Deoxynucleoside prodrugs for treating diseases caused by imbalanced nucleotide pools

Abstract

Provided herein are deoxynucleotide prodrugs for the treatment of diseases characterized by imbalanced nucleotide pools.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 656,861, filed April 12, 2018, which is incorporated herein by reference in its entirety.

[0002] The present invention relates generally to prodrugs for delivering deoxynucleosides and their use in the treatment of diseases caused by imbalanced nucleotide pools, including mitochondrial DNA depletion syndrome. [Background technology]

[0003] Mitochondrial diseases are clinically diverse disorders due to defects in the mitochondrial respiratory chain (RC) and oxidative phosphorylation, a biochemical pathway that converts electron energy into adenosine triphosphate (ATP). The respiratory chain is composed of four multi-subunit enzymes (complexes I-IV) that transfer electrons to generate a proton gradient across the inner membrane of mitochondria, and this proton flow drives ATP synthesis via complex V (Non-Patent Documents 1 and 2). Coenzyme Qio (CoQio) is an essential molecule that shuttles electrons from complexes I and II to complex III. The respiratory chain is unique to eukaryotic organisms, e.g., mammalian cells, because it is controlled by two genomes: mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). As a result, mutations in either genome can cause mitochondrial disease. Most mitochondrial diseases affect multiple body organs and are usually fatal in childhood or early adulthood. There are no proven effective treatments for mitochondrial diseases, only supportive therapies such as administration of CoQio and its analogues to increase respiratory chain activity and neutralize reactive oxygen species (ROS), which are toxic by-products of dysfunctional respiratory chain enzymes.

[0004] Mitochondrial DNA depletion syndrome (MDS), a subgroup of mitochondrial diseases, is the cause of many severe childhood encephalomyopathy cases, whose molecular characteristics are a reduction in mitochondrial DNA (mtDNA) copy number in tissues and insufficient synthesis of the mitochondrial RC complex (Non-Patent Document 3). Mutations in several nuclear genes, such as TK2, DGUOK, POLG, POLG2, SCLA25A4, MPV17, RRM2B, SUCLA2, SUCLG1, TYMP, OPA1, and ClOorfl (PEOl), have been identified as the cause of infantile MDS (Non-Patent Documents 4-12). Furthermore, mutations in these nuclear genes can also cause multiple deletions of mtDNA with or without mtDNA depletion (Non-Patent Documents 13-22).

[0005] One of these genes is TK2, which encodes thymidine kinase (TK2), a mitochondrial enzyme required for phosphorylation of pyrimidine nucleosides (thymidine and deoxycytidine) to generate deoxycytidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP) (Non-Patent Document 23). Mutations in TK2 impair the mitochondrial nucleoside / nucleotide salvage pathway required for the synthesis of deoxynucleotide triphosphates (dNTPs), components of mDNA replication and repair.

[0006] TK2 deficiency was first described in 2001 by Saada and coworkers in four affected children from four different families with severe devastating myopathy (24). After favorable early development, between 6 and 36 months of age, patients developed hyperCKemia, severe hypotonia, and then loss of spontaneous activity. The disease progressed rapidly, with two patients being placed on a ventilator by 3 years, whereas the other two had already died at the time of the report.

[0007] After the initial description, 60 additional patients were reported in the literature and at least 26 patients were further diagnosed but not reported (25-45), resulting in 90 patients, 53 men and 37 women. The 26 most recently diagnosed patients were identified by next-generation DNA sequencing. This large number of newly identified cases suggests that TK2 deficiency is an underdiagnosed disorder.

[0008] TK2 deficiency displays a wide clinical and molecular genetic spectrum, with the majority of patients presenting with a devastating clinical course in early childhood, whereas others show a slowly progressive decline over decades.

[0009] Like most MDS and mitochondrial disorders, treatment of TK2 deficiency has been limited to supportive care. Administration of deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP) has been shown to improve the condition of both TK2 knock-in mutant mice and human patients with TK2 deficiency (Patent Document 1, which is incorporated herein in its entirety), as has administration of deoxynucleosides (e.g., deoxythymidine (dT) or deoxycytidine (dC) or mixtures thereof) (Patent Document 2, which is also incorporated herein in its entirety). However, there remains a need for additional therapeutic interventions for TK2 deficiency.

[0010] Additionally, there is a need for treatment of other forms of MDS, and other diseases characterized by imbalanced nucleotide pools. For example, several Mendelian genetic diseases involving mtDNA depletion or multiple deletions, or both, are characterized by imbalanced deoxynucleotide triphosphate pools that lead to defective mtDNA replication. One such disorder, DGUOK mutations, impair the intramitochondrial enzyme deoxyguanosine kinase, which normally phosphorylates the deoxypurine nucleosides deoxyguanosine and deoxycytidine to produce deoxyguanosine monophosphate (dGMP) and deoxycytidine monophosphate (dCMP). Other nuclear genes that disrupt mitochondrial dNTP pools include TYMP, RRM2B, SUCLA2, SUCLG1, and MPV17. Therapies that restore balance to the dNTP pools would be similarly useful in treating these disorders. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent Application Serial No. 15 / 082,207 [Patent Document 2] International Publication No. 2016205671 Brochure [Non-patent literature]

[0012] [Non-Patent Document 1] DiMauro and Schon 2003; [Non-Patent Document 2] DiMauro and Hirano 2005 [Non-Patent Document 3] Hirano et al. 2001 [Non-Patent Document 4] Bourdon et al. 2007 [Non-Patent Document 5] Copeland 2008; [Non-Patent Document 6] Elpeleg, et al. 2005;

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[0013] [Means for solving the problem]

[0014] The present invention relates generally to prodrugs for the delivery of deoxynucleosides. In one aspect, the present invention provides a compound of formula I: [ka]

