Pharmaceutical composition for EGFR gene mutation-positive lung cancer

Abstract

An EGFR inhibitor is used for the treatment of EGFR gene mutation-positive lung cancer, but resistance is acquired over time, and the effect of the EGFR inhibitor disappears. There has been no effective measure against this.SOLUTION: A pharmaceutical composition for EGFR gene mutation-positive lung cancer, which comprises a first pharmaceutical composition for inhibiting EGFR and a second pharmaceutical composition for inhibiting at least one member selected from cGAS, STING and TBK1, and which can overcome resistance to EGFR inhibition and can be administered continuously while maintaining the effect of suppressing cell proliferation.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for lung cancer with EGFR gene mutation, and in particular to a pharmaceutical composition for lung cancer that has acquired resistance to EGFR inhibitors. [Background technology]

[0002] EGFR gene-mutated lung cancer is a genetic abnormality found in approximately one in three Japanese patients with non-small cell lung cancer. The standard treatment for this tumor is EGFR inhibitors, but their therapeutic efficacy is limited and they eventually become ineffective in all cases. Resistance to EGFR inhibitors develops. Chromosomal instability (CIN) in tumor cells has been found to be one of the causes of this resistance. However, a method for overcoming this resistance has yet to be identified, and new therapeutic agents (pharmaceutical compositions) are needed to provide better treatment.

[0003] Non-Patent Document 1 shows that the MET inhibitor PHA-665752 is effective against MET gene amplification-dependent resistance acquired when EGFR-TKIs are used for EGFR-mutated lung cancer. It also states that co-administration of a MET inhibitor is effective against resistance mediated by the MET bypass pathway due to MET gene amplification. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Jeffrey A. Engelman et.al:MET Amplification Leads to Gefitinib Resistance in Lung Cancer by Activating ERBB3 Signaling SCIENCE. VOL 316, 18 MAY, 2007, 1039-1043 Summary of the Invention [Problem to be solved by the invention]

[0005] Non-Patent Document 1 shows that when an EGFR inhibitor is used for EGFR-mutated lung cancer, the MET inhibitor is effective against resistance that is due to MET gene amplification. However, it does not provide any knowledge of a pharmaceutical composition that can effectively treat cancer that has acquired resistance due to other causes, such as EGFR gene mutation. [Means for solving the problem]

[0006] The present invention provides a pharmaceutical composition that overcomes resistance to non-small cell carcinoma that has acquired resistance to an EGFR inhibitor and exerts therapeutic effects such as shrinkage of cancer cells.

[0007] More specifically, the pharmaceutical composition for EGFR gene mutation-positive lung cancer according to the present invention comprises: A pharmaceutical composition for lung cancer positive for EGFR gene mutation, comprising: a first pharmaceutical composition that inhibits EGFR; It is characterized by comprising a second pharmaceutical composition that inhibits at least one of cGAS, STING, or TBK1. [Effects of the Invention]

[0008] The pharmaceutical composition of the present invention for EGFR gene mutation-positive lung cancer can suppress the proliferation of cancer cells caused by EGFR inhibitors and overcome the resistance that develops over time, allowing for the administration of EGFR inhibitors over a long period of time, thereby enabling effective treatment. [Brief explanation of the drawings]

[0009] [Figure 1] 10 is a photograph showing the results of a colony formation test for evaluating the sensitivity of H1975Cont and H1975dnMCAK to the EGFR inhibitor osimertinib. [Figure 2] 1 is a graph showing the results of a growth inhibition test in which H1975Cont and H1975dnMCAK were cultured with multiple concentrations of the EGFR inhibitor osimertinib. [Figure 3] 1 is a graph showing the results of gene signature analysis for H1975Cont and H1975dnMCAK. [Figure 4] This figure shows the results of a colony formation assay performed on H1975dnMCAK to evaluate the antitumor effects of single-agent and combined administration of the EGFR inhibitor osimertinib and an inhibitor of the cGAS-STING-TBK1 pathway activated by CIN. DETAILED DESCRIPTION OF THE INVENTION

[0010] The pharmaceutical composition for EGFR gene mutation-positive lung cancer according to the present invention will be described below with reference to drawings and examples. Note that the following description exemplifies one embodiment of the present invention and one example, and the present invention is not limited to the following description. The following description can be modified within the scope of the present invention.

