CN110664818B

CN110664818B - Medicine for treating lung cancer

Info

Publication number: CN110664818B
Application number: CN201910972043.9A
Authority: CN
Inventors: 陈良; 陈智鹏
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2019-10-14
Filing date: 2019-10-14
Publication date: 2021-03-19
Anticipated expiration: 2039-10-14
Also published as: CN110664818A

Abstract

The invention discloses a medicament for treating lung cancer, which consists of NG25 and trametinib in a mass ratio of 4: 1; the lung cancer is lung cancer with CLU gene function loss or down regulation. Further, the lung cancer is deficient or down-regulated in CLU gene function and is induced by KRAS^G12DOncogene driven lung cancer. In vitro experiments show that lung cancer cells with reduced CLU knock-out are more sensitive to NG25, compared with a control, NG25 can obviously inhibit the growth of the lung cancer cells, and when the cells are treated together with Trametinib, the lung cancer cells are more obviously inhibited. Similarly, in an animal model, the TAK1 inhibitor NG25 can inhibit the growth of CLU-knocked-down cell transplanted tumors and CLU-knocked-down in-situ lung cancer, and the tumor can be remarkably atrophied by combining with the treatment of Trametinib, so that the purpose of treating the type of lung cancer is achieved.

Description

Medicine for treating lung cancer

Technical Field

The invention belongs to the field of biological medicine, particularly relates to a medicine for treating lung cancer, and particularly relates to a combination of a TAK1 inhibitor NG25 and Trametinib (Trametinib) on KRAS accompanied with CLU gene function deficiency^G12DA therapeutic drug for cancer gene driven lung cancer.

Background

It is now known that the occurrence of lung cancer is mainly associated with changes in oncogenes. KRAS is a common driver gene mutation in lung cancer, with mutations in this gene being present in 10-30% of lung cancer patients. To date, there is a clinical lack of effective targeted therapies or methods for this type of lung cancer.

Although some effect of targeted therapy of lung cancer has been achieved, a significant proportion of patients still show no due therapeutic effect after taking the inhibitor against the oncogene mutated in tumors. There are many reasons for this, but the role of oncogenes is of concern. Intensive research also shows that the cancer suppressor genes have important influence on the development of lung cancer and the sensitivity of medicaments. Therefore, elucidation of the mechanism of action of oncogenes in lung cancer will help to provide an effective therapeutic strategy for the treatment of lung cancer caused by oncogene and oncogene mutations.

At present, the discovery of cancer suppressor genes of lung cancer is very limited, and besides the frequency of inactivation of the P53 gene, RB gene and CDKN2A gene in lung cancer is very high, a lot of genes with very low frequency such as SMAD2, SMAD4, TGFBR2, PTEN and the like are also discovered. In addition, low expression of some genes, such as the CLU gene, has been found to have an effect on the development of lung cancer.

The CLU gene is located on chromosome eight, contains 9 exons, and its protein Clusterin is widely distributed in tissues and is involved in a number of cellular processes, including: tissue differentiation, lipid transport, cell proliferation, apoptosis, and the like. Clusterin expression is regulated by growth factors. In addition, the protooncogenes H-Ras, c-Myc and N-Myc inhibit CLU expression. Therefore, it is considered that: inhibition of CLU expression is required for oncogene-dependent cell transformation. The study on the prostatic cancer shows that the mouse knockout of CLU can induce the generation of prostatic epithelial sarcoma (PIN), promote the cell proliferation and the enhancement of NF-kB signals, and can promote the malignant development of tumors. CLU was also found to exert a negative tumor-controlling effect in gliomas. From the Oncomine database, CLU was found to be down-regulated in mRNA levels in most primary tumors and is inversely proportional to the malignancy stage and extent of the cancer, including prostate, breast, lung. This suggests that loss of CLU function plays an important role in the development of lung cancer. However, current treatments for this type of lung cancer, even lung cancer that also simultaneously carries a KRAS mutation, lack effective treatment.

Disclosure of Invention

Based on the lack of guidance and effective treatment means aiming at CLU-deficient lung cancer, the invention aims to provide a composition for treating lung cancer. The invention constructs stable cells with reduced CLU, mouse transplantation tumor with reduced CLU and KRAS accompanied with CLU knockout^G12DAnimal models of mouse orthotopic lung cancer maximally mimic CLU-deficient lung cancers, and these models are then used to evaluate the therapeutic effect of TAK1 inhibitors on such lung cancers, and in combination with Trametinib, to treat such lung cancers that also carry KRAS mutations.

