KR20150083167A - Pharmaceutical composition comprising methylene blue for treating or preventing sensorineural hearing loss - Google Patents

Abstract

The present invention relates to a method for preventing and/or treating modified or damaged snail, vestibular mother cells and nerve of spiral ganglion by injecting methylene blue and, more specifically, a method for treating sensorineural hearing loss.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a pharmaceutical composition for treating or preventing sensory neural deafness including methylene blue,

The present invention relates to the development of medicines for the prevention and treatment of sensory neural deafness using methylene blue, which has been used as a conventional stain agent, and can contribute to drastically reducing the incidence of hearing loss patients in the medical field.

Hearing impairment is becoming a rapidly growing health problem worldwide. Noise-induced hearing loss (NIHL) is one of the most common occupational diseases in both developed and developing countries. It is one of the major causes of sensory-nerve deafness. About 5% of the world's population is exposed to high levels of noise and suffering from hearing loss. Numerous studies have been conducted for the prevention and treatment of NIHL by understanding the disease physiological mechanisms of cell death in woW caused by noise. The latest way to prevent NIHL is to wear hearing protection equipment. However, such a device has a problem that it is difficult to protect the hearing of the user when wearing the device improperly, and communication is difficult. Therefore, in order to overcome these problems, efforts are continuously made to develop pharmaceutical preparations.

A number of studies have reported that oxidative phosphorylation of the mitochondria and the resulting reactive oxygen species (ROS) and reactive nitrogen species (ROS) nitrogen species (RNS). Removal of reactive oxygen species in the body can be accomplished in two ways. In the first method, the generated active oxygen groups are nonspecifically removed using a scavenger. The second approach is to directly target the source of reactive oxygen species and block the process of generation or the process before it. In the former case, N-acetyl-cysteine, Ebselen, MnTBAP, and Tiron are used experimentally. In the latter case, pravastatin targeting ubiquinol or NADPH oxidase, which targets mitochondria, is experimentally used.

The methylene blue (3,7-bis (dimethylamino) phenazathionium chloride (MB)) is an aniline-based staining agent currently used in clinical practice as a methemoglobinemia and a phototherapy aid. Which is very low, so that oxidation and reduction of the electrons can be freely performed. When the function of the electron transport system complex of the mitochondrion is degraded due to this characteristic, the activity of the complex is increased by participating in the electron transport system when the methylene blue is administered. As a result, the electron leakage, which is the main cause of the active oxygen system, The cell homeostasis can be maintained in the normal range.

In the prior art, wearing of hearing protection devices (HPD) is the only preventive method, especially in case of noise exposure, but it is limited in practical industrial field because of the disadvantage that communication among workers is blocked under a noisy environment. Therefore, a number of researchers are continuing to investigate the possibility of orally administered drugs for the treatment and prevention of hearing loss in order to overcome such limitations. In addition, since oral administration drugs showing no definite effects have not been developed even in senile deafness caused by deterioration of mitochondrial function and nervous system degeneration caused by aging, it is required to find out drug preparations capable of overcoming these limitations.

The present invention relates generally to methods of preventing and / or treating damage or degeneration of wo (and vestibular) hair cells and spinal ganglia neurons by administering methylene blue. In particular, the invention relates to a method for treating sensory neural herniation loss.

As one embodiment of the present invention, there can be provided a pharmaceutical composition for treating or preventing sensory neuron degeneration comprising an effective amount of a compound represented by the following formula (1).

[Chemical Formula 1]

The sensory nerve impairment is characterized by noise-induced hearing loss or senile hearing loss.

In addition, the sensory neuron deafness is characterized by damage or denaturation of inner ear hair cells.

In addition, the pharmaceutical composition is characterized in that it acts on the mitochondria of inner ear.

In addition, the pharmaceutical composition is characterized by effectively reducing active oxygen groups in the mitochondria.

In addition, the pharmaceutical composition is characterized in that the expression of the neurotrophin is increased.

In addition, the pharmaceutical composition is characterized in that it is selected from oral solid pharmaceutical preparations comprising tablets, pills, capsules, powders and granules.

