Polymorphic forms of deoxycytidine, compositions containing same and uses

Abstract

Provided herein are polymorphic forms of deoxycytidine and methods for preparing same. Also provided are compositions comprising polymorphic forms of deoxycytidine and at least one residual solvent, and methods for treating mitochondrial diseases using the compositions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 888,893, filed August 19, 2019, which is incorporated herein in its entirety.

[0002] The present invention relates to polymorphic forms of deoxycytidine; compositions comprising polymorphic forms of deoxycytidine and at least one residual solvent at a concentration less than 10% of the ICH limit; and methods of treating diseases or disorders characterized by imbalanced nucleotide pools, particularly those found in mitochondrial DNA depletion syndromes, with compositions comprising polymorphic forms of deoxycytidine and at least one residual solvent at a concentration less than 10% of the ICH limit. [Background technology]

[0003] Polymorphs exist as two or more crystalline phases with different arrangements and / or different conformations of molecules within the crystal lattice. When solvent molecules are included within the crystal lattice, the resulting crystal is called a pseudopolymorph or solvate. When the solvent molecules within the crystal structure are water molecules, the pseudopolymorph / solvate is called a hydrate. Polymorphic and pseudopolymorphic solids exhibit different physical properties, including those due to packing, as well as various thermodynamic, spectroscopic, interfacial, and mechanical properties (see Non-Patent Document 1). Polymorphic and pseudopolymorphic forms of drug substances (also known as "active pharmaceutical ingredients" (APIs)) are well known when administered alone or formulated into dosage forms (final or finished dosage forms, or also known as pharmaceutical compositions), and can affect, for example, the solubility, stability, flowability, friability, and compressibility of the drug substance and the safety and efficacy of the formulation (see, for example, Non-Patent Document 2).

[0004] Deoxycytidine (2´-deoxycytidine, dC), when used in combination with deoxythymidine (dT), is useful for treating certain mitochondrial DNA depletion syndromes (MDS). Mitochondrial DNA depletion syndromes (MDS), a subgroup of mitochondrial diseases, are frequent causes of severe childhood encephalomyopathies characterized by reduced mitochondrial DNA (mtDNA) copy number in tissues and insufficient synthesis of the mitochondrial RC complex (Hirano, et al. 2001). Mutations in several nuclear genes, including TK2, DGUOK, POLG, POLG2, SCLA25A4, MPV17, RRM2B, SUCLA2, SUCLG1, TYMP, OPA1, and ClOorfl (PEOl), have been identified as causes of infantile MDS (Bourdon et al. 2007; Copeland 2008; Elpeleg et al. 2005; Mandel et al. 2001; Naviaux and Nguyen 2004; Ostergaard et al. 2007; Saada et al. 2003; Sarzi et al. 2007; Spinazzola et al. 2006). Furthermore, mutations in these nuclear genes can also cause multiple deletions of mtDNA with or without mtDNA depletion (Behin, et al. 2012; Garone, et al. 2012; Longley, et al. 2006; Nishino, et al. 1999; Paradas, et al. 2012; Ronchi, et al. 2012; Spelbrink, et al. 2001; Tyynismaa, et al. 2009; Tyynismaa, et al. 2012; Van Goethem, et al. 2001).

[0005] One of these genes is TK2, which encodes thymidine kinase (TK2), a mitochondrial enzyme required for the phosphorylation of pyrimidine nucleosides (thymidine and deoxycytidine) to generate deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP) (Saada, et al. 2001). Mutations in TK2 impair the mitochondrial nucleoside / nucleotide salvage pathway required for the synthesis of deoxynucleotide triphosphates (dNTPs), the building blocks for mRNA replication and repair.

[0006] Patients diagnosed with TK2 deficiency show improvement with the administration of a combination of deoxycytidine and deoxythymidine (Patent Document 1). The administered doses are relatively high, e.g., 400 mg / kg / day for each of deoxycytidine and deoxythymidine. Therefore, a 100 kg patient can receive a maximum of 80 g of active substance per day (40 g of deoxycytidine and 40 g of deoxythymidine). Therefore, it is important to minimize the amount of residual solvents resulting from the manufacture of active ingredients. Residual solvents include ICH Class 1 (i.e., solvents to be avoided), Class 2 (i.e., solvents to be restricted), and Class 3 (i.e., solvents with low toxicity). The classification of solvents according to ICH guidelines is well known. The ICH guidelines specify acceptable daily intakes (PDEs) and concentration limits for Class 2 and Class 3 residual solvents. Class 1 solvents should not be used in manufacturing unless unavoidable and unless the ICH guidelines limit the concentrations of these solvents to very low levels.

[0007] Thus, there is a need for improved manufacturing processes that provide deoxycytidine compositions with reduced residual solvent content compared to other methods or materials available from commercial chemical suppliers. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2016205671 [Non-patent literature]

[0009] [Non-Patent Document 1] H. Brittain, Polymorphism in Pharmaceutical Solids, Marcel Dekker, New York, NY, 1999, pp.1-2 [Non-patent document 2] Knapman,K Modem Drug Discovery,Mar.2000:53 Summary of the Invention

[0010] In one aspect, the present invention provides deoxycytidine form B. Deoxycytidine form B has the X-ray powder diffraction (XRPD) pattern shown in Figure 2. In one embodiment, deoxycytidine form B is characterized by XRPD peaks at 13.7°, 17.2°, 18.0°, 19.2°, and 22.8° 2θ (±0.2° 2θ). In a more specific embodiment, the XRPD pattern of form B further comprises one or more peaks at 11.5°, 11.8°, 13.7°, 17.2°, 18.0°, 19.2°, 20.2°, 21.1°, 21.4°, 21.8°, and 22.8° 2θ (±0.2° 2θ).

[0011] Deoxycytidine Form B of the present invention can be prepared by a large-scale manufacturing process and contains minimal residual solvents. In one embodiment, deoxycytidine Form B has less than about 100 ppm ethyl acetate and / or less than about 100 ppm n-heptane and / or less than about 200 ppm ethanol.

[0012] Form B deoxycytidine of the present invention is stable for at least about six months, for example, when stored in a sealed container at room temperature. Stability can be measured by several methods, including the conversion of Form A to deoxycytidine.

[0013] Form B deoxycytidine can be substantially pure, i.e., it can be a mixture comprising a form of deoxycytidine having about 95% by weight or more of Form B. In another embodiment, isolated Form B is provided.

[0014] In another aspect, the present invention provides a method for preparing deoxycytidine form B, the method comprising the steps of: (a) providing crude deoxycytidine; (b) contacting the crude deoxycytidine with ethanol to produce a first mixture; (c) distilling the ethanol from the first mixture to produce a residue; (d) contacting the residue with purified water to produce a second mixture; (e) heating the second mixture to an internal temperature of at least about 40° C.; (f) adding ethanol, ethyl acetate, and heptane to the second mixture to produce a third mixture; (g) cooling the third mixture to produce deoxycytidine form B crystals; and (h) separating the deoxycytidine form B crystals from the third mixture.

[0015] The manufacturing methods described herein provide deoxycytidine compositions with reduced residual solvents, for example, compared to commercial chemical suppliers or previous manufacturing methods. Indeed, the methods have been optimized to provide deoxycytidine compositions with less than about 10% of the Class 2 and Class 3 residual solvent concentrations permitted by ICH guidelines. The methods also provide deoxycytidine compositions with undetectable levels of certain Class 1 solvents (e.g., 1,2-dichloroethane) according to validated methods.

