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WO2023035201A1 - 五氟利多联合醋酸甲羟孕酮在制备治疗子宫内膜癌的药物中的应用 - Google Patents

五氟利多联合醋酸甲羟孕酮在制备治疗子宫内膜癌的药物中的应用 Download PDF

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Publication number
WO2023035201A1
WO2023035201A1 PCT/CN2021/117536 CN2021117536W WO2023035201A1 WO 2023035201 A1 WO2023035201 A1 WO 2023035201A1 CN 2021117536 W CN2021117536 W CN 2021117536W WO 2023035201 A1 WO2023035201 A1 WO 2023035201A1
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Prior art keywords
endometrial cancer
penfluridol
cells
medroxyprogesterone acetate
pharmaceutical composition
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English (en)
French (fr)
Inventor
王玉东
孙笑
毛斐
伍慧雯
刘小虎
李丽娟
李剑
李晓康
朱进
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International Peace Maternity And Child Health Hospital
East China University of Science and Technology
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International Peace Maternity And Child Health Hospital
East China University of Science and Technology
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Priority to PCT/CN2021/117536 priority Critical patent/WO2023035201A1/zh
Publication of WO2023035201A1 publication Critical patent/WO2023035201A1/zh
Anticipated expiration legal-status Critical
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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
    • A61K31/435—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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/451—Non condensed piperidines, e.g. piperocaine having a carbocyclic group directly attached to the heterocyclic ring, e.g. glutethimide, meperidine, loperamide, phencyclidine, piminodine
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • 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 invention relates to the technical field of medicine, in particular to the application of penfluridol combined with medroxyprogesterone acetate in the preparation of medicines for treating endometrial cancer.
  • Endometrial cancer is a group of epithelial malignant tumors that occur in the endometrium. It is one of the most common gynecological malignancies in the world, and its morbidity and mortality are increasing (Karen, Endometrial Cancer , The New England Journal of Medicine 2020, 383, 2053-2064).
  • EC Endometrial Cancer
  • the incidence of EC in young women has increased dramatically over the past decade, with younger and younger women being diagnosed with endometrial cancer, and the underlying cause of the increase is obesity
  • the prevalence and resulting hyperinsulinemia, and endometrial cancer rates are rising as obesity rates rise (Moore, Endometrial Cancer: Is This a New Disease? Am. Soc. Clin. Oncol. 2017, 37, 435-442).
  • 2020 there will be 420,000 new cases of endometrial cancer worldwide, including 80,000 new cases in China.
  • EC is usually divided into type I and type II.
  • type I EC is positive for progesterone receptor expression
  • type II EC is negative for progesterone receptor expression
  • progesterone receptor expression Bookhman, Gynecologic oncology, 1983, 15, 10-17 .
  • surgical treatment is a common method for the treatment of EC, but conservative treatment with progesterone therapy is required for patients who need to protect their reproductive function.
  • the main method of conservative treatment is hormone therapy with progesterone drugs (mainly medroxyprogesterone acetate (MPA)).
  • MPA medroxyprogesterone acetate
  • hormone therapy has limitations. It is only effective for patients with positive progesterone receptor expression and has a high recurrence rate, which is prone to drug resistance.
  • the purpose of the present invention is to provide a drug capable of treating EC, especially progesterone receptor-negative and progesterone-resistant EC.
  • composition comprising:
  • the pharmaceutical composition is used for treating and/or preventing endometrial cancer.
  • the endometrial cancer includes type I and type II endometrial cancer.
  • the endometrial cancer includes progesterone-sensitive endometrial cancer or progesterone-resistant endometrial cancer.
  • the endometrial cancer is progesterone receptor-negative and progesterone-resistant endometrial cancer.
  • the progestin is selected from the group consisting of medroxyprogesterone acid, megestrol acetate, progesterone caproate, or combinations thereof.
  • the progestin is medroxyprogesterone acetate.
  • the weight ratio of the first active ingredient to the second active ingredient is from 1:20 to 10:1, preferably from 1:15 to 1:1, more preferably from 1:10 to 1:5.
  • the content of the first active ingredient in the pharmaceutical composition is 0.01-90wt%, preferably 0.1-30wt%, more preferably 0.5-10wt%.
  • the content of the second active ingredient in the pharmaceutical composition is 0.01-90wt%, preferably 0.1-30wt%, more preferably 0.5-10wt%.
  • the first active ingredient and the second active ingredient account for 0.01-90wt% of the total mass of the pharmaceutical composition, preferably 0.1-50wt%, more preferably 0.5-30wt%.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the dosage form of the pharmaceutical composition includes injection dosage form and oral dosage form.
  • the dosage forms of the pharmaceutical composition include injections, tablets, capsules, pills, suspensions and emulsions.
  • the pharmaceutical composition further includes other anti-endometrial cancer drugs.
  • a kit which includes:
  • the progestin is selected from the group consisting of medroxyprogesterone acid, megestrol acetate, progesterone caproate, or combinations thereof.
  • the kit further includes instructions, which indicate that the first preparation and the second preparation are used in combination to treat and/or alleviate endometrial cancer.
  • the first preparation and the second preparation are administered simultaneously, separately or sequentially in the treatment of endometrial cancer.
  • the first preparation and the second preparation in the kit are independent of each other, or combined.
  • the first preparation and the second preparation in the kit are independent of each other.
  • the first preparation is an oral preparation or an injection preparation.
  • the dosage forms of the first preparation include injections, tablets, capsules, pills, suspensions and emulsions.
  • the second preparation is an oral preparation or an injection preparation.
  • the dosage forms of the second preparation include injections, tablets, capsules, pills, suspensions and emulsions.
  • a combination of active ingredients which includes:
  • the progestin is selected from the group consisting of medroxyprogesterone acid, megestrol acetate, progesterone caproate, or combinations thereof.
  • the fourth aspect of the present invention there is provided a use of the pharmaceutical composition described in the first aspect of the present invention, the kit described in the second aspect and/or the combination of active ingredients described in the third aspect, for the preparation of Medicines to treat and/or relieve endometrial cancer.
  • the present invention also provides the use of the pharmaceutical composition, the kit and/or the combination of active ingredients described in the present invention for treating and/or alleviating endometrial cancer.
  • the endometrial cancer includes type I and type II endometrial cancer.
  • the drug is also used for one or more of the following purposes:
  • the endometrial cancer cells are selected from the group consisting of ISK, KLE, or a combination thereof.
  • the endometrial cancer cells are ISK and/or KLE cells.
  • a method for inhibiting endometrial cancer cells in vitro comprising the steps of:
  • the method is non-therapeutic and non-diagnostic.
  • the inhibition is selected from the group consisting of:
  • the endometrial cancer cells are ISK and/or KLE cells.
  • a method for treating endometrial cancer comprising the step of: administering the pharmaceutical composition described in the first aspect of the present invention, the kit described in the second aspect or The combination of active ingredients described in the third aspect.
  • the subject is a patient with endometrial cancer.
  • the endometrial cancer includes type I and type II endometrial cancer.
  • the treatment method further includes administering other anti-endometrial cancer drugs to the subject in need.
  • Figure 1 is a schematic diagram of the inhibitory activity of penfluridol and medroxyprogesterone acetate on the proliferation of ISK and KLE cells.
  • A ISK cell proliferation inhibitory activity
  • B KLE cell proliferation inhibitory activity.
  • Figure 2 is a schematic diagram of the synergistic inhibitory effect of penfluridol and medroxyprogesterone acetate on ISK and KLE cells.
