Method for improving bioavailability of nucleoside drug substances

Abstract

The invention provides a method for improving the bioavailability of a nucleoside drug for treating mitochondrial depletion syndrome. In particular, the methods involve increasing the bioavailability of deoxycytidine and deoxythymidine by administering a therapeutically effective amount of deoxycytidine and deoxythymidine with food.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 388,470 filed on July 12, 2022, which is incorporated by reference in its entirety into this application. Field of the Invention

[0003] The present invention relates to the field of mitochondrial diseases. In certain aspects, the present invention relates to methods for increasing the bioavailability of nucleoside drug substances deoxycytidine and deoxythymidine in the treatment of mitochondrial depletion syndromes.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] Mitochondrial diseases are clinically heterogeneous disorders caused by defects in the mitochondrial respiratory chain (RC) and oxidative phosphorylation, i.e., the biochemical pathway that converts energy from electrons into adenosine triphosphate (ATP). The respiratory chain consists of four multi-subunit enzymes (complexes I-IV) that transfer electrons to generate a proton gradient across the inner mitochondrial membrane, which flows through complex V to drive ATP synthesis (DiMauro and Schon 2003; DiMauro and Hirano 2005). Coenzyme Q10 (CoQ10) is an essential molecule that transports electrons from complexes I and II to complex III. The respiratory chain is unique in eukaryotic (e.g., mammalian) cells in that it is controlled by two genomes, mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Therefore, mutations in either genome may lead to mitochondrial disease. Most mitochondrial diseases affect multiple organs of the body, with very high mortality rates in the most severely affected patients and a rapidly progressive course in patients with early onset. There are no approved treatments for mitochondrial diseases, only supportive therapies such as administration of CoQ10 and its analogs to enhance respiratory chain activity and detoxify reactive oxygen species (ROS), which are toxic byproducts of dysfunctional respiratory chain enzymes.

[0006] Mitochondrial diseases can be classified into mitochondrial depletion syndromes and mitochondrial DNA deletion syndromes. In some mitochondrial diseases, both mtDNA depletion and deletion occur simultaneously.

[0007] Mitochondrial DNA depletion syndromes (MDS) are a group of clinically and genetically heterogeneous, clinically distinct syndromes that are usually recessive, with early onset or teenage onset and are characterized by severe reductions in mitochondrial DNA content.

[0008] Mitochondrial DNA deletion syndrome is most commonly caused by spontaneous (de novo) gene deletions and is not inherited, however, de novo mutations in nuclear genes involved in mitochondrial DNA replication can also result in mtDNA deletions.

[0009] Both depletion and deletion syndromes result in low mitochondrial energy production, with symptoms manifesting in organs and tissues with high energy demands, such as the brain, liver, and muscle. The clinical manifestations of MDS can be divided into three major phenotypic forms, depending on the tissues affected and the mtDNA content of those tissues: encephalomyopathy, myopathy, and hepatic encephalopathy.

[0010] MDS is a common cause of severe childhood encephalomyopathy, characterized by reduced mitochondrial DNA (mtDNA) copy number in tissues and insufficient synthesis of mitochondrial RC complexes (Hirano et al., 2001). The mitochondrial genome contains 37 genes that encode two rRNAs, 22 tRNAs, and 13 protein subunits, all of which are involved in the oxidative phosphorylation process that serves as the "power plant" of animal and plant cells.

[0011] In contrast, many of the proteins involved in the replication and maintenance of adequate levels of functional mitochondrial DNA are encoded in the nuclear DNA. Nuclear genes that function in the nucleotide pools required for mitochondrial maintenance DNA synthesis are, by non-limiting example, TK2, SUCLA2, SUCLG1, RRM2B, DGUOK, and TYMP, while nuclear genes that function in mtDNA replication are, by non-limiting example, POLG and C10orf2. For example, one gene is thymidine kinase 2 (TK2), which is a nuclear gene product. TK2 deficiency (TK2d) is caused by an autosomal recessive defect in the thymidine kinase 2 gene and results in an extremely rare mitochondrial DNA depletion and deletion syndrome. [Domínguez-González C et al., Orphanet J Rare Dis. 2019;14(1):100; Hirano M et al. Essays Biochem. 2018;62(3):467-481.]

[0012] Normally, the expression product of TK2 translation is localized in the mitochondria, where the protein catalyzes the phosphorylation of deoxythymidine and, to some extent, deoxycytidine to form the corresponding monophosphates. [Saada A et al., Nat Genet. 2001; 29(3): 342-344]. Therefore, mutations in this gene impair the availability of these monophosphate precursors to form the final deoxyribonucleotide triphosphate (dNTP) components necessary for mitochondrial DNA replication.

[0013] The clinical presentation of TK2d is characterized by progressive proximal muscle weakness, respiratory insufficiency, and premature death in most patients. [Garone 2018natl hx / p2para5; Wang 2018natl hx / p5para3-4] Patients with TK2d and their caregivers report mobility, respiratory function, and hospitalization as the most significant health-related impacts, while fatigue has the most significant impact on quality of life. [Jensen MP et al. poster presented at the World Muscle Society Conference, 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]. There are currently no disease-specific therapies approved by regulatory authorities, so treatment is supportive or experimental.

[0014] Reports have been published on the use of chemical-grade pyrimidine deoxynucleotides (i.e., non-GMP grade (not produced in accordance with current good manufacturing practices)) and later deoxynucleosides to treat patients with TK2d under compassionate use protocols. [Domínguez-González C et al., Orphanet J Rare Dis. 2019;14(1):100]. Initially, patients were treated with the deoxynucleotides deoxythymidine monophosphate and deoxycytidine monophosphate (dCMP / dTMP) in an effort to bypass the defective TK2 enzyme and replenish deoxythymidine monophosphate and deoxycytidine monophosphate directly to the mitochondria. See, e.g., US10,292,996. Later, unexpectedly better results were obtained using a deoxynucleoside combination of deoxycytidine and deoxythymidine. See, e.g., US10,471,087, which describes the use of these pyrimidine nucleosides to treat TK2 deficiency. Combined nucleoside administration is important because an unbalanced nucleoside pool may lead to a loss of mtDNA replication fidelity, potentially introducing deleterious mutations.

[0015] An investigational product under development, referred to as MT1621, is a combination of deoxycytidine and deoxythymidine. MT1621 is a fixed-dose drug product containing equal weights of dC and dT (hereinafter referred to as the "fixed-dose product"), formulated as a powder for oral administration by reconstitution in water or juice. MT1621 provides a nucleoside therapy with an equal weight ratio of dC and dT to restore the dNTP pool through two mechanisms. [Blázquez-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. TK2deficiency). EbioMedicine.2019;46:356-367]. The first mechanism is to add nucleoside supplementation to maximize the residual TK2 activity in mitochondria and restore mtDNA replication. Second, MT1621 utilizes the thymidine kinase 1 (TK1) / deoxycytidine kinase (dCK) rescue pathway in the cytosol to restore the dNTP pool. Exogenous dT and dC drive the formation of deoxynucleotides dCMP and dTMP through TK1 / dCK-mediated dT and dC phosphorylation, which can enter mitochondria through membrane transporters, thereby restoring mtDNA replication by a mechanism that bypasses the TK2 enzyme.

