CN113786491A

CN113786491A - An anti-tumor combined preparation containing tetrandrine, dihydroquercetin or quercetin

Info

Publication number: CN113786491A
Application number: CN202111172123.XA
Authority: CN
Inventors: 何琪杨; 王金彩; 陈淑珍; 王爱民; 张娟; 陈阳
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2021-10-08
Filing date: 2021-10-08
Publication date: 2021-12-14
Anticipated expiration: 2041-10-08
Also published as: CN113786491B

Abstract

The invention relates to an anti-tumor combined preparation containing tetrandrine, dihydroquercetin or quercetin. The composition of tetrandrine and dihydroquercetin or quercetin and related salts thereof also comprise a targeting drug, wherein the targeting drug is oxitinib or a drug with similar action to the oxitinib. The tumor is preferably non-small cell lung cancer. The combined preparation of the invention has obvious functions of synergism and drug resistance reversion for the oxitinib.

Description

An anti-tumor combined preparation containing tetrandrine, dihydroquercetin or quercetin

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to an anti-tumor combined preparation containing tetrandrine, dihydroquercetin or quercetin.

Background

Tetrandrine (TET), also called tetrandrine, is derived from traditional Chinese medicines such as tetrandrine and has various pharmacological activities. In 1970 s, tetrandrine was marketed in China as an antihypertensive drug, and was found to have excellent silicosis-treating activity and to be widely used clinically. In 1992, Heqiyang et al first found that tetrandrine can reverse the resistance of CHO cells to the anti-tumor drug doxorubicin, and later, further research found that human leukemia HL-60 resistant cells are more sensitive to tetrandrine. Researches of other scholars find that the tetrandrine can block tumor cells at the G1 stage and enhance the activity of the targeted drug sorafenib on liver cancer. In 2020, tetrandrine is also found to have strong activity of inhibiting new corona virus.

Dihydroquercetin (DHQ), also known as taxifolin (taxifolin), is a natural product of flavonoids (flavanones), and is present in various vegetables and plant traditional Chinese medicines, for example: larch, onion, etc. Dihydroquercetin has low toxicity to human body and has various pharmacological activities, such as: has strong antioxidant activity and strong antitumor effect, and has stronger effect of resisting liver injury than quercetin (QUERetin, QUE) with similar structure. In 2019, the european union approved for food antioxidants. At the end of 2021, 4 months, dihydroquercetin approved by Weijian Commission of China is used as a new resource food material, and the daily dosage is not more than 100 mg.

Lung cancer is the most common tumor with the highest mortality in the world, as is the case in our country. Of these, 80-85% of lung cancers are non-small cell lung cancers, and of these, Epidermal Growth Factor Receptor (EGFR) mutations in approximately 30-40% of patients are suitable targets for targeted drug tyrosine kinase inhibitors. Oxitinib (OSI) is a third generation targeted drug against mutant EGFR, developed and marketed by asixican, england. At present, the drug is approved by the FDA and the drug administration in China as a second-line therapy, a first-line therapy and a post-operative adjuvant therapy for EGFR T790M mutant patients for the treatment of 3 indications, and is a most excellent targeted drug in the field of lung cancer treatment. Ametinib (ALM) is a third-generation targeted drug developed by Hoisen Biotechnology corporation in China and having similar activity to Oncinib, and is approved by the drug administration in China as a second-line therapy for EGFR T790M mutation. .

Disclosure of Invention

The invention firstly relates to an anti-tumor combined preparation, which comprises the following components:

(1) a therapeutically effective amount of tetrandrine (TET) or a salt thereof;

(2) a therapeutically effective amount of a flavonol or flavanonol compound; preferably, the flavonol compound is Quercetin (QUE) or its related salt; the flavanonol compound is Dihydroquercetin (DHQ) or its related salt;

(3) a therapeutically effective amount of a tyrosine kinase inhibitor; preferably, the tyrosine kinase inhibitor is a third generation tyrosine kinase inhibitor; more preferably, the tyrosine kinase inhibitor is Oxitinib (OSI) or Amitinib (ALM);

and (4) necessary pharmaceutical excipients;

the tumor is lung tumor; preferably, the tumor is non-small cell lung cancer; more preferably, the tumor is tyrosine kinase inhibitor drug-resistant non-small cell lung cancer caused by EGFR mutation.