[0016] In one embodiment, a composition comprises deoxycytidine Form B and at least one residual solvent selected from ICH Class 2 and Class 3 solvents, wherein the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for each residual solvent. Preferred Class 2 and Class 3 residual solvents include methanol, toluene, methylene chloride, ethanol, tert-butyl methyl ether (TBME), acetone, ethyl acetate, and n-heptane.

[0017] In a specific embodiment, the composition comprises deoxycytidine Form B and at least one of: (a) 1 ppm to about 300 ppm of methanol, (b) 1 ppm to about 89 ppm of toluene, (c) 1 ppm to about 600 ppm of methylene chloride, (d) 1 ppm to about 500 ppm of ethanol, (e) 1 ppm to about 500 ppm of TBME, (f) 1 ppm to about 500 ppm of acetone, (g) 1 ppm to about 500 ppm of ethyl acetate, and (h) 1 ppm to about 500 ppm of n-heptane.

[0018] In another specific embodiment, the composition comprises deoxycytidine Form B and 300 ppm or less of methanol, 89 ppm or less of toluene, 60 ppm or less of methylene chloride, 500 ppm or less of ethanol, 500 ppm or less of TBME, 500 ppm or less of acetone, 500 ppm or less of ethyl acetate, and 500 ppm or less of n-heptane.

[0019] In a preferred embodiment, the composition has undetectable levels of Class 1 solvents, particularly 1,2-dichloroethane.

[0020] In yet another embodiment, there is provided a pharmaceutical composition comprising substantially pure deoxycytidine Form B and at least one pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise deoxythymidine.

[0021] In one embodiment, a pharmaceutical composition comprises deoxycytidine Form B, at least one residual solvent, and at least one pharmaceutically acceptable carrier, wherein the at least one residual solvent is selected from ICH Class 2 and Class 3 solvents, and wherein the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for each residual solvent.

[0022] In a specific embodiment, the pharmaceutical composition comprises deoxycytidine Form B, at least one pharmaceutically acceptable carrier, and at least one of: (a) 1 ppm to about 300 ppm of methanol, (b) 1 ppm to about 89 ppm of toluene, (c) 1 ppm to about 60 ppm of methylene chloride, (d) 1 ppm to about 500 ppm of ethanol, (e) 1 ppm to about 500 ppm of TBME, (f) 1 ppm to about 500 ppm of acetone, (g) 1 ppm to about 500 ppm of ethyl acetate, and (h) 1 ppm to about 500 ppm of n-heptane.

[0023] In another specific embodiment, the pharmaceutical composition comprises less than or equal to 300 ppm methanol, less than or equal to 89 ppm toluene, less than or equal to 60 ppm methylene chloride, less than or equal to 500 ppm ethanol, less than or equal to 500 ppm TBME, less than or equal to 500 ppm acetone, less than or equal to 500 ppm ethyl acetate, and less than or equal to 500 ppm n-heptane.

[0024] In a preferred embodiment, the pharmaceutical composition has undetectable levels of Class 1 solvents, particularly 1,2-dichloroethane.

[0025] In another embodiment, a fixed-dose powder pharmaceutical composition is provided comprising deoxycytidine Form B, deoxythymidine, at least one residual solvent, and at least one pharmaceutically acceptable carrier, wherein the at least one residual solvent is selected from ICH Class 2 and Class 3 solvents, and the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for each residual solvent. The fixed-dose powder pharmaceutical composition preferably has undetectable levels of Class 1 solvents, particularly 1,2-dichloroethane.

[0026] In yet another aspect, provided is a method for treating the disease or disorder characterized by unbalanced nucleotide pool.The method comprises administering a therapeutically effective amount of the composition described herein to a subject in need thereof.In a specific embodiment, the disorder is thymidine kinase 2 deficiency characterized by mutation in TK2 gene.

[0027] In one embodiment, a composition comprising deoxycytidine Form B and at least one residual solvent is administered to a subject, wherein the at least one residual solvent is selected from ICH Class 2 and Class 3 solvents, and the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for each residual solvent. The composition may be a pharmaceutical composition, further comprising at least one pharmaceutically acceptable carrier.

[0028] The method may further comprise administering to the subject a therapeutically effective amount of a second composition comprising deoxythymidine. The second composition may be a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier. In such an embodiment, the combined residual solvent content in the first and second compositions for each residual solvent is less than about 10% of the ICH concentration limit value for each residual solvent.

[0029] In other embodiments, one composition administered comprises deoxycytidine Form B, deoxythymidine, and at least one residual solvent, wherein the at least one residual solvent is selected from ICH Class 2 and Class 3 solvents, and the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for each residual solvent. The composition may be a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier. The composition may be a fixed-dose powder pharmaceutical composition described herein. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 shows the X-ray diffraction pattern of a mixture of Form A and Form B deoxycytidine.

[0031] [Figure 2] FIG. 1 shows the X-ray diffraction pattern of deoxycytidine form B.

[0032] [Figure 3] FIG. 1 shows the X-ray diffraction pattern of deoxycytidine form A. DETAILED DESCRIPTION OF THE INVENTION

[0033] I. Forms of deoxycytidine The present invention provides crystalline forms of deoxycytidine and compositions comprising crystalline forms of deoxycytidine.

[0034] In one embodiment, deoxycytidine Form B is provided. The powder X-ray diffraction (XRPD) pattern of deoxycytidine Form B is shown in Figure 2. Deoxycytidine Form B is characterized by an XRPD pattern comprising peaks at 13.7°, 17.2°, 18.0°, 19.2°, and 22.8° 2θ (±0.2° 2θ). In a more specific embodiment, Form B is characterized by an XRPD pattern comprising peaks at 13.7°, 17.2°, 18.0°, 19.2°, and 22.8° 2θ (±0.1° 2θ). XRPD is preferably performed using CuK α1 It is done using radiation.

[0035] The XRPD pattern of Form B may further include one or more peaks at 11.5°, 11.8°, 20.2°, 21.1°, 21.4°, and 21.8° 2θ (±0.2° 2θ). In more specific embodiments, the XRPD pattern may further include one or more peaks at 11.5°, 11.8°, 20.2°, 21.1°, 21.4°, and 21.8° 2θ (±0.1° 2θ).

[0036] In a specific embodiment, deoxycytidine form B is characterized by an XRPD pattern comprising peaks at 11.5°, 11.8°, 13.7°, 17.2°, 18.0°, 19.2°, 20.2°, 21.1°, 21.4°, 21.8°, and 22.8° 2θ (±0.2° 2θ). In an even more specific embodiment, deoxycytidine form B is characterized by an XRPD pattern comprising peaks at 11.5°, 11.8°, 13.7°, 17.2°, 18.0°, 19.2°, 20.2°, 21.1°, 21.4°, 21.8°, and 22.8° 2θ (±0.1° 2θ).

[0037] In one embodiment, deoxycytidine Form B is provided in substantially pure form. "Substantially pure," as used herein, refers to a mixture of forms of deoxycytidine that contains about 95% or more by weight of Form B, about 96% or more by weight of Form B, about 97% or more by weight of Form B, about 98% or more by weight of Form B, or about 99% or more by weight of Form B.

[0038] Form B deoxycytidine may also be provided in isolated form, i.e., 100% Form B.

