Heteroplasmy suppressor and application thereof

Abstract

To provide a heteroplasmy suppressor that is capable of suppressing progression of heteroplasmy to suppress mitochondrial respiratory dysfunction.SOLUTION: The present invention relates to a heteroplasmy suppressor comprising an Ape1 inhibitor, and to a method for suppressing heteroplasmy using the suppressor. The present invention also relates to a therapeutic agent for a disease in which mitochondrial DNA heteroplasmy is implicated, the therapeutic agent comprising an Ape1 inhibitor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a heteroplasmy formation inhibitor, a therapeutic agent for diseases associated with heteroplasmy formation, and a method for inhibiting heteroplasmy formation. [Background technology]

[0002] Eukaryotic cells contain hundreds to thousands of mitochondrial DNAs (mtDNAs) in their mitochondria. Mitochondrial DNA (mtDNA) encodes factors essential for cellular respiration. Mitochondria are the major source of reactive oxygen species (ROS) within cells, and ROS act as central hubs in signal transduction networks.

[0003] Healthy humans are born with a single genotype of mtDNA in each cell of their entire body; in other words, all cells share the same wild-type base sequence (a state known as homoplasmy). However, because mtDNA is more prone to mutation than nuclear chromosomal DNA, mutant mtDNA coexists with wild-type mtDNA in cells in patients with mitochondrial disease and in adult human cells (a state known as heteroplasmy). Mutations and deletions in somatic mtDNA can expand clonally to harmful concentrations within cells, and clinical symptoms can develop when the ratio of mutant mtDNA exceeds a certain threshold. For example, large deletions have been reported at mtDNA position 3243 in patients with MELAS and cardiomyopathy, at position 8344 in patients with MERRF, and in patients with CPEO and Pearson's disease (Non-Patent Document 1). Furthermore, the A3243G substitution mutation in mtDNA is known to cause diabetes.

[0004] Furthermore, induced pluripotent stem cells (iPSCs) can proliferate indefinitely and differentiate into any type of cell in the body, but because the reprogramming process used to establish iPSCs does not reset mtDNA, the mtDNA in iPSCs remains heteroplasmic (Non-Patent Document 2). For this reason, preventing mtDNA mutations and removing mutated mtDNA are of great significance in the field of regenerative medicine.

[0005] As a method for promoting mtDNA homogeneity, the present inventors have developed a method for changing the proportion of mutant mtDNA in eukaryotic cells by contacting them with reactive oxygen species or chemical species that generate reactive oxygen species within the cells (Patent Document 1).The present inventors have also proposed a method for promoting mitochondrial DNA homogeneity, which involves expressing the Mhr1 gene product of budding yeast in yeast cells (Patent Document 2).

[0006] Ape1 (apurinic / apyrimidinic endonuclease I) is a base excision repair enzyme that promotes double-strand breaks when mtDNA bases are oxidatively damaged by reactive oxygen species or the like (Non-Patent Document 3). Known Ape1 inhibitors include 2,4,9-trimethylbenzo[b][1,8]naphthyridin-5-amine (Non-Patent Documents 4 to 6). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2011-125248 [Patent Document 2] Japanese Patent Application Laid-Open No. 2005-87150 [Non-patent literature]

[0008] [Non-Patent Document 1] Wallace, Annu Rev Genet.,39,359-407,2005 [Non-patent document 2] Cherry, AB et al., Stem Cells, 31, 1287-1297. [Non-patent document 3] ANTIOXIDANTS & REDOX SIGNALING Volume 20, Number 4, 2014 [Non-patent document 4] Cell Mol Life Sci. 2010 November; 67(21): 3621-3631 [Non-Patent Document 5] Probe Reports from the NIH Molecular Libraries Program [Internet]. https: / / www.ncbi.nlm.nih.gov / books / NBK133448 / (2024.1.16) [Non-patent document 6] Journal of Pharmacology and Experimental Therapeutics Volume: 334 Issue: 3 Pages: 988-998 (2010) Summary of the Invention [Problem to be solved by the invention]

[0009] In adult human cells, mutant mtDNA coexists with wild-type mtDNA. However, as heteroplasmy progresses and exceeds a threshold, mitochondrial respiratory function declines, resulting in a decrease in ATP production and cellular dysfunction. Cellular dysfunction can lead to aging and various diseases. Therefore, there is a need to establish a technology that can prevent cellular dysfunction by suppressing the progression of heteroplasmy to a level that does not exceed a threshold, i.e., a level that does not cause abnormalities in cellular respiratory function.

