CN111358952A

CN111358952A - Anti-tumor pharmaceutical composition, preparation and application thereof

Info

Publication number: CN111358952A
Application number: CN202010296042.XA
Authority: CN
Inventors: 刘然义; 岳欣; 韩辉; 刘听雨; 王雪涔
Original assignee: Sun Yat Sen University Cancer Center
Current assignee: Sun Yat Sen University Cancer Center
Priority date: 2020-04-15
Filing date: 2020-04-15
Publication date: 2020-07-03
Anticipated expiration: 2040-04-15
Also published as: CN111358952B

Abstract

The invention provides an anti-tumor pharmaceutical composition, a preparation and an application thereof, wherein at least one tyrosine kinase receptor inhibitor, especially nilotinib, is further added on the basis of the original sunitinib, so that a very obvious synergistic interaction effect can be generated with the sunitinib, the apoptosis of cancer cells can be promoted in a combined manner, the proliferation of the cancer cells can be obviously inhibited, and the anti-tumor pharmaceutical composition has very obvious killing efficiency on the cancer cells. The pharmaceutical composition provided by the invention can obviously improve the serious drug resistance and obviously reduced treatment effect caused by the simple adoption of sunitinib in the prior art, provides a new scheme for clinical treatment, and has very wide market prospect and extremely important social significance.

Description

Anti-tumor pharmaceutical composition, preparation and application thereof

Technical Field

The invention belongs to the technical field of tumor treatment, and particularly relates to an anti-tumor pharmaceutical composition, and a preparation and an application thereof.

Background

Malignant tumors occur because activation of oncogenes and/or inactivation of oncogenes allow cells to acquire the ability to divide continuously into cancer cells. The treatment of tumors includes surgery, radiotherapy and chemotherapy, wherein chemotherapy is further divided into traditional chemotherapy, targeted therapy, immunotherapy and the like. Different malignant tumors have different initiation factors and the heterogeneity of the tumors, which causes sensitivity and tolerance to different treatments, thus bringing great difficulty to clinical treatment of tumor diseases and being the key research direction of researchers.

Renal cancer (Kidney cancer) is one of the three major malignancies of the urinary system. Among them, Renal Cell Carcinoma (RCC) is a malignant tumor derived from tubular epithelial cells, accounts for 90% of all kidney cancers, and is the most common type of renal malignancy. Due to the application of screening means such as ultrasound and CT and the enhancement of health physical examination consciousness of people, the incidence rate of kidney cancer is increased, but early kidney cancer is treated by radical surgery, and the prognosis is better. The overall 5-year survival rate of kidney cancer is 49%, but the prognosis is very related to the clinical stage and pathological grade of the disease, and the prognosis of patients in clinical stage III and IV or

pathological grade

3 and 4 is very poor.

The recurrence of renal cancer refers to the regrowth of tumor in the operative site or nearby tissues after radical surgery; advanced kidney cancer refers to kidney cancer that reaches stage IV at first visit (i.e., the primary tumor reaches T4 or has developed distant metastases). At this time, tumor treatment should be mainly systemic treatment according to the recommendation of the 2020 NCCN (the National Comprehensive Cancer network) renal Cancer guideline.

Renal cancer is not sensitive to traditional chemotherapeutic drugs. Currently, there are mainly targeted therapies and immunotherapies for the systemic treatment of kidney cancer. The prognosis of renal cancer is associated with its clinical stage and pathological grade. The overall 5-year survival rate of kidney cancer was 49%, which is a significant improvement over that seen by 2006. The reasons for this may be that the increased detection rate of early renal cancer allows radical surgery to be performed and the use of targeted drugs. Especially, the advent of targeted drugs has greatly improved the prognosis of patients with advanced metastatic renal cancer. Even so, the Overall Survival rate (OS) of patients with advanced renal cancer is now only increased by around 2 months (29%) compared to before the absence of the targeting agent. The existence of drug resistance is a great part of reasons, and therefore, the problem of targeted drug resistance of the kidney cancer is urgently needed to be solved, so that the prognosis of the kidney cancer patient is further improved.

