Treating mitochondrial DNA depletion disorders

Abstract

To provide a method for treating mitochondrial DNA depletion syndrome.SOLUTION: Described is a method for treating mitochondrial DNA depletion syndrome by administering a therapeutic amount of a composition comprising a dinucleotide compound or a mixture thereof. Furthermore, compounds, compositions, and methods for treating TK2 deficiency are described herein.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 896,218, filed September 5, 2019, the disclosure of which is incorporated by reference in its entirety.

[0002] FIELD OF THE INVENTION The present disclosure describes methods for treating mitochondrial DNA depletion syndrome (MDS) by administering a therapeutic amount of a composition comprising a multinucleotide compound or a mixture thereof. Additionally, compounds, compositions, and methods for treating thymidine kinase 2 (TK2) deficiency are described herein. [Background technology]

[0003] background Mitochondria are known as the energy-producing organelles of cells. These double-membrane organelles function independently of the cell, reside within the cell, and have their own genome, separate from the nuclear genome. While the nuclear genome encodes several mitochondrial proteins, the mitochondrial genome specifically encodes proteins associated with energy-related metabolic processes, including the electron transport chain and ATP production. Mitochondrial DNA depletion syndrome (MDS) is an umbrella term used to describe a variety of disorders, all characterized by a significant reduction in cellular mitochondrial DNA. MDS results from mutations in nuclear genes involved in mitochondrial DNA nucleotide synthesis or replication and is associated with various genes, including TK2, SUCLA2, SUCLG1, POLG, DGUOK, MPV17, TYMP, and RRM2B (Chansprasert et al., 2017). Consequently, these autosomal recessive disorders result in extreme depletion of mitochondrial DNA (mtDNA), adversely affecting energy production (El-Hattab et al., 2013).

[0004] Symptoms typically appear in infancy or early childhood, primarily affecting muscle, liver, or brain tissue, 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 result in encephalomyopathic MDS, clinically manifesting as 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 also vary widely, ranging from mild symptoms to severe symptoms leading to death during infancy and childhood. Due to such heterogeneity in clinical manifestations, treatment options are primarily limited to symptom management via nutritional supplementation.

[0005] Thymidine kinase 2 (TK2) deficiency is a specific type of MDS. The thymidine kinase 2 enzyme is a nuclear-encoded enzyme involved in mitochondrial DNA (mtDNA) synthesis, which is involved in nucleotide recycling (Genetics Home Reference, 2013). Mutations in the TK2 gene reduce enzyme activity, impairing mtDNA nucleotide recycling and resulting in reduced levels of deoxythymidine monophosphate and deoxycytidine monophosphate. This deficiency in nucleotide pools affects mtDNA synthesis and ultimately leads to progressive myopathy, which can begin in early childhood and eventually results in loss of motor skills (Garone et al., 2018). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Chansprasert et al., 2017 [Non-patent document 2] El-Hattab et al., 2013 [Non-patent document 3] Genetics Home Reference, 2013 [Non-patent document 4] Garone et al., 2018 Summary of the Invention [Problem to be solved by the invention]

[0007] Currently, there are no disease-modifying therapies for TK2 disease, and treatment is primarily symptomatic. Oral supplementation with deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP) has demonstrated the ability to reverse mtDNA depletion and prolong overall survival in animal models, but such treatments require high doses of each deoxyribonucleoside monophosphate (dNMP) to achieve overall therapeutic benefit and are associated with significant side effects. [Means for solving the problem]

[0008] One embodiment described herein is a method of treating mitochondrial DNA depletion syndrome in a subject in need thereof, comprising administering to a subject a compound of Formula I [ka] (In the formula, R1 is H or OH; R2 is a purine derivative or a pyrimidine derivative; R3 is a purine derivative or a pyrimidine derivative, R4 is H or OH The method comprises administering to a subject a therapeutically effective amount of a multinucleotide composition comprising a compound of formula (I).

[0009] In one embodiment, 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. In another embodiment, 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.

[0010] In one embodiment, the composition is administered orally, enterally, intravenously, or subcutaneously, hi a further embodiment, the composition is administered intravenously.

[0011] In one embodiment, the plasma levels of each mononucleoside are higher after intravenous administration compared to the plasma levels after oral administration of the individual mononucleotides.

[0012] In another embodiment, intravenous administration of the composition provides a first AUC 0-24h and oral administration of the individual mononucleotides results in plasma levels having a second AUC 0-24h and the first AUC 0-24h and the second AUC 0-24h The ratio is between about 100 and about 400.

[0013] In yet another embodiment, when the composition is administered at a dosage between about 1 mg / kg and about 20 mg / kg, the plasma concentration of the corresponding nucleoside is from about 50 ng / ml to about 5000 ng / ml.

