Application of pentaflulide in the preparation of drugs for treating endometrial cancer

Abstract

This invention relates to the field of pharmaceutical technology, specifically to the application of pentaflulide in the preparation of drugs for treating endometrial cancer. Research in this invention shows that pentaflulide inhibits the proliferation, colony formation, and migration of ISK and KLE endometrial cancer cells, induces EC cell apoptosis, and inhibits the growth of subcutaneous xenografts in mice. Its effectiveness has been demonstrated in in vivo experiments in mice, indicating its potential application in the treatment of endometrial cancer.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of pentafluridone in the preparation of drugs for treating endometrial cancer. Background Technology

[0002] Endometrial cancer (EC) is a group of epithelial malignant tumors that occur in the uterine lining. It is one of the most common gynecological malignancies worldwide, with both incidence and mortality rates rising. Although EC is more common in postmenopausal women, its incidence has increased dramatically in younger women over the past decade, with an increasing number of younger women being diagnosed with endometrial cancer. The underlying cause of this increase is the obesity epidemic and the resulting hyperinsulinemia; as obesity rates rise, so does the incidence of endometrial cancer. In 2015, China reported approximately 63,400 new EC cases and 21,800 new EC deaths.

[0003] EC is generally classified into type I and type II. The main difference between the two is that type I EC is progesterone receptor positive, while type II EC is progesterone receptor negative. Currently, surgery is the common treatment for EC. However, for patients who need to preserve fertility, conservative treatment methods using progesterone therapy are necessary. The main method of conservative treatment is hormone therapy with progesterone-based drugs. However, hormone therapy has limitations; it is only effective for patients with positive progesterone receptor expression, has a high relapse rate, and is prone to developing drug resistance. Currently, the demand for fertility-preserving conservative treatment among EC patients is increasing.

[0004] Therefore, there is an urgent need in this field to develop a drug that can treat EC, especially progesterone receptor-negative and progesterone-resistant EC. Summary of the Invention

[0005] The purpose of this invention is to provide a medicine that can treat endometrial cancer, particularly progesterone receptor-negative and progesterone-resistant EC.

[0006] In a first aspect of the invention, there is provided the use of an active ingredient or a formulation containing said active ingredient in the preparation of a medicament for treating and / or preventing endometrial cancer, wherein said active ingredient is penicillin or a pharmaceutically acceptable salt thereof.

[0007] The present invention also provides the use of an active ingredient or a formulation containing said active ingredient for the treatment and / or prevention of endometrial cancer, wherein said active ingredient is penicillin or a pharmaceutically acceptable salt thereof.

[0008] In another preferred embodiment, the endometrial cancer includes type I and type II endometrial cancer.

[0009] In another preferred embodiment, the endometrial cancer includes progesterone-sensitive endometrial cancer or progesterone-resistant endometrial cancer.

[0010] In another preferred embodiment, the endometrial cancer is a progesterone receptor-negative and progesterone-resistant endometrial cancer.

[0011] In another preferred embodiment, the active ingredient or a formulation containing the active ingredient is used to prepare a medicament for one or more of the following purposes:

[0012] (a) Inhibits the proliferation of endometrial cancer cells;

[0013] (b) Inhibits the cloning of endometrial cancer cells;

[0014] (c) Inhibits the migration of endometrial cancer cells;

[0015] (d) Induces apoptosis in endometrial cancer cells.

[0016] In another preferred embodiment, the endometrial cancer cells are selected from the group consisting of ISK, KLE, HEC-1-A, HEC-1-B, and AN3CA, or combinations thereof.

[0017] In another preferred embodiment, the endometrial cancer cells are ISK and / or KLE cells.

[0018] In another preferred embodiment, the formulation is an oral formulation or a non-oral formulation.

[0019] In another preferred embodiment, the formulation is selected from the group consisting of: injections, inhalations, tinctures, powders, granules, capsules, oral liquids, tablets, pills, suspensions, emulsions, lozenges, or drops.

[0020] In another preferred embodiment, the preparation is administered orally or by injection.

[0021] In another preferred embodiment, the formulation also includes other anti-endometrial cancer drugs.

[0022] In another preferred embodiment, the anti-endometrial cancer drug is a progestin.

