CN115887664A - Pharmaceutical composition with anti-colorectal cancer effect and application thereof - Google Patents

Abstract

The invention relates to a medicinal composition with an anti-colorectal cancer effect and application thereof, belonging to the field of medicines. The present invention relates to a pharmaceutical combination comprising (a) isorhamnetin and (b) an autophagy inhibitor; pharmaceutical compositions, kits thereof, use of the pharmaceutical combinations, pharmaceutical compositions and kits in the treatment of proliferative diseases; and methods of treating an individual having a proliferative disease comprising administering the pharmaceutical combination.

Description

Pharmaceutical composition with anti-colorectal cancer effect and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a medicinal composition with an anti-colorectal cancer effect and application thereof. In particular, the present invention relates to a pharmaceutical combination comprising (a) isorhamnetin and (b) an autophagy inhibitor; pharmaceutical compositions, kits thereof, use of said pharmaceutical combinations, pharmaceutical compositions and kits in the treatment of proliferative diseases; and methods of treating an individual having a proliferative disease comprising administering the pharmaceutical combination.

Background

Colorectal cancer is one of the common malignant tumors at present, and seriously threatens human health. Surgery, chemotherapy, targeted therapy and immunotherapy are the main treatments at present. However, the survival rate of metastatic colorectal cancer is still not high at present. Therefore, the need for new effective, safe and low-cost anti-colorectal cancer drugs is urgent.

During tumorigenesis and development, autophagy is associated with apoptosis. Studies show that autophagy can mediate tumor cells to generate multidrug resistance, and inhibition of autophagy can enhance the sensitivity of cells to drugs resisting colorectal cancer. In clinic, radiotherapy and chemotherapy drugs are often combined with autophagy inhibitors to treat colorectal cancer. 3-methyladenine (3-methyladenine, 3-MA) is a commonly used early autophagy inhibitor that prevents the formation of autophagic vesicles and autophagosomes by inhibiting PI3K activity. It has been reported that 3-MA inhibits the growth of a variety of tumor cells.

Isorhamnetin (ISO) is a flavonoid compound widely existing in vegetables, fruits, traditional Chinese medicines and the like. Modern pharmacological research shows that isorhamnetin has the activities of resisting inflammation, resisting oxidation, resisting arrhythmia and resisting tumor. It is found that isorhamnetin can induce autophagy in colorectal cancer cells. No report that isorhamnetin is used together with an autophagy inhibitor for treating colorectal cancer exists.

Disclosure of Invention

In view of the above technical problems, the present invention relates to a pharmaceutical composition comprising: (1) Isorhamnetin or a derivative thereof, and (2) an autophagy inhibitor. In one embodiment, the present invention also provides a pharmaceutical combination comprising: (1) Isorhamnetin or a derivative thereof, and (2) an autophagy inhibitor for use in treating a proliferative disease in a subject in need thereof. In one embodiment, the present invention also provides a pharmaceutical combination, wherein: (1) Isorhamnetin or a derivative thereof, and (2) an autophagy inhibitor, are administered simultaneously, separately or sequentially.

In one aspect, the invention also relates to a pharmaceutical composition comprising (a) isorhamnetin and (b) an autophagy inhibitor, and (c) one or more pharmaceutically acceptable carriers.

In another aspect, the invention also relates to a kit comprising (a) isorhamnetin and (b) an autophagy inhibitor; and optionally (c) instructions for use.

In another aspect, the invention also relates to the use of a combination of isorhamnetin or a derivative thereof and an autophagy inhibiting agent for the preparation of a medicament for the treatment of a proliferative disease.

The isorhamnetin and the autophagy inhibitor in the pharmaceutical composition have obvious synergistic effect and good colorectal cancer resistance effect.

Drawings

FIG. 1 effects of ISO/3-MA combination on cell survival in human colorectal cancer HCT116 (A) and SW620 (B). Compared with the blank control group, the composition of the composition, ^** P<0.01; in comparison with the ISO/3-MA combination, ^# P<0.05, ^## P<0.01；&&，CDI<0.7, significant synergistic effect.

FIG. 2, effect of ISO/CQ combination on human colorectal cancer HCT116 cell survival. Compared with the blank control group, the composition of the composition, ^** P<0.01; in comparison with the ISO/3-MA combination, ^## P<0.01；&&，CDI<0.7, significant synergistic effect.

FIG. 3 effects of ISO/3-MA combination on apoptosis in human colorectal cancer HCT116 and SW620 cells. Compared with the blank control group, the composition of the composition, ^** P<0.01； ^## P<0.01。

FIG. 4 shows the effect of ISO/3-MA combination on tumor growth in nude mice bearing human colorectal cancer HCT116 cells subcutaneously. Compared with the blank control group, the composition of the composition, ^* P<0.05， ^** P<0.01； ^# P<0.05， ^## P<0.01。&，CDI<1, has synergistic effect.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete.

The specific experimental procedures not indicated in the following examples are generally performed according to conventional methods, such as Sambrook et al, molecular cloning: the method described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, but in the event of conflict, the definitions set forth herein shall control.

As used in the specification and in the claims, the singular form of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

All numbers or expressions referring to quantities of ingredients, process conditions, etc. used in the specification and claims are to be understood as modified in all instances by the term "about". The term "about" when referring to a quantity or a numerical range means that the quantity or numerical range so referred to is an approximation within experimental variability (or within statistical experimental error). In the present invention, the term "about" shall have a meaning within 10%, preferably within 5%, of the specified value or range.

All ranges directed to the same component or property are inclusive of the endpoints, and independently combinable. Because these ranges are continuous, they include every value between the minimum and maximum values. It should also be understood that any numerical range recited herein is intended to include all sub-ranges within that range.