  • A Synergistic inhibitory effect of combined drugs on ISK cells;
  • B Synergistic inhibitory effect of combined drugs on KLE cells.
  • Fig. 3 is a schematic diagram of the effect of penfluridol combined with medroxyprogesterone acetate on the migration ability of ISK and KLE cells.
  • A ISK cell scratch;
  • B KLE cell scratch;
  • C ISK cell wound healing distance percentage;
  • D KLE cell wound healing distance percentage.
  • Fig. 4 is a schematic diagram of the effect of penfluridol and medroxyprogesterone acetate on the migration ability of ISK and KLE cells.
  • A ISK cell crystal violet staining
  • B ISK cell crystal violet staining quantification
  • C KLE cell crystal violet staining
  • D KLE cell crystal violet staining quantification.
  • Fig. 5 is a schematic diagram showing the effects of penfluridol and medroxyprogesterone acetate on the apoptosis of ISK and KLE cells.
  • A Effect of combined drug on ISK cell apoptosis;
  • B Effect of combined drug on KLE cell apoptosis;
  • C Quantification of ISK cell apoptosis ratio;
  • D KLE cell apoptosis ratio quantification.
  • the present inventor unexpectedly found that the combination of antipsychotic drug penfluridol and medroxyprogesterone acetate (MPA) can significantly improve the therapeutic effect on endometrial cancer, and can significantly improve the therapeutic effect on endometrial cancer ISK and
  • the proliferation and migration abilities of KLE cells have a synergistic inhibitory effect, and can synergistically induce the apoptosis of endometrial cancer ISK and KLE cells.
  • the synergistic therapeutic effect of penfluridol and medroxyprogesterone acetate is obviously better than that of penfluridol or medroxyprogesterone acetate alone. On this basis, the present invention has been accomplished.
  • the terms “comprising”, “including”, and “containing” are used interchangeably to include not only closed definitions, but also semi-closed, and open definitions. In other words, the terms include “consisting of”, “consisting essentially of”.
  • pharmaceutically acceptable carrier refers to ingredients suitable for humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergic reactions), that is, with a reasonable benefit/risk ratio substance.
  • the term "therapeutically effective amount” refers to an amount that produces functions or activities on humans and/or animals and is acceptable to humans and/or animals. Those of ordinary skill in the art should understand that the “therapeutically effective amount” may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvant of the drug used, the severity of the disease, and the combination with other drugs. different.
  • Penfluridol Penfluridol
  • PFL the first active ingredient of the present invention
  • the penfluridol has the following structural formula:
  • Penfluridol is an oral long-acting antipsychotic drug approved by the U.S. Food and Drug Administration (FDA). For the treatment of various types of schizophrenia. Penfluridol is a potent inhibitor of dopamine D2 receptors and calcium channels.
  • penfluridol has a proliferation inhibitory effect on both endometrial cancer cells ISK (type I, progesterone-sensitive) and type II endometrial cancer cells KLE (progesterone-resistant). Further studies have found that its combined application with medroxyprogesterone acetate has a significant synergistic inhibitory effect on the two types of cells, can inhibit the proliferation and migration of the two types of EC cells, and induce EC cell apoptosis. Therefore, the present invention provides the use of penfluridol and medroxyprogesterone acetate in combination to prepare a medicine for treating endometrial cancer.
  • second active ingredient of the invention refers to a progestin.
  • the progestin is selected from the group consisting of medroxyprogesterone acid, megestrol acetate, progesterone caproate, or combinations thereof.
  • the progestogen is medroxyprogesterone acetate and pharmaceutically acceptable salts thereof.
  • endometrial cancer is a group of epithelial malignant tumors that occur in the endometrium, also known as uterine body cancer, and is one of the three most common malignant tumors of the female reproductive system. . Endometrial cancer is more likely to occur in perimenopausal and postmenopausal women. According to its pathogenesis and biological behavior characteristics, it can be divided into type I endometrial cancer (estrogen-dependent type) and type II endometrial cancer (non-estrogen-dependent type). hormone-dependent).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition is used for treating and/or preventing endometrial cancer.
  • the present invention also provides a kit, which includes:
  • the first preparation and the second preparation are different preparations or the same preparation.
  • the order of administration is not particularly limited, and they can be administered sequentially, together, or sequentially.
  • the progestin is selected from the group consisting of medroxyprogesterone acid, megestrol acetate, progesterone caproate, or combinations thereof.
  • the progestin is medroxyprogesterone acetate.
  • the weight ratio of the first active ingredient to the second active ingredient is preferably 1:20 to 10:1, more preferably 1:15 to 1:1, further preferably 1:10 to 1:1: 5.
  • the content of the first active ingredient in the pharmaceutical composition is preferably 0.01-90wt%, more preferably 0.1-30wt%, even more preferably 0.5-10wt%.
  • the content of the second active ingredient in the pharmaceutical composition is preferably 0.01-90wt%, more preferably 0.1-30wt%, even more preferably 0.5-10wt%.
  • the first active ingredient and the second active ingredient account for the total mass of the pharmaceutical composition, preferably 0.01-90 wt%, more preferably 0.1-50 wt%, further preferably 0.5-30 wt%.
  • the carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.
  • the compounds and pharmaceutical compositions provided by the present invention can be in various forms, such as tablets, injections, capsules, powders, syrups, solutions, suspensions and aerosols, etc., and can be present in suitable solid or liquid carriers or Diluent and appropriate sterile equipment for injection or infusion.
  • compositions of the present invention can be prepared according to conventional preparation methods in the field of pharmacy.
  • the pharmaceutical composition of the present invention can be clinically used in mammals, including humans and animals, and can be administered through oral, nasal, skin, lung or gastrointestinal tract and other routes. Oral administration is most preferred.
  • the most preferred daily dose is 0.01-400 mg/kg body weight, taken once, or 0.01-200 mg/kg body weight in divided doses. Regardless of the method of administration, the optimal dosage for an individual should depend on the specific treatment. Usually, start with a small dose and gradually increase the dose until you find the most suitable dose.
  • the drug or inhibitor of the present invention can be administered in various ways, for example, it can be introduced into the body by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemically mediated methods such as muscle, intradermal, subcutaneous, intravenous , mucosal tissue; or mixed or wrapped by other substances into the body.
  • the active ingredient of the present invention or the pharmaceutical composition containing it can be administered in unit dose form, and the route of administration can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, Eyes, lungs and respiratory tract, skin, vagina, rectum, etc.
  • the dosage form for administration may be a liquid dosage form, a solid dosage form or a semi-solid dosage form.
  • Liquid dosage form can be solution (including true solution and colloid solution), emulsion (including O/W type, W/O type and double emulsion), suspension, injection (including water injection, powder injection and infusion), eye drops Agents, nasal drops, lotions and liniments, etc.
  • solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, gas (powder) aerosols, sprays, etc.
  • semi-solid dosage forms can be ointments, Gels, pastes, etc.
  • the active ingredients of the present invention can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.
  • diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • the wetting agent can be water, ethanol, Isopropanol, etc.
  • the binder can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, arabic mucilage, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hypromellose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.
  • disintegrants can be dry starch, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • the wetting agent can be water, ethanol, Isopropanol, etc.
  • the binder can be starch
  • Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.
  • the active ingredient of the present invention can be mixed with a diluent and a glidant, and the mixture is directly placed in a hard capsule or a soft capsule.
  • the active ingredients can also be made into granules or pellets with diluents, binders, and disintegrants, and then placed in hard or soft capsules.
