CN106491603A - A kind of pharmaceutical composition and its application containing capsaicin and Sorafenib - Google Patents

Abstract

The invention belongs to field of antineoplastic medicaments, and in particular to a kind of pharmaceutical composition containing capsaicin and Sorafenib.In described pharmaceutical composition, the mol ratio of capsaicin and Sorafenib is 5:1～10:1.Sorafenib is unique hepatocarcinoma system chemotherapeutics in current global range, but its curative effect needs further to be improved.The pharmaceutical composition can substantially suppress hepatoma cell proliferation, inducing apoptosis of tumour cell, and both use in conjunction show good synergism.

Description

A kind of pharmaceutical composition containing capsaicin and Sorafenib and its application

technical field

The invention relates to the field of antitumor drugs, in particular to a pharmaceutical composition containing capsaicin and sorafenib and application thereof.

Background technique

Primary liver cancer is one of the most common malignant tumors in the world, and its morbidity and mortality are increasing year by year, seriously threatening human health. In China, the morbidity and mortality of liver cancer are particularly severe due to the presence of serious cancer-causing risk factors such as hepatitis B virus infection. According to a statistical report released by the World Health Organization in 2014, the number of new cases of liver cancer in China exceeded 400,000 in 2012, accounting for about 55% of the total number of new cases worldwide. The onset of liver cancer is hidden, and most patients are already in the middle and advanced stages when they are first diagnosed, and the best time for surgical treatment is lost. Local therapies such as radiofrequency ablation and hepatic arterial chemoembolization are also limited in addressing micrometastases. Therefore, systemic chemotherapy is an urgently needed treatment in clinic.

Sorafenib, a targeted drug, is a systemic chemotherapy drug for liver cancer and kidney cancer that has been approved for clinical use in the United States, the European Union, and China in recent years. Sorafenib plays an anti-tumor effect through a dual mechanism: on the one hand, it directly inhibits tumor growth by inhibiting the RAF/MEK/ERK signaling pathway of tumor cells; And platelet-derived growth factor receptor (PDGFR) to block the formation of tumor angiogenesis, and indirectly inhibit the growth of tumor cells. Sorafenib is a milestone discovery in the molecular targeted drug therapy of liver cancer, and it plays an irreplaceable role in the comprehensive treatment of advanced liver cancer. Nevertheless, the clinical efficacy of sorafenib is not quite satisfactory. Clinical practice has found that liver cancer has different degrees of congenital drug resistance to sorafenib, and only 43% of patients treated with sorafenib can be controlled in the short term. Therefore, it is of great significance to explore drugs that can enhance the curative effect of sorafenib, especially naturally occurring medicinal and food homologous substances that have little toxic and side effects on the human body.

CN103933039A discloses a composition of sorafenib and a natural medicine silybin, both of which have synergistic effect on inhibiting the growth and proliferation of liver cancer cells. CN102836160A discloses a composition of sorafenib and a chemically synthesized small molecular substance ABT-263, the combination of the two has a synergistic and synergistic effect on the anti-tumor effect.

Pepper is a vegetable and condiment widely eaten by people. Its pungentness comes from the capsaicinoids contained therein, including capsaicin, dihydrocasaicin, and nordihydrocapsaicin. Wait. Capsaicin is the component with the highest content in capsaicinoids, accounting for about 69% of the total. Capsaicin has many pharmacological activities and can be used for analgesia and anti-inflammation. Studies in recent years have shown that capsaicin also has certain anti-tumor activity, and can inhibit the proliferation and induce apoptosis of malignant tumor cells such as liver cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer and bladder cancer. The anti-tumor effect of capsaicin is related to the following factors: increasing cell membrane permeability, inhibiting mitochondrial respiration; up-regulating the expression of anti-cancer gene p53 and inhibiting the cell signal transduction pathway JNK; increasing the content of intracellular reactive oxygen species.

CN104447777A discloses a capsaicin-camptothecin anticancer drug conjugate, which can directly release capsaicin and camptothecin drugs in the body through hydrolysis, and the two have synergistic antitumor effects. CN104983900A discloses that the combined use of capsicum extract and procyanidin can prevent nausea and vomiting caused by cancer chemotherapy, and at the same time play an auxiliary role in inhibiting tumors.