Sunitinib (Sunitinib) is a highly potent, multi-targeted tyrosine kinase Receptor inhibitor, the targets of which include vascular endothelial

growth factor Receptor

1,2,3(VEGF Receptor, VEGFR-1,2,3), platelet growth factor Receptor α (PDGF Receptor, PDGFR- α), c-KIT, and the like, according to existing research results, Sunitinib exerts anti-Tumor effects mainly through three pathways, inhibiting Tumor angiogenesis, destroying Tumor vessels, and directly killing Tumor cells.

Although sunitinib slows the progression of metastatic renal cancer and improves patient prognosis, almost all patients eventually develop resistance and eventually progress again. According to the existing research reports, although the evaluation criteria of each research are slightly different, it can be seen that about 70% of patients are sensitive to sunitinib in the first line treatment, and 30% of patients show primary drug resistance to sunitinib. In sensitive patients, the effective period is usually only 6-15 months, after which acquired resistance and disease progression occurs. There are many studies on the mechanism of acquired drug resistance, and the mechanism can be generally classified into the following: activation of pro-angiogenic signals, alterations in the tumor microenvironment, lysosomal retention, action of non-coding RNAs, and activation of other signaling pathways.

The angiogenesis promoting signal activation mainly comprises the expression increase of Fibroblast Growth Factor (FGF), Interleukin 8 (IL-8), placenta Growth Factor (PIGF) and angiogenin (Ang), thereby resisting the angiogenesis and tumor vessel damage caused by sunitinib; fibroblasts, pericytes, endothelial cells, hematopoietic cells and the like of the tumor microenvironment may mediate resistance to sunitinib through secretion of chemical factors, cytokines and direct contact; it has been found that sunitinib from drug-resistant cells is accumulated in lysosomes, and therefore, although the concentration of sunitinib in cells is high, the levels of p-ERK and p-AKT are not affected; non-coding RNA was also involved in resistance to sunitinib, and the expression profiles of mirnas were found by researchers to compare poor prognosis (PSF <6 months) and good prognosis (PSF >6 months) in mRCC patients with sunitinib: the down regulation of miRNA-410, miRNA-1181 and miRNA-424 is related to the prolonging of drug reaction time, the expression reduction of miRNA-192, miRNA-193a-3p and miRNA-501-3p is related to poor prognosis, and recent research reports that long-chain non-coding RNA (lncARSR) can mediate the drug resistance of sunitinib through a Competitive endogenous RNA (ceRNA) mechanism and can enable sensitive cells to obtain the drug resistance through exosomes; in addition, activation of other signaling pathways, such as SK-1, may also be involved in the development of sunitinib resistance. Generally, resistance of kidney cancer to sunitinib relates to various molecular mechanisms, and although many researches are carried out at present, how to solve the problem of resistance of kidney cancer is still a hotspot and difficulty in the research field of kidney cancer.

Disclosure of Invention

In order to solve the problems and the defects, the invention provides an anti-tumor drug composition to solve the problems of serious drug resistance and obviously reduced treatment effect caused by the simple adoption of sunitinib in the prior art, so that more obvious clinical treatment effect and reduced sunitinib drug resistance are obtained.

The invention is realized by the following technical scheme:

in a first aspect, the present invention provides an anti-tumor pharmaceutical composition comprising sunitinib or a pharmaceutically acceptable salt thereof and at least one tyrosine kinase receptor inhibitor other than sunitinib or a pharmaceutically acceptable salt thereof.

Preferably, the tyrosine kinase receptor inhibitor is selected from one or more of Nilotinib (Nilotinib), Gefitinib (Gefitinib), Erlotinib (Erlotinib), Lapatinib (Lapatinib), Afatinib (Afatinib), Dacomitinib (Dacomitinib), Vandetanib (Vandetanib), Neratinib (Neratinib), oxitinib (osiritinib), Rociletinib, Olmutinib, Naquotinib, tervatinib, and nazertinib.

Preferably, the tyrosine kinase receptor inhibitor is selected from nilotinib.