[0014] In one embodiment, mitochondrial DNA depletion syndromes include 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. In a further embodiment, mitochondrial DNA depletion syndromes include thymidine kinase 2 deficiency.

[0015] In one embodiment, the subject is a human.

[0016] Another embodiment described herein is a method for treating thymidine kinase 2 (TK2) deficiency in a subject in need thereof, comprising the steps of: a) obtaining a nucleic acid sample from the subject; b) determining whether the subject has a TK2 deficiency; and c) detecting a nucleic acid sample comprising a nucleotide sequence corresponding to Formula 1: [ka] (In the formula, R1 is H or OH; R2 is a purine derivative or a pyrimidine derivative; R3 is a purine derivative or a pyrimidine derivative, R4 is H or OH and d) measuring the plasma level of the corresponding mononucleoside, wherein the plasma level of the corresponding nucleoside is from about 50 ng / mL to about 5000 ng / mL.

[0017] Another embodiment described herein is a compound of Formula I: [ka] (In the formula, R1 is H or OH; R2 is a purine derivative or a pyrimidine derivative; R3 is a purine derivative or a pyrimidine derivative, R4 is H or OH is a compound of

[0018] In one embodiment, the compound is substantially free of impurities. In another embodiment, R1 is H, R2 is thymine, R3 is cytosine, and R4 is OH.

[0019] In another embodiment, the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient and a therapeutically effective amount of any of the compounds described herein. In a further embodiment, the composition comprises a compound where 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.

[0020] One or more aspects and embodiments may be combined in different embodiments, even if not specifically stated, i.e., all aspects and embodiments may be combined in any way or combination. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows the structure and metabolism of test articles. [Figure 2] FIG. 1 shows plasma levels of dT and dC up to 48 hours after dosing, corrected for labeled vs. unlabeled amount of test article and normalized to the total dose of test article administered. [Figure 3] Figure 1 shows label-corrected and dose-normalized AUC exposures of total dT and dC after acute PO administration of labeled and unlabeled dCMP+dTMP and acute IV administration of labeled and unlabeled dC(P2)dT. The ratio of plasma AUC for IV vs. PO administration was approximately 350-fold and 110-fold higher for dT and dC, respectively. [Figure 4] FIG. 1 shows plasma levels of dT and dC up to 48 hours after a single IV dose and one and two SC doses of labeled and unlabeled dC(P2)dT. [Figure 5] FIG. 1 shows the AUC0-24h exposure of label-corrected total dT and dC after parenteral administration of dC(P2)dT. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present disclosure describes methods for treating mitochondrial DNA depletion syndrome (MDS). Also described herein are compounds and methods for treating thymidine kinase 2 (TK2) deficiency.

[0023] definition As used herein, the term "about" refers to any value, including both integer and fractional components, within a range of variation of up to ±10% of the value modified by the term "about." For example, the phrase "about 50%" corresponds to any value ≈50±10%, e.g., 44.6%, 45%, 46%, 47%, 48%, 49%, 49.5%, 50%, 50.3%, 51%, 52%, 53%, 54%, 55%, among others.

[0024] As used herein, "a" or "an" means one or more, unless otherwise specified. As used herein, an "effective amount" refers to an amount sufficient to achieve a therapeutic effect when administered to a patient in need of treatment.

[0025] "AUC 0→24 " as used herein refers to the area under the blood (plasma, serum, or whole blood) concentration versus time curve from 0 to 24 hours.

[0026] The term "derivative" as used herein refers to a compound derived from a purine or pyrimidine, including compounds formed from a purine or pyrimidine precursor, respectively.

[0027] The term "dosage" or "dose" refers to any form of formulation of an active ingredient that contains a sufficient amount to produce a therapeutic effect in a single administration. The dosage form used herein may be for oral, enteral or intravenous administration.

[0028] The term "formulation" or "composition," as used herein, refers to an active pharmaceutical ingredient or drug in combination with a pharmaceutically acceptable excipient. These include orally administrable formulations as well as formulations that can be administered by other means.

[0029] Terms such as "include," "including," "contain," "containing," "has," or "having" mean comprising.

[0030] The term "or" may be conjunctive or disjunctive.

[0031] The term "mononucleoside," as used herein, refers to a single purine or pyrimidine base attached to a sugar. Examples include deoxycytidine (dC) and deoxythymidine (dT).

[0032] The term "mononucleotide," as used herein, refers to a single purine or pyrimidine base attached to a sugar and phosphate group, either as the free acid or as any salt form, e.g., a disodium salt. Examples include deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP).

[0033] The term "dinucleotide," as used herein, refers to a compound containing two mononucleotides, either as the free acid or in any salt form, such as the disodium salt, e.g., deoxycytidine-deoxythymidine diphosphate dC(P2)dT or dC(P2)dT.Na2.

