CN107951888A - The drug regimen and its application of Afatinib and 10-hydroxycamptothecine - Google Patents

Abstract

The present invention relates to a pharmaceutical combination of Afatinib and 10-hydroxycamptothecin and its application, wherein the pharmaceutical composition contains Afatinib and 10-hydroxycamptothecin, and the afatinib and 10-hydroxycamptothecin The molar ratio of 10‑hydroxycamptothecin is 1:2. The present invention relates to a kind of medicine combination combination, and its described medicine composition contains afatinib and 10-hydroxycamptothecin. When the pharmaceutical composition of the invention is used to treat non-small cell lung cancer with EGFR positive mutation, it can obtain better drug effect than that used alone, has a sensitizing effect, and provides a scientific basis for developing new anticancer drugs.

Description

Drug Combination of Afatinib and 10-Hydroxycamptothecin and Its Application

technical field

The present invention relates to an antitumor pharmaceutical composition, relates to the pharmaceutical combination of Afatinib and HCPT and its preparation for treating lung cancer, pancreatic cancer, colon cancer, liver cancer, prostate cancer, kidney cancer, gastric cancer, brain tumor, sarcoma, ovarian cancer or breast cancer Application in cancer medicine.

Background technique

Cancer is one of the major diseases threatening human health. According to literature reports, in China, cancer has caused 18 million deaths in 2015, and the death toll will continue to rise. Most cancer deaths are caused by cancers of the lung, stomach, liver, colon and breast. Currently, there are mainly the following methods for cancer treatment, surgery, radiotherapy, chemotherapy and targeted therapy. Chemotherapy and targeted therapy are the main treatment methods. However, the drugs used in chemotherapy often restrict their application due to problems such as poor solubility and strong side effects. Targeted therapy often cannot obtain better therapeutic effects due to problems such as high price and drug resistance. Therefore, how to make drugs achieve the purpose of synergizing and reducing toxicity, enhancing targeting and reducing drug resistance has become the focus of cancer treatment. One of the most effective methods is to use drugs with different mechanisms of action and targets for combination therapy.

Afatinib is an oral, dual irreversible ErbB family blocker, which can block EGFR, Her2, ErbB3 and ErbB4 signaling pathways. It is a second-generation EGFR-TKI. Unlike the first-generation targeted drugs, it can irreversibly It selectively and completely inhibits ErbB receptor signaling and, more importantly, is able to antagonize HER-1 overexpression secondary to the point mutation T790M, which often results in resistance to gefitinib and erlotinib. Afatinib is suitable for the first-line treatment of advanced non-small cell lung cancer and HER2 mutation-positive advanced breast cancer patients.

HCPT is a trace alkaloid extracted from Camptotheca acuminate Decne, a specific inhibitor of DNA topoisomerase I (Topo-I), which can cause double-strand DNA breaks in vivo, thereby controlling DNA replication, blocking RNA synthesis, interfering with cell division cycle. The difference from camptothecin is that it is mainly excreted in bile and rarely excreted in urine, so urinary system toxicity is rare. HCPT has a broad anti-tumor spectrum, and has no cross-resistance with other commonly used anti-tumor drugs, and has a therapeutic effect on drug-resistant tumors.

So far, there is no report about the combined use of Afatinib and HCPT in anti-tumor and the synergistic effect of the two in anti-tumor.

Contents of the invention

The object of the present invention is to provide a highly active antitumor pharmaceutical composition and its application in the preparation of medicines for treating lung cancer.

The purpose of the present invention is achieved through the following technical solutions:

An antitumor pharmaceutical composition, the active ingredients of which include afatinib and 10-hydroxycamptothecin.

Moreover, the molar ratio of afatinib to 10-hydroxycamptothecin is 1:2.

Moreover, the concentrations of afatinib and 10-hydroxycamptothecin are 0.625-5 μM and 1.25-10 μM, respectively.

Moreover, the dilution solvent of the afatinib and 10-hydroxycamptothecin is dimethyl sulfoxide.

Moreover, the anti-tumor pharmaceutical composition is used for the preparation of pharmaceutical preparations for treating EGFR positive mutation non-small cell lung cancer.

Moreover, the application of the anti-tumor pharmaceutical composition in the preparation of medicines for treating lung cancer, pancreatic cancer, colon cancer, liver cancer, prostate cancer, kidney cancer, gastric cancer, brain tumor, sarcoma, ovarian cancer or breast cancer.

The advantages and effects of the present invention are as follows:

The pharmaceutical composition of the invention has remarkable synergistic effect in treating lung cancer, improves the curative effect of the medicine, reduces the dosage of medicine and reduces the occurrence of side effects.

The invention relates to a medicine combination combination, the medicine composition of which contains afatinib (Afatinib) and 10-hydroxycamptothecin (HCPT). When the pharmaceutical composition of the invention is used to treat non-small cell lung cancer with EGFR positive mutation, it can obtain better drug effect than that used alone, has a sensitizing effect, and provides a scientific basis for developing new anticancer drugs.

