Composition for reversing drug resistance of small cell lung cancer hepatometastatic ferroptosis inducer and application thereof

Abstract

The invention provides a composition for reversing drug resistance of a small cell lung cancer hepatic metastatic ferroptosis inducer and application of the composition, and belongs to the technical field of biological medicine. The composition for reversing the drug resistance of the small cell lung cancer hepatometastatic ferroptosis inducer provided by the invention comprises the ferroptosis inducer and a GPX4 inhibitor. The composition can remarkably reverse the drug resistance of a ferroptosis inducer induced by a liver microenvironment, reduce the dosage of the ferroptosis inducer and improve the detection sensitivity and reproducibility through the synergistic effect of improving the lipid peroxidation pressure and reducing the scavenging capacity, and the mechanism specificity is proved to have a good application prospect through a Fer-1 rescue test. The medicine is suitable for small cell lung cancer people with or with high-risk liver metastasis, and a new effective strategy is provided for clinical treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a composition for reversing resistance to ferroptosis-inducing agents in liver metastases of small cell lung cancer and its application. Background Technology

[0002] Small cell lung cancer (SCLC) progresses rapidly, metastasizes early, and has a high recurrence rate; liver metastasis is one of the common and challenging progression patterns. While standard first-line chemotherapy can achieve short-term remission, it suffers from low durable response rates and rapid drug resistance development. Immunotherapy has inconsistent efficacy in a significant proportion of patients and offers limited control over liver metastases. Clinically, there is an urgent need for a new strategy with a clearly defined mechanism, quantifiable validation, and parallel compatibility with existing regimens, specifically addressing drug tolerance issues related to the liver microenvironment.

[0003] Ferroptosis, a form of cell death centered on lipid peroxidation, has potential lethality against certain refractory tumors. Small molecules such as Erastin / IKE are known to induce lipid peroxidation stress; while GPX4 (glutathione peroxidase 4) inhibitors (ML210 / JKE-1674) further amplify lipid peroxidation stress by inhibiting detoxification. While single-drug studies and in vitro findings have been conducted, in the liver metastasis-related microenvironment, single-drug therapy often results in insufficient inhibition or unstable efficacy. Furthermore, the lack of a unified evaluation methodology and reproducible parameter windows hinders the development of transferable engineered solutions.

[0004] The existing technology has three main drawbacks: 1. Single drugs are easily "inactivated" in the liver microenvironment: Hepatocyte secretion factors and metabolic supply can significantly change the redox state of tumor cells. The efficacy of a single ferroptosis inducer or a single GPX4 inhibitor is insufficient, leading to increased tumor cell tolerance and limited endpoint inhibition effect.

[0005] 2. Lack of definition of "necessary parameters" for dosing sequence and dosage: Existing studies have not clarified whether ferroptosis inducers and GPX4 inhibitors need to be administered simultaneously, the upper and lower limits of their dosages, solvent ratios, and other key parameters. Once these parameters are exceeded, the synergistic effect may disappear or non-specific toxicity may occur.

[0006] 3. Lack of mechanism-specific "positive-negative" discrimination: Most studies did not set up Fer-1 (a specific inhibitor of ferroptosis) mechanism rescue experiments as a strong chain of evidence, resulting in conclusions that only remained at the phenomenological level and could not confirm that the effect originated from the ferroptosis pathway.

[0007] Based on the aforementioned clinical pain points and shortcomings of existing technologies, this invention proposes a technical solution combining ferroptosis inducers and GPX4 inhibitors in a simulated liver microenvironment scenario for small cell lung cancer, aiming to solve the problems of existing single-drug resistance, unclear parameters, and insufficient mechanism verification. Summary of the Invention

[0008] The purpose of this invention is to provide a composition for reversing resistance to ferroptosis inducers in liver metastases of small cell lung cancer, addressing the resistance problem of small cell lung cancer to ferroptosis inducers in the liver microenvironment, achieving resistance reversal and improving treatment efficacy.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a composition for reversing ferroptosis inducer resistance in liver metastases of small cell lung cancer, comprising a ferroptosis inducer and a GPX4 inhibitor.