[0023] In another preferred embodiment, the progestin is selected from: medroxyprogesterone acetate, medroxyprogesterone acetate, progesterone caproate, or combinations thereof.

[0024] In another preferred embodiment, the other anti-endometrial cancer drug is medroxyprogesterone acetate.

[0025] In a second aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0026] (A1) Penfluridone or a pharmaceutically acceptable salt thereof as the primary active ingredient;

[0027] (A2) Other anti-endometrial cancer drugs as the second active ingredient;

[0028] (B) Pharmaceutically acceptable carriers or excipients.

[0029] In another preferred embodiment, the anti-endometrial cancer drug is a progestin.

[0030] In another preferred embodiment, the progestin is selected from: medroxyprogesterone acetate, medroxyprogesterone acetate, progesterone caproate, or combinations thereof.

[0031] In another preferred embodiment, the other anti-endometrial cancer drug is medroxyprogesterone acetate.

[0032] In another preferred embodiment, the endometrial cancer includes type I and type II endometrial cancer.

[0033] In another preferred embodiment, the first active ingredient accounts for 0.1-99% of the total weight of the drug by mass.

[0034] In a third aspect of the present invention, a method for in vitro inhibition of endometrial cancer cells is provided, comprising the steps of:

[0035] (i) Endometrial cancer cells are cultured in the presence of penfluridone or a pharmaceutically acceptable salt thereof, thereby inhibiting the endometrial cancer cells.

[0036] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0037] In another preferred embodiment, the inhibition is selected from the group consisting of:

[0038] (a) Inhibits the proliferation of endometrial cancer cells;

[0039] (b) Inhibits the cloning of endometrial cancer cells;

[0040] (c) Inhibits the migration of endometrial cancer cells;

[0041] (d) Induces apoptosis in endometrial cancer cells;

[0042] (e) Any combination of (a) to (d) above.

[0043] In another preferred embodiment, the endometrial cancer cells are selected from the group consisting of ISK, KLE, HEC-1-A, HEC-1-B, and AN3CA, or combinations thereof.

[0044] In another preferred embodiment, the endometrial cancer cells are ISK and / or KLE cells.

[0045] In a fourth aspect of the invention, a method for treating endometrial cancer is provided, comprising the steps of administering to a subject in need a therapeutically effective amount of penfluridone or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described in the second aspect of the invention.

[0046] In another preferred embodiment, the subject is a patient with endometrial cancer.

[0047] In another preferred embodiment, the endometrial cancer includes type I and type II endometrial cancer.

[0048] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0049] Figure 1 This diagram illustrates the inhibitory activity of pendiflubenzuron on the proliferation of ISK and KLE cells after treatment for different durations. (A) Inhibitory activity of ISK cell proliferation; (B) Inhibitory activity of KLE cell proliferation.

[0050] Figure 2 This diagram illustrates the effects of pendiflubenzuron on colony formation in ISK and KLE cells. (A) Crystal violet staining of ISK cells; (B) Quantitative analysis of crystal violet staining in ISK cells; (C) Crystal violet staining of KLE cells; (D) Quantitative analysis of crystal violet staining in KLE cells.

[0051] Figure 3 This diagram illustrates the effect of pendiflubenzuron on the migration ability of ISK and KLE cells. (A) Crystal violet staining of ISK cells; (B) Quantitative analysis of crystal violet staining of ISK cells; (C) Crystal violet staining of KLE cells; (D) Quantitative analysis of crystal violet staining of KLE cells.

[0052] Figure 4 Schematic diagram of the effect of pendiflubenzuron on apoptosis of ISK and KLE cells. (A) Effect of pendiflubenzuron on apoptosis of ISK cells; (B) Effect of pendiflubenzuron on apoptosis of KLE cells; (C) Quantitative analysis of the apoptosis rate of ISK cells; (D) Quantitative analysis of the apoptosis rate of KLE cells.