In addition, the pharmaceutical composition is characterized in that it is selected from pharmaceutically acceptable aqueous solutions, suspensions, emulsions, syrups, pharmaceutical preparations dissolved in use, and oral liquid pharmaceutical preparations comprising elixirs.

In addition, the pharmaceutical composition is characterized by being an injectable pharmaceutical preparation for parenteral administration.

The present invention provides a pharmaceutical composition comprising methylene blue, which can be used for the treatment or prevention of hearing loss patients in the medical field.

FIG. 1 is a graph (A) showing an experimental schedule and a graph (BC) comparing hearing threshold value changes using electrophysiological tests.
FIG. 2 is a photograph and a graph showing the results of an experiment in which deletion of hair cells after noise exposure was evaluated using a confocal microscope (A, B) and a scanning electron microscope (C).
FIG. 3 is a graph quantifying the result of immunostaining and Western blotting showing the blocking effect of production of reactive oxygen species by pretreatment of MB in WoW after noise exposure.
FIG. 4 is a photograph showing immunostaining showing the effect of inhibiting the generation of an active nitrogen group by MB pretreatment in a wah after a noise exposure.
FIG. 5 is a graph showing experimental results of restoring the mitochondrial electron transport system function in which MB is reduced in cell (in vitro) and animal (in vivo) experiments.
FIG. 6 is a photograph and a graph showing the result of Western blotting to investigate changes in the expression level of neurotrophic factor according to the presence or absence of MB pretreatment in the wah after exposure to noise.
FIG. 7 is a photograph and a graph showing the difference in the expression patterns of synapsin-1 and PSD-95 according to presence or absence of MB pretreatment in the wah after noise exposure using a confocal microscope.

Although the medicines attempted to treat hearing loss in the existing studies are limited to targeting the already generated active oxygen groups, the present invention uses methylene blue selectively acting on the mitochondria that play a central role in the auditory organ cells, Is effectively reduced from the production stage and induces the improvement of intracellular energy production efficiency. In addition, the present invention suggests the utility of a pretreatment method for prevention of the development of hearing loss, and it is characterized by maintaining the normal function of the auditory system due to the neuroprotective effect by inducing an increased expression of neurotrophin-3 have.

The present invention improves the efficiency of the electron transport system in the mitochondrial respiratory chain by the active redox reaction occurring when the methylene blue, which is a dye, has a very low redox potential close to zero, And induces an increase in the expression of neurotrophic factor, thereby providing a preventive and therapeutic agent for the development of hearing loss through a mechanism of blocking the progression of key pathological physiology of the hearing loss.

As a specific description of the invention, the hearing-related diseases to be treated or prevented by the pharmaceutical composition to be provided by the present invention are specifically described. The pharmaceutical composition of the present invention has therapeutic or prophylactic effects on sensory nerve impairment. Examples of such sensory nerve impairment include hearing loss due to excessive noise and senile hearing loss.

&Quot; Sensory nerve impairment " It occurs when the components of the inner ear or accompanying neural components are affected, and may include nerve or sensory components if the brain's auditory or auditory pathways are affected. Sensory hearing loss can be hereditary, or it can be due to acoustic trauma (eg, very loud noises such as explosions), viral infections, drug-induced or Meniere's disease. Neurotic deafness can be caused by a brain tumor, infection or various brain and neurological disorders, such as stroke. Some genetic disorders, such as Levofloxacin (a defective accumulation of branched fatty acids), can also cause neurological disorders that affect hearing loss. The auditory nerve pathway may be selected from the group consisting of dehydrating diseases such as idiopathic inflammatory dehydratative diseases (including multiple sclerosis), transverse myelitis, Dick's disease, progressive multiseptic leukopenia, Guillain-Barre syndrome, chronic inflammatory dehydrative polyneuropathy, MAG is impaired by peripheral neuropathy.

&Quot; Noise-induced hearing loss " can result in hearing loss even when exposed to loud noises such as loud music, heavy equipment or machinery, airplanes, bombardment or other human noise for a long period of time. Deafness is caused by the destruction of the inner ear's hair cell receptors. Such hearing loss often involves tinnitus. Often, permanent hearing loss is diagnosed. Currently, there is no therapy for noise-induced hearing loss, but several therapies are being experimentally developed, including treatment with insulin-like growth factor-1 (IGF-1). Lee et al. Otol. Neurotol. (2007) 28: 976-981.