When the present invention is defined by ranges for physical properties such as molecular weight or for chemical properties, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term "comprising" (and related terms such as "comprising" or "including" or "having" or "including") includes embodiments that are, for example, any combination of materials, compositions, methods, or processes that "consist of or" consist essentially of the recited features.

As used in this specification and claims, "and/or" should be understood to mean "either or both" of the associated components, i.e., the components are present in combination in some cases and are present separately in other cases. A plurality of components listed with "and/or" should be understood in the same way, i.e., "one or more" of the associated component. In addition to the "and/or" clause-specific components, other components may optionally be present, whether related or unrelated to those specifically identified components. Thus, by way of non-limiting example, reference to "a and/or B," when used in connection with an open ended word such as "comprising," may refer in one embodiment to a alone (optionally including components other than B); in another embodiment, reference may be made to B alone (optionally including components other than a); in yet another embodiment, refers to a and B (optionally including other components), and the like.

In the invention, isorhamnetin (3, 5, 7-trihydroxy-2- (4-hydroxy-3-methoxyphenyl) benzopyran-4-ketone) is a flavonoid compound existing in vegetables, fruits, traditional Chinese medicines, medicines containing the compound, health products, functional foods and the like. The isorhamnetin has the following chemical structure:

the "isorhamnetin derivative" in the invention can be selected from: isorhamnetin-3-O-rhamnogalactide, isorhamnetin-3-O-glucoside, isorhamnetin-3-O-rhamnoglucoside, isorhamnetin-3-O-arabinoglucoside, isorhamnetin-3-O-glucosylglucoside, isorhamnetin-3-O-galactoside and isorhamnetin-7-O-rhamnoside.

The term "synergistic" or "synergy" as used herein refers to a therapeutic combination that is more effective than the additive effects of two or more individual agents. The determination of a synergistic interaction between isorhamnetin or a derivative thereof and one or more autophagy inhibitors may be based on the effect obtained from the assay described herein. Synergy is achieved when the active ingredients are: (1) Co-formulated in a combined unit dosage formulation and administered or delivered simultaneously; (2) delivery as separate formulations, alternately or in parallel; (3) by some other medication regimen. When delivered in alternating therapy, a synergistic effect may be achieved when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes. Typically, during alternation therapy, an effective dose of each active ingredient is administered sequentially, i.e., consecutively, whereas in combination therapy, an effective dose of two or more active ingredients is administered together.

As used herein, "autophagy inhibitor" means a substance that reduces the level of autophagy in a cell that undergoes autophagy in its presence as compared to the level of autophagy in a cell that undergoes autophagy in its absence. Autophagy is a catabolic process of massive solvent degradation and recycling of cytoplasmic material and organelles, characterized by the appearance of autophagic vacuoles in the cytoplasm, which result in the autodigestion of cytoplasmic organelles and other constituents in lysosomal compartments. Although autophagy can allow for critical cell killing when allowed to reach its limits, autophagy can provide a temporary survival mechanism for cells under stress conditions, but can also make cells vulnerable to several forms of cell death under specific circumstances. Depending on the conditions and agents used, inhibition of Autophagy may either promote or inhibit Cell death (Amaravadri, R.K., D.Yu, J.J.Lum, T.Bui, M.A.Christophorou, G.I.Evan, A.Thomas-Tikhonenko, and C.B.Thompson, (2007), autophagyi inhibition processes thermal-induced Apoptosis in a Myc-induced model of lymphoma. J.Clin invest.117:326-336 oemer, G.and M.Jaattela.2005. Lysogenes and Autophagy in Cell death control. Nat Rev cancer.885: 6-97 Levine, B.2005 and J.Ysosomen. Cell and Autophagy in Cell, apokinase, 36. J.26. Cell, cell J.2619, cell J.26. And 2679. Autophagy is a catabolic process with different stages. These phases include an induction phase, a sequestration phase (sequestration), a fusion phase and a degradation phase. An autophagy inhibiting agent may inhibit one or more of the stages. In some embodiments, the autophagy inhibiting agent can inhibit a later stage of autophagy. In some embodiments, the autophagy inhibitor can inhibit the sequestration, fusion, and degradation phases of autophagy. In some embodiments, the autophagy inhibitor can inhibit the fusion phase and degradation phase of autophagy. In some embodiments, the autophagy inhibitor can inhibit the degradation phase of autophagy. Useful autophagy inhibitors include siRNA; antisense RNA; an agent that inhibits the expression or function of LAMP2, LAMP1, or an autophagy (Atg) gene (e.g., atg1, atg4, atg8, atg5, atg7, or Atg 12); 3-methyladenine (3-MA), wortmannin; an anti-proteasome agent (which may also be an anti-parasitic agent), such as chloroquine, hydroxychloroquine (hydroxychloroquine), or suramin (suramin); vacuolar proton-atpase inhibitors, such as bafilomycin A1 (bafilomycin A1); agents acting on the circulatory system, such as Amiodarone (Amiodarone) or perhexiline (Perhexilene), cytotoxic agents, such as Vinblastine (Vinblastine), agents which influence lipid metabolism, antibiotics, such as monensin, or hormones, such as glucagon or estradiol; anti-lysosome agents, such as ammonium chloride, cAMP or methylamine; an ATPase inhibitor; a protease inhibitor; lysosomal protease inhibitors such as cathepsin inhibitors and cathepsin knock-down (cathepsin knock-down), as well as LAMP knock-down, such as LAMP1 and LAMP2.

As used herein, the term "combination drug" or "drug combination" and related terms refer to the simultaneous or sequential administration of a drug or therapeutic agent of the present invention.

The term "pharmaceutically effective amount" or "clinically effective amount" or "therapeutically effective amount" refers to an amount sufficient to provide an observable improvement in the signs and symptoms observed at clinical baseline of the disorder.

