Application of RAB7 agonists in the preparation of drugs for treating MASLD, CHB, or their comorbidities

Abstract

This invention belongs to the field of biomedical technology and discloses the application of RAB7 agonists in the preparation of drugs for treating MASLD, CHB, or their comorbidities. RAB7 agonists activate Rab7, simultaneously promoting HBV DNA and HBsAg degradation and autophagy-lysosomal function, achieving a dual effect of simultaneously targeting and improving both viral load and metabolism. This invention solves the technical challenge of existing treatment regimens failing to synergistically regulate viral replication and metabolic disorders, providing an efficient and safe treatment strategy for clinically refractory comorbidities.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of RAB7 agonists in the preparation of drugs for treating MASLD, CHB or their comorbidities. Background Technology

[0002] Chronic hepatitis B (CHB), often simply referred to as "hepatitis B," is caused by infection with the hepatitis B virus (HBV). When infection persists for more than 6 months, the liver develops varying degrees of inflammation, necrosis, and / or fibrosis. Early symptoms mainly include recurrent fatigue, dizziness, and loss of appetite. As the condition worsens, it may manifest as palmar erythema, spider angiomas, and hepatosplenomegaly. HBV is primarily transmitted through mother-to-child transmission, blood, broken skin and mucous membranes, and sexual contact. The primary goal of treatment for chronic hepatitis B is antiviral therapy, often employing a comprehensive and individualized approach, including adequate rest, a proper diet, and nutrition. Treatment and maintenance antiviral medication regimens should be adjusted according to the severity of the disease. Antiviral drugs include nucleoside (acid) analogues (such as emtricitabine and tenofovir) and interferon analogues (such as interferon and pegylated interferon).

[0003] Metabolic dysfunction-associated fatty liver disease (MASLD) is currently the most common chronic liver disease worldwide. 25% of patients with MASLD will progress to metabolic dysfunction-associated steatohepatitis (MASH), with fibrosis developing within 3 years. The development of MASLD is associated with multiple factors, including obesity, type 2 diabetes, hypertension, and metabolic syndrome such as dyslipidemia. MASLD can be broadly classified into two types: metabolic dysfunction-associated fatty liver (MAFL), i.e., non-progressive MASLD; and metabolic dysfunction-associated steatohepatitis (MASH), i.e., progressive MASLD. MAFL is hepatic steatosis, typically occurring in zone 3 of the liver lobule, with or without mild inflammation. The characteristic histological features of MAFL progressing to MASH are the presence of three main features: steatosis, lobular inflammation, and ballooning hepatocytes, possibly accompanied by fibrosis. The process of MASH reverting back to MAFL can be highly dynamic and may even occur within a short period. Abnormal lipid metabolism is a significant contributing factor to the development of MASLD, and the endoplasmic reticulum (ER), as a crucial organelle for lipid metabolism and protein synthesis, plays a key role in the regulation of MASLD. Inflammation is a critical factor in the progression of MASLD to MASH. ER stress promotes the release of inflammatory factors by activating signaling pathways such as NF-κB, which further exacerbate liver inflammation. While the pathogenesis of MAFLD has been extensively studied both domestically and internationally, the exact pathogenesis and treatment of MASLD remain unclear. Therefore, the search for highly effective, low-toxicity small-molecule drugs has become a new strategy for the clinical prevention and treatment of MASLD. Current treatments for metabolic dysfunction-related fatty liver disease (MASLD, formerly known as non-alcoholic fatty liver disease / NAFLD) primarily focus on improving metabolic disorders and anti-inflammatory and anti-fibrotic effects. Approved or extensively studied drugs include pioglitazone, vitamin E (α-tocopherol), semaglutide, tirzepatide, SGLT2 inhibitors (such as empagliflozin and dapagliflozin), resmetirom, and obeticholic acid (OCA).

