Use of chlorpromazine hydrochloride in the treatment of endometrial cancer

Abstract

The application relates to the medical technology field, in particular to application of chlorpromazine hydrochloride in treating endometrial carcinoma, wherein the endometrial carcinoma includes type I and type II endometrial carcinoma. It is found through experiments that chlorpromazine hydrochloride has inhibiting effects on the proliferation, clone formation and migration capacity of Ishikawa (ISK cell) and KLE cells of endometrial carcinoma, can induce cell apoptosis, up-regulate the expression of cell PRB, inhibit the phosphorylation of cell AKT, and can inhibit the growth of a subcutaneous transplanted tumor of a mouse, and the effectiveness is proved in a mouse experiment. Therefore, the application provides a new use of an antipsychotic drug chlorpromazine hydrochloride for treating endometrial carcinoma.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of chlorpromazine hydrochloride in the treatment of endometrial cancer. Background Technology

[0002] Endometrial cancer (EC) is one of the most prevalent malignant tumors of the female reproductive tract worldwide, and its incidence is increasing (Morice, The Lancet, 2016, 387, 1094–108), with up to 25% of cases occurring before menopause (Sanderson, Obstetrics, Gynaecology & Reproductive Medicine, 2019, 29, 225-232). According to the American Cancer Society, the United States is projected to have 65,620 new EC cases and 12,590 new EC deaths in 2020 (Siegel, CA: A Cancer Journal for Clinicians, 2020, 70, 7-30). In China, there were approximately 63,400 new EC cases and 21,800 new EC deaths in 2015 (Chen, CA: A Cancer Journal for Clinicians, 2016, 66, 115-132). In recent years, the age of onset of EC in China has also shown a trend of becoming younger (Xu Guorong, Chinese Journal of Misdiagnosis, 2009, 9, 5858-5859). With social development, the phenomenon of women marrying and having children later in life is becoming more and more common. In addition, with the opening of the two-child policy, conservative treatment that preserves fertility is more important for young EC patients who have fertility needs. Therefore, safe and effective conservative treatment for EC patients has become a hot topic of research.

[0003] EC is classified into type I and type II (Morice, The Lancet, 2016, 387, 1094-1108). Type I, also known as estrogen-dependent type, is associated with metabolic diseases such as obesity, hyperglycemia, and hyperlipidemia, and is often accompanied by atypical endometrial hyperplasia. The typical histological type is endometrioid adenocarcinoma. Type II, also known as non-estrogen-dependent type, is not related to high estrogen levels, has no endocrine metabolic disorders, is accompanied by atrophic endometrium, is poorly differentiated, and highly invasive. The typical histological types are serous carcinoma and clear cell carcinoma (Sanderson, Obstetrics, Gynaecology & Reproductive Medicine, 2019, 29, 225-232. Morice, The Lancet, 2016, 387, 1094-1108).

[0004] For patients with early-stage EC, the most common and effective conservative treatment is progestin therapy (Wei, Medicine. 2017, 96, e8034). However, long-term use of progestins inevitably induces drug resistance in EC, and progestins are only effective for EC patients with high progesterone receptor expression. There are currently no particularly effective drugs for patients with type II EC who have low progesterone receptor expression. Therefore, the development of new EC treatments, especially those for progestin-resistant and type II EC, is an urgent need.

[0005] Chlorpromazine hydrochloride is a drug used clinically to treat psychosis, and there are currently no literature reports on its use in treating endometrial cancer. However, as a known drug, it may have new applications in the treatment of endometrial cancer (EC). Summary of the Invention

[0006] The main objective of this invention is to develop a drug that can effectively treat EC, especially progesterone-resistant and type II EC. Specifically, this invention provides a new use of chlorpromazine hydrochloride in the treatment of EC.

[0007] In a first aspect of the invention, there is provided the use of an active ingredient or a formulation containing said active ingredient, said active ingredient being chlorpromazine hydrochloride or a pharmaceutically acceptable salt thereof.

[0008] Furthermore, the active ingredient or a formulation containing the active ingredient is used to prepare a drug for treating and / or alleviating endometrial cancer.

[0009] In another preferred embodiment, the endometrial cancer includes type I and type II endometrial cancer.

[0010] 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:

[0011] (a) Inhibits the proliferation of endometrial cancer cells;

[0012] (b) Inhibits the cloning of endometrial cancer cells;

[0013] (c) Inhibits the migration of endometrial cancer cells;

[0014] (d) Induces apoptosis in endometrial cancer cells.

[0015] In another preferred embodiment, the endometrial cancer cells are selected from the group consisting of ISK, KLE, HEC-1A, AN3CA, or combinations thereof.

