Composition for use in the treatment of diseases associated with mitochondrial dysfunction

Abstract

The present invention relates to a composition comprising a methyl group donor compound and an acetyl-CoA donor compound and used for the treatment and prevention of diseases associated with mitochondrial dysfunction, wherein the diseases associated with mitochondrial dysfunction are particularly nucleotide excision repair deficiency diseases such as xeroderma pigmentosum A, or ataxia telangiectasia due to DNA repair deficiency associated with mitochondrial dysfunction. In particular, the composition is used for the treatment of nucleotide excision repair deficiency diseases such as xeroderma pigmentosum A, B, C, D, E, F, G, V, etc., various forms of Cockayne syndrome (CS) such as Cockayne syndrome group B (CSB) and Cockayne syndrome group A (CSA), trichothiodystrophy (TTD) or ataxia telangiectasia due to DNA repair deficiency associated with mitochondrial dysfunction.

Description

Technical Field

[0001] The present invention relates to a composition containing a methyl group donor compound and an acetyl-CoA donor compound, and is particularly used for the treatment and prevention of diseases related to mitochondrial dysfunction such as nucleotide excision repair deficiency, ataxia telangiectasia caused by DNA repair deficiency related to mitochondrial dysfunction, mitochondrial diseases, or aging-related diseases. In particular, the composition is used for the treatment and prevention of nucleotide excision repair deficiencies such as xeroderma pigmentosum A, B, C, D, E, F, G, V, etc., various forms of Cockayne syndrome (CS) such as Cockayne syndrome group B (CSB) and Cockayne syndrome group A (CSA), trichothiodystrophy (TTD), ataxia telangiectasia caused by DNA repair deficiency related to mitochondrial dysfunction, MELAS (myopathy, encephalopathy, lactic acidosis, stroke-like episodes), MERRF (ragged red fiber-myoclonus epilepsy) syndrome, CPEO (chronic progressive external ophthalmoplegia), KSS (Kearns-Sayre syndrome), LS (Leigh encephalopathy), and LHON (Leber hereditary optic neuropathy) and other mitochondrial diseases, or aging-related diseases such as endogenous skin aging, exogenous skin aging, and juvenile skin aging.

Background Art

[0002] Xeroderma pigmentosum (XP) is an incurable hereditary disease caused by specific mutations in one of eight different genes, and is classified into eight different types depending on the location of the mutation. The particularly severe course of this disease is seen especially in association with mutations in the XPA gene. A typical symptom is that the skin becomes extremely sensitive to ultraviolet rays (UV-B). As a result, patients get sunburned even with very low levels of sunlight exposure and may develop skin cancer from childhood. In these patients, the risk of skin cancer increases by 1000-fold. Being very sensitive to sunlight, such patients usually only go outside at night, and are popularly called "moonlight children". In addition to being hypersensitive to ultraviolet rays and having an extremely high risk of skin cancer, patients show signs of accelerated aging of many processes. In addition to the skin, other organs such as the central nervous system are also affected. Therefore, xeroderma pigmentosum is also counted as one of the so-called progeroid syndromes.

[0003] Investigations into the cause of xeroderma pigmentosum have generally classified this disease as a so-called DNA repair deficiency disorder. Specifically, the above eight genes encode proteins that play important roles in nucleotide excision repair (NER) at different sites. NER is one of the most important endogenous DNA repair systems in humans. This becomes the central mechanism for repairing UVB-induced damage to the genetic material (nuclear DNA) of skin cells. Therefore, the breakdown of this repair mechanism is related to the increased risk of skin cancer.

[0004] However, this does not explain why affected individuals also exhibit neurological phenotypes or all the signs of premature aging. Therefore, it is speculated that XP proteins may have additional biological functions beyond their role in DNA repair.

[0005] This speculation is supported by the discovery of mitochondrial metabolic dysfunction in XPA-deficient human cells and nematodes. Fang et al (Fang et al, Defective mitophagy in XPA via PARP1 hyperactivation and NAD+ / SIRT1 reduction, Cell, 2014) proposed based on functional studies that the XPA protein is important for the activation of the sirtuin Sirt1 and the removal of damaged mitochondria by the mitophagy mechanism.

[0006] Chinese Patent Specification No. 109673857 describes a composition containing butyrate and betaine for reducing mitochondrial autophagy and oxidative stress in pigs.

[0007] U.S. Patent Application Publication No. 2018 / 256612 describes a composition containing exogenous ketone bodies and methyl donors for treating aging, stress-related diseases or disorders, diabetes (type I or II, obesity, neurodegenerative diseases (Alzheimer's disease, neurodegenerative diseases, etc.), cardiovascular diseases, muscle diseases, blood coagulation disorders, inflammation, cancer, eye diseases or mitochondrial abnormalities.

[0008] U.S. Patent Application Publication No. 2003 / 078269 describes a composition containing L-carnitine and choline for the treatment of insulin resistance and type II diabetes.

[0009] European Patent Application Publication No. 2792354 relates to the use of a composition containing acetyl-L-carnitine and betaine for the treatment of acute and chronic hepatic encephalopathy.

[0010] U.S. Patent Application Publication No. 2019 / 247326 describes a composition containing vitamin B6 and butyrate for the treatment of Wolf-Hirschhorn syndrome.

[0011] M. J. Smerdom et al. (1982), “Sodium Butyrate Stimulates DNA Repair in UV-irraditated Normal and Xeroderma Pigmentosum Fibroblasts”, The Journal of Biological Chemistry, 13441-13447 describes the use of sodium butyrate in the treatment of xeroderma pigmentosum.

[0012] M. Scheibye-Knudsen et al. (2014), “A High Fat Diet and NAD + Rescue Premature Aging in Cockayne Syndrome”, Cell Metab., 20, 5, 840-855 reports that a high-fat diet rescued Csb m / m phenotype mice at the metabolic, transcriptome and behavioral levels, that a high-fat diet increased β-hydroxybutyrate levels, and that β-hydroxybutyrate, PARP inhibitors or NAD + supplementation can activate SIRT1 and rescue CS-related phenotypes.

Prior Art Documents

Patent Documents

[0013]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0014] [Non-Patent Document 1] Fang et al., Defective mitophagy in XPA via PARP1 hyperactivation and NAD+ / SIRT1 reduction, Cell, 2014 [Non-Patent Document 2] M.J. Smerdom et al. (1982), “Sodium Butyrate Stimulates DNA Repair in UV-irraditated Normal and Xeroderma Pigmentosum Fibroblasts”, The Journal of Biological Chemistry, 13441-13447 [Non-Patent Document 3] M. Scheibye-Knudsen et al. (2014), “A High Fat Diet and NAD+ Rescue Premature Aging in Cockayne Syndrome”, Cell Metab., 20, 5, 840-855 [Non-Patent Document 4] F. Depeint et al. (2006), “Mitochondrial function and toxicity: Role of B vitamins on the one-carbon transfer pathways”, Chemico-Biological Interactions, 163, 113-132 [Non-Patent Document 5] E.N. Proshkina (2020), “Key Molecular Mechanisms of Aging, Biomarkers, and Potential Interventions”, Molecular Biology, 54, 6, 777-811 [Non-Patent Document 6] J.M.Frans Trijbels et al.(2004),“Chapter 5 - Biochemical Diagnosis of OXPHOS Disorders”,“Oxidative Phosphorylation in Health and Disease”,Eurekah.com and Kluwer Academic / Plenum Publishers

Summary of the Invention

Problems to be Solved by the Invention

[0015] There is still a need to provide pharmaceutical compositions that can be used for the treatment and / or prevention of diseases associated with mitochondrial dysfunction, such as nucleotide excision repair deficiency, ataxia telangiectasia caused by DNA repair deficiency associated with mitochondrial dysfunction, mitochondrial diseases, or aging-related diseases. Therefore, an object of the present invention is to provide a pharmaceutical composition for use in the treatment and prevention of diseases associated with mitochondrial dysfunction, particularly xeroderma pigmentosum. This problem can be achieved by targeting the mitochondrial metabolic dysfunction in the above-mentioned XPA-deficient human cells and nematodes.

Means for Solving the Problems

[0016] Surprisingly, the problem of the present invention is solved by a composition comprising a first compound A that is a methyl group donor and a second compound B that is an acetyl-CoA donor, and is used for the treatment and prevention of diseases associated with mitochondrial dysfunction, particularly diseases in group A of xeroderma pigmentosum.

