CN115245514A - Pharmaceutical composition for treating non-small cell lung cancer - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and relates to a pharmaceutical composition for treating lung cancer, in particular to a pharmaceutical composition for treating non-small cell lung cancer, which consists of An Luoti and/or trametinib. The experiment shows that the pharmaceutical composition intervention scheme of An Luo Tini combined with trametinib can inhibit the proliferation, clone formation, invasion and migration of KRAS cell line in vitro and induce G1 phase cycle retardation and apoptosis, and the pharmaceutical composition has the synergistic effect of inhibiting tumor; compared with single-drug intervention, the drug combination intervention scheme of An Luoti combined with trametinib can more obviously inhibit the growth of KRAS mutant mouse transplanted tumor model tumors, and cannot generate intolerable toxicity. The drug combination intervention scheme can be used for preparing novel therapeutic drugs for treating KRAS mutation non-small cell lung cancer, and provides a new way for clinical practice intervention on non-small cell lung cancer.

Description

Pharmaceutical composition for treating non-small cell lung cancer

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a pharmaceutical composition for treating lung cancer, in particular to a pharmaceutical composition for treating non-small cell lung cancer, which consists of An Luoti and/or trametinib.

Background

The prior art discloses that non-small cell lung cancer (NSCLC) is a malignant tumor derived from the bronchial or alveolar epithelium, one of the most common types of lung cancer. It has been investigated that most patients are aged 65 years or older, but the onset of disease tends to be younger in recent years. The non-small cell lung cancer can be divided into adenocarcinoma, squamous carcinoma, large cell carcinoma, adenosquamous carcinoma and salivary gland type tumor according to the pathological types, and the non-small cell lung cancer patient is usually treated by intervention procedures such as surgical excision, radiotherapy, chemotherapy, targeted therapy, immunotherapy and the like in clinical practice, so that the aims of relieving pain and prolonging the service life are fulfilled. It is well known to those skilled in the art that the population with smoking is the high incidence of lung cancer, and smoking cessation and non-smoking is one of the most important preventive measures. In addition, some people who contact radon and people who live in areas with serious air pollution are also easy to cause lung cancer. Studies have shown that KRAS is one of the most common driver genes for mutations in non-small cell lung cancer (NSCLC), ranging from 15% to 25% [1,3].

Studies have shown that direct targeting of KRAS proteins has been less successful due to high affinity for GTP/GDP and the lack of known allosteric regulatory sites [2,4]. Alternatively, a number of preclinical studies and clinical trials have focused on inhibiting downstream effector molecules [2,5]. MEK1/2 is the most critical and most extensively studied downstream molecule of KRAS, and the approved indications for the MEK inhibitor trametinib include: (1) BRAF V600E or V600K mutated unresectable/metastatic melanoma; (2) metastatic non-small cell lung cancer with BRAF V600E mutation.

Although trametinib was initially shown to be effective against KRAS mutant lung cancer cell lines and patients, clinical practice has shown that relapse inevitably occurs in a short period of time, primarily due to induction of multiple RTK genes or ligands [8-10]. Research on MEK inhibitor resistance mechanism in different tumor types shows that after resistance, the genes of FGFR1, FGFR2, VEGFRB, PDGFRB, DDR1, IGF1R, AXL, ERBB3 and the like are activated to express [5, 11]. The diversity of adaptive resistance mechanisms suggests that combination therapy of a MEK inhibitor and a single RTK inhibitor is impractical. However, researchers believe that by effectively blocking signals from activation of multiple RTKs may be a viable strategy to prevent drug resistance.

With the prevalence of combined anti-tumor strategies, combinations of MEK inhibitors and other therapeutic approaches (e.g., PI3K/AKT/mTOR inhibitors, BRAF inhibitors, chemotherapy) [2,9, 12] have been widely discovered, suggesting another possible option for future clinical trials, but existing combination strategies are limited by toxicity or poor efficacy [5, 12], and thus, strategies that effectively block signals from multiple active RTKs may lead to better efficacy and prevent adaptive drug resistance.