[0016] In another preferred embodiment, the endometrial cancer cells are ISK and / or KLE cells.

[0017] In another preferred embodiment, the formulation is an oral formulation or a non-oral formulation.

[0018] In another preferred embodiment, the formulation is selected from the group consisting of: injections, inhalations, tinctures, powders, granules, capsules, oral liquids, tablets, pills, suspensions, emulsion tablets, or drops.

[0019] In another preferred embodiment, the preparation is administered orally or by injection.

[0020] In another preferred embodiment, the formulation also includes other anti-endometrial cancer drugs.

[0021] In another preferred embodiment, the anti-endometrial cancer drug is a progestin.

[0022] In another preferred embodiment, the progestin is selected from: megestrol acetate, medroxyprogesterone acetate (MPA), progesterone caproate, or combinations thereof.

[0023] In another preferred embodiment, the other anti-endometrial cancer drug is MPA.

[0024] In a second aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0025] (A1) Chlorpromazine hydrochloride or a pharmaceutically acceptable salt thereof as the first active ingredient;

[0026] (A2) Other anti-endometrial cancer drugs as the second active ingredient;

[0027] (B) Pharmaceutically acceptable carriers or excipients.

[0028] In another preferred embodiment, the anti-endometrial cancer drug is a progestin.

[0029] In another preferred embodiment, the progestin is selected from: megestrol acetate, medroxyprogesterone acetate (MPA), progesterone caproate, or combinations thereof.

[0030] In another preferred embodiment, the other anti-endometrial cancer drug is MPA.

[0031] In another preferred embodiment, the endometrial cancer includes type I and type II endometrial cancer.

[0032] In another preferred embodiment, the first active ingredient accounts for 0.1-99% of the total weight of the drug by mass.

[0033] In a third aspect of the invention, a method for activating PRB expression in endometrial cancer cells in vitro without diagnosis or treatment is provided, comprising the step of contacting the endometrial cancer cells with a medically effective amount of chlorpromazine hydrochloride or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described in the second aspect.

[0034] In a fourth aspect of the invention, a non-diagnostic, non-therapeutic method for inhibiting AKT phosphorylation in endometrial cancer cells in vitro is provided, comprising the step of contacting the endometrial cancer cells with a medically effective amount of chlorpromazine hydrochloride or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described in the second aspect.

[0035] In another preferred embodiment, the endometrial cancer cells are ISK and / or KLE cells.

[0036] In a fifth aspect of the invention, a method for inhibiting endometrial cancer is provided, comprising the step of administering to a subject in need a medically effective amount of chlorpromazine hydrochloride or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described in the second aspect.

[0037] In another preferred embodiment, the subject is a patient with endometrial cancer.

[0038] In another preferred embodiment, the endometrial cancer includes type I and type II endometrial cancer.

[0039] 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

[0040] Figure 1 This is a schematic diagram illustrating the inhibitory effect of chlorpromazine hydrochloride on the proliferation of different cells. Figure 1 A shows the inhibitory effect of chlorpromazine hydrochloride on the proliferation of ISK and KLE cells. Figure 1 B shows the inhibitory effect of chlorpromazine hydrochloride on the proliferation of several other endometrial cancer cells.

[0041] Figure 2 This is a schematic diagram illustrating the effects of chlorpromazine hydrochloride and MPA on the colony formation of ISK and KLE cells.

[0042] Figure 3 This is a schematic diagram illustrating the effects of chlorpromazine hydrochloride and MPA on the migration ability of ISK and KLE cells.

[0043] Figure 4 This is a schematic diagram illustrating the effect of chlorpromazine hydrochloride on apoptosis in ISK and KLE cells.

[0044] Figure 5This is a schematic diagram illustrating the effect of chlorpromazine hydrochloride on PRB expression in ISK and KLE cells.

[0045] Figure 6 This is a schematic diagram illustrating the effect of chlorpromazine hydrochloride on AKT phosphorylation in ISK and KLE cells.

[0046] Figure 7 This is a schematic diagram illustrating the growth-inhibiting effect of chlorpromazine hydrochloride on subcutaneous xenograft tumors (ISK cells) in mice. Detailed Implementation

[0047] Through extensive and in-depth research, the inventors unexpectedly discovered that chlorpromazine hydrochloride can effectively treat endometrial cancer, including type I and type II endometrial cancer, and based on this discovery, the present invention was completed.

[0048] Specifically, this invention has found that chlorpromazine hydrochloride has an inhibitory effect on the proliferation, colony formation, and migration of endometrial cancer Ishikawa and KLE cells, and can induce apoptosis, upregulate cellular PRB expression, inhibit cellular AKT phosphorylation, and inhibit the growth of subcutaneous xenografts in mice. Its effectiveness has been demonstrated in mouse experiments, and therefore it can be used to prepare drugs for the treatment of endometrial cancer.