[0015] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; R 1 is an optionally substituted acyl, an optionally substituted O-linked amino acid, [ka] wherein X, Y and Z are each independently selected from O and S; R 2 , R 3 and R 4 are each hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, optionally substituted C 3-6Cycloalkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryl (C 1-6 ) alkyl, [ka] or R 2 and R 3 and can be taken together to form a cyclic moiety, R 5 , R 6 and R 7 are each optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, NR 20 R 21 , an optionally substituted N-linked amino acid, and an optionally substituted N-linked amino acid ester; R 8 , R 9 , R 11 and R 12 are each independently selected from hydrogen, optionally substituted C alkyl, and optionally substituted aryl; R 10 and R 13 are each independently selected from hydrogen, optionally substituted C1-24 alkyl and optionally substituted aryl, optionally substituted -O-C1-24 alkyl, optionally substituted -O-aryl, optionally substituted -O-heteroaryl, optionally substituted -O-monocyclic heterocyclyl; R 14 , R 15 and R 19 are each hydrogen, optionally substituted C 1-24 independently selected from alkyl, and optionally substituted aryl; R 16 and R17 are -CN, optionally substituted C 2-8 Organyl carbonyl, C 2-8 Alkoxycarbonyl and C 2-8 is independently selected from organylaminocarbonyl, R 18 is hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, and optionally substituted C 3-6 cycloalkenyl; R 20 and R 21 are each hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, and optionally substituted C 3-6 cycloalkenyl, and Each of n, m and p is independently selected from 0, 1, 2 or 3.

[0016] In another embodiment, the prodrug is a compound of formula II: [ka]

[0017] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; X is selected from S and O, and R 22 -O - , -OH, -O-alkyl, optionally substituted C 1-6 Alkoxy, [ka] , optionally substituted N-linked amino acids, and optionally substituted N-linked amino acid esters, wherein R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , n, m and p are defined as above.

[0018] In yet another embodiment, the prodrug is a compound of formula III: [ka]

[0019] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; R 1 and R 2 are independently selected from hydrogen, phosphate (including monophosphates, diphosphates or triphosphates of Formula I, and modified phosphates), linear, branched or cyclic alkyl, acyl, CO-alkyl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate ester, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, lipids, phospholipids, amino acids, carbohydrates, peptides and cholesterol).

[0020] In yet another aspect, the present invention further generally relates to a method of treating a disease or disorder characterized by an imbalanced nucleotide pool in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one prodrug of the present invention. The prodrug can be administered per se (i.e., alone) or in the form of a pharmaceutical composition.

[0021] Suitable diseases or disorders include, but are not limited to, TK2 deficiency, RRM2B deficiency, mutations in TYMP, SUCLA2 deficiency, SUCLG1 deficiency, MPV17 deficiency, and DGUOK mutations.

[0022] Administration can be via any route, including, but not limited to, intrathecal, parental, mucosal, and transdermal.

[0023] The dosage of the at least one prodrug or a composition comprising same can be from about 25 mg / kg / day to about 1,000 mg / kg / day. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description of the Invention I. Definition As used herein, "subject" refers to a mammal. Mammals include dogs, cats, rodents, cattle, horses, pigs, sheep and primates. Thus, the present invention can be used in veterinary medicine, for example, to treat companion animals, livestock, zoo laboratory animals, and wild animals. The present invention is particularly desirable for human medical use.

[0025] As used herein, "patient" refers to human subject.In some embodiments of the present invention, "patient" is known or suspected to have mitochondrial disease, mitochondrial DNA depletion syndrome or TK2 deficiency, which are diseases or disorders characterized by unbalanced nucleotide pools.

[0026] As used herein, a "therapeutically effective amount" refers to an amount sufficient to cause an improvement in a clinically significant condition in a subject, or to delay or minimize or alleviate one or more symptoms associated with a disease or disorder, or to effect a desired physiologically beneficial change in a subject.

[0027] As used herein, "treat," "treatment," and the like refer to a means of slowing, alleviating, ameliorating, or alleviating at least one symptom of a disease or disorder, or reversing a disease or disorder after onset.

[0028] As used herein, "prevent", "prevention" and the like refer to acting prior to the onset of an overt disease or disorder to prevent the onset of the disease or disorder, minimize the extent of the disease or disorder, or slow the course of its development.

[0029] As used herein, "in need thereof" refers to a subject known or suspected of having a mitochondrial disease, mitochondrial DNA depletion syndrome, or TK2 deficiency, which are diseases or disorders characterized by imbalanced nucleotide pools.

[0030] As used herein, "prodrug" refers to a derivative of a deoxynucleoside that is converted under conditions of use, such as in the body, to release a deoxynucleoside. Prodrugs are often, but not necessarily, pharmacologically inactive until converted to an active form. Prodrugs can be obtained by attaching a promoiety to a drug, usually via a functional group.

[0031] As used herein, a "promoiety" refers to a group, typically a functional group, attached to a deoxynucleoside via a bond that is cleavable under certain conditions of use. The bond between the drug and the promoiety may be cleaved by enzymatic or non-enzymatic means. Under conditions of use, for example after administration to a patient, the bond between the drug and the promoiety may be cleaved to release the parent drug. Cleavage of the promoiety may proceed spontaneously, such as via a hydrolysis reaction, or may be catalyzed or induced by another agent, such as an enzyme, light, acid, or by a change in, or exposure to, a physical or environmental parameter, such as a change in temperature, pH, or the like. The agent may be endogenous to the conditions of use, such as an enzyme present in the systemic circulation of the patient to whom the prodrug is administered, or to the acidic conditions of the stomach, or the agent may be provided exogenously.

[0032] As used herein, a "side effect" is an undesirable reaction caused by administration of a drug. In most cases, administration of deoxynucleosides has not caused any side effects. The most expected side effect is mild gastrointestinal intolerance.

