Methods and medications for treating endometriosis

Abstract

A method and agent for treating endometriosis in a subject requiring treatment for endometriosis are provided, comprising administering an effective amount of an integrin subunit beta-2 antagonist to the subject.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority benefit of International Application No. PCT / CN2023 / 112278, filed on August 10, 2023, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to the medical field, and particularly to a method for treating endometriosis.

Background Art

[0003] Endometriosis is a chronic inflammatory gynecological disease characterized mainly by the presence of ectopic endometrial glands and stroma in the pelvic peritoneum, ovaries, and rectovaginal septum. It is estimated that approximately 5 - 15% of women of reproductive age worldwide are affected by endometriosis (PMID: 27159755), and the prevalence can reach 35 - 80% in women with pelvic pain and / or infertility (PMID: 32212520, 33640070). Due to the diversity of symptoms and the confusion between the signs of endometriosis and those of other diseases, a delay in disease diagnosis of an average of 4 - 12 years is frequently reported (PMID: 8671344, 22990516, 12790847, 28440744). Since the etiology of endometriosis remains unclear, several hypotheses have been proposed to explain the cause of this disease (PMID: 30026507). Pathologically, genetics, environment, immune dysfunction, and estrogen fluctuations may contribute to the formation and maintenance of endometriosis lesions (PMID: 30026507). Among these, dysregulation of steroidogenesis is the most studied mechanism and can be reflected in the hormonal fluctuations that are common risk factors for this disease.

[0004] Due to its diverse pathogenesis, endometriosis remains incurable. Multiple therapeutic approaches have been employed to manage endometriosis and infertility, including surgery, drug therapy, and acupuncture. Hormone therapy is considered the first-line treatment for endometriosis. Approved hormones, Elagolix and MYFEMBREE, have been comprehensively demonstrated in various Phase III trials to reduce endometriosis-related pain without safety concerns (PMID: 28525302, 29889764, 34134684, 33066973, 35717987).

[0005] While endometriosis is not fatal, chronic pain and infertility significantly impact quality of life. The economic burden of endometriosis is estimated to reach $80 billion annually in the United States alone (PMID:35620300). Furthermore, there is growing evidence suggesting that endometriosis is a systemic disease that interferes with cardiovascular, neurological, metabolic, and immune functions (PMID:33640070). Patients may be at increased risk of developing several chronic conditions, including adenomyosis (PMID:24532217), ovarian cancer (PMID:28240000), and autoimmune diseases (PMID:31260048). All of this strongly indicates the need for innovative and effective treatments for endometriosis. To address this challenge, we utilized PandaOmics, an artificial intelligence (AI)-driven target discovery platform, to identify novel drug-worthy targets for treating endometriosis. As shown in Figure 1, PandaOmics used 11 endometriosis transcriptomics datasets to identify the top two genes, guanylate-binding protein 2 and hematopoietic kinase, as potential therapeutic targets. Meanwhile, integrin subunit beta 2 was proposed as a candidate for drug repurposing. Furthermore, upregulation of GBP2 and HCK was confirmed at both the mRNA and protein levels in human endometriosis samples. Both targets were validated through in vitro and in vivo experiments. Lifitegrast, an approved drug targeting ITGB2, significantly inhibited the progression of endometriosis, demonstrating its potential for repurposing in the treatment of endometriosis. This study is the first demonstration of applying AI to the search for therapeutic targets for endometriosis. [Overview of the project]

[0006] The object of the present invention is to provide a treatment method or agent for the treatment of endometriosis.

[0007] One aspect of the present invention provides a method for treating endometriosis in a subject requiring treatment for endometriosis, the method comprising administering an effective amount of an integrin subunit beta-beta2 antagonist to the subject.

[0008] In certain embodiments, the integrin subunit beta-2 antagonist is one or more selected from the group consisting of Rifitegrast, BMS-688521, NPC-15669, BI-1950, BMS-587101, XVA143, BIRT377, A-286982, RWJ50271, and their derivatives.

[0009] Another aspect of the present invention provides the use of integrin subunit beta-2 antagonists in the manufacture of agents for treating endometriosis.

[0010] In certain embodiments, the integrin subunit beta-2 antagonist is one or more selected from the group consisting of Rifitegrast, BMS-688521, NPC-15669, BI-1950, BMS-587101, XVA143, BIRT377, A-286982, RWJ50271, and their derivatives.

[0011] Another aspect of the present invention provides a drug for use in the treatment of endometriosis, comprising an integrin subunit beta-2 antagonist.

[0012] In certain embodiments, the integrin subunit beta-2 antagonist is one or more selected from the group consisting of Rifitegrast, BMS-688521, NPC-15669, BI-1950, BMS-587101, XVA143, BIRT377, A-286982, RWJ50271, and their derivatives. [Brief explanation of the drawing]

