WO2023190707A1

WO2023190707A1 - Composition for preventing or treating sensorineural hearing loss

Info

Publication number: WO2023190707A1
Application number: PCT/JP2023/012857
Authority: WO
Inventors: 昌一島田; 誠近藤; 秀典猪原; 和也大畠
Original assignee: 国立大学法人大阪大学
Priority date: 2022-03-30
Filing date: 2023-03-29
Publication date: 2023-10-05

Abstract

[Problem] To clarify the mechanism of the medial olivocochlear (MOC) feedback system and provide a composition for preventing or treating auditory disorders in which MOC neurons participate. To provide a method for screening a compound for preventing or treating auditory disorders in which MOC neurons participate. [Solution] A composition for preventing or treating sensorineural hearing loss that contains a serotonin 3 receptor agonist. Sensorineural hearing loss includes acoustic trauma hearing loss, age-related hearing loss, etc. Also provided is a composition for activating MOC neurons that contains a serotonin 3 receptor agonist. The serotonin 3 receptor agonist activates MOC neurons and thus enables the prevention and treatment of sensorineural hearing loss. Also provided are: a method for screening a compound for preventing or treating sensorineural hearing loss, said method including a step for measuring serotonin 3 receptor agonist activity; and a method for screening a compound activating MOC neurons.

Description

Composition for prevention or treatment of sensorineural hearing loss

The present invention relates to the prevention or treatment of hearing impairment.

The organ of Corti, which is the sensory epithelium in the cochlea, has inner and outer hair cells, and each has afferent nerves going to the center and efferent nerves going from the center to the hair cells. The afferent nerves are divided into spiral nerves type 1 and type 2, both of which project to the cochlear nucleus. On the other hand, one of the efferent nerves is called a MOC (medial olivocochlear) neuron, and the other is called a LOC (lateral olivocochlear) neuron. The input sound is adjusted by efferent nerves (efference feedback control).

The cochlear efferent feedback system conveyed by the olive cochlear pathway plays an important role in auditory processing (Non-Patent Document 1). MOC neurons are central in this neural pathway (Non-Patent Documents 2, 3), and the efferent feedback system via MOC neurons is called the MOC feedback system. The MOC feedback system adjusts the dynamic range of hearing (Non-patent Document 4), has the function of protecting against acoustic trauma (Non-patent Documents 5, 6), and mediates signal detection in noisy environments (Non-patent Documents 7). However, the cellular and molecular mechanisms of the MOC feedback system are not completely understood.

The serotonin 3 (5-HT3) receptor is the only ionotropic receptor in the serotonin receptor family (Non-Patent Document 8). The 5-HT3 receptor consists of two subunits (5-HT3A and 5-HT3B), and the 5-HT3A subunit is essential for the formation of a functional receptor (Non-Patent Document 8). It has been suggested that 5-HT3A receptors are widely expressed in the central and peripheral nervous systems and are related to various important neurological functions (Non-Patent Document 9). In a previous study, the present inventors reported that 5-HT3A receptors are expressed in the superior olivary complex (SOC) of the brainstem, where MOC neurons and LOC neurons are located (Non-patent Document 10 ). However, the relationship between the MOC feedback system and 5-HT3 receptors is unknown.

Acoustic trauma is damage to the auditory system in which the cochlea of the inner ear is damaged by powerful sound waves. Symptoms include hearing loss and tinnitus. Chronic acoustic trauma, also called noise-induced hearing loss, occurs when people are exposed to high-intensity noise in the environment for long periods of time. Chronic acoustic trauma (noise-induced hearing loss) progresses gradually and has few symptoms. Acute acoustic trauma includes hearing loss caused by instantaneous or extremely short-lived loud noises caused by explosions, firearms, airbags, etc., and hearing loss caused by exposure to loud noises from headphones, concerts, etc. over a period of several minutes to several hours. Includes disability. Most environments in which people are exposed to loud noises for long periods of time were mostly occupational, but with the improvement in the performance and portability of audio equipment in recent years, it has been pointed out that there is a danger that prolonged exposure to loud noises is becoming more common in everyday life. has been done.

As a serotonin 3 receptor agonist, for example, SR57227A (1-(6-chloropyridin-2-yl)piperidin-4-amine or its hydrochloride) is known (Patent Document 1).

Japanese Patent Application Publication No. 7-145166

The present invention aims to elucidate the mechanism of the MOC feedback system, provide compositions for preventing or treating hearing disorders involving MOC neurons, and compounds for preventing or treating hearing disorders involving MOC neurons. The objective is to provide a screening method for

As a result of conducting research to solve the above problems, the present inventors discovered that serotonin 3 receptors (hereinafter also referred to as 5-HT3 receptors) have a fundamental role in the MOC feedback system, and that 5-HT3 receptors have a fundamental role in the MOC feedback system. The inventors have discovered that a body agonist can solve the above problems, and have completed the present invention. That is, the present invention includes the following aspects.
1. A composition for preventing or treating sensorineural hearing loss containing a serotonin 3 receptor agonist.
2. 2. The composition according to item 1, wherein the sensorineural hearing loss is acoustic trauma or age-related hearing loss.
3. The symptoms of acoustic trauma or age-related hearing loss include hearing loss, insufficient speech discrimination in a noisy environment even with normal hearing, and insufficient speech discrimination in a noisy environment accompanied by hearing loss. The composition according to item 2 above, wherein the composition has at least one selected from the group consisting of:
4. 2. The composition according to item 1, wherein the sensorineural hearing loss is hearing loss due to exposure to loud sounds.
5. The serotonin 3 receptor agonist has the following formula (I):

[In the formula,
m is an integer from 1 to 4;
R ¹ is a hydrogen atom, a halogen atom, a methyl group optionally substituted with 1 to 3 halogen atoms, a methoxy group optionally substituted with 1 to 3 halogen atoms, 1 to 3 halogen atoms an ethoxy group which may be substituted with , a methylthio group which may be substituted with 1 to 3 halogen atoms, and a halogen atom, trifluoromethyl, C _1-3 alkyl, C _1-3 alkoxy, C _{1 -3} phenoxy groups optionally substituted with alkylthio or cyano groups, each independently selected from the group consisting of
The composition according to any one of items 1 to 4 above, which is a compound represented by or a pharmaceutically acceptable salt thereof.
6. 5. The composition according to item 5, wherein each R ¹ is independently a halogen atom.
7. 7. The composition according to item 6, wherein the halogen atom is a chlorine atom.
8. 8. The composition according to any one of items 1 to 7 above, which is a therapeutic composition.
9. A composition for activating MOC neurons containing a serotonin 3 receptor agonist.
10. 10. The composition according to item 9, which is used for the prevention or treatment of hearing disorders involving MOC neurons.
11. A method for screening a compound for prevention or treatment of sensorineural hearing loss, comprising the step of measuring serotonin 3 receptor agonist activity.
12. A method for screening MOC neuron activating compounds, the method comprising the step of measuring serotonin 3 receptor agonist activity.
13. 13. The screening method according to item 12, wherein the MOC neuron activating compound is a compound for preventing or treating hearing loss associated with MOC neurons.
14. A method of preventing or treating sensorineural hearing loss comprising administering a composition containing a serotonin 3 receptor agonist to a subject in need thereof.
15. A method of activating MOC neurons comprising administering a composition containing a serotonin 3 receptor agonist to a subject in need thereof.
16. A method for preventing or treating hearing impairment involving MOC neurons, the method comprising administering a composition containing a serotonin 3 receptor agonist to a subject in need thereof.
17. Use of a serotonin-3 receptor agonist for the preparation of a composition for the prevention or treatment of sensorineural hearing loss.
18. Use of a serotonin 3 receptor agonist for the preparation of a composition for activating MOC neurons.
19. Use of a serotonin 3 receptor agonist for the preparation of a composition for preventing or treating hearing disorders involving MOC neurons.

Stimulation of 5-HT3 receptors by 5-HT3 receptor agonists can activate MOC neurons and enhance or improve the function of the MOC feedback system via MOC neurons. 5-HT3 receptors have a fundamental role in the MOC feedback system. Sensorineural hearing loss, which is a hearing impairment or auditory condition involving impaired or inadequate functioning of MOC neurons, can be prevented or treated with 5-HT3 receptor agonists.

The MOC feedback system is a system that responds in real time to sounds that are too loud or difficult to hear by regulating the movement of outer hair cells and controlling the amplitude of the basilar membrane through its efferent neurons. One of the functions of the MOC feedback system is to protect the cochlea by suppressing the vibration of the basilar membrane in response to strong loud sounds and suppressing damage to hair cells and the like. Enhancement of MOC neuron activity via 5-HT3 receptors by 5-HT3 receptor agonists enhances cochlear protection and enables the prevention and treatment of hearing loss caused by exposure to high-intensity sounds. One of the other functions of the MOC feedback system is the ability to amplify and suppress basilar membrane vibrations that correspond to specific frequencies. For example, in the case of a small sound, the vibration of the basilar membrane is amplified, and when talking in noise, the vibration of the basilar membrane corresponding to the frequency range of the human voice is amplified, and the vibration of the basilar membrane is amplified to correspond to the frequency range of the surrounding noise. Suppresses the vibration of the basement membrane. Enhancement of MOC neuron activity via 5-HT3 receptors by 5-HT3 receptor agonists enables the prevention and treatment of hearing disorders such as age-related hearing loss and insufficient speech discrimination ability in noisy environments. .

