WO2020045980A1

WO2020045980A1 - Pharmaceutical composition for treating or preventing sensorineural hearing loss

Info

Publication number: WO2020045980A1
Application number: PCT/KR2019/010998
Authority: WO
Inventors: Jin Sei Jung; Jae Young Choi; Young Min Hyun
Original assignee: Industry-Academic Cooperation Foundation, Yonsei University
Priority date: 2018-08-28
Filing date: 2019-08-28
Publication date: 2020-03-05

Abstract

Disclosed are a pharmaceutical composition for treating or preventing sensorineural hearing loss, the composition including a polypeptide including a motif of YxxxSxxCxAAVHxGVI as an active ingredient, wherein x is any amino acid residue. Further, disclosed is a method for treating or preventing sensorineural hearing loss using the pharmaceutical composition.

Description

PHARMACEUTICAL COMPOSITION FOR TREATING OR PREVENTING SENSORINEURAL HEARING LOSS

The present disclosure relates to pharmaceutical compositions, and more particularly to pharmaceutical compositions that include, as an active ingredient, polypeptides composed of motifs effective for the treatment and prevention of sensorineural hearing loss, and chronic otitis media.

Hearing loss may be divided into conductive hearing loss and sensorineural hearing loss.

More specifically, the conductive hearing loss may be a hearing loss caused by a disorder of an organ that transmits sounds such as the outer ear, the eardrum, and the middle ear. The sensorineural hearing loss may result from an abnormality in the ability to detect sound due to the death of hair cells caused by an inflammatory response in the cochlea or result from abnormalities of the auditory nerve or the central nervous system that transmits sound stimuli to the brain.

The conductive hearing loss may be surgically correctable, and thus may be relatively easier to treat than the sensorineural hearing loss. Further, the sensorineural hearing loss may be coped with use of auxiliary means such as a hearing aid or cochlear implant surgery. However, the effect thereof may be less as the degree of the hearing loss increases. In addition, there is a lot of inconvenience in daily life due to difficult recovery of listening ability normally.

In order to develop effective therapies for the sensorineural hearing loss, the researchers have focused on excessive oxidative phosphorylation of mitochondria, and thus reactive oxygen species (ROS), and active nitrogen species (RNS) generation as one of the key etiologies. As a result, therapies for non-specific reactive oxygen species removal using reactive oxygen species scavengers such as N-acetyl-cysteine, Ebselen, MnTBAP, Tiron, or therapies that directly target the source of reactive oxygen species to block the developmental stage or earlier stage have emerged as new therapies for sensorineural hearing loss.

However, the therapeutic effects of the reactive oxygen species or reactive nitrogen species removal on the sensorineural hearing loss remain insignificant. Therefore, it has been continuously required to develop a new pharmaceutical composition, which can prevent damage of the cochlea caused by inflammation, and thus can substantially treat and prevent sensorineural hearing loss.

This Background section has been prepared to allow understanding of the present disclosure to be easier. Thus, it should not be understood that the matters described in this Background section is qualified as a prior art.

The inventors of the present disclosure recognized that removing the inflammation-causing pathogens in the cochlea or modulating the immune response may lead to a therapeutic or preventive effect of the sensorineural hearing loss.

In particular, the inventors of the present disclosure noted that induction of an immune response in the cochlea may suppress not only the invasion of pathogenic bacteria or viruses, but also protect the Corti organ which are essential sensory organs. More specifically, the inventors of the present disclosure have found that not only the immediate elimination of invading pathogens but also inflammatory changes that cause damage to the Corti organs may be induced by activity of phagocyte such as macrophage, and neutrophilic leukocyte as induced during the immune response progresses in the cochlea.

Meanwhile, the cochlin protein encoded by the COCH gene is expressed in the inner ear, eye, and spleen, and is composed of LCCL (Limulus factor C, Cochlin and Lgl1) domain, and vWFA (von Willebrand factor A-like) domain. Abundance of the cochlin proteins may play an important role in essential functions of the ear, such as maintaining hearing function.

In this connection, the inventors of the present disclosure noted the function of the LCCL domain. As a result, we could find out that the LCCL domain is associated with an immune response. More specifically, the inventors of the present disclosure have confirmed that the LCCL domain induces the aggregation of pathogens in the inner ear during infection of pathogens and induces the activity of immune cells.

In particular, the inventors of the present disclosure confirmed that when the peptide containing the LCCL domain is microinjected or topically applied to the middle ear or inner ear, aggregation of gram-positive bacteria is induced, and thus immune cells to remove these pathogens are stimulated.

Thus, the inventors of the present disclosure confirmed that as the LCCL domain preserves the structure, and auditory function of the sensory organs, this domain may be used for the treatment, and prevention of sensorineural hearing loss, particularly, as induced by chronic otitis media or as induced by infection of pathogens.

Furthermore, the inventors of the present disclosure could recognize that, considering the characteristics of the LCCL domains described above, peptides containing these domains may be used for the treatment and prevention of chronic otitis media induced by infection of pathogenic bacteria.

Meanwhile, the inventors of the present disclosure found that the LCCL domains are present not only in the cochlin proteins but also in proteins such as CRISLD1 (Cysteine-rich secretory protein LCCL domain-containing 1), CRISLD2 (Cysteine-rich secretory protein LCCL domain-containing 2), Vitrin DCBLD1 (Discoidin, CUB and LCCL domain-containing protein 1), and DCBLD2 (Discoidin, CUB and LCCL domain-containing protein 2).

In this connection, the inventors of the present disclosure have discovered that a conservative motif of YxxxSxxCxAAVHxGVI is common for LCCL domains derived from the above-mentioned proteins. It could be appreciated by the present inventors that the motif could be used for the treatment and prevention of sensorineural hearing loss, and chronic otitis media.

As a result, the present inventors have developed a pharmaceutical composition that includes a peptide composed of a conservative motif of YxxxSxxCxAAVHxGVI, which is effective in the treatment and prevention of sensorineural hearing loss, and chronic otitis media.

Therefore, an object of the present disclosure is to provide a pharmaceutical composition for treating or preventing sensorineural hearing loss including a polypeptide including a motif of YxxxSxxCxAAVHxGVI as an active ingredient.

Another object of the present disclosure is to provide a pharmaceutical composition for treating or preventing sensorineural hearing loss in an intratracheal microinjection or topical application dosage form.