[0013] [Figure 1]Figure 1 shows the pipeline for identifying therapeutic targets and reused drugs for endometriosis using PandaOmics. PandaOmics was input with 11 endometriosis datasets and prioritized endometriosis targets (target IDs) under various novel settings using a combination of Omics and text scores. DEG and pathway analyses were performed using this platform. After target IDs, the proposed novel targets were subjected to IHC validation. Only validated targets proceeded to in vitro and in vivo validation using siRNA approaches. Next, downstream effectors of the validated targets were predicted using both computational and experimental methods. Meanwhile, approved drugs associated with the proposed high-confidence targets were tested in vivo to determine the efficacy of reuse in endometriosis treatment. [Figure 2A]Figure 2A shows that integrin subunit beta-2 has been proposed as a highly reliable target for endometriosis. (Figure 2A) Screenshot of the PandaOmics target ID page for endometriosis meta-analysis. Integrin subunit beta-2 is shown as a highly reliable drug-worthy target for endometriosis. (Figure 2B) Box plot showing the expression of integrin subunit beta-2 in 11 comparisons related to endometriosis. (Figure 2C) Dysregulation pathways related to integrin subunit beta-2 in endometriosis comparisons are shown. Pathways were annotated by the Reactome database, and the degree of pathway dysregulation was determined by the iPANDA algorithm. Green and red bars indicate the number of comparisons with significant activation and inactivation of the corresponding pathway, respectively. Pathways labeled in blue are immune-related. (Figure 2D) The mean Spearman correlation coefficients for both composite case and control sets of 11 transcriptomics datasets are shown in a violin plot using LFC values ​​for integrin subunit beta-2 and its nine reported ligands. (Figure 2E) Box plots show the expression of cell adhesion molecule 1 and integrin subunit alpha-M in 11 comparisons associated with endometriosis. FDR < 0.05, ** FDR < 0.01, *** FDR < 0.001. FDR < 0.05 indicates significant differential expression. [Figure 2B]Figure 2B shows that integrin subunit beta-2 has been proposed as a reliable target for endometriosis. (Figure 2A) Screenshot of the PandaOmics target ID page for endometriosis meta-analysis. Integrin subunit beta-2 is shown as a reliable drug-worthy target for endometriosis. (Figure 2B) Box plot showing the expression of integrin subunit beta-2 in 11 comparisons related to endometriosis. (Figure 2C) Dysregulation pathways related to integrin subunit beta-2 in endometriosis comparisons are shown. Pathways were annotated by the Reactome database, and the degree of pathway dysregulation was determined by the iPANDA algorithm. Green and red bars indicate the number of comparisons with significant activation and inactivation of the corresponding pathway, respectively. Pathways labeled in blue are immune-related. (Figure 2D) The mean Spearman correlation coefficients for both composite case and control sets of 11 transcriptomics datasets are shown in a violin plot using LFC values ​​for integrin subunit beta-2 and its nine reported ligands. (Figure 2E) Box plots show the expression of cell adhesion molecule 1 and integrin subunit alpha-M in 11 comparisons associated with endometriosis. FDR < 0.05, ** FDR < 0.01, *** FDR < 0.001. FDR < 0.05 indicates significant differential expression. [Figure 2C]Figure 2C shows that integrin subunit beta-2 has been proposed as a highly reliable target for endometriosis. (Figure 2A) Screenshot of the PandaOmics target ID page for endometriosis meta-analysis. Integrin subunit beta-2 is shown as a highly reliable drug-worthy target for endometriosis. (Figure 2B) Box plot showing the expression of integrin subunit beta-2 in 11 comparisons related to endometriosis. (Figure 2C) Dysregulation pathways related to integrin subunit beta-2 in endometriosis comparisons are shown. Pathways were annotated by the Reactome database, and the degree of pathway dysregulation was determined by the iPANDA algorithm. Green and red bars indicate the number of comparisons with significant activation and inactivation of the corresponding pathway, respectively. Pathways labeled in blue are immune-related. (Figure 2D) The mean Spearman correlation coefficients for both composite case and control sets of 11 transcriptomics datasets are shown in a violin plot using LFC values ​​for integrin subunit beta-2 and its nine reported ligands. (Figure 2E) Box plots show the expression of cell adhesion molecule 1 and integrin subunit alpha-M in 11 comparisons associated with endometriosis. FDR < 0.05, ** FDR < 0.01, *** FDR < 0.001. FDR < 0.05 indicates significant differential expression. [Figure 2D]Figure 2D shows that integrin subunit beta-2 was proposed as a highly reliable target for endometriosis. (Figure 2A) Screenshot of the PandaOmics target ID page for endometriosis meta-analysis. Integrin subunit beta-2 is shown as a highly reliable drug-worthy target for endometriosis. (Figure 2B) Box plot showing the expression of integrin subunit beta-2 in 11 comparisons related to endometriosis. (Figure 2C) Dysregulated pathways related to integrin subunit beta-2 in endometriosis comparisons are shown. Pathways were annotated by the Reactome database, and the degree of pathway dysregulation was determined by the iPANDA algorithm. Green and red bars indicate the number of comparisons with significant activation and inactivation of the corresponding pathway, respectively. Pathways labeled in blue are immune-related. (Figure 2D) The mean Spearman correlation coefficients for both composite case and control sets of 11 transcriptomics datasets are shown in a violin plot using LFC values ​​for integrin subunit beta-2 and its nine reported ligands. (Figure 2E) Box plots show the expression of cell adhesion molecule 1 and integrin subunit alpha-M in 11 comparisons associated with endometriosis. FDR < 0.05, ** FDR < 0.01, *** FDR < 0.001. FDR < 0.05 indicates significant differential expression. [Figure 2E]Figure 2E shows that integrin subunit beta-2 has been proposed as a highly reliable target for endometriosis. (Figure 2A) Screenshot of the PandaOmics target ID page for endometriosis meta-analysis. Integrin subunit beta-2 is shown as a highly reliable drug-worthy target for endometriosis. (Figure 2B) Box plot showing the expression of integrin subunit beta-2 in 11 comparisons related to endometriosis. (Figure 2C) Dysregulated pathways related to integrin subunit beta-2 in endometriosis comparisons are shown. Pathways were annotated by the Reactome database, and the degree of pathway dysregulation was determined by the iPANDA algorithm. Green and red bars indicate the number of comparisons with significant activation and inactivation of the corresponding pathway, respectively. Pathways labeled in blue are immune-related. (Figure 2D) The mean Spearman correlation coefficients for both composite case and control sets of 11 transcriptomics datasets are shown in a violin plot using LFC values ​​for integrin subunit beta-2 and its nine reported ligands. (Figure 2E) Box plots show the expression of cell adhesion molecule 1 and integrin subunit alpha-M in 11 comparisons associated with endometriosis. FDR < 0.05, ** FDR < 0.01, *** FDR < 0.001. FDR < 0.05 indicates significant differential expression. [Figure 3A]Figure 3A shows the expression of integrin subunit beta-2 in two sets of endometriosis single-cell transcriptomics datasets. (Figure 3A) 31 endometrial samples from GSE179640 were classified into five groups: ovarian endometriosis tissue, peritoneal endometriosis tissue and adjacent tissue, normal endometrium, and control. Cells identified by scRNA-seq data were linked and clustered into 18 clusters. (Figure 3B) The expression intensity of integrin subunit beta-2 in each cell cluster is shown in a t-SNE plot. (Figure 3C) 20 menstrual secretion samples from GSE203191 were classified into a diagnostic group and a control group. Cells