Method for increasing the bioavailability of nucleoside drugs

Abstract

Provided is a method for increasing the bioavailability of nucleoside drugs in the treatment of mitochondrial depletion syndrome. In particular, the method relates to increasing the bioavailability of deoxycytidine and deoxythymidine by administering a therapeutically effective amount of deoxycytidine and deoxythymidine with food.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 388,470, filed Jul. 12, 2022, which is hereby incorporated by reference in its entirety.

[0002] The present invention relates to the field of mitochondrial diseases. In some aspects, the present invention is directed to methods for increasing the bioavailability of nucleoside agents, deoxycytidine, and deoxythymidine in the treatment of mitochondrial depletion syndrome.

Background Art

[0003] Mitochondrial diseases are clinically heterogeneous disorders resulting from deficiencies in the mitochondrial respiratory chain (RC) and oxidative phosphorylation, which are biochemical pathways that convert the energy in electrons into adenosine triphosphate (ATP). The respiratory chain consists of four multi - subunit enzymes (Complexes I - IV) that transfer electrons and generate a proton gradient across the inner mitochondrial membrane, and the flow of protons through Complex V promotes ATP synthesis (DiMauro and Schon 2003, DiMauro and Hirano 2005). Coenzyme Q10 (CoQ10) is an essential molecule that shuttles electrons from Complexes I and II to Complex III. The respiratory chain is specific in eukaryotic (e.g., mammalian) cells by being controlled by two genomes, mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). As a result, mutations in either genome can cause mitochondrial diseases. Most mitochondrial diseases affect multiple organs of the body, have a very high mortality rate in the most severely affected patients, and in early - onset patients, the disease progresses rapidly. There is no approved treatment for mitochondrial diseases, and there is only supportive therapy such as the administration of CoQ10 and its analogs for enhancing the activity of the respiratory chain and detoxifying reactive oxygen species (ROS), which are toxic by - products of dysfunctional respiratory chain enzymes.

[0004] Mitochondrial diseases can be classified as mitochondrial depletion syndromes and mitochondrial DNA deletion syndromes. In some mitochondrial diseases, both mtDNA depletion and deletions can occur.

[0005] Mitochondrial DNA depletion syndrome (MDS) is a clinically and genetically heterogeneous group of clinically distinct syndromes that are typically early- or juvenile-onset autosomal recessive disorders characterized by a severe reduction in mtDNA content.

[0006] Mitochondrial DNA deletion syndromes are mostly caused by spontaneous (de novo) gene deletions and are not hereditary, although de novo mutations in nuclear genes involved in mitochondrial DNA replication can also cause mtDNA deletions.

[0007] Both depletion syndromes and deletion syndromes result in reduced mitochondrial energy production and are associated with symptoms that develop in high-energy-requiring organs and tissues such as the brain, liver, and muscles. Based on the affected tissue and the mtDNA content of that tissue, the clinical picture of MDS can be classified into three main phenotypes: encephalomyopathy, myopathy, and hepatocerebral.

[0008] MDS is often the cause of severe pediatric encephalomyopathy characterized by a decrease in the copy number of mitochondrial DNA (mtDNA) in tissues and insufficient synthesis of mitochondrial RC complexes (Hirano, et al. 2001). The mitochondrial genome contains 37 genes encoding two rRNAs, 22 tRNAs, and 13 protein subunits, all of which are involved in the oxidative phosphorylation process that acts as the "powerhouse" of animal and plant cells.

[0009] In contrast, many of the replication and maintenance proteins involved in the maintenance of functional mtDNA at appropriate levels are encoded by nuclear DNA. Nuclear genes that function in the mitochondrial maintenance of the nucleotide pool required for DNA synthesis include, by way of non-limiting examples, TK2, SUCLA2, SUCLG1, RRM2B, DGUOK, and TYMP, and nuclear genes that function in mtDNA replication include, by way of non-limiting examples, POLG and C10orf2. One gene is, for example, thymidine kinase 2 (TK2), a nuclear gene product. TK2 deficiency (TK2d) is caused by an autosomal recessive genetic defect in the thymidine kinase 2 gene and results in a very rare DNA depletion and deletion syndrome [Dominguez-Gonzalez C, et al. Orphanet J Rare Dis. 2019;14(1):100, Hirano M, et al. Essays Biochem. 2018;62(3):467-481].

[0010] Typically, the expression product of TK2 translation localizes in the mitochondria, where the protein catalyzes the phosphorylation of deoxythymidine and, to some extent, forms each monophosphate to deoxycytidine [Saada A, et al. Nat Genet. 2001;29(3):342-344]. Thus, mutations in this gene ultimately impair the availability of these monophosphate precursors for forming the deoxyribonucleotide triphosphate (dNTP) components required for mtDNA replication.

[0011] The clinical picture of TK2d is characterized in most patients by progressive proximal muscle weakness, respiratory insufficiency, and early death. [Garone 2018 natl hx / p2para5; Wang 2018 natl hx / p5para3-4] TK2d patients and their caregivers have reported that mobility, respiratory function, and hospitalization are the most substantial health-related impacts, and that fatigue has the most substantial impact on quality of life. [Jensen MP, et al. Poster presented at World Muscle Society meeting, September 20-24, 2021 https: / / zogenix-pharmawrite.ipostersessions.com / Default.aspx?s=C5-40-1F-93-C3-22-F7-19-E2-D1-D2-97-D7-33-58-7A]. Currently, there are no disease-specific therapies with regulatory approval, and thus treatment is supportive or experimental.

[0012] Reports have been published on the use of chemically graded pyrimidine deoxynucleotides (i.e., non-GMP grade, not manufactured under Good Manufacturing Practice for Pharmaceuticals) and subsequent deoxynucleosides under an exceptional use protocol for treating patients with TK2d [Dominguez-Gonzalez C, et al. Orphanet J Rare Dis. 2019;14(1):100]. First, patients were treated with the deoxynucleotides deoxythymidine monophosphate and deoxycytidine monophosphate (dCMP / dTMP) with the aim of avoiding the defective TK2 enzyme and directly replenishing deoxythymidine monophosphate and deoxycytidine monophosphate in mitochondria. See, for example, U.S. Patent No. 10,292,996. Subsequently, unexpectedly, better results were obtained with a combination of deoxynucleosides of deoxycytidine and deoxythymidine. See, for example, U.S. Patent No. 10,471,087, which describes the treatment of TK2 deficiency with these pyrimidine nucleosides. Combined nucleoside administration is important because imbalanced nucleoside pools can lead to loss of fidelity in mtDNA replication and potentially introduce harmful mutations.

