Use of a preparation of acetic acid phenylalanylarginine vasopressin for the prevention and / or treatment of acute respiratory distress syndrome

Abstract

The application provides a use of a teriparatide acetate in preparation of a medicine for preventing and / or treating acute respiratory distress syndrome, and belongs to the technical field of medicines.The application finds that after a mouse with acute respiratory distress syndrome (ARDS) is injected with the teriparatide acetate, neutrophil infiltration can be effectively reduced, and lung hemorrhage, lung capillary hyperemia and lung capillary septum thickening and other lung injury symptoms can be reduced.In addition, the teriparatide acetate can effectively prevent the occurrence of ARDS, can reduce the lung injury degree of the mouse, can reduce the neutrophil infiltration, lung hemorrhage and lung capillary hyperemia and other symptoms, can reduce the level of inflammatory factors in serum, and can reduce lung edema.Therefore, the teriparatide acetate can not only be used for treating acute respiratory syndrome and acute lung injury, but also can prevent the occurrence and development of acute respiratory syndrome, has a remarkable effect, is high in safety, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of phenyllis vasopressin acetate in the preparation of medicaments for the prevention and / or treatment of acute respiratory distress syndrome. Background Technology

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

[0003] Currently, treatment methods for acute respiratory distress syndrome (ARDS) fall into two main categories: mechanical ventilation and non-mechanical ventilation. Mechanical ventilation is the primary treatment for ARDS patients. Depending on the method, it is divided into non-invasive and invasive ventilation. Non-invasive ventilation relies on a face mask, while invasive ventilation relies on endotracheal intubation or a tracheostomy tube. Although there are many non-mechanical ventilation methods for ARDS, their reliable efficacy has not yet been definitively established. Non-mechanical ventilation methods include: pulmonary edema clearance and fluid management, pulmonary surfactant supplementation, 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 a vasopressin 1 agonist. Currently, Felypressin acetate is widely used in dental procedures.

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

[0006] The purpose of this invention is to provide the use of phenyllis vasopressin acetate in the preparation of a medicament for the prevention and / or treatment of acute respiratory distress syndrome.

[0007] This invention provides the use of phenyllis vasopressin acetate in the preparation of medicaments for the prevention and / or treatment of acute respiratory distress syndrome.

[0008] The present invention also provides the use of phenyllis vasopressin acetate in the preparation of medicaments for the treatment and / or prevention of acute lung injury.

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

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

[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 or injectable preparation made with phenylvasopressin acetate as the active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients; preferably, the preparation is an injectable preparation.

[0013] Furthermore, the injectable formulation is an intravenous injection formulation, an intramuscular injection formulation, or a subcutaneous injection formulation.

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

[0015] The present invention also provides a medicament for the prevention and / or treatment of acute respiratory distress syndrome and / or acute lung injury, which is an oral or injectable preparation made of phenylvasopressin acetate as the active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients; preferably, the preparation is an injectable preparation.

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

[0017] This invention provides the use of phenylvasopressin acetate in the preparation of a medicament for the prevention and / or treatment of acute respiratory distress syndrome (ARDS). Experiments have shown that injection of phenylvasopressin acetate into ARDS model mice effectively reduces neutrophil infiltration, pulmonary hemorrhage, pulmonary capillary congestion, and thickening of the pulmonary capillary septa, among other lung injury symptoms. More importantly, pre-administration of phenylvasopressin acetate also effectively reduces the severity of lung injury in ARDS model mice, including reducing neutrophil infiltration, pulmonary hemorrhage, and pulmonary capillary congestion, while simultaneously lowering serum IL-6 and TNF-α levels and alleviating pulmonary edema. Therefore, phenylvasopressin acetate can be used not only to treat acute respiratory syndrome and acute lung injury but also for the prevention of these conditions, demonstrating significant efficacy, high safety, and broad application prospects.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1 The results of the experiment on phenylalanine acetate for the treatment of ARDS are as follows: A) Schematic diagram of the experimental procedure; B) Assessment results of lung injury in mice in each experimental group; C) H&E staining to observe lung tissue damage in mice; D) Immunofluorescence staining to observe neutrophil infiltration in lung tissue of mice in each group; E) Quantitative detection of neutrophils in lung tissue by flow cytometry.

