Application of phenylipressin acetate in preparation of medicine for preventing and / or treating acute respiratory distress syndrome

Abstract

The invention provides application of phenylipressin acetate in preparation of a medicine for preventing and / or treating acute respiratory distress syndrome, and belongs to the technical field of medicine. It is found that when phenylipressin acetate is injected into an acute respiratory distress syndrome (ARDS) mouse, neutrophil infiltration can be effectively reduced, and lung injury symptoms such as pulmonary hemorrhage, pulmonary capillary congestion and pulmonary capillary interval thickening can be reduced. Moreover, the phenylipressin acetate is given in advance, so that the occurrence of ARDS can be effectively prevented, the lung injury degree of mice can be reduced, symptoms such as neutrophil infiltration, pulmonary hemorrhage and pulmonary capillary congestion can be reduced, the level of inflammatory factors in serum can be reduced, and pulmonary edema can be relieved. Therefore, the phenylipressin acetate not only can be used for treating the acute respiratory syndrome and the acute lung injury, but also can be used for preventing the occurrence and development of the acute respiratory syndrome, and is remarkable in effect, high in safety and wide in application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to use of felypressin acetate in preparing a medicament for preventing and / or treating acute respiratory distress syndrome. Background Art

[0002] Acute lung injury (ALI) refers to the damage to pulmonary capillary endothelial cells and alveolar epithelial cells during non-cardiogenic diseases such as severe infection, shock, trauma and burns, resulting in diffuse pulmonary interstitial and alveolar edema, leading to acute hypoxic respiratory insufficiency or respiratory failure. When the damage reaches a certain level, acute respiratory distress syndrome (ARDS) may occur.

[0003] Currently, treatments for ARDS fall into two main categories: mechanical ventilation and non-mechanical ventilation. Mechanical ventilation is the primary treatment for ARDS. Depending on the type of mechanical ventilation, it is divided into non-invasive ventilation and invasive ventilation. Non-invasive ventilation relies on a mask, while invasive ventilation relies on an endotracheal tube or tracheostomy tube. Although there are many non-mechanical ventilation treatments for ARDS, their reliable efficacy has yet to be established. Non-mechanical ventilation treatments include: lung water removal and fluid management, alveolar surfactant replacement therapy, the use of antioxidants and enzyme inhibitors, blood purification therapy, and nutritional intervention.

[0004] Felypressin acetate (CAS: 914453-97-7) is a non-catecholamine vasoconstrictor and vasopressin 1 agonist. Felypressin acetate is currently widely used in dental surgery.

[0005] There are currently no reports on the use of felypressin acetate in the treatment of acute respiratory distress syndrome. Summary of the Invention

[0006] The present invention aims to provide a use of felypressin acetate in preparing a medicament for preventing and / or treating acute respiratory distress syndrome.

[0007] The present invention provides a use of felypressin acetate in preparing a medicament for preventing and / or treating acute respiratory distress syndrome.

[0008] The present invention also provides a use of felypressin acetate in preparing a medicament for treating and / or preventing acute lung injury.

[0009] Furthermore, the drug is a drug for reducing lung damage; the lung damage is pulmonary hemorrhage, pulmonary capillary congestion and / or pulmonary capillary septal thickening.

[0010] Furthermore, the drug is a drug that reduces lung neutrophil infiltration.

[0011] Furthermore, the drug is a drug that reduces the expression level of IL-6 in serum, or reduces the expression level of TNF-α, or alleviates pulmonary edema.

[0012] Furthermore, the drug is an oral preparation or an injection preparation prepared with felypressin acetate as the active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients; preferably, the preparation is an injection preparation.

[0013] Furthermore, the injection preparation is an intravenous injection preparation, an intramuscular injection preparation or a subcutaneous injection preparation,

[0014] Furthermore, the injection preparation is an intravenous injection preparation.

