HK1254882B - Combination therapies for treatment of cancer - Google Patents

Description

Combination therapies for treating cancer

background

Technical Field

The present invention generally relates to methods of treating cancer by administering a cyclin-dependent kinase inhibitor and a DNA methyltransferase inhibitor.

Description of related fields

Myelodysplastic syndrome (MDS) is a diverse group of bone marrow diseases characterized by the inability to produce healthy numbers of blood cells. MDS often (approximately 30%) progresses to acute myeloid leukemia (AML), which remains a largely incurable condition with relatively poor survival. One treatment option for MDS is the hypomethylating agents azacitidine and deoxyazacitidine (decitabine), which act through two main mechanisms: 1) inhibition of DNA methyltransferases, which leads to the activation of key tumor suppressor genes; and 2) direct damage to DNA after incorporation into replicating DNA chains. The second of these mechanisms activates a programmed cell death pathway (apoptosis), and this pathway has been shown to depend, in part, on the expression levels of key apoptosis regulatory proteins, including MCL-1, an anti-apoptotic member of the BH3 family of apoptosis regulatory proteins.

Cyclin-dependent kinases (CDKs) are important regulators that control the timing and coordination of the cell cycle. CDKs form reversible complexes with their obligate cyclin partners to control key moments in the transition through the cell cycle. In addition to regulating cell cycle progression, some CDK family members (e.g., CDK7 and CDK9) also play an active role in transcription. In particular, CDK9 directly phosphorylates RNA polymerase II and contributes to productive transcription. Agents that inhibit CDK9 have been shown to inhibit the expression of MCL-1, an important protein in the apoptosis pathway activated by DNA methyltransferase inhibitors. One such CDK inhibitor is avocidib, which is a potent and selective inhibitor of CDKs (e.g., CDK-9) and has anti-tumor activity against various tumor cell lines. Avocidib is also known to rapidly reduce the expression level of MCL-1.

To date, there have been no reports of combinations of DNA methyltransferase inhibitors and cyclin-dependent kinase inhibitors for the treatment of cancers such as MDS and/or AML. While progress has been made, there remains a need in the art for improved combination therapies for the treatment of various cancers. The present invention addresses this need and provides related advantages.

Summary of the Invention

In short, embodiments of the present invention provide methods for treating cancer comprising administering two different therapeutic agents. In one embodiment, the present disclosure provides a method for treating cancer in a mammal in need thereof, comprising administering to the mammal an effective amount of the following therapeutic agents:

i) cyclin-dependent kinase inhibitors, and

ii) DNA methyltransferase inhibitors.

Also provided are kits and pharmaceutical compositions useful in the disclosed methods, the kits comprising a cyclin-dependent kinase inhibitor, a DNA methyltransferase inhibitor, and instructions for administering the cyclin-dependent kinase inhibitor and the DNA methyltransferase inhibitor to a mammal in need of treatment for cancer.

These and other aspects of the invention will become apparent upon reference to the following detailed description. To this end, various references are set forth herein that describe in more detail certain background information, processes, compounds and/or compositions, and are each hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference numbers represent similar elements. The sizes and relative positions of elements in the drawings are not necessarily drawn to scale, and some of these elements are arbitrarily enlarged and positioned to improve the legibility of the drawings. In addition, the specific shapes of the elements drawn are not intended to convey any information about the actual shape of the specific elements and are selected solely for ease of identification in the drawings.

FIG1A provides the relative expression levels of MCL-1 compared to β-actin in MV 4-11 cells over a range of avosidic doses.

Figure 1B is a gel showing the relative expression levels of MCL-1 compared to β-actin in MV 4-11 cells over a range of time points.

FIG2 is a cellular graph showing the synergistic effect of combining avosidic acid with azacitidine to inhibit the growth of MV 4-11 cells.

Figure 3 presents caspase 3/7 activity relative to logarithmic scale concentration for the combination of avocidin and azacitidine that induces apoptosis in MV 4-11 cells.

Figures 4A-D are bar graphs providing data for MV 4-11 cells treated with avosidic acid or azacitidine alone or in combination at varying concentrations of azacitidine.

Figures 5A and 5B present data from combined treatment of avosidic and decitabine in MV4-11 cells.

Detailed Description of the Invention

In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the present invention. However, it will be understood by those skilled in the art that the present invention can be practiced without these details.

Throughout this specification and claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" are to be interpreted in an open, inclusive sense, i.e., "including but not limited to."

References throughout this specification to "one embodiment" or "an embodiment" mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features or characteristics may be combined in any suitable manner in one or more embodiments.

"DNA methyltransferase inhibitors" are agents that have dual activity as DNA methyltransferase inhibitors (i.e., hypomethylating agents) and activity as DNA damaging agents. Exemplary DNA methyltransferase inhibitors are incorporated into DNA (e.g., DNA in cancer cells), thereby inhibiting DNA methyltransferase and causing DNA damage and apoptosis. Exemplary DNA methyltransferase inhibitors include nucleoside analogs (such as azanucleosides).

