CN108187055B - Anticancer composition with synergistic effect - Google Patents

Abstract

The invention relates to an anti-cancer composition with a synergistic effect, in particular to a composition comprising effective amounts of at least one VEGF/VEGFR inhibitor and 2-position (containing an N-aryl piperazine structure) substituted quinazoline derivative. The composition of the invention can obviously inhibit the growth of tumor cells, reduce the use of anticancer drugs, reduce the treatment risk and the cost, and has wide application prospect.

Description

Anticancer composition with synergistic effect

Technical Field

The invention relates to an anticancer composition with a synergistic effect, a preparation method and application thereof.

Background

Cancer, also known as malignant tumor, is caused by abnormal proliferation of cells. These abnormally proliferating cells invade other parts of the body. Current traditional methods of treating cancer include surgery, radiation therapy, chemotherapy immunotherapy, gene therapy, and the like. The reasons influencing the occurrence and development of the tumor are very complex, and drug resistance and tumor recurrence are easy to generate in the treatment process, so that the tumor treatment difficulty is higher.

Only a small fraction of tumor cells in the whole tumor tissue are tumorigenic, i.e., tumor stem cells (CSCs), and the generation of CSCs is closely related to the self-renewal capacity of Cancer cells, traditional chemotherapy resistance, tumor recurrence and metastasis, and the like. Sunitinib (SN) is a small molecular TKI with multiple target points, and the target points include Vascular Endothelial Growth Factor (VEGFR), platelet Endothelial Growth Factor (PDGFR), etc., but SN has poor clinical effect on treating breast cancer, and SN induces hypoxia in tumor to increase the number of CSCs.

In recent years, research shows that DA can reduce the proportion of tumor stem cells by exciting Dopamine D1 receptor (D1Dopamine receptor, D1DR) so as to increase the efficacy of SN in treating tumors. 2-position (containing N-aryl piperazine structure) substituted quinazoline derivatives C2 and C17 have strong affinity with D1DR, but no relevant report of the effect of the compounds on tumors is found.

Disclosure of Invention

The inventor finds that 2-site (containing N-aryl piperazine structure) substituted quinazoline derivatives have strong affinity with D1DR in the process of tumor treatment, and the combination of the derivatives and SN can remarkably enhance the tumor inhibition effect of the SN.

The inventor develops the inhibition effect of the compound and SN on the in-vitro proliferation of two pancreatic cancer cells in the initial stage of research, and the result shows that the synergy index of the two medicines is less than 1 after the two medicines are combined, which indicates that the two medicines have the synergy effect (see example 1). And the combination of the two drugs can significantly reduce the colony forming ability of the tumor cells (see example 4). In vivo experiments, the compound in the human pancreatic cancer xenograft tumor model can be combined with SN to obviously enhance the anti-tumor effect of the SN, and has no obvious drug toxicity.

Accordingly, it is an object of the present invention to provide an anticancer composition having a synergistic effect, comprising an effective amount of at least one anticancer drug which is a VEGF/VEGFR inhibitor and an effective amount of a non-anticancer drug; the non-anticancer drug is a quinazoline derivative substituted at 2-position (containing an N-aryl piperazine structure); the 2-substituted quinazoline derivative (containing an N-aryl piperazine structure) can generate a synergistic effect on the treatment effect of the anti-cancer drug taking the vascular endothelial growth factor VEGF or the receptor thereof as a target.

According to one aspect of the present invention, the VEGF/VEGFR inhibitor is preferably a small molecule VEGFR inhibitor and a monoclonal antibody against VEGF, wherein the small molecule VEGFR inhibitor is preferably Sorafenib (Sorafenib, Bayer/Onyx, 2005), Sunitinib (Sunitinib, pFizer, 2006), Pazopanib (Pazopanib, GSK, 2009), Axitinib (Axitinib, pFizer, 2012) or one or more of their pharmaceutically acceptable salts, and most preferably Sunitinib (Sunitinib, SN) or its pharmaceutically acceptable salts. The monoclonal antibody to VEGF is preferably Bevacizumab (Bevacizumab, Genentech,2004) or Ranibizumab (Ranibizumab, Genentech, 2006).