[0011] Furthermore, embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. Furthermore, all documents described in this specification are incorporated herein by reference. In this specification, when a numerical range is described as "A to B," this means "greater than or equal to A and less than or equal to B."

[0012] The pharmaceutical composition for EGFR gene mutation-positive lung cancer according to the present invention is intended to treat cancers in which an EGFR gene mutation is detected. Cancers that are primarily candidates for treatment with the pharmaceutical composition for EGFR gene mutation-positive lung cancer according to the present invention include exon 19 deletion, in which a portion of exon 19 of the EGFR gene is deleted, and "L858R point mutation," in which the nucleotide sequence in exon 21 is rearranged. Furthermore, "treatment" here refers only to the inhibition of cancer cell proliferation, and does not necessarily mean the reduction or elimination of cancer cells.

[0013] The pharmaceutical composition for EGFR gene mutation-positive lung cancer according to the present invention comprises two drugs: an EGFR inhibitor (first pharmaceutical composition) and at least one drug selected from the group consisting of a cGAS inhibitor, a STING inhibitor, and a TBK1 inhibitor (second pharmaceutical composition).

[0014] The first pharmaceutical composition, EGFR (epidermal growth factor receptor), is an epidermal growth factor receptor. EGFR is a cell membrane-spanning receptor that transmits cell growth signals by binding to the growth factor EGF on the cell membrane. However, when a mutation occurs in the EGFR gene, the cell growth signal continues to be activated even without binding to growth factors, causing cancer cells to grow indefinitely. EGFR inhibitors stop this signal transduction and suppress cancer cell growth.

[0015] As EGFR inhibitors, in addition to antibody drugs such as cetuximab, panitumumab, necitumumab, and amivantamab, small molecule inhibitors such as gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, fumonertinib, brigatinib, nazartinib, and lazertinib can be suitably used.

[0016] The second pharmaceutical composition, cGAS (cyclic GMP-AMP synthase) and STING (stimulator of interferon genes), constitute the cGAS-STING pathway, which is known as a pathway in which non-self DNA enters cells, activates cGAS as a DNA sensor, and induces the production of inflammatory response substances such as IFN (interferon), IL-6 (interleukin-6), and TNF (tumor necrosis factor) through STING. Furthermore, the second pharmaceutical composition, TBK1 (TNAK binding kinase 1), induces a conformational change in STING in the cGAS-STING pathway, promoting its migration from the endoplasmic reticulum to the Golgi apparatus.

[0017] Suitable cGAS inhibitors include G140, G150, TDI-6670, TDI-8246, Compound 102, Ventus Therapeutic, cGAS-IN-2 (Compound 109, Ventus Therapeutic), PF-06928215, and Vent03. Suitable STING inhibitors include C-176, C-178, and H151. Suitable TBK1 inhibitors include BX-795, MRT67307, GSK8602, BAY-985, and Amiexanox.

[0018] The second pharmaceutical composition contains at least one drug selected from a cGAS inhibitor, a STING inhibitor, and a TBK1 inhibitor, and it is desirable to use a dosage of each drug that can exert its therapeutic effect (EGFR inhibitor, cGAS inhibitor, STING inhibitor, TBK1 inhibitor).

[0019] The forms of the first and second pharmaceutical compositions are not particularly limited, as long as they are in a form that allows them to be administered almost simultaneously. One may be in liquid form and the other in tablet, powder, or the like. However, it is most preferable if the first and second pharmaceutical compositions are in a mixed state from the beginning.

[0020] It is still unclear why EGFR, which is involved in cell proliferation signals, and cGAS, which is involved in intracellular autoimmunity, can overcome resistance to EGFR inhibitors. However, the following reasons are beginning to emerge: Activation of the cGAS-STING-TBK1 pathway induces epithelial-mesenchymal transition in cancer cells. Epithelial-mesenchymal transition is known to be the cause of resistance to EGFR inhibitors. Therefore, inhibition of the cGAS-STING-TBK1 pathway suppresses epithelial-mesenchymal transition, thereby overcoming resistance to EGFR inhibitors. Therefore, it is thought that the combination of an EGFR inhibitor and an inhibitor of the cGAS-STING-TBK1 pathway can overcome resistance to EGFR inhibitors.

