Pharmaceutical composition for treating TET2 deficiency related hepatic fibrosis and application

Abstract

The invention relates to hepatic fibrosis intervention, and provides a pharmaceutical composition for treating and / or preventing hepatic fibrosis related to TET2 function deficiency type myeloid cells and application of the pharmaceutical composition. The composition comprises: (a) an inhibitor or antagonist for inhibiting chemotactic signals mediated by CCL2 and / or CCL8 and CCR2 and / or CCR3; and (b) an IL-6 pathway inhibitor. Preferably, (a) is Bindarit or a pharmaceutically acceptable salt thereof, and (b) is an IL-6 neutralizing antibody or an IL-6 receptor antibody. According to the composition, by reducing mononuclear cell recruitment / proinflammatory mononuclear cell source macrophage infiltration and blocking IL-6 mediated hepatic stellate cell activation, collagen deposition is relieved, and fibrosis indexes such as alpha-SMA and Col1a1 are reduced. Animal experiments show that in a CCl4-induced myeloid Tet2 deletion mouse model and an old-age chimeric model, combined administration is superior to single administration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to intervention strategies for liver fibrosis, specifically to a pharmaceutical composition for treating and / or preventing liver fibrosis associated with TET2-deficient myeloid cells and its use. Background Technology

[0002] Liver fibrosis is a common pathological process in the progression of various chronic liver injuries to cirrhosis, mainly manifested as abnormal deposition of extracellular matrix, formation of fibrous septa, and remodeling of liver tissue structure. Current research generally believes that the activation of hepatic stellate cells (HSCs) and the resulting excessive production of matrix components such as collagen are one of the key links in the occurrence, development, and maintenance of liver fibrosis.

[0003] In chronic liver injury, intrahepatic immune cells participate in the coupling of inflammatory response and fibrosis formation, among which monocytes / macrophages play an important role in the pathological process. Damaged liver tissue can induce the expression of chemokines, recruiting peripheral monocytes into the liver and forming a population of pro-inflammatory monocyte-derived macrophages (pMDMs). This population releases inflammatory cytokines, amplifies the local inflammatory response, and promotes HSC activation and matrix deposition through paracrine mechanisms, thereby driving the fibrosis process.

[0004] Strategies targeting chemokines or their receptors have been proposed. However, the formation of liver fibrosis involves a continuous cascade of processes: inflammatory cell recruitment, inflammation amplification, and fibrotic effects, involving multiple pathways and cell types. Interventions targeting only the chemokine axis or a single receptor pathway often fail to simultaneously cover these continuous steps, resulting in insufficient blocking of fibrosis and limited therapeutic efficacy.

[0005] On the other hand, clonal hematopoiesis of indeterminate potential (CHIP) is associated with an increased risk of various chronic inflammatory diseases. Publicly available research suggests that myeloid mutations, such as TET2, may promote inflammatory responses; and in animal models, transplantation of Tet2-deficient hematopoietic cells can exacerbate liver inflammation and fibrosis. Given that TET2 mutations are more common in the elderly, liver fibrosis in the elderly may involve a specific pathological mechanism driven by TET2-deficient myeloid cells, thus making the etiological chain and intervention targets somewhat unique for these patients.

[0006] Therefore, from the perspective of existing technologies as a whole, there are at least the following shortcomings: First, there is a lack of targeted intervention strategies for liver fibrosis caused by "TET2-deficient myeloid cell-related / driven" fibrosis, making it difficult to achieve precise treatment based on population stratification; second, there is a lack of combination drug regimens that can simultaneously cover the two key links of "inflammatory cell recruitment (upstream)" and "HSC activation (downstream)" in the mechanistic chain, making it difficult to obtain more stable and reproducible anti-fibrotic effects in complex pathological networks. Based on the above shortcomings, it is necessary to propose an implementable and transferable drug combination strategy to improve the intervention effect on this type of liver fibrosis and meet clinical application needs. Summary of the Invention

[0007] Purpose of the invention Given that existing technologies for liver fibrosis intervention mostly focus on a single pathway or a single step, making it difficult to simultaneously cover the continuous cascade process of "inflammatory cell recruitment - inflammation amplification - fibrotic effect," and lacking population stratification and combined intervention programs for TET2-deficient myeloid cell-related liver fibrosis, the purpose of this invention is to provide an implementable and convertible pharmaceutical composition and its use for the treatment and / or prevention of TET2-deficient myeloid cell-related liver fibrosis, in order to achieve a more stable anti-fibrotic effect than single-pathway intervention.