Figure 1 shows that serotonin 3 receptors are expressed on MOC neurons that project to the cochlea. (A) Coronal section of mouse brainstem stained with hematoxylin-eosin is shown. (B) shows a coronal section of the brainstem of a 5-HT3AR-EGFP transgenic reporter mouse stained with enhanced green fluorescent protein (EGFP). (C) Coronal section of the brainstem of a wild-type mouse after intratympanic injection of Fluoro-Gold™ (hereinafter also referred to as FG). Left: Ipsilateral; Right: Contralateral. (D) Coronal section of the brainstem of a 5-HT3AR-EGFP transgenic reporter mouse after intratympanic FG injection is shown. Arrows indicate that FG-labeled neurons are in the same position as the EGFP signal. Top: low magnification; bottom: high magnification (A-D) The dotted lines indicate the approximate outlines of the MOC and LOC regions according to the mouse brain map. The scale bar indicates 200 μm. At least three independent experiments showed similar results. Figure 2 shows the results of immunohistochemical analysis of the cochlea of a 5-HT3AR-EGFP transgenic reporter mouse, showing the distribution of efferent fibers of 5-HT3 receptor-expressing MOC neurons in the cochlea. (A) Immunostaining for GFP and ChAT (choline acetyltransferase) and DAPI counterstaining in the basal, middle, and apical regions of a cochlear whole-mount specimen. (B) Immunostaining for GFP and ChAT and DAPI counterstaining of cochlear sections is shown. Arrowheads indicate EGFP signals on nerve fibers traversing the tunnel of Corti, and arrows indicate EGFP signals below OHCs (outer hair cells). GFP: Green, ChAT: Red, DAPI: Blue, but all are shown in gray in gray scale. Scale bars indicate 10 μm (A) and 20 μm (B). At least three independent experiments showed similar results. Figure 3 shows that 5-HT3 receptors are involved in the activation of MOC neurons by exposure to high-intensity sound. (A) Shows c-Fos immunostaining of a coronal section of the brainstem of a wild type mouse before exposure to high-intensity sound. (B) Immunostaining of c-Fos in the coronal section of the brainstem of a wild-type mouse injected with FG intratympanically 1 h after exposure to high-intensity sound. Arrows indicate c-Fos positive neurons labeled by FG. Top: low magnification; bottom: high magnification. (C) Immunostaining of c-Fos in a coronal section of the brain stem of a 5-HT3AR-EGFP transgenic mouse 1 h after exposure to high-intensity sound. Arrows indicate EGFP positive neurons coincident with c-Fos. (D, E) Shows the results of immunohistochemical analysis of c-Fos in the brainstem of wild type (WT) mice and Htr3a ^−/− (KO) mice 1 h after exposure to high-intensity sound. (D) Shows c-Fos immunostaining in MOC 1 h after exposure to high-intensity sound. (E) It is a graph showing the number of c-Fos positive cells in MOC (n=5 for each group) 1 h after exposure to strong sound, and shows the mean ± standard error. ^* P<0.05 (two-tailed Student's t-test); dotted line indicates the approximate outline of the MOC. Scale bar indicates 200 μm (A-D). ; at least three independent experiments showed similar results (AC). FIG. 4 shows that Htr3a ^−/− mice had impaired MOC function and hearing loss was exacerbated by exposure to loud sounds. (A) shows the degree of suppression of DPOAE by contralateral sounds in wild type (WT) mice and Htr3a ^−/− (KO) mice (n = 10 in each group). (B) shows the magnitude of DPOAE in WT mice and KO mice (n=10 in each group) under quiet conditions before and 7 days after exposure to loud noises. (C) ABR thresholds of WT mice and KO mice (n=6 in each group) before and 7 days after exposure to loud noises are shown. All graphs are presented as mean ± standard error. ^* P<0.05, ^** P<0.01, ^*** P<0.001 (one-way ANOVA, followed by the Holm-Sidak multiple comparison test) Figure 5 shows the results of immunohistochemical analysis of the organ of Corti in wild-type (WT) mice and Htr3a ^−/− (KO) mice before, 7 days after, and 14 days after exposure to high-intensity noise. Indicates hair cell damage. (A) Photograph of the base, middle, and apex of the cochlea stained with rhodamine phalloidin, with arrows indicating outer hair cell (OHC) loss. Scale bar is 10 μm. ( ^B ) Each region of the cochlea (base, The amount of OHC in the middle part and top part) is shown. The amount of OHC after exposure to high-intensity sound is expressed as a percentage (%) of the amount before exposure to high-intensity sound. All graphs are presented as mean ± standard error. Figure 6 shows that Htr3a ^−/− mice lose many ribbon synapses due to high-intensity sound. C-terminal-binding protein 2 (CtBP2) was used as a presynaptic marker of ribbon synapses, and GluA2 was used as a postsynaptic marker. The results of immunohistochemical analysis of ribbon synapses of inner hair cells (IHC) are shown. CtBP2-positive points are shown in green and GluA2-positive points are shown in red. On a gray scale, CtBP2-positive points are shown in white and GluA2-positive points are shown in gray. It is indicated by. (A) Immunostaining of CtBP2 and GluA2 in the cochlea. Arrows indicate parallel CtBP2/GluA2 positive points in IHC. The scale bar indicates 10 μm. (B) Shows the results of immunohistochemical analysis of CtBP2 and GluA2 in IHC of wild type (WT) and Htr3a ^−/− (KO) mice before, 1 hour, 24 hours, and 7 days after exposure to high-intensity sound. . The scale bar indicates 5 μm. The dotted line indicates the approximate outline of the IHC. Figure 7 shows that in Htr3a ^−/− mice, high-intensity sound causes loss of many inner hair cell (IHC) ribbon synapses in the cochlear base region. Immunohistochemical analysis results of IHC ribbon synapses in the cochlear base region of wild type (WT) and Htr3a ^−/− (KO) mice, showing the number of CtBP2-positive points (A) and the number of GluA2-positive points (B) of IHC. ) and the number of parallel CtBP2/GluA2 positive points (C) are shown. All graphs are presented as mean ± standard error. Each group n=5, ^* P<0.05, ^** P<0.01 (Student's t-test with Holm-Sidak correction) Figure 8 shows that in Htr3a ^−/− mice, high-intensity sound causes loss of many inner hair cell (IHC) ribbon synapses in the midcochlear region. Results of immunohistochemical analysis of IHC ribbon synapses in the midcochlear region of wild type (WT) and Htr3a ^−/− (KO) mice, showing the number of CtBP2-positive points (A), the number of GluA2-positive points ( B) and the number of parallel CtBP2/GluA2 positive points (C) are shown. All graphs are presented as mean ± standard error. Each group n=5, ^* P<0.05, ^** P<0.01 (Student's t-test with Holm-Sidak correction) Figure 9 shows the results of immunohistochemical analysis of IHC ribbon synapses in the apical cochlear region of wild type (WT) and Htr3a ^−/− (KO) mice, including the number of CtBP2-positive points (A) and GluA2-positive points of IHC. (B) and the number of parallel CtBP2/GluA2 positive points (C). All graphs are presented as mean ± standard error. Each group n=5 (Student's t-test with Holm-Sidak correction) FIG. 10 shows that 5-HT3 receptor agonists attenuate loud sound-induced hearing loss. (A) and (B) show cochlear function 7 days after exposure to high-intensity sound. Physiological saline (Sal) or SR57227A (Ag) was administered to mice 30 minutes before exposure to high-intensity sound. (A) Shows the results of DPOAE (distortion component otoacoustic emissions) in wild type (WT) and Htr3a ^−/− (KO) mice (n=10 in each group). (B) Shows the results of ABR (auditory brainstem response) of WT and KO mice (n=6 for each group). ^* P<0.05, ^** P<0.01, one-way ANOVA, followed by the Holm-Sidak multiple comparison test FIG. 11 shows that 5-HT3 receptor agonists attenuate loud sound-induced loss of ribbon synapses. (A) (B) and (C) IHC of the medial region of the cochlea of wild type (WT) and Htr3a ^−/− (KO) mice (n = 5 in each group) 24 hours after high-intensity sound exposure. The number of CtBP2-positive puncta (A), the number of GluA2-positive puncta (B), and the number of parallel CtBP2/GluA2-positive puncta (C) are shown. Mice were administered physiological saline (Sal), SR57227A (Ag), or ondansetron (Ant) 30 minutes before loud sound exposure. IHC: inner hair cells; NE: loud sound exposure (+: with exposure, -: without exposure) All graphs are shown as mean ± standard error. ^* P<0.05, ^** P<0.01, ^*** P<0.001; ns (not significant), one-way ANOVA, followed by Tukey's multiple comparison test

The serotonin 3 receptor agonist, which is the active ingredient of the composition of the present invention, is a substance that exerts a similar effect to serotonin on the serotonin 3 receptor, and is not limited thereto.

Suitable compounds for the serotonin 3 receptor agonist, which is an active ingredient of the composition of the present invention, include any one of the following formulas (I), (II), (III), (IV), (V) or (VI). Mention may be made of the indicated compounds or their pharmaceutically acceptable salts, or their hydrates or solvates.

The groups defined in the substituents of the structural formulas of the compounds of formulas (I), (II), (III), (IV), (V) and (VI) may be freely combined unless otherwise specified. I can do it.

In this specification, the number of carbon atoms in the definition of "substituent" may be expressed as "C _1-3 ", "C _1-6 ", etc., for example. Specifically, the notation "C _1-3 alkyl" is synonymous with a linear or branched alkyl group having 1 to 3 carbon atoms, and the notation "C _1-6 alkyl" has the same meaning as a straight-chain or branched alkyl group having 1 to 3 carbon atoms. It has the same meaning as 1 to 6 linear or branched alkyl groups.

The term "group" as used herein means a monovalent group. For example, "alkyl group" means a monovalent saturated hydrocarbon group. Furthermore, in the description of substituents in this specification, the term "group" may be omitted. Furthermore, unless otherwise specified, the description of each group also applies when the group is a part or substituent of another group.

Examples of the "halogen atom" include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

In one embodiment, the serotonin 3 receptor agonist that is the active ingredient of the present invention has the following formula (I):

[In the formula,
m is an integer from 1 to 4;
R ¹ is a hydrogen atom, a halogen atom, a methyl group optionally substituted with 1 to 3 halogen atoms, a methoxy group optionally substituted with 1 to 3 halogen atoms, 1 to 3 halogen atoms an ethoxy group which may be substituted with , a methylthio group which may be substituted with 1 to 3 halogen atoms, and a halogen atom, trifluoromethyl, C _1-3 alkyl, C _1-3 alkoxy, C _{1 -3} phenoxy groups optionally substituted with alkylthio or cyano groups, each independently selected from the group consisting of
or a pharmaceutically acceptable salt thereof.

m is an integer of 1 to 4, preferably an integer of 1 to 3, more preferably an integer of 1 to 2, particularly preferably 1.

The halogen atom in R ¹ is preferably a fluorine atom or a chlorine atom, with a chlorine atom being particularly preferred.

The methyl group optionally substituted with 1 to 3 halogen atoms in R ¹ is preferably a methyl group optionally substituted with 1 to 3 fluorine atoms, and methyl groups and trifluoromethyl groups are particularly preferred. preferable.

The methoxy group optionally substituted with 1 to 3 halogen atoms in R ¹ is preferably a methoxy group optionally substituted with 1 to 3 fluorine atoms, and methoxy groups and trifluoromethoxy groups are particularly preferred. preferable.

The ethoxy group optionally substituted with 1 to 3 halogen atoms in R ¹ is preferably an ethoxy group optionally substituted with 1 to 3 fluorine atoms, and ethoxy groups and 2,2,2- Particularly preferred is trifluoroethoxy group.

The methylthio group optionally substituted with 1 to 3 halogen atoms in R ¹ is preferably a methylthio group optionally substituted with 1 to 3 fluorine atoms, with methylthio groups and trifluoromethylthio groups being particularly preferred. preferable.

R ¹ can be a phenoxy group optionally substituted with a halogen atom, trifluoromethyl, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 alkylthio or cyano group. "C _1-3 alkyl", "C _1-3 alkoxy" and "C _1-3 alkylthio" are residues of saturated aliphatic hydrocarbons having 1, 2 or 3 carbon atoms (methyl, ethyl, propyl, isopropyl, etc. )including.

In one preferred embodiment, each R ¹ is independently a halogen atom, more preferably each R ¹ is a chlorine atom.

