KR101419816B1 - Compositions for preventing or treating non-small cell lung cancer having synergistically therapeutic effects - Google Patents

Abstract

The present invention provides 2-Deoxy-D-glucose for preventing or treating non-small cell lung cancer, a combined medication of irreversible EGFR TKI, and a combining method. The 2-Deoxy-D-glucose synergistically improves the sensitivity of the non-small cell lung cancer to the irreversible EGFR TKI, thereby the composition of the present invention can be usefully used to prevent and treat non-small cell lung cancer exhibiting tolerance to the non-small cell lung, especially the irreversible EGFR TKI.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating non-small cell lung cancer having a synergistic therapeutic effect,

The present invention relates to the prophylactic or therapeutic use of non-small cell lung cancer in a pharmaceutical combination comprising 2-Deoxy-D-glucose and an irreversible EGFR tyrosine kinase inhibitor.

EGFR (epidermal growth factor receptor), one of the HER family of tyrosine kinase receptors, mediates cell proliferation, neovascularization, invasion and metastasis (Harari PM, et al . J Clin Oncol . 2007; 25: 4057-65; Yarden Y, et al ., Nat Rev Mol Cell Biol ., 2: 127-37). Abnormal expression of EGFR is frequently observed in many tumors and is known to have strong oncogenic potential (Hirsch FR, et al . J Clin Oncol 2003; 21: 3798-807; Rubin Grandis J, et . al J Natl Cancer Inst 1998; 90:. 824-32).

The first-generation EGFR tyrosine kinase inhibitor (TKI) such as gefitinib and erlotinib reversibly inhibits the autophosphorylation of EGFR by binding to ATP cleft within the EGFR phosphorylase domain ( Mendelsohn J, et al J Clin Oncol 2003; 21: 2787-99). Although this EGFR TKI has been implicated in patients with advanced non-small cell lung cancer (NSCLC) with EGFR-activated mutations such as small in-frame deletions of exon 19 or L858R missense mutations of exon 21 (Pao W, et al . PLoS Med . 2005; 2: e73), although most patients showed resistance to the EGFR TKI through a secondary T790M mutation in EGFR exon 20. However, there is no standard treatment for patients who are resistant to the reversible EGFR TKI due to the acquisition of EGFR T790M (Riely GJ. J Thorac Oncol . 2008; 3: S146-9).

Afatinib (BIBW 2992) is one of the irreversible 2nd generation EGFR TKI. According to recent preclinical studies, apatinate has anti-tumor activity in in vitro and in vivo in non-small cell lung cancer with EGFR T790M. On the basis of these results, apatinate was expected to be the standard therapeutic option for non-small cell lung cancer patients with EGFR T790M (Li D, et al . Oncogene . 2008; 27: 4702-11). However, apapnib is 100-fold less effective in non-small cell lung cancer cells with EGFR T790M than in non-small cell lung cancer cells with EGFR-activated mutations (Takezawa K, et al . Mol Cancer Ther . 2010; 9: 1647-56 ). In addition, apatineib has shown little efficacy in recent clinical trials, suggesting the need to develop a new strategy to improve the efficacy of apathetic (Miller VA, et al . Lancet Oncol . 2012; 13: 528-38).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop an effective therapeutic strategy for non-small cell lung cancer, particularly non-small cell lung cancer that has acquired resistance to reversible EGFR TKI. As a result, the combination of 2-Deoxy-D-glucose and apatinate inhibited synergistically anti-tumor activity through down-regulation of Mcl-1 through alteration of the AMPK / mTOR signaling pathway in NSCLC , It is confirmed that the combined treatment and regimen of 2-Deoxy-D-glucose and irreversible EGFR TKI is a promising therapeutic strategy for the treatment of non-small cell lung cancer patients who have acquired resistance to reversible EGFR TKI. Thereby completing the invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating non-small cell lung cancer.

Another object of the present invention is to provide a composition for inhibiting resistance to non-small cell lung cancer.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) 2-Deoxy-D-glucose (2DG) or a pharmaceutically acceptable salt thereof; And (b) non-small cell lung cancer comprising afatinib, dacomitinib, CI-1033 (Canertinib), HKI-272 (Neratinib) or a pharmaceutically acceptable salt thereof as an active ingredient Or a pharmaceutically acceptable salt thereof.

The present inventors have sought to develop an effective therapeutic strategy for non-small cell lung cancer, particularly non-small cell lung cancer that has acquired resistance to reversible EGFR TKI. As a result, the combination of 2-Deoxy-D-glucose and apatinate inhibited synergistically anti-tumor activity through down-regulation of Mcl-1 through alteration of the AMPK / mTOR signaling pathway in NSCLC , It is confirmed that the combined treatment and regimen of 2-Deoxy-D-glucose and irreversible EGFR TKI is a promising therapeutic strategy for the treatment of non-small cell lung cancer patients who have acquired resistance to reversible EGFR TKI. Thereby completing the invention.