The pharmaceutically acceptable salts are synthesized by conventional chemical methods from the parent compound, which comprises a basic or acidic moiety. In general, the salts are prepared, for example, by reacting the free acid or base forms of these compounds with a stoichiometric equivalent of the appropriate base or acid in water or in an organic solvent or in a mixture of water and an organic solvent. Generally, nonaqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of the acid addition salts include inorganic acid addition salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and organic acid addition salts such as acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, analgin, and p-toluenesulfonate. Examples of base addition salts include inorganic salts such as sodium, potassium, calcium and ammonium salts, and organic alkali metal salts such as ethylenediamine, ethanolamine, N-dialkyleneethanolamine, triethanolamine and basic amino acid salts.

According to one aspect of the invention, the cancer is meant to encompass tumors, neoplasias and any other malignant tissue or cell. Specifically included are colon or rectal cancer, metastatic colorectal cancer, lung cancer, non-squamous non-small cell lung cancer, breast cancer, metastatic HER2 negative breast cancer, brain cancer, adult glioblastoma, childhood resistant glioblastoma, glioma, ependymoma, astrocytoma, medulloblastoma, childhood medulloblastoma, glioma, oligodendroglioma or meningioma, kidney cancer such as advanced renal cell carcinoma, bladder cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, gastric cancer, head and neck cancer, liver cancer, lung cancer (small cell lung cancer and non-small cell lung cancer), squamous non-small cell lung cancer, melanoma, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, salivary gland cancer, or spindle cell cancer.

According to one aspect of the invention, the effective amount of the at least one anti-cancer drug and the effective amount of the non-anti-cancer drug are administered at the same time or at different times. The components of the composition are used in the same or separate formulations when administered at the same time. Where used at different times, the components of the composition may be in the same or different dosage forms.

According to one aspect of the invention, the terms "combination" or "co-administration" as used throughout the specification refer to the use of therapeutic agents in the same or separate pharmaceutical preparations, either at the same time or at different times.

The invention also aims to provide an anticancer preparation which comprises the anticancer composition with the synergistic effect and pharmaceutically conventional auxiliary materials. The conventional auxiliary materials comprise a lubricant, a filling agent, a surfactant, a solubilizer, a cosolvent and the like.

According to one aspect of the present invention, each drug in the anticancer formulation may be a tablet, a capsule, a suspension, a solution, an injection, etc. individually or collectively.

The invention achieves the following beneficial effects:

(1) the combination of one or more anti-cancer drugs (VEGF/VEGFR inhibitors) and 2-bit (containing N-aryl piperazine structures) substituted quinazoline derivatives is used for treating certain tumors, the growth of tumor cells can be remarkably inhibited in vitro, the tumor volume can be remarkably inhibited in vivo, and the effect is remarkably better than that of the single anti-cancer drug.

(2) The combined drug can greatly reduce the use of the anticancer drugs on the basis of ensuring the same anticancer activity, and reduce the treatment risk and the toxic and side effect caused by the anticancer drugs under a larger dosage (the toxic and side effect of the anticancer drugs are larger usually).

(3) The 2-substituted quinazoline derivative (containing an N-aryl piperazine structure) can obviously reduce the proportion of tumor stem cells (closely related to tumor drug resistance), and the invention can be used as a good solution for solving the problem of the tumor drug resistance which is troublesome in the field of cancer treatment.

Drawings

FIG. 1 is a graph showing the effect of C2, C17 and SN on the inhibition of the proliferation of SW1990 and PANC-1 pancreatic cancer cells, respectively, by a single agent. A and B are C2 for inhibiting SW1990 and PANC-1 cell proliferation, respectively; c and D are C17 for the inhibition of SW1990 and PANC-1 cell proliferation, respectively; e and F are SN inhibition of SW1990 and PANC-1 cell proliferation, respectively.

