Therapeutic agent for cholesteatoma

Abstract

It was found that compounds acting as covalent menin inhibitors are effective for the treatment of cholesteatoma, and in particular, it was found that compounds having a specific structure among these compounds are particularly effective for suppressing cell proliferation in cholesteatoma. It was also found that a medicine containing the compound as an active ingredient is effective for the treatment of cholesteatoma.

Description

Cholesteatoma treatment drugs

[0001] The present invention relates to a therapeutic agent for cholesteatoma, which comprises a compound having a specific structure.

[0002] Middle ear cholesteatoma, a type of chronic otitis media, is composed of p63-positive tympanic membrane epithelial cells, neural crest-derived cells, and fibroblasts. Its tissues are distinct from benign and malignant tumors. This disease accounts for 25% of hearing loss cases, affecting 500,000 people in the United States, with an incidence rate of 3 per 100,000 children and 9.2 per 100,000 adults. It is a progressive, intractable disease that causes damage to the middle and inner ear, accompanied by hearing loss and dizziness, and requires therapeutic intervention. While surgical treatment is currently the only available treatment, recurrence and surgical complications are problematic. Therefore, there is a need for the development of therapeutic agents with high inhibitory effects on cholesteatoma, i.e., drugs that exert their therapeutic effects rapidly and at low concentrations.

[0003] The present inventors have previously conducted research into the relationship between cholesteatoma and the epigenetic gene expression mechanism known as epigenetics, which is a pathology of middle ear cholesteatoma, and reported that inhibiting histone H3K4 methylation in the promoter region has an inhibitory effect on cholesteatoma (Non-Patent Document 1). Furthermore, they demonstrated that controlling promoter- and enhancer-binding proteins is important for the complete cure of cholesteatoma (Non-Patent Document 2).

[0004] Menin is a protein encoded by the multiple endocrine neoplasia (MEN) gene. It forms a complex with the histone H3K4 methyltransferase MLL (Mixed-Lineage Leukemia) to promote gene expression and interacts with numerous transcription factors (Non-Patent Document 3). MLL, a histone H3K4 methyltransferase, is a member of the MLL1-MLL5 family, and forms an MLL histone methyltransferase complex with menin to catalyze the methylation of histone H3K4 in promoter regions (Non-Patent Document 4). Menin has been reported to interact with transcription factors JunD, Myc family, and FOX family that bind to enhancer regions (Non-Patent Document 3).

[0005] Non-covalent menin-MLL binding inhibitors are known to reversibly inhibit the binding of menin to the MLL protein complex in the promoter region. On the other hand, covalent menin inhibitors are known to form a covalent bond with the cysteine ​​Cys329 residue on menin and irreversibly inhibit the binding of the promoter region to the MLL protein complex, and are known to be useful in the treatment of cancer, etc. (Patent Document 1). Although the present inventors have demonstrated the cholesteatoma-inhibiting effect of non-covalent menin-MLL binding inhibitors in Non-Patent Document 1, the cholesteatoma-inhibiting effect of covalent menin inhibitors was unknown.

[0006] International Publication No. 2020 / 142557

[0007] Yamamoto-fukuda T et al, Int J Pediatr Otorhinolaryngol., 2021;140:110545Yamamoto-fukuda T et al, J Assoc Res Otolaryngol., 2021;22:405-424Matkar S et al, Trends Biochem Sci., 2013;38:394-402Schuettengruber et al, Cell, 2007;128:735-745Yamamoto-Fukuda T et al, J Assoc Res Otolaryngol., 2018;19:223-241Yamamoto-Fukuda T et al, Sci Rep., 2023;13:4532Yamamoto-Fukuda T et al, Eur Arch Otorhinolaryngol., 2015;272:2689-2696

[0008] An objective of the present invention is to provide a pharmaceutical agent that is effective in treating cholesteatoma.

[0009] In order to solve the above problems, the present inventors have searched for compounds that can be used as a fundamental therapeutic agent for cholesteatoma. As a result, they have found that compounds that act as covalent menin inhibitors are effective in treating cholesteatoma, and in particular, they have found that compounds having a specific structure among these compounds are particularly effective in treating cholesteatoma, thereby completing the present invention.