[0033] As used herein, "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as pharmaceutical formulation. For example, "about" can mean within 1 or more than 1 standard deviation, according to the practice in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. When a particular value is described in the application and claims, unless otherwise indicated, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0034] Whenever a group is described as "optionally substituted," the group may be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when "unsubstituted or substituted" is described, if the group is substituted, the substituents may be selected from one or more of the indicated substituents. When no substituents are indicated, the indicated "optionally substituted" or "substituted" group can be any of the following: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxy, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C- It means that the amino group may be substituted with one or more groups individually and independently selected from amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, azido, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, amino, mono-substituted amino groups, and di-substituted amino groups, and protected derivatives thereof.

[0035] As used herein, "Ca-Cb", where "a" and "b" are integers, refers to the number of carbon atoms in an alkyl, alkenyl, or alkynyl group, or the number of carbon atoms in a ring of a cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl group. That is, the alkyl, alkenyl, alkynyl, cycloalkyl ring, cycloalkenyl ring, aryl ring, heteroaryl ring, or heterocyclyl ring contains from "a" to "b" carbon atoms. Thus, for example, a "C1-C4 alkyl" group refers to all alkyl groups having 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-. When "a" and "b" are not specified for an alkyl, alkenyl, alkynyl, cycloalkylcycloalkenyl, aryl, heteroaryl, or heterocyclyl group, the broadest range set forth in these definitions is assumed.

[0036] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that contains a fully saturated (no double or triple bonds) hydrocarbon group. An alkyl group may have 1 to 20 carbon atoms (wherever it appears herein, a numerical range such as "1 to 20" refers to each integer within the given range. For example, "1 to 20 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., and may contain up to 20 carbon atoms, although this definition also applies when the term "alkyl" appears without a numerical range specified. An alkyl group may also be a medium-sized alkyl having 1 to 10 carbon atoms. An alkyl group may also be a lower alkyl having 1 to 6 carbon atoms. The alkyl groups of the compounds may be any of the following: A alkyl group may be designated as "C1-C4 alkyl" or a similar designation. By way of example only, "C1-C4 alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, and hexyl. Alkyl groups may be substituted or unsubstituted.

[0037] As used herein, "alkenyl" refers to an alkyl group that contains one or more double bonds in a straight or branched hydrocarbon chain. Examples of alkenyl groups include allenyl, vinylmethyl and ethenyl. Alkenyl groups can be unsubstituted or substituted.

[0038] As used herein, "alkynyl" refers to an alkyl group that contains one or more triple bonds in a straight or branched hydrocarbon chain. Examples of alkynyl include ethynyl and propynyl. Alkynyl groups can be unsubstituted or substituted.

[0039] As used herein, "cycloalkyl" refers to a monocyclic or polycyclic hydrocarbon ring system that is fully saturated (having no double or triple bonds). When composed of two or more rings, the rings may be fused together. Cycloalkyl groups can contain 3 to 10 atoms in the ring, or 3 to 8 atoms in the ring. Cycloalkyl groups can be unsubstituted or substituted. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0040] As used herein, "cycloalkenyl" refers to a monocyclic or polycyclic hydrocarbon ring system containing one or more double bonds in at least one ring, provided that if there are more than one, the double bonds cannot form a completely delocalized pi-electron system throughout all rings (otherwise the group would be "aryl" as defined herein). When composed of more than one ring, the rings may be fused together and connected. Cycloalkenyls may contain 3 to 10 atoms in the ring, or 3 to 8 atoms in the ring. Cycloalkenyl groups may be unsubstituted or substituted.

[0041] As used herein, "aryl" refers to a carbocyclic (all carbon) monocyclic or polycyclic aromatic ring system (including fused ring systems in which two carbon rings share a chemical bond) with a fully delocalized pi-electron system throughout all rings. The number of carbon atoms in an aryl group can vary. For example, an aryl group can be a C6-C14 aryl group, a C6-C10 aryl group, or a C6 aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene, and azulene. An aryl group can be substituted or unsubstituted.

[0042] As used herein, "heteroaryl" refers to monocyclic, bicyclic, and tricyclic aromatic ring systems (ring systems having a fully delocalized pi-electron system) containing one or more heteroatoms (e.g., 1-5 heteroatoms), i.e., elements other than carbon, such as, but not limited to, nitrogen, oxygen, and sulfur. The number of atoms in the rings of a heteroaryl group can vary. For example, a heteroaryl group can contain 4-14 atoms in the ring, 5-10 atoms in the ring, or 5-6 atoms in the ring. Additionally, the term "heteroaryl" includes fused ring systems in which two rings share at least one chemical bond, such as at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings. Examples of heteroaryl rings include, but are not limited to, furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole, 1,2,4-thiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, and triazine. Heteroaryl groups can be substituted or unsubstituted.

[0043] As used herein, "heterocyclyl" or "heteroalicyclyl" refers to monocyclic, bicyclic, and tricyclic ring systems in which 3, 4, 5, 6, 7, 8, 9, 10, and up to 18 members of carbon atoms, together with 1-5 heteroatoms, constitute the ring system. Heterocycles may optionally contain one or more unsaturated bonds arranged in such a way that a completely delocalized pi-electron system does not occur throughout all rings. Heteroatoms are elements other than carbon, such as, but not limited to, oxygen, sulfur, and nitrogen. Heterocycles may further contain one or more carbonyl or thiocarbonyl functional groups, and thus this definition is meant to include oxo and thio systems, such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. When composed of two or more rings, the rings may be fused together and joined. Additionally, the nitrogen of a heteroalicyclic may be quaternized. A heterocyclyl or heteroalicyclic group can be unsubstituted or substituted.Examples of such "heterocyclyl" or "heteroalicyclyl" groups include, but are not limited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiine, 1,3-oxathiolane, 1,3-dithiol, 1,3-dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine. , imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidine N-oxide, piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone, and their benzo-fused analogues (e.g., benzimidazolidinone, tetrahydroquinoline, and 3,4-methylenedioxyphenyl).

