WO2022188491A1 - Tumor chemotherapy pharmaceutical composition - Google Patents

Abstract

Disclosed is a tumor chemotherapy pharmaceutical composition, and specifically disclosed is the use of sildenafil as an anti-tumor drug sensitizer in the preparation of a tumor chemotherapy drug. It is found that sildenafil as a sensitizer is combined with antitumor drugs such as oxaliplatin, irinotecan or capecitabine by means of a carrier to prepare an antitumor composition, and the antitumor composition can be used for effectively killing colorectal cancer and other tumor cells or drug-resistant tumor cells. The sensitization effect of sildenafil on antitumor drugs is verified at the cellular level for the first time, the treatment synergistic effect of sildenafil as a sensitizer in colorectal cancer, gastric cancer, liver cancer, breast cancer, prostate cancer and other cancers is confirmed, and a new way and means are provided for effectively treating tumors.

Description

A kind of tumor chemotherapy drug composition technical field

The invention relates to the field of medicine, and relates to a tumor chemotherapy drug combination, in particular to the use of sildenafil as an antitumor drug sensitizer in the preparation of tumor chemotherapy drugs.

Background technique

Colorectal cancer, gastric cancer, liver cancer, breast cancer, prostate cancer and other tumors seriously threaten human life and health, and their morbidity and mortality are high. health problems. At present, the main treatment methods for colorectal cancer, gastric cancer, liver cancer, breast cancer, prostate cancer and other tumors include surgery, chemotherapy and radiation therapy. For patients with early detection, surgical resection is considered to be a better treatment method; however, when the tumor progresses to an advanced stage, surgery alone cannot cure the tumor. patient treatment. However, for the conventional chemotherapeutic drugs used in the related art, most chemotherapeutic drugs have certain toxicity and side effects, such as bone marrow suppression, reduction of white blood cell count, platelet inhibition, diarrhea, nausea, vomiting, etc. Hazardous. Therefore, it is of great clinical significance to seek to enhance the anticancer activity of traditional chemotherapeutic drugs and reduce their adverse reactions.

In addition, although research on tumor treatment is continuing, less than half of tumors are sensitive to chemotherapy, and more than 50% of tumors will rapidly develop resistance to chemotherapy drugs, that is, tumor multidrug resistance (MDR). . MDR means that tumor cells are not only resistant to one anti-tumor drug, but also have cross-resistance to other anti-tumor drugs with different structures and mechanisms of action. Therefore, finding a low-toxic and effective drug reversal is also of great significance for clinical tumor treatment.

SUMMARY OF THE INVENTION

The present invention aims to solve at least one of the technical problems existing in the prior art. The inventors discovered a new application of sildenafil, namely the application of sildenafil as an anti-tumor chemotherapeutic drug resistance sensitizer in the preparation of tumor chemotherapeutic drugs, and provided a combination of sildenafil and existing chemotherapeutic drugs Application of technical solutions for the treatment of cancer. At the same time, a pharmaceutical composition of sildenafil combined with chemotherapeutic drugs is provided.

A first aspect of the present invention provides a pharmaceutical composition comprising a tumor chemotherapy drug, sildenafil or a salt thereof.

The phosphodiesterase type 5 (PDE-5) inhibitor sildenafil (Sidenafil) is a prescription drug for erectile dysfunction (ED). In the related art, sildenafil has been found to have a certain antitumor activity, but there is no report on whether sildenafil has the property of reversing tumor multidrug resistance (MDR) and its related mechanism.

According to the first aspect of the present invention, in some embodiments of the present invention, the tumor chemotherapy drug includes at least one of platinum, fluorouracil, irinotecan and capecitabine.

In some preferred embodiments of the present invention, the tumor chemotherapy drug is at least one of platinum, fluorouracil, irinotecan and capecitabine.

In some more preferred embodiments of the present invention, the platinum agent includes at least one of cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin, and lobaplatin.

