Application of P4HB as a target in the preparation of drugs for the treatment of multiple types of cancer pleural and peritoneal effusion metastases

Abstract

This invention discloses the application of P4HB as a target in the preparation of therapeutic drugs for metastatic pleural and peritoneal effusions in multiple cancer types. Through combined single-cell transcriptomics and proteomics analysis, this invention revealed that P4HB is significantly overexpressed in metastatic pleural and peritoneal effusion lesions. Multiplex immunofluorescence and immunohistochemistry were used to perform in situ tissue validation in clinical samples of pleural and peritoneal effusions from breast cancer, lung cancer, gastric cancer, colorectal cancer, and ovarian cancer, clarifying the differential expression characteristics of P4HB in metastatic tumor cells and its correlation with poor prognosis. In patient-derived organoid models, targeting and inhibiting P4HB activity with small molecule inhibitors significantly reduced organoid growth capacity and survival rate, and showed a synergistic sensitizing effect on chemotherapeutic drugs. This invention reveals the use of P4HB as a therapeutic target for metastatic pleural and peritoneal effusions in multiple cancer types, providing a new target and direction for drug development in precision medicine.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and precision oncology treatment technology, specifically to the application of P4HB as a target in the preparation of drugs for the treatment of metastatic pleural and peritoneal effusions in multiple cancer types. Background Technology

[0002] In the progression of malignant tumors such as breast cancer, distant metastasis is a leading cause of death, with pleural and peritoneal effusion metastasis being one of the most representative end-stage manifestations lacking effective treatment. Clinical data shows that the median survival of breast cancer patients with pleural and peritoneal effusion metastasis is usually less than 6 months, and more than 70% of patients will relapse within three months, accompanied by progressive wasting, which greatly reduces the patient's quality of life. Pleural and peritoneal effusion not only leads to respiratory and circulatory failure, causing severe cachexia and multiple organ dysfunction, but is also a key marker of treatment failure and irreversible disease progression.

[0003] Currently, the clinical treatment strategy for metastatic pleural and peritoneal effusion remains primarily palliative, including intracavitary chemotherapy, anti-angiogenic therapy, cytokine inhibition, and adhesion blockade. However, these traditional interventions often involve significant toxic side effects and cannot specifically kill free tumor cells in the complex fluid microenvironment, resulting in extremely limited overall efficacy and an objective response rate generally below 30%. The fundamental reason is that current treatment decisions are mainly based on molecular testing of the primary lesion, failing to reflect the dynamic genetic evolution of metastatic lesions. Furthermore, under the cumulative effects of long-term, multi-line treatment pressures (such as chemotherapy, targeted therapy, or immunotherapy), tumor cell lineages undergo significant remodeling, with different clonal subsets competitively expanding, leading to secondary drug-resistant clones. Currently, there are no publicly reported clinical targets for treating metastatic pleural and peritoneal effusion in breast cancer, lung cancer, gastric cancer, colorectal cancer, or ovarian cancer. Therefore, there is an urgent need to find new therapeutic drugs specifically targeting metastatic pleural and peritoneal effusion. Summary of the Invention

[0004] The purpose of this invention is to provide an application of P4HB as a target in the preparation of therapeutic drugs for multiple types of cancer with pleural and peritoneal effusion. This invention, through the application of P4HB as a target in the preparation of therapeutic drugs for multiple types of cancer with pleural and peritoneal effusion, achieves precise inhibition of metastatic tumors in pleural and peritoneal effusion, filling a gap in the treatment options for this indication.

[0005] The technical solution provided by this invention is as follows: the application of P4HB as a target in the preparation of drugs for the treatment of metastatic pleural and peritoneal effusions in multiple cancer types.

[0006] In the above applications, the multiple cancer types are selected from one or more of breast cancer, lung cancer, gastric cancer, colorectal cancer, and ovarian cancer.

[0007] In the aforementioned applications, the drug is a substance that inhibits P4HB expression and / or activity.

[0008] In the aforementioned applications, the substance that inhibits P4HB expression and / or activity is selected from one or more small molecule compounds, antibodies, peptides, or nucleic acid molecules.

[0009] In the aforementioned applications, the small molecule compounds include PACMA31, KSC-34, or pharmaceutically acceptable salts thereof.