The invention also relates to an anti-tumor combined preparation, which comprises the following components in part by weight:

(1) a therapeutically effective amount of tetrandrine (TET) or a salt thereof;

(2) a therapeutically effective amount of a tyrosine kinase inhibitor; preferably, the tyrosine kinase inhibitor is a third generation tyrosine kinase inhibitor; more preferably, the tyrosine kinase inhibitor is Oxitinib (OSI) or Amitinib (ALM);

and (3) necessary pharmaceutical excipients;

(1) a therapeutically effective amount of a flavonol or flavanonol compound; preferably, the flavonol compound is Quercetin (QUE) or its related salt; the flavanonol compound is Dihydroquercetin (DHQ) or its related salt;

and (3) necessary pharmaceutical excipients;

The invention also relates to the application of tetrandrine (TET), Quercetin (QUE) or Dihydroquercetin (DHQ) in preparing an anti-tumor combined preparation, wherein the anti-tumor combined preparation at least comprises a tyrosine kinase inhibitor; preferably, the tumor is non-small cell lung cancer; more preferably, the tumor is tyrosine kinase inhibitor drug-resistant non-small cell lung cancer caused by EGFR mutation.

(1) 1-80% of tetrandrine;

(2) 1-80% of dihydroquercetin by mass ratio;

(3) a mass effective amount of oxitinib;

and (4) necessary pharmaceutic adjuvants.

Preferably, in the preparation, the content of tetrandrine: dihydroquercetin: the mass ratio of the oxitinib is 10: 10: 1.

the invention has the beneficial effects that:

1) the drug resistance of the lung cancer cells to the oxitinib is the most common problem in clinical treatment, and the drug resistance can be obviously reversed by applying the composition disclosed by the invention, so that the incidence rate of the drug resistance is reduced.

2) Although the clinical treatment effect of the oxitinib is good, part of patients are not sensitive to the oxitinib, the composition provided by the invention has the possibility of improving the curative effect of the oxitinib treatment, reducing the side effect, shortening the treatment period and prolonging the service life of the lung cancer patients.

3) The composition has a certain anti-tumor effect, and has a certain synergistic effect on other targeted drugs or cytotoxic chemotherapy drugs. Besides the oxitinib, the composition has the potential to be synergistic with more targeted drugs so as to improve the curative effect and survival rate of tumor patients.

4) The tetrandrine and the dihydroquercetin of the composition are both from natural products of traditional Chinese medicines, have low toxicity and high activity, are beneficial to the precise antitumor application of related traditional Chinese medicines, and expand the clinical application range.

Drawings

FIG. 1 the use of tetrandrine or dihydroquercetin alone to enhance the proliferation inhibitory effect of oxitinib on non-H1975 cells.

Figure 2 the combination of tetrandrine and dihydroquercetin enhances the proliferation inhibitory effect of oxitinib on non-H1975 cells.

FIG. 3 the combination of tetrandrine and dihydroquercetin reverses the proliferation inhibitory effect of HCC827/OR anti-Ottitinib cells.

FIG. 4 the composition of tetrandrine and dihydroquercetin enhances the proliferation inhibitory effect of Almetinib on H1975 cells.

FIG. 5 the combination of tetrandrine and quercetin enhances the proliferation inhibitory effect of oxitinib on H1975 cells.

FIG. 6 the composition of tetrandrine and dihydroquercetin enhances the proliferation inhibitory effect of oxitinib on NCI-H460 cells.

Figure 7 the composition of tetrandrine and dihydroquercetin enhances the anti-tumor effect of oxitinib in nude mice.

