KR20230158894A - Composition for preventing or treating EGFR-TKI resistant non-small cell lung cancer comprising AICDA and NF-kB inhibitor - Google Patents

Abstract

The present invention relates to a composition for preventing or treating EGFR-TKI resistant non-small cell lung cancer comprising an AICDA and NF-κB inhibitor, and more specifically, to perform simultaneous inhibition of AICDA and/or NFκB in advanced lung cancer that has failed EGFR-TKI treatment. This is about a new treatment strategy to restore sensitivity to EGFR-TKI.

Description

Composition for preventing or treating EGFR-TKI resistant non-small cell lung cancer comprising AICDA and NF-kB inhibitor {Composition for preventing or treating EGFR-TKI resistant non-small cell lung cancer comprising AICDA and NF-kB inhibitor}

The present invention relates to a composition for preventing or treating EGFR-TKI resistant non-small cell lung cancer comprising an AICDA and NF-kB inhibitor.

In treatment using an effective EGFR tyrosine kinase inhibitor (EGFR-TKI) for non-small cell lung cancer positive for an Epidermal Growth Factor Receptor (EGFR) gene mutation, in many cases, the target cancer may be treated while continuing treatment. Drug resistance to the inhibitor increases, and as a result, the disease progresses. Among cancers resistant to the first-generation EGFR-TKIs, Gefitinib and Erlotinib, and to the second-generation EGFR-TKI, Afatinib, about half have cancers on the EGFR gene. It is known to have the T790M mutation. Osimertinib, a third-generation EGFR-TKI, is known as an effective drug for non-small cell lung cancer that has been confirmed to have a positive EGFR T790M mutation. However, no intelligent agent is still approved for osimertinib-resistant non-small cell lung cancer. In addition, no intelligent drug has yet been approved for non-small cell lung cancer, which has been confirmed to be resistant to EGFR-TKI and negative for the EGFR T790M mutation.

Korean Patent Publication No. 2019-0120764 discloses a method of treating EGFR-TKI resistant non-small cell lung cancer by administering an anti-HER3 antibody-drug conjugate, and Korean Patent No. 2359214 discloses a method for treating non-small cell lung cancer of the lung and ovarian cancer. Disclosed is dianhydrogalactitol and its analogs or derivatives for treating .

However, a composition for preventing or treating EGFR-TKI resistant non-small cell lung cancer containing specific AICDA and NF-kB inhibitors as in the present invention has not been reported.

The purpose of the present invention is to provide a composition for preventing or treating EGFR-TKI resistant non-small cell lung cancer comprising an AICDA and NF-kB inhibitor. More specifically, the present invention can be a new treatment strategy to restore sensitivity to EGFR-TKI when concurrent inhibition of AICDA/NFκB is performed in advanced lung cancer that has failed EGFR-TKI treatment.

In order to achieve the above object, the present invention provides shRNA for inhibiting Activation Induced Cytidine Deaminase (AICDA) selected from the group consisting of SEQ ID NOs: 1 to 3.

In addition, the present invention provides a composition for preventing or treating lung cancer comprising shRNA for inhibiting Activation Induced Cytidine Deaminase (AICDA) selected from the group consisting of SEQ ID NOs: 1 to 3 as an active ingredient.

In one example of the present invention, the lung cancer may include all types of lung cancer, preferably non-small cell lung cancer, and more preferably EGFR-TKI (epidermal growth factor-tyrosine kinase inhibitor) resistant arsenic It may be cellular lung cancer, but is not limited thereto.

In another example of the present invention, the composition may further include one or more ingredients effective against lung cancer, and preferably may further include a NF-kB (nuclear factor-kB) inhibitor. Preferably, it may include BAY11-7082, but is not limited thereto.

The present invention provides a composition for preventing or treating EGFR-TKI (epidermal growth factor-tyrosine kinase inhibitor) resistant non-small cell lung cancer, comprising an Activation Induced Cytidine Deaminase (AICDA) inhibitor and a nuclear factor-kB (NF-kB) inhibitor as active ingredients. to provide.