[0021] When these agents are used as pharmaceutical compositions for EGFR gene mutation-positive lung cancer, they can be used alone or as salts by mixing with a pharmaceutically acceptable acid in a solvent such as water, methanol, ethanol, acetone, etc. Here, pharmaceutically acceptable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfate, phosphoric acid, and nitric acid, and organic acids such as acetic acid, propionic acid, oxalic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, maleic acid, fumaric acid, methanesulfonic acid, p-toluenesulfonic acid, and ascorbic acid.

[0022] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and may be either oral or parenteral. In addition, it can be prepared into an appropriate dosage form depending on the dosage form, for example, it can be prepared into various preparations such as injections, oral preparations such as capsules, tablets, granules, powders, pills, and fine granules, rectal preparations, oily suppositories, and aqueous suppositories. [Example]

[0023] <cell> We established a chromosomal instability (CIN) cell model using an EGFR gene mutation-positive lung cancer cell line (H1975). Specifically, we introduced the dominant-negative MCAK (dnMCAK) gene into the H1975 cell line according to previous literature to generate a cell line with severe CIN, H1975dnMCAK (hereafter referred to as "H1975dnMCAK"). A control cell line lacking the dnMCAK gene was also generated (hereafter referred to as "H1975Cont").

[0024] Next, a colony formation test was performed to evaluate sensitivity to the EGFR inhibitor osimertinib. The results are shown in Figure 1. In Figure 1(a) (H1975Cont) and Figure 1(b) (H1975dnMCAK), "-" indicates the case where osimertinib was not added, and "+" indicates the case where 100 nM osimertinib was added. The dark areas indicate the locations of cells in the H1975Cont and H1975dnMCAK strains.

[0025] Referring to Figure 1(a), in the control H1975Cont strain, exposure to the EGFR inhibitor osimertinib (Figure 1(a)+) resulted in no dark areas where cells existed, and cancer cell colony formation was suppressed. This is thought to be because EGFR inhibition prevented the generation of cell proliferation signals, resulting in the cessation of cell proliferation. On the other hand, in the H1975dnMCAK strain (Figure 1(b)), even when exposed to the EGFR inhibitor osimertinib (Figure 1(b)+), dark areas where cells existed were observed, and colony formation was relatively maintained.

[0026] This was further quantitatively investigated. 5,000 H1975Cont and H1975dnMCAK cell lines were seeded into each well of a 96-well plate. Various concentrations of osimertinib were added to the medium, or medium alone (control). Six wells were used for each concentration. After 48 hours, the number of viable cells in each well was determined using a colorimetric reagent, and the ratio of viable cells to the control was calculated.

[0027] The results are shown in Figure 2. Referring to Figure 2, the horizontal axis represents the osimertinib concentration (μM), and the vertical axis represents the cell survival rate (% of control) relative to the number of H1975Cont cells when osimertinib was not added. The horizontal axis is logarithmic. H1975Cont is shown by a dashed line, and H1975dnMCAK is shown by a solid line.

[0028] In the H1975Cont cell line (dashed line), the percentage of viable cells decreased to approximately 25% of the control with increasing concentrations of the EGFR inhibitor osimertinib, whereas in the H1975dnMCAK cell line, the percentage remained above 50%. This confirms that H1975dnMCAK is resistant to osimertinib compared to the H1975Cont cell line.

[0029] <Gene signature analysis> Gene signature analysis was performed on the H1975dnMCAK line to evaluate the activation of downstream signals of CIN (STING to interferon α / β). The results are shown in Figure 3. Figure 3(a) shows the signaling by STING, Figure 3(b) shows the signaling by IRF3, and Figure 3(c) shows the signaling by interferon α / β. The three blocks for H1975Cont and H1975dnMCAK each show the results of three clonal cells.

[0030] In Figures 3(a) to 3(c), the higher the activity, the lighter the color. The numbers below each color indicate the signal activity, with higher numbers indicating higher signal activity. The H1975dnMCAK strain showed more activated STING, IRF3, and interferon α / β signals than the H1975Cont strain.

[0031] A colony formation test was performed on H1975dnMCAK to evaluate the antitumor effects of single-agent and combined administration of the EGFR inhibitor osimertinib and an inhibitor of the cGAS-STING-TBK1 pathway downstream of CIN. Photographs of each plate and the results of quantifying the colonies are shown in Figure 4.