[0010] Preferably, the ferroptosis inducer includes Erastin and IKE; the GPX4 inhibitor includes ML210 and JKE-1674.

[0011] Preferably, the concentration ratio of the ferroptosis inducer to the GPX4 inhibitor is 5~10 μM:0.05~2 μM.

[0012] The present invention also provides the use of the composition thereof in the preparation of a medicament for reversing resistance to ferroptosis-inducing agents in liver metastases of small cell lung cancer.

[0013] The present invention also provides the use of the composition thereof in the preparation of a medicament for the treatment or prevention of small cell lung cancer.

[0014] Preferably, the small cell lung cancer is small cell lung cancer with liver metastasis or a high risk of liver metastasis.

[0015] Beneficial effects

[0016] 1. Significantly reverses liver microenvironment-induced tolerance to ferroptosis inducers: In a 0.4 μm Transwell co-culture model of THLE-2 and SCLC, this combination significantly reduced tumor cell survival / clonal formation ability compared to ferroptosis inducer monotherapy, with a reversal rate of 65.5%~86.4%. This is because GPX4 inhibitors weaken the cells' ability to clear lipid peroxidation, lower the ferroptosis threshold, and cause the "tolerance scaffold" provided by the liver microenvironment to fail.

[0017] 2. Clear synergistic effect: The combination index CI calculated with cell survival / lipid peroxidation as the endpoint is <1 (0.5-0.8), which proves that there is a synergistic effect between ferroptosis inducers and GPX4 inhibitors; the two types of drugs have complementary sites of action, one type increases lipid peroxidation stress, and the other type reduces clearance capacity, forming a closed loop of "attack + retreat".

[0018] 3. Reduce the dosage of ferroptosis inducers and broaden the therapeutic window: Under combined conditions, the IC50 of ferroptosis inducers... 50 Compared to monotherapy, the dose is reduced by 30% to 70%, which reduces the risk of potential non-specific toxicity while ensuring therapeutic effect. This is due to the synergistic amplification effect, which reduces the dose required for equivalent effect levels.

[0019] 4. High detection sensitivity and reproducibility: The signal-to-noise ratio of lipid peroxidation signal, cell death phenotype and clonal inhibition under combined treatment is higher and the inter-batch difference is smaller. It can be stably reproduced in different SCLC cell lines such as H446 and H196. The superposition of the two mechanisms improves the effect amplitude and reduces baseline fluctuation.

[0020] 5. Mechanism specificity has been rigorously verified: Fer-1 can completely or partially rescue combination-induced cell death / lipid peroxidation, confirming that the effect originates from the ferroptosis pathway itself, rather than other forms such as apoptosis / necrosis; Fer-1, as a specific inhibitor of ferroptosis, can block the lipid peroxidation chain reaction.

[0021] 6. Good applicability and scalability: Consistent trends were observed in multiple SCLC cell lines and liver microenvironment models; at the formulation level, it can be matched with pharmaceutically acceptable carriers / solvents such as cyclodextrin and PLGA microspheres, which facilitates subsequent dosage form development; the core reason is that it targets the common core node in the tumor spectrum of "lipid peroxidation-GPX4 clearance". Attached Figure Description

[0022] Figure 1 The absorbance test results for 0~24 h and 0~72 h are from Example 1; Figure 2 The absorbance test results for 0~24 h and 0~72 h are from Example 2; Figure 3 The cell counting results are from individual and co-culture in Example 3; Figure 4 The cell counting results are for individual and co-culture in Example 4. Detailed Implementation

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Experimental materials

[0025] 1. Cell lines: Human liver cell line THLE-2 (Chinese Academy of Sciences Cell Bank, mycoplasma negative); small cell lung cancer cells H446, H196, and DMS-53 (Chinese Academy of Sciences Cell Bank, mycoplasma negative).