[0010] That is, the present invention is as follows: [1] Formula I: A cytostatic agent for cholesteatoma, comprising a compound represented by the formula I or a pharmaceutically acceptable salt thereof, is unsubstituted or one or more H is independently replaced by a substituent, said substituent being an alkyl group having 1 to 3 carbon atoms; R 1 is a group selected from the group consisting of H, alkyl groups having 1 to 3 carbon atoms, halo groups, haloalkyl groups having 1 to 3 carbon atoms, and CN; R 2a ~R 2d and R 3a ~R 3c are each independently a group selected from the group consisting of H, an alkyl group having 1 to 3 carbon atoms, a halo group, a haloalkyl group having 1 to 3 carbon atoms, OR, and CN, where R is H or an alkyl group having 1 to 3 carbon atoms; R 4a ~R 4b are each independently H or an alkyl group having 1 to 3 carbon atoms; is unsubstituted or one or more H is independently replaced by a substituent, said substituent being an alkyl group having 1 to 3 carbon atoms; and R 5 is -C(=O)-CR 6a =C(R 6b )(R 6c ) or -C(=O)-OC(CH3)3, where R 6a ~R 6c are each independently a group selected from the group consisting of H, an alkyl group having 1 to 3 carbon atoms, halogen, a haloalkyl group having 1 to 3 carbon atoms, and CN. [2] is unsubstituted; R 1 is H or an alkyl group having 1 to 3 carbon atoms; R 2a ~R2d and R 3a ~R 3c are each independently H or an alkyl group having 1 to 3 carbon atoms; R 4a ~R 4b are H, respectively; is unsubstituted; and R 5 is -C(=O)-CR 6a =C(R 6b )(R 6c ) or -C(=O)-OC(CH3)3, where R 6a ~R 6c [3] The cell growth inhibitor according to [1], wherein R is independently H or an alkyl group having 1 to 3 carbon atoms. 1 is H; R 2a ~R 2d and R 3a ~R 3c are each H; and / or R 5 [4] The cell growth inhibitor according to [2], wherein is —C(═O)—CH═CH2 or —C(═O)—OC(CH3)3. or [5] The cell growth inhibitor according to any one of [1] to [4], wherein the cholesteatoma comprises p63-positive tympanic membrane epithelial cells. [6] A pharmaceutical composition for treating and / or preventing cholesteatoma, comprising the cell growth inhibitor according to any one of [1] to [5] and a pharmaceutically acceptable carrier. [7] The pharmaceutical composition according to [6], wherein the cholesteatoma is middle ear cholesteatoma.

[0011] The present invention provides a pharmaceutical agent effective for treating cholesteatoma. While surgical treatment, which is the current treatment method, has problems such as recurrence and postoperative complications, the present invention makes it possible to treat cholesteatoma with pharmaceutical therapy, thereby avoiding the risk of complications due to surgical invasion.

[0012] This figure shows the effect of BMF-219 on the in vitro proliferation of mouse p63-positive tympanic membrane epithelial cells. The vertical axis shows the cell proliferation rate (logarithm), and the horizontal axis shows the duration of compound addition. Black circles indicate no compound addition, black squares indicate compound addition (1.0 μM), and black triangles indicate compound addition (0.5 μM). Each group has an n=3 sample. Error bars indicate SD. This figure shows the effect of Menin-MLL inhibitor 20 (MI-20) on the in vitro proliferation of mouse p63-positive tympanic membrane epithelial cells. The vertical axis shows the cell proliferation rate (logarithm), and the horizontal axis shows the duration of compound addition. Black circles indicate no compound addition, black squares indicate compound addition (1.0 μM), and black triangles indicate compound addition (0.5 μM). Each group has an n=3 sample. Error bars indicate SD. This figure shows the effect of VTP50469 on the in vitro proliferation of mouse p63-positive tympanic membrane epithelial cells. The vertical axis indicates the cell proliferation rate (logarithm), and the horizontal axis indicates the duration of compound addition. Black circles indicate no compound addition, black squares indicate compound addition (1.0 μM), and black triangles indicate compound addition (0.5 μM). Each group consists of three cells. Error bars indicate SD. Figure 1 shows the effect of MI-463 on the in vitro proliferation of mouse p63-positive tympanic membrane epithelial cells. The vertical axis indicates the cell proliferation rate (logarithm), and the horizontal axis indicates the duration of compound addition. Black circles indicate no compound addition, black squares indicate compound addition (1.0 μM), and black triangles indicate compound addition (0.5 μM). Each group consists of three cells. Error bars indicate SD. Figure 1 shows the effect of MI-2 on the in vitro proliferation of mouse p63-positive tympanic membrane epithelial cells. The vertical axis indicates the cell proliferation rate (logarithm), and the horizontal axis indicates the duration of compound addition. The black circle symbol indicates no compound addition, the black square symbol indicates compound addition (1.0 μM), and the black triangle symbol indicates compound addition (0.5 μM). Each group had n=3, and error bars indicate SD. This photograph shows the results of Western blotting to examine the MYC inhibitory effect of a covalent menin inhibitor (BMF-219, Menin-MLL inhibitor 20) on mouse p63-positive tympanic membrane epithelial cells. Each group had n=3. The inhibitory effect of BMF-219 (solid line: 5.0 μM, dashed line: 50 μM) on mouse cholesteatoma is shown.The vertical axis shows the cholesteatoma area ratio relative to the pre-administration (Pre) value, taken as 100. The horizontal axis shows the dates of micro-CT imaging before (Pre) and after (Post: 7 days after administration). n = 3 for each group. Error bars indicate SD. Figure 1 shows the inhibitory effect of VTP50469 (solid line: 5.0 μM, dashed line: 50 μM) on mouse cholesteatoma. The vertical axis shows the cholesteatoma area ratio relative to the pre-administration (Pre) value, taken as 100. The horizontal axis shows the dates of micro-CT imaging before (Pre) and after (Post: 7 days after administration). n = 3 for each group. Error bars indicate SD. Figure 1 shows the inhibitory effect of MI-463 (solid line: 5.0 μM, dashed line: 50 μM) on mouse cholesteatoma. The vertical axis shows the cholesteatoma area ratio relative to the pre-administration (Pre) value, taken as 100. The horizontal axis shows the dates of micro-CT imaging before (Pre) and after (Post: 7 days after administration). n = 3 for each group. Error bars indicate SD. Each group had an n=3 sample, and error bars indicate SD. This shows the inhibitory effect of MI-2 (solid line: 5.0 μM, dashed line: 50 μM) on mouse cholesteatoma. The vertical axis shows the cholesteatoma shadow area ratio, with the value before administration (Pre) set at 100, and the horizontal axis shows the dates of micro-CT imaging before administration (Pre) and after administration (Post: 7 days after administration). Each group had an n=3 sample, and error bars indicate SD.