In one preferred embodiment, m is 1. In a more preferred embodiment, R ¹ is a halogen atom and m is 1.

In a preferred embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof has the following formula (I'):

[In the formula, R ¹ has the same meaning as above]
or a pharmaceutically acceptable salt thereof.

In a further preferred embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is of the following formula:

(Compound name: 1-(6-chloropyridin-2-yl)piperidin-4-amine; hereinafter also referred to as "Compound A") or a pharmaceutically acceptable salt thereof. Particularly preferred is SR57227A (compound A or its hydrochloride).

In another embodiment, the serotonin 3 receptor agonist that is the active ingredient of the present invention has the following formula (II):

[In the formula,
n is an integer from 1 to 4;
R ² is a hydrogen atom, a halogen atom, a hydroxy group, a cyano group, a methyl group optionally substituted with 1 to 3 halogen atoms, a methoxy group optionally substituted with 1 to 3 halogen atoms, and each independently selected from the group consisting of methylthio groups optionally substituted with 1 to 3 halogen atoms]
or a pharmaceutically acceptable salt thereof.

n is an integer of 1 to 4, preferably an integer of 1 to 3, more preferably an integer of 1 to 2, particularly preferably 1.

The halogen atom in R ² is preferably a fluorine atom or a chlorine atom, with a chlorine atom being particularly preferred.

The methyl group optionally substituted with 1 to 3 halogen atoms in R ² is preferably a methyl group optionally substituted with 1 to 3 fluorine atoms, and methyl groups and trifluoromethyl groups are particularly preferred. preferable.

The methoxy group optionally substituted with 1 to 3 halogen atoms in R ² is preferably a methoxy group optionally substituted with 1 to 3 fluorine atoms, and methoxy groups and trifluoromethoxy groups are particularly preferred. preferable.

The methylthio group optionally substituted with 1 to 3 halogen atoms in R ² is preferably a methylthio group optionally substituted with 1 to 3 fluorine atoms, with methylthio groups and trifluoromethylthio groups being particularly preferred. preferable.

In one embodiment, each R ² is independently a hydrogen atom or a halogen atom, preferably each R ² is independently a hydrogen atom or a chlorine atom.

In another embodiment, n is 1. In another embodiment, R ² is a hydrogen atom or a halogen atom and n is 1.

In another embodiment, the compound of formula (II) or a pharmaceutically acceptable salt thereof is of formula (II'):

[In the formula, R ² has the same meaning as above]
or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the compound of formula (II) or a pharmaceutically acceptable salt thereof has the following formula:

(Compound name: m-chlorophenyl biguanide; hereinafter also referred to as "Compound B") or a pharmaceutically acceptable salt thereof, and particularly preferably the hydrochloride of Compound B.

The compound represented by (compound name: 1-phenylbiguanide; hereinafter also referred to as "compound C") or a pharmaceutically acceptable salt thereof, and particularly preferably the hydrochloride of compound C.

In another embodiment, the serotonin 3 receptor agonist that is the active ingredient of the present invention has the following formula (III):

[In the formula,
o is an integer from 1 to 4;
R ³ is a hydrogen atom, a halogen atom, a hydroxy group, a cyano group, a methyl group optionally substituted with 1 to 3 halogen atoms, a methoxy group optionally substituted with 1 to 3 halogen atoms, and each independently selected from the group consisting of methylthio groups optionally substituted with 1 to 3 halogen atoms;
R ⁴ is selected from the group consisting of a hydrogen atom and a methyl group optionally substituted with 1 to 3 halogen atoms]
or a pharmaceutically acceptable salt thereof.

o is an integer of 1 to 4, preferably an integer of 1 to 3, more preferably an integer of 1 to 2, particularly preferably 1.

The halogen atom in R ³ is preferably a fluorine atom or a chlorine atom, with a chlorine atom being particularly preferred.

The methyl group optionally substituted with 1 to 3 halogen atoms in R ³ is preferably a methyl group optionally substituted with 1 to 3 fluorine atoms, with methyl groups and trifluoromethyl groups being particularly preferred. preferable.

The methoxy group optionally substituted with 1 to 3 halogen atoms in R ³ is preferably a methoxy group optionally substituted with 1 to 3 fluorine atoms, and methoxy groups and trifluoromethoxy groups are particularly preferred. preferable.

The methylthio group optionally substituted with 1 to 3 halogen atoms in R ³ is preferably a methylthio group optionally substituted with 1 to 3 fluorine atoms, and methylthio and trifluoromethylthio groups are particularly preferred. preferable.

The methyl group optionally substituted with 1 to 3 halogen atoms in R ⁴ is preferably a methyl group optionally substituted with 1 to 3 fluorine atoms, particularly preferably a methyl group.

In one embodiment, each R ³ is independently a hydrogen atom or a halogen atom, preferably each R ³ is independently a hydrogen atom or a chlorine atom.

In another embodiment, R ⁴ is a hydrogen atom or a methyl group.

In another embodiment, o is 1. In another embodiment, R ³ is a hydrogen atom or a halogen atom, R ⁴ is a hydrogen atom or a methyl group, and o is 1.

In another embodiment, the compound represented by formula (III) or a pharmaceutically acceptable salt thereof is represented by the following formula (III'):

[In the formula, R ⁴ has the same meaning as above]
or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the compound represented by formula (III) or a pharmaceutically acceptable salt thereof has the following formula:

(Compound name: N-methylquipazine; hereinafter also referred to as "Compound D") or a pharmaceutically acceptable salt thereof, and particularly preferred is the dimaleate of Compound D.

The compound represented by (compound name: quipazine; hereinafter also referred to as "compound E") or a pharmaceutically acceptable salt thereof, and particularly preferably the dimaleate of compound E.

In another embodiment, the serotonin 3 receptor agonist that is the active ingredient of the present invention has the following formula (IV):

[In the formula,
p is an integer from 1 to 4;
R ⁵ and R ⁶ are hydrogen atom, halogen atom, hydroxy group, cyano group, methyl group optionally substituted with 1 to 3 halogen atoms, methoxy optionally substituted with 1 to 3 halogen atoms and methylthio optionally substituted with 1 to 3 halogen atoms;
R ⁷ is selected from the group consisting of a hydrogen atom and a methyl group optionally substituted with 1 to 3 halogen atoms]
or a pharmaceutically acceptable salt thereof.

p is an integer of 1 to 4, preferably an integer of 1 to 3, more preferably an integer of 1 to 2, particularly preferably 1.

As the halogen atom in R ⁵ and R ⁶ , a fluorine atom and a chlorine atom are preferred, and a chlorine atom is particularly preferred.

The methyl group optionally substituted with 1 to 3 halogen atoms in R ⁵ and R ⁶ is preferably a methyl group optionally substituted with 1 to 3 fluorine atoms, and methyl groups and trifluoromethyl Particularly preferred are groups.

The methoxy group optionally substituted with 1 to 3 halogen atoms in R ⁵ and R ⁶ is preferably a methoxy group optionally substituted with 1 to 3 fluorine atoms, and methoxy groups and trifluoromethoxy Particularly preferred are groups.

The methylthio group optionally substituted with 1 to 3 halogen atoms in R ⁵ and R ⁶ is preferably a methylthio group optionally substituted with 1 to 3 fluorine atoms, and methylthio group and trifluoromethylthio group are preferred. Particularly preferred are groups.

The methyl group optionally substituted with 1 to 3 halogen atoms in R ⁷ is preferably a methyl group optionally substituted with 1 to 3 fluorine atoms, particularly preferably a methyl group.

In one embodiment, each R ⁵ is independently selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxy group, and a cyano group, and preferably each R ⁵ is a hydroxy group.

In another embodiment, R ⁶ is selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxy group, a cyano group, and a methyl group optionally substituted with 1 to 3 halogen atoms, preferably a methyl group. be.

In another embodiment, R ⁷ is a hydrogen atom or a methyl group, preferably a hydrogen atom.

In another embodiment, p is 1. In another embodiment, R ⁵ is selected from the group consisting of halogen atoms, hydroxy groups, and cyano groups, and R ⁶ is hydrogen atoms, halogen atoms, and methyl optionally substituted with 1 to 3 halogen atoms. R ⁷ is a hydrogen atom or a methyl group, and p is 1.

In another embodiment, the compound of formula (IV) or a pharmaceutically acceptable salt thereof is of formula (IV'):

[In the formula, R ⁵ , R ⁶ and R ⁷ have the same meanings as above]
or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the compound of formula (IV) or a pharmaceutically acceptable salt thereof has the following formula:

The compound represented by (compound name: 2-methyl-5-hydroxytryptamine; hereinafter also referred to as "compound F") or a pharmaceutically acceptable salt thereof, particularly preferably the hydrochloride of compound F.

In another embodiment, the serotonin 3 receptor agonist that is the active ingredient of the present invention has the following formula (V):

[In the formula,
q is an integer from 1 to 4;
R ⁸ and R ⁹ are hydrogen atom, halogen atom, hydroxy group, cyano group, methyl group optionally substituted with 1 to 3 halogen atoms, methoxy optionally substituted with 1 to 3 halogen atoms and methylthio optionally substituted with 1 to 3 halogen atoms;
R ¹⁰ is selected from the group consisting of a hydrogen atom and a methyl group optionally substituted with 1 to 3 halogen atoms]
or a pharmaceutically acceptable salt thereof.

q is an integer of 1 to 4, preferably an integer of 1 to 3, more preferably an integer of 1 to 2, particularly preferably 1.

As the halogen atom in R ⁸ and R ⁹ , a fluorine atom and a chlorine atom are preferred, and a chlorine atom is particularly preferred.

The methyl group optionally substituted with 1 to 3 halogen atoms in R ⁸ and R ⁹ is preferably a methyl group optionally substituted with 1 to 3 fluorine atoms, and methyl groups and trifluoromethyl Particularly preferred are groups.

The methoxy group optionally substituted with 1 to 3 halogen atoms in R ⁸ and R ⁹ is preferably a methoxy group optionally substituted with 1 to 3 fluorine atoms, and methoxy groups and trifluoromethoxy Particularly preferred are groups.

The methylthio group optionally substituted with 1 to 3 halogen atoms in R ⁸ and R ⁹ is preferably a methylthio group optionally substituted with 1 to 3 fluorine atoms, and methylthio group and trifluoromethylthio group are preferred. Particularly preferred are groups.

The methyl group optionally substituted with 1 to 3 halogen atoms in R ¹⁰ is preferably a methyl group optionally substituted with 1 to 3 fluorine atoms, particularly preferably a methyl group.

In one embodiment, each R ⁸ is independently selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxy group, and a cyano group, and preferably each R ⁸ is a hydrogen atom.

In another embodiment, R ⁹ is selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxy group, a cyano group, and a methyl group optionally substituted with 1 to 3 halogen atoms, preferably a hydrogen atom. be.

In another embodiment, R ¹⁰ is a hydrogen atom or a methyl group, preferably a methyl group.