As used herein, the term "non-small cell lung cancer" is used as is known to those skilled in the art. For example, non-small cell lung cancer is a malignant cell originating from the epithelial cells of the lung. The size of the tumor cell observed under a microscope by an observer, for example, a histopathologist, It is classified according to shape.

According to an embodiment of the present invention, the non-small cell lung cancer is squamous cell cancer, adenocarcinoma or large cell cancer.

The pharmaceutical composition of the present invention comprises 2DG as a first active ingredient and afacnib, dacomitinib, CI-1033 (Canertinib) or HKI-272 (Neratinib) as a second active ingredient. That is, the present invention relates to a novel combination preparation and therapy for irreversible EGFR TKI such as 2DG and apatineib, dakomitinib, CI-1033 and HKI-272 for the prevention and treatment of non-small cell lung cancer.

The first active ingredient, 2DG, is a glucose molecule whose 2-hydroxy group is substituted with hydrogen, and its molecular formula is C ₆ H ₁₂ O ₅ and is known to inhibit glycolysis.

(BIBW 2992), which is one of the second active ingredients, can be prepared by reacting compound 4 - [(3-chloro-4-fluorophenyl) amino] -6 - {[4- (N, -Oxo-2-buten-l-yl] amino} -7 - ((S) -tetrahydrofuran-3-yloxy) -quinazoline. Acapanib is a potent selective dual inhibitor of ErbB1 receptor (EGFR) and ErbB2 (Her2 / neu) receptor tyrosine kinase. In addition, apatinate is designed to covalently bind to EGFR and HER2 to irreversibly inactivate the receptor molecule to which apathi- nide binds. Such compounds, salts thereof (e.g. dimaleate salts) and their preparation are described in WO 02/50043 and WO 2005/037824, which are incorporated herein by reference.

(PF299804), another irreversible EGFR TKI like apatinib, is known to inhibit EFGR signaling and induce apoptosis in non-small cell lung cancer cells with the EGFR T790M mutation Small molecule compound.

One of the other second active ingredients, CI-1033, is known as irreversible TKI for EGFR, HER-2 and ErbB-4.

HKI-272, another of the second active ingredients, is known as a dual inhibitor of EGFR and HER-2 as a TKI.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable salt of the first active ingredient or the second active ingredient. As used herein, the term "pharmaceutically acceptable salt" means a formulation of a compound that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. The pharmaceutically acceptable salts are prepared by conventional methods well known in the art using pharmaceutically acceptable, substantially non-toxic organic and inorganic acids. The acid includes inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, and phosphoric acid; There may be mentioned sulfonic acids such as methanesulfonic acid, ethanesulfonic acid and p-toluenesulfonic acid, tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, , Lactic acid, fumaric acid, maleic acid, salicylic acid, and the like. Further, the compound of the present invention is reacted with a base to form an ammonium salt; Alkali metal salts such as sodium or potassium salts; Salts of alkaline earth metal salts such as calcium or magnesium salts; Salts of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, and tris (hydroxymethyl) methylamine; And amino acid salts such as arginine, lysine and the like.

According to one embodiment of the present invention, the non-small cell lung cancer has an EGFR activating mutation. The term "EGFR-activated mutation" means increasing the kinase activity by a change in the nucleotide sequence of the ErbBl gene encoding EGFR. Increased kinase activity is a direct consequence of changes in nucleic acids and is associated with proteins that the gene encodes.

In one particular example, the EGFR-activated mutation is a deletion mutation in exon 19 or a point mutation in exon 21. Examples of deletion mutants in exon 19 include E746-A750del, S752-I759del, K747-A750del and K747-E749 + A750. Examples of point mutations in exon 21 include L858R and L861Q point mutations .

According to one embodiment of the present invention, the non-small cell lung cancer additionally has an EGFR mutation in addition to EGFR activation mutation, which causes resistance to reversible EGFR TKI. In one particular example, the EGFR mutation that results in resistance to the EGFR TKI is T790M.

According to one embodiment of the present invention, the non-small cell lung cancer shows resistance to reversible EGFR TKI such as gefitinib or elotinib.

The reversible EGFR TKI is effective in the treatment of non-small cell lung cancer with EGFR-activated mutations such as E746-A750 deletion mutations and L858R point mutations. However, when secondary mutations such as T790M occur in non-small cell lung cancer, reversible EGFR TKI (Resistant) to become more ineffective in the treatment of lung cancer. The irreversible second generation EGFR TKI such as apatinib has been developed for the treatment of non - small cell lung cancer showing this resistance. However, the irreversible EGFR TKI has only limited effect on resistant non - small cell lung cancer, There was a problem.