Preferably, the tumor is one or more selected from renal cancer, lung cancer, intestinal cancer, gastric cancer, esophageal cancer, liver cancer, cervical cancer, breast cancer, leukemia, malignant lymphoma, nasopharyngeal cancer and pancreatic cancer.

Preferably, the tumor is selected from renal cancer.

The invention provides a preparation containing the anti-tumor pharmaceutical composition, which comprises sunitinib or a pharmaceutically acceptable salt thereof, at least one tyrosine kinase receptor inhibitor except for sunitinib or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable pharmaceutical excipients.

Preferably, the preparation dosage form is selected from one or more of tablets, capsules, pills, granules, injections, aerosols, sprays, films and suppositories.

Preferably, the pharmaceutically acceptable pharmaceutical excipients are selected from one or more of fillers, disintegrants, binders, lubricants, flavoring agents, preservatives, antioxidants and coloring agents.

Preferably, the tyrosine kinase inhibitor is selected from nilotinib.

Preferably, the tumor is selected from renal cancer.

The third aspect of the invention provides an application of the anti-tumor medicine composition in preparing a preparation for treating tumors.

Preferably, the tyrosine kinase inhibitor is selected from nilotinib.

Preferably, the tumor is selected from renal cancer.

Although sunitinib is widely used clinically for cancer treatment and can achieve relatively remarkable effect, sunitinib has been used as a first-line drug in the treatment of diseases such as kidney cancer. However, neither improvement in the patient's prognosis nor delay in the progression of the cancer prevents the development of resistance to sunitinib in the patient and ultimately leads to the disease progressing again. Researchers have conducted extensive studies on the development of resistance to drugs, and it was found that the causes of the development of the resistance are mainly the following: activation of pro-angiogenic signals, alterations in the tumor microenvironment, lysosomal retention, action of non-coding RNAs, and activation of other signaling pathways. Unfortunately, despite the resistance mechanism and discovery and demonstration, no better treatment regimen has emerged to improve the severe resistance of sunitinib, thereby compromising its clinical use and therapeutic efficacy in patients.

In this regard, the inventors have conducted extensive studies using a compound library from the ceramic company for screening, which includes 1374 FDA-approved drugs on the market, in hopes of obtaining compounds that improve clinical resistance to sunitinib and that can produce additive or even synergistic therapeutic effects therewith. In the screening process, it is found that the combination of at least one tyrosine kinase receptor inhibitor and the sunitinib, especially nilotinib, can generate a very significant synergistic effect (CI < 1).

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention improves the existing clinical cancer treatment scheme, screens a large amount of compounds, and obtains the compound which can be combined with sunitinib to obviously reduce the drug resistance of the sunitinib.

(2) On the basis of the original sunitinib, at least one tyrosine kinase receptor inhibitor, especially nilotinib, is further added, so that a very obvious synergistic interaction effect can be generated with the sunitinib, the apoptosis of cancer cells can be promoted in a combined manner, the proliferation of the cancer cells can be obviously inhibited, and the sunitinib has very obvious killing efficiency on the cancer cells.

(3) The pharmaceutical composition provided by the invention can obviously improve the serious drug resistance and obviously reduced treatment effect caused by the simple adoption of sunitinib in the prior art, provides a new scheme for clinical treatment, and has very wide market prospect and extremely important social significance.

Drawings

FIG. 1 shows the results of in vitro cell killing experiments.

FIG. 2 shows the results of in vitro inhibition of cell clonogenic capacity.

FIG. 3 shows the results of EDU experiments.

FIG. 4 is a schematic diagram of tumor size in an in vivo tumor growth inhibition experiment.

FIG. 5 is a tumor growth curve of in vivo tumor growth inhibition experiments.

FIG. 6 shows the results of the tumor inhibition rate experiments in vivo tumor growth inhibition experiments.

FIG. 7 shows the results of in vitro apoptosis experiments.

Fig. 8 shows the results of TUNEL experiments.

FIG. 9 shows the results of in vitro apoptosis double staining experiments.

