CN108186643B

CN108186643B - Pharmaceutical composition with synergistic osteosarcoma resistance effect and application thereof

Info

Publication number: CN108186643B
Application number: CN201810309050.6A
Authority: CN
Inventors: 赵蔚; 邹昌业
Original assignee: Xuzhou Weikang Biotechnology Co ltd
Current assignee: Xuzhou Weikang Biotechnology Co ltd
Priority date: 2018-04-09
Filing date: 2018-04-09
Publication date: 2021-04-20
Anticipated expiration: 2038-04-09
Also published as: CN108186643A

Abstract

A pharmaceutical composition with synergistic anti-osteosarcoma effect and its application are provided. The invention discloses a pharmaceutical composition containing VEGFR2 selective inhibitor Apatinib (Apatinib) and histone demethylase inhibitor GSK-J4 and application thereof. The Apatinib (Apatinib) and GSK-J4 are used in combination, have synergistic antitumor effect, can obviously inhibit the growth of osteosarcoma tumor cells, and can be used for treating tumors such as osteosarcoma.

Description

Pharmaceutical composition with synergistic osteosarcoma resistance effect and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a pharmaceutical composition with a synergistic anti-tumor effect and application thereof.

Background

Osteosarcoma is a primary malignant bone tumor that originates from mesenchymal cells of bone and is characterized by the proliferation of tumor cells directly forming immature bone or bone-like tissue. The clinical occurrence rate of the typical osteosarcoma is about 0.3/ten thousand, which accounts for about 0.2% of malignant tumor and 15% of primary bone tumor. The typical osteosarcoma is well developed in men, the onset age is 8-25 years, and the osteosarcoma is rare before 6 years and after 60 years; the hair-improving part is a long bone, generally the distal femur or the proximal tibia, and then the proximal humerus, and other parts are rarely seen. The blood circulation and metastasis occur early, the incidence is high, and the progress is rapid. The standard treatment for osteosarcoma was amputation before 1970, but 80% of patients had micrometastases at the time of diagnosis, the mean time from surgical treatment to the appearance of lung metastases was 8 months, the overall survival rate was low in 5 years, and most of patients with tumor lung metastases died within 1 year after diagnosis. Research shows that the 5-year overall survival rate of osteosarcoma patients in China in the last decade is 7.5-77.6%, the average survival rate is 64.0%, the 5-year tumor-free survival rate is 34.8-69.7%, and the average survival rate is 56.0%.

The existing methods for treating osteosarcoma mainly comprise chemotherapy, surgery, radiotherapy, gene therapy, immunotherapy and molecular targeted therapy. The limb protection surgery replaces the existing amputation surgery, but the surgical treatment has limitations, for example, the tumor section bone resection and the joint fusion surgery can cause the loss of joint functions, and the surgical treatment is less clinically applied at present; rotational molding has positive implications for preserving limbs, but its development is limited by its poor appearance. In the aspect of chemotherapy, the current radiotherapy method has poor effect on osteosarcoma and low survival rate of patients, and is generally only used for patients who have pain relieving and cannot excise osteosarcoma.

In the aspect of drug treatment, the chemotherapy field gradually forms a concept of neoadjuvant chemotherapy, but research results show that the neoadjuvant chemotherapy cannot improve the long-term survival rate on the basis of adjuvant chemotherapy. Conventional chemotherapeutic agents also have certain problems, firstly, small doses of conventional chemotherapeutic agents such as Methotrexate (MTX) are not effective in killing tumor cells; although good curative effect is achieved by high-dose chemotherapy, serious toxic and side effects caused by chemotherapy, such as serious granulocyte andor platelet drop and serious infection possibly caused by the serious toxic and side effects are not negligible; resistance problems are also a major cause of chemotherapy failure. Like other therapeutic drugs, molecular targeted therapeutic drugs also produce resistance and adverse reactions. There are mainly skin toxicity, gastrointestinal toxicity reactions, hypertension and adverse cardiac events, as well as effects on the blood, which can be manifested as neutropenia. In recent years, the treatment of osteosarcoma has a bottleneck, and particularly for the treatment of some patients with metastatic osteosarcoma and chemotherapy resistance, an effective osteosarcoma treatment drug is lacked, and a new drug and treatment strategy needs to be developed. Therefore, the development of new drugs and the application of new drug combinations are of great significance for the breakthrough of the progress of the treatment of osteosarcoma.