  • Various diluents, binders, wetting agents, disintegrants, and glidants used to prepare the tablets of the present invention can also be used to prepare the capsules of the present invention.
  • water, ethanol, isopropanol, propylene glycol or their mixture can be used as a solvent and an appropriate amount of commonly used solubilizers, cosolvents, pH regulators, and osmotic pressure regulators in the field can be added.
  • the solubilizer or co-solvent can be poloxamer, lecithin, hydroxypropyl- ⁇ -cyclodextrin, etc.
  • the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.
  • the osmotic pressure regulator can be Sodium chloride, mannitol, glucose, phosphate, acetate, etc.
  • mannitol, glucose, etc. can also be added as proppants.
  • coloring agents can also be added to the pharmaceutical preparations, if necessary.
  • the two active ingredients or compositions of the present invention can be taken together or sequentially, or further combined with other therapeutic drugs or symptomatic drugs.
  • the active ingredient of the present invention has a synergistic effect with other therapeutic drugs, its dose should be adjusted according to the actual situation.
  • the present invention found through experiments that penfluridol combined with medroxyprogesterone acetate has a synergistic inhibitory effect on the proliferation and migration of endometrial cancer ISK and KLE cells, and can synergistically induce the apoptosis of endometrial cancer ISK and KLE cells .
  • the present invention also found that the synergistic therapeutic effect of penfluridol and medroxyprogesterone acetate is significantly better than that of penfluridol or medroxyprogesterone acetate alone.
  • Penfluridol is an oral long-acting antipsychotic drug, so it has high safety. Combined use with medroxyprogesterone acetate also has higher safety and lower side effects.
  • Example 1 Inhibitory effect of penfluridol and medroxyprogesterone acetate on the proliferation of endometrial cancer cells
  • medroxyprogesterone acetate can inhibit the proliferation of endometrial cancer cells ISK, while the same dose of medroxyprogesterone acetate has no obvious inhibitory effect on KLE cells, and the combination of penfluridol and medroxyprogesterone acetate can significantly Inhibits the proliferation of endometrial cancer cells ISK and KLE in a concentration-dependent manner.
  • Endometrial cancer cells ISK and KLE were purchased from the American Type Culture Collection (ATCC); phosphate buffered saline (PBS) was purchased from Bio-channel; DMEM/F12 medium was purchased from Biosharp; fetal calf Serum (FBS) and trypsin were purchased from Gibco; CCK-8 was purchased from Biyuntian Biotechnology Co., Ltd.; penfluridol was from the old drug store of the laboratory; medroxyprogesterone acetate was purchased from Selleck.
  • PBS phosphate buffered saline
  • FBS fetal calf Serum
  • trypsin were purchased from Gibco
  • CCK-8 was purchased from Biyuntian Biotechnology Co., Ltd.
  • penfluridol was from the old drug store of the laboratory
  • medroxyprogesterone acetate was purchased from Selleck.
  • ISK and KLE cells were cultured in DMEM/F12 (containing 10% fetal bovine serum, 1% penicillin/streptomycin) medium and placed in an incubator at 37°C and 5% CO 2 .
  • the endometrial cancer cells basically covered the cell culture dish (10 cm)
  • the cells were digested with trypsin, and seeded in a 96-well plate at a cell density of 5000 cells/well, 100 ⁇ L per well.
  • DMEM/F12 medium containing different concentrations of PFL was added, 200 ⁇ L per well, and 3 replicate wells were set up for each group. Incubate them for 72 hours in a cell culture incubator.
  • Inhibition rate calculation formula: cell inhibition rate% [1-(administration group A value-blank group A value)/(control group A value-blank group A value)] ⁇ 100%, IC50 value through Graphpad Prism 8.0 software fit.
  • Table 1 and Figure 1 show the anti-proliferation data of the combination of penfluridol and medroxyprogesterone acetate on two kinds of EC cells.
  • the proliferative activity of endometrial cancer cells decreased with the increase of penfluridol concentration, indicating that the combination of penfluridol and medroxyprogesterone acetate can inhibit the proliferation of endometrial cancer cells ISK and KLE in a concentration-dependent manner.
  • the IC 50 of penfluridol and medroxyprogesterone acetate on the proliferation of ISK cells were 2.74 ⁇ M and 23.72 ⁇ M respectively;
  • the inhibitory activity IC 50 were 2.89 ⁇ M and 41.43 ⁇ M, respectively.
  • MPA medroxyprogesterone acetate
  • PFL penfluridol
  • Example 1 For the cultivation of ISK and KLE cells and the detection of CCK-8, see the experimental materials and methods in Example 1.
  • the concentration gradient of medroxyprogesterone acetate was set to 0, 5, 10, 15 and 20 ⁇ M, and the concentration gradient of penfluridol was 0, 0.5, 1, 2, 4, 8, 10, 20 and 50 ⁇ M.
  • the absorbance value A at 450nm was detected using a Bio-Tek multifunctional microplate reader, and the inhibition rate and IC50 value were calculated.
  • Inhibition rate calculation formula: cell inhibition rate% [1-(administration group A value-blank group A value)/(control group A value-blank group A value)] ⁇ 100%.
  • the obtained inhibition rate was analyzed by using SynergyFinder 2.0 to obtain the synergy scores of penfluridol and medroxyprogesterone acetate in ISK and KLE cells, respectively.
  • MPA medroxyprogesterone acetate
  • PFL penfluridol
  • the source of experimental materials is the same as in Example 1.
  • the EC cells in the logarithmic growth phase were digested with trypsin, prepared into a single cell suspension using serum-free DMEM/F12 medium, and seeded into a 6-well plate at a density of about 500,000 cells per well. After the cells adhered to the wall, they were divided into 6 groups, namely the control group, the MPA 5 ⁇ M group, the MPA 10 ⁇ M group, the PFL 1 ⁇ M group, the MPA 5 ⁇ M+PFL 1 ⁇ M group, and the MPA 10 ⁇ M+PFL 1 ⁇ M group, with 3 replicate wells in each group.
  • the percentage of wound healing distance between cell scratches was significantly lower than that in the single drug treatment group.
  • the vertical axis of the histogram indicates the percentage of wound healing distance of cells in each group, and the significant difference of the data was analyzed by One-way ANOVA method (Graphpad Prism8.0 software). Data are mean ⁇ SD: ***P ⁇ 0.001 vs Ctrl, ###P ⁇ 0.001 vs MPA 5 ⁇ M, $$$P ⁇ 0.001 vs MPA 10 ⁇ M.
  • the Transwell chamber was purchased from Costar Company; paraformaldehyde is a common reagent in the laboratory, commercially purchased without any treatment, and the sources of other experimental materials are the same as in Example 1.
  • the EC cells in the logarithmic growth phase were digested with trypsin, prepared into a single cell suspension using serum-free DMEM/F12 medium, and seeded on the top of the Transwell chamber at a density of about 100,000 cells per well. Add 600 ⁇ L of DMEM/F12 medium containing 20% FBS to the bottom of the small chamber, taking care to avoid the generation of air bubbles.
  • each group set 3 replicate wells.
  • 150 ⁇ L of serum-free DMEM/F12 medium containing different drugs was added to the upper chamber of the treatment group, and 150 ⁇ L of serum-free DMEM/F12 medium without drug was added to the upper chamber of the control group. Place in an incubator at 37°C with 5% CO 2 for 24 hours.