[0034] The term "multinucleotide" as used herein refers to a compound that contains more than one mononucleotide.For example, a multinucleotide compound may contain two nucleotides.The mononucleotides that make up a multinucleotide may all be the same or different.

[0035] The term "corresponding mononucleoside," as used herein, refers to a single mononucleoside corresponding to a mononucleotide, or a single mononucleoside component corresponding to a dinucleotide or multinucleotide. 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) In the case of nucleotides (nucleotides (dC) and (dT)), the corresponding mononucleosides are deoxycytidine (dC) and deoxythymidine (dT).

[0036] As used herein, the terms "subject" and "patient" are used interchangeably herein. In one embodiment, the subject is a human.

[0037] The term "substantially pure," as used herein, means having a level of purity that would be recognized as "pure" by one of ordinary skill in the art. This level of purity may be less than 100%.

[0038] When referring to the compounds disclosed herein, unless otherwise specified, the following terms have the following meanings.The following definitions are intended to clarify the defined terms, but are not intended to limit them.If a specific term used herein is not specifically defined, such term should not be considered unclear.Rather, the term is used within its acceptable meaning.

[0039] As used herein, the term "purine" refers to a heterocyclic aromatic organic compound consisting of a pyrimidine ring fused to an imidazole ring.

[0040] As used herein, the term "pyrimidine" refers to an aromatic heterocyclic organic compound.

[0041] As used herein, "substituted" refers to the replacement of a hydrogen atom, a hydrogen atom that would otherwise be present on a substituent. When discussing ring systems, any substitution is the replacement of a normally present hydrogen with typically one, two, or three substituents. However, when referring to straight-chain and branched moieties, the number of occurrences of substitution may be greater wherever a hydrogen is normally 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, mercapto (-SH), thioalkyl, halogen, cycloalkyl, heterocyclyl, aryl, or heteroaryl, where R' and R'' may be the same or different and each represent 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 from 4 to 6 ring atoms, where R ''' is alkyl or haloalkyl.

[0042] In certain cases, the substituents shown 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 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," and those that are non-superimposable mirror images of each other are termed "enantiomers." When a compound has an asymmetric center, for example, if it is bonded to four different groups, a pair of enantiomers is possible. Enantiomers may be characterized by the absolute configuration of their chiral centers and are designated by (R) or (S) according to the rules of Cahn and Prelog (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. Eng. 21:567-583; Mata and Lobo, 1993, Tetrahedron: Asymmetry 4:657-668), or by the way the molecule rotates the plane of polarized light, designated as dextrorotatory or levorotatory (i.e., as (+)- or (-)-isomers, respectively). Chiral compounds can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of enantiomers is called a "racemic mixture."

[0043] In certain embodiments, the compounds disclosed herein may have one or more asymmetric centers, and therefore, such compounds may be produced as individual (R)- or (S)-enantiomers or mixtures thereof. Unless otherwise indicated, for example, by designating the stereochemistry at any position in the formula, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers, mixtures thereof, racemic or otherwise. Methods for determining stereochemistry and separating stereoisomers are well known in the art. In certain embodiments, the stereoisomers of the compounds provided herein are depicted upon treatment with base.

[0044] In certain embodiments, the compounds disclosed herein are "stereochemically pure." A stereochemically pure compound has a level of stereochemical purity that would be recognized as "pure" by one of ordinary skill in the art. Of course, this level of purity can be less than 100%. In certain embodiments, "stereochemically pure" refers to a compound that is substantially free of alternative isomers, i.e., at least about 85% or more. In certain 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.

[0045] Additionally, pharmaceutically acceptable prodrugs of the compounds represented by Formula I are also included in the present invention. Pharmaceutically acceptable prodrugs refer to compounds having a group that can be converted by solvolysis or under physiological conditions to an amino group, a hydroxyl group, a carboxyl group, or the like. Examples of groups that form prodrugs include those described in Prog. Med., 5, pp. 2157-2161 (1985) or in "Pharmaceutical Research and Development" (Hirokawa Publishing Company, 1990), Vol. 7, Drug Design, pp. 163-198. The term prodrug is used throughout this specification to describe any pharmaceutically acceptable form of a compound that provides an active compound when administered to a patient. A pharmaceutically acceptable prodrug refers to a compound that is metabolized, for example, hydrolyzed or oxidized, in the host to form a compound of the present invention. Typical examples of prodrugs include compounds having biologically labile protecting groups on functional moieties of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce an active compound.

[0046] As used herein, "pharmaceutically acceptable salt" refers to any salt of a compound disclosed herein that retains its biological properties and is not toxic or undesirable for pesticidal, veterinary, or pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counterions known in the art. Such salts include: (1) salts of 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-ethane- Acid addition salts include those formed with acids such as disulfonic 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, cyclohexylsulfamic acid, quinic acid, and muconic acid.