[0013] <Cytostatic Agent> One embodiment of the present invention is a compound represented by Formula I: or a pharmaceutically acceptable salt thereof.

[0014] In formula I, is unsubstituted, or one or more H are independently substituted by a substituent, and the substituent is an alkyl group having 1 to 3 carbon atoms, preferably unsubstituted. The "one or more" is not particularly limited, but may be, for example, 1, 1 to 2, or 1 to 3. The alkyl group having 1 to 3 carbon atoms is not particularly limited, but may be, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, or a cyclopropyl group.

[0015] R 1is a group selected from the group consisting of H, an alkyl group having 1 to 3 carbon atoms, a halo group, a haloalkyl group having 1 to 3 carbon atoms, and CN, preferably H or an alkyl group having 1 to 3 carbon atoms, more preferably H. The alkyl group having 1 to 3 carbon atoms is not particularly limited, but may be, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, or a cyclopropyl group. The halogen constituting the halo group is not particularly limited, but may be, for example, bromine, chlorine, fluorine, or iodine. The haloalkyl group having 1 to 3 carbon atoms is a group in which at least one H contained in the above alkyl group is substituted with a halogen, and is not particularly limited, but may be, for example, a bromomethyl group, a chloromethyl group, a fluoromethyl group, or an iodomethyl group.

[0016] R 2a ~R 2d and R 3a ~R 3c are each independently selected from the group consisting of H, an alkyl group having 1 to 3 carbon atoms, a halo group, a haloalkyl group having 1 to 3 carbon atoms, OR, and CN, where R is H or an alkyl group having 1 to 3 carbon atoms, preferably each independently H or an alkyl group having 1 to 3 carbon atoms, more preferably H. The alkyl group having 1 to 3 carbon atoms is not particularly limited, but may be, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, or a cyclopropyl group. The halogen atom constituting the halo group is not particularly limited, but may be, for example, bromine, chlorine, fluorine, or iodine. The haloalkyl group having 1 to 3 carbon atoms is a group in which at least one H in the alkyl group is substituted with a halogen atom, and may be, for example, a bromomethyl group, a chloromethyl group, a fluoromethyl group, or an iodomethyl group. OR is not particularly limited, but may be, for example, a hydroxy group, a methoxy group, an ethoxy group, or a propoxy group.

[0017] R 4a ~R 4b are each independently H or an alkyl group having 1 to 3 carbon atoms;

[0018] is unsubstituted, or one or more H are independently substituted by a substituent, and the substituent is an alkyl group having 1 to 3 carbon atoms, preferably unsubstituted. The "one or more" is not particularly limited, but may be, for example, 1, 1 to 2, or 1 to 3. The alkyl group having 1 to 3 carbon atoms is not particularly limited, but may be, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, or a cyclopropyl group.

[0019] R 5 is -C(=O)-CR 6a =C(R 6b )(R 6c ) or -C(=O)-OC(CH3)3, where R 6a ~R 6c are each independently a group selected from the group consisting of H, an alkyl group having 1 to 3 carbon atoms, a halogen, a haloalkyl group having 1 to 3 carbon atoms, and CN; R 6a ~R 6c are preferably each independently H or an alkyl group having 1 to 3 carbon atoms. 5 is more preferably -C(=O)-CH=CH2 or -C(=O)-OC(CH3)3. The alkyl group having 1 to 3 carbon atoms is not particularly limited, and may be, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, or a cyclopropyl group. The halogen constituting the halo group is not particularly limited, and may be, for example, bromine, chlorine, fluorine, or iodine. The haloalkyl group having 1 to 3 carbon atoms is a group in which at least one H contained in the above alkyl group is substituted with a halogen, and is not particularly limited, and may be, for example, a bromomethyl group, a chloromethyl group, a fluoromethyl group, or an iodomethyl group.