  • the vertical axis of the histogram indicates the number of cells passing through the small chamber in each group, and the significant difference of the data was analyzed by One-way ANOVA method (Graphpad Prism8.0 software). Data are mean ⁇ SD: **P ⁇ 0.01, ***P ⁇ 0.001 vs Ctrl, ###P ⁇ 0.001 vs MPA 5 ⁇ M, $$$P ⁇ 0.001 vs MPA 10 ⁇ M.
  • Annexin V-FITC/PI cell apoptosis detection kit was used to test the effects of penfluridol and medroxyprogesterone acetate on the apoptosis of endometrial cancer cells ISK and KLE at the cellular level.
  • penfluridol can inhibit the apoptosis of endometrial cancer cells ISK and KLE, and its combination with medroxyprogesterone acetate can synergistically inhibit the apoptosis of endometrial cancer cells.
  • the Annexin V-FITC/PI Cell Apoptosis Detection Kit was purchased from Beyotime Biotechnology Co., Ltd., in which Annexin V-FITC Conjugate Solution, Annexin V-FITC, and Propidium Iodide (PI) are all reagents in the kit. All the other experimental materials are from the same sources as in Example 1.
  • the EC cells in the logarithmic growth phase were digested with trypsin to prepare a single cell suspension, seeded into a 6-well plate at a density of about 120,000 cells per well, and cultured overnight.
  • the cells adhered to the wall they were divided into 6 groups, namely the control group, the MPA 5 ⁇ M group, the MPA 10 ⁇ M group, the PFL 1 ⁇ M group, the MPA 5 ⁇ M+PFL 1 ⁇ M group, and the MPA 10 ⁇ M+PFL 1 ⁇ M group, with 3 replicate wells in each group. Incubate for 48 h at 37 °C in an incubator with 5% CO 2 .
  • the apoptosis ratios of ISK and KLE were 23.92% and 12.52%, respectively.
  • the apoptotic ratios of ISK and KLE were 22.97% and 35.16% when Lido combined with medroxyprogesterone acetate (10 ⁇ M).
  • the vertical axis of the histogram indicates the proportion of apoptosis in each group, and the significant difference of the data is analyzed by One-way ANOVA method (Graphpad Prism8.0 software).

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Abstract

五氟利多联合醋酸甲羟孕酮在制备治疗子宫内膜癌的药物中的应用。五氟利多与醋酸甲羟孕酮可协同抑制子宫内膜癌细胞的增殖与迁移，并促进子宫内膜癌细胞凋亡。

Description

五氟利多联合醋酸甲羟孕酮在制备治疗子宫内膜癌的药物中的应用 技术领域
本发明涉及医药技术领域，具体地涉及五氟利多联合醋酸甲羟孕酮在制备治疗子宫内膜癌的药物中的应用。
背景技术
子宫内膜癌(Endometrial Cancer，EC)是发生于子宫内膜的一组上皮性恶性肿瘤，是世界上最常见的妇科恶性肿瘤之一，其发病率和死亡率都在上升(Karen,Endometrial Cancer，The New England Journal of Medicine 2020,383,2053-2064)。虽然EC在绝经后妇女中更常见，但在过去的十年，年轻女性EC的发病率急剧增加，有越来越年轻的女性被诊断出子宫内膜癌，发病率增加的根本原因是肥胖的流行和由此引起的高胰岛素血症，随着肥胖率的上升，子宫内膜癌的发病率也在上升(Moore,Endometrial Cancer:Is This a New Disease？Am.Soc.Clin.Oncol.2017,37,435-442)。2020年全球子宫内膜癌新发病例42万，其中中国新发病例8万。
EC通常分为I型和II型，两者主要区别是在于I型EC孕激素受体表达呈阳性，II型EC孕激素受体表达呈阴性(Bokhman,Gynecologic oncology,1983,15,10-17)。目前手术治疗是治疗EC的常用方法，但是对于需要保护生育功能的患者需要采用孕激素治疗的保守治疗方法，保守治疗的主要方法是应用孕激素类药物的激素疗法(主要为醋酸甲羟孕酮(MPA))。然而激素疗法存在局限性，它只对孕激素受体表达呈阳性的患者有效并且复发率高，易产生耐药性。目前，EC患者对保留生育功能的保守治疗需求日益增加。
因此，本领域迫切需要开发能够治疗EC，特别是治疗孕激素受体阴性和孕激素耐药的EC的药物。
发明内容
本发明的目的是提供一种能够治疗EC，特别是治疗孕激素受体阴性和孕激素耐药的EC的药物。
在本发明的第一方面，提供了一种药物组合物，所述组合物包括：
(a)治疗有效量的第一活性成分，所述第一活性成分为五氟利多或其药学上可接受的盐；和
(b)治疗有效量的第二活性成分，所述第二活性成分为孕激素，
并且，所述的药物组合物用于治疗和/或预防子宫内膜癌。
在另一优选例中，所述子宫内膜癌包括I型和II型子宫内膜癌。
在另一优选例中，所述子宫内膜癌包括孕激素敏感型子宫内膜癌、或孕激素耐受型子宫内膜癌。
在另一优选例中，所述子宫内膜癌为孕激素受体阴性和孕激素耐药的子宫内膜癌。
在另一优选例中，所述孕激素选自下组：酸酸甲羟孕酮、醋酸甲地孕酮、己酸孕酮、或其组合。
在另一优选例中，所述孕激素为醋酸甲羟孕酮。
在另一优选例中，所述第一活性成分与第二活性成分的重量比为1:20至10:1，较佳地，1:15至1:1，更佳地，1:10至1:5。
在另一优选例中，所述第一活性成分在所述药物组合物中的含量为0.01-90wt％，较佳地，0.1-30wt％，更佳地，0.5-10wt％。
在另一优选例中，所述第二活性成分在所述药物组合物中的含量为0.01-90wt％，较佳地，0.1-30wt％，更佳地，0.5-10wt％。
在另一优选例中，所述第一活性成分和第二活性成分占所述药物组合物总质量0.01-90wt％，较佳地，0.1-50wt％，更佳地，0.5-30wt％。
