Application of ixazomib citrate in preparation of medicine for preventing or treating diseases related to pulmonary edema and lung inflammatory cell infiltration

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of ixazomib citrate in preparation of medicines for preventing or treating diseases related to pulmonary edema and lung inflammatory cell infiltration. The technical problem to be solved by the invention is to provide a new choice for prevention or treatment of pulmonary edema and lung inflammatory cell infiltration related diseases. The invention provides an application of ixazomib citrate in preparation of a medicine for treating or preventing pulmonary edema and lung inflammatory cell infiltration related diseases. It is found for the first time that the ixazomib citrate can significantly inhibit abnormal metabolism of lung HA, and the action mechanism of the ixazomib citrate is closely related to down-regulation of expression of HAS1 and HYAL1. The invention not only provides a new choice for preventing or treating diseases related to pulmonary edema and lung inflammatory cell infiltration, but also has a good application prospect; meanwhile, the indications of the ixazomib citrate are also expanded.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of ixazomib citrate in the preparation of a medicament for preventing or treating diseases related to pulmonary edema and pulmonary inflammatory cell infiltration. Background Art

[0002] Pulmonary edema and / or inflammatory cell infiltration of the lungs are common pathological features of a variety of acute and chronic diseases. These conditions typically present with lung parenchymal damage, impaired gas exchange, and varying degrees of hypoxemia. Severe cases can lead to respiratory failure and are associated with significant morbidity and mortality. Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are typical examples of these conditions. These conditions can be precipitated by a variety of etiologies, including infectious factors (e.g., bacteria, viruses, fungi, and parasites), immune-mediated diseases, cardiac or non-cardiac circulatory disorders, toxic insults, adverse drug reactions, and tumor-related pathologies. Inflammation is a common and core pathogenesis in these diseases. During the pathological process of disease development, alveolar structure is damaged and vascular permeability increases, leading to fluid extravasation and pulmonary edema. Simultaneously, a large number of immune cells (such as neutrophils, macrophages, and lymphocytes) infiltrate the lung tissue and release a large number of pro-inflammatory cytokines such as IL-2, TNF-α, IL-1β, and IL-6, further exacerbating lung damage. This inflammatory cascade not only impairs the barrier function of the alveolar epithelium and capillary endothelium but also induces lung tissue structural remodeling and functional deterioration, leading to complications such as pulmonary interstitial fibrosis and pulmonary hypertension.

[0003] Despite progress in research on these conditions, many candidate drugs targeting inflammatory pathways or immune cell activity are still in the preclinical or early clinical research stages. Currently, there are no clinically recognized, effective targeted therapeutics for the pathological mechanisms of pulmonary edema and inflammatory cell infiltration. Treatment primarily relies on supportive measures, including anti-infection, mechanical ventilation, and steroids, which have limited efficacy in controlling disease progression. Therefore, there is an urgent need to develop new therapeutic approaches with clear anti-inflammatory mechanisms that can regulate the lung immune microenvironment and alleviate lung tissue damage.

[0004] Hyaluronic acid (HA), a key component of the extracellular matrix, is the primary glycosaminoglycan in lung tissue. It plays a crucial role in maintaining lung homeostasis, regulating inflammatory responses, resisting infection, and repairing tissue damage. Studies have shown that HA degradation products produced by hyaluronidase and reactive oxygen species are associated with a variety of diseases, including infectious factors (such as bacteria, viruses, fungi, and parasites), immune-mediated diseases, cardiac and non-cardiac circulatory disorders, toxic injuries, adverse drug reactions, and tumor-related diseases. During the pathological processes of these diseases, HA levels are significantly elevated, participating in the regulation of inflammatory cell infiltration, increasing vascular permeability, and promoting the formation of alveolar and interstitial edema.

[0005] HA levels are regulated by its synthesis and degradation metabolic pathways. HA is primarily synthesized by HA synthases, of which hyaluronan synthase 1 (HAS1) is a key enzyme regulating inflammation-related HA synthesis in the lungs. Its expression is upregulated or dysregulated in various lung diseases, such as pulmonary hypertension, asthma, and COPD. Hyaluronidase 1 (HYAL1) is involved in the degradation of HA, generating low-molecular-weight HA fragments with proinflammatory activity that can further exacerbate lung inflammation and tissue damage.