identified by scRNA-seq data from each group were linked and clustered into 19 clusters. (Figure 3D) The expression intensity of integrin subunit beta-2 within each cell cluster is shown in a t-SNE plot. [Figure 3B] Figure 3B shows the expression of integrin subunit beta-2 in two sets of endometriosis single-cell transcriptomics datasets. (Figure 3A) 31 endometrial samples from GSE179640 were classified into five groups: ovarian endometriosis tissue, peritoneal endometriosis tissue and adjacent tissue, normal endometrium, and control. Cells identified by scRNA-seq data were linked and clustered into 18 clusters. (Figure 3B) The expression intensity of integrin subunit beta-2 in each cell cluster is shown in a t-SNE plot. (Figure 3C) 20 menstrual secretion samples from GSE203191 were classified into a diagnostic group and a control group. Cells identified by scRNA-seq data from each group were linked and clustered into 19 clusters. (Figure 3D) The expression intensity of integrin subunit beta-2 within each cell cluster is shown in a t-SNE plot. [Figure 3C]Figure 3C shows the expression of integrin subunit beta-2 in two sets of endometriosis single-cell transcriptomics datasets. (Figure 3A) 31 endometrial samples from GSE179640 were classified into five groups: ovarian endometriosis tissue, peritoneal endometriosis tissue and adjacent tissue, normal endometrium, and control. Cells identified by scRNA-seq data were linked and clustered into 18 clusters. (Figure 3B) The expression intensity of integrin subunit beta-2 in each cell cluster is shown in a t-SNE plot. (Figure 3C) 20 menstrual secretion samples from GSE203191 were classified into a diagnostic group and a control group. Cells identified by scRNA-seq data from each group were linked and clustered into 19 clusters. (Figure 3D) The expression intensity of integrin subunit beta-2 within each cell cluster is shown in a t-SNE plot. [Figure 3D] Figure 3D shows the expression of integrin subunit beta-2 in two sets of endometriosis single-cell transcriptomics datasets. (Figure 3A) 31 endometrial samples from GSE179640 were classified into five groups: ovarian endometriosis tissue, peritoneal endometriosis tissue and adjacent tissue, normal endometrium, and control. Cells identified by scRNA-seq data were linked and clustered into 18 clusters. (Figure 3B) The expression intensity of integrin subunit beta-2 in each cell cluster is shown in a t-SNE plot. (Figure 3C) 20 menstrual secretion samples from GSE203191 were classified into a diagnostic group and a control group. Cells identified by scRNA-seq data from each group were linked and clustered into 19 clusters. (Figure 3D) The expression intensity of integrin subunit beta-2 within each cell cluster is shown in a t-SNE plot. [Figure 4] Figure 4 is an explanatory diagram of the mechanism of action of Refitegrast. [Figure 5A]Figure 5A shows that subcutaneous and intraperitoneal administration of refitegrast improved the incidence of endometriosis. The efficacy of refitegrast in the treatment of endometriosis was determined by subcutaneous (Figures 5A-5F) and intraperitoneal (Figures 5G-5L) administration of refitegrast in an endometriosis mouse model (n=4). (Figures 5A, 5G) The volume and weight of the excised lesions were recorded and compared. mRNA expression (Figures 5B, 5H) and protein expression (Figures 5C, 5I) of integrin subunit beta 2 in mouse endometriosis xenografts were analyzed by qPCR and IHC. Cell proliferation (Figures 5D, 5J) and apoptotic status (Figures 5E, 5K) of xenografts treated with refitegrast were analyzed by Ki-67 IHC staining and TUNEL assay, respectively. (Figures 5F, 5L) Representative images of paraffin-embedded xenografts stained with hematoxylin and eosin, anti-integrin subunit beta-2 antibody, Ki-67 IHC staining, and TUNEL assay are shown for each experimental group. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. P<0.05 is considered statistically significant. [Figure 5B]Figure 5B shows that subcutaneous and intraperitoneal administration of Refitegrast improved the incidence of endometriosis. The efficacy of Refitegrast in the treatment of endometriosis was determined by subcutaneous (Figures 5A-5F) and intraperitoneal (Figures 5G-5L) administration of Refitegrast in an endometriosis mouse model (n=4). (Figures 5A, 5G) The volume and weight of the excised lesions were recorded and compared. mRNA expression (Figures 5B, 5H) and protein expression (Figures 5C, 5I) of integrin subunit beta 2 in mouse endometriosis xenografts were analyzed by qPCR and IHC. Cell proliferation (Figures 5D, 5J) and apoptotic status (Figures 5E, 5K) of xenografts treated with Refitegrast were analyzed by Ki-67 IHC staining and TUNEL assay, respectively. (Figures 5F, 5L) Representative images of paraffin-embedded xenografts stained with hematoxylin and eosin, anti-integrin subunit beta-2 antibody, Ki-67 IHC staining, and TUNEL assay are shown for each experimental group. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. P<0.05 is considered statistically significant. [Figure 5C]Figure 5C shows that subcutaneous and intraperitoneal administration of refitegrast improved the incidence of endometriosis. The efficacy of refitegrast in the treatment of endometriosis was determined by subcutaneous (Figures 5A-5F) and intraperitoneal (Figures 5G-5L) administration of refitegrast in an endometriosis mouse model (n=4). (Figures 5A, 5G) The volume and weight of the excised lesions were recorded and compared. mRNA expression (Figures 5B, 5H) and protein expression (Figures 5C, 5I) of integrin subunit beta 2 in mouse endometriosis xenografts were analyzed by qPCR and IHC. Cell proliferation (Figures 5D, 5J) and apoptotic status (Figures 5E, 5K) of xenografts treated with refitegrast were analyzed by Ki-67 IHC staining and TUNEL assay, respectively. (Figures 5F, 5L) Representative images of paraffin-embedded xenografts stained with hematoxylin and eosin, anti-integrin subunit beta-2 antibody, Ki-67 IHC staining, and TUNEL assay are shown for each experimental group. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. P<0.05 is considered statistically significant. [Figure 5D]Figure 5D shows that subcutaneous and intraperitoneal administration of refitegrast improved the incidence of endometriosis. The efficacy of refitegrast in the treatment of endometriosis was determined by subcutaneous (Figures 5A-5F) and intraperitoneal (Figures 5G-5L) administration of refitegrast in an endometriosis mouse model (n=4). (Figures 5A, 5G) The volume and weight of the excised lesions were recorded and compared. mRNA expression (Figures 5B, 5H) and protein expression (Figures 5C, 5I) of integrin subunit beta 2 in mouse endometriosis xenografts were analyzed by qPCR and IHC. Cell proliferation (Figures 5D, 5J) and apoptotic status (Figures 5E, 5K) of xenografts treated with refitegrast were analyzed by Ki-67 IHC staining and TUNEL assay, respectively. (Figures 5F, 5L) Representative images of paraffin-embedded xenografts stained with hematoxylin and eosin, anti-integrin subunit beta-2 antibody, Ki-67 IHC staining, and TUNEL assay are shown for each experimental group. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. P<0.05 is considered statistically significant. [Figure 5E]Figure 5E shows that subcutaneous and intraperitoneal administration of refitegrast improved the incidence of endometriosis. The efficacy of refitegrast in the treatment of endometriosis was determined by subcutaneous (Figures 5A-5F) and intraperitoneal (Figures 5G-5L) administration of refitegrast in an endometriosis mouse model (n=4). (Figures 5A, 5G) The volume and weight of the excised lesions were recorded and compared. mRNA expression (Figures 5B, 5H) and protein expression (Figures 5C, 5I) of integrin subunit beta 2 in mouse endometriosis xenografts were analyzed by qPCR and IHC. Cell proliferation (Figures 5D, 5J) and apoptotic status (Figures 5E, 5K) of xenografts treated with refitegrast were analyzed by Ki-67 IHC staining and TUNEL assay, respectively. (Figures 5F, 5L) Representative images of paraffin-embedded xenografts stained with hematoxylin and eosin, anti-integrin subunit beta-2 antibody, Ki-67 IHC staining, and TUNEL assay are shown for each experimental group. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. P<0.05 is considered statistically significant. [Figure 5F]Figure 5F shows that subcutaneous and intraperitoneal administration of refitegrast improved the incidence of endometriosis. The efficacy of refitegrast in the treatment of endometriosis was determined by subcutaneous (Figures 5A-5F) and intraperitoneal (Figures 5G-5L) administration of refitegrast in an endometriosis mouse model (n=4). (Figures 5A, 5G) The volume and weight of the excised lesions were recorded and compared. mRNA expression (Figures 5B, 5H) and protein expression (Figures 5C, 5I) of integrin subunit beta 2 in mouse endometriosis xenografts were analyzed by qPCR and IHC. Cell proliferation (Figures 5D, 5J) and apoptotic status (Figures 5E, 5K) of xenografts treated with refitegrast were analyzed by Ki-67 IHC staining and TUNEL assay, respectively. (Figures 5F, 5L) Representative images of paraffin-embedded xenografts stained with hematoxylin and eosin, anti-integrin subunit beta-2 antibody, Ki-67 IHC staining, and TUNEL assay are shown for each experimental group. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. P<0.05 is considered statistically significant. [Figure 5G]Figure 5G shows that subcutaneous and intraperitoneal administration of refitegrast improved the incidence of endometriosis. The efficacy of refitegrast in the treatment of endometriosis was determined by subcutaneous (Figures 5A-5F) and intraperitoneal (Figures 5G-5L) administration of refitegrast in an endometriosis mouse model (n=4). (Figures 5A, 5G) The volume and weight of the excised lesions were recorded and compared. mRNA expression (Figures 5B, 5H) and protein expression (Figures 5C, 5I) of integrin subunit beta 2 in mouse endometriosis xenografts were analyzed by qPCR and IHC. Cell proliferation (Figures 5D, 5J) and apoptotic status (Figures 5E, 5K) of xenografts treated with refitegrast were analyzed by Ki-67 IHC staining and TUNEL assay, respectively. (Figures 5F, 5L) Representative images of paraffin-embedded xenografts stained with hematoxylin and eosin, anti-integrin subunit beta-2 antibody, Ki-67 IHC staining, and TUNEL assay are shown for each experimental group. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. P<0.05 is considered statistically significant. [Figure 5H]Figure 5H shows that subcutaneous and intraperitoneal administration of Refitegrast improved the incidence of endometriosis. The efficacy of Refitegrast in the treatment of endometriosis was determined by subcutaneous (Figures 5A-5F) and intraperitoneal (Figures 5G-5L) administration of Refitegrast in an endometriosis mouse model (n=4). (Figures 5A, 5G) The volume and weight of the excised lesions were recorded and compared. mRNA expression (Figures 5B, 5H) and protein expression (Figures 5C, 5I) of integrin subunit beta 2 in mouse endometriosis xenografts were analyzed by qPCR and IHC. Cell proliferation (Figures 5D, 5J) and apoptotic status (Figures 5E, 5K) of xenografts treated with Refitegrast were analyzed by Ki-67 IHC staining and TUNEL assay, respectively. (Figures 5F, 5L) Representative images of paraffin-embedded xenografts stained with hematoxylin and eosin, anti-integrin subunit beta-2 antibody, Ki-67 IHC staining, and TUNEL assay are shown for each experimental group. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. P<0.05 is considered statistically significant. [Figure 5I]Figure 5I shows that subcutaneous and intraperitoneal administration of Refitegrast improved the incidence of endometriosis. The efficacy of Refitegrast in the treatment of endometriosis was determined by subcutaneous (Figures 5A-5F) and intraperitoneal (Figures 5G-5L) administration of Refitegrast in an endometriosis mouse model (n=4). (Figures 5A, 5G) The volume and weight of the excised lesions were recorded and compared. mRNA expression (Figures 5B, 5H) and protein expression (Figures 5C, 5I) of integrin subunit beta 2 in mouse endometriosis xenografts were analyzed by qPCR and IHC. Cell proliferation (Figures 5D, 5J) and apoptotic status (Figures 5E, 5K) of xenografts treated with Refitegrast were analyzed by Ki-67 IHC staining and TUNEL assay, respectively. (Figures 5F, 5L) Representative images of paraffin-embedded xenografts stained with hematoxylin and eosin, anti-integrin subunit beta-2 antibody, Ki-67 IHC staining, and TUNEL assay are shown for each experimental group. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. P<0.05 is considered statistically significant. [Figure 5J]Figure 5J shows the improvement of endometriosis onset by subcutaneous and intraperitoneal administration of refiglutide. The effectiveness of refiglutide in the treatment of endometriosis was determined by subcutaneous (Figs. 5A-5F) and intraperitoneal (Figs. 5G-5L) administration of refiglutide in an endometriosis mouse model (n = 4). (Figs. 5A, 5G) The volume and weight of the excised lesions were recorded and compared. The mRNA expression (Figs. 5B, 5H) and protein expression (Figs. 5C, 5I) of integrin subunit beta 2 in mouse endometriosis xenografts were analyzed by qPCR and IHC. The cell proliferation (Figs. 5D, 5J) and apoptotic status (Figs. 5E, 5K) of refiglutide-treated xenografts were analyzed by Ki-67 IHC staining and TUNEL assay, respectively. (Figs. 5F, 5L) Representative images of paraffin-embedded xenografts stained with hematoxylin and eosin, anti-integrin subunit beta 2 antibody, Ki-67 IHC staining, and TUNEL assay for each experimental group are shown. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. P<0.05 is considered statistically significant. [Figure 5K]Figure 5K shows that subcutaneous and intraperitoneal administration of refitegrast improved the incidence of endometriosis. The efficacy of refitegrast in the treatment of endometriosis was determined by subcutaneous (Figures 5A-5F) and intraperitoneal (Figures 5G-5L) administration of refitegrast in an endometriosis mouse model (n=4). (Figures 5A, 5G) The volume and weight of the excised lesions were recorded and compared. mRNA expression (Figures 5B, 5H) and protein expression (Figures 5C, 5I) of integrin subunit beta 2 in mouse endometriosis xenografts were analyzed by qPCR and IHC. Cell proliferation (Figures 5D, 5J) and apoptotic status (Figures 5E, 5K) of xenografts treated with refitegrast were analyzed by Ki-67 IHC staining and TUNEL assay, respectively. (Figures 5F, 5L) Representative images of paraffin-embedded xenografts stained with hematoxylin and eosin, anti-integrin subunit beta-2 antibody, Ki-67 IHC staining, and TUNEL assay are shown for each experimental group. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. P<0.05 is considered statistically significant. [Figure 5L]Figure 5L shows the improvement of the onset of endometriosis by subcutaneous and intraperitoneal administration of refiglutide. The efficacy of refiglutide in the treatment of endometriosis was determined by subcutaneous (Figures 5A - 5F) and intraperitoneal (Figures 5G - 5L) administration of refiglutide in an endometriosis mouse model (n = 4). (Figures 5A, 5G) The volume and weight of the excised lesions were recorded and compared. The mRNA expression (Figures 5B, 5H) and protein expression (Figures 5C, 5I) of integrin subunit beta 2 in mouse endometriosis xenografts were analyzed by qPCR and IHC. The cell proliferation (Figures 5D, 5J) and apoptotic state (Figures 5E, 5K) of xenografts treated with refiglutide were analyzed by Ki - 67 IHC staining and TUNEL assay, respectively. (Figures 5F, 5L) Representative images of paraffin - embedded xenografts stained with hematoxylin and eosin, anti - integrin subunit beta 2 antibody, Ki - 67 IHC staining, and TUNEL assay for each experimental group are shown. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P < 0.05 is considered statistically significant.