[0017] In particular, the problem of the present invention is solved by a composition comprising a first compound A that is a methyl group donor and a second compound B that is an acetyl-CoA donor, and is used for the treatment and prevention of diseases associated with mitochondrial dysfunction, wherein the diseases associated with mitochondrial dysfunction are particularly nucleotide excision repair deficiencies such as xeroderma pigmentosum A, or ataxia telangiectasia caused by DNA repair deficiency associated with mitochondrial dysfunction.

[0018] Surprisingly, mitochondrial dysfunction plays an important role in the pathophysiology of xeroderma pigmentosum, and it has been found that a combination of a methyl group donor compound and an acetyl-CoA donor compound is effective in at least partially compensating for the mitochondrial dysfunction by increasing adenosine triphosphate (ATP) and, most often, acetyl-CoA production in cells. The characteristics specific to diseases such as CS, XPA, and TTD are the most prominent transcriptional arrests that occur after induction of DNA damage by exposure to UV irradiation or other harmful substances. Therefore, it is particularly interesting that the combined treatment with a methyl group donor and an acetyl-CoA donor can at least partially overcome the loss of mRNA transcripts found in many genes in XPA fibroblasts with or without ultraviolet radiation. Apart from enhancing mRNA transcription or stabilization, the combined therapy can also increase the cellular abundance of individual proteins by improving protein stability or translation efficiency. Furthermore, surprisingly, it has been found that the compositions of the present invention can be widely used for the treatment and prevention of any diseases associated with mitochondrial dysfunction.

[0019] As compound A, an acceptable methyl group donor known from the prior art may be employed. The terms "methyl group donor" or "methyl donor" are known to those skilled in the art. In the art, this term is used to represent a compound that can supply a methyl group in the human body, for example, in metabolic pathways or DNA synthesis. Examples include F. Depeint et al. (2006), "Mitochondrial function and toxicity: Role of B vitamins on the one-carbon transfer pathways", Chemico-Biological Interactions, 163, 113-132, and E. N. Proshkina (2020), "Key Molecular Mechanisms of Aging, Biomarkers, and Potential Interventions", Molecular Biology, 54, 6, 777-811. Typical methyl group donors include trimethylammonium compounds such as choline and betaine, methionine, folic acid, folate, 5-methyltetrahydrofolate, vitamins B2, B6 and B12, sarcosine, serine, trimethylamine-N-oxide, and S-adenosylmethionine (SAM). In a preferred embodiment, the methyl group donor may be selected from the group consisting of compounds containing a trimethylamine group, sarcosine, trimethylamine-N-oxide, serine, and mixtures of two or more thereof. Preferably, the methyl group donor may be selected from trimethylammonium compounds and mixtures of two or more thereof. Preferred compounds containing a trimethylamine group are choline and betaine. A particularly preferred option for the methyl group donor is choline and betaine. As compound A, choline is most preferred.

[0020] In another preferred embodiment, the first compound A is selected from the group consisting of choline, betaine, sarcosine, trimethylamine-N-oxide, serine, and mixtures of two or more thereof, particularly selected from choline and betaine.

[0021] As compound B, an acceptable acetyl-CoA donor known from the prior art may be employed. The term "acetyl-CoA donor" is known to those skilled in the art. In this technical field, this term is used to represent a substance or compound that can generate acetyl-CoA in the human body, or a substance that inhibits a compound such as an enzyme that inhibits the generation of acetyl-CoA. Examples are described in J.M.Frans Trijbels et al. (2004), "Chapter 5 - Biochemical Diagnosis of OXPHOS Disorders", "Oxidative Phosphorylation in Health and Disease", Eurekah.com and Kluwer Academic / Plenum Publishers. A mixture of two or more different acetyl-CoA donors can also be employed. In the context of this application, an acetyl-CoA donor is a substance and compound that can generate acetyl-CoA in the human body, or a substance that inhibits a compound such as an enzyme that inhibits the generation of acetyl-CoA. Typical acetyl-CoA donors include fatty acids, fatty acid derivatives, fatty acid salts, fatty acid derivative salts, triglycerides, triglyceride derivatives, dicarboxylic acid derivatives, and pyruvate dehydrogenase kinase inhibitors.

[0022] Compounds capable of generating acetyl-CoA are, for example, fatty acids and their salts. In a preferred embodiment, the acetyl-CoA donor is selected from fatty acids and their salts, and acetate or butyrate, i.e., salts of acetic acid or butyric acid, are particularly preferred. Particularly preferably, compound B is acetate.

[0023] In the context of the present invention, a fatty acid is a carboxylic acid having from 2 to 28 carbon atoms, particularly from 10 to 25 carbon atoms. Its chain is aliphatic and may be saturated or unsaturated, and may be straight-chain or branched. In the context of the present application, both acetic acid and butyric acid are fatty acids. Pharmaceutically acceptable salts of the above-mentioned fatty acids known from the prior art may also be employed. Suitable salts include, for example, sodium salts of fatty acids, potassium salts of fatty acids, ammonium salts of fatty acids, calcium salts of fatty acids or magnesium salts of fatty acids.

[0024] Compounds that inhibit the production of acetyl-CoA are, for example, pyruvate dehydrogenase kinase (PDK). Pyruvate dehydrogenase kinase is a kinase enzyme that inactivates the enzyme pyruvate dehydrogenase by phosphorylating it using ATP. Four isozymes of human PDK are known, all of which exhibit the same catalytic action. Therefore, as compound B, inhibitors of pyruvate dehydrogenase kinase, such as dichloroacetic acid, AZD7545 ((R)-4-(3-chloro-4-(3,3,3-trifluoro-2-hydroxy-2-methylpropanamide)phenylsulfonyl)-N,N-dimethylbenzamide), PS10 (2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol), dicumarol, JX06 (bis(morpholinothio-carbonyl)disulfide), reiram or VER-246608 (N-[4-(2-chloro-5-methyl-4-pyrimidinyl)phenyl]-N-[[4-[[(2,2-difluoroacetyl)amino]methyl]phenyl]methyl]-2,4-dihydroxy-benzamide) are also suitable.

[0025] In a particularly preferred embodiment of the present invention, the second compound B may be selected from fatty acids, fatty acid derivatives, fatty acid salts, fatty acid derivative salts, triglycerides, triglyceride derivatives and dicarboxylic acid derivatives, particularly from acetate, butyrate, triheptanoin and dimethyl α-ketoglutarate, or may be selected from pyruvate dehydrogenase kinase inhibitors. Triheptanoin is an example of a triglyceride that may be used as compound B in the present invention. Dimethyl α-ketoglutarate is an example of a dicarboxylic acid derivative that may be used in the present invention. Preferably, the second compound B may be selected from acetate, butyrate, triheptanoin and dimethyl α-ketoglutarate, more preferably may be selected from acetate and butyrate. Particularly preferably, compound B is acetate.

[0026] The fatty acid derivative in the meaning of the present invention is a modified fatty acid such as oxylipin, hydroxy fatty acid, diol, alkenone and wax ester. β-Hydroxybutyric acid is an example of a fatty acid derivative that may be used in the present invention. The fatty acid derivative salt in the meaning of the present invention is a pharmaceutically acceptable fatty acid derivative salt. Suitable salts include, for example, sodium salt of fatty acid derivative, potassium salt of fatty acid derivative, ammonium salt of fatty acid derivative, calcium salt of fatty acid derivative or magnesium salt of fatty acid derivative.

[0027] In a preferred embodiment, the composition for use in the present invention may consist of compound A and compound B.

[0028] A particularly preferred composition for use in the present invention contains choline as compound A and contains acetate as compound B, or is composed of these.

[0029] Another particularly preferred composition for use in the present invention contains choline as compound A and contains butyrate as compound B, or is composed of these.

[0030] Yet another particularly preferred composition for use in the present invention contains choline as compound A and triheptanoin as compound B, or consists of these.

[0031] Yet another particularly preferred composition for use in the present invention contains choline as compound A and dimethyl α-ketoglutarate as compound B, or consists of these.

[0032] In a preferred embodiment, the molar ratio of compound A to compound B is in the range of 10:1 to 1:10. More preferably, the above ratio is in the range of 8:1 to 1:8 or 5:1 to 1:5, particularly in the range of 2:1 to 1:2, and particularly 1:1.

