CN113274394A

CN113274394A - Pharmaceutical composition for treating tyrosine kinase inhibitor drug-resistant non-small cell lung cancer

Info

Publication number: CN113274394A
Application number: CN202110690308.3A
Authority: CN
Inventors: 朱虹; 杨波; 何俏军; 曾晨鸣; 袁涛
Original assignee: Institute Of Intelligent Innovative Medicine Zhejiang University
Current assignee: Institute Of Intelligent Innovative Medicine Zhejiang University
Priority date: 2021-06-22
Filing date: 2021-06-22
Publication date: 2021-08-20
Anticipated expiration: 2041-06-22
Also published as: CN113274394B

Abstract

The invention discloses a pharmaceutical composition for treating tyrosine kinase inhibitor drug-resistant non-small cell lung cancer, belonging to the technical field of medicines. The active ingredients of the pharmaceutical composition are oxitinib and cystic fibrosis transmembrane conductance regulator agonist, the oxitinib and CFTR agonist are combined, the inhibition rate of TKIs-resistant multiple NSCLC cells is remarkably higher than that of single drugs, and the combined drugs of the oxitinib and the CFTR agonist have synergistic inhibition effect on TKIs-resistant multiple NSCLC cells. The experimental result of the invention provides theoretical basis for clinical combination of the oxitinib and the CFTR agonist and an effective scheme for treating TKIs (TKIs resistant NSCLC), and has wide application prospect in the field of medicine and pharmacology.

Description

Pharmaceutical composition for treating tyrosine kinase inhibitor drug-resistant non-small cell lung cancer

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a pharmaceutical composition for treating tyrosine kinase inhibitor-resistant non-small cell lung cancer and application thereof.

Background

Lung cancer is the first malignant tumor in both incidence and mortality in China. Epidermal Growth Factor Receptor (EGFR) mutations have been detected in many Non-small cell lung cancers (NSCLC), accounting for approximately 15-40% of lung adenocarcinomas. The medicine targeting EGFR mutation lung cancer becomes an important means in the process of treating lung cancer. Tyrosine Kinase Inhibitors (TKIs) are small molecule inhibitors that inhibit malignant proliferation of lung cancer cells by blocking the kinase activity of EGFR by competing with ATP for the intracellular Tyrosine kinase catalytic site of EGFR.

However, a large number of clinical and experimental data indicate that most patients develop acquired resistance after treatment with EGRF-TKIs. Studies have shown that the acquired T790M mutation is the most common cause of drug resistance in patients with advanced EGFR mutations, with most patients presenting the T790M mutation after first-line use of EGFR-TKIs. Currently, a number of EGFR mutation selective EGFR-TKIs have been developed to treat patients with acquired resistance due to the T790M mutation, such as ocitinib. Oxitinib (Osimetinib) is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor with a molecular formula of C₂₈H₃₃N₇O₂The structural formula is as follows:

however, patients receiving third generation EGFR-TKI treatment develop acquired resistance despite a better initial response.

For the drug resistance of third-generation TKIs, the secondary drug resistance mechanism which is discovered at present mainly comprises the following four aspects: (1) secondary mutations are generated. The T790M mutation is found to be the important reason for the drug resistance of the first and second generation TKIs. The secondary mutation of the EGFR gene is also an important reason for causing the drug resistance of third-generation TKIs (such as oxitinib), and potential drug resistance mutation sites discovered at present are C797S, L718, G796, L792 and the like; (2) compensatory activation of other signal pathways. The EGFR-TKIs block the EGFR pathway of the tumor cells, so that the abnormal proliferation of the tumor cells is inhibited. Compensatory activation of the bypass pathway has been found to cause secondary drug resistance. For example, MET gene amplification abnormally activates AKT, facilitating activation of the downstream mTOR pathway, thereby promoting proliferation of tumor cells. In addition, abnormal up-regulation of HER2, compensatory activation of the AXL pathway, IGF1R mediated AKT activation also play important roles in secondary resistance to third-generation TKIs; (3) EGFR-TKIs mediated apoptosis pathway inhibition; (4) transformation of small-cell lung cancer (SCLC).

