WO2023035200A1 - Application of pentafluorite in preparation of drug for treating endometrial cancer - Google Patents
Application of pentafluorite in preparation of drug for treating endometrial cancer Download PDFInfo
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- WO2023035200A1 WO2023035200A1 PCT/CN2021/117534 CN2021117534W WO2023035200A1 WO 2023035200 A1 WO2023035200 A1 WO 2023035200A1 CN 2021117534 W CN2021117534 W CN 2021117534W WO 2023035200 A1 WO2023035200 A1 WO 2023035200A1
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- Prior art keywords
- endometrial cancer
- cells
- penfluridol
- active ingredient
- kle
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Images
Classifications
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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
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- 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 belongs to the technical field of medicines, and in particular relates to the application of penfluridol 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. Although EC is more common in postmenopausal women, 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 have risen along with obesity rates. In 2015, there were about 63,400 new EC cases and about 21,800 new EC deaths 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.
- 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, and the main method of conservative treatment is hormone therapy with progesterone drugs.
- 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 endometrial cancer, especially progesterone receptor-negative and progesterone-resistant EC.
- an active ingredient or a preparation containing the active ingredient in the preparation of a drug for treating and/or preventing endometrial cancer wherein the active ingredient is penfluridol or its Pharmaceutically acceptable salts.
- the present invention also provides an active ingredient or a preparation containing the active ingredient for treating and/or preventing endometrial cancer, wherein the active ingredient is penfluridol or a pharmaceutically acceptable salt thereof.
- 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 active ingredient or the preparation containing the active ingredient is used to prepare medicines for one or more of the following purposes:
- the endometrial cancer cells are selected from the group consisting of ISK, KLE, HEC-1-A, HEC-1-B and AN3CA or combinations thereof.
- the endometrial cancer cells are ISK and/or KLE cells.
- the preparation is an oral preparation or a non-oral preparation.
- the preparation is selected from the group consisting of injection, inhalation, tincture, powder, granule, capsule, oral liquid, tablet, pill, suspension, emulsion, lozenge, or drop pill.
- the administration of the preparation is oral or injection.
- the preparation further includes other anti-endometrial cancer drugs.
- the anti-endometrial cancer drug is progesterone.
- the progestin is selected from: megestrol acetate, medroxyprogesterone acid, progesterone caproate, or combinations thereof.
- the other anti-endometrial cancer drug is medroxyprogesterone acid.
- composition which comprises:
- the anti-endometrial cancer drug is progesterone.
- the progestin is selected from: megestrol acetate, medroxyprogesterone acid, progesterone caproate, or combinations thereof.
- the other anti-endometrial cancer drug is medroxyprogesterone acid.
- the endometrial cancer includes type I and type II endometrial cancer.
- the first active ingredient accounts for 0.1-99% by mass of the total weight of the drug.
- 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:
- the endometrial cancer cells are selected from the group consisting of ISK, KLE, HEC-1-A, HEC-1-B and AN3CA or combinations thereof.
- the endometrial cancer cells are ISK and/or KLE cells.
- a method for treating endometrial cancer comprising the step of administering to a subject in need a therapeutically effective amount of penfluridol or a pharmaceutically acceptable salt thereof or the second aspect of the present invention
- the pharmaceutical composition comprising the step of administering to a subject in need a therapeutically effective amount of penfluridol or a pharmaceutically acceptable salt thereof or the second aspect of the present invention
- the pharmaceutical composition comprising the step of administering to a subject in need a therapeutically effective amount of penfluridol or a pharmaceutically acceptable salt thereof or the second aspect of the present invention.
- the subject is a patient with endometrial cancer.
- the endometrial cancer includes type I and type II endometrial cancer.
- Fig. 1 is a schematic diagram of the proliferation inhibitory activity of penfluridol on ISK and KLE cells after acting on them for different periods of time.
- A ISK cell proliferation inhibitory activity
- B KLE cell proliferation inhibitory activity.
- Figure 2 is a schematic diagram of the effect of penfluridol on the clone formation 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.
- Figure 3 is a schematic diagram of the effect of penfluridol 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. 4 is a schematic diagram showing the effect of penfluridol on the apoptosis of ISK and KLE cells.
- A Effect of penfluridol on apoptosis of ISK cells;
- B Effect of penfluridol on apoptosis of KLE cells;
- C Quantification of apoptosis ratio of ISK cells;
- D Quantification of apoptosis ratio of KLE cells.
- Figure 5 shows the inhibitory effect of penfluridol on the growth of subcutaneous KLE cell transplanted tumors in mice.
- A Change curve of mouse body weight with administration time;
- B Tumor volume after administration for 14 days;
- C Tumor pictures of mice after administration for 14 days;
- D Tumor weight after administration for 14 days.
- the inventor unexpectedly discovered a new application of the antipsychotic drug penfluridol in the preparation of a drug for treating endometrial cancer.
- the inventors found through experiments that penfluridol has inhibitory effects on the proliferation, clone formation and migration of endometrial cancer ISK and KLE cells, and can induce apoptosis of EC cells.
- Experiments in mice showed that penfluridol can inhibit the growth of subcutaneous transplanted tumors in mice. 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.
- PFL Penfluridol
- active ingredient of the present invention active ingredient of the present invention
- compound of the present invention is used interchangeably, and all refer to penfluridol and its pharmaceutically acceptable salts.
- 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 endometrial cancer cell ISK (type I, progesterone-sensitive), and further research found that it has an inhibitory effect on type II endometrial cancer cell KLE (progesterone-sensitive type).
- Drug-resistant type has an inhibitory effect on proliferation, can inhibit the formation and migration of two EC cell clones, induce EC cell apoptosis, and can inhibit the growth of subcutaneous xenograft tumors in nude mice in vivo. Therefore, the penfluridol of the present invention can be applied to the preparation of medicines for treating endometrial cancer.
- 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 capable of effectively treating endometrial cancer, comprising:
- the anti-endometrial cancer drug is progesterone.
- the progesterone is selected from: megestrol acetate (MA), medroxyprogesterone acetate (MPA), progesterone caproate (HPC), or a combination thereof.
- the other anti-endometrial cancer drug is MPA.
- the endometrial cancer includes type I and type II endometrial cancer.
- the pharmaceutical composition provided by the present invention preferably contains the first active ingredient in a weight ratio of 0.1-99wt%, and the rest is the second active ingredient, pharmaceutically acceptable carrier, diluent or solution or saline solution.
- 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.
- the unit dose of the preparation formula usually contains 0.05-1000 mg of the active compound of the present invention, preferably, the unit dose of the preparation formula contains 1 mg-500 mg of the active compound of the present invention.
- 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.
- binders 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.
- binders 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 that are used to prepare the tablet of the present invention can also be used to prepare the capsule 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 this 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 active ingredient or composition of the present invention can be taken alone, or used in combination 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 invention relates to the application of penfluridol in the preparation of medicine for treating 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 drug for treating endometrial cancer is penfluridol.
- the drug for treating endometrial cancer is a single component of penfluridol or a combination of penfluridol and other pharmaceutically acceptable components.
- the mass percentage of the active component penfluridol is 0.1-99%.
- the other pharmaceutically acceptable ingredients are anti-endometrial cancer drugs.
- the anti-endometrial cancer drug is progesterone.
- the progestin is one of megestrol acetate, medroxyprogesterone acid or progesterone caproate.
- the dosage form of the drug is one of injection, tablet, capsule, pill, suspension or emulsion.
- the drug formulation is administered orally or injected.