The term "administration" or "administering" as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the subject, cell, tissue, organ, or biological fluid.

The term "treating" as used herein includes treatment that alleviates, alleviates or reduces at least one symptom of the individual or affects the delay in progression of the disease. For example, the treatment may be to eliminate one or several symptoms of the disorder or to completely eradicate the disorder (e.g., cancer). Within the meaning of the present invention, the term "treatment" also means preventing, delaying onset (i.e. the time before clinical manifestation of the disease occurs) and/or reducing the risk of development or worsening of the disease.

The "subject" or "individual in need" to be administered includes, but is not limited to: a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, a child, an adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an older adult)) and/or a non-human animal, e.g., a mammal, e.g., a primate (e.g., a cynomolgus monkey, a rhesus monkey), a cow, a pig, a horse, a sheep, a goat, a rodent, a cat, and/or a dog. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human", "patient", "individual in need thereof" and "subject" are used interchangeably herein.

The term "proliferative disorder" refers to a disorder associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.

"tumor" as used herein refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous (pre-cancerous) and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, colorectal cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial diffuse melanoma, lentigo melanoma, and combinations thereof acromelanoma, nodular melanoma, multiple myeloma and B cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL), small Lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-nucleated NHL, storage disease (bulkydisease) NHL, mantle cell lymphoma, AIDS related lymphoma, and Waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, brain tumors, and brain cancers, and head and neck cancer, and associated metastases. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include breast cancer, colorectal cancer, large bowel cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma (carcinoid sarcoma), head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from: large bowel cancer, small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Also, in some embodiments, the cancer is selected from: large bowel cancer, non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer, including metastatic forms of those cancers.

"disease," "disorder," and "condition" are used interchangeably herein.

As used herein, unless otherwise specified, the term "treatment" includes the effect that occurs when a subject has a particular disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or delays or slows the progression of the disease, disorder or condition ("therapeutic treatment"), and also includes the effect that occurs before the subject begins to have the particular disease, disorder or condition ("prophylactic treatment").

As used herein, "pharmaceutical composition" includes both bulk compositions and individual dosage units comprising more than one (e.g., two) pharmaceutically active agent including isorhamnetin or a derivative thereof and one or more autophagy inhibiting agents, as well as any pharmaceutically inactive excipients, diluents, carriers, or glidants. The bulk composition and individual dosage units may contain a fixed amount of the pharmaceutically active agent described above. The bulk composition is a substance that has not yet been formed into individual dosage units. The dosage units used as an illustration are oral dosage units such as tablets, pills, capsules and the like. Similarly, the methods of treating a patient by administering a pharmaceutical composition of the invention described herein are also intended to include administration of both the bulk composition and the individual dosage units.

The term "pharmaceutically acceptable" indicates that the substance or composition is chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the subject being treated therewith.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In one aspect, the present invention provides a pharmaceutical combination (also referred to as a "combination drug") comprising isorhamnetin or a derivative thereof and one or more autophagy inhibitors.

In some embodiments, the present invention also provides a pharmaceutical combination comprising (a) isorhamnetin

And

(b) An autophagy inhibiting agent.

In some embodiments, the present invention also provides a pharmaceutical combination consisting of isorhamnetin and an autophagy inhibitor.

In some embodiments, the above pharmaceutical combination is for use in treating a proliferative disease in an individual in need thereof.

In some embodiments, the isorhamnetin and the autophagy inhibitor in the above pharmaceutical combination are administered simultaneously, separately or sequentially.

In some embodiments, the autophagy inhibiting agent in the above pharmaceutical combination is selected from the group consisting of: pan-PI3K inhibitors, anti-proteasome agents, vacuolar proton-atpase inhibitors, protease inhibitors, and lysosomal protease inhibitors; preferably, the autophagy inhibitor is selected from the group consisting of: 3-methyladenine, baveromycetin A1, wortmannin, MRT67307, lys05, chloroquine, hydroxychloroquine, suramin, amiodarone, perhexiline, vinblastine and combinations thereof; more preferably, the autophagy inhibiting agent is selected from the group consisting of chloroquine, 3-methyladenine and combinations thereof.

In some embodiments, the molar ratio of isorhamnetin to autophagy inhibitor in the above pharmaceutical combination ranges from about 1; preferably, the molar ratio is 1.2, 1; preferably, the molar ratio is 1.2, 1. More preferably, the molar ratio is 1.2, 1.

In some embodiments, the above pharmaceutical combination wherein the isorhamnetin is present at about 0.1mg/kg to 5.5mg/kg (subject body weight); preferably 0.1mg/kg to 5mg/kg, 0.1mg/kg to 4mg/kg, 0.1mg/kg to 3mg/kg, 0.1mg/kg to 2mg/kg, 0.1mg/kg to 1.1mg/kg; more preferably 1.1 mg/kg.

In some embodiments, the autophagy inhibitor in the above pharmaceutical combination is administered at about 0.055mg/kg to 5.5mg/kg (subject body weight); preferably 0.055 to 5mg/kg, 0.055 to 4mg/kg, 0.055 to 3mg/kg, 0.055 to 2mg/kg, 0.055 to 1mg/kg, 0.055 to 0.11mg/kg; more preferably 0.11 mg/kg.

In some embodiments, the unit dose of the isorhamnetin in the above pharmaceutical combination is about 6mg to 165mg, preferably 10mg to 150mg, 20mg to 140mg, 30mg to 130mg, 40mg to 120mg, 50mg to 110mg, 60mg to 100mg, 60mg to 90mg, 60mg to 80mg, 60mg to 70mg; more preferably 60mg to 70mg.