[0008] Technical solution To achieve the above objectives, the present invention provides the use of a pharmaceutical composition in the preparation of a medicament for treating and / or preventing liver fibrosis, wherein the liver fibrosis is liver fibrosis associated with TET2 loss-of-function myeloid cells. The pharmaceutical composition comprises at least: a chemokine axis inhibitor and an IL-6 pathway inhibitor; wherein the chemokine axis inhibitor is used to inhibit chemotactic signaling mediated by CCL2 and / or CCL8 and CCR2 and / or CCR3; and the IL-6 pathway inhibitor is used to inhibit IL-6-mediated inflammatory signaling pathways.

[0009] Based on observations of a Tet2-deficient myeloid cell-associated liver fibrosis model, the inventors found that in this type of liver fibrosis, enhanced monocyte recruitment mediated by chemokines (represented by CCL2 / CCL8) and enhanced signaling of inflammatory factors (represented by IL-6) jointly participate in the fibrotic process. Based on this understanding, this invention, by jointly inhibiting the aforementioned chemokine axis and IL-6 pathway, provides a combined intervention on monocyte recruitment and downstream inflammation-fibrosis effects, thereby helping to enhance the control of liver fibrosis progression and improve the adaptability and stability of treatment regimens (e.g., Figure 7 (As shown).

[0010] In some embodiments, the chemotactic axis inhibitor is selected from one or more of the following: (i) Inhibitors that suppress the expression of CCL2 and / or CCL8; (ii) Antibodies or antigen-binding fragments thereof that neutralize CCL2 and / or CCL8; (iii) CCR2 antagonists; (iv) CCR3 antagonists.

[0011] Preferably, the chemokine axis inhibitor is an inhibitor of MCP subfamily chemokine synthesis, wherein the MCP subfamily chemokines include at least CCL2 and CCL8. More preferably, the inhibitor of MCP subfamily chemokine synthesis is Bindarit or a pharmaceutically acceptable salt thereof.

[0012] In some embodiments, the IL-6 pathway inhibitor includes an IL-6 neutralizing antibody and / or an IL-6 receptor antibody. The antibody may be a full-length antibody or its antigen-binding fragment, and the antigen-binding fragment includes, but is not limited to, Fab, F(ab) ' 2. scFv. Optionally, the IL-6 pathway inhibitor also includes other pharmaceutically acceptable inhibitors that inhibit IL-6 signaling.

[0013] In some embodiments, the pharmaceutical composition may be formulated for simultaneous, separate, or sequential administration. Preferably, the chemokine axis inhibitor is formulated as an oral formulation, and the IL-6 pathway inhibitor is formulated as an injectable formulation. In some embodiments, the chemokine axis inhibitor and / or the IL-6 pathway inhibitor are administered at a therapeutically effective amount; the route of administration includes, but is not limited to, oral, intravenous, subcutaneous, or intraperitoneal administration; the frequency and duration of administration may be adjusted according to the subject's weight, disease severity, and combination therapy regimen. Examples of dosing parameters in animal experiments are given in Examples 4 and 6.

[0014] In some embodiments, the TET2-deficient myeloid cells are myeloid cells carrying TET2 inactivation mutations and / or reduced TET2 expression, preferably monocytes and / or macrophages; accordingly, the use is applicable to subjects carrying the TET2 inactivation mutations and / or reduced TET2 expression.

[0015] In one optional implementation, peripheral blood and / or bone marrow samples may be collected from the subject and myeloid cells isolated. In vitro detection of TET2 inactivation mutations may be performed using sequencing (e.g., NGS) or digital PCR (e.g., ddPCR), and / or transcriptional / protein detection methods may be used to assess TET2 expression levels for subject stratification or medication regimen selection. The detection threshold can be set according to the detection platform, sample quality, and clinical needs (e.g., VAF ≥ 5% for TET2 inactivation mutations). The above detection methods are optional for in vitro sample testing and subject stratification and do not constitute a limitation on diagnostic or therapeutic procedures performed on humans / animals.

[0016] Beneficial effects Compared with the prior art, the present invention has at least the following beneficial effects: (1) Dual-target synergistic coverage of key links. Chemotactic axis inhibitors are used to reduce monocyte recruitment and inhibit pMDMs expansion, while IL-6 pathway inhibitors are used to block HSC activation mediated by myeloid-derived IL-6. The combination of the two can cover key nodes in the "recruitment-amplification-fibrotic effect" chain of liver fibrosis, thereby improving the intensity and stability of intervention on the fibrosis process.

