WO2023016321A1 - 恩沙替尼或其盐在治疗携带met 14外显子跳跃突变的疾病中的用途 - Google Patents

恩沙替尼或其盐在治疗携带met 14外显子跳跃突变的疾病中的用途 Download PDF

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WO2023016321A1
WO2023016321A1 PCT/CN2022/110160 CN2022110160W WO2023016321A1 WO 2023016321 A1 WO2023016321 A1 WO 2023016321A1 CN 2022110160 W CN2022110160 W CN 2022110160W WO 2023016321 A1 WO2023016321 A1 WO 2023016321A1
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ensartinib
day
salt
cancer
met
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PCT/CN2022/110160
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English (en)
French (fr)
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WO2023016321A8 (zh
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刘莉嘉
郭晶
王钰
赵向东
陈洁
汪洋
袁晓玢
季东
孔里程
丁列明
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贝达药业股份有限公司
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Priority to US18/682,227 priority Critical patent/US20240342169A1/en
Priority to CN202280051617.9A priority patent/CN117897156A/zh
Priority to EP22855304.6A priority patent/EP4385510A1/en
Publication of WO2023016321A1 publication Critical patent/WO2023016321A1/zh
Publication of WO2023016321A8 publication Critical patent/WO2023016321A8/zh

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    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/501—Pyridazines; Hydrogenated pyridazines not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This application relates to the technical field of new uses of Ensartinib, in particular to the use of Ensartinib or its salts in the treatment of diseases carrying MET 14 exon skipping mutations.
  • c-MET is a transmembrane receptor encoded by the MET gene, which belongs to receptor tyrosine kinase (RTK), and is related to various oncogene products and regulatory proteins, and is an important factor for cell proliferation, differentiation and motility.
  • RTK receptor tyrosine kinase
  • Abnormal activation of the c-MET pathway mainly includes three types: MET exon 14 skipping mutation, MET gene amplification and c-MET protein overexpression (Organ S L, Tsao M S.An overview of the c-MET signaling pathway[J ]. Therapeutic Advances in Medical Oncology, 2011, 3 (1 Suppl).).
  • Exon 14 of MET corresponds to encoding 141 amino acids, and its juxtamembrane domain is the key negative regulatory region of c-MET, including the E3 ubiquitin ligase c-Cbl tyrosine binding site (Y1003), involved in Ubiquitination and degradation of c-MET protein.
  • the skipping mutation of MET exon 14 is mainly due to the fact that exon 14 is spliced together with the introns at both ends during the splicing process after the transcription of the MET gene, forming a mature mRNA that is 141 bp less than the normal transcription splicing, namely Form the mature mRNA that produces MET exon 14, and then translate through the ribosome to obtain the c-Met receptor protein (L964-D1010) of 47 amino acid series Pilotto S, Gkountakos A, Carbognin L, et al.MET exon 14 juxtamembrane splicing mutations: Clinical and therapeutic perspectives for cancer therapy [J]. Annals of Translational Medicine, 2017, 5(1):2-2.).
  • This mutation results in the deletion of the juxtamembrane domain containing the binding site of E3 ubiquitin ligase c-Cbl, resulting in the inability of c-Cbl to bind to c-met protein, thereby limiting the ubiquitination and degradation of c-MET protein, c-
  • the increased stability of MET leads to the continuous activation of MET downstream signals, and eventually becomes one of the driving genes of tumors.
  • NSCLC non-small cell lung cancer
  • drugs that can be used to treat NSCLC include osimertinib for EGFR T790M gene mutation; Takeda Pharmaceutical TAK-788 for EGFR exon 20 insertion mutation, etc.; different Targeted drugs have a good therapeutic effect on cancers caused by specific targets and mutations, but it is unpredictable whether they have the same or similar effects on similar cancers caused by other gene mutations; MET exon 14 skipping mutations occur in 3-4
  • the prognosis of this type of cancer is very poor in 1% of newly diagnosed advanced NSCLC cases, and more drugs that can target advanced NSCLC with MET exon 14 skipping mutations, as well as other diseases carrying MET exon 14 skipping mutations, are needed.
  • Ensartinib is a marketed drug for the treatment of ALK-positive non-small cell lung cancer, whether it can be used to treat diseases carrying MET 14 exon skipping mutations, such as advanced stage patients carrying MET 14 exon skipping mutations NSCLC is not currently being studied.
  • the inventors of the present application unexpectedly found in the research that Ensartinib or its salts have the effect of treating diseases carrying MET 14 exon skipping mutations, and completed the present application based on this.
  • the first aspect of the present application provides ensartinib or its salt, the composition comprising ensartinib or its salt in the treatment of diseases carrying MET 14 exon skipping mutation, wherein, said ensartinib Has the structure shown in formula I:
  • the second aspect of the present application provides ensartinib or a salt thereof, and a composition comprising ensartinib or a salt thereof in the preparation of a medicine for treating a disease carrying a MET 14 exon skipping mutation, wherein the Ensartinib has a structure shown in formula I:
  • the third aspect of the present application provides a method for treating a disease carrying a MET 14 exon skipping mutation, which administers an effective dose of Ensartinib or a salt thereof, or comprises Ensartinib or a salt thereof, to a subject in need.
  • the composition of salt, wherein, described ensartinib has the structure shown in formula I:
  • the ensartinib or its salt and the composition comprising ensartinib or its salt provided by the application can effectively inhibit the kinase activity of the MET 14 exon skipping mutation, and significantly inhibit the human gastric cancer Hs746T carrying the MET 14 exon skipping mutation Proliferation of cells showed good anti-tumor activity in vivo against xenograft tumor models carrying MET 14 exon skipping mutations, and demonstrated significant efficacy in clinical trials.
  • Ensartinib, salts of Ensartinib and compositions comprising Ensartinib or its salts can be used for the treatment of diseases carrying MET 14 exon skipping mutations, and then can be used for the preparation of treatments for MET14 exon skipping Drugs for diseases caused by mutations.
  • Figure 1 is the relationship curve between the kinase inhibition rate of MET 14 exon skipping mutation and the concentration of ensartinib.
  • Figure 2 is the relationship curve between the survival rate of Hs746T cells and the concentration of ensartinib.
  • Figure 3 is the tumor volume growth curve in mice treated with different doses of ensartinib hydrochloride.
  • administration and “treatment” refer to the combination of exogenous drugs, therapeutic agents, diagnostic agents or compositions when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids. Contact with animals, humans, subjects, cells, tissues, organs or biological fluids.