[0010] Therefore, an objective of the present invention is to provide a heteroplasmy formation inhibitor that can suppress the progression of heteroplasmy formation and thereby suppress the decline in mitochondrial respiratory function.A further objective of the present invention is to provide a therapeutic agent for diseases associated with heteroplasmy formation and a method for suppressing heteroplasmy formation, using the heteroplasmy formation inhibitor. [Means for solving the problem]

[0011] Examples of specific embodiments of the present invention are given below.

[0012] [1] Heteroplasmy inhibitors, including Ape1 inhibitors. [2] The heteroplasmy inhibitor according to [1], wherein the Ape1 inhibitor is 2,4,9-trimethylbenzo[b][1,8]naphthyridin-5-amine or a salt thereof. [3] The heteroplasmy formation inhibitor according to [1] or [2], which is intended to maintain mitochondrial function. [4] The heteroplasmy formation inhibitor according to any one of [1] to [3], which is intended to prevent or treat a disease caused by mitochondrial dysfunction. [5] The heteroplasmy formation inhibitor according to any one of [1] to [4], which is a pharmaceutical product, a food or drink product, or a cosmetic product. [6] A therapeutic agent for diseases involving mitochondrial DNA heteroplasmy, including an Ape1 inhibitor. [7] The therapeutic agent according to [6], wherein the Ape1 inhibitor is 2,4,9-trimethylbenzo[b][1,8]naphthyridin-5-amine or a salt thereof. [8] A method for suppressing heteroplasmy, comprising contacting a cell with an Ape1 inhibitor. [9] The method for suppressing heteroplasmy formation according to [8], wherein the Ape1 inhibitor is 2,4,9-trimethylbenzo[b][1,8]naphthyridin-5-amine or a salt thereof.

[10] The method for suppressing heteroplasmy formation according to [8] or [9], wherein the cells are human-derived cells.

[11] The method for suppressing heteroplasmy formation according to [8] or [9], wherein the cells are iPS cells.

[0013] [A] An agent comprising an Ape1 inhibitor for use in treating heteroplasmy suppression. [B] Use of an agent, including an Ape1 inhibitor, for the manufacture of a heteroplasmy inhibitor. [Effects of the Invention]

[0014] According to the present invention, a heteroplasmy inhibitor that can suppress the progression of heteroplasmy and thereby suppress the decline in mitochondrial respiratory function can be provided. Furthermore, according to the present invention, a therapeutic agent for a disease associated with heteroplasmy and a method for suppressing heteroplasmy can be provided. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows the results of measuring the OCR, ECAR, and OCR / ECAR ratio of PD-iPSCs cultured for 4 days in the presence of 2 μM AR03. [Figure 2] Figure 2 shows the results of measuring the OCR, ECAR, and OCR / ECAR ratio of BJ primary cells and MELAS cells cultured for 4 days in the presence of various concentrations of AR03, as well as the results of measuring the OCR, ECAR, and OCR / ECAR ratio of BJ primary cells cultured for 0 to 7 days in the presence of 1.95 μM and 2.0 μM AR03. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below. The following description may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0017] (heteroplasmy inhibitor) This embodiment relates to a heteroplasmy inhibitor containing an Ape1 inhibitor. In this embodiment, the heteroplasmy inhibitor containing an Ape1 inhibitor can suppress heteroplasmy in cells having wild-type mtDNA, or can suppress further progression of heteroplasmy in cells in which heteroplasmy has progressed to a certain extent. As a result, a decline in mitochondrial respiratory function is suppressed, and mitochondrial function is maintained.

[0018] Homoplasmy refers to a state in which the mtDNA base sequence is uniform at the cellular or individual level. In contrast, heteroplasmy refers to a state in which the mtDNA base sequence is not uniform at the cellular or individual level. For example, if the mtDNA base sequence contained in a cell (or individual) is uniform in all mitochondrial genome copies (if it contains only wild-type mtDNA), the cell (or individual) is a "homoplasmic cell (or individual)." If a cell (or individual) contains both mutant mtDNA and wild-type mtDNA, the cell (or individual) is a "heteroplasmic cell (or individual)." In this specification, the term "homoplasmy" simply refers to a state in which only wild-type mtDNA is present. Note that even if a mutation occurs in mtDNA, if the base sequence at the mutated site is uniform at the cellular or individual level, the state is homoplasmic with respect to the specific mutation, and this state is called "mutation homoplasmy." In this specification, the term "mutation homoplasmy" refers to a state in which only mutant mtDNA is present. The state in which wild-type mtDNA and mutant mtDNA coexist is called "heteroplasmy."

