WO2022127788A1 - 乐伐替尼和Aurora-A激酶抑制剂在制备抑制癌症的药物中的应用 - Google Patents

乐伐替尼和Aurora-A激酶抑制剂在制备抑制癌症的药物中的应用 Download PDF

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WO2022127788A1
WO2022127788A1 PCT/CN2021/137957 CN2021137957W WO2022127788A1 WO 2022127788 A1 WO2022127788 A1 WO 2022127788A1 CN 2021137957 W CN2021137957 W CN 2021137957W WO 2022127788 A1 WO2022127788 A1 WO 2022127788A1
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aurora
kinase
lenvatinib
cancer
gene
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French (fr)
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覃文新
金浩杰
王思颖
郑幸玲
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上海市肿瘤研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention belongs to the field of oncology, and more particularly, the present invention relates to the application of lenvatinib and Aurora-A kinase inhibitor in the preparation of drugs for inhibiting cancer.
  • Cancer is a major disease that seriously affects human health, and its mortality has always occupied the first place in the mortality rate of human diseases, threatening the physical and mental health of patients to a great extent. Although the level of cancer diagnosis and treatment has been getting higher and higher in recent years, a lot of research work is still needed to further improve the survival rate of patients and the living standard after the disease.
  • Hepatocellular carcinoma is one of the most important causes of cancer death worldwide, and China is a country with a high incidence of HCC. It is estimated that more than 500,000 new cases of liver cancer are diagnosed worldwide each year. At the same time, in developed countries and regions such as the United States, Canada and Europe, the incidence of liver cancer is increasing year by year.
  • Lenvatinib (Lenvatinib, also translated as: lenvatinib) is a first-line treatment drug approved in recent years for patients with advanced HCC.
  • Lenvatinib is an orally active inhibitor of multiple receptor tyrosine kinases (VEGFR 1-3, FGFR 1-4, PDGFRa, RET and KIT).
  • VEGFR 1-3, FGFR 1-4, PDGFRa, RET and KIT multiple receptor tyrosine kinases
  • OS 13.6 vs 12.3 months
  • sorafenib in the first-line treatment of patients with advanced HCC, becoming one of the treatment options for advanced HCC. an effective alternative treatment.
  • the objective response rate of Lenvatinib is only about 18.8%, which is far from meeting the clinical needs.
  • the purpose of the present invention is to provide the application of lenvatinib and Aurora-A kinase inhibitor in the preparation of drugs for inhibiting cancer.
  • an Aurora-A kinase inhibitor and lenvatinib for the preparation of a mixture, a pharmaceutical composition or a kit for inhibiting cancer.
  • the inhibiting cancer includes: preventing, relieving and/or treating cancer.
  • the cancer includes liver cancer, kidney cancer or thyroid cancer; preferably liver cancer.
  • the liver cancer includes advanced liver cancer.
  • the liver cancer includes hepatocellular carcinoma.
  • the Aurora-A kinase inhibitor includes: a small molecule compound that specifically inhibits Aurora-A kinase; an interfering molecule that specifically interferes with Aurora-A kinase gene expression; specifically knocks out Aurora-A Gene editing reagents for kinase genes; or antibodies or ligands that specifically bind to the protein encoded by the Aurora-A kinase gene.
  • the Aurora-A kinase inhibitor is a small molecule compound that specifically inhibits Aurora-A kinase, including: Alisertib (MLN 8237), MLN8054.
  • the Aurora-A kinase inhibitor includes: an interfering molecule that specifically interferes with Aurora-A kinase gene expression; an agent that mutates Aurora-A kinase with loss of function; specific knockout of Aurora-A kinase A gene editing reagent for the A kinase gene; or an antibody or ligand that specifically binds to Aurora-A kinase.
  • the Aurora-A kinase inhibitor is a gene editing reagent that specifically knocks out the Aurora-A kinase gene (such as an expression construct/expression plasmid containing gRNA), and the nucleotides of the gRNA are The sequence is shown in SEQ ID NO:1.
  • the gene editing reagent for specifically knocking out Aurora-A kinase gene is a gene editing reagent for conditionally knocking out Aurora-A kinase gene induced by doxycycline.
  • a pharmaceutical composition for inhibiting cancer which comprises an Aurora-A kinase inhibitor and lenvatinib, and a pharmaceutically acceptable carrier.
  • a cocktail for inhibiting cancer consisting of an Aurora-A kinase inhibitor and lenvatinib.
  • the Aurora-A kinase inhibitor is a small molecule compound Alisertib (MLN 8237) that specifically inhibits Aurora-A kinase, and its mass ratio to the lenvatinib is: (2 ⁇ 20): 1 (such as 4: 1, 5: 1, 6: 1, 8: 1, 10: 1, 15: 1, 18: 1, etc.); preferably (2.5-15): 1; more The ground is (3 ⁇ 12):1.
  • MN 8237 small molecule compound Alisertib
  • the Aurora-A kinase inhibitor is a gene editing reagent that specifically knocks out the Aurora-A kinase gene (such as a gRNA-containing expression construct/expression plasmid), and the nucleotide sequence of its gRNA is as shown in SEQ ID NO: 1; preferably, the gene editing reagent for specifically knocking out Aurora-A kinase gene is a gene editing reagent for conditionally knocking out Aurora-A kinase gene induced by doxycycline.
  • a gene editing reagent that specifically knocks out the Aurora-A kinase gene such as a gRNA-containing expression construct/expression plasmid
  • the gene editing reagent for specifically knocking out Aurora-A kinase gene is a gene editing reagent for conditionally knocking out Aurora-A kinase gene induced by doxycycline.
  • the dosage form of the pharmaceutical composition is: injection, infusion, tablet, capsule, pill; preferably injection.
  • kits for inhibiting cancer contains an Aurora-A kinase inhibitor and lenvatinib.
  • kits for inhibiting cancer contains the pharmaceutical composition.
  • kits for inhibiting cancer contains the mixture.
  • the kit also contains: instructions for use, explaining the method of inhibiting cancer.
  • the Aurora-A kinase inhibitor is a small molecule compound Alisertib (MLN 8237) that specifically inhibits Aurora-A kinase, which is combined with the lenvatinib Distributed in different containers (such as syringes) in the kit; and the Alisertib (MLN 8237) and the lenvatinib according to the mass ratio: (2-20): 1 (such as 4: 1, 5:1, 6:1, 8:1, 10:1, 15:1, 18:1, etc.); preferably (2.5-15):1; more preferably (3-12):1.
  • MSN 8237 small molecule compound Alisertib
  • a method for screening potential substances that promote lenvatinib to inhibit cancer comprising:
  • the system containing Aurora-A kinase is selected from: a cell (culture) system, a subcellular (culture) system, a tissue (culture) system or an animal system.
  • the improvement or promotion is a statistical improvement or promotion, for example, compared with the control or the base, the improvement or promotion is more than 10% or 20%, preferably the improvement or promotion is 30% or 50%. % or more, more preferably 80% or 90% or more.
  • the candidate substances include (but are not limited to): regulatory molecules (such as but not limited to up-regulators, interfering molecules, nucleic acid inhibitors) designed for Aurora-A kinase or its upstream or downstream proteins or genes , binding molecules (such as antibodies or ligands), CRISPR constructs, small molecule compounds, compounds from compound libraries.