Yet another object of the present disclosure is to provide pharmaceutical compositions that are effective in the treatment and prevention of sensorineural hearing losses other than hearing loss as induced by mutation of cochlin proteins.

Yet another object of the present disclosure is to provide a method of preventing or treating sensorineural hearing loss, the method including administering a pharmaceutical composition for treating or preventing sensorineural hearing loss to a subject suspected of sensorineural hearing loss, excluding humans.

Yet another object of the present disclosure is to provide a pharmaceutical composition for the treatment or prevention of chronic otitis media, the composition including a polypeptide including the motif of YxxxSxxCxAAVHxGVI as an active ingredient.

The purposes of the present disclosure are not limited to those mentioned above. Other purposes not mentioned may be clearly understood from the description below.

In order to achieve the above object, a pharmaceutical composition for treating or preventing sensorineural hearing loss is provided, the composition including a polypeptide including a motif of YxxxSxxCxAAVHxGVI as an active ingredient, according to one embodiment of the present disclosure.

As used herein, the term "sensorineural hearing loss" refers to hearing loss resulting from an abnormality in the ability to detect sound due to the death of hair cells caused by an inflammatory response in the cochlea, or resulting from abnormalities of the auditory nerve or the central nervous system that transmits sound stimuli to the brain.

According to one embodiment of the present disclosure, sensorineural hearing loss may be at least one selected from the group consisting of viral infection based Corti organ damage-induced hearing loss, tympanitis-induced hearing loss, presbycusis, noise-induced hearing loss, sudden sensorineural hearing loss, autoimmune hearing loss, ischemia-induced hearing loss, head injury-induced hearing loss, ototoxcity medication-induced hearing loss, and hereditary hearing loss. Preferably, the sensorineural hearing loss disclosed herein may be, but is not limited to, viral infection based Corti organ damage-induced hearing loss, chronic otitis media-induced hearing loss, or noise-induced hearing loss. For example, the sensorineural hearing loss may encompass any hearing loss that differs from the hearing loss induced by mutations of the cochlin protein.

As used herein, the term "motif of YxxxSxxCxAAVHxGVI" may mean a peptide motif formed by peptide bonds of 17 amino acids. In this connection, x may be any amino acid residue.

Meanwhile, as used herein, the term "polypeptide including a motif of YxxxSxxCxAAVHxGVI" may mean a peptide motif itself of YxxxSxxCxAAVHxGVI or a polypeptide including a sequence of a peptide motif of YxxxSxxCxAAVHxGVI with respect to the whole sequence.

The polypeptide including the motif of YxxxSxxCxAAVHxGVI may induce an immune response in the cochlea, and thus may not only prevent invading of pathogenic bacteria or viruses, but also protect a Corti organ as an essential sensory organ. Accordingly, the polypeptide including the motif of YxxxSxxCxAAVHxGVI may be used as a pharmaceutical composition for the treatment and prevention of sensorineural hearing loss.

According to one embodiment of the present disclosure, the polypeptide may be at least one selected from the group consisting of a peptide including an LCCL domain of cochlin protein, a peptide including an LCCL domain of CRISLD1 (Cysteine-rich secretory protein LCCL domain-containing 1), a peptide including an LCCL domain of CRISLD2 (Cysteine-rich secretory protein LCCL domain-containing 2), a peptide including an LCCL domain of Vitrin protein, a peptide including an LCCL domain of DCBLD1 (Discoidin, CUB and LCCL domain-containing protein 1), and a peptide including an LCCL domain of DCBLD2 (Discoidin, CUB and LCCL domain-containing protein 2). More specifically, the polypeptide may be a polypeptide including a protein-derived LCCL domain named COCH_HUMAN, CRLD1_HUMAN/B7Z929_HUMAN, CRLD2_HUMAN, B5MD45_HUMAN, Q6UXI7, Q8N8Z6, Q96PD2, Q14089_HUMAN according to UniProtKB (UniProt Knowledgebase).

Preferably, the polypeptide may be, but is not limited to, a polypeptide including the LCCL domain of a human cochlin protein. Furthermore, the cochlin protein itself containing the LCCL domain may be used as the pharmaceutical composition in accordance with the present disclosure. Further, as used herein, the polypeptide may have the same meaning as "a LCCL fragment", "a peptide composed of a LCCL domain", "a polypeptide including a LCCL domain", but is not limited thereto.

According to another embodiment of the present disclosure, the polypeptide may include at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

Preferably, the polypeptide may be, but is not limited to, a peptide having an amino acid sequence of SEQ ID NO: 1.

Meanwhile, according to one embodiment of the present disclosure, the polypeptide may induce aggregation of Gram-negative bacteria, and may stimulate immune cells for removal of Gram-negative bacteria.

According to another embodiment of the present disclosure, a content of the polypeptide may be 0.1 mg/ml to 100 mg/ml with respect to the pharmaceutical composition. However, it is not limited thereto. For example, the content of the polypeptide may vary based on a number of factors, including a type and extent of a response to be achieved by an administration of the pharmaceutical composition to treat or prevent the sensorineural hearing loss, a type of subject to be administered, a corrected state, a tooth state, an age, a weight, a general state of health, a symptom or severity of disease, a sex, a diet, excretion, a drug used concurrently or at different times toward the subject or similar factors well known in the art. Furthermore, one of ordinary skill in the art may readily determine and prescribe a content of a polypeptide including a motif of YxxxSxxCxAAVHxGVI effective for the desired treatment.

According to another embodiment of the present disclosure, a pharmaceutical composition for treating or preventing sensorineural hearing loss may have an intratracheal microinjection form or a topical application dosage form.

For example, when a pharmaceutical composition for treating or preventing sensorineural hearing loss is microinjected or topically applied to the middle ear or inner ear, activity of phagocyte such as macrophage, and neutrophilic leukocyte may be induced during the immune response progresses in the cochlea due to the invasion of the pathogens. Thus, not only the immediate elimination of invading pathogens but also inflammatory changes that cause damage to the Corti organ may be induced.

For example, a density of Gram-negative bacteria in the inner ear or middle ear of a subject suspected of a sensorineural hearing loss administered with the pharmaceutical composition in accordance with the present disclosure may be 2 to 3 times lower than a density of Gram-negative bacteria in the inner ear or middle ear of a subject suspected of a sensorineural hearing loss not administered with the pharmaceutical composition in accordance with the present disclosure.