However, although various mechanisms of resistance have been discovered, the mechanism of secondary resistance that generates third-generation TKIs is still unclear in about one-third of cases. Because the single medicine has higher probability of generating drug resistance of a human body to a specific medicine and unsatisfactory curative effect, the combined chemotherapy of the antitumor medicine, an antibody, an inhibitor, a sensitizer and the like is usually adopted clinically, so that the toxic and side effect of the chemotherapeutic medicine is reduced, and the antitumor effect of the medicine is improved. To date, there has been no report on the use of a combination of oxitinib and a CFTR agonist for the treatment of non-small cell lung cancer.

Disclosure of Invention

The invention aims to provide a medicinal composition for treating tyrosine kinase inhibitor-resistant non-small cell lung cancer, which aims to solve the problems of poor curative effect, drug resistance and the like of non-small cell lung cancer patients in the treatment process in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

use of oxitinib in combination with a cystic fibrosis transmembrane conductance regulator agonist for the manufacture of a medicament for the treatment of tyrosine kinase inhibitor-resistant non-small cell lung cancer.

The research of the invention shows that the cystic fibrosis transmembrane conductance regulator (CFTR) agonist can enhance the application of the ocitinib in the treatment of non-small cell lung cancer (NSCLC) with acquired drug resistance of Tyrosine Kinase Inhibitors (TKIs), and the combination of the cystic fibrosis transmembrane conductance regulator (CFTR) agonist and the TKIs enhances the curative effect of the ocitinib, thereby providing a basis for the new application of old drugs.

Further, the tyrosine kinase inhibitor-resistant non-small cell lung cancer is an oxitinib secondary drug-resistant non-small cell lung cancer.

Preferably, the cystic fibrosis transmembrane conductance regulator agonist is ivacator. Ivacaftor (Ivacaftor) is an orally administrable CFTR synergist named N- (2, 4-di-tert-butyl-5-hydroxyphenyl) -1, 4-dihydro-4-oxo-3-quinolinecarboxamide with the molecular formula C₂₄H₂₈N₂O₃The structural formula is as follows:

preferably, the molar ratio of the ivacaiton to the oxitinib in the medicine is 1.5-384: 1.

further preferably, the molar ratio of the ivakato to the oxitinib in the medicine is 3-16: 1.

more preferably, the molar ratio of ivacaiton to oxitinib in the medicament is 8: 1.

further, the invention provides a pharmaceutical preparation, which comprises the active ingredients of the medicine and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier is a filler, wetting agent, binder, disintegrant, or lubricant.

Preferably, the preparation form of the medicament is oral preparation, and the specific preparation form can be, but is not limited to, oral tablets, granules, injections and capsules.

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

the research of the invention finds that the combined use of the oxitinib and the CFTR agonist has a significantly higher inhibition rate on TKIs-resistant multiple NSCLC cells than that of the single use of the oxitinib and the CFTR agonist, and the combined use of the oxitinib and the CFTR agonist has a synergistic inhibition effect on TKIs-resistant multiple NSCLC cells. The combined medication not only can reduce the toxic and side effect caused by the medication, but also has the advantage of incomparable medication, can achieve the curative effect caused by the medication by using a smaller dosage of the medication, reduces the drug resistance of tumor cells, and has wider application prospect compared with the medication by using the medication alone.

Drawings

Fig. 1 shows the inhibition of PC-9-OR cell proliferation by esitinib in combination with ivacaiton at different concentrations (n-3).

Fig. 2 shows the inhibition of PC-9-OR cell proliferation by fixed concentrations of ivacaiton in combination with varying concentrations of ocitinib (n-3).

FIG. 3 is a graph of the inhibition of proliferation of NCI-H1975 Oxitinib-resistant cells by fixed concentrations of Evacatuo in combination with varying concentrations of Oxitinib (n ═ 3).

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The kit materials used in the following examples are all commercially available unless otherwise specified.

Example 1 inhibition of proliferation of Oxitinib in combination with Ivacatuo on Oxitinib-resistant Lung cancer PC-9 cells

1. Experimental materials

(1) PC-9 cells, provided by the stem cell bank of the Chinese academy of sciences; PC-9 Oxititinib-resistant cells (PC-9-OR) were constructed by the laboratory according to the concentration gradient increasing method.

(2) Oxitinib, available from MedChemexpress (MCE).

(3) Ivakato, available from Selleck.