[0010] In the aforementioned applications, the drug is used to treat patients with pleural and peritoneal effusion metastases and high expression of P4HB.

[0011] In the aforementioned applications, the drug may also include chemotherapy drugs and / or targeted drugs, forming a combination therapy.

[0012] In the aforementioned applications, the chemotherapy drug is selected from one or more of paclitaxel and cisplatin.

[0013] In the aforementioned applications, the route of administration of the drug includes intrapleural injection or intraperitoneal injection.

[0014] In the aforementioned applications, the drug is prepared as an injection, a lyophilized powder for injection, or a suspension.

[0015] Compared with existing technologies, this invention systematically reveals for the first time the application of P4HB as a therapeutic target for pleural and peritoneal effusion metastases in breast cancer, lung cancer, gastric cancer, colorectal cancer, and ovarian cancer. Single-cell transcriptomics and proteomics combined analysis revealed significantly high expression of P4HB in pleural and peritoneal effusion metastases. Multiplex immunofluorescence and immunohistochemistry validated its differential expression characteristics in metastatic tumor cells, clearly demonstrating that high P4HB expression is significantly associated with poor patient prognosis. In a patient-derived organoid (PDO) model, targeted inhibition of P4HB activity significantly reduced organoid growth capacity and survival rate, and showed a synergistic sensitizing effect against existing chemotherapy / targeted therapy. In a mouse model of breast cancer pleural effusion metastases, targeted intervention with P4HB significantly inhibited tumor growth without significant toxic side effects. This invention provides new targets and drug development directions for the precision treatment of pleural and peritoneal effusion metastases in the above-mentioned cancers, and has promising clinical translation prospects. Attached Figure Description

[0016] Figure 1 Venn diagram for bi-omics intersection analysis;

[0017] Figure 2 Bar chart of Metascape pathway enrichment analysis for core molecule sets;

[0018] Figure 3 A protein-protein interaction network diagram of 24 core molecules;

[0019] Figure 4This is an image showing the immunohistochemical staining results of P4HB in metastatic and primary lesions of breast cancer, lung cancer, gastric cancer, colorectal cancer, and ovarian cancer in pleural and peritoneal effusions according to an embodiment of the present invention.

[0020] Figure 5 Correlation analysis between high P4HB expression and progression-free survival (PFS) in breast cancer patients;

[0021] Figure 6 Correlation analysis between high P4HB expression and progression-free survival in lung cancer patients;

[0022] Figure 7 Correlation analysis between high P4HB expression and progression-free survival in gastric cancer patients;

[0023] Figure 8 Correlation analysis between high P4HB expression and progression-free survival in colorectal cancer patients;

[0024] Figure 9 Drug sensitivity curves of PDO from different patient sources to PACMA31;

[0025] Figure 10 Heatmap of IC50 values ​​for each PDO to PACMA31;

[0026] Figure 11 Statistical comparison of IC50 values ​​between the MPE group and the PT group, and Pearson correlation scatter plot of P4HB protein expression level and PACMA31 IC50 value;

[0027] Figure 12 Drug sensitivity curves of PDO from different patient sources to KSC-34;

[0028] Figure 13 Heatmap of IC50 values ​​for each PDO to KSC-34;

[0029] Figure 14 Statistical comparison of IC50 values ​​between the MPE and PT groups and Pearson correlation scatter plot of P4HB expression level and KSC-34 IC50 value;

[0030] Figure 15 This is a comparison of tumor growth curves between the PACMA31-treated group and the control group in a mouse breast cancer pleural and peritoneal effusion metastasis model according to an embodiment of the present invention. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present invention in any way. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0032] Example 1: This example provides the application of P4HB as a target in the preparation of drugs for the treatment of metastatic pleural and peritoneal effusions in multiple cancer types.