"Cyclin-dependent kinase inhibitors" are agents that inhibit the activity of cyclin-dependent kinases (CDKs), including CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, and CDK11. Exemplary CDK inhibitors inhibit the expression of MCL-1. Exemplary CDK inhibitors include, but are not limited to, avosidide, dinaciclib, olomoucine, roscovitine, purvalanol, paullone, palbociclib, thio/oxoflavopiridols, oxindoles, aminothiazoles, benzocarbazoles, pyrimidines, and seliciclib.

Avoside (also known as flavopiridol) is a synthetic flavonoid with the following structure:

"Azanucleosides" are naturally occurring nucleoside analogs in which at least one carbon atom has been replaced by a nitrogen atom. Exemplary azanucleosides include azacitidine and decitabine, each of which has the following structures:

"Mammal" includes humans as well as both domestic animals such as laboratory animals and household pets (eg, cats, dogs, pigs, cows, sheep, goats, horses, rabbits) and non-domestic animals (such as wild animals, etc.).

"Pharmaceutical composition" refers to a preparation of a compound of the present invention and a medium generally accepted in the art for delivering the biologically active compound to a mammal (e.g., a human). Such a medium includes all pharmaceutically acceptable carriers, diluents, or excipients thereof.

"Effective amount" or "therapeutically effective amount" refers to an amount of a compound of the present invention that is sufficient, when administered to a mammal, preferably a human, to achieve the effect defined below in treating cancer in a mammal, preferably a human. The amount of a compound of the present invention that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art, given his knowledge and this disclosure.

As used herein, "Treating" or "treatment" encompasses treating a target disease or condition in a mammal, preferably a human, having a target cancer, disease or condition, and includes:

(i) preventing a cancer, disease or condition from occurring in a mammal, particularly when such mammal is prone to the condition but has not yet been diagnosed with the condition;

(ii) inhibiting the cancer, disease or condition, i.e., preventing its development;

(iii) alleviate the cancer, disease or condition, i.e., cause regression of the disease or condition; or

(iv) Relief of symptoms caused by the cancer, disease, or condition, i.e., relief of pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably, or may be distinct, in that a particular disease or condition may not have a known pathogen (and thus the cause has not been addressed), and therefore it is not yet considered a disease, but rather an adverse disease condition or syndrome in which clinicians have identified a group of more or less specific symptoms.

I. Methods

In certain embodiments, the methods can be used to treat cancer cells and/or prevent, treat or ameliorate cancer and/or its symptoms in mammals, preferably humans. Thus, in one embodiment, the present disclosure provides a method for treating cancer in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of the following therapeutic agent:

i) cyclin-dependent kinase inhibitors, and

ii) DNA methyltransferase inhibitors.

In certain embodiments, the cyclin-dependent kinase inhibitor inhibits cyclin-dependent kinase (Cdk) proteins, such as Cdk4, Cdk6, Cdk7, Cdk8, Cdk9, Cdk10, and/or Cdk11. In some embodiments, the cyclin-dependent kinase inhibitor inhibits Cdk7, Cdk9, or both. In other embodiments, the cyclin-dependent kinase inhibitor inhibits the expression of MCL-1. In some embodiments, the cyclin-dependent kinase inhibitor is avosidic or denacitabine, or a pharmaceutically acceptable salt thereof. For example, in some more specific embodiments, the cyclin-dependent kinase inhibitor is avosidic or a pharmaceutically acceptable salt thereof. In some other specific embodiments, the cyclin-dependent kinase inhibitor is denacitabine, or a pharmaceutically acceptable salt thereof.

In some embodiments, DNA methyltransferases are capable of incorporating into RNA and/or DNA. In other embodiments, DNA methyltransferase inhibitors activate apoptosis due to DNA damage. In various embodiments, the DNA methyltransferase inhibitor is a nucleoside analog. In further embodiments, the DNA methyltransferase inhibitor is an azanucleoside, such as azacitidine or decitabine or a pharmaceutically acceptable salt thereof. In some more specific embodiments, the DNA methyltransferase inhibitor is azacitidine or a pharmaceutically acceptable salt thereof. In some other embodiments, the DNA methyltransferase inhibitor is decitabine or a pharmaceutically acceptable salt thereof.

In another embodiment of the foregoing, the cyclin-dependent kinase inhibitor is avosidic or a pharmaceutically acceptable salt thereof, and the DNA methyltransferase inhibitor is azacitidine or a pharmaceutically acceptable salt thereof. In other different embodiments, the cyclin-dependent kinase inhibitor is avosidic or a pharmaceutically acceptable salt thereof, and the DNA methyltransferase inhibitor is decitabine or a pharmaceutically acceptable salt thereof.