[0053] Figure 5 The inhibitory effect of penfluridone on the growth of subcutaneous KLE cell xenografts in mice. (A) Curve showing the change in mouse body weight over time; (B) Tumor volume 14 days after administration; (C) Image of mouse tumor 14 days after administration; (D) Tumor weight 14 days after administration. Detailed Implementation

[0054] Through extensive and in-depth research, the inventors unexpectedly discovered a novel use for the antipsychotic drug pentafluridone in the preparation of drugs for treating endometrial cancer. Experiments revealed that pentafluridone inhibits the proliferation, colony formation, and migration of ISK and KLE endometrial cancer cells and can induce apoptosis in EC cells. In vivo experiments in mice showed that pentafluridone can inhibit the growth of subcutaneous xenografts. Based on these findings, this invention was completed.

[0055] the term

[0056] 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 pertains.

[0057] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0058] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0059] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0060] Pentafluoride

[0061] As used herein, the terms “penfluridol” (PFL), “active ingredient of the invention”, and “compound of the invention” are used interchangeably and refer to penfluridol and its pharmaceutically acceptable salts.

[0062] The pentafluoride has the following structural formula:

[0063]

[0064] Fluphenazine is an oral, long-acting antipsychotic approved by the U.S. Food and Drug Administration (FDA). It belongs to the diphenylbutylpiperidine class of drugs and has a strong and long-lasting antipsychotic effect; a single oral dose can last for several days to a week. It is used to treat various types of schizophrenia. Fluphenazine is a potent inhibitor of dopamine D2 receptors and calcium channels.

[0065] Through extensive screening studies, this invention unexpectedly discovered that pendiflubenzuron has an inhibitory effect on the proliferation of ISK (type I, progesterone-sensitive) endometrial cancer cells. Further research revealed that it also inhibits the proliferation of KLE (progesterone-resistant) type II endometrial cancer cells, inhibits the formation and migration of both EC cell clones, induces EC cell apoptosis, and inhibits the growth of subcutaneous xenografts in nude mice in vivo. Therefore, pendiflubenzuron of this invention can be used to prepare drugs for the treatment of endometrial cancer.

[0066] Endometrial cancer

[0067] As used in this article, the term "endometrial cancer (EC)" refers to a group of epithelial malignant tumors that occur in the uterine lining, also known as uterine body cancer. It is one of the three most common malignant tumors of the female reproductive system. Endometrial cancer is more common in perimenopausal and postmenopausal women. Based on its pathogenesis and biological behavior, it can be divided into type I endometrial cancer (estrogen-dependent) and type II endometrial cancer (non-estrogen-dependent).

[0068] Pharmaceutical Composition

[0069] The present invention also provides a pharmaceutical composition that can effectively treat endometrial cancer, comprising:

[0070] (A1) Penfluridone or a pharmaceutically acceptable salt thereof as the primary active ingredient;

[0071] (A2) Other anti-endometrial cancer drugs as the second active ingredient;

[0072] (B) Pharmaceutically acceptable carriers or excipients.

[0073] In another preferred embodiment, the anti-endometrial cancer drug is a progestin.

[0074] In another preferred embodiment, the progestin is selected from: megestrol acetate (MA), medroxyprogesterone acetate (MPA), progesterone caproate (HPC), or combinations thereof.

[0075] In another preferred embodiment, the other anti-endometrial cancer drug is MPA.

[0076] In another preferred embodiment, the endometrial cancer includes type I and type II endometrial cancer.

[0077] The pharmaceutical composition provided by the present invention preferably contains 0.1-99 wt% of a first active ingredient, with the remainder being a second active ingredient, a pharmaceutically acceptable carrier, a diluent or solution or a salt solution.

[0078] When necessary, one or more pharmaceutically acceptable carriers may be added to the drug of this invention. These carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., which are conventional in the pharmaceutical field.

[0079] The compounds and pharmaceutical compositions provided by this invention can be in various forms, such as tablets, injections, capsules, powders, syrups, solutions, suspensions, and aerosols, and can be present in suitable solid or liquid carriers or diluents and in suitable sterilization devices for injection or infusion.

[0080] Various dosage forms of the pharmaceutical compositions of the present invention can be prepared according to conventional pharmaceutical preparation methods. The dosage form typically contains 0.05-1000 mg of the active compound of the present invention per unit volume, preferably 1-500 mg per unit volume.