[0013] The investigational drug, designated as MT1621, is a combination of deoxycytidine and deoxythymidine. MT1621 is a fixed-dose pharmaceutical product containing equal weights of dC and dT, formulated as a powder for oral administration by reconstitution in water or secretions. MT1621 provides nucleoside therapy with equal weight ratios of dC and dT to restore the dNTP pool by two mechanisms. [Blazquez-Bermejo C, et al. Age-related metabolic changes limit efficacy of deoxynucleoside-based therapy in thymidine kinase 2-deficient mice. EBioMedicine. 2019;46:342-355, Lopez-Gomez C, et al. Deoxycytidine and Deoxythymidine Treatment for Thymidine Kinase 2 Deficiency. Annals of Neurology. 2017;81(5):641-652, Lopez-Gomez C, et al. Bioavailability and cytosolic kinases modulate response to deoxynucleoside therapy in TK2 deficiency. EBioMedicine. 2019;46:356-367]. The first mechanism is the addition of nucleoside supplementation to maximize the remaining TK2 activity in mitochondria and restore mtDNA replication. Second, MT1621 utilizes the thymidine kinase 1 (TK1) / deoxycytidine kinase (dCK) salvage pathway in the cytosol to restore the dNTP pool. Exogenous dT and dC promote the formation of deoxynucleotides dCMP and dTMP by TK1 / dCK-mediated phosphorylation of dT and dC that can enter mitochondria via membrane transporters, thus restoring mtDNA replication by a mechanism that bypasses the TK2 enzyme.

[0014] Administering a sufficient dose of nucleosides can be limited by possible dose-limiting side effects such as diarrhea and gastrointestinal disorders, so the several grams amount of nucleosides that need to be administered daily is not always possible, and nucleoside replacement therapy poses a challenge for many TK2d patients. One strategy to avoid side effects was to administer three times a day in a smaller amount per administration as a result. For example, improved dosing to reduce side effects is still needed.

Summary of the Invention

[0015] Certain aspects of the invention described herein provide an improved method of increasing drug absorption and reducing dose-limiting side effects.

[0016] In some aspects, the present disclosure provides a method of reducing the dosage of deoxycytidine (dC) and deoxythymidine (dT).

[0017] In some aspects, the present disclosure provides a method of increasing the absorption and tolerance of deoxycytidine (dC) and deoxythymidine (dT) by administering to a patient dC or dT or both nucleosides, along with instructions to the patient to take the drug combination immediately before, during, or immediately after a meal.

[0018] The structures of 2'-deoxycytidine (β-D-2'-deoxycytidine) and 2'-deoxythymidine (β-D-2'-deoxythymidine) are shown below. Endogenous nucleosides (such as β-D-2'-deoxycytidine) are referred to as standard nucleosides and have the same molecular formula and opposite stereochemical structures as non-endogenous nucleosides (such as β-L-2'-deoxycytidine).

Chemical

Brief Description of the Drawings

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BRIEF DESCRIPTION OF THE INVENTION

[0025] Certain aspects of the present invention are based on the surprising discovery that deoxythymidine and deoxycytidine significantly alter pharmacokinetic measures of absorption rate and plasma exposure when administered with food. The International Nonproprietary Name (INN) and United States Adopted Name (USAN) of deoxythymidine is doxorib thymidine, and the INN and USAN of deoxycytidine is doxecytidine.

[0026] Definitions The terms used herein generally have their ordinary meaning in the context of the present invention and in the particular context in which each term is used. Specific terms are discussed below or elsewhere in this specification to provide additional guidance to the practitioner in explaining the methods of the present invention and how to use them. Moreover, it will be understood that the same thing can be said in more than one way.

[0027] Accordingly, alternative languages and synonyms may be used for any one or more of the terms discussed herein, and no special significance should be placed on whether a term is elaborated or discussed herein. Synonyms for particular terms are provided. The listing of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any of the terms discussed herein, is for illustration only and in no way limits the scope and meaning of the present invention or any of the exemplified terms. Similarly, the present invention is not limited to its preferred embodiments.

[0028] Abbreviations of pharmacokinetic parameters calculated in dosing studies and brief definitions explaining them as used herein are as follows:

[0029] “Tmax” refers to the time to the highest concentration in a body fluid such as plasma.

[0030] “Cmax” refers to the highest concentration observed in a body fluid such as plasma.

[0031] “Kel” refers to the slope of the terminal linear portion of the plasma concentration / time curve.

[0032] “T1 / 2” refers to the half-life of the parent drug in a body fluid such as plasma.

[0033] “AUC(0-t)” or “AUC0-t” refers to the AUC for which concentrations were measured from time 0 to the last measurable time point.

[0034] “AUC(last)” or “AUClast” refers to the area under the curve to the quantifiable concentration at the last time point.

[0035] “AUC(0-inf)”, “AUC0-inf”, or “AUC(0-infinity)” refers to the AUC value with infinity inserted.

[0036] The phrase “area under the curve” or “AUC” refers to a pharmacokinetic statistic used to describe the total exposure to a drug in a body fluid such as plasma or tissue. More specifically, this is the time-averaged concentration of the drug circulating in the analyzed body fluid (such as plasma, blood, or serum). The standard calculation of AUC requires using non-compartmental techniques to calculate the AUC of the concentration from time 0 to the last measurable time point (AUC 0·t ) and represents the overall observed exposure to the drug.

[0037] As used herein, the phrase “healthy subject” refers to a human who does not have mitochondrial depletion syndrome.

[0038] The term "therapeutically effective amount" refers to an amount of a compound that, when administered to a subject for treating a disease, is sufficient to alleviate to some extent one or more of the symptoms of the disease being treated, or to inhibit the progression of the disease or bring about at least a partial improvement. The "therapeutically effective amount" can vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject being treated.

[0039] The term "bioavailability" refers to the amount of a drug that is absorbed systemically and is thereby capable of producing a biological effect.

[0040] The fraction of oral bioavailability is F oral =F ABS ×F G ×F H and this is the fraction of the oral dose that reaches the circulation in the active unchanged form. F oral is less than 100% of the active ingredient in the oral dose for three reasons: because the drug is not absorbed through the gastrointestinal tract and is excreted in the feces; because the drug is biotransformed by the cells of the intestine (into inactive metabolites); or because the drug is removed by the cells of the liver either by biotransformation and / or transport into the bile. Thus, "oral bioavailability" is the product of the fraction of the oral dose that is absorbed (F ABS ), the fraction of the absorbed dose that successfully reaches the blood side of the gastrointestinal tract (F G ), and the fraction of the drug in the blood supply of the gastrointestinal system that reaches the heart side of the liver (F H ).

[0041] The term "food effect" refers to an unpredictable phenomenon in which food or a particular type of food can affect the absorption of a drug from the gastrointestinal tract after oral administration. In some embodiments, the food effect as used herein is when an active substance or its formulation, such as a tablet or capsule, is orally administered to a human subject simultaneously with food, or in other words, administered in a fed state, compared to when the same formulation is administered in a fasting state, at least a 20% relative difference in the AUC (area under the curve), Cmax (maximum plasma concentration), and / or Tmax (time to maximum concentration) of the substance. The criteria and guidelines of the US Food and Drug Administration and the European Medicines Agency define the administration of a large calorie intake of about 850 - 1000 kcal to confirm the variability of rapid food supply in the oral bioavailability of the test drug. This meal should supply approximately 150 kcal of protein, 250 kcal of carbohydrates, and about 500 - 600 kcal of fat. See Rangaraj, N. et al, Pharmaceutics 14(9):1807(2022).

[0042] The food effect can be shown as negative when absorption is decreased, or positive when absorption is increased and appears as an increase in oral bioavailability (as reflected in the total exposure).