The cyclin-dependent kinase inhibitor and the DNA methyltransferase inhibitor may be administered simultaneously or sequentially. For example, in some embodiments, the cyclin-dependent kinase inhibitor is administered and, after a sufficient period of time, the DNA methyltransferase inhibitor is administered. In some other embodiments, the DNA methyltransferase inhibitor is administered and, after a sufficient period of time, the cyclin-dependent kinase inhibitor is administered. One of ordinary skill in the art can derive an appropriate dosing regimen based on conventional techniques and knowledge.

In some embodiments, the cyclin-dependent kinase inhibitor is administered to the mammal prior to administration of the DNA methyltransferase inhibitor. For example, in some embodiments, the DNA methyltransferase inhibitor is administered within about 24 to 48 hours after administration of the cyclin-dependent kinase inhibitor. In some other embodiments, the DNA methyltransferase inhibitor is administered to the mammal prior to administration of the cyclin-dependent kinase inhibitor.

A wide variety of cancers, including solid tumors and leukemias (e.g., acute myeloid leukemia) are suitable for the methods disclosed herein. Cancer types that can be treated in various embodiments include, but are not limited to, breast adenocarcinoma, prostate adenocarcinoma, and colon adenocarcinoma; all forms of lung bronchogenic carcinoma, medullary carcinoma, melanoma, liver cancer, neuroblastoma, papilloma, amine precursor uptake and decarboxylation cell tumor, bud tumor, branchial progenitor tumor, malignant carcinoid syndrome, carcinoid heart disease, and carcinomas (e.g., Walker's carcinoma, basal cell carcinoma, basosquamous cell carcinoma, Brown-Pearce carcinoma, ductal carcinoma, Ehrlich tumor, Krebs 2 carcinoma, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, scirrhous carcinoma, bronchiolar carcinoma, bronchial carcinoma, squamous cell carcinoma, and transitional cell carcinoma). Other types of cancer that may be treated include histiocytic disease, leukemia, malignant histiocytosis, Hodgkin's disease, immunoproliferative small, non-Hodgkin's lymphoma, plasmacytoma, reticuloendothelial proliferation, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumor of bone, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, chordoma, craniopharyngioma, dysgerminoma, hamartoma, stromal tumor, mesonephroblastoma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, and trophoblastic tumor. In addition, the following types of cancer are also considered suitable for treatment: adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, ovarian gynecoma, hepatocarcinoma, sweat gland adenocarcinoma, islet cell tumor, Leydig cell tumor, papilloma, Sertoli cell tumor, thecoma, leiomyoma, leiomyosarcoma, myoblastoma, myoma, sarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, non-pheochromocytic paraganglioma. Types of cancer that may be treated also include, but are not limited to, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angiomasclerosing, angiomatosis, glomus tumor, hemangioendothelioma, hemangioma, hemangiopericytoma, angiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, angiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, sarcoma, myxosarcoma, ovarian cancer, rhabdomyosarcoma, sarcoma, neoplasm, neurofibromatosis, and cervical dysplasia.

In certain more specific embodiments, the cancer is a blood cancer. For example, in some embodiments, the blood cancer is selected from: acute myeloid leukemia (AML), multiple myeloma, follicular lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma. In other embodiments, the blood cancer is acute myeloid leukemia (AML). In some further embodiments, the blood cancer is myelodysplastic syndrome (MDS).

II. Kits and Pharmaceutical Compositions

Other embodiments relate to kits for implementing the disclosed methods. For example, in some embodiments, a kit is provided that comprises a cyclin-dependent kinase inhibitor, a DNA methyltransferase inhibitor, and instructions for administering the cyclin-dependent kinase inhibitor and the DNA methyltransferase inhibitor to a mammal in need of cancer treatment. In some embodiments, the cyclin-dependent kinase inhibitor present in the kit is as defined in any of the preceding embodiments. In other embodiments, the DNA methyltransferase inhibitor as part of the kit is as defined in any of the preceding embodiments. In further embodiments, the cancers treatable with the kit are as defined in any of the preceding embodiments.

In other embodiments, pharmaceutical compositions are provided. For example, in some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient and the following therapeutic agent:

i) cyclin-dependent kinase inhibitors, and

ii) DNA methyltransferase inhibitors.

The therapeutic agent present in the pharmaceutical composition can be any of those described herein or known in the art. In some embodiments, the composition comprises avocidin. In some other embodiments, the composition comprises azacitidine. In some embodiments, the composition comprises decitabine. In some other embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient, avocidin, and azacitidine. In various embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient, avocidin, and decitabine.

In some embodiments, the pharmaceutical composition is formulated for oral administration. In other embodiments, the pharmaceutical composition is formulated for injection.

Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, spray, parenteral, ocular, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration. In addition, parenteral delivery includes, by way of example only, intramuscular, subcutaneous, intravenous, intramedullary, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.