In some embodiments, the unit dose of the autophagy inhibiting agent in the above pharmaceutical combination is about 3.3mg to 165mg, preferably 3.3mg to 150mg, preferably 3.3mg to 140mg, preferably 3.3mg to 130mg, preferably 3.3mg to 120mg, preferably 3.3mg to 110mg, preferably 3.3mg to 100mg, preferably 3.3mg to 90mg, preferably 3.3mg to 80mg, preferably 3.3mg to 70mg, preferably 3.3mg to 60mg, preferably 3.3mg to 50mg, preferably 3.3mg to 40mg, preferably 3.3mg to 30mg, preferably 3.3mg to 20mg, preferably 3.3mg to 10mg, preferably 3.3mg to 6.6mg; more preferably 3.3mg to 6.6mg.

In some embodiments, the isorhamnetin and autophagy inhibitor described above can be administered in a particular dose at multiple intervals, e.g., 2 times per day, 1 time per day, 6 times per week, 5 times per week, or less. Preferably, the isorhamnetin is administered 1-2 times per day and the autophagy inhibiting agent is administered 1-2 times per day.

In some embodiments, the isorhamnetin and autophagy inhibitor described above can be administered by the same route of administration or by different routes of administration. In some embodiments, isorhamnetin is administered by inhalation, intranasal, intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, oral administration, preferably intravenous infusion. In some embodiments, the autophagy inhibitor is administered by inhalation, intranasally, intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, orally, preferably by intravenous infusion.

In some embodiments, the proliferative disease in the above pharmaceutical combination is a cancer, in particular, the cancer is a solid tumor or a hematological cancer, such as large intestine cancer, non-small cell lung cancer, renal cell cancer, colorectal cancer, colon cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, merkel cell carcinoma, and other hematological malignancies, such as Classical Hodgkin's Lymphoma (CHL), primary mediastinal large B cell lymphoma, T-cell/histiocytic B cell lymphoma, and EBV-related Diffuse Large B Cell Lymphoma (DLBCL), plasmacytoma, extranodal NK/T cell lymphoma, nasopharyngeal cancer, and HHV 8-related primary effusion lymphoma, hodgkin's lymphoma, central nervous system tumors (CNS), such as primary central nervous system tumors, spinal axis tumors, spinal cord axis tumors; preferably, the cancer is selected from the group consisting of large intestine, melanoma, kidney, prostate, breast, colon, lung, bone, pancreas, skin, head or neck, cutaneous or intraocular malignant melanoma, uterine, ovarian and rectal cancer; more preferably, the cancer is selected from the group consisting of colorectal cancer, colorectal cancer and colon cancer.

In some embodiments, the above pharmaceutical composition comprises (a) isorhamnetin

And

(b) An autophagy inhibitor, wherein the autophagy inhibitor is chloroquine.

In some embodiments, the above pharmaceutical composition comprises (a) isorhamnetin

And

(b) An autophagy inhibitor, wherein the autophagy inhibitor is 3-methyladenine.

In one aspect, the present invention provides a pharmaceutical composition comprising (a) isorhamnetin or a derivative thereof and (b) an autophagy inhibitor as described herein above, and optionally (c) one or more pharmaceutically acceptable carriers, diluents or excipients.

In some embodiments, the pharmaceutical composition comprises (a) isorhamnetin and (b) an autophagy inhibitor, and optionally (c) one or more pharmaceutically acceptable carriers, diluents, or excipients.

In other embodiments, the pharmaceutical composition comprises (a) isorhamnetin and (b) an autophagy inhibitor, and optionally (c) one or more pharmaceutically acceptable carriers, diluents, or excipients, wherein the autophagy inhibitor is chloroquine.

In other embodiments, the pharmaceutical composition comprises (a) isorhamnetin and (b) an autophagy inhibitor, and optionally (c) one or more pharmaceutically acceptable carriers, diluents, or excipients, wherein the autophagy inhibitor is 3-methyladenine.

Suitable carriers, diluents and excipients are well known to those skilled in the art and include substances such as carbohydrates, waxes, water-soluble and/or swellable polymers, hydrophilic or hydrophobic substances, gelatin, oils, solvents, water and the like. The particular carrier, diluent or excipient employed will depend upon the mode and objective in which the compounds of the present invention will be useful. The solvent is generally selected based on a solvent (GRAS) deemed safe for administration to a mammal by one skilled in the art. Generally, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG400, PEG 300), and the like, and mixtures thereof. The formulations may also include one or more buffering agents, stabilizing agents, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, fragrances, flavors, and other known additives to provide an elegant appearance of the drug (i.e., a compound of the present invention or a pharmaceutical composition thereof) or to aid in the manufacture of the drug (i.e., medicament).

The pharmaceutical compositions may be prepared using conventional dissolution and mixing procedures. For example, the bulk drug substance (i.e., a stable form of the active substance in the composition of the invention (e.g., a complex with a cyclodextrin derivative or other known complexing agent)) is dissolved in a suitable solvent in the presence of one or more of the excipients described above. The compounds of the present invention are typically formulated in pharmaceutical dosage forms to provide easily controllable dosages of the drug and to enable patient compliance with prescribed regimens.

The pharmaceutical composition (or formulation) for administration may be packaged in a variety of ways depending on the method used to administer the drug. Typically, the article for dispensing comprises a container in which a pharmaceutical formulation in a suitable form has been placed. Suitable containers are well known to those skilled in the art and include substances such as bottles (plastic or glass), sachets, ampoules, plastic bags, metal cylinders and the like. The container may also include tamper-proof means to prevent inadvertent access to the contents of the package. In addition to this, the container has placed thereon a label describing the contents of the container. The tag may also include an appropriate warning.