[0004] The coexistence of chronic hepatitis B (CHB) and fatty liver disease (especially metabolic-associated fatty liver disease, MASLD) has become an increasingly prominent clinical challenge and research hotspot in the field of hepatology. Numerous epidemiological data show that with the global prevalence of obesity and metabolic syndrome, the prevalence of fatty liver in CHB patients has significantly increased. Studies have shown that fatty liver may not only accelerate the progression of liver fibrosis in CHB patients, increasing the risk of cirrhosis and even hepatocellular carcinoma, but may also affect the response and efficacy of antiviral therapy, and have complex impacts on liver-related and all-cause mortality. However, due to limited existing research, the specific molecular mechanisms of their interaction are not fully elucidated, and precise treatment for these patients is lacking. Therefore, in-depth exploration of the pathophysiological mechanisms and treatment strategies of CHB and MASLD comorbidity is an urgent need and an important direction for current hepatology research.

[0005] Current clinical treatment of CHB and MASLD as single or comorbid conditions faces significant limitations. Existing regimens rely on a simple combination of antiviral drugs, such as nucleoside (nucleotide) analogs, and peg-IFN, along with metabolic interventions (such as lifestyle modifications and GLP-1 receptor agonists). This approach is a simple combination of single-disease treatments, lacking a dual-treatment strategy that simultaneously targets viral clearance and metabolic / inflammatory regulation. This results in insufficient synergistic intervention for comorbid patients and makes it difficult to effectively halt disease progression.

[0006] The interaction mechanism between CHB and MASLD is complex, and the lack of a shared core therapeutic target makes synergistic intervention with single drugs difficult. Furthermore, the pathological processes of the two diseases influence each other; viral activity promotes metabolic disorders, which in turn affect antiviral immunity, further increasing the difficulty of treatment. Current clinical practice lacks large clinical cohorts of patients with both diseases, and basic research is still inadequate, making it difficult to find compounds that can simultaneously and effectively target both viral and metabolic pathways.

[0007] Rab7, a key GTPase regulating the late fusion of endosomes / autophagosomes and lysosomes, is crucial for mediating the lysosomal degradation of substrates such as abnormal proteins, damaged organelles (e.g., mitochondria), and exogenous pathogens. Studies have shown that in chronic hepatitis B (CHB), HBV infection significantly impairs Rab7 function, leading to excessive lysosomal activity depletion and homeostasis imbalance, thereby weakening viral component clearance and promoting disease progression. In liver inflammatory lesions (MASLD), impaired autophagic flux, characterized by a significant decrease in autophagosome-lysosome fusion efficiency, is a core pathological link in triggering hepatic lipid metabolism disorders and inflammatory damage. Based on these mechanisms, Rab7 agonists theoretically represent an innovative intervention. These agonists, by specifically enhancing Rab7 activity, can effectively restore inhibited Rab7 function in the CHB model and significantly improve impaired autophagic flux and promote efficient autophagosome-lysosome fusion in the MASLD model. It can target and intervene in the core pathological mechanisms of CHB and MASLD at multiple levels, namely, enhancing viral clearance in CHB and promoting lipid degradation and inhibiting inflammatory response in MASLD. Rab7 agonists can provide a unified molecular theoretical basis for the treatment of these two diseases, especially their coexistence (comorbidity). Summary of the Invention

[0008] The purpose of this invention is to provide the application of RAB7 agonists in the preparation of drugs for treating MASLD, CHB or their comorbidities, to solve the technical problem that existing treatment regimens cannot synergistically regulate viral replication and metabolic disorders, and to provide an efficient and safe treatment strategy for clinically refractory comorbidities.

[0009] 1. Treatment of CHB alone cannot improve metabolic disorders, leading to the continuous progression of liver damage.

[0010] 2. MASLD alone has no effect on HBV infection, and patients with comorbidities need to use multiple drugs in combination, resulting in poor compliance.

[0011] 3. Comorbid treatment lacks targets that synergistically regulate viral replication and metabolic inflammation, resulting in low treatment efficiency.