In certain embodiments, one or more compounds as disclosed herein (i.e., cyclin-dependent kinase inhibitors and/or DNA methyltransferase inhibitors, referred to herein as "compounds") are administered in a local rather than systemic manner (e.g., via direct injection of the compound into an organ), typically in the form of a long-acting or sustained-release formulation. In specific embodiments, the long-acting formulation is administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. In addition, in other embodiments, one or more compounds are delivered in a targeted drug delivery system (e.g., in a liposome coated with an organ-specific antibody). In such embodiments, the liposome is targeted to an organ and selectively taken up by the organ. In other embodiments, one or more compounds are provided in the form of a rapid-release formulation, in the form of a sustained-release formulation, or in the form of an intermediate-release formulation. In other embodiments, one or more compounds are administered locally.

The compound is effective over a wide range of dosages. For example, in the treatment of adults, examples of dosages used in some embodiments are: a dosage of 0.01 to 1000 mg per day, a dosage of 0.5 to 100 mg per day, a dosage of 1 to 50 mg per day, and a dosage of 5 to 40 mg per day. An exemplary dosage is 10 to 30 mg per day. The exact dosage will depend on the route of administration, the form of the compound administered, the individual to be treated, the weight of the individual to be treated, and the preference and experience of the attending physician.

In some embodiments, one or more compounds are administered in a single dose. Typically, such administration is performed by injection (e.g., intravenous injection) to allow for rapid introduction of the agent. However, other routes may be used where appropriate. A single dose of a compound may also be used to treat acute conditions.

In some embodiments, one or more compounds are administered in multiple doses. In some embodiments, administration is about once a day, twice, three times, four times, five times, six times or more. In other embodiments, administration is about once a month, once every two weeks, once a week or once every other day. In another embodiment, the compounds are administered together from about once a day to about 6 times a day. In another embodiment, the administration of the compound continues for less than about 7 days. In another embodiment, administration continues for more than about 6 days, 10 days, 14 days, 28 days, 2 months, 6 months or 1 year. In some cases, continuous administration can be achieved and maintained as long as necessary.

Administration of the compound can continue as long as necessary. In some embodiments, the compound is administered for more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, or 28 days. In some embodiments, the compound is administered for less than 28 days, 14 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the compound is administered on an ongoing basis for a long period of time, for example, to treat a chronic effect.

In some embodiments, the compound is administered in a dose. Due to the inter-individual variability of compound pharmacokinetics, a personalized dosing regimen is provided in certain embodiments. According to the present disclosure, the administration of the compound can be found by routine experimentation, and/or the administration of the compound can be obtained by those of ordinary skill in the art.

In some embodiments, the compound is formulated as a pharmaceutical composition. In specific embodiments, the pharmaceutical composition is formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and adjuvants that facilitate processing of the active compound into a pharmaceutically usable formulation. The appropriate formulation depends on the selected route of administration. Any pharmaceutically acceptable technique, carrier, and excipient are suitably used to formulate the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, 19th ed. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed. (Lippincott Williams & Wilkins 1999).

As used herein, a pharmaceutical composition refers to a mixture of one or more compounds and other chemical components (such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and/or excipients). In certain embodiments, the pharmaceutical composition facilitates the administration of the compound to an organism. In some embodiments, the treatment or use methods provided herein are implemented, and the therapeutically effective amount of a cyclin-dependent kinase inhibitor and/or a DNA methyltransferase inhibitor provided herein is administered to a mammal suffering from a cancer, disease, disease or medical condition to be treated in the form of a pharmaceutical composition. In a specific embodiment, the mammal is a human. In certain embodiments, the therapeutically effective amount varies according to the severity of the cancer, the severity of the disease, the age and relative health of the individual, the efficacy of the compound used, and other factors. The compounds are used in combination with each other or as components of a mixture.

In one embodiment, the cyclin-dependent kinase inhibitor component and/or the DNA methyltransferase inhibitor component are formulated in an aqueous solution. In specific embodiments, the aqueous solution is selected from, by way of example only, a physiologically compatible buffer such as Hank's solution, Ringer's solution, or a physiological saline buffer. In other embodiments, the cyclin-dependent kinase inhibitor and/or the DNA methyltransferase inhibitor are formulated for transmucosal administration. In specific embodiments, the transmucosal formulation includes a penetrant appropriate to the barrier to be permeated. In other embodiments, where the compound is formulated for other parenteral injections, suitable formulations include aqueous or non-aqueous solutions. In specific embodiments, such solutions include physiologically compatible buffers and/or excipients.

In another embodiment, the compound is formulated for oral administration. The compound is formulated by combining the compound with, for example, a pharmaceutically acceptable carrier or excipient. In various embodiments, the compound is formulated into an oral dosage form, which includes, by way of example only, tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions, and the like.