[0016] Nucleoside supplementation therapy presents a challenge for many TK2d patients because it is not always feasible to take several grams of nucleoside per day, as providing adequate doses of nucleoside may be limited by the possibility of dose-limiting side effects, such as diarrhea and gastrointestinal discomfort. One strategy to avoid side effects is to give the drug three times per day, so that the amount given each time is lower. There is still a need to improve dosing, for example, to reduce side effects.

[0017] Overview of the Disclosure

[0018] Certain aspects of the invention described in this application provide improved methods for increasing drug absorption and reducing dose-limiting side effects.

[0019] In some aspects, the present disclosure provides methods of reducing the amount of deoxycytidine (dC) and deoxythymidine (dT) administered.

[0020] In some aspects, the present disclosure provides methods for increasing the absorption and tolerability of deoxycytidine (dC) or deoxythymidine (dT), or both nucleosides, by providing a patient with instructions to take the drug combination before, during, or shortly after a meal.

[0021] The structures of 2'-deoxycytidine (β-D-2'-deoxycytidine) and 2'-deoxythymidine (β-D-2'-deoxythymidine) are shown below. Endogenous nucleosides (e.g., (β-D-2'-deoxycytidine) are called canonical nucleosides, and they have the same molecular formula and opposite stereochemical structure as non-endogenous nucleosides (e.g., (β-L-2'-deoxycytidine).

[0022]

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The extent of dT and dC absorption (expressed as AUC) for the four treatment regimens described in this application is shown. 0-t Measurements) graph (see also Table 2). Dashed line = deoxythymidine (ng*h / mL) exposure; solid line = deoxycytidine exposure (ng*h / mL) for each treatment group.

[0025] Figure 2 The extent of dT and dC absorption (expressed as AUC) for the four treatment regimens described in this application is shown. 0-inf Measurements) graph (see also Table 3). Dashed line = deoxythymidine (ng*h / mL) exposure; solid line = deoxycytidine exposure (ng*h / mL) for each treatment group.

[0026] Figure 3Baseline-adjusted arithmetic mean plasma dC concentration-time profiles following administration of a single dose of 133.3 mg / kg MT1621 (i.e., 133.3 mg / kg dC and 133.3 mg / kg dT) either fasted or fed are shown on a linear scale.

[0027] Figure 4 The baseline-adjusted arithmetic mean plasma dT concentration-time curves after administration of a single dose of 43.3 mg / kg MT1621 (i.e., 43.3 mg / kg dC and 43.3 mg / kg dT) while fasting, 86.7 mg / kg MT1621 (i.e., 86.7 mg / kg dC and 86.7 mg / kg dT) while fasting, 133.3 mg / kg MT1621 (i.e., 133.3 mg / kg dC and 133.3 mg / kg dT) while fasting, and 133.3 mg / kg MT1621 (i.e., 133.3 mg / kg dC and 133.3 mg / kg dT) while fed are shown on a linear scale.

[0028] Figure 5 Shown is a graph of nominal time (hours) post-dose versus plasma concentration (ng / mL) of deoxycytidine (dC) measured in TK2-deficient subjects as described in Example 2.

[0029] Figure 6 Shown is a graph of nominal time (hours) post-dose versus plasma concentration (ng / mL) of deoxythymidine (dT) measured in TK2-deficient subjects as described in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0031] 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 taken with food. The International Nonproprietary Name (INN) and United States Adopted Name (USAN) of deoxythymidine is doxribtimine, and the INN and USAN of deoxycytidine is doxecitine.

[0032] definition

[0033] The terms used in this specification generally have their ordinary meaning in the art, in the context of the present invention, and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to practitioners to describe the methods of the present invention and how to use these methods. In addition, it should be recognized that the same thing can be expressed in multiple ways.

[0034] Therefore, alternative language and synonyms may be used for any one or more terms discussed in this application, and no special meaning is given to whether a term is elaborated or discussed in detail in this application. Synonyms for certain terms are provided. The description of one or more synonyms does not exclude the use of other synonyms. The examples used anywhere in this specification (including examples of any terms discussed in this application) are only illustrative and in no way limit the scope and meaning of the present invention or any exemplified terms. Similarly, the present invention is not limited to its preferred embodiments.

[0035] The abbreviations for the pharmacokinetic parameters calculated in the dosing studies and the brief definitions used in this application to describe them are:

[0036] "Tmax" refers to the time at which maximum concentration is reached in body fluids (such as plasma).

[0037] "Cmax" refers to the maximum concentration observed in body fluids (eg, plasma).

[0038] "Kel" refers to the slope of the terminal linear portion of the plasma concentration / time curve.

[0039] "T1 / 2" refers to the half-life of a drug substance in body fluids (eg, plasma).

[0040] “AUC(0-t)” or “AUC 0-t ” refers to the AUC measured from time 0 to the last measurable concentration.

[0041] "AUC(last)" or AUC last It refers to the area under the curve of the final quantifiable concentration.

[0042] “AUC(0-inf)”, “AUC 0-inf ” or “AUC (0-infinity)” refers to the AUC value extrapolated to infinity.

[0043] 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 (e.g., plasma) or tissue. More specifically, it is the time-averaged concentration of a drug circulating in the body fluid being analyzed (e.g., plasma, blood, or serum). The standard calculation of AUC involves using a non-compartmental technique to calculate the AUC from time 0 to the last measurable concentration (AUC 0.t , and represents the total observed exposure to the drug.

[0044] The phrase "healthy subject" as used in this application refers to a person who does not have mitochondrial depletion syndrome.

[0045] The phrase "therapeutically effective amount" refers to the amount of a compound that is sufficient to alleviate to some extent one or more symptoms of the disease being treated or to inhibit or at least partially reverse the progression of the disease when administered to a subject for the treatment of the disease. The "therapeutically effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0046] The term "bioavailability" refers to the extent to which a drug is absorbed systemically and therefore available to produce a biological effect.

[0047] The oral bioavailability fraction is F 口服 =F ABS x F G x F H , which is the fraction of an oral dose that enters the blood circulation in active, unchanged form. 口服 Less than 100% of the active ingredient in an oral dose for three reasons: the drug is not absorbed from the gastrointestinal tract and is eliminated in the feces; the drug is biotransformed by enterocytes (to inactive metabolites); or the drug is eliminated by hepatocytes, either by biotransformation and / or transport into the bile. Therefore, "oral bioavailability" is the fraction of an oral dose that is absorbed (F ABS ), the fraction of absorbed dose that successfully reaches the blood side of the gastrointestinal tract (F G ) and the fraction of drug in the gastrointestinal blood supply on the cardiac side of the liver (F H )

[0048] The phrase "food effect" refers to an unpredictable phenomenon in which food or certain types of food affect the absorption of a drug from the gastrointestinal tract after oral administration. In some aspects, the food effect used in this application refers to when an active substance or its formulation (e.g., tablets or capsules) is orally administered to a human subject simultaneously with food, or in other words, when the same formulation is administered in a fed state compared to a fasted state, the AUC (area under the curve), Cmax (maximum plasma concentration) and / or Tmax (maximum concentration time) of the active substance are at least 20% relative differences. The specifications and guidelines of the U.S. Food and Drug Administration and the European Medicines Agency provide for the administration of a large caloric intake of about 850-1000 kcal to examine the fasting-fed variability of the oral bioavailability of the drug under study. This meal provides about 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).

[0049] A food effect can be designated as a negative effect when absorption is decreased, or a positive effect when absorption is increased and manifests as an increase in oral bioavailability (reflected by total exposure).