[0081] The pharmaceutical compositions of the present invention can be used clinically in mammals, including humans and animals, via routes of administration such as mouth, nose, skin, lungs, or gastrointestinal tract. Oral administration is most preferred. The most preferred daily dose is 0.01-400 mg / kg body weight, taken as a single dose, or 0.01-200 mg / kg body weight, taken in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment. Generally, a low dose is started, and the dose is gradually increased until the most suitable dose is found.

[0082] The drugs or inhibitors of the present invention can be administered in various ways, such as by injection, spray, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods, into the body such as muscles, intradermal, subcutaneous, veins, and mucous membranes; or by being mixed with or encapsulated by other substances and introduced into the body.

[0083] Typically, the active ingredient of the present invention or a pharmaceutical composition containing it can be administered in unit dose form via enteral or non-enteric routes, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0084] Dosage forms can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including O / W, W / O, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc.

[0085] The active ingredients of this invention can be formulated into ordinary formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0086] In order to formulate the active ingredient of the present invention into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, croscarmellose, croscarmellose sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0087] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0088] To formulate the drug delivery unit into capsules, the active ingredient of this invention can be mixed with a diluent and a flow aid, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient can be first formed into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. The diluents, binders, wetting agents, disintegrants, and flow aids used to prepare the tablets of this invention can also be used to prepare the capsules of this invention.

[0089] To prepare the active ingredient of this invention into an injection, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, co-solvents, pH adjusters, and osmotic pressure regulators can be added. Solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure regulators can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing a lyophilized powder injection, mannitol, glucose, etc., can also be added as a support agent.

[0090] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0091] The active ingredients or compositions of the present invention can be taken alone or in combination with other therapeutic or symptomatic drugs.

[0092] When the active ingredient of this invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0093] Pentaflulide is used in the preparation of drugs for treating endometrial cancer.

[0094] This invention relates to the use of pentaflulide in the preparation of medicaments for treating endometrial cancer.

[0095] In another preferred embodiment, the endometrial cancer includes type I and type II endometrial cancer.

[0096] In another preferred embodiment, the endometrial cancer includes progesterone-sensitive endometrial cancer or progesterone-resistant endometrial cancer.

[0097] In another preferred embodiment, the endometrial cancer is a progesterone receptor-negative and progesterone-resistant endometrial cancer.

[0098] In another preferred embodiment, the drug for treating endometrial cancer is penicillin.

[0099] In another preferred embodiment, the medicament for treating endometrial cancer is a single component of penfluridone or a combination of penfluridone and other pharmaceutically acceptable components.

[0100] In another preferred embodiment, the composition of the pentafluridone with other pharmaceutically acceptable ingredients comprises an active ingredient pentafluridone at a mass percentage of 0.1-99%.

[0101] In another preferred embodiment, the other pharmaceutically acceptable ingredient is an anti-endometrial cancer drug.

[0102] In another preferred embodiment, the anti-endometrial cancer drug is a progestin.

[0103] In another preferred embodiment, the progestin is one of medroxyprogesterone acetate, medroxyprogesterone acetate, or progesterone caproate.

[0104] In another preferred embodiment, the dosage form of the drug is one of injection, tablet, capsule, pill, suspension or emulsion.

[0105] In another preferred embodiment, the drug formulation is administered orally or by injection.

[0106] The main advantages of this invention include:

[0107] This invention provides the application of pentaflulide in the preparation of drugs for treating endometrial cancer, including type I and type II endometrial cancer. Through experiments, this invention has found that pentaflulide inhibits the proliferation, colony formation, and migration of ISK and KLE endometrial cancer cells, and can inhibit the growth of subcutaneous xenografts in mice. Its effectiveness has been confirmed in in vivo experiments in mice, thus revealing an unexpected therapeutic effect of pentaflulide on endometrial cancer. The main advantages of this invention include:

[0108] 1) A novel use of pentaflulide in the treatment of endometrial cancer was discovered for the first time.

[0109] 2) It was discovered for the first time that pendiflubenzuron has an inhibitory effect on the proliferation, colony formation, and migration of endometrial cancer ISK and KLE cells.

[0110] 3) It was discovered for the first time that pendiflubenzuron can inhibit the growth of subcutaneous endometrial cancer cell xenografts in mice.