Based on the current situation of the prior art, the inventor of the application intends to provide a pharmaceutical composition for treating lung cancer, in particular to a pharmaceutical composition for treating non-small cell lung cancer, so as to provide a new path for clinical practice intervention on non-small cell lung cancer.

References relevant to the present invention are:

1.Singh H,Longo DL,Chabner BA.Improving Prospects for Targeting RAS. Journal of Clinical Oncology Official Journal of the American Society of Clinical Oncology.JCO.2015.62.1052.

2.Tomasini P,Walia P,Labbe C,Jao K,Leighl NB.Targeting the KRAS Pathway in Non-Small Cell Lung Cancer.Oncologist.theoncologist.2015-0084.

3.Wood K,Hensing T,Malik R,Salgia R.Prognostic and Predictive Value in KRAS in Non-Small-Cell Lung Cancer:A Review.2016；2(6):805.

4.Ostrem JM,Peters U,Sos ML,Wells JA,Shokat KM.K-Ras(G12C)inhibitors allosterically control GTP affinity and effector interactions. Nature.503(7477):548-51.

5.Anderson GR,Winter PS,Lin KH,Nussbaum DP,Wood KC.A Landscape of Therapeutic Cooperativity in KRAS Mutant Cancers Reveals Principles for Controlling Tumor Evolution.Cell Reports.2017；20(4):999-1015.

6.Rao G,In-Kyu K,Fabio C,Jing L,Zhang YW,Giuseppe G.Dasatinib sensitises KRAS-mutant cancer cells to mitogen-activated protein kinase kinase inhibitor via inhibition of TAZ activity.European Journal of Cancer.2018；99:37-48.

7.Manchado E,Weissmueller S,Morris JP,Chen C-C,Wullenkord R,Lujambio A, et al.A combinatorial strategy for treating KRAS-mutant lung cancer.Nature.

8.Blumenschein GR,Smit EF,Planchard D,Kim D-W,Cadranel J,De Pas T,et al.A randomized phase II study of the MEK1/MEK2 inhibitor trametinib(GSK1120212) compared with docetaxel in KRAS-mutant advanced non-small-cell lung cancer(NSCLC). Annals of Oncology Official Journal of the European Society for Medical Oncology.26(5):894-901.

9.Abdel-Rahman,O.Targeting the MEK signaling pathway in non-small cell lung cancer(NSCLC)patients with RAS aberrations.Ther Adv Respir Dis.1753465816632111.

10.Gilmartin AG,Bleam MR,Groy A,Moss KG,Minthorn EA,Kulkarni SG,et al. GSK1120212(JTP-74057)Is an Inhibitor of MEK Activity and Activation with Favorable Pharmacokinetic Properties for Sustained In Vivo Pathway Inhibition. Clinical Cancer Research An Official Journal of the American Association for Cancer Research.17(5):989-1000.

11.Fedele C,Ran H,Diskin B,Wei W,Jen J.SHP2 Inhibition Prevents Adaptive Resistance to MEK inhibitors in Multiple Cancer Models.Cancer Discov.

12.

PA,Shaw AT,Pereira JR,Jeannin G,CrinòL.Selumetinib plus docetaxel for KRAS-mutant advanced non-small-cell lung cancer:A randomised, multicentre,placebo-controlled,phase 2 study.Lancet Oncology.2012；14(1).

13.Lin B,Song X,Yang D,Bai D,Yao Y,Lu N.Anlotinib inhibits angiogenesis via suppressing the activation of VEGFR2,PDGFRβand FGFR1. Gene.S0378111918301550.

14.Lu J,Zhong H,Chu T,Zhang X,Li R,Sun J,et al.Role of anlotinib-induced CCL2 decrease in anti-angiogenesis and response prediction for non-small cell lung cancer therapy.European Respiratory Journal.

15.Si X,Zhang L,Wang H,Zhang X,Wang M,Han B,et al.Management of anlotinib-related adverse events in patients with advanced non-small cell lung cancer:Experiences in ALTER-0303.Thorac Cancer.2019；10(3):551-6.