[0044] As used herein, "aralkyl" and "aryl(alkyl)" refer to an aryl group connected as a substituent through a lower alkylene group. The lower alkylene and aryl groups of the aryl(alkyl) may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenyl(alkyl), 3-phenyl(alkyl) and naphthyl(alkyl).

[0045] As used herein, "heteroaralkyl" and "heteroaryl(alkyl)" refer to a heteroaryl group connected as a substituent via a lower alkylene group. The lower alkylene and heteroaryl groups of heteroaryl(alkyl) may be substituted or unsubstituted. Examples include, but are not limited to, 2-thienyl(alkyl), 3-thienyl(alkyl), furyl(alkyl), thienyl(alkyl), pyrrolyl(alkyl), pyridyl(alkyl), isoxazolyl(alkyl), imidazolyl(alkyl), and their benzo-fused analogs.

[0046] As used herein, "alkoxy" refers to the formula -OR, where R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaryl(alkyl) or heterocyclyl(alkyl) as defined herein. A non-limiting list of alkoxy is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, phenoxy, and benzoxy. Alkoxy may be substituted or unsubstituted.

[0047] As used herein, "acyl" refers to hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaryl(alkyl) or heterocyclyl(alkyl) connected as a substituent through a carbonyl group. Examples include formyl, acetyl, propanoyl, benzoyl, and acryl. Acyl may be substituted or unsubstituted.

[0048] As used herein, the term "heterocyclic base" refers to an optionally substituted nitrogen-containing heterocyclyl that can be attached to an optionally substituted pentose moiety or a modified pentose moiety. In some embodiments, the heterocyclic base can be selected from an optionally substituted purine base, an optionally substituted pyrimidine base, and an optionally substituted triazole base (e.g., 1,2,4-triazole). The term "purine base" is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. A non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine). An example of an optionally substituted triazole base is 1,2,4-triazole-3-carboxamide. Other non-limiting examples of heterocyclic bases include diaminopurine, 8-oxo-N 6 -Alkyl adenines (e.g., 8-oxo-N 6 -methyladenine), 7-deazaxanthine, 7-deazaguanine, 7-deazaadenine, N 4 ,N 4 -Ethanocytosin, N 6 ,N 6 Heterocyclic bases include -ethano-2,6-diaminopurine, 5-halouracils (e.g., 5-fluorouracil and 5-bromouracil), pseudoisocytosine, isocytosine, isoguanine, and other heterocyclic bases described in U.S. Patent Nos. 5,432,272 and 7,125,855, which are incorporated herein by reference for the limited purpose of disclosing additional heterocyclic bases. In some embodiments, the heterocyclic bases may be optionally substituted with amine or enol protecting groups.

[0049] As used herein, "-N-linked amino acid" refers to an amino acid that is linked to the indicated moiety through a main chain amino or monosubstituted amino group. When an amino acid is linked to an -N-linked amino acid, one of the hydrogens that is part of the main chain amino or monosubstituted amino group is absent, and the amino acid is linked through a nitrogen. An N-linked amino acid may be substituted or unsubstituted.

[0050] As used herein, "-N-linked amino acid ester" refers to an amino acid in which the backbone carboxylic acid group has been converted to an ester group. In some embodiments, the ester group has a formula selected from alkyl-OC(=O)-, cycloalkyl-OC(=O)-, aryl-OC(=O)-, and aryl(alkyl)-OC(=O)-. A non-limiting list of ester groups includes substituted and unsubstituted versions of the following: methyl-OC(=O)-, ethyl-OC(=O)-, n-propyl-OC(=O)-, isopropyl-OC(=O)-, n-butyl-OC(=O)-, isobutyl-OC(=O)-, tert-butyl-OC(=O)-, neopentyl-OC(=O)-, cyclopropyl-OC(=O)-, cyclobutyl-OC(=O)-, cyclopentyl-OC(=O)-, cyclohexyl-OC(=O)-, phenyl-OC(=O)-, benzyl-OC(=O)-, and naphthyl-OC(=O)-. N-linked amino acid ester derivatives can be substituted or unsubstituted.

[0051] As used herein, "-O-linked amino acid" refers to an amino acid that is attached to the indicated moiety through a hydroxy from its backbone carboxylic acid group. When an amino acid is attached to an -O-linked amino acid, the hydrogen that is part of the hydroxy from its backbone carboxylic acid group is not present, and the amino acid is attached through the oxygen. O-linked amino acids may be substituted or unsubstituted.

[0052] As used herein, the term "amino acid" refers to any amino acid (both standard and non-standard amino acids), including, but not limited to, α-amino acids, β-amino acids, γ-amino acids, and δ-amino acids. Examples of suitable amino acids include, but are not limited to, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Additional examples of suitable amino acids include, but are not limited to, ornithine, hypusine, 2-aminoisobutyric acid, dehydroalanine, γ-aminobutyric acid, citrulline, beta-alanine, alpha-ethyl-glycine, alpha-propyl-glycine, and norleucine.

[0053] The term "amino acid" includes natural and synthetic β, γ or δ amino acids, which may be in the D or L configuration. The amino acid may be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleucinyl, β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl, or β-histidinyl.

[0054] As used herein, the term "deoxynucleoside" refers to any nucleoside that contains a deoxy sugar, i.e., any compound formally derived from a sugar by replacing a hydroxy group with a hydrogen atom, e.g., deoxyribose. As used herein, the term "dNTP" refers to deoxyribonucleotide triphosphate. Each dNTP is composed of a phosphate group, a deoxyribose sugar, and a nitrogenous base. There are four different dNTPs, which can be divided into two groups: purines and pyrimidines. dATP (deoxyadenosine 5'-triphosphate) and dGTP (deoxyguanosine 5'-triphosphate) constitute the purines, while dTTP (deoxythymidine 5'-triphosphate) and dCTP (deoxycytidine 5'-triphosphate) constitute the pyrimidines. The purine-characteristic bases adenine and guanine both have a double ring structure, while the purine-characteristic bases thymine and cytosine both have a single ring structure.