GSK-J4, with structure shown in formula I, is a histone demethylase inhibitor. The GSK-J4 is thought to inhibit the proliferation of glioma and breast cancer cells by inhibiting the activity of demethylase UTX (also called KDM6A) and JMJD3 (also called KDM6B) of 27 th amino acid of histone H3.

Apatinib, the structure of which is shown in formula I, is also called Einstam, is the only oral preparation in the existing late gastric cancer targeted drugs, and is also an oral drug which is proved to be safe and effective after the late gastric cancer standard chemotherapy fails. A large number of clinical researches show that the apatinib can remarkably prolong the survival time of patients with advanced gastric cancer by inhibiting the generation of new blood vessels in tumor tissues.

Disclosure of Invention

The present invention aims to provide a combination drug having a superior anticancer effect, low toxicity, low side effects, and maximizing efficiency. In view of the above, the present invention provides a pharmaceutical composition and an application thereof.

The invention provides the following technical scheme:

the invention provides a pharmaceutical composition which comprises VEGFR2 selective inhibitor apatinib and histone demethylase inhibitor GSK-J4.

The apatinib and the GSK-J4 are used in combination, the drug effect of the combined use is larger than that of the single drug effect, and the combined use has obvious inhibition effect on the development of osteosarcoma and osteosarcoma.

Tests prove that the technical scheme provided by the invention can obviously inhibit the growth of osteosarcoma cells and has better anti-tumor efficacy.

In some embodiments of the invention, the molar ratio of apatinib to GSK-J4 is (1-3): (1-3).

In some embodiments of the invention, the apatinib is used at a concentration of 10-100 mg/kg; the GSK-J4 is used at a concentration of 5-50 mg/kg.

The invention also provides application of the pharmaceutical composition in preparing a medicament for treating cancer.

In some embodiments of the invention, the osteosarcoma cells are 143B cells and SJSA1 cells.

The invention does not limit the dosage form of the medicine, and all the dosage forms accepted in the field of medicine are in the protection scope of the invention.

Drawings

FIG. 1 shows the inhibition of the survival rate of osteosarcoma cells by apatinib and GSK-J4, respectively, as determined by cell viability assay;

FIG. 2 shows that there is a synergistic effect of the inhibition of the survival of osteosarcoma cells by the two drugs; FIG. 2A is a graph showing the determination of synergy index. FIG. 2B is a graph showing the inhibition of osteosarcoma cell survival by the combination of two agents compared to the inhibition of osteosarcoma cell survival by the combination of two agents alone.

FIGS. 3 to 5 show the detection of the apoptosis-inducing effect of the combination of two drugs and the single drugs of apatinib and GSK-J4 on bone and muscle cells, respectively; wherein, the effects of the drugs on the 143B cells of osteosarcoma cell line are shown in FIG. 3A and FIG. 4, which are a control group, a GSK-J4 group, an apatinib group and a combination group from left to right. FIGS. 3B and 5 show the effect of the drug on the osteosarcoma cell line SJSA1 cells, which are a control group, a GSK-J4 group, an apatinib group and a combination group from left to right.