[0020] The compound of formula I is preferably BMF-219 or Menin-MLL inhibitor 20 (MI-20).

[0021] BMF-219 is a compound represented by the following formula:

[0022] Menin-MLL inhibitor 20 is a compound represented by the following formula:

[0023] The compounds of the present invention are covalent menin inhibitors that exhibit irreversible menin-MLL inhibitory activity and / or irreversible MYC inhibitory activity by covalently binding to menin, preferably covalent menin inhibitors that exhibit both irreversible menin-MLL inhibitory activity and irreversible MYC inhibitory activity by covalently binding to menin. Examples of such covalent menin inhibitors include BMF-219 and menin-MLL inhibitor 20. Specifically, menin-MLL inhibitory activity inhibits the binding of promoter regions to MLL protein complexes, thereby inhibiting histone H3K4 methylation and suppressing the expression of disease-related genes. Specifically, MYC inhibitory activity inhibits the binding of menin to transcription factors that bind to enhancer regions, such as MYC. Meanwhile, menin-MLL inhibitors (e.g., VTP50469, MI-463, and MI-2) are reversible inhibitors that bind to menin via noncovalent bonds, such as hydrogen bonds. Covalent menin inhibitors with irreversible menin-MLL inhibitory activity and / or irreversible MYC inhibitory activity, preferably covalent menin inhibitors with both irreversible menin-MLL inhibitory activity and irreversible MYC inhibitory activity, are expected to provide stronger inhibitory effects than menin-MLL inhibitors with reversible inhibitory activity.

[0024] The menin-MLL inhibitory activity can be measured by known methods. For example, the amount of histone H3K4 methylation inhibition can be measured using immunohistochemical staining or immunoblotting, and the degree of expression suppression can be measured by measuring the expression level of a target disease-related gene using PCR or other methods. The menin-MLL inhibitory activity can be evaluated based on the results of these measurements. The menin-MLL inhibitory activity may be, for example, a reduction in the amount of histone H3K4 methylation or the expression level of a target disease-related gene when a compound of the present invention is added compared to when a compound of the present invention is not added. When the degree of reduction can be quantified, it may be, for example, a reduction of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. Furthermore, it may be, for example, a reduction in the amount of histone H3K4 methylation or the expression level of a target disease-related gene when a compound of the present invention is added compared to when a control compound is added. When the degree of the decrease can be quantified, it may be, for example, a decrease of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0025] The MYC inhibitory effect can be measured by known methods. Without limitation, for example, the expression level of cyclin D1 (CCND1), a downstream gene of MYC, can be measured. The expression level of CCND1 can be measured, for example, by immunohistochemical staining or immunoblotting, or by PCR, and the gene expression level can be measured. The MYC inhibitory effect can be evaluated based on the results of these measurements. The MYC inhibitory effect can be, for example, a reduction in the expression level of cyclin D1 (CCND1), a downstream gene of MYC, when the compound of the present invention is added, compared to when the compound of the present invention is not added. When the degree of reduction can be quantified, it can be, for example, a reduction of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. Furthermore, for example, a reduction in the expression level of cyclin D1 (CCND1), a downstream gene of MYC, when the compound of the present invention is added, compared to when a control compound is added, can be evaluated. When the degree of the decrease can be quantified, it may be, for example, a decrease of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0026] The cells whose proliferation is inhibited by the compound of the present invention are not particularly limited as long as they are cells that constitute cholesteatoma. For example, cholesteatoma preferably contains cells expressing p63, which is known as a marker for epithelial stem cells and / or their progenitor cells in middle ear cholesteatoma tissue, and more preferably contains p63-positive tympanic membrane epithelial cells.

[0027] The inhibition of cell proliferation may be, for example, a decrease in the cell proliferation rate or cell number when the compound of the present invention is added compared to when the compound of the present invention is not added. When the degree of decrease can be quantified, it may be, for example, a decrease of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. Furthermore, it may be, for example, a decrease in the cell proliferation rate or cell number when the compound of the present invention is added compared to when a control compound is added. When the degree of decrease can be quantified, it may be, for example, a decrease of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0028] salts of hydrohalic acids such as hydrofluoric acid, hydrochloric acid, hydrobromic acid, and hydroiodic acid; salts of inorganic acids such as sulfuric acid, nitric acid, phosphoric acid, perchloric acid, and carbonic acid; salts of organic carboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, hydroxyacetic acid, propionic acid, lactic acid, citric acid, tartaric acid, oxalic acid, benzoic acid, mandelic acid, butyric acid, fumaric acid, succinic acid, maleic acid, and malic acid; salts of acidic amino acids such as aspartic acid and glutamic acid; salts of basic amino acids such as arginine and lysine; ammonium salts; salts of aliphatic amines such as trimethylamine, triethylamine, and ethanolamine; and salts of sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and toluenesulfonic acid.