Platinum agents include first-generation cisplatin, second-generation carboplatin, nedaplatin, cycloplatin, third-generation oxaliplatin, and lobaplatin. Replication and Transcription. Among them, the first-generation platinum agent: cisplatin is the first-line drug for a variety of solid tumors. It can be used for advanced ovarian cancer, osteosarcoma, neuroblastoma, and is effective for head and neck, cervical, esophageal and urinary tract tumors. Nephrotoxicity and gastrointestinal toxicity. Second-generation platinum agent: carboplatin is mainly used for small cell lung cancer, ovarian cancer, testicular tumor, head and neck squamous cell carcinoma, etc. It can also be used for non-small cell lung cancer, bladder cancer, cervical cancer, pleural mesothelioma, melanoma , endometrial cancer, etc., but it is serious for bone marrow suppression and thrombocytopenia; nedaplatin is mainly used for esophageal cancer, non-small cell lung cancer, small cell lung cancer, it also has serious problems of bone marrow suppression and thrombocytopenia, and also Reduce the number of white blood cells; Cycloplatin is mainly used for the treatment of urogenital malignancies such as testicular cancer, ovarian cancer, head and neck cancer, lung cancer, bladder cancer, prostate cancer, etc. It also has the side effect of bone marrow suppression. The third-generation platinum agent: Oxaliplatin is mainly used for the first- and second-line treatment and postoperative adjuvant treatment of advanced colorectal cancer, as well as ovarian cancer, breast cancer, gastric cancer, pancreatic cancer, non-small cell lung cancer, melanoma, lymphoma However, oxaliplatin has certain neurotoxicity and digestive tract reactions, which limits its clinical application; lobaplatin is mainly used for the treatment of breast cancer, small cell lung cancer, and chronic myeloid leukemia. At the same time, the reduction of platelets is also the strongest.

Irinotecan (Irinotecan) is a semi-synthetic water-soluble camptothecin derivative, which is also a first-line drug for the treatment of colorectal cancer. Irinotecan mainly forms a complex with topoisomerase I and DNA, which can cause DNA single-strand breaks, prevent DNA replication and inhibit RNA synthesis, and has specific anti-cancer effects for the S phase of the cell cycle. However, irinotecan also has adverse reactions such as delayed diarrhea and neutropenia.

Capecitabine is rapidly absorbed through the intestinal mucosa after oral administration, converted into an inactive intermediate 5'-deoxy-5'fluorocytidine by carboxylesterase in the liver, and then deaminated by cytidine in the liver and tumor tissue. The action of the enzyme is converted into 5'-deoxy-5' fluorouridine, which is finally catalyzed by thymidine phosphorylase to fluorouracil (5-FU) in tumor tissue. Capecitabine is mainly used for the treatment of advanced breast cancer and colorectal cancer. However, capecitabine will produce more severe diarrhea, nausea, vomiting, gastritis and other adverse reactions.

In some preferred embodiments of the present invention, the tumor includes colorectal cancer, gastric cancer, liver cancer, breast cancer and prostate cancer.

Of course, those skilled in the art can reasonably select the corresponding tumor according to the indications of the actually used tumor chemotherapy drugs.

In some more preferred embodiments of the present invention, the tumor is at least one of colorectal cancer, gastric cancer, liver cancer, breast cancer or prostate cancer.

In some preferred embodiments of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable carrier or adjuvant.

In some more preferred embodiments of the present invention, the pharmaceutically acceptable carrier or adjuvant includes diluent, absorbent, wetting agent, binder, disintegrant, lubricant, flavoring agent or transdermal absorption one or more of the accelerators.

Of course, those skilled in the art can reasonably add other carriers or adjuvants to improve the efficacy of the pharmaceutical composition according to actual use requirements.

According to the first aspect of the invention, in some embodiments of the invention, the salt is sildenafil citrate.

The second aspect of the present invention provides a medicament containing the pharmaceutical composition of the first aspect of the present invention.

According to the second aspect of the present invention, in some embodiments of the present invention, the dosage forms of the medicament include oral preparations, injection preparations and transdermal preparations.

Of course, those skilled in the art can select different preparation methods to obtain other dosage forms according to actual use requirements.

The third aspect of the present invention provides the use of sildenafil or a salt thereof as an antitumor drug sensitizer in preparing a pharmaceutical composition for treating tumors.

The inventors found that, in practical use, 800nM sildenafil is resistant to some representative drug-resistant tumor cell lines, such as SGC7901/DDP, MCF-7/DDP, HepG2/DDP, PC-3/DDP, HCT -116/L-OHP, HT-29/CPT-11 and SW620/CAP had only mild inhibitory effects on cell proliferation. However, the combined use of sildenafil and chemotherapeutic drugs can significantly improve the inhibitory effect of chemotherapeutic drugs on the above-mentioned drug-resistant cell lines, and the combined administration can reduce the adverse reactions caused by high-dose chemotherapeutic drugs. In addition, the inventors also found that sildenafil-related sensitization mechanisms include down-regulating P-gp, MDR1, and MRP1 in drug-resistant cell lines, that is, reducing P-gp to pump chemotherapeutic drugs out of cells and enhancing tumor resistance to chemotherapeutic drugs. susceptibility, and to promote apoptosis in drug-resistant cell lines. Therefore, in the clinical application mainly based on chemotherapy, sildenafil can be used as a sensitizer to improve the therapeutic effect of chemotherapy drugs.