The present invention has solved the problems of the conventional effects of irreversible EGFR TKI by using 2DG synergistically improving the irreversible EGFR TKI and its therapeutic effect synergistically. As demonstrated in the following examples, the combined use of irreversible EGFR TKI and 2DG significantly inhibited the growth and cell death of non-small cell lung cancer cells with T790M mutations in vitro and in vivo , respectively, than when using them individually (Figures 2 and 3) 10).

According to one embodiment of the present invention, the first active ingredient, 2DG, synergistically increases the effect of the second active ingredient on non-small cell lung cancer, such as:

(i) the sensitivity of the non-small cell lung cancer to the second active ingredient,

(ii) the anti-tumor activity of the second active ingredient, or

(iii) Inhibitory effect of the second active ingredient on the corresponding process of non-small cell lung cancer cells.

From this point of view, the first active ingredient of the present invention functions as an anticancer sensitizer. The term "anti-cancer sensitization" is used in the same sense as chemical sensitization. When the cancer is treated with a chemical anti-cancer agent, the anticancer activity of the anti-cancer agent is further enhanced or increased when there is no anti-cancer sensitizer (chemical sensitizer) , Which means that resistance to anticancer drugs such as anticancer drug resistance is reduced and the effect of anticancer drugs is better displayed. In the present invention, 2DG, which is an anti-cancer sensitizer, sensitizes non-small cell lung cancer cells against irreversible EGFR TKI, thereby reducing the resistance of cancer cells to anti-cancer drugs and enhancing the effect of anti-cancer drugs.

According to one embodiment of the present invention, the composition of the present invention can contain the first active ingredient and the second active ingredient in the same container or each separately contained container.

The pharmaceutical composition of the present invention comprises a first effective ingredient and a second active ingredient in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, This includes an amount that induces relief of the symptoms of the disease or disorder being treated. Effective dose levels are well known to those skilled in the art, including the type and severity of the patient ' s disease, age, sex, drug activity, drug sensitivity, time of administration, route of administration and rate of release, Can be determined according to the element.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like. The first active ingredient and the second active ingredient of the present invention can be administered simultaneously or sequentially.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The daily dose of the pharmaceutical composition of the present invention is, for example, 0.0001-100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. Here, the formulations may be in the form of solutions, suspensions, syrups or emulsions in an oil or aqueous medium, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) 2DG or a pharmaceutically acceptable salt thereof; And (b) an EGFR tyrosine kinase inhibitor comprising apapnibe, dakomitinib, CI-1033 (Canertinib), HKI-272 (Neratinib) or a pharmaceutically acceptable salt thereof as an active ingredient. A composition for inhibiting resistance is provided.

Since the active ingredient contained in the composition for inhibiting tolerance is the same as the active ingredient contained in the pharmaceutical composition of the present invention, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

As used herein, the term "resistant" indicates that non-small cell lung cancer cells are resistant to anticancer agents such as reversible EGFR TKI. When treating patients with lung cancer using EGFR TKI such as gefitinib, Initially, cancer treatment was effective, but the cancer treatment effect is lost (decreased) in the continuous treatment process. Thus, it is well known that the effect of chemotherapeutic agents on cancer patients is gradually reduced when they are continuously administered to cancer patients. In particular, in the case of non-small cell lung cancer, mutation of EGFR is known to obtain resistance against reversible EGFR TKI.

According to one embodiment of the present invention, the non-small cell lung cancer shows resistance to EGFR TKI by mutation of T790M of exon 20 of EGFR. In one specific example, the EGFR TKI is goitinib or elotinib.

2DG synergistically enhances the sensitivity to irreversible EGFR TKI of non-small cell lung cancer that is resistant to reversible EGFR TKI, so that the composition for inhibiting resistance of the present invention is useful as a non-small cell lung cancer patient exhibiting no or low reactivity to reversible EGFR TKI And the like.

The features and advantages of the present invention are summarized as follows:

(i) The present invention provides a combined preparation and a combined use of 2-Deoxy-D-glucose and irreversible EGFR TKI for the prevention or treatment of non-small cell lung cancer.

(ii) 2-Deoxy-D-glucose synergistically enhances the sensitivity of non-small cell lung cancer to irreversible EGFR TKI. Thus, the composition of the present invention is useful for non-small cell lung cancer, non-small cell lung cancer Can be usefully used for prevention and treatment.