[0050] Alternatively, the food effect may refer to a change in maximum concentration or time to reach maximum concentration, independent of overall absorption. Therefore, it is recommended that some drugs be taken in a fasting or fed state to achieve optimal effect. For example, patients may be instructed to take medication during meals, before meals (e.g., one hour before meals), or after meals (e.g., two hours after meals). The pharmacokinetics of many drugs are not affected by food, and they can be taken in a fasting or fed state without significant effects on pharmacokinetic parameters (such as absorption rate and extent and exposure such as measurements of AUC, Cmax, Tmax, and T1 / 2).

[0051] The term "fasting" used in this application refers to administration at least 4 hours after a meal. In addition, the fasting state also requires continued fasting for at least 2 hours after administration.

[0052] The term "high fat" as used herein refers to the intake of foods that provide at least 500 kcal of fat.

[0053] The phrase "with food" refers to the state of eating solid food having sufficient volume and fat content and not being rapidly dissolved and absorbed in the stomach. In some embodiments, the food is a meal, such as breakfast, lunch or dinner.

[0054] Based on previously reported studies with telbivudine, no food effect is expected for pyrimidine nucleosides. Telbivudine is β-L-2-deoxythymidine, a non-natural enantiomer of deoxythymidine (β-D-2'-deoxythymidine); it has been approved by the FDA and EMA for the treatment of hepatitis B virus infection. Research publications report that Cmax, Tmax, and AUC values ​​are comparable when telbivudine (600 mg oral dose) is taken under fed and fasted conditions, and that telbivudine absorption measured as Cmax, Tmax, AUC(0-t), and AUC(0-infinity) is not altered by food intake immediately prior to oral administration. [Zhou XJ et al., J Clin Pharmacol. 2006 Mar;46(3):275-81].

[0055] In some embodiments, the Cmax of dC is increased by about 50% to about 100% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered under fasting conditions. In some embodiments, the Cmax of dC is increased 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 100% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered under fasting conditions. In some embodiments, the Cmax of dC is increased 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 under fasting conditions. In some embodiments, the Cmax of dC is increased by at least about 79% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered under fasting conditions.

[0056] In some embodiments, when deoxycytidine and deoxythymidine are administered with food, the AUC of dC is increased by 1.1% compared to when deoxycytidine and deoxythymidine are administered under fasting conditions. 0-t In some embodiments, the AUC0-t of dC is increased 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 160% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered under fasting conditions. In some embodiments, when deoxycytidine and deoxythymidine are administered with food, the AUC of dC is increased by 1.1% compared to when deoxycytidine and deoxythymidine are administered under fasting conditions. 0-t In some embodiments, the AUC of dC is increased 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% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered under fasting conditions. 0-t An increase of at least about 137%.

[0057] In some embodiments, the Cmax of dT is increased by about 10% to about 60% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered under fasting conditions. In some embodiments, the AUC of dC is increased by about 10% to about 60% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered under fasting conditions. 0-t In some embodiments, the Cmax of dT is increased 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 under fasting conditions. In some embodiments, the Cmax of dT is increased by at least about 27% when deoxycytidine and deoxythymidine are administered with food compared to when deoxycytidine and deoxythymidine are administered under fasting conditions.

[0058] In some embodiments, the AUC of dT is greater when deoxycytidine and deoxythymidine are administered with food than when deoxycytidine and deoxythymidine are administered under fasting conditions. 0-t In some embodiments, when deoxycytidine and deoxythymidine are administered with food, the AUC of dT is increased by about 50% to about 100% compared to when deoxycytidine and deoxythymidine are administered under fasting conditions. 0-t From about 50% to about 100%, from about 50% to about 90%, from about 50% to about 80%, from about 50% to about 70%, from about 50% to about 60%, from about 60% to about 100%, from about 60% to about 90%, from about 60% to about 80%, from about 60% to about 70%, from about 70% to about 100%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 100%, from about 80% to about 90%, or from about 90% to about 100%. In some embodiments, when deoxycytidine and deoxythymidine are administered with food, the AUC of dT is increased compared to when deoxycytidine and deoxythymidine are administered under fasting conditions. 0-t In some embodiments, the AUC of dT is increased 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 under fasting conditions. 0-tAn increase of at least about 74%.

[0059] One aspect of the present invention is to increase the bioavailability of deoxycytidine in human patients receiving deoxycytidine and deoxythymidine therapy when nucleoside combination is used together with food. In one embodiment, deoxycytidine and deoxythymidine therapy are used substantially simultaneously with meals. In another embodiment, deoxycytidine and deoxythymidine therapy are used within 30 minutes before meals. In certain aspects, administration to patients is carried out together with food or within 30 minutes or less before food is eaten (for example, 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) is carried out. In another embodiment, deoxycytidine and deoxythymidine therapy are used within 2 hours after meals. In some aspects, administration to patient occurs between about 1 hour before consuming food and about 2 hours after consuming food, between about 1 hour before consuming food and about 1.5 hours after consuming food, between about 1 hour before consuming food and about 1 hour after consuming food, between about 1 hour before consuming food and about 30 minutes after consuming food, between about 30 minutes before consuming food and about 2 hours after consuming food, between about 1.5 hours before consuming food, between about 30 minutes before consuming food and about 1 hour after consuming food, or between about 30 minutes before consuming food and about 30 minutes after consuming food. In some aspects, administration to patient occurs when food is eaten or immediately after food is eaten, until 1 hour (e.g., 0 minute to 1 hour) after described consumption.

[0060] Another aspect of the invention is a method of increasing the rate of deoxycytidine absorption in a human patient receiving deoxycytidine and deoxythymidine therapy when a nucleoside combination is administered with food. In one embodiment, deoxycytidine and deoxythymidine therapy is administered substantially simultaneously with a meal. In another embodiment, deoxycytidine and deoxythymidine therapy is administered within 30 minutes before a meal. In another embodiment, deoxycytidine and deoxythymidine therapy is administered within 2 hours after a meal. In one embodiment, the meal is a high-fat, high-calorie meal. In another embodiment, the meal is adapted to the digestive health of the individual patient, aiming to provide as much fat and caloric content as possible.

[0061] Another aspect of the invention is a method of increasing the bioavailability of deoxythymidine in a human patient receiving deoxycytidine and deoxythymidine therapy when the nucleoside combination is administered with food. In one embodiment, the deoxycytidine and deoxythymidine therapy is administered substantially simultaneously with a meal. In another embodiment, the deoxycytidine and deoxythymidine therapy is administered within 30 minutes before a meal. In another embodiment, the deoxycytidine and deoxythymidine therapy is administered within 2 hours after a meal.

[0062] Another aspect of the invention is to provide a method of increasing the rate and extent of absorption as measured by the concentration of the drug achieved in the bloodstream over time in a patient receiving a drug via oral dosing, the method comprising administering to the patient a therapeutically effective amount of deoxycytidine with food.

[0063] Another aspect of the present invention is to provide a method of treating a patient with deoxycytidine and deoxythymidine therapy and reducing or eliminating gastrointestinal side effects by administering the therapy with food. In one embodiment, the deoxycytidine and deoxythymidine therapy is administered substantially simultaneously with a meal. In another embodiment, the deoxycytidine and deoxythymidine therapy is administered within 30 minutes before a meal. In another embodiment, the deoxycytidine and deoxythymidine therapy is administered within 2 hours after a meal. In another embodiment, the gastrointestinal side effects are one or more of nausea, vomiting, and diarrhea.

