WO2023035201A1

WO2023035201A1 - Application of penfluridol combined with medroxyprogesterone acetate in preparation of drug for treating endometrial cancer

Info

Publication number: WO2023035201A1
Application number: PCT/CN2021/117536
Authority: WO
Inventors: 王玉东; 孙笑; 毛斐; 伍慧雯; 刘小虎; 李丽娟; 李剑; 李晓康; 朱进
Original assignee: 中国福利会国际和平妇幼保健院; 华东理工大学
Priority date: 2021-09-09
Filing date: 2021-09-09
Publication date: 2023-03-16

Abstract

An application of penfluridol combined with medroxyprogesterone acetate in the preparation of a drug for treating endometrial cancer. Penfluridol and medroxyprogesterone acetate can synergistically inhibit the proliferation and migration of endometrial cancer cells, and promote the apoptosis of the endometrial cancer cells.

Description

Application of penfluridol combined with medroxyprogesterone acetate in the preparation of drugs for the treatment of endometrial cancer

technical field

The invention relates to the technical field of medicine, in particular to the application of penfluridol combined with medroxyprogesterone acetate in the preparation of medicines for treating endometrial cancer.

Background technique

Endometrial cancer (Endometrial Cancer, EC) is a group of epithelial malignant tumors that occur in the endometrium. It is one of the most common gynecological malignancies in the world, and its morbidity and mortality are increasing (Karen, Endometrial Cancer , The New England Journal of Medicine 2020, 383, 2053-2064). Although EC is more common in postmenopausal women, the incidence of EC in young women has increased dramatically over the past decade, with younger and younger women being diagnosed with endometrial cancer, and the underlying cause of the increase is obesity The prevalence and resulting hyperinsulinemia, and endometrial cancer rates are rising as obesity rates rise (Moore, Endometrial Cancer: Is This a New Disease? Am. Soc. Clin. Oncol. 2017, 37, 435-442). In 2020, there will be 420,000 new cases of endometrial cancer worldwide, including 80,000 new cases in China.

EC is usually divided into type I and type II. The main difference between the two is that type I EC is positive for progesterone receptor expression, and type II EC is negative for progesterone receptor expression (Bokhman, Gynecologic oncology, 1983, 15, 10-17 ). At present, surgical treatment is a common method for the treatment of EC, but conservative treatment with progesterone therapy is required for patients who need to protect their reproductive function. The main method of conservative treatment is hormone therapy with progesterone drugs (mainly medroxyprogesterone acetate (MPA)). However, hormone therapy has limitations. It is only effective for patients with positive progesterone receptor expression and has a high recurrence rate, which is prone to drug resistance. At present, there is an increasing demand for conservative treatment for fertility preservation in patients with EC.

Therefore, there is an urgent need in the art to develop drugs capable of treating EC, especially progesterone receptor-negative and progesterone-resistant EC.

Contents of the invention

The purpose of the present invention is to provide a drug capable of treating EC, especially progesterone receptor-negative and progesterone-resistant EC.

In a first aspect of the present invention, a pharmaceutical composition is provided, the composition comprising:

(a) a therapeutically effective amount of a first active ingredient which is penfluridol or a pharmaceutically acceptable salt thereof; and

(b) a therapeutically effective amount of a second active ingredient which is a progestogen,

Moreover, the pharmaceutical composition is used for treating and/or preventing endometrial cancer.

In another preferred example, the endometrial cancer includes type I and type II endometrial cancer.

In another preferred example, the endometrial cancer includes progesterone-sensitive endometrial cancer or progesterone-resistant endometrial cancer.

In another preferred example, the endometrial cancer is progesterone receptor-negative and progesterone-resistant endometrial cancer.

In another preferred embodiment, the progestin is selected from the group consisting of medroxyprogesterone acid, megestrol acetate, progesterone caproate, or combinations thereof.

In another preferred example, the progestin is medroxyprogesterone acetate.

In another preferred example, the weight ratio of the first active ingredient to the second active ingredient is from 1:20 to 10:1, preferably from 1:15 to 1:1, more preferably from 1:10 to 1:5.

In another preferred example, the content of the first active ingredient in the pharmaceutical composition is 0.01-90wt%, preferably 0.1-30wt%, more preferably 0.5-10wt%.

In another preferred example, the content of the second active ingredient in the pharmaceutical composition is 0.01-90wt%, preferably 0.1-30wt%, more preferably 0.5-10wt%.

In another preferred example, the first active ingredient and the second active ingredient account for 0.01-90wt% of the total mass of the pharmaceutical composition, preferably 0.1-50wt%, more preferably 0.5-30wt%.