[0033] In another preferred embodiment, the molar ratio of compound A to compound B is in the range of 5:1 to 10:1.

[0034] In a preferred embodiment, the concentration of compound A is in the range of 5 to 250 mM, preferably in the range of 15 to 150 mM, more preferably in the range of 20 to 120 mM, and the concentration of compound B is in the range of 1 to 100 mM, preferably in the range of 5 to 50 mM, more preferably in the range of 7 to 15 mM.

[0035] In another preferred embodiment, the concentration of compound A is in the range of 25 to 100 mM and the concentration of compound B is 10 mM.

[0036] The dosage of the composition of the present invention and its components varies depending on the type of desired effect, the weight, age, gender of the subject, and the administration method. Generally, the composition for use can be administered in an amount based on the average weight of the subject. In a preferred embodiment, the composition for use in the present invention is administered in an amount such that compound A is 1 to 250 mg / kg / d, preferably 2 to 240 mg / kg / d, more preferably 5 to 220 mg / kg / d, and compound B is 1 to 250 mg / kg / d, preferably 10 to 220 mg / kg / d, more preferably 50 to 200 mg / kg / d.

[0037] In a further preferred embodiment, the composition of the present invention is administered in an amount such that choline as compound A is 1 to 25 mg / kg / d, preferably 2 to 20 mg / kg / d, more preferably 5 to 10 mg / kg / d, still more preferably 7 to 8 mg / kg / d, and compound B is 1 to 250 mg / kg / d, preferably 10 to 220 mg / kg / d, more preferably 50 to 200 mg / kg / d. Preferably, compound B is an acetate.

[0038] In a further preferred embodiment, the composition of the present invention is administered in an amount such that betaine as compound A is 100 to 250 mg / kg / d, preferably 120 to 240 mg / kg / d, more preferably 130 to 220 mg / kg / d, and compound B is 1 to 250 mg / kg / d, preferably 10 to 220 mg / kg / d, more preferably 50 to 200 mg / kg / d. Preferably, compound B is an acetate.

[0039] In a preferred embodiment, the composition for use of the present invention is administered daily.

[0040] The composition for use of the present invention may be administered by a suitable method known from the prior art. In a preferred embodiment, the composition for use of the present invention is administered orally, intravenously or topically. The composition for use of the present invention may be administered topically, for example, as a compound in a sunscreen product or alone. Depending on the administration, the composition for use according to the present invention may contain suitable additives known to those skilled in the art, such as solvents. For topical application, antioxidants, DNA repair enzymes, vitamins, UV filters, prebiotics or probiotic substances may be included.

[0041] The following further explains the principles of the present invention, and details how mitochondrial dysfunction leads to xeroderma pigmentosum (XP), and how the compositions for the use of the present invention alleviate mitochondrial dysfunction. It is understood that these theoretical findings are merely illustrative, and the present invention is not intended to be limited by the following theories.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0043] The present inventors have thoroughly studied mitochondrial dysfunction in primary human skin fibroblasts from XPA patients (i.e., patients suffering from group A of XP in which the mutation is localized in the XPA gene). Surprisingly, the following was observed.

[0044] (i) The XPA protein is localized in the mitochondria of human fibroblasts. (ii) The mitochondrial synthesis of ATP, which is required for numerous enzymatic processes (e.g., DNA repair) and corresponds to the universal energy truly necessary for cell survival, is greatly altered in XPA cells.

[0045] In healthy cells, the production of ATP in mitochondria occurs via the respiratory chain and is linked to the consumption of oxygen. Alternatively, ATP can also be obtained via the metabolic pathway glycolysis, but it is far less efficient in comparison, so if oxygen supply is sufficient, it is secondary in the production of ATP in healthy cells. In addition to the synthesis of ATP, the formation of acetyl-CoA, a high-energy metabolite, is also caused by the enzyme pyruvate dehydrogenase (PDH) in mitochondria. Acetyl-CoA plays various roles in cells. For example, by being inserted into the citric acid cycle, it can supply electrons to the mitochondrial respiratory chain, enabling the synthesis of ATP. Alternatively, acetyl-CoA can function as a substrate for post-translational modification of proteins through the reversible transfer (acetylation) of acetyl groups, thus changing its function. The second pathway of post-translational modification of proteins is also through the transfer (methylation) of methyl groups, which is reversible, and mitochondria are also involved in supplying the necessary metabolites for this. Acetylation and methylation have been extensively studied, especially using histones, which are nuclear DNA-binding proteins. Due to the complexity of the dynamic patterns of these post-translational modifications at the histone level, histones are also called the epigenetic code, controlling all DNA-related processes such as transcription, replication, and repair, and fundamentally affecting the fate of cells. Thus, these nucleus-related functions are controlled, at least indirectly, via a retrograde signaling pathway through mitochondria.

[0046] Surprisingly, the following was observed in XPA-deficient cells (e.g., skin fibroblasts from XPA patients). Consistent with mitochondrial dysfunction, XPA-deficient fibroblasts showed a decrease in histone acetylation and methylation (Figure 4B), which was particularly prominent after UVB irradiation. In addition, XPA-deficient fibroblasts showed an increase in mitochondrial oxygen consumption, suggesting an increased ATP demand (Figure 2C). Similarly, an increase in phosphorylation of PDH was observed in these cells, inactivating the enzyme. Therefore, the hypothesis that in XPA-deficient cells, there is less acetyl-CoA available for acetylation of proteins such as histones and for supplying electrons for ATP synthesis in the mitochondrial respiratory chain is reasonable. Also, our data show that in XPA-deficient fibroblasts after UVB exposure (Figure 2A), mitochondrial ATP production is significantly reduced. By increasing the glycolysis rate, the cells attempt to compensate for this loss of energy equivalent, but total ATP production remains significantly reduced compared to unirradiated XPA-deficient cells. Furthermore, immunofluorescent staining with an ATP-specific antibody revealed a loss of ATP in the nuclei of XPA-deficient fibroblasts with UVB irradiation (Figure 2B). At the same time, as a result of treatment with UVB irradiation or the DNA-damaging agent cisplatin (XPA Sham and XPA UVB in Figure 1A, and XPA Control and XPA Cisplatin in Figure 1B), apoptosis (cell death) induction increases in XPA-deficient fibroblasts, accompanied by the loss of many proteins essential for cell function.

[0047] Based on these results, we consider that the loss of energy equivalents such as ATP and acetyl-CoA and other mitochondrial metabolites that affect methylation may be an important factor involved in the development of, for example, XP.

[0048] The present invention proposes the following paradigm shift. The xeroderma pigmentosum protein is not uniquely or exclusively involved in the repair of DNA damage in the nucleus, but is extremely important for maintaining the normal function of mitochondria. Its main function is to supply the cell with energy equivalents, and this function is significantly impaired in XP cells. As a result, there is a significant deficiency of acetyl-CoA, leading to widespread effects on many cellular functions. This deficiency and the associated effects are most pronounced, for example, when cells are damaged by UV irradiation.

[0049] Based on these results, the inventors treated XPA cells for the purpose of increasing ATP and acetyl-CoA production in the cells. Surprisingly, it was proven that this is in principle possible. In this regard, combining two active substances was the most effective.

[0050] By administering a composition for use of the present invention containing a methyl group donor and an acetyl-CoA donor, preferably a combination of high-concentration choline and acetate, surprisingly, the mitochondrial phenotype of XPA cells was improved. In unirradiated XPA-deficient fibroblasts, the increased mitochondrial oxygen consumption and the elevated mitochondrial ATP production rate decreased upon treatment. In contrast, in XPA-deficient fibroblasts with UV irradiation, there was an increase in the mitochondrial ATP production and oxygen consumption rate (Figures 2A and 2C). At the same time, a significant increase in glycolysis-dependent ATP production was observed, and overall, more ATP was supplied to the cells (Figures 2A and 2B; Figure 7B).

[0051] Surprisingly, the composition for use in the present invention comprising a methyl group donor and an acetyl-CoA donor reduced the elevated apoptosis rate of XPA-deficient fibroblasts with UVB irradiation or cisplatin treatment (FIGS. 1A and 1B), and protected the cells from the loss of essential proteins (FIG. 4; FIG. 8). Along with the increase in ATP production by the activation of glycolysis, the anti-apoptotic effect of the combined treatment with a methyl group donor and an acetyl-CoA donor is partially dependent on the glycolytic flux because the glycolytic flux can be reduced by the excessive supply of 2-deoxy-glucose. This is further related to the increase in the expression of the glucose transporter GLUT1, which ensures sufficient fuel for glycolysis (FIG. 4A).