Further, when a pharmaceutical composition for treating or preventing sensorineural hearing loss is microinjected or topically applied to the middle ear or inner ear, a damage to auditory cells due to noise may be prevented.

Meanwhile, a dosage form of the pharmaceutical composition in accordance with the present disclosure is not limited thereto. For example, the pharmaceutical composition in accordance with the present disclosure may be formulated in the form of microinjections into the organ, as well as may be formulated in sterile injection solutions for intravenous, intramuscular, subcutaneous or intradermal injection. Further, the pharmaceutical composition in accordance with the present disclosure may be formulated in an external application preparation for intratracheal and skin topical application. Furthermore, the pharmaceutical composition in accordance with the present disclosure may be formulated in various forms depending on the route of administration, and mode of administration, as long as the polypeptide composed of the motif of YxxxSxxCxAAVHxGVI reaches a desired site.

When the pharmaceutical composition in accordance with the present disclosure is formulated in the form of a sterile injectable solution for microinjection into a target organ, suspensions, solubilizing agents, stabilizers, isotonization agents, preservatives, adsorption inhibitors, surfactants, diluents, pH adjusters, analgesics, buffers, containing sulfur-reducing agents, antioxidants may be further included in the composition.

Further, when the pharmaceutical composition in accordance with the present disclosure is formulated in an external application preparation for topical application to the target organ, the pharmaceutical composition may be applied to the organ or skin as a separate therapeutic agent or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents.

In the administration of the pharmaceutical composition for treating or preventing sensorineural hearing loss according to various embodiments of the present disclosure, it may be important to administer an amount that may achieve the maximum effect in a minimum amount without side effect with consideration of all the above factors. Furthermore, the application amount and dosage of the pharmaceutical composition may be readily determined by one skilled in the art as described above.

According to one embodiment of the present disclosure, the pharmaceutical composition in accordance with the present disclosure may further include calcium ions or calcium releasing materials.

For example, a polypeptide composed of a motif of YxxxSxxCxAAVHxGVI in the pharmaceutical composition in accordance with the present disclosure may induce aggregation of pathogenic bacteria in a calcium content dependent manner. Accordingly, the pharmaceutical composition further including calcium ions or calcium releasing materials may effectively induce aggregation of pathogenic bacteria.

Further, the polypeptide including the motif of YxxxSxxCxAAVHxGVI that provides a therapeutic or prophylactic effect on sensorineural hearing loss may be provided as a food composition for the prevention or amelioration of hearing loss caused by bacterial infection.

Thus, in order to solve the problems as described above, a food composition for preventing or improving hearing loss caused by bacterial infection according to another embodiment of the present disclosure may include a polypeptide including a motif of YxxxSxxCxAAVHxGVI as an active ingredient, wherein x is any amino acid residue.

Meanwhile, in order to solve the problems as described above, a method for treatment or prevention of sensorineural hearing loss in accordance with an embodiment of the present disclosure may include administering a pharmaceutical composition for treating or preventing sensorineural hearing loss to a subject suspected of sensorineural hearing loss, excluding humans.

As used herein, the term "a subject suspected of sensorineural hearing loss" may mean a subject with an abnormal symptom in the ability to detect sound due to hair cell death caused by an inflammatory response occurring in the cochlea, or with an abnormal symptom of the auditory nerve or the central nervous system to transmit a sound stimulus to the brain. In this connection, the subject suspected of sensorineural hearing loss may be any mammal, except humans.

According to one embodiment of the present disclosure, administering the composition may include microinjecting or topically applying the pharmaceutical composition for treating or preventing sensorineural hearing loss into the inner ear or middle ear of a subject suspected of sensorineural hearing loss.

According to another embodiment of the present disclosure, a density of Gram-negative bacteria in the inner ear or middle ear of the subject suspected of sensorineural hearing loss administered with the pharmaceutical composition in accordance with the present disclosure may be 2 to 3 times lower than a density of Gram-negative bacteria in the inner ear or middle ear of the subject suspected of sensorineural hearing loss not administered with the pharmaceutical composition in accordance with the present disclosure.

Meanwhile, the polypeptide including a motif of YxxxSxxCxAAVHxGVI may induce an immune response in cochlea as described above. Thus, the polypeptide including a motif of YxxxSxxCxAAVHxGVI may not only suppress the invasion of pathogenic bacteria or viruses, but also protect the Corti organ which are essential sensory organs. Thus, the polypeptide including a motif of YxxxSxxCxAAVHxGVI may be used for the treatment and prevention of chronic otitis media.

Thus, in order to solve the above problems, there is provided a pharmaceutical composition for treating or preventing chronic otitis media, including a polypeptide including a motif of YxxxSxxCxAAVHxGVI, according to another embodiment of the present disclosure.

In this connection, the term used herein, "chronic otitis media" may refer to all inflammatory changes occurring in a tympanic cavity. In this connection, the chronic otitis media may be perforated chronic otitis media that shows signs of perforation in the eardrum, or chronic otitis media with cholesteatoma in which the formation of the cholesteatoma occurs with or without the perforation of the eardrum. However, the present disclosure is not limited thereto. The chronic otitis media disclosed herein may collectively refer to all inflammatory responses induced by invasion of pathogenic bacteria or viruses into the middle ear.

As described above, when a polypeptide including a motif of YxxxSxxCxAAVHxGVI is administered to the middle ear or inner ear, this may induce aggregation of pathogens such as gram-negative bacteria and substantially lower the density of these pathogens. Thus, the polypeptide including a motif of YxxxSxxCxAAVHxGVI may be effective in the treatment and prevention of chronic otitis media.

Hereinafter, the present disclosure will be described in more detail with reference to the following examples. However, these examples are only intended to illustrate the present disclosure by way of example, and the scope of the present disclosure should not be construed as being limited by these examples.

In accordance with the present disclosure, by providing a pharmaceutical composition for treating or preventing sensorineural hearing loss including a polypeptide including a motif of YxxxSxxCxAAVHxGVI as an active ingredient, the polypeptide including a motif of YxxxSxxCxAAVHxGVI may induce an immune response in cochlea. Thus, the polypeptide including a motif of YxxxSxxCxAAVHxGVI may suppress the invasion of pathogenic bacteria or viruses, and protect the Corti organ.