  • treating means administering Ensartinib or a salt thereof, or comprising Ensartinib to a subject having a disease carrying a MET 14 exon skipping mutation or diagnosed with a disease carrying a MET 14 exon skipping mutation
  • a combination of tinib or a salt thereof, to achieve at least one positive therapeutic effect such as, when the disease is cancer, reducing the number of cancer cells, reducing tumor size, reducing the rate of cancer cell infiltration into peripheral organs, or reducing The rate of tumor metastasis, or tumor growth.
  • Treatment may include: stopping or delaying progression of disease with MET exon 14 skipping mutations, stabilization of disease with MET exon 14 skipping mutations, amelioration or abrogation of disease with MET exon 14 skipping mutations reduce the severity or duration of clinical symptoms of disease with MET exon 14 skipping mutations, prolong the survival of patients relative to the expected survival of untreated similar patients, and induce MET exon 14 Complete or partial remission of a disease with jumping mutations; for example, when the disease is cancer, "treatment” may include one or more of the following: reducing the amount of one or more tumor markers, suspending or delaying tumor progression growth, inhibiting the growth or survival of tumor cells, eliminating or reducing the size of one or more cancerous lesions or tumors, reducing the level of one or more tumor markers, inducing complete or partial remission of a cancerous condition.
  • an effective dose refers to an amount sufficient to reduce or ameliorate the severity, duration or development of the disorder to be treated, prevent Progression, causing regression of the disorder being treated, or enhancing or improving the prophylactic or therapeutic effect of another therapy.
  • the term "subject” is defined herein to include animals such as mammals, including but not limited to primates (such as humans), cows, sheep, goats, horses, dogs, cats, Rabbits, rats, mice, etc. In specific embodiments, the subject is a human.
  • the terms "subject” and “patient” are used interchangeably herein when referring to, for example, a mammalian subject such as a human.
  • “about” refers to numerical variations that may occur, for example, through typical measuring, handling and sampling procedures involved in the preparation, characterization and/or use of a substance or composition; through inadvertent error in these procedures; through making or Differences in the composition of use or the manufacture, source, or purity of ingredients used to practice a procedure; etc.
  • "about” can mean ⁇ 0.1%, ⁇ 0.5%, ⁇ 1%, ⁇ 2%, ⁇ 3%, ⁇ 4%, ⁇ 5%, ⁇ 6%, ⁇ 7%, ⁇ 8%, ⁇ 9%, or ⁇ 10% variation.
  • the first aspect of the present application provides Ensartinib (Ensartinib) or a salt thereof, and a composition comprising Ensartinib or a salt thereof in the treatment of a disease carrying a MET 14 exon skipping mutation, wherein the Ensartinib Satinib has a structure shown in formula I:
  • the second aspect of the present application provides ensartinib or a salt thereof, and a composition comprising ensartinib or a salt thereof in the preparation of a medicine for treating a disease carrying a MET 14 exon skipping mutation, wherein the Ensartinib has a structure shown in formula I:
  • the diseases carrying MET 14 exon skipping mutation include lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, kidney cancer, ovarian cancer, At least one of gastric cancer, skin cancer, bone cancer, glioma, papillary renal cell carcinoma, or squamous cell carcinoma of the head and neck.
  • the disease carrying the MET 14 exon skipping mutation is non-small cell lung cancer.
  • composition of the present application which includes an effective dose of ensartinib, or if applicable, the pharmaceutically acceptable salt of the compound, polymorphic form of the salt, solvate, hydrate, polymorphic form or a prodrug; and an acceptable carrier.
  • salts formed by alkali ions and free acids such as hydrochloride, phosphate, formate, acetate, fumarate, oxalate salt, maleate, citrate, etc.
  • compositions of the present application are formulated for pharmaceutical use (“pharmaceutical compositions"), wherein the carrier is a pharmaceutically acceptable carrier.
  • the carrier must be "acceptable” with regard to being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not injurious to the recipient thereof in amounts typically used in pharmaceuticals.
  • Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical compositions of the present application include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, Buffer substances such as phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, Zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymerization substances, polyethylene glycol and lanolin.
  • ion exchangers aluminum oxide, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • Buffer substances such as phosphat
  • compositions of the present application include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the ensartinib herein is administered transdermally (eg, using a transdermal patch).
  • Other formulations may conveniently be presented in unit dosage form, for example, tablets and sustained release capsules, and in liposomes, and may be prepared by any methods known in the art of pharmacy. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA (17th ed., 1985).
  • compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, liposomes or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present application suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as powders or granules; as liquids in aqueous or non-aqueous liquids. or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or encapsulated in liposomes, and as a pill, etc.
  • Soft gelatin capsules may be used to contain suspensions, which may beneficially increase the rate of absorption of the compounds.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by mixing the active ingredient in a free-flowing form such as powder or granules, optionally with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent, in a suitable machine. Compress to prepare. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.
  • compositions of such pharmaceutically active ingredients such as those herein and other compounds known in the art are known in the art and described in several issued U.S. patents, Some of these include, but are not limited to, US Patent Nos. 4,369,172 and 4,842,866, and references cited therein. Coatings can be used to deliver compounds to the intestine (see, eg, US Patent Nos. 6,638,534, 5,217,720, and 6,569,457, 6,461,631, 6,528,080, 6,800,663 and references cited therein).
  • a useful formulation for the compounds of the present application is in the form of enteric pellets, wherein the enteric layer comprises hydroxypropylmethylcellulose acetate succinate.
  • carriers which are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • compositions suitable for topical administration include lozenges, which include the ingredients in a flavored base, usually sucrose and acacia or tragacanth; and pastilles, which include the ingredients in an inert base.
  • the active ingredient in an inert base is, for example, gelatin and glycerin, or sucrose and acacia.
  • compositions suitable for parenteral administration include aqueous and nonaqueous sterile injectable solutions, which may contain antioxidants, buffers, bacteriostats, and solutes to render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous Sterile suspensions may contain suspending and thickening agents.
  • the formulations can be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier immediately before use, such as for injection use water.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets.
  • Such injection solutions may be in the form of, for example, sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (eg, Tween 80) and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol.
  • a nontoxic parenterally acceptable diluent or solvent for example a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents there may be employed mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.
  • compositions of the present application may be administered in the form of suppositories for rectal administration.
  • These compositions can be prepared by mixing a compound of the present application with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and will thus melt in the rectum to release the active ingredient.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
  • compositions of the present application can be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as a solution in saline with benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and/or or other solubilizers or dispersants known in the art.
