Treating mitochondrial DNA deletion disorder

Abstract

The present disclosure describes methods for treating mitochondrial DNA deletion syndrome by administering a therapeutic amount of a composition comprising a dinucleotide compound or a mixture thereof. Also described herein are compounds, compositions and methods for treating TK2 deficiency.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 896,218, filed September 5, 2019, the disclosure of which is incorporated by reference in its entirety. Field of the Invention

[0003] The present disclosure describes methods for treating mitochondrial DNA deletion syndrome (MDS) by administering a therapeutic amount of a composition comprising a polynucleotide compound or a mixture thereof. Also described herein are compounds, compositions and methods for treating thymidine kinase 2 (TK2) deficiency.

[0004] background

[0005] Mitochondria are known as the energy-generating organelles of cells. These double-membrane organelles function independently of the cells in which they are located, and even have their own genome, which is separate from the nuclear genome. Although the nuclear genome encodes some mitochondrial proteins, the mitochondrial genome specifically encodes proteins related to energy-related metabolic processes (including the production of the electron transport chain and ATP). Mitochondrial DNA deletion syndrome (MDS) is a general term used to describe a wide range of disorders characterized by a high reduction in mitochondrial DNA in cells. MDS is the result of a nuclear gene mutation involving nucleotide synthesis or replication of mitochondrial DNA, and is associated with a variety of genes, including TK2, SUCLA2, SUCLG1, POLG, DGUOK, MPV17, TYMP and RRM2B. (Chansprasert et al., 2017). Therefore, these autosomal recessive disorders lead to extreme loss of mitochondrial DNA (mtDNA), which in turn has a negative impact on energy production. (El-Hattab et al., 2013).

[0006] Symptoms appear in infancy or early childhood and primarily affect tissues of muscle, liver, or brain, and typically manifest as muscle weakness, organ and nerve dysfunction, and weight loss. However, these disorders are phenotypically heterogeneous. For example, mutations in SUCLA2, SUCLG1, or RRM2B lead to encephalomyopathy MDS, with clinical manifestations of hypotonia and neurological problems. Mutations in DGUOK, MPV17, or POLG manifest as early-onset liver dysfunction. (El-Hattab et al., 2013). Mutations in TYMP are associated with progressive gastrointestinal dysmotility and peripheral neuropathy. (El-Hattab et al., 2013). The severity and progression of these disorders are also highly variable, ranging from mild manifestations to severe symptoms leading to death in infancy and childhood. Due to this heterogeneity in clinical manifestations, treatment options are limited to managing symptoms primarily through nutritional supplementation.

[0007] Thymidine kinase 2 (TK2) deficiency is a specific type of MDS. The enzyme thymidine kinase 2 is a nuclear-encoded enzyme involved in mitochondrial DNA (mtDNA) synthesis that participates in the recycling of nucleotides. (Genetics Home Reference, 2013). Mutations in the TK2 gene reduce enzyme activity and impair mtDNA nucleotide recycling, resulting in low levels of deoxythymidine monophosphate and deoxycytidine monophosphate. This shortage in the nucleotide pool affects mtDNA synthesis and ultimately leads to a progressive myopathy that may begin in early childhood, ultimately resulting in loss of motor skills. (Garone et al., 2018).

[0008] Currently, there are no disease-modifying therapies for TK2 disease and treatment is primarily supportive. Although oral supplementation with deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP) has been shown to restore mtDNA loss and improve overall survival in animal models, such treatments require high doses of each deoxyribonucleoside monophosphate (dNMP) to achieve overall therapeutic benefit and are associated with significant side effects.

[0009] Overview

[0010] One embodiment described herein is a method of treating mitochondrial DNA deletion syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polynucleotide composition comprising a compound of Formula I

[0011]

[0012] in:

[0013] R1 is H or OH;

[0014] R2 is a purine derivative or a pyrimidine derivative;

[0015] R3 is a purine derivative or a pyrimidine derivative; and

[0016] R4 is H or OH.

[0017] In one aspect, the composition comprises a compound of formula I, wherein R1 is H, R2 is 5-methyl-2H-1λ2-pyrimidine-2,4(3H)-dione, R3 is 4-amino-2H-1λ2-pyrimidine-2-one, and R4 is OH. In another aspect, the composition is a mixture further comprising a compound of formula I wherein R1 is OH, R2 is 9λ2-purine-6-amine, R3 is 2-amino-9λ2-purine-6(1H)-one, and R4 is OH.

[0018] In one aspect, the composition is administered orally, enterally, intravenously or subcutaneously. In another aspect, the composition is administered intravenously.

[0019] In one aspect, the plasma level of each mononucleoside is higher following intravenous administration compared to the plasma level following oral administration of the individual mononucleotides.

[0020] In another aspect, intravenous administration of the composition results in a first AUC 0-24h and oral administration of a single mononucleotide resulted in a plasma level of 0-24h The plasma level of 0-24h With the second AUC 0-24h The ratio is about 100 to about 400.

[0021] In yet another aspect, when the composition is administered at a dose of about 1 mg / kg to about 20 mg / kg, the plasma concentration of the corresponding nucleoside is about 50 ng / ml to about 5000 ng / ml.

[0022] In one aspect, mitochondrial DNA deletion syndrome comprises thymidine kinase 2 deficiency, succinyl CoA synthetase deficiency, deoxyguanosine kinase deficiency, succinyl CoA ligase deficiency, ribonucleotide-diphosphate reductase subunit M2 B enzyme deficiency, thymidine phosphorylase deficiency and polymerase gamma deficiency. In another aspect, mitochondrial DNA deletion syndrome comprises thymidine kinase 2 deficiency.

[0023] In one aspect, the subject is a human.