[0064] On the other hand, the dosage range administered with food is about 50 mg / kg / day (that is, about 50 mg / kg / day dC and about 50 mg / kg / day dT) to about 500 mg / kg / day (that is, about 500 mg / kg / day dC and 500 mg / kg / day dT). Dosage can be adjusted so that the effect in the subject is the best. For example, the deoxynucleoside can be administered with 100 mg / kg / day, then increased to 200 mg / kg / day, 400 mg / kg / day, 800 mg / kg / day, until 1000 mg / kg / day with 30 mg / kg / day over time, depending on the reaction and tolerance of the subject. In one embodiment, the deoxynucleoside can be administered with about 200 mg / kg / day to 600 mg / kg / day. In one embodiment of the invention, the daily dose is up to a target dose or a maintenance dose of 400 mg / kg / day of a substantially equal mixture of dC and dT (weight to weight), administered with food, and optionally reduced based on the patient's tolerance to the dose. In another embodiment, the dose is divided into about one-third of the preferred daily dose and administered three times a day (TID) with food. Any adverse reactions of the subject can also be monitored, such as gastrointestinal intolerance, such as diarrhea, and the improvement is monitored when the treatment is administered with food. In some embodiments, one or more doses are administered with food every day. In some embodiments, two or more doses are administered with food every day. In some embodiments, three doses are administered with food every day. In some embodiments, when three doses are administered every day, at least one dose is administered with food. In some embodiments, when three doses are administered every day, at least two doses are administered with food. In some embodiments, when three doses are administered every day, three doses are administered with food. In some embodiments, one dose is administered with food every day. In some embodiments, two doses are administered with food every day. In some embodiments, three doses are administered with food every day.

[0065] In some embodiments, the total daily dose of dC and dT comprises equal weight portions of dC and dT. In some embodiments, the total daily dose of dC and dT comprising equal weight portions of the combined 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 150 mg / kg, about 50 mg / kg to about 200 mg / kg, about 50 mg / kg to about 300 mg / kg, about 50 mg / kg to about 400 mg / kg, about 50 mg / kg to about 500 mg / kg, about 50 mg / kg to about 500 mg / kg, about 50 mg / kg to about 200 mg / kg, about 50 mg / kg to about 300 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 300 mg / kg, about 50 mg / kg to about 500 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 200 mg / kg, about 50 mg / kg to about 300 mg / kg, about 50 mg / kg to about 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 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, g / 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.

[0066] In some embodiments, the total daily dose is equally divided into three separate doses of dC and dT, wherein each dose of dC and dT is administered separately within 24 hours. In some embodiments, each separate dose contains equal weight portions of dC and dT, and dC and dT are administered separately but substantially simultaneously. In some embodiments, each dose contains equal weight portions of dC and dT, and dC and dT are combined before administration. In some embodiments, each dose contains equal weight portions of dC and dT, and dC and dT are combined into a fixed dose combination pharmaceutical composition in a single package.

[0067] In some embodiments, the total daily dose includes three separate doses of dC and dT, each dose administered at a different time during a 24-hour period, wherein each dose contains equal weight portions of dC and dT. In some embodiments, each dose contains dC and dT in combined weight form, wherein dC and dT are equal weight portions, between about 15 mg / kg and about 330 mg / kg, between about 15 mg / kg and about 260 mg / kg, between about 15 mg / kg and about 200 mg / kg, between about 15 mg / kg and about 160 mg / kg, between about 15 mg / kg and about 133 mg / kg, between about 15 mg / kg and about 70 mg / kg , between about 15 mg / kg and about 33 mg / kg, between about 33 mg / kg and about 330 mg / kg, between about 33 mg / kg and about 260 mg / kg, between about 33 mg / kg and about 200 mg / kg, between about 33 mg / kg and about 160 mg / kg, between about 33 mg / kg and about 133 mg / kg, between about 33 mg / kg and about 70 mg / kg, between about 70 mg / kg and about 330 mg / kg / kg, between about 70 mg / kg and about 260 mg / kg, between about 70 mg / kg and about 200 mg / kg, between about 70 mg / kg and about 160 mg / kg, between about 70 mg / kg and about 133 mg / kg, between about 133 mg / kg and about 330 mg / kg, between about 133 mg / kg and about 260 mg / kg, between about 133 mg / kg and about 200 mg / kg, between about 133 mg / kg and about 160 mg / kg The invention relates to an oral administration of at least about 150 mg / kg to about 200 mg / kg of steroids, wherein three doses are administered over a 24 hour period. In some embodiments, each dose contains dC and dT in combined weight form, wherein dC and dT are equal weight portions, 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, wherein three doses are administered over a 24 hour period.

[0068] In one aspect, the efficacy of treatment for MDS patients is improved by administering a target daily dose of dC and dT with food.

[0069] On the other hand, the dosage range of each of the nucleosides can be reduced to take into account the increased bioavailability of the deoxynucleosides when taken with food. In one embodiment, reducing the daily dose of the deoxynucleosides reduces gastrointestinal intolerance. On the other hand, the dosage of each of the nucleosides can be reduced to take into account the increased bioavailability of the deoxynucleosides when taken with food.

[0070] Route of administration

[0071] In one embodiment, the deoxycytidine and deoxythymidine therapy is administered orally. In another embodiment, the deoxycytidine and deoxythymidine therapy is dissolved in water or juice to provide a solution for oral administration. In one embodiment, the solution is administered through a dispensing device, which can be a syringe or a graduated pipette, which can be used to measure different doses and deliver doses of the drug product solution. In one embodiment, the dispensing device is used as a dosing device, which is capable of dispensing a fixed volume of the drug product solution. In one embodiment, the deoxycytidine and deoxythymidine therapy is administered through a feeding tube. In some embodiments, the feeding tube is a nasogastric feeding tube or an enteral feeding tube.

[0072] Dosage form / dosing frequency

[0073] In the MT1621 clinical development program, two formulations were considered. The first formulation consisted of dC and dT supplied in different packages, which were mixed before administration. The second formulation, i.e., the formulation in which MT1621 therapy is provided for clinical studies, consists of dC and dT in a fixed dose combination supplied in one package. Pharmacokinetic (PK) considerations in the clinical development program include the frequency of administration of 3 divided doses per day, the absorption rate and extent of dC and dT, the effect of food on PK and tolerability, the dose ratio of the two formulations, and renal clearance.

[0074] Example 1. Design of an open-label, fixed-sequence, single ascending-dose study in healthy adult subjects to evaluate the safety, tolerability, PK, and food effect of MT1621

[0075] A Phase 1 study was conducted as an open-label, fixed-sequence, single ascending dose study in healthy adult subjects designed to evaluate the safety, tolerability, pharmacokinetics (PK), and food effects of MT1621 (dC and dT). To evaluate the pharmacokinetics of MT1621, the combination was administered at three different doses. Treatment A was 43.3 mg / kg of MT1621 (i.e., 43.3 mg / kg of dC and 43.3 mg / kg of dT (each equivalent to approximately 1 dose of 130 mg / kg daily dose three times a day [TID]) in healthy male and female subjects under fasting conditions; Treatment B was 86.7 mg / kg of MT1621 (i.e., 86.7 mg / kg of dC and 86.7 mg / kg of dT (each equivalent to approximately 1 dose of 260 mg / kg daily dose three times a day)) in healthy male and female subjects under fasting conditions; and Treatment C was 86.7 mg / kg of MT1621 (i.e., 86.7 mg / kg of dC and 86.7 mg / kg of dT (each equivalent to approximately 1 dose of 260 mg / kg daily dose three times a day)) in healthy male and female subjects under fasting conditions. Treatment D was a 133.3 mg / kg dose of MT1621 in healthy male and female subjects under fed conditions (i.e., a 133.3 mg / kg dose of dC and a 133.3 mg / kg dose of dT (each approximately equivalent to a 400 mg / kg daily dose taken three times a day)). In addition, Treatment D was a 133.3 mg / kg dose of MT1621 in healthy male and female subjects under fed conditions (i.e., a 133.3 mg / kg dose of dC and a 133.3 mg / kg dose of dT (each approximately equivalent to a 400 mg / kg daily dose taken three times a day)) to determine the effect of a high-fat meal on the single-dose PK of MT1621.