  • Topical administration of the pharmaceutical compositions of the present application is particularly useful when the desired treatment involves an area or organ readily accessible by topical application.
  • the pharmaceutical composition should be formulated in a suitable ointment containing the active components suspended or dissolved in a carrier.
  • Carriers for topical administration of the compounds of this application include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compounds suspended or dissolved in a carrier.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol , Benzyl Alcohol and Water.
  • the pharmaceutical compositions of the present application may also be administered topically to the lower intestinal tract by rectal suppositories or in the form of suitable enema formulations. Topically transdermal patches and iontophoresis are also included in this application.
  • the ensartinib or the salt thereof of the present application is present in an effective dose.
  • the correlation of doses in animals and humans is described in Freireich et al., (1966) Cancer Chemother Rep 50:219.
  • Body surface area can be roughly determined from the patient's height and weight. See, eg, Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537.
  • the effective amount of Ensartinib or its salt of the present application can be from about 5 mg/day to about 500 mg/day, preferably 10 mg/day to 400 mg/day, more preferably 25 mg/day to 250 mg/day.
  • the effective amount of Ensartinib of the present application can be 10mg/day, 20mg/day, 25mg/day, 30mg/day, 50mg/day, 75mg/day, 90mg/day, 100mg/day, 120mg/day, 150mg/day, 180mg/day, 200mg/day, 225mg/day, 250mg/day, 300mg/day, 350mg/day, 400mg/day, etc.
  • Effective dosages may also vary, as understood by those skilled in the art, depending on the disease being treated, the severity of the disease, the route of administration, the sex, age and general health of the patient, excipient usage, and other therapeutic Potential for method co-use (eg, use of other agents), and the judgment of the treating physician.
  • the third aspect of the present application provides a method for treating a disease carrying a MET 14 exon skipping mutation, which administers an effective dose of Ensartinib or a salt thereof, or comprises Ensartinib or a salt thereof, to a subject in need.
  • the composition of salt, wherein, described ensartinib has the structure shown in formula I:
  • the subject in need thereof is administered an effective dose of said ensartinib or a salt thereof, or comprising ensartinib or its salt composition.
  • the effective dose is based on Ensartinib or its salt, and the dose is about 5 mg/day to 500 mg/day, preferably 10 mg/day to 400 mg/day, and more preferably 25 mg/day to 250 mg/day, More preferably 225 mg/day.
  • the fourth aspect of the present application provides a kind of Ensartinib or its salt for the treatment of diseases carrying MET 14 exon skipping mutation, or a composition comprising Ensartinib or its salt, wherein the Ensartinib Nylon has a structure shown in formula I:
  • ADP adenosine diphosphate
  • ATP adenosine triphosphate
  • HPMC hydroxypropyl methylcellulose
  • DMSO dimethyl sulfoxide
  • FBS fetal bovine serum
  • PBS Phosphate Buffered Saline
  • NSCLC non-small cell lung cancer
  • DCR disease control rate
  • CTG reagent CellTiter-Glo reagent
  • TGI (%) tumor growth inhibition rate.
  • ADP-Glo Kinase Assay Kit was used to evaluate the inhibitory effect of Ensartinib on MET exon 14 deletion (MET ex14Del) kinase.
  • the test was carried out in a 384-well plate.
  • the compound to be tested ensartinib hydrochloride
  • DMSO dimethyl sulfoxide
  • the compound solution to be tested was added to a 384-well plate, 0.1 ⁇ L per well.
  • the RLU (relative luminescence unit) value was read using the Envision multifunctional plate reader, and the RLU value was used to characterize the degree of reaction between the enzyme and the substrate.
  • XLfit 5.3.1 was used to fit the IC 50 of the compound from a nonlinear regression equation.
  • CellTiter-Glo TM live cell detection kit method was used to evaluate the growth inhibitory effect of ensartinib on human tumor cell line Hs746T.
  • Human gastric cancer cell line Hs746T (carrying MET 14 exon skipping mutation, sourced from ATCC (American Type Culture Collection), grown in DMEM+10% FBS medium);
  • Fetal bovine serum FBS (Gibco Cat#10099-141);
  • Luminescent cell viability assay reagent Promega, Cat#G7573
  • Ensartinib hydrochloride (Betta Pharmaceuticals, Z170401T) was prepared into a drug stock solution with a concentration of 10 mM in DMSO.
  • the cells in the 96-well plate to which the drug has been added are placed at 37° C., 5% CO 2 , and 95% humidity to continue culturing for 168 hours, and then conduct CTG analysis.
  • GraphPad Prism 7.0 software was used to analyze the data, and the nonlinear S-curve regression was used to fit the data to obtain a dose-effect curve, and the IC50 value was calculated accordingly.
  • Cell survival rate (%) (Lum test drug -Lum culture solution control )/(Lum cell control -Lum culture solution control ) ⁇ 100%.
  • Fig. 2 The relational curve of Hs746T cell survival rate and ensartinib concentration is shown in Fig. 2, and test result shows, the ensartinib dose-dependently inhibiting the proliferation of Hs746T cell of the present application, IC 50 is 31nM, shows that ensartinib is to carrying Human gastric cancer cells with MET exon 14 skipping mutations are significantly suppressed.
  • mice SPF animals, 6-8 weeks old, weighing 18-23 grams; sex: female; quantity: 18 mice; provided by Shanghai Sipro-Bikay Laboratory Animal Co., Ltd.;
  • Double antibody penicillin, streptomycin: (Gibco, 15240-062).
  • solvent control group aqueous solution containing 0.5% HPMC and 0.4% Tween 80.
  • Hs746T cells (carrying MET exon 14 skipping mutation, sourced from ATCC (American Type Culture Collection)) to establish Hs746T xenograft tumor animal models.
  • Experiments were divided into solvent control group and ensartinib hydrochloride Group (in terms of ensartinib, dosage 30mg/kg), every group of 6 experimental animals.
  • the test compound was administered by intragastric administration from the day of grouping, twice a day, and administered for 18 days altogether. According to the changes in animal body weight and The safety evaluation was carried out for the death situation, and the curative effect evaluation was carried out according to the relative tumor inhibition rate (TGI%).
  • Human gastric cancer Hs746T cells were cultured in a monolayer in vitro.
  • the culture conditions were DMEM medium plus 10% fetal bovine serum, 1% double antibody, and cultured in a CO 2 incubator at 37°C. Conventional passaging was performed twice a week. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected, counted, and inoculated.