[0049] the term

[0050] Chlorpromazine hydrochloride

[0051] As used herein, "active ingredient of the present invention" and "compound of the present invention" are used interchangeably and refer to chlorpromazine hydrochloride and its pharmaceutically acceptable salts, which are effective in treating endometrial cancer.

[0052] The chemical structural formula of chlorpromazine hydrochloride is shown below:

[0053]

[0054] Endometrial cancer

[0055] Endometrial cancer (EC) is a group of epithelial malignant tumors that occur in the endometrium of the uterus. Also known as uterine corpus cancer, it is one of the three most common malignant tumors of the female reproductive system. Endometrial cancer is most 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).

[0056] Pharmaceutical Composition

[0057] The present invention also provides a pharmaceutical composition that can effectively treat endometrial cancer, comprising:

[0058] (A1) Chlorpromazine hydrochloride or a pharmaceutically acceptable salt thereof as the first active ingredient;

[0059] (A2) Other anti-endometrial cancer drugs as the second active ingredient;

[0060] (B) Pharmaceutically acceptable carriers or excipients.

[0061] In another preferred embodiment, the anti-endometrial cancer drug is a progestin.

[0062] In another preferred embodiment, the progestin is selected from: megestrol acetate, medroxyprogesterone acetate (MPA), progesterone caproate, or combinations thereof.

[0063] In another preferred embodiment, the other anti-endometrial cancer drug is MPA.

[0064] In another preferred embodiment, the endometrial cancer includes type I and type II endometrial cancer.

[0065] 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.

[0066] 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., commonly used in the pharmaceutical field.

[0067] 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.

[0068] 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.

[0069] 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 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, divided into multiple 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.

[0070] 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.

[0071] 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.

[0072] 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, droplets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0079] The active ingredients or compositions of the present invention can be taken alone or in combination with other therapeutic or symptomatic drugs.

[0080] 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.

[0081] The main advantages of this invention include:

[0082] (a) A novel use of chlorpromazine hydrochloride in the treatment of endometrial cancer has been discovered for the first time.

[0083] (b) For the first time, chlorpromazine hydrochloride was found to inhibit the proliferation, colony formation, and migration of Ishikawa (ISK) and KLE endometrial cancer cells, and to induce apoptosis. Chlorpromazine hydrochloride achieves its therapeutic effect on endometrial cancer by upregulating cellular PRB expression and inhibiting cellular AKT phosphorylation.

[0084] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope 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.

[0085] Example 1: Inhibitory effect of chlorpromazine hydrochloride on the proliferation of endometrial cancer cells ISK (ISK, type I, progesterone sensitive), KLE cells (type II, progesterone resistant), HEC-1A (type II), and AN3CA (type I, FGFR2 mutation).

[0086] 1.1 Experimental Materials and Methods

[0087] Endometrial cancer cells ISK, KLE, HEC-1A, and AN3CA were purchased from the American Tissue Culture Bank (ATCC); phosphate-buffered saline (PBS) and DMEM / F12 medium were purchased from Hyclone; serum (FBS) was purchased from Gibco; trypsin and CCK8 were purchased from Beyotime Biotechnology Co., Ltd.; chlorpromazine hydrochloride (CPZ) and medroxyprogesterone acetate (MPA) were obtained from the laboratory's existing drug library. Specific experimental methods are as follows:

[0088] 1) Take cells in the logarithmic growth phase, discard the culture medium in the culture dish, and add 2 mL of PBS to wash the cells twice.

[0089] 2) Add 1 mL of trypsin and digest at room temperature for 2 min.

[0090] 3) Add 2 mL of culture medium containing 10% FBS to neutralize the trypsin, and gently pipette to form a single-cell suspension. The pipetting force should be such that no air bubbles are produced.

[0091] 4) Transfer the single-cell suspension to a 15mL centrifuge tube, centrifuge at 1000rpm for 3min, and discard the supernatant.

[0092] 5) Add 2 mL of cell culture medium containing 10% FBS and pipette the suspended cells into a single-cell suspension.

[0093] 6) Pipette 10 μL of cell suspension into a cell counting chamber and count the cells. Adjust the concentration of the single cell suspension to 80,000 cells / mL.

[0094] 7) Add the single-cell suspension to a 96-well plate, 100 μL per well (about 8000 cells), with 3 replicates per group. Incubate the 96-well plate in an incubator for 24 h (37℃, 5% CO2) to allow the cells to adhere.