According to the third aspect of the present invention, in some embodiments of the present invention, the salt is sildenafil citrate.

According to the third aspect of the present invention, in some embodiments of the present invention, the antitumor drug comprises at least one of platinum, fluorouracil, irinotecan and capecitabine.

In some preferred embodiments of the present invention, the antitumor drug is at least one of platinum, fluorouracil, irinotecan and capecitabine.

In some more preferred embodiments of the present invention, the platinum agent includes at least one of cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin, and lobaplatin.

In some preferred embodiments of the present invention, the tumor includes colorectal cancer, gastric cancer, liver cancer, breast cancer and prostate cancer.

Of course, those skilled in the art can reasonably select the corresponding tumor according to the indications of the actually used tumor chemotherapy drugs.

In some more preferred embodiments of the present invention, the tumor is at least one of colorectal cancer, gastric cancer, liver cancer, breast cancer or prostate cancer.

The fourth aspect of the present invention provides the use of sildenafil or a salt thereof in combination with a tumor chemotherapy drug in the preparation of a drug for treating tumors.

The present invention proposes for the first time a technical scheme of combining sildenafil with platinum, irinotecan, capecitabine and other chemotherapeutic drugs through a pharmaceutically acceptable carrier. It provides new ways and means in the effective treatment of tumors.

According to the fourth aspect of the present invention, in some embodiments of the present invention, the antitumor drug includes at least one of platinum, fluorouracil, irinotecan and capecitabine.

In some preferred embodiments of the present invention, the antitumor drug is at least one of platinum, fluorouracil, irinotecan and capecitabine.

In some more preferred embodiments of the present invention, the platinum agent includes at least one of cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin, and lobaplatin.

In some preferred embodiments of the present invention, the tumor includes colorectal cancer, gastric cancer, liver cancer, breast cancer and prostate cancer.

Of course, those skilled in the art can reasonably select the corresponding tumor according to the indications of the actually used tumor chemotherapy drugs.

In some more preferred embodiments of the present invention, the tumor is at least one of colorectal cancer, gastric cancer, liver cancer, breast cancer or prostate cancer.

According to the fourth aspect of the present invention, in some embodiments of the present invention, the dosage of sildenafil or its salt is 0.5-20 mg/day.

In some preferred embodiments of the present invention, the administration mode of sildenafil or its salt includes injection administration, and the injection administration includes but is not limited to intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection, etc.

The beneficial effects of the present invention are:

1. The present invention has verified for the first time that sildenafil is used as an anti-tumor drug sensitizer at the cellular level. The killing effect of chemotherapeutic drugs such as capecitabine on drug-resistant cell lines.

2. The present invention discloses the sensitization mechanism related to sildenafil, and finds that sildenafil can reduce P-gp, MDR1, and MRP1 in drug-resistant cell lines, that is, attenuate P-gp to pump chemotherapeutic drugs out of cells. , enhance the sensitivity of tumors to chemotherapeutic drugs, and promote the apoptosis of drug-resistant cell lines, thereby effectively improving the therapeutic effect of chemotherapeutic drugs.

3. The present invention proposes for the first time a technical scheme of combining sildenafil with platinum, irinotecan, capecitabine and other chemotherapeutic drugs through a pharmaceutically acceptable carrier. It can avoid the adverse reactions caused by the administration of high-dose chemotherapy drugs, and provide new ways and means for improving the effective treatment of tumors by chemotherapy drugs.

Description of drawings

Fig. 1 is the detection result of the inhibitory effect of sildenafil on colorectal cancer HCT-116 cells and DLD-1 cells in the embodiment of the present invention;

Fig. 2 is a graph showing the results of sildenafil significantly enhancing the killing effect of oxaliplatin and irinotecan on human colon cancer cells (HCT-116) in vivo in the embodiment of the present invention; wherein, A is a physical image of a nude mouse tumor, and B In Figure 2, group A is the control group, group B is the oxaliplatin single-use group, group C is the irinotecan single-use group, and group D is the oxaliplatin and sildena group The non-combination group and group E are the combination group of irinotecan and sildenafil;

Fig. 3 is a graph showing the results of sildenafil significantly enhancing the killing effect of oxaliplatin on human colon cancer oxaliplatin-resistant cells (HCT-116/L-OHP) in vivo in the embodiment of the present invention; wherein, A is naked The physical map of the mouse tumor, B is the tumor growth curve of the nude mouse; in Figure 2, group A is the control group, group B is the oxaliplatin single-use group, and group C is the oxaliplatin and sildenafil combination group.