[0006] Ixazomib citrate is the first FDA-approved oral proteasome inhibitor, a small molecule compound from the boronate class. Rapidly converted to its active form in the body, ixazomib is a well-accepted and well-tolerated proteasome inhibitor currently used primarily for the treatment of multiple myeloma. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a new option for the prevention or treatment of diseases related to pulmonary edema and pulmonary inflammatory cell infiltration.

[0008] The present invention provides use of ixazomib citrate in preparing a medicament for treating or preventing pulmonary edema and diseases related to pulmonary inflammatory cell infiltration.

[0009] Specifically, the diseases related to pulmonary edema and pulmonary inflammatory cell infiltration are ALI and / or ARDS.

[0010] Furthermore, the ALI is induced by LPS.

[0011] Furthermore, the ALI is caused by HA accumulation.

[0012] Furthermore, the ARDS is induced by LPS.

[0013] Furthermore, the ARDS is caused by HA accumulation.

[0014] The present invention also provides the use of ixazomib citrate in preparing a substance for inhibiting the expression of HA, HAS1 and / or HYAL1.

[0015] Beneficial effects of the present invention: The present invention discovered for the first time that ixazomib citrate can significantly inhibit the abnormal metabolism of HA in the lungs, and its mechanism of action is closely related to downregulating the expression of HAS1 and HYAL1. Experiments have shown that ixazomib citrate can significantly inhibit pulmonary edema, pulmonary infiltration of macrophages and neutrophils, reduce vascular permeability, and reduce the production of inflammatory factors, thereby inhibiting the occurrence of pulmonary edema and improving lung function; it has good preventive and therapeutic effects on a variety of pulmonary edema and inflammatory injuries caused by infectious, immune, cardiogenic or other causes. The present invention not only provides a new option for the prevention or treatment of diseases related to pulmonary edema and pulmonary inflammatory cell infiltration, and has good application prospects; it also expands the indications of ixazomib citrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 (A) UMAP dimensionality reduction embedding of bronchoalveolar lavage fluid (BALF) samples from pneumonia patients from a public database. (B) Expression levels of HAS1 and HYAL1 in samples from healthy individuals, patients with moderate pneumonia, and patients with severe pneumonia. (C) Two-dimensional (left) and three-dimensional (right) molecular docking images of ixazomib citrate (HY-10452) with HAS1.

[0017] Figure 2 (A) Liver and kidney function indicators were measured in the control group, LPS-induced ALI / ARDS model mice group, and LPS-treated ALI / ARDS model mice group. (B) HE staining of mouse lung tissue, 100× and 400× magnifications, respectively; Smith lung injury score; and statistical graph of Smith lung injury scores in mouse lung tissue.

[0018] Figure 3 (A) Macroscopic observation of mouse lung tissue; statistical diagram of the dry-to-wet ratio of mouse lung tissue. (B) Proportion of macrophages in mouse lung tissue samples (n=6); proportion of neutrophils in mouse lung tissue samples (n=6); proportion of neutrophils in mouse blood samples (n=8); proportion of immune cells expressing IL-2 in mouse lung tissue samples (n=6).

[0019] Figure 4(A) ELISA detection of HA expression levels in mouse bronchoalveolar lavage fluid (BALF) samples (n=6). (B) ELISA detection of HAS1 expression levels in mouse bronchoalveolar lavage fluid (BALF) samples (n=6). (C) ELISA detection of HYAL1 expression levels in mouse bronchoalveolar lavage fluid (BALF) samples (n=6). DETAILED DESCRIPTION

[0020] In order to screen drugs with therapeutic effects on lung infection, we re-analyzed the single-cell transcriptome sequencing data of alveolar lavage fluid cells in a previously published article (title: Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19; DOI: 10.1038 / s41591-020-0901-9) Figure 1 A) It was found that the expression of hyaluronan-related enzymes was significantly increased in patients with severe lung infection, and the expression of HAS1 and HYAL1 was the most obvious ( Figure 1 B). Therefore, a computer-generated virtual drug screening was conducted using HAS1 or HYAL1 as the target. After extensive screening, the top-ranked compound, ixazomib citrate (HY-10452), was obtained. The chemical structure of ixazomib citrate is as follows: Furthermore, a series of validation experiments were conducted using an LPS-induced ALI / ARDS disease model. These experiments demonstrated that ixazomib citrate significantly reduced the production of HA, HAS1, and inflammatory factors, and prevented the infiltration of macrophages and neutrophils. It also significantly inhibited pathological changes in the lung tissue of mice with pulmonary edema and diseases associated with pulmonary inflammatory cell infiltration, indicating that ixazomib citrate can be used to prevent and / or treat diseases associated with pulmonary edema and pulmonary inflammatory cell infiltration, as well as related conditions.