[0052] Another characteristic of XPA-deficient cells is that they widely inhibit RNA neosynthesis after UVB irradiation because the accurate reading of information is hindered by unrepaired DNA damage. Surprisingly, after administration of the composition for use in the present invention (e.g., choline / acetic acid salt; choline / DMKG; choline / butyric acid salt), an increase in the RNA concentration of respiratory chain-related genes was observed in XPA-deficient fibroblasts with UVB irradiation, along with phenomena affecting genes encoded by mitochondrial DNA and genes encoded by nuclear DNA (FIGS. 3; 9; 10). This increase in RNA is also reflected in the increase in the protein levels of proteins involved in the mitochondrial electron transport chain, the tricarboxylic acid cycle, and glycolysis.

[0053] At the histone level, an increase in trimethylation was also observed in H3K4, a marker indicating epigenetic modification related to active gene expression. Treatment also increased the trimethylation of H3K9 (FIG. 4B).

[0054] All of these effects were only observed in the combination of the two active substances of the composition for use in the present invention, and were not observed when treating cells with only a methyl group donor (e.g., choline) or only an acetyl-CoA donor (e.g., acetate).

[0055] In summary, these data show that administration of the compositions for use in the present invention, such as choline / acetic acid salts, etc., can (i) significantly improve or normalize the mitochondrial phenotype of XPA fibroblasts, (ii) improve epigenetic changes, (iii) at least partially overcome transcriptional repression by stimulating gene expression of genes involved in energy metabolism or enhancing the stability of respective mRNAs. Further, (iv) the abundance of proteins involved in specific energy metabolism increases by enhancing protein stability or improving translation efficiency by choline / acetic acid salt treatment. The loss of ATP is considered to be an important factor that causes XPA-deficient fibroblasts to undergo apoptosis after UVB exposure, and (v) administration of the compositions for use in the present invention, such as choline / acetic acid salts, etc., stabilizes energy metabolism and promotes an increase in ATP production, thereby protecting cells.

[0056] Surprisingly, it has been found that the therapeutic approach outlined above is also applicable to the treatment and prevention of other diseases, particularly those associated with mitochondrial dysfunction within the respiratory chain. This is because, similar to XPA, such diseases may also be causally related to deficiencies in ATP and other high-energy metabolites. After administration of the compositions for use in the present invention (e.g., choline / acetic acid salt or choline / triheptanoin), a synergistic increase in protein levels after UVB irradiation was observed in primary CSB-deficient human fibroblasts derived from Cockayne syndrome group B patients (Figure 5), and an increase in the expression of mitochondrial-related genes was also observed in primary CSB-deficient human fibroblasts (Figure 6; Figure 12; Figure 13). Furthermore, treatment with the composition of the present invention (e.g., choline / acetic acid salt) resulted in an increase in ATP production in primary human CSB-deficient fibroblasts (Figure 11A and Figure 11B).

[0057] In addition, treatment with the composition of the present invention (e.g., a combination of Compound A and Compound B, particularly choline and acetic acid salt) resulted in an increase in ATP production, particularly in the cell nucleus, in primary normal human skin fibroblasts from elderly donors (Figure 14).

[0058] In one embodiment of the present invention, the disease associated with mitochondrial dysfunction is nucleotide excision repair deficiency, ataxia telangiectasia due to DNA repair deficiency associated with mitochondrial dysfunction, mitochondrial disease, or an aging-related disease. Preferably, the nucleotide excision repair deficiency is various forms of xeroderma pigmentosum (XP) such as XP-A, XP-B, XP-C, XP-D, XP-E, XP-F, XP-G, XP-V, Cockayne syndrome (CS) groups B and A, and trichothiodystrophy (TTD). Preferably, the mitochondrial disease is MELAS (myopathy, encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy with ragged red fibers) syndrome, CPEO (chronic progressive external ophthalmoplegia), KSS (Kearns-Sayre syndrome), LS (Leigh encephalopathy), and LHON (Leber hereditary optic neuropathy). Preferably, the aging-related disease is endogenous skin aging, exogenous skin aging, and juvenile skin aging.

[0059] In a preferred embodiment, the disease associated with mitochondrial dysfunction is ataxia telangiectasia due to nucleotide excision repair deficiency or DNA repair deficiency associated with mitochondrial dysfunction.

[0060] Preferably, the nucleotide excision repair deficiency is various forms of xeroderma pigmentosum such as XP-A, XP-B, XP-C, XP-D, XP-E, XP-F, XP-G, XP-V, Cockayne syndrome group B, Cockayne syndrome group A, or trichothiodystrophy (TTD).

[0061] In another preferred embodiment of the present invention, the nucleotide excision repair deficiency is XP-A or Cockayne syndrome group B.

[0062] DNA repair deficiency is a progeria and shares many characteristics with normal aging, but those characteristics are more prominent and progress faster. This is particularly evident, for example, in skin aging. In fact, skin fibroblasts isolated from healthy elderly individuals exhibit mitochondrial dysfunction. Thus, in another preferred embodiment, the disease associated with the mitochondrial dysfunction is an aging-related disease, particularly endogenous skin aging, exogenous skin aging, or juvenile skin aging. Endogenous skin aging is mainly caused by internal factors only. This is sometimes referred to as chronological aging and is an inherent degenerative process due to the decline of physiological functions and capabilities. Exogenous skin aging is a characteristic degenerative process particularly caused by external factors such as ultraviolet radiation, smoking, and air pollution. Such an aging process includes qualitative and quantitative changes and may include a decrease or deficiency in the synthesis of collagen and elastin in the dermis. The characteristic of skin aging is a progressive decline in mitochondrial function. In particular, in the skin of elderly donors, it has been found that the markers of mitochondrial energy metabolism are decreased. Specifically, fibroblasts in aged skin usually show a decrease in ATP production ability. Since many cellular processes deeply involved in maintaining tissue homeostasis, including DNA repair, transcription, replication, and translation, require a large amount of ATP, the loss of mitochondrial function and ATP production is thought to be involved in the expression of aging phenotypes in the skin and other organs. Therefore, it is considered very important to identify compounds that can improve mitochondrial function and ATP production in order to slow down or alleviate the aging process. Surprisingly, the application of the composition for use in the present invention (for example, choline / acetate, or choline / triheptanoin) restored ATP production in aged skin fibroblasts and a reversal of their aging phenotype was observed (Figure 14). In particular, the composition for use may be applied topically for the topical treatment of aging-related diseases such as endogenous skin aging, exogenous skin aging, or juvenile skin aging, for example, as a component of sunscreen products or anti-aging cosmetics.

[0063] Another preferred embodiment is the cosmetic use of the above composition comprising a first compound A which is a methyl group donor and a second compound B which is an acetyl-CoA donor for the treatment of endogenous skin aging, exogenous skin aging or juvenile skin aging, in particular endogenous skin aging or exogenous skin aging. The above composition may be applied topically, for example, as a component of a sunscreen product or an anti-aging cosmetic. The above composition may contain suitable additives known to those skilled in the art such as solvents. For topical application, antioxidants, DNA repair enzymes, vitamins, UV filters, prebiotics or probiotic substances may be included.

[0064] In another preferred embodiment, the diseases associated with the above mitochondrial dysfunction are mitochondrial disease MELAS (myopathy, encephalopathy, lactic acidosis, stroke-like episodes), MERRF (red ragged fiber myoclonic epilepsy) syndrome, CPEO (chronic progressive external ophthalmoplegia), KSS (Kearns-Sayre syndrome), LHON (Leber hereditary optic neuropathy) or LS (Leigh encephalopathy).