[0017] (2) Stronger matching of the pathogenesis chain. This invention targets the upstream chemotactic abnormalities and downstream cytokine effects in TET2-deficient myeloid cell-related liver fibrosis, which is more conducive to effectively blocking the complex pathological network than a single pathway-targeting approach.

[0018] (3) Combination therapy is superior to monotherapy, supported by experimental evidence. See Examples 1-6 and... Figures 1-6 As shown, in the CCl4-induced myeloid-specific Tet2-deficient mouse liver fibrosis model and related chimeric models, the combined drug significantly reduced collagen deposition in liver tissue and fibrosis markers such as α-SMA and Col1a1, and reduced serum type IV collagen and hyaluronic acid levels, while also reducing IL-6, CCL2, and CCL8 levels, demonstrating a synergistic anti-fibrotic effect.

[0019] (4) Possesses potential for translational accessibility. The Bindarit and IL-6 pathway inhibitors used in this invention have publicly available clinical research or application foundations, providing conditions for subsequent pharmaceutical development and clinical translation. Attached Figure Description

[0020] Figure 1 The results show the detection of Tet2 deficiency mice with different tissue specificity in a CCl4-induced liver fibrosis model; among them, Figure 1 Image A shows the gross appearance of liver fibrosis. Figure 1B represents the liver weight / body weight ratio; Figure 1 C~ Figure 1 F represents the mRNA expression of fibrosis-related genes Acta2, Col1a1, Pdgfr, and Timp1; Figure 1 G represents the immunoblotting results of α-SMA and type I collagen; Figure 1 H represents a collagen-related histological staining image of liver tissue; Figure 1 I am Figure 1 Quantitative analysis of H; Figure 1 J represents the immunohistochemical images of α-SMA and type I collagen; Figure 1 K~ Figure 1 L is Figure 1 Quantitative analysis of J; Figure 1 M~ Figure 1 N represents the serum type IV collagen and hyaluronic acid test results.

[0021] Figure 2 This document outlines the construction process of a Tet2-deficient myeloid cell chimeric mouse model and the detection results under CCl4 treatment conditions; among them, Figure 2 A illustrates the process of constructing a chimeric model; Figure 2 B is an image of collagen-related histological staining in liver tissue; Figure 2 C is Figure 2 Quantitative analysis of B; Figure 2 D represents the immunohistochemical results of type I collagen and α-SMA; Figure 2 E represents the immunofluorescence detection results of mononuclear macrophages in liver tissue; Figure 2 F represents the results of flow cytometry analysis of the proportion of donor-derived mononuclear macrophages; Figure 2 G represents the results of in vitro assays related to the proliferation capacity of macrophages derived from mononuclear cells.

[0022] Figure 3 The effect of clophosphate liposomes on the clearance of intrahepatic macrophages on a CCl4-induced liver fibrosis model; among which, Figure 3 A shows the results of immunofluorescence detection of mononuclear macrophages in liver tissue; Figure 3 B is Figure 3 Quantitative analysis of A; Figure 3 C represents the results of flow cytometry analysis of the clearance effect of chlorphospholiposomes on the hepatic mononuclear-derived macrophage-related cell population. Figure 3 D is Figure 3 Quantitative analysis of C; Figure 3 E is a histological image of Sirius red staining related to the detection of collagen deposition in liver tissue; Figure 3 F represents the immunohistochemical results of type I collagen and α-SMA.

[0023] Figure 4 The results of RNA sequencing screening and validation of chemokine-related findings and the effects of Bindarit intervention were used; among them, Figure 4 A is a heatmap of differentially expressed genes; Figure 4 B represents the mRNA expression of Ccl2 and Ccl8 in mononuclear macrophages; Figure 4 C represents the immunohistochemical results of Ccl2 and Ccl8 in liver tissue; Figure 4 D is a Sirius red staining image of collagen-related histology in liver tissue; Figure 4 E represents the immunohistochemical results of type I collagen and α-SMA; Figure 4 F~ Figure 4 G is Figure 4 Quantitative analysis of E; Figure 4 H represents the detection results of the level of intrahepatic mononuclear macrophage-related infiltration.

[0024] Figure 5 The results of the detection of Tet2 deletion and its correlation with Ccl2 / Ccl8 mRNA stability and RNA-protein interactions; among them, Figure 5 A~ Figure 5 B represents the stability test results of Ccl2 and Ccl8 mRNA; Figure 5 C represents the immunoblotting results of RNA-protein binding detection; Figure 5 D represents the results of RIP-qPCR detection of the enrichment of RNA-binding proteins with Ccl2 / Ccl8 mRNA; Figure 5 E~ Figure 5 F represents the half-life of Ccl2 and Ccl8 mRNA under the Tet2 gene enzyme activity mutation condition.