[0024] Another embodiment described herein is a method for treating thymidine kinase 2 (TK2) deficiency in a subject in need thereof, comprising: a) obtaining a nucleic acid sample from the subject; b) determining whether the subject has TK2 deficiency; c) administering a therapeutically effective amount of a composition comprising a polynucleotide compound of Formula 1:

[0025]

[0026] in:

[0027] R1 is H or OH;

[0028] R2 is a purine derivative or a pyrimidine derivative;

[0029] R3 is a purine derivative or a pyrimidine derivative; and

[0030] R4 is H or OH; and d) determining the plasma level of the corresponding mononucleoside;

[0031] wherein the plasma level of the corresponding nucleoside is about 50 ng / mL to about 5000 ng / mL.

[0032] Another embodiment described herein is a compound of Formula I:

[0033]

[0034] in

[0035] R1 is H or OH;

[0036] R2 is a purine derivative or a pyrimidine derivative;

[0037] R3 is a purine derivative or a pyrimidine derivative; and

[0038] R4 is H or OH.

[0039] In one aspect, the compound is substantially free of impurities. In another aspect, R1 is H, R2 is thymine, and R3 is cytosine, and R4 is OH.

[0040] In another aspect, the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient and a therapeutically effective amount of any one of the compounds described herein. In another aspect, the composition comprises a compound wherein R1 is H, R2 is 5-methyl-2H-1λ2-pyrimidine-2,4(3H)-dione, R3 is 4-amino-2H-1λ2-pyrimidine-2-one, and R4 is OH.

[0041] Even though not specifically described, one or more aspects and embodiments may be incorporated into different embodiments. That is, all aspects and embodiments may be combined in any manner or combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Characterize the structure and metabolism of the test article.

[0044] Figure 2 Plasma levels of dT and dC are depicted up to 48 h post-dose, corrected for the amount of labeled vs. unlabeled test article and calibrated for the total dose of test article administered.

[0045] Figure 3 Plots of the AUC of label-corrected and dose-adjusted total dT and dC after acute oral administration of labeled and unlabeled dCMP+dTMP and acute intravenous administration of labeled and unlabeled dC(P2)dT 0-24h Exposure. Plasma AUC after intravenous vs oral administration 0-24h The ratios are approximately 350-fold and 110-fold higher for dT and dC, respectively.

[0046] Figure 4 Plasma levels of dT and dC up to 48 h after a single intravenous administration and one and two subcutaneous administrations of labeled and unlabeled dC(P2)dT are depicted.

[0047] Figure 5 Plotting the AUC of label-corrected total dT and dC after parenteral administration of dC(P2)dT 0-24h Exposed.

[0048] Detailed Description

[0049] The present disclosure describes methods of treating mitochondrial DNA deletion syndrome (MDS). Also described herein are compounds and methods for treating thymidine kinase 2 (TK2) deficiency.

[0050] definition

[0051] As used herein, the term "about" refers to any value including integers and fractional parts within a range of variation of up to ±10% of the value modified by the term "about". For example, the phrase "about 50%" is equivalent to any value of ≈50±10%, such as 44.6%, 45%, 46%, 47%, 48%, 49%, 49.5%, 50%, 50.3%, 51%, 52%, 53%, 54%, 55%, etc.

[0052] Unless otherwise indicated, "a" or "an" as used herein refers to one or more. As used herein, "effective amount" refers to an amount sufficient to achieve a therapeutic effect when administered to a patient in need of treatment.

[0053] As used herein, the term "AUC 0→24 ” refers to the area under the blood (plasma, serum or whole blood) concentration versus time curve from 0 to 24 hours.

[0054] As used herein, the term "derivative" refers to a compound derived from a purine or pyrimidine, including compounds formed from purine or pyrimidine precursors, respectively.

[0055] The term "dosage" or "dose" refers to any form of a formulation containing an amount of active ingredient sufficient to produce a therapeutic effect with a single administration. The dosage form used herein can be for oral, enteral or intravenous administration.

[0056] As used herein, the term "formulation" or "composition" refers to a combination of an active pharmaceutical ingredient or drug and a pharmaceutically acceptable excipient. This includes orally administrable formulations as well as formulations that can be administrated by other means.

[0057] Terms such as "include," "including," "contain," "containing," "has," or "having" mean "comprising."

[0058] The term "or" may be conjunctive or disjunctive.

[0059] As used herein, the term "mononucleoside" refers to a single purine or pyrimidine base linked to a sugar. Examples include deoxycytidine (dC) and deoxythymidine (dT).

[0060] The term "mononucleotide" as used herein refers to a single purine or pyrimidine base attached to a sugar and a phosphate group, in the free acid or any salt form (such as the disodium salt). Examples include deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP).

[0061] As used herein, the term "dinucleotide" refers to a compound containing two mononucleotides in the form of a free acid or any salt (such as a disodium salt), for example, deoxycytidine-deoxythymidine diphosphate dC(P2)dT or dC(P2)dT.Na2.

[0062] As used herein, the term "polynucleotide" refers to a compound containing more than one mononucleotide. For example, a polynucleotide compound may contain two nucleotides. The mononucleotides constituting the polynucleotide may all be the same or different.

[0063] As used herein, the term "corresponding mononucleoside" refers to a single mononucleoside corresponding to a mononucleotide, or a single mononucleoside component corresponding to a dinucleotide or polynucleotide. For example, in the mononucleotide deoxycytidine monophosphate (dCMP), the corresponding mononucleoside is deoxycytidine (dC). In another example, the dinucleotide deoxycytidine-deoxythymidine diphosphate (dC(P 2) dT), the corresponding single nucleosides are deoxycytidine (dC) and deoxythymidine (dT).