Detailed Description

Experimental Material

Oxitinib (S7297), tetrandrine (S2403), quercetin (S2347) and dihydroquercetin (S2366) were purchased from SELLECK Chemical company.

Amatinib (S8817) was purchased from SELLECK Chemical company.

EGFR wild type non-small cell lung cancer NCI-H460 cell and non-small cell lung cancer H1975 cell were purchased from the cell center of the institute of basic medicine of Chinese academy of medical sciences.

The non-small cell lung cancer HCC827/OR cells resistant to oxiginib are gifted by Drucker, Shipu, university of Xian traffic.

RPMI-1640 medium (C11875500BT), fetal bovine serum (# 10099-.

60mm plates (430166) and 96-well plates (3599) for cell culture were purchased from Corning. CCK-8 reagent (B34302) was purchased from Bimake, USA.

Example 1 Sinomenine or Dihydroquercetin alone enhances the proliferation inhibitory effect of Oxecitinib on non-Small cell Lung cancer H1975 cells

1.1 CCK-8 method for detecting cell proliferation rate change

1) H1975 cells in logarithmic growth phase were digested with 0.0625% trypsin at 3X 10³The density of each well was seeded in 96-well plates and placed in a 5% CO₂Culturing in a constant-temperature moisturizing incubator at 37 ℃ for 24 hours;

2) adding different concentrations of drug to treat the cells, and setting 3 multiple wells for each concentration;

3) after the drug treatment is carried out for 72h, 20 mu L of CCK-8 reagent is directly added into the cell culture solution, after the mixture is fully mixed, the 96-well plate is put back into the cell culture box again, and the incubation is continued for 1 h;

4) the 96-well plate was placed on an microplate oscillator and shaken for 30s for thorough mixing, and the absorbance value (OD) was measured at a wavelength of 450nm by a microplate reader.

The cell survival rate of each group was calculated by the following formula, in which the cell survival rate of the control group was set to 100%. The calculation formula is as follows: cell survival (%), (OD value-blank OD value of administration group)/(OD value of control group-blank OD value) x 100%.

1.2 results of the experiment

The combination of tetrandrine (TET) with concentration (1,2 μ M) which does not have obvious inhibition effect on cell proliferation and Oxitinib (OSI) can detect that TET has obvious effect of enhancing oxitinib, and the inhibition effect of oxitinib is increased along with the increase of TET concentration (figure 1). Similarly, Dihydroquercetin (DHQ) with concentration (50,100 mu M) without obvious inhibition effect on cell proliferation is selected to be used together with oxitinib, so that the synergistic effect is obvious, and especially the synergistic effect of the high-concentration 100nM OSI effect group is most obvious. The results of this example show that: the single use of TET or DHQ can obviously improve the antitumor activity of the oxitinib.

Example 2 the combination of tetrandrine and dihydroquercetin enhances the proliferation inhibitory effect of oxitinib on non-small cell lung carcinoma H1975 cells

2.1 Experimental methods

The cell proliferation inhibitory effect after the action of the drug was examined by the CCK-8 method, which was the same as in example 1.

2.2 results of the experiment

From the results of fig. 2, it can be seen that: the single TET and DHQ medicines have no obvious inhibition effect on cell proliferation, but can obviously enhance OSI effect after being used together, and have concentration correlation. For example: the combination of 2 μ M TET and 100 μ M DHQ can reduce the cell survival rate of 100nM OSI from 47% to 19.3%, with obvious synergistic effect.

Example 3A combination of tetrandrine and Dihydroquercetin reverses the proliferation inhibitory effects of HCC827/OR Ottitinib cells

3.1 Experimental methods

3.2 results of the experiment

HCC827/OR resistant cells were strongly resistant to oxitinib, therefore, OSI concentrations of 0.1, 1 μ M were selected, and the effect of the composition alone (2 μ M TET +100 μ M DHQ) had almost no effect on cell survival, but significantly enhanced the OSI proliferation inhibitory effect (fig. 3). The composition is proved to have the effect of reversing the drug resistance of the oxitinib.