FIG. 2 is a graph showing the effect of C2 in combination with SN on the inhibition of proliferation of human pancreatic cancer cells. A and B are the inhibition effect of single use of two medicines and combination of different concentrations on SW1990 and PANC-1 cell proliferation respectively; c and D are synergistic indexes of the two drugs in SW1990 and PANC-1 cells at different concentrations, and the synergistic index is less than 1, which represents that the two drugs have synergistic action.

FIG. 3 is a graph showing the effect of C17 in combination with SN on the inhibition of proliferation of human pancreatic cancer cells. A and B are the inhibition effect of single use of two medicines and combination of different concentrations on SW1990 and PANC-1 cell proliferation respectively; c and D are synergistic indexes of the two drugs in SW1990 and PANC-1 cells at different concentrations, and the synergistic index is less than 1, which represents that the two drugs have synergistic action.

FIG. 4 is a graph of the effect of C2 and C17 on the inhibition of colony forming ability of human pancreatic cancer cells. A and B are the inhibitory effects of C2 at different concentrations on the colony forming ability of SW1990 and PANC-1 cells, respectively; c and D are the inhibitory effect of C17 on the colony forming ability of SW1990 and PANC-1 cells at different concentrations, respectively.

FIG. 5 is a graph showing the effect of C2 in combination with SN on the in vitro colony formation inhibition of the doxorubicin-resistant human breast cancer cell line MCF-7/Adr.

FIG. 6 is a graph showing the effect of C2 and C17 on inhibiting tumor stem cells in human pancreatic cancer cells PANC-1.

FIG. 7 is a graph of the effect of C2 in combination with SN on tumor growth inhibition in an in vivo pancreatic cancer transplant tumor model. A, female nu/nu nude mice respectively give a growth curve of tumor volume when a blank control, gemcitabine GEM 15mg/kg, SN10mg/kg, C2100 mg/kg, SN10mg/kg + C2100 mg/kg; b, photographing results of tumors in each group; c, weight change of mice in each group; d, on day 28 of administration, animals are sacrificed, mice are dissected, the heart, liver, spleen, lung and kidney are taken and weighed, and the weight of each organ relative to the net weight of the mice and the organ index are calculated; e, day 28 of dosing, mice orbital whole blood assay blood routine results.

FIG. 8 is a graph of the effect of C2 in combination with SN on tumor growth inhibition in a human transplanted tumor model of pancreatic cancer in vivo. A, growth curves of tumor volume when NOD/SCID mice are given blank control, SN10mg/kg, C2100 mg/kg, SN10mg/kg + C2100 mg/kg respectively; b, photographing results of tumors in each group; c, weight change of mice in each group; d, on day 28 of administration, animals are sacrificed, mice are dissected, the heart, liver, spleen, lung and kidney are taken and weighed, and the weight of each organ relative to the net weight of the mice and the organ index are calculated; e, day 28 of dosing, mice orbital whole blood assay blood routine results.

Detailed Description

For a better understanding of the present invention, the inventors provide the following examples, which, however, are given for illustrative purposes only and are not to be construed as limiting the present invention, as many variations thereof are possible without departing from the spirit and scope thereof. While the compositions and methods of this invention have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept and scope of the invention. More specifically, it will be apparent that other pharmacological agents which are chemically and biologically related may be substituted for the agents described herein while the same or similar effects are achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope and concept of the invention.

The invention researches the in-vitro proliferation, colony formation and antitumor effects of a VEGFR inhibitor SN and a quinazoline derivative substituted at the 2-position (containing an N-aryl piperazine structure) on tumor cells in combination and on two pancreatic cancer xenograft tumor models.