[0019] As used herein, "mutated mtDNA" refers to mtDNA with a sequence that differs from the wild-type mtDNA sequence. The number of mutations present per mtDNA is one or more, preferably one to three, and more preferably one. Furthermore, the "mutation" in the mutant mtDNA is not particularly limited and includes all mutations that differ from the wild-type mtDNA sequence, and may result in a mutation that causes some disease. Examples of such "mutations" include substitution mutations at position 3243 (the causative mutation of MELAS) and position 8344 (the causative mutation of MERRF) in the mtDNA (see Non-Patent Document 1, etc.).

[0020] The proportion of mutant mtDNA present in cells gradually increases with growth, and in adult human cells, mutant mtDNA coexists with wild-type mtDNA within the cells. It is known that heteroplasmy progresses from this state, and when the threshold is exceeded, mitochondrial dysfunction, such as a decrease in ATP production capacity due to a decrease in mitochondrial respiratory function, occurs, resulting in cellular dysfunction. As used herein, "normal cells" refer to cells in which wild-type mtDNA is present in a homoplasmic state within the cells (such normal cells are also referred to as homoplasmic cells), or cells in which heteroplasmy has progressed to a degree that does not exceed the threshold, resulting in the coexistence of mutant mtDNA and wild-type mtDNA within the cells. In other words, "normal cells" as used herein refer to cells in which mitochondrial dysfunction does not occur. In normal cells herein, the intracellular ratio of wild-type mtDNA copies with the same nucleotide sequence is preferably 60% or more (40% or less mutant mtDNA), more preferably 70% or more (30% or less mutant mtDNA), even more preferably 80% or more (20% or less mutant mtDNA), and particularly preferably 90% or more (10% or less mutant mtDNA). The proportion of mutant mtDNA present in cells can be easily determined, for example, using restriction enzymes with different cleavage sensitivities depending on the presence or absence of mutations (see, for example, Goto et al., Nature, 348, 651-653, 1990). Other methods that can be used include allele-specific PCR, quantitative PCR, the Invader method, and next-generation sequencer analysis. As used herein, heteroplasmy refers to increasing the proportion of mutant mtDNA in cells and decreasing the proportion of normal cells in an individual. On the other hand, a state in which heteroplasmy formation is suppressed refers to a state in which the rate of increase in the proportion of mutant mtDNA is moderate or no increase in the proportion of mutant mtDNA is observed compared to when the heteroplasmy formation inhibitor of the present invention is not administered (added), and within an individual, this refers to a state in which the rate of normal cells is maintained or its decrease is moderate or no increase in the proportion of heteroplasmic cells is observed.

[0021] The heteroplasmy inhibitor of this embodiment is an agent for suppressing an increase in the proportion of heteroplasmic cells by minimizing the reduction in the proportion of wild-type mtDNA present in cells, maintaining mitochondrial function at the time of administration, preferably in a normal state, and suppressing an increase in the proportion of heteroplasmic cells. By maintaining cells, preferably normal cells, that have wild-type mtDNA, it is possible to suppress a decline in mitochondrial respiratory function, which is expected to provide effects such as the treatment and prevention of diseases associated with heteroplasmy and the prevention of aging.

[0022] The heteroplasmy formation inhibitor of this embodiment preferably inhibits the heteroplasmy of cells that were originally normal cells (cells that were homoplasmic from birth). Alternatively, the heteroplasmy formation inhibitor of this embodiment may inhibit the heteroplasmy of normal cells obtained by converting heteroplasmic cells into homoplasmy.

[0023] As described above, by suppressing heteroplasmy and maintaining cells, preferably normal cells, that have wild-type mtDNA, mitochondrial protection, mitochondrial dysfunction suppression, and mitochondrial function maintenance effects are expected in humans and other organisms. That is, the heteroplasmy inhibitor of this embodiment can also be called a mitochondrial respiratory function decline suppressor or a mitochondrial function maintainer. Furthermore, the heteroplasmy inhibitor of this embodiment is expected to be used as a drug for treating or preventing mitochondrial disorders and mitochondrial diseases caused by mitochondrial dysfunction, controlling the progression of disease, and slowing the progression of aging. Examples of organisms other than humans include invertebrates, birds, fish, amphibians, reptiles, and vertebrates such as mammals.