16.Han B,Li K,Wang Q,Zhang L,Shi J,Wang Z,et al.Effect of Anlotinib as a Third-Line or Further Treatment on Overall Survival of Patients With Advanced Non-Small Cell Lung Cancer:The ALTER 0303 Phase 3 Randomized Clinical Trial.JAMA Oncol.2018；4(11):1569-75.

17.Shen G,Zheng F,Ren D,Du F,Dong Q,Wang Z,et al.Anlotinib:a novel multi-targeting tyrosine kinase inhibitor in clinical development.J Hematol Oncol. 2018；11(1):120.

18.Han B,Li K,Zhao Y,Li B,Cheng Y,Zhou J,et al.Anlotinib as a third-line therapy in patients with refractory advanced non-small-cell lung cancer:a multicentre,randomised phase II trial(ALTER0302).Br J Cancer. 2018；118(5):654-61.。

disclosure of Invention

The invention aims to provide a novel pharmaceutical composition for treating lung cancer, in particular to a pharmaceutical composition for treating non-small cell lung cancer based on the current situation of the prior art, so as to provide a novel path for clinical practice intervention on the non-small cell lung cancer.

The invention provides a pharmaceutical composition for treating non-small cell lung cancer, which consists of An Luoti and/or trametinib.

Based on the prior art that An Luo Tini is a broad-spectrum drug, can inhibit activation of angiogenesis receptor tyrosine kinases (VEGFR 1, 2, 3/PDGFR A, B/FGFR1, 2, 3, 4) and several tumor-related receptor tyrosine kinases (c-Kit), ret, aurora-B, c-FMS, DDR 1) [13, 14], the drug combination of An Luo Tini and trametinib is adopted to intervene and treat non-small cell lung cancer, more specifically, animal in vivo experiments are carried out, and experimental results show that the drug combination of An Luoti and trametinib can inhibit proliferation, clone formation, invasion and migration of KRAS cell lines in vitro, and induce G1 phase cycle arrest and apoptosis, and the drug combination has a synergistic tumor inhibition effect; compared with single-drug intervention, the drug combination intervention scheme of An Luoti combined with trametinib can more obviously inhibit the growth of KRAS mutant mouse transplanted tumor model tumors, and cannot generate intolerable toxicity.

In the invention, preferably, the active ingredients of the pharmaceutical composition are An Luoti and trametinib.

In the invention, preferably, in the pharmaceutical composition, the ratio of An Luoti nylon and trametinib is (50-300): 1, molar ratio.

In the present invention, preferably, the pharmaceutical composition is selected from the following drugs:

drugs for inhibiting proliferation, clone formation, invasion and migration of non-small cell lung cancer cells;

drugs that induce non-small cell lung cancer G1 phase cycle arrest and apoptosis;

a medicament for inhibiting the growth of non-small cell lung cancer mouse transplantation tumor model tumors.

Furthermore, the medicine composition and the intervention scheme thereof can be used for preparing a novel therapeutic medicine for treating KRAS mutation non-small cell lung cancer, and provide a new way for clinical practice intervention on non-small cell lung cancer.

The invention further provides application of the novel medicinal composition for treating the non-small cell lung cancer.

The invention provides application of a pharmaceutical composition containing An Luoti and trametinib, wherein the pharmaceutical composition is a medicament for treating non-small cell lung cancer.

Preferably, the pharmaceutical composition has one or more of the following properties:

inhibiting proliferation, clone formation, invasion and migration of the non-small cell lung cancer cell line;

inducing non-small cell lung cancer cell G1 phase cycle arrest and apoptosis, and having synergistic anti-tumor effect;

obviously inhibit the growth of non-small cell lung cancer mouse transplanted tumor model tumor, and does not generate additional intolerable toxicity.