FIG. 6 shows the inhibitory effect of the combination of two drugs on the tumorigenesis of osteosarcoma 143B cells and SJSA1 cells in experimental animals compared to the corresponding single drug; wherein, FIG. 6A shows 143B cell group, which comprises control group, GSK-J4 group (50mg/kg), apatinib group (100mg/kg), and combination group (GSK-J450 mg/kg plus apatinib 100mg/kg) from top to bottom. FIG. 6B shows SJSA1 group, which is composed of control group, GSK-J4 group (50mg/kg), apatinib group (100mg/kg), and combination group (GSK-J450 mg/kg plus apatinib 100mg/kg) from top to bottom.

Detailed Description

The invention discloses a pharmaceutical composition and application thereof, and can be realized by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

In the following examples, the inhibition rate + survival rate was 1.

The invention is further illustrated by the following examples:

1. example 1

Apatinib (from sel leck, cat # S2221) and GSK-J4 (from sel leck, cat # S7070) were prepared at different concentrations and were ready for use.

The effect of testing different concentrations of the drugs on the proliferation of human osteosarcoma cells 143B and SJSA1 was examined by MTT assay. 143B (ATCC number CRL-8303. TM., human, epithelial-like adherent growth) and SJSA1 cells (ATCC number CRL-2098. TM., human, epithelial-like adherent growth) in exponential growth phase were trypsinized, blown up into single cell suspensions, counted and plated into 96-well plates. Culturing for 24h in an incubator, adding test drugs with different concentrations after the cells adhere to the walls, stopping culturing after 48h, adding 20 μ l MTT (5mg/mL) into each well, and incubating for 4h at 37 ℃ in a dark place. Then, the liquid in each well is discarded, 150 mu L DMSO is added, the mixture is shaken for 15min, crystals are fully dissolved, and the optical density value of the cells is measured under the 490nm wavelength of a microplate reader.

The MTT method is adopted to respectively detect the inhibition effect of apatinib and GSK-J4 on cells of two cell lines of osteosarcoma. The calculation method of the action relationship of the medicine comprises the following steps: survival rate ═ experimental group number-blank number)/(control group number-blank number)

The experimental results are as follows:

the results show that apatinib and GSK-J4 have certain inhibitory effects on 143B and SJSA1 cells alone (fig. 1).

2. Example 2

And further examining the proliferation inhibition effect of the combination of apatinib and GSK-J4 on 143B and SJSA1 cells, and obtaining a drug interaction index CI through calculation.

Determination by MTT method

Apatinib and GSK-J4, and the combination of the above two drugs, have inhibitory effects on 143B and SJSA1 cell proliferation.

Grouping experiments: experimental groups 143B and SJSA1 cell lines were treated as follows: 1) blank control group: no drug treatment is added; 2) each single medicine group: apatinib group: culturing for 48h under the action of 40, 20, 10, 5, 2.5, 1.25 and 0.625 mu mol/L apatinib; group GSK-J4: culturing for 48h under the action of 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 μmol/LGSK-J4; 3) a combination of drugs: apatinib + GSK-J4 group: 40. 20, 10, 5, 2.5, 1.25 and 0.625 mu mol/L apatinib are respectively cultured for 48 hours under the action of 20, 10, 5, 2.5, 1.25, 0.625 and 0.3125 mu mol/LGSK-J4; each group was set with 3 replicate wells, the experiment was repeated 3 times, and the average was taken as the final result.

Cell viability assay in the same manner as in example 1, Calcusyn software (Bio-soft, Ferguson, Mo., USA) was used in combination with the index assay to analyze synergy index (Combi-indication index, CI), and the results showed that CI <1 is synergistic, 1 is additive and 1 is antagonistic

As a result, when the apatinib and the GSK-J4 are used in combination, synergistic inhibition effect on osteosarcoma cells can be shown under different concentrations (0.1-100 mu g/ml) (figure 2A).

For the SJSA1 cell line, CI >1 when the inhibition rate (fa) < 20%, both had antagonistic effects, with a synergistic effect for fa > 20%; for 143B, the synergistic effect was most pronounced in the fa range, which was around 100% with the lowest CI value (fig. 2A).