[0033] Specifically, this embodiment first constructs a complete chain of evidence from "target discovery" to "clinical relevance verification" using high-throughput screening technology, establishing a causal link between P4HB and the treatment of pleural and peritoneal effusion metastases. This embodiment details the process of screening P4HB through combined single-cell transcriptomics and proteomics analysis. Figures 1-3 The results of P4HB target screening and functional analysis according to an embodiment of the present invention are shown in the figure. Figure 1 The Venn diagram for the intersection analysis of the two omics is shown. The blue circles represent differentially expressed proteins screened by quantitative proteomics (n=256), and the red circles represent MPE tumor cell-specific upregulated genes screened by single-cell transcriptomics (l=687). The 24 molecules overlapping in the middle are high-confidence core driver molecules of MPE. Figure 2 The Metascape pathway enrichment analysis bar chart, which is based on the core molecular set, shows the top 10 pathways with the highest enrichment levels. The results show that upregulation of the endoplasmic reticulum protein folding pathway is the most significant biological feature of MPE tumor cells. Figure 3 The diagram shows the protein-protein interaction network of 24 core molecules. Nodes represent proteins, and lines represent known interactions (based on the STRING database). Six key candidate targets (P4HB, ERO1A, VAMP8, GSDMD, ALDOA, and EZR) are highlighted in red based on node connectivity.

[0034] The applicant selected pathologically confirmed high-abundance paired breast cancer pleural effusion cell deposits and primary lesion tissue for quantitative proteomics sequencing analysis. The results showed significant spatial separation between malignant pleural effusion (MPE) samples and their primary lesions in the overall protein expression profile, successfully identifying 256 differentially expressed proteins with significant upregulation in pleural effusion tumor cells. Simultaneously, to eliminate background interference from stromal components such as immune cells and mesothelial cells, the applicant further integrated relevant single-cell sequencing resources from GEO databases (such as GSE208532 and GSE262288), specifically extracting tumor cell subsets expressing epithelial markers EPCAM and KRT8 for differential expression analysis, obtaining 687 MPE tumor cell-specific upregulated genes. Intersection analysis of the two sets of data revealed 24 core driver molecules that were stably and significantly upregulated at both the transcriptional and proteomic levels in pleural effusion tumor cells. Subsequently, pathway enrichment analysis of these 24 intersection genes was performed using the Metascape database, showing that these molecules were highly enriched in pathways related to protein folding, endoplasmic reticulum function regulation, and stress response. Using the STRING database, a protein-protein interaction network was constructed to analyze the interactions of core molecules. The results showed that these 24 candidate molecules formed a tightly linked functional module. Surprisingly, the applicant discovered that P4HB (prolyl 4-hydroxylase β subunit) not only occupies a central hub position in the network, but multiple validations also indicated that it plays a key executor role in endoplasmic reticulum stress adaptation in MPE tumor cells. Compared to other candidate molecules (ERO1A, VAMP8, GSDMD, ALDOA, EZR), P4HB showed the most significant differences in expression abundance and node connectivity, thus identifying P4HB as a specific therapeutic target for subsequent targeted intervention studies. This screening process eliminated interference from other non-critical molecules, precisely identifying the key target driving pleural and peritoneal effusion metastasis.

[0035] To further translate the aforementioned bioinformatics screening results into in situ tissue-level experimental validation, the applicant used multiplex immunofluorescence staining to simultaneously detect the differential expression of the six key candidate targets (P4HB, ERO1A, VAMP8, GSDMD, ALDOA, and EZR) in breast cancer pleural and peritoneal effusion metastases and paired primary tumor tissues. Compared with the vast majority of paired primary tumor tissues, P4HB showed the most significant differential expression in pleural and peritoneal effusion metastases, with its fluorescence signal intensity significantly enhanced in the metastatic tumor cells. While other candidate molecules also showed some degree of upregulation, the magnitude of the difference was not as significant as that of P4HB. This multiplex immunofluorescence validation result confirms in situ tissue that P4HB is the most characteristic differentially expressed molecule in pleural and peritoneal effusion metastases, highly consistent with previous multi-omics screening conclusions, further solidifying the experimental basis for identifying P4HB as a specific therapeutic target.