In accordance with the present disclosure, more particularly, activity of phagocyte such as macrophage, and neutrophilic leukocyte may be induced during the immune response progresses in the cochlea. Thus, the immediate elimination of invading pathogens may be induced. Furthermore, the present disclosure may provide therapeutic and preventive effects on hearing loss induced by viral infection, as the composition may induce inflammatory changes that cause damage to the Corti organ.

Furthermore, the composition in accordance with the present disclosure may be effective in the inflammatory response induced in the middle ear by viral infection, and thus may be used for the treatment, and prevention of chronic otitis media.

Further, the pharmaceutical composition for treating or preventing sensorineural hearing loss in accordance with the present disclosure may protect the Corti organ and thus contribute to preventing or treating noise-induced hearing loss caused by noise.

In accordance with the present disclosure, the pharmaceutical composition induces aggregation of Gram-negative bacteria in the site of infection, for example the cochlea, and reduce the density of these pathogens by 2 to 3 times.

Thus, the composition in accordance with the present disclosure has a therapeutic or prophylactic effect on Corti organ damage-induced hearing loss, or tympanitis-induced hearing loss, as induced by viral infections, which is different from hearing loss induced by mutation of cochlin proteins.

The present disclosure has the effect of providing an effective amount and dosage form of the polypeptide composed of a motif of YxxxSxxCxAAVHxGVI effective for the treatment or prevention of sensorineural hearing loss, and chronic otitis media.

The present disclosure provides the pharmaceutical composition that further includes calcium, which regulates the activity of a polypeptide composed of a motif of YxxxSxxCxAAVHxGVI, for example, a polypeptide including the LCCL domain of the cochlin protein. Thus, the pharmaceutical composition may be more effective in the aggregation of pathogens that cause inflammation.

In addition to the effects as described above, specific effects of the present disclosure are described together with specific details for carrying out the invention.

FIGs. 1A to 1C show auditory changes depending on whether a coding gene of the cochlin protein that provides the polypeptide in the pharmaceutical composition in accordance with various examples of the present disclosure is knocked out and whether the pathogenic bacteria infection occurs.

FIG. 1D shows CFU of a cochlear explant depending on whether a coding gene of the cochlin protein that provides the polypeptide in the pharmaceutical composition in accordance with various examples of the present disclosure is knocked out and whether the pathogenic bacteria infection occurs.

FIG. 1E shows a level of inflammatory cytokines depending on whether a coding gene of the cochlin protein that provides the polypeptide in the pharmaceutical composition in accordance with various examples of the present disclosure is knocked out and whether the pathogenic bacteria infection occurs.

FIG. 1F shows a level of inflammatory cytokines depending on whether a wild-type mouse model (Coch^+/+) in which a coding gene of the cochlin protein that provides the polypeptide in the pharmaceutical composition in accordance with various examples of the present disclosure is not knocked out is infected with pathogenic bacteria and whether the wild-type mouse model is treated with MMP inhibitor (matrix metalloproteinase III inhibitor MMP_inh).

FIG. 2A shows a result of detection of a cochlin protein-derived LCCL domain-containing fragment used as a polypeptide in the pharmaceutical compositions of various examples, depending on whether the pathogenic bacteria infection occurs.

FIG. 2B shows a result of hearing ability change after treatment of a cochlin protein-derived LCCL-containing fragment used as a polypeptide in the pharmaceutical compositions of various examples of the present disclosure.

FIG. 2C shows a growth curve of pathogenic bacteria after treatment of a cochlin protein-derived LCCL-containing fragment used as a polypeptide in the pharmaceutical compositions of various examples of the present disclosure.

FIGs. 2D to 2F show changes in a cluster of pathogenic bacteria after treatment of a cochlin protein-derived LCCL-containing fragment used as a polypeptide in the pharmaceutical compositions of various examples of the present disclosure.

FIG. 2G shows a level of pathogenic bacteria on a plate after treatment of a cochlin protein-derived LCCL-containing fragment used as a polypeptide in the pharmaceutical compositions of various examples of the present disclosure.

FIGs. 2H and 2I show aggregation of pathogenic bacteria after treatment of a cochlin protein-derived LCCL-containing fragment used as a polypeptide in the pharmaceutical compositions of various examples of the present disclosure.

FIGs. 3A, and 3B show aggregation of pathogenic bacteria, and a density of pathogenic bacteria per unit area, depending on a treatment content of a cochlin protein-derived LCCL-containing fragment used as a polypeptide in the pharmaceutical compositions of various examples of the present disclosure.

FIG. 4 shows a result of detection of a cochlin protein-derived LCCL domain-containing fragment used as a polypeptide in a pharmaceutical composition to treat or prevent sensorineural hearing loss according to one embodiment of the present disclosure following noise exposure.

Advantages, features, and methods of achieving the present disclosure will be apparent with reference to the embodiments described below in detail with the accompanying drawings. However, the present disclosure is not limited to the embodiments disclosed below, but may be implemented in various different forms. The present embodiments merely allow the disclosure of the present disclosure to be complete and is provided to completely inform the scope of the invention to those of ordinary skill in the technical field to which the present disclosure belongs. The present disclosure is only defined by the scope of the claims.

Hereinafter, with reference to Example 1 to Example 3, the effects of pharmaceutical compositions for treating or preventing sensorineural hearing loss, and chronic otitis media according to various embodiments of the present disclosure will be described. In this connection, a peptide fragment consisting of a cochlin protein-derived LCCL domain was used as a polypeptide including the motif of YxxxSxxCxAAVHxGVI. However, the effect of the present disclosure is not limited thereto. For example, polypeptides including the LCCL domain of CRISLD1, the LCCL domain of CRISLD2, the LCCL domain of Vitrin protein, the LCCL domain of DCBLD1, and the LCCL domain of DCBLD2 may provide effects resulting from the pharmaceutical composition treatment of the present disclosure, which will be described later.

Example 1: Cochlin protein maintains auditory function, and has antibacterial effect

In a following experiment, a cochlin gene knockout mouse model (Coch^-/-), and a wild-type mouse model (Coch^+/+) as a control were prepared to observe the hearing ability change according to the cochlin gene deletion. Then, PAO1 strains of Pseudomonas aeruginosa were inoculated to a middle ear of each mouse model, such that the strains were infected to the inner ear. Then, hearing ability changes according to the cochlin deletion were evaluated. In this connection, mouse models inoculated with culture media as a control are prepared.