在另一优选例中，所述的药物组合物还包括药学上可接受的载体。
在另一优选例中，所述的药物组合物的剂型包括注射剂型和口服剂型。
在另一优选例中，所述的药物组合物的剂型包括注射剂、片剂、胶囊剂、丸剂、悬浮剂和乳剂。
在另一优选例中，所述药物组合物还包括其他抗子宫内膜癌药物。
在本发明的第二方面，提供了一种药盒，所述药盒包括：
(a)含有五氟利多或其药学上可接受的盐的第一制剂；和
(b)含有孕激素的第二制剂。
在另一优选例中，所述孕激素选自下组：酸酸甲羟孕酮、醋酸甲地孕酮、己酸孕酮、或其组合。
在另一优选例中，所述的药盒还包括说明书，所述的说明书中注明将所述第一制剂和第二制剂联用，从而治疗和/或缓解子宫内膜癌。
在另一优选例中，所述第一制剂和第二制剂在治疗子宫内膜癌中同时给药、分别给药或顺序给药。
在本发明所述的药盒中，所述的药盒中第一制剂和第二制剂是相互独立的，或是合并的。
在另一优选例中，所述的药盒中第一制剂和第二制剂是相互独立的。
在另一优选例中，所述的第一制剂为口服制剂或注射用制剂。
在另一优选例中，所述的第一制剂的剂型包括注射剂、片剂、胶囊剂、丸剂、悬浮剂和乳剂。
在另一优选例中，所述的第二制剂为口服制剂或注射用制剂。
在另一优选例中，所述的第二制剂的剂型包括注射剂、片剂、胶囊剂、丸剂、悬浮剂和乳剂。
在本发明的第三方面，提供了一种活性成分组合，所述活性成分组合包括：
(a)治疗有效量的第一活性成分，所述第一活性成分为五氟利多或其药学上可接受的盐；和
(b)治疗有效量的第二活性成分，所述第二活性成分为孕激素。
在另一优选例中，所述孕激素选自下组：酸酸甲羟孕酮、醋酸甲地孕酮、己酸孕酮、或其组合。
在本发明的第四方面，提供了一种本发明第一方面所述的药物组合物、第二方面所述的药盒和/或第三方面所述的活性成分组合的用途，用于制备治疗和/或缓解子宫内膜癌的药物。
本发明还提供了本发明所述的药物组合物、所述的药盒和/或所述的活性成分组合用于治疗和/或缓解子宫内膜癌的用途。
在另一优选例中，所述子宫内膜癌包括I型和II型子宫内膜癌。
在另一优选例中，所述药物还用于以下一种或多种用途：
(a)抑制子宫内膜癌细胞增殖；
(b)抑制子宫内膜癌细胞迁移；和
(c)诱导子宫内膜癌细胞凋亡。
在另一优选例中，所述子宫内膜癌细胞选自下组：ISK、KLE、或其组合。
在另一优选例中，所述子宫内膜癌细胞为ISK和/或KLE细胞。
在本发明的第五方面，提供了一种体外抑制子宫内膜癌细胞的方法，包括步骤：
(i)在五氟利多或其药学上可接受的盐和孕激素存在下，培养子宫内膜癌细胞，从而抑制所述的子宫内膜癌细胞。
在另一优选例中，所述的方法是非治疗性和非诊断性的。
在另一优选例中，所述的抑制选自下组：
(a)抑制子宫内膜癌细胞增殖；
(b)抑制子宫内膜癌细胞迁移；
(c)诱导子宫内膜癌细胞凋亡；
(d)上述(a)至(c)的任意组合。
在另一优选例中，所述子宫内膜癌细胞为ISK和/或KLE细胞。
在本发明的第六方面，提供了一种治疗子宫内膜癌的方法，包括步骤：给有需要的对象施用本发明第一方面所述的药物组合物、第二方面所述的药盒或第三方面所述的活性成分组合。
在另一优选例中，所述对象为子宫内膜癌患者。
在另一优选例中，所述子宫内膜癌包括I型和II型子宫内膜癌。
在另一优选例中，所述治疗方法进一步包括给有需要的对象施用其他抗子宫内膜癌药物。
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。限于篇幅，在此不再一一累述。
附图说明
图1为五氟利多与醋酸甲羟孕酮对ISK和KLE细胞的增殖抑制活性示意图。(A)ISK细胞增殖抑制活性；(B)KLE细胞增殖抑制活性。
图2为五氟利多与醋酸甲羟孕酮对ISK和KLE细胞的协同抑制作用示意图。(A)联合用药对ISK细胞的协同抑制作用；(B)联合用药对KLE细胞的协同抑制作用。
图3为五氟利多联合醋酸甲羟孕酮对ISK和KLE细胞的迁移能力的影响示意图。(A)ISK细胞划痕；(B)KLE细胞划痕；(C)ISK细胞伤口愈合距离百分比；(D)KLE细胞伤口愈合距离百分比。
图4为五氟利多与醋酸甲羟孕酮对ISK和KLE细胞的迁移能力的影响示意图。(A)ISK 细胞结晶紫染色；(B)ISK细胞结晶紫染色定量；(C)KLE细胞结晶紫染色；(D)KLE细胞结晶紫染色定量。
图5为五氟利多与醋酸甲羟孕酮对ISK和KLE细胞的凋亡的影响示意图。(A)联合用药对ISK细胞凋亡的影响；(B)联合用药对KLE细胞凋亡的影响；(C)ISK细胞凋亡比例定量；(D)KLE细胞凋亡比例定量。
具体实施方式
本发明人经过广泛而深入的研究，意外地发现了抗精神病药物五氟利多和醋酸甲羟孕酮(MPA)联用能够明显提高对子宫内膜癌的治疗效果，对子宫内膜癌ISK和KLE细胞的增殖、迁移能力具有协同抑制作用，且能够协同诱导子宫内膜癌ISK和KLE细胞的凋亡。五氟利多和醋酸甲羟孕酮的协同治疗效果明显优于五氟利多或醋酸甲羟孕酮的单独使用。在此基础上，完成了本发明。
术语
除非另有定义,否则本文中所用的所有技术和科学术语的含义与本发明所属领域普通技术人员普遍理解的含义相同。
如本文所用，术语“包含”、“包括”、“含有”可互换使用，不仅包括封闭式定义，还包括半封闭、和开放式的定义。换言之，所述术语包括了“由……构成”、“基本上由……构成”。
如本文所用，术语“药学上可接受的载体”的成分是指适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)的，即有合理的效益/风险比的物质。
如本文所用，术语“治疗有效量”，是指对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。本领域的普通技术人员应该理解，所述的“治疗有效量”可随着药物组合物的形式、给药途径、所用药物的辅料、疾病的严重程度以及与其他药物联合用药等情况的不同而有所不同。
五氟利多
如本文所用，术语“五氟利多”(Penfluridol，PFL)、“本发明的第一活性成分”可互换使用，均指五氟利多及其药学上可接受的盐。
所述五氟利多具有如下结构式：
Figure PCTCN2021117536-appb-000001
五氟利多是美国食品药品监督局(FDA)批准的一种口服长效抗精神病药，属于二苯基丁基哌啶类药物，抗精神病作用强而持久，口服一次可维持数天至一周，用于治疗各型精神分裂症。五氟利多是多巴胺D2受体和钙通道的强效抑制剂。
本发明人经大量筛选研究，意外地发现五氟利多对子宫内膜癌细胞ISK(I型，孕激素敏感)与II型子宫内膜癌细胞KLE(孕激素耐药)均具有增殖抑制作用，进一步研究发现其与醋酸甲羟孕酮联合应用对两种细胞有明显的协同抑制作用，能够抑制两种EC细胞的增殖与迁移能力，并诱导EC细胞凋亡。因此，本发明提供了五氟利多和醋酸甲羟孕酮联合制备治疗子宫内膜癌的药物的用途。
第二活性成分
如本文所用，术语“本发明的第二活性成分”指孕激素。
在另一优选例中，所述孕激素选自下组：酸酸甲羟孕酮、醋酸甲地孕酮、己酸孕酮、或其组合。
在另一优选例中，所述孕激素为醋酸甲羟孕酮及其药学上可接受的盐。
醋酸甲羟孕酮的结构式如下：
Figure PCTCN2021117536-appb-000002
子宫内膜癌
如本文所用，术语“子宫内膜癌(endometrial cancer,EC)”是发生于子宫内膜的一组上皮性恶性肿瘤，又称子宫体癌，是女性生殖系统最常见的三大恶性肿瘤之一。子宫内膜癌好发于围绝经期及绝经后女性，根据其发病机制和生物学行为特点，可分为I型子宫内膜癌(雌激素依赖型)与II型子宫内膜癌(非雌激素依赖型)。
药物组合物和药盒
本发明还提供了一种药物组合物，所述组合物包括：
(a)治疗有效量的第一活性成分，所述第一活性成分为五氟利多或其药学上可接受的盐；和
(b)治疗有效量的第二活性成分，所述第二活性成分为孕激素，
并且，所述的药物组合物用于治疗和/或预防子宫内膜癌。
本发明还提供了一种药盒，所述药盒包括：
(a)含有五氟利多或其药学上可接受的盐的第一制剂；和
(b)含有孕激素的第二制剂。
在另一优选例中，所述的第一制剂和第二制剂是不同的制剂、或同一制剂。
当联用两种活性成分(或其相应制剂)时，服用的次序没有特别限制，可以先后、一起、或依次施用。