[0043] Alternatively, the food effect can refer to a change in the maximum concentration, or the time to reach the maximum concentration, independent of overall absorption. As a result, some drugs are recommended to be taken either in a fasting or fed state to achieve optimal effect. For example, a patient may be instructed to take the drug with a meal, before the meal (e.g., 1 hour before the meal), or after the meal (e.g., 2 hours after the meal). The pharmacokinetics of many drugs are not affected by food and can be taken in either a fasting or fed state without significantly affecting measures of absorption rate and amount such as AUC, Cmax, Tmax, and T1 / 2, and pharmacokinetic parameters such as exposure.

[0044] As used herein, the term "fasting" refers to administration at least 4 hours after a meal. Further, a fasting state also requires continuation of fasting for at least 2 hours after administration.

[0045] As used herein, the term "high fat" refers to the ingestion of food that supplies at least 500 kcal of fat.

[0046] The phrase "with food" refers to the state of ingesting a solid meal having sufficient bulk and fat content that is not rapidly dissolved and absorbed in the stomach. In some embodiments, the food is a meal such as breakfast, lunch, or dinner.

[0047] The food effect of pyrimidine nucleosides was not predicted based on the previously reported study of telbivudine. Telbivudine is β-L-2-deoxythymidine, a non-natural enantiomer of deoxythymidine (β-D-2'-deoxythymidine), and is FDA-approved and EMA-approved for the treatment of hepatitis B virus infection. The published study reported that the values of Cmax, Tmax, and AUC were equivalent when telbivudine (oral dose of 600 mg) was administered in the fed and fasting states, and that the absorption of telbivudine measured by Cmax, Tmax, AUC(0-t), and AUC(0-infinity) was not changed by food intake immediately before oral administration. [Zhou XJ, et al., J Clin Pharmacol. 2006 Mar;46(3):275-81].

[0048] In some embodiments, the Cmax of dC increases by about 50% to about 100% when deoxycytidine and deoxythymidine are administered with food, compared to when deoxycytidine and deoxythymidine are administered in a fasting state. In some embodiments, the Cmax of dC increases by about 50% - about 100%, about 50% - about 90%, about 50% - about 80%, about 50% - about 70%, about 50% - about 60%, about 60% - about 100%, about 60% - about 90%, about 60% - about 80%, about 60% - about 70%, about 70% - about 100%, about 70% - about 90%, about 70% - about 80%, about 80% - about 100%, about 80% - about 90%, or about 90% - about 100% when deoxycytidine and deoxythymidine are administered with food, compared to when deoxycytidine and deoxythymidine are administered in a fasting state. In some embodiments, the Cmax of dC increases by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% when deoxycytidine and deoxythymidine are administered with food, compared to when deoxycytidine and deoxythymidine are administered in a fasting state. In some embodiments, the Cmax of dC increases by at least about 79% when deoxycytidine and deoxythymidine are administered with food, compared to when deoxycytidine and deoxythymidine are administered in a fasting state.

[0049] In some embodiments, the AUC of dC 0-t increases by about 110% to about 160% when deoxycytidine and deoxythymidine are administered with food, compared to when deoxycytidine and deoxythymidine are administered in a fasting state. In some embodiments, the AUC of dC 0-tWhen deoxycytidine and deoxythymidine are administered with food, it increases by about 110% to about 160%, about 110% to about 150%, about 110% to about 140%, about 110% to about 130%, about 110% to about 120%, about 120% to about 160%, about 120% to about 150%, about 120% to about 140%, about 120% to about 130%, about 130% to about 160%, about 130% to about 150%, about 130% to about 140%, about 140% to about 160%, about 140% to about 150%, or about 150% to about 160% compared to when deoxycytidine and deoxythymidine are administered in a fasting state. In some embodiments, the AUC of dC 0-t When deoxycytidine and deoxythymidine are administered with food, it increases by at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, or at least about 160% compared to when deoxycytidine and deoxythymidine are administered in a fasting state. In some embodiments, the AUC of dC 0-t When deoxycytidine and deoxythymidine are administered with food, it increases by at least about 137% compared to when deoxycytidine and deoxythymidine are administered in a fasting state.

[0050] In some embodiments, the Cmax of dT increases by about 10% to about 60% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered in a fasting state. In some embodiments, the AUC of dC 0-tWhen deoxycytidine and deoxythymidine are administered with food, it increases by about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 60%, about 40% to about 50%, or about 50% to about 60% compared to when deoxycytidine and deoxythymidine are administered in a fasting state. In some embodiments, the Cmax of dT increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered in a fasting state. In some embodiments, the Cmax of dT increases by at least about 27% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered in a fasting state.

[0051] In some embodiments, the AUC of dT 0-t increases by about 50% to about 100% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered in a fasting state. In some embodiments, the AUC of dT 0-t increases by about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 100%, about 70% to about 90%, about 70% to about 80%, about 80% to about 100%, about 80% to about 90%, or about 90% to about 100% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered in a fasting state. In some embodiments, the AUC of dT 0-tWhen deoxycytidine and deoxythymidine are administered with food, they increase by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to when deoxycytidine and deoxythymidine are administered in a fasting state. In some embodiments, the AUC of dT 0-t When deoxycytidine and deoxythymidine are administered with food, it increases by at least about 74% compared to when deoxycytidine and deoxythymidine are administered in a fasting state.

[0052] One aspect of the invention is a method of increasing the bioavailability of deoxycytidine in a human patient taking a deoxycytidine and deoxythymidine therapeutic when the combination of nucleosides is administered with food. In one embodiment, the deoxycytidine and deoxythymidine therapeutic is administered substantially simultaneously with a meal. In another embodiment, the deoxycytidine and deoxythymidine therapeutic is administered within 30 minutes before a meal. In some aspects, administration to the patient is with food intake or within a range of 30 minutes or less before food intake (e.g., within 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 8, 7, 6, 5, 4, 3, 2, or 1 minute). In another embodiment, the deoxycytidine and deoxythymidine therapeutic is administered within 2 hours after a meal. In some aspects, administration to the patient is about 1 hour before to about 2 hours after food intake, about 1 hour before to about 1.5 hours after food intake, about 1 hour before to about 1 hour after food intake, about 1 hour before to about 30 minutes after food intake, about 30 minutes before to about 2 hours after food intake, about 30 minutes before to about 1.5 hours after food intake, about 30 minutes before to about 1 hour after food intake, or about 30 minutes before to about 30 minutes after food intake. In some aspects, administration to the patient is with food intake or from immediately after food intake to 1 hour after said intake (e.g., 0 minutes to 1 hour).

[0053] Another aspect of the present invention is a method of increasing the absorption rate of deoxycytidine in a human patient who is taking deoxycytidine and deoxythymidine therapeutic agents when the combination of nucleosides is administered with food. In one embodiment, the deoxycytidine and deoxythymidine therapeutic agents are administered substantially simultaneously with a meal. In another embodiment, the deoxycytidine and deoxythymidine therapeutic agents are administered within 30 minutes before a meal. In another embodiment, the deoxycytidine and deoxythymidine therapeutic agents are administered within 2 hours after a meal. In one embodiment, the meal is a high-fat and high-calorie meal. In another embodiment, the meal is adapted to the gastrointestinal health of the individual patient for the purpose of supplying as much fat and calorie content as possible.

