Composition containing decitabine, 5-azacytidine and tetrahydrouridine and use thereof

Abstract

Disclosed herein is a composition for treating cancer, comprising decitabine and tetrahydrouridine or 5-azacytidine and tetrahydrouridine. The composition in the form of a pill is administered to a human subject sequentially or alternately by measuring predictive biomarkers.

Description

[0001] Related application data

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 262,839 filed on December 3, 2015 and Serial No. 62 / 429,292 filed on December 2, 2016. Technical Field

[0003] The present application generally relates to compositions containing decitabine (DEC), 5-azacytidine (5AZA) and tetrahydrouridine (THU) and methods of using the compositions to treat cancer and blood disorders in subjects. Specifically, the compositions in the form of pills include DEC and THU, or 5AZA and THU, or a combination of DEC, 5AZA and THU as non-cytotoxic treatments for cancer or other blood disorders in subjects. Background of the Invention

[0005] Radiation therapy and drug therapy for cancer aim to connect physiological pathways that can stop cell growth and division. The specific physiological pathway that almost all cancer drugs try to connect is the p53 / p16-mediated cell apoptosis pathway (cytotoxicity). Because p53 and p16 are the two most commonly inactivated genes in cancer, this dependence of cancer drugs and radiation on p53 / p16 is the fundamental basis for treatment resistance and recurrence. Therefore, new treatment methods that do not use the p53 / p16 system to stop cancer cell growth and division are needed.

[0006] Preclinical and clinical studies have shown that non-cytotoxic depletion of the enzyme DNA methyltransferase 1 (DNMT1) by decitabine (DEC) and 5-azacytidylserine (5AZA) can be an effective form of treatment that does not require or rely on the p53 / p16 system (Negrotto S. et al., 2011 Cancer Res 71(4):1431-1441). However, neither 5AZA nor DEC alone is curative in preclinical in vivo models of cancer and in the clinic. Therefore, it is necessary to exploit the resistance mechanisms of cancer to treatments that are independent of p53 with 5AZA or DEC. SUMMARY OF THE INVENTION

[0007] The present disclosure has identified that cancer resistance to p53-independent treatment with 5AZA or DEC can be effectively overcome by a rational, non-toxic and clinically applicable solution. Specifically, the present disclosure provides compositions of non-cytotoxic THU-DEC / THU-5AZA, and methods of overcoming cancer resistance by administering a composition comprising non-cytotoxic levels of THU-DEC and administering the composition using an alternating regimen (i.e., alternating intake of a composition containing THU-DEC and THU-5AZA, which may be in the form of a pill or tablet, for a specified period of time).

[0008] Thus, in one embodiment, an oral composition is provided for inducing a safe, non-cytotoxic (p53 / pl6-independent) differentiation-mediated cell cycle exit of cancer cells.

[0009] The orally administered composition comprises a nucleoside analog drug that irreversibly binds to and consumes the enzyme DNA methyltransferase 1 (DNMT1), a potent cytosine deaminase (CDA) inhibitor, and one or more pharmaceutically acceptable excipients, wherein the nucleoside analog drug and the potent cytosine deaminase inhibitor are present in a therapeutically effective amount sufficient to induce differentiation of refractory and resistant cancer cells without cytotoxicity to normal tissues. The nucleoside analog drug that consumes the DNMT1 enzyme present in the composition is DEC. In one embodiment, the nucleoside analog drug may also be 5AZA. The CDA inhibitor in the composition may be tetrahydrouridine (THU) or certain tetrahydrouridine derivatives, such as 2'-fluorinated tetrahydrouridine derivatives. In this embodiment, when the nucleoside analog drug that consumes DNMT1 is DEC, it may be present in an amount ranging from 1 to 10 mg / m 2 When the nucleoside analog drug present in the oral composition is 5AZA, its amount may be in the range of 10-100 mg / m 2 The CDA inhibitor THU may be present in an amount of about 400 mg / m 2 About 500 mg / m 2 within the range.

[0010] The therapeutic composition for inducing non-cytotoxic differentiation can be in the form of a pill or tablet or capsule. In one embodiment, the therapeutic composition can include nucleoside analog drugs DEC and THU and one or more pharmaceutically acceptable carriers. In another embodiment, the therapeutic composition can include 5AZA, THU and one or more pharmaceutically acceptable carriers. In some embodiments, the therapeutic composition can include a therapeutically effective amount of a nucleoside analog drug (DEC or 5AZA) and a combination of THU in a single pill.

[0011] The therapeutic composition can be administered to the subject after measuring predictive biomarkers (including but not limited to DCK in cancer cells). The development of predictive biomarkers can help the treating physician choose the right treatment regimen to more effectively treat the subject's especially refractory and aggressive metastatic cancer. In this embodiment, the predictive biomarker can be the pyrimidine metabolic enzyme deoxycytidine kinase (DCK) of the cancer cell. It is known that the DCK enzyme phosphorylates and captures DEC in the cell and several cancer cells rely on the alternative pyrimidine metabolic enzyme UCK2 to avoid DEC-inducible DNMT-1 consumption, which makes the cancer cell susceptible to the drug 5-5AZA. The DCK of the cancer cell can be measured by functional molecular imaging with positron emission tomography (PET) or by QRT-PCR before, during or after the administration of the oral composition.