[0025] Figure 6 The results included assays for Tet2-deficient pMDMs and their association with IL-6, as well as assays for combined intervention with Bindarit and IL-6 pathway inhibitors; among which, Figure 6 A represents serum IL-6 levels; Figure 6 B shows a schematic diagram of co-culturing pMDMs and HSCs; Figure 6 C represents the IL-6 related detection results using ELISA in the co-culture system; Figure 6 D represents collagen-related histological staining images of liver tissue under different drug administration conditions; Figure 6 E is Figure 6 Quantitative analysis of D; Figure 6 F is a schematic diagram of the chimeric model construction and the timeline of CCl4 treatment and drug administration; Figure 6G represents the detection results related to pMDMs in the chimeric model; Figure 6 H represents the detection results of inflammatory factors / chemokines in the chimeric model; Figure 6 Image I shows histological images of liver tissue stained with Sirius red under different treatment conditions.

[0026] Figure 7 This diagram illustrates the "targeted elimination of MDMs combined with IL-6 neutralization" therapy, providing the first precision treatment strategy that can effectively block the progression of liver fibrosis in patients with myeloid-specific Tet2 mutations. Detailed Implementation

[0027] The following examples are provided to further illustrate the present invention, but should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, conventional conditions in the art or conditions recommended by the reagent manufacturer may be used.

[0028] Terms and Definitions TET2-deficient myeloid cells (Tet2) ΔMye TET2 (or TET2-related mutations) refers to cells in myeloid lineage cells where the TET2 gene is deleted or inactivated, or where TET2 expression levels and / or enzyme activity are reduced, resulting in decreased TET2-related functions.

[0029] Pro-inflammatory monocyte-derived macrophages (pMDMs): These are macrophage populations recruited from peripheral monocytes, entering the liver and differentiating to possess a pro-inflammatory phenotype; their identification can be based on a combination of flow cytometry and / or immunomarkers. For example, MDMs can be defined as CD45.2... + CD11b + F4 / 80 + Ly6c + Cells; pMDMs can be defined as CD68 + MDMs; activated MDMs can be further defined as iNOS + MDMs. The determination method can be flow cytometry (e.g., BD FACS Melody sorting) or immunofluorescence (IF) labeling (e.g., CD45.2, F4 / 80, Ly6c, CD68, iNOS, etc.), and the results can be expressed as cell frequency (%) or percentage of positive staining area (%).

[0030] Chemotactic axis inhibitors: These are drugs or biological agents that can inhibit chemotactic signals mediated by CCL2 and / or CCL8 and their receptors CCR2 and / or CCR3, thereby reducing monocyte recruitment and / or pMDMs expansion.

[0031] IL-6 pathway inhibitors: refer to drugs or biological agents that can inhibit the IL-6 signaling pathway (including classical and trans signaling).

[0032] MCP subfamily chemokines: refers to chemokines of the monocyte chemoattractant protein (MCP) subfamily, preferably including CCL2 and CCL8; MCP subfamily chemokine synthesis inhibitors refer to drugs that can inhibit the expression of the above-mentioned chemokines, preferably Bindarit or a pharmaceutically acceptable salt thereof.

[0033] Pharmaceutically acceptable salts: These are salts formed with pharmaceutically acceptable acids or bases that are safe under administration conditions and retain the pharmacological activity of the active ingredient.

[0034] Variant allele frequency (VAF): refers to the proportion of sequencing reads carrying the target variant to the total number of reads in a sample being tested, usually expressed as a percentage.

[0035] Example Example 1: Myeloid-specific Tet2 deficiency exacerbates CCl4-induced liver fibrosis (1) Animals and Genotypes: Systemic Tet2 deletion mice, myeloid-specific Tet2 conditional deletion mice, and hepatocyte-specific Tet2 conditional deletion mice were constructed, and corresponding littermate controls were set up. The myeloid-specific Tet2 conditional deletion mice were defined as Tet2^f / f; LysM-Cre + Mice (hereinafter also referred to as Tet2^ΔMye mice); the hepatocyte-specific Tet2 conditionally deficient mice are Tet2^f / f; Alb-Cre + Mice (hereinafter also referred to as Tet2) ΔAlb Mice). The mouse strains used included: C57BL / 6J mice; Alb-Cre transgenic mice (B6.Cg-Speer6-ps1Tg(Alb-cre)21Mgn / J); LysM-Cre mice (also known as Lyz2-Cre, B6.129P2-Lyz2tm1(cre)Ifo / J); Tet2^f / f mice (B6;129S-Tet2tm1.1aai / J).