[0047] Salts also include, by way of example only, salts of non-toxic organic or inorganic acids such as halides, e.g., chlorides and bromides, sulfuric acid, phosphoric acid, sulfamic acid, nitric 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 (mesylic acid), ethanesulfonic acid, ... Examples of sulfonic acid salts include those of sulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid (besylic 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 sulfate, gluconic acid, benzoic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, cyclohexylsulfamic acid, quinic acid, and muconic acid.

[0048] The present disclosure includes all pharmaceutically acceptable isotopically labeled compounds of the present invention in which one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes suitable for inclusion in compounds of the present invention include hydrogen isotopes, e.g. 2 H and 3 H, carbon isotopes, e.g. 11 C. 13 C and 14 C, chlorine isotopes, e.g. 36 Cl, fluorine isotopes, e.g. 18 F, iodine isotopes, e.g. 123 I and 125 I, nitrogen isotopes, e.g. 13 N and 15 N, oxygen isotopes, e.g. 15 O. 17 O and 18 O, phosphorus isotopes, e.g. 32P, and sulfur isotopes, e.g. 35 Certain isotopically labeled compounds of the present invention, for example 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 useful for this purpose given its ease of incorporation and ready means of detection. 2 Substitution with H can confer certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances. Positron-emitting isotopes, such as 11 C. 18 F, 15 O and 13 Substitution at N may be useful in positron emission tomography (PET) studies to investigate substrate receptor occupancy. Isotopically labeled compounds of the present invention may generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the accompanying Examples and Preparations, using appropriate isotopically labeled reagents in place of previously employed non-labeled reagents.

[0049] The present disclosure provides a method for treating mitochondrial DNA depletion syndrome. Mitochondrial DNA depletion syndrome occurs when mtDNA levels are severely depleted, negatively affecting cellular energy production (El-Hattab et al., 2013). Therefore, without intending to be bound by any particular theory, it is believed that the compounds provided herein are metabolized to their corresponding mononucleosides, thus replenishing the nucleoside pool required to provide building blocks for mtDNA synthesis. Compounds contemplated by the present disclosure include, but are not limited to, the exemplary compounds provided herein and their salts.

[0050] One embodiment described herein is a method for treating mitochondrial DNA depletion syndrome, comprising administering a compound of Formula I or a pharmaceutically acceptable salt thereof: [ka] (In the formula, R1 is H or OH; R2 is a purine derivative or a pyrimidine derivative; R3 is a purine derivative or a pyrimidine derivative, R4 is H or OH and administering a therapeutically effective amount of a multinucleotide composition comprising:

[0051] Examples of purine groups include, but are not limited to, adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid, and isoguanine. In one embodiment, the purine is adenine or guanine.

[0052] Examples of pyrimidine groups include, but are not limited to, thymine, cytosine and uracil. In one embodiment, the pyrimidine is thymine or cytosine.

[0053] The multinucleotide composition may comprise more than one mononucleotide, hi one embodiment, the multinucleotide is a dinucleotide.

[0054] In one embodiment, the multinucleotide composition comprises a compound of Formula I where 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. In another embodiment, the composition comprises a mixture further comprising a compound of Formula I where R1 is OH, R2 is 9λ2-purin-6-amine, R3 is 2-amino-9λ2-purin-6(1H)-one, and R4 is OH.

[0055] In one aspect, the compound is substantially free of impurities. A substantially pure compound has a level of purity that would be recognized by one of ordinary skill in the art as "pure." Of course, this level of purity may be less than 100%. In certain aspects, "substantially pure" refers to a compound that is substantially free of other compounds, i.e., at least about 85% or more. In certain 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 compounds.

[0056] In one aspect, described herein are compositions that include one or more compounds of Formula I described herein and one or more pharmaceutically acceptable carriers.

[0057] The term "composition," as used herein, is intended to encompass a product containing the specified ingredients in the specified amounts, as well as any product that results directly or indirectly from combining the specified ingredients in the specified amounts. "Pharmaceutically acceptable" means the carrier, diluent, or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0058] The pharmaceutical composition for administering the compound of the present disclosure can be conveniently presented in unit dosage form and can be prepared by any method known in the art of pharmacy.All methods include the step of combining the active ingredient with the carrier that constitutes one or more accessory ingredients.Generally, pharmaceutical compositions are prepared by uniformly and thoroughly combining the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.In pharmaceutical compositions, the target active compound is contained in an amount sufficient to produce the desired effect on the process or condition of disease.

