JP2018090497A - Medical treatment for inner ear disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel treatment based on inner ear disease mechanism.SOLUTION: An agent for preventing or treating an inner ear disease which contains a necrosis inhibitor as an active ingredient is provided. The necrosis inhibitor is selected from the group consisting of Necrostatin-1, Necrostatin-2, Necrostatin-3, Necrostatin-4, Necrostatin-5, Necrostatin-7, Ponatinib, and a pharmaceutically acceptable salt, a solvate or prodrug thereof.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a novel therapeutic agent for inner ear diseases, and more specifically to a therapeutic agent for diseases and symptoms caused by inner ear disorders such as deafness such as sudden deafness, dizziness and tinnitus.

  Sudden deafness suddenly causes severe tinnitus and deafness in one ear, and various factors such as blood flow disorders are considered as onset factors. At present, viral infection theory is dominant. According to an epidemiological survey conducted by the Ministry of Health, Labor and Welfare's Acute Deafness Research Group every 10 years, the number of patients treated nationwide was 24,000 per year in 1993 and 35,000 per year in 2001. According to an estimation by epidemiological studies conducted in Ehime, Aichi and Iwate Prefectures by a research group, the number of cases was reported to be 60.9 per 100,000 population (Survey on acute severe hearing loss (2013) 2013)).

  Sound enters the ear canal as vibrations of the air, is transmitted from the eardrum to the ossicles, and further becomes a vibration of lymph filled in the inner ear, which is converted into an electrical signal by hair cells on the basement plate of the cochlea, via the auditory nerve It is transmitted to the auditory center of the brain. When this hair cell is damaged, it becomes impossible to feel the sound, but in the case of sudden deafness, the sensory hair in the hair cell falls off and causes hair cell death due to the study using human temporal bone pathology It is known.

  Currently, corticosteroids, diuretics, vitamins, blood flow improvers, etc. are used for drug treatment of sudden deafness, and interferon is used as a therapeutic agent for deafness diseases including sudden deafness. Such proteins and small molecule compounds have been proposed (Patent Documents 1 to 5).

  On the other hand, cell death has traditionally been differentiated between pre-programmed apoptosis and accidental necrosis associated with changes in the environment surrounding the cell. It has become clear that there is something that is highly controlled, as is the case with necrotosis. Although the mechanism of necrotosis has not been fully elucidated, a necrotosis inhibitor of a small molecule compound has been reported and includes a compound named necrostatin-1 (Non-patent Documents 1 to 3).

  A method for promoting axonal regeneration in CNS neurons using a necrosis inhibitor (Patent Document 6), or a pharmaceutical composition for preventing or treating a necrotosis-related disease using a necrotosis inhibitor (Patent Document 7) is disclosed. However, the relationship with inner ear disease is not disclosed.

Patent No. 2759050 JP2004-99537 JP 2004-123713 A JP 2007-99675 JP 2012-148995 Special table 2014-530881 International Publication WO 2014/126127

Nature Chemical Biology 1, 112-119 (2005) Cell Death and Differentiation 20, 185-187 (2013) Cell Death and Differentiation 20, 366 (2013)

  As described above, corticosteroids, diuretics, vitamins, blood flow improving drugs, and the like have been used for the treatment of sudden deafness, but there is no established treatment with evidence. It is desired to elucidate the mechanism of inner ear diseases including sudden deafness and to develop a new treatment based on the mechanism.

  In the mouse cochlear tissue infected with the Tyler mouse encephalomyelitis virus (TEMV), the present inventors have damaged hair cells, causing loss of sensory hair and hair cell death. It was also found for the first time that this hair cell death was due to necroptosis. This phenomenon can be used as an unprecedented inner ear disease model system for elucidating the mechanism of disease, screening for therapeutic agents, and the like.

  The present inventors further studied the effects of various compounds using the above-mentioned inner ear disease model, and surprisingly, as a result, the signaling molecule RIP kinase 1 (RIPK1: receptor), which is said to hold the key to necroptosis, is surprisingly found. It was found that hair cell death is suppressed by a compound having a selective inhibitory action of interacting serine / threonine kinase 1).