[0054] Another aspect of the present invention is a method of increasing the bioavailability of deoxythymidine in a human patient who is taking deoxycytidine and deoxythymidine therapeutic agents when the combination of nucleosides is administered with food. In one embodiment, the deoxycytidine and deoxythymidine therapeutic agents are administered substantially simultaneously with a meal. In another embodiment, the deoxycytidine and deoxythymidine therapeutic agents are administered within 30 minutes before a meal. In another embodiment, the deoxycytidine and deoxythymidine therapeutic agents are administered within 2 hours after a meal.

[0055] Another aspect of the present invention provides a method of increasing the absorption rate and absorption amount measured by the drug concentration achieved in the bloodstream over time in a patient being administered a drug via oral administration, the method comprising administering to the patient a therapeutically effective amount of deoxycytidine with food.

[0056] Another aspect of the present invention provides a method of treating a patient with a deoxythymidine and deoxythymidine therapeutic agent and reducing or eliminating gastrointestinal side effects by administering the therapeutic agent with food. In one embodiment, the deoxythymidine and deoxythymidine therapeutic agent is administered substantially simultaneously with a meal. In another embodiment, the deoxythymidine and deoxythymidine therapeutic agent is administered within 30 minutes before a meal. In another embodiment, the deoxythymidine and deoxythymidine therapeutic agent is administered within 2 hours after a meal. In another embodiment, the gastrointestinal side effects are one or more of nausea, vomiting, and diarrhea.

[0057] In another embodiment, the dosage range for administration with food is from about 50 mg / kg / day (i.e., about 50 mg / kg / day of dC and about 50 mg / kg / day of dT) to about 500 mg / kg / day (i.e., about 500 mg / kg / day of dC and 500 mg / kg / day of dT). The dosage can be adjusted to optimize the effect in the subject. For example, the deoxynucleoside can be started at a dosage of 100 mg / kg / day and then increased over time from 30 mg / kg / day to 200 mg / kg / day, 400 mg / kg / day, 800 mg / kg / day, up to a maximum of 1000 mg / kg / day, depending on the response and tolerance of the subject. In one embodiment, the deoxynucleoside can be administered from about 200 mg / kg / day to 600 mg / kg / day. In one embodiment of the present invention, the daily dosage is up to a target or maintenance dosage of 400 mg / kg / day of a substantially equal mixture (equal weight) of dC and dT, administered with food and optionally decreased based on the tolerance of the patient's dosage. In a further embodiment, the dosage is divided into about one-third of the preferred daily dosage and administered three times a day (TID) with food. Also, the subject can be monitored for any adverse effects such as gastrointestinal intolerance, e.g., diarrhea, and improvement when the therapeutic agent is administered with food can also be monitored. In some embodiments, a dosage of more than once a day is administered with food. In some embodiments, a dosage of more than twice a day is administered with food. In some embodiments, a dosage of three times a day is administered with food. In some embodiments, when a dosage of three times a day is administered, at least one dosage is administered with food. In some embodiments, when a dosage of three times a day is administered, at least two dosages are administered with food. In some embodiments, when a dosage of three times a day is administered, all three dosages are administered with food. In some embodiments, a dosage of once a day is administered with food. In some embodiments, a dosage of twice a day is administered with food. In some embodiments, a dosage of three times a day is administered with food.

[0058] In some embodiments, the total daily dose of dC and dT comprises equal parts by weight of dC and dT. In some embodiments, the total daily dose of the combined weight of dC and dT containing equal parts by weight of dC and dT is about 50 mg / kg to about 1000 mg / kg, about 50 mg / kg to about 800 mg / kg, about 50 mg / kg to about 600 mg / kg, about 50 mg / kg to about 500 mg / kg, about 50 mg / kg to about 400 mg / kg, about 50 mg / kg to about 200 mg / kg, about 50 mg / kg to about 100 mg / kg, about 100 mg / kg to about 1000 mg / kg, about 100 mg / kg to about 800 mg / kg, about 100 mg / kg to about 600 mg / kg, about 100 mg / kg to about 500 mg / kg, about 100 mg / kg to about 400 mg / kg, about 100 mg / kg to about 200 mg / kg, about 200 mg / kg to about 1000 mg / kg, about 200 mg / kg to about 800 mg / kg, about 200 mg / kg to about 600 mg / kg, about 200 mg / kg to about 500 mg / kg, about 200 mg / kg to about 400 mg / kg, about 400 mg / kg to about 1000 mg / kg, about 400 mg / kg to about 800 mg / kg, about 400 mg / kg to about 600 mg / kg, about 400 mg / kg to about 500 mg / kg, about 500 mg / kg to about 1000 mg / kg, about 500 mg / kg to about 800 mg / kg, about 500 mg / kg to about 600 mg / kg, about 600 mg / kg to about 1000 mg / kg, about 600 mg / kg to about 800 mg / kg, or about 800 mg / kg to about 1000 mg / kg.

[0059] In some embodiments, the total daily dose of dC and dT is equally divided into three separate doses, with dC and dT of each dose administered separately over 24 hours. In some embodiments, each separate dose comprises equal parts by weight of dC and dT, and dC and dT are administered separately but substantially simultaneously. In some embodiments, each dose comprises equal parts by weight of dC and dT, and dC and dT are combined prior to administration. In some embodiments, each dose comprises equal parts by weight of dC and dT, and dC and dT are combined as a fixed-dose combination pharmaceutical composition in a single packet.

[0060] In some embodiments, the total daily dose comprises three separate doses of dC and dT, each dose being administered at a different time during a 24-hour period, and each dose comprising equal parts by weight of dC and dT. In some embodiments, each dose comprises the combined weight of dC and dT where dC and dT are equal parts by weight, and is about 15 mg / kg to about 330 mg / kg, about 15 mg / kg to about 260 mg / kg, about 15 mg / kg to about 200 mg / kg, about 15 mg / kg to about 160 mg / kg, about 15 mg / kg to about 133 mg / kg, about 15 mg / kg to about 70 mg / kg, about 15 mg / kg to about 33 mg / kg, about 33 mg / kg to about 330 mg / kg, about 33 mg / kg to about 260 mg / kg, about 33 mg / kg to about 200 mg / kg, about 33 mg / kg to about 160 mg / kg, about 33 mg / kg to about 133 mg / kg, about 33 mg / kg to about 70 mg / kg, about 70 mg / kg to about 330 mg / kg, about 70 mg / kg to about 260 mg / kg, about 70 mg / kg to about 200 mg / kg, about 70 mg / kg to about 160 mg / kg, about 133 mg / kg to about 330 mg / kg, about 133 mg / kg to about 260 mg / kg, about 133 mg / kg to about 200 mg / kg, about 133 mg / kg to about 160 mg / kg, about 160 mg / kg to about 330 mg / kg, about 160 mg / kg to about 260 mg / kg, about 160 mg / kg to about 200 mg / kg, about 200 mg / kg to about 330 mg / kg, about 200 mg / kg to about 260 mg / kg, or about 260 mg / kg to about 330 mg / kg, and the three doses are administered over 24 hours. In some embodiments, each dose comprises the combined weight of dC and dT where dC and dT are equal parts by weight, and is about 330 mg / kg, about 260 mg / kg, about 200 mg / kg, about 160 mg / kg, about 133 mg / kg, about 70 mg / kg, about 33 mg / kg, or about 15 mg / kg, and the three doses are administered over 24 hours.