[0021] In summary, the present invention has obtained, through extensive screening, a compound, ixazomib citrate, which has excellent preventive and / or therapeutic effects on diseases causing pulmonary edema and pulmonary inflammatory cell infiltration and related conditions; and also expands the scope of indications of the compound ixazomib citrate.

[0022] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0023] Example 1: Using hyaluronic acid-related enzymes as targets, effective hyaluronic acid-related enzyme inhibitors in the US FDA drug database were screened by computer virtual screening. Based on the single-cell transcriptome sequencing data of bronchoalveolar lavage fluid cells in the previously published article (title: Single-cell landscape of bronchoalveolarimmune cells in patients with COVID-19; DOI: 10.1038 / s41591-020-0901-9), we re-analyzed the data. Figure 1 A) It was found that the expression of hyaluronan-related enzymes was significantly increased in patients with severe lung infection, and the expression of HAS1 and HYAL1 was the most obvious ( Figure 1 B). Therefore, we conducted virtual drug screening targeting HAS1 or HYAL1. Literature research revealed that a high proportion of AlphaFold-predicted three-dimensional structures of Human HAS1 scored 70 or higher, demonstrating the reliability of AlphaFold's predicted structures. Therefore, we selected the substrate binding pocket of Human HAS1 (reference PDB ID: 7SP8, the binding pocket of UD1) predicted by AlphaFold and conducted Schrödinger Maestro 11.4 virtual screening, hoping to identify small molecules with strong binding to the target protein.

[0024] The specific steps are as follows: Download the predicted three-dimensional structure of Human HAS1 from the AlphaFold website (AlphaFold ID: AF-Q92839-F1). Hydrogenate the protein using the Protein Preparation Wizard module. Energy optimization is then performed (OPLS2005 force field, RMSD 0.30 Å). A grid file is generated using the Receptor Grid Generation module, centered around ASP237 / TYR104 / LYS215 / TRP488 / GLN375, with a box size of 20 Å × 20 Å × 20 Å. A 2D format of the FDA-Approved Drug Library (containing 2.6K compounds) is processed using the Schrödinger software LigPrep Module for hydrogenation, energy optimization, and output as a 3D structure for virtual screening. The Virtual Screening Workflow module was used for virtual screening. The prepared compounds were imported and molecular docking was performed using the Glide module. This involves docking the receptor and ligand molecules through geometric and energy matching. Compounds from the FDA-Approved Drug Library were docked with the target protein Human HAS1 in the standard (SP) mode. The top 20% of small molecule compounds were then selected for a second round of screening using the standard (SP) mode to obtain a ranking of the small molecule compounds. Finally, the binding affinity between the target and the compound, the compound structure, and other factors were manually reviewed to select Ixazomib citrate (HY-10452), the compound ranked first in the FDA-Approved Drug Library. Figure 1 The docking score for ixazomib citrate and human HAS1 is -8.67. In the 3D image, the human HAS1 protein C backbone is shown in green, nitrogen atoms in blue, oxygen atoms in bright red, hydrogen atoms in white, and HY-10452 as a light blue stick. Hydrogen bond lengths are shown as red dashed lines; longer bond lengths indicate weaker hydrogen bonds. Ixazomib citrate can form 6 hydrogen bonds and 1 halogen bond with Human HAS1 protein: two amide bonds can form 4 hydrogen bonds with ARG378, TRP379, GLN375, and ASP237, with distances of 2.0 Å, 2.3 Å, 2.5 Å, and 2.2 Å, respectively; one carboxyl group can form 2 hydrogen bonds with TYR104 and ARG493, with distances of 1.7 Å and 2.4 Å, respectively; one chlorine atom can form 1 halogen bond with ARG340, with a distance of 2.3 Å ( Figure 1C).

[0025] Example 2 Ixazomib citrate has an effective therapeutic effect on lung pathological changes in ALI / ARDS mice Experimental animals: Female C57BL / 6 mice, SPF grade, 8-10 weeks old, weighing approximately 20-25 g, were purchased from Ruisiyuan Biotechnology Co., Ltd., Zhaoqing City, Guangdong Province. No known murine pathogens were found in parasite detection, bacterial detection, and serological examinations. Animal experiments were conducted after the mice had been acclimated to the environment for 1 week.