The present invention has been completed based on these findings. That is, the present invention provides the following.
1. A preventive or therapeutic agent for inner ear diseases, comprising a necrotosis inhibitor as an active ingredient.
2. 2. The preventive or therapeutic agent according to 1 above, wherein the necrotosis inhibitor is a selective inhibitor of RIP kinase 1 (RIPK1: receptor interacting serine / threonine kinase 1).
3. 3. The preventive or therapeutic agent according to 1 or 2 above, which suppresses sensory hair loss and / or hair cell death in the inner ear.
4). Necrostatin inhibitors include necrostatin-1, necrostatin-2, necrostatin-3, necrostatin-4, necrostatin-5, necrostatin-7, ponatinib, and their pharmaceutically 4. The prophylactic or therapeutic agent according to any one of 1 to 3 above, which is selected from acceptable salts, solvates or prodrugs.
5. 5. The preventive or therapeutic agent according to any one of 1 to 4 above, wherein the inner ear disease is sensorineural hearing loss or mixed hearing loss.
6). 5. The preventive or therapeutic agent according to any one of 1 to 4 above, wherein the inner ear disease is inner ear deafness.
7). The preventive or therapeutic agent according to any one of 1 to 6 above, which is administered alone or in combination with another drug.

  The present invention provides a new therapeutic agent based on a mechanism different from that of conventionally used or proposed therapeutic agents for hearing loss. Therefore, the therapeutic agent of the present invention can be applied alone or in combination with other drugs.

Sensory hairs of hair cells in mouse cochlear tissues infected with Tyler mouse encephalomyelitis virus (TEMV) were stained with phalloidin (Invitrogen), and at the same time, myosin 7a (myosin7a) was stained as a marker for hair cells. . OHC: outer hair cell, IHC: inner hair cell. A: After 24 hours from virus infection, loss of sensory hair and hair cell death stained with phalloidin staining were observed in most hair cells. B: The result of adding 20 μM of necrostatin-1 at the time of virus infection is shown. C: The result of adding 200 μM of necrostatin-1 at the time of virus infection is shown. D: shows the result of adding 400 μM of necrostatin-1 at the time of virus infection. Depending on the amount of necrostatin-1 added, hair cell death and sensory hair loss are reduced, and yellow (red and green are dyed simultaneously = sensory hair + hair cells) is often observed. It shows that the decrease of both outer hair cells (A) and inner hair cells (B) is suppressed depending on the concentration of necrostatin-1 added. The result of staining the mouse cochlear tissue infected with TEMV with DAPI fluorescent dye and examining the phosphorylation of MLKL is shown. It is shown that drop of sensory hair and hair cell death are suppressed by adding liposome (Clophosome) containing clodronic acid at the time of virus infection. It shows that the decrease of both outer hair cells (A) and inner hair cells (B) is suppressed in the presence of liposome containing clodronic acid (Clophosome). It shows that both sensory hair loss and hair cell death are suppressed depending on the concentration of ponatinib added. A: Ponatinib 5.0 μM, B: Ponatinib 0.5 μM. It shows that the decrease of both outer hair cells (A) and inner hair cells (B) is suppressed depending on the concentration of ponatinib added.

  The present invention provides a preventive or therapeutic agent for inner ear diseases, which contains a necrotosis inhibitor as an active ingredient. In this specification, the necrotosis inhibitor is specifically a selective inhibitor of RIP kinase 1 (RIPK1: receptor interacting serine / threonine kinase 1).

  The present inventors have found that the preventive or therapeutic agent described above suppresses sensory hair loss and / or hair cell death in the inner ear.

  Necrostatin inhibitors preferably used in the present invention are necrostatin-1, necrostatin-2, necrostatin-3, necrostatin-4, necrostatin-5, necrostatin-7, ponatinib, and their pharmaceuticals. Selected from pharmaceutically acceptable salts, solvates or prodrugs.

  These compounds are synthesized based on the description of, for example, Nature Chemical Biology 1, 112-119 (2005); Cell Death and Differentiation 20, 185-187 (2013); Cell Death and Differentiation 20, 366 (2013). It is also possible to obtain a commercial product.

  For example, necrostatin-1, which is an example of a necrotosis inhibitor, is a compound represented by the following formula. It has the IUPAC name of 5-((1H-indol-3-yl) methyl) -3-methyl-2-thioxoimidazolidin-4-one and is also referred to as methylhydantoin- (DL-) tryptophan. Necrostatin-1 can be synthesized, and can also be obtained from Funakoshi Co., Ltd., Abcam Co., etc.

  Necrostatin-1 has been reported as a specific inhibitor of necrotosis. Regarding necroptosis, phosphorylation is continuously induced from RIPK (RIP kinase) 1 → RIPK3 → MLKL (Mixed lineage kinase domain-like, adapter molecule with pseudokinase domain), and phosphorylated MLKL is expressed in the cell membrane. A model has been proposed that necrotosis is performed by forming pores (Vanden Berghe et al., Nat. Rev. Mol. Cell Biol., 15 (2), 135-147, 2014; Pasparakis and Vandenabeele, Nature 517, 311-320, 2015). Necrostatin-2, necrostatin-3, necrostatin-4, necrostatin-5, and necrostatin-7 are also selected for RIP kinase 1 (RIPK1: receptor interacting serine / threonine kinase 1), similar to necrostatin-1. It is said that necrotosis is inhibited by a mechanical inhibition mechanism.