- the invention provides the application of penfluridol in the preparation of medicine for treating endometrial cancer, and the endometrial cancer includes type I and type II endometrial cancer.
- the present invention finds through experiments that penfluridol has an inhibitory effect on the proliferation, clone formation, and migration of endometrial cancer ISK and KLE cells, and can inhibit the growth of subcutaneous transplanted tumors in mice, which has been proved to be effective in mice in vivo experiments, thereby Penfluridol was found to have an unexpected effect in the treatment of endometrial cancer.
- the main advantages of the present invention include:
- penfluridol can inhibit the proliferation, colony formation and migration of endometrial cancer ISK and KLE cells.
- penfluridol can inhibit the growth of subcutaneous endometrial cancer xenografts in mice.
- 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 library of the laboratory.
- ATCC American Type Culture Collection
- PBS phosphate buffered saline
- DMEM/F12 medium was purchased from Biosharp
- FBS fetal calf Serum
- trypsin were purchased from Gibco
- CCK-8 was purchased from Biyuntian Biotechnology Co., Ltd.
- penfluridol was from the old drug library of the laboratory.
- 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 in cell culture incubator for 24h, 48h or 72h respectively.
- 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.
- Penfluridol inhibited the proliferation of ISK cells at incubation times of 24h, 48h, and 72h with IC 50 of 4.65 ⁇ M, 3.48 ⁇ M, and 2.77 ⁇ M; penfluridol inhibited the proliferation of KLE cells at incubation times of 24h, 48h, and 72h
- the active IC 50 were 5.86 ⁇ M, 3.14 ⁇ M, 2.88 ⁇ M, respectively.
- penfluridol can inhibit the proliferation of endometrial cancer cells ISK and KLE in a time-dependent and concentration-dependent manner.
- penfluridol can inhibit the colony formation ability of endometrial cancer cells ISK and KLE, and can inhibit the colony formation of endometrial cancer cells in a concentration-dependent manner.
- 0.5% crystal violet dye solution was purchased from Beyontian Biotechnology Co., Ltd. Methanol is a common reagent in the laboratory and was purchased commercially without any treatment. 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 1000 cells per well, and cultured overnight. After the cells adhered to the wall, they were divided into 4 groups, namely the control group, the PFL 2 ⁇ M group, the PFL 3 ⁇ M group and the PFL 4 ⁇ M group, with 3 replicate wells in each group.
- the drug-administered group was cultured with 2 mL of DMEM/F12 medium containing different concentrations of drugs for 48 hours, and then replaced with a drug-free medium to continue culturing for about 8 days. After the cells grow into a cell group visible to the naked eye, place them on ice, and wash the cells twice with pre-cooled PBS for 3 minutes each time. Cells were then fixed with pre-cooled methanol for 10 min at -20°C. Suction methanol, add crystal violet staining solution 1mL, stain for 30min. Remove the crystal violet dye solution and wash with water until the dye solution is completely eluted. Invert the 6-well plate and wait for it to dry, take pictures with a gel imager, count manually with Image J software, and count the number of clones formed in each well.
- the vertical axis of the histogram indicates the number of cell communities in each group, and the significant difference of data was analyzed by One-way ANOVA method (Graphpad Prism 8.0 software). Data are mean ⁇ SD: *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001 vs Ctrl.
- penfluridol has an inhibitory effect on the colony formation of ISK and KLE cells, and can inhibit the colony formation of endometrial cancer cells ISK and KLE in a concentration-dependent manner.
- penfluridol can inhibit the migration of endometrial cancer cells ISK and KLE, and can inhibit the migration of endometrial cancer cells in a concentration-dependent manner.
- 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.
- penfluridol has the ability to inhibit the migration of ISK and KLE cells, and can inhibit the migration of endometrial cancer cells in a concentration-dependent manner.
- the Annexin V-FITC/PI cell apoptosis detection kit was used to test the effect of penfluridol on the apoptosis of endometrial cancer cells ISK and KLE at the cellular level.
- the research results show that penfluridol can inhibit the apoptosis of endometrial cancer cells ISK and KLE, and can inhibit the apoptosis of endometrial cancer cells in a concentration-dependent manner.
- 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 4 groups, namely the control group, the PFL 2 ⁇ M group, the PFL 3 ⁇ M group and the PFL 4 ⁇ M group, with 3 replicate wells in each group.
- the treatment group was added with 2 mL of DMEM/F12 medium containing different concentrations of PFL, and the control group was added with 2 mL of drug-free DMEM/F12 medium. Incubate for 48 h at 37°C in an incubator with 5% CO 2 .
- the percentages of apoptosis were 12.11%, 27.34% and 31.83% when the concentration of penfluridol was 2 ⁇ M, 3 ⁇ M and 4 ⁇ M, respectively.
- 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). Data are mean ⁇ SD: *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001 vs Ctrl.
- penfluridol has the ability to induce apoptosis of ISK and KLE cells, and can induce the apoptosis of ISK and KLE cells in a concentration-dependent manner.
- mice of SPF grade 5-6 weeks old female BALB/C nude mice of SPF grade were selected as experimental animals, which were purchased from Shanghai Slack Animal Experiment Co., Ltd.; cisplatin (DDP) was obtained from the old drug storehouse of the laboratory.
- the compound preparation method is to take a certain amount of compound, dissolve it in DMSO and castor oil (the final concentration of DMSO is 5%, and the final concentration of castor oil is 8%), then dilute it into the required concentration with normal saline, and the corresponding solvent is given to the control group. .
- Compounds are formulated and used immediately prior to administration.
- mice were implanted subcutaneously in the right armpit of 5-6 week-old nude mice, and about 5 million cells were implanted in each mouse. After the tumor grew to 800-1000 mm 3 , the mice were sacrificed, and then the tumor tissue was cut into tumor tissue pieces of uniform size, which were transplanted into new 5-6 week-old nude mice under the skin of the right armpit. When the newly grown tumor volume reached an average of 300-400 mm 3 , the mice were randomly divided into 4 groups: control group, positive drug DDP 2 mg/kg, PFL 2 mg/kg administration group, and PFL 5 mg/kg administration group. Inject 0.1 mL intraperitoneally according to different drug concentrations, and weigh the mice every day.
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Abstract
The present invention relates to the technical field of drugs, and in particular, to an application of pentafluorite in the preparation of a drug for treating endometrial cancer. Studies of the present invention show that pentafluorite has the effects of inhibiting proliferation, clone formation and migration ability of endometrial cancer ISK and KLE cells, can induce apoptosis of EC cells, can inhibit growth of subcutaneous transplanted tumors of mice, prove effective in experiments in mice, and have application potential in treating endometrial cancer.
Description
本发明属于药物技术领域，具体涉及五氟利多在制备治疗子宫内膜癌的药物中的应用。The invention belongs to the technical field of medicines, and in particular relates to the application of penfluridol in the preparation of medicines for treating endometrial cancer.
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。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。2015年中国新增EC病例约63400例，新增EC死亡病例约21,800例。Endometrial cancer (EC) 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. Although EC is more common in postmenopausal women, 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 have risen along with obesity rates. In 2015, there were about 63,400 new EC cases and about 21,800 new EC deaths in China.
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。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。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。EC is usually divided into type I and type II. The main difference between the two is that type I EC is positive for progesterone receptor expression, and type II EC is negative for progesterone receptor expression. At present, 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, and the main method of conservative treatment is hormone therapy with progesterone drugs. However, 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. At present, there is an increasing demand for conservative treatment for fertility preservation in patients with EC.