[0012] In a therapeutic method for inducing non-cytotoxic differentiation of cancer cells in any mammalian tissue, a first therapeutic composition comprising DEC, THU and one or more pharmaceutically acceptable carriers and a second therapeutic composition comprising 5-5AZA, THU and one or more pharmaceutically acceptable carriers can be administered to a patient in need thereof, either sequentially, alternately or simultaneously. In this embodiment, tetrahydrouridine can also include derivatives, such as 2'-fluorinated tetrahydrouridine derivatives. The first and second compositions can be administered after, during or before measuring predictive biomarkers in cancer cells.

[0013] In one embodiment, the first and second therapeutic compositions may each be in the form of a pill or they may be combined in a single pill. When the composition of the present disclosure is in the form of a pill, THU or a THU derivative may be specifically located on the surface of the pill, and DEC or 5AZA may be located inside the pill. This differential position of the nucleoside analog drug DEC or 5AZA and THU on the pill ensures that THU or a THU derivative may be bioavailable from about 1 minute to 180 minutes or 15 minutes to about 180 minutes before DEC or 5AZA, and more preferably bioavailable between 30 to about 60 minutes before DEC or 5AZA. In another embodiment, DEC or 5AZA may be coated or embedded in a polymer to help delay drug release compared to the release of THU or a THU derivative coated or embedded with a polymer.

[0014] The combination of DEC and THU can increase oral bioavailability by at least about 10-fold after oral administration of the therapeutic composition, achieve the low C concentration required for non-cytotoxic depletion of DNMT-1 by DEC or 5AZA, and provide a therapeutic composition that is effective in reducing the risk of DNMT-1 depletion. max and multiple hours T max). Because of the known off-target or toxic effects of DEC and 5AZA, optimizing DEC or 5AZA to deplete DNMT1 in vivo can have powerful therapeutic benefits. Thus, the therapeutic composition can produce a peak plasma DEC level of about 0.05 μM to about 0.5 μM, or a peak plasma 5AZA level of about 0.5 μM to about 5 μM in a human subject. In this embodiment, the treating physician can prescribe a pill containing DEC and THU (or)-5AZA and THU (sequentially or intermittently) in a certain way so that the cancer patient first takes a pill containing DEC and THU, followed by a pill containing 5AZA and THU a few days or weeks later, or vice versa.

[0015] In one embodiment, a method for treating any tumor, metastatic tumor or combination thereof in a subject's tissue or a blood malignancy or blood disorder of a subject is provided. The method comprises the following steps: (i) administering a therapeutically effective amount of a first composition in the form of a pill, the first composition comprising a first nucleoside analog drug, a cytosine deaminase inhibitor and a pharmaceutically acceptable carrier, (ii) administering a second composition in the form of a pill, the second composition comprising a second nucleoside analog drug, a cytosine deaminase inhibitor and a pharmaceutically acceptable carrier, wherein the nucleoside analog drug in the first composition may be DEC, the nucleoside analog drug in the second composition may be 5AZA, and the cytosine deaminase inhibitor in the first composition and the second composition is THU or a THU derivative, such as a 2'-fluorinated tetrahydrouridine derivative. In this embodiment, the first and second compositions may be administered sequentially or alternately or simultaneously. In addition, the first and second compositions may be administered before, during or after measuring the level of a predictive biomarker in a subject's cancer cell.

[0016] Treatment options include determining the level of DCK in a patient in need of treatment, and prescribing a combination of an appropriate nucleoside analog drug and THU (i.e., DEC and THU or 5AZA and THU or both), which is carried out in a manner that the cancerous tissue is diverted into a differentiation pathway. A prescription for a pharmaceutical composition in pill form may be prescribed to a patient suffering from a cancer or hematological malignancy or disorder, once a week or once every two weeks or once to three times a week. In yet other embodiments, a prescription for a pharmaceutical composition in pill form may be prescribed to a patient at risk of developing a hematological or solid malignancy, or at risk of recurrence of a previously diagnosed hematological or solid malignancy or hematological or blood disorder, once every two weeks to three times a week.

[0017] In one embodiment, a kit for treating a tumor or refractory tumor or hematological tumor or blood disease is provided. The kit may include: (i) a pill comprising DEC, THU or a THU derivative and a carrier, (ii) a pill comprising 5AZA, THU or a THU derivative and a carrier, and (ii) an instruction manual. In another embodiment, the kit may include (i) a capsule comprising a mixture of DEC / THU microspheres or microparticles and 5AZA / THU microspheres or microparticles, and (ii) an instruction manual. The pills or capsules may be formulated in a certain manner so that THU or a THU derivative is always bioavailable at least 1 minute to about 60 minutes before the nucleoside analog drug DEC or 5AZA. In one embodiment, THU or a THU derivative may be bioavailable 1 minute to about 15 minutes, or 15 minutes to 30 minutes, 30 minutes to 45 minutes, 45 minutes to 60 minutes before DEC or 5AZA. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic representation of CDKN2A and TP53 deletions / mutations predicted in renal cell carcinoma (RCC).