Example 1

Inhibition of pancreatic cancer cell proliferation by C2, C17 and SN alone

Cells in the logarithmic growth phase were taken, digested with 0.25% pancreatin-0.53 mmol/L EDTA solution, centrifuged, resuspended and counted. SW1990 cells and PANC-1 cells were seeded at 6000 cells/well in 96-well cell culture plates. After 24h of incubation, dosing was performed. For SW1990 cells, the final concentrations of the single drug administration group of C2 were: 0.1, 1, 2.5, 5, 10, 15, 50, 100 μ M; for PANC-1 cells, the final concentrations of the C2 single drug dose group were: 0.1, 1,2, 6, 8, 10, 20, 50 μ M. In both cell lines, the final concentrations of the single dose group of C17 were: 1.5, 10, 15, 20, 25, 30, 50, 100. mu.M. In both cell lines, the final concentrations of the SN single drug dosing groups were: 0.1, 0.5, 1, 5, 7, 10, 15, 50, 100. mu.M. Each group was provided with 6 parallel holes. Incubation was continued for 48 h. After the incubation is finished, removing the culture medium, adding 100 mu L of 10% trichloroacetic acid (TCA, w/v) solution pre-cooled into each hole, and fixing for 1h at 4 ℃; discarding TCA solution, washing with tap water for 5 times, and naturally drying; adding 0.4% sulforhodamine B (SRB) dye into each hole by 100 mul, and dyeing for 30min at room temperature; discarding the SRB dye, washing with 1% acetic acid solution for 5 times, and naturally drying; mu.l of 10mmol/L Tris solution (pH10.5) was added to each well, and SRB bound to the basic amino acid residues of tumor cells was dissolved by shaking on a shaker for 10min, and absorbance at a wavelength of 540nm was measured by a microplate reader. The absorbance of the blank control group and the drug-treated group was expressed as OD_control,540And OD_sample,540. One of the 96-well cell culture plates was not dosed and was plated for 24h and then treated as described above. The average absorbance was OD_0h，540. Cell viability was calculated according to equation 1.1:

the results show (see fig. 1): IC50 of C2 was 4.32. mu.M and 6.40. mu.M in both SW1990 and PANC-1 cell lines (FIG. 1A, B), IC50 of C17 was 12.56. mu.M and 10.53. mu.M in both SW1990 and PANC-1 cell lines (FIG. 1C, D), IC50 of SN was 2.41. mu.M and 8.26. mu.M in both SW1990 and PANC-1 cell lines (FIG. 1E, F),

example 2

C2 and SN coupleInhibition of human pancreatic cancer cell proliferation

Cells in the logarithmic growth phase were taken, digested with 0.25% pancreatin-0.53 mmol/L EDTA solution, centrifuged, resuspended and counted. SW1990 cells and PANC-1 cells were seeded at 6000 cells/well in 96-well cell culture plates. After 24h of incubation, dosing was performed. For the three parallel 96-well plates of SW990 cells, the SN concentrations were 0, 1, 5. mu. mol/L in this order, and for the four parallel 96-well plates of PANC-1 cells, the SN concentrations were 0,2, 5, 10. mu. mol/L in this order, and in each 1 96-well plate, the C2 concentrations were 0, 1,2, 4. mu. mol/L, with 6 parallel wells in each group. Incubation was continued for 48 h. The absorbance at 540nm of each well was measured by the SRB method. The magnitude of the synergy index CI (CI) was calculated using literature reported methods. And (3) calculating theoretical absorbance of the combined administration group according to a formula 2.1, wherein the ratio of the actually measured absorbance to the theoretical value is the synergy index (formula 2.2). CI values less than, equal to, and greater than 1 indicate synergy, addition, and antagonism of C2 and SN, respectively.

The results show (see fig. 2): for SW1990 cells (figure 2A, C) and PANC-1 cells (figure 2B, D), C2 remarkably enhances the inhibition effect of SN on cancer cells, and when C2 is 2 and 4 mu mol/L, the synergy index (CI) of the combination of the two drugs is less than 1, which indicates that the combination of the two drugs has the synergy effect.