Modes for Carrying Out the Invention

[0014] It should be understood that the present invention is not limited to the specific embodiments described herein. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials for which they are cited.

[0016] Where a range of values ​​with one or two limits is provided, it is understood that the narrower range between any intermediate value within that range and any of the limit values ​​within that range is included in the present invention. Where the described range includes one or two limits, the range excluding one or both of those limits is also included in the present invention.

[0017] term It should be noted that the singular forms “a,” “an,” and “the” as used herein and in the appended claims include multiple referents unless the context clearly indicates otherwise. Furthermore, it should be noted that claims may be constructed to exclude any desired element. Accordingly, this statement is intended to be a premise for using the exclusive terms “solely,” “only,” and similar terms, or for using “negative” restrictions, in relation to the description of the elements of a claim.

[0018] Unless otherwise stated, the terms “comprise,” “include,” and “contain,” and their variations (such as “comprising,” “comprises,” and “comprised”) are not intended to exclude any further members, components, integers, or processes. These terms also encompass the meaning of “consist of” or “consisting of.” The terms “consist of” or “consisting of” are specific embodiments of the term “comprise,” excluding any other unspecified members, components, integers, or processes.

[0019] The term "approximately" refers to a range equal to a specific value ±10 percent (±10%).

[0020] The term "and / or" refers to any, any, or all of the elements connected by that term.

[0021] As used herein, the term “endometriosis” refers to a condition in which tissue containing typical endometrial granules and stromal components abnormally develops in various locations in the pelvic cavity or other areas of the body (most commonly the abdominal cavity).

[0022] As used herein, the terms “treat,” “treating,” or “treatment” refer to reducing, inhibiting, and / or reversing the progression of a disease (such as endometriosis). The term “treating” includes all indicators of success in treating or improving the disease, and may include objective or subjective parameters such as relief, remission, reduction of symptoms, or improved tolerance of injury, disease, or condition to the subject, or slowing or reducing the rate of progression. Measurement of treatment or improvement may be based, for example, on the results of physical examinations, pathological examinations, and / or diagnostic examinations known in the art. Treating may also refer to reducing the incidence or onset of the disease, or reducing relapses (such as extending the remission period), compared to no intervention. Clinically, such treatment can also be considered prevention.

[0023] As used herein, the term "activator" refers to a pharmaceutically active chemical substance that produces some pharmacological effect and is used to treat or prevent diseases such as endometriosis.

[0024] As used herein, the terms “inhibitor” and “antagonist” are interchangeable and refer to any molecule that partially or completely blocks or inhibits the activity of a target (such as a protein used as a target in this invention). As used herein, “inhibitor” and “antagonist” may also refer to “activator.”

[0025] As used herein, the term “derivative” of a compound refers to any pharmaceutically acceptable molecule that is derived from (i.e., structurally related to) a compound, has similar or substantially the same activity as the compound, and, when administered to a subject, can provide (directly or indirectly) the compound of the activator or its active metabolite. Examples of derivatives include, but are not limited to, pharmaceutically acceptable salts, hydrates, solvates, prodrugs, or metabolites.

[0026] As used herein, the term “pharmaceutically acceptable salt” refers to a relatively non-toxic inorganic or organic acid salt of the compound of the present invention. These salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free form with a suitable organic or inorganic acid and isolating the salt thus formed. Typical acid salts include acetate, adipine, aspartate, benzoate, besilate, bicarbonate / carbonate, bisulfate, borate, cansilate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, and lactic acid. Examples of pharmaceutically acceptable salts include, but are not limited to, salts, malates, maleates, malons, mesylates, methylsulfates, naphthylates, 2-napsylates, nicotinates, nitrates, orotates, oxalates, palmitates, pamoates, phosphates / hydrogen phosphates / dihydrogen phosphates, pyroglutamates, saccharates, stearates, succinates, tannates, tartrates, tosylates, trifluoroacetates, and xinafoates. In one embodiment, pharmaceutically acceptable salts are hydrochlorides / chloride salts.

[0027] As used herein, the term “solvate” refers to a variable stoichiometric complex formed by a solute (e.g., the activator of the present invention) and a solvent. For the purposes of the present invention, such a solvent may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol, and acetic acid.

[0028] As used herein, the term “prodrug” refers to a precursor that, when administered to a biological system, results in the formation of the aforementioned compound. For example, a prodrug may have the structure X-drug, where X is an inactive carrier moiety and drug is the active compound. As used herein, the term “metabolite” refers to a molecule produced by modification or treatment of a compound after it has been administered to a subject. The term “metabolite” may also refer to a modified or treated drug that retains at least some of the activity of the parent compound.

[0029] As used herein, “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form suitable for contact with the tissue in question, within the bounds of sound medical judgment, without undue toxicity, irritation, allergic reaction, or other problematic complications commensurate with a reasonable benefit / risk ratio.

[0030] As used herein, the term “pharmacologically acceptable carrier” refers to any carrier that, when administered to a subject, does not cause substantially long-term or permanent adverse effects, such as stabilizers, diluents, excipients, adjuvants, and similar substances. “Pharmacologically acceptable carrier” refers to a pharmaceutically inactive substance that is substantially biologically inactive and constitutes a major component of the formulation.

[0031] As used herein, the term “subject” refers to any organism to which the activator of the composition of the present invention may be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, and rabbits; non-human primates such as chimpanzees and other apes and monkeys; and humans). The subjects are mammals, particularly humans, and include males or females, and include neonates, infants, boys, adolescents, adults, or elderly persons, and further include various races and ethnicities.

[0032] As used herein, the terms “therapeutic effective dose” or “effective dose” may be used interchangeably with “therapeutic effective amount” or “effective amount,” and refer to an amount that is effective in treating a disease (such as endometriosis) as can be determined through clinical trials and evaluations, patient observations, and / or similar means. Furthermore, “effective amount” may also specify an amount that causes a detectable change in biological or chemical activity. A detectable change can be detected and / or further quantified by those skilled in the art with respect to the relevant mechanism or process. Furthermore, “effective amount” may specify an amount that maintains a desirable physiological state, i.e., reduces or prevents significant decline, and / or promotes improvement of the state.

