阅读说明：本技术 组蛋白去乙酰化酶抑制剂与蛋白激酶抑制剂之组合及其制药用途 (Combination of histone deacetylase inhibitor and protein kinase inhibitor and pharmaceutical application thereof ) 是由 鲁先平 宁志强 周游 辛利军 王雅楠 王世刚 潘德思 山松 于 2018-08-17 设计创作，主要内容包括：本发明涉及组蛋白去乙酰化酶抑制剂与蛋白激酶抑制剂之组合在制备用于治疗或预防肿瘤的药物中的用途,本发明还涉及包含组蛋白去乙酰化酶抑制剂和蛋白激酶抑制剂作为活性成分的药物组合物以及联用组蛋白去乙酰化酶抑制剂和蛋白激酶抑制剂治疗或预防癌症的方法。(The present invention relates to the use of a combination of a histone deacetylase inhibitor and a protein kinase inhibitor for the preparation of a medicament for the treatment or prevention of tumors, a pharmaceutical composition comprising the histone deacetylase inhibitor and the protein kinase inhibitor as active ingredients, and a method for treating or preventing cancer using the histone deacetylase inhibitor and the protein kinase inhibitor in combination.)

1. Use of a combination of a histone deacetylase inhibitor and a protein kinase inhibitor for the preparation of a medicament for the treatment or prevention of a tumour.

2. The use of claim 1, wherein the protein kinase inhibitor is selected from the group consisting of a serine inhibitor, a threonine kinase inhibitor, and a tyrosine kinase inhibitor.

3. The use according to claim 1 or 2, wherein the protein deacetylase inhibitor is cidalimine or a pharmaceutically acceptable salt thereof.

4. The use according to any one of claims 1 to 3, wherein the protein kinase inhibitor is seolonide or a pharmaceutically acceptable salt thereof.

5. The use according to any one of claims 1 to 4, wherein the protein deacetylase inhibitor is cidaplamide and the kinase inhibitor is seolonide.

6. A pharmaceutical composition comprising as pharmaceutically active ingredients a histone deacetylase inhibitor and a protein kinase inhibitor, and optionally a pharmaceutically acceptable excipient and/or carrier.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutically active ingredient consists of seoroni and cidaproamine.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable excipients and/or carriers comprise povidone, copovidone, hydroxypropylmethylcellulose and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer.

9. The pharmaceutical composition according to claim 7 or 8, wherein the content of xiprole is 5-100mg and the content of xioloni is 5-100mg, preferably 5-60mg and 10-100mg, in unit dose.

10. The pharmaceutical composition according to any one of claims 7 to 9, in the form of granules, solid dispersions, capsules or tablets.

11. The pharmaceutical composition according to claim 10, comprising a pharmaceutically acceptable excipient and/or carrier selected from the group consisting of: microcrystalline cellulose, povidone, copovidone, lactose, mannitol, crospovidone, and sodium carboxymethylcellulose.

12. A kit for treating or preventing cancer comprising as active ingredients a histone deacetylase inhibitor and a protein kinase inhibitor for simultaneous or sequential administration.

13. The kit according to claim 12, comprising as active ingredients xidalbenamine or a pharmaceutically acceptable salt thereof and xioroni or a pharmaceutically acceptable salt thereof for simultaneous or sequential administration.

14. The kit according to claim 13, comprising as active ingredients a first administered xidalbenamine or a pharmaceutically acceptable salt thereof and a second administered xioroni or a pharmaceutically acceptable salt thereof.

Technical Field

The invention relates to the technical field of micromolecule targeted anti-tumor medicines, in particular to application of a combination of a histone deacetylase inhibitor and a kinase inhibitor in preparing a medicine for treating or preventing tumors, and a pharmaceutical composition containing the histone deacetylase inhibitor and the kinase inhibitor as active ingredients of the medicine. The present invention particularly relates to a pharmaceutical composition comprising seoroni or a pharmaceutically acceptable salt thereof and cidentamine or a pharmaceutically acceptable salt thereof as active ingredients and a method for treating or preventing cancer by combining seoroni or a pharmaceutically acceptable salt thereof and cidentamine or a pharmaceutically acceptable salt thereof.