The pharmaceutical composition is preferably sterile. In particular, formulations to be used for in vivo administration must be sterile. Such sterilization is readily accomplished by filtration through sterile filtration membranes.

The pharmaceutical compositions can generally be stored as solid formulations, lyophilized formulations or as aqueous solutions.

The pharmaceutical compositions of the present invention will be formulated and administered in a manner consistent with good medical practice (i.e., amounts, concentrations, schedules, procedures, vehicles, and routes of administration). Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to medical practitioners.

Acceptable diluents, carriers, excipients, and stabilizers are nontoxic to subjects at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, phenylethylamine chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbonsA hydrate comprising glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zn-protein complexes); and/or nonionic surfactants such as TWEEN ^TM 、PLURONICS ^TM Or polyethylene glycol (PEG). The active pharmaceutical ingredient may also be entrapped in the prepared microcapsules, for example hydroxymethylcellulose or gelatin microcapsules and poly- (methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, for example by coacervation techniques or by interfacial polymerization. These techniques are disclosed in Remington's Pharmaceutical Sciences 18 th edition, (1995) Mack publication.

The pharmaceutical composition of the present invention may be prepared as a sustained release formulation. Suitable examples of sustained-release preparations include semipermeable matrices of pharmaceutical compositions of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels [ e.g., poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol) ], polylactides (US 3773919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON deep ^TM (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D (-) 3-hydroxybutyrate.

The dosage form used in the present invention includes, for example, tablets, capsules, granules, suspensions, oral liquids, injections, or ointments. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are commonly found in Remington's Pharmaceutical Sciences 18 th edition (1995) Mack Publishing co., easton, PA. These methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. Typically the formulation is prepared by the following method: 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.

The pharmaceutical compositions of the present invention may be prepared as discrete units such as pills, hard or soft capsules, e.g., gelatin capsules, cachets, lozenges, troches, aqueous or oily suspensions, dispersible powders or granules, emulsions, syrups or elixirs. These pharmaceutical combinations or pharmaceutical compositions may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide a palatable preparation. Compressed tablets may be prepared by the following method: the active ingredient in free-flowing form (e.g., powder or granules), optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersant, is compressed in a suitable machine. Molded tablets may be prepared by the following method: the mixture of powdered active ingredient moistened with an inert liquid diluent is moulded in a suitable machine. The tablets may optionally be coated or scored and optionally formulated so as to provide slow or controlled release of the active ingredient therefrom.

Tablet excipients described in the present invention may include: a filler (or diluent) to increase the volume capacity of the powdered drug constituting the tablet; a disintegrant which, when ingested, promotes the breaking up of the tablet into small fragments, ideally individual drug particles, and promotes rapid dissolution and absorption of the drug; a binder to ensure that granules and tablets can be formed with the desired mechanical strength and to hold the tablets together after they have been compressed to prevent them from breaking into their component powders during packaging, shipping and daily handling; glidants to improve the flowability of the powders making up the tablet during production; lubricants to ensure that the tableting powders do not adhere to the equipment used for tableting during manufacture. They improve the flow of the powder mixture through the press and minimize friction and breakage when the finished tablet is discharged from the apparatus; antiadherents, having functions similar to those of glidants, which reduce adhesion between the powder constituting the tablet and the machine used to punch the tablet shape during manufacture; flavors incorporated into the tablets to impart a more pleasant taste thereto or to mask an unpleasant taste; and colorants to aid in identification and patient compliance.

Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques, including microencapsulation, to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

Excipients suitable for the manufacture of aqueous suspensions described herein include suspending agents, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; and dispersing or wetting agents such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., sorbitan polyoxyethylene ether monooleate). The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose and saccharin.

The pharmaceutical compositions of the present invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. The suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol or as a solution prepared from a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils (sterile fixed oils) may be employed as a solvent or suspending medium. Any bland fixed oil may also be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Pharmaceutical combinations or compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

Pharmaceutical compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoring agent, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

The pharmaceutical compositions of the present invention may be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water, immediately prior to use for injections. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose of the active ingredient, or an appropriate fraction thereof, as detailed herein above.

The pharmaceutical compositions of the present invention may be administered by any route suitable for the condition being treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, inhalation, intradermal, intrathecal, epidural and infusion techniques), transdermal, rectal, nasal, topical (including buccal and sublingual), vaginal, intraperitoneal, intrapulmonary and intranasal. Topical administration may also involve the use of transdermal administration, such as transdermal patches or iontophoresis devices. Pharmaceutical formulations are discussed in Remington's Pharmaceutical Sciences, 18 th edition (1995) Mack Publishing co. Examples of other Pharmaceutical formulations can also be found in Liberman, h.a. and Lachman, l., eds., pharmaceutical Dosage Forms, marcel Decker, volume 3, second edition, new York, NY.

In some embodiments, the invention also relates to a kit comprising (a) isorhamnetin or a derivative thereof and (b) an autophagy inhibitor, optionally, the kit may further comprise a package insert. In other embodiments, the above kit comprises (a) isorhamnetin or a derivative thereof and (b) an autophagy inhibitor, optionally, the kit may further comprise a package insert, wherein the autophagy inhibitor is chloroquine. In other embodiments, the above-described kit comprises (a) isorhamnetin or a derivative thereof and (b) an autophagy inhibitor, optionally, the kit may further comprise instructions for a pharmaceutical product, wherein the autophagy inhibitor is 3-methyladenine.