  Ponatinib is a compound represented by the following formula. 3- (2- {Imidazo [1,2-b] pyridazin-3-yl} ethynyl) -4-methyl-N- {4-[(4-methylpiperazin-1-yl) methyl] -3- (tri It has the IUPAC name of fluoromethyl) phenyl} -benzamide and is sometimes referred to as AP24534. Ponatinib can be synthesized or can be obtained from Selleckchem. Ponatinib has been reported to be effective as a therapeutic agent for chronic myelogenous leukemia, but it has not been known that it can be used as a prophylactic or therapeutic agent for inner ear diseases by necrotosis inhibitory action.

  Also, pharmaceutically acceptable salts, solvates, and prodrugs of necrostatin-1, necrostatin-2, necrostatin-3, necrostatin-4, necrostatin-5, necrostatin-7, and ponatinib Can be used in the same manner as long as they are converted into these compounds in vivo after administration. Examples of the pharmaceutically acceptable salt include, but are not limited to, hydrochloride, sulfate, phosphate, succinate, fumarate, mesylate, tosylate, hydrobromide , Acetate, trifluoroacetate and the like can be preferably used. Examples of solvates include, but are not limited to, hydrates. Examples of the prodrug include esters that are decomposed by an enzyme in the body to produce the above compound.

  The present inventors have found that necrotosis inhibitors suppress the loss of sensory hairs and cell death of hair cells in mammalian cochlear tissues in a concentration-dependent manner. This effect was observed in both outer hair cells (OHC) and inner hair cells. Moreover, the loss of sensory hair and cell death of the hair cells are due to phosphorylation of MLKL, which is a downstream molecule of the above phosphorylation pathway, and the supporting cells around the hair cells are changed to macrophages. It was also suggested that hair cells are damaging by Trail (TNF-related apoptosis-inducing factor).

  Therefore, based on the knowledge of the present invention, the necrotosis inhibitor can be used as an agent for preventing or treating inner ear diseases. Examples of such compounds include necrostatin-1, necrostatin-2, necrostatin-3, necrostatin-4, necrostatin-5, necrostatin-7, and ponatinib.

  The inner ear is an organ that controls hearing and balance functions, but in any function, hair cells convert physical stimuli into neural stimuli and transmit them to the spiral ganglion cells. Once damaged, hair cells are extremely difficult to regenerate. When hair cells are lost, ganglion cells are gradually lost and inner ear damage progresses irreversibly. When the inner ear is damaged, it causes symptoms such as dizziness and tinnitus in addition to hearing loss.

  Therefore, the inner ear diseases in the present invention include sudden hearing loss, noise-induced hearing loss, senile deafness, genetic deafness, bilateral sensorineural hearing loss, unilateral sensorineural hearing loss, bilateral mixed hearing loss, unilateral mixed hearing loss, drug-related hearing loss Deafness as well as ear vascular disorders, Meniere's disease, benign paroxysmal positional vertigo, vestibular neuritis or other peripheral vertigo, other vestibular dysfunction, tinnitus, or auditory neuropathy.

  Alternatively, hearing loss can be caused by differences in impaired organs from the outer ear to the middle ear (sound transmitter), sound-induced deafness, inner ear or auditory nerve, or sensory sensory deafness, It is classified as mixed hearing loss with both sound and sound sensor impairments. Therefore, the target disease of the preventive or therapeutic agent of the present invention is sensorineural hearing loss or mixed hearing loss, and particularly targets inner ear hearing loss.

  Necrotosis inhibitors, more specifically necrostatin-1, necrostatin-2, necrostatin-3, necrostatin-4, necrostatin-5, necrostatin-7, and ponatinib, or pharmaceutically acceptable thereof The salts, solvates or prodrugs used are used for the prevention or treatment of the aforementioned inner ear diseases. As used herein, “treatment” refers to complete or partial cure of a disease, relief of symptoms, prevention of progression of symptoms in patients who already have symptoms, and “prevention” refers to the possibility of a disease. It refers to the suppression of onset in patients who have.

  The compound of the above formula, or a pharmaceutically acceptable salt, solvate or prodrug thereof can be administered as it is as a prophylactic or therapeutic agent, but it can be used as a carrier or excipient normally used in pharmaceutical compositions. A dosage form, preservative, oxidation stabilizer and the like can be added as appropriate to administer as a pharmaceutical composition.