[0095] 8) Set up a control group and CPZ-treated groups with different concentrations. The control group was given 200 μL of DME / F12 medium, and the treated groups were given 200 μL of DME / F12 medium containing different concentrations of CPZ. After incubation for 24, 48, and 72 h, the medium was removed, and 100 μL of serum-free medium containing 10% CCK-8 was added to each well (including blank wells). Incubation continued for 1 h, and the absorbance at 450 nm was measured using a microplate reader. 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 software.

[0096] 1.2 Experimental Results

[0097] like Figure 1 As shown, Figure 1 This diagram illustrates the inhibitory effect of chlorpromazine hydrochloride on the proliferation of ISK, KLE, HEC-1A, and AN3CA cells. As can be seen from the diagram... Figure 1 The image above (A) is a schematic diagram illustrating the inhibitory effect of chlorpromazine hydrochloride on the proliferation of ISK cells. Figure 1 The following diagram (A) illustrates the inhibitory effect of chlorpromazine hydrochloride on the proliferation of KLE cells. The horizontal axis represents concentration, and the vertical axis represents inhibition rate. Chlorpromazine hydrochloride inhibits the proliferation of both ISK and KLE cells. The 24, 48, and 72-hour half-maximal inhibitory concentrations (IC50) for ISK cells are shown in the diagram. 50 The half-maximal inhibitory concentrations (IC50) of KLE at 24, 48, and 72 h were 33.14±2.28 μM, 22.09±3.27 μM, and 22.75±0.89 μM, respectively. 50 The values ​​were 35.62±3.18μM, 25.54±1.70μM, and 9.97±1.22μM.

[0098] Chlorpromazine hydrochloride has inhibitory effects on the proliferation of several other types of endometrial cancer cells, such as... Figure 1 As shown in Figure B, CPZ inhibits the proliferation of ISK, KLE, HEC-1A, and AN3CA cells. The 72-hour half-maximal inhibitory concentration (IC50) for HEC-1A and AN3CA cells is [data missing]. 50 The effective concentrations were 18.77±2.86μM and 22.15±0.93μM, respectively, both of which showed superior activity compared to the progestin drug MPA.

[0099] Example 2: Effects of chlorpromazine hydrochloride and MPA on colony formation in ISK and KLE cells

[0100] 2.1 Experimental Materials and Methods

[0101] Crystal violet was purchased from Beyotime Biotechnology Co., Ltd.; methanol was a commonly used laboratory reagent, commercially purchased, and untreated. The sources of other experimental materials were the same as in Example 1. The specific experimental methods are as follows:

[0102] 1) Take cells in the logarithmic growth phase, digest them with trypsin to prepare a single-cell suspension, serially dilute them, and seed them into 6-well plates at a density of about 800 cells per well, and let them adhere overnight.

[0103] 2) Five experimental groups were set up: control group; MPA 10μM group; MPA 20μM group; CPZ 5μM group; CPZ 10μM group; and CPZ 20μM group. The drug-treated groups were cultured in 1mL of DME / F12 medium containing different drug concentrations for 3 days, then switched to drug-free medium and cultured for another 7 days before staining and counting. The control group was cultured in 2mL of drug-free DME / F12 medium for 10 days (with medium changed every three days) before staining and counting.

[0104] 3) Place the cells to be stained on ice and wash them twice with pre-cooled PBS at 4°C for 3 minutes each time.

[0105] 4) Fix cells at -20°C for 10 min using pre-cooled methanol.

[0106] 5) Remove the methanol, add enough 0.5% crystal violet staining solution to cover (about 1 mL), and incubate for 10 min.

[0107] 6) Remove the crystal violet staining solution and rinse the stained cells with water until the staining solution is completely washed away.

[0108] 7) Count and take photos.

[0109] 2.2 Experimental Results

[0110] like Figure 2 As shown, Figure 2This diagram illustrates the effects of chlorpromazine hydrochloride and MPA on colony formation in ISK and KLE cells. The double-negative group represents the control group, and each black dot in the image represents a cell community. It can be observed that, compared to the control group, the cell communities in the MPA and CPZ groups were significantly reduced in both ISK and KLE cells, especially in the CPZ 20μM group, where only a very small number of cell communities were present (p<0.0001). The bar chart represents the number of cell communities in each group. Significant differences were analyzed using the t-test method (Graphpad Prism software). *Comparison between the treated group and the control group, ***p<0.001, ****p<0.0001.

[0111] This experiment demonstrates that chlorpromazine hydrochloride inhibits the colony formation of both ISK and KLE cells, and its inhibitory effect is superior to that of the progestin drug MPA.