因此，本领域迫切需要开发一种能够治疗EC，特别是治疗孕激素受体阴性和孕激素耐药的EC的药物。Therefore, there is an urgent need in the art to develop a drug capable of treating EC, especially progesterone receptor-negative and progesterone-resistant EC.
发明内容Contents of the invention
本发明的目的是提供一种能够治疗子宫内膜癌，特别是治疗孕激素受体阴性和孕激素耐药的EC的药物。The purpose of the present invention is to provide a drug capable of treating endometrial cancer, especially progesterone receptor-negative and progesterone-resistant EC.
在本发明的第一方面，提供了一种活性成分或含所述活性成分的制剂在制备治疗和/或预防子宫内膜癌的药物中的用途，其中所述活性成分为五氟利多或其药学上可接受的盐。In the first aspect of the present invention, there is provided an active ingredient or a preparation containing the active ingredient in the preparation of a drug for treating and/or preventing endometrial cancer, wherein the active ingredient is penfluridol or its Pharmaceutically acceptable salts.
本发明还提供了一种活性成分或含所述活性成分的制剂用于治疗和/或预防子宫内膜癌的用途，其中所述活性成分为五氟利多或其药学上可接受的盐。The present invention also provides an active ingredient or a preparation containing the active ingredient for treating and/or preventing endometrial cancer, wherein the active ingredient is penfluridol or a pharmaceutically acceptable salt thereof.
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。In another preferred example, the endometrial cancer includes type I and type II endometrial cancer.
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。In another preferred example, the endometrial cancer includes progesterone-sensitive endometrial cancer or progesterone-resistant endometrial cancer.
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。In another preferred example, the endometrial cancer is progesterone receptor-negative and progesterone-resistant endometrial cancer.
在另一优选例中，所述活性成分或含所述活性成分的制剂用于制备以下一种或多种用途的药物：In another preferred example, the active ingredient or the preparation containing the active ingredient is used to prepare medicines for one or more of the following purposes:
(a)抑制子宫内膜癌细胞增殖；(a) inhibit endometrial cancer cell proliferation;
(b)抑制子宫内膜癌细胞克隆；(b) inhibit endometrial cancer cell clones;
(c)抑制子宫内膜癌细胞迁移；(c) inhibit endometrial cancer cell migration;
(d)诱导子宫内膜癌细胞凋亡。(d) Induction of apoptosis in endometrial cancer cells.
在另一优选例中，所述子宫内膜癌细胞选自下组：ISK、KLE、HEC-1-A、HEC-1-B和AN3CA或其组合。In another preferred example, the endometrial cancer cells are selected from the group consisting of ISK, KLE, HEC-1-A, HEC-1-B and AN3CA or combinations thereof.
在另一优选例中，所述子宫内膜癌细胞为ISK和/或KLE细胞。In another preferred example, the endometrial cancer cells are ISK and/or KLE cells.
在另一优选例中，所述制剂为口服制剂或非口服制剂。In another preferred example, the preparation is an oral preparation or a non-oral preparation.
在另一优选例中，所述制剂选自下组：注射剂、吸入剂、酊剂、粉剂、颗粒剂、胶囊剂、口服液、片剂、丸剂、悬浮剂、乳剂、含片、或滴丸。In another preferred embodiment, the preparation is selected from the group consisting of injection, inhalation, tincture, powder, granule, capsule, oral liquid, tablet, pill, suspension, emulsion, lozenge, or drop pill.
在另一优选例中，所述制剂的给药方式为口服或注射。In another preferred example, the administration of the preparation is oral or injection.
在另一优选例中，所述制剂还包括其他抗子宫内膜癌药物。In another preferred example, the preparation further includes other anti-endometrial cancer drugs.
在另一优选例中，所述抗子宫内膜癌药物为孕激素。In another preferred example, the anti-endometrial cancer drug is progesterone.
在另一优选例中，所述孕激素选自：醋酸甲地孕酮、酸酸甲羟孕酮、己酸孕酮、或其组合。In another preferred embodiment, the progestin is selected from: megestrol acetate, medroxyprogesterone acid, progesterone caproate, or combinations thereof.
在另一优选例中，所述其他抗子宫内膜癌药物为酸酸甲羟孕酮。In another preferred example, the other anti-endometrial cancer drug is medroxyprogesterone acid.
在本发明的第二方面，提供了一种药物组合物，所述药物组合物包含：In a second aspect of the present invention, a pharmaceutical composition is provided, which comprises:
(A1)作为第一活性成分的五氟利多或其药学上可接受的盐；(A1) Penfluridol or a pharmaceutically acceptable salt thereof as the first active ingredient;
(A2)作为第二活性成分的其他抗子宫内膜癌药物；(A2) Other anti-endometrial cancer drugs as the second active ingredient;
(B)药学上可接受的载体或赋形剂。(B) A pharmaceutically acceptable carrier or excipient.
在另一优选例中，所述抗子宫内膜癌药物为孕激素。In another preferred example, the anti-endometrial cancer drug is progesterone.
在另一优选例中，所述孕激素选自：醋酸甲地孕酮、酸酸甲羟孕酮、己酸孕酮、或其组合。In another preferred embodiment, the progestin is selected from: megestrol acetate, medroxyprogesterone acid, progesterone caproate, or combinations thereof.
在另一优选例中，所述其他抗子宫内膜癌药物为酸酸甲羟孕酮。In another preferred example, the other anti-endometrial cancer drug is medroxyprogesterone acid.
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。In another preferred example, the endometrial cancer includes type I and type II endometrial cancer.
在另一优选例中，所述第一活性成分占药物总重量的质量百分含量为0.1-99％。In another preferred example, the first active ingredient accounts for 0.1-99% by mass of the total weight of the drug.
在本发明的第三方面，提供了一种体外抑制子宫内膜癌细胞的方法，包括步骤：In a third aspect of the present invention, there is provided a method for inhibiting endometrial cancer cells in vitro, comprising the steps of:
(i)在五氟利多或其药学上可接受的盐存在下，培养子宫内膜癌细胞，从而抑制所述的子宫内膜癌细胞。(i) culturing endometrial cancer cells in the presence of penfluridol or a pharmaceutically acceptable salt thereof, thereby inhibiting said endometrial cancer cells.
在另一优选例中，所述的方法是非治疗性和非诊断性的。In another preferred embodiment, the method is non-therapeutic and non-diagnostic.
在另一优选例中，所述的抑制选自下组：：In another preferred example, the inhibition is selected from the group:
(a)抑制子宫内膜癌细胞增殖；(a) inhibit endometrial cancer cell proliferation;
(b)抑制子宫内膜癌细胞克隆；(b) inhibit endometrial cancer cell clones;
(c)抑制子宫内膜癌细胞迁移；(c) inhibit endometrial cancer cell migration;
(d)诱导子宫内膜癌细胞凋亡；(d) inducing apoptosis of endometrial cancer cells;
(e)上述(a)至(d)的任意组合。(e) Any combination of the above (a) to (d).
在另一优选例中，所述子宫内膜癌细胞选自下组：ISK、KLE、HEC-1-A、HEC-1-B和AN3CA或其组合。In another preferred example, the endometrial cancer cells are selected from the group consisting of ISK, KLE, HEC-1-A, HEC-1-B and AN3CA or combinations thereof.
在另一优选例中，所述子宫内膜癌细胞为ISK和/或KLE细胞。In another preferred example, the endometrial cancer cells are ISK and/or KLE cells.