[0026] 2. Culture media and excipients: IMDM, RPMI-1640, Waymouth medium (source culture); FBS (Gibco, Australian brand); penicillin-streptomycin combination antibiotic (Synthetium arabinose, 1% (v / v)).

[0027] 3. Drugs and solvents: Erastin, IKE, ML210, JKE-1674, Fer-1 (all molecular biology grade); DMSO (molecular biology grade, final concentration ≤0.1% (v / v) when preparing working solution).

[0028] 4. Consumables and equipment: 24-well plates, 96-well plates; 0.4μm Transwell chambers (Corning 3401); 0.22μm filter membranes; sterile pipette tips.

[0029] 5. Equipment: CO2 incubator (37℃, 5% C) (); inverted fluorescence microscope; flow cytometer; clean bench; benchtop centrifuge.

[0030] Example 1: Inhibitory effect and accelerated onset of action of Erastin+ML210 in H196 single-culture model

[0031] H196 small cell lung cancer cells were tested negative for mycoplasma before use. They were routinely cultured in RPMI-1640 medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibody), with H196 cells in logarithmic growth phase sporadically at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 µL per well in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. Drug administration was performed according to the following groups (T0 being the start time of administration): DMSO control group, Erastin 5 μM single-drug group, Erastin 5 μM + ML210 50 nM combination group, and Erastin 5 μM + ML210 50 nM + Fer-1 10 μM rescue group; ensuring that the final DMSO concentration in each well was consistent and ≤0.1%. Each group had 3 replicates. The GI index was calculated and CCK-8 absorbance was measured in two windows: 0 / 8 / 16 / 24 h and 0 / 24 / 48 / 72 h. The results are shown below. Figure 1 As shown. The normalized GI(t) = [(OD_t / OD_0) treatment group] / [(OD_t / OD_0) control group] × 100% was used to calculate rAUC. 0–T(Relative area under the curve), T 50 (GI = 50% of the time) and I_max (the maximum suppression amplitude at the endpoint = 100% - GI_end).

[0032] Results showed that from 0 to 24 hours, the rAUC of the Erastin monotherapy group was: 0–24h The percentage was 92.8%, and T was not achieved. 50 GI_end was 70.7% (I_max = 29.3%); the combined group rAUC 0–24h It was 78.0%, T 50 =22.8h, GI_end=45.2% (I_max=54.8%); rescue team rAUC 0–24h The percentage is 96.2%, and GI_end = 99.9%.

[0033] 0-72h: Erastin monotherapy group rAU –72h =71.2%, =57.5 h, GI_end = 31.1% (I_max = 68.9%); combined group rAUC 0–72h It is 35.5%, T 50 =17.6h, GI_end=12.0% (I_max=88.0%); rescue team rAUC 0–72h ≈100.9%, GI_end≈102.4%.

[0034] It is evident that in the H196 single-culture model, the Erastin+ML210 combination significantly reduced rAUC and shortened T compared to single-drug administration. 50 The increase of I_max and the significant rescue effect of Fer-1 confirm that it is a ferroptosis-related mechanism.

[0035] Example 2: Inhibitory effect and endpoint enhancement of Erastin+ML210 in H446 single-culture model

[0036] H446 small cell lung cancer cells were routinely cultured in IMDM medium (containing 20% ​​fetal bovine serum and 1% penicillin-drug antibody) and administered drugs according to the following groups (T0 being the start time of drug administration): DMSO control group, Erastin 5μM single drug group, Erastin 5μM + ML2 10 50nM combination group, and Erastin 5μM + ML2 10 50nM + Fer-1 10μM rescue group. CCK-8 absorbance was measured at 0, 6, 12, 18, 24, 48, and 72 h. The results are as follows: Figure 2As shown. Using the normalized caliber GI(t) = [(OD_t / OD_0)_group] / [(OD_t / OD_0)_DMSO] × 100%, rAUC is calculated. 0–T (Relative area under the curve), T 50 (GI = 50% of the time) and I_max (the maximum suppression amplitude at the endpoint = 100% - GI_end).