[0036] Furthermore, to demonstrate the clinical value of P4HB as a target, this embodiment combines... Figures 5-8 This study elucidates the correlation between high P4HB expression and poor prognosis. The applicant used the KMplotter online bioinformatics database to perform Kaplan-Meier survival analysis on survival data from a large sample of patients with breast cancer, lung cancer, gastric cancer, and colorectal cancer. Patients were divided into high-expression and low-expression groups based on P4HB gene expression levels for long-term survival prediction comparisons. Figure 5 Correlation analysis between high P4HB expression and progression-free survival (PFS) in breast cancer patients; Figure 6 Correlation analysis between high P4HB expression and progression-free survival in lung cancer patients; Figure 7 Correlation analysis between high P4HB expression and progression-free survival in gastric cancer patients; Figure 8 Correlation analysis between high expression of P4HB and progression-free survival (PFS) in colorectal cancer patients. Figures 5-8 It is evident that the survival probability of patients in the P4HB high-expression group was significantly lower than that in the P4HB low-expression group, with a mortality hazard ratio of 1.13 (95% confidence interval 1.02–1.25), and the difference was statistically significant (P = 0.016). This result conclusively demonstrates that high P4HB expression is significantly positively correlated with poor patient survival, suggesting that P4HB is not only a specifically highly expressed molecule in metastatic tumor cells in pleural and peritoneal effusions, but also possesses significant clinical translational value as a biomarker for assessing patient clinical outcomes and poor prognosis. Inhibiting P4HB expression or activity may potentially block the survival advantage of tumor cells in the pleural and peritoneal effusion microenvironment, thereby improving patient prognosis.

[0037] Based on the above findings, this embodiment explicitly states that the mechanism of action of the drug is to inhibit P4HB expression or activity, thereby interfering with tumor cell survival. P4HB, as a key molecule in endoplasmic reticulum stress adaptation, endows tumor cells with anti-apoptotic ability in the unique fluid microenvironment of pleural and peritoneal effusions. By preparing drugs that can inhibit P4HB expression and / or activity, the protein folding homeostasis of tumor cells can be effectively disrupted, inducing apoptosis, thereby achieving the goal of treating pleural and peritoneal effusion metastasis. This provides a solid theoretical basis and target foundation for the development of specific inhibitors (such as small molecule compounds, antibodies, etc.) in subsequent embodiments.

[0038] Example 2: Based on Example 1, this example further verifies the universality of P4HB as a target in the treatment of pleural and peritoneal effusion metastases in various specific cancer types, and establishes a patient screening strategy based on P4HB expression levels.

[0039] Specifically, the multiple cancer types are selected from one or more of breast cancer, lung cancer, gastric cancer, colorectal cancer, and ovarian cancer. The applicant collected clinical samples of the above five cancer types, including primary lesion tissue and paired pleural and peritoneal metastatic lesion tissue (such as malignant pleural effusion sediment and ascites sediment). The expression level of P4HB protein was detected using immunohistochemistry (IHC). Figure 4 These are immunohistochemical staining results of P4HB in metastatic lesions and primary lesions of breast cancer, lung cancer, gastric cancer, colorectal cancer, and ovarian cancer, according to embodiments of the present invention. They respectively show the comparison between the strong positive diffuse expression of P4HB in metastatic lesions of the five cancer types and the low basal expression in the paired primary lesions. Figure 4 As can be seen, P4HB protein showed significant strong positive expression in pleural and peritoneal effusion metastases originating from breast cancer, lung cancer, gastric cancer, colorectal cancer, and ovarian cancer. The staining was mainly localized in the cytoplasm and endoplasmic reticulum of tumor cells, exhibiting a diffuse brownish-red distribution, particularly evident in tumor cell clusters (such as typical glandular structures or dense "cannonball" spherical clusters). In contrast, in paired primary tumor tissues, P4HB protein showed only low basal expression or focal weak positivity (H-score mostly below 80). The applicant introduced a standardized image quantitative analysis workflow, extracting pure DAB channels separately to calculate the average optical density and histological score (H-score). Quantitative analysis results showed that the mean P4HB histological score (H-score) of metastatic tissues for each cancer type was significantly higher than that of primary tumor tissues (H-score of metastatic lesions was above 150, P < 0.001). This result definitively demonstrates that high expression of P4HB is not specific to a single cancer type, but rather a common characteristic widely present in the metastasis of pleural and peritoneal effusions in various epithelial malignancies. This cross-cancer-type common high expression characteristic makes P4HB a potential therapeutic target for pan-cancer pleural and peritoneal effusion metastases, thus providing a solid pathological basis for the generalization of the "multi-cancer" scope.