[0020] Figure 2 Schematic representation of the genes VHL, PBRM1, and Pax8 that are amplified or deleted in RCC.

[0021] Figure 3 Figure 2 is a graphic representation showing that WT1 promoter CpGs are hypermethylated in RCC compared to unpaired noncancerous kidneys.

[0022] Figure 4 Schematic representation of the high expression of differentiation drivers and paradoxical epigenetic repression of key epithelial differentiation target genes (e.g., WT1 and GATA3) in RCC.

[0023] Figure 5 A comprehensive record of the coactivator-corepressor interactions of Pax8 obtained by LC / MSMS.

[0024] Figure 6 Schematic representation of the interactions of PAX8 with coactivators and corepressors resulting from depletion of DNMT1 at non-cytotoxic concentrations of DEC.

[0025] Figure 7 Graph showing DCK and UCK2 expression levels associated with DEC and 5AZA sensitivity, respectively.

[0026] Figure 8 Schematic representation of relapse of MDS / AML cells from patients treated with Dec, where DCK levels were low and UCK2 levels were high, and vice versa in patients treated with 5-Aza.

[0027] Fig. 9 Graph showing the efficacy of three times weekly THU-DEC alternating with three times weekly THU-55AZA in a xenograft model of patient-derived AML.

[0028] Fig.10 Figure 2 is a flow chart showing how biomarkers can guide rational measures to augment drug response and remedy relapse. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present disclosure relates to compositions containing a combination of a nucleoside analog drug with a potent cytosine deaminase inhibitor and a pharmaceutically acceptable excipient. The nucleoside analog drug present in the composition can be any known nucleoside deoxycytidine analog drug that is also a DNA demethylating agent, including but not limited to DEC, 5AZA, gemcitabine, etc.

[0031] The term "nucleoside analog" refers to any nucleoside containing a nucleic acid analog and a sugar, which acts as a metabolite inhibitor when phosphorylated. Specifically, the nucleoside analog in the present disclosure refers to a cytosine nucleoside analog, including but not limited to DEC with unmodified sugar, and 5AZA. DEC (5-5-aza-2'-deoxycytidine) mimics the natural components of DNA and is relatively unique in the large family of nucleoside analog drugs in that it can irreversibly bind to and consume the enzyme DNA methyltransferase 1 (DNMT1), and unmodified sugars do not terminate DNA chain extension, thereby achieving the consumption of DNMT1 without cytotoxicity. In cancer cells, the consumption of DNMT1 by the drug DEC prevents the inhibition of differentiation genes and renews cancer cell differentiation-the abnormal growth of cancer cells is caused by the obstruction of their normal differentiation process, which is alleviated by DEC. It is particularly noted that the consumption of DNMT1 in normal stem cells increases their self-renewal; that is, the consumption of DNMT1 increases the number of normal stem cells, which is contrary to its effect on cancer cells. Therefore, depletion of DNMT1 by DEC could be an effective and extremely safe well-tolerated cancer therapy.

[0032] In one embodiment of the invention, the pharmacological goal of non-cytotoxic therapy (defined as a treatment that does not induce cytotoxicity (apoptosis) in normally dividing cells) is to maximize the in vivo time-above-threshold concentration of DEC or 5AZA for depletion of DNMT1 (≥0.05-0.2 μM for DEC; ≥0.5-2 μM for 5AZA) while avoiding high peak levels of damaged DNA (≥0.5-1 μM for DEC; ≥5-10 μM for 5AZA). This low C max But the longer T maxExposure will also occur in solid tissues where there is minimal or even negligible distribution of the present DEC and 5AZA formulations due to high CDA expression in solid tissues, including liver, kidney, intestine, and brain. Because continuous exposure to DEC or 5AZA can result in accumulation of DEC or 5AZA in normal cells to levels that induce cytotoxicity, the DNMT1 depletion effect of DEC or 5AZA is expected to be intermittent in the present disclosure. For example, currently known routes of administration, regimens, and formulations of DEC or 5AZA can produce higher peak drug levels that are known to kill normal cells in sensitive tissues such as bone marrow through cytotoxicity, but produce extremely short above-threshold time concentrations to deplete DNMT1 in the bone marrow, but especially in solid tissues that highly express CDA. Moreover, currently known routes of administration, regimens, and formulations of DEC or 5AZA do not increase and distribute exposure time to increase DNMT1 depletion while avoiding continuous or sustained exposure that would cause DEC or 5AZA to accumulate in normal bone marrow cells to levels that cause cytotoxicity. In fact, these current methods mimic the traditional regimens that utilize pulsatile cyclic administration, which is intended for cytotoxic therapeutic purposes. Importantly, the destruction of DEC and 5AZA by the enzyme cytosine deaminase (CDA) results in a shortened in vivo half-life of <20 minutes (although the in vitro half-life is 5 to 9 hours) (Liu, Z. et al., 2006 Rapid Common Mass Spectrum 20: 1117-1126); and especially for solid tissue cancers present in tissues that intrinsically express high levels of CDA, this drastic reduction in half-life is a significant obstacle to the effective in vivo translation of in vitro observations. High peak levels of DEC or 5AZA in plasma and bone marrow are also caused by pharmacogenomic variations in CDA (Gilbert, JA et al., 2006 Clin. Cancer Res 12, 1794-1803; Kirch, HC et al., 1998 Exp. Hematol. 26, 421-425; Yue, L. et al., 2003 Pharmacogenetics 13, 29-38), which can lead to large inter-individual differences in pharmacokinetics (PK) and clinical effects.Moreover, malignant cells can develop resistance due to the destruction of DEC by CDA (Ohta, T. et al., 2004 Oncol Rep 12: 1115-1120; Hubeek, I. et al., 2005 Br J Cancer 93: 1388-1394; Huang, Y. et al., 2004 Cancer Res 64: 4294-4301), and malignant cells can be protected from the therapeutic effects of DEC by existing in tissues with high levels of CDA (Ebrahem, Q., 2012 Oncotarget 3(10): 1137-45).