In another preferred example, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

In another preferred example, the dosage form of the pharmaceutical composition includes injection dosage form and oral dosage form.

In another preferred example, the dosage forms of the pharmaceutical composition include injections, tablets, capsules, pills, suspensions and emulsions.

In another preferred example, the pharmaceutical composition further includes other anti-endometrial cancer drugs.

In a second aspect of the present invention, a kit is provided, which includes:

(a) a first formulation containing penfluridol or a pharmaceutically acceptable salt thereof; and

(b) A second formulation comprising a progestin.

In another preferred example, the kit further includes instructions, which indicate that the first preparation and the second preparation are used in combination to treat and/or alleviate endometrial cancer.

In another preferred example, the first preparation and the second preparation are administered simultaneously, separately or sequentially in the treatment of endometrial cancer.

In the kit of the present invention, the first preparation and the second preparation in the kit are independent of each other, or combined.

In another preferred example, the first preparation and the second preparation in the kit are independent of each other.

In another preferred example, the first preparation is an oral preparation or an injection preparation.

In another preferred example, the dosage forms of the first preparation include injections, tablets, capsules, pills, suspensions and emulsions.

In another preferred example, the second preparation is an oral preparation or an injection preparation.

In another preferred example, the dosage forms of the second preparation include injections, tablets, capsules, pills, suspensions and emulsions.

In a third aspect of the present invention, a combination of active ingredients is provided, which includes:

(b) A therapeutically effective amount of a second active ingredient which is a progestin.

In the fourth aspect of the present invention, there is provided a use of the pharmaceutical composition described in the first aspect of the present invention, the kit described in the second aspect and/or the combination of active ingredients described in the third aspect, for the preparation of Medicines to treat and/or relieve endometrial cancer.

The present invention also provides the use of the pharmaceutical composition, the kit and/or the combination of active ingredients described in the present invention for treating and/or alleviating endometrial cancer.

In another preferred example, the drug is also used for one or more of the following purposes:

(a) inhibit endometrial cancer cell proliferation;

(b) inhibit endometrial cancer cell migration; and

(c) Induction of apoptosis in endometrial cancer cells.

In another preferred example, the endometrial cancer cells are selected from the group consisting of ISK, KLE, or a combination thereof.

In another preferred example, the endometrial cancer cells are ISK and/or KLE cells.

In a fifth aspect of the present invention, a method for inhibiting endometrial cancer cells in vitro is provided, comprising the steps of:

(i) culturing endometrial cancer cells in the presence of penfluridol or a pharmaceutically acceptable salt thereof and progesterone, thereby inhibiting said endometrial cancer cells.

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

In another preferred example, the inhibition is selected from the group consisting of:

(a) inhibit endometrial cancer cell proliferation;

(b) inhibit endometrial cancer cell migration;

(c) inducing apoptosis of endometrial cancer cells;

(d) Any combination of the above (a) to (c).

In the sixth aspect of the present invention, there is provided a method for treating endometrial cancer, comprising the step of: administering the pharmaceutical composition described in the first aspect of the present invention, the kit described in the second aspect or The combination of active ingredients described in the third aspect.

In another preferred example, the subject is a patient with endometrial cancer.

In another preferred example, the treatment method further includes administering other anti-endometrial cancer drugs to the subject in need.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 is a schematic diagram of the inhibitory activity of penfluridol and medroxyprogesterone acetate on the proliferation of ISK and KLE cells. (A) ISK cell proliferation inhibitory activity; (B) KLE cell proliferation inhibitory activity.

Figure 2 is a schematic diagram of the synergistic inhibitory effect of penfluridol and medroxyprogesterone acetate on ISK and KLE cells. (A) Synergistic inhibitory effect of combined drugs on ISK cells; (B) Synergistic inhibitory effect of combined drugs on KLE cells.

Fig. 3 is a schematic diagram of the effect of penfluridol combined with medroxyprogesterone acetate on the migration ability of ISK and KLE cells. (A) ISK cell scratch; (B) KLE cell scratch; (C) ISK cell wound healing distance percentage; (D) KLE cell wound healing distance percentage.

Fig. 4 is a schematic diagram of the effect of penfluridol and medroxyprogesterone acetate on the migration ability of ISK and KLE cells. (A) ISK cell crystal violet staining; (B) ISK cell crystal violet staining quantification; (C) KLE cell crystal violet staining; (D) KLE cell crystal violet staining quantification.