  • regulatory molecules such as but not limited to up-regulators, interfering molecules, nucleic acid inhibitors
  • binding molecules such as antibodies or ligands
  • CRISPR constructs small molecule compounds, compounds from compound libraries.
  • Figure 1 Acquisition of combined action targets to enhance the therapeutic effect of Lenvatinib in vivo.
  • A CRISPR-Cas9 gene knockout technology based on kinomics (including 5960 gRNAs targeting 503 different kinases) in nude mouse MHCC97H liver cancer tumor model to screen for combined targets that can improve the therapeutic effect of Lenvatinib in vivo;
  • B analysis Results of deep sequencing of tumor genome gRNAs in 3 biological replicates in vivo experiments identified a series of enriched or lost gRNAs;
  • C Analysis of sequencing results showed that multiple gRNAs targeting Aurora-A kinase were significantly increased after treatment with Lenvatinib. lost.
  • Figure 2 In vitro experiments combined with Lenvatinib and Alisertib inhibit tumor.
  • A MHCC97H liver cancer cells were cultured, and the cells were stained to detect the antitumor effect of the drugs alone and in combination;
  • B MHCC97H liver cancer cells were cultured, and the cell viability was measured to detect the antitumor effects of the drugs alone and in combination.
  • FIG. 3 Combination of Lenvatinib and Alisertib inhibits tumors.
  • Nude mouse models of subcutaneous xenografts were constructed using liver cancer cell lines MHCC97H (A, C) and SK-Hep1 (B, D).
  • the tumor volume reached about 200mm 3
  • the nude mice were randomly divided into a placebo group (Ctr), a Lenvatinib treatment group (Lenvatinib), an Aurora-A kinase inhibitor treatment group (Alisertib) and a combination group (Combination), and were treated respectively. From 19 to 27 days, the volume change (A, B) and the body weight change (C, D) of nude mice were recorded respectively.
  • FIG. 4 Combination of Lenvatinib and Alisertib inhibits tumors.
  • liver cancer PDXs were constructed using tumor tissue derived from liver cancer patients, and the tumor volume reached about 200 mm 3 .
  • the nude mice were randomly divided into a placebo group (Ctr), a Lenvatinib-treated group (Lenvatinib), and an Aurora-A kinase inhibitor-treated group (Alisertib).
  • Combination the combination group
  • FIG. 5 Combination of Lenvatinib and Alisertib inhibits tumors.
  • Trp53 sgRNA plasmid and Myc overexpression plasmid were injected into the tail vein to construct a liver cancer model. After 2-3 weeks of injection, the primary liver tumor was detected by MRI, and the mice were randomly divided into a placebo group (Ctr) after tumor formation.
  • AURKA Aurora-A kinase
  • Aurora-A kinase inhibitor includes Aurora-A kinase activity or function inhibitors, and also includes Aurora-A kinase nucleic acid inhibitors, antagonists, inhibitors, blockers, blockers, etc. , as long as they can down-regulate the expression level of Aurora-A kinase, inhibit the activity or function of Aurora-A kinase.
  • They can be chemical compounds, small chemical molecules, biomolecules.
  • the biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.
  • the Aurora-A kinase inhibitor can be a variety of substances that can reduce the activity of Aurora-A kinase, reduce the stability of Aurora-A kinase, down-regulate the expression of Aurora-A kinase, and reduce the effective action time of Aurora-A kinase, All of these substances can be used in the present invention as useful substances for down-regulating Aurora-A kinase, thereby alleviating or treating cancer.
  • the Aurora-A kinase inhibitor can be: nucleic acid inhibitor, protein inhibitor, antibody, ligand, compound, nuclease, nucleic acid binding molecule, etc., provided that it can down-regulate the expression of Aurora-A kinase, inhibit the its activity or function.
  • Described nucleic acid inhibitor includes: the shRNA with Aurora-A kinase coding gene or its transcript as the target of inhibition or silencing, antisense nucleic acid, small interfering RNA, microRNA, or can express or form described shRNA, antisense. Nucleic acid, small interfering RNA, microRNA constructs.
  • the Aurora-A kinase inhibitor is a small molecule compound that specifically inhibits Aurora-A kinase, including: Alisertib and MLN8054. After extensive screening and comparison, the inventors found that the small molecule compound has particularly ideal effect when used in combination with Lenvatinib.
  • the small molecule compound can be a compound in pure form, or a compound with a purity greater than 85% (preferably greater than 90%, such as greater than 95%, 98%, 99%).
  • the small molecule compound can be obtained by chemical synthesis.
  • the present invention also includes the precursor of the compound, the "precursor" refers to the compound which is metabolized or chemically reacted in the body of the patient to be converted into the active compound when administered by an appropriate method.
  • the Aurora-A kinase inhibitor is an inhibitor targeting the mutation, gene editing or gene recombination of the Aurora-A kinase gene.
  • the Aurora-A kinase is converted into a mutant thereof so that it no longer functions.
  • the CRISPR/Cas9 system is used for gene editing. Appropriate gRNA target sites will bring higher gene editing efficiency, so before proceeding with gene editing, suitable target sites can be designed and found.
  • a common method for knocking out the Aurora-A gene includes: co-transferring a gRNA or a nucleic acid capable of forming the gRNA, Cas9 mRNA or a nucleic acid capable of forming the Cas9 mRNA into a targeted region or into a targeted cell. After the target site is determined, the gRNA and Cas9 can be introduced into the cell using known methods.
  • the nucleic acid that can form the gRNA is a nucleic acid construct or an expression vector
  • the nucleic acid that can form the Cas9 mRNA is a nucleic acid construct or an expression vector, and these expression vectors are introduced into the cell, thereby in the cell. Active gRNA and Cas9 mRNA are formed in the body.
  • the method of homologous recombination can be used to specifically target RNF130 to cause its expression defect or lack of expression. Cre and loxp methods can also be used to selectively knock out, reduce or inactivate related genes in the genome of animals or cells.
  • the Aurora-A kinase inhibitor is an interfering molecule that specifically interferes with the expression of Aurora-A kinase gene.
  • RNA interference technology is a technique for silencing gene expression. The principle of RNA interference technology is that long double-stranded RNA is cut and processed into 21-23nt small interfering RNA composed of sense and antisense strands by specific nuclease Dicer. Small interfering RNAs then unwind into single strands to form an RNA-induced silencing complex (RISC). The antisense strand guides the silencing complex to specifically bind to the target mRNA through base pairing, resulting in the breakdown of the mRNA.
  • RISC RNA-induced silencing complex
  • Small hairpin RNA is an RNA sequence that forms a sharp turn structure, which can silence genes through RNA interference.
  • the interfering molecule that specifically interferes with Aurora-A kinase gene expression can be a shRNA molecule directed against Aurora-A kinase, or an siRNA molecule directed against Aurora-A kinase.
  • Lenvatinib (Lenvatinib, also translated as: lenvatinib) is an oral multi-target tyrosine kinase receptor inhibitor. Its mechanism of action is to control tumors by inhibiting the growth of tumor blood vessels.
  • isomers, solvates, precursors, or salts of the compound Lenvatinib may also be included.
  • the present invention provides a method for combined medication, including a method for combining Lenvatinib with an inhibitor targeting Aurora-A kinase.
  • Aurora-A kinase has been associated with tumors, and targeted inhibition of Aurora-A kinase has been found to have an inhibitory effect on some tumors.