[0029] The compound of this embodiment may be used either in vivo or in vitro.

[0030] The compound of this embodiment may be a commercially available compound, or a compound prepared by a method known to those skilled in the art.

[0031] Regarding the cell proliferation inhibitor, the content of the compound represented by formula I or a pharmaceutically acceptable salt thereof as an active ingredient, dosage form, administration site, target disease, etc. can be found in the section <Pharmaceutical composition for treating cholesteatoma> below.

[0032] Another aspect of the present invention is the use of a compound represented by formula I or a pharmaceutically acceptable salt thereof in the manufacture of a cytostatic agent for cholesteatoma. Another aspect of the present invention is the use of a compound represented by formula I or a pharmaceutically acceptable salt thereof for cytostatic treatment of cholesteatoma. Another aspect of the present invention is a compound represented by formula I or a pharmaceutically acceptable salt thereof for use in cytostatic treatment of cholesteatoma. Another aspect of the present invention is a method for cytostatic treatment of cholesteatoma, comprising administering a compound represented by formula I or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0033] <Pharmaceutical Composition for Treating Cholesteatoma> Another embodiment of the present invention is a pharmaceutical composition for treating and / or preventing cholesteatoma, comprising the above-mentioned cytostatic agent and a pharmaceutically acceptable carrier.

[0034] Histopathologically, cholesteatoma is an epidermoid cyst and is found not only in the head and neck region, including middle ear cholesteatoma and external auditory canal cholesteatoma, but also throughout the body, including the skull and body surface. The type and site of cholesteatoma treated and / or prevented by the pharmaceutical composition of this embodiment are not particularly limited. For example, cholesteatoma occurring in the head and neck region, such as middle ear cholesteatoma or external auditory canal cholesteatoma, or cholesteatoma occurring outside the head and neck region, such as the entire body surface tissue, may be treated. Cholesteatoma occurring in the head and neck region, such as middle ear cholesteatoma or external auditory canal cholesteatoma, is preferred, and middle ear cholesteatoma is more preferred. The stage of progression of cholesteatoma is not particularly limited. Either congenital or acquired cholesteatoma may be treated, and either primary or recurrent cholesteatoma may be treated. Prevention may be prevention of primary cholesteatoma or prevention of recurrent cholesteatoma.

[0035] Regarding the therapeutic effect, for example, if it is confirmed that the treatment with a pharmaceutical composition for treating cholesteatoma results in inhibition of cholesteatoma growth, a reduction in the number of cells constituting the cholesteatoma, or a reduction in the size of the cholesteatoma, compared to a subject before treatment with the pharmaceutical composition for treating cholesteatoma or a control that was not treated with the pharmaceutical composition for treating cholesteatoma, the therapeutic effect can be determined to be effective. The analytical method is not particularly limited, and can be performed by any method known to those skilled in the art. Furthermore, the therapeutic effect can also be predicted based on cytobiological methods. For example, by comparing a sample after treatment with a pharmaceutical composition for treating cholesteatoma with a sample before treatment with the pharmaceutical composition for treating cholesteatoma, the therapeutic effect of a therapeutic drug in vivo can be predicted. The cytobiological analysis is not particularly limited, and examples include cell proliferation assays and Western blotting, and can be performed by any method known to those skilled in the art.

[0036] Regarding the preventive effect, for example, when a subject at high risk of developing cholesteatoma is treated with a pharmaceutical composition for treating cholesteatoma, the preventive effect can be determined by the following: the subject does not develop cholesteatoma for a predetermined period of time; the growth of cholesteatoma is suppressed compared to a subject at high risk of developing cholesteatoma who is not treated with the pharmaceutical composition for treating cholesteatoma; etc. The analytical method is not particularly limited, and can be performed by a method known to those skilled in the art.

[0037] The content of the compound represented by Formula I or a pharmaceutically acceptable salt thereof, which is the active ingredient in the pharmaceutical composition for treating cholesteatoma, is not particularly limited as long as it is an amount effective for treating and / or preventing cholesteatoma. Although it generally varies depending on the dosage form, the content of the active ingredient in the pharmaceutical composition for treating cholesteatoma can be set, for example, within the range of about 0.01% by mass to about 99.9% by mass. For example, the amount of the active ingredient relative to the total amount of the pharmaceutical composition may be 1 ng / mL to 1 mg / mL, 1 ng / mL to 100 μg / mL, 1 ng / mL to 10 μg / mL, 1 ng / mL to 1000 ng / mL, 1 ng / mL to 100 ng / mL, 1 ng / mL to 10 ng / mL, or, for example, 1 ng / mg to 1 mg / mg, 1 ng / mg to 100 μg / mg, 1 ng / mg to 10 μg / mg, 1 ng / mg to 1000 ng / mg, 1 ng / mg to 100 ng / mg, or 1 ng / mg to 10 ng / mg.