  • 0.1 mL (2 ⁇ 10 6 ) of Hs746T cells were subcutaneously inoculated on the right back of each mouse, and administered in groups when the tumor volume reached about 128 mm 3 .
  • Tumor diameters were measured twice a week with vernier calipers.
  • TGI (%) [(1-(average tumor volume at the end of administration of a treatment group-average tumor volume at the beginning of administration of this treatment group))/(average tumor volume at the end of treatment of the solvent control group Volume-vehicle control group (average tumor volume at the beginning of treatment)] ⁇ 100%.
  • the average value of tumor volume at each time point of each group is shown in Table 1, and the tumor growth curve is shown in Figure 3.
  • the experimental results show that the tumor-bearing mice given different doses of ensartinib hydrochloride can significantly inhibit the growth of tumors, and obvious tumor regression can be observed, and the ensartinib of the present application has obvious anti-tumor effect in vivo. active.
  • Ensartinib Hydrochloride of the present application can significantly inhibit MET (ex14Del) kinase activity in vitro, inhibit the proliferation of cells carrying MET 14 exon skipping, and show significant anti-tumor activity in vivo, so it can be used for For the treatment of diseases carrying MET 14 exon skipping mutations, ensartinib or salts thereof and compositions comprising ensartinib or salts thereof can be used to prepare medicaments for treating diseases carrying MET 14 exon skipping mutations.
  • Embodiment 4 clinical research
  • age 18 years old, male or female; histologically or cytologically confirmed stage IIIB-IV NSCLC patients; MET exon 14 skipping mutation; EGFR/ALK wild type; at least one evaluable lesion.
  • Treatment plan Ensartinib hydrochloride 225 mg orally once a day until disease progression or unacceptable toxicity occurs.

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Abstract

本申请实施例提供了恩沙替尼或其盐以及包含恩沙替尼或其盐的组合物在制备治疗携带MET 14外显子跳跃突变的疾病的药物中的用途。

Description

恩沙替尼或其盐在治疗携带MET 14外显子跳跃突变的疾病中的用途
本申请要求于2021年8月10日提交国际局、国际申请号为PCT/CN2021/111878、发明名称为“恩沙替尼或其盐在治疗MET14外显子跳跃突变引起的疾病中的用途”的国际申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及恩沙替尼新用途技术领域，特别是涉及恩沙替尼或其盐在治疗携带MET 14外显子跳跃突变的疾病中的用途。
背景技术
MET基因位于7号染色体(7q21-q31)，由21个外显子组成。c-MET是由MET基因编码的跨膜受体，属于酪氨酸激酶受体(RTK)，与多种癌基因产物和调节蛋白相关，是细胞增殖、分化和运动的重要因素。c-MET通路异常激活主要包括MET 14外显子跳跃突变、MET基因扩增和c-MET蛋白过表达3种类型(Organ S L,Tsao M S.An overview of the c-MET signaling pathway[J].Therapeutic Advances in Medical Oncology,2011,3(1Suppl).)。
MET 14外显子对应编码141个氨基酸，其所在的近膜结构域是c-MET的关键负性调控区，包含着E3泛素连接酶c-Cbl酪氨酸结合位点(Y1003)，参与c-MET蛋白的泛素化和降解。MET 14外显子跳跃突变主要是由于在MET基因在转录后，剪接过程中14号外显子随两端内含子一同被剪接，形成了较正常转录剪接少141bp碱基对的成熟mRNA，即形成产生MET 14号外显子的成熟mRNA，进而通过核糖体翻译得到少47个氨基酸系列的c-Met受体蛋白(L964-D1010)Pilotto S,Gkountakos A,Carbognin L,et al.MET exon 14 juxtamembrane splicing mutations:Clinical and therapeutical perspectives for cancer therapy[J].Annals of Translational Medicine,2017,5(1):2-2.)。该突变使得含有E3泛素连接酶c-Cbl结合位点的近膜结构域缺失，从而导致c-Cbl无法结合c-met蛋白，进而c-MET蛋白的泛素化和降解受限、c-MET的稳定性增加，引起MET下游信号的持续激活，最终成为肿瘤的驱动基因之一。