  • the development of drugs that inhibit Aurora-A kinase is still less studied, and its inhibitory effect needs to be improved.
  • the medication situation of Lenvatinib is also not optimistic, and its objective response rate is only about 18.8%, which is far from meeting the clinical needs.
  • the inventors found that the combined application of Aurora-A kinase inhibitor and Lenvatinib has an extremely excellent inhibitory effect on inhibiting cancer.
  • the present invention provides the use of an Aurora-A kinase inhibitor and Lenvatinib for the preparation of a mixture, a pharmaceutical composition or a kit for treating cancer.
  • Aurora-A kinase inhibitor can be used to down-regulate the expression or activity of Aurora-A kinase first, and then Lenvatinib can be used to inhibit it; or it can be performed simultaneously. It is to be understood that various modes of administration are encompassed by the present invention.
  • the invention provides a mixture of small molecule compounds, which contains: Alisertib and Lenvatinib, small molecule compounds that specifically inhibit Aurora-A kinase, as active components.
  • the mass ratio of the small molecule compound Alisertib that specifically inhibits Aurora-A kinase and the Lenvatinib is: (2-20):1 (such as 4:1, 5:1). 1, 6:1, 8:1, 10:1, 15:1, 18:1, etc.); preferably (2.5-15):1; more preferably (3-12):1.
  • the present invention provides a pharmaceutical composition, comprising: (a) an effective amount of a small molecule compound that specifically inhibits Aurora-A kinase; (b) an effective amount of Lenvatinib; and (c) a pharmaceutically acceptable carrier or excipient Form.
  • the term “comprising” means that the various ingredients can be used together in the mixture or composition of the present invention.
  • the terms “consisting essentially of” and “consisting of” are encompassed by the term “comprising”.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or animals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier is a pharmaceutically acceptable solvent, suspending agent or excipient for delivering the active ingredient of the present invention to animals or humans.
  • pharmaceutically acceptable carriers can be liquid or solid.
  • the pharmaceutical compositions or mixtures of the present invention can be prepared in any conventional formulation by conventional methods.
  • the dosage form may be various, as long as the dosage form can effectively reach the mammalian body of the active ingredient.
  • it can be selected from: injections, infusions, tablets, capsules, and pills.
  • the active ingredient may be present in a suitable solid or liquid carrier or diluent.
  • the mixture or pharmaceutical composition of the small molecule compound that specifically inhibits Aurora-A kinase and Lenvatinib of the present invention can also be stored in a sterile device suitable for injection or instillation.
  • the small molecule compounds Alisertib and Lenvatinib that specifically inhibit Aurora-A kinase can account for 0.01-20% of the total weight of the pharmaceutical composition as active ingredients, and the rest can be pharmaceutically acceptable carriers .
  • the effective dose of the small molecule compound that specifically inhibits Aurora-A kinase and lenvatinib used can vary with the mode of administration and the severity of the disease to be treated.
  • the small-molecule compounds Alisertib and Lenvatinib that specifically inhibit Aurora-A kinase can also be administered in combination with other active ingredients or drugs.
  • the present invention also provides a kit for treating tumors, the kit contains: a container 1, and the Aurora-A kinase inhibitor Alisertib placed in the container 1; and a container 2 and placed in the container 2 Lenvatinib in.
  • the Aurora-A kinase inhibitor and Lenvatinib are both small molecule compounds, so the kit can also contain a mixture of the Aurora-A kinase inhibitor and Lenvatinib, wherein the Aurora-A kinase inhibitor and Lenvatinib are mixed.
  • the content of Lenvatinib is as described above.
  • the medicine box may also contain some materials for auxiliary medicine, such as injection needles and the like.
  • kit may also contain instructions for use, explaining the method for inhibiting cancer by using the combination drug method of the present invention.
  • the technical solution of the present invention is the combined application of Aurora-A kinase inhibitor and Lenvatinib. Although in clinical practice, the actual effects of these two drugs are ignored. For example, the phase 3 clinical trial of alisertib single drug ended in failure, while the clinical ORR of lenvatinib is only about 18.8%. However, in the present invention, it is found that the combined application of the two has excellent tumor suppressing effect, which greatly exceeds the expectations of ordinary persons/physicians in the art.
  • Example 1 Acquisition of the combined action target for improving the therapeutic effect of Lenvatinib in vivo
  • the present inventors used the CRISPR-Cas9 gene knockout technology based on kinomics (including about 6000 gRNAs targeting about 500 different kinases) to screen the combined target that can improve the therapeutic effect of Lenvatinib in the nude mouse MHCC97H liver cancer tumor model point.
  • FIG. 1A The specific screening strategy is shown in Figure 1A: the inventors firstly transfected the kinase-targeting gRNA library into liver cancer cells MHCC97H (human highly metastatic liver cancer cells), and then inoculated the transfected cells into nude mice subcutaneously after puromycin screening. About 1 week after inoculation, nude mice were randomly divided into placebo group (3 ⁇ 3 biological replicates) and Lenvatinib medication group (3 ⁇ 3 biological replicates). About 1 month after drug treatment, the tumor tissue of each nude mouse was collected, its genome was extracted and deep sequencing technology was used to detect the abundant changes of each gRNA in the genome (Fig. 1B).
  • MHCC97H human highly metastatic liver cancer cells
  • Fig. 1C shows that multiple gRNAs targeting Aurora-A kinase were significantly lost after treatment with Lenvatinib compared to the placebo-treated group, suggesting that targeted inhibition of Aurora-A kinase can be Enhanced liver cancer treatment efficacy of Lenvatinib in vivo.
  • MHCC97H hepatoma cells human highly metastatic hepatoma cells
  • the cells were cultured in suspension to form 3D cell spheroids, and then 5 ⁇ M Lenvatinib, 300nM Alisertib and "5 ⁇ M Lenvatinib+
  • the cells were treated with 300nM Alisertib "Combination", cultured for about 1 week, then transferred to conventional culture, and fixed and stained after adherence.
  • the inventors inoculated MHCC97H liver cancer cells into a culture plate with an ultra-low adsorption surface, treated the cells with 5 ⁇ M Lenvatinib, 300nM Alisertib and the combination of "5 ⁇ M Lenvatinib+300nM Alisertib", and used CellTiter after culturing for about 4 days.
  • -Glo cell proliferation detection kit was used to detect the changes of cell viability in each group.
  • the inventors constructed nude mice subcutaneously transplanted tumor models using human liver cancer cell lines MHCC97H and SK-Hep1, respectively.
  • the tumor volume reached about 200mm 3
  • the nude mice were randomly divided into a placebo group (Ctr), a Lenvatinib treatment group (Lenvatinib), an Aurora-A kinase inhibitor treatment group (Alisertib) and a combination group (Combination), and were treated respectively. From 19 to 27 days, the changes in tumor volume and body weight were recorded.
  • the dosage of Lenvatinib was 5 mg/Kg body weight; the dosage of Alisertib was 25 mg/Kg body weight; when combined, the dosage of the two drugs was 5 mg/Kg body weight Lenvatinib + 25 mg/Kg body weight Alisertib.
  • the dosage of Lenvatinib was 4 mg/Kg body weight; the dosage of Alisertib was 25 mg/Kg body weight; when combined, the dosage of the two drugs was 4 mg/Kg body weight Lenvatinib + 25 mg/Kg body weight Alisertib.