[0040] Furthermore, the drug is used to treat patients with pleural and peritoneal effusion metastases exhibiting high P4HB expression. To achieve precision treatment, this embodiment clarifies the criteria for determining "high P4HB expression." In this embodiment, the H-score scoring system is used to quantify the P4HB expression level. The specific calculation formula is: H-score = (1 × percentage of weakly positive cells) + (2 × percentage of moderately positive cells) + (3 × percentage of strongly positive cells), with a scoring range of 0-300 points. When the H-score ≥ 150 points, it is determined to be high P4HB expression; when the H-score < 150 points, it is determined to be low P4HB expression. It should be understood that the above thresholds are only illustrative examples, and those skilled in the art can make appropriate adjustments based on actual detection conditions, antibody titers, and sample types.

[0041] The applicant found a significant positive correlation between P4HB expression levels and patient sensitivity to P4HB inhibitors. Based on the drug sensitivity assessment results, patient-derived organoids (PDOs) were divided into high-expression and low-expression groups according to P4HB expression levels, and treated with the P4HB inhibitors PACMA31 or KSC-34, respectively. Figure 9 Drug sensitivity curves of PDO from different patient sources to PACMA31; Figure 10 Heatmap of IC50 values ​​for each PDO to PACMA31; Figure 11 Statistical comparison of IC50 values ​​between the MPE group and the PT group (P=0.044) and Pearson correlation scatter plot of P4HB protein expression level and PACMA31 IC50 value (R=-0.81, P=0.00043). Figure 12 Drug sensitivity curves of PDO from different patient sources to KSC-34; Figure 13 Heatmap of IC50 values ​​for each PDO to KSC-34; Figure 14 Statistical comparison of IC50 values ​​between the MPE group and the PT group (P=0.00041) and Pearson correlation scatter plot of P4HB expression level and KSC-34 IC50 value (R=-0.68, P=0.0069). Figures 9-14 The results showed that the half-maximal inhibitory concentration (IC50) of PDO in the high-expression P4HB group was significantly lower than that in the low-expression group (PACMA31 group P=0.044, KSC-34 group P=0.00041), indicating that the high-expression group was more sensitive to inhibitors. Based on the endoplasmic reticulum stress adaptation mechanism of P4HB described in Example 1, tumor cells with high P4HB expression exhibit an "addictive" dependence on this pathway. Therefore, targeted inhibition of P4HB can more effectively disrupt its protein folding homeostasis and induce apoptosis. Conversely, tumor cells with low P4HB expression have a weaker dependence on this pathway, and the inhibitory effect is relatively limited. Therefore, screening patients with high expression by detecting P4HB expression levels can significantly improve the response rate and effectiveness of treatment, realizing the transformation from "blind treatment" to "precision treatment." This also establishes the clinical value of P4HB as a companion diagnostic biomarker.

[0042] Example 3: This example further specifies the specific material form of the drug based on the above examples. The drug is a substance that inhibits the expression and / or activity of P4HB. Specifically, based on the key driving role of P4HB in metastatic tumor cells in pleural and peritoneal effusions, the survival pathway of tumor cells can be effectively blocked by intervening in the protein level or enzyme activity of P4HB.

[0043] In a preferred embodiment, the substance inhibiting P4HB expression and / or activity is selected from one or more small molecule compounds, antibodies, peptides, or nucleic acid molecules. The applicant has discovered that various pharmacological forms can be used to intervene against the target P4HB. For example, nucleic acid molecules (such as siRNA, shRNA, or antisense oligonucleotides) can specifically degrade P4HB mRNA, inhibiting P4HB expression at the post-transcriptional level; antibodies or specifically binding peptides can recognize P4HB protein surface antigen epitopes, blocking their interaction with other proteins or inducing antibody-dependent cell-mediated cytotoxicity (ADCC); small molecule compounds can penetrate the cell membrane to enter the cell and directly bind to the active pocket of the P4HB protein, inhibiting its enzymatic activity. All of the above-mentioned inhibitors are within the scope of protection of this invention, aiming to construct a multi-layered defense depth.