[0064] As used herein, the terms "subject" and "patient" are used interchangeably herein. In one embodiment, the subject is a human.

[0065] As used herein, the term "substantially pure" refers to having a purity level that can be recognized as "pure" by one skilled in the art. Such purity levels may be less than 100%.

[0066] When referring to the compounds disclosed herein, unless otherwise indicated, the following terms have the following meanings. The following definitions are intended to illustrate, but not to limit, the defined terms. If a particular term is not specifically defined as used herein, it should not be considered that the term is undefined. Instead, the term is used within its generally accepted meaning.

[0067] The term "purine" as used herein refers to a heterocyclic aromatic organic compound consisting of a pyrimidine ring fused to an imidazole ring.

[0068] As used herein, the term "pyrimidine" refers to an aromatic heterocyclic organic compound.

[0069] As used herein, "substituted" refers to the replacement of a hydrogen atom which may otherwise be present on a substituent. When discussing ring systems, optional substitutions are typically 1, 2 or 3 substituents replacing the normally present hydrogens. However, when referring to straight and branched moieties, the number of substitutions may be multiple, occurring anywhere hydrogen would normally be present. The substitutions may be the same or different. Exemplary substitutions include nitro, -NR'R", cyano, -NR'COR"', alkyl, alkenyl, -C(O), -SO2R"', -NR'SO2R"', -SO2NR'R", -CONR'R", -CONHC6H5, hydroxy, alkoxy, alkylsulfonyl, haloalkyl, haloalkenyl, haloalkoxy, thiol (-SH), thioalkyl, halogen, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein R' and R" are the same or different and each represents hydrogen or alkyl; or when R' and R" are each attached to a nitrogen atom, they may form a saturated or unsaturated heterocyclic ring containing 4-6 ring atoms, and wherein R"' is alkyl or haloalkyl.

[0070] In some cases, the depicted substituents may contribute to optical and / or stereoisomerism. Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space are termed "isomers". Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of one another are termed "diastereomers", while stereoisomers that are non-superimposable mirror images of one another are termed "enantiomers". When a compound has an asymmetric center, such as when it is bonded to four different groups, a pair of enantiomers may occur. Enantiomers can be characterized by the absolute configuration of their asymmetric centers and are designated (R) or (S) according to the Cahn and Prelog rules (Cahn et al., 1966, Angew. Chem. 78:413-447, Angew. Chem., Int. Ed. Engl. 5:385-414 (errata: Angew. Chem., Int. Ed. Engl. 5:511); Prelog and Helmchen, 1982, Angew. Chem. 94:614-631, Angew. Chem. Internat. Ed. Engl. 21:567-583; Mata and Lobo, 1993, Tetrahedron: Asymmetry 4:657-668), or by the manner in which the molecules rotate the plane of polarized light and are referred to as dextrorotatory or levorotatory (i.e., (+)- or (-)-isomers, respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a "racemic mixture."

[0071] In certain embodiments, the compounds disclosed herein may have one or more asymmetric centers; and such compounds may thus be produced as individual (R)- or (S)-enantiomers or as mixtures thereof. Unless otherwise indicated, for example, by specifying the stereochemistry at any position of the formula, the description or naming of a particular compound in the specification and claims is intended to include individual enantiomers and mixtures thereof, racemic or otherwise. Methods for determining stereochemistry and separating stereoisomers are well known in the art. In specific embodiments, the stereoisomers of the compounds provided herein are described when treated with a base.

[0072] In certain embodiments, the compounds disclosed herein are "stereochemically pure". A stereochemically pure compound has a stereochemical purity level that can be recognized as "pure" by those skilled in the art. Of course, this purity level can be less than 100%. In certain embodiments, "stereochemically pure" means a compound that is substantially free (i.e., at least about 85% or more) of alternative isomers. In specific embodiments, the compound is at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9% free of other isomers.

[0073] In addition, the pharmaceutically acceptable prodrug of the compound represented by Formula I is also included in the present invention. Pharmaceutically acceptable prodrug refers to a compound having a group that can be converted into an amino, hydroxyl, carboxyl, etc. by solvolysis or under physiological conditions. Examples of groups forming prodrugs include those described in Prog.Med., 5, 2157-2161 (1985) or "Pharmaceutical Research and Development" (Hirokawa Publishing Company, 1990), Vol. 7, Drug Design, 163-198. The term prodrug is used throughout this specification to describe any pharmaceutically acceptable form of a compound, which provides an active compound when applied to a patient. Pharmaceutically acceptable prodrug refers to a compound that is metabolized (e.g., hydrolyzed or oxidized) in a host to form a compound of the present invention. Typical examples of prodrugs include compounds having biologically unstable protective groups on the functional portion of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrated, alkylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylated to produce the active compound.

[0074] As used herein, "pharmaceutically acceptable salt" refers to any salt of a compound disclosed herein that retains the biological properties of the compound and is nontoxic or otherwise undesirable for insecticidal, veterinary or pharmaceutical use. Such salts can be derived from various organic and inorganic counterions known in the art. Such salts include: (1) acid addition salts formed with organic or inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, glutaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, picric acid, cinnamic acid, mandelic acid, phthalic ... Acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphoric acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, benzoic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, cyclohexylaminosulfonic acid, quinic acid, muconic acid and the like.

[0075] Salts also include, by way of example only, salts of nontoxic organic or inorganic acids such as halides (such as chlorides and bromides), sulfates, phosphates, sulfamates, nitrates, acetates, trifluoroacetates, trichloroacetates, propionates, hexanoates, cyclopentylpropionates, glycolates, glutarates, pyruvates, lactates, malonates, succinates, sorbates, ascorbates, malates, maleates, fumarates, tartrates, citrates, benzoates, 3-(4-hydroxybenzoyl)benzoate, picrates, cinnamates, mandelates, phthalates, Laurate, methanesulfonate (mesylate), ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (benzenesulfonate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylaminosulfonate, quinate, muconate, etc.