[0015] The present invention also provides a drug for preventing and / or treating acute respiratory distress syndrome and / or acute lung injury, which is an oral preparation or an injectable preparation prepared with felypressin acetate as the active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients; preferably, the preparation is an injectable preparation.

[0016] Furthermore, the injection preparation is an intravenous injection preparation, an intramuscular injection preparation or a subcutaneous injection preparation, preferably an intravenous injection preparation.

[0017] The present invention provides a use of felypressin acetate in the preparation of a medicament for preventing and / or treating acute respiratory distress syndrome. The present invention has found through experiments that injection of felypressin acetate into ARDS model mice can effectively reduce neutrophil infiltration, reduce lung hemorrhage, pulmonary capillary congestion, and lung capillary septal thickening and other lung injury symptoms. More importantly, pre-administration of felypressin acetate can also effectively reduce the degree of lung injury in ARDS model mice, including reducing symptoms such as neutrophil infiltration, pulmonary hemorrhage, and pulmonary capillary congestion, while reducing the levels of IL-6 and TNF-α in serum, and reducing pulmonary edema. Therefore, felypressin acetate can not only be used to treat acute respiratory syndrome and acute lung injury, but also can be used to prevent acute respiratory syndrome and acute lung injury, with significant effect, high safety, and broad application prospects.

[0018] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0019] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Results of an experiment using felypressin acetate to treat ARDS. A) Schematic diagram of the experimental process; B) Lung injury assessment results for mice in each experimental group; C) H&E staining to observe lung tissue damage in mice; D) Immunofluorescence staining to observe neutrophil infiltration in the lung tissue of mice in each group; E) Flow cytometry to quantitatively detect neutrophils in lung tissue.

[0021] Figure 2 Experimental results of felypressin acetate in preventing ARDS. A) Schematic diagram of the experimental process; B) IL-6 expression levels in the serum of mice in each group; C) TNF-α expression levels in the serum of mice in each group; D) Myeloperoxidase (MPO) expression levels in mouse lung tissue; E) Lung injury assessment results; F) H&E staining results of mouse lung tissue; G) Fluorescent staining results of neutrophils in mouse lung tissue; H) Quantitative detection of neutrophils in lung tissue by flow cytometry; I) Dry-to-wet ratio of lung tissue (pulmonary edema results).

[0022] Figure 3 Results of the in vivo safety assessment. A) Alanine aminotransferase (ALT) level in mouse blood; B) Albumin (ALB) level in mouse blood; C) Aspartate aminotransferase (AST) level in mouse blood; D) Alkaline phosphatase (ALP) level in mouse blood; E) Total protein (TP) level in mouse blood; F) Creatinine (CREA) level in mouse blood. G) H&E staining of the mouse heart, liver, spleen, and kidney. DETAILED DESCRIPTION

[0023] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0024] Felypressin acetate used in the experiment was purchased from MCE (https: / / www.medchemexpress.cn / felypressin-acetate.html) with the catalog number: HY-A0182A (CAS No. 56-59-7). LPS was purchased from Shanghai R&D Systems Inc.

[0025] Example 1: Felypressin acetate in the treatment of ARDS

[0026] 1. Experimental Methods

[0027] (1) Establishment of ARDS model

[0028] Lipopolysaccharide (LPS) was used to induce ARDS model in mice. The specific operation was as follows: 6-8 week-old Balb / c male mice (purchased from Beijing Huafukang Biotechnology Co., Ltd., Beijing, China) were placed on a fixed rack, and 4 mg / kg LPS was aerosolized and injected into the airways of Balb / c mice using a nebulizer (AP-1AirPump; Beijing Yuansen Kaide; HY-LWH02).