[0044] To verify the effectiveness of the small molecule compounds, PACMA31 and KSC-34 were selected as representative inhibitors for detailed description in this embodiment. The small molecule compounds include PACMA31, KSC-34, or pharmaceutically acceptable salts thereof. The inhibitory effects of these compounds on tumor growth were evaluated using patient-derived organoids (PDO) models of breast and lung cancer. The specific experimental methods are as follows: mature PDOs were digested into single cells or microclusters, seeded in matrix gel, and after organoid formation, different concentration gradients (e.g., 0.11 μM to 81 μM) of PACMA31 or KSC-34 were added for treatment, with DMSO solvent used as a control group. After a certain period of culture (e.g., 5-7 days), cell viability was detected by CellTiter-Glo luminescence assay, and dose-response curves were fitted using GraphPad Prism software to calculate the half-maximal inhibitory concentration (IC50).

[0045] like Figure 9 , Figure 10 , Figure 12 and Figure 13As shown, both PACMA31 and KSC-34 exhibited significant antitumor activity. Compared to the control group, the treated groups showed significantly smaller organoid volume, fewer organoids, and significantly lower cell viability. Quantitative analysis revealed that the growth inhibition of PDO by PACMA31 and KSC-34 was significantly concentration-dependent. Specifically, the IC50 values ​​of PACMA31 against some highly sensitive PDO samples were as low as micromolar or even nanomolar levels, indicating its potent killing ability. Furthermore, to explore the intrinsic relationship between P4HB expression abundance and targeted drug sensitivity, the applicant integrated quantitative P4HB expression data from the proteomics of each PDO sample with the corresponding drug IC50 values ​​for Pearson correlation analysis. The results showed a highly significant negative correlation between P4HB protein expression level in MPE-PDO and its IC50 value against PACMA31 (R=-0.81, P=0.00043), and a significant negative correlation between P4HB expression level and the IC50 value against KSC-34 (R=-0.68, P=0.0069). These Pearson correlation analysis results further validated the positive correlation between P4HB expression level and inhibitor sensitivity found in Example 2, confirming that targeted intervention with P4HB can directly disrupt the survival homeostasis of pleural and peritoneal effusion tumor cells, and that P4HB expression abundance can serve as a reliable biomarker for prospectively predicting the efficacy of this type of targeted drug. This result conclusively demonstrates that by targeting and inhibiting P4HB activity with small molecule compounds, the growth homeostasis of metastatic tumor cells in pleural and peritoneal effusions can be effectively disrupted, inducing their death.

[0046] Furthermore, the applicant also detected changes in downstream signaling pathways using Western blotting experiments. The results showed that treatment with PACMA31 or KSC-34 significantly downregulated the expression levels of key proteins in the downstream proliferative signaling pathway of P4HB, while upregulating the expression of apoptosis markers (such as Cleaved Caspase-3). This further confirms the scientific rationale for the anti-tumor effect of small molecule inhibitors by inhibiting P4HB activity at the molecular mechanism level. It should be understood that although this embodiment uses PACMA31 and KSC-34 as examples for verification, those skilled in the art, based on the protein structural characteristics of P4HB, can obtain other small molecule compounds with similar inhibitory activities through reasonable drug design (such as structure-based drug design or high-throughput screening). These should all be considered equivalent substitutions for the technical solutions of this invention.

[0047] Example 4: This example further provides a combination therapy regimen for the drug based on the above examples. The drug also includes chemotherapy drugs and / or targeted drugs, forming a combination therapy. The applicant found that although P4HB inhibitors can effectively inhibit tumor growth when used alone, in clinical practice, patients often face complex situations such as high tumor heterogeneity and strong drug resistance. By combining P4HB inhibitors with other anti-tumor drugs, unexpected synergistic effects can be produced, significantly improving the treatment effect.

[0048] In a preferred embodiment, the chemotherapeutic drug is selected from one or more of paclitaxel and cisplatin. To verify the efficacy of the combination therapy, the applicant conducted detailed in vitro pharmacodynamic experiments using patient-derived organoids (PDO) models of breast and lung cancer. The specific experimental method is as follows: mature PDOs were digested into single cells or microclusters, seeded in matrix gel, and after organoid formation, they were divided into a control group, a P4HB inhibitor monotherapy group, a chemotherapeutic drug monotherapy group, and a combination therapy group. The P4HB inhibitor used was PACMA31 or KSC-34, and the chemotherapeutic drug used was paclitaxel or cisplatin. After a certain period of culture, cell viability was detected by CellTiter-Glo chemiluminescence assay, and the half-maximal inhibitory concentration (IC50) of each drug was calculated.