  The preventive or therapeutic agent of the present invention or the above pharmaceutical composition can be administered orally and parenterally, for example, oral; intravenous, intramuscular, transdermal, subcutaneous, intradermal, intraperitoneal injection or infusion. There is no particular limitation, however. A person skilled in the art can appropriately determine a suitable administration route for administration of the preventive or therapeutic agent of the present invention or the pharmaceutical composition.

  In one embodiment, the prophylactic or therapeutic agent of the present invention or the pharmaceutical composition is administered systemically. In one embodiment, the prophylactic or therapeutic agent of the present invention or the pharmaceutical composition is administered topically. In particular, for the purpose of preventing or treating inner ear diseases, and also from the viewpoint of safety, it is preferable to administer locally to the inner ear.

  In the case of local administration to the inner ear, for example, a method in which a small amount of drug is included in a sponge-like product that has already been sold and used as a sponzel and placed in a round window that is the entrance of the inner ear. Can be used.

  The dose of the prophylactic or therapeutic agent of the present invention to human depends on the age, weight, symptoms, etc. of the patient to be administered, and is not particularly limited, but for example, 100 μg / kg body weight to 100 mg / kg body weight per day, preferably Can be in the range of 500 μg / kg body weight to 50 mg / kg body weight, more preferably 1 mg / kg to 30 mg / kg body weight. It can be administered once to several times a day, every 2 days, every 3 days, every week, every 2 weeks, every month, every 2 months, every 3 months, etc., for example, until the symptoms are alleviated.

  The prophylactic or therapeutic agent of the present invention or the above pharmaceutical composition can be administered alone or in combination with other drugs. Other drugs are one or more drugs selected from, but not limited to, steroid drugs, diuretics, vitamin drugs, and blood flow improving drugs, which may be effective by different mechanisms for inner ear disease It can be. Examples of the steroid drug include betamethasone, prednisolone, methylprednisolone, hydrocortisone, dexamethasone and the like. Examples of diuretics include isobaide, menilet and the like. Examples of vitamin drugs include vitamin B12 and Adephos as an ATP preparation. Examples of the blood flow improving drug include kallikrein, prostaglandin, low molecular weight dextran, blood coagulation inhibitor and the like.

  Alternatively, the other drug may be a drug administered for other symptoms of the subject patient. The prophylactic or therapeutic agent of the present invention and other drugs may be administered by the same administration route or by different administration routes. For example, the preventive or therapeutic agent of the present invention can be administered locally, and other drugs can be administered systemically, for example, orally.

  Therefore, the prophylactic or therapeutic agent of the present invention may be administered simultaneously with the other drugs described above, continuously or separately. In the case of simultaneous administration, the prophylactic or therapeutic agent of the present invention and the other drugs described above may be administered as separate compositions or as the same composition.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Example 1]
Tyler mouse encephalomyelitis virus belongs to the genus Cardeovirus of the Picornaviridae family and has been reported to cause encephalomyelitis and immune demyelination in mice and can be a model animal for multiple sclerosis (eg, Shinshu Medical Journal, 43 (5): 405-415, 1995).

  The present inventors have found that in mice infected with Tyler mouse encephalomyelitis virus, sensory hair loss and hair cell death are observed, and thus can be a model animal for inner ear diseases such as sudden deafness. Study was carried out.

Tissue mouse encephalomyelitis virus (TEMV) 3.0 x 10 ⁷ pfu in order to reproduce the pathology of sudden hearing loss / ml was infected.

  After infection, the cells were fixed with a 4% paraformaldehyde solution at multiple time points, and the tissues were evaluated by immunostaining. Specifically, hair cells were stained red with phalloidin (Invitrogen) that stains sensory hair. At the same time, myosin 7a (myosin 7a) was stained with myosin 7a antibody (Proteus Biosciences) and a fluorescently labeled secondary antibody (488-conjugated anti-rabbit goat IgG, Invitrogen) as a marker for hair cells. As a result, 24 hours after the start of virus infection, sensory hair loss and hair cell death of most sensory cells (hair cells) were observed (FIG. 1A).

On the other hand, when the mouse cochlear tissue was similarly infected with TEMV 3.0 × 10 ⁷ pfu / ml, necrostatin-1 (20 μM / 200 μM / 400 μM, Abcam), a necrotosis inhibitor, was simultaneously added to the medium. Tissue was fixed with 4% paraformaldehyde solution 24 hours after administration, and tissue evaluation was carried out by immunostaining. As a result, it was observed that sensory hair loss and hair cell death were suppressed depending on the concentration of necrostatin-1. (FIGS. 1B-1D).