[0038] The pharmaceutical composition for treating cholesteatoma may be formulated in any dosage form. Examples include parenteral dosage forms such as topical preparations, ointments, creams, ear drops, liquids, suspensions, and injections, and oral dosage forms such as tablets, capsules, powders, and granules. The mode of administration is not particularly limited, but in the case of parenteral dosage forms, examples include local administration to the affected area of ​​the ear or its surrounding tissues, or administration to the entire body surface tissue affected by cholesteatoma or its surrounding tissues.

[0039] Examples of pharmaceutically acceptable carriers include commonly used excipients, binders, disintegrants, and lubricants, as well as various carriers, stabilizers, surfactants, plasticizers, solubilizers, reducing agents, buffers, sweeteners, bases, adsorbents, flavorings, suspending agents, antioxidants, glossing agents, coating agents, shells, humectants, wetting agents, wetting regulators, fillers, antifoaming agents, refreshing agents, colorants, flavoring agents, flavorings, sugar-coating agents, isotonic agents, softeners, emulsifiers, thickening agents, foaming agents, pH adjusters, diluents, dispersants, disintegration aids, disintegration extenders, fragrances, moisture-proofing agents, preservatives, solubilizers, solubilizers, solvents, fluidizing agents, antistatic agents, bulking agents, moisturizing agents, and wetting agents.

[0040] The pharmaceutical composition of this embodiment may contain other active ingredients, if desired.

[0041] The subject of administration is a mammal that has developed cholesteatoma, such as a human, mouse, dog, or cat, and preferably a human.

[0042] The dosage, frequency, and duration of administration of the pharmaceutical composition for treating cholesteatoma are not particularly limited, as long as the amount of the active ingredient, a compound represented by Formula I or a pharmaceutically acceptable salt thereof, is effective for the treatment and / or prevention of cholesteatoma. These dosages can be adjusted appropriately depending on the age, sex, weight, symptoms, therapeutic effect, area of ​​the treatment site, dosage form, and mode of administration of the subject. For example, in the case of a human adult (body weight 60 kg), the dosage may be set within the range of about 0.001 mg to about 5000 mg per day. For example, the dosage may be about 0.01 mg to about 1000 mg. The pharmaceutical composition may be administered in the above dosage amounts once per day or in multiple divided doses per day. The administration period may be, for example, one day or more and up to one year, or may be continued for a longer period.

[0043] The description of the cytostatic agent and its active ingredient, the compound represented by formula I or a pharmaceutically acceptable salt thereof, can be found in the above section <Cytostatic Agent>.

[0044] Another aspect of the present invention is the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating and / or preventing cholesteatoma. Another aspect of the present invention is the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, for treating and / or preventing cholesteatoma. Another aspect of the present invention is a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment and / or prevention of cholesteatoma. Another aspect of the present invention is a method of treating cholesteatoma, comprising administering a compound of Formula I, or a pharmaceutically acceptable salt thereof, to a subject with cholesteatoma.

[0045] The examples are set forth for purposes of disclosure and are not intended to limit the scope of the invention.

[0046] The efficacy of the covalent menin inhibitor against middle ear cholesteatoma was demonstrated in cell proliferation inhibition experiments (cell experiments), MYC downstream gene expression inhibition experiments (Western blotting), and cholesteatoma inhibition experiments (cholesteatoma animal model experiments).

[0047] Example 1: Cell Proliferation Inhibition Experiment This study was conducted using p63-positive tympanic membrane epithelial cells, which have been reported to proliferate in cholesteatoma (Non-Patent Document 5). CnT-PR basal medium (Cellntec, Bern, Switzerland) was used as the culture medium for mouse p63-positive tympanic membrane epithelial cells. p63-positive tympanic membrane epithelial cells (passage 12) established from tympanic membrane tissue of mice on a C57BL / 6 background (Jackson Laboratories, Bar Harbor, ME; Charles River Laboratories, Wilmington, MA) were used (Non-Patent Document 6). Each compound listed in Table 1 was diluted with dimethyl sulfoxide (#472301, SIGMA-ALDORICH) and further diluted with 1x D-PBS (#14249-24, Nacalai Tesque) to 500 μM or 250 μM (from 10 mM or 50 mM). The compounds were stored at -30°C and thawed prior to use.