In another embodiment, q is 1. In another embodiment, R ⁸ is a hydrogen atom or a hydroxy group, R ⁹ is a hydrogen atom or a methyl group, R ¹⁰ is a hydrogen atom or a methyl group, and q is 1.

In another embodiment, the compound represented by formula (V) or a pharmaceutically acceptable salt thereof has the following formula (V'):

[In the formula, R ⁸ , R ⁹ , and R ¹⁰ have the same meanings as above]
or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the compound of formula (V) or a pharmaceutically acceptable salt thereof has the following formula:

The compound represented by (RS56812; compound name: (R)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2-(1-methyl-1H-indol-3-yl)- 2-(1-methyl-1H-indol-3-yl)-2-oxoacetamide; hereinafter also referred to as "Compound G") or a pharmaceutically acceptable salt thereof, particularly preferably the hydrochloride of Compound G. be.

In another embodiment, the active ingredient of the present invention, a serotonin 3 receptor agonist, has the following formula (VI):

[In the formula, Q is the following formula (a) to (c):

(In the formula, R ¹¹ represents a hydrogen atom or a C _1-6 alkyl group;
R ¹² and R ¹³ are the same or different and represent a hydrogen atom or a C _1-6 alkyl group;
Alternatively, they may be combined with the carbon atoms to which they are bonded to form a 3- to 8-membered cycloalkane ring;
R ¹⁴ and R ¹⁵ are the same or different and represent a hydrogen atom or a C _1-6 alkyl group;
Alternatively, they may be combined with the nitrogen atom to which they are bonded to form a 3- to 8-membered cyclic amine;
n represents 0, 1, 2, 3, 4, or 5;
Here, when R ¹⁴ and R ¹⁵ are both hydrogen atoms, R ¹² represents a group represented by any one of C _2-6 alkyl group]
or a pharmaceutically acceptable salt thereof.

Preferably, Q is a group of formula (a) or (b). More preferably, Q is a group represented by formula (a).

Preferably n is 1, 2 or 3.

Preferably R ¹² is a hydrogen atom. Preferably, R ¹³ is a hydrogen atom or a C _1-6 alkyl group. More preferably, R ¹² is a hydrogen atom, and R ¹³ is a hydrogen atom or a C _1-6 alkyl group.

Preferably, R ¹⁴ is a hydrogen atom or a C _1-6 alkyl group. Suitably R ¹⁵ is a C _1-6 alkyl group. More preferably, R ¹⁴ is a hydrogen atom or a C _1-6 alkyl group, and R ¹⁵ is a C _1-6 alkyl group.

Preferably, R ¹¹ is a hydrogen atom. Preferably, R ¹¹ and R ¹² are hydrogen atoms. Preferably, R ¹¹ is a hydrogen atom, and R ¹³ is a hydrogen atom or a C _1-6 alkyl group. More preferably, R ¹¹ and R ¹² are hydrogen atoms, and R ¹³ is a hydrogen atom or a C _1-6 alkyl group.

Preferably, R ¹¹ is a hydrogen atom, and R ¹⁴ is a hydrogen atom or a C _1-6 alkyl group. Preferably, R ¹¹ is a hydrogen atom and R ¹⁵ is a C _1-6 alkyl group. More preferably, R ¹¹ is a hydrogen atom, R ¹⁴ is a hydrogen atom or a C _1-6 alkyl group, and R ¹⁵ is a C _1-6 alkyl group.

"C _1-6 alkyl group" means a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms. Preferably it is a "C _1-4 alkyl group". Specific examples of "C _1-6 alkyl group" include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl , 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl and the like.

Examples of the "cycloalkane ring" in which R ¹² and R ¹³ "may form a 3- to 8-membered cycloalkane ring together with the carbon atom to which they are bonded" include, for example, cyclopropane. ring, cyclobutane ring, cyclopentane ring, cyclohexane ring, cycloheptane ring, cyclooctane ring, etc.

A specific example of Q in the case where R ¹² and R ¹³ "may be combined with the carbon atom to which they are bonded to form a 3- to 8-membered cycloalkane ring" is represented by the following formula: Examples include groups such as

"3- to 8-membered cyclic amine" means a 3- to 8-membered saturated or unsaturated cyclic amine. Examples of the "cyclic amine" in which R ¹⁴ and R ¹⁵ "may form a 3- to 8-membered cyclic amine together with the nitrogen atom to which they are bonded" include, for example, an aziridine ring, an azetidine ring, pyrrolidine ring, piperidine ring, azepane ring, azocane ring and the like.

A specific example of Q in the case where R ¹⁴ and R ¹⁵ "may be combined with the nitrogen atom to which they are bonded to form a 3- to 8-membered cyclic amine" is represented by the following formula: Examples include groups.

Group represented by formula (a):

Examples include 2-aziridinyl group, 3-azetidinyl group, 3-pyrrolidinyl group, and 3-piperidinyl group. Preferred are 3-azetidinyl group, 3-pyrrolidinyl group, and 3-piperidinyl group.

Group represented by formula (b):

Examples include aziridin-2-ylmethyl group, azetidin-2-ylmethyl group, pyrrolidin-2-ylmethyl group, piperidin-2-ylmethyl group, and the like. Preferably it is a pyrrolidin-2-ylmethyl group.

In a preferred embodiment, the compound of formula (VI) or a pharmaceutically acceptable salt thereof has the following formula:

(Compound name: (2-aminophenyl)(azetidin-3-yl)methanone; hereinafter also referred to as "compound H") or a pharmaceutically acceptable salt thereof.

(Compound name: 1-(2-aminophenyl)-3-(methylamino)propan-1-one; hereinafter also referred to as "Compound I") or a pharmaceutically acceptable salt thereof. Particularly preferred is the hydrochloride of compound I.

(Compound name: (2-aminophenyl)(pyrrolidin-3-yl)methanone; hereinafter also referred to as "Compound J") or a pharmaceutically acceptable salt thereof. Particularly preferred is the hydrochloride of Compound J.

(Compound name: (2-aminophenyl)(piperidin-3-yl)methanone; hereinafter also referred to as "compound K") or a pharmaceutically acceptable salt thereof.

(Compound name: 1-(2-aminophenyl)-2-(pyrrolidin-2-yl)ethanone; hereinafter also referred to as "Compound L") or a pharmaceutically acceptable salt thereof. Particularly preferred is the hydrochloride of compound L.

(Compound name: 1-(2-aminophenyl)-3-(methylamino)butan-1-one; hereinafter also referred to as "compound M") or a pharmaceutically acceptable salt thereof.

(Compound name: 3-amino-1-(2-aminophenyl)butan-1-one; hereinafter also referred to as "compound N") or a pharmaceutically acceptable salt thereof.

(Compound name: (-)-(2-aminophenyl)(pyrrolidin-3-yl)methanone; hereinafter also referred to as "compound O") or a pharmaceutically acceptable salt thereof. Particularly preferred is the hydrochloride of compound O.

(Compound name: (+)-(2-aminophenyl)(pyrrolidin-3-yl)methanone; hereinafter also referred to as "compound P") or a pharmaceutically acceptable salt thereof. Particularly preferred is the hydrochloride of compound P.

In one embodiment, the serotonin 3 receptor agonist that is the active ingredient of the present invention is represented by any of formulas (I), (II), (III), (IV), (V) or (VI). compound or a pharmaceutically acceptable salt thereof. In another embodiment, the serotonin 3 receptor agonist that is the active ingredient of the present invention is a compound represented by any one of formula (I), (II), (IV), (V) or (VI) or a pharmaceutical thereof. It is an acceptable salt. In another embodiment, the active ingredient of the present invention, a serotonin 3 receptor agonist, has the formula (I'), (II'), (III'), (IV'), or (V'). The indicated compound or a pharmaceutically acceptable salt thereof. In another embodiment, the serotonin 3 receptor agonist that is the active ingredient of the present invention is a compound represented by any of formulas (I'), (II'), (IV'), or (V') or its It is a pharmaceutically acceptable salt. In another embodiment, the active ingredient of the present invention, a serotonin 3 receptor agonist, is a compound A, B, C, D, E, F, G, H, I, J, K, L, M, N, O or P or a pharmaceutically acceptable salt thereof. In another embodiment, the active ingredient of the present invention, a serotonin 3 receptor agonist, is a compound A, B, C, F, G, H, I, J, K, L, M, N, O or P or its It is a pharmaceutically acceptable salt.

In a preferred embodiment, the serotonin 3 receptor agonist that is the active ingredient of the present invention is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof. In a more preferred embodiment, the serotonin 3 receptor agonist that is the active ingredient of the present invention is a compound represented by formula (I') or a pharmaceutically acceptable salt thereof. In a further preferred embodiment, the serotonin 3 receptor agonist that is the active ingredient of the present invention is Compound A or a pharmaceutically acceptable salt thereof.

The serotonin 3 receptor agonist, which is an active ingredient of the present invention, such as a compound represented by any of formulas (I), (II), (III), (IV), (V) or (VI), can be used in free form. However, it may also be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, formate, acetate, propionate, fumarate, Oxalate, malonate, succinate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, maleate, dimaleate, lactate, malate, tartrate , acid addition salts such as citrate, pamoate and trifluoroacetate; metal salts such as lithium, potassium, calcium, magnesium, sodium, zinc and aluminum salts; and ammonium salts, diethanolamine. salts, base addition salts such as ethylenediamine salts, triethanolamine salts, and triethylamine salts.

The serotonin 3 receptor agonist which is the active ingredient of the present invention, such as a compound represented by any one of formulas (I), (II), (III), (IV), (V) or (VI) or its pharmaceutical Acceptable salts include any of their internal salts, adducts, solvates or hydrates, co-crystals, and the like.

The serotonin 3 receptor agonist, which is the active ingredient of the present invention, for example, a compound represented by formula (I), (II), (III), (IV), (V) or (VI), has a When it has an asymmetric carbon atom, it may exist as a plurality of stereoisomers (i.e., diastereoisomers, optical isomers) based on the asymmetric carbon atom, and the active ingredient of the present invention does not contain any of these stereoisomers. It includes both stereoisomers and mixtures thereof.

In addition, the serotonin 3 receptor agonist, which is the active ingredient of the present invention, such as a compound represented by any one of formulas (I), (II), (III), (IV), (V) or (VI), may be The isomers may include cis and trans isomers, and if the molecule has axial asymmetry, it may include isomers based on axial asymmetry, and any one of these isomers or its Both include mixtures.

The serotonin 3 receptor agonist which is the active ingredient of the present invention, for example, a compound represented by any one of formulas (I), (II), (III), (IV), (V) or (VI), is an isotope ( For example, compounds labeled with ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F, ³² P, ³⁵ S, ¹²⁵ I, etc.) and deuterium converters are included.

The invention also includes prodrugs of the compounds of the invention or pharmaceutically acceptable salts thereof. The prodrugs are functional derivatives of the compounds of the invention that can be easily converted in vivo to the required compound.