[0039] Form B deoxycytidine contains minimal residual solvents from manufacture.

[0040] In one embodiment, deoxycytidine Form B contains less than about 100 ppm ethyl acetate, e.g., less than about 90 ppm, less than about 80 ppm, less than about 60 ppm, or less than about 50 ppm ethyl acetate. In another embodiment, deoxycytidine Form B contains from 1 ppm to about 100 ppm ethyl acetate, e.g., from 1 ppm to about 90 ppm, from 1 ppm to about 80 ppm, from 1 ppm to about 60 ppm, and from 1 ppm to about 50 ppm ethyl acetate.

[0041] Deoxycytidine Form B contains less than about 100 ppm n-heptane, preferably less than about 90 ppm, less than about 80 ppm, less than about 70 ppm, less than about 60 ppm, less than about 50 ppm, less than about 40 ppm, or less than about 30 ppm n-heptane. In another embodiment, deoxycytidine Form B contains from 1 ppm to about 100 ppm n-heptane, e.g., from 1 ppm to about 90 ppm, from 1 ppm to about 80 ppm, from 1 ppm to about 70 ppm, from 1 ppm to about 60 ppm, from 1 ppm to about 50 ppm, from 1 ppm to about 40 ppm, and from 1 ppm to about 30 ppm n-heptane.

[0042] Deoxycytidine Form B contains less than about 200 ppm ethanol, preferably less than about 150 ppm ethanol. In another embodiment, deoxycytidine Form B contains from 1 ppm to about 200 ppm ethanol, e.g., from 1 ppm to about 150 ppm, from 1 ppm to about 125 ppm, from 1 ppm to about 100 ppm, from 1 ppm to about 75 ppm, from 1 ppm to about 50 ppm, and from 1 ppm to about 25 ppm ethanol.

[0043] In a specific embodiment, deoxycytidine Form B contains less than about 100 ppm ethyl acetate, less than about 100 ppm n-heptane, and less than about 200 ppm ethanol. Deoxycytidine Form B can contain 1 ppm to about 100 ppm ethyl acetate, 1 ppm to about 100 ppm n-heptane, and 1 ppm to about 200 ppm ethanol. In another embodiment, deoxycytidine Form B has at least one of: (a) 1 ppm to about 100 ppm ethyl acetate, (b) 1 ppm to about 100 ppm n-heptane, or (c) 1 ppm to about 200 ppm ethanol.

[0044] In another specific embodiment, deoxycytidine Form B contains less than about 70 ppm ethyl acetate, less than about 50 ppm n-heptane, and less than about 200 ppm ethanol. Deoxycytidine Form B can contain 1 ppm to about 70 ppm ethyl acetate, 1 ppm to about 50 ppm n-heptane, and 1 ppm to about 200 ppm ethanol. In another embodiment, deoxycytidine Form B contains at least one of: (a) 1 ppm to about 70 ppm ethyl acetate, (b) 1 ppm to about 50 ppm n-heptane, or (c) 1 ppm to about 200 ppm ethanol.

[0045] Deoxycytidine form B is stable for at least about 6 months, for example, when stored in a sealed container at room temperature, and more particularly, deoxycytidine form B is stable for at least about 12 months or at least about 18 months.

[0046] "Stability," as used herein, can be measured by examining the XRPD pattern of Form B of a material after a predetermined storage time. Conversion of Form B deoxycytidine to Form A deoxycytidine, as determined by XRPD, indicates instability. Stability can also be measured by examining compliance with test attribute criteria, as outlined in Example 4. Test attributes include physical appearance, IR trace, HPLC trace of active substance and impurities, water content, residual solvent content, elemental impurities, and microbial growth. One or more of these attributes can be measured over a predetermined period of time, e.g., 6 months, 12 months, or longer, and under various conditions, e.g., 25±2°C / 60±5% relative humidity or 40±2°C / 75±5% relative humidity.

[0047] A method for preparing Form B of deoxycytidine is provided in Example 1.

[0048] In one embodiment, a method for preparing deoxycytidine form B includes the steps of: (a) providing crude deoxycytidine; (b) contacting the crude deoxycytidine with ethanol to produce a first mixture; (c) distilling the ethanol from the first mixture to produce a residue; (d) contacting the residue with purified water to produce a second mixture; (e) heating the second mixture to an internal temperature of at least about 40° C.; (f) adding ethanol, ethyl acetate, and heptane to the second mixture to produce a third mixture; (g) cooling the third mixture to produce deoxycytidine form B crystals; and (h) separating the deoxycytidine form B crystals from the third mixture.

[0049] The method can be used for large scale manufacturing, such as to produce batches of deoxycytidine greater than about 75 kg, e.g., greater than about 100 kg, greater than about 125 kg, greater than about 150 kg, or greater than about 200 kg.

[0050] Crude deoxycytidine may comprise a mixture of polymorphic forms, Form A and Form B, in amounts equal to about 100%. In one embodiment, crude deoxycytidine comprises at least about 5% Form B, at least about 10% Form B, at least about 20% Form B, at least about 30% Form B, at least about 40% Form B, at least about 50% Form B, at least about 60% Form B, at least about 70% Form B, at least about 80% Form B, or at least about 10% Form B. In another embodiment, crude deoxycytidine comprises at least about 5% Form A, at least about 10% Form A, at least about 20% Form A, at least about 30% Form A, at least about 40% Form A, at least about 50% Form A, at least about 60% Form A, at least about 70% Form A, at least about 80% Form A, or at least about 10% Form A.

[0051] The distillation in step (c) is preferably carried out at a temperature not exceeding about 70°C, for example at a temperature of from about 40°C to less than about 70°C.

[0052] The amount of water used in (d) is preferably about 0.10 to about 0.5 volumes, for example, 0.25 volumes.

[0053] The internal temperature of the second mixture in (e) is preferably about 40°C to about 50°C.

[0054] The solvents in the addition step (f) can be added in any order, and can be added all at once or in multiple portions. In one embodiment, ethanol is added first, followed by a mixture of ethanol, ethyl acetate, and n-heptane. The amounts of ethanol, ethyl acetate, and n-heptane can vary. The amount of ethanol can be 1 volume, 2 volumes, or 3 volumes. The amount of ethyl acetate can be 1 volume, 2 volumes, or 3 volumes. The amount of n-heptane can be 1 volume, 2 volumes, or 3 volumes. The solvent or mixture of solvents is generally added dropwise or slowly enough to prevent crash precipitation.

[0055] The third mixture in (g) can be cooled in a stepwise or gradient manner. In one embodiment, the third mixture is cooled to an internal temperature of about 20°C to about 30°C, maintained at that temperature for a period of time with stirring, and then further cooled. In a more specific embodiment, the third mixture is stirred for at least about 1 hour, at least about 3 hours, at least about 6 hours, at least about 12 hours, or at least about 24 hours, and then further cooled. After stirring, the mixture can be further cooled to an internal temperature of about 0°C to about 10°C.

[0056] The separation step (h) can be carried out by any suitable separation means, for example by centrifugal filtration, gravity filtration or vacuum filtration.

[0057] The method provides a composition comprising substantially pure Form B deoxycytidine.

[0058] Form B crystals obtained by the methods herein may contain at least one residual solvent and therefore may be classified as a composition of matter.