[0061] In one aspect, the efficacy of treatment for patients with MDS is increased by administering the target doses of dC and dT per day with food.

[0062] In another aspect, the dosage range of each nucleoside can be decreased taking into account the increased bioavailability of deoxynucleoside when administered with food. In one embodiment, the decrease in the daily dosage of deoxynucleoside reduces gastrointestinal intolerance. In another aspect, the dosage of each nucleoside can be decreased taking into account the increased bioavailability of deoxynucleoside when administered with food.

[0063] Route of administration In one embodiment, the deoxycytidine and deoxythymidine therapeutic agents are administered orally. In another embodiment, the deoxycytidine and deoxythymidine therapeutic agents are dissolved in water or a secretion to obtain a solution for oral administration. In one embodiment, the solution is administered via a dispensing device that can measure various dosages and can be a syringe or a graduated pipette useful for delivering the dosage of the pharmaceutical solution. In one embodiment, the dispensing device is used as a metered-dose device that can dispense a fixed amount of the pharmaceutical solution. In one embodiment, the deoxycytidine and deoxythymidine therapeutic agents are administered by a feeding tube. In some embodiments, the feeding tube is a nasogastric feeding tube or an enteral feeding tube.

[0064] Dosage form / Administration frequency In the MT1621 clinical development program, two formulations were considered. The first formulation consists of dC and dT supplied in separate packets, which are combined prior to administration. The second formulation is the formulation in which the MT1621 therapeutic agent is supplied for clinical trials and consists of a fixed dosage combination of dC and dT supplied in a single packet. Considerations for pharmacokinetics (PK) in the clinical development program include a three-divided dosage / day administration frequency, the absorption rate and absorption amount of dC and dT, the effect of food on PK and tolerability, the dose proportionality of both formulations, and renal clearance.

Examples

[0065] Open-label, single ascending dose study in healthy adult subjects designed to evaluate the safety, tolerability, PK, and food effect of MT1621 The Phase I study was conducted as an open-label, single ascending dose study in healthy adult subjects designed to evaluate the safety, tolerability, PK, and food effect of MT1621 (dC and dT). To evaluate the PK of MT1621, the combination was administered at three different doses. Treatment A was MT1621 at a dose of 43.3 mg / kg in healthy male and female subjects in the fasting state (i.e., 43.3 mg / kg of dC and 43.3 mg / kg of dT, each approximately equal to one-third of a 130 mg / kg daily dose [TID]), Treatment B was MT1621 at a dose of 86.7 mg / kg in healthy male and female subjects in the fasting state (i.e., 86.7 mg / kg of dC and 86.7 mg / kg of dT, each approximately equal to one-third of a 260 mg / kg daily dose [TID]), and Treatment C was MT1621 at a dose of 133.3 mg / kg in healthy male and female subjects in the fasting state (i.e., 133.3 mg / kg of dC and 133.3 mg / kg of dT, each approximately equal to one-third of a 400 mg / kg daily dose [TID]). In addition, Treatment D was MT1621 at a dose of 133.3 mg / kg in healthy male and female subjects in the fed state to determine the effect of a high-fat meal on the single-dose PK of MT1621 (i.e., 133.3 mg / kg of dC and 133.3 mg / kg of dT, each approximately equal to one-third of a 400 mg / kg daily dose [TID]).

[0066] Methods of a Phase I study of single-dose escalating dosing and food effect by open-label fixed-sequence method. Fourteen (14) healthy adult male and female subjects were enrolled with an approximately equal distribution between male and female subjects (at least 40% of each sex).

[0067] Subject screening was conducted within 28 days prior to the first administration.

[0068] On the first day of each period, the subject took MT1621 at a one-third dose level in a fasting state as a gradually increasing single-dose administration over three periods in Part A (Treatment A, B, and C in Periods 1, 2, and 3 respectively), and took the maximum dose in a fed state in Part B (Treatment D in Period 4). Of the 14 subjects who completed Part A (Periods 1 - 3), 4 were unable to complete Part B (Treatment D in Period 4) during the first restraint. These subjects were then returned to the clinical department and the study was continued to complete Part B (Treatment D in Period 4), and 2 of the 4 subjects completed Part B.

[0069] Each treatment was evaluated by pre-dose and post-dose blood and urine PK sampling up to 48 hours to determine dC and dT in plasma and urine PK, and possible future analysis of blood for PD biomarkers.

[0070] The dose escalation to the next dose level (i.e., the next period) was not done until sufficient safety and tolerability were demonstrated at the previous dose level to allow progression to the next dose level.

[0071] At least a 2-day washout period without drug administration was maintained between each treatment. Fourteen subjects were enrolled in the study and 12 subjects completed the study. Data from 14 subjects were included in the PK analysis for Periods 1, 2, and 3, and data from 12 subjects were included in the PK analysis for Period 4.

[0072] The test drug administered in Example 1 was 0.5 g of dC and 0.5 g of dT powder for solution, and was reconstituted in secretion or water.

[0073] After reconstitution with a certain amount of water or secretion, the solution was administered in a measured volume to obtain the amounts specified in Tests A, B, C, and D.

[0074] The active ingredients and excipients in the pharmaceutical in development are as shown in Table 1.

[0075] Supply the target's daily dose, and the packet that is dissolved in water or secretion once a day and the volume corresponding to mg / kg required for the divided dose are withdrawn for multiple administrations.

Table 1

[0076] In Treatments A, B, and C, MT1621 was orally administered at hour 0 on day 1 after an overnight fast. In Treatment D, MT1621 was orally administered within 5 minutes after ingestion of a standardized high-fat breakfast (including eggs, bacon, and toast with butter).

[0077] Pharmacokinetics: Blood samples for determination of plasma dC and dT concentrations were collected before dosing (0 hour) and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 24, and 48 hours after dosing of the test drug on day 1 of each period. In Period 4, blood was collected for the 48-hour PK sample. In the case of Periods 1-3, the 48-hour PK sample was collected simultaneously with the pre-dose PK sample of the next period, and thus only one sample was collected for both PK time points. It should be noted that Periods 1-3 used sampling up to 24 hours at most for PK, while Period 4 used a complete 48-hour profile.

[0078] Urine samples for determination of dC and dT concentrations were collected before dosing (up to 2 hours before dosing) and at 0-4 hours, 4-8 hours, 8-12 hours, 12-24 hours, and 24-48 hours after dosing.

[0079] The following non-compartmental PK parameters for dC and dT in plasma were determined: area under the curve from time zero to the last non-zero concentration observed / measured (AUC0-t), area under the concentration-time curve extrapolated from time zero to infinity (AUC0-inf), area under the concentration-time curve for each dosing interval (AUCtau), maximum observed concentration (Cmax), time to reach Cmax (Tmax), apparent terminal-phase elimination rate constant (Kel) representing the fraction of drug eliminated per unit time, apparent terminal elimination-phase half-life of the drug in plasma (t1 / 2), apparent total plasma clearance after extravascular administration (CL / F), and apparent volume of distribution during the terminal elimination phase after extravascular administration (Vz / F).