[0049] The results are as follows Figure 11 and Figure 14 As shown, the combination therapy group exhibited a significant synergistic inhibitory effect. Specifically, the combination of PACMA31 and paclitaxel significantly enhanced the growth inhibition of PDO compared to the combined effect of either drug alone. Quantitative analysis revealed that the IC50 value of the combination therapy group was significantly lower than that of the single-drug group, with reductions ranging from 30% to 50%. Similarly, the same synergistic trend was observed when KSC-34 was combined with cisplatin. These results definitively demonstrate that P4HB inhibitors can significantly enhance the sensitivity of tumor cells to traditional chemotherapy drugs, thereby achieving the same or even better anti-tumor effects while reducing the dosage of chemotherapy drugs. This is of great significance for reducing the toxic side effects caused by high-dose chemotherapy in patients.

[0050] The applicant conducted an in-depth investigation into the aforementioned synergistic mechanism, using Western blotting experiments to detect changes in downstream signaling pathways. The results showed that treatment with P4HB inhibitors significantly activated endoplasmic reticulum stress-related signaling pathways in tumor cells, while simultaneously downregulating the expression levels of anti-apoptotic proteins (such as Bcl-2 and Mcl-1). This suggests that P4HB inhibitors weaken the defense capabilities of tumor cells against external stresses (such as chemotherapy drug attacks) by disrupting their protein folding homeostasis, thus making tumor cells more susceptible to chemotherapy-induced apoptosis. In other words, P4HB inhibitors act as "sensitizers," breaking down the tolerance barrier of tumor cells to chemotherapy drugs.

[0051] It should be understood that although this embodiment uses paclitaxel and cisplatin as examples, the scope of protection of this invention is not limited thereto. Based on the same synergistic mechanism, the chemotherapeutic drugs can also be selected from other antimicrotubule drugs (such as vincristine, docetaxel) or platinum drugs (such as carboplatin, oxaliplatin). In addition, the targeted drugs can be selected from one or more of epidermal growth factor receptor (EGFR) inhibitors, vascular endothelial growth factor (VEGF) inhibitors, or immune checkpoint inhibitors. By rationally combining P4HB inhibitors with the above-mentioned drugs, it is hoped that personalized combination therapy regimens for patients with pleural and peritoneal effusion metastases of different cancer types and molecular subtypes can be constructed, further improving clinical benefits.

[0052] Example 5: Based on the above examples, this example further verifies the efficacy and safety of P4HB inhibitors in treating pleural and peritoneal effusion metastases through in vivo animal experiments, and specifically defines the route of administration and formulation of the drug.

[0053] Specifically, the drug can be administered via intrapleural or intraperitoneal injection. The applicant constructed a mouse model of breast cancer metastasis in the pleural and peritoneal effusion zones for validation. Six- to eight-week-old female BALB / c nude mice were selected, and fluorescently labeled MDA-MB-231 breast cancer cell lines (e.g., luciferase-labeled) were injected intraperitoneally into the mice to establish the pleural and peritoneal effusion metastasis model. After successful model establishment, the mice were randomly divided into a control group (solvent control) and a treatment group (PACMA31). The treatment group received PACMA31 via intrapleural injection at a dose of 2 mg / kg / day, administered every two days for 21 consecutive days. The advantage of choosing intrapleural or intraperitoneal injection is that the metastatic lesions in the pleural and peritoneal effusion zones are mainly located within body cavities. Local administration allows the drug to act directly on the lesion site, significantly increasing the local drug concentration, while avoiding the first-pass effect and reducing drug loss in systemic circulation and toxic side effects on normal tissues. This is particularly important for patients with end-stage pleural and peritoneal effusion metastases, enabling precise targeting and reducing the burden on patients.