[0033] In order to address these multiple basic pharmacological limitations of DEC and 5AZA, an oral route of administering DEC or 5AZA in combination with the CDA inhibitor THU is preferred herein. Compared with the current parenteral administration route, this oral administration of DEC or 5AZA in combination with THU substantially reduces the peak level while substantially increasing the above-threshold time concentration for consuming DNMT1; the oral administration can achieve long-term, frequent but not daily (i.e., rhythmic) therapy to maintain a lifelong therapeutic effect while allowing cell division and avoiding cytotoxicity; the oral combined administration enables DEC or 5AZA to be distributed to solid tissues as cancer sites and eliminates the shelter of cancer cells in CDA-rich organs such as the liver. In short, the combination can achieve the treatment of solid tissue cancer. In addition, oral administration contributes to the global widespread use of drugs.

[028] Cytosine deaminase (CDA) is an enzyme that is highly expressed in the liver and intestines and rapidly destroys DEC and 5AZA in the body. In humans, the CDA gene has non-synonymous single nucleotide polymorphisms that produce cytosine deaminase variants with 3-fold or greater differences in enzyme activity (Gilbert, JA et al., 2006 Clin Cancer Res 12, 1794-1803; Kirch, HC et al., 1998 Exp Hematol 26, 421-425; Yue, L. et al., 2003 Pharmacogenetics 13, 29-38).

[0034] THU is known to inhibit the enzyme cytosine deaminase or CDA and has many desirable characteristics, including but not limited to: displaying a favorable toxicity profile, a well-characterized PK, the ability to overcome intestinal and hepatic first-pass barriers to oral bioavailability of DEC and 5AZA, producing more predictable effects of DEC or 5AZA doses across individuals, increasing the time-to-threshold concentration of DEC or 5AZA for depletion of DNMT1, removing sanctuary sites that shield malignant cells from the therapeutic effects of DEC or 5AZA, etc. Thus, in one embodiment the invention provides a composition for oral administration, wherein the composition comprises about 1-10 mg / m2 DEC, or 10-100 mg / m 2 range of 5AZA, and an amount of about 400 mg / m 2 About 500 mg / m 2 The CDA inhibitor THU within the scope of the present invention. Although the CDA inhibitor used in the composition of the present invention is THU, those skilled in the art will appreciate the fact that other CDA inhibitors such as zebularine can be used instead of THU. In some other embodiments, the CDA inhibitor used in the therapeutic composition may be a commonly known THU derivative, such as a 2'-fluorinated tetrahydrouridine derivative, which is synthesized as an inhibitor of CDA and described in Ferraris et al., 2014 J Med Chem. 57(6): 2582-88 or U.S. Pat. No. 8,329,665, or EP2447272.

[0035] Because malignant cells can produce resistance to the DEC-THU combination by moving pyrimidine metabolism from DCK remedy to UCK2 remedy, or by moving pyrimidine metabolism from UCK2 remedy to DCK remedy to 5AZA-THU combination, it is expected that these two drugs will be used alternately or sequentially to rationally address their respective resistance mechanisms. In another embodiment, the administration of the composition produces a DEC plasma concentration of about 0.05 μM to about 0.5 μM or a 5AZA concentration of about 0.5 μM to about 5 μM. In yet another embodiment, the composition is administered to a patient suffering from metastatic cancer once a week or once every two weeks. In yet another embodiment, the composition is administered to a patient suffering from cancer once to three times a week. In some embodiments, once every two weeks to three times a week, so frequently to a patient at risk of producing a hematological or solid malignancy, or at risk of recurrence of a previously diagnosed hematological or solid malignancy, administer the composition of the present invention.

[0036] 5AZA is incorporated into RNA to a greater extent than DNA, which incorporation results in inhibition of protein production. When 5AZA is incorporated into DNA, it covalently binds to DNA methyltransferases that prevent DNA synthesis and subsequent cytotoxicity. In a preferred embodiment, the DEC and 5AZA used in the composition for oral administration are in salt-free free drug form.

