Pharmaceutical compositions of eplerenone and anti-pd-1 antibodies and their use in the manufacture of a medicament for the treatment of hepatocellular carcinoma

Abstract

The application belongs to the technical field of tumor immunotherapy, new use of drugs and combined therapy, and particularly relates to a pharmaceutical composition of eplerenone and anti-PD-1 antibody and application thereof in preparation of a drug for treating hepatocellular carcinoma. It is found for the first time that MASH-HCC exists an immune suppression mechanism driven by aldosterone of adrenal origin. It is proved for the first time that eplerenone can reverse aldosterone-mediated CD8 + T cell exhaustion, restore anti-tumor immunity, and confirm MR as a new target for immune regulation. The MASH-HCC immune sensitization scheme of eplerenone combined with anti-PD-1 antibody is established for the first time, which significantly overcomes immune resistance. It is confirmed that CD8 + T cell MR is an essential medium for aldosterone-induced immune suppression, providing a basis for precise intervention. Preclinical evidence of new use of old drugs is provided, and eplerenone is safe, low in cost, and suitable for MASH-HCC immunotherapy sensitization.

Description

Technical Field

[0001] This invention belongs to the fields of tumor immunotherapy, new uses of drugs and combination therapy, specifically relating to a pharmaceutical composition of eplerenone and anti-PD-1 antibody and its application in the preparation of drugs for the treatment of hepatocellular carcinoma. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, ranking among the top in both incidence and mortality. With the prevalence of metabolic diseases such as obesity and type 2 diabetes, the incidence of hepatocellular carcinoma caused by metabolic dysfunction-associated steatohepatitis (MASH-HCC) has been rising rapidly in recent years, becoming an important subtype of HCC. [1] Currently, HCC treatments include surgical resection, hepatic artery chemoembolization, radiofrequency ablation, and molecular targeted therapy, but overall efficacy is limited, with a five-year survival rate of only about 18%. Immune checkpoint inhibitors (ICIs), represented by anti-PD-1 / PD-L1 antibodies, have brought new breakthroughs to HCC treatment, with an objective response rate of approximately 20%–30% for single-agent therapy. However, MASH-HCC has a unique immunosuppressive microenvironment with significant CD8+ inhibition. + T cell depletion, poor efficacy of immunotherapies such as anti-PD-1 antibodies, and particularly prominent issues of immunotherapy resistance are all problems. [2] .

[0003] Metabolic inflammation originating from a diseased liver can transmit endocrine and paracrine signals, remodeling distal tissues, including adipose tissue, the vascular system, and the immune system. [3] However, little is known about the systemic activation of the endocrine axes closely related to cardiovascular and renal physiology during the progression of MASH-HCC. Steroid hormones, in particular, have long been studied for their roles in systemic metabolism, stress adaptation, and electrolyte balance, but have received relatively little attention in cancer research. Among steroid hormones, aldosterone is the dominant mineralocorticoid, with circulating concentrations less than 10% of glucocorticoids under physiological conditions. Traditionally, aldosterone has been considered a regulator of sodium balance and blood pressure. Therefore, aldosterone signaling has been largely overlooked in oncology, while glucocorticoids dominate mechanistic studies and clinical considerations. Notably, recent clinical observations have shown elevated plasma aldosterone levels, rather than elevated non-cortisol levels, in patients with metabolic syndrome and MASH. [4-6] Despite these findings, it remains unclear whether MASH-HCC is associated with changes in adrenal metabolism, and whether such changes affect tumor immunity and treatment response.

[0004] Eplerenone is a second-generation, orally administered selective mineralocorticoid receptor antagonist (MRA). It competitively blocks the binding of aldosterone to the mineralocorticoid receptor (MR / NR3C2), thereby inhibiting MR-mediated electrolyte homeostasis, blood pressure regulation, and non-classical inflammation and oxidative stress-related signaling. [7] Compared to first-generation MRAs like spironolactone, eplerenone has a lower affinity for androgen and progesterone receptors, thus exhibiting higher receptor selectivity and fewer sex hormone-related adverse reactions. [8] Pharmacokinetic studies show that eplerenone can be absorbed orally and is mainly metabolized by CYP3A4. In clinical use, attention should be paid to the safety issues related to hyperkalemia and renal function. However, its overall pharmacological characteristics are clear and its clinical controllability is good. [9, 10] In clinical applications, eplerenone has been used to treat hypertension, left ventricular dysfunction complicated with heart failure after acute myocardial infarction, and chronic systolic heart failure. [11-13] The large randomized controlled clinical trial EPHESUS showed that eplerenone can reduce mortality and the risk of cardiovascular events in patients with left ventricular dysfunction and heart failure after acute myocardial infarction.