  • patient-derived tumor xenogeneic animal models can provide better preclinical drug efficacy testing and analysis. Therefore, the inventors established 4 liver cancer PDXs to detect the therapeutic effect of combining Lenvatinib and Alisertib.
  • liver cancer PDX Fresh tumor tissue from liver cancer patients was cut into small pieces with a size of about 10-20 mm 3 under sterile conditions, and it was quickly implanted into immunodeficient mice. When the tumor volume reached about 500-1000 mm 3 Tumors were passaged in vivo; then rapidly thawed passaged tumor tissue blocks were subcutaneously transplanted into immunodeficient mice through a trocar, and tumor growth was monitored regularly.
  • the test drugs were administered respectively.
  • the dosage of Lenvatinib was 4 mg/Kg body weight; the dosage of Alisertib was 25 mg/Kg body weight; when combined, the dosage of the two drugs was 4 mg/Kg body weight Lenvatinib + 25 mg/kg body weight. Kg body weight Alisertib.
  • Example 5 The extremely significant effect of combining Lenvatinib and Alisertib on tumor inhibition
  • the inventors constructed a liver cancer model with a complete immune system by injecting Trp53 sgRNA plasmid and Myc overexpression plasmid through tail vein hypertension.
  • the primary liver tumors were detected by MRI 2 to 3 weeks after the injection of tail vein hypertension in mice, and then the mice were randomly divided into a placebo group (Ctr), a Lenvatinib-treated group (Lenvatinib), and an Aurora-A kinase inhibitor-treated group (Alisertib). ) and the combination group (Combination), and the corresponding drug treatment was performed (Fig. 5A).
  • the dosage of Lenvatinib is 4mg/Kg body weight; the dosage of Alisertib is 25mg/Kg body weight; when combined, the dosage of the two drugs is 4mg/Kg body weight Lenvatinib + 25mg/Kg body weight Alisertib.
  • the inventors further calculated and drawn the tumor growth curve ( FIG. 5C ) and the survival curve ( FIG. 5D ) of the animals according to the survival conditions of the animals.
  • the results further show that Lenvatinib combined with Alisertib can significantly inhibit the growth of liver cancer and greatly prolong the survival of mice.
  • Example 6 Knockout of Aurora-A (AURKA) kinase in MHCC97H liver cancer cells in vivo can enhance the therapeutic effect of Lenvatinib on liver cancer
  • the MHCC97H liver cancer cell line with conditional knockout of AURKA induced by doxycycline was constructed to verify the synergistic therapeutic effect of knockout of AURKA and Lenvatinib.
  • MHCC97H cells were transfected with Doxycycline-inducible Cas9 (iCas9) lentiviral plasmid to construct MHCC97H-iCas9 cells.
  • the gRNA targeting the AURKA gene was cloned into the LentiGuide-puro plasmid, and the pCMV-VSV-G, pRSV-Rev and MDLg/pRRE lentiviral plasmid packaging systems were used for viral packaging in 293T cells, and then the AURKA gRNA-containing plasmid was transfected. into MHCC97H-iCas9 cells to construct a MHCC97H cell line that can conditionally knock out AURKA by doxycycline.
  • iCas9 Doxycycline-inducible Cas9
  • the gRNA is: 5'-ATTCTGAACCGGCTTGTGAC-3' (SEQ ID NO: 1).
  • a nude mouse model of subcutaneous xenografts was constructed using the aforementioned human hepatoma cell line MHCC97H.