[0076] Methods for an Open-label, Phase 1, Fixed-Sequence, Single-Dose, Dose-Escalation, and Food-Effect Study Fourteen (14) healthy adult male and female subjects were recruited with approximately equal distribution of male and female subjects (at least 40% of each sex).

[0077] Screening of subjects was performed within 28 days before the first dose of medication.

[0078] On the first day of each phase, subjects received 1 of 3 dose levels of MT1621 under fasting conditions as a gradually increasing single dose in the 3 phases of Part A (treatments A, B, and C in phases 1, 2, and 3, respectively), and received the highest dose under fed conditions in Part B (treatment D in phase 4). Four of the 14 subjects who completed Part A (phases 1 to 3) were unable to complete Part B (treatment D in phase 4) during the initial restriction period. These subjects subsequently returned to the clinical unit and continued the study to complete Part B (treatment D in phase 4); 2 of the 4 subjects completed Part B.

[0079] Each treatment was evaluated by collecting blood and urine PK samples before and up to 48 hours after dosing for determination of dC and dT in plasma and urine PK and possible future analysis of PD biomarkers in blood.

[0080] The dose will not be increased to the next dose level (ie, the next phase) until it is determined that adequate safety and tolerability has been demonstrated with respect to the previous dose level to allow for progression to the next dose level.

[0081] A washout period of at least 2 days without drug was maintained between each treatment administration. Fourteen subjects participated in the study, and 12 subjects completed the study. Data from 14 subjects were included in the PK analysis of Phases 1, 2, and 3, and data from 12 subjects were included in the PK analysis of Phase 4.

[0082] The study drugs administered in Example 1 were 0.5 g dC and 0.5 g dT powders for solution and reconstituted in juice or water.

[0083] After reconstitution in a fixed amount of water or juice, the solutions were administered via metered volumes to provide the amounts specified in Studies A, B, C, and D.

[0084] The active ingredients and excipients in the drug products under development are shown in Table 1.

[0085] Dissolve the packet providing the target daily dose in water or juice once daily and remove the volume corresponding to the mg / kg required for divided doses for multiple dosing.

[0086] Table 1

[0087]

[0088] MT1621 was administered orally at hour 0 on day 1 after an overnight fast for treatments A, B, and C, and within 5 minutes of consuming a standardized high-fat breakfast consisting of eggs, bacon, and buttered toast for treatment D.

[0089] Pharmacokinetics:

[0090] Blood samples for determination of plasma dC and dT concentrations were collected on day 1 of each phase before dosing (0 hours) and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 24, and 48 hours after study drug administration. In phase 4, blood was collected for 48-hour PK samples; for phases 1 to 3, 48-hour PK samples were collected simultaneously with the pre-dose PK samples of the next phase, so only 1 sample was collected for these two PK time points. It should be noted that phases 1-3 used up to 24 hours of PK sampling, while phase 4 used a full 48-hour profile.

[0091] Urine samples 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 for determination of dC and dT concentrations.

[0092] The following non-compartmental PK parameters were determined for plasma dC and dT: area under the curve (AUC) from time zero to the last observed / measured non-zero concentration. 0-t ), the area under the concentration-time curve (AUC 0-inf ), the area under the concentration-time curve (AUC tau ), maximum observed concentration (C max ), reach C max Time (T max ), the apparent terminal elimination rate constant (Kel) representing the fraction of drug eliminated per unit time, the apparent terminal elimination half-life of the drug in plasma (t1 / 2), the apparent total plasma clearance after extravascular administration (CL / F), and the apparent volume of distribution during the terminal elimination phase after extravascular administration (Vz / F).

[0093] Urinary PK parameters for dC and dT are provided: drug concentration in urine during the urine collection interval (Cur), urine volume collected during the entire urine collection interval (Vur), amount of unchanged drug excreted in urine (Ae), cumulative amount of unchanged drug excreted in urine (Ae,Cum), fraction (percent) 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.

[0094] For calculation of PK parameters, raw (unadjusted) plasma dC and dT concentrations below the limit of quantitation (BLQ) were set to 1 / 2 the assay limit of quantitation (LOQ) throughout the PK profile if the majority of subjects showed measurable pre-dose (endogenous baseline) concentrations. Since all subjects had measurable pre-dose (endogenous baseline) concentrations, Kel and related PK parameters for the unadjusted data were not calculated.

[0095] Methods for baseline adjustment of plasma pharmacokinetic parameters

[0096] Predose blood samples at each dose level (each treatment period) were used to confirm adequate washout between dose levels and also to determine the amount of endogenous dC and dT. Each subject had 3 predose samples (-2, -1, and -0.25 [-0.583] hours). All subjects had baseline adjustments regardless of the magnitude of their predose values. Given the expected short T1 / 2 of dC and dT, no carryover effects from exogenous MT1621 administration were expected in this single-dose study.

[0097] Prior to calculation of adjusted PK parameters, baseline adjustment was performed by subtracting the arithmetic mean baseline for each subject during the treatment period (the mean of all 3 predose concentrations) from each postdose plasma dC and dT concentration. This was done on a per-subject and per-period basis. All negative values ​​after baseline adjustment were set to zero.

[0098] Bioanalytical procedures

[0099] Plasma: dC and dT were quantified using validated high-performance liquid chromatography tandem mass spectrometry (LC-MS / MS) methods. dC and dT were extracted from plasma samples by a single solid phase extraction procedure. The extracts were injected once using Syneos Health High-Performance Liquid Chromatography (HPLC) Method TM.2541 for dC and again using Syneos Health HPLC Method TM.2547 for dT. Both methods for the determination of dC and dT in human plasma were validated according to the 2018 FDA Guidance on Bioanalytical Method Validation and the 2011 EMA Guidance. The method demonstrated acceptable sensitivity, selectivity, precision, and accuracy for the quantification of dC and dT in the range of 0.500 to 200 ng / mL for each analysis (lower limit of quantification [LLOQ] of 0.5 ng / mL) in 0.200 mL of tetrahydrouridine and sodium citrate-stabilized dipotassium ethylenediaminetetraacetic acid (K2EDTA)-treated human plasma by liquid chromatography electrospray ionization tandem mass spectrometry (LC / ESI / MS / MS) in positive ion mode using [15N3]-deoxycytidine and [D3]-deoxythymidine, respectively, as internal standards (IS).