[0033] As used herein, the term “unit dosage form” refers to a physically distinct unit (such as a capsule, tablet, or filled syringe cylinder) suitable as a unit dose for a subject, each unit containing a predetermined amount of activator calculated to produce the desired therapeutic effect in conjunction with the required drug carrier.

[0034] As used herein, the term "unit dose" refers to the dose of a substance (such as the activator of the present invention) in a unit dosage form.

[0035] Activating agents for treating endometriosis Through extensive research, the inventors discovered integrin subunit beta-2 (ITGB2) as a therapeutic target for endometriosis. Antagonists of this target can be used to treat endometriosis.

[0036] Integrin subunit beta-2 (ITGB2), also known as CD18, is an integrin subunit beta-chain protein encoded by the human integrin subunit beta-2 gene. When bound to one of its numerous alpha chains, integrin subunit beta-2 can form multiple heterodimers, playing crucial roles in cell adhesion and cell surface signaling, as well as in immune responses. Integrin subunit beta-2 also exists in a soluble ligand-bound form. Deficiency in integrin subunit beta-2 expression can lead to impaired adhesion of circulating leukocytes in humans, potentially reducing the immune system's ability to fight exogenous pathogens.

[0037] The active agents used to treat endometriosis may be any one selected from the group consisting of integrin subunit beta-2 antagonists, including but not limited to GB-2175, Lifitegrast, BMS-688521, NPC-15669, BI-1950, BMS-587101, XVA143, BIRT 377, A-286982, RWJ 50271, and their derivatives.

[0038] The structure of the integrin subunit beta-2 antagonist exemplified is shown below. [Table 1-1] [Table 1-2]

[0039] The amount of each active ingredient in a unit dosage form can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 900, 1000 mg, or any range between any two of the above specific values.

[0040] Administration Each of the active agents of the present invention can be administered to a subject via oral, buccal, sublingual, rectal, vaginal, parenteral, intradermal or intranasal, or parenteral routes. Parenteral administration includes intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intracranial, intrathecal, intratumoral, percutaneous, transmucosal, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, or intracranial injection or infusion.

[0041] The activators used herein may be formulated for administration in pharmaceutical compositions according to known techniques. See, for example, Remington: The Science and Practice of Pharmacy (9th edition, 1995). In the preparation of pharmaceutical compositions according to the present invention, the activator is typically mixed with a pharmaceutically acceptable carrier, among other things. Of course, the carrier must be acceptable in the sense that it is compatible with the other components in the formulation and must not be harmful to the patient. The carrier may be solid, liquid, or both, and is preferably formulated together with the compound as a unit-dose formulation, such as a tablet, which can contain the active agent in an amount of 0.01% by weight or 0.5% to 95% by weight or 99% by weight. The formulations of the present invention may contain one or more activators, and the formulations may be prepared by any known pharmaceutical technique, which includes mixing the components, and may optionally contain one or more auxiliary components and / or excipients. In some embodiments, any of the compositions, carriers, auxiliary components / excipients, and / or formulations of the present invention may contain components from natural or non-natural sources. In other embodiments, any component of the composition, carrier, auxiliary components / excipients, and / or formulation of the present invention may be provided in a sterile form. Non-limiting examples of sterile carriers include endotoxin-free water or pyrogen-free water.

[0042] In some embodiments, the pharmaceutical compositions of the present invention are provided as part of a sterile composition / formulation comprising the activator of the present invention and a pharmaceutically acceptable carrier and / or excipient.

[0043] Suitable dosage forms for oral administration include tablets, capsules, powders, pills, granules, suspensions, solutions or preconcentrates of solutions, emulsions or preconcentrates of emulsions. Pharmaceutically acceptable carriers that can be used for oral dosage forms include water, glycols, oils, alcohols, fragrances, preservatives, colorants, and the like. Oral solid dosage forms such as powders, capsules, or tablets can be prepared using carriers such as starch, sugars, microcrystalline cellulose, diluents, fillers, flow enhancers, granulators, lubricants, binders, stabilizers, disintegrants, and the like.

[0044] Examples of diluents include, but are not limited to, microcrystalline cellulose, mannitol, powdered sugar, compressed sugar, dextran, dextrin, spinose, lactose, cellulose powder, sorbitol, sucrose, talc powder, or combinations thereof. The diluent may be 5% to 90%, preferably 10% to 80%, 20% to 70%, 30% to 60%, or 40% to 50%, based on the total weight of the oral composition.

[0045] Disintegrants include, but are not limited to, cellulose, alginates, gums, cross-linked polymers (such as cross-linked polyvinylpyrrolidone or crospovidone), croscarmellose sodium, croscarmellose calcium, soybean polysaccharides, sodium glycolate starch, guar gum, or any combination thereof. The disintegrant may be present in an amount of about 1% to 15%, preferably 2% to 10%, based on the total weight of the oral composition.

[0046] Examples of binders include, but are not limited to, starch, cellulose, or derivatives thereof (such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropyl methylcellulose), sucrose, dextrose, corn syrup, polysaccharides, gelatin, or any combination thereof. The binder may be present in an amount of 0.01% to 10%, preferably 1% to 10%, based on the total weight of the composition.

[0047] Examples of flow promoters include, but are not limited to, colloidal silicon dioxide, magnesium trisilicate, cellulose powder, talc powder, or combinations thereof. The flow promoter may be present in an amount of 0.1% to 10%, preferably 0.1% to 0.5%, based on the total weight of the composition.

[0048] The dosage form may be, for example, tablets or capsules, and the effective dose may be provided in one or more tablets, capsules, or of the same kind, and may be provided once daily or throughout the day, at intervals of, for example, 4, 8, or 12 hours. For example, a tablet or capsule may contain, for example, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 1,250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, 1,100 mg, or 1,250 mg of the active ingredient. For example, administration of the active agent of the present invention to human subjects may include daily doses in the range of 100-1,250 mg, 150-1,000 mg, 200-800 mg, or 250-750 mg, and this daily dose can be administered entirely once a day, or in portions throughout the day at intervals. Liquid formulations can also be prepared to allow for easy and convenient dispensing of any dosage.

[0049] Parenteral dosage forms are preferably sterilized or can be sterilized before administration to the subject. Examples of parenteral dosage forms include, but are not limited to, solutions for injection, dried products dissolved or suspended in a pharmaceutically acceptable carrier for injection, suspensions for injection, and emulsions.