在本发明的第四方面，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治疗有效量的五氟利多或其药学上可接受的盐或本发明第二方面所述的药物组合物。In the fourth aspect of the present invention, there is provided a method for treating endometrial cancer, comprising the step of administering to a subject in need a therapeutically effective amount of penfluridol or a pharmaceutically acceptable salt thereof or the second aspect of the present invention The pharmaceutical composition.
在另一优选例中，所述对象为子宫内膜癌患者。In another preferred example, the subject is a patient with endometrial cancer.
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。In another preferred example, the endometrial cancer includes type I and type II endometrial cancer.
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。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.
图1为五氟利多对ISK和KLE细胞作用不同时间后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。Fig. 1 is a schematic diagram of the proliferation inhibitory activity of penfluridol on ISK and KLE cells after acting on them for different periods of time. (A) ISK cell proliferation inhibitory activity; (B) KLE cell proliferation inhibitory activity.
图2为五氟利多对ISK和KLE细胞克隆形成的影响示意图。(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。Figure 2 is a schematic diagram of the effect of penfluridol on the clone formation 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.
图3为五氟利多对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。Figure 3 is a schematic diagram of the effect of penfluridol 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.
图4为五氟利多对ISK和KLE细胞的凋亡的影响示意图。(A)五氟利多对ISK细胞凋 亡的影响；(B)五氟利多对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。Fig. 4 is a schematic diagram showing the effect of penfluridol on the apoptosis of ISK and KLE cells. (A) Effect of penfluridol on apoptosis of ISK cells; (B) Effect of penfluridol on apoptosis of KLE cells; (C) Quantification of apoptosis ratio of ISK cells; (D) Quantification of apoptosis ratio of KLE cells.
图5为五氟利多对小鼠皮下KLE细胞移植瘤的生长抑制作用。(A)小鼠体重随给药时间的变化曲线；(B)给药14天后肿瘤体积；(C)给药14天后小鼠肿瘤图片；(D)给药14天后肿瘤重量。Figure 5 shows the inhibitory effect of penfluridol on the growth of subcutaneous KLE cell transplanted tumors in mice. (A) Change curve of mouse body weight with administration time; (B) Tumor volume after administration for 14 days; (C) Tumor pictures of mice after administration for 14 days; (D) Tumor weight after administration for 14 days.
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在制备治疗子宫内膜癌的药物中的新用途。本发明人通过实验发现，五氟利多对子宫内膜癌ISK和KLE细胞的增殖、克隆形成、迁移能力具有抑制作用，能诱导EC细胞凋亡。小鼠体内实验表明，五氟利多能够抑制小鼠皮下移植瘤的生长。在此基础上，完成了本发明。After extensive and in-depth research, the inventor unexpectedly discovered a new application of the antipsychotic drug penfluridol in the preparation of a drug for treating endometrial cancer. The inventors found through experiments that penfluridol has inhibitory effects on the proliferation, clone formation and migration of endometrial cancer ISK and KLE cells, and can induce apoptosis of EC cells. Experiments in mice showed that penfluridol can inhibit the growth of subcutaneous transplanted tumors in mice. On this basis, the present invention has been accomplished.
术语the term
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。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
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”。As used herein, 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".
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***反应)的，即有合理的效益/风险比的物质。As used herein, the term "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.
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。As used herein, 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.
五氟利多Penfluridol
如本文所用，术语“五氟利多”(Penfluridol，PFL)、“本发明的活性成分”、“本发明的化合物”可互换使用，均指五氟利多及其药学上可接受的盐。As used herein, the terms "Penfluridol (PFL), "active ingredient of the present invention" and "compound of the present invention" are used interchangeably, and all refer to penfluridol and its pharmaceutically acceptable salts.
所述五氟利多具有如下结构式：The penfluridol has the following structural formula:
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***症。五氟利多是多巴胺D2受体和钙通道的强效抑制剂。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.
本发明经大量筛选研究，意外地发现五氟利多对子宫内膜癌细胞ISK(I型，孕激素敏感型)具有增殖抑制作用，进一步研究发现其对II型子宫内膜癌细胞KLE(孕激素耐药型)的增殖具有抑制作用，并能够抑制两种EC细胞克隆形成和迁移，诱导EC细胞凋亡，以及能够体内抑制裸鼠皮下移植瘤的生长。因此，本发明五氟利多可应用于制备治疗子宫内膜癌的药物。After a large number of screening studies, the present invention unexpectedly found that penfluridol has a proliferation inhibitory effect on endometrial cancer cell ISK (type I, progesterone-sensitive), and further research found that it has an inhibitory effect on type II endometrial cancer cell KLE (progesterone-sensitive type). Drug-resistant type) has an inhibitory effect on proliferation, can inhibit the formation and migration of two EC cell clones, induce EC cell apoptosis, and can inhibit the growth of subcutaneous xenograft tumors in nude mice in vivo. Therefore, the penfluridol of the present invention can be applied to the preparation of medicines for treating endometrial cancer.
子宫内膜癌endometrial cancer
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***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(***依赖型)与II型子宫内膜癌(非***依赖型)。As used herein, the term "endometrial cancer (EC)" 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).
药物组合物pharmaceutical composition
本发明还提供了一种能有效治疗子宫内膜癌的药物组合物，包含：The present invention also provides a pharmaceutical composition capable of effectively treating endometrial cancer, comprising:
(A1)作为第一活性成分的五氟利多或其药学上可接受的盐；(A1) Penfluridol or a pharmaceutically acceptable salt thereof as the first active ingredient;
(A2)作为第二活性成分的其他抗子宫内膜癌药物；(A2) Other anti-endometrial cancer drugs as the second active ingredient;
(B)药学上可接受的载体或赋形剂。(B) A pharmaceutically acceptable carrier or excipient.
在另一优选例中，所述抗子宫内膜癌药物为孕激素。In another preferred example, the anti-endometrial cancer drug is progesterone.
在另一优选例中，所述孕激素选自：醋酸甲地孕酮(MA)、酸酸甲羟孕酮(MPA)、己酸孕酮(HPC)、或其组合。In another preferred embodiment, the progesterone is selected from: megestrol acetate (MA), medroxyprogesterone acetate (MPA), progesterone caproate (HPC), or a combination thereof.
在另一优选例中，所述其他抗子宫内膜癌药物为MPA。In another preferred example, the other anti-endometrial cancer drug is MPA.
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。In another preferred example, the endometrial cancer includes type I and type II endometrial cancer.
本发明所提供的药物组合物优选含有重量比为0.1-99wt％的第一活性成份，其余部分为第二活性成分、药学可接受的载体、稀释液或溶液或盐溶液。The pharmaceutical composition provided by the present invention preferably contains the first active ingredient in a weight ratio of 0.1-99wt%, and the rest is the second active ingredient, pharmaceutically acceptable carrier, diluent or solution or saline solution.
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。When necessary, one or more pharmaceutically acceptable carriers can also be added to the medicine of the present invention. The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.
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。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.
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。其制剂配方的单位计量中通常包含0.05-1000mg本发明的活性化合物，优选地，制剂配方的单位计量中包含1mg-500mg本发明的活性化合物。Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the field of pharmacy. The unit dose of the preparation formula usually contains 0.05-1000 mg of the active compound of the present invention, preferably, the unit dose of the preparation formula contains 1 mg-500 mg of the active compound of the present invention.
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。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.
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。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.
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、***、直肠等。Typically, 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.
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。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.
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***。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.
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、***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。In order to make the active ingredient of the present invention into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluent 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.; binders 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, low-substituted hydroxypropyl cellulose, cross-linked poly Vinylpyrrolidone, croscarmellose sodium, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecylsulfonate, etc.; lubricant and flow aid The agent can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol and the like.