In certain embodiments, the pharmaceutical preparation for oral use is obtained by the following steps: one or more solid excipients are mixed with one or more compounds, the resulting mixture is optionally ground, and the granular mixture is processed to obtain tablets or dragee cores after adding suitable adjuvants (if necessary). Suitable excipients are, in particular, fillers such as sugars (including lactose, sucrose, mannitol or sorbitol), cellulose preparations (such as: for example corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose), or other substances (such as: polyvinyl pyrrolidone (PVP or povidone) or calcium phosphate). In a specific embodiment, a disintegrant is optionally added. By way of example only, disintegrants include cross-linked croscarmellose sodium, polyvinyl pyrrolidone, agar or alginic acid or a salt thereof (such as sodium alginate).

In one embodiment, dosage form (such as dragee core and tablet) is provided with one or more applicable coatings.In a specific embodiment, concentrated sugar solution is used for dosage form coating.Sugar solution optionally contains other composition, such as only for example gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solution and applicable organic solvent or solvent mixture.Colorant and/or pigment also optionally join in the coating for identification purpose.In addition, colorant and/or pigment pigment are optionally used for characterizing the different combinations of compound dosage.

In certain embodiments, a therapeutically effective amount of the compound is formulated into other oral dosage forms. Oral dosage forms include push-fit capsules made of gelatin, and soft sealed capsules made of gelatin and a plasticizer (such as glycerol or sorbitol). In specific embodiments, the push-fit capsules contain the active ingredient blended with one or more fillers. By way of example only, fillers include lactose, binders (such as starch) and/or lubricants (such as talc or magnesium stearate), and optionally stabilizers. In other embodiments, soft capsules contain one or more active compounds dissolved or suspended in a suitable liquid. By way of example only, suitable liquids include one or more fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers are optionally added.

In other embodiments, a therapeutically effective amount of the compound is formulated for buccal or sublingual administration. By way of example only, formulations suitable for buccal or sublingual administration include tablets, lozenges, or gels. In other embodiments, the compound is formulated for parenteral injection, including formulations suitable for bolus injection or continuous infusion. In specific embodiments, the formulation for injection is presented in unit dosage form (such as in an ampoule) or in a multidose container. A preservative is optionally added to the injection formulation. In other embodiments, the pharmaceutical composition is formulated as a sterile suspension, solution, or emulsion in an oily or aqueous vehicle in a form suitable for parenteral injection. Parenteral injection formulations optionally contain preparatoners, such as suspending agents, stabilizers, and/or dispersants. In specific embodiments, pharmaceutical formulations for parenteral administration include aqueous solutions of the active compound in water-soluble form. In another embodiment, a suspension of the compound is prepared into an appropriate oily injection suspension. By way of example only, suitable lipophilic solvents or vehicles for use in the pharmaceutical compositions described herein include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate or triglycerides) or liposomes. In certain specific embodiments, aqueous injection suspensions include substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension includes a suitable stabilizer or an agent that increases the solubility of the compound to allow the preparation of a highly concentrated solution. Alternatively, in other embodiments, before use, the active ingredient is in a powder form for being constituted with a suitable vehicle (such as sterile pyrogen-free water).

In other embodiments, a therapeutically effective amount of the compound is administered topically. In some embodiments, the compound is formulated into various topically administrable compositions such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, or ointments. Such pharmaceutical compositions optionally contain solubilizers, stabilizers, tonicity enhancing agents, buffers, and preservatives.

In other embodiments, the compound of therapeutically effective amount is formulated for transdermal administration. In a specific embodiment, transdermal formulations adopt transdermal delivery devices and transdermal delivery patches, and can be lipophilic emulsions or buffered aqueous solutions, dissolved and/or dispersed in polymers or adhesives. In various embodiments, such patches are constructed for continuous, pulsatile or on-demand delivery of medicaments. In other embodiments, the transdermal delivery of the compound of therapeutically effective amount is achieved by means of iontophoresis patches, etc. In certain embodiments, transdermal patches provide controlled delivery of the compound of therapeutically effective amount as described herein. In a specific embodiment, the absorption rate is slowed down by using a rate-controlling membrane or by trapping the compound in a polymer matrix or gel. In an optional embodiment, absorption is increased using an absorption enhancer. Absorption enhancers or carriers include pharmaceutically acceptable solvents that help to absorb through the skin. For example, in one embodiment, the transdermal device is in the form of a bandage, the bandage comprising a backing member, a reservoir comprising a compound optionally having a carrier, a rate-controlling barrier that optionally delivers the compound to the host's skin at a controlled and predetermined rate over a long period of time, and an instrument for fixing the device to the skin.

In other embodiments, a therapeutically effective amount of the compound is formulated for administration by inhalation. Various forms suitable for administration by inhalation include, but are not limited to, aerosols, mists, or powders. A pharmaceutical composition containing a therapeutically effective amount of the compound is conveniently delivered in the form of an aerosol spray from a pressurized package or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In a specific embodiment, the dosage unit of the pressurized aerosol is determined by providing a valve that delivers a metered amount. In certain embodiments, capsules and cartridges such as, by way of example only, gelatin for use in an inhaler or insufflator are formulated to contain a powder mixture of the compound and a suitable powder base (such as lactose or starch).