在癌症的治疗中，针对特定肿瘤驱动基因的靶向药物发挥着极其重要的 作用。以非小细胞肺癌(NSCLC)为例，目前已知的可用于治疗NSCLC的药物包括针对EGFR T790M基因突变的奥西替尼；针对EGFR 20外显子插入突变的武田制药TAK-788等；不同的靶向药物针对特定靶点及其突变引起的癌症具有好的治疗效果，而对其他基因突变引起的同类癌症是否具有相同或相似的效果不可预知；MET 14外显子跳跃突变发生在3-4％的新诊断晚期NSCLC病例中，此类癌症预后非常差，尚需开发更多能够针对MET 14外显子跳跃突变的晚期NSCLC，以及其他携带MET 14外显子跳跃突变的疾病的药物。
恩沙替尼是一种已上市的用于治疗ALK阳性非小细胞肺癌的药物，其是否能够用于治疗携带MET 14外显子跳跃突变的疾病，例如携带MET 14外显子跳跃突变的晚期NSCLC目前并没有研究。
发明内容
本申请的发明人在研究中意外地发现，恩沙替尼或其盐具有治疗携带MET 14外显子跳跃突变的疾病的作用，并基于此完成了本申请。
本申请第一方面提供了恩沙替尼或其盐、包含恩沙替尼或其盐的组合物在治疗携带MET 14外显子跳跃突变的疾病中的用途，其中，所述恩沙替尼具有式I所示的结构：
Figure PCTCN2022110160-appb-000001
本申请第二方面提供了恩沙替尼或其盐、包含恩沙替尼或其盐的组合物在制备用于治疗携带MET 14外显子跳跃突变的疾病的药物中的用途，其中所述恩沙替尼具有式I所示的结构：
Figure PCTCN2022110160-appb-000002
本申请第三方面提供了一种治疗携带MET 14外显子跳跃突变的疾病的方法，其向有需要的对象施以有效剂量的恩沙替尼或其盐，或包含恩沙替尼或其盐的组合物，其中，所述恩沙替尼具有式I所示的结构：
Figure PCTCN2022110160-appb-000003
本申请提供的恩沙替尼或其盐、包含恩沙替尼或其盐的组合物能够有效抑制MET 14外显子跳跃突变激酶活性，显著抑制携带MET 14外显子跳跃突变的人胃癌Hs746T细胞的增殖，在体内对携带MET 14外显子跳跃突变的异种移植瘤模型展示出良好的抗肿瘤活性，并在临床试验中展示出显著的疗效。因此恩沙替尼、恩沙替尼的盐以及包含恩沙替尼或其盐的组合物能够用于治疗携带MET 14外显子跳跃突变的疾病，进而能够用于制备治疗MET14外显子跳跃突变引起的疾病的药物。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一种实施方式，对于本领域普通技术人员来讲，还可以根据这些附图获得其他的实施方式。
图1为MET 14外显子跳跃突变激酶抑制率与恩沙替尼浓度的关系曲线。
图2为Hs746T细胞存活率与恩沙替尼浓度的关系曲线。
图3为不同剂量盐酸恩沙替尼治疗下小鼠体内肿瘤体积生长曲线。
具体实施方式
为使本申请的目的、技术方案、及优点更加清楚明白，以下参照附图并举实施例，对本申请进一步详细说明。显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。本领域普通技术人员基于本申请中的实施例所获得的所有其他实施例，都属于本申请保护的范围。
本申请中，“施用”和“治疗”在其应用至动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体接触。如本文所用，“治疗”意指向具有携带MET 14外显子跳跃突变的疾病或诊断具有携带MET 14外显子跳跃突变的疾病的受试者施用恩沙替尼或其盐，或包含恩沙替尼或其盐的组合物，以实现至少一种阳性治疗效果，例如当所述疾病为癌症时，减少癌细胞数量，减小肿瘤大小，降低癌细胞浸润至外周器官中的速率，或降低肿瘤转移或肿瘤生长的速率。“治疗”可以包括：中止或延迟与携带MET 14外显子跳跃突变的疾病的进展，携带MET 14外显子跳跃突变的疾病的稳定化，改善或废除携带MET 14外显子跳跃突变的疾病的临床表现，降低携带MET 14外显子跳跃突变的疾病的临床症状的严重程度或持续时间，相对于未治疗的类似患者的预期存活期延长患者的存活期，和诱导携带MET 14外显子跳跃突变的疾病的完全或部分缓解；例如当所述疾病为癌症时，“治疗”可以包括以下中的一项或多项：减少一种或多种肿瘤标志物的数量，中止或延迟肿瘤的生长，抑制肿瘤细胞的生长或存活，消除或减小一个或多个癌性病变或肿瘤的大小，降低一种或多种肿瘤标志物的水平，诱导癌症病况的完全或者部分缓解。
如本文使用的术语“有效剂量”是指这样的量，当以合适的给药方案施用时，该量足够降低或改善要治疗的紊乱的严重度、持续时间或发展，防止要治疗的紊乱的进展，引起要治疗的紊乱的消退，或增强或改善另一种疗法的预防或治疗效果。
如本文所用，且除非另有说明，否则术语“对象”在本文定义为包括动物例如哺乳动物，包括但不限于灵长类动物(例如人)、牛、绵羊、山羊、马、 狗、猫、兔、大鼠、小鼠等。在具体的实施方案中，对象是人。在提及例如哺乳动物对象(例如人)时，术语“对象”和“患者”在本文中互换使用。
术语“约”是指可能发生的数值的变异，例如，通过参与物质或组合物的制备、表征和/或使用的典型的测量、处理和取样程序；通过这些程序中的无意错误；通过制作或使用组合物或实施程序所用的成分的制造、来源或纯度的差异；等。在某些实施方案中，“约”可以意指±0.1％、±0.5％、±1％、±2％、±3％、±4％、±5％、±6％、±7％、±8％、±9％或±10％的变异。
本申请第一方面提供了恩沙替尼(Ensartinib)或其盐、包含恩沙替尼或其盐的组合物在治疗携带MET 14外显子跳跃突变的疾病中的用途，其中，所述恩沙替尼具有式I所示的结构：
Figure PCTCN2022110160-appb-000004
本申请第二方面提供了恩沙替尼或其盐、包含恩沙替尼或其盐的组合物在制备用于治疗携带MET 14外显子跳跃突变的疾病的药物中的用途，其中所述恩沙替尼具有式I所示的结构：
Figure PCTCN2022110160-appb-000005