Detailed ways

In order to make the invention purpose, technical solutions and technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention, rather than for limiting the present invention.

The experimental materials and reagents used, unless otherwise specified, are conventional consumables and reagents that can be obtained from commercial sources.

Experimental Materials

The cell lines used in the following examples include: colorectal cancer HCT-116 cells and DLD-1 cells, human cisplatin-resistant gastric cancer cells (SGC7901/DDP), and human breast cancer cisplatin-resistant cell lines (MCF-7 /DDP), human liver cancer-resistant cell line (HepG2/DDP), human prostate cancer cisplatin-resistant cell line (PC-3/DDP), human colon cancer oxaliplatin-resistant cell line (HCT-116/L- OHP), human colorectal cancer irinotecan-resistant cells (HT-29/CPT-11), and human colon cancer capecitabine-resistant cells (SW620/CAP).

The main drugs and reagents used in the following examples are: Sildenafil (SIGMA), fetal bovine serum (GIBCO), RPMI-1640 culture medium (GIBCO), 0.25% pancreatin (GIBCO), MTT (SIGMA), DMSO (Shanghai Sinopharm).

The main instruments involved in the following examples are: WATER JACKET carbon dioxide incubator (Japan ASTEC company); BSC-1600IIA2 biological safety cabinet (Sujing Group Suzhou Antai Air Technology Co., Ltd.); XDS-1B inverted microscope (Chongqing Photoelectric Instrument Co., Ltd. Company); AcculAB ALC-210.3 electronic balance (Aikeler, Germany); Synergy2 multifunctional microplate reader (BioTek Instrument Co., Ltd., USA).

In vitro inhibition test of sildenafil on non-drug-resistant cell lines

In this example, the non-drug-resistant cell line colorectal cancer HCT-116 cells and DLD-1 cells were used as the objects, and the in vitro inhibitory effect of sildenafil on the non-drug-resistant cell line was detected.

The specific experimental methods are as follows:

Take HCT-116 and DLD-1 cells and inoculate them in 100mm culture dishes respectively, culture for 72h, and remove the culture medium. Then digested with 0.25% trypsin, collected the cells, counted the cells, prepared a single-cell suspension with a concentration of 10,000 cells/mL, and inoculated 0.2 mL of the single-cell suspension in each well into a 96-well plate, and the total number of cells in each well was 2,000. After culturing for 24h, add sildenafil (final concentrations of 25, 50, 100, 200, 400, 800, 1600nM) for 72h, remove the supernatant, add 0.2mL DMSO, and measure each well with a microplate reader at 570nm absorbance value (OD). Cell viability was calculated based on the OD value.

The results are shown in Figure 1. After 72h of 800nM sildenafil treatment, the survival rates of HCT-116 and DLD-1 cells were both greater than 85%. This shows that when the concentration of sildenafil is lower than 800nM, treatment for 72h has no obvious inhibitory effect on the non-drug-resistant cell lines HCT-116 and DLD-1.

In vitro test of inhibition effect of sildenafil on different drug-resistant cell lines

In this example, the MTT method was used to detect the in vitro inhibitory effect of sildenafil on different drug-resistant cell lines, and the specific experimental method was as follows:

Take SGC7901/DDP, MCF-7/DDP, HepG2/DDP, PC-3/DDP, HCT-116/L-OHP, HT-29/CPT-11 and SW620/CAP cell lines and inoculate them in 100mm petri dishes respectively , cultured for 72h, and removed the culture medium. Digest with 0.25% trypsin, collect the cells, count the cells, and prepare a single-cell suspension with a concentration of 10,000 cells/mL. The prepared single cell suspension was seeded into a 96-well plate at 0.2 mL/well, and the number of cells in each well was 2000. After culturing for 24h, different concentrations of sildenafil (final concentrations were 25, 50, 100, 200, 400, 800, 1600nM) were added for treatment for 72h, the control group was added with an equal volume of RPIM 1640 culture medium, and a blank group ( RPIM 1640 culture medium without cells), cultured at 37°C (the culture time is determined according to the type of cell line), after the culture is completed, add 20 μL of MTT solution (final concentration 0.5mg/mL), and continue to culture for 4h. The supernatant was discarded, 150 μL DMSO was added to each well, and the absorbance value (OD) of each well was measured at 570 nm with a microplate reader. Cell viability was calculated based on the OD value.