[0076] The present disclosure includes all pharmaceutically acceptable isotopically labeled compounds of the present invention in which one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention include isotopes such as hydrogen, 2H and 3 H, carbon, such as 11 C. 13 C and 4 C, chlorine, such as 36 Cl, fluorine, such as 18 F, iodine, such as 123 I and 125 I, nitrogen, such as 13 N and 15 N, oxygen, such as 15 O. 17 O and 18 O, phosphorus, such as 32 P, and sulfur, such as 35 S. Certain isotopically labeled compounds of the invention, such as those incorporating a radioactive isotope, may be useful in drug or substrate tissue distribution studies. The radioactive isotope tritium, i.e. 3 H, and carbon-14, i.e. 14 C is particularly suitable for this purpose due to its ease of incorporation and readily available detection methods. 2 H substitution may offer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and thus may be preferred in some circumstances. 11 C. 18 F. 15 O and 13 N substitution can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples and preparations, using an appropriate isotopically labeled reagent in place of the unlabeled reagent previously employed.

[0077] The present disclosure provides a method for treating mitochondrial DNA deficiency syndrome. When mtDNA levels are extremely deficient and have a negative impact on cellular energy production, mitochondrial DNA deficiency syndrome can result. (El-Hattab et al., 2013). Therefore, it is not expected to be bound by any particular theory, and it is believed that the compounds provided herein are metabolized into their corresponding mononucleosides, and thus supplement the nucleoside pool required for providing building blocks for mtDNA synthesis. The compounds contemplated by the present disclosure include, but are not limited to, the exemplary compounds provided herein and their salts.

[0078] One embodiment described herein is a method for treating mitochondrial DNA deletion syndrome comprising administering a therapeutically effective amount of a polynucleotide composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof:

[0079]

[0080] in

[0081] R1 is H or OH;

[0082] R2 is a purine derivative or a pyrimidine derivative;

[0083] R3 is a purine derivative or a pyrimidine derivative; and

[0084] R4 is H or OH.

[0085] Examples of purine groups include, but are not limited to, adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid, isoguanine. In one aspect, the purine is adenine or guanine.

[0086] Examples of pyrimidine groups include, but are not limited to, thymine, cytosine, and uracil. In one aspect, the pyrimidine is thymine or cytosine.

[0087] The polynucleotide composition may comprise more than one mononucleotide. In one aspect, the polynucleotide is a dinucleotide.

[0088] In one aspect, the polynucleotide composition comprises a compound of formula I, wherein R1 is H, R2 is 5-methyl-2H-1λ2-pyrimidine-2,4(3H)-dione, R3 is 4-amino-2H-1λ2-pyrimidine-2-one, and R4 is OH. In another aspect, the composition comprises a mixture, the mixture further comprising a compound of formula I wherein R1 is OH, R2 is 9λ2-purine-6-amine, R3 is 2-amino-9λ2-purine-6(1H)-one, and R4 is OH.

[0089] In one aspect, compound is substantially free of impurities.Substantially pure compound has a purity level that can be identified as "pure" by those skilled in the art.Certainly, this purity level can be less than 100%.In some aspects, "substantially pure" means a compound that is substantially free of (i.e., at least about 85% or more) other compounds.In a specific embodiment, the compound is at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or about 99.9% free of other compounds.

[0090] One aspect described herein is a composition comprising one or more of the compounds of Formula I as described herein and one or more pharmaceutically acceptable carriers.

[0091] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. By "pharmaceutically acceptable" is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0092] The pharmaceutical composition for administering the compound of the present disclosure can conveniently exist in the form of unit dosage form and can be prepared by any method well-known in the pharmaceutical field. All methods include the step of mixing the active ingredient with a carrier constituting one or more auxiliary components. Usually, the pharmaceutical composition is prepared by uniformly and closely mixing the active ingredient with a liquid carrier or a finely divided solid carrier or both of them, and then, if necessary, the product is molded into a desired preparation. In the pharmaceutical composition, the active target compound is included in an amount sufficient to produce a desired effect on the course of the disease or the condition.

[0093] The pharmaceutical composition containing the active ingredient can be in a form suitable for oral use, for example, tablets, tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions and self-emulsifiers (as described in U.S. Pat. No. 6,451,339), hard or soft capsules, or syrups or elixirs. Compositions intended for oral use can be prepared according to any method known in the art for preparing pharmaceutical compositions. Such compositions can contain one or more agents selected from sweeteners, flavoring agents, coloring agents and preservatives to provide pharmaceutically elegant and palatable preparations. Tablets contain a mixture of active ingredients and other non-toxic pharmaceutically acceptable excipients suitable for making tablets. These excipients can be, for example, inert diluents such as cellulose, silicon dioxide, aluminum oxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate or sodium phosphate; granulating and disintegrants such as corn starch or alginic acid; binders such as PVP, cellulose, PEG, starch, gelatin or gum arabic, and lubricants such as magnesium stearate, stearic acid or talc. Tablets can be uncoated, or they can be enteric coated or otherwise coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period of time. For example, a time-delay material such as monostearate or distearate can be used. They can also be coated by the techniques described in U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for controlled release.

[0094] Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oily medium (e.g., peanut oil, liquid paraffin or olive oil). In addition, emulsions may be prepared with water-immiscible ingredients such as oils and stabilized with surfactants such as mono-diglycerides, PEG esters, and the like. In addition, the polynucleotides described herein may be formulated as prodrugs to allow for the synthesis of compounds in a manner that allows for direct intracellular delivery of the polynucleotides to cells.

