative bacterial infections. Although they are the most widely used antibiotics due to their high efficacy and low cost, major side effects such as nephrotoxicity and ototoxicity have been reported. Since drug-induced ototoxicity is one of the major causes of acquired hearing loss, it can cause damage to cochlear hair cells by three aminoglycosides (amikacin, kanamycin, and gentamicin). However, due to the lack of understanding of ototoxic hearing loss caused by aminoglycoside drugs, the development of treatments for this has been slow, and there is currently no clear preventive drug.

[Prior art literature]

[Patent Document]

(Patent Document 1) 1. Republic of Korea Publication Patent No. 10-2018-0020229

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating ototoxic hearing loss caused by an aminoglycoside drug containing berberine chloride as an active ingredient.

Another object of the present invention is to provide a combination antimicrobial agents comprising berberine chloride and an aminoglycoside series drug.

Another object of the present invention is to provide a health functional food composition for preventing or improving ototoxic hearing loss caused by an aminoglycoside drug, which contains berberine chloride as an active ingredient.

To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating ototoxic hearing loss caused by an aminoglycoside drug containing berberine chloride as an active ingredient.

In addition, the present invention provides a combination antimicrobial agents comprising berberine chloride and an aminoglycoside series drug.

In addition, the present invention provides a health functional food composition for preventing or improving ototoxic hearing loss caused by an aminoglycoside drug containing berberine chloride as an active ingredient.

According to the present invention, since it has been confirmed that berberine chloride exhibits an effect of preventing and treating damage to auditory cells from ototoxic drugs such as aminoglycoside drugs, a composition containing berberine chloride as an active ingredient can be provided as a pharmaceutical composition and health food for preventing or treating ototoxic hearing loss.

Figure 1 shows the protective effect of berberine chloride (hereinafter referred to as BC) on mouse hair cells from ototoxicity.

Figure 2 shows the inhibitory effect of BC treatment on the death of hair cells induced by treatment with three aminoglycoside drugs [amikacin (hereinafter referred to as AK), kanamycin (hereinafter referred to as KM), and gentamicin (hereinafter referred to as GM)].

Figure 3 shows the inhibitory effect of BC on mitochondrial reactive oxygen species (ROS) accumulation in cochlear hair cells.

Figure 4 shows that BC shows the protective effect of aminoglycoside series drugs on cochlear hair cells by mitochondrial membrane potential.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for preventing or treating ototoxic hearing loss caused by an aminoglycoside drug containing berberine chloride as an active ingredient.

The above aminoglycoside series drugs may be selected from the group consisting of amikacin, kanamycin, and gentamicin, but are not limited thereto.

The above pharmaceutical composition can inhibit apoptosis of auditory cells and increase the number of hair cells.

The above pharmaceutical composition can inhibit mitochondrial reactive oxygen species (ROS) accumulation.

In another embodiment of the present invention, the pharmaceutical composition may further comprise one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, glidants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersing agents, surfactants, binders and lubricants commonly used in the manufacture of pharmaceutical compositions.

Specifically, carriers, excipients and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

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

The dosage of the effective ingredient according to the present invention may vary depending on the condition and weight of the subject, the type and degree of the disease, the drug form, the route and period of administration, and may be appropriately selected by a person skilled in the art, and the daily dosage may be 0.01 mg/kg to 200 mg/kg, preferably 0.1 mg/kg to 200 mg/kg, and more preferably 0.1 mg/kg to 100 mg/kg. Administration may be once a day or divided into several times, and the scope of the present invention is not limited thereby.

In addition, the present invention provides a combination antimicrobial agents comprising berberine chloride and an aminoglycoside series drug.

The above aminoglycoside series drugs may be selected from the group consisting of amikacin, kanamycin, and gentamicin, but are not limited thereto.

The above combination antibiotics can alleviate or improve ototoxic hearing loss caused by aminoglycoside drugs.

In addition, the present invention provides a health functional food composition for preventing or improving ototoxic hearing loss caused by an aminoglycoside drug containing berberine chloride as an active ingredient.

The above aminoglycoside series drugs may be selected from the group consisting of amikacin, kanamycin, and gentamicin, but are not limited thereto.

The above health functional food may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

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

In addition, the above health functional food may additionally contain food additives, and its suitability as a “food additive” shall be determined by the standards and criteria for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified.

Examples of items listed in the above “Food Additives Codex” include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kohlrabi pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.

At this time, the content of the effective ingredient added to the food during the process of manufacturing the health functional food can be appropriately increased or decreased as needed, and preferably, it can be added so as to be included in an amount of 1 to 90 parts by weight per 100 parts by weight of the food.

Hereinafter, in order to help understand the present invention, examples and the like will be given to explain in detail. However, the following examples and the like only illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples and the like. The examples and the like of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.