[0100] Statistical methods:

[0101] Plasma and urine dC and dT concentrations for all subjects in the PK population group were listed and summarized by treatment and time point. Summary statistics were calculated for all nominal concentration time points and PK parameters, including sample size (N), arithmetic mean (Mean), standard deviation (SD), coefficient of variation (CV%), minimum value (Min), median, maximum value (Max), geometric mean (GM), and geometric CV% (GMCV[%]).

[0102] Food effects were assessed only for data from subjects who completed the study or had sufficient data for pairwise comparisons.

[0103] The plasma PK parameter AUC was transformed using the natural logarithm (ln) for raw and baseline-adjusted plasma dC and dT concentrations. 0-t Analysis of variance (ANOVA) was performed on the Cmax.

[0104] The ANOVA model included dietary condition (fasted, fed) as a fixed effect and subject as a random effect. Each ANOVA included calculation of the least squares mean (LSM), the difference between regimen LSMs, and the standard error associated with this difference.

[0105] Ratios of LSM were calculated from analyses of the natural logarithms converted to fasting conditions using exponentiation of the LSM.

[0106] Crude and baseline-adjusted plasma dC and dT, AUC, and 0-inf , AUC 0-t and dose proportionality of Cmax parameters.

[0107] AUC 0-t and Cmax. These ratios are expressed as percentages relative to fasting conditions (ie, Treatment D / Treatment C).

[0108] Consistent with the two-sided test, the ln-transformed AUC 0-t The CIs obtained for the differences between the regimen LSMs obtained from the analysis of Cmax and Cmax were exponentiated to give the 90% confidence interval (CI) for the ratio. The CIs are expressed as percentages relative to the fasting condition.

[0109] Crude and baseline-adjusted plasma dC and dTAUC were evaluated after single-dose administration (fasting treatments A, B, and C). 0-inf , AUC 0-t and dose proportionality of Cmax parameters.

[0110] Food Impact Assessment

[0111] Food effects were assessed only for data from subjects who completed the study or had sufficient data for pairwise comparisons.

[0112] Baseline-corrected natural logarithm (ln) transformed plasma PK parameter AUC 0-t Analysis of variance (ANOVA) was performed on the Cmax. The ANOVA model included dietary regimen (fasting, fed) as a fixed effect and subject as a random effect. Each ANOVA included calculation of the least squares mean ("LSM"), the difference between regimen LSMs, and the standard error associated with this difference.

[0113] Using the AUC from the ln transformation 0-t The ratios of LSMs were calculated by exponentiation of the LSMs of the analyses of Cmax and Cmax. These ratios were expressed as percentages relative to the fasting condition (i.e., Treatment D / Treatment C).

[0114] Consistent with the two-sided test, the ln-transformed AUC 0-t The CI obtained for the difference between the analyzed regimen LSMs of Cmax and Cmax was exponentiated to obtain the 90% confidence interval (CI) for the ratio. The CI is expressed as a percentage relative to the fasting condition (ie, Treatment D / Treatment C).

[0115] If baseline adjustment was appropriate (i.e., predose dC and dT concentrations were measurable for most subjects), the baseline-adjusted PK parameter AUC was also calculated. 0-t , AUC 0-inf The analysis was performed using the Cmax.

[0116] Dose proportionality analysis

[0117] Raw and baseline-corrected plasma dC and dT AUCs were evaluated after single-dose administration (fasting treatments A, B, and C). 0-t Dose-proportional relationships of the Cmax and Cmax parameters. To assess dose proportionality, linear regression and power model methods were used.

[0118] First, the PK parameter AUC was fitted using a regression including linear (β1) and quadratic (β2) effect terms. 0-t The linear relationship between Cmax and dose:

[0119] Y=α+β1*Dose+β2*Dose2+ε,

[0120] Where Y represents the PK parameter AUC 0-t , AUC 0-inf and the natural logarithm of Cmax, with ε representing the measurement error of the independent variable. If β2 or α deviates from zero, a dose proportionality relationship is not claimed. If β2 is not significantly different from zero, the linear regression simplifies to:

[0121] Y=α+β*dose+ε

[0122] The slope β measures the dose proportionality relationship between the dose and the dose-dependent PK parameter. A dose proportionality relationship requires β=1.

[0123] Therefore, in the second step, the 95% CI of the slope β corresponding to the ln transformed PK parameter was calculated. 0-t and Cmax) are 1, then the dose proportionality relationship holds.

[0124] Since baseline adjustment was performed (i.e., predose dC and dT concentrations were measurable in most subjects), the baseline-adjusted PK parameter AUC was then 0-t This analysis was also performed for Cmax.

[0125] Table 2. Summary of Baseline-Adjusted Plasma dC Pharmacokinetic Parameters Following Single Dose Administration of 43.3 mg / kg MT1621, Fasted (A); 86.7 mg / kg MT1621, Fasted (B); 133.3 mg / kg MT1621, Fasted (C); and 133.3 mg / kg MT1621, Fed (D) - Part 2 (PK Evaluable Population)

[0126]

[0127] Treatment A: (43.3 mg / kg dose), day 1, hour 0, after overnight fast

[0128] Treatment B: (86.7 mg / kg dose), day 1, hour 0, after overnight fast

[0129] Treatment C: (dose 133.3 mg / kg), day 1, hour 0, after overnight fasting

[0130] Treatment D: (133.3 mg / kg) dose, within 5 minutes of consuming a standard high-fat breakfast, at hour 0 on day 1

[0131] AUC and Cmax are expressed as geometric mean (geometric coefficient of variation % (CV%))

[0132] Tmax is expressed as median (minimum, maximum)

[0133] Other parameters are expressed as arithmetic mean ± SD

[0134] Table 3. Summary of baseline-adjusted plasma dT pharmacokinetic parameters following single-dose administration of 43.3 mg / kg MT1621, fasting (A); 86.7 mg / kg MT1621, fasting (B); 133.3 mg / kg MT1621, fasting (C); and 133.3 mg / kg MT1621, fed (D) Part 2 (PK evaluable population)

[0135]

[0136] Treatment A: (43.3 mg / kg dose), day 1, hour 0, after overnight fast

[0137] Treatment B: (86.7 mg / kg dose), day 1, hour 0, after overnight fast

[0138] Treatment C: (133.3 mg / kg dose), day 1, hour 0, after overnight fast

[0139] Treatment D: 133.3 mg / kg dose), within 5 minutes of consuming a standard high-fat breakfast, day 1, hour 0

[0140] NC = Not Calculated

[0141] AUC and Cmax are expressed as geometric mean (geometric coefficient of variation % (CV%))

[0142] Tmax is expressed as median (minimum, maximum)

[0143] Other parameters are expressed as arithmetic mean ± SD

[0144] Table 4. Statistical Comparison of Baseline-Adjusted Plasma dC Pharmacokinetic Parameters: 133.3 mg / kg MT1621, Fed vs. 133.3 mg / kg MT1621, Fasted - Part 2 (PK Population)

[0145]

[0146] Treatment D: 133.3 mg / kg dose of MT1621 within 5 minutes of consuming a standard high-fat breakfast, day 1, hour 0 (test)

[0147] Treatment C: MT1621 at a dose of 133.3 mg / kg at hour 0 on day 1 after an overnight fast (reference)

[0148] Parameters were ln transformed before analysis.

[0149] Geometric least square means (LSM) were calculated by exponentiating the LSM from ANOVA.

[0150] Geometric mean ratio (GMR) = 100*(test / reference)

[0151] The within-subject CV% was calculated as 100*square root(exp[MSE]-1), where MSE=residual variance from ANOVA.