In other embodiments, a therapeutically effective amount of the compound is formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, gel suppositories, or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides and synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. In suppository forms of the compositions, a low melting wax such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter, is first melted.

In certain embodiments, the pharmaceutical composition is formulated in any conventional manner using one or more physiologically acceptable carriers including excipients and adjuvants that facilitate the processing of the compound into a pharmaceutically useful formulation. The appropriate formulation depends on the chosen route of administration. Any pharmaceutically acceptable technique, carrier, and excipient are optionally used where appropriate. Pharmaceutical compositions comprising an inhibitor targeting at least two super enhancer components are manufactured in a conventional manner, such as, by way of example only, by conventional mixing processes, dissolution processes, granulation processes, dragee making processes, milling processes, emulsification processes, encapsulation processes, embedding processes, or compression processes.

The pharmaceutical composition includes at least one pharmaceutically acceptable carrier, diluent or excipient and one or more compounds as a therapeutically effective amount of an active ingredient. The active ingredient is in the form of a free acid or free base, or in the form of a pharmaceutically acceptable salt. In addition, the methods and pharmaceutical compositions described herein include the use of N-oxides, crystalline forms (also referred to as polymorphs) and active metabolites of these compounds with the same type of activity. All tautomers of the compound are included within the scope of the compound presented herein. In addition, the compound includes an unsolvated form and a solvated form with a pharmaceutically acceptable solvent (such as water, ethanol, etc.). The solvated form of the compound of a therapeutically effective amount is also considered to be disclosed herein. In addition, the pharmaceutical composition optionally includes other pharmaceutical agents or medicaments, carriers, adjuvants (such as preservatives, stabilizers, wetting agents or emulsifiers), solution promoters, salts for regulating osmotic pressure, buffers and/or other substances of therapeutic value.

Methods for preparing compositions comprising a therapeutically effective amount of a compound include formulating the compound with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semisolid, or liquid. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include solutions in which the compound is dissolved, emulsions containing the compound, or solutions containing liposomes, micelles, or nanoparticles containing a compound as disclosed herein. Semisolid compositions include, but are not limited to, gels, suspensions, and creams. The pharmaceutical compositions described herein may be in the form of liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to use, or as emulsions. These compositions may also optionally contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, and the like.

In some embodiments, pharmaceutical compositions comprising a therapeutically effective amount of a compound described herein illustratively take the form of a liquid wherein the agent is present as a solution, a suspension, or both. Typically, when the composition is administered as a solution or a suspension, a first portion of the agent is present in solution and a second portion of the agent is present as particles in suspension in a liquid matrix. In some embodiments, the liquid composition comprises a gel formulation. In other embodiments, the liquid composition is aqueous.

In certain embodiments, useful aqueous suspensions include one or more polymers as suspending agents. Useful polymers include water-soluble polymers (such as cellulosic polymers such as hydroxypropyl methylcellulose) and water-insoluble polymers (such as cross-linked carboxyl-containing polymers). Certain pharmaceutical compositions described herein include mucoadhesive polymers selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate, and dextran.

Optionally, useful pharmaceutical compositions also include a solubilizing agent to aid in the solubility of a therapeutically effective amount of a compound as described herein. The term "solubilizing agent" generally includes agents that result in the formation of micellar or true solutions of an agent. Certain acceptable nonionic surfactants, such as polysorbate 80, can be used as solubilizing agents, as can ophthalmologically acceptable glycols, polyglycols (e.g., polyethylene glycol 400), and glycol ethers.

In addition, useful pharmaceutical compositions optionally include one or more pH adjusting agents or buffers, including acids (such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid), bases (such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris), and buffers (such as citrate/dextrose, sodium bicarbonate, and ammonium chloride). Such acids, bases, and buffers are included in amounts required to maintain the pH of the composition within an acceptable range.

Other useful pharmaceutical compositions optionally include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances (such as phenylmercuric and thimerosal), stabilized chlorine dioxide, and quaternary ammonium compounds (such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride).

Other useful compositions include one or more surfactants to enhance physical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils (e.g., polyoxyethylene (60) hydrogenated castor oil) and polyoxyethylene alkyl ethers and alkylphenyl ethers (e.g., octoxynol 10, octoxynol 40).

Other useful compositions include one or more antioxidants to enhance chemical stability, where desired. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite.

In certain embodiments, aqueous suspension compositions are packaged in single-dose non-reclosable containers. Alternatively, multiple-dose reclosable containers are used, in which case a preservative is typically included in the composition.