[0024] The heteroplasmy inhibitor of this embodiment includes an Ape1 (apurinic / apyrimidinic endonuclease I) inhibitor. Ape1 is a base excision repair enzyme that promotes double-strand breaks when mtDNA bases are oxidatively damaged by reactive oxygen species or the like. This results in the removal of the oxidatively damaged bases, and it is believed that the double-strand breaks promote mtDNA heteroplasmy. The heteroplasmy inhibitor of this embodiment targets the active site of Ape1 and inhibits the enzymatic activity of Ape1, thereby suppressing mtDNA cleavage and, as a result, is believed to be able to suppress mtDNA heteroplasmy.

[0025] Examples of Ape1 inhibitors include, but are not limited to, AR03 (2,4,9-trimethylbenzo[b][1,8]naphthyridin-5-amine), CRT (CRT0044876: (7-nitroindole-2-carboxylic acid)), ML199 (N-[3-(1,3-benzothiazol-2-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-2-yl]acetamide), allylstibonic acids, 6-hydroxy-DL-DOPA (rac-(R*)-2-amino-3-(2,4,5-trimethylbenzo[b][1,8]naphthyridin-5-amine), and the like. hydroxyphenyl)propionic acid), Reactive Blue 2 (2-anthracenesulfonic acid), myricetin (3,3',4',5,5',7-hexahydroxyflavone), aurintricarboxylic acid (ATA), lucanthone (1-[[2-(diethylamino)ethyl]amino]-4-methyl-9H-thioxanthen-9-one), hycanthone (1-[[2-(diethylamino)ethyl]amino]-4-(hydroxymethyl)-9H-thioxanthen-9-one), methoxyamine, etc., or salts thereof.

[0026] Among these, the Ape1 inhibitor is preferably at least one selected from AR03 and CRT, and particularly preferably AR03. By using AR03 as the Ape1 inhibitor, the progression of heteroplasmy can be more effectively suppressed in cells having wild-type mtDNA, preferably normal cells, thereby suppressing the decline in mitochondrial respiratory function and maintaining mitochondrial function.

[0027] The salt contained in the Ape1 inhibitor is not particularly limited as long as it is a pharmaceutically acceptable salt, and examples thereof include inorganic acid salts such as hydrochloride and sulfate, organic salts such as acetate, oxalate, fumarate, and succinate, salts with amino acids, metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, ammonium salt, etc. Ape1 inhibitors may be used alone or in combination.

[0028] The form of the heteroplasmy formation inhibitor of this embodiment is not particularly limited, and may be granules, powder, solid, or liquid. The heteroplasmy formation inhibitor of this embodiment may consist solely of an Ape1 inhibitor, or may contain optional ingredients in addition to the Ape1 inhibitor.

[0029] The heteroplasmy formation inhibitor of this embodiment may be a pharmaceutical, food or beverage, cosmetic, or the like. When the heteroplasmy formation inhibitor is formulated into a pharmaceutical (including quasi-drugs), the Ape1 inhibitor may be prepared into the desired form either directly or in combination with other additives. Specific examples of pharmaceuticals include oral pharmaceuticals such as tablets, pills, powders, fine granules, granules, capsules (including hard capsules and soft capsules), troches, chewable tablets, extracts (including soft extracts, dry extracts, etc.), jellies, syrups, spirits, elixirs, and liposome preparations; topical pharmaceuticals such as ointments, creams, lotions, gels, sprays, patches, emulsions, suspensions, poultices, liniments, aerosols, ointments, packs, inhalants, and suppositories; and injections. The heteroplasmy formation inhibitor of this embodiment can also be further combined with other pharmaceuticals within the scope of the present invention, provided that the effects of the present invention are not impaired.