[0080] The following urinary PK parameters were presented for dC and dT: urinary drug concentration for each urine collection interval (Cur), volume of urine collected over the entire urine collection interval (Vur), amount of unchanged drug excreted in urine (Ae), cumulative amount of unchanged drug excreted in urine (Ae, Cum), percentage of the dose excreted in urine (fe), and renal clearance (CLr) calculated as the ratio of the cumulative amount excreted in urine to the area under the plasma curve.

[0081] For the calculation of PK parameters, when most subjects showed measurable pre-dose (endogenous baseline) concentrations, the raw (unadjusted) plasma dC and dT concentrations below the lower limit of quantification (BLQ) were set to 1 / 2 of the limit of quantification (LOQ) of the assay throughout the PK profile. Since all subjects had measurable pre-dose (endogenous baseline) concentrations, Kel and related PK parameters were not calculated for the unadjusted data.

[0082] Method for baseline adjustment of plasma pharmacokinetic parameters Pre-dose blood samples for each dose level (each treatment period) served as confirmation of sufficient washout between dose levels and were also used to determine the amount of endogenous dC and dT. Each subject had three pre-dose samples (-2, -1, and -0.25 [-0.583] hours). Baseline adjustment was performed in all subjects regardless of the magnitude of their pre-dose values. Given the expected short T1 / 2 of dC and dT, carryover effects from exogenous MT1621 administration were not expected in this single-dose study.

[0083] Baseline adjustment was performed by subtracting the arithmetic mean baseline (average of all three pre-dose concentrations) for each subject in each treatment period from the post-dose plasma dC and dT concentrations for each subject prior to calculating the adjusted PK parameters. This was done for each subject and each period. All negative values after baseline adjustment were set to zero.

[0084] Bioanalytical procedures Plasma: dC and dT were quantified using valid high-performance liquid chromatography and tandem mass spectrometry (LC-MS / MS) methods. dC and dT were extracted from plasma samples by a single solid-phase extraction method. For dC, the extract was injected once using the Syneos Health high-performance liquid chromatography (HPLC) method TM.2541, and for dT, it was injected again using the Syneos Health HPLC method TM.2547. The two methods for determining dC and dT in human plasma were validated according to the 2018 FDA guidance and 2011 EMA guidance on the validation of biological analytical methods. These methods demonstrated the sensitivity, selectivity, precision, and accuracy acceptable for the quantification of dC and dT over the range of 0.500 to 200 ng / mL for each analyte in human plasma treated with potassium ethylenediaminetetraacetate (K2EDTA) by liquid chromatography electrospray ionization tandem mass spectrometry (LC / ESI / MS / MS) in positive ion mode, using [15N3]-deoxycytidine and [D3]-deoxythymidine as internal standards (IS) respectively (lower limit of quantification [LLOQ] of 0.5 ng / mL in 0.200 mL of tetrahydrofuridine and sodium citrate).

[0085] Statistical methods: The plasma and urine concentrations of dC and dT were tabulated and summarized for each treatment and time point for all subjects in the PK population cohort. Summary statistics including sample size (N), arithmetic mean (Mean), standard deviation (SD), coefficient of variation (CV%), minimum (Min), median, maximum (Max), geometric mean (GM), and geometric CV% (GMCV [%]) were calculated for all nominal concentration time points and PK parameters.

[0086] The assessment of the food effect was performed only on data from subjects who completed the study or had sufficient data for a pairwise comparison.

[0087] Analysis of variance (ANOVA) was performed on the natural logarithm (ln)-transformed plasma PK parameters AUC0-t and Cmax for the original plasma dC and dT concentrations and the baseline-adjusted plasma dC and dT concentrations.

[0088] The ANOVA model included diet condition (fasting, fed) as a fixed effect and subject as a random effect. Each ANOVA included calculation of least-squares means (LSM), differences between regimen LSMs, and standard errors associated with these differences.

[0089] The ratio of LSMs was calculated using the exponentiation of the LSMs from the analysis of the natural logarithm transformation for the fasting condition.

[0090] Dose proportionality was evaluated for the original and baseline-adjusted plasma dC and dT, AUC0-inf, AUC0-t, and Cmax parameters following administration of single doses (treatment A, B, and C in the fasting state).

[0091] AUC0-t and Cmax. These ratios were expressed as percentages relative to the fasting condition (i.e., treatment D / treatment C).

[0092] Consistent with two one-sided tests, the 90% confidence interval (CI) for the ratio was derived by exponentiating the CI obtained for the difference between regimen LSMs from the analysis of the ln-transformed AUC0-t and Cmax. The CI was expressed as a percentage relative to the fasting condition.

[0093] Dose proportionality was evaluated for the original and baseline-adjusted plasma dC and dT, AUC0-inf, AUC0-t, and Cmax parameters following administration of single doses (treatment A, B, and C in the fasting state).

[0094] Assessment of the food effect Assessment of the food effect was performed only on data from subjects who had completed the study or had sufficient data for a pairwise comparison.

[0095] Analysis of variance (ANOVA) was performed on the baseline-corrected natural logarithm (ln) transformed plasma PK parameters AUC0-t and Cmax. The ANOVA model included diet regimen (fasted, fed) as a parametric effect and subject as a random effect. Each ANOVA included calculation of the least-squares mean value (“LSM”), the difference between regimen LSMs, and the standard error associated with this difference.

[0096] The ratio of LSMs was calculated using the powers of the LSMs from the analysis of ln-transformed AUC0-t and Cmax. These ratios were expressed as percentages relative to the fasted state (i.e., treatment D / treatment C).

[0097] Consistent with two one-sided tests, the 90% confidence interval (CI) for the ratio was derived by taking the powers of the CIs obtained for the difference between regimen LSMs from the analysis of ln-transformed AUC0-t and Cmax. The CIs were expressed as percentages relative to the fasted state (i.e., treatment D / treatment C).

[0098] When baseline adjustment was appropriate (i.e., pre-dose dC and dT concentrations were measurable in most subjects), this analysis was also performed on the baseline-adjusted PK parameters AUC0-t, AUC0-inf, and Cmax.

[0099] Dose proportionality analysis Dose proportionality was evaluated for the raw and baseline-corrected plasma dC and dT, AUC0-t, and Cmax parameters following administration of single doses (treatment A, B, and C in the fasted state). To evaluate dose proportionality, linear regression and power model approaches were used.

[0100] First, a regression including both first (β1) and second (β2) effect terms: Y = α + β1 * dose + β2 * dose2 + ε (In the formula, Y represents the natural logarithm of the PK parameters AUC0-t, AUC0-inf, and Cmax, and ε explains the measurement error of the independent variable.) Using , the linear relationship between the PK parameters AUC0-t and Cmax and the dose was fitted. When β2 or α deviated from zero, dose proportionality was not shown. When β2 was not significantly different from zero, the linear regression was simplified as follows: Y = α + β * dose + ε

[0101] The slope β measured the dose proportionality between the dose and the dose-dependent PK parameter. Dose proportionality required β = 1.