Example 3

Inhibition of human pancreatic cancer cell proliferation by C17 in combination with SN

Cells in the logarithmic growth phase were taken, digested with 0.25% pancreatin-0.53 mmol/L EDTA solution, centrifuged, resuspended and counted. SW1990 cells and PANC-1 cells were seeded at 6000 cells/well in 96-well cell culture plates. After 24h of incubation, dosing was performed. For four parallel 96-well plates of two cells, the concentration of C17 was 0, 1, 4, 10. mu. mol/L in sequence, and the concentration of SN was 0, 1,2, 5. mu. mol/L in each 1 96-well plate, with 6 parallel wells for each group. Incubation was continued for 48 h. The absorbance at 540nm of each well was measured by the SRB method. The magnitude of the synergy index CI (CI) was calculated using literature reported methods. And (3) calculating theoretical absorbance of the combined administration group according to a formula 3.1, wherein the ratio of the actually measured absorbance to the theoretical value is the synergy index (a formula 2.2). CI values less than, equal to, and greater than 1 indicate synergy, addition, and antagonism of C17 and SN, respectively.

The results show (see fig. 3): for SW1990 cells (FIG. 3A, C) and PANC-1 cells (FIG. 3B, D), C17 significantly enhanced the inhibition of SN on cancer cells at higher doses, and the synergy index (CI) of the combination of the two drugs was less than 1 at C17 of 10. mu. mol/L, indicating that the combination of the two drugs has a synergistic effect.

Example 4

Effect of C2 and C17 on in vitro colony formation of human pancreatic cancer cells

This example explored the effect of C2 and C17 on the ability of human pancreatic cancer cells to form colonies in vitro.

SW1990 cells and PANC-1 cells in the logarithmic growth phase were taken, digested with 0.25% pancreatin-0.53 mmol/L EDTA solution, centrifuged, resuspended and counted. SW1990 cells and PANC-1 cell suspensions were diluted to 2000 and 1500 cells/mL with medium, respectively, and 1mL of medium and 1mL of cell suspension were added to 6-well plates per well to give 2000 and 1500 cells per well, respectively. The inoculated 6-well plate was placed in a cell incubator. After 24h of incubation, dosing was performed. For SW1990 cells, drug-containing media at concentrations of C2 of 0.5. mu. mol/L and 2. mu. mol/L were prepared, and drug-containing media at concentrations of C17 of 0.5. mu. mol/L and 0.75. mu. mol/L were prepared. For PANC-1 cells, a drug-containing medium was prepared at a concentration of 0.5. mu. mol/L and 1. mu. mol/L for C2, and at a concentration of 0.5. mu. mol/L and 1. mu. mol/L for C17. The blank medium was supplemented with an equal volume of DMSO, and the DMSO concentration in all systems was 0.5%.

After 48h of dosing, the medium was aspirated from each well, rinsed once per well with 0.5mL PBS, and 2mL fresh medium was added to each well, and the medium was changed every 2 days. After 10 days of inoculation, the medium was aspirated off, 0.5mL PBS was added to each well and rinsed once, 1mL methanol was added to each well after the PBS was aspirated off, and the mixture was fixed at room temperature for 10 min. The methanol was removed by suction and stained with 1mL of 0.5% crystal violet solution for 10 min. The crystal violet solution was discarded, and the 6-well plate was rinsed with tap water to wash away unbound dye, and air dried naturally. The groups were compared by taking pictures of 6-well plates using Amersham Imager600 and counting colony formation with Image Quant TL 7.0.

The results show (see fig. 4A, B, C, D): the colony forming ability of the human pancreatic cancer cells SW1990 and PANC-1 in vitro is obviously influenced by C2 and C17, and the higher the concentration is, the stronger the effect of inhibiting the tumor cell colony formation is.

Example 5

Effect of C2 and SN combined on in-vitro colony formation of human breast cancer cells

This example explored the effect of C2 in combination with SN on the ability of human breast cancer cells to form colonies in vitro.

MCF-7/Adr cells in logarithmic growth phase were taken, digested with 0.25% pancreatin-0.53 mmol/L EDTA solution, centrifuged, resuspended and counted. MCF-7/Adr cell suspensions were diluted to 1000 cells/mL with medium and 1mL of cell suspension were added to 6-well plates per well to make 1000 cells per well. The inoculated 6-well plate was placed in a cell incubator. After 24h of incubation, dosing was performed. The dosing groups were set as follows: c22. mu. mol/L, SN 0.5. mu. mol/L, C22. mu. mol/L + SN 0.5. mu. mol/L. The blank medium was supplemented with an equal volume of DMSO, and the DMSO concentration in all systems was 0.5%.