In selectable embodiments, other delivery systems for hydrophobic pharmaceutical compounds are employed. Liposomes and emulsions are examples of delivery vehicles or carriers used herein. In certain embodiments, organic solvents such as N-methylpyrrolidone are also employed. In other embodiments, sustained-release systems (such as semipermeable matrices of solid hydrophobic polymers comprising therapeutic agents) are used to deliver the compounds described herein. Various sustained-release materials are useful herein. In some embodiments, sustained-release capsules release compounds for several weeks, up to 100 days or more. Depending on the chemical properties and biological stability of the therapeutic agent, other strategies for protein stabilization are employed.

In certain embodiments, the formulations described herein comprise one or more antioxidants, metal chelators, thiol-containing compounds, and/or other general stabilizers. Examples of such stabilizers include, but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparin analogs, (m) divalent cations (such as magnesium and zinc); or (n) combinations thereof.

In some embodiments, the concentration of one or more compounds provided in the pharmaceutical compositions of the present invention is less than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.1%, 0.2%, 0.3%, 0.4%, 0.1%, 0.09%, 0.08%, 0.07%, 0.1%, 0.2%, 0.3%, 0.4%, 0.2 ... 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of one or more compounds is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75%, 7.50%, 7. .25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 1.25%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0. 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of one or more compounds is in the range of about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, about 1% to about 10% w/w, w/v, or v/v.

In some embodiments, the concentration of one or more compounds is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v.

In some embodiments, the amount of one or more compounds is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0. 0.004g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

In some embodiments, the amount of one or more compounds is greater than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007g, 0.007 5g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0.08g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.45g, 0 .5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or 10g.

In some embodiments, the amount of one or more compounds is in the range of 0.0001-10 g, 0.0005-9 g, 0.001-8 g, 0.005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-4 g, or 1-3 g.

Example

Example 1

Avosidic acid reduces MCL-1 expression in a time- and dose-dependent manner

The AML cell line MV-4-11 expresses MCL-1. MCL-1 is a key anti-apoptotic protein in MV-4-11 cells. It has been well documented that avosidic therapy, a CDK9 inhibitor, reduces the expression of MCL-1. Avosidic therapy downregulates MCL-1 in a dose-dependent manner (Figure 1A). Dose-dependence was observed by monitoring the relative expression of MCL-1 compared to β-actin when cells were dosed with avosidic therapy at concentrations ranging from 0 to 8 μM. MV4-11 cells were treated with avosidic therapy in vitro for 16 hours. After treatment, cells were harvested and prepared for immunodetection of MCL-1 expression and β-actin expression using standard Western blotting techniques. A 0.1 μM dose of avosidic therapy reduced MCL-1 expression, while 1 μM avosidic therapy completely blocked expression at 16 hours.

Furthermore, avosidide also downregulated MCL-1 in a time-dependent manner (Figure 1B). The time dependence was observed by monitoring the relative expression of MCL-1 compared to β-actin over time. After administration of 80 nM avosidide, the relative expression of MCL-1 was measured at time points of 0.5–48 hours.

MV4-11 cells were treated in vitro with 80 nM avocidin for the indicated times. Following treatment, cells were harvested and prepared for immunodetection of MCL-1 and β-actin expression using standard Western blotting techniques. Avocidin reduced MCL-1 expression as early as 1 hour after treatment, with MCL-1 expression almost completely blocked by 16 hours. This inhibition of expression was maintained for up to 48 hours after treatment. Relative expression of MCL-1 demonstrates that avocidin reduces MCL-1 expression in MV4-11 cells.

Example 2

Treatment with a combination of avosidic and azacitidine and its effects on cell viability

Avocidin and azacitidine synergistically inhibited AML cell proliferation. Avocidin was used alone and in combination with azacitidine to determine the effects of the single agents and combinations on cell viability. Significant changes in cell viability were observed when the cells were treated with the combination of avocidin and azacitidine compared to when the cells were treated with azacitidine alone.

First, MV 4-11 cells were dosed with avosidide (80 nM) or DMSO for 24 hours, followed by a wash step. After the wash step, the cells were dosed again with azacitidine (30 μM to 0.001 μM) for 72 hours. Cell viability was measured 96 hours from the initial dose (72 hours after the wash step) and normalized to data collected for cells dosed with avosidide alone. Cell viability was then assessed in the Cell titer-glo assay according to the manufacturer's protocol. Cell viability was then plotted against concentration on a logarithmic scale (Figure 2). When normalized to avosidide treatment alone, the addition of 80 nM avosidide resulted in a left shift in the IC50 curve, indicating synergistic activity on the efficacy of 5-azacytidine when using this combination. The resulting EC50 for cells dosed with azacitidine alone was reduced from 1.779 μM, compared to the EC50 of 0.6408 μM for cells also dosed with avosidide.

These results provide evidence of increased activity (eg, synergy) of the combination of avosidic and azacitidine relative to the single agents.