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。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.
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。In order to make the administration unit into a capsule, 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 that are used to prepare the tablet of the present invention can also be used to prepare the capsule of the present invention.
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。In order to make the active ingredient of the present invention into an injection, 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 this field can be added. The solubilizer or co-solvent can be poloxamer, lecithin, hydroxypropyl-β-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. For preparation of freeze-dried powder injection, mannitol, glucose, etc. can also be added as proppants.
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。In addition, coloring agents, preservatives, fragrances, flavoring agents or other additives can also be added to the pharmaceutical preparations, if necessary.
本发明的活性成分或组合物可单独服用，或与其他治疗药物或对症药物合并使用。The active ingredient or composition of the present invention can be taken alone, or used in combination with other therapeutic drugs or symptomatic drugs.
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。When 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.
五氟利多应用于制备治疗子宫内膜癌的药物Application of Penfluridol in the Preparation of Drugs for Treating Endometrial Cancer
本发明涉及五氟利多在制备治疗子宫内膜癌的药物中的应用。The invention relates to the application of penfluridol in the preparation of medicine for treating endometrial cancer.
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。In another preferred example, the endometrial cancer includes type I and type II endometrial cancer.
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。In another preferred example, the endometrial cancer includes progesterone-sensitive endometrial cancer or progesterone-resistant endometrial cancer.
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。In another preferred example, the endometrial cancer is progesterone receptor-negative and progesterone-resistant endometrial cancer.
在另一优选例中，所述治疗子宫内膜癌的药物为五氟利多。In another preferred example, the drug for treating endometrial cancer is penfluridol.
在另一优选例中，所述治疗子宫内膜癌的药物是以五氟利多单一成分作为药物或五氟利多与其他药学上可接受的成分构成组合物作为药物。In another preferred example, the drug for treating endometrial cancer is a single component of penfluridol or a combination of penfluridol and other pharmaceutically acceptable components.
在另一优选例中，所述五氟利多与其他药学上可接受的成分构成组合物中，活性组分 五氟利多的质量百分含量为0.1-99％。In another preferred embodiment, in the composition composed of the penfluridol and other pharmaceutically acceptable components, the mass percentage of the active component penfluridol is 0.1-99%.
在另一优选例中，所述其他药学上可接受的成分为抗子宫内膜癌药物。In another preferred example, the other pharmaceutically acceptable ingredients are anti-endometrial cancer drugs.
在另一优选例中，所述抗子宫内膜癌药物为孕激素。In another preferred example, the anti-endometrial cancer drug is progesterone.
在另一优选例中，所述孕激素为醋酸甲地孕酮、酸酸甲羟孕酮或己酸孕酮中的一种。In another preferred embodiment, the progestin is one of megestrol acetate, medroxyprogesterone acid or progesterone caproate.
在另一优选例中，所述药物的剂型为注射剂、片剂、胶囊剂、丸剂、悬浮剂或乳剂中的一种。In another preferred example, the dosage form of the drug is one of injection, tablet, capsule, pill, suspension or emulsion.
在另一优选例中，所述药物的制剂的给药方式为口服或注射。In another preferred example, the drug formulation is administered orally or injected.
本发明的主要优点包括：The main advantages of the present invention include:
本发明提供了一种五氟利多在制备治疗子宫内膜癌药物中的应用，所述子宫内膜癌包括Ⅰ型和Ⅱ型子宫内膜癌。本发明通过实验发现五氟利多对子宫内膜癌ISK和KLE细胞的增殖、克隆形成、迁移能力具有抑制作用，且能够抑制小鼠皮下移植瘤的生长，在小鼠体内实验中证实有效，从而发现了五氟利多具有意料不到的治疗子宫内膜癌的效果。本发明主要优点包括：The invention provides the application of penfluridol in the preparation of medicine for treating endometrial cancer, and the endometrial cancer includes type I and type II endometrial cancer. The present invention finds through experiments that penfluridol has an inhibitory effect on the proliferation, clone formation, and migration of endometrial cancer ISK and KLE cells, and can inhibit the growth of subcutaneous transplanted tumors in mice, which has been proved to be effective in mice in vivo experiments, thereby Penfluridol was found to have an unexpected effect in the treatment of endometrial cancer. The main advantages of the present invention include:
1)首次发现了五氟利多在治疗子宫内膜癌中的新用途。1) Discovered for the first time the new use of penfluridol in the treatment of endometrial cancer.
2)首次发现了五氟利多对子宫内膜癌ISK和KLE细胞的增殖、克隆形成、迁移能力具有抑制作用。2) For the first time, it was found that penfluridol can inhibit the proliferation, colony formation and migration of endometrial cancer ISK and KLE cells.
3)首次发现了五氟利多能够抑制小鼠皮下子宫内膜癌细胞移植瘤的生长。3) For the first time, it was discovered that penfluridol can inhibit the growth of subcutaneous endometrial cancer xenografts in mice.
为了更清楚地说明本发明，下面结合优选实施例对本发明做进一步的说明。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。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。In order to illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not limit the protection scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
实施例1 五氟利多对子宫内膜癌细胞ISK和KLE的增殖抑制Example 1 Inhibition of penfluridol on proliferation of endometrial cancer cells ISK and KLE
本实施例在细胞水平上通过CCK-8实验测试五氟利多对子宫内膜癌细胞ISK和KLE的增殖抑制作用。研究结果表明，五氟利多能够抑制子宫内膜癌细胞ISK和KLE的增殖，并且具有时间依赖性和浓度依赖性。In this example, the inhibitory effect of penfluridol on the proliferation of endometrial cancer cells ISK and KLE was tested by CCK-8 experiment at the cellular level. The research results showed that penfluridol could inhibit the proliferation of endometrial cancer cells ISK and KLE in a time-dependent and concentration-dependent manner.
1、实验材料和方法1. Experimental materials and methods
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。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 library of the laboratory.
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24h、48h或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。
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 . After 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 μL per well. After culturing overnight, after the cells adhered to the wall, DMEM/F12 medium containing different concentrations of PFL was added, 200 μL per well, and 3 replicate wells were set up for each group. Incubate in cell culture incubator for 24h, 48h or 72h respectively. Then remove the medium, add 100 μL of serum-free medium containing 10% CCK-8 to each well, incubate in the incubator at 37°C for 1 hour, use a Bio-Tek multi-functional microplate reader to detect the absorbance value A at 450 nm, and calculate Inhibition rate and IC 50 value. 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.
2、实验结果2. Experimental results
五氟利多对两种EC细胞不同时间的增殖抑制活性数据如表1和图1所示。结果表明，五氟利多对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，并且随着时间延长这种影响更加显著。五氟利多对ISK细胞在24h、48h和72h的孵育时间下增殖抑制活性IC
50分别为4.65μM、3.48μM、2.77μM；五氟利多对KLE细胞在24h、48h和72h的孵育时间下增殖抑制活性IC
50分别为5.86μM、3.14μM、2.88μM。
The data of penfluridol's proliferation inhibitory activity on two kinds of EC cells at different time are shown in Table 1 and Figure 1. The results showed that penfluridol had a significant inhibitory effect on the proliferation of endometrial cancer cells. The proliferative activity of endometrial cancer cells decreased with the increase of penfluridol concentration, and this effect became more significant with time. Penfluridol inhibited the proliferation of ISK cells at incubation times of 24h, 48h, and 72h with IC 50 of 4.65μM, 3.48μM, and 2.77μM; penfluridol inhibited the proliferation of KLE cells at incubation times of 24h, 48h, and 72h The active IC 50 were 5.86 μM, 3.14 μM, 2.88 μM, respectively.