Figure 1 shows that 2DG sensitizes non-small cell lung cancer cells with EGFR T790M to apapnibe. [A] cells were incubated with each drug for 72 hours. Cell viability was measured by MTT assay. * p < 0.05, ** p < 0.01, *** p < 0.001. [B] cells were treated with 2 mM 2DG, 100 nM apatapnib or a combination thereof for 24 hours. Dead cells were identified by Annexin V / PI staining and FACS analysis.
Fig. 2 shows that the combination treatment of 2DG and apatinate causes selective cytotoxicity to cancer cells. 2 mM 2DG, 100 nM apatanide, or a combination thereof was treated with non-small cell lung cancer cells (H1975 and PC9-GR) and normal cells (NHBE and MRC5) for the time indicated in the figure.
Figure 3 shows that the combination treatment of 2DG and apatineib synergistically inhibits the metabolism of the process. [A and B] cells were treated with 2 mM 2DG, 100 nM apatanide, or a combination thereof. After 48 hours, ATP or lactate levels were measured. ** p < 0.01, *** p < 0.001.
Fig. 4 shows that apapanib inhibits ATP production through inhibition of Akt activity. After 24 h of transfection of [A and B] mock or myr-Akt vectors, cells were treated with 2 mM 2DG, 100 nM apatinate, or a combination thereof. After 48 hours, the cells were harvested for ATP production assay or Western blot analysis.
Figure 5a shows that cytotoxicity by combination treatment of 2DG and apatinate is mediated through modulation of the AMPK / mTOR / Mcl-1 signaling pathway. [A] cells were treated with 2 mM 2DG, 100 nM apatanide or a combination thereof for 24 hours and the cells were recovered. [B] cells were treated with 1 [mu] M of Compound C for 1 hour and incubated for 72 hours with 2 mM 2DG, 100 nM apatanide or a combination thereof. After treatment of [C] AICAR for 72 h, cell viability was determined by MTT assay. [D] cells were treated with 1 [mu] M of Compound C for 1 hour and incubated with 2 mM 2DG, 100 nM apatanide or a combination thereof for 24 hours. [E and F] cells were treated with AICAR or rapamycin for 24 hours and cells were harvested. *** p < 0.001.
Figure 5b shows that cytotoxicity by combination treatment of 2DG and apatinate is mediated through modulation of the AMPK / mTOR / Mcl-1 signaling pathway. [A] cells were treated with 2 mM 2DG, 100 nM apatanide or a combination thereof for 24 hours and the cells were recovered. [B] cells were treated with 1 [mu] M of Compound C for 1 hour and incubated for 72 hours with 2 mM 2DG, 100 nM apatanide or a combination thereof. After treatment of [C] AICAR for 72 h, cell viability was determined by MTT assay. [D] cells were treated with 2 mM 2DG, 100 nM apatinate, or a combination thereof for 24 hours. The levels of Mcl-1, Bcl-xL and Bcl-2 were evaluated by Western blot. [E] cells were treated with 1 [mu] M of compound C for 1 hour and incubated for 24 hours with 2 mM 2DG, 100 nM apatanide or a combination thereof. [F and G] cells were treated with AICAR or rapamycin for 24 hours and the cells were recovered. *** p < 0.001.
FIG. 6 shows that Mcl-1 knockdown shows similar cytotoxic effects to the combination treatment of 2DG and apatinate. After transfection with Mcl-1 specific siRNA or control for 24 hours, control siRNA transduced cells were treated with 2 mM 2DG, 100 nM apatinate, or a combination thereof. After 72 hours of incubation, cell viability was assessed by MTT. *** p < 0.001 vs control siRNA Transduced cells.
Figure 7 shows that inhibition of glucose metabolism by combination treatment of 2DG and apatinate does not affect the transcription of Mcl-1. Cells were treated with 2 mM 2DG, 100 nM apatinate, or a combination thereof for 24 hours. Mcl-1 mRNA expression was measured by qRT-PCR. The results were expressed as the ratio of Mcl-1 level to control.
FIG. 8 shows that the down-regulation of Mcl-1 by combination treatment of 2DG and apatineib occurs through translation inhibition of cells. [A and B] cells were treated with 2 mM 2DG, 100 nM apatanide or a combination thereof for 24 hours and treated with 20 μM MG132 for 3 hours or 100 μg / ml CHX for 1 hour. The quantification of Mcl-1 was normalized with [beta] -actin and the results were expressed as the ratio of the level of Mcl-1 to the control. [C] cells were pretreated with 1 [mu] M compound C for 1 hour and incubated with 2 mM 2DG, 100 nM apatanide or a combination thereof for 24 hours. methyl ^7- GTP pull down assay, the association between 4E-BPI and eIF4E was identified by Western blot analysis.
FIG. 9 shows that the down-regulation of Mcl-1 by combination treatment of DG and apatineib occurs through translation inhibition of cells. PC9-GR cells were treated with 2 mM 2DG, 100 nM apatanide, or a combination thereof for 24 hours. The relative amounts of Mcl-1, Bcl-1 and [beta] -actin mRNA in each fraction were expressed as a percentage of total mRNA in the polysome gradient.
Figure 10 shows that 2DG increases anti-tumor activity by apathi- nide in the PC9-GR tumor xenograft model. [A] Mice with PC9-GR xenografts were dosed daily with the drug. Data were expressed as mean ㅁ SE. *** p <0.001 vs control, ### p <0.0001 vs 2DG, +++ p <0.001 vs apathetic. [B] After 5 days of drug treatment, tumor tissues were analyzed by IHC.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