[0030] When preparing the heteroplasmy inhibitor in the form of a food or beverage, the Ape1 inhibitor may be prepared as is or in combination with other food ingredients or additives to form the desired form. Examples of such foods and beverages include general foods and beverages, as well as foods with health claims (including foods for specified health uses, foods with nutrient functions, and foods with functional claims), and foods for patients. The form of these foods and beverages is not particularly limited, but specific examples include beverages such as tea drinks, energy drinks, fruit juice drinks, carbonated drinks, and lactic acid drinks; supplements such as capsules (soft capsules and hard capsules), tablets, granules, powders, jellies, and liposome preparations; and luxury items such as gummies, candies, and jellies.

[0031] When the heteroplasmy formation inhibitor is formulated into a cosmetic product, the Ape1 inhibitor may be formulated into a desired form either directly or in combination with other additives, etc. Specific examples of cosmetic products include creams, lotions, gels, emulsions, liquids, ointments, and packs.

[0032] Among these, the heteroplasmy inhibitor is preferably a pharmaceutical agent, and is particularly preferably a therapeutic agent for a disease associated with mitochondrial DNA heteroplasmy. That is, this embodiment may relate to a therapeutic agent for a disease associated with mitochondrial DNA heteroplasmy, including an Ape1 inhibitor. In this case, the Ape1 inhibitor is preferably AR03. Diseases associated with mitochondrial DNA heteroplasmy include mitochondrial diseases. Mitochondrial diseases are a general term for pathological conditions in which symptoms appear due to decreased mitochondrial function. Use of the heteroplasmy inhibitor of this embodiment is expected to prevent the onset of various symptoms associated with mitochondrial disease or alleviate the symptoms. Note that the therapeutic agent in this specification also includes agents for preventing the onset of diseases associated with mitochondrial DNA heteroplasmy, as well as agents for delaying the onset or suppressing the severity of the onset.

[0033] The therapeutic agent for diseases associated with heteroplasmy of mitochondrial DNA may be further combined with other ingredients or medicines within the scope that does not impair the effects of the present invention.

[0034] The heteroplasmy inhibitor of this embodiment can suppress the progression of heteroplasmy in cells having wild-type mtDNA, preferably normal cells, and as a result, can suppress the decline in mitochondrial respiratory function and maintain mitochondrial function. Furthermore, maintaining mitochondrial function also makes it possible to prevent or treat diseases caused by mitochondrial dysfunction and suppress the progression of the condition. In other words, the heteroplasmy inhibitor of this embodiment is a mitochondrial function maintainer. Furthermore, the heteroplasmy inhibitor of this embodiment is preferably intended for the prevention or treatment of diseases caused by mitochondrial dysfunction.

[0035] (Method for suppressing heteroplasmy) This embodiment relates to a method for inhibiting heteroplasmy formation, which includes contacting cells with an Ape1 inhibitor. However, the method for inhibiting heteroplasmy formation of this embodiment may exclude human therapeutic methods. This embodiment may also relate to a method for inhibiting heteroplasmy formation, which includes introducing an Ape1 inhibitor into cells in vitro. The method for inhibiting heteroplasmy formation can inhibit the progression of heteroplasmy formation in cells having wild-type mtDNA, preferably normal cells, thereby suppressing the decline in mitochondrial respiratory function and maintaining mitochondrial function. In other words, the method for inhibiting heteroplasmy formation of this embodiment can also be called a method for inhibiting the decline in mitochondrial respiratory function or a method for maintaining mitochondrial function.

[0036] The cells used in the heteroplasmy suppression method are eukaryotic cells. Eukaryotic cells are not particularly limited, but include, for example, mammalian cells, particularly cells derived from humans and non-human organisms. Examples of non-human cells include cells derived from mice, rats, cows, horses, pigs, sheep, monkeys, dogs, cats, and birds. Of these, human-derived eukaryotic cells are preferred. Furthermore, the eukaryotic cells used in the heteroplasmy suppression method are not particularly limited as long as they have wild-type mtDNA, and may be cells having only wild-type mtDNA, or cells in which mutant mtDNA and wild-type mtDNA coexist within the cells.