Example 4 composition of tetrandrine and Dihydroquercetin enhances the proliferation inhibitory effect of Almetinib on H1975 cells

Amitinib (ALM) is a third-generation tyrosine kinase inhibitor with similar effects as oxitinib, and this example observes whether the composition enhances the proliferation inhibitory effect of ALM.

4.1 Experimental methods

4.2 results of the experiment

The inhibition effect of the Almetinib alone on H1975 cells is obviously stronger than that of the Oxitinib, so that the low concentrations of 1nM and 5nM are selected to be combined with the composition, the cell survival rate can be obviously reduced, and the concentration dependence exists (figure 4). For example: wherein the cell survival rate of 1nM ALM can be reduced from 64% to 21.9% by the combined concentration (2 muM TET +100 muM DHQ), and the synergistic effect is very obvious.

Example 5 the combination of tetrandrine and Quercetin enhances the proliferation inhibitory effect of Oxitinib on H1975 cells

Quercetin (QUE) is a compound with similar chemical structure and effect to dihydroquercetin, and the experimental example shows whether the composition of tetrandrine and quercetin can enhance the proliferation inhibition effect of oxitinib.

5.1 Experimental methods

5.3 results of the experiment

The single-drug inhibitory effect of quercetin is significantly stronger than that of dihydroquercetin, and in this example, 10, 20 μ M and TET were selected for combined use. As shown in FIG. 5, the composition of TET and QUE at each concentration clearly showed the growth inhibitory effect of OSI, and had a certain concentration dependence. In particular, the combined concentration (2. mu.M TET + 20. mu.M QUE) can reduce the single-acting cell survival rate of 10nM OSI to 46% to 14.4%, and the synergistic effect is very obvious.

Example 6A composition of tetrandrine and Dihydroquercetin enhances the proliferation inhibitory effect of Oxecitinib on NCI-H460 cells

While oxitinib is primarily used for the treatment of EGFR-mutated non-small cell lung cancer, this example shows whether this composition enhances the proliferation inhibition of EGFR wild-type NCI-H460 cells by oxitinib.

6.1 Experimental methods

6.3 results of the experiment

The NCI-H460 cells were not OSI-sensitive, so higher concentrations of OSI were selected for use with the compositions. The results show that the composition has obvious synergistic effect and has concentration dependence, and especially the combined concentration (4 mu M TET +200 mu M DHQ) can reduce the cell survival rate of 0.1 mu M OSI from 99% to 26.7%, and the synergistic effect is obvious (figure 6).

Example 7 composition of tetrandrine and Dihydroquercetin enhances the anti-tumor effects of Oxecitinib in nude mice

To better evaluate the results of the in vivo effect of the composition, H1975 cells were inoculated into nude mice for tumor formation at a dose of 10mg/kg TET and 10mg/kg DHQ, and the tumor-inhibiting effect of the composition in combination with oxitinib in vivo was examined by administering the same dose.

7.1 Experimental materials

Oxitinib (S7297) and tetrandrine (S2403) were obtained from SELLECK chemical, dihydroquercetin was obtained from Jianwei Natural Biotech Co., Ltd, Jilin province, and the source of H1975 cells was the same as in example 1.

Experimental animals: 18-22g female BALB/c nude mice: purchased from sbefu (beijing) biotechnology limited, certification No.: 110324211103078355.

experimental equipment: scalpel, big forceps, flat head scissors, flat head forceps, trocar, 1ml syringe, normal saline, collodion cotton, alcohol cotton ball, etc.