Cell culture

NCI-H1975 cells (EGFR L858R / T790M) were purchased from the American Type Culture Collection (Manassas, Va.). PC9-GR cells (EGFR delE746_A750 / T790M) were obtained from Lee JC (Korea Atomic Energy Academy). The presence of the EGFR T790M mutation in PC9-GR cells was confirmed by direct sequencing. The two cells were cultured in RPMI 1640 (HyClone, Logan, UT) containing 10% fetal bovine serum.

Reagents and Antibodies

Acapaine was obtained from Boehringer Ingelheim Pharma (Boehringer Ingelheim Pharma GmbH & Co KG, Germany). AICAR (5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside) and cycloheximide were obtained from Sigma. Anti-β-actin antibodies were obtained from Santa Cruz Biotechnology, and all other antibodies were purchased from Cell Signaling. All other reagents were purchased from Calbiochem.

Cell survival analysis

After incubation with the drug for 72 hours, 0.5 mg / ml of MTT (Amresco, Solon, OH) was added to the medium. The formazan crystals of viable cells were dissolved in 100 [mu] l DMSO. The absorbance value of MTT formazan product was confirmed at 565 nm with a microplate reader. All experiments were repeated three times.

Analysis of apoptosis using annexin V / propidium iodide staining

Annexin V / propidium iodide (PI) double staining was performed according to the manufacturer's instructions (BD Pharmigen, San Diego, Calif.). Briefly, cells were incubated with Annexin V / PI in 1X binding buffer for 15 minutes and analyzed with a flow cytometer (BD Biosciences, San Jose, Calif.). Data was processed using WinMDI 2.9 software (Salk Institute, La Jolla, Calif.).

Intracellular ATP analysis

Intracellular ATP was measured using the ATP colorimetric assay kit (Abcam, Cambridge, Mass.) According to the manufacturer's instructions. Briefly, cell lysates were incubated with the ATP reaction mixture for 30 minutes after centrifugation (13,000 rpm, 5 min, 4 ° C). The absorbance values of the mixtures in each well were confirmed at 570 nm with a microplate reader. ATP concentration was calculated using a standard curve and normalized to the number of cells.

Lactate production analysis

Lactate production was measured according to the manufacturer's instructions using a lactate assay kit (Biovision, Mountain View, Calif.). Briefly, after centrifugation (13,000 rpm, 15 min, 4 ° C), the cell culture medium was diluted with lactate assay buffer and mixed with the lactate reaction mixture for 30 minutes. The absorbance values of the mixtures in each well were confirmed at 570 nm with a microplate reader. The lactate concentration was calculated using a standard curve and normalized to the number of cells.

Transient transfection

The pUseAkt-CA (myristoylated constitutively active Akt) plasmid was obtained from Lee JC (Korea Atomic Science Institute). Transformation was performed using lipofectamine ^TM 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, cells were transfected with DNA (1.5 μg / well) using the above reagents and replaced with fresh growth medium. After 24 hours, the cells were treated with the drug.

Western blot analysis

Cell lysates were prepared, equal amounts of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane (BioRAD). Membranes were blocked with 5% skim milk and incubated with the antibody overnight at 4 &lt; 0 &gt; C. Proteins were detected using an HRP-conjugated secondary antibody and an ECL chemiluminescence detection system (Amersham-Pharmacia Biotech).

Methyl ⁷ -GTP sepharose 4B pull-down analysis

450 [mu] g of cell lysate was incubated with 50 [mu] l of methyl ^7- GTP sepharose 4B bead (Amersham Biosciences) at 4 [deg.] C for two hours. The beads were washed and boiled in 2X sample buffer. After SDS-PAGE separation, the association of 4E-BPI with eIF4E was detected by Western blot.

qRT-PCR

QRT-PCR was performed on a 7500 real-time PCR system (Applied Biosystems) using the SYBR green detection protocol. The primer sequences used were as follows:

Mcl-1: (F) 5'-GGACATCAAAAACGAAGACG-3 '(SEQ ID No. 1), (R) 5'-GCAGCTTTCTTGGTTTATGG-3' (SEQ ID No. 2);

Bcl-2: (F) 5'-ATGTGTGTGGAGAGCGTCAACC-3 '(SEQ ID No. 3), (R) 5'-TGAGCAGAGTCTTCAGAGACAGCC-3' (SEQ ID No. 4);

? -actin: (F) 5'-CTGGAACGGTGAAGGTGACA-3 '(SEQ ID No. 5); (R) 5'-AAGGGACTTCCTGTAACAATGCA -3 '(SEQ ID No. 6).