[0152] Table 5. Statistical Comparison of Baseline-Adjusted Plasma dT Pharmacokinetic Parameters: 133.3 mg / kg MT1621, Fed vs. 133.3 mg / kg MT1621, Fasted - Part 2 (PK Population)

[0153]

[0154] Treatment D: 133.3 mg / kg dose of MT1621 within 5 minutes of consuming a standard high-fat breakfast, day 1, hour 0 (test)

[0155] Treatment C: MT1621 at a dose of 133.3 mg / kg, at hour 0 on day 1, after an overnight fast (reference) Parameters were ln transformed prior to analysis.

[0156] Geometric least square means (LSM) were calculated by exponentiating the LSM from ANOVA.

[0157] Geometric mean ratio (GMR) = 100*(test / reference)

[0158] The within-subject CV% was calculated as 100*square root exp[MSE]-1), where MSE=residual variance from ANOVA.

[0159] The pharmacokinetic results of this study demonstrated that MT1621 was absorbed into the systemic circulation; more specifically, maximum dC and dT plasma concentrations (Cmax) were reached at a median of 1.0 to 1.25 hours (Tmax) and 1.0 hour (Tmax) under fasting conditions, and at 1.0 and 3.0 hours under fed conditions. The geometric mean elimination half-life values ​​for dC were 2.16 hours, 1.44 hours, and 3.01 hours at the 43.3 mg / kg, 86.7 mg / kg, and 133.3 mg / kg dose levels, respectively. PK variability was high, with >30% for both dC and dT. Baseline-corrected dC Cmax increased in a less-than-dose-proportional manner across the 3 fasting dose levels. Total exposure to dC (measured as AUC 0-t and AUC 0-inf measurements) increased in a less dose-proportional manner.

[0160] Plasma exposures of dC and dT were consistently higher at all 3 dose levels after MT1621 administration compared to endogenous (baseline) concentrations. This study demonstrated significant increases in systemic exposures of dC and dT after a single dose of MT1621.

[0161] Administration of 133.3 mg / kg MT1621 under fed conditions increased the baseline-adjusted Cmax and AUC of plasma dC by 79% and 137%, respectively, and the baseline-adjusted Cmax and AUC of plasma dT by 27% and 74%, respectively, compared with fasting conditions, indicating that food has a significant effect on MT1621.

[0162] Dose proportionality analysis of plasma dC showed that while AUC appeared to be proportional to dose (95% CI for slope [b] included 1), Cmax was not. Dose proportionality analysis of plasma dT showed that neither AUC nor Cmax was proportional to dose.

[0163] Example 2. Open-label safety and efficacy study of MT1621 in study participants with TK2 deficiency

[0164] A Phase 2 study was conducted in participants with TK2 deficiency as an open-label continuation of MT1621 treatment to evaluate the safety, tolerability, and PK of MT1621 (fixed-dose combination of dC and dT supplied in a single package). Forty-seven (47) participants were enrolled in the study. Upon enrollment, participants switched from their current chemical grade 2'-deoxycytidine monophosphate / 2'-deoxythymidine monophosphate (dCMP / dTMP) to deoxycytidine and deoxythymidine or continued on deoxycytidine and deoxythymidine. At enrollment, participants who were stable on a maintenance dose of 400 mg / kg / day were treated three times daily (TID) with the same dose of deoxycytidine and deoxythymidine. Study participants who received a total dose of <400 mg / kg / day were switched to 260 mg / kg / day (130 mg / kg / day deoxycytidine and 130 mg / kg / day deoxythymidine) TID, 520 mg / kg / day (260 mg / kg / day deoxycytidine and 260 mg / kg / day deoxythymidine) TID, or 800 mg / kg / day (400 mg / kg / day deoxycytidine and 400 mg / kg / day deoxythymidine) TID, depending on which dose was closest to the participant's previous dose.

[0165] Summary statistics of key covariates for participants at baseline:

[0166] Age (years); N = 47; mean (SD) = 18.4 (18.7); median (minimum, maximum) = 9.90 (0.90, 75.6);

[0167] Body weight (kg): N = 47; mean (SD) = 37.0 (22.6); median (minimum, maximum) = 31.5 (6.00, 99.5);

[0168] Absolute eGFR (mL / min): N = 46; mean (SD) = 285 (124); median (minimum, maximum) = 248 (111, 606);

[0169] Creatinine clearance (mL / min): N = 47; mean (SD) = 203 (120); median (minimum, maximum) = 173 (42.9, 561);

[0170] Gender: Male = 27 (57.4%); Female = 20 (42.6%);

[0171] Race: Unknown = 0 (0%); Latino or Latino = 14 (29.8%); Not Latino or Latino = 33 (70.2%).

[0172] The early protocol for the administration of deoxycytidine and deoxythymidine was to have participants take the drug product without regard to food. In addition, the study did not carefully record the food status of the participants when they took their daily dose and on the day of PK sampling. Later in the program, when the unexpected effect of food on PK was noted, the protocol was modified to instruct participants to take the drug with food. By then, the planned PK samples had been collected in most of the participants. After the first day of the study, a total of 3 blood samples were collected from each individual to measure dC plasma concentrations. Sparse PK blood sampling and PD endpoint collection were performed according to the following schedule: at month 1, sampling was performed at 0.5, 1, 2 or 3 hours after dosing; and at month 3, sampling was performed at 8, 10, 12 or 14 hours after dosing. During the sampling period, it was recorded whether food was consumed from 1 hour before dosing to 2 hours after dosing. And, the lower limit of quantification (LLOQ) of both dC and dT was 0.5ng / mL.

[0173] For calculation of PK parameters, raw (unadjusted) plasma dC and dT concentrations below the limit of quantitation (BLQ) were set to 1 / 2 the limit of quantitation (LOQ) of the assay. Since no pre-dose plasma samples were collected for the study, the plasma concentrations of the participants could not be adjusted for baseline. Since all participants had measurable pre-dose concentrations, Kel and related PK parameters were not calculated for the unadjusted data.

[0174] PK data from five (5) studies (including the studies described in Example 1 and Example 2) were pooled, including three (3) Phase 1 studies in healthy volunteers (HV) and two (2) open-label Phase 2 studies that included adult and pediatric study participants with TK2d. 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 ranging in age from 0.9 to 75.6 years.

[0175] The pooled population PK analysis confirmed that fed state was a statistically significant covariate for the PK of dC, including participants with TK2 deficiency. Table 6 reflects the analysis of all dC PK data, including data for participants with TK2 deficiency. The magnitude of the positive effect of food on the oral bioavailability of dC was 1.44, or a 144% increase. This is a correlation value that estimates the effect of food on dC exposure. It suggests that, in general, fed state is associated with increased bioavailability of dC, including for participants with TK2 deficiency. The reduction in dC absorption rate by food (parameter Ka in Table 6) is thought to be caused by slower gastric / intestinal motility in the fed state. It is likely that the effect of food on dT concentrations in participants with TK2 deficiency was masked by high variability and limited PK sampling.

[0176] Table 6. Population parameter estimates and bootstrap results for the final pharmacokinetic model of dC

[0177]

[0178]

[0179] BSL = baseline; Ka = absorption rate constant; F = relative bioavailability; CL / F = apparent oral clearance; V / F = apparent oral volume of distribution; RUV = residual unexplained variability

[0180] IIV = inter-individual variability; IOV = inter-occasion variability; %RSE = relative standard error expressed as a percentage, calculated as (standard error) / (estimate)*100; IIV and IOV are both expressed as CV%, calculated as sqrt(ω 2 )*100.