[0036] (2) Breeding strategy: Tet2^f / f mice were crossed with Alb-Cre mice or LysM-Cre mice to obtain Tet2^f / f; Alb-Cre + Or Tet2^f / f; LysM-Cre + Offspring; Cre-negative mice with the genotype Tet2^f / f served as controls.

[0037] (3) Genotyping: Genomic DNA was extracted from tail tip tissue, and PCR was used to identify Tet2-related alleles and Cre transgenes. The PCR amplification procedure is shown in Table 1; the PCR primer sequences and amplification fragment lengths were set according to the official instructions or supplier recommendations for the corresponding strain.

[0038] (4) Assessment indicators: Record liver / body weight ratio; detect the expression of liver fibrosis-related genes (e.g., Acta2, Col1a1, Pdgfr, Timp1, etc.); perform Sirius red and / or Masson staining to assess collagen deposition; detect α-SMA and collagen deposition; detect serum Col IV, HA, and ALT / AST levels. In the CCl4-induced liver fibrosis model, CCl4 was prepared as a 20% (v / v) working solution with olive oil and administered intraperitoneally at 5 µL / g (based on working solution) twice a week for 7–8 weeks; the control group received an equal volume of olive oil.

[0039] Transcriptional levels of liver fibrosis-related genes were detected by RT-qPCR, with Gapdh as an internal control. Sirius red staining was performed according to standard procedures, and the percentage of positive area was analyzed using Image-Pro Plus 6.0 software. Serum Col IV (#20024, Ruixin), HA (#20067, Ruixin), and IL-6 (#KE10091, Proteintech) levels were detected by ELISA. Serum ALT and AST levels were detected using kits (Leidu, #S03030, #S03040). Experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using two-way ANOVA combined with Tukey's multiple comparison test. P < 0.05 was considered statistically significant.

[0040] Table 1 PCR amplification program Result: As Figure 1 As shown in A-1N, compared with the control, myeloid-specific Tet2 deficiency (Tet2^ΔMye) mice exhibited a higher liver / body weight ratio, upregulated expression of fibrosis marker genes, increased collagen and α-SMA deposition, and elevated serum Col IV, HA, and ALT / AST levels; in contrast, hepatocyte-specific deficiency did not cause significant fibrotic changes. These results indicate that Tet2 deficiency in myeloid cells is an important factor driving the progression of liver fibrosis.

[0041] Comparative Example 1: Fibrosis phenotype in hepatocyte-specific Tet2-deficient mice under identical modeling conditions Following the CCl4-induced liver fibrosis modeling conditions and detection methods described in Example 1, hepatocyte-specific Tet2-deficient mice (Tet2^ΔAlb, genotype Tet2^f / f; Alb-Cre^) were selected. + ) and its littermate control mice (Tet2^f / f; Alb-Cre^ - The results were compared. Except for the genotype difference, the other modeling conditions, sampling time points, and detection indicators were consistent with those in Example 1.

[0042] The test results showed that, under the modeling conditions described above, there were no significant differences between the Tet2^ΔAlb group and the control group in terms of liver weight / body weight ratio, expression of fibrosis-related genes, histological staining / immunoassay, and serum fibrosis markers (see [link to study]). Figure 1 The comparison results between the "hepatocyte knockout group" and its control group are shown in the figure. This comparison is used to illustrate that the loss of Tet2 in hepatocytes is not the main determinant of the aggravation of fibrosis, thus highlighting the specificity of the aggravation of fibrosis caused by myeloid-specific Tet2 loss in Example 1.

[0043] Example 2: Validating the Cell Autonomy of Tet2-Deficient Myeloid Cells Using a Chimeric Mouse Model Chimeric model establishment: Bone marrow cells derived from Tet2-deficient myeloid cells were transplanted into wild-type recipient mice to construct a chimeric model.

[0044] The donor was CD45.2. + Tet2⁻ / ⁻ mice, with CD45.1 as the receptor. + Wild-type mice were irradiated with 9.5 Gy X-rays and then intravenously transplanted with 2 × 10⁶ mice. 6 CD45.2 + CD11b + Cells (divided into 4 doses, 5 × 10⁶ cells each time) 5 Chimerism was assessed 3 weeks post-transplantation (flow cytometry analysis of CD45.2). + Cell ratio).