Xenograft studies

All female athymic BALB-c / nu mice (5-6 weeks of age, Orient Bio, Korea) were treated according to the guidelines of the Animal Research Council of the Yonsei University College of Medicine and all facilities were accredited by AAALAC ). PC9-GR cells (5 × 10 ⁶ ) were subcutaneously injected into mice. After the tumoral volume reached about 70 mm ³ , mice were divided into 6 groups of vehicle control, apatinate-naïve group, 2DG-alone group, or combination of apatinib and 2DG in each group. Afpanib was suspended in 0.5% (w / v) methylcellulose containing 0.4% Tween 80 and administered orally 5 mg / kg once a day with gavage. 2DG was dissolved in saline and administered intraperitoneally at a daily dose of 500 mg / kg. Tumor size was measured at 2-day intervals using a caliper. The average tumor volume of each group was calculated according to the following prolate spheroid formula and expressed as mm ³ .

[Mathematical Expression]

Tumor volume = 0.523 x large diameter x (small diameter) ²

Immunohistochemistry (IHC)

The tumor was cut with paraffin pore (4 쨉 m). Tissue fractions were deparaffinized and wetted with ethanol, microwaved in 0.01 mol / l sodium citrate buffer (pH 6.0) and incubated in 3% H ₂ O ₂ for 10 min. After incubation in PBS containing 1% BSA for 10 minutes, the fragments were incubated with mouse anti-PCNA monoclonal antibody (1: 300 dilution), rabbit anti-p-AMPKa-T172 monoclonal antibody (1: p-mTOR-S2448 monoclonal antibody (1: 100 dilution) and rabbit anti-Mcl-1 monoclonal antibody (1: 100 dilution) overnight at 4 占 폚. After incubation with a peroxidase-conjugated secondary antibody, peroxidase activity was confirmed using diaminobenzidine.

Statistical analysis

In vitro results were expressed as mean ± SD, and in vivo results were expressed as mean ± SE. Student's t-test was performed to determine statistical significance between groups, and p <0.05 was considered to be statistically significant.

Experiment result

Inhibition of this process enhances the sensitivity of apoptinib to non-small cell lung cancer cells with the EGFR T790M mutation.

The present inventors first confirmed by MTT analysis that inhibition of glucose metabolism can promote apapnibe-induced cytotoxicity. 2DG (2-deoxy-D-glucose) was used to inhibit the process. 2DG is a non-metabolic form of glucose and is known as an inhibitor of the primary rate-controlling step of the process (Wu M, et al ., Am J Physiol Cell Physiol . 2007; 292: C 125-36). As a result, 2DG treatment reduced cell survival in a dose-dependent manner and markedly enhanced sensitivity to apatinate in H1975 and PC9-GR cells (Fig. 1A). In addition, 2DG treatment enhanced apoptotic cell death (Fig. 1B).

Furthermore, as shown in Fig. 2, cell growth was inhibited in a time-dependent manner by 2DG or apatinate alone treatment in non-small cell lung cancer cells including H1975 and PC9-GR. The combination of 2DG and apatinate inhibited cell growth synergistically in a time-dependent manner and also reduced the number of cells. Contrary to cancer cells, in normal cells containing NHBE and MRC5, cell growth was hardly inhibited by 2DG or apatinate alone treatment. The inhibitory effect of the combination on cell growth was much lower than that observed in cancer cells. The above experimental results indicate that the combination treatment of 2DG and apatinebine selectively cytotoxic to cancer cells.

The combination of 2DG and apatinate inhibits the metabolism of cancer cells and induces ATP depletion.

Intracellular ATP analysis and lactate (product of aerobic equivalent) analysis were performed to confirm that 2DG treatment inhibited the process. 2DG alone resulted in a significant reduction in intracellular ATP levels and lactate production in H1975 and PC9-GR cells. Interestingly, apatinate alone treatment also reduced intracellular ATP levels and lactate levels in both cells. Combined treatment of 2DG and apatinate resulted in synergistic induction of ATP depletion and reduced lactate production (FIG. 3). These experimental results show that the combined treatment of 2DG and apatineib can effectively inhibit glucose metabolism in cancer cells.

Next, we examined how apapanib suppresses glucose metabolism in cancer cells. The PI3K / Akt signaling pathway is known to be a positive regulator of the process (Elstrom RL, et al . Cancer Res . 2004; 64: 3892-9). It is known that apapnib has an inhibitory effect on Akt activity through blockade of the ErbB family (Takezawa K, et al . Mol Cancer Ther . 2010; 9: 1647-56). Thus, we investigated whether Akt is associated with inhibition of the corresponding process induced by apapanib. As shown in Fig. 4, in the H1975 and PC9-GR cells, Akt was significantly inactivated by treatment with apatinate, but not with 2DG alone. Induction of normal Akt activation by enhanced expression of myr-AKT in both cells resulted in the inhibition of apoptinib alone and in combination with apatinib and 2DG in Akt activation and ATP production. These results indicate that the inhibition of the process by apapanib is mediated through the inhibition of Akt activity.