The term "package insert" is used to refer to instructions customarily included in commercial packages of pharmaceutical products, that contain information about the indications, usage, dosage, method of administration, contraindications and/or warnings concerning the use of such pharmaceutical products. Suitable containers include, for example, bottles, vials, syringes, blister packs and the like. The container may be formed from a variety of materials such as glass or plastic. The container may contain a pharmaceutical composition or pharmaceutical formulation of the invention and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). The instructions indicate that the medicament is for treating a selected condition, such as colorectal cancer. The kit may also comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial or user standpoint, including other buffers, diluents, filters, injection needles and syringes.

In some embodiments, the kit may further comprise a container for holding separate active components of the composition, such as a separate bottle or a separate foil package; however, the active components of the separate compositions may also be contained in a single, undivided container. The kit comprises directions for administration of the separate active ingredients. The kit form is particularly advantageous when the separate active ingredients are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when the prescribing physician desires a titration of the individual components of the composition.

In some embodiments, the present invention relates to the use of the above-described pharmaceutical combination for the preparation of a medicament for the treatment of a proliferative disease.

In some embodiments, the present invention relates to the use of the above pharmaceutical composition for the preparation of a medicament for the treatment of a proliferative disease.

In some embodiments, the present invention relates to the use of the above-described kit for the preparation of a medicament for the treatment of a proliferative disease.

In some embodiments, the present invention relates to a method of treating a proliferative disease comprising administering to a subject in need thereof the above pharmaceutical combination, pharmaceutical composition or kit.

In some embodiments, the proliferative disease is colorectal cancer.

In another aspect, the present invention also relates to the following embodiments:

embodiment 1. A pharmaceutical combination comprising:

(a) Isorhamnetin

And

(b) 3-methyladenine.

Embodiment 2. A pharmaceutical composition comprising (a) isorhamnetin and (b) 3-methyladenine, and (c) one or more pharmaceutically acceptable carriers.

Embodiment 3. A kit comprising (a) isorhamnetin and (b) 3-methyladenine; and optionally (c) instructions for use.

Embodiment 4. The pharmaceutical combination, pharmaceutical composition or kit of any one of embodiments 1 to 3, wherein the molar ratio of isorhamnetin to 3-methyladenine is about 1.

Embodiment 5. The pharmaceutical combination, pharmaceutical composition or kit of any one of embodiments 1 to 4, wherein the unit dose of isorhamnetin is from about 60mg to about 70mg, preferably 66mg; the unit dose of 3-methyladenine is preferably 3.3mg to 6.6mg, preferably 6.6mg.

Embodiment 6 use of the pharmaceutical combination, pharmaceutical composition or kit of any one of embodiments 1 to 5 in the manufacture of a medicament for the treatment of a proliferative disease.

Embodiment 7 the use of embodiment 6 wherein the proliferative disease is colorectal cancer.

Embodiment 8. A pharmaceutical combination comprising:

(a) Isorhamnetin

And

(b) And (3) chloroquine.

Embodiment 9 a pharmaceutical composition comprising (a) isorhamnetin and (b) chloroquine, and (c) one or more pharmaceutically acceptable carriers.

Embodiment 10. A kit comprising (a) isorhamnetin and (b) chloroquine; and optionally (c) instructions for use.

Embodiment 11 the pharmaceutical combination, pharmaceutical composition or kit of any one of embodiments 8 to 10, wherein the molar ratio of isorhamnetin to chloroquine is in the range of about 1.

Embodiment 12 use of the pharmaceutical combination, pharmaceutical composition or kit of any one of embodiments 8 to 11 in the manufacture of a medicament for the treatment of a proliferative disease.

Embodiment 13 the use of embodiment 12 wherein the proliferative disease is colorectal cancer.

The present invention will be further described with reference to specific examples so that those skilled in the art may better understand the present invention, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above-described contents of the present invention belong to the scope of the present invention.

Example 1

In this example, the MTT method was used to evaluate the effect of the isorhamnetin/3-methyladenine (ISO/3-MA) combination on the survival rate of colon cancer cells. 3-MA (3-methyladenine ) is an autophagy inhibitor.

Human colorectal cancer HCT116 and SW620 cells were cultured in DMEM medium containing 1% of twin antibiotics (penicillin/streptomycin) and 5% of fetal bovine serum at 37 ℃ with 5% of CO ₂ Culturing in a cell culture box.

The above cells were digested with trypsin and then cultured in complete medium to give cells in an amount of 5X 10 cells/ml ⁴ Perml of the suspension, inoculated into a 96-well plate at 100. Mu.L per well, charged at 37 ℃ and 5% CO ₂ Culturing in a cell culture incubator for 24 hours, discarding the cell culture solution, adding complete medium containing 0.1% DMSO to the blank control group, and adding 50 μ M ISO, 5mM 3-MA and 50 μ M/5mM ISO/3-MA combination to the administration group of HCT116, respectively; the SW620 dosing group was added with 50. Mu.M ISO, 5mM 3-MA and 50. Mu.M/5 mM ISO/3-MA combination, respectively. Each concentration was provided with 3 multiple wells. After 48 hours of incubation, 10 μ L of MTT solution (5 mg/mL, PBS solution preparation) is added into each well, after 2 hours of culture, the supernatant is absorbed by a pipette, then 100 μ L of DMSO is respectively added into each well, the mixture is shaken on a shaker in the dark until crystals are completely dissolved, finally, the absorbance value of each well is measured by referring to 630nm at the wavelength of 570nm under an ultraviolet spectrophotometer, and the survival rate of each group of cells is calculated. See FIG. 1 for results.