MOC (medial olivocochlear) neurons are nerve cells that project from the superior olivary nucleus of the brain stem to the cochlea. In the present invention, activating MOC neurons means increasing the neural activity of MOC neurons by stimulating the 5-HT3 receptors expressed in MOC neurons with its agonist. The cochlear efferent feedback system conveyed by the olivocochlear pathway plays an important role in auditory processing. MOC neurons, which project to the cochlea from the superior olivary nucleus of the brainstem, have a central role in the cochlear efferent feedback system. The efferent feedback system via MOC neurons is called the MOC feedback system. Neural activity of MOC neurons includes adjustment of the dynamic range of hearing, protection from acoustic trauma, and signal detection in a noisy environment, and MOC neuron activation includes activation of these neural activities. It will be done.

When sound (physical air vibrations) enters the ear canal, it is converted into vibrations of endolymph fluid in the eardrum and ossicles, and in the inner ear, the vibrations of endolymph fluid cause a membrane called the basilar membrane to vibrate. The vibration of the basilar membrane further vibrates the sensory hairs of the hair cells of the organ of Corti on the basilar membrane, and the basilar membrane of the corresponding region vibrates in response to the frequency of the sound. When the sensory hairs of hair cells vibrate, ion channels that convert physical signals into electrical signals open and close. The electrical signals generated there transmit sound information to the brain as signals from afferent nerves.

In the process of sound conveying information to the brain, there are two main functions of the MOC feedback system via MOC neurons. One of its functions is to suppress the vibration of the basement membrane so that hair cells and the like are not damaged due to the excessive vibration of the basement membrane in response to strong and loud noises. Another function is to amplify and suppress vibrations of the basement membrane that correspond to specific frequencies. For example, for conversation, the vibrations of the basilar membrane that correspond to the frequency range of the human voice are amplified, and the vibrations of the basilar membrane that correspond to the frequency range of noise such as the sounds of surrounding cars are suppressed.

By activating the MOC feedback system via MOC neurons, the composition of the present invention improves the function of the system, making it possible to prevent or treat hearing disorders involving MOC neurons. In the present invention, a hearing disorder involving MOC neurons is a disorder due to decreased or insufficient function of the MOC feedback system via MOC neurons. Hearing disorders involving MOC neurons include sensorineural hearing loss due to acoustic trauma, aging, etc. Sensorineural hearing loss includes hearing loss, insufficient speech discrimination ability in a noisy environment even if hearing is normal, insufficient speech discrimination ability in a noisy environment accompanied by hearing loss, tinnitus, hyperacusis, etc. included. The compositions of the present invention enable the prevention or treatment of sensorineural hearing loss, acoustic trauma, age-related hearing loss, and poor speech discrimination ability in a noisy environment even when hearing is normal. It can be suitably used.

In the present invention, hearing loss is a state in which hearing ability is inferior to normal, and a state in which the hearing threshold is increased. Hearing is considered normal if the average hearing level is less than 25 dB.

Conductive hearing loss caused by damage to the middle ear is caused by chronic otitis media, ossicular malformation, and the like, and these hearing losses can be treated by surgery or the like. On the other hand, sensorineural hearing loss caused by damage to the inner ear is caused by noise, aging, etc., and treatment is symptomatic and a complete cure is difficult. Hearing disorders due to decreased or insufficient function of the MOC feedback system via MOC neurons include sensorineural hearing loss caused by damage to the inner ear.

The compositions of the invention allow prevention or treatment of sensorineural hearing loss. Sensorineural hearing loss is hearing loss caused by damage to the inner ear. Sensorineural hearing loss includes acoustic trauma, age-related hearing loss, etc. Symptoms include hearing loss, insufficient speech discrimination ability in a noisy environment even with normal hearing ability, insufficient speech discrimination ability in a noisy environment accompanied by hearing loss, tinnitus, and hyperacusis. These symptoms are largely related to decreased or insufficient function of the MOC feedback system mediated by MOC neurons.

The compositions of the invention enable the prevention or treatment of acoustic trauma. Acoustic trauma is a disease that causes sensorineural hearing loss due to exposure to powerful sounds. Acoustic trauma in the present invention includes chronic acoustic trauma and acute acoustic trauma. Chronic acoustic trauma is also called noise-induced hearing loss. Symptoms of acoustic trauma include hearing loss, poor speech discrimination in noisy environments, tinnitus, and hyperacusis. Acoustic trauma (hearing loss due to exposure to loud noises) includes noise-induced hearing loss, hearing loss due to audio equipment such as headphones and earphones, hearing loss due to concerts and live performances, and hearing loss due to explosion sounds, etc. The composition of the present invention can be suitably used for the prevention and treatment of hearing loss due to exposure to loud sounds, that is, hearing loss due to acoustic trauma.

The compositions of the invention enable the prevention or treatment of age-related hearing loss. Symptoms of age-related hearing loss include hearing loss, insufficient speech discrimination ability in noisy environments, inability to hear small sounds and loud and unpleasant sounds (narrow dynamic range), tinnitus, and hyperacusis. Symptoms included. Furthermore, even in cases where hearing ability has not deteriorated significantly, the ability to discriminate speech in a noisy environment is often impaired. The composition of the present invention can be suitably used for the prevention or treatment of hearing loss due to age-related hearing loss. Furthermore, the composition of the present invention can be suitably used for the prevention or treatment of insufficient speech discrimination ability in a noisy environment even when hearing is normal due to age-related hearing loss. Furthermore, the composition of the present invention can be suitably used for the prevention or treatment of insufficient speech discrimination ability in a noisy environment accompanied by hearing loss due to age-related hearing loss.

In the present invention, insufficient speech discrimination ability in a noisy environment refers to symptoms such as not being able to understand conversations well in noise, or being unable to understand the content although hearing voices, and in cases where there is no hearing loss ( cases with normal hearing) and cases with hearing loss. The MOC feedback system via MOC neurons has the function of amplifying the vibrations of the basilar membrane corresponding to the frequency range of human voices for conversation, and suppressing the vibrations of the basilar membrane corresponding to the frequency range of noise such as the sound of surrounding cars. Therefore, activation of MOC neurons by a 5-HT3 receptor agonist can improve the insufficient speech discrimination ability in a noisy environment. The composition of the present invention can be suitably used for the prevention or treatment of insufficient speech discrimination ability in a noisy environment, and can be suitably used for preventing or treating insufficient speech discrimination ability in a noisy environment even if hearing is normal. It can be further suitably used for treatment.

5-HT3 receptors play a fundamental role in the MOC feedback system and are involved in hearing loss due to exposure to high-intensity sounds. Stimulation of 5-HT3 receptors attenuates hearing loss and loss of ribbon synapses due to high-intensity sound exposure and enhances cochlear protection by the MOC feedback system. Therefore, pharmacological enhancement of MOC neuron activity via 5-HT3 receptors allows for the prevention and treatment of hearing loss caused by exposure to high-intensity sounds. Patients with functional hearing loss (e.g., loud sound exposure hearing loss and age-related hearing loss) with normal hearing often have poor speech discrimination in noisy environments, including tinnitus and hyperacusis. The patient exhibits auditory symptoms such as paresthesia. Cochlear synaptopathy has been suggested to be related to the pathophysiology of these hearing disorders, and dysfunction of the MOC feedback system is implicated as one of the causes (Guinan JJ Jr. Ear Hear. 2006;27:589- 607, Maison SF et al. J Neurosci. 2013;33:5542-5552). Therefore, 5-HT3 receptors may be an important therapeutic target for the treatment of auditory symptoms associated with dysfunction of the MOC feedback system, allowing for prevention and treatment with 5-HT3 receptor agonists.

The prophylactic or therapeutic composition in the present invention includes a prophylactic composition, a therapeutic composition, and a prophylactic and therapeutic composition.

Prevention or treatment in the present invention includes prevention of onset, improvement of symptoms, suppression of exacerbation of symptoms, prevention of recurrence of symptoms, early recovery of symptoms, etc. with respect to one or more symptoms related to hearing impairment. The composition of the invention may preferably be a therapeutic composition. A therapeutic composition is a composition that is administered for the purpose of preventing the onset of symptoms, improving symptoms, suppressing exacerbation of symptoms, preventing recurrence of symptoms, early recovery of symptoms, and the like.

The composition of the present invention may contain two or more types of serotonin 3 receptor agonists as active ingredients. The composition of the present invention may contain a hearing impairment therapeutic other than a serotonin 3 receptor agonist. Furthermore, the composition of the present invention may contain drugs other than the hearing impairment therapeutic.

The composition of the present invention can include a serotonin 3 receptor agonist as an active ingredient and a pharmaceutically acceptable carrier. Such carriers include excipients (for example, sugar derivatives such as mannitol and sorbitol; starch derivatives such as corn starch and potato starch; or cellulose derivatives such as crystalline cellulose), lubricants (for example, stearic acid metal stearates such as magnesium; or talc, etc.), binders (e.g., hydroxypropylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone, etc.), disintegrants (e.g., cellulose derivatives such as carboxymethylcellulose, carboxymethylcellulose calcium, etc.), Water, preservatives (e.g. paraoxybenzoic acid esters such as methylparaben, propylparaben; or alcohols such as chlorobutanol, benzyl alcohol, etc.), pH adjusters (e.g. inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid, acetic acid) , organic acids such as succinic acid, fumaric acid, or malic acid, or their salts), and commonly used carriers for pharmaceutical preparations, such as diluents (e.g., water for injection, etc.), singly or in combination of two or more. It can be blended. The composition of the present invention includes a solution in which the active ingredient is dissolved in water.

The serotonin 3 receptor agonist, which is the active ingredient of the present invention, is mixed with the above-mentioned carrier as necessary, and then prepared into tablets, granules, capsules, powders, solutions, suspensions, emulsions, etc. It can be administered orally in the form of a suppository, injection, intravenous infusion, transdermal, transmucosal, or inhaled parenterally.

The serotonin 3 receptor agonist, which is the active ingredient of the present invention, is formulated into the above-mentioned dosage form and then administered to a subject in need thereof, such as a human or an animal, preferably a human.

The composition of the present invention can be administered to a subject in need thereof as a medicine, food, or drink. Preferably, it can be administered as a medicine.

The dosage and frequency of administration of the serotonin 3 receptor agonist of the present invention can be changed as appropriate depending on conditions such as the severity of symptoms, age, weight, sex of the patient, type of drug, dosage form, and route of administration. When administered to humans, the active ingredient is administered parenterally, such as subcutaneously, intravenously, intraperitoneally, intramuscularly, or intrarectally, at a dose of about 0.01 to 10 mg/kg body weight, preferably about 0.01 to 10 mg/kg body weight per administration. about 0.1 to 5 mg/kg body weight, particularly preferably about 0.3 to 3 mg/kg body weight, and orally about 0.01 to 100 mg/kg body weight, preferably about 0.1 to 50 mg/kg body weight. , particularly preferably about 1 to 30 mg/kg body weight. The frequency of administration may also be one or more times per day, such as 1 to 3 times, 1 to 2 times, or once per day.