"Elderly hearing loss" is an age-related hearing loss that occurs as part of normal aging and results from the degeneration of receptor cells in the inner ear's cortical spinal cord. Another cause may be the loss of flexibility of the basilar membrane of the womb, as well as the decrease in the number of nerve fibers in the vestibular nerve. There is currently no known cure for permanent hearing loss due to senile hearing loss or excessive noise.

The present invention relates to drugs related to ear diseases, and provides a description of terms of ear structure.

"Wow" is the inner ear associated with hearing. The wah is a spiral tube-like structure wrapped in a snail-like shape. The inside of the wah is divided into three areas, and is divided by the location of the vestibular and basilar membrane. The area above the vestibule is the anterior chamber, which contains the outer lymph fluid, which extends from the innermost to the apex of the wah and has a low potassium content and a high sodium content. The basement membrane confines the hyperreal area, which extends from the vertex of the wah to the gill window and also contains the outer lymph fluid. The basement membrane contains a large number of stiff fibers, which gradually increase in length from the window to the apex of the wah. The fibers of the basement membrane are vibrated when activated by sound. The cochlea is located between the anterior and the posterior, and the distal end of the cochlea is closed at the apex of the wah. The cochlea contains the lymphatic fluid, which is similar to cerebrospinal fluid and has a high potassium content.

The Corti organ is located on the basement membrane and extends upwardly into the cochlea. The Corti organ contains hair cells with hairy protrusions extending from the free surface and contacts the gelatin surface, called the cover membrane. They do not have axons, but they are surrounded by sensory nerve fibers that form the womb of the vestibular nerve (cranial nerve VIII).

A "hair cell" is a hair-like cell attached to the basement membrane within the ear of the ear, and is a receptive cell with a cap at one end. When the basal membrane is moved, the hair cells are shaken and the nervous excitement occurs.

"Mitochondria" is one of the organelles called mitochondria. It is a device that produces the energy required for the cell's life function in the form of ATP in an aerobic manner. It is present in all cells except the red blood cells and shows the shape of ??,? The electron microscope shows that the mitochondria are surrounded by inner and outer mitochondrial membranes, and many wrinkles (facial features) protrude inwards from the inner membranes. The endocardium and ridge contain oxygen involved in the electron transport system and oxidative phosphorylation, and the mitochondrial matrix of the cyanobacteria contains the oxygen bacteria of the citric acid circuit.

The pharmaceutical composition according to the present invention can be administered in vivo as various formulations for the treatment or prevention of diseases.

As oral solid pharmaceutical preparations intended for oral administration, for example, tablets, pills, capsules, powders and granules may be mentioned. Capsules include hard and soft capsules.

In such oral solid pharmaceutical preparations, one or more of the active ingredients may be mixed or granulated by themselves or in combination with additives (e.g., via agitation granulation, fluidized bed granulation, dry granulation, tumble stirred fluid bed granulation, etc.) Then, the preparation is made into a pharmaceutical preparation (for example, encapsulation, tablet pressing, etc.) according to a conventional method. One or more additives can be suitably formulated. The additives include, for example, excipients such as lactose, mannitol, glucose, microcrystalline cellulose, corn starch, etc., binders such as hydroxypropylcellulose, polyvinylpyrrolidone, magnesium meta silicoaluminate, Stabilizers, solubilizing agents (for example, glutamic acid, aspartic acid, etc.), water-soluble polymers (for example, corn starch and the like), disintegrating agents (for example, calcium fibrinoglycolate and the like), lubricants (for example, magnesium stearate and the like) (E.g., cellulose) (e.g., hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, etc.), synthetic polymers (e.g., polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, etc.) Sucrose, fructose, glucose, lactose, hydrogenated maltose starch syrup, powdered hydrogenated maltose starch syrup, glucose Rukto trehalose syrup, honey, sorbitol, Marti tolyl, mannitol, xylitol, erythritol, aspartame, saccharin, saccharin sodium, etc.) are included. The solid pharmaceutical formulation may be coated with a coating agent (e.g., white sugar, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.) or optionally coated with two or more layers. In addition, solid pharmaceutical preparations include capsules made up of an absorbable material, such as gelatin.