[0012] To achieve the above objectives, this application adopts the following technical solution:

[0013] This invention provides the use of RAB7 agonists in the preparation of medicaments for treating liver diseases, namely, metabolic dysfunction-related fatty liver disease, chronic hepatitis B, or comorbidities.

[0014] In the above technical solution, the RAB7 agonist is ML-098.

[0015] In the above technical solutions, in the preparation of a drug for treating fatty liver disease related to metabolic dysfunction, the drug uses a RAB7 agonist as the active ingredient; or

[0016] The drug contains a RAB7 agonist and a STING inhibitor as its active ingredients.

[0017] In the above technical solutions, the STING inhibitor is one of H-151, compound 18, SN-011, 4-OI, BB-Cl-amidine, and LB244.

[0018] In the above technical solutions, in the preparation of a drug for treating chronic hepatitis B, the drug uses a RAB7 agonist as the active ingredient; or

[0019] The drug contains RAB7 agonist and STING agonist as active ingredients.

[0020] In the above technical solutions, the STING agonist is one of DMXAA, ADU-S100, E7766, IMSA101, MV-626, compound 31, SR-8541A, TXN10128, RBS2418, diABZI, GSK3745417, CRD3874-SI, and KL340399.

[0021] In the above technical solutions, in the preparation of a drug for treating comorbidities of chronic hepatitis B and metabolic dysfunction-related fatty liver disease, the drug uses a RAB7 agonist as the active ingredient.

[0022] In the above technical solution, the RAB7 agonist promotes HBV virus degradation and liver autophagy by activating Rab7 protein, and simultaneously reduces HBV DNA and HBsAg and improves blood lipids and blood sugar.

[0023] In the above technical solution, the RAB7 agonist reduces the levels of liver tissue inflammatory factors IL-6, IL-1β, TNF-α, and serum ALT and AST, thereby alleviating liver fibrosis.

[0024] Compared with the prior art, the outstanding advantages of the present invention are reflected in the following aspects:

[0025] 1. Multi-target synergistic regulation

[0026] Traditional antiviral drugs (such as entecavir) only inhibit HBV polymerase and have no effect on metabolic disorders; RAB7 agonists (ML-098) activate Rab7, thereby promoting the degradation of HBV DNA and HBsAg and autophagy-lysosomal function, achieving a dual effect of simultaneously targeting and improving both the virus and metabolism.

[0027] 2. Clinical advantages of comorbidity treatment

[0028] Current treatment for comorbid patients involves antiviral therapy combined with MASLD treatment. However, there are currently no effective drugs for MASLD, and this simple combination therapy increases the incidence of adverse reactions. This invention, using the RAB7 agonist (ML-098) as monotherapy five times a week, showed no adverse reactions such as diarrhea or hypoglycemia in mice during experiments, and the short dosing cycle provides strong evidence for clinical translation. Attached Figure Description

[0029] Figure 1 Schematic diagram of the experimental design of CHB combined with MASLD mice treated with ML-098.

[0030] Figure 2 Schematic diagram of the experimental design of MASLD mice treated with ML-098 / H-51.

[0031] Figure 3 Schematic diagram of the experimental design for treating HBV-tg mice with ML-098 / DMXAA.

[0032] Figure 4 ML-098 improves lipid metabolism in CHB-MASLD mice; (A) Representative images of Oil Red O staining of control group, pAAV / HBV1.2+HFD mice, and pAAV / HBV1.2+HFD+ML-098 mice, scale bar: 50 μm; (B) Expression of serum TC, TG, and NEFA; (C) Subcutaneous fat weight, epididymal fat weight, and fasting blood glucose levels in mice.

[0033] Figure 5 ML-098 improves liver inflammation in CHB-induced MASLD mice; (A) Representative images of H&E and Sirius red staining in control group, pAAV / HBV1.2+HFD mice, and pAAV / HBV1.2+HFD+ML-098 mice, scale bar: 50 μm; (B) Serum HBV DNA and HBsAg levels in mice; (C) Serum ALT and AST expression in mice; (D) Expression levels of liver inflammatory factors TNFα, IL-1β, and IL-6 in mice.