[0095] Aqueous suspensions contain a mixture of the active substance and excipients suitable for the manufacture of aqueous suspensions, such as saline salts or buffers. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic; dispersants or wetting agents may be naturally occurring phosphatides, for example lecithin, or condensation products of alkylene oxides with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain fatty alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitolanhydride, for example polyethylene sorbitan monooleate. The aqueous suspension may also contain one or more preservatives (for example ethyl or n-propyl p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents and one or more sweetening agents (such as sucrose or saccharin).

[0096] Oily suspensions can be prepared by suspending the active ingredient in a vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil or in a mineral oil such as liquid paraffin. Oily suspensions can contain thickeners such as beeswax, hard paraffin or cetyl alcohol. Sweeteners and flavoring agents such as those described above can be added to provide a palatable oral preparation. These compositions can be preserved by adding antioxidants such as ascorbic acid.

[0097] Dispersible powders and granules suitable for preparing aqueous suspensions by adding water provide a mixture of the active ingredient with a dispersant or wetting agent, a suspending agent and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are exemplified by those listed above. Other excipients, such as sweeteners, flavoring agents and coloring agents, may also be present.

[0098] The pharmaceutical composition of the present disclosure can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin or a mixture of these. Suitable emulsifiers can be naturally occurring gums, such as gum arabic or tragacanth, naturally occurring phospholipids, such as soybeans, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of the partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion can also contain sweeteners and flavoring agents.

[0099] Syrups and elixirs can be formulated with sweeteners such as glycerol, propylene glycol, sorbitol or sucrose. Such preparations may also contain demulcents, preservatives, and flavoring and coloring agents. Oral solutions may be prepared in combination with, for example, cyclodextrins, PEGs, and surfactants.

[0100] In one aspect, the composition can be administered intravenously. The pharmaceutical composition can be in the form of a sterile injectable aqueous or oily suspension. This suspension can be prepared according to known techniques using those suitable dispersants or wetting agents and suspending agents cited above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injections.

[0101] As demonstrated in the Examples, intravenous administration of a composition as described herein at significantly lower doses compared to oral administration showed improved plasma concentrations of the corresponding single nucleosides compared to oral administration of a single nucleotide therapy alone (Example 1).

[0102] In another aspect, the composition can be administered subcutaneously.

[0103] The compounds of the present disclosure may also be administered in the form of suppositories for rectal administration of the drug. These compositions may be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and will melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycol.

[0104] The dosage form can contain about 1 mg / kg-about 20 mg / kg of the polynucleotide composition described herein. In one aspect, the composition is administered at a dosage of about 1 mg / kg-about 5 mg / kg. In another aspect, the composition is administered at a dosage of about 3 mg / kg-about 8 mg / kg. In another aspect, the composition is administered at a dosage of about 5 mg / kg-about 10 mg / kg. In another aspect, the composition is administered at a dosage of about 8 mg / kg-about 13 mg / kg. In one aspect, the composition is administered at a dosage of about 10 mg / kg-about 15 mg / kg. In yet another aspect, the composition is administered at a dosage of about 13 mg / kg-about 18 mg / kg. In one aspect, the composition is administered at a dosage of about 15 mg / kg-about 20 mg / kg.

[0105] The compounds of formula I are used to treat mitochondrial DNA deletion syndrome. Examples of such syndromes include, but are not limited to, thymidine kinase 2 deficiency, succinyl CoA synthetase deficiency, deoxyguanosine kinase deficiency, succinyl CoA ligase deficiency, ribonucleotide-diphosphate reductase subunit M2 B enzyme deficiency, thymidine phosphorylase deficiency, and polymerase gamma deficiency. In one aspect described herein, the mitochondrial DNA deletion syndrome is thymidine kinase 2 deficiency.

[0106] The compound of formula I can be combined with one or more agents having the same activity range, for example, to increase activity, or with a material having another activity range, for example, to broaden the activity range. Any of the agents listed separately can be used in conjunction with the compound of formula I and any other one or more agents listed independently.

[0107] Suitable agents for combination therapy include those wherein one or more compounds of Formula I may be used as such or in the form of a preparation or formulation in combination with one or more other pharmaceutically active substances, such as, for example, therapeutic agents for treating the symptoms of a particular form of MDS, inhibitors of ubiquitous nucleoside metabolizing enzymes, including, but not limited to, tetrahydrouridine, triacetyluridine, N-acetylcysteine, N-acetylcysteine ​​amide, vitamin E, immucillin H, and tipiraci. The combination may be part of the same formulation or may be administered separately or sequentially.

[0108] The pharmaceutical preparation for delivery to humans or other mammals comprising a compound of Formula I or a pharmaceutically acceptable salt thereof is preferably in unit dosage form, wherein the preparation is further divided into unit doses containing an appropriate amount of active ingredient. The unit dosage form can be a packaged preparation containing discrete amounts of preparations, such as packaged tablets, capsules, and powders in vials or ampoules. In addition, the unit dosage form can be a capsule, tablet, or injection, or can be any of these in an appropriate number of packaged forms.

[0109] The amount of active ingredient in a unit dose preparation may be varied or adjusted between about 0.1 mg to about 1000 mg, depending on the particular application and the potency of the active ingredient. If desired, the composition may also contain other compatible therapeutic agents.