FIGs. 1A to 1C show auditory changes depending on whether a coding gene of the cochlin protein that provides the polypeptide in the pharmaceutical composition to treat or prevent sensorineural hearing loss and chronic otitis media according to one embodiment of the present disclosure is knocked out and whether the pathogenic bacteria infection occurs.

Referring to (a), (b), (c), and (d) of FIG. 1A, in the auditory brainstem response test, the cochlin gene knockout mouse models (Coch^-/-) and the wild-type mouse models (Coch^+/+) as inoculated with pathogenic bacteria of the PAO1 strain (Pseudomonas) may have a higher hearing ability threshold than that in a control. That is, the both models show hearing loss due to infection with the PAO1 strain. In particular, the cochlin gene knockout mouse model (Coch^-/-) may have a higher hearing ability threshold than the wild-type mouse model (Coch^+/+). These results may indicate a significant hearing loss following infection of pathogenic bacteria in the cochlin gene knockout mouse model (Coch^-/-). This may mean that the cochlin protein contributes to hearing function protection.

Referring to FIG. 1B, in the cochlin gene knockout mouse models (Coch^-/-) and the wild-type mouse models (Coch^+/+) as inoculated with pathogenic bacteria of the PAO1 strain (Pseudomonas), the cochlin gene knockout mouse model (Coch^-/-) may have a higher hearing ability threshold than the wild-type mouse model (Coch^+/+). This result may mean that hearing loss according to infection of pathogenic bacteria is large due to cochlin deletion in the cochlin gene knockout mouse model (Coch^-/-). Furthermore, this may mean that cochlin protein contributes to hearing function protection.

Referring to FIG. 1C, the results of the ABR evaluation performed at 4000, 8000, 16,000, and 32,000 Hz are shown. More specifically, the cochlin gene knockout mouse model (Coch^-/-) has a high level hearing ability threshold of about 100 Db after the infection by pathogenic bacteria of the PAO1 strain (Pseudomonas). On the other hand, the wild-type mouse model without cochlin gene deletion (Coch^+/+) may have a hearing ability threshold similar to that of the control mouse model, even when pathogenic bacteria are infected thereto. That is, this result may mean that hearing loss according to pathogenic bacteria infection is large due to cochlin deletion in the cochlin gene knockout mouse model (Coch^-/-). Furthermore, this may mean that cochlin protein contributes to hearing function protection.

In a following experiment, to evaluate the function of cochlin protein in ex vivo cochlear explant, after inoculating PAO1 strain through the inner ear for each of the cochlin gene knockout mouse model (Coch^-/-), and the wild-type mouse model (Coch^+/+), a cochlear explant of the inner ear of each model is obtained as anatomical procedures acquire. In this connection, after inoculating the culture medium to the cochlin gene knockout mouse model (Coch^-/-), and the wild-type mouse model (Coch^+/+), the cochlear explants obtained from the both models are set as controls.

FIG. 1D shows CFU of a cochlear explant depending on whether a coding gene of the cochlin protein that provides the polypeptide in the pharmaceutical composition to treat or prevent sensorineural hearing loss or the pharmaceutical composition to treat or prevent chronic otitis media according to one embodiment of the present disclosure is knocked out and whether the pathogenic bacteria infection occurs. FIG. 1E shows a level of inflammatory cytokines depending on whether a coding gene of the cochlin protein that provides the polypeptide in the pharmaceutical composition to treat or prevent sensorineural hearing loss according to one embodiment of the present disclosure is knocked out and whether the pathogenic bacteria infection occurs.

Referring to FIG. 1D, cochlear explants respectively isolated from the cochlin gene knockout mouse model (Coch^-/-) and wild-type mouse model (Coch^+/+) inoculated with PAO1 strain were cultured in DMEM medium and then culturing result is shown. More specifically, the number of bacteria in the cochlear explant derived from the wild-type mouse model (Coch^+/+) may be about 1.5 times lower than that of the cochlin gene knockout mouse model (Coch^-/-) derived cochlear explant. That is, bacteria show a significantly higher growth rate in the inner ear of the cochlin gene knockout mouse model (Coch^-/-) than in the inner ear of the wild-type mouse model (Coch^+/+). These results may mean that the cochlin protein in the inner ear is involved in inhibiting bacterial growth.

Referring to (a), (b), (c), and (d) of FIG. 1E, expression levels of inflammatory cytokines measured in culture media of cochlear explants respectively isolated from the cochlin gene knockout mouse models (Coch^-/-) and wild-type mouse models (Coch^+/+) inoculated with PAO1 strains or culture media are shown. More specifically, the levels of IL-1b, and IL-6 among cytokines upon infection of pathogenic bacteria (Pseudomonas) may be significantly higher in the cochlear explant derived from the wild-type mouse model (Coch^+/+) than in the cochlin gene knockout mouse model (Coch^-/-) derived cochlear explant. These results may indicate that increased expression of cochlin in the inner ear during pathogenic bacterial infection may result in increased secretion of inflammatory cytokines, particularly IL-1b, and IL-6. Thus, the immune response may be increased by cochlin. Furthermore, this result may indicate that cochlin proteins may be involved in the immune response following infection of pathogenic bacteria in the inner ear.

FIG. 1F shows a level of inflammatory cytokines depending on whether a wild-type mouse model (Coch^+/+) in which a coding gene of the cochlin protein that provides the polypeptide in the pharmaceutical composition to treat or prevent sensorineural hearing loss according to one embodiment of the present disclosure is not knocked out is infected with pathogenic bacteria and whether the wild-type mouse model is treated with MMP inhibitor (matrix metalloproteinase III inhibitor MMP_inh).

Referring to (a), and (b) of FIG. 1F, expression levels of IL-1b, and IL-6 based on absence or presence of MMP inhibitor treatment to the cochlear explant of cochlin non-knockout mouse (Coch^+/+), and based on absence or presence of pathogenic bacterial infection are shown. In this connection, MMP inhibitors may act to block the function of protease. That is, the MMP inhibitor may inhibit the protease of MMP which has a function of cleaving fragments of the LCCL domain with respect to the cochlin protein. More specifically, depending on absence or presence of the pathogenic bacterial infection, the expression level of IL-1b, and IL-6 in the cochlea of mice without gene knockout (Coch^+/+) may be increased by the expression of cochlin. In contrast, however, when MMP inhibitors are treated, the expression levels of IL-1b, and IL-6 may be lowered to levels similar to that of the control. This result may indicate that as the MMP inhibitor treatment reduced the cleavage of LCCL domain fragments from the cochlin protein despite the expression of cochlin, the immune response increase does not occur following infection with pathogenic bacteria. As a result, increased expression levels of inflammatory cytokines such as IL-1b, and IL-6 in the inner ear, i.e. increased immune response, may be deeply associated with the LCCL domain of the cochlin protein. Therefore, the LCCL domain of the cochlin protein increases the immune response following invasion of pathogenic bacteria, and may contribute to protecting hearing function. Thus, the LCCL domain may be used for the treatment and prevention of sensorineural hearing loss, in particular hearing loss induced by bacterial infection or tympanitis.

