Application of irbesartan in preparation of medicine for preventing or treating osteoarthritis

Abstract

The invention discloses application of irbesartan in preparation of a medicine for preventing or treating osteoarthritis, and belongs to the technical field of biological medicine. Osteoarthritis single cell transcriptome data are integrated and analyzed, differential expression genes are screened, and irbesartan is screened by using a relocation method and Mendel randomization analysis to serve as a medicine for treating osteoarthritis; analysis results show that irbesartan can significantly reduce the risk of osteoarthritis. An irbesartan administration experiment is carried out by using a zebra fish bone arthritis model, and the experiment result shows that irbesartan obviously increases the joint gap of anal fins of zebra fish and has an obvious protection effect on articular cartilage; a transcriptome sequencing result shows that irbesartan plays the role by inhibiting the MAPK signal channel. The invention provides the effect of irbesartan in the development of osteoarthritis for the first time, provides a new strategy for prevention and treatment of osteoarthritis, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of irbesartan in the preparation of a drug for preventing or treating osteoarthritis. Background Art

[0002] Osteoarthritis is a chronic degenerative joint disease characterized by progressive destruction of articular cartilage, synovial inflammation, subchondral bone sclerosis, and osteophyte formation, and is mainly manifested as joint pain, stiffness, and limited mobility. Among them, osteoblasts that mediate bone remodeling, chondrocytes that maintain cartilage matrix metabolism, and synovial cells that regulate pro-inflammatory factors play important roles in the occurrence and development of osteoarthritis. Due to the complex etiology of osteoarthritis, there is still no effective treatment method to reverse the pathological features within the joints of osteoarthritis so far, and the current treatment goal of osteoarthritis is only to relieve pain and prevent the loss of normal joint function.

[0003] Drugs clinically applied to patients with osteoarthritis include: non-steroidal anti-inflammatory drugs that relieve pain by inhibiting inflammatory factors, intra-articular injection drugs such as hyaluronic acid and glucocorticoids, and disease-modifying drugs such as diacerein or chondroitin sulfate. However, the research and development of new drugs requires an investment of 2-3 billion US dollars, and the research and development cycle is as long as 13-15 years. However, the above drugs all have certain defects to a certain extent. Although non-steroidal anti-inflammatory drugs can inhibit inflammation and relieve pain, they cannot reverse cartilage degeneration, and long-term use has gastrointestinal and cardiovascular risks. The efficacy of intra-articular injection drugs is short-lived and may accelerate cartilage degradation. The clinical effects of disease-modifying drugs are limited, and they lack the targeting of specific repair of chondrocytes. For drugs screened based on network pharmacology, they do not combine single-cell sequencing to analyze the heterogeneity of different cell types and lack causal verification. Existing technologies mostly rely on traditional pathological mechanism assumptions to screen drugs, or mine candidate molecules through high-throughput data, but they do not accurately target osteoblasts, chondrocytes, and synovial cells, and also lack systematic verification of epidemiology and in vivo models.

[0004] Based on the above content, there is an urgent need to explore the new therapeutic potential of existing drugs or candidate compounds beyond their original therapeutic fields, so as to reduce the time and economic costs of drug development and reduce the probability of clinical development failure. Summary of the Invention

[0005] The object of the present invention is to provide the application of irbesartan in the preparation of a drug for preventing or treating osteoarthritis, so as to solve the problems existing in the above-mentioned prior art. The present invention first proposes the application of irbesartan in the preparation of a drug for reducing the risk of osteoarthritis and treating osteoarthrosis, providing a new strategy for the prevention and treatment of osteoarthritis.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Starting from the single-cell level, this invention conducts an integrated analysis of osteoarthritis single-cell transcriptome data, characterizing cell types in arthritis. Differential genes in osteoblasts, chondrocytes, and synovial cells of osteoarthritis are screened. Candidate drugs are screened using a drug repurposing method based on network proximity, and drugs are screened by Mendelian randomization analysis of the association between drug targets and osteoarthritis. A zebrafish osteoarthritis model induced by triclocarban is constructed and drug-treated. The effects of the drugs are verified by analyzing zebrafish phenotype data. Meanwhile, transcriptome sequencing is performed, and functional enrichment analysis of differentially expressed genes after drug treatment is carried out to screen the signaling pathways they regulate.

[0008] This invention provides the use of irbesartan in the preparation of a drug for preventing and / or treating osteoarthritis.