[12] The EMPHASIS-HF study further demonstrated that eplerenone can reduce the risk of cardiovascular death and hospitalization for heart failure in patients with mild systolic heart failure.

[13] These studies suggest that eplerenone, as an MR blocker, has a well-established safety profile, pharmacokinetic basis, and clinical translational advantages. However, current applications of eplerenone and other mineralocorticoid receptor antagonists are primarily focused on hypertension, heart failure, and kidney damage-related diseases; their potential to reverse CD8+ in the tumor microenvironment has not yet been proposed or validated. + T cell exhaustion. The main limitations of existing technology are: first, MASH-HCC exhibits significant CD8+ depletion. + T cell exhaustion leads to low response rates to immunotherapy, particularly anti-PD-1 antibodies, and a lack of targeted sensitization strategies.

[14] Second, MASH-HCC is a systemic disease involving abnormal inter-organ communication.

[15] However, whether MASH-HCC is related to changes in adrenal metabolism and whether such changes affect tumor immunity and treatment response still lacks direct evidence; third, MRAs have been widely used in the field of clinical cardiovascular diseases and have the advantages of good safety, low cost and broad medical insurance coverage, but whether MRAs can enhance anti-tumor immunity by regulating immune cell function, especially whether they can overcome immunotherapy resistance in MASH-HCC, is currently unclear and lacks preclinical evidence.

[0005] References: 1 Wang, X., et al. (2025) Molecular mechanisms in MASLD / MASH-related HCC. Hepatology 82, 1303-1324 2 Ju, Y., et al. (2026) Metabolic-immune microenvironment crosstalk mediating ICI resistance in MASH-HCC. Trends Endocrinol Metab 37, 262-276 3 Targher, G., et al. (2024) MASLD: a systemic metabolic disorder with cardiovascular and malignant complications. Gut 73, 691-702 4 Shen, D., et al. (2024) Associating plasma aldosterone concentration with the prevalence of MAFLD in hypertensive patients: insights from a large-scale cross-sectional study. Front Endocrinol (Lausanne) 15, 1451383 5 Hu, J., et al. (2023) Relationship Between Plasma Aldosterone Concentrations and Non-Alcoholic Fatty Liver Disease Diagnosis in Patients with Hypertension: A Retrospective Cohort Study. Diabetes Metab Syndr Obes 16, 1625-1636 6 Hubel, J.M., et al. (2015) Influence of plasma cortisol and otherlaboratory parameters on nonalcoholic Fatty liver disease. Horm Metab Res 47,479-484 7 Moore, T.D., et al. (2003) Eplerenone: a selective aldosteronereceptor antagonist for hypertension and heart failure. Heart Dis 5, 354-363 8 Barnes, B.J. and Howard, P.A. (2005) Eplerenone: a selectivealdosterone receptor antagonist for patients with heart failure. AnnPharmacother 39, 68-76 9 Cook, C.S., et al. (2003) Pharmacokinetics and metabolism of [14C]eplerenone after oral administration to humans. Drug Metab Dispos 31, 1448-1455 10 Sica, D.A. (2005) Pharmacokinetics and pharmacodynamics ofmineralocorticoid blocking agents and their effects on potassium homeostasis.Heart Fail Rev 10, 23-29 11 Weinberger, M.H., et al. (2002) Eplerenone, a selectivealdosterone blocker, in mild-to-moderate hypertension. Am J Hypertens 15,709-716 12 Pitt, B., et al. (2003) Eplerenone, a selective aldosteroneblocker, in patients with left ventricular dysfunction after myocardialinfarction. N Engl J Med 348, 1309-1321 13 Zannad, F., et al. (2011) Eplerenone in patients with systolicheart failure and mild symptoms. N Engl J Med 364, 11-21 14 Pfister, D., et al. (2021) NASH limits anti-tumour surveillance inimmunotherapy-treated HCC. Nature 592, 450-456 15 Llovet, JM, et al. (2023) Nonalcoholic steatohepatitis-relatedhepatocellular carcinoma: pathogenesis and treatment. Nat Rev GastroenterolHepatol 20, 487-503 Summary of the Invention This invention addresses the shortcomings of existing technologies, such as low response rates in MASH-HCC immunotherapy, unclear adrenal-endocrine immune regulation mechanisms, and lack of effective sensitization methods. It aims to provide a pharmaceutical composition of eplerenone and anti-PD-1 antibody and its application in the preparation of drugs for the treatment of hepatocellular carcinoma.

[0006] In a first aspect, the present invention provides a pharmaceutical composition comprising eplerenone and an anti-PD-1 antibody.