The purpose of the invention is realized by the following technical scheme:

a medicine for treating lung cancer comprises NG25(N- [4- [ (4-ethyl-1-piperazinyl) methyl ] -3- (trifluoromethyl) phenyl ] -4-methyl-3- (1H-pyrrolo [2,3-B ] pyridin-4-yloxy) benzamide) and trametinib;

preferably, in the medicament, the mass ratio of NG25 to trametinib is 4: 1;

preferably, the lung cancer is lung cancer with functional deletion or down-regulation of CLU gene;

preferably, the lung cancer is deficient or down-regulated in CLU gene function and is produced by KRAS^G12DOncogene driven lung cancer;

further, the lung cancer includes carcinoma in situ and transplantation carcinoma;

the medicine can also contain more than one pharmaceutically acceptable carrier; the carrier is preferably a sustained-release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant and the like;

the medicine can be further prepared into various forms such as injection, tablets, granules or capsules, and the medicines of various formulations can be prepared according to the conventional method in the pharmaceutical field.

The invention discovers that the deletion of the CLU function can promote the activation of the TAK1 pathway, thereby activating the downstream NFKB signal and promoting the cell growth; and the inhibition of the activation of the TAK1 signal can inhibit the growth of the lung cancer cells caused by the down-regulation of Clusterin expression. These studies suggest that the TAK1 signal is a potential target for lung cancer therapy on which Clusterin downregulation depends.

N- [4- [ (4-ethyl-1-piperazinyl) methyl ] -3- (trifluoromethyl) phenyl ] -4-methyl-3- (1H-pyrrolo [2,3-B ] pyridin-4-yloxy) benzamide (NG25 trihydrochloride), NG25 for short, is a dual inhibitor of TAK1 and MAP4K 2. NG25 selectively binds to TAK1 and MAP4K2 to inhibit their activity, thereby inhibiting the activation of phosphorylation of proteins downstream thereof, such as IKK, p38 and JNK.

Compared with the prior art, the invention has the following advantages and effects:

the application discovers through a great deal of creative work: the TAK1 signaling pathway plays an important role in the growth of KRAS-mutated lung cancer with CLU loss of function, inhibition of TAK1 signaling can effectively prevent the growth of such lung cancer cells, and combination with the MEK inhibitor Trametinib can more effectively inhibit the growth of lung cancer. In vitro experiments found that lung cancer cells with reduced CLU knockdown were more sensitive to NG25, NG25 significantly inhibited lung cancer cell growth compared to controls, and when cells were co-treated with Trametinib, lung cancer cells were more significantly inhibited. Similarly, in an animal model, the TAK1 inhibitor NG25 can inhibit the growth of CLU-knocked-down cell transplanted tumors and CLU-knocked-down in-situ lung cancer, and the tumor can be remarkably atrophied by combining with the treatment of Trametinib, so that the purpose of treating the type of lung cancer is achieved.

Drawings

FIG. 1 is a graph of CLU-knockdown lung cancer cell Hop62 susceptibility to NG 25; wherein A is a protein immunoblot evaluation chart of the CLU knockdown effect of Hop62 cells; b is half inhibitory concentration (IC50) of the control group Hop62-shGFP cell to NG 25; c is half inhibitory concentration of CLU knockdown cell Hop62-shCLU to NG25 (IC 50); d is the plate clone result of the NG25 processed Hop62-shGFP and Hop62-shCLU cells, and the right is a statistical chart of the cell clone number; e is a graph of the degree of inhibition of NG25 on cell clones.

FIG. 2 is a graph showing the synergistic evaluation of NG25 and Trametinib on the inhibition of cell growth; wherein A is the result of CCK8 experiment evaluation on the growth influence of the cells of NG25 and Trametinib which are treated independently and cooperatively; b is the result of the influence of the drug and the synergistic treatment on the clone formation shown in the plate clone evaluation, and the right graph is the statistics of the cell clone number.

FIG. 3 is a graph showing the evaluation of the synergistic effect of the drugs on the growth inhibition of tumors in subcutaneous transplantable tumors in nude mice; wherein A is the recorded result of the change of the tumor size in the two-week process of the drug and the synergistic treatment; b is a comparison of tumor size taken two weeks after drug treatment, and the right is a statistical plot of tumor weight.