[0059] The composition can further contain one or more polymorphic forms of deoxycytidine, e.g., other polymorphic forms, including Form A or hydrated polymorphic forms, in addition to Form B. In one embodiment, the composition contains up to about 10% by weight of one or more other polymorphic forms of deoxycytidine, e.g., about 0.5% to about 10% by weight, about 1% to about 10% by weight, about 1% to about 5% by weight, or about 1% to about 3% by weight of one or more other polymorphic forms of deoxycytidine.

[0060] Compositions containing deoxycytidine Form B prepared by the methods described herein contain at least one residual solvent. The solvent may belong to ICH Class 2 (i.e., solvents to be restricted) or Class 3 (i.e., solvents of low toxicity). The ICH maximum permitted daily intake (PDE) and concentration limits for Class 2 solvents are listed in the table below. Class 3 solvents are considered less toxic and pose a lower risk to human health. The ICH guidelines for Class 3 solvents are 5,000 ppm or less, or 50 mg / day or less.

[0061] TIFF0007769603000001.tif203170

[0062] Class 1 solvents are solvents that should be avoided. The ICH guidelines state that Class 1 solvents should not be used in the manufacture of drug substances due to unacceptable toxicity or adverse environmental effects. However, they are permitted in limited amounts if unavoidable. The ICH concentration limits for Class 1 solvents are shown in the table below.

[0063] TIFF0007769603000002.tif46170

[0064] The manufacturing methods described herein provide deoxycytidine compositions with significantly reduced residual solvent concentrations, compared to, for example, commercial chemical suppliers or previously known manufacturing methods. Indeed, the methods have been optimized to provide deoxycytidine compositions containing (a) about 10% or less of the concentrations / amounts of Class 2 and Class 3 residual solvents allowed by ICH guidelines, and (b) undetectable levels of ICH Class 1 solvents (particularly 1,2-dichloroethane).

[0065] "Undetectable" as used herein refers to a concentration of a substance that is below the detection limit of that substance when measured using a standard validated method. Such methods are known in the art, including, for example, the gas chromatography (GC) method specified in the United States Pharmacopoeia (USP) Chapter on Residual Solvents (467) and the European Pharmacopoeia (Ph.Eur). The detection limit of 1,2-dichloroethane is, for example, 0.2 ppm.

[0066] In one embodiment, the composition comprises deoxycytidine Form B and at least one residual solvent, such as one residual solvent, two residual solvents, three residual solvents, four residual solvents, five residual solvents, or six or more residual solvents.

[0067] In one embodiment, the composition comprises deoxycytidine Form B and at least one residual solvent, wherein the at least one residual solvent is selected from ICH Class 2 and Class 3 solvents, and the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for each residual solvent. In a more specific embodiment, the composition consists of deoxycytidine Form B and at least one residual solvent, wherein the at least one residual solvent is selected from ICH Class 2 and Class 3 solvents, and the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for the particular residual solvent.

[0068] Preferred Class 2 and Class 3 residual solvents include methanol, toluene, methylene chloride, ethanol, tert-butyl methyl ether, acetone, ethyl acetate, and n-heptane.

[0069] Methanol is an ICH Class 2 solvent with a permissible daily intake (PDE) of 30 mg or a concentration limit of 3,000 ppm. The compositions of the present invention may contain methanol at a concentration of 1 ppm to about 300 ppm, e.g., 1 ppm to about 250 ppm, 1 ppm to about 200 ppm, 1 ppm to about 150 ppm, 1 ppm to about 100 ppm, 1 ppm to about 50 ppm, or 1 ppm to about 25 ppm.

[0070] Toluene is an ICH Class 2 solvent with a PDE of 8.9 mg / day or a concentration limit of 890 ppm. The compositions of the present invention may contain toluene at a concentration of from 1 ppm to about 89 ppm, e.g., from 1 ppm to about 45 ppm, from 1 ppm to about 25 ppm, and from 1 ppm to about 10 ppm.

[0071] Methylene chloride (dichloromethane) is an ICH Class 2 solvent with a PDE of 6.0 mg / day or a concentration limit of 600 ppm. The compositions of the present invention may contain methylene chloride at a concentration of 1 ppm to about 60 ppm, e.g., 1 ppm to about 50 ppm, 1 ppm to about 40 ppm, 1 ppm to about 30 ppm, 1 ppm to about 20 ppm, 1 ppm to about 10 ppm, and 1 ppm to about 5 ppm.

[0072] Ethanol is an ICH Class 3 solvent with a PDE of 50 mg / day or a concentration limit of 5,000 ppm. The compositions of the present invention may contain ethanol at a concentration of 1 ppm to about 500 ppm, for example, 1 ppm to about 400 ppm, 1 ppm to about 300 ppm, 1 ppm to about 200 ppm, 1 ppm to about 100 ppm, and 1 ppm to about 50 ppm.

[0073] Tert-butyl methyl ether (TBME) is an ICH Class 3 solvent with a PDE of 50 mg / day or a concentration limit of 5,000 ppm. The compositions of the present invention may contain tert-butyl methyl ether at a concentration of 1 ppm to about 500 ppm, e.g., 1 ppm to about 400 ppm, 1 ppm to about 300 ppm, 1 ppm to about 200 ppm, 1 ppm to about 100 ppm, and 1 ppm to about 50 ppm.

[0074] Acetone is an ICH Class 3 solvent with a PDE of 50 mg / day or a concentration limit of 5,000 ppm. The compositions of the present invention may contain acetone at a concentration of from 1 ppm to about 500 ppm, e.g., from 1 ppm to about 400 ppm, from 1 ppm to about 300 ppm, from 1 ppm to about 200 ppm, from 1 ppm to about 100 ppm, and from 1 ppm to about 50 ppm.

[0075] Ethyl acetate is an ICH Class 3 solvent with a PDE of 50 mg / day or a concentration limit of 5,000 ppm. The compositions of the present invention may contain ethyl acetate at a concentration of 1 ppm to about 500 ppm, e.g., 1 ppm to about 400 ppm, 1 ppm to about 300 ppm, 1 ppm to about 200 ppm, 1 ppm to about 100 ppm, and 1 ppm to about 50 ppm.

[0076] n-Heptane is an ICH Class 3 solvent with a PDE of 50 mg or a limit of 5,000 ppm. The compositions of the present invention may contain n-heptane at a concentration of 1 ppm to about 500 ppm, e.g., 1 ppm to about 400 ppm, 1 ppm to about 300 ppm, 1 ppm to about 200 ppm, 1 ppm to about 100 ppm, and 1 ppm to about 50 ppm.

[0077] In a specific embodiment, the composition comprises deoxycytidine Form B and at least one of Class 2 and / or Class 3 ICH solvents: (a) 1 ppm to about 300 ppm methanol, (b) 1 ppm to about 89 ppm toluene, (c) 1 ppm to about 60 ppm methylene chloride, (d) 1 ppm to about 500 ppm ethanol, (e) 1 ppm to about 500 ppm TBME, (f) 1 ppm to about 500 ppm acetone, (g) 1 ppm to about 500 ppm ethyl acetate, and (h) 1 ppm to about 500 ppm n-heptane.

[0078] 1,2-Dichloroethane (1,2-DCE) is an ICH Class 1 solvent with a concentration limit of 5 ppm. The compositions of the present invention may contain 1,2-DCE at a concentration of 0.2 ppm to about 0.2 ppm to about 0.5 ppm, preferably less than 0.2 ppm, such that the 1,2-DCE is undetectable.