FIG. 10 shows the results of detection of key proteins in the apoptotic pathway.

FIG. 11 shows the pathological conditions and apoptosis pathway-related protein expression in vivo.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Cell lines listed in the context of the present invention, including 786-O, A498 and Caki-1, were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured according to ATCC guidelines, unless otherwise specified. All cell lines were identified by short tandem repeat analysis of the chinese typical culture collection (wuhan) and verified for the presence of mycoplasma contamination using a PCR assay kit (shanghai Biothrive Sci) while being cryopreserved in liquid nitrogen and used for subsequent experiments. The reagents used in the present invention are commercially available.

Representative results from selection of biological replicates are presented in the context figures, and data are presented as mean ± SD and mean ± SEM as specified in the figure. All in vitro experiments were repeated at least three times and animal experiments were repeated twice. Data were analyzed using GraphPad Prism 5.0 or SPSS 20.0 software. And comparing the difference of the mean values of two or more groups by using a t test or an analysis of variance. p < 0.05 was considered a significant difference.

Example 1 in vitro cell proliferation inhibition assay

Independent drug administration is selected to carry out in-vitro cell proliferation experiments on 786-O, A498 and Caki-1 cells, and the specific experimental method is as follows:

cell proliferation assay: (1) planting cells in logarithmic growth phase in a 96-well plate according to a certain density, abandoning an old culture medium after 24h, adding culture media containing different drugs, wherein the drug concentration is sunitinib (2 mu M) and nilotinib (2 mu M), and each group is provided with 3 multiple wells;

(2) culturing for 48h, collecting one plate every day, discarding the liquid medicine, adding culture medium containing 5% CCK8, incubating at 37 deg.C for 3h, and detecting OD value of 450nm with microplate reader.

Based on the DMSO group, the growth rate of each group of cells = (drug addition OD value-blank OD value)/(DMSO OD value-blank OD value) × 100% per day.

Clone formation experiments: (1) laying 500 cells/well in a six-well plate, adding corresponding drugs after 24h, wherein the selected drug concentrations are sunitinib (80nM) and nilotinib (200 nM);

(2) after further culturing for 14 days, the medium was discarded, washed 2 times with PBS, stained with 0.5% crystal violet methanol solution, and the number of clones (> 50 cells were counted as one clone) for each group of cells was counted, and the plate clone formation rate = (number of clones formed/number of plated cells) × 100 was calculated as 100%.

As a result, it was found that the single drug showed no significant proliferation inhibitory effect in all three cell species compared to the control group (DMSO), while the combined drug showed significant inhibition of cell proliferation and even cell killing effect (as shown in FIG. 1). Then, a clone formation experiment was performed using 786-O cells, and as a result, it was found that the clone formation ability of 786-O cells was not affected by the administration of the drug alone, while the clone number of cells was significantly reduced and the clone area was significantly reduced in the combination group, as compared with the control group (as shown in FIG. 2).

Further, EDU experiments were performed on three cell lines, and the specific experimental methods were as follows:

(1) taking tumor cells in logarithmic growth phase, and pressing according to 1 × 10⁵The density of the hole is that the cells are paved in a culture dish with a glass bottom, the cells are incubated overnight, and the cell density reaches about 60 percent after the cells are adhered to the wall overnight on the next day;

(2) and after the cells adhere to the wall, replacing a fresh culture medium containing different medicines to culture the cells for 24 hours. The drug concentration is sunitinib (4 mu M) and nilotinib (4 mu M), and the combined administration group is sunitinib (2 mu M) and nilotinib (2 mu M);

(3) after continuously culturing for 24h, adding corresponding reagents according to the EDU experiment kit instructions, and detecting the experiment result by a confocal microscope

In the EDU experiment, more severe experimental conditions are set, namely the concentrations of two medicaments of the combined medicament group are reduced by half on the basis of a single medicament group. The results showed that the proportion of proliferating cells was significantly reduced in the combination group, while the individual groups were not significantly different from the control group (results are shown in fig. 3).