FIG. 4 shows LSL-KRAS^G12DThe inhibition effect diagram of the drugs and the synergistic treatment on the primary tumor of the lung cancer is evaluated in a lung cancer transgenic mouse model; wherein A is CT images before and after treatment of the mice; and B is a quantitative graph of the change of the tumor size after two weeks of drug treatment.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

In the following examples NG25 is an inhibitor of TAK1 and Trametinib is an inhibitor of MEK

Example 1

CLU-knockdown lung cancer cells are more sensitive to the TAK1 inhibitor NG25

First, experiment method

(1) Hop62 cell construction for stable CLU knockdown cell line construction: the expression vector pLKO.1-shCLU (Sigma, TRCN0000078611, sequence: CCGGGCTAAAGTCCTACCAGTGGAACTCGAGTTCCACTGGTAGGACTTTAGCTTTTTG) was first lentivirally packaged with 293T cells cotransformed with the viral backbone vector psPAX and PM2.G, with pLKO.1-shGFP (Sigma, SHC004) as a control. Hop62 cells were infected with the packaged shCLU and shGFP viruses, respectively, and then 2ug/mL of Puromycin was used to select cell lines stably knocked down for CLU. The knockdown effect of the cell line was evaluated by immunoblotting.

(2) The sensitivity of Hop62-shCLU cells to NG25 was examined by the method of CCK 8: adding Hop62-shGFP and Hop62-shCLU cells (800 cells/well) into a 96-well plate, treating the cells with NG25 at concentrations of 0, 0.01625, 0.3125, 0.625, 1.25, 2.5, 5 and 10 μ M for 6 days, detecting the cell activity by the method of CCK8, and finally calculating the half-inhibitory concentration of the drug to the cells by the GraphPad software (IC50)

(3) Plate colony formation assay CLU knockdown cells were evaluated for sensitivity to NG 25: 800/well of Hop62-shGFP and Hop62-shCLU cells were seeded in 6-well plates and treated with different concentrations of NG25, respectively. After 7 days of cell culture, fixation and crystal violet staining were performed, and statistics and analysis of changes in cell clone numbers were performed.

Second, experimental results and analysis

(1) CLU expression in a Hop62-shCLU stable cell line was shown to be significantly down-regulated compared to the control Hop62-shGFP by immunoblotting methods (fig. 1A).

(2) The half-inhibitory concentration of Hop62-shGFP cells on NG25 was higher than that of Hop62-shCLU cells (FIG. 1B and FIG. 1C) as detected by CCK8 cell activity, indicating that Hop62 cells are more sensitive to the drug NG25 after CLU knockdown.

(3) In the evaluation of the colony forming ability of the plate, it was found that the ability of the Hop62-shCLU cell plate clone was significantly reduced compared to the control cell under the treatment of NG25 at a concentration of 2.5. mu.M (FIG. 1D). From the analysis of the degree of drug effect, the number of shGFP clones in the control cells was reduced by 8.9%, while that in the Hop62-shCLU cells was reduced by 36.2% (FIG. 1E). These results also indicate that Hop62-shCLU cells are more sensitive to NG 25.

Example 2

NG25 synergistic with Trametinib can obviously inhibit CLU down-regulated lung cancer cell growth

First, experiment method

(1) Hop62-shCLU cells were seeded in 96-well plates at 800cells per well, and then the cells were treated with four ways of DMSO (control), NG25 (1.25. mu.M), Trametinib (Tram,25nM) and NG25 in combination with Tram (1.25. mu.M +25nM), respectively. The growth activity of the cells was measured with CCK8 every day, and the growth of the cells was plotted after 6 days.

(2) Plate clonogenic evaluation the effect of NG25 in combination with Tram on cell growth: the 6-well plates were seeded with 400 cells/well and the cells were treated with four different combinations of DMSO (control), NG25 (1.25. mu.M), Tram (25nM) and NG25 in combination with Tram (1.25. mu.M +25 nM). After 7 days of culture, cells were fixed, stained, and statistically analyzed.

Second, experimental results and analysis

(1) In the cell growth assay, NG25 slightly inhibited cell growth at this concentration treatment, and Tram had a significant inhibitory effect on cells compared to the DMSO group, whereas when the two drugs were combined, cell growth was significantly inhibited, with a significantly better effect than the treatment with the two drugs alone (fig. 2A).

(2) In a plate clone formation experiment, the single drugs of NG25 and Tram are also found to have obvious inhibition effect on the clonogenic capacity of Hop62-shCLU cells. When the two drugs act in combination, the clonogenic capacity of the cells is greatly inhibited (FIG. 2B).