[0065] Therefore, the present invention discloses the following items. 1. A composition comprising a first compound A which is a methyl group donor and a second compound B which is an acetyl-CoA donor, for use in the treatment and prevention of diseases associated with mitochondrial dysfunction, particularly xeroderma pigmentosum A. 2. The composition for use according to item 1, wherein the first compound A is selected from the group consisting of compounds comprising a trimethylamine group, sarcosine, trimethylamine-N-oxide, serine and mixtures of two or more thereof, particularly selected from choline and betaine. 3. The composition for use according to item 1 or item 2, wherein the second compound B is selected from fatty acids and fatty acid salts, particularly from acetate and butyrate, or from pyruvate dehydrogenase kinase inhibitors. 4. The molar ratio of the above compound A to the above compound B is from 10:1 to 1:10, particularly from 8:1 to 1:8, preferably from 5:1 to 1:5, and particularly preferably from 2:1 to 1:2 such as 1:1, for the use according to any one of items 1 to 3. 5. The composition according to any one of items 1 to 4 for the use, wherein the composition is administered orally, intravenously or topically, particularly as a compound in a sunburn prevention product or topically administered alone. 6. The composition according to any one of items 1 to 5 for the use, wherein the concentration of the above compound A is in the range of 5 to 250 mM and the concentration of the above compound B is in the range of 1 to 100 mM. 7. The composition according to any one of items 1 to 6 for the use, wherein the disease associated with the above mitochondrial dysfunction is ataxia telangiectasia due to nucleotide excision repair deficiency or DNA repair deficiency associated with mitochondrial dysfunction. 8. The composition for the use according to item 7, wherein the nucleotide excision repair deficiency is xeroderma pigmentosum A, B, C, D, E, F, G, V, Cockayne syndrome group B, Cockayne syndrome group A or trichothiodystrophy (TTD). 9. The composition according to any one of items 1 to 6 for the use, wherein the disease associated with the above mitochondrial dysfunction is mitochondrial disease MELAS (myopathy, encephalopathy, lactic acidosis, stroke-like episodes), MERRF (red ragged fiber - myoclonic epilepsy) syndrome, CPEO (chronic progressive external ophthalmoplegia), KSS (Kearns - Sayre syndrome), LHON (Leber hereditary optic neuropathy) or LS (Leigh encephalopathy). 10. The composition according to any one of items 1 to 6 for the use, wherein the disease associated with the above mitochondrial dysfunction is an aging - related disease, particularly endogenous skin aging, exogenous skin aging or juvenile skin aging.

[0066] Hereinafter, the present invention will be disclosed by way of examples, but is not limited thereto.

Examples

[0067] [Basic Procedure] <Cell Culture and Treatment> Primary human fibroblasts obtained from patients with xeroderma pigmentosum group A (XPA) and Cockayne syndrome group B (CSB) were purchased from the Coriell Institute (USA). Normal human fibroblasts (NHF) were isolated from skin samples obtained from healthy age-matched donors who underwent dermatologic surgery. The cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% glutamine, antibiotics·antifungal agents (growth medium), and 15% fetal bovine serum (FCS) at 37 °C in a humidified atmosphere containing 5% CO2. For experiments, the cells were detached from the culture flask by treatment with a 0.05% trypsin solution (5 min / 37 °C). After blocking trypsin activity by adding growth medium containing 10% FCS, the cells were sedimented, resuspended in growth medium (15% FCS), and plated in appropriate culture dishes. The cells were grown until they reached a confluence of approximately 80%. Thereafter, the medium was removed, the cells were washed with phosphate-buffered saline (PBS), and growth medium containing 1% FCS was provided and the cells were grown for 24 hours.

[0068] The next day, the cells were treated with choline chloride (referred to as choline here), sodium acetate (referred to as acetate here), or both (both compounds were purchased from Sigma-Aldrich) as compounds. For treatment, an appropriate amount of the above compounds was dissolved in growth medium (1% FCS) sterile-filtered through a membrane with a pore size of 0.22 μm. Control growth medium (1% FCS) without the addition of compounds was also sterile-filtered in the same manner. The old growth medium was removed from the cells and replaced with the filtered control growth medium or growth medium containing the compounds. The cells were grown for an additional 24 hours before UVB irradiation.

[0069] Irradiation was performed using a self-constructed device equipped with a TL20W / 12RS SLV bulb (Philips) that emits UVB rays (radiation range: 290 - 320 nm, peak: 302 nm). The irradiation dose in all experiments was 20 mJ / cm 2It was. The output of the device was periodically tested with an ultraviolet intensity meter (Waldmann), and the required irradiation time was calculated. For irradiation, the growth medium was removed and the cells were washed with PBS. Before placing the cells in the irradiation device, fresh PBS was given to the cells and they were exposed to UVB for an appropriate time to reach the desired dose. Unirradiated control cells (referred to as Sham here) were treated in the same manner except that they were not subjected to UVB irradiation. After irradiation, the cells were removed from the UVB device, the PBS was aspirated, and fresh growth medium containing each compound or fresh control growth medium (the same type of medium as that given before irradiation) was given to all the cells. The cells were cultured for 24 hours before being used for various analyses described in later examples.

[0070] [Example 1] [Apoptosis measurement] NHF and XPA-deficient fibroblasts were grown in a culture dish in a growth medium containing 15% FCS. Forty-eight hours before UVB irradiation, the medium was removed, the cells were washed with PBS, and a growth medium containing 1% FCS was given. After 24 hours, the medium was removed, and a growth medium (1% FCS) containing different concentrations of choline (25, 50, 75, or 100 mM) and a constant concentration of acetate (10 mM) was given to the cells. The control cells were given a normal growth medium supplied with 1% FCS. After 24 hours, the cells were either exposed to UVB or not irradiated (Sham). After irradiation, the PBS was removed, and each growth medium containing choline / acetate or the control growth medium was given to the cells, and they were cultured for another 24 hours. For treatment with the DNA-damaging agent cisplatin, the medium was removed, and the cells were treated with a medium containing cisplatin (50 μM) (Medchem Express) dissolved in each growth medium supplied with choline / acetate or the control growth medium, and they were cultured for another 24 hours. Apoptosis measurement was performed according to the manufacturer's procedure using a kit based on flow cytometry (Biolegend). Briefly, the medium was removed, the cells were washed with PBS, and they were separated from the culture dish using trypsin by culturing at 37 °C for 5 minutes. The trypsin was blocked by adding PBS containing 10% FCS. Each cell suspension was pooled with the respective removed medium and PBS to also recover the previously separated cells. The cells were sedimented and resuspended in annexin V binding buffer containing annexin V conjugated to the fluorescent dye APC and propidium iodide. The cells were analyzed using a FACSCanto flow cytometry system. The percentage of early apoptotic cells defined as annexin V(+) and propidium iodide(−) was determined.

[0071] The results of apoptosis measurement are shown in Figure 1. In Figure 1A, the cells are either with UVB irradiation (UVB) or without irradiation (Sham). In Figure 1B, the cells are either treated with the DNA-damaging compound cisplatin (Cisplatin) or untreated (Control). Figure 1 shows that primary XPA-deficient human fibroblasts are protected from apoptosis induced by UVB and cisplatin by co-treatment with choline and acetate.

[0072] [Example 2] <Measurement of ATP production rate and oxygen consumption rate> Primary NHF and XPA-deficient fibroblasts were grown in growth medium containing 1% FCS in a dedicated culture chamber designed for the Seahorse XFp Analyzer (Agilent) for 24 hours before UVB irradiation, the medium was removed, and growth medium (1% FCS) containing choline (100 mM) and acetate (10 mM) was given to the cells. Control cells were given normal growth medium supplemented with 1% FCS. After 24 hours, the medium was removed, the cells were washed with PBS, and irradiated with UVB in fresh PBS. Unirradiated control cells (Sham) grown in a second chamber were treated in basically the same way except that they were not exposed to UVB. After irradiation, PBS was removed, and the cells were treated with choline / acetate or control medium. After 24 hours, the mitochondrial and glycolytic ATP production rates were measured using the Seahorse ATP Real-Time Rate Assay Kit (Agilent) according to the manufacturer's procedure.

[0073] The basal oxygen consumption rate was measured using the Seahorse Mitostress Kit (Agilent) according to the manufacturer's procedure. To normalize the results, the cells were fixed with 2% paraformaldehyde and stained with the nuclear fluorescent dye DAPI after the analysis was completed. The cells were counted using a fluorescence microscope (Zeiss), and the results of the analysis were normalized according to the number of cell counts in each well.

[0074] The results are shown in Figures 2A and 2C. Figure 2A shows the measurement results of the ATP production rates of glycolysis (glyco) and mitochondria (mito) using the Seahorse technology. Figure 2C shows the measurement results of the basal oxygen consumption rate (OCR) of NHF and XPA fibroblasts obtained using the Seahorse technology.