表1 五氟利多对不同EC细胞株以及孵育不同时间的增殖抑制活性Table 1 Proliferation inhibitory activity of penfluridol on different EC cell lines and incubation time
本实施例说明了五氟利多能够时间依赖性和浓度依赖性地抑制子宫内膜癌细胞ISK和KLE的增殖。This example illustrates that penfluridol can inhibit the proliferation of endometrial cancer cells ISK and KLE in a time-dependent and concentration-dependent manner.
实施例2 五氟利多对子宫内膜癌细胞ISK和KLE克隆形成能力的影响Example 2 Effect of penfluridol on the clone formation ability of endometrial cancer cells ISK and KLE
本实施例在细胞水平上通过平皿克隆形成实验测试五氟利多对子宫内膜癌细胞ISK和KLE克隆形成能力的影响。研究结果表明，五氟利多能够抑制子宫内膜癌细胞ISK和KLE 的克隆形成能力，并且能够浓度依赖性地抑制子宫内膜癌细胞的克隆形成。In this example, the effect of penfluridol on the colony formation ability of endometrial cancer cells ISK and KLE was tested by a plate colony formation experiment at the cell level. The research results show that penfluridol can inhibit the colony formation ability of endometrial cancer cells ISK and KLE, and can inhibit the colony formation of endometrial cancer cells in a concentration-dependent manner.
1、实验材料和方法1. Experimental materials and methods
0.5％的结晶紫染液购自于碧云天生物科技有限公司；甲醇为实验室常用试剂，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制备成单细胞混悬液，以每孔约1000个细胞的密度接种至6孔板，过夜培养。待细胞贴壁后，分成4组，分别为对照组、PFL 2μM组、PFL 3μM组和PFL 4μM组，每组设3个复孔。给药组用含不同浓度药物的DMEM/F12培养基2mL培养48h，之后换成无药的培养基继续培养8天左右。待细胞长成肉眼可见的细胞群落后，置于冰上，用预冷的PBS清洗细胞两次，每次3min。然后用预冷的甲醇，于-20℃固定细胞10min。吸除甲醇，加入结晶紫染液1mL，染色30min。移除结晶紫染液，清水清洗至染液被洗脱完毕。倒扣6孔板待其晾干，用凝胶成像仪拍照，Image J软件手动计数，统计各个孔的克隆形成数。0.5% crystal violet dye solution was purchased from Beyontian Biotechnology Co., Ltd. Methanol is a common reagent in the laboratory and was purchased commercially without any treatment. 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 1000 cells per well, and cultured overnight. After the cells adhered to the wall, they were divided into 4 groups, namely the control group, the PFL 2 μM group, the PFL 3 μM group and the PFL 4 μM group, with 3 replicate wells in each group. The drug-administered group was cultured with 2 mL of DMEM/F12 medium containing different concentrations of drugs for 48 hours, and then replaced with a drug-free medium to continue culturing for about 8 days. After the cells grow into a cell group visible to the naked eye, place them on ice, and wash the cells twice with pre-cooled PBS for 3 minutes each time. Cells were then fixed with pre-cooled methanol for 10 min at -20°C. Suction methanol, add crystal violet staining solution 1mL, stain for 30min. Remove the crystal violet dye solution and wash with water until the dye solution is completely eluted. Invert the 6-well plate and wait for it to dry, take pictures with a gel imager, count manually with Image J software, and count the number of clones formed in each well.
2、实验结果2. Experimental results
实验结果如图2所示，图中Ctrl为对照组，照片中的每一个黑点表示一个细胞群落。结果表明，对于ISK细胞，与Ctrl相比，给药组的细胞群落数量减少，且随着五氟利多浓度的增加而明显减少，尤其是PFL 4μM组，只有极少数细胞群落存在(p<0.0001)。对于KLE细胞，与对照组相比，给药组的细胞群落数量也有所减少，且随着五氟利多浓度的增加而明显减少，其中PFL 3μM组和PFL 4μM组的细胞群落显著减少(PFL 3μM组:p<0.0001；PFL 4μM组:p<0.0001)。柱状图纵坐标表示各组细胞群落的数目，数据显著性差异采用One-way ANOVA法分析(Graphpad Prism 8.0软件)。数据为平均值±SD:*P<0.05,**P<0.01,***P<0.001,****P<0.0001vs Ctrl。The experimental results are shown in Figure 2, where Ctrl is the control group, and each black dot in the photo represents a cell community. The results showed that for ISK cells, compared with Ctrl, the number of cell colonies in the administration group decreased, and it decreased significantly with the increase of penfluridol concentration, especially in the PFL 4μM group, only a very small number of cell colonies existed (p<0.0001 ). For KLE cells, compared with the control group, the number of cell colonies in the administration group also decreased, and it decreased significantly with the increase of penfluridol concentration, and the cell colonies in the PFL 3 μM group and the PFL 4 μM group decreased significantly ( group: p<0.0001; PFL 4 μM group: p<0.0001). The vertical axis of the histogram indicates the number of cell communities in each group, and the significant difference of data was analyzed by One-way ANOVA method (Graphpad Prism 8.0 software). Data are mean±SD: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Ctrl.
本实施例说明了五氟利多对ISK和KLE细胞的克隆形成具有抑制作用，并且能够浓度依赖性地抑制子宫内膜癌细胞ISK和KLE的克隆形成。This example illustrates that penfluridol has an inhibitory effect on the colony formation of ISK and KLE cells, and can inhibit the colony formation of endometrial cancer cells ISK and KLE in a concentration-dependent manner.
实施例3 五氟利多对子宫内膜癌细胞ISK和KLE迁移能力的影响Example 3 Effect of penfluridol on migration ability of endometrial cancer cells ISK and KLE
本实施例在细胞水平上通过Transwell实验测试五氟利多对子宫内膜癌细胞ISK和KLE迁移能力的影响。研究结果表明，五氟利多能够抑制子宫内膜癌细胞ISK和KLE迁移，并且能够浓度依赖性地抑制子宫内膜癌细胞的迁移。In this example, the effect of penfluridol on the migration ability of endometrial cancer cells ISK and KLE was tested by Transwell experiment at the cellular level. The research results show that penfluridol can inhibit the migration of endometrial cancer cells ISK and KLE, and can inhibit the migration of endometrial cancer cells in a concentration-dependent manner.
1、实验材料和方法1. Experimental materials and methods
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4组，分别为对照组、PFL 2μM组、PFL 3μM组和PFL 4μM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。
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 μL of DMEM/F12 medium containing 20% FBS to the bottom of the small chamber, taking care to avoid the generation of air bubbles. They were divided into 4 groups, namely the control group, PFL 2μM group, PFL 3μM group and PFL 4μM group, and each group had 3 replicate wells. 150 μL of serum-free DMEM/F12 medium containing different drugs was added to the upper chamber of the treatment group, and 150 μL 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. Then carefully remove the small chamber with tweezers, blot the liquid in the upper chamber, move it to a 24-well plate with about 800 μL of pre-cooled PBS in advance, and wash it twice for 5 minutes each time. Take out the small chamber, move it to a 24-well plate pre-added with about 800 μL of paraformaldehyde solution, and fix it at room temperature for 30 minutes. Take out the small chamber, blot dry the fixative in the upper chamber, transfer to a 24-well plate pre-added with about 800 μL of crystal violet staining solution, and stain at room temperature for 30 minutes. Gently rinse with water several times, and carefully wipe off the cells on the upper chamber surface with a wet cotton swab. Five fields of view were randomly selected to take pictures under a microscope at 200 times, and the number of cells in each field of view was counted manually by Image J software.