[0102] Therefore, as the second step, the 95% CI of the slope β corresponding to the ln-transformed PK parameter was calculated. When the 95% CI included the value of 1 for the dose-dependent parameters (AUC0-t and Cmax), dose proportionality was established.

[0103] Since baseline adjustment was performed (i.e., because the pre-dose dC and dT concentrations were measurable in most subjects), this analysis was also performed for the baseline-adjusted PK parameters AUC0-t and Cmax.

Table 2-1

Table 2-2

Table 3-1

Table 3-2

Table 4

Table 5

[0104] The pharmacokinetic results from this study showed that MT1621 was absorbed into the systemic circulation, and more specifically, that the maximum dC and dT plasma concentrations (Cmax) were achieved at median (Tmax) of 1.0 - 1.25 hours and 1.0 hour (Tmax) in the fasting state, and 1.0 and 3.0 in the fed state. The values of the geometric mean elimination half-life of dC were 2.16 hours, 1.44 hours, and 3.01 hours at dose levels of 43.3 mg / kg, 86.7 mg / kg, and 133.3 mg / kg, respectively. PK variability was high, >30% for both dC and dT. The baseline-corrected dC Cmax increased at a lower than dose-proportional rate across the three fasting dose levels. The total exposure of dC (measured by AUC0-t and AUC0-inf) increased at a lower than dose-proportional rate.

[0105] Plasma exposure to dC and dT following MT1621 administration was consistently high at all three dose levels compared to the endogenous (baseline) concentration. This study demonstrated a significant increase in the systemic exposure of dC and dT following single-dose administration of MT1621.

[0106] Administration of 133.3 mg / kg of MT1621 in the fed state showed a significant food effect on MT1621, with a 79% and 137% increase in baseline-adjusted Cmax and AUC, respectively, for dC in plasma, and a 27% and 74% increase for dT in plasma, respectively, compared to the fasting state.

[0107] The dose-proportionality analysis of dC in plasma showed that the AUC appeared to be proportional to the dose (the 95% CI of the slope [b] included 1), but Cmax was not. The results of the dose-proportionality analysis of dT in plasma showed that neither AUC nor Cmax was dose-proportional.

[0108] Example 2. Open-label safety and efficacy study of MT1621 in study participants with TK2 deficiency The Phase II trial was conducted as an open-label continuous treatment with MT1621 (a combination of a fixed dose of dC and dT supplied in a single packet) in participants with TK2d, designed to evaluate the safety, tolerability, and PK of MT1621. Forty-seven participants were enrolled in the trial. After enrollment, participants switched from current chemical grade 2'-deoxycytidine monophosphate / 2'-deoxythymidine monophosphate (dCMP / dTMP) to deoxycytidine and deoxythymidine, or continued the use of deoxycytidine and deoxythymidine. At enrollment, participants who were at a maintenance dose of 400 mg / kg / day were treated three times a day (TID) with the same dose of deoxycytidine and deoxythymidine. Study participants who were at a total dose <400 mg / kg / day switched to either 260 mg / kg / day (130 mg / kg / day of deoxycytidine and 130 mg / kg / day of deoxythymidine) TID, 520 mg / kg / day (260 mg / kg / day of deoxycytidine and 260 mg / kg / day of deoxythymidine) TID, or 800 mg / kg / day (400 mg / kg / day of deoxycytidine and 400 mg / kg / day of deoxythymidine) TID, depending on which was closest to the participant's previous dose.

[0109] Summary statistics of important covariates of participants at baseline:

[0110] Age (years); N = 47; Mean (SD) = 18.4 (18.7); Median (Minimum, Maximum) = 9.90 (0.90, 75.6);

[0111] Weight (kg): N = 47; Mean (SD) = 37.0 (22.6); Median (Minimum, Maximum) = 31.5 (6.00, 99.5);

[0112] Absolute eGFR (mL / min): N = 46; Mean (SD) = 285 (124); Median (Minimum, Maximum) = 248 (111, 606);

[0113] Creatinine Clearance (mL / min): N = 47; Mean (SD) = 203 (120); Median (Minimum, Maximum) = 173 (42.9, 561);

[0114] Gender: Male = 27 (57.4%); Female = 20 (42.6%); and

[0115] Ethnicity: Unknown = 0 (0%); Hispanic or Latino = 14 (29.8%); Non-Hispanic or Latino = 33 (70.2%).

[0116] The initial protocol for deoxycytidine and deoxythymidine administration was for participants to take the drug regardless of food. Moreover, the study did not carefully record the participants' dietary status when they took the daily dose and on PK sampling days. In the second half of the program, when an unexpected food effect on PK was observed, the protocol was amended to instruct participants to take the drug with food. By that point, the planned PK samples had been collected for most participants. After the first day of the study, a total of three blood samples were collected from each individual and the plasma concentration of dC was measured. A small amount of PK blood sampling and PD endpoint collection were performed according to the following schedule: at 1 month, sampling was done 0.5, 1, 2, or 3 hours after dosing; at 3 months, sampling was done 8, 10, 12, or 14 hours after dosing. During sampling, it was confirmed whether food was ingested from 1 hour before dosing to 2 hours after dosing. Also, the lower limit of quantification (LLOQ) was 0.5 ng / mL for both dC and dT.

[0117] For the calculation of PK parameters, the raw (unadjusted) plasma concentrations of dC and dT below the lower limit of quantification (BLQ) were set to 1 / 2 of the assay limit of quantification (LOQ). Since the study did not sample pre-dose plasma, the plasma concentration of the participants could not be adjusted to the baseline. Since all participants had measurable pre-dose concentrations, Kel and related PK parameters were not calculated for the unadjusted data.

[0118] The PK data analysis was integrated from five studies, including three Phase I studies in healthy volunteers (HV) and two non-blind Phase II studies including both adult and pediatric study participants with TK2d (including those described in Examples 1 and 2). A total of 119 study participants were included, consisting of 44 healthy study participants, 16 study participants with renal impairment (moderate or severe), and 59 participants with TK2d (including participants aged 0.9 - 75.6 years).

[0119] Population PK analysis confirmed that the fed state was a statistically significant covariate for the PK of dC, including in participants with TK2 deficiency. Table 6 reflects the analysis of the PK data of all dC, including data from participants with TK2 deficiency. The strength of the positive food effect on the oral bioavailability of dC was 1.44, i.e., a 144% increase. This is the relevant value for estimating the food effect on dC exposure. This indicates that the fed state was generally associated with an increase in the bioavailability of dC, including in participants with TK2 deficiency. The decrease in the absorption rate of dC absorbed with food (parameter Ka in Table 6) is thought to be due to slower gastric / gastrointestinal motility in the fed state. The food effect on dT concentration in participants with TK2 deficiency may have been masked by high variability and limited PK sampling.