进一步地，所述携带MET 14外显子跳跃突变的疾病包括携带MET 14外显子跳跃突变的肺癌、结肠癌、乳腺癌、前列腺癌、肝癌、胰腺癌、脑癌、肾癌、卵巢癌、胃癌、皮肤癌、骨癌、神经胶质瘤、乳头状肾细胞癌或头颈部鳞状细胞癌中的至少一种。
进一步地，所述携带MET 14外显子跳跃突变的疾病为非小细胞肺癌。
本申请所说的组合物，其包括有效剂量的恩沙替尼，或如果适用，所述化合物的药学可接受的盐、盐的多晶型物、溶剂合物、水合物、多晶型物或前药；以及可接受的载体。
本申请所说的“药学可接受的盐”、“盐”包括碱根离子与自由酸形成的盐，例如包括盐酸盐、磷酸盐、甲酸盐、乙酸盐、富马酸盐、草酸盐、马来酸盐、柠檬酸盐等。
优选地，本申请的组合物为药用用途而配制(“药物组合物”)，其中所述载体是药学可接受的载体。考虑到与制剂其他成分相容、并且在药学可接受的载体的情况下，所述载体必须是“可接受的”，在典型用于药物中的量下不损害其接受者。
可用于本申请的药物组合物的药学可接受的载体、佐剂和赋形剂包括但不限于，离子交换剂、氧化铝、硬脂酸铝、卵磷脂、例如人血清白蛋白的血清蛋白、例如磷酸盐的缓冲物质、甘氨酸、山梨酸、山梨酸钾、饱和植物脂肪酸的偏甘油酯混合物、水、盐或电解质，例如硫酸鱼精蛋白、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐、胶体二氧化硅、三硅酸镁、聚乙烯吡咯烷酮、纤维素基物质、聚乙二醇、羧甲基纤维素钠、聚丙烯酸酯、蜡、聚乙烯-聚氧化丙烯-嵌段聚合物、聚乙二醇和羊毛脂。
本申请的药物组合物包括适合于口服、直肠、鼻、局部(包括口腔和舌下)、阴道或胃肠外(包括皮下、肌内、静脉内和皮内)给药的那些。在某些实施方式中，本文的恩沙替尼透皮(例如，利用透皮贴片)给药。其他制剂可以方便地以单位剂型存在，例如，片剂和持续释放胶囊，以及在脂质体中存在，并可以通过药学领域中已知的任何方法来制备。参见，例如Remington’s Pharmaceutical Sciences(雷明顿制药科学)，Mack Publishing Company，Philadelphia，PA(第17版，1985)。
这样的制备方法包括使例如构成一种或多种辅助成分的载体的成分与要施用的分子相缔合的步骤。一般地，通过将活性成分与液体载体、脂质体或细分散的固体载体或两者进行均匀地和紧密地缔合，然后如有必要使产品成型，来制备组合物。
在某些优选的实施方式中，所述化合物口服给药。适合于口服给药的本申请的组合物可以作为离散单位存在，例如各自含有预定量的活性成分的胶囊、小袋(sachet)或片剂；作为粉剂或颗粒剂；作为水性液体或非水性液体中的溶液或悬浮液；或作为水包油液体乳剂或油包水液体乳剂，或包裹在脂质体中，以及作为丸剂等等。软明胶胶囊可用于含有这样的悬浮液，其可以有益地提高化合物的吸收率。
片剂可以通过压缩或模压，任选地与一种或多种辅助成分一起来制成。压缩的片剂可以通过将自由流动形式的活性成分例如粉剂或颗粒剂，任选地与粘合剂、润滑剂、惰性稀释剂、防腐剂、表面活性剂或分散剂混合，在适合的机器中压缩来制备。模压的片剂可以通过在适合的机器中模压用惰性液体稀释剂湿润的粉末状化合物的混合物来制成。片剂任选地可以被包衣或刻痕，并且可以被配制以提供其中活性成分的缓慢或受控释放。配制这种药学活性成分(例如本文的那些和本领域已知的其他化合物)的缓慢或受控释放的组合物的方法的是本领域已知的，并且在一些授权的美国专利中描述过，其中一些包括但不限于美国专利第4,369,172号和第4,842,866号、以及其中引用的参考文献。可以使用包衣将化合物递送到肠(参见，例如，美国专利第6,638,534、5,217,720、和6,569,457、6,461,631、6,528,080、6,800,663号和其中引用的参考文献)。对于本申请的化合物的有用的制剂是肠丸粒的形式，其中肠层包含羟丙基甲基纤维素醋酸琥珀酸盐。
在口服用途的片剂的情况下，常用的载体包括乳糖和玉米淀粉。典型地也添加润滑剂，例如硬脂酸镁。对于以胶囊剂形式口服给药，有用的稀释剂包括乳糖和干玉米淀粉。当口服施用水性悬浮液时，活性成分与乳化剂和悬浮剂结合。如果需要，可以添加某些甜味剂和/或调味剂和/或着色剂。
适合于局部给药的组合物包括锭剂(lozenge)，其包括在调味基质中的成分，调味基质通常为蔗糖和阿拉伯胶或黄耆胶；和软锭剂(pastille)，其包括在惰性基质中的活性成分，惰性基质是例如明胶和甘油、或蔗糖和阿拉伯胶。
适合于肠胃外给药的组合物包括水性和非水性无菌注射溶液，其可以含有抗氧化剂、缓冲剂、抑菌剂和使得制剂与预定接受者的血液等渗的溶质；以及水性和非水性无菌悬浮液，其可以包括悬浮剂和增稠剂。制剂可以存在 于单位剂量或多剂量容器中，例如，密封的安瓿瓶和小瓶，并可以保存在冷冻干燥的(冻干)条件中，仅需要在使用前即刻添加无菌的液体载体，例如注射用水。临时的注射溶液和悬浮液可以从无菌的粉剂、颗粒剂和片剂来制备。
这样的注射溶液可以是例如无菌可注射的水性或油性悬浮液的形式。这些悬浮液可以根据本领域已知的技术使用适合的分散剂或润湿剂(例如，Tween80)和悬浮剂来配制。无菌可注射制剂也可以是在无毒的胃肠外可接受的稀释剂或溶剂中，例如1，3-丁二醇溶液中的无菌可注射溶液或悬浮液。在可接受的赋形剂和溶剂之中，可以使用甘露醇、水、Ringer’s溶液和等渗氯化钠溶液。此外，无菌的不挥发性油通常用作溶剂或悬浮介质。为此目的，可以使用任何温和的不挥发性油，包括合成的单甘油酯或二甘油酯。脂肪酸，例如油酸和它的甘油酯衍生物在注射剂的制备中是有用的，其是天然的药学可接受的油，例如橄榄油或蓖麻油，特别是它们的聚氧乙烯化形式也是一样。这些油溶液或悬浮液也可含有长链醇类稀释剂或分散剂。
本申请的药物组合物可以以用于直肠给药的栓剂形式来施用。这些组合物可以通过将本申请的化合物与适合的无刺激性赋形剂混合来制备，所述赋形剂在室温下是固体但在直肠温度下是液体，因而将在直肠中融化而释放出活性组分。这种材料包括但不限于，可可脂、蜂蜡和聚乙二醇。
本申请的药物组合物可以通过鼻气雾剂或吸入剂来给药。根据药物制剂领域公知的技术来制备这种组合物，并且可以将其制备为盐水的溶液，并使用苯甲醇或其他适合的防腐剂、提高生物利用度的吸收促进剂、氟碳化合物、和/或本领域已知的其他增溶剂或分散剂。
当希望的治疗涉及通过局部施用容易达到的区域或器官时，本申请的药物组合物的局部给药是特别有用的。为了局部用于皮肤，药物组合物应配制为含有悬浮于或溶解于载体的活性组分的适合的软膏剂。用于局部施用本申请的化合物的载体包括但不限于，矿物油、液体石油、白石油、丙二醇、聚氧乙烯聚氧丙烯化合物、乳化蜡和水。或者，药物组合物可以用适合的洗液或乳膏剂配制，其含有悬浮于或溶解于载体中的活性化合物。适合的载体包括但不限于，矿物油、单硬脂酸山梨糖醇酯、聚山梨醇酯60、十六烷基酯蜡、 鲸腊硬脂(cetearyl)醇、2-辛基十二烷醇、苯甲醇和水。本申请的药物组合物也可以通过直肠栓剂或以适合的灌肠制剂的形式局部地施用到低位肠道。局部透皮贴片和离子电渗给药也包括在本申请中。