As a result of Example 1 above, the cochlin protein, particularly, the LCCL domain used in various embodiments of the present disclosure of the cochlin proteins may protect hearing loss during bacterial infection, and has antibacterial effect against pathogenic bacteria, and may enhance the immune response. Accordingly, the "polypeptide including the motif of YxxxSxxCxAAVHxGVI" used in various embodiments of the present disclosure may be a polypeptide including the LCCL domain. Furthermore, the polypeptide composed of the LCCL domain may be used as an active ingredient of a composition for treating or preventing sensorineural hearing loss, especially hearing loss induced by bacterial infection or tympanitis.

Example 2: Auditory function maintenance and antibacterial effect by polypeptides including LCCL domain

In the following experiments, to evaluate the detection of LCCL domain-containing fragments according to the presence or absence of pathogenic bacterial infection in ex vivo cochlear explant, after inoculating PAO1 strain through the inner ear of each of the cochlin gene knockout mouse model (Coch^-/-), and the wild-type mouse model (Coch^+/+), a cochlear explant of the inner ear is obtained as anatomical procedures. In this connection, after inoculating the culture medium for the cochlin gene knockout mouse model (Coch^-/-), and the wild-type mouse model (Coch^+/+), cochlear explants obtained from both models are set as controls.

FIG. 2A shows a result of detection of a cochlin protein-derived LCCL domain-containing fragment used as a polypeptide in the pharmaceutical compositions to treat or prevent sensorineural hearing loss according to one embodiment of the present disclosure, depending on whether the pathogenic bacteria infection occurs.

Referring to (a) of FIG. 2A, cochlin protein is composed of LCCL domain and vWFA domain. In this connection, LCCL domain-containing fragments resulting from cleavage of the cochlin protein may be detected by an N-terminal antibody (N-terminal Ab). vWFA domain-containing fragments may be detected by C-terminal antibodies (C-terminal Ab).

Referring to (b) of FIG. 2A, under a condition (basal condition) free of pathogenic bacteria of PAO1 strain (Pseudomonas), for cochlea tissue, cochlin protein with a size of about 60 kDa may have been detected. In the meantime, in cochlear tissues derived from a wild-type mouse model (Coch^+/+) after infection with pathogenic bacteria, LCCL domain-containing fragments and vWFA domain-containing fragments of 8, and 18 kDa, were detected following cochlin protein cleavage. In contrast, cochlin protein, LCCL domain fragments and vWFA domain fragments were not detected in cochlear tissue derived from a cochlin gene knockout mouse model (Coch^-/-) after infection with pathogenic bacteria. This result may mean that the cochlin protein was cleaved due to infection of pathogenic bacteria in cochlear tissue, and thus the LCCL domain-containing fragment was detected.

Referring to (c) of FIG. 2A, cochlin protein was not detected in cochlea perilymph before and after infection of pathogenic bacteria of PAO1 strain (Pseudomonas). Meanwhile, LCCL domain-containing fragments are observed in cochlea perilymph derived from wild-type mouse model (Coch^+/+) after infection of pathogenic bacteria. These results may mean that the LCCL domain-containing fragments secreted in the cochlear tissues migrated to the cochlear perilymph following infection of the pathogenic bacteria.

In the following experiments, the infection of the pathogenic bacteria into the inner ear of the cochlin gene knockout mouse model (Coch^-/-) was induced. Hearing ability changes according to absence or presence of the addition of a peptide having the sequence of the LCCL domain are evaluated. In this connection, the peptide may be topically applied or microinjected into the inner ear of the cochlin gene knockout mouse model (Coch^-/-). Further, in this experiment, an uninfected cochlin gene knockout mouse model (Coch^-/-) was set as a control.

FIG. 2B shows a result of hearing ability change after treatment of a cochlin protein-derived LCCL-containing fragment used as a polypeptide in the pharmaceutical compositions to treat or prevent sensorineural hearing loss according to one embodiment of the present disclosure.

Referring to FIG. 2B, the cochlin gene knockout mouse model (Coch^-/-) shows a high hearing ability threshold of about 80 dB following infection with pathogenic bacteria. In contrast, the mouse model treated with the peptide having the sequence of LCCL domain shows a hearing ability threshold of about 60 dB, similar to that of the control. These results may indicate that the hearing ability of the cochlin gene knockout mouse model (Coch^-/-) was restored upon treatment of the peptide.

In the following experiments, pathogenic bacteria of GFP (green fluorescent protein) labeled PAO1 (GFP-PAO1) were prepared to assess the changes in pathogenic bacteria following peptide treatment with sequences in the LCCL domain. In this connection, pathogenic bacteria may tinged with fluorescent green at certain wavelengths, for example at 655 nm by GFP.

FIG. 2C shows a growth curve of pathogenic bacteria after treatment of a cochlin protein-derived LCCL-containing fragment used as a polypeptide in the pharmaceutical compositions to treat or prevent sensorineural hearing loss according to one embodiment of the present disclosure. FIGs. 2D to 2F show changes in a cluster of pathogenic bacteria after treatment of a cochlin protein-derived LCCL-containing fragment used as a polypeptide in the pharmaceutical compositions to treat or prevent sensorineural hearing loss according to one embodiment of the present disclosure.

Referring to FIG. 2C, there was no significant change in bacterial growth according to the treatment of the peptide consisting of LCCL domain.

In the meantime, referring to FIG. 2D, GFP-PAO1 showed different colony formation according to LCCL fragment treatment. More specifically, only GFP-PAO1 before treatment of the peptide of the LCCL domain is applied may form a single colony. In contrast, when the LCCL domain was applied, the aggregation of the pathogenic bacterium PAO1 appeared. These results may mean that the LCCL domain induces the aggregation of pathogenic bacteria.