在另一优选例中，所述孕激素选自下组：酸酸甲羟孕酮、醋酸甲地孕酮、己酸孕酮、或其组合。
在另一优选例中，所述孕激素为醋酸甲羟孕酮。
本发明的药物组合物中，第一活性成分与第二活性成分的重量比优选为1:20至10:1，更优选为1:15至1:1，进一步优选为1:10至1:5。
本发明的药物组合物中，第一活性成分在所述药物组合物中的含量优选为0.01-90wt％，更优选为0.1-30wt％，进一步优选为0.5-10wt％。
本发明的药物组合物中，第二活性成分在所述药物组合物中的含量优选为0.01-90wt％，更优选为0.1-30wt％，进一步优选为0.5-10wt％。
本发明的药物组合物中，第一活性成分和第二活性成分占所述药物组合物总质量优选0.01-90wt％，更优选地，0.1-50wt％，进一步优选地，0.5-30wt％。
需要的时候，在本发明药物中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。
本发明所提供的化合物和药物组合物可以是多种形式，如片剂、注射剂、胶囊、粉剂、糖浆、溶液状、悬浮液和气雾剂等，并可以存在于适宜的固体或液体的载体或稀释液中和适宜的用于注射或滴注的消毒器具中。
本发明的药物组合物的各种剂型可按照药学领域的常规制备方法制备。
本发明的药物组合物可对哺乳动物临床使用，包括人和动物，可以通过口、鼻、皮肤、肺或者胃肠道等的给药途径。最优选为口服。最优选日剂量为0.01-400mg/kg 体重，一次性服用，或0.01-200mg/kg体重分次服用。不管用何种服用方法，个人的最佳剂量应依据具体的治疗而定。通常情况下是从小剂量开始，逐渐增加剂量一直到找到最适合的剂量。
本发明的药物或抑制剂可通过各种不同方式施用，例如可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、静脉、粘膜组织；或是被其他物质混合或包裹导入机体。
典型地，本发明活性成分或含有它的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、静脉注射、肌肉注射、皮下注射、鼻腔、口腔粘膜、眼、肺和呼吸道、皮肤、阴道、直肠等。
给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括O/W型、W/O型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等；固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、凝胶剂、糊剂等。
本发明活性成分可以被制成普通制剂、也可以制成缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
为了将本发明活性成分被制成片剂，可以广泛使用本领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；润湿剂可以是水、乙醇、异丙醇等；黏合剂可以是淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、微晶纤维素、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、聚乙烯吡咯烷酮、聚乙二醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与枸橼酸、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠等；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
为了将给药单元制成胶囊剂，可以将有效成分本发明活性成分与稀释剂、助流剂混合，将混合物直接置于硬胶囊或软胶囊中。也可将有效成分先与稀释剂、黏合剂、崩解剂制成颗粒或微丸，再置于硬胶囊或软胶囊中。用于制备本发明片剂的各稀释剂、 黏合剂、润湿剂、崩解剂、助流剂品种也可用于制备本发明的胶囊剂。
为将本发明活性成分制成注射剂，可以用水、乙醇、异丙醇、丙二醇或它们的混合物作溶剂并加入适量本领域常用的增溶剂、助溶剂、PH调剂剂、渗透压调节剂。增溶剂或助溶剂可以是泊洛沙姆、卵磷脂、羟丙基-β-环糊精等；PH调剂剂可以是磷酸盐、醋酸盐、盐酸、氢氧化钠等；渗透压调节剂可以是氯化钠、甘露醇、葡萄糖、磷酸盐、醋酸盐等。如制备冻干粉针剂，还可加入甘露醇、葡萄糖等作为支撑剂。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂或其他添加剂。
本发明的两种活性成分或组合物可一起服用或先后服用，或进一步地与其他治疗药物或对症药物合并使用。
当本发明的活性成分与其他治疗药物存在协同作用时，应根据实际情况调整它的剂量。
本发明的主要优点包括：
1、本发明通过实验发现五氟利多联合醋酸甲羟孕酮对子宫内膜癌ISK和KLE细胞的增殖、迁移能力具有协同抑制作用，且能够协同诱导子宫内膜癌ISK和KLE细胞的凋亡。
2、本发明还发现五氟利多和醋酸甲羟孕酮的协同治疗效果明显优于五氟利多或醋酸甲羟孕酮的单独使用。
3、五氟利多是一种口服长效抗精神病药，因此安全性高。与醋酸甲羟孕酮联用也具有较高的安全性和较低的副作用。
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，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1五氟利多与醋酸甲羟孕酮对子宫内膜癌细胞增殖的抑制作用
本实施例中，在细胞水平上通过CCK-8实验测试五氟利多与醋酸甲羟孕酮对子宫内膜癌细胞ISK和KLE的增殖抑制作用。
研究结果表明，醋酸甲羟孕酮能够抑制子宫内膜癌细胞ISK的增殖，而相同剂量 的醋酸甲羟孕酮对KLE细胞没有明显抑制作用，五氟利多与醋酸甲羟孕酮联用能够显著抑制子宫内膜癌细胞ISK和KLE的增殖，并且具有明显浓度依赖性。
1、实验材料和方法
子宫内膜癌细胞ISK和KLE购自于美国模式菌种收集中心(ATCC)；磷酸盐缓冲盐溶液(PBS)购自于Bio-channel公司；DMEM/F12培养基购自于Biosharp公司；胎牛血清(FBS)和胰蛋白酶购自于Gibco公司；CCK-8购自于碧云天生物科技有限公司；五氟利多来源于实验室老药库；醋酸甲羟孕酮购自于Selleck公司。
ISK和KLE细胞用DMEM/F12(含10％胎牛血清，1％青霉素/链霉素)培养基，并置于37℃，5％CO 2的培养箱中培养。待子宫内膜癌细胞基本长满细胞培养皿(10cm)后，用胰蛋白酶将细胞消化下来，并以5000个/孔的细胞密度种到96孔板中，每孔100μL。培养过夜，待细胞贴壁后，分别加入含不同浓度PFL的DMEM/F12培养基，每孔200μL，每组设3个复孔。在细胞培养箱中分别孵育72h。随后去除培养基，每孔加入100μL含10％CCK-8的无血清培养基，在培养箱中37℃孵育1h后，使用Bio-Tek多功能酶标仪检测在450nm处的吸光值A，计算抑制率和IC 50值。
抑制率计算公式：细胞抑制率％＝[1-(给药组A值-空白组A值)/(对照组A值-空白组A值)]×100％，IC 50值通过Graphpad Prism 8.0软件拟合。
2、实验结果
五氟利多与醋酸甲羟孕酮联用对两种EC细胞的增殖抑制活性数据如表1和图1所示。
结果表明，醋酸甲羟孕酮对子宫内膜癌细胞ISK的增殖具有显著的抑制作用，而相同剂量醋酸甲羟孕酮对KLE细胞的增殖无明显影响。五氟利多与醋酸甲羟孕酮联用对两种子宫内膜癌细胞的增殖具有显著的抑制作用。
子宫内膜癌细胞的增殖活性随着五氟利多浓度的增加而减弱，说明五氟利多与醋酸甲羟孕酮联合能够浓度依赖性地抑制子宫内膜癌细胞ISK和KLE的增殖。
五氟利多和醋酸甲羟孕酮对ISK细胞在72h的孵育时间下增殖抑制活性IC 50分别为2.74μM、23.72μM；五氟利多和醋酸甲羟孕酮对KLE细胞在72h的孵育时间下增殖抑制活性IC 50分别为2.89μM、41.43μM。
表1五氟利多和醋酸甲羟孕酮对不同EC细胞株的增殖抑制活性
Figure PCTCN2021117536-appb-000003
MPA：醋酸甲羟孕酮；PFL：五氟利多.