[0111] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive and should not be construed as limiting the scope of protection of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0112] Example 1: Inhibition of proliferation of ISK and KLE endometrial cancer cells by pendiflubenzuron

[0113] This study tested the inhibitory effect of penfluridine on the proliferation of endometrial cancer cells ISK and KLE at the cellular level using a CCK-8 assay. The results showed that penfluridine could inhibit the proliferation of ISK and KLE endometrial cancer cells in a time- and concentration-dependent manner.

[0114] 1. Experimental Materials and Methods

[0115] Endometrial cancer cells ISK and KLE were purchased from the American Type Culture Collection Center (ATCC); phosphate-buffered saline (PBS) was purchased from Bio-channel; DMEM / F12 medium was purchased from Biosharp; fetal bovine serum (FBS) and trypsin were purchased from Gibco; CCK-8 was purchased from Beyotime Biotechnology Co., Ltd.; and pendiflubenzuron was obtained from the laboratory's old drug library.

[0116] ISK and KLE cells were cultured in DMEM / F12 medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) at 37°C in a 5% CO2 incubator. After the endometrial cancer cells had largely filled the culture dish (10 cm), the cells were digested with trypsin and seeded at a density of 5000 cells / well (100 μL per well). After overnight culture and cell attachment, 200 μL of DMEM / F12 medium containing different concentrations of PFL was added to each well (3 replicates per group). Cells were incubated for 24 h, 48 h, or 72 h. The medium was then removed, and 100 μL of serum-free medium containing 10% CCK-8 was added to each well. After incubation at 37°C for 1 h, the absorbance (A) at 450 nm was measured using a Bio-Tek multi-plate reader, and the inhibition rate and IC50 were calculated. 50 Inhibition rate calculation formula: Cell inhibition rate % = [1 - (A value of drug-treated group - A value of blank group) / (A value of control group - A value of blank group)] × 100%, IC50 value. 50 The values ​​were fitted using Graphpad Prism 8.0 software.

[0117] 2. Experimental Results

[0118] Data on the inhibitory activity of pendiflubenzuron on the proliferation of two EC cell lines at different time points are shown in Table 1 and... Figure 1 As shown in the figure. The results indicate that pendiflubenzuron significantly inhibits the proliferation of endometrial cancer cells. The proliferative activity of endometrial cancer cells decreases with increasing pendiflubenzuron concentration, and this effect becomes more pronounced with prolonged incubation time. The IC50 value of pendiflubenzuron on the proliferation inhibition activity of ISK cells at incubation times of 24h, 48h, and 72h is [value missing]. 50The concentrations of pentafluridone were 4.65 μM, 3.48 μM, and 2.77 μM, respectively; the IC50 values ​​of pentafluridone against KLE cells at incubation times of 24 h, 48 h, and 72 h were [values ​​missing]. 50 The values ​​were 5.86 μM, 3.14 μM, and 2.88 μM, respectively.

[0119] Table 1. Inhibitory activity of pendiflubenzuron against different EC cell lines and at different incubation times.

[0120]

[0121] This example demonstrates that pendiflubenzuron can inhibit the proliferation of endometrial cancer cells ISK and KLE in a time- and concentration-dependent manner.

[0122] Example 2: Effect of pentaflulide on the clonogenic ability of ISK and KLE endometrial cancer cells

[0123] This study investigated the effect of penflurazole on the clonogenic ability of ISK and KLE endometrial cancer cells at the cellular level using a plate colony formation assay. The results showed that penflurazole inhibited the clonogenic ability of ISK and KLE endometrial cancer cells, and that this inhibition was concentration-dependent.

[0124] 1. Experimental Materials and Methods

[0125] 0.5% crystal violet staining solution was purchased from Beyotime Biotechnology Co., Ltd.; methanol was a commonly used laboratory reagent, commercially purchased, and untreated. Other experimental materials were sourced the same as in Example 1. Logarithmic growth phase EC cells were digested with trypsin to prepare single-cell suspensions, which were seeded into 6-well plates at a density of approximately 1000 cells per well and cultured overnight. After cell attachment, the cells were divided into four groups: control group, PFL 2μM group, PFL 3μM group, and PFL 4μM group, with three replicates per group. The drug-treated groups were cultured in 2mL of DMEM / F12 medium containing different drug concentrations for 48h, then replaced with drug-free medium for approximately 8 days. Once the cells had grown into a visible cell population, they were placed on ice and washed twice with pre-chilled PBS for 3 min each time. Then, the cells were fixed with pre-chilled methanol at -20℃ for 10 min. The methanol was removed, and 1mL of crystal violet staining solution was added for staining for 30 min. Remove the crystal violet stain and rinse with water until the stain is completely washed away. Invert the 6-well plate and allow it to air dry. Take a picture using a gel imaging system and manually count the number of clones formed in each well using ImageJ software.