[0059] Pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, and self-emulsifying formulations described in U.S. Pat. No. 6,451,339, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art of pharmaceutical composition manufacture. Such compositions may contain one or more agents selected from sweeteners, flavoring agents, coloring agents, and preservatives to provide a pharmaceutically elegant and palatable preparation. Tablets contain the active ingredient mixed with other non-toxic pharmaceutically acceptable excipients suitable for tablet manufacture. These excipients may 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 disintegrating agents such as corn starch or alginic acid; binders such as PVP, cellulose, PEG, starch, gelatin, or acacia; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or enteric coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate may be used. They may 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.

[0060] Formulations for oral use may also be prepared 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 oil medium, such as peanut oil, liquid paraffin, or olive oil. Furthermore, emulsions may be prepared using water-immiscible ingredients, such as oils, and stabilized with surfactants, such as mono- and diglycerides, PEG esters, and the like. Furthermore, the multinucleotides described herein may be formulated as prodrugs, such that the compounds are synthesized in a manner that allows for direct intracellular delivery of the multinucleotide into cells.

[0061] Aqueous suspensions contain the active material mixed with excipients suitable for the manufacture of aqueous suspensions, such as saline or buffer solutions. Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, and the dispersing or wetting agent may be a naturally occurring phosphatide, such as lecithin, or a condensation product of an alkylene oxide with a fatty acid, such as polyoxyethylene stearate, or a condensation product of ethylene oxide with a long-chain aliphatic alcohol, such as heptadecaethyleneoxycetanol, or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol, such as polyoxyethylene sorbitol monooleate, or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, such as polyethylene sorbitan monooleate. The aqueous suspensions 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.

[0062] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as peanut oil, olive oil, sesame oil, or coconut oil, or in liquid paraffin, such as mineral oil. Oily suspensions may contain thickening agents, such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners, such as those listed above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by adding antioxidants, such as ascorbic acid.

[0063] Dispersible powders and granules suitable for preparation of an aqueous suspension by adding water provide the active ingredient mixed with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweeteners, flavorings, and coloring agents, may also be present.

[0064] The pharmaceutical composition of the present disclosure may be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin, or a mixture thereof. Suitable emulsifiers may be naturally occurring gums, such as acacia gum or tragacanth gum, naturally occurring phosphatides, such as soybean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsifying agent may also contain sweeteners and flavoring agents.

[0065] Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol, or sucrose. Such formulations may also contain demulcents, preservatives, and flavoring and coloring agents. Oral solutions may be prepared in combination with, for example, cyclodextrin, PEG, and surfactants.

[0066] In one embodiment, the composition may be administered intravenously. The pharmaceutical composition may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known techniques using dispersing or wetting agents and suitable suspending agents as described above. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a 1,3-butanediol solution. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any sterile, fixed oil may be used, including synthetic mono- or diglycerides. Additionally, fatty acids, such as oleic acid, have found use in the preparation of injectables.

[0067] As demonstrated by the Examples, intravenous administration of the compositions described herein at significantly lower dosages compared to oral administration has been demonstrated to improve plasma concentrations of the corresponding mononucleosides compared to oral administration of the individual mononucleotide therapies (Example 1).

[0068] In another embodiment, the composition may be administered subcutaneously.

[0069] The compounds of the present disclosure may 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, so that it melts in the rectum and releases the drug. Such materials include cocoa butter and polyethylene glycol.

[0070] The dosage form may contain between about 1 mg / kg and about 20 mg / kg of the multinucleotide composition described herein. In one embodiment, the composition is administered at a dosage of between about 1 mg / kg and about 5 mg / kg. In another embodiment, the composition is administered at a dosage of between about 3 mg / kg and about 8 mg / kg. In another embodiment, the composition is administered at a dosage of between about 5 mg / kg and about 10 mg / kg. In another embodiment, the composition is administered at a dosage of between about 8 mg / kg and about 13 mg / kg. In one embodiment, the composition is administered at a dosage of between about 10 mg / kg and about 15 mg / kg. In yet another embodiment, the composition is administered at a dosage of between about 13 mg / kg and about 18 mg / kg. In one embodiment, the composition is administered at a dosage of between about 15 mg / kg and about 20 mg / kg.

[0071] The compound of formula I is useful for treating mitochondrial DNA depletion syndrome.Examples of such syndrome 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 M2B enzyme deficiency, thymidine phosphorylase deficiency and polymerase gamma deficiency.In one embodiment described herein, the mitochondrial DNA depletion syndrome is thymidine kinase 2 deficiency.

[0072] A compound of formula I may be used in combination with one or more agents having the same spectrum of activity, e.g., to enhance activity, or with substances having a different spectrum of activity, e.g., to broaden the spectrum of activity. Any of the individually listed agents may be used individually in combination with a compound of formula I and one or more of the other listed agents.