Referring to FIG. 2E, a peptide composed of a LCCL domain tinged with red. In this connection, aggregation of GFP-PAO1 may be caused by peptides composed of LCCL domains. That is, the LCCL domain may induce aggregation through direct interaction with the pathogenic bacteria of PAO1.

Referring to (a), and (b) of FIG. 2F, the density of GFP-PAO1 decreased when LCCL was added. In addition, GFP-PAO1 having a diameter of 20 mm or more increased with LCCL addition, unlike the control containing GFP-PAO1 alone. This may be due to the formation of aggregated clusters following the aggregation of pathogenic bacteria of PAO1 by the LCCL.

Referring to (a), and (b) of FIG. 2G, colonies of GFP-PAO1 cultured in LB medium, and colonies of GFP-PAO1 cultured in LB medium with LCCL added thereto are shown. More specifically, the number of single colonies of GFP-PAO1 cultured in LB medium with LCCL added thereto may be smaller than that when LCCL was not added thereto.

Referring to FIG. 2H, an image showing the growth pattern of GFP-PAO1 following LCCL fragment treatment as obtained by electron microscope, is shown. More specifically, LCCL induces aggregation of pathogenic bacteria (see white arrows), and may aggregate bacteria by enclosing the bacteria in the form of traps (see yellow arrows) or forming complex beads, and strings (see red arrows).

Referring to FIG. 2I, the result of treatment after LCCL is added to the culture medium of GFP-PAO1 and then EDTA is added thereto is shown. More specifically, the aggregation of GFP-PAO1 formed by LCCL may dissociate upon treatment with ethylene diamine tetra acetic acid (EDTA). In this connection, EDTA may cause calcium depletion. That is, the aggregation of pathogenic bacteria by LCCL may be calcium content dependent. For example, if calcium content increases, the aggregation of pathogenic bacteria by LCCL may be further improved. Accordingly, the pharmaceutical composition used in various embodiments of the present disclosure may further include calcium.

As a result of the Example 2, when the polypeptide including the LCCL domain used in various embodiments of the present disclosure is micro-injected or topically applied to the inner ear or middle ear, this stimulates neutrophils/monocytes to induce an immune response by the secretion of inflammatory cytokines, and thus induces aggregation of pathogenic bacteria, in particular Gram-negative bacteria. That is, the polypeptide including the LCCL domain induces an immune response and induces bacterial aggregation to protect hearing function, when bacterial infection into the cochlea occur.

Accordingly, the pharmaceutical composition according to various embodiments of the present disclosure, particularly the pharmaceutical composition including the polypeptide including the LCCL domain used as an active ingredient may have a therapeutic or prophylactic effect on Corti organ damage-induced hearing loss, or tympanitis-induced hearing loss, induced by viral infection, which is different from the hearing loss induced by mutation of the cochlin protein.

Furthermore, the present disclosure may provide a pharmaceutical composition that further includes calcium to regulate bacterial aggregation activity by polypeptides containing the LCCL domain and thus be more effective in the aggregation of pathogens cause inflammation.

In the meantime, the effect and use of the polypeptide including the motif of YxxxSxxCxAAVHxGVI, particularly the polypeptide including the LCCL domain used in various embodiments of the present disclosure are not limited thereto. For example, the polypeptide including the LCCL domain may be provided as a food composition for preventing or improving the hearing loss caused by bacterial infection.

Example 3: Antibacterial effect according to the content of polypeptide including LCCL domain

In the following experiments, pathogenic bacteria of GFP labeled PAO1 (PAO1-GFP) were prepared to evaluate the growth change of pathogenic bacteria according to the treatment content of the peptide including the LCCL domain. In this connection, the treatment contents of peptides containing the LCCL domain are 0.06 mM (LCCL 10), 0.18 mM (LCCL 30) and 0.6 mM (LCCL 100). Furthermore, in this experiment, PAO1-GFP not subjected to the peptide treatment is set as a control.

FIGs. 3A, and 3B show aggregation of pathogenic bacteria, and a density of pathogenic bacteria per unit area, depending on a treatment content of a cochlin protein-derived LCCL-containing fragment used as a polypeptide in the pharmaceutical compositions to treat or prevent sensorineural hearing loss according to one embodiment of the present disclosure.

Referring to FIG. 3A, the levels of green fluorescent light decreased in the three groups treated with LCCL domain-containing peptides, which is not the case in the control without LCCL domain-containing peptides (PAO1-GFP). In particular, the experimental group treated with 0.6 mM LCCL domain-containing peptide (PAO1-GFP + LCCL 100) showed a lower level of green fluorescent light than the other two groups (PAO1-GFP + LCCL 10, and PAO1-GFP + LCCL 30). This result may mean a decrease in growth of PAO1 with increasing LCCL content.

Referring to FIG. 3B, the density of PAO1 per unit area was decreased in the three experimental groups treated with LCCL domain-containing peptides, which is not the case in the control that were not treated with LCCL domain-containing peptides (PAO1-GFP). In particular, the experimental group treated with 0.6 mM LCCL domain-containing peptide (PAO1-GFP + LCCL 100) had a lower density of PAO1 per unit area than the other two groups (PAO1-GFP + LCCL 10, and PAO1-GFP + LCCL 30). This result may mean a decrease in growth of PAO1 with increasing LCCL content.

As a result of the Example 3, the content of the polypeptide including the LCCL domain used in various embodiments of the present disclosure may be in range of 0.1 mM to 1 mM with respect to the entire content of the pharmaceutical composition. Preferably, the content of the polypeptide including the LCCL domain to be used may be in a range of 0.06 mM to 0.6 mM based on the entire content of the pharmaceutical composition. However, the content of the polypeptide including the LCCL domain is not limited thereto. The content of the polypeptide including the LCCL domain may be set in various ways as long as the pharmaceutical composition for treating or preventing sensorineural hearing loss according to various embodiments of the present disclosure has the maximum effect in a minimum amount thereof without side effects.

Example 4: LCCL domain secretion based on noise exposure

Hereinafter, referring to FIG. 4, the result of secretion of LCCL domain by noise exposure is explained. In this evaluation, the mouse model was exposed, for 3 hours, to the noise at 115 dB, and then the levels of the LCCL domains in the right (Rt) and left (Lt) inner ear after 3 hours, 1 day, and 3 days were evaluated. In this connection, the LCCL domain may be detected by an N-terminal antibody (N-terminal Ab).