[0075] <Immunofluorescence analysis> Primary NHF and XPA-deficient fibroblasts were grown on chamber slides (Sarstedt) in growth medium containing 15% FCS. Forty-eight hours before UVB irradiation, the medium was removed, the cells were washed with PBS, and growth medium containing 1% FCS was given. After 24 hours, the medium was removed, and growth medium (1% FCS) containing choline (100 mM) and acetate (10 mM) was given to the cells. Control cells were given normal growth medium supplied with 1% FCS. After 24 hours, the medium was removed, the cells were washed with PBS, and irradiated with UVB in fresh PBS. Unirradiated control cells (Sham) grown in a second chamber were treated basically in the same way except that they were not exposed to UVB. After irradiation, PBS was removed, and the cells were treated with choline / acetate or control growth medium. After 24 hours, the medium was removed, the cells were washed with PBS, and fixed with 2% paraformaldehyde. For immunofluorescent staining of ATP and the ATP5A subunit of mitochondrial ATP synthase, the cells were permeabilized with PBS containing 0.1% Triton X-100 and cultured overnight at 4°C using an anti-ATP antibody generated in rabbits (MyBioSource) and an anti-ATP5A antibody generated in mice (Abcam). The next day, the antibody was removed, and the cells were washed three times with PBS containing 0.1% Triton X-100. The cells were cultured at room temperature for 2 hours using anti-mouse and anti-rabbit secondary antibodies labeled with either a green (Alexa Fluor488, Thermo Fisher) or red fluorescent dye (Alexa Fluor594, Thermo Fisher). The antibody was removed, and the cells were washed three times with PBS containing 0.1% Triton X-100. The slides were mounted using anti-fading mounting medium (Southern Biotech) and analyzed with a fluorescence microscope (Zeiss).

[0076] The results are shown in Fig. 2B. ATP concentration was visualized by staining cells with an ATP-specific antibody (middle panel) and an antibody against the ATP5A subunit of mitochondrial ATP synthase (upper panel) and performing fluorescence microscopy analysis. The staining shows the accumulation of ATP in the cell nuclei during treatment with Chol / Ac.

[0077] Fig. 2 shows that co-treatment with choline and acetate increases ATP production in primary human XPA-deficient and normal fibroblasts.

[0078] Similar to the description of Fig. 2B, primary normal human skin fibroblasts (NHF) and XPA-deficient fibroblasts were further grown on chamber slides (Sarstedt) in growth medium containing 15% FCS. The medium was removed, and the cells were given growth medium (1% FCS) containing either choline (100 mM), acetate (10 mM), or a combination of choline (100 mM) and acetate (10 mM) (Chol / Ac). Control cells were given normal growth medium supplemented with 1% FCS. After 24 hours, fresh medium containing each compound was given to the cells, and they were cultured for an additional 24 hours. The cells were washed with PBS and fixed with 2% paraformaldehyde. For immunofluorescence staining of ATP and the ATP5A subunit of mitochondrial ATP synthase, the cells were permeabilized with PBS containing 0.1% Triton X-100 and cultured overnight at 4 °C using an anti-ATP antibody raised in rabbits (MyBioSource) and an anti-ATP5A antibody raised in mice (Abcam). The next day, the antibodies were removed, and the cells were washed three times with PBS containing 0.1% Triton X-100. The cells were cultured for 2 hours at room temperature using anti-mouse and anti-rabbit secondary antibodies labeled with either a green (Alexa Fluor488, Thermo Fisher) or a red fluorescent dye (Alexa Fluor594, Thermo Fisher). The antibodies were removed, and the cells were washed three times with PBS containing 0.1% Triton X-100. The slides were mounted using a mounting medium for anti-fade (Southern Biotech) and analyzed with a fluorescence microscope (Zeiss).

[0079] The results are shown in Fig. 7. ATP concentration was visualized by staining cells with an ATP-specific antibody (middle panel; Figs. 7A and 7B) and an antibody against the ATP5A subunit of mitochondrial ATP synthase (upper panel; Figs. 7A and 7B), followed by fluorescence microscopy analysis. The staining showed the accumulation of ATP in the cell nuclei during treatment with choline, which could be further increased by adding acetate in both NHF (Fig. 7A) and XPA-deficient fibroblasts (Fig. 7B). Acetate alone had no effect on the intracellular ATP content. The combination of choline and acetate (Chol / Ac) synergistically increased the intracellular ATP content.

[0080] <Example 2.1 - Fluorescence Microscopic Analysis of CSB-Deficient Fibroblasts> Similar to the description of Figure 2B, primary NHF and CSB-deficient fibroblasts were grown on chamber slides (Sarstedt) in growth medium containing 15% FCS. Forty-eight hours before UVB irradiation, the medium was removed, the cells were washed with PBS, and growth medium containing 1% FCS was given. After 24 hours, the medium was removed, and the cells were given growth medium (1% FCS) containing either choline (100 mM), acetate (10 mM), or a combination of choline (100 mM) and acetate (10 mM) (Chol / Ac). Control cells were given normal growth medium supplemented with 1% FCS. After 24 hours, the medium was removed, the cells were washed with PBS, and irradiated with UVB in fresh PBS (Figure 11B). Unirradiated control cells (Sham) grown in a second chamber (Figure 11A) were treated basically the same way except that they were not exposed to UVB. After irradiation, the PBS was removed, and the cells were treated again with each compound or control growth medium. After 24 hours, the medium was removed, the cells were washed with PBS, and fixed with 2% paraformaldehyde. For immunofluorescent staining of ATP and the ATP5A subunit of mitochondrial ATP synthase, the cells were permeabilized with PBS containing 0.1% Triton X-100 and incubated overnight at 4 °C with anti-ATP antibody generated in rabbit (MyBioSource) and antibody against mitochondrial marker TOM20 (generated in mouse, Santa Cruz, USA). The next day, the antibody was removed, and the cells were washed three times with PBS containing 0.1% Triton X-100. The cells were incubated for 2 hours at room temperature with anti-mouse and anti-rabbit secondary antibodies labeled with either green (Alexa Fluor488, Thermo Fisher) or red fluorescent dye (Alexa Fluor594, Thermo Fisher). The antibody was removed, and the cells were washed three times with PBS containing 0.1% Triton X-100. The slides were mounted with anti-fade mounting medium (Southern Biotech) and analyzed with a fluorescence microscope (Zeiss).

[0081] The results are shown in FIGS. 11A and 11B. Cells having an ATP-specific antibody (middle row in both the NHF and CSB experiments) and an antibody against the mitochondrial marker TOM20 (top row in both the NHF and CSB experiments) were stained, and fluorescence microscopy analysis was performed to visualize the ATP concentration. The staining shows the accumulation of ATP in the cell nucleus during treatment with choline, and can be further increased by adding acetate in both NHF and XPA-deficient fibroblasts. The combination of choline and acetate synergistically increases the intracellular ATP content.

[0082] <Example 2.2 - Fluorescence Microscopic Analysis of Primary Normal Human Skin Fibroblasts from Elderly Donors> Similar to the description of FIG. 2B, primary normal human dermal fibroblasts (NHF) obtained from a volunteer healthy subject (age: 52 years) were grown on a chamber slide (Sarstedt) in a growth medium containing 15% FCS. The medium was removed, and a growth medium (1% FCS) containing a combination of choline (100 mM) and acetate (10 mM) (Chol / Ac) was given to the cells. Control cells (Control) were given a normal growth medium supplied with 1% FCS. After 24 hours, fresh medium containing each compound or normal medium was given to the cells respectively, and the cells were cultured for another 24 hours. The cells were washed with PBS and fixed with 2% paraformaldehyde. For immunofluorescent staining of ATP and the ATP5A subunit of mitochondrial ATP synthase, the cells were permeabilized with PBS containing 0.1% Triton X-100 and cultured overnight at 4°C using an anti-ATP antibody generated in rabbits (MyBioSource) and an anti-ATP5A antibody generated in mice (Abcam). The next day, the antibodies were removed, and the cells were washed three times with PBS containing 0.1% Triton X-100. The cells were cultured for 2 hours at room temperature using anti-mouse and anti-rabbit secondary antibodies labeled with either a green (Alexa Fluor488, Thermo Fisher) or a red fluorescent dye (Alexa Fluor594, Thermo Fisher). The antibodies were removed, and the cells were washed three times with PBS containing 0.1% Triton X-100. The slides were mounted using an anti-fading mounting medium (Southern Biotech) and analyzed with a fluorescence microscope (Zeiss).