The serotonin 3 receptor agonist of the present invention can be produced according to known methods. For example, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be produced by the method described in Patent Document 1. Moreover, compound H, I, J, K, L, M, N, O or P can be manufactured by the method described in International Publication No. WO2016/027757. Commercially available compounds can also be used as compounds A, B, C, D, E, F, or G.

The present invention also relates to a method for screening a compound for prevention or treatment of sensorineural hearing loss, which comprises a step of measuring serotonin 3 receptor agonist activity. The compound for preventing or treating sensorineural hearing loss is preferably a compound for preventing or treating acoustic trauma or age-related hearing loss. More preferably, it is a compound for preventing or treating hearing loss due to exposure to loud noises.

Furthermore, the present invention relates to a method of screening for a MOC neuron activating compound or a compound for preventing or treating a hearing disorder involving MOC neurons, which comprises the step of measuring serotonin 3 receptor agonist activity.

The screening method of the present invention includes, for example, the step of measuring serotonin 3 receptor agonist activity for a compound library. The compound library may be known or unknown. Known compound libraries include compound libraries that have already been approved by food (e.g., U.S. Food and Drug Administration (FDA)) or drugs (e.g., European Medicines Agency (EMEA)) (e.g., PRESTWICK). Chemical libraries) (this is a collection of compounds whose patent terms have expired), and compound libraries that are a collection of compounds that have not yet been approved for food or medicine.

In addition, as a method for measuring serotonin 3 receptor agonist activity, cells (Xenopus oocytes, HEK293 cells, etc.) are made to express cDNA of serotonin 3 receptor subunit A, or A and B. , a method of electrophysiologically measuring the current flowing into cells (Nakamura Y et al. Biochem Biophys Res Commun. 415(2) (2011) 416-20) and fluorescent membrane-poten Fluorescence intensity using tial sensitive dye Examples include a method of measuring the current flowing into the cell by measuring the current flowing into the cell (Lummis et al. Neuropharmacology 73 (2013) 241-246). In either method, serotonin 3 receptor agonist activity can be measured by administering the compound to cells and examining the response obtained.

The screening method of the present invention may further include the step of selecting compounds based on the measured serotonin 3 receptor agonist activity.

The compounds obtained by the screening method of the present invention have serotonin 3 receptor agonist activity and can activate MOC neurons, and also prevent or treat hearing disorders involving MOC neurons and sensorineural hearing loss. It can be used for prevention or treatment.

The present invention will be explained in more detail below with reference to Examples and Test Examples, but the present invention is not limited thereto.

<Example 1>
SR57227A (TOCRIS, hydrochloride of compound A) was dissolved in physiological saline to prepare an injection preparation (0.5 mg/ml).

1. Materials and methods

<1.1 Animals>
5-HT3A receptor-deficient (Htr3a ^−/− ) mice (RRID:IMSR_JAX:005251) (Zeitz et al. J Neurosci. 2002;22:1010-1019) and 5-HT3AR-EGFP (Enhanced green fluorescent protein) transgenic Reporter mice (RRID: MMRRC_000273-UNC) (Chittajallu et al. Nat Neurosci. 2013;16:1598-1607) were incubated at Jackson Laboratory (Bar Harbor, ME, USA) and Mutant Mouse Regional Resource Center (Chapel Hill, NC), respectively. , USA). Htr3a ^−/− mice were backcrossed with C57BL/6J mice for at least 10 generations as previously reported (Kondo et al. Mol Psychiatry. 2018;23:833-842). Since 5-HT3A is essential for the formation of a functional receptor, Htr3a ^−/− mice are mice deficient in 5-HT3 receptor function. We used 7-week-old male wild-type (C57BL/6J) and Htr3a ^−/− mice and 7-12-week-old male 5-HT3AR-EGFP transgenic reporter mice. Our previous study showed that EGFP expression reflects normal 5-HT3 receptor expression in 5-HT3AR-EGFP transgenic reporter mice (Koyama et al. Sci Rep. 2017;7:42884). All mice were kept at 23-25°C under a controlled light-dark cycle and had standard laboratory chow and water ad libitum. All mice were randomly assigned to various treatment groups in each experiment.

<1.2 Strong sound exposure>
Animals were exposed to a sound pressure of 95 dB for 4 hours in a specially designed soundproof box (50 x 50 x 55 cm) with ceiling-mounted speakers (MF1-M, Tucker-Davis Technologies, Alachua, FL, USA). Subjects were exposed to broadband high-intensity sound from 1 to 50 kHz (SPL). During loud sound exposure, animals were awake and unrestrained in their cages. 1-4 mice were placed in each cage and one or two cages were exposed simultaneously. The electric Gaussian loud sound was transmitted through the loudspeaker after power amplification (SA1 Stereo power amp, Tucker-Davis Technologies). SPL was calibrated immediately before each sound exposure. SPL varied within 1 dB within the cage within the box.

<1.3 Cochlea function test>
Auditory brainstem response (ABR): Mice were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. Needle electrodes were inserted into the top of the head and pinnae, and a ground electrode was placed near the tail. TDT hardware for ABR (Tucker-Davis Technologies) was used for stimulus generation and biosignal acquisition. We used alternating polarity click stimuli of 100 μs duration, tones of 8 kHz and 16 kHz, and bursts with 0.1 ms linear rising and falling envelopes. The tone burst duration was 1 ms. An algorithm for automated determination of ABR amplitude based on the above definition was developed by the BioResearch Center (Nagoya, Japan) (Hanada et al. Sci Rep. 2018;8:11491). Thresholds were determined by decreasing the stimulus intensity in 5 dB steps (from 80 dB SPL to less than 5 dB) until the waveform lost its reproducible morphology. At each sound level, 1024 responses were averaged.

Distortion Product Otoacoustic Emissions (DPOAEs): The ear canal of each mouse was coupled to a small microphone used to detect changes in sound pressure representing DPOAEs. Using DP-2000 (Morita, Kyoto, Japan), 13 f2 frequencies between 4000 Hz and 16000 Hz (frequency ratio f2/f1 = 1.2) with constant levels of f1 = 65 dB SPL and f2 = 55 dB SPL. The DPOAE for the primary tone was recorded. The DPOAE response at 2f1-f2 and the amplitude of the surrounding loud sound floor were extracted from ear canal sound pressure measurements (Kujawa et al. J Neurosci. 2009;29:14077-14085). For contralateral suppression (CS) of DPOAE, a wideband high-intensity sound (3-30 kHz bandwidth) was delivered to the contralateral ear at 55 dB. A level of 55 dB was chosen because 55 dB is a level below the middle ear reflex of mice and other rodents. The magnitude of the CS was calculated and defined by subtracting the distortion component (DP) (dBSPL) under quiet conditions from the distortion component (DP) (dBSPL) associated with the contralateral sound at each test frequency.

For drug stimulation of 5-HT3 receptors, mice were treated with saline or 5 mg/kg of SR57227A [4-amino-1-(6-chloro-2-pyridyl)-piperidine hydrochloride, Sigma, USA; Cat # S1688] (selective 5-HT3 receptor agonist) was administered intraperitoneally 30 minutes before exposure to loud noise. Then, 7 days after the exposure to intense sound, DPOAE and ABR tests were conducted as described above.

<1.4 Preparation of cochlear whole mount and immunostaining>

The isolated cochlea was fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PBS) for 2 h and decalcified with 10% EDTA for 4 days (Hanada et al. Sci Rep. 2018;8: 11491). The basement membrane containing the organ of Corti was microdissected and divided into three regions: basal, middle, and apical (Boero et al. J Neurosci.2018;38:7440-7451 ). Microdissected sections were treated with antibodies against GFP (chicken; Abcam, UK; 1:1000; Cat# ab13970, RRID: AB_300798) and choline acetyltransferase (ChAT) (goat; Merck Millipore, USA; 1:200; Immunostained with Cat# AB144P, RRID: AB_2079751). Sections were also stained with the nuclear dye 4',6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, USA; Cat# D1306, RRID: AB_2629482). Images of the specimens were acquired with an LSM880 confocal laser scanning microscope (Carl Zeiss, Jena, Germany). Images were collected in a 1024×1024 raster using a high resolution oil immersion objective (63×, numerical aperture 1.3). Images were loaded into image processing software (ZEN, Carl Zeiss). The green, red, and blue channels were analyzed separately to generate the maximum projection. The maximum projections from each channel were merged and converted into triplicate images.

<1.5 Dissection and immunostaining of the cochlea>
Mice were anesthetized and perfused transcardially with 4% paraformaldehyde in 0.1 M PBS. The temporal bone was then removed, postfixed, and decalcified (Whitlon et ai. Brain Res Brain Res Protoc. 2001;6:159-166). Cochlear samples were cut into 10 μm thick sections using a cryostat and immunostained with antibodies against GFP and ChAT. The sections were counterstained with DAPI.

<1.6 FG label of retrocochlear auditory pathway>

Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). Under the microscope, the tympanic membrane was visualized by making a small incision in the ear canal cartilage. Using a 5 μL Hamilton syringe with a 32G blunt needle, add 3 μL of 5% Fluorogold (FG) (Sigma-Aldrich, USA; Ca# 223769-64-0) dissolved in 0.9% saline. ) was slowly injected through the inferior posterior quadrant of the tympanic membrane (Dean et al. Otol Neurotol. 2012;33:1085-1091). Mice were sacrificed 5 days after FG injection, and whole brains were removed for immunofluorescence staining. In a study by Dean et al., intratympanic injection of FG was performed because intratympanic injection of a neuron tracer was reported to be more specific and less invasive than intracochlear injection for tracking cochlear efferent neurons. Ta. For quantitative analysis of EGFP (Fig. 1D) and c-Fos (Fig. 3B) expression in FG-labeled MOC neurons, 50 FG-labeled MOC neurons from three mice were evaluated.

<1.7 Immunostaining and analysis of c-Fos expression>
Mice were anesthetized and perfused transcardially with 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The whole brain of each mouse was removed and fixed in 4% paraformaldehyde overnight at 4°C, then placed in 30% sucrose in 0.1 M PBS at 4°C (Kondo et al. Neuron. 2012;73: 743-757). The brains were then embedded in OCT compound and frozen. Brain samples were cut into 20 μm coronal sections by cryostat. After washing with 0.1M PBS, sections were incubated with 0.3% Triton X-100/5% bovine serum albumin in 0.1M PBS at room temperature (Kondo et al. Mol Psychiatry. 2018;23:833- 842). Sections were subsequently incubated with primary antibodies overnight at 4°C. Sections were then washed with 0.1 M PBS and incubated with appropriate secondary antibodies for 1 hour at room temperature. Images were acquired with a BX53 microscope (Olympus, Tokyo, Japan) and a BZ-X700 microscope (Keyence, Osaka, Japan). Sections were also stained with hematoxylin and eosin. The location of MOC neurons was determined according to the mouse brain map (Franklin & Paxinos 2007). To quantify c-Fos-positive cells in the MOC feedback system, every fourth coronal section from the brain of each mouse was evaluated (total of 10 sections per mouse) (Figure 3E) (Ueda et al. Biochem Biophys Res Comm . 2018;506:498-503). To quantitatively analyze c-Fos expression in EGFP-positive MOC neurons after high-intensity sound exposure, 50 EGFP-positive cells from three 5-HT3AR-EGFP transgenic reporter mice were evaluated (Fig. 3C). . The following primary antibodies were used: anti-GFP (chicken; Abcam; 1:1000; Cat# ab13970, RRID: AB_300798) and anti-c-Fos (rabbit; Santa Cruz Biotechnology, USA; 1:1000; Cat# sc-52 , RRID: AB_2106783).