  • nude mice were randomly divided into placebo group (Control), AURKA conditional knockout group (Doxycycline), Lenvatinib treatment group (Lenvatinib), AURKA conditional knockout combined with Lenvatinib treatment group (combination) , were treated for 24 to 59 days, and the changes in tumor volume were recorded.
  • the dosage of Lenvatinib was 4 mg/Kg body weight; the feeding method of Doxycycline was 2.0 mg/ml doxycycline and 10 mg/ml sucrose dissolved in drinking water for the mice to drink freely.
  • the two drugs were treated with the same batch of mice according to the single-drug administration method described above.

Abstract

本发明提供了一种乐伐替尼和Aurora-A激酶抑制剂在制备抑制癌症的药物中的应用。本发明人发现靶向抑制Aurora-A激酶(AURKA)可以极为显著地提高Lenvatinib的癌症治疗效果，由此提供一种Aurora-A激酶抑制剂和Lenvatinib联合应用的用药方案。

Description

乐伐替尼和Aurora-A激酶抑制剂在制备抑制癌症的药物中的应用 技术领域
本发明属于肿瘤学领域，更具体地，本发明涉及乐伐替尼和Aurora-A激酶抑制剂在制备抑制癌症的药物中的应用。
背景技术
癌症是严重影响人类健康的重大疾病，其死亡率一直占据人类疾病死亡率的前位，极大程度地威胁着患病人员身心健康。尽管近几年来癌症的诊治水平越来越高，但是还需要大量的研究工作来进一步地提高患者的生存率以及患病后的生活水平。
肝细胞肝癌(HCC)是全球癌症最重要的死亡原因之一，中国更是HCC的高发国家。据估计，全球每年有超过50万的新发肝癌病例被诊断。同时，在美国，加拿大和欧洲等发达国家和地区，肝癌的发病率呈逐年上升趋势。
乐伐替尼(Lenvatinib，又译为：仑伐替尼)是近年来被批准用于晚期HCC患者的一线治疗药物。Lenvatinib是多种受体酪氨酸激酶(VEGFR 1-3，FGFR 1-4，PDGFRa，RET和KIT)的口服活性抑制剂。在一项III期多中心REFLECT临床试验中，Lenvatinib在晚期HCC患者的一线治疗中显示出与Sorafenib相似的总生存率获益(OS：13.6vs 12.3个月)，成为晚期HCC治疗方案中的一种有效替代治疗方法。然而，Lenvatinib的客观缓解率却仅约为18.8％，远不能满足临床需求。
因此，本领域亟需寻找可提高Lenvatinib临床治疗效果的联合用药新策略。
发明内容
本发明的目的在于提供乐伐替尼和Aurora-A激酶抑制剂在制备抑制癌症的药物中的应用。
在本发明的第一方面，提供Aurora-A激酶抑制剂和乐伐替尼的用途，用于制备抑制癌症的混合物、药物组合物或药盒。
在一个优选例中，所述的抑制癌症包括：预防、缓解和/或治疗癌症。
在另一优选例中，所述的癌症包括肝癌、肾癌或甲状腺癌；较佳地为肝癌。
在另一优选例中，所述的肝癌包括晚期肝癌。
在另一优选例中，所述的肝癌包括肝细胞癌。
在另一优选例中，所述的Aurora-A激酶抑制剂包括：特异性抑制Aurora-A激酶的小分子化合物；特异性干扰Aurora-A激酶基因表达的干扰分子；特异性敲除Aurora-A激酶基因的基因编辑试剂；或特异性与Aurora-A激酶基因编码的蛋白结合的抗体或配体。
在另一优选例中，所述的Aurora-A激酶抑制剂是特异性抑制Aurora-A激酶的小分子化合物，包括：Alisertib(MLN 8237)，MLN8054。
在另一优选例中，所述的Aurora-A激酶抑制剂包括：特异性干扰Aurora-A激酶基因表达的干扰分子；将Aurora-A激酶进行功能丧失性突变的试剂；特异性敲除Aurora-A激酶基因的基因编辑试剂；或特异性与Aurora-A激酶结合的抗体或配体。
在另一优选例中，所述的Aurora-A激酶抑制剂是特异性敲除Aurora-A激酶基因的基因编辑试剂(如为含有gRNA的表达构建体/表达质粒)，其gRNA的核苷酸序列如SEQ ID NO:1所示。
在另一优选例中，所述的特异性敲除Aurora-A激酶基因的基因编辑试剂是强力霉素诱导的条件性敲除Aurora-A激酶基因的基因编辑试剂。
在本发明的另一方面，提供一种用于抑制癌症的药物组合物，所述的药物组合物中包括Aurora-A激酶抑制剂和乐伐替尼，以及药学上可接受的载体。
在本发明的另一方面，提供一种用于抑制癌症的混合物，所述混合物由Aurora-A激酶抑制剂和乐伐替尼组成。
在一个优选例中，所述的Aurora-A激酶抑制剂是特异性抑制Aurora-A激酶的小分子化合物Alisertib(MLN 8237)，其与所述乐伐替尼的按照质量比为：(2～20):1(如4:1，5:1，6:1，8:1，10:1，15:1，18:1等)；较佳地为(2.5～15):1；更佳地为(3～12):1。较佳地，所述的Aurora-A激酶抑制剂是特异性敲除Aurora-A激酶基因的基因编辑试剂(如为含有gRNA的表达构建体/表达质粒)，其gRNA的核苷酸序列如SEQ ID NO:1所示；较佳地，所述的特异性敲除 Aurora-A激酶基因的基因编辑试剂是强力霉素诱导的条件性敲除Aurora-A激酶基因的基因编辑试剂。
在另一优选例中，所述的药物组合物的剂型是：注射剂，输液剂，片剂，胶囊剂，丸剂；较佳地为注射剂。
在本发明的另一方面，提供一种用于抑制癌症的药盒，所述的药盒中含有Aurora-A激酶抑制剂，以及乐伐替尼。
在本发明的另一方面，提供一种用于抑制癌症的药盒，所述的药盒中含有所述的药物组合物。
在本发明的另一方面，提供一种用于抑制癌症的药盒，所述的药盒中含有所述的混合物。
在另一优选例中，所述的药盒中还含有：使用说明书，说明抑制癌症的方法。
在另一优选例中，所述的药盒中，所述的Aurora-A激酶抑制剂是特异性抑制Aurora-A激酶的小分子化合物Alisertib(MLN 8237)，其与所述乐伐替尼被分置于所述药盒中的不同容器(如注射器)中；且所述Alisertib(MLN 8237)与所述乐伐替尼按照质量比为：(2～20):1(如4:1，5:1，6:1，8:1，10:1，15:1，18:1等)；较佳地为(2.5～15):1；更佳地为(3～12):1。
在本发明的另一方面，提供一种筛选促进乐伐替尼抑制癌症的潜在物质(即，可与乐伐替尼联合应用于抑制癌症的潜在物质)的方法，所述方法包括：
(1)用候选物质处理一表达体系，该体系表达Aurora-A激酶；和
(2)检测所述体系中Aurora-A激酶的表达或活性；若所述候选物质在统计学上降低Aurora-A激酶的表达或活性，则表明该候选物质是促进乐伐替尼抑制癌症的潜在物质(如目前未被披露的抑制该激酶的物质)。
在一个优选例中，所述的含有Aurora-A激酶的体系选自：细胞(培养物)体系、亚细胞(培养物)体系、组织(培养物)体系或动物体系。
在另一优选例中，所述的提高或促进为统计学上的提高或促进，如与对照或基底相比，提高或促进10％或20％以上，较佳地提高或促进30％或50％以上，更佳地提高或促进80％或90％以上。
在另一优选例中，所述的候选物质包括(但不限于)：针对Aurora-A激酶或 其上游或下游蛋白或基因设计的调控分子(如但不限于上调剂、干扰分子、核酸抑制物、结合分子(如抗体或配体))，CRISPR构建物，小分子化合物，来自化合物库的化合物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案，这些组合的方案也被包含在本发明中。
附图说明