Fig. 5 is a schematic diagram showing the effects of penfluridol and medroxyprogesterone acetate on the apoptosis of ISK and KLE cells. (A) Effect of combined drug on ISK cell apoptosis; (B) Effect of combined drug on KLE cell apoptosis; (C) Quantification of ISK cell apoptosis ratio; (D) KLE cell apoptosis ratio quantification.

Detailed ways

After extensive and in-depth research, the present inventor unexpectedly found that the combination of antipsychotic drug penfluridol and medroxyprogesterone acetate (MPA) can significantly improve the therapeutic effect on endometrial cancer, and can significantly improve the therapeutic effect on endometrial cancer ISK and The proliferation and migration abilities of KLE cells have a synergistic inhibitory effect, and can synergistically induce the apoptosis of endometrial cancer ISK and KLE cells. The synergistic therapeutic effect of penfluridol and medroxyprogesterone acetate is obviously better than that of penfluridol or medroxyprogesterone acetate alone. On this basis, the present invention has been accomplished.

the term

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the terms "comprising", "including", and "containing" are used interchangeably to include not only closed definitions, but also semi-closed, and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

As used herein, the term "pharmaceutically acceptable carrier" refers to ingredients suitable for humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergic reactions), that is, with a reasonable benefit/risk ratio substance.

As used herein, the term "therapeutically effective amount" refers to an amount that produces functions or activities on humans and/or animals and is acceptable to humans and/or animals. Those of ordinary skill in the art should understand that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvant of the drug used, the severity of the disease, and the combination with other drugs. different.

Penfluridol

As used herein, the terms "Penfluridol" (Penfluridol, PFL) and "the first active ingredient of the present invention" are used interchangeably, and both refer to penfluridol and its pharmaceutically acceptable salts.

The penfluridol has the following structural formula:

Penfluridol is an oral long-acting antipsychotic drug approved by the U.S. Food and Drug Administration (FDA). For the treatment of various types of schizophrenia. Penfluridol is a potent inhibitor of dopamine D2 receptors and calcium channels.

After extensive screening studies, the inventors unexpectedly found that penfluridol has a proliferation inhibitory effect on both endometrial cancer cells ISK (type I, progesterone-sensitive) and type II endometrial cancer cells KLE (progesterone-resistant). Further studies have found that its combined application with medroxyprogesterone acetate has a significant synergistic inhibitory effect on the two types of cells, can inhibit the proliferation and migration of the two types of EC cells, and induce EC cell apoptosis. Therefore, the present invention provides the use of penfluridol and medroxyprogesterone acetate in combination to prepare a medicine for treating endometrial cancer.

second active ingredient

As used herein, the term "second active ingredient of the invention" refers to a progestin.

In another preferred embodiment, the progestogen is medroxyprogesterone acetate and pharmaceutically acceptable salts thereof.

The structural formula of medroxyprogesterone acetate is as follows:

endometrial cancer

As used herein, the term "endometrial cancer (EC)" is a group of epithelial malignant tumors that occur in the endometrium, also known as uterine body cancer, and is one of the three most common malignant tumors of the female reproductive system. . Endometrial cancer is more likely to occur in perimenopausal and postmenopausal women. According to its pathogenesis and biological behavior characteristics, it can be divided into type I endometrial cancer (estrogen-dependent type) and type II endometrial cancer (non-estrogen-dependent type). hormone-dependent).

Pharmaceutical compositions and kits

The present invention also provides a pharmaceutical composition comprising:

The present invention also provides a kit, which includes:

(b) A second formulation comprising a progestin.

In another preferred example, the first preparation and the second preparation are different preparations or the same preparation.

When two active ingredients (or their corresponding preparations) are used in combination, the order of administration is not particularly limited, and they can be administered sequentially, together, or sequentially.

In another preferred example, the progestin is medroxyprogesterone acetate.

In the pharmaceutical composition of the present invention, the weight ratio of the first active ingredient to the second active ingredient is preferably 1:20 to 10:1, more preferably 1:15 to 1:1, further preferably 1:10 to 1:1: 5.

In the pharmaceutical composition of the present invention, the content of the first active ingredient in the pharmaceutical composition is preferably 0.01-90wt%, more preferably 0.1-30wt%, even more preferably 0.5-10wt%.

In the pharmaceutical composition of the present invention, the content of the second active ingredient in the pharmaceutical composition is preferably 0.01-90wt%, more preferably 0.1-30wt%, even more preferably 0.5-10wt%.

In the pharmaceutical composition of the present invention, the first active ingredient and the second active ingredient account for the total mass of the pharmaceutical composition, preferably 0.01-90 wt%, more preferably 0.1-50 wt%, further preferably 0.5-30 wt%.