[0037] The cells used in the method for suppressing heteroplasmy are also preferably iPS cells (iPSCs). iPS cells are cells that have acquired pluripotency equivalent to that of ES cells by introducing several types of transcription factor genes and proteins that confer pluripotency, and compounds that induce pluripotency (collectively referred to as pluripotency factors) into somatic cells. Many pluripotency factors have already been reported, including, but not limited to, the Oct family (e.g., Oct3 / 4), the SOX family (e.g., SOX2, SOX1, SOX3, SOX15, and SOX17), the Klf family (e.g., Klf4 and Klf2), the MYC family (e.g., c-MYC, N-MYC, and L-MYC), NANOG, and LIN28. iPS cells can proliferate indefinitely and differentiate into any type of cell in the body, but because the reprogramming process to establish iPS cells does not reset mtDNA, the mtDNA in iPS cells is in a heteroplasmic state. Therefore, suppressing further heteroplasmy in iPS cells by exposing them to an Ape1 inhibitor is useful in the field of regenerative medicine.

[0038] When Ape1 inhibitors contact cells, they inhibit the enzymatic activity of Ape1 within the cells, thereby enhancing mitochondrial respiratory function. Mitochondrial respiratory function is measured by measuring the OCR / ECAR ratio, and a higher ratio indicates better mitochondrial respiratory function. OCR is the oxygen consumption rate during mitochondrial oxidative phosphorylation, which produces ATP, and ECAR is the extracellular acidification rate (ECR), which is an indicator of glycolysis.

[0039] "Contacting" an Ape1 inhibitor with a eukaryotic cell means making these components present in the medium in which the cells are growing, etc., and treating the cells so that the Ape1 inhibitor can enter the target cells and these components can come into contact with the cells.

[0040] The Ape1 inhibitor is preferably at least one selected from AR03 and CRT, and particularly preferably AR03. When an Ape1 inhibitor is added to a medium in which cells are growing in the method for suppressing heteroplasmy formation, the amount added is preferably 0.1 to 100 μM, more preferably 0.5 to 50 μM, and even more preferably 1 to 20 μM. By adding the Ape1 inhibitor in an amount within the above range, heteroplasmy formation can be more effectively suppressed.

[0041] The time for contacting eukaryotic cells with an Ape1 inhibitor can be calculated as the treatment time during which these components are present in the cell culture medium. This treatment time is a time that does not damage the cells used and is effective for treating the cells with these components. The treatment time is, for example, preferably 1 to 500 hours, more preferably 5 to 240 hours, and even more preferably 24 to 150 hours. In a preferred embodiment, for example, the Ape1 inhibitor is added to the cell culture medium and then cultured for 2 to 6 days. By setting the treatment time within the above range, heteroplasmy formation can be more effectively suppressed.

[0042] The heteroplasmy suppression method of this embodiment can suppress heteroplasmy in cells having wild-type mtDNA, preferably normal cells, and as a result, can suppress the decline in mitochondrial respiratory function. Therefore, the heteroplasmy suppression method of this embodiment can also be called a method for maintaining mitochondrial function.

[0043] (Method for preparing normal cells and normal cells) One embodiment of the present invention is a method for preparing normal cells using the above-described method for suppressing heteroplasmy. Specifically, this embodiment is a method for preparing cells in which wild-type mtDNA exists in a homoplasmic state, or cells in which mutant mtDNA and wild-type mtDNA coexist within the cells at levels not exceeding a threshold. Another embodiment of the present invention is a normal cell prepared by this method. The normal cells prepared are cells in which wild-type mtDNA exists in a homoplasmic state, or cells in which mutant mtDNA and wild-type mtDNA coexist within the cells at levels not exceeding a threshold.

[0044] This embodiment may be a method for preparing homoplasmic cells using the above-described method for suppressing heteroplasmy. Another embodiment may be a homoplasmic cell prepared by the method. Homoplasmic cells are cells in which wild-type mtDNA exists in a homoplasmic state.

[0045] When selecting and preparing normal or homoplasmic cells, cells treated with the heteroplasmy inhibitor described above are allowed to form single colonies by an appropriate method (e.g., limiting dilution, etc.), and DNA samples are prepared from cell clones derived from the resulting single colonies. Cell clones with the desired abundance ratio can be selected by confirming the intracellular abundance ratio of mtDNA mutations present in the prepared DNA sample. When selecting normal or homoplasmic cells, selection can be made more easily in the presence of an appropriate agent that inhibits glycolysis (e.g., sodium fluoride, iodoacetic acid, etc.).