[0007] Preferably, the anti-PD-1 antibody is at least one of pembrolizumab, nivolumab, sintilimab, camrelizumab, tislelizumab, toripalimab, cepalimumab, slulimab, cimiprimab, and dotalimab.

[0008] Preferably, it also includes a pharmaceutically acceptable carrier.

[0009] The "pharmaceutically acceptable carrier" as described in this invention refers to conventional, non-toxic, inert solid or liquid fillers, diluents, excipients, or encapsulating materials used in the pharmaceutical field. The carrier includes, but is not limited to: (1) Fillers / diluents: lactose (including lactose monohydrate and anhydrous lactose), microcrystalline cellulose, starch (including corn starch and potato starch), pregelatinized starch, calcium sulfate, dicalcium phosphate, mannitol, sorbitol, sucrose, glucose, dextrin, cyclodextrin, etc. (2) Adhesives: Hydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose, ethylcellulose, sodium carboxymethyl cellulose, povidone, gelatin, starch paste, sodium alginate, gum arabic, tragacanth, etc. (3) Disintegrants: croscarmellose sodium cellulose, croscarmellose, croscarmellose sodium starch, low-substituted hydroxypropyl cellulose, alginate, microcrystalline cellulose, starch (including pregelatinized starch), etc. (4) Lubricants / flow aids / anti-sticking agents: stearic acid, magnesium stearate, calcium stearate, zinc stearate, talc, colloidal silica, micronized silica gel, polyethylene glycol, hydrogenated vegetable oil, sodium lauryl sulfate, sodium lauryl sulfate, sodium benzoate, etc. (5) Solubilizers / surfactants: Sodium dodecyl sulfate, polysorbates (including polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80), poloxamer (including poloxamer 188, poloxamer 407), polyethylene glycol (including PEG 400, PEG 4000, PEG 6000), lecithin, phosphatidylcholine, polyoxyethylene castor oil, vitamin E polyethylene glycol succinate, etc. (6) Coating materials: hydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose, ethylcellulose, povidone, polyethylene glycol, titanium dioxide, talc, acrylic resins (including Eutec E100 and Eutec L30D-55), gastric-soluble coating materials, enteric-soluble coating materials, etc. (7) Solvents / liquid carriers: water, ethanol, propylene glycol, glycerol, polyethylene glycol (including PEG 400), dimethyl sulfoxide, N-methylpyrrolidone, benzyl benzoate, oils for injection (including soybean oil, sesame oil, olive oil), medium-chain triglycerides, etc. (8) Preservatives / antioxidants: Methylparaben, Propylparaben, Sodium benzoate, Potassium sorbate, Chlorobutanol, Benzyl alcohol, Benzalkonium chloride, Sodium thiosulfate, Sodium bisulfite, Sodium metabisulfite, Vitamin C, Vitamin E, Butylated hydroxyanisole, Butylated hydroxytoluene, etc. (9) pH adjusters / buffers: citric acid, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, acetic acid, sodium acetate, tartaric acid, sodium tartrate, sodium hydroxide, hydrochloric acid, phosphoric acid, triethanolamine, etc. (10) Isotonic regulators: sodium chloride, glucose, glycerol, mannitol, etc.; (11) Flavoring agents / coloring agents: sucrose, sodium saccharin, aspartame, menthol, flavoring, iron oxide red, iron oxide yellow, titanium dioxide, sunset yellow, lemon yellow, etc.; (12) Complexing agents / encapsulating agents: cyclodextrins (including α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin), ethylenediaminetetraacetic acid and its salts, etc.; (13) Other commonly used excipients: any other inert carriers or excipients not listed above but known to those skilled in the art that can be used in pharmaceutical formulations.

[0010] In a second aspect, the present invention provides the use of the pharmaceutical composition described herein in the preparation of a medicament for treating hepatocellular carcinoma.

[0011] Preferably, the hepatocellular carcinoma is hepatocellular carcinoma caused by metabolic dysfunction-related steatohepatitis.

[0012] This invention reveals that MASH-HCC can induce CD8 by activating adrenal aldosterone synthesis. + T cell function is exhausted, leading to an immunosuppressive microenvironment; mineralocorticoid receptor antagonists can block the aldosterone-MR signaling pathway and restore CD8+. + T cells enhance the anti-tumor function of cells, thereby improving the response to immunotherapy.

[0013] Eplerenone can be used to enhance the efficacy of MASH-HCC immunotherapy. The mechanism of action of this drug is as follows: MASH-HCC involves excessive aldosterone synthesis in the adrenal glands; and elevated plasma aldosterone levels are related to CD8. + The binding of mineralocorticoid receptor (MR) on the surface of T cells activates the downstream SGK1-JNK-STAT6-NOX signaling cascade, inducing CD8+. + The accumulation of ROS (reactive oxygen species) in T cells, mitochondrial dysfunction, and metabolic disorders lead to CD8+ oxidase buildup. + T cell depletion and loss of cytotoxicity ultimately mediate tumor immune escape.