[0048]

[0049] Cells were prepared in CnT-PR medium at a concentration of 40,000 cells / mL and seeded (500 μL / well) into 12-well tissue culture plates (#3815-012, IWAKI). After 24 hours, cell adhesion was confirmed, and 1 μL / well of each compound (final concentration 1.0 μM or 0.5 μM) or PBS was added (day 0). The cells were then cultured for an additional 1, 3, or 5 days. One day after compound addition (day 1), three days after compound addition (day 3), and five days after compound addition (day 5), 250 μL / well of 0.25% trypsin / EDTA (#25200-072, Gibco) was added to each well and incubated at 37°C for 10 minutes. Then, 50 μL / well of fetal bovine serum (Lot 08020002, 35-0790CV, Corning) was added. Cell numbers were counted (n=3) using a hemocytometer (#DHC-N01, NanoEntek) under a 10x magnification with a T1-SM Nikon Microscope (Nikon). The results are shown in Figures 1–5.

[0050] In cell experiments using p63-positive tympanic membrane epithelial cells that form cholesteatoma, the cell proliferation inhibitory effects of covalent menin inhibitors (BMF-219 and menin-MLL inhibitor 20) were observed at concentrations of 0.5 μM or higher on day 1 after administration, and the cell proliferation inhibitory effects were concentration- and duration-dependent (Figs. 1 and 2). Furthermore, the cell proliferation inhibitory effects of covalent menin inhibitors (BMF-219 and menin-MLL inhibitor 20) were more effective than those of menin-MLL binding inhibitors (VTP50469, MI-463, and MI-2) that only exhibit reversible menin-MLL inhibitory activity similar to that of a menin-MLL binding inhibitor (MI-503) previously reported to inhibit middle ear cholesteatoma (Non-Patent Document 1) (Figs. 3 and 5).

[0051] [Example 2] MYC downstream gene expression suppression experiment (Western blotting) Mouse p63-positive tympanic membrane epithelial cells were maintained and cultured in CnT-PR basal media at 37°C in a 5% CO2 incubator. 2 x 10 mouse p63-positive tympanic membrane epithelial cells were cultured in a CnT-PR basal media culture medium at 37°C in a 5% CO2 incubator.5Cells were prepared in CnT-PR medium at a concentration of 1.0 μM / mL and seeded (500 μL / well) into 12-well tissue culture plates (#3815-012, IWAKI). After 24 hours, cell adhesion was confirmed. Then, 1 μL / well of each compound (final concentration: 1.0 μM or 0.5 μM) or PBS was added and the plates were cultured for an additional day (n=3). After incubation, the cells were washed with PBS and lysed in 75 μL of 4°C lysis solution [1x Laemmli buffer (Bio-Rad, Hercules, CA), 2-mercaptoethanol (Wako), protein / phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA)]. After incubation at 95°C for 5 minutes, 10 μL of each solution was electrophoresed on a 4-12% SDS-polyacrylamide gel (Thermo Fisher Scientific, Dreiech, Germany). After electrophoresis, proteins in the gel were transferred to a PVDF membrane (Millipore, Bedford, MA) using an i-blot electrophoresis system (Invitrogen). The membrane was then immersed in 5% skim milk / 0.1% Tween 20 in Tris-buffered saline (TBST) solution for 1 hour at room temperature with gentle agitation to block nonspecific binding. The PVDF membrane was then incubated overnight at 4°C with primary antibodies: anti-CCND1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, mouse, sc-8396) and anti-β-actin antibody (Cell Signaling Technology, Danvers, MA, rabbit, #4970) diluted in 2.5% skim milk / 0.1% TBST. After three 5-minute washes with 0.1% TBST, the membrane was immersed in peroxidase-polymerized anti-mouse IgG antibody dilution or peroxidase-polymerized anti-rabbit IgG antibody dilution for 1 hour.After washing three times with 0.1% TBST solution for 5 minutes, the transferred PVDF membrane was immersed in ECL (GE Healthcare Biosciences, Pittsburgh, PA) detection solution, and luminescence was detected using an LAS 4000 Chemiluminescence Imager (GE Healthcare). The results are shown in Figure 6.

[0052] After 1 day of culture with 1.0 μM or 0.5 μM concentrations of a covalent menin inhibitor (BMF-219, menin-MLL inhibitor 20), the protein expression of cyclin D1 (CCND1), a downstream gene of MYC, decreased in a concentration-dependent manner. The protein levels of β-actin, used as an internal standard, were similar in all samples. Thus, the covalent menin inhibitor (BMF-219, menin-MLL inhibitor 20) concentration-dependently suppressed the expression of cyclin D1 (CCND1), a downstream gene of MYC. These results demonstrate that the administration of the covalent menin inhibitor (BMF-219, menin-MLL inhibitor 20) inhibits cell proliferation by inhibiting the interaction between menin and MYC.