Cytotoxicity by combination of 2DG and apatinate is mediated by modulation of the AMPK / mTOR / Mcl-1 signaling pathway.

AMPK is known to be activated by stimulation that increases the AMP / ATP ratio of cells (El Mjiyad N, et al . Oncogene . 2011; 30: 253-64). Therefore, we investigated whether ATP depletion induced by 2DG and apatinate could activate AMPK. In H1975 and PC-9GR cells, the combination of 2DG and apatinate induced significant activation of AMPK (FIGS. 5A and 5B). Treatment of the AMPK inhibitor (compound C) prevented the reduction of cell survival induced by the combination of 2DG and apatinate in the two cells, suggesting that synergistic cytotoxicity of 2DG and apathetic combination was mediated by AMPK activation (Figs. 5A and 5B). AICAR and AMPK inhibitors were used to determine whether AMPK activation could mediate cytotoxicity of tumor cells. In H1975 and PC9-GR cells, AICAR treatment significantly decreased cell viability in a dose-dependent manner (C in Figure 5). The above experimental results show that the combination treatment of 2DG and apatinate causes cytotoxicity through AMPK activation in NSCLC cells with EGFR T790M.

It is known that the maintenance of anti-apoptotic Bcl-2 family protein plays an important role in the survival under metabolic stress (Wensveen FM, et al . Apoptosis . 2011; 16: 708-21). Thus, we investigated whether the combination of 2DG and apatapnib affect the expression levels of anti-apoptotic Bcl-2 family proteins such as Mcl-1, Bcl-2 and Bcl-xL. 2DG alone or treatment with apatinate reduced Mcl-1 levels in PC9-GR cells, and the combination of 2DG and apatinate reduced synergistic Mcl-1 levels. However, the treatment with 2DG and apatinate alone or in combination did not affect Bcl-2 or Bcl-xL levels (Fig. 5). To confirm that maintenance of Mcl-1 is important for cell survival, we investigated whether siRNA-1 knockdown could reduce the survival rate of cancer cells. MTT assay significantly inhibited cell growth similar to the combination treatment of 2DG and apatinate in H1975 and PC9-GR cells by siMcl-1 treatment (Fig. 6). These results indicate that down-regulation of Mcl-1 by combination treatment of 2DG and apatineib is important for inhibiting the growth of NSCLC cells with EGFR T790M.

Next, we examined whether down-regulation of Mcl-1 is mediated through changes in the AMPK / mTOR signaling pathway following treatment with 2DG and apatanav. As shown in FIG. 5, the combination treatment of 2DG and apatinate induced synergistic AMPK activation and down-regulation of Mcl-1 in H1975 and PC9-GR cells. In addition, mTOR inhibition was shown by combination treatment of 2DG and apatinate. MTOR inhibition and Mcl-1 downregulation by 2DG and apatinate were significantly restored in the presence of compound C. In these two cells, AICAR treatment inhibited mTOR in a dose-dependent manner and decreased Mcl-1 levels (Fig. 5A E and Fig. 5B F). In addition, rapamycin (mTOR inhibitor) treatment reduced Mcl-1 levels in H1975 and PC9-GR cells (F in Figure 5a and G in Figure 5b). These results indicate that AMPK activation and mTOR inhibition result in down-regulation of Mcl-1, but not down-expression of other Bcl-2 anti-apoptotic factors, by inhibition of the process by the combination of 2DG and apatinate .

Mcl-1 is down-regulated through the translational mechanism by the inhibition of the corresponding process by the combination treatment of 2DG and apatinate.

As shown in FIG. 7, the level of Mcl-1 mRNA is not affected by treatment with 2DG or apatinate alone or in combination, which means that the level of Mcl-1 is not regulated at the transcription level. (Zhong Q, et al . Cell . 2005; 121: 1085-95), that the central mechanism of Mcl-1 regulation is ubiquitin-mediated proteasome degradation Lt; RTI ID = 0.0 &gt; Mcl-1 &lt; / RTI &gt; In H1975 and PC9-GR cells, the relative change in Mcl-1 levels after treatment with MG132, although the level of Mcl-1 protein was lower than in MG132 treatment in combination with 2DG and apatinate, There was no difference in the combination treatment (Fig. 8A). In addition, the half-life of Mcl-1 by 2DG, apatinate or its combination treatment was not accelerated in the presence of a protein biosynthesis inhibitor (B in Fig. 8B). The above experimental results show that a significant decrease in Mcl-1 levels by combination treatment of 2DG and apatinate is not due to transcriptional and post-translational regulation.