[0009] Preferably, the treatment of osteoarthritis includes maintaining the joint space and protecting articular cartilage.

[0010] Preferably, the irbesartan plays the role of maintaining the joint space and protecting articular cartilage by inhibiting the MAPK signaling pathway.

[0011] This invention also provides a drug for preventing and / or treating osteoarthritis, which uses irbesartan as the only active ingredient.

[0012] Preferably, it also includes pharmaceutically acceptable excipients.

[0013] Preferably, the dosage form of the drug is an oral preparation or an injection.

[0014] Preferably, the treatment of osteoarthritis includes maintaining the joint space and protecting articular cartilage.

[0015] Preferably, the irbesartan plays the role of maintaining the joint space and protecting articular cartilage by inhibiting the MAPK signaling pathway.

[0016] This invention discloses the following technical effects:

[0017] This invention conducts an integrated analysis of osteoarthritis single-cell transcriptome data, screens differentially expressed genes, and uses a repurposing method and Mendelian randomization analysis to screen irbesartan as a drug for treating osteoarthritis; the analysis results show that irbesartan can significantly reduce the risk of developing osteoarthritis. This invention uses a zebrafish osteoarthritis model for irbesartan administration experiments. The experimental results show that irbesartan significantly increases the hip fin joint space of zebrafish and has a significant protective effect on articular cartilage; the transcriptome sequencing results show that irbesartan plays the above role by inhibiting the MAPK signaling pathway. This invention first proposes the role of irbesartan in the progression of osteoarthritis, provides a new strategy for the prevention and treatment of osteoarthritis, and has broad application prospects. Brief Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1 It is a cell annotation result diagram of osteoarthritis single-cell transcriptome data; among them, a is the single-cell data analysis of the femoral head tissue source; b is the single-cell annotation information of the cartilage tissue source; c is the single-cell annotation information of the synovial tissue source

[0020] Figure 2 It is a volcano plot of differential gene analysis of osteoblasts (a), chondrocytes (b) and synoviocytes (c) in the control group and the osteoarthritis group;

[0021] Figure 3 It is a screening result diagram of drug repositioning analysis; among them, a is a Venn diagram of drug screening results in osteoblasts, chondrocytes and synoviocytes; b is the IVW analysis result; c is the leave-one-out cross-validation result;

[0022] Figure 4 It is a result diagram of the pharmacoepidemiological analysis of irbesartan in different gender groups; among them, a is the comparison between irbesartan and α-adrenergic receptor blockers; b is the comparison between irbesartan and other angiotensin II receptor antagonists; c is the comparison between irbesartan and angiotensin-converting enzyme inhibitors;

[0023] Figure 5 It is an experimental result diagram of the effect of irbesartan on zebrafish osteoarthritis; a is the observation result of the staining of the hip fin joint space of zebrafish in the control group (Control), the osteoarthritis model group (TCC) and the drug treatment group (TCC+Irb), and the joint space is marked by a red arrow; b is the quantification result of the joint space of each group;

[0024] Figure 6 It is a result diagram of the gene analysis of zebrafish after irbesartan treatment; among them, a is the analysis result of the transcriptome sequencing data of the zebrafish hip fin part; b is the result of the GO enrichment analysis of the significantly decreased genes. Detailed Embodiments

[0025] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention's specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0029] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0030] The chemical formula of irbesartan of the present invention is C 25 H 28 N6O, and the CAS registration number is 138402-11-6.

[0031] Example 1 Integration and Analysis of Single-Cell Transcriptome Data of Osteoarthritis

[0032] The present invention first obtained the femoral head tissues discarded after hip replacement surgery from 1 case of osteoarthritis group and 3 cases of fracture patients, used magnetic bead sorting to enrich osteoblasts, and then performed single-cell transcriptome sequencing. The obtained single-cell transcriptome data was integrated and analyzed with the dataset GSE147390 in the GEO database using the R package Seurat, and cell annotation was performed by searching the literature and using the CellMarker software to obtain marker genes of different cell types.

[0033] As Figure 1As shown in Figure a, single-cell data analysis of femoral head tissue sources annotated cell types including neutrophils (NE), osteoblasts (OB), chondrocytes (CHON), macrophages (Mac), smooth muscle cells (SMC), endothelial cells (EC), erythrocytes (Er), natural killer cells (NK), and plasma cells (PC). Subsequently, osteoblasts and chondrocytes will be extracted for subsequent analysis.