图1、提高Lenvatinib体内治疗效果的联合作用靶点的获得。A，基于激酶组学(包括针对503个不同激酶的5960个gRNAs)的CRISPR-Cas9基因敲除技术在裸鼠MHCC97H肝癌肿瘤模型中筛选可以提高Lenvatinib体内治疗效果的联合作用靶点；B，分析3个生物学重复体内实验中肿瘤基因组gRNA深度测序的结果，鉴定发现一系列富集或丢失的gRNA；C，分析测序结果显示，多个靶向Aurora-A激酶的gRNA在用Lenvatinib处理后明显丢失。
图2、体外实验联合Lenvatinib和Alisertib抑制肿瘤。A，培养MHCC97H肝癌细胞，细胞染色以检测药物单用与联用的抑瘤效果；B，培养MHCC97H肝癌细胞，细胞测活以检测药物单用与联用的抑瘤效果。
图3、联合Lenvatinib和Alisertib抑制肿瘤。利用肝癌细胞株MHCC97H(A，C)和SK-Hep1(B，D)构建了裸鼠皮下移植瘤模型。等肿瘤体积达到200mm 3左右，将裸鼠随机分为安慰剂组(Ctr)，Lenvatinib处理组(Lenvatinib)，Aurora-A激酶抑制剂处理组(Alisertib)和联合用药组(Combination)，分别进行处理19-27天，分别记录裸鼠皮下移植瘤的体积变化(A，B)和裸鼠体重变化(C，D)。
图4、联合Lenvatinib和Alisertib抑制肿瘤。利用肝癌病人来源肿瘤组织构建4个肝癌PDX，等肿瘤体积达到200mm 3左右，将裸鼠随机分为安慰剂组(Ctr)，Lenvatinib处理组(Lenvatinib)，Aurora-A激酶抑制剂处理组(Alisertib)和联合用药组(Combination)，分别进行药物处理并定期记录裸鼠皮下移植瘤的体 积变化。
图5、联合Lenvatinib和Alisertib抑制肿瘤。A，通过尾静脉高压注射Trp53的sgRNA质粒和Myc过表达质粒构建肝癌模型，注射2-3周后通过MRI检测肝脏原发肿瘤，确定成瘤后将小鼠随机分为安慰剂组(Ctr)，Lenvatinib处理组(Lenvatinib)，Aurora-A激酶抑制剂处理组(Alisertib)和联合用药组(Combination)，并进行相应的药物处理；B，药物处理14天后，MRI扫描各组小鼠肝脏中的肿瘤灶情况；C-D，药物处理期间，各组小鼠的肿瘤体积变化(C)和生存期情况(D)。
图6、体内敲除MHCC97H肝癌细胞中Aurora-A(AURKA)激酶，可增强Lenvatinib的肝癌治疗效果。
具体实施方式
本发明人经过广泛而深入的研究和筛选，发现靶向抑制Aurora-A激酶(AURKA)可以提高Lenvatinib的癌症治疗效果；所述的癌症可以包括在肝癌、肾癌或甲状腺癌。将Aurora-A激酶抑制剂和Lenvatinib联合应用，对于抑制肝癌具有极其优异的效果。
Aurora-A激酶抑制剂
所述的“Aurora-A激酶抑制剂”包括了Aurora-A激酶的活性或功能抑制剂，也包括了Aurora-A激酶的核酸抑制物、拮抗剂、抑制剂、阻滞剂、阻断剂等，只要它们能够下调Aurora-A激酶的表达水平、抑制Aurora-A激酶的活性或功能。它们可以是化合物、化学小分子、生物分子。所述的生物分子可以是核酸水平(包括DNA、RNA)的，也可以是蛋白水平的。
所述的Aurora-A激酶抑制剂可以是多种可降低Aurora-A激酶的活性、降低Aurora-A激酶的稳定性、下调Aurora-A激酶的表达、减少Aurora-A激酶有效作用时间的物质，这些物质均可用于本发明，作为对于下调Aurora-A激酶有用的物质，从而可用于缓解或治疗癌症。例如，所述的Aurora-A激酶抑制剂可 以是：核酸抑制物、蛋白抑制剂、抗体、配体、化合物、核酸酶、核酸结合分子等，前提是其能够下调Aurora-A激酶的表达、抑制其活性或功能。所述的核酸抑制物包括：以Aurora-A激酶的编码基因或其转录本为抑制或沉默靶标的shRNA，反义核酸、小干扰RNA、微小RNA，或能表达或形成所述shRNA，反义核酸、小干扰RNA、微小RNA的构建物。
作为本发明的优选方式，所述的Aurora-A激酶抑制剂是特异性抑制Aurora-A激酶的小分子化合物，包括：Alisertib和MLN8054。经过大量的筛选比较，本发明人发现，该小分子化合物当与Lenvatinib联合应用时，效果特别理想。
在本发明中，所述的小分子化合物可以是纯净形式存在的化合物，或纯度大于85％(较佳地大于90％，例如大于95％，98％，99％)的化合物。
在得知其化学结构的情况下，所述的小分子化合物可通过化学合成的方式获得。本发明还包括化合物的前体，所述的“前体”指当用适当的方法服用后，该化合物的前体在病人体内进行代谢或化学反应而转变成具有活性的该化合物。
作为本发明的优选方式，所述的Aurora-A激酶抑制剂是靶向于Aurora-A激酶基因的突变、基因编辑或基因重组的抑制剂。作为一种更为具体的实施例方式，藉由上述任一的方法，使Aurora-A激酶转变为其突变体，从而使其不再发挥作用。作为一种更为具体的实施例方式，采用CRISPR/Cas9系统进行基因编辑。合适的gRNA靶位点，会带来更高的基因编辑效率，所以在着手进行基因编辑前，可以设计并找到合适的靶位点。
常见的敲除Aurora-A基因的方法包括：将gRNA或能形成所述gRNA的核酸、Cas9 mRNA或能形成所述Cas9 mRNA的核酸共转到靶向区域或靶向细胞中。在确定了靶位点之后，可以采用已知的方法来使得gRNA及Cas9被引入到细胞内。所述的能形成所述gRNA的核酸为核酸构建体或表达载体，或所述的能形成所述Cas9 mRNA的核酸为核酸构建体或表达载体，将这些表达载体导入到细胞内，从而在细胞内形成有活性的gRNA及Cas9 mRNA。
作为本发明的一种可选的方式，可采用同源重组的方法，特异性地靶向于RNF130，使之发生表达缺陷或缺失表达。也可应用Cre和loxp方法使动物或 细胞的基因组中相关基因选择性的敲除，表达降低或失活。
作为本发明的另一可选方式，所述的Aurora-A激酶抑制剂是特异性干扰Aurora-A激酶基因表达的干扰分子。RNA干扰技术是一种沉默基因表达的技术。RNA干扰技术的原理是较长双链RNA被特异核酸酶Dicer切割加工成21-23nt的由正义和反义链组成的小干扰RNA。小干扰RNA随后形成沉默复合体(RNA-induced silencing complex，RISC)解旋成单链。反义链引导该沉默复合体通过碱基配对特异性结合到目标mRNA上，使mRNA分解。小发夹RNA(short hairpin RNA，shRNA)是一种形成急转弯结构的RNA序列，可以经由RNA干扰使基因沉默。所述的特异性干扰Aurora-A激酶基因表达的干扰分子可以是针对Aurora-A激酶的shRNA分子，也可以是针对Aurora-A激酶的siRNA分子。
Lenvatinib
乐伐替尼(Lenvatinib，又译为：仑伐替尼)是一种口服型多靶点酪氨酸激酶受体抑制剂。它的作用机制是通过抑制肿瘤血管生长来达到控制肿瘤的目的。
本发明中，还可包括化合物Lenvatinib的异构体、溶剂合物、前体，或其盐。
Aurora-A激酶抑制剂和Lenvatinib的联合应用
本发明提供了一种联合用药的方法，包括利用靶向Aurora-A激酶的抑制剂联合Lenvatinib的用药方法。
本领域中，已经将Aurora-A激酶与肿瘤相关联，发现靶向抑制Aurora-A激酶对于有的肿瘤具有抑制作用。然而抑制Aurora-A激酶的药物开发还研究得较少，且其抑制效果还有待改进。Lenvatinib的用药情况也不容乐观，其客观缓解率却仅约为18.8％，远不能满足临床需求。而本发明人经过大量的研究筛选后发现，将Aurora-A激酶抑制剂和Lenvatinib联合应用，对于抑制癌症具有极其优异的抑制效果。
因此，本发明提供了Aurora-A激酶抑制剂和Lenvatinib的用途，用于制备治疗癌症的混合物、药物组合物或药盒。
在给药时，可以先利用Aurora-A激酶抑制剂下调Aurora-A激酶的表达或 活性，再以Lenvatinib加以抑制；或者也可同时进行。应理解，多种给药方式均被包含在本发明中。