[0034] Figure 6 ML-098 and / or H-151 improve lipid metabolism in MASLD mice; (A) Representative images of Oil Red O staining of control group, MASLD mice, and MASLD (different drug administration groups) mice, scale bar: 50 μm; (B) Expression of serum TC, TG, and NEFA; (C) Subcutaneous fat weight, epididymal fat weight, and fasting blood glucose levels in mice.

[0035] Figure 7ML-098 and / or H-151 improve liver inflammation in MASLD mice; (A) Representative images of H&E and Sirius red staining in control group, MASLD mice, and MASLD (different drug administration groups) mice, scale bar: 50 μm; (B) Expression of ALT and AST in mouse serum; (C) Expression levels of inflammatory factors TNFα, IL-1β and IL-6 in mouse liver.

[0036] Figure 8 ML-098 or DMXAA reduced HBV virological levels in mice; (A) expression of HBV DNA in mouse serum; (B) expression of HBsAg in mouse serum. Detailed Implementation

[0037] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0038] This invention provides the use of RAB7 agonists in the preparation of medicaments for treating liver diseases, namely, metabolic dysfunction-associated fatty liver disease (MASLD), chronic hepatitis B (CHB), or their comorbidities.

[0039] RAB7 agonists promote HBV viral degradation and hepatic autophagy by activating the Rab7 protein, while simultaneously reducing HBV DNA and HBsAg levels and improving blood lipids and blood glucose. RAB7 agonists also reduce the levels of liver tissue inflammatory factors IL-6, IL-1β, TNF-α, and serum ALT and AST, thus alleviating liver fibrosis.

[0040] Preferably, the RAB7 agonist is ML-098. The chemical structural formula of ML-098 is as follows, and its CAS No. is 878978-76-8.

[0041]

[0042] In addition, it should be noted that other RAB7 agonists besides ML-098 can also be used, and they have the same therapeutic effect.

[0043] Preferably, in the preparation of a medicament for treating metabolic dysfunction-associated fatty liver disease (MASLD), the medicament uses a RAB7 agonist as the active ingredient; or

[0044] The drug contains a RAB7 agonist and a STING inhibitor as its active ingredients.

[0045] STING inhibitors include:

[0046] (1) Inhibitors of LBD

[0047] Utilizing small molecules targeting LBD to compete with ligands for binding sites is one of the drug design strategies for STING inhibitors. Compound 18 inhibits STING activation through competitive action. SN-011 is another small molecule STING inhibitor.

[0048] (2) Polymerization inhibitors

[0049] H-151 is a nitrofuran derivative that can inhibit palmitoylation of human STING by covalently modifying the Cys91 site. Despite its short half-life, intraperitoneal injection of this inhibitor significantly reduces IFN-β levels in Trex1- / - mice.

[0050] 4-Octylcis-O-I can directly alkylate Cys91, thereby inhibiting the activation of the cGAS / STING signaling pathway.

[0051] (3) BB-Cl-amidine inhibits palmitoylation of STING by covalently modifying Cys148, thereby weakening STING polymerization, IFN-I signaling, and autophagy. Subsequently, through further screening of BB-Cl-amidine analogs, LB244, with higher activity and better selectivity, was discovered. Intraperitoneal injection of LB244 significantly attenuated diABZI-induced cytokine secretion. However, oral administration of LB244 resulted in low bioavailability and high clearance, and this instability limited its drug delivery method.

[0052] Preferably, the STING inhibitor is one of H-151, compound 18, SN-011, 4-OI, BB-Cl-amidine, or LB244.

[0053] Preferably, the STING inhibitor is H-151. The chemical structural formula of H-151 is as follows, and its CAS No. is 941987-60-6.

[0054]

[0055] Preferably, in the preparation of a drug for treating chronic hepatitis B (CHB), the drug uses a RAB7 agonist as the active ingredient; or

[0056] The drug contains RAB7 agonists and STING agonists as active ingredients.