[0079] Compositions provided herein comprising deoxycytidine Form B and at least one residual solvent are stable for a period of at least 6 months, e.g., at least 9 months, at least 12 months, or at least 24 months. The stability of the compositions can be measured according to Example 4, i.e., by verifying compliance with test attributes when subjected to storage conditions.

[0080] In another embodiment, deoxycytidine Form A is provided. The XRPD pattern of deoxycytidine Form A is shown in Figure 3. Deoxycytidine Form A is characterized by an XRPD pattern including peaks at 8.4°, 12.9°, 14.2°, 16.8°, 18.4°, 19.4°, 28.4°, 30.0°, and 30.6° 2θ (±0.2° 2θ). In a more specific embodiment, Form A is characterized by an XRPD pattern including peaks at 8.4°, 12.9°, 14.2°, 16.8°, 18.4°, 19.4°, 28.4°, 30.0°, and 30.6° 2θ (±0.1° 2θ). XRPD is preferably performed using CuK α1 It is done using radiation.

[0081] The XRPD pattern of Form A may further include one or more peaks at 21.3°, 21.6°, and 22.2° 2θ (±0.2° 2θ). In more specific embodiments, the XRPD pattern may further include one or more peaks at 21.3°, 21.6°, and 22.2° 2θ (±0.1° 2θ).

[0082] In one embodiment, deoxycytidine Form A is provided in substantially pure form. "Substantially pure," as used herein, refers to a mixture of forms of deoxycytidine that contains about 95% or more by weight of Form A, about 96% or more by weight of Form A, about 97% or more by weight of Form A, about 98% or more by weight of Form A, or about 99% or more by weight of Form A.

[0083] II. Pharmaceutical Compositions The present invention also provides pharmaceutical compositions comprising deoxycytidine in a substantially pure form as described herein as an active ingredient, and at least one pharmaceutically acceptable carrier.

[0084] In one embodiment, the pharmaceutical composition comprises substantially pure Form B deoxycytidine as the active ingredient, at least one residual solvent, and at least one pharmaceutically acceptable carrier.

[0085] In another embodiment, the pharmaceutical composition comprises a substantially pure mixture of Form B deoxycytidine and deoxythymidine, wherein deoxythymidine also acts as the active ingredient, at least one residual solvent, and at least one pharmaceutically acceptable carrier.

[0086] In yet another embodiment, the pharmaceutical composition comprises a composition comprising deoxycytidine Form B as described herein and at least one pharmaceutically acceptable carrier.

[0087] In yet another embodiment, the pharmaceutical composition comprises a composition comprising deoxycytidine Form B described herein, a composition comprising deoxythymidine, and at least one pharmaceutically acceptable carrier.

[0088] A composition comprising deoxythymidine contains deoxythymidine in an amount of at least 97% by weight, for example, about 97-99% by weight deoxythymidine or 98-99% by weight deoxythymidine, the remainder being one or more impurities, including at least one residual solvent.

[0089] Such pharmaceutical compositions comprise a therapeutically effective amount of deoxycytidine Form B (and, if applicable, a therapeutically effective amount of deoxythymidine) and at least one pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerated, typically do not cause allergic or similar adverse reactions, such as stomach upset, dizziness, etc., when administered to humans, and are approved by federal or state regulatory agencies or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, more specifically humans. A "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids, such as water or saline solutions in oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0090] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel (anhydrous colloidal silica), magnesium stearate, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0091] The pharmaceutical composition may contain at least one fluidizing agent.The fluidizing agent is a substance that counteracts the poor flowability of powder during the manufacturing process.Exemplary fluidizing agents include, but are not limited to, colloidal silica, for example, colloidal silicon dioxide, for example, AEROSIL, magnesium trisilicate (Mg), powdered cellulose, starch, talc and tricalcium phosphate.

[0092] The pharmaceutical composition may contain at least one lubricant. A lubricant is a substance that reduces friction between particles. Exemplary lubricants include, but are not limited to, magnesium stearate, aluminum (Al) stearate or calcium stearate, PEG 4000-8000, talc, hydrogenated castor oil, stearic acid and its salts, glycerol esters, sodium stearyl fumarate, and hydrogenated cottonseed oil.

[0093] In a specific embodiment, the fixed-dose pharmaceutical composition comprises deoxycytidine Form B, deoxythymidine, at least one residual solvent, at least one glidant, and at least one lubricant, wherein the composition is in the form of a powder.

[0094] The amount of deoxycytidine Form B in the pharmaceutical composition may be from 1,000 mg to about 5,000 mg, for example, from about 1,000 mg to about 4,000 mg, from about 1,000 mg to about 3,000 mg, or from about 1,000 mg to about 2,000 mg.

[0095] The amount of deoxythymidine in the pharmaceutical composition may be 1,000 mg to about 5,000 mg, for example, about 1,000 mg to about 4,000 mg, about 1,000 mg to about 3,000 mg, or about 1,000 mg to about 2,000 mg.

[0096] In a preferred embodiment, both Form B deoxycytidine and deoxythymidine are present at about 2,000 mg.

[0097] The ratio of deoxycytidine to deoxythymidine can vary, for example, it can be a 50 / 50 ratio, or 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.

[0098] The at least one fluidizing agent may be present in an amount of about 0.1% to about 10% by weight, such as about 1% to about 5% or about 1% to about 3%. In a preferred embodiment, the fluidizing agent is colloidal silicon dioxide (e.g., AEROSIL 200).

[0099] The at least one lubricant may be present in an amount of about 0.1% to about 1% by weight, for example, about 0.1% to about 0.5% or about 0.3% to about 0.7%. In a preferred embodiment, the lubricant is magnesium stearate.

[0100] Deoxycytidine Form B can be present in an amount of about 40% to about 60% by weight, e.g., about 40% to about 50% by weight, about 50% to about 60% by weight, or about 45% to about 55% by weight.

[0101] Similarly, deoxythymidine can be present in an amount of about 40% to about 60% by weight, such as about 40% to about 50% by weight, about 50% to about 60% by weight, or about 45% to about 55% by weight.

[0102] The powdered pharmaceutical composition can be packaged in any suitable form, such as a bottle, a pouch, or a sachet. In a preferred embodiment, the pharmaceutical composition is packaged in a pouch made of laminated PET, aluminum, and low-density polyethylene. Then, before administration, the powdered pharmaceutical composition is dissolved in water to prepare an oral solution.

[0103] At least one residual solvent may be carried over from the manufacture of either Form B deoxycytidine (as described above), the pharmaceutically acceptable carrier, or deoxythymidine, if applicable.

[0104] The pharmaceutical composition contains at least one residual solvent, such as two residual solvents, three residual solvents, four residual solvents, or five or more residual solvents.

[0105] The at least one residual solvent is selected from ICH Class 2 and Class 3 solvents, and the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for each residual solvent.

[0106] Preferred Class 2 and Class 3 residual solvents include methanol, toluene, methylene chloride, ethanol, tert-butyl methyl ether, acetone, ethyl acetate, and n-heptane.

[0107] Methanol is an ICH Class 2 solvent with a permissible daily intake (PDE) of 30 mg or a concentration limit of 3,000 ppm. The pharmaceutical composition of the present invention may contain methanol at a concentration of 1 ppm to about 300 ppm, for example, 1 ppm to about 250 ppm, 1 ppm to about 200 ppm, 1 ppm to about 150 ppm, 1 ppm to about 100 ppm, 1 ppm to about 50 ppm, or 1 ppm to about 25 ppm.