[0046] One embodiment of this may relate to iPS cells (iPSCs) or somatic cells prepared from the above method. Many publications have been published regarding methods for establishing iPS cells, and these can be used as reference (e.g., Takahashi et al., Cell 2006, 126:663-676; Okita et al., Nature 2007, 448:313-317; Wernig et al., Nature 2007, 448:318-324; Maherali et al., Cell Stem Cell 2007, 1:55-70; Park et al., Nature 2007, 451:141-146; Nakagawa et al., Nat Biotechnol 2008, 26:101-106; Wernig et al., Cell Stem Cell 2008, 10:10-12; Yu et al., Science 2007, 318:1917-1920; Takahashi et al., Cell 2007,131:861-872; Stadtfeld et al., Science 2008 322:945-949, etc.).

[0047] The type of somatic cell may be any somatic cell, including, but not limited to, fibroblasts, osteoblasts, adipocytes, chondrocytes, myoblasts, bone marrow stromal cells, tenocytes, blood cells (e.g., peripheral blood mononuclear cells, T cells, etc.), hepatocytes, pancreatic cells, intestinal epithelial cells, glial cells, nerve cells, cardiac muscle cells, smooth muscle cells, vascular endothelial cells, and lymphatic endothelial cells. [Example]

[0048] The features of the present invention will be explained in more detail below with reference to examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0049] <Method> <Cell culture> BJ foreskin fibroblasts and MELAS cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS, 100 units / mL penicillin, and 100 μg / mL streptomycin. PD-iPSCs were grown in Stem Fit AKO2N medium (Ajinomoto Co., Inc.) containing 10 μM of the ROCK inhibitor Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) at 37°C and 5% CO2.

[0050] <Induction of iPSCs into somatic cells> To induce differentiation of iPSCs into somatic cells, we used the Cellartis iPS Cell to Hepatocyte Differentiation System. Hep-iPSCs were cultured for 21 days using the Cellartis Definitive Endoderm Differentiation (DEF)-Counter-Strike (CS) Culture System, followed by 11 days of culture using the Cellartis Definitive Endoderm Differentiation (DEF) Kit. All kits were purchased from Takara Bio.

[0051] <Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)> The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) represent mitochondrial function and glycolytic flux in cells, respectively. For each cell line, at least three independent measurements of OCR and ECAR were performed using an Agilent Seahorse Bioscience XF-96 Extracellular Flux Analyzer (Agilent / Seahorse Bioscience) (Dranka, B.P., et al., (2011) Assessing bioenergetic function in response to oxidative stress by metabolic profiling. Free Radic Biol Med, 51, 1621-1635; Schneider, L., et al., (2011) Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress. Free Radic Biol Med, 51, 2007-2017; and Divakaruni, A.S., et al., (2014) Analysis and interpretation of microplate-based oxygen consumption and pH data. Methods Enzymol, 547, 309-354). Cell numbers were determined using a Countless II FL automated cell counter (Thermo Fisher Scientific Inc.). The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) are established indicators of mitochondrial function and glycolytic flux. The OCR / ECAR ratio is an indicator of the change in metabolic phenotype between mitochondrial respiration and glycolysis for ATP production.

[0052] <Statistical analysis> Data are shown as mean ± standard deviation. Student's t-test and Tukey's multiple comparison analysis were used to compare values ​​between two groups. P < 0.05 was considered significant.

[0053] Treatment with Ape1 inhibitor (1) 1×10 , , ,

[0055] , 4 , 4 , , ,

[0054] , , Four days of culture were performed on 1×10 individual PD-iPSCs (iPS cells derived from Parkinson's disease patients) in the presence of 2 μM of AR03. Similarly, 1×10 4 individual BJ primary cells (skin cells derived from a baby) were cultured for 4 days in the presence of 1.95 μM or 2.0 μM of AR03. Also in the same way, 1×10 4 individual MELAS cells (somatic cells derived from MELAS patients) were cultured for 4 days in the presence of 1.95 μM, 2.0 μM or 2.1 μM of CRT. The conditions for treating and culturing the cells in the presence of AR03 or CRT were 37 °C and 5% CO2 conditions. Also, the medium used in the above-mentioned <Cell culture> was used as it was. After culturing for 4 days, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured by the method described above, and the OCR and OCR / ECAR ratios were calculated. Note that since the height of the OCR value measured using an extracellular flux analyzer is generally known to depend on the cell number, in this measurement system, the OCR values were compared between samples with adjusted cell numbers to the same extent.