Background

Tumors are a serious disease threatening human health, and the treatment of tumors has been of great concern all over the world. Traditional chemotherapeutic drugs non-specifically block cell division to cause cell death, killing tumor cells while destroying normal cells of the body. In addition, many cytotoxic drugs have a limited therapeutic range, are prone to cause adverse reactions, and cause drug resistance problems after long-term administration.

Disclosure of Invention

The inventor unexpectedly finds out in the research that: the synergistic anti-tumor effect can be realized by combining a histone deacetylase inhibitor and a protein kinase inhibitor.

To this end, in a first aspect, the present invention provides the use of a combination of a histone deacetylase inhibitor and a protein kinase inhibitor for the preparation of a medicament for the treatment or prevention of a tumour.

In another aspect, the present invention provides a method for treating or preventing a tumor, comprising administering a histone deacetylase inhibitor and a protein kinase inhibitor to a subject in need thereof.

Protein kinases are a family of enzymes that catalyze the phosphorylation of specific residues in proteins, broadly classified as tyrosine and serine/threonine kinases, and represent a large family of proteins that play an important role in the regulation of a variety of cellular processes and in the maintenance of cellular function. Protein kinases are enzymatic components of signal transduction pathways that catalyze the transfer of the terminal phosphate of ATP to the hydroxyl group of tyrosine, serine, and/or threonine residues of proteins. Overexpression or inappropriate expression of normal or mutant protein kinases in mammals has been the subject of extensive research and has been shown to play a significant role in the development of many diseases, including cancer. A partial non-limiting list of these kinases includes: non-receptor tyrosine kinases, such as the Janus kinase family (Jak1, Jak2, Jak3 and Tyk 2); receptor tyrosine kinases such as platelet derived growth factor receptor kinase (PDGFR); and serine/threonine kinases such as b-RAF. Abnormal kinase activity is observed in a number of disease states, including benign and malignant proliferative disorders as well as diseases resulting from inappropriate activation of the immune and nervous systems.

Protein kinases, a large family of structurally related enzymes, are responsible for controlling a variety of signal transduction processes in cells (see, e.g., Hardie and Hanks, The Protein Kinase enzymes Book, I and II, Academic Press, san diego, calif., 1995). Protein kinases are thought to have evolved from common ancestral genes due to conservation of their structure and catalytic function. Almost all kinases contain a similar catalytic domain with 250-300 amino acids. Kinases can be classified into families according to their phosphorylation receptors (e.g., protein-tyrosine, protein-serine/threonine, lipids, etc.). Sequence motifs that generally correspond to each of these families have been identified (see, e.g., Hanks and Hunter, (1995), FASEB J.9: 576-.

Inappropriate kinase activity due to mutations, over-expression or inappropriate regulation, dysregulation or dysregulation, and over-or under-production of growth factors or cytokines can be implicated in a number of diseases including, but not limited to, a variety of diseases such as cancer. Protein kinases have become an important class of enzymes that are targets for therapeutic intervention. In particular, over-activation of the tyrosine kinase cKit is associated with hematologic malignancies and is a target for cancer therapy (Heinrich, Griffith et al, Blood2000,96(3): 925-32). Similarly, JAK3 signaling is implicated in leukemias and lymphomas and is currently used as a potential therapeutic target (Heinrich, Griffith et al, 2000)). Protein kinases also play a central role in cell cycle regulation. It has been found that defects in various components of the signal transduction pathway can cause a variety of diseases, including various forms of cancer (Gaestel et al, Current medical Chemistry, (2007)14: 2214-. In recent years, protein kinases involved in oncogenic signaling pathways have become important drug targets in the treatment of a variety of diseases, including many types of cancer. Various protein kinase inhibitors are also used as antitumor agents.

In the present invention, preferably, the protein kinase inhibitor is selected from the group consisting of serine inhibitors, threonine kinase inhibitors and tyrosine kinase inhibitors. Preferably, the protein kinase inhibitor is seoronib or a pharmaceutically acceptable salt thereof.

In the present invention, preferably, the histone deacetylase inhibitor is cidapolamine or a pharmaceutically acceptable salt thereof.

In a particularly preferred aspect of the invention, the histone deacetylase inhibitor is cidamide or a pharmaceutically acceptable salt thereof, and the kinase inhibitor is seolonib or a pharmaceutically acceptable salt thereof. Particularly preferably, the histone deacetylase inhibitor is cidalimine and the kinase inhibitor is seoronide.