Examples of oral liquid pharmaceutical preparations intended for oral administration include pharmaceutically acceptable aqueous solutions, suspensions and emulsions, syrups, pharmaceutical preparations (e.g., dry syrups) and elixirs that dissolve upon use. In such liquid pharmaceutical preparations, one or more active ingredients are dissolved, suspended or emulsified in a diluent commonly used (e.g., purified water, ethanol, or a mixture thereof).

In addition, such liquid pharmaceutical preparations may contain wetting agents, suspending agents, emulsifying agents, sweetening agents, flavoring agents, flavoring agents, preservatives, buffers and the like. Oral liquid pharmaceutical preparations also include pharmaceutical preparations (for example, dry syrups) which dissolve in use when taken in the form of a solution of the aforementioned solid oral pharmaceutical preparations.

Injectable pharmaceutical preparations intended for parenteral administration include solutions, suspensions, emulsions and solid injectable pharmaceutical preparations which are dissolved or suspended in the solvent in use. Injectable pharmaceutical preparations are prepared by dissolving, suspending or emulsifying one or more active ingredients in a solvent. As such a solvent, for example, distilled water for injection, physiological saline, vegetable oil, alcohol such as propylene glycol, polyethylene glycol and ethanol, and the like, and a combination thereof can be used. Such injectable pharmaceutical preparations may further contain stabilizers, solubilizing agents (e.g., glutamic acid, aspartic acid, Polysolvate 80 (trade name), etc.), suspending agents, emulsifying agents, pain relieving agents, buffers, preservatives and the like. Injectable pharmaceutical preparations can be sterilized in the final step or prepared by aseptic handling processes. Alternatively, sterile solid pharmaceutical preparations (e. G., Lyophilisates) are made and used by dissolving them in sterile distilled water for injection or other solvents prior to use.

The inventors of the present invention have investigated how methylene blue has a therapeutic or protective effect in relation to sensory nerve impairment, and the results of the study on the specific mechanism will be described as follows.

The present inventors have evaluated the prevention of acoustic damage by methylene blue. This will be described with reference to Fig. Specifically, in order to evaluate whether methylene blue prevents noise-induced injury (co-induced injury) of cochlea, experiments were conducted as shown in FIG. 1A. First, in order to confirm the efficacy of MB treatment, changes in hearing threshold were calculated based on the difference between auditory brainstem response (ABR) threshold values before and after noise exposure of each animal. Baseline ABR threshold (-1 day) was not significantly different between the experimental groups. The compound threshold shift (CTS) was measured on the first day after noise exposure and the permanent threshold shift (PTS) was measured on the 14th day after noise exposure. Figures 1B and C show that the threshold change patterns are similar at 16 and 32 kHz in both the noise exposed group and the MB pretreated group (pre-MB). In the noise exposed group, the average CTS was more than 40 dB at 16 and 32 kHz. On the other hand, the pre-MB group significantly reduced the mean CTS compared to the noise-exposed group (FIG. 1B). When the PTS was measured in the pre-MB group, the threshold shift was reduced by about 50% as compared with the noise-only group (FIG. 1C). MB was administered to the animals (post-MB group) for 3 consecutive days after the noise exposure to assess whether the MB post-treatment had a protective effect after the noise exposure. MB posttreatment alleviated the PTS by about 20%, but did not observe additional relaxation of the noise-induced threshold change as compared to the MB pretreatment group (FIG. 1C). This shows that MB preprocessing is more effective after MB. We tested the efficacy of pretreatment and post-treatment (pre + post-MB group) combined with MB for 7 consecutive days before and after noise exposure, but no further decrease in threshold change was observed. These results demonstrate that MB treatment alleviates noise-induced threshold shifts and that the prophylactic efficacy of MB on noise-induced threshold migration is superior to its therapeutic efficacy.