在本申请的组合物中，本申请的恩沙替尼或其盐以有效剂量存在。在Freireich等，(1966)Cancer Chemother Rep50：219中描述了剂量对动物和人的相互关系(根据毫克每平方米体表面积)。体表面积可以根据患者的身高和体重大致地确定。参见，例如，Scientific Tables，Geigy Pharmaceuticals(科学表格，Geigy药物)，Ardley，N.Y.，1970，537。本申请的恩沙替尼或其盐的有效量可以从约5mg/天到约500mg/天，优选10mg/天至400mg/天，再优选25mg/天至250mg/天。在一些实施方式中，本申请的恩沙替尼的有效量可以为10mg/天、20mg/天、25mg/天、30mg/天、50mg/天、75mg/天、90mg/天、100mg/天、120mg/天、150mg/天、180mg/天、200mg/天、225mg/天、250mg/天、300mg/天、350mg/天、400mg/天等。有效的剂量也可以变化，如本领域技术人员所了解的，取决于所治疗的疾病、疾病的严重度、给药途径、患者的性别、年龄和一般健康状况、赋形剂用法、与其他治疗方法共同使用的可能性(例如使用其他试剂)、以及治疗医师的判断。
本申请第三方面提供了一种治疗携带MET 14外显子跳跃突变的疾病的方法，其向有需要的对象施以有效剂量的恩沙替尼或其盐，或包含恩沙替尼或其盐的组合物，其中，所述恩沙替尼具有式I所示的结构：
Figure PCTCN2022110160-appb-000006
在一些实施方式中，通过口服、直肠、鼻、局部、阴道或胃肠外给药，向有需要的对象施以有效剂量的所述恩沙替尼或其盐，或包含恩沙替尼或其盐的组合物。
在一些实施方式中，所述有效剂量以恩沙替尼或其盐计，剂量为约5mg/天至500mg/天，优选10mg/天至400mg/天，再优选25mg/天至250mg/天，更优 选为225mg/天。
本申请第四方面提供了一种用于治疗携带MET 14外显子跳跃突变的疾病的恩沙替尼或其盐，或包含恩沙替尼或其盐的组合物，其中所述恩沙替尼具有式I所示的结构：
Figure PCTCN2022110160-appb-000007
实施例中使用了下列缩略语：
ADP：二磷酸腺苷；
ATP：三磷酸腺苷；
HPMC：羟丙基甲基纤维素；
DMSO：二甲基亚砜；
FBS：胎牛血清；
PBS：磷酸盐缓冲盐溶液；
NSCLC：非小细胞肺癌；
CR：完全缓解；
PR：部分缓解；
SD：疾病稳定；
DCR：疾病控制率；
ORR：客观缓解率；
CTG试剂：CellTiter-Glo试剂；
TGI(％)：肿瘤生长抑制率。
实施例1激酶抑制活性测定
采用ADP-Glo激酶检测试剂盒测试评价恩沙替尼对MET 14外显子缺失(即MET ex14Del)激酶的抑制作用。
实验材料：
MET(ex14Del)激酶(Sinalchem，M52-12PG)；
ADP-Glo激酶检测试剂盒(Promega，V9102)；
测试在384孔板中进行，将待测化合物盐酸恩沙替尼从300μM开始按照1:3的体积比在DMSO中连续稀释9次，共10个浓度梯度的100×化合物工作液，稀释后的待测化合物溶液加入384孔板中，每孔0.1μL。向384孔板中加入5μL酶工作液，1000rpm离心1min，25℃孵育15分钟。向上述孔板中加入5μL/孔的底物工作液，得到化合物终浓度为最大3000nM、3倍稀释共10个浓度梯度的1×化合物工作液，引发反应，然后25℃孵育60min。向上述孔板中加入10μL ADP-Glo试剂(Promega，V9102)，1000rpm离心1分钟，25℃孵育60min，而后每孔加入20μL激酶检测试剂，1000rpm离心1min。使用Envision多功能读板机读取RLU(相对发光单位)值，RLU值大小用于表征酶与底物反应程度，用XLfit 5.3.1由非线性回归方程拟合化合物的IC 50。
MET 14跳跃突变激酶抑制率与恩沙替尼浓度的关系曲线如图1所示，可见本申请的恩沙替尼对MET(ex14Del)激酶的IC 50(半抑制浓度)为7.6nM，说明恩沙替尼对MET 14外显子跳跃突变激酶具有优异的抑制作用。
实施例2细胞增殖抑制活性
采用CellTiter-Glo TM活细胞检测试剂盒方法，评价恩沙替尼对人肿瘤细胞Hs746T细胞株的生长抑制作用。
实验材料与细胞系：
人胃癌细胞Hs746T细胞株(携带MET 14外显子跳跃突变，来源为ATCC(American Type Cultural Collection)，在DMEM+10％FBS的培养基中生长)；
高糖培养基DMEM(Gibco，11995-065)；
胎牛血清FBS(Gibco Cat#10099-141)；
Figure PCTCN2022110160-appb-000008
发光细胞活力检测试剂(Promega,Cat#G7573)；
盐酸恩沙替尼(贝达制药，Z170401T)，以DMSO配制成浓度为10mM的药物储备液。
实验方法：
一、细胞培养和接种
1.收获处于对数生长期的细胞并采用血小板计数器进行细胞计数。用台盼蓝排斥法检测细胞活力，确保细胞活力在90％以上。
2.调整细胞浓度，分别添加180μL细胞悬液至96孔板中，细胞接种密度1200个/孔。
3.将96孔板中的细胞置于37℃、5％CO 2、95％湿度条件下培养过夜。
二、T0读板
1.在接种有细胞的96孔板中每孔加入20μL PBS。
2.融化CTG试剂并平衡细胞板至室温30分钟。
3.每孔加入100μL的CTG溶液。
4.在定轨摇床上振动5分钟使细胞裂解。
5.将细胞板放置于室温20分钟以稳定冷光信号。
6.读取T0冷光值Lum。
三、药物稀释和加药
在相应的接种有细胞的96孔板中每孔加入20μL药物溶液，恩沙替尼浓度分别为10000nM、3333nM、1111nM、370nM、124nM、41nM、14nM、4.6nM、1.5nM：每个药物浓度设置三个复孔，DMSO终浓度为0.1％。
3.将已加药的96孔板中的细胞置于37℃、5％CO 2、95％湿度条件下继续培养168小时，之后进行CTG分析。
四、终点读板
1.融化CTG试剂并平衡细胞板至室温30分钟。
2.每孔加入100μL的CTG试剂。
3.在定轨摇床上振动5分钟使细胞裂解。
4.将细胞板放置于室温20分钟以稳定冷光信号。
5.读取终点冷光值Lum。
数据处理与实验结果：