[0034] For the single-cell data of cartilage tissue, the present invention obtained the GSE220243 dataset from the GEO database, which included 6 cases of knee cartilage tissue without joint injury and 6 cases of osteoarthritis cartilage tissue after total knee arthroplasty. The R package Seurat was also used for integrated analysis, and marker genes of different cell types were obtained through literature search and the use of the CellMarker software for cell annotation.

[0035] As Figure 1 shown in Figure b, single-cell annotation information of cartilage tissue sources annotated cell types including effector chondrocytes (EC), prehypertrophic chondrocytes (preHTC), regulatory chondrocytes (RegC), fibrocartilage cells (FC), homeostatic chondrocytes (HomC), chondrocytes with high metallothionein expression (MTC), prefibrocartilage cells (preFC), reparative chondrocytes (RepC), hypertrophic chondrocytes (HTC), and proliferative chondrocytes (ProC). Subsequently, two types of chondrocytes, proliferative chondrocytes and reparative chondrocytes (Chon), will be extracted for subsequent analysis.

[0036] For the single-cell data of synovial tissue, the present invention integrated the synovial single-cell sequencing datasets shared by GSE152805 and the Department of Orthopaedic Surgery of the First Affiliated Hospital of Jinan University, including a total of single-cell sequencing data of 3 non-OA samples and 5 OA patients' synovial tissues. The R package Seurat was also used for integrated analysis and cell annotation.

[0037] As Figure 1 shown in Figure c, single-cell annotation information of synovial tissue sources annotated cell types including synoviocytes (SF) containing subsynovial fibroblasts (SSF) and synovial intimal fibroblasts (SIF), macrophages (MAC), endothelial cells (EC), plasma cells (PC), lymphocytes (LYC), mast cells (MC), dendritic cells (DC), and pericytes (PIC). Subsequently, two types of synoviocytes, subsynovial fibroblasts and synovial intimal fibroblasts (SF), will be extracted for subsequent analysis.

[0038] Example 2 Differential Gene Analysis

[0039] The FindMarkers function was used to evaluate the differentially expressed genes (DEGs) between osteoblasts, chondrocytes, and synoviocytes in the control group and the osteoarthritis group to identify the DEGs of osteoblasts. In the present invention, the differentially expressed genes with significant differences were screened with the criteria of |avg_log2FC| value greater than 0.5 and P < 0.05 after correction; the upregulated genes in the osteoarthritis group were defined according to P < 0.05 after correction and log2[foldchange(FC)] > 0.5.

[0040] The volcano plots of differential genes of osteoblasts, chondrocytes, and synoviocytes in the control group and the osteoarthritis group are shown as Figure 2 a - c in

[0041] Example 3 Drug Repurposing Analysis

[0042] In the present invention, a network - based method was adopted for drug repurposing analysis. The core idea is: mapping known drug targets to the human proteome and further associating them with disease modules. First, the differentially expressed genes in osteoblasts, chondrocytes, and synoviocytes in the osteoarthritis group were obtained. Subsequently, these differentially expressed genes were extracted from the human interactome to construct a protein - protein interaction network as the disease module. After completing the construction of the drug - target network, the network proximity between the drug targets and the disease module was calculated. A higher network proximity (i.e., a lower Z - score) indicates a stronger network association between the two.

[0043] As Figure 3 shown in a, 46 drugs were simultaneously screened in the three types of cells after drug repurposing analysis using the differentially expressed genes in osteoblasts, chondrocytes, and synoviocytes. After Mendelian randomization (MR) analysis through the summary data of gene expression quantitative trait loci of drug targets and genome - wide association studies of osteoarthritis, it was found that four drugs: choline, acetaminophen, tilmicosin, and irbesartan were causally related to osteoarthritis. By screening instrumental variables for candidate drug targets, 39 SNPs in the cis - eQTL of the choline target SLC22A1, 2 SNPs in the cis - eQTL of the acetaminophen target TRPV1, and 6 SNPs in the cis - eQTL of the irbesartan target AGTR1 for tilmicosin and irbesartan were respectively included in the MR analysis. Since acetaminophen has been used as an analgesic drug for arthritis clinically and the inhibitory effect of choline on osteoarthritis has been reported, and tilmicosin is an antitumor drug, the present invention selected irbesartan for further research.