[0110] In the therapeutic use for treating mitochondrial DNA deficiency syndrome in humans or other mammals, the compound used in the method of treatment is administered at an initial dose of about 0.1 mg / kg-about 100 mg / kg per interval. Preferred intervals may be daily, weekly, semi-monthly, monthly, bimonthly, quarterly, every three years, semi-annually or annually. The dosage may be administered once a day, twice a day, three times a day or as many times as necessary as determined by the physician. The dosage may vary according to the needs of the patient, for example, the size of the person or mammal being treated, the severity of the condition being treated, the route of administration and the efficacy of the compound used. The appropriate dosage and route of administration for a particular situation are determined within the physician's skill range. Typically, treatment will begin with a smaller dose that is less than the optimal dose of the compound, which may be increased in small increments until the optimal effect in a particular situation is achieved. For convenience, if desired, the total daily dose may be administered in several portions during the day.

[0111] In order to understand the metabolism of compositions as herein described, the plasma levels of the polynucleotide metabolism into its corresponding mononucleosides are measured after administration. In one aspect as herein described, the plasma levels of the corresponding mononucleosides are higher than the plasma levels of the single mononucleotides used. In another aspect, the plasma levels of the corresponding nucleosides are about 50ng / mL-about 5000ng / mL. In another aspect, the plasma levels of the corresponding nucleosides are about 75ng / mL-about 2500ng / mL.

[0112] For intravenous administration, the area under the curve (AUC) for plasma levels from 0 to about 24 hours is measured. 0-24h ) is about 5000 ng-h / mL to about 120,000 ng-h / mL.

[0113] Another embodiment described herein is a method for treating thymidine kinase 2 in a subject, comprising obtaining a nucleic acid sample from the subject, determining whether the subject has TK2 deficiency, administering a therapeutically effective amount of a composition comprising a compound of Formula I:

[0114]

[0115] in

[0116] R1 is H or OH;

[0117] R2 is a purine derivative or a pyrimidine derivative;

[0118] R3 is a purine derivative or a pyrimidine derivative;

[0119] R4 is H or OH; and

[0120] The plasma level of the corresponding single nucleoside is determined, wherein the plasma level of the corresponding nucleoside is about 50 ng / mL to about 5000 ng / mL.

[0121] Patients who present with phenotypic TK2 deficiency, including the most typical manifestations of a progressive myopathy characterized by generalized hypotonia, proximal muscle weakness, loss of previously acquired motor skills, poor feeding, and dyspnea, can be tested to confirm the diagnosis of the disorder.

[0122] Molecular genetic testing can be performed using a panel of genes known to cause mtDNA deletion syndrome. (Chanprasert, et al., 2012). Testing can be performed by sequence analysis of the TK2 coding region. Further testing can also be performed to confirm the diagnosis of TK2 deficiency, including testing serum creatine kinase concentration, electromyography, histopathology of skeletal muscle, mitochondrial DNA content (copy number), and electron transport chain (ETC) activity in skeletal muscle. Example

[0123] Preparation of compounds of formula I

[0124] The preparation of the compounds of the present disclosure or their pharmaceutically acceptable salts can be accomplished via the routes and intermediates shown below.

[0125] Solution 1

[0126]

[0127] Scheme 1 describes a general method for preparing compounds of Formula I. Diphosphate nucleotides are synthesized by coupling the corresponding nucleotide tributylammonium salts using carbonyldiimidazole as a coupling reagent to give the desired products. In the process of forming such dinucleotides, asymmetric compounds such as (dC(P2)dC) and (dT(P2)dT) are also formed. The currently used process is sufficient for small-scale preparation (1-5 g), but better methods are desired on a larger scale (100 g).

[0128] Example 1

[0129] To determine the systemic exposure of two mononucleotides (dCMP and dTMP) and their corresponding mononucleosides (dC and dT) following oral administration of the two mononucleotides (dCMP & dTMP) vs. intravenous administration of a dinucleotide (dC(P2)dT). The test article was prepared as follows:

[0130] Unlabeled test article:

[0131] dC=deoxycytidine (nucleoside)

[0132] dT = deoxythymidine (nucleoside)

[0133] dCMP = deoxycytidine monophosphate (mononucleotide)

[0134] dTMP = deoxythymidine monophosphate (mononucleotide)

[0135] dC(P2)dT or dT(P2)dC = deoxycytidine-deoxythymidine diphosphate (dinucleotide)

[0136] Labelled test article:

[0137] dC*= 15 N3 labeled dC (labeled nucleoside)

[0138] dT**= 13 C 10 and 15 N2 labeled dT (labeled nucleoside)

[0139] The corresponding labeled nucleotides are dC*MP and dT**MP

[0140] The corresponding labeled dinucleotides are dC*(P2)dT** or dT**(P2)dC*.

[0141] The structure and metabolism of these test products are Figure 1 as shown in .

[0142] The dose intensity of a single intravenous injection of dC(P2)dT was determined to result in comparable systemic exposures of dC and dT compared to a single oral administration of dCMP+dTMP, where systemic exposure was quantified as AUC 0-24h .

[0143] Nucleosides (dC and dT) and their corresponding monophosphate nucleotides (dCMP and dTMP) are naturally produced in vivo. To distinguish between naturally occurring vs exogenously administered dC / dT and dCMP / dTMP, a mixture of labeled dC*MP / dT**MP and unlabeled dCMP / dTMP was administered via the oral route, and a mixture of labeled dC*(P2)dT** and unlabeled dC(P2)dT was administered via the intravenous route. The labeled test articles were used as tracers to determine systemic levels of exogenous dC / dT and dCMP / dTMP after administration.

[0144] The plasma samples were then analyzed for the following analytes: dT**, dC*, dT**MP, dC*MP, and dC*(P2)dT**. Only dT** and dC* were detectable, indicating that dT**MP, dC*MP, and dC*(P2)dT** were rapidly metabolized in vivo. The results are shown in Table 1.