When necessary, one or more pharmaceutically acceptable carriers can also be added to the medicine of the present invention. The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.

The compounds and pharmaceutical compositions provided by the present invention can be in various forms, such as tablets, injections, capsules, powders, syrups, solutions, suspensions and aerosols, etc., and can be present in suitable solid or liquid carriers or Diluent and appropriate sterile equipment for injection or infusion.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the field of pharmacy.

The pharmaceutical composition of the present invention can be clinically used in mammals, including humans and animals, and can be administered through oral, nasal, skin, lung or gastrointestinal tract and other routes. Oral administration is most preferred. The most preferred daily dose is 0.01-400 mg/kg body weight, taken once, or 0.01-200 mg/kg body weight in divided doses. Regardless of the method of administration, the optimal dosage for an individual should depend on the specific treatment. Usually, start with a small dose and gradually increase the dose until you find the most suitable dose.

The drug or inhibitor of the present invention can be administered in various ways, for example, it can be introduced into the body by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemically mediated methods such as muscle, intradermal, subcutaneous, intravenous , mucosal tissue; or mixed or wrapped by other substances into the body.

Typically, the active ingredient of the present invention or the pharmaceutical composition containing it can be administered in unit dose form, and the route of administration can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, Eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

The dosage form for administration may be a liquid dosage form, a solid dosage form or a semi-solid dosage form. Liquid dosage form can be solution (including true solution and colloid solution), emulsion (including O/W type, W/O type and double emulsion), suspension, injection (including water injection, powder injection and infusion), eye drops Agents, nasal drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, gas (powder) aerosols, sprays, etc.; semi-solid dosage forms can be ointments, Gels, pastes, etc.

The active ingredients of the present invention can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

In order to make the active ingredient of the present invention into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, Isopropanol, etc.; the binder can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, arabic mucilage, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hypromellose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked poly Vinylpyrrolidone, croscarmellose sodium, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecylsulfonate, etc.; lubricant and flow aid The agent can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol and the like.

Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

In order to make the administration unit into a capsule, the active ingredient of the present invention can be mixed with a diluent and a glidant, and the mixture is directly placed in a hard capsule or a soft capsule. The active ingredients can also be made into granules or pellets with diluents, binders, and disintegrants, and then placed in hard or soft capsules. Various diluents, binders, wetting agents, disintegrants, and glidants used to prepare the tablets of the present invention can also be used to prepare the capsules of the present invention.

In order to make the active ingredient of the present invention into an injection, water, ethanol, isopropanol, propylene glycol or their mixture can be used as a solvent and an appropriate amount of commonly used solubilizers, cosolvents, pH regulators, and osmotic pressure regulators in the field can be added. The solubilizer or co-solvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be Sodium chloride, mannitol, glucose, phosphate, acetate, etc. For preparation of freeze-dried powder injection, mannitol, glucose, etc. can also be added as proppants.

In addition, coloring agents, preservatives, fragrances, flavoring agents or other additives can also be added to the pharmaceutical preparations, if necessary.

The two active ingredients or compositions of the present invention can be taken together or sequentially, or further combined with other therapeutic drugs or symptomatic drugs.

When the active ingredient of the present invention has a synergistic effect with other therapeutic drugs, its dose should be adjusted according to the actual situation.

The main advantages of the present invention include:

1. The present invention found through experiments that penfluridol combined with medroxyprogesterone acetate has a synergistic inhibitory effect on the proliferation and migration of endometrial cancer ISK and KLE cells, and can synergistically induce the apoptosis of endometrial cancer ISK and KLE cells .

2. The present invention also found that the synergistic therapeutic effect of penfluridol and medroxyprogesterone acetate is significantly better than that of penfluridol or medroxyprogesterone acetate alone.

3. Penfluridol is an oral long-acting antipsychotic drug, so it has high safety. Combined use with medroxyprogesterone acetate also has higher safety and lower side effects.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1 Inhibitory effect of penfluridol and medroxyprogesterone acetate on the proliferation of endometrial cancer cells

In this example, the inhibitory effect of penfluridol and medroxyprogesterone acetate on the proliferation of endometrial cancer cells ISK and KLE was tested by CCK-8 experiment at the cellular level.

The research results show that medroxyprogesterone acetate can inhibit the proliferation of endometrial cancer cells ISK, while the same dose of medroxyprogesterone acetate has no obvious inhibitory effect on KLE cells, and the combination of penfluridol and medroxyprogesterone acetate can significantly Inhibits the proliferation of endometrial cancer cells ISK and KLE in a concentration-dependent manner.