The results are shown in Table 1.

Table 1 Effects of different concentrations of sildenafil on the survival rate of drug-resistant cell lines (%, n=5)

As can be seen from Table 1, after adding 800nM sildenafil for 72h, SGC7901/DDP, MCF-7/DDP, HepG2/DDP, PC-3/DDP, HCT-116/L-OHP, HT-29/CPT The survival rates of -11 and SW620/CAP were both greater than 85%. This shows that when the concentration of sildenafil is lower than 800nM, after 72h of treatment, there is no obvious inhibitory effect on the above-mentioned drug-resistant cell lines. Therefore, in the following examples, 800nM sildenafil was selected as the highest concentration of the sensitizer, and used in combination with chemotherapy drugs such as cisplatin, oxaliplatin, and irinotecan, respectively, to detect the sensitizing effect of sildenafil, In order to avoid the interference caused by the anti-tumor effect of sildenafil itself.

Experiment of Sildenafil Enhanced Inhibitory Effect of Chemotherapy Drugs on Non-resistant Cell Lines

The DLD-1 cell line was taken and inoculated into 100mm petri dishes, cultured for 72h, and the culture medium was removed. Digest with 0.25% trypsin, collect the cells, count the cells, and prepare a single-cell suspension with a concentration of 10,000 cells/mL. The prepared single cell suspension was seeded into a 96-well plate at 0.2 mL/well, and the number of cells in each well was 2000. After culturing for 24 hours, different drugs were added, and the specific additions were as follows:

(1) Add 10, 20, 50, 100, 200, 400, 1000, 2000, 4000 nM oxaliplatin separately;

(2) Add 5, 10, 20, 50, 100, 200, 400, 1000, 2000 nM irinotecan separately;

(3) 10, 20, 50, 100, 200, 400, 1000, 2000, 4000nM oxaliplatin + 800nM sildenafil;

(4) 5, 10, 20, 50, 100, 200, 400, 1000, 2000 nM irinotecan + 800 nM sildenafil.

At the same time, a blank group (RPIM 1640 medium without cells) and a control group (an equal volume of RPIM 1640 medium) were set up at 37°C for incubation (the incubation time was determined according to the type of cell line). After the incubation, 20 μL of MTT solution ( The final concentration was 0.5 mg/mL), and the culture was continued for 4 h. The supernatant was discarded, 150 μL of DMSO was added to each well, and the absorbance value (OD) of each well was measured at 570 nm with a microplate reader. The half cell inhibitory concentration ( _IC50 ) was calculated.

The results are shown in Table 2.

Table 2 Effects of different drug treatments on IC ₅₀ of non-drug-resistant cell lines (nM, n=15)

The results showed that 800nM sildenafil alone had only a weak inhibitory effect on the cell proliferation of colorectal cancer DLD-1 cell line. When 800 nM sildenafil was administered in combination with oxaliplatin and irinotecan, the _IC50 of oxaliplatin and irinotecan on DLD-1 cells was significantly lower than that of oxaliplatin and irinotecan alone. _IC50 for DLD-1 cells. The above results indicate that the combination of 800nM sildenafil with oxaliplatin and irinotecan can significantly reduce the IC ₅₀ concentrations of oxaliplatin and irinotecan, and enhance the killing effect of these two chemotherapy drugs on DLD-1 cells. .

Experiment of Sildenafil to Enhance the Inhibitory Effect of Chemotherapy Drugs on Drug-resistant Cell Lines

Take SGC7901/DDP, MCF-7/DDP, HepG2/DDP, PC-3/DDP, HCT-116/L-OHP, HT-29/CPT-11 and SW620/CAP cell lines and inoculate them in 100mm petri dishes respectively , cultured for 72h, and removed the culture medium. Digest with 0.25% trypsin, collect the cells, count the cells, and prepare a single-cell suspension with a concentration of 10,000 cells/mL. The prepared single cell suspension was seeded into a 96-well plate at 0.2 mL/well, and the number of cells in each well was 2000. After culturing for 24 hours, different drugs were added according to the cell line type. The specific additions were as follows:

(1) SGC7901/DDP, MCF-7/DDP, HepG2/DDP, PC-3/DDP: add different concentrations of cisplatin (final concentrations are 20, 50, 100, 200, 400, 1000, 2000, 4000, 8000nM) and different concentrations of sildenafil (final concentrations were 50, 200, 800nM);

(2) HCT-116/L-OHP: Add different concentrations of oxaliplatin (final concentrations are 10, 20, 50, 100, 200, 400, 1000, 2000, 4000nM) and different concentrations of sildenafil (final concentrations were 50, 200, and 800 nM, respectively);

(3) HT-29/CPT-11: adding different concentrations of irinotecan (final concentrations were 5, 10, 20, 50, 100, 200, 400, 1000, 2000 nM) and different concentrations of sildenafil ( The final concentrations were 50, 200, and 800 nM, respectively);

(4) SW620/CAP: add capecitabine at different concentrations (final concentrations are 20, 50, 100, 200, 400, 1000, 2000, 4000, 8000 nM) and sildenafil at different concentrations (final concentrations respectively 50, 200, 800 nM).