[0014] Eplerenone and anti-PD-1 antibody work by acting on CD8 + Different levels of T cell dysfunction produce synergistic anti-tumor effects: Eplerenone blocks CD8 + It inhibits aldosterone-MR signaling in T cells, suppresses downstream oxidative stress and mitochondrial metabolic damage, reduces the expression of exhaustion-related molecules such as TIM-3 and CD39, and restores CD8+ expression. +T cell proliferation, metabolic adaptation, cytotoxicity, and IFN-γ and GZMB secretion capabilities can thereby enhance the infiltration of CD8+ cells into tumor cells that were previously in a state of functional suppression. + T cells regain their anti-tumor effects; based on this, anti-PD-1 antibodies further deactivate PD-1-mediated inhibitory immune checkpoint signals and promote the recovery of CD8 function. + T cells remain continuously activated and effectively kill tumor cells. Therefore, eplerenone primarily corrects oxidative stress and metabolic dysfunction that limit the efficacy of anti-PD-1 therapy, while anti-PD-1 therapy relieves residual receptor-mediated inhibition; both work together to achieve CD8 activation. + T cell functional reprogramming reverses the MASH-HCC immunosuppressive microenvironment and enhances its response to immune checkpoint blockade therapy.

[0015] This invention reveals a novel endocrine-immune regulatory mechanism, filling a gap in the field: existing studies mostly focus on the local metabolic microenvironment of the liver, while this invention elucidates for the first time that MASH-HCC drives abnormal aldosterone synthesis in the adrenal glands through IGFBP7, thereby inducing CD8. + The endocrine-immune axis of T cell depletion has been identified, clarifying that aldosterone-MR signaling is a key link connecting systemic metabolic disorders and tumor immunosuppression. This breakthrough in the traditional understanding of local immune regulation provides a new theoretical framework for research on metabolism-related tumor immunity.

[0016] Reversing immune resistance and significantly improving the efficacy of immunotherapy: Current immune checkpoint inhibitors have a response rate of less than 20% to MASH-HCC. This invention demonstrates that eplerenone can block the aldosterone-MR pathway and inhibit CD8. + T cell oxidative stress and mitochondrial damage restore their proliferative capacity, cytotoxic activity, and IFN-γ and GZMB secretion functions, reduce the expression of exhaustion markers such as TIM3 and CD39, effectively reverse immune resistance, and significantly improve the response rate of immunotherapy.

[0017] Establishing a new drug repurposing program with high clinical translation value: Eplerenone is a commonly used antihypertensive drug in clinical practice. It has good safety, low toxicity and side effects, low price, and high accessibility. It does not require complex synthesis processes, thus avoiding the problems of long development cycles, high costs, and high risks associated with new drug development. This invention provides a new use for it in MASH-HCC immunosensitization, which can be quickly translated into clinical practice and reduce the treatment burden on patients.

[0018] Providing a universal treatment approach for metabolism-driven tumors: This invention demonstrates the key role of the aldosterone-MR pathway in the immunosuppression of metabolism-related tumors. Its intervention strategy is not only applicable to MASH-HCC, but also provides a universal target and intervention direction for the immunotherapy of tumors such as prostate cancer and pancreatic cancer related to metabolic abnormalities, with broad application prospects.

[0019] Thirdly, this invention provides an eplerenone preparation for reversing aldosterone-mediated CD8 reversal. + Application in drugs for T cell depletion.

[0020] Preferably, the reversal of aldosterone-mediated CD8 + T cell exhaustion includes: (1) Downregulate the expression of exhaustion markers T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) and CD39; and / or restore CD8 expression. + The proliferation capacity, cytotoxicity, and secretion functions of T cells, including interferon-gamma (IFN-γ) and granzyme B (GZMB).

[0021] Fourthly, the present invention provides the use of eplerenone in the preparation of a medicament for treating hepatocellular carcinoma.

[0022] Preferably, the hepatocellular carcinoma is MASH-HCC.

[0023] The beneficial effects of this invention are as follows: (1) The first discovery of an adrenal aldosterone-driven immunosuppressive mechanism in MASH-HCC.

[0024] (2) First demonstration that eplerenone can reverse aldosterone-mediated CD8 + T cell depletion and restoration of anti-tumor immunity have identified it as a new target for immune regulation.