Description of drawings

Figure 1 is a graph showing the inhibition of the proliferation of H1975 cells when the two drugs are used alone or in combination at a ratio of Afatinib:HCPT (1:1).

Figure 2 shows the CI value of the combined ratio of Afatinib:HCPT (1:1) on H1975 cells.

Figure 3 is a graph showing the inhibition of the proliferation of H1975 cells when the two drugs are used alone or in combination at the ratio of Afatinib:HCPT (2:1).

Figure 4 shows the CI value of the combination ratio of Afatinib:HCPT (1:2) acting on H1975 cells.

Figure 5 is a graph showing the inhibition of the proliferation of H1975 cells when the two drugs are used alone or in combination at a ratio of Afatinib:HCPT (1:2).

Figure 6 shows the CI value of the combined ratio of Afatinib:HCPT (1:2) acting on H1975 cells.

Figure 7 is a graph showing the inhibition of the proliferation of H1650 cells when the two drugs are used alone or in combination at the ratio of Afatinib:HCPT (5:1).

Figure 8 shows the CI value of the combined ratio of Afatinib:HCPT (5:1) on H1650 cells.

Figure 9 is a graph showing the inhibition of the proliferation of H1650 cells when the two drugs are used alone or in combination at the ratio of Afatinib:HCPT (10:1).

Figure 10 shows the CI value of the combined ratio of Afatinib:HCPT (10:1) on H1650 cells.

Figure 11 is a graph showing the inhibition of the proliferation of H1650 cells when the two drugs are used alone or in combination at a ratio of Afatinib:HCPT (1:2).

Figure 12 shows the CI value of the combined ratio of Afatinib:HCPT (1:2) on H1650 cells.

Detailed ways

Below in conjunction with embodiment the present invention will be further elaborated, but the present invention is not limited thereto, under the situation of not departing from the idea and purpose of the present invention, any change that the technical scheme of the present invention is done should fall into the present invention. within the scope defined by the claims.

Antitumor pharmaceutical composition of the present invention contains and Afatinib and HCPT

The structural formulas of Afatinib and HCPT are shown in I and II

The molar ratio of Afatinib to HCPT is preferably 0.625-5:10.

In the antitumor pharmaceutical composition of the present invention, preferably, the effective concentrations of the Afatinib and HCPT are 0.625-5 μM and 1.25-10 μM, respectively.

In the antitumor pharmaceutical composition of the present invention, preferably, the dilution solvent of the Afatinib and HCPT is dimethyl sulfoxide.

The antitumor pharmaceutical composition of the invention can be applied to the treatment of lung cancer.

For the application of the anti-tumor pharmaceutical composition, preferably, the Afatinib and HCPT are added simultaneously, and more preferably, they act for 48 hours after the simultaneous addition.

Afatinib or its derivatives in the antineoplastic pharmaceutical composition of the present invention can be directly mixed with HCPT or its derivatives to make preparations; respectively mixed with corresponding auxiliary materials to make preparations, and then packaged or combined in a conventional manner in the art Together, or separately mixed with the corresponding auxiliary materials, and then mixed to make a preparation. The adjuvant used in the preparation can be the conventional adjuvant in the field, provided that it does not react with the pharmaceutical composition of the present invention or affect the curative effect of the pharmaceutical composition of the present invention.

According to different preparation forms and preparation specifications, the content of the pharmaceutical composition of the present invention in the preparation can be adjusted between 1-99wt%, preferably 10-99wt%. In addition, the dosage of the pharmaceutical composition of the present invention can be appropriately changed according to the subject of administration, the route of administration or the formulation of the drug, but the premise is to ensure that the pharmaceutical composition can achieve an effective blood concentration in mammals.

The biological material, medicine and experimental method used in the specific embodiment are as follows:

Cells: Human lung adenocarcinoma cells H1650 and human lung adenocarcinoma cells H1975 were purchased from Peking Union Medical College Cell Resource Center.

Drugs: Accurately weigh HCPT standard products (Dalian Meilun Biotechnology Co., Ltd.), Afatinib standard products (Dalian Meilun Biotechnology Co., Ltd.). Dissolve them in dimethyl sulfoxide respectively to prepare a stock solution with a concentration of HCPT of 100mmol/L and a concentration of Afatinib of 100mmol/L, and store them at -20°C. Dilute to the appropriate concentration listed in the table for use.

MTT detection of cell proliferation: cells were cultured in RPMI1640 cell culture medium containing 1% penicillin-streptomycin solution (double antibody) and 10% fetal bovine serum in a 5% CO2 incubator at 37°C. . The cells were trypsinized and counted with a hemocytometer. Inoculate 96-well cell culture plate (concentration: 5×104cell/mL) according to the volume of each well of 100 μL, cultivate in 37°C, 5% CO2 incubator for 24h, then add different concentration gradient samples to be tested, place at 37°C, 5 Cultivate for 48 hours in a %CO2 constant temperature incubator. Add 20 μL of 5 mg/mL MTT solution (prepared in PBS, sterilized by filtration with a 0.22 μm filter) to each well, place in a 37°C, 5% CO2 constant temperature incubator and continue to incubate for 4 hours to terminate the culture. Carefully remove the culture supernatant in the wells, add 100 μL DMSO to each well, and place at 37°C for 10 min to fully dissolve the purple crystals, and measure the absorbance (OD) value of each well with a microplate reader (492nm, 630nm).