[0044] Example 4 Mendelian Randomization

[0045] After obtaining the candidate drugs, search for drug targets in the Drugbank database, obtain the SNPs of the targets in the openGWAS database and the gtex database as exposure factors, and use the GWAS data (ebi-a-GCST90038686) of hip OA from openGWAS as the outcome. Through association analysis in the exposure data, select SNPs strongly associated with the exposure factors as instrumental variables, with the filtering condition of P<5e-08.

[0046] After removing linkage disequilibrium, perform Mendelian randomization analysis, and judge whether it is a positive result according to the Inversevariance weighted (IVW) method. In addition, evaluate heterogeneity through IVW and MR-Egger tests. A pvalue>0.05 indicates that there is no heterogeneity in the study. Finally, use the Leave-one-outanalysis method for cross-validation, gradually removing SNPs to judge the impact of each SNP on the results of Mendelian randomization analysis.

[0047] As Figure 3 Shown in b and Table 1, the results of the IVW analysis showed that the expression of the drug target gene AGTR1 of irbesartan had a significant positive causal effect on osteoarthritis (P = 0.004, OR = 1.002). As shown in Table 2, the p-value of the Cochran’s Q test was greater than 0.05, indicating no heterogeneity, and the MR-Egger intercept analysis did not detect horizontal pleiotropy.

[0048] Table 1 Results of Mendelian randomization analysis of the causal effect of drug targets on osteoarthritis

[0049]

[0050] Note: Or: Odds Ratio, the odds ratio; or_lci95: 95%Lower Confidence Interval, the lower limit of the 95% confidence interval of the odds ratio; or_uci95: 95%Upper Confidence Interval, the upper limit of the 95% confidence interval of the odds ratio.

[0051] Table 2 Causal effect of AGTR1 on osteoarthritis by Cochran's Q test and MR-Egger intercept test

[0052]

[0053] As Figure 3As shown in Figure c, the leave-one-out cross-validation results indicated that removing any one SNP could yield a positive causal effect of AGTR1 on osteoarthritis. Irbesartan has antagonistic activity against the angiotensin II type 1 receptor encoded by AGTR1, suggesting a potential therapeutic effect of irbesartan on osteoarthritis.

[0054] Example 5 Pharmacoepidemiological Verification

[0055] Pharmacoepidemiological analysis was performed using the electronic medical record data of UK Biobank. To verify the therapeutic effect of irbesartan on osteoarthritis, a total of three antihypertensive drugs with different mechanisms of action were used as control groups and compared with irbesartan. The three control groups were: α-adrenergic receptor blockers, other angiotensin II receptor antagonists except irbesartan, and angiotensin-converting enzyme inhibitors. The final cohort populations of each group were obtained through three steps: extraction, filtration, and matching of the electronic health record data. All patients taking medications recorded in UK Biobank were extracted according to the International Classification of Diseases (ICD 10) codes, BNF, and Read2 drug codes. The earliest medication date was set as the index date, and data on whether osteoarthritis developed after the index date were obtained. Data diagnosed with osteoarthritis before the index date were filtered out. The propensity score matching algorithm was used to control potential age and gender confounding variables to reduce the interference of confounding bias. Finally, compared with the control group drugs, a logistic regression model was used to infer the OR and 95% CI of the risk of developing osteoarthritis with the use of irbesartan.

[0056] Through the gender and age propensity score matching algorithm, 372 people were matched between irbesartan and α-adrenergic receptor blockers. Among them, 10 people in the irbesartan group had osteoarthritis, and 22 people in the α-adrenergic receptor blocker group had osteoarthritis. 506 people were matched between irbesartan and other angiotensin II receptor antagonists. Among them, 16 people in the irbesartan group had osteoarthritis, and 18 people in the centrally acting antihypertensive drug group had osteoarthritis. 506 people were matched between irbesartan and the angiotensin-converting enzyme inhibitor group. Among them, 16 people in the irbesartan group had osteoarthritis, and 23 people in the angiotensin-converting enzyme inhibitor group had osteoarthritis.

[0057] The results are as Figure 4 shown in Figure a. Compared with the cohort population of α-adrenergic receptor blockers, the use of irbesartan was significantly associated with a reduced risk of osteoarthritis, which could reduce the disease risk by 56% (OR = 0.44, 95% CI = 0.21 - 0.94, P = 0.034). Among men, the use of irbesartan was also significantly associated with a reduced risk of osteoarthritis (OR = 0.12, 95% CI = 0.01 - 0.97, P = 0.047).