As shown in FIG. 1, ISO,3-MA, and ISO/3-MA combinations all reduced colorectal cancer cell survival (. About.P) compared to placebo<0.05，**P<0.01)。ISO/3-MAThe effect of the combination on reducing the cell survival rate is significantly stronger than that of the ISO and 3-MA single group ( ^## P<0.01). ISO/3-MA can synergistically inhibit proliferation of HCT116 and SW620 cells in colorectal cancer&&，CDI<0.7; the synergistic effect is remarkable). The CDI (coefficient of drug interaction) is calculated by the following formula: CDI = AB/(a × B). Wherein AB is the survival rate of colorectal cancer cells of the two-medicine combination group compared with the survival rate of the control group; a or B is the survival rate of colon cancer cells of the group with each drug alone compared with the survival rate of colon cancer cells of the control group. When CDI<At 0.7, the synergistic effect is remarkable.

Example 2

In this example, the MTT method was used to evaluate the effect of isorhamnetin/chloroquine (ISO/CQ) combinations on the survival rate of colorectal cancer cells. CQ (Chloroquine) is an autophagy inhibitor.

The culture system of human colorectal cancer HCT116 cells adopts DMEM medium containing 1% double antibiotics (penicillin/streptomycin) and 5% fetal bovine serum, and the content of CO is 5% at 37 DEG C ₂ Culturing in a cell culture box.

The above cells were digested with trypsin and then cultured in complete medium to give cells in an amount of 5X 10 cells/ml ⁴ Perml of the suspension, inoculated into a 96-well plate at 100. Mu.L per well, charged at 37 ℃ and 5% CO ₂ After culturing for 24 hours in the cell incubator, the cell culture solution was discarded, the blank control group was added with complete medium containing 0.1% DMSO, and the administration group was added with 50. Mu.M ISO, 50. Mu.M CQ, and 50. Mu.M/50. Mu.M ISO/CQ combination, respectively. 3 multiple wells were set for each concentration. After 48 hours of incubation, 10 μ L of MTT solution (5 mg/mL, PBS solution preparation) is added into each well, after 2 hours of culture, the supernatant is absorbed by a pipette, then 100 μ L of DMSO is respectively added into each well, the mixture is shaken on a shaker in the dark until crystals are completely dissolved, finally, the absorbance value of each well is measured by referring to 630nm at the wavelength of 570nm under an ultraviolet spectrophotometer, and the survival rate of each group of cells is calculated. See figure 2 for results.

As shown in FIG. 2, ISO, CQ, and ISO/CQ combinations all reduced colorectal cancer cell survival (. About.P) compared to placebo<0.01). The effect of the ISO/CQ combination on reducing cell viability is stronger than that of the ISO and CQ single group ( ^## P<0.01, compared to the ISO/CQ group). The ISO/CQ combination can synergistically inhibit the proliferation of HCT116 cells of colorectal cancer: (&&，CDI<0.7; the synergistic effect is remarkable).

Example 3

To determine whether the reduced cell survival rate of the ISO/3-MA combination is related to apoptosis, the present example investigated the effect of the ISO/3-MA combination on apoptosis in colorectal cancer cells by flow cytometry.

The culture system of human colorectal cancer HCT116 and SW620 cells was characterized by using DMEM medium containing 1% of bis-antibiotic (penicillin/streptomycin) and 5% of fetal bovine serum at 37 ℃ in 5% CO ₂ Culturing in a cell culture box.

The cells are digested with trypsin and then cultured in complete culture medium to obtain cells with a cell content of 5 × 10/ml ⁵ The suspension/mL was inoculated into a 6-well plate at 2mL per well, charged at 37 ℃ with 5% CO ₂ After 24 hours of culture in the cell incubator, the cell culture solution was discarded, the complete medium containing 0.1% DMSO was added to the blank control group, and 50. Mu.M of ISO, 5mM of 3-MA and 50. Mu.M/5 mM of ISO/3-MA combination were added to the administration group, respectively. After 48 hours, digesting with trypsin, centrifuging at 1000rpm/min for 5min, washing with PBS buffer for 2 times, collecting cells, centrifuging at 1000rpm/min for 5min, discarding the supernatant, adding 100. Mu.L of 1 × binding buffer, adding 5. Mu.L of Annexin V-FITC (phospholipid binding protein labeled with fluorescent probe FITC having green fluorescence), adding 5. Mu.L of PI (propidium iodide), mixing, keeping out of the sun at room temperature for 15min, adding 400. Mu.L of 1 × binding buffer, and detecting on a flow cytometer within 1 hour. See figure 3 for results.

As shown in FIG. 3, the apoptosis rate of ISO/3-MA group was significantly higher than that of ISO or 3-MA administered group alone ( ^## P<0.01 ISO and 3-MA are suggested to have synergistic tumor cell killing effects.

Example 4

This example establishes a human colorectal cancer HCT116 xenograft tumor model to evaluate the in vivo anti-colorectal cancer effect of the ISO/3-MA combination.

Male BALB/c-nu/nu nude mice (about 8 weeks in size) weighed about 24g and were provided by the animal testing center, university of Chinese, hong Kong. In a constant-temperature constant-pressure sterile environment of the experimental animal center of hong Kong university of Diploc, a human colorectal cancer HCT116 xenograft tumor model is established after 1 week of free drinking water intake to evaluate the in-vivo anti-colorectal cancer curative effect of the ISO/3-MA combination.

Taking HCT116 cells according to the ratio of 4X 10 ⁶ 0.1 mL/mouse was inoculated subcutaneously on the back. One week later, mice were randomly divided into 4 groups of 5 mice by tumor size, administered by intraperitoneal injection for two weeks. The blank control group was dosed with equal amounts of solvent (30% PEG400,5 % Tween 80,5% dimethyl sulfoxide, 60% PBS); the single dosage of ISO is 10mg/kg/d,6 times per week and 0.1 mL/mouse; the single dosage of the 3-MA is 1mg/kg/d,6 times per week and 0.1 mL/piece; the dosage of the ISO/3-MA combination is 10mg/kg/d of ISO +1mg/kg/d of 3-MA,6 times per week. After the start of administration, the tumor volume (length. Times. Width. Times. Height, mm) was measured every 2 to 3 days ³ ) And animal body weights (g) were recorded. Two weeks later the animals were euthanized, stripped of solid tumors, weighed (g) and recorded by photography. The results are shown in FIG. 4.