[0025] (3) The first MASH-HCC immunosensitization regimen combining eplerenone with anti-PD-1 antibody was established, which significantly overcame immune resistance.

[0026] (4) Confirming CD8 + T-cell MR is an essential mediator of aldosterone-induced immunosuppression, providing a basis for precise intervention.

[0027] (5) Provides preclinical evidence for the repurposing of old drugs. Eplerenone has good safety and low cost, making it suitable for sensitizing metabolism-driven tumor immunotherapy. Attached Figure Description

[0028] Figure 1 The diagram illustrates the efficacy of MASH-HCC immunotherapy by targeting the aldosterone / MR axis. Figure 2The diagram shows the results of adrenal hyperplasia and aldosterone synthesis activation in MASH-HCC. Figure a shows CT images of the adrenal glands and medial collateral ramus thickness measurements in healthy controls, MASH, non-MASH-HCC, and MASH-HCC patients. Figure b shows the flowchart and H&E staining results of liver tissue in the DEN+HFHC diet-based MASH-HCC mouse model. Figure c shows the dynamic detection results of plasma aldosterone concentration in each group of mice in the DEN+HFHC model. Figure d shows the relative aldosterone content in the adrenal glands and liver of each group of mice in the DEN+HFHC model. Figure e shows the immunofluorescence detection results of the zona glomerulosa in the adrenal cortex in the DEN+HFHC model. Figure f shows the flowchart and H&E staining results of liver tissue in the DEN+MCD diet-based MASH-HCC mouse model. Figure g shows the dynamic detection results of plasma aldosterone concentration in each group of mice in the DEN+MCD model. Figure h shows the relative aldosterone content in the adrenal glands and liver of each group of mice in the DEN+MCD model. Figure 3 The image shows aldosterone-induced CD8. + The figure shows the experimental results of T cell function suppression and exhaustion-related phenotypes, and the reversal of this effect by eplerenone. In this figure, a represents OT-I CD8. + Flowchart of the experimental procedure for T cell isolation, activation, drug treatment, and co-culture with Hepa1-6-OVA tumor cells; b shows the OT-I CD8 groups under different treatments. + Statistical results of T cell killing rate against Hepa1-6-OVA tumor cells; c represents CD8 in different treatment groups. + Flow cytometry results of IFN-γ, GZMB, TIM-3 and CD39 expression in T cells; d represents CD8 expression in different treatment groups. + The relative content of total ROS in T cells was detected; e represents CD8 in different treatment groups. + Results of T cell mitochondrial ROS fluorescence detection; Figure 4 The figure shows the in vivo antitumor efficacy validation of eplerenone combined with anti-PD-1 antibody in treating MASH-HCC. Specifically, a) is the drug administration flowchart of the DEN+HFHC-induced MASH-HCC mouse model, gross liver images at the endpoint for each group of mice, and H&E staining results; b) is the statistical results of the number of tumor nodules in each group of mice; c) is the statistical results of the maximum tumor diameter in each group of mice; d) is the Ki-67 proliferation index, TUNEL apoptosis detection, and CD8α immunohistochemical results of tumor tissue in each group; e) is the CD8α infiltration of tumor tissue in each group. + Flow cytometry results of IFN-γ, GZMB, TIM-3 and CD39 expression in T cells. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used in the embodiments, unless otherwise specified, are all commercially available conventional products.

[0030] Figure 1 The diagram illustrates how targeting the aldosterone / MR axis enhances the efficacy of MASH-HCC immunotherapy. MASH-HCC can induce functional activation of zona glomerulosa cells in the adrenal cortex and promote increased aldosterone production. The elevated aldosterone further acts on CD8 cells via circulation. + T cells, enabling CD8 + T cells exhibit enhanced oxidative stress, mitochondrial dysfunction, decreased cytotoxicity, and increased exhaustion-related phenotypes, thereby weakening their killing effect on HCC cells and creating an immunosuppressive state conducive to the progression of MASH-HCC. The combined use of eplerenone and anti-PD-1 antibody can block aldosterone-mediated CD8 activation. + T cell function is suppressed, oxidative stress and mitochondrial damage are reduced, and CD8+ is restored. + It enhances the anti-tumor effect of T cells and improves the therapeutic effect of ICIs on MASH-HCC.

[0031] Example 1: MASH-HCC exhibits adrenal hyperplasia and activated aldosterone synthesis. (1) Clinical CT imaging assessment of the adrenal glands ( Figure 2 a) CT imaging was used to examine the adrenal morphology of healthy controls, MASH patients, non-MASH-HCC patients, and MASH-HCC patients, and the thickness of the medial adrenal ramus was measured. A medial ramus thickness greater than 5 mm was used as the criterion for adrenal hyperplasia. Results showed that the medial adrenal ramus thickness was lower in healthy controls and non-MASH-HCC patients, while it was significantly thickened in MASH and MASH-HCC patients, with the thickening being more pronounced in MASH-HCC patients. These results suggest that MASH-HCC patients exhibit significant adrenal hyperplasia-like changes.