[0058] AsFigure 4 As shown in Figure b, compared with other angiotensin II receptor antagonist cohort populations, in men, the use of irbesartan was significantly associated with a reduced risk of osteoarthritis, with a 90% reduced risk of disease (OR = 0.1, 95% CI = 0.01 - 0.76, P = 0.026).

[0059] As Figure 4 As shown in Figure c, compared with the angiotensin-converting enzyme inhibitor cohort population, in men, the use of irbesartan was significantly associated with a reduced risk of osteoarthritis, with a 90% reduced risk of disease (OR = 0.1, 95% CI = 0.01 - 0.76, P = 0.026).

[0060] The above results indicate that the use of irbesartan can significantly reduce the risk of developing osteoarthritis, especially in men.

[0061] Example 6 Animal Experiment

[0062] Wild-type TU zebrafish were used, and their embryos and adult fish were both raised in an aquatic circulation system at a temperature of 28.5 °C and a light / dark cycle of 14 h light / 10 h dark. According to the established guidelines for zebrafish laboratory breeding, adult zebrafish were provided with standard feeding twice a day. Adult zebrafish (6 months old) were randomly divided into 5 groups (n = 15): 0.25% DMSO as the control group; 2 μM triclocarban (TCC) as the osteoarthritis model group; 2 μM TCC + 10 μg / mL irbesartan as the drug treatment group. The culture water was changed every 3 days, and on the 21st day, the zebrafish were anesthetized and the hip fin joints of the stained zebrafish were observed using a stereomicroscope.

[0063] The results were as Figure 5 As shown in Figures a and b, the results of alcian blue-alizarin red double staining showed that compared with the control group of zebrafish (Control), the hip fin joint space of the zebrafish in the osteoarthritis model group (TCC) was significantly narrowed, indicating successful modeling; while the hip fin joint space of the zebrafish in the drug treatment group (TCC + Irb) treated with irbesartan was significantly widened compared with the osteoarthritis model group, indicating that irbesartan significantly alleviated the osteoarthritis symptoms of zebrafish and had a significant protective effect on articular cartilage.

[0064] Example 7 Transcriptome Sequencing and Data Analysis

[0065] Total RNA was extracted from the pelvic fins of zebrafish in the osteoarthritis model group and the drug treatment group. After electrophoresis detection to confirm the RNA quality, the samples were subjected to machine detection using the BGI platform. Referring to the database of RNA-seq data analysis results, significantly changed genes were screened for GO enrichment analysis. Gene enrichment analysis for GO was performed using the ClusterProfiler R package to explore the comprehensive molecular biological mechanisms and pathways involved by the differential genes. GO analysis of the differential genes was identified based on pcutoffvalue < 0.05 using the enrichGO function in ClusterProfiler.

[0066] After analyzing the transcriptome sequencing data of the pelvic fins of zebrafish in the osteoarthritis model group and the drug treatment group, differential genes ( Figure 6 as shown in a) were obtained. Genes significantly decreased in the drug treatment group were used for GO enrichment analysis, and it was found that the genes inhibited by irbesartan were significantly enriched in the inflammatory and immune-related signaling pathways of "inflammatory response", "activation of immune response", "immune response-activating signaling pathway", and the positive regulation of the MAPK signaling pathway of "positive regulation of MAPK cascade" ( Figure 6 as shown in b). The MAPK signaling pathway is activated in osteoarthritis, and the genes inhibited by irbesartan positively regulate the MAPK pathway, indicating that irbesartan can inhibit the MAPK signaling pathway and play a role by inhibiting MAPK.

[0067] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of irbesartan in the preparation of a drug for preventing and / or treating osteoarthritis.

2. The application according to claim 1, characterized in that The treatment of osteoarthritis includes maintaining the joint space and protecting articular cartilage.

3. The application according to claim 2, characterized in that The irbesartan plays the role of maintaining the joint space and protecting articular cartilage by inhibiting the MAPK signaling pathway.

4. A drug for preventing and / or treating osteoarthritis, characterized in that, The drug uses irbesartan as the sole active ingredient.

5. The drug according to claim 4, characterized in that, It also includes pharmaceutically acceptable excipients.

6. The drug according to claim 5, wherein, The dosage form of the drug is an oral preparation or an injection.

7. The drug according to any one of claims 4 to 6, characterized in that, The treatment of osteoarthritis includes maintaining the joint space and protecting articular cartilage.

8. The drug according to claim 7, characterized in that, The irbesartan plays the role of maintaining the joint space and protecting articular cartilage by inhibiting the MAPK signaling pathway.

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