2. Cell culture medium

500mL of RPMI-1640 culture solution is subpackaged into 45 mL/part in an ultra-clean workbench, 5mL of fetal bovine serum is added into each part, 1% of mixed antibiotics of penicillin and streptomycin are added, and the mixture is stored in a refrigerator at 4 ℃ for later use.

3. Determination of cell proliferation inhibition by SRB method

Taking PC-9 oxicetinic drug-resistant cells in logarithmic growth phase, and adding 100 mu L of culture solution into each well of a 96-well plate to obtain 2X 10³And (4) placing the individual cells into a cell culture box for culturing for 24 hours to make the cells in the 96-well plate adhere to the wall. Experiment ofDifferent concentrations of Oxitritinib (0.0625, 0.125, 0.25, 0.5, 1, 2 mu mol/L), different concentrations of Evacantol (1, 2,4, 6, 8, 10 mu mol/L) and different concentrations of combination drug (0.0625 mu mol/L Oxitrinib +1 mu mol/L Evacantol, 0.125 mu mol/L Oxitrinib +2 mu mol/L Evacantol, 0.25 mu mol/L Oxitrinib +4 mu mol/L Evacantol, 0.5 mu mol/L Oxitrinib +6 mu mol/L Evacantol, 1 mu mol/L Oxitrinib +8 mu mol/L Evacantol, 2 mu mol/L Oxitrinib +10 mu mol/L Evacantol); using culture medium containing DMSO (concentration less than 0.1%) as control group; medium served as blank.

After 72 hours of action, the stock culture solution is discarded, 200. mu.L of 10% trichloroacetic acid solution is added into each well, and the mixture is placed in a refrigerator at 4 ℃ for fixation for 30 min. Taking out, washing with deionized water for 5 times, and air drying at room temperature. After the 96-well plate is dried at room temperature, 70 microliter of SRB dye solution (prepared by 1% acetic acid) of 0.4% (w/v) is added into each well, the dye solution is poured out after dyeing for 30min, the dye solution is washed for 5 times by 1% (v/v) acetic acid, unbound dye is removed, and the plate is dried at room temperature. The dye bound to the cell protein was solubilized in 100. mu.L of unbuffered Tris-base lye (10mM, pH 10.5), shaken on a horizontal shaker for 20min, and the light absorption (OD) was measured using a microplate reader at 540 nm. The inhibition rate of cell proliferation was calculated as follows:

the inhibition rate is 1- (the OD value of the drug addition group-the OD value of the blank group)/(the OD value of the control group-the OD value of the blank group) × 100%.

The results of the experiment are shown in table 1 and fig. 1.

TABLE 1 inhibition of proliferation of PC-9 Oxitinib resistant cells (PC-9-OR) by Oxitinib in combination with Evacatuo at different concentrations (n ═ 3)

As can be seen from the experimental results in Table 1 and FIG. 1, after the Oxovitinib and Ivakato act on the PC-9 Oxovitinib drug-resistant cells (PC-9-OR) alone for 72h, the effect of inhibiting cell proliferation is obviously enhanced along with the increase of the drug concentration, and the effect of inhibiting the proliferation of the PC-9-OR is in a concentration-dependent relationship.

And Oxitinib (0.015625 mu mol/L, 0.03125 mu mol/L, 0.0625 mu mol/L, 0.125 mu mol/L, 0.25 mu mol/L, 0.5 mu mol/L, 1 mu mol/L and 2 mu mol/L) with different concentrations is selected to be respectively combined with the Ivacator 6 mu mol/L to act on the PC-9-OR, and the experimental results are shown in Table 2 and figure 2.

TABLE 2 fixed concentration of Ivacatuo in combination with varying concentrations of oxitinib for inhibition of proliferation of PC-9-OR cells (n ═ 3)

The experimental results in table 2 and fig. 2 show that the inhibition rate of the combined drug of oxitinib and ivakatom on the PC-9 oxitinib drug-resistant cells is obviously higher than that of the single drug group and the IC of the single oxitinib group₅₀Oxititinib IC with value of 2.339. mu.M, combination group₅₀The value is 0.2303 μ M, i.e. the combination of oxitinib and ivacaiton has a synergistic inhibitory effect on PC-9-OR.