In another aspect of the present invention, a pharmaceutical composition is provided, which comprises a histone deacetylase inhibitor and a protein kinase inhibitor as pharmaceutically active ingredients, and optionally pharmaceutically acceptable excipients and/or carriers.

In the pharmaceutical composition of the present invention, preferably, the protein kinase inhibitor is selected from the group consisting of serine inhibitors, threonine kinase inhibitors and tyrosine kinase inhibitors. Particularly preferably, the protein kinase inhibitor is seoroni or a pharmaceutically acceptable salt thereof. Particularly preferably, the pharmaceutically active ingredient consists of seoroni or a pharmaceutically acceptable salt thereof and cidapolamine or a pharmaceutically acceptable salt thereof. In a particular embodiment, the pharmaceutically active ingredient consists of seoroni and cidaproamine.

In a particularly preferred embodiment of the invention, in the pharmaceutical composition, the content of xidapipramine is 5-100mg, the content of xiolonide is 5-100mg, in unit dose; more preferably, the content of the xidapipramine is 5-60mg, and the content of the xioroni is 10-100 mg.

In some embodiments of the invention, in the pharmaceutical composition, the pharmaceutically acceptable excipients and/or carriers include povidone, copovidone, hydroxypropylmethylcellulose and polyvinylcaprolactam-polyvinylacetate-polyethylene glycol graft copolymer (e.g. under the trade name Soluplus); in other embodiments, the pharmaceutically acceptable excipients and/or carriers include microcrystalline cellulose, povidone, copovidone, lactose, mannitol, crospovidone, and sodium carboxymethylcellulose.

The pharmaceutical composition of the present invention is preferably in the form of granules, solid dispersions, capsules or tablets.

In still another aspect of the present invention, there is provided a kit for treating or preventing cancer or tumor, comprising a histone deacetylase inhibitor and a protein kinase inhibitor, which are administered simultaneously or sequentially as active ingredients.

In the kit of the present invention, it is preferable to contain, as active ingredients, xidapamide or a pharmaceutically acceptable salt thereof and xioroni or a pharmaceutically acceptable salt thereof, for simultaneous or sequential administration. In a particularly preferred aspect, the kit of the invention comprises as active ingredients a first administration of xidanbenamine or a pharmaceutically acceptable salt thereof and a second administration of xioroni or a pharmaceutically acceptable salt thereof.

In another aspect of the present invention, there is provided a method for preventing or treating tumor, comprising administering a histone deacetylase inhibitor and a protein kinase inhibitor to a subject in need thereof simultaneously or sequentially.

The invention unexpectedly discovers that the combination of the histone deacetylase inhibitor and the protein kinase inhibitor has obvious synergistic antitumor effect, which is shown in that the combination of the histone deacetylase inhibitor and the protein kinase inhibitor can synergistically induce the apoptosis of cancer cells and synergistically inhibit the clone formation of the cancer cells, and the synergistic antitumor effect is proved in a nude mouse test. The inventor also unexpectedly finds that the pretreatment by using the histone deacetylase inhibitor can enhance the sensitivity of cells to the protein kinase inhibitor and more effectively enhance the anti-tumor effect of the protein kinase inhibitor.

The invention discovers that the combined administration of a histone deacetylase inhibitor and a protein kinase inhibitor can synergistically induce the cycle inhibition and apoptosis of hepatoma cell strains Bel-7404 and Bel-7402 through MTS, clone formation, flow cell cycle detection, Caspase 3/7 and other experiments. Meanwhile, the synergistic antitumor effect of the combination of the histone deacetylase inhibitor and the protein kinase inhibitor is verified on a Bel-7404 nude mouse transplantation tumor model.