[0032] (2) Construction of the MASH-HCC mouse model ( Figure 2 (b, f) Two MASH-HCC animal models were established using male C57BL / 6J mice (Wuhan Wanqianjiaxing Biotechnology Co., Ltd.). At 2 weeks of age, mice were divided into groups and administered a single intraperitoneal injection of normal saline (Normal Saline, NS, solarbio, IN9000) or diethylnitrosamine (DEN, MedChemExpress, HY-N7434). The DEN dosage was 5 mg / kg.

[0033] The first model is the DEN combined with HFHC diet (high-fat, high-cholesterol diet, 40% energy from fat, 43% from carbohydrates, 17% from protein, supplemented with 0.2% (w / w) cholesterol; Research Diets, D12079B). This model has four groups: NS+ND group, NS+HFHC group, DEN+ND group, and DEN+HFHC group. In the NS+ND group: at 2 weeks of age, a single intraperitoneal injection of saline was administered, followed by feeding the entire diet with ND diet (normal maintenance diet: 15% energy from protein, 9% from fat, 76% from carbohydrates, energy density 3.81 kcal / g; Research Diets, D12079B). Diets (D10012M); NS+HFHC group: single intraperitoneal injection of saline at 2 weeks of age, fed HFHC feed from 8 weeks of age; DEN+ND group: single intraperitoneal injection of DEN at 2 weeks of age, fed ND feed throughout the period; DEN+HFHC group: single intraperitoneal injection of DEN at 2 weeks of age, fed HFHC feed from 8 weeks of age. The second model is the DEN combined with MCD diet (methionine-choline deficient diet, without added methionine and choline, 16% of energy from protein, 21% from fat, and 63% from carbohydrates, with an energy density of 4.2 kcal / g; Research Diets, A02082002B) model, which sets up 4 groups: NS+MCS: A single intraperitoneal injection of saline was administered at 2 weeks of age, followed by feeding an MCS diet (methionine-choline-sufficient control diet, supplemented with L-methionine and choline tartrate, with 17% energy from protein, 21% from fat, and 62% from carbohydrates, energy density 4.16 kcal / g, ResearchDiets, A02082003B) was used. NS+MCD: A single intraperitoneal injection of saline was administered at 2 weeks of age, followed by feeding an MCD diet at 8 weeks of age. DEN+MCS: A single intraperitoneal injection of DEN was administered at 2 weeks of age, followed by feeding an MCS diet at 8 weeks of age. DEN+MCD: A single intraperitoneal injection of DEN was administered at 2 weeks of age, followed by feeding an MCD diet at 8 weeks of age. Liver tissue H&E staining results showed that both dietary interventions could induce MASH-HCC-related liver pathological changes in the context of DEN treatment.

[0034] (3) Activation of aldosterone synthesis and hyperplasia of the zona glomerulosa in the DEN+HFHC model Figure 2 (China) In the DEN+HFHC model, plasma aldosterone concentrations in mice of different groups were dynamically measured. Results showed that plasma aldosterone concentrations in the DEN+HFHC group mice continuously increased with disease progression. Further tissue analysis at the endpoint revealed a significant increase in the relative content of adrenal aldosterone in the DEN+HFHC group mice, along with marked hyperplasia of the zona glomerulosa of the adrenal cortex.

[0035] (4) Activation verification of aldosterone synthesis in the DEN+MCD model ( Figure 2 (g, h) To verify whether the above changes are common characteristics of MASH-HCC-related pathological states, the DEN+MCD model was further used for verification. The results showed that mice in the DEN+MCD group also showed elevated plasma aldosterone levels and increased relative adrenal aldosterone content, suggesting that the activation of aldosterone synthesis related to MASH-HCC is not a diet-specific phenomenon of HFHC.

[0036] In summary, clinical CT imaging and two animal models of MASH-HCC jointly demonstrate that adrenal hyperplasia and activated aldosterone synthesis exist in MASH-HCC. The pathological changes are manifested as thickening of the medial adrenal limbs, elevated circulating aldosterone levels in MASH-HCC mice, increased adrenal aldosterone content, and hyperplasia of the zona glomerulosa of the adrenal cortex, suggesting that the elevated aldosterone levels associated with MASH-HCC primarily originate from the adrenal glands.