1. Experimental materials and methods

Endometrial cancer cells ISK and KLE were purchased from the American Type Culture Collection (ATCC); phosphate buffered saline (PBS) was purchased from Bio-channel; DMEM/F12 medium was purchased from Biosharp; fetal calf Serum (FBS) and trypsin were purchased from Gibco; CCK-8 was purchased from Biyuntian Biotechnology Co., Ltd.; penfluridol was from the old drug store of the laboratory; medroxyprogesterone acetate was purchased from Selleck.

ISK and KLE cells were cultured in DMEM/F12 (containing 10% fetal bovine serum, 1% penicillin/streptomycin) medium and placed in an incubator at 37°C and 5% CO ₂ . After the endometrial cancer cells basically covered the cell culture dish (10 cm), the cells were digested with trypsin, and seeded in a 96-well plate at a cell density of 5000 cells/well, 100 μL per well. After culturing overnight, after the cells adhered to the wall, DMEM/F12 medium containing different concentrations of PFL was added, 200 μL per well, and 3 replicate wells were set up for each group. Incubate them for 72 hours in a cell culture incubator. Then remove the medium, add 100 μL of serum-free medium containing 10% CCK-8 to each well, incubate in the incubator at 37°C for 1 hour, use a Bio-Tek multi-functional microplate reader to detect the absorbance value A at 450 nm, and calculate Inhibition rate and IC ₅₀ value.

Inhibition rate calculation formula: cell inhibition rate%=[1-(administration group A value-blank group A value)/(control group A value-blank group A value)]×100%, _IC50 value through Graphpad Prism 8.0 software fit.

2. Experimental results

Table 1 and Figure 1 show the anti-proliferation data of the combination of penfluridol and medroxyprogesterone acetate on two kinds of EC cells.

The results showed that medroxyprogesterone acetate had a significant inhibitory effect on the proliferation of endometrial cancer cell ISK, while the same dose of medroxyprogesterone acetate had no significant effect on the proliferation of KLE cells. The combination of penfluridol and medroxyprogesterone acetate has a significant inhibitory effect on the proliferation of two endometrial cancer cells.

The proliferative activity of endometrial cancer cells decreased with the increase of penfluridol concentration, indicating that the combination of penfluridol and medroxyprogesterone acetate can inhibit the proliferation of endometrial cancer cells ISK and KLE in a concentration-dependent manner.

The IC ₅₀ of penfluridol and medroxyprogesterone acetate on the proliferation of ISK cells were 2.74 μM and 23.72 μM respectively; The inhibitory activity IC ₅₀ were 2.89 μM and 41.43 μM, respectively.

Table 1 Proliferation inhibitory activity of penfluridol and medroxyprogesterone acetate on different EC cell lines

MPA: medroxyprogesterone acetate; PFL: penfluridol.

This example shows that the combination of penfluridol and medroxyprogesterone acetate can inhibit the proliferation of endometrial cancer cells ISK and KLE in a concentration-dependent manner.

Example 2 Synergistic inhibitory effect of penfluridol combined with medroxyprogesterone acetate on endometrial cancer cells

In this example, the synergistic inhibitory effect of penfluridol combined with medroxyprogesterone acetate on endometrial cancer cells ISK and KLE was tested by CCK-8 experiment at the cellular level.

The results of the study showed that penfluridol and medroxyprogesterone acetate could synergistically inhibit the growth of endometrial cancer cells ISK and KLE.

1. Experimental materials and methods

For endometrial cancer cells and reagents, see Experimental Materials and Methods in Example 1.

For the cultivation of ISK and KLE cells and the detection of CCK-8, see the experimental materials and methods in Example 1. According to the above-mentioned inhibitory activity of penfluridol and medroxyprogesterone acetate on the proliferation of endometrial cancer cells, the concentration gradient of medroxyprogesterone acetate was set to 0, 5, 10, 15 and 20 μM, and the concentration gradient of penfluridol was 0, 0.5, 1, 2, 4, 8, 10, 20 and 50 μM. The absorbance value A at 450nm was detected using a Bio-Tek multifunctional microplate reader, and the inhibition rate and _IC50 value were calculated. Inhibition rate calculation formula: cell inhibition rate%=[1-(administration group A value-blank group A value)/(control group A value-blank group A value)]×100%.

The obtained inhibition rate was analyzed by using SynergyFinder 2.0 to obtain the synergy scores of penfluridol and medroxyprogesterone acetate in ISK and KLE cells, respectively.