In the invention, the following method and steps are adopted to intervene and inhibit the proliferation, clone formation, invasion and migration of the non-small cell lung cancer cell line; inducing non-small cell lung cancer cell G1 phase cycle arrest and apoptosis; inhibit the growth of non-small cell lung cancer mouse graft tumor model tumors without generating additional intolerable toxicity:

obtaining non-small cell lung cancer cells, and culturing in vitro in a cell culture medium;

an Luoti nylon and trametinib are added into the cell culture medium of the non-small cell lung cancer cells for incubation.

In embodiments of the invention, the non-small cell lung cancer cell is a KRAS mutated non-small cell lung cancer cell.

In the embodiment of the invention, the concentrations of An Luoti nylon and trametinib are not lower than 0.5 mu mol/L and 5nmol/L respectively.

In yet another aspect, the present invention provides a method of inhibiting the growth of non-small cell lung cancer cells cultured in vitro, said use comprising the steps of:

obtaining non-small cell lung cancer cells, and culturing in vitro in a cell culture medium;

an Luoti nylon and trametinib are added into the cell culture medium of the non-small cell lung cancer cells for incubation. Typically, at least overnight incubation occurs.

The invention provides a An Luoti combination trametinib pharmaceutical composition. The experimental result of the invention shows that the KRAS mutant cells have good overall response to An Luoti ni treatment and are a feasible treatment means for KRAS non-small cell lung cancer patients; a synergistic effect of the two-drug combination was observed in KRAS mutant cells; the double-drug scheme of An Luoti combined with trametinib can inhibit proliferation, clone formation, invasion and migration of KRAS cell lines in vitro, induce G1 phase cycle block and apoptosis, and has a synergistic anti-tumor effect; the dual-drug regimen using An Luoti ni in combination with trametinib inhibited the growth of KRAS mutant mouse graft tumor model tumors more significantly than single drug, without additional intolerable toxicity. An Luoti ni and trametinib are effective novel therapeutic drugs for treating KRAS mutant non-small cell lung cancer, and a new way is provided for treating non-small cell lung cancer.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1, drug reactivity of different KRAS mutant non-small cell lung cancer cell lines to An Luoti ni, wherein,

reactivity of kras mutant non-small cell lung cancer cell lines to different concentrations of An Luoti ni;

b. KRAS site mutation information of non-small cell lung cancer cell lines.

FIG. 2, an Luoti ni and trametinib synergistically inhibit the growth of KRAS mutant non-small cell lung cancer cell lines,

wherein the content of the first and second substances,

a.A549 reactivity to different concentrations An Luoti ni and trametinib combinations,

b.analysis of synergy of trametinib and An Luoti ni in A549 cells,

reactivity of H23 to combinations of An Luoti nylon and trametinib at different concentrations,

analysis of synergy effects of trametinib and An Luoti ni in H23 cells,

CI <1, synergistic effect; CI =1, additive effect; CI >1, antagonism.

FIG. 3, the combined action of An Luo Tini and trametinib inhibits A549 cell clonogenic.

Figure 4, a Luo Tini and trimetinib combined induced a549 apoptosis and G1 phase cell cycle arrest.

FIG. 5, an Luoti as combined with trametinib inhibits A549 cell migration and invasion, wherein,

a-b. An Luoti as combined with trametinib inhibits migration of A549 cells,

c-d. An Luoti as combined with trametinib inhibits a549 cell invasion.

FIG. 6, an Luoti ni in combination with trametinib inhibited mouse graft tumor growth,

wherein the content of the first and second substances,

a. a tumor tissue picture of a mouse transplanted tumor model,

b. counting the size of the tumor of the mouse transplantation tumor model,

c. counting the body weight of the transplanted tumor mice,

d. mice transplanted tumor tissue HE and Ki67 histochemical staining.

Detailed Description

The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Example 1 drug responsiveness of different KRAS mutant non-small cell lung cancer cell lines to An Luoti ni

The invention firstly tests the inhibition of An Luoti ni with different gradient concentrations on different KRAS mutant NSCLC cell strains for 72 hours on cell proliferation, as shown in figure 1, a and table 1 show that An Luoti ni has similar inhibition on different KRAS cell lines, has no relation with mutation sites, has good overall response on An Luoti ni treatment, and has an IC50 value range of 2.35uM to 7.22uM.