In one example of the present invention, the AICDA inhibitor includes all ingredients capable of inhibiting all types of AICDA known in the art to which the present invention pertains, and is preferably one selected from natural products, compounds, antibodies, and gene inhibitors. It may include the above, and more preferably may include AICDA shRNA selected from the group consisting of SEQ ID NOs: 1 to 3, but is not limited thereto.

In another example of the present invention, the NF-kB inhibitor includes all components capable of inhibiting all types of NF-kB known in the art to which the present invention pertains, preferably natural products, compounds, antibodies, and gene inhibitors. It may include one or more selected from, and more preferably may include BAY11-7082, but is not limited thereto.

Additionally, the present invention provides a method for preventing or treating lung cancer in non-human animals, comprising administering shRNA for inhibiting Activation Induced Cytidine Deaminase (AICDA) selected from the group consisting of SEQ ID NOs: 1 to 3.

In addition, prevention or treatment of EGFR-TKI (epidermal growth factor-tyrosine kinase inhibitor)-resistant non-small cell lung cancer in non-human animals, including the step of administering an Activation Induced Cytidine Deaminase (AICDA) inhibitor and a nuclear factor-kB (NF-kB) inhibitor. Provides a method.

In the present invention, the term “prevention” or “prevention” means suppressing or delaying the occurrence of a disease from its cause.

In this specification, “treatment” means stopping the progression of damage by suppressing the progression and/or worsening of symptoms even if not completely cured, or improving some or all of the symptoms and leading toward healing.

In the present invention, the term “improvement” refers to any action that improves or beneficially changes symptoms.

The composition of the present invention can be manufactured in any form by further including appropriate carriers, excipients and diluents commonly used in the manufacture of compositions, and is preferably manufactured in the form of a pharmaceutical composition or health functional food composition. , but is not limited to this.

The pharmaceutical composition of the present invention can be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions according to conventional methods. , tablets, capsules, injections and liquid formulations are more preferable. This formulation can be performed by a method commonly performed in the pharmaceutical field, and can be preferably formulated according to each disease or ingredient using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

When formulated, it can be prepared by additionally using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( It is prepared by mixing lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous agents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween, cacao, laurin, glycerogeratin, etc. can be used.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, for desirable effects, the pharmaceutical composition of the present invention may be included in an amount of 0.01 to 99.9% by weight, preferably 0.1 to 99% by weight, per day. The daily dosage may be about 0.1 to 1,000 mg/kg, preferably 100 to 300 mg/kg.

Health functional foods to which the composition can be added include, for example, various general foods, beverages, gum, tea, vitamin complexes, etc.

Additionally, the composition can be added to food or beverages for the purpose of preventing diseases. At this time, the amount of the extract in the food or beverage can be added at 0.01 to 15% by weight of the total weight of the food, and the health drink composition can be added at a rate of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 g. there is.

The health functional drink composition of the present invention has no particular restrictions on other ingredients other than containing the extract as an essential ingredient in the indicated ratio, and may contain additional ingredients such as various flavoring agents or natural carbohydrates like a conventional beverage. there is. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Flavoring agents other than those mentioned above include natural flavoring agent thaumatin, stevia extract, such as rebaudioside A, glycyrrhizin, etc.; and synthetic flavoring agents such as saccharin, aspartame, etc. can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 g of the composition of the present invention. In addition to the above, the extract of the present invention contains various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavoring agents, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, and protective properties. It may contain colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the extracts of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. These ingredients can be used independently or in combination. At this time, the ratio of the additive is not very important, but is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

According to the present invention, a composition for preventing or treating EGFR-TKI resistant non-small cell lung cancer comprising an AICDA and NF-kB inhibitor can be provided. More specifically, the present invention provides a higher therapeutic effect and acquisition of EGFR-TKI resistance when simultaneous inhibition of EGFR-TKI and AICDA/NFκB is performed as the first standard treatment for EGFR-mutant non-small cell lung cancer, in addition to advanced lung cancer resistant to EGFR-TKI treatment. A delaying effect can be expected.