At the same time, a blank group (RPIM 1640 medium without cells) and a control group (an equal volume of RPIM 1640 medium) were set up at 37°C (the culture time was determined according to the type of cell line). After the culture was completed, 20 μL of MTT solution ( The final concentration was 0.5 mg/mL), and the culture was continued for 4 h. The supernatant was discarded, 150 μL of DMSO was added to each well, and the absorbance value (OD) of each well was measured at 570 nm with a microplate reader. The half cell inhibitory concentration ( _IC50 ) was calculated.

The results are shown in Table 3.

Table 3 Effects of different drug treatments on IC ₅₀ of drug-resistant cell lines (nM, n=15)

As can be seen from Table 3, 800 nM of sildenafil alone was used for SGC7901/DDP, MCF-7/DDP, HepG2/DDP, PC-3/DDP, HCT-116/L-OHP, HT-29/CPT -11 and SW620/CAP had only weak inhibitory effects on cell proliferation. After combining 800nM sildenafil with cisplatin, oxaliplatin, irinotecan, and capecitabine, the IC _{50 of the corresponding drug-resistant cell lines was significantly lower than the IC 50 of} chemotherapy drugs alone, that is, 800 nM Sildenafil combined with chemotherapeutic drugs can significantly reduce the IC ₅₀ concentration of the latter and enhance the killing effect of chemotherapeutic drugs on the above-mentioned drug-resistant cell lines.

Investigating the sensitization mechanism of sildenafil

This example takes the HT-29/CPT-11 cell line as an example to illustrate the sensitization mechanism of sildenafil, but it should be noted that the sensitization mechanism described in this example is not limited to HT-29/CPT-11 cell line. CPT-11 cell line.

In this example, P-glycoprotein (P-gp), multidrug resistance gene 1 (MDR1) and multidrug resistance gene 1 (MDR1) were detected in the sildenafil-treated HT-29/CPT-11 cell line (the treatment method was the same as that in the above example). ), multidrug resistance-related protein 1 (MRP1), and apoptosis to obtain the sensitization mechanism of sildenafil.

The specific experimental steps include:

(1) Detection of P-glycoprotein (P-gp):

The content of P-glycoprotein (P-gp) was detected by Western blot method. The specific steps were as follows: inoculate HT-29/CPT-11 in 6-well plates and divide them into 4 groups (blank group, sildenafil group with different concentrations ( The final concentrations of sildenafil were respectively 50, 200, and 800 nM)). After incubation for 24 h, the cells were washed with PBS, and fully lysed by adding a cell lysate containing PMSF and phosphatase inhibitors to extract the total protein. After protein quantification, use SDS polyacrylamide gel electrophoresis to separate the target protein, wet transfer membrane, block, incubate the primary antibody (mouse anti-human antibody P-gp (Abcam) by conventional method, 4 ℃ overnight, add the secondary antibody (spicy). Root peroxidase-labeled goat anti-mouse IgG (EARTHOX) was incubated for 1 h, developed and fixed, washed with X-ray film, air-dried, scanned, and the target bands and corresponding internal reference bars were analyzed by Image J 1.44p image processing software. The ratio of optical densities of bands (with GAPDH as an internal reference).

(2) Detection of multidrug resistance gene 1 (MDR1) and multidrug resistance related protein 1 (MRP1):

Western blot was used to detect multidrug resistance gene 1 (MDR1) and multidrug resistance-related protein 1 (MRP1). The specific steps were the same as the detection of P-glycoprotein (P-gp). The primary antibodies were mouse anti-human antibodies. MDR1 and mouse anti-human antibody MRP1.