[0112] Example 3: Effects of chlorpromazine hydrochloride and MPA on the migration ability of ISK and KLE cells

[0113] 3.1 Experimental Materials and Methods

[0114] Paraformaldehyde is a commonly used laboratory reagent, commercially purchased, and untreated. The other experimental materials were sourced from the same place as in Example 1. The specific experimental methods are as follows:

[0115] 1) Add 100 μL (approximately 200,000 cells) of serum-free ISK / KLE cell suspension to the upper chamber of a Transwell chamber. The culture medium used for the cell suspension is serum-free DME / F12 medium.

[0116] 2) Add 600 μL of DME / F12 medium containing 20% ​​FBS to the lower chamber, taking care to avoid the formation of air bubbles.

[0117] 3) Three experimental groups were set up: a control group; an MPA 5μM group; and a CPZ 5μM group. 100μL of serum-free DME / F12 medium containing the different drugs was added to the upper chamber of the drug-treated groups. 100μL of drug-free and serum-free DME / F12 medium was added to the upper chamber of the control group. The mixtures were incubated for 24 h (37℃, 5% CO2).

[0118] 4) Carefully remove the chamber with tweezers, aspirate the liquid in the upper chamber, and transfer it to a 24-well plate pre-filled with approximately 800 μL of pre-cooled PBS. Wash twice, 5 min each time.

[0119] 5) Remove the chamber and transfer it to a 24-well plate pre-filled with approximately 800 μL of paraformaldehyde solution. Fix at room temperature for 30 minutes.

[0120] 6) Remove the chamber, aspirate the fixative from the upper chamber, and transfer it to a 24-well plate pre-filled with approximately 800 μL of 0.5% crystal violet staining solution. Stain at room temperature for 30 min.

[0121] 7) Remove the chamber, gently rinse several times with clean water, and carefully wipe away the cells on the membrane surface at the bottom of the upper chamber with a damp cotton swab. Observe the cells in 5 random fields of view under a 200x microscope and count them.

[0122] 3.2 Experimental Results

[0123] like Figure 3 As shown, Figure 3 This diagram illustrates the effects of chlorpromazine hydrochloride and MPA on the migration ability of ISK and KLE cells. The double-negative group in the diagram represents the control group, and each black dot in the image represents a cell that has passed through the chamber. It can be observed that, compared with the control group, the number of cells passing through the chamber in the MPA group was not reduced, but the number of cells passing through the chamber in the CPZ group was significantly reduced (p<0.0001). The bar chart represents the number of cells passing through the chamber in each group. Significant differences in data were analyzed using the t-test method (Graphpad Prism software). *Comparison between the treated group and the control group, ****p<0.0001.

[0124] This experiment demonstrates that chlorpromazine hydrochloride has the ability to inhibit the migration of ISK and KLE cells, while MPA does not have the ability to inhibit the migration of ISK and KLE cells.

[0125] Example 4: Effects of chlorpromazine hydrochloride on apoptosis in ISK and KLE cells

[0126] 4.1 Experimental Materials and Methods

[0127] This experiment was conducted using the Annexin V-FITC / PI apoptosis detection kit, purchased from Beyotime Biotechnology Co., Ltd. The kit contained Annexin V-FITC binding solution, Annexin V-FITC, and propidium iodide (PI). All other experimental materials were obtained from the same source as in Example 1. The specific experimental methods are as follows:

[0128] 1) Transfer ISK / KLE cells into 6-well plates, with approximately 300,000 cells per well, and let them adhere overnight.

[0129] 2) Three experimental groups were set up: a control group; a CPZ 5μM group; a CPZ 10μM group; and a CPZ 20μM group. The drug-treated groups were given 4 mL of DME / F12 medium containing CPZ, while the control group was given 4 mL of drug-free DME / F12 medium. The cultures were incubated for 72 h (37℃, 5% CO2).

[0130] 3) Aspirate the cell culture medium into a 10ml centrifuge tube, wash the adherent cells once with PBS, and add 300μL of trypsin to digest the cells for 2min. Aspirate the trypsin, add the cell culture medium collected in step 1, gently pipette the cells off, transfer them to a centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, collect the cells, gently resuspend the cells in PBS and count them.

[0131] 4) Take 50,000 to 100,000 cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, and gently resuspend the cells in 195 μL of Annexin V-FITC binding solution.

[0132] 5) Add 5 μL Annexin V-FITC and mix gently.

[0133] 6) Add 10 μL of propidium iodide (PI) staining solution and mix gently.

[0134] 7) Incubate at room temperature in the dark for 20 minutes, and then immediately detect the cells using a flow cytometer.