Figure 1 shows NF-κB and AICDA protein expression in PC9 and PC9/GR and expression changes according to Gefitinib administration.
Figure 2 shows the recovery of Gefitinib treatment effect upon AICDA inhibition in PC9/GR cells.
Figure 3 shows the results of cell viability analysis when NFkB (bay 11-7082) and AICDA were simultaneously inhibited in PC9/GR cells.
Figure 4 shows that the combination of NFκB inhibitor (B. Bay11-7082) and EGFR-TKI (G. gefitinib) shows a dose-dependent therapeutic effect in PC9/GR (vector), but when combined with AICDA inhibition (ShAICDA), cell survival rate is maximum. The cell activity analysis results for the decrease are shown (*** p <0.0001).

Below, examples are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

<Example 1> Cell culture

PC9 (EGFR-exon 19 deletion mutant lung adenocarcinoma cell line) and PC9/GR (EGFR-T790M mutant EGFR-TKI resistant lung adenocarcinoma cell line) were used in the experiment. The cell line was cultured at 37°C in 5% CO ₂ in RPMI-1640 medium (WELGENE) containing 10% fetal bovine serum (FBS) (WELGENE).

Tyrosine kinase inhibitor (Gefitinib) was purchased from Tocris (Iressa, 184475-35-2).

<Example 2> Western blotting

The collected proteins were suspended in protein lysis buffer (Translab, Korea) and then measured using the Bio-Rad protein assay (Bio-Rad, cat.no.500-81 0006). 30 μg of protein was separated on a 10% SDS-PAGE gel, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) 82, and then probed with the following antibody (company name) and measured.

anti-β-actin (sc-47778, Santa Cruz Biotechnology), anti-AICDA (ZA001, Invitrogen), anti-p-NF-kB (p-p65) (#3033, Cell Signaling Technology), anti-NF-kB (p65)(#8242, Cell Signaling Technology), anti-NF-kB(p105/50)(ab7549, 86 Abcam), anti-NF-kB(p100/52) (#4882, Cell Signaling Technology), anti- RelB (#10544, Cell 87 Signaling Technology)

<Example 3> AICDA gene suppression

PC9 and PC9GR cells were infected with 105 lentiviruses to suppress the human AICDA gene. The expression-quiescent lentiviral 106 vector encoding control AICDA shRNA was used provided by Sigma 107 (TRCN0000412426, TRCN0000050344, and TRCN0000424739).

Virus supernatant was added to cells (70-80% confluence) along with polybrene (containing 2 ng/ml). 109 uninfected cells were removed by puromycin selection.

The sequence of shRNA is as follows:

shAICDA#1 (SEQ ID NO: 1)

5'-CCGGACCACGAAAGAACTTTCAAAGCTCGAGCTTTGAAAGTTCTTTCGTGGTTTTTTTG-3'

shAICDA#2 (SEQ ID NO: 2)

5'-CCGGCATTTCGTACTTTGGGACTTTCTCGAGAAAGTCCCAAAGTACGAAATGTTTTTG-3'

shAICDA#3 (SEQ ID NO: 3)

5'-CCGGCATTTCGTACTTTGGGACTTTCTCGAGAAAGTCCCAAAGTACGAAATGTTTTTG-3'

<Example 4> NFkB inhibition in PC9/GR cell line

Bay11-7082 (D5556) from Sigma was added commercially to PC9 and PC9GR cells.

<Example 5> Cell survival experiment

It was measured using the CCK-8 assay kit (Dojindo Labor-77 atories).

<Test Example 1> Basic expression forms of NF-κB and AICDA in cell lines and expression changes according to Gefitinib administration

In the PC9/GR cell line, NF-kBp-p65, NF-kB p65, NF-kB p50, and NF-kB AICDA were expressed more, and NF-kB p100 and NF-kB p105 were expressed less.