2、实验结果2. Experimental results
结果如图3所示，图中Ctrl为对照组，照片中的每一个黑点表示一个穿过小室的细胞。结果表明，在ISK和KLE细胞中，与Ctrl组相比，给药组穿过小室的细胞数量减少，且随着五氟利多浓度的增加而明显减少，尤其是PFL 4μM组，只有少量细胞穿过(p<0.0001)。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<0.05,**P<0.01,***P<0.001,****P<0.0001vs Ctrl。The results are shown in Figure 3, where Ctrl is the control group, and each black dot in the photo represents a cell passing through the small chamber. The results showed that in ISK and KLE cells, compared with the Ctrl group, the number of cells passing through the small chamber in the administration group decreased, and it decreased significantly with the increase of penfluridol concentration, especially in the PFL 4 μM group, only a small number of cells passed through the chamber. over (p<0.0001). 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<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Ctrl.
本实施例说明了五氟利多具有抑制ISK和KLE细胞迁移的能力，并且能够浓度依赖性地抑制子宫内膜癌细胞的迁移。This example illustrates that penfluridol has the ability to inhibit the migration of ISK and KLE cells, and can inhibit the migration of endometrial cancer cells in a concentration-dependent manner.
实施例4 五氟利多对子宫内膜癌细胞ISK和KLE凋亡的影响Example 4 Effect of Penfluridol on Apoptosis of Endometrial Cancer Cells ISK and KLE
本实施例在细胞水平上采用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，并且能够浓度依赖性地抑制子宫内膜癌细胞的凋亡。In this example, the Annexin V-FITC/PI cell apoptosis detection kit was used to test the effect of penfluridol on the apoptosis of endometrial cancer cells ISK and KLE at the cellular level. The research results show that penfluridol can inhibit the apoptosis of endometrial cancer cells ISK and KLE, and can inhibit the apoptosis of endometrial cancer cells in a concentration-dependent manner.
1.实验材料和方法1. Experimental materials and methods
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4组，分别为对照组、PFL 2μM组、PFL 3μM组和PFL 4μM组，每组设3个复孔。给药组加入含不同浓度PFL的DMEM/F12培养基2mL，对照组加入无药物DMEM/F12培养基2mL。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。
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. After the cells adhered to the wall, they were divided into 4 groups, namely the control group, the PFL 2 μM group, the PFL 3 μM group and the PFL 4 μM group, with 3 replicate wells in each group. The treatment group was added with 2 mL of DMEM/F12 medium containing different concentrations of PFL, and the control group was added with 2 mL of drug-free DMEM/F12 medium. Incubate for 48 h at 37°C in an incubator with 5% CO 2 . Then suck out the cell culture solution into a 10ml centrifuge tube, wash the adherent cells once with PBS, add 300 μL of trypsin to digest the cells for 2 minutes, blow the cells down gently, transfer them to the corresponding centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, and collect the cells. Gently resuspend cells in PBS and count. Take 50,000-100,000 resuspended cells, centrifuge at 1000rpm for 5min, discard the supernatant, add 195μL Annexin V-FITC binding solution to gently resuspend the cells, add 5μL Annexin V-FITC and 10μL propidium iodide (PI) staining solution, gently Mix lightly. Incubate at room temperature in the dark for 20 min, and immediately detect with a Beckman Coulter (cytoFLEX LX) flow cytometer.
2、实验结果2. Experimental results
结果如图4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能够显著诱导ISK和KLE细胞凋亡，并且具有浓度依赖性。对于ISK细胞，五氟利多浓度在2μM、3μM和4μM时凋亡的比例分别为13.73％、34.16％和47.10％。对于KLE细胞，五氟利多浓度在2μM、3μM和4μM时凋亡的比例分别为12.11％、27.34％和31.83％。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<0.05,**P<0.01,***P<0.001,****P<0.0001vs Ctrl。The results are shown in Figure 4, where Ctrl is the control group, the first quadrant in the schematic diagram of apoptosis is late apoptotic cells, the fourth quadrant is early apoptotic cells, and the apoptotic ratio is the sum of the ratios of the first and fourth quadrants. Compared with the control group, PFL can significantly induce the apoptosis of ISK and KLE cells in a concentration-dependent manner. For ISK cells, the percentages of apoptosis were 13.73%, 34.16% and 47.10% when the concentration of penfluridol was 2μM, 3μM and 4μM, respectively. For KLE cells, the percentages of apoptosis were 12.11%, 27.34% and 31.83% when the concentration of penfluridol was 2μM, 3μM and 4μM, respectively. 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). Data are mean±SD: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Ctrl.
本实施例说明了五氟利多具有诱导ISK和KLE细胞凋亡的能力，并且能够浓度依赖性地诱导ISK和KLE细胞的凋亡。This example illustrates that penfluridol has the ability to induce apoptosis of ISK and KLE cells, and can induce the apoptosis of ISK and KLE cells in a concentration-dependent manner.
实施例5 五氟利多对子宫内膜癌细胞(KLE)移植瘤生长的影响Example 5 Effect of Penfluridol on Growth of Endometrial Cancer Cell (KLE) Transplanted Tumors
本实施例通过裸鼠体内皮下成瘤实验测试了五氟利多对子宫内膜癌细胞(KLE)移植瘤生长的影响。研究结果表明，五氟利多能够显著抑制小鼠皮下KLE细胞移植瘤的生长。In this example, the effect of penfluridol on the growth of endometrial cancer cell (KLE) xenografts was tested by the in vivo subcutaneous tumor formation experiment in nude mice. The results of the study showed that penfluridol could significantly inhibit the growth of subcutaneous KLE cell transplanted tumors in mice.
1、实验材料和方法1. Experimental materials and methods
实验动物选择5-6周龄的SPF级的雌性BALB/C裸鼠，购自上海斯莱克动物实验有限公司；顺铂(DDP)来源于实验室老药库。化合物配制方法是称取一定量的化合物，溶于DMSO和蓖麻油中(DMSO终浓度为5％，蓖麻油终浓度为8％)，再用生理盐水稀释成所需浓度，对照组给予相应溶剂。化合物给药前现配现用。5-6 weeks old female BALB/C nude mice of SPF grade were selected as experimental animals, which were purchased from Shanghai Slack Animal Experiment Co., Ltd.; cisplatin (DDP) was obtained from the old drug storehouse of the laboratory. The compound preparation method is to take a certain amount of compound, dissolve it in DMSO and castor oil (the final concentration of DMSO is 5%, and the final concentration of castor oil is 8%), then dilute it into the required concentration with normal saline, and the corresponding solvent is given to the control group. . Compounds are formulated and used immediately prior to administration.