[Example 1] Organotypic cochlear explants

Postnatal day 3 ICR (Institute for Cancer Research) mice were purchased from Hyochang Science (Daegu, South Korea) for cochlear transplantation. Dissected cochleae were cultured in high-glucose DMEM (Dulbecco's Modified Eagle's Medium; Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (Hyclone) and ampicillin (10 μg/mL; Life Technologies, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere of 5% _CO2 . After 16 h of culture, cochleae were first treated with 0.1 μM or 1 μM BC for 1 h, and then one of three aminoglycoside antibiotics (1.2 mM KM, 3 mM AK, or 100 μM GM) was added to the culture medium. BC concentrations were optimized for each aminoglycoside (0.1 μM for KM and 1 μM for AK and GM).

All animal experiments were approved by the Animal Experiment Ethics Committee and followed the guidelines of the Kyungpook National University Animal Experiment Practice Committee.

[Example 2] Phalloidin staining

To evaluate the morphology of inner hair cells (IHCs) and outer hair cells (OHCs) of the organ of Corti, specimens were cultured for 48 h and fixed for 15 min with 4% paraformaldehyde (PFA, pH 7.4) in phosphate-buffered saline (PBS). After washing three times with PBS, stereocilia of the explants were stained with Alexa Fluor® 488- or 555-conjugated phalloidin (1:1,000; Invitrogen-Molecular Probes, Eugene, OR) in PBS for 1 h at room temperature (RT). Specimens were mounted on glass slides using Fluoromount (Sigma-Aldrich, St. Louis, MO) and visualized using an Axio Imager A2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

[Example 3] Immunohistochemical and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis

Immunohistochemistry and TUNEL assay for active caspase-3 were used to determine whether cell death in the organ of Corti occurred via apoptosis. Specimens were fixed with 4% poly(phenylene sulfonyl) FA, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS-Tx) for 30 min at room temperature (RT), blocked with 5% normal goat serum for 1 h at room temperature (RT), and incubated overnight with anti-active caspase-3 antibody (1:1,000; Cell Signaling Technology, Bevery, MA) diluted in blocking solution.

To detect DNA fragmentation leading to apoptosis, a TUNEL assay kit (Promega, Madison, WI) was used according to the manufacturer's protocol. Specimens were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature (RT) and treated with 0.1% PBS-Tx in 0.1% sodium citrate for 30 min at 37 °C. Fragmented DNA from cells was stained using TUNEL working solution for 30 min at 37 °C. F-actin was labeled with Alexa Fluor 555-conjugated phalloidin in PBS-Tx for 3 h at room temperature (RT) in the dark and visualized using a Zeiss Axio Imager A2 fluorescence microscope.

[Example 4] Measurement of mitochondrial damage caused by antibiotic-induced oxidative stress

To analyze AK or KM-induced mitochondrial oxidative stress, cochlear explants were examined by MitoSOX Red assay and JC-1 assay. Mitochondrial reactive oxygen species (ROS) levels were detected by staining with 5 μM MitoSOX Red (Invitrogen-Molecular Probes) for 5 min at 37 °C in a humidified atmosphere of 5% _CO2 . Specimens were washed with PBS and visualized using a Zeiss Axio Imager A2 fluorescence microscope.

Mitochondrial membrane potential was measured using the cationic fluorescent dye MitoProbe ^TM JC-1 (Invitrogen-Molecular Probes). After 30 h of incubation, the specimens were washed with PBS and stained with JC-1 (2 μM) for 2 h at 37 °C, 5% CO ₂ in the dark. To verify the sensitivity of the JC-1 assay, 50 μM CCCP (carbonyl cyanide 3-chlorophenylhydrazone) was added to the untreated specimens before JC-1 staining, and then the specimens were incubated for 5 min at 37 °C, 5% CO ₂ to activate CCCP. JC-1 fluorescence was split into red and green, and the segmented images were converted to grayscale and evaluated using ImageJ software (http:/imageJ.nih.gove/ij/).

[Example 5] Quantification and statistical analysis of maternal cells

For quantitative analysis of hair cell damage, IHCs and OHCs with intact v-shaped stereopsis along a 200 μm length of basilar membrane were individually counted in the apical (30%), middle (50%), and basal (70%) regions of each cochlear probe. All experiments were performed independently at least three times. Data were analyzed using a two-tailed Student’s t-test, with a P-value < 0.05 considered statistically significant.

[Experimental Example 1] Protective effect of BC on drug-induced hair cell damage

To determine whether BC protects hair cells, we compared the severity of antibiotic-induced hair cell damage between BC-treated and untreated cochlear explants. In the untreated control group, IHCs and OHCs with well-shaped stereocilia were intact from the apical to the basal cochlear turn. However, antibiotic treatment disrupted the stereocilia and hair cell arrangement (Fig. 1a). KM not only altered the ciliary morphology of hair cells, but also disrupted cell arrangement and induced cell loss in a complex manner. These changes were more severe in OHCs than in IHCs. Hair cells treated with AK had most of their cilia collapsed and lost, making them unrecognizable. Similar to KM, GM caused more severe damage to OHCs than to IHCs, with hair cell loss. All three antibiotics caused severe hair cell damage, which increased in severity from the apical to the basal end, consistent with the high-frequency range loss typical of ototoxic loss. However, treatment with 0.1 or 1 μM BC before antibiotic (KM, AK, or GM) treatment attenuated stereocilia degeneration and hair cell loss in all cochlear regions, suggesting a protective effect of BC against antibiotic-induced cytotoxicity in hair cells.