From the above results, it can be seen that even in the 786-O, A498 and Caki-1 cells, which are not sensitive to sunitinib alone, sunitinib in combination with nilotinib still produces a very significant in vitro proliferation inhibitory effect.

Example 2 in vivo tumor growth inhibition assay

The in vivo tumor growth inhibition experiment is carried out on the nude mice about 7 weeks, and the specific experimental method is as follows:

(1) 786-O cells are cultured in vitro, and cells in a logarithmic phase are collected, centrifuged and suspended;

(2) injecting the cell suspension to the subcutaneous part of 3 nude mice, taking out tumor bodies after the tumor bodies are formed, and dividing the tumor bodies into 5mm³Small tumor masses of left and right size;

(3) the obtained small tumor mass is transplanted to the subcutaneous part of 20 new nude mice again;

(4) after the tumor formation, randomly dividing 20 nude mice into 4 groups, each group comprises 5 mice, and the numbers are 1-5 in sequence, and performing intragastric administration; wherein group 1 was given sunitinib 10 mg/kg. d, group 2 was given with nilotinib 10 mg/kg. d, group 3 was given with sunitinib 5 mg/kg. d + nilotinib 5 mg/kg. d, group 4 was given with DMSO for 5 consecutive days, with drug withdrawal for 2 days, for one cycle, for a total of 4 cycles, tumor volume was measured once a week, mice were sacrificed and tumor masses were removed after treatment was completed.

The result shows that the tumor mass of the combined medicine group is obviously smaller than that of the control group and the single medicine group by naked eyes; the size of the tumor mass of the sunitinib group is slightly smaller than that of the control group; the nilotinib group was not significantly different from the control group. According to the continuous measurement result of the tumor volume, the tumor volume of the combined administration group is obviously reduced compared with that of the control group and that of the single administration group, the difference is reflected 1 week after the administration, the difference is gradually increased in the last observation, and the final statistical results are obviously different. In addition, the tumor volume of the sunitinib group was also reduced to some extent compared with the control group, and the difference between the nilotinib group and the control group was not statistically significant (the results are shown in fig. 4-fig. 5).

The analysis result of the tumor weight shows that the change trend of the tumor weight is basically consistent with the result shown by the tumor volume. The tumor inhibition rate of the combined administration group is higher than that of the sunitinib group and nilotinib group, and the combined administration group has statistical significance (the results are shown in figure 6).

From the above results, it can be seen that sunitinib and nilotinib administered in combination can inhibit proliferation of renal cancer cells

Example 3 in vitro apoptosis promotion experiment

The results of in vitro apoptosis experiments on 786-O, A498 and Caki-1 cells selected by single drug show that the cells in the combined drug administration group are obviously shriveled and the number of adherent cells is reduced, while the morphology and the density of the cells are not obviously influenced when the drugs with corresponding concentrations are treated separately (the results are shown in figure 7).

TUNEL experiments were subsequently performed after drug treatment in 786-O, A498 and Caki-1 cells as follows:

(3) after further culturing for 24h, according to the instructions of the TUNEL test kit, the corresponding reagent is added for treatment, and then the test result is detected by a confocal microscope.

As a result, when the drug concentration was reduced by half and then the drug concentration was combined in the single drug administration group, the cells still showed positive results in the TUNEL test, (A498 and Caki-1 cell test results are not shown) (results are shown in FIG. 8).

Further, apoptosis double staining experiments were performed in three cells, and the specific experimental methods were as follows:

(1) taking tumor cells in logarithmic growth phase, and pressing according to 1 × 10⁵The density of the hole is that the cells are paved into a 6-hole plate, the cells are incubated overnight, and the cell density reaches about 60 percent after the cells are adhered to the wall overnight on the next day;

(2) and after the cells adhere to the wall, replacing a fresh culture medium containing different medicines to culture the cells for 24 hours. The drug concentration at this time was sunitinib (2 μ M) and nilotinib (2 μ M);

(3) and after 24h, collecting cells according to the specification of the apoptosis double-staining reagent counting box, adding corresponding reagents, and detecting the proportion of apoptotic cells by a flow cytometer.