Preparing experimental medicines: 5mg of OSI is weighed and dissolved in 100. mu.l of ethanol to prepare a 50mg/ml mother liquor, 3.2. mu.l of normal saline is added into 1596.8. mu.l each time to be mixed evenly, and the dosage is 1 mg/kg. 1.6mg of TET was suspended in 1.6ml of physiological saline at a dose of 10 mg/kg. 10mg of DHQ is weighed and dissolved in 100 mul of ethanol to prepare a mother solution of 100mg/ml, 16 mul of the mother solution is added with 1584 mul of physiological saline each time and mixed evenly, and the dosage is 10 mg/kg.

7.2 Experimental methods

Feeding nude mice in specific germ-free environment, and making the volume reach 1000mm when the mice are adapted to environment and recovered³The H1975 tumor mass of (1) was taken out of the body of the mouse and cut into 1mm with a sterilized scalpel³The small blocks were inoculated to the right forelimb of mice and randomly grouped, and 5 were kept in one group.

When the tumor mass grows to a palpable extent (after about one week), the mice are marked and randomly grouped on average according to the body weight and the tumor mass size, a solvent control group, an OSI, TET, DHQ single drug group, and three drug combination group are set, and the drug is administered once every two days, and simultaneously weighed, and the long diameter and the short diameter of the tumor are measured by a vernier caliper to estimate the tumor volume, which is (long diameter × short diameter 2) ÷ 2. The administration mode is intragastric administration, and 200 μ l/tube.

Seven times after administration, animals were sacrificed, tumor masses were taken and weighed, and the tumor inhibition rate (%) of each group was calculated as (average tumor weight of control group-average tumor weight of administration group)/average tumor weight of control group x 100%.

7.3 results of the experiment

According to the results of periodic measurements of the change in tumor volume (FIG. 7A), 1mg/kg OSI, 10mg/kg TET and 10mg/kg DHQ, when administered alone, all had some inhibitory effect, with OSI being the most significant. The combination of the three medicines shows obvious in-vivo tumor inhibition effect, and the tumor volume is hardly increased obviously. The last dose gave results on tumor weight (fig. 7C, D), similar to the volume change, with OSI single drug inhibition of 52.8%, stronger than single drug TET (33.7%) and single drug DHQ (6.1%); the tumor inhibition rate of the combined composition is as high as 86.6 percent, and the synergistic effect is very obvious. During the dosing period, we monitored the body weight changes of the mice, and the body weight changes of the mice of each group were not significantly different (fig. 7B), indicating that the toxicity of the composition was low.

Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not used to limit the protection scope of the present invention.

Claims

1. An anti-tumor combined preparation, which comprises:

(1) a therapeutically effective amount of tetrandrine (TET) or a salt thereof;

and (4) necessary pharmaceutic adjuvants.

2. An anti-tumor combined preparation, which comprises:

(1) a therapeutically effective amount of tetrandrine (TET) or a salt thereof;

and (3) necessary pharmaceutic adjuvants.

3. An anti-tumor combined preparation, which comprises:

and (3) necessary pharmaceutic adjuvants.

4. The anti-tumor combination according to any one of claims 1 to 3, wherein the tumor is a lung tumor;

preferably, the tumor is non-small cell lung cancer;

more preferably, the tumor is tyrosine kinase inhibitor drug-resistant non-small cell lung cancer caused by EGFR mutation.

5. The application of tetrandrine (TET), Quercetin (QUE) or Dihydroquercetin (DHQ) in preparing an anti-tumor combined preparation, wherein the anti-tumor combined preparation at least comprises a tyrosine kinase inhibitor.

6. The use of claim 5, wherein the tumor is non-small cell lung cancer; preferably, the tumor is tyrosine kinase inhibitor drug-resistant non-small cell lung cancer caused by EGFR mutation.

7. An anti-tumor combined preparation, which comprises:

(1) 1-80% of tetrandrine;

(2) 1-80% of dihydroquercetin by mass ratio;

(3) a mass effective amount of oxitinib;

and (4) necessary pharmaceutic adjuvants.

8. The combined preparation for resisting tumors according to claim 7, wherein the content of tetrandrine: dihydroquercetin: the mass ratio of the oxitinib is 10: 10: 1.