After 48h of dosing, the medium was aspirated from each well, rinsed once per well with 0.5mL PBS, and 2mL fresh medium was added to each well, and the medium was changed every 2 days. After 10 days of inoculation, the medium was aspirated off, 0.5mL PBS was added to each well and rinsed once, 1mL methanol was added to each well after the PBS was aspirated off, and the mixture was fixed at room temperature for 10 min. The methanol was removed by suction and stained with 1mL of 0.5% crystal violet solution for 10 min. The crystal violet solution was discarded, and the 6-well plate was rinsed with tap water to wash away unbound dye, and air dried naturally. The groups were compared by taking pictures of 6-well plates using Amersham Imager600 and counting colony formation with Image Quant TL 7.0.

The results show (see fig. 5): SN 0.5. mu. mol/L has no significant effect on the in vitro colony forming ability of human breast cancer MCF-7/Adr, but C22. mu. mol/L in combination with SN 0.5. mu. mol/L significantly reduces the colony forming ability of tumor cells.

Example 6

Effect of C2 and C17 on human pancreatic cancer Stem cell ratio

PANC-1 cells in the logarithmic growth phase were taken, digested with 0.25% pancreatin-0.53 mmol/L EDTA solution, centrifuged, resuspended and counted. Dilution of PANC-1 cell suspension to 6X 10 with culture Medium⁵Each flask was filled with 5mL of medium and 1mL of cell suspension at 25cm²In the cell culture flask, the number of cells per flask was 6X 10⁵And (4) respectively. And placing the inoculated culture bottle in a cell culture box. After 24h of incubation, dosing was performed. The C2 and C17 dosing groups were set as follows: DMSO, C22. mu. mol/L, C24. mu. mol/L, C172. mu. mol/L, C174. mu. mol/L, the DMSO concentration in all systems being 1%.

After 48h administration, the cells were digested into a single cell suspension, centrifuged at 300 Xg for 7min, the supernatant discarded, 10mL of PBS pre-cooled in a refrigerator at 4 ℃ was added to resuspend the cells, centrifuged at 300 Xg for 7min, the supernatant discarded, and 1mL of ALDH buffer equilibrated to room temperature was added to resuspend the cells. Viable cells were counted using trypan blue staining. Cells were diluted to 1X 10 with ALDH buffer⁶one/mL. 0.5mL of cell suspension was taken from each sample and placed in 2mL centrifuge tubes, which were designated as the control tube and the detection tube for the sample. mu.L of a 1.5mmol/L stock of DEAB (diethylaminobenzaldehyde, specific inhibitor for ALDH, control for background fluorescence) was added to the control tube. Add 2.5. mu.L of activated ALDH substrate to control and test tubes, respectively, and mix immediately. Each control tube and detection tube was incubated in a 37 ℃ water bath for 45 min. Centrifugation was carried out at 300 Xg at 4 ℃ for 5min, the supernatant was discarded, and 0.5mL of ALDH buffer pre-cooled on ice was added to resuspend the cells. And (4) screening the cell by using a 200-mesh cell screen, and carrying out sample detection on a flow cytometer.

The results show (see fig. 6): both C2 and C17 can reduce the proportion of tumor stem cells in PANC-1 cells, and the higher the concentration is, the stronger the effect of inhibiting the proportion of tumor stem cells is.