Drawings

FIG. 1: the antitumor effect of the histone deacetylase inhibitor (xidapamide) synergistically enhanced protein kinase inhibitor (xiolonide);

FIG. 2: crystal violet staining shows that the combination of histone deacetylase inhibitor and protein kinase inhibitor can synergistically inhibit the clonogenic capacity of tumor cells;

FIG. 3: flow cytometry detection of PI staining shows that the combination of histone deacetylase inhibitor and protein kinase inhibitor can enhance the inhibition of tumor cell cycle;

FIG. 4: caspase 3/7 enzyme activity detection shows that the combination of histone deacetylase inhibitor and protein kinase inhibitor can enhance the induction of tumor apoptosis;

FIG. 5: sequential administration experiments show that the tumor inhibition effect of a protein kinase inhibitor (seolomide) can be enhanced by the pretreatment of a histone deacetylase inhibitor (sidapamide);

FIG. 6: synergistic antitumor effect of combined administration of histone deacetylase inhibitor (sidapamide) and protein kinase inhibitor (seoroni) in nude mouse animal experiment.

Detailed Description

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "includes," including, "" has, "" contains, "or variants thereof are used in the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term" comprising.

The terms "treatment," "alleviating," and "improving," as used herein, are used interchangeably. These terms refer to a method for obtaining a beneficial or intended effect, including but not limited to a therapeutic benefit and/or a prophylactic benefit.

The term "anti-tumor" as used herein refers to the treatment, alleviation or amelioration of a "neoplastic condition". The term "neoplastic condition" refers to the presence of cells with abnormal growth characteristics such as uncontrolled proliferation, unlimited proliferation, metastatic potential, rapid growth and proliferation rate, disorganized oncogenic signaling, and certain characteristic morphological characteristics. This includes, but is not limited to, the growth of the following cells: (1) benign or malignant cells (e.g., tumor cells) associated with overexpression of histone deacetylase, tyrosine, or serine/threonine kinases; (2) benign or malignant cells (e.g., tumor cells) associated with abnormally high levels of histone deacetylase, tyrosine, or serine/threonine kinase activity.

It will be appreciated by the skilled person that in the pharmaceutical combination or pharmaceutical composition of the invention the pharmaceutically active ingredient is used in an effective or therapeutically effective amount. The term "effective amount" or "therapeutically effective amount" refers to an amount of an inhibitor described herein sufficient to effect the intended use (including but not limited to treatment of disease) as defined below. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the mode of administration, etc., which can be readily determined by one of ordinary skill in the art. The term also applies to doses that will induce a particular response (e.g., a reduction in proliferation or down-regulation of target protein activity) in the target cells. The specific dosage will vary depending upon the particular compound selected, the dosage regimen to be followed, whether or not it is administered in combination with other compounds, the timing of administration, the tissue to be administered, and the physical delivery system in which it is carried.

"synergistic" or "synergistic effect" means that when used in combination with an effective amount of another pharmaceutically active ingredient or therapy, it produces a better effect than when the two pharmaceutically active ingredients are used alone. In some embodiments, synergistically effective therapeutic amounts of the pharmaceutically active ingredients or therapies produce better effects when used in combination than the additive effects of each of the two pharmaceutically active ingredients or therapies used alone.

The term "inhibitor" refers to a compound that has the ability to inhibit the biological function of a target protein by inhibiting the activity or expression of the target protein. The biological activity of the inhibitor is associated with the development, growth or spread of tumors or undesirable immune responses manifested in autoimmune diseases.

The terms "co-administration", "co-administration" and grammatical equivalents thereof include administration of two or more pharmaceutically active ingredients to a subject such that the two pharmaceutically active ingredients and/or their metabolites are present simultaneously within the animal. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present. The pharmaceutically active ingredients to be co-administered may be present in the same formulation, in different formulations, and also in the same product, for example as a kit.

"therapeutic effect" as used herein includes a therapeutic benefit and/or a prophylactic benefit as described above. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, stopping, or reversing the progression of a disease or condition, or any combination thereof.

The term "pharmaceutically acceptable salts" refers to salts derived from a variety of organic and inorganic counterions well known in the art. Pharmaceutically acceptable salts include pharmaceutically acceptable acid addition salts and base addition salts. Acid addition salts may be formed using inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed using inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine, among others. In some embodiments, the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts.

"pharmaceutically acceptable excipients and/or carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents in pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions of the invention is contemplated. Supplementary active ingredients may also be incorporated into the composition.