[0037] Example 2: Aldosterone mediates CD8 receptors via mineralocorticoid receptors + T cell function suppression and exhaustion-like phenotype, which can be reversed with eplerenone. (1) Original OT-I CD8 + T cell isolation, activation and drug treatment ( Figure 3 a) Take C57BL / 6J (OT-I) (OT-I: OVA specific CD8) + TCR transgenic mice (male mice) were used, employing mouse CD8... + The T-cell magnetic bead sorting kit (Selleckchem, B90011) uses magnetically activated cell sorting (MACS) technology to isolate primary OT-I CD8 cells from mouse spleen or tumor tissue. +T cells were sorted and resuspended in RPMI 1640 complete medium containing 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics, and 50 μM β-mercaptoethanol. SIINFEKL peptide (200 ng / mL) and recombinant IL-2 (20 ng / mL) were added, and the cells were activated in vitro at 37 ℃ in a 5% CO2 incubator for 72 h.

[0038] Activated OT-I CD8 + T cells were divided into a DMSO control group, an aldosterone treatment group, an eplerenone treatment group, and an aldosterone combined with eplerenone treatment group. The aldosterone treatment group was set with concentration gradients of 0.5 nM, 1 nM, and 2 nM; the eplerenone treatment group received 5 μM eplerenone; and the aldosterone combined with eplerenone treatment group received both 2 nM aldosterone and 5 μM eplerenone. After 24 hours of intervention, the treated OT-I CD8 cells were... + T cells were co-cultured with Hepa1-6-OVA tumor cells at different effector-target ratios. Figure 3 (a)

[0039] (2) CD8 + T cell killing function test ( Figure 3 (b) OT-I CD8 + The cytotoxicity of T cells co-cultured with Hepa1-6-OVA tumor cells was evaluated. Results showed that after aldosterone treatment, OT-I CD8... + The killing rate of T cells against Hepa1-6-OVA tumor cells decreased, and this decrease further increased with increasing aldosterone concentration; however, the aldosterone-induced decrease in cell killing function was significantly restored upon the addition of eplerenone. These results suggest that aldosterone can inhibit CD8... + Eplerenone can reverse the inhibitory effect of T cells' anti-tumor killing function.

[0040] (3) CD8 + Detection of T cell effector molecules and exhaustion-related biomarkers ( Figure 3 c) CD8 counts in each group were detected by flow cytometry. + Expression of IFN-γ, GZMB, TIM-3, and CD39 in T cells. Results showed that after aldosterone treatment, CD8... + IFN-γ in T cells + and GZMB + The cell ratio decreased, while TIM-3 + and CD39 +The proportion of cells increased; eplerenone combined treatment restored IFN-γ and GZMB expression and reduced the expression of exhaustion-related markers such as TIM-3 and CD39. These results indicate that aldosterone can induce CD8+ expression. + Eplerenone can reverse the decline in T cell effector function accompanied by an enhanced exhaustion-related phenotype.

[0041] (4) CD8 + T cell ROS accumulation and mitochondrial damage detection ( Figure 3 (d, e) Further testing of CD8 in each group + Intracellular ROS levels and mitochondrial ROS fluorescence signal in T cells were measured. Results showed that aldosterone treatment significantly increased CD8+ levels. + Treatment with eplerenone significantly reduced intracellular ROS levels and enhanced mitochondrial ROS fluorescence signal; however, combined treatment with eplerenone resulted in a marked decrease in intracellular ROS accumulation and mitochondrial ROS signal. These results suggest that aldosterone can induce CD8+ oxidase activity. + T cell oxidative stress and mitochondrial dysfunction can be effectively blocked by eplerenone.

[0042] Example 3: Validation of the antitumor effect of eplerenone combined with anti-PD-1 therapy in the treatment of MASH-HCC (1) Model construction and dosing regimen ( Figure 4 a) Two-week-old male C57BL / 6 mice (Wuhan Wanqian Jiaxing) were given a single intraperitoneal injection of 2 mg / mL DEN dissolved in sterile PBS (phosphate-buffered saline) at a dose of 5 mg / kg. From six weeks of age, they were continuously fed an HFHC diet. At 24 weeks, the mice were randomly assigned to an IgG isotype control group, an anti-PD-1 monotherapy group, an eplerenone group, and an eplerenone combined with anti-PD-1 treatment group. Anti-PD-1 antibody (RMP1-14, MedChemExpress, HY-P99144) was administered with an initial dose of 25 mg / kg, followed by 10 mg / kg every two weeks via intraperitoneal injection. Eplerenone (MedChemExpress, HY-B0251) was administered by gavage at a dose of 100 mg / kg / day. Mice in the combined treatment group were administered eplerenone daily by gavage, and both drug interventions were completed on the same day as the anti-PD-1 antibody injection. Each group continued treatment until 32 weeks of age, after which they were sacrificed for tissue collection, including liver tumor tissue and immune cells, for testing.