[0055] The term "mitochondrial DNA depletion syndrome" as used herein refers to a class of phenotypically diverse diseases and disorders characterized by a severe reduction in mitochondrial DNA (mtDNA) content in affected tissues and organs, e.g., muscle, liver, brain, and / or gastrointestinal tract. The reduction or depletion may result from an imbalance in the mitochondrial nucleotide pool available for mtDNA replication, and from abnormalities in mitochondrial replication. Based on the age of onset, two subtypes are distinguished: congenital (or early onset) and infantile (or late onset). Although the later onset type results in a longer survival time, the syndrome is fatal in almost all patients, and no effective treatment currently exists.

[0056] I. Prodrugs The present invention provides deoxynucleotide prodrugs. "Deoxynucleoside" refers to 2'-deoxynucleosides, such as deoxycytidine (dC, shown below), deoxythymidine (dT), deoxyadenosine (dA) and deoxyguanosine (dG). The full length names and common abbreviations of each are used interchangeably.

[0057] In certain embodiments, the bases are selected from cytosine, thymine, guanine, and adenine.

[0058] Thus, the prodrugs described herein are deoxycytidine prodrugs (dC prodrugs), deoxythymidine prodrugs (dT prodrugs), deoxyguanosine prodrugs (dG prodrugs) and deoxyadenosine prodrugs (dA prodrugs).

[0059] [ka]

[0060] The prodrugs are preferably in the natural β-D-configuration.

[0061] In one embodiment, the prodrug strategy involves masking reactive groups, such as charged -OH and phosphate groups, in vivo to allow crossing of cell membranes.

[0062] In one embodiment, the present invention provides a prodrug of formula I: [ka]

[0063] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; R 1 is an optionally substituted acyl, an optionally substituted O-linked amino acid, [ka] is selected from the group consisting of Each of X, Y, and Z is independently selected from O and S; R 2 , R 3 and R 4 are each hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24Alkynyl, optionally substituted C 3-6 Cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryl (C 1-6 ) alkyl, [ka] or R 2 and R 3 and can be taken together to form a cyclic moiety, R 5 , R 6 and R 7 are each optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, NR 20 R 21 , an optionally substituted N-linked amino acid, and an optionally substituted N-linked amino acid ester; R 8 , R 9 , R 11 and R 12 are each independently selected from hydrogen, optionally substituted C alkyl, and optionally substituted aryl; R 10 and R 13 are each independently selected from hydrogen, optionally substituted C1-24 alkyl and optionally substituted aryl, optionally substituted -O-C1-24 alkyl, optionally substituted -O-aryl, optionally substituted -O-heteroaryl, optionally substituted -O-monocyclic heterocyclyl; R 14 , R 15 and R 19 are each hydrogen, optionally substituted C 1-24independently selected from alkyl, and optionally substituted aryl; R 16 and R 17 are -CN, optionally substituted C 2-8 Organyl carbonyl, C 2-8 Alkoxycarbonyl and C 2-8 is independently selected from organylaminocarbonyl, R 18 is hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, and optionally substituted C 3-6 cycloalkenyl; R 20 and R 21 are each hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, and optionally substituted C 3-6 cycloalkenyl, and Each of n, m and p is independently selected from 0, 1, 2 or 3.

[0064] In certain embodiments, the prodrug is a compound of formula Ia: [ka]

[0065] In the formula, R 1 , R 2 and R 3 is hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6Cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted aryl (C 1-6 ) alkyl.

[0066] In certain embodiments, R 1 is aryl, and R 2 is C 1-24 alkyl, and R 3 is C 1-24 It is an alkyl.

[0067] In another particular embodiment, R 2 is an amino acid side chain.

[0068] In more particular embodiments, the prodrug is selected from one of the following compounds: [ka]

[0069] In another embodiment, the present invention provides a prodrug of formula II: [ka]

[0070] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; X is selected from S and O, and R 22 -O - , -OH, -O-alkyl, optionally substituted C 1-6 Alkoxy, [ka] , optionally substituted N-linked amino acids and optionally substituted N-linked amino acid esters, 8, R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , n, m and p are defined as above.

[0071] In certain embodiments, the prodrug is a compound of formula IIa: [ka]

[0072] In the formula, R is C 1-4 It is an alkyl.

[0073] In yet another particular embodiment, the prodrug is selected from one of the following compounds: [ka]

[0074] In yet another embodiment, the present invention provides a prodrug of formula III: [ka]

[0075] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; R 1 and R 2 are independently selected from hydrogen, phosphate (including monophosphates, diphosphates, or triphosphates of Formula I, and modified phosphates), linear, branched or cyclic alkyl, optionally substituted acyl, CO-alkyl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate ester, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, lipids, phospholipids, amino acids, carbohydrates, peptides and cholesterol).

[0076] In one embodiment, R 2 is an amino acid, i.e., the compound of formula III is a 3'-amino acid ester, e.g., the compound of formula IIIa: [ka]

[0077] where R is the side chain of an amino acid.

[0078] In certain embodiments, the amino acid is valine (i.e., R is CH(CH3)2).

[0079] In a more particular embodiment, the prodrug is a compound of formula IIIb: [ka]

[0080] In the formula, base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group.

[0081] In even more particular embodiments, the prodrug is selected from one of the following compounds: [ka]

[0082] In yet another particular embodiment, the prodrug is selected from one of the following compounds: [ka]

[0083] In yet another embodiment, the prodrug is a compound of formula IIIc: [ka]

[0084] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; Ra is a straight, branched or cyclic alkyl and R is the side chain of an amino acid.

[0085] In certain embodiments, Ra is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl, and R is CH(CH3)2).

[0086] II. How to use The present invention provides methods for treating a disease or disorder characterized by imbalanced nucleotide pools in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one prodrug described herein.

[0087] In one embodiment, the prodrugs described above may be utilized in the present methods.