组合物或混合物
本发明提供了一种小分子化合物的混合物，含有：特异性抑制Aurora-A激酶的小分子化合物Alisertib和Lenvatinib作为活性组分。较佳地，所述的混合物中，所述的特异性抑制Aurora-A激酶的小分子化合物Alisertib与所述Lenvatinib的按照质量比为：(2～20):1(如4:1，5:1，6:1，8:1，10:1，15:1，18:1等)；较佳地为(2.5～15):1；更佳地为(3～12):1。
本发明提供了一种药物组合物，含有：(a)有效量的特异性抑制Aurora-A激酶的小分子化合物；(b)有效量的Lenvatinib；以及(c)药学上可接受的载体或赋形剂。
本发明中，术语“含有”表示各种成分可一起应用于本发明的混合物或组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。
本发明中，“药学上可接受的”成分是适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)即有合理的效益/风险比的物质。
本发明中，“药学上可接受的载体”是用于将本发明的活性组分传送给动物或人的药学上可接受的溶剂、悬浮剂或赋形剂。“药学上可接受的载体”可以是液体或固体。
本发明的药物组合物或混合物可以通过常规方法制成任何常规的制剂形式。剂型可以是多种多样的，只要是能够使活性成分有效地到达哺乳动物体内的剂型都是可以的。比如可选自：注射剂，输液剂，片剂，胶囊剂，丸剂。其中活性组分可以存在于适宜的固体或液体的载体或稀释液中。
本发明的特异性抑制Aurora-A激酶的小分子化合物和Lenvatinib的混合物或药物组合物也可储存在适宜于注射或滴注的消毒器具中。通常，在本发明的药物组合物中，特异性抑制Aurora-A激酶的小分子化合物Alisertib和Lenvatinib作为活性成分可以占药物组合物总重量的0.01-20％，其余可以为药学上可接受的载体。
所用的特异性抑制Aurora-A激酶的小分子化合物和Lenvatinib的有效剂量可随给药的模式和待治疗的疾病的严重程度而变化。必要的时候，特异性抑制Aurora-A激酶的小分子化合物Alisertib和Lenvatinib还可与其它活性成分或药物联合给药。
药盒
本发明还提供了一种用于治疗肿瘤的药盒，所述的药盒中，含有：容器1，以及置于容器1中的Aurora-A激酶抑制剂Alisertib；以及容器2以及置于容器2中的Lenvatinib。
所述的Aurora-A激酶抑制剂和Lenvatinib均为小分子化合物，因此所述的药盒中，也可含有所述的Aurora-A激酶抑制剂和Lenvatinib的混合物，其中Aurora-A激酶抑制剂和Lenvatinib的含量如前述。
此外，所述的药盒中还可以还有一些辅助用药的材料，例如注射用针管等。
此外，所述的药盒中还可含有使用说明书，说明利用本发明的联合用药方法来抑制癌症的方法。
本发明的技术方案，联合应用Aurora-A激酶抑制剂和Lenvatinib。尽管在临床实践中，该两种药物的单药实际效果都不理，例如Alisertib单药的3期临床以失败告终，而lenvatinib的临床ORR只有约18.8％。但本发明中，发现两者的联合应用具有优异的肿瘤抑制作用，这大大超出了本领域一般人员/医师的预料。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、提高Lenvatinib体内治疗效果的联合作用靶点的获得
本发明人运用基于激酶组学(包括针对约500个不同激酶的约6000个 gRNAs)的CRISPR-Cas9基因敲除技术，在裸鼠MHCC97H肝癌肿瘤模型中筛选可以提高Lenvatinib体内治疗效果的联合作用靶点。
具体筛选策略如图1A：本发明人首先将靶向激酶的gRNA文库转染至肝癌细胞MHCC97H(人高转移性肝癌细胞)中，嘌呤霉素筛选后将转染的细胞接种到裸鼠皮下。接种大概1周后将裸鼠随机分为安慰剂组(3只×3次生物学重复)和Lenvatinib用药组(3只×3次生物学重复)。用药处理1个月左右，收集每只裸鼠的肿瘤组织，提取其基因组并用深度测序技术检测每个gRNA在基因组中的丰富变化(图1B)。
经过广泛的分析测序，本发明人发现(图1C)，相比安慰剂处理组，用Lenvatinib处理后，多个靶向Aurora-A激酶的gRNA明显丢失，提示靶向抑制Aurora-A激酶可在体内提高Lenvatinib的肝癌治疗效果。
实施例2、体外实验联合Lenvatinib和Alisertib抑制肿瘤
1、培养MHCC97H肝癌细胞，细胞染色以检测药物单用与联用的抑瘤效果
将MHCC97H肝癌细胞(人高转移性肝癌细胞)接种到超低吸附表面的培养皿中，使细胞处于悬浮培养状态，从而形成3D细胞球，然后再分别使用5μM Lenvatinib，300nM Alisertib及“5μM Lenvatinib+300nM Alisertib”两药联用(Combination)处理细胞，培养1周左右再将各组细胞转移到常规培养中，待贴壁后进行固定和染色。
结果如图2A，可见单用Lenvatinib或单用Alisertib对于MHCC97H的抑制作用是非常微小的，通过定量细胞灰度值得出抑制率，抑制率分别约为3％和27％。而若将两者联合应用，则对该MHCC97H的抑制作用极为显著地提升，抑制率达到86％。
2、培养MHCC97H肝癌细胞，细胞测活以检测药物单用与联用的抑瘤效果
本发明人将MHCC97H肝癌细胞接种到超低吸附表面的培养板中，分别使用5μM Lenvatinib，300nM Alisertib及“5μM Lenvatinib+300nM Alisertib” 两药联用(Combination)处理细胞，培养4天左右后使用CellTiter-Glo细胞增殖检测试剂盒检测各组细胞活力变化。
结果如图2B，结果显示，单用Lenvatinib或Alisertib的肿瘤抑制作用非常有限，而联合使用两药可极为显著地提高肿瘤抑制效果。
实施例3、联合Lenvatinib和Alisertib抑制肿瘤(移植瘤动物模型)
Alisertib(MLN 8237)是一种口服活性和选择性的Aurora A激酶抑制剂(IC50＝1.2nM)，其能与Aurora A激酶结合，导致细胞周期阻滞、细胞凋亡与自噬。
为了验证前述实施例1的体内筛选结果，本发明人分别利用人肝癌细胞株MHCC97H和SK-Hep1构建了裸鼠皮下移植瘤模型。等肿瘤体积达到200mm 3左右，将裸鼠随机分为安慰剂组(Ctr)，Lenvatinib处理组(Lenvatinib)，Aurora-A激酶抑制剂处理组(Alisertib)和联合用药组(Combination)，分别进行处理19～27天，并记录肿瘤体积变化和体重变化。其中，对于MHCC97H移植瘤模型，Lenvatinib用量为5mg/Kg体重；Alisertib用量为25mg/Kg体重；组合用药时，两种药的用量为5mg/Kg体重Lenvatinib+25mg/Kg体重Alisertib。对于SK-Hep1移植瘤模型，Lenvatinib用量为4mg/Kg体重；Alisertib用量为25mg/Kg体重；组合用药时，两种药的用量为4mg/Kg体重Lenvatinib+25mg/Kg体重Alisertib。
结果显示，单药Lenvatinib一定程度抑制肝癌肿瘤的生长，而进一步联合Aurora-A激酶抑制Alisertib可完全遏制肿瘤的皮下生长(图3A-B)。
同时，本发明人的结果也显示(图3C-D)，联合Lenvatinib和Alisertib并不影响裸鼠的体重，提示该联合用药方式毒副作用低，可耐受性良好。
实施例4、联合Lenvatinib和Alisertib抑制肿瘤(PDX模型)
相比传统细胞系异种动物模型，病人来源肿瘤异种动物模型(PDX)能提供更好的临床前药物疗效测试及分析。因此，本发明人建立了4种肝癌PDX来检测联合Lenvatinib和Alisertib的治疗效果。