[0083] The results are shown in FIG. 14. By staining cells with an ATP-specific antibody and an antibody against the ATP5A subunit of mitochondrial ATP synthase and performing fluorescence microscopy analysis, the ATP concentration was visualized. The staining shows the accumulation of ATP, particularly in the cell nucleus, during treatment with Chol / Ac.

[0084] [Example 3] [RNA Isolation and Real-Time PCR] Primary NHF, XPA-deficient fibroblasts (Figure 3) or CSB-deficient fibroblasts (Figure 6) were grown in a culture dish in growth medium containing 15% FCS. Forty-eight hours before UVB irradiation, the medium was removed, the cells were washed with PBS, and growth medium containing 1% FCS was provided. After 24 hours, the medium was removed, and the cells were treated with choline (100 mM), acetate (10 mM), or choline (100 mM) and acetate (10 mM) (XPA-deficient fibroblasts; Figure 3), or a combination of different concentrations of choline (25, 50, 75, or 100 mM) with a constant concentration of acetate (10 mM) (CSB-deficient fibroblasts; Figure 6) in growth medium (1% FCS). Control cells were provided with normal growth medium supplemented with 1% FCS. After 24 hours, the medium was removed, the cells were washed with PBS, and irradiated with UVB in fresh PBS. After irradiation, the PBS was removed, and the cells were treated with choline, acetic acid, choline / acetate, or control growth medium. After 24 hours, the medium was removed, the cells were washed with PBS, and RNA was extracted using the Direct-zol RNA kit (Zymo Research) according to the manufacturer's protocol and stored at -80°C. 200 ng of purified RNA was used for cDNA synthesis using SuperScript reverse transcriptase (Thermo Fisher). The expression levels of the genes PKM1, PKM2, MT-ATP6, MT-ATP8, MT-ND1, MT-ND3, TFAM, SOD2, AKR1B1, HSPA2, and TFPI2 were analyzed using a CFX Connect real-time PCR analysis system and SYBR Green Supermix (both from Bio-Rad). The expression levels of these genes were normalized relative to the housekeeping gene 18S (for experiments using XPA-deficient fibroblasts) or β2-microglobulin (for experiments using CSB-deficient fibroblasts).

[0085] The results for primary XPA-deficient human fibroblasts are shown in Figure 3. Figure 3 shows that co-treatment with choline and acetate induces synergistic changes in gene expression in primary XPA-deficient human fibroblasts. The RNA concentrations of pyruvate kinase 1 (PKM1) [A] and pyruvate kinase 2 (PKM2) [B], which are ATP-producing glycolytic enzymes, ATP synthase subunits MT-ATP6 [C] and MT-ATP8 [D] encoded by mitochondrial DNA, mitochondrial antioxidant enzyme SOD2 [E], glucose-converting enzyme AKR1B1 [F], chaperone HSPA2 [G], and tumor suppressor gene TFPI2 [H] are shown. Acetate alone does not increase the RNA concentration after UVB, whereas when both compounds are given simultaneously, the choline-induced gene expression can be increased.

[0086] The results for primary CSB-deficient human fibroblasts are shown in Figure 6. Figure 6 shows that co-treatment with choline and acetate increases the expression of mitochondrial-related genes in primary CSB-deficient human fibroblasts (CS1AN). The RNA concentrations of genes MT-ATP6 [A], MT-ATP8 [B], MT-ND1 [C], MT-ND3 [D] encoded by mitochondrial DNA and mitochondrial transcription factor gene TFAM [E] encoded by nuclear DNA are shown.

[0087] <Example 3.1 - Changes in Gene Expression in Primary XPA-Deficient Human Fibroblasts> As described for Figure 3, primary NHF and XPA-deficient fibroblasts were grown in a growth medium containing 15% FCS in culture dishes. Forty-eight hours before UVB irradiation, the medium was removed, the cells were washed with PBS, and a growth medium containing 1% FCS was provided. After 24 hours, the medium was removed, and the cells were treated with either choline (100 mM), dimethyl α-ketoglutarate (DMKG) (10 mM) or a combination of choline (100 mM) and DMKG (10 mM) (Figure 9), or choline (100 mM), butyrate (5 mM) or a combination of choline (100 mM) and butyrate (5 mM) (Figure 10) in a growth medium (1% FCS). Control cells were provided with a normal growth medium supplied with 1% FCS. After 24 hours, the medium was removed, the cells were washed with PBS, and irradiated with UVB in fresh PBS. After irradiation, the PBS was removed, and the cells were treated again with either choline, DMKG or choline / DMKG, choline, butyrate or choline / butyrate, or the control growth medium. After 24 hours, the medium was removed, the cells were washed with PBS, and RNA was extracted using the Direct-zol RNA kit (Zymo Research) according to the manufacturer's protocol and stored at -80 °C. 200 ng of purified RNA was used for cDNA synthesis using SuperScript reverse transcriptase (Thermo Fisher). The expression levels of the selected genes were analyzed using a CFX Connect real-time PCR analysis system and SYBR Green Supermix (both from Bio-Rad). The expression levels of the selected genes were normalized relative to the housekeeping gene 18S.

[0088] The results of the experiments using choline and dimethyl α-ketoglutarate (DMKG) are shown in Figure 9. The expression of the selected genes was analyzed 24 hours after UVB irradiation. The RNA concentrations of mitochondrial heat shock proteins CRYAB [A], HSPA2 [B] and the enzyme ISYNA1 [C] involved in inositol metabolism are shown. DMKG alone has little (if any) effect on most RNA concentrations, whereas when both compounds are provided simultaneously, the choline-induced gene expression of CRYAB, HSPA2 and ISYNA1 can be increased synergistically.

[0089] The results of the experiment using choline and butyrate are shown in Fig. 10. The expression of the selected gene was analyzed 24 hours after UVB irradiation. The RNA concentration of glucose-converting enzyme AKR1B1 is shown. Butyrate alone has little effect on the RNA concentration of AKR1B1, while when given simultaneously with choline, it can synergistically increase gene expression.

[0090] <Example 3.2 - Changes in Gene Expression in Primary CSB-Deficient Human Fibroblasts> Similar to the description of Fig. 6, primary NHF or CSB-deficient fibroblasts were grown in a growth medium containing 15% FCS in a culture dish. 48 hours before UVB irradiation, the medium was removed, the cells were washed with PBS, and a growth medium containing 1% FCS was given. After 24 hours, the medium was removed, and the cells were given a growth medium (1% FCS) containing either choline (100 mM), acetate (10 mM), or a combination of choline (100 mM) and acetate (10 mM) (Fig. 12), or choline (Chol) (100 mM), triheptanoin (Trihep) (100 μM), or a combination of choline (100 mM) and Trihep (100 μM) (Chol / Trihep) (Fig. 13). The control cells were given a normal growth medium supplied with 1% FCS. After 24 hours, the medium was removed, the cells were washed with PBS, and irradiated with UVB in fresh PBS. After irradiation, the cells were treated with either choline, acetate, or choline / acetate, choline, triheptanoin, or choline / triheptanoin, or the control growth medium.

[0091] In the case of treatment with choline and acetate, the expression of the selected gene was analyzed 48 hours after UVB irradiation. In the case of treatment with choline and triheptanoin, the expression of the selected gene was analyzed 24 hours after UVB irradiation. In both cases, the medium was removed, the cells were washed with PBS, and RNA was extracted using the Direct-zol RNA kit (Zymo Research) according to the manufacturer's procedure and stored at -80 °C. 200 ng of purified RNA was used for cDNA synthesis using SuperScript reverse transcriptase (Thermo Fisher). The expression levels of the selected genes were analyzed using a CFX Connect real-time PCR analysis system and SYBR Green Supermix (both from Bio-Rad). The expression levels of the selected genes were normalized relative to the housekeeping gene 18S β2-microglobulin.