In addition, the present inventors evaluated the reduction of noise-induced hair cell loss by methylene blue pretreatment. This will be described with reference to FIG. Specifically, two types of hair cell morphology assays were performed. First, by using surface preparation, OHC loss was detected and quantitated at the basal turn, which is the most sensitive area of hearing damage, at 14 days after noise exposure. Second, SEM analysis was performed at the basal turn site. SEM images showed considerable damage to the stereocilia of OHC in the noise-only group compared with the control group. In the pre-MB group, the damage to the stereocilia of the OHC was significantly reduced (Fig. 2C). These results are consistent with the results of the surface treatment, indicating that MB pretreatment effectively alleviates hair cell loss following acoustical overstimulation.

In addition, the present inventors have tested the inhibition of noise-induced active oxygen species (ROS) and active nitrogen group (RNS) generation in the worm by methylene blue pretreatment, which will be described with reference to FIGS. 3 and 4. Excessive ROS and RNS production due to excessive noise stimulation is known to be associated with hair cell deletion (Henderson et al., 2006; Le Prell et al., 2007). 4-hydroxynonenal (4-HNE) is a membrane peroxidation product that is formed by free radical reaction in the plasma membrane. To determine whether MB pretreatment alleviated ROS production after excessive noise stimulation, serial immunohistochemistry of 4-HNE was performed on samples with and without MB pretreatment. After 4-HNE stimulation, 4-HNE expression was significantly increased in the noise-only group (stria vascularis, spiral ligament, and organ of Corti (OC) compared to the control group Respectively. In the case of MB pretreatment, 4-HNE expression was significantly increased compared to the control group, but a significant reduction in 4-NHE expression was seen in both the lateral wall and OC compared to the noise-only group (Fig. 3A -C). The expression level of 4-HNE-adhered protein was measured in a homogenate of mouse woW by Western blotting. Indeed, the concentration of 4-NHE after noise injury was significantly reduced in the pre-MB group (Figures 3D and E). This indicates that lipid peroxidation was alleviated by MB pretreatment. 3-nitrotyrosine (3-NT) can be used as a marker for oxidation and nitrification products, reflecting the reaction of peroxynitrite (ONOO-). Immunostaining for 3-NT showed that 3-NT was strongly detected in the lateral wall of the wah and in the OC in the noise-exposed group (Figs. 4A and 4B) compared to the control group. 3-NT expression was alleviated in the pre-MB group compared to the noise-only exposed group (FIG. 4C). This was similar to the result of 4-HNE. These results suggest that MB pretreatment effectively alleviates oxidative stress in the wah after acoustical overstimulation.

In addition, the present inventors have confirmed that methylene blue pretreatment selectively enhances mitochondrial electron transport system function in both cell experiments and animal vivo experiments. Specifically, referring to FIG. 5, blockade of the mitochondrial complex by selective inhibitors induces electronic leakage, which results in cytotoxicity induced by ROS production and ATP depletion. UB-OC1 cells, a cortical organ cell line, were first used to solve the molecular mechanism that explains the protective effect of MB on noise-induced woW damage. UB-OC1 cells have been used to study mechanisms of sensory auditory loss. The mitochondrial ATP depletion and damage induced by the complex inhibition of the mitochondrial electron transport chain has been used as an in vitro model of noise-induced hearing loss. Therefore, the present inventors inhibited complexes I, III and V, respectively, using rothenone, antimycin A and oligomycin in the MB treatment group and the untreated group of UB-OC 1 cells, respectively. Cell viability was significantly increased in rotenone and MB co-treated cells compared to rotenone treated cells (Fig. 5A). This protective efficacy was also observed in complex III inhibitors, antimycin A and MB co-treated cells (Fig. 5B). Contrary to these results obtained in complex I and III inhibitors, MB did not protect UB-OC 1 cells in complex V inhibition by oligomycin (FIG. 5C). In addition, the inventors measured the effects of rotenone, antimycin A and oligomycin under MB treatment or non-treatment for mitochondrial ATP production. After 12 hours of treatment with rottenone, antimycin A and oligomycin, respectively, a significant reduction in intracellular ATP concentration was observed. However, MB concurrent treatment significantly restored reduced ATP levels by both rotenone and antimycin A. However, cells treated with MB at the same time did not restore the decreased ATP concentration by oligomycin (Fig. 5E). Cell viability and mitochondrial ATP production were not only affected by MB treatment (Figures 5D and E). These results show that the improvement of damaged mitochondrial function by MB is due to a reduction in the specific leakage of electrons in complex I / III. In addition, MB has been reported to enhance the activity of complex IV. Thus, the present inventors have tested whether the MB pretreatment in vitro improves the activity of the Wow mitochondrial complex IV. Complex IV activity was directly suppressed by about 20% after noise exposure in the noise exposed group. MB, on the other hand, blocked significant reductions in complex IV activity after noise exposure (Fig. 5F). However, MB pretreatment did not have a significant effect on complex IV activity.