Next, we examined whether the down-regulation of Mcl-1 by 2DG and apatamin was regulated at the translation level. The possibility of down-regulation of Mcl-1 by translational inhibition was supported by an experiment using a methyl ^7- GTP sepharose 4B bead similar to the 5'cap structure of mRNA. As shown in FIG. 8C, methyl ^7- GTP pull-down analysis showed that the binding of 4E-BP1, a translational inhibitor, was synergistically increased by the combination treatment of 2DG and apatinate, This result implies that the inhibition of translation by the combination of 2DG and apatineib occurs in an AMPK-dependent manner. In order to confirm whether the inhibition of the process specifically blocked Mcl-1 translation, the polysome distribution of Mcl-1 was monitored by 2DG or apatinate alone or in combination with PC9-GR cells . As shown in Fig. 9, the amount of Mcl-1 mRNA associated with polysomes was significantly reduced by the combination treatment of 2DG and apatinate. Under the same conditions, the distribution of the polysomes of control mRNA (beta -actin) or Bcl-2 was not significantly affected compared to Mcl-1. These results indicate that Mcl-1 in the Bcl-2 family is specifically down-regulated at the translation level by the combination of 2DG and apatinate.

2DG additionally synergistically enhances the anti-tumor activity of apapanib in the PC9-GR xenograft model.

In order to investigate anti-tumor activity following combination therapy of 2DG and apatinate, PC9-GR transplanted anhid nude mice were treated with control, 2DG, apatinate, or a combination thereof. Treatment with apatinate alone for 30 days delayed tumor growth compared with the control group. 2DG monotherapy showed no significant anti-tumor activity. In particular, the combination treatment of 2DG and apatinate showed significant tumor suppression (Fig. 10A). In addition, synergistic anti-tumor activity of 2DG and apapanibe combination treatment was confirmed by IHC staining for PCNA (cell proliferation marker) (Fig. 10B). Similar to in vitro results, the combined treatment of 2DG and apatinate resulted in a marked decrease in staining for mTOR and Mcl-1, but a marked increase in p-AMPK staining. The above in vitro and in vivo experimental results show that glucose metabolism is a promising therapeutic target for enhancing the susceptibility of apapnib in non-small cell lung cancer with EGFR T790M mutation.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Industry-Academic Cooperation Foundation, Yonsei University          JE UK Inc. <120> Compositions for preventing or treating non-small cell lung          cancer having synergistically therapeutic effects <130> PN120693 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> foward primer for Mcl-1 <400> 1 ggacatcaaa aacgaagacg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Mcl-1 <400> 2 gcagctttct tggtttatgg 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> foward primer for Bcl-2 <400> 3 atgtgtgtgg agagcgtcaa cc 22 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Bcl-2 <400> 4 tgagcagagt cttcagagac agcc 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> foward primer for beta-actin <400> 5 ctggaacggt gaaggtgaca 20 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for beta-actin <400> 6 aagggacttc ctgtaacaat gca 23

Claims (9)

(a) 2DG (2-Deoxy-D-glucose) or a pharmaceutically acceptable salt thereof; And (b) non-small cell lung cancer comprising afatinib, dacomitinib, CI-1033 (Canertinib), HKI-272 (Neratinib) or a pharmaceutically acceptable salt thereof as an active ingredient &Lt; / RTI &gt;
The composition of claim 1, wherein the non-small cell lung cancer has a deletion mutation in exon 19 or an exon 21 mutation in epidermal growth factor receptor (EGFR).
3. The composition of claim 2, wherein the non-small cell lung cancer additionally has a T790M mutation in exon 20 of EGFR.
The composition of claim 1, wherein the non-small cell lung cancer is resistant to gefitinib or erlotinib.
The composition of claim 1, wherein the non-small cell lung cancer is squamous cell carcinoma, adenocarcinoma or large cell carcinoma.
2. The method according to claim 1, wherein the 2DG has a sensitivity to non-small cell lung cancer to apatnib, dakomitinib, CI-1033 (Canertinib), HKI-272 (Neratinib) or a pharmaceutically acceptable salt thereof Gt; synergistically &lt; / RTI &gt;
(a) 2DG (2-Deoxy-D-glucose) or a pharmaceutically acceptable salt thereof; And (b) an EGFR tyrosine kinase inhibitor comprising apapnibe, dakomitinib, CI-1033 (Canertinib), HKI-272 (Neratinib) or a pharmaceutically acceptable salt thereof as an active ingredient. / RTI &gt;
8. The composition of claim 7, wherein said non-small cell lung cancer is resistant to a reversible EGFR tyrosine kinase inhibitor by the T790M mutation of exon 20 of EGFR.
9. The composition of claim 8, wherein the reversible EGFR tyrosine kinase inhibitor is goitinib or erotinib.

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