  In the above experiment, the number of hair cells protected by the addition of necrostatin-1 was measured. Both outer hair cells: OHC (FIG. 2A) and inner hair cells: IHC (FIG. 2B) It was confirmed that the decrease in the number of cells due to virus infection was suppressed depending on the concentration of statin-1.

[Example 2]
In the same manner as in Example 1, mouse cochlear tissue 2 days old was infected with TEMV 3.0 × 10 ⁷ pfu / ml, and after 19 hours, the tissue was fixed with 4% paraformaldehyde solution. In order to confirm the importance of necroptosis in sensory hair loss and hair cell death, rabbit monoclonals were tested for the presence of phosphorylation of the regulatory molecule MLKL downstream of phosphorylase RIPK1 and RIPK3, proteins that induce necroptosis. Tissue evaluation by immunostaining was performed using an anti-p-Mlkl antibody (phospho S345, Abcam) and a fluorescently labeled secondary antibody (488-conjugated anti-rabbit goat IgG, Invitrogen).

  As a result, phosphorylation of MLKL was confirmed in cochlear hair cells, and it was revealed that hair cell death caused by virus infection is due to necrotosis (FIG. 3).

[Example 3]
In the same manner as in Example 1, 2-day-old mouse cochlea tissue was infected with TEMV 3.0 x 10 ⁷ pfu / ml, and at the same time, liposome containing clodronic acid that killed phagocytic macrophages (Clophosome, Funakoshi Co., Ltd.) 91 μg / ml was added. Twenty-four hours after administration of Clophosome, the tissue was fixed with 4% paraformaldehyde solution, and tissue evaluation was performed by immunostaining. As a result, sensory hair loss and suppression of hair cell death were observed (FIG. 4). .

  In the above experiment, when the number of hair cells protected by Clophosome administration was measured, both inner hair cells: IHC (FIG. 5A) and outer hair cells: OHC (FIG. 5B) were transformed into macrophages. The degree of damage caused by the supporting cells was reduced.

  From the above results, it was confirmed that the supporting cells, that is, the group of cells present around the hair cells, changed to macrophages and secreted Trail to damage the hair cells.

[Example 4]
In the same manner as in Example 1, when infecting mouse cochlear tissue with TEMV 3.0 × 10 ⁷ pfu / ml, a necrotosis inhibitor ponatinib (0.5 μM / 5 μM, Selleckchem) was simultaneously added to the medium. Tissue was fixed with 4% paraformaldehyde solution 24 hours after administration, and tissue evaluation was performed by immunostaining. As a result, it was observed that sensory hair loss and hair cell death were suppressed depending on the concentration of ponatinib (Fig. 6A and B).

  In the above experiment, the number of hair cells protected by the addition of ponatinib was measured, and both outer hair cells: OHC (FIG. 7A) and inner hair cells: IHC (FIG. 7B) depended on the concentration of ponatinib. It was confirmed that the decrease in the number of cells due to viral infection was suppressed.

  The number of patients suffering from inner ear diseases including deafness is very large worldwide, and the incidence of inner ear diseases is increasing as the society ages. In particular, the 21st century is an era in which the improvement of quality of life is required to be called “the era of sensory organs”, and there are effective therapeutic agents for intractable inner ear diseases such as deafness. It has been desired. The present invention provides a novel therapeutic method that effectively suppresses sensory hair loss and cell death in hair cells and is extremely effective in the treatment and prevention of inner ear diseases.

Claims (7)

  A preventive or therapeutic agent for inner ear diseases, comprising a necrotosis inhibitor as an active ingredient.   The prophylactic or therapeutic agent according to claim 1, wherein the necrotosis inhibitor is a selective inhibitor of RIP kinase 1 (RIPK1: receptor interacting serine / threonine kinase 1).   3. The preventive or therapeutic agent according to claim 1 or 2, which suppresses sensory hair loss and / or hair cell death in the inner ear.   Necrostatin inhibitors include necrostatin-1, necrostatin-2, necrostatin-3, necrostatin-4, necrostatin-5, necrostatin-7, ponatinib, and their pharmaceutically The prophylactic or therapeutic agent according to any one of claims 1 to 3, which is selected from an acceptable salt, solvate or prodrug.   The preventive or therapeutic agent according to any one of claims 1 to 4, wherein the inner ear disease is sensorineural hearing loss or mixed hearing loss.   The preventive or therapeutic agent according to any one of claims 1 to 4, wherein the inner ear disease is inner ear hearing loss.   The prophylactic or therapeutic agent according to any one of claims 1 to 6, which is administered alone or in combination with another drug.

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