首先在5-6周龄的裸鼠右侧腋下皮下植入KLE细胞，每只小鼠约植入500万个细胞。待肿瘤长至800-1000mm
3后处死小鼠，再将肿瘤组织切成大小均匀的肿瘤组织块，通过手术移植到新的5-6周龄裸鼠右侧腋下皮下。待新长出的肿瘤体积达到平均300-400mm
3时，将小鼠随机分4组：对照组、阳性药DDP 2mg/kg、PFL 2mg/kg给药组、PFL 5mg/kg给药组。按不同的药物浓度腹腔注射0.1mL，每天称量小鼠体重。每天给药，连续给药2周后，处死小鼠，剥离肿瘤，拍照。解剖当天用游标卡尺测量肿瘤长L(mm)和宽W(mm)，肿瘤体积计算公式V(mm
3)＝0.5×L(mm)×W(mm)
2。
Firstly, KLE cells were implanted subcutaneously in the right armpit of 5-6 week-old nude mice, and about 5 million cells were implanted in each mouse. After the tumor grew to 800-1000 mm 3 , the mice were sacrificed, and then the tumor tissue was cut into tumor tissue pieces of uniform size, which were transplanted into new 5-6 week-old nude mice under the skin of the right armpit. When the newly grown tumor volume reached an average of 300-400 mm 3 , the mice were randomly divided into 4 groups: control group, positive drug DDP 2 mg/kg, PFL 2 mg/kg administration group, and PFL 5 mg/kg administration group. Inject 0.1 mL intraperitoneally according to different drug concentrations, and weigh the mice every day. After administration every day for 2 weeks, the mice were sacrificed, the tumors were peeled off, and photographed. On the day of dissection, the tumor length L (mm) and width W (mm) were measured with a vernier caliper, and the tumor volume was calculated by the formula V (mm 3 )=0.5×L (mm)×W (mm) 2 .
2、实验结果2. Experimental results
实验结果如图5所示，图中Vehicle为对照组。其中A图为小鼠体重随给药时间的变化曲线，横坐标为给药天数，纵坐标为小鼠体重，从图中可以看到，五氟利多给药组体重平稳，顺铂DDP组体重有明显下降。B、C、D图可以看出给药结束后，与对照组相比，五氟利多给药组的肿瘤生长均受到抑制，PFL 2mg/kg和PFL 5mg/kg均能显著抑制小鼠肿瘤的生长，且与阳性药顺铂组效果相当。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<0.05,**P<0.01,***P<0.001,****P<0.0001vs Ctrl。The experimental results are shown in Figure 5, in which Vehicle is the control group. Among them, graph A is the change curve of the body weight of the mice with the time of administration, the abscissa is the days of administration, and the ordinate is the body weight of the mice. As can be seen from the figure, the body weight of the penfluridol administration group is stable, and the body weight of the cisplatin DDP group is stable. There is a significant decline. Figures B, C, and D show that after the administration, compared with the control group, the tumor growth of the penfluridol administration group was inhibited, and both PFL 2mg/kg and PFL 5mg/kg could significantly inhibit the tumor growth in mice Growth, and the effect is equivalent to the positive drug cisplatin group. The significant difference of the data was analyzed by One-way ANOVA method (Graphpad Prism8.0 software). Data are mean±SD: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Ctrl.
本实施例表明，五氟利多能够抑制小鼠皮下KLE细胞移植瘤的生长且对体重没有影响，其药效与阳性药顺铂相当，毒性小于顺铂。This example shows that penfluridol can inhibit the growth of subcutaneous KLE cell transplanted tumors in mice without affecting body weight, and its efficacy is equivalent to that of the positive drug cisplatin, and its toxicity is less than that of cisplatin.
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。All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
Claims (10)
- 一种活性成分或含所述活性成分的制剂的用途，其特征在于，所述活性成分为五氟利多或其药学上可接受的盐，A use of an active ingredient or a preparation containing the active ingredient, characterized in that the active ingredient is penfluridol or a pharmaceutically acceptable salt thereof,并且所述活性成分或含所述活性成分的制剂用于制备治疗和/或预防子宫内膜癌的药物。And the active ingredient or the preparation containing the active ingredient is used to prepare a medicine for treating and/or preventing endometrial cancer.
- 如权利要求1所述的用途，其特征在于，所述子宫内膜癌包括I型和II型子宫内膜癌。The use according to claim 1, characterized in that the endometrial cancer includes type I and type II endometrial cancer.
- 如权利要求1所述的用途，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。The use according to claim 1, characterized in that the endometrial cancer comprises progesterone-sensitive endometrial cancer or progesterone-resistant endometrial cancer.
- 如权利要求1所述的用途，其特征在于，所述活性成分或含所述活性成分的制剂用于制备以下一种或多种用途的药物：The use according to claim 1, wherein the active ingredient or the preparation containing the active ingredient is used for the preparation of one or more of the following medicines:(a)抑制子宫内膜癌细胞增殖；(a) inhibit endometrial cancer cell proliferation;(b)抑制子宫内膜癌细胞克隆；(b) inhibit endometrial cancer cell clones;(c)抑制子宫内膜癌细胞迁移；(c) inhibit endometrial cancer cell migration;(d)诱导子宫内膜癌细胞凋亡。(d) Induction of apoptosis in endometrial cancer cells.
- 如权利要求1所述的用途，其特征在于，所述制剂还包括其他抗子宫内膜癌药物。The use according to claim 1, characterized in that the preparation also includes other anti-endometrial cancer drugs.
- 一种药物组合物，其特征在于，所述药物组合物包含：A pharmaceutical composition, characterized in that the pharmaceutical composition comprises:(A1)作为第一活性成分的五氟利多或其药学上可接受的盐；(A1) Penfluridol or a pharmaceutically acceptable salt thereof as the first active ingredient;(A2)作为第二活性成分的其他抗子宫内膜癌药物；(A2) Other anti-endometrial cancer drugs as the second active ingredient;(B)药学上可接受的载体或赋形剂。(B) A pharmaceutically acceptable carrier or excipient.
- 如权利要求6所述的药物组合物，其特征在于，所述抗子宫内膜癌药物为孕激素。The pharmaceutical composition according to claim 6, wherein the anti-endometrial cancer drug is progesterone.
- 如权利要求6所述的药物组合物，其特征在于，所述第一活性成分占药物总重量的质量百分含量为0.1-99％。The pharmaceutical composition according to claim 6, wherein the mass percentage of the first active ingredient in the total weight of the medicine is 0.1-99%.
- 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 method for inhibiting endometrial cancer cells in vitro, comprising the steps of:(i)在五氟利多或其药学上可接受的盐存在下，培养子宫内膜癌细胞，从而抑制所述的子宫内膜癌细胞。(i) culturing endometrial cancer cells in the presence of penfluridol or a pharmaceutically acceptable salt thereof, thereby inhibiting said endometrial cancer cells.
- 如权利要求9所述的方法，其特征在于，所述子宫内膜癌细胞选自下组：ISK、KLE、HEC-1-A、HEC-1-B和AN3CA或其组合。The method of claim 9, wherein the endometrial cancer cells are selected from the group consisting of ISK, KLE, HEC-1-A, HEC-1-B and AN3CA or combinations thereof.
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| Title |
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| HE SUQIAN, CHENG YU-XIA; QI HUANG-PENG; ZHOU PING; FANG LEI; SUN QING: "Analysis of Clinicopathological Features and Prognosis Factors in 198 Patients with Endometrial Cancer", CHINESE JOURNAL OF CANCER PREVENTION AND TREATMENT, vol. 26, no. 20, 31 October 2019 (2019-10-31), pages 1562 - 1568, XP093046401, DOI: 10.16073/j.cnki.cjcpt.2019.20.13 * |
| HENDOUEI NARJES, SAGHAFI FATEMEH, SHADFAR FAEZEH, HOSSEINIMEHR SEYED JALAL: "Molecular mechanisms of anti-psychotic drugs for improvement of cancer treatment", EUROPEAN JOURNAL OF PHARMACOLOGY, ELSEVIER SCIENCE, NL, vol. 856, 1 August 2019 (2019-08-01), NL , pages 172402, XP093046402, ISSN: 0014-2999, DOI: 10.1016/j.ejphar.2019.05.031 * |
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