[0088] A prodrug can be administered by itself (i.e., alone) or in the form of a pharmaceutical composition. A pharmaceutical composition containing one or more prodrugs for administration can include a therapeutically effective amount of the prodrug and a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically acceptable and do not normally produce allergic reactions or similar adverse reactions, such as acute gastric peristalsis, dizziness, etc., when administered to humans, and that are approved by federal or state government regulatory agencies or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more specifically in humans. A "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as saline, and oils, including oils of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil. When the pharmaceutical composition is administered intravenously, saline is the preferred carrier. Physiological saline and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, especially for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc.The composition can also contain small amounts of wetting or emulsifying agents, or pH buffering agents, if necessary.

[0089] In certain embodiments, the disclosed methods can be used to treat mitochondrial DNA (mtDNA) depletion syndrome (MDS). Each nucleated cell contains hundreds of mitochondria. Mitochondria are unique organelles that are under two strands of genome control. Although mitochondria contain their own DNA, mtDNA, most of the mitochondrial proteins, including all proteins required for mtDNA replication, transcription, and repair, are encoded by nuclear genes.

[0090] Maintenance of mtDNA requires proteins essential for mtDNA synthesis, maintenance of mitochondrial nucleotide pools, and mediating mitochondrial fusion. Enzymes that synthesize mtDNA require a balanced supply of intramitochondrial nucleotides. These are supplied through the mitochondrial nucleotide salvage pathway and through import of nucleotides from the cytosol via specific transporters. The amounts of these enzymes must be properly balanced to function properly in mtDNA synthesis. Proteins known to be required for mtDNA synthesis are encoded by nuclear genes. When pathogenic variants disrupt the function of any one of the proteins encoded by these genes, mtDNA synthesis is impaired, resulting in quantitative defects in mtDNA (mtDNA depletion) or qualitative defects in mtDNA (multiple mtDNA deletions). To date, pathogenic variants in more than 20 nuclear genes are known to be associated with defects in mtDNA maintenance.

[0091] In one embodiment, mtDNA depletion syndrome is nuclear DNA-based mtDNA depletion syndrome.In certain embodiments, nuclear DNA encodes the protein involved in nucleotide metabolism.The imbalance of free nucleotide concentration leads to the impairment of mtDNA replication, and as a result, leads to the reduction of mtDNA copy number.

[0092] In certain embodiments, the method of the present invention is useful for treating diseases or disorders caused by imbalance of deoxyribonucleoside triphosphate (dNTP) pool.dTNP is the precursor used by DNA polymerase for the replication and repair of nuclear and mitochondrial DNA in animal cells.The concentration of dNTP depends on the balance of synthesis, consumption and degradation.

[0093] Accurate DNA synthesis requires adequate amounts of each dNTP, and properly balanced dNTP pools. The size of the total cellular pools ranges from 10 to 100 pmol / million cells of each dNTP during S-phase, with the mitochondrial pool accounting for up to 10% of the total. In quiescent or differentiated cells, the pools are approximately 10 times smaller in both the cytosol and mitochondria. Contrary to what would be expected on the basis that the four nitrogenous bases in DNA are in roughly equimolar amounts, the four dNTPs are present in the pools in different ratios, with pyrimidines often exceeding purines. Individual cell lines may exhibit different pool compositions, even when derived from the same animal species. An increase in the concentration of one dNTP usually leads to the depletion of another dNTP.

[0094] The mtDNA depletion syndrome may be associated with a particular tissue or organ, such as muscle, liver, brain, and / or gastrointestinal tract. In certain embodiments, the mtDNA depletion syndrome is a myopathy syndrome or a hepatocerebral syndrome.

[0095] Exemplary disorders characterized by imbalanced nucleotide pools include, but are not limited to, TK2 deficiency, RRM2B-related deficiency (encoding p53R2, the p53-inducible small subunit of ribonucleotide reductase, RNR), and mutations in TYMP (encoding thymidine phosphorylase, TP) that cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Additional nuclear genes that disrupt mitochondrial dNTP pools include, but are not limited to, SUCLA2, SUCLG1, and MPV17. Parallel defects in deoxyguanosine kinase (dGK), due to autosomal recessive mutations in DGUOK with dGMP and dAMP deficiencies, cause mtDNA depletion that usually manifests as an early childhood-onset hepatocerebral disease (Mandel, et al. 2001). Disorders associated with these genes can also be treated with the methods herein.

[0096] In certain embodiments, the disorder is thymidine kinase 2 (TK2) deficiency. TK2 is a mitochondrial enzyme involved in salvaging the deoxyribonucleotides required for mitochondrial DNA (mtDNA) replication. TK2 catalyzes the first rate-limiting step of the deoxypyrimidine salvage pathway. Autosomal recessive TK2 mutations cause a spectrum of infantile to adult onset diseases that primarily manifest as myopathy.

[0097] As the mechanisms of other forms of MDS and other disorders become elucidated, the appropriate deoxynucleoside for treatment can be determined by the skilled practitioner.

[0098] For example, a patient with a TK2 deficiency is administered a prodrug of dC and / or dT. In another example, a patient with a DGUOK deficiency is administered a prodrug of dG and / or dA.

[0099] In one embodiment, the method further comprises identifying the patient with disease or disorder characterized by unbalanced nucleotide pool.In one embodiment, the disease or disorder is TK2 deficiency.Patients who show the above phenotype of TK2 deficiency, including the most typical symptoms of progressive muscle disease characterized by generalized hypotonia, proximal muscle weakness, loss of previously acquired motor skills, poor feeding, and difficulty breathing, can be tested for definitive diagnosis of disease.

[0100] Molecular genetic testing using a panel of genes known to cause mtDNA depletion syndromes should be performed (Chanprasert, et al. 2012) and can also be used to identify patients with diseases or disorders characterized by imbalanced nucleotide pools.