使用GraphPad Prism 7.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算IC 50值。
细胞存活率(％)＝(Lum 待测药-Lum 培养液对照)/(Lum 细胞对照-Lum 培养液对照)×100％。
Hs746T细胞存活率与恩沙替尼浓度的关系曲线见图2，试验结果表明，本申请的恩沙替尼剂量依赖性地抑制Hs746T细胞的增殖，IC 50为31nM，说明恩沙替尼对携带MET 14外显子跳跃突变的人胃癌细胞具有显著的抑制作用。
实施例3体内抗肿瘤活性测定
实验动物及实验材料：
BALB/c裸小鼠：SPF级动物，6-8周龄，体重18-23克；性别：雌性；数量：入组小鼠18只；由上海西普尔-必凯实验动物有限公司提供；
双抗(青霉素，链霉素)：(Gibco，15240-062)。
溶剂对照组给药：含0.5％HPMC和0.4％吐温80的水溶液。
实验方法：
1.建模
BALB/c裸小鼠皮下接种Hs746T细胞(携带MET 14外显子跳跃突变，来源为ATCC(American Type Cultural Collection)，建立Hs746T异种移植肿 瘤动物模型。实验分为溶剂对照组和盐酸恩沙替尼组(以恩沙替尼计，给药量30mg/kg)，每组6只实验动物。测试化合物从分组当天开始灌胃给药，每天两次，共给药18天。根据动物体重变化和死亡情况进行安全性评价，根据相对肿瘤抑制率(TGI％)进行疗效评价。
2.细胞培养
人胃癌Hs746T细胞体外单层培养，培养条件为DMEM培养基中加10％胎牛血清，1％双抗，37℃CO 2孵箱培养。一周两次进行常规处理传代。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数，接种。将0.1mL(2×10 6个)Hs746T细胞皮下接种于每只小鼠的右后背，待肿瘤体积约达到128mm 3进行分组给药。
3.肿瘤测量和实验指标
每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。
在实验结束后将检测肿瘤重量，并计算TGI(％)，以反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝[(1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积))/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)]×100％。
数据处理与实验结果：
每个组的每个时间点的肿瘤体积的平均值见表1，肿瘤生长曲线见图3。由实验结果表明，荷瘤小鼠给予不同剂量的盐酸恩沙替尼，能够显著抑制肿瘤的生长，且均可观察到明显的肿瘤消退现象，本申请的恩沙替尼体内具有明显的抗肿瘤活性。
表1各组不同时间点的瘤体积
Figure PCTCN2022110160-appb-000009
Figure PCTCN2022110160-appb-000010
综合以上实施例，本申请的盐酸恩沙替尼体外能够显著抑制MET(ex14Del)激酶活性、抑制携带MET 14外显子跳跃突细胞的增殖，体内表现出显著的抗肿瘤活性，因此能够用于治疗携带MET 14外显子跳跃突变的疾病，进而恩沙替尼或其盐以及包含恩沙替尼或其盐的组合物能够用于制备治疗携带MET 14外显子跳跃突变的疾病的药物。
实施例4临床研究
关键入组标准：年龄≥18周岁，男女不限；组织学或细胞学确诊的IIIB-IV期NSCLC患者；携带MET 14外显子跳跃突变；EGFR/ALK野生型；至少有一个可评估病灶。
治疗方案：每天口服一次225mg盐酸恩沙替尼直至疾病进展或出现无法耐受的毒性。
临研究结果：在17名患者中，出现1例CR、11例PR、4例SD。DCR达到94％(16/17)，ORR达到71％(12/17)；平均肿瘤缩小率达到-37.7％。上述结果表明本申请所述的恩沙替尼在携带MET 14外显子跳跃突变的疾病中具有显著的疗效。
以上所述仅为本申请的较佳实施例而已，并不用以限制本申请，凡在本 申请的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本申请保护的范围之内。

Claims (10)

  1. 恩沙替尼或其盐、包含恩沙替尼或其盐的组合物在治疗携带MET 14外显子跳跃突变的疾病中的用途，其中，所述恩沙替尼具有式I所示的结构：
    Figure PCTCN2022110160-appb-100001
  2. 恩沙替尼或其盐、包含恩沙替尼或其盐的组合物在制备用于治疗携带MET 14外显子跳跃突变的疾病的药物中的用途，其中所述恩沙替尼具有式I所示的结构：
    Figure PCTCN2022110160-appb-100002
  3. 根据权利要求1或2所述的用途，其中，所述携带MET 14外显子跳跃突变的疾病包括携带MET 14外显子跳跃突变的肺癌、结肠癌、乳腺癌、前列腺癌、肝癌、胰腺癌、脑癌、肾癌、卵巢癌、胃癌、皮肤癌、骨癌、神经胶质瘤、乳头状肾细胞癌或头颈部鳞状细胞癌中的至少一种。
  4. 根据权利要求1或2所述的用途，其中，所述恩沙替尼或其盐、包含恩沙替尼或其盐的组合物通过口服、直肠、鼻、局部、阴道或胃肠外给药的方式施用于有需要的对象。
  5. 根据权利要求1或2所述的用途，其中，向有需要的对象施以恩沙替尼或其盐，或包含恩沙替尼或其盐的组合物，以恩沙替尼计，剂量为5mg/天至500mg/天，优选10mg/天至400mg/天，再优选25mg/天至250mg/天。
  6. 根据权利要求1-5中任一项所述的用途，其中，所述恩沙替尼的盐为盐 酸盐、磷酸盐、甲酸盐、乙酸盐、富马酸盐、草酸盐、马来酸盐、柠檬酸盐中的一种，优选为盐酸盐。
  7. 一种治疗携带MET 14外显子跳跃突变的疾病的方法，其包括向有需要的对象施以有效剂量的恩沙替尼或其盐，或包含恩沙替尼或其盐的组合物，其中，所述恩沙替尼具有式I所示的结构：
    Figure PCTCN2022110160-appb-100003
  8. 根据权利要求7所述的方法，其中，通过口服、直肠、鼻、局部、阴道或胃肠外给药。
  9. 根据权利要求7所述的方法，其中，所述有效剂量以恩沙替尼计，为5mg/天到500mg/天，优选10mg/天至400mg/天，再优选25mg/天至250mg/天。
  10. 根据权利要求7-9中任一项所述的方法，其中所述恩沙替尼的盐为盐酸盐、磷酸盐、甲酸盐、乙酸盐、富马酸盐、草酸盐、马来酸盐、柠檬酸盐中的一种，优选为盐酸盐。
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