Example 3

Effects of combined treatment on caspase 3/7 activity and cell apoptosis

Avocidin was used in combination with azacitidine to treat MV4-11 cells. The combination treatment was compared with azacitidine alone. This comparison was used to assess the synergistic effects of the combination on caspase 3/7 activity and apoptosis. Adding 80 nM avocidin to 5-azacytidine treatment resulted in a synergistic increase in caspase activity in MV4-11 cells.

First, MV 4-11 cells were dosed with avosidic for 24 hours, followed by a wash step. Afterward, the cells were dosed with azacitidine for 24 hours. Caspase 3/7 activity was measured 48 hours after the initial dose of avosidic (24 hours after the wash step and azacitidine administration). Normalized caspase 3/7 activity in cells treated with a combination of avosidic (FVP) and azacitidine was plotted against a range of azacitidine concentrations (see Figure 3). The dose of avosidic was kept constant (80 nM) across the azacitidine dosing range (0.3 μM–30 μM). Cell apoptosis was then assessed in a Caspase-glo assay according to the manufacturer's protocol.

To measure the synergistic effect of the avocidin and azacitidine combination, caspase 3/7 activity in untreated cells was plotted alongside cells treated with either single agent or the combination of avocidin and azacitidine. Cells treated with the single agents were treated with avocidin (80 nM) or azacitidine + DMSO (0.3 μM–30 μM) for 48 hours. Cells treated with the combination of avocidin and azacitidine were initially treated with avocidin (80 nM) for 24 hours. Cells were then washed and treated with azacitidine (0.3 μM–30 μM) for 24 hours. Figures 4A-4D provide the results for various concentrations of azacitidine. Comparison demonstrates that, at the tested concentrations of azacitidine and avocidin, the combination regimen resulted in a synergistic increase in caspase 3/7 activity/apoptosis compared to untreated cells or cells treated with either agent alone.

Example 4

Treatment with avosidic and decitabine combination and its effect on cell viability

Figures 5A and 5B present data from the combined treatment of avocidib and decitabine in MV4-11 cells. At the concentrations shown in Figure 5A, MV4-11 cells were first treated with avocidib + decitabine for 96 hours according to the general procedure of Example 2. A moderate decrease in IC50 (μM) was observed with the addition of avocidib. Alternatively, MV4-11 cells were pretreated with avocidib for 24 hours and then treated with decitabine for an additional 72 hours (96 hours total, Figure 5B). Here, when avocidib was administered before decitabine, a nearly 10-fold change in the IC50 (μM) of decitabine was observed with the addition of avocidib (100 nM).

All U.S. patents, specifically U.S. patent application No. 62/200,499, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety to the extent this specification is consistent with this specification.

From the foregoing it will be understood that although specific embodiments of the present invention have been described herein for illustrative purposes, various modifications can be made without departing from the spirit and scope of the present invention. Accordingly, the present invention is not to be restricted except as by the appended claims.

Claims (9)

1. The use of (a) a therapeutically effective amount of avozidil and (b) a therapeutically effective amount of a DNA methyltransferase inhibitor selected from azacitidine and decitabine in the preparation of a pharmaceutical combination, said pharmaceutical combination being used sequentially to treat acute myeloid leukemia in a subject with such need, wherein avozidil is administered to the subject prior to administration of the DNA methyltransferase inhibitor to the subject. 2. The use as described in claim 1, wherein the DNA methyltransferase inhibitor is decitabine. 3. The use as described in claim 1, wherein the DNA methyltransferase inhibitor is azacitidine. 4. The use as described in claim 1, wherein the DNA methyltransferase inhibitor is administered within 24-48 hours after administration of avodia syringe. 5. (a) the use of a therapeutically effective amount of avozepine or a pharmaceutically acceptable salt thereof, and (b) the use of a therapeutically effective amount of decitabine or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical combination, said pharmaceutical combination being used in sequence to treat acute myeloid leukemia in a subject in need, wherein avozepine or a pharmaceutically acceptable salt thereof is administered to the subject prior to administration of decitabine or a pharmaceutically acceptable salt thereof to the subject. 6. The use as claimed in claim 5, wherein the decitabine or a pharmaceutically acceptable salt thereof is administered within 24-48 hours after administration of avozidine or a pharmaceutically acceptable salt thereof. 7. (a) the use of a therapeutically effective amount of avozidine or a pharmaceutically acceptable salt thereof, and (b) the use of a therapeutically effective amount of azacitidine or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical combination, said pharmaceutical combination being used sequentially to treat acute myeloid leukemia in a subject in need, wherein avozidine or a pharmaceutically acceptable salt thereof is administered to the subject prior to administration of azacitidine or a pharmaceutically acceptable salt thereof to the subject. 8. The use as claimed in claim 7, wherein the azacitidine or a pharmaceutically acceptable salt thereof is administered within 24-48 hours after administration of avozidil or a pharmaceutically acceptable salt thereof. 9. The use as claimed in any one of claims 1-8, wherein the object is a human being.