[0021] Figure 2 Experimental results of phenylvasopressin acetate in the prevention of ARDS. A) Schematic diagram of experimental procedure; B) Expression level of IL-6 in the serum of mice in each group; C) Expression level of TNF-α in the serum of mice in each group; D) Detection results of myeloperoxidase (MPO) expression level in mouse lung tissue; E) Assessment results of mouse lung injury; 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) Results of lung tissue dry-wet ratio (pulmonary edema results).

[0022] Figure 3 Results of in vivo safety assessment. A) Detection of alanine aminotransferase (ALT) levels in mouse blood; B) Detection of albumin (ALB) levels in mouse blood; C) Detection of aspartate aminotransferase (AST) levels in mouse blood; D) Detection of alkaline phosphatase (ALP) levels in mouse blood; E) Detection of total protein (TP) levels in mouse blood; F) Detection of creatinine (CREA) levels in mouse blood; G) H&E staining results of mouse heart, liver, spleen, and kidneys. Detailed Implementation

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

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

[0025] Example 1: Treatment of ARDS with phenyllisotrine acetate

[0026] I. Experimental Methods

[0027] (1) ARDS model establishment

[0028] A mouse model of ARDS was established using lipopolysaccharide (LPS). The specific procedure was as follows: 6-8 week old male Balb / c mice (purchased from Beijing Huafukang Biotechnology Co., Ltd., Beijing, China) were placed on a fixation frame, and 4 mg / kg LPS was nebulized into the airways of the Balb / c mice through a nebulizer (AP-1 Air Pump; Beijing Yuansen Kaide; HY-LWH02).

[0029] (2) Experimental Grouping

[0030] Mice were divided into three groups (n=6 per group): a control group (CTL), an ARDS group, and a phenylvasopressin acetate group (HY-A0182A group). Control group (CTL) mice had free access to water and a normal diet, fed a standard maintenance diet without any special treatment. ARDS and HY-A0182A group mice were induced to develop ARDS by inhaling 4 mg / kg LPS. HY-A0182A group: 1 h and 6 h after inhalation of 4 mg / kg LPS, phenylvasopressin acetate was administered intravenously at a dose of 5 mg / kg. Twelve h after modeling, lung tissue and blood from each group were isolated for pharmacodynamic evaluation and mechanistic studies.

[0031] (3) H&E staining

[0032] Mouse lung tissue was 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 and 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 regions. (5) Immunofluorescence detection of neutrophil infiltration in lung tissue

[0035] In cases of acute lung injury (ALI) or acute respiratory syndrome (ARDS), neutrophil infiltration is a key characteristic of the pathological changes. Immunofluorescence assays are used to detect the number of neutrophils in lung tissue to assess disease severity. The antibody used in this experiment was a neutrophil-specific Ly-6G antibody. First, mouse lung tissue was collected and frozen sections were prepared. The sections were then washed three times with PBS for 3 minutes each time, followed by fixation with 4% paraformaldehyde (prepared in PBS) for 15 minutes, and then washed three times with PBS for 3 minutes each time. Next, 300 μL of 0.5% Triton X-100 (prepared in PBS) was added to each well, and the slides were permeated at room temperature for 20 minutes. The slides were then washed three times again with PBS for 3 minutes each time, the PBS was blotted dry with absorbent paper, and normal goat serum or 5% BSA was added to the slides. The slides were then blocked at room temperature for 30 minutes. Subsequently, the blocking solution was absorbed with absorbent paper, and without washing, a sufficient amount of diluted primary antibody (1:200) was added to each slide for immersion, and the slides were placed in a humidified chamber (primary antibody prepared with 1% BSA) and incubated overnight at 4°C. The next day, the slides were washed three times with PBST (1L PBS added to 1ml Soft Water), 3 minutes each time. After absorbing excess liquid from the slides with absorbent paper, diluted fluorescent secondary antibody (1% BSA prepared with 1:200) was added, and the slides were incubated in a humidified chamber at 20-37°C (room temperature) for 1 hour. Then, the slides were washed three times with PBST, 3 minutes each time, and the images were observed and acquired under a fluorescence microscope.

[0036] (6) Flow cytometry detection of neutrophil count in lung tissue

[0037] Accurate counting and analysis of neutrophils in mouse lung tissue was achieved using flow cytometry combined with specific monoclonal antibody labeling. Specifically, mouse lung tissue was first obtained through dissection and then ground on a filter containing phosphate-buffered saline (PBS) to prepare a cell suspension. Neutrophils were specifically labeled and counted using three monoclonal antibodies (anti-CD45-FITC, anti-CD11b-APC, and anti-Ly6G-BV421). CD45 is a common surface marker of leukocytes, while CD11b and Ly6G are specific markers of neutrophils; the combination of these markers ensures accurate identification and counting of neutrophils. The stained cell suspension was then analyzed using a flow cytometer.