[0135] 2. Experimental Results

[0136] like Figure 4 As shown, Figure 4 This diagram illustrates the effect of chlorpromazine hydrochloride on apoptosis in ISK and KLE cells. The double-negative group in the diagram is the control group. In the apoptosis diagram, the first quadrant represents late-apoptotic cells, and the fourth quadrant represents early-apoptotic cells. The percentage of the sum of the first and fourth quadrants represents the apoptosis rate.

[0137] Compared with the control group, both 10 μM and 20 μM CPZ significantly induced apoptosis in ISK cells, with apoptosis rates of 9.49% and 17.69%, respectively (control group: 2.84%). 20 μM CPZ significantly induced apoptosis in KLE cells, with an apoptosis rate of 15.74% (control group: 2.74%). The bar chart represents the apoptosis rate in each group. Significant differences were analyzed using the t-test method (GraphpadPrism software). *Comparison between the treated group and the control group, *p<0.05, **p<0.01.

[0138] This experiment demonstrates that chlorpromazine hydrochloride can induce apoptosis in ISK and KLE cells.

[0139] Example 5: Effects of chlorpromazine hydrochloride on PRB expression and AKT phosphorylation in ISK and KLE cells

[0140] 5.1 Experimental Materials and Methods

[0141] Tris(hydroxymethyl)aminomethane, glycine, sodium dodecyl sulfate, NaCl, HCl, KCl, KH₂PO₄, NaCl, Na₂HPO₄·2H₂O, benzyl sulfonyl fluoride, skim milk powder, etc., are all commonly used laboratory reagents, purchased commercially, and without any processing. RIPA lysis buffer, 5×SDS loading buffer, developing solutions (solutions A and B), BCA protein concentration assay kit (including BCA reagent A, BCA reagent B, and bovine serum albumin (BSA) standard), and solid bovine serum albumin (BSA) were purchased from Shanghai Yisheng Biotechnology Co., Ltd.; PVDF membranes were purchased from Merck Millipore. Primary antibodies included PRB antibody, AKT antibody, and p-AKT. 473 The antibodies and GAPDH antibody were purchased from Cell Signaling Technology (CST). The secondary antibody was a mouse antibody, also purchased from CST. The other experimental reagents were obtained from the same sources as in Example 1. The specific experimental methods are as follows:

[0142] 1) Preparation of reagents for Western blotting

[0143] a) 8% SDS-PAGE adhesive sheet.

[0144] b) SDS-PAGE electrophoresis buffer: 3.03g tris(hydroxymethyl)aminomethane (Tris), 14.4g glycine, 1g sodium dodecyl sulfate (SDS), add double-distilled water to a final volume of 1000mL, dissolve and store at room temperature.

[0145] c) Transfer buffer: 3.03g Tris, 14.4g glycine, 200mL methanol, add double-distilled water to a final volume of 1000mL, dissolve and store at room temperature.

[0146] d) TBST buffer: 10 mL of 1 mol / L Tris·HCl (pH = 7.5), 8.8 g of NaCl, dissolved in triple-distilled water and brought to a final volume of 1000 mL to prepare TBS buffer, then add 0.1% Tween 20.

[0147] e) PBS buffer: KCl 0.2g, KH2PO4 0.2g, NaCl 8.0g, Na2HPO4·2H2O 1.56g, add double-distilled water to a final volume of 1000mL, dissolve and store at room temperature.

[0148] f) Blocking solution: 2.5g skim milk powder, 45mL TBST buffer, dissolved and stored at 4℃.

[0149] g) Antibody dilution solution: 2.5g solid BSA, 45mL TBST buffer, dissolved and stored at 4℃.

[0150] 2) Extraction of total cellular protein

[0151] a) ISK / KLE cells were seeded in 6-well plates, with approximately 100,000 cells per well, and incubated overnight until they adhered to the plates. The samples were divided into two groups: a control group and a CPZ 10μM group, and cultured for 48 hours.

[0152] b) Remove the culture medium, and wash each well of the 6-well plate three times with 1 mL of pre-chilled PBS at 4°C. After removing the PBS, place the culture dish on ice.

[0153] c) Add 100 μL of lysis buffer containing benzyl sulfonyl fluoride (PMSF) to each well of a 6-well plate (add 10 μL of 100 mM PMSF to 1 mL of RIPA lysis buffer) and lyse on ice for 5 min.

[0154] d) After lysis, scrape the cells to one side with a clean scraper, and then transfer the cell debris and lysis buffer to a 1.5 mL centrifuge tube; centrifuge at 12000 rpm for 15 min at 4 °C, and transfer the supernatant to a clean 1.5 mL centrifuge tube.