TABLE 1

Cell line	Exon	Mutation
			H23
	2	G12C
			Calu-1	2	G12C
A549
		2	G12S
SW1573
		2	G12C
H460				3	Q61H
	SW900
		2	G12V
	H358
		2	G12C

Example 2 An Luoti Ni and trametinib synergistically inhibit KRAS mutant non-small cell lung cancer cell line growth

The inhibition effect of the dual drug combination of trametinib and An Luoti ni in two KRAS mutant cell lines sensitive to An Luoti ni is tested, and the results of a combination experiment of two drug doses prove that the combined intervention of trametinib and An Luoti ni can obviously inhibit the proliferation of cells with KRAS mutation (shown as a and c in FIG. 2); in addition, CI values were calculated using the CompuSyn software program and a synergistic effect was observed for the a549 and H23 cells on An Luoti and trametinib intervention (CI < 1), which was more pronounced in a549 cells.

The results are shown in fig. 2, in which,

a.A549 reactivity to different concentrations An Luoti ni and trametinib combinations,

b.analysis of synergy of trametinib and An Luoti ni in A549 cells,

reactivity of H23 to combinations of An Luoti nylon and trametinib at different concentrations,

(ii) analysis of the synergistic effect of trametinib and An Luoti ni in H23 cells, wherein,

CI <1, synergistic effect; CI =1, additive effect; CI >1, antagonism.

Example 3

Further evaluation of the synergistic inhibitory effect of An Luoti ni in combination with trametinib, a clone formation survival assay was performed on a549 cells exposed to trametinib (10 nM), anlotinib (1 um,2 um) or a combination of both for 14 days, as shown in fig. 3, a-b, with low concentration of An Luoti ni inhibiting colony formation and significantly reduced colony size and number in the 10nM concentration of trametinib-treated group; however, the combined pharmaceutical intervention almost completely eliminates the colonies of cancer cells; after the composition intervenes in the induced antitumor effect, whether the effect is caused by cell cycle arrest and apoptosis is studied by using flow cytometry, as shown in fig. 4, a-bs, A549 cells are exposed to trametinib and/or An Luoti ni for 48h, and the result shows that the proportion of the cells treated by the combined intervention scheme in the G1 phase is obviously increased, and the cells are arrested in the G1 phase; furthermore, the combined drug intervention resulted in a significant increase in apoptosis of KRAS mutant cancer cells (in fig. 4, c-d); the result shows that the drug combination intervention mode of the invention can obviously inhibit the proliferation and growth of KRAS mutant cell lines.

Example 4 An Luoti ni in combination with trametinib inhibits A549 cell migration and invasion

The test shows that the cell migration rate of the drug combination intervention treatment group of 24h is obviously reduced compared with that of a single drug intervention group (in figure 5, a-b), and simultaneously, the drug combination intervention effect also obviously inhibits the capacity of A549 cells, and the number of the cells penetrating out of a small chamber is obviously reduced; the result shows that the combined intervention of the medicine combination inhibits the invasion and migration functions of KRAS mutant cells.

Example 5 in vivo experiments to explore the effects of drug combination intervention on kras-mutant NSCLC cells

In view of the significant anti-tumor effect of the drug combination combined intervention in vitro experiments, an Luoti ni in combination with trametinib was subsequently evaluated for anti-tumor activity in KRAS mutant lung cancer xenografts; when tumors reached approximately 150m3, a549 cell xenograft tumor mice were intervened with clinically relevant doses of trametinib 0.3mg/kg body weight and An Luoti ni 1.5mg/kg body weight, although intervention with either trametinib or anloninib alone had only minor anti-tumor effects, combined intervention of trametinib and An Luoti ni was effective at inhibiting tumor growth and generally resulted in tumor regression (as shown in figure 6, a-b); meanwhile, the mental state and body weight of the mice were observed to be relatively stable during the administration period, and the mice in the single-drug intervention group and the two-drug combination intervention group were not significantly different from the control group (as shown in fig. 6, c), indicating that the drug combination intervention did not cause significant toxicity.