[0045] Fibrosis induction and evaluation: CCl4-induced fibrosis was performed after chimeric reconstruction and evaluated according to the indicators in Example 1.

[0046] The CCl4 induction conditions in this instruction manual are consistent: CCl4 is prepared with olive oil as a 20% (v / v) working solution and administered intraperitoneally at 5 µL / g (based on the working solution) twice a week for 7 weeks; in Examples 2 and 6 (5), 2 µL / g is used for 6 weeks.

[0047] Three weeks post-transplantation, patients were treated with CCl4 (2 µL / g, based on 20% (v / v) working solution, with olive oil as the carrier, administered intraperitoneally twice weekly for 6 weeks). The endpoint was determined by Sirius red staining of liver tissue, IHC (α-SMA, Collagen I), and serum Col IV and HA levels. Each group had n=4 patients.

[0048] like Figure 2 As shown in A-2G, liver fibrosis was significantly aggravated in mice transplanted with Tet2-deficient myeloid cells, supporting the cell-autonomous pathogenic role of Tet2-deficient myeloid cells in fibrosis.

[0049] Example 3: Clearing intrahepatic macrophages to verify the key role of MDMs in fibrosis Macrophage clearance: In fibrosis models of Tet2^ΔMye mice and control mice, chlorophosphonate liposomes were used to clear intrahepatic macrophages.

[0050] Chlorophosphonate liposomes (Yeasen, 40337ES08) were administered intravenously at a dose of 150 µL per animal, once a week for a total of 7 weeks, starting one week after CCl4 treatment. PBS liposomes served as the control.

[0051] Assessment: Compare collagen deposition, fibrosis marker expression, and serum fibrosis indicators before and after clearance.

[0052] Test results are as follows Figure 3 As shown in A-3F, clearing MDMs can reduce collagen deposition and decrease the expression of fibrosis markers, and improve serum fibrosis indicators; this effect is particularly pronounced in Tet2^ΔMye mice, suggesting that liver fibrosis caused by Tet2 deficiency is highly dependent on the infiltration and activation of intrahepatic macrophages.

[0053] Example 4: Intervention targeting the CCL2 / CCL8 chemotactic axis and its anti-fibrotic effect Differential expression screening: RNA sequencing was performed on liver tissue or sorted intrahepatic macrophages to screen for differentially expressed genes and validate them.

[0054] The samples were liver tissues from Tet2^WT, Tet2^ΔMye, Tet2^WT-CCl4, and Tet2^ΔMye-CCl4 mice (n=4-5). RNA sequencing was performed on the Novaseq platform (Illumina), with differentially expressed genes defined as |log2FC|>1 and FDR<0.05 as thresholds. qPCR used Gapdh as an internal control; ELISA was used to detect CCL2 (Fantai MG9180) and CCL8 (Ruixin RX27820) in serum and liver tissue.

[0055] Key findings: such as Figure 4As shown in A-4B, CCL2 and CCL8 expression was upregulated in Tet2^ΔMye mouse samples; combined with immunological detection results, this suggests that CCL2 / CCL8 and their related receptor pathways are involved in the recruitment and expansion of monocytes / macrophages to the liver.

[0056] Drug intervention: Bindarit, a dual inhibitor of CCL2 / CCL8, was administered.

[0057] Bindarit (MCE, AF283) was dissolved in PBS and administered orally by gavage at a dose of 50 mg / kg once a week for a total of 7 weeks, starting one week after CCl4 treatment.

[0058] Test results are as follows Figure 4 As shown in C-4H, Bindarit can reduce serum and intrahepatic CCL2 / CCL8 levels, decrease macrophage infiltration, and inhibit collagen deposition and fibrosis marker expression, demonstrating that targeting CCL2 / CCL8 signaling can block [certain pathways]. Tet2 Macrophage-dependent fibrosis process caused by deficiency.

[0059] Example 5: Validation of the mechanism by which Tet2 deletion enhances the stability of Ccl2 / Ccl8 mRNA 5hmC-related modification detection: Detection of the conversion level of 5mC to 5hmC in the 3'UTR region of Ccl2 / Ccl8 mRNA and analysis of RNA-binding protein interactions.

[0060] The binding of RNA-binding proteins to Ccl2 / Ccl8 mRNA was detected by RIP-qPCR. Antibodies: anti-Elavl1 (Proteintech, 11910-1-AP), anti-Zfp36 (Proteintech, 12737-1-AP), and anti-Ybx1 (Proteintech, 20339-1-AP). Results are expressed as %input or relative enrichment fold, n=3.