[0155] 3) BCA method for determining protein concentration

[0156] a) Calculate the total required volume of BCA working solution. Total BCA working solution volume = (number of standards + number of samples to be tested) × number of replicates × required volume of BCA working solution per sample.

[0157] b) Prepare BCA working solution: Add 1 volume of BCA reagent B to 50 volumes of BCA reagent A (A:B = 50:1) and mix thoroughly.

[0158] c) Prepare diluted BSA standard solution: Dilute the BSA protein standard in the BCA protein concentration assay kit to a specific concentration with PBS, setting nine concentrations: 0 μg / mL, 25 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 750 μg / mL, 1000 μg / mL, 1500 μg / mL, and 2000 μg / mL.

[0159] d) Add 180 μL of BCA working solution to each well of the 96-well plate.

[0160] e) Take 20 μL of diluted BSA standard solution and add it to the test sample (protein extracted in step 2) into the working solution of the microplate, and incubate at 37°C for 30 min.

[0161] f) Cool to room temperature, measure the absorbance at 562 nm wavelength using an ELISA reader, and calculate the protein concentration.

[0162] 4) SDS-PAGE electrophoresis

[0163] a) After measuring the protein content, adjust the protein concentration to be consistent, add 5×SDS loading buffer to one-quarter of the sample volume, and boil the sample in boiling water for 5 minutes to denature the protein.

[0164] b) After adding electrophoresis buffer to the electrophoresis tank, start loading the sample. The amount of protein loaded per well is 20-30 μg (depending on protein expression).

[0165] c) During electrophoresis, the voltage is set to 80V. When the sample is pressed into a straight line in the stacking gel, the voltage is adjusted to 120V. Electrophoresis is stopped when the bromophenol blue just runs out, and then the membrane is transferred.

[0166] 5) Transfer membrane

[0167] a) Take a PVDF membrane and immerse it in methanol for 5 minutes to activate it.

[0168] b) Place the transfer clamp, two sponge pads, a glass rod, filter paper, and the soaked membrane in an enamel tray containing transfer solution; open the clamp to keep the black side horizontal. Place a sponge pad on top and use the glass rod to roll back and forth several times to remove air bubbles. Place a layer of filter paper on the sponge pad, then place the PVDF membrane, another layer of filter paper, and then cover with another sponge pad. Close the clamp.

[0169] c) Place the clamp into the transfer tank, ensuring the black side of the clamp faces the black side of the tank and the white side faces the red side of the tank. Heat will be generated during electrotransfer; place an ice block on one side of the tank to cool it down. Perform a constant current transfer at 300mA for 1 hour.

[0170] d) After the transfer, stain the membrane with Ponceau S for 5 minutes and shake it on a destaining shaker. Then rinse off the unstained stain with deionized water to see the protein on the membrane.

[0171] 6) Immune response

[0172] a) Transfer the membrane to a petri dish containing the blocking solution and block it on a decolorizing shaker at room temperature for 1 hour.

[0173] b) Dilute the primary antibody with antibody dilution buffer at a volume ratio of 1:1000. Remove the membrane from the blocking buffer, blot off any residual liquid with filter paper, cut the membrane, and place it in the diluted primary antibody. Incubate at 4°C for 24 hours. Remove the membrane and wash it three times with TBST for 5 minutes each time at room temperature.

[0174] c) Dilute the secondary antibody with antibody dilution buffer at a volume ratio of 1:1000. Place the membrane in the diluted secondary antibody and incubate at room temperature for 1 hour. Remove the membrane and wash it three times with TBST for 5 minutes each time.

[0175] 7) Development

[0176] Mix developer A and developer B at a volume ratio of 1:1. Place the PVDF membrane on a gel imaging system and add the developer. Adjust the exposure time according to the signal strength to achieve the best results.

[0177] 3.2 Experimental Results

[0178] like Figure 5 As shown, Figure 5 This diagram illustrates the effect of chlorpromazine hydrochloride on PRB expression in ISK and KLE cells. As shown in the figure, chlorpromazine hydrochloride upregulates PRB expression in KLE cells. Figure 6 As shown, Figure 6 This diagram illustrates the effect of chlorpromazine hydrochloride on AKT phosphorylation in ISK and KLE cells. As shown in the figure, chlorpromazine hydrochloride inhibits AKT phosphorylation in both ISK and KLE cells.

[0179] Example 6: Growth inhibitory effect of chlorpromazine hydrochloride on mouse subcutaneous xenograft tumors (ISK cells)

[0180] 6.1 Laboratory Animals

[0181] Nude mice, approximately 20g, 10 weeks old, female, SPF grade, purchased from Shanghai Slack Animal Laboratory Co., Ltd.; cisplatin (DDP) was sourced from the laboratory's old drug storage.