[0043] (2) Tumor burden and histopathological assessment Figure 4 (c) Macroscopic observation of the liver and H&E staining at the endpoint showed that, compared with the IgG isotype control group, the anti-PD-1 monotherapy group had no significant inhibitory effect on the tumor burden of MASH-HCC, while the eplerenone group reduced the number of tumor nodules and the maximum tumor diameter. The number of tumor nodules and the maximum tumor diameter were further reduced in the eplerenone combined with anti-PD-1 treatment group, suggesting that eplerenone can enhance the in vivo anti-tumor effect of anti-PD-1.

[0044] (3) Detection of tumor cell proliferation and apoptosis ( Figure 4 (d) Eplerenone combined with anti-PD-1 therapy can reduce the Ki-67 positive proliferation index, increase the proportion of TUNEL positive cells, and increase CD8+ in tumor tissue. + The combination therapy significantly improved T cell infiltration levels, while the anti-PD-1 monotherapy group showed almost no effect. This indicates that combination therapy can inhibit tumor cell proliferation, promote tumor cell death, and improve the tumor immune microenvironment.

[0045] (4) Tumor microenvironment CD8 + T cell function analysis ( Figure 4 (e) Flow cytometry further evaluated the tumor-infiltrating CD8. + T cell functional status. Compared with either monotherapy, eplerenone combined with anti-PD-1 therapy significantly improved CD8 function. + The combination therapy not only enhanced the proportion of IFN-γ and GZMB-positive T cells, but also reduced the proportion of TIM-3 and CD39-positive cells, indicating that it not only enhanced CD8 but also reduced the proportion of T cells positive. + Eplerenone enhanced T cell effector cytokine production and cytotoxicity, and also attenuated their terminal exhaustion-related phenotype, while anti-PD-1 monotherapy had almost no effect. Mechanistically, eplerenone works by blocking CD8... + T cell aldosterone-MR signaling alleviates persistent oxidative stress and mitochondrial metabolic dysfunction downstream of MR, thereby reducing the dysfunction of CD8 cells. + T cells regain their effector response capabilities; anti-PD-1 antibodies further relieve PD-1-mediated immune checkpoint inhibition, promoting the recovery of these functions by CD8 cells. + T cells are continuously activated and kill tumor cells. Therefore, the two treatments target CD8 restriction. + The metabolic inhibition and receptor-mediated inhibition of T cell anti-tumor activity form a complementary mechanism of "metabolic function repair - immune checkpoint deactivation," thereby increasing intratumoral CD8. + T cell infiltration and activity promote tumor cell apoptosis and produce a synergistic anti-tumor effect superior to monotherapy.

[0046] The anti-mouse PD-1 antibody used in this embodiment of the invention is used to block the mouse PD-1 immune checkpoint pathway. Both human PD-1 and mouse PD-1 belong to the immunoglobulin superfamily / CD28-CTLA-4 family-related inhibitory immune receptors. They have similar domain structures and both bind to PD-L1 and / or PD-L2 through extracellular immunoglobulin-like domains, and recruit SHP-1 / SHP-2 phosphatases through intracellular tyrosine motifs, inhibiting TCR and / or CD28-related activation signals, thereby negatively regulating T cell function. The immunosuppressive pathway mediated by human and mouse PD-1 / PD-L1 / PD-L2 and its downstream SHP2-related signaling axis show clear functional conservation in both humans and mice. Therefore, the synergistic effect of eplerenone combined with anti-mouse PD-1 antibodies observed in animal models can be used as a treatment for human MASH-HCC by combining eplerenone with clinical anti-human PD-1 antibodies (pembrolizumab, nivolumab, sintilimab, camrelizumab, tislelizumab, toripalimab, cepalimumab, slulimumab, cimiprimab, dotalimumab or their functional equivalents).

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, It contains eplerenone and anti-PD-1 antibody.

2. The pharmaceutical composition according to claim 1, characterized in that, The anti-PD-1 antibody is at least one of pembrolizumab, nivolumab, sintilimab, camrelizumab, tislelizumab, toripalimab, cepalimumab, slulimab, cimiprimab, and dotalimab.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, It also includes pharmaceutically acceptable carriers.

4. Use of the pharmaceutical composition according to any one of claims 1-3 in the preparation of a medicament for treating hepatocellular carcinoma.

5. The application according to claim 4, characterized in that, The hepatocellular carcinoma mentioned is hepatocellular carcinoma caused by metabolic dysfunction-related fatty liver disease.

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