[0145] Table 1

[0146]

[0147]

[0148] As shown in Table 1, 10% of the total test article (TA) administered by the oral route was labeled, and 20% of the total TA administered by the parenteral route (intravenous) was labeled. To correct for the total TA administered (labeled + unlabeled), the plasma levels of the labeled analytes were multiplied by 10 and 5 for the oral and intravenous treatment groups, respectively. In addition, the total TA doses were 1000 mg / kg (900 mg / kg unlabeled + 100 mg / kg labeled) (administered by the oral route) and 10 mg / kg (8 mg / kg unlabeled and 2 mg / kg labeled) for the oral and intravenous treatment groups, respectively. To calibrate by the unit dose of TA administered, the plasma levels of the analytes were divided by 1000 and 10 for the oral and intravenous treatment groups, respectively. The results of the label correction and dose calibration levels of dT and dC for the oral and intravenous administration routes are shown in Table 1. Figure 2 as shown in .

[0149] Figure 3 Plasma exposure to the two analytes in the first 24 h after dosing is shown by Figure 2The area under the total dose-corrected dT and dC plasma levels was calculated (AUC 0-24 h) to determine the intravenous vs oral AUCs of the two analytes 0-24h The difference was statistically significant (p<0.01, t-test). Intravenous / oral AUC (0-24h) The ratio is approximately 350 for dT and approximately 110 for dC. Therefore, based on label-corrected and dose-calibrated PK analyses, these data suggest that to deliver comparable plasma exposures for dT and dC, we may need to orally administer approximately 350-fold and approximately 110-fold higher doses of dTMP and dCMP, respectively, compared to intravenous dC(P2)dT.

[0150] Example 2

[0151] Systemic exposure of dC and dT following a single intravenous injection of dC(P2)dT was compared with one and two subcutaneous (SC) injections of dC(P2)dT, with the second SC injection being administered 24 h after the first. The study design is summarized in Table 2.

[0152] Table 2

[0153]

[0154] Plasma samples were analyzed for the following analytes: dT**, dC*, dT**MP, dC*MP, and dC*(P2)dT**, and as in Example 1, only dT** and dC* were detectable. Plasma levels of labeled-calibrated dT and dC levels after a single intravenous and one and two subcutaneous administrations of labeled and unlabeled dC(P2)dT groups were as follows: Figure 4 as shown in . Figure 5 Plasma exposures of the two analytes in the three treatment groups during the first 24 h after dosing are shown by comparing the plasma Figure 4 The mean area under the total dose-corrected dT and dC plasma levels were calculated (AUC 0-24 h) to determine the mean AUC in the three treatment groups. 0-24h The values ​​were not statistically different, indicating that intravenous and subcutaneous administration of dC(P2)dT resulted in comparable exposures of dT and dC in plasma.

[0155] All publications, patents, and patent applications cited in this specification are incorporated herein by reference for the purposes of the teachings of using such references.

[0156] The specific responses observed may vary and depend upon the particular active compound selected or the presence or absence of a carrier, as well as the type of formulation and mode of administration employed, and such expected variations or differences in results are contemplated by the practice of the present invention.

[0157] Although specific embodiments of the present invention are illustrated and described in detail herein, the present invention is not limited thereto. The above detailed description is provided as an example of the present invention and should not be construed as constituting any limitation to the present invention. Variations will be apparent to those skilled in the art, and all variations that do not depart from the spirit of the present invention are intended to be included within the scope of the appended claims.

[0158] References

[0159] Chansprasert, S. and Craigen WJ, (2017) Mitochondrial Disorders Causing Cardioskeletal Myopathies in Childhood. Cardioskeletal Myopathies in Children and Young Adults.

[0160] El-Hattab, AW and Scaglia, F., (2013) Mitochondrial DNA depletionsyndromes: review and updates of genetic basis, manifestations, and therapeutic options. Neurotherapeutics. Apr:10(2):186-198.

[0161] Garone,C.,Taylor,RW,Nascimento,A,Poulton,J,Fratter,C,Dominguez-Gonzalez,C,Evans,JC,Loos,M,Isohanni,P,Suomalainen,A,Ram,D,Hughes,MI,McFarland,R,Barca,E,Lopez Gomez,C.,Jayawant,S.,Thomas,ND,Manzur,AY,Kleinsteuber,K,Martin,MA,Kerr,T,Gorman,GS,Sommerville,EW,Chinnery,PF,Hofer,M.,Karch,C.,Ralph,J.,Chamber,Y.,Madruga-Garrid o,M.,Dominguez-Carral.J.,Ortez,C.,Emperor,S.,Montoya,J.,Chakrapani,A.,Kriger,JF,Schoenaker,R.,Levin,B.,Thompson,JLP,Long,Y.,Rahman,S.,Donati,MA,DiMauro.S.and Hirano M.,(2018)Retrospective natural history of thymidine kinase2deficiency.J Med Genet.Aug:55(8):515-521.

[0162] TK2-related mitochondrial DNA depletion syndrome,myopathic form:National Library of Medicine(US).Genetics Home Reference[Internet].Bethesda(MD):The Library:9 / 9 / 2013[9 / 2013 / 9 / 2013 / Retrieved / 9 / 9 / 19 / 2013]4 [6 pages].Available at:https: / / ghr.nlm.nih.gov / condition / cystic-fibrosis

Claims

1. Use of a compound of formula I in the preparation of a pharmaceutical composition for treating mitochondrial DNA deletion syndrome in a subject in need thereof: in: R1 is H or OH; R2 is a purine derivative or a pyrimidine derivative; R3 is a purine derivative or a pyrimidine derivative; and R4 is H or OH, wherein the purine derivative is selected from adenine, guanine, hypoxanthine, theobromine, caffeine, uric acid, isoguanine, 9λ2-purine-6-amine or 2-amino-9λ2-purine-6(1H)-one; and The pyrimidine derivative is selected from thymine, cytosine, uracil, 5-methyl-2H-1λ2-pyrimidine-2,4(3H)-dione or 4-amino-2H-1λ2-pyrimidine-2-one.