[0054] During the experiment, changes in mouse body weight, abdominal circumference, and tumor fluorescence intensity were monitored regularly. At the end of the experiment, mice were sacrificed, ascites fluid was collected, and tumor tissue was dissected and weighed. Figure 15 This is a comparison of tumor growth curves between the PACMA31-treated group and the control group in a mouse breast cancer pleural and peritoneal effusion metastasis model according to an embodiment of the present invention. It shows that the tumor fluorescence intensity in the control group increased significantly over time, while the growth in the treated group was significantly inhibited. At the end of the experiment, the fluorescence intensity in the treated group was only 32% of that in the control group (P<0.01), and no obvious toxic side effects were observed in the mice in the treated group. Figure 15The results showed that the tumor fluorescence intensity in the control group mice increased significantly over time, indicating rapid tumor growth; while the increase in tumor fluorescence intensity in the PACMA31-treated group mice was significantly inhibited, with the fluorescence intensity value in the treated group at the experimental endpoint being only 32% of that in the control group (P<0.01). Simultaneously, throughout the experiment, the treated group mice did not exhibit significant weight loss, reduced activity, hair loss, or other toxic side effects, and the weight change curve was not significantly different from that of the control group. This result conclusively demonstrates that targeted inhibition of P4HB can effectively suppress the growth of metastatic breast cancer tumors in pleural and peritoneal effusions in vivo, with good safety and tolerability. The drug has the potential to inhibit tumor cell growth, reduce tumor cell survival rate, or reduce the amount of pleural and peritoneal effusion.

[0055] Furthermore, the drug is prepared into an injection, a lyophilized powder for injection, or a suspension. To meet the needs of clinical administration, this embodiment provides specific methods for preparing the drug formulation. Taking an injection as an example, PACMA31 or KSC-34 is dissolved in an appropriate amount of solvent (such as a physiological saline solution containing 5% DMSO, 40% PEG300, and 5% Tween-80), sterilized by filtration through a 0.22 μm filter membrane, and then dispensed. Taking a lyophilized powder for injection as an example, PACMA31 or KSC-34 is mixed with a lyophilization protectant (such as mannitol or trehalose), dissolved in water for injection, sterilized by filtration, dispensed into vials, and then freeze-dried to obtain the lyophilized powder for injection. The powder is reconstituted with physiological saline before use. Taking suspension as an example, PACMA31 or KSC-34 is micronized and dispersed in physiological saline containing a suspending agent (such as sodium carboxymethyl cellulose) and a wetting agent (such as Tween 80). Homogenization is then performed to ensure uniform dispersion of the drug particles, resulting in a suspension formulation suitable for intrapleural or intraperitoneal injection. It should be understood that the above formulation is merely illustrative, and those skilled in the art can add pharmaceutically acceptable carriers, excipients, or other materials to prepare other dosage forms suitable for intrapleural or intraperitoneal injection based on actual clinical needs. Through the above formulation process, the stability, solubility, and bioavailability of the drug can be guaranteed, thereby ensuring optimal antitumor effects in vivo.

[0056] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Use of P4HB as a target in the preparation of a drug for treating multiple cancer ascites metastasis.

2. Use according to claim 1, characterized in that, The multiple cancer is selected from one or more of breast cancer, lung cancer, gastric cancer, colorectal cancer and ovarian cancer.

3. Use according to claim 1, characterized in that, The drug is a substance that inhibits the expression and / or activity of P4HB.

4. Use according to claim 3, characterized in that, The substance that inhibits the expression and / or activity of P4HB is selected from one or more of a small molecule compound, an antibody, a polypeptide or a nucleic acid molecule.

5. Use according to claim 4, characterized in that, The small molecule compound includes PACMA31, KSC-34 or a pharmaceutically acceptable salt thereof.

6. Use according to claim 1, characterized in that, The drug is used to treat a patient with P4HB highly expressed ascites metastasis.

7. Use according to claim 1, characterized in that, The drug further comprises a chemotherapeutic drug and / or a targeted drug, forming a combination drug.

8. Use according to claim 7, characterized in that, The chemotherapeutic drug is selected from one or more of paclitaxel and cisplatin.

9. The use according to claim 1, characterized in that, The administration route of the drug includes intrathoracic injection or intraperitoneal injection.

10. The use according to claim 1, characterized in that, The drug is prepared into an injection, a freeze-dried powder injection or a suspension.

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