[0029] (2) Experimental groups

[0030] Mice were divided into three groups (n=6 per group): a control group (CTL), an ARDS group, and a felypressin acetate group (HY-A0182A group). Mice in the CTL control group had free access to water, a normal diet, and a standard maintenance diet without any special treatment. Mice in the ARDS and HY-A0182A groups inhaled 4 mg / kg LPS to establish an ARDS model. In the HY-A0182A group, felypressin acetate was injected intravenously 1 and 6 hours after inhalation of 4 mg / kg LPS. Twelve hours after modeling, lung tissue and blood were isolated from each group for pharmacodynamic evaluation and mechanism studies.

[0031] (3) H&E staining

[0032] Mouse lung tissues were fixed in 10% neutral buffered formalin solution at 4°C for 48 h, then embedded in paraffin and cut into 5 μm thick sections. The sections were stained with hematoxylin-eosin (H&E) for pathological evaluation and analyzed using an Olympus CKX53 microscope and CaseViewer Native Windows application software.

[0033] (4) Lung injury assessment

[0034] Lung injury in mice was evaluated by H&E staining, and the severity of injury was scored as follows: based on four independent indicators, namely, pulmonary hemorrhage, neutrophil infiltration, pulmonary capillary congestion, and septal thickening, the severity of lung injury was divided into grades 0 to 4; grade 0: normal; grade 1: mild injury (<25% injury); grade 2: moderate injury (25% to 50% injury); grade 3: severe injury (50% to 75% injury); grade 4: extremely severe injury (>75% injury). The score for each mouse was calculated as the average of five randomly selected areas. (5) Immunofluorescence detection of neutrophil infiltration in lung tissue

[0035] In the case of acute lung injury (ALI) or acute respiratory syndrome (ARDS), neutrophil infiltration is an important feature of pathological changes. The number of neutrophils in lung tissue is detected by immunofluorescence to assess the severity of the disease. The antibody used in this experiment is a neutrophil-specific Ly-6G antibody. First, mouse lung tissue is taken and frozen. Then, it is washed with PBS three times for 3 minutes each time, followed by fixing the slides with 4% paraformaldehyde (prepared in PBS) for 15 minutes, and then the slides are washed with PBS three times for 3 minutes each time. Next, 0.5% Triton X-100 (prepared in PBS) is added to each well, and permeabilization is carried out at room temperature for 20 minutes. Then, the slides are washed again with PBS three times for 3 minutes each time, the PBS is absorbed with absorbent paper, and normal goat serum or 5% BSA is added to the slides and blocked at room temperature for 30 minutes. Subsequently, the blocking solution was removed with absorbent paper. Without washing, a sufficient amount of diluted primary antibody (1:200) was added to each slide and placed in a humidified chamber (primary antibody prepared in 1% BSA) for overnight incubation at 4°C. The next day, the slides were washed three times with PBST (1L PBS plus 1ml of PBST) for 3 minutes each time. After blotting the excess liquid on the slides with absorbent paper, the diluted fluorescent secondary antibody (1:200 in 1% BSA) was added and incubated in a humidified chamber at 20-37°C (room temperature) for 1 hour. The sections were then washed three times with PBST for 3 minutes each time and observed under a fluorescence microscope.

[0036] (6) Flow cytometry to detect the number of neutrophils in lung tissue

[0037] By using flow cytometry technology, combined with specific monoclonal antibody labeling, the neutrophils in the mouse lung tissue were accurately counted and analyzed. Specifically, the lung tissue of the mouse was first dissected and then placed on a filter containing phosphate buffered saline (PBS) for grinding to prepare a cell suspension. Three monoclonal antibodies (anti-CD45-FITC, anti-CD11b-APC and anti-Ly6G-BV421) were used for staining to specifically label and count neutrophils. CD45 is a common surface marker of leukocytes, while CD11b and Ly6G are unique markers of neutrophils. The combined use of these markers can ensure the accurate identification and counting of neutrophils. The stained cell suspension was then sent to a flow cytometer for analysis.