2. The use of claim 1, wherein the composition comprises a compound of formula I, wherein R1 is H, R2 is 5-methyl-2H-1λ2-pyrimidine-2,4(3H)-dione, R3 is 4-amino-2H-1λ2-pyrimidin-2-one, and R4 is OH.

3. The use of claim 1, wherein the composition is a mixture further comprising a compound of formula I wherein R1 is OH, R2 is 9λ2-purin-6-amine, R3 is 2-amino-9λ2-purin-6(1H)-one, and R4 is OH.

4. The use according to any one of claims 1 to 3, wherein the composition is prepared in a dosage form for oral, enteral, intravenous or subcutaneous administration.

5. The use according to any one of claims 1 to 3, wherein the composition is prepared in a dosage form for intravenous administration.

6. The use of claim 4, wherein the composition is prepared in a dosage form for intravenous administration.

7. The use of claim 5, wherein the plasma level of each single nucleoside is higher after intravenous administration compared to the plasma level after oral administration of the single single nucleotide.

8. The use of claim 6, wherein the plasma level of each single nucleoside is higher after intravenous administration compared to the plasma level after oral administration of the single single nucleotide.

9. The use of any one of claims 1-3 and 6-8, wherein intravenous administration of the composition produces a drug having a first AUC 0-24h , and oral administration of a single mononucleotide produced plasma levels with a second AUC 0-24h The plasma level of 0-24h With the second AUC 0-24h The ratio is 100-400.

10. The use of claim 4, wherein intravenous administration of the composition produces a drug having a first AUC 0-24h , and oral administration of a single mononucleotide produced plasma levels with a second AUC 0-24h The plasma level of 0-24h With the second AUC 0-24h The ratio is 100-400.

11. The use of claim 5, wherein intravenous administration of the composition produces a drug having a first AUC 0-24h , and oral administration of a single mononucleotide produced plasma levels with a second AUC 0-24h The plasma level of 0-24h With the second AUC 0-24h The ratio is 100-400.

12. The use according to any one of claims 1-3, 6-8 and 10-11, wherein when the composition is administered at a dose of 1 mg / kg-20 mg / kg, the plasma concentration of the corresponding nucleoside is between 50 ng / ml and 5000 ng / ml.

13. The use of claim 4, wherein when the composition is administered at a dose of 1 mg / kg-20 mg / kg, the plasma concentration of the corresponding nucleoside is between 50 ng / ml and 5000 ng / ml.

14. The use of claim 5, wherein when the composition is administered at a dose of 1 mg / kg-20 mg / kg, the plasma concentration of the corresponding nucleoside is between 50 ng / ml and 5000 ng / ml.

15. The use of claim 9, wherein when the composition is administered at a dose of 1 mg / kg-20 mg / kg, the plasma concentration of the corresponding nucleoside is between 50 ng / ml and 5000 ng / ml.

16. The use of any one of claims 1-3, 6-8, 10-11 and 13-15, wherein the mitochondrial DNA deletion syndrome comprises thymidine kinase 2 deficiency, succinyl CoA synthetase deficiency, deoxyguanosine kinase deficiency, succinyl CoA ligase deficiency, ribonucleotide-diphosphate reductase subunit M2 B enzyme deficiency, thymidine phosphorylase deficiency and polymerase gamma deficiency.

17. The method of claim 4, wherein the mitochondrial DNA deletion syndrome comprises thymidine kinase 2 deficiency, succinyl CoA synthetase deficiency, deoxyguanosine kinase deficiency, succinyl CoA ligase deficiency, ribonucleotide-diphosphate reductase subunit M2 B enzyme deficiency, thymidine phosphorylase deficiency, and polymerase gamma deficiency.

18. The method of claim 5, wherein the mitochondrial DNA deletion syndrome comprises thymidine kinase 2 deficiency, succinyl CoA synthetase deficiency, deoxyguanosine kinase deficiency, succinyl CoA ligase deficiency, ribonucleotide-diphosphate reductase subunit M2 B enzyme deficiency, thymidine phosphorylase deficiency, and polymerase gamma deficiency.

19. The method of claim 9, wherein the mitochondrial DNA deletion syndrome comprises thymidine kinase 2 deficiency, succinyl CoA synthetase deficiency, deoxyguanosine kinase deficiency, succinyl CoA ligase deficiency, ribonucleotide-diphosphate reductase subunit M2 B enzyme deficiency, thymidine phosphorylase deficiency, and polymerase gamma deficiency.

20. The method of claim 12, wherein the mitochondrial DNA deletion syndrome comprises thymidine kinase 2 deficiency, succinyl CoA synthetase deficiency, deoxyguanosine kinase deficiency, succinyl CoA ligase deficiency, ribonucleotide-diphosphate reductase subunit M2B enzyme deficiency, thymidine phosphorylase deficiency, and polymerase gamma deficiency.

21. The use of claim 16, wherein the mitochondrial DNA deletion syndrome comprises thymidine kinase 2 deficiency.

22. The use of any one of claims 17-20, wherein the mitochondrial DNA deletion syndrome comprises thymidine kinase 2 deficiency.

23. The use of any one of claims 1-3, 6-8, 10-11, 13-15, 17-20 and 21, wherein the subject is a human.

24. The use of claim 4, wherein the subject is a human.

25. The use of claim 5, wherein the subject is a human.

26. The use of claim 9, wherein the subject is a human.

27. The use of claim 12, wherein the subject is a human.

28. The use of claim 16, wherein the subject is a human.

29. The use of claim 22, wherein the subject is a human.

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