Table 6 - 1

Table 6 - 2

[0120] The PK of dC was described by a one-compartment model of first-order absorption and first-order elimination, and the following was found: · The endogenous dC baseline was the same across the studies and the population. · Body weight was found to have a positive correlation with apparent oral clearance (CL / F) and apparent volume of distribution in the mouth (V / F) using fixed allometric exponents of 0.75 and 1, respectively. · Age was not determined to be a significant covariate. · Relative bioavailability (F) increased by approximately 150% with food intake (standard or high-fat diet). · Absorption rate constant (Ka) decreased by approximately 44% with food intake (standard or high-fat diet). · Latino / Hispanic ethnicity was not determined to be a significant covariate. · No difference in F was detected between HV participants and TK2-deficient participants.

[0121] The PK of dT was described by a one-compartment model with two parallel (with lag time) first-order absorption and first-order elimination, and the following was found: · Endogenous dT baseline varied throughout the study (range of 0.220 - 1.14 ng / mL). · Body weight was not determined to be a significant covariate for CL / F or V / F. · Age was not determined to be a significant covariate. · Approximately 83% of relative bioavailability (F) was attributed to the first depot compartment. · F increased by approximately 120% with a high-fat diet. · F decreased by approximately 0.4-fold at a dose of 43.3 mg / kg and approximately 0.7-fold at a dose of 86.6 mg / kg relative to the F value at a dose of 133.3 mg / kg in healthy study participants. Previously, another analysis of the Phase I trial in HV showed that exposure to dT after a single escalating dose in this dose range increased more than a proportional increase with dose. · The absorption rate constant (K12) from the first input compartment and the lag time (ALAG3) from the second input compartment both decreased by approximately 80% with a high-fat diet. · F decreased by approximately 50% in the Latino / Hispanic population. ·F decreased (70% - 90%) in healthy subjects compared to participants with TK2 deficiency.

[0122] The dC model was able to explain the PK of dC with good accuracy, with limited or no bias. The population PK model provided a robust fit to the observed data using stable PK parameter estimates associated with high accuracy. The dC model was considered suitable for explaining dC exposure in patients with TK2d for further pharmacokinetic / pharmacodynamic analysis or exposure - response analysis.

[0123] The dT model was able to moderately explain the PK of dT with good accuracy and limited bias in HV, test participants with renal dysfunction, and participants with TK2d. However, the unexplained residual variability in the model suggests that while it may be suitable for the current dataset, it has limitations for use in model predictions. Therefore, dT exposure in participants with TK2 deficiency derived from the model should be interpreted with caution.

[0124] In conclusion, the PK changes induced by a standardized high-fat and high-calorie diet reflect the maximum effect on gastrointestinal physiology. The resulting maximum effect on oral bioavailability of drugs is generalizable to all subjects, including healthy subjects and those with TK2 deficiency. Regulations instruct that the food effect on drug PK be evaluated in a standardized high-fat meal in healthy adult participants with an appropriate sample size under controlled conditions, preferably with dense / rich PK sampling. To the best of our knowledge, there is no physiological basis to predict that underlying disease (TK2 deficiency) would impart different food effects on the bioavailability of dC and dT compared to healthy subjects. Thus, the results from a dedicated study in healthy subjects (Example 1) provide definitive and reliable findings regarding the food effect on the oral PK of dC and dT that are fully applicable to the target patient population. The ability of the population PK model to independently identify food and (in the case of dT in healthy participants) high-fat meal as significant covariates of PK provides further support.

Claims

**Claim 1** A method for increasing the oral bioavailability of deoxycytidine (dC) and deoxythymidine (dT) in a patient undergoing treatment for mitochondrial depletion syndrome (MDS), the method comprising administering a therapeutically effective amount of dC and dT to the patient in need thereof with food, or instructing a caregiver of the patient to administer a therapeutically effective amount of dC and dT with food. **Claim 2** A method for increasing the amount of deoxycytidine (dC) absorbed by a patient's body, the method comprising administering a dose containing equal amounts of dC and dT, wherein the combined weight of dC and deoxythymidine (dT) is from about 100 mg / kg to about 200 mg / kg, and wherein the administration is carried out with food. **Claim 3** The method according to claim 2, wherein the dose, wherein the combined weight of dC and dT is from about 100 mg / kg to about 200 mg / kg, is administered three times a day. **Claim 4** The method according to claim 1, wherein the therapeutically effective amount of the combined weight of dC and dT is from about 200 mg / kg / day to about 800 mg / kg / day. **Claim 5** The method according to claim 1 or 4, wherein the therapeutically effective amount of the combined weight of dC and dT is 800 mg / kg / day. **Claim 6** The method according to claim 1 or 2, wherein the dC and dT are administered as a combined pharmaceutical composition in a fixed dose. **Claim 7** The method according to claim 1 or 2, wherein the dC and dT are administered after combining two pharmaceutical compositions, one containing dC and the other containing dT, in equal parts by weight. **Claim 8** The method according to claim 1 or 2, which increases the absorption rate of dC and dT. **Claim 9** The method according to claim 1 or 2, which increases the absorption amount of dC and dT. **Claim 10** The method according to claim 1 or 2, which increases the circulating half-life of dC and dT. **Claim 11** The method according to claim 1 or 2, wherein the dC and dT are administered simultaneously with the oral administration of food to up to about 30 minutes after the oral administration of food. **Claim 12** The method according to claim 1 or 2, wherein the food is a high-fat, high-calorie diet. **Claim 13** The method according to claim 1 or 2, wherein the dose of dC and dT is administered one to three times a day, and at least one dose is administered with food. **Claim 14** When dC and dT are administered with food, the mean maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC0-t) of deoxycytidine and deoxythymidine in the subject in need thereof are increased as compared to when dC and dT are administered in a fasting state, the method according to claim 1 or 2.

15. The method according to claim 14, wherein the Cmax of dC is increased by at least about 79%.

16. The method according to claim 14, wherein the AUC0-t of dC is increased by at least about 137%.

17. The method according to claim 14, wherein the Cmax of dT is increased by at least about 27%.

18. The method according to claim 14, wherein the AUC0-t of dT is increased by at least about 74%.

19. The method according to claim 2, wherein the patient has mitochondrial depletion syndrome (MDS).

20. The method according to claim 1 or 19, wherein the MDS is a TK2 deficiency disorder or a POLG deficiency disorder.

21. A method for treating a patient with mitochondrial depletion syndrome, comprising administering to the patient a therapeutically effective amount of deoxycytidine (dC) and a therapeutically effective amount of deoxythymidine (dT), and inducing the patient to ingest the dC and dT with food.

22. The method according to claim 21, wherein the administration to the patient is carried out substantially simultaneously with the ingestion of the food.

23. The method according to claim 21, wherein the administration to the patient is carried out from about 30 minutes before to about 2 hours after food ingestion.

24. The method according to claim 21, wherein the administration to the patient is carried out immediately after food ingestion to 1 hour after food ingestion.

25. The method according to claim 21, wherein the dC and dT are in the form of a powder composition for reconstitution in water or a secretion.

26. The method according to claim 25, wherein the powder is in the form of a combined pharmaceutical composition of a fixed dose of dC and dT.

27. The dC and dT are in the form of a composition, and when administered with food, it results in an increase in the maximum plasma concentration (Cmax) and the absorption amount (AUC(last)) as compared to administration without food, and a container comprising a printed label recommending that the composition should be ingested with food, the method according to claim 1.

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