[0032] Referring to Figure 4, Figure 4(a) shows H1975Cont, and Figures 4(b) to 4(d) show H1975dnMCAK. Figure 4(a) shows the results of adding 100 nM of the EGFR inhibitor (osimertinib) alone, Figure 4(b) shows the results of adding 10 μM of the cGAS inhibitor (G150) to 100 nM of the EGFR inhibitor (osimertinib). Figure 4(c) shows the results of adding 1 μM of the STING inhibitor (C-176) to 100 nM of the EGFR inhibitor (osimertinib), and Figure 4(d) shows the results of adding 1 μM of the TBK1 inhibitor (MRT67307) to 100 nM of the EGFR inhibitor (osimertinib).

[0033] In each figure, "-" indicates no addition, and "+" indicates addition. The following shows the addition and non-addition of the first pharmaceutical composition (EGFR inhibitor) and the second pharmaceutical composition (cGAS inhibitor, STING inhibitor, TBK1 inhibitor), in that order. For example, in Figure 4(b), the addition of osimertinib without the addition of G150 is represented as "Figure 4(b) (+, -)." The numbers above the squares represent the colony formation efficiency, with the colony formation efficiency without the addition of an inhibitor set at 100.

[0034] Referring to Figure 4(a), in the osimertinib-sensitive H1975Cont cell line, exposure to osimertinib (Figure 4(a)(+)) reduced the colony formation rate to 24% compared to the medium-only control (Figure 4(a)(-)). On the other hand, referring to Figure 4(b), in the osimertinib-resistant H1975dnMCAK cell line, colony formation was only 44% of the control even with exposure to osimertinib (Figure 4(b)(+,-)).

[0035] Furthermore, single-agent administration of G150 (cGAS inhibitor) (Figure 4(b) (-, +)), C-176 (STING inhibitor) (Figure 4(c) (-, +)), and MRT67307 (TBK1 inhibitor) (Figure 4(d) (-, +)) had limited inhibitory effects on colony formation (64%, 28%, and 38%, respectively).

[0036] On the other hand, when these second pharmaceutical compositions were used in combination with osimertinib (first pharmaceutical composition) (Figure 4(b)(+,+), Figure 4(c)(+,+), Figure 4(d)(+,+)), the colony formation rate was below 24%, and colony formation was strongly inhibited. [Industrial Applicability]

[0037] The pharmaceutical composition for EGFR gene mutation-positive lung cancer according to the present invention can be suitably used as a therapeutic agent for EGFR gene mutation-positive lung cancer, and can effectively treat non-small cell lung cancer, which is a cancer that frequently undergoes EGFR gene mutation, by overcoming resistance and inhibiting cancer cell proliferation.

Claims

1. A pharmaceutical composition for lung cancer positive for EGFR gene mutation, comprising: a first pharmaceutical composition that inhibits EGFR; and A pharmaceutical composition for EGFR gene mutation-positive lung cancer, comprising a second pharmaceutical composition that inhibits at least one of cGAS, STING, or TBK1.

2. The first pharmaceutical composition is at least one drug selected from the group consisting of antibody drugs such as cetuximab, panitumumab, necitumumab, and amivantamab, as well as gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, fumonertinib, brigatinib, nazartinib, and lazertinib. The pharmaceutical composition for EGFR gene mutation-positive lung cancer according to claim 1.

3. 3. The pharmaceutical composition for EGFR gene mutation-positive lung cancer according to claim 1 or 2, wherein the cGAS inhibitor is at least one drug selected from the group consisting of G140, G150, TDI-6670, TDI-8246, Compound 102, Ventus Therapeutic, cGAS-IN-2 (Compound 109, Ventus Therapeutic), PF-06928215, and Vent 03.

4. The pharmaceutical composition for EGFR gene mutation-positive lung cancer according to claim 1 or 2, wherein the STING inhibitor is at least one of C-176, C-178, and H151.

5. The pharmaceutical composition for EGFR gene mutation-positive lung cancer according to claim 1 or 2, wherein the TBK1 inhibitor is at least one drug selected from the group consisting of BX-795, MRT67307, GSK8602, BAY-985, and Amiexanox.

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