In addition, methylene blue pretreatment confirmed upregulation of the neurotrophin-3 (NT-3) neurotrophin. MB treatment has been reported to improve the in vivo concentration of neurotrophic factor brain-derived neurotrophic factor (BDNF), which leads to improved behavior in the Huntington's disease model. Acoustic hyperactivity is associated with changes in the capillary nerve endings of Inner Hair Cells and a lack of nutritional support in the central auditory pathway. Thus, we investigated the expression of two representative woW-related neurotrophic factors NT-3 and BDNF. MT pretreatment in the absence of noise exposure had little effect on the expression of BDNF and NT-3 (Fig. 6A-C). However, MB pretreatment significantly increased NT-3 expression in acute conditions of noise-induced woW damage (FIGS. 6D and E). Conversely, noise exposure or MB pretreatment did not significantly affect BDNF expression (Figures 6F and G).

In addition, in the present invention, it was confirmed that NT-3 upregulation of the neurotrophin induced by methylene blue pretreatment protects the afferent and efferent synaptic structures of WoW. To this end, we examined the neuroprotective effect of MB on wah synaptic structure. The synaptic vascular membrane protein synapsin-1 is used to mark presumptive efferent nerve endings that develop outer and inner mammary cells. On the second day after the end of the noise exposure, the present inventors confirmed that the expression of synapsin-1 was significantly reduced in the group exposed only to noise compared with the control group. However, damage to synapse-1 expression was restored by MB pretreatment (Fig. 7A, C). The present inventors also observed postsynaptic density-95 (PSD-95) expression to detect the expression of the afferent nerve endings of the post-synapse between the inner mammary gland and the spiral ganglion neurons (SGN). On the second day after the end of noise exposure, nonspecific increases in PSD-95 expression were observed around the primary synaptic neurons in both the MB pre-treatment group and the noise-only exposed group compared to the control group (Fig. 7B). However, the region of distribution of PSD-95 positive puncta within the synaptic region between IHC and primary synaptic neurons was reduced in the noise exposed group only. On the other hand, MB conserve the distribution area of afferent synapses around the inner mammary glands compared with the noise exposed group (Fig. 7B). In addition, the average number of PSD-95 spots per IHC was quantified (Figure 7D). MB pretreatment was effectively conserved in PSD-95 spots compared to noise exposed groups. These results indicate that the possible mechanism to explain the protective effect of MB is not only related to the mitochondrial function improvement of hair cells, but also to the preservation or regeneration of wah synaptic components.

The present invention uses an already established hearing loss animal model method, and this method has an advantage that it is very easy to be applied to clinical trials since the mammalian auditory system including humans has the same anatomical structure. In addition, short-induced hearing loss time (auditory system damaged 2 weeks after a permanent hearing loss after the change) in the pharmacological mechanism that is used to verify an existing vitro There is an advantage that a model system can be used in combination. The methylene blue concentration (2 mg / kg) used in the present invention is proved to be a level that is not toxic, but it can be administered for a long time due to the low unit price of methylene blue. In humans, no clinically significant side effects have been reported at doses of 1-2 mg / kg, and dosing at doses less than 7 mg / kg is recommended.

As an example of the present invention, a process for verifying auditory organ protection effect by methylene blue pretreatment will be described. The scope of the present invention is not limited to the scope of the present invention. For ease of explanation, description will be made in accordance with the experimental time sequence, and the implementation of the hearing protection effect of methylene blue and the verification method using the same will be described below together with experimental data.