[0038] 2. Experimental Results

[0039] (1) Lung injury results

[0040] The results of H&E staining are as follows Figure 1As shown in C, the ARDS group showed significantly more severe pathological manifestations such as pulmonary hemorrhage, neutrophil infiltration, pulmonary capillary congestion, and septal thickening than the control group, indicating that the ARDS model was successfully established by LPS. The HY-A0182A group showed significantly less pathological manifestations than the ARDS group, and the lung injury score was significantly reduced ( Figure 1 B).

[0041] Experimental results showed that felypressin acetate significantly reduced lung damage in ARDS model mice, including pulmonary hemorrhage, neutrophil infiltration, pulmonary capillary congestion, and septal thickening. Felypressin acetate is a promising treatment for acute respiratory syndrome and acute lung injury, with excellent efficacy.

[0042] (2) Neutrophil infiltration results

[0043] Immunofluorescence detection of neutrophil infiltration in lung tissue Figure 1 As shown in D, the results of flow cytometry detection of neutrophil count in lung tissue are as follows Figure 1 E. It can be seen that the neutrophil infiltration of ARDS group mice was significantly higher than that of the control group, indicating that the ARDS model mice were successfully established, and the neutrophil infiltration of the HY-A0182A group was significantly lower than that of the ARDS group.

[0044] The experimental results showed that felypressin acetate can significantly reduce neutrophil infiltration in ARDS model mice and can be used to treat acute respiratory syndrome and acute lung injury with excellent efficacy.

[0045] In summary, felypressin acetate can effectively reduce neutrophil infiltration in ARDS model mice, reduce lung damage such as pulmonary hemorrhage, pulmonary capillary congestion, and thickening of pulmonary capillary septa, and can be used to treat acute respiratory syndrome and acute lung injury with excellent results.

[0046] Example 2: Preventive Effect of Felypressin Acetate on ARDS

[0047] 1. Experimental Methods

[0048] (1) Animal grouping and modeling

[0049] Mice were randomly divided into three groups (n=6 per group): a control group (CTL), an ARDS group, and a felypressin acetate group (HY-A0182A group). The CTL group received free access to water, a normal diet, and a standard maintenance diet without any special treatment. Mice in the ARDS and HY-A0182A groups received 4 mg / kg LPS inhalation to establish an ARDS model.

[0050] The HY-A0182A group received three intravenous injections of felypressin acetate at a dose of 5 mg / kg: 12 hours, 6 hours, and 6 hours before modeling. Twelve hours after modeling, lung tissue was isolated from mice in each group, and blood was analyzed.

[0051] (2) Lung injury assessment

[0052] The method is the same as part (4) of Example 1.

[0053] (3) Detection of inflammatory factors by enzyme-linked immunosorbent assay

[0054] The expression levels of inflammatory cytokines TNF-α (88-7324-22) and IL-6 (BMS603-2) in mouse serum were detected by ELISA kits (eBioscience Co, Invitrogen, San Diego, CA).

[0055] A 96-well plate (Corning Costar 9018) was coated with 100 μL / well of capture antibody and incubated at 4°C overnight. Horseradish peroxidase (HRP)-conjugated antibody was added, and the absorbance was read at 450 nm to analyze the expression level of inflammatory cytokines.

[0056] (4) Detection of myeloperoxidase (MPO) expression levels in lung tissue

[0057] Myeloperoxidase (MPO) is a heme protein primarily found in neutrophils. LPS stimulation can recruit and activate neutrophils to release MPO. Measuring MPO expression can be used to determine the number of neutrophils in lung tissue. Higher MPO expression indicates greater neutrophil activation and infiltration in the lungs. MPO is an important biomarker for acute respiratory syndrome and acute lung injury.

[0058] 100 mg of mouse lung tissue was taken, 1 mL of reaction solution (50 mM hexadecyltrimethylammonium bromide, 50 mM KH2PO4 solution at pH 6.0, 0.5 mM EDTA solution) was added, the lung tissue was homogenized, and the supernatant was removed after centrifugation at 12000 r / min and 4°C for 15 min. 100 μL of buffer (0.167 mg / mL O-dianisidine solution, 50 mM KH2PO4 solution at pH 6.0, 0.0005% mM H2O2 solution) was added, and the OD value was measured at 460 nm to analyze the expression of MPO in neutrophils.