[0061] RNA-protein interactions: Verify enhanced binding of stabilizing factor YBX1 / ELAVL1 to Ccl2 / Ccl8 mRNA and weakened binding of degradation factor ZFP36 (e.g., RIP-qPCR).

[0062] Table 2 shows the primer sequences and amplification fragments used to detect the enrichment of the 3'UTR region of Ccl2 and Ccl8 mRNA for genotyping PCR; RIP-qPCR results are preferably expressed as %input.

[0063] Table 2. Primer sequences and amplification fragment information for genotyping PCR mRNA stability: The half-life of Ccl2 / Ccl8 mRNA was measured to verify that Tet2 deletion can improve its stability.

[0064] Macrophages were treated with 5 µg / mL actinomycin D, and samples were taken at 0, 2, 4, 6, 8, and 10 h. The remaining amount of Ccl2 / Ccl8 mRNA was detected by RT-qPCR, and the fitted half-life (t) was calculated. 1 / 2 ) Enzyme activity-dependent verification: Experiments related to Tet2 catalytic inactivation mutation were conducted to verify the role of Tet2 enzyme activity in regulating the stability of Ccl2 / Ccl8 mRNA.

[0065] The experimental parameters for the Tet2 catalytic inactivation mutant were as follows: The TET2 used was mouse-derived (Gene ID: 214133), and the catalytic inactivation mutant Tet2^Mut (H1382Y) was constructed. The point mutation was introduced into THP-1 cells via CRISPR-Cas9 plasmid transfection and then screened. TET2 expression was verified by Western blot; catalytic inactivation was confirmed by Dot blot detection of 5 hmC levels. mRNA stability was determined by treatment with actinomycin D (5 µg / mL), with samples taken at 0, 2, 4, 6, 8, and 10 h. The remaining Ccl2 / Ccl8 mRNA levels were detected by RT-qPCR, with GAPDH as the internal control. Data are presented as relative decay curves and fitted using a linear approximation.

[0066] Test results are as follows Figure 5 As shown in A-5F, Tet2 deletion leads to upregulation of the 5hmC level in the 3'UTR region of Ccl2 / Ccl8 mRNA, enhancing its binding to YBX1 / ELAVL1 and weakening its binding to ZFP36, thereby improving mRNA stability and providing an epigenetic explanation for the abnormality of upstream chemokines.

[0067] Example 6: Synergistic anti-fibrotic effect of blocking the IL-6 pathway and combination therapy IL-6 Levels and Sources: IL-6 levels in serum and liver tissue were measured, and CD45.2 levels were obtained through sorting. + pMDMs were used to detect IL-6 secretion in order to identify the primary source of IL-6.

[0068] In vitro co-culture: Tet2-deficient pMDMs were co-cultured with hepatic stellate cells to detect HSC activation and collagen production; IL-6 neutralizing antibody was added to verify the blocking effect.

[0069] In vivo combination therapy: In a CCl4-induced Tet2^ΔMye liver fibrosis model, PBS, Bindarit, anti-IL-6 antibody, and Bindarit were administered respectively. + Anti-IL-6 antibody.

[0070] Combination therapy is superior to monotherapy in reducing collagen deposition, HSC activation markers, and serum fibrosis indicators, and is accompanied by hepatic pMDMs infiltration and a decrease in IL-6, CCL2, and CCL8 levels (e.g., Figure 6 A-6E shown).

[0071] Aged chimeric model: Tet2^ΔMye bone marrow cells were transplanted into aged recipient mice to simulate aged CHIP, and combined drug administration was validated after fibrosis induction (e.g. Figure 6 (As shown in F-6I).

[0072] The receptor is CD45.1 in 18-month-old infants. + Mice, transplanted CD45.2 + Tet2^ΔMye BMCs(20%) + CD45.1 + Tet2^ + / + BMCs (80%) were reconstituted and then induced with CCl4 (2 µL / g, based on 20% (v / v) working solution, with olive oil as the carrier, administered intraperitoneally twice weekly for 6 weeks) after 6 weeks, and were given Bindarit. + Combination therapy with anti-IL-6 antibodies. Each group has n=4, with a chimerism rate of approximately 50%.

[0073] Test results are as follows Figure 6 As shown in F-6H, in the elderly model, Tet2-deficient myeloid cells showed more significant expansion and a higher degree of fibrosis; the combination therapy was still able to effectively inhibit the progression of fibrosis, suggesting that this strategy is applicable to elderly patients with CHIP-related liver fibrosis.