[0037] In one embodiment, the present disclosure provides an oral administration composition in the form of a pill, the composition comprising THU and DEC or THU and -5AZA, wherein THU or a THU derivative (2'-fluorinated tetrahydrouridine derivative) is released faster than DEC or 5AZA. It will be appreciated by those skilled in the art that such differential and / or dual release of the drugs of the present disclosure can be achieved by any known means in the art. 'Differential or dual release' refers to the release of 'drugs' in a pill containing more than one drug at different time points, and / or the bioavailability of the drug at different time points after ingestion. For example, in a differential release formulation, THU may have a faster dissolution rate, or THU may be located on the surface of the pill, while DEC or 5AZA is located inside the pill. Because of the positioning of THU or THU being coated or embedded in a polymer or suitable vehicle, THU or THU derivatives are bioavailable from about 1 minute to about 180 minutes before DEC or 5AZA; in another case, from about 15 minutes to about 60 minutes before DEC or 5AZA; in another case, from about 30 minutes or 60 minutes before DEC or 5AZA. THU can also be administered separately and continuously, first THU, then DEC or 5AZA. As mentioned herein, the term "bioavailable" means that the active agent (THU or DEC or 5AZA) can be absorbed and used by the body. "Orally bioavailable" means that the agent has been orally administered and can be absorbed and used by the body.

[0038] In another embodiment, the composition for oral administration may be a single pill (also used herein as 'capsule or tablet'), which contains nucleoside analog drugs DEC and 5AZA in addition to THU or THU derivatives and at least one pharmaceutically acceptable excipient. In this embodiment, DEC or 5AZA is located on the pill differently from THU so that THU or THU derivatives are bioavailable at least 1 minute to about 180 minutes before DEC or 5AZA. In this embodiment, the drugs DEC and 5AZA may each be in the form of coated or embedded microspheres or microparticles, which help the drug to be released after THU or THU derivatives. This composition comprising two cytosine nucleoside analog drugs is administered to patients with aggressive and refractory cancers once to three times a week for a specified time period. In another embodiment, the present composition is administered to patients at risk of developing hematological or solid malignancies, or at risk of recurrence of previously diagnosed hematological or solid malignancies, as frequently as once every two weeks to three times a week.

[0039] The term "pharmaceutically acceptable excipient" as used in the composition refers to a carrier and vehicle that is compatible with the active ingredient (such as the compound of the composition) of the pharmaceutical composition of the present invention (and preferably capable of stabilizing it) and harmless to the subject to be treated. For example, a solubilizing agent that forms a more soluble specific complex with the compound of the present invention can be used as a pharmaceutical excipient for delivering the compound. Known suitable carriers and vehicles are those known to those skilled in the art. As used herein, the term "excipient" covers all such carriers, adjuvants, diluents, solvents or other inactive additives. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solutions, alcohols, vegetable oils, polyethylene glycols, gelatin, lactose, straight-chain starch, magnesium stearate, talc, silicic acid, viscous paraffin, aromatic oils, fatty acid monoglycerides and diglycerides, petroleum hydrocarbon fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical composition of the present invention can also be sterilized and, if necessary, mixed with auxiliary agents that do not deleteriously react with the active compounds of the present invention, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavoring agents and / or aromatic substances.

[0040] In another embodiment, the composition of the present invention is stored with a desiccant. This can be used to extend the shelf life of the composition of the present invention and facilitate its distribution and use worldwide.

[0041] As used herein, the term "treatment" refers to changing the course of the disease of the subject being treated. The therapeutic efficacy includes but is not limited to preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

[0042] Pharmaceutical composition

[0043] The pharmaceutical compositions of the present disclosure may be prepared as described in U.S. Applications 13 / 141,669 (issued as US9,259,469) and 13 / 414,546 (issued as U.S. Pat. No. 9,265,785), which are incorporated herein in their entirety. In one embodiment, the pharmaceutical compositions and non-toxic dosage forms of the present invention comprise about 0.1 mg / kg to about 0.5 mg / kg CDEC or 1 mg / kg to about 5 mg / kg 5AZA and about 10 mg / kg to about 50 mg / kg THU and a pharmaceutically acceptable excipient in relative amounts and formulated in a manner such that a given pharmaceutical composition or dosage form releases inhibition of differentiation pathways in tumor cells or reduces abnormal inhibition of differentiation-related genes in tumor cells. The therapeutically effective amount of the pharmaceutical compositions and dosage forms of the present invention may represent or result in one or more of the following: (i) a peak level of DEC or 5AZA in the patient's plasma below a threshold above which damage to DNA in normal dividing cells is sufficient to cause apoptosis (cytotoxicity), (ii) an exposure time sufficient to deplete DNMT1 in cancer cells (DEC or 5AZA alone has a plasma exposure time of several minutes) extending from several minutes to several hours (Tmax extended 5 to 20 times), but below a threshold that results in accumulation of intracellular levels of DEC or 5AZA to a level that induces damage to DNA in normal dividing cells that is sufficient to cause apoptosis (cytotoxicity). In short, an exposure time optimized for non-cytotoxic depletion of DNMT1: (iii) a 5 to 20-fold increase in oral bioavailability of DEC or 5AZA, (iv) distribution of DEC or 5AZA to CDA-rich tissues where cancer cells would otherwise be protected from the therapeutic effects of DEC or 5AZA. The same principle applies to oral bioavailability, i.e. the ability of DEC or 5AZA to now distribute through the intestine and liver: (v) induction of expression of genes and proteins necessary for cell cycle exit (p53 / p16-independent cell cycle exit) by cancer cells through differentiation, and (v) absence of cytotoxic mechanisms of action, which would avoid normally dividing cells. Avoiding toxicity in this way would facilitate frequent intermittent treatments to increase the fraction of cancer exposed to DNMT1 depletion.