[0108] Toluene is an ICH Class 2 solvent with a PDE of 8.9 mg / day or a concentration limit of 890 ppm. The pharmaceutical compositions of the present invention may contain toluene at a concentration of 1 ppm to about 89 ppm, for example, 1 ppm to about 45 ppm, 1 ppm to about 25 ppm, and 1 ppm to about 10 ppm.

[0109] Methylene chloride (dichloromethane) is an ICH Class 2 solvent with a PDE of 6.0 mg / day or a concentration limit of 600 ppm. The pharmaceutical compositions of the present invention may contain methylene chloride at a concentration of 1 ppm to about 60 ppm, for example, 1 ppm to about 50 ppm, 1 ppm to about 40 ppm, 1 ppm to about 30 ppm, 1 ppm to about 20 ppm, 1 ppm to about 10 ppm, and 1 ppm to about 5 ppm.

[0110] Ethanol is an ICH Class 3 solvent with a PDE of 50 mg / day or a concentration limit of 5,000 ppm. The pharmaceutical composition of the present invention may contain ethanol at a concentration of 1 ppm to about 500 ppm, for example, 1 ppm to about 400 ppm, 1 ppm to about 300 ppm, 1 ppm to about 200 ppm, 1 ppm to about 100 ppm, and 1 ppm to about 50 ppm.

[0111] Tert-butyl methyl ether (TBME) is an ICH Class 3 solvent with a PDE of 50 mg / day or a concentration limit of 5,000 ppm. The pharmaceutical composition of the present invention may contain tert-butyl methyl ether at a concentration of 1 ppm to about 500 ppm, for example, 1 ppm to about 400 ppm, 1 ppm to about 300 ppm, 1 ppm to about 200 ppm, 1 ppm to about 100 ppm, and 1 ppm to about 50 ppm.

[0112] Acetone is an ICH Class 3 solvent with a PDE of 50 mg / day or a concentration limit of 5,000 ppm. The pharmaceutical composition of the present invention may contain acetone at a concentration of 1 ppm to about 500 ppm, for example, 1 ppm to about 400 ppm, 1 ppm to about 300 ppm, 1 ppm to about 200 ppm, 1 ppm to about 100 ppm, and 1 ppm to about 50 ppm.

[0113] Ethyl acetate is an ICH Class 3 solvent with a PDE of 50 mg / day or a concentration limit of 5,000 ppm. The pharmaceutical composition of the present invention may contain ethyl acetate at a concentration of 1 ppm to about 500 ppm, for example, 1 ppm to about 400 ppm, 1 ppm to about 300 ppm, 1 ppm to about 200 ppm, 1 ppm to about 100 ppm, and 1 ppm to about 50 ppm.

[0114] n-Heptane is an ICH Class 3 solvent with a PDE of 50 mg or a limit of 5,000 ppm. The pharmaceutical compositions of the present invention may contain n-heptane at a concentration of 1 ppm to about 500 ppm, e.g., 1 ppm to about 400 ppm, 1 ppm to about 300 ppm, 1 ppm to about 200 ppm, 1 ppm to about 100 ppm, and 1 ppm to about 50 ppm.

[0115] In specific embodiments, the pharmaceutical compositions described herein comprise at least one of: (a) 1 ppm to about 300 ppm of methanol; (b) 1 ppm to about 89 ppm of toluene; (c) 1 ppm to about 60 ppm of methylene chloride; (d) 1 ppm to about 500 ppm of ethanol; (e) 1 ppm to about 500 ppm of TBME; (f) 1 ppm to about 500 ppm of acetone; (g) 1 ppm to about 500 ppm of ethyl acetate; and (h) 1 ppm to about 500 ppm of n-heptane.

[0116] In another specific embodiment, the pharmaceutical composition comprises deoxycytidine Form B, deoxythymidine, at least one pharmaceutically acceptable carrier, and at least one of: (a) 1 ppm to about 300 ppm of methanol, (b) 1 ppm to about 89 ppm of toluene, (c) 1 ppm to about 60 ppm of methylene chloride, (d) 1 ppm to about 500 ppm of ethanol, (e) 1 ppm to about 500 ppm of TBME, (f) 1 ppm to about 500 ppm of acetone, (g) 1 ppm to about 500 ppm of ethyl acetate, and (h) 1 ppm to about 500 ppm of n-heptane.

[0117] 1,2-Dichloroethane (1,2-DCE) is an ICH Class 1 solvent with a concentration limit of 5 ppm. The compositions of the present invention may contain 1,2-DCE at a concentration of 0.2 ppm to about 0.5 ppm, preferably less than 0.2 ppm, such that the 1,2-DCE is undetectable.

[0118] Oral administration is the preferred method of administration. The active ingredient can be added to any form of liquid that the patient will consume, including, but not limited to, milk from both bovine and human breasts, infant formula, and water.

[0119] Furthermore, pharmaceutical compositions suitable for oral administration may be 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 its derivatives, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, stearic acid or its salts. Soft gelatin capsules may contain vegetable oils, waxes, fats, semisolid or liquid polyols. Solutions and syrups may contain water, polyols, and sugars. Active agents intended for oral administration may be coated with or mixed with a material that delays disintegration and / or absorption of the active agent in the gastrointestinal tract. Thus, sustained release can be achieved over an extended period of time, and, if necessary, the active agent can be protected from degradation in the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate release of the active agent at specific gastrointestinal locations due to specific pH or enzymatic conditions.

[0120] To overcome the problem of crossing the blood-brain barrier, intrathecal administration is a more preferred form of administration. Intrathecal administration involves injecting a drug into the spinal canal, more specifically the subarachnoid space, so that it reaches the cerebrospinal fluid. This method is commonly used for spinal anesthesia, chemotherapy, and analgesics. Intrathecal administration can be performed by lumbar puncture (bolus injection) or a port-catheter system (bolus or infusion). The catheter is most commonly inserted between the lumbar lamina, and the tip penetrates the intrathecal cavity to the desired level (generally L3-L4). Intrathecal formulations most commonly use water and saline as excipients, although EDTA and lipids have also been used.

[0121] A more preferred administration mode is parenteral administration, including intravenous administration. Pharmaceutical compositions adapted for parenteral administration, including intravenous administration, include aqueous and non-aqueous sterile injection solutions or suspensions, 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 parenteral administration may be provided in single-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile carrier immediately before 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 present 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, 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.

[0122] Additionally, because some patients may be receiving enteral nutrition by the time deoxynucleoside treatment is initiated, dN can be administered through a gastric feeding tube or other enteral nutritional means.

[0123] Further modes of administration include mucosal administration, such as nasal, sublingual, vaginal, buccal or rectal administration, or transdermal administration to a subject.

[0124] 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 deep inhalation or by introduction through a mouthpiece. These compositions may contain aqueous or oil solutions of the active ingredient. Compositions for inhalation may be delivered in specially adapted devices, including, but not limited to, pressurized aerosols, nebulizers, or inhalers, which may be configured to provide a predetermined dose of the active ingredient.

[0125] 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.

[0126] 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.

[0127] III. Treatment Methods The present invention also provides a method of 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 a composition described herein comprising deoxycytidine Form B.