In FIG. 6, d shows that immunohistochemical examination of tumor tissues obtained from transplanted tumor mice shows that the area of necrosis of the combined intervention group of the pharmaceutical composition is larger and the expression of Ki67 is significantly reduced, i.e., the proliferation is reduced, compared with the single-drug intervention group, and the results show that the combined intervention using the pharmaceutical composition can significantly inhibit the proliferation of tumors and cause necrosis.

The experimental results of the invention show that:

the KRAS mutant cells have good overall response to An Luoti ni treatment, and are a feasible treatment means for patients with KRAS non-small cell lung cancer;

the synergistic effect of the combination of drugs was observed in KRAS mutant cells;

the drug combination dual-drug scheme of An Luoti ni combined with trametinib can inhibit proliferation, clone formation, invasion and migration of KRAS cell lines in vitro, induce G1 phase cycle block and apoptosis, and has synergistic anti-tumor effect;

the dual-drug regimen using An Luoti ni in combination with trametinib inhibited the growth of KRAS mutant mouse graft tumor model tumors more significantly than single drug, without additional intolerable toxicity.

Experimental results show that the pharmaceutical composition provided by the invention provides an effective novel intervention scheme for KRAS mutant non-small cell lung cancer.

The above description is only for the specific embodiments of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present disclosure should be covered within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims (9)

1. The pharmaceutical composition for treating non-small cell lung cancer is characterized by consisting of An Luoti and/or trametinib, wherein the ratio of An Luoti to trametinib is 50-300: 1.

2. the pharmaceutical composition of claim 1, wherein the pharmaceutical composition is one or more of the following drugs:

drugs for inhibiting proliferation, clone formation, invasion and migration of non-small cell lung cancer cells;

inducing non-small cell lung cancer G1 phase cycle arrest;

drugs that induce apoptosis of non-small cell lung cancer cells; or alternatively

A medicament for inhibiting the growth of non-small cell lung cancer mouse transplantation tumor model tumors.

3. Use of the pharmaceutical composition of any one of claims 1-2 in the preparation of a medicament for the treatment of non-small cell lung cancer.

4. The use according to claim 3, wherein the medicament has one or more of the following characteristics:

inhibiting proliferation, clone formation, invasion and migration of the non-small cell lung cancer cell line;

inducing G1 phase cycle arrest and apoptosis of non-small cell lung cancer cells, and having synergistic anti-tumor effect;

obviously inhibit the growth of non-small cell lung cancer mouse transplanted tumor model tumor, and does not generate additional intolerable toxicity.

5. The use according to claim 4, wherein the pharmaceutical composition inhibits proliferation, clonogenic, invasion, migration of a non-small cell lung cancer cell line by the methods and procedures described below; inducing non-small cell lung cancer cell G1 phase cycle arrest and apoptosis; inhibit the growth of non-small cell lung cancer mouse graft tumor model tumors without generating additional intolerable toxicity:

obtaining non-small cell lung cancer cells, and culturing in vitro in a cell culture medium;

an Luoti nylon and trametinib are added into the cell culture medium of the non-small cell lung cancer cells for incubation.

6. The use of claim 4, wherein said non-small cell lung cancer cell is a KRAS-mutated non-small cell lung cancer cell.

7. The use of claim 4, wherein the medicament is a combination of An Luoti and trametinib, wherein the concentrations of An Luoti and trametinib are no less than 0.5 μmol/L and 5nmol/L, respectively.

8. A method of inhibiting the growth of non-small cell lung cancer cells cultured in vitro comprising the steps of:

obtaining non-small cell lung cancer cells, and culturing in vitro in a cell culture medium;

an Luoti nylon and trametinib are added into the cell culture medium of the non-small cell lung cancer cells for incubation.

9. The method of claim 8, wherein said incubation is for a period of not less than 12 hours.

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