[0044] For example, in some embodiments, the pharmaceutical compositions and non-toxic dosage forms of the present invention comprise about 1 mg / kg to about 5 mg / kg 5AZA or 0.1 mg / kg to about 0.5 mg / kg DEC and about 10 mg / kg to about 50 mg / kg THU and a pharmaceutically acceptable excipient in relative amounts and formulated in a manner such that a given pharmaceutical composition or dosage form releases inhibition of a differentiation pathway in a tumor cell or reduces aberrant inhibition of a differentiation-related gene in a tumor cell.

[0045] The pharmaceutical composition or dosage form may also include additional active agents, including chemotherapeutic agents known in the art. The pharmaceutical composition may also be administered alone or in combination with other known compositions for treating cancer in mammalian subjects. The term "combination" may include administering the composition of the present disclosure simultaneously or sequentially. Sequential administration may include administering the disclosed therapeutic composition followed by any known composition, or vice versa.

[0046] The composition may be administered in single or multiple doses. Generally, the therapeutically effective compounds of the present disclosure are present in dosage forms at concentration levels ranging from about 5.0% by weight to about 70% by weight. In some embodiments, a subject in need of treatment for a tumor may receive a tablet or capsule containing 10 mg / kg THU and 0.2 mg / kg DEC or 2 mg / kg 5AZA twice a week on consecutive days or once a week or once every three days for about one or two weeks.

[0047] In order to achieve differential release of THU or THU derivatives and nucleoside analog drugs, a sustained release composition can be formulated in which the active component is derived with a coating that can be distinguished and degraded (e.g., by microencapsulation, multilayer coating). In some embodiments, nanoparticles of the pharmaceutical composition can be prepared as described in the U.S. published application 14 / 046,343, which is incorporated herein as a whole. These compositions ensure that THU is bioavailable from about 1 minute to about 180 minutes before DEC and / or -5AZA. In another embodiment, microformulations or nanoformulations ensure that THU is bioavailable from about 15 minutes to about 60 minutes or 30 minutes to about 60 minutes before DEC and / or -5AZA.

[0048] The compositions of the present disclosure may also additionally include other agents that reduce the rate of decomposition of the active compound. These agents may include stabilizers, including but not limited to antioxidants, ascorbic acid, pH buffers or salt buffers.

[0049] Predictive biomarkers

[0050] The development of "predictive or companion" biomarkers to guide treatment selection is a powerful and effective tool for selecting meaningful individualized treatment regimens (such as dosage and duration) for any subject in need of treatment. Therefore, predictive biomarkers in the form of enzyme profiles are measured during the treatment regimen to help the treating physician determine the appropriate treatment regimen for the subject. DNA synthesis and turnover of genetic material are essential for cell proliferation, and high expression of the pyrimidine salvage enzyme DCK is a pan-cancer hall mark for proliferation. Therefore, nucleoside analogs of radioactive markers can be used to image tumor proliferation through the salvage pathway of DNA synthesis. 1-(2'-deoxy-2'-[ 18[F]-fluoro-β-D-arabinofuranosyl)cytosine (TFAC), a deoxycytidine analog like DEC, is a radiotracer. Because active DEC requires activation by DCK to exert its DNMT1-depleting effects, cancer DCK expression reliably predicts sensitivity to DEC across the full range of cancer histologies and genetics. Therefore, TFAC-positron emission tomography (PET) can be used to measure DCK-dependent cancer cell proliferation.

[0051] In some embodiments, predictive bioassays can be performed before, during, and after treatment with the disclosed compositions. Assays can be performed to select an appropriate treatment regimen. For example, if assays before or during treatment indicate high FAC uptake, which is associated with a response to oral THU-DEC, the treating physician may continue to prescribe pills or tablets containing THU-DEC. If FAC uptake is low or decreased with increasing tumor size, indicating that cancer cells that use the alternative pyrimidine metabolizing enzyme UCK2 (a resistance mechanism to DEC) predominate, the physician may prescribe tablets or capsules containing THU-5AZA.

[0052] Crucially, these measures not only provide scientific insights into resistance mechanisms, but also guide the application of rational clinical measures to overcome resistance and amplify responses: (i) Failure to achieve molecular PD in the absence of adverse effects of this non-cytotoxic therapy may guide rational escalation of the dose or frequency of oral THU-DEC ( Fig. 9 (ii) Resistance to increased variant allele frequency burden concurrent with loss of molecular PD and shift in pyrimidine metabolic enzymes may guide a rational switch to oral THU-5AZA in therapy ( Fig. 9 Optimal use would likely be with one oral administration of THU-DEC, alternating with oral THU-55AZA administered the following week, for long term administration ( Figure 7-9 ).