本实施例说明，五氟利多与醋酸甲羟孕酮联合能够浓度依赖性地抑制子宫内膜癌细胞ISK和KLE的增殖。
实施例2五氟利多与醋酸甲羟孕酮联用对子宫内膜癌细胞的协同抑制作用
本实施例在细胞水平上通过CCK-8实验测试五氟利多与醋酸甲羟孕酮联合应用对子宫内膜癌细胞ISK和KLE的协同抑制作用。
研究结果表明，五氟利多与醋酸甲羟孕酮能够协同抑制子宫内膜癌细胞ISK和KLE的生长。
1、实验材料和方法
子宫内膜癌细胞与试剂见实施例1中实验材料和方法。
ISK和KLE细胞的培养与CCK-8检测见实施例1中实验材料和方法。根据前述五氟利多与醋酸甲羟孕酮对子宫内膜癌细胞的增值抑制活性，分别设置醋酸甲羟孕酮浓度梯度为0、5、10、15与20μM，五氟利多浓度梯度为0、0.5、1、2、4、8、10、20与50μM。使用Bio-Tek多功能酶标仪检测在450nm处的吸光值A，计算抑制率和IC 50值。抑制率计算公式：细胞抑制率％＝[1-(给药组A值-空白组A值)/(对照组A值-空白组A值)]×100％。
将所得抑制率使用SynergyFinder 2.0分析获得五氟利多与醋酸甲羟孕酮分别在ISK与KLE细胞中的协同分数。
2、实验结果
实验结果如图2和表2所示，五氟利多与醋酸甲羟孕酮都能够剂量依赖地抑制子宫内膜癌细胞ISK与KLE的生长，低浓度(5μM)醋酸甲羟孕酮对子宫内膜癌细胞ISK和KLE的抑制作用弱，抑制率分别为9.63％和2.64，低剂量(1μM)五氟利多对两种细胞的抑制率也较弱，分别为27.98％和32.39％，而两者联合应用对两种细胞的抑制率 可达到44.45％和43.39％，表现出显著的协同效应。同时，无论是与低剂量，还是与高剂量醋酸甲羟孕酮联用，五氟利多皆可剂量依赖地增加其对子宫内膜癌细胞的抑制作用。五氟利多与醋酸甲羟孕酮在子宫内膜癌细胞ISK中的协同抑制分数为10.07，在KLE细胞中的协同抑制分数为14.44。
表2五氟利多与醋酸甲羟孕酮对ISK和KLE细胞的协同分数
细胞类型 药物组合 协同分数 最大协同面积分数 方法
Ishikawa细胞 PFL-MPA 10.07 16.87 ZIP
KLE细胞 PFL-MPA 14.44 21.3 ZIP
注：MPA：醋酸甲羟孕酮；PFL：五氟利多.
本实施例说明了五氟利多与MPA联用可增强对子宫内膜癌细胞的抑制作用，该作用对五氟利多和MPA均表现出剂量依赖性。
实施例3抑制子宫内膜癌细胞迁移
本实施例在细胞水平上通过细胞划痕实验测试五氟利多对子宫内膜癌细胞ISK和KLE细胞迁移能力的影响。
研究结果表明，低剂量的五氟利多或醋酸甲羟孕酮抑制子宫内膜癌细胞ISK和KLE迁移的能力有限，但低剂量五氟利多与醋酸甲羟孕酮联用可显著抑制ISK和KLE细胞的迁移能力。
1、实验材料和方法
实验材料来源同实施例1。取对数生长期的EC细胞，用胰蛋白酶消化下来，使用不含血清的DMEM/F12培养基制备成单细胞混悬液，以每孔约50万个细胞的密度接种至6孔板。待细胞贴壁后，分成6组，分别为对照组、MPA 5μM组、MPA 10μM组、PFL 1μM组、MPA 5μM+PFL 1μM组、MPA 10μM+PFL 1μM组，每组设3个复孔。第二天使用20μL枪头，垂直于细胞平面，沿着前一天划在平板背面的线在细胞层上进行划痕。随后使用无菌PBS洗细胞3次，去除不贴壁的细胞，更换新鲜无血清培养基继续培养48h。在0h与48h取出细胞，显微镜下观察并拍照记录。使用ImageJ每张图随机划取6条水平线，计算细胞间距离均值，以48h细胞间距与0h细胞间距比值测得各组细胞伤口愈合距离百分比。
2、实验结果
结果如图3所示，图中Control为对照组，在ISK和KLE细胞中，与对照组相比，低剂量五氟利多组细胞划痕间距明显增加，说明五氟利多具有抑制ISK和KLE细胞迁移的能力，同时低剂量醋酸甲羟孕酮(5μM)仅抑制ISK细胞的迁移，对KLE细胞迁移 能力无明显影响，而10μM醋酸甲羟孕酮处理能够同时抑制两种子宫内膜癌细胞的迁移。
在联合用药组，细胞划痕间伤口愈合距离百分比明显低于单药处理组。柱状图纵坐标表示各组细胞伤口愈合距离百分比，数据显著性差异采用One-way ANOVA法分析(Graphpad Prism8.0软件)。数据为平均值±SD:***P<0.001vs Ctrl,###P<0.001vs MPA 5μM，$$$P<0.001vs MPA 10μM。
本实验说明，五氟利多与MPA联合用药(优选的用量比例为1:10至1:5)具有抑制ISK和KLE细胞迁移的能力，且联合用药抑制细胞迁移的能力显著优于分别应用单药。
实施例4五氟利多与醋酸甲羟孕酮对子宫内膜癌细胞迁移的抑制
在本实施例中，在细胞水平上通过Transwell实验测试五氟利多与醋酸甲羟孕酮对子宫内膜癌细胞ISK和KLE迁移能力的影响。研究结果表明，五氟利多与醋酸甲羟孕酮能够协同抑制子宫内膜癌细胞ISK和KLE迁移。
1、实验材料和方法
Transwell小室购自于Costar公司；多聚甲醛为实验室常用试剂，商业购买，未经任何处理，其余实验材料来源同实施例1。取对数生长期的EC细胞，用胰蛋白酶消化下来，使用不含血清的DMEM/F12培养基制备成单细胞混悬液，以每孔约10万个细胞的密度接种至Transwell小室上方。小室下方加入600μL含20％FBS的DMEM/F12培养基，注意避免气泡产生。分成6组，分别为对照组、MPA 5μM组、MPA 10μM组、PFL 1μM组、MPA 5μM+PFL 1μM组、MPA 10μM+PFL 1μM组，每组设3个复孔。给药组的上室加入含不同药物的无血清DMEM/F12培养基150μL，对照组上室加入无药物无血清的DMEM/F12培养基150μL。置于37℃，5％CO 2的培养箱培养24h。随后用镊子小心取出小室，吸干上室液体，移到预先加入约800μL预冷PBS的24孔板中，清洗2次，每次5min。取出小室，移到预先加入约800μL多聚甲醛溶液的24孔板中，室温固定30min。取出小室，吸干上室固定液，移到预先加入约800μL结晶紫染液的24孔板中，室温染色30min。轻轻用清水冲洗数次，用湿棉棒小心擦去上室膜表面上的细胞。200倍显微镜下随机取5个视野拍照，Image J软件手动计数，统计各个视野下的细胞数。
2、实验结果