<1.8 Quantification of OHC loss>
Cochlear whole-mount samples were prepared as previously described. Microdissected fragments were stained with rhodamine phalloidin (Cytoskeleton; 1:500; Cat# PHDR1). Images of the specimens were acquired with an LSM880 confocal laser scanning microscope. Images were collected in a 1024×1024 raster using a high resolution oil immersion objective (63×, numerical aperture 1.3). Images were loaded into ZEN image processing software. Outer hair cell (OHC) loss was assessed in a 200 μm field. The number of OHCs after exposure to high-intensity sound was expressed as a percentage of the number of OHCs before exposure to high-intensity sound (Maison et al. J Neurosci. 2013;33:5542-5552).

<1.9 Quantification of ribbon synapses>
Isolated cochleae were postfixed with 4% paraformaldehyde in 0.1 M PBS for 2 hours and then decalcified with 10% EDTA for 4 days. Microdissected sections of the cochlea were treated with anti-CtBP2 antibody (mouse; BD Biosciences, USA; 1:200; Cat# 612 044, RRID: AB_399431) and anti-GluA2 antibody (mouse, Millipore; 1:2000; Cat# MAB397, RRID: AB_2113875) and immunostained with appropriate secondary antibodies. Images of the specimens were acquired with an LSM880 confocal laser scanning microscope. Z-stack images are obtained from each part of the cochlear surface preparation (basal, middle, and apical) from the inner spiral nerve bundle downward to the synaptic poles of approximately 16 inner hair cells. , with a z step of 0.25 μm. Images were collected in a 1024×1024 raster using a high-resolution oil immersion objective (63×, numerical aperture 1.3), and digital zoom (×2). Images were loaded into ZEN image processing software and inner hair cells (IHCs) were identified by CtBP2-stained nuclei. The green and red channels were analyzed separately and the maximum projection was generated to quantify CtBP2 and GluA2 positive puncta. Maximum projections from each channel were merged and converted to a binary image. The number of ribbon synapses identified by parallel CtBP2-positive puncta and GluA2-positive puncta was manually counted by colocalization of CtBP2 and GluA2. For pharmacological stimulation or blockade of 5-HT3 receptors, mice were treated with saline, 5 mg/kg SR57227A (selective 5-HT3 receptor agonist), or 3 mg/kg ondansetron (Tokyo Kasei Kogyo, Tokyo). Japan; Cat# O0470) (selective 5-HT3 receptor antagonist) was injected intraperitoneally 30 minutes before loud sound exposure. These doses were selected according to previous reports (Kondo M. et al. Mol Psychiatry. 2018;23:833-842). Next, 24 hours after exposure to intense sound, microdissection of the central cochlea was performed as described above.

<1.10 Data and statistical analysis>
Mice were randomly assigned to various treatment groups in each experiment. Analysis of cochlear function tests was automated to eliminate bias. Data analysis of other experiments was blinded. Statistical analysis is explained in the figure legends. The number of c-Fos positive cells in the MOC feedback system was analyzed using an unpaired two-tailed Student's t-test. Data from cochlear function tests (ABR and DPOAE) were analyzed by one-way ANOVA followed by Holm-Sidak multiple comparison test. Quantification of ribbon synapses was analyzed by Student's t-test followed by Tukey's multiple comparison test using Holm-Sidak correction and one-way ANOVA. Quantification of OHC loss was analyzed by Student's t-test using Holm-Sidak correction. All statistical analyzes were performed using GraphPad Prism software version 7 (GraphPad Software, Inc. San Diego, CA, USA). The mean and standard error of the data are displayed on each graph. For all analyses, P<0.05 was considered statistically significant.

2. result

<2.1 5-HT3 receptors are expressed in MOC efferent neurons that innervate the OHC of the cochlea>

Both OHC and IHC are innervated by afferent and efferent nerve fibers. Afferent fibers are the dendrites of the cochlear nerve, and their neuronal cell bodies are located in the spiral ganglion. In contrast, efferent fibers originate in the superior olivary nucleus (SOC) of the brainstem, where MOC and LOC neurons are located. MOC neurons innervate the OHC, whereas LOC neurons innervate afferent fibers that contact the IHC. In a recent study, we revealed that 5-HT3 receptors are expressed in SOC neurons (Koyama et al. Sci Rep. 2017;7:42884).

Using 5-HT3AR-EGFP transgenic reporter mice, we investigated the expression pattern of 5-HT3 receptors in SOC neurons in more detail. Immunohistochemical analysis of reporter mice showed EGFP signals only in MOC neurons (Fig. 1A,B). To confirm efferent innervation by MOC neurons, we performed retrograde neuronal tracing of the cochlear auditory pathway by intratympanic administration of FG to 5-HT3AR-EGFP reporter mice. FG labeling was detected in both MOC and LOC neurons ipsilateral to the injection, whereas contralaterally, FG labeling was observed only in MOC neurons (Fig. 1C). This is consistent with previous reports on the branching pattern of olivocochlear neurons. Analysis of reporter mice showed that approximately 95% of FG-labeled MOC neurons colocalized with EGFP signals (47 out of 50 FG-labeled MOC neurons from 3 mice were EGFP-positive neurons). There was; Figure 1D). This indicates that MOC neurons expressing 5-HT3 receptors project efferent axons to the cochlea.

The efferent fibers of MOC neurons pass through the spiral ganglion, pass through the tunnel of Corti, and form synaptic connections with OHCs. Since 5-HT3AR-EGFP reporter mice show EGFP signals throughout neurons, including axons and dendrites expressing 5-HT3 receptors, we further hypothesized that OHC in the cochlea by 5-HT3 receptor-expressing MOC neurons. We investigated the pattern of efferent innervation to. Whole-mount immunostaining of the cochlea of reporter mice, including the basal, middle, and apical regions, clearly showed EGFP signals in nerve fibers passing through the tunnel of Corti and projecting to the OHC. (Figure 2A). Furthermore, the distribution of EGFP-positive fibers was confirmed by immunostaining of cochlear sections (Fig. 2B). Since cochlear efferent fibers are cholinergic, ChAT is used as a marker for efferent fibers. Our analysis showed that ChAT signals were located in fibers extending downwards in the OHC across the tunnel of Corti (Fig. 2A,B). Furthermore, double immunostaining revealed that in the cochlea, EGFP-positive fibers and large terminals of OHCs colocalized with ChAT signals (Fig. 2A,B), indicating that EGFP-positive fibers project to OHCs It was suggested that these were sexual fibers. The EGFP signal observed in IHC (Fig. 2A,B) may be a bundle of MOC fibers in the inner spiral bundle rather than LOC fibers innervating afferent fibers contacting IHC. These results indicated that 5-HT3 receptors are expressed in MOC efferent neurons that innervate the OHC of the cochlea.

<2.2 MOC neurons are activated by exposure to loud sounds>

It has been reported that the activity of the MOC feedback pathway plays an important role in protection from acoustic trauma. To examine the activation of MOC neurons, we performed immunohistochemical analysis of c-Fos, a neuronal activation marker, in wild-type mice after exposure to high-intensity sound. No c-Fos-positive cells were detected when not exposed to high-intensity sound (Fig. 3A). In contrast, c-Fos expression was detected in some MOC neurons 1 h after high-intensity sound exposure (Fig. 3B). Further analysis in combination with FG labeling revealed that almost all FG-labeled MOC neurons were c-Fos positive 1 h after loud sound exposure (50 FG-labeled MOCs from 3 mice). Fifty of the neurons were c-Fos positive neurons; Fig. 3B). These results indicated that MOC neurons innervating the OHC were activated by high-intensity sound exposure.

<2.3 5-HT3 receptors are involved in the activation of MOC neurons by exposure to high-intensity sounds>

Next, we investigated the relationship between activation of MOC neurons by exposure to high-intensity sound and 5-HT3 receptors. Immunohistochemical analysis of 5-HT3AR-EGFP transgenic reporter mice showed c-Fos expression in approximately 70% of EGFP-positive MOC neurons after high-intensity sound exposure (50 EGFP-positive from 3 mice Thirty-six of the cells were c-Fos positive cells; Figure 3C). Next, we further investigated the role of 5-HT3 receptors in MOC neuron activation by high-intensity sound exposure using 5-HT3 receptor-deficient (Htr3a ^−/− ) mice. Interestingly, the number of c-Fos-positive MOC cells was significantly lower in Htr3a ^−/− mice than in wild-type mice at 1 h after high-intensity sound exposure (P=0.0209; Fig. 3D,E). There was no significant difference in the total number of cells in the MOC area between wild-type and Htr3a ^−/− mice (wild type, 105.6 ± 3.70 cells/section; Htr3a ^−/− , 98.4 ± 3. 76 cells/section, n=5 in each group, P=0.21, two-tailed Student's t-test). These results suggest that 5-HT3 receptors played an important role in the activation of MOC neurons by exposure to high-intensity sound.

<2.4 Htr3a ^−/− mice show impaired MOC function and worsening of hearing loss due to exposure to loud sounds>

Figures 1 to 3 show that 5-HT3 receptors are expressed in MOC neurons activated by high-intensity sounds. MOC neurons are neurons that innervate the OHC. These results suggested that 5-HT3 receptors influenced MOC function. DPOAE is a sound generated by the distortion of output sound for two types of input sound through the sensory epithelium, which is converted into mechanical movement, amplified by OHC through MOC, and then back-propagated to the eardrum. Can be measured with a microphone. Therefore, DPOAE is used to assess OHC function and the regulatory effects of MOC neurons on these cells. It has been reported that MOC function can be analyzed by DPOAE measurement with contralateral high-intensity stimulation that induces MOC activity (Zhu X. et al. J Comp Neurol. 2007;503:593-604). Therefore, to investigate the role of 5-HT3 receptors in MOC function, we measured contralateral suppression (CS) of DPOAE in Htr3a ^−/− mice. Interestingly, Htr3a ^−/− mice had less contralateral suppression (CS) compared to wild-type mice (P = 0.04 at 11 kHz; P = 0.04 at 14 kHz; Fig. 4A). This indicates that MOC function is impaired in Htr3a ^−/− mice. Next, in order to examine changes in OHC function due to high-intensity sound, we measured the magnitude of DPOAE in wild-type and Htr3a ^−/− mice under quiet conditions after exposure to high-intensity sound. After 7 days of loud sound exposure, Htr3a ^−/− mice showed lower DPOAEs at several f2 frequencies compared to wild-type mice (P = 0.02 at 12 kHz; P = 0.04 at 14 kHz; P = 0.07 at 16 kHz) (Fig. 4B). No significant difference was observed in DPOAE amplitude before loud sound exposure between these mice (Fig. 4B). These results indicate that loud sound-induced OHC dysfunction is greater in Htr3a ^−/− mice.