[0053] Treatment

[0054] In one embodiment, a pill containing the therapeutic composition of the present disclosure may be administered to a cancer patient in need of treatment. The cancer may be a solid or liquid cancer or may be an invasive and refractory cancer of any known tissue of the subject. The therapeutic composition of the present disclosure may also be effective in treating hematological malignancies or blood disorders, such as, but not limited to, sickle cell disease, hemoglobinopathy, thalassemia, or any related condition as described in detail in U.S. Patent Application 13 / 414,546 (now granted as U.S. Patent 9,265,785), which is incorporated herein in its entirety. 'Treatment' refers to any known physical and / or biochemical and / or molecular change in cancer tissue. For example, treatment may refer to improving tumor size or known cancer biomarker proteins in normal and malignant cells, such as DNMT1 levels, or may be molecular therapy, such as in the de-repression of specific differentiation genes or all of the above. Treatment methods include measuring predictive biomarkers before, during, or after treatment. Predictive biomarkers can inform the treating physician of the appropriate dose and schedule to be prescribed to the patient. For example, a patient may receive a drug of the present disclosure as follows: THU 10 mg / kg orally, followed by DEC 0.2 mg / kg orally 60 minutes after THU, twice a week on consecutive days. Treatment may include administering a pill comprising THU-DEC or THU-5AZA or a pill containing DEC and 5-5AZA, wherein the THU or THU derivative is bioavailable from about 1 minute to about 180 minutes or 15 minutes to about 180 minutes before DEC and / or 5AZA.

[0055] The treatment method may include administering a first composition comprising DEC, THU or a THU derivative and at least one pharmaceutically acceptable excipient, followed by alternating administration of a second composition comprising 5AZA, THU or a THU derivative and at least one pharmaceutically acceptable excipient a few days or weeks apart. This sequential or alternating treatment method may be followed for a few weeks to several months.

[0056] In some aspects, as described in U.S. published application Ser. No. 14 / 046,343, which is incorporated herein in its entirety, the method of treatment includes additional treatment modalities including, but not limited to, radiation therapy or cytotoxic chemotherapeutic agents such as antimetabolites, doxorubicin, HDAC inhibitors, antiviral agents, antiretroviral agents, or any combination of these agents. Example

[0057] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0058] Embodiment 1:

[0059] The need for treatments that are mechanistically different from current options

[0060] In the past, most drugs evaluated in RCC attempted to use the p53 / p16 apoptosis system (cytotoxicity) to terminate proliferation. For decades, these treatments have been shown to be toxic and inefficient because RCC cells, like most cancer cells, have apoptosis resistance relative to normal cells through genetic abnormalities in key p53 / p16 system genes such as CDKN2A. The first drugs approved by the FDA for the treatment of RCC inhibit signal transduction pathways (such as AKT / mTOR) that stabilize the MYC protein, which is the main coordinator of cell growth and division. Therefore, these drugs directly hinder RCC growth by acting on the MYC protein and also indirectly by inhibiting angiogenesis required to support RCC growth. Although these therapies have made significant progress, there are few lasting responses (median survival of 18 to 30 months), and p53 / p16 variations still affect responses, which are robust to multivariate analysis ( Figure 1 ). In addition, the durability of responses in the second or third cell lines was substantially inferior to that in the first cell line, suggesting some overlap in the mechanisms of resistance to approved drugs. Mechanistically distinct treatments that use pathways other than p53 / p16 to terminate RCC cell growth and division are therefore indicated and suitable for evaluation.

[0061] PBRM1 inactivation and epithelial differentiation therapy in RCC

[0062] An unexpected finding from RCC genomic studies is the essentially universal inactivating variants in genes of coactivator subunits, particularly PBRM1, but also others (SETD2, SMARCC1, SMARCA2, ARID1A) ( Figure 2 ). These observations are recent, and the pathway functions of these coregulators in normal renal cells, or the pathways whose loss of function confers clonal advantage, are unknown. We speculate that a possible link is the observation that key epithelial terminal differentiation genes (e.g., GATA3, WT1) are epigenetically repressed in RCC ( Figure 3 , 4 This epigenetic repression of late differentiation genes is particularly interesting because RCC cells express unmutated master transcription factors that drive epithelial differentiation (e.g., PAX8, PAX2) at relatively preserved mRNA and high protein levels ( Figure 4). Another related observation is that increased chromatin repressive marks are associated with poor prognosis in multiple RCC studies. In the last funding cycle, we experimentally linked these observations and found that PBRM1 / BAF mediates transcriptional activation by PAX8, a master promoter of mesenchymal-epithelial transition11-16 that is highly expressed in RCC17-22. PBRM1 loss causes an unbalanced recruitment of PBRM1 / BAF corepressor counterparts (e.g., DNMT1) to PAX8, thereby repressing rather than activating proliferation-terminating epithelial differentiation target genes ( Figure 5 Importantly, inhibition of these specific corepressors (e.g., DNMT1) restored expression of multiple PAX8 target genes, thereby renewing epithelial differentiation and physiological, p53 / p16-independent cell cycle exit ( Figure 6 ).