[0038] II. Experimental Results

[0039] (I) Lung Injury Outcome

[0040] H&E staining results are as follows Figure 1As shown in Figure C, the ARDS group exhibited significantly more severe pathological manifestations, including pulmonary hemorrhage, neutrophil infiltration, pulmonary capillary congestion, and septal thickening, compared to the control group, indicating the successful establishment of a mouse ARDS model using LPS. The HY-A0182A group showed significantly reduced pathological manifestations and a significantly lower lung injury score compared to the ARDS group. Figure 1 B).

[0041] Experimental results show that phenylvasopressin acetate can significantly reduce lung injury in ARDS model mice, reducing pathological conditions such as pulmonary hemorrhage, neutrophil infiltration, pulmonary capillary congestion, and septal thickening. Phenylvasopressin acetate can be used to treat acute respiratory syndrome and acute lung injury with excellent efficacy.

[0042] (II) Results of neutrophil infiltration

[0043] Immunofluorescence detection results of neutrophil infiltration in lung tissue are as follows Figure 1 As shown in Figure D, the results of flow cytometry analysis of neutrophil count in lung tissue are as follows: Figure 1 As shown in E, the neutrophil infiltration in the ARDS group was significantly higher than that in the control group, indicating that the ARDS model mouse was successfully constructed. Furthermore, the neutrophil infiltration in the HY-A0182A group was significantly lower than that in the ARDS group.

[0044] Experimental results show that phenyl lisiostatin 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, phenylvasopressin acetate can effectively reduce neutrophil infiltration, lung hemorrhage, pulmonary capillary congestion, and thickening of pulmonary capillary septa in ARDS model mice, and can be used to treat acute respiratory syndrome and acute lung injury with excellent results.

[0046] Example 2: The preventive effect of phenylpressin acetate on ARDS

[0047] I. 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 phenylethylamine acetate group (HY-A0182A group). Control group (CTL) mice had free access to water and a normal diet, fed a standard maintenance diet, and received no special treatment. ARDS and HY-A0182A group mice were induced to develop an ARDS model by inhaling 4 mg / kg LPS.

[0050] In the HY-A0182A group, mice were intravenously injected three times at a dose of 5 mg / kg at 12 hours, 6 hours, and 6 hours after modeling. Twelve hours after modeling, lung tissue and blood from each group of mice were separated and 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 (ELISA)

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

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

[0056] (4) Detect the expression level of myeloperoxidase (MPO) in lung tissue.

[0057] Myeloperoxidase (MPO) is a heme protein primarily found in neutrophils. LPS stimulation can aggregate and activate neutrophils, releasing MPO. The number of neutrophils in lung tissue can be determined by detecting MPO expression levels. Higher MPO expression levels indicate greater activation and infiltration of neutrophils 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 and 1 mL of reaction solution (50 mM cetyltrimethylammonium bromide, 50 mM pH 6.0 KH2PO4 solution, 0.5 mM EDTA solution) was added. The lung tissue was homogenized and centrifuged at 12000 r / min and 4 °C for 15 min. The supernatant was removed and 100 μL of buffer (0.167 mg / mL O-dianisidine solution, 50 mM pH 6.0 KH2PO4 solution, 0.0005% mM H2O2 solution) was added. The OD value was measured at 460 nm to analyze the expression level of MPO in neutrophils.

[0059] (5) Detection of pulmonary edema

[0060] Mouse lung tissue was collected, and its wet weight (W) was measured. The lung tissue was then dried in a 60°C oven for 72 hours. The sample was then weighed again to obtain the dry weight (D) of the lung tissue, and the wet-to-dry weight ratio (W / D) was calculated. The degree of pulmonary edema was assessed using the wet-to-dry weight ratio.

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

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

[0063] (7) Flow cytometry detection of neutrophil count in lung tissue

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

[0065] II. Experimental Results

[0066] (1) Detection results of pro-inflammatory factors in serum

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

[0068] (II) Results of myeloperoxidase (MPO) expression level detection

[0069] The results of myeloperoxidase (MPO) expression level detection are as follows: Figure 2 As shown in Figure D, the MPO expression level in the serum of ARDS mice was significantly higher than that in the control group, indicating that the ARDS model mouse was successfully constructed. In contrast, the MPO expression level in the serum of the HY-A0182A group was significantly lower than that in the ARDS group and close to that in the control group, reflecting that phenyl lisiostatin acetate can prevent neutrophil infiltration caused by acute lung injury.