[0053] Example 3: Evaluation of inhibition of cholesteatoma in a mouse model. KGF-induced middle ear cholesteatoma was induced in both ears of ICR mice (male, 6 weeks old) and examined (Non-Patent Document 7). Each compound listed in Table 1 was diluted with dimethyl sulfoxide (#472301, SIGMA-ALDORICH) and then further diluted with 1x D-PBS (#14249-24, Nacalai Tesque) (adjusted from 10 mM or 50 mM to 5.0 μM or 50 μM). The compound was stored at -30°C until use and thawed prior to use. KGF-induced middle ear cholesteatoma was confirmed in both ears of male ICR mice (6 weeks old) using a 0° endoscope (AVS Co., Tokyo, Japan) or a micro-CT (LaTheta LCT-200; Hitachi Aloka Medical Ltd., Tokyo, Japan). Each compound was administered intracanalically every 24 hours for 7 consecutive days at a dose of 30 μL per ear (right ear: 5.0 μM, left ear: 50 μM, n = 3). Micro-CT images were taken before and 7 days after administration. The acquired temporal bone data were converted to bone terms (gray scale, 400-2000) using the micro-CT system's internal software, and images were acquired using Adobe Photoshop. Cholesteatomas were visualized as soft tissue shadows in the temporal bone images. For all mice, coronal slices depicting the cochlear middle turn were used for quantitative analysis. Quantitative analysis was performed using ImageJ software version 15.1 (NIH, https: / / imagej.nih.gov / ij / ). The results are shown in Figures 7 to 10.

[0054] In an animal model of cholesteatoma, the covalent menin inhibitor (BMF-219) showed a suppressive effect on mouse cholesteatoma at a lower concentration (5.0 μM) and for a shorter period (7 days) compared with the suppressive effect of a known menin-MLL inhibitor (MI-503, 50 μM) administered for 14 days (Fig. 7). Furthermore, compared with the results of animal model experiments using compounds with the same menin-MLL inhibitory activity (VTP50469, MI-463, and MI-2) as the known menin-MLL inhibitor (MI-503) (Figs. 8-10), the covalent menin inhibitor (BMF-219) showed a more potent suppressive effect on cholesteatoma (Fig. 7).

[0055] The results of the above cell proliferation inhibition experiment, MYC downstream gene expression inhibition experiment, and cholesteatoma inhibition experiment demonstrated the medicinal use of covalent menin inhibitors (BMF-219 and Menin-MLL inhibitor 20) or their pharmaceutically acceptable salts, as well as pharmaceutical compositions containing them, as agents for treating or preventing recurrence of middle ear diseases, including middle ear cholesteatoma. In particular, the covalent menin inhibitors demonstrated the advantage of exerting early therapeutic effects at low concentrations against middle ear cholesteatoma.

Claims

1. Formula I: or a pharma- ceutical acceptable salt thereof, wherein in formula I, is unsubstituted or one or more H are independently replaced by a substituent, said substituent being an alkyl group having 1 to 3 carbon atoms; R 1 is a group selected from the group consisting of H, an alkyl group having 1 to 3 carbon atoms, a halo group, a haloalkyl group having 1 to 3 carbon atoms, and CN; R 2a ~R 2d and R 3a ~R 3c are each independently a group selected from the group consisting of H, an alkyl group having 1 to 3 carbon atoms, a halo group, a haloalkyl group having 1 to 3 carbon atoms, OR, and CN, where R is H or an alkyl group having 1 to 3 carbon atoms; R 4a ~R 4b are each independently H or an alkyl group having 1 to 3 carbon atoms; is unsubstituted or one or more H are independently replaced by a substituent, said substituent being an alkyl group having 1 to 3 carbon atoms; and R 5 is -C(=O)-CR 6a =C(R 6b )(R 6c ) or -C(=O)-OC(CH3)3, where R 6a ~R 6c are each independently a group selected from the group consisting of H, an alkyl group having 1 to 3 carbon atoms, a halogen, a haloalkyl group having 1 to 3 carbon atoms, and CN.

2. is unsubstituted; R 1 is H or an alkyl group having 1 to 3 carbon atoms; R 2a ~R 2d and R 3a ~R 3c are each independently H or an alkyl group having 1 to 3 carbon atoms; R 4a ~R 4b are each H; is unsubstituted; and R 5 is -C(=O)-CR 6a =C(R 6b )(R 6c ) or -C(=O)-OC(CH3)3, where R 6a ~R 6c The cell proliferation inhibitor according to claim 1, wherein each of the groups independently represents H or an alkyl group having 1 to 3 carbon atoms.

3. R 1 is H; R 2a ~R 2d and R 3a ~R 3c are each H; and / or R 5 The cell proliferation inhibitor according to claim 2, wherein is -C(=O)-CH=CH2 or -C(=O)-OC(CH3)3.

4. The compound, or The cell proliferation inhibitor according to claim 1, 5. The cell proliferation inhibitor described in claim 1, wherein the cholesteatoma contains p63-positive tympanic membrane epithelial cells.

6. A pharmaceutical composition for treating and / or preventing cholesteatoma, comprising the cell proliferation inhibitor according to any one of claims 1 to 5 and a pharma- ceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, wherein the cholesteatoma is middle ear cholesteatoma.

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