[0074] In summary, the experimental results of Examples 1 to 6 show that in liver fibrosis involving TET2-deficient myeloid cells, upstream chemokines, represented by CCL2 / CCL8, abnormally mediate CCR2 / CCR3-dependent recruitment and expansion of inflammatory cells, while downstream inflammatory factors, represented by IL-6, jointly promote hepatic stellate cell activation and extracellular matrix deposition. These two processes constitute a mutually amplified cascade of fibrotic pathways. While blocking the chemokine axis or inhibiting IL-6 signaling alone can alleviate the fibrotic process to some extent, the inhibitory effect remains limited or lacks stability.

[0075] This invention, by jointly blocking the CCL2 / CCL8-CCR2 / CCR3 chemotactic axis and inhibiting the IL-6 pathway, synergistically intervenes in the above-mentioned cascade process of "inflammatory cell recruitment - inflammation amplification - fibrotic effect". It can simultaneously weaken the source of pro-fibrotic signals and its amplification links at multiple levels, thereby significantly reducing the infiltration and expansion of macrophages derived from inflammatory monocytes in the liver, inhibiting the activation of hepatic stellate cells, reducing collagen deposition and abnormal accumulation of extracellular matrix, and showing an anti-fibrotic effect superior to single-pathway intervention.

[0076] Furthermore, in a bone marrow chimeric model simulating the elderly CHIP state, the above-mentioned combined intervention strategy can also effectively inhibit the proliferation of Tet2-deficient myeloid cells and the progression of liver fibrosis mediated by them. This indicates that the combined treatment method described in this invention is not only applicable to general inflammatory liver fibrosis models, but also to complex pathological conditions related to age-related and clonal hematopoietic abnormalities, and has good stability and applicability.

[0077] Therefore, the combined treatment strategy provided by this invention has clear technical effects and application prospects in the prevention and treatment of TET2-deficient myeloid cell-related liver fibrosis, and can provide new technical means for the intervention of related diseases.

Claims

1. The use of a pharmaceutical composition in the preparation of a medicament for treating and / or preventing liver fibrosis, characterized in that, The pharmaceutical composition comprises: (a) an inhibitor or antagonist of chemotactic signaling mediated by CCL2 and / or CCL8 and CCR2 and / or CCR3; and (b) an IL-6 pathway inhibitor; wherein the liver fibrosis is liver fibrosis associated with TET2-deficient myeloid cells.

2. The use as described in claim 1, characterized in that, The chemotactic axis inhibitor is selected from one or more of the following: (i) inhibitors that inhibit the expression of CCL2 and / or CCL8; (ii) antibodies or antigen-binding fragments thereof that neutralize CCL2 and / or CCL8; (iii) CCR2 antagonists; (iv) CCR3 antagonists.

3. The use as described in claim 1 or 2, characterized in that, The chemokine axis inhibitor is an inhibitor of the synthesis of MCP subfamily chemokines, and the MCP subfamily chemokines include at least CCL2 and CCL8.

4. The use as described in claim 3, characterized in that, The MCP subfamily chemokine synthesis inhibitor is Bindarit or a pharmaceutically acceptable salt thereof.

5. The use as described in any one of claims 1-4, characterized in that, The IL-6 pathway inhibitor is an IL-6 neutralizing antibody and / or an IL-6 receptor antibody.

6. The use as described in any one of claims 1-5, characterized in that, The TET2-deficient myeloid cells are myeloid cells carrying TET2 inactivation mutations and / or reduced TET2 expression, and the myeloid cells are monocytes and / or macrophages.

7. The use as described in claim 6, characterized in that, The intended use applies to subjects carrying the TET2 inactivation mutation and / or with reduced TET2 expression.

8. The use as described in any one of claims 1-7, characterized in that, The pharmaceutical composition is formulated for simultaneous, separate, or sequential administration.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a) an inhibitor or antagonist that inhibits chemotactic signaling mediated by CCL2 and / or CCL8 and CCR2 and / or CCR3; (b) an IL-6 pathway inhibitor; and (c) a pharmaceutically acceptable carrier or excipient; wherein the pharmaceutical composition is used to treat and / or prevent liver fibrosis associated with TET2-deficient myeloid cells.

10. A reagent kit, characterized in that, The kit includes a first container and a second container; the first container contains a chemotactic axis inhibitor, the second container contains an IL-6 pathway inhibitor, and is accompanied by instructions for combined administration. The kit is used to treat and / or prevent liver fibrosis associated with TET2-deficient myeloid cells.

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