[0126] 2. Experimental Results

[0127] Experimental results are as follows Figure 2As shown in the figure, Ctrl represents the control group, and each black dot in the image represents a cell community. The results indicate that for ISK cells, compared to the Ctrl group, the number of cell communities in the treatment groups was reduced, and this reduction was significant with increasing pentafluridone concentration, especially in the PFL 4μM group, where only a very small number of cell communities were present (p<0.0001). For KLE cells, compared to the control group, the number of cell communities in the treatment groups was also reduced, and this reduction was significant with increasing pentafluridone concentration, with significantly reduced cell communities in the PFL 3μM and PFL 4μM groups (PFL 3μM group: p<0.0001; PFL 4μM group: p<0.0001). The vertical axis of the bar chart represents the number of cell communities in each group. Significant differences in data were analyzed using one-way ANOVA (Graphpad Prism 8.0 software). Data are mean ± SD: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Ctrl.

[0128] This example demonstrates that pendiflubenzuron inhibits the clonogenicity of ISK and KLE cells, and can inhibit the clonogenicity of endometrial cancer cells ISK and KLE in a concentration-dependent manner.

[0129] Example 3: Effect of pentaflulide on the migration ability of ISK and KLE endometrial cancer cells

[0130] This study investigated the effect of pendiflubenzuron on the migration ability of ISK and KLE endometrial cancer cells at the cellular level using a Transwell assay. The results showed that pendiflubenzuron can inhibit the migration of ISK and KLE endometrial cancer cells in a concentration-dependent manner.

[0131] 1. Experimental Materials and Methods

[0132] Transwell chambers were purchased from Costa Biotech; paraformaldehyde (PFL) was a commonly used laboratory reagent, commercially purchased, and untreated; other experimental materials were sourced from the same sources as in Example 1. Logarithmic growth phase EC cells were digested with trypsin and prepared into single-cell suspensions using serum-free DMEM / F12 medium. These suspensions were seeded at a density of approximately 100,000 cells per well onto the upper part of the Transwell chamber. 600 μL of DMEM / F12 medium containing 20% ​​FBS was added to the bottom of the chamber, taking care to avoid air bubble formation. The cells were divided into four groups: control group, PFL 2 μM group, PFL 3 μM group, and PFL 4 μM group, with three replicates per group. 150 μL of serum-free DMEM / F12 medium containing different drugs was added to the upper chamber of the drug-treated groups, while 150 μL of drug-free and serum-free DMEM / F12 medium was added to the upper chamber of the control group. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h. The chamber was then carefully removed with tweezers, the liquid in the upper chamber was aspirated, and the cells were transferred to a 24-well plate pre-filled with approximately 800 μL of pre-chilled PBS. The plate was washed twice, 5 min each time. The chamber was then removed and transferred to a 24-well plate pre-filled with approximately 800 μL of paraformaldehyde solution, and fixed at room temperature for 30 min. The chamber was then removed, the fixative in the upper chamber was aspirated, and the cells were transferred to a 24-well plate pre-filled with approximately 800 μL of crystal violet staining solution, and stained at room temperature for 30 min. The cells were gently rinsed several times with water, and the cells on the upper chamber membrane surface were carefully wiped away with a damp cotton swab. Five fields of view were randomly selected and photographed under a 200x microscope. The cells were manually counted using ImageJ software, and the cell count in each field of view was recorded.