[0026] Experimental Methods: In this example, LPS-induced acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) disease models (ALI / ARDS models) were used as representative of numerous diseases associated with pulmonary edema and inflammatory cell infiltration. Mice were randomly divided into three groups: a control group, an ALI / ARDS model group, and an ALI / ARDS model group treated with ixazomib citrate. LPS (Sigma, Escherichia coli 055:B5) at a concentration of 5 mg / kg was injected intraperitoneally to induce the ALI / ARDS model. Two hours later, ixazomib citrate was administered via oral gavage at a concentration of 0.5 mg / kg in a volume of 100 μL. The control and ALI / ARDS model groups were gavaged with an equal volume of the drug solvent (2% DMSO + 40% PEG 300 + 5% Tween 80 + 53% PBS). On the second and third days, the drug was administered orally in the same manner. On the fourth day, the mice were euthanized and lung tissue was obtained. Peripheral blood was collected from the mouse eyeballs to test liver and kidney function indicators, including alanine aminotransferase, aspartate aminotransferase, creatinine, urea nitrogen, and uric acid. Figure 2 As shown in A, the results of the five indicators showed no statistical difference, which indicates that the drug has no liver and kidney toxicity. The right posterior lobe was fixed with neutral formaldehyde, embedded in paraffin, sliced ​​and stained with HE, and the lung damage was observed under an optical microscope. Figure 2 As shown in B, the magnification on the left is 100×, and the magnification on the right is 400×. Based on the photographic results, the pathological changes of ALI / ARDS lung tissue were scored. The pathological scoring was completed by professional technicians. Figure 2 The right side of B is the Smith lung injury score scale; the lower side is the Smith lung injury score statistical chart. The results show that the lung injury of the ALI / ARDS model mouse group is significantly aggravated compared with the control group, indicating that the model is successful, while the lung injury of the model + treatment group is significantly alleviated compared with the ALI / ARDS model mouse group, indicating that ixazomib citrate can effectively treat lung injury diseases. The left lung of the mouse was taken and the wet weight was immediately weighed. It was dried in an oven at 55℃ for 48 hours and the dry weight was weighed. Figure 3As shown in Figure A, the left side shows the macroscopic view of mouse lung tissue, and the right side shows the dry-to-wet ratio, which is used to measure the degree of pulmonary edema in mice. As shown in the statistical results, the pulmonary edema in the ALI / ARDS model mice group was significantly increased compared to the control group, while the pulmonary edema in the model + treatment group was significantly alleviated.

[0027] The data were expressed as mean ± standard error (Mean ± SEM). The differences among the groups were analyzed statistically using Unpaired-T test and One-way ANOVA. A significant difference was considered when p < 0.05.

[0028] Example 3 Ixazomib citrate effectively inhibits the increase of inflammatory cells in the lungs and peripheral blood of ALI / ARDS mice Experimental animals: Female C57BL / 6 mice, SPF grade, 8-10 weeks old, weighing approximately 20-25 g, were purchased from Ruisiyuan Biotechnology Co., Ltd., Zhaoqing City, Guangdong Province. No known murine pathogens were found in parasite detection, bacterial detection, and serological examinations. Animal experiments were started after the mice had been acclimated to the environment for 1 week.

[0029] Experimental Methods: In this example, the LPS-induced ALI / ARDS model was used as a representative model of many diseases associated with pulmonary edema and inflammatory cell infiltration. Mice were randomly divided into three groups: a control group, an ALI / ARDS model group, and an ALI / ARDS model group treated with ixazomib citrate. LPS (Sigma, Escherichia coli 055:B5) at a concentration of 5 mg / kg was injected intraperitoneally to induce the ALI / ARDS model. Two hours later, the first oral administration of ixazomib citrate was performed at a concentration of 0.5 mg / kg in a volume of 100 μL. The control and ALI / ARDS model groups were gavaged with an equal volume of the drug solvent (2% DMSO + 40% PEG 300 + 5% Tween 80 + 53% PBS). On the second and third days, the drug was administered by gavage in the same manner. On the fourth day, the mice were anesthetized and euthanized. The eyeballs were removed and venous blood was collected. The mice were dissected and the intact lung tissue was removed and minced. The lung tissue was digested with a digestion solution (10 mL 1640 + 1 mg collagenase IV + DNase I (final concentration of 5 μg / mL) + 2% FBS). Each lung tissue sample was added to a Percoll separation buffer (Cytiva) to separate immune cells. After lysing the red blood cells, the remaining cells were stained on ice in the dark with Ly6G (APC-Cy7 fluorescent, Biolegend) flow cytometry antibody, CD11b (FITC fluorescent, Biolegend) flow cytometry antibody, F4 / 80 (PE-Cy7 fluorescent, Biolegend) flow cytometry antibody, and IL-2 (APC fluorescent, Biolegend) flow cytometry antibody. Finally, the flow cytometer was used to analyze the cells. Figure 3As shown in B, macrophages are CD11b + f / 4 / 80 + neutrophils are CD11b + Ly6G + IL-2 + Statistical results showed that after LPS modeling, the proportion of macrophages and neutrophils in the mouse lungs, the proportion of immune cells that release the inflammatory factor IL-2 in the lungs, and the proportion of neutrophils in the blood significantly increased, and ixazomib citrate had an effective inhibitory effect on these.