Example 7

Inhibition of tumor growth in a pancreatic cancer transplant tumor model in vivo by a combination of SN and C2

Taking SW1990 cells in logarithmic growth phase, digesting with 0.25% pancreatin-0.53 mmol/L EDTA solution to prepare single cell suspension, centrifuging at 1,000rpm for 5min, discarding supernatant, adding a small amount of serum-free 1640 medium to blow and mix the cells again, counting, adjusting cell density to 1.75 × 10 with serum-free 1640 according to counting result⁷one/mL. The diluted cell suspension was inoculated under aseptic conditions to the right underarm of 5-week-old female nu/nu nude mice, and 0.2mL (approximately containing 3.5X 10. sup. th of cells) was injected into each mouse⁶Individual cells), the inoculation was observed. The long diameter (Dmax) and the short diameter (Dmin) of the tumor were measured with a vernier caliper, and the tumor volume V (V ═ Dmax × Dmin) was calculated²/2)。

After inoculation, the tumor grows to 50-100 mm³Mice were randomly divided into 5 groups of 5 mice each. The dosing regimen for each group was: (1) blank control group: intragastric administration of 0,2ml of 1, 2-propanediol solution every day; (2) and GEM group: injecting 0.1mL of normal saline containing GEM into tail vein every three days, wherein the administration dosage of the GEM is 15 mg/kg; (3) SN group: intragastrically administering 0.2ml SN1, 2-propylene glycol solution daily at a dose of 10 mg/kg; (4) group C2: intragastrically administering 0.2ml of C2 1, 2-propylene glycol solution per day at a dose of 100 mg/kg; (5) combination group: gavage 0.1mL SN in 1, 2-propylene glycol solution and 0.1mL C2 in 1, 2-propylene glycol solution daily, SN administered at 10mg/kg and C2 administered at 100 mg/kg. Wherein, the blank control group is a stealth control group, the GEM is gemcitabine, and the GEM is a positive control group.

Starting on the 0 th day of administration, the tumor size and body weight of the animals were measured every other day, and the survival status of the animals was observed for the presence or absence of adverse reactions. After 28 days of administration, 20. mu.L of whole blood was collected from the orbit of the mouse, quickly added to 2mL of the diluted solution, mixed well by gentle shaking, and then subjected to routine blood measurement. The animals are sacrificed, the mice are dissected, tumors are taken out, the mice are placed according to groups and then photographed, the heart, the liver, the spleen, the lung and the kidney are taken out and soaked in physiological saline, the mice are dried by filter paper and then weighed, and the visceral organ index of the individual visceral organs of the animals is calculated. The calculation formula is as follows:

the results show (fig. 7): the C2 alone has no obvious inhibition effect on the tumor volume, but the combined use of the C2 and the SN can obviously reduce the tumor size and has slightly better drug effect than the positive control group GEM (figure 7A, B), the weight of the nude mice has no obvious change during the administration period (figure 7C), main organs have no obvious damage (figure 7D), and the business is obvious blood toxicity (figure 7E). The results show that C2 can significantly increase the tumor inhibition effect of SN on xenograft tumor inoculated with human pancreatic cancer cells, and the combination of C2 and SN has good safety.

Example 8

Inhibition of tumor growth in a human transplanted tumor model of pancreatic cancer in vivo by a combination of SN and C2

Pancreatic cancer tissue obtained by surgical resection from a clinical Patient is partially cut into small pieces of about 2mm × 2mm × 3mm, and injected subcutaneously under the right abdomen of an NOD/SCID mouse of about 6 weeks old using a sterilized bone-penetrating needle, which is called a first generation human-derived xenograft (PDX) model mouse (F1); until the tumor grows to about 500mm³When the tumor is to be removed, the mouse is sacrificed and the tumor is taken out quickly, and a part of the tumor is placed in a cryopreservation tube, added with FBS containing 10% DMSO and placed in liquid nitrogen for cryopreservation; another part of the tumor was cut into 2 mm. times.2 mm. times.3 mm pieces and inoculated subcutaneously into NOD/SCID mice as described above, referred to as second generation PDX model mice (F2), and so on. The PDX model mouse used in this experiment was third generation (F3).

After the third-generation inoculation, the tumor grows to 80-100 mm³Mice were randomly divided into 4 groups of 5 mice each. The dosing schedule for each group was: (1) blank control group: intragastric administration of 0,2ml of 1, 2-propanediol solution every day; (2) SN group: intragastrically administering 0.2ml SN of 1, 2-propylene glycol solution every day with administration dosage of 10 mg/kg; (3) group C2: intragastrically administering 0.2ml of C2 1, 2-propylene glycol solution per day at a dose of 100 mg/kg; (4) combination group: gavage 0 every day1ml of 1, 2-propanediol solution of SN and 0.1mLC2 of 1, 2-propanediol solution, the dosage of SN being 10mg/kg and the dosage of C2 being 100 mg/kg.