[0133] 2. Experimental Results

[0134] The results are as follows Figure 3 As shown in the figure, Ctrl represents the control group, and each black dot in the image represents a cell that has passed through the chamber. The results indicate that in ISK and KLE cells, compared with the Ctrl group, the number of cells passing through the chamber was reduced in the drug-treated groups, and this reduction was significant with increasing pentafluphenone concentration, especially in the PFL 4μM group, where only a small number of cells passed through (p<0.0001). The vertical axis of the bar chart represents the number of cells passing through the chamber in each group. Significant differences in data were analyzed using one-way ANOVA (Graphpad Prism 8.0 software). Data are presented as mean ± SD: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Ctrl.

[0135] This example demonstrates that pentaflulide 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.

[0136] Example 4: Effect of pentaflulide on apoptosis of ISK and KLE endometrial cancer cells

[0137] This study investigated the effect of pendiflubenzuron on apoptosis in ISK and KLE endometrial cancer cells at the cellular level using the Annexin V-FITC / PI apoptosis detection kit. The results showed that pendiflubenzuron inhibited apoptosis in both ISK and KLE endometrial cancer cells in a concentration-dependent manner.

[0138] 1. Experimental Materials and Methods

[0139] The Annexin V-FITC / PI apoptosis detection kit was purchased from Beyotime Biotechnology Co., Ltd. Annexin V-FITC binding solution, Annexin V-FITC, and propidium iodide (PI) were all reagents included in the kit. Other experimental materials were sourced the same as in Example 1. Logarithmic growth phase EC cells were digested with trypsin to prepare a single-cell suspension, which was seeded into 6-well plates at a density of approximately 120,000 cells per well and cultured overnight. After cell attachment, the cells were divided into four groups: control group, PFL 2μM group, PFL 3μM group, and PFL 4μM group, with three replicates per group. The drug-treated groups were added 2 mL of DMEM / F12 medium containing different concentrations of PFL, while the control group was added 2 mL of drug-free DMEM / F12 medium. The cells were incubated at 37°C in a 5% CO2 incubator for 48 h. The cell culture medium was then aspirated into 10 ml centrifuge tubes. Adherent cells were washed once with PBS, and 300 μL of trypsin was added to digest the cells for 2 min. The cells were gently pipetted off and transferred to the appropriate centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were collected. The cells were gently resuspended in PBS and counted. 50,000-100,000 cells were resuspended, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and 195 μL of Annexin V-FITC binding buffer was added to gently resuspend the cells. 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI) staining solution were added, and the mixture was gently mixed. The cells were incubated at room temperature in the dark for 20 min, and immediately analyzed using a Beckman Coulter (cytoFLEX LX) flow cytometer.

[0140] 2. Experimental Results

[0141] The results are as follows Figure 4As shown in the figure, Ctrl represents the control group. In the apoptosis diagram, the first quadrant represents late-apoptotic cells, and the fourth quadrant represents early-apoptotic cells. The apoptosis rate is the sum of the rates in the first and fourth quadrants. Compared with the control group, PFL significantly induced apoptosis in ISK and KLE cells in a concentration-dependent manner. For ISK cells, the apoptosis rates at PFL concentrations of 2 μM, 3 μM, and 4 μM were 13.73%, 34.16%, and 47.10%, respectively. For KLE cells, the apoptosis rates at PFL concentrations of 2 μM, 3 μM, and 4 μM were 12.11%, 27.34%, and 31.83%, respectively. The vertical axis of the bar chart represents the apoptosis rate in each group. Significant differences were analyzed using one-way ANOVA (Graphpad Prism 8.0 software). Data are presented as mean ± SD: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Ctrl).

[0142] This example demonstrates that pendiflubenzuron has the ability to induce apoptosis in ISK and KLE cells, and that it can induce apoptosis in ISK and KLE cells in a concentration-dependent manner.

[0143] Example 5: Effect of pentaflulide on the growth of endometrial cancer cell (KLE) xenografts

[0144] This study investigated the effect of pendiflubenzuron on the growth of endometrial cancer cell (KLE) xenografts in nude mice using a subcutaneous tumorigenesis assay. The results showed that pendiflubenzuron significantly inhibited the growth of subcutaneous KLE cell xenografts in mice.