[0059] (5) Detection of pulmonary edema

[0060] The lung tissue of mice was taken and the wet weight (W) was measured. The lung tissue was dried in a 60°C oven for 72 hours. The sample was then reweighed to obtain the dry weight (D) of the lung tissue and the dry-wet ratio (W / D) was calculated. The wet-to-dry weight ratio was used to detect pulmonary edema.

[0061] (6) Immunofluorescence detection of neutrophil infiltration in lung tissue

[0062] The method is the same as that in part (5) of Example 1.

[0063] (7) Flow cytometry to detect the number of neutrophils in lung tissue

[0064] The method is the same as that in part (6) of Example 1.

[0065] 2. Experimental Results

[0066] (1) Results of serum pro-inflammatory factor detection

[0067] The levels of IL-6 and TNF-α in mouse serum were Figure 2 B and Figure 2 As shown in Figure C, the ARDS group had significantly higher levels of IL-6 and TNF-α in their serum compared to the control group, while the HY-A0182A group had significantly lower levels of IL-6 and TNF-α in their serum compared to the ARDS group. This suggests that HY-A0182A can effectively reduce pro-inflammatory factors in the blood, thereby preventing the development of ARDS.

[0068] (2) Myeloperoxidase (MPO) expression level detection results

[0069] Myeloperoxidase (MPO) expression level test results Figure 2 As shown in D, it can be seen that the MPO expression level in the serum of ARDS group mice was significantly higher than that of the control group, indicating that the ARDS model mice were successfully constructed, while the MPO expression level in the serum of the HY-A0182A group was significantly lower than that of the ARDS group and close to that of the control group, reflecting that felypressin acetate can prevent neutrophil infiltration caused by acute lung injury.

[0070] (3) Lung injury test results

[0071] Microscopic observation of H&E staining results Figure 2 As shown in F; compared with the control group, the ARDS group had an increase in granulocytes, defects in alveolar structure, thickening of alveolar walls and congestion of pulmonary capillaries. The HY-A0182A group had significantly alleviated lung pathological changes, a decrease in granulocytes, improved alveolar structural integrity, reduced alveolar wall thickening and congestion of pulmonary capillaries compared with the ARDS group. The lung injury index was as shown in Figure 2 As shown in E.

[0072] (IV) Pulmonary edema test results

[0073] The wet-to-dry weight ratio of the lungs in the ARDS group was significantly increased compared with the control group, that is, pulmonary edema occurred in the ARDS group. The wet-to-dry weight ratio of the lungs in the HY-A0182A group was significantly reduced compared with the ARDS group. Felypressin acetate can reduce pulmonary edema in ARDS patients. Figure 2 As shown in I.

[0074] (V) Neutrophil infiltration test results

[0075] Immunofluorescence and flow cytometry were used to detect neutrophil infiltration in lung tissue. Figure 2 As shown in Figures G and 2H, the neutrophil infiltration in the ARDS group was significantly higher than that in the control group, indicating that the ARDS model mouse model was successfully established. Neutrophil infiltration in the HY-A0182A group was significantly lower than that in the ARDS group, indicating that felypressin acetate can effectively inhibit neutrophil infiltration caused by acute lung injury. This result is consistent with the results of MPO expression in lung tissue.

[0076] In summary, pre-administration of felypressin acetate can effectively reduce neutrophil infiltration in ARDS model mice, reduce lung damage such as pulmonary hemorrhage, pulmonary capillary congestion, and thickening of pulmonary capillary septa, reduce the levels of IL-6 and TNF-α in serum, and reduce pulmonary edema. It can be used to prevent acute respiratory syndrome and acute lung injury with excellent results.