[0037] Results showed that from 0 to 24 hours, the rAUC of the Erastin monotherapy group was: 0–24h The percentage was 92.5%, and the target was not achieved. 50 GI_end was 69.1% (I_max = 30.9%); the combined group rAUC 0–24h It is 84.5%, T 50 =23.7h, GI_end=48.7% (I_max=51.3%); rescue team rAUC 0–24h The percentage is 93.6%, and GI_end = 92.2%.

[0038] 0-72h: rAUC of Erastin monotherapy group 0–72h The figure was 64.0%, and T was not achieved. 50 GI_end=54.7% (I_max=45.3%); combined group rAUC 0–72h It is 52.4%, T 50 =20.4h, GI_end=40.3% (I_max=59.8%); rescue team rAUC0 –72h ≈86.1%, GI_end≈87.7%.

[0039] It is evident that in the H446 single-culture model, the combination also exhibits a reduction in rAUC and a shortening of T. 50 The effect of improving I_max is that Fer-1 can partially / significantly rescue, indicating that the inhibitory effect originates from the ferroptosis pathway.

[0040] Example 3: Resistance reversal effect of Erastin+ML210 in the H446 co-culture model (liver microenvironment)

[0041] Human immortalized hepatocytes THLE-2 and small cell lung cancer cells H446 were cultured in a non-contact co-culture system using a Transwell® (Corning) plate with a 0.4 µm pore size. The lower chamber (bottom of a 24-well plate) was seeded with H446 cells at a density of 1.8 × 10⁻⁶ cells / well. 5 Cells / well, volume 400 µL. Upper chamber (Transwell chamber): seeded with THLE-2 cells at a density of 3.5 × 10⁻⁶ cells / well. 5Cells / compartment, volume 200 µL. Co-culture medium: Use IMDM complete medium corresponding to H446 cells. Co-culture conditions: 37℃, 5% C The treatment lasted for 24 hours. Two systems were set up: H446 cells cultured alone and co-cultured with THLE-2 cells. Groups were assigned as follows: DMSO control group, Erastin 5μM single-drug group, Erastin 5μM + ML210 50nM combination group, and Erastin 5μM + ML210 50nM + Fer-1 10μM rescue group; the final DMSO concentration was ≤0.1%. After 24 hours of co-culture (T0), each group was administered the appropriate drug. 24 hours after drug treatment, the number of viable H446 cells in the lower chamber was counted using 0.4% trypan blue staining. Viable cells were counted in 9 fields per well. The results are as follows: Figure 3 As shown, mean ± SD (n=9) was calculated and normalized to relative survival rate using the DMSO group of each system.

[0042] The results showed that, in the case of single culture (H446-mono): with DMSO group as 100%, the relative survival rate of Erastin single drug group was 7.66% (SD≈4.72%, 95% CI 4.04%~11.29%), the combined group was 3.28% (SD≈2.60%, 95% CI 1.29%~5.28%), and the rescue group was 79.26% (SD≈20.29%, 95% CI 63.63%~94.79%).

[0043] Co-culture (H446-CO, liver microenvironment): DMSO group 100%, Erastin monotherapy group 36.51% (SD≈8.43%, 95% CI 30.01%~42.96%), combination group 4.94% (SD≈4.23%, 95% CI 1.69%~8.20%), rescue group 99.05% (SD≈30.44%, 95% CI 75.62%~122.47%).