Then fixing the proportion and the concentration of the two medicines, and detecting the synergistic effect of the combination. The experimental groups were as follows: 1) blank control group: no drug treatment is added; 2) each single medicine group: apatinib group: culturing for 24h, 48h, and 72h under the action of 10 μmol/L apatinib; group GSK-J4: culturing for 24h, 48h and 72h under the action of 5 mu mol/LGSK-J4; 3) a combination of drugs: the Apatinib 10 mu mol/L and 5 mu mol/LGSK-J4 were cultured for 24h, 48h, and 72 h. For cell viability assays at 24h, 48h, and 72h time points, apatinib in combination with GSK-J4 had a stronger proliferation inhibitory effect on 143B and SJSA1 cells than each single drug group (P <0.05) (fig. 2B).

In order to determine whether the combined use of apatinib and GSK-J4 has stronger apoptosis effect on osteosarcoma cells, the apoptosis induction effect of 143B and SJSA1 cells treated by apatinib and GSK-J4 single drugs and the combination of the two drugs for 12 hours is detected by a flow cytometer by adopting an Annexin V-FITC/PI double staining method.

The experimental process is that different groups of medicaments act on 10 respectively⁵ Centrifuging 143B or SJSA1 cells for 12h to obtain cells, washing, staining with a double staining reagent annexinV-FITC/PI for 15-20 min, centrifuging, washing, re-suspending with staining Buffer, and performing flow cytometryAnd (3) measuring a fluorescence channel corresponding to FITC/PI, wherein normal cells are FITC-/PI-, cells at the early apoptosis stage are FITC +/PI-, and cells at the late apoptosis stage and necrotic stages are FITC +/PI +.

The results show that, compared with the single drug group, the combined use of apatinib and GSK-J4 has obviously enhanced apoptosis induction effect on osteosarcoma 143B and SJSA1 cells, and the difference has extremely obvious statistical significance (P < 0.01). (FIGS. 3A and 3B, FIGS. 4 and 5).

3. Example 3

Culturing osteosarcoma 143B and SJSA1 cells, digesting with pancreatin when the cells are in exponential growth stage, collecting cells, and preparing into 1 × 10 with sterile physiological saline⁷one/mL of the cell suspension was injected subcutaneously into the left underarm position of male nude mice, 0.2mL each. The condition of the mice was observed every day, and the major diameter (a) and the minor diameter (b) of the tumor were measured with a vernier caliper according to the formula V (mm)³)＝(a×b²) The tumor volume was calculated. When the tumor volume reaches 80-100 mm³At the time, animals were randomly divided into 4 groups of 5 animals each. Respectively as follows: control group: physiological saline; apatinib group: 100 mg/kg; ③ GSK-J4 group: 50 mg/kg; fourthly, combination group: apatinib 100mg/kg + GSK-J450 mg/kg. The control group, the apatinib group, the GSK-J4 group and the combination group are administrated intraperitoneally once every other day for 5 times continuously. After 27 days, the mice were sacrificed by cervical dislocation, and the tumors were peeled off, weighed, and compared for the size of the tumor growth.

Results of the experiment

As shown in fig. 6, when the two were used in combination, the growth of the transplanted tumor was inhibited to a greater extent. The results show that the combined administration group obviously reduces the sarcoma volume and the tumor weight of the mice.

Claims

1. A pharmaceutical composition for treating osteosarcoma, which comprises VEGFR2 selective inhibitor apatinib and histone demethylase inhibitor GSK-J4, wherein the molar ratio of apatinib to GSK-J4 is 2: 1.

2. The pharmaceutical composition according to claim 1, wherein apatinib is used in a concentration of 10-100 mg/kg; the GSK-J4 is used at a concentration of 5-50 mg/kg.

3. Use of a pharmaceutical composition according to claim 1 or 2 for the preparation of a medicament for the treatment of osteosarcoma.