结果如图4所示，图中control为对照组，照片中的每一个黑点表示一个穿过小室的细胞。
结果显示，在ISK细胞中，与control组相比，醋酸甲羟孕酮组和五氟利多组穿过 小室的细胞数量减少，且五氟利多与醋酸甲羟孕酮联用组穿过小室的细胞数量进一步减少，差异具有统计学意义(p<0.0001)，说明五氟利多与醋酸甲羟孕酮具有协同抑制ISK细胞迁移的能力。
在KLE细胞中，与Ctrl组相比，醋酸甲羟孕酮组穿过小室的细胞数量无明显改变，五氟利多组穿过小室的细胞数量减少，同时，与后两组相比，五氟利多与醋酸甲羟孕酮联用组发生迁移细胞数目显著减少，说明五氟利多与醋酸甲羟孕酮联用对KLE细胞迁移能力具有明显的协同抑制作用。
柱状图纵坐标表示各组穿过小室细胞数目，数据显著性差异采用One-way ANOVA法分析(Graphpad Prism8.0软件)。数据为平均值±SD:**P<0.01,***P<0.001vs Ctrl,###P<0.001vs MPA 5μM，$$$P<0.001vs MPA 10μM。
该实验说明了五氟利多与醋酸甲羟孕酮联用(优选的用量比例为1:10至1:5)对ISK和KLE细胞迁移能力具有明显的协同抑制作用。
实施例5五氟利多与醋酸甲羟孕酮对子宫内膜癌细胞凋亡的影响
在本实施例中，在细胞水平上采用Annexin V-FITC/PI细胞凋亡检测试剂盒测试五氟利多与醋酸甲羟孕酮对子宫内膜癌细胞ISK和KLE凋亡的影响。研究结果表明，五氟利多能够抑制子宫内膜癌细胞ISK和KLE凋亡，并且其与醋酸甲羟孕酮联用能够协同抑制子宫内膜癌细胞的凋亡。
1.实验材料和方法
Annexin V-FITC/PI细胞凋亡检测试剂盒购自于碧云天生物科技有限公司，其中Annexin V-FITC结合液、Annexin V-FITC、碘化丙啶(PI)均为试剂盒中试剂。其余实验材料来源同实施例1。取对数生长期的EC细胞，用胰蛋白酶消化下来制备成单细胞混悬液，以每孔约12万个细胞的密度接种至6孔板，过夜培养。待细胞贴壁后，分成6组，分别为对照组、MPA 5μM组、MPA 10μM组、PFL 1μM组、MPA 5μM+PFL 1μM组、MPA 10μM+PFL 1μM组，每组设3个复孔。在37℃，5％CO 2的培养箱孵育48h。随后将细胞培养液吸出至10ml离心管内，PBS清洗贴壁细胞一次，加入300μL胰酶消化细胞2min，将细胞轻轻吹打下来，转移到相应离心管内，1000rpm离心5min，弃上清，收集细胞，用PBS轻轻重悬细胞并计数。取5-10万重悬的细胞，1000rpm离心5min，弃上清，加入195μL Annexin V-FITC结合液轻轻重悬细胞，加入5μL Annexin V-FITC和10μL碘化丙啶(PI)染色液，轻轻混匀。室温避光孵育20min，立即用Beckman Coulter(cytoFLEX LX)流式细胞仪检测。
2、实验结果
结果如图5所示，图中Ctrl为对照组，在凋亡示意图中的第一象限为晚凋细胞，第四象限为早凋细胞，凋亡比例为第一、四象限比例之和。与对照组相比，PFL能够诱导ISK细胞凋亡，对KLE细胞凋亡无明显影响，MPA能够诱导ISK和KLE细胞凋亡，但对ISK作用更为显著。五氟利多联合醋酸甲羟孕酮则可显著诱导ISK与KLE细胞凋亡，五氟利多联合醋酸甲羟孕酮(5μM)时ISK与KLE凋亡的比例分别为23.92％和12.52％，五氟利多联合醋酸甲羟孕酮(10μM)时ISK与KLE凋亡的比例分别为22.97％和35.16％。柱状图纵坐标表示各组细胞凋亡比例，数据显著性差异采用One-way ANOVA法分析(Graphpad Prism8.0软件)。数据为平均值±SD:**P<0.01,***P<0.001vs Ctrl,##P<0.01,###P<0.001vs MPA 5μM，$$$P<0.001vs MPA 10μM。
该实验说明了，五氟利多联合MPA(优选的用量比例为1:10至1:5)可显著诱导ISK与KLE细胞凋亡，效果显著优于分别应用单药。
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 (10)

  1. 一种药物组合物，其特征在于，所述组合物包括：
    (a)治疗有效量的第一活性成分，所述第一活性成分为五氟利多或其药学上可接受的盐；和
    (b)治疗有效量的第二活性成分，所述第二活性成分为孕激素。
  2. 如权利要求1所述的药物组合物，其特征在于，所述孕激素选自下组：酸酸甲羟孕酮、醋酸甲地孕酮、己酸孕酮、或其组合。
  3. 如权利要求1所述的药物组合物，其特征在于，所述第一活性成分与第二活性成分的重量比为1:20至10：1，较佳地，1:15至1:1，更佳地，1:10至1:5。
  4. 如权利要求1所述的药物组合物，其特征在于，所述的药物组合物用于治疗和/或预防子宫内膜癌。
  5. 如权利要求4所述的药物组合物，其特征在于，所述子宫内膜癌包括I型和II型子宫内膜癌。
  6. 如权利要求5所述的药物组合物，其特征在于，所述子宫内膜癌包括孕激素敏感型子宫内膜癌、或孕激素耐受型子宫内膜癌。
  7. 一种药盒，其特征在于，所述药盒包括：
    (a)含有五氟利多或其药学上可接受的盐的第一制剂；和
    (b)含有孕激素的第二制剂。
  8. 一种活性成分组合，其特征在于，所述活性成分组合包括：
    (a)治疗有效量的第一活性成分，所述第一活性成分为五氟利多或其药学上可接受的盐；和
    (b)治疗有效量的第二活性成分，所述第二活性成分为孕激素。
  9. 一种如权利要求1所述的药物组合物、如权利要求7所述的药盒和/或如权利要求8所述的活性成分组合的用途，用于制备治疗和/或缓解子宫内膜癌的药物。
  10. 一种体外抑制子宫内膜癌细胞的方法，其特征在于，包括步骤：
    (a)在五氟利多或其药学上可接受的盐和孕激素存在下，培养子宫内膜癌细胞，从而抑制所述的子宫内膜癌细胞。
PCT/CN2021/117536 2021-09-09 2021-09-09 五氟利多联合醋酸甲羟孕酮在制备治疗子宫内膜癌的药物中的应用 Ceased WO2023035201A1 (zh)

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