[0077] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0078] Experimental Example 3: In vivo safety assessment of felypressin acetate

[0079] 1. Experimental Methods

[0080] (1) Animal grouping

[0081] BALB / C male mice (6-8 weeks old) were randomly divided into two groups, each consisting of six mice. The control group (CTL) received free access to water and a normal diet, along with a standard maintenance diet, without any special treatment. The felypressin acetate group (HY-A0182A group) received an intravenous injection of 110 μg of felypressin acetate. Twenty-four hours after injection, blood was collected from the orbital venous plexus, and organs such as the heart, spleen, liver, and kidney were harvested for histochemical analysis.

[0082] (2) Mouse blood biochemical test

[0083] The expression levels of alanine aminotransferase (ALT), albumin (ALB), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP) and creatinine (CREA) in the blood of mice in each group were detected by Siemens automatic biochemical analyzer.

[0084] (3) H&E staining of mouse organs

[0085] Except for the differences between the tissues, the H&E staining method was the same as that in Example 1 (3) H&E staining.

[0086] 2. Experimental Results

[0087] ALT (a marker of liver and gallbladder damage in the blood) Figure 3 A),ALB( Figure 3 B),AST( Figure 3 C), ALP( Figure 3 D), TP( Figure 3 E) and creatinine ( Figure 3 F), the results showed that there was no significant difference between the HY-A0182A group and the control group. In addition, H&E staining results showed that there was no inflammation and tissue damage in the heart, spleen, liver and kidney of mice in the HY-A0182A group, and there was no significant difference compared with the control group ( Figure 3 G).

[0088] In summary, felypressin acetate will not cause damage or inflammatory response to important organs such as the heart, spleen, liver and kidneys, and felypressin acetate is highly safe.

[0089] In summary, the present invention provides a use of felypressin acetate in the preparation of a medicament for preventing and / or treating acute respiratory distress syndrome. The present invention has found through experiments that injection of felypressin acetate into ARDS model mice can effectively reduce neutrophil infiltration, reduce lung hemorrhage, pulmonary capillary congestion, and lung injury symptoms such as thickening of the pulmonary capillary septum. More importantly, pre-administration of felypressin acetate can also effectively reduce the degree of lung injury in ARDS model mice, including reducing symptoms such as neutrophil infiltration, pulmonary hemorrhage, and pulmonary capillary congestion, while reducing the levels of IL-6 and TNF-α in serum, and reducing pulmonary edema. Therefore, felypressin acetate can not only be used to treat acute respiratory syndrome and acute lung injury, but also can be used to prevent acute respiratory syndrome and acute lung injury, with significant effect, high safety, and broad application prospects.

Claims

1. Use of felypressin acetate in the preparation of a medicament for preventing and / or treating acute respiratory distress syndrome.

2. Use of felypressin acetate in the preparation of a medicament for treating and / or preventing acute lung injury.

3. The use according to claim 1 or 2, characterized in that: The drug is a drug for reducing lung damage; the lung damage is pulmonary hemorrhage, pulmonary capillary congestion and / or pulmonary capillary septal thickening.

4. The use according to claim 3, characterized in that: The drug is a drug for reducing pulmonary neutrophil infiltration.

5. The use according to claim 3, characterized in that: The drug is a drug that reduces the expression level of IL-6 in serum, reduces the expression level of TNF-α, and alleviates pulmonary edema.

6. The use according to any one of claims 1 to 5, characterized in that: The medicine is an oral preparation or injection preparation prepared by taking felypressin acetate as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

7. The use according to claim 6, characterized in that: The preparation is an injection preparation.

8. The use according to claim 7, characterized in that: The injection preparation is an intravenous injection preparation.

9. The use according to claim 7, characterized in that: The injection preparation is an intramuscular injection preparation.

10. The use according to claim 7, characterized in that: The injection preparation is a subcutaneous injection preparation.

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