[0073] Suitable agents for combination therapy include, for example, therapeutic agents for treating the symptoms of particular forms of MDS, including, but not limited to, inhibitors of ubiquitous nucleosidases, including tetrahydrouridine, triacetyluridine, N-acetylcysteine, N-acetylcysteine ​​amide, vitamin E, isomucillin H, and tipiracil, whereby one or more compounds of Formula I can be used in combination with one or more other pharmaceutically active substances, either on their own or in the form of preparations or formulations thereof. The combinations can be part of the same formulation, or can be administered separately or sequentially.

[0074] The pharmaceutical preparations containing the compound of formula I or its pharmaceutically acceptable salt for delivery to humans or other mammals are preferably in unit dosage form, and the preparations are subdivided into unit doses containing appropriate amounts of active ingredients.The unit dosage form may be a packaged preparation containing individual amounts of the preparation, for example, tablets, capsules, and powders packaged in vials or ampoules.The unit dosage form may also be a capsule, tablet, or injection, or any of the appropriate number of these in packaged form.

[0075] The quantity of active ingredient in a unit dose preparation may be varied or adjusted from about 0.1 mg to about 1000 mg depending on the particular application and potency of the active ingredient. If desired, the composition may also contain other compatible therapeutic agents.

[0076] In therapeutic use for treating mitochondrial DNA depletion syndrome in humans or other mammals, the compounds utilized in the treatment method are administered at an initial dosage of about 0.1 mg / kg to about 100 mg / kg per interval. Preferred intervals may be daily, weekly, semimonthly, monthly, bimonthly, quarterly, triennially, semiannually, or annually. Dosages may be administered once daily, twice daily, three times daily, or as needed as determined by the practitioner. Dosages may vary depending on the patient's requirements, such as the size of the human or mammal being treated, the severity of the condition being treated, the route of administration, and the potency of the compound used. Determining the appropriate dosage and route of administration for a particular situation is within the skill of the practitioner. Generally, treatment will be initiated with a small dosage, which is less than the optimal dose of the compound, and may be increased by small increments until the optimal effect under the particular circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions throughout the day as needed.

[0077] In order to understand the metabolism of the compositions described herein, the plasma level of the multinucleotide metabolized into its corresponding mononucleoside is measured after administration.In one embodiment described herein, the plasma level of the corresponding mononucleoside is higher than that of the administration of individual mononucleotides.In another embodiment, the plasma level of the corresponding nucleoside is about 50ng / mL to about 5000ng / mL.In another embodiment, the plasma level of the corresponding nucleoside is about 75ng / mL to about 2500ng / mL.

[0078] The area under the curve (AUC) for plasma levels over time from 0 to approximately 24 hours 0-24h ) measured between about 5000 ng-h / mL and about 120,000 ng-h / mL for intravenous administration.

[0079] 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 a TK2 deficiency; and administering to the subject a therapeutically effective amount of a compound of Formula I: [ka] (In the formula, R1 is H or OH; R2 is a purine derivative or a pyrimidine derivative; R3 is a purine derivative or a pyrimidine derivative, R4 is H or OH administering a composition comprising a compound of formula (I); measuring the plasma level of the corresponding mononucleoside, wherein the plasma level of the corresponding nucleoside is from about 50 ng / mL to about 5000 ng / mL; The method includes:

[0080] Patients who exhibit phenotypic TK2 deficiency, including the most classic symptoms of a progressive muscle disease characterized by generalized hypotonia, proximal muscle weakness, loss of previously acquired motor skills, poor feeding, and respiratory distress, may be tested to confirm the diagnosis of the disease.

[0081] Molecular genetic testing may be performed using a panel of genes known to cause mtDNA depletion syndrome (Chanprasert et al., 2012). Testing may be performed by sequence analysis of the coding region of TK2. Further testing may be performed to confirm a TK2 deficiency diagnosis, including testing serum creatine kinase levels, electromyography, skeletal muscle histopathology, mitochondrial DNA content (copy number), and electron transport chain (ETC) activity in skeletal muscle. [Example]

[0082] Preparation of Compounds of Formula I The preparation of the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may be achieved via the routes and intermediates set out herein below. Scheme 1

[0083] [ka]

[0084] Scheme 1 describes a general method for preparing compounds of Formula I. Synthesis of diphosphate nucleotides was achieved by coupling the individual nucleotide tributylammonium salts using carbonyldiimidazole as the coupling agent to give the desired products. In forming these dinucleotides, asymmetric compounds such as (dC(P2)dC) and (dT(P2)dT) are also formed; currently used methods are sufficient for small-scale production (1-5 g), but better methods are desirable for large-scale production (hundreds of grams).