Cell survival rate (%)=(experimental group OD-blank group OD)/(control group OD-blank group OD)×100%

Calculation of Combined Index (CI): Calculated by CompuSyn software

Example 1

Dilute Afatinib and HCPT with dimethyl sulfoxide respectively to the concentrations in the table. The experimental results of the inhibitory effect on proliferation of different concentrations of Afatinib and HCPT when used alone are shown in the figure and table.

Table 1 Calculation of H1650 cell viability at different dosages of Afatinib.

Concentration of Afatinib (μM) Cell viability (100%) 0.625 90.57±6.41 1.25 86.07±4.68 2.5 73.03±4.84 5 39.31±5.74 Concentration of Afatinib (μM) Cell viability (100%) 0.125 101.16±3.45 0.25 101.31±2.62 0.5 93.49±4.03 1 87.66±4.22 2 77.50±3.45

Table 2 Calculation of H1650 cell viability at different dosages of HCPT.

HCPT concentration (μM) Cell viability (100%) 0.025 101.34±4.5 0.05 94.94±2.03 0.1 68±5.55 0.2 59.35±0.96

Table 3 Cell survival rate and CI value when Afatinib was combined with HCPT.

Combination drugs can have synergistic effect, antagonistic effect and additive effect. Generally, the combination index CI is used to judge the specific effect. CI<1 means that the combination of the two drugs has a synergistic effect; CI=1 means that the combination of the two drugs has an additive effect; CI>1 means that the combination of the two drugs has an antagonistic effect. It can be seen from the above results that when Afatinib and HCPT are administered at the same time, the CI value can reach <1 at a certain dose ratio, and at this time, the combined use of the two has a better synergistic effect on the killing effect of H1650 cells.

Example 2

Dilute Afatinib and HCPT with dimethyl sulfoxide respectively to the concentrations in the table. The experimental results of the inhibitory effect on proliferation of different concentrations of Afatinib and HCPT when used alone are shown in the figure and table.

Table 1 Calculation of the H1975 cell viability of Afatinib at different dosages.

Concentration of Afatinib (μM) Cell viability (100%) 0.625 77.39±7.57 1.25 51.22±4.51 2.5 27.37±3.3 5 16.88±1.51

Concentration of Afatinib (μM) Cell viability (100%) 0.25 85.94±3.83 0.5 79.02±1.5 1 51.44±3.84 2 42.94±1.45 4 20.37±3.31

Table 2 Calculation of H1975 cell viability at different dosages of HCPT.

HCPT concentration (μM) Cell viability (100%) 1.25 52.02±3.1 2.5 36.96±5.24 5 28.24±2.72 10 22.48±2.49

HCPT concentration (μM) Cell viability (100%) 0.25 71.41±3.72 0.5 66.18±3.81 1 57.66±3.67 2 42.04±6.7

Table 3 Cell survival rate and CI value when Afatinib was combined with HCPT.

Combination drugs can have synergistic effect, antagonistic effect and additive effect. Generally, the combination index CI is used to judge the specific effect. CI<1 means that the combination of the two drugs has a synergistic effect; CI=1 means that the combination of the two drugs has an additive effect; CI>1 means that the combination of the two drugs has an antagonistic effect. It can be seen from the above results that when Afatinib and HCPT are administered at the same time, the CI value can reach <1 at a certain dose ratio. At this time, the combined use of the two has a better synergistic effect on the killing effect of H1975 cells.

Claims (6)

1. An antitumor pharmaceutical composition, characterized in that: the active ingredient of the composition comprises afatinib and 10-hydroxycamptothecin. 2. The antitumor pharmaceutical composition according to claim 1, characterized in that: the molar ratio of the afatinib to 10-hydroxycamptothecin is 1:2. 3. The antitumor pharmaceutical composition according to claim 1, characterized in that: the concentrations of the afatinib and 10-hydroxycamptothecin are 0.625-5 μM and 1.25-10 μM, respectively. 4. The antineoplastic pharmaceutical composition according to claim 1, characterized in that: the dilution solvent of the afatinib and 10-hydroxycamptothecin is dimethyl sulfoxide. 5. The application of the antitumor pharmaceutical composition according to any one of claims 1-4 for preparing a pharmaceutical preparation for treating non-small cell lung cancer with EGFR positive mutation. 6. The antitumor pharmaceutical composition described in any one of claims 1-4 is used in the preparation treatment of lung cancer, pancreatic cancer, colon cancer, liver cancer, prostate cancer, kidney cancer, gastric cancer, brain tumor, sarcoma, ovarian cancer or breast cancer application in medicines.