[0063] Embodiment 2:

[0064] The need for mechanism-based predictive biomarkers

[0065] DNA synthesis and turnover of genetic material are essential for cell proliferation, and high expression of the pyrimidine salvage enzyme DCK is a pan-cancer hallmark of proliferation. Therefore, radiolabeled nucleoside analogs have been developed for imaging tumor proliferation through the salvage pathway of DNA synthesis. 1-(2'-deoxy-2'-[18F]-fluoro-β-D-arabinofuranosyl)cytosine (FAC) such as Dec (a deoxycytidine analog) is one such radiotracer. It has been documented that FAC, similar to Dec, is not well distributed into solid tissues such as the liver and kidney due to rapid deamination by CDA. It was then discovered that co-injection of THU-FAC (TFAC) solved this problem, resulting in high FAC uptake in hepatocellular carcinomas on the background liver in the woodchuck animal model of viral infection-induced primary hepatocellular carcinoma. As Fig.10 As shown in , the use of predictive biomarker assays will help determine whether the frequency or dose of THU-Dec should be increased.

[0066] In this way, TFAC-PET can suggest reasonable salvage therapies. If validated, it could also play a role in selecting primary therapy between THU-DEC versus THU-5Aza. Additionally, it is possible that TFAC-PET does capture a direction in RCC biology that correlates well with responses to and outcomes of other RCC drugs (sunitinib, etc.). In other words, it is conceivable that TFAC-PET could enable widespread use of predictive biomarkers that FDG-PET has not yet met. The potential utility described herein may justify the evaluation of this broader purpose.

[0067] Embodiment 3:

[0068] Treatment-resistant malignant cells from patients treated with DEC upregulated UCK2 and downregulated DCK, and in patients treated with 5AZA Vice versa in patients

[0069] Bone marrow aspirate samples were obtained from patients before and at the time of relapse while on treatment with DEC or 5AZA, and the expression of the pyrimidine metabolic enzymes DCK and UCK2 was measured by quantitative real-time PCR. Figure 8 As shown in , during relapse, MDS / AML cells from patients treated with Dec had lower DCK levels and higher UCK2, and vice versa in patients treated with 5Aza.

[0070] Embodiment 4:

[0071] In a xenograft model of patient-derived AML, DEC alone or THU-DEC alone was associated with a significantly higher rate of death compared with DEC alone. The non-cytotoxic treatment of 3x / week THU-DEC alternating with 3x / week THU-5AZA dramatically amplified the response.

[0072] Primary AML cells (1x10 6 5 cells) xenografts were transplanted into NSG mice. Five days after inoculation, mice were treated with vehicle, DEC alone (0.2 mg / kg, subcutaneously, 3 times / week), THU-DEC (THU 8 mg / kg and DEC 0.1 mg / kg, subcutaneously, 3 times / week), or 3 times / week THU-DEC alternating with 3 times / week THU-5AZA (THU 8 mg / kg and 5AZA 1 mg / kg). n=5 per treatment group. Fig. 9 Clearly, alternating regimens of THU-Dec and Thu-5Aza significantly improved survival in these xenograft models of patient-derived AML.

Claims

1. Use of a combination of a first composition comprising decitabine, tetrahydrouridine and a pharmaceutically acceptable carrier and a second composition comprising 5-azacytidine, tetrahydrouridine and a pharmaceutically acceptable carrier in the preparation of a medicament for treating cancer in a subject, wherein the first composition and the second composition are used to be administered alternately to the subject, Wherein the cancer is selected from hematological malignancies.

2. The use according to claim 1, wherein the first composition comprises 1 to 10 mg / m 2 of decitabine and 400 to 500 mg / m 2 of tetrahydrouridine.

3. The use according to claim 1, wherein the second composition comprises 10 to 100 mg / m 2 5-azacytidine and 400 to 500 mg / m 2 of tetrahydrouridine.

4. The use according to claim 1, wherein administration of the first composition and the second composition produces a peak decitabine plasma concentration of less than 0.5 μM and a peak 5-azacytidine plasma concentration of no more than 5 μM in the subject.

5. The use according to claim 1, wherein the tetrahydrouridine in the first composition and the second composition is bioavailable 1 minute to 180 minutes before decitabine or 5-azacytidine.

6. The use according to claim 1, wherein the tetrahydrouridine in the first composition and the second composition is a 2'-fluorinated tetrahydrouridine derivative and is bioavailable 15 minutes to 180 minutes before decitabine or 5-azacytidine.

7. The use according to claim 1, wherein the first composition is present in a first pellet and the second composition is present in a second pellet.

8. The use according to claim 1, wherein the cancer comprises benign, metastatic or refractory cancer.

9. The use according to claim 1, wherein the cancer is acute myeloid leukemia (AML).

10. The use according to any one of claims 1 to 9, wherein the first composition and the second composition are for administration after measuring the predictive biomarkers in the subject.

11. The use according to claim 10, wherein the measurement of the predictive biomarker comprises the level of deoxycytidine kinase in the subject.

12. The use according to claim 11, wherein the subject is a mammal.

13. The use according to claim 10, wherein the subject is a mammal.

14. The use according to any one of claims 1 to 9, wherein the subject is a mammal.

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