[0085] [Example 1] The systemic exposure of two mononucleotides (dCMP and dTMP) and their corresponding mononucleosides (dC and dT) was determined after oral administration of the two mononucleotides (dCMP and dTMP) and intravenous administration of the dinucleotide (dC(P2)dT). The test articles were prepared as follows: Unlabeled Articles to be Tested: dC = deoxycytidine (nucleoside) dT = deoxythymidine (nucleoside) dCMP = deoxycytidine monophosphate (mononucleotide) dTMP = deoxythymidine monophosphate (mononucleotide) dC(P2)dT or dT(P2)dC = deoxycytidine-deoxythymidine diphosphate (dinucleotide) Marked article to be tested: dC * = 15 N3 labeled dC (labeled nucleoside) dT ** = 13 C 10 and 15 N2 labeled dT (labeled nucleoside) The corresponding labeled nucleotide is dC * MP and dT ** MP The corresponding labeled dinucleotide is dC *(P2)dT ** or dT ** (P2)dC * is.

[0086] The structures and metabolism of these test articles are shown in FIG.

[0087] The dose intensity of a single IV injection of dC(P2)dT compared to that of a single PO administration of dCMP+dTMP was determined, resulting in comparable systemic exposure of dC and dT, and systemic exposure was calculated using the AUC 0-24h was quantified as

[0088] Nucleosides (dC and dT) and their corresponding monophosphate nucleotides (dCMP and dTMP) are naturally produced in the body. To distinguish between naturally occurring and exogenously administered dC / dT and dCMP / dTMP, labeled dC * MP / dT ** A mixture of MP and unlabeled dCMP / dTMP was administered via oral route, and labeled dC * (P2)dT ** A mixture of unlabeled dC(P2)dT and unlabeled dC(P2)dT was administered via the IV route. The labeled test article served as a tracer to determine the systemic levels of exogenous dC / dT and dCMP / dTMP after administration.

[0089] Plasma samples were then analyzed for the following analytes: dT ** , dC * , dT ** MP, dC * MP and dC * (P2)dT ** .dT ** and dC * Only dT is detectable in vivo. ** MP, dC * MP and dC * (P2)dT ** The results are shown in Table 1.

[0090] [Table 1]

[0091] As shown in Table 1, 10% of all test articles (TA) administered via the PO route were labeled, and 20% of all TA administered via the parenteral (IV) route were labeled. To correct for the total TA administered (labeled + unlabeled), plasma levels of labeled analytes were multiplied by 10 and 5 for the PO and IV treatment groups, respectively. Additionally, the total TA dose administered was 1000 mg / kg (900 mg / kg unlabeled + 100 mg / kg labeled) and 10 mg / kg (8 mg / kg unlabeled and 2 mg / kg labeled) for the PO and IV treatment groups, respectively. To normalize by the unit dose of TA administered, plasma levels of analytes were divided by 1000 and 10 for the PO and IV treatment groups, respectively. The results of the label-corrected and dose-normalized levels of dT and dC for the PO and IV administration routes are shown in Figure 2.

[0092] FIG. 3 shows the area under the curve (AUC) for total dose-normalized dT and dC plasma levels from FIG. 0-24h ) for both analytes. 0-24h The difference was statistically different (p<0.01, t-test). (0-24h) is approximately 350 for dT and approximately 110 for dC. Thus, based on label-corrected and dose-normalized PK analysis, these data indicated that approximately 350-fold and 110-fold higher doses of oral dTMP and dCMP, respectively, would be required to achieve comparable plasma exposures of dT and dC compared to intravenous dC(P2)dT.

[0093] [Example 2] The systemic exposure of dC and dT was compared after a single IV injection of dC(P2)dT versus one and two subcutaneous (SC) injections of dC(P2)dT, with the second SC injection administered 24 hours after the first. The study design is summarized in Table 2.

[0094] [Table 2]

[0095] The following analytes: dT ** , dC * , dT ** MP, dC * MP and dC * (P2)dT ** Plasma samples were analyzed for dT as in Example 1. ** and dC * Plasma levels of label-normalized dT and dC levels after a single IV dose and after one and two SC doses for the labeled and unlabeled dC(P2)dT groups are shown in Figure 4. Figure 5 shows the plasma exposure for the first 24 hours post-dose for the two analytes for the three treatment groups, which are the mean area under the curve (AUC) for total dose-normalized dT and dC plasma levels from Figure 4. 0-24 h) was determined by calculating the mean AUC across the three treatment groups. 0-24h The values ​​were not statistically different, indicating that IV and SC administration of dC(P2)dT resulted in comparable exposure of dT and dC in plasma.

[0096] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference for the teachings to which such citations are applied.

[0097] The specific responses observed may vary according to and depending on the particular active compound selected or the presence or absence of carriers present, as well as the type of formulation and mode of administration used, and such expected variations or differences in results are contemplated by the practice of the present invention.