The results showed that the apoptosis ratio of the combination group was significantly increased compared to the group administered alone (the results are shown in fig. 9). Subsequently, the critical proteins including clear caspase-3, clear caspase-7 and clear PARP-1 in the apoptosis pathway were detected, and as a result, it was found that the levels of these apoptosis-related proteins were significantly up-regulated in the combination-administered group, but not detected in the control group and the single-administered group (the results are shown in FIG. 10).

Finally, the tumor of the in vivo experiment is made into pathological section and then H & E and immunohistochemical staining is carried out. As shown in FIG. 11, the levels of the apoptotic effector proteins, cleared cassette-3 and cleared PARP-1, were significantly increased in the tumors of the combination-administered group as compared to the control group and the single-administered group.

From the above results, it can be known that the combination of sunitinib and nilotinib can promote the apoptosis of renal cancer cells.

On the basis of the original sunitinib, at least one tyrosine kinase receptor inhibitor, especially nilotinib, is further added, so that a very obvious synergistic interaction effect can be generated with the sunitinib, the apoptosis of cancer cells can be promoted in a combined manner, the proliferation of the cancer cells can be obviously inhibited, and the sunitinib has very obvious killing efficiency on the cancer cells. The pharmaceutical composition provided by the invention can obviously improve the serious drug resistance and obviously reduced treatment effect caused by the simple adoption of sunitinib in the prior art, provides a new scheme for clinical treatment, and has very wide market prospect and extremely important social significance.

The above detailed description section specifically describes the analysis method according to the present invention. It should be noted that the above description is only for the purpose of helping those skilled in the art better understand the method and idea of the present invention, and not for the limitation of the related contents. The present invention may be appropriately adjusted or modified by those skilled in the art without departing from the principle of the present invention, and the adjustment and modification also fall within the scope of the present invention.

Claims

1. An anti-tumor pharmaceutical composition comprising sunitinib or a pharmaceutically acceptable salt thereof and at least one tyrosine kinase receptor inhibitor other than sunitinib or a pharmaceutically acceptable salt thereof.

2. The antineoplastic pharmaceutical composition as claimed in claim 1, wherein said tyrosine kinase receptor inhibitor is selected from one or more of nilotinib, gefitinib, erlotinib, lapatinib, afatinib, dacomitinib, vandetanib, neratinib, oxitinib, Rociletinib, Olmutinib, Naquotinib, Tesevatinib and nazurttinib.

3. The antineoplastic pharmaceutical composition as claimed in claim 2, wherein said tyrosine kinase inhibitor is selected from nilotinib.

4. The antitumor pharmaceutical composition as claimed in claim 1, wherein the tumor is selected from one or more of renal cancer, lung cancer, intestinal cancer, gastric cancer, esophageal cancer, liver cancer, cervical cancer, breast cancer, leukemia, malignant lymphoma, nasopharyngeal cancer, and pancreatic cancer.

5. The antitumor pharmaceutical composition according to claim 4, wherein said tumor is selected from renal cancer.

6. An antitumor pharmaceutical preparation comprising the antitumor pharmaceutical composition according to any one of claims 1 to 5 and a pharmaceutically acceptable pharmaceutical excipient.

7. The antitumor drug preparation as claimed in claim 6, wherein the dosage form is selected from one or more of tablets, capsules, pills, granules, injections, aerosols, sprays, films and suppositories.

8. The antitumor pharmaceutical preparation according to claim 6, wherein said pharmaceutically acceptable pharmaceutical excipients are selected from one or more of fillers, disintegrants, binders, lubricants, flavors, preservatives, antioxidants, and colorants.

9. Use of the antitumor pharmaceutical composition according to any one of claims 1 to 5 for the preparation of a formulation for treating tumors.

10. The use according to claim 9, wherein the tumor is selected from one or more of renal cancer, lung cancer, intestinal cancer, gastric cancer, esophageal cancer, liver cancer, cervical cancer, breast cancer, leukemia, malignant lymphoma, nasopharyngeal cancer, pancreatic cancer.