Methylene blue  Pretreatment and noise exposure

In accordance with the experimental schedule of FIG. 1A, administration of methylene blue is started at the same time every day from 3 days before exposure to 8-week-old male BALB / c mice, and final administration is performed immediately before the noise exposure (total 4 doses). Methylene blue (66720, Sigma Aldrich, St. Louis, Mo.) dissolved in distilled water is orally administered at a dose of 2 mg / kg. Noise exposure is exposed using a noise generator for 3 hours at a sound pressure of 112 decibels (dB) (dB SPL). Immediately after the end of the noise exposure, methylene blue is finally administered to the mice. Control animals are not exposed to noise and do not receive MB. The noise-only group was treated with distilled water at the same time.

Hearing threshold  Change measurement

One day before the noise exposure, the auditory brainstem response (ABR) (pre-ABR), which is an electrophysiological test, is used to exclude the animals with hearing problems. Hearing status was measured using the Biosig 32 ABR system (Tucker-Davis Technologies, Gainesville, Fla.). Each mouse was anesthetized with an intraperitoneal injection of chloral hydrate. ABR was recorded subcutaneously using a sterile electrode needle. The reference electrode was inserted under the ear of the ear and beneath the opposite ear, and the active electrode was inserted under the skin above the head. Acoustic stimulation was given through earphones placed directly on the ear canal. After selecting the hearing frequency, the measurement was started after the pre-computed stimulus of 16 or 32 kHz was applied at 75 dB until the ABR waveform disappeared and the response at the stimulation level of 10 dB was confirmed automatically at a sound pressure reduced by 5 dB . The average of 1,000 stimuli at each frequency was obtained, and the results for each sound pressure level were recorded and stored. The hearing threshold was determined by the lowest intensity that could be derived with reproducible and visually detectable wavelengths. ABR for 16 and 32 kHz stimulation was recorded on days 1 and 14 after noise exposure as well as before noise exposure. To assess acute phase hearing loss, the inventors measured the change in hearing threshold (compound threshold shift) (CTS) 1 day after the end of noise exposure. To assess the permanent hearing loss, changes in the hearing threshold after the end of the 14th exposure (permanent threshold shift; PTS) were measured.

Confirming the inhibitory effect of active oxygen groups and active nitrogen groups

One week after the noise exposure, the mice were harvested from the inner ear tissues in the control, noise-only, and noise-exposed groups (pre-MB) after methylene blue pretreatment to prepare paraffin sections. Specifically, animals are anesthetized and perfused through the heart with saline containing 0.5% sodium nitrate and heparin (10 U / ml). And treated with 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer (pH 7.2) for tissue fixation. The temporal bone was dissected from both sides and fixed in 4% PFA at 4 ° C for 12 hours to obtain wah. The immobilized wau was washed three times with phosphate buffered saline (PBS) and put in a 5% ethylenediaminetetraacetic acid (EDTA) solution to remove the calcification for 3 days. After calcination, the sample was fixed with paraffin. Four successive 5 mu m thick sections were obtained using a sliding microtome (Leica, Wetzlar, Germany). Serial sections were placed on gelatin-coated slides, rinsed three times with PBS, treated with 3% H ₂ O ₂ for 5 minutes, and rinsed with PBS containing 0.2% Triton X-100 (PBST). Non-specific binding was blocked with 1% bovine serum albumin (BSA) dissolved in PBST. The sections were incubated with 4-hydroxynoninals (ab46545, 4-HNE; 1: 100 dilution; Abcam, Cambridge, UK) and 3-nitrotyrosine (ALX-804-505, 3-NT; , Farmingdale, NY) was incubated with the primary antibody at 4 ° C for 12 hours. After rinsing with PBST, the sections were incubated with biotinylated secondary antibody (BA-1000, Vector Laboratories, Burlingame, CA) for 1 hour and with avidin / biotin system (Vector Laboratories) for 1 hour, Was visualized using a solution of 3'-diaminobenzidine (D5637, DAB, Sigma Aldrich) (0.05% DAB and 0.003% hydrogen peroxide in 0.1 M PB). The nucleus was contrasted with hematoxylin-QS (H-3404, Vector Laboratories). The light field image was acquired using PictureFrame Application 2.3 software. The expression levels of 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-nitrotyrosine) were measured by immunohistochemistry (IHC) (Fig. 3 and Fig. 4). A further decrease in the amount of 4-HNE-tagged protein by methylene blue is further verified using the Western blot (WB) method (FIG. 3).