As shown in FIG. 4, ISO/3-MA inhibited the growth of colorectal cancer in nude mice; the tumor weight and volume of the ISO/3-MA combination administration group are significantly smaller than those of the blank control group: ( ^* P<0.05， ^** P<0.01 And ISO or 3-MA administration group ( ^# P<0.05， ^## P<0.01 ); ISO/3-MA synergistically reduces tumor weight in mice&，CDI<1, the two drugs have synergistic effect) and reduce the tumor volume: (&，CDI<1) The function of (1). Animals in all treatment groups did not show significant weight loss or behavioral abnormalities.

The dosage administered in ISO mice in the examples is 10mg/kg, with possible doses ranging from 1mg/kg to 50mg/kg, and an effective dose range converted to 60kg for adults ranging from about 6mg to 330mg per day. In the examples, the dose of 3-MA administered to mice is 1mg/kg, the possible dose ranges are 0.5mg/kg to 50mg/kg, and the effective dose converted to a 60kg adult ranges from about 3.3mg to 330mg per day. The results of the animal experiments in example 4 suggest that the injection of isorhamnetin and 3-methyladenine of about 66 mg/day and 6.6 mg/day into a 60kg adult can effectively inhibit the growth of colorectal cancer cells in vivo. The specific drug dosage and the administration scheme need to be adjusted according to the clinical test results. The pharmaceutical composition of the present invention should contain 6mg-165mg ISO,3.3mg-165mg 3-MA per unit dose, and the treatment regimen is 1-2 doses per day.

Various technical features of the above embodiments may be combined arbitrarily, and for brevity, all possible combinations of the technical features in the above embodiments are not described. The combination of these features should be considered as the scope of the present specification unless there is any contradiction.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (12)

1. A pharmaceutical combination comprising:

(a) Isorhamnetin

And

(b) An autophagy inhibitor.

2. The pharmaceutical combination of claim 1 for use in the treatment of a proliferative disease in an individual in need thereof.

3. The pharmaceutical combination of any one of claims 1-2, wherein the isorhamnetin and the autophagy inhibitor are administered simultaneously, separately or sequentially.

4. A pharmaceutical composition comprising (a) isorhamnetin and (b) an autophagy inhibitor and (c) one or more pharmaceutically acceptable carriers.

5. A kit comprising (a) isorhamnetin and (b) an autophagy inhibitor; and optionally (c) instructions for use.

6. Use of a pharmaceutical combination, pharmaceutical composition or kit according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of a proliferative disease.

7. The pharmaceutical combination, pharmaceutical composition, kit or use of any one of claims 1-6, wherein the autophagy inhibitor is selected from the group consisting of: pan-PI3K inhibitors, anti-proteasome agents, vacuolar proton-atpase inhibitors, protease inhibitors, and lysosomal protease inhibitors; preferably, the autophagy inhibitor is selected from the group consisting of 3-methyladenine, baverromycin A1, wortmannin, MRT67307, lys05, chloroquine, hydroxychloroquine, suramin, amiodarone, perhexiline, vinblastine and combinations thereof; more preferably, the autophagy inhibiting agent is selected from the group consisting of chloroquine, 3-methyladenine and combinations thereof.

8. The pharmaceutical combination, pharmaceutical composition, kit or use of any one of claims 1-7, wherein the autophagy inhibitor is chloroquine or 3-methyladenine.

9. The pharmaceutical combination, pharmaceutical composition, kit or use according to any one of claims 1 to 8, wherein the molar ratio of isorhamnetin to autophagy inhibiting agent is in the range of from about 1.02 to about 1; preferably, the molar ratio is 1.

10. The pharmaceutical combination, pharmaceutical composition, kit or use according to any one of claims 1 to 9, wherein the unit dose of isorhamnetin is from about 6mg to about 165mg, preferably from 60mg to 70mg; the unit dose of the autophagy inhibiting agent is from about 3.3mg to about 165mg, preferably from 3.3mg to 6.6mg.

11. The pharmaceutical combination, pharmaceutical composition, kit or use according to any one of claims 1 to 10, wherein the isorhamnetin is administered by inhalation, intranasal, intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, oral administration, preferably intravenous infusion; the autophagy inhibitor is administered by inhalation, intranasally, intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, orally, preferably by intravenous infusion.

12. The pharmaceutical combination, pharmaceutical composition, kit or use according to any one of claims 1 to 11, wherein the proliferative disease is a cancer, in particular, a solid or hematological cancer, such as large intestine cancer, non-small cell lung cancer, renal cell cancer, colorectal cancer, colon cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, merkel cell carcinoma and other hematological malignancies, such as Classical Hodgkin Lymphoma (CHL), primary mediastinal large B cell lymphoma, T-cell/histiocytic abundant B cell lymphoma, and EBV-related Diffuse Large B Cell Lymphoma (DLBCL), plasmacytoma, extranodal NK/T cell lymphoma, cancer and HHV 8-related primary effusion lymphoma, hodgkin lymphoma, central nervous system tumor (CNS), such as primary spinal cord lymphoma, spinal cord axis glioma; preferably, the cancer is selected from the group consisting of large intestine, melanoma, kidney, prostate, breast, colon, lung, bone, pancreas, skin, head or neck, cutaneous or intraocular malignant melanoma, uterine, ovarian and rectal cancer; more preferably, the cancer is selected from the group consisting of colorectal cancer, colorectal cancer and colon cancer.

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