[0182] 2. Compound preparation

[0183] Weigh a certain amount of the compound, dissolve it in DMSO (final concentration of 10%), shake to dissolve the compound, and then dilute it with PBS to the required concentration. The control group was given the corresponding solvent. The compound should be prepared fresh before administration.

[0184] 3. Experimental Design

[0185] 1) ISK cells were implanted into the right axilla of 10-week-old mice, with approximately 5 million cells implanted in each mouse.

[0186] 2) Administer the medication 22 days after tumor transplantation (average tumor size 300-400 mm). 3 Mice were randomly divided into 5 groups: control group; positive control group (DDP 4 mg / kg); MPA 40 mg / kg; CPZ 3 mg / kg; and CPZ 15 mg / kg.

[0187] 3) Administer 0.1 mL intraperitoneally at different drug concentrations, daily.

[0188] 4) Measure the tumor length L (mm) and width W (mm) every two days using vernier calipers. The tumor volume is calculated using the formula V (mm²). 3= 0.5 × L (mm) × W (mm) 2 .

[0189] 5) Weigh the mice every two days.

[0190] 6) After 22 days of continuous administration, the mice were sacrificed, the tumors were removed, and photographs were taken.

[0191] 6.2 Experimental Results

[0192] like Figure 6 As shown, Figure 6 This diagram illustrates the inhibitory effect of chlorpromazine hydrochloride on the growth of subcutaneous xenograft tumors (ISK cells) in mice. The Vehicle in the diagram represents the control group. Figure A shows the change in relative tumor volume in mice, with the horizontal axis representing the number of days of administration and the vertical axis representing relative tumor volume. As can be seen from the figure, tumor growth in all treatment groups was inhibited compared to the control group. CPZ at 3 mg / kg and 15 mg / kg significantly inhibited tumor growth in mice, while MPA at 40 mg / kg showed some inhibitory effect, but the difference was not statistically significant. Significant differences were analyzed using one-way ANOVA (GraphpadPrism software). *Comparison between treatment groups and the control group, ****p<0.0001. Figure B shows a photograph of the mouse tumor after removal following administration.

[0193] This experiment demonstrates that chlorpromazine hydrochloride can inhibit the growth of subcutaneous xenograft tumors (ISK cells) in mice, and its efficacy is superior to that of MPA.

[0194] discuss:

[0195] This invention is the first to discover that chlorpromazine hydrochloride is effective in treating endometrial cancer, and therefore can be used to prepare drugs for treating and / or alleviating endometrial cancer (type I and type II endometrial cancer). Experiments of this invention demonstrate that chlorpromazine hydrochloride achieves its anti-type II endometrial cancer effect (progesterone-insensitive endometrial cancer) by upregulating PRB expression levels in progesterone-insensitive KLE cells.

[0196] It is known that MPA also exerts its anti-endometrial cancer effect mainly through PRB (Nakamura, Cancer Letters, 2013, 336(1):68-75.), which inspired the inventors to study the regulatory effect of chlorpromazine hydrochloride on MPA in KLE cells. Through experiments, it was demonstrated that the compound of the present invention can also activate MPA expression and exert a therapeutic effect on progesterone-insensitive endometrial cancer.

[0197] 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 chlorpromazine hydrochloride or a pharmaceutically acceptable salt thereof. Furthermore, the active ingredient or a formulation containing the active ingredient is used to prepare a drug for treating and / or alleviating endometrial cancer; In the formulation, chlorpromazine hydrochloride or a pharmaceutically acceptable salt thereof is the sole active ingredient or the active ingredient is composed of chlorpromazine hydrochloride or a pharmaceutically acceptable salt thereof and medroxyprogesterone acetate (MPA). The treatment and / or relief of endometrial cancer includes: inhibiting the migration of endometrial cancer cells; The endometrial cancer mentioned is a type II endometrial cancer that is insensitive to progesterone.

2. The use as described in claim 1, characterized in that, The progestin is selected from: medroxyprogesterone acetate, medroxyprogesterone acetate (MPA), progesterone caproate, or combinations thereof.

3. The use as described in claim 1, characterized in that, The preparation is selected from the group consisting of: injections, inhalers, tinctures, powders, granules, capsules, oral liquids, tablets, pills, suspensions, emulsions, lozenges, or drops.

4. The use as described in claim 1, characterized in that, Used in the preparation of drugs 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) Induces apoptosis in endometrial cancer cells.

5. The use as described in claim 1, characterized in that, The preparation can be an oral preparation or a non-oral preparation.

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