Referring to FIG. 4, one day after exposure to noise, fragments containing LCCL domains of 8, and 18 kDa were detected after cleavage of the cochlin protein. These results may mean that the cleavage of the cochlin protein occurs due to the stimulation of the inner ear, especially the cochlea, due to noise, and thus the LCCL domain-containing fragment may be detected. Furthermore, this indicates that the LCCL domain-containing fragments are closely related to the pathogenesis of noise-induced hearing loss and thus, secretion control thereof may lead to treating, and preventing noise-induced hearing loss.

Accordingly, a pharmaceutical composition according to various embodiments of the present disclosure, particularly a pharmaceutical composition including a polypeptide including an LCCL domain as an active ingredient may have a therapeutic or preventive effect on noise-induced hearing loss caused by noise.

Meanwhile, the effect and use of the polypeptide including the motif of YxxxSxxCxAAVHxGVI, particularly, the polypeptide including the LCCL domain used in various embodiments of the present disclosure are not limited thereto.

Although the embodiments of the present disclosure have been described in more detail with reference to the accompanying drawings, the present disclosure is not necessarily limited to these embodiments. Various modifications may be made thereto without departing from the technical spirit of the present disclosure. Accordingly, the embodiments disclosed in the present disclosure are not intended to limit the technical spirit of the present disclosure, but to describe the technical spirit of the present disclosure. The scope of the technical spirit of the present disclosure is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of protection of the present disclosure should be interpreted by the following claims. All technical ideas falling within equivalent scope should be interpreted as being included in the scope of the present disclosure.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY

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SENSORINEURAL HEARING LOSS AND CHRONIC OTITIS MEDIA

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[National R & D Projects Supporting This Invention 1]Task identification number:2017R1D1A1B03030046, Name of government department:Ministry of Education, Research Management Specialized Organization:National Research Foundation of Korea, Name of Research Project:Personal Science Basic Research Support Project, Project name:Identification of the deafness effect of LCCL peptide in cochlea, Contribution rate: 7/10, Managing department:Yonsei University Industry-Academic Cooperation Foundation, Research Period: 2018.03.01 ~ 2019.02.28

[National R & D Projects Supporting This Invention 2]Task identification number:2017M3A9E8029714, Name of government department:Ministry of Science and ICT, Research Management Specialized Organization:National Research Foundation of Korea, Name of Research Project:Biomedical Technology Development Program Clinical Scientists Research Capacity Enhancement Project, Project name:Drugs based on genome-specific induced pluripotent stem cells in KoreanScreening for Aging Hearing Loss Treatment, Contribution rate: 3/10, Managing department:Yonsei University Industry-Academic Cooperation Foundation, Research Period: 2017.04.01 ~ 2021.03.31

Claims

A pharmaceutical composition for treating or preventing sensorineural hearing loss, the composition comprising a polypeptide including a motif of YxxxSxxCxAAVHxGVI as an active ingredient, wherein x is any amino acid residue.
The pharmaceutical composition of claim 1, wherein the polypeptide includes at least one selected from the group consisting of a peptide including an LCCL domain of cochlin protein, a peptide including an LCCL domain of CRISLD1 (Cysteine-rich secretory protein LCCL domain-containing 1), a peptide including an LCCL domain of CRISLD2 (Cysteine-rich secretory protein LCCL domain-containing 2), a peptide including an LCCL domain of Vitrin protein, a peptide including an LCCL domain of DCBLD1 (Discoidin, CUB and LCCL domain-containing protein 1), and a peptide including an LCCL domain of DCBLD2 (Discoidin, CUB and LCCL domain-containing protein 2), respectively.
The pharmaceutical composition of claim 2, wherein the polypeptide is composed of the LCCL domain of the cochlin protein.
The pharmaceutical composition of claim 1, wherein the polypeptide having at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
The pharmaceutical composition of claim 4, wherein the polypeptide includes a peptide having the amino acid sequence of SEQ ID NO: 1.
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition has an intratracheal microinjection form or a topical application dosage form.
The pharmaceutical composition of claim 1, wherein sensorineural hearing loss includes at least one selected from the group consisting of viral infection based Corti organ damage-induced hearing loss, chronic otitis media-induced hearing loss, presbycusis, noise-induced hearing loss, sudden sensorineural hearing loss, autoimmune hearing loss, ischemia-induced hearing loss, head injury-induced hearing loss, ototoxcity medication-induced hearing loss, and hereditary hearing loss.
The pharmaceutical composition of claim 1, wherein sensorineural hearing loss includes at least one of viral infection based Corti organ damage-induced hearing loss, chronic otitis media-induced hearing loss, or noise-induced hearing loss.
The pharmaceutical composition of claim 1, wherein the sensorineural hearing loss differs from hearing loss induced by mutation of a cochlin protein.
The pharmaceutical composition of claim 1, wherein the polypeptide induces aggregation of Gram-negative bacteria, and stimulates immune cells for removal of the gram-negative bacteria.
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises calcium ions or calcium releasing materials.
The pharmaceutical composition of claim 1, wherein a content of the polypeptide is in a range of 0.1 mg/ml to 100 mg/ml based on a content of the pharmaceutical composition.
A method for preventing or treating sensorineural hearing loss, the method comprising administering the pharmaceutical composition according to one of claims 1 to 12 to a subject suspected of sensorineural hearing loss, excluding a human.
The method of claim 13, wherein administering the pharmaceutical composition comprises microinjecting or topically applying the pharmaceutical composition to an inner ear or middle ear of the subject suspected of sensorineural hearing loss.
The method of claim 13, wherein a density of Gram-negative bacteria in the inner ear or middle ear of the subject suspected of a sensorineural hearing loss administered with the pharmaceutical composition is 2 to 3 times lower than a density of Gram-negative bacteria in an inner ear or middle ear of a subject suspected of a sensorineural hearing loss not administered with the pharmaceutical composition.
A food composition for preventing or improving hearing loss caused by bacterial infection, the composition comprising a polypeptide including a motif of YxxxSxxCxAAVHxGVI as an active ingredient, wherein x is any amino acid residue.
A pharmaceutical composition for treating or preventing chronic otitis media, the composition comprising a polypeptide including a motif of YxxxSxxCxAAVHxGVI as an active ingredient, wherein x is any amino acid residue.
The pharmaceutical composition of claim 17, wherein the polypeptide induces aggregation of Gram-negative bacteria, and stimulates immune cells for removal of the gram-negative bacteria.