[0145] 1. Experimental Materials and Methods

[0146] Female BALB / c nude mice, aged 5-6 weeks and SPF grade, were purchased from Shanghai Slack Animal Laboratory Co., Ltd. Cisplatin (DDP) was sourced from the laboratory's old drug storage. The compound was prepared by weighing a specific amount of the compound, dissolving it in DMSO and castor oil (DMSO final concentration 5%, castor oil final concentration 8%), and then diluting it with physiological saline to the required concentration. The control group received the corresponding solvent. The compound was prepared and used immediately before administration.

[0147] First, KLE cells were subcutaneously implanted into the right axilla of 5-6 week old nude mice, with approximately 5 million cells implanted per mouse. The tumors were then allowed to grow to 800-1000 mm. 3 The mice were then euthanized, and the tumor tissue was cut into uniformly sized pieces and surgically transplanted subcutaneously into the right axilla of new 5-6 week old nude mice. The newly grown tumors were allowed to grow until they reached an average size of 300-400 mm². 3Mice were randomly divided into four groups: a control group, a positive control group treated with DDP 2 mg / kg, a PFL group treated with PFL 2 mg / kg, and a PFL group treated with PFL 5 mg / kg. Mice were intraperitoneally injected with 0.1 mL of each drug at different concentrations, and their body weight was measured daily. After two weeks of daily administration, the mice were sacrificed, the tumors were dissected, and photographs were taken. On the day of dissection, the tumor length L (mm) and width W (mm) were measured using calipers. The tumor volume was calculated using the formula V (mm²). 3 = 0.5 × L (mm) × W (mm) 2 .

[0148] 2. Experimental Results

[0149] Experimental results are as follows Figure 5 As shown in the figure, Vehicle represents the control group. Graph A shows the change in mouse body weight over time, with the horizontal axis representing the number of days of administration and the vertical axis representing mouse body weight. The graph shows that the body weight in the penfluridone group remained stable, while the body weight in the cisplatin (DDP) group decreased significantly. Graphs B, C, and D show that after the administration period, compared with the control group, tumor growth in the penfluridone group was inhibited. Both PFL 2 mg / kg and PFL 5 mg / kg significantly inhibited mouse tumor growth, and the effect was comparable to that of the positive control drug cisplatin. Significant differences in data were analyzed using one-way ANOVA (GraphpadPrism 8.0 software). Data are presented as mean ± SD: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Ctrl.

[0150] This embodiment demonstrates that pendiflubenzuron can inhibit the growth of subcutaneous KLE cell xenografts in mice without affecting body weight. Its efficacy is comparable to that of the positive control drug cisplatin, but its toxicity is less than that of cisplatin.

[0151] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of an active ingredient or a formulation containing said active ingredient, characterized in that, The active ingredient is penicillin or a pharmaceutically acceptable salt thereof. Furthermore, the active ingredient or a preparation containing the active ingredient is used to prepare a drug for treating and / or preventing endometrial cancer.

2. The use as described in claim 1, characterized in that, The endometrial cancer includes type I and type II endometrial cancer.

3. The use as described in claim 1, characterized in that, The endometrial cancers include progesterone-sensitive endometrial cancer or progesterone-resistant endometrial cancer.

4. The use as described in claim 1, characterized in that, The endometrial cancer mentioned is progesterone receptor-negative and progesterone-resistant endometrial cancer.

5. The use as described in claim 1, characterized in that, The active ingredient or a formulation containing the active ingredient is used to prepare a drug for one or more of the following purposes: (a) Inhibits the proliferation of endometrial cancer cells; (b) Inhibits the cloning of endometrial cancer cells; (c) Inhibits the migration of endometrial cancer cells; (d) Induces apoptosis in endometrial cancer cells.

6. The use as described in claim 5, characterized in that, The endometrial cancer cells are ISK and / or KLE cells.

7. The use as described in claim 1, characterized in that, The formulation also includes other anti-endometrial cancer drugs.

8. The use as described in claim 7, characterized in that, The anti-endometrial cancer drug is a progestin.

9. The use as described in claim 7, characterized in that, The anti-endometrial cancer drug is medroxyprogesterone acetate.

10. A method for inhibiting endometrial cancer cells in vitro, characterized in that, Including the following steps: (i) Endometrial cancer cells are cultured in the presence of penfluridone or a pharmaceutically acceptable salt thereof to inhibit the endometrial cancer cells selected from the group consisting of ISK and KLE.

Images