[0044] As can be seen, compared with the Erastin monotherapy group, the combination group further reduced the relative survival rate (7.66%→3.28%), which significantly rebounded to 79.26% after the addition of Fer-1, suggesting that the inhibitory effect is related to ferroptosis. The relative survival rate of Erastin monotherapy in the co-culture system was 36.51%, significantly higher than its survival rate in the single culture system, indicating that the hepatocyte microenvironment had a significant protective effect on SCLC cells, reducing their sensitivity to ferroptosis inducers. In the co-culture system, the combination group inhibited cell survival rate to 4.94%, and compared with the Erastin monotherapy group (36.51%), the reversal rate (calculated as: [(Erastin monotherapy group survival rate - combination group survival rate) / Erastin monotherapy group survival rate]×100%) was as high as 86.4%. This shows that the composition of the present invention can effectively overcome the protection provided by the liver microenvironment. The survival rate of the rescue group in the co-culture system recovered to 99.05%, further demonstrating that the effect of the composition in the complex microenvironment is still dominated by the ferroptosis mechanism.

[0045] Example 4: Resistance reversal effect of Erastin+ML210 in the H196 co-culture model (liver microenvironment)

[0046] Human immortalized hepatocytes THLE-2 and small cell lung cancer cells H196 were cultured in a non-contact co-culture system using a Transwell® (Corning) plate with a 0.4 µm pore size. The lower chamber (bottom of a 24-well plate) was seeded with H196 cells at a density of 1.8 × 10⁶ cells / well. 5 Cells / well, volume 400 µL. Upper chamber (Transwell chamber): seeded with THLE-2 cells at a density of 3.5 × 10⁻⁶ cells / well. 5 Cells / compartment, volume 200 µL. Co-culture medium: RPMI-1640 complete medium corresponding to H196 cells. Co-culture conditions: 37℃, 5% C The treatment lasted for 24 hours. Two systems were set up: H196 cells cultured alone and co-cultured with THLE-2. Groups were assigned as follows: DMSO control group, Erastin 5μM single-drug group, Erastin 5μM + ML210 50nM combination group, and Erastin 5μM + ML210 50nM + Fer-1 10μM rescue group; the final DMSO concentration was ≤0.1%. After 24 hours of co-culture (T0), each group was administered the corresponding drug. 24 hours after drug treatment, the number of viable H196 cells in the lower chamber was counted using 0.4% trypan blue staining. Viable cells were counted in 9 fields per well. The results are as follows: Figure 4 As shown, the relative survival rate (%) was normalized to the DMSO group of each system, and the mean ± SD (n=9) and 95% CI were reported.

[0047] The results showed that, in the H196-mono culture (with DMSO group as 100%), the relative survival rates were 51.61% in the Erastin monotherapy group (SD≈11.76%, 95% CI 42.57%~60.65%), 29.45% in the combination group (SD≈7.53%, 95% CI 23.67%~35.24%), and 68.26% in the rescue group (SD 23.78%, 95% CI 49.96%~86.56%).

[0048] Co-culture (H196-CO): DMSO group 100%, Erastin monotherapy group 55.55% relative survival (SD≈18.50%, 95% CI 41.37%~69.75%), combination group 19.21% (SD≈6.52%, 95% CI 14.20%~24.22%), rescue group 54.48% (SD≈22.61%, 95% CI 37.09%~71.87%).

[0049] It is evident that the liver microenvironment reduced the inhibitory effect of Erastin monotherapy (relative survival rate of 55.55% in the co-culture group vs. 51.61% in the monoculture group); the Erastin+ML210 combination showed a reversal of approximately 65.5% under co-culture conditions, and Fer-1 significantly rescued the combination effect, confirming the specificity of ferroptosis.

[0050] Example 5: Co-culture of H446 liver microenvironment

[0051] The inhibitory effects of existing single-agent therapy (Erastin) and the proposed solution (Erastin+ML210) in the liver microenvironment were compared to verify whether tolerance could be reversed; Fer-1 was used as the mechanism for rescue.

[0052] Cells and systems: H446 and THLE-2 cells were co-cultured non-contactly in a 0.4 µm Transwell; lower chamber (H446) 1.8 × 10⁻⁶ cells. 5 / well, 400 µL; upper chamber (THLE-2) 3.5 × 10 5 / small chamber, 200 µL; uniformly used IMDM + 20% FBS + 1% double antibiotics; 37℃, 5% C .