Identification of changes in neuronal expression level and observation of synaptic structure

Phalloidin staining was performed to detect changes in hair cells. After the ABR analysis on the 14th day, the temporal bone was dissected. The cut woof was perfused with 4% PFA in 0.1 M PB and kept overnight at 4 ° C. The wahs were washed three times for 10 minutes in PBS and decalcified for 3 days in 5% EDTA solution. After decalcification, the optical and optical samples were removed, and under the optical microscope the lateral wall, Reissner? The membrane and tectorial membrane were removed. Corti organ (OC) was stained with Texas Red VR-X Paloidin (T7471; 200 U / mL methanol diluted 1: 100 in PBS; Invitrogen, Eugene, OR) for 1 hour. Then, the entire sample was rinsed with PBS and cut into microdissected into individual turns, and 4 ', 6-diamidino-2-phenylindole (H-1200; 1.5 μg / ml; DAPI; Vector Laboratories ) Was placed on a glass slide. Slides were incubated for a minimum of 15 minutes. OC hair cells were observed with a confocal microscope (LSM510, Carl Zeiss, Jena, Germany). The test area was 32kHz corresponding to the upper part of the hook region and was visually measured and measured in a plurality of samples obtained from 4 mice per experiment group.

The criteria for loss of hair cell loss was determined to be missing signals (missing OHC) using DAPI and Texas Red VR-X phalloidin. Missing OHC was observed as a black dot under a microscope. In immunofluorescence, the end of the centrifugal nerve was labeled with anti-synapsin-1 (SC-55774; 1: 100 dilution; Santa Cruz Biotechnology, Santa Cruz, Calif.), 028, PSD-95; 1: 400 dilution; UC Davis / NIH NeuroMab Facility, Davis, CA) to label post-synaptic density in primary wah neurons. The cleaved OCs were incubated with the primary antibody for 12 hours. After rinsing with PBST, samples were incubated with Alexa Fluor 568 conjugated secondary antibody (A11057, A11031, Invitrogen) for 1 hour. The number of PSD-95 spots in the dendrites directly in contact with the oil-resistant cells was visually measured. Each IHC and PSD-95 spots within the region corresponding to 32 kHz were measured. Each measured region has a length corresponding to 12 to 15 oil-resistant cells.

To avoid underestimation of spots due to stacking of stacked images, a three-dimensional image was created with the Zeiss LSM Image Browser and rotated ( xy plane projection image) to evaluate accurately. Three mice in the control group, six mice in the noise-only exposed group and six mice in the MB pretreated group were used on the second day after the noise exposure.

The mouse inner ear tissues were collected from 3 hours after the end of the noise exposure to 24 hours and 72 hours, and the change in the expression level of neurotrophic factor (NTF) was observed using the WB method (FIG. 6).

In addition, 48 hours after the end of the noise exposure, the mouse inner ear tissues were collected and surface preparation was performed to observe changes in the expression levels of synaptic markers synapsin-1 and PSD-95 protein (FIG. 7).

Claims (9)

A pharmaceutical composition for treating or preventing sensory neuron degeneration comprising a compound represented by the following formula (1).
[Chemical Formula 1]

The pharmaceutical composition according to claim 1, wherein the sensory neuron deafness is either noise-induced hearing loss or senile hearing loss. 2. The pharmaceutical composition according to claim 1, wherein said sensory neural deafness is due to hair cell damage or denaturation of inner ear. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition acts on mitochondria of inner ear. 5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition effectively reduces active oxygen groups in the mitochondrion. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition induces an increased expression of a neurotrophic factor. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is selected from oral solid pharmaceutical preparations comprising tablets, pills, capsules, powders and granules. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is selected from the group consisting of pharmaceutically acceptable aqueous solutions, suspensions, emulsions, syrups, pharmaceutical preparations dissolved in use and elixirs. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is an injectable parenteral pharmaceutical preparation.

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