[0057] STING agonists are mainly classified into cyclic dinucleotide (CDN) agonists, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitors, and non-CDN agonists.

[0058] (1) CDN agonists

[0059] ADU-S100, clinical trial number: NCT02675439.

[0060] E7766's Phase I clinical trial (NCT04144140) was terminated early. As of December 2024, IMSA101 (NCT06601296 and CTR20211689) is undergoing clinical studies to fully evaluate its safety and efficacy.

[0061] (2) ENPP1 inhibitors: ENPP1 can degrade extracellular 2',3'-cGAMP. By inhibiting ENPP1, the concentration of 2',3'-cGAMP in the blood can be increased, thereby activating STING.

[0062] MV-626 is a selective ENPP1 inhibitor. In a mouse pancreatic cancer model, injection of MV-626 alone or in combination with radiotherapy enhanced the anti-tumor immune response and improved the overall survival rate of mice. (See: He X, Wedn A, Wang J, ...) et al . IUPHAR ECR review: the cGAS- STING pathway: novel functions beyond innate immune and emerging therapeutic opportunities[J / OL]. Pharmacol Res , 2024, 201: 107063[2024-12-30]. https: / / pubmed.ncbi.nlm.nih.gov / 38216006 / . DOI: 10.1016 / j.phrs.2024.107063.

[0063] Compound 31 is another inhibitor targeting the ENPP1 catalytic pocket. In a mouse model of breast cancer, this compound significantly reduced the number of lung metastatic nodules and intraperitoneal nodules, while the mice's body weight did not change significantly, indicating that this inhibitor has good antitumor activity and safety. See reference: Sun Y, Chen M, Han Y, et al. Discovery of pyrido[2,3- d ]pyrimidin- 7-one derivatives as highly potent and efficient ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitors for cancer treatment[J]. JMed Chem, 2024, 67(5): 3986-4006.

[0064] Currently, ENPP1 inhibitors that have entered Phase I clinical trials include SR-8541A ​​(NCT06063681), TXN10128 (NCT05978492), and RBS2418 (NCT05270213).

[0065] (3) Non-CDN agonists

[0066] Linked amidobenzimidazole (diABZI): Induces type I interferon and nuclear factor κB pathways by binding to the STING protein and triggering downstream TBK1 kinase activation.

[0067] GSK3745417 is currently in Phase I clinical trials (NCT03843359).

[0068] CRD3874-SI has entered a Phase I clinical trial to evaluate its efficacy in patients with acute myeloid leukemia (NCT06626633) or advanced / metastatic malignant solid tumors (NCT06021626).

[0069] In addition, the non-CDN-type STING agonist KL340399 has also entered the clinical trial stage (NCT05549804 and NCT05387928).

[0070] Preferably, the STING agonist is one of DMXAA, ADU-S100, E7766, IMSA101, MV-626, compound 31, SR-8541A, TXN10128, RBS2418, diABZI, GSK3745417, CRD3874-SI, and KL340399.

[0071] Preferably, the STING agonist is DMXAA. The chemical structural formula of DMXAA is as follows, and its CAS No. is 117570-53-3.

[0072]

[0073] Preferably, in the preparation of a medicament for treating comorbidities of chronic hepatitis B and fatty liver disease associated with metabolic dysfunction, the medicament uses a RAB7 agonist as the active ingredient.

[0074] Drug preparation procedure in mice:

[0075] Taking the preparation of 1 ml of ML-098, DMXAA, or H-151 solution with a concentration of 5 mg / ml as an example, the specific operation is as follows: First, accurately weigh 5 mg of the target drug (ML-098, DMXAA, or H-151) and place it in a sterile centrifuge tube; then, add 100 μl of DMSO (10% of the final volume) and vortex until the drug is completely dissolved, at which point the solution is clear; next, slowly add 400 μl of PEG 300 (40% of the final volume), gently inverting the centrifuge tube to mix while adding, avoiding the formation of air bubbles and ensuring full integration with the previous solution; then, add 50 μl of Tween-80 (5% of the final volume) and continue vortexing for 30 seconds to evenly disperse the surfactant and prevent drug precipitation; finally, add 450 μl of physiological saline (45% of the final volume) and vortex again for 1 minute to form a homogeneous solution with a total volume of 1 ml, wherein the ratio of each component is strictly 10% DMSO and 40% PEG. Prepare 300, 5% Tween-80 and 45% physiological saline to precisely maintain the drug concentration at 5 mg / ml. After preparation, it can be used immediately for experimental administration or stored briefly at 4°C protected from light (not exceeding 24 hours) to ensure stability.