[0050] Suitable carriers that can be used to provide the parenteral dosage forms described herein include, but are not limited to, water for injection; aqueous media such as sodium chloride injection, Ringer's injection, and glucose injection (but are not limited to these); water-miscible carriers such as ethyl alcohol, polyethylene glycol, and polypropylene glycol (but are not limited to these); and non-aqueous carriers such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate (but are not limited to these).

[0051] Compounds that enhance the solubility of one or more activators disclosed herein can also be incorporated into the parenteral dosage forms provided herein. For example, cyclodextrins and their derivatives can be used to enhance the solubility of the activators of the present invention.

[0052] It should be understood that the effective therapeutic dose can be determined by the physician depending on the type, stage, and / or severity of the disease, the patient's condition, age, weight, sex, and response, as well as the route of administration.

[0053] The therapeutically effective dose is the amount sufficient to achieve plasma concentrations of approximately 0.01 μg / mL to 100 μg / mL, approximately 0.1 μg / mL to 10 μg / mL, and approximately 1 μg / mL to 5 μg / mL when administered to a target subject.

[0054] When administering the activators of the present invention to a target, the therapeutically effective dose of each activator may generally be within the range of approximately 0.5 to approximately 250 mg / kg, approximately 1 to approximately 250 mg / kg, approximately 2 to approximately 200 mg / kg, approximately 3 to approximately 120 mg / kg, approximately 5 to approximately 250 mg / kg, approximately 10 to approximately 200 mg / kg, or approximately 20 to approximately 120 mg / kg, depending on the activator of the present invention. In some embodiments, the therapeutically effective dose may be 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 20 mg / kg, 25 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 75 mg / kg, 100 mg / kg, 120 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, or 300 mg / kg.

[0055] Each of the activators of the present invention may be administered once or twice daily, or once every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or once every 1, 2, or 3 weeks. In some embodiments, each of the activators of the present invention may be administered in a five-times-a-week dosing schedule. In a five-times-a-week dosing schedule, administration may be given for five consecutive days (once daily), followed by two consecutive days of rest.

[0056] As used herein, the term “kit” refers to the package and, in principle, the instructions for use. The active ingredients or pharmaceutical compositions within the kit may be in any of a variety of forms suitable for distribution within the kit. Such forms include liquids, powders, tablets, suspensions, and similar forms. Two or more active ingredients may be provided in separate containers suitable for separate administration, or as a composition in a single container within the package. The kit may contain sufficient quantities for one or more doses of the drug, depending on the treatment method. The instructions for use generally include a description of how to treat a disease (such as ALS) using the drug in the kit.

[0057] It should be understood that the combination or pharmaceutical composition of the present invention may include, in addition to the activator of the present invention, other therapeutic agents or therapies such as biological therapeutic agents and / or chemotherapeutic agents. The method may include, in addition to the administration of the activator of the present invention, the administration of other therapeutic agents or therapies such as biological therapeutic agents and / or chemotherapeutic agents. The other therapeutic agents or therapies may be administered simultaneously with, separately from, or sequentially with the therapeutic agent of the present invention. [Examples]

[0058] Example 1. Identification of therapeutic targets and drug reuse candidates for the endometrium using the AI-powered biological target discovery platform "PandaOmics®". 1. Method Data sources and availability This analysis utilized data from both bulk transcriptomics, single-cell transcriptomics, and proteomics. A total of 36 endometriosis-related bulk transcriptomics datasets, including microarrays and RNA sequencing series from various tissue sources obtained from Gene Expression Omnibus (GEO) and ArrayExpress, were available for direct downstream analysis and target identification using PandaOmics. Two single-cell transcriptomics datasets, GSE179640 and GSE203191, were also extracted from GEO.

[0059] Datasets and Comparison Selection Since the disease etiology begins with orthotopic endometrium, endometrial tissue datasets were selected for downstream analysis and target identification. Eleven case-control comparisons were generated from eleven bulk transcriptomics datasets, and these are listed in Table 1. Both orthotopic endometrial tissue from healthy individuals and orthotopic endometrial tissue from endometriosis patients were selected as controls and labeled "Healthy" and "Orthotopic Endometrium," respectively, in the "Experimental Design" column of Table 1. [Table 2]

[0060] Target identification using PandaOmics PandaOmics was a cloud-based target discovery platform that incorporated multiple deep learning models and AI algorithms into its target prioritization process. Twenty-three target prioritization models were developed, encompassing Omics, text-based, economic, and key opinion leader (KOL) data, to predict the association between target genes and specific indications. Each model's score was expressed on a normalized scale from 0 to 1, with higher scores indicating a greater relevance to the target disease predicted by the model. These models were validated using a Time Machine approach to confirm their target identification capabilities. Adjustable filters for drugability, tissue specificity, target family, and development status were also available to refine the target list.

[0061] To identify potential targets for endometriosis, a meta-analysis was conducted using case-control bulk transcriptomics comparisons. Target prioritization was modified by customizing filter and scoring settings to identify viable targets with varying levels of novelty. The analysis retained only targets belonging to drug-potential protein classes and not considered essential genes as defined by the Therapeutic Targets Database (TTD). This resulted in three lists of highly reliable, moderately and highly novel drug-potential targets, with the top 50 targets further analyzed.

[0062] Processing and analysis of single-cell transcriptomics data For GSE203191, menstrual secretion samples were collected from 11 women with diagnosed endometriosis, 13 women with symptoms (undiagnosed but exhibiting symptoms of chronic endometriosis), and 9 control women. Samples for GSE179640 were isolated from five regions: healthy endometrium, orthotopic endometriosis from endometriosis patients, ectopic endometriotic tissue from the ovaries and peritoneum, and tissue adjacent to endometriotic lesions in the peritoneum.

[0063] For GSE203191, the sample data matrix generated by Cell Ranger was obtained from the GEO database, and gene and cell filtering and cell cluster annotation were applied as defined in the original paper (PMID:36104692). For GSE179640, processed data including normalized expression, cell cluster annotation, sample metadata, and dimensionality-reduced coordinates were provided by the inventors and obtained from https: / / singlecell.jax.org / datasets / endometriosis-2022. Differential gene expression between different cell clusters or different sample groups was calculated by Wilcoxon rank-sum test using log-normalized count data.

[0064] Pathway analysis The activation state of signaling pathways was assessed using PandaOmics' proprietary single-network model, iPANDA (PMID:27848968). iPANDA robustly identified biologically relevant pathway signature sets from the input data while significantly reducing noise by combining differential gene expression data and the degree of pathway topology decomposition. iPANDA values ​​of 1 and -1 indicate pathway activation and repression, respectively. The hierarchical structure of signaling pathways was curated in the Reactome database.