At present, there is no report on the combination of capsaicin and sorafenib in the treatment of malignant tumors.

Contents of the invention

Purpose of the invention: In order to solve the problems in the prior art, the present invention proposes a pharmaceutical composition containing capsaicin and Sorafenib and its application, which improves the antitumor effect.

Technical scheme: in order to realize above-mentioned technical purpose, the present invention proposes a kind of pharmaceutical composition containing capsaicin and Sorafenib, wherein, the mol ratio of capsaicin and Sorafenib in described pharmaceutical composition is 5:1 ~10:1.

As a preference, in the pharmaceutical composition containing capsaicin and sorafenib, the molar ratio of capsaicin and sorafenib is 10:1.

Wherein, the pharmaceutical composition containing capsaicin and sorafenib also includes a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier may be a pharmaceutically acceptable carrier known to those skilled in the art.

The present invention also proposes the use of the above pharmaceutical composition in the preparation of medicines for treating malignant tumors.

Wherein, the malignant tumor is any one of primary liver cancer and renal cancer.

The pharmaceutical composition of the present invention can be administered in various oral dosage forms commonly used in pharmacy. The above-mentioned various oral dosage forms commonly used in pharmacy can be tablets, capsules, granules, oral liquids and the like.

In addition, the single active drug or the pharmaceutical composition in the pharmaceutical composition can also be prepared into sustained and controlled release preparations, such as sustained release tablets, pellets or controlled release tablets, according to conventional methods.

Pharmacological tests show that the pharmaceutical composition containing capsaicin and sorafenib of the present invention has the function of synergistically treating malignant tumors such as primary liver cancer, and the malignant tumors are preferably primary liver cancer and renal cancer.

Beneficial effect: Compared with the prior art, the anti-tumor effect of the medicinal composition containing capsaicin and Sorafenib of the present invention is significantly stronger than that of a single drug, and the combined use of capsaicin and Sorafenib can make the drug effect produce synergy. In particular, when the molar ratio of capsaicin and sorafenib is 10:1, the synergistic effect of the two is the best.

Description of drawings

Figure 1 Inhibitory effect of sorafenib combined with capsaicin on tumor cell proliferation

Figure 2 Effect of sorafenib combined with capsaicin on tumor cell morphology

Figure 3 Effect of sorafenib combined with capsaicin on tumor cell Caspase 3/7 activity

detailed description

The sources of tumor cell lines used in the following examples are as follows: liver cancer cells PLC/PRF/5 and kidney cancer cells OS-RC-2 are from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences.

Drugs and reagents: Sorafenib was purchased from Selleck Chemicals in the United States; capsaicin was purchased from Tocris in the United Kingdom. Both drugs were dissolved in dimethyl sulfoxide (DMSO), stored at -80°C, and diluted to working concentrations during experiments (DMSO concentration <0.5%). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Hoechst33258, Propidium Iodide (PI) and Caspase 3/7 detection reagents were purchased from ThermoFisher, USA.

Example 1 Determination of the half inhibitory concentration (IC ₅₀ ) of sorafenib and capsaicin on tumor cells.

In this example, MTT method was used to measure the inhibitory effect of capsaicin and sorafenib on liver cancer cells PLC/PRF/5 and kidney cancer cells OS-RC-2. The cells were inoculated in a 96-well culture plate at a concentration of 5×10 ⁴ /mL, 100 μL per well, and the drug was added after culturing overnight for the cells to adhere to the wall. Five drug concentrations were set for sorafenib, which were 0.3, 1, 3, 10, and 30 μM; five drug concentrations were set for capsaicin, which were 10, 30, 100, 300, and 1000 μM. After the cells and the drug solution were incubated in the incubator for 72 hours, add 10 μL of MTT solution (5 mg/mL) to each well, incubate at 37°C for 4 hours, discard the medium, add 150 μL DMSO to each well, place at 37°C for 1 hour, and then use the enzyme Measure the absorbance (OD) of each well at 490nm with a standard instrument, and calculate the inhibitory rate of the drug on the cells. The calculation formula of cell inhibition rate is: inhibition rate (%)=(OD value of control group-OD value of test group)/(OD value of control group-OD value of blank group)×100%.