[0054] Treatment with Ape1 inhibitor (2) 1×10 4 individual BJ primary cells were cultured in a medium containing 2.0 μM of AR03 (2,4,9-trimethylbenzo[b][1,8]naphthyridin-5-amine) for 0 to 7 days. After culturing for 3 to 7 days, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured by the method described above, and the OCR / ECAR ratio was calculated.

[0055] Treatment with Ape1 inhibitor (3) BJ primary cells cultured by the treatment with the above-mentioned <Ape1 inhibitor (2)> were analyzed using an extracellular flux analyzer for the state of aerobic respiration and glycolysis by mitochondria, which is the main energy metabolic pathway of cells, in the presence of oligomycin A (OMA), an ATP synthesis inhibitor, FCCP, a compound that inhibits the coupling of both the electron transport system and the ATP synthesis reaction without inhibiting either reaction, rotenone (RTN), a mitochondrial complex I inhibitor, and antimycin A (AMA), a mitochondrial complex III inhibitor. Specifically, about 15 minutes after starting to measure the values of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), oligomycin A (OMA) was injected to perform a reaction that inhibits ATP synthesis for about 15 minutes. Then, FCCP was injected to perform a reaction that inhibits the coupling of the electron transport system and the ATP synthesis reaction for about 15 minutes. Next, RTN and AMA were injected to perform a reaction that inhibits mitochondrial complex I for 15 minutes. OMA, FCCP, RTN / AMA were added so that their final concentrations became 0.125 mM, 1 mM, and 1 mM, respectively.

[0056] <Results> In PD-iPSCs cultured for 4 days in the presence of 2 μM AR03, the OCR and the OCR / ECAR ratio were significantly increased (Figs. 1A - C). From this, it was found that specific inhibition of human Ape1 prevents the generation of heteroplasmy. It was suggested that the suppression of heteroplasmy improves the potential of mitochondrial oxidative phosphorylation (OXPHOS).

[0057] Also, AR03 and CRT significantly increased the OCR and the OCR / ECAR ratio without giving a detectable change to the ECAR in BJ primary cells (Figs. 2A - C) and MELAS cells (Figs. 2D - F). The OCR and the OCR / ECAR ratio of BJ primary cells increased with the treatment time up to the 6th day while being treated with 2 μM AR03 for 3 - 7 days (Figs. 2G and H).

[0058] Taken together, we demonstrate that inhibiting human Ape1 to prevent heteroplasmy improves mitochondrial oxidative phosphorylation (OXPHOS) function in human cells. [Industrial Applicability]

[0059] According to the present invention, heteroplasmy of wild-type mitochondrial DNA can be suppressed. This reduces the proportion of heteroplasmic cells and maintains the proportion of normal cells. In light of the current situation where a cure for mitochondrial diseases caused by mtDNA mutations has not yet been discovered, the present invention contributes to the protection of mitochondria, the suppression of mitochondrial damage, and the maintenance of mitochondrial function.

Claims

1. Heteroplasmy suppressors, including Ape1 inhibitors.

2. The heteroplasmy formation inhibitor according to claim 1, wherein the Ape1 inhibitor is 2,4,9-trimethylbenzo[b][1,8]naphthyridin-5-amine or a salt thereof.

3. The heteroplasmy formation inhibitor according to claim 1 or 2, which is for maintaining mitochondrial function.

4. The heteroplasmy formation inhibitor according to claim 1 or 2, which is used for preventing or treating a disease caused by mitochondrial dysfunction.

5. The heteroplasmy formation inhibitor according to claim 1 or 2, which is a pharmaceutical, food or beverage, or cosmetic.

6. A therapeutic agent for a disease associated with heteroplasmy of mitochondrial DNA, comprising an Ape1 inhibitor.

7. The therapeutic agent according to claim 6, wherein the Ape1 inhibitor is 2,4,9-trimethylbenzo[b][1,8]naphthyridin-5-amine or a salt thereof.

8. A method for suppressing heteroplasmy, comprising contacting a cell with an Ape1 inhibitor.

9. The method for suppressing heteroplasmy formation according to claim 8, wherein the Ape1 inhibitor is 2,4,9-trimethylbenzo[b][1,8]naphthyridin-5-amine or a salt thereof.

10. The method for suppressing heteroplasmy formation according to claim 8 or 9, wherein the cells are human-derived cells.

11. The method for suppressing heteroplasmy formation according to claim 8 or 9, wherein the cells are iPS cells.

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