[0065] Efficacy testing of recycled drugs Functional analysis of the proposed drugs was performed in a mouse model mimicking endometriosis. The endometriosis-mimicking mouse model was established by transplanting endometrial tissue excised from the uterine horn of a healthy female mouse subcutaneously or intraperitoneally into another healthy female mouse. Two weeks after transplantation, the mice were divided into two groups and subjected to one of the following treatments: subcutaneous or intraperitoneal administration of either refitegrast or DMSO daily for one week, depending on the transplantation site. On day 7, the mice were sacrificed and the extrauterine endometrial lesions were excised. The volume and weight of the lesions were recorded. The xenografts underwent expression verification of the target organism by qPCR and IHC, followed by Ki-67 IHC staining and TUNEL assay to examine the effects of the drugs on cell proliferation and apoptosis.

[0066] 2.Results Identification of integrin subunit beta 2 as a potential therapeutic target for drug reuse by PandaOmics Based on expression correlation results, an endometriosis meta-analysis including nonspecific comparisons of 11 subtypes and cycles was used for target identification. To identify potential therapeutic targets for endometriosis, 141 unique genes with three levels of novelty were screened based on rankings calculated by PandaOmics, consistency of dysregulation expression across all comparisons included in the meta-analysis, statistical significance of dysregulation, and literature support regarding their potential role in the underlying mechanisms that activate or promote endometriosis. Integrin subunit beta2 was identified as a potential therapeutic candidate for endometriosis. It ranked among the top 10 targets in all six meta-analyses under high confidence settings and received a score of 0.8 or higher in seven of the 13 Omics models in PandaOmics (Figure 2A). In human ectopic endometrial lesion samples, integrin subunit beta-2 was upregulated in 8 out of 11 bulk transcriptome comparisons (72.7%), and in 4 of these upregulations, it was determined to be a differentially expressed gene (Figure 2B). Two single-cell transcriptomics datasets from endometriosis (GSE179640 and GSE203191) were also used to study cell-specific expression profiles. Integrin subunit beta-2 was specifically enriched in bone marrow cells and lymphocyte clusters in both datasets (Figures 3A-3D). Integrin subunit beta-2 was significantly elevated in one bone marrow cell cluster within a peritoneal lesion in GSE179640 (Figure 3B), and in one natural killer cell and two bone marrow cell clusters in menstrual secretions collected from patients diagnosed with endometriosis in GSE203191 (Figure 3D). In addition to expression dysregulation analysis, pathway enrichment analysis was performed to investigate the hidden role of integrin subunit beta-2 in endometriosis. Nine of the eleven altered pathways associated with integrin subunit beta-2 were involved in the immune response (Figure 2C). With the exception of the adaptive immune system, all immune signaling pathways associated with integrin subunit beta-2 were activated.

[0067] Expression correlation analysis was performed on nine ligands of integrin subunit beta-2. The expression of integrin subunit alpha-L, cell adhesion molecule 1, integrin subunit alpha-M, and integrin subunit alpha-X showed a positive correlation with integrin subunit beta-2 expression in both the case and control groups, with a mean Spearman r < 0.5 (Figure 2D). Of these four ligands, cell adhesion molecule 1 and integrin subunit alpha-M were overexpressed in ectopic endometrial tissue samples in comparisons of 8 and 10, with five of them showing overexpression with an FDR < 0.05 (Figure 2E).

[0068] ITGB2 inhibition with lifitegrast improved the development of endometriosis. Given that integrin subunit beta-2 has been identified as a highly reliable target, we investigated the relationship between integrin subunit beta-2 and FDA-approved drugs. Lifitegrast, an antagonist of lymphocyte function-associated antigen 1, is indicated for dry eye disease and targets integrin subunit beta-2 and integrin subunit alpha-L, which interact to form the lymphocyte function-associated antigen 1 complex. As shown in Figure 4, intercellular adhesion molecule 1 attached to mature antigen-presenting cells, damaged epithelium, or endothelium binds to lymphocyte function-associated antigen 1 on T lymphocytes, triggering cytokine release during inflammation. Lifitegrast competes with intercellular adhesion molecule 1 for lymphocyte function-associated antigen 1 and inhibits their interaction. In this study, lifitegrast was administered subcutaneously or intraperitoneally at three doses (0.25 mg / kg, 0.50 mg / kg, and 0.75 mg / kg) (Figure 5) to evaluate its efficacy in treating endometriosis. Subcutaneous administration of Refitegrast at both 0.25 mg / kg and 0.50 mg / kg significantly reduced the volume and weight of endometriotic lesions in mice (Figure 5A, first column of Figure 5F). Despite the fact that all three tested doses reduced integrin subunit beta-2 expression in endometriotic tissue at both mRNA and protein levels, the effects of Refitegrast at 0.50 mg / kg and 0.75 mg / kg were statistically significant (Figures 5B-C, second row of Figure 5F). Functionally, at doses of 0.50 mg / kg and above, Refitegrast significantly suppressed the proliferative capacity of endometriotic cells (Figure 5D, third row of Figure 5F). The use of both 0.50 mg / kg and 0.75 mg / kg significantly increased the amount of apoptotic cells in endometriotic lesions compared to the control group (Figure 5E, fourth row of Figure 5F).

[0069] Intraperitoneal administration of Lifitegrast at a dose of 0.75 mg / kg significantly reduced the volume and weight of endometriotic lesions in mice (Figure 5G, first row of Figure 5L), and suppressed the expression of integrin subunit beta 2 at both mRNA and protein levels (Figures 5H to I, second row of Figure 5L). Functionally, administration of 0.75 mg / kg of Lifitegrast significantly suppressed the proliferative capacity of endometriotic cells (Figure 5J, third row of Figure 5L), and increased the amount of apoptotic cells in endometriotic lesions compared to the control group (Figure 5K, fourth row of Figure 5L).

Claims

1. A method for treating endometriosis in a subject requiring treatment for endometriosis, comprising administering an effective amount of an integrin subunit beta-2 antagonist to the subject.

2. The method according to claim 1, wherein the integrin subunit beta 2 antagonist is one or more selected from the group consisting of GB-2175, Rifitegrast, BMS-688521, NPC-15669, BI-1950, BMS-587101, XVA143, BIRT377, A-286982, RWJ50271, and derivatives thereof.

3. Use of integrin subunit beta-2 antagonists in the manufacture of drugs for treating endometriosis.

4. The use according to claim 3, wherein the integrin subunit beta 2 antagonist is one or more selected from the group consisting of GB-2175, Rifitegrast, BMS-688521, NPC-15669, BI-1950, BMS-587101, XVA143, BIRT377, A-286982, RWJ50271, and derivatives thereof.

5. A drug for use in the treatment of endometriosis, comprising an integrin subunit beta-2 antagonist.

6. The agent for use according to claim 5, wherein the integrin subunit beta 2 antagonist is one or more selected from the group consisting of GB-2175, Rifitegrast, BMS-688521, NPC-15669, BI-1950, BMS-587101, XVA143, BIRT377, A-286982, RWJ50271, and derivatives thereof.

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