To further clarify the role of 5-HT3 receptors in changes in auditory function due to exposure to high-intensity sound, ABR of Htr3a ^−/− mice was recorded after exposure to high-intensity sound. Prior to exposure to high-intensity sound, no significant differences in ABR thresholds were observed between wild-type and Htr3a ^−/− mice (Fig. 4C). Seven days after high-noise exposure, ABR thresholds of both wild-type and Htr3a ^−/− mice were increased, but importantly, at higher frequencies, they were higher in Htr3a ^−/− mice compared with wild-type mice. Thresholds were observed (P = 0.06 at 16 kHz; P = 0.007 at 24 kHz; P < 0.001 at 32 kHz; Fig. 4C).

Taken together, cochlear function test data (DPOAE and ABR) suggested that Htr3a ^−/− mice exhibit impaired MOC function and exacerbation of hearing loss with loud sound exposure. On the other hand, high-intensity sound exposure in our experiments caused similar OHC loss after 7 and 14 days in wild-type and Htr3a ^−/− mice (Fig. 5).

<2.5 Htr3a ^−/− mice lose many ribbon synapses due to intense sound>

It has been reported that high-intensity sound exposure causes extensive ribbon synapse loss between the IHC and the cochlear nerve. Loss of ribbon synapses, known as cochlear synaptopathy, has been reported to be involved in hearing loss due to exposure to high-intensity sounds. Therefore, we performed immunohistochemical analysis to examine ribbon synapse loss after loud sound exposure in Htr3a ^−/− mice. In order to confirm ribbon synapses between IHC and cochlear nerves after exposure to high-intensity sound, CtBP2-positive points (presynaptic), The number of GluA2-positive puncta (postsynaptic) and parallel CtBP2/GluA2-positive puncta was quantified (Fig. 6A, B). Before exposure to high-intensity sound, CtBP2-positive points, ^GluA2 -positive points, or parallel No difference was observed in the number of CtBP2/GluA2 positive puncta (Figures 7-9). High-intensity sound exposure clearly decreased the number of positive points in both wild-type and Htr3a ^−/− mice (Figures 7-9). This observation of synaptic loss upon high-intensity sound exposure in wild-type mice was consistent with previous reports. However, at 1 h, 24 h, and 7 days after high-intensity sound exposure, the number of parallel CtBP2/GluA2-positive puncta in the base and middle cochlear regions of Htr3a ^−/− mice was significantly lower than that of wild-type mice. (Base: 1 hour P = 0.0159; 24 hours P = 0.0079; 7 days P = 0.0238 (Fig. 7C); Middle part: 1 hour P = 0.03; 24 hours P = 0.001 ; 7 days P = 0.03 (Fig. 8C). Furthermore, after exposure to high-intensity sound, the number of CtBP2-positive puncta and GluA2-positive puncta in the basal and medial regions of the cochlea of Htr3a ^−/− mice was lower than that of wild-type mice. Comparatively less was observed (CtBP2 intermediate: 7 days P = 0.042 (Figure 8A); GluA2 base: 1 hour P = 0.04; 24 hours P = 0.001 (Figure 7B); intermediate P = 0.0013 (Fig. 8B). For the reduction in the number of CtBP2-positive puncta, GluA2-positive puncta, and parallel CtBP2/GluA2-positive puncta in the cochlear apex region after high-intensity sound exposure, wild type and Htr3a ^{− /−} mice (FIG. 9). These results indicated that lack of 5-HT3 receptors resulted in more loss of ribbon synapses after high-intensity sound exposure.

<2.6 5-HT3 receptor agonists reduce hearing loss and loss of ribbon synapses due to exposure to high-intensity sounds>

In order to further elucidate the role of 5-HT3 receptors in cochlear dysfunction caused by exposure to high-intensity sounds, we investigated the effects of pharmacological stimulation of 5-HT3 receptors on hearing loss due to exposure to high-intensity sounds. High-intensity sound exposure decreased DPOAE levels in wild-type mice at several f2 frequencies, but administration of SR57227A (selective 5-HT3 receptor agonist) (5 mg/kg) 30 minutes before high-intensity sound exposure significantly reduced DPOAE levels in wild-type mice. The decrease in DPOAE value due to sound exposure was reduced (Sal vs. Ag: P = 0.02 at 10 kHz; P = 0.14 at 11 kHz; P = 0.09 at 12 kHz; P = 0.11 at 14 kHz; P at 16 kHz =0.04; Figure 10A). In addition, SR57227A administration reduced the threshold increase in ABR caused by loud sound exposure in wild-type mice (Sal vs. Ag: P = 0.03 at 4 kHz; P = 0.09 at 8 kHz; P = 0.004 at 16 kHz). ; P = 0.13 at 24 kHz; P = 0.04 at 32 kHz; Figure 10B). These results indicate that 5-HT3 receptor stimulation with a 5-HT3 receptor agonist alleviated hearing loss caused by exposure to high-intensity sound.

To investigate the role of 5-HT3 receptors in ribbon synapse loss induced by high-intensity sound exposure, we investigated the role of 5-HT3 receptor pharmacological treatment ( 5-HT3 receptor agonist administration) was investigated (FIGS. 11A-C). Since the maximal effect of ribbon synapse loss due to 5-HT3 receptor knockout was observed 24 hours after high-intensity sound exposure (FIG. 8C), the effects of 5-HT3 receptor agonists were evaluated at this time point. After 24 h of loud sound exposure, the number of parallel CtBP2/GluA2 positive puncta decreased in wild-type mice, and a significantly lower number of positive puncta was observed in Htr3a ^−/− mice (Fig. 11C), which is shown in Fig. 8C. It was consistent with the results. Administration of ondansetron (a selective 5-HT3 receptor antagonist) 30 minutes before loud sound exposure exacerbated loud sound exposure-induced loss of ribbon synapses in wild-type mice (FIG. 11C). This was consistent with the level of synaptic loss due to severe loud sound exposure observed in 5-HT3 receptor-deficient (Htr3a ^−/− ) mice (FIG. 8C). Interestingly, administration of SR57227A 30 minutes before loud sound exposure significantly attenuated loud sound-induced loss of ribbon synapses in wild-type mice (NE+Sal vs NE+Ag: P=0.0374; Figure 11C). This neuroprotective effect of SR57227A was not observed in Htr3a ^−/− mice (FIG. 11C). In wild-type mice not exposed to high-intensity sound, neither SR57227A nor ondansetron affected the number of ribbon synapses (FIG. 11C). Data regarding the number of CtBP2-positive and GluA2-positive points (FIGS. 11A, B) supported the results shown in FIG. 11C and were consistent with the results shown in FIG. 8. These results indicated that 5-HT3 receptor stimulation with a 5-HT3 receptor agonist attenuated the loss of ribbon synapses due to high-intensity sound exposure. Taken together, these protective effects of 5-HT3 receptor agonists suggested that activation of 5-HT3 receptors enhanced cochlear protection by the MOC feedback system.

In this study, expression of 5-HT3 receptors was found in MOC neurons innervating OHCs (Figures 1 and 2). Furthermore, 5-HT3 receptors were involved in the activation of MOC neurons by exposure to high-intensity sound (Figure 3). Furthermore, Htr3a ^−/− mice have impaired MOC function (smaller contralateral inhibition of DPOAE; Fig. 4A ) and worsened hearing loss due to high-intensity sound exposure (reduced DPOAE values and increased ABR thresholds due to high-intensity sound exposure; 4B,C) were shown. Furthermore, in Htr3a ^−/− mice, loss of many ribbon synapses was observed after exposure to high-intensity sound (FIGS. 6-9). Furthermore, 5-HT3 receptor stimulation with a 5-HT3 receptor agonist attenuated hearing loss and loss of ribbon synapses due to high-intensity sound exposure (Figures 10-11). The above results indicated that 5-HT3 receptor plays a fundamental role in MOC function and contributes to cochlear protection against high-intensity sound exposure. Our findings demonstrate a novel 5-HT3 receptor-mediated mechanism, which is the mechanism underlying the MOC feedback system.

The present invention contributes to the prevention and treatment of hearing impairment.

Claims

A composition for preventing or treating sensorineural hearing loss containing a serotonin 3 receptor agonist.
2. The composition of claim 1, wherein the sensorineural hearing loss is acoustic trauma or age-related hearing loss.
The symptoms of acoustic trauma or age-related hearing loss include hearing loss, insufficient speech discrimination in a noisy environment even with normal hearing, and insufficient speech discrimination in a noisy environment accompanied by hearing loss. The composition according to claim 2, wherein the composition has at least one selected from the group consisting of:
2. The composition of claim 1, wherein the sensorineural hearing loss is hearing loss due to exposure to high-intensity sound.
The serotonin 3 receptor agonist has the following formula (I):

[In the formula,
m is an integer from 1 to 4;
R 1 is a hydrogen atom, a halogen atom, a methyl group optionally substituted with 1 to 3 halogen atoms, a methoxy group optionally substituted with 1 to 3 halogen atoms, 1 to 3 halogen atoms an ethoxy group which may be substituted with , a methylthio group which may be substituted with 1 to 3 halogen atoms, and a halogen atom, trifluoromethyl, C 1-3 alkyl, C 1-3 alkoxy, C 1 -3 phenoxy groups optionally substituted with alkylthio or cyano groups, each independently selected from the group consisting of
The composition according to any one of claims 1 to 4, which is a compound represented by or a pharmaceutically acceptable salt thereof.
6. The composition according to claim 5, wherein each R 1 is independently a halogen atom.
The composition according to claim 6, wherein the halogen atom is a chlorine atom.
A composition according to any one of claims 1 to 4, which is a therapeutic composition.
A composition for activating MOC neurons containing a serotonin 3 receptor agonist.
The composition according to claim 9, which is used for the prevention or treatment of hearing disorders involving MOC neurons.
A method for screening a compound for prevention or treatment of sensorineural hearing loss, comprising the step of measuring serotonin 3 receptor agonist activity.
A method for screening MOC neuron activating compounds, the method comprising the step of measuring serotonin 3 receptor agonist activity.
13. The screening method according to claim 12, wherein the MOC neuron activating compound is a compound for preventing or treating hearing disorders involving MOC neurons.