2. Experimental results

The experimental results are shown in Figure 2 and Table 2. Both penfluridol and medroxyprogesterone acetate can inhibit the growth of endometrial cancer cells ISK and KLE in a dose-dependent manner. The inhibitory effect of membranous cancer cells ISK and KLE was weak, and the inhibition rates were 9.63% and 2.64, respectively. The combined application can achieve 44.45% and 43.39% inhibition rate of the two kinds of cells, showing a significant synergistic effect. At the same time, whether combined with low dose or high dose of medroxyprogesterone acetate, penfluridol can dose-dependently increase its inhibitory effect on endometrial cancer cells. The synergistic inhibition score of penfluridol and medroxyprogesterone acetate in endometrial cancer cell ISK was 10.07, and the synergistic inhibition score in KLE cell was 14.44.

Table 2 Synergistic scores of penfluridol and medroxyprogesterone acetate on ISK and KLE cells

细胞类型cell type	药物组合drug combination	协同分数synergy score	最大协同面积分数maximum synergy area fraction	方法method
Ishikawa细胞Ishikawa cells	PFL-MPAPFL-MPA	10.0710.07	16.8716.87	ZIPZIP
KLE细胞KLE cells	PFL-MPAPFL-MPA	14.4414.44	21.321.3	ZIPZIP

Note: MPA: medroxyprogesterone acetate; PFL: penfluridol.

This example illustrates that the combined use of penfluridol and MPA can enhance the inhibitory effect on endometrial cancer cells, and the effect is dose-dependent on both penfluridol and MPA.

Example 3 Inhibition of migration of endometrial cancer cells

In this example, the effect of penfluridol on the migration ability of endometrial cancer cells ISK and KLE cells was tested by cell scratch experiment at the cell level.

The results of the study showed that low-dose penfluridol or medroxyprogesterone acetate had limited ability to inhibit the migration of endometrial cancer cells ISK and KLE, but the combination of low-dose penfluridol and medroxyprogesterone acetate could significantly inhibit ISK and KLE cell migration ability.

1. Experimental materials and methods

The source of experimental materials is the same as in Example 1. The EC cells in the logarithmic growth phase were digested with trypsin, prepared into a single cell suspension using serum-free DMEM/F12 medium, and seeded into a 6-well plate at a density of about 500,000 cells per well. After the cells adhered to the wall, they were divided into 6 groups, namely the control group, the MPA 5 μM group, the MPA 10 μM group, the PFL 1 μM group, the MPA 5 μM+PFL 1 μM group, and the MPA 10 μM+PFL 1 μM group, with 3 replicate wells in each group. The next day, use a 20 μL pipette tip, perpendicular to the cell plane, to scratch the cell layer along the line drawn on the back of the plate the day before. Subsequently, the cells were washed 3 times with sterile PBS to remove non-adherent cells, and replaced with fresh serum-free medium to continue culturing for 48 h. The cells were taken out at 0h and 48h, observed under a microscope and photographed for record. Six horizontal lines were randomly drawn from each image using ImageJ, and the average distance between cells was calculated, and the percentage of wound healing distance of cells in each group was measured by the ratio of the distance between cells at 48h and the distance between cells at 0h.

2. Experimental results

The results are shown in Figure 3. Control in the figure is the control group. In the ISK and KLE cells, compared with the control group, the distance between the cell scratches in the low-dose penfluridol group was significantly increased, indicating that penfluridol has the ability to inhibit ISK and KLE cells. At the same time, low-dose medroxyprogesterone acetate (5 μM) only inhibited the migration of ISK cells, but had no significant effect on the migration ability of KLE cells, while 10 μM medroxyprogesterone acetate treatment could simultaneously inhibit the migration of two endometrial cancer cells. migrate.

In the combination drug group, the percentage of wound healing distance between cell scratches was significantly lower than that in the single drug treatment group. The vertical axis of the histogram indicates the percentage of wound healing distance of cells in each group, and the significant difference of the data was analyzed by One-way ANOVA method (Graphpad Prism8.0 software). Data are mean±SD: ***P<0.001 vs Ctrl, ###P<0.001 vs MPA 5 μM, $$$P<0.001 vs MPA 10 μM.

This experiment shows that the combination of penfluridol and MPA (the preferred dosage ratio is 1:10 to 1:5) has the ability to inhibit the migration of ISK and KLE cells, and the ability of the combined drug to inhibit cell migration is significantly better than that of the single drug alone. .

Example 4 Penfluridol and Medroxyprogesterone Acetate Inhibit the Migration of Endometrial Cancer Cells

In this embodiment, the effects of penfluridol and medroxyprogesterone acetate on the migration ability of endometrial cancer cells ISK and KLE were tested at the cellular level by Transwell experiments. The results of the study showed that penfluridol and medroxyprogesterone acetate could synergistically inhibit the migration of endometrial cancer cells ISK and KLE.