[0128] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a second composition comprising deoxythymidine.

[0129] In other embodiments, one composition containing both deoxycytidine and deoxythymidine is administered.

[0130] In one embodiment, a composition comprising deoxycytidine Form B and at least one residual solvent is administered to a subject, wherein the at least one residual solvent is selected from ICH Class 2 and Class 3 solvents, and the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for each residual solvent.

[0131] In a more specific embodiment, a composition consisting of deoxycytidine Form B and at least one residual solvent is administered to a subject, wherein the at least one residual solvent is selected from ICH Class 2 and Class 3 solvents, and the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for each residual solvent. Any composition, including the pharmaceutical compositions described above, may be administered.

[0132] In a specific embodiment, the composition comprises deoxycytidine Form B and at least one of: (a) 1 ppm to about 300 ppm of methanol, (b) 1 ppm to about 89 ppm of toluene, (c) 1 ppm to about 60 ppm of methylene chloride, (d) 1 ppm to about 500 ppm of ethanol, (e) 1 ppm to about 500 ppm of TBME, (f) 1 ppm to about 500 ppm of acetone, (g) 1 ppm to about 500 ppm of ethyl acetate, and (h) 1 ppm to about 500 ppm of n-heptane.

[0133] In a preferred embodiment, ICH Class 1 solvents, particularly 1,2-dichloroethane, are undetectable in the composition.

[0134] In another embodiment, a pharmaceutical composition is administered comprising deoxycytidine Form B, at least one residual solvent, and a pharmaceutically acceptable carrier described herein, wherein the residual solvents are selected from ICH Class 2 and Class 3 solvents, and the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for each residual solvent.

[0135] In a more specific embodiment, a pharmaceutical composition consisting of deoxycytidine Form B, deoxythymidine, at least one residual solvent, and at least one pharmaceutically acceptable carrier as described herein is administered, wherein the residual solvents are selected from ICH Class 2 and Class 3 solvents, and the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for each residual solvent. In a specific embodiment, the pharmaceutical composition is a fixed-dose powder composition as described above.

[0136] In yet another embodiment, a method for treating a disease or disorder characterized by imbalanced nucleotide pools in a subject in need thereof comprises administering to the subject therapeutically effective amounts of (1) a first composition comprising deoxycytidine Form B and at least one residual solvent and (2) a second composition comprising deoxythymidine and, optionally, at least one residual solvent, wherein the at least one residual solvent is selected from ICH Class 2 and Class 3 solvents, and wherein the concentration of the at least one residual solvent is less than about 10% of the ICH concentration limit for each residual solvent. In such an embodiment, the combined residual solvent amount of (1) and (2) is less than about 10% of the ICH concentration limit for that particular residual solvent.

[0137] In a specific embodiment, the first composition (i.e., (1)) and / or the second composition (i.e., (2)) is a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

[0138] Regardless of whether deoxycytidine and deoxythymidine are administered in the same or separate pharmaceutical compositions, the ratio of deoxycytidine to deoxythymidine can vary. For example, they can be 50 / 50, or 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.

[0139] Diseases or disorders characterized by unbalanced nucleotide pools that can be treated by the method of the present invention include, but are not limited to, those characterized by mutations in the following genes: TK2; DGUOK; TYMP; RRM2B; SUCLA2; SUCLGl; and MPV17.In a preferred embodiment, the disorder is mitochondrial DNA depletion syndrome (MDS).In a more preferred embodiment, MDS includes myopathy type characterized by mutations in TK2, encephalomyopathic type characterized by mutations in SUCLA2, neurogastrointestinal encephalopathy type characterized by mutations in TYMP, and hepatopathy type characterized by mutations in DGUOK, POLG, and MPV17.In the most preferred embodiment, the disorder is thymidine kinase 2 deficiency characterized by mutations in TK2 gene.

[0140] Administration of the pharmaceutical composition should begin as soon as a disorder characterized by an imbalanced nucleotide pool, such as MDS, is suspected and should continue throughout the patient's life. Tests for diagnosing such disorders, including TK2 deficiency, are known in the art.

[0141] For example, dT and dC are administered in equal amounts in a mixture for TK2 deficiency. The selection of a therapeutically effective dose is determined by those skilled in the art, taking into account several factors known to those skilled in the art. Such factors include the specific form of the deoxynucleoside and its pharmacokinetic parameters, such as bioavailability, metabolism, and half-life, which are established during the normal development procedures typically used to obtain regulatory approval for pharmaceutical compounds. Additional factors to consider when considering dosage include the condition or disease being treated or the benefit achieved in normal individuals, the patient's weight, the route of administration (whether acute or chronic), concomitant medications, and other factors known to affect the effectiveness of the administered pharmaceutical. Therefore, the exact dose should be determined according to standard clinical techniques, depending on the judgment of the skilled artisan and the circumstances of each patient.

[0142] A preferred dose ranges from about 100 mg / kg / day to about 1,000 mg / kg / day. A more preferred dose ranges from about 200 mg / kg / day to about 800 mg / kg / day. An even more preferred dose ranges from about 250 mg / kg / day to about 400 mg / kg / day. These dosages are for individual deoxynucleosides or compositions containing a mixture of two or more deoxynucleosides, such as dT and dC. For example, a dose can include 400 mg / kg / day of dT alone. In another example, a dose can include 200 mg / kg / day of dT and 200 mg / kg / day of dC. In another example, a dose can include 400 mg / kg / day of a mixture of dT and dC.

[0143] Administration of the deoxynucleoside can be once daily, twice daily, three times daily, four times daily, five times daily, or up to six times daily, preferably periodically. For example, if the deoxynucleoside is administered four times daily, dosing would be at 8:00 AM, 12:00 PM, 4:00 PM, and 8:00 PM.

[0144] If administered intravenously or intrathecally, the dose may be lower.

[0145] A preferred dosage range for such administration is from about 50 mg / kg / day to about 500 mg / kg / day.

[0146] Dosage can be adjusted to optimize efficacy in a subject. For example, a deoxynucleoside can be initiated at 100 mg / kg / day and then increased over time to 200 mg / kg / day, 400 mg / kg / day, 800 mg / kg / day, and up to 1000 mg / kg / day, depending on the subject's response and tolerance.

[0147] Before increasing dosage, the patient can be monitored for improvement of their condition.The response of the patient to the therapeutic administration of deoxynucleoside can be monitored by observing the changes in the patient's muscle strength and control, mobility, and 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, the dosage of deoxynucleoside can be increased.

[0148] Deoxynucleosides may be co-administered with other drugs. Such drugs include therapeutic agents for treating the symptoms of certain forms of MDS. In particular, in the case of TK2 deficiency, dT and dC may be co-administered with inhibitors of ubiquitous nucleoside degrading (catabolic) enzymes, including, but not limited to, tetrahydrouridine (an inhibitor of cytidine deaminase), immucillin 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 several cancers. [Example]

[0149] Example 1: Preparation of deoxycytidine form B

[0150] Deoxycytidine Form B was produced by the following 130 kg engineering process.

[0151] Step 1: The concentrated residue (crude deoxycytidine) of reactor R-6424 was sealed by vacuum. 924 kg of ethanol (99.5%) was introduced through a dedicated pipe by passing it through a filter housing.

[0152] Step 2: The reactants in reactor R-6426 were stirred for approximately 30 minutes.