[0101] The TK2 gene is the only gene whose mutations are known to cause TK2-related mitochondrial DNA depletion syndrome. This testing may include sequence analysis of the entire coding region and exon / intron junction regions of TK2 for sequence variants and deletions / duplications. If compound heterozygous or homozygous deleterious mutations are identified in the sequence analysis, the diagnosis of TK2 deficiency is established and therefore the subject is likely to benefit from deoxynucleoside therapy. If the sequence analysis does not identify two compound heterozygous or homozygous deleterious mutations, deletion / duplication analysis should be considered to determine and / or establish the diagnosis of TK2 deficiency.

[0102] Further testing to determine and / or establish a diagnosis of TK2 deficiency includes testing for serum creatine kinase (CK) concentrations, electromyography, skeletal muscle histopathology, mitochondrial DNA (mtDNA) content (copy number), and skeletal muscle electron transport chain (ETC) activity. TK2 deficiency is determined and / or established if these tests find one or more of the following: Elevated CK concentrations compared to healthy controls may indicate TK2 deficiency. A skeletal muscle biopsy can be performed, followed by skeletal muscle mtDNA content analysis. A diagnosis of TK2 deficiency can be determined and / or established when skeletal muscle biopsies show marked variation in fiber size, variable sarcoplasmic vacuoles, variable increased connective tissue, and irregular red fibers, as well as increased succinate dehydrogenase (SDH) activity and low to absent cytochrome c oxidase (COX) activity, and significantly reduced mtDNA copy number (usually less than 20% of age- and tissue-matched healthy controls) (Chanprasert, et al. 2012).

[0103] Furthermore, TK2 deficiency is inherited in an autosomal recessive manner, therefore, siblings of an affected individual can be tested as soon as possible after birth to diagnose the disease.

[0104] Administration can be via any route, including, but not limited to, intrathecal, parental, mucosal, and transdermal.

[0105] In one embodiment, administration is oral. Exemplary oral dosage forms include, but are not limited to, capsules, tablets, powders, granules, solutions, syrups, suspensions (in non-aqueous or aqueous liquids), or emulsions. Tablets or hard gelatin capsules may contain lactose, starch or derivatives thereof, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatin capsules may contain vegetable oils, waxes, fats, semi-solid or liquid polyols. Solutions and syrups may contain water, polyols, and sugars. The prodrugs described herein may be added to any form of liquid ingested by a patient, including, but not limited to, cow's milk, breast milk, both cow's and human, infant formula, and water. The prodrugs may be coated with or mixed with materials that delay disintegration and / or absorption in the digestive tract. Thus, sustained release may be achieved over many hours.

[0106] In another embodiment, administration is intrathecal. Intrathecal administration involves injecting a drug into the spinal canal, more specifically into the subarachnoid space to reach the cerebrospinal fluid. This method is commonly used for spinal anesthesia, chemotherapy, and analgesics. Intrathecal administration can be done by lumbar puncture (bolus injection) or by a port catheter system (bolus or infusion). The catheter is most commonly inserted between the lamina of the lumbar spine and the tip is threaded into the intrathecal cavity to the desired level (usually L3-L4). Water or saline are most commonly used as excipients for intrathecal formulations, but EDTA and lipids have been used as well.

[0107] In yet another embodiment, the administration is parenteral, including intravenous administration. Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile solutions or suspensions for injection, which may contain antioxidants, buffers, bacteriostats, and solutes that render the composition substantially isotonic with the subject's blood. Other components that may be present in such compositions include water, alcohol, polyols, glycerin, and vegetable oils. Compositions adapted for parental administration may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile carrier immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include: Water for Injection USP; aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as ethyl alcohol, polyethylene glycol and polypropylene glycol; and non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate. Additionally, since some patients may be enterally fed by the time treatment is initiated, the prodrug may be administered via a gastronomy feeding tube or other enteral feeding means.

[0108] Pharmaceutical compositions adapted for nasal and pulmonary administration may contain a solid carrier, such as a powder, which can be administered by rapid inhalation through the nose. Compositions for nasal administration may contain a liquid carrier, such as a spray or droplets. Alternatively, direct inhalation into the lungs can be achieved by inhaling deeply or by introducing through a mouthpiece. These compositions may contain aqueous or oily solutions of the active ingredient. Compositions for inhalation may be delivered by specially adapted devices, including but not limited to pressurized aerosols, nebulizers or inhalers, which may be constructed to provide a predetermined dose of the active ingredient.

[0109] Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0110] Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.

[0111] The dosage of at least one prodrug or a composition comprising the same may be about 25 mg / kg / day to about 1,000 mg / kg / day. More preferred dosages are in the range of about 200 mg / kg / day to about 800 mg / kg / day. More preferred dosages are in the range of about 100 mg / kg / day to about 600 mg / kg / day, for example, about 250 mg / kg / day to about 500 mg / kg / day, about 300 mg / kg / day to about 500 mg / kg / day, or about 400 mg / kg / day to about 500 mg / kg / day.

[0112] In one embodiment, the dose is about 20% equimolar to about 100% equimolar to a standard nucleoside (dT, dC, dG, dA), for example, about 20% to about 80%, about 20% to about 50%, about 50% to about 80%, or about 50% to about 100%. In a particular embodiment, the dose is about 20% equimolar to a standard nucleoside. In another particular embodiment, the dose is about 50% equimolar to a standard nucleoside. In yet another embodiment, the dose is about 100% equimolar to a standard nucleoside.

[0113] Administration of at least one prodrug or composition comprising same can be once a day, twice a day, three times a day, four times a day, five times a day, up to six times a day, preferably at regular intervals. When administered intravenously or intrathecally, the dose may be reduced. The preferred dose range for such administration is about 50 mg / kg / day to about 500 mg / kg / day.