[0181] Clearance and volume terms were allometrically scaled using fixed exponents of 0.75 and 1, respectively, and body weight was centered around 70 kg.

[0182] a :Ka Fed =Ka*(1+the effect of food on Ka)

[0183] b : FFed =F*(1+the effect of food on F)

[0184] c : Summary of 734 bootstrapped minimizations from 1000 successful minimizations.

[0185] The dC PK was described by a one-compartment model with first-order absorption and first-order elimination and found that:

[0186] Endogenous dC baseline was the same across studies and populations;

[0187] Body weight was found to be positively correlated with apparent oral clearance (CL / F) and apparent oral volume of distribution (V / F), using fixed allometric exponents of 0.75 and 1, respectively;

[0188] Age was not identified as a significant covariate;

[0189] The relative bioavailability (F) increases by ∼150% with food intake (standard or high-fat meal);

[0190] The absorption rate constant (Ka) decreases by 44% with food intake (standard or high-fat meal);

[0191] Latino / Latino race was not identified as a significant covariate; and

[0192] No differences in F values ​​were detected between HV- and TK2-deficient participants.

[0193] The dT PK was described by a one-compartment model with 2 parallel first-order absorptions (with lag time) and first-order elimination and found that:

[0194] Endogenous dT baseline varied among studies (ranging from 0.220 to 1.14 ng / mL);

[0195] Body weight was not found to be a significant covariate of CL / F, nor was body weight found to be a significant covariate of V / F;

[0196] Age was not determined to be a significant covariate;

[0197] About 83% of the relative bioavailability (F) is attributed to the first reservoir compartment;

[0198] · With a high-fat meal, F increases by ~120%;

[0199] Relative to F values ​​at 133.3 mg / kg in healthy study participants, F decreased by ~0.4-fold at 43.3 mg / kg and by ~0.7-fold at 86.6 mg / kg; separate analyses of previous Phase 1 studies in HV showed that dT exposure increased in a more than dose-proportional manner after single ascending doses within this dose range;

[0200] The high-fat meal reduced both the absorption rate constant from the first input compartment (K12) and the lag time from the second input compartment (ALAG3) by ∼80%;

[0201] -50% reduction in F in the Latino / Latino population; and

[0202] • The F values ​​of healthy volunteers were lower than those of TK2-deficient participants (70% vs. 90%).

[0203] The dC model was able to describe the PK of dC with good accuracy and limited or no bias. The population PK model provided a robust fit to the observed data with stable PK parameter estimates with high precision. The dC model was considered appropriate to describe dC exposure in patients with TK2d for further pharmacokinetic / pharmacodynamic or exposure-response analyses.

[0204] The dT model was able to reasonably describe the PK of dT in HV, study participants with impaired renal function, and participants with TK2d, with good precision and limited bias. However, the high residual unexplained variability in the model suggests that, while it may be applicable to the current data set, the model has limited predictive utility. Therefore, dT exposures derived from the model in participants with TK2 deficiency should be interpreted with caution.

[0205] In summary, the PK changes caused by a standardized high-fat, high-calorie meal reflect the greatest impact on gastrointestinal physiology. The greatest impact on the oral bioavailability of the drug can be generalized to all subjects, including healthy subjects and subjects with TK2 deficiency. Regulations require that the effect of food on drug PK be evaluated with a standardized high-fat meal, preferably in healthy adult participants of sufficient sample size, under controlled conditions with intensive / rich PK sampling. To our knowledge, there is no physiological basis to expect that the underlying disease (TK2 deficiency) will cause food to have a different effect on the bioavailability of dC and dT compared to healthy subjects. Therefore, the results of a dedicated study of healthy subjects (Example 1) provide clear and reliable findings on the effect of food on the oral PK of dC and dT, which are fully applicable to the target patient population. The ability of the population PK model to independently identify food (for dC in healthy and TK2-deficient participants) and a high-fat meal (for dT in healthy participants) as significant covariates of PK provides additional support.

Claims

1. A method for increasing the oral bioavailability of deoxycytidine (dC) and deoxythymidine (dT) in a patient being treated for mitochondrial depletion syndrome (MDS), the method comprising administering to a patient in need thereof a therapeutically effective amount of dC and dT with food, or instructing a caregiver of the patient to administer the therapeutically effective amount of dC and dT with food.

2. A method of increasing the amount of deoxycytidine (dC) absorbed by a patient's body, the method comprising administering a dose of about 100 mg / kg to about 200 mg / kg of a combined weight of dC and deoxythymidine (dT), containing equal parts of dC and dT, wherein the administration is with food.

3. The method of claim 2, wherein the combined weight of dC and dT is administered three times a day at a dosage of about 100 mg / kg to about 200 mg / kg.

4. The method of claim 1, wherein the therapeutically effective amount of dC and dT, by combined weight, is between about 200 mg / kg / day and about 800 mg / kg / day.

5. The method of claim 1 or 4, wherein the therapeutically effective amount of dC and dT, by combined weight, is 800 mg / kg / day.

6. The method of claim 1 or 2, wherein dC and dT are administered as a fixed dose combination pharmaceutical composition.

7. The method of claim 1 or 2, wherein dC and dT are administered after combining two pharmaceutical compositions in equal parts by weight, wherein one pharmaceutical composition comprises dC and one pharmaceutical composition comprises dT.

8. The method of claim 1 or 2, wherein the absorption rates of dC and dT are increased.

9. The method of claim 1 or 2, wherein the extent of absorption of dC and dT is increased.

10. The method of claim 1 or 2, wherein the circulating half-life of dC and dT is increased.

11. The method of claim 1 or 2, wherein dC and dT are administered simultaneously with oral administration of food to about 30 minutes after oral administration of food.

12. The method of claim 1 or 2, wherein the food is a high-fat, high-calorie meal.

13. The method of claim 1 or 2, wherein the dC and dT doses are administered one to three times daily, and one or more of the doses are administered with food.

14. The method of claim 1 or 2, wherein the mean maximum plasma concentration (Cmax) and area under the plasma concentration time curve (AUC) of deoxycytidine and deoxythymidine in subjects in need thereof are increased when dC and dT are administered with food compared to when dC and dT are administered under fasting conditions. 0-t )Increase.

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

16. The method of claim 14, wherein the AUC of dC 0-t An increase of at least about 137%.

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

18. The method of claim 14, wherein the AUC of dT is 0-t An increase of at least about 74%.

19. The method of claim 2, wherein the patient suffers from mitochondrial depletion syndrome (MDS).

20. The method of claim 1 or 19, wherein the MDS is TK2 deficiency or POLG deficiency.

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

22. The method of claim 21, wherein the administering to the patient occurs substantially simultaneously with consumption of food.

23. The method of claim 21, wherein the administering to the patient occurs between about 30 minutes before and about 2 hours after consuming food.

24. The method of claim 21, wherein the administering is performed to the patient immediately after consuming food to 1 hour after consuming food.

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

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

27. The method of claim 1, wherein dC and dT are in the form of a composition and provided with a container having a printed label suggesting that administration with food results in an increase in maximum plasma concentration (Cmax) and extent of absorption (AUC(last)) compared to administration without food and that the composition should be administered with food.

Images