[0153] Step 3: The reactant in reactor R-6426 was distilled under reduced pressure at 70°C or less.

[0154] Step 4: The concentrated residue in reactor R-6426 was opened using nitrogen. 98 L (0.25 vol) of purified water was passed through a dedicated pipe by passing it through a filter housing.

[0155] Step 5: The reactants in reactor R-6426 were stirred for approximately 10 minutes.

[0156] Step 6: The internal temperature of reactor R-6426 was increased to about 48°C.

[0157] Step 7: Maintain internal temperature and stir until mixture is visibly dissolved.

[0158] Step 8: Reactor R-6425 was placed under vacuum. 308 kg (0.70 vol) of ethanol (99.5%) was charged through a dedicated pipe and then released using nitrogen vacuum.

[0159] Step 9: The internal temperature of reactor R-6426 was maintained between 40° C. and 50° C. Pressurized ethanol (99.5%) from reactor R-6425 was slowly added to reactor R-6426 using nitrogen to prevent precipitation of crystals.

[0160] Step 10: 616 kg (1.58 vol) of ethanol (99.5%), 702 kg (1.8 vol) of ethyl acetate, and 730 kg (1.36 vol) of heptane were charged under vacuum to reactor R-6425. Nitrogen was used to release the vacuum. The mixture was stirred.

[0161] Step 11: The internal temperature of R-6426 was maintained between 40° C. and 50° C. The pressurized mixture from reactor R-6425 was slowly added to reactor R-6426.

[0162] Step 12: The internal temperature of reactor R-6426 was cooled to between 20°C and 30°C.

[0163] Step 13: The mixture was stirred for 1 hour.

[0164] Step 13: The internal temperature of reactor R-6426 was cooled to between 0°C and 10°C.

[0165] Step 14: The mixture was stirred for about 1 hour.

[0166] Step 15: The internal temperature of reactor R-6426 was maintained between 0°C and 10°C.

[0167] Step 16: The crystals were collected using a centrifugal filter and washed with ethyl acetate. The crystals were characterized as deoxycytidine Form B by XRPD analysis (see Example 2).

[0168] Example 2: Lot test results

[0169] Production of dC at a 4 kg scale yielded dC form B (Figure 2). Scaling up the manufacturing yielded a mixture of dC forms A and B (Figure 1) and increased the residual solvent content of the final API.

[0170] Lot 2DC(7) / SP-17001 was manufactured at a 4 kg scale.

[0171] Lots 2DC(7)-6-18001, 2DC(7)-6-18002, and 2DC(7)-6-18003 in Table 1 below were 55 kg manufacturing lots prepared in a different manner than that described in Example 1 herein, producing a mixture of Forms A and B as shown in Figure 1.

[0172] Lots 2DC(7)-E-6-19001 and 2DC(7)-E-6-19002 were 130 kg manufacturing runs using the process described in Example 1 and providing the Form B polymorph (Figure 2).

[0173] TIFF0007769603000003.tif244170

[0174] XRPD data for the mixture of A and B dC isoforms (2DC(A+B mix)), the B isoform produced in a small-scale manufacturing run (2DC(7) / SP-17001 initial), and the isolated A isoform are shown in Table 2. Stable form B was confirmed by determining the XRPD after 12 months of storage (2DC(7) / SP-17001 12 months).

[0175] TIFF0007769603000004.tif124170

[0176] Example 3: Residual Solvent Content of Commercially Available Materials

[0177] Samples of dC and dT purchased from a commercial supplier (Carbosynth) were analyzed by GS-MS to determine residual solvent content. The results are shown in the table below:

[0178] TIFF0007769603000005.tif60170

[0179] Example 4: Stability Study of Deoxycytidine Form B

[0180] A study was conducted to determine the stability over a 12-month period of deoxycytidine Form B prepared by the method of Example 1. Four lots of material were tested: (1) and (2) packaged in polyethylene (PE) bags sealed with cable ties, (3) packaged in a PE bag containing silica gel sealed with cable ties, and (4) packaged in a PE drum sealed with a lid.

[0181] The following test attributes were measured: Appearance (white to off-white powder) IR tracing HPLC ○Purity 99.0% or more Less than 1.0% total impurities ○0.5% or less cytosine Less than 0.05% α-anomer ●Water content 0.5% or less ●Specific rotation (+57.0°~+60.0°) ●Ignition residue ○0.5% or less ●Residual solvent (GC) ○MeOH: 3,000ppm or less Toluene: 890 ppm or less Methylene chloride: 600 ppm or less ○MTBE: 5,000ppm or less Ethyl acetate: 5,000 ppm or less n-heptane 5,000 ppm or less Ethanol: 5,000 ppm or less Elemental impurities ○Cd: 5ppm or less ○Pb: 5ppm or less ○As: 15ppm or less ○Hg:30ppm or less ○Co:50ppm or less ○V: 100ppm or less ○Ni: 200ppm or less ○Ti: 8ppm or less ○Au: 100ppm or less ○Pd: 100ppm or less ○Ir: 100ppm or less ○Os: 100ppm or less ○Rh: 100ppm or less ○Ru: 100ppm or less ○Se: 150ppm or less ○Ag: 150ppm or less ○Pt: 100ppm or less ○Mo: 3,000ppm or less ○Cu: 3,000ppm or less ○Cr: 11,000ppm or less ●Microbial content ○ Total aerobic microbial count: 100 CFU / g or less Total yeast and mold count: 100 CFU / g or less

[0182] Two sets of conditions were applied: (1) long-term storage (25±2°C / 60±5% relative humidity) and (2) accelerated storage (40±2°C / 75±5% relative humidity).

[0183] All attributes were measured initially (0 months) to confirm compliance with the above criteria. Appearance, IR, HPLC, and moisture content were then measured at 3, 6, 9, and 12 months to confirm compliance. Microbial content was measured again at 12 months. No significant changes were observed from initial to 12 months across all test attributes measured.

[0184] Example 5: Fixed-dose pharmaceutical composition

[0185] A fixed-dose powder for oral solution containing 2'-deoxycytidine (2.0 g) and 2'-deoxythymidine (2.0 g) was prepared in the amounts shown in the table below. The powder for oral solution was packaged in foil pouches (stick packs) composed of laminated polyethylene terephthalate, aluminum, and low-density polyethylene. The oral solution was prepared by dissolving the powder contents in water prior to administration to patients.

[0186] TIFF0007769603000006.tif74170

Claims

1. 1. A method for preparing substantially pure deoxycytidine Form B, comprising: a. providing crude deoxycytidine comprising a mixture of Form A and Form B polymorphic forms; b. contacting the crude deoxycytidine with ethanol to produce a first mixture; c. distilling ethanol from the first mixture to produce a residue; d. contacting the residue with purified water to form a second mixture; e. heating the second mixture to an internal temperature of at least about 40°C; f. adding ethanol, ethyl acetate, and heptane to the second mixture to form a third mixture; g. cooling the third mixture to produce substantially pure Form B deoxycytidine crystals; h. separating the substantially pure Form B crystals from the third mixture; A method comprising:

2. In (f), a first portion of ethanol is added to the second mixture, followed by the addition of a solution comprising a second portion of ethanol, ethyl acetate, and n-heptane. The method of claim 1.

3. the third mixture in (g) is cooled to an internal temperature of about 0°C to about 10°C; The method of claim 1.

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