[0070] (III) Lung Injury Detection Results

[0071] Microscopic observation of H&E staining results as follows Figure 2 As shown in F; the ARDS group showed increased granulocytes, alveolar structural defects, alveolar wall thickening, and pulmonary capillary congestion compared to the control group. The HY-A0182A group showed significantly reduced lung pathological changes, with decreased granulocytes, improved alveolar structural integrity, and reduced alveolar wall thickening and pulmonary capillary congestion compared to the ARDS group. The lung injury index was as follows: Figure 2 As shown in E.

[0072] (iv) Results of pulmonary edema detection

[0073] The lung wet-to-dry weight ratio was significantly increased in the ARDS group compared to the control group, indicating pulmonary edema in the ARDS group. The lung wet-to-dry weight ratio was significantly decreased in the HY-A0182A group compared to the ARDS group, suggesting that topical phenylvasopressin acetate can reduce pulmonary edema in ARDS patients. Figure 2 As shown in Figure I.

[0074] (V) Results of Neutrophil Infiltration Detection

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

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

[0077] The following experimental examples demonstrate the beneficial effects of the present invention.

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

[0079] I. Experimental Methods

[0080] (1) Grouping of animals

[0081] Male BALB / c mice (6-8 weeks old) were randomly divided into two groups of six each. The control group (CTL) mice had free access to water and a normal diet, fed a standard maintenance diet without any special treatment. The phenylethylamine acetate group (HY-A0182A group) mice were intravenously injected with 110 μg of phenylethylamine acetate. Twenty-four hours after injection, blood was collected from the orbital venous plexus, and organs such as the heart, spleen, liver, and kidneys were collected for histochemical analysis.

[0082] (2) Blood biochemistry detection in mice

[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 using a Siemens fully automated biochemical analyzer.

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

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

[0086] II. Experimental Results

[0087] ALT (a blood indicator reflecting liver and gallbladder damage) Figure 3 A),ALB( Figure 3 B),AST( Figure 3 C), ALP Figure 3 D), TP Figure 3 E) and creatinine, which reflects kidney damage ( Figure 3 F), the results showed no significant difference between the HY-A0182A group and the control group. Furthermore, H&E staining results showed no inflammation or tissue damage in the heart, spleen, liver, and kidneys of the HY-A0182A group mice, with no significant difference compared to the control group. Figure 3 G).

[0088] In conclusion, phenyl lisiostatin acetate does not cause damage or inflammatory reactions to vital organs such as the heart, spleen, liver, and kidneys, and has a high safety profile.

[0089] In summary, this invention provides the use of phenylvasopressin acetate in the preparation of a medicament for the prevention and / or treatment of acute respiratory distress syndrome (ARDS). Experiments conducted by this invention have shown that injection of phenylvasopressin acetate into ARDS model mice effectively reduces neutrophil infiltration, and decreases lung injury symptoms such as pulmonary hemorrhage, pulmonary capillary congestion, and thickening of pulmonary capillary septa. More importantly, pre-administration of phenylvasopressin acetate also effectively reduces the severity of lung injury in ARDS model mice, including reducing symptoms such as neutrophil infiltration, pulmonary hemorrhage, and pulmonary capillary congestion, while simultaneously lowering serum levels of IL-6 and TNF-α, and alleviating pulmonary edema. Therefore, phenylvasopressin acetate can be used not only to treat acute respiratory syndrome and acute lung injury, but also for the prevention of these conditions, with significant efficacy, high safety, and broad application prospects.

Claims

1. Use of phenyllisotrol acetate in the preparation of medicines for the prevention and / or treatment of acute respiratory distress syndrome.

2. The use according to claim 1, characterized in that: The drug in question is a medication that reduces lung damage.

3. The use according to claim 2, characterized in that: The drug in question is one that reduces neutrophil infiltration in the lungs.

4. The use according to any one of claims 1 to 3, characterized in that: The drug is an injectable formulation prepared with phenyl lisiostatin acetate as the active ingredient and pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that: The injectable preparation is an intravenous injection preparation.

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