The results showed that both sorafenib and capsaicin could inhibit the proliferation of tumor cells in a dose-dependent manner. The half maximal inhibitory concentrations of sorafenib to PLC/PRF/5 and OS-RC-2 cells were 7.6 and 17.0 μM, respectively. The half inhibitory concentrations of capsaicin on the proliferation of PLC/PRF/5 and OS-RC-2 cells were 137.0 and 169.8 μM, respectively.

Example 2 Inhibitory effect of sorafenib combined with capsaicin on tumor cell proliferation.

The PLC/PRF/5 and OS-RC-2 cells in the logarithmic growth phase were inoculated in a 96-well culture plate at a concentration of 5×10 ⁴ /mL, 100 μL per well, and the drugs were added after the cells were cultured overnight. The experimental groups were as follows: negative control group, capsaicin group, sorafenib group, capsaicin and sorafenib combined administration group. The capsaicin group was given 10, 50, and 100 μM capsaicin respectively; the sorafenib group was given 10 μM sorafenib; Rafenib (combined drug group 1: capsaicin 10 μM + sorafenib 10 μM; combined drug group 2: capsaicin 50 μM + sorafenib 10 μM; combined drug group 3: capsaicin 100 μM + sorafenib 10 μM). After the cells and the drug solution were incubated in the incubator for 72 hours, add 10 μL of MTT solution (5 mg/mL) to each well, incubate at 37°C for 4 hours, discard the medium, add 150 μL DMSO to each well, place at 37°C for 1 hour, and then use the enzyme The absorbance of each well was measured by a standard instrument at 490 nm, and the inhibitory rate of the drug on tumor cell proliferation was calculated. The type of interaction between Sorafenib and capsaicin (antagonism, additive or synergistic effect) was determined by Chou-Talalay combination index method. CompuSyn software was used to calculate the drug combination index (combination index, CI). When the CI value was in the range of 0.90-1.10, it was judged as additive effect, when the CI value was less than 0.90, it was judged as synergistic effect, and when the CI value was greater than 1.1, it was judged as synergistic effect. antagonism.

The experimental results are shown in Figure 1. When the concentration of capsaicin was 10 μM, it could not significantly enhance the inhibitory effect of Sorafenib on PLC/PRF/5 and OS-RC-2 cells (combined drug group 1, p>0.05), but in At 50 and 100 μM, the antiproliferative effect of sorafenib can be significantly enhanced (combined drug group 2 and group 3, p<0.01), suggesting that when the molar ratio of capsaicin to sorafenib is 5:1-10:1 , the curative effect of the pharmaceutical composition can be significantly better than that of a single drug. The combined effect index CI of the two drugs was further calculated, and the results were as follows: in PLC/PRF/5 cells, when the molar ratio of capsaicin and Sorafenib was 5:1 and 10:1, the CI values were 1.09 and 0.55, respectively, It is suggested that the former is an additive effect and the latter is a synergistic effect; in OS-RC-2 cells, when the molar ratio of capsaicin and sorafenib is 5:1 and 10:1, the CI values are 0.43 and 0.32, respectively, suggesting that in The drug interactions at these two ratios are synergistic effects, but the synergistic effect of the latter is more obvious. The above results show that when the molar ratio of capsaicin and sorafenib is 5:1, an additive or synergistic anti-tumor effect can be obtained, and when the molar ratio is 10:1, a synergistic antitumor effect can be obtained on both cells. tumor effect.

Example 3 Effect of capsaicin combined with sorafenib on tumor cell morphology.

The PLC/PRF/5 and OS-RC-2 cells in the logarithmic growth phase were inoculated in a 96-well culture plate at a concentration of 1×10 ⁵ /mL, 100 μL per well, and the drugs were added after the cells were cultured overnight. The experimental groups were as follows: negative control group, capsaicin group, sorafenib group, capsaicin and sorafenib combined administration group. The capsaicin group was given 100 μM capsaicin; the sorafenib group was given 10 μM sorafenib; the combination group was given 100 μM capsaicin and 10 μM sorafenib. After the cells were incubated with the drug for 24 hours in the incubator, the morphology of the cells was observed with an inverted phase-contrast microscope.