1. Experimental materials and methods

The Transwell chamber was purchased from Costar Company; paraformaldehyde is a common reagent in the laboratory, commercially purchased without any treatment, and the sources of other experimental materials are the same as in Example 1. The EC cells in the logarithmic growth phase were digested with trypsin, prepared into a single cell suspension using serum-free DMEM/F12 medium, and seeded on the top of the Transwell chamber at a density of about 100,000 cells per well. Add 600 μL of DMEM/F12 medium containing 20% FBS to the bottom of the small chamber, taking care to avoid the generation of air bubbles. Divided into 6 groups, namely control group, MPA 5 μM group, MPA 10 μM group, PFL 1 μM group, MPA 5 μM+PFL 1 μM group, MPA 10 μM+PFL 1 μM group, each group set 3 replicate wells. 150 μL of serum-free DMEM/F12 medium containing different drugs was added to the upper chamber of the treatment group, and 150 μL of serum-free DMEM/F12 medium without drug was added to the upper chamber of the control group. Place in an incubator at 37°C with 5% CO ₂ for 24 hours. Then carefully remove the small chamber with tweezers, blot the liquid in the upper chamber, move it to a 24-well plate in which about 800 μL of pre-cooled PBS has been added, and wash twice, 5 min each time. Take out the small chamber, move it to a 24-well plate pre-added with about 800 μL of paraformaldehyde solution, and fix it at room temperature for 30 minutes. Take out the small chamber, blot dry the fixative in the upper chamber, transfer to a 24-well plate pre-added with about 800 μL of crystal violet staining solution, and stain at room temperature for 30 minutes. Gently rinse with water several times, and carefully wipe off the cells on the surface of the upper chamber with a wet cotton swab. Five fields of view were randomly selected under a 200X microscope to take pictures, and Image J software was used to manually count the number of cells in each field of view.

2. Experimental results

The results are shown in Figure 4, the control in the figure is the control group, and each black dot in the picture represents a cell passing through the small chamber.

The results showed that in ISK cells, compared with the control group, the number of cells passing through the small chamber in the medroxyprogesterone acetate group and penfluridol group decreased, and the number of cells passing through the small chamber in the penfluridol combined with medroxyprogesterone acetate group The number of cells further decreased, and the difference was statistically significant (p<0.0001), indicating that penfluridol and medroxyprogesterone acetate have the ability to synergistically inhibit the migration of ISK cells.

In KLE cells, compared with the Ctrl group, the number of cells passing through the small chamber in the medroxyprogesterone acetate group had no significant change, and the number of cells passing through the small chamber in the penfluridol group decreased. The number of migrating cells in the combination group of lidocaine and medroxyprogesterone acetate was significantly reduced, indicating that the combination of penfluridol and medroxyprogesterone acetate had an obvious synergistic inhibitory effect on the migration ability of KLE cells.

The vertical axis of the histogram indicates the number of cells passing through the small chamber in each group, and the significant difference of the data was analyzed by One-way ANOVA method (Graphpad Prism8.0 software). Data are mean±SD: **P<0.01, ***P<0.001 vs Ctrl, ###P<0.001 vs MPA 5 μM, $$$P<0.001 vs MPA 10 μM.

This experiment shows that the combination of penfluridol and medroxyprogesterone acetate (the preferred dosage ratio is 1:10 to 1:5) has an obvious synergistic inhibitory effect on the migration ability of ISK and KLE cells.

Example 5 Effect of Penfluridol and Medroxyprogesterone Acetate on Endometrial Cancer Cell Apoptosis

In this example, Annexin V-FITC/PI cell apoptosis detection kit was used to test the effects of penfluridol and medroxyprogesterone acetate on the apoptosis of endometrial cancer cells ISK and KLE at the cellular level. The results of the study showed that penfluridol can inhibit the apoptosis of endometrial cancer cells ISK and KLE, and its combination with medroxyprogesterone acetate can synergistically inhibit the apoptosis of endometrial cancer cells.