Upon administration of gefitinib, the expression of AICDA, NFkB p50, NF-kB p105, and NF-kB p-p65 proteins increased in a TKI dose-dependent manner in PC9 and PC9/GR cell lines, and the expression of NF-kB p100 and NF-kB RELB proteins increased. decreased over time (Figure 1).

<Test Example 2> Effect of AICDA inhibition

When AICDA expression was suppressed (shAICDA) in an EGFR-TKI-resistant non-small cell lung cancer cell line (PC9/GR), treatment sensitivity to EGFR-TKI (gefitinib) was restored and cell survival rate was reduced in a statistically significant dose-dependent manner (Figure 2; * p < 0.005, ** p < 0.001).

<Test Example 3> Effect of NF-kB inhibition alone or combined AICDA inhibition

In PC9/GR, NFκB inhibition had the effect of reducing cell viability even without AICDA inhibition (vector), but when AICDA inhibition was combined (ShAICDA), cell viability was further reduced in a dose-dependent manner (Figure 3).

In PC9/GR, combined treatment with EGFR-TKI and inhibition of NFkB and AICDA resulted in a maximum decrease in cell survival (measured at the maximum treatment effect, Figure 4).

In conclusion, combining AICDA/NFκB simultaneous inhibition with EGFR-TKI treatment may be a new treatment strategy for EGFR-TKI resistant lung cancer.

<110>CNU <120> Composition for preventing or treating EGFR-TKI resistant Non-small cell lung cancer comprising AICDA and NF-kB inhibitor <130> M22-9151 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> shAICDA#1 <400> 1 ccggaccacg aaagaacttt caaagctcga gctttgaaag ttctttcgtg gtttttttg 59 <210> 2 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> shAICDA#2 <400> 2 ccggcatttc gtactttggg actttctcga gaaagtccca aagtacgaaa tgtttttg 58 <210> 3 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> shAICDA#3 <400> 3 ccggcatttc gtactttggg actttctcga gaaagtccca aagtacgaaa tgtttttg 58

Claims (11)

shRNA for inhibiting Activation Induced Cytidine Deaminase (AICDA) selected from the group consisting of SEQ ID NOs: 1 to 3. A composition for preventing or treating lung cancer comprising shRNA for inhibiting Activation Induced Cytidine Deaminase (AICDA) selected from the group consisting of SEQ ID NOs: 1 to 3 as an active ingredient. The composition for preventing or treating lung cancer according to claim 2, wherein the lung cancer is non-small cell lung cancer. The composition for preventing or treating lung cancer according to claim 2, wherein the lung cancer is EGFR-TKI (epidermal growth factor-tyrosine kinase inhibitor) resistant non-small cell lung cancer. The composition for preventing or treating lung cancer according to claim 2, further comprising an NF-kB (nuclear factor-kB) inhibitor. The composition for preventing or treating lung cancer according to claim 5, wherein the NF-kB inhibitor is BAY11-7082. A composition for preventing or treating EGFR-TKI (epidermal growth factor-tyrosine kinase inhibitor)-resistant non-small cell lung cancer containing an AICDA (Activation Induced Cytidine Deaminase) inhibitor and an NF-kB (nuclear factor-kB) inhibitor as active ingredients. The composition of claim 7, wherein the AICDA inhibitor comprises an AICDA shRNA selected from the group consisting of SEQ ID NOs: 1 to 3. The composition for preventing or treating EGFR-TKI (epidermal growth factor-tyrosine kinase inhibitor) resistant non-small cell lung cancer according to claim 7, wherein the NF-kB inhibitor is BAY11-7082. Method for preventing or treating lung cancer in non-human animals comprising administering shRNA for inhibiting Activation Induced Cytidine Deaminase (AICDA) selected from the group consisting of SEQ ID NOs: 1 to 3 Method for preventing or treating EGFR-TKI (epidermal growth factor-tyrosine kinase inhibitor) resistant non-small cell lung cancer in non-human animals comprising administering an AICDA (Activation Induced Cytidine Deaminase) inhibitor and an NF-kB (nuclear factor-kB) inhibitor.