The protective effect of BC against hair cell damage induced by KM, AK or GM was statistically confirmed in three cochlear regions: apical, middle and basal turns, by counting surviving hair cells with V-shaped stereocilia (Fig. 1b). We measured the cytotoxicity of BC and found that it had no detrimental effect on IHCs or OHCs. IHCs were virtually destroyed by AK, but were more resistant to KM and GM-induced cytotoxicity. Compared with IHCs, OHCs were much more susceptible to antibiotic-induced cytotoxicity. Importantly, pretreatment of both IHCs and OHCs with BC reduced the antibiotic-induced damage to almost normal levels. Therefore, BC could effectively prevent antibiotic-induced hair cell loss in cochlear tissue.

[Experimental Example 2] Inhibition of Cell Death by BC in Mouse Cochlear Explants

Because BC attenuated antibiotic-induced ototoxicity in mouse cochlear explants, we examined whether this effect was due to inhibition of apoptotic hair cell death. Two hallmarks of intrinsic apoptosis, caspase-3 activation and DNA fragmentation, were tested by immunohistochemical staining. Organs of Corti treated with KM, AK, or GM exhibited significantly increased levels of cleaved caspase-3 compared with untreated controls, whereas BC treatment prior to antibiotics decreased cleaved caspase-3 (Fig. 2a). Similarly, apoptotic DNA fragmentation in cells from the organ of Corti, as measured by TUNEL labeling, was significantly reduced by BC pretreatment to near control levels compared with KM, AK, or GM treatment (Fig. 2b). In both assays, signal intensities for cleaved caspase-3 and fragmented DNA were stronger toward the basal turn, consistent with the pattern of cell loss and ciliary damage depicted in Fig. 1 . Therefore, these results suggested that hair cell damage induced by KM, AK, or GM in the mouse cochlea resulted in cell death, which was effectively inhibited by BC.

[Experimental Example 3] Prevention of oxidative stress and mitochondrial damage in mouse cochlear hair cells by aminoglycoside drugs by BC

Since excessive accumulation of intracellular ROS leads to mitochondrial dysfunction, the accumulation of mitochondrial ROS induced by aminoglycoside drugs was measured using the MitoSOX Red fluorescence assay. As shown in Fig. 3a, all three drug treatments resulted in excessive accumulation of ROS, which was shown by a higher fluorescence intensity. In contrast, the BC pretreatment group showed a significant decrease in fluorescence intensity. The reduced fluorescence intensity of the integrated density of red fluorescence in the BC treatment group was statistically significant compared with the AK, KM, and GM groups (Fig. 3b). This suggests that BC inhibits mitochondrial ROS accumulation in hair cells, thereby inhibiting apoptotic cell death caused by oxidative stress.

Mitochondria are known to be a major source of intracellular ROS but are also vulnerable to ROS damage. This is because depolarization of the mitochondrial membrane potential causes mitochondrial dysfunction. The mitochondrial membrane potential of cochlear explants treated with aminoglycoside drugs was compared with or without BC treatment using MitoProbeTM JC-1 analysis. JC-1 monomers that emit green fluorescence signals have the characteristic of aggregating in mitochondria and generating red fluorescence in mitochondria with high membrane potential. Therefore, the mitochondrial membrane potential was measured by the red/green fluorescence ratio. As a result, according to Fig. 4a, hair cells treated only with KM, AK, or GM showed a significant decrease in the red/green ratio along with a loss of the red signal, which confirmed that this was mitochondrial damage induced by aminoglycosides. However, the red/green fluorescence ratio was confirmed to be increased in BC-pretreated mammary cells, confirming that the protective effect of BC was statistically significant (n = 3 per group, *P < 0.05 and **P < 0.01; Fig. 4b). These results showed that BC prevented aminoglycoside-induced loss of mitochondrial membrane potential.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (9)

A pharmaceutical composition for preventing or treating ototoxic hearing loss caused by an aminoglycoside drug containing berberine chloride as an active ingredient. A pharmaceutical composition according to claim 1, characterized in that the aminoglycoside series drug is selected from the group consisting of amikacin, kanamycin, and gentamicin. A pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition inhibits apoptosis of auditory cells and increases the number of hair cells. A pharmaceutical composition according to claim 1, characterized in that it inhibits mitochondrial reactive oxygen species (ROS) accumulation in hair cells. A combination of antimicrobial agents containing berberine chloride and aminoglycoside drugs. A combination antibiotic (antimicrobial agents) according to claim 5, characterized in that the aminoglycoside series drug is selected from the group consisting of amikacin, kanamycin, and gentamicin. In claim 5, the combination antibiotic is a combination antibiotic (antimicrobial agents) characterized in that it alleviates or improves ototoxic hearing loss caused by aminoglycoside series drugs. A health functional food composition for preventing or improving hearing loss caused by an aminoglycoside drug containing berberine chloride as an active ingredient. A health functional food composition according to claim 8, characterized in that the aminoglycoside series drug is selected from the group consisting of amikacin, kanamycin, and gentamicin.

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