In the present invention, the tumor includes the following diseases or conditions: breast cancer; ovarian cancer; uterine cancer; cervical cancer; prostate cancer; bladder cancer; leukemias, including Acute Myeloid Leukemia (AML), acute lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, myelodysplasia, myeloproliferative disorders, Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), mastocytosis, Chronic Lymphocytic Leukemia (CLL), Multiple Myeloma (MM), and myelodysplastic syndrome (MDS); bone cancer; lung cancer; skin cancer, including basal cell carcinoma, melanoma, squamous cell carcinoma; retinoblastoma of the eye; cutaneous or intraocular (ocular) melanoma; primary liver cancer; kidney cancer; thyroid cancer; AIDS-associated lymphomas such as diffuse large B-cell lymphoma, B-cell immunoblastic lymphoma and small anaplastic lymphoma; kaposi's sarcoma; central nervous system cancers, such as primary brain tumors, including gliomas; peripheral nervous system cancers including neurofibromas and schwannoma, malignant fibrous cell tumor, malignant fibrous histiocytoma, malignant meningioma, malignant mesothelioma, and malignant mixed miller tumor; oral and oropharyngeal cancer; gastric cancer; testicular cancer; thymus gland cancer; rectal cancer and colon cancer.

The combination therapy according to the invention is effective over a wide dosage range. For example, in the treatment of adults, the daily dose of the histone deacetylase inhibitor and the protein kinase inhibitor is 1 to 100mg, preferably 5 to 100mg, respectively. In a specific embodiment, a combination of xidanamine and xioroni is used, wherein the daily dose of xidanamine is 5 to 60mg and the daily dose of xioroni is 10 to 100 mg. The exact dosage will depend upon the pharmaceutical active ingredient selected, the route of administration, the form of the compound administered, the subject to be treated, the weight of the subject to be treated and the preferences and experience of the attending physician and can be readily determined by one of skill in the art.

The pharmaceutical composition of the invention can also be used together with other pharmaceutically active ingredients with anti-tumor activity; accordingly, the pharmaceutical composition or kit of the present invention may further comprise other pharmaceutically active ingredients having anti-tumor activity.

In some embodiments, the pharmaceutical composition may be a pharmaceutical composition suitable for oral administration, e.g., in the form of granules, capsules, or tablets, and the like. Pharmaceutical compositions of the invention suitable for oral administration may be in discrete dosage forms, such as capsules or tablets, or liquids or sprays, each containing a predetermined amount of the active ingredient, as powders or in granules, solutions, or suspensions in aqueous or non-aqueous liquids, oil-in-water emulsions or water-in-oil liquid emulsions, including liquid dosage forms (e.g. suspensions or slurries) and oral solid dosage forms (e.g. tablets or bulk powders). The term "tablet" as used herein generally refers to tablets, caplets, capsules (including soft gelatin capsules) and lozenges. Oral dosage forms may be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by the individual or patient to be treated. Such dosage forms may be prepared by any pharmaceutical method. Suitable excipients may be fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired form. For example, tablets may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with excipients such as, but not limited to, binders, lubricants, inert diluents, and/or surfactants or dispersants. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed as the carrier, in the case of oral liquid preparations (e.g., suspensions, solutions, and elixirs) or aerosols, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like; or in the case of oral solid preparations, in some embodiments where lactose is not used, carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents may be used. For example, suitable carriers for solid oral formulations include powders, capsules and tablets. Tablets may be coated, if desired, by standard aqueous or non-aqueous techniques.

The composition may further comprise one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, but are not limited to, detackifiers, antifoams, buffers, polymers, antioxidants, preservatives, chelating agents, viscosity modifiers, tonicity modifiers, flavoring agents, coloring agents, flavoring agents, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.

Experimental part

Experimental materials:

human hepatoma cell strains Bel-7402 and Bel-7404 were obtained from cell resource center of Shanghai Life science research institute of Chinese academy of sciences at 37 deg.C and 5% CO₂Culturing under conventional conditions, wherein the culture solution is RPMI-1640(Gibco) containing 10% Fetal bovine serum (FBS; Gibco) and 1% Penicilin-streptomycin (HyClone); trypsin (Trypsin) was purchased from Gibco. Crystal violet, RNase A (10mg/mL) solution, propidium iodide (Propidi)um Iodide, PI), Triton X-100 from Biotechnology engineering (Shanghai) Inc.; MTS cell activity assay kit, Caspase-Glo3/7 assay kit was purchased from Promega. Nude mice were purchased from the medical laboratory animal center in Guangdong province.