[0076] Intraperitoneal injection of the drug in mice: Mice were administered the drug at a dose of 10 mg / kg. First, the mice were gently fixed in place with their abdomens facing upwards to expose the injection area. The skin of the mouse abdomen was disinfected with a 75% alcohol swab. Then, holding a 1 ml syringe, the prepared drug solution was drawn and inserted into the skin at a 30°-45° angle to the abdominal wall on the left or right side of the mouse abdomen (avoiding the bladder and vital organs). The drug was slowly injected, and the needle was quickly withdrawn after the injection. A dry cotton ball was used to gently press the injection site for a moment to prevent leakage of the drug solution.

[0077] Dosage frequency:

[0078] 1. ML-098 single-drug intraperitoneal injection, 10 mg / kg, 5 times a week (5 consecutive days of injection), for a total of 2 weeks.

[0079] 2. H-151 single-drug intraperitoneal injection, dose 10 mg / kg, once a week for a total of 4 weeks.

[0080] 3. Combination regimen: ML-098 (10 mg / kg) + STING inhibitor H-151 (10 mg / kg) are administered intraperitoneally, with the same dosing frequency and cycle as the single-drug regimen.

[0081] 4. DMXAA single-drug intraperitoneal injection, dose 10 mg / kg, once every 3 days, for a total of 2 weeks.

[0082] 5. Combination regimen: ML-098 (10 mg / kg) + STING agonist DMXAA (10 mg / kg) intraperitoneally, with the same dosing frequency and cycle as the single-drug regimen.

[0083] Animal tissue sampling:

[0084] 1. After reaching the modeling endpoint, mice in each group were fasted but allowed free water overnight;

[0085] 2. After recording the final weight of the mice at the end of the modeling process using an electronic balance, the mice were anesthetized with ether, and blood was collected from the mice using the ocular blood collection method into sterile EP (Eppendorf) tubes. The tubes were left at room temperature for 2 hours to allow the blood to coagulate, and then the mice were centrifuged at 3500 rpm for 10 minutes to collect the mouse serum, which was then stored in a -80℃ freezer.

[0086] 3. Mice were euthanized by cervical dislocation. The mice were then disinfected by immersing them in 75% alcohol, and their limbs were fixed in the euthanasia table. Ophthalmic scissors and forceps were used to separate the abdominal skin and subcutaneous fat, fully exposing the liver. The hepatic ligaments were carefully separated, the gallbladder removed, and the removed liver was rinsed in PBS and then blotted dry with filter paper.

[0087] 4. Take two pieces of tissue the size of mung beans and fix them in 4% paraformaldehyde and OCT embedding medium respectively for subsequent experiments. Cut the remaining liver tissue into small pieces and place them in sterile cryovials, quick-freeze in liquid nitrogen, and finally transfer them to a -80℃ freezer for storage.

[0088] 5. For collecting intraocular venous blood from mice: After anesthetizing the mice with ether, gently press the skin of the head and neck with the thumb and forefinger of the left hand to slightly protrude the eyeball. Hold the capillary tube in the right hand and insert it at a 30° angle towards the posterior orbit through the conjunctival fornix between the inner canthus and the eyeball. When a noticeable loss of sensation is felt, gently lift the capillary tube to collect blood using negative pressure siphon. Immediately after withdrawing the tube, apply pressure with a sterile cotton ball for 10 seconds to stop the bleeding.