[0114] In embodiments where the composition comprises two or more prodrugs, the ratio of the prodrugs can vary.For example, when two prodrugs are administered, they can be in a ratio of 50 / 50, or in a ratio of about 5 / 95, 10 / 90, 15 / 85, 20 / 80, 25 / 75, 30 / 70, 35 / 65, 40 / 60, 45 / 55, 55 / 45, 60 / 40, 65 / 35, 70 / 30, 75 / 25, 80 / 20, 85 / 15, 90 / 10 and 95 / 5.

[0115] In one embodiment, the method further comprises monitoring the subject for improvement of the subject's condition before increasing dosage.The subject's response to therapeutic administration can be monitored by observing the changes in the subject's muscle strength and control, and mobility, as well as height and weight.If one or more of these parameters increase after administration, treatment can be continued.If one or more of these parameters remain the same or decrease, dosage can be increased.

[0116] In another embodiment, the method further comprises monitoring the subject for side effects prior to reducing the dosage. Exemplary side effects include, but are not limited to, diarrhea, abdominal bloating, and other gastrointestinal symptoms.

[0117] The prodrugs of the present invention can also be co-administered with other drugs. Such drugs include therapeutic drugs for treating the symptoms of certain forms of MDS. In particular, in the case of TK2 deficiency, other drugs include ubiquitous inhibitors of nucleoside catabolism, including, but not limited to, tetrahydrouridine (an inhibitor of cytidine deaminase) and enzyme inhibitors such as imucillin H (an inhibitor of purine nucleoside phosphorylase) and tipiracil (an inhibitor of thymidine phosphorylase). Such inhibitors are known and are used in the treatment of some cancers. EXAMPLES

[0118] Example 1: Mouse model of TK2 deficiency The efficacy of the prodrugs described herein was accessed through a mouse model. A homozygous Tk2 H126N knock-in mutant (Tk2 - / - ) mice have been reported previously (Akman, et al. 2008). Between 10 and 13 days after birth, Tk2 - / - Mice rapidly develop a fatal encephalomyopathic disease characterized by reduced locomotion, unsteady gait, coarse tremor, growth retardation, and rapid progression to early death at 14-16 days of age. Molecular and biochemical analysis of mouse models indicates that the pathogenesis of the disease is due to loss of enzyme activity and an imbalance in dNTP pools with reduced levels of dTTP in the brain and both dTTP and dCTP in the liver, leading to depletion of mtDNA and defects in respiratory chain enzymes containing mtDNA-encoded subunits, most notably in the brain and spinal cord.

[0119] The prodrug was administered daily by oral gavage in 50 μl of Esbilac small pet formula (Pet-Ag) to Tk2 H126N knock-in mice (Tk2 - / - ) and age-matched controls (Tk2 + A 50 / 50 mixture of 3 and 4 was administered at 580 mg / kg / day. A 50 / 50 mixture of 5 and 6 was administered at 120 mg / kg / day to 430 mg / kg / day.

[0120] All treatments were administered from postnatal day 4 to 29. At 21 days of age, mice were separated from their mothers and treatments were continued by oral gavage. + The mice were weighed and closely observed for comparison.

[0121] Mice were followed and weighed daily (it has been previously observed that the inability to gain weight is the first sign of disease).

[0122] The prodrug mixture extended survival of tk2- / - mice in a manner comparable to standard nucleosides and demonstrated non-statistically significant improvements in body weight.

[0123] Example 2: Fibroblasts from a dGK-deficient patient The prodrugs were tested in fibroblasts from dGK-deficient patients to determine their effect on mtDNA copy number. The advantage of using dGK-deficient fibroblasts is that after quiescence is induced, mtDNA is spontaneously depleted without the addition of DNA damaging agents. It has been previously demonstrated that supplementing the cell culture medium with dGuo (50 μM) is sufficient to prevent this depletion. Cells were grown to confluence. Quiescence was induced by reducing FBS in the medium to 0.1%. After 3 days (day 0), we supplemented the cell culture medium with 50 μM dGuo (deoxyguanosine) or the prodrugs of the present invention. Cells were maintained in the same conditions until day 18, with regular addition of fresh medium. The copy number of mtDNA was evaluated at various time points (days 4, 9 and 18) throughout the experiment. Results are expressed as the mean + SD of duplicate experiments and plotted as mtDNA / nDNA ratio relative to the mean value obtained for three untreated healthy controls cultured in parallel.

[0124] Equimolar concentrations of dGuo and other available dG prodrugs (50 µM) were tested in parallel under similar conditions. Results showed that a 50 / 50 mixture of 7 and 8 at 50 µM prevented mtDNA depletion.

Claims

1. 1. A pharmaceutical composition for treating mitochondrial DNA depletion syndrome, said pharmaceutical composition comprising a prodrug, said prodrug being a compound of formula IIIa, and said treatment comprising administering in a subject in need thereof a therapeutically effective amount of said prodrug: 【Chemistry 9】 During the ceremony, the base is selected from the group consisting of cytosine, 5-methylcytosine, thymine, guanine, and adenine; R １ is hydrogen and R is the side chain of an amino acid.

2. 2. The pharmaceutical composition of claim 1, wherein the prodrug is selected from the following: 【Chemistry 11】

3. The method of claim 1 , wherein the treatment comprises administration of at least two prodrugs.

4. 3. The pharmaceutical composition of claim 2, wherein the treatment comprises administration of two prodrugs selected from (i) compound 5 and compound 6 or (ii) compound 7 and compound 8.

5. 5. The pharmaceutical composition according to claim 3, wherein the weight ratio of one prodrug to another is 50 / 50.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the mitochondrial DNA depletion syndrome is selected from the group consisting of TK2 deficiency, RRM2B deficiency, TYMP mutation, SUCLA2 deficiency, SUCLG1 deficiency, MPV17 deficiency and DGUOK mutation.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein said administration is oral.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the dosage of said prodrug administered is from about 200 mg / kg / day to about 1,000 mg / kg / day.

9. The pharmaceutical composition according to any one of claims 1 to 8, which is administered at least once a day.

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