The photos of liver cancer PLC/PRF/5 cells treated with different drugs are shown in Figure 2A: the cells in the negative control group spread naturally on the bottom of the well, with a soft and plump appearance and normal shape; in the sorafenib treatment group, the cells shrank and some cells Detachment, black granular substances in the cytoplasm increased and vacuoles appeared, suggesting that the cells showed signs of apoptosis under the action of drugs; in the capsaicin treatment group, the granular substances in the cells increased, and some cell membranes shrank; In the combination treatment group of glucoside and sorafenib, a large number of cells detached and became round, and some cells were broken, suggesting that the cells undergo apoptosis. The morphological changes of kidney cancer OS-RC-2 cells after drug treatment were similar to those of PLC/PRF/5 cells. The results of this experiment showed that capsaicin combined with sorafenib had the greatest effect on the appearance of tumor cells, and suggested that cells may undergo apoptosis under drug treatment.

Example 4 Sorafenib combined with capsaicin induces tumor cell apoptosis.

Hoechst 33258 and PI double staining were used to detect the effects of sorafenib, capsaicin and their combination on the apoptosis of PLC/PRF/5 cells. The PLC/PRF/5 cells were inoculated in a 24-well culture plate at a concentration of 5×10 ⁴ /mL, 2 mL per well, cultured overnight, and the drug was added after the cells adhered to the wall. The dosing regimen was designed as follows: the capsaicin group was given 100 μM capsaicin; the sorafenib group was given 10 μM sorafenib; the combined drug group was given 100 μM capsaicin and 10 μM sorafenib. The cells were incubated with the drug in the incubator for 24 hours, the cells were collected, stained with Hoechst 33258 and PI, respectively, and then observed with an immunofluorescence microscope, randomly photographed in 5 different areas, counted the number of apoptotic cells and the total number of cells, Calculate the apoptosis rate.

The results of Hoechst 33258 and PI double staining experiments showed that the apoptosis rate of negative control group was 0.41%, that of capsaicin group was 4.22%, that of sorafenib group was 9.76%, and that of capsaicin and Sola The apoptosis rate of the fenib combined group was 30.58%. The apoptosis rate of the combined drug group was significantly higher than that of the sorafenib group and the capsaicin group (p<0.05).

Example 5 Effect of capsaicin combined with sorafenib on the activity of apoptosis-related protein Caspase 3/7

Caspase protein cascade activation can directly lead to apoptosis. In this example, fluorescence microscopy was used to detect the effect of capsaicin combined with sorafenib on the activity of Caspase 3/7 in liver cancer PLC/PRF/5 cells. The PLC/PRF/5 cells were inoculated in a 24-well culture plate at a concentration of 2.5×10 ⁵ /mL, 400 μL per well, cultured overnight, and the drug was added after the cells adhered to the wall. The dosing regimen was designed as follows: the drug concentration of the capsaicin group was 100 μM; the drug concentration of the sorafenib group was 10 μM; the combined drug group added both capsaicin and sorafenib (same concentration as above). Cells and drugs were incubated in an incubator for 24 hours, the medium was discarded, and Caspase 3/7 detection reagent CellEvent ^TM Caspase-3/7Green Detection Reagent was added, incubated at 37°C for 20 minutes, and observed under a fluorescence microscope. Cells activated by Caspase 3/7 showed green fluorescence, and 5 fields of view were randomly selected from each well, counted and averaged.

The results are shown in Figure 3, the number of cells showing green fluorescence in the combined drug group was significantly more than that in the sorafenib or capsaicin alone group (p<0.05), suggesting that the combined drug can more effectively activate the PLC/PRF/5 cells. Caspase 3/7, thereby inducing cell apoptosis.

Claims (5)

1. a kind of pharmaceutical composition containing capsaicin and Sorafenib, it is characterised in that in described pharmaceutical composition capsaicin and The mol ratio of Sorafenib is 5:1~10:1.

2. the pharmaceutical composition described in claim 1, it is characterised in that the mol ratio of wherein capsaicin and Sorafenib is 10: 1.

3. the pharmaceutical composition described in claim 1 and 2, it is characterised in that described pharmaceutical composition also includes pharmaceutically connecing The carrier that receives.

4. the pharmaceutical composition described in claim 1 and 2 prepare treatment malignant tumor medicine on purposes.

5. the pharmaceutical composition described in claim 1, prepare for treat primary hepatocarcinoma and renal carcinoma medicine on purposes.