1. Experimental materials and methods

The Annexin V-FITC/PI Cell Apoptosis Detection Kit was purchased from Beyotime Biotechnology Co., Ltd., in which Annexin V-FITC Conjugate Solution, Annexin V-FITC, and Propidium Iodide (PI) are all reagents in the kit. All the other experimental materials are from the same sources as in Example 1. The EC cells in the logarithmic growth phase were digested with trypsin to prepare a single cell suspension, seeded into a 6-well plate at a density of about 120,000 cells per well, and cultured overnight. After the cells adhered to the wall, they were divided into 6 groups, namely the control group, the MPA 5 μM group, the MPA 10 μM group, the PFL 1 μM group, the MPA 5 μM+PFL 1 μM group, and the MPA 10 μM+PFL 1 μM group, with 3 replicate wells in each group. Incubate for 48 h at 37 °C in an incubator with 5% CO ₂ . Then suck out the cell culture solution into a 10ml centrifuge tube, wash the adherent cells once with PBS, add 300 μL of trypsin to digest the cells for 2 minutes, blow the cells down gently, transfer them to the corresponding centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, and collect the cells. Gently resuspend cells in PBS and count. Take 50,000-100,000 resuspended cells, centrifuge at 1000rpm for 5min, discard the supernatant, add 195μL Annexin V-FITC binding solution to gently resuspend the cells, add 5μL Annexin V-FITC and 10μL propidium iodide (PI) staining solution, gently Mix lightly. Incubate at room temperature in the dark for 20 min, and immediately detect with a Beckman Coulter (cytoFLEX LX) flow cytometer.

2. Experimental results

The results are shown in Figure 5, where Ctrl is the control group, the first quadrant in the schematic diagram of apoptosis is the late apoptotic cells, the fourth quadrant is the early apoptotic cells, and the apoptotic ratio is the sum of the ratios of the first and fourth quadrants. Compared with the control group, PFL can induce the apoptosis of ISK cells, but has no obvious effect on the apoptosis of KLE cells. MPA can induce the apoptosis of both ISK and KLE cells, but the effect on ISK cells is more significant. Penfluridol combined with medroxyprogesterone acetate can significantly induce the apoptosis of ISK and KLE cells. When penfluridol combined with medroxyprogesterone acetate (5μM), the apoptosis ratios of ISK and KLE were 23.92% and 12.52%, respectively. The apoptotic ratios of ISK and KLE were 22.97% and 35.16% when Lido combined with medroxyprogesterone acetate (10μM). The vertical axis of the histogram indicates the proportion of apoptosis in each group, and the significant difference of the data is analyzed by One-way ANOVA method (Graphpad Prism8.0 software). Data are mean±SD: **P<0.01, ***P<0.001 vs Ctrl, ##P<0.01, ###P<0.001 vs MPA 5 μM, $$$P<0.001 vs MPA 10 μM.

This experiment shows that penfluridol combined with MPA (the preferred dosage ratio is 1:10 to 1:5) can significantly induce the apoptosis of ISK and KLE cells, and the effect is significantly better than that of single drugs respectively.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

A pharmaceutical composition, characterized in that the composition comprises:

(a) a therapeutically effective amount of a first active ingredient which is penfluridol or a pharmaceutically acceptable salt thereof; and

(b) A therapeutically effective amount of a second active ingredient which is a progestin.
The pharmaceutical composition according to claim 1, wherein the progesterone is selected from the group consisting of medroxyprogesterone acid, megestrol acetate, progesterone caproate, or combinations thereof.
The pharmaceutical composition according to claim 1, wherein the weight ratio of the first active ingredient to the second active ingredient is 1:20 to 10:1, preferably 1:15 to 1:1, More preferably, 1:10 to 1:5.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is used for treating and/or preventing endometrial cancer.
The pharmaceutical composition according to claim 4, wherein the endometrial cancer includes type I and type II endometrial cancer.
The pharmaceutical composition according to claim 5, wherein the endometrial cancer comprises progesterone-sensitive endometrial cancer or progesterone-resistant endometrial cancer.
A medicine box, is characterized in that, described medicine box comprises:

(a) a first formulation containing penfluridol or a pharmaceutically acceptable salt thereof; and

(b) A second formulation comprising a progestin.
A combination of active ingredients, characterized in that the combination of active ingredients includes:

(a) a therapeutically effective amount of a first active ingredient which is penfluridol or a pharmaceutically acceptable salt thereof; and

(b) A therapeutically effective amount of a second active ingredient which is a progestin.
A pharmaceutical composition as claimed in claim 1, a kit as claimed in claim 7 and/or an active ingredient combination as claimed in claim 8, for the preparation of treatment and/or alleviation of endometrial cancer Drug.
A method for inhibiting endometrial cancer cells in vitro, comprising the steps of:

(a) culturing endometrial cancer cells in the presence of penfluridol or a pharmaceutically acceptable salt thereof and progesterone, thereby inhibiting said endometrial cancer cells.