Example 2 inhibition of proliferation of Oxitinib in combination with Evacatuo on Oxitinib-resistant Lung cancer NCI-H1975 cells

1. Experimental materials

(1) NCl-H1975 cells, provided by the stem cell bank of the Chinese academy of sciences; NCI-H1975 Oxititinib drug-resistant cells, which are constructed by the laboratory according to a concentration gradient increasing method;

(2) oxitinib, available from MedChemexpress (MCE).

(3) Ivakato, available from Selleck.

2. Cell culture medium

3. Determination of cell proliferation inhibition by SRB method

Taking NCl-H1975 Oxititinib drug-resistant cells in logarithmic growth phase, and adding 100 μ L of culture solution into each well of 96-well plate to obtain a mixture of 2 × 10³And (4) placing the individual cells into a cell culture box for culturing for 24 hours to make the cells in the 96-well plate adhere to the wall. The experimental components are respectively added with different concentrations of Oxicitinib (0.015625 mu mol/L, 0.03125 mu mol/L, 0.0625 mu mol/L, 0.125 mu mol/L, 0.25 mu mol/L, 0.5 mu mol/L, 1 mu mol/L, 2 mu mol/L and 4 mu mol/L), fixed concentration of Evacatol (6 mu mol/L), and combined medicines (0.015625 mu mol/L Oxicitinib +6 mu mol/L Evacatol, 0.03125 mu mol/L Oxicitinib +6 mu mol/L Evacatol, 0.0625 mu mol/L Oxicitinib +6 mu mol/L Evacatol, 0.125 mu mol/L Oxicitinib +6 mu mol/L Evacatol, 0.25 mu mol/L Oxicitinib +6 mu mol/L Evacatuo, 0.5 mu mol/L Evacatol +6 mu mol/L Evacatol, 1 mu mol/L of Oxitinib +6 mu mol/L of Evakato, 2 mu mol/L of Oxitinib +6 mu mol/L of Evakato, 4 mu mol/L of Oxitinib +6 mu mol/L of Evakato); using culture medium containing DMSO (concentration less than 0.1%) as control group; medium served as blank.

The results of the experiment are shown in table 3 and fig. 3.

TABLE 3 fixed concentration of Ivacatuo in combination with varying concentrations of Oxitinib inhibition of proliferation of NCI-H1975 Oxitinib resistant cells (n ═ 3)

As can be seen from the experimental results in Table 3 and FIG. 3, the inhibition rate of the combination of Oxitinib and Evacatu on NCI-H1975 Oxitinib drug-resistant cells is significantly higher than the IC of the single drug group and the single Oxitinib group₅₀Oxitinib IC with value of 0.6627 μ M, combination group₅₀The value is 0.0976 mu M, namely the combination of the oxitinib and the ivakato has synergistic inhibition effect on NCl-H1975 oxitinib drug-resistant cells.

The experimental result provides theoretical basis for clinical combination of the oxitinib and the CFTR agonist and an effective scheme for treating TKIs (TKIs resistant NSCLC), and has wide application prospect in the field of medicine and pharmacology.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications or equivalents may be made to the technical solution without departing from the principle of the present invention, and these modifications or equivalents should also be regarded as the protection scope of the present invention.

Claims

1. Use of oxitinib in combination with a cystic fibrosis transmembrane conductance regulator agonist for the manufacture of a medicament for the treatment of tyrosine kinase inhibitor-resistant non-small cell lung cancer.

2. The use of claim 1, wherein the tyrosine kinase inhibitor-resistant non-small cell lung cancer is an oxitinib secondary-resistant non-small cell lung cancer.

3. The use of claim 1, wherein the cystic fibrosis transmembrane conductance regulator agonist is ivacator.

4. The use according to claim 3, wherein the molar ratio of ivacaiton to oxitinib in the medicament is from 1.5 to 384: 1.

5. the use according to claim 4, wherein the molar ratio of ivacaiton to oxitinib in the medicament is 3-16: 1.

6. the use of claim 5, wherein the molar ratio of ivacaiton to oxitinib in the medicament is 8: 1.

7. the use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier.

8. The use of claim 7, wherein the pharmaceutically acceptable carrier is a filler, wetting agent, binder, disintegrant, or lubricant.

9. The use according to claim 7, wherein the medicament is formulated in the form of oral tablets, granules, injections or capsules.