[0030] Example 4 Ixazomib citrate alleviates inflammation in the lungs and peripheral blood of ALI / ARDS mice by targeting HAS1 Experimental animals: Female C57BL / 6 mice, SPF grade, 8–10 weeks old, weighing approximately 20–25 g, were purchased from Ruisiyuan Biotechnology Co., Ltd., Zhaoqing City, Guangdong Province. No known murine pathogens were found in parasite, bacterial, and serological tests. Animal experiments were conducted after the mice had been acclimated to the environment for 1 week.

[0031] Experimental Methods: In this example, the LPS-induced ALI / ARDS model was used as a representative model of many diseases associated with pulmonary edema and inflammatory cell infiltration. Mice were randomly divided into three groups: a control group, an ALI / ARDS model group, and an ALI / ARDS model group treated with ixazomib citrate. LPS (Sigma, Escherichia coli 055:B5) at a concentration of 5 mg / kg was injected intraperitoneally to induce the ALI / ARDS model. Two hours later, the first oral administration of ixazomib citrate was performed at a concentration of 0.5 mg / kg in a volume of 100 μL. The control and ALI / ARDS model groups were gavaged with an equal volume of the drug solvent (2% DMSO + 40% PEG 300 + 5% Tween 80 + 53% PBS). On the second and third days, the drug was gavage-administered in the same manner. On the fourth day, the mice were anesthetized and euthanized, and their eyeballs were removed to obtain venous blood. The mice were dissected to expose the lung cavity and trachea. A thin thread was prepared and passed through the mouse trachea. A small inverted V-shaped opening was cut on the mouse trachea. A small gavage needle was inserted into the opening and the trachea was tightened with a thin thread (but be careful not to break the thin thread). 0.5 mL of PBS was injected for lavage and then aspirated. The lungs were rinsed slowly and gently 3 to 4 times, and the alveolar lavage fluid was placed in an ep tube on ice. After taking the alveolar lavage fluid from all mice, it was centrifuged at 3000 rpm and 4°C for 10 minutes. The supernatant was aspirated into a new ep tube for Elisa detection. The expression levels of HA, HAS1, and HYAL1 were tested for each sample, such as Figure 4 As shown, HA in bronchoalveolar lavage fluid samples of ALI / ARDS model mice group ( Figure 4 A) and HAS1 ( Figure 4 The expression of HYAL1 (HA) increased in the model + treatment group, while its expression was significantly lower in the ALI / ARDS model group compared with the ALI / ARDS model group, indicating that ixazomib citrate is effective in alleviating the accumulation of HA and HAS1 in the lungs. While no statistical difference was observed for HYAL1, the overall trend was consistent with that of HA and HAS1. These results suggest that ixazomib citrate exerts its anti-inflammatory effects by regulating the HA-HAS1 axis.

[0032] The data were expressed as mean ± standard error (Mean ± SEM). The differences among the groups were analyzed statistically using Unpaired-T test and One-way ANOVA. A significant difference was considered when p < 0.05.

[0033] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Use of ixazomib citrate in the preparation of drugs for treating or preventing pulmonary edema and diseases related to pulmonary inflammatory cell infiltration.

2. The application according to claim 1, characterized in that: The diseases related to pulmonary edema and pulmonary inflammatory cell infiltration are ALI and / or ARDS.

3. The application according to claim 2, characterized in that: The ALI was induced by LPS.

4. The application according to claim 2, characterized in that: The ALI is caused by HA accumulation.

5. The application according to claim 2, characterized in that: The ARDS was induced by LPS.

6. The application according to claim 2, characterized in that: The ARDS is caused by HA accumulation.

7. Use of ixazomib citrate in the preparation of a substance for inhibiting the expression of HA, HAS1 and / or HYAL1.

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