(3) Detection of cell apoptosis:

HT-29/CPT-11 was inoculated in 96-well plate and divided into 5 groups (blank group, irinotecan group (final concentration 10 μM), sildenafil + irinotecan group (irinotecan final concentration 10 μM, western Denafil final concentration was 50, 200, 800nM)), incubate for 24h, collect cells, wash twice with PBS, collect about 5×10 ⁵ cells, add 500μL of Binding Buffer to suspend, add 5μL of Annexin V-FITC and mix well , and finally add 5 μL Propidium Iodide, gently shake and mix to resuspend the cells, and detect the cell apoptosis by flow cytometry, repeat 6 times, and calculate the average apoptosis rate.

The results are shown in Tables 4 and 5.

Table 4 The effect of sildenafil on the expression of P-gp, MDR1 and MRP1 in HT-29/CPT-11 (n=6)

group	P-gp/GAPDH	MDR1/GAPDH	MRP1/GAPDH
blank group	0.95±0.19	0.79±0.09	0.66±0.11
50nM Sildenafil	0.70±0.15	0.71±0.11	0.58±0.14
200nM Sildenafil	0.59±0.16	0.63±0.10	0.50±0.06
800nM Sildenafil	0.33±0.10	0.52±0.08	0.45±0.07

Note: Compared with the blank group, *P<0.05 in each group.

MDR-related molecular mechanisms are extremely complex, including increased drug efflux, intracellular drug accumulation and redistribution, increased drug detoxification or altered drug target molecules, enhanced DNA damage repair, and inhibition of drug-induced apoptosis. P-gp is a member of the ABC transporter superfamily, which can pump its substrates out of the cell, thereby weakening the drug effect. The high activation of P-gp is the main cause of multidrug resistance in tumor cells; in addition, drug resistance Activation and increased expression of genes such as MDR1 and MRP1 have also been shown to be associated with tumor resistance. According to the results in Table 3, compared with the blank group, 50, 200 and 800 nM sildenafil can significantly reduce the expression of P-gp, and 200 and 800 nM sildenafil can significantly reduce the expression of MDR1, MDR1, HT-29 and CPT-11. The expression of MRP1, the difference was statistically significant (P<0.05). It shows that sildenafil can significantly down-regulate P-gp, MDR1, and MRP1 in HT-29/CPT-11 cells, that is, attenuate P-gp's pumping of chemotherapeutic drugs out of cells, and enhance tumor sensitivity to chemotherapeutic drugs.

Table 5 Comparison of the average apoptosis rate of different groups (%, n=6)

group	Apoptosis rate
blank group	0.66±0.09*
Irinotecan group	23.35±3.24
50nM sildenafil + irinotecan group	25.47±4.12
200nM Sildenafil + Irinotecan Group	30.58±6.49*
800nM Sildenafil + Irinotecan Group	37.43±5.34*

Note: *P<0.05 compared with irinotecan group

As shown in Table 5, compared with the irinotecan group, the addition of 200 and 800 nM sildenafil could significantly increase the apoptosis rate of HT-29/CPT-11, and the differences were statistically significant (P<0.05). It indicated that sildenafil could enhance the apoptosis of HT-29/CPT-11 induced by irinotecan.

In summary, it can be found that sildenafil can achieve drug-resistant cells by reducing the expression of P-glycoprotein, multidrug resistance gene 1 (MDR1), multidrug resistance-related protein 1 and inducing apoptosis. Strain sensitizers to drugs.

Sildenafil enhances the killing effect of oxaliplatin and irinotecan on non-drug-resistant cell line HCT-116 in vivo

In this example, 4-5 week old athymic nude mice inoculated with colorectal cancer cell line HCT-116 were used as animal models to verify that sildenafil enhances the killing effect of oxaliplatin and irinotecan in vivo.

The specific experimental steps are:

HCT-116 was inoculated into a 100 mm petri dish, cultured for 72 h, and the culture medium was removed; digested with 0.25% trypsin, the cells were collected with PBS, and the concentration was adjusted to 1×10 ⁷ /ml. The collected cells were injected into the bilateral armpits of athymic nude mice. One day after the injection, the experimental nude mice were randomly divided into 5 groups with different treatment regimens:

(1) blank control group;

(2) Use oxaliplatin alone, 10 mg/kg, by tail vein injection;

(3) Use irinotecan alone, 20 mg/kg, by tail vein injection;

(4) Sildenafil (10mg/kg) combined with oxaliplatin (10mg/kg), tail vein injection;

(5) Sildenafil (10mg/kg) combined with irinotecan (20mg/kg), injected via tail vein.

Injections were repeated every 3 days for a total of 6 cycles. Tumor diameter (width and length) and mouse body weight were measured every 3 days until animals were sacrificed. After animals were sacrificed, tumor tissue was excised and weighed.