Example 3

NG25 combined with Trametinib can effectively inhibit growth of CLU (CLU-downregulated lung cancer cell) subcutaneous transplantation tumor

(1) Resuspending CLU-knockdown Lung cancer cell Hop62-shCLU in BD matrixIn matrigel (3X 10 per graft tumor required)⁶Cell count, resuspend cells in 100 μ Ι _ BD Matrigel). The above cell and matrix mixture was inoculated into 100. mu.L each of the left and right flank of the dorsal subcutaneous skin of nude mice.

(2) After the nude mice are inoculated with the fixed tumors for about 6 days, the nude mice are divided into 4 groups, namely a blank group (no treatment), an NG25 treatment group (intraperitoneal injection, 4mg/kg/Day), a Tram treatment group (intragastric administration, 1mg/kg/Day) and a combined treatment group of NG25 and Tram (two medicaments are simultaneously administered). Hop62-shGFP was used as a control group of cells.

(3) Tumor size was measured every two days from the start of treatment according to the formula: length x width²Count size of/2. Nude mice were sacrificed 2 weeks after treatment and tumors were dissected.

Second, experimental results and analysis

Compared with the shGFP control, the Hop62-shCLU tumor grows faster and has larger tumor volume. Compared with untreated tumors, NG25 and Tram were single drugs having inhibitory effect on the growth of Hop62-shCLU tumors, whereas when NG25 and Tram were combined to treat nude mice, it was found that the growth of tumors was very significantly inhibited (fig. 3A) and the tumor volume and tumors were significantly reduced (fig. 3B).

Example 4

NG25 combined with Trametinib can effectively treat in-situ tumor

The transplanted tumor treatment model can conveniently observe and measure the size change of tumor growth in the evaluation of the drug effect, has a single system and can clearly explain the problem. But has the disadvantage of not simulating the complex canceration, tumor formation process, and interaction between tumor and stromal cells of primary lung cancer. Thus, the ability to predict treatment of a drug in a patient is poor. To this end, the present invention further utilizes LSL-KRAS^G12DLung cancer orthotopic tumor model of transgenic mice the therapeutic effect of NG25 in combination with Tram was evaluated.

First, experiment method

(1) About 8 weeks LSL-KRAS^G12DThe mouse of (2) was nasally infected with the virus of pSECC-sgCLU (the virus contains Cre, Cas9 and sgCLU, sequence: GGAGATTCAGAACGCCGTCC), and the virus was allowed to enter the mouse lung from the trachea and thenTo induce KRAS in lung epithelial cells^G12DThe activation and knocking-out of CLU gene in the cell simulates the occurrence of KRAS mutation-induced lung cancer under the condition of CLU down regulation. Mice developed lung cancer more rapidly half a year later due to CLU knockout.

(2) Computed Tomography (CT) records the size and severity of the tumor.

(3) Mice with lung cancer were divided into three groups, namely, an NG25 treatment group (intraperitoneal injection, 4mg/kg/Day), a Tram treatment group (intragastric administration, 1mg/kg/Day) and a NG25 and Tram combined treatment group (two drugs are simultaneously administered), and administration treatment was performed for 12 days. And (5) recording the change condition of the tumor size by CT detection after treatment.

Second, experimental results and analysis

After 12 days of treatment, the lung tumor status of mice in different treatment groups was scanned by CT. As a result, NG25 monotherapy resulted in a slight reduction in lung tumor area (about 38.5% reduction compared to pre-use), Tram alone resulted in a reduction of about 46.5% in tumor size, whereas NG25 combined with Tram had a very significant tumor-inhibiting effect, with a reduction of about 76.4% (fig. 4A and 4B). The therapeutic effect of the orthotopic tumor model also indicates that NG25 in combination with Tram has a significant therapeutic effect on CLU downregulated lung cancer.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A medicament for treating lung cancer is characterized by comprising NG25 and trametinib according to the mass ratio of 4: 1;

the structural formulas of the NG25 and the trametinib are shown as follows:

the lung cancer is CLU gene function loss orDown-regulated by KRAS^G12DOncogene driven lung cancers, including carcinoma in situ and transplanted cancers.

2. The medicament of claim 1, wherein: the medicine also contains more than one carrier.

3. The medicament of claim 2, wherein: the carrier is a sustained release agent, a filling agent, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant.

4. The medicament of claim 1, wherein: the medicine is injection, tablet, granule or capsule.