4种肝癌PDX的建立：将肝癌患者的新鲜肿瘤组织在无菌条件下切成大小约10-20mm 3的小块，迅速植入免疫缺陷小鼠，当肿瘤体积达500-1000mm 3左 右，对肿瘤进行体内传代；然后将快速解冻的传代肿瘤组织块通过套管针皮下移植到免疫缺陷小鼠，定期监测肿瘤生长情况，当肿瘤体积达200mm 3时再分组给予药物处理。
对于上述建立的4种肝癌PDX，分别给予受试药物处理，Lenvatinib用量为4mg/Kg体重；Alisertib用量为25mg/Kg体重；组合用药时，两种药的用量为4mg/Kg体重Lenvatinib+25mg/Kg体重Alisertib。
结果显示(图4)，在4种肝癌PDX中，相比单药治疗组，Lenvatinib联合Aurora-A激酶抑制Alisertib都可明显进一步抑制肝癌PDX的生长。
实施例5、联合Lenvatinib和Alisertib对于抑制肿瘤的促进作用的极其显著性
为了进一步评估免疫系统对该联合治疗策略的影响，本发明人通过尾静脉高压注射Trp53的sgRNA质粒和Myc过表达质粒构建免疫系统完整的肝癌模型。
小鼠尾静脉高压注射2～3周后通过MRI检测肝脏原发肿瘤，然后将小鼠随机分为安慰剂组(Ctr)，Lenvatinib处理组(Lenvatinib)，Aurora-A激酶抑制剂处理组(Alisertib)和联合用药组(Combination)，并进行相应的药物处理(图5A)。Lenvatinib用量为4mg/Kg体重；Alisertib用量为25mg/Kg体重；组合用药时，两种药的用量为4mg/Kg体重Lenvatinib+25mg/Kg体重Alisertib。
用药14天后，MRI检测结果显示，相对单药Lenvatinib组和单药Alisertib组，两药联合可明显减少小鼠肝脏中肿瘤灶的数量和大小(图5B)。
本发明人进一步根据动物生存情况，统计和绘制了动物的肿瘤生长曲线(图5C)和生存期曲线(图5D)。结果进一步显示，Lenvatinib联合Alisertib可极为显著地遏制肝癌的生长和并大大延长小鼠的生存期。
实施例6、体内敲除MHCC97H肝癌细胞中Aurora-A(AURKA)激酶，可增强Lenvatinib的肝癌治疗效果
构建强力霉素(Doxycycline)诱导的条件性敲除AURKA的MHCC97H肝癌细胞系，验证敲除AURKA与Lenvatinib的协同治疗效果。
MHCC97H细胞转染Doxycycline-inducible Cas9(iCas9)慢病毒质粒，构建MHCC97H-iCas9细胞。将靶向AURKA基因的gRNA克隆到LentiGuide-puro质粒中，在293T细胞中利用pCMV-VSV-G、pRSV-Rev和MDLg/pRRE慢病毒质粒包装系统进行病毒包装，然后将含AURKA gRNA的质粒转入MHCC97H-iCas9细胞，构建可通过doxycycline条件性敲除AURKA的MHCC97H细胞系。
所述gRNA为：5’-ATTCTGAACCGGCTTGTGAC-3’(SEQ ID NO:1)。
利用前述人肝癌细胞株MHCC97H构建了裸鼠皮下移植瘤模型。等肿瘤体积达到200mm 3左右，将裸鼠随机分为安慰剂组(Control)，AURKA条件性敲除组(Doxycycline)，Lenvatinib处理组(Lenvatinib)，AURKA条件性敲除联合Lenvatinib处理组(组合)，分别进行处理24～59天，并记录肿瘤体积变化。其中，对于MHCC97H移植瘤模型，Lenvatinib用量为4mg/Kg体重；Doxycycline给喂方式为2.0mg/ml doxycycline和10mg/ml蔗糖一起溶解于饮用水，供小鼠自由饮用。组合用药时，两种药分别按上述单药给药方式处理同一批小鼠。
结果如图6。结果显示，单药Lenvatinib一定程度抑制肝癌肿瘤的生长，而敲除AURKA可极为显著地增强Lenvatinib对皮下移植瘤的生长抑制作用，肿瘤体积大大减小。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. Aurora-A激酶抑制剂和乐伐替尼的用途，用于制备抑制癌症的混合物、药物组合物或药盒。
  2. 如权利要求1所述的用途，其特征在于，所述的Aurora-A激酶抑制剂包括：特异性抑制Aurora-A激酶的小分子化合物；特异性干扰Aurora-A激酶基因表达的干扰分子；特异性敲除Aurora-A激酶基因的基因编辑试剂；或特异性与Aurora-A激酶基因编码的蛋白结合的抗体或配体。
  3. 如权利要求1所述的用途，其特征在于，所述的Aurora-A激酶抑制剂是特异性抑制Aurora-A激酶的小分子化合物，包括：Alisertib，MLN8054。
  4. 如权利要求1所述的用途，其特征在于，所述的Aurora-A激酶抑制剂包括：特异性干扰Aurora-A激酶基因表达的干扰分子；将Aurora-A激酶进行功能丧失性突变的试剂；特异性敲除Aurora-A激酶基因的基因编辑试剂；或特异性与Aurora-A激酶结合的抗体或配体。
  5. 如权利要求4所述的用途，其特征在于，所述的Aurora-A激酶抑制剂是特异性敲除Aurora-A激酶基因的基因编辑试剂(如为含有gRNA的表达构建体/表达质粒)，其gRNA的核苷酸序列如SEQ ID NO:1所示。
  6. 如权利要求5所述的用途，其特征在于，所述的特异性敲除Aurora-A激酶基因的基因编辑试剂是强力霉素诱导的条件性敲除Aurora-A激酶基因的基因编辑试剂。
  7. 如权利要求1所述的用途，其特征在于，所述的癌症包括肝癌、肾癌或甲状腺癌；较佳地为肝癌。
  8. 一种用于抑制癌症的药物组合物，所述的药物组合物中包括Aurora-A激酶抑制剂和乐伐替尼，以及药学上可接受的载体。
  9. 一种用于抑制癌症的混合物，所述混合物由Aurora-A激酶抑制剂和乐伐替尼组成。
  10. 如权利要求8或9所述，其特征在于，所述的Aurora-A激酶抑制剂是特异性抑制Aurora-A激酶的小分子化合物Alisertib，其与所述乐伐替尼的按照质量比为：(2～20):1；较佳地为(2.5～15):1；更佳地为(3～12):1。
  11. 如权利要求8或9所述，其特征在于，所述的Aurora-A激酶抑制剂是特异性敲除Aurora-A激酶基因的基因编辑试剂(如为含有gRNA的表达构建体/表达质粒)，其gRNA的核苷酸序列如SEQ ID NO:1所示；较佳地，所述的特异性敲除Aurora-A激酶基因的基因编辑试剂是强力霉素诱导的条件性敲除Aurora-A激酶基因的基因编辑试剂。
  12. 如权利要求8或9所述，其特征在于，所述的癌症包括肝癌、肾癌或甲状腺癌；较佳地为肝癌。
  13. 一种用于抑制癌症的药盒，所述的药盒中含有Aurora-A激酶抑制剂，以及乐伐替尼；或
    所述的药盒中含有权利要求8所述的药物组合物；或
    所述的药盒中含有权利要求9所述的混合物。
  14. 如权利要求13所述的药盒，其特征在于，所述的Aurora-A激酶抑制剂是特异性抑制Aurora-A激酶的小分子化合物Alisertib，其与所述乐伐替尼被分置于所述药盒中的不同容器中；且所述Alisertib与所述乐伐替尼按照质量比为：(2～20):1；较佳地为(2.5～15):1；更佳地为(3～12):1。
  15. 一种筛选促进乐伐替尼抑制癌症的潜在物质的方法，所述方法包括：
    (1)用候选物质处理一表达体系，该体系表达Aurora-A激酶；和
    (2)检测所述体系中Aurora-A激酶的表达或活性；若所述候选物质在统计学上降低Aurora-A激酶的表达或活性，则表明该候选物质是促进乐伐替尼抑制癌症的潜在物质。
PCT/CN2021/137957 2020-12-14 2021-12-14 乐伐替尼和Aurora-A激酶抑制剂在制备抑制癌症的药物中的应用 WO2022127788A1 (zh)

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