[0089] Mouse tissues and serum were used for subsequent metabolic, inflammatory, and virological testing.

[0090] Example 1

[0091] This embodiment verifies the therapeutic effect of ML-098 through in vivo animal experiments. The specific technical solution is as follows:

[0092] (I) Establishment of experimental model

[0093] 1. Comorbidity model of CHB and MASLD: 10 μg pAAV / HBV1.2 plasmid was injected into mice via high-pressure hydrodynamic injection through the tail vein. Mice with HBsAg>500 mIU / ml that successfully developed the model were then selected and randomly fed HFD for 20 weeks.

[0094] 2. Simple MASLD model: 6-8 week old male C57BL / 6 mice were selected and fed a high-fat diet (HFD, 60% fat calories) for 20 weeks.

[0095] 3. Simple HBV infection model: 6-8 week old male HBV-tg transgenic mice (C57BL / 6 background, stably expressing the entire HBV (type A) genome) were selected, with a baseline serum HBV DNA level of 1×10⁻⁶. 8 -2×10 8 IU / mL, baseline HBsAg is 15000-25000 IU / mL.

[0096] (II) Dosing regimen design:

[0097] Drug dissolution protocol: For the dissolution of ML-098, DMXAA and H-151, add 10% DMSO, 40% PEG 300 and 5% Tween-80 in sequence, and finally prepare a 45% solution with physiological saline.

[0098] 1. Treatment of CHB combined with MASLD:

[0099] Monotherapy regimen: ML-098 intraperitoneal injection, 10 mg / kg, 5 times a week (5 consecutive days), for a total of 2 weeks. Figure 1 )

[0100] 2. Simple MASLD treatment:

[0101] Monotherapy regimen: ML-098 intraperitoneal injection, dose 10 mg / kg, 5 times a week (5 consecutive days), for a total of 2 weeks. H-151 intraperitoneal injection, dose 10 mg / kg, once a week, for a total of 4 weeks.

[0102] Combination regimen: ML-098 (10 mg / kg) + STING inhibitor H-151 (10 mg / kg) co-administered intraperitoneally, with the same dosing frequency and cycle as the monotherapy regimen. Figure 2 )

[0103] 3. Treatment of CHB alone:

[0104] Monotherapy regimen: ML-098 intraperitoneal injection, dose 10 mg / kg, 5 times a week (5 consecutive days), for a total of 2 weeks. DMXAA intraperitoneal injection, dose 10 mg / kg, once every 3 days, for a total of 2 weeks.

[0105] Combination regimen: ML-098 (10 mg / kg) + STING agonist DMXAA (10 mg / kg) intraperitoneally, with the same dosing frequency and cycle as the monotherapy regimen. Figure 3 )

[0106] (III) Detection Indicators and Methods:

[0107] 1. Antiviral effect:

[0108] Serum HBV DNA and serum HBsAg.

[0109] 2. Metabolic indicators:

[0110] Hepatic steatosis: H&E staining of paraffin sections of liver tissue.

[0111] Blood lipid (TC, TG, NEFA) testing.

[0112] Fasting blood glucose (FBG): Blood is drawn from the tail vein and measured with a blood glucose meter.

[0113] Fat weight: The epididymal fat (eWAT) and subcutaneous fat (iWAT) were dissected and weighed using an electronic balance.

[0114] 3. Liver damage and inflammation:

[0115] Liver enzymes (ALT, AST) were detected using a biochemical analyzer.

[0116] Inflammatory factors (IL-6, IL-1β, TNF-α) ELISA detection kit.

[0117] Liver fibrosis: Sirius red trichrome staining was used to observe the area of ​​collagen deposition.

[0118] (iv) Experimental results:

[0119] 1. Treatment efficacy for comorbidities

[0120] 1) Improves glucose and lipid metabolism disorders: After ML-098 treatment, lipid deposition in the liver is reduced ( Figure 4 A); triglycerides (TG), cholesterol (TC), and free fatty acids (NEFA) decreased ( Figure 4 B), subcutaneous fat weight, epididymal fat weight decreased, fasting blood glucose level decreased ( Figure 4 C).