[0092] The results of the experiment using choline and acetate are shown in Fig. 12. The expression of the selected gene was analyzed 48 hours after UVB irradiation. The RNA concentrations of mitochondrial antioxidant enzyme SOD2 [A] and mitochondrial heat shock protein HSPA2 [B] are shown. Acetate has little or no effect on the RNA concentrations of the genes shown alone, whereas when given together with choline, its expression can be synergistically increased.

[0093] The results of the experiment using choline (Chol) and triheptanoin (Trihep) are shown in Fig. 13. The expression of the selected gene was analyzed 24 hours after UVB irradiation. The RNA concentrations of glucose-converting enzyme AKR1B1 [A] and mitochondrial antioxidant enzyme SOD2 [B] are shown. Triheptanoin has little or no positive effect on the RNA concentrations of AKR1B1 and SOD2 (after UVB irradiation) alone, whereas when given together with choline, the expression of these genes can be synergistically increased.

[0094] [Example 4] <Protein Extraction and Western Blot Analysis> Primary NHF, XPA-deficient fibroblasts (Figure 4) and CSB-deficient fibroblasts (Figure 5) were grown in a culture dish in growth medium containing 15% FCS. Forty-eight hours before UVB irradiation, the medium was removed, the cells were washed with phosphate-buffered saline (PBS), and growth medium containing 1% FCS was given. After 24 hours, the medium was removed, and growth medium (1% FCS) containing either choline (100 mM), acetate (10 mM), or choline (100 mM) and acetate (10 mM) was given to the cells. Control cells were given normal growth medium supplied with 1% FCS. After 24 hours, the medium was removed, the cells were washed with PBS, and irradiated with UVB in fresh PBS. After irradiation, the PBS was removed, and the cells were treated with either choline, acetate, choline / acetate, or control growth medium. After 24 hours, the medium was removed, the cells were washed with ice-cold PBS, and covered with fresh ice-cold PBS. The cells were detached from the culture dish using a cell lifter, transferred to an Eppendorf tube, sedimented, and resuspended in ice-cold RIPA lysis protein extraction buffer supplied with protease and phosphatase inhibitors. The samples were held on ice for 20 minutes and then sedimented at 14,000 rpm and 4 °C for 15 minutes. The supernatant containing solubilized protein was recovered, snap-frozen in liquid nitrogen, and stored at -80 °C. The pellet containing RIPA-insoluble protein including histone was also stored at -80 °C. Extraction of histone was performed using the EpiQuik Total Histone Extraction kit (Epigentek). The pellet was dissolved in ice-cold lysis buffer and placed on ice for 30 minutes. The samples were sedimented, the supernatant was recovered, and neutralized with balance buffer supplied with DTT. The histone samples were stored at -80 °C. For Western blot detection, protein aliquots were separated by SDS polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane using the Trans-Blot Turbo transfer system (Bio-Rad). The membrane sections were incubated overnight at 4 °C on a shaker using the indicated antibodies. After washing, the membrane sections were incubated for 2 hours at room temperature using an appropriate secondary antibody conjugated with horseradish peroxidase. After washing, the membrane sections were incubated using an ECL Western blotting substrate, and protein bands were detected using an Odyssey XF imaging system (LI-COR).

[0095] Results for primary XPA-deficient human fibroblasts are shown in Figure 4. Figure 4A shows the results of Western blot analysis of cell-soluble proteins. Figure 4B shows the results of Western blot analysis of DNA-related histones. Figure 4 shows that co-treatment with choline and acetate induces synergistic changes in protein abundance, intracellular signaling, and epigenetic markers in primary XPA-deficient human fibroblasts. MT-ND1 and AKR1B1 show synergistic accumulation with or without UVB irradiation, whereas MT-ATP8 accumulates particularly after UVB irradiation, and ErbB2 accumulates particularly without UVB irradiation. GLUT1 levels increase similarly with choline alone or choline + acetate treatment. AMPK, which functions as an energy sensor that stabilizes and phosphorylates in response to the energy requirements of cells, shows a synergistic downregulation of total protein levels and a decrease in phosphorylation particularly after UVB, suggesting a high abundance of energy equivalents with choline + acetate treatment.

[0096] Results for primary CSB-deficient human fibroblasts are shown in Figure 5. Figure 5 shows that co-treatment with choline and acetate induces synergistic changes in protein abundance in primary CSB-deficient human fibroblasts. The synergistic increase in protein levels with choline + acetate treatment is particularly prominent after UVB irradiation.

[0097] Similar to the description of Figure 4, primary NHF and XPA-deficient fibroblasts were grown in a growth medium containing 15% FCS in a culture dish. After reaching confluence, XPA-deficient fibroblasts were treated with a growth medium (1% FCS) containing either choline (100 mM), dimethyl α-ketoglutarate (10 mM), or a combination of choline (100 mM) and dimethyl α-ketoglutarate (10 mM). Control cells were given a normal growth medium supplemented with 1% FCS. After 24 hours, the medium was removed, and the cells were treated with either choline, dimethyl α-ketoglutarate, choline / dimethyl α-ketoglutarate, or the control growth medium. At 24 hours of culture, the medium was removed, the cells were washed with ice-cold PBS, and covered with fresh ice-cold PBS. The cells were detached from the culture dish using a cell lifter, transferred to an Eppendorf tube, pelleted, and resuspended in ice-cold RIPA lysis protein extraction buffer supplemented with protease and phosphatase inhibitors. The samples were held on ice for 20 minutes and then pelleted at 14,000 rpm and 4 °C for 15 minutes. The supernatant containing the solubilized protein was collected, snap-frozen in liquid nitrogen, and stored at -80 °C. For Western blot detection, protein aliquots were separated by SDS polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane using a Trans-Blot Turbo transfer system (Bio-Rad). The membrane strips were incubated overnight at 4 °C on a shaker using the indicated antibodies. After washing, the membrane strips were incubated for 2 hours at room temperature using an appropriate secondary antibody conjugated to horseradish peroxidase. After washing, the membrane strips were incubated with an ECL Western blotting substrate, and protein bands were detected using an Odyssey XF imaging system (LI-COR).

[0098] The results are shown in Figure 8. During treatment with both compounds, choline and dimethyl α-ketoglutarate, the mitochondrial proteins CRYAB, SOD2, IF1, MT-ATP8, IDH2, and to a lesser extent TFAM, showed synergistic accumulation.

Claims

1. A composition comprising a first compound A which is a methyl group donor and a second compound B which is an acetyl CoA donor, for use in the treatment and prevention of diseases associated with mitochondrial dysfunction, wherein the disease associated with mitochondrial dysfunction is particularly a nucleotide excision repair deficiency such as xeroderma pigmentosum A, or ataxia telangiectasia due to DNA repair deficiency associated with mitochondrial dysfunction.

2. The composition for use according to claim 1, wherein the first compound A is selected from the group consisting of compounds containing a trimethylamine group, sarcosine, trimethylamine-N-oxide, serine and mixtures of two or more thereof, preferably selected from trimethylammonium compounds and mixtures of two or more thereof, particularly selected from choline and betaine.

3. The composition for use according to claim 1 or claim 2, wherein the second compound B is selected from fatty acids, fatty acid derivatives, fatty acid salts, fatty acid derivative salts, triglycerides, triglyceride derivatives and dicarboxylic acid derivatives, particularly selected from acetate, butyrate, triheptanoin and dimethyl α-ketoglutarate, or from pyruvate dehydrogenase kinase inhibitors.

4. The composition for use according to any one of claims 1 to 3, wherein the molar ratio of the compound A to the compound B is from 10:1 to 1:10, particularly from 8:1 to 1:8, preferably from 5:1 to 1:5, and particularly preferably from 2:1 to 1:2 such as 1:

1.

5. The composition for use according to any one of claims 1 to 4, wherein the composition is administered orally, intravenously or topically, particularly as a compound in a sunscreen product or topically alone.

6. The composition for use according to any one of claims 1 to 5, wherein the concentration of the compound A is in the range of 5 to 250 mM and the concentration of the compound B is in the range of 1 to 100 mM.

7. The composition for use according to any one of claims 1 to 6, wherein the nucleotide excision repair deficiency is xeroderma pigmentosum A, B, C, D, E, F, G, V, Cockayne syndrome group B, Cockayne syndrome group A or trichothiodystrophy (TTD).

8. The composition for use according to any one of claims 1 to 7, wherein the nucleotide excision repair deficiency is xeroderma pigmentosum group A or Cockayne syndrome group B.

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