[0098] Although specific embodiments of the present invention have been shown 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 of the present invention. Modifications will be obvious to those skilled in the art, and all modifications that do not depart from the spirit of the present invention are intended to be included within the scope of the appended claims.

[0099] References Chansprasert, S. and Craigen WJ, (2017) Mitochondrial Disorders Causing Cardioskeletal Myopathies in Childhood. Cardioskeletal Myopathies in Children and Young Adults. El-Hattab, AW and Scaglia, F., (2013) Mitochondrial DNA depletion syndromes: review and updates of genetic basis, manifestations, and therapeutic options. Neurotherapeutics. Apr:10(2):186-198. 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. , Camara , Y. , Madruga-Garrido , 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 kinase 2 deficiency. J Med Genet. Aug; 55(8):515-521. TK2-related mitochondrial DNA depletion syndrome, myopathic form: National Library of Medicine (US). Genetics Home Reference [Internet]. Bethesda (MD): The Library; 2013 Sep; [reviewed 2013 Sep; cited 2013 Sep 19]; [about 6 screens] Available from: https: / / ghr.nlm.nih.gov / condition / cystic-fibrosis

Claims

1. 1. A method of treating mitochondrial DNA depletion syndrome in a subject in need thereof, comprising administering to a subject a compound of formula I 【Chemistry 1】 (In the formula, R 1 is H or OH, R 2 is a purine derivative or a pyrimidine derivative, R 3 is a purine derivative or a pyrimidine derivative, R 4 is H or OH) administering to a subject a therapeutically effective amount of a multinucleotide composition comprising a compound of formula (I).

2. The composition is R 1 is H and R 2 is 5-methyl-2H-1λ2-pyrimidine-2,4(3H)-dione, and R 3 is 4-amino-2H-1λ2-pyrimidin-2-one, and R 4 10. The method of claim 1, comprising a compound of formula I, wherein is OH.

3. The composition is R 1 is OH and R 2 is 9λ2-purin-6-amine, and R 3 is 2-amino-9λ2-purin-6(1H)-one, and R 4 10. The method of claim 1, wherein the mixture further comprises a compound of formula I wherein is OH.

4. 4. The method of any one of claims 1 to 3, wherein the composition is administered orally, enterally, intravenously, or subcutaneously.

5. 5. The method of any one of claims 1 to 4, wherein the composition is administered intravenously.

6. 6. The method of claim 5, wherein the plasma level of each mononucleoside is higher after intravenous administration compared to the plasma level after oral administration of the individual mononucleotide.

7. Intravenous administration of the composition results in a first AUC 0-24h and oral administration of the individual mononucleotides results in plasma levels having a second AUC 0-24h and the first AUC 0-24h and the second AUC 0-24h 7. The method of claim 1, wherein the ratio of 0 to 100 is between about 100 and about 400.

8. 8. The method of any one of claims 1 to 7, wherein when the composition is administered at a dosage of between about 1 mg / kg and about 20 mg / kg, the plasma concentration of the corresponding nucleoside is between about 50 ng / ml and about 5000 ng / ml.

9. 9. The method of any one of claims 1 to 8, wherein the mitochondrial DNA depletion 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.

10. 10. The method of claim 9, wherein the mitochondrial DNA depletion syndrome comprises thymidine kinase 2 deficiency.

11. 11. The method of any one of claims 1 to 10, wherein the subject is a human.

12. 1. A method for treating thymidine kinase 2 (TK2) deficiency in a subject in need thereof, comprising: a) obtaining a nucleic acid sample from a subject; b) determining whether the subject has a TK2 deficiency; c) Equation 1: 【Chemistry 2】 (In the formula, R 1 is H or OH, R 2 is a purine derivative or a pyrimidine derivative, R 3 is a purine derivative or a pyrimidine derivative, R 4 is H or OH) administering a therapeutically effective amount of a composition comprising a multinucleotide compound of d) measuring the plasma level of the corresponding mononucleoside, wherein the plasma level of the corresponding nucleoside is from about 50 ng / mL to about 5000 ng / mL; A method comprising:

13. Compounds of Formula I: 【Transformation 3】 (In the formula, R 1 is H or OH, R 2 is a purine derivative or a pyrimidine derivative, R 3 is a purine derivative or a pyrimidine derivative, R 4 is H or OH).

14. 14. The compound of claim 13, substantially free of impurities.

15. R 1 is H and R 2 is thymine and R 3 is cytosine, R 4 15. The compound of claim 13 or 14, wherein is OH.

16. 16. A pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and a therapeutically effective amount of a compound according to any one of claims 13 to 15.

17. R 1 is H and R 2 is 5-methyl-2H-1λ2-pyrimidine-2,4(3H)-dione, and R 3 is 4-amino-2H-1λ2-pyrimidin-2-one, and R 4 The composition of claim 16, comprising a compound wherein is OH.

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