Starting on the 0 th day of administration, the tumor size and body weight of the animals were measured every other day, and the survival status of the animals was observed for the presence or absence of adverse reactions. After 28 days of administration, 20. mu.L of whole blood was collected from the orbit of the mouse, quickly added to 2mL of the diluted solution, mixed well by gentle shaking, and then subjected to routine blood measurement. The animals are sacrificed, the mice are dissected, the tumors are taken out, the animals are arranged in anzu for photographing, the heart, the liver, the spleen, the lung and the kidney are taken out and soaked in physiological saline, the animals are weighed after being dried by filter paper, and the visceral organ index of the individual visceral organs of the animals is calculated.

The results show (see fig. 8): c2 alone did not significantly inhibit tumor volume, but the combination of C2 and SN significantly reduced tumor size (fig. 8A, B), no significant change in body weight of nude mice during administration (fig. 8C), no significant damage to major organs (fig. 8D) and no significant hematologic toxicity (fig. 8E). The results show that C2 can significantly increase the tumor inhibition effect of SN on the xenograft tumor inoculated by human tissues, and the combination of C2 and SN has good safety.

Discussion: from the in vitro results, it can be seen that C2, C17, in combination with SN, can significantly increase the inhibition effect of SN on the growth of tumor cells, and C2, C17 can significantly reduce the colony forming ability of tumor cells and the proportion of pancreatic cancer stem cells. In vivo experiments, C2 can significantly increase the antitumor effect of SN. In two pancreatic cancer transplantation tumor models, the combination of C2 and SN can obviously increase the antitumor effect, and has no obvious blood toxicity and systemic toxicity, which indicates that the combination scheme has wide application prospect.

Claims (7)

1. An anticancer composition with synergistic effect, which comprises effective amounts of at least one VEGF/VEGFR inhibitor and 2-substituted quinazoline derivatives C2 or C17 containing N-aryl piperazine structure; wherein the VEGF/VEGFR inhibitor is selected from one or more of sunitinib, pazopanib, axitinib, sorafenib and pharmaceutically acceptable salts thereof;

wherein the 2-substituted quinazoline derivative C2 or C17 containing the N-aryl piperazine structure has the following structures:

C2：

C17：

2. the synergistic anticancer composition as claimed in claim 1, wherein the VEGF/VEGFR inhibitor is sunitinib or a pharmaceutically acceptable salt thereof.

3. The synergistic anticancer composition as claimed in claim 1, wherein said cancer is a solid cancer.

4. The anticancer composition with synergistic effect as claimed in claim 1, wherein the cancer is one or more selected from breast cancer, bladder cancer, cervical cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, melanoma, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocellular cancer, nasopharyngeal cancer, neuroendocrine cancer, or salivary gland cancer;

wherein the lung cancer is one or more of small cell lung cancer, non-small cell lung cancer and non-squamous non-small cell lung cancer, the sarcoma includes osteosarcoma, leiomyosarcoma, liposarcoma, the skin cancer includes squamous cell carcinoma, spindle cell carcinoma, and the renal cancer includes advanced renal cell carcinoma.

5. The synergistic anticancer composition as claimed in claim 1, wherein the cancer is one or both of pancreatic cancer and breast cancer.

6. A pharmaceutical preparation with anticancer effect, comprising the anticancer composition of any one of claims 1 to 5 and conventional pharmaceutical adjuvants, wherein the dosage form of the pharmaceutical preparation is conventional in the pharmaceutical field, wherein different kinds of drugs are packaged separately or together, and different kinds of drugs are in the same dosage form or in different dosage forms.

7. Use of the anticancer composition with synergistic effect of any one of claims 1 to 5 in the preparation of anticancer drugs for breast cancer and pancreatic cancer.