The results are shown in Figure 2. In the in vivo experiment, 10mg/kg sildenafil did not significantly inhibit the growth of non-drug-resistant tumor cells HCT-116 xenografts, and 10mg/kg oxaliplatin alone had no effect on colon cancer HCT. The inhibition rate of -116 cells was 50.7%, while that of the combination of oxaliplatin and sildenafil was 86.9%. The inhibition rate of 20mg/kg irinotecan alone on colon cancer HCT-116 cells was 47.2%, while the inhibition rate of irinotecan and sildenafil combined was 87.4%. The data prove that the combination of sildenafil and oxaliplatin or irinotecan can significantly enhance the killing effect of oxaliplatin or irinotecan on HCT-116 cells in vivo.

Sildenafil enhances the killing effect of oxaliplatin on drug-resistant cell line HCT-116/L-OHP in vivo

In this example, 4-5 weeks old athymic nude mice inoculated with human colon cancer oxaliplatin-resistant cells (HCT-116/L-OHP) were used as animal models to verify that sildenafil enhances oxaliplatin in vivo killing effect.

The specific experimental steps are:

The HCT-116/L-OHP was inoculated into a 100 mm petri dish, cultured for 72 h, and the culture medium was removed; digested with 0.25% trypsin, the cells were collected with PBS, and the concentration was adjusted to 1×10 ⁷ /ml. The collected cells were injected into the bilateral armpits of athymic nude mice. One day after the injection, the experimental nude mice were randomly divided into 3 groups with different treatment regimens:

(1) blank control group;

(2) Use oxaliplatin alone, 10 mg/kg, by tail vein injection;

(3) Sildenafil (10mg/kg) combined with oxaliplatin (10mg/kg), injected via tail vein.

Injections were repeated every 3 days for a total of 6 cycles. Tumor diameter (width and length) and mouse body weight were measured every 3 days until animals were sacrificed. After animals were sacrificed, tumor tissue was excised and weighed.

The results are shown in Figure 3. In the in vivo experiment, 10mg/kg sildenafil did not significantly inhibit the growth of multidrug-resistant tumor cell HCT-116 xenografts, and 10mg/kg oxaliplatin alone had no effect on colon cancer HCT. The inhibition rate of -116/L-OHP cells was 50.7%, while that of the combination of oxaliplatin and sildenafil was 86.9%. The data prove that the combination of sildenafil and oxaliplatin can significantly enhance the killing effect of oxaliplatin on HCT-116/L-OHP cells in vivo.

In summary, in vitro and in vivo studies have shown that sildenafil can significantly reverse the ADR of a variety of tumor-resistant cell lines, and significantly reduce the effect of cisplatin, oxaliplatin, irinotecan, capecitabine and other chemotherapeutic drugs. The mechanism of IC ₅₀ of drug-resistant tumor cell lines may be related to the effects of sildenafil on down-regulating drug-resistant genes and enhancing apoptosis of drug-resistant tumor cells.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

Claims (10)

1. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises a tumor chemotherapy drug, sildenafil or a salt thereof.
2. The pharmaceutical composition according to claim 1, wherein the tumor chemotherapy drug is selected from at least one of platinum, fluorouracil, irinotecan and capecitabine; the tumor chemotherapy drug is preferably platinum , at least one of fluorouracil, irinotecan and capecitabine; the platinum agent is selected from at least one of cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin, and lobaplatin.
3. The pharmaceutical composition according to claim 1 or 2, wherein the tumor is selected from at least one of colorectal cancer, gastric cancer, liver cancer, breast cancer and prostate cancer.
4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant; the pharmaceutically acceptable adjuvant preferably comprises a diluent, an absorbent, One or more of wetting agents, binders, disintegrating agents, lubricants, flavoring agents or transdermal absorption enhancers.
5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the dosage form of the pharmaceutical composition is an oral preparation, an injection preparation or a transdermal preparation.
6. Use of sildenafil or a salt thereof as an antitumor drug sensitizer in preparing a pharmaceutical composition for treating tumors.
7. The use according to claim 6, wherein the salt of sildenafil is sildenafil citrate.
8. The use according to claim 6, wherein the antitumor drug is selected from at least one of platinum, fluorouracil, irinotecan and capecitabine; the antitumor drug is preferably platinum, fluorouracil , at least one of irinotecan and capecitabine; the platinum agent is selected from at least one of cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin, and lobaplatin.
9. Use of the pharmaceutical composition according to any one of claims 1-5 in the preparation of a medicament for treating tumors.
10. The use according to claim 9, wherein the dosage of sildenafil or its salt is 0.5-20 mg/day.

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