[0121] 2) Improvement in liver inflammation: Reduced infiltration of inflammatory cells ( Figure 5 A); Decreased liver function ALT and AST levels ( Figure 5 C); Inflammatory factors: TNF-α, IL-1β, and IL-6 decreased ( Figure 5 D).

[0122] 3) Decreased levels of HBV DNA and HBsAg ( Figure 5 B).

[0123] 4) Improvement in liver fibrosis: Reduced collagen deposition area ( Figure 5 A).

[0124] 2. Treatment efficacy of MASLD alone

[0125] 1) Improvement of glucose and lipid metabolism disorders: Reduced lipid deposition in the liver was observed in the ML-098 monotherapy group, the H-151 monotherapy group, and the ML-098+H-151 combination therapy group. Figure 6 A), decreases in TG, TC, and NEFA ( Figure 6 B) and the reduction in subcutaneous fat and epididymal fat weight ( Figure 6 C). Only the ML-098 monotherapy group and the ML-098+H-151 combination group could improve fasting blood glucose levels; the H-151 monotherapy group could not improve blood glucose levels in MASLD mice. Figure 6 C).

[0126] 2) Inflammation and liver injury: Reduced infiltration of inflammatory cells in the liver was observed in the ML-098 monotherapy group, the H-151 monotherapy group, and the ML-098+H-151 combination therapy group. Figure 7 A), while only the ML-098 monotherapy group improved ALT and AST in MASLD mice (A). Figure 7 B). The ML-098 monotherapy group, the H-151 monotherapy group, and the ML-098+H-151 combination group all reduced the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in MASLD mice. Figure 7 C).

[0127] 3. Treatment efficacy of CHB alone

[0128] 1) Both the ML-098 monotherapy group and the DMXAA monotherapy group could reduce HBV DNA levels in HBV-tg mice, while the ML-098+DMXAA combination therapy group was less effective at reducing HBV DNA than the monotherapy group. Figure 8 A).

[0129] 2) The single-drug DMXAA group and the combination drug ML-098+DMXAA group were less effective in reducing HBsAg levels in mice than the single-drug ML-098 group. Figure 8 B).

[0130] This invention provides a RAB7 agonist monotherapy or combination therapy that significantly reduces HBV replication markers (HBV DNA, HBsAg); improves hepatic steatosis, dyslipidemia, and glycemic disorders in patients with metastatic liver disease (MASLD); simultaneously inhibits liver inflammation and fibrosis, and reduces liver damage markers; and provides an integrated treatment plan for patients with chronic hepatitis B (CHB) and MASLD comorbidity, reducing adverse reactions caused by combination drug therapy.

[0131] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The use of a RAB7 agonist in the preparation of a medicament for treating liver diseases, wherein the liver disease is chronic hepatitis B or a comorbidity of chronic hepatitis B and metabolic dysfunction-related fatty liver disease; wherein the RAB7 agonist is ML-098.

2. The application according to claim 1, characterized in that: In the preparation of a drug for treating chronic hepatitis B, the drug uses a RAB7 agonist as the active ingredient; or The drug has RAB7 agonist and STING agonist as active ingredients, and the STING agonist is DMXAA.

3. The application according to claim 1, characterized in that: In the preparation of a medicament for treating comorbidities of chronic hepatitis B and metabolic dysfunction-related fatty liver disease, the medicament uses a RAB7 agonist as the active ingredient.

4. The application according to claim 1, characterized in that: The RAB7 agonist promotes HBV virus degradation and liver autophagy by activating the Rab7 protein, while simultaneously reducing HBV DNA and HBsAg and improving blood lipids and blood glucose.

5. The application according to claim 1, characterized in that: The RAB7 agonist reduces the levels of liver tissue inflammatory factors IL-6, IL-1β, TNF-α, and serum ALT and AST, thereby alleviating liver fibrosis.

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