[0053] Grouping and administration: DMSO ≤ 0.1%; Erastin 5 µM; Erastin + ML210 50 nM; Erastin + ML210 + Fer-1 10 µM (added simultaneously).

[0054] Readings and aperture: 24 h after drug administration, viable cells were counted in the lower chamber with 0.4% trypan blue; n=9 per group (9 fields of view per well); relative viability was normalized to % by DMSO of each system and reported as mean ± SD, 95% CI.

[0055] Evaluation indicators: Key indicators: relative survival % (of DMSO), reversal rate %.

[0056] Supporting evidence: Can Fer-1 significantly boost the combined effect (mechanism rescue)?

[0057] The results are as follows: Relative survival % (mean ± SD; 95% CI) Erastin monotherapy: 36.51% (SD≈8.43; 95% CI 30.01~42.96); Erastin+ML210: 4.94% (SD≈4.23; 95%CI 1.69~8.20); Erastin+ML210+Fer-1: 99.05% (SD≈30.44; 95%CI 75.62~122.47).

[0058] Reversal magnitude % (CO): [(36.51-4.94) / 36.51]×100≈86.4%; Effect difference (Δ)=31.57%; Proportional decrease (combination / E)≈0.135.

[0059] Statistical analysis and judgment: Erastin monotherapy showed a significant difference compared to combination therapy (Welch t, ​​two-sided, α=0.05); Fer-1 significantly improved the combination therapy, which was determined to be specific to ferroptosis.

[0060] Conclusion: Single drugs are insufficient to inhibit liver function in the liver microenvironment; the combination of the present invention significantly “reverses tolerance” and is a ferroptosis effect that can be rescued by Fer-1.

[0061] Example 6: Co-culture of H196 liver microenvironment

[0062] The conditions and methods were the same as in Example 5, except that the culture medium was changed to RPMI-1640 + 10% FBS + 1% double antibiotics, and the rest were the same.

[0063] The results are as follows: Relative survival % (mean ± SD; 95% CI) Erastin monotherapy: 55.55% (SD≈18.50; 95% CI 41.37~69.75); Erastin+ML210: 19.21% (SD≈6.52; 95%CI 14.20~24.22); Erastin+ML210+Fer-1: 54.48% (SD≈22.61; 95%CI 37.09~71.87); Reversal magnitude % (CO): [(55.55-19.21) / 55.55]×100≈65.5%; Δ=36.34%; Proportional decrease (combination / E)≈0.346.

[0064] Significant differences were observed; Erastin monotherapy showed a significant difference compared to combination therapy, and Fer-1 brought the combination therapy back to levels close to Erastin monotherapy, which was determined to be specific to ferroptosis.

[0065] Conclusion: The trend of "this invention is superior to single drugs" was reproduced across cell lines, demonstrating its universality and mechanism specificity.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition for reversing ferroptosis-inducing agent resistance in liver metastases of small cell lung cancer, characterized in that, Including ferroptosis inducers and GPX4 inhibitors.

2. The composition according to claim 1, characterized in that, The ferroptosis inducers include Erastin and IKE; the GPX4 inhibitors include ML210 and JKE-1674.

3. The composition according to claim 1, characterized in that, The concentration ratio of the ferroptosis inducer to the GPX4 inhibitor is 5-10 μM: 0.05-2 μM.

4. The use of the composition of claim 1 in the preparation of a medicament for reversing resistance to ferroptosis-inducing agents in liver metastases of small cell lung cancer.

5. Use of the composition of claim 1 in the preparation of a medicament for treating or preventing small cell lung cancer.

6. The application as described in claim 5, characterized in that, The small cell lung cancer mentioned refers to small cell lung cancer with liver metastasis or a high risk of liver metastasis.

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